| Co-Investigator(Kenkyū-buntansha) |
MOMOTA Ryusuke Okayama University, Dept.of Mol.Biol.& Biochem.Instructor, 医学部, 助手 (80263557)
OOHASHI Toshitaka Okayama University, Dept.of Mol.Biol.& Biochem.Assistant Professor, 医学部, 助手 (50194262)
YOSHIOKA Hidekatsu Okayama University, Dept.of Mol.Biol.& Biochem.Associate Professor, 医学部, 教授 (00222430)
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| Research Abstract |
To characterize the structure of mouse col4a6 gene, we screened mouse 129 genomic library and isolated a fragment of TSg6, approximately 18.3kb in size. Fine analysis of the genomic fragment and comparison of the structure to that of the cDNA revealed that alternative transcripts that are observed in the human gene do not exist when the mouse is transcribed. In order to create null mouse for col4a6 gene, we manipulated genomic fragment and inserted NcoR gene into the exon 2 of col4a6 gene. The vector was transfected into the R1-Es cells derived from 129v mouse and positive ES cells screened by G418. The positive ES cells were inserted into blastocysts, then the blastocysts were moved uterus of pseudopregnant mice. Chimera mice, heterozygotes and homozygotes are now being checked if any phenotypes are noticed. When homozygote mice were stained by a6 specific monoclonal antibodies, no positive signals was obtained from any tissues, indicating that a6 polypeptide is not expressed in the m
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ice.
While collagen IV molecules composed of alpha1 and alpha2 chains are broadly distributed, molecules comprising combinations of the other four chains, alpha3-alpha6, are important components of specialized basement membranes. The precise chain composition of triple-helical molecules assembled from the alpha3-alpha6 chains is not entirely clear, but it is hypothesized that alpha3-alpha5 chains and alpha5 and alpha6 chains form heterotrimeric molecules. Several pieces of evidence indicate that alpha3/alpha4/alpha5 molecules and alpha5/alpha6 molecules are components of the basement membrane network.
We identified an DL/AS deletion and determined the breakpoint wequences of the Japanese case. The results demonstrated that a deletion clininates the first coding exon of COL4A5 and the first two of COL4A6. They also showed that the breakpoints share the same sequence, which is in turn closely homologous to the consensuses of topoisomerases I and II.Additional DNA evidence suggestetd that the male patient is a somatic mosaic for the mutation. Immunohistochemical analysis using alpha chain-specific monoclonal antibodies supportedthis conclusion in that they revealed the absence of the alpha5 (IV) and alpha6 (IV) collagen chains in most but not all of basement membranes of the smooth muscle cell tumor. This study is greatly relevant to the understanding of DL pathogenesis and its etiology. Less
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