WO1998059057A1 - Ga4 homologue dna, protein and methods of use - Google Patents

Abstract

The invention relates to GA4 homologue (GA4H) DNA and proteins encoded by GA4H DNA. GA4H is believed to be a member of the family of enzymes involved in the biosynthesis of the gibberellin family (GA) of plant growth hormones that promote various growth and developmental processes in higher plants, such as seed germination, stem elongation, flowering and fruiting. More specifically, the protein encoded by the GA4H loci may have similar function(s) to β-hydroxylases. The invention also relates to vectors containing the DNA and the expression of the protein encoded by the DNA of the invention in a host cell. Additional aspects of the invention are drawn to host cells transformed with the DNA or antisense sequence of the invention, the use of such host cells for the maintenance, or expression or inhibition of expression of the DNA of the invention and to transgenic plants containing DNA of the invention. Finally, the invention also relates to the use of the GA4 homologues to alter aspects of plant growth.

Description

GA4 Homologue DNA, Protein and Methods of Use

Field of the Invention

The invention relates to the field of molecular biology and plant growth hormones, and especially to gibberellin synthesis.

Background of the Invention

Gibberellins (GA) are a large family of tetracyclic triterpenoid plant growth hormones that promote various growth and developmental processes in higher plants. These processes include promotion of cell division and extension, seed germination, stem elongation, flowering and fruiting (Stowe, B.B. et al, Annu. Rev. Plant Physiol. 5:181-216 (1957), Graebe, J.E. Annu. Rev. Plant. Physiol. 55,419-465 (1987), Phillips etal, Plant. Physiol. 705:1049-1057 (1995), Xu et al. , Proc. Natl. Acad.. Sci. USA 92:6640-6444 ( 1995), Martin et al, Plant

200:159-166 (1996)). Genes that can alter GA biosynthesis or sensitivity have had an impact on the development of new plant species and on agriculture in general.

A number of GA responsive dwarf mutants have been isolated from various plant species, such as maize, pea, and Arabidopsis (Phinney, B.O. et al,

"Chemical Genetics and the Gibberellin Pathway" in Zea mays L. in Plant

Growth Substance, ed., P.F. Waering, New York: Academic (1982) pp. 101-110;

Ingram, T.J. et al, Plant 750:455-463 (1984); Koornneef, M., Arabidopsis Inf. Serv. 75:17-20. (1978)). The dwarf mutants of maize (dwarf -I, dwarf -2, dwarf -3, dwarf-5) have been used to characterize the maize GA biosynthesis pathway by determining specific steps leading to biologically important metabolites (Phinney, B.O. et al, "Chemical Genetics and the Gibberellin Pathway" in Zea mays L. in Plant Growth Substance, ed., P.F. Waering, New York: Academic (1982) pp.

101-110; Fujioka, S. et al, Plant Physiol. 55:1367-1372 (1988)). Similar studies have been done with the dwarf mutants from a pea (Pisum sativum L.) (Ingram, TJ. et al, Plant 760:455-463 (1984)). GA deficient mutants have also been isolated from Arabidopsis (gal, ga2, ga3, ga4, ga5) (Koornneef, M., et al, Theor. Appl. Genet. 55:257-263 (1980)). The Arabidopsis ga4 mutant, induced by ethyl methanesulfonate (EMS) mutagenesis, is a germinating, GA responsive, semidwarf plant whose phenotype can be restored to wild type by repeated application of exogenous GA (Koornneef, M. etal, Theor. Appl. Genet. 55:257- 263 (1980)). The GA4 gene encodes a β-hydroxylase in Arabidopsis thaliana. A mutant allele (ga4) blocks the conversion of 3-β-hydroxy GAs, reducing the endogenous levels of GA,, GA₈ and GA₄ and increasing the endogenous levels of GA_]9, GA₂₀ and GA₉ (Talon, M. et al, Proc. Natl. Acad. Sci. USA 57:7983- 7987 (1990)). The reduced levels of the 3-β-hydroxy GAs is the cause of the semidwarf phenotype of the ga4 mutant. It has been suggested that the pea le mutant also encodes an altered form of 3-β-hydroxylase (Ross, J.J. et al, Physiol. Plant. 76: 173- 176 (1989)). The pea deactivation mutant, sin, causes an elongated slender phenotype (Ross et al, Plant J. 7:512-523 (1995)). Thus, β-hydroxylase is clearly implicated in the process of plant growth. Homologues of the GA4 gene (GA4H) that encode GA4-homologue proteins (GA4H) are described in this application. Two specific homologues, GA4H1 and GA4H2 are exemplified. High levels of sequence homology between the GA4H1, GA4H2 and GA4 genes, as well as between the proteins encoded by these genes suggest that at least these two homologue proteins (GA4H1 and GA4H2) may have similar functions or catalyze similar reactions in plants to that of GA4. Thus, the GA4H proteins should be useful for plant growth modulation.

Summary of the Invention

The invention provides genes involved in gibberellin biosynthesis from which one can express and obtain proteins useful for the regulation of plant growth. Additionally, the invention provides for new DNA probes useful for obtaining additional GA4 homologue genes and proteins. Lastly, this invention provides methods of regulating plant growth.

The invention is first directed to GA4H DNA and proteins encoded by GA4H DNA.

The invention is further directed to GA4H antisense DNA, and to the GA4H antisense RNA transcribed from it.

The invention is further directed to vectors containing GA4H encoding DNA and to the expression of GA4H proteins encoded by GA4H DNA in a host cell.

The invention is further directed to vectors containing GA4H antisense DNA and to the expression of GA4H antisense RNA by the GA4H antisense DNA in a host cell.

The invention is further directed to host cells transformed with a GA4H encoding DNA of the invention, and to the use of such host cells for the maintenance of GA4H DNA or expression of a GA4H protein of the invention.

The invention is further directed to host cells transformed with a GA4H antisense DNA of the invention, and to the use of such host cells for the maintenance of the GA4H DNA or expression of the GA4H antisense RNA of the invention, as inhibitors of the expression of endogenous GA4H.

The invention is further directed to transgenic plants containing a GA4H- encoding or GA4H antisense DNA of the invention. The invention is further directed to a method for altering plant growth, using a GA4H encoding or GA4H antisense DNA of the invention

The invention is further directed to a method for altering plant growth, using a recombinantly made GA4H protein of the invention. Preferably, each of the above embodiments is directed to GA4H1 or

GA4H2 or the cDNA or genomic DNA encoding the GA4 homologues, as well as the antisense DNA OΪGA4H1 or GA4H2.

Brief Description of the Drawings

Figure 1: Sequence of the GA4 cDNA (Chiang, H.H., et al., Plant Cell 7:195-201 (1995)) (SEQ ID Nos. 1, 2, 3 and 4). The figure shows the locations from which DNA probes were generated. The underlined nucleotides (Unique probes) (SEQ ID No. 3) indicate the region specific to the GA4 gene that was used as a probe. Probes (Homologous probes) (SEQ ID No. 4) generated from boxed nucleotides were used for isolation of the GA4 homologues.

Figure 2 A-2C : DNA gel blots of Arabidopsis genomic DNA . Figure 2 A shows a blot that was hybridized to probes derived from the homologous region of the GA4 gene (Figure 1) at low stringency (42°C). Figure 2B shows a blot that was hybridized at low stringency to probes derived from the unique region of the GA4 gene (Figure 1). Figure 2C shows a blot that was hybridized at high stringency to probes derived from p3-l, GA4H1 gene (Figure 3), DNA. DNA in lanes 1, 2 and 3 was digested with Hindlll, BamUl, and EcøRI, respectively. The predicted size (in kilobase pairs; kbp) of the three major hybridizing bands are shown on the left.

Figure 3: The restriction map of the genomic clone, pL VN103 (ATCC accession no. 98436; Deposited at the American Type Culture Collection, 10801

University Boulevard, Manasas, VA 201 10-2209, U.S.A.) under the terms of the Budapest Treaty), containing two linked homologues of GA4. The plasmid pLNN103 contains the entire genomic insert from λ3 but was cloned into pBSKS(+). Plasmid p3-l is a subclone of λ3 and carries the 2.1 kb Hindlϊl fragment. This subclone contains most of the coding region of the GA4H1 gene. The region containing both GA4H1 and GA4H2 genes are shown in more detail on the bottom of the figure. The arrows indicate the direction of transcription of these genes. The line indicates the noncoding area, and rectangular boxes represent the coding region of the DNA. Abbreviations: B, BamHl; H, HindUI.

Figure 4A-4B: Physical mapping the GA4H1 and GA4H2 genes by anchoring to mapped YACs. PCR amplification of the GA4H1 (with GA-P2 and GA-P6 primers) (See Figure 6) and GA4H2 (with GA-P19 and GA-P20 primers) (See Figure 8) genes (For the primer sequences, see Example 1). Figure

4A shows an ethidium bromide stained gel of the PCR product. Figure 4B shows an Autoradiograph of a DNA blot of the gel in Figure 4A using probes derived from the genomic clone pLVN103. Primers GA-P19 and GA-P20 were used in lanes 1-2 and 4-6, while primers GA-P2 and GA-P6 were used in lanes 7-8 and

10-12. Molecular weight markers (1 kb DNA ladder) were loaded in lanes 3 and

9. DNA templates are: genomic clone pLV 103 (lanes 2 and 8); YAC CIC6C3

(lanes 1 and 7) of chromosome 2; CIC1E4 (lanes 4 and 10); CIC6C10 (lanes 5 and 11); and CIC10A11 (lanes 6 and 12).

Figure 5: Nucleotide sequence (SEQIDNo. 5) of the GA4H1 RT-PCR product (cDNA). The predicted start (ATG) and stop (taa) codons are present at nucleotide nos. 44 and 1109, respectively. The intron is located at nucleotide no. 513 and is represented by a filled triangle (T). Underlined nucleotides indicate the start (ATG) and stop (taa) codons. Lower case nucleotides represent 5' and

3' untranslated regions. A "G" at nucleotide no. 1059, indicated with an asterisk (*), does not agree with the genomic DNA at this position. The number on the left indicates the nucleotide position. Figure 6: The genomic sequence of the GA4H1 gene (SEQ ID No. 6). Upper and lower case letters represent the coding and noncoding regions of the gene, respectively. The predicted translated protein sequence (SEQ ID No. 7) is shown below its corresponding nucleotide sequence. Arrows represent primers used in either PCR or RT-PCR analyses. The nucleotide and the amino acid positions are shown on the right.

Figure 7: Nucleotide sequence of the GA4H2 RT-PCR product (cDNA)

(SEQ ID No. 8). The predicted start (ATG) and stop (taa) codons are present at sequence nos. 49 and 1190, respectively. The intron is located at sequence no. 518. The number on the left indicates the nucleotide position.

Figure 8: Genomic sequence of the GA4H2 gene (SEQ ID No. 9). Upper and lower case letters represent the coding and noncoding regions of the gene, respectively. The predicted translated protein sequence (SEQ ID No. 10) is shown below its corresponding nucleotide sequence. Arrows represent primers used in either PCR or RT-PCR analyses. The position of the nucleotide and the amino acid are shown on the right.

Figure 9: Alignment of GA4, GA4H1 and GA4H2 proteins. Both Pileup and Prettybox (Genetics Computer Group, Wisconsin, MA, U.S.A.) commands were used to generate this alignment. The position of the amino acid is shown on the right.

Figure 10: Amino acid sequence identity and similarity between GA4 (SEQ ID No. 2), GA4H1 (SEQ ID No. 7), GA4H2 (SEQ ID No. 10) and some other related 2-oxoacid-dependent dioxygenases (2-ODD). The percentage of sequence identity and similarity (in parenthesis) were generated using the GAP software of the GCG package. Shaded boxes indicate the putative GA4 gene family in Arabidopsis. Abbreviations: GA5, Arabidopsis GA₂₀-oxidase (accession number X83379); F3H, Zeamaize flavanone-3-β-hydroxylase (accession number U04434); FLS, potato flavanol synthase (accession number X92178); ANS, apple anthocyanidin hydroxylase (accession number S33144); EFE, tobacco ethylene forming enzyme (accession number Z29529). Accession number refer to GENBANK.

Figure 11A-11B: GA4H1 gene expressed in the flowers and shoot meristems. One-tenth of the PCR product of each sample was electrophoresed on an agarose gel and then stained with ethidium bromide (Figure 11 A). A DNA blot of the gel in Figure 1 1A was probed with GA4H1 specific DNA (Figure 1 IB). Primers, G A-P 13 and GA-P 17, were used to amplify the 220 bp cDN A and 630 bp genomic DNA of the GA4H1 gene. Primers Tua4F/ Tua4R were used as an internal control that amplified the 320 bp cDNA of the α-tubulin 4 gene (TUA4). DNA templates of pLVN115 (lane 1), pCD7 (lane 2), and pLVN103 (lane 3) were used in the PCR amplification. First strand cDNA templates of floral shoots (lane 5), leaves (lane 6), roots (lane 7), and siliques (lane 8) were subjected to

RT-PCR. The 123 bp BRL DNA marker is present in lane 4.

Figure 12A-12B: GA4H2 gene expressed predominantly in the roots. One-tenth of the PCR product from each sample was separated on agarose gel and then stained with ethidium bromide (Figure 12A). The DNA gel blot shown in Figure 12A was probed with the GA4H2 specific probes (Figure 12B). Primers,

GA-P 18 and GA-P20, were used to amplify the 440 bp cDNA and 860 bp genomic DNA of the GA4H2 gene. The same primer pair of the TUA4 gene was also used as an internal control during the RT-PCR. RNA templates of siliques (lane 1), roots (lane 2), leaves (lane 3), and floral shoots (lane 4) were subjected to RT-PCR. DNA templates of pLVNl 03 (lane 6), pCD7 (lane 7), and pLVNl 07

(lane 8) were used in the PCR amplification. The 123 bp BRL DNA marker is present in lane 5. Figure 13. Phenotype of transgenic plants expressing the sense and antisense of the GA4H1 gene.

Definitions

"GA_n" (with a number subscripted), refers to the "gibberellin A_n" compound. The chemical structures of some of the gibberellin A_n's are presented in Moritz, T. et al, Plant 795:1-8 (1994). GA without a subscript, e.g. GA1 refers to enzymes presumably involved in the gibberellin biosynthetic pathway.

Italicized, uppercase names, such as "GA4 or GA4H, " refer to the wild type gene. Italicized, lowercase names such as "ga4" refer to the mutant gene. Uppercase names, such as "GA4H," refer to the protein, DNA or RNA encoded by a GA4H gene, while lowercase names, such as "ga4," refer to the protein, DNA or RNA encoded by a mutant, such as the mutant ga4 gene.

GA4H refers to any GA4 homologue, while GA4H1 and GA4H2 refers to the homologues of GA4 shown in figures 6 and 8, or minor variations of these homologues or their cDNAs (Figures 5 and 7) . Such minor variations may include, but are not limited to substitution of conservative amino acids or degenerate substitutions in the DNA encoding the amino acid sequence of GA4H1 and GA4H2. Such variation may also be referred to as "substantially similar" molecules. A unique probe should be understood to be a probe that contains a DNA sequence unique to GA4 DNA and that can be used to pull out the GA4 DNA. A "unique" probe sequence is indicated in Figure 1 by underlining. A homologue probe contains a DNA sequence homologous to a sequence found in GA4 homologue DNA. A "homologous" probe sequence is indicated in Figure 1 by the boxed nucleotide sequence and can be used to obtain GA4H DNA.

Plant should be understood as referring to a multicellular differentiated organism capable of photosynthesis including angiosperms (monocots and dicots) and gymnosperms. Plant cell should be understood as referring to the structural and physiological unit of plants. The term "plant cell" refers to any cell which is either part of or derived from a plant. Some examples of cells encompassed by the present invention include differentiated cells that are part of a living plant; differentiated cells in culture; undifferentiated cells in culture; the cells of undifferentiated tissue such as callus or tumors.

Plant cell progeny should be understood as referring to any cell or tissue derived from plant cells including callus; plant parts such as stems, roots, fruits, leaves or flowers; plants; plant seed; pollen; and plant embryos. Propagules should be understood as referring to any plant material capable of being sexually or asexually propagated, or being propagated in vivo or in vitro. Such propagules preferably consist of the protoplasts, cells, calli, tissues, embryos or seeds of the regenerated plants.

