CN1920040A - Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production - Google Patents

Abstract

Isolated nucleic acid molecules, designated SMP nucleic acid molecules, which encode novel SMP proteins from Corynebacterium glutamicum are described. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing SMP nucleic acid molecules, and host cells into which the expression vectors have been introduced. The invention still further provides isolated SMP proteins, mutated SMP proteins, fusion proteins, antigenic peptides and methods for the improvement of production of a desired compound from C. glutamicum based on genetic engineering of SMP genes in this organism.

Description

The Corynebacterium glutamicum gene of the protein of coding involved in carbon metabolism and power generation

The application is to be on June 23rd, 2000 applying date, and application number is 00811819.1, and denomination of invention is divided an application for the application for a patent for invention of " Corynebacterium glutamicum gene of the protein of coding involved in carbon metabolism and power generation ".

Related application

The application requires the priority of the United States Patent (USP) provisional application sequence number 60/143208 of applying in the U.S. Provisional Patent Application serial number 60/141031 of application on June 25th, 1999, on July 9th, 1999 and the United States Patent (USP) provisional application sequence number 60/151572 of applying on August 31st, 1999. This application also requires the priority of following patent application: the German patent application of on July 8th, 1999 application numbers 19931412.8, the German patent application of on July 8th, 1999 application numbers 19931413.6, the German patent application of on July 8th, 1999 application numbers 19931419.5, the German patent application of on July 8th, 1999 application numbers 19931420.9, the German patent application of on July 8th, 1999 application numbers 19931424.1, the German patent application of on July 8th, 1999 application numbers 19931428.4, the German patent application of on July 8th, 1997 application numbers 19931431.4, the German patent application of on July 8th, 1999 application numbers 19931433.0, the German patent application of on July 8th, 1999 application numbers 19931434.9, the German patent application of on July 8th, 1999 application numbers 19931510.8, the German patent application of on July 8th, 1999 application numbers 19931562.0, the German patent application of on July 8th, 1999 application numbers 19931634.1, the German patent application of on July 9th, 1999 application numbers 19932180.9, the German patent application of on July 9th, 1999 application numbers 19932227.9, the German patent application of on July 9th, 1999 application numbers 19932230.9, the German patent application of on July 14th, 1999 application numbers 19932924.9, the German patent application of on July 14th, 1999 application numbers 19932973.7, the German patent application of on July 14th, 1999 application numbers 19933005.0, the German patent application of on August 27th, 1999 application numbers 19940765.7, the German patent application of on September 3rd, 1999 application numbers 19942076.9, the German patent application of on September 3rd, 1999 application numbers 19942079.3, the German patent application of on September 3rd, 1999 application numbers 19942086.6, the German patent application of on September 3rd, 1999 application numbers 19942087.4, the German patent application of on September 3rd, 1999 application numbers 19942088.2, the German patent application of on September 3rd, 1999 application numbers 19942095.5, the German patent application of the German patent application of on September 3rd, 1999 application number application on September 3rd, 19942123.4 and 1999 numbers 19942125.0. The full content of all above cited application is all incorporated herein by reference especially.

Background of invention

Some product of the metabolic process of natural generation and accessory substance have practicality in multiple industry in cell, and described industry comprises food industry, feed industry, cosmetics industry and pharmacy industry. These molecules are referred to as " fine chemicals ", comprise amino acid, nucleotides and nucleosides, lipid and aliphatic acid, dihydroxylic alcohols, carbohydrate, aromatic compounds, vitamin and co-factor and enzyme that organic acid, proteinogenous and nonprotein generate. Their production is to need the large-scale culture of the bacterium of molecule to carry out by exploitation in order to produce and to secrete a large amount of one or more the most easily. A kind of useful especially biology that is used for this purpose is Corynebacterium glutamicum (Corynebacterium glutamicum), and this is a kind of gram-positive non-pathogenic bacteria. By the selection of bacterial strain, a series of saltants that need compound of many generations have been developed. Yet being chosen in the improved bacterial strain in specific molecular production aspect is a time-consuming and difficult process.

Brief summary of the invention

The invention provides the novel bacterial nucleic acid molecules that serves many purposes. These purposes comprise that evaluation can be used for producing the microorganism of fine chemicals, adjusting Corynebacterium glutamicum or Related Bacteria and produce fine chemicals, typing or evaluation Corynebacterium glutamicum or Related Bacteria, is used as the reference point of Corynebacterium glutamicum gene group mapping and is used as the mark that transforms. These novel nucleic acids molecule encodings are called (SMP) protein of albumen of glycometabolism and oxidative phosphorylation (sugar metabolism and_oxidative phosphorylation) herein.

Corynebacterium glutamicum is a kind of Gram-positive aerobic bacteria, is generally used for the miscellaneous fine chemicals of large-scale industrial production, the oxidation that also is used for degradable carbon hydrogen compound (for example for the treatment of the oil spill thing) and is used for terpenoid. Therefore, SMP nucleic acid molecules of the present invention can be used for identifying the microorganism that can for example produce fine chemicals by sweat. The modification of the adjusting of SMP expression of nucleic acid of the present invention or SMP sequence of nucleic acid molecules of the present invention can be in order to the production (for example improving productive rate or the output of one or more fine chemicals of various excellent bacillus or brevibacterium) of one or more fine chemicals of regulating microorganism.

SMP nucleic acid of the present invention also can be used for identifying the biology of Corynebacterium glutamicum or its closely related bacterial classification, or is used for identifying that whether mixed microorganism colony Glutamic Acid rod bacillus or its relevant bacterial classification exist. The invention provides the nucleotide sequence of multiple Corynebacterium glutamicum gene; By the probe with the section of crossing over the peculiar gene of Corynebacterium glutamicum, under stringent condition, survey the genomic DNA of the extraction of peculiar Microbial consortium culture or mixed microorganism culturing in groups thing, people can determine whether this biology exists. Although Corynebacterium glutamicum itself is non-pathogenic, it with the mankind in pathogenic species for example corynebacterium diphtheriae (Corynebacterium diphtheriae) (diphtheria pathogen) is relevant; Detection to this class biology has important clinical significance.

SMP nucleic acid molecules of the present invention also can be as the reference point of Corynebacterium glutamicum gene group or associated biomolecule genomic mapping. Equally, these molecules and variant thereof or its part can be as the marks of genetic engineering rod bacillus or brevibacterium bacterial classification.

The SMP albumen of novel nucleic acids molecule encoding of the present invention can for example be carried out the function that relates to carbon compound (for example sugar) metabolism in Corynebacterium glutamicum, or execution relates to by the function such as oxidative phosphorylation process produce power molecule. The known U.S. Patent number 4 that obtains cloning vector such as the Sinskey etc. for Corynebacterium glutamicum, 649,119 disclosed cloning vectors and be used for Corynebacterium glutamicum and the Genetic Manipulative Technology of relevant brevibacterium bacterial classification (such as brevibacterium (Brevibacterium lactofermentum)) (Yoshihama etc., J.Bacteriol.162:591-597 (1985); Katsumata etc., J.Bacteriol.159:306-311 (1984); With Santamaria etc., J.Gen.Microbiol.130:2237-2246 (1984)), then nucleic acid molecules of the present invention can be used for the genetic engineering of this biology, so that it becomes the better or more effective production bacterium of one or more fine chemicals. It may be because the direct effect of genetic manipulation of the present invention causes that this fine chemicals output or production efficiency improve, and perhaps may be that the indirect effect by this generic operation causes.

SMP albumen of the present invention changes may directly affect productive rate, output and/or the production efficiency that the Corynebacterium glutamicum strain that mixes this change albumen produces fine chemicals by number of mechanisms. Such as the high-energy carbon molecular degradation of sugar and such as NADH and FADH₂Compound be converted into the compound that contains energy-rich phosphate bond by oxidative phosphorylation and can produce multiple compounds, the fine chemicals that these compounds may need exactly itself is such as pyruvic acid, ATP, NADH and many intermediate sugar compounds. Kinetomeres (such as ATP) and reducing equivalent thing (such as NADH or NADPH) that cell can utilize these metabolic pathways to produce drive disadvantageous reaction on energy. Such adverse effect comprises many fine chemicals biosynthesis pathways. People utilize by improving cell specific sugar ability (for example to coding participate in cell degradation should sugar and the gene that is translated into the enzyme of energy operate) can improve energy so that cell can carry out metabolic response (for example biosynthesis needs fine chemicals) unfavorable but that need

One or more SMP genes of the present invention of mutagenesis also may produce the SMP albumen of activity change, and the SMP albumen remote-effects Corynebacterium glutamicum of activity change produces one or more needs fine chemicals. For example, people utilize one or more sugared efficient (being converted into useful kinetomeres thereby strengthen described sugar) or improve the efficient (for example active by improving oxidative phosphorylation efficient or strengthening atp synthase) that the reducing equivalent thing is converted into useful kinetomeres by improving Corynebacterium glutamicum, can increase these energy-rich compound amounts, for the common disadvantageous metabolic process of cellular driven. Such process comprise make up cell membrane, transcribe, translation and essential compound (such as nucleotides, amino acid, vitamin, lipid etc.) (Lengeler etc. (1999) the Biology of Prokaryotes of biosynthesis cell division, Thieme Verlag:Stuttgart, 88-109,913-918,875-899 page or leaf). By improving the Growth and reproduction of these engineering cells, both can improve the cell viability in the large-scale culture thing, also can improve its division rate, the more substantial cell of in the fermentation tank culture thing, can surviving like this. At least because exist more generation to need the living cells of fine chemicals and improve productive rate, output or production efficiency. In addition, many catabolites of cell utilization sugar sweat generation need precursor or the intermediate of product such as fine chemicals as producing other. Thereby, owing to strengthened the ability of cellular metabolism sugar, thus the catabolite quantity for other process of cell also can be increased.

The invention provides coding this paper and be called the novel nucleic acids molecule of SMP albumen, described SMP albumen can for example be carried out the carbohydrate metabolism that participates in Corynebacterium glutamicum such as sugar and by the function such as oxidative phosphorylation process produce power molecule. The nucleic acid molecules of coding SMP albumen is referred to herein as the SMP nucleic acid molecules. In a preferred embodiment, described SMP albumen participation carbon molecule and catabolite thereof are converted into the energy that cell is used for metabolic process. The example of this albuminoid comprises the albumen that is provided gene code by table 1.

Therefore, one aspect of the present invention relates to the nucleic acid molecules (for example cDNA, DNA or RNA) of separation, the nucleic acid molecules of described separation comprises the nucleotide sequence of coding SMP albumen or its biologically-active moiety, also relates to being suitable as detecting or the primer of amplification SMP code nucleic acid (for example DNA or mRNA) or the nucleic acid fragment of hybridization probe. In particularly preferred embodiments, the nucleic acid molecules of described separation comprises code area or its complementary series of one of one of nucleotide sequence described in the sequence table odd number SEQ ID NO (such as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7...) or these nucleotide sequences. In other particularly preferred embodiment, the nucleic acid molecules of separation of the present invention comprises such nucleotide sequence or its part: described nucleotide sequence and a kind of sequence table odd number SEQ ID NO (such as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7...) nucleotide sequence hybridization or have at least about 50% with it, preferably at least about 60%, more preferably at least about 70%, 80% or 90% even more preferably at least about 95%, 96%, 97%, 98%, 99% or higher homology. In other preferred embodiment, a kind of sequence table even number of the nucleic acid molecule encoding of described separation SEQ ID NO (such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8...) amino acid sequence. It is active that the preferred SMP albumen of the present invention also preferably has at least a SMP as herein described.

In another embodiment, a kind of protein of the nucleic acid molecule encoding of described separation or its part, wherein said albumen or its part comprise the amino acid sequence with the enough homologies of amino acid sequence of the present invention (as having the sequence of sequence table even number SEQ ID NO), for example with the enough homologies of amino acid sequence of the present invention, so that described protein or its part keep the SMP activity. Preferably the protein of described nucleic acid molecule encoding or its part keep to carry out participate in the ability such as the carbohydrate metabolism of sugar and the function by oxidative phosphorylation process produce power molecule (for example ATP) of Corynebacterium glutamicum. In one embodiment, by the homology of the protein of described nucleic acid molecule encoding and amino acid sequence of the present invention (for example being selected from the complete amino acid sequence with sequence table even number SEQ ID NO) be at least about to 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, most preferably at least about 95%, 96%, 97%, 98% or 99% or higher. In another preferred embodiment, described protein is and complete amino acid sequence of the present invention (by the open read frame as shown in the corresponding odd number SEQ of the sequence table ID NO (such as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7...) coding) the total length Corynebacterium glutamicum protein of homology basically.

In another preferred embodiment, the nucleic acid molecules of described separation derives from Corynebacterium glutamicum, and coding comprises the protein (for example SMP fusion) in a biologically active structure territory, described biologically active structure territory and a kind of amino acid sequence of the present invention (for example a kind of even number SEQ ID NO sequence in the sequence table) have at least about 50% or higher homology, and can carry out the carbohydrate metabolism that participates in Corynebacterium glutamicum such as sugar and the function of passing through oxidative phosphorylation process produce power molecule (for example ATP), or having one or more activity that table 1 provides, described biologically active structure territory also comprises the heterologous nucleic acid sequence of coding heterologous polypeptide or regulatory region.

In another embodiment, the nucleic acid molecules of described separation is to 15 nucleotides of the youthful and the elderly, and under stringent condition with the making nucleic acid molecular hybridization that comprises nucleotide sequence of the present invention (for example sequence table odd number SEQ ID NO sequence). The nucleic acid molecules of preferred described separation is equivalent to naturally occurring nucleic acid molecules. The more preferably nucleic acid coding of described separation naturally occurring Corynebacterium glutamicum SMP albumen or its biologically-active moiety.

Another aspect of the present invention relates to the carrier that contains nucleic acid molecules of the present invention, and for example recombinant expression carrier relates to the host cell that imports this class carrier. In one embodiment, a kind of like this host cell is used for producing SMP albumen by cultivate described host cell in suitable culture medium. Then from described culture medium or described host cell, isolate described SMP albumen.

The microorganism that relates in one aspect to again the hereditary change that has wherein imported SMP gene or change SMP gene of the present invention. In one embodiment, by importing the nucleic acid molecules of the present invention as genetically modified encoding wild type or saltant SMP sequence, changed the genome of described microorganism. In another embodiment, the endogenous SMP gene in the described microbial genome is by being changed for example functional fracture with the SMP homologous recombination that changes. In another embodiment, SMP gene endogenous or that introduce is changed by one or more point mutation, disappearance or inversion in the microorganism, but still encoding function SMP albumen. In an embodiment again, one or more regulatory regions of SMP gene in the microorganism (for example promoter, repressor protein or inducer) are changed (for example by disappearance, brachymemma, inversion or point mutation), so that the expression of described SMP gene is adjusted. A preferred embodiment, described microorganism belongs to Corynebacterium or brevibacterium, particularly preferably Corynebacterium glutamicum. In a preferred embodiment, described microorganism is also for the production of the needs compound, for example amino acid, particularly preferably lysine.

On the other hand, the invention provides the method that whether has corynebacterium diphtheriae or its active situation among a kind of experimenter of evaluation. The method comprises one or more nucleotide sequences of the present invention or the amino acid sequence (for example sequence table SEQ ID NO 1-782 sequence) that detects in the subject, detects thus whether have corynebacterium diphtheriae or its active situation in the described subject.

Another aspect of the present invention relates to SMP albumen or its part, for example biologically-active moiety of separation. In a preferred embodiment, described separation SMP albumen or its part can be carried out the carbohydrate metabolism that participates in Corynebacterium glutamicum such as sugar and the function of passing through oxidative phosphorylation process produce power molecule (for example ATP). In another preferred embodiment, the enough homologies of the SMP albumen of described separation or its part and amino acid sequence of the present invention (for example sequence table even number SEQ ID NO sequence) are carried out the carbohydrate metabolism that participates in Corynebacterium glutamicum such as sugar and the ability of passing through the function of oxidative phosphorylation process produce power molecule (for example ATP) so that described protein or its part keep.

The present invention also provides the separation preparation of SMP albumen. In preferred embodiments, described SMP albumen comprises amino acid sequence of the present invention (for example sequence table even number SEQ ID NO sequence). In another preferred embodiment, the present invention relates to and complete amino acid sequence of the present invention (for example sequence table even number SEQ ID NO sequence) (by corresponding odd number SEQ ID NO open read frame coding in the sequence table) full length protein that separates of homology basically. In an embodiment again, the homology of described protein and complete amino acid sequence of the present invention (for example sequence table even number SEQ ID NO sequence) is at least about to 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, most preferably at least about 95%, 96%, 97%, 98% or 99% or higher. In other embodiments, the SMP albumen of described separation comprises with the homology of a kind of amino acid sequence of the present invention (for example sequence table even number SEQ ID NO sequence) at least about 50% or higher amino acid sequence, and can carry out the carbohydrate metabolism that participates in Corynebacterium glutamicum such as sugar and the function of passing through oxidative phosphorylation process produce power molecule (for example ATP), or have one or more activity that table 1 provides.

Perhaps, the SMP albumen of described separation can comprise so nucleotide sequence coded amino acid sequence: described nucleotides sequence be listed under the stringent condition with sequence table in a kind of odd number SEQ ID NO nucleotide sequence hybridization, or with sequence table in a kind of odd number SEQ ID NO nucleotide sequence homology at least about to 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, most preferably at least about 95%, 96%, 97%, 98% or 99% or higher. Also the preferred form of preferred SMP albumen also has one or more SMP biologically actives described herein.

Described SMP polypeptide or its biologically-active moiety can effectively be connected with a kind of non-SMP polypeptide, form fusion. In preferred embodiments, this fusion has the activity that is different from independent described SMP albumen. In other preferred embodiment, this fusion is carried out the carbohydrate metabolism that participates in Corynebacterium glutamicum such as sugar and the function of passing through oxidative phosphorylation process produce power molecule (for example ATP). In particularly preferred embodiments, this fusion is incorporated in the host cell, and the compound that needs of regulating described cell produces.

On the other hand, the invention provides the method for regulating the molecule of SMP protein active for screening, described molecule is by interacting with SMP albumen itself, perhaps interact by substrate or binding partners with described SMP albumen, perhaps by regulating transcribing or translating of SMP nucleic acid molecules of the present invention, regulate the SMP protein active.

Another aspect of the present invention relates to the method for the production of fine chemicals. The method comprises cultivates the cell that comprises the carrier that instructs SMP nucleic acid molecules expression of the present invention, so that can produce fine chemicals. In a preferred embodiment, the method comprises that also acquisition contains the step of the cell of this carrier, wherein with the carrier transfectional cell that instructs the SMP expression of nucleic acid. In another preferred embodiment, the method also comprises the step that reclaims described fine chemicals from culture. In an especially preferred embodiment, described cell derives from Corynebacterium or brevibacterium, or is selected from the bacterial strain described in the table 3.

Another aspect of the present invention relates to for the method for regulating by the micro-organisms molecule. These class methods comprise makes described cell contact with the factor of regulating SMP protein active or SMP protein expression, so that the cell related activity is changed for the described activity when lacking the described factor. In a preferred embodiment, regulate one or more Corynebacterium glutamicum carbon metabolic pathways of described cell, or regulate described cell by the process produce power such as oxidative phosphorylation, so that improve productive rate or the productive rate that this micro-organisms needs fine chemicals. The factor of regulating the SMP protein active can be the factor that stimulates SMP protein active or SMP expression of nucleic acid. The example of the factor of stimulation SMP protein active or SMP expression of nucleic acid comprises little molecule, active SMP albumen and has imported the nucleic acid of the coding SMP albumen of described cell. The example of the factor that suppresses the SMP activity or express comprises little molecule and antisense SMP nucleic acid molecules.

Another aspect of the present invention relates to the method that needs the compound productive rate of regulating cell, and described method comprises in wild type or the saltant SMP gene transfered cell, perhaps is retained on the independent plasmid or is incorporated in the genome of host cell. If be incorporated in the genome, then this integration can be at random, or it can occur by homologous recombination, so that described natural gene is replaced by the copy that is imported, the cell that causes regulating produces the described compound that needs. In a preferred embodiment, described productive rate improves. In another preferred embodiment, described chemicals is a kind of fine chemicals. In an especially preferred embodiment, described fine chemicals is a seed amino acid. In an especially preferred embodiment, described amino acid is 1B.

Detailed Description Of The Invention

The invention provides SMP nucleic acid molecules and SMP protein molecular, described molecule participates in the carbon compound metabolism of Corynebacterium glutamicum such as sugar and passes through oxidative phosphorylation process produce power molecule. Molecule of the present invention can be used for regulating for example Corynebacterium glutamicum generation fine chemicals of microorganism, this can have a direct impact (for example overexpression or optimization glycolytic pathway albumen have a direct impact productive rate, output and/or the production efficiency of for example pyruvic acid of improvement Corynebacterium glutamicum), maybe can produce remote-effects, remote-effects still make the productive rate that needs compound, output and/or production efficiency improve (for example regulating the energy change that the albumen that participates in oxidative phosphorylation causes finishing for cell necessary metabolic process and other cell function (such as nucleic acid and protein biosynthesis and transcribe and translate)). Below further describe various aspects of the present invention.

I. fine chemicals

Term " fine chemicals " is well known in the art, comprises the molecule that has multiple application in various industry (such as but not limited to pharmacy industry, agricultural and cosmetics industry) by biogenic. This compounds comprises organic acid, for example tartaric acid, itaconic acid and diaminopimelic acid; The amino acid of proteinogenous and non-formation protein; Purine and pyrimidine bases, nucleosides and nucleotides (as being described in for example Kuninaka, A. (1996) nucleotides and related compound are stated from Biotechnology, the 6th volume, the 561-612 page or leaf, Rehm etc. write, VCH:Weinheim and the list of references that wherein contains); Lipid, saturated and unrighted acid (for example arachidonic acid); Dihydroxylic alcohols (for example propane diols and butanediol); Carbohydrate (for example hyaluronic acid and trehalose); Aromatic compounds (for example aromatic amine, vanillic aldehyde and indigo); Vitamin and co-factor (as are described in Ullmann ' s Encyclopedia of Industrial Chemistry, A27 volume, " vitamin ", 443-613 page or leaf (1996) VCH:Weinheim and list of references wherein; And Ong, A.S., Niki, E. and Packer, L. (1995) " Nutrition; Lipids, Health, and Disease " Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research-Asia, hold AOCS Press, (1995) at Malaysian Penang in 1-3 day in September, 1994); Enzyme; Polyketide (Cane etc. (1998) Science 282:63-68); And Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, all other compounds that ISBN:0818805086 and list of references are wherein introduced. Some metabolism and application in these fine chemicals below is described in further detail.

A. amino acid metabolism and application

Amino acid is the basic structural unit of all proteins, is that all biological normal cell functions are requisite therefore. Term " amino acid " is known in the art. The amino acid that forms protein has 20 kinds, and as the construction unit of protein, they connect by peptide bond in protein; But not the amino acid (known hundreds of described amino acid) that forms protein is not present in the protein usually (referring to Ulmann ' s Encyclopedia of Industrial Chemitry, the A2 volume, 57-97 page or leaf VCH:Weinheim (1985)). Amino acid can have D-or L-optical configuration, although L-amino acid generally is the unique type of finding in the naturally occurring protein. 20 kinds of amino acid each biosynthesis and degradation pathway in prokaryotic and eukaryotic that form protein fully characterize (referring to for example Stryer, L.Biochemistry, the 3rd edition, 578-590 page or leaf (1988)). " essential " amino acid (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine) is name so, because their biosynthesis is complicated, generally be to need in the nutrition, described " essential " amino acid is converted into all the other 11 kinds of " nonessential " amino acid (alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine) by simple biosynthesis pathway easily. Higher mammal has kept some the amino acid whose ability in synthetic these amino acid really, but essential amino acid must supply from diet, so that it is synthetic to carry out normal protein.

Except the function of these amino acid in the protein biosynthesis, these amino acid self or interesting chemicals have been found that wherein many amino acid have various uses in food industry, feed industry, chemical industry, cosmetics industry, agricultural and pharmacy industry. Lysine is not only the important amino acid of human nutrition, and is the important amino acid of nonruminant (for example poultry and pig) nutrition. Glutamic acid is the most frequently used to be made flavouring additive (monosodium glutamate MSG), and is widely used in food industry, and aspartic acid, phenylalanine, glycine and cysteine also are like this. Glycine, METHIONINE and tryptophan all are used for pharmacy industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are valuable on pharmacy industry and cosmetics industry. Threonine, tryptophan and D/L-methionine are the feed addictives of commonly using. (Leuchtenberger, W. (1996) amino acid-production technology and application are stated from (writing) Biotechnology such as Rehm, the 6th volume, the 14a chapter, the 466-502 page or leaf, VCH:Weinheim). In addition, have been found that these amino acid can be used as the precursor of synthetic synthesizing amino acid and protein, for example N-acetylcystein, S-carboxymethyl-Cys, (S)-5HTP and Ulmann ' s Encyclopedia of Industrial Chemistry, the A2 volume, the 57-97 page or leaf, VCH:Weinheim, other synthesizing amino acid and the protein introduced in 1985.

Fully characterized the biosynthesis of these amino acid in can produce these natural amino acid whose biologies (for example bacterium) (the synthetic and summary of regulating of relevant bacterium amino acid bio referring to Umbarger, H.E. (1978) Ann.Rev.Biochem.47:533-606). By the synthetic glutamic acid of the reductive amination of the intermediate KG in the citrate cycle. Produce respectively glutamine, proline and arginine by glutamic acid subsequently. The biosynthesis of serine is three step process, with 3-phoshoglyceric acid (glycolysis intermediate) beginning, behind oxidation, transamination and hydrolysing step, produces this seed amino acid. Cysteine and glycine are all produced by serine, and the former produces by homocysteine and serine condensation, and the latter produces by the side chain beta carbon being transferred to tetrahydrofolic acid at one in by the reaction of serine transhydroxymethylase catalysis. Phenylalanine and tyrosine are synthetic by the 9 step biosynthesis pathways that glycolysis and pentose phosphate pathway precursor erythrose-4-phosphate and phosphoenolpyruvate begin, and the biosynthesis pathway of this two seed amino acid anti-latter two steps behind synthetic prephenic acid is different. Tryptophan is also produced by these two kinds of starting molecules, but its synthetic be 11 step approach. Tyrosine also can be synthetic by phenylalanine in by the reaction of PAH catalysis at one. Alanine, valine and leucine all are the biosynthetic products of glycolysis end-product-pyruvic acid. Aspartic acid is generated by the intermediate oxaloacetic acid of citrate cycle. Asparagine, methionine, threonine and lysine all are that the conversion by aspartic acid produces. Isoleucine is generated by threonine. 9 step approach of a complexity are that ribose 5-phosphate-1-pyrophosphoric acid produces histidine by a kind of activation sugar.

The amino acid that exceeds the synthetic demand of cell protein can not be stored, but be degraded, (relevant summary is referring to Stryer for the main metabolic pathway of cell provides intermediate, L.Biochemistry, the 3rd edition, the 21st chapter, " amino acid degradation and urea cycle ", 495-516 page or leaf (1988)). Although cell can be converted into useful Metabolic Intermediate with unwanted amino acid, with regard to the necessary enzyme of energy, precursor molecule and synthesizing amino acid, amino acids production is expensive. Therefore, amino acid bio is synthetic, and to be subjected to feedback inhibition be N/R, in feedback inhibition, the existence of specific amino acids play a part to slow down or stop himself producing fully (summary of the feedback mechanism in the relevant amino acid biosynthetic pathway, referring to Stryer, L.Biochemistry, the 3rd edition, the 24th chapter, " biosynthesis of amino acid and ferroheme ", 575-600 page or leaf (1988)). Therefore, the output of any specific amino acids is subjected to the amino acid quantitative limitation that exists in the cell.

B. the metabolism of vitamin, co-factor and nutritional drugs and application

Vitamin, co-factor and nutritional drugs consist of the other component that higher mammal has been lost synthesis capability and therefore must take in, and for example bacterium is synthetic but they are easily by other biology. These molecules or itself are bioactivators, or can be as the precursor as the bioactivator of the intermediate of electron carrier or multiple metabolic pathway. These compounds are except having nutritive value, and the essential industry that also has as colouring agent, antioxidant and catalyst or other processing aid is worth. (about the general introduction of structure, activity and the commercial Application of these compounds, referring to for example Ullman ' s Encyclopedia of Industrial Chemistry, " vitamin ", A27 volume, 443-613 page or leaf, VCH:Weinheim, 1996). Term " vitamin " is well known in the art, comprises biological normal function needs but the biological nutrients that self can not synthesize. Vitamins can comprise co-factor and nutritional drugs compound. Term " co-factor " comprises the active required non-albumen compound of generation normal enzyme. This compounds can be organic compound or inorganic compound; Co-factor molecule of the present invention is organic molecule preferably. Term " nutritional drugs " is included in the food additives that have health advantages among plant and animal, the particularly mankind. The example of this quasi-molecule is vitamin, antioxidant and some lipid (for example polyunsaturated fatty acid).

Characterized in a large number these molecules for example biosynthesis in the bacterium of the biology that can produce it (Ullman ' s Encyclopedia of Industrial Chemistry, the A27 volume, " vitamin " 443-613 page or leaf, VCH:Weinheim, 1996; Michal, G. (1999) Biochemical Pathways:An Atlas of Biochemistry and Molecular Biology, John Wiley ﹠ Sons; Ong, A.S., Niki, E. and Packer, L. (1995) " Nutrition, Lipids, Health; and Disease " Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research-Asia holds AOCS Press:Champaign in 1-3 day in September, 1994 at Malaysian Penang, IL X, 374S).

By with pyrimidine and the chemical coupling of thiazole part, produce thiamine (Cobastab₁). Riboflavin (Cobastab₂) synthetic by guanosine-5 '-triphosphoric acid (GTP) and ribose-5 '-phosphoric acid. Riboflavin and then be used for synthetic FMN (FMN) and flavin adenine dinucleotide (FAD) (FAD). Be referred to as " Cobastab₆" compound family (for example puridoxine hydrochloride of pyridoxol, pyridoxamine, P5P and commercial use) all be the derivative with common structure unit 5-hydroxyl-6-picoline. Pantothenic acid ((R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxo butyl)-Beta-alanine) can or pass through fermenting and producing by chemical synthesis. Last step in the pantothenic acid biosynthesis is comprised of Beta-alanine and the pantoic acid condensation that ATP drives. The enzyme of being responsible for being converted into pantoic acid, Beta-alanine and condensation and being the biosynthesis step of pantothenic acid is known. The pantothenic acid of metabolic activity form is coacetylase, and its biosynthesis is carried out with 5 enzymatic steps. Pantothenic acid, pyridoxal-5 '-phosphoric acid, cysteine and ATP are the precursors of coacetylase. These enzymes are the generation of catalysis pantothenic acid not only, and catalysis (R)-pantoic acid, (R)-pantolacton, (R)-panthenol (provitamin B₅), the generation of pantetheine (and derivative) and coacetylase.

Study the biotin biosynthesis that is begun by precursor molecule heptanedioyl coacetylase in the microorganism in great detail, and identified related some genes. It is synthetic to have been found that many corresponding protein also participate in iron bunch, and is the member of nifS proteinoid. Lipoic acid is derived from sad, and as the coenzyme in the energetic supersession, it becomes the part of pyruvate dehydrogenase complex and ketoglurate dehydrogenase complex in energetic supersession. The folic acid class is the material of a class folic acid derivatives, and folic acid is again derived from Pidolidone, p-aminobenzoic acid and 6-methylpterin. At length studied the biosynthesis of folic acid and derivative thereof in certain micro-organisms, described biosynthesis originates in Metabolic Intermediate guanosine-5 '-triphosphoric acid (GTP), Pidolidone and p-aminobenzoic acid.

Corrinoid (for example cobalamin, particularly Cobastab₁₂) and porphyrin belong to the chemical substance that a category feature is the tetrapyrrole ring system. Cobastab₁₂Biosynthesis enough complicated so that not yet identified fully, but now known many related enzymes and substrate. Nicotinic acid and niacinamide are pyridine derivates, are also referred to as " niacin ". Nicotinic acid is important coenzyme NAD (NADH) and the precursor of NADP (nicotinamide-adenine dinucleotide phosphate) and reduction form thereof.

The large-scale production of these compounds mainly relies on acellular chemical synthesis always, although some in these chemicals also cultivates to produce for example riboflavin, Cobastab by extensive microorganism₆, pantothenic acid and biotin. Cobastab only₁₂Because its synthetic complexity is passed through fermenting and producing. In-vitro method spends large content of starting materials and time, and ordinary disbursements is huge.

C. the metabolism of purine, pyrimidine, nucleosides and nucleotides and application

Purine and pyrimidine metabolic gene and corresponding protein thereof are the important treatment targets of tumor disease and virus infections. Term " purine " or " pyrimidine " comprise nitrogenous base, and they are components of nucleic acid, coenzyme and nucleotides. Term " nucleotides " comprises the basic structural unit of nucleic acid molecules, they by a nitrogenous base, (in the situation of RNA, described sugar is ribose to a pentose; In the situation of DNA, described sugar is the D-deoxyribose) and the phosphoric acid formation. Term " nucleosides " comprises as nucleotide precursor but lacks the phosphoric acid molecule partly that nucleotides has. By the biosynthesis that suppresses these molecules or the conversion that suppresses its formation nucleic acid molecules, might suppress the synthetic of RNA and DNA; Suppress this activity by the mode with target cancer cell, can inhibition tumor cell division and the ability that copies. In addition, have and do not form nucleic acid molecules but as energy storage (being AMP) or as the nucleotides of coenzyme (being FAD and NAD).

Several publications have been described by affecting purine and/or pyrimidine metabolic, these chemical substances are used for these medical science indications (Christopherson for example, R.I. and Lyons, S.D. (1990) " as pyrimidine and from the beginning biosynthetic establishment agent of purine of chemotherapeutic ", Med.Res. Reviews 10:505-548). The research of the enzyme that participates in purine and pyrimidine metabolic is concentrated on always can be as for example as the exploitation (Smith of the new drug of immunodepressant or antiproliferative agents, J.L., (1995) " enzyme during nucleotides is synthetic ", Curr.Opin.Struct.Biol.5:752-757; (1995) Biochem Soc.Transact.23:877-902). Yet, purine and pyrimidine bases, nucleosides and nucleotides have other purposes: as the biosynthetic intermediate of several fine chemicals (for example thiamine, S-adenosine-methionine, folic acid or riboflavin), as the energy carrier (for example ATP or GTP) of cell with for chemicals self, be typically used as flavoring agent (for example IMP or GMP) or be used for some medical usages (referring to for example Kuninaka, A. (1996) Nucleotides and Related Compounds in Biotechnology, the 6th volume, Rehm etc. write, VCH:Weinheim, the 561-612 page or leaf). In addition, participate in the enzyme of purine, pyrimidine, nucleosides or nucleotide metabolism more and more as the target of exploitation for the protection of the chemicals of crop, described chemicals comprises fungicide, herbicide and pesticide.

(relevant summary is referring to for example Zalkin to have identified the metabolism of these compounds in bacterium, H. and Dixon, J.E. (1992) " from the beginning biosynthesis of purine nucleotides ", be stated from: Progress in Nucleic Acid Research and Molecular Biology, the 42nd volume, Academic Press, the 259-287 page or leaf; And Michal, G. (1999) " nucleotides and nucleosides " is stated from: Biochemical Pathways:An Atlas of Biochemistry and Molecular Biology, the 8th chapter, Wiley: New York). Purine metabolism is the theme of broad research always, and is absolutely necessary for the normal function of cell. Purine metabolism is impaired in higher mammal can cause serious disease, for example gout. Purine nucleotides is begun to synthesize by ribose-5-phosphoric acid, series of steps by midbody compound inosine-5 '-phosphoric acid (IMP), cause the generation of GMP (GMP) or AMP (AMP), generate easily the triphosphoric acid form that is used as nucleotides by GMP or AMP. These compounds are also as the energy storage thing, so its many different Biochemical processes that are degraded in the cell provide energy. The pyrimidine biosynthesis is undertaken by forming uracil-5 '-a phosphoric acid (UMP) by ribose-5-phosphoric acid. UMP so be converted into Cytidine-5 '-triphosphoric acid (CTP). The deoxidation form of all these nucleotides produced in the reduction reaction in a step, was the diphosphonic acid deoxyribose form of described nucleotides from the diphosphonic acid ribose form reduction reaction of described nucleotides. After phosphorylation, it is synthetic that these molecules can participate in DNA.

D. the metabolism of trehalose and application

Trehalose is by with α, α-1, and two molecule glucoses that 1-connects form. Trehalose is typically used as sweetener in food industry, namely be used for a kind of additive of dried foods or frozen food and beverage. Yet it also has many application (referring to such as Nishimoto etc., (1998) U.S. Patent number 5,759,610 in pharmacy industry, cosmetics industry and biotechnological industries; Singer, M.A. and Lindquist, S. (1998) Trends Biotech.16:460-467; Paiva, C.L.A. and Panek, A.D. (1996) Biotech.Ann.Rev.2:293-314; And Shiosaka, M. (1997) J.Japan 172:97-102). Trehalose is produced by enzyme by many microorganisms, and natural being discharged in the surrounding medium, can collect trehalose from described surrounding medium with methods known in the art.

II. utilization and the oxidative phosphorylation of sugar and carbon molecule

Carbon is the important element that forms all organic compounds, so it is not only the nutritional requirement of Corynebacterium glutamicum growth division, and is the nutritional requirement of the excessive generation fine chemicals of this microorganism. Carbohydrate such as monosaccharide and disaccharide or polysaccharide is good especially carbon source, so the standard growth culture medium contains one or more following sugar usually: glucose, fructose, mannose, galactolipin, ribose, sorbose, ribulose, lactose, maltose, sucrose, gossypose, starch or cellulose (Ullmann ' s Encyclopedia of Industrial Chemistry (1987) A9 volume, " enzyme ", VCH:Weinheim). On the other hand, more compound sugar can be used for described culture medium, for example other byproduct in molasses or the sugar refining. As an alternative carbon source of described sugar other compound in addition be can use, alcohols (for example ethanol or methyl alcohol), paraffinic, sugar alcohol, aliphatic acid and organic acid (for example acetic acid or lactic acid) comprised. About carbon source and cultivate microorganism to the summary of their utilization, referring to: Ullman ' s Encyclopedia of Industrial Chemistry (1987) A9 volume, " enzyme ", VCH:Weinheim; Stoppok, E. and Buchholz, K. (1996) " the sugared type raw material that fermentation is used " is stated from: Biotechnology (Rehm, H.J. writes) the 6th volume, VCH:Weinheim, 5-29 page or leaf; Rehm, H.J. (1980) Industrielle Mikrobiologie, Springer:Berlin; Bartholomew, W.H. and Reiman, H.B. (1979), zymotechnique economics is stated from: Peppler, H.J. and Perlman, D. writes, Microbial Technology the 2nd edition, the 2nd volume, the 18th chapter, Academic Press: New York; And Kockova-Kratachvilova, the feature of A. (1981) industrial microorganism is stated from: Rehm, and H.J. and Reed, G. writes, Handbook of Biotechnology, the 1st volume, the 1st chapter, Verlag Chemie:Weiheim.

After these carbon molecule absorption that are rich in energy enter cell, must process processing, can be by a kind of main glycometabolism approach degraded. This approach directly produces useful catabolite, for example ribose 5-phosphate and phosphoenolpyruvate, and phosphoenolpyruvate can be converted into pyruvic acid subsequently. 3 kinds of most important bacterium glycometabolism approach comprise Embdeh-Meyerhoff-Pamas (EMP) approach (being also referred to as glycolytic pathway or diphosphofructose approach), hexose monophosphoric acid (HMP) approach (being also referred to as pentose shunt approach or pentose phosphate pathway) and Entner-Doudoroff (ED) approach, and (summary is referring to Michal, G. (1999) Biochemical Pathways:An Atlas of Biochemistry and Molecualr Biology, Wiley: New York and Stryer, L. (1988) Biochemistry, the 13-19 chapter, Freeman: New York, and the list of references of wherein quoting).

EMP Embden Meyerbof Parnas pathway makes the hexose molecule be converted into pyruvic acid, and this process produces 2 molecule ATP and 2 molecule NADH. (can from culture medium, directly absorb with Cori ester, perhaps can be produced by glycogen, starch or cellulose) be raw material, make the glucose molecule isomery turn to fructose-6-phosphate, make its phosphorylation, be cracked into 2 three carbon molecule glyceraldehyde-3-phosphates. In dehydrogenation, phosphorylation with after resetting continuously, obtain pyruvic acid.

It is reducing equivalent thing such as NADPH that the HMP approach makes conversion of glucose, and produces pentose and tetrose compound, and they are necessary intermediate and precursors of many other metabolic pathways. In the HMP approach, G-6-P is converted into ribulose-5-phosphoric acid by 2 continuous dehydrogenation enzyme reactions (it also discharges 2 molecule NADPH) and 1 carboxylated step. Ribulose-5-phosphoric acid also can be converted into xylulose-5-phosphoric acid and ribose-5-phosphoric acid; The former can be converted into G-6-P through a series of biochemical steps, and G-6-P can enter EMP Embden Meyerbof Parnas pathway, and the latter is typically used as the intermediate of its biosynthesis pathway of cell.

The ED approach begins with compound glucose or gluconic acid, and it is phosphorylation and dehydrogenation formation 2-dehydrogenation-3-deoxidation-6-P-gluconic acid subsequently. Glucuronic acid and galacturonic acid also can be converted into by more complicated biochemical route 2-dehydrogenation-3-deoxidation-6-P-gluconic acid. This product molecule is cracked into glyceraldehyde-3-P and pyruvic acid subsequently; Glyceraldehyde-3-P itself also can be converted into pyruvic acid.

EMP and HMP approach have many features, comprise intermediate and enzyme. EMP Embden Meyerbof Parnas pathway provides ATP amount at most, but it does not produce ribose-5-phosphoric acid, and ribose-5-phosphoric acid is the important as precursors of Nucleic acid for example, does not also produce E4P, and erythrose is synthetic to amino acid bio to be important. Therefore can only utilize the microorganism of glucose EMP Embden Meyerbof Parnas pathway not grow as the simple culture media of sole carbon source at glucose. This microorganism is called fastidious microorganism, and its growth needs adds organochromium compound, for example sees the organochromium compound of yeast extract.

On the contrary, the HMP approach produces nucleic acid and synthetic the two the essential whole precursor of amino acid bio, but only produces half ATP energy of EMP Embden Meyerbof Parnas pathway. The HMP approach also produces NADPH, and NADPH can be used for the reduction reaction of biosynthesis pathway. Yet the HMP approach does not directly produce pyruvic acid, so these microorganisms also must have this part of EMP Embden Meyerbof Parnas pathway. So countless microorganisms, especially facultative anaerobe are evolved and have this two kinds of approach, this is not unexpected.

The ED approach only sees bacterium at present. Although this approach is relevant with people HMP approach part on the opposite direction that precursor forms, the ED approach directly forms acetonate by aldolase cracking 3-ketone group deoxidation-6-phosphogluconic acid. The ED approach can independently exist, for most of strictly anaerobic Institute of Micro-biology utilizes. Final result is similar to the HMP approach, but only has when carbon atom is converted into acetonate rather than precursor molecule, just produces 1 mole of ATP.

The pyruvate molecules that produces by above arbitrary approach can be converted into energy by Krebs circulation (being also referred to as citrate cycle, citrate cycle or tricarboxylic acid cycle (TCA circulation)) at an easy rate. In the Krebs circulation, at first pyruvic acid decarboxylation produces 1 molecule NADH, 1 molecule acetyl-CoA and 1 molecule carbon dioxide. The acetyl group of acetyl-CoA subsequently with the reaction of the oxaloacetic acid (oxaolacetate) of 4 carbosilane units, form 6 carbon organic acid citric acids. Dehydration and discharge again 2 molecule carbon dioxide. At last, produce again oxaloacetic acid and can be used as again the acetyl group acceptor, so far finish circulation. The electronics that intermediate oxidation in the TCA circulation discharges is transferred to NAD+, produces NADH.

In the respiratory, the NADH electronics is transferred to oxygen molecule or other terminal electron acceptor. This process is by respiratory chain catalysis, and respiratory chain is the electron transport system that comprises integral protein and embrane-associated protein. There are two basic roles in this system: the first, and accept the electronics of electron donor and it is transferred to electron acceptor, the second, preserve electronics by synthetic ATP and shift the portion of energy that discharges. Known several oxidoreducing enzyme and electron transport albumen participate in this process, comprise nadh dehydrogenase, contain flavine electron carrier, iron-sulfur protein and cytochromes. Nadh dehydrogenase is positioned at the kytoplasm face of plasma membrane, and it is transferred to flavoprotein with hydrogen atom from NADH, then accepts the electronics of NADH. Flavoprotein is one group of electron carrier with flavine prothetic group, alternately reduction and oxidation when its acceptance and metastatic electron. Known 3 kinds of flavine participate in these reactions: riboflavin, flavine-adenine-dinucleotide (FAD) and FMN (FMN). Iron-sulfur protein contains cluster iron atom and sulphur atom, they not with the ferroheme bonding, but still can participate in dehydration and rehydration is reacted. Succinate dehydrogenase and aconitase are typical iron-sulfur proteins; Its iron-sulfur complex plays a part to accept and metastatic electron as the part of whole electron transport chain. Cytochromes are the albumen that contains heme group (ferroheme). Many dissimilar cytochromes are arranged, and their reduction potential is different. On function, these cytochromes consist of the approach that electronics wherein can be transferred to other cytochromes that positive reduction potential constantly increases progressively. The non-albumen electron carrier of other known type has: fat-soluble quinone (for example ubiquinone). These molecules also play hydrogen atom acceptor and electron donor.

