Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-10-13
2004-01-27
Caputa, Anthony C. (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024330, C435S320100, C435S325000
Reexamination Certificate
active
06683168
ABSTRACT:
This work was supported by United States Public Health Service grant AI40181 awarded by the National Institutes of Health and a grant from the Arthritis Foundation, the United States government has certain rights in this invention.
FIELD OF THE INVENTION
This invention generally relates to the nucleic acid sequences of a novel gene called the Fas Apoptosis Inhibitory Molecule (faim) that encodes an apoptosis inhibiting protein. Furthermore, this invention relates to methods of identifying and testing antagonists of FAIM activity and to methods to assay for FAIM expression.
BACKGROUND OF THE INVENTION
Programmed cell death (PCD) is mediated by a process called apoptosis. Although the investigation of cell death is a relatively new field of study, it has become readily apparent that many disease states are manifested due to the aberrant control of programmed cell death. Recent evidence suggest that the failure of cells to undergo apoptotic cell death might be involved in the pathogenesis of a variety of human diseases including cancer, autoimmune diseases and viral infections. The understanding of survival pathways would be critical in disease states where excessive cell numbers, such as in various cancers, are the result of cell death rather than cell proliferation. The screening of potential therapeutics has been hindered by both a lack of understanding of the physiological basis of cell death and by a dearth of reagents specific for critical points in the cell death signaling pathways. Additionally, much work has focused on the delineation of the death-inducing pathway and reagents that may block it and not on pathways that prevent apoptosis or confer survival signals. What is needed are reagents and methodologies that allow for the identification and testing of agonists and antagonists of apoptosis inhibiting pathways or cell survival pathways.
SUMMARY OF THE INVENTION
The present invention generally relates to compositions and methods of identifying and testing FAIM pathway agonists and antagonists. The product of the gene faim is responsible for preventing apoptosis in Bal-17 B lymphoma cells and in various murine cells. When ectopically expressed, FAIM inhibits apoptosis. In addition, the invention relates to methods to identify other members of the FAIM signal pathway, methods to identify homologs of FAIM which are native to other tissue or cell types and methods to generate reagents derived from the invention. Additionally, the invention relates to methods to assay for FAIM expression in various cell types including, but not limited to, cancer cells, autoimmune cells and diseased cells.
The present invention is not limited by the method of the employed screen. In one embodiment, the present invention contemplates screening suspected compounds in a system utilizing transfected cell lines. In one embodiment the cells may be transfected transiently. In another embodiment the cells may be stably transfected. In yet another embodiment, transgenic animals may be generated with the transgene under the control of an inducible, tissue specific promotor.
The present invention may also be used to identify new constituents of the FAIM signaling pathway. In one embodiment antibodies generated to translation products of the invention may be used in immunoprecipitation experiments to isolate novel FAIM pathway constituents or natural mutations thereof. In another embodiment the invention may be used to generate fusion proteins that could be used to isolate novel FAIM pathway constituents or natural mutations thereof. In yet another embodiment screens may be conducted using the yeast two-hybrid system.
The present invention may also be used to identify new homologs of FAIM or natural mutations thereof. The present invention contemplates screening for homologs using standard molecular procedures. In one embodiment screens are conducted using Northern and Southern blotting.
The present invention may also be used to determine the expression level of FAIM in various cell types including, but not limited to, pathological cells. In one embodiment, assays are conducted using FAIM antibodies to precipitate expressed FAIM. In another embodiment, assays are conducted using PCR primers or oligonucleotides that recognize FAIM RNA. In yet another embodiment, assays are conducted using various blotting techniques such as, but not limited to. Northern, Southern and Western blotting. The adaptation of such assays to high throughput screening techniques is also contemplated by the present invention.
The present invention further provides a composition comprising DNA having an oligonucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or portions thereof. The present invention further includes isolated RNA transcribed from any of the DNA sequences listed above, isolated protein translated from the RNA, and isolated antibodies produced from the proteins. The present invention further provides expression constructs comprising any of the above listed DNA and cells comprising said expression constructs.
The present invention also provides a method of screening a compound comprising: providing, in any order, cells containing a recombinant expression vector, wherein the vector comprises at least a portion of the oligonucleotide sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or variants or homologs thereof, and a compound suspected of having the ability to alter FAIM activity; and contacting the cells with the compound. In preferred embodiments, the method further comprises the steps of detecting programmed cell death modulation effects of the compound; and/or detecting the appropriate marker if a reporter construct was utilized for detection of compound interaction.
The present invention further provides a method of screening for homologs, said method comprising: providing, in any order 1) DNA, wherein DNA comprises at least a portion of the oligonucleotide sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or variants or homologs thereof, and 2) DNA libraries from cells or tissues suspected of having the homologs; and contacting the first DNA with the second DNA; and isolating, purifying, and sequencing the DNA suspected of coding for the homologs.
The present invention also provides methods of screening for interactive peptides, said method comprising: providing, in any order, 1) peptides, wherein the peptide comprises at least a portion of a peptide sequence of an isolated protein translated from RNA which is transcribed from the oligonucleotide sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or variants or homologs thereof, wherein the peptide has been modified with a GST or other suitable fusion protein for purification purposes, and 2) extracts from cells or tissues suspected of having the homolog; and contacting the extracts with the peptides; and isolating, purifying, and sequencing the homolog.
The present invention also provides methods of assaying for FAIM activity, said method comprising: providing, in any order, 1) antibodies produced from proteins translated from RNA which is transcribed from the oligonucleotide sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or variants or homologs thereof, and 2) extracts from cells suspected of expressing FAIM; contacting said extracts with said antibodies; and detecting said expressed FAIM.
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Zhong X, et al. Mol Immunol Jan. 2001; 38 (1): 65-72.*
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Hillier, L, et al, 1996, Genome Research, vol. 6, No. 9, pp. 807-828 (Database GenBank Accession No. AA099194).*
NCI-CGA
Donohoe Terrence J.
Rothstein Thomas L.
Schneider Thomas J.
Boston Medical Center Corporation
Caputa Anthony C.
Nixon & Peabody LLP
Rawlings Stephen L.
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