CA2567357A1 - Anther-specific cdna sequences, genomic dna sequences and recombinant dna sequences - Google Patents

Abstract

cDNA sequences are disclosed which are expressed specifically in the anther of a plant.
Genomic DNA sequences corresponding to the cDNA clones are obtained using the cDNA clones as hybridization probes. Recombinant, or chimeric, DNA sequences are constructed in which the promoter sequence from anther-specific genomic clones are operatively linked to a DNA sequence coding for a desired polypeptide.
Transgenic plants are made in which the chimeric DNA sequences are expressed in the anther of the transgenic plant. In a preferred embodiment, the coding DNA sequence expresses a polypeptide which will disrupt formation of viable pollen, resulting in a male-sterile plant.

Description

ANTHER-SPECIFIC cDNA SEQUENCES, GENOMIC DNA
SEQUENCES AND RECOMBINANT DND SEQUENCES
This application is a division of application 2,099,482 filed June 30, 1993.
The present invention is in the field of plant genetic engineering. It primarily relates to novel DNA sequences which function as promoters of anther-specific transcription of associated expressible and preferably coding DNA sequences in recombinant or chimeric DNA sequences. The present invention also relates to recombinant or chimeric DNA
sequences which are expressed specifically in the anther of a plant. The said recombinant or chimeric DNA sequences may be used to create transgenic plants, but especially transgenic male-sterile plants. The invention further relates to anther-specific cDNA
sequences and genomic DNA sequences which may be suitably used in the isolation of the anther-specific promoter sequences according to the invention.
The creation of male sterile plants is of economic interest in the production of hybrid seeds. Male sterility prevents self pollination which otherwise occurs in many plant species and hinders breeding and hybrid seed production.
Transcription of many plaJlt genes is controlled in a temporal and spatial manner.
Regulation of gene activity is mediated by the interaction of traps acting factors and cis regulatory elements in the promoter region of a gene.
Of particular interest are genes which are expressed primarily or exclusively in the sexual tissue of the plant, such as anther or pollen tissue. Such genes can be used to express polypeptides that are not naturally produced in the anther or pollen. For example, the promoter region from an anther specific gene may be used to express a polypeptide which will disrupt formation of vi;~ble pollen when expressed in the anther cells, resulting in a male sterile plant.
European Patent Application 0 420 819 Al describes the use of wunl gene to produce male sterile plants.

United States Patent 5,086,169 describes the isolation of the promoter region from the Zml3 clone of a pollen-specific gene of corn, and its use to express genes in pollen.
PCT WO 89/10396 describes the use of male-sterility DNAs and anther-specific cDNAs TA13, TA26 and TA29. The developmental expression profiles of TA13 and TA29 matched two cDNA clones isolated by the applicants, ANTS and ANT45, respectively.
PCT WO 90/08825 describes three gene sequences, pMS 10, pMS 14 and pMS 18, and their use in recombinant DNA sequences with the GUS reporter gene. No evidence of expression is given.
PCT WO 90/08831 describes a disrupter gene known as the mammalian uncoupling protein (UCP) gene which inhibits respiration in cells.
PCT WO 90/08828 describes molecular methods of hybrid seed production in which recombinant DNA molecules using pollen specific promoters are used to control the production of fertile pollen in plants.
It is therefore the main object of the present invention to provide DNA
sequences which are capable of directing the anther-specific expression of genes in plants. It is a further object of the invention to provide recombinant or chimeric DNA sequences which are expressed specifically in the anther of a plant and vectors comprising the said recombinant or chimeric DNA sequences. The said DNA sequences or vectors may be used to create transgenic plants, but especially transgenic male-sterile plants.
This object could be surprisingly achieved within the scope of the present invention by isolating anther-specific cDNA sequences and genomic DNA sequences, which may be suitably used for the preparation of anther-specific promoter DNA sequences and ligating the said promoter sequences in an operable manner to expressible DNA sequences that encode a desired expression product.
Thus, according to the present invention, novel DNA sequences are provided which are expressed specifically in the anther of a plant. In particular, several cDNA
sequences are provided which may be used as probes to isolate anther-specific genomic DNA
sequences.
In a specific embodiment of the present invention, two genomic DNA sequences are 300~~-41 provided which are expressed specifically in the anther of a plant. Anther-specific promoter DNA sequences are isolated from these genomic clones and are ligated to expressible DNA sequences, which preferably encode a desired protein product, to provide chimeric vectors that: are specifically expressed in the anther of a plant.
The present invention thus further relates to anther-specific cDNA sequences and genomic DNA sequences.
In one embodiment, the present invention comprises an isolated nucleotide sequence consisting essentially of an anther-specific cDNA sequence. The cDNA sequence may be obtained by differential screening of cDNA libraries and selecting those cDNA
clones which are observed to be expressed in a highly specific manner in anther tissue. The cDNA clones of the present invention may have the DNA sequences of SEQ LD No.
1, SEQ. ID No. 3, SEQ.117 No. 5, SEQ.117 No. 7, SEQ. ID No. 9, SEQ. ID No. 11, SEQ. 1D
No. 13, SEQ. 1D No. 14 and SEQ. m No. 20.
In another embodiment, the present invention comprises an isolated genomic DNA
sequence which essenrially consists of a DNA sequence that corresponds to an anther-specific cDNA clone; and is thus capable of hybridizing with the said anther-specific cDNA. The genonvc DNA sequences of the present invention may thus be preferably obtained by hybridization of a genomic library with the anther-specific cDNA
sequence used as a probe. The genomic DNA sequences of the present invention may have the DNA sequences of SEQ. ID No. 16 and SEQ. ID No. 18, or they may be obtained by hybridization with a cDNA having the DNA sequence of SEQ. ID No. 1, SEQ. ID
No.
3, SEQ. ll7 No. 5, SEQ. ID No. 7, SEQ. ll~ No. 9, SEQ. ID No. 11, SEQ. ID No.
13, SEQ.
ID No. 14 and SEQ. m No. 20.

- 3a -These genomic DNA clones are being preferably used as a source for the isolation of DNA sequences which function as promoters of anther-specific transcription of associated expressible and preferably coding DNA sequences in recombinant or chimeric DNA constructs.
It goes without saying that the DNA sequences according to the invention, once having been identified, do not have to be newly isolated each time from a suitable source but can of course be synthesised very easily at any time by means of kncwn chemical processes. Suitable processes for the synthesis of short DNA oligonucleotides, for example the phosphotriester or phosphite method, are known to the person skilled in the art. Today, the majority of oligonucleotide syntheses are mechanised and automated, so that short DNA fragments can be produced in a short period of time.
By deletion, insertion or substitution of one or more base pairs in the above-mentioned DNA sequences according to the invention, therefore, variants or mutants of those sequences can very easily be produced and checked for their suitability as an anther-specific promoter sequence.
An especially suitable method of producing specific mutants is so-called oligonucleotide-mediated mutagenesis. In that method, short oligonucleotide fragments are synthesised which, although substantially homologous to the wild-type sequence, differ therefrom in individual nucleotides. The said differences may be insertions, deletions, inversions or a substitution of one or more nucleotides, or they may be a combination of the above-mentioned procedures. These mutated fragments are then substituted for the homologous counterparts on the wild-type gene by generally known methods. The final construct can then, as described above, be cloned into a suitable plant expression vector and transformed into a plant.
However, the mutation of certain DNA fragments can also preferably be carried out using the polymerise chain reaction [PCR]. In this in vitro process there are used chemically synthesised oligonucleotides which generally originate from the peripheral regions of the DNA fragment to be mutated and are strand-specific. Under suitable conditions, hybridisation of the oligonucleotides with the complementary regions on the DNA single strands produced by denaturing occurs. The double-stranded regions produced in this manner are used as primers for the subsequent polymerise reaction.
In this process there may be used in addition to DNA polymerises from E. coli especially heat-stable polymerises from thermophilic bacteria, for example Thermos aquaricus.
The present invention is therefore not limited to those promoter sequences that can be derived from the genomic DNA sequences according to the invention, but also includes all mutants and/or variants of those DNA sequences that can be produced by deletion or insertion of one or more bases or, especially, by the substitution of one or more bases, and that still have the specific properties, according to the invention, of the starting sequences.
In yet another embodiment, the present invention comprises isolated recombinant, or chimeric, DNA sequences in which a promoter region obtainable from an anther-specific genomic DNA sequence is operatively linked to an expressible DNA sequence which preferably encodes a protein which is desired to be expressed in the anther cells. For example, the isolated recombinant DNA sequences of the present invention may comprise, in a 5' to 3' direction, a promoter region obtainable from an anther-specific genomic DNA
sequence operatively linked to a DNA sequence which encodes a polypeptide which will disrupt formation of viable pollen when expressed in the anther cells. The resulting plant will not be able to produce viable pollen cells, and hence will be male sterile. Examples of such a recombinant DNA sequence include chimeric vectors in which an anther-specific promoter is operatively linked to a DNA sequence which encodes a polypeptide selected from the group consisting of the coding sequence from the DTA, TURF-13, pectate lyase, gin recombinase, iaaL or cytA toxin genes.
The present invention thus further relates to a process for the production of a recombinant DNA molecule comprising, :in a 5' to 3' direction, an anther-specific promoter region operatively linked to an expressible DNA sequence, which process comprises (a) first isolating from a suitable source or synthesising by means of known processes a promoter region responsible for the anther-specific expression of an associated expressible DNA sequence; and (b) operably linking the said anther-specific DNA sequence in a 5' to 3' direction to an expressible DNA sequence.
In order to specifically direct the location of the peptide encoded by the recombinant DNA
sequence within the plant cell, the recombinant DNA sequence of the present invention may comprise, in a 5' to 3' direction, an anther-specific promoter region operably linked to a signal sequence, which is operably linked to a coding DNA sequence.

- 5a -In another aspect, the invention provides a process for the production of transformed plant material, including whole plants, comprising a recombinant DNA molecule comprising, in a 5' to 3' direction, an anther-specific promoter region operatively linked to an expressible DNA sequence, which process essentially comprises: (a) first isolating from a suitable source or synthesising by means of known processes a promoter region responsible for the anther-specific expression of an associated expressible DNA sequence; (b) operably linking the said anther-specific DNA sequence in a 5' to 3' direction to an expressible DNA sequence;
(c) cloning the final construct into a plant expression vector under the control of expression signals active in plants; (d) transforming the said expression vector into plant material by means of known processes and expressing it therein; and optionally (e) regenerating the plant material transformed according to step (d) to a whole and preferably phenotypically normal plant.
Another embodiment of the present invention comprises plasmids containing anther-specific promoter sequences of the present invention but especially recombinant, or chirrleric, DNA sequences in which a promoter region obtainable from an anther-specific genomic DNA sequence is operatively linked to an expressible DNA sequence which ~>referably encodes a protein which is desired to be expre~;sed in the anther cells. These plasmids include, but are not. limited to, pCIB3132, pCIB3132B, pCIB3178, pCIB3179, and pLC251. The present invention also includes promoter fragments that may be derived from the plasmids of the pre~;ent invention, which still exhibit those characteristics that. are essential for carrying out the invention, that is, which are capable of driving the expression of an associated DNA sequence in an anther-specific manner.
For the purpose of the present invention, the term "derived from" a plasmid refers to the physical isolation of a nucleotide sequence or fragment from a plasmid, as well as the physical isolation of a nucleotide sequence or fragment using a probe homologous to one of the above plasmids, or a synthetic nucleotide sequence prepared by using some or all of the nucleotide sequences of the above plasmids.
Another embodiment of the present invention comprises transgenic: plants and the sexual and/or asexual progeny thereof, which plants have been transformed with a recombinant, or chimeric, DNA sequence comprising an anther-specific promoter sequence according to the invention and, optionally, a signal sequence operatively linked in a 5' to 3' orientation to an expressible DNA sequence, but preferably to a coding DNA sequence. Such transgenic plants will express the pol.ypeptide coded by the chimeric DNA sequence only in the anther of the plant. When the polypeptide encoded is a polypeptide which will disrupt formation of viable pollen when expressed in the anther cells, the transgeni.c plant will not be able to produce viable pollen cells, and hence will be male sterile. For example, such transgenic plants may encode for DTA, TURF-13, pectate lyase, gin recombinase, iaaL or cytA toxin.
It is thus still a further embodiment of the present invention to provide male-sterile plants transformed with one of the above recombinant, or chimeric, DNA sequences.

