| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 26.03.2002 |
| PATENT TITLE |
Compositions and methods for production of male-sterile plants |
| PATENT ABSTRACT |
The present invention provides plant calcium/calmodulin-dependent protein kinase (CCaMK) nucleic acids, polypeptides, antibodies, and related methods. CCaMK genes are expressed in anthers in a developmental stage-specific manner. Suppression of CCaMK expression, e.g., by an antisense transgene, results in male-sterility. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | February 25, 1999 |
| PATENT REFERENCES CITED |
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Liu et al., "Reduced expression of an anther-specific calcium/calmodulin-dependent protein kinase gene induces male sterility," Annual Meeting of the American Society of Plant Physiologists: San Antonio, TX 111:58, 1996. Tirlapur et al., "Changes in calcium and calmodulin levels during microsporogenesis pollen development and germination in gasteria-verrucosa Mill. H. Duval," Plant Sexual Reproduction 5:214-223, 1992. (Abstract only). Watillon et al., "Structure of a calmodulin-binding protein kinase gene from apple," Plant Physiology 108:847-848, 1995. |
| PATENT GOVERNMENT INTERESTS |
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT This invention was made with government support under National Science Foundation grant number DCB 91-4586. The government has certain rights in this invention. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. An isolated promoter, comprising a nucleic acid sequence selected from the group consisting of: (a) a nucleic acid sequence as shown in SEQ ID NO: 11; (b) a nucleic acid sequence comprising nucleic acid residues 1101-1701 of the sequence shown in SEQ ID NO: 11; and (c) a nucleic acid sequence that shares at least 90% sequence identity with the nucleic acid sequences shown in (a) or (b), wherein the nucleic acid sequence is capable of promoting transcription. 2. The promoter of claim 1, wherein the promoter comprises nucleic acid residues 1101-1701 of the sequence shown in SEQ ID NO: 11. 3. The promoter of claim 1, wherein the promoter comprises a nucleic acid sequence as shown in SEQ ID NO: 11. 4. The promoter of claim 1, wherein the promoter is anther specific. 5. A method for expressing a nucleic acid sequence, comprising operably linking the promoter of claim 1 to a recombinant nucleic acid sequence, wherein the promoter drives the expression of the recombinant nucleic acid sequence. 6. The method according to claim 5, wherein the expression of the recombinant nucleic acid sequence occurs in vivo. 7. The method of claim 6, wherein the expression occurs in a plant cell. 8. The method according to claim 5, wherein the expression of the recombinant nucleic acid sequence occurs in vitro. 9. The method according to claim 5, wherein the recombinant nucleic acid sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 10, the complement of SEQ ID NO: 1, and the complement of SEQ ID NO: 10. |
| PATENT DESCRIPTION |
TECHNICAL FIELD This invention relates to plant calcium/calmodulin-dependent protein kinases, particularly anther-specific calcium/calmodulin-dependent protein kinases. BACKGROUND OF THE INVENTION Calcium and calmodulin regulate diverse cellular processes in plants (Poovaiah and Reddy, CRC Crit. Rev. Plant Sci. 6:47-103, 1987, and CRC Crit. Rev. Plant Sci. 12:185-211, 1993; Roberts and Harmon, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:375-414, 1992; Gilroy and Trewavas, BioEssays 16:677-682, 1994). Transient changes in intracellular Ca.sup.2+ concentration can affect a number of physiological processes through the action of calmodulin (CaM), a ubiquitous Ca.sup.2+ -binding protein. Ca.sup.2+ /calmodulin-regulated protein phosphorylation plays a pivotal role in amplifying and diversifying the action of Ca.sup.2+ -mediated signals (Veluthambi and Poovaiah, Science 223:167-169, 1984; Schulman, Curr. Opin. in Cell. Biol. 5:247-253, 1993). Extracellular and intracellular signals regulate the activity of protein kinases, either directly or through second messengers. These protein kinases in turn regulate the activity of their substrates by phosphorylation (Cohen, Trends Biochem. Sci. 17:408-413, 1992; Stone and Walker, Plant Physiol. 108:451-457, 1995). In animals, Ca.sup.2+ /calmodulin-dependent protein kinases play a pivotal role in cellular regulation (Colbran and Soderling, Current Topics in Cell. Reg. 31:181-221, 1990; Hanson and Schulman, Annu. Rev. Biochem. 61:559-601, 1992; Mayford et al., Cell 81:891-904, 1995). Several types of CaM-dependent protein kinases (CaM kinases, phosphorylase kinase, and myosin light chain kinase) have been well characterized in mammalian systems (Fujisawa, BioEssays 12:27-29, 1990; Colbran and Soderling, Current Topics in Cell. Reg. 31:181-221,1990; Klee, Neurochem. Res. 16:1059-1065, 1991; Mochizuki et al., J. Biol. Chem. 268:9143-9147, 1993). Although little is known about Ca.sup.2+ /calmodulin-dependent protein kinases in plants (Poovaiah et al., in Progress in Plant Growth Regulation, Karssen et al., eds., Dordrecht, The Netherlands: Kluwer Academic Publishers, 1992, pp. 691-702; Watillon et al., Plant Physiol. 101:1381-1384, 1993), Ca.sup.2+ -dependent, calmodulin-independent protein kinases (CDPKs) have been identified (Harper et al., Science 252:951-954, 1991; Roberts and Harmon, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:375-414, 1992). Male gametophyte formation in the anther is a complex developmental process involving many cellular events. During microsporogenesis, microsporocytes undergo meiosis to form tetrads of microspores that are surrounded by a callose wall composed of .beta.