AU2007224398A1 - Plant regulatory sequences for control of gene expression - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 REGULATION 3.2 Name of Applicant: Actual Inventor/s: MONSANTO TECHNOLOGY LLC HEATHER M. ANDERSON, CATHERINE A. CHAY, GUILAN CHEN and TIMOTHY W. CONNER Address for Service: E.F. WELLINGTON

CO.,

Patent and Trade Mark Attorneys, 312 St. Kilda Road, Melbourne, Southbank, Victoria, 3006.

Invention Title: "PLANT REGULATORY SEQUENCES FOR CONTROL OF GENE EXPRESSION" Details of Associated Provisional Applications Nos: The following statement is a full description of this invention including the best method of performing it known to us.

-1 S-1A- O PLANT REGULATORY SEOUENCES FOR CONTROLOF GENE EXPRESSION in FIELD OF THE INVENTION The present invention relates to the isolation and use of nucleic acid molecules for control 00 of gene expression in plants, specifically novel plant promoters.

s BACKGROUND OF THE INVENTION N One of the goals of plant genetic engineering is to produce plants with agronomically important characteristics or traits. Recent advances in genetic engineering have provided the Cl requisite tools to transform plants to contain and express foreign genes (Kahl et al. (1995) World Journal of Microbiology and Biotechnology 11:449-460). Particularly desirable traits or o0 qualities of interest for plant genetic engineering would include but are not limited to resistance to insects and other pests and disease-causing agents, tolerances to herbicides, enhanced stability, yield, or shelf-life, environmental tolerances, and nutritional enhancements. The technological advances in plant transformation and regeneration have enabled researchers to take pieces of DNA, such as a gene or genes from a heterologous source, or a native source, but modified to is have different or improved qualities, and incorporate the exogenous DNA into the plant's genome. The gene or gene(s) can then be expressed in the plant cell to exhibit the added characteristic(s) or trait(s). In one approach, expression of a novel gene that is not normally expressed in a particular plant or plant tissue may confer a desired phenotypic effect. In another approach, transcription of a gene or part of a gene in an antisense orientation may produce a desirable effect by preventing or inhibiting expression of an endogenous gene.

Isolated plant promoters are useful for modifying plants through genetic engineering to have desired phenotypic characteristics. In order to produce such a transgenic plant, a vector that includes a heterologous gene sequence that confers the desired phenotype when expressed in the plant is introduced into the plant cell. The vector also includes a plant promoter that is operably linked to the heterologous gene sequence, often a promoter not normally associated with the heterologous gene. The vector is then introduced into a plant cell to produce a transformed plant cell, and the transformed plant cell is regenerated into a transgenic plant. The promoter controls expression of the introduced DNA sequence to which the promoter is operably linked and thus affects the desired characteristic conferred by the DNA sequence.

Since the promoter is a 5' regulatory element that plays an integral part in the overall expression of a gene or gene(s), it would be advantageous to have a variety of promoters to tailor -2- Q gene expression such that a gene or gene(s) is transcribed efficiently at the right time during plant growth and development, in the optimal location in the plant, and in the amount necessary to produce the desired effect. In one case, for example, constitutive expression of a gene product 00 may be beneficial in one location of the plant, but less beneficial in another part of the plant. In other cases, it may be beneficial to have a gene product produced at a certain developmental S stage of the plant, or in response to certain environmental or chemical stimuli. The commercial S development of genetically improved germplasm- has also advanced to the stage of introducing S multiple traits into crop plants, often referred to as a gene stacking approach. In this approach, multiple genes conferring different characteristics of interest can be introduced into a plant. It is important when introducing multiple genes into a plant, that each gene is modulated or controlled for optimal expression and that the regulatory elements are diverse, to reduce the potential of gene silencing which can be caused by recombination of homologous sequences. In light of these and other considerations, it is apparent that optimal control of gene expression and regulatory element diversity are important in plant biotechnology.

The proper regulatory sequences must be present and in the proper location with respect to the DNA sequence of interest, for the newly inserted D)NA to be transcribed and thereby, if desired translated into a protein in the plant cell. These regulatory sequences include but are not limited to a promoter, a 5' untranslated leader, and a 3' polyadenylation sequence. The ability to select the tissues in which to transcribe such foreign DNA, and the time during plant growth in which to obtain transcription of such foreign DNA is also possible through the choice of appropriate promoter sequences that control transcription of these genes.

A variety of different types or classes of promoters can be used for plant genetic engineering. Promotcrs can be classified on the basis of range or tissue specificity. For example, promoters referred to as constitutive promoters are capable of transcribing operatively linked DNA sequences efficiently and expressing said DNA sequences in multiple tissues.

Tissue-enhanced or tissue-specific promoters can be found upstream and operatively linked to l)NA sequences normally transcribed in higher levels in certain plant tissues or specifically in certain plant tissues. Other classes of promoters would include but are not limited to inducible promoters which can be triggered by external stimuli such as chemical agents, developmental Stimuli, or environmental stimuli. Thus, the different types of promoters desired can be obtained -3- O by isolating the upstream 5' regulatory regions of DNA sequences which are transcribed and tn expressed in a constitutive, tissue-enhanced, or inducible manner.

The technological advances of high-throughput sequencing and bioinformatics has oO provided additional molecular tools for promoter discovery. Particular target plant cells, tissues, c s or organs at a specific stage of development, or under particular chemical, environmental, or N physiological conditions can be used as source material to isolate the mRNA and construct cDNA libraries. The cDNA libraries are quickly sequenced and the expressed sequences catalogued electronically. Using sequence analysis software, thousands of sequences can be analyzed in a short period, and sequences from selected cDNA libraries can be compared. The o combination of laboratory and computer-based subtraction methods allows researchers to scan and compare cDNA libraries and identify sequences with a desired expression profile. For example, sequences expressed preferentially in one tissue can be identified by comparing a cDNA library from one tissue to cDNA libraries of other tissues and electronically "subtracting" common sequences to find sequences only expressed in the target tissue of interest. The tissue s enhanced sequence can then be used as a probe or primer to clone the corresponding full-length cDNA. A genomic library of the target plant can then be used to isolate the corresponding gene and the associated regulatory elements, including promoter sequences.

Multiple promoter sequences which confer a desired expression profile such as promoters capable of regulating expression of operably linked genes in multiple tissues can be isolated by selectively comparing cDNA libraries of target tissues of interest with non-target or background cDNA libraries to find the 5' regulatory regions associated with the expressed sequences in those target libraries. The isolated promoter sequences can be used for selectively modulating expression of any operatively linked gene and provide additional regulatory element diversity in a plant expression vector in gene stacking approaches.

SUMMARY OF THE INVENTION The present invention provides nucleic acid sequences, which comprise regulatory sequences located upstream of the 5' end of plant DNA structural coding sequences that are transcribed in multiple tissues of interest and shown in SEQ ID NOS: 53-75.

In one aspect, the present invention provides nucleic acid sequences comprising a sequence selected from the group consisting of SEQ ID NOS: 53-75 or any fragments, regions, S-4- O or cis elements of the sequence that are capable of regulating transcription of operably linked _3 DNA sequences.

The present invention also provides nucleic acid sequences comprising a sequence 00 selected from the group consisting of SEQ ID NOS: 53-75 that are promoters.

Another aspect of the present invention relates to the use of at least one fragment, region, or cis element thereof of the disclosed 5' promoter sequences that can be combined to create S novel promoters or used in a novel combination with another heterologous regulatory sequence to create a hybrid or chimeric promoter capable of modulating transcription of an operably linked DNA sequence.

Hence, the present invention relates to the use of nucleic acid sequences disclosed in SEQ ID NOS: 53-75, or any fragment, region, or cis elements of the disclosed sequences that are capable of regulating transcription of a DNA sequence when operably linked to the DNA sequence. Therefore, the invention not only encompasses the sequences as disclosed in SEQ ID NOS: 53-75, but also includes any truncated or deletion derivatives, or fragments or regions is thereof which are capable of functioning independently as a promoter including cis elements that are capable of functioning as regulatory sequences in conjunction with one or more regulatory sequences when operably linked to a transcribable sequence.

The present Invention thus encompasses a novel promoter or a hybrid or chimeric promoter comprising a nucleic acid of SEQ ID NOS: 53-75. The hybrid or chimeric promoters can consist of any length fragments, regions, or cis elements of the disclosed sequences.of

SEQ

ID NOS: 53-75 combined with any other transcriptionally active minimal or full-length promoter. For example, a promoter sequence selected from SEQ ID NOS: 53-75 may be combined with a CaMV 35S or other promoter to construct a novel hybrid promoter. A minimal promoter can also be used in combination with the nucleic acid sequences of the present invention. A novel promoter also comprises any promoter constructed by engineering the nucleic acid sequences disclosed in SEQ ID NOS: 53-75 or any fragment, region, or cis element of the disclosed sequences in any manner sufficient to transcribe an operably linked DNA sequence.

Another aspect of the present invention relates to the ability of the promoter sequences of SEQ ID NOS: 53-75, or fiagments, regions, or cis elements thereof to regulate transcription of operably linked transcribable sequences in multiple tissues. Fragments, regions, or cis elements 0 O ofSEQ ID NOS: 53-75 that are capable of regulating operably linked DNA sequences in certain _t tissues may be isolated from the disclosed nucleic acid sequences of SEQ ID NOS: 53-75 and used to engineer novel promoters that confer a desired expression profile.

00 The present invention also encompasses DNA constructs comprising the disclosed s sequences as shown in SEQ ID NOS: 53-75 or any fragments, regions, or cis elements thereof, N including novel promoters generated using the disclosed sequences or any fragment, region, or cis element of the disclosed sequences.

SThe present invention also includes any cells and transgenic plants containing the DNA disclosed in the sequences as shown in SEQ ID NOS: 53-75, or any fragments, regions, or cis to elements thereof.

The present invention also provides a method of regulating transcription of a DNA sequence comprising operably linking the DNA sequence to any promoter comprising a nucleic acid comprising all or any fragment, region or cis element of a sequence selected from the group consisting of SEQ ID NOS: 53-75.

s1 In a another embodiment the present invention provides a method regulating expression of DNA sequences in multiple tissues by operably linking a sequence selected from the group consisting of SEQ ID NOS:- 53-75, or any fragment, region, or cis element of the disclosed sequences to any transcribable DNA sequence. The fragments, regions, or cis elements of the disclosed promoters as shown in SEQ ID NOS: 53-75 can be engineered and used independently in novel combinations including multimers, or truncated derivatives and the novel promoters can be operably linked with a transcribable DNA sequence. Alternatively the disclosed fragments, regions, or cis elements of the disclosed sequences can be used in combination with a heterologous promoter including a minimal promoter to create a novel hybrid or chimeric promoter and the novel hybrid promoter can be operably linked to a transcribable

DNA

sequence.

The present invention also provides a method of making a transgenic plant by introducing into the cell of a plant a DNA construct comprising: a promoter comprising a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOS: 53-75, or fragments, regions, or cis elements thereof, and operably linked to the promoter, (ii) a transcribable

DNA

sequence and (iii) a 3' untranslated region.

-6- O The present invention also provides a method of isolating at least one 5' regulatory sequence of a desired expression profile from a target plant of interest by evaluating a collection of nucleic acid sequences of expressed sequence tags (ESTs) derived from one or more cDNA 00 libraries prepared from a plant cell type of interest, comparing EST sequences from at least one s target plant cDNA library and one or more non-target cDNA libraries of ESTs from a different S plant cell type, subtracting common EST sequences found in both target and non-target libraries, designing gene-specific primers from the remaining ESTs after the subtractions which are representative of the targeted expressed sequences, and isolating the corresponding 5' flanking and regulatory sequences, which includes promoter sequences from a genomic library prepared 0o from the target plant using the gene specific primers.

The foregoing and other aspects of the invention will become more apparent from the following detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS is Figure 1 is a plasmid map of pMON 19469 Figure 2 is a plasmid map ofpMON43623 Figure 3 is a plasmid map of pMON42410 Figure 4 is a plasmid map of pMON42411 Figure 5 is a plasmid map ofpMON25455 Figure 6 is a plasmid map of pMON46124 Figure 7 is a plasmid map ofpMON43644 Figure 8 is a plasmid map ofpMON45361 Figure 9 is a plasmid map ofpMON43716 Figure 10 is a plasmid map ofpMON43738 Figure 11 is a plasmid map ofpMON45362 Figure 12 is a plasmid map of pMON43722 Figure 13 is a plasmid map ofpMON43739 Figure 14 is a plasmid map ofpMON45360 0 0 -7- O DETAILED DESCRIPTION OF THE INVENTION This application claims the benefit of U.S. Provisional Application No. 60/154,182 filed 09/16/99.

0 0 Definitions and Methods The following definitions and methods are provided to better define the present invention S and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary S -skill in the relevant art.. The nomenclature for DNA bases as set forth at 37 CFR 1.822 is used. The standard one- and three-letter nomenclature for amino acid residues is used.

to "Nucleic acid (sequence)" or "polynucleotide (sequence)" refers to single- or doublestranded DNA or RNA of genomic or synthetic origin, a polymer of deoxyribonucleotide or ribonucleotide bases, respectively, read from the 5' (upstream) end to the 3' (downstream) end.

The nucleic acid can represent the sense or complementary (antisense) strand.

"Native" refers to a naturally occurring ("wild-type") nucleic acid sequence.

s1 "Heterologous" sequence refers to a sequence which originates from a foreign source or species or, if from the same source, is modified from its original form.

An "isolated" nucleic acid sequence is substantially separated or purified away from other nucleic acid sequences with which the nucleic acid is normally associated in the cell of the organism in which the nucleic acid naturally occurs, other chromosomal or extrachromosomal DNA. The term embraces nucleic acids that are biochemically purified so as to substantially remove contaminating nucleic acids and other cellular components. The term also embraces recombinant nucleic acids and chemically synthesized nucleic acids.

The term "substantially purified, as used herein, refers to a molecule separated from substantially all other molecules normally associated with it in its native state. More preferably, a substantially purified molecule is the predominant species present in a preparation.

A

substantially purified molecule may be greater than 60% free, preferably 75% free, more preferably 90% free from the other molecules (exclusive of solvent) present in the natural mixture. The term "substantially purified" is not intended to encompass molecules present in their native state.

A first nucleic acid sequence displays "substantially identity" to a reference nucleic acid sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions totaling CN -8rless than 20 percent of the reference sequence over the window of comparison) with the other nucleic acid (or its complementary strand), there is at least about 75% nucleotide sequence S identity, preferably at least about 80% identity, more preferably at least about 85% identity, and most preferably at least about 90% identity over a comparison window of at least 20 nucleotide s positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide I positions, and most preferably over the entire length of the first nucleic acid. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology I algorithm of Smthh and Waterman Adv. Appl. Math. 2:482, 1981; by the homology alignment algorithm of Needleman and Wunsch, J. Ml. Biol. 48:44, 1970; by the search for similarity o method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; preferably by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI. The reference nucleic acid may be a full-length molecule or a portion of a longer molecule. Alternatively, two nucleic acids are have substantial identity if one hybridizes is to the other under stringent conditions, as defined below.

A first nucleic acid sequence is "operably linked" with a second nucleic acid sequence when the sequences are so arranged that the first nucleic acid sequence affects the function of the second nucleic-acid sequence. Preferably, the two sequences are part of a single contiguous nucleic acid molecule and more preferably are adjacent. For example, a promoter is operably linked to a gene if the promoter regulates or mediates transcription ofthe gene in a cell.

A "recombinant" nucleic acid is made by an artificial combination of two otherwise separated segments of sequence, by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. Techniques for nucleic-acid manipulation are well-known (see, Sambrook ct al., Molecular Cloning, A Laboratory 2s Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989, and Ausubel ct al., 1992).

Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22:859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc.

103:3185, 1981. Chemical synthesis f nucleic acids can be perfbrmed, for example, on commercial automated oJigonucleotide synthesizers.

-9- O A "synthetic nucleic acid sequence" can be designed and chemically synthesized for enhanced expression in particular host cells and for the purposes of cloning into appropriate vectors. Host cells often display a preferred pattern of codon usage (Murray et al., 1989).

0 Synthetic DNAs designed to enhance expression in a particular host should therefore reflect the pattern of codon usage in the host cell. Computer programs are available for these purposes including but not limited to the "BestFit" or "Gap" programs of the Sequence Analysis Software 0 Package, Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, c, Madison, WI 53711.

"Amplification" of nucleic acids or "nucleic acid reproduction refers to the production to of additional copies of a nucleic acid sequence and is carried out using polymerase chain reaction (PCR) technologies. A variety of amplification methods are known in the art and are described, inter alia, in U.S. Patent Nos. 4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed. Innis et al., Academic Press, San Diego, 1990. In PCR, a primer refers to a short oligonucleotide of defined sequence which is annealed to a DNA template to s1 initiate the polymerase chain reaction.

