Genes encoding several poly(ADP-ribose) glycohydrolase (PARG) enzymes, the proteins and fragments thereof, and antibodies immunoreactive therewith

Abstract

The isolation and characterization of cDNAs encoding poly(ADP-ribose) glycohydrolase (PARG) enzymes and the amino acid sequences of PARGs from several species are described. PARG is involved in the cellular response to DNA damage and its proper function is associated with the body's response to neoplastic disorder inducing agents and oxidative stress. Expression vectors containing the cDNAs and cells transformed with the vectors are described. Probes and primers that hybridize with the cDNAs are described. Expression of the cDNA in E. coli results in an enzymatically active protein of about 111 kDa and an active fragment of about 59 kDa. Methods for inhibiting PARG expression or overexpressing PARG in a subject for therapeutic benefit are described. Exemplary of PARG inhibitors are anti-sense oligonucleotides. The invention has implications for treatment of neoplastic disorder, heart attack, stroke, and neurodegenerative diseases. Methods for detecting a mutant PARG allele are also described. Antibodies immunoreactive with PARGs and fragments thereof are described.

1 - 35 . (canceled)  
     
     
         36 . A method of preventing, treating, or ameliorating a disease condition in an individual in need thereof comprising administering a therapeutically effective amount of a PARG modulator to the individual.  
     
     
         37 . The method of  claim 36 , wherein the disease state is a neoplastic disorder, a myocardial infarction, a vascular stroke or a neurodegenerative disorder.  
     
     
         38 . The method of  claim 36 , wherein the PARG modulator is an anti-sense oligonucleotide, which hybridizes in-vivo to messenger RNA encoded by a PARG gene.  
     
     
         39 . The method of  claim 36  wherein the PARG modulator is a vector which expresses an antisense nucleotide message.  
     
     
         40 . (canceled)  
     
     
         41 . A method of identifying a mutant PARG allele in an individual comprising: 
 (i) obtaining genomic material from the individual;    (ii) digesting the genomic material with a restriction enzyme having a recognition site inclusive of said mutant allele;    (iii) fractionating the restriction fragments obtained from said digesting; and    (iv) comparing the fractionation pattern with that obtained for a normal allele, thereby determining the presence of absence of the mutant allele.    
     
     
         42 . The method of  claim 41 , wherein said fractionation is performed with electrophoresis.  
     
     
         43 . A method of screening candidate molecules for PARG modulating activity, comprising the steps of: 
 providing a purified PARG enzyme;    assaying the enzyme in the presence of a candidate molecule to be screened; and    comparing the activity of the PARG enzyme in the presence of the molecule to the activity of the PARG enzyme in the absence of the molecule.    
     
     
         44 . A method for gene therapy comprising the step of delivering to a cell to be treated an oligonucleotide having a sequenc complementary to at least a portion of a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.  
     
     
         45 . The method of  claim 44  wherein the oligonucleotide has a sequence complementary to a sequence encoding a C-terminal portion of a PARG enzyme.  
     
     
         46 . The method of  claim 44 , wherein the oligonucleotide is an RNA and further comprises a ribozyme.  
     
     
         47 . A method of sensitizing a cell to a chemotherapeutic agent, comprising the step of contacting the cell with a molecule that modulates an enzymatic activity of a PARG enzyme.  
     
     
         48 . The method of  claim 47 , wherein the molecule is an oligonucleotide having a sequence complementary to at least a portion of a polynucleotide encoding a PARG enzyme.  
     
     
         49 . The method of  claim 47 , wherein the oligonucleotide has a sequence complementary to a sequence encoding a C-terminal portion of a PARG enzyme.  
     
     
         50 . The method of  claim 47 , wherein the oligonucleotide further comprises a ribozyme.  
     
     
         51 . A method of treating a diseased cell characterized by the presence of DNA strand breaks, comprising the step of contacting the cell with a molecule that modulates an enzymatic activity of a PARG enzyme.  
     
     
         52 . The method of  claim 51 , wherein the molecule is an oligonucleotide having a sequence complementary to at least a portion of a polynucleotide encoding a PARG enzyme.  
     
     
         53 . The method of  claim 51 , wherein the oligonucleotide has a sequence complementary to a sequence encoding a C-terminal portion of a PARG enzyme.  
     
     
         54 . The method of  claim 51 , wherein the oligonucleotide further comprises a ribozyme.  
     
     
         55 - 58 . (canceled)  
     
     
         59 . A pharmaceutical composition comprising an nucleic acid molecule having a sequence complementary to at least a portion of a polynucleotide encoding a PARG enzyme.  
     
     
         60 . The pharmaceutical composition of  claim 59 , wherein the oligonucleotide has a sequence complementary to a sequence encoding a C-terminal portion of a PARG enzyme.  
     
     
         61 . The pharmaceutical composition of  claim 59 , wherein the oligonucleotide further comprises a ribozyme.  
     
     
         62 . A transgenic knockout mouse comprising a homozygous disruption in its endogenous PARG gene, wherein said disruption prevents the expression of a PARG protein, and further wherein the phenotype of said knockout mouse relative to a mouse having a wild type PARG gene comprises: 
 an absence of PARG activity.    
     
     
         63 . The knockout mouse of  claim 62 , wherein the disruption comprises an insertion into a coding region of the PARG gene.  
     
     
         64 . The knockout mouse of  claim 62 , wherein the insertion replaces DNA at the start of the coding region of the PARG gene.  
     
     
         65 . (canceled)  
     
     
         66 . (canceled)