Respiratory chain plays a role and produces the cross-cell membrane proton gradient, produces proton motive force. Cell utilizes this power by the synthetic ATP of cross-film enzyme atp synthase. The ATP enzyme is a kind of compound of polyprotein, wherein H+ molecule subunit's physical property rotation in cell membrane transporter makes cell, (summary is referring to Fillingame to be accompanied by ADP phosphorylation formation ATP, R.H. and Divall, S. (1999) Novartis Found.Symp.221:218-229,229-234).

Non-hexose carbon substrate also can be as carbon source and the energy source of cell. This substrate at first is converted into the hexose of gluconeogenesis approach, cell synthesis of glucose at first wherein, the produce power of then degrading. The raw material of this reaction is phosphoenolpyruvate (PEP), and phosphoenolpyruvate is a kind of important intermediate of glycolytic pathway. PEP can be formed by the substrate beyond the sugar, and for example acetic acid perhaps forms by oxaloacetic acid (this is as the intermediate of TCA circulation) decarboxylation. Reverse glycolytic pathway (utilization is different from the cascade enzyme of original glycolytic pathway) and can form G-6-P. Pyruvic acid is converted into glucose need to utilize 6 energy-rich phosphate bonds, and glycolysis is that pyruvic acid only produces 2 ATP with conversion of glucose. Yet, glucose complete oxidation (glycolysis, pyruvic acid are converted into acetyl-CoA, citrate cycle and oxidative phosphorylation) produces 36-38 ATP, the energy-rich phosphate bond that carries out like this gluconeogenesis net loss produces such energy-rich phosphate bond and generally increase by oxidizing glucose, thereby is compensated.

III. element of the present invention and method

The present invention is at least part of based on having found that this paper is called the novel molecular of SMP nucleic acid and protein molecular, the sugar that described SMP molecule participates in Corynebacterium glutamicum is converted into useful catabolite and energy (for example ATP), perhaps produces the useful molecule that is rich in energy (for example ATP) by other process such as oxidative phosphorylation. In one embodiment, the SMP molecule participates in the carbon compound metabolism of Corynebacterium glutamicum such as sugar and by the process produce power molecule (for example ATP) such as oxidative phosphorylation. In a preferred embodiment, participating in Corynebacterium glutamicum carbon metabolism and energy-producing SMP molecular activity of the present invention needs fine chemicals to exert an influence to this biology generation. In an especially preferred embodiment, regulate the activity of SMP molecule of the present invention, so that Corynebacterium glutamicum metabolic pathway and energy pathway that SMP albumen of the present invention participates in regulated in productive rate, output and/or production efficiency, this directly or the indirect regulation Corynebacterium glutamicum produce productive rate, output and/or the production efficiency that needs fine chemicals.

Term " SMP albumen " or " SMP polypeptide " comprise can carry out the carbohydrate metabolism that participates in Corynebacterium glutamicum such as sugar and by the protein such as the energy-producing function of process of oxidative phosphorylation. SMP albumen example comprises the protein of the SMP gene code that is provided by table 1 and the protein of being encoded by odd number SEQ ID NO. Term " SMP gene " or " SMP nucleotide sequence " comprise the nucleotide sequence of coding SMP albumen, and this nucleotide sequence is comprised of a code area and corresponding untranslated 5 ' and 3 ' sequence area. The example of SMP albumen comprises the albumen that table 1 provides. Term " output " or " production capacity " are well known in the art, are included in tunning (for example the needing fine chemicals) concentration that obtains in preset time and the given fermentation volume (for example per hour every liter kg product). Term " production efficiency " comprises the required time of the specific level of production that reaches (for example how long cell reaches specific fine chemicals output capacity needs). Term " productive rate " or " product/carbon productive rate " are well known in the art, comprise that carbon source is converted into the efficient of product (being fine chemicals). This is typically expressed as for example kg product of every kg carbon source. By productive rate or the output that increases described compound, can increase this compound in preset time, specified rate culture the recovery molecular amounts or effectively reclaim molecular amounts. Term " biosynthesis " or " biosynthesis pathway " are well known in the art, comprise that cell begins synthetic compound, organic compound preferably take the process that may as multi-step, highly regulate from midbody compound. Term " degraded " or " degradation pathway " are well known in the art, comprise that cell is take the process that may as multi-step, highly regulate with compound, preferably organic compound is decomposed into catabolite (molecule that generally speaking, be less or complexity is lower). Term " catabolite " is well known in the art, comprises the catabolite of compound. Described product itself may be as essential precursor (starting point) or intermediate molecule when synthetic other compound of cell biological. Term " metabolism " is well known in the art, comprises biological esoteric all biological chemical reaction. The metabolism of the specific compound amino acid whose metabolism of glycine (for example such as) comprises then that all biological relevant with this compound in the cell is synthetic, modification and degradation pathway.

In another embodiment, SMP molecule of the present invention can be regulated microorganism for example needs for example generation of fine chemicals of molecule in the Corynebacterium glutamicum. SMP albumen of the present invention changes may directly affect productive rate, output and/or the production efficiency that the Corynebacterium glutamicum strain that mixes this change albumen produces fine chemicals by number of mechanisms. Degrade the high-energy carbon molecule such as sugar and make compound such as NADH and FADH2 be converted into more useful form by oxidative phosphorylation and can produce chemical compound lot, the fine chemicals that these compounds may need exactly itself, for example pyruvic acid, ATP, NADH and multiple intermediate sugar compounds. And then kinetomeres (such as ATP) and reducing equivalent thing (such as NADH or NADPH) that cell can utilize these metabolic pathways to produce drive disadvantageous reaction on energy. Such adverse effect comprises many fine chemicals biosynthesis pathways. People utilize the ability (for example operate coding participates in described cell degradation and transforms the gene that this sugar is the enzyme of energy) of specific sugar can improve energy by improving cell, so that cell can carry out metabolic response (for example biosynthesis needs fine chemicals) unfavorable but that need

One or more SMP genes of the present invention of mutagenesis also may produce the SMP albumen of activity change, and the SMP albumen remote-effects Corynebacterium glutamicum of activity change produces one or more needs fine chemicals. For example, people can increase these energy-rich compound amounts by the efficient (for example active by improving oxidative phosphorylation efficient or strengthening atp synthase) that raising utilizes one or more sugared efficient (being converted into useful kinetomeres thereby strengthen described sugar) or raising reducing equivalent thing to be converted into useful kinetomeres, for the common disadvantageous metabolic process of cellular driven. Such process comprise make up cell membrane, transcribe, translation and essential compound (such as nucleotides, amino acid, vitamin, lipid etc.) (Lengeler etc. (1999) the Biology of Prokaryotes of biosynthesis cell division, Thieme Verlag:Stuttgart, 88-109,913-918,875-899 page or leaf). By improving the Growth and reproduction of these engineering cells, both can improve the cell viability in the large-scale culture thing, also can improve its division rate, more substantial like this cell can be survived in the fermentation tank culture thing. At least improve productive rate, output or production efficiency because there being the living cells of the required fine chemicals of more generation. In addition, a large amount of degradeds of glycometabolism generation and midbody compound are essential precursor and the intermediate of whole other biosynthesis pathway of cell. For example many amino acid are directly by the compou nd synthesis (for example serine by glycolysis intermediate 3-phoshoglyceric acid synthetic) of common generation from glycolysis or TCA circulation. Thereby, by having strengthened the sugared efficient that is converted into the useful energy molecule, also can increase useful catabolite quantity.

The nucleotide sequence of separation of the present invention is included in the genome of Corynebacterium glutamicum strain, and described bacterial strain can obtain by American type culture collection, and preserving number is ATCC 13032. The nucleotide sequence of the Corynebacterium glutamicum SMP DNA of described separation and the predicted amino acid sequence of Corynebacterium glutamicum SMP albumen are shown among sequence table odd number SEQ ID NO and the even number SEQ ID NO. Carry out Computer Analysis, with the classification of these nucleotide sequences and/or be accredited as that coding has the carbon compound metabolism that participates in Corynebacterium glutamicum such as sugar and by the sequence such as the protein of the function of the process produce power molecule of oxidative phosphorylation.

The amino acid that the present invention also relates to have and amino acid sequence of the present invention (for example sequence table even number SEQ ID NO sequence) be the protein of homology basically. The amino acid sequence that has used herein and selected amino acid sequence be the protein of homology basically, with selected amino acid sequence (for example described complete selected amino acid sequence) at least about 50% homology. The amino acid sequence that has and selected amino acid sequence be the protein of homology basically, also can with selected amino acid sequence have at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90% or 90-95%, most preferably at least about 96%, 97%, 98%, 99% or higher homology.

SMP albumen of the present invention or its biologically-active moiety or fragment can participate in the carbon compound metabolism of Corynebacterium glutamicum such as sugar and by such as the process produce power molecule (for example ATP) of oxidative phosphorylation, or have one or more activity that table 1 provides.

Various aspects of the present invention are described in following trifle in further detail.

A. the nucleic acid molecules that separates

One aspect of the present invention relates to the nucleic acid molecules and being suitable as of the separation of coding SMP polypeptide or its biologically-active moiety to be identified or the hybridization probe of amplification SMP code nucleic acid (for example SMP DNA) or the nucleic acid fragment of primer. Term used herein " nucleic acid molecules " will refer to comprise DNA or the RNA analog of dna molecular (for example cDNA or genomic DNA) and RNA molecule (for example mRNA) and employing nucleotide analog deposits yields. This term also comprises the non-translated sequence that is positioned at gene coding region 3 ' end and 5 ' end: sequence and the gene coding region 3 ' at least about 100 nucleotides of 5 ' end upstream, code area hold the downstream at least about the sequence of 20 nucleotides. Described nucleic acid molecules can be strand or two strands, but double-stranded DNA preferably. " separation " nucleic acid molecules is the nucleic acid molecules that separates with other nucleic acid molecules of existing in the described nucleic acid natural origin. Preferably " separation " nucleic acid molecules does not contain the sequence (namely being positioned at the sequence of described nucleic acid 5 ' end and 3 ' end) of the described nucleic acid of natural adjacency in the biological genomic DNA in described nucleic acid source. For example, in various embodiments, the SMP nucleic acid molecules of separation can contain the nucleotide sequence of nucleic acid molecules as described in the natural adjacency in the genomic DNA of the cell (such as the Corynebacterium glutamicum cell) in described nucleic acid source less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb. In addition, " separation " nucleic acid molecules, for example dna molecular can be substantially free of other cellular material, does not maybe contain culture medium when cultivating by recombinant technique, or do not contain precursor or other chemical substance when chemical synthesis.

Nucleic acid molecules of the present invention (nucleic acid molecules that for example has sequence table odd number SEQ ID NO nucleotide sequence) or its part can learn a skill with standard biological and separate with sequence information provided herein. For example, can be with the full sequence of one of sequence table odd number SEQ ID NO sequence or partial sequence as hybridization probe, with the standard hybridization technique (for example adopt, be described in the technology with Publication about Document: Sambrook, J., Fritsh, E.F. and Maniatis, T.Molecular Cloning:A Laboratory Manual. the 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), from the Corynebacterium glutamicum library, isolate Corynebacterium glutamicum SMP DNA. In addition, the nucleic acid molecules that comprises all or part of sequence of one of nucleotide sequence of the present invention (for example odd number SEQ ID NO), can adopt the Oligonucleolide primers based on this sequences Design, by the PCR separate (nucleic acid molecules that for example, comprises all or part of sequence of one of nucleotide sequence of the present invention (such as sequence table odd number SEQ ID NO) can adopt based on as described in the Oligonucleolide primers of design of sequence separate by the PCR). For example, can be from normal endothelial cell separating mRNA (such as the guanidine thiocyanate extraction method by (1979) Biochemistry 18:5294-5299 such as Chirgwin), then use reverse transcriptase (Moloney MLV reverse transcriptase for example, can derive from Gibco/BRL, Bethesda, MD; Or the AMV reverse transcriptase, can derive from Seikagaku America, Inc., St. Petersburg, FL) preparation DNA. Can based on one of nucleotide sequence shown in the sequence table, be designed for the synthetic oligonucleotide primer thing of PCR amplification. As template, with suitable Oligonucleolide primers, according to Standard PC R amplification technique, nucleic acid of the present invention can increase with cDNA or genomic DNA. The nucleic acid clone of so amplification in suitable carrier, and is identified by dna sequence analysis. In addition, can pass through the standard synthetic technology corresponding to the oligonucleotides of SMP nucleotide sequence, for example prepare with automatic dna synthesizer.

In a preferred embodiment, the nucleic acid molecules of separation of the present invention comprises the nucleotide sequence shown in a kind of sequence table. Nucleotide sequence of the present invention shown in the sequence table is equivalent to Corynebacterium glutamicum SMP DNA of the present invention. This DNA comprises sequence (being " code area " shown in each odd number SEQ ID NO of sequence table) and 5 ' non-translated sequence and the 3 ' non-translated sequence of coding SMP albumen, and described non-translated sequence also is shown among the sequence table odd number SEQ ID NO. Perhaps, described nucleic acid molecules can only comprise the code area of the arbitrary nucleotide sequence of sequence table.

In order to carry out this application, people know, every kind of nucleotide sequence shown in the sequence table and amino acid sequence have identification RXA, RXN or RXS numbering, meet identifier RXA, RXN or the RXS (being RXA01626, RXN00043 or RXS0735) of 5 bit digital after described numbering has. Every kind of described nucleotide sequence comprises at the most three parts: a 5 ' upstream, a code area and a catchment. Each district in these three districts differentiates by identical RXA, RXN or RXS identifier, to avoid confusion. Narration " one of sequence table odd number sequence " then refers to the arbitrary nucleotide sequence of sequence table, and it also can be distinguished according to its different RXA, RXN or RXS identifier. The code area of each sequence in these sequences is translated into corresponding amino acid sequence, and described amino acid sequence also is shown in the sequence table, as being right after corresponding nucleic sequence even number SEQ ID NO afterwards. For example, the code area of RXA02735 is shown among the SEQ ID NO:1, and the amino acid sequence of its coding is shown among the SEQ ID NO:2. The sequence of nucleic acid molecules of the present invention is differentiated according to RXA, RXN or the RXS identifier identical with their coded amino acid moleculars, so that can easily they be interrelated. For example, the amino acid sequence of RXA00042 by name is the nucleotide sequence coded district translation of RXA00042 nucleic acid molecules. Corresponding relation between the SEQ ID NO of RXA, RXN and RXS nucleotide sequence and amino acid sequence and its appointment sees Table 1.

Some genes of the present invention are " F marker gene ". The F marker gene comprises the gene that had " F " before the RXA identifier shown in the table 1. For example, the SEQ ID NO:11 of as shown in table 1 being called " F RXA01312 " is a kind of F marker gene, SEQ ID NO:29,33 and 39 (being called " F RXA02803 " in table 1, " F RXA02854 ", " F RXA01365 ") also is like this.

In one embodiment, nucleic acid molecules of the present invention does not comprise the nucleic acid molecules of table 2 inediting. With regard to the dapD gene, the sequence of this gene is published in Wehrmann, A. etc. (1998) J.Bacteriol.180 (12): among the 3159-3165. Yet the sequence that the present application people obtains significantly is longer than the form of announcing. Think that described announcement form is because of incorrect initiation codon, therefore only represents a fragment of true code area.

In another preferred embodiment, the nucleic acid molecules of separation of the present invention comprises kernel of complementary sequence acid molecule or its part of a kind of nucleotide sequence of the present invention (for example sequence table odd number SEQ ID NO sequence). With a kind of nucleic acid molecules of complementation of nucleotide sequence of the present invention be and one of nucleotide sequence shown in the sequence table (odd number SEQ ID NO for example: sequence) enough complementary so that can and therefore form the nucleic acid molecules of stablizing duplex with the hybridization of one of nucleotide sequence of the present invention.

In a preferred embodiment again, the nucleic acid molecules of separation of the present invention comprises such nucleotide sequence or its part: the homology of described nucleotide sequence and nucleotide sequence of the present invention (for example sequence table odd number SEQ ID NO sequence) is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% or 91%, 92%, 93%, 94%, even more preferably at least about 95%, 96%, 97%, 98%, 99% or higher. The present invention also comprises the scope and sign numerical value (for example 70-90% is identical or 80-95% identical) between the above-mentioned numerical value. For example, comprise that the above-mentioned value that adopts any combination is as the ident value scope of the upper limit and/or lower limit. In another preferred embodiment, the nucleic acid molecules of separation of the present invention comprises nucleotide sequence or its part with the hybridization of one of nucleotide sequence of the present invention, for example hybridize under stringent condition.

In addition, nucleic acid molecules of the present invention can only comprise the part in a kind of odd number SEQ ID NO sequential coding district in the sequence table, for example can be used as the fragment of the biologically-active moiety of the fragment of probe or primer or the SMP albumen of encoding. According to the nucleotide sequence of measuring from Corynebacterium glutamicum clone SMP gene, so that can be for the preparation of probe or the primer of the SMP homologue of identifying and/or cloning SMP homologue in other cell type or the biology and other excellent bacillus or relevant bacterial classification. Described probe/primer comprises the oligonucleotides of basic purifying usually. Described oligonucleotides comprises a kind of nucleotide sequence district usually, described nucleotide sequence district under stringent condition with the antisense sequences of one of the sense strand of one of nucleotide sequence of the present invention (such as sequence table odd number SEQ ID NO sequence), these sequences or its naturally occurring mutant at least about 12, preferred about 25, more preferably from about 40,50 or 75 continuous nucleotides hybridization. Primer based on nucleotide sequence of the present invention can be used for the PCR reaction, with clone SMP homologue. Can be used for detecting transcript or the genome sequence of the same albumen of coding or homologous protein based on the probe of described SMP nucleotide sequence. In preferred embodiments, described probe also comprises a connected labelling groups, and for example described labelling groups can be radio isotope, fluorescent chemicals, enzyme or enzyme cofactor. This class probe can be as the part of diagnostic test kit, to identify the cell of unconventionality expression SMP albumen, for example by measuring the level of SMP code nucleic acid in the cell sample, for example detect SMP mRNA level or detect genome SMP gene and whether suddenly change or lack.

In one embodiment, nucleic acid molecule encoding of the present invention is a kind of to comprise protein or its part with the amino acid sequence of the enough homologies of amino acid sequence of the present invention (for example sequence table even number SEQ ID NO sequence), so that described protein or its part keep the carbon compound metabolism that participates in Corynebacterium glutamicum such as sugar and by the process produce power molecule (ability of (for example ATP) such as oxidative phosphorylation. Term used herein " enough homology " refers to a kind of like this protein or its part: the amino acid sequence that described protein or its part have comprises the identical with amino acid sequence of the present invention of minimal amount or is equal to (amino acid residue that the side chain that for example has is similar to the amino acid residue in the sequence table even number SEQ ID NO sequence) amino acid residue, so that described protein or its part can participate in the carbon compound metabolism of Corynebacterium glutamicum such as sugar and pass through process produce power molecule (for example ATP) such as oxidative phosphorylation. The albumen member of this class glycometabolism approach as herein described or energy production system may worked aspect the production of one or more fine chemicals and the secretion. The example of this class activity has also been described in this article. Therefore, " function of SMP albumen " affects productive rate, output and/or the production efficiency of one or more fine chemicals directly or indirectly. SMP protein active example sees Table 1.

In another embodiment, the homology of described protein and complete amino acid sequence of the present invention (for example sequence table even number SEQ ID NO sequence) is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, 90-95%, most preferably at least about 96%, 97%, 98%, 99% or higher.

The part of the protein that SMP nucleic acid molecules of the present invention is coded is a kind of biologically-active moiety of described SMP albumen preferably. Term used herein " biologically-active moiety of SMP albumen " comprises the part of the SMP albumen of the carbon compound metabolism that participates in Corynebacterium glutamicum such as sugar and production capacity approach, domain/motif for example, or have a kind of activity that table 1 provides. In order to determine that whether SMP albumen or its biologically-active moiety can participate in the metabolism of Corynebacterium glutamicum carbon compound and produce the molecule that is rich in energy, can carry out the mensuration of enzymatic activity. This class assay method is that those skilled in the art are well-known, such as what describe in detail among the embodiment 8 of embodiment part.

Be prepared as follows the nucleic acid fragment of other coding SMP protein biological activity part: a part of separating a kind of amino acid sequence of the present invention (for example sequence table even number SEQ ID NO sequence), express the coded portion (for example passing through in-vitro recombination expression) of described SMP albumen or peptide, and estimate the activity of the coded portion of described SMP albumen or peptide.

The present invention also comprises the nucleic acid molecules that is different from a kind of nucleotide sequence of the present invention (for example sequence table odd number SEQ ID NO sequence) (with its part) and the SMP albumen that therefore coding is identical with the coded albumen of nucleotide sequence of the present invention owing to the degeneracy of genetic code. In another embodiment, the nucleotide sequence coded protein with amino acid sequence shown in the sequence table (for example even number SEQ ID NO) that has of the nucleic acid molecules of separation of the present invention. In an embodiment again, nucleic acid molecule encoding of the present invention and amino acid sequence of the present invention (by the coding of the open read frame shown in the sequence table odd number SEQ ID NO) be the total length Corynebacterium glutamicum albumen of homology basically.

One skilled in the art will appreciate that in one embodiment sequence of the present invention does not comprise the sequence of prior art, the Genbank sequence shown in the table 2 or 4 that for example before the present invention, just obtains. In one embodiment, the present invention includes the homogeneity percentage that has with nucleotide sequence of the present invention or amino acid sequence greater than nucleotide sequence and the amino acid sequence of the homogeneity percentage of the sequence of prior art (for example Genbank sequence of narration in the table 2 or 4 protein of this sequential coding (or by)). For example, the present invention includes: with the homogeneity of the nucleotide sequence of RXA00014 by name (SEQ ID NO:41) greater than 58% and/or be at least 58% nucleotide sequence; With the homogeneity of the nucleotide sequence of RXA00195 by name (SEQ ID NO:399) greater than % and/or be at least the nucleotide sequence of %; And with the homogeneity of the nucleotide sequence of RXA00196 by name (SEQ ID NO:401) greater than 42% and/or be at least 42% nucleotide sequence. The homogeneity fractions that those skilled in the art calculate by detecting the GAP shown in the highest table 4 that hits each of three of any given sequences of the present invention, and deduct the homogeneity percentage that the highest GAP calculates with 100%, can calculate the lower limit of described given sequence homogeneity percentage of the present invention. Those skilled in the art also can know, the present invention comprises that also homogeneity percentage is greater than the lower limit that so calculates (for example at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% or 91%, 92%, 93%, 94%, even more preferably at least about 95%, 96%, 97%, 98%, 99% or higher) nucleotide sequence and amino acid sequence.

Except the Corynebacterium glutamicum SMP nucleotide sequence shown in the sequence table odd number SEQ ID NO, one skilled in the art will appreciate that and in colony (for example Corynebacterium glutamicum colony), may have the dna sequence polymorphism that causes SMP Argine Monohydrochloride sequence to change. Because natural variation, the genetic polymorphism in the described SMP gene of this class may be present in the individuality of colony. Term used herein " gene " and " recombination " refer to comprise coding SMP albumen, the nucleic acid molecules of the open read frame of Corynebacterium glutamicum SMP albumen preferably. This class natural variation causes the variation of 1-5% in the described SMP gene nucleotide series usually. Because the result of natural variation and do not change any or all this class nucleotide diversity of SMP protein function activity and the amino acid polymorphism that produces is included in the scope of the present invention.

According to the natural variation body that is equivalent to Corynebacterium glutamicum SMP DNA of the present invention and the nucleic acid molecules of non-glutamic acid rod bacillus homologue and the homology of Corynebacterium glutamicum SMP nucleic acid disclosed herein, adopt described Corynebacterium glutamicum DNA or its part as hybridization probe under stringent hybridization condition according to the standard hybridization technique, can isolate described nucleic acid molecules. Therefore, in another embodiment, at least 15 nucleotides of the nucleic acid molecules of separation of the present invention are long, and under stringent condition with comprise the making nucleic acid molecular hybridization of sequence table odd number SEQ ID NO nucleotide sequence. In one embodiment, the length of described nucleic acid is at least 30,50,100,250 or more nucleotides. Term used herein " hybridize under stringent condition " is intended to describe hybridization and the wash conditions that the nucleotide sequence of at least 60% homology each other keeps the phase mutual cross usually. Preferably described condition so that each other homology be at least about 65%, more preferably at least about 70% in addition more preferably at least about 75% or higher sequence usually keep the phase mutual cross. This class stringent condition is well known by persons skilled in the art, and can be at Ausubel etc., Current Protocols in Molecular Biology, and John Wiley ﹠ Sons, N.Y. (1989) finds among the 6.3.1-6.3.6. A preferred limiting examples of stringent hybridization condition be in 6X sodium chloride/sodium citrate (SSC) in about 45 ℃ of hybridization, then at 0.2 * SSC, carry out one or many washing in 50-65 ℃ among 0.1% SDS. Preferably under stringent condition, be equivalent to naturally occurring nucleic acid molecules with the isolated nucleic acid molecule of the present invention of nucleotide sequence hybridization of the present invention. " naturally occurring " used herein nucleic acid molecules refers to have naturally occurring nucleotide sequence RNA or the dna molecular of (native protein of for example encoding). In one embodiment, a kind of natural Corynebacterium glutamicum SMP albumen of described nucleic acid coding.

Except the naturally occurring variant of the described SMP sequence that in colony, may exist, those skilled in the art also know, can import change by in nucleotide sequence of the present invention, suddenling change, cause thus the amino acid sequence of coded SMP albumen to change, and do not change the Functional Capability of described SMP albumen. For example, can in nucleotides of the present invention, cause replacing at the nucleotides of " nonessential " amino acid residue upper amino acid replacement. " nonessential " amino acid residue is can be changed in the wild-type sequence (for example sequence table even number SEQ ID NO) of described SMP albumen and do not change the residue of described SMP protein active, and " essential " amino acid residue is SMP protein active needs. Yet other amino acid residue (for example conservative in the domain with SMP activity or semiconservative residue only) may not be active essential, therefore may be fit to change and does not change the SMP activity.

Therefore, another aspect of the present invention relates to the nucleic acid molecules of coding SMP albumen, and the active nonessential amino acid residue of the SMP of described SMP albumen changes. This class SMP albumen is different from sequence table even number SEQ ID NO sequence at amino acid sequence, but still it is active to keep at least a SMP as herein described. In one embodiment, the nucleic acid molecules of described separation comprises the nucleotide sequence of coded protein, wherein said protein comprises and the amino acid sequence of amino acid sequence of the present invention at least about 50% homology, and can participate in carbon compound metabolism and the biosynthesis energy-rich compound of Corynebacterium glutamicum such as sugar, or have one or more activity that table 1 provides. Preferably in the protein of described nucleic acid molecule encoding and the sequence table a kind of even number SEQ ID NO amino acid sequence at least about the 50-60% homology, more preferably with one of these sequences at least about the 60-70% homology, more preferably with one of these sequences at least about 70-80%, 80-90%, 90-95% homology, most preferably with a kind of amino acid sequence of the present invention at least about 96%, 97%, 98% or 99% homology.

In order to determine the percent homology of two seed amino acid sequences (for example a kind of amino acid sequence of the present invention and its mutant) or two kinds of nucleic acid, carry out best sequence contrast (for example can introduce the room in the sequence of a kind of protein or nucleic acid, to carry out the optimal sequence contrast with another kind of protein or nucleic acid) relatively. Then more corresponding amino acid position or nucleotide position upper amino acid residue or nucleotides. When a position in the sequence (such as a kind of amino acid sequence of the present invention) was occupied by the same amino acid residue of relevant position in another sequence (mutant of amino acid sequence for example) or nucleotides, then described two kinds of molecules were homology (being that amino acid used herein or nucleic acid " homology " are equal to amino acid or nucleic acid " homogeneity ") in this position. Percent homology between these two sequences is the function (being percent homology=same position number/total number of positions * 100) that described sequence is shared the same position number.

Coding can make up by following steps with the nucleic acid molecules that separates of the SMP albumen of protein sequence of the present invention (for example sequence table even number SEQ ID NO sequence) homology: in nucleotide sequence of the present invention, import one or more nucleotides and replace, add or disappearance, so that in coded protein, import one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, interpolation or disappearance. Can adopt standard technique, for example sudden change of direct mutagenesis and PCR mediation, will suddenly change imports in a kind of nucleotide sequence of the present invention. Preferably the nonessential amino acid residue in one or more predictions carries out the conserved amino acid replacement. " conserved amino acid replacement " is the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor that wherein said amino acid residue is had the amino acid residue replacement of similar side chain. Determined in the art to have the amino acid residue classification of similar side chain. These classifications comprise the amino acid (for example lysine, arginine, histidine) with basic side chain, the amino acid (for example aspartic acid, glutamic acid) with acid side-chain, the amino acid (for example glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) with uncharged polar side chain, the amino acid (for example alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) with non-polar sidechain, the amino acid (for example threonine, valine, isoleucine) with β-branched building block and the amino acid (for example tyrosine, phenylalanine, tryptophan, histidine) with aromatic side chains. Therefore, the nonessential amino acid residue of predicting in the SMP albumen is preferably replaced by the another kind of amino acid residue from same side chain classification. Perhaps, in another embodiment, can import at random sudden change along all or part SMP coded sequence, for example import sudden change by saturation mutagenesis, and can screen the mutant that produces according to SMP activity as herein described, to identify the mutant that keeps the SMP activity. After the nucleotide sequence mutagenesis to one of sequence table odd number SEQ ID NO, can recombinant expressed coded protein, then can adopt mensuration as herein described (referring to the embodiment 8 of embodiment part) for example to measure the activity of described protein.

Except the nucleic acid molecules of above-mentioned coding SMP albumen, another aspect of the present invention relates to the nucleic acid molecules of the separation of antisense. " antisense " nucleic acid comprises the nucleotide sequence with " justice is arranged " nucleic acid complementation of coded protein, for example complementary with the coding strand of double-stranded DNA or with the nucleotide sequence of mRNA sequence complementation. Therefore, antisensenucleic acids can form hydrogen bond with phosphorothioate odn is arranged. Described antisensenucleic acids and complete SMP coding strand or only complementary with its part. In one embodiment, antisense nucleic acid molecule is the antisense nucleic acid molecule of " code area " of the nucleotide sequence coded chain of coding SMP albumen. Term " code area " refers to comprise the nucleotide sequence district (for example the complete coding region of SEQ ID NO:3 (RXA01626) comprises nucleotides 1-345) of the codon that is translated into amino acid residue. In another embodiment, described antisense nucleic acid molecule is the antisense nucleic acid molecule of the nucleotide sequence coded chain " noncoding region " of coding SMP. Term " noncoding region " refers in abutting connection with described code area, is not translated into amino acid whose 5 ' sequence and 3 ' sequence (namely being also referred to as 5 ' and 3 ' non-translational region).

The coding strand sequence (for example sequence shown in the sequence table odd number SEQ ID NO) of known coding SMP disclosed herein therefore can according to Waston and Crick basepairing rule, design antisensenucleic acids of the present invention. Described antisense nucleic acid molecule can be complementary with the complete coding region of SMP mRNA, but only be more preferably the oligonucleotides with a part of antisense of SMP mRNA code area or noncoding region. For example, described ASON can be complementary with the section around the SMP mRNA translation initiation site. For example, described ASON can be for example about 5,10,15,20,25,30,35,40,45 or 50 nucleotides long. The enzyme coupled reaction that can adopt chemical synthesis and adopt methods known in the art makes up antisensenucleic acids of the present invention. For example, can synthesize antisensenucleic acids (such as ASON) with the nucleotides chemistry of naturally occurring nucleotides or various modifications, the nucleotides design of described modification is in order to increase described molecular biology stability or to increase described antisensenucleic acids and formed duplex physical stability between the phosphorothioate odn is arranged, the nucleotides that for example can use phosphorothioate analogue and acridine to replace. The example of nucleotides that can be used for producing the modification of described antisensenucleic acids comprises 5 FU 5 fluorouracil, 5-bromouracil, the 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, the 4-acetylcytosine, 5-(carboxyl hydroxymethyl) uracil, 5-carboxymethylamino methyl-2-thio uridine, 5-carboxymethylamino methyluracil, dihydrouracil, β-D-galactosyl Q nucleosides (queosine), inosine, the N6-isopentenyl gland purine, the 1-methyl guanine, M1I, 2, the 2-dimethylguanine, the 2-methyl adenine, the 2-methyl guanine, the 3-methylcystein, the 5-methylcystein, the N6-adenine, the 7-methyl guanine, 5-methylamino methyluracil, 5-methoxyl group amino methyl-2-thiouracil, β-D-MANNOSE base Q nucleosides, 5 '-methoxyl group carboxyl methyluracil, the 5-methoxyuracil, 2-methyl mercapto-N6-isopentenyl gland purine, uracil-5-glycolic acid (v), wybutoxosine, pseudouracil, the Q nucleosides, 2-sulfo-cytimidine, 5-methyl-2-thiouracil, the 2-thiouracil, the 4-thiouracil, methyl uracil, uracil-5-hydroxy methyl acetate, uracil-5-glycolic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxylic propyl group) uracil, (acp3) w and 2,6-diaminopurine. On the other hand, (RNA that is namely gone out by the transcribed nucleic acid that is inserted will have the antisense orientation of interested target nucleic acid can to have adopted with the antisense orientation subclone a kind of nucleic acid, in following trifle, further describe) expression vector, produce described antisensenucleic acids with biological method.

Usually give cell with antisense nucleic acid molecule of the present invention or its original position is produced, so that they are hybridized or combination with cell mRNA and/or the genomic DNA of coding SMP albumen, to suppress thus described protein expression, for example transcribe and/or translate by inhibition and suppress to express. Can hybridize by the nucleotides complementarity of routine, forming stable duplex, or for example in the situation of the antisense nucleic acid molecule of being combined with DNA duplex, interact by the specificity in the double helix tap drain and to hybridize. Can modify described antisense molecule, so that it is for example by described antisense nucleic acid molecule is connected with the peptide that is incorporated into cell surface receptor or antigen or antibody, and be incorporated into specifically acceptor or the antigen of selected cell surface expression. Can described antisense nucleic acid molecule be passed to cell with carrier described herein. In order to obtain the described antisense molecule of enough intracellular concentrations, preferred wherein said antisense nucleic acid molecule places the vector construct under strong prokaryotes, virus or the promoter in eukaryote control.

In an embodiment again, antisense nucleic acid molecule of the present invention is α-different nucleic acid molecules. α-different nucleic acid molecules and complementary RNA form specific double-stranded crossbred, and be wherein opposite with common β unit, described chain (Gaultier etc. (1987) Nucleic Acids.Res. 15:6625-6641) parallel to each other. Described antisense nucleic acid molecule also can comprise 2 '-o-methyl ribonucleotides (Inoue etc. (1987) Nucleic Acids Res.15:6131-6148) or chimeric RNA-DNA analog (Inoue etc. (1987) FEBS Lett.215:327-330).

In yet another embodiment, antisensenucleic acids of the present invention is a kind of ribozyme. Ribozyme is the catalytic RNA molecule with ribalgilase, and they can cut the single-chain nucleic acid that has complementary district with it, for example mRNA. Therefore, can (for example hammerhead ribozyme (being described in Haselhoff and Gerlach (1988) Nature 334:585-591) comes catalyze cleavage SMP mRNA transcript, suppresses thus the translation of SMP mRNA with ribozyme. Can (be SEQ ID NO.3 (RXA01626), design has specific ribozyme to the SMP code nucleic acid according to the nucleotide sequence of SMP cDNA disclosed herein. For example, make up the derivative of a kind of tetrahymena (Tetrahymena) L-19 IVS RNA, wherein the nucleotide sequence of active site and SMP coding among the mRNA nucleotide sequence to be cut complementary. Referring to the U.S. Patent number 5,116,742 such as the U.S. Patent number 4,987,071 of Cech etc. and Cech etc. Perhaps, can use SMP mRNA, from the RNA library of molecules, select to have the catalytic RNA of specific ribonuclease activity. Referring to for example Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.

On the other hand, the nucleotide sequence by target and SMP nucleotides regulatory region (for example SMP promoter and/or enhancer) complementation to form the triple-helix structure that stops SMP genetic transcription in the target cell, can suppress the expression of SMP gene. Generally referring to Helene, C. (1991) Anticancer Drug Des.6 (6): 569-84; Helene, C. etc. (1992) Ann.N.Y.Acad. Sci.660:27-36; And Maher, L.J. (1992) Bioassays 14 (12): 807-15.

B. recombinant expression carrier and host cell

Another aspect of the present invention relates to the carrier of the nucleic acid that contains coding SMP albumen (or its part), preferred expression carrier. Term used herein " carrier " refers to transport the nucleic acid molecules of connected another kind of nucleic acid. One type carrier is " plasmid ", and plasmid refers to wherein can connect the circular double stranded DNA ring of other DNA section. The carrier of another kind of type is viral vectors, wherein other DNA section can be connected in the viral genome. Some carrier can be in the host cell that they import self-replicating (bacteria carrier and the additive type mammal carrier that for example have the bacterium origin of replication). ((for example non-add type mammal carrier) is incorporated into after importing to host cell in the genome of host cell other carrier, therefore copies with host genome. In addition, some carrier can instruct the expression with its gene that effectively is connected. This class carrier is referred to herein as " expression vector ", and generally speaking, the expression vector that uses in the recombinant DNA technology is the carrier of plasmid form normally. In this manual, " plasmid " and " carrier " can Alternate, because plasmid is the carrier format that uses most. Yet, the present invention includes the expression vector of this other form of class, viral vectors (for example replication defect type retroviruse, adenovirus and adeno-associated virus) for example, the function that their performances are equal to.

Recombinant expression carrier of the present invention comprises the nucleic acid of the present invention that is suitable for expressing the form of nucleic acid of the present invention in host cell, this refers to that described recombinant expression carrier comprises one or more adjusting sequences of selecting according to the host cell that is used for expressing, and described adjusting sequence effectively is connected in nucleotide sequence to be expressed. In recombinant expression carrier, " effectively connecting " refers to interested nucleotide sequence is connected in described adjusting sequence in the mode that allows described nucleotide sequence to express (for example in in-vitro transcription/translation system maybe when with described carrier importing host cell in described host cell). Term " adjusting sequence " comprises promoter, enhancer and other expression control element (for example polyadenylation signal). This class is regulated sequence description in for example Goeddel; Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulate sequence and be included in the adjusting sequence that instructs the adjusting sequence of nucleotide sequence constitutive expression in many type host cells and only in some host cell, instruct described nucleotide sequence to express. The preferred sequence of regulating is for example promoter, for example cos-, tac-, trp-, tet-, trp-tet, lpp-, lac-, lpp-lac, lacI^q、T7-、T5-、 T3-、gal-、trc-、ara-、SP6-、arny、SPO2、λ-P _R-or λ P_L, they are preferred in the bacterium. Other regulates sequence is for example promoter of yeast and fungi, for example ADC1, MF α, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH; Plant promoter, for example CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, no or ubiquitin promoter or phaseolin promoter. Also can use artificial promoter. The design that one skilled in the art will appreciate that described expression vector is depended on selection such as host cell to be transformed, is needed the factor of protein expression level etc. Expression vector of the present invention can be imported in the host cell, produce thus protein or peptide by nucleic acid coding as herein described, comprise fusion or peptide (such as the SMP albumen of SMP albumen, mutant form, fusion etc.).

Can design recombinant expression carrier of the present invention is used at prokaryotic or eukaryotic expression SMP albumen. For example, can be at following cells SMP gene: bacterial cell for example Corynebacterium glutamicum, insect cell (use rhabdovirus expression vector), yeast and other fungal cell (referring to Romanos, M.A. etc. (1992) " exogenous gene expression in the yeast: summary ", Yeast 8:423-488; Van den Hondel, C.A.M.J.J. etc. (1991) " allogeneic gene expression in filamentous fungi ", be stated from: More Gene Manipulations in Fungi, J.W. Bennet and L.L.Lasure write, the 396-428 page or leaf: Academic Press:San Diego; With van den Hondel, C.A.M.J.J. and Punt, P.J. (1991), " be used for the gene transfer system of filamentous fungi and the exploitation of carrier ", be stated from: Applied Molecular Genetics of Fungi, Peberdy, J.F. wait and write, the 1-28 page or leaf, Cambridge University Press:Cambridge), algae and metaphyte cell are (referring to Schmidt, L. (1988) " arabidopsis (Arabidopsis thaliana) of efficient Agrobacterium tumefaciems (Agrobacterium tumefaciens) mediation and the conversion of cotyledon explant " Plant Cell Rep.:583-586) or mammalian cell R. and Willmitzer. Suitable host cell is at Goeddel, Gene Expression Technology:Methods in Enzymology 185, and Academic Press, San Diego, CA has further description in (1990). Perhaps, described recombinant expression carrier can for example transcribe and translate with T7 promoter adjusting sequence and T7 polymerase external.

Expressing protein is the most frequently used in prokaryotes contains the composing type that instructs fusion or non-expressing fusion protein or the carrier of inducible promoter carries out. Fusion vector adds to a plurality of amino acid wherein on the coded albumen, usually adds to the amino terminal of described recombinant protein, but also adds to the C end, or merge in the suitable district in described protein. This class fusion vector has three effects usually: 1) increase the expression of recombinant protein; 2) dissolubility of the described recombinant protein of increase; With 3) by help the purifying of described recombinant protein as the part in the affinity purification. Usually, in fusion expression vector, introduce a protease cutting site at the contact place of merging part and recombinant protein, so that can behind the described fusion of purifying, recombinant protein be separated with fusion section. This fermentoid and relevant recognition sequence thereof comprise factor Xa, fibrin ferment and enterokinase.

Typical fusion expression vector comprises pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), they merge glutathione S-transferase (GST), maltose E respectively in conjunction with albumen or A albumen and target recombinant protein. In one embodiment, the coded sequence of described SMP albumen is cloned in the pGEX expression vector, to produce the carrier of encoding fusion protein, described fusion comprises GST-fibrin ferment cleavage site-X protein from N end to C end. Described fusion can pass through affinitive layer purification with glutathione-agarose resin. The restructuring SMP albumen with the GST fusion can not reclaim by cut described fusion with fibrin ferment.

The example of the non-fusion coli expression carrier of suitable induction type comprises pTrc (Amann etc., (1988) Gene 69:301-315), pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λ gt11, pBdC1 and pET 11d (Studier etc., Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; Write (1985) Cloning Vectors. Elsevier with Pouwels etc.: New York IBSN 0 444 904018). The expression of pTrc carrier target gene depends on the host RNA polymerase of hybrid trp-lac promoter, fusion and transcribes. The expression of the target gene of pET11d carrier depends on transcribing of viral rna polymerase (T7 gnl) T7 gn10-lac promoter, fusion by co expression. This varial polymerases is by host strain BL21 (DE3) or HMS174 (DE3) supply of residence (resident) the λ prophage that contains the T7 gnl gene under the control of lacUV5 promoter transcription. About the conversion of other bacterium mutation, can select suitable carrier. For example, known plasmid pIJ101, pIJ364, pIJ702 and pIJ361 can be used for transforming streptomyces (Streptomyces), and plasmid pUB110, pC194 or pBD214 are applicable to transforming bacillus and belong to (Bacillus) bacterial classification. Comprise pHM1519, pBL1, pSA77 or pAJ667 (Pouwels etc. write (1985) Cloning Vectors.Elsevier: New York IBSN 0,444 904018) for several plasmids of hereditary information being transferred to Corynebacterium.

Making the maximized a kind of strategy of expression of recombinant proteins is to express described albumen (Gottesman in the impaired host bacteria of the described recombinant protein ability of proteolytic cleavage, S., Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Californiia (1990) 119-128). Another kind of strategy is to change the nucleotide sequence that is inserted into the nucleic acid in the expression vector, so that each amino acid whose each codon is the preferential codon (Wada etc. (1992) Nucleic Acids Res.20:2111-2118) that uses of bacterium (such as Corynebacterium glutamicum) that is used for expression selected. The change of this nucleotide sequence of the present invention can be carried out with the standard DNA synthetic technology.

In another embodiment, described SMP protein expression vector is a kind of Yeast expression carrier. Example for the carrier of expressing at yeast saccharomyces cerevisiae (S.cerevisiae) comprises pYepSecl (Baldari etc. (1987) Embo J.6:229-234), 2 μ, pAG-1, Yep6, Yep13, pEMBLYe23, pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz etc., Gene 54:113-123) and pYES2 (Invitrogen Corporation (1987), San Diego, CA). Be applicable to other fungi for example the carrier of filamentous fungi and carrier construction method be included in carrier and method to describe in detail in the Publication about Document: van den Hondel, C.A.M.J.J. and Punt, P.J. (1991) " are used for the gene transfer system of filamentous fungi and the exploitation of carrier ", be stated from: Applied Molecular Genetics of Fungi, J.F.Peberdy etc. write, the 1-28 page or leaf, Cambridge University Press:Cambridge, and Pouwels etc. writes (1985) Cloning Vectors.Elsevier: New York (IBSN 0,444 904018).

On the other hand, SMP albumen of the present invention can be with rhabdovirus expression vector in expressed in insect cells. The baculovirus vector that is used in the middle expressing protein of insect cell (such as the Sf9 cell) of cultivation comprises pAc series (Smith etc. (1983) Mol.Cell Biol.3:2156-2165) and pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In another embodiment, SMP albumen of the present invention can be expressed at one-celled plants cell (for example algae) or in the plant cell of higher plant (for example seed plant, such as crop). The example of plant expression vector comprises the expression vector that is specified in in the Publication about Document: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) " near the novel plant binary vector that has selected marker on the left side circle ", Plant Mol.Biol.20:1195-1197; And Bevan, M.W. (1984) " the double base agrobacterium vector that is used for Plant Transformation ", Nucl.Acid.Res.12:8711-8721, and comprise pLGV23, pGHlac+, pBIN19, pAK2004 and pDH51 (Pouwels etc. write (1985) Cloning Vectors.Elsevier: New York IBSN 0,444 904018).