"Transformed", "transfected", or "transgenic" refers to a cell, tissue, organ, or organism into Which has been introduced a foreign nucleic acid, such as a recombinant vector. Preferably, the introduced nucleic acid is integrated into the genomic DNA of the recipient cell, tissue, organ or organism such that the introduced nucleic acid is inherited by subsequent progeny.

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"transgenic" or "transformed" cell or organism also includes progeny of the cell or organism and progeny produced from a breeding program employing such a "transgenic" plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a recombinant construct or vector.

The term "gene" refers to chromosomal DNA, plasmid DNA, c DNA, synthetic DNA, or B other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequence involved in the regulation of expression. Some genes can be transcribed into mRNA and translated into polypeptides (structural genes); other genes can be transcribed into RNA rRNA, tRNA); and other types of gene function as regulators of expression (regulator genes).

"Expression" of a gene refers to the transcription of a gene to produce the corresponding mRNA and translation of this nmRNA to produce the corresponding gene product, a peptide, O O polypeptide, or protein. Gene expression is controlled or modulated by regulatory elements including 5' regulatory elements such as promoters.

"Genetic component" refers to any nucleic acid sequence or genetic element that may Salso be a component or part of an expression vector. Examples of genetic components include, but are not limited to promoter regions, 5' untranslated leaders, introns, genes, 3' untranslated regions, and other regulatory sequences or sequences which affect transcription or translation of one or more nucleic acid sequences.

O The terms "recombinant DNA construct", "recombinant vector", "expression vector" or expression cassette" refer to any agent such as a plasmid, cosmid, virus, BAC (bacterial to artificial chromosome), autonomously replicating sequence, phage, or linear or circular singlestranded or double-stranded DNA or RNA nucleotide sequence, derived from any source, capable ofgenomic integration or autonomous replication, comprising a DNA molecule in which one or more DNA sequences have been linked in a functionally operative manner.

"Complementary" refers to the natural association of nucleic acid sequences by base- 1s pairing (A-G-T pairs with the complementary sequence Complementarity between two single-stranded molecules may be partial, if only some of the nucleic acids pair are complementary; or complee, if all bases pair are complementary. The degree of complementarity affects the efficiency and strength of hybridization and amplification reactions. "Homology" refers to the level of similarity between nucleic acid or amino acid sequences in terms of percent nucleotide or amino acid positional identity, respecively, i.e., sequence similarity or identity. Homology also refers to the concept of similar functional properties among different nucleic acids or proteins.

"Ss"from a or Expressed Sequence Tags are short sequences of randomly selected clones from a cDNA (or complementary DNA) library which are representative of the cDNA inserts of these randomly selected clones (McCombie, et al., Nature Genetics, 1:124, 1992; Kurata, et al., Nature Genetics, 8: 365,1994; Okubo, et al., Nature Genetics, 2: 173, 1992).

The term "electronic Northern" refers to a computer-based sequence analysis which allows sequences from multiple cDNA libraries to be compared electronically based on parameters the researcher identifies including abundance in EST populations in multiple eDNA ~0 libraries, or exclusively to EST sets from One or co minations of libraries.

Ii O "Subsetting" refers to any method of comparing nucleic acid sequences from different or i multiple sources which can be used to identify the profile of the nucleic acid sequences which reflects gene transcription activity and message stability in a particular tissue, at a particular 00 time, or under particular conditions.

"Promoter" refers to a nucleic acid sequence located upstream or 5' to a translational start N codon of an open reading frame (or protein-coding region) of a gene and that is involved in r recognition and binding of RNA polymerase II and other proteins (trans-acting transcription 0 factors) to initiate transcription. A "plant promoter" is a native or non-native promoter that is functional in plant cells. Constitutive promoters are functional in most or all tissues of a plant to throughout plant development. Tissue-, organ- or cell-specific promoters are expressed only or predominantly in a particular tissue, organ, or cell type, respectively. Rather than being expressed "specifically" in a given tissue, organ, or cell type, a promoter may display "enhanced" expression, a higher level of expression, in one part cell type, tissue, or organ) of the plant compared to other parts of the plant. Temporally regulated promoters are is functional only or predominantly during certain periods of plant development or at certain times of day, as in the case of genes associated with circadian rhythm, for example. Inducible promoters selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals. Inducible or regulated promoters include, for example, promoters regulated by light, heat, stress, flooding or drought, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.

Any plant promoter can be used as a 5' regulatory sequence for modulation expression of a particular gene or genes. One preferred promoter would be a lant RNA polymerase promoter. Plant RNA polymerase 1I promoters, like those of other higher eukaryotes, have complex structures and are comprised of several distinct elements. One such element is the **rg-Hogness box, which is rqe for -c TATA box or Goldberg-Hogness box, which is required for correct expression of eukaryotic genes in vitro and accurate, efficient initiation of transcription in vivo. The TATA box is typically positioned at approximately -25 to -35 that is, at 25 to 35 basepairs (bp) upstream of the transcription initiation site, or cap site, which is defined as position +1 (Breathnach and Chambon, Ann. Rev. Biochem. 50:349-383, 1981; Messing et al., In: Genetic Engineering of O -12- O Plants, Kosuge et al., eds., pp. 211-227, 1983). Another common element, the CCAAT box, is r located between -70 and -100 bp. In plants, the CCAAT box may have a different consensus sequence than the functionally analogous sequence of mammalian promoters (the plant analogue 00 has been termed the "AGGA box" to differentiate it from its animal counterpart; Messing et al., e s In: Genetic Engineering of Plants, Kosuge et al., eds., pp. 211-227, 1983). In addition, virtually all promoters include additional upstream activating sequences or enhancers (Benoist and Chambon, Nature 290:304-310, 1981; Gruss et al., Proc. Nat. Acad. Sci. USA 78:943-947, 1981; and Khoury and Gruss, Cell 27:313-314, 1983) extending from around -100 bp to -1,000 bp or more upstream of the transcription initiation site.

When fused to heterologous DNA sequences, such promoters typically cause the fused sequence to be transcribed in a manner that is similar to that of the gene sequence with which the promoter is normally associated. Promoter fragments that include regulatory sequences can be added (for example, fused to the 5' end of, or inserted within, an active promoter having ts own partial or complete regulatory sequences (Fluhr et al., Science 232:1106-1112, 1986; Ellis et al., 1S EMBO J. 6:11-16, 1987; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986-8990, 1987; Poulsen and Chua, Mol. Gen. Genet. 214:16-23, 1988; Comai et al., Plant Mol. Biol. 15:373- 381, 1991). Alternatively, heterologous regulatory sequences can be added to the 5' upstream region of an inactive, truncated promoter, a promoter including only the core TATA and, sometimes, the CCAAT elements (Fluhr et al., Science 232:1106-1112, 1986; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986-8990, 1987; Aryan etal., Mol. Gen. Genet. 225:65-71, 1991). 7 A ry an et al M ol G e Genet. 225:65-71, Promoters are typically comprised of multiple distinct "cis-acting transcriptional regulatory elements," or simply "cis-elements," each of which appears to confer a different aspect of the overall control of gene expression (Strittmatter and Chua, Proc. Nat. Acad. Sci.

USA 84:8986-8990, 1987; Ellis et al., EMBO J. 6:11-16, 1987; Benfey et al., EMBO J. 9:1677- 1684, 1990)." cis elements" bind trans-acting protein factors that regulate transcription. Some cis elements bind more than one fctor, and trans-acting transcription factrs may interact with different affinities with more than one cis element (Johnson and McKnight, Ann. Rev. Bioche.

58:799-839, 1989). Plant transcription fIctors, corresponding cis elements, and analysis of their interaction are discussed, for example, in: Martin Curr. Opinions Biotech. 7:130-138, 196; Mural, In: Methods in Plant Biochemistry and Molecula Biology, Dashek ed., CRC Press, O -13- -q- O 1997, pp. 397-422; and Methods in Plant Molecular Biology, Maliga et al., eds., Cold Spring t Harbor Press, 1995, pp. 233-300. The promoter sequences of the present invention can contain "cis elements" which can confer or modulate gene expression.

o Cis elements can be identified by a number of techniques, including deletion analysis, S 5 deleting one or more nucleotides from the 5' end or internal to a promoter; DNA binding protein analysis using DNase I footprinting, methylation interference, electrophoresis mobilityshift assays, in vivo genomic footprinting by ligation-mediated PCR, and other conventional Sassays; or by sequence similarity with known cis element motifs by conventional sequence comparison methods. The fine structure of a cis element can be further studies by mutagenesis to (or substitution) of one or more nucleotides or by other conventional methods. See, Methods in Plant Biochemistry and Molecular Biology, Dashek, ed., CRC Press, 1997, pp. 397-422; and Methods in Plant Molecular Biology, Maliga et al., eds., Cold Spring Harbor Press, 1995, pp.

233-300.

Cis elements can be obtained by chemical synthesis or by cloning from promoters that includes such elements, and they can be synthesized with additional flanking sequences that contain useful restriction enzyme sites to facilitate subsequence manipulation. In one embodiment, the promoters are comprised of multiple distinct "cis-acting transcriptioial regulatory elements," or simply "cis-elements," each of which appears to confer a different aspect of the overall control of gene expression (Strittmatter and Chua, Proc. Nat. Acad. Sci.

USA 84:8986-8990, 1987; Ellis et al., EMBO J. 6:11-16, 1987; Benfey et al., EMBO J. 9:1677- 1684, 1990). In a preferred embodiment sequence regions comprising "cis elements" of the nucleic acid sequences of SEQ ID NOS: 53-75 are identified using computer programs designed specifically to identify cis elements, or domains or motifs within sequences.

The present invention includes cis elements of SEQ ID NOS: 53-75 or homologues of cis elements known to effect gene regulation that show homology with the nucleic acid sequences of the present invention. A number of such elements are known in the literature, such as elements which are regulated by numerous factors such as light, heat, or stress; elements which arc r t o i dght, eat, or stress; elements which are regulated or induced by pathogens or chemicals, and the like. Such elements may either positively or negatively regulated gene expression, depending on the conditions. Examples of cis elements would include but are not limited to oxygen responsive elements (Cowen et al., J.

Biol. Chem. 268(36):26904, 1993), light regulatory clements (see for example, Bruce and Quail), S-14- S Plant Cell 2(11): 1081. 1990, and Bruce e a l EMBO 10:3015, 1991, acis element reponsive to methyl jasmonate treatment (Beaudoin and Rothstein, Plant Mol. Biol. 33:835, 1997, salicylic acid responsive elements (Strange et al., Plant J. 11:1315, 1997, heat shock response elements (Pelham et al., Trends Genet. 1:31, 1985, elements responsive to wounding and abiotic stress 0 (Loace et al., Proc. Natl. Acad. Sci. U. S. A. 89:9230, 1992; Mhiri et al., Plant Mol. Biol. 33:257, 1997), low temperature elements (Baker et al., Plant Mol. Biol. 24:701, 1994; Jiang qt al., Plant Mol. Biol. 30:679, 1996; Nordin et al., Plant Mol. Biol. 21:641, 993; Zhou et al., J. Biol. Chem.

267:23515, 1992), and drought responsive elements, (Yamaguchi et al., Plant Cell 6:251-264 1994; Wang et al., Plant Mol. Biol. 28:605, 1995; Bray E. A. Trends in Plant Science 2:48, io 1997).

The present invention therefore encompasses fragments or "cis elements" of the disclosed nucleic acid molecules and the nucleic acid fragments can include any region of the disclosed sequences. The promoter regions or partial promoter regions of the present invention as shown in SEQ ID NOS: 53-75 can contain one or more regulatory elements including but not limited to "cis elements" or domains which are capable of regulating transcription of operably linked

DNA

sequences in multiple tissues.

Plant promoters can include promoters produced through the manipulation of known promoters to produce synthetic, chimeric, or hybrid promoters. Such promoters can also combine cis elements fom oneor more promoters, fbr example, by. adding a heterologous regulatory sequence to an active promoter with its own partial or complete regulatory sequences (Ellis et al., EMBO J. 6:11-16, 1987; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986- 8990, 1987; Poulsen and Chua, Mol. Gen. Genet. 214:16-23, 1988; Comai et al., Plant. Mol.

Biol. 15:373-381, 1991). Chimeric promoters have also been developed by adding a heterologous regulatory sequence to the 5' upstream region of an inactive, truncated promoter, 2 a a promoter that includes only the core TATA and, optionally, the CCAAT elements (Fluhr et al., Science 232:106-1112, 1986; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986- 8990, 1987; Aryan et al., Mol. Gen. Genet. 225:65-71, 1991). fThe design, construction, and use of chimeric or hybrid promoters comprising at least one cis element of SEQ ID) NOS: 53-75 for modulating he expression of operably linked nucleic acid sequences is also encompassed by the present invention.

0 The promoter sequences, fragments, regions or cis elements thereof of SEQ ID NOS: 53it 75 are capable of transcribing operably linked DNA sequences in multiple tissues and can selectively regulate expression of genes in these tissues. For a number of agronomic traits, oo expression of a gene or genes of interest is desirable in multiple tissues in order to confer the 5 desired characteristic(s). The availability of suitable promoters which regulate transcription of operably linked genes in selected target tissues of interest is important since it may not be desirable to have expression in every tissue, but only in certain tissues. For example, if one 0desires to control an insect pest which targets particular tissues, it would be of interest to express the desired gene product(s) in those tissues. For herbicide tolerance, it may be desirable to have io a promoter which transcribes operably linked genes in a manner which confers herbicide tolerance at the desired levels in both vegetative and reproductive tissues. Consequently, it is important to have a wide variety of choices of 5' regulatory elements for any plant biotechnology strategy.

The advent of genomics, which comprises molecular and bioinformatics techniques, has is resulted in rapid sequencing and analyses of a large number of DNA samples from a vast number of targets, including but not limited to plant species of agronomic importance. To identify the nucleic acid sequences of the present invention from a database or collection of cDNA sequences, the first step involves constructing cDNA libraries from specific plant tissue targets of interest. Briefly, the cDNA libraries are first constructed from these tissues that are harvested at a particular developmental stage, or under particular environmental conditions. By identifying differentially expressed genes in plant tissues at different developmental stages, or under different conditions, the corresponding regulatory sequences of those genes can be identified and isolated. Transcript imaging enables the identification of tissue-preferred sequences based on specific imaging of nucleic acid sequences from a cDNA library. By transcript imaging as used herein is meant an analysis which compares the abundance of expressed genes in one or more libraries. The clones contained within a cDNA library are sequenced and the sequences compared with sequences from publicly available databases. Computer-based methods allows the researcher to provide queries which compare sequences from multiple libraries. The process enables quick identification of clonesf enables quick identification of clones of interest compared with conventional hybridization subtraction methods known to those of skill in the art.

O -16- Using conventional methodologies, cDNA libraries can be constructed from the mRNA (messenger RNA) of a given tissue or organism using poly dT primers and reverse transcriptase (Efstratiadis, et al., Cell 7:279, 1976; Higuchi, et al., Proc. Natl. Acad. Sci. 73:3146, 0 1976; Maniatis, et al., Cell 8:163, 1976; Land et al., Nucleic Acids Res. 9:2251, 1981; S 5 Okayama, et al., Mol. Cell. Biol. 2:161, 1982; Gubler, et al., Gene 25:263, 1983).

Several methods can be employed to obtain full-length cDNA constructs. For example, C terminal transferase can be used to add homopolymeric tails of dC residues to the free 3' O hydroxyl groups (Land, et al., Nucleic Acids Res. 9:2251, 1981). This tail can then be hybridized by a poly dG oligo which can act as a primer for the synthesis of full length second to strand cDNA. Okayama and Berg, report a method for obtaining full length cDNA constructs.

This method has been simplified by using synthetic primer-adapters that have both homopolymeric tails for priming the synthesis of the first and second strands and restriction sites for cloning into plasmids (Coleclough, et al., Gene 34:305, 1985) and bacteriophage vectors (Krawinkel, et al., Nucleic Acids Res. 14:1913, 1986; and Han, et al., Nucleic Acids Res.

Is 15:6304, 1987).

These strategies can be coupled with additional strategies for isolating rare mRNA populations. For example, a typical mammalian cell contains between 10,000 and 30,000 different mRNA sequences. Davidson, Gene Activity in Early Development, 2nd ed., Academic Press, New York, 1976. The number of clones required to achieve a given probability that a low-abundance mRNA will be present in a cDNA library is N where N is the number of clones required, P is the probability desired, and 1/n is the fractional proportion of the total mRNA that is represented by a single rare mRNA (Sambrook, et al., 1989).