-1,3-glucan. The callose wall is subsequently degraded by callase, which is secreted by cells of the tapetum (Steiglitz, Dev. Biol. 57:87-97, 1977), a specialized anther tissue that produces a number of proteins and other substrates that aid in pollen development or become a component of the pollen outer wall (Paciani et al., Plant Syst. Evol. 149:155-185, 1985; Bedinger, Plant Cell 4:879-887, 1992; Mariani et al., Nature 347:737-741, 1990). The timing of callase secretion is critical for microspore development. Male sterility has been shown to result from premature or delayed appearance of callase (Worral et al., Plant Cell 4:759-771, 1992; Tsuchiya et al., Plant Cell Physiol. 36:487-494, 1995). Induction of male sterility in plants can provide significant cost savings in hybrid plant production, enable production of hybrid plants where such production was previously difficult or impossible, and allow the production of plants with reduced pollen formation to reduced the tendency of such plants to elicit allergic reactions or to extend the life of flowers that senesce upon pollination (e.g., orchids). Several strategies have been developed for the production of male-sterile plants (Goldberg et al., Plant Cell 5:1217-1229, 1993), including: selective destruction of the tapetum by fusing the ribonuclease gene to a tapetum-specific promoter, TA29 (Mariani et al., Nature 347:737-741, 1990); premature dissolution of the callose wall in pollen tetrads by fusing glucanase gene to tapetum-specific A9 or Osg6B promoters (Worrall et al., Plant Cell 4:759-771, 1992; Tsuchiya et al., Plant Cell Physiol. 36:487-494, 1995); antisense inhibition of flavonoid biosynthesis within tapetal cells (Van der Meer et al., Plant Cell 4:253-262, 1992); tapetal-specific expression of the Agrobacterium rhizogenes rolB gene (Spena et al., Theor. Appl. Genet. 84:520-527, 1992); and overexpression of the mitochondrial gene atp9 (Hernould et al., Proc. Natl. Acad. Sci. USA 90:2370-2374, 1993). SUMMARY OF THE INVENTION Genes encoding plant calcium/calmodulin-dependent protein kinases (CCaMKs) have been cloned and sequenced. Expression of CCaMK genes is highly organ- and developmental stage-specific. When CCaMK antisense constructs were expressed in plants, the plants were rendered male-sterile. The availability of CCaMK CDNA and genomic DNA sequences makes possible the production of a wide variety of male-sterile plants, including monocotyledonous, dicotyledonous, and other plant varieties. CCaMK promoters are also useful for targeted expression of heterologous genes, as is described in greater detail below. Accordingly, the present invention provides isolated nucleic acids based on the cloned CCaMK sequences. Nucleic acids that include at least 15 contiguous nucleotides of a native lily or tobacco CCaMK gene and hybridize specifically to a CCaMK sequence under stringent conditions are useful, for example, as CCaMK-specific probes and primers. CCaMK promoter sequences are useful for the expression of heterologous genes in anthers of transgenic plants in a developmental stage-specific manner. Isolated CCaMK nucleic acids can be expressed in host cells to produce recombinant CCaMK polypeptide or fragments thereof, which in turn can be used, for example, to raise CCaMK-specific antibodies that are useful for CCaMK immunoassays, for purification of CCaMK polypeptides, and for screening expression libraries to obtain CCaMK homologs from other plant species. The native CCaMK sequence can be altered, e.g., by silent and conservative substitutions, to produce modified forms of CCaMK that preferably retain calcium/calmodulin-dependent protein kinase activity. Alternately, CCaMK polypeptides can be obtained from plant tissue by standard protein purification techniques, including the use of CCaMK-specific antibodies. The foregoing and other objects and advantages of the invention will become more apparent from the following detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1B show the nucleotide and deduced amino acid sequences of lily CCaMK. Diagnostic sequences (GKGGFS, DLKPEN, SIDYVSPE) for serine/threonine kinases are underlined. Sequences corresponding to two PCR primers (DLKPEN and FNARRKL) are indicated by arrows. The calmodulin-binding domain is double-underlined, Ca.sup.2+ -binding EF-hand motifs are boxed, the putative autophosphorylation sites (RXXS/T) are indicated by asterisks, and the hatched region indicates the putative biotin-binding site (LKAMKMNSLI). FIG. 2 shows a comparison of the deduced amino-acid sequence of the C-terminal region (amino acid residues 338-520) of lily CCaMK to neural visinin-like Ca.sup.2+ -binding proteins. Conserved amino acids are boxed; Ca.sup.2+ -binding domains (I-III) are indicated by solid lines; putative autophosphorylation site is indicated by an asterisk; and the putative biotin-binding site (B) is indicated by a hatched box. Abbreviations: Rahc1, rat hippocalcin (Gen2:Ratp23K); Ravl3, rat neural visinin-like protein (Gen2:Ratnvp3); Bovl1, bovine neurocalcin (Gen1:Bovpcaln); Ravl1 rat neural visinin-like protein (Gen2:Ratnvp1); Chvl1 chicken visinin-like protein (Gen2:Ggvilip); Ravl2, rat neural visinin-like protein (Gen2:Ratnvp2); Drfr1, Drosophila frequenin (Gen2: Drofreq). FIG. 3 is a schematic representation of structural features of the lily CCaMK polypeptide. FIG. 4A shows SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of lily CCaMK polypeptide expressed in E. coli after induction by IPTG. Lanes: 1, IPTG-induced cell extract; 2, uninduced cell extract. Protein size is marked on the left. FIG. 4B shows binding of lily CCaMK protein (250 ng) to .sup.35 S-labeled calmodulin (50 nM) in the presence of either 5 mM EGTA or 1 mM CaCl.sub.2. The histogram shows radioactivity (cpm) on the nitrocellulose filter. Columns: 1, 5 mM EGTA; 2, 1 mM CaCl.sub.2 ; 3, 1 mM CaCl.sub.2 plus 2.5 .mu.M unlabeled calmodulin. An autoradiogram is shown on top of each corresponding column. FIG. 4C shows Ca.sup.2+ -binding to lily CCaMK. 