Transgenic plant should be understood as referring to a plant having stably incorporated exogenous DNA (i.e. DNA not normally found) in its genetic material. The term also includes exogenous DNA which may be introduced into a cell or protoplast in various forms, including, for example, naked DNA in circular, linear or supercoiled form, DNA contained in nucleosomes or chromosomes or nuclei or parts thereof, DNA complexed or associated with other molecules, DNA enclosed in liposomes, spheroplasts, cells or protoplasts.

Purified as it refers to preparations made from biological cells or hosts should be understood to mean any cell extract containing the indicated DNA or protein including a crude extract of the DNA or protein of interest. For example, in the case of a protein, a purified preparation can be obtained following an individual technique or a series of preparative or biochemical techniques and the

DNA or protein of interest can be present at various degrees of purity in these preparations. The procedures may include for example, but are not limited to, ammonium sulfate fractionation, gel filtration, ion exchange change chromatography, affinity chromatography, density gradient centrifugation and electrophoresis. A preparation of DNA or protein that is "pure" or "isolated" should be understood to mean a preparation free from naturally occurring materials with which such DNA or protein is normally associated in nature. "Essentially pure" should be understood to mean a "highly" purified preparation that contains at least 95% of the DNA or protein of interest.

A ce77 extract that contains the DNA or protein of interest should be understood to mean a homogenate preparation or cell-free preparation obtained from cells that express the protein or contain the DNA of interest. The term "cell extract" is intended to include culture media, especially spent culture media from which the cells have been removed.

Afragment of a molecule should be understood as referring to a shortened sequence of an amino acid or nucleotide sequence that retains one or more desired chemical or biological properties of the full-length sequence such that use of the full-length sequence. A functional derivative of GA4H (or GA4) should be understood as referring to a protein, or DNA encoding a protein, that possesses a biological activity that is substantially similar to the biological activity of GA4H (or GA4). A functional derivative may or may not contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function. The term "functional derivative" is intended to include the "fragments," "variants, " "analogues," or "chemical derivatives" of a molecule. The derivative retains at least one of the naturally-occurring functions of the parent gene or protein. The function can be any of the regulatory gene functions or any of the function(s) of the finally processed protein. The degree of activity of the function need not be quantitatively identical as long as the qualitative function is substantially similar.

A mutation should be understood as referring to a detectable change in the genetic material which may be transmitted to daughter cells and possibly even to succeeding generations giving rise to mutant cells or mutant organisms. If the descendants of a mutant cell give rise only to somatic cells in multicellular organisms, a mutant spot or area of cells arises. Mutations in the germ line of sexually reproducing organisms may be transmitted by the gametes to the next generation resulting in an individual with the new mutant condition in both its somatic and germ cells. A mutation may be any (or a combination of) detectable, unnatural change affecting the chemical or physical constitution, mutability, replication, phenotypic function, or recombination of one or more deoxyribonucleotides; nucleotides may be added, deleted, substituted for, inverted, or transposed to new positions with and without inversion. Mutations may occur spontaneously and can be induced experimentally by application of mutagens. A mutant variation of a nucleic acid molecule results from a mutation. A mutant polypeptide may result from a mutant nucleic acid molecule.

A species should be understood as referring to a group of actually or potentially interbreeding natural populations. A species variation within a nucleic acid molecule or protein is a change in the nucleic acid or amino acid sequence that occurs among species and may be determined by DNA sequencing of the molecule in question.

A preparation that is substantially free of other A. thaliana DNA (or protein) should be understood as referring to a preparation wherein the only A. thaliana DNA (or protein) is that of the recited A. thaliana DNA (or protein).

Though proteins may be present in the sample which are homologous to other y thaliana proteins, the sample is still said to be substantially free of such other y thaliana DNA (or protein) as long as the homologous proteins contained in the sample are not expressed from genes obtained from A. thaliana. A DNA construct should be understood as referring to a recombinant, man-made DNA, linear or circular.

T-DNA (transferred DNA) should be understood as referring to a segment or fragment of Ti (tumor-inducing) plasmid DNA which integrates into the plant nuclear DNA. Stringent hybridization conditions should be understood to be those conditions normally used by one of skill in the art to establish at least a 90% homology between complementary pieces of DNA or DNA and RNA. Lesser homologies, such as at least 70% homology or preferably at least 80% may also be desired and obtained by varying the hybridization conditions.

There are only three requirements for hybridization to a denatured strand of DNA to occur. (1) There must be complementary single strands in the sample. (2) The ionic strength of the solution of single-stranded DNA must be fairly high so that the bases can approach one another; operationally, this means greater than 0.2M. (3) The DNA concentration must be high enough for intermolecular collisions to occur at a reasonable frequency. The third condition only affects the rate, not whether renaturation/hybridization will occur.

Conditions routinely used by those of skill in the art are set out in readily available procedure texts, e.g., Current Protocols in Molecular Biology, Vol. I, Chap. 2.10, John Wiley & Sons, Publishers (1994) or Sambrook et al. , Molecular

Cloning, Cold Spring Harbor (1989), incorporated herein by reference. As would be known by one of skill in the art, the ultimate hybridization stringency reflects both the actual hybridization conditions as well as the washing conditions following the hybridization, and one of skill in the art would know the appropriate manner in which to change these conditions to obtain a desired result.

For example, a prehybridization solution should contain sufficient salt and nonspecific DNA to allow for hybridization to non-specific sites on the solid matrix, at the desired temperature and in the desired prehybridization time. For example, for stringent hybridization, such prehybridization solution could contain 6x sodium chloride/sodium citrate (lxSSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5x Denhardt's solution, 0.05%) sodium pyrophosphate and 100 μg per ml of herring sperm DNA. An appropriate stringent hybridization mixture might then contain 6x SSC, lx Denhardt's solution, 100 μg per ml of yeast tRNA and 0.05%) sodium pyrophosphate. Alternative conditions for DNA-DNA analysis could entail the following : 1 ) prehybridization at room temperature and hybridization at 68 °C;

2) washing with 0.2x SSC/0.1% SDS at room temperature;

3) as desired, additional washes at 0.2x SSC/0.1%) SDS at 42°C (moderate-stringency wash); or 4) as desired, additional washes at O.lx SSC/0.1%) SDS at 68 °C

(high stringency). Known hybridization mixtures, e.g., that of Church and Gilbert, Proc. Natl. Acad. Sci. USA 57:1991-1995 (1984), comprising the following composition may also be used: 1 % crystalline grade bovine serum albumin/1 mM EDTA/0.5M NaHPO₄, pH 7.2/7% SDS. Additional, alternative but similar reaction conditions can also be found in Sambrook et al. , Molecular Cloning, Cold Spring Harbor (1989). Formamide may also be included in prehybridization/hybridization solutions as desired.

It should be understood that these conditions are not meant to be definitive or limiting and may be adjusted as required by those of ordinary skill in the art to accomplish the desired objective.

A vector should be understood to be a DNA element used as a vehicle for cloning or expressing a desired sequence, such as a gene of the invention, in a host. A host or host cell should be understood to be a cell in which a recombinant sequence, such as a sequence encoding a GA4H DNA of the invention, is incorporated and expressed. A GA4H gene of the invention or the antisense of the gene may be introduced into a host cell as part of a vector by transformation. Both the sense and the antisense DNA sequences are present in the same host cell since DNA is double stranded. The direction of transcription, however, as directed by an operably linked promoter as designed by the artisan, dictates which of the two strands is ultimately transcribed into RNA.

Detailed Description The process for genetically engineering GA4H protein sequences, according to the invention, is facilitated through the cloning of genetic sequences that are capable of encoding GA4H proteins and through the expression of such genetic sequences. As used herein, the term "genetic sequence" is intended to refer to a nucleic acid sequence (preferably DNA). Genetic sequences that are capable of encoding GA4H proteins can be derived from a variety of sources.

These sources include genomic DNA, RNA, cDNA, synthetic DNA, and combinations thereof. The preferred source of the GA4H genomic DNA is a plant genomic library and most preferably an Arabidopsis genomic library. A more preferred source of the GA4H cDNA is a plant cDNA library and most preferably an Arabidopsis cDNA library made from silique mRNA, although the message is ubiquitously expressed in the root, leaf and flower of plants. This invention, however, is not meant to be limited to GA4H homologues from only the plant genus Arabidopsis.

Methods for obtaining and screening genomic libraries are well known in the art. An example of obtaining and screening a genomic library which is not meant to be limiting follows. Additional methods may be found in Example 1 of the specification.

One may begin with a CsCl DNA preparation and partially digests it with Sau3AI. After digestion, a partial fill-in reaction is performed. The reaction mixture for the partial fill-in is as follows:

40 μl DNA

6 μl Sau3AI buffer (1 OX)

2.5 μl 0.1 M DTT

1 μl lOO mM dATP 1 μl lOO mM dGTP

5 μl Klenow enzyme

4.5 μl H₂O

After 30 minutes at 37 °C the reaction is terminated with phenol-chloroform and the DNA is obtained. The DNA is then loaded on a 0.7% low melting point agarose gel and after electrophoresing, bands between 10 and 23 kb are cut out from the gel. The gel with the cut-out bands is then melted at 67 °C. The isolated DNA is then placed in the following ligation mixture:

2 μl Lambda Fix II, pre-digested arms (2 μg) 1 μg genomic DNA, partial fill-in 0.5 μl 1 Ox ligation buffer

0.5 μl 10 mM ATP (pH 7.05)

0.5 μl T4 DNA ligase

- 1.5 μl H₂O (to 5μl final volume)

Following ligation overnight at 4°C, the DNA is packaged using GIGAPACK II GOLD.

Plaque lifts are made using Hybond filters (Amersham Corp.), which were then autoclaved for 2 min. Filters were hybridized with probes as described for DNA and RNA gel blot analysis below.

Bacteriophage λ DNA is prepared from ER1458 ly sates according to the mini-prep method of Grossberger, D., Nucl. Acids. Res. 15:6737 (1987). DNA fragments are subcloned into pBluescript KS^" vectors (Stratagene) and used to transform JM 109.

Double stranded DNA is isolated from plasmid clones and purified by CsCl banding. Sequencing is performed using the ABI PRISM dye terminator cycle sequencing kit and the products are separated and detected on the ABI 377

(Perkin Elmer). Sequence analysis is performed using the Sequence Analysis Software package (Genetics Computer Group, Inc., Madison, WI) and the Blast network service of the National Center for Biotechnology Information (Bethesda, MD). Electrophoresis of DNA is in Tris-Acetate-EDTA buffer with subsequent transfer in 25 mM NaHPO₄ to Biotrans filters (International Chemical and Nuclear Corp.). Electrophoresis of RNA samples is in agarose gels containing RNAase inhibitor using MOPS/EDTA buffer and transferred to filters as for DNA. Filters were UV-crosslinked using a Stratalinker (Stratagene) and baked for l hr at 80°C. Radioactive probes are separated from unincorporated nucleotides using a 1-ml Sephadex G-50 spin column and denatured in a microwave oven (Stroop, W.G. et al., Anal. Biochem. 182:222-225 (1989)). Prehybridization for 1 hr and hybridization overnight is performed at 65 °C in the hybridization buffer described by Church, G.M. et al., Proc. Natl. Acad.. Sci. USA 81 : 1991-1995

(1984)). Filters are washed once for 15 min in 2xSSC at room temperature, then two times for 30 min in O.lxSSC and 0.1%SDS at 60°C. The damp filters are autoradiographed at -80°C using intensifying screens. Filters are stripped twice in 2mM Tris-HCl, pH8.0, ImM EDTA, 0.2% SDS at 70°C for 30 min prior to reprobing (Church, G.M. et al, Proc. Natl. Acad.. Sci. USA 81 :1991-1995

(1984)).

The recombinant GA4H cDNA of the invention will not include naturally occurring introns if the cDNA is made using mature GA4H mRNA as a template. Genomic DNA may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with the homologous (isolated from the same source; native) 5' promoter region of the GA4H gene sequences and/or with the homologous 3 ' transcriptional termination region. Further, such genomic DNA may be obtained in association with the genetic sequences that provide the homologous 5 ' non-translated region of the GA4 mRNA and/or with the genetic sequences which provide the homologous 3 ' non-translated region.

Due to the degeneracy of nucleotide coding sequences, and to the fact that the DNA code is known, all other DNA sequences which encode the same amino acid sequence as depicted for example, in Figure 6 [SEQ ID No. 7] can be determined and used in the practice of the present invention. Additionally, those sequences that hybridize to for example, to a GA4H sequence such as SEQ. ID

Nos. 5 or 6, under stringent conditions are also useful in the practice of the present invention.

A DNA sequence encoding GA4H protein or GA4H antisense RNA can be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. In one embodiment of the invention, expression vectors are provided that are capable of expressing GA4H mRNA or antisense

RNA. Vectors for propagating a given sequence in a variety of host systems are well known and can readily be altered by one of skill in the art such that the vector will contain DNA or RNA encoding the desired genetic sequence and will be propagated in a desired host. Such vectors include plasmids and viruses and such hosts include eukaryotic organisms and cells, for example plant, yeast, insect, plant, mouse or human cells, and prokaryotic organisms, for example E coli and B. subtilus. Shuttle vectors in which the desired genetic sequence is "maintained" in an available form before being extracted and transformed into a second host for expression are also useful DNA constructs envisioned as carrying the DNA of the invention.

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide or antisense sequence if it contains a nucleotide sequence that encodes such polypeptide or antisense sequence and transcriptional and, if necessary, translational regulatory information operably linked to the nucleotide sequences that encode the polypeptide or antisense sequence.

Two DNA sequences (such as a promoter region sequence and the GA4H gene encoding or antisense sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired sequence, or

(3) interfere with the ability of the desired sequence to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a desired DNA sequence if the promoter were capable of effecting transcription of that DNA sequence. In one embodiment of the invention, a vector is employed that is capable of integrating the desired gene sequences into the host cell chromosome. Cells that have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. In another embodiment, the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

The present invention also encompasses the expression of the GA4H protein (or a functional derivative thereof) in either prokaryotic or eukaryotic cells. Preferred prokaryotic hosts include bacteria such as E. coli, Bacillus,

Streptomyces, Pseudomonas, Salmonella, Serratia, etc. The most preferred prokaryotic host is E. coli. Bacterial hosts of particular interest include E coli Kl 2 strain 294 (ATCC 31446), E. coli χ 1776 (ATCC 31537), E coli W3110 (P, lambda^", prototrophic (ATCC 27325)), and other enterobacterium such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species. Under such conditions, the GA4H gene product will not be glycosylated. The procaryotic host must be compatible with the replicon and control sequences in the expression plasmid.

Hosts can be utilized for production of the desired genetic sequence, or GA4H protein, using conventional methods, such as by growth in shake flasks, fermentors, tissue culture plates or bottles. Alternatively, multi cellular organisms such as a plant might be used.

DNA encoding the desired protein is preferably operably linked to a promoter region, a transcription initiation site, and a transcription termination sequence, functional in plants. Any of a number of promoters which direct transcription in a plant cell is suitable. The promoter can be either constitutive or inducible. Some examples of promoters functional in plants include the nopaline synthase promoter and other promoters derived from native Ti plasmids, viral promoters including the 35S and 19S RNA promoters of cauliflower mosaic virus (Odell et al, Nature 575:810-812 (1985)), and numerous plant promoters.

Alternative promoters that may be used include nos, ocs, and CaMV promoters. Overproducing plant promoters may also be used. Such promoters, operably linked to the GA4H gene, should increase the expression of the GA4 protein. Overproducing plant promoters that may be used in this invention include the promoter of the small subunit (ss) of ribulose-l,5-biphosphate carboxylase from soybean (Berry-Lowe etal.,J. Molecular and App. Gen. 7:483- 498 (1982), and the promoter of the chlorophyll a/b binding protein. These two promoters are known to be light-induced in eukaryotic plant cells (see, for example, Genetic Engineering of Plants, an Agricultural Perspective, A. Cashmore, Plenum, New York 1983, pages 29-38; Corruzi, G. et al, J. of Biol.

Chem. 255:1399 (1983); and Dunsmuir, P. et al, J. of Mol and Applied Genet. 2:285 (1983)).

To express the GA4H gene (or a functional derivative thereof) in a prokaryotic cell (such as, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.), it is necessary to operably link the GA4H gene encoding sequence to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage λ, the bla promoter of the β-lactamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pBR325, etc. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage λ (P_L and P_R), the trp, recA, lacZ, lacl, and gal promoters of E. coli, the α-amylase (Ulmanen, I., etal, J. Bacteriol 162:176-182 (1985)) and the ς-28-specific promoters of B. subtilis (Gilman, M.Z., et al, Gene sequence 52:11-20 (1984)), the promoters of the bacteriophages of Bacillus

(Gryczan, T.J., In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward, J.M., et al, Mol. Gen. Genet. 205:468-478 (1986)).

