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
1998-10-19
2004-06-29
Bugaisky, Gabriele (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...
C435S320100, C435S252300, C435S252330, C435S244000, C435S325000, C435S410000, C536S063000
Reexamination Certificate
active
06756212
ABSTRACT:
BACKGROUND OF THE INVENTION
Apoptosis or programmed cell death is an essential physiological process of selective elimination of cells in multicellular organisms. This process is invoked during normal organ development and tissue homeostasis and also during certain pathological conditions that result in degenerative diseases. Programmed cell death plays an important role in regulating the multiplication of and pathogenesis by a number of viruses. In virus-infected cells, apoptotic paradigms are initiated as a cellular defensive mechanism to eliminate infected cells. However, a number of viruses encode proteins that suppress apoptosis resulting in efficient viral replication and pathogenesis. Human adenovinises have evolved multiple strategies employing proteins coded by three different gene blocks, E1B, E3, and E4 to overcome the effect of apoptosis of infected permissive cells. One of the proteins coded by the E1B region, the 19K protein confers a survival fiction in adenovirus-infected cells and prevents premature cell death. Adenovirus mutants defective in the 19K gene produced large clear plaques on infected cell monolayers. Several of these mutants induce an enhanced cytopathic effect in infected cells resulting in cellular destruction as well as fragmentation of cellular and viral DNA. The DNA fragmentation induced by 19K mutants is reminiscent of that observed during apoptosis. Although it has not yet been determined whether DNA fragmentation induced by 19K mutants occurs by an apoptotic mechanism, it is clear that the 19 kDa protein protects against a cell death program induced by viral infection, thus facilitating efficient virus replication. In addition, the 19 kDa protein suppresses the cytotoxic effects of certain external stimuli such as the tumor necrosis factor &agr; and anti-Fas antibody. Both of these agents cause cell death through apoptosis. Similarly, the 19 kDa protein protects cells against the effects of DNA-damaging agents such as the anti-cancer drug cisplatin and ultraviolet radiation induced cell death through a p53-dependent apoptotic pathway. Cells expressing the 19 kDa protein efficiently suppress cell death induced by overexpression of p53. Thus, the 19 kDa protein provides a survival function in virus-infected cells and also protects cells against certain other death-inducing stimuli.
The survival function provided by E1B 19K is similar to the activity of the cellular protooncogene Bcl-2. The Bcl-2 oncogene was isolated from a follicular lymphoma and has been shown to enhance the survival of hematopoietic B and T cells by blocking apoptosis. In addition, overexpression of Bcl-2 protein inhibits apoptosis induced by exposure to diverse stimuli possibly through different pathways. Although the effect of 19 kDa protein expression on cell death induced by diverse stimuli has not been extensively examined, the Bcl-2 protein can clearly substitute for the 19 kDa protein during adenovirus infection. Characteristic fragmentation of cellular DNA induced by infection with adenovirus type 2 (Ad2) 19K mutants is efficiently suppressed in cells ectopically expressing the human Bcl-2 protein. Similarly, expression of Bcl-2 by an Ad2-Bcl-2 recombinant virus (Ad-Bcl2) can fully substitute for the 19K function. The Ad-Bcl12 virus does not induce DNA fragmentation in infected cells and forms small plaques on cell monolayers. Rao et al. (1992)
Proc. Natl. Acad. Sci
. U.S.A. 89:7742-46 have reported that Bcl-2 can substitute for 19K in transformation of primary rat kidney cells in cooperation with E1A. Although these results do not clarify whether the 19 kDa and Bcl-2 proteins function by similar mechanisms, they indicate that these two proteins can provide cell survival function against certain stimuli.
The mechanism by which the 19K gene and the Bcl-2 protooncogene protect against cell death is not known. These proteins may mediate cell survival by interacting with certain cellular proteins. Boyd J. M. et al. (1994)
Cell
79:341-351, have identified three proteins, NIP1, NIP2, and NIP3, which interact with both the adenovirus E1B 19 kDa protein and the Bcl-2 protein. The NIP1, NIP2, and NIP3 proteins are believed to play a role in the ability of the E1B 19 kDa protein to provide a cell survival function.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of novel NIP2 family members, referred to herein as “NIP2b, NIP2cL, and NIP2cS” nucleic acid and protein molecules. The NIP2b, NIP2cL, and NIP2cS molecules of the present invention are useful as modulating agents for regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding NIP2b, NIP2cL, and NIP2cS proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of NIP2b, NIP2cL, and NIP2cS-encoding nucleic acids.
In one embodiment, a NIP2b, NIP2cL, or NIP2cS nucleic acid molecule of the invention is at least 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1, 3, 4, 6, 7, or 9, or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-369 of SEQ ID NO: 1. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1483-3076 of SEQ ID NO:1. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:1 or 3. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 535 nucleotides (e.g., 535 contiguous nucleotides) of the nucleotide sequence of SEQ ID NO:1 or 3, or a complement thereof.
In another preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:4 or 6, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:6 and nucleotides 1-22 of SEQ ID NO:4. In another embodiment, the nucleic acid molecule includes SEQ ID NO:6 and nucleotides 989-4235 of SEQ ID NO:4. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:4 or 6. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 3225 nucleotides (e.g., 3225 contiguous nucleotides) of the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:6, or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:7 or 9, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:9 and nucleotides 1-55 of SEQ ID NO:7. In another embodiment, the nucleic acid molecule includes SEQ ID NO:9 and nucleotides 434-2966 of SEQ ID NO:7. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:7 or 9. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 461 nucleotides (e.g., 461 contiguous nucleotides) of the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:9, or a complement thereof.
In another embodiment, a NIP2b, NIP2cL, and NIP2cS nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, 5, or 8. In a preferred embodiment, a NIP2b, NIP2cL, and NIP2cS nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the entire length of the amino acid sequence of SEQ ID NO:2, 5, or 8.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human NIP2b, NIP2cL, and NIP2cS. In yet another preferred embodiment, the nucleic acid mo
Curtis Rory A. J.
Glucksmann M. Alexandra
Bugaisky Gabriele
Lahive & Cockfield LLP
Millennium Pharmaceuticals Inc.
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