HK1022719B - HAMSTER EF-1α TRANSCRIPTIONAL REGULATORY DNA - Google Patents

Description

hamster EF-1 alpha transcriptional regulatory DNA

Background

Transcription of any given eukaryotic gene is performed by one of three RNA polymerases, each of which acts on a certain specific subset of genes. If it is said that transcription of large ribosomal RNAs is performed by RNA polymerase I and small ribosomal RNAs and tRNAs are transcribed by RNA polymerase III, the DNA sequence encoding the protein and most of the small nuclear RNAs are transcribed by RNA polymerase II. For each type of gene, transcription requires interaction of the polymerase with the gene promoter sequence and formation of a stable transcription initiation complex. Generally, transcription of any of the three polymerases also requires some binding factor to interact with the promoter sequence and recognition of the bound factor by a second factor, thereby allowing the polymerase to interact with the gene sequence. If it is said that this mechanism is the minimum requirement for transcription by RNA polymerases I and III, the process leading to transcription by RNA polymerase II is more complicated.

It is speculated that due to the large amount of gene sequences transcribed by RNA polymerase II and due to the fact that the regulatory patterns of these genes vary widely within the same cell as well as between different cells, transcription by RNA polymerase II is affected by the binding of many transcription factors in the initiation complex in addition to the interaction of other binding proteins with regulatory DNA sequences other than the promoter. Together, these other binding proteins may function to activate transcription above basal levels or to repress transcription. Repressor binding may also be considered as a means of preventing activation, based on the observation that basic transcription is generally low in higher eukaryotes. On the other hand, activation is often a terminal response to certain physiological signals and requires removal of repressor binding proteins or alteration of chromatin structure in order for an active transcription initiation complex to form.

At the core of transcription complex formation (and a prerequisite for substantial levels of gene expression) is a promoter sequence called the "TATA" box, which is located upstream from the polymerase II transcription start. The TATA box is the binding site for a ubiquitous transcription factor designated TFIID, but as mentioned, transcription of promoter sequences in most genes is predominantly affected by additional regulatory DNA sequences that enhance or repress gene transcription. This type of DNA transcription element is located at various positions relative to the coding sequence in the gene and relative to the TATA box. These additional transcriptional regulatory elements often function in a tissue or cell specific manner.

Of particular importance in recombinant protein expression is the screening for regulatory DNA, which includes promoter TATA sequences and other regulatory elements compatible with the host cell transcription machinery. For this purpose, it is generally preferred to select DNA endogenous to the host cell. Alternatively, the use of regulatory DNA from viral genomic sequences has been significantly successful due to the broad host range of viruses in general and the demonstrated activity of viral regulatory DNA in different cell types. For example, well known and commonly utilized viral regulatory DNA for recombinant protein expression include the SV40 early gene promoter/enhancer [ Dijkema et al, EMBO j.4: 761(1985), Rous sarcoma virus long terminal repeat DNA [ Gorman et al, Proc. Natl. Acad. Sci. (USA) 79: 6777(1982b) ], bovine papilloma virus fragments [ Sarver et al, mo. cell. biol.1: 486(1981) and the human cytomegalovirus promoter/enhancer element [ boshirt et al, Cell 41: 521(1985)]. Although the range of cell types in which viral regulatory DNA has been demonstrated to be functional is wide, it is possible that non-viral promoter/enhancer DNA elements are present which allow for increased expression of recombinant proteins in specific cell lines by more efficient use of host cell transcription machinery.

There is therefore a need in the art to identify promoter/enhancer regulatory DNA sequences that are functional in both homologous and heterologous cell types to increase recombinant protein expression and provide high yields of the desired protein product. Of particular importance is the need to identify the type of promoter/enhancer regulatory DNA that can be most effectively utilized in mammalian cells to increase the in vitro yield of recombinant proteins that are glycosylated in a manner similar to the glycosylation pattern produced by in vivo protein expression. Proteins expressed in this manner and administered therapeutically or prophylactically are less likely to be antigenic and more likely to be physiologically active. Regulatory DNA sequences of this type are also readily inserted into host cells to enhance expression of genes endogenous to the host cell or to enhance expression of genes previously introduced into the genome of the host cell by techniques well known and routinely employed in the art.

Summary of The Invention

The present invention relates to purified and isolated polynucleotides, which are derived from hamster cell regulated gene transcription. The polynucleotide comprises a regulatory DNA sequence 5' of the translational region of Chinese hamster ovary elongation factor-1 alpha (EF-1 alpha). The preferred DNA of the present invention is designated CHEF1 regulatory DNA and comprises about 3.7kb of DNA extending from the SpeI restriction enzyme recognition site to the initiating methionine (ATG) codon of the EF-1. alpha. protein. The invention also includes polynucleotides of less than 3.7kb, as small as the smaller fragments are capable of enhancing transcription from operably linked genes. Active fragments of the DNA of the invention defined as being capable of modulating (i.e., enhancing) transcription of a gene can be readily identified by deletion studies that are well known in the art and are routinely employed. Regulatory DNA of the invention includes polynucleotides isolated from natural sources, such as cell culture, and polynucleotides produced enzymatically or by purely chemical synthesis. Thus, in one embodiment, the invention provides for the preparation of CHEF1DNA from a genomic library. Alternatively, the DNA may be constructed by enzymatic synthesis, using, for example, the Polymerase Chain Reaction (PCR), or pure chemical synthesis, wherein single nucleotides are added sequentially or overlapping oligonucleotides are hybridized and ligated. The most preferred embodiment has the DNA sequence set forth in SEQ ID NO: 1 is given. The invention further includes a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 1, or a DNA sequence to which the DNA given in 1 hybridizes. Stringent conditions include washing at 65 ℃ in a buffer containing about 2 XSSC and about 0.1% SDS or equivalent.

The invention further includes plasmid DNA comprising a CHEF1 regulatory polynucleotide. The plasmids of the invention may also include a DNA sequence encoding a protein of interest or an RNA product of interest operably linked to the CHEF1 polynucleotide sequence. The invention further encompasses cells transformed, transfected and or electroporated with a polynucleotide or plasmid of the invention. The plasmid DNA of the present invention may be integrated into the genome of the host cell or may be present in the cell in the form of a closed circular plasmid. Preferred plasmids of the invention are particularly susceptible to insertion into the desired DNA sequences, including plasmids pDEF14 and pDEF 2. Bacterial host cells transformed with pDEF14 and pDEF2 were stored by the type culture Collection of 12301ParklawnDrive, Rockville, Maryland 20852 at 1997, month 4, day 29, and 1994, month 3, day 4 and designated accession numbers 98398 and 98343, respectively.

The invention also includes linear vector DNA comprising the CHEF1 polynucleotide. The vectors of the invention may be obtained from viral sources or may be synthesized in vitro. The invention further includes host cells transfected or electroporated with the linear vector sequences. This type of DNA is particularly useful for expressing heterologous gene sequences operably linked to the CHEF1DNA sequence or for site-directed homologous recombination where the CHEF1 sequence can be inserted into the genome of a host cell to modulate the expression of the operably linked sequence.

The invention further provides chimeric recombinantDNA molecules in which CHEF1DNA is operably linked (i.e., initiates gene transcription) to a gene sequence encoding a desired protein product. Chimeric molecules are generally those containing functional domains that are not normally found in a wild-type environment to bind; in the present invention, the chimeric DNA may include a portion or all of the CHEF1 regulatory DNA that binds to a DNA sequence other than the gene encoding the hamster EF-1. alpha. protein. Protein products encoded by the chimeric molecules include physiologically active proteins, portions or subunits of active proteins, and marker (or reporter) proteins. The polynucleotide sequence encoding the protein product may be obtained from complementary DNA (cDNA), genomic DNA, synthetic DNA, or DNA derived from a combination of these types of molecules. The protein product may be endogenous to Chinese Hamster Ovary (CHO) cells (i.e., normally found in the CHO genome without DNA introduced by transformation, transfection, etc.). In addition, the protein product may be encoded by an exogenous source, an exogenous source other than the CHO cell genome including, for example, synthetic DNA. Preferred chimeric molecules of the invention include those comprising CHEF1DNA operably linked to DNA encoding: (i) heavy chain of anti-ICAM 3 antibody ICM3, (ii) light chain of ICM (iii) anti-CD 11/CD18 antibody hu23F2G heavy chain, (iv) hu23F2G light chain, (v) chitinase, (vi) platelet activating factor acetylhydrolase (PAF-AH), and (vii) Macrophage Derived Chemokine (MDC). Bacterial host cells transformed with plasmid DNA containing the chimeric molecule were stored in the type culture collection on 1/4 of 1997 and designated accession numbers (i)98381, (ii)98382, (iii)98393, (iv)98384, (v)98385, (vi)98386, and (vii)98387, respectively.

The present invention further relates to host cells transformed or transfected with the CHEF1 DNAs of the present invention. Preferred host cells of the invention are obtained from the chinese hamster (Cricetulus griseus), where CHEF1 regulatory DNA is inferred to provide high levels of protein expression. However, the invention encompasses other cell types of alternative breed origin, including, for example, animal cell types derived from the american hamster (Cricetulus migtoris) or syrian hamster (syrinctus) as well as from other genera. The cells may be derived from lung, ovarian, peritoneal, muscle, spleen, kidney, melanoma, or somatic cell sources, as well as from whole fetuses or whole embryos. The host cells listed above, as well as other cell types of the invention such as myeloma cell lines (in which CHEF1DNA is believed to provide high levels of transcription) may be obtained from the American type culture Collection or may be cultured directly from animal sources.

Recombinant molecules of the invention, including CHEF1 regulatory DNA, will provide for increased mRNA expression levels of operably linked heterologous (i.e., not normally found in the host genome into which the polynucleotide is introduced without transfection, transformation, etc.) polynucleotides. Depending on the nature of the polynucleotide being ligated to the CHEF1 polynucleotide sequence, increased transcription will ultimately result in increased polypeptide levels, or increased levels of various RNAs, in these cases where the ligated polynucleotide encodes, for example, transfer RNA, ribosomal RNA, small nuclear RNA, and the like. The species of RNA may also include antisense RNA complementary to mRNA transcribed from the endogenous or exogenous gene sequence. The increase in translation of the polynucleotide will depend essentially on the presence of appropriate translation signals present in the mRNA.

The invention further includes methods of inserting CHEF1DNA into a specific site in the genomic DNA of a host cell to increase the level of transcription of an endogenous gene sequence. Homologous recombination can be used to insert all or part of the CHEF1 DNA. In a host cell modified in this manner, the CHEF1DNA is operably linked to the encoding DNA sequence. See, e.g., PCT International publication Nos. WO 94/12650; PCT international publication numbers WO 92/20808; and PCT International publication No. WO 91/09955. The present invention necessarily comprises altered genomic DNA sequences in which the CHEF1DNA has been inserted.

Alternatively, the invention encompasses host cells in which the exogenous DNA is inserted into the vicinity and the site of manipulation relative to the CHEF1DNA present in the genome. Such host cells include CHO and the inserted sequence replaces the DNA encoding EF-1 α or is inserted between the CHEF1 regulatory DNA and the DNA encoding EF-1 α. The invention also encompasses host cells that are not CHO cells in which the CHEF1DNA has been previously inserted into the genome and additional DNA is subsequently inserted into an operably linked site.

The invention further provides a method of increasing transcription of a desired gene comprising the step of introducing into a host cell a polynucleotide comprising a hamster EF-1 alpha regulatory sequence integrated into the host genomic DNA at a position operably linked to the desired gene. The invention further comprises a method of enhancing transcription of a desired gene comprising the step of introducing into a host cell a polynucleotide comprising a hamster EF-1 α regulatory sequence and a leader peptide constructed in a manner such that the regulatory sequence at one operable linkage site is integrated into the desired gene encoding a protein other than EF-1 α. The methods of the invention include methods of increasing transcription of a gene that is endogenous to a CHO cell and methods of increasing transcription of a gene that is exogenous to a CHO cell.