Prokaryotic promoters are reviewed by Glick, B.R., (J. Ind. Microbiol 7:277-282 (1987)); Cenatiempo, Y. (Biochimie 65:505-516 (1986)); and

Gottesman, S. (Ann. Rev. Genet. 75:415-442 (1984)).

Proper expression in a prokaryotic cell also requires the presence of a ribosome binding site upstream of the gene sequence-encoding sequence. Such ribosome binding sites are disclosed, for example, by Gold, L., et al. (Ann. Rev. Microbiol. 55:365-404 (1981)).

Preferred eukaryotic hosts include yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture. Mammalian cells that can be useful as hosts include cells of fibroblast origin such as VERO or CHO-Kl , or cells of lymphoid origin, such as the hybridoma SP2/O-AG14 or the myeloma P3x63Sg8, and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332 that may provide better capacities for correct post-translational processing.

For a mammalian host, several possible vector systems are available for the expression of the GA4H gene. A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, etc., may be employed. Transcriptional initiation regulatory signals may be selected that allow for repression or activation, so that expression of the gene sequences can be modulated. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.

Yeast provides substantial advantages in that it can also carry out post- translational peptide modifications. A number of recombinant DNA strategies exist that utilize strong promoter sequences and high copy number plasmids that can be utilized for production of the desired proteins in yeast. Yeast recognizes leader sequences on cloned mammalian gene sequence products and secretes peptides bearing leader sequences (i.e., pre-peptides).

Any of a series of yeast gene sequence expression systems incorporating promoter and termination elements from the actively expressed gene sequences coding for glycolytic enzymes produced in large quantities when yeast are grown in medium rich in glucose can be utilized. Known glycolytic gene sequences can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene sequence can be utilized.

Another preferred host is insect cells, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used (Rubin, G.M., Science 2 0:1453-1459 (1988)). Alternatively, baculovirus vectors can be engineered to express large amounts of the GA1 gene in insects cells (Jasny, B.R., Science 255:1653 (1987); Miller, D.W., et al, in Genetic Engineering (1986), Setlow, J.K., et al, eds., Plenum, Vol. 8, pp. 277- 297).

As discussed above, expression of the GA4H gene in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include the promoter of the mouse metallothionine I gene sequence (Hamer, D., et al. , J. Mol. Appl. Gen. :273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 57:355-365 (1982)); the SV40 early promoter (Benoist, C, et al, Nature (London) 290:304- 310 (1981)); the yeast gal4 gene sequence promoter (Johnston, S.A., etal, Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P.A., et al, Proc. Natl. Acad. Sci. (USA) 57:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at the codon that encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence that encodes the GA4H gene (or a functional derivative thereof) does not contain any intervening codons that are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in the formation of a fusion protein (if the AUG codon is in the same reading frame as the GA4H gene encoding DNA sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the GA1 gene encoding sequence). The GA 4H gene encoding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non- replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the GA4H gene may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced sequence into the host chromosome.

In one embodiment, a vector is employed that is capable of integrating the desired gene sequences into the host cell chromosome. Cells that have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers that allow for selection of host cells which contain the expression vector. The marker can provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements can also be needed for optimal synthesis of single chain binding protein mRNA. These elements can include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama, H., Molec. Cell. Biol. 5:280 (1983). Inapreferred embodiment, the introduced sequence is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors can be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector can be recognized and selected from those recipient cells that do not contain the vector; the number of copies of the vector that are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColΕl, pSClOl, pACYC 184, πVX. Such plasmids are, for example, disclosed by Maniatis, T., et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)). Bacillus plasmids include pC194, pC221 , pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: 77ze Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include pIJlOl

(Kendall, K.J., et al, J. Bacteriol 7(59:4177-4183 (1987)), and streptomyces bacteriophages such as φC31 (Chater, K.F., et al, In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John, J.F., et al. (Rev. Infect. Dis. 5:693-704 (1986)), and Izaki, K. (Jpn. J. Bacteriol. 55:729-742

(1978)).

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Bot- stein, D.. et al, Miami Wntr. Symp. 79:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, J.R., Ce7/ 25:203-204 (1982); Bollon, D.P., etal, J. Clin. Hematol Oncol. 70:39- 48 (1980); Maniatis, T., In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene sequence Expression, Academic Press, NY, pp. 563-608 (1980)). Once the vector or DNA sequence containing the construct(s) has been prepared for expression, the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means: transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate- precipitation, direct microinjection, etc. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the GA4H gene, or fragments thereof. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like).

Following expression in an appropriate host, the GA4H protein can be readily isolated using standard techniques such as immunochromatography or HPLC to produce GA4H protein free of other A. thaliana proteins.

Genetic sequences comprising the desired gene or antisense sequence operably linked to a plant promoter may be joined to secretion signal sequences and the construct ligated into a suitable cloning vector. In general, plasmid or viral (bacteriophage) vectors containing replication and control sequences derived from species compatible with the host cell are used. The cloning vector will typically carry a replication origin, as well as specific genes that are capable of providing phenotypic selection markers in transformed host cells, typically antibiotic resistance genes.

General methods for selecting transgenic plant cells containing a selectable marker are well known and taught, for example, by Herrera-Estrella, L. and Simpson, J. (1988) "Foreign Gene Expression in Plants" in Plant Molecular Biology, A Practical Approach, Ed. CH. Shaw, IRL Press, Oxford, England, pp. 131-160.

In another embodiment, the present invention relates to a transformed plant cell comprising exogenous copies of DNA (that is, copies that originated outside of the plant) encoding a GA4 gene expressible in the plant cell wherein said plant cell is free of other foreign marker genes (preferably, other foreign selectable marker genes); a plant regenerated from the plant cell; progeny or a propagule of the plant; and seed produced by the progeny.

Plant transformation techniques are well known in the art and include direct transformation (which includes, but is not limited to: microinjection

(Crossway, Mol. Gen. Genetics 202:179-185 (1985)), polyethylene glycol transformation (Krens et al, Nature 296:72-74 (1982)), high velocity ballistic penetration (Klein et al, Nature 527:70-73 (1987)), fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies (Fraley et al, Proc. Natl Acad. Sci. USA 79:1859-1863 (1982)), electroporation (Fromm et al, Proc. Natl. Acad. Sci. USA 52:5824 (1985)) and techniques set forth in U.S. Patent No. 5,231,019)) and Agrobacterium tumefaciens mediated transformation as described herein and in (Hoekema et al, Nature 505:179 (1983), de Framond et al, Bio/technology 7:262 (1983), Fraley et al. WO84/02913, WO84/02919 and WO84/02920, Zambryski et al. EP

1 16,718. Jordan et al, Plant Cell Reports 7:281-284 (1988), Leple et al. Plant Cell Reports 77: 137-141 (1992), Stomp et al, Plant Physiol. 92:1226-1232 (1990), andKnauf etal, Plasmid 5:45-54 (1982), Chiang etal, Plant Cell 7:195- 201 (1995)). Another method of transformation is the leaf disc transformation technique as described by Horsch et al. Science 227: 1229- 1230 (1985), Bechtold et al, Acad. Sci. Paris 316:1194-1199 (1993).

The transformation techniques can utilize DNA encoding a GA4H amino acid sequence of, including the GA4H cDNA sequence, the GA4H genomic sequence, fragments thereof or the antisense sequence, or degenerate variants of said sequences such that they are expressible in plants. Included within the scope of a gene encoding a GA4H amino acid sequence are functional derivatives of the GA4H sequences of the invention, as well as variant, analog, species, allelic and mutational derivatives.

The preparation of functional derivatives can be achieved, for example, by site-directed mutagenesis. (Sambrook et al. , Molecular Cloning: A Laboratory

Manual, Cold Spring Harbor Press (1989)). Site-directed mutagenesis allows the production of a functional derivative through the use of a specific oligonucleotide that contains the desired mutated DNA sequence. One skilled in the art will recognize that the functionality of the derivative can be evaluated by routine screening assays.

As used herein, modulation of GA4H expression entails the enhancement or reduction of the naturally occurring levels of the protein. Specifically, the translation of RNA encoding GA4H can be reduced using the technique of antisense cloning. In general, antisense cloning entails the generation of an expression module which encodes an RNA complementary (antisense) to the RNA encoding GA4H (sense). By expressing the antisense RNA in a cell which expresses the sense strand, hybridization between the two RNA species will occur resulting in the blocking of translation. Alternatively, overexpression of a GA4H protein might be accomplished by use of appropriate promoters, enhancers, and other modifications. Those of skill in the art would be aware of references describing the use of antisense genes in plants (van der Krol et al, Gene 72:45-50 (1988); van der Krol et al, Plant Mol. Biol. 74:467-486 (1990); Zhang et al, Plant Cell 4:1575-1588 (1992)). Other foreign marker genes (i.e., exogenously introduced genes) typically used include selectable markers such as a neo gene (Potrykus et al, Mol. Gen. Genet 799: 183-188 (1985)) which codes for kanamycin resistance; a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al, Bio/technology (5:915-922 (1988)) which encodes glyphosate resistance; a nitrilase gene which confers resistance to bromoxynil (Stalker et al, J. Biol. Chem. 2(55:6310-6314 (1988)); a mutant acetolactate synthase gene (ALS) which confers imidazolinone or sulphonylurea resistance (EP application number 154,204); a methotrexate resistant DHFR gene (Thillet et al, J. Biol. Chem. 263: 12500- 12508) and screenable markers which include β-glucuronidase (GUS) or an R-locus gene, alone or in combination with a C-locus gene (Ludwig et al,

Proc. Natl. Acad. Sci. USA 86:7092 (1989); Paz-Ares et al, EMBO J. 6:3553 (1987)).

Alternatively, the genetic construct for expressing the desired protein can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. The genetic material may also be transferred into plant cells using polyethylene glycol to form a precipitation complex with the genetic material that is taken up by cells. (Paszkowski et al. , EMBOJ. 5:2717-22 (1984)). The desired gene may also be introduced into plant cells by electro- poration. (Fromm et al, "Expression of Genes Transferred into Monocot and Dicot Plant Cells by Electroporation," Proc. Nat'l. Acad. Sci. U.S.A. 52:5824

(1985)). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the desired genetic construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of plasmids. Electroporated plant protoplasts reform cell walls, divide, and form plant calli. Selection of the transformed plant cells expressing the desired gene can be accomplished using phenotypic markers as described above.

Another method of introducing the desired gene into plant cells is to infect the plant cells with Agrobacterium tumefaciens transformed with the desired gene. Under appropriate conditions well-known in the art, transformed plant cells are grown to form shoots, roots, and develop further into plants. The desired genetic sequences can be joined to the Ti plasmid of Agrobacterium tumefaciens. The Ti plasmid is transmitted to plant cells on infection by Agrobacterium tumefaciens and is stably integrated into the plant genome. Horsch et al, "Inheritance of Functional Foreign Genes in Plants," Science 233: 496-498 (1984); Fraley et al., Proc. Nat'l Acad. Sci. U.S.A. 80: 4803 (1983)); Feldmann, K.A. etal, Mol. Gen. Genet, 208: 1-9 (1987); Walden. R. et al, Plant J., 7: 281- 288 (1991).

Presently there are several different ways to transform plant cells with Agrobacterium: (1) co-cultivation of Agrobacterium with cultured, isolated protoplasts, or (2) transformation of cells or tissues with Agrobacterium. Method (1) requires an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts. Method (2) requires that the plant cells or tissues can be transformed by Agrobacterium and that the transformed cells or tissues can be induced to regenerate into whole plants. In the binary system, to have infection, two plasmids are needed: a T-DNA containing plasmid and a vir plasmid.

Routinely, however, one of the simplest methods of plant transformation is explant inoculation, which involves incubation of sectioned tissue with

Agrobacterium containing the appropriate transformation vector (Plant Genetic Transformation and Gene Expression, A Laboratory Manual, Oxford: Blackwell Scientific Publications (1988); Walden, Genetic Transformation in Plants, Milton Koynes: Open University Press (1988)). All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be used for the expression of the desired gene. Suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis,

Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura. Additional plant genera that may be transformed by Agrobacterium include Ipomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Santalum, Allium, Lilium, Narcissus, Ananas, Arachis, Phaseolus, and Pisum.

Plant regeneration techniques are well known in the art and include those set forth in the Handbook of Plant Cell Culture, Volumes 1-3, Eds. Evans et al. Macmillan Publishing Co., New York, NY (1983, 1984, 1984, respectively);

Predieri and Malavasi, Plant Cell, Tissue, and Organ Culture 77: 133- 142 (1989); James, D.J., et α7., J. Plant Physiol. 752:148-154 (1988); Fasolo, F., et al, Plant Cell, Tissue, andOrgan Culture 7(5:75-87 (1989); Valobra and James, Plant Cell, Tissue, andOrgan Culture 21:51 -54 (1990); Srivastava, P.S., etal. , Plant Science 42:209-214 (1985); Rowland and Ogden, Hort. Science 27:1127-1129 (1992);

Park and Son, Plant Cell, Tissue, and Organ Culture 75:95-105 (1988); Noh and Minocha, Plant Cell Reports 5:464-467 (1986); Brand and Lineberger, Plant Science 57:173-179 (1988); Boτ koγ, ?.W., etal, Plant Cell Reports 77:386-389 (1992); Kvaalen and von Arnold, Plant Cell, Tissue, andOrgan Culture 27:49-57 (1991); Tremblay and Tremblay, Plant Cell, Tissue, and Organ Culture 27:95-

103 (1991); Gupta and Pullman, U.S. Patent No. 5,036,007; Michler and Bauer, Plant Science 77: 111 - 118 (1991 ); Wetzstein, H. Y., et al. , Plant Science 64: 193- 201 (\989); McGranah , G.ll., etaL, Bio/Technology 6:800-804 (1988); Gingas, V.M., Hort. Science 26:1217-1218 (1991); Chalupa, V., Plant Cell Reports 9:398-401 ( 1990); Gingas and Lineberger, PlantCell, Tissue, and Organ Culture

77:191-203 (1989); Bureno, M.A., et al, Phys. Plant. 55:30-34 (1992); and Roberts, D.R., et al, Can. J. Bot. 65:1086-1090 (1990).

Plant regeneration from cultured protoplasts is described in Evans et al. , "Protoplast Isolation and Culture," in Handbook of Plant Cell Culture 1 : 124-176 (MacMillan Publishing Co., New York, 1983); M.R. Davey, "Recent

Developments in the Culture and Regeneration of Plant Protoplasts," Protoplasts, 1983 - Lecture Proceedings, pp. 19-29 (Birkhauser, Basel, 1983); P.J. Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," in Protoplasts 1983 - Lecture Proceedings, pp. 31 -41 (Birkhauser, Basel, 1983); and H. Binding, "Regeneration of Plants," in Plant Protoplasts, pp. 21-37 (CRC Press, Boca Raton, 1985).

Techniques for the regeneration of plants varies from species to species but generally, a suspension of transformed protoplasts containing multiple copies of the desired gene is first provided. Embryo formation can then be induced from the protoplast suspensions, to the stage of ripening and germination as natural embryos. The culture media will generally contain various amino acids and hormones, such as auxins and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Mature plants, grown from transformed plant cells, are selfed to produce an inbred plant. The inbred plant produces seed containing the recombinant DNA sequences promoting increased expression of GA4H.

Parts obtained from regenerated plants, such as flowers, seeds, leaves, branches, fruit, and the like are covered by the invention provided that these parts comprise the herbicidal tolerant cells. Progeny and variants, and mutants of the regenerated plants are also included within the scope of this invention. As used herein, variant describes phenotypic changes that are stable and heritable, including heritable variation that is sexually transmitted to progeny of plants, provided that the variant still comprises a herbicidal tolerant plant through enhanced rate of acetylation. Also, as used herein, mutant describes variation as a result of environmental conditions, such as radiation, or as a result of genetic variation in which a trait is transmitted meiotically according to well-established laws of inheritance.

Plants which contain the GA4H encoding DNA of the invention and no other foreign marker gene are advantageous in that removal of the foreign marker gene, once inserted into the plant, may be impossible without also removing the GA4H gene. Absence of the foreign marker gene is sometimes desired so as to minimize the number of foreign genes expressed. This can be achieved by providing the GA4H-encoding DNA between Ti-plasmid borders. The GA4H gene product may have similar function(s) to 3-β-hydroxylase. 3 -β-hydroxylase is critical for controlling stem growth (Ingram et al, Plant 160: 455-463 (1984). Accordingly, the GA4H of the invention may be applied to crops to enhance and facilitate such stem elongation, flowering and fruiting. Alternatively, the DNA encoding GA4H may be genetically inserted into the plant host to produce a similar effect.