1, bovine serum albumin (2 .mu.g); 2, calmodulin (2 .mu.g); 3, CCaMK (2 .mu.g). FIG. 5 shows results of an RNase protection assay using total RNA (20 .mu.g) from various parts of lily. Lanes: 1, leaf; 2, stem; 3, anthers from phase II; 4, sepals and petals from phase III; 5 anthers from phase III; 6, sepals and petals from phase III; 7, yeast tRNA control. FIG. 6 shows a Southern blot of lily genomic DNA digested with restriction enzymes and probed with a lily CCaMK cDNA probe. Lanes: 1, DraI; 2, EcoRI; 3, EcORV; 4, HindIII, 5, PstI; 6, XbaI; 7, XhoI. FIG. 7A shows Ca.sup.2+ /calmodulin-dependent phosphorylation of histone IIAS by lily CCaMK in the presence of 0.5 mM CaCl.sub.2 and increasing amounts of calmodulin (.mu.M). CCaMK activity is presented as nmol phosphate/min/mg CCaMK. FIG. 7B shows the time course of phosphorylation of histone IIAS by lily CCaMK in the presence of 2.5 mM EGTA (.circle-solid.) or 0.5 mM CaCl.sub.2 (.DELTA.) or 0.5 mM CaCl.sub.2 and 1 .mu.M calmodulin (.largecircle.). CCaMK activity is represented as nmol of phosphate per mg CCaMK. FIG. 8 shows a saturation curve of .sup.35 S-calmodulin binding to lily CCaMK. The amount of bound calmodulin at each point is represented as percent of maximal binding. Inset: Scatchard plot analysis (bound/free and bound calmodulin are expressed as B/F and B, respectively). FIG. 9A shows the results of calmodulin binding assays using wild-type and truncated forms of lily CCaMK in order to determine the calmodulin binding site. Left: Schematic diagram of wild-type and truncation mutants of CCaMK used for .sup.35 S-calmodulin binding assays. Right: Autoradiograms showing calmodulin binding corresponding to the wild-type and mutant CCaMKs. FIG. 9B shows a comparison of amino acid sequences surrounding the putative calmodulin-binding sites of lily CCaMK and a subunit of mammalian calmodulin kinase II (CaMKII). FIG. 10 shows calmodulin binding to synthetic peptides from the calmodulin-binding region (residues 311-340). A. Schematic diagram of wild-type and truncation mutants of CCaMK used for .sup.35 S-calmodulin binding assays are shown on the left. The mutants 1-356 and 1-322 represent CCaMK lacking the visinin-like domain and both visinin-like and calmodulin-binding domains. The autoradiogram is shown on the right of each diagram (boxed area). The radioactivity (cpm) of bound 35S-calmodulin was 11,600 for wild-type, 12,500 for the mutant 1-356 and 99 for the mutant 1-322, respectively. B. Comparison of amino acid sequences surrounding the putative calmodulin-binding sites of CCaMK and the .alpha. subunit of CaMK II. C. Calmodulin binding to the synthetic peptides in a gel mobility-shift assay. Non-denaturing gel electrophoresis performed in the presence of 0.5 mM CaCl.sub.2 using calmodulin alone (lane 1) or a mixture of calmodulin and each of the following peptides: CCaMK 328-340 (lane 2); CCaMK 322-340 (lane 3); 317-340 (lane 4); and 311-340 (lane 5). The bands of calmodulin (free CaM) or CaM-peptide complex were visualized by Coomassie Brilliant Blue. FIG. 11 shows a helical wheel projection of calmodulin-binding sequences in lily CCaMK (left) and animal CaMKII.alpha. (right). Hydrophobic amino acid residues are boxed. Basic amino acid residues are marked with (+). FIG. 12 shows a time course of autophosphorylation of lily CCaMK in the presence of 2.5 mM EGTA (.circle-solid.) or 0.5 mM CaCl.sub.2 (.DELTA.) or 0.5 mM CaCl.sub.2 and 1 .mu.M calmodulin (.largecircle.). The autophosphorylation is presented as pmol .sup.32 P incorporated per 21.4 pmol of CCaMK. FIG. 13A shows the effect of calmodulin on Ca.sup.2+ -dependent autophosphorylation of lily CCaMK in the presence of CaCl.sub.2 (0.5 mM) and increasing concentrations of calmodulin. Lane 1, +CaCl.sub.2, (0.5 mM); lanes 2-6, +CaCl.sub.2 (0.5 mM) and 60, 120, 240, 360, and 480 nM of calmodulin respectively. FIG. 13B shows phosphoamino acid analysis of autophosphorylated lily CCaMK (200 ng) either in the presence of 2.5 mM EGTA (-Ca), 0.5 mM CaCl2 (+Ca), or 0.5 mM CaCl.sub.2 plus 1 .mu.M calmodulin (+Ca/CaM). The positions of phosphoserine (S) and phosphothreonine (T) are marked. FIG. 14A shows amino acid sequences of the three EF-hand motifs in the visinin-like domain of lily CCaMK. Six Ca.sup.2+ -ligating residues denoted as x, y, z, -y, -x, -z, respectively, are marked. Site-directed mutants were prepared by substituting the amino acid residues at the -x position with alanine (A). FIG. 14B shows the Ca.sup.2+ -dependent mobility shift of wild-type CCaMK and CCamKs mutated in the visinin-like domain in the presence of 2.5 mM EGTA (lane 1) or 0.5 mM CaCl.sub.2 (lanes 2-6). Wild-type protein (lanes 1 and 2), proteins mutated in the EF hand I (lane 3), EF-hand II (lane 4), EF-hand III (lane 5), and all three EF hands (lane 6) are shown. FIGS. 15A-15D show a comparison of enzyme activity of wild-type (A and B) and a truncated lily CCaMK mutant (1-356) (C and D) with respect to Ca.sup.2+ -dependent autophosphorylation (A and C) and Ca.sup.2+ /calmodulin-dependent histone IIAS phosphorylation (B and D). The assays were carried out in the presence of 2.5 mM EGTA (-Ca), 0.5 mM CaCl.sub.2 (+Ca) or 0.5 mM CaCl.sub.2 and 1 .mu.M calmodulin (+Ca/CaM). FIG. 16A shows the effect of increasing concentrations of calmodulin on the GS peptide phosphorylation by autophosphorylated lily CCaMK. FIG. 16B shows the effect of CCaMK autophosphorylation on Ca.sup.2+ /calmodulin-dependent and calmodulin-independent activity. Column 1, CCaMK autophosphorylated in the presence of 0.5 mM CaCl.sub.2 and used for Ca.sup.2+ /calmodulin-dependent GS peptide phosphorylation (hatched bar). Column 2, unphosphorylated enzyme used for Ca.sup.2+ /calmodulin-dependent GS peptide phosphorylation (hatched bar). Solid bars represent the activity of autophosphorylated CCaMK (column 1) and unphosphorylated CCaMK (column 2) in the presence of 2.5 mM EGTA. FIGS. 17A-l7B show the effects of increasing amounts of synthetic peptides derived from the CCaMK autoinhibitory domain (amino acid residues 311-340) on the activity of the constitutive mutant 1-322. FIGS. 18A-18B show models describing the regulation of CCaMK by Ca.sup.2+ and Ca.sup.2+ /calmodulin (A) and the autoinhibitory domain (B). FIGS. 19A-19C show cross-sections of lily anthers demonstrating progressive development of tapetal cells and microspores. A. Pollen mother cell stage (2.0 cm bud), 97.times.. B. Meiosis stage I (2.5 cm bud), 97.times.. C. Uninucleate microspore stage (3.5 cm bud), 97.times.. Abbreviations: E, epidermis; En, endothecium; M, middle layers; PMC, pollen mother cells; DMS, dividing microspores; UMS, uninucleate microspores; T, tapetal cells. FIGS. 19D-19E show the results of in situ hybridization showing localization of CCaMK in tapetal cells. Abbreviations: Exine (Ex), microspore (MS) (3.5 cm bud). Magnification: 19D, 865.times.; 19E, 1600.times.. Arrows indicate hybridization signals. FIG. 20 shows Ca.sup.2+ /calmodulin-dependent phosphorylation of heat-inactivated lily anther proteins in the presence (+) or absence of CCaMK (-) at different stages of development. Numbers on top indicate the sizes of flower buds in cm from which the anthers were used for protein extraction. Molecular weight markers (kDa) are indicated on the left. Arrow indicates 24 kDa protein showing high levels of phosphorylation when buds are 1.0-3.0 cm. FIG. 21 shows the expression pattern of CCaMK-binding proteins at different stages of lily anther development. The numbers indicate the sizes of flower buds in cm from which the anthers were used for protein extraction. Molecular weight markers (kDa) are indicated on the left. FIG. 22 shows the nucleotide sequence and deduced amino-acid sequence of the tobacco CCaMK cDNA. FIGS. 23A-23B show a comparison of deduced amino acid sequences of tobacco and lily CCaMKs. Eleven major conserved subdomains of serine/threonine protein kinases are marked. Hatched region indicates calmodulin-binding domain, the three Ca.sup.2+ -binding EF-hands are boxed. FIG. 23C is a diagram showing the kinase domain, calmodulin-binding domain, and visinin-like Ca.sup.2+ -binding domain of the lily and tobacco CCaMK polypeptides. Three Ca.sup.2+ -binding sites within the visinin-like binding domain are indicated by Roman numerals I, II, and III. FIG. 24 shows the results of RT-PCR showing the expression pattern of tobacco CCaMK at different stages of anther development. Numbers on top of each lane indicate bud size in cm, "M" indicates mature anther. Lower band in each lane shows calmodulin (CaM) control. FIG. 25 shows a Southern blot of tobacco genomic DNA digested with various restriction enzymes and probed with CCaMK. Lanes: 1, EcoRI; 2, EcoRV; 3, HindIII. Sizes in kb are shown on the right. FIG. 26 shows the nucleotide sequence of the promoter region of the tobacco CCaMK genomic clone. The putative TATA box is underlined and the start codon is boxed. FIGS. 27A-27H show tobacco flowers from wild-type and male sterile antisense plants. A. Wild-type flower before dehiscence. B. Antisense flower before dehiscence. (Note: there are no obvious differences between wild-type and antisense flower prior to dehiscence.) C. Wild-type flower after dehiscence. D. Antisense flower after dehiscence. Note: the wild-type anthers are white and fluffy, while the antisense anthers are bare. E. Enlarged view of the wild-type anther, 5.times.. F. Enlarged view of antisense anther, 5.times.. G. Enlarged view of wild-type anther, 20.times.. H. Enlarged view of antisense anther, 20.times.. FIGS. 28A-28D shows scanning electron micrographs showing wild-type and antisense pollen grains. A. Wild-type 300.times.. B. Antisense, 300.times.. C. Wild-type, 100,000.times.. D. Antisense, 100,000.times.. Note: antisense pollen grains are smaller and shriveled in contrast to wild-type. The whitish granular structures on the right of FIGS. 24B and 24D are remnants of pollen grains that have failed to develop. FIGS. 29A-29H show histochemical localization of callose in the outer walls of pollen grains from wild-type and antisense plants. On the left are the bright field images. On the right are the same respective views under blue excitation to highlight callose. A, B. Wild-type pollen grains, 300.times.. C, D. Antisense pollen grains, 300.times.. E, F. Wild-type pollen grains, 480.times.. G, H. Antisense pollen grains, 480.times.. Note: the pollen wall is not smooth and callose granules are unevenly distributed in antisense plants. FIG. 30A-30B shows pollen germination. A. Wild-type pollen, 139.times.. B. Antisense pollen, 139.times.. FIG. 31A shows the results of slot-blot analysis demonstrating suppression of CCaMK mRNA in antisense plants (A3, A4, A14, and A17), wild-type plants (WT), and transgenic plants carrying vector alone (C). FIG. 31B shows calmodulin control (the same filter re-hybridized with calmodulin). FIG. 32 shows GUS and antisense CCaMK constructs for transformation of plants. I. Transcriptional fusion of the tobacco CCaMK promoter to the .beta.-glucuronidase (GUS) reporter gene. II. Transcriptional fusion of a truncated version of the tobacco CCaMK promoter to GUS. III. Translational fusion of the tobacco CCaMK promoter to the tobacco CCaMK coding region and GUS. IV. Transcriptional fusion of the CCaMK promoter to the tobacco CCaMK in an antisense orientation. V. Transcriptional fusion of the TA29 promoter to antisense tobacco CCaMK. VI. Transcriptional fusion of the cauliflower mosaic virus (CaMV) 35S promoter to antisense tobacco CCaMK. All constructs include the Agrobacterium tumefaciens nopaline synthase terminator sequence (Nos-ter). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions and Methods The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Definitions of common terms in molecular biology may also be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994. The nomenclature for DNA bases as set forth at 37 CFR .sctn. 1.822 is used. The standard one- and three letter nomenclature for amino acid residues is used. Nucleic Acids "CCaMK Gene". The term "CCaMK gene" refers to a native CCaMK nucleic acid sequence or a fragment thereof, e.g., the native lily or tobacco CCaMK cDNA or genomic sequences and alleles and homologs thereof. The term also encompasses variant forms of a native CCaMK nucleic acid sequence or fragment thereof as discussed below, preferably a nucleic acid that encodes a polypeptide having CCaMK biological activity. Native CCaMK sequences include cDNA sequences and the corresponding genomic sequences (including flanking or internal sequences operably linked thereto, including regulatory elements and/or intron sequences). "Native". The term "native" refers to a naturally-occurring ("wild-type") nucleic acid or polypeptide. "Homolog". A "homolog" of a lily or tobacco CCaMK gene is a gene sequence encoding a CCaMK polypeptide isolated from an organism other than lily or tobacco. "Isolated". An "isolated" nucleic acid is one that has been substantially separated or purified away from other nucleic acid sequences in the cell of the organism in which the nucleic acid naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, by conventional nucleic acid-purification methods. The term also embraces recombinant nucleic acids and chemically synthesized nucleic acids. Fragments., Probes, and Primers. A fragment of a CCaMK nucleic acid is a portion of a CCaMK nucleic acid that is less than full-length and comprises at least a minimum length capable of hybridizing specifically with a native CCaMK nucleic acid under stringent hybridization conditions. The length of such a fragment is preferably at least 15 nucleotides, more preferably at least 20 nucleotides, and most preferably at least 30 nucleotides of a native CCaMK nucleic acid sequence. Nucleic acid probes and primers can be prepared based on a native CCaMK gene sequence. A "probe" is an isolated DNA or RNA attached to a detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme. "Primers" are isolated nucleic acids, generally DNA oligonucleotides 15 nucleotides or more in length, preferably 20 nucleotides or more, and more preferably 30 nucleotides or more, that are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e.g., a DNA polymerase. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other conventional nucleic-acid amplification methods. Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 (hereinafter, "Sambrook et al., 1989"); Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1987 (with periodic updates) (hereinafter, "Ausubel et al., 1987); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR-primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, .circle-w/dot. 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Substantial Similarity. A first nucleic acid is "substantially similar" to a second nucleic acid if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is at least about 75% nucleotide sequence identity, preferably at least about 85% identity, and more preferably at least about 90% identity. Sequence similarity can be determined by comparing the nucleotide sequences of two nucleic acids using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wis. Alternatively, two nucleic acids are substantially similar if they hybridize under stringent conditions, as defined below. Operably Linked. A first nucleic-acid sequence is "operably" linked with a second nucleic-acid sequence when the first nucleic-acid sequence is placed in a functional relationship with the second nucleic-acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. "Recombinant". A "recombinant" nucleic acid is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. Techniques for nucleic-acid manipulation are well-known (see, e.g., Sambrook et al., 1989, and Ausubel et al., 1987). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of nucleic acids can be performed, for example, on commercial automated oligonucleotide synthesizers. Preparation of Recombinant or Chemically Synthesized Nucleic acids; Vectors, Transformation, Host cells. Natural or synthetic nucleic acids according to the present invention can be incorporated into recombinant nucleic-acid constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct preferably is a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. For the practice of the present invention, conventional compositions and methods for preparing and using vectors and host cells are employed, as discussed, inter alia, in Sambrook et al., 1989, or Ausubel et al., 1987. A variety of well-known promoters or other sequences useful in constructing expression vectors are available for use in bacterial, yeast, mammalian, insect, amphibian, avian, or other host cells. A cell, tissue, organ, or organism into which has been introduced a foreign nucleic acid, such as a recombinant vector, is considered "transformed", "transfected", or "transgenic." A "transgenic" or "transformed" cell or organism also includes (1) progeny of the cell or organism and (2) progeny produced from a breeding program employing such a "transgenic" plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of the recombinant CCaMK DNA construct. Nucleic-Acid Hybridization; "Stringent Conditions"; "Specific". The nucleic-acid probes and primers of the present invention hybridize under stringent conditions to a target DNA sequence, e.g., to a CCaMK gene. The term "stringent conditions" is functionally defined with regard to the hybridization of a nucleic-acid probe to a target nucleic acid (i.e., to a particular nucleic-acid sequence of interest) by the hybridization procedure discussed in Sambrook et al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52, 9.56-9.58; Kanehisa, Nucl. Acids Res. 12:203-213, 1984; and Wetmur and Davidson, J. Mol. Biol. 31:349-370, 1968. Regarding the amplification of a target nucleic-acid sequence (e.g., by PCR) using a particular amplification primer pair, "stringent conditions" are conditions that permit the primer pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and preferably to produce a unique amplification product. The term "specific for (a target sequence)" indicates that a probe or primer hybridizes under stringent conditions only