One method to enrich preparations of mRNA for sequences of interest is to fractionate by size. One such method is to fractionate by electrophoresis through an agarose gel (Pennica, et 2s al., Nature 301:214, 1983). Another such method employs sucrose gradient centrifugation in the presence of an agent, such as methylmercuric hydroxide, that denatures secondary structure in RNA (Schweinfest, et al., Proc. Natl. Acad. Sci. 79:4997-5000, 1982).

A frequently adopted method is to construct equalized or normalized cDNA libraries (Ko, Nucleic Acids Res. 18:5705, 1990; Patanjali, S. R. et al., Proc. Natl. Acad. Sci. o 88:1943, 1991). Typically, the cDNA population is normalized by subtractive hybridization.

Schmid, et al., J. Neurochem. 48:307, 1987; l"Fgnoli, et al., Anal. Biochem. 187:364, 1990; 17- O Travis, et al., Proc. Natl. Acad. Sci 85:1696, 1988; Kato, Eur. J. Neurosci. 2:704, 1990; and Schweinfest, et al., Genet. Anal. Tech. Appl. 7:64, 1990). Subtraction represents another method for reducing the population of certain sequences in the cDNA library (Swaroop, et al., OO Nucleic Acids Res. 19:1954, 1991). Normalized libraries can be constructed using the Soares s procedure (Soares et al., Proc. Natl. Acad. Sci. S. 91:9228, 1994). This approach is designed to reduce the initial 10,000-fold variation in individual cDNA frequencies to achieve abundances within one order of magnitude while maintaining the overall sequence complexity of I the library. In the normalization process, the prevalence of high-abundance cDNA clones decreases dramatically, clones with mid-level abundance are relatively unaffected, and clones for rare transcripts are effectively increased in abundance.

ESTs can be sequenced by a number of methods. Two basic methods can be used for DNA sequencing, the chain termination method of Sanger et al., Proc. Natl. Acad. Sci. 74: 5463, 1977 and the chemical degradation method of Maxam and Gilbert, Proc. Nat. Acad.

Sci. 74: 560, 1977. Automation and advances in technology such as the replacement of is radioisotopes with fluorescence-based sequencing have reduced the effort required to sequence DNA (Craxton, Methods, 2: 20, 1991; Ju et al., Proc. Natl. Acad. Sci. 92: 4347, 1995; Tabor and Richardson, Proc. Natl. Acad. Sci. 92: 6339, 1995). Automated sequencers are available from a number of manufacturers, for example, Pharmacia Biotech, Inc., Piscataway, New Jersey (Pharmacia ALF), LI-COR, Inc., Lincoln, Nebraska (LI-COR 4,000) and Millipore, Bedford, Massachusetts (Millipore BaseStation).

ESTs longer than 150 bp have been found to be useful for similarity searches and mapping. (Adams, et al., Science 252:1651, 1991. EST sequences normally range from 150-450 bases. This is the length of sequence information that is routinely and reliably generated using single run sequence data. Typically, only single run sequence data is obtained from the cDNA library, Adams, et al., Science 252:1651, 1991. Automated single run sequencing typically results in an approximately 2-3% error or base ambiguity rate. (Boguski, et al., Nature Genetics, 4:332, 1993).

EST databases have been constructed or partially constructed from, for example, C.

elegans (McCombrie, et al., Nature Genetics 1:124, 1992, human liver cell line HepG2 (Okubo, et al., Nature Genetics 2:173, 1992); human brain RNA (Adams, et al., Science 252:1651, 1991; Adams, et al., Nature 355:632, 1992); Arabidopsis, (Newman, et al., Plant Physiol. 106:1241, 1-18- 1994); and rice (Kurata, et al., Nature Genetics 8:365, 1994). The present invention uses ESTs t1 from a number of libraries prepared from multiple corn tissues as a tool for the identification of genes expressed in desired tissues which then facilitates the isolation of 5' regulatory sequences 00 such as promoters which regulate the genes.

Computer-based sequence analyses can be used to identify differentially expressed Ssequences including but not limited to those sequences expressed in one tissue compared with another tissue. For example, a different set of sequences can be found from cDNA isolated from Splant tissue isolated from root tissue versus leaf tissue. Accordingly, sequences can be compared from cDNA libraries prepared from plants grown under different environmental or physiological to conditions. Once the preferred sequences are identified from the cDNA library of interest, the genomic clones can be isolated from a genoric library prepared from the plant tissue, and corresponding regulatory sequences including but not limited to 5' regulatory sequences can be identified and isolated.

In one preferred embodiment, expressed sequence tags (EST) sequences from a variety of is cDNA libraries are catalogued in a sequence database. This database is used to identify promoter targets from a particular tissue of interest. The selection of expressed sequence tags for subsequent promoter isolation is reflective of the presence of one or more sequences among the representative ESTs from a random sampling of an individual cDNA library, or a collection of cDNA libraries. For example, the identification of regulatory sequences that regulate the expression of transcripts in leaf and root tissue is conducted by identifying ESTs found in leaf and root cDNA libraries and absent or in lower abundance in other cDNA libraries and the expression profile for a given EST is assessed. By abundance as used herein is meant the number of times a clone or cluster of clones appears in a library. The sequences that are enhanced or in high abundance in a specific tissue or organ which represent a target expression profile are identified in this manner and primers can be designed from the identified

EST

sequences. A PCR-based approach can be used to amplify flanking regions from a genoric library of the target plant of interest. A number of methods are known to those of skill in the art to amplify unknown DNA sequences adjacent to a core region of known sequence. Methods include but are not limited to inverse PCR (IPCR), vectorette PCR, Y-shaped PCR and genome walking approaches.

S- 19- In a preferred embodiment, genomic DNA ligated to an adapter is subjected to a primary C round of PCR amplification with a gene-specific primer and a primer which anneals to the adapter sequence. The PCR product is next used as the template for a nested round of PCR oO amplification with a second gene-specific primer and second adapter. The resulting fragments s from the nested PCR reaction are then isolated, purified and subcloned into an appropriate vector. The fragments are sequenced and the translational start sites can be identified when the EST is derived from a truncated cDNA. The fragments can be cloned into plant expression 0 vectors as transcriptional or translational fusions with a reporter gene such as P-glucuronidase (GUS). The constructs can be tested in transient analyses and subsequently the 5' regulatory o0 regions are operably linked to other genes and regulatory sequences of interest in a suitable plant transformation vector and the transformed plants are analyzed for the expression of the gene(s) of interest by any number of methods known to those of skill in the art.

Any plant can be selected for the identification of genes and regulatory sequences.

Examples of suitable plant targets for the isolation of genes and regulatory sequences would include but are not limited to Acadia, alfalfa, apple, apricot, Arabidopsis, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus, clementines, clover, coconut, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, linseed, mango, melon, mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive, onion, orange, an ornamental plant, palm, papaya, parsley, parsnip, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radiscchio, radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, 'soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf, turnip, a vine, watermelon, wheat, yams, and zucchini.

Particularly preferred plant targets would include corn, cotton, soybean, and wheat.

The nucleic acid molecules of the present invention are isolated from corn (Zea mays).

The corn plant develops about 20-21 leaves, silks about 65 days post-emergence, and matures about 125 days post-emergence. Normal corn plants follow a general pattern of development, O O but the time interval between different stages and morphology varies between different hybrids, ngrowth and environmental conditions.

There are a number of identifiable stages in corn plant development. The stages are oO defined as vegetative and reproductive stages. Subdivisions of the V stages are s numerically designated as VI, V2,V3, etc., through V(n) where represents the last leaf stage N before tasseling (VT) and the first V stage is the emergence (VE) stage. For example, VE is the emergence from the soil of a seedling leaf, VI represents the first true leaf, V2 represents the Ssecond leaf, etc. The reproductive stages include the first appearance of silk to the mature seed and are represented as follows: RI is silking, R2 is blistering, R3 is the milk stage, R4 is the to dough stage, R5 is the dent stage, and R6 is physiological maturity (see for example, Ritchie SW et al. (1986) How a Corn Plant Develops, Iowa State University of Science and Technology Cooperative Exension Service, Ames, IA 48: 1-21).

Any type of plant tissue can be used as a target tissue for the identification of genes and associated regulatory elements, including but not limited to promoter sequences. For the present 1s invention multiple tissues of corn are used. More preferably, corn meristem, immature tassel, leaf, root, culm, etiolated seedlings, sheath, primary shoot, ear, silk and endosperm are the target tissues for identification of promoter sequences.

Any method which allows a differential comparison between different types or classes of sequences can be used to isolate genes or regulatory 'sequences of interest. For example in one differential screening approach, a cDNA library from mRNA isolated from a particular tissue can be prepared in a bacteriophage host using a commercially available cloning kit. The plaques are spread onto plates containing lawns of a bacterial host such as E. coli to generate bacteriophage plaques. About 105 106 plaques can be lifted onto DNA binding membranes. Duplicate membranes are probed using probes generated fiom mRNA from the target and non-target or 2s background tissue. The probes are labeled to facilitate detection after hybridization and development. Plaques that hybridize to target tissue -derived probes but not to non-target tissue derived probes that display a desired differential pattern of expression can be selected for further analysis. Genomic DNA libraries can also be prepared from a chosen species by partial digestion with a restriction enzyme and size selecting the DNA fiagments within a particular size range. The genomic D)NA can be cloned into a suitable vector including but not limited to a S-21o bacteriophage, and prepared using a suitable kit as described earlier (see for example, Stratagene, i La Jolla, CA or Gibco BRL, Gaithersburg, MD).

Differential hybridization techniques as described are well known to those of skill in the 0 art and can be used to isolate a desired class of sequences. By classes of sequences as used s herein is meant sequences that can be grouped based on a common identifier including but not Slimited to sequences isolated from a common target plant, a common library, or a common plant tissue type. In a preferred embodiment, sequences of interest are identified based on sequence O analyses and querying of a collection of diverse cDNA sequences from libraries of different tissue types. The disclosed method provides an example of a differential screening approach based on electronic sequence analyses of plant ESTs derived from diverse cDNA libraries.

A number of methods used to assess gene expression are based on measuring the mRNA level in an organ, tissue, or cell sample. Typical methods include but are not limited to RNA blots, ribonuclease protection assays and RT-PCR. In another preferred embodiment, a highthroughput method is used whereby regulatory sequences are identified from a transcript is profiling approach. The development of cDNA microarray technology enables the systematic monitoring of gene expression profiles for thousands of genes (Schena et al, Science, 270: 467, 1995). This DNA chip-based technology arrays thousands of cDNA sequences on a support surface. These arrays are simultaneously hybridized to a -multiple of labeled cDNA probes prepared from RNA samples of different cell or tissue types, allowing direct comparative analysis of expression. This technology was first demonstrated by analyzing 48 Arabidopsis genes for differential expression in roots and shoots (Schena et al, Science, 270:467, 1995).

More recently, the expression profiles of over 1400 genes were monitored using cDNA microarrays (Ruan et al, The Plant Journal 15:821, 1998). Microarrays provide a highthroughput, quantitative and reproducible method to analyze gene expression and characterize gene function. The transcript profiling approach using microarrays thus provides another valuable tool for the isolation of regulatory sequences such as promoters associated with those genes.

The present invention uses high throughput sequence analyses to form the foundation of rapid computer-based identification of sequences of interest. Those of skill in the art are aware of the resources available for sequence analyses. Sequence comparisons can be undertaken by determining the similarity of the test or query sequence with sequences in publicly available or 0D -22- O proprietary databases ("similarity analysis") or by searching for certain motifs ("intrinsic sequence analysis") cis elements) (Coulson, Trends in Biotechnology, 12:76, 1994; Birren, et al., Genome Analysis, 1:543, 1997).

0 The nucleotide sequences provided in SEQ ID NOS: 53-75 or fragments thereof, or complements thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical M even more preferably 99% or 100% identical to the sequence provided in SEQ ID NOS: 53-75 or r fragment thereof, or complement thereof, can be "provided" in a variety of mediums to facilitate Suse. Such a medium can also provide a subset thereof in a form that allows one of skill in the art to examine the sequences.

In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media. As used herein, "computer readable media" refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc, storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM is and ROM; and hybrids of these categories such as magnetic/optical storage media. One of skill in the art can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.

By providing one or more of nucleotide sequences of the present invention, those of skill in the art can routinely access the sequence information for a variety of purposes. Computer software is publicly available which allows one of skill in the art to access sequence information provided in a computer readable medium. Examples of public databases would include but is not limited to the DNA Database of Japan (DDBJ) (bttp://www.ddbj.nig.ac.jp/);Genbank (http://www.ncbi.nlm.nih.gov/web/Genbank/Index.html); and the European Molecular Biology Laboratory Nucleic Acid Sequence Database (EMBL)(http://www.ebi.ac.uk/ebi docs/ embl_db.html) or versions thereof. A number of different search algorithms have been developed, including but rot limited to the suite of programs referred to as BLAST programs.

There are five implementations of BLAST, three designed for nucleotide sequence queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology, 12:76-80, 1994; Birren, et al., Genome Analysis, 1:543, 1997).

-23- O -23 -q- Q Any program designed for motif searching also has utility in the present invention.

Sequence analysis programs designed for motif searching can be used for identification of cis elements. Preferred computer programs would include but are not limited to MEME, SIGNAL o SCAN, and GENESCAN. Meme is a program that identifies conserved motifs (either nucleic s acid or peptide) in a group of unaligned sequences. Meme saves these motifs as a set of profiles.

These profiles can be used to search a database of sequences. A MEME algorithm (version 2.2) can be found in version 10.0 of the GCG package; MEME Bailey and C. Elkan, Machine Learning, 21(1-2):51-80,1995 and the location of the website is as follows: (http://www.sdsc.edu/MEME/meme/website/COPYRIGHT.html.). SignalScan is a program that to identifies known motifs in the test sequences using information from other motif databases (Prestridge, CABIOS 7, 203-206 (1991). SignalScan version 4.0 information is available at the following website: http://biosci.cbs.umn.edu/software/sigscan.html. The ftp site for Signal Scan is ftp://biosci.cbs.umn.edu/software/sigscan.html. Databases used with Signal Scan include PLACE (http://www.dna.affrc.go.ip/htdocs/PLACE (Higo et al., Nucleic Acids Research 27(1):297-300 (1999) and TRANSFAC (Heinemeye, X. et al., Nucleic Acid Research 27(1):318- 322) which can be found at the following website: http://transfac.gbf.de/. GeneScan is another suitable program for motif searching (Burge, C and Karlin, S. J. Mol. Biol. 268, 78-94 (1997) and version 1.0 information is available at the following website: http://gnomic.stanford.edu/GENESCANW.html. As used herein, "a target structural motif', or "target motif' refers any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known to those of skill in the art.

Protein target motifs include but are not limited to, enzymatic active sites and signal sequences.

Preferred target motifs of the present invention would include but are not limited to promoter sequences, cis elements, hairpin structures and other expression elements such as protein binding sequences.

As used herein, "search means" refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the sequences of the present invention that match a particular target sequence or target motif. Also, multiple sequences can be compared in order to identify Cg -24- 0 common regions or motifs which may be responsible for specific functions. For example, cis elements or sequence domains which confer a specific expression profile can be identified when multiple promoter regions of similar classes of promoters are aligned and analyzed by certain 0 software packages.

The present invention further provides systems, particularly computer-based systems, which contain the sequence information described herein. As used herein, a "computer-based system" refers to the hardware means, software means, and data storage means used to analyze S the nucleotide sequence information of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), .o input means, output means, and data storage means. Those of skill in the art can appreciate that any one of the available computer-based systems are suitable for use in the present invention.

SEQ ID NOS: 6-52 are primers designed from the cDNA sequences identified from the computer-based sequence comparisons. These sequences are used to extend the nucleic acid sequence using polymerase chain reaction (PCR) amplification techniques (see for example, is Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1986; Erlich, et al., European Patent Appln. 50,424; European Patent Appln. 84,796, European Patent Appln. 258,017, European Patent Appln. 237,362; Mullis, European Patent Appln. 201,184; Mullis, et al., U. S.

Patent No. 4,683,202; Erlich, U. S. Patent 4,582,788; and Saiki, et al., U. S. Patent No.

4,683,194). A number of PCR amplification methods are known to those of skill in the art, and are used to identify nucleic acid sequences adjacent to a known sequence. For example, inverse per (IPCR) methods to amplify unknown DNA sequences adjacent to a core region of known sequence have been described. Other methods are also available such as capture PCR (Lagerstrom et al., PCR Methods Applic. 1:111, 1991, and walking PCR (Parker, JD et al.,Nucleic Acids Res 19:3055, 1991). A number of manufacturers have also developed kits based on modifications of these methods for the purposes of identifying sequences of interest.