All plants which can be transformed are intended to be hosts included within the scope of the invention (preferably, dicotyledonous plants). Such plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot,

Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Sencia, Salpiglossis, Cucumis, Browalia, Glycine, Lolium, Zea, Triticum, Sorghum, Malus, Apium,

Datura, the le mutant in peas, the ga4 mutant in Arabadopsis, and the dwarf-1 mutant in Monocotyledonous plants such as corn.

Examples of commercially useful agricultural plants useful in the methods of the invention as transgenic hosts containing the GA4 DNA or antisense sequence of the invention include grains, legumes, vegetables and fruits, including but not limited to soybean, wheat, corn, barley, alfalfa, cotton, rapeseed, rice, tobacco, rye, tomatoes, beans, peas, celery, grapes, cabbage, oilseed, apples, strawberries, mulberries, potatoes, cranberries and lettuce.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified. Examples

Example 1 Isolation of The GA4 Homologue Genes

The presence of a Gv44-homologue gene (GA4H) was first determined by low stringency hybridization using a probe made from the GA4 sequence. The probe was designed based on the DNA sequence of a conserved amino acid region between GA4 and similar proteins (i.e. β-hydroxylases).

Methods

"Plant and Nucleic Acid Sources and Preparation" A ga4-l (an ethyl methanesulfonate, EMS, induced mutant) mutant was obtained from M Koornneef (Agricultural University, Wageningen, The

Netherlands). Plants were grown under a 16-hr light/ 8-hr dark cycle. For genomic

DNA isolation, rossette leaves of 3-4 week old plants were harvested and frozen in liquid nitrogen. For RNA isolation, tissues from matured flowering plants of either ga4-l or Lansberg erecta were collected and immediately frozen in liquid nitrogen. pCD7 DNA containing the GA4 cDNA has been described previously (Chiang, H.H., et al, Plant Cell 7:195-201 (1995)). The cloning vectors were either pBSKS(-) or pBSKS(+) of Stratagene (La Jolla, CA, U.S.A.). DNA markers, 1 Kb and 123 bp, are from Gibco BRL (Gaithersburg, MD, U.S.A.).

Restriction and modifying enzymes were from New England Biolab (Cambridge, MA, U.S.A.).

Genomic DNA of yeast strains carrying YAC DNA was isolated according to Ausubel, F.M., et al, Current Protocols in Molecular Biology, New York: Greene Publishing Association and Wiley-Interscience (1987). Plant genomic DNA was isolated by the method of Watson, J.C., and Thompson, W.F., Methods in Enzymology 118:57-75 (1986). RNA was isolated using the Tri-Reagent (Molecular Research Center, Cincinnati, OH, U.S.A.). "Oligonucleotides and Sequence Analysis "

Oligonucleotides were synthesized by the DNA Synthesis Core Facility of the Molecular Biology/ Endocrine Departments of Massachusetts General

Hospital (MGH) (Boston, MA, U.S.A.). In the following oligonucleotides the underlined nucleotides indicate the restriction recognition site shown in parenthesis. The name and sequence of the oligonucleotides are as follows:

Homol : 5'-GTGGTTAGCACTAAATTCAC-3' (SEQ ID No. 11)

Homo2: 5'-GACCCATGGCTCGGTCCGGT-3' (SEQ ID No. 12)

GA-P 1 X: 5 ' -GCTCTAGAGAGTATTTGAGAAGG-3 ' (SEQ ID No. 13) (Xbal)

GA-P2: 5'-GTTTACTATTGCCGATGACT-3' (SEQ ID No. 14) GA-P6: 5'-CAATACCAAAAATGAAAAGC-3'(SEQ ID No. 15) GA-P13: 5'-CTCCTACCGCAACCATTTC-3' (SEQ ID No. 16)

GA-P14S: 5'-TCCCCCGGGTTTATGTGATGAGCATCCC-3'(SEQ ID No. 17)

(Smal)

GA-P15: 5'-CCAAAGTAATTGTTTATGTG-3' (SEQ ID No. 18)

GA-P 16: 5'-AATTTAGGTTTTTCATTAAG-3' (SEQ ID No. 19

GA-P 17: 5'-GTAGTGGTTTAGTCGTATGG-3' (SEQ ID No. 20) GA-P18: 5'-AAAACTTGGAGACCGGCGG-3' (SEQ ID No. 21)

GA-P19: 5'-TATCATGTAATCTTTTTGG-3' (SEQ ID No. 22)

GA-P20: 5'-CCGGCTTCCCGTACAGCGG-3' (SEQ ID No. 23)

GA-P21 : 5'-AATCAAGAAATTCAGTCGG-3' (SEQ ID No. 24)

GA-P27E: 5 ' -GGAATTC AT ACC AAA AAC ATAA AGCC-3 ' (SEQ ID No. 25) (Ec RI)

Tua4F: 5'-CTAGTTTCTTTCTTCCACG-3' (SΕQ ID No. 26) Tua4R: 5'-TAGCTGCATCTTCTTTACC-3' (SΕQ ID No. 27)

DNA sequences were determined by the DNA Sequencing Core Facility of the Department of Molecular Biology at Massachusetts General Hospital. Sequence analyses were performed using the software package of the Genetics Computer Group (GCG; Madison, WI, U.S.A.). Blast searches were conducted through the National Center for Biotechnology Information (NCBI), (Bethesda, MD, U.S.A.) using the algorithm of Altschul, S.F., et al, J. Mol. Biol. 215:403-10 (1990).

"Polymerase Chain Reaction"

PCR was performed using the Peltier Thermal Cycler (PTC-200) of MJ

Research (Watertown, MA, U.S.A.). A DNA fragment containing a conserved region on the second exon of the GA4 gene (Chiang, H.H., et al, Plant Cell 7:195-201 (1995)) was generated by PCR using Homol and Homo2 primers.

Probes prepared from this fragment (Homologous probes) were used for the genomic DNA gel blot and for screening the genomic library. The PCR reaction was carried out in 100 μl total volume and contained 0.4 ng of pCD7 DNA, 200 μM of dNTP, 15 μM of each primer, and 2.5 units of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN, U.S.A.). The PCR temperature profile was 35 cycles of 1 minute at 94°C, 1 minute at 50°C , and 3 minutes at 72°C. Preparation of the Unique probes were described earlier (Chiang, H.H., et al., Plant Cell 7:195-201 (1995)).

In the mapping study, 1 μg of each YAC DNA was used as templates for PCR amplification of the two homologous genes. The GA4H1 gene was amplified using GA-P2 and GA-P6 primers. The GA4H2 gene was amplified using GA-P 19 and GA-P20 primers. Each PCR reaction was carried out in 25 μl total volume and contained 80 μM of dNTPs, 10 μM of each primer, and 2 units of Taq DNA polymerase (Boehringer Mannheim). The PCR was performed using 35 cycles of 40 seconds at 92°C, 40 seconds at 55°C, and 40 seconds at 72°C. One fifth of the PCR product was separated on 0.8%> agarose gel.

"RT-PCR Conditions"

First strand cDNA synthesis was performed according to Sambrook, J., et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor: Cold Spring Harbor Laboratory (1989). The reaction volume was 25 μl and it contained 1 μg of total RNA, 9 μM of (dT)₂₀, 1.2 mM dNTP, 136 units of RNASE inhibitor (Amersham, Arlington Heights, IL, U.S.A.), and 9.5 units of avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI,

U.S.A.). The reaction was incubated at 42°C for one hour and then at 72°C for 15 minutes. Eight microliters of the first strand cDNA was used as templates in the PCR amplification. The reaction was in 50 μl and used 63 μM of dNTP, 0.6 μM of homologous gene specific primer, 0.4 μM of tubulin primer, and 2.5 units of Taq DNA polymerase (Boerhinger Mannheim). The thermal profile was 40 cycles of 45 seconds at 94°C, 45 seconds at 55°C, and 45 seconds at 72°C. When amplifying the full length cDNA, tubulin primers were not included and the extension time of 45 seconds at 72°C was increased to 1.5 minutes. One-tenth of the PCR product was analyzed on an agarose gel.

"Genomic Library Screening"

An Arabidopsis genomic library made from ecotype C24 was kindly provided by Dr. Lin Sun (Nemapharm, Cambridge, MA, U.S.A.). This library was constructed using the Sau3A partial digested genomic DNA and subsequently cloned into theXhol site the λFIX-II vector (Stratagene). Screening of the library was performed according to the manufacturer's protocol

(Stratagene). Plaques were transferred and crosslinked to Biotrans nylon membrane by autoclaving for 2 minutes. Homologous probes was prepared and the hybridization conditions were as described in Chiang, H.H., et al, Plant Cell 7:195-201 (1995), except that Homol and Homo2 primers were used and filters were hybridized at 42°C (low stringency). Filters were washed once in 2X SSC

( 1 X= 0.15 M NaCl, 0.015 M sodium citrate) for 15 minutes at room temperature and twice in 0.1X SSC, 0.1% SDS for 30 minutes at 42°C (low stringency). "DNA Gel Blot Analysis"

In the genomic Southern, Arabidopsis (ecotype Lansberg erecta) genomic DNA was digested with appropriate restriction enzymes, separated by agarose gel electrophoresis, and transferred to Biotrans membrane (ICN Biomedical Inc., Aurora, OH USA) as described in Chiang, H.H., et al, Plant Cell 7:195-201

(1995). For the homologous and unique GA4 gene probes, the hybridization and washing conditions were the same as the library screening above (low stringency). The DNA gel blot analysis using the GA4H1 gene, p3-l , probes was performed as described in Chiang, H.H., et al, Plant Cell 7: 195-201 (1995). The hybridization and washing conditions were performed at 65°C (high stringency).

DNA blot analyses for the mapping and RT-PCR products were performed as described (Cheng, C.L., et al, Proc. Natl. Acad.. Sci. U.S.A.

59: 1861 -4 ( 1992)). In the mapping of homologous genes by PCR, probes specific to these genes were generated by PCR. Probes were prepared using a 4.4 kbp BglU/ Xhol genomic DNA fragment, containing these two genes, as templates with four primers (GA-P2, GA-P6, GA-P 19, and GA-P20). The reaction was in 50 μl, and it contained 5 ng of DNA template, 100 μM each of dCTP, dGTP, and dTTP, 5 μM dATP, 50 μCuries of α-³²P dATP (Dupont NEN, Wilmington, DE, U.S.A.) 0.4 μM each primer, and 2.5 units of Taq DNA polymerase (Boerhinger Mannheim). The thermal profile was 30 cycles of 40 seconds at 94°C, 30 seconds at 55°C, and 30 seconds at 72°C.

In the RT-PCR DNA gel blot, the same PCR method as above was used to prepare the GA4H1 and GA4H2 specific probes, except that different primers were employed. Primers pairs of GA-P 13/ GA-P 17 and GA-P 18/ GA-P20 were used to prepare GA4H1 and GA4H2 gene probes, respectively.

Results

To isolate the DNA sequences with similar sequence to the GA4 gene

(ATCC accession nos. 98393 and 98394), low stringency hybridization (see Materials and Methods) to Arabidopsis genomic DNA was performed with Homologous probes (SEQ ID No. 2) prepared from a conserved region the GA4 gene (Figure 1), compared to GA5 and other β-hydroxylases. Results from the blot of this genomic DNA, isolated from ecotype Lansberg erecta, are shown in Figure 2A. Beside a strong 3.2 kbp size band in the Hindlll digested DNA, a less intense 2.1 kbp band is visible and assumed to contain DNA similar to the GA4 gene (Figure 2A, lane 1). Similarly, there is a light 2.8 kbp band in the BamHI digested DNA (Figure 2 A, lane 2).

To identify the GA4 gene, a similar blot was hybridized at low stringency to a Unique probe (Figure 1 - SEQ ID No. 3) derived from a less conserved region of the GA4 gene. This probe would hybridize specifically to the GA4 gene, and results are shown in Figure 2B.

In the Hindlll digestion, the GA4 specific probes hybridized strongly to the 3.2 kbp size band, and no detectable signal was found at the 2.1 kbp size (Figure 2B, lane 1). Similarly, the 2.8 kbp band in the BamHI digested DNA was not visible, indicating that the 2.1 kbp Hindlll and the 2.8 kbp BamHI fragments contain a homologous sequence to the GA4 DNA (Figure 2B, lane 2). DNA digested with the EcoRI enzyme resulted in only high molecular weight bands being visible when either Homologous or Unique probes were used (Figure 2A and 2B, lane 3).

The homologous probes were also used to screen a genomic library (ecotype C24) at low stringency conditions as described above. In addition to the GA4 genomic clones, one other genomic clone (λ3) that contained the 2.1 kbp Hindlll fragment was isolated. This 2.1 kbp fragment of λ3 was subcloned into pBSKS(-) to produce p3-l (Figure 3). The whole genomic insert in λ3 was also cloned into pBSKS(+) using the NotI sites flanking the insert to generate pLVΝ103. To confirm this genomic clone, the p3-l DNA was used as a probe and hybridized at high stringency to the same genomic blot above. As shown in Figure 2C, both the 2.1 kbp Hindlll and 2.8 kbp BamHI fragments are present (lane 1 and 2). The predicted high molecular weight fragment in EcoRI digested DNA is also present (lane 3). These results indicated that the predicted homologue of the GA4 gene had been isolated.

Clones p3-l and part of pLVN103 DNAs were sequenced, and the homologue gene was named G A4H 1. Further sequencing in the 5 ' flanking of the GA4H1 gene revealed a second gene, named GA4H2, that also has sequence similarity to the GA4 as well as to the GA4H1 genes. The genome organization of these two linked genes is represented in Figure 3. When compared to the GA4 gene, both the GA4H1 and GA4H2 genes also possess a single intron that is located at a similar position in the gene. Transcription of both genes is in the same direction, and they are separated by a 1 kbp spacer region (Figure 3).

The plasmid designated pLVN103 comprising the genomic sequence of both the GA4H 1 and GA4H2 genes was deposited at the ATCC (Rockville, MD.) under the terms of the Budapest Treaty and has been granted accession number 98436.

Example 2

Chromosomal Location of the Homologue Genes

It was determined that both homologue genes are located on chromosome

1. Since many continuous overlapping DNA clones of Yeast Artificial Chromosomes (YAC) containing Arabidopsis genomic DNA had been placed on the five linkage groups, the GA4H1 and GA4H2 genes can be mapped by anchoring them to YACs of known position.

Probes derived from the genomic clone p3-l were hybridized to the CIC YAC library (Creusot, F., et al, Plant Journal 5:763-70 (1995)), and three YAC clones (CIC1E4, CIC6C10 and CIC10A11) were isolated (data not shown).The intensity of the hybridization was higher in CIC1E4 and CIC6C10 than in CIC 10A 11 (data not shown). These putative YACs were subsequently confirmed by PCR amplification using primers specific to these two genes.

Two specific primer sets (GA-P2/GA-P6 and GA-P19/GA-P20 for GA4H1 and GA4H2 genes, respectively) were used to amplify a short region in these genes. The predicted amplified products for GA4H1 and GA4H2 genes are 480 bp and 410 bp, respectively. The analysis of PCR products is shown in Figure 4A. For the GA4H2 gene, the predicted PCR product of 410 bp was present in both the control pLVN103 DNA (lane 2) and in two of the three putative YACs, CIC1E4 (lane 4) and CIC6C10 (lane 5). However, CIC10A11

YAC did not appear to carry the GA4H2 gene, since the 410 bp size band was not present (lane 6).

