Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-07-23
2002-10-29
Bugaisky, Gabrielle (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023400, C536S023500, C435S320100, C435S252300, C435S254110, C435S325000, C435S410000
Reexamination Certificate
active
06472170
ABSTRACT:
BACKGROUND OF THE INVENTION
Members of the family of BCL-2-related proteins serve as regulators of programmed cell death, or apoptosis (Cory, 1995
Annu. Rev. Immunol
. 13, 513-543; Hockenbery, 1995
Nature
348, 334-336; Nunez et al., 1994
Nature
348, 334-336; Reed, 1994
. J. Cell Biol
. 124, 1-6; Akbar et al., 1993
. Immunology Today
14, 526-532). Apoptosis has been shown to be involved in several immune processes, including intrathymic deletion of autoreactive cells, elimination of peripheral T cells during the response to viral and bacterial superantigens and lysis of target cells by cytotoxic T lymphocytes. There is increasing evidence that clonal expansion of antigen-specific T-cells is determined by the relative level of cellular proliferation and apoptosis following TCR ligation (Zinkernagel et al. 1993
. Immunol. Rev
. 131:199). However, the genetic mechanisms responsible for regulating these response phenotypes are not well understood.
The first gene to be identified which encoded a protein in this family, bcl-2, was cloned from the chromosomal breakpoint oft(14;18)-bearing B-cell lymphomas (Tsujimoto et al., 1984. Science 226:1097) and shown to inhibit cellular susceptibility to apoptosis (Cory, supra).
Several genes with homology to bcl-2 have subsequently been characterized, including the following: Al, which encodes an 80-amino acid protein that is rapidly induced in macrophages in response to GM-CSF or LPS (Lin et al., 1993
. J. Immunol
. 151, 1979-1988); MCL1, an early response gene in myeloid cell lines which undergo macrophage differentiation (Kozopas et al., 1993
. Proc. Natl. Acad. Sci. USA
90, 3516-3520); and Bak, a BCL-2 homologue that may enhance apoptosis (Chittenden et al., 1995. Nature 374:733; Kiefer et al., 1995. Nature 374:736).
The bcl-x gene product, closely related to the BCL-2-related protein family, also protects cells from apoptosis. Analysis of mice deficient in BCL-x has suggested that its function is to support the viability of immature cells during development of the nervous and hematopoietic systems (Motoyama et al., 1995
. Science
267, 1506-1510; Ma et al., 1995
. Proc. Natl. Acad. Sci. USA
92, 4763-4767). Alternative splicing of human bcl-x may result in at least two distinct BCL-x mRNA species. The predominant protein product (233 amino acids) of the larger BCL-x mRNA, BCL-xL, inhibits cell death upon growth factor withdrawal (Boise et al., 1993
. Cell
74, 597-608) and its transgenic expression alters thymocyte maturation leading to increased numbers of mature thymocytes (Chao et al., 1995
. J. Exp. Med
. 182, 821-828; Grillot et al., 1995
. J. Exp. Med
. 182, 1973-1983). After co-ligation of CD3 and CD28 in murine T-cells, enhanced BCL-xL expression may confer protection from apoptosis (Boise et al., 1995
. Immunity
3, 87-98; Radvanyi et al., 1996
. J. Immunol
. 156, 1788-1798; Mueller et al., 1996
. J. Immunol
156, 1764-177 1). The contribution of other isoforms of this gene to activation-induced death in T-cells is less well-defined (Gonzalez-Garcia et al., 1994
. Development
120, 3033-3042; Fang et al., 1994
J. Immunol
. 153, 4388-4398). A second human BCL-x isoform, BCL-xS, encodes a smaller protein of 170 amino acids which may enhance apoptosis, suggesting that different members of the BCL-x family may have opposing functions. Additional murine BCL-x isoforms, termed BCL-x&bgr; and BCL-x&Dgr;TM, have been defined. The &bgr; isoform may inhibit apoptosis in neurons (Gonzalez-Garcia et al., 1995
. Proc. Natl. Acad. Sci. U.S.A
. 92, 4304-4308) and the &Dgr;TM isoform may inhibit apoptosis in B-cells (Fang et al., supra).
Several proteins which interact with BCL-2 proteins have also been identified including bax, Nip1, Nip2, Nip 3, bad, and bag-1. These various BCL-2 binding proteins have different effects on apoptosis. For example, bak and bax function as inducers of apoptosis, whereas bag increases the resistance of cells to apoptosis (Farrow and Brown. 1996
. Curr. Opin. Genetics and Devel
. 6:45).