Technical advances including improvements in primer and adapter design, improvements in the polymerase enzyme, and thermocycler capabilities have facilitated quicker, efficient methods for isolating sequences of interest.

In a preferred embodiment, the flanking sequences containing the 5' regulatory elements of the present invention are isolated using a genome-walking approach (Universal GenomeWalker .M Kit, CLONTECH Laboratories, Inc., Palo, Alto, CA). In brief, the purified O genomic DNA is subjected to a restriction enzyme digest which produces genomic DNA fragments with ends that are ligated with GenomeWalkerM adapters. GenomeWalker'M primers are used along with gene specific primers in two consecutive PCR reactions (primary and nested oo PCR reactions) to produce PCR products containing the 5' regulatory sequences which are c 5 subsequently cloned and sequenced.

C In addition to their use in modulating gene expression, the promoter sequences of the present invention also have utility as probes or primers in nucleic acid hybridization experiments.

The nucleic-acid probes and primers of the present invention can hybridize under stringent conditions to a target DNA sequence. The term "stringent hybridization conditions" is defined to as conditions under which a probe or primer hybridizes specifically with a target sequence(s) and not with non-target sequences, as can be determined empirically. The term "stringent conditions" is functionally defined with regard to the hybridization of a nucleic-acid probe to a target nucleic acid to a particular nucleic-acid sequence of interest) by the specific hybridization procedure discussed in Sambrook et al., 1989, at 9.52-9.55. See also, Sambrook et is al., 1989 at 9.47-9.52, 9.56-9.58; Kanehisa, Nucl. Acids Res. 12:203-213, 1984; and Wetmur and Davidson, J. Mol. Biol. 31:349-370, 1968. Appropriate stringency conditions which promote DNA hybridization are, for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 450 C, followed by a wash of 2.0 x SSC at 50 0 C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley Sons, 1989, 6.3.1-6.3.6. For 2o example, the salt concentration in the wash step can be selected from a low stringency of about x SSC at 50°C to a high stringency of about 0.2 x SSC at 50 0 C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65 0 C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed.

For example, hybridization using DNA or RNA probes or primers can be performed at in 6x SSC, 0.5% SDS, 5x Denhardt's, 100 [.tg/mL nonspecific DNA sonicated salmon sperm DNA) with washing at 0.5x SSC, 0.5% SDS at 65 0 C, for high stringency.

It is contemplated that lower stringency hybridization conditions such as lower hybridization and/or washing temperatures can be used to identify related sequences having a lower degree of sequence similarity if specificity of binding of the probe or primer to target 26- O sequence(s) is preserved. Accordingly, the nucleotide sequences of the present invention can be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments. Detection of DNA segments via hybridization is well-known to those of skill in the OO art, and thus depending on the application envisioned, one will desire to employ varying M s hybridization conditions to achieve varying degrees of selectivity of probe towards target sequence and the method of choice will depend on the desired results.

The nucleic acid sequences in SEQ ID NOS: 53-75 and any variants thereof, are capable S of hybridizing to other nucleic acid sequences under appropriately selected conditions of stringency. As used herein, two nucleic acid molecules are said to be capable of specifically 0o hybridizing to one another if the two molecules are capable of forming an anti-parallel, doublestranded nucleic acid structure. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if they exhibit complete complementarity. As used herein, molecules are said to exhibit "complete complementarity" when every nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be is "minimally complementary" if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional "low stringency" conditions. Similarly, the molecules are said to be "complementary" is they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional "high stringency" conditions. Conventional stringency conditions are described by Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989, and by Haymes et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC, 1985.

In a preferred embodiment, the nucleic acid sequences, SEQ ID NOS: 53-75, or a fragment, region, cis element, or oligomer of any of these sequences, may be used in hybridization assays of other plant tissues to identify closely related or homologous genes and associated regulatory sequences. These include but are not limited to Southern or northern hybridization assays on any substrate including but not limited to an appropriately prepared plant tissue, cellulose, nylon, or combination filter, chip, or glass slide. Such methodologies are well known in the art and are available in a kit or preparation which can be supplied by commercial vendors.

0 -27- O Of course, fragments can also be obtained by other techniques such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer. Also, fragments can be obtained by application of nucleic acid 0 reproduction technology, such as the PCR'" (polymerase chain reaction) technology by S 5 recombinant DNA techniques generally known to those of skill in the art of molecular biology.

M Regarding the amplification of a target nucleic- acid sequence by PCR) using a particular amplification primer pair, "stringent PCR conditions" refer to conditions that permit the primer 0pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and preferably to produce a to unique amplification product.

A fragment of a nucleic acid as used herein refers to any portion of the nucleic acid that is less than full-length. For example, for the present invention any length ofnucleotide sequence that is less than the disclosed nucleotide sequences of SEQ ID NOS: 53-75 is considered to be a fragment. A fragment can also comprise at least a minimum length capable of hybridizing is specifically with a native nucleic acid under stringent hybridization conditions as defined above.

The length of such a minimal fragment is preferably at least eight nucleotides, more preferably nucleotides, even more preferably at least 20 nucleotides, and most preferably at least nucleotides of a native nucleic acid sequence.

The nucleic acid sequences of the present invention can also be used as probes and primers. Nucleic acid probes and primers can be prepared based on a native gene sequence. A "probe" is an isolated nucleic acid to which is attached a conventional detectable label or reporter molecule, a radioactive isotope, ligand, chemiluminescent agent, or enzyme. "Primers" are isolated nucleic acids that are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, a DNA polymerase. Primer pairs can be used for amplification of a nucleic acid sequence, by the polymerase chain reaction (PCR) or other conventional nucleic-acid amplification methods.

Probes and primers are generally 15 nucleotides or more in length, preferably nucleotides or more, more preferably 25 nucleotides, and most preferably 30 nucleotides or 3o more. Such probes and primers hybridize specifically to a target DNA or RNA sequence under high stringency hybridization conditions and hybridize specifically to a target native sequence of -28- 0 another species under lower stringency conditions. Preferably, probes and primers according to the present invention have complete sequence similarity with the native sequence, although probes differing from the native sequence and that retain the ability to hybridize to target native 0 sequences may be designed by conventional methods. Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., C vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 (hereinafter, "Sambrook et al., 1989"); Current Protocols in Molecular Biology, ed.

S Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates) (hereinafter, "Ausubel et al., 1992); and Innis et al., PCR Protocols: A Guide to io Methods and Applications, Academic Press: San Diego, 1990. PCR-primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA). Primers and probes based on the native promoter sequences disclosed herein can be used to confirm and, if necessary, to modify the disclosed sequences by conventional methods, e.g., is by re-cloning and re-sequencing.

In another embodiment, the nucleotide sequences of the promoters disclosed herein tan be modified. Those skilled in the art can create DNA molecules that have variations in the nucleotide sequence. The nucleotide sequences of the present invention as shown in SEQ ID NOS: 53-75 may be modified or altered to enhance their control characteristics. One preferred method of alteration of a nucleic acid sequence is to use PCR to modify selected nucleotides or regions of sequences. These methods are known to those of skill in the art. Sequences can be modified, for example by insertion, deletion or replacement of template sequences in a PCRbased DNA modification approach. "Variant" DNA molecules are DNA molecules containing changes in which one or more nuclcotides of a native sequence is deleted, added, and/or substituted, preferably while substantially maintaining promoter function. In the case of a promoter fragment, "variant" DNA can include changes affecting the transcription of a minimal promoter to which it is operably linked. Variant DNA molecules can be produced, for example, by standard DNA mutagenesis techniques or by chemically synthesizing the variant DNA molecule or a portion thereof.

In another embodiment, the nucleotide sequences as shown in SEQ ID NOS: 53-75 includes any length of said nucleotide sequences which is capable of regulating an operably 2 9 O linked DNA sequence. For example, the sequences as disclosed in SEQ ID NOS: 53-75 may be S truncated or portions deleted and still be capable of regulating transcription of an operably linked DNA sequence. In a related embodiment, a cis element of the disclosed sequences may confer a 00 particular specificity such as conferring enhanced expression of operably linked DNA sequences in certain tissues and therefore is also capable of regulating transcription of operably linked DNA C, sequences. Consequently, any sequence fragments, portions, or regions of the disclosed S sequences of SEQ ID NOS: 53-75 can be used as regulatory sequences, including but not limited C, to cis elements or motifs of the disclosed sequences. For example, one or more base pairs may be deleted from the 5' or 3' end of a promoter sequence to produce a "truncated" promoter. One to or more base pairs can also be inserted, deleted, or substituted internally to a promoter sequence.

Promoters can be constructed such that promoter fragments or elements are operably linked, for example, by placing such a fragment upstream of a minimal promoter. A minimal or basal promoter is a piece of DNA which is capable of recruiting and binding the basal transcription machinery. One example of basal transcription machinery in eukaryotic cells is the RNA 1i polymerase II complex and its accessory proteins. The enzymatic components of the basal transcription machinery are capable of initiating and elongating transcription of a given gene, utilizing a minimal or basal promoter. That is, there are not added cis-acting sequences in the promoter region which are capable of recruiting and binding transcription factors that modulate transcription, enhance, repress, render transcription hormone-dependent, etc. Substitutions, deletions, insertions or any combination thereof can be combined to produce a final construct.

Native or synthetic nucleic acids according to the present invention can be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a host cell. In one preferred embodiment, the nucleotide sequences of the present invention as shown in SEQ ID NOS: 53-75 or fragments, variants, or derivatives thereof are incorporated into an expression vector cassette which includes the promoter regions of the present invention operably linked to a genetic component such as a selectable, screenable, or scorable marker gene. The disclosed nucleic acid sequences of the present invention are preferably operably linked to a genetic component such as a nucleic acid which confers a desirable characteristic associated with plant morphology, physiology, growth and development 3" yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. These genetic components such as marker genes or agronomic genes of interest can 0 0 0 0 function in the identification of a transbfrmed plant cell or plant, or a produce a product of agronomic utility.

In a preferred embodiment, one genetic component produces a product which serves as a OO selection device and functions in a regenerable plant tissue to produce a compound which would confer upon the plant tissue resistance to an otherwise toxic compound. Genes of interest for use as a selectable, screenable, or scorable marker would include but are not limited to the coding sequence for -glucuronidase (GUS), the coding sequence for green fluorescent protein (GFP), S the coding sequence for luciferase (LUX), antibiotic or herbicide tolerance genes. Examples of transposons and associated antibiotic resistance genes include the transposons Tns (bla), io (nptlI), Tn7 (dhfr), penicillins, kanamycin (and neomycin, G418, bleomycin); methotrexate (and trimethoprim); chloramphenicol; kanamycin and tetracycline.

Characteristics useful for selectable markers in plants have been outlined in a report on the use of microorganisms (Advisory Committee on Novel Foods and Processes, July 1994).

These include stringent selection with minimum number of nontransformed tissues, large is numbers of independent transformation events with no significant interference with the regeneration, application to a large number of species, and availability of an assay to score the tissues for presence of the marker.

A number of selectable marker genes are known in the art and several antibiotic resistance markers satisfy these criteria, including those resistant to kanamycin (nptll), hygromycin B (aph IV) and gentamycin (aac3 and aacC4). Useful dominant selectable marker genes include genes encoding antibiotic resistance genes resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin); and herbicide resistance genes phosphinothricin acetyltransferase). A useful strategy for selection of transformants for herbicide resistance is described, in Vasil, Cell Culture and Somatic Cell Genetics of Plants, Vols. I-III, Laboratory Procedures and Their Applications Academic Press, New York, 1984. Particularly preferred selectable marker genes for use in the present invention would genes which confer resistance to compounds such as antibiotics like kanamycin and herbicides like glyphosate (Della-Cioppa et al., Bio/fechnology 1987, U. S. Patent 5,463,175, U. S. Patent 5,633,435). Other selection devices can also be implemented and would still fall within the scope of the present invention.

0 3 1 O For the practice of the present invention, conventional compositions and methods for Spreparing and using vectors and host cells are employed, as discussed, inter alia, in Sambrook et al., 1989). In a preferred embodiment, the host cell is a plant cell. A number of vectors suitable 0O for stable transfection of plant cells or for the establishment of transgenic plants have been s described in, Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987); N Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990; and R. R. D. Croy, Plant Molecular Biology LabFax, BIOS Scientific Publishers, 1993. Plant expression vectors can include, for example, one or more cloned plant genes under the transcriptional control of and 3' regulatory sequences. They can also can include a selectable marker as described to allow selection of host cells containing the expression vector. Such plant expression vectors also typically contain a promoter regulatory region a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing is signal, a transcription termination site, and a polyadenylation signal. Other sequences, of bacterial origin are also included to allow the vector to be cloned in a bacterial host. The vector will also typically contain a broad host range prokaryotic origin of replication. In a particularly preferred embodiment, the host cell is a plant cell and the plant expression vector comprises a promoter region as disclosed in SEQ ID NOS: 53-75, an operably linked transcribable sequence, and a transcription termination sequence. Other regulatory sequences can also be included such as 5' non-translated leaders, in addition to restriction enzyme sites for cloning purposes.

A number of promoters have utility for plant gene expression for any gene of interest including but not limited to selectable markers, scorable markers, genes for pest tolerance, disease tolerance, nutritional enhancements and any other gene of agronomic interest.. Examples of constitutive promoters useful for plant gene expression include but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, Odel et al., Nature 313:810, 1985), including monocots (see, Dekeyser et al., Plant Cell 2:591, 1990; Terada and Shimamoto, Mol. Gen.

Genet. 220:389, 1990); the nopaline synthase promoter (An et al., Plant Physiol. 88:547, 1988) and the octopine synthase promoter (Fromm et al., Plant Cell 1:977, 1989) and the figwort mosaic virus (FMV) promoter.

0 -32-

C.)

O A variety of plant gene promoters that are regulated in response to environmental, t hormonal, chemical, and/or developmental signals can be used for expression of an operably linked gene in plant cells, including promoters regulated by heat (Callis et al., Plant Physiol.

OO 88:965, 1988), light pea rbcS-3A promoter, Kuhlemeier et al., Plant Cell 1:471, 1989; s maize RbcS promoter, Schaffner and Sheen, Plant Cell 3:997, 1991; or chlorophyll a/b-binding N protein promoter, Simpson et al., EMBO J. 4:2723, 1985), hormones, such as abscisic acid (Marcotte et al., Plant Cell 1:969, 1989), wounding wund, Siebertz et al., Plant Cell 1:961, 1989); or chemicals such as methyl jasmonate, salicylic acid, or Safener. It may also be advantageous to employ organ-specific promoters Roshal et al., EMBO J. 6:1155, to 1987; Schernthaner et al., EMBO J. 7:1249, 1988; Bustos et al., Plant Cell 1:839, 1989).

The promoters of the present invention are plant promoters that are capable of transcribing operatively linked DNA sequences in multiple tissues and can be operably linked to any gene of interest in an expression vector.

Plant expression vectors can include RNA processing signals, introns, which may be positioned upstream or downstream of a polypeptide-encoding sequence in the transgene. In addition, the expression vectors may include additional regulatory sequences from the 3'untranslated region of plant genes (Thornburg et al., Proc. Natl. Acad. Sci. USA 84:744 (1987); An et al., Plant Cell 1:115 (1989), a 3' terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3' terminator regions. 5' non-translated regions of a mRNA can play an important role in translation initiation and can also be a genetic component in a plant expression vector. For example, non-translated 5' leader sequences derived from heat shock protein genes have been demonstrated to enhance gene expression in plants (see, for example U. S. Patent 5,362,865 herein incorporated by reference in its entirety). These additional upstream and downstream regulatory sequences may be derived from a source that is native or heterologous with respect to the other elements present on the expression vector.

