AU714454B2 - Recombinant Gibberellin DNA and uses thereof - Google Patents

Description

P00011 Regulation 3.2

AUSTRALIA

Patents Act, 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT TO BE COMPLETED BY THE APPLICANT NAME OF APPLICANT: ACTUAL INVENTOR(S): ADDRESS FOR SERVICE: INVENTION TITLE: THE GENERAL HOSPITAL CORPORATION TAI-PING SUN HOWARD M GOODMAN FREDERICK M AUSUBEL Peter Maxwell Associates Level 6 Pitt Street SYDNEY NSW 2000 RECOMBINANT GIBBERELLIN DNA AND USES THEREOF DETAILS OF ASSOCIATED PROVISIONAL APPLICATION NO(S): The following statement is a full description of this invention including the best method of performing it known to me:- -1a- The invention pertains to recombinant DNA technology. Specifically, the invention relates to cDNA and genomic DNA corresponding to the GA 1 locus of Arabidopsis thaliana which encodes ent-kaurene synthetase, expression vectors containing such genes, hosts transformed with such vectors, the regulatory regions of the GA 1 gene, the use of such regulatory regions to direct the expression of operably-linked heterologous genes in transgenic plants, the GA 1 protein substantially free of other A. thaliana proteins, 1111___1 L1I antibodies capable of binding to the GAl protein, and to methods of assaying for the expression of the GA1 gene and the presence of GAl protein in plant cells and tissues.

Background of the Invention A. Gibberellins Gibberellins (GAs) are a family of diterpenoid plant growth hormones required for seed germination, leaf expansion, stem elongation, flowering, and S. fruit set. GAs have been the subject of many physiological, and biochemical studies, and a variety of plant mutants with altered patterns of GA biosynthesis 10 or response have been studied (Graebe, Ann. Rev. Plant Physiol. 38:419- 465 (1987)). However, none of the genes involved in GA synthesis have yet been cloned.

One of the most extensive genetic studies of GA mutants has been carried out by Koornneef et al. (Theor. Appl. Genet. 58:257-263 (1980); 15 Koornneef et al., Genet. Res. Camb. 41:57-68 (1983)) in the small crucifer, Arabidopsis thaliana. Using ethylmethanesulfonate (EMS) and fast neutron Smutagenesis, Koornneef has isolated nine alleles mapping to the GAl locus of A. thaliana (Koornneef et al. (Theor. Appl. Genet. 58:257-263 (1980); Koornneef et al., Genet. Res. Camb. 41:57-68 (1983)).

A. thaliana gal mutants are non-germinating, GA-responsive, malesterile dwarfs whose phenotype can be converted to wild-type by repeated application of GA (Koornneef and van der Veen, Theor. Appl. Genet. 58:257- 263 (1980)). Koornneef et al. used three independent alleles generated by fast neutron bombardment (31.89, 29.9 and 6.59) and six independent alleles (NG4, NG5, d69, A428, d352 and Bo27) generated by ethyl methane sulfonate mutagenesis to construct a fine-structure genetic map of the A. thaliana GAl locus (Figure 2A). One of the fast-neutron-generated mutants, 31.89, failed to recombine with the six alleles indicated in Figure 2A, and was classified as an intragenic deletion (Koornneef et al., Genet. Res. Camb. 41:57-68 (1983)).

The enzyme encoded by the GA1 gene is involved in the conversion of geranylgeranyl pyrophosphate to ent-kaurene (Barendse and Koornneef, Arabidopsis Inf. Serv. 19:25-28 (1982); Barendse et al., Physiol. Plant.

67:315-319 (1986); Zeevaart, in Plant Research '86, Annual Report of the MSU-DOE Plant Research Laboratory, 130-131 (East Lansing,

MI,

1986)), a key intermediate in the biosynthesis of GAs (Graebe, Ann.

10 Rev. Plant Physiol. 38:419-465 (1987)).

*L

synthetase I IQ and a recombination rate of 10 cM per nucleotide was determined for this region of the A. thaliana genome. (Koornnetase has beef, Genet. Res. Comb. 41:57-68 (1983)).

Although gal mutants have been available for some time, the cloning of the GAi gene has remained elusive. The difficulty associated ith cloning 1 1ICi;il_;__l 1 I:I; the GAl gene and other genes involved in GA biosynthesis is most likely caused by the unavailability of efficient transformation/ selection systems as well as the lack of available protein sequences.

B. Gene Cloning The ability to identify and clone a particular, desired gene sequence from a virus, prokaryote or eukaryote is of tremendous significance to molecular biology. Such cloned gene sequences can be used to express a desired gene product and therefore can potentially be used for applications ranging from catalysis to gene replacement.

10 A variety of methods have been developed for isolating and cloning desired gene sequences. Early methods permitted only the identification and isolation of gene sequences which possessed a unique property such as proximity to a prophage integration site, capacity for self-replication, distinctive molecular weight, extreme abundance, etc. (The Bacteriophage Lambda, A.D. Hershey, ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1971); Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY (1972); Molecular Biology of the .Gene, Watson, J.D. et al., (4th ed.) Benjamin/Cummings, Menlo Park, CA (1987); Darnell, J. et al. Molecular Biology, Scientific American Books, NY, NY (1986)). Because these methods relied upon distinctive properties of a gene sequence, they were largely (or completely) unsuitable for identifying and cloning most gene sequences.

In order to identify desired gene sequences which lacked a distinctive property, well characterized genetic systems (such as Escherichia coli, Saccharomyces cerevisiae, maize, mammalian cells, etc.) have been exploited.

In accordance with this methodology, cells are mutagenized by chemicals, such as UV light, hydroxylamine, etc. (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY (1972)), or by genetic means, such as transposon tagging (Davis, R.W. et al. A Manual for Genetic Engineering, Advanced Bacterial Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY (1980)), to produce mutants having discernible genetic deficiencies. A desired gene sequence is then identified by its capacity to complement remedy) the genetic deficiencies of such mutant cells.

Such genetic identification permitted the genetic characterization of the gene sequences, and the construction of genetic maps which localized the gene sequence to a region of a particular chromosome (Taylor, Bacteriol. Rev.

34:155 (1970)). With the advent of recombinant DNA technologies, it became possible to clone to physically isolate) such genetically characterized gene sequences. Random fragments of a genome could be introduced into selfreplicating vectors to produce gene libraries, each of whose members contain a unique DNA fragment (Maniatis, T. et al., In: Molecular Cloning, a 15 Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)). By screening the members of such libraries for those capable of Scomplementing the deficiency of a mutant cell, it was possible to clone the desired gene sequence.

Although these methods permit the identification and cloning of many 20 gene sequences, they may be employed only where a host cell exists which has a mutation conferring a discernible deficiency, and the gene sequence can be cloned into a gene sequence delivery system (such as a vector) capable of entering the host cell and being expressed.

The capacity to physically isolate certain gene sequences has led to the development of methods which are capable of isolating a desired gene sequence even in the absence of mutations or vectors.

In one such technique, known as "chromosome walking," a desired sequence can be obtained by isolating a gene sequence which is capable of hybridizing to a particular reference sequence. This isolated gene sequence is then employed as a reference sequence in a subsequent hybridization experiment in order to clone a gene sequence which is adjacent to, and which partially overlaps, the originally isolated sequence. This newly isolated sequence will be physically closer to the desired gene sequence than was the originally isolated sequence. Tlhis process is repeated until the desired gene sequence has been obtained. As will be appreciated, the ability to clone a gene sequence, in the absence of genetic mutants or vectors, requires some initial information concerning the nucleotide sequence or restriction endonuclease digestion profile of the desired sequence.