In an embodiment again, nucleic acid of the present invention is expressed in mammalian cell with mammalian expression vector. The example of mammalian expression vector comprises pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman etc. (1987) EMBO J.6:187-195). When being used for mammalian cell, the control function of described expression vector is provided by viral regulating element usually. For example, promoter commonly used derives from polyomavirus, adenovirus 2, cytomegalovirus and simian virus 40. For being used for prokaryotic and eukaryotic other suitable expression system, referring to Sambrook, J., Fritsh, E.F. and Maniatis, the 16th and 17 chapters that T.Molecular Cloning:A Laboratory Manual. is the 2nd edition, Cold Spring Harbor Lab oratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

In another embodiment, recombinant mammalian expression vector can instruct described nucleic acid preferentially to express (for example expressing described nucleic acid with the tissue specificity regulating element) in specific cell type. The tissue specificity regulating element is known in the art. The limiting examples of suitable tissue-specific promoter comprises albumin promoter (liver specificity; Pinkert etc. (1987) Genes Dev.1:268-277), lymph (lymphoid) specificity promoter (Calame and Eaton (1988) Adv.Immunol.43:235-275), the particularly promoter of φt cell receptor (Winoto and Baltimore (1989) EMBO J.8:729-733) and immunoglobulin promoter (Banerji etc. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuronal specificity promoter (neurofilament promoter for example; Byrne and Ruddle (1989) PNAS 86:5473-5477), pancreas specificity promoter (Edlund etc. (1985) Science 230:912-916) and mammary gland-specific promoter (whey promoter for example; U.S. Patent number 4,873,316 and European Patent Application No. 264,166). Also comprise and grow adjustment type promoter, for example muroid hox promoter (Kessel and Gruss (1990) Science 249:374-379) and afp promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

The present invention also provides and comprises the recombinant expression carrier of being cloned into the DNA molecule of the present invention in the expression vector with antisense orientation. That is described dna molecular with regulate sequence and effectively be connected, its connected mode provides the expression (by transcribing described dna molecular) of the antisense rna molecule of SMP mRNA. Can select effectively to be connected in antisense orientation clone's nucleic acid, the adjusting sequence of guidance described antisense rna molecule of continuous expression in the various kinds of cell type, for example viral promotors and/or enhancer perhaps can select to instruct the adjusting sequence of composing type, tissue specificity or cell type specificity antisence RNA. Antisense expression vector can be the form of recombinant vector, phasmid or attenuated virus, and wherein antisensenucleic acids produces under the control of efficient regulatory region, and its activity can be determined by the cell type that described carrier imports. For the discussion of the gene expression adjusting of using antisense gene, referring to Weintraub, H. etc., as the antisense RNA of genetic analysis molecular tool, Reviews-Trends in Genetics, the 1st (1) volume 1986.

Another aspect of the present invention relates to the host cell that imports recombinant expression carrier of the present invention. Term " host cell " and " recombinant host cell " are used interchangeably in this article. Self-evident, this term refers to that not only specifically topic is stated cell, and refers to the filial generation of this cell or potential filial generation. Because in the successive generation since sudden change or ambient influnence and some modification may occur so this class filial generation in fact may be incomplete same with parental cell, but still be included in the term scope as herein described.

Host cell can be any prokaryotic or eukaryotic. For example, SMP albumen can for example be expressed in Corynebacterium glutamicum, insect cell, yeast cells or the mammalian cell (for example Chinese hamster ovary cell (CHO) or COS cell) at bacterial cell. Other suitable host cell is well known by persons skilled in the art. Microorganism relevant with Corynebacterium glutamicum, that can be easily used as the host cell of nucleic acid of the present invention and protein molecular is shown in table 3.

Can transform or rotaring dyeing technology by conventional, carrier DNA is imported in prokaryotic or the eukaryotic. Term used herein " conversion " and " transfection ", " joint " and " transduction " refer to the multiple technologies for exogenous nucleic acid (such as the nucleic acid (for example plasmid, bacteriophage, phasmid (phasmid), phasmid (phagemid), transposons or other DNA) of linear DNA or RNA (such as linearized vector or do not comprise the gene constructs of carrier) or carrier format) being imported host cell well known in the art, comprise transfer or the electroporation of transfection, the fat transfection of calcium phosphate or calcium chloride co-precipitation, the mediation of DEAE-glucan, natural competence, chemical substance mediation. The appropriate method that is used for conversion or transfection host cell can be at (Molecular Cloning:A Laboratory Manual. the 2nd edition such as Sambrook, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and in other laboratory manual find.

About the stable transfection of mammalian cell, known to used expression vector and rotaring dyeing technology, only the sub-fraction cell can be incorporated into foreign DNA in its genome. In order to identify and select these integrons, the gene with codes selection mark (such as antibiotic resistance) imports in the host cell with genes of interest usually. Preferred selected marker comprises gives the medicine selected marker of the resistance of G418, hygromycin and amethopterin for example. The nucleic acid of codes selection mark can be imported host cell in the identical carrier of coding SMP albumen, perhaps it can be imported at independent carrier. Can identify by medicament selection the cell (cell that has for example mixed selectable marker gene will be survived, and other cell death) of the nucleic acid stability transfection that imports.

In order to obtain the homologous recombination microorganism, preparation contains the carrier of at least a portion SMP gene, has introduced disappearance in described SMP Gene Partial, add or replaced to change thus (for example functional destruction) described SMP gene. Preferably this SMP gene is Corynebacterium glutamicum SMP gene, but it can be from Related Bacteria or even the homologue in mammal, yeast or insect source. In a preferred embodiment, design described carrier, so that after homologous recombination, described endogenous SMP gene is by functional destruction (i.e. encoding function albumen no longer; Be also referred to as " knocking out " carrier). Perhaps, can design described carrier, so that after homologous recombination, described endogenous SMP gene is suddenlyd change or is changed, but still encoding function albumen (for example can change the upstream regulation district, to change thus described endogenous SMP protein expression). In described homologous recombination vector, the change of described SMP gene part is at the nucleic acid of its 5 ' end and the another kind of SMP gene of 3 ' end adjacency, makes it possible between the entrained external source SMP gene of described carrier and the endogenous SMP gene in the microorganism homologous recombination occur. Described another kind of flank SMP nucleic acid has the length that is enough to endogenous gene success homologous recombination. Usually, the flanking DNA (5 ' end and 3 ' end) (description of relevant homologous recombination vector, referring to for example Thomas, K.R. and Capecchi, M.R. (1987) Cell 51:503) that in described carrier, comprises thousands of bases. Described carrier is imported in the microorganism (for example passing through electroporation) the SMP gene that then wherein imports with the choice of technology known in the art and the cell of endogenous SMP homologous recombination.

In another embodiment, can produce the recombinant microorganism that contains selecting system, described selecting system provides the expression of modulated institute's quiding gene. For example, be included in the SMP gene that places under the control of lac operon at carrier, allow described SMP gene only when IPTG exists, to express. This class regulating system is well-known in the art.

In another embodiment, destroy the endogenous SMP gene (for example by homologous recombination or other genetic method known in the art) in the host cell, so that the expression of its protein product does not occur. In another embodiment, by one or more point mutation, disappearance or inversion, changed SMP gene endogenous or that import in the host cell, but its encoding function SMP albumen still. In an embodiment again, one or more regulatory regions of SMP gene in the microorganism (for example promoter, repressor protein or inducer) (for example by disappearance, brachymemma, inversion or point mutation) have been changed, so that regulate the expression of described SMP gene. One skilled in the art will appreciate that the described SMP gene and the protein modified host cell that contain more than one can adopt method of the present invention easily to produce, and be included in the present invention.

Host cell of the present invention (for example prokaryotic in the culture or eukaryotic) can be used for producing (namely expressing) SMP albumen. Therefore, the present invention also provides the method for using host cell of the present invention to produce SMP albumen. In one embodiment, described method is included in cultivation host cell of the present invention in the suitable culture medium and (has wherein imported the recombinant expression carrier of coding SMP albumen, or in its genome, imported the gene of the SMP albumen of encoding wild type or change), until produce SMP albumen. In another embodiment, described method also comprises separation SMP albumen from culture medium or from host cell.

C. the SMP albumen that separates

Another aspect of the present invention relates to SMP albumen and the biologically-active moiety thereof of separation. " separation " or " purifying " albumen or its biologically-active moiety are substantially free of cellular material when producing by recombinant DNA technology, or are substantially free of precursor or other chemical substance when chemical synthesis. Term " is substantially free of cellular material " and comprises the SMP albumen prepared product that cellular component that natural generation in the wherein smp protein and cell or restructuring produce separates. In one embodiment, term " be substantially free of cellular material " and comprise contained non-SMP albumen (being also referred to as " contaminating protein " herein) be lower than about 30% (with dry weight basis), more preferably less than 20%, again more preferably less than about 10%, most preferably be lower than about 5% SMP albumen prepared product. When restructuring produced described SMP albumen or its biologically-active moiety, it did not preferably contain culture medium substantially yet. It is about 20% to be that culture medium is less than, and more preferably less than about 10%, most preferably is less than about 5% albumen prepared product amount. Term " is substantially free of precursor or other chemical substance " and comprises the SMP albumen prepared product that the precursor synthetic with relating to described albumen of SMP albumen wherein or other chemical substance are separated. In one embodiment, term " be substantially free of precursor or other chemical substance " and comprise the precursor that has or non-SMP chemical substance be lower than about 30% (with dry weight basis), more preferably less than 20%, again more preferably less than about 10%, most preferably be lower than about 5% SMP albumen prepared product. In preferred embodiments, the protein of separation or its biologically-active moiety do not have the contaminating protein from the same biology that obtains described SMP albumen. Usually, Corynebacterium glutamicum SMP albumen produces this albuminoid by for example for example expressing in the Corynebacterium glutamicum in microorganism.

The SMP albumen of separation of the present invention or its part can participate in the carbon compound metabolism of Corynebacterium glutamicum such as sugar, or produce to be used for driving the energy compound (for example by oxidative phosphorylation) of unfavorable metabolic pathway, or have described one or more activity of table 1. In preferred embodiments, described protein or its part comprise the amino acid sequence with the enough homologies of amino acid sequence of the present invention (for example sequence table even number SEQ ID NO sequence), so that described protein or its part keep and carry out the carbon compound metabolism that participates in Corynebacterium glutamicum such as sugar and by the ability such as the process produce power molecule of oxidative phosphorylation. The described part of described albumen is biologically-active moiety as herein described preferably. In another preferred embodiment, SMP albumen of the present invention has in the sequence table amino acid sequence shown in the even number SEQ ID NO. In a preferred embodiment again, described SMP albumen has amino acid sequence coded with the nucleotide sequence of nucleotide sequence of the present invention (for example sequence table odd number SEQ ID NO sequence) hybridization under stringent condition. In another preferred embodiment, described SMP albumen has by following nucleotide sequence coded amino acid sequence or its part, the homology of wherein said nucleotide sequence and a kind of nucleotide sequence of the present invention is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% or 91%, 92%, 93%, 94%, even more preferably at least about 95%, 96%, 97%, 98%, 99% or higher. The present invention also comprises the scope and sign numerical value (for example 70-90% is identical or 80-95% identical) between the above-mentioned numerical value. For example, comprise that the above-mentioned value that adopts any combination is as the ident value scope of the upper limit and/or lower limit. It is active that the preferred SMP albumen of the present invention also preferably has at least a SMP as herein described. For example, preferred SMP albumen of the present invention comprise by with the nucleotide sequence coded amino acid sequence of a kind of nucleic acid array hybridizing of the present invention, for example hybridize under stringent condition, and can participate in the carbon compound metabolism of Corynebacterium glutamicum such as sugar and by the process produce power molecule (for example ATP) such as oxidative phosphorylation, perhaps have one or more activity that table 1 provides.

In other embodiments, described SMP albumen and a kind of amino acid sequence of the present invention (for example sequence table even number SEQ ID NO sequence) be homology basically, and the functional activity that keeps the albumen of one of amino acid sequence of the present invention, but because natural variation or mutagenesis are different at amino acid sequence, as describing in detail in the above I trifle. Therefore, in another embodiment, described SMP albumen is a kind of like this protein: the amino acid sequence that described protein comprises and a kind of homology of complete amino acid sequence of the present invention are at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% or 91%, 92%, 93%, 94%, even more preferably at least about 95%, 96%, 97%, 98%, 99% or higher, and has at least a SMP activity as herein described. The present invention also comprises the scope and sign numerical value (for example 70-90% is identical or 80-95% identical) between the above-mentioned numerical value. For example, comprise that the above-mentioned value that adopts any combination is as the ident value scope of the upper limit and/or lower limit. In another embodiment, the present invention relates to and a kind of complete amino acid sequence of the present invention Corynebacterium glutamicum albumen of the total length of homology basically.

The biologically-active moiety of SMP albumen comprise comprise derived from the amino acid sequence of SMP Argine Monohydrochloride sequence (for example sequence table even number SEQ ID NO: amino acid sequence) or with the peptide of the Argine Monohydrochloride sequence of SMP albumen homology, its amino acid that comprises is less than total length SMP albumen or with the full length protein of SMP albumen homology and have at least a SMP protein active. Usually, biologically-active moiety (peptide, for example length for example is 5,10,15,20,30,35,36,37,38,39,40,50,100 or more amino acid whose peptide) comprises domain or the motif of the activity with at least a SMP albumen. In addition, other biologically-active moiety of other district's disappearance of wherein said protein can prepare by recombinant technique, and estimates according to one or more activity as herein described. Preferably the biologically-active moiety of SMP albumen comprises and has bioactive one or more selected domain/motif or its part.

SMP albumen preferably produces by recombinant DNA technology. For example, the cloned nucleic acid molecule of code for said proteins in expression vector (as mentioned above), is imported host's (as mentioned above) with described expression vector, then in described host cell, express described SMP albumen. Then can adopt standard protein purification technique by suitable purification scheme, from described cell, isolate described SMP albumen. As recombinant expressed alternative, can use the standard peptide synthetic technology, chemical synthesis SMP albumen, polypeptide or peptide. In addition, can for example adopt by standard technique, the anti-SMP antibody that utilizes SMP albumen of the present invention or its fragment to produce, separating natural SMP albumen from described cell (for example endothelial cell).

The present invention also provides SMP chimeric protein or fusion. SMP used herein " chimeric protein " or " fusion " comprise the SMP polypeptide that effectively is connected with non-SMP polypeptide. " SMP polypeptide " refers to have the polypeptide of the amino acid sequence that is equivalent to SMP albumen, and " non-SMP polypeptide " refers to that the amino acid sequence that has is equivalent to and the described SMP albumen polypeptide of the protein of homology not basically, for example is different from described SMP albumen and derives from same biology or different biological protein. In described fusion, term " effective connection " refers to that described SMP polypeptide and described non-SMP polypeptide merge by frame each other. Described non-SMP polypeptide can merge to the N end of described SMP polypeptide or C terminal. For example, in one embodiment, described fusion is that wherein the SMP sequence merges to the GST-SMP fusion of the C end of GST sequence. This class fusion can be so that purification of Recombinant SMP albumen. In another embodiment, described fusion is the SMP albumen that contains an allos burst at the N end. At some host cell (for example in the mammalian host cell), can increase SMP protein expression and/or secretion by utilizing the allos burst.

Preferably SMP chimeric protein of the present invention or fusion adopt the standard recombinant dna technology to produce. For example, according to routine techniques, for example the end by utilizing flush end or stagger end to connect, carry out Restriction Enzyme to digest to provide suitable, fill cohesive end suitable the time, carry out alkaline phosphatase treatment to avoid undesirable connection, then carry out enzyme and connect, the dna fragmentation of the different peptide sequences of coding is linked together with meeting frame. In another embodiment, by routine techniques, comprise automatic dna synthesizer, can synthesize described fusion. Perhaps, adopt anchor primer to carry out the pcr amplification of genetic fragment, described anchor primer produces complementary jag between two consecutive gene fragments, subsequently with its annealing and again amplification, produce chimeric gene sequence (referring to for example Current Protocols in Molecular Biology, Ausubel etc. write, John Wiley ﹠ Sons:1992). In addition, many expression vectors are commercially available, and encoded one merges part (for example gst polypeptide). The SMP code nucleic acid can be cloned in a kind of like this expression vector, merge so that described fusion part meets frame ground with described SMP albumen.

By described SMP albumen is carried out mutagenesis, for example discrete point mutation or brachymemma can produce the homologue of described SMP albumen. Term used herein " homologue " refers to the variant form as the described SMP albumen of the activator of described SMP protein active or antagonist. The activator of SMP albumen can keep the identical biologically active of described SMP albumen or the biologically active of a subclass basically. The antagonist of SMP albumen comprises the glycan molecule metabolism cascade of SMP albumen or downstream or the upstream member of power generation approach by for example competitive binding, can suppress one or more activity of native form SMP albumen.

In an alternate embodiment, have the mutant combinatorial libraries of truncated mutant for example of the described SMP albumen of SMP protein agonist or antagonist activities by screening, can identify the homologue of described SMP albumen. In one embodiment, by carrying out associating mutagenesis in nucleic acid level, producing a piebald library of SMP variant, is encoded by a piebald gene library in described piebald library. By for example enzyme process synthetic oligonucleotide mixture is connected in the gene order, so that the potential SMP sequence of one group of degeneracy can act on single expression of polypeptides, perhaps as the one group of larger expressing fusion protein that wherein contains one group of SMP sequence (for example being used for phage display), can obtain SMP variant piebald library. There is several different methods to produce potential SMP homologue library with the oligonucleotide sequence of cause degeneracy. Can in the automated DNA synthesizer, carry out the chemical synthesis of degeneracy gene order, then described synthetic gene is connected in the suitable expression vector. Use one group of degeneracy gene, so that the sequence that can in a kind of mixture, provide all to encode one group of required potential SMP sequence. The method of synthetic degenerate oligonucleotide be known in the art (referring to for example Narang, S.A. (1983) Tetrahedron 39:3; Itakura etc. (1984) Annu.Rev.Biochem.53:323; Itakura etc. (1984) Science 198:1056; Ike etc. (1983) Nucleic Acid Res.11:477).

In addition, the library of the fragment of described SMP encoding histone can be used for producing the piebald colony of SMP fragment, is used for screening and selects subsequently the homologue of SMP albumen. In one embodiment, the library of following generation coded sequence fragment: the double-stranded PCR fragment with nuclease treatment S MP coded sequence approximately only occurs at each molecule under the condition of a breach, make described double-stranded DNA sex change, the right double-stranded DNA of sense/antisense that described DNA renaturation is formed to comprise different breach products, from the duplex that again forms, remove the strand part by processing with the S1 nuclease, then the fragment library that produces is connected in the expression vector. In this way, can obtain an expression library, N terminal fragment, C terminal fragment and the interior segments of the described SMP albumen of described expression library coding all size.

Several technology known in the art is used for screening and seeks the gene outcome with selected characteristic by gene outcome and the screening cDNA library of the combinatorial libraries of point mutation or brachymemma preparation. This class technology is applicable to the gene library that rapid screening produces by combinatorial mutagenesis SMP homologue. Be suitable for high throughput analysis, be used for screening the most widely used technology of large gene library, comprise described gene library is cloned in the science expression vector, transform suitable cell with the vector library that produces, and described combination gene is expressed under the condition that the detection of required activity helps carrier to separate, and the detected gene of its gene outcome of described vector encoded. The overall mutagenesis (REM) that circulates is a kind of new technology that improves functional form mutant frequency in the library, can be combined with (Arkin and Yourvan (1992) PNAS 89:7811-7815 with the Screening test of identifying the SMP homologue; Delgrave etc. (1993) Protein Engineering 6 (3): 327-331).

In another embodiment, can adopt method well known in the art, use based on the mensuration of cell and analyze piebald SMP library.

D. application of the present invention and method

Nucleic acid molecules as herein described, protein, protein homologs, fusion, primer, carrier and host cell can be used for one or more following methods: identify Corynebacterium glutamicum and associated biomolecule; The genomic mapping of the biology relevant with Corynebacterium glutamicum; Identify and location purpose Corynebacterium glutamicum sequence; Study on Evolution; The SMP protein region that measurement function is required; Regulate the SMP protein active; Regulate one or more glycometabolisms; Regulate cell and produce high energy molecule (being ATP, NADPH); And the generation of adjusting cell needs compound (for example fine chemicals).

SMP nucleic acid molecules of the present invention serves many purposes. At first, they can be used for identifying Corynebacterium glutamicum and its closely-related bacterial classification. In addition, whether they can be used in the Identifying micro-organisms population mixture the relevant bacterial classification with it of Corynebacterium glutamicum. The invention provides the nucleotide sequence of many Corynebacterium glutamicum genes; By under stringent condition, using the probe of crossing over the peculiar gene region of Corynebacterium glutamicum, survey the genomic DNA that a kind of micropopulation and mixed microorganism culturing in groups thing extract, thereby people can determine whether to exist the glutamic acid bacillus.

Although Corynebacterium glutamicum self is non-pathogenic, for example corynebacterium diphtheriae is relevant with pathogenic species for it. Corynebacterium diphtheriae is the diphtheria pathogen, and diphtheria is that a kind of febris acuta sexuality that relates to the fast development of local and systematic pathology is dyed. In this disease, local patholoic change betides the upper respiratory tract, and relates to the damage of epithelial cell gangrenosum acne; Corynebacterium diphtheriae excretes poison, the distant place susceptible tissue of toxin from the local patholoic change disperse to body. The protein of various tissues synthesizes and the generation sex change because suppress, and causes the diphtheria systematic pathology, and described tissue comprises heart, muscle, peripheral nerve, adrenal gland, kidney, liver and spleen. Diphtheria comprises that in many areas, the world incidence of disease is always very high in the many independent states of Africa, Asia, European east and the former Soviet Union. Since nineteen ninety, a diphtheria in latter two area is popular to cause 5,000 people dead.

In one embodiment, the invention provides a kind of method that whether has corynebacterium diphtheriae or its active situation in the subject of identifying. This method comprises identifies one or more nucleotide sequences of the present invention or amino acid sequence (for example being respectively the sequence shown in sequence table odd number or the even number SEQ ID NO) in the subject, detects thus whether have Corynebacterium glutamicum or active situation in the described subject. Corynebacterium glutamicum and corynebacterium diphtheriae are Related Bacteria, and therefore the many nucleic acid in the Corynebacterium glutamicum and the nucleic acid of protein molecule and corynebacterium diphtheriae and protein molecule homology can be used for detecting the corynebacterium diphtheriae in the subject.

Nucleic acid molecules of the present invention and protein molecule also can be as the marks of genome given zone. This not only can be applicable to also can be applicable to the functional study of Corynebacterium glutamicum albumen in the genomic mapping. For example, for the zone of specific Corynebacterium glutamicum DBP institute combination in the identified gene group, can be with the digestion of Corynebacterium glutamicum gene group, then with fragment with described DBP incubation. Fragment in conjunction with described albumen can be surveyed with nucleic acid molecules of the present invention in addition, preferably surveys with the mark that can detect easily; The combination of this nucleic acid molecules and described genomic fragment make it possible to above-mentioned fragment be positioned on the Genome Atlas of Corynebacterium glutamicum, and when carrying out repeatedly with different enzymes, help the Fast Measurement of the nucleotide sequence of described albumen institute combination. In addition, nucleic acid molecules of the present invention may have enough homologys with the sequence of relevant bacterial classification, so these nucleic acid molecules can be as the mark that for example makes up Genome Atlas in Related Bacteria in the brevibacterium.

SMP nucleic acid molecules of the present invention also can be used for Study on Evolution and protein structure research. Metabolism and energy release process that molecule of the present invention participates in are utilized by various prokaryotes and eukaryotic cells; By the sequence of nucleic acid molecules of comparison sequence of nucleic acid molecules of the present invention to the similar enzyme of other biology of coding, can estimate biological evolution correlation. Equally, does not guard in guard in which district of the described sequence of this relatively permission evaluation, which district, and this helps definite necessary protein region of enzyme function. Research is valuable to such mensuration for protein engineering, and can provide protein and can tolerate and the index of not loss of function aspect mutagenesis.

Operation to SMP nucleic acid molecules of the present invention can cause producing the SMP albumen that is different from wild type SMP albumen on the function. But these protein energy efficiency aspects or active aspect are improved, and the quantity that exists in cell may be greater than common quantity, but or energy efficiency or activity decreased.

The invention provides screening technique, be used for screening by with the substrate of protein self or described SMP albumen or binding partners interacts or transcribe or translate the molecule of regulating the SMP protein active by what regulate SMP nucleic acid molecules of the present invention. In these class methods, the microorganism of expressing one or more SMP albumen of the present invention is contacted with one or more test compounds, and estimate every kind of test compound to the impact of described SMP protein active or expression.

SMP albumen of the present invention change can by number of mechanisms directly the impact Corynebacterium glutamicum strain that mixes this change albumen produce productive rate, output and/and the production efficiency of fine chemicals. Such as the high-energy carbon molecular degradation of sugar and be converted into more useful form such as the compound of NADH and FADH2 by oxidative phosphorylation and can produce multiple compounds, the fine chemicals that these compounds may need exactly itself is such as pyruvic acid, ATP, NADH and multiple intermediate sugar compounds. And then kinetomeres (such as ATP) and reducing equivalent thing (such as NADH or NADPH) that cell can utilize these metabolic pathways to produce drive disadvantageous reaction on energy. Such adverse effect comprises many fine chemicals biosynthesis pathways. People utilize the ability (for example operate coding gene that to participate in described cell degradation invert sugar be the enzyme of energy) of specific sugar by improving cell, can improve energy, but so that cell can carry out the unfavorable metabolic response that still needs (for example biosynthesis needs fine chemicals).

In addition, can optimize the energy that the invert sugar molecule contains to the regulating of one or more approach of involved in sugar utilization, in order to produce the fine chemicals that one or more need. For example participate in for example enzymatic activity of gluconeogenesis by reduction, can obtain more ATP for the required biochemical reaction of cellular driven (for example fine chemicals biosynthesis). In addition, also can regulate sugared total energy molecule and produce to guarantee that cell is by the energy maximization of each glycan molecule generation. Invalid sugar utilization may produce excessive carbon dioxide and excessive energy, and excessive energy may produce useless metabolic cycles. By improving the glycan molecule metabolism, cell is functionating more effectively, and needs less carbon molecule. This also should obtain improved fine chemicals: glycan molecule is than (improve carbon productive rate), and can reduce by hundred large scale fermentations and cultivate the sugar amount that must add in this engineering Corynebacterium glutamicum in the culture medium.

One or more SMP genes of the present invention of mutagenesis also may produce the SMP albumen of activity change, and the SMP albumen remote-effects Corynebacterium glutamicum of activity change produces one or more needs fine chemicals. For example, people utilize one or more sugared efficient (being converted into useful kinetomeres thereby strengthen described sugar) or improve the efficient (for example active by improving oxidative phosphorylation efficient or strengthening atp synthase) that the reducing equivalent thing is converted into useful kinetomeres by improving Corynebacterium glutamicum, can increase these energy-rich compound amounts, for the common disadvantageous metabolic process of cellular driven. Unfavorable process like this comprise make up cell membrane, transcribe, translation and essential compound (such as nucleotides, amino acid, vitamin, lipid etc.) (Lengeler etc. (1999) the Biology of Prokaryotes of biosynthesis cell division, Thieme Verlag:Stuttgart, 88-109,913-918,875-899 page or leaf). By improving the Growth and reproduction of these engineering cells, both can improve the cell viability in the large-scale culture thing, also can improve its division rate, more substantial like this cell can be survived in the fermentation tank culture thing. At least because exist more generation to need the living cells of fine chemicals and improve productive rate, output or production efficiency.

In addition, cell itself utilizes many catabolites of glycometabolism generation as the precursor or the intermediate that produce countless other useful compounds (wherein part is fine chemicals). For example pyruvic acid is converted into amino acid alanine, and ribose-5-phosphoric acid is a part of nucleic acid molecule for example. Therefore, glycometabolic amount and efficient have appreciable impact to the validity of these catabolites in cell. By (for example engineering improvement participates in the enzyme of these approach in the ability that strengthens cellular metabolism sugar aspect the efficient of existing approach, thereby optimize its activity), perhaps participate in the validity (for example increasing the quantity of these enzymes that exist in the cell) of the enzyme of these approach by raising, also can improve the validity of these catabolites in cell, produce many different other and need compound (for example fine chemicals) thereby strengthen again cell.

The above-mentioned SMP albumen mutagenesis strategy that Corynebacterium glutamicum fine chemicals productive rate is improved is not restrictive; It is apparent for those skilled in the art that these strategies are changed. Employ these tactics and add mechanism disclosed herein, can utilize nucleic acid molecules of the present invention and protein molecule to produce Corynebacterium glutamicum or the Related Bacteria bacterial classification of expressing saltant SMP nucleic acid molecules and protein molecule, so that need productive rate, output and/or the production efficiency of compound to be improved. This compound that needs can be the spawn of Corynebacterium glutamicum, comprise the end-product of biosynthesis pathway and the intermediate of naturally occurring metabolic pathway, and be not molecule naturally occurring but that produced by Corynebacterium glutamicum strain of the present invention in the Corynebacterium glutamicum metabolism.

Use following examples and further illustrate the present invention, it should be interpreted as limiting the present invention. The content of patent application, form and the sequence table of all lists of references of quoting in this application, patent application, patent, announcement is all incorporated herein by reference.

Table 1: the gene among the application

HMP：
							Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
1   3   5   7	2   4   6   8	RXS02735   RXA01626   RXA02245   RXA01015	VV0074   GR00452   GR00654   GR00290	14576   4270   13639   346	15280   3926   14295   5	6-phosphogluconic acid enzyme lactonase L-ribulose-phosphatase 24-epimerase ribulose-phosphoric acid 3-epimerase (EC 5.1.3.1) ribose 5-phosphoric acid isomerase (EC 5.3.1.6)
							TCA.
Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							9   11   13   15   17   19   21	10   12   14   16   18   20   22	RXN01312   F RXA01312   RXN00231   RXA01311   RXA01535   RXA00517   RXA01350	VV0082   GR00380   VV0083   GR00380   GR00427   GR00131   GR00392	20803   2690   15484   1611   1354   1407   1844	18785   1614   14015   865   2760   2447   2827	Succinate dehydrogenase flavoprotein subunit of succinate dehydrogenase flavoprotein subunit (EC 1.3.99.1) (EC 1.3.99.1) butanedioic acid-hemiacetal dehydrogenase (NADP+) (EC 1.2.1.16) succinate dehydrogenase iron-sulfur protein (EC 1.3.99.1) fumaric acid hydrase precursor (EC 4.2.1.2.) malic dehydrogenase (EC 1.1.1.37) (EC 1.1.1.82) malic dehydrogenase (EC 1.1.1.37)
The EMB-approach
							Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
23   25   27   29   31   33   35   37   39   41   43   45   47   49   51   53   55   57	24   26   28   30   32   34   36   38   40   42   44   46   48   50   52   54   56   58	RXA02149   RXA01814   RXN02803   F RXA02803   RXN03076   F RXA02854   RXA00511   RXN01365   F RXA01365   RXA00098   RXA01989   RXA00340   RXA02492   RXA00381   RXA02122   RXA00206   RXA01243   RXA01882	GR00639   GR00515   VV0086   GR00784   VV0043   GR10002   GR00129   VV0091   GR00397   GR00014   GR00578   GR00059   GR00720   GR00082   GR00636   GR00032   GR00359   GR00538	17786   2571   1   1   1624   1588   1   1476   897   6525   1   1549   2201   1451   6511   6171   2302   1165	18754   910   657   400   35   5   513   103   4   8144   630   2694   2917   846   5813   5134   3261   2154	( EC 2.7.1.2 ) ( EC 5.4.2.2. ) / ( EC 5.4.2.8 ) ( EC 5.4.2.2. ) / ( EC 5.4.2.8 ) ( EC 5.4.2.2. ) / ( EC 5.4.2.8 ) ( EC 5.4.2.2. ) / ( EC 5.4.2.8 ) ( EC 5.4.2.2. ) / ( EC 5.4.2.8 ) ( EC 5.4.2.2. ) / ( EC 5.4.2.8 ) ( EC 5.4.2.2. ) / ( EC 5.4.2.8 ) ( EC 5.4.2.2. ) / ( EC 5.4.2.8 ) -6- ( GPI ) ( EC 5.3.1.9 ) -6-A ( GPIA ) ( EC 5.3.1.9 ) ( EC 5.4.2.1 ) ( EC 5.4.2.1 ) ( EC 5.4.2.1 ) ( EC 5.4.2.1 ) 6- ( EC 2.7.1.11 ) 1- ( EC 2.7.1.56 ) 1- ( EC 2.7.1.56 )

Table 1 (continuing)

Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							59   61   63   65   67   69   71   73   75   77   79   81   83   85   87   89   91   93   95   97   99   101   103   105   107   109   111   113   115   117   119   121   123   125   127   129   131   133   135   137	60   62   64   66   68   70   72   74   76   78   80   82   84   86   88   90   92   94   96   98   100   102   104   106   108   110   112   114   116   118   120   122   124   126   128   130   132   134   136   138	RXA01702   RXA02258   RXN01225   F RXA01225   RXA02256   RXA02257   RXA00235   RXA01093   RXN02675   F RXA02675   F RXA02695   RXA00682   RXA00683   RXN00635   F RXA02807   F RXA00635   RXN03044   F RXA02852   F RXA00268   RXN03086   F RXA02887   RXN03043   F RXA02897   RXN03083   F RXA02853   RXA02259   RXN02326   F RXA02326   RXN02327   F RXA02327   RXN02328   F RXA02328   RXN01048   F RXA01048   F RXA00290   RXA02694   RXN00296   F RXA00296   RXA01901   RXN01952	GR00479   GR00654   VV0064   GR00354   GR00654   GR00654   GR00036   GR00306   VV0098   GR00754   GR00755   GR00179   GR00179   VV0135   GR00788   GR00167   VV0019   GR00852   GR00041   VV0049   GR10022   VV0019   GR10039   VV0047   GR10001   GR00654   VV0047   GR00668   VV0047   GR00668   VV0047   GR00668   VV0079   GR00296   GR00046   GR00755   VV0176   GR00048   GR00544   VV0105	1397   26451   6382   5302   23934   25155   2365   1552   72801   2   2949   5299   6440   22708   88   3   1391   3   125   2243   411   1   1291   88   89   27401   4500   5338   3533   6305   1842   7783   12539   3   4693   1879   35763   3   4158   9954	366   27227   4943   6741   24935   26369   1091   122   70945   364   4370   3401   5349   20972   552   923   2221   281   955   2650   4   1362   5   1110   1495   30172   5315   4523   4492   5346   3437   6401   11316   290   5655   2820   38606   2837   5417   11666	( EC 4.1.2.13 ) ( EC 5.3.1.1 ) -3- ( EC 1.2.1.12 ) -3- -3- ( EC 1.2.1.12 ) ( EC 2.7.2.3 ) ( EC 4.2.1.11 ) ( EC 2.7.1.40 ) ( EC 2.7.1.40 ) ( EC 2.7.1.40 ) ( EC 2.7.1.40 ) ( EC 2.7.9.2 ) ( EC 2.7.9.2 ) ( ) ( EC 1.2.2.2 ) ( ) ( EC 1.2.2.2 ) ( ) ( EC 1.2.2.2 ) E1 ( EC 1.2.4.1 ) E1 ( EC 1.2.4.1 ) E1 ( EC 1.2.4.1 ) E1 ( EC 1.2.4.1 ) E1 ( EC 1.2.4.1 ) E1 ( EC 1.2.4.1 ) E1 ( EC 1.2.4.1 ) ( EC 1.8.1.4 ) ( EC 1.8.1.4 ) ( EC 4.1.1.31 ) ( EC 6.4.1.1 ) ( EC 6.4.1.1 ) ( EC 6.4.1.1 ) ( EC 6.4.1.1 ) EC 1.1.1.39 ) ( EC 1.1.1.39 ) ( EC 1.1.1.39 ) L- ( EC 1.1.1.27 ) D- ( ) ( EC 1.1.2.4 ) D- ( ) ( EC 1.1.2.4 ) L- ( ) ( EC 1.1.2.3 ) D- ( EC 1.1.1.28 )

Table 1 (continuing)

Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							139   141   143   145   147   149   151   153   155   157   159   161   163   165   167   169   171   173   175   177   179   181   183	140   142   144   146   148   150   152   154   156   158   160   162   164   166   168   170   172   174   176   178   180   182   184	F RXA01952   F RXA01955   RXA00293   RXN01130   F RXA01130   RXN03112   F RXA01133   RXN00871   F RXA00871   RXN02829   F RXA02829   RXN01468   F RXA01468   RXA00794   RXN02920   F RXA02379   RXN02688   RXN03087   RXN03186   RXN03187   RXN02591   RXS01260   RXS01261	GR00562   GR00562   GR00047   VV0157   GR00315   VV0085   GR00316   VV0127   GR00239   VV0354   GR00816   VV0019   GR00422   GR00211   VV0213   GR00690   VV0098   VV0052   VV0377   VV0382   VV0098   VV0009   VV0009	1   4611   2645   6138   2   509   568   3127   2344   287   287   7474   1250   3993   6135   1390   59053   3216   310   3   14370   3477   3703	216   6209   1734   5536   304   6   1116   2240   3207   559   562   8298   2074   2989   5224   686   58385   3428   519   281   12541   2296   3533	D-lactic acid dehydrogenase (EC 1.1.1.28) D-lactic acid dehydrogenase (EC 1.1.1.28) D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) IOLB protein I OLB albumen: D-Fructose 1,6-diphosphonic acid=glycerosone-CC phosphoric acid+D-glyceraldehyde 3 phosphate. IOLS protein I OLS albumen NAGD albumen is inferred (EC4.1.1.32) the diaphorase component (E3) (EC 1.8.1.4) of diaphorase component (E3) (EC 1.8.1.4) branched-chain alpha-ketoacid dehydrogenase complex of branched-chain alpha-ketoacid dehydrogenase complex of N-glyceraldehyde-2-phosphotransferase GLPX protein D-3-phosphoglyceric acid dehydroenase (EC 1.1.1.95) D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) phosphoglycerate phosphomutase (EC 5.4.2.1) pyruvate carboxylase (EC 6.4.1.1) pyruvic dehydrogenase E1 component (EC 1.2.4.1) pyruvic dehydrogenase E1 component (EC 1.2.4.1) phosphoenolpy ruvate carboxy kinase [GTP]
Glycerol metabolism
							Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
185   187   189   191   193   195   197   199   201   203	186   188   190   192   194   196   198   200   202   204	RXA02640   RXN01025   F RXA01025   RXA01851   RXA01242   RXA02288   RXN01891   F RXA01891   RXA02414   RXN01580	GR00749   VV0143   GR00293   GR00525   GR00359   GR00661   VV0122   GR00541   GR00703   VV0122	1400   5483   939   3515   1526   992   24949   1736   3808   22091	2926   4488   1853   1830   2302   147   24086   918   3062   22807	The aerobic glycerol-3-phosphate dehydrogenase of glycerokinase (EC 2.7.1.30) glycerol-3-phosphate dehydrogenase (NAD (P)+) (EC 1.1.1.94) glycerol-3-phosphate dehydrogenase (NAD (P)+) (EC 1.1.1.94) (NAD (P)+) (EC 1.1.99.5) glycerol-3-phosphate is regulated sub-repressor protein glycerol-3-phosphate and is regulated sub-repressor protein glycerol-3-phosphate-in conjunction with periplasm protein precursor glycerol-3-phosphate-participate in conjunction with periplasm protein precursor uncharacterized protein (homologue of fruit bat rhomboid) the phosphoglycerol diester phosphodiesterase of glycerol metabolism

Table 1 (continuing)

Acetate metabolism
							Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
205   207   209   211   213   215   217   219   221   223   225   227   229   231   233   235	206   208   210   212   214   216   218   220   222   224   226   228   230   232   234   236	RXA01436   RXA00686   RXA00246   RXA01571   RXA01572   RXA01758   RXA02539   RXN03061   RXN03150   RXN01340   RXN01498   RXN02674   RXN00868   RXN01143   RXN01146   RXN01144	GR00418   GR00179   GR00037   GR00438   GR00438   GR00498   GR00726   VV0034   VV0155   VV0033   VV0008   VV0315   VV0127   VV0077   VV0264   VV0077	2547   8744   4425   1360   1928   3961   11676   108   10678   3   1598   15614   2230   9372   243   8237	1357   7941   3391   1959   2419   2945   10159   437   10055   860   3160   14163   320   8254   935   7722	Acetokinase (EC 2.7.2.1) acetic acid operon repressor protein alcohol dehydrogenase (EC 1.1.1.1) alcohol dehydrogenase (EC 1.1.1.1) alcohol dehydrogenase (EC 1.1.1.1) alcohol dehydrogenase (EC 1.1.1.1) acetaldehyde dehydrogenase (EC acetaldehyde dehydrogenase (EC 1.2.1.3) acetaldehyde dehydrogenase (EC 1.2.1.3) acetaldehyde dehydrogenase (EC 1.2.1.3) acetaldehyde dehydrogenase (EC 1.2.1.3) acetaldehyde dehydrogenase (EC 1.2.1.3) acetolactate synthase large subunit (EC 4.1.3.18) acetolactate synthase large subunit (EC 4.1.3.18) the little subunit of acetolactate synthase large subunit (EC 4.1.3.18) acetolactate synthase (EC 4.1.3.18)
							Butanediol, diacetyl and 3-Hydroxybutanone form
Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							237   239   241	238   240   242	RXA02474   RXA02453   RXS01758	GR00715   GR00710   VV0112	8082   6103   27383	7309   5351   28399	(S, S)-butane-2,3-two alcohol dehydrogenase (EC 1.1.1.76) 3-Hydroxybutanones (diacetyl) reductase (EC 1.1.1.5) alcohol dehydrogenase (EC 1.1.1.1)
The HMP-circulation
							Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
243   245   247   249   251   253	244   246   248   250   252   254	RXA02737   RXA02738   RXA02739   RXA00965   RXN00999   F RXA00999	GR00763   GR00763   GR00763   GR00270   VV0106   GR00283	3312   4499   6769   1232   2817   3012	1771   3420   4670   510   1366   4448	G-6-P 1-dehydrogenase (EC 1.1.1.49) transaldolase (EC 2.2.1.2) transketolase (EC 2.2.1.1) 6-phosphogluconate dehydrogenase, decarboxylation (EC 1.1.1.44) 6-phosphogluconate dehydrogenase, decarboxylation (EC 1.1.1.44) 6-phosphogluconate dehydrogenase, decarboxylation (EC 1.1.1.44)
							The nucleotide sugar conversion
Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							255   257	256   258	RXN02596   F RXA02596	VV0098   GR00742	48784   1	47582   489	UDP-galactopyranose mutase (EC 5.4.99.9) UDP-pyrrole rings galactolipin mutase (EC 5.4.99.9)

Table 1 (continuing)

Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							259   261   263   265   267   269   271   273   275   277   279   281   283   285	260   262   264   266   268   270   272   274   276   278   280   282   284   286	F RXA02642   RXA02572   RXA02485   RXA01216   RXA01259   RXA02028   RXA01262   RXA01377   RXA02063   RXN00014   F RXA00014   RXA01570   RXA02666   RXA00825	GR00749   GR00737   GR00718   GR00352   GR00367   GR00616   GR00367   GR00400   GR00626   VV0048   GR00002   GR00438   GR00753   GR00222	5383   2   2345   2302   987   573   8351   3935   3301   8848   4448   427   7260   222	5880   646   3445   1202   130   998   7191   5020   4527   9627   5227   1281   6493   1154	UDP- ( EC 5.4.99.9 ) UDP-6- ( EC 1.1.1.22 ) UDP-N- ( EC 1.1.1.1.58 ) UDP-N- ( EC 2.7.7.23 ) UTP--1- ( EC 2.7.7.9 ) UTP--1- ( EC 2.7.7.9 ) GDP-6- ( EC 1.1.1.132 ) -1- ( EC 2.7.7.13 ) -1- ( EC 2.7.7.27 ) -1- ( EC 2.7.7.24 ) -1- ( EC 2.7.7.24 ) -1- ( EC 2.7.7.24 ) D--5- ( EC 2.7.7.40 ) DTDP-4,6- ( EC 4.2.1.46 )
Inositol and ribitol metabolism
							Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
287   289   291   293   295   297   299   301   303   305   307   309   311   313	288   290   292   294   296   298   300   302   304   306   308   310   312   314	RXA01887   RXN00013   F RXA00013   RXA01099   RXN01332   F RXA01332   RXA01632   RXA01633   RXN01406   RXN01630   RXN00528   RXN03057   F RXA02902   RXA00251	GR00539   VV0048   GR00002   GR00306   VV0273   GR00388   GR00454   GR00454   VV0278   VV0050   VV0079   VV0028   GR10040   GR00038	4219   7966   3566   6328   579   552   2338   3380   2999   48113   23406   7017   10277   931	3209   8838   4438   5504   4   4   3342   4462   1977   47037   22318   7688   10948   224	Inositol 2-dehydrogenase (EC 1.1.1.18) inositol-1 (or 4)-one phosphatase 1 (EC 3.1.3.25) inositol-1 (or 4)-one phosphatase 1 (EC 3.1.3.25) inositol monophosphate phosphatase inositol 2-dehydrogenase (EC 1.1.1.18) inositol 2-dehydrogenase (EC 1.1.1.18) inositol 2-dehydrogenase (EC 1.1.1.18) inositol 2-dehydrogenase (EC 1.1.1.18) inositol 2-dehydrogenase (EC 1.1.1.18) inositol 2-dehydrogenase (EC 1.1.1.18) inositol-1-phosphate synthase (EC 5.5.1.4) inositol 2-dehydrogenase (1.1.1.18) glucose-fructose redox enzyme precursor (EC 1.1.99.28) ribitol 2-dehydrogenase (EC 1.1.1.56)
							Utilization about sugar
Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							315   317   319   321	316   318   320   322	RXN02654   F RXA02654   RXN01049   F RXA01049	VV0090   GR00752   VV0079   GR00296	12206   7405   9633   1502	13090   8289   11114   492	Glucose 1-dehydrogenase (EC 1.1.1.47) glucose 1-dehydrogenase II (EC 1.1.1.47) gluconokinase (EC 2.7.1.12) gluconokinase (EC 2.7.1.12)