The promoter sequences of the present invention are used to control gene expression in plant cells. The disclosed promoter sequences are genetic components which are part of vectors used in plant transformation. The promoter sequences of the present invention can be used with any suitable plant transformation plasmid or vector containing a selectable or screenable marker and associated regulatory elements, as described, along with one or more nucleic acids expressed O' -33- O in a manner sufficient to confer a particular desirable trait. Examples of suitable structural genes of agronomic interest envisioned by the present invention would include but are not limited to one or more genes for insect tolerance, such as Bacillus thuringiensis., pest tolerance such as 00 genes for fungal disease control, herbicide tolerance such as genes conferring glyphosate tolerance, and genes for quality improvements such as yield, nutritional enhancements, Cl environmental or stress tolerances, or any desirable changes in plant physiology, growth, 0 development, morphology or plant product(s). Particularly preferred genes would include any CN herbicide tolerance gene which confers tolerance to the herbicide glyphosate. These genes would include but are not limited to AGRTU.aroA:CP4 and GOX genes as described in U. S.

to Patent Nos. 5,463,175 herein incorporated by reference in its entirety and 5,633,435 herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can effect these phenotypes by encoding a nontranslatable RNA molecule that causes the targeted inhibition of expression of an endogenous gene, for example via antisense- or cosuppression-mediated mechanisms (see, for example, Bird s1 et al., Biotech. Gen. Engin. Rev. 9:207,1991). The RNA could also be a catalytic RNA molecule a ribozyme) engineered to cleave a desired endogenous mRNA product (see for example, Gibson and Shillitoe, Mol. Biotech. 7:125,1997). Thus, any gene which produces a protein or mRNA which expresses a phenotype or morphology change of interest are useful for the practice of the present invention.

In addition to regulatory elements or sequences located upstream or within a DNA sequence, there are downstream sequences which affect gene expression and thus the term regulatory sequence as used herein refers to any nucleotide sequence located upstream, within, or downstream to a DNA sequence which controls, mediates, or affects expression of a gene product in conjunction with the protein synthetic apparatus of the cell.

2s The promoter sequences of the present invention may be modified, for example for expression in other plant systems. In another approach, novel hybrid promoters can be designed or engineered by a number of methods. Many promoters contain upstream sequences which activate, enhance or define the strength and/or specificity of the promoter (Atchison, Ann. Rev.

Cell Biol. 4:127, 1988). T-DNA genes, for example contain "TATA" boxes defining the site of transcription initiation and other upstream elements located upstream of the transcription initiation site modulate transcription levels (Gelvin, In Transgenic Plants (Kung, And Us, O 34- -q- 0 eds), San Diego: Academic Press, pp.49-87, 1988). Another chimeric promoter combined a trimer of the octopine synthase (ocs) activator to the mannopine synthase (mas) activator plus promoter and reported an increase in expression of a reporter gene (Min Ni et al., The Plant 00 Journal 7:661, 1995). The upstream regulatory sequences of the present invention can be used s for the construction of such chimeric or hybrid promoters. Methods for construction of variant promoters of the present invention include but are not limited to combining control elements of Sdifferent promoters or duplicating portions or regions of a promoter (see for example U. S.

Ie Patent 5,110732 herein incorporated by reference in its entirety and U. S. Patent 5,097,025 herein incorporated by reference in its entirety). Those of skill in the art are familiar with the to standard resource materials which describe specific conditions and procedures for the construction, manipulation and isolation of macromolecules DNA molecules, plasmids, etc.), generation of recombinant organisms and the screening and isolation of genes, (see for example Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989; Mailga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press, 1995; is Birren et al., Genome Analysis: volume 1, Analyzing DNA, (1997); volume 2, Detecting Genes, (1998); volume 3, Cloning Systems, (1999), volume 4, Mapping Genomes, (1999), Cold Spring Harbor, New York)..

The promoter sequences of the present invention can be incorporated into an expression vector using screenable or scorable markers as described and tested in transient analyses which provide an indication of gene expression in stable plant systems. Methods of testing gene expression in transient assays are known to those of skill in the art. Transient expression of marker genes has been reported using a variety of plants, tissues and DNA delivery systems. For example, types of transient analyses can include but are not limited to direct gene delivery via electroporation or particle bombardment of tissues in any transient plant assay using any plant species of interest. Such transient systems would include but are not limited to protoplasts from suspension cultures in wheat (Zhou et al., Plant Cell Reports 12:612. 1993), electroporation of leaf protoplasts of wheat (Sethi et al., J. Crop Sci. 52: 152, 1983); electroporation of protoplast prepared from corn tissue (Sheen, J. The Plant Cell 3: 225, 1991), or particle bombardment of specific tissues of interest. The present invention encompasses the use of any transient expression system to evaluate regulatory sequences operatively linked to selected reporter genes, marker genes or agronomic genes of interest. Examples of plant tissues envisioned to test in 0 O transients via an appropriate delivery system would include but are not limited to leaf base tissues, callus, cotyledons, roots, endosperm, embryos, floral tissue, pollen, and epidermal tissue.

Any scorable or screenable marker can be used in a transient assay. Preferred marker 0 genes for transient analyses of the promoters or 5' regulatory sequences of the present invention s include a GUS gene (coding sequence for P-glucuronidase) or a GFP gene (coding sequence for Cl green fluorescent protein). The expression vectors containing the 5' regulatory sequences C operably linked to a marker gene are delivered to the tissues and the tissues are analyzed by the N, appropriate mechanism, depending on the marker. The quantitative or qualitative analyses are used as a tool to evaluate the potential expression profile of the 5' regulatory sequences when operatively linked to genes of agronomic interest in stable plants. Ultimately, the promoter sequences of the present invention are directly incorporated into suitable plant transformation expression vectors comprising the 5' regulatory sequences operatively linked to selectable markers and genes of interest, transformed into plants and the plants analyzed for the desired expression profile conferred by the 5' regulatory sequences.

Those of skill in the art are aware of the vectors and suitable for plant transformation.

Suitable vectors would include but are not limited to disarmed Ti-plasmids for Agrobacteriummediated methods. These vectors can contain a resistance marker, 1-2 T-DNA borders, and origins of replication for E. coli and Agrobacterium along with one or more genes of interest and associated regulatory regions. Those of skill in the art are aware that for Agrobacteriummediated approaches a number of strains and methods are available. Such strains would include but are not limited to Agrobacterium strains C58, LBA4404, EHA101 and EHA105. Particularly preferred strains are Agrobacterium tumefaciens strains. Other DNA delivery systems for plant transformation are also known to those of skill in the art and include but is not limited to particle bombardment of selected plant tissues.

2s Exemplary nucleic acids which may be introduced by the methods encompassed by the present invention include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical reproduction or breeding techniques. However, the term exogenous, is also intended to refer to genes which are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally O 36- 0 present yet which one desires, to have over-expressed. Thus, the term "exogenous" gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA 0 included in the exogenous DNA can include DNA which is already present in the plant cell, Ss DNA from another plant, DNA from a different organism, or a DNA generated externally, such l as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

SThe plant transformation vectors containing the promoter sequences of the present invention may be introduced into plants by any plant transformation method. Several methods 0o are available for introducing DNA sequences into plant cells and are well known in the art.

Suitable methods include but are not limited to bacterial infection, binary bacterial artificial chromosome vectors, direct delivery of DNA via PEG-mediated transformation, desiccationlinhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers, and acceleration of DNA coated particles (reviewed in Potrykus, Ann. Rev. Plant Physiol.

is Plant Mol. Biol., 42: 205, 1991).

Methods for specifically transforming dicots primarily use Agrobacterium lumefaciens.

For example, transgenic plants reported include but are not limited to cotton S. Patent No.

5,004,863; U. S. Patent No. 5,159,135; U. S. Patent No. 5,518,908, WO 97/43430), soybean (U.

S. Patent No. 5,569,834; U. S. Patent No. 5,416,011; McCabe et al., Bio/Technology, 6:923, 1988; Christou et al., Plant Physiol., 87:671, 1988); Brassica S. Patent No. 5,463,174), and peanut (Cheng et al., Plant Cell Rep., 15: 653, 1996).

Similar methods have been reported in the transformation of monocots. Transformation and plant regeneration using these methods have been described for a number of crops including but not limited to asparagus (Asparagus officinalis; Bytebier et al., Proc. Natl. Acad. Sci. U.S.A., 84: 5345, 1987); barley (Hordeum vulgarae; Wan and Lemaux, Plant Physiol., 104: 37, 1994); maize (Zea mays; Rhodes, et al., Science, 240: 204, 1988; Gordon-Kamm, et al., Plant Cell, 2: 603, 1990; Fromm, et al., Bio/Technology, 8: 833, 1990; Koziel, et al., Bio/Technology, 11: 194, 1993); oats (Avena sativa; Somers, ct al., Bio/Technology, 10: 1589, 1992); orchardgrass (Dactylis glomerata; Horn, et al., Plant Cell Rep., 7: 469, 1988); rice (Oryza sativa, including indica and japonica varieties, Toriyama, el: al., Bio/Technology, 6: 10, 1988; Zhang, et al., Plant Cell Rep., 7: 379, 1988; Luo and Wu, Plant Mol. Biol. Rep., 6: 165, 1988; Zhang and 0 O -37- O Wu, Theor. Appl. Genet., 76: 835, 1988; Christou, et al., Bio/Technology, 9: 957, 1991); sorghum (Sorghum bicolor; Casas, et al., Proc. Natl. Acad. Sci. 90: 11212, 1993); sugar cane (Saccharum spp.; Bower and Birch, Plant 2: 409, 1992); tall fescue (Festuca S arundinacea; Wang, Z.Y. et al., Bio/Technology, 10: 691, 1992); turfgrass (Agrostis palustris; Zhong et al., Plant Cell Rep., 13: 1, 1993); wheat (Trilicum aestivum; Vasil et al., S Bio/Technology, 10: 667, 1992; Weeks et al., Plant Physiol., 102: 1077, 1993; Becker, et al., S Plant, J. 5: 299, 1994), and alfalfa (Masoud, et al., Transgen. Res., 5: 313, 1996). It is CN apparent to those of skill in the art that a number of transformation methodologies can be used and modified for production of stable transgenic plants from any number of target crops of to interest.

The transformed plants are analyzed for the presence of the genes of interest and the expression level and/or profile conferred by the promoter sequences of the present invention.

Those of skill in the art are aware of the numerous methods available for the analysis of transformed plants. A variety of methods are used to assess gene expression and determine if the is introduced gene(s) is integrated, functioning properly, and inherited as expected. For the present invention the promoters can be evaluated by determining the expression levels of genes to which the promoters are operatively linked. A preliminary assessment of promoter function can be determined by a transient assay method using reporter genes, but a more definitive promoter assessment can be determined from the analysis of stable plants. Methods for plant analysis include but are not limited to Southern blots or northern blots, PCR-based approaches, biochemical analyses, phenotypic screening methods, field evaluations, and immunodiagnostic assays.

The methods of the present invention including but not limited to cDNA library preparation, genomic library preparation, sequencing, sequence analyses, PCR technologies; vector construction, transient assays, and plant transformation methods are well known to those of skill in the art and are carried out using standard techniques or modifications thereof.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are 0 -38- O disclosed and still obtain a like or similar result without departing from the spirit and scope of V¢ the invention, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

00 M 5 EXAMPLES SEXAMPLE 1.

SPlant Material. DNA Isolation and Library Construction A number of tissues and plant developmental stages are selected for preparation of the to corn libraries. Those of skill in the art are aware of the variations in tissue selection and preparation that occur from one tissue sampler to the next. The following are the conditions for the target libraries: Plant Growth Conditions. Seeds are planted at a depth of about 3 cm in soil into 2"-3" pots containing Metro Mix 200 growing medium and transplanted into larger 10" pots containing the same soil after 2-3 weeks. Peters 15-16-17 fertilizer is applied abut 3 times per week after transplanting, at a strength of 150 ppm N. 2-3 times during the life of the plant from transplanting to flowering. A total of about 900 mg Fe is added to each pot. Corn plants are grown in the greenhouse in 15 hr day/9 hr night cycles. The daytime temperature is 80 0 F and the night temperature is 70F. Lighting is provided by 1000W sodium vapor lamps.

Tissue Isolation. The corn immature tassel cDNA library (SATMONO21) is generated from immature maize (DK604, DEKALB Genetics, Dekalb Illinois, tassel at the V8 plant developmental stage. The tassel is an immature tassel at about 2-3 cm in length. The tassels are collected and frozen in liquid nitrogen and the harvested tissue is stored at -80 0 C until the RNA is prepared.

The corn root cDNA library (SATMON010) is generated from maize (DK604, DEKALB Genetics, Dekalb Illinois, U. S. root tissue at the V8 plant developmental stage. The root tissue is collected when the corn plant is at the 8-leaf stage. The root system is cut from the plant and rinsed with water to remove the soil. The tissue is frozen in liquid nitrogen and the harvested tissue is stored at -80°C until the RNA is prepared.

The corn leaf cDNA library (SATIMON004) is generated from maize (B73 x Mol7, Illinois Foundation Seeds, Champaign, Illinois IU. S. total leaf tissue at the V6 plant

O

O -39- O developmental stage. The older leaves in the basal position and the younger leaves in the upper it positions are all cut at the leaf base. The leaves are pooled and immediately frozen in liquid nitrogen and crushed. The harvested tissue is stored at -80 0 C until the RNA is prepared.

00 The corn primary root cDNA library (SATMON007) is generated from the primary root s tissue of maize (DK604, DEKALB Genetics, Dekalb, Illinois U. S. A) 5 day old seedlings. The seeds are planted on a moist filter paper on a covered tray kept in the dark until germination (one day).. After germination, the tray containing the moist filter paper is moved to the greenhouse at a 15 hour day/9 hour night cycle and grown until the seedlings are 5 days old. The day temperature is 80 0 F and the night temperature is 70°F. The tissue is collected when the seedlings 0o are 5 days old. At this stage, the primary root (radicle) has pushed through the coleorhiza which has pushed through the seed coat. The primary root which is about 2-3 cm long is cut and frozen in liquid nitrogen. The harvested tissue is stored at -80 0 C until the RNA is prepared.

The corn primary shoot cDNA library (SATMON008) is generated from the primary shoot (coleoptile 2-3 cm) of maize (DK604, DEKALB Genetics, Dekalb, Illinois U. S. A.) seedlings which are approximately 5 days old (same conditions described for primary root library). The coleoptile is dissected away from the rest of the seedling and immediately frozen in liquid nitrogen and stored at -80 0 C until the RNA is prepared.

The corn undeveloped leaf cDNA library (SATMON011) is generated from maize (DK604, DEKALB Genetics, Dekalb, Illinois U. S. The tissue is collected when the corn plant is at a 6-leaf developmental stage. The second youngest leaf which is at the base of the apical leaf of the V6 corn plant is cut at the base and immediately transferred to liquid nitrogen containers and the leaves are crushed. The harvested tissue is stored at -80 0 C until the RNA is prepared.

The corn meristem cDNA library (SATMON013) is generated from maize (DK604, DEKALB Genetics, Dekalb, Illinois U. S. The corn plant is at a 4-leaf stage. The leaf at the apex of theV4 stage corn plant is known as the meristem founder. This apical meristem founder is cut and immediately frozen in liquid nitrogen and crushed. The harvested tissue is then stored at -80 0 C until the RNA is prepared.

The corn sheath cDNA library (SATMON016) is generated from maize (DK604, DEKALB Genetics, Dekalb, Illinois U. S. Sheath tissue is collected from the corn plant at the V8 stage. When the maize plants are at the V8 stage the 5th and 6th leaves from the base of C- 0 0 the plant have fully developed leaf blades. At the base of these leaves, the ligule is differentiated and the leaf blade is joined to the sheath. The sheath is dissected away from the base of the leaf and frozen in liquid nitrogen crushed. The tissue is stored at -80°C until RNA preparation.

The corn culm (stem) cDNA library (SATMON019) is generated from maize (DK604, s DEKALB Genetics, Dekalb, Illinois U. S. The corn plant is at a V8 developmental stage ,I and the 5th and 6th leaves from the bottom have fully developed leaf blades. The region 0 between the nodes of the 5th and 6th leaves firom the bottom is the region of the stem that is cI collected. The leaves are pulled out and the sheath is also torn away from the stem. This stem tissue is completely freed of any leaf and sheath tissue. The tissue is frozen in liquid nitrogen to and stored at -80 0 C until the RNA is prepared.

The corn immature tassel cDNA library (SATMON001) is generated from maize (B73, Illinois Foundation Seeds, Champaign, Illinois U. S. Corn immature tassels are collected at the V6 developmental stage. Tassels which are 2-3 cm in length are collected and frozen in liquid nitrogen. The harvested tissue is stored at -80 0 C until the RNA is prepared.

Is The corn ear cDNA library (SATMON023) is generated from maize (DK604, DEKALB Genetics, Dekalb, Illinois U. S. Tissue is collected when the plant is in the V8 stage. The length of the ear that is harvested is about 10-15 cm and the silks are just exposed (approximately one inch). The ear along with the silks is frozen in liquid nitrogen and the tissue is stored at 80 0 C until the RNA is prepared.

The corn etiolated seedling cDNA library (SATMON029) is generated from maize (DK604, DEKALB Genetics, Dekalb, Illinois U. S. Seeds are planted on a moist filter paper on a covered tray and kept in the dark for 4 days. The daytime temperature is about 80°F and the nighttime temperature is about 70 0 F. The tissue is collected when the seedlings are 4 days old.