The recombinant molecules of the invention can be used to produce transgenic animals in which chimeric recombinant DNA comprising CHEF1DNA operably linked to a DNA sequence of interest is introduced into developing sperm or somatic cells of an animal. For example, the chimeric recombinant molecule can be introduced by microinjection, and when performed with sperm cells, the resulting cells in the animal can all comprise the chimeric recombinant DNA of the invention. Alternatively, the chimeric recombinant DNA of the present invention may be introduced into embryonic cells, and a large number of cells of the resulting animal may contain the DNA of the present invention.

CHEF1DNA can also be used to identify those closely related regulatory sequences that can increase gene expression beyond that permitted by CHEF 1. Similarly, knowledge of the CHEF1DNA sequence allows the construction of synthetic DNAs (whether from de novo synthesis or single or multiple modifications of CHEF1 DNAs) that have sequences similar to CHEF1 but that are capable of promoting higher gene transcription levels.

Detailed Description

The invention is illustrated by the following examples. Example 1 describes the cloning of the hamster EF-1. alpha. gene and the corresponding CHEF1 regulatory DNA. Example 2 relates to subcloning and sequence analysis of CHEF1 regulatory sequences. Example 3 provides features of the CHEF1 promoter polynucleotide, and example 4 provides a description of the method of construction of various expression vectors comprising CHEF1 DNA. Example 5 describes a transfection assay for determining the efficacy of CHEF1 regulatory DNA. Example 6 details comparison of recombinant protein expression levels using CHEF1 regulatory DNA or Cytomegalovirus (CMV) promoter. Example 7 differences in the regulatory ability of different lengths of CHEF1DNA were examined.

Example 1

CHEF1 clone

The cDNA encoding human EF-1. alpha. was used to screen the Chinese hamster ovary genomic DNA library (CHO-K1) (Stratagene, La Jolla, Calif.) in order to isolate the hamster homolog of the human EF-1. alpha. gene.

CHO-K1 library (Lambda)Partial digest of Sau3A in the cloning vector II) was obtained from Stratagene (La Jolla, Calif.) in the form of host cell lines XL-1-Blue MRA and Xll-Blue MRA (P2). The host cells were prepared according to the manufacturer's suggested protocol with the following modifications.

Briefly, cells from glycerol stocks were streaked onto antibiotic-free LB platesOn the board. Screening of monoclonals for inoculation of liquid LB Medium and culture to late logarithmic phase and OD₆₀₀About 0.9. At this point, the cells were stored according to the manufacturer's recommendations or immediately used as follows.

In preparing plated cultures, single colonies were picked from the plates prepared as described above for inoculation and cells were grown in 50 ml of LB medium supplemented with 0.5ml of 1M magnesium sulfate and 1 ml of 1% maltose in deionized water. Following overnight growth at 30 ℃, cells were collected by centrifugation in a tabletop centrifuge (2000rpm, room temperature, 5 minutes) and resuspended in 10 mM magnesium sulfate, OD₆₀₀Is 0.5.

Lambda phage (supplied by the manufacturer) was diluted in SM buffer (1 l stock was prepared containing 5.8 g sodium chloride, 2.0 g magnesium sulfate monohydrate, 50 ml1M Tris-HCI, pH7.5, and 5ml 2% [ w/v ]]Gelatin) concentration range of 10-fold to 10-fold⁵Two fold, and 2. mu.l of each dilution was added to 400. mu.l of host cells (OD) in 10 mM magnesium sulfate₆₀₀0.5). The resulting mixtures were incubated at 37 ℃ for 15 minutes to allow the phage to adsorb to the cells, top agar (0.75% LBM agarose, 48 ℃) was added, and each mixture was plated in duplicate on LBM agarose plates. The results showed a titer of 3X 10⁶Plaque forming units (pfu)/microliter of phage stock.

Fresh host cells were prepared as described above prior to screening the library. About 50,000 phage pfu was added to 600. mu.l of host cells (OD)₆₀₀Ca.0.5) and 6.5ml, 0.75% LBM in 48 ℃ agarose. The mixture was plated on an agarose plate and then incubated at 37 ℃ for about 8 hours. The plate was then cooled at 4 ℃ for 5 hours to prevent the top agarose from sticking to the nitrocellulose cover. BA-85, 0.45 micron pore size, (S + S, Keene, NH) membranes were spread on the plates and transferred for 2 minutes. The membrane was removed from the plate and the transferred DNA was denatured in 1.5M NaCl/0.5 MnaOH for 2 min. The filters were neutralized in 1.5M NaCI/1.0M Tris-HCI (pH8.0), blotted onto Whatman3-MM filter paper, and vacuum dried at 80 ℃ for about 1.5 to 2 hours.

Human EF-1. alpha. cDNA sequence [ Uetsuki, et al J.biol, chem.264: 5791-5798(1989) ] (previously shown to be 95% identical to the CHO EF-1. alpha. coding region [ Hayashi et al, J.biochem.106: 560-5631) was used as a probe for screening libraries and was prepared as follows. The 1.4kb human EF-1. alpha. probe was obtained from plasmid M0107, a human EF-1. alpha. cDNACRc/CMV (Invitrogen, San Diego, CA) vector containing the insertion at the recognition site of EcoR 1. To preliminary confirm that plasmid M0107 contains the desired human cDNA, the insert DNA was sequenced using the 3 'and 5' vector primer pairs.

94-17AGGCACAGTCGAGGCTGATC(SEQ ID NO：2)

94-18TTCCAGGGTCAAGGAAGGCA(SEQ ID NO：3)

The first 263bp of the insert shows greater than 90% identity to the published human EF-1 alpha coding sequence, while its 3' end sequence is difficult to determine accurately, and small extension fragments can be aligned with the desired sequence. Taken together, these sequence alignments indicate that the plasmid encodes the desired sequence.

The entire human insert was removed from the plasmid by EcoRI digestion and a 1.4kb cDNA band gel purified using the QIAGEN QIA quick gel extraction kit according to the manufacturer's recommendations. The DNA was eluted into 50. mu.l TE, the resulting solution was concentrated to 25. mu.l in Microcon-10 and labeled with the Boehringer Mannheim Random Primed DNA labeling kit, according to the manufacturer's protocol, in order to obtain a DNA fragment of the desired size³²P-alpha-dTTP and³²P-alpha-dCTP was used to label one portion of the reaction solution. The labeled probe was purified using a G50 spin column to remove unincorporated nucleotides and a comparison of the purified probe with the reaction solution before incorporation showed 46% incorporation of the radiolabel, counted 5X 10⁵cpm/min/microliter.

The nitrocellulose membrane prepared as above was probed as follows. Pre-hybridization/hybridization stock buffers were prepared, which included 22.5ml of 20 XSSC, 30.0ml of 50 XDenhardt's solution, 3.0ml of 1M phosphate buffer (pH 6.8) (69ml of 1M sodium dihydrogen phosphate, 31 ml of 1 molar disodium hydrogen phosphate), 0.75ml of 20% SDS, and 78.75 ml of distilled water. For prehybridization, 1.4ml of 10mg/ml salmon sperm DNA (Stratagene) was boiled with 0.6 ml of distilled water for 5 minutes, and then 7 ml of distilled water and 72 ml of stock buffer were added. The filter paper was incubated at 65 ℃ for a minimum of 2 hours in the prehybridization buffer.

For hybridization, 30. mu.l of the probe, 200. mu.l of 10mg/ml salmon sperm DNA and 770. mu.l of distilled water were combined, boiled for 5 minutes, and 36 ml of a stock buffer containing 3 ml of distilled water was added. The pre-hybridization solution was removed from the filter, hybridization buffer was added and the filter was allowed to stand overnight at 65 ℃. After hybridization, 3 times of total washes with 2 XSSC and 0.1% SDS-containing buffer at 65 ℃ for 3 hours, followed by autoradiography for 48 hours.

47 positive clones were identified and picked into 1 ml SM buffer containing 20. mu.l chloroform. To eliminate possible pseudogenes missing one or more introns, pairs of PCR primers were designed flanking each of the two introns: primers 95-136(SEQ ID NO: 4) and 95-137(SEQ ID NO: 5) flank introns 2 and 3; primers 95-138(SEQ ID NO: 6) and 95-139(SEQ ID NO: 7) flank introns 3 and 4; primers 95-140(SEQ ID NO: 8) and 95-141(SEQ ID NO: 9) flank introns 4 and 5; primers 95-142(SEQ ID NO: 10) and 95-143(SEQ ID NO: 11) flank introns 6 and 7.

95-136(SEQ ID NO：4)GCCACCTGATCTACAAATGT

95-137(SEQ ID NO：5)GAGATACCAGCCTCAAATTC

95-138(SEQ ID NO：6)ATGTGACCATCATTGATGCC

95-140(SEQ ID NO：8)GTTGGAATGGTGACAACATG

95-141(SEQ ID NO：9)CAGGTTTTAAAACACCAGTC

95-142(SEQ ID NO：10)AATGACCCACCAATGGAAGC

95-143(SEQ ID NO：11)ACAGCAACTGTCTGCCTCAT

Use ofThe expected size of the PCR product of the CHO EF-1. alpha. template DNA is based on the size and position of the intron in the published human EF-1. alpha. sequence. Each PCR reaction solution contained 2. mu.l of phage, 2.5. mu.l of each appropriate primer pair (100. mu.g/ml), 2. mu.l of a 2 mM dNTP mix, 2.5. mu.l of a 10 XPCR buffer (Roche Mol ecll ar Systems, Branchburg, N.J.), 1.5. mu.l of 25 mM magnesium chloride, 0.125. mu.l of Taq polymerase (5 units/l) (Perkin Elmer) and 11.8. mu.l of distilled water. The amplification reaction was performed at 94 ℃ for 4 minutes, followed by 30 cycles of 90 ℃ for 1 minute, 50 ℃ for 2 minutes, and 72 ℃ for 4 minutes. The amplification products were separated on a 1.2% agarose gel at 1 XTAE. Three of the 47 positive plaques were found to encode true genes containing all introns (nos. 2, 7 and 40) and the three positive samples were subjected to the following tertiary screening. According to a ratio of 10 to 10⁵Dilution the stock prepared with each of the three positive samples was used to prepare plate cultures as described above. Screening 20 to 50 plaques isolated from each stock by PCR gave the following results. Clone No. 2 produced two positive plaques (designated 2.12 and 2.17) from 20 selected plaques, clone No. 7 produced one positive plaque (designated 7.44) from 50 selected plaques, and clone No. 40 produced one positive plaque (designated positive 40.24) from 40 screens. Phage DNA was isolated from each of the four samples as follows.

XL-1Blue MRA (P2) was streaked onto LB agarose plates and grown overnight at 37 ℃. A single clone was isolated to inoculate 50 ml of LB medium (0.2% maltose and 10 mM magnesium sulfate) and the culture was grown overnight at 30 ℃. Cells were harvested by centrifugation at 2000rpm for 5 minutes at room temperature in a bench top centrifuge and resuspended in 50 ml 10 mmol magnesium sulfate. The resuspended host cells (50. mu.l) were mixed with 100. mu.l of each phage stock and incubated at 37 ℃ for 15 minutes to allow phage adsorption to the cells. About 500. mu.l of LBM medium was added and mixed at 37 ℃ with shaking for 2 hours. An additional 200. mu.l of host cells were added and incubation continued at 37 ℃ for an additional 15 minutes. Top agar (8 ml of 0.75% LBM agarose, 48 ℃) was added, after which the mixture was plated and grown overnight at 37 ℃.