The CIC6C3 YAC, located on the bottom of chromosome 2, was used as a negative control. As expected, no PCR product was present in CIC6C3, indicating the specificity of these primers (lane 1). Similar results were also obtained for the GA4H1 gene where the predicted PCR product is 480 bp in size. The 480 bp size band was present in the pLVN103 control (lane 8) as well as in CIC1E4 and 6C10 (lanes 10 and 11). Again, the 480 bp size band was absent in CIC10A11. These results were further confirmed by the DNA gel blot. Probes, generated using the same 4 primers with the genomic clone

(pLVNl 03), were hybridized to the DNA blot, and the results are shown in Figure 4B. All predicted PCR products of 410 bp and 480 bp in size (for GA4H2 and GA4H1 genes, respectively) were hybridized to the probes. Since both CIC1E4 and CIC6C10 were previously anchored to the bottom of chromosome 1, it was concluded that concluded that GA4H1 and GA4H2 genes are located at about

159-cM (on the physical map) of chromosome 1 (http://cbil.humgen.upenn.edu/~atgc/ATGCUP.html; http://cbil.humgen.upenn.edu/~atgc/physical-mapping/xlchl_pt4.html). CIC10A11 has overlapping regions to those two YACs above, and it hybridized weakly to probes prepared from p3-l . However, no PCR product was amplified when CIC10A11 was used as a template DNA. These results suggest that the edge of CIC10A11 DNA may end shortly after the Hindlll site, located in the 3' flanking of the GA4H1 gene (see Figure 3). Example 3 Cloning ofGA4Hl and GA4H2 cDNAs By RT-PCR

To determine whether the GA4H1 gene is expressed, probes derived from the clone p3-l containing most of the GA4H1 coding region were used to hybridize to RNA isolated from flowers, shoot meristems, leaves, roots and siliques. However, no visible signal was present in the RNA blot (data not shown). Another attempt to isolate the cDNA by screening a yeast expression library (Minet, M., et al, Plant Journal 2:417-422 (1992)) using probes derived from p3-l also failed. Furthermore, searching the Arabidopsis EST database using the GA4H1 sequence no match was found to any known EST, indicating that the GA4H1 gene may be expressed at very low levels or only in a specific developmental stage of the plant. Therefore, isolation of the GA4H1 cDNA by reverse-transcriptase PCR (RT-PCR) was undertaken.

The ga4 mutant was used as a source of RNA since the expression of the GA4 gene is under feedback regulation resulting in the induction of its mRNA

(Chiang, H.H., et al, Plant Cell 7:195-201 (1995)). If the expression of the GA4H1 gene is regulated by the same or a similar mechanism, i.e. a higher level of GA4H1 mRNA in the ga4 mutant than wild type, then one has a better chance of obtaining the cDNA in the ga4 mutant background. RT-PCR was performed using RNAs isolated from whole seedlings of ga4-l (EMS) and ga4-2 (T-DNA) mutants grown in liquid and from leaves and inflorescences of soil grown ga4-l plants. Inflorescences contain the shoot meristems, flowers and siliques. A predicted PCR product was observed only in RNA isolated from inflorescence tissues (data not shown). Therefore, inflorescences were used as a source of RNA for cloning the GA4H1 cDNA.

Primers GA-P 15 and GA-P 16 were used in PCR following the reverse transcription. A nested PCR using GA-P IX and GA-P14S primers was performed, and the product was subsequently cloned into pBSKS(+) at the Smal and Xbal sites. Since Taq DNA polymerase, a low fidelity enzyme, was used in the PCR amplification, three independent RT-PCR clones (pLVN107a, b, c) were sequenced. The consensus sequence of this cDNA clone, labeled as pLVN107, is shown in Figure 5 (SEQ ID No. 5). The cDNA contains 43 and 22 nucleotides in the 5' and 3' untranslated regions of the gene, respectively. Four of the nine nucleotides in the sequence surrounding the predicted start codon (ATG) are identical to the consensus sequence (Joshi, C. P., Nucleic Acids Res. 75:6643-53 (1987)). The intron occurs at a similar position relative to the GA4 gene. The GA4H1 genomic DNA sequence (SEQ ID No. 6), along with its deduced amino acid sequence (SEQ ID

No. 7), are shown in Figure 6. The gene possesses a single 409 bp intron, and it follows the intron's GT/AG consensus rule. This gene encodes a protein of 355 amino acids long.

Comparison between the RT-PCR sequence (pLVN 107) and the genomic sequence (pLVNl 03) revealed one nucleotide mismatch at the position no. 1059 of the cDNA sequence (Figure 5) (SEQ ID No. 5). The cDNA has a "G" at this position while the genomic DNA has an "A". This mismatch may arise from differences in the Lansberg erecta (L. er.) and C24 ecotypes from which cDNA and genomic sequences were derived, respectively. To resolve this, the genomic DNA of the L. er. ecotype was cloned by

PCR amplification with a high fidelity enzyme, Pfu (Stratagene), using GA-P IX and GA-P14S primers. The sequence of this clone, pLVNl 10, is identical to the genomic clone in C24 ecotype, pLVN103 (data not shown). Therefore, the mismatch at this position could not be resolved by current data. Similar RT-PCR conditions were used to isolate the GA4H2 cDNA except that GA-P27E and GA-P21 primers (SEQ ID Nos. 11 and 14 respectively) were used, and the RNA source was of Lansberg erecta. One cDNA was cloned, pLVNl 15, and its sequence (SEQ ID No. 8) is shown in Figure 7.

Similar to GA4 and GA4H1 gene, there is a single intron present at a conserved position in the gene. The sequence surrounding the predicted ATG show 3/9 matches against the consensus sequence. The genomic sequence of the gene is shown in Figure 8 (SEQ ID No. 9). Sequence comparison between this cDNA and its genomic DNA shows a perfect match. The GA4H2 gene encodes a protein of 347 amino acids long (SEQ ID No. 10).

Example 4

Sequence Analysis of the GA4H1 and GA4H2 Proteins

Each of the protein sequences (SEQ ID Nos. 7 and 10) of the GA4H1 and GA4H2 genes was searched against the protein Genbank database, and each time the GA4 protein was found to be the best match. This is not surprising, since these genes were isolated by hybridization to probes prepared from the GA4

DNA sequence.

The predicted proteins encoded by the GA4, GA4H1 , and GA4H2 genes were compared and the results of this comparison are shown in Figure 9. As expected, many conserved regions are present throughout these proteins. However, GA4H2 protein has higher homology to GA4 than does GA4H1.

Amino acid sequence identity was calculated among these proteins using GAP software of the GCG package and the results are shown in Figure 10. GA4H2 and GA4 share 76%> and 85%> amino acid identity and similarity, respectively. Compared to this, GA4H1 and GA4 only share 57% and 73% amino acid identity and similarity, respectively. Results of comparison between GA4H 1 and GA4H2 are similar to those between GA4H1 and GA4.

Several enzymes, including GA4 (β-hydroxylase), in the gibberellin biosynthesis pathway belong to a group of non-heme iron-containing enzymes called 2-oxoacid-dependent dioxygenases (2-ODD). A binary comparison between these proteins and the three proteins described above is shown in Figure

10. Proteins of different functions often share around 30% amino acid identity, while those from a multigene family in the same species show greater than 50% amino acid identity (Prescott, A.G., and John, R.,Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:245-271 (1996)). Results in Figure 10 appear to support this observation with the exception of the GA4, GA4H1 and GA4H2. These three proteins share greater than 50%) amino acid identity, which indicates that they belong to the same family and/ or may have similar enzyme activities.

Example 5 Differential Expression ofGA4Hl and GA4H2 Genes

Since the expression of the GA4 gene is primarily in the silique, the expression levels of ^'GA4H1 and GA4H2 genes in various organs was investigated to determine whether a similar expression pattern occurred. RT-PCR using Arabidopsis (Lansberg er) RNAs isolated from liquid grown roots, soil grown rosette leaves, floral shoots (including flowers), and siliques was performed.

GA-P 13/ GA-P 17 and GA-P 18/ GA-P20 primer pairs were used to amplify the GA4H1 and GA4H2 genes, respectively. Primers in each pair, located on separated exons were used to differentiate between cDNA and genomic DNA. The predicted RT-PCR products of GA4H1 and GA4H2 genes are 220 bp and 440 bp, respectively. The predicted PCR products of GA4H1 and GA4H2 genomic DNAs (containing the intron sequence) are 630 bp and 860 bp, respectively.

Primers from the α-tubulin 4 gene, TUA4 (Kopczak, S.D., et al, Plant Cell 4:539-47 (1992)), were used as an internal control along with GA4 homologue gene specific primers. The α-tubulin primers generates a 320 bp

RT-PCR product. Results of RT-PCR analysis are shown in Figure 11A.

To confirm the PCR products, a DNA gel blot analysis was performed using probes derived from the GA4H1 gene (Figure 1 IB). The GA4H1 gene was mainly expressed in the flowers and shoot meristems, with smaller amounts in the siliques (Figures 11 A and 1 IB, lanes 5 and 8). In addition, GA4H1 gene was barely detected in the root tissues (Figures 11 A and 1 IB, lane 7). However, there was no detectable level of GA4H1 gene in the rosette leaves (Figures 11A and 1 IB, lane 6). Similar to the polymerase chain reaction control, the 630 bp product was present in pLVN103 containing the genomic clone (lane 3). There was a small amount of genomic DNA present in the RNA preparation, as indicated by the presence of the 630 bp size band in all tissue types. pCD7 (GA4 cDNA clone) and pLVN 1 15 (GA4H2 cDNA clone) were also used as templates to demonstrate the specificity of GA-P 13 and GA-P 17 primer pair.

Although some unspecific PCR products (Figure 11 A, lanes 1 and 2) were present, these primers amplified neither the GA4 nor the GA4H2 gene (Figure 1 1 B, lanes 1 and 2). The internal RT-PCR control (α-tubulin 4 gene) was present evenly in different tissue types with the exception of siliques (Figure 11 A, lane

8). This may indicate that less silique RNA was used in this experiment, suggesting that the expression level of these genes in siliques was underestimated.

A similar experiment was performed on the GA4H2 gene where GA-P 18 and GA-P20 primers were used in the amplification. Again, there was less silique RNA used, as indicated in Figure 12A (lanes 1-4). Unlike the GA4H1 gene,

GA4H2 transcripts were more abundant in the root tissues, while lower levels were present in the flowers and shoot meristems (Figure 1 1 A and 1 IB, lanes 2 and 4). In addition, GA4H2 expression is barely detected in siliques but not in leaves (Figure 11 A and 1 IB, lane 1 and 3). Again, the expression level of GA4H2 gene in siliques was underestimated when compared to other tissues. A genomic

DNA clone (pLVN103) was used as the control, and it possess the predicted 860 bp size band (Figure 11A and 1 IB, lane 6). Similar to the GA4H1 RT-PCR result, primers used in this experiment were specific to the GA4H2 gene (Figure 11 A and 1 IB, lanes 7 and 8).

Example 6

Expression of Antisense GA4H RNA

An expression vector is constructed using methods well known in the art, such that it expresses an RNA complementary to the sense strand GA4H RNA. The antisense GA4H RNA is expressed in a constitutive fashion using promoters that are constitutively expressed in a given host plant, for example, the cauliflower mosaic virus 35S promoter. Alternatively, the antisense RNA is expressed in a tissue specific fashion using tissue specific promoters. As described earlier, such promoters are well known in the art. In one example, the antisense construct pPO35 (Oeller et al, Science

254:437-439 (1991)) is cut with BamHI and Sacl to remove the tACC2 cDNA sequence. After removing the tACC2 cDNA, the vector is treated with the Klenow fragment of E. coli DNA polymerase I to fill in the ends, and the sequence described in Figure 6 or 8 is blunt end ligated into the vector such that the strand operably linked to the promoter is that which transcribes the GA1 antisense RNA sequence. The ligated vector is used to transform an appropriate E coli strain.

Colonies containing the ligated vector are screened using colony hybridization or Southern blotting to obtain vectors which contain the GA4H cDNA in the orientation which will produce antisense RNA when transcribed from the 35S promoter contained in the vector.

The antisense GA4H vector is isolated from a colony identified as having the proper orientation and the DNA is introduced into plant cells by one of the techniques described earlier, for example, electroporation or AgrobacteriumlT plasmid mediated transformation.

Plants regenerated from the transformed cells express antisense GA4H RNA. The expressed antisense GA4H RNA binds to sense strand GA4H RNA and thus prevents translation.

In an initial experiment the phenotypes of transgenic plants epressing the antisense of the GA4H1 gene were examines. Constructs carrying the sense and antisense of the GA4H1 cDNA, under transcriptional control of the cauliflower mosaic virus 35S promoter, were transferred into Arabidopsis thalian ecotype Lansberg erecta via Agrobacterium ediated transformation (Bechtold et al. , Acad. Sci. Paris 576:1194-1199 (1993)). These constructs contained a neomycin phosphotranf erase (NPT-II) gene whos proudct confers resistance to kanamycin. Transgenic seed were harvested and subsequently germinated on MS medium supplemented with 50 mg/L kanamycin. Resistant seedlings (TI generation) were transplanted to soil and the height was measured on mature plants. Untransformed plants, Lansberg erecta ecotype, were grown similarly but in the absence of kanamycin.

Results of transgenic plants carrying the sense or antisense cDNA of the GA4H1 gene are shown in Figure 13. Overexpression of the GA4H1 cDNA in the sense orientation does not seem to alter the plant's height. However, several plants carrying the antisense of the GA4H1 cDNA exhibit dwarf phenotype. These preliminary results require further validation, especially in the subsequent generation. These results suggest that one can use the GA4H1 gene inthe antisense orientation to generate dwarf plants.

EXAMPLE 7 GA4H Protein Level in Wild-Type and Transgenic Lines

Agrobacterium tumefaciens-mediated transformation of Arabidopsis root explants.

The transformation procedure is described previously (Valvekens et al.,

1988) with slight modifications (Sun et al, Plant Cell 4: 119-128 (1992)). Sense or anti-sense DNA is introduced into Agrobacterium LBA4404 by electroporation

(Ausubel et al., Current Protocols in Molecular Biology (New York: Green Publishing Associates/Wiley-Interscience) (1990). Stability of the insert of the plasmid in LBA4404 is tested by restriction digestion and gel electrophoresis of plasmid DNA purified by NaOH/SDS minipreparation procedure (Ausubel et al, Current Protocols in Molecular Biology (New York: Green Publishing

Associates/Wiley-Interscience) (1990).

A fresh overnight culture of LBA4404 carrying individual plasmids is used to infect root explants of four- week-old wild-type plants. Km^r transgenic plants are regenerated as described (Valvekens et al. , Proc. Natl. Acad.. Sci. USA 55:5536-5540 (1988)). Seeds of transgenic plants are germinated on MS agar plates containing kanamycin (50 μg/ml). Non-germinating seeds after 8 days were transferred onto MS plates containing 100 μM GA₃and 50 μg/ml kanamycin to score for GA7Km^r and GA^"/Km^s segregation.

The levels of GA4H proteins in both sense and antisense transgenic Arabidopsis plants are compared to the level in wild-type plants (ecotype

Landsberg erecta) by immunoblot analysis. Supernatant fractions, are obtained by tissue extraction and centrifugation (Bensen and Zeevaart, J. Plant Growth

Regul 9:237-242 (1990)).

The expression of a gene in a plant is directed such that the gene has the same temporal and spatial expression pattern of GA4H. The gene is operably linked to the regulatory sequences of GA4H DNA to create an expression module, and a plant is then transformed with the expression module. One can examine the pattern of expression of the endogenous GA4H gene using a promoter-glucuronidase (GUS) gene fusion. The data from this analysis is used to design plant organ-specific promoters and cDNA gene fusions in order to manipulate the GA biosynthesis in specific plant organs.

Immunoblot Analyses

Proteins from 2-week-old Arabidopsis seedlings are extracted and fractionated by centrifugation at 10,000 g for 10 min and then at 100,000 g for 90 min at 4°C (Bensen and Zeevaart, J. Plant Growth Regul. 9:237-242, 1990). The

100,000 g supernatant fractions (50 mg each) are loaded on an 8%> SDS-PAGE gel, electrophoresed and transferred to a GeneScreen membrane (Du Pont-New England Nuclear). Immunoblot analysis is carried out as described (Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1989). The membrane is incubated with a GA4H antisera

(primary antibody), then with 2500-fold diluted peroxidase-conjugated goat anti-rabbit antisera (secondary antibody, Sigma), and detected using the enhanced chemiluminescence reagent (ECL, Amersham) followed by autoradiography. EXAMPLE 8

Over-Expression of GA4H Proteins in E. coli and the Procedure for Generating GA4H Antibodies

Methods for heterologous expression of DNA clones in E. coli are known in the art (Chiang et al, Plant Cell 7: 195-201 (1995), Phillips et al, 705:1049-

1057 (1995), Wu et al, Plant Physiol. 770:547-554 (1996), Yamaguchi et al, Plant J. 70:203-213 (1996)). Plasmids containing DNA encoding a GA4H protein are transformed into DE3 lysogenic E. coli strain BL21(DE3) (Studier et al, Methods Enzymol. 755:60-89 (1990). The expression of the GA4H cDNA is induced by the addition of 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at absorbance (600 nm)=0.8 with 2 hour incubation at 37°C. Thirty ml of cell cultures are harvested by centrifugation, washed and resuspended in 10 ml of 50 mM Tris (pH 8.0), 2 mM EDTA. The cells are sonicated on ice with a Branson microtip at a setting of 4, with four 20-sec pulses. The sonicate is mixed with 1 % Triton X-100, incubated on ice for 5 min and then centrifuged at 12000 g for 10 min at 4°C to isolate inclusion bodies (Marston, DNA Cloning: A Practical Approach, Oxford England: IRL Press, 1987, with slight modification).