All the structures of the seedlings appear thin and elongated. The primary root has penetrated the colerhiza and is about 4-5 cm in length. Secondary lateral roots have also appeared. The coleoptile has also pushed through the seed coat and is about 4-5 cm long. The seedlings are then frozen in liquid nitrogen and crushed. The harvested tissue is then stored at -80°C until the RNA is prepared.

The corn endospern eDNA library (twenty-two days after pollination or 22-DAP) (SATMON036) is generated from maize RX601. The growth conditions are as fllows: Corn seeds are sterilized for Iminute in 10% Clorox solution, rolled in germination paper, and 0 0 -41- O germinated in 0.5 mM calcium sulfate for two days at 30 0 C. The seedlings are transplanted into _t a peat mix media in 3" pots at the rate of three seedlings per pot. The seedlings are grown in the greenhouse and twenty pots are placed in a high C02 environment (-1000 ppm C02) and twenty oO plants are grown under about 450 ppm C02. The plants are hand watered and lightly fertilized s with Peters 20-20-20. At 10 days after planting, the shoots from both atmospheres are placed in liquid nitrogen and light ground by hand. The roots are rinsed in distilled water to remove most of the support media, and frozen in liquid nitrogen. The tissues are stored at -80°C until RNA is prepared.

For preparation of the cDNA libraries, the RNA is purified using Trizol reagent available o1 from Life Technologies (Gaithersburg, Maryland) essentially as recommended by the manufacturer. Poly A+ RNA (mRNA) is purified using magnetic oligo dT beads essentially as recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake Success, New York).

Construction of cDNA libraries is well-known in the art and a number of cloning strategies exist. A number of cDNA library construction kits are commercially available. The Superscript TM Plasmid System for cDNA synthesis and Plasmid Cloning (Gibco BRL, Life Technologies, Gaithersburg, MD) is used, following the conditions suggested by the manufacturer.

The cDNA libraries are plated on LB agar containing the appropriate antibiotics for selection and incubated at 37 0 C for a sufficient time to allow the growth of individual colonies.

Single colonies are individually placed in each well of a 96-well microtiter plate containing LB liquid including selective antibiotics. The plates are incubated overnight at approximately 37 0

C

with gentle shaking to promote growth of the cultures. The plasmid DNA is isolated from each clone using Qiaprep Plasmid Isolation kits, using the conditions recommended by the manufacturer (Qiagen Inc., Santa Clara, CA).

Template plasmid DNA clones are used for subsequent sequencing. For sequencing, the ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS, is used (PE Applied Biosystems, Foster City, CA).

EXAMPLE 2.

Promoter Identification -42- The database of EST sequences dlerived from the cI)NA libraries prepared from various corn tissues is used to identify the promoter candidates for expression of operably linked

DNA

sequences in multiple tissues. The sequences art: also used as query sequences against GenBank 00 databases which contain previously identified and annotated sequences and searched for regions C* of homology using BLAST prograins. The selection of expressed sequence tags (ESTs) for subsequent promoter isolation is reflective of the presence of one or more sequences among the representative ESTs from a random sampling of ani individual cDNA library or collection of cDNA libraries. To identifyi regulatory sequences that regulate the expression of transcripts in the target tissues of interest fr-om EST 'sequences in the database, a subsetting function is done, requesting ESTs found in target libraries such as leaf, ear, cuim, tassel, nmeristem, root and seedling. For promoters for expression of glyphosate tolerance genes, a search is performed particularly selecting libraries from reproductivc and rapidly dividing tissues, since suitable regulatory sequences for vegetative and reproductive tolerance can be identified. The chosen targets are subjected to an electronic northern function in which the putative tissue expression is profile and abundance levels in a library for a given EST is displayed. Target ESTs with the desired expression profile and abundance levels are identified and gene specific primers are designed based on the identified EiST sequence. Percent abundance relates to the number of -times the members of a group of related sequences appears in a library the EST is derived from.

A number of parameters can be selected to obtain the selected ESTs and will depend upon the EiST database and computer programs available for sequence analyses and queries. For the promoters or 5' regulatory sequences of SEQ I1) NOS: 53-75, a percent abundance in the selected libraries of about 0.05 to 0.50 is used.

Table I provides background clone 11) (EiST) information, library sources, and GenBank identifier information I-r the LiSTs used for subsequent isolation of the promoter sequences of SEQ ID NOS: 53-75. Sequence annotaion) is listed f"or clone IDs based on a Genlank BLAST search with a p-value cut-off of 10-8. The information is subject to change as new sequences ar-e submitted to the sequence databases. The annotations for the LiSrs are listed as follows with the annotation information in parentheses: Clone ID 700333814 (corn mRNA Rhr cysteine proleinase, clone CCP, cornpicic cds); Clone ID '700806028 (maize mRNA for enoluse); Clonc ID 700210506 (1ii. vulgare 'uRNA for IJDP pyrophosphoryiase); Clone 11) 700043360 (Maize pyruvate, orthopho~sphate dikinasc niRNiX, complete cds); Clone ID 700094769 (Wheat 0 43 .mRNA for cytosolic phosphoglycerate kinase); Clone ID) 700150082 (Z mnays strain B73 Beta- 1)-glucosidase mRNA, nuclear gene encoding chioroplast protein, complete cds); Clone ID 700094721 (Wheat mnRNA for cytosolic phosphoglycerate kinase); Clone ID 700164347 (Z.

mays W-22 histone H2A mRNA complete cds), two clones (SEQ I.D NO:53 and SEQ ID NO:54 which contains about 2.0 kb of additional sequence); Clone ID 700154443 mays elongation factor 1-alpha); Clone ID 700017196 mays gene for cyclophilin); Clone ID 700342364 (Z.

mays mRNA for translation initiation factor 5SA); Clone 11) 7003425890 (27 mnays MNB Ib mRNA for DNA-binding protein); Clone ID 700342746 mays ribosomal protein S8 mRNA, complete cds); Clone ID 700164205 (Z mays IDnaJ-related protein ZMDJ1 (mdii) gene, io complete cds); Clone 11) 700343485 (Sorghum bicolor heat shock protein 70 cognate mRNA, partial cds); Clone ID 70034555'7 (Z mays ubiquitin conjugating enzyme (UBC) mRNA, complete cds); Clone ID 700345819 (Z mays superoxide dismutase 4A (sod.4A) gene, complete cds); Clone ID 700345704 (V fiaba guanine nucleotide regulatory protein mRNA, complete cds).

is TABLE I1. Prompter Summary Information SEQ ID NO Clone ID Number Library SouM.

57 700046047 leaf 62 700333814 culm (stem) 58 700806028- endosperm, 22-DAII 61 700210506 sheath 59 700043360 leaf 700094769 primary shoot 63 700150082 primary root 64 700094721 primary shoot 53 700164347 meristem 54 700164347 meristemn 700154443 primary root 56 700017196 immnature tassel 700342364 immature tassel 72 700342580 immature tassel GenI~ank Idyntifier (g) none g643596 g22272 g212995 g168579 g21834 g4096601 g21834 g473602 g473602 g2282583 g829147 g1546918 g3 97395 44- 0 73 700342976 immature tassel none 66 700342746 immature tassel g1498052 700350638 ear, growing silks none oO 67 700342649 immature tassel none 68 700164205 meristem g2984708 C 69 700343485 immature tassel gl 181672 700345557 immature tassel g2668743 71 700345819 immature tassel g1899026 74 700345704 immature tassel g395071 EXAMPLE 3.

Genomic Library Construction, PCR Amplification and Promoter Isolation For genomic libraries, corn DNA (from maize hybrid Fr27 X FrMol7) is isolated by a CsCI purification protocol according to Ausubel et al., 1992, by a CTAB purification method (Rogers and Bendich, Plant Mol. Biol., 5:69, 1988) or a similar DNA isolation method suitable for the isolation of plant DNA. Reagents are available commercially (see, for example Sigma Chemical Co., St. Louis, MO). The libraries are prepared according to manufacturer instructions (GENOME WALKER, a trademark of CLONTECH Laboratories, Inc, Palo Alto, CA). In to separate reactions, genomic DNA is subjected to restriction enzyme digestion overnight at 37 0

C

with the following blunt-end endonucleases: EcoRV, Seal, Dral, Pvull, or Stul (CLONTECH Laboratories, Inc. Palo Alto, CA). The reaction mixtures are extracted with phenol:chloroform, ethanol precipitated, and resuspended in Tris-EDTA buffer. The purified blunt-ended genomic DNA fragments are then ligated to the GenomeWalkerrM adaptors and ligation of the resulting is DNA fragments to adaptors were done according to manufacturer protocol. The GenomeWalkerm sublibraries are aliquoted and stored at -20 0

C.

Genomic DNA ligated to the GenomeWalkerM adaptor (above) is subjected to a primary round of PCR amplification with gene-specific primer 1 (GSPI) and a primer which anneals to the adaptor sequence, adaptor primer 1 (API) SEQ ID NO:1. A diluted (1:50) aliquot of the primary PCR reaction is used as the input DNA for a nested round of PCR amplification with gene-specific primer 2 (GSP2) and adaptor primer 2 (AP2) SEQ ID NO:2, or adaptor primer 3 (AP3) SEQ ID NO:3. The annealing temperatures of the GenomeWalker'r primary primer (AP1) and nested primer (AP2) are 59 0 C and 71 0 C, respectively. Generally, gene specific primers are designed to have the following characteristics: 26-30 nucleotides in length, GC content of 40-60% with resulting temperatures for most of the gene specific primers in the high 0 C range or about 70 0 C. The Taq polymerase used is Amplitaq Gold'

M

available through s Perkin-Elmer Biosystems (Branchbury, New Jersey). A number of temperature cycling instruments and reagent kits are commercially available for performing PCR experiments and include those available from PE Biosystems (Foster City, CA), Strategene (La Jolla, CA), and MJ Research Inc. (Watertown, MA). Following the primary PCR reaction, an aliquot is taken (10-15pl) for agarose gel analysis. Isolation of each unknown sequence required amplification to from 5 sub-genomic libraries and a negative control (without DNA).

The PCR components and conditions generally used are outlined below: PRIMARY PCR (Method 1) Component Amount/Volume required is Sub-library aliquot 1l1 Gene-specific primer 1 I pl (100 pmol) GenomeWalker'" Adaptor primer 1 (AP 1 l dNTP mix (10 mM of each dNTP) 11 DMSO 2.5pl (or 2-5% final concentration) 1OX PCR buffer (containing MgCI2) 5p1 (final concentration of IX) Amplitaq Gold T M Distilled Water For final reaction volume of Reaction Conditions for Primary PCR: A. 9 minutes at 95 0

C

B. 94°C for 2 seconds, 70 0 C for 3 minutes; repeat 94 0 C/70°C cycling for total of 7 times C. 94 0 C for 2 seconds, 65 0 C for 3 minutes; repeat 94 0 C/65°C cycling for total of 36 times D. 65 0 C for 4 minutes as a final extension E. 100C for an extended incubation -46- NESTED PCR (secondary PCR reaction) Component 1:50 dilution of the primary PCR reaction Gene-specific primer 2 GenomeWalkrTm Adaptor primer 2 or 3 (AP2 or A 13)) dNTP mix (10 mM of each dNTP) DMS0 ILOX PCR buffer (containing MgC12) Amplitaq GoldTM io Distilled water Amount/Volume Required I Pjd I .Ll Spd (final concentration of I X) tl to final reaction volume of Reaction Conditions for Nested PCR: A. 9 minutes at B. 94'C for 2 seconds, 70'C for 3 minutes; repeat 94'C/70*C cycling for total of 5 times C. 94'C for 2 seconds, 65'C for 3 minutcs;*rcpeat 94'(./65'C cycling for total of 24 times D. 65'C for 4 minutes as a final extension E. 1 0 0 C for an extended incubation Those of skill in the art are aware of the variations in PCR conditions including choice of polymerase, cycling conditions and concentrations of the reaction components. Other modifications to the above procedure include the use of" Expand High Fidelity polymerase (Boehringer Mannheim, Indianapolis, IN), 'Faq polymerase (Promega Corp., Madison, WI) and slight variations in temperature are outlined below: MODIFICATION 1: Polymerase (Expand High Fidelity, Boehringer Mannheimn,

IN)

Primary PCR reaction step I 95'C 2mmn step 2 94'C 2sec step 3 72"C 3min step 4 repeat step 2 and 3, 7 times o step 5 94 0 C 2 sc step 6 68 0 C 3 min step 7 repeat steps 5 and 6, 36 times 00 M~f 5 Secondary PCR reaction c-K1step 1, step 2, step 3 (samne as primary PCR reaction) step 4 repeat step 2 and 3, 5 times step 5 and step 6 (same as primary PCR reaction) step 7 repeat step 5 and step 6, 24 times MODIFICATION 2: Polymerase (Amplitaq (iold

TM

Perkin Elmner, Foster City, CA) Primary PCR reaction st~p I 9511C step 2 94'C 2 sec step 3 701C 3 min step 4 repeat step 2 and 3, 7 times step 5 94'C 2 sec step 6 68"C 3 min step 7 repeat step 5 and step 6, 24 times M01)1FICATION 3: Polymerase enzyme is Taq (Promega Corp., Madison,

WI)

same cycle conditions as Modification 2 except step I is 2 minutes.

MODIFICATION 4: Polymerase enzyme is Accu~aq (Sigma, St. Louis,

MO)

same cycle conditions as Modification 3.

MODIFICATION 5: Polymerase is Expand High Fidelity (13M) Mixture Primary PCR Secondary

PCR

l OX PCR buffer 2 2p]J I jl dNTP l~ 11 0 -48 adaptor primer (IOm M) llLd API gene specific primer (I10mM) I pi GSPI Polymerase 2.5 units template DNA GenomeWalke.1m library I J-1.

to 201d i i1 A 12 I j1l GSP2 units 1:50 of primary PCR product to 50 iI Modification 5: I'CR Cycling Conditions: step I 94-C I min step 2 94"C 2 sec step 3 701C 3 mini step 4 repeat step 2 another 5 cycles step 5 94 0 C 2 sec step 6 68 0 C 3min step 7 repeat step 5 another 34 cycles step 8 68'C 10 min step 9 10 0 'C hold MODIFICATION 6: PCR Conditions step I 95'C 2 min step 2 94"C 30 sec step 3 65'C 30 sec, decrease 1 0 C each cycle for 14 cycles step 4 72'C 3 minutes step 5 repeat 2-4, 14 times step 6 step 7 72'C step 8 repeat 6-7, 15 times Modification 5 is used for the isolation oil promoter sequence SEQ ID NO: 53 (clone ID#700 164347). SEQ ID NO: I is combined with GSPI SEQ ID NO: 6 in the primary PCR -49- O reaction. For the nested PCR reaction SEQ I1) NO: 2 is combined with GSP2 SEQ ID NO: 7 in St the secondary PCR reaction and a PCR product is isolated.

Modification 5 is used for the isolation of promoter sequence SEQ ID NO: 54 (clone

ID

O #700164347 that contains additional 2 kb of 5' sequence). SEQ ID NO:1 is combined with GSPI SEQ ID NO: 6 in the primary PCR reaction. For the nested PCR reaction, SEQ ID No. 2 is combined with GSP2 SEQ ID NO: 7 in the secondary PCR reaction and a PCR product is isolated. the secondary PCR Modification 5 is used Ibr the isolation of promoter sequence SEQ ID NO: 55 (clone

ID

#700154443). SEQ ID NO: 1 is combined with GSPI SEQ ID NO: 8 in the primary

PCR

io reaction. For the nested PCR reaction, SEQ ID NO: 2 is combined with GSP2 SEQ ID No. 9 in the secondary PCR reaction and a PCR product is isolated.

Modification 5 is used fbr the isolation of promoter sequence SEQ ID NO: 56 (clone

ID

#700017196). SEQ ID No. 1 is combined with GSPI SEQ ID NO: 10 in the primary

PCR

reaction. For the nested PCR reaction, SEQ ID No. 2 is combined With GSP2 SEQ ID NO: 11 in is the secondary PCR reaction and a PCR product is isolated.

PCR method I is used for the isolation of promoter sequence SEQ ID NO:62 (clone

ID

#700333814) SEQ ID NO:I is combined with GSPI SEQ ID NO: 22 in the primary

PCR

reaction. For the nested PCR reaction, SEQ ID NO: 2 i combined with GSP2 SEQ ID NO: 23 in the secondary PCR reaction and a PCR product is isolated.