Alternatively, the chromosome of a virus or cell can be characterized to produce a physical map based on either nucleotide sequence or restriction endonuclease cleavage data an RFLP map). Using such a map, restriction fragments of the chromosome can be cloned without any prior determination as to their genetic function.

15 More recently, gene cloning has been achieved by producing synthesizing oligonucleotide molecules whose sequence has been deduced from the amino acid sequence of an isolated protein, by forming cDNA copies of S'.isolated RNA transcripts, by differential colony or library subtractive hybridizations using either two different RNA sources, or cDNA and RNA.

20 Although these methods may be employed even in the absence of either mutants or a gene sequence delivery system, they permit a desired gene sequence to be identified and cloned only if sequences naturally linked to the desired sequence have been characterized and isolated, or if the sequence or restriction map of such sequences has been obtained. Since such data is often unavailable, these methods are often incapable of use in identifying and cloning a desired gene sequence.

Two general approaches have been described for cloning sequences that are present in one strain and absent in another. The first approach, differential screening, has been used to clone the esc gene from Drosophila.

Using genomic DNA from strains with and without a deletion to probe replicas of a genomic library poses technical difficulties which become daunting for large genomes. In addition, the deletion must cover at least one entire insert in the genomic library which does not contain any repeated sequences.

The second approach, competitive hybridization, provides an elegant alternative to differential screening. This technique was used by Lamar et al.

(Cell 37:171-177 (1984)) to isolate clones specific for the human Y chromosome. In accordance with this method, an excess of sheared DNA from a human female is denatured and reannealed along with a small amount of DNA from a male (the male-derived DNA having been previously treated to have sticky ends). Most of the male DNA reassociated with the sheared DNA yielding unclonable fragments lacking sticky ends. Fragments unique to the Y chromosome, however, could only reassociate with the complementary restricted strand (derived from the Y chromosome). Such reassociation thus formed clonable fragments with sticky ends. This technique 15 has also been used successfully to clone DNA corresponding to deletions in the Duchenne muscular dystrophy locus, and choroideremia.

Unfortunately, the competitive hybridization method does not provide a large enough degree of enrichment. For example, enrichments of about one hundred fold were obtained for the sequences of interest in the above 20 experiments. With enrichments of such low magnitude, the technique is practical only when dealing with large deletions. Indeed, even if the deletion covered 0.1 of the genome, many putative positive clones have to be tested individually by labeling and probing genomic Southern blots (Southern, J.

Molec. Biol. 98:503-517 (1975)). The method as it stands, then, is not practical for deletions on the order of 1 kbp (kilobasepair) unless one is dealing with a small prokaryotic genome.

Thus, in summary, the ability to clone DNA corresponding to a locus defined only by a mutation is a relatively simply matter when working with E. coli, S. cerevisiae or other organisms in which transformation and complementation with genomic libraries is feasible. Chromosome walking techniques may be used in other organisms to clone genetically defined loci if the mutant was obtained by transposon tagging, if the locus can be linked to markers in an RFLP map, or if an ordered library for the genome exists.

Unfortunately, there are numerous organisms in which mutants with interesting phenotypes have been isolated but for which such procedures have not been developed, such as the GA synthesis mutants of A. thaliana. Thus, many gene sequences cannot be isolated using the above methods.

C. Transgenic and Chimeric Plants Recent advances in recombinant DNA and genetic technologies have made it possible to introduce and express a desired gene sequence in a recipient plant. Through the use of such methods, plants have been engineered to contain gene sequences that are not normally or naturally present in an unaltered plant. In addition, these techniques have been used to produce plants which exhibit altered expression of naturally present gene sequences.

The plants produced through the use of these methods are known as either "chimeric" or "transgenic" plants. In a "chimeric" plant, only some of the plant's cells contain and express the introduced gene sequence, whereas other cells remain unaltered. In contrast, all of the cells of a "transgenic" plant contain the introduced gene sequence.

Transgenic plants generally are generated from a transformed single plant cell. Many genera of plants have been regenerated from a single cell.

(Friedt, W. etal. Prog. Botany 49:192-215 (1987); Brunold, C. etal., Molec.

Gen. Genet. 208:469-473 (1987); Durand, J. et al., Plant Sci. 62:263-272 (1989) which references are incorporated herein by reference).

Several methods have been developed to deliver and express a foreign gene into a plant cell. These include engineered Ti plasmids from the soil bacterium A. tumefaciens (Czako, M. et al., Plant Mol. Biol. 6:101-109 (1986); Jones, J.D.G. et al., EMBO J. 4:2411-2418 (1985), engineered plant viruses such as the cauliflower mosaic virus (Shah, D.M. et al., Science 233:478-481 (1986)); Shewmaker, C.K. et al., Virol. 140-281-288 (1985)), microinjection of gene sequences into a plant cell (Crossway, A. et al., Molec.

Gen. Genet. 202:179-185 (1986); Potrykus, I. et al., Molec. Gen. Genet.

199:169-177 (1985)), electroporation (Fromm, M.E. et al., Nature 319:791- 793 (1986); Morikawa, H. et al., Gene 41:121-124 (1986)), and DNA coated particle acceleration (Bolik, M. et al. Protoplasma 162:61-68 (1991)).

The application of the technologies for the creation of transgenic and *o.

10 chimeric plants has the potential to produce plants which cannot be generated using classical genetics. For example, chimeric and transgenic plants have substantial use as probes of natural gene expression. When applied to food crops, the technologies have the potential of yielding improved food, fiber, etc.

15 Chimeric and transgenic plants having a specific temporal and spatial pattern of expression of the introduced gene sequence can be generated. The expression of an introduced gene sequence can be controlled through the selection of regulatory sequences to direct transcription and or translation in a temporal or spatial fashion.

S. o 20 Summary of the Invention The invention is directed to isolated genomic DNA and cDNA corresponding to the GA1 locus of A. thaliana, vectors containing such DNA, hosts transformed with such vectors, the regulatory regions that control the expression of the GA1 protein, and the use of such regulatory sequences to direct the expression of a heterologous gene.

The invention further concerns the GAI protein, substantially free of other A. thaliana proteins, antibodies capable of binding the GA1 protein, and the use of such GAl protein and antibodies thereto.

The invention further concerns chimeric and transgenic plants transformed with the GAl encoding DNA sequence, or transformed with a heterologous gene controlled by the regulatory sequences of the GAl gene.

The invention further concerns the use of sequences encoding the GAI protein and antibodies capable of binding to the GAl protein to detect the expression of GAl and to isolate the regulatory proteins which bind to GAI

C

10 gene sequences.

Brief Description of the Figures Figure 1. A diagram of the enrichment and cloning method of the preferred embodiment of the present invention. DNA is depicted as a solid line; biotinylated DNA is depicted as a striped black/white line; Sau3a adaptors are 15 shown as an open line; avidin beads are shown as speckled circles; radiolabelled fragments are shown with asterisks.

Sc.* ""Figure 2. Genetic and physical maps of the A. thaliana GAl locus.

A: Genetic map in cM X 10 2 of nine A. thaliana ga-I alleles (29.9, NG4, d69, A428, d325, 6.59, Bo27, 31.89) (Koornneef et al., Genet. Res.

Camb. 41:57-68 (1983)). The presumptive deletion in 31.89 is indicated by the horizontal line.

B: Physical map of the GAl region. The heavy horizontal line is a HindII restriction map of the Landsberg erecta DNA encompassing the GA-1 locus.