Table 1 (continuing)

Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							323   325   327   329   331   333   335   337   339   341   343   345   347   349   351   353   355   357   359   361   363   365   367   369   371   373   375   377   379   381   383   385   387   389   391   393   395   397   399   401	324  326  328  330  332  334  336  338  340  342  344  346  348  350  352  354  356  358  360  362  364  366  368  370  372  374  376  378  380  382  384  386  388  390  392  394  396  398  400  402	F RXA01050   RXA00202   RXN00872   F RXA00872   RXN00799   F RXA00799   RXA00032   RXA02528   RXN00316   F RXA00309   RXN00310   F RXA00310   RXA00041   RXA02026   RXA02061   RXN01369   F RXA01369   F RXA01373   RXA02611   RXA02612   RXN01884   F RXA01884   RXA01111   RXN01550   F RXA01550   RXN02100   F RXA02100   F RXA02113   RXA02147   RXA01478   RXA01888   RXN01927   FRXA01927   RXA02729   RXA02797   RXA02730   RXA02551   RXA01325   RXA00195   RXA00196	GR00296   GR00032   VV0127   GR00240   VV0009   GR00214   GR00003   GR00725   VV0006   GR00053   VV0006   GR00053   GR00007   GR00615   GR00626   VV0124   GR00398   GR00399   GR00743   GR00743   VV0184   GR00539   GR00306   VV0143   GR00431   VV0318   GR00631   GR00633   GR00639   GR00422   GR00539   VV0127   GR00555   GR00762   GR00778   GR00762   GR00729   GR00385   GR00030   GR00030	1972   1216   6557   565   58477   1   12028   6880   7035   316   6616   735   1246   725   1842   595   3   595   1   1793   1   3   16981   14749   3   2   3   2   15516   10517   4366   50623   3   747   1739   1768   2193   5676   543   1094	1499   275   5604   1086   56834   1584   10520   7854   8180   5   7050   301   5   6   349   1776   503   1302   1752   3985   1890   1475   17427   16260   1346   2326   920   1207   16532   12352   4923   49244   1118   4   2641   731   2552   5005   1103   1708	( EC 2.7.1.12 ) D- ( EC 2.7.1.4 ) ( EC 2.7.1.4 ) β-/β- ( EC 3.2.1.21 ) ( EC 3.2.1.37 ) β-/β- ( EC 3.2.1.21 ) ( EC 3.2.1.37 ) 2- ( EC 1.1.1.67 ) ( EC 1.1.1.-) - ( EC 1.1.99.28 ) - ( EC 1.1.99.28 ) - ( EC 1.1.99.28 ) -6- ( EC 3.2.1.26 ) -6- ( EC 3.2.1.26 ) -6- ( EC 3.2.1.26 ) -6- ( EC 5.3.1.8 ) -6- ( EC 5.3.1.8 ) -6- ( EC 5.3.1.8 ) 1； 4-α- ( EC 2.4.1.18 ) 1,4-α- ( EC 2.4.1.18 ) ( EC 24.1.25 ) ( EC 3.2.1.33 ) ( EC 24.1.25 ) ( EC 3.2.1.33 ) GLGX ( EC 3.2.1.-) ( EC 2.4.1.1 ) ( EC 2.4.1.1 ) ( EC 2.4.1.1 ) ( EC 2.4.1.1 ) ( EC 2.4.1.1 ) α- ( EC 3.2.1.1 ) G1G2 ( EC 3.2.1.3 ) ( EC 2.7.1.17 ) ( EC 2.7.1.17 ) ( EC 2.7.1.15 ) ( EC 2.7.1.15 ) 5--β- ( EC 3.2.1.86 ) - ( EC 4.1.2.4 ) 1--D-5- ( EC 1.1.1.-) 1--D-5- ( EC 1.1.1.-)

Table 1 (continuing)

Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							403   405   407   409   411   413   415   417   419   421   423   425   427   429   431   433   435   437   439   441   443   445   447   449   451   453   455   457   459   461   463   465   467   469   471   473   475   477   479	404   406   408   410   412   414   416   418   420   422   424   426   428   430   432   434   436   438   440   442   444   446   448   450   452   454   456   458   460   462   464   466   468   470   472   474   476   478   480	RXN01562   F RXA01562   F RXA01705   RXN00879   F RXA00879   RXN00043   F RXA00043   RXN01752   F RXA01839   RXA01859   RXA00042   RXA01482   RXN03179   F RXA02872   RXN03180   F RXA02873   RXA02292   RXA02666   RXA00202   RXA02440   RXN01569   F RXA01569   F RXA02055   RXA00825   RXA02054   RXN00427   F RXA00427   RXA00327   RXA00328   RXA00329   RXN01554   RXN03015   RXN03056   RXN03030   RXN00401   RXN02125   RXN00200   RXN01175   RXN01376	VV0191   GR00436   GR00480   VV0099   GR00242   VV0119   GR00007   VV0127   GR00520   GR00529   GR00007   GR00422   VV0336   GR10013   VV0337   GR10014   GR00662   GR00753   GR00032   GR00709   VV0009   GR00438   GR00624   GR00222   GR00624   VV0112   GR00098   GR00057   GR00057   GR00057   VV0135   VV0063   VV0028   VV0009   VV0025   VV0102   VV0181   VV0017   VV0091	1230   2   971   8763   5927   3244   3244   35265   1157   1473   2037   17271   2   675   672   672   1611   7260   1216   5097   41086   2   7122   222   6103   7004   1591   10263   11147   12390   28686   289   6258   57006   12427   23242   1679   39688   5610	3137   1039   1573   6646   3828   2081   2081   33805   510   547   1279   15397   667   4   163   163   2285   6493   275   4258   42444   427   8042   1154   7119   6219   2022   9880   10656   11167   26545   8   6935   56443   11489   22442   5116   38303   4750	1-deoxy-D-xylulose sugar-5-phosphate synthase 1-deoxy-D-xylulose sugar-5-phosphate synthase 1-deoxy-D-xylulose sugar-5-phosphate synthase 4-alpha-Glucanotransferase (EC 2.4.1.25) 4-alpha-Glucanotransferase (EC 2.4.1.25), amylomaltase N-acetyl-glucosamine-6-phosphoric acid deacetylase (EC 3.5.1.25) N-acetyl-glucosamine-6-phosphoric acid deacetylase (EC 3.5.1.25) N-acetyl-glucosamine transferase (EC 2.4.1.-) N-acetyl-glucosamine transferase (EC 2.4.1.-) N-acetyl-glucosamine transferase (EC 2.4.1.-) glucosamine-6-phosphate isomerase (EC 5.3.1.10) aminoglucose-fructose-6-phosphate aminopherase (isomerization) (EC 2.6.1.16) uronic isomerase (EC 5.3.1.12) uronic isomerase, glucuronate isomerase (EC 5.3.1.12) uronic isomerase (EC 5.3.1.12) uronic isomerase, glucuronate isomerase (EC 5.3.1.12) galactoside O-transacetylase (EC 2.3.1.18) D-ribose alcohol-5-cytidine phosphate acyltransferase (EC 2.7.7.40) D-ribose-in conjunction with periplasm protein precursor D-ribose-in conjunction with periplasm protein precursor dTDP-4-dehydrogenation rhamnose reductase (EC 1.1.1.133) DTDP-4-dehydrogenation rhamnose reductase (EC 1.1.1.133) DTDP-4-dehydrogenation rhamnose reductase (EC 1.1.1.133) DTDP-glucose 4,6-dehydratase (EC 4.2.1.46) DTDP-glucose 4,6-dehydratase (EC 4.2.1.46) dTDP-rhamnose transferase RFBF (EC 2.-.-.-) DTDP-rhamnose transferase RFBF (EC 2.-.-.-) a-protein RAJ a-protein RAJ a-protein RAJ glucan inscribe-1.3-β-glucosyl enzym A1 precursor (EC 3.2.1.39) UDPG 6-dehydrogenase (EC 1.1.1.22) is inferred ketohexose-6-phosphoric acid isomerase (EC 5.-.-.-) pericentral siphon β-glucosyl enzym/xylobiase precursor (EC 3.2.1.21) (EC 3.2.1.37) 5-Dehydro-4-deoxyglucarate dehydratase (EC 4.2.1.41) aldose reductase (EC 1.1.1.21) the arabinose transferase B of subunit (EC 2.4.2.-) phospho-2-dehydro-3-deoxyheptonate aldolase (EC 4.1.2.15) and is inferred glycosyl transferase WBIF

Table 1 (continuing)

Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							481  483  485  487  489  491  493  495  497  499  501  503  505  507  509  511  513  515  517  519  521  523  525  527  529  531  533  535  537  539  541  543	482   484   486   488   490   492   494   496   498   500   502   504   506   508   510   512   514   516   518   520   522   524   526   528   530   532   534   536   538   540   542   544	RXN01631   RXN01593   RXN00337   RXS00584   RXS02574   RXS03215   F RXA01915   RXS03224   F RXA00038   RXC00233   RXC00236   RXC00271   RXC00338   RXC00362   RXC00412   RXC00526   RXC01004   RXC01017   RXC01021   RXC01212   RXC01306   RXC01366   RXC01372   RXC01659   RXC01663   RXC01693   RXC01703   RXC02254   RXC02255   RXC02435   F RXA02435   RXC03216	VV0050   VV0229   VV0197   VV0323   GR00549   GR00006   GR00709	47021   13274   20369   5516   1   1417   825	46143   12408   21418   6640   1008   260   268	-6- ( EC 5.-.-.-) NAGD ( EC 2.7.1.6 ) -2--3- ( EC 4.1.2.15 ) β-A ( EC 3.2.1.52 ) - ( EC 1.1.99.28 ) - ( EC 1.1.99.28 ) ( EC 3.2.1.54 ) ( EC 3.2.1.54 ) ABCATP ABCATP ABCATP ( rhomboid )
The TCA-circulation
							Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
545  547	546   548	RXA02175   RXA02621	GR00641   GR00746	10710   2647	9418   1829	Citrate synthase (EC 4.1.3.7) citric acid lyase β chain (EC 4.1.3.6)

Table 1 (continuing)

Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							549   551   553   555   557   559   561   563   565   567   569   571   573   575   577   579   581   583   585   587	550  552  554  556  558  560  562  564  566  568  570  572  574  576  578  580  582  584  586  588	RXN00519   F RXA00521   RXN02209   F RXA02209   RXN02213   F RXA02213   RXA02056   RXA01745   RXA00782   RXA00783   RXN01695   F RXA01615   F RXA01695   RXA00290   RXN01048   F RXA01048   F RXA00290   RXN03101   RXN02046   RXN00389	VV0144   GR00133   VV0304   GR00648   VV0305   GR00649   GR00625   GR00495   GR00206   GR00206   VV0139   GR00449   GR00474   GR00046   VV0079   GR00296   GR00046   VV0066   VV0025   VV0025	5585   2   1   3   1378   1330   3   2   3984   5280   11307   8608   4388   4693   12539   3   4693   2   15056   11481	3372   1060   1671   1661   2151   2046   2870   1495   3103   4009   12806   9546   4179   5655   11316   290   5655   583   14640   9922	( NADP ) ( EC 1.1.1.42 ) ( NADP ) ( EC 1.1.1.42 ) ( EC 4.2.1.3 ) ( EC 4.2.1.3 ) ( EC 4.2.1.3 ) ( EC 4.2.1.3 ) 2-E1 ( EC 1.2.4.2 ) 2- ( E2 ) ( EC 2.3.1.61 ) -CoAα ( EC 6.2.1.5 ) -CoAβ ( EC 6.2.1.5 ) L- ( ) ( EC 1.1.99.16 ) L- ( ) ( EC 1.1.99.16 ) L- ( ) ( EC 1.1.99.16 ) ( EC 1.1.1.39 ) ( EC 1.1.1.39 ) ( EC 1.1.1.39 ) ( EC 1.1.1.39 ) 2- ( E2 ) ( EC 2.3.1.61 ) 2- ( EC 2.3.1.61 ) ( EC 1.2.1.-)
Glyoxylate bypass
							Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
589   591   593   595   597   599	590  592  594  596  598  600	RXN02399   F RXA02399   RXN02404   F RXA02404   RXA01089   RXA01886	VV0176   GR00699   VV0176   GR00700   GR00304   GR00539	19708   478   20259   3798   3209   3203	18365   1773   22475   1663   3958   2430	Isocitratase (EC 4.1.3 1) isocitratase (EC 4.1.3.1) malate synthase (EC 4.1.3.2) malate synthase (EC 4.1.3.2) glyoxalic acid inducible protein glyoxalic acid inducible protein
							The methylcitrate approach
Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							600   601   603   605	602  604  606  608	RXN03117   F RXA00406   F RXA00514   RXA00512	VV0092   GR00090   GR00130   GR00130	3087   978   1983   621	1576   4   1576   4	2-methyl isocitrate lyase (EC 5.3.3.-) 2-methyl isocitrate lyase (EC 5.3.3.-) 2-methyl isocitrate lyase (EC 5.3.3.-) 2-Methylcitrate synthase (EC 4.1.3.3.1)

Table 1 (continuing)

Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							607   609   611   613   615   617   619   621   623	610  612  614  616  618  620  622  624  626	RXA00518   RXA01077   RXN03144   F RXA02322   RXA02329   RXA02332   RXN02333   F RXA02333   RXA00030	GR00131   GR00300   VV0141   GR00668   GR00669   GR00671   VV0141   GR00671   GR00003	3069   4647   2   415   607   1906   901   2120   9590	2773   6017   901   5   5   764   1815   1902   9979	2-Methylcitrate synthase (EC 4.1.3.31) 2-methyl isocitrate lyase (EC 5.3.3.-) 2-methyl isocitrate lyase (EC 5.3.3.-) 2-methyl isocitrate lyase (EC 5.3.3.-) 2-methyl isocitrate lyase (EC 5.3.3.-) 2-Methylcitrate synthase (EC 4.1.3.31) isocitric acid methyl esters lyase (EC 4.1.3.30) isocitric acid methyl esters lyase (EC 4.1.3.30) lactylglutathion lyase (EC 4.4.1.5)
Methyl-malonyl-CoA-mutase
							Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
625   627   629	628  630  632	RXN00148   F RXA00148   RXA00149	VV0167   GR00023   GR00023	9849   2002   3856	12059   5   2009	Methylmalonyl-CoA mutase α-subunit (EC 5.4.99.2) methylmalonyl-CoA mutase α-subunit (EC 5.4.99.2) methylmalonyl-CoA mutase beta subunit (EC 5.4.99.2)
							Other
Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							631   635   637   639	634  636  638  640	RXN00317   F RXA00317   RXA02196   RXN02461	VV0197   GR00055   GR00645   VV0124	26879   344   3956   14236	27532   6   3264   14643	Phosphoglycolate phosphatase (EC 3.1.3.18) phosphoglycolate phosphatase (EC 3.1.3.18) phosphoglycolate phosphatase (EC 3.1.3.18) phosphoglycolate phosphatase (EC 3.1.3.18)
The redox chain
							Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
641   643   645   647   649   651   653   655   657   659	642  644  646  648  650  652  654  656  658  660	RXN01744   F RXA00055   F RXA01744   RXA00379   RXA00385   RXA01743   RXN02480   F RXA01919   F RXA02480   F RXA02481	VV0174   GR00008   GR00494   GR00082   GR00083   GR00494   VV0084   GR00550   GR00717   GR00717	2350   11753   2113   212   773   806   31222   288   1449   1945	812   11890   812   6   435   6   29567   4   601   1334	Biological biological PROTEIN C CDA cytochromes D ubiquinol oxidase subunit II (EC 1.10.3.-) cytochrome C oxidase polypeptide I (EC 1.9.3.1) cytochrome C oxidase subunit I (EC 1.9.3.1) cytochrome C oxidase polypeptide I (EC 1.9.3.1) the cytochrome C oxidase polypeptide I (EC 1.9.3.1) of generation of PROTEIN C CDA cromoci type that occur of cytochromes D ubiquinol oxidase subunit I (EC 1.10.3.-) cytochromes D ubiquinol oxidase subunit I (EC 1.10.3.-) cytochromes D ubiquinol oxidase subunit I (EC 1.10.3.-) cromoci type

Table 1 (continuing)

Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							661   663   665   667   669   671   673   675   677   679   681   683   685   687   689   691   693   695   697   699   701   703   705   707   709   711   713   715   717   719   721   723   725   727   729   731   733   735	662  664  666  668  670  672  674  676  678  680  682  684  686  688  690  692  694  696  698  700  702  704  706  708  710  712  714  716  718  720  722  734  726  728  730  732  734  736	RXA02140   RXA02142   RXA02144   RXA02740   RXA02743   RXA01227   RXA01865   RXA00680   RXA00679   RXA00224   RXA00225   RXN00606   F RXA00606   RXN00595   F RXA00608   RXA00913   RXA00909   RXA00700   RXN00483   F RXA00483   RXA01534   RXA00288   RXA02741   RXN02560   F RXA02560   RXA01311   RXN03014   F RXA00910   RXN01895   F RXA01895   RXA00703   RXN00705   F RXA00705   RXN00388   F RXA00388   F RXA00386   RXA00945   RXN02556	GR00639   GR00639   GR00639   GR00763   GR00763   GR00355   GR00532   GR00179   GR00179   GR00032   GR00032   VV0192   GR00160   VV0192   GR00160   GR00249   GR00247   GR00182   VV0086   GR00119   GR00427   GR00046   GR00763   VV0101   GR00731   GR00380   VV0058   GR00248   VV0117   GR00543   GR00183   VV0005   GR00184   VV0025   GR00085   GR00084   GR00259   VV0101	7339   9413   11025   7613   13534   1199   436   2632   2302   24965   25783   11299   121   8642   2253   3   2552   846   44824   19106   1035   2646   9585   9922   6339   1611   1273   3   955   2   2556   6111   1291   2081   969   514   1876   5602	8415   10063   12248   8542   12497   1519   122   2315   1037   24015   24998   9026   1869   7113   3017   2120   3406   43   46287   20569   547   1636   8620   10788   7160   865   368   1259   5   817   271   5197   407   3091   667   5   2847   6759	CII ( EC 1.9.3.1 ) CI ( EC 1.9.3.1 ) RIESKE C AA3 VI -NAD ( + ) ( EC 1.18.1.3 ) α- β- NADHIL ( EC 1.6.5.3 ) NADHIL ( EC 1.6.5.3 ) NADHIM ( EC 1.6.5.3 ) NADHIM ( EC 1.6.5.3 ) NADHIL ( EC 1.6.5.3 ) NADHIL ( EC 1.6.5.3 ) NADH-2 NADH-39KD ( EC 1.6.5.3 ) ( EC 1.6.99.3 ) NADH-39KD ( EC 1.6.5.3 ) ( EC 1.6.99.3 ) NADHFMN ( EC 1.6.5.5 ) ( EC 1.6.5.5 ) NADPH- ( EC 1.6.99.-) NADPH- ( EC 1.6.99.-) ( EC 1.3.99.1 ) NADHIM ( EC 1.6.5.3 ) NADH ( EC 1.6.99.3 ) α ( EC 1.2.1.2 ) FDHD FDHD CCSA c RESC,c / ( EC 1.6.99.7 )

Table 1 (continuing)

Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							737   739   741   743   745   747   749   751   753	738   740   742   744   746   748   750   752   754	F RXA02556   RXA01392   RXA00800   RXA02143   RXN03096   RXN02036   RXN02765   RXN02206   RXN02554	GR00731   GR00408   GR00214   GR00639   VV0058   VV0176   VV0317   VV0302   VV0101	2019   2297   2031   10138   405   32683   3552   1784   4633	3176   3373   3134   11025   4   33063   2794   849   4010	Flavine hemoprotein glutathione S-transferase (EC 2.5.1.18) glutathione dependence formaldehyde dehydrogenase (EC 1.2.1.1) QCRC albumen: menadione alcohol: cytochrome c oxidoreducing enzyme nadh dehydrogenase I chain M (EC 1.6.5.3) NADH-ubiquinone oxide-reductase enzyme chain 4 (EC 1.6.5.3) supposition oxidoreducing enzyme supposition oxidoreducing enzyme supposition oxidoreducing enzyme (EC 1.1.1.-)
The ATP-synthase
							Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
755   757   759   761   763   765   767   769   771   773   775   777	756   758   760   762   764   766   768   770   772   774   776   778	RXN01204   F RXA01204   RXA01201   RXN01193   F RXA01193   F RXA01203   RXN02821   F RXA02821   RXA01200   RXA01194   RXA01202   RXN02434	VV0121   GR00345   GR00344   VV0175   GR00343   GR00344   VV0121   GR00802   GR00344   GR00343   GR00344   VV0090	1270   394   675   5280   15   3355   324   139   2   770   2375   4923	461   1155   2315   3832   755   3993   85   318   610   1141   3349   3274	Atp synthase A chain (EC 3.6.1.34) atp synthase A chain (EC 3.6.1.34) atp synthase α chain (EC 3.6.1.34) atp synthase β chain (EC 3.6.1.34) atp synthase β chain (EC 3.6.1.34) atp synthase β chain (EC 3.6.1.34) atp synthase C chain (EC 3.6.1.34) atp synthase C chain (EC 3.6.1.34) atp synthase δ chain (EC 3.6.1.34) atp synthase ε chain (EC 3.6.1.34) atp synthase γ chain (EC 3.6.1.34) ATP is in conjunction with albumen
							The cytochromes metabolism
Nucleic acid SEQ ID NQ	Amino acid SEQ ID NQ	Identification code	Contig	NT is initial	NT stops	Function
							779   781	780   782	RXN00684   RXN00387	VV0005   VV0025	29864   1150	28581   2004	The biological albumen that occurs of Cytochrome P450 116 (EC 1.14.-.-) supposition cytochrome c

The gene that table 2-gets rid of
				The GenBankTM registration number	The gene title	Gene function	List of references
A09073	PPg	Phosphoenolpyruvate carboxylase	Bachmann, B. etc. " dna fragmentation of Orynebacterium carboxylase, carry the recombinant DNA of described fragment, with the bacterial strain of described recombinant DNA and adopt described bacterial strain to produce the amino acid whose method of L-", patent: EP0358940-A03/21/90
				A45579，  A45581，  A45583，  A45585  A45587		Threonine dehydratase	Moeckel, B. etc. " utilization has the recombinant microorganism of regulating threonine dehydratase and produces ILE " patent: WO 9519442-A 507/20/95
AB003132	murC；ftsQ；ftsZ		Kobayashi, M. etc. " excellent bacillus ftsZ gene cloning, order-checking and evaluation " Biochem.Biophys.Res. Commun., 236 (2): 383-388 (1997)
				AB015023	murC；ftsQ		Wachi, M. etc. " the marC gene of excellent bacillus " Appl.Microbiol.Biotechnol., 51 (2): 223-228 (1999)
AB018530	dtsR		Kimura, E. etc. " to the molecular cloning of rescue derived from the novel gene dtsR of the detergent sensitiveness of the mutant of brevibacterium ", Biosci.Biotechnol.Biochem., 60 (10): 1565-1570 (1996)
				AB018531	dtsR1；dtsR2
AB020624	murl	The D-Glu racemase
				AB023377	tkt	Transketolase

Table 2 (continuing)
				AB024708	gltB；gltD	The large subunit of glutamine 2-oxoglutaric acid aminopherase and small subunit
AB025424	acn	Aconitase
				AB027714	rep	Replication protein
AB027715	rep；aad	Replication protein; Aminoglycoside adenylate transferase
				AF005242	argC	N-acetylglutamat-5-half aldehyde dehydrogenase
AF005635	glnA	Glutamine synthelase
				AF030405	hisF	Cyclase
AF030520	argG	Argininosuccinate synthetase
				AF031518	argF	OCT
AF036932	aroD	The 3-dehydroquinate dehydratase
				AF038548	pyc	Pyruvate carboxylase
AF038651	dciAE；apt；rel	Dipeptides is in conjunction with albumen; Adenine phosphoribosyl transferase; The GTP pyrophosphokinase	Wehmeier, L. etc. " effect of Corynebacterium glutamicum rel gene in (p) ppGpp metabolism ", Microbiology, 144:1853-1862 (1998)
				AF041436	argR	The arginine repressor protein
AF045998	impA	The inositol monophosphate phosphatase
				AF048764	argH	The argininosuccinic acid lyase

Table 2 is (continuous
				AF049897	argC；argJ；argB；   argD；argF；argR；   argG；argH	N-acetyl glutamy phosphoric acid reduction enzyme; The ornithine acetyltransferase; The N-acetylglutamat kinases; Acetyl-ornithine transaminase; OCT; The arginine repressor protein; The argininosuccinic acid synthase; The argininosuccinic acid lyase
AF050109	inhA	Enol-acyl carrier protein reductase
				AF050166	hisG	ATP phosphoribosyltransferase
AF051846	hisA	Ribose phosphate formyl imino group-5-amino-ribose 1-phosphate base-4-Imidazole carboxamide isomerase
				AF052652	metA	Homoserine O-acetyltransferase	Park, S. etc. " separation and analysis of the methionine biosynthesis gene of coding homoserine acetyltransferase in metA-Corynebacterium glutamicum ", Mol.Cell., 8 (3): 286-294 (1998)
AF053071	aroB	Dehydroquinate synthase
				AF060558	hisH	The glutamine transamidase
AF086704	hisE	Phosphoribosyl-ATP-pyrophosphohydrolase
				AF114233	aroA	5-enol pyruvic acid shikimic acid 3-phosphate synthase
AF116184	panD	L-Aspartic acid-α-decarboxylase precursor	Dusch, N. etc. " expression of the Corynebacterium glutamicum panD gene of coding L-Aspartic acid-α-decarboxylase causes Escherichia coli excessively to produce pantothenic acid ", Appl.Environ.Microbiol., 65 (4) 1530-1539 (1999)
				AF124518	aroDaroE	3-dehydroquinase (dehydroquinase); Shikimate dehydrogenase

Table 2 (continuing)
				AF124600	aroC；aroK；aroB；   pepQ	Chorismate synthase; Shikimate kinase; 3-Dehydroquinate synthase; The kytoplasm peptase of inferring
AF145897	inhA
				AF145898	inhA
AJ001436	ectP	The transhipment of ectoine, betaine, proline	Peter, H. etc. " Corynebacterium glutamicum has the Second support of four kinds of coupling solutes: evaluation, order-checking and the sign of proline/ectoine shooting system ProP and ectoine/ proline/betaine carrier EctP ", J. Bacteriol., 180 (22): 6005-6012 (1998)
				AJ004934	dapD	Tetrahydrochysene 2, dipicolimic acid 2 succinylation enzyme (incomplete i)	Wehrmann, A. etc. " the synthetic and effect aspect the cell membrane integrality of the diaminopimelic acid of different mode: the research of carrying out with Corynebacterium glutamicum ", J.Bacteriol., 180 (12); 3159-3165 (1998)
AJ007732	ppc；secG；amt；ocd；   soxA	Phosphoenolpyruvate carboxylase; The high affinity ammonium is taken in albumen; The ornithine of inferring-ring decarboxylase; Sarcosine oxidase
				AJ010319	ftsY，glnB，glnD；srp；   amtP	Participate in cell division; PH albumen; Uridyl transferase (uridine acyl group removing enzyme); Signal recognition particle; Low affinity ammonium is taken in albumen	Jakoby, M. etc. " regulate by the nitrogen in the Corynebacterium glutamicum; Relate to the separation of gene of the Biochemical Identification of respective egg white matter ", FEMS Microbiol., 173 (2): 303-310 (1999)
AJ132968	cat	Chloramphenicol acetyltransferase
				AJ224946	mqo	L MALIC ACID; Quinone oxidoreductase	Molenaar, D. etc. " Corynebacterium glutamicum film be correlated with the biochemistry of malic dehydrogenase (acceptor) and genetics characteristics identify ", Eur.J.Biochem., 254 (2): 395-403 (1998)
AJ238250	ndh	Nadh dehydrogenase

Table 2 (continuing)
				AJ238703	porA	Porin	Lichtinger, T. etc. " biochemistry of Corynebacterium glutamicum cell envelope PFP and biophysics characterization: this passage is formed by low molecular weight polypeptide ", Biochemistry, 37 (43): 15024-15032 (1998)
D17429		Transposable element IS31831	Vertes etc. " derive from separation and the characterization of the transposable element IS31831 of Corynebacterium glutamicum ", Mol. Microbiol., 11 (4): 739-746 (1994)
				D84102	odhA	OdhA	Usuda, Y. etc. " molecular cloning of the Corynebacterium glutamicum of encoding novel odhA (brevibacterium AJ12036) odhA gene ", Microbiology, 142:3347-3354 (1996)
E01358	hdh；hk	Homoserine dehydrogenase; Homoserine kinase	Katsumata, R. etc. " production of L-threonine and ILE ", patent: JP 1987232392-A 2 10/12/87
				E01359		The upstream of homoserine kinase gene start codon	Katsumata, R. etc. " production of L-threonine and ILE ", patent t:JP 1987232392-A 2 10/12/87
E01375		Tryptophan operon
				E01376	trpL；trpE	Leader peptide; The o-amino benzoyl acid synthase	Matsui, K. etc. " tryptophan operon, the peptides and proteins by its coding, the utilization of Trp Operon Gene expression and the production of tryptophan ", patent: JP 1987244382-A 1 10/24/87
E01377		The promoter of tryptophan operon and operator region	Matsui, K. etc. " tryptophan operon, the peptides and proteins by its coding, the utilization of Trp Operon Gene expression and the production of tryptophan ", patent: JP 1987244382-A 1 10/24/87
				E03937		The biotin synthase	Hatakeyama, K. etc. dna fragmentation and the application thereof of gene of element synzyme " contain can encoding human ", patent: JP 1992278088-A 1 10/02/92
E04040		Diamino pelargonic amino transferase	Kohama, K. etc. " gene and the application thereof of coding diaminopimelic acid aminopherase and dethiobiotin synthetase ", patent: JP 1992330284-A 1 11/18/92

Table 2 (continuing)
				E04041		Dethiobiotin synthetase	Kohama, K. etc. " gene and the application thereof of coding diaminopimelic acid aminopherase and dethiobiotin synthetase ", patent: JP1992330284-A 1 11/18/92
E04307		The Flavum Aspartase	Kurusu, Y. etc. " gene DNA and the application thereof of coding Aspartase ", patent: JP 1993030977-A 1 02/09/93
				E04376		Isocitratase	Katsumata, R. etc. " gene shows controlled DNA ", patent: JP 1993056782-A 3 03/09/93
E04377		Isocitratase N terminal fragment	Katsumata, R. etc. " gene shows controlled DNA ", patent: JP 1993056782-A 3 03/09/93
				E04484		Prephenate dehydratase	Sotouchi, N. etc. " by the fermenting and producing L-Phe ", patent: JP 1993076352-A 2 03/30/93
E05108		Aspartokinase	Fugono, N. etc. " gene DNA and the application thereof of coding aspartokinase ", patent: JP 1993184366-A 1 07/27/93
				E05112		Dihydro-dipichorinate synzyme	Hatakeyama, K. etc. " coding dihydro 2, the gene DNA of dipicolimic acid 2 synzyme and application thereof ", patent: JP 1993184371-A 1 07/27/93
E05776		Diaminopimelate dehydrogenase	Kobayashi, M. etc. " gene DNA and the application thereof of coding diaminopimelate dehydrogenase ", patent: JP 1993284970-A 1 11/02/93
				E05779		Threonine synthase	Kohama, K. etc. " gene DNA and the application thereof of coding threonine synthase ", patent: JP 1993284972-A 1 11/02/93
E06110		Prephenate dehydratase	Kikuchi, T. etc. " produce L-Phe by fermentation method ", patent: JP1993344881-A 1 12/27/93
				E06111		The saltant prephenate dehydratase	Kikuchi, T. etc. " produce L-Phe by fermentation method ", patent: JP 1993344881-A 1 12/27/93

Table 2 (continuing)
				E06146		Acetohydroxy acid synthetase	Inui, M. etc. " gene and the application thereof of the acetohydroxy acid synthetase of can encoding ", patent: JP 1993344893-A 1 12/27/93
E06825		Aspartokinase	Sugimoto, M. etc. " saltant aspartokinase gene ", patent: JP 1994062866-A 1 03/08/94
				E06826		Saltant aspartokinase α subunit	Sugimoto, M. etc. " saltant aspartokinase gene ", patent: JP 1994062866-A 1 03/08/94
E06827		Saltant aspartokinase α subunit	Sugimoto, M. etc. " saltant aspartokinase gene ", patent: JP 1994062866-A 1 03/08/94
				E07701	secY		Honno, N. etc. " participate in the gene DNA that memebrane protein is integrated into film ", patent: JP 1994169780-A 1 06/21/94
E08177		Aspartokinase	Sato, Y. etc. " can encoder feedback suppress hereditary DNA and the application thereof of the aspartokinase that discharges ", patent: JP 1994261766-A 1 09/20/94
				E08178，   E08179，   E08180，   E08181，   E08182		The aspartokinase that feedback inhibition discharges	Sato, Y. etc. " can encoder feedback suppress hereditary DNA and the application thereof of the aspartokinase that discharges ", patent: JP 1994261766-A 1 09/20/94
E08232		Acetohydroxy acid isomeroreductase	Inui, M. etc. " gene DNA of coding acetohydroxy acid isomeroreductase ", patent: JP 1994277067-A 10/04/94

Table 2 (continuing)
				E08234	secE		Asai, Y. etc. " gene DNA of coded protein transporting mechanism ", patent: JP 1994277073-A 1 10/04/94
E08643		FT aminopherase and dethiobiotin synthetase promoter region	Hatakeyama, K. etc. " dna fragmentation that has promoter function in the excellent bacillus ", patent: JP 1995031476-A 1 02/03/95
				E08646		The biotin synzyme	Hatakeyama, K. etc. " dna fragmentation that has promoter function in the excellent bacillus ", patent: JP 1995031476-A 1 02/03/95
E08649		Aspartase	Kohama, K. etc. " dna fragmentation that has promoter function in the excellent bacillus ", patent: JP 1995031478-A 1 02/03/95
				E08900		Dihydro 2, the dipicolimic acid 2 reductase	Madori, M. etc. " contain coding dihydro 2, the dna fragmentation of the gene of dipicolimic acid 2 reductase and application thereof ", patent: JP 1995075578-A 1 03/20/95
E08901		Diaminapimelate decarboxylase	Madori, M. etc. " contain dna fragmentation and the application thereof of the gene of coding dihydro pimelic acid decarboxylase ", patent: JP 1995075579-A 1 03/20/95
				E12594		Serine hydroxymethylase	Hatakeyama, K. etc. " production of L-Trp ", patent: JP 1997028391-A 1 02/04/97
E12760，   E12759，   E12758		Transposase	Moriya, M. etc. " adopt the artificial transposon amplification gene ", patent: JP 1997070291-A 03/18/97
				E12764		Arginyl-tRNA synthetase; Diaminapimelate decarboxylase	Moriya, M. etc. " adopt the artificial transposon amplification gene ", patent: JP 1997070291-A 03/18/97
E12767		Dihydro 2, the dipicolimic acid 2 synzyme	Moriya, M. etc. " adopt the artificial transposon amplification gene ", patent: JP 1997070291-A 03/18/97
				E12770		Aspartokinase	Moriya, M. etc. " adopt the artificial transposon amplification gene ", patent: JP 1997070291-A 03/18/97

Table 2 (continuing)
				E12773		Dihydro 2, the dipicolimic acid 2 reductase	Moriya, M. etc. " adopt the artificial transposon amplification gene ", patent: 1997070291-A 03/18/97
E13655		Glucose-6-phosphate dehydrogenase (G6PD)	Hatakeyama, K. etc. " glucose-6-phosphate dehydrogenase (G6PD) and its DNA of can encoding ", patent: JP 1997224661-A 1 09/02/97
				L01508	IlvA	Threonine dehydratase	Moeckel, B. etc. " the function and structure analysis of Corynebacterium glutamicum threonine dehydratase ", J.Bacteriol., 174:8065-8072 (1992)
L07603	EC 4.2.1.15	AroG	Chen, C. etc. " Corynebacterium glutamicum aroG gene cloning and nucleotide sequence ", FEMS Microbiol.Lett., 107:223-230 (1993)
				L09232	IlvB；ilvN；ilvC	The large subunit acetohydroxy acid synthase of acetohydroxy acid synthase small subunit; Acetohydroxy acid isomeroreductase	Keilhauer, C. etc. " isoleucine in the Corynebacterium glutamicum is synthetic: the analysis of molecules of ilvB-ilvN-ilvC operon ", J.Bacteriol., 175 (17): 5595-5603 (1993)
L18874	PtsM	The phosphoenolpyruvate sugar phosphotransferase	Fouet, A etc. " the sucrose enzyme-specific II of hay bacillus phosphotransferase system: Expression in Escherichia coli and with the homology of enterobacteria enzyme II ", PNAS USA, 84 (24): 8773-8777 (1987); Lee, J.K. etc. " analysis of the nucleotide sequence of the gene of coding Corynebacterium glutamicum seminase II and the protein sequence of derivation ", FEMS Microbiol.Lett., 119 (1-2): 137-145 (1994)
				L27123	aceB	Malate synthase	Lee, H-S. etc. " characterization of molecules of the gene of coding malate synthase is identified in aceB-Corynebacterium glutamicum ", J.Microbiol.Biotechnol., 4 (4): 256-263 (1994)
L27126		Pyruvate kinase	Jetten, M.S. etc. " structure of Corynebacterium glutamicum pyruvate kinase and functional analysis ", Appl. Environ.Microbiol., 60 (7): 2501-2507 (1994)
				L28760	aceA	Isocitratase
L35906	dtxr	The diphtheria toxin repressor protein	Oguiza, J.A. etc. " from molecular cloning, dna sequence analysis and the characterization of the corynebacterium diphtheriae dtxR of brevibacterium ", J.Bacteriol., 177 (2): 465-467 (1995)

Table 2 (continuing)
				M13774		Prephenate dehydratase	Follettie, M.T. etc. " molecular cloning and the nucleotide sequence of Corynebacterium glutamicum pheA gene ", J. Bacteriol., 167:695-702 (1986)
M16175	5S rRNA		Park, Y-H. etc. " analyze the kind system that excellent bacillus carries out by 56 kinds of rRNA sequences ", J. Bacteriol., 169:1801-1806 (1987)
				M16663	trpE	The o-amino benzoyl acid synthase, 5 ' end	Sano, K. etc. " a kind of glutamic acid is produced the 26S Proteasome Structure and Function of bacterium-brevibacterium trp operon control zone ", Gene, 52:191-200 (1987)
M16664	trpA	Tryptophan synthetase, 3 ' end	Sano, K. etc. " a kind of glutamic acid is produced the 26S Proteasome Structure and Function of bacterium-brevibacterium trp operon control zone ", Gene, 52:191-200 (1987)
				M25819		Phosphoenolpyruvate carboxylase	O ' Regan, M. etc. " clone and the nucleotide sequence of the phosphoenolpyruvate carboxylase encoding gene of Corynebacterium glutamicum ATCC13032 ", Gene, 77 (2): 237-251 (1989)
M85106		23S rRNA gene insetion sequence	Roller, C. etc. " there be common the insertion in being characterized as of gram-positive bacterium with high DNA G+C content in its 23S rRNA gene ", J.Gen.Microbiol., 138:1167-1175 (1992)
				M85107，   M85108		23S rRNA gene insetion sequence	Roller, C. etc. " there be common the insertion in being characterized as at its 23S rRNA gene of gram-positive bacterium with high DNA G+C content ", J.Gen.Microbiol., 138:1167-1175 (1992)
M89931	aecD；brnQ；yhbw	β C-S lyase; Branched-chain amino acid is taken in carrier; The albumen yhbw that supposes	Rossol, I. etc. " a kind of α with degraded aminoethylcysteine of Corynebacterium glutamicum aecD gene code, the C-S lyase of β-elimination activity ", J.Bacteriol., 174 (9): 2968-2977 (1992); Tauch, A. etc. " isoleucine among the Corynebacterium glutamicum ATCC 13032 is taken in by the control of brnQ gene outcome ", Arch.Microbiol., 169 (4): 303-312 (1998)

Table 2 (continuing)
				S59299	trp	Leading gene (promoter)	Herry, D.M. etc. " the trp gene cluster of clone Corynebacterium glutamicum Soviet Union tryptophan superior strain: identify the sudden change of trp targeting sequencing ", Appl.Environ.Microbiol., 59 (3): 791-799 (1993)
U11545	trpD	Anthranilate phosphoribosyl transferase	O ' Gara, J.P. and Dunican, L.K. (1994) " the complete nucleotide sequence of Corynebacterium glutamicum ATCC 21850tpD gene ", Thesis, Microbiology Department, University College Galway, Ireland.
				U13922	cgIIM；cgIIR；clgIIIR	The II type 5-cytosine methyltransferase of inferring; The II type restriction endonuclease of inferring; The I type of inferring or III type restriction endonuclease	Schafer, A. etc. " from the clone in the DNA district of the encoding stress sensitiveness restriction system of Corynebacterium glutamicum ATCC 13032 and characterization and with colibacillary genus between function analysis aspect engaging ", J.Bacteriol., 176 (23): 7309-7319 (1994); " the cgIIM gene of the 5-cytimidine in the Corynebacterium glutamicum coding McrBC defective escherichia coli bacterial strain ", Gene, 203 (2): 95-101 (1997)
U14965	recA
				U131224	ppx		Ankri, S. etc. " sudden change in the Corynebacterium glutamicum biosynthesis pathway: the natural bypass of proA step ", J.Bacteriol., 178 (15): 4412-4419 (1996)
U31225	proC	L-PROLINE: NADP+5-oxidoreducing enzyme	Ankri, S. etc. " sudden change in the Corynebacterium glutamicum biosynthesis pathway: the natural bypass of proA step ", J.Bacteriol., 178 (15): 4412-4419 (1996)
				U31230	obg；proB；unkdh	The gamma-glutamyl kinases; Similar to the specific 2-carboxylic acid of D-isomers dehydratase	Ankri, S. etc. " sudden change in the Corynebacterium glutamicum biosynthesis pathway: the natural bypass of proA step ", J.Bacteriol., 178 (15): 4412-4419 (1996)
U31281	bioB	The biotin synthase	Serebriiskii, I.G., " two newcomers of bio B ultrasonic family: bio B gene cloning, order-checking and the expression of Methylobacillus flagellatum and Corynebacterium glutamicum ", Gene, 175:15-22 (1996)

Table 2 (continuing)
				U35023	thtR；accBC	Thiosulfate sulfurtransferase; The acyl-CoA carboxylase	Jager, W. etc. " coding is similar to the Corynebacterium glutamicum gene of two domain proteins of biotin carboxylase and biotin carboxyl carrier protein ", Arch.Microbiol., 166 (2); 76-82 (1996)
U43535	cmr	The multi-medicament resistance protein	Jager, W. etc. " give the Corynebacterium glutamicum gene of multi-medicament resistance " in the heterologous host Escherichia coli, J.Bacteriol., 179 (7): 2449-2451 (1997)
				U43536	clpB	Heat shock ATP is in conjunction with albumen
U53587	aphA-3	3 ' 5 "-aminoglycoside phosphotransferase
				U89648		Participate in the not evaluation sequence of histidine biosynthesis in the Corynebacterium glutamicum, partial sequence
X04960	TrpA；trpB；trpC；trpD；   trpE；trpG；trpL	Tryptophan operon	Matsui, K. etc. " the complete nucleotide sequence of brevibacterium tryptophan operon and the amino acid sequence of derivation ", Nucleic Acids Res., 14 (24): 10113-10114 (1986)
				X07563	lys A	The DAP decarboxylase (in-diaminapimelate decarboxylase, EC 4.1.1.20)	Yeh, P. etc. " nucleotide sequence of the lysA gene of Corynebacterium glutamicum with and mechanism that express to regulate ", Mol.Gen.Genet., 212 (1): 112-119 (1988)
X14234	EC 4.1.1.31	Phosphoenolpyruvate carboxylase	Eikmanns, B.J. etc. " the phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence and expression ", Mol.Gen.Genet., 218 (2): 330-339 (1989); Lepiniec, L etc. " jowar phosphoenolpyruvate carboxylase gene family: structure, function and molecular evolution ", Plant Mol.Biol., 21 (3): 487-502 (1993)
				X17313	fda	Fructosediphosphate aldolase	Vonder Osten, C.H. etc. " molecular cloning, nucleotide sequence and the fine-structure distribution of Corynebacterium glutamicum fda gene: the structure of Corynebacterium glutamicum ester of Harden Young aldolase and I class and II class aldolase is relatively ", Mol.Microbiol.,