to the target sequence in a sample comprising the target sequence. Nucleic-Acid Amplification. As used herein, "amplified DNA" refers to the product of nucleic-acid amplification of a target nucleic-acid sequence. Nucleic-acid amplification can be accomplished by any of the various nucleic-acid amplification methods known in the art, including the polymerase chain reaction (PCR). A variety of amplification methods are known in the art and are described, inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed. Innis et al., Academic Press, San Diego, 1990. Methods of Obtaining Alleles and Homologs of Lily and Tobacco CCaMK. Based upon the availability of the lily CCaMK cDNA and tobacco CCaMK cDNA and genomic sequences disclosed herein, alleles and homologs can be readily obtained from a wide variety of plants by cloning methods known in the art, e.g., by screening a cDNA or genomic library with a probe that specifically hybridizes to a native CCaMK sequence under stringent conditions or by PCR or another amplification method using a primer or primers that specifically hybridize to a native CCaMK sequence under stringent conditions. Cloning of a CCaMK Genomic Sequence. The availability of a CCaMK cDNA sequence enables the skilled artisan to obtain a genomic clone corresponding to the cDNA (including the promoter and other regulatory regions and intron sequences) and the determination of its nucleotide sequence by conventional methods. Both monocots and dicots possess CCaMK genes. Primers and probes based on the native lily and tobacco CCaMK sequences disclosed herein can be used to confirm (and, if necessary, to correct) the CCaMK sequences by conventional methods. Nucleotide-Sequence Variants of Native CCaMK Nucleic Acids and Amino Acid Sequence Variants of Native CCaMK Proteins. Using the nucleotide and the amino-acid sequence of the CCaMK polypeptides disclosed herein, those skilled in the art can create DNA molecules and polypeptides that have minor variations in their nucleotide or amino acid sequence. "Variant" DNA molecules are DNA molecules containing minor changes in a native CCaMK sequence, i.e., changes in which one or more nucleotides of a native CCaMK sequence is deleted, added, and/or substituted, preferably while substantially maintaining a CCaMK biological activity. Variant DNA molecules can be produced, for example, by standard DNA mutagenesis techniques or by chemically synthesizing the variant DNA molecule or a portion thereof. Such variants preferably do not change the reading frame of the protein-coding region of the nucleic acid and preferably encode a protein having no change, only a minor reduction, or an increase in CCaMK biological function. Amino-acid substitutions are preferably substitutions of single amino-acid residues. DNA insertions are preferably of about 1 to 10 contiguous nucleotides and deletions are preferably of about 1 to 30 contiguous nucleotides. Insertions and deletions are preferably insertions or deletions from an end of the protein-coding or non-coding sequence and are preferably made in adjacent base pairs. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a final construct. Preferably, variant nucleic acids according to the present invention are "silent" or "conservative" variants. "Silent" variants are variants of a native CCaMK sequence or a homolog thereof in which there has been a substitution of one or more base pairs but no change in the amino-acid sequence of the polypeptide encoded by the sequence. "Conservative" variants are variants of the native CCaMK sequence or a homolog thereof in which at least one codon in the protein-coding region of the gene has been changed, resulting in a conservative change in one or more amino acid residues of the polypeptide encoded by the nucleic-acid sequence, i.e., an amino acid substitution. A number of conservative amino acid substitutions are listed below. In addition, one or more codons encoding cysteine residues can be substituted for, resulting in a loss of a cysteine residue and affecting disulfide linkages in the CCaMK polypeptide. Original Residue Conservative Substitutions Ala ser Arg lys Asn gln, his Asp glu Cys ser Gln asn Glu asp Gly pro His asn; gln Ile leu, val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu Substantial changes in function are made by selecting substitutions that are less conservative than those listed above, e.g., causing changes in: (a) the structure of the polypeptide backbone in the area of the substitution; (b) the charge or hydrophobicity of the polypeptide at the target site; or (c) the bulk of an amino acid side chain. Substitutions generally expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine. A CCaMK gene sequence can be modified as follows: (1) To improve expression efficiency and redirect the targeting of the expressed polypeptide: For expression in non-plant hosts (or to direct the expressed polypeptide to a different intracellular compartment in a plant host), an appropriate transit or secretion peptide sequence can be added to the protein-coding region of the native gene sequence. In addition, one or more codons can be changed, for example, to conform the gene to the codon usage bias of the host cell or organism for improved expression. Enzymatic stability can be altered by removing or adding one or more cysteine residues, thus removing or adding one or more disulfide bonds. (2) To alter substrate and ligand binding and CCaMK enzymatic activity: One or more amino acid residues in a substrate or ligand binding domain (e.g., the calmodulin-binding domain) can be mutagenized to affect the strength or specificity of the interaction between CCaMK and its polypeptide substrates or to affect control of CCaMK activity by Ca.sup.2+ and/or calmodulin. The autoinhibitory domain can also be mutagenized or an autophosphorylation site removed or added to affect CCaMK activity. Further targets, including the visinin-like Ca.sup.2+ -binding domain or one or more EF-hand