Following overnight growth, 12 ml of lambda diluent (10 mmol Tris-HCI, pH7.5, 10 mmol magnesium sulfate) was added to each plate and the plates were shaken gently for 2 hours. The diluent was removed and centrifuged at 4000 Xg for 10 minutes in a bench top centrifuge. To the supernatant, 1 mg/ml each of RNase A and DNase I was added 1. mu.l and incubated at 37 ℃ for 15 minutes. An equal volume of precipitation buffer (20% PBG8000, 2M sodium chloride, 20 mmol Tris-HCI, ph7.5, 10 mmol magnesium sulfate) was added, followed by ice incubation for 1 hour, after which the mixture was centrifuged at 8000 xg for 20 minutes, the supernatant was discarded, and the pellet was air-dried at room temperature for 10 minutes. The sediment was resuspended in 500. mu.l of TE, centrifuged briefly to remove particles, and the supernatant was transferred to a clean 1.65 ml microcentrifuge tube. 2.5. mu.l of 20% SDS (incubation at 65 ℃ for 5 minutes) were added, followed by 2.5. mu.l of 10mg/ml proteinase K (incubation at 65 ℃ for 1 hour) and 10. mu.l of 5 mol sodium chloride. The mixture was extracted 1 time with an equal volume of phenol to chloroform and 1 time with an equal volume of chloroform. An equal volume of isopropanol was added, followed by 3 hours of incubation at-70 ℃, after which the mixture was centrifuged in a microcentrifuge for 15 minutes at maximum speed. The resulting sediment was washed with 70% ethanol, air dried, and resuspended in 100. mu.l of TE.

Example 2

Secondary cloning of EF-1 alpha regulatory sequences

To determine the size of the insert EF-1. alpha. DNA, the phage DNA prepared as in example 1 was digested with NotI and the resulting restriction fragments were separated on a 0.6% 1 × TAE agarose gel. Clones 2.12 and 2.17 showed the same enzymatic banding pattern, with bands of 11kb and 4.5kb in addition to the expected 19kb and 10kb lambda fragment flanks. Clones 7.44 and 40.24 also showed the same band pattern with 12kb and 7kb insert bands, which together with the digestion data of clones 2.12 and 2.17 indicated the presence of an internal NotI restriction enzyme recognition site in the EF-1. alpha. DNA. Inserts derived from clones 2.12 and 7.44 were subcloned as follows.

Phage DNA (60. mu.l) prepared as described in example 1 was digested with NotI, after which the digested DNA was precipitated by addition of 20. mu.l of 3M sodium acetate and 400. mu.l of 100% ethanol. The precipitated DNA was collected by centrifugation, washed in 200. mu.l of 40% ethanol, air-dried for 15 minutes, resuspended in 20. mu.l of TE and heated to 65 ℃ for 10 minutes, and then 2. mu.l of loading buffer was added for electrophoresis. The DNA was separated by agarose gel electrophoresis and gel sections of 4.5kb, 7kb, 11kb and 12kb were excised. DNA was extracted from each gel section using the QLAGEN QIA Rapid gel extraction kit according to the manufacturer's recommendations. The purity and concentration of the bands were assessed by separating the fragments separated on a 5 microliter aliquot of 0.6% 1 × TAE agarose gel.

Each fragment was separately ligated to NotI-digested pBluescript SW⁺. During ligation with the 11 and 12kb fragments, the linearized vector was treated with bovine alkaline phosphatase before introduction of the insert. Two microliters of each linker was used to electroporate 40 microliters of XL-1Blue electrocompetent cells. Transformed cells were plated on LBM/carb agarose plates containing 40. mu.l of 5% X-gal Dimethylformamide (DMF) and 20. mu.l of 0.1 molar IPTG per plate. Cells were cultured overnight at 37 ℃ and plates were moved to 4 ℃ in the morning to increase the intensity of the blue color.

In a second round of screening, white colonies were streaked onto LBM/carb agarose plates containing X-gal and IPTG as described above and on the following day still white colonies were further grown overnight at 37 ℃ in 3 ml LBM/carb. Plasmid DNA was prepared from overnight grown white colonies using the WIZARD Plus miniprep DNA purification System (Promega, Madison, Wis.) according to the manufacturer's recommendations.

Restriction enzyme analysis of the isolated plasmid DNA showed that the 4.5kb, 7kb and 12kb fragments were successfully ligated into pBluescript SW⁺In the vector, and the resulting plasmids were digested pSK/EF1.4.5, pSK/EF1.7 and pSK/EF1.12, respectively.

Each plasmid was then prepared using a QIAGEN Medium preparation (midi prep) kit according to the manufacturer's recommendations. PCR was performed on each new vector, template DNA was titrated and the coding region primers (SEQ ID NOs: 4 to 11, used in pairs as described above to screen out pseudogenes lacking one or more of the various introns) were used. After all three fragments were found (at high concentrations) to contain the entire EF-1 coding region, suggesting that there could be cross-contamination between the three plasmid preparations, titration was performed. In the titration, PCR was performed in a reaction solution containing 2.5. mu.l of template DNA at a concentration of 0.001, 0.01, 0.1, 1 or 10 ng/ul and containing 2.5. mu.l of 10 XPCR buffer (Perkin Elmer), 2.0. mu.l of 2 mM dNTP mix, 1.5. mu.l of 25 mM magnesium chloride, 0.125. mu.l of Taq polymerase (Perkin Elmer) and 11.4. mu.l of distilled water. Amplification was performed under the following conditions: 94 ℃ for 4 minutes followed by 30 cycles of 90 ℃ for 1 minute, 50 ℃ for 2 minutes, 72 ℃ for 4 minutes. The results indicated that the entire EF-1. alpha. coding region was located within the 4.5kb and 7kb fragments.

By design with Lamda FIXThe Stratagene FLASHNoradioactive Gene Mapping Kit used with the vector was restriction-enzyme mapped to each insert. However, instead of using phage DNA, plasmid DNA cut out from the above pSK vector by digestion with NotI was used. The mapping method is basically recommended by the manufacturer. Briefly, M13(SEQ ID NO: 12) and M13 reverse (SEQ ID NO: 13) primers (in contrast to the pBluescriptSW)⁺Region complementary within the polyclonal region) to locate T3(SEQ ID NO: 14) and T7(SEQ ID NO: 15) a primer sequence.

M13 GTAAAACGACGGCCAGT (SEQ ID NO：12)

M13rev GGAAACAGCTATGACCATG (SEQ ID NO：13)

T3 AATTAACCCTCACTAAAGGG (SEQ ID NO：14)

T7 GTTAATACGACTCACTATAGGGC (SEQ ID NO：15)

The T7 and 73 primers were used as probes in the gene mapping method. Since the EF-1. alpha. insert was shown to contain an internal NotI recognition site, the pair of fragments (4.5kb/11kb and 7kb/12kb) were considered to include this or that sequence in the primer sequence, and thus it was determined that the 4.5 and 12kb inserts contained the T7 primer sequence, while the 7kb insert contained the T3 sequence.

The 4.5kb and 7kb inserts containing the EF-1. alpha. coding region were excised from the vector by NotI digestion. The digested DNA was separated on an agarose gel, the separated fragments were excised from the gel, and DNA was isolated from each gel slice using the QIAGEN QIA Rapid gel extraction kit according to the manufacturer's recommendations. Mapping was performed in partial restriction lysates using seven different enzymes. The reaction products were separated on an agarose gel, and the DNA was transferred to a Duralon-UV Nylon membrane and probed with T3 or T7 oligonucleotide. The band is sized and a restriction map is formed.

Plasmid Psk/EF1.7 was sequenced using the internal primers originally designed for the PCR screening (SEQ ID NOs: 4 to 11[ primers 95-136-95-143 described previously ]) to ensure that the gene sequence was the previously identified protein sequence. The coding regions were oriented and additional primers designed to allow sequencing to reach the internal NotI recognition site. The entire coding region was sequenced and then compared to the expected cDNA sequence described in Hayashi et al [ supra ]. Sequence analysis confirmed that the isolated DNA did indeed encode the same EF-1. alpha. sequence as described in Hayashi [ supra ]. Furthermore, the first exon sequence was identical to the previously disclosed hamster EF-1. alpha. cDNA sequence located at the 5' end and the seven introns identified gave a structure similar to known human homologous sequences.

The sequence and restriction map obtained from the 7kb fragment indicated that a portion of the 5 'intron and the promoter were located in the 12kb fragment 5' to the internal NotI recognition site. A SpeI/NotI 3kb fragment 5' of the internal NotI site was excised from the 12kb insert and subcloned into pBluescriptSK digested beforehand with the same enzymes⁺The resulting plasmid was designated pSK/EF 1.3. To the 3kb pieces separatelyThe fragments were mapped with KpnI and ClaI as described above to confirm restriction sites and sequenced using the ClaI and KpnI sites which create 5 'and 3' overhangs using an Erase-a-Base kit (Promega, Madison, Wis.). Sequence analysis indicated that the promoter, TATA box, and the human gene were involved [ Uetsuki, 1989, supra ]]A region of 0.9kb intron in the sequence flanking the 5' untranslated region of the same length as the first intron. The CHEF1 promoter sequence and 5' intron are set forth in SEQ ID NO: 1, the intron includes nucleotides 2699 to 3641.

Example 3

Features of CHEF regulatory DNA

Upstream of the sequence of the hamster EF-1 α gene and including the ATG start code at SEQ ID NO: 1. Most of the identifiable transcription factor binding sites that appear are located 3 'of the upstream SacI site, which is located 5', 1.56kb of the initiating ATG codon of the EF-1. alpha. gene. However, the expression vector including the CHEF1 sequence described below also contained 2kb of CHEF1 sequence 5' of the SacI site.

Sequencing indicated the presence of a complete consensus TATA box of about 1kb located 5' to the initiation ATG initiation codon, and the region between the upstream SacI site and TATA box contains multiple potential transcription factor binding sites [ boulukas, crit. 117-321(1994) ], including the Sp1 site (SEQ ID NOs: 16 or 17), the ATF site (SEQ ID NO: 18), and the NF-1 site (SEQ ID NO: 19).

GGCGGG SEQ ID NO：16

GGGCGG SEQ ID NO：17

TGACGY(C/A)R SEQ ID NO：18

TTGGCN_5-6(T/G)CCR SEQ ID NO：19

Similar to the human EF-1. alpha. gene, [ Uetsuki et al, supra ], there is a 943bp intron in the 5' untranslated region (UTR) of the hamster EF-1. alpha. gene, apparently from the site of the splice donor and acceptor sequences. However, using the Geneworks DNA analysis program, intron sequences in the 5' UTR were found to be only 62% identical to the corresponding sequences in the human gene. The 5 'intron includes a number of potential transcription factor binding sites, roughly equal to the number of binding sites between the SacI site and the TATA box, indicating that the 5' intron may be important for optimal transcription from the EF-1. alpha. promoter.

Using the Geneworks DNA analysis program, the CHEF1 sequence of the upstream SacI restriction site downstream of (but not including) the 5' intron sequence was found to be 64% identical to the human EF-1. alpha. sequence. A comparison of the positions of transcription factor binding sites in humans and hamsters is shown in table 1. The complete human EF-1 alpha gene sequence [ Uetsuli et al, supra ] is shown in SEQ ID NO: 29 is given in.

TABLE 1

Distance from Sp1 site to TATA box

Hamster human

Nucleotide position

-424 -335

-304 -220

-183 -208

-171 432

-151 476

-135 573

-26 581

63 690

156

168

257

261

425

495

589

594

688

Example 4

Construction of expression plasmids

A plurality of plasmids used in the following examples were constructed as follows.

The plasmid pSV2-DHFR (ATCC accession No. 37146) was digested with SphI/BamHI and the 1.8kb fragment (fragment 1) encoding dihydrofolate reductase (DHFR) was purified. The DHFR-encoding fragment also includes the SV40 promoter/operator and polyadenylation sequences located 5 'and 3', respectively, associated with the DHFR gene. Plasmid pSL1190(Pharmacia) was digested with HindIII, the ends of the overhang were filled in with Klenow, and the blunt-ended DNA was religated to disrupt the HindIII site to give plasmid pSL1190H, which was then digested with SphI/BamHI and the 3.4kb fragment (fragment 2) was purified. The pSV2-dhfr1.8kb fragment (fragment 1) was ligated to the pSL11903.4kb fragment (fragment 2) to give plasmid pSL 1190H-dhfr.