HindIII restriction sites are depicted by vertical ticks extending below the ;I horizontal line. The numbers immediately below the heavy horizontal line represent the size, in kilobase pairs, of the respective HindlII restriction fragments. The location of the deletion in 31.89 is indicated by the hatched box. The horizontal lines above the restriction map indicate the extent of the sequences contained in the X clone XGA1-3, the plasmid pGA1-2 (deposited January 7, 1993 pursuant to the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms For The Purposes of Procedure (Budapest Treaty) with the American Type Culture Collection (ATCC) in Rockville, Maryland, U.S.A. 20852, and identified by ATCC 10 Accession No. 75394), and the cosmid clone pGA1-4 (deposited January 7, 1993 pursuant to the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms For The Purposes of Procedure (Budapest Treaty) with the American Type Culture Collection (ATCC) in S" Rockville, Maryland, U.S.A. 20852, and identified by ATCC Accession No.

15 75395). The diagram below the horizontal lines depicts the location of introns (lines) and exons (open boxes) of the GA1 gene within the 1.2 kb HindIII restriction fragment and the locations of the insertion mutation in allele 6.59 and the point mutations in alleles, d352, A428 and Bo27.

Figure 3. Detection of deletions and insertions in 31.89 and 6.59 DNA, 20 respectively. Autoradiograms are shown for Southern blots probed with (A) the 250 bp Sau3A fragment from pGAl-1 (see Example and the 6 kb fragment from pGA1-2 (ATCC No. 75394) that covers the entire deleted region in 31.89 (Figure 1B). Both blots A and B contain HindIII-digested DNA isolated from Landsberg erecta (lane and three ga-1 mutants, 31.89 (lane 29.9 (lane and 6.59 (lane The arrows in panel B indicate altered HindIII fragments in 31.89 (4.2 kb) and 6.59 (1.3 and 3.3 kb).

I

-12- Figure 4. Photograph and Southern blots of wild-type and transgenic plants containing GAl gene.

Photograph of six-week-old A. thaliana Landsberg erecta plants. Left to right: a ga-1 mutant (31.89), a transgenic ga-1 mutant (31.89) plant containing the 20 kb insert from pGA1-4 (ATCC No. 75395), a wild-type Landsberg erecta plant. Autoradiograms are shown for Southern blots probed with the 6 kb fragment from pGA1-2 (ATCC No. 75394), and pOCA18 DNA which is the vector for pGAl-4 (ATCC No. 75395) (see Figure Blots B and C contain HindIII-digested DNA from Landsberg erecta (lane 1 in B), S 10 Columbia (lane 2 in B, lane 1 in 31.89 (lane 3 in B, lane 2 in and two T3 generation transgenic ga-1 (31.89) plants transformed with pGA1-4 (ATCC No. 75395) (lane 4,5 in B; lane 3,4 in C).

Figure 5. Detection of a 2.8 kb mRNA using GA1 cDNA probes.

Autoradiogram of an RNA blot probed with a "P-labelled 0.9 kb GAl cDNA 15 or cab cDNA (chlorophyll a/b-binding protein gene). RNA was from wild type four-week-old plants (lane five-week-old wild type plants, (lane 2), immature wild-type siliques (lane and four week-old ga-1 mutant 31.89 plants (lane 4).

Figure 6. Partial cDNA sequence of the GAl gene (Sequence ID No. The GA1 DNA strand complementary to GAL mRNA is shown in a 5'-3' orientation. The GAl variant d352 has the identical sequence to that shown except for the substitution of an A for the G at position 425. The GA1 variant A428 has the identical sequence to that shown except for the substitution of a T for the C at position 420. The GA1 variant Bo27 has the identical sequence to that shown except for the substitution of a T for the C at position 246.

I i Figure 7. Partial cDNA sequence of the GA1 gene (Sequence ID No. 2).

The GA1 DNA strand shown is analogous to GA1 mRNA and complementary to the strand shown in Figure 6. The GA1 variant d352 has the identical sequence to that shown except for the substitution of a T for the C at position 479. The GA1 variant A428 has the identical sequence to that shown except for the substitution of an A for the G at position 484. The GA1 variant Bo27 has the identical sequence to that shown except for the substitution of an A for the G at position 658.

Description of the Preferred Embodiments 10 Using genomic subtraction, a gene involved in the synthesis of GA has Sbeen isolated. Genomic subtraction is a method for enriching, and clonally isolating a gene sequence present in one nucleic acid population but absent in another. Following the procedures outlined herein that demonstrate the cloning of the GAl gene, it is now also possible to isolate other genes involved in GA synthesis.

A. The GA-1 gene from A. thaliana Using the technique of genomic subtraction, a gene involved in the synthesis of GA, encoded by the GAl locus of A. thaliana, has been cloned (hereinafter the GAl gene, Example 1).

In one embodiment of the present invention, vectors containing genomic or cDNA encoding the GAl protein (Sequence ID No. or a fragment thereof, are provided. Specifically, such vectors are capable of generating large quantities of the GAl sequence, substantially free of other A.

thaliana DNA.

-14- 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 the GAl 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 yeast, insect, plant, mouse or human cells, and prokaryotic organisms, for example E. coi and B. sutillus.

As used herein, a sequence is said to be "substantially free of other A.

thlana DNA" when the only A. thalana DNA present in the sample or vector is of a specific sequence.

As used herein, a "DNA construct" refers to a recombinant, man-made

DNA.

As used herein, "a fragment thereof" relates to any polynucleotide subset of the entire GAl sequence. The most preferred fragments are those containing the active site of the enzyme encoded by GAl, or the regulatory regions of the GAl protein and gene respectively.

In a further embodiment of the present invention, expression vectors are described which are capable of expressing and producing large quantities of the GAl protein, substantially free of other A. thatiana proteins.

As used herein, a protein is said to be "substantially free of other A.

thalana proteins" when the only A. thaliana protein present in the sample is the expressed protein. Though proteins may be present in the sample which are homologous to other A. thalana proteins, the sample is still said to be substantially free as long as the homologous proteins contained in the sample are not expressed from genes obtained from A. thaliana.

A nucleic acid mole cule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression. The precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of gene synthesis. Such regions will normally include those coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.

10 If desired, the non-coding region 3' to the gene sequence coding for the GA1 gene may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3'-region naturally contiguous to the DNA sequence coding for the GA1 gene, the transcriptional 15 termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.

Two DNA sequences (such as a promoter region sequence and the GA1 gene encoding sequence) are said to be operably linked if the nature of the 20 linkage between the two DNA sequences does not result in the introduction "i of a frame-shift mutation, interfere with the ability of the promoter region sequence to direct the transcription of the GA1 gene sequence, or interfere with the ability of the GA1 gene sequence to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.

Thus, to express the GA1 gene transcriptional and translational signals recognized by an appropriate host are necessary.

-16- The present invention encompasses the expression of the GA1 gene 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 K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli W3110 lambda-- prototrophic (ATCC 27325)), and other enterobacterium such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species. Under such conditions, the GAl gene will not be glycosylated. The procaryotic host must be compatible with the replicon and control sequences in the expression plasmid.

To express the GA1 gene (or a functional derivative thereof) in a S.prokaryotic cell (such as, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.), it is necessary to operably link the GA1 gene encoding 15 sequence to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable inducible or derepressible).

Examples of constitutive promoters include the int promoter of bacteriophage X, the bla promoter of the B-lactamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of 20 pPR325, etc. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage X (PL and the trp, recA, lacZ, lad, and gal promoters of E. coli, the a-amylase (Ulmanen, et al., J.

Bacteriol. 162:176-182 (1985)) and the sigma-28-specific promoters of B.

subtilis (Gilman, et al., Gene 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward, et al., Mol. Gen. Genet. 203:468-478 (1986)).

