International Mammalian Genome Society


The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

E32 Isolation, Characterization and Analysis of p140mRhoGAP

Michael R. Crowley1, Karen E. Slon1,3, Norma J. Nowak2, Kazutoyo Osoegawa2, Pieter DeJong2 and Bonnie B. Asch1. 1Departments of Experimental Pathology and 2Cancer Genetics Roswell Park Cancer Institute, Buffalo, NY, USA; 3Department of Biology, University of Connecticut, Storrs, CT, USA

Endogenous retroviral like elements can be agents of genetic variation within an organism. Expression of retroelements can lead to reintegration of the proviral genome back into the host's genome causing several possible outcomes for the organism. One possible result of reintegration is the overexpression of a gene either directly from the powerful promoter of the retroviral long terminal repeats (LTRs) or indirectly from the LTRs participating as enhancers. BALB/c mice harbor a single endogenous copy of the ecotropic murine leukemia virus (MuLV), an endogenous retroviral element. Overexpression of the ecotropic MuLV in the hormonally induced BALB/c D2 HAN and the resulting D2 tumor has led to reintegration of the provial element into new locations within the mouse genome. Our group isolated several of the novel integration sites from D2 tumors using inverse PCR. Sequence analysis of flanking DNA suggests that the 8 clones isolated are new integrations. Genetic backcross analysis localized 4 of the 8 integrations to mouse chromosomes 13 (two), 2 and 6. Southern anaylsis of the EN31 integration site has demonstrated ecotropic MuLV integration in several D2 tumors and one D2 HAN. Several mouse PAC and BAC clones were isolated for two integration sites (EN21 and EN31). Sequence analysis of subclones of the EN31 integration site uncovered an exon having 93% homology with a partial human cDNA, KIAA0411. Through database searches, RT-PCR and 5' RACE we have identified an 8.4 knt transcript corresponding to the mouse homologue of hKIAA0411. Sequence analysis has identified a 2.9 kb open reading frame which encodes a 963 amino acid protein with RhoGAP and SH3 domains. SDS-PAGE analysis of a His6 tagged bacterially expressed fusion protein resolved a protein of ~140 KDa. We now suggest the name of p140mRhoGAP. Northern analysis showed approximately 2.8 to 7.5 fold overexpression of the mouse gene in D2 tumors compared to normal mammary gland. Several mammary tumors induced via viral (C3H) or hormonal (D1, D2) mechanisms demonstrated overexpression of p140mRhoGAP. Interestingly, DMBA-induced mammary tumors showed no overexpression. p140mRhoGAP was expressed in the virgin mammary gland and at similar levels in mammary glands from pregnant and lactating mice but shows a reduction in involution. The p140mRhoGAP transcript was highly expressed in the mouse brain, but greatly reduced or not detectable in all other tissues analyzed. Dissection of the mouse brain demonstrated a slightly elevated level of expression in the cerebellum compared to the cerebral cortex, and midbrain. Developmentally, expression of p140mRhoGAP was observed at E11.5 (the earlist time point examined) but expression became restriced primarily to the developing brain from E12.5 through birth. Intracellular localization to the membrane was discovered through expression of a p140mRhoGAP/GFP fusion construct in COS-7 cells. The transformation potential of p140mRhoGAP is currently being assessed.

 


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