Alternatively, full-length cDNA clones may be expressed as fusion proteins similar to Phillips et al. (Plant Physiol. 108:1049-1057, 1995) by using for example, an Invitrogen (San Diego, CA) Xpress Kit.

The GA4H proteins are purified from the inclusion body fraction of E. coli extracts by SDS-polyacrylamide gel electrophoresis, and electroelution with the Electro-separation system (Schleicher & Schuell). Other methods routinely used by those of skill in the art protein purification can also be used. The purified proteins are detected as single bands on SDS-polyacrylamide gels by Coomassie Blue staining. Rabbit antibodies to GA4H proteins are obtained by subcutaneous injection of gel-purified proteins in complete Freund's adjuvant (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1988). For N-group analysis, proteins are fractionated by SDS-polyacrylamide gel electrophoresis and then transferred to Immobilon membrane (Millipore) in Tris-Glycine and 10% methanol. The membrane is first stained with Ponceau S, destained in deionized water and the appropriate protein bands excised for N-group analysis. The antibodies obtained should be useful for identifying cells or tissues expressing GA4H. A method to accomplish this objective comprises the steps of: a) incubating said cells or said tissues with an agent capable of binding to t h e GA4H protein or the RNA encoding GA4H; and b) detecting the presence of the bound agent.

Example 9

Modulating the Translation of RNA Encoding GA4H Protein

The translation of RNA encoding GA4H protein in a plant is modulated by generating an expression vector encoding antisense GA4 HRNA. The plant is then transfected with the expression vector encoding the antisense GA4H RNA.

Example 10

Cloning DNA Encoding GA4H Protein

A DNA molecule encoding the GA4H protein is cloned by hybridizing a desired DNA molecule to the sequences or antisense sequences of for example, DNA SEQ ID No. 5 or DNA SEQ ID No.6 under stringent hybridization conditions. Those DNA molecules hybridizing to the probe sequences are selected and transformed into a host cell. The transformants that express GA4H are selected and cloned.

One possible set of hybridization conditions for the cloning of the DNA encoding GA4H protein is as follows: 1) prehybridizing for 1 hour;

2) hybridizing overnight at 65 °C in the hybridization buffer; and

3) washing once for 15 minutes in 2xSSC at room temperature, then two times for 30 minutes in O.lxSSC and 0.1% SDS at 60°C. Example 11 Stimulating Plant Stem Elongation

Plant stem elongation is stimulated by inserting a DNA construct encoding the amino acid sequence of a GA4H protein into a transgenic plant. The transgenic plant is produced by any of several methods known in the art including those previously described in this specification.

The stem elongation may be stimulated in Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Major ana,

Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Sencia, Salpiglossis, Cucumis, Brow alia, Gly cine, Lolium, Zea, Triticum, Sorghum, Malus, Apium, and Datura.

Example 12

Producing Dwarf Plants

Dwarf plants are produced by blocking the GA4H gene by homologous recombination, or by transforming with a GA4H anti-sense DNA in order to produce transgenic plants. A cDNA sequence can be used to construct the antisense construct which is then transformed into a plant by using an

Agrobacterium vector (Zhang et al, Plant Cell 4: 1575-1588 (Dec. 1992)). Even partial antisense sequences can be used as antisense and can interfere with the cognate endogenous genes (van der Krol et al, Plant Mol. Biol. 14: 457-466 (1990)). The plant is transformed with the antisense construct according to the protocol of Valvekens et al, Proc. Natl. Acad, Sci, USA 55:5536-5540 (1988).

Dwarf plants are known to be commercially valuable. For example, dwarf trees for apples, cherries, peaches, pears and nectarines are commercially available (Burpee Gardens Catalogue 1994, pages 122-123). Example 13 Molecular Weight Markers

The GA4H1 and GA4H2 proteins produced recombinantly are purified by routine methods in the art (Current Protocol in Molecular Biology, Vol. 2, Chap. 10, John Wiley & Sons, Publishers (1994)). Because the deduced amino acid sequence is known, the molecular weight of these proteins can be precisely determined, and the proteins can be used as molecular weight markers for gel electrophoresis. The calculated molecular weightsof the GA4H1 and GA4H2 proteins based on the deduced amino acid sequences are 39086 daltons and 38740 daltons respectively.

Conclusions

A genomic clone, comprising the sequences encoding the GA4H1 and GA4H2 proteins was obtained. The GA4H1 and GA4H2 proteins are homologues of the GA4 protein. It is believed that the GA4 locus encodes an hydroxylase involved in gibberellin biosynthesis.

All references mentioned herein are fully incorporated by reference into the disclosure.

Having now fully described the invention by way of illustration and example for purposes of clarity and understanding, it will be apparent to those of ordinary skill in the art that certain changes and modifications may be made in the disclosed embodiments, and such modifications are intended to be within the scope of the present invention. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(l) APPLICANT: THE GENERAL HOSPITAL CORPORATION FRUIT STREET BOSTON, MA 02114 UNITED STATES OF AMERICA

APPLICANT/INVENTOR: GOODMAN, HOWARD M.

NGUYEN, LONG V. CHIANG, HUI-HWA

(n) TITLE OF INVENTION: GA4 HOMOLOGUE DNA, PROTEIN AND METHODS OF USE

(m) NUMBER OF SEQUENCES: 29

(IV) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.

(B) STREET: 1100 NEW YORK AVE . , SUITE 600

(C) CITY: WASHINGTON

(D) STATE: DC

(E) COUNTRY: USA

(F) ZIP: 20005

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy d sk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: To be assigned

(B) FILING DATE: Herewith

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/050,615

(B) FILING DATE: 24-JUN-1997

(vm) ATTORNEY/AGENT INFORMATION:

(A) NAME: CIMBALA, MICHELE A.

(B) REGISTRATION NUMBER: 33,851

(C) REFERENCE/ DOCKET NUMBER: 0609. 39PC01/MAC/LBB

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202)371-2600

(B) TELEFAX: (202)371-2540

(2) INFORMATION FOR SEQ ID NO : 1 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1228 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA ( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 67..1140

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATAAGAAAAA AAACACAAAC ATCTATCAAA TTTACAAAGT TTTAAAACTA ATTAAAAAAG 60

AGCAAG ATG CCT GCT ATG TTA ACA GAT GTG TTT AGA GGC CAT CCC ATT 108 Met Pro Ala Met Leu Thr Asp Val Phe Arg Gly His Pro He 1 5 10

CAC CTC CCA CAC TCT CAC ATA CCT GAC TTC ACA TCT CTC CGG GAG CTC 156 His Leu Pro His Ser His He Pro Asp Phe Thr Ser Leu Arg Glu Leu 15 20 25 30

CCG GAT TCT TAC AAG TGG ACC CCT AAA GAC GAT CTC CTC TTC TCC GCT 204 Pro Asp Ser Tyr Lys Trp Thr Pro Lys Asp Asp Leu Leu Phe Ser Ala 35 40 45

GCT CCT TCT CCT CCG GCC ACC GGT GAA AAC ATC CCT CTC ATC GAC CTC 252 Ala Pro Ser Pro Pro Ala Thr Gly Glu Asn He Pro Leu He Asp Leu 50 55 60

GAC CAC CCG GAC GCG ACT AAC CAA ATC GGT CAT GCA TGT AGA ACT TGG 300 Asp His Pro Asp Ala Thr Asn Gin He Gly His Ala Cys Arg Thr Trp 65 70 75

GGT GCC TTC CAA ATC TCA AAC CAC GGC GTG CCT TTG GGA CTT CTC CAA 348 Gly Ala Phe Gin He Ser Asn His Gly Val Pro Leu Gly Leu Leu Gin 80 85 90

GAC ATT GAG TTT CTC ACC GGT AGT CTC TTC GGG CTA CCT GTC CAA CGC 396 Asp He Glu Phe Leu Thr Gly Ser Leu Phe Gly Leu Pro Val Gin Arg 95 100 105 110

AAG CTT AAG TCT GCT CGG TCG GAG ACA GGT GTG TCC GGC TAC GGC GTC 444 Lys Leu Lys Ser Ala Arg Ser Glu Thr Gly Val Ser Gly Tyr Gly Val 115 120 125

GCT CGT ATC GCA TCT TTC TTC AAT AAG CAA ATG TGG TCC GAA GGT TTC 492 Ala Arg He Ala Ser Phe Phe Asn Lys Gin Met Trp Ser Glu Gly Phe 130 135 140

ACC ATC ACT GGC TCG CCT CTC AAC GAT TTC CGT AAA CTT TGG CCC CAA 540 Thr He Thr Gly Ser Pro Leu Asn Asp Phe Arg Lys Leu Trp Pro Gin 145 150 155

CAT CAC CTC AAC TAC TGC GAT ATC GTT GAA GAG TAC GAG GAA CAT ATG 588 His His Leu Asn Tyr Cys Asp He Val Glu Glu Tyr Glu Glu His Met 160 165 170

AAA AAG TTG GCA TCG AAA TTG ATG TGG TTA GCA CTA AAT TCA CTT GGG 636 Lys Lys Leu Ala Ser Lys Leu Met Trp Leu Ala Leu Asn Ser Leu Gly 175 180 185 190

GTC AGC GAA GAA GAC ATT GAA TGG GCC AGT CTC AGT TCA GAT TTA AAC 684 Val Ser Glu Glu Asp He Glu Trp Ala Ser Leu Ser Ser Asp Leu Asn 195 200 205

TGG GCC CAA GCT GCT CTC CAG CTA AAT CAC TAC CCG GTT TGT CCT GAA 732 Trp Ala Gin Ala Ala Leu Gin Leu Asn His Tyr Pro Val Cys Pro Glu 210 215 220

CCG GAC CGA GCC ATG GGT CTA GCA GCT CAT ACC GAC TCC ACC CTC CTA 780 Pro Asp Arg Ala Met Gly Leu Ala Ala His Thr Asp Ser Thr Leu Leu 225 230 235

ACC ATT CTG TAC CAG AAC AAT ACC GCC GGT CTA CAA GTA TTT CGC GAT 828 Thr He Leu Tyr Gin Asn Asn Thr Ala Gly Leu Gin Val Phe Arg Asp 240 245 250

GAT CTT GGT TGG GTC ACC GTG CCA CCG TTT CCT GGC TCG CTC GTG GTT 876 Asp Leu Gly Trp Val Thr Val Pro Pro Phe Pro Gly Ser Leu Val Val 255 260 265 270

AAC GTT GGT GAC CTC TTC CAC ATC CTA TCC AAT GGA TTG TTT AAA AGC 924 Asn Val Gly Asp Leu Phe His He Leu Ser Asn Gly Leu Phe Lys Ser 275 280 285

GTG TTG CAC CGC GCT CGG GTT AAC CAA ACC AGA GCC CGG TTA TCT GTA 972 Val Leu His Arg Ala Arg Val Asn Gin Thr Arg Ala Arg Leu Ser Val 290 295 300

GCA TTC CTT TGG GGT CCG CAA TCT GAT ATC AAG ATA TCA CCT GTA CCG 1020 Ala Phe Leu Trp Gly Pro Gin Ser Asp He Lys He Ser Pro Val Pro 305 310 315

AAG CTG GTT AGT CCC GTT GAA TCG CCT CTA TAC CAA TCG GTG ACA TGG 1068 Lys Leu Val Ser Pro Val Glu Ser Pro Leu Tyr Gin Ser Val Thr Trp 320 325 330

AAA GAG TAT CTT CGA ACA AAA GCA ACT CAC TTC AAC AAA GCT CTT TCA 1116 Lys Glu Tyr Leu Arg Thr Lys Ala Thr His Phe Asn Lys Ala Leu Ser 335 340 345 350

ATG ATT AGA AAT CAC AGA GAA GAA TGATTAGATA ATAATAGTTG TGATCTACTA 117C Met He Arg Asn His Arg Glu Glu 355

GTTAGTTTGA TTAATAAATT GTTGTAAATG ATTTCAGCAA TATGATTTGT TTGTCCTC 122S

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 358 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Met Pro Ala Met Leu Thr Asp Val Phe Arg Gly His Pro He His Leu 1 5 10 15

Pro His Ser His He Pro Asp Phe Thr Ser Leu Arg Glu Leu Pro Asp 20 25 30

Ser Tyr Lys Trp Thr Pro Lys Asp Asp Leu Leu Phe Ser Ala Ala Pro 35 40 45 Ser Pro Pro Ala Thr Gly Glu Asn He Pro Leu He Asp Leu Asp His 50 55 60

Pro Asp Ala Thr Asn Gin He Gly His Ala Cys Arg Thr Trp Gly Ala 65 70 75 80

Phe Gin He Ser Asn His Gly Val Pro Leu Gly Leu Leu Gin Asp He 85 90 95

Glu Phe Leu Thr Gly Ser Leu Phe Gly Leu Pro Val Gin Arg Lys Leu 100 105 110

Lys Ser Ala Arg Ser Glu Thr Gly Val Ser Gly Tyr Gly Val Ala Arg 115 120 125

He Ala Ser Phe Phe Asn Lys Gin Met Trp Ser Glu Gly Phe Thr He 130 135 140

Thr Gly Ser Pro Leu Asn Asp Phe Arg Lys Leu Trp Pro Gin His His 145 150 155 160

Leu Asn Tyr Cys Asp He Val Glu Glu Tyr Glu Glu His Met Lys Lys 165 170 175

Leu Ala Ser Lys Leu Met Trp Leu Ala Leu Asn Ser Leu Gly Val Ser 180 185 190

Glu Glu Asp He Glu Trp Ala Ser Leu Ser Ser Asp Leu Asn Trp Ala 195 200 205

Gin Ala Ala Leu Gin Leu Asn His Tyr Pro Val Cys Pro Glu Pro Asp 210 215 220

Arg Ala Met Gly Leu Ala Ala His Thr Asp Ser Thr Leu Leu Thr He 225 230 235 240

Leu Tyr Gin Asn Asn Thr Ala Gly Leu Gin Val Phe Arg Asp Asp Leu 245 250 255

Gly Trp Val Tnr Val Pro Pro Pne Pro Gly Ser Leu Val Val Asn Val 260 265 270

Gly Asp Leu Phe His He Leu Ser Asn Gly Leu Phe Lys Ser Val Leu 275 280 285

His Arg Ala Arg Val Asn Gin Thr Arg Ala Arg Leu Ser Val Ala Phe 290 295 300

Leu Trp Gly Pro Gin Ser Asp He Lys He Ser Pro Val Pro Lys Leu 305 310 315 320

Val Ser Pro Val Glu Ser Pro Leu Tyr Gin Ser Val Thr Trp Lys Glu 325 330 335

Tyr Leu Arg Tnr Lys Ala Thr His Phe Asn Lys Ala Leu Ser Met He 340 345 350

Arg Asn His Arg Glu Glu 355

(2) INFORMATION FOR SEQ ID NO : 3 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 159 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

CTTAAGTCTG CTCGGTCGGA GACAGGTGTG TCCGGCTACG GCGTCGCTCG TATCGCATCT 60

TTCTTCAATA AGCAAATGTG GTCCGAAGGT TTCACCATCA CTGGCTCGCC TCTCAACGAT 120

TTCCGTAAAC TTTGGCCCCA ACATCACCTC AACTACTGC 159 (2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 140 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : GTGGTTAGCA CTAAATTCAC TTGGGGTCAG CGAAGAAGAC ATTGAATGGG CCAGTCTCAG 60 TTCAGATTTA AACTGGGCCC AAGCTGCTCT CCAGCTAAAT CACTACCCGG TTTGTCCTGA 120 ACCGGACCGA GCCATGGGTC 140

(2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1133 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

GTTTATGTGA TGAGCATCCC ATTCTCTCAT TAGTTCACAA GTCATGCCTT CACTAGCAGA 60

AGAGATATGT ATTGGTAACT TAGGCAGTCT CCAAACACTC CCCGAGTCGT TCACCTGGAA 120

ACTCACAGCC GCCGACTCCC TTCTGCGTCC CTCCTCCGCC GTCTCATTCG ACGCAGTGGA 180

AGAGTCCATT CCTGTGATCG ACCTCTCTAA TCCTGACGTT ACCACCCTCA TTGGAGATGC 240 CTCCAAAACA TGGGGAGCGT TTCAGATAGC CAACCACGGG ATTTCTCAGA AGCTTCTCGA 300