Table 2 (continuing)
				X53993	dapA	L-2,3-dihydro 2, dipicolimic acid 2 synzyme (EC 4.2.1.52)	Bonnassie, S. etc. " nucleotide sequence of Corynebacterium glutamicum dapA gene ", NucleicAcids Res., 18 (21): 6421 (1990)
X54223		The AttB related locus	Cianciotto, N. etc. " the dna sequence dna homology between the attP site of corynebacterium diphtheriae, ulcer rod bacillus (Corynebacterium ulcerans) and Corynebacterium glutamicum att B related locus and λ β-cory-nephage ", FEMS, Microbiol.Lett., 66:299-302 (1990)
				X54740	argS；lysA	Arginyl-tRNA synthetase; Diaminapimelate decarboxylase	Marcel, T. etc. " nucleotide sequence in Corynebacterium glutamicum lysA upstream region of gene district and group structure ", Mol. Microbiol., 4 (11): 1819-1830 (1990)
X55994	trpL；trpE	The leader peptide of inferring; O-amino benzoyl acid synthase component I	Heery, D.M. etc. " nucleotide sequence of Corynebacterium glutamicum trpE gene ", Nucleic Acids Res., 18 (23): 7138 (1990)
				X56037	ThrC	Threonine synthase	Han, K.S. etc. " molecular structure of Corynebacterium glutamicum threonine synthase gene ", Mol.Microbiol., 4 (10): 1693-1702 (1990)
X56075	The attB-related locus	Attachment site	Ciancotto, N. etc. " the dna sequence dna homology between the attP site of corynebacterium diphtheriae, ulcer rod bacillus (Corynebacterium ulcerans) and Corynebacterium glutamicum att B related locus and λ β-cory-nephage ", FEMS.Microbiol.Lett., 66:299-302 (1990)
				X57226	lysC-alpha；lysC-beta；   asd	Aspartokinase α subunit; Aspartokinase β subunit; Aspartic acid β half aldehyde dehydrogenase	Kalinowski, J. etc. " heredity of Corynebacterium glutamicum aspartokinase and biochemical analysis ", Mol. Microbiol., 5 (5): 1197-1204 (1991); Kalinowski, J. etc. " aspartokinase gene lysC α and lysC β are overlapping and adjacent with aspartic acid β half aldehyde dehydrogenase gene asd in Corynebacterium glutamicum ", Mol.Gen.Genet., 224 (3): 317-324 (1990)

Table 2 (continuing)
				X59403	gap；pgk；tpi	Glyceraldehyde-3-phosphate; Phosphoglycerokinase; Triose-phosphate isomerase	Eikmanns, B.J. " Corynebacterium glutamicum gene of a kind of coding three kinds of glycolytic ferment glyceraldehyde-3-phosphate dehydrogenases, glycerol 3-phosphate acid kinase and triose-phosphate isomerases bunch evaluation, sequence analysis and expression ", J.Bacteriol., 174 (19): 6076-6086 (1992)
X59404	gdh	Glutamte dehydrogenase	Bormann, E.R. etc. " analysis of molecules of the Corynebacterium glutamicum gdh gene of coding glutamte dehydrogenase ", Mol.Microbiol., 6 (3): 317-326 (1992)
				X60312	lysI	The 1B permease	Seep-Feldhaus, A.H etc. " participate in the analysis of molecules of the Corynebacterium glutamicum lysl gene of lysine absorption ", Mol.Microbiol., 5 (12): 2995-3005 (1991)
X66078	copI	PsI albumen	Joliff, G. etc. " cspl gene cloning and the nucleotide sequence of one of two kinds of kDa major secretory proteins of coding Corynebacterium glutamicum PSl: the N end region of inferring of PS1 and Mycobacterium antigen 85 composite bulk phases are seemingly ", Mol.Microbiol., 6 (16): 2349-2362 (1992)
				X66112	glt	Citrate synthase	Eikmanns, B.J. etc. " Corynebacterium glutamicum gltA gene cloning sequence, expression and the transcription analysis of coding citrate synthase ", Microbiol., 140:1817-1828 (1994)
X67737	dapB	Dihydro 2, the dipicolimic acid 2 reductase
				X69103	csp2	S-layer proteins PS2	Peyret, J.L. etc. " characterization of the cspB gene of a kind of orderly S-layer proteins PS2 in the coding Corynebacterium glutamicum ", Mol.Microbiol., 9 (1): 97-109 (1993)
X69104		The IS3 insertion element of being correlated with	Bonamy, C. etc. " evaluation of the relevant insetion sequence IS 1206 of a kind of Corynebacterium glutamicum IS3 and kind system analyze ", Mol.Microbiol., 14 (3): 571-581 (1994)
				X70959	leuA	Isopropylmalate synthase	Patek, M. etc. " leucine in the Corynebacterium glutamicum is synthetic: the impact that the enzymatic activity of leuA, structure and leuA inactivation are synthetic on lysine ", Appl.Environ.Microbiol., 60 (1): 133-140 (1994)

Table 2 (continuing)
				X71489	icd	Isocitric dehydrogenase (NADP+)	Eikmanns, B.J. etc. " biochemical character of the sequence analysis of Corynebacterium glutamicum icd gene cloning, expression and inactivation and this enzyme of coding isocitric dehydrogenase is identified ", J.Bacteriol., 177 (3): 774-782 (1995)
X72855	GDHA	Glutamte dehydrogenase (NADP+)
				X75083，   X70584	mtrA	The 5-methyl tryptophan resistance	Heery, D.M. etc. " sequence of the coding 5-methyl tryptophan resistance of Corynebacterium glutamicum tryptophan superior strain ", Biochem.Biophys.Res.Commun., 201 (3): 1255-1262 (1994)
X75085	recA		Fitzpatrick, R. etc. " structure and the characterization of the recA mutant strain of Corynebacterium glutamicum and brevibacterium ", Appl.Microbiol.Biotechnol., 42 (4): 575-580 (1994)
				X75504	aceA；thiX	The part isocitratase;	Reinscheid, D.J. etc. " characterization of Corynebacterium glutamicum isocitratase gene and the biochemical analysis of this enzyme ", J.Bacteriol., 176 (12): 3474-3483 (1994)
X76875		ATP enzyme β subunit	Ludwig, W. etc. " the bacterium kind system based on EF-T u and the comparative analysis of atp synthase beta subunit gene is related ", Antonie Van Leeuwenhoek, 64:285-305 (1993)
				X77034	tuf	EF-T u	Ludwig, W. etc. " the bacterium kind system based on EF-T u and the comparative analysis of atp synthase beta subunit gene is related ", Antonie Van Leeuwenhoek, 64:285-305 (1993)
X77384	recA		Billman-Jacobe, H. " nucleotide sequence of Corynebacterium glutamicum recA gene ", DNA Seq., 4 (6): 403-404 (1994)
				X78491	aceB	Malate synthase	Reinscheid, D.J. etc. " malate synthase of the Corynebacterium glutamicum pta-ack operon of coding phosphotransacetylase: sequence analysis ", Microbiology, 140:3099-3108 (1994)

Table 2 (continuing)
				X80629	16S rDNA	The 16S rRNA	Rainey, F.A. etc. " the kind system generation analysis of red bacterium genus (Rhodococcus) and Nocardia (Norcardia) and Nocard's bacillus are from the evidence of the evolution origin of red bacterium radiation ", Microbiol., 141:523-528 (1995)
X81191	gluA；gluB；gluC；   gluD	The glutamic acid shooting system	Kronemeyer, W. etc. " gluABCD bunch structure of coding Corynebacterium glutamicum glutamic acid shooting system ", J.Bacteriol., 177 (5): 1152-1158 (1995)
				X81379	dapE	The succinyl diaminopimelic acid takes off the succinyl group enzyme	Wehrmann, A. etc. " analyses of the different dna fragmentations of the Corynebacterium glutamicum of complementary Escherichia coli dapE ", Microbiology, 40:3349-56 (1994)
X82061	16S rDNA	The 16S rRNA	Ruimy, R. etc. " the kind system by the Corynebacterium of small subunit ribosomal dna sequence analytical derivation occurs ", Int.J.Syst.Bacteriol., 45 (4): 740-746 (1995)
				X82928	asd；lysC	Aspartate-semialdehyde dehydrogenase;	Serebrijski, I. etc. " the osmotic stress dependence complementation that is caused by allos proA in the multicopy inhibition of asd gene and the proA mutant ", J.Bacteriol., 177 (24): 7255-7260 (1995)
X82929	proA	Gamma-glutamyl phosphoric acid reduction enzyme	Serebrijski, I. etc. " the osmotic stress dependence complementation that is caused by allos proA in the multicopy inhibition of asd gene and the proA mutant ", J.Bacteriol., 177 (24): 7255-7260 (1995)
				X84257	16S rDNA	The 16S ribose RNA	Pascual, C. etc. " the kind system based on the Corynebacterium of 16S rRNA gene order analyzes ", Int. J.Syst.Bacteriol., 45 (4): 724-728 (1995)
X85965	aroP；dapE	The ArAA permease;	Wehrmann etc. " there is the aroP of coding aromatic amino acid transport protein in the functional analysis explanation of the flanking sequence of Corynebacterium glutamicum proline dapE ", J.Bacteriol., 177 (20): 5991-5993 (1995)

Table 2 (continuing)
				X86157	argB；argC；argD；   argF；argJ	Acetylglutamate kinase; N-acetyl-gamma-glutamyl phosphoric acid reduction enzyme; Ncg12355; OCT; Glutamic acid N-acetyltransferase	Sakanyan, V. etc. " gene and the enzyme of the circulation of Arginine biosynthesis acetyl group in the Corynebacterium glutamicum: the enzyme in the early stage step of arginine pathway is evolved ", Microbiology, 142:99-108 (1996)
X89084	pta；ackA	The phosphoric acid acetyltransferase; Acetokinase	Reinscheid, D.J. etc. " clone, sequence analysis, expression and the inactivation of the Corynebacterium glutamicum pta-ack operon of coding phosphotransacetylase and acetokinase ", Microbiology, 145:503-513 (1999)
				X89850	attB	Attachment site	Le Marrec, C. etc. " hereditary feature of the phi AAU2 site-directed integration function of infection " golden yellow arthrobacterium (Arthrobacter aureus) C70 " is identified ", J.Bacteriol., 178 (7): 1996-2004 (1996)
X90356		Promoter fragment F1	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)
				X90357		Promoter fragment F2	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)
X90358		Promoter fragment F10	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)
				X90359		Promoter fragment F13	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)
X90360		Promoter fragment F22	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)
				X90361		Promoter fragment F34	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)

Table 2 (continuing)
				X90362		Promoter fragment F37	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)
X90363		Promoter fragment F45	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)
				X90364		Promoter fragment F64	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)
X90365		Promoter fragment F75	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)
				X90366		Promoter fragment PF101	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)
X90367		Promoter fragment PF104	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)
				X90368		Promoter fragment PF109	Patek, M. etc. " Corynebacterium glutamicum promoter: the search of clone, analysis of molecules and consensus motif ", Microbiology, 142:1297-1309 (1996)
X93513	amt	The ammonium movement system	Siewe, R.M. etc. " Corynebacterium glutamicum (methyl) ammonium is taken in function and the hereditary feature of carrier and identified ", J. Biol.Chem., 271 (10): 5398-5403 (1996)
				X93514	betP	The betaine movement system	Pater, H. etc. " Corynebacterium glutamicum betP Gene Isolation, characterization and the expression of codes match solute betaine movement system ", J.Bacteriol., 178 (17): 5229-5234 (1996)

Table 2 (continuing)
				X95649	orf4		Patek, M. etc. " coding participates in evaluation and the transcription analysis of the Corynebacterium glutamicum dapB-ORF2-dapA-ORF4 operon of two kinds of synthetic enzymes of 1B ", Biotechnol.Lett., 19:1113-1117 (1997)
X96471	lysE；lysG	Lysine output albumen; Albumen is regulated in lysine output	Vflljic, M etc. " the new type carting albumen with novel cell function: the 1B output of Corynebacterium glutamicum ", Mol.Microbiol., 22 (5): 815-826 (1996)
				X96580	panB；panC；xylB	3-methyl-2-hydroxybutyric acid hydroxymethyl transferases; Pantoate-beta-alanine ligase; Xylulokinase	Sahm, H. etc. " the synthetic and excessive production that panBC and the synthetic gene of coding Valine is applied to Pantothenic acid of the Pantothenic acid in the Corynebacterium glutamicum ", Appl.Environ.Microbiol., 65 (5): 1973-1979 (1999)
X96962		Insetion sequence IS1207 and transposase
				X99289		Elongation factors P	Ramos, A. etc. " coded amino acid is produced gene cloning, order-checking and the expression of elongation factors P in the bacterium brevibacterium (Corynebacterium glutamicum ATCC 13869) ", Gene, 198:217-222 (1997)
Y00140	thrB	Homoserine kinase	Mateos, L.M. etc. " nucleotide sequence of the homoserine kinase of brevibacterium (thrB) gene ", Nucleic Acids Res., 15 (9): 3922 (1987)
				Y00151	ddh	In-diaminopimelic acid D-dehydrogenase (EC 1.4.1.16)	Ishino, S. etc. " Corynebacterium glutamicum in-nucleotide sequence of diaminopimelic acid D-dehydrogenase gene ", Nucleic Acids Res., 15 (9): 3917 (1987)
Y00476	thrA	Homoserine dehydrogenase	Mateos, L.M. etc. " nucleotide sequence of the homoserine dehydrogenase of brevibacterium (thrA) gene ", Nucleic Acids Res., 15 (24): 10598 (1987)
				Y00546	hom；thrB	Homoserine dehydrogenase; Homoserine kinase	Peoples, O.P. etc. " nucleotide sequence and the fine-structure distribution of Corynebacterium glutamicum hom-thrB operon ", Mol.Microbiol., 2 (1): 63-72 (1988)

Table 2 (continuing)
				Y08964	murC；ftsQ/divD；ftsZ	UPD-N-acetylmuramic acid-alanine ligase; Divide initial albumen or Cyclin; Cyclin	Honrubia, M.P. etc. " evaluation of brevibacterium ftsZ gene, sign and genome structure ", Mol.Gen.Genet., 259 (1): 97-104 (1998)
Y09163	putP	High affinity proline movement system	Peter, H. etc. " characterization of the low affinity shooting system of the separation of Corynebacterium glutamicum putP gene and coupling solute ", Arch.Microbiol., 168 (2): 143-151 (1997)
				Y09548	pyc	Pyruvate carboxylase	Peters-Wendisch, P.G. etc. " Corynebacterium glutamicum pyruvate carboxylase: the characterization of pyc gene, expression and inactivation ", Microbiology, 144:915-927 (1998)
Y09578	leuB	3-Isopropylmalate dehydrogenase	Patek, M. etc. " analysis of Corynebacterium glutamicum leuB gene ", Appl.Microbiol.Biotechnol., 50 (1): 42-47 (1998)
				Y12472		Attachment site bacteriophage Phi-16	Moreau, S. etc. " site-directed integration of excellent bacillus bacteriophage Phi-16: the structure of integration vector ", Microbiol., 145:539-548 (1999)
Y12537	proP	Proline/ectoine shooting system albumen	Peter, H. etc. " Corynebacterium glutamicum has the Second support of four kinds of coupling solutes: evaluation, order-checking and the characterization of proline/ectoine shooting system ProP and ectoine/ proline/betaine carrier EctP ", J.Bacteriol., 180 (22): 6005-6012 (1998)
				Y13221	glnA	Glutamine synthelase I	Jakoby, M. etc. " separation of the Corynebacterium glutamicum glnA gene of coding glutamate synthetase I ", FEMS Microbiol.Lett., 154 (1): 81-88 (1997)
Y16642	lpd	Dihydrolipoamide dehydrogenase
				Y18059		Attachment site rod bacillus bacteriophage 304L	Moreau, the “ ﹠phi such as S; The analysis of 304L integration function: a kind of integrase assembly in the excellent bacillus bacteriophage ", Virology, 255 (1): 150-159 (1999)

Table 2 (continuing)
				Z21501	argS；lysA	Arginyl-tRNA synthetase; Diaminapimelate decarboxylase (part)	Oguiza, J.A. etc. " gene of coding Arginyl-tRNA synthetase is positioned at the upstream of brevibacterium lysA gene: arginine is to the adjusting of argS-lysA bunch of expression ", J.Bacteriol., 175 (22): 7356-7362 (1993)
Z21502	dapA；dapb	Dihydro 2, the dipicolimic acid 2 synthase; Dihydro 2, the dipicolimic acid 2 reductase	Pisabarro, A. etc. " the gene cluster coding dihydro 2 of three genes of brevibacterium (dapA, orf2 and dapB); the third polypeptide of dipicolimic acid 2 reductase and a kind of Unknown Function ", J.Bacteriol., 175 (9): 2743-2749 (1993)
				Z29563	thrC	Threonine synthase	Malumbres, M. etc. " analysis and the expression of the thrC gene of coding threonine synthase ", Appl. Environ.Microbiol., 60 (7) 2209-2219 (1994)
Z46753	16S rDNA	The gene of 16S rRNA
				Z49822	sigA	The SigA sigma factor	Oguiza, J.A. etc. " a plurality of sigma factor genes in the brevibacterium: the characterization of sigA and sigB ", J.Bacteriol., 178 (2): 550-553 (1996)
Z49823	galE；dtxR	Catalytic activity UDP-galactolipin 4-epimerase; Diphtheria toxin is regulated albumen	Oguiza, J.A. etc. " galE gene and the dmdR genetic transcription coupling of Corynebacterium glutamicum coding UDP-galactolipin 4-epimerase ", Gene, 177:103-107 (1996)
				Z49824	orfl；sigB	"; The SigB sigma factor	Oguiza, J.A. etc. " a plurality of sigma factor genes in the brevibacterium: the characterization of sigA and sigB ", Gene, 178 (2): 550-553 (1996)
Z66534		Transposase	Correia, A. etc. " clone and the characterization that are present in the IS sample element among the brevibacterium ATCC 13869 ", Gene, 170 (1): 91-94 (1996)
				1The sequence of this gene is published in the specified list of references. Yet the sequence that the present inventor obtains obviously is longer than the form of announcing. Think that the form of announcing depends on incorrect initiation codon, therefore only represent the fragment of true code area.

Table 3: can be used for implementing Corynebacterium of the present invention and brevibacterium bacterial strain

Bacterial classification	ATCC	FRRM	NRRL	CECT	NCIMB	CBS	NCTC	DSMZ
									Brevibacterium ammoniagene (Brevibacterium ammoniagenes)	21054
Brevibacterium ammoniagene	19350
									Brevibacterium ammoniagene	19351
Brevibacterium ammoniagene	19352
									Brevibacterium ammoniagene	19353
Brevibacterium ammoniagene	19354
									Brevibacterium ammoniagene	19355
Brevibacterium ammoniagene	19356
									Brevibacterium ammoniagene	21055
Brevibacterium ammoniagene	21077
									Brevibacterium ammoniagene	21553
Brevibacterium ammoniagene	21580
									Brevibacterium ammoniagene	39101
Brevibacterium ammoniagene (Brevibacterium butanicum)	21196
									Brevibacterium ammoniagene (Brevibacterium divaricatum)	21792	P928
Deep yellow brevibacterium (Brevibacterium flavum)	21474
									Deep yellow brevibacterium	21129
Deep yellow brevibacterium	21518
									Deep yellow brevibacterium			B11474
Deep yellow brevibacterium			B11472
									Deep yellow brevibacterium	21127
Deep yellow brevibacterium	21128
									Deep yellow brevibacterium	21427
Deep yellow brevibacterium	21475
									Deep yellow brevibacterium	21517
Deep yellow brevibacterium	21528
									Deep yellow brevibacterium	21529
Deep yellow brevibacterium			B11477
									Deep yellow brevibacterium			B11478
Deep yellow brevibacterium	21127
									Deep yellow brevibacterium			B11474
Xi Shi brevibacterium (Brevibacterium healii)	15527
									Ketoglutaric acid brevibacterium (Brevibacterium ketoglutamicum)	21004
The ketoglutaric acid brevibacterium	21089
									The ketoglutaric acid brevibacterium	21914
Brevibacterium				70
									Brevibacterium				74
Brevibacterium				77
									Brevibacterium	21798
Brevibacterium	21799
									Brevibacterium	21800
Brevibacterium	21801
									Brevibacterium			B11470
Brevibacterium			B11471

Bacterial classification	ATCC	FRRM	NRRL	CECT	NCIMB	CBS	NCTC	DSMZ
									Brevibacterium	21086
Brevibacterium	21420
									Brevibacterium	21086
Brevibacterium	31269
									Extension brevibacterium (Brevibacterium linens)	9174
Extension brevibacterium	19391
									Extension brevibacterium	8377
Brevibacterium  paraffinolyticum					11160
									Brevibacterium (Brevibacterium spec)						717.73
Brevibacterium						717.73
									Brevibacterium	14604
Brevibacterium	21860
									Brevibacterium	21864
Brevibacterium	21865
									Brevibacterium	21866
Brevibacterium	19240
									Corynebacterium acctoacidophlum (Corynebacterium acetoacidophilum)	21476
Corynebacterium acctoacidophlum	13870
									Have a liking for vinegar paddy rod bacillus			B11473
Have a liking for vinegar paddy rod bacillus			B11475
									Have a liking for vinegar paddy rod bacillus	15806
Have a liking for vinegar paddy rod bacillus	21491
									Have a liking for vinegar paddy rod bacillus	31270
Have a liking for acetyl rod bacillus (Corynebacterium acetophilum)			B3671
									Brevibacterium ammoniagene	6872						2399
Brevibacterium ammoniagene	15511
									Corynebacterium fujiokense	21496
Corynebacterium glutamicum	14067
									Corynebacterium glutamicum	39137
Corynebacterium glutamicum	21254
									Corynebacterium glutamicum	21255
Corynebacterium glutamicum	31830
									Corynebacterium glutamicum	13032
Corynebacterium glutamicum	14305
									Corynebacterium glutamicum	15455
Corynebacterium glutamicum	13058
									Corynebacterium glutamicum	13059
Corynebacterium glutamicum	13060
									Corynebacterium glutamicum	21492
Corynebacterium glutamicum	21513
									Corynebacterium glutamicum	21526
Corynebacterium glutamicum	21543
									Corynebacterium glutamicum	13287
Corynebacterium glutamicum	21851
									Corynebacterium glutamicum	21253
Corynebacterium glutamicum	21514
									Corynebacterium glutamicum	21516

Bacterial classification	ATCC	FRRM	NRRL	CECT	NCIMB	CBS	NCTC	DSMZ
									Corynebacterium glutamicum	21299
Corynebacterium glutamicum	21300
									Corynebacterium glutamicum	39684
Corynebacterium glutamicum	21488
									Corynebacterium glutamicum	21649
Corynebacterium glutamicum	21650
									Corynebacterium glutamicum	19223
Corynebacterium glutamicum	13869
									Corynebacterium glutamicum	21157
Corynebacterium glutamicum	21158
									Corynebacterium glutamicum	21159
Corynebacterium glutamicum	21355
									Corynebacterium glutamicum	31808
Corynebacterium glutamicum	21674
									Corynebacterium glutamicum	21562
Corynebacterium glutamicum	21563
									Corynebacterium glutamicum	21564
Corynebacterium glutamicum	21565
									Corynebacterium glutamicum	21566
Corynebacterium glutamicum	21567
									Corynebacterium glutamicum	21568
Corynebacterium glutamicum	21569
									Corynebacterium glutamicum	21570
Corynebacterium glutamicum	21571
									Corynebacterium glutamicum	21572
Corynebacterium glutamicum	21573
									Corynebacterium glutamicum	21579
Corynebacterium glutamicum	19049
									Corynebacterium glutamicum	19050
Corynebacterium glutamicum	19051
									Corynebacterium glutamicum	19052
Corynebacterium glutamicum	19053
									Corynebacterium glutamicum	19054
Corynebacterium glutamicum	19055
									Corynebacterium glutamicum	19056
Corynebacterium glutamicum	19057
									Corynebacterium glutamicum	19058
Corynebacterium glutamicum	19059
									Corynebacterium glutamicum	19060
Corynebacterium glutamicum	19185
									Corynebacterium glutamicum	13286
Corynebacterium glutamicum	21515
									Corynebacterium glutamicum	21527
Corynebacterium glutamicum	21544
									Corynebacterium glutamicum	21492
Corynebacterium glutamicum			B8183
									Corynebacterium glutamicum			B8182
Corynebacterium glutamicum			B12416
									Corynebacterium glutamicum			B12417
Corynebacterium glutamicum			B12418
									Corynebacterium glutamicum			B11476
Corynebacterium glutamicum	21608

Bacterial classification	ATCC	FRRM	NRRL	CECT	NCIMB	CBS	NCTC	DSMZ
									Lily hedysarum scoparium bacillus (Corynebacterium lilium)		P973
Corynebacterium nitrilophilus	21419				11594
									Rod bacillus (Corynebacterium spec.)		P4445
The rod bacillus		P4446
									The rod bacillus	31088
The rod bacillus	31089
									The rod bacillus	31090
The rod bacillus	31090
									The rod bacillus	31090
The rod bacillus	15954							20145
									The rod bacillus	21857
The rod bacillus	21862
									The rod bacillus	21863

ATCC: American type culture collection (American Type Culture Collection, Rockville, MD, USA).

FERM: Japanese fermentation research institute (Fermentation Research Institute, Chiba, Japan).

NRRL: american agriculture research institution preservation center (ARS Culture Collection, Northern Regional Research Laboratory, Peoria, USA).

CECT: Spain typical case culture collection center (Coleccion Espanola de Cultivos Tipo, Valencia, Spain).

NCIMB: the state-run industry of Britain and marine microorganism preservation Co., Ltd (National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, UK).

CBS: Dutch fungi strain preservation center (Centraalbureau voor Schimmelcultures, Baarn, NL).

NCTC: Britain state-run typical culture collection center (National Collection of Type Cultures, London, UK).

DSMZ: Germany microbial preservation center (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany).

List of references is seen: Sugawara, (1993) World directory of collections of cultures of microorganisms:Bacteria such as H., fungi and yeasts (4th edn), World federation for culture collections world data center on microorganisms, Saimata, Japan.

Table 4: sequence alignment result

ID#	Length N T	Genank hits	Length	Registration number	The title that Genbank hits	The source that Genbank hits	Homology % (GAP)	Date of entry
									rxa00013             rxa00014           rxa00030             rxa00032                        rxa00041         rxa00042	996             903           513             1632                     1342           882	GB_GSS4：AQ713475     GB_HTG3：AC007420     GB_HTG3：AC007420     GB_BA1：MTCY3A2   GB_BA1：MLCB 1779     GB_BA1：SAPURCLU   S   GB_EST21：C89713     GB_EST28：AI497294     GB_EST21：C92167     GB_BA2：AF010496     GB_BA2：AF018073           GB_BA2：AF045245       EM_PAT：E11760       GB_PAT：I26124   GB_IN1：LMFL5883   EM_PAT：E11760       GB_PAT：I26124	581     130583     130583     25830   43254     9120     767     484     637     189370     9810           5930       6911       6911   31934   6911       6911	AQ713475     AC007420     AC007420     Z83867   Z98271     X92429     C89713     AI497294     C92167     AF010496     AF018073           AF045245       E11760       I26124   AL117384   E11760       I26124	HS_5402_B2_A12_T7A RPCI-11 male sex BAC library human genome clone flat board=978 row=24 row=B, genome summary sequence. Drosophila melanogaster chromosome 2 clone BACR07M10 (D630) RPCI-98 07.M.10map 24A-24D bacterial strain y; Cn bw sp, the * * * * * * that checking order, 83 non-sequential segments. Drosophila melanogaster chromosome 2 clone BACR07M10 (D630) RPCI-98 07.M.10map 24A-24D bacterial strain y; Cn bw sp, the * * * * * * that checking order, 83 non-sequential segments. Mycobacterium tuberculosis H37Rv complete genome group; Sections 136/162 Mycobacterium leprae clay B1779 is black streptomycete (S.albonigrt) napH, pur7, pur10, pur6, pur4, pur5 and pur3 gene C 89713 Dictyostelium discoideum SS (H.Urushihara) Dictyostelium discoideum cDNA clone SSG229 in vain, mRNA sequence fb63g03.y1 Zebrafish WashU MPING EST Danio rerio cDNA 5 ' is similar to SW:AFP4_MYOOCP80961 antifreeze protein LS-12; MRNA sequence C 92167 Dictyostelium discoideum SS (H.Urushihara) Dictyostelium discoideum cDNA clone SSD179, mRNA sequence pod membrane Rhodobacter strain SB1003, the red bacterium operon of portion gene category ball is regulated albumen (smoC), the pericentral siphon sorbierite is transported ATP binding transport albumen (smoK), SDH (smoS), mannitol dehydrogenase (mtlK) and pericentral siphon sweet mellow wine in conjunction with albumen (smoM) gene, complete cds in conjunction with albumen (smoE), sorbierite/sweet mellow wine transhipment inner membrane protein (smoF), sorbierite/sweet mellow wine transhipment inner membrane protein (smoG), sorbierite/sweet mellow wine. Friedlanders bacillus D-R alcohol transport protein (dalT), D-R alcohol kinases (dalK), D-R alcohol dehydrogenase (dalD) and repressor protein (dalR) gene, complete cds. The sequence 4 Leishmania maior Friedlin chromosomes 23 clay L5883 of saccharase gene base sequence United States Patent (USP) 5556776, the sequence 4 of complete sequence saccharase gene base sequence United States Patent (USP) 5556776	The unknown Leishmania major of people (Homo sapiens) Drosophila melanogaster (Drosophila melanogaster) Drosophila melanogaster Much's bacillus Mycobacterium leprae (Mycobacterium leprae) ring streptomycete (Streptomyces anulatus) the Dictyostelium discoideum Danio rerio Dictyostelium discoideum pod membrane red bacterium of red bacterium (Rhodobacter capsulatus) class ball (Rhodobacter sphaeroides) Friedlanders bacillus (Klebsiella pneumoniae) Corynebacterium glutamicum Corynebacterium glutamicum is unknown	37,148     34,568     34,568     58,140   57,589     55,667     45,283     42,991     44,444     39,689     48,045           38,514       99,031       99,031   43,663   94,767       94,767	99-07-13     99-09-20     99-09-20     98-06-17   97-08-08     96-02-28     98-04-20     99-03-11     99-07-12     98-05-12     97-10-22           98-07-16       97-10-08   (Rel.52，   Created)   96-10-07   99-10-21   97-10-08   (Rel.52，   Created)   96-10-07

Table 4 (continuing)

rxa00043           rxa00098           rxa00148       rxa00149       rxa00195         rxa00196       rxa00202               rxa00206

1287   1743   2334   1971   684   738   1065   1161

GB_INI：CEU33051   GB_PAT：E6124   EM_PAT：E11760   GB_PR3：AC005174   GB_BA1：MSU88433   GB_BA1：SC5A7   GB_BA1：MTCY10D7   GB_BA1：MTCY277   GB_BA1：MSGY456   GB_BA1：MSGY175   GB_BA1：MSGY456   GB_BA1：MSGY175   GB_BA1：MTCY277   GB_BA1：MTCY274   GB_BA1：MSGB 1529C   S   GB_BA1：MTCY274   GB_BA1：MTCY274   GB_BA1：MTCY274   GB_RO：RATCBRQ   GB_EST11：AA253618   GB_EST26：AI390284   GB_EST26：AI390280   GB_BA1：MLCB637   GB_BA1：MTV012

4899   6911   6911   39769   1928   40337   39800   38300   37316   18106   37316   18106   38300   39991   36985   39991   39991   39991   10752   313   490   467   44882   70287

U33051   I26124   E11760   AC005174   U88433   AL031107   Z79700   Z79701   AD000001   AD000015   AD000001   AD000015   Z79701   Z74024   L78824   Z74024   Z74024   Z74024   M55532   AA253618   AI390284   AI390280   Z99263   AL021287

Caenorhabditis elegans sur-2mRNA, complete cds. The sequence 4 of United States Patent (USP) 5556776. The saccharase gene base sequence. The people clones UWGC:g1564a012,7p14-15, complete sequence. Smegma mycobacterium phosphogvlucoisomerase gene, complete cds. Streptomyces coelicolor clay 5A7. Mycobacterium tuberculosis H37Rv complete genome group; Sections 44/162. Mycobacterium tuberculosis H37Rv complete genome group; Sections 65/162. The Much's bacillus sequence of clone y456. The Much's bacillus sequence of clone y175. The Much's bacillus sequence of clone y456. The Much's bacillus sequence of clone y175. Mycobacterium tuberculosis H37Rv complete genome group; Sections 65/162. Mycobacterium tuberculosis H37Rv complete genome group; Sections 126/162. Mycobacterium leprae clay B1529DNA sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 126/162. Mycobacterium tuberculosis H37Rv complete genome group; Sections 126/162. Mycobacterium tuberculosis H37Rv complete genome group; Sections 126/162. Rat carbohydrate bind receptor gene, complete cds. Mw95c 10.rl Soares mouse NML house mouse cDNA clones IMAGE:678450 5 ', mRNA sequence. Mw96a03.71 Soares mouse NML house mouse cDNA clones IMAGE:678508 5 ', is similar to the TR:O09171 betaine homocysteine methyl transferase; The mRNA sequence. Mw95c 10.yl Soares mouse NML house mouse cDNA clones IMAGE:678450 5 ', mRNA sequence. Mycobacterium leprae clay B637. Mycobacterium tuberculosis H37Rv complete genome group; Sections 132/162.

Unknown Corynebacterium glutamicum people smegma mycobacterium (Mycobacterium smegmatis) streptomyces coelicolor (Streptomyces coelicolor) Much's bacillus Much's bacillus Much's bacillus Much's bacillus Much's bacillus Much's bacillus Much's bacillus Much's bacillus Mycobacterium leprae Much's bacillus Much's bacillus Much's bacillus brown rat (Rattus norvegicus) house mouse (Mus musculus) the house mouse house mouse of Caenorhabditis elegans Mycobacterium leprae Much's bacillus

40,276   97,591   97,591   35,879   62,658   37,638   36,784   67,457   40,883   67,457   35,883   51,001   51,001   35,735   57,014   41,892   41,841   36,599   36,212   38,816   42,239   37,307   58,312   36,632

96-01-23   96-10-07   97-10-   08(Rel.52，   Created)   98-06-24   97-04-19   98-07-27   98-06-17   98-06-17   96-12-03   96-12-10   96-12-03   96-12-10   98-06-17   98-06-19   96-06-15   98-06-19   98-06-19   98-06-19   93-04-27   97-03-13   99-02-02   99-02-02   97-09-17   99-06-23

Table 4 (continuing)

rxa00224           rxa00225           rxa00235                   rxa00246           rxa00251       rxa00288           rxa00293

1074           909           1398                   1158           831       1134           1035

GB_BA1：SC6E10   GB_BA1：BJU32230     GB_BA1：PDEETFAB     GB_HTG3：AC009689   GB_RO：AF060178   GB_GSS 11：AQ325043     GB EST31：AI676413     GB_BA1：MTCY10G2   GB_BA2：AF061753     GB_BA2：AF086791             GB_BA2：AF012550     GB_PAT：E03856     GB_BA1：BACADHT   GB_BA1：MTCY20G9   GB_BA1：MTV004   GB_BA1：MTV004   GB_BA2：AF050114     GB_GSS3：B16984     GB_IN2：AF144549   GB_EST1：T28483

23990   1769     2440     177954   2057   734     551     38970   3721     37867             2690     1506     1688   37218   69350   69350   1038     469     7887   313

AL109661   U32230     L14864     AC009689   AF060178   AQ325043     AI676413     Z92539   AF061753     AF086791             AF012550     E03856     D90421   Z77162   AL009198   AL009198   AF050114     B16984     AF144549   T28483

Streptomyces coelicolor clay 6E10. Soybean is the little subunit of living rhizobium electron transfer flavoprotein (etfS) and large subunit (etfL) gene slowly, complete cds. Paracoccus denitrificans electron transfer flavoprotein α and β subunit gene, complete cds. Human chromosome 4 clone 104_F_7map 4, the low sequence of samples of passing through. House mouse Heparan sulfate 2-sulfotransferase (Hs2st) mRNA, complete cds. Mgxb0020J0lr CUGI Rice Blast BAC library Magnaporthe grosea genomic clone mgxb0020J01r, genome summary sequence. EtmEST0167 EtH1 eimeria avium (Eimeria tenella) cDNA clones etmc074 5 ', mRNA sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 47/162. Nitrosomonas europaea CTP synthase (pyrG) gene, part cds; And enolase (eno) gene, complete cds. The Zymomonasmobilis bacterial strain ZM4 clone little subunit of 67E10 carbamylphosphate synthetase (carA), carbamylphosphate synthetase large subunit (carB), transcriptional elongation factor (greA), enolase (eno), pyruvic dehydrogenase alpha subunit (pdhA), pyruvic dehydrogenase β subunit (pdhB), ribonuclease H (rnh), homoserine kinase homologue, alcohol dehydrogenase II (adhB) and exonuclease ABC subunit A (uvrA) gene, complete cds; And unknown gene. Acinetobacter calcoaceticus BD413 ComP (comP) gene, complete cds. The gDNA of coding alcohol dehydrogenase. The adhT gene of bacillus stearothermophilus alcohol dehydrogenase. Mycobacterium tuberculosis H37Rv complete genome group; Sections 25/162. Mycobacterium tuberculosis H37Rv complete genome group; Sections 144/162. Mycobacterium tuberculosis H37Rv complete genome group; Sections 144/162. Pseudomonad W7 alginic acid lyase, complete cds. 344A14.TVC CIT978SKA1 human genome clone A-344A14, genome summary sequence. Aedes albopictus (Aedes albopictus) ribosomal protein L 34 (rpl34) gene, complete cds. EST46182 people's kidney people cDNA 3 ' end is similar to the monooxygenase 1 (HT:1956) that contains flavoprotein, the mRNA sequence.

Streptomyces coelicolor A3 (2) soybean is living rhizobium (Bradyrhizobium japonicum) Paracoccus denitrificans (Paracoccus denitrificans) people house mouse Magnaporthe grisea eimeria avium Much's bacillus Nitrosomonas europaeas (Nitrosomonas europaea) Zymomonas mobilis acinetobacter calcoaceticus BD413 (Acinetobacter sp BD413) bacillus stearothermophiluses (Bacillus stearothermophilus) bacillus stearothermophilus Much's bacillus Much's bacillus Much's bacillus pseudomonas bacterium (Pseudomonas sp.) W7 people's aedes albopictus people slowly

38,616   48,038     48,351     38,756   39,506   38,333     35,542     65,759   58,941     61,239             53,726     51,688     51,602   42,875   40,380   41,789   49,898     39,355     36,509   42,997

99-08-05   96-05-25     93-10-27     99-08-28   98-06-18   99-01-08     99-05-19     98-06-17   98-08-31     98-11-04             99-09-27     97-09-29     99-02-07   98-06-17   98-06-18   98-06-18   99-03-03     98-06-04     99-06-03   95-09-06

Table 4 (continuing)

rxa00296             rxa00310         rxa00317           rxa00327     rxa00328       rxa00329       rxa00340         rxa00379

2967             558         777           507     615       1347       1269         307

GB_PR1：HUMFMO1   GB_EST32：AI734238       GB_HTG6：AC011069     GB_EST15：AA531468     GB_HTG6：AC011069     GB_VI：VMVY16780   GB_VI：VARCG     GB_VI：VVCGAA   GB_HTG3：AC009571     GB_HTG3：AC009571     GB_PR3：AC005697   GB_BA1：LCATPASEB   GB_BA1：LCATPASEB   GB_BA1：STYPUTPE   GB_BA1：STYPUTPF   GB_BA1：STYPUTPI   GB_PR3：AC004691   GB_PR4：AC004916   GB_PR3：AC004691   GB_BA1：MTCY427   GB_GSS12：AQ412290     GB_PL2：AF112871   GB_HTG1：CEY56A3     GB_HTG1：CEY56A3

2134   512       168266     414     168266     186986   186103     185578   159648     159648     174503   1514   1514   1887   1887   1889   141990   129014   141990   38110   238     2394   224746     224746

M64082   AI734238       AC011069     AA531468     AC011069     Y16780   L22579     X69198   AC009571     AC009571     AC005697   X64542   X64542   L01138   L01139   L01142   AC004691   AC004916   AC004691   Z70692   AQ412290     AF112871   AL022280     AL022280

The people contains monooxygenase (FMO1) mRNA of flavoprotein, complete cds. Zb73c05.y5Soares_ Fetal Lung _ NbHL 19W people cDNA clones IMAGE:3092245 ', is similar to gb:M64082 dimethyl propylene propylhomoserin monooxygenase (people); The mRNA sequence. Drosophila melanogaster chromosome X clone BACR11H20 (D881) RPCI-98 11.H.20map 12B-12C bacterial strain y; Cn bw sp, the * * * * * * that checking order, 92 non-sequential segments. Nj63d12.sl NCI_CGAP_Pr10 people cDNA clones IMAGE:997175, mRNA sequence. Drosophila melanogaster chromosome X clone BACR11H20 (D881) RPCI-98 11.H20map 12B-12C bacterial strain y; Cn bw sp, the * * * * * * that checking order, 92 non-sequential segments. Milk-pox virus (Variola minor virus) complete genome group. Variola major virus (Variola major virus) (bacterial strain Bangladesh-1975) complete genome group. Variola virus (Variola virus) DNA complete genome group. Human chromosome 4 clone 57_A_22map 4, the * * * * * * that checking order, 8 non-sequential segments. Human chromosome 4 clone 57_A_22map 4, the * * * * * * that checking order, 8 non-sequential segments. Human chromosome 17, clone hRPK.138_P_22, complete sequence. Lactobacillus casei ATP enzyme β subunit gene. Lactobacillus casei ATP enzyme β subunit gene. Salmonella (S2980) proline permease (putP) gene, 5 ' end. Salmonella (S2983) proline permease (putP) gene, 5 ' end. Salmonella (S3015) proline permease (putP) gene, 5 ' end. The pac clone DJ0740D02 of people 7p14-p15, complete sequence. The people clones DJ0891L14, complete sequence. The pac clone DJ0740D02 of people 7p14-p15, complete sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 99/162. RPCI-11-195H2.TV RPCI-11 human genome clone RPCI-11-195H2, genome summary sequence. The little subunit of Astasia longa ribosomal RNA gene, complete sequence. Caenorhabditis elegans chromosome III clones Y56A3, the * * * * * * that checking order, non-sequential segments. Caenorhabditis elegans chromosome III clones Y56A3, the * * * * * * that checking order, non-sequential segments.

Everybody bacillus tuberculosis typus humanus people Astasia longa Caenorhabditis elegans Caenorhabditis elegans of everybody Lactobacillus casei (Lactobacillus casei) Lactobacillus casei Salmonella bacterium (Salmonella sp.) Salmonella bacterium Salmonella bacterium of everybody Drosophila melanogaster people Drosophila melanogaster milk-pox virus variola major virus variola virus people

37,915   41,502       33,890     40,821     30,963     35,883   34,664     36,000   36,988     36,988     36,340   34,664   39,308   39,623   39,623   42,906   38,142   38,549   35,865   38,940   36,555     36,465   35,179     35,179

94-11-08   99-06-14       99-12-02     97-08-20     99-12-02     99-09-02   95-01-12     96-12-13   99-09-29     99-09-29     98-10-09   92-12-11   92-12-11   96-05-09   96-05-09   96-05-09   98-05-16   99-07-17   98-05-16   99-06-24   99-03-23     99-06-28   99-09-06     99-09-06

Table 4 (continuing)

rxa00381                 rxa00385           rxa00388         rxa00427         rxa00483           rxa00511     rxa00512

729                 362           1134         909         1587           615     718

GB_PR2：HS 134O19   GB_GSS4：AQ730532     GB_EST23：AI120939       GB_EST23：AI120939       GB_EST32：AI726450     GB_GSS4：AQ740856     GB_PR1：HSPAIP   GB_BA1：MTY25D10   GB_BA1：MSGY224   GB_HTG1：AP000471     GB_BA1：MSGY126   GB_BA1：MTY13D12   GB_HTG1：CEY48C3     GB_PR2：HSAF001550   GB_BA1：LLCPJW565     GB_HTG2：AC006754     GB_PR3：HSE127C11   GB_PR3：HSE127C11   GB_BA1：MTCY22G8

86897   416     561       561       565     768     1587   40838   40051   72466     37164   37085   270193     173882   12828     206217     38423   38423   22550

AL034555   AQ730532     AI120939       AI120939       AI726450     AQ740856     X91809   Z95558   AD000004   AP000471     AD000012   Z80343   Z92855     AF001550   Y12736     AC006754     Z74581   Z74581   Z95585

The human DNA sequence of the clone 134O19 of chromosome lp36.11-36.33, complete sequence. The human genome sperm library D human genome clone flat board of HS_2149_A1_C06_T7C CIT approval=2149, row=11, OK=and E, genome summary sequence. Ub74f05.r1 Soares mammary gland of mouse NMLMG house mouse cDNA clones IMAGE:13834895 ', is similar to gb:J04046 calmodulin (people); Gb:M19381 mouse calmodulin (mouse); The mRNA sequence. Ub74f05.r1 Soares mammary gland of mouse NMLMG house mouse cDNA clones IMAGE:1383489 5 ', is similar to gb:J04046 calmodulin (people); Gb:M19381 mouse calmodulin (mouse); The mRNA sequence. Six days cotton fiber upland cotton of BNLGHi5857 cDNA 5 ' is similar to (AF015913) Skb1Hs[people], the mRNA sequence. The human genome sperm library D human genome clone flat board of HS_2274_A2_A07_T7C CIT approval=2274, row=14, OK=and A, genome summary sequence. The mRNA of people GAIP albumen. Mycobacterium tuberculosis H37Rv complete genome group; Sections 28/162. The sequence of Much's bacillus clone y224. Human chromosome 21 clone B2308H15map 21q223, the * * * * * * that checking order, non-sequential segments. The sequence of Much's bacillus clone y126. Mycobacterium tuberculosis H37Rv complete genome group; Sections 156/162. Caenorhabditis elegans chromosome II clones Y48C3, the * * * * * * that checking order, non-sequential segments. Human chromosome 16BAC clone CIT987SK-334D11 complete sequence. Lactococcus lactis subsp plasmid pJW565DNA, abiiM, abiiR gene and orfX Caenorhabditis elegans clone Y40B10, the * * * * * * that checking order, 5 non-sequential segments. The human DNA sequence of the clay E127C11 of Chromosome 22q11 .2-q end is contained STS. The human DNA sequence of the clay E127C11 of Chromosome 22q11 .2-q end is contained STS. Mycobacterium tuberculosis H37Rv complete genome group; Sections 49/162.