Despite the apparent importance of BCL-x in development and function of T-cells, none of the BCL-x isoforms described so far displays restricted expression with respect to this lineage; all four isoforms of BCL-x are ubiquitously expressed in a wide variety of tissues (Gonzalez-Garcia et al., 1994
. Development
120, 3033-3042; Fang et al., supra). This may be because previous studies have isolated most of BCL-x isoforms (BCL-xL, BCL-xS and BCL-x&Dgr;TM) after screening cDNA libraries from tissues other than T-cells (Gonzalez-Garcia et al., 1994 supra; Fang et al., supra). The physiologic expression of these BCL-x isoforms is not sufficient to confer resistance to apoptosis following TCR ligation, since they are expressed equally well in apoptotic and non-apoptotic T-cell blasts. Moreover, overexpression of Bcl-xL does not affect thymocyte selection (Grillot et al. 1995
. J. Exp. Med
. 182:1973).
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as “BCL-x&ggr;” nucleic acid and protein molecules. The BCL-x&ggr; molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes.
Analysis of the BCL-x&ggr; protein indicates that it contains a BH1 and a BH2 domain which are found in other BCL-x family members. However, the BCL-x&ggr; protein also contains a novel &ggr; domain (shown in amino acids 185-235 of SEQ ID NO:2, which includes an ankyrin domain, e.g., amino acids 185-217 of SEQ ID NO:2). The &ggr; domain (C-terminal amino acids 185-235) of the deduced BCL-x&ggr; protein lacks homology with the C-termini of previously described murine BCL-x&ggr; isoforms, including BCL-xL, BCL-x&bgr; or BCL-x&Dgr;TM. Since BCL-x&ggr; does not contain an apparent hydrophobic domain flanked by charged residues it is unlikely to be membrane-bound, similar to the murine BCL-x&Dgr;TM isoform (Gonzalez-Garcia et al., 1994, supra; Fang et al., supra) but in contrast to both, human and murine, BCL-xL and BCL-xS isoforms (Boise et al., 1993) whose C-termini contain sequences that may serve as membrane-anchoring domains (Chen-Levy et al., (1989)
Mol. Cell Biol
. 9, 701-710; Hochenbery et al., 1990 348, 334-336; Nguyen et al., 1993
J. Biol. Chem
. 268, 25265). The BCL-x&ggr; protein has a calculated molecular weight of approximately 26,122 and migrates at approximately 33 kD. The murine amino acid sequence is shown in Seq. ID No. 2.
In contrast to BCL-xL, BCL-x&bgr;, and BCL-x&Dgr;TM, which are expressed in all tissues tested, including brain, eyes, intestine, kidney, liver, lung, lymph nodes, and thymus, the BCL-x&ggr; isoform was detected selectively in thymus, lymph nodes, lung, and eye, but not in heart, intestine, kidney, liver, or brain. BCL-x&ggr; has been found to be expressed only in T-lymphocytes since its message is detected in lymph nodes from BALB/c control but not from BALB/c nu
u mice or from Rag-2 deficient mice. BCL-x&ggr; is expressed in the less mature, cortisone-sensitive fraction of thymocytes. In addition, BCL-x&ggr; has not been detected in thymuses from class I or class II MHC-deficient B6 mice, implying that expression of this Bcl-x isoform may normally depend on an interaction between the TCR and MHC/peptide complexes. The fact that BCL-x&ggr; has been detected in double positive thymocytes indicates that it plays a role in thymic selection not played by other BCL molecules. Thus, unlike previously described forms of BCL-x molecules, BCL-x&ggr; proteins of the invention are specifically connected to TCR ligation and are essential for resistance to TCR-dependent apoptosis.
In one aspect, the invention features an isolated nucleic acid molecule comprising a nucleotide sequence encoding a naturally occurring BCL-x&ggr;. In one embodiment a BCL-x&ggr; nucleic acid molecule encodes mouse BCL-x&ggr;. In another embodiment a BCL-x&ggr; nucleic acid molecule encodes human BCL-x&ggr;. In a preferred embodiment an isolated BCL-x&ggr; nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:2.
I
Cantor Harvey
Weber Georg F.
Yang Xiao-Feng
Bugaisky Gabrielle
Dana-Farber Cancer Institute
Lahive & Cockfield LLP
Mandragouras Esq. Amy E.
Smith, Esq. DeAnn F.
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