I

-17- Prokaryotic promoters are reviewed by Glick, Ind. Microbiol.

1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442 (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, et al. (Ann.

Rev. Microbiol. 35:365-404 (1981)).

Preferred eukaryotic hosts include yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture. Mammalian cells which may be 10 useful as hosts include cells of fibroblast origin such as VERO or CHO-K1, 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.

15 For a mammalian host, several possible vector systems are available for the expression of the GAI 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, 20 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 which 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.

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-18- Yeast provides substantial advantages in that it can also carry out posttranslational peptide modifications. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids which 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 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 mediums 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 15 larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used. Rubin, Science 240:1453-1459 (1988).

Alternatively, baculovirus vectors can be engineered to express large amounts of the GA1 gene in insects cells (Jasny, Science 238:1653 (1987); Miller, et al., in Genetic Engineering (1986), Setlow, et al., eds., 20 Plenum, Vol. 8, pp. 277-297).

As discussed above, expression of the GAl 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 metallothionein I gene sequence (Hamer, et al., J. Mol. Appl. Gen. 1:273- 288 (1982)); the TK promoter of Herpes virus (McKnight, Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, et al., Nature (London) 290:304-310 (1981)); the yeast gal4 gene sequence promoter (Johnston,

S.A.,

-19et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the GA1 gene (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine AUG). The presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as the GAl 10 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 I 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 15 molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the GAl 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.

20 In one embodiment, a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which 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, 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 may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may 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. 3:280 (1983).

In a preferred 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 10 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. Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, 15 ColE1, pSC101, pACYC 184, rVX. Such plasmids are, for example, disclosed by Maniatis, 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: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include plJ101 (Kendall, et al., J. Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages such as 4C31 (Chater, et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John, et al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki, K. (Jpn.

J. Bacteriol. 33:729-742 (1978)).

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Botstein, et al., Miami Wntr. Symp. 19:265-274 (1982); Broach, In: -21- The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p.

445-470 (1981); Broach, Cell 28:203-204 (1982); Bollon, et al., J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, 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, 10 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 GAI gene, or fragments thereof.

15 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 GAI protein can be readily isolated using standard techniques such as immunochromatography or 20 HPLC to produce GAl protein free of other A. thaliana proteins.

By employing chromosomal walking techniques, one skilled in the art can readily isolate other full length genomic copies of GAI as well as clones containing the regulatory sequences 5' of the GAI coding region.

As used herein, "full length genomic copies" refers to a DNA segment which contains a protein's entire coding region.

As used herein, "regulatory sequences" refers to DNA sequences which are capable of directing the transcription and/or translation of an operably linked DNA sequence. Such regulatory sequences may include, but are not limited to, a promoter, ribosome binding site, and regulatory protein binding -22site. One skilled in the art can readily identify certain regulatory sequences by comparing sequences found 5' to a coding region with known regulatory sequence motifs, such as those recognized by the computer programs "motif" j and "consensus".

In detail, the GAl DNA sequences disclosed herein were used to screen an A. thaliana genomic DNA library via chromosome walking. Genomic DNA libraries for A. thaliana are commercially available (Clontech Laboratories Inc, and American Type Culture Collection) or can be generated using a variety of techniques known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989)). -By :isolating clones which overlap and occur 5' or 3' to the sequences disclosed herein, sequences hybridizing to Sequence ID No. 1 were identified and isolated. Such sequences are contained in the vectors PGA1-4 (ATCC No.

75395) and XGA1-3.

Regulatory sequences are those which occur 5' to a coding region.

The preferred regulatory sequences of the present invention are those which appear from about -2 kb 0 bp 5' of the GAl starting codon (AGTIMet). Thie more preferred sequences appear from about -500 bp -O bp, the most preferred being sequences from about -250 bp 0 bp.

20 Using techniques known in the art and the clones described herein, it *.is now possible to generate functional derivatives of the GA1 gene aswell as the regulatory sequence of this gene. Such derivatives allow one skilled in the art to associate a given biological activity with a specific sequence and/or structure and then design and generate derivatives with an altered biological or physical property.

The preparation of a functional derivative of the GAl sequence can be achieved 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 ___i1011 -23of a specific oligonucleotide which contains the desired mutated DNA sequence.

While the site for introducing a sequence variation is predetermined, the mutation perse need not be predetermined. For example, to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at a target region and the newly generated sequences can be screened for the optimal combination of desired activity.

The functional derivatives created this way may exhibit the same qualitative biological activity as the naturally occurring sequence when 10 operably linked to a heterologous gene. The derivative may however, differ

S

substantially in such characteristics as to the level of induction in response to phytohormones.

It is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so. One skilled in the art will recognize that the 15 functionality of the derivative can be evaluated by routine screening assays.

For example, a functional derivative made by site-directed mutagenesis can be operably linked to a reporter gene, such as /-glucuronidase (GUS), and the achimeric gene can then be quantitatively-screened for phytohormone responsiveness in chimeric or transgenic plants, or in a transient expression 20 system.

Using a reporter gene and the GA1 regulatory elements, mutations which alter tissue specificity and strength of the GA1 promoter can be generated. By analyzing the sequence of the GAl regulatory elements, one skilled in the art will recognize the various protein binding motifs present in the GAl promoter, and direct mutagenesis activity to these regions.

In another embodiment of the present invention, antibodies which bind the GA1 protein are provided.

In detail, an antibody which binds to the GAl protein can be generated in a variety of ways using techniques known in the art. Specifically, in one _Ir__l such method, GA1 protein purified from either an expression host or from plant tissue is used to immunize a suitable mammalian host. One skilled in the art will readily adapt known procedures in order to generate both polyclonal and monoclonal anti-GA1 antibodies. (Harlow, Antibodies, Cold Spring Harbor Press (1989)).

Alternatively, anti-GAl antibodies can be generated using synthetic peptides. Using the deduced amino acid sequence encoded by the GA1 gene described herein, a synthetic peptide can be made, such that when administered to an appropriate host, antibodies will be generated which bind 10 to the GAl protein.

In a further embodiment of the present invention, a procedure is described for detecting the expression of the GA1 gene or the presence of the GAl protein in a cell or tissue.

Specifically, using the antibodies and DNA sequences of the present 15 invention, one skilled in the art can readily adapt known assay formats such as in situ hybridization, ELISA, and protein or nucleic acid blotting Stechniques, in order to detect the presence of RNA encoding GA1, or the GAI protein itself. Utilizing such a detection system, it is now possible to identify the specific tissues and cells which transcribe or translate the GA1 gene.

9.

20 B. Transgenic or chimeric plants containing genes whose expression mimics the GAl gene.

In another embodiment of the invention, a method for creating a chimeric or transgenic plant is described in which the plant contains one or more exogenously supplied genes which are expressed in the same temporal and spatial manner as GAI.

In detail, a chimeric or transgenic plant is generated such that it contains an exogenously supplied expression module. The expression module comprises the regulatory elements of the GA1 gene, operably linked to a heterologous gene.

As described earlier, the regulatory region of the GA1 gene is contained in the region from about -2 kb to 0 bp, 5' to the GAl start codon (Met). One skilled in the art can readily generate expression modules containing this region, or a fragment thereof.

Methods for linking a heterologous gene to a regulatory region and the subsequent expression of the heterologous gene in plants are well known in the art. (Weissbach et al., Methods for Plant Molecular Biology, Academic Press, 10 San Diego, CA (1988)). One skilled in the art will readily adapt procedures for plant cell transformation, such as electroporation, Ti plasmid mediated transformation, particle acceleration, and plant regeneration to utilize the GAl regulatory elements. In an expression module all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can 15 be transformed with the expression module of the present invention. The efficacy of expression will vary between plant species depending on the plant Sutilized. However, one skilled in the art can readily determine the plant varieties in which the GAl regulatory elements will function.

