Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
1999-09-09
2002-04-09
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S252300, C435S419000, C435S325000, C435S320100
Reexamination Certificate
active
06368848
ABSTRACT:
FIELD OF INVENTION
This invention relates to bioassay techniques utilizing plant proteins expressed in a host cell system to identify their functionality in a plant G protein-coupled system, and is particularly useful in identifying plant G protein subunits and plant proteins that function in a manner similar to such G protein subunits and G protein coupled receptors. Other embodiments of the invention relate to host cells expressing such plant proteins, and especially plant G protein subunits, vectors useful for making such cells, and methods of making and using same.
BACKGROUND OF THE INVENTION
In animal systems, the actions of many extracellular signals, for example: neurotransmitters, hormones, odorants and light, are mediated by receptors with seven transmembrane domains (G protein-coupled receptors) and heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins). G proteins are comprised of three subunits: a guanyl-nucleotide binding &agr; subunit; a &bgr; subunit; and a &ggr; subunit [for review, see Conklin, B. R and Bourne, H. R. (1993) Cell 73, 631-641]. G proteins cycle between two forms, depending on whether GDP or GTP is bound to the &agr; subunit. When GDP is bound, the G protein exists as a heterotrimer, the G&agr;&bgr;&ggr; complex. When GTP is bound, the &agr; subunit disassociates, leaving a G&bgr;&ggr; complex. Importantly, when a G&agr;&bgr;&ggr; complex operatively associates with an activated G protein coupled receptor in a cell membrane, the rate of exchange of GTP for bound GDP is increased and, hence, the rate of disassociation of the bound G&agr; subunit from the G&bgr;&ggr; complex increases. The free G&agr; subunit and G&bgr;&ggr; complex are capable of transmitting a signal to downstream elements of a variety of signal transduction pathways. This fundamental scheme of events forms the basis for a multiplicity of different cell signaling phenomena. For additional review, see H. G. Dohlman, J. Thorner, M. Caron, and R. J. Lefkowitz, Ann. Rev. Biochem, 60, 653-688 (1991).
In plants, there is evidence that a number of plant signal transduction pathways, including red and blue light signaling (Warpeha et al., 1991; Romero and Lam, 1993; Neuhaus et al, 1993), K+ channel regulation of stomatal opening (Fairley-Grenot and Assmann, 1991; Li and Assmann, 1993; Armstrong and Blatt, 1995), and auxin signal transduction (Zaina et al., 1990) are regulated through G-protein intermediates. More specifically, in certain of these studies, red light-dependent responses in the tomato mutant aureus were measured after microinjection of either the GTP analogue GTP&ggr;S or cholera toxin, either of which cause constitutive activation of a G-protein, and were found to produce the same effects as microinjecting phytochrome and exposing the plant to red light. Injection of inhibitors GDP&bgr;S or pertussis toxin were effective in blocking red light-dependent responses.
It has also been suggested that an inhibitory G-protein modulates blue light-dependent K+ channel opening on the basis of electrophysiological studies using GTP&ggr;S, GDP&bgr;S and toxins. In separate experiments it was shown that blue light can activate a GTP-binding protein in the plasma membrane of peas. In rice, the binding of GTP&ggr;S to vesicles in vitro is increased by auxin, while binding of GTP&ggr;S decreased binding of auxin. This binding relationship suggests that auxin activation of a G-protein stimulates cell elongation. In addition, there is evidence for G-protein mediation of plant defense responses (Legendre et al., 1992, Vera-Estralla et al., 1994; Beffa et al., 1995).
G&agr;-like proteins, having molecular weights close to that of the animal G&agr; subunits and recognized by antibodies against animal G&agr; subunits, have been detected in a large number of plant species (for reviews see Ma, 1994 and Kaufman, 1994) and three genes encoding G protein-&agr; subunits have been identified. The first was cloned from
Arabidopsis thaliana
(Ma et al. 1990) using PCR primers based on sequences known to be conserved between animal G protein &agr;-subunits. The predicted protein has all of the consensus sequences for guanine nucleotide binding and hydrolysis that are characteristic of GTP-binding proteins and shows 36% identity with rat Gi(1-3) and bovine transducin. Using the
Arabidopsis thaliana
gene as a probe, genes were identified in both tomato (Ma et al., 1991) and soybean (Kim et al., 1995). The
Arabidopsis thaliana
tomato and soybean genes share over 80% identity, suggesting that plant G-protein &agr;-subunits may be highly conserved. Both
Arabidopsis thaliana
and tomato DNA appear to have single genes based on Southern blot analysis, whereas multiple genes may be present in soybean. Single genes encoding G protein &bgr; subunits have also been cloned from maize and
Arabidopsis thaliana
(Weiss et al., 1994). The predicted protein sequences shares 76% identity with each other and 41% identity with mammalian G protein &bgr; subunits.
Although the sequence of the plant G &agr; subunits have been conserved relative to that of the mammalian G &agr; subunits, there is no published data that demonstrates that the function of the plant protein is also conserved. Although physiological studies implicate G protein mediated responses in a number of pathways based on sensitivity of the response to cholera or pertussis toxins or the enhancement of the GTP binding to membranes by specific stimuli, this evidence is indirect. Prior to the work as detailed herein, the art has failed to provide direct evidence that these effects occur by the same mechanism by which they occur in other systems. In fact, it has recently been reported by one of the leaders in the field of plant G-protein molecular biology, that it is “ . . . unlikely that the plant G &agr;s are functional homologues of any of the non-plant ones.” . . . (ibid. Hong Ma 1994).
Protein-mediated signaling systems are present in organisms as divergent as yeast, plant, and man. The yeast
Saccharomyces cerevisiae
is utilized as a model eukaryotic organism. Due to the ease with which one can manipulate the genetic constitution of the yeast
Saccharomyces cerevisiae,
researchers have developed a detailed understanding of many complex biological pathways. It has been demonstrated in numerous systems that the evolutionary conservation of protein structure is such that many heterologous proteins can substitute for their yeast equivalents. For example, mammalian G&agr; proteins can form heterotrimeric complexes with yeast G&bgr;&ggr; proteins [Kang, Y.-S., Kane, J., Kuijan, J., Stadel, J. M., and Tipper, D. J. (1990) Mol. Cell. Biol. 10, 2582-2590]. Screening assays utilizing yeast strains genetically modified to accommodate functional expression of plant G proteins offer significant advantages in research involving the identification of plant proteins that function in intracellular signaling systems, such as G-protein coupled systems.
SUMMARY OF THE INVENTION
A first aspect of the present invention is directed to a method of identifying a plant protein that functions in an intracellular signaling system such as a G protein coupled cellular signaling system. This is accomplished by providing a host cell with a nucleotide sequence encoding the plant protein it is desired to identify, which may be a plant protein suspected of being or functioning in a manner similar to a G protein subunit or a plant protein that is suspected of being or functioning in a manner similar to a G protein coupled receptor that functions in the intracellular signaling systems of mammals, insects (such as mammalian G protein coupled systems and insect G protein coupled systems), and the like. In certain preferred embodiments, an endogenous corollary component of a G protein coupled cellular signaling pathway, such as a G&agr; protein subunit, is rendered inoperative in the host cell. The host cell is transformed with a nucleotide sequence encoding the plant protein it is desired to identify and analyze, and the transformed host
Kemmerer Elizabeth
Sutherland & Asbill & Brennan LLP
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