Functional Analysis of Collagen IV by Gene Targeting

Col 4a6null mice were created by introducing Neo I D1R菜单D1 gene into exon II of col4a6gene.We analyzed if the gene is transcribed and translated intoα6(IV)polypeptide by in site hybridization，Northern-blot hybridization，and immunohistochemical staining usingαchain-specific monoclonal antibodies.The results demonstrated that theα6(IV)polypeptide was not stained at all in any tissues or organs in col4a6null mice。We also analyzed if the mice have some phenotypes，but we have not detected ant differences between wild type and col4a6null mice so far.We predicted the presence of the three molecular forms of type IV collagen；[α1(IV)]2α2(IV)，α3(IV)α4(IV)α5(IV)，and[α5(IV)]2α6α(IV)by use of double staining of the monoclonal antibodies specific forα(IV)chains.The[α1(IV)]2α2(IV)form is present in all basement membranes，whereas theα3(IV)α4(IV)α5(IV)form is localized in glomeruler and alveolar basement membranes and the[α5(IV)]2α6(IV)form is in the Bowman‘s capsules of the kidney and dermal basement membrane regions。Heterogeneous distribution of the three molecular forms indicated that they may have specific roles in different basement membranes。To describe the biological roles of the molecules，we should think of how the three molecules are incorporated into the supramolecular aggregates of each basement membrane.We identified and characterized the breakpoint sequences of the deletion of DNA from a patient of diffuse leiomyomatosis associated with Alport syndrome.The results demonstrated that a deletion eliminates the first coding exon of COL4A5and 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 suggested that the male patient is a somatic for the mutation。This study is greatly relevant to the understanding of DL pathogenesis and its etiology.

Col4a6 null mice were created by introducing NeoィイD1RィエD1 gene into exon II of col4a6 gene. We analyzed if the gene is transcribed and translated into α6(IV) polypeptide by in site hybridization, Northern-blot hybridization, and immunohistochemical staining using α chain-specific monoclonal antibodies. The results demonstrated that the α6(IV) polypeptide was not stained at all in any tissues or organs in col4a6 null mice. We also analyzed if the mice have some phenotypes, but we have not detected ant differences between wild type and col4a6 null mice so far.We predicted the presence of the three molecular forms of type IV collagen ; [α1(IV)]2α2(IV), α3(IV)α4(IV)α5(IV), and [α5(IV)]2α6α(IV) by use of double staining of the monoclonal antibodies specific for α(IV) chains. The [α1(IV)]2α2(IV) form is present in all basement membranes, whereas the α3(IV)α4(IV)α5(IV) form is localized in glomeruler and alveolar basement membranes and the [α5(IV)]2α6(IV) form is in the Bowman's capsules of the kidney and dermal basement membrane regions. Heterogeneous distribution of the three molecular forms indicated that they may have specific roles in different basement membranes. To describe the biological roles of the molecules, we should think of how the three molecules are incorporated into the supramolecular aggregates of each basement membrane.We identified and characterized the breakpoint sequences of the deletion of DNA from a patient of diffuse leiomyomatosis associated with Alport syndrome. The results demonstrated that a deletion eliminates 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 suggested that the male patient is a somatic for the mutation. This study is greatly relevant to the understanding of DL pathogenesis and its etiology.