Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-05-28
2003-07-22
Bugaisky, Gabrielle (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S501000, C530S350000, C514S002600, C514S012200
Reexamination Certificate
active
06596495
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of the invention is cell death.
Programmed cell death is a physiological process that has been conserved through evolution (Ellis, Ann. Rev. Cell Biol. 7:663-698, 1991; Raff, Nature 356:397-400, 1992). Genetic analyses of the cell-death process in
C. elegans
have defined a genetic pathway for programmed cell death (reviewed by Horvitz et al., Cold Spring Harbor Symposia on Quantitative Biology LIX, 377-385, 1994). Mutations in three genes, ced-9, ced-4, and ced-3 (ced, cell death abnormal), affect, most if not all, of the 131 somatic cell deaths that occur during the development of the
C. elegans
hermaphrodite (Sulston and Horvitz, Dev. Biol. 56:110-156, 1977; Sulston et al., Dev. Biol. 100:64-119, 1983). Loss-of-function (lf) mutations in ced-3 or ced-4 result in the survival of cells that normally die, indicating that these genes are required for the killing process (Ellis and Horvitz, Cell:44:817-829, 1986). ced-9, by contrast, is a negative regulator of programmed cell death. A gain-of-function (gf) mutation in ced-9 prevents most if not all programmed cell deaths, and loss-of-function (lf) mutations in ced-9 cause embryonic lethality as a consequence of ectopic cell death (Hengartner et al., Nature 356:494-499, 1992). This lethality is suppressed by loss-of-function mutations in ced-3 or ced-4, indicating that ced-3 and ced-4 act downstream of, or in parallel to, ced-9 (Hengartner et al., Nature 356:494-499, 1992). ced-4 is likely to act upstream of ced-3, since cell death induced by ced-4 overexpression is greatly reduced in the absence of ced-3 activity (Shaham and Horvitz, Genes Dev. 10:578-591, 1996). The ced-9, ced-4, and ced-3 central cell-death machinery is thought to be regulated by cell-type-specific regulators, which includes the cell-death specification genes ces-1 and ces-2; these two genes specify the life-versus-death decisions of a subset of cells, including the sisters of the NSM neurons in the pharynx (Ellis and Horvitz, Development 112:591-603, 1991).
The genetically established interactions among ced-9, ced-4, and ced-3 may reflect direct physical interactions of the protein products of these genes. The CED-9 protein binds to the CED-4 protein (Spector et al., Nature 385:653-656, 1997; Chinnaiyan et al., Science 275:1122-1126, 1997; Wu et al., Science 275:1126-1129, 1997; James et al., Curr. Biol. 7:246-252, 1997; Ottilie et al., Cell Death and Differentiation 4:526-533, 1997), which, in turn, can bind to the CED-3 protein (Chinnaiyan et al., Science 275:1122-1126, 1997; Wu et al., J. Biol. Chem. 272:21449-21454,1997). Furthermore, the interaction of CED-4 with CED-3 appears to lead to the activation of CED-3 and the initiation of cell death (Seshagiri and Miller, Curr. Biol. 7:455-460, 1997; Chinnaiyan et al., Nature 388:728-729, 1997; Wu et al., J. Biol. Chem. 272:21449-21454,1997).
ced-9 and ced-3 have mammalian counterparts also shown to be involved in programmed cell death. ced-9 encodes a protein structurally and functionally similar to the mammalian cell-death inhibitor Bcl-2 (Hengartner and Horvitz, Cell 76:665-676, 1994), the prototype of a family of Bcl-2-like molecules that act as regulators of cell death in mammals (reviewed by White, Genes Dev. 10:1-15, 1996; Rinkenberger and Korsmeyer, Curr. Op. Gen. Dev. 7:589-596, 1997). CED-3 is a member of a family of invertebrate and mammalian cysteine proteases, collectively called caspases, that are cell-death effectors acting mainly downstream of Bcl-2-like cell-death regulators (reviewed by Fraser and Evan, Cell 85:781-784, 1996; Nicholson and Thomberry, Trends Biochem. Sci. 22:299-306, 1997).