Plasmid pSL1190H-dhfr was modified to remove several restriction sites as follows. Plasmid pSL1190-dhfr was first digested with XbaI/NheI (to generate complementary overhang ends) and the linear plasmids were religated to remove the XbaI and NheI sites. In a second procedure, pSL1190H-dhfr was digested with HindIII, the overhang ends were filled with Klenow, and the linear plasmids were religated to remove the HindIII site. In a third procedure, pSL1190H-dhfr was digested with BglII, the overhang ends were filled with Klenow, and the linear plasmids were religated to remove the BglII site. The final result of these three steps yielded plasmid pSL1190H-dhfr/NXHB, in which the XbaI, Nhe I, HindIII, and BglII sites were disrupted. Plasmid Psl1190-dhfr/NXHB was then digested with EcoRI/BamHI and inserted into an NPB1/NPB2 linker (from the annealed oligonucleotides NPB and NPB2[ SEQ ID NOs20 and 21]) to generate plasmid pSL/dhfr/NotI containing unique NotI restriction sites.

NPB1 GATCGCGGCCGCGTTTAAACGGATCC (SEQ ID NO：20)

NPB2 AATTGGATCCGTTTAAACGCGGCCGC (SEQ ID NO：21)

The resulting plasmid pSL/DHFR/NotI was digested with Asp718/BamHI and the 1.8kb fragment encoding DHFR (fragment 3) was purified. Plasmid pRc/CMV (Invitrogen, San Diego, Calif.) was digested with Asp718/BglII to remove the CMV promoter and Bovine Growth Hormone (BGH) polyadenylated DNA and to purify the 3.7kb fragment (fragment 4). A1.8 kb pSL/DHFR/NotI fragment encoding DHFE carrying the SV40 promoter/operator and polyadenylation sequences (fragment 3) was ligated to the 3.7kb pRc/CMV fragment (fragment 4) to generate plasmid pRc/DHFR/NotI.

Plasmid pRc/CMV was digested with Asp718/XbaI and a 0.8kb fragment encoding BGH polyadenylation DNA was purified (fragment 5). Plasmid pRc/DHFR/NotI was digested with BamHI/Asp718 and the 4kb fragment containing DHFR, SV40 promoter/operator and polyadenylation sequences was purified (fragment 6). Plasmid pCEP4(Invitrogen, San Diego, Calif.) was digested with BglII/SacI and the 0.7kb fragment encoding the CMV promoter (fragment 7) was purified. The 0.8kbpRc/CMV fragment (fragment 5), the 4kb pRc/DHFR/NotI fragment (fragment 6), the 0.7kbpCEP4 fragment (fragment 7), and the synthetic SacI/XbaI adaptor fragment were ligated in a 4-way (4-way) ligation reaction to generate plasmid pDC 1. Adaptors are constructed by annealing oligonucleotides SXP1 and SXP 2.

SXP1(SEQ ID NO：22)

5' -CGTTTAGTGAACCGTCAGATCTACATAAC from CATTCTC

CTCTAAAGAAGCCCCAAGCTTGATATCTGCAGAATTCT-3′

SXP(SEQ ID NO：23)

5′-CTAGAGAATTCTGCAGATATCAAGCTTGGGGCTTCTTTAGAG

GAGGAATGTTGTTATGTAGATCTGACGGTTCACTAAACGAGCT-3′

The plasmid pDC1 thus comprises the CMV promoter, a polylinker region, a polyadenylation site from BGH, and the dhfr gene under the control of the SV40 promoter as well as the SV40 splice/polyadenylation sequence.

Plasmid pDC1 was digested with XhoI, and a 4.5kb fragment lacking the CMV promoter and BHG polyadenylation DNA was isolated and ligated to yield plasmid pDCi 1. Plasmid pDCI1 was then digested with BamHI/XhoI and the 4.5kb fragment (fragment 8) was purified. This 4.5kb fragment was ligated into a BamHI/SalI digested PCR fragment encoding part of the CMV promoter sequence generated with primers 96-13 and 96-14 (plasmid pDC1 as template) to generate plasmid pDCI 2.

Primer 96-13(SEQ ID NO: 24)

5′-AGTTCAGTCGACGGCGCGCCAACCCGGGAATCC

GGACGGGATCTATACATTGAATCAATATTGGCA-3′

Primer 96-14(SEQ ID NO: 25)

ATGTCAGGATCCACGCGGAACTCCATATATGGGCTATGAACT

Plasmid pDCi2 was digested with Asp718/SpeI and purified to encode a DHFR4.2kb fragment with SV40 promoter/operator and polyadenylation sequences (fragment 9), pDC1 was digested with Asp718/SpeI and a 1.5kb fragment containing part of the CMV promoter DNA was purified with the BGH polyadenylation signal (fragment 10). The 4.2kb pDCI2 fragment (fragment 9) was ligated to the 1.5kb pDC1 fragment (fragment 10) to generate plasmid pDC 31. Plasmid pDC31 differs from pDC1 in that several new restriction sites were created, including SmaI and AscI 5' to the CMV promoter. Plasmid pDC31 was digested with NotI, DNA ends were hung flush with T4 polymerase and the plasmid was religated to remove the NotI site and produce plasmid pDC 36. Plasmid pDC36 was identical to pDC31 except that the NotI was disrupted.

Plasmid pDC36 was digested with ApaI/BamHI, the overhang ends were filled in, and the plasmid was religated to yield pDC 38. pDC38 was identical to plasmid pDC36 except that the ApaI/BamHI fragment containing the bovine growth hormone polyadenylation sequence had been removed. Plasmid pDC38 was digested with SmaI/HindIII and the 4.6kb fragment lacking CMV promoter DNA (fragment 11) was purified.

Plasmid pSK/EF1.3 was digested with EcoRV/NotI/PvuI and the 2.9kb fragment (fragment 12) encoding the CHEF1 promoter and upstream DNA sequences was purified. Digestion of Bluescript SW with NotI⁺II(pSK⁺) And the 2.9kb fragment (fragment 13) was purified. The 7kb NotI fragment from bacteriophage lambda 7.4 was ligated into 2.9pSK⁺Fragment (fragment 13) to generate plasmid pSK/EF 1.7.

Plasmid pSK was digested with HindIII/NotI⁺And the 2.9kb fragment (fragment 14) was purified. The pSK/EF1.7 plasmid was digested with NotI/NcoI and a 620bp fragment encoding part of the intron of the untranslated region of CHEF 15' was purified (fragment 15). A123 bp HidIII/NcoI digested PCR fragment encoding the remainder of the 5' intron was generated with primers 96-36 and 96-45 and pSK/EF1.12 was purified as template (fragment 16).

Primer 96-36(SEQ ID NO: 26)

GGCTTAGCTCCGAGGAGGG

Primer 96-45(SEQ ID NO: 27)

CGTGACAAGCTTGGTTTTCACAACAC

Ligation of the 2.9kb pSK⁺Fragment (fragment 14), the 620bp pSK/EF1.7 fragment (fragment 15), and the 123bp HindIII/Ncoi fragment (fragment 16) to generate plasmid pSK/5 'EF-1 containing the complete CHEF 15' intron sequence.

Plasmid Psk/5 'EF-1 was digested with HindIII/NotI and the 0.7kb fragment encoding the 5' intron (fragment 17) was purified. A4.6 kb pDC38 fragment lacking the CMV promoter (fragment 11), a 2.9kbpSK/EF1.3 fragment with the CHEF1 promoter and upstream sequences (fragment 12), and a 0.7kb pSK/5 ' EF-1 fragment encoding the entire 5 ' intron (fragment 17) were ligated in a 3-way (3-way) ligation reaction to produce plasmid pDEF1, whereby this plasmid contained the entire 5 ' CHEF1 regulatory DNA sequence, the dhfr gene, and the SV40 origin of replication, which enabled replication in SV40T antigen-transformed cell lines to very high copy levels.

Plasmid pDEF1 was digested with HindIII, the overhang ends were filled in, and the plasmid was digested with NotI and a 2.6kb fragment containing the CHEF1 promoter and the upstream DNA sequence in addition to part of the 5' intron sequence (fragment 18) was purified. Plasmid pDEF1 was digested with NotI/Asp718 and the 1.3kb fragment encoding the remainder of the 5' intron (fragment 19) was purified. Plasmid pDC38 was digested with SmaI/Asp718 and purified to encode a 4kb fragment (fragment 20) carrying the SV40 promoter/operator and DNA. The 2.6kb pDEF1 fragment (fragment 18), the 1.3kb pDEF1 fragment (fragment 19), and the 4kb pDC38 fragment (fragment 20) were ligated in a 3-way (3-way) ligation reaction to yield plasmid pDEF 2. The plasmid pDEF2 in E.coli strain XL-1Blue was stored on 4.4.1997 at the American type culture Collection of 12301Parklawn Drive, Rockville, MD 20852 and designated accession number 98343. Plasmid pDEF2 differs from pDEF1 in that the 0.3kb HindIII/speI fragment 2.3kb upstream of the TATA box of CHEF1 was removed and contained only one HindIII site in the polylinker region. In the case where the gene to be expressed includes its own polyadenylation site, pDEF2 was used.

The plasmid pDEF2 was digested with Asp718/XbaI to linearize the plasmid, in addition to which the origin of replication sequence was removed and the 7.4kb fragment (fragment 21) was purified. Plasmid pDC1 was digested with Asp718/XbaI and a 0.8kb fragment (fragment 22) comprising the replacement origin or replication DNA in addition to the BGH polyadenylation sequence was purified. The 7.4kb pDEF2 fragment (fragment 21) and the 0.8kbpDC21 fragment (fragment 22) were combined to produce plasmid pDEF10, which differs from pDEF1 in the same manner as pDEF 2. Plasmid pDEF10 differs from pDEF2 in that the polyadenylation site of the bovine growth hormone gene is located 3' to the polylinker region of pDEF 10.

Plasmid pDEF10 was digested with HindIII/XbaI to linearize the plasmid and purify the 8.2kb fragment (fragment 23). Plasmid pDC1/MDC, a plasmid encoding a human chemokine, macrophage-derived chemokine (MDC), was digested with HindIII/XbaI and the 0.4kb fragment encoding MDC was purified (fragment 24). The 8.2kbpDEF10 fragment (fragment 23) was ligated with the 0.4kbpDC1/MDC fragment (fragment 24) to generate plasmid pDEF 10/MDC.1.

The plasmid pRc/CMV was digested with BamHI, the overlapping ends were filled with Klenow, the plasmid was digested with Asp718, and the 1.5kb fragment (fragment 25) was purified. Plasmid pDC1 was digested with NotI, the overlapping ends were filled with Klenow, the plasmid was digested with Asp, and the 3.9kb fragment (fragment 26) was purified. The 1.5kb pRc/CMV fragment (fragment 25) was ligated with the 3.9kb fragment (fragment 26) to generate plasmid pNCX.

Plasmid pNCX is similar to the pDC1, except that the dhfr gene is replaced by a neomycin resistance gene (NeoR). Plasmid pNCX was digested with Asp718/Pvu and the 2.1kb fragment (fragment 27) was purified. Plasmid pDEF1 was digested with Asp718/PvuI and the 5.9kb fragment (fragment 28) was purified. The 2.1kb pNXC fragment (fragment 27) was ligated to the 5.9kb fragment (fragment 28) to generate plasmid pNEF 1. pNEF1 differs from pDEF1 in that the plasmid carries a bacterial neomycin resistance gene encoding neomycin or G418. The gene inserted into pNEF1 is generally a HindIII/XbaI fragment inserted following partial digestion with HindIII or 3-way (3-way) ligation, since the plasmid has two HindIII sites.