- 6a -The present invention further comprises a process for the production of transformed plant material, including whole plants, comprising a recombinant DNA molecule comprising, in a 5' to 3' direction, an anther-specific promoter region operatively linked to an expressible DNA sequence, which process essentially comprises:
(a) first of all isolating from a suitable source or synthesising by means of known processes a promoter region responsible for the anther-specific expression of an associated expressible DNA sequence;
(b) operably linking the said anther-specific DNA sequence in a 5' to 3' direction to an expressible DNA sequence;
(c) cloning the final construct into a plant expression vector under the control of expression signals active in plants;

(d) transforming the said expression vector into plant material by means of known processes and expressing it therein; and optionally (e) regenerating the plant material transformed according to step (d) to a whole and preferably phaenotypically normal plant.
Tla~ ni tinn c "Anther-specific" is used to describe cDNAs, genomic DNAs, messenger RNAs, promoter DNA sequences and genes which are associated with anther tissue. In the case of cDNAs, genomic DNAs and messenger RNAs, "anther-specific" describes the fact that, when assayed through northern blot hybridization, the mRNA corresponding to the cDNA, genomic DNA or mRNA sequence is present in anther tissue in concentrations at least about 100-fold that observed in other tissues. In the case of promoter DNA
sequences, "anther-specific" describes a regulatory sequence which directs the transcription of associated coding sequences so that the corresponding messenger RNA is present in anther tissue in concentrations at least about 100-fold that observed in other tissues. In the case of a gene, "anther-specific" describes a gene which is expressed in a manner so that the gene product is present in anther tissue in concentrations at least about 100-fold that observed in other tissues. Because anther and pollen tissue are both involved in the male sexual function of a plant, a DNA sequence or gene may be considered to be "anther-specific" for the purpose of the present invention if it is expressed specifically in pollen as well as in anther tissues.
"Recombinant" and "chimeric" are both used to indicate that a DNA sequence, vector or gene is comprised of more than one DNA sequence of distinct origin which have been fused or ligated together, resulting in a DNA sequence, vector or gene which does not occur naturally. For example, the ligation of a promoter DNA sequence from an anther-specific gene with the coding DNA sequence of a different gene of heterologous origin is said to be "recombinant" or "'chimeric".
In the following description, a number of expressions are used that are customary in recombinant DNA technology and in plant genetics. In order to ensure a clear and uniform understanding of the description and the claims and also of the scope to be accorded to the said expressions, the following definitions are listed.
Plant material: Parts of plants that are viable in culture or that are viable as such, such as _8_ protoplasts, cells, callus, tissue, embryos, plant organs, buds, seeds, etc., and also whole plants.
Plant cell: Structural and physiological unit of the plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or as a part of higher organized unit such as, for example, a plant tissue, a plant organ or a whole plant.
Protoplast: "Naked" plant cell that has no cell wall and has been isolated from plant cells or plant tissues and has the potential to regenerate to a cell clone or a whole plant.
Plant tissue: Group of plant cells organised in the form of a structural and functional unit.
Plant organ: Structural and functional unit comprising several tissues, for example root, stem, leaf or embryo.
Heterologous ~ene(s) or DNA_ A DNA sequence that codes for a specific product or products-or fulfils a biological function and that originates from a species other than that into which the said gene is to be inserted; the said DNA sequence is also referred to a.s a foreign gene or foreign DNA.
Homologous genes) or DNA: A DNA sequence that codes for a specific product or products or fulfils a biological function and that originates from the same species as that into which the said gene is to be inserted.
Synthetic ~ene(s) or DNA: A DNA sequence that codes for a specific product or products or fulfils a biological function and that is produced by synthetic means.
The present invention relates to anther-specific nucleotide sequences especially to recombinant, or chimeric, DNA sequences which are expressed in much higher amounts in the anther of a plant than in other tissue. The said recombinant, or chimeric, DNA
sequences comprise a promoter region obtainable from an anther-specific genomic DNA
sequence, which is operatively linked to an expressible DNA sequence which preferably encodes a protein desired to be expressed in the anther cells.
In a especially preferred embodiment, the coding DNA sequence encodes a polypeptide which, when expressed in the anther cells, will disrupt formation of viable pollen.

Preferred as the coding DNA sequence are sequences which encode a polypeptide selected from the group consisting of DTA, TURF-13, pectate lyase, gin recombinase, iaaL and cytA toxin.
The anther-specific promoter DNA sequences to be used in the recombinant, or chimeric, construct according to the invention are preferably isolated from genomic clones and are ligated in a 5' to 3' orientation to expressible DNA sequences, which preferably encode a desired protein product, to provide chimeric vectors that are specifically expressed in the anther of a plant.
The invention thus further relates to anther-specific genomic DNA sequences.
In a specific embodiment, the present invention comprises isolated anther-specific genomic DNA
clones which correspond to the anther-specific cDNA clones of SEQ. ID Nos. l, 3, 5, 7, 9, 1 l, 13, 14 and 20 above.
The anther-specific genomic clones may be obtained by using anther-specific cDNA
clones as probes to pull out the corresponding genomic DNA clones.
Corresponding genomic DNA clones are those which are transcribed to form a messenger RNA
which is complementary to and transcribed into a given cDNA. In a particular embodiment, the present invention comprises the isolated anther-specific genomic DNA clones ant32 and ant43D, the sequences of which are provided at SEQ. ID No. 16 and SEQ. 117 No.
18, respectively.
Several cDNA sequences are provided which may be used as probes to isolate anther-specific genomic DNA sequences.
In one embodiment, the present invention comprises isolated anther-specific cDNA clones ant32, ant43D, ant9, ant52, ;ant59, ant66, ant67, ant68 and ant43C, corresponding to the sequences of SEQ ID No. 1, SEQ. ID No. 3, SEQ. ID No. 5, SEQ. ID No. 7, SEQ.
ll~ No.
9, SEQ. ID No. 11, SEQ.117 No. 13, SEQ. ID No. 14 and SEQ. ID No. 20, respectively.
In a preferred embodiment of the present invention, the anther-specific cDNA
sequence of the present invention is obtained by preparing cDNA libraries from anther tissue and leaf tissue. Single stranded DNA from the leaf is photobiotinylated and hybridized to the anther DNA. Photobiotinylated DNA is removed, leaving a library enriched for anther-specific cDNA sequences. (Example 3). Anther-specific cDNAs are identified by differential screening (Example 4)). The anther-specific cDNAs are cross-hybridized to identify unique cDNAs. (Example 4). Anther-specific expression is verified by RNA blot hybridization with various plant tissues and in situ hybridization. (Examples 5 and 8).
Developmental expression, sequences and gene copy number of the anther-specific cDNA
clones is also determined. (Examples 6 and 7 and 9).
The cDNA sequences of the present invention can be used to isolate genomic DNA
sequences. Where a partial cDNA has been obtained, the partial cDNA is used as a probe to screen the anther cDNA library in order to isolate a full length cDNA
clone.
Hybridizing clones are purified, restriction mapped and sequenced. A full length clone will be near message size as well as having a complete open reading frame. To isolate a genomic clone, the full length anther cDNA is used as a probe to screen a genomic library.
By restriction mapping and hybridization to the anther cDNA, the coding region of the genomic clone is identified. The area upstream from the coding area of the clone is the anther promoter region.
The anther-specific promoter region may be more precisely mapped through deletion analysis. 5' deletions of an anther promoter are made by introducing restriction sites by PCR using oligonucleotide primers with restriction sites at the 5' ends and anther promoter sequences at the 3' ends. The PCR products are digested, purified, and cloned into pBI101 (Clontech). The deletion mutants contain the 5' untranslated leader sequence fused to the translational start site of the GUS gene. Internal and 3' deletions of anther promoters are made by PCR in a similar manner. The PCR fragments are fused to a GUS
vector containing the CAM'V 35S minimal promoter [-46 to +1; Benfey et al, 1990].
Transgenic plants are tested with the GUS fluorometric and histochemical assay.
The anter-specific promoter region may be suitably used within the scope of the present invention for the preparation of recombinant, or chimeric, DNA constructs comprising an expressible DNA sequence but preferable a coding DNA sequence, that is specifically expressed in the anther of a plant.
The present invention thus further comprises recombinant DNA sequences comprising, in a 5' to 3' direction, a promoter region obtainable from an anther-specific genomic DNA
sequence, which is operatively linked to a coding DNA sequence. The recombinant DNA
sequences result in anther-specific expression of the associated expressible DNA, which preferably is a coding DNA sequence which encodes for a structural gene.

The coding DNA sequence of the present invention may be any DNA sequence encoding for a desired polypeptide. Preferred within the scope of the present invention are, for example, all those structural genes which lead to a protective effect in the transformed plant cells and also in the tissues developing therefrom and especially in the plants, for example increased resistance to pathogens (for example to phytopathogenic fungi, bacteria, viruses, etc.); resistance to chemicals (for example to herbicides (e.g. triazines, sulfonylureas, imidazolinones, triazole pyrimidines, bialaphos, : glyphosate, etc.), insecticides or other biocides); resistance to adverse environmental factors (for example to heat, cold, wind, adverse soil conditions, moisture, dryness, etc.).
Further coding DNA sequences to be used within the scope of the present invention may be those that lead to the production of desirable and useful compounds in the~plant cell.
Genes that may also be used within the scope of the present invention include, for example, those which lead to increased formation of reserve or stored substances in leaves, seeds, tubers, roots, stems, etc: or in the protein bodies of seeds.
The desirable substances that can be produced by transgenic plants include, for example, proteins, carbo-.
hydrates, amino acids, vitamins, alkaloids, flavins, perfumes, colourings, fats, etc..
There may also be associated with the DNA sequence according to the invention structural genes that code for pharmaceutically acceptable active substances, for example hormones, immunomodulators and other physiologically active substances.
Especially preferred for use in the present invention are, however, coding DNA
sequences which encode the production of a polypeptide which, when expressed in anther tissue, will result in the inability of the-plant to produce viable pollen. Examples of such coding DNA
sequences include the genes which are described in the following references.
a) Diptheria toxin A-chain gene (DTA), which inhibits protein synthesis, Greenfield et al, 1983; Paliniter et al, 1987.
b) Pectate lyase gene pelf from Erwinia chrysanthemi EC16, which degrades pectin, causing cell lysis. Keen et a~, 1986.

c) T-urfl3 (TURF-13) gene from cms-T maize mitochondria) genomes; this gene encodes a polypeptide designated URF13 which disrupts mitochondria) or plasma membranes.
Braun et al, 1990; Dewey et al, 1987; and Dewey et al, 1986.
d) Gin recombinase gene from phage Mu gene, which encodes a site-specific DNA
recombinase which will cause genome rearrangements and loss of cell viability when expressed in cells of plants. Maeser et al, 1991.
e) Indole acetic acid-lysine synthetase gene (iaaL) from Pseudomonas syringae, which encodes an enzyme that conjugates lysine to indoleacetic acid (IAA). When expressed in the cells of plants, it causes altered development due to the removal of IAA
from the cell via conjugation. Romano et al, 1991; Spena et al, 1991; Roberto et al, 1990.
f) CytA toxin gene from Bacillus thuringiensis Israeliensis which encodes a protein that is mosquitocidal and hemolytic. When expressed in plant cells, it causes death of the cell due to disruption of the cell membrane. McLean et al, 1987; Ellar et al, United States Patent No. 4,918,006 (1990).
The recombinant DNA sequences of the present invention may further comprise, in a 5' to 3' direction, a promoter region obtainable from an anther-specific genomic DNA
sequence, operatively linked to a signal sequence, which is operatively linked to a coding DNA sequence. The signal sequence is responsible for specialized transport of the associated peptide within the plant cell.
The signal sequence of the present invention may be any DNA sequence which is able to direct the transport of and associated polypeptide into one or more of the cellular compartments. The signal sequence is preferably a sequence which is translated into a signal peptide, which becomes separated from the peptide after transit of the peptide is complete. Signal sequences are useful for directing the polypeptide product of the coding DNA sequence to a desired location within the cell, such as to the mitochondria or to the endoplasmic reticulum, or to direct extracellular transport outside of the cell. Among the signal sequences useful for the present invention are, for example, the signal sequence from the pathogenesis-related gene (PR-1) of tobacco, which is described in Cornellisen et al, 1986; the yeast mitochondria) presequence; Schmitz et al, 1989; the signal sequence from plant mitochondria) Rieske iron-sulfur protein, Huang et al, 1991;
mitochondria) and chloroplast targeting peptides, von Heijne et al, 1989. The identification of other leader sequences is known in the art. See Della-Cioppa et al, 1987; Schekman, 1985.
The various sections of sequence can be linked to one another by methods known per se to form a complete coding DNA sequence. Suitable methods include, for example, the in vivo recombination of DNA sequences having homologous sections and the in vitro linking of restriction fragments.
As cloning vectors there are generally used plasmid or virus (bacteriophage) vectors having replication and control sequences originating from species that are compatible with the host cell.
The cloning vector generally carries an origin of replication, especially an origin of replication that is capable of functioning in E. coli, in Agrobacterium or in both, and, in addition, specific genes that lead to phenotypic selection features in the transformed host cell, especially to resistance to antibiotics or to specific herbicides. The transformed vectors can be selected on the basis of those phenotypic markers after transformation in a host cell.
Selectable phenotypic markers that may be used within the scope of this invention include, for example, resistance to ampicillin, tetracycline, hygromycin, kanamycin, methotrexate, 6418 and neomycin, but this list, which is given by way of example, is not intended to limit the subject of the invention.
Suitable host cells within the scope of this invention are prokaryotes, including bacterial hosts, for example A. tumefaciens, E. coli, S. typhimurium and Serratia marcescens, and also cyanobacteria. Eukaryotic hosts, such as yeasts, mycelium-forming fungi and plant cells, may also be used within the scope of this invention.
The splicing of the hybrid gene construction according to the invention into a suitable cloning vector is carried out using standard methods, such as those described, for example, in Maniatis et al (1982) or Sambrook et al (1989).
The cloning vectors and the host cell transformed with those vectors are generally used to increase the number of copies of the constructs cloned therein. With an increased number of copies it is possible to isolate the vector carrying the hybrid gene construction and prepare it, for example, for insertion of the chimaeric gene sequence into a plant cell.