Everybody is house mouse house mouse upland cotton (Gossypium hirsutum) people bacillus tuberculosis typus humanus Much's bacillus bacillus tuberculosis typus humanus's Much's bacillus Caenorhabditis elegans people's lactococcus lactis subsp (Lactococcus lactis subsp.cremoris) Caenorhabditis elegans people bacillus tuberculosis typus humanus

40,604   35,766     41,113       41,113       41,152     41,360     36,792   51,852   51,852   36,875     60,022   60,022   28,013     38,226   37,492     36,648     39,831   36,409   56,232

99-11-23   99-07-15     98-09-02       98-09-02       99-06-11     99-07-16     96-03-29   98-06-17   96-12-03   99-09-13     96-12-10   98-06-17   99-05-29     97-08-22   99-03-01     99-02-23     99-11-23   99-11-23   98-06-17

Table 4 (continuing)

rxa00517             rxa00518           rxa00606       rxa00635       rxa00679       rxa00680       rxa00682

1164             320           2378       1860       1389       441       2022

GB_BA1：MSGLTA   GB_BA2：ECU73857   GB_HTG2：AC006911     GB-HTG2：AC006911     GB_EST29：AI602158     GB_BA2：ECU73857   GB_BA2：STU51879       GB_BA2：AE000140   GB_EST32：AU068253   GB_EST13：AA363046   GB_EST32：AU068253   GB_BA1：PAORF1     GB_BA1：PAORF1   GB_PL2：AC010871   GB_PL1：AT81KBGEN   GB_PL2：AC010871   GB_PR3：AC004058   GB_PL1：AT81KBGEN   GB_PL1：AB026648   GB_HTG3：AC010325     GB_HTG3：AC010325     GB_PR4：AC008179

1776   128824   298804     298804     481     128824   8371       12498   376   329   376   1440     1440   80381   81493   80381   38400   81493   43481   197110     197110     181745

X60513   U73857   AC006911     AC006911     AI602158     U73857   U51879       AE000140   AU068253   AA363046   AU068253   X13378     X13378   AC010871   X98130   AC010871   AC004058   X98130   AB026648   AC010325     AC010325     AC008179

Smegma mycobacterium citrate synthase gltA gene. Escherichia coli minute chromosome 6-8. Caenorhabditis elegans clones Y94H6x, the * * * * * * that checking order, 15 non-sequential segments. Caenorhabditis elegans clones Y94H6x, the * * * * * * that checking order, 15 non-sequential segments. U1-R-AB0-vy-a-01-0-U1.s2U1-R-AB0 brown rat cDNA clone U1-R-AB0-vy-a-01-0-U13 ', the mRNA sequence. Escherichia coli minute chromosome 6-8. Salmonella typhimurium propionic acid catabolism operon: RpoN activator protein homologue (prpR), carboxyl phosphoenolpyruvate transphosphorylase homologue (prpB), citrate synthase homologue (prpC), prpD and prpE gene, complete cds. E. coli k-12 MG1655,30 of the selections of 400 complete genome groups. AU068253 Rice callus rice cDNA clones C12658_9A, mRNA sequence. EST72922 OvaryII people cDNA 5 ' end, the mRNA sequence. AU068253 Rice callus rice cDNA clones C12658_9A, mRNA sequence. Starch skin pseudomonad ORF 1 DNA. Starch skin pseudomonad ORF 1 DNA. Arabidopsis thaliana chromosome IIIBAC T16O11 genome sequence, complete sequence. Arabidopsis 81kb genome sequence. Arabidopsis thaliana chromosome IIIBAC T16O11 genome sequence, complete sequence. Human chromosome 4 clone B241P19map 4q25, complete sequence. Arabidopsis 81kb genome sequence. Arabidopsis thaliana genomic dna, chromosome 3, P1 clone: MLJ15, complete sequence. Human chromosome 19 clone clone CITB-E1_2568A17, the * * * * * * that checking order, 40 non-sequential segments. Human chromosome 19 clone clone CITB-E1_2568A17, the * * * * * * that checking order, 40 non-sequential segments. The people clones NH0576F01, complete sequence.

Smegma mycobacterium Escherichia coli Caenorhabditis elegans Caenorhabditis elegans brown rat Escherichia coli salmonella typhimuriums (Salmonella typhimurium) Escherichia coli rice (Oryza sativa) people's rice starch skin pseudomonads (Pseudomonas amyloderamosa) starch skin pseudomonad arabidopsis (Arabidopsis thaliana) arabidopsis arabidopsis people arabidopsis arabidopsis people everybody

56,143   48,563   37,889     37,889     40,833     49,688   50,313       49,688   41,333   34,347   41,899   53,912     54,422   38,244   36,091   37,135   36,165   38,732   38,732   37,976     37,976     37,143

91-09-20   99-07-14   99-02-24     99-02-24     99-04-21     99-07-14   99-08-05       98-11-12   99-06-07   97-04-21   99-06-07   95-07-14     95-07-14   99-11-13   97-03-12   99-11-13   98-09-30   97-03-12   99-05-07   99-09-15     99-09-15     99-09-28

Table 4 (continuing)

rxa00683             rxa00686             rxa00700                 rxa00703       rxa00705             rxa00782         rxa00783           rxa00794

1215             927             927                 2409       1038             1005         1395           1128

GB_BA2：AE000896       GB_IN1：DMBR7A4   GB_EST35：AV163010     GB_HTG2：HSDJ137K   2   GB_HTG2：HSD1137K   2   GB_EST12：AA284399     GB_EST34：AI785570       GB_EST25：AI256147       GB_BA1：CARCG12     GB_BA1：SC7H2   GB_BA1：MTCY274   GB_BA2：REU60056   GB_GSS15：AQ604477     GB_EST11：AA224340     GB_EST5：N30648     GB_BA1：MTCY10D7   GB_BA1：MLCL373   GB_BA2：AF128399     GB_HTG2：AC008158     GB_HTG2：AC008158     GB_PR3：AC005017   GB_BA1：MTV017

10707       212734   273     190223     190223     431     454       684       2079     42655   39991   2520   505     443     291     39800   37304   2842     118792     118792     137176   67200

AE000896       AL109630   AV163010     AL049820     AL049820     AA284399     AI785570       AI256147       X14979     AL109732   Z74024   U60056   AQ604477     AA224340     N30648     Z79700   AL035500   AF128399     AC008158     AC008158     AC005017   AL021897

The base 1189349-1200055 of hot autotrophic methane bacteria complete genome group (102/148 part (section 102/148)). Drosophila melanogaster clone BACR7A4. 13 days embryo house mouses of AV163010 house mouse C57BL/6J cDNA clones 3110006J22, mRNA sequence. Human chromosome 6 clone RP1-137K2map q251-25.3, the * * * * * * that checking order, non-sequential segments. Human chromosome 6 clone RP1-137K2map q25.1-25.3, the * * * * * * that checking order, non-sequential segments. Zs57b04.r1 NCI_CGAP_GCB1 people cDNA clones IMAGE:7015515 ', mRNA sequence. Uj44d03.x1 Sugano mouse liver mlia house mouse cDNA clones IMAGE:1922789 3 ', is similar to gb:Z28407 60S ribosomal protein L 8 (people); The mRNA sequence. Ui95e12.x1 Sugano mouse liver mlia house mouse cDNA clones IMAGE:1890190 3 ', is similar to gb:Z28407 60S ribosomal protein L 8 (people); The mRNA sequence. Chloroflexus aurantiacus reaction center gene 1 and 2, streptomyces coelicolor clay 7H2. Mycobacterium tuberculosis H37Rv complete genome group; Sections 126/162. Ralstonia eutropha hydrogenlyase sample albumen (cbbBc) gene, complete cds. The human genome sperm library D human genome clone flat board of HS_2116_B1_G07_MR CIT approval=2116, row=13, OK=and N, genome summary sequence. Zr14e07.s1 Stratagene hNT neuron (#937233) people cDNA clones IMAGE:648804 3 ', mRNA sequence. Yw77b02.s1 Soares_ placenta _ 8-9 week _ 2NbHP 8-9 week people cDNA clone IMAGE:2582193 ', the mRNA sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 44/162. Mycobacterium leprae clay L373. Pseudomonas aeruginosa succinyl CoA synthetase β subunit (sucC) and succinyl CoA synthetase alpha subunit (sucD) gene, complete cds. Human chromosome 17 clone hRPK.42_F_20map 17, the * * * * * * that checking order, 14 non-sequential segments. Human chromosome 17 clone hRPK.42_F_20map 17, the * * * * * * that checking order, 14 non-sequential segments. The BAC clone GS214N13 of people 7p14-p15, complete sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 48/162.

Everybody bacillus tuberculosis typus humanus Mycobacterium leprae pseudomonas aeruginosa (Pseudomonas aeruginosa) people bacillus tuberculosis typus humanus of everybody house mouse house mouse Chloroflexus aurantiacus (Chloroflexus aurantiacus) streptomyces coelicolor A3 (2) Much's bacillus Ralstonia eutropha of hot autotrophic methane bacteria (Methanobacterium thermoautotrophicum) Drosophila melanogaster house mouse people

38,429       36,454   41,758     38,031     38,031     39,205     41,943       40,791       37,721     56,646   37,369   51,087   39,617     35,129     43,986     63,327   62,300   53,698     35,135     35,135     35,864   40,331

97-11-15       99-07-30   99-07-08     99-12-03     99-12-03     97-08-14     99-07-02       98-11-12       91-04-23     99-08-02   98-06-19   96-10-16   99-06-10     98-03-11     96-01-05     98-06-17   99-08-27   99-03-25     99-07-28     99-07-28     98-08-08   99-06-24

Table 4 (continuing)

rxa00799           rxa00800         rxa00825         rxa00871   rxa00872           rxa00879       rxa00909           rxa00913

1767           1227         1056           1077           2241       955           2118

GB_BA1：MLCB1222   GB_PR2：HS151B14         GB PL2：AF016327   GB_HTG2：HSDJ319M   7   GB_HTG2：HSDJ319M   7   GB_BA1：MTV022   GB_BA1：AB019513   GB_PL1：SCSFAARP     GB_BA1：MTY15C10   GB_BA1：MLCB2548   GB_BA2：AF169031       GB_IN1：CEF23H12   GB_HTG2：AC007263     GB_HTG2：AC007263     GB_BA1：MTV049   GB_PL2：CDU236897   GB_PL1：CAACT1A   GB_BA2：AF010496   GB_BA1：RMPHA     GB_EST16：C23528     GB_HTG2：AC007734

34714   128942         616   128208     128208     13025   4417   7008     33050   38916   1141       35564   167390     167390     40360   1827   3206   189370   7888     317     188267

AL049491   Z82188         AF016327   AL079341     AL079341     AL021925   AB019513   X68020     Z95436   AL023093   AF169031       Z74472   AC007263     AC007263     AL022021   AJ236897   X16377   AF010496   X93358     C23528     AC007734

Mycobacterium leprae clay B1222. The human DNA sequence of the clone 151B14 of chromosome 22 is contained growth hormone 3 receptors (SS3R) gene, the pseudogene that is similar to the L39 of ribosomal protein, RAC2 (the C3 botulin toxin substrate 2 (P21-RAC2) that RAS is relevant) gene EST, STS, GSS and CpG island, complete sequence. Hordeum vulgare Barperml (perml) mRNA, part cds. Human chromosome 6 clone RP1-319M7map p211-21.3, the * * * * * * that checking order, non-sequential segments. Human chromosome 6 clone RP1-319M7map p21.1-21.3, the * * * * * * that checking order, non-sequential segments. Mycobacterium tuberculosis H37Rv complete genome group; Sections 100/162. Streptomyces coelicolor alcohol dehydrogenase gene and abc transport albumen, complete cds. Saccharomyces Cerevisiae in S FA and ARP gene. Mycobacterium tuberculosis H37Rv complete genome group; Sections 154/162. Mycobacterium leprae clay B2548. Xanthomonas Oryzae Pv. Oryzae is inferred nucleotides epimerase/dehydrase gene, part cds. Caenorhabditis elegans clay F23H12, complete sequence. Human chromosome 14 clone BAC 79J20map 14q31, the * * * * * * that checking order, 5 sequential segments. Human chromosome 14 clone BAC 79J20map 14q31, the * * * * * * that checking order, 5 sequential segments. Mycobacterium tuberculosis H37Rv complete genome group; Sections 81/162. Candida dubliniensis ACT1 gene, exons 1-2. Candida albicans actin act1 gene. Pod membrane Rhodobacter strain SB1003, the portion gene group. Rhizobium meliloti pha[A, B, C, D, E, F, G] gene. C23528 Japanese flounder spleen Paralichthys olivaceus cDNA clones HB5 (2), mRNA sequence. Human chromosome 18 clone hRPK.44_O_I_map 18, the * * * * * * that checking order, 18 non-sequential segments.

The red bacterium Sinorhizobium meliloti of Mycobacterium leprae people Hordeum vulgare people bacillus tuberculosis typus humanus's streptomyces coelicolor saccharomyces cerevisiae (Saccharomyces cerevisiae) Much's bacillus Mycobacterium leprae Xanthomonas Oryzae Pv. Oryzae (Xanthomonas oryzae pv.oryzae) Caenorhabditis elegans people bacillus tuberculosis typus humanus Candida dubliniensis candida albicans (Candida albicans) pod membrane (Sinorhizobium meliloti) Paralichthys olivaceus people

61,170   37,455         41,311   36,845     36,845     63,101   41,312   36,288     39,980   39,435   46,232       34,502   35,714     35,714     36,981   38,716   36,610   51,586   48,367     41,640     34,457

99-08-27   99-06-16         97-10-01   99-11-30     99-11-30     98-06-17   98-11-13   94-11-29     98-06-17   99-08-27   99-09-14       99-10-08   99-05-24     99-05-24     98-06-19   99-09-01   93-04-10   98-05-12   99-03-12     99-09-28     99-06-05

Table 4 (continuing)

rxa00945           rxa00965   rxa00999       rxa01015     rxa01025         rxa01048         rxa01049             rxa01077         rxa01089

1095             1575       442     1119         1347         1605             1494         873

GB_HTG2：AC007734     GB_EST18：AA709478     GB_HTG4：AC010351     GB_HTG4：AC010351     GB_BA1：MTCY05A6     GB_PAT：E13660   GB_BA1：MTCY359   GB_BA1：MLCB1788   GB_BA1：MTV008   GB_BA1：MTV008   GB_BA1：SC7A1   GB_BA1：MSGB1723C   S   GB_BA1：MLCB637   GB_BA2：AF017444   GB_BA1：BSUB0013   GB_VI：HSV2HG52     GB_HTG2：AC002518     GB_HTG2：AC002518     GB_HTG2：AC002518     GB_PR3：HSDJ653C5     GB_BA1：ECU29579   GB_BA1：ECU29579   GB_GSS8：AQ044021

188267     406     220710     220710     38631     1916   36021   39228   63033   63033   32039   38477     44882   3067   218470   154746     131855     131855     131855     85237     72221   72221   387

AC007734     AA709478     AC010351     AC010351     Z96072     E13660   Z83859   AL008609   AL021246   AL021246   AL034447   L78825     Z99263   AF017444   Z99116   Z86099     AC002518     AC002518     AC002518     AL049743     U29579   U29579   AQ044021

Human chromosome 18 clone hRPK.44_O_1map 18, the * * * * * * that checking order, 18 non-sequential segments. Vv34a05.r1 Stratagene house mouse heart (#937316) house mouse cDNA clones IMAGE:12242725 ', mRNA sequence. Human chromosome 5 clone CITB-H1_2022B6, the * * * * * * that checking order, 68 non-sequential segments. Human chromosome 5 clone CITB-H1_2022B6, the * * * * * * that checking order, 68 non-sequential segments. Mycobacterium tuberculosis H37Rv complete genome group; Sections 120/162. The gDNA of coding 6-phosphoric acid grape saccharic acid dehydrogenase. Mycobacterium tuberculosis H37Rv complete genome group; Sections 84/162. Mycobacterium leprae clay B1788. Mycobacterium tuberculosis H37Rv complete genome group; Sections 108/162. Mycobacterium tuberculosis H37Rv complete genome group; Sections 108/162. Streptomyces coelicolor clay 7A1. Mycobacterium leprae clay B1723DNA sequence. Mycobacterium leprae clay B637. Sinorhizobium meliloti NADP dependence malate dehydrogenase (tme) gene, complete cds. Bacillus subtilis complete genome group (13/21 part): 2395261-2613730. Herpes simplex types 2 virus (bacterial strain HG52), the complete genome group. Human chromosome X clones bWXD20, the * * * * * * that checking order, 11 non-sequential segments. Human chromosome X clones bWXD20, the * * * * * * that checking order, 11 non-sequential segments. Human chromosome X clones bWXD20, the * * * * * * that checking order, 11 non-sequential segments. The human DNA sequence of the clone 653C5 of chromosome 1p21.3-22.3 is contained CA repetitive sequence (D1S435), STS and GSS, complete sequence. The e. coli k-12 genome, about 61-62 minute. The e. coli k-12 genome, about 61-62 minute. CIT-HSP-2318C18.TRCIT-HSP human genome clone 2318C18, genome summary sequence.

People house mouse people bacillus tuberculosis typus humanus Corynebacterium glutamicum Much's bacillus Mycobacterium leprae Much's bacillus Much's bacillus streptomyces coelicolor Mycobacterium leprae Mycobacterium leprae Sinorhizobium meliloti bacillus subtilis (Bacillus subtilis) herpes simplex types 2 virus (human herpesvirus 2) everybody everybody esherichia coli people

34,457     42,065     36,448     36,448     36,218     98,349   38,520   64,355   39,860   39,120   55,287   56,847     56,676   53,660   37,255   38,081     35,647     35,647     26,180     36,462     41,808   36,130   36,528

99-06-05     97-12-24     99-10-31     99-10-31     98-06-17     98-06-24   98-06-17   99-08-27   98-06-17   98-06-17   98-12-15   96-06-15     98-09-17   97-11-02   97-11-26   98-12-04     97-09-02     97-09-02     97-09-02     99-11-23     95-07-01   95-07-01   98-07-14

Table 4 (continuing)

rxa01093         rxa01099         rxa01111       rxa01130       rxa0193           rxa01194           rxa01200

1554         948         541       687       1572           495

GB_GSS8：AQ042907     GB_GSS8：AQ044021     GB_BA1：CORPYK1   GB_BA1：MTCY01B2   GB_BA1：MIU65430     GB_BA2：AF045998   GB_BA2：AF051846     GB_GSS1：FR0005503   GB_PR3：AC004063   GB_PR3：HS1178121   GB_HTG3：AC009301   GB_HTG3：AC009444   GB_HTG3：AC009444   GB_IN1：DMC66A1   GB_BA1：CGASO19   EM_PAT：E09634       GB_BA1：MLU15186   EM_PAT：E09634       GB_BA1：CGASO19   GB_VI：HEPCRE4B

392     387     2795   35938   1439     780   738     619   177014   62268   163369   164587   164587   34127   1452   1452       36241   1452       1452   414

AQ042907     AQ044021     L27126   Z95554   U65430     AF045998   AF051846     Z89313   AC004063   AL109852   AC009301   AC009444   AC009444   AL031227   X76875   E09634       U15186   E09634       X76875   X60570

CIT-HSP-2318D17.TR CIT-HSP human genome clone 2318D17, genome summary sequence. CIT-HSP-2318C18.TR CIT-HSP human genome clone 2318C18, genome summary sequence. Rod bacillus pyruvate kinase gene, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 72/162. Mycobacterium intracellulare pyruvate kinase (pykF) gene, complete cds. Corynebacterium glutamicum inositol monophosphate phosphatase (impA) gene, complete cds. Corynebacterium glutamicum ribose phosphate formimino group-5-amino-ribose 1-phosphate-4-Imidazole carboxamide isomerase (hisA) gene, complete cds. F.rubripes GSS sequence, clone 079B16aE8, genome summary sequence. Human chromosome 4 clone B3218, complete sequence. The human DNA sequence of the clone RP5-1178121 of chromosome x, complete sequence. The people clones NH0062F14, the * * * * * * that checking order, 5 non-sequential segments. The people clones 1_O_3, the * * * * * * that checking order, 8 non-sequential segments. The people clones 1_O_3, the * * * * * * that checking order, 8 non-sequential segments. Drosophila melanogaster clay 66A1. Corynebacterium glutamicum (ASO 19) ATP enzyme beta subunit gene. Brevibacterium flavum UncD gene, its gene outcome participates in. Mycobacterium leprae clay L471. Brevibacterium flavum UncD gene, its gene outcome participates in. Corynebacterium glutamicum (ASO 19) ATP enzyme beta subunit gene. HCV is inferred the geneome RNA (RE4B separator) of envelope protein.

Everybody Drosophila melanogaster Corynebacterium glutamicum Corynebacterium glutamicum Mycobacterium leprae Corynebacterium glutamicum Corynebacterium glutamicum HCV of everybody people of everybody Corynebacterium glutamicum Much's bacillus Mycobacterium intracellulare (Mycobacterium intracellulare) Corynebacterium glutamicum Corynebacterium glutamicum Fugu rubripes

35,969     44,545     100,00   63,771   67,061     99,615   100,000     37,785   35,835   37,873   37,240   38,416   38,416   38,416   99,931   99,242       39,153   100,000       100,000   36,769

98-07-14     98-07-14     94-12-07   98-06-17   96-12-23     98-02-19   98-03-12     97-03-01   98-07-10   99-12-01   99-08-13   99-08-22   99-08-22   98-10-05   94-10-27   97-10-   07(Rel.52   Created)   95-03-09   97-10-   07(Rel.52   Created)   94-10-27   92-04-05

Table 4 (continuing)

rxa01201       rxa01202         rxa01204       rxa01216           rxa01225                 rxa01227         rxa01242

1764       1098         933       1124           1563                 444         900

GB_BA1：SLATPSYNA   GB_BA1：MTCY373   GB_BA1：MLU15186   GB_BA1：SLATPSYNA   GB_BA1：SLATPSYNA   GB_BA1：MCSQSSHC     GB_PL1：AP000423   GB_HTG6：AC009762   GB_HTG6：AC009762   GB_BA1：MTCY10G2   GB_BA2：AF017435     GB_BA1：CCRFLBDB   A   GB_BA2：AF058302     GB_HTG3：AC007301       GB_HTG3：AC007301       GB_BA1：SERFDXA     GB_BA1：MTV005   GB_BA1：MSGY348   GB_PR3：AC005697   GB_HTG3：AC010722   GB_HTG3：AC010722

8560   35516   36241   8560   8560   5538     154478   164070   164070   38970   4301     4424     25306     165741       16541       3869     37840   40056   174503   160723   160723

Z22606   Z73419   U15186   Z22606   Z22606   Y09978     AP000423   AC009762   AC009762   Z92539   AF017435     M69228     AF058302     AC007301       AC007301       M61119     AL010186   AD000020   AC005697   AC010722   AC010722

S.lividansi albumen and atp synthase gene. Mycobacterium tuberculosis H37Rv complete genome group; Sections 57/162. Mycobacterium leprae clay L417. S.lividansi albumen and atp synthase gene. S.lividansi albumen and atp synthase gene. Capsule methyl coccus orfx, orfy, orfz, sqs and shc gene. The arabidopsis chloroplast genomic dna, complete sequence, bacterial strain: Columbia people clones RP11-114I16, the * * * * * * that checking order, 39 non-sequential segments. The people clones RP11-114I16, the * * * * * * that checking order, 39 non-sequential segments. Mycobacterium tuberculosis H37Rv complete genome group; Sections 47/162. Turn round demethylation bacillus methanol oxidation gene, glmU sample gene, part cds and orfL2, orfL1, orfR gene, complete cds. Crescent handle bacillus flagellin gene promoter region. Rose yellow streptomycete frenolicin biosynthesis gene family, complete sequence. Drosophila melanogaster chromosome 2 clone BACR04B09 (D576) RPCI-9804.B.9map 43E12-44F1 bacterial strain y; Cn bw sp, the * * * * * * that checking order, 150 non-sequential segments. Drosophila melanogaster chromosome 2 clone BACR04B09 (D576) RPCI-9804.B.9map 43E12-44F1 bacterial strain y; Cn bw sp, the * * * * * * that checking order, 150 non-sequential segments. Red saccharopolyspora ferrodoxins (fdxA) gene, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 51/162. The sequence of Much's bacillus clone y348. Human chromosome 17, clone hRPK.138_P_22, complete sequence. The people clones NH0122L09, the * * * * * * that checking order, 2 non-sequential segments. Enter to clone NH0122L09, the * * * * * * that checking order, 2 non-sequential segments.

Streptomyces lividans Much's bacillus Mycobacterium leprae Streptomyces lividans Streptomyces lividans pod membrane methyl coccus (Methylococcus capsulatus) Chloroplast arabidopsis people bacillus tuberculosis typus humanus turn round demethylation bacillus (Methylobacterium exorquens) crescent handle bacillus (Caulobacter crescentus) rose yellow streptomycete (Streptomyces roseofulvus) Drosophila melanogaster Drosophila melanogaster red saccharopolyspora (Saccharopolyspora erythraea) Much's bacillus Much's bacillus people everybody

66,269   65,437   39,302   57,087   38,298   37,626     38,395   35,459   36,117   39,064   42,671     41,054     36,205     39,922       39,922       64,908     62,838   61,712   35,373   39,863   39,863

95-05-01   98-06-17   95-03-09   95-05-01   95-05-01   98-05-26     99-09-15   99-12-04   99-12-04   98-06-17   98-03-10     93-04-26     98-06-02     99-08-17       99-08-17       96-03-13     98-06-17   96-12-10   98-10-09   99-09-25   99-09-25

Table 4 (continuing)

rxa01243         rxa01259           rxa01262                   rxa01311       rxa01312       rxa01325       rxa01332

1083         981           1284                   870       2142       795       576

GB_GSS10：AQ255057     GB_IN1：CEK05D4   GB_IN1：CEK05D4   GB_BA1：CGLPD   GB_HTG4：AC010567     GB_HTG4：AC010567     GB_BA2：AF172324             GB_BA2：ECU78086     GB_BA1：D90841   GB_PR3：AC004103   GB_HTG3：AC007383   GB_HTG3：AC007383   GB_BA2；AE000487   GB_BA1：MTV016   GB_BA1：U00022   GB_HTG4：AC009245   GB_HTG4：AC009245   GB_HTG4：AC009245   GB_HTG6：AC007186     GB：HTG6：AC007147

583     19000   19000   1800   143287     143287     14263             4759     20226   144368   215529   215529   13889   53662   36411   215767   215767   215767   225851     202291

AQ255057     Z92804   Z92804   Y16642   AC010567     AC010567     AF172324             U78086     D90841   AC004103   AC007383   AC007383   AE000487   AL021841   U00022   AC009245   AC009245   AC009245   AC007186     AC007147

Mgxb0008N01r CUGI Rice Blast BAC library Magnaporthe grisea genomic clone mgxb0008N01r, genome summary sequence. Caenorhabditis elegans clay K05D4, complete sequence. Caenorhabditis elegans clay K05D4, complete sequence. Corynebacterium glutamicum 1pd gene, complete CDS. Drosophila melanogaster chromosome 3L/69C1 clone RPCI98-1 1N6, the * * * * * * that checking order, 70 non-sequential segments. Drosophila melanogaster chromosome 3L/69C1 clones RPCI98-11N6, the * * * * * * that checking order, 70 non-sequential segments. Escherichia coli GalF (galF) gene, part cds; The transport protein Wzx of O-antigen recurring unit (wzx), WbnA (wbnA), O-antigen polymerase Wzy (wzy), WbnB (wbnB), WbnC (wbnC), WbnD (wbnD), WbnE (wbnE), UDP-Glc-4-epimerase GalE (galE), 6-Phosphogluconic dehydrogenase Gnd (gnd), UDP-Glc-6-dehydrogenase Ugd (ugd) and WbnF (wbnF) gene, complete cds; And chain length determines albumen Wzz (wzz) gene, part cds. Escherichia coli supposition UDP-Glc dehydrogenase (ugd) and O-chain length are regulated albumen (wzz) gene, complete cds. Genome of E.coli DNA, Kohara clone #351 (45.1-45.5 minute) people Xp22BACGS-619J3 (genome system people BAC library) complete sequence. The people clones NH0310K15, the * * * * * * that checking order, 4 non-sequential segments. The people clones NH0310K15, the * * * * * * that checking order, 4 non-sequential segments. 377/400 part of e. coli k-12 MG1655 complete genome group. Mycobacterium tuberculosis H37Rv complete genome group; Sections 143/162. Mycobacterium leprae clay L308. Human chromosome 7, the * * * * * * that checking order, 24 non-sequential segments. Human chromosome 7, the * * * * * * that checking order, 24 non-sequential segments. Human chromosome 7, the * * * * * * that checking order, 24 non-sequential segments. Drosophila melanogaster chromosome 2 clone BACR03D06 (D569) RPCI-9803.D.6map 32A-32A bacterial strain y; Cn bw sp, the * * * * * * that checking order, 91 non-sequential segments. Drosophila melanogaster chromosome 2 clone BACR19N18 (D572) RPCI-9819.N.18map 32A-32A bacterial strain y; Cn bw sp, the * * * * * * that checking order, 22 non-sequential segments.

Everybody Drosophila melanogaster Drosophila melanogaster of everybody people Escherichia coli Much's bacillus Mycobacterium leprae people of Magnaporthe grisea Caenorhabditis elegans Caenorhabditis elegans Corynebacterium glutamicum Drosophila melanogaster Drosophila melanogaster esherichia coli Escherichia coli

38,722     35,448   35,694   100,000   37,178     37.178     59,719             59,735     37,904   37,340   36,385   36,385   39,494   46,252   46,368   36,016   36,016   39,618   35,366     35,366

98-10-23     98-11-23   98-11-23   99-02-01   99-10-16     99-10-16     99-10-29             97-11-05     97-03-21   98-04-18   99-09-25   99-09-25   98-11-12   99-06-23   94-03-01   99-11-02   99-11-02   99-11-02   99-12-07     99-12-07

Table 4 (continuing)

rxa01350             rxa01365             rxa01369         rxa01377           rxa01392             rxa01436         rxa001468

1107             1497             1305         1209           1200             1314         948

GB_HTG3：AC010207   GB_BA2：AF109682     GB_HTG2：AC006759     GB_HTG2：AC006759     GB_BA1：MTY20B11   GB_BA1：XANXANAB       GB_GSS10：AQ194038     GB_BA1：MTY20B11   GB_GSS3：B10037   GB_GSS3：B09549     GB_BA1：MTCY71   GB_HTG5：AC007547     GB_HTG5：AC007547     GB_BA2：AF072709       GB_BA1：CGLYSEG   GB_PR4：AC005906     GB_BA1：CGPTAACK   A   GB_BA1：D90861   GB_PAT：E02087   GB_GSS1：HPU60627   GB_EST31：AI701691

207890   990     103725     103725     36330   3410       697     36330   974   1097     42729   262181     262181     8366       2374   185952     3657     14839   1200   280   349

AC010207   AF109682     AC006759     AC006759     Z95121   M83231       AQ194038     Z95121   B10037   B09549     Z92771   AC007547     AC007547     AF072709       X96471   AC005906     X89084     D90861   E02087   U60627   AI701691

The people clones RPCI11-375I20, the * * * * * * that checking order, 25 non-sequential segments. Arctic water spirillum malic dehydrogenase (MDH) gene, complete cds. Caenorhabditis elegans clones Y40G12, the * * * * * * that checking order, 8 non-sequential segments. Caenorhabditis elegans clones Y40G12, the * * * * * * that checking order, 8 non-sequential segments. Mycobacterium tuberculosis H37Rv complete genome group; Sections 139/162. Xanthomonas campestris phosphoglucomutase and mannose-phosphate mutase (xanA) and phosphomannose isomerase and GDP-mannose pyrophosphorylase (xanB) gene, complete cds. RPCI11-47D24.TJ RPCI-11 human genome clone RPCI-11-47D24, genome summary sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 139/162. T27A19-T7 TAMU arabidopsis gene group clone T27A19, genome summary sequence. T27A19-T7.1 TAMU arabidopsis gene group clone T21A19, genome summary sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 141/162. The people clones RP11-252O18, WORKING DRAFT sequence, 121 non-sequential segments. The people clones RP11-252O18, WORKING DRAFT sequence, 121 non-sequential segments. Streptomyces lividans amplification element AUD4: the supposition transcript regutation protein, suppose ferrodoxins, suppose cytochrome P450 reductase and supposition oxide-reductase gene, complete cds; And unknown gene. Corynebacterium glutamicum lysE and lysG gene. People 12p13.3 BAC RPCI11-429 A20 (Roswell Park ICR people BAC library) complete sequence. Corynebacterium glutamicum pta gene and ackA gene. Genome of E.coli DNA, Kohara clones #405 (52.0-5.23 minute). The DNA of coding Escherichia coli acetokinase albumen. Helicobacter pylori feoB sample dna sequence dna, genome summary sequence. We81c04.xl Soares_NFL_T_GBC_S1 people cDNA clones IMAGE:2347494 3 ', is similar to gb:L19686_ma1 macrophage migration inhibitory factor (people); The mRNA sequence.

Excellent bacillus esherichia coli helicobacter pylori (Helicobacter pylori) people of everybody Streptomyces lividans Corynebacterium glutamicum human glutamic acid of people arctic water spirillum (Aquaspirillum arcticum) Caenorhabditis elegans Caenorhabditis elegans Much's bacillus xanthomonas campestris (Xanthomonas campestris) bacillus tuberculosis typus humanus's arabidopsis arabidopsis Much's bacillus

34,821   58,487     37,963     37,963     38,011   47,726       36,599     36,940   35,284   38,324     39,778   32,658     38,395     55,221       100,000   36,756     100,000     53,041   54,461   39,286   39,412

99-09-16   99-10-19     99-02-25     99-02-25     98-06-17   93-04-26       99-04-20     98-06-17   97-05-14   97-05-14     99-02-10   99-11-16     99-11-16     98-07-08       97-02-24   99-01-30     99-03-23     97-05-29   97-09-29   97-04-09   99-06-03

Table 4 (continuing)

rxa01478       rxa01482       rxa01534   rxa01535         rxa01550         rxa01562   rxa01569         rxa01570

1959       1998         1530         1635           1482         978

GB_EST15：AA480256     GB_BA1：SCI51   GB_BA1：SCE36   GB_BA1：CGU43535   GB_BA1：SC6G4   GB_BA1：U00020   GB_BA1：MTCY77     GB_BA1：MLCB1222   GB_BA1：MTV017   GB_BA1：PAU72494     GB_BA1：D90907     GB_IN2：AF073177   GB_IN2：AF073179     GB_BA1：D78182   GB_BA2：AF079139     GB_BA2：AF087022   GB_BA1：MTCY63   GB_BA2：AF097519

389     40745   12581   2531   41055   36947   22255     34714   67200   4368     132419     9534   3159     7836   4342     1470   38900   4594

AA480256     AL109848   AL049763   U43535   AL031317   U00020   Z95389     AL049491   AL021897   U72494     D90907     AF073177   AF073179     D78182   AF079139     AF087022   Z96800   AF097519

Ne3 1f04.sl NCI_CGAP_Co3 people cDNA clones IMAGE:8989753 ', is similar to gb:L19686ma1 macrophage migration inhibitory factor (people); The mRNA sequence. Streptomyces coelicolor clay 151. Streptomyces coelicolor clay E36. Corynebacterium glutamicum multi-drug resistance albumen (cmr) gene, complete cds. Streptomyces coelicolor clay 6G4. Mycobacterium leprae clay B229. Mycobacterium tuberculosis H37Rv complete genome group; Sections 146/162. Mycobacterium leprae clay B1222. Mycobacterium tuberculosis H37Rv complete genome group; Sections 48/162. Pseudomonas aeruginosa fumarase (fumC) and the super oxygen thing of Mn enzyme mutase (sodA) gene, complete cds. Collection born of the same parents cyanobacteria belongs to bacterium PCC6803 complete genome group, 9/27,1056467-1188885. Drosophila melanogaster glycogen phosphorylase (GlyP) gene, complete cds. Drosophila melanogaster glycogen phosphorylase (Glpl) mRNA, complete cds. The Streptococcus mutans DNA of dTDP-rhamnose route of synthesis, complete cds. The auspicious interior slide fastener mould pikCD operon of entrusting, complete sequence. Auspicious interior slide fastener fungal cell cytochrome p 450 monooxygenase (picK) gene of entrusting, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 16/162. Friedlanders bacillus dTDP-D-glucose 4,6 dehydratases (rmlB), Cori ester thymidine transferase (rmlA), dTDP-4-ketone-L-rhamnose reductase (rmlD), dTDP-4-ketone-6-DDG 3,5-epimerase (mlC) and rhamnosyltransferase (wbbL) gene, complete cds.

People's streptomyces coelicolor A3 (2) streptomyces coelicolor Corynebacterium glutamicum streptomyces coelicolor Mycobacterium leprae Much's bacillus Mycobacterium leprae Much's bacillus pseudomonas aeruginosa collection born of the same parents cyanobacteria belongs to bacterium (Synechocystis sp. ) the auspicious interior slide fastener mould Much's bacillus Friedlanders bacillus of Drosophila melanogaster Drosophila melanogaster Streptococcus mutans (Streptococcus mutans) the auspicious interior slide fastener mould of committee (Streptomyces venezuelae) committee

39,574     54,141   38,126   41,852   62,149   38,303   38,179     66,208   38,553   52,690     56,487     55,100   56,708     44,050   38,587     38,621   59,035   59,714

97-08-14     99-08-16   99-05-05   97-04-09   98-08-20   94-03-01   98-06-18     99-08-27   99-06-24   96-10-23     99-02-07     99-07-01   99-04-27     99-02-05   98-10-28     98-10-15   98-06-17   98-11-04

Table 4 (continuing)

rxa01571         rxa01572         rxa01606               rxa01626       rxa01632                 rxa01633

723         615         2799               468       1128                 1206

GB_BA2：NGOCPSPS       GB_BA1：AB011413     GB_BA1：AB011413     GB_BA1：AB011413     GB_BA1：AB011413     GB_VI：CFU72240       GB_GSS10：AQ213248     GB GSS8：AQ070145     GB_PR4：AF152510   GB_PR4：AF152323   GB_PR4：AF152509   GB_HTG4：AC006590       GB_HTG4：AC006590       GB_GSS8：B99182     GB_BA1：BSUB0009   GB_BA1：BSUB0009   GB_HTG2：AC006247

8905       12070     12070     12070     12070     4783       408     285     2490   4605   2712   127171       127171       415     208780   208780   174368

L09189       AB011413     AB011413     AB011413     AB011413     U72240       AQ213248     AQ070145     AF152510   AF152323   AF152509   AC006590       AC006590       B99182     Z99112   Z99112   AC006247

Meningitis naphthalene Se Shi coccus dTDP-D-glucose 4,6-dehydratase (rfbB), Cori ester thymidine transferase (rfbA) and rfbC gene, complete cds and UPD-glucose-4-epimerase (galE) pseudogene. The gene of streptococcus cinereus Orf2, Orf3, Orf4, Orf5, AfsA, Orf8, part and complete cds. The gene of streptococcus cinereus Orf2, Orf3, Orf4, Orf5, AfsA, Orf8, part and complete cds. The gene of streptococcus cinereus Orf2, Orf3, Orf4, Orf5, AfsA, Orf8, part and complete cds. The gene of streptococcus cinereus Orf2, Orf3, Orf4, Orf5, AfsA, Orf8, part and complete cds. Choristoneura fumiferana nuclear polyhedrosis virus ETM albumen homologue, 79kDa albumen homologue, 15kDa albumen homologue and GTA albumen homologue gene, complete cds. The human genome sperm library D human genome clone flat board of HS_3249_B1_A02_MR CIT approval=3249 row=3 row=B, genome summary sequence. The human genome sperm library D human genome clone flat board of HS_3207_B1_H02_MR CIT approval=3207 row=3 row=P, genome summary sequence. The short type albumen of people protocadherin γ A3 (variable region sequences of PCDH-γ-A3), complete cds. People protocadherin γ A3 (mRNA of PCDH-γ-A3), complete cds. People PCDH-γ-A3 gene, aberrant splicing, mRNA sequence. Drosophila melanogaster chromosome 2 clone BACR13N02 (D543) RPCI-9813.N.2map 36E-36E bacterial strain y; Cn bw sp, the * * * * * * that checking order, 101 non-sequential segments. Drosophila melanogaster chromosome 2 clone BACR13N02 (D543) RPCI-98 13.N.2map 36E-36E bacterial strain y; Cn bw sp, the * * * * * * that checking order, 101 non-sequential segments. With CIT-HSP-2280I13.TR CIT-HSP human genome clone 2280I13, genome summary sequence. Bacillus subtilis complete genome group (9/21 part): 1598421-1807200. Bacillus subtilis complete genome group (9/21 part): 1598421-1807200. Drosophila melanogaster chromosome 2 clone BACR48I10 (D505) RPCI-98 48.1.10map 49E6-49F8 bacterial strain y; Cn bw sp, the * * * * * * that checking order, 17 non-sequential segments.

Everybody Drosophila melanogaster Drosophila melanogaster people bacillus subtilis bacillus subtilis Drosophila melanogaster of everybody people of meningitis naphthalene Se Shi coccus (Neisseria meningitidis) streptococcus cinereus (Streptomyces griseus) streptococcus cinereus streptococcus cinereus streptococcus cinereus choristoneura fumiferana nuclear polyhedrosis virus (Choristoneura fumiferana nucleopolyhedrovirus)

58,384       57,500     35,655     57,843     38,119     37,115       34,559     40,351     34,298   34,298   34,298   33,812       33,812       36,111     36,591   34,941   37,037

96-07-30       98-08-07     98-08-07     98-08-07     98-08-07     99-01-29       98-09-18     98-08-05     99-07-14   99-07-22   99-07-14   99-10-19       99-10-19       98-06-26     97-11-26   97-11-26   99-08-02

Table 4 (continuing)

rxa001695       rxa01702       rxa01743           rxa01744         rxa01745       rxa01758         rxa01814       rxa01851

1623       1155       901           1662         836       1140         1785       1809

GB_BA1：CGA224946   GB_BA1：MTCY24A1   GB_IN1：DMU15974   GB_BA1：CGFDA   GB_BA1：MTY13E10   GB_BA1：MLCB4   GB_IN2：CELC27H5   GB_EST24：AI167112     GB_GSS9：AQ102635     GB_BA1：MTCY01B2   GB_GSS1：AF009226     GB_BA1：SCD78   GB_BA1：MTCY190   GB_BA1：MLCB22   GB_BA2：AE000175   GB_PR3：HS57G9     GB_PL2：YSCH9666   GB-PL2：YSCH9986   GB_BA1：ABCCELB   GB_BA1：MTCY22D7   GB_BA1：MTCY22D7   GB_GSS9：AQ142579     GB_IN2：AC005889     GB_GSS1：AG008814

2408   20270   2994   3371   35019   36310   35840   579     347     35938   665     36224   34150   40281   15067   113872     39057   41664   2058   31859   31859   529     108924     637

AJ224946   Z95207   U15974   X17313   Z95324   AL023514   U14635   AI167112     AQ102635     Z95554   AF009226     AL034355   Z70283   Z98741   AE000175   Z95116     U10397   U00027   L24077   Z83866   Z83866   AQ142579     AC005889     AG008814

Corynebacterium glutamicum L MALIC ACID: quinone oxidoreductase DNA. Mycobacterium tuberculosis H37Rv complete genome group; Sections 124/162. Drosophila melanogaster kinesin sample albumen (klp68d) mRNA, complete cds. Corynebacterium glutamicum fructosediphosphate aldolase fda gene (EC 4.1.2.13). Mycobacterium tuberculosis H37Rv complete genome group; Sections 18/162. Mycobacterium leprae clay B4. Caenorhabditis elegans clay C27H5. Xylem.est.878 willow xylem λ ZAPII library Populus balsamifera subsp. trichocarpa cDNA 5 ', the mRNA sequence. The human genome sperm library D human genome clone flat board of HS_3048_B1_F08_MF CIT approval=3048 row=15 row=L, genome summary sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 72/162. Much's bacillus cytochromes D oxidase subunit I (appC) gene, partial sequence, genome summary sequence. Streptomyces coelicolor clay D78. Mycobacterium tuberculosis H37Rv complete genome group; Sections 98/162. Mycobacterium leprae clay B22. 65/400 part of e. coli k12 MG1655 complete genome group. The human DNA sequence of the BAC 57G9 of chromosome 22 q12.1 is contained EST, CA repetitive sequence, GSS. Saccharomyces cerevisiae chromosome VIII clay 9666. Saccharomyces cerevisiae chromosome VIII clay 9986. Acetobacter xylinum phosphoglucomutase (celB) gene, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 133/162. Mycobacterium tuberculosis H37Rv complete genome group; Sections 133/162. The human genome sperm library D human genome clone flat board of HS_2222_B1_H03_MR CIT approval=2222 row=5 row=P, genome summary sequence. Drosophila melanogaster, chromosome 2L, 30A3-30A6 district, P1 clone DS06958 and DS03097, complete sequence. The human gene group DNA, 21q district, clone: B137B7BB68, genome summary sequence.

