Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-12-04
2001-04-03
Kemmerer, Elizabeth (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S300000
Reexamination Certificate
active
06211339
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of pest control and molecular biology and, in particular, to genetic control of programmed cell death (PCD), or apoptosis in insect and human cells.
2. Description of Related Art
Programmed cell death (PCD) or apoptosis is an essential regulator of tissue differentiation and cellular maintenance as animals develop and age [Saunders and Fallon, 1967; Truman, 1984; Hurle, 1988; Ellis, et al., 1991; Oppenheim, 1991; Raff, 1992]. Recent studies have implicated the misregulation of apoptosis in the pathophysiology of several human diseases including AIDS [Meyaard, et al., 1992; Gougen and Montagnier, 1993], neurodegenerative disease [Roy, et al., 1995; Liston, et al., 1996; Vito, et al., 1996] and cancer [reviewed in Williams, 1991].
Several lines of evidence suggest that the physiology of apoptosis is quite highly conserved. First, the morphological changes associated with programmed cell'deaths are stiringly similar in both vertebrates and invertebrates [Kerr, et al., 1972; Wyllie, et al., 1980; Kerr and Harmon, 1991; Abrams, et al, 1993]. Second, at least two essential cell death genes in
Caenorhabditis elegans
, ced-3 and ced-9, are members of gene families that encode apoptotic functions in vertebrates [reviewed in Steller, 1995; White, 1996). Third, viral proteins that suppress apoptosis in their hosts (p35 and, crmA) can exhibit potent anti-apoptotic activity in a wide range of heterologous species Rabizadeh, et al., 1993; Hay, et al., 1994; Sugimoto, et al., 1994; Grether, et al, 1995; Pronk, et al., 1996; White, et al., 1996].
In
Drosophila melanogaster
, a genomic interval defined by the H99 deletion mutation is required for embryonic programmed cell death [Abrams, et al., 1993; White, et al., 1994; Abrams, 1996]. This region spans ~300 kilobases of DNA that includes at least two cell death genes, reaper (rpr) [White, et al., 1994] and head involution defective. (hid) [Grether, et al., 1995]. The former gene product is thought to share similarities to the “death domain” of the FAS/TNFR1 protein family [Cleveland and Ihle, 1995; Golstein, et al., 1995; Golstein, et al., 1995] whereas the latter shares no extensive sequence similarity to known proteins. Although the distribution of RNA from both genes generally corresponds to embryonic patterns of apoptosis, only rpr appears to be selectively expressed in all cells that will later undergo programmed cell death.
Although no apoptosis occurs in embryos bearing homozygous deletions of the entire H99 interval [Abrams, et al., 1993; White, et al., 1994], null mutations at hid display only mild cell death defects [Grether, et al., 1995] and, to date, no single-gene mutants of the rpr have been identified. Therefore, the precise number of cell death genes uncovered by H99 is not known. In fact, phenotypes associated with two informative deletions in the region, X14 and X25 [White, et al., 1994; Grether, et al., 1995], raised the possibility that perhaps one or more additional cell death genes might reside between hid and rpr. Both strains partially uncover the H99 interval from the distal boundary thereby eliminating hid yet preserving rpr. However, although both deletions exhibit mild and indistinguishable PCD phenotypes as homozygotes [Grether, et al., 1995], X25 uncovers a far more severe phenotype when placed in trans to H99 than does X14. Whereas X14/H99 embryos show subtle cell death defects similar to those observed for hid null alleles, X25/H99 transheterozygotes exhibit a severe reduction in apoptosis frequency that can be easily visualized by staining with acridine orange [Grether, 1994]. Since the relevant breakpoint of X25 is ~60kb more proximal than that of X14 [Grether, 1994], it is possible that one or more additional cell death functions map to the interval bounded by these breakpoints.
There is still a need therefore, for methods of controlling insect pests without the use of pesticides that remain in the environment and contribute to pesticide resistance, or for methods of controlling apoptosis in cells that have a disrupted apoptotic function by restoration of apoptosis genes. These needs may be met by the discovery of apoptosis controlling genes that are active in insect species and that also may be active in other species, including mammalian and even human cells.
SUMMARY OF THE INVENTION
The present invention seeks to overcome certain drawbacks inherent in the prior art by providing the isolation of a novel Drosophila apoptosis gene, designated as grim. The isolation of this gene allows the production of high levels of the GRIM protein as well as providing methods of inducing apoptosis in various types of cells, including, but not limited to, insect cells, such as insect embryo cells, and human cells. Of particular advantage is the use of the present discovery in conjunction with an inducible promoter so that the apoptosis gene may be inserted into a particular cell line and remain silent until the inducing condition is encountered, at which time the expression of GRIM causes programmed cell death in the recombinant or transgenic cell.
In a certain broad aspect, the present invention is an isolated nucleic acid segment and particularly a nucleic acid segment that encodes a Drosophila GRIM polypeptide. The encoded Grim polypeptide of the present invention may comprise an amino acid sequence of SEQ ID NO:2 or may in certain embodiments have the amino acid sequence of SEQ ID NO:2. The invention may also be described in a certain broad aspect as an isolated nucleic acid segment that comprises a nucleic acid sequence including the coding region of SEQ ID NO:1 or its complement, or even as an isolated nucleic acid segment having a sequence as set forth in SEQ ID NO:1 or its complement. The nucleic acid segments so described may be fused to other functional nucleic acid sequences such as those encoding leader sequences, fusion proteins, epitope tags, ribosomal binding sites, polyadenylation sites, genetic linkers containing restriction enzyme recognition sequences, promoters, selectable markers and a variety of other segments well known in the art.
The complement of a DNA or RNA sequence is well known in the art and is based on the Watson-Crick pairing of nucleic acid polymers. The complement of a nucleic acid segment is generated by converting all “G” residues to “C” residues, all “C” residues to “G” residues, all “A” residues to “T” (in the case of DNA) or “U” (in the case of RNA) and all “T” or “U” residues to “A”, and then reversing the 5′ to 3′ orientation of the generated sequence. As used herein therefore, the term “complement” defines a second strand of nucleic acid which will hybridize to a first strand of nucleic acid to form an antiparallel duplex molecule in which base pairs are matched as G:C, C:G, A:T/U or T/U:A.
The present invention may also be described in certain embodiments as a nucleic acid segment that is hybridizable to the nucleic acid segment of SEQ ID NO:1 or its complement under stringent conditions. Hybridizable is understood to mean the formation of a double stranded molecule or a molecule with partial double stranded structure. Stringent conditions are those that allow hybridization between two nucleic acid sequences with some degree of homology, but precludes hybridization of random sequences. The degree of homology would depend on the length of the sequences to be hybridized. A sequence of from 10 to 14 or even about 20 bases in length would likely tolerate no internal mismatches at high stringency, but longer sequences of up to 50 or 100 bases, for example would tolerate some mismatches as long as stretches of 17-20 or more bases contained within the longer sequences hybridized without mismatches. For example, hybridization at low temperature and/or high ionic strength is termed low stringency and hybr
Abrams John M.
Chen Po
Nordstrom William
Board of Regents , The University of Texas System
Fulbright & Jaworski
Kemmerer Elizabeth
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