Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide contains a tissue – organ – or cell...
Reexamination Certificate
1999-02-25
2002-03-26
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide contains a tissue, organ, or cell...
C800S278000, C536S023600, C536S024100, C435S194000, C435S468000
Reexamination Certificate
active
06362395
ABSTRACT:
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
2+
concentration can affect a number of physiological processes through the action of calmodulin (CaM), a ubiquitous Ca
2+
-binding protein. Ca
2+
/calmodulin-regulated protein phosphorylation plays a pivotal role in amplifying and diversifying the action of Ca
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
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
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
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 &bgr;-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.
REFERENCES:
patent: 0 329 308 (1989-08-01), None
patent: 0 344 029 (1989-11-01), None
patent: 0 513 884 (1992-11-01), None
patent: WO 94/13809 (1994-06-01), None
Ames et al, “Amino-terminal myristoylation induces cooperative calcium binding to recoverin,”J. Biol. Chem., 270:4526-4533, 1995.
Botella et al, “Differential expression of two calmodulin genes in response to physical and chemical stimuli,”Plant Mol. Biol., 24:757-766, 1994.
Braam et al, “Rain-, wind-, and touch-induced expression of calmodulin and calmodulin-related genes in Arabidopsis,”Cell, 60:357-364, 1990.
Brickey et al, “Mutational analysis of the autoinhibitory domain of calmodulin kinase II,”J. Biol. Chem., 269:29047-29054, 1994.
Bush, “Regulation of cytosolic calcium in plants,”Plant Physiol., 103:7-13, 1993.
Cohen, “Signal integration at the level of protein kinases, protein phospatases and their substrates,”TIBS, 17:408-413, 1992.
Colbran et al, “Calcium/calmodulin-dependent protein kinase II,”Biochem. J., 258:313-325, 1989.
Colbran et al, “Calcium/calmodulin-dependent protein kinase II,”Curr. Top. Cell. Reg., 31:181-221, 1990.
Colbran, “Inactivation of Ca2+/calmodulin-dependent protein kinase II by basal autophosphorylation,”J. Biol. Chem., 268:7163-7170, 1993.
Enslen et al, “Characterization of Ca2+/calmodulin-dependent protein kinase IV,”J. Biol. Chem., 269:15520-15527,
Liu Zhihua
Patil Shameekumar
Poovaiah Bachettira W.
Takezawa Daisuke
Fox David T.
Klarquist & Sparkman, LLP
Washington State University Research Foundation
LandOfFree
Compositions and methods for production of male-sterile plants does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Compositions and methods for production of male-sterile plants, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Compositions and methods for production of male-sterile plants will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2878416