PCR method I is used for the isolation of promoter sequence SEQ ID NO: 59 (clone ID #700043360). SEQ ID NO: 1 is combined with GSPI SEQ ID NO: 16 in the primary

PCR

reaction. For the nested PCR reaction, SEQ ID NO: 2 is combined with GSP2 SEQ ID NO: 17 in the secondary PCR reaction and a PCR product is isolated.

PCR method I is used for the isolation of promoter sequence SEQ ID NO: 60 (clone

ID

#700094769) SEQ 1D NO: 1 is combined with GSP I SEQ ID NO: 18 in the primary

PCR

reaction. For the nested PCR reaction, SEQ ID NO: 2 is combined with GSP2 SEQ ID NO: 19 in the secondary PCR reaction and a PCR product is isolated.

PCR method 1 is used for the isolation of promoter sequence SEQ ID NO: 61 (clone

ID

#700210506) SEQ ID NO: 1 is combined with (iSII SEQ ID) NO: 20 in a primary PCR reaction.

o3 For the nested PCR reaction, SEQ ID NO: 2 is combined with GSP2 SEQ ID NO: 21 in the secondary PCR reaction and a PCR product is isolated.

O O PCR method I is used for the isolation of promoter sequence SEQ ID NO: 57 (clone ID #700046047) SEQ ID NO: 1 is combined with GSPI SEQ ID NO:12 in the primary

PCR

reaction. For a nested PCR reaction, SEQ ID NO: 2 is combined with GSP2 SEQ ID NO: 13 in 0 the secondary PCR reaction and the PCR product is isolated.

M 5 PCR method I is used for the isolation of promoter sequence SEQ ID NO: 58 (clone ID N #700806028). SEQ ID NO: I is combined with SEQ ID NO: 14 in the primary PCR reaction.

For the nested PCR reaction, SEQ ID NO: 2 is combined with SEQ ID NO: 15 in the secondary PCR reaction and a PCR product is isolated.

PCR method 1 is used for the isolation of promoter sequence SEQ ID NO: 63 (clone ID io #700150082). SEQ ID NO: 1 is combined with GSP1 SEQ ID NO: 24 in the primary

PCR

reaction. For the nested PCR reaction, SEQ ID NO: 2 is combined with GSP2 SEQ ID NO: in the secondary PCR reaction and a PCR product is isolated.

PCR method 1 is used for the isolation of promoter sequence SEQ ID NO: 64 (clone ID #70094721). SEQ ID NO: 1 is combined with GSPI SEQ ID NO: 26 in the primary

PCR

is reaction. For the nested PCR reaction, SEQ ID NO: 2 is combined with GSP2 SEQ ID NO: 27 in the secondary PCR reaction and a PCR product is isolated.

For the isolation of promoter sequence SEQ ID NO: 65 (clone ID #700342364),

GSPI

primer SEQ ID NO: 28 is combined with SEQ ID NO: 1 in a primary PCR reaction according to PCR modification 1. For the secondary PCR reaction nested primer SEQ ID NO: 2. is combined with GSP2 primer SEQ ID NO: 29 and a PCR product is isolated, cloned into a pGEM-T easy vector (Promega, Madison, WI), and transformed into E. coli HB101. To introduce a StuI restriction site at the 3' end and a HindlIl site at the 5' end of the promoter, the pGEM-T promoter clone is used as the template in a PCR reaction using method 6 with SEQ ID NO: 3 and GSP primer SEQ ID NO: For the isolation of promoter sequence SEQ ID NO: 66 (clone ID #700342746),

GSPI

SEQ ID NO: 31 is combined with SEQ ID NO: 1 in a primary PCR reaction according to PCR modification 1. For the secondary PCR reaction nested primer SEQ ID NO: 2 is combined with GSP2 SEQ ID NO: 32 and a PCR product is isolated kb PCR product using the Dral digested genomic library). To introduce a Stul site at the 3' end and a Hindll site at the 5' end of the promoter the promoter clone is used as a template in a PCR reaction using method 6 with 0 -51 O adapter primer SEQ ID NO: 3 and GSP primer SEQ ID NO: 33. The PCR product is isolated Sand cloned into pGEM-T easy (Promega Corp., Madison,

WI).

For the isolation of promoter sequence SEQ ID NO: 67 (clone ID #700342649),

GSPI

00 SEQ ID NO: 34 is combined with SEQ ID NO: 1 in a primary PCR reaction according to PCR Ss Modification 2. For the secondary PCR reaction nested primer SEQ ID NO: 2 is combined with S GSP2 SEQ ID NO: 35 and the PCR product is isolated. To introduce a StuI site at the 3' end and a HindII site at the 5' end, the promoter clone is used as the template in a PCR reaction using 0 method 6 with adapter primer SEQ ID NO: 3 and GSP primer SEQ ID NO: 33.

For the isolation of SEQ ID NO: 68 (clone ID#700164205), GSPI SEQ ID NO: 37 is to combined with SEQ ID NO: 1 in a primary PCR reaction according to PCR Modification 3. For the secondary PCR reaction nested primer SEQ ID NO: 3. is combined with GSP2 primer

SEQ

ID NO: 38 and a PCR product is isolated kb PCR product using the StuI digested genomic library) and cloned into pGEM-T Easy (Promega Corp., Madison,

WI).

For the isolation of promoter sequence SEQ ID NO: 69 (clone ID #700343485),

GSPI

Is SEQ ID NO: 39 is combined with SEQ ID NO: 1 in a primary PCR reaction according to PCR Modification 3. For the secondary PCR reaction nested primer SEQ ID NO: 3 is combined with GSP2 SEQ ID NO: 40 and the PCR product is isolated and cloned into pGEM-T Easy (Promega Corp., Madison,

WI).

For the isolation of promoter SEQ ID NO: 70 (clone ID #700345557), GSPI SEQ ID NO: 41 is combined with SEQ ID NO: 1 in a primary PCR reaction according to PCR Modification 3. For the secondary PCR reaction nested primer SEQ ID NO: 3 is combined with GSP2 SEQ ID NO:42 and the PCR product is isolated.

For the isolation of promoter sequence SEQ ID NO:71, (clone ID# 700345819)

GSPI

SEQ ID NO: 43 is combined with SEQ ID NO: 1 in a primary PCR reaction according to PCR Modification 3. For the secondary PCR reaction nested primer SEQ ID NO: 3 is combined with GSP2 SEQ ID NO: 44 and a PCR product is isolated using the EcoRV digested library).

For the isolation of promoter sequence SEQ ID NO: 72 (clone ID #700342580),

GSPI

SEQ ID NO: 45 is combined with SEQ ID NO: in a primary PCR reaction according to PCR Modification 4. For the secondary PCR reaction nested primer SEQ ID NO: 3 is combined ith 3o GSP2 SEQ ID NO: 46. An -2.2 kb PCR product is isolated using the Scal digested library.

N -52 O For the isolation of promoter sequence SEQ ID NO: 73 clone ID# 700342976),

OSPI

SEQ ID NO: 47 is combined with SEQ ID NO: I according to PCR Modification 4. For the secondary PCR reaction nested primer SEQ ID NO: 3 is combined with GSP2 SEQ ID NO: 48.

oO An 4.0 kb PCR product is isolated using the Scal digested library.

S 5 For the isolation of promoter sequence SEQ ID NO: 74 (clone ID# 700345704),

GSPI

SEQ ID NO: 49 is combined with SEQ ID NO: I in a primary PCR reaction according to PCR.

Modification 4. For the secondary PCR reaction nested primer SEQ ID NO: 3 is combined with GSP2 SEQ ID NO: 50. An -1.3kb PCR product is isolated using the EcoRV digested library.

For the isolation of promoter sequence SEQ ID NO: 75 (clone ID# 700350638),

GSPI

SEQ ID NO: 51 is combined with SEQ ID NO: 1 in a primary PCR reaction according to PCR Modification 4. For the secondary PCR reaction nested primer SEQ ID NO: 3 is combined with GSP2 SEQ ID NO: 52. An -1.0kb PCR product is isolated using the Stul digested library (PCR Method 4).

1 EXAMPLE 4.

Promoter Isolation and Clonijn The DNA fragments resulting from the nested PCR amplification described above are isolated and gel purified. A 40pl aliquot of the secondary PCR is run on an agarose gel. The DNA fragment of the secondary PCR product is purified from the agarose gel using the BIO101 Geneclean II Kit (Midwest Scientific, Valley Park, MO) following the conditions suggested by the manufacturer. The purified DNA is ligated to pGEM-T Easy vector (pGEM-T Easy Vector System I, Promega Corp., Madison, WI) following the conditions recommended by the manufacturer. An aliquot of the ligation reaction is transformed into a suitable E. coli host such as DHI0B and the cells plated on selection medium (for DH1IOB, 100 Pg/ml carbenicilli).

Bacterial transformants are selected grown in liquid culture, and the plasmid DNA isolated using a commercially available kit such as the Qiaprep Spin Microprep Kit (Qiagen Corp., Valencia, CA). Purified plasmid containing the predicted insert size based on restriction enzyme analysis are sequenced using the dye terminator method in both directions using the M 13 forward and reverse primers shown in SEQ II) NO: 4 (M 13 forward primer) and SEQ ID NO: (MI3 reverse primer). Restriction enzymes arc available from a number of manufacturers (see for example, Boehringer Mannheim (Indianapolis, IN). The 5' flanking region containing the promoter sequence is determined and shown in SEQ ID NOS: 53-75. Engineering restriction O -53- O sites for cloning into suitable vectors generally use the PCR method outlined in method 6, using V standard molecular biology techniques known to those of skill in the art.

00 EXAMPLE SPlantcll An sis of Promoter Activity SFor plant cell expression, promoter fragments are cloned into expression vectors such S pMON19469 as shown in Figure 1. Plasmid pMON19469 is an expression vector consisting of the following genetic components: P-CaMV.35S:en is the promoter for the 35S RNA from Cauliflower mosaic virus containing a duplication of the -90 to -300 region; I-Zm.Hsp70 is an to intron of the maize heat shock protein as described in U. S. Patent Nos. 5,593,874 herein incorporated by reference in its entirety and 5,859,347 herein incorporated by reference in its entirety; Ec.GUS:1 (GUS:1) is the coding region for -gl ucuronidase; T-AGRTU.nos is the termination signal from the nopaline synthase gene isolated from Agrobacterium tumefaciens; ori-M13 and ori-pUC are origins of replication; AMP is the regulatory region for ampicillin selection. If an AUG of a target promoter is identified, the fragment is cloned into pMON19469 in place of the P-CaMV.35S:en genetic element. If an AUG is not identified, the promoter fragment is cloned into an expression vector modified to enable translational fusions with a reporter gene such as P-glucuronidase (GUS) (Jefferson et al., EMBO 6:3901, 1987) or green fluorescent protein (GFP) as described in Pang et al., Plant Physiol. 112:893 (1996).

pMON25455 (Figure 5) is a basic expression vector consisting of the rice actin promoter from Actl gene, the GUS:I coding sequence and the T-AGRTU.nos terminator. Additional basic expression vectors containing various expression elements include but are not limited to pMON42410 (Figure pMON42411 (Figure and pMON46124 (Figure The promoter DNA fragments of the present invention can replace the promoter elements of the basic expression vector to determine the promoter activity of the DNA fragments.

The promoter DNA fragments that are clone II) #s 700342746 (SEQ ID NO.66), 700342364 (SEQ II) NO. 65), 700350638 (SEQ II) NO. 75), 700164205 (SEQ ID NO. 68), 700343485 (SEQ ID NO. 69), 700345557 (SEQ ID NO. 70), 700345819 (SEQ ID NO. 71), 7003442580 (SEQ ID NO. 72), 700342976 (SEQ ID NO. 73), 700345704 (SEQ 1) NO. 74), are cloned ittnt the S containing basic xpession constructs by restriction enzyme digestion and DNA ligs trtmnt ofthe fragments under the appropriate conditions well known by those of 0 -54o skill in the art of molecular biology. The purified plasmid DNA of the resulting expression cassettes are tested in a corn protoplast electroporation assay for GUS activity. A number of assays are available and known to those of skill in the art. Analysis of reporter genes in a S protoplast system can be used to assess the activity of a regulatory element, such as a promoter operably linked to the reporter gene. A leaf protoplast isolation and electroporation protocol is S followed essentially as described by Sheen, (Plant Cell 3:225-245, 1991) with the following modifications: The seed used is FR27RHM X FRMol7RHM from Illinois Foundation Seeds.

S The seed is surface sterilized for 2 minutes in 95% ethanol, rinsed twice with sterile water, minutes in 50% bleach (Clorox) plus 2 drops of Tween-20, three rinses in sterile water followed 'o by a 5 minute soak in benlate/captan solution to prevent fungal growth. The seeds are germinated in phytotrays containing 100 mis V 2 MS media (2.2g/L MS salts, 0.25% gelrite), 7 seeds per phytotray. The seeds are grown 5 days at 26 0 C in 16/8 hour day/night photoperiod and 7 days in the dark at 28 0 C. The second leaf from each plant is sliced longitudinally using Feather no. 11 surgical blades. Digestion time is two hours and 10 minutes in the light at 26 0

C.

s After digestion, the plates are swirled two times at 80-100 rpm for 20 seconds each and the protoplast/enzyme solution is pipetted through a 190ptm tissue collector. Protoplasts are counted using a hemacytometer counting only protoplasts that are intact and circular. Ten to fifty micrograms of DNA containing the vector of interest is added per cuvette. Final protoplast densities at electroporation range from 3 x 106/ml to 4.5 x 106/ml. Electroporations are performed in the light using Bio-rad Gene pulser cuvettes (Bio/Rad Hercules, CA) with a 0.4 cm gap and a maximum volume of 0.8 ml at 125 pFarads capacitance and 260 volts. The protoplasts are incubated on ice after resuspension in electroporation buffer and are kept on ice in cuvettes until 10 minutes after electroporation. The protoplasts arc kept at room temperature for ten minutes before adding 7 mis of protoplast growth media. The protoplast culture media has been described (Fromm et al., Methods in Enzymology 153, 351-366, 1987). Culture plates are layered with growth media and 1.5% SeaPlaque agarose (FMC BioProducts, Rockland, ME) to prevent protoplast loss. Samples are cultured in the light at 26 0 C, 16/8 day/night cycle, until harvested for the assay (typically 18-22 hours after electroporation). Samples are pipetted from the petri plates to 15 ml centrifuge tubes and harvested by centrifugation at 800-1000 rpm. The :0 supernatant is removed and samples are assayed immediately for the gene of interest. Samples can also be frozen for later analysis.

O The expression of GUS by the DNA promoters of the present invention is measured as a 3 percentage of the activity observed when the basic expression vector, pMON25455

(P-

Os.Actl/GUS/AGRTU.nos) is electroporated into corn protoplasts (Table The MUG assays oo provided a quantitative analysis of the GUS expression in the transgenic plant cells. Total protein s was extracted from each sample. The MUG assay used 5001l of GUS extraction buffer added to N the tissues, and tissues were ground with a teflon pestle in 1.5 ml eppendorf tube and centrifuged S at 10K RPM for 5min at 4 degree (Beckman GS-15R). 4 0 0 pl of supernatant was transferred to a S fresh 96-deep well plate. The extracts are frozen on dry ice and stored at -80 till use. The MUG assay consisted of generating a standard curve of activity with a serial dilution of 4-methyl to umbelliferone Sigma Chemical Co M1381) from 31.2 pmoles to 2000 pmoles. 5pl of each extract was added to a flat bottom 9 6-well plate (Falcon 3872) in duplicate after the plant was preread for blanking the background. 200pl of GUS assay solution (0.1M KPO 4 pH7.8, EDTA, 5% glycerol, 10.0mM DTT, 2mM 4-methyl umbelliferyl glucuronide Fluka 69602) was added to each well and mixed with the samples by pipetting. The Plate was read kinetically on a is F-max (Molecular Devices) at 37 °C with the filter pair: excitiation-355/emission-460. A typical read consists of 21 readings at 3 min intervals and last 1 hour. GUS activity (pmol/min/pg protein) was calculated base on MUG results and protein results of each sample. Total protein was assayed using Bio-Rad Protein Assay kit Serial dilutions of BSA protein from 0.05mg/ml to were used for the standard curve. 1.Spl of extracts was added to flat bottom 96-well plate (Falcon) in duplicate. 200ul of diluted dye reagent was added and mixed with the samples.

The absorbance at 595nm was measured in Spectromax 250 (Molecular Devices) at room temperature after 5 min incubation at room temperature.