motifs thereof, can also be mutagenized. Nucleic Acids Attached to a Solid support. The nucleic acids of the present invention can be free in solution or covalently or noncovalently attached by conventional means to a solid support, such as a hybridization membrane (e.g., nitrocellulose or nylon), a bead, etc. Polypeptides "CCaMK Protein". The term "CCaMK protein" (or polypeptide) refers to a protein encoded by a CCaMK nucleic acid, including alleles, homologs, and variants of a native CCaMK nucleic acid, for example. A CCaMK polypeptide can be produced by the expression of a recombinant CCaMK nucleic acid or be chemically synthesized. Techniques for chemical synthesis of polypeptides are described, for example, in Merrifield, J. Amer. Chem. Soc. 85:2149-2156, 1963. Polypeptide Sequence Homology. Ordinarily, CCaMK polypeptides encompassed by the present invention are at least about 70% homologous to a native CCaMK polypeptide, preferably at least about 80% homologous, and more preferably at least about 95% homologous. Such homology is considered to be "substantial homology," although more important than shared amino-acid sequence homology can be the common possession of characteristic structural features and the retention of biological activity that is characteristic of CCaMK, preferably CCaMK catalytic activity. Polypeptide homology is typically analyzed using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology center, Madison, Wis.). Polypeptide sequence analysis software matches homologous sequences using measures of homology assigned to various substitutions, deletions, substitutions, and other modifications. "Isolated," "Purified," "Homogeneous" Polypeptides. A polypeptide is "isolated" if it has been separated from the cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) that naturally accompany it. Such a polypeptide can also be referred to as "pure" or "homogeneous" or "substantially" pure or homogeneous. Thus, a polypeptide which is chemically synthesized or recombinant (i.e., the product of the expression of a recombinant nucleic acid, even if expressed in a homologous cell type) is considered to be isolated. A monomeric polypeptide is isolated when at least 60-90% by weight of a sample is composed of the polypeptide, preferably 95% or more, and more preferably more than 99%. Protein purity or homogeneity is indicated, for example, by polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band upon staining the polyacrylamide gel; high pressure liquid chromatography; or other conventional methods. Protein Purification. The polypeptides of the present invention can be purified by any of the means known in the art. Various methods of protein purification are described, e.g., in Guide to Protein Purification, ed. Deutscher, Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982. Variant and Modified Forms of CCaMK Polypeptides. Encompassed by the CCaMK polypeptides of the present invention are variant polypeptides in which there have been substitutions, deletions, insertions or other modifications of a native CCaMK polypeptide. The variants substantially retain structural characteristics and biological activities of a corresponding native CCaMK polypeptide and are preferably silent or conservative substitutions of one or a small number of contiguous amino acid residues. A native CCaMK polypeptide sequence can be modified by conventional methods, e.g., by acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, and labeling, whether accomplished by in vivo or in vitro enzymatic treatment of a CCaMK polypeptide or by the synthesis of a CCaMK polypeptide using modified amino acids. Labeling. There are a variety of conventional methods and reagents for labeling polypeptides and fragments thereof. Typical labels include radioactive isotopes, ligands or ligand receptors, fluorophores, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al., 1989 and Ausubel et al., 1987. Polypeptide Fragments. The present invention also encompasses fragments of a CCaMK polypeptide that lacks at least one residue of a native full-length CCaMK polypeptide. Preferably, such a fragment retains Ca.sup.2+ /calmodulin-dependent kinase activity or possession of a characteristic functional domain (e.g., a calmodulin-binding domain, Ca.sup.2+ -binding EF-hand motif(s), autophosphorylation site(s), etc.), or an immunological determinant characteristic of a native CCaMK polypeptide (and thus able to elicit production of a CCaMK-specific antibody in a mouse or rabbit, for example or to compete with CCaMK for binding to CCaMK-specific antibodies) that is therefore useful in immunoassays for the presence of a CCaMK polypeptide in a biological sample. Such immunologically active fragments typically have a minimum size of 7 to 17 or more amino acids. The terms "biological activity", "biologically active", "activity" and "active" refer primarily to the characteristic enzymatic activity or activities of a native CCaMK polypeptide, including, but not limited to, Ca.sup.2+ /calmodulin-dependent kinase activity. Fusion Polypeptides. The present invention also provides fusion polypeptides including, for example, heterologous fusion polypeptides, i.e., a CCaMK polypeptide sequence or fragment thereof and a heterologous polypeptide sequence, e.g., a sequence from a different polypeptide. Such heterologous fusion polypeptides thus exhibit biological properties (such as substrate or ligand binding, enzymatic activity, antigenic determinants, etc.) derived from each of the fused sequences. Fusion partners include, for example, .beta.