TGATATCGAG TCTCTGTCCA AAACCCTATT CGACATGCCG TCAGAGAGGA AGCTTGAAGC 360

GGCTTCCTCC GATAAAGGAG TTAGTGGCTA CGGAGAACCT CGAATCTCCC CCTTTTTCGA 420

GAAGAAAATG TGGTCTGAAG GGTTTACTAT TGCCGATGAC TCCTACCGCA ACCATTTCAA 480

TACTCTTTGG CCTCATGATC ACACCAAGTA CTGCGGTATA ATCCAAGAAT ACGTGGACGA 540

AATGGAAAAA TTAGCAAGCA GACTTCTGTA TTGCACATTA GGCTCACTTG GTGTCACCGT 600

GGAAGACATT GAATGGGCTC ACAAGCTAGA GAAATCTGGA TCAAAAGTGG GCAGAGGCGC 660

CATACGACTA AACCACTACC CGGTTTGTCC TGAACCAGAA CGAGCCATGG GTCTAGCCGC 720

TCATACAGAC TCCACTATCC TAACCATTCT GCACCAGAGC AACACGGGAG GGCTACAAGT 780

GTTCAGGGAA GAGTCCGGTT GGGTCACGGT TGAGCCGGCT CCTGGTGTCC TCGTGGTCAA 840

CATGGGTGAT CTCTTTCACA TCTTATCGAA CGGGAAAATC CCAAGCGTGG TTCATCGAGC 900

CAAAGTTAAC CATACTCGGT CAAGAATTTC GATTGCGTAC TTATGGGGTG GTCCAGCTGG 960

TGATGTGCAA ATCGCACCTA TCTCTAAGTT AACCGGTCCG GCTGAACCGT CTCTTTACCG 1020

GTCAATTACA TGGAAAGAGT ATCTCCAAAT AAAGTATGGG GTTTTCGACA AGGCCATGGA 1080

CGCAATTAGG GTCGTTAATC CCACCAATTA AATCTCCTTC TCAAATACTC TCT 1133 (2) INFORMATION FOR SEQ ID NO : 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1610 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 86..556

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 966..1559

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

ACATATGTGT GTAGTATCTA TGCATATATA TCCAAAGTAA TTGTTTATGT GATGAGCATC 60

CCATTCTCTC ATTAGTTCAC AAGTC ATG CCT TCA CTA GCA GAA GAG ATA TGT 112

Met Pro Ser Leu Ala Glu Glu He Cys 1 5

ATT GGT AAC TTA GGC AGT CTC CAA ACA CTC CCC GAG TCG TTC ACC TGG 160 He Gly Asn Leu Gly Ser Leu Gin Thr Leu Pro Glu Ser Phe Thr Trp 10 15 20 25 AAA CTC ACA GCC GCC GAC TCC CTT CTG CGT CCC TCC TCC GCC GTC TCA 208 Lys Leu Thr Ala Ala Asp Ser Leu Leu Arg Pro Ser Ser Ala Val Ser 30 35 40

TTC GAC GCA GTG GAA GAG TCC ATT CCT GTG ATC GAC CTC TCT AAT CCT 256 Phe Asp Ala Val Glu Glu Ser He Pro Val He Asp Leu Ser Asn Pro 45 50 55

GAC GTT ACC ACC CTC ATT GGA GAT GCC TCC AAA ACA TGG GGA GCG TTT 304 Asp Val Thr Thr Leu He Gly Asp Ala Ser Lys Thr Trp Gly Ala Phe 60 65 70

CAG ATA GCC AAC CAC GGG ATT TCT CAG AAG CTT CTC GAT GAT ATC GAG 352 Gin He Ala Asn His Gly He Ser Gin Lys Leu Leu Asp Asp He Glu 75 80 85

TCT CTG TCC AAA ACC CTA TTC GAC ATG CCG TCA GAG AGG AAG CTT GAA 400 Ser Leu Ser Lys Thr Leu Phe Asp Met Pro Ser Glu Arg Lys Leu Glu 90 95 100 105

GCG GCT TCC TCC GAT AAA GGA GTT AGT GGC TAC GGA GAA CCT CGA ATC 448 Ala Ala Ser Ser Asp Lys Gly Val Ser Gly Tyr Gly Glu Pro Arg He 110 115 120

TCC CCC TTT TTC GAG AAG AAA ATG TGG TCT GAA GGG TTT ACT ATT GCC 496 Ser Pro Phe Phe Glu Lys Lys Met Trp Ser Glu Gly Phe Thr He Ala 125 130 135

GAT GAC TCC TAC CGC AAC CAT TTC AAT ACT CTT TGG CCT CAT GAT CAC 544 Asp Asp Ser Tyr Arg Asn His Phe Asn Thr Leu Trp Pro His Asp His 140 145 150

ACC AAG TAC TGG TAACGTCTAT TACACACACA TATATATATT TTTTGCTTAT 596

Thr Lys Tyr Trp 155

TTCGCAAAAG TGTGGCAAAG GAAATTGCAC ACTTTTTTTT TGCACTAAGA CTTAGTTATT 656

ATTAAAAGTG TTTAAATGTT TTTTTCTGTT CATAAAAAAG TGTTTATATG TTCCGAGTAA 716

TTGATGTTTA TGATTAGTGA TAACTGATAA CACATAGAGT GTAGCCTTCA AAGTTTCTAA 776

TTAAATAGTT TGAGCAACAT CCTTATATTT TATGAAGTAG TACTTCTTAT TGCATATTAC 836

AGCAAATTAA AGTACCAAAG TCTCTATGAA ATGTGATAAT TTGGCTAATG TCGAGGTCTT 896

AACATTAGAT TACCAAAAAC CTTAATTACT GTAAATTGTA TTTGCTTTTC ATTTTTGGTA 956

TTGTGCAGC GGT ATA ATC CAA GAA TAC GTG GAC GAA ATG GAA AAA TTA 1004

Gly He He Gin Glu Tyr Val Asp Glu Met Glu Lys Leu 1 5 10

GCA AGC AGA CTT CTG TAT TGC ACA TTA GGC TCA CTT GGT GTC ACC GTG 1052 Ala Ser Arg Leu Leu Tyr Cys Thr Leu Gly Ser Leu Gly Val Thr Val 15 20 25

GAA GAC ATT GAA TGG GCT CAC AAG CTA GAG AAA TCT GGA TCA AAA GTG 1100 Glu Asp He Glu Trp Ala His Lys Leu Glu Lys Ser Gly Ser Lys Val 30 35 40 45

GGC AGA GGC GCC ATA CGA CTA AAC CAC TAC CCG GTT TGT CCT GAA CCA 1148 Gly Arg Gly Ala He Arg Leu Asn His Tyr Pro Val Cys Pro Glu Pro 50 55 60

GAA CGA GCC ATG GGT CTA GCC GCT CAT ACA GAC TCC ACT ATC CTA ACC 1196 Glu Arg Ala Met Gly Leu Ala Ala His Thr Asp Ser Thr He Leu Thr 65 70 75

ATT CTG CAC CAG AGC AAC ACG GGA GGG CTA CAA GTG TTC AGG GAA GAG 1244 He Leu His Gin Ser Asn Thr Gly Gly Leu Gin Val Phe Arg Glu Glu 80 85 90

TCC GGT TGG GTC ACG GTT GAG CCG GCT CCT GGT GTC CTC GTG GTC AAC 1292 Ser Gly Trp Val Thr Val Glu Pro Ala Pro Gly Val Leu Val Val Asn 95 100 105

ATG GGT GAT CTC TTT CAC ATC TTA TCG AAC GGG AAA ATC CCA AGC GTG 1340 Met Gly Asp Leu Phe His He Leu Ser Asn Gly Lys He Pro Ser Val 110 115 120 125

GTT CAT CGA GCC AAA GTT AAC CAT ACT CGG TCA AGA ATT TCG ATT GCG 1388 Val His Arg Ala Lys Val Asn His Thr Arg Ser Arg He Ser He Ala 130 135 140

TAC TTA TGG GGT GGT CCA GCT GGT GAT GTG CAA ATC GCA CCT ATC TCT 1436 Tyr Leu Trp Gly Gly Pro Ala Gly Asp Val Gin He Ala Pro He Ser 145 150 155

AAG TTA ACC GGT CCG GCT GAA CCG TCT CTT TAC CGG TCA ATT ACA TGG 1484 Lys Leu Thr Gly Pro Ala Glu Pro Ser Leu Tyr Arg Ser He Thr Trp 160 165 170

AAA GAG TAT CTC CAA ATA AAG TAT GAG GTT TTC GAC AAG GCC ATG GAC 1532 Lys Glu Tyr Leu Gin He Lys Tyr Glu Val Phe Asp Lys Ala Met Asp 175 180 185

GCA ATT AGG GTC GTT AAT CCC ACC AAT TAAATCTCCT TCTCAAATAC 1579

Ala He Arg Val Val Asn Pro Thr Asn 190 195

TCTCTTAATG AAAAACCTAA ATTAAATGCG A 1610

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 157 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Met Pro Ser Leu Ala Glu Glu He Cys He Gly Asn Leu Gly Ser Leu 1 5 10 15

Gin Thr Leu Pro Glu Ser Phe Thr Trp Lys Leu Thr Ala Ala Asp Ser 20 25 30

Leu Leu Arg Pro Ser Ser Ala Val Ser Phe Asp Ala Val Glu Glu Ser 35 40 45

He Pro Val He Asp Leu Ser Asn Pro Asp Val Thr Thr Leu He Gly 50 55 60

Asp Ala Ser Lys Thr Trp Gly Ala Phe Gin He Ala Asn His Gly He 65 70 75 80

Ser Gin Lys Leu Leu Asp Asp He Glu Ser Leu Ser Lys Thr Leu Phe 85 90 95

Asp Met Pro Ser Glu Arg Lys Leu Glu Ala Ala Ser Ser Asp Lys Gly 100 105 110

Val Ser Gly Tyr Gly Glu Pro Arg He Ser Pro Phe Phe Glu Lys Lys 115 120 125

Met Trp Ser Glu Gly Phe Thr He Ala Asp Asp Ser Tyr Arg Asn His 130 135 140

Phe Asn Thr Leu Trp Pro His Asp His Thr Lys Tyr Trp 145 150 155

(2) INFORMATION FOR SEQ ID NO: 8:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 198 amino acids

(B) TYPE: ammo acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Gly He He Gin Glu Tyr Val Asp Glu Met Glu Lys Leu Ala Ser Arg 1 5 10 15

Leu Leu Tyr Cys Thr Leu Gly Ser Leu Gly Val Thr Val Glu Asp He 20 25 30

Glu Trp Ala His Lys Leu Glu Lys Ser Gly Ser Lys Val Gly Arg Gly 35 40 45

Ala He Arg Leu Asn His Tyr Pro Val Cys Pro Glu Pro Glu Arg Ala 50 55 60

Met Gly Leu Ala Ala His Thr Asp Ser Thr He Leu Thr He Leu His 65 70 75 80

Gin Ser Asn Thr Gly Gly Leu Gin Val Phe Arg Glu Glu Ser Gly Trp 85 90 95

Val Thr Val Glu Pro Ala Pro Gly Val Leu Val Val Asn Met Gly Asp 100 105 110

Leu Phe His He Leu Ser Asn Gly Lys He Pro Ser Val Val His Arg 115 120 125

Ala Lys Val Asn His Thr Arg Ser Arg He Ser He Ala Tyr Leu Trp 130 135 140

Gly Gly Pro Ala Gly Asp Val Gin He Ala Pro He Ser Lys Leu Thr 145 150 155 160 Gly Pro Ala Glu Pro Ser Leu Tyr Arg Ser He Thr Trp Lys Glu Tyr 165 170 175

Leu Gin He Lys Tyr Glu Val Phe Asp Lys Ala Met Asp Ala He Arg 180 185 190

Val Val Asn Pro Thr Asn 195

(2) INFORMATION FOR SEQ ID NO : 9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1105 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

TCATACCAAA AACATAAAGC CAAAATATAA ACACATAAGC CTTTTAGCAT GAGTTCAACG 60

TTGAGCGATG TGTTTAGATC GCATCCCATT CACATCCCAC TCTCAAACCC ACCTGACTTC 120

AAATCTCTCC CGGATTCTTA CACGTGGACT CCTAAAGATG ATCTCCTCTT CTCCGCCTCC 180

GCCTCCGACG AAACCCTGCC GCTCATCGAC CTCTCCGATA TCCACGTGGC CACTCTTGTG 240

GGCCATGCTT GTACCACGTG GGGAGCGTTC CAGATCACCA ACCACGGCGT CCCCTCGCGA 300

CTTCTCGACG ACATTGAGTT CCTCACCGGA AGTCTTTTCC GGCTTCCCGT ACAGCGGAAG 360

CTCAAGGCGG CTCGGTCAGA GAATGGCGTC TCCGGCTACG GCGTAGCTCG TATTGCTTCG 420

TTCTTTAATA AGAAGATGTG GTCCGAAGGT TTCACCGTTA TTGGCTCTCC CCTCCACGAT 480

TTCCGTAAAC TCTGGCCCAG CCACCACCTC AAATACTGTG AAATTATTGA AGAGTATGAA 540

GAACATATGC AAAAGTTGGC AGCCAAGTTG ATGTGGTTCG CATTAGGTTC ACTGGGAGTT 600

GAAGAAAAGG ACATACAATG GGCCGGGCCT AATTCAGACT TTCAAGGAAC CCAAGCAGCT 660

ATCCAACTAA ACCATTATCC AAAATGTCCA GAACCAGACA GAGCCATGGG CCTCGCAGCC 720

CAT CAGACT CGACCCTCAT GACCATTCTG TACCAGAACA ACACCGCCGG TCTCCAAGTT 780

TTCCGGGATG ACGTGGGCTG GGTTACCGCG CCACCTGTCC CTGGCTCGCT GGTGGTCAAC 840

GTCGGTGACT TGCTCCACAT TTTAACCAAC GGAATCTTCC CGAGCGTGCT TCACCGAGCC 900

AGGGTTAACC ACGTCCGATC TCGGTTCTCA ATGGCTTACC TGTGGGGTCC ACCATCCGAT 960

GTAATGATCT CTCCACTTCC CAAACTGGTT GATCCTCTCC AATCTCCTCT CTACCCATCT 1020

CTCACTTGGA AACAATACCT TGCTACCAAA GCTACTCATT TTAATCAATC TCTTTCCATT 1080

ATTAGAAATT AACTGTCTTC CGACT 1105 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1690 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 95..565

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 986..1555

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

TCACCGATCT ATAAATACAC TCCTCTTCTC CACCAAAAGT ATCATATCAT ACCAAAAACA 60

TAAAGCCAAA ATATAAACAC ATAAGCCTTT TAGC ATG AGT TCA ACG TTG AGC 112

Met Ser Ser Thr Leu Ser 1 5

GAT GTG TTT AGA TCG CAT CCC ATT CAC ATC CCA CTC TCA AAC CCA CCT 160 Asp Val Phe Arg Ser His Pro He His He Pro Leu Ser Asn Pro Pro 10 15 20

GAC TTC AAA TCT CTC CCG GAT TCT TAC ACG TGG ACT CCT AAA GAT GAT 208 Asp Phe Lys Ser Leu Pro Asp Ser Tyr Thr Trp Thr Pro Lys Asp Asp 25 30 35

CTC CTC TTC TCC GCC TCC GCC TCC GAC GAA ACC CTG CCG CTC ATC GAC 256 Leu Leu Phe Ser Ala Ser Ala Ser Asp Glu Thr Leu Pro Leu He Asp 40 45 50

CTC TCC GAT ATC CAC GTG GCC ACT CTT GTG GGC CAT GCT TGT ACC ACG 304 Leu Ser Asp He His Val Ala Thr Leu Val Gly His Ala Cys Thr Thr 55 60 65 70

TGG GGA GCG TTC CAG ATC ACC AAC CAC GGC GTC CCC TCG CGA CTT CTC 352 Trp Gly Ala Phe Gin He Thr Asn His Gly Val Pro Ser Arg Leu Leu 75 80 85