In a further process step, these; plasmids are used to insert the structural genes coding for a desired gene product or non-coding DNA sequences having a regulatory function into a plant cell and, optionally, to integrate them into the plant genome.
The present invention therefore relates also to the production of recipient plant cells that comprise the said structural gene or other desirable genes or gene fragments or other useful DNA sequences incorporated in their genome.
A number of very efficient processes have come into existence for introducing DNA into plant cells, which processes are based on the use of gene transfer vectors or on direct gene transfer processes.
One possible method comprises, for example, bringing plant cells into contact with viruses or with Agroba c t eri um. This may be achieved by infecting sensitive plant cells or by co-cultivating protoplasts derived from plant cells. Within the scope of this invention, Cauliflower Mosaic Virus (CaMV) may also be used as a vector for the insertion of the chimaeric genetic construction according to the invention into a plant.
Another method of inserting the chimaeric gene construction into a cell makes use of the infection of the plant cell with Agrobacterium tumefaciens and/or Agrobacterium rhizogenes, which has been transformed with the said gene construction. The transgenic plant cells are then cultured under suitable culture conditions known to the person skilled in the art, so that they form shoots and roots and whole plants are finally formed.
Using newly developed transformation techniques, it has also become possible in principle to transform in vitro plant species that are not natural host plants for Agrobacterium [Grimsley NH et al (1987)]. For example, monocotyledonous plants, especially the cereal species and various grasses, are not natural hosts for Agrobacterium.
Preferred within the scope of this invention is so-called leaf disk transformation using Agrobacterium [Horsch et al (1985)]. Sterile leaf disks from a suitable target plant are incubated with Agrobacterium cells comprising one of the chimaeric gene constructions according to the invention, and are then transferred into or onto a suitable nutrient medium. Especially suitable, and therefore preferred within the scope of this invention, are LS media that have been solidified by the addition of agar and enriched with one or more of the plant growth regulators customarily used, especially those selected from the group of the auxins consisting of a-naphthylacetic acid, picloram, 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, indole-3-butyric acid, indole-3-lactic acid, indole-3-succinic acid, indole-3-acetic acid and p-chlorophenoxyacetic acid, and from the group of the cytokinins consisting of kinetin, 6-benzyladenine, 2-isopentenyladenine and zeatin. The preferred concentration of auxins and cytokinins is in the range of from 0.1 mg/1 to 10 mg/1.
After incubation for several days, but preferably after incubation for 2 to 3 days at a temperature of from 20°C to 40°C, preferably from 23°C to 35°C and more especially at 25°C and in diffuse light, the leaf disks are .transferred to a suitable medium for the purpose of shoot induction. Especially preferred for the selection of the transformants is an LS medium that does not contain auxin but contains cytokinin instead, and to which a selective substance has been added. The cultures are kept in the light and are transferred to fresh medium at suitable intervals, but preferably at intervals of one week. Developing green shoots are cut out and cultured further in a medium that induces the shoots to form roots. Especially preferred within the scope of this invention is an LS medium that does not contain auxin or cytokinin but to which a selective substance has been added for the selection of the transformants.
In addition to Agrobacterium-mediated transformation, within the scope of this invention it is possible to use direct transformation methods for the insertion of the gene constructions according to the invention into plant material.
For example, the genetic material contained in a vector can be inserted directly into a plant cell, for example using purely physical procedures, for example by microinjection using finely drawn micropipettes [Neuhaus et al (1987)] or by bombarding the cells with microprojectiles that are coated with the transforming DNA ["Microprojectile Bombardment"; Wang Y-C et al (1988)].
Other possible methods for the direct transfer of genetic material into a plant cell comprise the treatment of protoplasts using procedures that modify the plasma membrane, for example polyethylene glycol treatment, heat shock treatment or electroporation, or a combination of those procedures [Shillito et al (1985)].
A further method for the direct introduction of genetic material into plant cells, which is based on purely chemical pr<xedures and which enables the transformation to be carried out very efficiently and rapidly, is described in Negrutiu I et al (1987) and in Goodall G et al.
Also suitable for the transformation of plant material is direct gene transfer using co-transformation (Schocher RJ et al 1986).
The list of possible transformation methods given above by way of example does not claim to be complete and is not intended to limit the subject of the invention in any way.
The present invention therefore also comprises.transgenic plant material, selected from the group consisting of protoplasts, cells, calli, tissues, organs, seeds, embryos, ovules, zygotes, etc. and, especially, whole and preferably male-sterile plants, that has been transformed by means of the processes described above and comprises the recombinant DNA according to the invention in expressible form, and processes for the production of the said transgenic plant material.
The process for the production of transformed plant material, including whole plants, comprising an expression product that is expressed in an anther-specific manner essentially comprises:
(a) first of all isolating from a suitable source or synthesising by means of known processes a DNA sequence responsible for the anther-specific expression of an associated expressible DNA sequence;
(b) operably linking the said anther-specific DNA sequence in a 5' to 3' direction to an expressible DNA sequence;
(c) cloning the final construct into a plant expression vector under the control of expression signals active in plants;
(d) transforming the said expression vector into plant material by means of known processes and expressing it therein; and optionally (e) regenerating the plant material transformed according to step (d) to a whole and preferably phaenotypically normal plant.
The present invention thus also comprises transgenic plants and the sexual and/or asexual progeny thereof, which have been transformed with a recombinant DNA sequence comprising the promoter region from an anther-specific genomic DNA sequence.

The expression "asexual or sexual progeny of transgenic plants" includes by definition according to the invention all mutants and variants obtainable by means of known processes, such as for example cell fusion or mutant selection and which still exhibit the characteristic properties of the initial transformed plant, together with all crossing and fusion products of the transformed plant material.
Another object of the invention concerns the proliferation material of transgenic plants.
The proliferation material of transgenic plants is defined relative to the invention as any plant material that may be propagated sexually or asexually in vivo or in vitro.
Particularly preferred within the scope of the present invention are protoplasts, cells, calli, tissues, organs, seeds, embryos, egg cells, zygotes, together with any other propagating material obtained from transgenic plants.
Description of the Sequences Sequence 1 is the nucleotide sequence of anther-specific cDNA clone ant32.
Sequence 2 is the amino acid sequence of the polypeptide encoded by the ant32 nucleotide sequence of Sequence 1.
Sequence 3 is the nucleotide sequence of anther-specific cDNA clone ant43D.
Sequence 4 is the amino acid sequence of the polypeptide encoded by the ant43D
nucleotide sequence of Sequence 3.
Sequence 5 is the nucleotide sequence of anther-specific cDNA clone ant9.
Sequence 6 is the amino acid sequence of the polypeptide encoded by the ant9 nucleotide sequence of Sequence 5.
Sequence 7 is the nucleotide sequence of anther-specific cDNA clone ant52.
Sequence 8 is the amino acid sequence of the polypeptide encoded by the ant52 nucleotide sequence of Sequence 7.

Sequence 9 is the nucleotide sequence of anther-specific cDNA clone ant59.
Sequence 10 is the amino acid sequence of the polypeptide encoded by the ant59 nucleotide sequence of Sequence 9.
Sequence 11 is the nucleotide sequence of anther-specific cDNA clone ant66.
Sequence 12 is the amino acid sequence of the polypeptide encoded by the ant66 nucleotide sequence of Sequence 11.
Sequence 13 is the nucleotide sequence of anther-speck cDNA clone ant67.
Sequence 14 is the nucleotide sequence of anther-specific cDNA clone ant68.
Sequence 15 is the amino acid sequence of the polypeptide encoded by the ant68 nucleotide sequence of Sequence 14.
Sequence 16 is the nucleotide sequence of the Ant32 genomic clone. This sequence shows the nucleotide sequence of the ant32 gene, including 2.0 kb of S' flanking sequence.
The TATA box is found at bases 1971 to 1975. The putative transcription start site is found at base 2009. Bases 2009 to 2075 comprise the untranslated leader sequence. The ATG translational initiation codon is found at bases 2076 to 2078. No introns are present.
The TGA stop codon is found at bases 3420 to 3422.
Sequence 17 is the amino acid sequence of the polypeptide encoded by the Ant32 nucleotide sequence of Sequence 16.
Sequence 18 is the nucleotide sequence of the Ant43D genomic clone. This sequence shows the nucleotide sequence of the ant43D gene, including approximately 1.2 kb of S' flanking sequence. The putative transcriptional start site is found at base 1167. An unusually long TATA box is found at bases 1089 to 1147. The sequence "TA" is repeated 29 times. The untranslated leader is found between bases 1167 and 1229. The translational initiation codon occurs at bases 1230 to 1232. Translated sequences are shown in uppercase. One intron occurs at bases 1571 to 1668.

