Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-10-07
2001-09-25
Hauda, Karen M. (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S069100, C435S320100, C435S325000, C536S023100, C536S023500, C530S350000
Reexamination Certificate
active
06294384
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to an isoform of p53 and to polynucleotides encoding this isoform. The invention also encompasses a variety of compositions and methods relating to the isoform and polynucleotides.
BACKGROUND OF THE INVENTION
P53 is a tumor suppressor that plays a central role in arresting cell growth and inducing programed cell death. Mutations of p53 are the most common genetic abnormality identified in human malignancies. Despite the significance of p53 as a regulator of cellular growth/death and its apparent role in human disease, the details regarding its biological actions remain relatively obscure.
One mechanism by which p53 is believed to act is through the “transactivation,” or the coordinated turning on, of genes in response to stimuli such as cellular stress. Transactivation depends upon the ability of the p53 protein to bind to specific regions of DNA (p53 responsive elements) that are within promoter sequences adjacent to target genes. This binding may lead to either an enhancement or inhibition of target gene expression. In addition, it is believed that p53 may mediate changes in cell behavior through other mechanisms that are independent of its ability to bind DNA.
The p53 protein is itself regulated both by changes in its state of phosphorylation and by intracellular localization. In addition, it has recently become clear that there are a family of proteins, structurally and functionally related to p53, that may play a role in regulating its activity. Among these proteins are at least two truncated forms of the protein that result from alternative splicing of the messenger RNA (mRNA) encoding the C-terminal end of the native protein. Identifying new isoforms of p53 and defining how they affect cellular activity may lead to new ways of regulating cell growth and, eventually, to new diagnostic and therapeutic procedures.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery of a new isoform of p53 that is truncated to eliminate a substantial portion of the C-terminal end of the protein. The isoform is useful as a marker of myocardial infarction and in the control of cellular proliferation both in vitro and in vivo.
In its first aspect, the invention is directed to a substantially purified isoform of p53 (designated “p35”) consisting essentially of the amino acid sequence shown in
FIG. 1
(SEQ ID NO:1). The term “consisting essentially of” is meant to encompass proteins having exactly the same amino acid sequence as that shown in the figure, as well as proteins with differences that are not substantial as evidenced by their retaining the basic, qualitative functional properties of p35. A “substantially purified” isoform is one that has been separated from other accompanying biological components and will typically comprise at least 85% of a sample, with greater percentages being preferred. Many means are available for assessing the purity of a protein within a sample, including analysis by polyacrylamide gel electrophoresis, chromatography and analytical centrifugation. One preferred method for assessing the purity of p53 is by performing Western blots using an antibody directed against epitopes of p53 retained in p35. The present isoform should appear in such blots as a band at a position characteristic of a protein with a molecular weight of about 35,000.
The invention also encompasses antibodies that are made by a process involving the injection of a preparation of the p35 isoform into an animal capable of antibody production. These antibodies may be either polyclonal or monoclonal. In the latter case, it is preferred that antibody be made by a process involving the injection of a pharmaceutically acceptable preparation into a mouse, followed by fusing mouse spleen cells with myeloma cells.
The invention is also directed to a substantially pure polynucleotide consisting essentially of a nucleotide sequence encoding the p35 isoforn having the amino acid sequence shown in
FIG. 2
(SEQ ID NO:2); expression vectors comprising a distinct coding element consisting essentially of such polynucleotides; and host cells transformed with such vectors. A “distinct coding element” refers to the portion of an expression vector responsible for determining the amino acid sequence of an expressed protein. The invention includes all such elements producing proteins corresponding to the amino acid sequence shown in
FIG. 1
as well as other proteins that do not differ substantially in terms of structure and function. Also included in the invention is the recombinant p53 isoform made by the host cells described above. The recombinant protein may be isolated using standard techniques, including affinity chromatography with antibodies against epitopes of p53 retained in p35. In a preferred embodiment, the polynucleotide encoding p35 consists essentially of the nucleotide sequence shown in FIG.
2
. This polynucleotide may be used in vectors for expressing the isoform, in host cells transformed with such vectors, and in the production of recombinant p35.
In another embodiment, the present invention is directed to a substantially purified human p53 isoform in which the C-terminal portion of the protein has been eliminated. The deleted amino acids correspond to those encoded by exon 7 and by exons corresponding to amino acids lying C-terminal to exon 7. Preferably, the human isoform consists essentially of the amino acid sequence shown in
FIG. 3
(SEQ ID NO:3). Antibodies made by injecting a preparation of this isoform into an animal capable of producing antibodies are included in the invention, as are substantially purified polynucleotides encoding the isoform. A polynucleotide encoding human p35 may be used as a distinct coding element in an expression vector. The preferred polynucleotide consists essentially of the nucleotide sequence shown in
FIG. 4
(SEQ ID NO:4). Also preferred are vectors which incorporate this particular sequence as a distinct coding element and the host cells and recombinant p53 produced using these vectors.
In another embodiment, the present invention is directed to methods for controlling the proliferation of cells using any of the p35 isoforms or polynucleotides discussed above. The preferred method for controlling proliferation is to transfect cells with a vector capable of expressing p35 in the target cells. Thus, the vector should typically have a p35 coding sequence operably linked to a promoter responsive in the cells being transfected. The procedure may be used either in vitro or in vivo. When used in vitro, any means of transfection may be used including calcium phosphate precipitation, liposome-mediated transfection, electroporation, microinjection, etc. Preferred methods for introducing DNA into cells in vivo include transfection using cationic liposomes (e.g., as described in U.S. Pat. No. 5,676,954 or U.S. Pat. No.4,897,355); using a particular cationic lipid (e.g., as discussed in 5,703,055); or by injecting DNA directly into tissue (e.g., as discussed in U.S. Pat. No. 5,589,466 or U.S. Pat. No. 5,580,859).
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Dell'Acqua Giorgio
Dzau Victor J.
Mann Michael J.
Hauda Karen M.
Pillsbury & Winthrop LLP
Sanzo Michael A.
Schnizer Richard
The Brigham and Women's Hospital Inc.
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