Recently, a new group of mammalian cell-death activators has been identified. These proteins, which include Bik, Bid, Harakiri, and Bad, interact with Bcl-2-like proteins and can induce cell death when overexpressed (Yang et al., Cell 80:285-291, 1995; Boyd et al. Oncogene 11:1921-1928, 1995; Han et al., Mol. Cell. Biol. 16:5857-5864, 1996; Wang et al., 1996; Inohara et al., EMBO J. 16:1686-1694, 1997; Zha et al., J. Biol. Chem. 272:24101-24104, 1997; Kelekar et al., Mol. Cell. Biol. 17:7040-7046, 1997; Ottilie et al., J. Biol. Chem. 272:30866-30872, 1997). The amino acid sequences of these cell-death activators are dissimilar, except for a nine amino acid stretch similar to one of the four Bcl-2 homology (BH) domains, the BH3 domain, and particularly similar to the BH3 domain of the Bcl-2-like cell-death activators Bax and Bak (Chittenden et al., EMBO J. 14:5589-5596, 1995; 1995; Han et al., Genes Dev. 10:461-477,1996 1996b; Zha et al., J. Biol. Chem. 271:7440-7444, 1996; Hunter and Parslow, J. Biol. Chem. 271:8521-8524, 1996). As in the cases of Bax and Bak (Chittenden et al., EMBO J. 14:5589-5596, 1995; Han et al., Genes Dev. 10:461-477,1996), the BH3 domains of this new group of cell-death activators are important both for their interaction with Bcl-2-like molecules and for their ability to induce cell death (Wang et al., Genes Dev. 10:2859-2869, 1996; Inohara et al., EMBO J. 16:1686-1694, 1997; Zha et al., J. Biol. Chem. 272:24101-24104, 1997; Kelekar et al., Mol. Cell. Biol. 17:7040-7046, 1997; Ottilie et al., J. Biol. Chem. 272:30866-30872, 1997).
It would be useful to identify and clone additional genes in the
C. elegans
cell death pathways. Due to the conservation between nematode and mammalian cell death pathways, identification of such genes and their encoded proteins could allow detection of therapeutic targets, therapeutic compounds, and novel cell death genes.
SUMMARY OF THE INVENTION
We have discovered and cloned a new
C. elegans
cell death gene, egl-1 (egl, egg-laying defective) that encodes a protein that interacts with CED-9 and that contains a region similar to the BH3 domains of BH3-containing cell-death activators. Gain-of-function mutations in egl-1, such as egl-1(n1084 n3082), cause the two HSN neurons, which are required for egg laying, to inappropriately undergo programmed cell death; these mutants were identified in screens for egg-laying defective (Egl) mutations (Trent et al., Genetics 104:619-647, 1983). By isolating a dominant suppressor of the egl-1 Egl phenotype, we identified a loss-of-function mutation in the egl-1 gene, egl-1(n1084 n3082). This mutation prevents not only the ectopic deaths of the HSNs but most if not all normally occurring programmed cell deaths, indicating that egl-1 is a cell-death activator and encodes a component of the general cell-death machinery in
C. elegans.
In the first aspect, the invention features substantially pure nucleic acid encoding EGL-1 polypeptide. Such nucleic acid is defined by its ability to complement any of the egl-1(n1084 n3082) mutations provided herein or by the ability to suppress mutations in egl-1(n1084). Preferably, specifically excluded is the exact wild-type nucleic acid sequence provided at the ced(n3082) map position in the
C. elegans
Genome Consortium database on May 28, 1997; conservative substitutions of this sequence are, however, preferably included. In a related aspect, the invention features egl-1 nucleic acids which have deletions. Fusions of additional nucleic acids to the egl-1 gene are also included, as are fragments sufficient for use as primers, probes, or synthesis of epitopes for antibody preparation. Homologs of
C. elegans
egl-1 from other species (and fragments, fusions, deletions, and other mutations therein) are also a related aspect of the invention. Homologs are defined as having at least 50%, preferably 90% identity over at least 100 base pairs, or as being able to complement at least one egl-1(n1084 n3082) allele and having at least 20% identity over the entire gene. Identity is preferably determined using the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wis. 53705, on the default settings.
In a related aspect the invention provides polypeptide encoded by the egl-1 gene. Proteins encoded by egl-1 nucleic acids which are fragmented, deleted or fused to other sequences are
Conradt Barbara
Horvitz H. Robert
Bieker-Brady Kristina
Bugaisky Gabrielle
Clark & Elbing LLP
Massachusetts Institute of Technology
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