Sequence 19 is the amino acid sequence of the polypeptide encoded by the Ant43D
nucleotide sequence of Sequence 18.
Sequence 20 is the nucleotide sequence of anther-specific cDNA clone ant43C.
Sequence 21 is the amino acid sequence of the polypeptide encoded by the ant43C
nucleotide sequence of Sequence 20.
Description of the Fi ores Figure 1: Restriction map of Ant32 genomic clone pCIB950. The arrow indicates the location of the ant32 gene, as well as its 5' to 3' orientation in genomic subclone pCIB950. The promoter region extends from the upstream PstI site to the coding region.
Figure 2: Restriction map of Ant43D genomic clone pCIB952. The arrow indicates the location of the ant43D gene, as well as its S' to 3' orientation in genomic subclone pCIB952. The promoter region extends from the upstream EcoRI site to the coding region.
Figure 3: Site-specific mutagenesis via PCR resulting in insertion of a XbaI
site before the start of translation of Ant32 (Figure 3A) and Ant43D (Figure 3B).
In Figure 3A, the drawing on the top left shows the 3' end of the PstI-SacI
ant32 genomic subclone containing the promoter. Underneath it is the sequence at the ATG
before which an XbaI site was inserted as described in Example 16.
In Figure 3B, the drawing on the top left shows the 3' end of the EcoRI ant43D
genomic subclone containing the promoter. Underneath it is the sequence at the ATG
before which an XbaI site was inserted as described in Example 18.
Figure 4: Plasmid maps of Ant32-GUS fusions pCIB3132 (2.0 kb promoter - Figure 4A) and pCIB3132B (600 by promoter - Figure 4B), pCIB3132 has been deposited with the USDA Agricultural Research Service Culture Collection, Northern Regional Research Center (NRRL) at 1815 North University Street, Peoria, Ill. 61604, on June 16, 1992 and has been accorded deposit no. NRRL B-18977.
Figure 5: Plasmid map of Ant32-DTA fusion pLC251 Figure 6: Plasmid map of Ant43D-GUS fusion pCIB3178. pCIB3178 has been deposited with the USDA NRRL on June 16, 1992 and has been accorded deposit no.
NRRL B-18978.
Figure 7: Plasmid map of Ant43D-DTA fusion pCIB3179.
The following examples further describe the materials and methods used in carrying out the invention. They are offered by way of illustration, and their recitation should not be considered as a limitation of the claimed invention.
NON-LIMITING EXAMPLES
General recombinant DNA techniques Since many of the recombinant DNA techniques employed in this invention are a matter of routine for the person skilled in the art, it is better to give a short description of these generally used techniques here rather than to describe them every time they occur. Except where there is a specific indication to the contrary, all these procedures are described in the Maniatis et al (1982) reference.
A. Cleaving with restriction endonucleases A reaction batch typically contains about 50 to 500 ~,g/ml of DNA in the buffer solution recommended by the manufacturer, New England Biolabs, Beverly, MA.. 2 to 5 units of restriction endonucleases are added for each pg of DNA and the reaction batch is incubated for from one to three hours at the temperature recommended by the manufacturer. The reaction is terminated by heating at 65°C for 10 minutes or by extraction with phenol, followed by precipitation of the DNA with ethanol.
This technique is also described on pages 104 to 106 of the Maniatis et al (1982) reference.
B. Treatment of DNA with polymerase in order to produce blunt ends 50 to 500 ~.g/ml of DNA fragments are added to a reaction batch in the buffer recommended by the manufacturer, New England Biolabs. The reaction batch contains all ~0o41-4z four deoxynucleotide triphosphates in concentrations of 0.2 mM. The reaction takes place over a period of 30 minutes at 15°C and is then terminated by heating at 65°C for 10 minutes. For fragments obtained by cleaving with restriction endonucleases that produce 5'-projecting ends, such as EcoRI and BamHI, the large fragment, or HIenow fragment, of DNA polymerise is used For fragments obtained by means of endonucleases that produce 3'-projecting ends, such as PstI and SacI, the T4 DNA polymerise is used. The use of these two enzymes is described on pages 113 to 121 of the Maniatis et al (1982) reference.
C. Agarose gel electrophoresis and purification of DNA fragments from gels Agarose gel electrophoresis is carried out in a horizontal apparatus, as described on pages 150 to 163 of the Maniatis et al reference. The buffer used is the tris-borate buffer described therein. The DNA fragments are stained using 0.5 ~tg/ml of ethidium bromide which is either present in the gel or tank buffer during electrophoresis or is added after electrophoresis. The DNA is made visible by illumination with long-wave ultraviolet light. If the fragments are to be separated from the gel, an agarose is used that gels at low temperature and is obtainable from Sigma Chemical, St. Louis, Missouri. After the electrophoresis, the desired fragment is cut out, placed in a plastics test tube, heated at 65°C for about 15 minutes, extracted three times with phenol and precipitated twice with ethanol. This procedure is slightly different from that described by Maniatis et al (1982) on page 170.
As an alternative, the DNA can be isolated from the agarose with the aid of the Genecleari kit (Bio 101 Inc., La Jolla, CA, USA).
D. Addition of synthetic linker fragments to DNA ends If it is desired to add a new endonuclease cleavage site to the end of a DNA
molecule, the molecule is optionally first treaied with DNA-polymerise in order to produce blunt ends, as described in the section above. About 0.1 to 1.0 ~tg of this fragment is added to about ng of phosphorylated linker DNA, obtained from New England Biolabs, in a volume of to 30 ~.l with 2 ~.1 of T4 DNA ligase from New England Biolabs, and 1 mM ATP
in the buffer recommended by the manufacturer. After incubation overnight at 15°C, the reaction is terminated by heating at 65°C for 10 minutes.
*Trade-mark The reaction batch is diluted to about 100 ~.l in a buffer appropriate for the restriction endonuclease that cleaves the synthetic linker sequence. About 50 to 200 units of this endonuclease are added. The mixture is incubated for 2 to 6 hours at the appropriate temperature, then the fragment is subjected to agarose gel electrophoresis and purified as described above. The resulting fragment will then have ends with endings that were produced by cleaving with the restriction endonuclease. These ends are usually cohesive, so that the resulting fragment can then readily be linked to other fragments having the same cohesive ends.
E. Removal of 5'-terminal phosphates from DNA fragments During the plasmid cloning steps, treatment of the vector plasmid with phosphatase reduces the recircularisation of the vector (discussed on page 13 of the Maniatis et al reference). After cleavage of the DNA with the correct restriction endonuclease, one unit of calf intestinal alkaline phosphatase obtained from Boehringer-Mannheim, Mannheim, is added. The DNA is incubated at 37°C for one hour and then extracted twice with phenol and precipitated with ethanol.
F. Linking of DNA fragments If fragments having complementary cohesive ends are to be linked to one another, about 100 ng of each fragment are incubated in a reaction mixture of 20 to 40 p.1 containing about 0.2 unit of T4 DNA ligase from New England Biolabs in the buffer recommended by the manufacturer. Incubation is carried out for 1 to 20 hours at 15°C. If DNA
fragments having blunt ends are to be linked, they are incubated as above except that the amount of T4 DNA ligase is increased to 2 to 4 units.
G. Transformation of DNA into E. coli E. coli strain HB 101 is used for most of the experiments. DNA is introduced into E. coli using the calcium chloride method, as described by Maniatis et al (1982), pages 250 and 251.
H. Screening of E. coli for plasmids After transformation, the resulting colonies of E. coli are tested for the presence of the desired plasmid by means of a rapid plasmid isolation process. Two customary processes are described on pages 366 to 369 of the Maniatis et al (1982) reference.
I. Large-scale isolation of plasmid DNA
Processes for the isolation of plasmids from E. coli on a large scale are described on pages 88 to 94 of the Maniatis et al (1982) reference.
J. Cloning in Ml3 phage vectors In the following description it is to be understood that the double-stranded replicative form of the phage M13 derivatives is used for routine processes, such as cleaving with restriction endonuclease, linking etc..
Unless there is a specific indication to the contrary, enzymes can be obtained from Boehringer, Biolabs (BRL). They are used in accordance with the manufacturer's instructions unless otherwise indicated.
K. Southern blot analysis The extracted DNA is first treated with restriction enzymes, then subjected to electrophoresis in a 0.8 % to 1 % agarose gel, transferred to a nitrocellulose membrane [Southern EM (1975)] and hybridised with the DNA to be detected which has previously been subjected to nick-translation (DNA-specific activities of 5 x 108 to 10 x c.p.m./~.g). The filters are washed three times for 1 hour each time with an aqueous solution of 0.03M sodium citrate and 0.3M sodium chloride at 65°C. The hybridised DNA
is made visible by blackening an X-ray film over a period of 24 to 48 hours.
Examples Example 1: Plant Material and Growth Conditions Tobacco plants (Nicotiana tabacum cv Xanthi) are grown from seed in a greenhouse under a 16-hour light/ 8-hour dark light regime.

