Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers resistance to heat or cold
Reexamination Certificate
1999-02-12
2001-07-31
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide confers resistance to heat or cold
C800S320100, C800S317000, C800S320000, C800S306000, C800S298000, C800S278000, C435S320100, C435S468000, C435S419000, C536S023100, C536S023600, C536S024100
Reexamination Certificate
active
06268548
ABSTRACT:
BACKGROUND OF THE INVENTION
The effect of environmental stress on agronomic plants has been a major focus of plant research. Plant productivity is related to the ability of plants to respond to and adapt to environmental stress (Sachs and Ho 1986). The proteins produced by higher plants in response to stress have been well-characterized (Key et al., 1981; Cooper and Ho, 1983; Sachs and Ho, 1986). Many stress proteins have recently been found to be chaperones, a class of proteins involved in the folding of newly synthesized proteins (Ellis and van der Vies, 1991; Gething and Sambrook, 1992; Craig et al., 1993). The chaperones have been proposed to function during stress in binding partially denatured proteins, thus preventing their degradation. Additionally, the chaperones assist in the refolding of these partially denatured proteins into their native structure in an ATP-dependent manner following the relief of stress (Rochester et al., 1986; Ellis and Hemmingsen, 1989; Hendrick and Hartl, 1993; Schröder et al., 1993). The two most extensively studied classes of chaperones are heat shock protein (HSP) 70 homologs and cpn60 homologs.
HSP70 homologs have been found in higher plant cytoplasm (Giorini and Galili, 1991), endoplasmic reticulum (Denecke et al., 1991), chloroplasts (Marshall et al., 1990; Ko et al., 20 1992; Marshall and Keegstra, 1992; Madueño et al., 1993; Wang et al., 1993), and mitochondria (Watts et al., 1992; Neuman et al., 1993). Genes for mitochondrial HSP70 are nuclearly encoded and have been isolated from pea (Watts et al., 1992), potato and tomato (Neuman et al., 1993).
The cpn60s are a group of ubiquitous proteins with a subunit size of approximately 60 kDa that share a functional and structural similarity to the tetradecameric
E. coli
GroEL complex (Gatenby, 1992). The maize and
Arabidopsis thaliana
mitochondrial cpn60 genes have been isolated and found to be encoded in the nucleus (Prasad and Stewart, 1992). The maize cpn60 was hypothesized to aid in the assembly of new mitochondrial protein complexes during the rapid organelle biogenesis of seedling germination and heterotrophic growth (Prasad and Stewart, 1992).
Neither the HSP70 homologs nor the cpn 60 homologs are very effective indicators of a heat resistant plant's ability to tolerate heat stress. However, there is another group of heat shock proteins that may be effective indicators. This group is the low molecular mass (17-30 kDa) HSPs (Waters et al., 1996). Recent reports have established that the cytosolic forms of plant small HSPs (sHSPs) can function as molecular chaperones in vitro (Lee et al., 1995a). Lenne and Douce (1994) identified a mitochondrial matrix-localized low molecular mass HSP identified as HSP22. Pea leaf mitochondrial HSP22 is conditionally expressed only at high temperatures and the protein level remained high for at least three days following heat stress (Lenne and Douce, 1994). A cDNA for pea mitochondrial HSP22 has been identified and establishes this protein as a member of the sHSP superfamily (Lenne et al., 1995). cDNAs for mitochondrial sHSPs have also been characterized in soybean (La Fayette et al., 1996),
A. thaliana
(Willett et al., 1996), and
Chenopodium rubrum
(Lenne et al., 1995; Waters et al., 1996). However, there has not been an sHSP isolated from a mitochondria of a heat resistant plant. For example, maize is a heat-resistant plant and is one of the world's greatest food sources. The isolation of an sHSP from maize would allow for testing of plants to determine their ability to tolerate heat stress resistance. This testing could take several forms if a sHSP were isolated. Additionally, vectors could be constructed containing the nucleic acid molecule for the sHSP.
There is a need for the discovery of an isolated sHSP protein from a heat resistant plant. This sHSP could be utilized to indicate a heat resistant plant's ability to tolerate heat stress. Additionally, if a sHSP were isolated from a heat resistant plant and cloned into an expression vector, there would be large quantities of HSP for research, the production of antibodies, and the generation of nucleic acid probes.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide a method for determining a plant specie's ability to tolerate heat stress by comparing levels of HSP22 expression.
A further object of the present invention is to provide a combination comprising the necessary materials needed to determine a plant specie's ability to tolerate heat stress by comparing levels of HSP22 expression.
A further object of the present invention is to provide an isolated nucleic acid molecule encoding a protein that is expressed during heat stress conditions and that hybridizes, under stringent conditions, to SEQ. ID. NO. 7.
A further object of the present invention is to provide isolated protein comprising SEQ. ID. NO. 6.
A further object of the present invention is to provide an isolated nucleic acid molecule encoding a protein that is expressed during heat stress conditions and that hybridizes under stringent conditions to a nucleic acid molecule corresponding to an amino acid molecule of SEQ. ID. NO. 6.
A further object of the present invention is to provide fragments of the nucleic acid molecule encoding HSP22 that hybridize to SEQ. ID. NO. 7 and that code for products that have substantially the same physical characteristics as HSP22. These fragments can be either recombinant, synthetic, or a combination thereof.
A further object of the present invention is to provide a recombinant vector comprising a nucleic acid molecule encoding a protein that has physical characteristics substantially similar to HSP22. The definition of a vector for the purposes of this invention is any nucleic acid molecule into which a foreign nucleic acid molecule may be inserted wherein the nucleic acid molecule containing the foreign nucleic acid molecule may be used to introduce the foreign nucleic acid molecule into a host cell. This vector may also comprise regulatory elements operably linked to the nucleic acid molecule.
A further object of the present invention is to provide various cells transformed with a vector comprising a nucleic acid molecule encoding a protein that has physical characteristics substantially equal to HSP22. Addtionally, these cells could be plant cells capable of producing plants that have an increased ability to survive in heat stress conditions.
A further object of the present invention is to provide a methodology for the production of various products of the present invention. Examples include, but are not limited to, isolated nucleic acid molecules encoding HSP22, isolated HSP22, isolated SEQ. ID. NO. 7, isolated SEQ. ID. NO. 6, and the protein products of SEQ. ID. NO. 7.
Another object of the present invention is to provide novel and purified antibodies to HSP22. The antibodies can be polyclonal, monoclonal, or fragments thereof.
It is a further object of the present invention to provide a hybridoma for the production of HSP22 monoclonal antibodies.
It is a further object of the present invention to provide a method for the production of the hybridoma capable of producing monoclonal antibodies for HSP22.
By providing the above-stated objects, several advantages are realized. For example, plants can be screened for their ability to tolerate heat stress by comparing levels of HSP22 expression.
Another advantage realized by the present invention is the ability to screen plants with either antibodies to HSP22 or with nucleic acid probes that bind to HSP22 mRNA.
Still another advantage realized by the present invention is the ability to produce large quantities of HSP22, HSP22 antibodies, and HSP22 nucleic acids, for both commercial and scientific purposes.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention.
The mouse cel
Bhattramakki Dinakar
Elthon Thomas E.
Lund Adrian A.
Rhoads David
Board of Regents of University of Nebraska
Fox David T.
Ibrahim Medina A.
Marsh, Jr. James H.
Shook Hardy & Bacon LLP
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