Corynebacterium glutamicum Much's bacillus Drosophila melanogaster Corynebacterium glutamicum Much's bacillus Mycobacterium leprae Caenorhabditis elegans Populus balsamifera subsp. trichocarpa bacillus tuberculosis typus humanus's Much's bacillus streptomyces coelicolor Much's bacillus Mycobacterium leprae Escherichia coli people's saccharomyces cerevisiae saccharomyces cerevisiae acetobacter xylinums (Acetobacter xylinus) Much's bacillus Much's bacillus people Drosophila melanogaster people

100,000   38,626   36,783   99,913   38,786   38,238   35,334   39,222     40,653     36,650   63,438     53,088   62,081   61,364   52,323   39,209     40,021   34,375   62,173   39,749   40,034   38,068     36,557     35,316

98-08-11   98-06-17   95-07-18   93-09-12   98-06-17   99-08-27   95-07-13   98-12-03     98-08-27     98-06-17   97-07-31     98-11-26   98-06-17   97-08-22   98-11-12   99-11-23     97-09-05   97-08-29   94-09-21   98-06-17   98-06-17   98-09-24     98-10-30     99-02-07

Table 4 (continuing)

rxa01859                     rxa01865       rxa01882           rxa01884       rxa01886           rxa01887             rxa01888

1050                     438       1113           1913       897           1134             658

GB_BA2：AF183408         GB_HTG5：AC008031     GB_BA2：AF183408         GB_BA1：SERFDXA   GB_BA1：MTV005   GB_BA1：MSGY348   GB_PR1：HUMADRA2   C   GB_PR4：HSU72648   GB_GSS3：B42200     GB_BA1：MTCY48   GB_BA1：SCO001206   GB_BA1：D90908   GB_GSS9：AQ116291     GB_BA2：AE001721   GB_EST16：AA567090     GB_HTG6：AC008147   GB_HTG6：AC008147   GB_BA2：ALW243431         GB_HTG2：AC008197

63626         158889     63626         3869   37840   40056   1491     4850   387     35377   9184   122349   572     17632   596     303147   303147   26953         125235

AF183408         AC008031     AF183408         M61119   AL010186   AD000020   J03853     U72648   B42200     Z74020   AJ001206   D90908   AQ116291     AE001721   AA567090     AC008147   AC008147   AJ243431         AC008197

Verdigris note of the ancient Chinese capsule cyanobacteria dna polymerase i II β subunit (dnaN) gene, part cds; Microcystin synthase gene bunch, complete sequence; Uma1 (uma1), Uma2 (uma2), Uma3 (uma3), Uma4 (uma4) and Uma5 (uma5) gene, complete cds and Uma6 (uma6) gene, part cds. Bu Lusishi trypanosome chromosome II clones RPCI93-25N14, the * * * * * * that checking order, 2 non-sequential segments. Microcystis aeruginosa dna polymerase i II β subunit (dnaN) gene, part cds; Microcystin synthase gene bunch, complete sequence; Uma1 (uma1), Uma2 (uma2), Uma3 (uma3), Uma4 (uma4) and Uma5 (uma5) gene, complete cds and Uma6 (uma6) gene, part cds. Red saccharopolyspora ferrodoxins (fdxA) gene, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 51/162. The sequence of Much's bacillus clone y348. People's kidney α-2-adrenergic receptor gene, complete cds. People's kidney α 2-C4-adrenergic receptor gene, complete cds. Human genome sperm library C human genome clone flat board=CT 777 row=5 row=B of HS_1055_B1_A03_MR.abi CIT approval, genome summary sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 69/162. Streptomyces coelicolor A3 (2), glycogen metabolism bunch II. Collection born of the same parents cyanobacteria belongs to bacterium PCC6803 complete genome group, 10/27,1188886-1311234. RPCI11-49P6.TK.1 RPCI-11 human genome clone RPCI-11-49P6, genome summary sequence. 33/136 part of Thermotoga maritima complete genome group. GM01044.5prime GM Drosophila melanogaster ovary BlueScript Drosophila melanogaster cDNA clone GM01044 5prime, the mRNA sequence. The people clones RP3-405J10, the * * * * * * that checking order, 102 non-sequential segments. The people clones RP3-405J10, the * * * * * * that checking order, 102 non-sequential segments. Acinebobacter lwoffi wzc, wzb, wza, weeA, weeB, wceC, wzx, wzy, weeD, weeE, weeF, weeG, weeH, weeI, weeJ, weeK, glaU, ugd, pgi, galE, pgm (part) and mip (part) gene (1 bunch of emulsion adhesive biosynthesis gene), bacterial strain RAG-1. Drosophila melanogaster chromosome 3 clone BACR02L12 (D753) RPCI-9802.L.12map 94B-94C bacterial strain y; The cnbw sp*** * * * that checking order, 113 non-sequential segments.

Verdigris note of the ancient Chinese capsule cyanobacteria (Mycrocystis aeruginosa) Bu Lusishi trypanosome (Trypanosoma brucei) Microcystis aeruginosa red saccharopolyspora Much's bacillus Much's bacillus people bacillus tuberculosis typus humanus streptomyces coelicolor collection born of the same parents cyanobacteria belongs to the bacterium sea of faces everybody Acinebobacter lwoffi (Acinetobacter lwoffii) Drosophila melanogaster of thermobacillus (Thermotoga maritima) Drosophila melanogaster of dwelling

36,364         35,334     36,529         59,862   61,949   59,908   36,899     36,899   34,805     37,892   40,413   47,792   43,231     39,306   42,807     36,417   37,667   39,640         32,969

99-10-03         99-11-15     99-10-03         96-03-13   98-06-17   96-12-10   93-04-27     98-11-23   97-10-18     98-06-17   99-03-29   99-02-07   99-04-20     99-06-02   98-11-28     99-12-03   99-12-03   99-10-01         99-08-02

Table 4 (continuing)

rxa01891       rxa01895       rxa01901       rxa01927       rxa01952           Rxa01989         rxa02026         rxa02028

887       1051       1383       1503       1836           630         720         526

GB_HTG2：AC008197       GB_EST36：A1881527     GB_VI：HIV232971   GB_PL1：AFCHSE   GB_PR3：AF064858   GB_BA1：CGL238250   GB_BA2：AF038423   GB_BA1：MTCY359   GB_BA1：MSG38COS   GB_BA1：SCE63   GB_PR3：AF093117   GB_BA1：CGPAN   GB_BA1：ASXYLA   GB_HTG3：AC009500   GB_BA2：AE000739   GB_EST28：AI519629     GB_EST21：AA949396     GB_BA1：BSPGIA   GB_BA1：BSUB0017   GB_BA2：AF132127     GB_BA1：SXSCRBA     GB_BA1：BSUB0020   GB_BA1：BSGENR   GB_BA1：MTCI237

12535       598     621   6158   193387   1593   1376   36021   37114   37200   147216   2164   1905   176060   13335   612     767     1822   217420   8452     3161     212150   97015   27030

AC008197       A1881527     AJ232971   Y09542   AF064858   AJ238250   AF038423   Z83859   L01095   AL035640   AF093117   X96580   X59466   AC009500   AE000739   AI519629     AA949396     X16639   Z99120   AF132127     X67744     Z99123   X73124   Z94752

Drosophila melanogaster chromosome 3 clone BACR02L12 (D753) RPCI-9802.L.12map 94B-94C bacterial strain y; Cn bw sp, the * * * * * * that checking order, 113 non-sequential segments. 606070C09.y1 606-Schmidt lab maize cDNA and fringe are organized cDNA library, the mRNA sequence. 1 type human immunodeficiency virus C hypotype nef gene, patient MP83. Aspergillus fumigatus chsE gene. Human chromosome 21q22.3BAC 28F9, complete sequence. Corynebacterium glutamicum ndh gene. Smegma mycobacterium nadh dehydrogenase (ndh) gene, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 84/162. The Mycobacterium leprae genomic dna sequence, clay B35 bfr gene, complete cds. Streptomyces coelicolor clay E63. Human chromosome 7qtelo BACE3, complete sequence. Corynebacterium glutamicum panB, panC ﹠ xylB gene. The xylA gene people of Arthrobacter bacterium N.R.R.L.B3728D-wood sugar (D-Glucose) isomerase clones NH0511A20, the * * * * * * that checking order, 6 non-sequential segments. 71/109 part of Aquifex aeolicus complete genome group. LD39282.5primeLD Drosophila melanogaster embryo pOT2 Drosophila melanogaster cDNA clone LD39282 5prime, the mRNA sequence. LD28277.5primeLD Drosophila melanogaster embryo pOT2 Drosophila melanogaster cDNA clone LD28277 5prime, the mRNA sequence. The pgiA gene of bacillus stearothermophilus glucose phosphate isomerase A (EC 5.3.1.9). Bacillus subtilis complete genome group (17/21 part): 3197001-3414420. Pure phosphoenolpyruvate is ploughed on the Streptococcus mutans mountain: sugar phosphotransferase operon, complete sequence and unknown gene. Staphylococcus xylosus scrB and scrR gene. Bacillus subtilis complete genome group (20/21 part): 3798401-4010550. Bacillus subtilis genome section (325-333) Mycobacterium tuberculosis H37Rv complete genome group; Sections 46/162.

Drosophila melanogaster maize (Zeamays) 1 type human immunodeficiency virus aspergillus fumigatus (Aspergillus fumigatus) human glutamic acid rod bacillus smegma mycobacterium Much's bacillus Mycobacterium leprae streptomyces coelicolor human glutamic acid rod bacillus Arthrobacter bacterium (Arthrobacter sp.) people Aquifex aeolicus Drosophila melanogaster Drosophila melanogaster bacillus stearothermophilus bacillus subtilis Streptococcus mutans staphylococcus xylosus (Staphylococcus xylosus) bacillus subtilis bacillus subtilis Much's bacillus

32,969       43,617     40,040   37,844   37,136   100,000   65,254   40,058   59,551   39,468   39,291   38,384   56,283   37,593   36,309   41,941     39,855     66,292   37,255   63,607     67,778     35,574   51,826   54,476

99-08-02       99-07-21     99-03-05   97-04-01   98-06-02   99-04-24   98-05-05   98-06-17   94-09-06   99-03-17   98-10-02   99-05-11   92-05-04   99-08-24   98-03-25   99-03-16     98-11-25     95-04-20   97-11-26   99-09-28     96-11-28     97-11-26   93-11-02   98-06-17

Table 4 (continuing)

rxa02054         rxa02056       rxa02061         rxa02063           rxa02100       rxa02122           rxa02140           rxa02142

1140         2891       1617         1350           2348       822           1200           774

GB_PL2：SCE9537   GB_GSS13：AQ501177     GB_BA1：MLCB1222   GB_BA1：MTY13E12   GB_BA1：MTU43540     GB_PAT：E14601   GB_BA1：D84102   GB_BA1：MTV006   GB_HTG7：AC005883     GB_PL2：ATAC003033   GB_PL2：ATAC002334   GB_BA1：SCGLGC   GB_GSS4：AQ687350     GB_EST38：AW028530     GB_BA1：MSGY151   GB_BA1：MTCY130   GB_BA1：SCO001205   GB_BA1：D90858   GB_EST37：AI948595     GB_HTG3：AC010387     GB_BA1：MSGB1551C   S   GB_BA1：MSGB1554C   S   GB_RO：AF093099   GB_BA1：MTCY190

66030   767     34714   43401   3453     4394   4394   22440   211682     84254   75050   1518   786     444     37036   32514   9589   13548   469     220665     36548     36548     2482   34150

U18778   AQ501177     AL049491   Z95390   U43540     E14601   D84102   AL021006   AC005883     AC003033   AC002334   X89733   AQ687350     AW02853   0   AD000018   Z73902   AJ001205   D90858   AI948595     AC010387     L78813     L78814     AF093099   Z70283

Saccharomyces cerevisiae chromosome V clay 9537,9581,9495,9867 and λ clone 5898. V26G9mTn-3xHA/lacZ inserts library genes of brewing yeast group 5 ', genome summary sequence. Mycobacterium leprae clay B1222. Mycobacterium tuberculosis H37Rv complete genome group; Sections 147/162. Much's bacillus rfbA, rhamnose biosynthesis albumen (rfbA) and rm1C gene, complete cds. Brevibacterium α-ketone group glutamte dehydrogenase. Corynebacterium glutamicum 2-oxo glutamte dehydrogenase DNA, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 54/162. Human chromosome 17 clone RP11-958E11map17, the * * * * * * that checking order, 2 sequential segments. Arabidopsis thaliana chromosome IIBAC T21L14 genome sequence, complete sequence. Arabidopsis thaliana chromosome IIBACF25I18 genome sequence, complete sequence. The DNA of streptomyces coelicolor glgC gene. Nbxb0074H11r CUGI rice BAC library rice genomic clone nbxb0074H11r, genome summary sequence. Wv27f10.x1 NCI_CGAP_Kid11 people cDNA clones IMAGE:25307953 ', is similar to WP:T03G11.6 CE04874; The mRNA sequence. The sequence of Much's bacillus clone y151. Mycobacterium tuberculosis H37Rv complete genome group; Sections 59/162. Streptomyces coelicolor A3 (2) glycogen metabolism bunch I. Genome of E.coli DNA, Kohara clone #401 (51.3-51.6 minute) wq07d12.x1 NCI_CGAP_Kid12 people cDNA clone IMAGE:24705833 '; The mRNA sequence. Human chromosome 5 clone CITB-H1_2074D8, the * * * * * * that checking order, 77 non-sequential segments. Mycobacterium leprae clay B1551DNA sequence. Mycobacterium leprae clay B1554DNA sequence. House mouse transcription factor TBLYM (Tblym) mRNA, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 98/162.

Everybody Mycobacterium leprae Mycobacterium leprae house mouse Much's bacillus of saccharomyces cerevisiae saccharomyces cerevisiae Mycobacterium leprae Much's bacillus Much's bacillus Corynebacterium glutamicum Corynebacterium glutamicum Much's bacillus people arabidopsis arabidopsis streptomyces coelicolor rice bacillus tuberculosis typus humanus Much's bacillus streptomyces coelicolor Escherichia coli

36,100   32,039     61,896   59,964   59,659     98,928   98,928   39,265   37,453     37,711   37,711   56,972   40,696     36,795     40,156   55,218   38,475   38,586   37,259     38,868     51,399     51,399     36,683   57,292

97-08-01   99-04-29     99-08-27   98-06-17   97-08-14     99-07-28   99-02-06   98-06-18   99-12-08     97-12-19   98-03-04   99-07-12   99-07-01     99-10-27     96-12-10   98-06-17   99-03-29   97-05-29   99-09-06     99-09-15     96-06-15     96-06-15     99-10-01   98-06-17

Table 4 (continuing)

rxa02143           rxa02144           rxa02147             rxa02149           rxa02175       rxa02196         rxa02209

1011           1347           1140             1092           1416       816         1694

GB_BA1：SC6G10   GB_BA1：AB016787     GB_BA1：MTCY190   GB_BA1：MSGB1551C   S   GB_BA1：MSGB1554C   S   GB_BA1：MTCY190   GB_HTG3：AC011500   0   GB_HTG3：AC011500   0   GB_EST28：AI492095     GB_EST10：AA15767     GB_EST10：AA15767     GB_PR3：HSBK277P6   GB_BA2：EMB065R07   5   GB_EST34：AI789323     GB_BA1：CGGLTG   GB_BA1：MTCY31   GB_BA1：MLCB57   GB_RO：RATDAPRP   GB_GSS8：AQ012162     GB_RO：RATDAPRP   GB_BA1：AB025424   GB_BA2：AF002133

36734   5550     34150   36548     36548     34150   300851     300851     485     376     376     61698   360     574     3013   37630   38029   2819   763     2819   2995   15437

AL049497   AB016787     Z70283   L78813     L78814     Z70283   AC011500     AC011500     AI492095     AA157467     AA157467     AL117347   AF116423     AI789323     X66112   Z73101   Z99494   M76426   AQ012162     M76426   AB025424   AF002133

Streptomyces coelicolor clay 6G10. Gene and the 2ORF of pseudomonas putida cytochromes o ubiquinol oxidizing ferment A-E, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 98/162. Mycobacterium leprae clay B1551DNA sequence. Mycobacterium leprae clay B1554DNA sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 98/162. Human chromosome 19 clone CIT978SKB_60E11, the * * * * * * that checking order, 246 non-sequential segments. Human chromosome 19 clone CIT978SKB_60E11, the * * * * * * that checking order, 246 non-sequential segments. Tg07a01.x1 NCI_CGAP_CLL1 people cDNA clones IMAGE:21080403 '; The mRNA sequence. Zo50e01.rl Stratagene endothelial cell 937223 people cDNA clone IMAGE:590328 5 '; The mRNA sequence. Zo50e01.rl Stratagene endothelial cell 937223 people cDNA clone IMAGE:5903285 '; The mRNA sequence. The human DNA sequence of the clone 277P6 of chromosome lq25.3-31.2, complete sequence. Rhizobium etli mutant MB045 RosR-transcriptional regulatory sequences. Uk53g05.yl Sugano mouse kidney mkia house mouse cDNA clones IMAGE:19727605 ', is similar to WP:K11H12.8 CE12160; The mRNA sequence. Corynebacterium glutamicum citrate synthase glt gene and ORF. Mycobacterium tuberculosis H37Rv complete genome group; Sections 41/162. Mycobacterium leprae clay B57. Brown rat two peptidyl aminopeptidase GAP-associated protein GAP (dpp6) mRNA, complete cds. The red bacterial genomes clone of 127PB037070197 chromosome II cosmid library class ball 127PB037070197, genome summary sequence. Brown rat two peptidyl aminopeptidase GAP-associated protein GAP (dpp6) mRNA, complete cds. Corynebacterium glutamicum aconitase gene, part cds. Mycobacterium avium bacterial strain GIR10 transcript regutation protein (mav81) gene, part cds, aconitase (acn), invasion 1 (invl), invasion 2 (inv2), transcript regutation protein (moxR), ketoacyl base reductase (fabG), enoyl reductase (inhA) and ferrochelatase (mav272) gene, complete cds.

Streptomyces coelicolor pseudomonas putida (Pseudomonas putida) Much's bacillus Mycobacterium leprae Mycobacterium leprae Much's bacillus everybody everybody everybody Rhizobium etli house mouse Corynebacterium glutamicum Much's bacillus Mycobacterium leprae brown rat class ball red bacterium brown rat Corynebacterium glutamicum mycobacterium avium (Mycobacterium avium)

35,058   47,403     57,317   38,159     38,159     55,530   39,659     39,659     39,798     36,436     36,436     36,872   43,175     39,715     100,000   64,331   62,491   38,791   40,044     37,312   99,173   40,219

99-03-24   99-08-05     98-06-17   96-06-15     96-06-15     98-06-17   00-02-18     00-02-18     99-03-30     96-12-11     96-12-11     99-11-23   99-12-06     99-07-02     95-02-17   98-06-17   99-02-10   95-05-31   98-06-04     95-05-31   99-04-03   98-03-26

Table 4 (continuing)

rxa02213             rxa02245                 rxa02256         rxa02257           rxa02258         rxa02259

874             780                 1125         1338           900         2895

GB_BA1：MTV007   GB_BA1：AB025424   GB_BA1：MTV007   GB_BA2：AF002133         GB_BA2：RCU23145           GB_BA1：ECU82664   GB_HTG2：AC007922     GB_BA1：CGGAPPGK     GB_BA1：SCC54   GB_BA1：MTCY493   GB_BA1：CGGAPPGK     GB_BA1：MTCY493   GB_BA2：MAU82749     GB_BA1：CGGAPPGK     GB_BA1：CORPEPC   GB_PAT：A09073   GB_BA1：CORPEPC   GB_PAT：A09073   GB_BA1：CGPPC

32806   2995   32806   15437         5960           139818   158858     3804     30753   40790   3804     40790   2530     3804     4885   4885   4885   4885   3292

AL021184   AB025424   AL021184   AF002133         U23145           U82664   AC007922     X59403     AL035591   Z95844   X59403     Z95844   U82749     X59403     M25819   A09073   M25819   A09073   X14234

Mycobacterium tuberculosis H37Rv complete genome group; Sections 64/162. Corynebacterium glutamicum aconitase gene, part cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 64/162. Mycobacterium avium bacterial strain GIR10 transcript regutation protein (mav81) gene, part cds, suitable achilleic acid enzyme (acn), invasion 1 (inv1), invasion 2 (inv2), transcript regutation protein (moxR), ketoacyl base reductase (fabG), enoyl reductase (inhA) and ferrochelatase (mav272) gene, complete cds. The red bacterium Calvin cycle of pod membrane carbon dioxide fixation operon: fructose-1,6-/sedoheptulose-1,7-bisphosphate aldolase (cbbA) gene, part cds, II type ribulose-1,5-bisphosphate, 5-diphosphonic acid Carboxylase/oxygenase (cbbM) gene, complete cds and Calvin cycle operon: pentose-5-phosphoric acid-3-epimerase (cbbE), phosphoglycerate phosphatase (cbbZ) and cbbY gene, complete cds. Escherichia coli 9-11 minute genome sequence. Human chromosome 18 clone hRPK.178_F_10map 18, the * * * * * * that checking order, 11 sequential segments. The gap of Corynebacterium glutamicum glyceraldehyde-3-phosphate, phosphoglyceric kinase and triose-phosphate isomerase, pgk and tpi gene. Streptomyces coelicolor clay C54. Mycobacterium tuberculosis H37Rv complete genome group; Sections 63/162. The gap of Corynebacterium glutamicum glyceraldehyde-3-phosphate, phosphoglyceric kinase and triose-phosphate isomerase, pgk and tpi gene. Mycobacterium tuberculosis H37Rv complete genome group; Sections 63/162. Mycobacterium avium glyceraldehyde-3-phosphate, dehydrogenase homologue (gapdh) gene, complete cds; And phosphoglyceric kinase gene, part cds. The gap of Corynebacterium glutamicum glyceraldehyde-3-phosphate, phosphoglyceric kinase and triose-phosphate isomerase, pgk and tpi gene. Corynebacterium glutamicum phosphoenolpyruvate carboxylase gene. Corynebacterium glutamicum phosphoenolpyruvate carboxylase ppg gene. Corynebacterium glutamicum phosphoenolpyruvate carboxylase gene, complete cds. Corynebacterium glutamicum phosphoenolpyruvate carboxylase ppg gene. Corynebacterium glutamicum phosphoenolpyruvate carboxylase gene (EC 4.1.1.31).

The red bacteria Escherichia coli human glutamic acid rod of Much's bacillus Corynebacterium glutamicum Much's bacillus mycobacterium avium pod membrane bacillus streptomyces coelicolor Much's bacillus Corynebacterium glutamicum Much's bacillus mycobacterium avium Corynebacterium glutamicum Corynebacterium glutamicum Corynebacterium glutamicum Corynebacterium glutamicum Corynebacterium glutamicum Corynebacterium glutamicum

38,253   99,096   34,937   36,885         48,701           39,119   33,118     99,289     36,951   64,196   98,873     61,273   61,772     99,667     100,000   100,000   100,000   100,000   99,827

98-06-17   99-04-03   98-06-17   98-03-26         97-10-28           97-01-11   99-06-26     92-10-05     99-06-11   98-06-19   92-10-05     98-06-19   98-01-06     92-10-05     95-12-15   93-08-25   95-12-15   93-08-25   93-09-12

Table 4 (continuing)

rxa02288       rxa02292           rxa02322     rxa02326       rxa02327       rxa02328       rxa02332         rxa02333

969       798           511     939       1083       1719       1266         1038

GB_PR3：HSDJ94E24   GB_HTG3：AC010091   GB_HTG3：AC010091   GB_BA2：AF125164     GB_GSS5：AQ744695     GB_EST14：AA381925   GB_BA1：MTCY22G8   GB_BA1：MTCY22G8   GB_BA1：CGPYC   GB_BA2：AF038548   GB_BA1：MTCY349   GB_BA1：CGPYC   GB_BA2：AF03848   GB_BA1：MTCY349   GB_BA1：CGPYC   GB_BA2：AF038548   GB_PL2：AF097728   GB_BA1：MSGLTA   GB_BA2：ABU85944     GB_BA2：AE000175   GB_BA1：MSGLTA   GB_PR4：HUAC00229   9

243145   159526   159526   26443     827     309   22550   22550   3728   3637   43523   3728   3637   43523   3728   3637   3916   1776   1334     15067   1776   171681

AL050317   AC010091   AC010091   AF125164     AQ744695     AA381925   Z95585   Z95585   Y09548   AF038548   Z83018   Y09548   AF038548   Z83018   Y09548   AF038548   AF097728   X60513   U85944     AE000175   X60513   AC002299

The human DNA sequence of the clone RP1-94E24 of chromosome 20q12, complete sequence. The people clones NH0295A01, the * * * * * * that checking order, and 4 non-sequential segments people clone NH0295A01, the * * * * * * that checking order, 4 non-sequential segments bacteroides fragilis 638R Polysaccharide Bs (PS B2) biosynthesis gene seat, complete sequence; And unknown gene. HS_5505_A2_C06_SP6 RPCI-11 male sex BAC library human genome clone flat board=1081 row=12 row=E, genome summary sequence. EST95058 activating T cell people cDNA 5 ' end, the mRNA sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 49/162. Mycobacterium tuberculosis H37Rv complete genome group; Sections 49/162. Corynebacterium glutamicum pyc gene. Corynebacterium glutamicum pyruvate carboxylase (pyc) gene, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 131/162. Corynebacterium glutamicum pyc gene. Corynebacterium glutamicum pyruvate carboxylase (pyc) gene, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 131/162. Corynebacterium glutamicum pyc gene. Corynebacterium glutamicum pyruvate carboxylase (pyc) gene, complete cds. Aspergillus terreus pyruvate carboxylase (Pyc) mRNA, complete cds. Smegma mycobacterium citrate synthase gltA gene. Antarctic bacteria DS2-3R citrate synthase (cisy) gene, complete cds. 65/400 part of e. coli k-12 MG1655 complete genome group. Smegma mycobacterium citrate synthase gltA gene. Human chromosome 16BAC clones CIT987-SKA-113A6～complete genome group sequence, complete sequence.

Everybody bacteroides fragilis (Bacteroides fragilis) people bacillus tuberculosis typus humanus Much's bacillus Corynebacterium glutamicum Corynebacterium glutamicum Much's bacillus Corynebacterium glutamicum Corynebacterium glutamicum Much's bacillus Corynebacterium glutamicum Corynebacterium glutamicum Aspergillus terreus (Aspergillus terreus) smegma mycobacterium Antarctic bacteria (Antarctic bacterium) DS2-3R Escherichia coli smegma mycobacterium people of people

36,039   35,331   35,331   39,747     39,185     35,922   57,677   37,143   100,000   100,000   37,363   99,259   99,259   41,317   100,000   100,000   52,248   58,460   57,154     38,164   58,929   33,070

99-12-03   99-09-11   99-09-11   99-12-01     99-07-16     97-04-21   98-06-17   98-06-17   98-05-08   97-12-24   98-06-17   98-05-08   97-12-24   98-06-17   98-05-08   97-12-24   98-10-29   91-09-20   97-09-23     98-11-12   91-09-20   99-11-23

Table 4 (continuing)

rxa02399       rxa02404         rxa02414       rxa02435           rxa02440           rxa02453         rxa02474           rxa02480

1467       2340         870       681           963           876         897           1779

GB_HTG2：AC007889       GB_BA1：CGACEA   GB_BA1：CORACEA   GB_PAT：I13693   GB_BA1：CGACEB   GB_BA1：CORACEB   GB_BA1：PFFC2     GB_PR4：AC007102   GB_HTG3：AC011214   GB_HTG3：AC011214   GB_BA2：AF101055     GB_OM：RABPKA   GB_OM：RABPLASIS   M   GB_EST14：AA417723     GB_EST11：AA215428     GB_BA1：MTCY77   GB_EST14：AA426336     GB_BA1：STMAACC8   GB_PR3：AC004500   GB_BA1：AB009078     GB_OM：BTU71200   GB_EST2：F12685     GB_BA1：MTV012

127840       2427   1905   2135   3024   2725   5588     176258   183414   183414   7457     4441   4458     374     303     22255   375     1353   77538   2686     877   287     70287

AC007889       X75504   L28760   I13693   X78491   L27123   Y11998     AC007102   AC011214   AC011214   AF101055     J03247   M64656     AA417723     AA215428     Z95389   AA426336     M55426   AC004500   AB009078     U71200   F12685     AL021287

Drosophila melanogaster chromosome 3 clone BACR48E12 (D695) RPCI-98 48.E.12map 87A-87B bacterial strain y; Cn bw sp, the * * * * * * that checking order, 86 non-sequential segments. Corynebacterium glutamicum aceA gene and thiX gene (part). Corynebacterium glutamicum isocitratase (aceA) gene. The sequence 3 of United States Patent (USP) 5439822. Corynebacterium glutamicum (ATCC 13032) aceB gene. Corynebacterium glutamicum malate synthase (aceB) gene, complete cds. Pseudomonas fluorescens FC2.1, FC2.2, FC2.3c, FC2.4 and FC2.5c frame. Human chromosome 4 clone C0162P16map 4p16, complete sequence. The people clones 5_C_3, the low sequence of samples of passing through. The people clones 5_C_3, the low sequence of samples of passing through. Clostridium acetobutylicum atp operon, complete sequence. Rabbit phosphorylase kinase (alpha subunit) mRNA, complete cds. Oryctolagus cuniculus phosphorylase kinase alpha subunit mRNA, complete cds. Zv01b12.sl NCI_CGAP_GCB1 people cDNA clones IMAGE:7462073 ', is similar to and contains the Alu repeat element; Contain element L1 repeat element; The mRNA sequence. Zr95a07.sl NCI_CGAP_GCB1 people cDNA clones IMAGE:6834123 ', is similar to and contains the Alu repeat element; The mRNA sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 146/162. Zv53g02.sl Soares testis NHI people cDNA clones IMAGE:7573943 ', mRNA sequence. Streptomyces fradiae glucosaminide acetyltransferase (aacC8) gene, complete cds. Human chromosome 5, P1 clones 1076B9 (LBNLH14), complete sequence. Brevibacterium saccharolyticum L-2, the gene of 3-butanediol dehydrogenation enzyme, complete cds. Bos taurus 3-Hydroxybutanone reductase mRNA, complete cds. HSC3DA031 normalization baby brain cDNA people cDNA clones c-3da03, mRNA sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 132/162.

Everybody clostridium acetobutylicum (Clostridium acetobutylicum) Oryctolagus cuniculus Oryctolagus cuniculus people's bacillus tuberculosis typus humanus people's streptomyces fradiae (Streptomyces fradiae) people's Brevibacterium saccharolyticum (Brevibacterium saccharolyticum) Bos taurus bacillus tuberculosis typus humanus of unknown Corynebacterium glutamicum Corynebacterium glutamicum Pseudomonas fluorescens (Pseudomonas fluorescens) people of Drosophila melanogaster Corynebacterium glutamicum Corynebacterium glutamicum

34,897       100,000   100,000   99,795   99,914   99,786   63,539     35,069   36,885   36,885   39,605     36,061   36,000     38,770     39,934     38,889   38,043     37,097   33,256   96,990     51,659   41,509     36,737

99-08-02       94-09-09   95-02-10   95-09-26   95-01-13   95-06-08   97-07-11     99-06-02   99-10-03   99-10-03   99-03-03     93-04-27   98-06-22     97-10-16     97-08-13     98-06-18   97-10-16     93-05-05   98-03-30   99-02-13     97-10-08   95-03-14     99-06-23

Table 4 (continuing)

rxa02485   rxa02492       rxa02528               rxa02539               rxa02551           rxa02556       rxa02560

840       1098               1641               483           1281       990

GB_BA1：SC6G10   GB_BA1：AP000060     GB_BA1：STMPGM   GB_BA1：MTCY20G9   GB_BA1：U00018   GB_PR2：HS161N10   GB_HTG2：AC008235       GB_HTG2：AC008235       GB_BA2：RSU17129     GB_BA1：MTV038   GB_BA2：AF068264         GB_BA1：BACHYPTP   GB_BA1：BACHUTWA   PA   GB_BA1：BSGBGLUC     GB_HTG3：AC008128   GB_HTG3：AC008128   GB_PL2：AC005292   GB_IN1：CEF07A11   GB_EST32：AI731605     GB_IN1：CEF07A11

36734   347800     921   37218   42991   56075   136017       136017       17425     16094   3152         17057   28954     4290     335761   335761   99053   35692   566     35692

AL049497   AP000060     M83661   Z77162   U00018   AL008707   AC008235       AC008235       U17129     AL021933   AF068264         D29985   D31856     Z34526     AC008128   AC008128   AC005292   Z66511   AI731605     Z66511

Streptomyces coelicolor clay 6G10. Aeropyrum pernix genome grinds DNA, 3/7 part. Streptomyces coelicolor phosphoglycerate phosphomutase (PGM) gene, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 25/162. Mycobacterium leprae clay B2168. The human DNA sequence of the PAC 161N10 of chromosome x q25 is contained EST. Drosophila melanogaster chromosome 3 clone BACR15B19 (D995) RPCI-9815.B.19map 94F-95A bacterial strain y; Cn bw sp, the * * * * * * that checking order, 125 non-sequential segments. Drosophila melanogaster chromosome 3 clone BACR15B19 (D995) RPCI-9815.B.19map 94F-95A bacterial strain y; Cnbw sp, the * * * * * * that checking order, 125 non-sequential segments. Rhodococcus erythropolis ThcA (thcA) gene, complete cds; And unknown gene. Mycobacterium tuberculosis H37Rv complete genome group; Sections 24/162. Pseudomonas aeruginosa quinoprotein alcohol dehydrogenase (exaA) gene, part cds; Cytochrome c 550 precursors (exaB), NAD+ dependence acetaldehyde dehydrogenase (exaC) and PQQ synthesize A (pqqA) gene, complete cds; And synthetic B (pqqB) gene of PQQ, part cds. The bacillus subtilis wall is in conjunction with albumen and wapA and the orf gene of inferring albumen. The bacillus subtilis genome that contains hut and wapA locus. The gene of bacillus subtilis (Marburg 168) β-glucoside permease and β-glucosyl enzym. People, the * * * * * * that checking order, 106 non-sequential segments. People, the * * * * * * that checking order, 106 non-sequential segments. The genome sequence of arabidopsis BACF26F24, complete sequence. Caenorhabditis elegans clay F07A11, complete sequence. 6 days cotton fiber upland cotton of BNLGHi10201 cDNA 5 ' is similar to (AC004684) putative protein [arabidopsis], the mRNA sequence. Caenorhabditis elegans clay F07A11, complete sequence.

Everybody arabidopsis Caenorhabditis elegans upland cotton Caenorhabditis elegans of streptomyces coelicolor Aeropyrum pernix streptomyces coelicolor Much's bacillus Mycobacterium leprae people's Drosophila melanogaster Drosophila melanogaster Rhodococcus erythropolis (Rhodococcus erythropolis) Much's bacillus pseudomonas aeruginosa bacillus subtilis bacillus subtilis bacillus subtilis

35,511   48,014     65,672   61,436   37,893   37,051   36,822       36,822     66,117     65,174   65,448         53,602   53,602     53,602     34,022   34,022   33,858   36,420   38,095     33,707

99-03-24   99-06-22     93-04-26   98-06-17   94-03-01   99-11-23   99-08-02       99-08-02       99-07-16     98-06-17   99-03-18         99-02-07   99-02-07     95-07-03     99-08-22   99-08-22   99-04-16   99-09-02   99-06-11     99-09-02

Table 4 (continuing)

rxa02572         rxa02596       rxa02611       rxa02612         rxa02621         rxa02640           rxa02654         rxa02666

668         1326       1775       2316         942         1650           1008         891

GB_BA1：MTCY63   GB_BA1：MTCY63   GB_HTG1：HS24H01     GB_BA1：MTV026   GB_BA2：AF026540   GB_BA2：MTU96128   GB_BA1：MTCY130   GB_BA1：MSGY151   GB_BA1：U00014   GB_BA1：MTCY130   GB_BA1：MSGY151   GB_BA1：STMGLGEN     GB_BA1：CGL133719   GB_IN1：CEM106   GB_EST29：AI547662     GB_BA1：MTV025   GB_BA1：PAU49666     GB_BA1：AB015974     GB_EST6：N65787   GB_PL2：T17H3   GB_RO：MMU58105     GB_PR3：AC004643

38900   38900   46989     23740   1778   1200   32514   37036   36470   32514   37036   2557     1839   39973   377     121125   4495     1641     512   65839   88871     43411

Z96800   Z96800   AL121632     AL022076   AF026540   U96128   Z73902   AD000018   U00014   Z73902   AD000018   L11647     AJ133719   Z46935   AI547662     AL022121   U49666     AB015974     N65787   AC005916   U58105     AC004643

Mycobacterium tuberculosis H37Rv complete genome group; Sections 16/162. Mycobacterium tuberculosis H37Rv complete genome group; Sections 16/162. Human chromosome 21 clone LLNLcl 16H0124map 21q21, the * * * * * * that checking order, non-sequential segments. Mycobacterium tuberculosis H37Rv complete genome group; Sections 157/162. Much's bacillus UDP-galactopyranose mutase (glf) gene, complete cds. Much's bacillus UDP-galactopyranose mutase (glf) gene, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 59/162. The sequence of Much's bacillus clone y151. Mycobacterium leprae clay B1549. Mycobacterium tuberculosis H37Rv complete genome group; Sections 59/162. The sequence of Much's bacillus clone y151. Streptomyces aureofaciens glycogen branching enzyme (glgB) gene, complete cds. Corynebacterium glutamicum yjcc gene, amtR gene and citE gene, part. Caenorhabditis elegans clay M106, complete sequence. U1-R-C3-sz-h-03-0-UI.sl UI-R-C3 brown rat cDNA clone UI-R-C3-sz-h-03-0-UI 3 ', the mRNA sequence. Mycobacterium tuberculosis H37Rv complete genome group; Sections 155/162. Pseudomonas aeruginosa (orfX), glycerine disperse promote albumen (glpF), glycerokinase (glpK) and Glp repressor protein (glpR) gene, complete cds and (orfK) gene, part cds. The glpK gene of holder Laplace pseudomonad glycerokinase, complete cds. 20827 λ-PRL2 arabidopsis cDNA clones 232B7T7, mRNA sequence. Arabidopsis thaliana chromosome 1 BAC T17H3 sequence, complete sequence. House mouse Btk locus, α-d-galactosidase (Ags), ribosomal protein (L44L) and Bruton EGFR-TK (Btk) gene, complete cds. Human chromosome 16, clay clone 363E3 (LANL), complete sequence.

Much's bacillus Much's bacillus bacillus tuberculosis typus humanus Much's bacillus Much's bacillus Much's bacillus Much's bacillus Mycobacterium leprae Much's bacillus Much's bacillus streptomyces aureofaciens (Streptomyces aureofaciens) Corynebacterium glutamicum Caenorhabditis elegans brown rat Much's bacillus pseudomonas aeruginosa holder Laplace pseudomonads (Pseudomonas tolaasil) arabidopsis arabidopsis house mouse people

61,677   37,170   19,820     36,957   67,627   70,417   38,532   60,575   57,486   38,018   58,510   57,193     36,858   37,608   50,667     39,187   59,273     58,339     39,637   33,735   35,431     38,851

98-06-17   98-06-17   99-09-29     99-06-24   98-10-30   98-03-25   98-06-17   96-12-10   94-09-29   98-06-17   96-12-10   95-05-25     99-08-12   99-09-02   99-07-03     99-06-24   97-05-18     99-08-28     98-01-05   99-08-05   97-02-13     98-05-01

Table 4 (continuing)

rxa02675           rxa02694         rxa02729         rxa02730           rxa02737       rxa02738       rxa02739

1980           1065         844         1161           1665       1203       2223

GB_PR3：AC004643   GB_BA2：AF049897         GB_BA1：PDENQOUR   F     GB_BA1：MTCY339   GB_BA1：MXADEVRS   GB_BA1：BACLDH     GB_BA1：BACLDHL   GB_PAT：A06664   GB_EST15：AA494626     GB_EST15：AA494626     GB_EST19：AA758660     GB_EST15：AA494626     GB_PR4：AC006285   GB_PAT：E13655   GB_BA1：MTCY493   GB_BA1：SC5A7   GB_PAT：E13655   GB_BA1：SCC22   GB_BA1：SC5A7   GB_BA1：AB023377

43411   9196         10425       42861   2452   1147     1361   1350   121     121     233     121     150172   2260   40790   40337   2260   22115   40337   2572

AC004643   AF049897         L02354       Z77163   L19029   M19394     M14788   AO6664   AA494626     AA494626     AA758660     AA494626     AC006285   E13655   Z95844   AL031107   E13655   AL096839   AL031107   AB023377

Human chromosome 16, clay clone 363E3 (LANL), complete sequence. Corynebacterium glutamicum N-acetyl glutamy phosphoric acid reduction enzyme (argC), ornithine acetyltransferase (argJ), N-acetylglutamat kinases (argB), acetyl-ornithine transaminase (argD), ornithine carbamyl based transferase (argF), arginine repressor protein (argR), argininosuccinic acid synthase (argG) and argininosuccinic acid lyase (argH) gene, complete cds. Paracoccus denitrificans nadh dehydrogenase (URF4), (NQO8), (NQO9), (URF5), (URF6), (NQO10), (NQO11), (NQO12), (NQO13) and (NQO14) gene, complete cds; Biotin [acetyl-CoA carboxyl] ligase (birA) gene, complete cds. Mycobacterium tuberculosis H37Rv complete genome group; Sections 101/162. Myxococcus xanthus devR and devS gene, complete cds. Thermosol bacillus lactic dehydrogenase (LDH) gene, complete cds. The lct gene of bacillus stearothermophilus coding LDH, complete cds. Bacillus stearothermophilus lct gene. Fa09d04.rl Zebrafish ICRFzfls Danio rerio cDNA clones 11,A22 5 ', is similar to TR:G1171163 G1171163 G/T mispairing in conjunction with albumen; The mRNA sequence. Fa09d04.rl Zebrafish ICRFzfls Danio rerio cDNA clones 11,A22 5 ', is similar to TR:G1171163 G1171163 G/T mispairing in conjunction with albumen; The mRNA sequence. Ah67d06.sl Soares_ testis _ NHT people cDNA clone 13206833 ', mRNA sequence. Fa09d04.rl Zebrafish ICRFzfls Danio rerio cDNA clones 11,A22 5 ', is similar to TR:G1171163 G1171163 G/T mispairing in conjunction with albumen; The mRNA sequence. The people, complete sequence. The gDNA of coding glucose-6-phosphate dehydrogenase (G6PD). Mycobacterium tuberculosis H37Rv complete genome group; Sections 63/162. Streptomyces coelicolor clay 5A7. The gDNA of coding glucose-6-phosphate dehydrogenase (G6PD). Streptomyces coelicolor clay C22. Streptomyces coelicolor clay 5A7. Corynebacterium glutamicum transketolase tkt gene, complete cds.

Human glutamic acid rod bacillus Paracoccus denitrificans (Paracoccus denitrificans) Much's bacillus Myxococcus xanthus (Myxococcus xanthus) thermosol bacillus (Bacillus caldolyticus) bacillus stearothermophilus bacillus stearothermophilus Danio rerio Danio rerio people Danio rerio human glutamic acid rod bacillus Much's bacillus streptomyces coelicolor Corynebacterium glutamicum streptomyces coelicolor streptomyces coelicolor Corynebacterium glutamicum

41,599   40,413         40,735       36,471   38,477   57,371     57,277   57,277   50,746     36,364     37,059     42,149     37,655   99,580   38,363   39,444   98,226   60,399   36,426   99,640

98-05-01   98-07-01         93-05-20       98-06-17   94-01-27   93-04-26     93-04-26   93-07-29   97-06-27     97-06-27     98-12-29     97-06-27     99-11-15   98-06-24   98-06-19   98-07-27   98-06-24   99-07-12   98-07-27   99-02-20

Table 4 (continuing)

rxa02740                 rxa02741       rxa02743       rxa02797             rxa02803           rxa02821

1053                 1089       1161       1026             680           363

GB_BA1：MLCL536   GB_BA1：U00013   GB_HTG2：AC006247       GB_HTG2：AC006247       GB_HTG3：AC007150     GB_HTG2：AC004951   GB_HTG2：AC004951   GB_IN1：AB006546   GB_BA1：MLCL536   GB_BA1：U00013   GB_HTG2：AC007401   GB_BA1：CGBETPGE   N   GB_GSS9：AQ148714     GB_BA1：BFU64514     GB_BA1：U00020   GB_BA2：PSU85643       GB_BA1：SC6G4   GB_HTG2：AC008105     GB_HTG2：AC008105     GB_EST33：AV117143

36224   35881   174368       174368       121474     129429   129429   931   36224   35881   83657   2339     405     3837     36947   4032       41055   91421     91421     222

Z99125   U00013   AC006247       AC006247       AC007150     AC004951   AC004951   AB006546   Z99125   U00013   AC007401   X93514     AQ148714     U64514     U00020   U85643       AL031317   AC008105     AC008105     AV117143

Mycobacterium leprae clay L536. Mycobacterium leprae clay B1496. Drosophila melanogaster chromosome 2 clone BACR48I10 (D505) RPCI-9848.1.10map 49E6-49F8 bacterial strain y; Cn bw sp, the * * * * * * that checking order, 17 non-sequential segments. Drosophila melanogaster chromosome 2 clone BACR48I10 (D505) RPCI-98 48.1.10map 49E6-49F8 bacterial strain y; Cn bw sp, the * * * * * * that checking order, 17 non-sequential segments. Drosophila melanogaster chromosome 2 clone BACR16P13 (D597) RPCI-98 16.P.13map 49E-49F bacterial strain y; Cn bw sp, the * * * * * * that checking order, 87 non-sequential segments. The people clones DJ1022I14, the * * * * * * that checking order, 14 non-sequential segments. The people clones DJ1022I14, the * * * * * * that checking order, 14 non-sequential segments. The mRNA of Ephydatia fluviatilis G albumen a subunit 4, part cds. Mycobacterium leprae clay L536. Mycobacterium leprae clay B1496. The people clones NH0501O07, the * * * * * * that checking order, 3 non-sequential segments. Corynebacterium glutamicum betP gene. The human genome sperm library D human genome clone flat board of HS 3136 A1 A03 MR CIT approval=3136 row=5 row=A, genome summary sequence. Bacillus firmus dppABC operon, dipeptides transport protein dppA gene, part cds and dipeptides transport protein dppB and dppC gene, complete cds. The dihydrofolate synthase gene is inferred in the pathogenic mutation of Mycobacterium leprae clay B229 pseudomonas syringae cloves, part cds, regulate albumen MrsA (mrsA), triose-phosphate isomerase (tpiA), transport protein SecG (secG), tRNA-Leu, tRNA-Met and 15kDa GFP, complete cds. Streptomyces coelicolor clay 6G4. Human chromosome 17 clone 2020_K_17map 17, the * * * * * * that checking order, 12 non-sequential segments. Human chromosome 17 clone 2020_K_17map 17, the * * * * * * that checking order, 12 non-sequential segments. C57BL/6J10 days embryo house mouses of AV117143 house mouse cDNA clones 26102O0J17, mRNA sequence.