In another embodiment of the present invention, a method of i 20 modulating the translation of RNA encoding GAl in a chimeric or transgenic *i plant is described.

As used herein, modulation entails the enhancement or reduction of the naturally occurring levels of translation.

Specifically, the translation of GAl encoding RNA can be reduced using the technique of antisense cloning. Antisense cloning has been demonstrated to be effective in plant systems and can be readily adapted by one of ordinary skill to utilize the GA1 gene. (Oeller et al., Science 254:437- 439 (1991)).

In general, antisense cloning entails the generation of an expression module which encodes an RNA complementary (antisense) to the RNA encoding GA1 (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.

In another embodiment of the present invention, a method of modulating the activity of the GAl protein is described.

Specifically, the activity of GAl can be suppressed in a transgenic or chimeric plant by transforming a plant with an expression module which 10 encodes an anti-GAl antibody. The expressed antibody will bind the free GA1 *and thus impair the proteins ability to function.

One skilled in the art will recognize that DNA encoding an anti-GAl antibody can readily be obtained using techniques known in the art. In general, such DNA is obtained as cDNA, generated from mRNA which has been 15 isolated from a hybridoma producing anti-GAl antibodies. Methods of obtaining such a hybridoma are described earlier.

A system for the study of gene expression in plants In another embodiment of the present invention, a method is described to identify the molecular interaction and the proteins responsible for the induction of the GA1 gene.

In detail, using the regulatory sequences of the GA1 gene, it is now possible to isolate the proteins which bind to these sequences.

Procedures for the isolation of regulatory factors capable of binding to a specific DNA sequence are well known in the art. One such method is affinity chromatography. DNA containing the regulatory sequence is immobilized on an appropriate matrix, such as Sepharose, and used as an affinity matrix in chromatography (Arcangioli et al., Eur. J. Biochem.

179:359-364 (1989)).

-27- Proteins which bind the GAl regulatory element can be extracted from plant tissues expressing the GAl gene. A protein extract obtained in such a fashion, is applied to a column which contains immobilized GAl regulatory region. Proteins which do not bind to the DNA sequence are washed off the column and proteins which bind to the DNA sequence are removed from the column using a salt gradient. The DNA binding protein obtained this way can be further purified using procedures such as ion exchange chromatography, high performance liquid chromatography, and size exclusion chromatography.

During the purification of the DNA binding protein, the protein can be 10 readily assayed using a gel retardation assay (Garner, M.M. etal., Nucl. Acid Res. 9:3047 (1981) and Fried, M. et al., Nucl. Acid Res. 9:6506 (1981)).

Once the DNA binding protein has been purified, a partial amino acid sequence can be obtained from the N-terminal of the protein. Alternatively, the protein can be tryptically mapped and the amino acid sequence of one of 15 the fragments can be determined.

Next, the deduced amino acid sequence is used to generate an oligonucleotide probe. The probe's sequence can be based on codons which are known to be more frequently used by the organism (codon preference), or, alternatively, the probe can consist of a mixture of all the possible codon 20 combination which could encode the polypeptide (degenerate).

Such a probe can be used to screen either a cDNA or genomic library for sequences which encode the DNA binding protein. Once the gene encoding the DNA binding protein has been obtained, the sequence of the DNA encoding the binding protein can be determined, the gene can be used to obtain large amounts of the protein using an expression system, or in mutational analysis can be performed to further define the functional regions within the protein which interacts with the DNA.

Alternatively, proteins which bind to the GAl regulatory elements can be isolated by identifying a clone expressing such a protein using the technique of Southwestern blotting (Sharp, Z.D. eta!., Biochzim BiophysActa, 1048:306.

309 (1990), Gunther, C. V. et Genes Dev. 4: 667-679 (1990), and Walker, M.D. et Nucleic Acids Res. 18:1159-1166 (1990)). In a Southwestern blot, a labeled DNA sequence is used to screen a cDNA expression library whose expressed proteins have been immobilized on a filter via colony or plaque transfer. The labeled DNA sequences will bind to colonies or plaques which express a protein capable of binding to the particular DNA sequence.

Clones expressing a protein which binds to the labeled DNA sequence can be :purified and the cDNA insert which encodes the DNA binding protein can be isolated and sequenced. The isolated DNA can be used to express large amounts of the protein for further purification and study, to isolate the genomnic sequences corresponding to the cDNA, or to generate functional derivative of the binding protein.

D. DNA Homologous to GAl Isolated From Other Plant Secies Using the DNA sequences isolated from A. thaliana thus far described, it is now possible to isolated homologous sequences from other plant varieties.

*5 Specifically, using the GAl DNA sequence of Sequence ID No. 1, or a fragment thereof, one skilled in the art can use routine procedures and screen either genomic or cDNA libraries from other plant varieties in order to obtain homologous DNA sequences. By obtaining homologous sequences, it is now possible to study the evolution of the GAl gene within the plant kingdom.

Additionally, by examining the differences in enzymatic activity of GA 1 isolated from a variety of sources and correlation the differences with -29sequence divergence, it is now possible to associate specific functional variations with regions within the protein.

The invention thus far described has been directed to the GAI gene.

One skilled in the art will recognize that the procedures described herein can be used to obtain DNA encoding other enzymes responsible for GA synthesis.

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.

S 10

EXAMPLES

EXAMPLE 1 *Genomic subtraction between A. thaliana Landsberg erecta DNA and g: al 31.89 DNA was performed as described previously (Straus and Ausubel, Proc. Natl. Acad. Sci. USA 87:1889-1893 (1990)) with the following 15 modifications.

9 A. thaliana Landsberg erecta DNA and gal mutant (31.89) DNA were isolated and purified by CsCI gradient centrifugation as described (Ausubel *et al., in Current Protocols in Molecular Biology, Vol. 1 (Greene Publishing Associates/Wiley-Interscience, New York, 1990)). In the first cycle of subtraction, 0.25 g of Landsberg erecta DNA digested with Sau3A was hybridized with 12.5 of the gal mutant 31.89 DNA that had been sheared and photobiotinylated. 10 pg of biotinylated 31.89 DNA was added in each additional cycle. Hybridizations were carried out for at least 20 hours at a concentration of 3 j/g DNA//l at 65"C. After five cycles of subtraction, the amplified products were ligated to Sau3A adaptors, amplified by PCR and ligated into the Smal site of pUC 13.

I

After five cycles of subtractive hybridization, the remaining DNA fragments were enriched for sequences present in wild-type DNA but missing from 31.89 DNA. These DNA fragments were amplified by the polymerase chain reaction (PCR) and cloned. One of six clones examined (pGAI-1) contained a 250 bp Sau3A fragment that was deleted from 31.89 DNA.

1 jig HindII-digested DNA from Landsberg erecta and gal alleles 31.89, 29.9, and 6.59 was fractionated on a 1 agarose gel, transferred to GeneScreen membrane (New England Nuclear), and probed with the 250 bp and 6 kb inserts in pGAl-1 and pGA1-2 (ATCC No. 75394) that had been 10 gel-purified and labelled with Figure 3. Hybridization conditions were the same as described in Church and Gilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995 (1984).

The insert in pGA1-1 hybridized to a 1.4 kb HindIII fragment in DNA samples isolated from wild-type Landsberg erecta and from the gal mutants 15 29.9 and 6.59 but did not hybridize to 31.89 DNA (Figure 3A).