.x0041-41 Example 2: Anther and Leaf mRNA Isolation Total RNA is isolated from anthers from 0 to lOmm pistil length flower buds and from 5 week old seedlings by the Phenol/SDS method described by Ausubel et a1, Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1987). PolyA+RNA is purified from total RNA as described by Maniatis et a1, (1982).
Example 3: Construction of Subtracted cDNA Libraries Anther and seedling cDNA libraries are made using INVIT'ROGEN's Libraviani II
ldt [1NVITROGEN CORD, 3985B Sorrento Valley. Blvd, San Diego, CA; Cat No L1958-15].
Double-stranded cDNA is synthesized from anther and leaf polyA+ RNA,- BstXI
non-palindromic linkers are ligated on and the cDNA is cloned into a BstXt cut pTZlBR-B- vector, which is available from INVfTROGENE CORP. [3985B Sorrento Valley Blvd., San Diego, CA]. Transformation is into E. coli DhilaF' cells, which can also be purchased from 1NVITROGENE CORD. A subtraction cDNA library is~ madc using INVITROGEN's Subtractor kit [BV1~ITROGEN CORD, 3985B Sorrento Valley Blvd, San Diego, CA; Cat No K4320-01]. Single-stranded DNA is isolated from the anther and leaf cDNA libraries. The leaf single-stranded DNA is photobiotinylated and hybridized to the anther single-stranded DNA. Both hybridized and unhybridized photobiotinylated sequences are removed with streptavidin and phenol extraction. The remaining DNA is convened tv double-sirande.d form with Klenow and transformed into E. coli DHlaF' cells.
Example 4: Isolation of Anther-Specific cDNA Clones Anther-specific clones are identified by differential screening of the anther subtraction cDNA library. 20,000 clones are replica plated onto nitrocellulose filters and differentially screened to identify colonies hybridizing to radioactively labeled first strand cDNA from anther polyA+ RNA but not to first strand cDNA from seedling polyA+.
The cDNAs insened into the cloning vector are cut out with a restriction enzyme and the inserts of althogether 70 cDNAs are differentially screened again by Southern blot.
Northern blots of anther, pistil and leaf total RNA are probed with the cDNAs to confirm tissue specificity. All anther-specific cDNAs are cross-hybridized to identify unique cDNAs. Unique cDNA clones are purified and subcloned into bluescript vector. A
full-length cDNA clone of ant32 is isolated by screening the anther cDNA lib_ nary with a .9 *Trade-mark kb partial cDNA. The two cDNAs are 95% homologous at the sequence level, and are therefore closely related members of the same gene family.
Example 5: Verification of Expression Pattern by RNA Blot Hybridization Northern blots are done using nitrocellulose filters as described in Maniatis (1982). 20wg of anther, pistil and leaf total RNA are loaded per lane. Prehybridizations are done at 68°C for 4 hours in 3X SSC, 5X Denhardt's, 20mM Tris pH 7, .1% SDS, 2mM
EDTA and 1005g/ml sheared denatured salmon sperm DNA. Hybridizations are done at 42°C
overnight in 6X SSC, 5X Denhardt's, .1% SDS, 500 wg/ml salmon sperm DNA, 8%
dextron sulfate and 50% formamide to which 5.5 x 106 cpm/ml of probe is added.
Probes are synthesized using PHARMACIA's oligolabelling kit [PHARMACIA LKB
BIOTECHNOLOGY, 800 Centennial Avenue, Piscataway, NJ; Cat No 27-9250-O1] in accordance with the manufacturer's instructions. Expression of the cDNAs is seen only in anther RNA. Expression in pollen is also seen with the ant66 cDNA.
PolyA+ RNA is isolated from anther, pistil, leaf, petal, stem and root tissue.
15g of each along with 205g of seed and sepal total RNA are run on a Northern and probed with the ant32 and ant43D cDNAs. Expression of both cDNAs is seen only in anther RNA, demonstrating that the ant32 and ant43D cDNAs are tightly regulated and expressed only in anther tissue.
Example 6: Developmental Expression of Anther-Specific cDNA Clones Total RNA is isolated from anthers from 6 stages of flower bud lengths. Slot blots are probed with the anther-specific cDNAs to determine developmental expression.
Slot hybridization is done as in Maniatis (1982) using 105g of RNA. Table 1 contains the developmental expression profile of the cDNAs. Ant9, 32, 43C, 59, and 68 are expressed only early in anther development, whereas ant43D, 52 and 67 are expressed throughout development. Ant66 is expressed only late in development.
Example 7: Sequencing of Anther-Specific cDNA Clones DNA is sequenced using the dideoxy chain-termination method of Sanger et al, 1977, using double-stranded plasmid DNA as a template. All DNA sequence analysis is carned out on a Digital Vax 8530 computer using the University of Wisconsin Computer Genetics Group software, which is commerically available from GENETICS COMPUTER
GROUP, INC [University Research Park 575 Schience Drive, Madison, WI]. The oligonucleotide primers are synthesized on an Applied Biosystems Model 380A
Synthesizer.
Table 1 contains a comparison of message size to insert size of the anther-specific cDNAs.
The ant32 and ant43D cDNAs are close to the size expected for full length copies of the mRNAs. The rest of the cDNAs are incomplete clones. Ant32, 43C, 43D, 52, 59, 66 and 68 encode a single open reading frame. The ant32 cDNA is a near full-length clone of 1542 bases (SEQUENCE 1D NO. 1). The sequence contains a large open reading frame which extends from nucleotide 66 to 1412, encoding a complete polypeptide of 448 amino acids. The open reading frame is flanked by 5' and 3' non-coding regions of 65 and 130 bases respectively. A polyadenylation signal, AATAAA, occurs at position 1502.
The ant43D cDNA is a near full-length clone of 552 bases (SEQUENCE 117 NO. 3).
The sequence contains a complete open reading frame of 118 amino acids, extending from bases 41 to 397. The open reading frame is flanked by 40 bases on the 5' end and 155 bases on the 3' end. A polyadenylation signal is found starting at position 437.
Ant43C is an incomplete cDNA of 437 bases (SEQUENCE ID NO. 20). A partial polypeptide of 90 amino acids is encoded by nucleotides 167 to 436. The ant43C
cDNA
and the ant43D cDNA are 90% homologous at the sequence level.
Ant52, an incomplete cDNA clone of 96 bases (SEQUENCE ID NO. 7) contains an open reading frame of 31 amino acids.
Ant59 is an incomplete cDNA clone of 1201 bases (SEQUENCE ID NO. 9). An open reading frame extending from nucleotide 1 to 1119 encodes a partial polypeptide of 372 amino acids. The open reading frame is flanked by a 3' non-coding region of 82 bases.
Ant66 is an incomplete cDNA clone of 952 bases (SEQUENCE ID NO. 11). A partial polypeptide of 236 amino acids is encoded by nucleotides 1 to 711. The open reading frame is flanked by a 3' region of 241 bases. The sequence contains a polyA
tail of 15 bases.
Ant68 is an incomplete cDNA clone of 445 bases (SEQUENCE 117 NO. 20. An open reading frame of 148 amino acids is encoded by the sequence.
Ant67 is an incomplete cDNA clone of 305 bases (SEQUENCE ID NO. 13). It is unknown which strand is the sense strand since a single large open reading frame was not found. This clone contains the 3' end of an open reading frame and a 3' flanking region in translations of both strands.
Ant9 is an incomplete, chimeric cDNA of 612 bases (SEQUENCE ID NO. 5).
Northerns of anther, pistil, and leaf tissue are probed with 5' and 3' regions of the chimeric cDNA to determine the anther-specific region of the cDNA clone. Northerns probed with bases 1 to 325 hybridize to anther, pistil and leaf tissue. This region of the cDNA
encodes an open reading frame. Northerns probed with bases 326 to 612 hybridize exclusively to anther tissue. This region is identified as the anther-specific region of the chimeric cDNA. A
partial polypeptide of 32 amino acids is encoded by nucleotides 344 to 442. A
polyadenylation signal starts at position 461.
Each deduced amino acid sequence is compared to sequences in GenBank Genetic Sequence Data Bank, a computer data base of DNA sequences. The ant66 cDNA had a 74% overall amino acid identity with a plasma membrane proton ATPase (H+-ATPase) from Arabadopsis thaliana. Harper et al, 1989. The ant68 cDNA encodes a glycine-rich protein.
Example 8: Verification of Ant32 Expression Pattern by In-Situ Hybridization In situ hybridization studies with paraffin-embedded anther sections from l2mm long flower buds are carried out as described by Perez-Grau et al, 1989. 35S-RNA
probes used for in situ hybridizations are synthesized using STRATAGENE's RNA
Transcription Kit [STRATAGENE CLONING SYSTEMS, 11099 North Torrey Pines Rd, La Jolla, CA; Cat No 200340]. Cross sections and longitudinal sections are probed with ant32 antisense and sense RNA probes. Expression is localized in the tapetal cell layer of the anther with the antisense probe.
Example 9: Gene Copy Number In order to determine how many genes in the tobacco genome hybridize with the anther-specific genes, Xanthi genomic DNA was digested with XbaI, HindIII, EcoRI, and BamHI. Southern blots are probed with the cDNA clones. The blots probed with ant32, 43, 52, 59 and 67 had 2 bands hybridizing in each digest, indicating that these cDNAs are single copy genes or members of small gene families. More bands per digest hybridized in the blots probed with ant9, 66 and 68, indicating that these cDNAs are members of larger gene families.
Example 10: Southern Blots Southern blots are done with nitrocellulose as described in Maniatis (1982).
Prehybridizations are in 6X SSC, lOX Denhardt's, .2% SDS, and 75 ug/ml salmon sperm DNA at 68°C for 4 to 6 hours. Hybridizations are done at 68°C
in 6X SSC, SX
Denhardt's, .5% SDS and 125 ~g/ml salmon sperm DNA to which 1 x 106 cpm/ml DNA
probe is added. Washes are as described in Maniatis (1982). Genomic Southern blots are done with Duralon-UV membranes (Stratagene) and hybridization conditions are as in the manufacturer's directions.
Example 11: Construction of Tobacco Genomic DNA Libraries Tobacco DNA is isolated from leaves using the method of Shure et al, (1983).
Sau3AI
partial digests of Xanthi genomic DNA are cloned into the BamHI site of Stratagene's Lambda DashII vector and the library is amplified. Another genomic library is made using PROMEGA's LambdaGEM-l I XhoI Half Site Arms Cloning System [PROMEGA, 2800 Woods Hollow Road, Madison, WI; Cat No B1960]. Partially filled-in Sau3AI
digested genomic DNA is cloned into partially filled-in XhoI LambdaGEM-11 arms.
Example I2: Isolation and Sequencing of the Ant32 Genomic Clone The amplified Stratagene genomic library is screened with ant32 as a probe, yielding 4 hybridized placques. All four clones are purified and restriction mapped. When probed with ant32, one EcoRI fral,~nent from each clone hybridized. The EcoRI
fragments are subcloned in plasmid pBS SK+, which is available from STRATAGENE CLONING
SYSTEMS [11099 North Torrey Pines Road, La Jolla, CA]. Subcloning and mapping of the EcoRI fragments from the 4 clones showed that 2 are identical. Figure 1 contains the map of EcoRI subclone pCIB950. Fragments from pCIB950 are then subcloned for sequencing. 2.0 kb of promoter, the entire coding region, and .28 kb of 3' untranslated region is sequenced. The 2.0 kb promoter fragment from ant32 is functional. As shown in *Trade-mark Example 16, a .6kb fragment from ant32 is sufficient to confer anther specific activity.
Example 13: Isolation and Sequencing of the Ant43D Genomic Clone The LambdaGEM-11 primary library is screened with ant43D as a probe.
Hybridizing placques are rescreened by PCR to distinguish between ant43D and ant43C, a closely related cDNA. PCR fragments generated from the placques are digested to distinguish between genomics correlating to the 2 cDNAs. Two genomic clones correspond to ant43D, and they are purified and mapped. A 6.6 kb SacI band from both hybridizes to an ant43D probe. Subcloning and mapping of both SacI bands shows that they are identical.
Figure 2 contains the map of the SacI subclone, pCIB9S2. Fragments from pCIB9S2 are subcloned and sequenced. 1.2 kb of promoter, the entire coding region including one intron, and .22 kb of 3' untranslated region is sequenced. The 1.2 kb promoter fragment contains the entire ant43D promoter.
Example 14: Primer Extension The primer is end-labeled using [~,-32P] ATP (6000 Ci/mmole, Amersham) and T4 polynucleotide kinase. 20wg of anther total RNA is mixed with .O1 pmole of primer in 20w1 of reverse transcriptase buffer (50mM Tris pH8.3, 7SmM KCI, 3mM MgCl2).
The mixture is heated at 80°C for 10 min., annealed by slowly cooling to 40°C, and hybridized overnight at 40°C. To each 20N1 reaction is added 30N1 of SmM DTT, .lmg/ml BSA, 1mM
each of dATP, dCTP, dGTP, and dTTP in reverse transcriptase buffer containing 200 units of RNAsin (Promega) and 400 units of MMLV reverse transcriptase (BRL). Primer extension is carried out at 40°C for 60 min. The DNA/RNA hybrid is extracted once with phenol:chloroform and ethanol precipitated in the presence of carrier DNA. The pellet is dissolved in sequencing loading dye and analyzed on a 6% acrylamide-urea sequencing gel.
The primers used for the primer extension experiments are as follows:
for Ant 32 ~ - AT IO1M03 - S'-GGC TTC ACT ACC CAG TGG TG-3' for Ant 43D --~ - AT 12SM03 - S'-CAA CGC GCC CTT CTT TGA AA-3' .10041-41 Example 15: Mapping the Transcript Start Site by Primer Extension The start of transcription of the ant32 cDNA and the ant43D cDNA are mapped using primer extension. The largest primer extension product falls within a few base pairs of the end of the ant32 cDNA. The largest primer extension product falls 23 base pairs upstream of the end of the ant43D cDNA.
Example 16: Fusions of the ant32 promoter sequence to the GUS gene The 2.0 kb S' flanking region of pCIB950 containing the ant32 promoter is fused to the bacterial reporter gene for glucuronidase (GUS) in order to characterize the promoter of the anther-specific gene in transgenic plants. An XbaI site is inserted before the ATG by PCR as follows:
A 350 by XhoI-XbaI fragment (top right of figure 3A) is synthesized using polymerise chain reaction (PCR) technology [see Mullis et al, 1987; Erlich 1989] to copy the ant32 promoter sequence from 55 by before the unique XhoI site to two by before the ATG.
One of the PCR primers inserts an XbaI site 2bp before the ATG. A full-length ant32 promoter consisting of the PstI-XhoI fragment from the original clone and the XhoI-XbaI
PCR cassette is reassembled in a 3-way ligation into the PstI-XbaI sites of the Bluescript vector pBluescript SK (Stratagene). This promoter clone can be used for transcriptonal fusions to coding sequences.
The resulting promoter is excised as a SalI - XbaI fragment and fused to the GUS gene in pBI101 (Clontech). A 600 base pair ant32 promoter - GUS fusion is constructed by deleting a 1.4 kb HindBI fragment from the bluescript promoter clone. The deleted promoter is excised as a SalI - XbaI fragment and fused to the GUS gene in pBI101. The 2.0 kb promoter-GUS fusion is designated pCIB3132 and the 0.6 kb promoter-GUS
fusion is designated pCIB3132B. The 0.6 kb promoter fragment from ant32 is sufficient to confer anther specific activity.
Example 17: Fusion of the ant32 promoter sequence to the DTA gene A chimeric gene is constructed using a 5' ant32 promoter sequence and the Diptheria toxin A-chain (DTA) coding sequence [Palmiter et al, 1987]. The GUS coding sequence is excised from pCIB3132B with SmaI and SacI, the SacI site is filled in, and the plasmid is *Trade-mark religated back together (pLC250). The DTA coding sequence is ligated as a BgIII
fragment, cut out of plasmid p72 [Palmiter et al, 1987], into the BamHI site of pLC250, resulting in pLC251. The DTA coding sequence is fused in the opposite orientation in pLC252.
Example 18: Fusion of the ant43D promoter sequence to the GUS gene The 1.2 kb 5' flanking region of the ant43D gene is fused to GUS. An XbaI site is inserted before the ATG by PCR as follows:
A 210 by Earl-XbaI fragment (top right of figure 3B) is synthesized by PCR in order to copy the ant43D promoter from 39 by before the Earl site to 22 by before the ATG. One of the PCR primers inserts an XbaI site 22 by before the ATG. A full-length ant43D
promoter consisting of the EcoRI-Earl fragment from the original clone and the Earl-XbaI
PCR cassette is reassembled in a 3-way ligation into the EcoRI-XbaI sites of bluescript.
This promoter clone can be used for transcriptional fusions to coding sequences.
The resulting promoter is excised as a HindIII-XbaI fragment and fused to the GUS gene in PBI101 (pCIB3178). Figure 3B demonstrates how the 1.2 kb flanking region of the ant43D gene is obtained. The 1.2 kb promoter fragment is sufficient to confer anther specific activity.
Example 19: Fusion of the ant43D promoter sequence to the DTA gene The I.2 kb ant32 promoter is excised from pLC251 with HindIII-XbaI and replaced with a HindIII-XbaI ant43D promoter fragment. The resulting plasmid is designated pCIB3179.
The DTA coding sequence is fused in the opposite orientation in pCIB3188. The ant32 promoter is excised with HindIII and Xbal from pLC252 and replaced with the ant43D
promoter.
Example 20: Production of Transgenic Plants Tobacco leaf discs are transformed with the ant32-GUS (pCIB3132 (2 kb promoter) and pCIB3132B (.6 kb promoter)), ant32-DTA (pLC251 and antisense control (pLC252)), ant43D-GUS (pCIB3I78), and ant43D-DTA (pCIB3I79 and antisense control(pCIB3188)) constructions and mature transformed plants selected as in Horsch et al, (1985). The presence of transforming DNA is confirmed using PCR.
Example 21: GUS Analysis of ant32 Transgene Expression Transformants are tested by the GUS histochemical assay as in Koltunow et al, (1990) and fluorometrically as in Jefferson, (1987). In the histochemical assay, GUS
expression is seen in the tapetal cell layer of the anthers of flower buds 10 to 20 mm long.
Expression is also seen in pollen to a lesser extent. Anther, pistil, pollen, leaf and stem tissue are assayed fluorometrically and GUS activity is limited to anther and pollen tissue.
Example 22: Analysis of ant32-DTA Transgenic Plants The flower morphology of 13 transgenic plants containing pLC251 and 15 plants of pLC252 is observed. The plants containing pLC251 all had brown, withered anthers and no pollen shed. In contrast, pLC252 transgenic plants had normal anthers and pollen shed.
Selfs and backcrosses are done on all plants. In the pLC251 plants, no self pollinations are obtained, but seeds are obtained from backcrosses. Fertility in self and backcross pollinations is normal for pLC252 plants.
Anthers from 14-l6mm and 25-30mm long flower buds are fixed, embedded in paraffin, and sections are stained with toluidine blue. The tapetum and pollen sac are destroyed in pLC251 plants, whereas pLC252 plants had normal morphology.
Example 23: GUS Analysis of ant43D Transgene Expression Transformants are tested by the GUS histochemical and fluorometric assays. In the histochemical assay, GUS expression is seen in the tapetal cell layer of anthers of buds 14 to 16 mm long, in microspores, and increasingly in the connective and wall tissue of the anther. Anther, pollen, pistil, leaf, sepal, stem and root tissue are assayed fluorometrically. GUS activity is limited to anther and pollen tissue.
Example 24: Analysis of ant43D-DTA Transgenic plants The flower morphology of 8 pCIB3179 plants and eight plants of the control pCIB3188 (DTA in antisense orientation) is observed. The pCIB3179 transgenic plants all had nonfunctional anthers as no pollen was shed. Anther size among different plants ranged from normal to shrunken, anther color from green to brown, and anthers from dehiscent to nondehiscent. The control pCIB3188 transgenic plants had normal anther morphology and pollen shed. Selfs and backcrosses are done on the plants. In pCIB3179 plants, pollinations from backcrosses are obtained, but self pollinations are not.
Fertilization in pCIB3188 plants is normal.
Anthers from 8-lOmm, 10-l2mm, 14-l6mm and 25-30mm long flower buds are fixed, embedded in paraffin, and sections are stained with toluidine blue.
Microspores are absent from pCIB3179 plants as early as in 8-lOmm long buds.
DEPOSIT
The following deposits have been made at the the USDA Agricultural Research Service Culture Collection, Northern Regional Research Center (NRRL) at 1815 North University Street, Peoria, Ill. 61604 in accordance with the requirements of the Budapest Treaty:
Plasmid Date of depositDate of viabilityDeposition statement No pCIB 3178 June 16, 1992 June 22, 1992 NRRL B-18978 pCIB 3132 June 16, 1992 June 22, 1992 NRRL B-18977 b o .n H