-glucuronidase, immunoglobulins, beta galactosidase, trpE, protein A, beta lactamase, alpha amylase, alcohol dehydrogenase, yeast alpha mating factor, and various signal and leader sequences which, e.g., can direct the secretion of the polypeptide. Fusion polypeptides are preferably made by the expression of recombinant nucleic acids produced by standard techniques. Polypeptide Sequence Determination. The sequence of a polypeptide of the present invention can be determined by various methods known in the art. In order to determine the sequence of a polypeptide, the polypeptide is typically fragmented, the fragments separated, and the sequence of each fragment determined. To obtain fragments of a CCaMK polypeptide, the polypeptide can be digested with an enzyme such as trypsin, clostripain, or Staphylococcus protease, or with chemical agents such as cyanogen bromide, o-iodosobenzoate, hydroxylamine or 2-nitro-5-thiocyanobenzoate. Peptide fragments can be separated, e.g., by reversed-phase high-performance liquid chromatography (HPLC) and analyzed by gas-phase sequencing. Polypeptide Coupling to a solid Phase Support. The polypeptides of the present invention can be free in solution or coupled to a solid-phase support, e.g., nitrocellulose, nylon, column packing materials (e.g., Sepharose beads), magnetic beads, or glass wool, by conventional methods. Antibodies The present invention also encompasses polyclonal and/or monoclonal antibodies capable of specifically binding to a CCaMK polypeptide and/or fragments thereof. Such antibodies are raised against a CCaMK polypeptide or fragment thereof and are capable of distinguishing a CCaMK polypeptide from other polypeptides. An immunological response is usually assayed with an immunoassay. Normally such immunoassays involve some purification of a source of antigen, for example, produced by the same cells and in the same fashion as the antigen was produced. For the preparation and use of antibodies according to the present invention, including various immunoassay techniques and applications, see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, 2d ed, Academic Press, New York, 1986; and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988. CCaMK-specific antibodies are useful, for example in: purifying a CCaMK polypeptide from a biological sample, such as a host cell expressing recombinant a CCaMK polypeptide; in cloning a CCaMK allele or homolog from an expression library; as antibody probes for protein blots and immunoassays; etc. CCaMK polypeptides and antibodies can be labeled by joining, either covalently or noncovalently, to a substance which provides for a detectable signal by conventional methods. A wide variety of labels and conjugation techniques are known. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles, etc. Plant Transformation and Regeneration Various nucleic acid constructs that include a CCaMK nucleic acid are useful for producing male-sterile plants. As detailed in the Examples below, transgenic plants containing as a transgene a nucleic acid construct in which a CCaMK nucleic acid is expressed in an antisense orientation are male sterile. CCaMK nucleic acids can be expressed in plants or plant cells in a sense or antisense orientation under the control of an operably linked promoter that is capable of expression in a cell of a particular plant. Various promoters suitable for expression of heterologous genes in plant cells are well known, including constitutive promoters (e.g. the CaMV 35S promoter), organ- or tissue-specific promoters (e.g., the tapetum-specific TA29, A9 or Osg6B promoters), and promoters that are inducible by chemicals such as methyl jasminate, salicylic acid, or Safener, for example. In addition to antisense expression of CCaMK in transgenic plants, as discussed below (see also, U.S. Pat. No. 5,283,184), the availability of CCaMK genes permits the use of other conventional methods for interfering with CCaMK gene expression, including triplex formation, production of an untranslatable plus-sense CCaMK RNA, etc. A CCaMK promoter can be used to drive the expression of a CCaMK antisense transgene and also to express other nucleic acids in transgenic plants in an organ- and developmental stage-specific manner. For example, a CCaMK promoter can be used to drive the expression in transcriptional or translational fusions of antisense versions of nucleic acids encoding polypeptides necessary for male fertility, e.g., antisense inhibition of flavonoid biosynthesis (Van der Meer et al., Plant Cell 4:253-262, 1992), or to express, in a sense orientation, genes that interfere with male fertility, e.g., ribonuclease (Mariani et al., Nature 347:737-741, 1990); glucanase (Worrall et al., Plant Cell 4:759-771, 1992; Tsuchiya et al., Plant Cell Physiol. 36:487-494, 1995); Agrobacterium rhizogenes rolB (Spena et al., Theor. Appl. Genet. 84:520-527, 1992); and mitochondrial gene atp9 (Hernould et al., Proc. Natl. Acad. Sci. USA 90:2370-2374, 1993). Any well-known method can be employed for plant cell transformation, culture, and regeneration in the practice of the present invention with regard to a particular plant species. Methods for introduction of foreign DNA into plant cells include, but are not limited to: transfer involving the use of Agrobacterium tumefaciens and appropriate Ti vectors, including binary vectors; chemically induced transfer (e.g., with polyethylene glycol); biolistics; and microinjection. See, e.g., An et al., Plant Molecular Biology Manual A3: 1-19, 1988. The term "plant" encompasses any higher plant and progeny thereof, including monocots (e.g., lily, corn, rice, wheat, etc.), dicots (e.g., tobacco, potato, apple, tomato, etc.), gymnosperms, etc., and includes parts of plants, including reproductive units of a plant, fruit, flowers, wood, etc. A "reproductive unit" of a plant is any totipotent part or tissue of the plant from which one can obtain a progeny of the plant, including, for example, seeds, cuttings, tubers, buds, bulbs, somatic embryos, cultured cell (e.g., callus or suspension cultures), etc. The invention will be better understood by reference to the following Examples, which are intended to merely illustrate the best mode now known for practicing the invention. The scope of the invention is not to be considered limited thereto, however. |
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