Example 5

Transfection and Productivity assay of DG44 cells

For transfection of host DG44 cells with a single plasmid, 50-100 micrograms of plasmid were typically linearized by restriction enzyme PvuI or AscI digestion. For transfection in which both plasmids were introduced into CHO cells, the plasmids were still undigested. Before transformation, ethanol precipitation of plasmid and 70% ethanol washing twice. The DNA pellet was briefly dried and resuspended in 400. mu.L sterile, distilled water. To the resuspended DNA was added 400. mu.l of sterile 2X HeBS (40 mM HEPES-NaOH, pH 7.0; 274 mM NaCI; 10 mM KCI; 1.4 mM disodium hydrogen phosphate; 12 mM glucose). Untransfected DG44 cells were cultured in DMEM/F-12 medium (also referred to as "HT") supplemented with hypoxanthine (0.01 mM final concentration) and thymidine (0.0016 mM final concentration).To grow both untransfected and transfected DG44 cells, dialyzed FBS was added to the medium to produce final concentrations of 5 to 10% by volume. DG44 cells were prepared for transfection by growing cultures to 50% or less of a confluent treated 150 square centimeter tissue culture polystyrene bottle (Corning). The cells were removed by first aspirating the medium, washed once with calcium-free magnesium phosphate buffered saline (CMF-PBS: 2.7 mM KCI; 1.5 mM potassium dihydrogen phosphate; 137 mM NaCI; 8.1 mM dipotassium hydrogen phosphate), added with 4ml of CMF-PBS containing 0.0125% radiant trypsin (Worthington Biochemical Corporation) and incubated at 37 ℃ for several minutes. The cell concentration was measured by adding a medium (4 ml) containing Fetal Bovine Serum (FBS), and the cell concentration was determined by centrifugation to 2X 10 portions⁷The cell of (1). Each cell pellet was washed once in CMF-PBS and resuspended in 0.8 mL of HeBS-containing solution along with the desired plasmid DNA. The resuspended cells were transferred to a 0.4 cm Gene Pulser cuvette (Bio-Rad) at room temperature and placed in a Bio-Rad Genepulser electroporator. At room temperature, 290 volts and 960 μ FD (9 to 11.5msec pulse) was discharged with a capacitor. The cells were kept in the sample cell for about 10 minutes and then added to 10 ml of 5-10% dialyzed FBS and HT supplemented DMEM/F-12. Lethality was measured at this point (trypan blue excluded) and would typically be found to be about 50%. The cells were then pelleted by centrifugation, resuspended in 2 ml of DMEM/F-12 with 5-10% dialyzed FBS and HT ("non-selective medium") and plated into 10 cm polystyrene tissue culture plates. After two days of growth (usually at this point confluent) the cells were removed from the plates as described above with trypsin and plated into various dilutions of DMEM/F-1210 square centimeter plates supplemented with 5-10% dialysis FBS without HT ("selective media"). After 5 and 9 days, the cells were fed with selective medium. After about two weeks, transfected cell colonies (typically more than 1,000 per transfection) were removed from the plates with trypsin as described above and the concentration of the cells was determined after addition of selective media. At least two 2X 10 aliquots were taken from each transfection⁶Cells were added to 10 cm plates containing 10 ml of selective medium. Culturing the cells to extinction (extint)ion), i.e., when most cells have fallen off the plate due to overcrowding. Supernatants from the extinct cultures were tested by ELISA to determine the mean product titer.

Example 6

Production of recombinant proteins using CHEF1

DG44 cells described in example 5 were transfected with plasmids carrying the genes given in table 3 operably linked to CHEF1 regulatory DNA. Bacterial strains transformed with plasmids encoding each of the genes used in the assay were stored to the american type culture collection of 12301ParklawnDrive, Rockville, Maryland 20852 on 1/4 1997 and designated as accession numbers as given in table 2.

TABLE 2

Accession number of plasmid

Encoded gene Nomenclature of plasmids Login number

Antibody ICM3 heavy chain pDEF1/ICM 3H.298381

Antibody ICM3 light chain pNEF1/ICM 3L.398382

Antibody 23F2G heavy chain pDEF 1/F2GH.198383

Antibody 23F2G light chain pNEF 1/F2GL.198384

Chitinase pDEF 1/CTN.198385

Platelet activating factor pDEF 2/HPH.498386

Acetyl hydroxylase

(PAF-AH)

Macrophage derived pDEF 10/MDC.198387

Chemokines (MDC)

After selection of transfectants, pools of cell colonies (over 200) from each transfection were removed from each plate with trypsin and a consistent number of cells from each transfection were replated and grown to extinction. Supernatants were removed and tested for protein expression using ELI SA or using an enzyme assay. The results are shown in table 3.

TABLE 3

Protein expression using CHEF1 and CMV regulatory sequences

Protein titer

Transfected gene CMV CHEF1

ICM3H+L 554 1862

ICM3H+L 288 2153

Hu23F2G H+L 337 1848

MDC 360 2100

PAF-AH 590 6574

Chitinase-132002300

Chitinase-2890012850

In addition to the first experiments to examine chitinase expression (designated chitinase-1 in Table 3), the use of CHEF1 to regulate DNA resulted in significantly higher levels of protein production, ranging from 3-fold to 11-fold higher than expression with the CMV promoter.

To determine whether the increased expression of chitinase observed in the test is an abnormality or unique to chitinase and/or a reproducible conclusive feature thereof, the experiment was performed a second time, but using two separate transfections, as opposed to the single transfection used in the first experiment which produced an abnormally low number of transfectants. In addition, a different chitinase activity test method was used, which proved to be more accurate than the method used in the first experiment.

The number of transfectants produced by transfection in the second experiment was more consistent with the results previously observed with the pDEF2 plasmid, which in additional experiments produced more than 150% of the number of transfectants using the CMV plasmid. The results obtained in the second experiment (chitinase-2, named in Table 3) are essentially identical, providing further evidence that even the observed increase in protein expression over that expressed with the CMV promoter (about 1.4 fold) is lower than that observed for other proteins previously tested.

Example 7

Production of recombinant proteins with varying lengths of CHEF1 regulatory DNA

An expression vector containing an additional 8kb of EF-1. alpha. gene 5' flanking DNA was also constructed and designated pDEF 14. Plasmid pDEF14 differs from pDEF2 in that pDEF14 contains 11.7kb of hamster EF-1. alpha.5' flanking DNA, whereas pDEF2 contains only 3.3kb of the same sequence. Plasmid pDEF14 also included 4kb hamster 3 'flanking DNA adjacent to the 3' end of the DHFR expression cassette. The larger pDEF14 plasmid was constructed as follows.

Briefly, the 2.6kb AscI/NotI CHEF1 fragment was removed from pDEF2 and an 11kb AscI/NotI CHEF fragment was inserted in its place. Insertion of the larger sequence first requires modification of the Xbal site located 5' 11.7kb of the ATG starting from EF-1. alpha. to the AscI site. In addition, a 4kb blunt NsiI/SalI fragment (containing the CHEF1 flanking sequence starting at the NsiI site 3 ' 118bp from the EF-1. alpha. termination codon) was inserted into the PmeI/SalI site of the DHER expression cassette 3 ' to the 3 ' end of the polylinker region into which the gene to be expressed was inserted. The complete 3' DNA sequence (starting with the stop codon of the EF-1. alpha. gene) is shown in SEQ ID NO: and 28. Bacteria transformed with this plasmid were stored at 9.4.1997 in the American type culture Collection 12301ParklawnDrive, Rokville, Maryland 20852 and designated accession number 98398. The resulting plasmid contains unique XbaI and NotI sites along with multiple HindIII sites, thus 3-way (3-way) ligation is necessary to insert a gene into the plasmid for expression. For example, a gene can be inserted into the plasmid by ligating the HindIII/XbaI fragment containing the desired gene with an 737bp NotI/HindIII fragment from pDEF14 and a 19.7kb XbaI/NotI fragment from pDEF 14.

Protein expression using this vector was compared to expression using pDEF2 containing a smaller portion of the EF-1. alpha.5' side chain region. In the primary experiment, DNA encoding both the heavy and light chains of antibody 23F2G were secondary cloned into pDEF2 and pDEF14 vectors and expression levels were determined after transfection into DG44 cells as described in example 5 above. The genes encoding the two antibody chains were arranged linearly in the vector, and expression of both genes was driven by the CHEF1 sequence in each plasmid. The coding regions of both strands in each plasmid were separated by the same 4.3kb DNA from the untranslated region of human IgG 43'.

The results show that the expression of 23F2G from the larger CHEF1 sequence in pDEF14 was 4-fold higher than the antibody expression from the smaller pDEF2 sequence. This result was unexpected because the majority of identifiable pDEF14 transcription binding factor sites were also located in the DNA sequence in pDEF 2. Thus, there may be one or more additional unidentified transcription factor binding sites or enhancer sequences located in the CHEF1DNA of the pDEF14 plasmid. Alternatively, larger CHEF1DNA may provide the facility to increase transcription as a result of certain properties of the 3' DNA sequence.

It is expected that one skilled in the art will be able to encounter many modifications and variations of the invention as described in the illustrative examples above. Accordingly, the invention should be limited only as appears in the appended claims.

Sequence listing

(1) General data:

(i) the applicant: allison, Daniel S.

(ii) The invention provides a subject: hamster EF-1 alpha regulatory DNA

(iii) Sequence number: 29

(iv) Contact address:

(A) address: marshall, O' Toole, Gerstein, Murray & Borun

(B) Street: 233 South Wacker Drive, 6300 search Tower

(C) City: chicago

(D) State: illinois

(E) The state is as follows: USA

(F) And E, postcode: 60606

(v) A computer-readable form:

(A) type of medium: flexible disk

(B) A computer: IBM PC compatibility

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: patentin Release #1.0, #1.30 edition

(vi) The current application information:

(A) application No.: US

(B) Submission date:

(C) and (4) classification:

(vii) client/agent profile

(D) Name: williams jr., Joseph a.

(E) Registration number: 38,659

(F) Reference/summary number: 27866/33537

(viii) Telecommunication data:

(G) telephone: 312-474-6300

(H) Electric transmission: 312-474-0448

(2) The SEQ ID NO data: 1:

(i) sequence characteristics:

(A) length: 3678 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 1:

ACTAGTTCCA AAGATGAATT ACTAACCAGT GTTTCCAAGG AAATAAATGA AAGCAGAGAG 60

ATTAGTTCTA TTGCTAGTGT TTCATTTTCG TATATTTCTT ACAATTTCTC TTGTTACAAA 120

TAGGCACTAG GGTATCAAGA TAATTTTAAC GACTGGCTGA GAACCCTAGA AAATCTCTGT 180

GAAAAAGGGA TTTGTGAAAT GAGAGAGGGT AATGTGGCCA TTATAGAAAA GGCTTTTGTG 240

TGCCTTGCAT GCATAGACCC TGTGTTTGAT CTCTTAACAC CCTCCTTGAC CAGAAAAAGC 300

TTCTGTGGAT AGAAAATGAT TAGTTATATA TACTTTTAGG GAAACGTAGT TCTGGATTCT 360

TTGGTTACAA TTAACAGAAT TAAGTGCAAA CAAAGCCAGA AACCTCCTGA TAAATGAGAA 420

AACCTGCTTG TAGAAGGTTG TAAGGCTCTG TAATATAGGA ATTAGGAGAA AAGAAACCTG 480

TGTGGTGGGG CACGTCTGTA ATCCCAGCAT TGGGAAGTAG AGGTAGAAGA TTAGAAATCA 540

AAGGCCAGCC TCAGCAACAC AGTGAGTTTG AGGCCACCCT GAACTACATC AGGTTCTGTC 600

TCCTTTCTTT TTTTTTTTTT TTTCTTTTCT TTTTTTGGTT TCTCTGTGTA GTTTTGGAGC 660

CTATCCTGGC ACTAGCTCTG AAGAGCAGGC TGGCCTCGAA CTCAGAGATC AGCCAGCCTC 720

TGCTGGGATT AAAGGTATGC ACCACCAACG CCCCAGGTTT TGTCTCAAAC AAACAAAAAT 780

AACATCAGGA GGTGGTGAGA GGGCTCAGTG GTCACAGGCA TTCTCTGCAA AGCCTGACTC 840

TGAGTTGGAT CCTTTAGAGC TACATGGTTG AGGGAAGAGA ACTGACTCCT GGAAGGTGTC 900

CTCTGGTCCC CACACATAGC TATACACAGC ATGTGCATTC ACACACACTA AATAATGCTA 960

TTTTTAAAAA AATTAAAAAC AACAACAGTT TGGGTTGTGA AAACTAGAAC TAGATAATAG 1020

GTAAGAATCA AGTATCATGT AAATTTGCTT TCAACTCATC CCAAAATTTG TTTTATATTT 1080

CAGTTTTTTT CCTTCCTAGC TTGACTGTGG AGTCTTGTCC GGAAGCAAAT AGTTCCTTTG 1140

CAGATCCCAC ATGTGGACAC CGGACAGTAG GTCCTCAAAT GCTCCTTATT AGGTTGGTTC 1200

AATAATATCA ATTGTTTGTT ACTAGGCAGT GATGTTGTAC ATCTGGAGGA GATCTCTTGA 1260

GCCCATAATC AGGTTATTAG GAATAAATAC TCTAAGGCTA AAAATGTAGC TTAGTGATAA 1320

GAGTGCTTGC CTGGTGTGCT GAGACCCTCG GTTCCATCTC CACAACCCCA TATTCCATTA 1380

CAAAATACCT TTTCACCGTC CCTAGCATTA AGAAACAAAA CAACAAAGAA GTTTTTCTTT 1440

CTTCTGAGAT CCTGCCCGGA GAGGCATTTA AAACTGGCCA GGGCCAAAAA AAAAAAAAAA 1500

AAAAGAAAAA AAAGAAAAGA AAACAGGCTA GGGCCGGCAT GGTGGCGCAC GCCTTTAATC 1560

CCAGCACGCA GGAGGCAGAG GCAGGGCGGA TCTCTGTGAG TTTGAGGTCA GCCTGGTCTA 1620

CCTAGTGAGT TTCAGGGCAC CCAGGGCTAA AGAGACTGTC TCAAAAACAA AACAGCCACA 1680

CAATCAGAAC CACAGCAAAA CGCAGTTATG ATCCTTGGAA CTGTAGGAAT GACAAGCATT 1740

TAAATAATAG GACGAGCCAT TTTTGAGAAG CTCTGATTTC ACAAGTGTCA GGGATGGGCT 1800

CTGGGCGAGT AAGATTGCTA ATGCTGGCCT CTAAATGAGA CCACGTGGAG TTGATTAGAT 1860

TCTTTTCATG TTCCTCGTGC TCTATCAAAT AACTGTACCC AAATACACAC ACACACACAC 1920

ACACACACAA TGCGCGCACA CACAAAATCC TTTTTTAGCT TAAGAAGCCC AGAATCAGAA 1980

GTAAAGCTAA CTGTGGGACT TAAGTATTAT TCTGAACGGA ACTCCCAGGG CGTGAAGCGC 2040

GCTTCAGGCT TCCAGAGAAG CAGCTGGCGC TGGATGGAAT GAACCAAGAG GCCAGCACAG 2100

GGGCAGATCC GTCGAGCTCT CGGCCACCGA GCTGAGCCCT TAGGTTCTGG GGCTGGGAAG 2160

GGTCCCTAGG ATTGTGCACC TCTCCCGCGG GGGACAAGCA GGGGATGGCG GGGCTGACGT 2220

CGGGAGGTGG CCTCCACGGG AAGGGACACC CGGATCTCGA CACAGCCTTG GCAGTGGAGT 2280

CAGGAAGGGT AGGACAGATT CTGGACGCCC TCTTGGCCAG TCCTCACCGC CCCACCCCCG 2340

ATGGAGCCGA GAGTAATTCA TACAAAAGGA GGGATCGCCT TCGCCCCTGG GAATCCCAGG 2400

GACCGTCGCT AAATTCTGGC CGGCCTCCCA GCCCGGAACC GCTGTGCCCG CCCAGCGCGG 2460

CGGGAGGAGC CTGCGCCTAG GGCGGATCGC GGGTCGGCGG GAGAGCACAA GCCCACAGTC 2520

CCCGGCGGTG GGGGAGGGGC GCGCTGAGCG GGGGCCCGGG AGCCAGCGCG GGGCAAACTG 2580

GGAAAGTGGT GTCGTGTGCT GGCTCCGCCC TCTTCCCGAG GGTGGGGGAG AACGGTATAA 2640

AAGTGCGGTA GTCGCGTTGG ACGTTCTTTT TCGCAACGGG TTTGCCGTCA GAACGCAGGT 2700

GAGTGGCGGG TGTGGCCTCC GCGGGCCCGG GCTCCCTCCT TTGAGCGGGG TCGGACCGCC 2760

GTGCGGGTGT CGTCGGCCGG GCTTCTCTGC GAGCGTTCCC GCCCTGGATG GCGGGCTGTG 2820

CGGGAGGGCG AGGGGGGGAG GCCTGGCGGC GGCCCCGGAG CCTCGCCTCG TGTCGGGCGT 2880

GAGGCCTAGC GTGGCTTCCG CCCCGCCGCG TGCCACCGCG GCCGCGCTTT GCTGTCTGCC 2940

CGGCTGCCCT CGATTGCCTG CCCGCGGCCC GGGCCAACAA AGGGAGGGCG TGGAGCTGGC 3000

TGGTAGGGAG CCCCGTAGTC CGCATGTCGG GCAGGGAGAG CGGCAGCAGT CGGGGGGGGG 3060

ACCGGGCCCG CCCGTCCCGC AGCACATGTC CGACGCCGCC TGGACGGGTA GCGGCCTGTG 3120

TCCTGATAAG GCGGCCGGGC GGTGGGTTTT AGATGCCGGG TTCAGGTGGC CCCGGGTCCC 3180

GGCCCGGTCT GGCCAGTACC CCGTAGTGGC TTAGCTCCGA GGAGGGCGAG CCCGCCCGCC 3240

CGGCACCAGT TGCGTGCGCG GAAAGATGGC CGCTCCCGGG CCCTGTAGCA AGGAGCTCAA 3300

AATGGAGGAC GCGGCAGCCC GGCGGAGCGG GGCGGGTGAG TCACCCACAC AAAGGAAGAG 3360

GGCCTTGCCC CTCGCCGGCC GCTGCTTCCT GTGACCCCGT GGTGTACCGG CCGCACTTCA 3420

GTCACCCCGG GCGCTCTTTC GGAGCACCGC TGGCCTCCGC TGGGGGAGGG GATCTGTCTA 3480

ATGGCGTTGG AGTTTGCTCA CATTTGGTGG GTGGAGACTG TAGCCAGGCC AGCCTGGCCA 3540

TGGAAGTAAT TCTTGGAATT TGCCCATTTT GAGTTTGGAG CGAAGCTGAT TGACAAAGCT 3600

GCTTAGCCGT TCAAAGGTAT TCTTCGAACT TTTTTTTTAA GGTGTTGTGA AAACCACCGC 3660

TAATTCAAAT CCAACATG 3678

(2) SEQ ID NO: 2: data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 2:

AGGCACAGTC GAGGCTGATC 20

(2) SEQ ID NO: 3: data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 3:

TTCCAGGGTC AAGGAAGGCA 20

(2) SEQ ID NO: 4: data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 4:

GCCACCTGAT CTACAAATGT 20

(2) SEQ ID NO: 5: data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 5:

GAGATACCAG CCTCAAATTC 20

(2) SEQ ID NO: 6: data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 6:

ATGTGACCAT CATTGATGCC 20

(2) SEQ ID NO: 7: data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 7:

GTGACTTTCC ATCCCTTGAA 20

(2) SEQ ID NO: 8: data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 8:

GTTGGAATGG TGACAACATG 20

(2) SEQ ID NO: 9: data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 9:

CAGGTTTTAA AACACCAGTC 20

(2) SEQ ID NO: 10: data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 10:

AATGACCCAC CAATGGAAGC 20

(2) SEQ ID NO: 11: data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 11:

ACAGCAACTG TCTGCCTCAT 20

(2) SEQ ID NO: 12: data:

(i) sequence characteristics:

(A) length: 17 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 12:

GTAAAACGAC GGCCAGT 17

(2) SEQ ID NO: 13: data:

(i) sequence characteristics:

(A) length: 19 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 13:

GGAAACAGCTATGACCATG 19

(2) SEQ ID NO: 14: data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 14:

AATTAACCCT CACTAAAGGG 20

(2) SEQ ID NO: 15: data:

(i) sequence characteristics:

(A) length: 23 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 15:

GTTAATACGA CTCACTATAG GGC 23

(2) SEQ ID NO: 16: data:

(i) sequence characteristics:

(A) length: 6 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 16:

GGCGGG 6

(2) SEQ ID NO: 17: data:

(i) sequence characteristics:

(A) length: 6 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 17:

GGGCGG 6

(2) SEQ ID NO: 18: data:

(i) sequence characteristics:

(A) length: 8 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 18:

TGACGYMR 8

(2) SEQ ID NO: 19: data:

(i) sequence characteristics:

(A) length: 13 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO 19:

TGGCNNNNNKCCR

13

(2) SEQ ID NO: 20: data:

(i) sequence characteristics:

(A) length: 26 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 20:

GATCGCGGCC GCGTTTAAAC GGATCC 26

(2) SEQ ID NO: 21: data:

(i) sequence characteristics:

(A) length: 26 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 21:

AATTGGATCC GTTTAAACGC GGCCGC 26

(2) SEQ ID NO: 22: data:

(i) sequence characteristics:

(A) length: 77 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 22:

CGTTTAGTGA ACCGTCAGAT CTACATAACA ACATTCCTCC TCTAAAGAAG CCCCAAGCTT 60

GATATCTGCA GAATTCT 77

(2) SEQ ID NO: 23: data:

(i) sequence characteristics:

(A) length: 85 base pair

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 23:

CTAGAGAATT CTGCAGATAT CAAGCTTGGG GCTTCTTTAG AGGAGGAATG TTGTTATGTA 60

GATCTGACGG TTCACTAAAC GAGCT 85

(2) SEQ ID NO: 24: data:

(i) sequence characteristics:

(A) length: 66 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 24:

AGTTCAGTCG ACGGCGCGCC AACCCGGGAA TCCGGACGGG ATCTATACAT TGAATCAATA 60

TTGGCA 66

2) SEQ ID NO: 25: data:

(i) sequence characteristics:

(A) length: 42 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 25:

ATGTCAGGAT CCACGCGGAA CTCCATATAT GGGCTATGAA CT 42

(2) SEQ ID NO: 26: data:

(i) sequence characteristics:

(A) length: 19 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 26:

GGCTTAGCTC CGAGGAGGG 19

(2) SEQ ID NO: 27: data:

(i) sequence characteristics:

(A) length: 26 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 27:

CGTGACAAGC TTGGTTTTCA CAACAC 26

(2) SEQ ID NO: 28: data:

(i) sequence characteristics:

(A) length: 4263 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 28:

TGAATATTAC CCCTAACACC TGCCACCCCA GTCTTAATCA GTGGTGGAAG AACGGTCTCA 60

GAACTGTTTG TCTCAATTGG CCATTTAAGT TTAATAGTGA AAGACTGGTT AATGATAACA 120

ATGCATCGGA AAACCTTCAG GAGGAAAGGA GAATGTTTTG TGGAACATTT TTGTGTGTGT 180

GGCAGTTTTA AGTTATTAGT TTTCAAAATC AGTACTTTTT AATGGAAACA ACTTGACCAA 240

AAATCTGTCA CAGAATTTTG AGACCCATTA AAATACAAGT TTAATGAGAA GTCTGTCTCT 300

GTTAATGCTG AAGTCATTAC TAAGTGCTTA GCTTAGCAAG GTATGTGGAT GCCCATTTGT 360

GTTCCAAGGG ATTGGACTGT TCATCAGGAC CCAGAGCTGA GTTTCAAGGG CTCAAGAGAT 420

GGCTTATTAC CTGTGGGTGT CTTGAAGGTT CTGGTTGGGA CAAATTAGGA ATGTTTTTGG 480

CAGACATGGT GACTACCTTC ATCTGGGTGA GTTCAGTTGA TTTGTCTTGA GCCTTTGGGG 540

TTTACACAAG TAAATGACAT CATACAGTTA GTGTATTGTT AGTGAATATT AATATATGAG 600

GCAGGCTTTG CTCTAGCAAT TTTAGAACTA GTTTTCAGGA AAGGGTTCAT CTTGTGCATT 660

GGATGTTTGA TTCTATCACT TAGAGTTTAA ACTGAAAGTG CTCAAGAGGT TTTATTTAGG 720

CTGGGATATA AATAAGCCTT TCTGTAGCTT GTAATGGTAT CAGGAATTTA AAAGGCCATC 780

TGGGGCACAA AGATTAAGCA GAAAAGGTAG AAGGTGAGAT TGGGGGACTT TGAGTACTTC 840

ACACACTTTA ATGTGTGAGT GCTTTAGTGC ATATAGTACA ACTGCCAGAT AAGGGCATCC 900

ACATCTGATT GTTTGGAAGG CACCTTGTGG TTTCTGGGAA TTCAGAATTG GGAGAAAAAT 960

GCTCCCAACC GCTGAAGCCC TTGGTAATCT GCAGGGTGTT TATTTAGCAG GAGATAAGGA 1020

CAAAAAGTTA TAGTGTGGAG TTGGTTGAGT TGGTAGATGT CATTACAACA GGTGGTCTTA 1080

AATTGGGTTA GGAGTCACTT TGAAATACCT GGGCCATAAG CAAAGTGGCA TTTTCACCTT 1140

TCAGGAGAAA CTGGTACACT TATCCATTCT ATAGTGCATG CTTGTTCAAT TGGGCTGATG 1200

ACTAAACCGG TGACTAAAGG TTTGTCAGTA TAAATGGATG GGTTGTAGGC AGACGGTGAG 1260

GAATTACTAT ACCTGCAAGG AGTCATTGCC TGATCTGCCT GGAAAGGGGC AGGATTGAGT 1320

CTCAGAACGT GTACACCATA GGATATGGAA AAATTTGTCA CGCCTAGCAT TCAACTTAGT 1380

GGTGTAGCGC CACCTACTGG CACTTTAAAA GCTTAGCATA GAGGAGCATG TGTGTTAGGA 1440

GCTCGGATGG GATCCAGGGC CTCAAGGTTT GCATGTAAAT AAAAGCCCTT TACCAAATTA 1500

ACTACATACC AGCATACATC AGTCCTTTAG TGTTGAAAAA CAGAAGGGAA AGCTAATATA 1560

TATAGTGCTT GCTTTATTTA AGTCTAGCTG ATTACGTGTT TGGTTGCCAG TGTGACTAGT 1620

CTGGAGTTGA ATTTGTCCTC AGACACGTAA AATGGAATTT GGGATTCACA ACACTCTAGT 1680

ATGAGGGACC TAATGGCCTG TACCAGGCAC AAACGTGTCT ATAAACTACA CAAAACGAAG 1740

GAATTTACAG GAATTAGGAA GGTATTCTTA ACATTAAAAC ATTATGGGCA TTTTAAAAAA 1800

AGCTTTGACA GGATTTCTTT GTCATGGCTG TCCTGGAGCT AGTTGTGTAG ACCAGGCTGG 1860

GCTGAAATCT TGTCTGCCTG CCTGGCTTGG ACACTTTTTT ATTATGTATA CAACATTCTG 1920

CTTCCATGTA TATCTGCACA TTAGAAGACG GCACCAGATC TCCTAATGGA TGGTTGTGAG 1980

CCACCATGTG GTTGCTGGGA ATTGAACTCA GGACCTCTGG AAGAGCAGTG CTCTTAACCT 2040

CTGAGCCATC TCCAGCCCCA GCTTGGGCAC ATTTTTAATG GCTGGGAAAT CAAACCCCCT 2100

AGGCCTTCTG TCAGTAATGA AGGGCTTTTG GCTACCGAGA GTAGGATTTA AGGTTATTCG 2160

GAGCTGCAGG TCTGCCTCAG TGCAGGTTTG GGAGTCCAGC ATCTTAGAAA ATGCAGTGAA 2220

GCCAAGCTGA GCTATATTTT GTTTAAAAAA AAAATAAGTG GGTAAAGTGC TGCTGAGCCT 2280

GATGACCAAG CTGGGACACA AGTAGAAGAA CATAGGCCAA TGCTCTATAT TAAAAGCATG 2340

GGTCATTTTT AATGCTCTTG AGAAGGCTAT GCCTACACTA CTCTCAGCCA CCGCAGCGTG 2400

TTTAAATTAA ACTAGTTTGG AAATTTTCTT TGGGGGTAAG CTATTTAACC TAGTGCCTTG 2460

GCAGGTATAC TACTGAACTC TCCTCCTCAT TCCTTTTTGT TTTTTAAGAA TTTCAGTCAG 2520

GCTCAGGCAG CCCTTAAACT TGTGATTAAG CCTGAGAACA GTTACGATTA TGAGCCTATT 2580

AGTATACCGA TCAATATGTG AATTTTTTTG GGATGGGGGT CAGGCCTCCC TGCCTCCCAA 2640

ATACTGGGAC TAAAGGCTGC ACCACCACAA CCTGGCTCTT GAAATACTTT TCTACATTTT 2700

TTGGGGGGCA TGGGTGGGAG AGCAGGGTTT CTCTGTATTA GCCCTGGCTC TCCTGGAACT 2760

CTGTAGACCA GGCTATTCTT GAGCTCAGAT TAGCCTGTCT CTGCCTCCTA AATTCTGGGA 2820

TTAAAGGTGT GTGCTACTGC TGCCTGGCTA CAAAGACATT TTTTTTTTTC TTAAATTTAA 2880

AAACAAAAGT GGTTCTTTTA GAAGGGTGGT TGGTGTTGGC ACATACTCCA AGCACTCAGG 2940

TTTTGAGTTT GTCCCAGGAA TGAAGACTGC ATTACTGCCG CCCCTCCCTG GTAAGGGCTA 3000

CACAGAGAAA TCCTATTTGG AGCCTATCCT GGTAACTCGC TCTGTAGACC AGGCTGGCCT 3060

CGAACTCAAG AGAACCACCT GCCTCTGAAT GCTGGTATTA AGGGCAGGCA CCACCAACAC 3120

CCAGCCTAAA AAATGTCTTT TTTTTAAAGA TTTTTTTTTT TTTTTTTACA GAATAAACAT 3180

TCTGTTTACA ATATTCTGCT TCTATGTATA TCTGCACACT AGAAGAGGGC ACCCGATCTC 3240

ATAATGGATG GTTGTGAGCC ACCAAGTGGT TGCTGGGAAT TGAACTCAGA ACCTCTGGAA 3300

GAGCAGTCAG TGCTCTTAAC CTCTGAGCCA TCTCTCCAGC CCCTAAAAAT GGCTCTTGAG 3360

ATAGGGTCTC AAGTAGTTTG AGACTGAGTT GGCTATATAA ACAAGGCTGG CACATAGCAC 3420

CATGTACAGC TGGGTTTAGT TTACATGGGG TGTTTTTGTC TCTGGAGGCA GGAGGATCAT 3480

TTGAGCATAG GGAGTTAATA GTGAGGTCAT GTTTTATCTA CTCTTCTGAA TTGAGAACTA 3540

AGCTGATGCA AAGCAAGTTT GACTGAAGAA GTCCAGTTTA TGAGAACAAG GGTGGAAACT 3600

AATGTGTCAA AGATGGCCTT GCATGTGTTT TAGATGATGA CCCAGTCACT TGGGAATTAC 3660

TGGATGTGTA AGACCTATAT CTTGACAGGA GTGAACAGTG TCTTATAGGT CCTATATGAA 3720

AGAAATGAGA CATACCCATT TTGTTTCCCC TAAGAATTCA CTTTTCCTAA CCTGGTTCAT 3780

GCTATTTAGG TTATTTTACT TGCAAATCCT AGGTGCTCCC TTACCCAGTA TTGCTTATGT 3840

GGCACCAAAG TCACTCACTC CCATGATTTG CAAGTCTCTG GGAACTTCCA TGACAACCTA 3900

GAATAGCAAC TCAAATACAT TTTCTCAGTA CCAATTTTGA AGAAAAAATA TTTTGCAAAA 3960

TAGCTGTATG GATGGGTACT AAATAGTGAG GTTATCTCCA GAAGGCCTAT GAAGAATTAA 4020

GGTTGAGTTC AGTTGAGTTC AGCAGCAAGT TTAAGGTTCA TCCATTTTTG TACAGTGTTT 4080

TCCTATTACG GTAAGTGTTT TGCCTGCAGG AATATCTGTA CCACATGCTT GCCTGGTACC 4140

TATATCGGCC AGAAGAGGGC TTTGGATCCT CTGGACTTGA ATTACAGATG GGTATTAGCC 4200

ACCATTTAGG TGCTGGGAAT TGAAACCAAG TCCTCTGGAA GAACAGCAAG TGATCGAGTC 4260

GAC 4263

(2) SEQ ID NO: 29: data:

(i) sequence characteristics:

(A) length: 4695 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) Topological structure: line shape

(ii) Molecular type: DNA

(xi) Description of the sequence: SEQ ID NO: 29:

