Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-12-22
2004-09-14
Nashed, Nashaat (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C530S300000, C530S321000, C530S326000, C435S069100, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06790648
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns the discovery of a DNA fragmentation factor that triggers nuclear changes during apoptosis. Methods and compositions for making and using the same are disclosed.
2. Description of Related Art
Apoptosis, or programmed cell death, is executed through a “suicide” program that is built into all animal cells (reviewed by White, 1996; Wyllie, 1995). Cells undergoing apoptosis show distinctive morphological changes, including membrane blebbing, cytoplasmic and nuclear condensation, chromatin aggregation and formation of apoptotic bodies (Wyllie, 1980). The biochemical hallmark of apoptosis is the cleavage of chromatin into nucleosomal fragments (Wyllie et al., 1980). Multiple lines of evidence indicate that apoptosis can be triggered by the activation of a family of cysteine proteases with specificity for aspartic acid residues, including CED-3 of
C. elegans
, CPP32/Yama/Apopain of humans, and DCP-1 of Drosophila (Yuan et al., 1993; Xue et al., 1996; Femandes-Alnemri, et al., 1994; Tewari, et al., 1995; Nicholson, et al., 1995; Song, et al., 1997). Recently, these proteins have been designated as caspases (Alnemri et al., 1996).
The most intensively studied apoptotic caspase is caspase-3, previously called CPP32/Yama/Apopain (Fernandes-Alnemri, et al., 1994; Tewari, et al., 1995; Nicholson, et al., 1995). Caspase-3 normally exists in the cytosolic fraction of cells as an inactive precursor that is activated proteolytically when cells are signaled to undergo apoptosis (Schlegel et al., 1996; Wang et al., 1996). Multiple apoptotic signals, including serum withdrawal, activation of Fas, treatment with granzyme B, ionizing radiation, and a variety of pharmacological agents, activate caspase-3 (Chinnaiyan et al., 1996; Darmon, et al., 1996; Datta et al., 1996, I997; Erhardt and Cooper, 1996; Hasegawa et al., 1996; Jacobsen et al., 1996; Martin et al., 1996; Schlegel et al, 1996).
A caspase-3-specific tetrapeptide inhibitor, Ac-DEVD-CHO, can abolish the ability of cytosol from apoptotic cells to induce apoptosis in normal nuclei and block the initiation of the cellular apoptotic program m response to apoptotic stimuli (Nicholson et al., 1995; Dubrez, et al., 1996; Jacobsen et al., 1996). Deletion of caspase-3 from the mouse genome through homologous recombination results in excessive accumulation of neuronal cells, owing to a lack of apoptosis in the brain (Kuida et al., 1996). Addition of active caspase-3 to normal cytosol activates the apoptotic program (Enari et al., 1996). These data indicate that caspase-3 is both necessary and sufficient to trigger apoptosis.
The identified substrates of caspase-3 include poly(ADP-ribose) polymerase (PARP) (Tewari et al., 1995; Nicholson et al., 1995), sterol-regulatory element binding proteins (SREBPs) (Wang et al., 1995; 1996), the U1 associated 70 kDa protein (Caciola-Rosen et al., 1996), D4GDI (Na et al., 1996), huntingtin (Goldberg et al., 1996), and the DNA-dependent protein Kinase (Casciola-Rosen et al., 1996; Song et al., 1996). It is not known whether the cleavage of any of these substrates plays a causal role in apoptosis.
Given that apoptosis is tightly regulated and has been linked to pathways that are dysregulated in a variety of diseases including cancer, it is important to identify mechanisms by which to control this process.
