Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1995-06-07
2003-05-27
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S069100, C435S325000, C435S455000, C435S254200, C435S254110
Reexamination Certificate
active
06569617
ABSTRACT:
BACKGROUND OF THE INVENTION
Presently, there are several methods available for cloning mammalian genes. The standard approach to cloning mammalian genes requires obtaining purified protein, determining a partial amino acid sequence of the purified protein, using the partial amino acid sequence to produce degenerate oligonucleotide probes, and screening cDNA libraries with these probes in order to obtain cDNA encoding the protein. This method is time consuming and, because of the degeneracy of the probes used, may identify sequences other than those encoding the protein(s) of interest. Many mammalian genes have been cloned this way, including the cGMP phosphodiesterase expressed in retina (Ovchinnikov, Y-A. et al.,
FEB
223: 169 (1987)).
A second approach to cloning genes encoding a protein of interest is to use a known gene as a probe to find homologs. This approach is particularly useful when members of a gene family or families are sufficiently homologous. It is reasonable to expect that members of a given gene family can be so cloned once one member of the family has been cloned. The
D. melanogaster
dunce phosphodiesterase gene was used, for example to clone rat homologs. (Davis, R. L. et al.;
Proc. Natl. Acad. Sci. USA
86: 3604 (1989); Swinnen, J. V. et al.,
Proc. Natl. Acad. Sci. USA
86: 5325 (1989)). Although members of one family of phosphodiesterase genes might be cloned once a member of that family has been cloned, it is unclear whether the nucleotide sequences of genes belonging to different phosphodiesterase gene families exhibit sufficient homology to use probes derived from one family to identify members of another family.
It would be useful to have a method which could be used to clone genes which does not have the limitations of presently available techniques.
SUMMARY OF THE INVENTION
The present invention relates to a method of cloning mammalian genes encoding proteins which can function in microorganisms, particularly yeast, and can modify, complement, or suppress a genetic defect associated with an identifiable phenotypic alteration or characteristic in the microorganism. It further relates to mammalian genes cloned by the present method, as well as to products encoded by such genes and antibodies which can bind the encoded proteins. More specifically, the present invention relates to a method of cloning mammalian genes which encode products which modify, complement or suppress a genetic defect in a biochemical pathway in which cAMP participates or in a biochemical pathway which is controlled, directly or indirectly, by a RAS protein, to products (RNA, proteins) encoded by the mammalian genes cloned in this manner and to antibodies which can bind the encoded proteins. As described herein, the present method has been used to identify novel mammalian genes which encode cAMP, phosphodiesterases and proteins which interact with RAS proteins. These genes, and others that can be derived by the claimed method, are part of this invention, as are the proteins which they encode.
The present invention further relates to a method of identifying agents which alter (i.e., reduce or stimulate) the activity of the protein products of such mammalian genes expressed in microorganisms, such as yeast. Identification of such agents can be carried out using two types of screening procedures: one based on biochemical assays of mammalian proteins of known enzymatic function and one based on phenotypic assays for proteins of unknown function. In the former case, if the encoded proteins are cAMP phosphodiesterases, pharmacological screens include the assay for agents which alter (i.e., reduce or stimulate) phosphodiesterase activity. In the latter case, if the encoded proteins interact with RAS proteins, pharmacological screens include the assay for agents which reduce or stimulate interactions with RAS proteins.
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Colicelli John J.
Wigler Michael H.
Cold Spring Harbor Laboratory
Guzo David
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