To determine the extent of the deletion in 31.89 DNA identified by pGAI-1, pGA-1 DNA was used as a hybridization probe to isolate larger genomic fragments corresponding to the deletion in 31.89. These cloned fragments are shown in Figure 2B.

20 XGA1-3 was isolated from a Landsberg erecta genomic library constructed in XFIX (Voytas et al., Genetics 126:713-721 (1990)) using "Plabelled pGA-1 as probe. pGA1-2 (ATCC No. 75394) was obtained by ligating a 6 kb Sall-EcoRI fragment from XGA1-3 into the XhoI and EcoRI sites of pBluescriptlI SK (Stratagene). pGA1-4 (ATCC No. 75395) was isolated from a genomic library of A. thaliana ecotype Columbia DNA constructed in the binary vector pOCA18 (Olszewski et al., Nucl. Acid Res.

16:10765-10782 (1988)) which contains the T-DNA borders required for efficient transfer of cloned DNA into plant genomes (Olszewski et al., Nucl.

Acid Res. 16:10765-10782 (1988)).

-31- Plasmid pGAl-2 (ATCC No. 75394) containing a 6 kb fragment spanning the insert in pGAl-1 (Figure 2B), was used to probe a Southern blot containing HindIII-digested DNA from wild-type A. thaliana and from several gal mutants. As shown in Figures 3B and 4B, pGA1-2 (ATCC No. 75394) hybridized to four HindIII fragments (1.0 kb, 1.2 kb, 1.4 kb and 5.6 kb) in wild-type DNA that were absent in DNA from 31.89 mutants. The deletion mutation produces an extra HindIII fragment (4.2 kb) in 31.89 DNA. These results and additional restriction mapping (not shown) showed that the deletion in 31.89 DNA is 5 kb, corresponding to 0.005% of the A. thaliana genome 10 (5 kb/10 s kb) (See Figure 2B).

Three lines of evidence indicate that the characterized 5.0 kb deletion in mutant 31.89 corresponds to the GAl locus. First, RFLP mapping analysis carried out by the procedure detailed in Nam, H.G. et al. Plant Cell 1:699- 705 (1989), using XGA1-3 (Figure 2B) as a hybridization probe showed that 15 XGA1-3 maps to the telomere proximal region at the top of chromosome 4, consistent with the location to which the GAI locus had been mapped previously by Koornneef et al. Hered. 74:265-272 (1983)).

Second, a cosmid clone pGAl-4 (ATCC No. 75395) (Figure 2B), which contains a 20 kb insert of wild-type (Columbia) DNA spanning the 20 deletion in 31.89, complemented the ga-1 mutation in 31.89 as determined by the phenotype of Agrobacterium tumefaciens-mediated transformants (Figure 4A).

Agrobacterium tumefaciens strain LBA4404 containing pGA1-4 was used to infect root explants of gal mutant 31.89 and kanamycin-resistant (Km') transgenic plants were selected as described (Valvekens et al., Proc. Natl.

Acad. Sci. USA 85:5536-5540 (1988)). 130 Km' plants were regenerated which set seeds in the absence of exogenous GA (T1 generation). 50 to 300 seeds from each of 4 different T1 plants showed 100% linkage of the gal and -32- Km'phenotypes which segregated approximately 3:1 to the gal/Km" phenotype (T2 generation).

Seeds of transgenic gal and wild-type plants were germinated on agarose plates containing 1X Murashige Skoog salts and 2% sucrose with or without kanamycin (MS plates). Seeds of the gal mutant 31.89 were soaked in 100 pM GA 3 for 4 days before being germinated on MS plates. Seven-dayold seedlings were transferred to soil.

To show the dwarf phenotype, no additional GA 3 was given to the mutant 31.89 after germination. Southern blot analyses were carried out as 10 described for Figure 3. The insert in pGAl-4 (ATCC No. 75395) was isolated from the Columbia ecotype. As seen in lanes 1 and 2 in panel B, pGA1-2 (ATCC No. 75394) detected an RFLP between the Landsberg (5.6 kb) and Columbia (5.0 kb) DNAs. The DNA in lanes 1, 2, and 3 in panel B was purified by CsCI density gradient centrifugation whereas the DNA in lanes 15 4 and 5 in panel B was purified by a miniprep procedure. This explains the minor differences in mobilities of the hybridizing bands in lanes 1, 2, and 3 compared to lanes 4 and Several independent T2 generation transgenic plants, containing the insert of pGA1-4 (ATCC No. 75395) integrated in the 31.89 genome, did not 20 require exogenous GA for normal growth. Germination, stem elongation, and seed set were the same in the transgenic plants as in the wild-type plants without exogenous GA treatment. Southern blot analysis, using the 6 kb fragment from pGA1-2 (ATCC No. 75394) as a probe, indicated that both the endogenous gal 31.89 locus (4.2 kb) and wild-type GAl DNA 1.4, 1.2 and 1.0 kb HindIII fragments) were present in two independent T3 generation transgenic plants (Figure 4B).

Further Southern blot analysis, using the vector pOCA18 which contains the T-DNA border sequences as a probe, showed that only two border fragments were present in the genomes of both transgenic plants (Figure 4C). These results indicated that the wild-type GAI DNA was integrated at a single locus in the genomes of both transgenic plants.

Third, to obtain unequivocal evidence that the 5.0 kb deletion in 31.89 corresponds to the GAI locus, we showed that four additional gal alleles contain alterations from the wild-type sequence within the region deleted in 31.89 in the order predicted by the genetic map. To aid in this analysis, a partial GAI cDNA clone (0.9 kb) (Sequence ID No. containing poly A and corresponding to the 1.2 kb HindIII fragment (Figure 2B), was isolated from a cDNA library constructed from RNA isolated from siliques (seed pods) of 10 A. thaliana ecotype Columbia. Four exons and three introns in the 1.2 kb HindIII fragment were deduced by comparison of the cDNA and genomic DNA sequences (Figure 2B, sequence data not shown). The identification of this cDNA clone showed that the 1.2 kb HindIII fragment is located at the 3' end of the GAI gene and suggested that the mutations in the gal alleles 31.89, 15 Bo27, 6.59, d352, and A428 should also be located at the 3' end of the GAI gene.

In addition to the 31.89 allele, two other gal alleles, 6.59 and 29.9, were induced by fast neutron mutagenesis (Koorneef et al., Genet. Res.

Camb. 41:57-68 (1983)). As shown in Figure 3B, the 1.2 kb HindIII 20 fragment in 6.59 DNA was replaced by two new fragments of 1.3 kb and 3.3 kb without alteration of the adjacent 1.4 kb and 5.6 kb fragments. Further Southern blot analysis and direct DNA sequencing of PCR products from 6.59 DNA templates indicated that the 6.59 allele contains a 3.4 kb or larger insertion in the 1.2 kb HindII fragment in the last intron defined by the cDNA clone (Figure 2B). Southern blot analyses, using pGA1-2 (ATCC No. 75394) (Figure 2B) and pGAl-4 (ATCC No. 75395) as probes, showed that there are no visible deletions or insertions in 29.9 DNA. Three additional gal alleles, A428, d352 and Bo27, are located at or near the 6.59 allele on the genetic map (Figure 2A). Direct sequencing of PCR products amplified from Bo27, A428, and d352 mutant DNA templates revealed single nucleotide changes within the last two exons in the 1.2 kb HindIII fragment in all three mutants (Figure 2B). Mutant Bo27, which defines one side of the genetic map, contained a single nucleotide change in the most distal GAl exon. The single nucleotide changes in mutants Bo27, A428, and d352 result in missense mutations, consistent with the leaky phenotypes of mutants A428 and d352 (Koornneef et al., Genet. Res. Camb. 41:57-68 (1983)). It is unlikely that the base changes observed in mutants Bo27, A428, and d352 are PCR artifacts or are due to the highly polymorphic nature of the GAl locus because the 1.2 kb 10 HindIII fragment amplified and sequenced from mutants NG4 and NG5 both had the wild-type sequence. Moreover, the PCR products were sequenced directly and the sequence analysis was carried out twice using the products of two independent amplifications for each allele examined.