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REFERENCE LISTING
Benfey et al, EMBO 9: 1677-1684, 1990 Braun et al, Plant Cell 2: 153, 1990 Cornellisen et al, EMBO 5: 37-40, 1986 Dells-Cioppa et al, Plant Physiology 84: 965-968, 1987 Dewey et al, CeII 44: 439, 1986 Dewey et al, Proc Natl Acad Sci USA, 84: 5374, 1987 Erlich (Fd.), PCR Technoloay, Stockton Press (New York 1989) Goodall G et a1, Methods in Enzymology 181: 148-161,1990 Greenfield et al, Proc Natl Acad Sci USA, 80: 6853, 1983 Gri.msley NH et al, Nature 325: 177-179, 1987 Harper et al, Proc Natl Acad Sci, USA 86: 1234-1238 Heijne et al, Eur J Biochem, 180: 535-545, 1989 Horsch et al, Science 227: 1229-1231, 1985 Huang et al, Proc Nat Acad Sci USA, 88: 10716-10720, 1991 Jefferson, Plant Mol Biol Rep 5: 387-405, 1987 Keen et al, J. Bacteriology 168: 595, 1986 Koltunow et al, Plant Cell 2: 1201-1224, 1990 Maeser et al, Mol Gen Genet 230: 170-176, 1991 Maniatis et al, Molecular Cloning: A Laborator~r Manual, Cold Spring Harbor Laboratory Press, NY (1982) McLean et al, J Bacteriology 169: 1017-1023, 1987 Mullis et al, Meth Enzymology 155: 335-350, 1987;
Negrutiu I et a1, Plant Mol Biol 8: 363-373, 1987 Neuhaus a t a 1, Theor Appl. Genet 74: 30 -36, 19 8 7 Palmiter et al, Cell 50: 435-443, 1987 Perez-Grau et al, Plant Cell 1: 1095-1109, 1989 Roberto et al, Pro Natl Acad Sci USA, 87: 5795-5801, 1990 Romano et al, Genes and Development 5: 438-446, 1991 Sambrook J et al, Molecular Cloning, A Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press, 1989 Sanger et al Proc Natl Acad Sci USA, 74: 5463-5467, 1977 Schekman, TIBS, 188, 1985 Schmitz et al, Plant Cell, l: '783-791, 1989 Schocher RJ et al, Bio/Technology 4: 1093-1096,1986 Shillito et a1, Bio/Technology 3: 1099-1103, 1985 Spena et al, Mol Gen Genet 227: 205-212, 1991 Wang Y-C et al, Plant Mol Biol 1 l: 433-439, 1988 Patent Literature US-P 4,918,006 US-P 5,086,169 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: CIBA-GEIGY AG
(B) STREET: Klybeckstr. 141 (C) CITY: Basel (E) COUNTRY: SCHWEIZ
(F) POSTAL CODE (ZIP): 4002 (G) TELEPHONE: +41 61 69 11 11 (H) TELEFAX: + 41 61 696 79 76 (I) TELEX: 962 991 (ii) TITLE OF INVENTION: Anther-specific cDNA Sequences, Genomic DNA
Sequences and Recombinant DNA Sequences (iii) NUMBER OF SEQUENCES: 21 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1542 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (C) INDIVIDUAL ISOLATE: Ant32 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 66..1412 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

Met Lys Val Ser Leu Lys His His Trp Val Val Lys Pro Ala Glu Ala Thr Trp Asn Gly Thr Val Ser Leu Ser Glu Cys Asp Gln Thr Phe Ala Val Thr His Val Pro Thr Ile Tyr Tyr Tyr Arg Phe Cys His Asp Cys Leu Pro Ser Thr Asp Asn Ile Ile Lys Thr Leu Arg Thr Ser Leu Ser Lys Ala Leu Val His Phe Tyr Pro Leu Ser Gly Arg Leu Arg Trp Ile Ala Gly Ser Arg Leu Glu Leu Asp Cys Asn Ala Ser Gly Ile Val Leu Met Glu Ala Glu Thr Glu Ala Lys Leu Asp Asp Leu Gly Asp Phe Ser Pro Ser Pro Asp Leu Asn Ser Leu Phe Pro Arg Val Asp Tyr Thr Ile Pro Ile Asp Glu Leu Pro Leu Leu Phe Val Gln Leu Thr Lys Phe Gln Cys Gly Gly Ile Ala Leu Ser Phe Ala Ile Ser His Ala Val Val Asp Gly Gln Ser Ala Leu Tyr Phe Leu Thr Glu Trp Ala Ser Leu Ala Arg Gly Glu Pro Leu Gly Asn Glu Pro Phe His Asp Arg Lys Phe Leu Arg Ala Gly Glu Pro Pro Ile Ala Tyr Pro Thr Phe Glu His Leu Gln Phe Asn Pro Pro Pro Leu Leu Leu Gly Gln Ser Ser Ser Glu Glu Glu Lys Lys Asn Glu Thr Lys Gly Ser Met Leu Lys Leu Thr Lys His Gln Val Glu Met Leu Arg Lys Lys Ala Asn Gln Gly Asn Gln Gly Arg Ser Tyr Thr Arg Tyr Glu Val Val Thr Ala His Ile Trp Arg Cys Ala Cys Lys Ala Arg Gly His Lys Phe Glu Gln Pro Thr Asn Leu Cys Ile Cys Val Asn Ile Arg Asn Ile Met Gln Pro Pro Leu Pro Lys Ser Tyr Phe Gly Asn Ala Ile Val Asp Val Ile Ala Asn Gly Val Ser Gly Asp Ile Thr Ser Arg Pro Leu Glu Tyr Val Ala Arg Arg Val Arg Ala Ala Ile Lys Met Val Thr Ser Asp Tyr Ala Asn Ser Thr Ile Asp Phe Leu Lys Asn Gln Glu Asp Leu Ser Lys Tyr Gln Asp Ile His Ala Phe Arg Ser Lys Glu Gly Pro Phe Tyr Gly Asn Pro Asn Leu Gly Val Ile Ser Trp Ile Ser Leu Pro Leu Leu Gly Leu Asp Phe Gly Trp Gly Lys Glu Ile His Met Ser Pro Gly Thr His Glu Tyr Asp Gly Asp Cys Val Ile Leu Pro Gly Lys Glu Gly Asp Gly Ser Leu Thr Val Ala Ile Ile Leu Gln Ala Val His Val Asp Ala Phe Lys Asn Phe Phe Tyr Glu Glu Ile Glu Cys (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 448 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Lys Val Ser Leu Lys His His Trp Val Val Lys Pro Ala Glu Ala Thr Trp Asn Gly Thr Val Ser Leu Ser, Glu Cys Asp Gln Thr Phe Ala Val Thr His Val Pro Thr Ile Tyr Tyr Tyr Arg Phe Cys His Asp Cys Leu Pro Ser Thr Asp Asn Ile Ile Lys Thr Leu Arg Thr Ser Leu Ser Lys Ala Leu Val His Phe Tyr Pro Leu Ser Gly Arg Leu Arg Trp Ile Ala Gly Ser Arg Leu Glu Leu Asp Cys Asn Ala Ser Gly Ile Val Leu Met Glu Ala Glu Thr Glu Ala Lys Leu Asp Asp Leu Gly Asp Phe Ser Pro Ser Pro Asp Leu Asn Ser Leu Phe Pro Arg Val Asp Tyr Thr Ile Pro Ile Asp Glu Leu Pro Leu Leu Phe Val Gln Leu Thr Lys Phe Gln Cys Gly Gly Ile Ala Leu Ser Phe Ala Ile Ser His Ala Val Val Asp Gly Gln Ser Ala Leu Tyr Phe Leu Thr Glu Trp Ala Ser Leu Ala Arg Gly Glu Pro Leu Gly Asn Glu Pro Phe His Asp Arg Lys Phe Leu Arg Ala Gly Glu Pro Pro Ile Ala Tyr Pro Thr Phe Glu His Leu Gln Phe Asn Pro Pro Pro Leu Leu Leu Gly Gln Ser Ser Ser Glu Glu Glu Lys Lys Asn Glu Thr Lys Gly Ser Met Leu Lys Leu Thr Lys His Gln Val Glu Met Leu Arg Lys Lys Ala Asn Gln Gly Asn Gln Gly Arg Ser Tyr Thr Arg Tyr Glu Val Val Thr Ala His Ile Trp Arg Cys Ala Cys Lys Ala Arg Gly His Lys Phe Glu Gln Pro Thr Asn Leu Cys Ile Cys Val Asn Ile Arg Asn Ile Met Gln Pro Pro Leu Pro Lys Ser Tyr Phe Gly Asn Ala Ile Val Asp Val Ile Ala Asn Gly Val Ser Gly Asp Ile Thr Ser Arg Pro Leu Glu Tyr Val Ala Arg Arg Val Arg Ala Ala Ile Lys Met Val Thr Ser Asp Tyr Ala Asn Ser Thr Ile Asp Phe Leu Lys Asn Gln Glu Asp Leu Ser Lys Tyr Gln Asp Ile His Ala Phe Arg Ser Lys Glu Gly Pro Phe Tyr Gly Asn Pro Asn Leu Gly Val Ile Ser Trp Ile Ser Leu Pro Leu Leu Gly Leu Asp Phe Gly Trp Gly Lys Glu Ile His Met Ser Pro Gly Thr His Glu Tyr Asp, Gly Asp Cys Val Ile Leu Pro Gly Lys Glu Gly Asp G1y Ser Leu Thr Val Ala Ile Ile Leu Gln Ala Val His Val Asp Ala Phe Lys Asn Phe Phe Tyr Glu Glu Ile Glu Cys (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 552 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum (C) INDIVIDUALISOLATE: Ant43D
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 41..397 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