CCCGGGCTGG GCTGAGACCC GCAGAGGAAG ACGCTCTAGG GATTTGTCCC GGACTAGCGA 60

GATGGCAAGG CTGAGGACGG GAGGCTGATT GAGAGGCGAA GGTACACCCT AATCTCAATA 120

CAACCTTTGG AGCTAAGCCA GCAATGGTAG AGGGAAGATT CTGCACGTCC CTTCCAGGCG 180

GCCTCCCCGT CACCACCCCC CCCAACCCGC CCCGACCGGA GCTGAGAGTA ATTCATACAA 240

AAGGACTCGC CCCTGCCTTG GGGAATCCCA GGGACCGTCG TTAAACTCCC ACTAACGTAG 300

AACCCAGAGA TCGCTGCGTT CCCGCCCCCT CACCCGCCCG CTCTCGTCAT CACTGAGGTG 360

GAGAAGAGCA TGCGTGAGGC TCCGGTGCCC GTCAGTGGGC AGAGCGCACA TCGCCCACAG 420

TCCCCGAGAA GTTGGGGGGA GGGGTCGGCA ATTGAACCGG TGCCTAGAGA AGGTGGCGCG 480

GGGTAAACTG GGAAAGTGAT GTCGTGTACT GGCTCCGCCT TTTTCCCGAG GGTGGGGGAG 540

AACCGTATAT AAGTGCAGTA GTCGCCGTGA ACGTTCTTTT TCGCAACGGG TTTGCCGCCA 600

GAACACAGGT AAGTGCCGTG TGTGGTTCCC GCGGGCCTGG CCTCTTTACG GGTTATGGCC 660

CTTGCGTGCC TTGAATTACT TCCACGCCCC TGGCTGCAGT ACGTGATTCT TGATCCCGAG 720

CTTCGGGTTG GAAGTGGGTG GGAGAGTTCG AGGCCTTGCG CTTAAGGAGC CCCTTCGCCT 780

CGTGCTTGAG TTGAGGCCTG GCCTGGGCGC TGGGGCCGCC GCGTGCGAAT CTGGTGGCAC 840

CTTCGCGCCT GTCTCGCTGC TTTCGATAAG TCTCTAGCCA TTTAAAATTT TTGATGACCT 900

GCTGCGACGC TTTTTTTCTG GCAAGATAGT CTTGTAAATG CGGGCCAAGA TCTGCACACT 960

GGTATTTCGG TTTTTGGGGC CGCGGGCGGC GACGGGGCCC GTGCGTCCCA GCGCACATGT 1020

TCGGCGAGGC GGGGCCTGCG AGCGCGGCCA CCGAGAATCG GACGGGGGTA GTCTCAAGCT 1080

GGCCGGCCTG CTCTGGTGCC TGGCCTCGCG CCGCCGTGTA TCGCCCCGCC CTGGGCGGCA 1140

AGGCTGGCCC GGTCGGCACC AGTTGCGTGA GCGGAAAGAT GGCCGCTTCC CGGCCCTGCT 1200

GCAGGGAGCT CAAAATGGAG GACGCGGCGC TCGGGAGAGC GGGCGGGTGA GTCACCCACA 1260

CAAAGGAAAA GGGCCTTTCC GTCCTCAGCC GTCGCTTCAT GTGACTCCAC GGAGTACCGG 1320

GCGCCGTCCA GGCACCTCGA TTAGTTCTCG AGCTTTTGGA GTACGTCGTC TTTAGGTTGG 1380

GGGGAGGGGT TTTATGCGAT GGAGTTTCCC CACACTGAGT GGGTGGAGAC TGAAGTTAGG 1440

CCAGCTTGGC ACTTGATGTA ATTCTCCTTG GAATTTGCCC TTTTTGAGTT TGGATCTTGG 1500

TTCATTCTCA AGCCTCAGAC AGTGGTTCAA AGTTTTTTTC TTCCATTTCA GGTGTCGTGA 1560

AAACTACCCC TAAAAGCCAA AATGGGAAAG GAAAAGACTC ATATCAACAT TGTCGTCATT 1620

GGACACGTAG ATTCGGGCAA GTCCACCACT ACTGGCCATC TGATCTATAA ATGCGGTGGC 1680

ATCGACAAAA GAACCATTGA AAAATTTGAG AAGGAGGCTG CTGAGGTATG TTTAATACCA 1740

GAAAGGGAAA GATCAACTAA AATGAGTTTT ACCAGCAGAA TCATTAGGTG ATTTCCCCAG 1800

AACTAGTGAG TGGTTTAGAT CTGAATGCTA ATAGTTAAGA CCTTACTTAT GAAATAATTT 1860

TGCTTTTGGT GACTTCTGTA ATCGTATTGC TAGTGAGTAG ATTTGGATGT TAATAGTTAA 1920

GATCCTACTT ATAAAAGTTT GATTTTTGGT TGCTTCTGTA ACCCAAAGTG ACCAAAATCA 1980

CTTTGGACTT GGAGTTGTAA AGTGGAAACT GCCAATTAAG GGCTGGGGAC AAGGAAATTG 2040

AAGCTGGAGT TTGTGTTTTA GTAACCAAGT AACGACTCTT AATCCTTACA GATGGGAAAG 2100

GGCTCCTTCA AGTATGCCTG GGTCTTGGAT AAACTGAAAG CTGAGCGTGA ACGTGGTATC 2160

ACCATTGATA TCTCCTTGTG GAAATTTGAG ACCAGCAAGT ACTATGTGAC TATCATTGAT 2220

GCCCCAGGAC ACAGAGACTT TATCAAAAAC ATGATTACAG GGACATCTCA GGTTGGGATT 2280

AATAATTCTA GGTTTCTTTA TCCCAAAAGG CTTGCTTTGT ACACTGGTTT TGTCATTTGG 2340

AGAGTTGACA GGGATATGTC TTTGCTTTCT TTAAAGGCTG ACTGTGCTGT CCTGATTGTT 2400

GCTGCTGGTG TTGGTGAATT TGAAGCTGGT ATCTCCAAGA ATGGGCAGAC CCGAGAGCAT 2460

GCCCTTCTGG CTTACACACT GGGTGTGAAA CAACTAATTG TCGGTGTTAA CAAAATGGAT 2520

TCCACTGAGC CACCCTACAG CCAGAAGAGA TATGAGGAAA TTGTTAAGGA AGTCAGCACT 2580

TACATTAAGA AAATTGGCTA CAACCCCGAC ACAGTAGCAT TTGTGCCAAT TTCTGGTTGG 2640

AATGGTGACA ACATGCTGGA GCCAAGTGCT AACGTAAGTG GCTTTCAAGA CCATTGTTAA 2700

AAAGCTCTGG GAATGGCGAT TTCATGCTTA CACAAATTGG CATGCTTGTG TTTCAGATGC 2760

CTTGGTTCAA GGGATGGAAA GTCACCCGTA AGGATGGCAA TGCCAGTGGA ACCACGCTGC 2820

TTGAGGCTCT GGACTGCATC CTACCACCAA CTCGTCCAAC TGACAAGCCC TTGCGCCTGC 2880

CTCTCCAGGA TGTCTACAAA ATTGGTGGTA AGTTGGCTGT AAACAAAGTT GAATTTGAGT 2940

TGATAGAGTA CTGTCTGCCT TCATAGGTAT TTAGTATGCT GTAAATATTT TTAGGTATTG 3000

GTACTGTTCC TGTTGGCCGA GTGGAGACTG GTGTTCTCAA ACCCGGTATG GTGGTCACCT 3060

TTGCTCCAGT CAACGTTACA ACGGAAGTAA AATCTGTCGA AATGCACCAT GAAGCTTTGA 3120

GTGAAGCTCT TCCTGGGGAC AATGTGGGCT TCAATGTCAA GAATGTGTCT GTCAAGGATG 3180

TTCGTCGTGG CAACGTTGCT GGTGACAGCA AAAATGACCC ACCAATGGAA GCAGCTGGCT 3240

TCACTGCTCA GGTAACAATT TAAAGTAACA TTAACTTATT GCAGAGGCTA AAGTCATTTG 3300

AGACTTTGGA TTTGCACTGA ATGCAAATCT TTTTTCCAAG GTGATTATCC TGAACCATCC 3360

AGGCCAAATA AGCGCCGGCT ATGCCCCTGT ATTGGATTGC CACACGGCTC ACATTGCATG 3420

CAAGTTTGCT GAGCTGAAGG AAAAGATTGA TCGCCGTTCT GGTAAAAAGC TGGAAGATGG 3480

CCCTAAATTC TTGAAGTCTG GTGATGCTGC CATGTTGATA TGGTTCCTG GCAAGCCCAT 3540

GTGTGTTGAG AGCTTCTCAG ACTATCCACC TTTGGGTAAG GATGACTACT TAAATGTAAA 3600

AAAGTTGTGT TAAAGATGAA AAATACAACT GAACAGTACT TTGGGTAATA ATTAACTTTT 3660

TTTTTAATAG GTCGCTTTGC TGTTCGTGAT ATGAGACAGA CAGTTGCGGT GGGTGTCATC 3720

AAAGCAGTGG ACAAGAAGGC TGCTGGAGCT GGCAAGGTCA CCAAGTCTGC CCAGAAAGCT 3780

CAGAAGGCTA AATGAATATT ATCCCTAATA CCTGCCACCC CACTCTTAAT CAGTGGTGGA 3840

AGAACGGTCT CAGAACTGTT TGTTTCAATT GGCCATTTAA GTTTAGTAGT AAAAGACTGG 3900

TTAATGATAA CAATGCATCG TAAAACCTTC AGAAGGAAAG GAGAATGTTT TGTGGACCAC 3960

TTTGGTTTTC TTTTTTGCGT GTGGCAGTTT TAAGTTATTA GTTTTTAAAA TCAGTACTTT 4020

TTAATGGAAA CAACTTGACC AAAAATTTGT CACAGAATTT TGAGACCCAT TAAAAAAGTT 4080

AAATGAGAAA CCTGTGTGTT CCTTTGGTCA ACACCGAGAC ATTTAGGTGA AAGACATCTA 4140

ATTCTGGTTT TACGAATCTG GAAACTTCTT GAAAATGTAA TTCTTGAGTT AACACTTCTG 4200

GGTGGAGAAT AGGGTTGTTT TCCCCCCACA TAATTGGAAG GGGAAGGAAT ATCATTTAAA 4260

GCTATGGGAG GGTTTCTTTG ATTACAACAC TGGAGAGAAA TGCAGCATGT TGCTGATTGC 4320

CTGTCACTAA AACAGGCCAA AAACTGAGTC CTTGGGTTGC ATAGAAAGCT TCATGTTGCT 4380

AAACCAATGT TAAGTGAATC TTTGGAAACA AAATGTTTCC AAATTACTGG GATGTGCATG 4440

TTGAAACGTG GGTTAAAATG ACTGGGCAGT GAAAGTTGAC TATTTGCCAT GACATAAGAA 4500

ATAAGTGTAG TGGCTAGTGT ACACCCTATG AGTGGAAGGG TCCATTTTGA AGTCAGTGGA 4560

GTAAGCTTTA TGCCATTTTG ATGGTTTCAC AAGTTCTATT GAGTGCTATT CAGAATAGGA 4620

ACAAGGTTCT AATAGAAAAA GATGGCAATT TGAAGTAGCT ATAAAATTAG ACTAATTACA 4680

TTGCTTTTCT CCGAC 4695

Claims (31)

1. Purified and isolated hamster EF-1 α transcriptional regulatory DNA selected from the group consisting of the following regulatory DNA sequences:

(a) DNA consisting of SEQ ID NO: 1; and

(d) 11.7Kb of hamster EF-1 alpha regulatory DNA sequence of plasmid pDEF14 deposited under accession number ATCC 98398.

2. Purified and isolated hamster EF-1 α transcriptional regulatory DNA consisting of the amino acid sequence of SEQ ID NO: 1, 5' 1.56Kb of ATG initiation codon.

3. A host cell comprising the DNA of claim 1 or 2, wherein said DNA is not operably linked to an EF-1 a coding sequence.

4. A vector comprising the DNA of claim 1 or 2, wherein said DNA is not operably linked to an EF-1 α coding sequence.

5. A host cell transformed or transfected with the vector of claim 4.

6. A chimeric polynucleotide comprising the regulatory DNA of claim 1 or 2 operably linked to a DNA sequence encoding a protein, said DNA sequence encoding a protein other than hamster EF-1 α protein.

7. The chimeric polynucleotide of claim 6, wherein the regulatory DNA sequence comprises the nucleotide sequence of SEQ ID NO: 1.

8. The chimeric polynucleotide of claim 6, wherein the regulatory DNA comprises the nucleotide sequence of SEQ ID NO: 1 from position 2114 to position 3656.

9. The chimeric polynucleotide of claim 6, wherein the protein-encoding DNA sequence encodes a non-EF-1 α protein that is endogenous to hamster cells.

10. The chimeric polynucleotide of claim 6, wherein the DNA sequence encodes a protein heterologous to hamster cells.

11. A host cell transformed or transfected with the chimeric polynucleotide of any one of claims 6 to 10.

12. An expression plasmid comprising the chimeric polynucleotide of any one of claims 6 to 10.

13. The expression plasmid of claim 12, further comprising SEQ ID NO: 28, or a nucleotide sequence as set forth in seq id no.

14. The expression plasmid of claim 13, wherein the expression plasmid according to SEQ ID NO: 28 is located 3' to the chimeric polynucleotide.

15. A host cell transformed or transfected with the expression plasmid of claim 12.

16. A host cell transformed or transfected with the expression plasmid of claim 13 or 14.

17. Plasmid pDEF2 deposited under accession number ATCC 98343.

18. Plasmid pDEF14 deposited under accession number ATCC 98398.

19. Plasmid pDEF1/ICM3H.2 with deposit number ATCC 98381.

20. Plasmid pNEF1/ICM3L.3 deposited under accession number ATCC 98382.

21. Plasmid pDEF1/F2GH.1 with deposit number ATCC 98383.

22. Plasmid pNEF1/F2GL.1, deposited under accession number ATCC 98384.

23. Plasmid pDEF1/CTN.1 with deposit number ATCC 98385.

24. Plasmid pDEF2/HPH.4 with deposit number ATCC 98386.

25. Plasmid pDEF10/MDC.1 with deposit number ATCC 98387.

26. A host cell transformed or transfected with an expression plasmid according to any one of claims 17 to 25.

27. A method for increasing transcription of a target gene in a host cell, comprising the step of integrating DNA comprising the hamster EF-1 α regulatory DNA of claim 1 or 2 into genomic DNA of said host cell at a location operably linked to the target gene.

28. The method of claim 27, wherein the integrated DNA further comprises a leader peptide.

29. The method of claim 27, wherein the host cell is a chinese hamster ovary cell.

30. The method of claim 29, wherein the gene of interest is endogenous to a chinese hamster ovary host cell.

31. The method of claim 27, wherein the target gene is heterologous to the chinese hamster ovary cells and is stably integrated into the chinese hamster ovary cell genomic DNA.