GAC GAC ATT GAG TTC CTC ACC GGA AGT CTT TTC CGG CTT CCC GTA CAG 400 Asp Asp He Glu Phe Leu Thr Gly Ser Leu Phe Arg Leu Pro Val Gin 90 95 100

CGG AAG CTC AAG GCG GCT CGG TCA GAG AAT GGC GTC TCC GGC TAC GGC 448 Arg Lys Leu Lys Ala Ala Arg Ser Glu Asn Gly Val Ser Gly Tyr Gly 105 110 115

GTA GCT CGT ATT GCT TCG TTC TTT AAT AAG AAG ATG TGG TCC GAA GGT 496 Val Ala Arg He Ala Ser Phe Phe Asn Lys Lys Met Trp Ser Glu Gly 120 125 130

TTC ACC GTT ATT GGC TCT CCC CTC CAC GAT TTC CGT AAA CTC TGG CCC 544 Phe Thr Val He Gly Ser Pro Leu His Asp Phe Arg Lys Leu Trp Pro 135 140 145 150

AGC CAC CAC CTC AAA TAC TGG TATCTTTTTC AATGGTTCAT TTTATCAACG 595

Ser His His Leu Lys Tyr Trp 155

TTAAGACCAT ATTAACGTAA CGTAACTTAT CTTTGTATGA AAAAAAAAAA AAAAACTGTG 655

GACGTTAGTA CAGTTGACTA TTCAATTGAT ATAGATTCGG GAATAATACG AAAAGGGTAA 715

AGTAGAAACC ATTTTTTGCC ATGTCGTAGT TAGTAAAAAG CACAATGAAA ACTCATGGAC 775

CCACCAAAAA GATTACATGA TATAATATAT ATATATATAT TTATATAAAT ATTATATAAT 835

ATATTTATAT AATATTATGT GCAAAAATTA AATGAAAATA AATATTATCA GGAGAATGTG 895

AAATACAGTA TAAGATTTTC CTTTGGCTAC ATGACGATTT CTATAGATTT GAAGGTTAAG 955

ATACTAATTT CATATTATCG ATTCAACAGT GAA ATT ATT GAA GAG TAT GAA GAA IOC 9

Glu He He Glu Glu Tyr Glu Glu 1 5

CAT ATG CAA AAG TTG GCA GCC AAG TTG ATG TGG TTC GCA TTA GGT TCA 105^"1 His Met Gin Lys Leu Ala Ala Lys Leu Met Trp Phe Ala Leu Gly Ser 10 15 20

CTG GGA GTT GAA GAA AAG GAC ATA CAA TGG GCC GGG CCT AAT TCA GAC 1105 Leu Gly Val Glu Glu Lys Asp He Gin Trp Ala Gly Pro Asn Ser Asp 25 30 35 40

TTT CAA GGA ACC CAA GCA GCT ATC CAA CTA AAC CAT TAT CCA AAA TGT 1153 Phe Gin Gly Thr Gin Ala Ala He Gin Leu Asn His Tyr Pro Lys Cys 45 50 55

CCA GAA CCA GAC AGA GCC ATG GGC CTC GCA GCC CAT ACA GAC TCG ACC 1201 Pro Glu Pro Asp Arg Ala Met Gly Leu Ala Ala His Thr Asp Ser Thr 60 65 70

CTC ATG ACC ATT CTG TAC CAG AAC AAC ACC GCC GGT CTC CAA GTT TTC 1249 Leu Met Thr He Leu Tyr Gin Asn Asn Thr Ala Gly Leu Gin Val Phe 75 80 85

CGG GAT GAC GTG GGC TGG GTT ACC GCG CCA CCT GTC CCT GGC TCG CTG 1297 Arg Asp Asp Val Gly Trp Val Thr Ala Pro Pro Val Pro Gly Ser Leu 90 95 100

GTG GTC AAC GTC GGT GAC TTG CTC CAC ATT TTA ACC AAC GGA ATC TTC 1345 Val Val Asn Val Gly Asp Leu Leu His He Leu Thr Asn Gly He Phe 105 110 115 120

CCG AGC GTG CTT CAC CGA GCC AGG GTT AAC CAC GTC CGA TCT CGG TTC 1393 Pro Ser Val Leu His Arg Ala Arg Val Asn His Val Arg Ser Arg Phe 125 130 135

TCA ATG GCT TAC CTG TGG GGT CCA CCA TCC GAT GTA ATG ATC TCT CCA 1441 Ser Met Ala Tyr Leu Trp Gly Pro Pro Ser Asp Val Met He Ser Pro 140 145 150

CTT CCC AAA CTG GTT GAT CCT CTC CAA TCT CCT CTC TAC CCA TCT CTC 1489 Leu Pro Lys Leu Val Asp Pro Leu Gin Ser Pro Leu Tyr Pro Ser Leu 155 160 165 ACT TGG AAA CAA TAC CTT GCT ACC AAA GCT ACT CAT TTT AAT CAA TCT 1537 Thr Trp Lys Gin Tyr Leu Ala Thr Lys Ala Thr His Phe Asn Gin Ser 170 175 180

CTT TCC ATT ATT AGA AAT TAACTGTCTT CCGACTGAAT TTCTTGATTT 1585

Leu Ser He He Arg Asn 185 190

TCAGATTTTA CTATTTATTT TCTTAGTAAT ATGATGATAT CTATTACTGT TTCGATTTTA 1645

GATGAGTGGT TCTTCAAATT CACAATTAGT AGCTTAATAT TGATT 1690

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 157 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Ser Ser Thr Leu Ser Asp Val Phe Arg Ser His Pro He His He 1 5 10 15

Pro Leu Ser Asn Pro Pro Asp Phe Lys Ser Leu Pro Asp Ser Tyr Thr 20 25 30

Trp Thr Pro Lys Asp Asp Leu Leu Phe Ser Ala Ser Ala Ser Asp Glu 35 40 45

Thr Leu Pro Leu He Asp Leu Ser Asp He His Val Ala Thr Leu Val 50 55 60

Gly His Ala Cys Thr Thr Trp Gly Ala Phe Gin He Thr Asn His Gly 65 70 75 80

Val Pro Ser Arg Leu Leu Asp Asp He Glu Phe Leu Thr Gly Ser Leu 85 90 95

Phe Arg Leu Pro Val Gin Arg Lys Leu Lys Ala Ala Arg Ser Glu Asn 100 105 110

Gly Val Ser Gly Tyr Gly Val Ala Arg He Ala Ser Phe Phe Asn Lys 115 120 125

Lys Met Trp Ser Glu Gly Phe Thr Val He Gly Ser Pro Leu His Asp 130 135 140

Phe Arg Lys Leu Trp Pro Ser His His Leu Lys Tyr Trp 145 150 155

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 190 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Glu He He Glu Glu Tyr Glu Glu His Met Gin Lys Leu Ala Ala Lys 1 5 10 15

Leu Met Trp Phe Ala Leu Gly Ser Leu Gly Val Glu Glu Lys Asp He 20 25 30

Gin Trp Ala Gly Pro Asn Ser Asp Phe Gin Gly Thr Gin Ala Ala He 35 40 45

Gin Leu Asn His Tyr Pro Lys Cys Pro Glu Pro Asp Arg Ala Met Gly 50 55 60

Leu Ala Ala His Thr Asp Ser Thr Leu Met Thr He Leu Tyr Gin Asn 65 70 75 80

Asn Thr Ala Gly Leu Gin Val Phe Arg Asp Asp Val Gly Trp Val Thr 85 90 95

Ala Pro Pro Val Pro Gly Ser Leu Val Val Asn Val Gly Asp Leu Leu 100 105 110

His He Leu Thr Asn Gly He Phe Pro Ser Val Leu His Arg Ala Arg 115 120 125

Val Asn His Val Arg Ser Arg Phe Ser Met Ala Tyr Leu Trp Gly Pro 130 135 140

Pro Ser Asp Val Met He Ser Pro Leu Pro Lys Leu Val Asp Pro Leu 145 150 155 160

Gin Ser Pro Leu Tyr Pro Ser Leu Thr Trp Lys Gin Tyr Leu Ala Thr 165 170 175

Lys Ala Thr His Phe Asn Gin Ser Leu Ser He He Arg Asn 180 185 190

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GTGGTTAGCA CTAAATTCAC 20

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GACCCATGGC TCGGTCCGGT 20

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GCTCTAGAGA GTATTTGAGA AGG (2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GTTTACTATT GCCGATGACT (2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17 CAATACCAAA AATGAAAAGC (2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

CTCCTACCGC AACCATTTC 19

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS: (A; LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TCCCCCGGGT TTATGTGATG AGCATCCC 28

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid (C STRANDEDNESS: both (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

CCAAAGTAAT TGTTTATGTG 20

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B; TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: AATTTAGGTT TTTCATTAAG (2) INFORMATION FOR SEQ ID NO: 22:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GTAGTGGTTT AGTCGTATGG (2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: AAAACTTGGA GACCGGCGG (2) INFORMATION FOR SEQ ID NO: 24:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: TATCATGTAA TCTTTTTGG (2) INFORMATION FOR SEQ ID NO:25:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: CCGGCTTCCC GTACAGCGG 19

(2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: AATCAAGAAA TTCAGTCGG 19

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27 GGAATTCATA CCAAAAACAT AAAGCC (2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2. CTAGTTTCTT TCTTCCACG (2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 29 : TAGCTGCATC TTCTTTACC

70/1

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule \3bis)

A. The indications made below relate to the microorganism referred to in the description on page line 25-26

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet U

Name of depositary institution

American Type Culture Collection

Address of depositary institution (including postal code and countrv)

12301 Parklawn Drive Now at 10801 University Boulevard

Rockville, Maryland 20852 Manassas. Virginia 20110-2209 United States of America United States of America

Date of deposit Accession Number

May 20, 1997 ATCC 98436

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet tJ

Arabidopsis thaliana genomic DNA of GA4H1 and GA4H2 genes cloned into pBSKS(+) (Stratagene) vector pLVN103 in DH5

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS ./__..<■ blank ,/ ™, applicable)

The indications listed below w ill be submitted to the international Bureau later (specify the general nature of the indications, e g "Accession Number of Deposit")

Claims

-71-What Is Claimed Is:

1. A purified DNA molecule comprising a DNA sequence encoding the amino acid sequence of a GA4 homologue.

2. The DNA molecule of claim 1 encoding the amino acid sequence of GA4H1 in Figure 6 (SEQ. ID. No. 7).

3. The DNA molecule of claim 1 encoding the amino acid sequence of GA4H2 in Figure 8 (SEQ. ID. No. 10).

4. The DNA molecule of claim 1 , wherein said DNA is selected from the group consisting of the genomic DNA's, SEQ ID No. 6 in Figure 6, SEQ ID

No. 9 in Figure 8, cDNAs having SEQ ID No. 5 in Figure 5, SEQ. ID. No. 8 in Figure 7 and a degenerate variant of any of said sequences.

5. A DNA molecule comprising a sequence with at least 95% homology to the DNA sequence in any one of claims 1-4.

6. A vector comprising the sequence of claim 5.

7. A host transformed with the vector of claim 6.

8. The host of claim 7, wherein said host is selected from the group consisting of bacteria, yeast, plants, insects or mammals.

9. The host of claim 8, wherein said host is a plant cell.

10. The host of claim 9, wherein said plant cell is a dicotyledonous plant cell. -72-

11. A plant regenerated from the plant cell of claim 10.

12. Progeny of the plant of claim 11.

13. A propagule of the plant of claim 11.

14. A seed produced by the progeny of claim 11.

15. Purified GA4H protein.

16. The protein of claim 15, wherein said GA4H protein is an Arabidopsis protein.

17. The GA4H protein of claim 15, wherein said GA4H protein is selected from the group consisting of GA4H1 comprising the amino acid sequence shown in Figure 6 (SEQ. ID NO. 7), GA4H2 comprising the amino acid sequence shown in Figure 8 (SEQ. ID NO. 10) and a functional derivative of said sequences.

18. The GA4H protein of any one of claims 15-17, wherein said

GA4H protein is substantially free of other A. thaliana proteins.

19. A cell extract comprising a GA4H protein.

20. The cell extract of claim 21, wherein said GA4H protein is an Arabidopsis protein.

21. The cell extract of claim 26, wherein said GA4H protein comprises the amino acid sequence selected from the group consisting of Figure 6 (SEQ. ID NO. 7) , Figure 8 (SEQ. ID. NO. 10) and a functional derivative of said sequences. -73-

22. The cell extract of any one of claims 19-21 wherein said cell is a prokaryotic cell or a eukaryotic cell.

23. The cell extract of claim 22, wherein said prokaryotic cell is an E. coli.

24. The cell extract of claim 22, wherein said eukaryotic cell is ayeast, fungal, insect, mammalian or transgenic plant cell.

25. A cell extract comprising thaliana GA4H protein, wherein said cell is not thaliana.

26. A method of making GA4H protein wherein said GA4H protein is substantially free of other A. thaliana proteins, said method comprising: a) transforming a prokaryotic or eukaryotic cell with a GA4H recombinant expression vector encoding a GA4H protein or a functional derivative of a GA4H protein, b) expressing said GA4H protein, and c) isolating said GA4H protein substantially free of other A. thaliana proteins.

27. The method of claim 26 wherein said GA4H protein is isolated from E coli inclusion bodies.

28. A method ofdirecting the expression ofa gene in a plant, such that said gene has the same temporal and spatial expression pattern of a GA4H, said method comprising the steps of:

1) operably linking said gene to the regulatory sequences of GA4H to create an expression module, and 2) transforming said plant with said expression module of part (l). -74-

29. A method of modulating the translation of RNA encoding GA4H in a plant comprising the steps of:

1) generating an expression vector encoding antisense GA4H RNA;

2) transfecting said plant with said expression vector of part

(1).

30. An isolated DNA construct wherein said construct consists essentially of a nucleic acid sequence, and wherein said nucleic acid sequence: 1) encodes GA4H polypeptide, and

2) hybridizes to the sense or antisense sequence of the GA4H DNA when hybridization is performed under stringent hybridization conditions.

31. An isolated DNA molecule encoding a GA4H protein, said DNA molecule prepared by a process comprising:

1) hybridizing a desired DNA molecule to the sense or antisense sequence of a GA4H DNA sequence, wherein the hybridization is performed under stringent hybridization conditions; 2) selecting those DNA molecules of said population that hybridize to said sequence; and

3) selecting DNA molecules of part (2) that encode said GA4H protein.

32. An isolated DNA molecule encoding a GA4H protein as claimed in claims 30 or 31 , said DNA molecule prepared by a process comprising:

1) prehybridizing for 1 hour;

2) hybridizing overnight at 65 ┬░ C in the hybridization buffer; and -75-

3) washing once for 15 minutes in 2xSSC at room temperature, then two times for 30 minutes in O.lxSSC and 0.1% SDS at 60┬░C.

33. A method of cloning a DNA molecule that encodes a GA4H protein, said method comprising:

1) hybridizing a desired DNA molecule to the sense or antisense sequence of GA4H DNA wherein the Γûá hybridization is performed under stringent hybridization conditions;

2) selecting those DNA molecules of said population that hybridize to said sequence;

3) transforming said DNA of part (2) into a host cell; and 4) selecting transformants that express said GA4.

34. The method of claim 33 wherein the hybridization conditions consist essentially of:

1) prehybridizing for 1 hour;

2) hybridizing overnight at 65 ┬░ C in the hybridization buffer; and

3) washing once for 15 minutes in 2xSSC at room temperature, then two times for 30 minutes in O.lxSSC and 0.1% SDS at 60┬░C.

35. A method of altering stem elongation, said method comprising inserting a DNA construct encoding the amino acid sequence ofa GA4H protein into a transgenic plant.

36. A method of producing a transgenic dwarf plant said method comprising transforming a plant with the antisense or sense construct ofa GA4H gene or cDNA. -76-

37. A method of making GA4H protein wherein said GA4H protein is substantially free of other A. thaliana proteins, comprising: a) transforming a prokaryotic or eukaryotic cell with a GA4H recombinant expression vector encoding a GA4H protein, b) expressing said GA4H protein, and c) purifying said GA4H protein substantially free of other A. thaliana proteins.

38. An antibody or fragment thereof, capable of binding a GA4H protein.

39. A method of identifying cells or tissues expressing GA4H comprising the steps of: a) incubating said cells or said tissues with an agent capable of binding to the GA4H protein or the RNA encoding GA4H; and b) detecting the presence of the bound agent.

40. The method of claim 39 wherein said agent capable of binding to the GA4H protein is an antibody or fragment thereof.