We used the recombination frequency between different gal alleles 15 reported by Koornneef et al. (Genet. Res. Camb. 41:57-68 (1983)) to calculate that the recombination frequency per base pair is approximately 10 5 cM within the GAl locus. This calculation is based on the reported recombination frequency of 0.5 X 10.2 cM between gal alleles A428 or d352 and Bo27 (Figure 2A) and our observation that the mutations in d352 and Bo27 and in 20 A428 and Bo27 are separated by 432 and 427 bp, respectively. This calculation suggests that the extent of the entire GAl locus defined by mutants 29.9 and Bo27 is approximately 7 kb. The predicted size of this locus can accommodate the 2.8 kb mRNA detected in wild-type plants using the GAl cDNA as a hybridization probe (Figure Poly(A)+RNA of four-week-old and five-week-old plants was prepared from the entire plant except the roots and silique RNA was prepared from immature siliques plus some flower buds and stems as previously described (Ausubel et al., in Current Protocols in Molecular Biology, Vol. 1 (Greene Publishing Associates/Wiley-Interscience, New York, 1990); Maniatis et al., in Molecular Cloning: A Laboratory Manual, 197-201 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982)). Approximately 2 micrograms of RNA of each sample was size-fractionated on a 1 agarose gel (Maniatis et al., in Molecular Cloning: A Laboratory Manual, 197-201 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982)), transferred to GeneScreen membrane, and hybridized with a 3 P-labelled 0.9 kb EcoRI DNA fragment from the GAl cDNA (Figure The RNA blot was also hybridized with a "P-labelled 1.65 kb EcoRI fragment containing the A. thaliana cab gene (AB 165) (Leutwiler et al., Nucl. Acid Res. 14:4051-4064 -(1986)).

10 Decreased hybridization of the cab probe in lane 3 reflects the fact that the cab gene is not highly expressed in siliques.

As expected, the 2.8 kB RNA could not be detected in the deletion mutant (figure The linkage map ofA. thaliana is approximately 600 cM and the genome size is approximately 1.08 x 10' bp (Goodman et at., 15 unpublished results). This is equivalent to approximately 6 x 10 6 cM per base pair, in good agreement with the observed recombination frequency in the GAl locus.

Cloning the A. thaliana GAl gene presented a variety of experimental opportunities to investigate the regulation and the site of GA biosynthesis.

20 Because ent-kaurene is the first committed intermediate in GA biosynthesis, it is likely that the GAl gene, required for the formation of ent-kaurene, is a point of regulation for GA biosynthesis (Graebe, Ann. Rev. Plant Physiol. 38:419-465 (1987); Moore, in Biochemistry and Physiology of Plant Hormones, 113-135 (Springer-Verlag, New York, 1989)). Indeed, the biosynthesis of ent-kaurene has been shown to occur preferentially in rapidly developing tissues, such as immature seeds, shoot tips, petioles, and stipules near the young elongating internodes (Moore, in Biochemistry and Physiology of Plant Hormones, 113-135 (Springer-Verlag, New York, 1989); Chung and Coolbaugh, Plant Physiol. 80:544-548 (1986)).

-36- Genomic subtraction is not labor intensive and the results reported here indicate that genomic subtraction could be routinely used to clone other nonessential A. thaliana genes if a method were available for generating deletions at high frequency. In addition to the gal deletion in mutant 31.89 induced by fast neutron mutagenesis (Koornneef et al., Genet. Res. Camb. 41:57-68 (1983); Dellaert, "X-ray- and Fast Neutron-Induced Mutations in Arabidopsis thaliana, and the Effect of Dithiothreitol upon the Mutant Spectrum," Ph.D. thesis, Wageningen (1980); Koornneef et al., Mutation Research 93:109-123 (1982)), X-ray- and -y-ray- irradiation have also been *10 shown to induce short viable deletions in A. thaliana at the chl-3 (Wilkinson and Crawford, Plant Cell 3:461-471 (1991)), tt-3 Shirley and H. M.

Goodman, unpublished result) and gl-1 loci Marks, personal communication).

9* 4 EXAMPLE 2 9* 15 Expression of Antisense GA1 RNA.

.i An expression vector is constructed as previously described such that it expresses an RNA complementary to the sense strand GA1 RNA. The antisense GA1 RNA is expressed in a constitutive fashion using promoters which 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 BamH1 and SAC1 to remove the tACC2 cDNA sequence. After removing the tACC2 cDNA, the vector is treated with ^pr- the Klenow fragment of E. coli DNA polymerase I to fill in the ends, and the sequence described in SEQ ID. NO. 1 is blunt end ligated into the vector.

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 GA1 cDNA in the orientation which will produce antisense RNA when transcribed from the 35S promoter contained in the vector.

The antisense GA1 vector is isolated from a colony identified as having the proper orientation and the DNA is introduced into plant cells by one of the 10 techniques described earlier, for example, electroporation or Agrobacterium/Ti plasmid mediated transformation.

Plants regenerated from the transformed cells express antisense GA1 RNA. The expressed antisense GA1 RNA binds to sense strand GA1 RNA and thus prevent translation.

*o*o 15 CONCLUSION While the invention has been described in connection with specific

O

embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

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NAME: Cimbala, Michele

A.

REGISTRATION NUMBER: 33,851 REFERENCE/DOCKET NUMBER: 0609.37S0004 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (202) 466-0800 TELEFAX: (202) 833-8716 INFORMATION FOR SEQ ID NO:l: SEQUENCE

CHARACTERISTICS:

LENGTH: 903 base pairs TYPE: nucleic acid STRANDEDNESS- both TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CTGCAGGAAT TCCTTTTTTT

TATATATACT

CCAACACAcA

AGAATCGTAC

TACTTCGAAT

CCGGAAXAC

AACGACGCAT

CCAAGAACCA

AGTTTTATCT

TTCCCCGTCC

ATTACTATTA

ATGTATTAGA

TTGAAATATA

AACCTTGTAA

GGTACATCAA

CCGATrGGCA

GTGATTCCTT

GCAAGGGTAT

TCGGAAAGTT

CCGGCATCGA

GTTAGGTTTC

CTTCCAACAC

GGCAAATCAT

TTT1TITTTT

TTCATATAAA

GCTTTAGCTC

TGTTTATTCC

TATGCTCATC

TGTTGCATTG

TGATGAGACG

GGTTCTCGGC

TCAATGCAAC

TCAAGATCGT

TAATCTGAGG

GTTGAACCTC

TGGCTTTGAG

AATAGAATGT

TrTCTTGGCG

TCGAGCTATC

ATTTTCGTCT

ATGAGGAAAC

ATCATGATAA

GATCCATTTC

CCAAGCTGTA

TTTCACACTC

AGCATCTTCT

TTGAGAATTA

TGAAGTACAT

TCAAATGTAT

TATATATCrI'

TCAAGCAACG

TCTAGCTTCC

AGATTCCATG

GAGAAGAGAT

ACGGCGGAGG

TCGTAAGCCG

TTCACTGCTT

CCTTGAAGCT

ATGTATTCTT

AGGACCCATC

ATTTTTTGCc

TTAAGACCGG

ATGGGAATGC

CAATATrrrc

ATCTTAGAGC

ACGCATCTCC

GAAACGCCGG

ATATCGTAAT

CTTTGAATc

GTTGCCACTC

GAGTTCGAAG

120 180 240 300 360 420 480 540 600 720 CTTTGAACAAJ TGTATGGAAC CG;CITTCTGCA -rTTGTCTCTA GCGACATTGA

GAGGAGATCC

39 TGAGATGGTA AGGAAJAGAAG AAGATATTGT TG;TTTTAGTA GAACTGAGAA AGGTGTACT TGGAATGGAG TTTAGAACAT GATACTGAAG AGACATGGCT TTAAAAAAAp AAAAA.AGGAA

T'TC

INFORMATION FOR SEQ ID NO:.2: SEQUENCE CHARACTERISTICS: LENGTH: 903 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GAATTCrCTTT 7TTTTTTTTT

S.

