Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-06-02
2003-11-25
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023100
Reexamination Certificate
active
06653126
ABSTRACT:
FIELD OF THE INVENTION
This invention generally relates to the nucleic acid sequences (and corresponding translated products) of novel mutant forms of the Drosophila DIAP1 gene and methods of identifying and testing agonists and antagonists of DIAP1 that enhance or reduce the apoptotic process.
BACKGROUND
Essentially all animal cells have the ability to activate an intrinsic cell suicide program, called programmed cell death (Steller, H. “Mechanisms and Genes of Cellular Suicide”,
Science
267:1445-1446, 1995; White E. “Life, death and the pursuit of apoptosis”,
Genes Dev.
10:1-15, 1996; Jacobson, M. D., et al. “Programmed Cell Death in Animal Development”,
Cell
88:347-354, 1997). The execution of this program leads to a morphologically distinct form of cell death termed apoptosis (Kerr et al. “Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics”,
Br. J. Cancer
26:239-257, 1972; Wyllie et al. “Cell Death: the significance of apoptosis”,
Int. Rev. Cytol.”
68:251-306, 1980). It is now generally accepted that apoptosis is of central importance for the development and homeostasis of metazoan animals. The roles of apoptosis include the sculpting of structures during development, deletion of unneeded cells and tissues, regulation of growth and cell number, and the elimination of abnormal and potentially dangerous cells. In this way, apoptosis provides a stringent and highly effective “quality control mechanism” that limits the accumulation of harmful cells, such as self-reactive lymphocytes, virus-infected cells and tumor cells (Reed “Regulation of apoptosis by bcl-2 family proteins and its role in cancer and chemoresistance”,
Curr. Opin. Oncol
7:541-546, 1995; Thompson “Apoptosis in the Pathogenesis and Treatment of Disease”,
Science
267:1456-1462, 1995; Naik et al. “The rise and fall of apoptosis during multistage tumorigenesis: down-modulation contributes to tumor progression from angiogenic progenitors”
Genes Dev.
10:2105-2116, 1996; Morin et al. “Apoptosis and APC in colorectal tumorigenesis”,
Proc. Natl. Acad. Sci
. USA 93:7950-7954, 1996; White “Life, death and the pursuit of apoptosis”
Genes Dev.
10:1-15. 1996). On the other hand, inappropriate apoptosis is associated with a wide variety of diseases, including AIDS, neurodegenerative disorders, and ischemic stroke (Martinou et al. “Over-expression of Bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia”
Neuron
13:1017-1030, 1994; Thompson “Apoptosis in the Pathogenesis and Treatment of Disease”,
Science
267:1456-1462, 1995; Pettmann and Henderson “Neuronal Cell Death”
Neuron
20:808-810, 1998).
Because it is now clear that apoptosis is the result of an active, gene-directed process, it should be possible to manipulate this form of death by developing drugs that interact with cell death proteins. Prior attempts at drug screening have been hampered by the lack of reagents that allow for the identification of compounds that interact with known regulatory constituents of the cell death mechanism. There is a pressing need for new reagents that help identify cell death agonistic or antagonistic compounds that act with specificity at known cell death modulating proteins. Knowing specifically where the compounds interact in the cell death pathway will allow for the modification of those compounds found to be agonistic or antagonistic thereby allow for the development of improved versions of the compound.
SUMMARY OF THE INVENTION
The present invention generally relates to compositions and methods of identifying and testing DIAP1 pathway agonists and antagonists. In addition, the invention relates to methods to identify other members of the DIAP1 signal pathway, methods to identify homologs of DIAP1 which are native to other tissue or cell types, methods to identify tissues that may harbor tumors expressing similar or homologous genes with simular mutations and methods to generate reagents derived from the invention.
The present invention contemplates employing novel mutant forms of the wild-type Drosophila DIAP1 gene (SEQ ID NO:1) in these screening methods. In one embodiment, the present invention contemplates generating chemically induced mutants that modulate the partial eye abolation phenotype of the GMRreaper and/or GMRHid of trangenic
Drosophila melanogaster
. In this way it is possible to screen for gof and lof mutations. In one embodiment, the present invention contemplates a composition comprising isolated and purified DNA having an oligonucleotide sequence selected from the group consisting of: DIAP1
6-3S
cDNA having the nucleotide sequence of SEQ ID NO: 2; DIAP1
45-2S
cDNA having the nucleotide sequence of SEQ ID NO: 3; DIAP1
23-4S
cDNA having the nucleotide sequence of SEQ ID NO: 4; DIAP1
11-3E
cDNA having the nucleotide sequence of SEQ ID NO: 5; DIAP1
22-8S
cDNA having the nucleotide sequence of SEQ ID NO: 6; DIAP1
21-4S
cDNA having the nucleotide sequence of SEQ ID NO: 7; DIAP1
33-1S
cDNA having the nucleotide sequence of SEQ ID NO: 8; DIAP1
21-2S
cDNA having the nucleotide sequence of SEQ ID NO: 9; DIAP1
41-8S
cDNA having the nucleotide sequence of SEQ ID NO: 10. Such DNA may readily be inserted into expression constructs and the present invention contemplates such constructs as well as their use. The present invention also contemplates RNA transcribed from the above-indicated cDNAs as well as protein (typically purified protein) translated from this RNA. Moreover, the present invention contemplates antibodies produced from immunizing with this translated protein.
The present invention also contemplates transgenic animals comprising the above-indicated DNA (i.e. the “transgene”) or portions thereof. In a particular embodiment, the transgenic animal of the present invention may be generated with the transgene contained in an inducible, tissue specific promotor.
The present invention also contemplates using the above-named compositions in screening assays. The present invention is not limited by the particular method of screening. In one embodiment insect cells are used such as, but not limited to, Drosophila SL2 cells. In another embodiment mammalian cells may be used. The present invention is not limited to the nature of the transfection construct. The transfection constructs utilized will be the optimal constructs available for the cell line chosen at the time of setting up the assay. 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 translation products of the invention may be used in a cell-free assay system. In yet another embodiment, antibodies generated to the translation products of the invention may be used in immunoprecipitation assays.
The present invention may also be used to screen for tumors which manifest mutations in genes similar to, or homologous with, the cDNA encoding the invention. In, one embodiment cDNA encoding the invention may be used in microchip assays. The present invention contemplates a method of screening, comprising: a) providing in any order: i) a first solid support (e.g. microchip) comprising cDNA encoding at least a portion of the oligonucleotide sequence of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9 or 10, ii) a second solid support (e.g. a second microchip) comprising at least a portion of the wild type Drosophila DAIP1 gene oligonucleotide sequence (SEQ ID NO:1), and iii) sample DNA from at least one tissue sample suspected of having mutations in genes similar to (or homologous with) SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9 or 10; b) contacting said first and second microassay microchips with said sample DNA under conditions such that hybridization can take place.
The present invention may also be used to identify new constituents of the DIAP1 signaling pathway. In one embodiment, antibodies generated to translation products of the invention may be u
Agapite Julie
Goyal Lakshmi
McCall Kim
Steller Hermann
Katcheves Konstantina
Ketter James
Massachusetts Insititute of Technology
Medlen & Carroll LLP
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