Met Ala Arg Phe Leu Val Phe Leu Ala Leu Ala Leu Val Ile Ile Ser Lys Lys Gly Ala Leu Gly Ala Pro Pro Ser Cys Pro Thr Val Thr Thr Gln Leu Ala Pro Cys Leu Ser Tyr Ile Gln Gly Gly Gly Asp Pro Ser Val Pro Cys Cys Thr Gly Ile Asn Asn Ile Tyr Glu Leu Ala Lys Thr Lys Glu Asp Arg Val Ala Ile Cys Asn Cys Leu Lys Thr Ala Phe Thr His Ala Gly Asn Val Asn Pro Thr Leu Val Ala Gln Leu Pro Lys Lys Cys Gly Ile Ser Phe Asn Met Pro Pro Ile Asp Lys Asn Tyr Asp Cys Asn Thr Ile Ser Met Tyr (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Ala Arg Phe Leu Val Phe Leu Ala Leu Ala Leu Val Ile Ile Ser 1' 5 10 15 Lys Lys Gly Ala Leu Gly Ala Pro Pro Ser Cys Pro Thr Val Thr Thr Gln Leu Ala Pro Cys Leu Ser Tyr Ile Gln Gly Gly Gly Asp Pro Ser Val Pro Cys Cys Thr Gly Ile Asn Asn Ile Tyr Glu Leu Ala Lys Thr Lys Glu Asp Arg Val Ala Ile Cys Asn Cys Leu Lys Thr Ala Phe Thr His Ala Gly Asn Val Asn Pro Thr Leu Val Ala Gln Leu Pro Lys Lys Cys Gly Ile Ser Phe Asn Met Pro Pro Ile Asp Lys Asn Tyr Asp Cys Asn Thr Ile Ser Met Tyr (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 612 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (C) INDIVIDUAL ISOLATE: Ant9 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 344..442 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:

Met Pro Ser Leu Ser Val Gln Ser Ser Ser Pro Leu Leu Cys Gly Lys Leu Ser Leu Met Gly Ser Val Pro Thr Ser Ser Gln Ser Leu Gly Glu (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTF~RISTICS:

(A) LENGTH: 32 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Pro Ser Leu Ser Val Gln Ser Ser Ser Pro Leu Leu Cys Gly Lys Leu Ser Leu Met Gly Ser Val Pro Thr Ser Ser Gln Ser Leu Gly Glu (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (C) INDIVIDUAL ISOLATE: Ant52 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..95 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

His Asn Cys Leu Asn Cys Asn Ser Lys Arg Gln Gln Asp Ser Tyr Phe Phe Thr Asp Pro Met Lys Ala Gln Ser Ile Val Gly Thr Val Thr (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
His Asn Cys Leu Asn Cys Asn Ser Lys Arg Gln Gln Asp Ser Tyr Phe Phe Thr Asp Pro Met Lys Ala Gln Ser Ile Val Gly Thr Val Thr (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1201 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (C) INDIVIDUALISOLATE: Ant59 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1119 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:

Ile Phe Ser Ser Gln Ile Trp Thr Gln Pro Asn Ser Glu Met Asn Asn Asp Leu Val Ile Pro Ala Ile Phe Asn His Glu Lys Leu Arg Thr Ile Ser Arg Glu Cys Asp Pro Lys Arg Lys Leu Ala Glu Ser Asn Ser Gly Asp Ile Met Gly Glu Val Lys Lys Thr His Gln Ala Ile Gln Ser Leu Asp Lys Ser Met Ser Thr Leu Glu Asn Glu Leu Ala Ile Ala Arg Thr Arg Gln Thr Ile Ser His Asn Ala Lys Glu Asn Arg Ala Ser Asn His Thr Thr Pro Asn Lys Ala Phe Ile Val Val Gly Ile Asn Thr Ala Phe Ser Ser Arg Lys Arg Arg Asp Ser Leu Arg Glu Thr Trp Met Pro Lys Gly Asp Lys Leu Arg Lys Leu Glu Lys Glu Lys Gly Ile Val Ile Arg Phe Val Ile Gly His Ser Ala Thr Arg Gly Gly Val Leu Asp Arg Ala Ile Asp Ser Glu Asp Ala Gln Tyr Lys Asp Phe Leu Arg Leu Asp His Val Glu Gly Tyr His Glu Leu Ser Thr Lys Thr Arg Leu Tyr Phe Ser Lys Ala Val Ser Ile Trp Asp Ala Asp Phe Tyr Val Lys Val Asp Asp Asp Val His Leu Asn Leu Gly Met Leu Ala Asn Thr Leu Ala Lys Tyr Lys Ser Lys Pro Arg Val Tyr Ile Gly Cys Met Lys Ser Gly Pro Val Leu Ser Gln Lys Gly Val Arg Tyr Tyr Glu Pro Glu Tyr Trp Lys Phe Gly Glu Glu Gly Asn Lys Tyr Phe Arg His Ala Thr Gly Gln Ile Tyr Gly Ile Ser Arg Asp Leu Ala Ser Tyr Ile Ser Ile Asn Ser Gly Ile Leu His Arg Tyr Ala Asn Glu Asp Val Ser Leu Gly Ser Trp Leu Ile Gly Leu Glu Val Glu His Val Asp Glu Arg Ser Met Cys Cys Gly Thr Pro Pro Asp Cys Glu Trp Lys Ala Lys Gly Gly Asn Ile Cys Val Ala Ser Phe Asp Trp Ser Cys Ser Gly Ile Cys Lys Ser Val Glu Arg Met Lys Asp Val His His Ser Cys Gly Glu Gly Asp Ala Ala Leu Trp Asn Val Pro Leu Ser (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 372 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ile Phe Ser Ser Gln Ile Trp Thr Gln Pro Asn Ser Glu Met Asn Asn Asp Leu Val Ile Pro Ala Ile Phe Asn His Glu Lys Leu Arg Thr Ile Ser Arg Glu Cys Asp Pro Lys Arg Lys Leu Ala Glu Ser Asn Ser Gly Asp Ile Met Gly Glu Val Lys Lys Thr His Gln Ala Ile Gln Ser Leu Asp Lys Ser Met Ser Thr Leu Glu Asn Glu Leu Ala Ile Ala Arg Thr Arg Gln Thr Ile Ser His Asn Ala Lys Glu Asn Arg Ala Ser Asn His Thr Thr Pro Asn Lys Ala Phe Ile Val Val Gly Ile Asn Thr Ala Phe Ser Ser Arg Lys Arg Arg Asp Ser Leu Arg Glu Thr Trp Met Pro Lys Gly Asp Lys Leu Arg Lys Leu Glu Lys, Glu Lys Gly Ile Val Ile Arg Phe Val Ile Gly His Ser Ala Thr Arg Gly Gly Val Leu Asp Arg Ala Ile Asp Ser Glu Asp Ala Gln Tyr Lys Asp Phe Leu Arg Leu Asp His Val Glu Gly Tyr His Glu Leu Ser Thr Lys Thr Arg Leu Tyr Phe Ser Lys Ala Val Ser Ile Trp Asp Ala Asp Phe Tyr Val Lys Val Asp Asp Asp Val His Leu Asn Leu Gly Met Leu Ala Asn Thr Leu Ala Lys Tyr Lys Ser Lys Pro Arg Val Tyr Ile Gly Cys Met Lys Ser Gly Pro Val Leu Ser Gln Lys Gly Val Arg Tyr Tyr Glu Pro Glu Tyr Trp Lys Phe Gly Glu Glu Gly Asn Lys Tyr Phe Arg His Ala Thr Gly Gln Ile Tyr Gly Ile Ser Arg Asp Leu Ala Ser Tyr Ile Ser Ile Asn Ser Gly Ile Leu His Arg Tyr Ala Asn Glu Asp Val Ser Leu Gly Ser Trp Leu Ile Gly Leu Glu Val Glu His Val Asp Glu Arg Ser Met Cys Cys Gly Thr Pro Pro Asp Cys Glu Trp Lys Ala Lys Gly Gly Asn Ile Cys Val Ala Ser Phe Asp Trp Ser Cys Ser Gly Ile Cys Lys Ser Val Glu Arg Met Lys Asp Val His His Ser Cys Gly Glu Gly Asp Ala Ala Leu Trp Asn Val Pro Leu Ser (2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 952 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (C) INDIVIDUALISOLATE: Ant66 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..711 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:

Ile Met Thr Ile Ser Lys Asp Arg Val Lys Pro Ser Pro Leu Pro Asp Ser Trp Lys Leu Asn Glu Ile Phe Ala Thr Gly Ile Val Leu Gly Thr Tyr Gln Ala Ile Met Thr Val Val Phe Phe Tyr Leu Ala Ala Asp Thr Asp Phe Phe Thr Glu Lys Phe Asn Val Lys Ser Ile Arg Asp Asn Pro Tyr Glu Leu Thr Ala Ala Val Tyr Leu Gln Val Ser Ile Ile Ser Gln 65 '70 75 80 Ala Leu Ile Phe Val Thr Arg Ser Arg Ser Trp Ser Phe Leu Glu Arg Pro Gly Phe Leu Leu Val Thr Ala Phe Leu Leu Ala Gln Phe Val Ala Thr Leu Ile Ala Val Tyr Ala Asn Trp Lys Phe Ala Arg Ile His Gly Ile Gly Trp Gly Trp Ala Gly Ile Ile Trp Ile Tyr Thr Ile Ile Thr Tyr Ile Pro Leu Asp Ile Leu Lys Phe Ile Ser Arg Tyr Thr Leu Ser Gly Glu Ala Trp Asn Ser Met Ile Gln Asn Lys Thr Ala Phe Thr Thr Lys Lys Asp Tyr Gly Lys Gly Glu Arg Glu Ala Gln Trp Ala Val Ala Gln Arg Thr Leu His Gly Leu Gln Thr Ala Glu Ser Asn Gly Leu Phe His Asp Lys Asn Tyr Arg Glu Leu Asn Glu Ile Ala Glu Gln Ala Lys Arg Arg Ala Glu Val Ala Lys Tyr Thr His Glu Pro (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 236 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Ile Met Thr Ile Ser Lys Asp Arg Val Lys Pro Ser Pro Leu Pro Asp Ser Trp Lys Leu Asn Glu Ile Phe Ala Thr Gly Ile Val Leu Gly Thr Tyr Gln Ala Ile Met Thr Val Val Phe Phe Tyr Leu Ala Ala Asp Thr Asp Phe Phe Thr Glu Lys Phe Asn Val Lys Ser Ile Arg Asp Asn Pro Tyr Glu Leu Thr Ala Ala Val Tyr Leu Gln Val Ser Ile Ile Ser Gln Ala Leu Ile Phe Val Thr Arg Ser Arg Ser Trp Ser Phe Leu Glu Arg Pro Gly Phe Leu Leu Val Thr Ala Phe Leu Leu Ala Gln Phe Val Ala Thr Leu Ile Ala Val Tyr Ala Asn Trp Lys Phe Ala Arg Ile His Gly Ile Gly Trp Gly Trp Ala Gly Ile Ile Trp Ile Tyr Thr Ile Ile Thr Tyr Ile Pro Leu Asp Ile Leu Lys Phe Ile Ser Arg Tyr Thr Leu Ser Gly Glu Ala Trp Asn Ser Met Ile Gln Asn Lys Thr Ala Phe Thr Thr Lys Lys Asp Tyr Gly Lys Gly Glu Arg Glu Ala Gln Trp Ala Val Ala Gln Arg Thr Leu His Gly Leu Gln Thr Ala Glu Ser Asn Gly Leu Phe His Asp Lys Asn Tyr Arg Glu Leu Asn Glu Ile Ala Glu Gln Ala Lys Arg Arg Ala Glu Val Ala Lys Tyr Thr His Glu Pro 225 2.30 235 (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 305 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (C) INDIVIDUAL ISOLATE: Ant67 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:

(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 445 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (C) INDIVIDUALISOLATE: Ant68 (ix) FEATURE:
(A} NAME/KEY: CDS
(B) LOCATION: 2..445 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:

Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala Tyr Gly Ser Gly Cys Gly Glu Asn Gly Cys Asn Tyr Pro Pro Val Val Pro Gly Pro Pro Gln Thr Gly Glu Asn Pro Tyr Cys Met Pro Gly Cys Gly Val Gly Gly Gly Gly Val Gly Gly Ser Asn Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Tyr Gly Ser Gly Tyr Gly Glu Asn Gly Asn Cys Asn Tyr Pro Pro Val Ile Pro Gly Pro Pro Gln Thr Ile Gly Pro Ile Cys Asn Cys Pro Ile Thr Gln Pro Thr Phe Pro Phe Arg Cys Pro Tyr Gly Cys Gln Pro Pro Pro Ser Tyr Gly Cys Pro Asn Gly Asn Ser Arg Leu Thr His Asp Lys Glu Lys Gln Asn His Gln Pro Lys Thr Thr Ala Ser (2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 148 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala Tyr Gly Ser Gly Cys Gly Glu Asn Gly Cys Asn Tyr Pro Pro Val Val Pro Gly Pro Pro Gln Thr Gly Glu Asn Pro Tyr Cys Met Pro Gly Cys Gly Val Gly Gly Gly Gly Val Gly Gly Ser Asn Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Tyr Gly Ser Gly Tyr Gly Glu Asn Gly Asn Cys Asn Tyr Pro Pro Val Ile Pro Gly Pro Pro Gln Thr Ile Gly Pro Ile Cys Asn Cys Pro Ile Thr Gln Pro Thr Phe Pro Phe Arg Cys Pro Tyr Gly Cys Gln Pro Pro Pro Ser Tyr Gly Cys Pro Asn Gly Asn Ser Arg Leu Thr His Asp Lys Glu Lys Gln Asn His Gln Pro Lys Thr Thr Ala Ser (2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3705 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (C) INDIVIDUAL ISOLATE: Ant32 genomic clone (vii) IMMEDIATE SOURCE:
(B) CLONE: pCIB950 (ix) FEATURE:
(A) NAME/KEY: TATA signal (B) LOCATION: 1971..1975 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2076..3422 (ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 2009 (D) OTHERINFORMATION: /note= "Putative transcription start site"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:

Met Lys Val Ser Leu Lys His His Trp Val Val Lys Pro Ala Glu Ala Thr Trp Asn Gly Thr Val Ser Leu Ser Glu Cys Asp Gln Thr Phe Ala Val Thr His Val Pro Thr Ile Tyr Tyr Tyr Arg Phe Cys His Asp Cys Leu Pro Ser Thr Asp Asn Ile Ile Lys Thr Leu Arg Thr Ser Leu Ser Lys Ala Leu Val His Phe Tyr Pro Leu Ser Gly Arg Leu Arg Trp Ile Ala Gly Ser Arg Leu Glu Leu Asp Cys Asn Ala Ser Gly Ile Val Leu Met Glu Ala Glu Thr Glu Ala Lys Leu Asp Asp Leu Gly Asp Phe Ser Pro Ser Pro Asp Leu Asn Ser Leu Phe Pro Arg Val Asp Tyr Thr Ile Pro Ile Asp Glu Leu Pro Leu Leu Phe Val Gln Leu Thr Lys Phe Gln Cys Gly Gly Ile Ala Leu Ser Phe Ala Ile Ser His Ala Val Val Asp Gly Gln Ser Ala Leu Tyr Phe Leu Thr Glu Trp Ala Ser Leu Ala Arg Gly Glu Pro Leu Gly Asn Glu Pro Phe His Asp Arg Lys Phe Leu Arg Ala Gly Glu Pro Pro Ile Ala Tyr Pro Thr Phe Glu His Leu Gln Phe Asn Pro Pro Pro Leu Leu Leu Gly Gln Ser Ser Ser Glu Glu Glu Lys Lys Asn Glu Thr Lys Gly Ser Met Leu Lys Leu Thr Lys His Gln Val GIu Met Leu Arg Lys Lys Ala Asn Gln Gly Asn Gln Gly Arg Ser Tyr Thr Arg Tyr Glu Val Val Thr Ala His Ile Trp Arg Cys Ala Cys Lys Ala Arg Gly His Lys Phe Glu Gln Pro Thr Asn Leu Cys Ile Cys Val Asn Ile Arg Asn Ile Met Gln Pro Pro Leu Pro Lys Ser Tyr Phe Gly Asn Ala Ile Val Asp Val Ile Ala Asn Gly Val Ser Gly Asp Ile Thr Ser Arg Pro Leu Glu Tyr - 70 _ Val Ala Arg Arg Val Arg Ala Ala Ile Lys Met Val Thr Ser Asp Tyr Ala Asn Sex Thr Ile Asp Phe Leu Lys Asn Gln Glu Asp Leu Ser Lys Tyr Gln Asp Ile His Ala Phe Arg Ser Lys Glu Gly Pro Phe Tyr Gly Asn Pro Asn Leu Gly Val Ile Ser Trp Ile Ser Leu Pro Leu Leu Gly Leu Asp Phe Gly Trp Gly Lys Glu Ile His Met Ser Pro Gly Thr His Glu Tyr Asp Gly Asp Cys Val Ile Leu Pro Gly Lys Glu Gly Asp Gly Ser Leu Thr Val Ala Ile Ile Leu Gln Ala Val His Val Asp Ala Phe Lys Asn Phe Phe Tyr Glu Glu Ile Glu Cys (2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 448 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Met Lys Val Ser Leu Lys His His Trp Val Val Lys Pro Ala Glu Ala Thr Trp Asn Gly Thr Val Ser Leu Ser Glu Cys Asp Gln Thr Phe Ala Val Thr His Val Pro Thr Ile Tyr Tyr Tyr Arg Phe Cys His Asp Cys Leu Pro Ser Thr Asp Asn Ile Ile Lys Thr Leu Arg Thr Ser Leu Ser Lys Ala Leu Val His Phe Tyr Pro Leu Ser Gly Arg Leu Arg Trp Ile Ala Gly Ser Arg Leu Glu Leu Asp Cys Asn Ala Ser Gly Ile Val Leu Met Glu Ala Glu Thr Glu Ala Lys Leu Asp Asp Leu Gly Asp Phe Ser Pro Ser Pro Asp Leu Asn Ser Leu Phe Pro Arg Val Asp Tyr Thr Ile Pro Ile Asp Glu Leu Pro Leu Leu Phe Val Gln Leu Thr Lys Phe Gln Cys Gly Gly Ile Ala Leu Ser Phe Ala Ile Ser His Ala Val Val Asp Gly Gln Ser Ala Leu Tyr Phe Leu Thr Glu Trp Ala Ser Leu Ala Arg Gly Glu Pro Leu Gly Asn Glu Pro Phe His Asp Arg Lys Phe Leu Arg Ala Gly Glu Pro Pro Ile Ala Tyr Pro Thr Phe Glu His Leu Gln Phe Asn Pro Pro Pro Leu Leu Leu Gly Gln Ser Ser Ser Glu Glu Glu Lys Lys Asn Glu Thr Lys Gly Ser Met Leu Lys Leu Thr Lys His Gln Val Glu Met Leu Arg Lys Lys Ala Asn Gln Gly Asn Gln Gly Arg Ser Tyr Thr Arg Tyr Glu Val Val Thr Ala His Ile Trp Arg Cys Ala Cys Lys Ala Arg Gly His Lys Phe Glu Gln Pro Thr Asn Leu Cys Ile Cys Val Asn Ile Arg Asn Ile Met Gln Pro Pro Leu Pro Lys Ser Tyr Phe Gly Asn Ala Ile Val Asp Val Ile Ala Asn Gly Val Ser Gly Asp Ile Thr Ser Arg Pro Leu Glu Tyr Val Ala Arg Arg Val Arg Ala Ala Ile Lys Met Val Thr Ser Asp Tyr Ala Asn Ser Thr Ile Asp Phe Leu Lys Asn Gln Glu Asp Leu Ser Lys Tyr Gln Asp Ile His Ala Phe Arg Ser Lys Glu Gly Pro Phe Tyr Gly Asn Pro Asn Leu Gly Val Ile Ser Trp Ile Ser Leu Pro Leu Leu Gly Leu Asp Phe Gly Trp Gly Lys Glu Ile His Met Ser Pro Gly Thr His Glu Tyr Asp Gly Asp Cys Val Ile Leu Pro Gly Lys Glu Gly Asp Gly Ser Leu Thr Val Ala Ile Ile Leu Gln Ala Val His Val Asp Ala Phe Lys Asn Phe Phe Tyr Glu Glu Ile Glu Cys (2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1906 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (C) INDIVIDUAL ISOLATE: Ant43D
(vii) IMMEDIATE SOURCE:
(B) CLONE: pCIB952 (ix) OFEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(1230..1570, 1669..1684) (ix) FEATURE:
(A) NAME/KEY: intron (B) LOCATION: 1571..1668 (ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 1167 (D) OTHER INFORMATION: /note= "Putative transcriptional start site"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:

Met Ala Arg Phe Leu Val Phe Leu Ala Leu Ala Leu Val Ile Ile Ser Lys Lys Gly Ala Leu Gly Ala Pro Pro Ser Cys Pro Thr Val Thr Thr Gln Leu Ala Pro Cys Leu Ser Tyr Ile Gln Gly Gly Gly Asp Pro Ser Val Pro Cys Cys Thr Gly Ile Asn Asn Ile Tyr Glu Leu Ala Lys Thr Lys Glu Asp Arg Val Ala Ile Cys Asn Cys Leu Lys Thr Ala Phe Thr His Ala Gly Asn Val Asn Pro Thr Leu Val Ala Gln Leu Pro Lys Lys Cys Gly Ile Ser Phe Asn Met Pro Pro Ile Asp Lys Asn Tyr Asp Cys Asn Thr Ile Ser Met Tyr (2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Met Ala Arg Phe Leu Val Phe Leu Ala Leu Ala Leu Val Ile Ile Ser Lys Lys Gly Ala Leu Gly Ala Pro Pro Ser Cys Pro Thr Val Thr Thr Gln Leu Ala Pro Cys Leu Ser Tyr Ile Gln Gly Gly Gly Asp Pro Ser Val Pro Cys Cys Thr Gly Ile Asn Asn Ile Tyr Glu Leu Ala Lys Thr Lys Glu Asp Arg Val Ala Ile Cys Asn Cys Leu Lys Thr Ala Phe Thr His Ala Gly Asn Val Asn Pro Thr Leu Val Ala Gln Leu Pro Lys Lys Cys Gly Ile Ser Phe Asn Met Pro Pro Ile Asp Lys Asn Tyr Asp Cys Asn Thr Ile Ser Met Tyr (2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 437 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (C) INDIVIDUAL ISOLATE: Ant43C
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 167..436 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:

Met Ala Arg Phe Leu Ala Leu Ala Leu Val Val Ile Ala Leu Ser Asn Asp Ala Leu Gly Ala Pro Pro Ser Cys Gln Thr Val Thr Thr Gln Leu Ala Pro Cys Leu Ser Tyr Ile Gln Asn Arg Val Lys Gly Gly Gly Asn Pro Ser Val Pro Cys Cys Thr Gly Ile Asn Asn Ile Tyr Glu Leu Ala Lys Thr Lys Glu Asp Arg Val Ala Ile Cys Asn Cys Leu Lys Asn Ala Phe Ile His Ala Gly Asn Val Asn Pro Thr (2) INFORMATION FOR SEQ ID N0:21:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 90 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Met Ala Arg Phe Leu Ala Leu Ala Leu Val Val Ile Ala Leu Ser Asn Asp Ala Leu Gly Ala Pro Pro Ser Cys Gln Thr Val Thr Thr Gln Leu Ala Pro Cys Leu Ser Tyr Ile Gln Asn Arg Val Lys Gly Gly Gly Asn Pro Ser Val Pro Cys Cys Thr Gly Ile Asn Asn Ile Tyr Glu Leu Ala Lys Thr Lys Glu Asp Arg Val Ala Ile Cys Asn Cys Leu Zys Asn Ala Phe Ile His Ala Gly Asn Val Asn Pro Thr

Claims (2)

1. A process for the production of a recombinant DNA molecule comprising, in a 5' to 3' direction, an anther-specific promoter region operatively linked to an expressible DNA sequence, which process comprises (a) first isolating from a suitable source or synthesising by means of known processes a promoter region responsible for the anther-specific expression of an associated expressible DNA sequence; and (b) operably linking the said anther-specific DNA sequence in a 5' to 3' direction to an expressible DNA sequence.

2. A process for the production of transformed plant material, including whole plants, comprising a recombinant DNA molecule comprising, in a 5' to 3' direction, an anther-specific promoter region operatively linked to an expressible DNA sequence, which process essentially comprises:
(a) first isolating from a suitable source or synthesising by means of known processes a promoter region responsible for the anther-specific expression of an associated expressible DNA sequence;
(b) operably linking the said anther-specific DNA sequence in a 5' to 3' direction to an expressible DNA sequence;
(c) cloning the final construct into a plant expression vector under the control of expression signals active in plants;

(d) transforming the said expression vector into plant material by means of known processes and expressing it therein; and optionally (e) regenerating the plant material transformed according to step (d) to a whole and preferably phenotypically normal plant.