CCAAGTACAA

TCAGGATCTC

AAGCTTCGAA

CATGACGC

A.ATGCATTCA

GAAATTACGA

ACTCCGGCGT

TGGGGAGATG

GTTGCTCTAA

CGGGAAAATA

GTAGCATTCC

TCTCCGGTCT

TGGGCCAAAA

ATAGATGGGT

CAG

CCTTTCTCAG

CTCTC.AATGT

CTCAAGAATA

AACAGCTTCA

AAGAAGCAGT

TATCGGCTTA

TTCCCTCCGC

CGTATCTCTT

GATCATGGAA

TTGGGAAGCT

CATCGTTGCT

TAAAAGATAT

AATATACATT

CCTATGTACT

TAAACCCATG

TTCTACTAA

CGCTAGAGAC

CATTAATrCT

AGGAGAAGAT

GAAGAGTGTG

CGATACAGCT

CGTGAAATGG

CTCTTATCAT

TCTCTTC

AGAAGACAA

TGAGATAGCT

ATACGCCAAG

TGAACATTCT

TCACTCAAAG

TCTCTTCAGT

ACAACAATAT

AAATCCAGAA

CAAGAGGTTC

GCTCCTCAGA

AAAACGATCT

TGGGTrGCAT

ATCGCCGAGA

GATCGTCTCA

CATCAATGCA

AATGATGAGc CGAG43AATAA

AAAGAGCTAA

ATTTTTATAT

CCAAAAAAAA

ATCATGTTCT AAACTCCATT CTTCTTCTTT CCTTACCATC GCGGTTCCAT ACATTGTTCA

AACATGATTT

TTAGTGTTGG

TGAGAAACCT

TGATCGATGC

ACChACTTC

TCAATACCCT

ACAAAGGAAT

ATATGCCAAT

ACATTGATGT

AGCTTACAAG

GAATATATT

AAAAAAAAAA

GCCTCTAATA

AAGTAATAGT

AACGGACGGG

CGGAGATAAA

CGATGGTTCT

TGCATGCGTC

CACGTTTTTC

CGGATTCGAA

ACCGTACGAT

GTTrGTGTGT

CAAAGTATAT

GGAATTCCTG

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description on page 11 ,line B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet Name of depositary institution American Type Culture Collection Address of depositary institution (including postal code and country) 12301 Parklawn Drive Rockville, Maryland 20852 United States of America Date of deposit Accession Number 07 January 1993 75394 o

I

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet Plasmid DNA, pGAl-2 00 In respect of those designations in which a European Patent is sought a sample of the deposited microorganism will be made available until the publication of :0 the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28(4) EPC) D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if theindications are not forall designatedStates) e E. SEPARATE FURNISHING OF INDICATIONS (leave blank ifna applicable) The indications listed belowwill be submitted to the International Bureau later (spify he gencral naure oftheindications 'Accession Number of Deposit') For receiving Office use only For International Bureau use only [P This sheet was received with the international application j Thissheet was received by the International Bureau on: Authoi d officer Form l'CT/RO/134 (July 1992) Authorized officer -41- INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description on page 11 ,line B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet 0 Name of depositary institution American Type Culture Collection Address of depositary institution (including postal code and country) 12301 Parklawn Drive Rockville, Maryland 20852 United States of America DNA, pGA-4 Date of deposit Accession Number 07 January 1993 75395 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet |jJ Cosmid DNA, pGAl-4 In respect of those designations in which a European Patent is sought a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28(4) EPC).

S D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (iftlie indications are not for all designated Slates) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications 'Accession Number of Deposit') For receiving Office use only For International Bureau use only This sheet was received with the international application I This sheet was received by the International Bureau on: Authorized officer Authorized officer li:rm il'Cf/RO/134 (Jul i9Q 2)

Claims (24)

1. A DNA construct comprising the DNA sequence of the GAI gene of Arabidopsis thaliana.

2. The regulatory sequences of the GAI gene, substantially free of DNA sequence encoding the GAI protein.

3. A vector containing the sequences of claims 1 or 2.

4. A host transformed with one of the vectors of claim 3.

5. The host of claim 4, wherein said host is selected from the group consisting of bacteria, yeast, plants, insects or mammals.

6. A method of directing the expression of a gene in a plant, such that said gene has the same temporal and spatial expression pattern of GAI, said method comprising the steps of: 1) operably linking said gene to the regulatory sequences of GAI to create an expression module, and 2) transforming said plant with said expression module

7. The method of claim 6 wherein said regulatory sequences comprise the sequences from about -2 kb to 0 bp, 5' of the GAI coding region. -43-

8. The method of claim 6 wherein said regulatory sequences comprise the sequences from about -500 bp to 0 bp, 5' of the GA1 coding region.

9. The method of olaim 6 wherein said regulatory sequences comprise the sequences from about -250 bp to 0 bp, 5' of the GA1 coding region.

A method of modulating the translation of RNA encoding GAl in a plant comprising the steps of: 1) generating an expression vector encoding antisense GA1 RNA; 2) transfecting said plant with said expression vector .4*o

11. A method of modulating the activity of the GA1 protein in a plant comprising the steps of 1) generating an expression vector encoding an antibody, or fragment thereof, capable of binding to the GAI protein; 2) transforming a plant with said expression vector. g*

12. An antibody, or fragment thereof, capable of binding the GA1 protein.

13. A protein capable of binding to the regulatory region of the GAI gene.

14. A method of identifying cells or tissues expressing GAl comprising the steps of: IF] 1) incubating said cells or said tissues with an agent capable of binding to the GA 1 protein or the RNA encoding GA 1; and 2) detecting the presence of bound agent.

The method of claim 14 wherein said agent capable of binding to the GA 1 protein is an antibody, or fragment thereof.

16. The method of claim 14 wherein said agent capable of binding to the RNA encoding GA 1 is selected from the group consisting of RNA and DNA.

17. The regulatory sequences of the GA1 gene of claim 2 wherein the GA1 gene is of Arabidopsis.

18. The method of claim 6 wherein the GA1 is of Arabidopsis.

19. The method of claim 10 wherein the RNA encoding GA1 is of Arabidopsis.

The method of claim 11 wherein the GA1 protein is of Arabidopsis.

21. The antibody, or fragment thereof, of claim 12 wherein the GA1 protein is of Arabidopsis.

22. The protein of claim 13 wherein the GA1 gene is of Arabidopsis.

23. The method of claim 14 wherein the GA1 is of Arabidopsis. 28/10/99

24. A DNA construct comprising the DNA sequence of the GA1 gene of Arabidopsis thaliana substantially as hereinbefore described with reference to the accompanying drawings. Dated this 28th day of October 1999 THE GENERAL HOSPITAL CORPORATION Patent Attorneys for the Applicant PETER MAXWELL ASSOCIATES a.