Three experiments 2, and 3) with 2 replications each were conducted to assay the expression of the 10 promoter sequences as shown in Table 2. This analysis identified 4 promoter sequences that are equal to or higher than that observed from the rice actin I promoter sequence, these expression cassettes are contained in pMON43716 (Figure pMON43722 (Figure 12), pMON43734 (P-Zm.700342976/l-Os.Actl/GUS/T-Ta.Hsp 17), and pMON43736

(P-

Zm.700345 70 4 /I-Os.Act /GUS/T-Ta.Hsp 17).

-56- Table 2. Corn prtPaSt analIsIS Of DNA promoe Scunedrig-Mxression of GUS: I 2 -7 700342746 SEQ -ID NO.66 700342364 SEQ ID N6.6-5 700350638 SEL-Q -IDNO.75 700164205 SEQ ID NO.68 700343485 SEQ ID NO.69 7 00 3 4 5-557 SQ ID NO.7-0 700345819 SEQ ID NO.71 700342580 SEQ ID NO.72 70042976 SEQ [D N-O7 700345704 SEQ ID NO.74 CaM V.3595 Os.Act i pMON 43710 4330 43720 43722 43726 43728 43732 43:5734 43 736 46-124 2T54-5 5 FPromo--itr GUS Os.Actl/GUS) Fragment, 1 2 3 -Kb 1.2 1.4,2.4 20 66,145 126, 111 1.024 ,2 0.5 16,17 1.3 232,275 1.5 26,2 0.6 47, 54 2.2 7, NA 1.2 118.2, 100 4 300, 409 84,573 8 31,682- 1445.5,1081.8 100 10 100 EXAMPLE 6 Tranisformation of Wheat with iantt xprzessincos~pii2D Apooe eiecso the present invention.

Wheat cells are transformed with DNA vectors containing the DNA promoter sequences of the present invention expressing GUS:1 reporter coding sequence. pMON43644 (clone ID#700154443) Figure 7, pMON43738 (clone 1D4700342364) Figure 10 and pMON43739 (clone ID#700343485) Figure 13 express the GUS gene in the leaves, ovaries and anthers of transgenic wheat plants. Wheat plants are transformed with DNA promoter sequences of the present invention expressing the AGRTU.aroA:CP4 (CP4) coding sequerice of a glyphosate EPSPS enzyme for conferring glyphosate tolerance to transgenic wheat plants. Wheat plants containing pMON45 360 (P-Zm.70034348 S/1-Os.ActICP4/Ta.hsp 17:: P-CaMV.35 S/Zm.Hisp7O/ CP4rF-AGRTU.nos) Figure 14, a double expression cassette vector with the P- 1s Zm.700343485 promoter driving expression of the C134 EPSPS coding sequence and the Ppromoter driving expression of an additional copy of the CP4 EPSPS coding sequcnce; pMON4536 1 (1P-Zm.7001I5 4 443!VIa.CAB/I -Os.AcIICP4/TFa.Hsp 17: :P-CaMV.3

SS/

Zm.H~sp7O/C.P4/Tf-A(GRTtJnos) Figure 8, a double expression cassette vector with the P- Zm.700 15443 promoter driving expression of the CP4 EPSPS coding sequence and the P-

C.)

promoter driving expression of an additional copy of' the CP4 EPSPS coding sequence; pMON4 5362 (P-Zm.7003423 6 4 /1-Os.Acr/CP4/TIa.Hsp 17:: P-CaM V.35S/Zm.H4sp7o/ 00 CP4/T-AGRTU.nos) Figure 11, a double expression cassette vector with the P-Zm.700342364 promoter driving expression of the CP4 EPSPS coding sequence and the P1-CaM V.35S promoter s driving expression of an additional copy of the CP4 EPSPS coding sequence; pMON43623

(P-

r- 7 00 l 6 4 3 4 7'a.CAB/1-Os.Act/CPI4/I'a.1spl 7) Figure 2, a single expression cassette vector with the P-Zm.700164347 promoter driving expression of' the CP4 EIISPS coding sequence. These Cl vectors and control vectors, pMON4241 I (Figure pMON4241O (Figure 3) and pMON30167 (P-Os;.Act/Is.Act/CP4/'AGRrUflos) are transformed into wheat cells by the following i0 method.

Immature embryos of wheat (Triuicum aestivum L) cv Bobwhite are isolated from the immature caryopsis 13-15 days after pollination, and cultured on CM4C (Table 3) for 3-4 days.

The embryos showing active cell division, but no apparent callus formation are selected for Agrobacterium infection.

Table 3. Supplemental Components in Basal Media' -CD0-fment CM 4 CM4C MMUIC, M M 2,4-D (mg/I) 0.5 0.5 0.2 PiChloram (mg/I)7 2.2 2.2 Maltose 40.0 40.0 40.0 40.0 Ghztamine 0.5 Magnesium Chloride 0.75 0.7 Casein I-ydrolysate 0.1 0. 1 MES 1.95 1.95 1.95 Ascorbic Acid (mg/I) 2 100.0 10(0.0 100.0 Gelling Arnt 3~l )2 AllI media contain basal salts (MS basal salts) and vitamlins (MS vitamiin,) fro n 2 Mirashige and Skoog (1962). 'he pH- inl each medium is adJusted to 5.8.

3Filter-sterilized and added to the medium after autoclaving.

3 PhytagelM1 or Gelrite®(G).

O

cI -58 0 A disarmed Agrobacteriun strain C58 (ABI) harboring a binary vector of interest oO (pMON45367, Figure 5) is used for all the experiments. The pMON vectors are transferred into SAgrobacterium by a triparental mating method (Ditta et al., Proc. Natl. Acad. Sci. 77:7347s 7351). Liquid cultures of Agrobacterium are initiated from glycerol stocks or from a freshly streaked plate and grown overnight at 260C-28oC with shaking (approximately 150 rpm) to mid- O log phase (OD 660 in liquid LB medium, pH 7.0 containing 50 mg/1 kanamycin, 50 mg/1 streptomycin and spectinomycin and 25 mg/l chlorarmphenicol with 200iM acetosyringone

(AS).

The Agrobacterium cells are resuspended in the inoculation medium (liquid CM4C) and the 0o density is adjusted to OD660 of 1. The immature embryos cultured in CM4C medium are transferred into sterile petri plates (16 x 20 mm) or wells of a 6-well cell culture plate (Costar Corporation, Cambridge, MA) containing 10 ml of inoculation medium per petri plate or 5 ml per cell culture cluster plate. An equal amount of the Agrobacterium cell suspension is added such that the final concentration ofAgrobacterium cells is an OD,oo of 0.5. In most experiments, is pluronic F68 is added to the inoculation mixture at a final concentration of 0.01%. The ratio between the Agrobacterium and immature embryos (IEs) is about 10 ml: 20-200 IEs. The inoculation is allowed to proceed at 23°C-26 0 C from 5-60 minutes. After the inoculation period, the remaining Agrobacterium cells are removed from the explants by using the in-house vacuum equipment. A piece of sterile Whatman No. 1 filter paper (to fit the size of the petri plate) is placed in each of 60 x 15 or 60 x 20 mm petri dishes. Two hundred ll of sterile water is placed in the middle of the filter paper. After 2-3 minutes, the inoculated immature embryos are placed in the plates. Usually, 20-50 explants are grouped as one stack (about 1 cm in size and 60-80 mg/stack), with 4-5 stacks on each plate. The plates are immediately covered with Parafilm® and then co-cultivated in the dark at 24°C-26°C for 2-3 days.

The co-cultivated PCIEs are transferred CM4C+500 mg/l carbenicillin medium (delay medium) at dark. After 7 days on the delay medium, the immature embryos are transferred to CM4C supplemented with 2 mM glyphosate (not Roundup formulation) and 500 mg/1 carbenicillin for selection for one week. Then calli are transferred to MMS0.2C+0.1mM glyphosate (not Roundup® formulation) +250 mg/1 carbenicillin medium for 2 weeks under light for further selection. Embryogenic calli are transferred to a second regeneration medium MMSOC with lower glyphosate (not Roundup® formulation) concentration (0.02 mM) and 500 C -59- 0 mg/L carbenicillin for plant regeneration. Tose embryoenic cai ar transferred nto fresh medium every two week. Regenerated plantlets are transferred to Sundae cups (Sweetheart Cup Company, Chicago, IL) containing the second regeneration medium for further growth and 00 selection. When roots are well established from transgenic plants the plants are transferred to 5 soil for further evaluation.

The evaluation of transgenic plant tolerance to glyphosate was conducted on the RO O transgenic plants. Glyphosate is administered through the use of a linear track sprayer set to C deliver a 64 oz./A rate of glyphosate. Plants are sprayed once or twice. Vegetative tolerance to the glyphosate is visually evaluated a week after spray based on a scale of 0 to 5 (0 No io observable/vegetative effect of glyphosate; 1 Chlorosis observed; 2 Advanced chlorosis, minor necrosis; 3 Advanced chlorosis, moderate necrosis; 4 Advanced chlorosis, severe necrosis; 5 No live tissue remaining). The R 0 plants produced are allowed to self, and fertility rated as a percent of unsprayed check plants. Plants that show vegetative tolerance and greater than or equal to 80% fertility are advanced to a screen of the R progeny using spray applications 1s of glyphosate and the rating system as described for the Ro screen. The efficacy of a promoter is evaluated by this method. Effective transgenic tolerance to glyphosate herbicide and other systemic herbicides is often a function of the constitutive nature of the promoter expression pattern. In the promoter evaluation shown in Table 3, the percentage of the plants produced that show vegetative and reproductive tolerance isa measure of te utility of the promoter for constitutive expression. The prooters P-Zm.700343485, and P-Zm.70015443, in combination with a P-CaMV.35S expression cassette, demonstrated an increase in the vegetative and reproductive tolerance of transgenic wheat plants to glyphosate herbicide. P-Zm.700342364 showed an increase in vegetative tolerance, however, a reduction in the reproductive tolerance was observed. P-Zm.700164347 as a single expression cassette promoter was effective in conferring vegetative and fertility tolerance to transgenic wheat plants. These promoters in combination with regulatory elements of the expression cassette different than those tested in these demonstrations such as, introns, leaders, transit peptides and 3' untranslated terminator regions can further express desirable expression patterns that can be selected from by those skilled in the art of plant science. For example, as shown in Table 3, transgenic glyphosate o3 tolerant plant lines can be selected at a higher frequency that demonstrate both vegetative and reproductive tolerance when the CaMV.35S promoter is operably linked to the Os.Actl intron

C.)

O

0 0-0 a>.

O l

(-N

60 compared to the Zm.Hsp70 intron. These promoters are effective for use in producing herbicide tolerance crop plants, they are as well useful for the expression of insect resistance genes, disease resistance genes, stress resistance genes and yield traits, which is an aspect of this invention.

Table 3. Transgenic Wheat RO Spray Results pMON# Promoter Elements RllO Plants Vegetative lolerance 42411 P-CaMV.35S/I-Zm.Hsp70 71- 26 (37%) 42410 P-CaMV.35S/1-Os.Actl 150 78(52%) 30167 P-Os.Act/I-Os.Act 63 4 45360 P-Zm.700343485 25 20 (80%) 45361 P-Zm.70015443 26 22 (85/) 45362 P-Zm.700342364 23 14 (61%) 43623 P-Zm.700164347 166 95 (57%) Plants Fertility Advanced 1 1 38 2 3 14(70%) 56 20(91%) 77- 5 22 20 12 EXAMPLE 7.

Identification ofCis acting elements Cis acting regulatory elements necessary for proper promoter regulation can be identified o by a number of means. In one method, deletion analysis is carried out to remove regions of the promoter and the resulting promoter fragments are assayed for promoter activity.

DNA

fragments are considered necessary for promoter regulation if the activity of the truncated promoter is altered compared to the original promoter fragment. Through this deletion analysis, small regions of DNA can be identified which are necessary for positive or negative regulation of transcription. Promoter sequence motifs can also be identified and novel promoters engineered to contain these cis elements for modulating expression of operably linked transcribable sequences. See for example U. S. Patent No. 5,223,419, herein incorporated by reference in its entirety, U. S. Patent No. 4,990,607 herein incorporated by reference in its entirety, and U. S. Patent No. 5,097,025 herein incorporated by reference in its entirety.

An alternative approach is to look for similar sequences between promoters with similar expression profiles. Promoters with overlapping patterns of activity can have common regulatory mechanisms. Several computer programs can be used to identify conserved, sequence c) -61 0 motifs between promoters, including but not limited to MEME, SNA SCAN or GENE SCANlimited to MEME, SNAL SCAN, or GENE SCAN. These motifs can represent binding sites for transcriptions factors which act to regulate the promoters. Once the sequence motifs are identified, their imction can be assayed. For N exampe, te moamc eir unc t on can b ased o the motif sequences can be deleted fom the promoter to determine if the motif is C 5 necessary fbr proper promoter function. Alternatively, the motif can be added to a minimal lpromoter to test whether it is sufficient to activate transcription. Suspected negative regulatory elements can be tested lbr sufficiency by adding to an active promoter and testing for a reduction in promoter activity. Some cis acting regulatory elements may require other elements to function. Therefore, multiple elements can be tested in various combinations by any number of i0 methods known to those of skill in the art.

Once functional promoter elements have been identified, promoter elements can be modified at the nucleotide level to affict protein binding. The modifications can cause either higher or lower affinity binding which would affect the level of transcription from that promoter.

Promoter elements can act additively or synergistically to affect promoter activity. In this regard, promoter elements from different 5' regulatory regions can be placed in tandem to obtain a promoter with a different spectrum of activity or different expression profile. Accordingly, combinations of promoter elements from heterlogous sources or duplication of similar element or the same element sources o r duplication of similar elements or the same element c onfr a higher level of expression of operably linked transcribable sequences For example, a promoter element can be multimerized to increase levels of expression specifically in the pattern affected by that promoter element.

he technical methods needed for constructing expressionvectors containing the novel engineered 5' regulatory elements are known to those of skill in the art. The engineered promoters are tested in e se of he promoters are tested in expression vectors and tested transiently by operably linking the novel promoters to a suitable reporter gene such as GUS and testing in a transient plant assay. The 2s novel promoters are operably linked to one or more genes of interest and incorporated into a plant transformation vector along with one or more additional regulatory elements and transformed into a target plant of interest by a suitable DNA delivery system. The stably transformed plants and subsequent progeny are evaluated by any number of molecular, immunodiagnostic, biochemical, phenotypic, or field methods suitable for assessing the desired agronomic characteristic(s).

O

-62- 0 It Having illustrated and described the principles of the present invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail 00 without departing from such principles. We claim all modifications that are within the spirit and oM scope of the appended claims.

Nc 5 All publications and published patent documents cited in this specification are >r incorporated herein by reference to the same extent as if each individual publication or patent 0 application is specifically and individually indicated to be incorporated by reference.

With reference to the use of the word(s) "comprise" or "comprises" or "comprising" in the foregoing description and/or in the following claims, unless the context requires 0 otherwise, those words are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that each of those words is to be so interpreted in construing the foregoing description and/or the following claims.

Claims (9)

1. An isolated DNA molecule that is capable of regulating transcription of an operably linked DNA coding sequence, comprising a promoter polynucleotide selected from O the group consisting of SEQ ID NO:56-68 or SEQ ID NO:70-75 or a fragment thereof having at least 20 nucleotides. (N C

2. The isolated DNA molecule of claim 1, further comprising a second polynucleotide a molecule operably linked to said promoter polynucleotide.

3. The isolated DNA molecule of claim 2, wherein the promoter polynucleotide confers expression of said second polynucleotide molecule in transgenic plant tissue.

4. The isolated DNA molecule of claim 2, wherein the polynucleotide is a hybrid promoter.

The isolated DNA molecule of claim 4, further comprising a minimal promoter sequence.

6. A recombinant DNA construct comprising SEQ ID NO:56-68 or SEQ ID NO:70-75, or a fragment thereof having at least 20 nucleotide that is capable of regulating transcription of an operably linked DNA sequence, wherein said DNA construct is operably linked to a heterologous polynucleotide and a 3'non-translated region.

7. A transgenic plant or plant cell comprising the recombinant DNA construct of claim 6.

8. A transgenic plant of claim 7, wherein said plant is a monocot plant.

9. A transgenic plant of claim 7 or 8, wherein said recombinant DNA construct confers herbicide tolerance to said plant. A method of making a transgenic plant comprising: introducing into a plant cell a DNA construct comprising a promoter comprising a polynucleotide comprising SEQ ID NO:56-68 or SEQ ID NO:70-75 or a fragment thereof having at least nucleotides, wherein the promoter is operably linked to a heterologous polynucleotide and a 3' non-translated region; (ii) selecting said plant cell; and (iii) regenerating said plant cell into a plant. BW7202/8