SUMMARY OF THE INVENTION
The present invention is directed towards the identification of factors involved in apoptosis. Thus in a preferred embodiment, there is provided an isolated polypeptide encoding a DFF40 DNA fragmentation factor. In particularly preferred embodiment, the DNA fragmentation factor has the amino acid sequence as set forth in SEQ ID NO:2. Also provided by the present invention is an isolated peptide having between about 10 and about 50 consecutive residues of a DFF40 DNA fragmentation factor. In certain defined aspects, the DNA fragmentation factor has an amino acid sequence of about 10 to about 50 consecutive residues of SEQ ID NO:2. In particular embodiments, the peptide is conjugated to a carrier molecule. In preferred embodiments, the carrier molecule is selected from the group consisting of KLH and BSA.
The present invention further provides a monoclonal antibody that binds immunologically to a DFF40 DNA fragmentation factor. In certain embodiments, the antibody does not bind immunologically to other human polypeptides. In particular embodiments, the antibody further comprises a detectable label. The detectable label may be selected from the group consisting of a fluorescent label, a chemiluminescent label, a radiolabel and an enzyme.
The present invention contemplates a hybridoma cell that produces a monoclonal antibody that binds immunologically to a DFF40 DNA fragmentation factor. In preferred aspects the antibody does not bind immunologically to other human polypeptides.
Also contemplated is a polyclonal antisera, antibodies of which bind immunologically to a DFF40 DNA fragmentation factor. In defined embodiments, the antisera may be derived from an animal other than a human.
Also provided by the present invention is an isolated nucleic acid comprising a region, or the complement thereof, encoding a DFF40 DNA fragmentation factor or an allelic variant thereof. In a preferred embodiment, the DFF40 DNA fragmentation factor is human. In other preferred embodiments, the DNA fragmentation factor has the amino acid sequence of SEQ ID NO:2. In other preferred embodiments, the nucleic acid sequence comprises the coding region having the sequence of SEQ ID NO:1 or the complement thereof. It is contemplated that the nucleic acid may be selected from the group consisting of genomic DNA, complementary DNA and RNA.
In particularly preferred embodiments, the nucleic acid is a complementary DNA and further comprises a promoter operably linked to the region, or the complement thereof, encoding the DNA fragmentation factor. In further embodiments, the nucleic acid further comprises a polyadenylation signal operably linked to the region encoding the DNA fragmentation factor. In still further embodiments, the nucleic acid farther comprises an origin of replication. In other aspects of this embodiment, the nucleic acid may be defined as a viral vector selected from the group consisting of retrovirus, adenovirus, herpesvirus, vaccinia virus and adeno-associated virus. In particularly preferred embodiments, the nucleic acid is packaged in a virus particle. In other alternative embodiments, the nucleic acid is packaged in a liposome.
The present invention further provides an isolated oligonucleotide of between about 15 and about 50 consecutive bases of a nucleic acid, or complement thereof, encoding a DFF40 DNA fragmentation factor. In preferred embodiments, the DNA fragmentation factor is human. In particularly preferred embodiments, the nucleic acid is the coding region of SEQ ID NO:1. In other preferred embodiments, the oligonucleotide is about 15 bases in length, is about 17 bases in length is about 20 bases in length is about 25 bases in length, is about 50 bases in length. Of course longer oligonucleotides also are contemplated.
Another aspect of the present invention provides a plasmid construct comprising a first nucleic acid encoding a DFF40 DNA fragmentation factor. In particular embodiments, the construct further comprises a first promoter active in eukaryotic cells positioned 5′ to the first nucleic acid. In yet additional embodiments, the construct, further comprises a second nucleic acid encoding a DFF45 DNA fragmentation factor. In yet another embodiment the construct further comprises an internal ribosome entry site (IRES), wherein the IRES is positioned 3′ to the upstream nucleic acid and 5′ to the downstream nucleic acid. In other embodiments, the construct further comprises a second promoter functional in eukaryotic cells, wherein the second promoter is positioned 5′ to the second nucleic acid.
It is contem
Liu Xuesong
Wang Xiaodong
Board of Regents , The University of Texas System
Fulbright & Jaworski L.L.P.
Moore William W.
Nashed Nashaat
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