Everybody Ephydatia fluviatilis Mycobacterium leprae Mycobacterium leprae human glutamic acid of Mycobacterium leprae Mycobacterium leprae Drosophila melanogaster Drosophila melanogaster Drosophila melanogaster rod bacillus people's bacillus firmus (Bacillus firmus) Mycobacterium leprae pseudomonas syringae cloves everybody house mouse of mutation (Pseudomonas syringae pv.syringae) streptomyces coelicolor of causing a disease

61,573   61,573   37,105       37,105       38,728     33,116   33,116   36,379   48,401   48,401   37,128   38,889     34,321     38,072     34,462   50,445       59,314   37,607     37,607     40,157

98-12-04   94-03-01   99-08-02       99-08-02       99-09-20     98-06-12   98-06-12   99-06-23   98-12-04   94-03-01   99-06-26   97-09-08     98-10-08     97-02-01     94-03-01   97-04-09       98-08-20   99-07-22     99-07-22     99-06-30

Table 4 (continuing)

rxa02829             rxa03216               rxa03215           rxa03224

373             1141               1038           1288

GB_HTG1：HSU9G8     GB_HTG1：HSU9G8     GB_PR3：HSU85B5     GB_HTG3：AC008184       GB_EST15：AA477537       GB_EST26：AI330662   GB_BA1：SC3F9   GB_BA1：SLLINC   GB_HTG5：AC009660       GB_PR3：AC004076   GB_PL2：SPAC926     GB_BA2：AE001081

48735     48735     39550     151720       411       412   19830   36270   204320       41322   23193     11473

AL008714     AL008714     Z69724     AC008184       AA477537       AI330662   AL023862   X79146   AC009660       AC004076   AL110469     AE001081

Human chromosome X clones LL0XNC01-9G8, the * * * * * * that checking order, non-sequential segments. Human chromosome X clones LL0XNC01-9G8, the * * * * * * that checking order, non-sequential segments. The human DNA sequence of clay U85B5 is between chromosome x mark DXS366 and the DXS87. Drosophila melanogaster chromosome 2 clone BACR04D05 (D540) RPCI-98 04.D.5map 36E5-36F2 bacterial strain y; Cn bw sp, the * * * * * * that checking order, 27 non-sequential segments. Zu36g 12.rl Soares ovarian neoplasm NbHOT people cDNA clones IMAGE:740134 5 ', is similar to and contains the Alu repeat element; Contain element HGR repeat element; The mRNA sequence. Fa91d08.yl zebrafish fin regeneration in 1 day Danio rerio cDNA 5 ', the mRNA sequence. Streptomyces coelicolor clay 3F9. Streptomyces lincolnensis (78-11) lincomycin produces gene. Human chromosome 15 clone RP11-424J10map15, the * * * * * * that checking order, 41 non-sequential segments. Human chromosome 19, clay R30217, complete sequence. Chestnut wine fission yeast chromosome 1 clay c926. Archaeoglobus fulgidus complete genome group 26/172 part.

Everybody trembles wine fission yeast (Schizosaccharomyces pombe) Archaeoglobus fulgidus everybody Drosophila melanogaster people Danio rerio streptomyces coelicolor A3 (2) streptomyces lincolnensis (Streptomyces lincolnensis) of people

41,595     41,595     41,595     39,600       37,260       37,805   48,657   39,430     35,151     37,788   38,474     35,871

99-11-23     99-11-23     99-11-23     99-08-02       97-11-09       98-12-28   99-02-10   96-05-15     99-12-04     98-01-29   99-09-02     97-12-15

The embodiment part

Embodiment 1: the preparation of the total genomic dna of Corynebacterium glutamicum ATCC13032

Allow Corynebacterium glutamicum (ATCC 13032) culture under 30 ℃ in BHI culture medium (Difco) oscillating growth spend the night. By centrifugal collecting cell, abandoning supernatant is resuspended in 5ml buffer solution I (the volume bacterium of all appointments of 5%--of culture initial volume is all calculated according to the 100ml culture volume) with cell. The composition of buffer solution I: 140.34g/l sucrose, 2.46 g/l MgSO₄×7H ₂O、10ml/l KH ₂PO ₄Solution (100g/l transfers to pH 6.7 with KOH), 50ml/l M12 concentrate (10g/l (NH4)₂SO ₄、1g/l NaCl、2g/l MgSO ₄×7H ₂O、 0.2g/l CaCl ₂, 0.5g/l yeast extract (Difco), 10ml/l trace element mixture (200mg/l FeSO₄×H ₂O、10mg/l ZnSO ₄×7H ₂O、3mg/l MnCl ₂×4H ₂O、30mg/l H3BO3、20mg/l CoCl ₂×6H ₂O、1mg/l NiCl ₂×6H ₂O、3mg/l  Na ₂MoO ₄×2H ₂, 500mg/l complexing agent (EDTA or citric acid), 100mg/l vitamin mixtures (0.2 mg/l biotin, 0.2mg/l folic acid, 20mg/l p-aminobenzoic acid, 20mg/l riboflavin, 40mg/l calcium pantothenate (ca-panthothenate), 140mg/l nicotinic acid, 40mg/l pyridoxal hydrochloride, 200mg/l inositol). Lysozyme is added in the supernatant to final concentration 2.5mg/ml. After about 4 hours, cell membrane is degraded in 37 ℃ of incubations, the protoplast that produces by centrifugal results. Precipitation is with 5ml buffer solution I washing 1 time, with 5ml TE buffer solution (10mM Tris-HCl, 1mM EDTA, pH 8) washing 1 time. Precipitation is resuspended in the 4ml TE buffer solution, adds 0.5ml SDS solution (10%) and 0.5ml NaCl solution (5M). Adding Proteinase K to final concentration 200 μ g/ml, the night that will suspend was in about 18 hours of 37 ℃ of incubations. Adopt standard method, by using phenol, phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol extracting, purify DNA. Then, by 3M sodium acetate and 2 volume ethanol that add 1/50 volume, then in-20 ℃ of incubations 30 minutes, in supercentrifuge, with SS34 rotor (Sorvall) with 12,000rpm centrifugal 30 minutes, precipitate described DNA. DNA is dissolved in the TE buffer solution that 1ml contains 20 μ g/ml RNaseAs, in 4 ℃ to 1000ml TE buffer solution dialysis at least 3 hours. During this period, exchange buffering liquid is 3 times. In the dna solution through dialysing of 0.4ml equal portions, add 0.4ml 2M LiCl and 0.8ml ethanol. After 30 minutes, collect DNA by centrifugal (13,000rpm, Biofuge Fresco, Heraeus, Hanau, Germany) in-20 ℃ of incubations. The DNA precipitation is dissolved in the TE buffer solution. DNA with the method preparation can be used for all purposes, comprises the structure of southern blotting technique analysis or genomic library.

Embodiment 2: make up the genomic library of Corynebacterium glutamicum ATCC13032 in Escherichia coli

Adopt the DNA of embodiment 1 described preparation, according to known clear and definite method (referring to for example Sambrook, J. etc., (1989) " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Laboratory Press, or Ausubel, F.M. etc. (1994) " Current Protocols in Molecular Biology ", John Wiley ﹠ Sons.) structure cosmid library and plasmid library.

Can use any plasmid or clay. The concrete plasmid that uses is plasmid pBR322 (Sutcliffe, J.G. (1979) Proc.Natl.Acad.Sci.USA, 75:3737-3741), pACYC177 (Change ﹠ Cohen (1978) J.Bacteriol 134:1141-1156), pBS plasmid series (pBSSK+, pBSSK-etc.; Stratagene, LaJolla, USA) or clay such as SuperCosl (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J., Rosenthal A. and Waterson, R.H. (1987) Gene 53:283-286). Use plasmid pSL109 can make up the gene library (Lee, H.-S. and A.J.Sinskey (1994) J. Microbiol.Biotechnol.4:256-263) that is used in particular for Corynebacterium glutamicum.

Embodiment 3:DNA order-checking and computing function analysis

The genomic library that Application Example 2 is described is according to standard method, especially utilize the chain termination method of ABI377 sequenator (referring to for example Fleischman, R.D. etc. (1995) " at random genome sequencing and assembling of haemophilus influenzae Rd. ", Science, 269:496-512) carry out dna sequencing. Use has the sequencing primer of following nucleotide sequence: 5 '-GGAAACAGTATGACCATG-3 ' or 5 '-GTAAAACGACGGCCAGT-3 '.

Embodiment 4: mutagenesis in vivo

Plasmid (or other carrier) DNA is gone down to posterity by impaired Escherichia coli or other biology (for example Bacillus bacillus or yeast such as saccharomyces cerevisiae) of ability that keeps the hereditary information integrality, thus mutagenesis Corynebacterium glutamicum in vivo. There is sudden change (such as muHLS, mutD, mutT etc. in the DNA repair system gene of typical case's mutator; List of references is seen Rupp, and W.D. (1996) DNA repair mechanism is stated from: Escherichia coli and Salmonella, and the 2277-2294 page or leaf, ASM:Washington.). This class bacterial strain is that those skilled in the art are known. Greener for example, A. and Callahan have introduced the use of described bacterial strain among M. (1994) the Strategies 7:32-34.

Embodiment 5: the DNA between Escherichia coli and the Corynebacterium glutamicum shifts

Some Corynebacteriums and brevibacterium species contain the endogenous plasmid (for example pHM1519 or pBL1) of self-replicating (summarizing referring to for example Martin J.F. etc. (1987) Biotechnology, 5:137-146). Adopt the Escherichia coli standard vector (Sambrook that wherein adds Corynebacterium glutamicum origin of replication and Corynebacterium glutamicum appropriate flags, J. etc. (1989), " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Laboratory Press or Ausubel, F.M. etc. (1994) " Current Protocols in Molecular Biology ", John Wiley ﹠ Sons), can easily make up the shuttle vector of Escherichia coli and Corynebacterium glutamicum. Described origin of replication is preferably taken from the endogenous plasmid that separates from Corynebacterium and brevibacterium species. The transformation marker of concrete these species that use is kalamycin resistance gene (for example kalamycin resistance gene of Tn5 or Tn903 transposons generation) or chloramphenicol resistance gene (Winnacker, E.L. (1987), From Genes to Clones-Introduction to Gene Technology, VCH, Weinheim). Enumerated a large amount of about making up the example of various shuttle vectors in the described document, described shuttle vector can copy and can be used for some purposes in Escherichia coli and Corynebacterium glutamicum, (list of references is referring to for example Yoshihama to comprise gene overexpression, (1985) J.Bacteriol.162:591-597 such as M., (1987) Biotechnology such as Martin J.F., 5:137-146 and Eikmanns, B.J. etc. (1991) Gene, 102:93-98).

The application standard method can be cloned into genes of interest a kind of above-mentioned shuttle vector and this heterozygosis carrier is imported in the Corynebacterium glutamicum strain. Can adopt following methods to transform Corynebacterium glutamicum: protoplast transformation (Kastsumata, (1984) J.Bacteriol.159306-311 such as R.), electroporation (Liebl, (1989) the FEMS Microbiol.Letters such as E., 53:399-303), if use special carrier, also can adopt joint method (seeing for example Sch  fer, A etc. (1990) J. Bacteriol.172:1663-1666). Be transformed into Escherichia coli by preparation Corynebacterium glutamicum DNA (utilizing the known standard method in this area) and with it, also can make shuttle vector transfer to Escherichia coli from Corynebacterium glutamicum. Adopt standard method can carry out this step, but preferably use Mcr defective coli strain such as NM522 (Gough ﹠ Murray (1983) J.Mol. Biol.166:1-19).

Can use and comprise pCG1 (U.S. Patent number 4,617,267) or its fragment and the optional TN903 kalamycin resistance gene (Grindley that comprises, N.D. and Joyce, C.M. (1980) Proc. Natl.Acad.Sci.USA 77 (12): plasmid 7176-7180) is the overexpression range gene in Corynebacterium glutamicum strain. In addition, also can use plasmid pSL109 overexpression range gene (Lee, H.-S. and A.J.Sinskey (1994) J.Microbiol. Biotechnol.4:256-263) in Corynebacterium glutamicum strain.

Except using the science plasmid, also can realize gene overexpression by being integrated into genome. Utilize known method can be implemented in genome conformity in Corynebacterium glutamicum or other Corynebacterium or the brevibacterium species, for example with the fusion (REMI) (referring to for example DE patent 19823834) of the homologous recombination in genome district, restriction endonuclease mediation or use transposons. The activity that also can take following measures to modify regulatory region (for example promoter, repressor and/or enhancer) and regulate genes of interest: utilize fix-point method (for example homologous recombination) or carry out sequence modification, insertion or disappearance based on the method (for example transposon mutagenesis or REMI) of chance event. Also can insert the nucleotide sequence of transcription terminator effect at 3 ' of the one or more gene coding regions of the present invention; Described terminator is that this area is known, for example at Winnacker, E.L. (1987) From Genes to Clones-Introduction to Gene Technology.VCH:Weinheim) in its introduction is arranged.

Embodiment 6: estimate the expression of mutain

Mutain activity in the observation transformed host cell depends on such fact: mutain reaches with similar scale in the mode similar to wild-type protein. The process useful of a kind of definite mutator transcriptional level (can translate into the index of the mRNA amount of gene outcome) is to carry out the RNA trace (list of references is seen such as (1988) Current Protocols in Molecular Biology such as Ausubel, Wiley: New York), wherein use the design primer of detectable label (being generally radioactivity or chemiluminescence) mark binding purpose gene, so that when the total RNA that extracts described organism culture, on gel electrophoresis, be transferred on the stabilized matrix and during with described probe incubation, the combination of described probe and binding capacity can illustrate the mRNA amount that has described gene and indicate this gene. Such information is the evidence that mutator is transcribed degree. Using the known method in some this areas can prepare total cell RNA from Corynebacterium glutamicum, Bormann for example, the method that E.R. etc. (1992) Mol.Microbiol.6:317-326 introduces.

Whether exist or relative quantity in order to estimate described mRNA translation albumen, but Application standard technology such as Western blotting (referring to such as (1988) Current Protocols in Molecular Biology such as Ausubel, Wiley: New York). In this process, extract total cell protein, separate through gel electrophoresis, be transferred to as needing probe such as the antibody incubation of albumen on the matrix of celluloid and with specific binding. General chemiluminescence or the colorimetric marker mark with detecting easily of this probe. Observe label existence and label amount mutain and the amount thereof that existence needs in the described cell is described.

Embodiment 7: cultivation-culture medium of the Corynebacterium glutamicum of genetic improvement and condition of culture

With synthetic or spontaneous growth medium culture genetic improvement rod bacillus. The many different growth mediums that are used for excellent bacillus are well-known and easy acquisition (Lieb etc. (1989) Appl. Microbiol.Biotechnol., 32:205-210; (1998) the Biotechnology Letters such as von der Osten, 11:11-16; Patent DE 4,120, and 867; Liebl (1992) " Corynebacterium " is stated from: The Procaryotes, and the II volume, Balows, A. etc. write, Springer-Verlag). The composition of these culture mediums is: one or more carbon sources, nitrogenous source, inorganic salts, vitamin and trace element. Preferred carbon source is that sugar is such as monose, disaccharides or polysaccharide. For example glucose, fructose, mannose, galactolipin, ribose, sorbose, ribulose, lactose, maltose, sucrose, gossypose, starch or cellulose are extraordinary carbon sources. Also can provide sugar for culture medium by other byproduct of complex chemical compound such as molasses or refined sugar. The mixture that different carbon sources are provided also may be useful. Other possible carbon source is alcohols and organic acid, for example methyl alcohol, ethanol, acetic acid or lactic acid. Nitrogenous source is generally the organic or inorganic nitrogen compound, perhaps contains the material of these compounds. Typical case's nitrogenous source comprises ammonia or ammonium salt, such as ammonium chloride or ammonium sulfate, ammonium hydroxide, nitrate, urea, amino acid or compound nitrogen source such as corn steep liquor, soy meal, soybean protein, yeast extract, meat extract etc.

The inorganic salt compound that culture medium can contain comprises hydrochloride, phosphate or the sulfate of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Can add chelate compound in the described culture medium, to keep the metal ion in the solution. Especially effectively chelate compound comprises dihydric phenol (for example catechol or catechol hydrochlorate) or organic acid (for example citric acid). Described culture medium also routine contains other growth factor, for example vitamin or growth promoter, and the example comprises biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid and pyridoxol. Growth factor and salt are derived from the complex medium component usually, such as yeast extract, molasses, corn steep liquor etc. The culture medium compound really is cut into minute closely related with direct experiment and specifically depends on each particular case. Can be at textbook " Applied Microbiol.Physiology; obtain among the A Practical Approach (P.M.Rhodes, P.F.Stanbury writes, IRL Press (1997); 53-73 page or leaf, ISBN 0 19 9635773) about the information of medium optimization. Also can be from commercial Supplier Selection growth medium, standard No. 1 (Merck) or BHI (grain heart infusion, DIFCO) etc. for example.

All nutrient media componentses are by heating (in 1.5 handkerchiefs and 121 ℃ 20 minutes) or aseptic filtration sterilization. Described component can be sterilized together, can sterilize respectively if need. Just exist when all nutrient media componentses can be cultivated beginning, perhaps can choose wantonly continuously or in batches adding.

Each experiment separate provision condition of culture. Temperature should be 15 ℃-45 ℃. Cultivation temperature can keep constant or experiment in change. The pH of culture medium should be 5-8.5, preferred about 7.0, can add buffer in culture medium and keep pH. The typical buffer that is used for this purpose is potassium phosphate buffer agent. But choice for use or the synthetic buffer of simultaneously use such as MOPS, HEPES, ACES etc. Also can add in the training period NaOH or ammonium hydroxide and keep constant cultivation pH. If use complex medium component such as yeast extract, may reduce the needs of extra buffer, because in fact many complex chemical compounds have high buffer capacity. If use the described microorganism of fermentation tank culture, also can use ammonia control pH.

Incubation time is generally a few hours to a couple of days. Select incubation time so that the product amount of fluid nutrient medium accumulation is maximum. Available various container carries out disclosed use, for example glass or the metal fermentation tank of microtiter plate, glass tube, vial or different sizes. In order to screen a large amount of clones, use microtiter plate, glass tube or shaking flask (with or do not have a baffle plate). The preferred 100ml shaking flask of using wherein fills the growth medium that needs with 10% (volume). Blake bottle should be upper with the jolting of 100-300rpm velocity interval at shaking table (amplitude 25mm). Keeping moistening atmosphere makes the evaporation forfeiture minimum; Perhaps should carry out accurate correction evaporation loss.

If test heredity improves the clone, also should test the contrast clone of not improvement or contain contrast clone without any the basic plasmid of Insert Fragment. Utilize in the agar plate of 30 ℃ of incubations such as the cell inoculation medium on the CM dull and stereotyped (10g/l glucose, 2.5g/l sodium chloride, 2g/l urea, the poly-peptone of 10g/l, 5g/l yeast extract, 5g/l meat extract, 22g/l sodium chloride, 2g/l urea, the poly-peptone of 10g/l, 5g/l yeast extract, 5g/l meat extract, 22g/l agar transfer to pH6.8 with 2M NaOH) to OD₆₀₀Be 0.5-1.5. Add the salt water slurry of the Corynebacterium glutamicum cell derive from the CM flat board or add the liquid preculture thing of this bacterium, thereby finish culture medium inoculated.

Embodiment 8: the function of analyzed in vitro mutain

This area has clear and definite enzymatic activity and Determination of Kinetic Parameters method. Must make the experiment of measuring any given change enzymatic activity be fit to the specific activity of wild-type enzyme, this is fully in persons skilled in the art limit of power. Be found in for example with Publication about Document about the general introduction of enzyme and the detail and the many enzyme assay examples that relate to structure, dynamics, principle, method, application: Dixon, M. and Webb, E.C., (1979) Enzymes, Longmans:London; Fersht, (1985) Enzyme Structure and Mechanism, Freeman: New York; Walsh, (1979) Enzymatic Reaction Mechanisms.Freeman:San Francisco; Price, N.C., Stevens, L. (1982) Fundamentals of Enzymology.Oxford Univ.Press:Oxford; Boyer, P.D. write (1983) The Enzymes, the third edition, Academic Press: New York; Bisswanger, H., (1994) Enzymkinetik, second edition, VCH:Weinheim (ISBN 3527300325); Bergmeyer, H.U., Bergmeyer, J., Gra β l, M. write (1983-1986) Methods of Enzymatic Analysis, the third edition, I-XII volume, Verlag Chemie:Weinheim; And Ullmann ' s Encyclopedia of Industrial Chemistry (1987), A9 volume, " Enzymes ", VCH:Weinheim, 352-363 page or leaf.

Can use the displacement of several clear and definite methods such as DNA band and measure (being also referred to as gel blocking measures) mensuration in conjunction with the protein active of DNA. Available reporter is measured (Kolmar for example, H. etc. (1995) EMBO J.14:3895-3904 and the mensuration introduced of citing document wherein) and is measured described albumen to the impact of other developed by molecule. The reporter test macro is well-known and clearly is used for prokaryote and eukaryotic cells, utilizes enzyme such as beta galactosidase, green fluorescent protein etc.

According to for example Gennis, R.B. (1989) " hole, passage and transport protein ", be stated from: Biomembranes, Molecular Structure and Function, Springer:Heidelberg, the method that 85-137,199-234 and 270-322 page or leaf are introduced can be measured the activity of protein called membrane transporters.

Embodiment 9: analyze the impact of mutain on needing product to produce

The heredity that can followingly detect Corynebacterium glutamicum improves needing the impact of compound (for example amino acid) generation: the improvement microorganism is grown under appropraite condition (for example above-mentioned condition), and the output that needs product (being amino acid) of then analyzing in described culture medium and/or the cellular component increases situation. Described analytical technology is that persons skilled in the art are known, comprise that spectrum detection method, thin-layer chromatography, various decoration method, enzyme and micro-biological process and analytical chromatography method such as high performance liquid chroma-tography are (referring to for example Ullman, Encyclopedia of Industrial Chemistry, the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. etc., (1987) " HPLC application in biochemistry " is stated from: Laboratory Techniques in Biochemistry and Molecular Biology, the 17th volume; Rehm etc. (1993) Biotechnology, the 3rd volume, III chapter: " retrieve and purification of product ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) Bioseparations:downstream processing for biotechnology, John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) biochemistry is separated, and is stated from: Ulmann ' s Encyclopedia of Industrial Chemistry, B3 volume, Chapter 11,1-27 page or leaf, VCH:Weinheim; And Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).

Except detecting the fermentation end-product, also can analyze other component that needs the metabolic pathway of product in order to generation, for example intermediate and accessory substance are to determine to produce the overall efficiency of described compound. Analytical method comprises the gas that produces between nutrient level (for example sugar, hydro carbons, nitrogenous source, phosphate and other ion), detection of biological amount composition and the growth of measuring in the culture medium, the output of analyzing biosynthesis pathway common metabolic thing and measurement yeast phase. The standard method of these detections is summarized in Applied Microbial Physiology, A Practical Approach, P.M. Rhodes and P.F.Stanbury write, IRL Press, 103-129,131-163 and 165-192 pages (ISBN:0199635773) and the list of references of wherein quoting.

Embodiment 10: need product from Corynebacterium glutamicum culture purifying

The known the whole bag of tricks in available this area reclaims from the Corynebacterium glutamicum cell of above-mentioned culture or supernatant needs product. Secretion out can be by low-speed centrifugal from the culture harvesting from cell if need product, application standard method cell lysis, for example mechanical force or ultrasonic. Centrifugal removal cell fragment keeps the supernatant that contains described soluble protein and partly supplies to be further purified needed compound. If product from Corynebacterium glutamicum emiocytosis out, is then removed cell by low-speed centrifugal from described culture, keep supernatant partly for being further purified.

So that carry out chromatography from the supernatant of arbitrary purification process part in appropriate resin, perhaps need molecule to be retained on the chromatographic resin and the most of impurity in the sample are washed out, perhaps impurity is retained on the resin and needs a minute subsample to be washed out. Can repeat described chromatographic step as required, use identical or different chromatographic resin. Persons skilled in the art are being selected suitable chromatographic resin and described resin are applied to be proficient in very much aspect the specific molecular to be purified most effectively. Purified product can by filtering or ultrafiltration concentration, be housed under the most stable state of temperature of product.

Many purification process known in the art are arranged, and above-mentioned purification process is not restrictive. Such purification process is described in for example Bailey, J.E.﹠ Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The standard technique of using this area can detect identity and the purity of separating compound. Described standard technique comprises high performance liquid chromatography (HPLC), spectroscopic methodology, decoration method, thin-layer chromatography, NIRS, enzyme assay or microbiological assay. The summary of described analytical method is seen: Patek etc. (1994) Appl. Environ.Microbiol.60:133-140; Malakhova etc. (1996) Biotekhnologiya 11:27-32; And (1998) the Bioprocess Engineer such as Schmidt, 19:67-70; Ulmann ' s Encyclopedia of Industrial Chemistry, (1996) A27 volume, VCH:Weinheim, 89-90,521-540,540-547,559-566,575-581 and 581-587 page or leaf; Michal, G. (1999) Biochemical Pathways:An Atlas of biochemistry and Molecular Biology, John Wiley and Sons; Fallon, the application of A. etc. (1987) HPLC in biochemistry is stated from: Laboratory Techniques in Biochemistry and Molecular Biology, the 17th volume.

Embodiment 11: analyze gene order of the present invention

Comparative sequences also determines that the homology percentage of two kinds of sequences is technology known in the art, can adopt mathematical algorithm to finish, for example the algorithm of Karlin and Altschul ((1990) Proc. Natl.Acad.Sci.USA 87:2264-68) and modified algorithm (Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-77 thereof. This algorithm has added NBLAST and the XBLAST program (2.0 editions) of (1990) J.Mol.Biol.215:403-10 such as Altschul. Can use the NBLAST program, marking=100, the retrieval of BLAST nucleotides is carried out in word string length=12, so that the nucleotide sequence of acquisition and SMP nucleic acid molecules homology of the present invention. Can use the XBLAT program, marking=50, the retrieval of BLAST protein is carried out in word string length=3, so that the amino acid sequence of acquisition and SMP protein molecular homology of the present invention. In order to obtain the band room sequence contrast of comparison purpose, can be according to Altschul etc., (1997) Nucleic Acids Res 25 (17): 3389-3402 is described, uses Gapped BLAST. When using BLAST and Gapped BLAST program, one skilled in the art will know that program (for example XBLAST and the NBLAST) parameter of how optimizing for analyzing particular sequence.

Another mathematical algorithm example that is used for the sequence comparison is Meyers and Miller algorithm ((1988) Comput.Appl.Biosci.4:11-17). This algorithm has added ALIGN program (2.0 editions), and the ALIGN program is the part of GCG sequence comparison software bag. When using ALIGN program comparision amino acid sequence, can use PAM120 weight residue table, room length point penalty 12 and gap penalty 4. Other sequence analysis algorithm is known in the art, comprises ADVANCE and ADAM that Torelli and Robotti (1994) Comput.Appl.Biosci.10:3-5 introduces; And the FASTA of Pearson and Lipman (1988) P.N.A.S.85:2444-8 introduction.

Also can use GAP program in the GCG software kit (canhttp://www.gcg.comObtain), selecting Blosum 62 matrixes or PAM250 matrix and room weight is 12,10,8,6 or 4 and the length weight is 2,3 or 4, the homology percentage that obtains two seed amino acid sequences calculates. Use the GAP program in the GCG software kit, the Application standard parameter, for example room weight 50 and length weight 3 can obtain the homology percentage of two kinds of nucleotide sequences. Adopt methods known in the art (to write (1998) Bioinformatics:A Practical Guide to the Analysis of Genes and Proteins referring to for example Bexevanis and Ouellette, John Wiley and Sons: New York), can carry out the comparative analysis of gene order of the present invention and Genbank sequence. Compare gene order of the present invention and Genbank sequence with 3 step method. The first step is carried out BLASTN to each sequence of the present invention and Genbank nucleotide sequence and is analyzed (for example local contrast analysis), and keeping front 500, to hit sequence for further analysis. Second step hits sequence to these 500 and carries out FASTA retrieval (for example associating is local and the Comprehensive Comparison of global sequence, wherein arranges contrast sequence localized area). At last, adopt the GAP program (Application standard parameter) in the GCG software kit, each gene order of the present invention and front 3 FASTA are hit in the sequence each carry out sequence of overall importance and contrast. In order to obtain correct result, use the known method in this area and make the sequence length that takes out from Genbank be adjusted into search sequence length. Analysis result sees Table 4. To analyze the data that obtain identical with separately each gene of the present invention and each reference sequences of Genbank being carried out GAP (overall situation) for the data that obtain, but compare with such database-wide GAP (overall situation) analysis, need to computing time significantly reduce. The sequence of the present invention that does not have acquisition to be higher than the sequence contrast of threshold value is denoted as in table 4 does not have the sequence comparative information. And then those skilled in the art should be appreciated that the GAP sequence contrast homology percentage under table 4 gauge outfit " homology (%) (GAP) " lists with European number format, and wherein ", " represents decimal point. For example the numerical value " 40,345 " in this hurdle refers to " 40.345% ".

Embodiment 12: make up and the operation dna microarray

In addition, sequence of the present invention can be used for making up and operating dna microarray, and (design of DNA array, methodology and use are well-known in the art, its introduction: Schena is for example arranged, M. etc. (1995) Science 270:467-470 in Publication about Document; Wodicka, L. etc. (1997) Nature Biotechnology 15:1359-1367; DeSaizieu, A. etc. (1998) Nature Biotechnology 16:45-48; And DeRisi, J.L. etc. (1997) Science 278:680-686).

Dna microarray is solid or flexible holder, is comprised of celluloid, nylon, glass, silicone or other material. The orderly combination of nucleic acid molecules and support surface. Suitably behind the mark, other nucleic acid or mixtures of nucleic acids can be hybridized with immobilized nucleic acid molecule, and label can be used to monitor and measure each signal strength signal intensity of the hybrid molecule in the definite area. But relative quantity or the absolute magnitude of whole or selected nucleic acid in the method nucleic acid samples that simultaneous quantitative adds or the mixture. Therefore, but the expression of dna microarray parallel analysis a plurality of (nearly 6800 or more) nucleic acid (referring to for example Schena, M. (1996) BioEssays 18 (5): 427-431).

Sequence of the present invention can be used to the design oligonucleotides primer, can increase by the nucleic acid amplification reaction such as the PCR regulation zone of one or more Corynebacterium glutamicum genes of this Oligonucleolide primers. Choice and design 5 ' or 3 ' Oligonucleolide primers or suitable joint can make the PCR product of generation and above-mentioned holder dielectric surface covalently bound (in addition, referring to for example Schena, M. etc. (1995) Science 270:467-470 introduces).

According to Wodicka, L. etc. (1997) Nature Biotechnology 15:1359-1367 introduces, also can be by the synthetic nucleic acid microarray that makes up of original position oligonucleotides. Use photolithographic process and make the exposure of matrix explication district. Thus exciting light unstable protection group, and adding nucleotides, any change then can not occur in the lucifuge district. Protection subsequently is with the photoactivation circulation so that at the synthetic different oligonucleotides of definition. Can limit the district at microarray gene synthetic of the present invention by the solid phase oligonucleotides is synthetic.

The nucleic acid molecules of the present invention that exists in sample or the mixture of ribonucleotides can with described microarray hybridization. But according to these nucleic acid molecules of standard method mark. In brief, when for example reverse transcription or DNA are synthetic by mixing isotope or fluorescence labeling nucleotides, thereby marker nucleic acid molecule (for example mRNA molecule or dna molecular). Existing introduction about labeling nucleic acid and microarray hybridization (for example be stated from: Schena, M etc. (1995) are the same; Wokicka, L etc. (1997), the same; And DeSaizieu A. etc. (1998), the same). The detection of hybrid molecule and quantitative palpus are fit to the specific label that mixes. For example according to Schena, M etc. (1995) (the same) but introduce the detection of radioactive labels thing, and can detect fluorescent marker such as the method according to ((1996) Genome Research 6:639-645) such as Shalon.

As mentioned above, sequence of the present invention is used for the different strains that the dna microarray technology can relatively be analyzed Corynebacterium glutamicum or other Corynebacterium. For example, the nucleic acid array methodology helps according to distribute in the research bacterial strain variation and identifying for specific and/or required strain characteristic such as pathogenic, production capacity and the important gene of stress tolerance of each transcript. In addition, utilize the nucleic acid array method can compare the expression and distribution of gene of the present invention between yeast phase.

Embodiment 13: analysis of cells albumen group motion mechanics (proteomics (Proteomics))

Gene of the present invention, composition and method can be used for studying the mutual effect of albumen faciation and dynamics, are called " proteomics ". The destination protein group includes but not limited to Corynebacterium glutamicum total protein group (for example and the albumen group of other organism relatively), under specific environment or metabolism condition (for example high temperature between yeast phase or low temperature, perhaps high pH or low pH) activated albumen of tool, cultivating and the activated albumen of development given period tool.

The method that available this area is known such as gel electrophoresis analysis protein group. For example can obtain cell protein by cracking or extraction, use various electrophoretic techniques the albumen group is separated from each other. Lauryl sodium sulfate gel electrophoresis (SDS-PAGE) is mainly according to the molecular weight of albumen protein isolate. Isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) is by albumen isoelectric point (it not only reflects amino acid sequence, and modifies after reflecting described protein translation) protein isolate. The sequential combination that another preferred analysis of protein method is IEF-PAGE and SDS-PAGE is called the 2-D-gel electrophoresis (such as seeing following Introduction of Literatures: Hermann etc. (1998) Electrophoresis 19:3217-3221; Fountoulakis etc. (1998) Electrophoresis 19:1193-1202; Langen etc. (1997) Electrophoresis 18:1184-1192; Antelmann etc. (1997) Electrophoresis 18:1451-1463). Also can use other analysis of protein method to carry out Separation of Proteins, for example Capillary Electrophoresis; Described technology is the known technology in this area.

The available standards technology manifests the albumen that described method is separated such as dyeing or mark. Suitable dye is known in the art, comprises Coomassie brilliant blue, silver-colored dyestuff or fluorescent dye such as Sypro Ruby (Molecular Probes). (for example in the Corynebacterium glutamicum culture medium, contain radiolabeled amino acids or other amyloid protein precursor³⁵The S-methionine,³⁵The S-cysteine,¹⁴The C-labeled amino acid,¹⁵The N-labeled amino acid,¹⁵NO ₃Or¹⁵NH ₄ ⁺Or¹³The C-labeled amino acid) can be before it separates the albumen of these cells of mark. Can use fluorescent marker equally. Can extract according to preceding method, separately and separate described labelled protein.

By measuring used dyestuff or label amount, can further analyze the albumen that described technology manifests. But adopt for example given protein content of optical means quantitative assay, and can with identical gel or other gel on other protein content relatively. For example adopt optics comparison, spectroscopic methodology, scanning of image and gel analysis or by using photographic film and screen, can carry out protein ratio at gel. Such technology is that this area is known.

In order to determine the identity of any given albumen, can adopt direct Sequencing or other standard technique. Can use for example N-and/or C-end amino acid order-checking (for example Edman degraded), also can use mass spectrography (especially MALDI or ESI technology (referring to such as (1997) Electrophoresis 18:1184-1192 such as Langen)). Protein sequence provided by the invention can be used for by described technical appraisement Corynebacterium glutamicum albumen.

The albumen of the Information Availability that described method obtains between the different samples of more various biotic factors (such as different biologies, different fermentations time point, the different biotopes of different culture media conditioned disjunction etc.) exists pattern, protein active or protein modified. These experiments can be used for various uses separately or with data that other technical tie-up obtains, for example behavior of various biologies in more given (for example metabolism) situation, improve the production capacity of the bacterial strain of producing fine chemicals or improve the productive rate of producing fine chemicals.

Be equal to embodiment

Those skilled in the art will know that or only adopt many embodiments that are equal to of the specific embodiments that normal experiment just can determine that the present invention introduces. Following claims comprise such embodiment that is equal to.

Claims (34)

1. the nucleic acid molecules of a separation or its complementary series, described nucleic acid molecules comprises each the odd number nucleotide sequence that is selected from SEQ ID NO:1 to 781, and prerequisite is that described nucleic acid molecules be can't help the genomic constitution of any F sign that table 1 provides.

2. the nucleic acid molecules of a separation or its complementary series, described nucleic acid molecule encoding comprises the polypeptide of each the even number amino acid sequence that is selected from SEQ ID NO:2 to 782, and prerequisite is that described nucleic acid molecules be can't help the genomic constitution of any F sign that table 1 provides.

3. the nucleic acid molecules of a separation or its complementary series, the naturally occurring allelic variation body of described nucleic acid molecule encoding one peptide species, described polypeptide comprises each the even number amino acid sequence that is selected from SEQ ID NO:2 to 782, and prerequisite is that described nucleic acid molecules be can't help the genomic constitution of any F sign that table 1 provides.

4. the nucleic acid molecules of a separation or its complementary series, described nucleic acid molecules comprises the nucleotide sequence that the complete nucleotide sequence of any odd number with SEQ ID NO:1 to 781 has at least 50% homogeneity, and prerequisite is that described nucleic acid molecules be can't help the genomic constitution of any F sign that table 1 provides.

5. the nucleic acid molecules of a separation or its complementary series, described nucleic acid molecules comprises the fragment of at least 15 continuous nucleotides of any odd number nucleotide sequence that is selected from SEQ ID NO:1 to 781, and prerequisite is that described nucleic acid molecules be can't help the genomic constitution of any F sign that table 1 provides.

6. the nucleic acid molecules of a separation, described nucleic acid molecules comprise each nucleic acid molecules and a kind of nucleotide sequence of the heterologous polypeptide of encoding among the claim 1-5.

7. carrier, described carrier comprise among the claim 1-6 each nucleic acid molecules.

8. the carrier of claim 7, described carrier is expression vector.

9. host cell, described host cell are the host cells with the expression vector transfection of claim 8.

10. the host cell of claim 9, wherein said cell is microorganism.

11. the host cell of claim 10, wherein said cell belong to Corynebacterium (Corynebacterium) or brevibacterium (Brevibacterium).

12. the host cell of claim 9, the expression of wherein said nucleic acid molecules cause regulating producing fine chemicals by described cell.

13. the host cell of claim 12, wherein said fine chemicals is selected from: organic acid, the amino acid that generates protein and amino acid, purine and pyrimidine bases, nucleosides, nucleotides, lipid, saturated and unrighted acid, dihydroxylic alcohols, carbohydrate, aromatic compounds, vitamin, co-factor, polyketide and the enzyme of non-generation protein.

14. a method of producing polypeptide, the method are included in the host cell of cultivating claim 9 in the suitable culture medium, produce thus described polypeptide.

15. the polypeptide of a separation, described polypeptide comprise each the even number amino acid sequence that is selected from SEQ ID NO:2 to 782, prerequisite is that described amino acid sequence be can't help the gene code of any F sign that table 1 provides.

16. the polypeptide of a separation, described polypeptide comprises a kind of naturally occurring allelic variation body that contains the polypeptide of each the even number amino acid sequence that is selected from SEQ ID NO:2 to 782, and prerequisite is that described amino acid sequence be can't help the gene code of any F sign that table 1 provides.

17. the polypeptide of a separation, described polypeptide is by a kind of nucleic acid molecule encoding, described nucleic acid molecules comprises the nucleotide sequence that the complete nucleotide sequence of any odd number with SEQ ID NO:1 to 781 has at least 50% homogeneity, and prerequisite is that described nucleic acid molecules be can't help the nucleic acid molecules of any F sign that table 1 provides and formed.

18. the polypeptide of a separation, described polypeptide comprise the amino acid sequence that has 50% homogeneity with any even number complete amino acid sequence of SEQ ID NO:2 to 782 at least, prerequisite is that described amino acid sequence be can't help the gene code of any F sign that table 1 provides.

19. the polypeptide of a separation, described polypeptide comprises the fragment of polypeptide of any even number amino acid sequence of a kind of SEQ of containing ID NO:2 to 782, prerequisite is that described amino acid sequence be can't help the gene code of any F sign that table 1 provides, and wherein said polypeptide fragment keeps comprising the biologically active of the polypeptide of amino acid sequence.

20. the polypeptide of a separation, described polypeptide be by the nucleic acid molecule encoding of any odd number nucleotide sequence that comprises SEQ ID NO:1 to 781, prerequisite is that described nucleic acid molecules be can't help the nucleic acid molecules of any F sign that table 1 provides and formed.

21. each isolated polypeptide among the claim 15-20, described polypeptide also comprises the allogeneic amino acid sequence.

22. a method of producing fine chemicals, described method comprises the cell of cultivating claim 9, produces thus described fine chemicals.

23. the method for claim 22, wherein said method also comprises the step that reclaims described fine chemicals from described culture.

24. the method for claim 22, wherein said cell belongs to Corynebacterium or brevibacterium.

25. the method for claim 22, wherein said cell is selected from: Corynebacterium glutamicum (Corynebacterium glutamicum), man of great strength's rod bacillus (Corynebacterium herculis), lily hedysarum scoparium bacillus (Corynebacterium lilium), Corynebacterium acctoacidophlum (Corynebacterium acetoacidophilum), vinegar paddy rod bacillus (Corynebacterium acetoglutamicum), have a liking for acetyl rod bacillus (Corynebacterium acetophilum), produce ammonia rod bacillus (Corynebacterium ammoniagenes), Corynebacterium fujiokense, Corynebacterium nitrilophilus, brevibacterium ammoniagene (Brevibacterium ammoniagenes), Brevibacterium butanicum, Corynebacterium glutamicum (Brevibacterium divaricatum), brevibacterium flavum (Brevibacterium flavum), Xi Shi brevibacterium (Brevibacterium healii), ketoglutaric acid brevibacterium (Brevibacterium ketoglutamicum), Brevibacterium ketosoreductum, brevibacterium (Brevibacterium lactofermentum), extension brevibacterium (Brevlbacterium linens), the bacterial strain that provides in Brevibacterium paraffinolyticum and the table 3.

26. the method for claim 22 wherein causes production to described fine chemicals to produce from the expression of the described nucleic acid molecules of described carrier and regulates.

27. the method for claim 22, wherein said fine chemicals is selected from: organic acid, the amino acid that generates protein and amino acid, purine and pyrimidine bases, nucleosides, nucleotides, lipid, saturated and unrighted acid, dihydroxylic alcohols, carbohydrate, aromatic compounds, vitamin, co-factor, polyketide and the enzyme of non-generation protein.

28. the method for claim 22, wherein said fine chemicals are amino acid.

29. the method for claim 28, wherein said amino acid is selected from: lysine, glutamic acid, glutamine, alanine, aspartic acid, glycine, serine, threonine, methionine, cysteine, valine, leucine, isoleucine, arginine, proline, histidine, tyrosine, phenylalanine and tryptophan.

30. a method of producing fine chemicals, described method comprise the cell of cultivating its genomic DNA and changing because containing among the claim 1-6 each nucleic acid molecules.

31. existence or its active method of diagnosing corynebacterium diphtheriae in curee's body, described method comprises at least a in the peptide molecule that detects the nucleic acid molecules that whether there is claim 1-5 in described curee's body or claim 15-20, diagnoses thus existence or the activity of corynebacterium diphtheriae in described curee's body.

32. a host cell, described host cell comprise each the odd number nucleic acid molecules that is selected from SEQ ID NO:1 to 781, wherein said nucleic acid molecules is destroyed.

33. a host cell, described host cell comprise each the odd number nucleic acid molecules that is selected from SEQ ID NO:1 to 781, wherein said nucleic acid molecules is compared with any odd number sequence of SEQ ID NO:1 to 781 and is comprised one or more nucleic acid modifications.

34. a host cell, described host cell comprise each the odd number nucleic acid molecules that is selected from SEQ ID NO:1 to 781, wherein for the wild type regulatory region of described nucleic acid molecules, the regulatory region of described nucleic acid molecules is modified.