RESUMO
BACKGROUND: Recognition that mutation of the protein nephrin, encoded by the NPHS1 gene, singly results in the cellular alterations that result in foot process effacement, and nephrotic range proteinuria emphasizes the pivotal role that this protein plays in regulating glomerular filter integrity. This article reports the development of reagents necessary to study the biology of nephrin in mouse, and describes the initial characterization of the nephrin protein. METHODS: A cDNA including the full-length mouse nephrin open reading frame was cloned and sequenced. Immuno-affinity purified polyclonal antiserum directed against the cytoplasmic domain of mouse nephrin was developed. RESULTS: Nephrin identified in mouse glomerular extract was found to be a glycoprotein with an apparent molecular mass of 185 kDa. As detected by indirect immunofluorescence microscopy and immunogold electron microscopy, nephrin was located only in visceral glomerular epithelial cells, where it was targeted to intercellular junctions of mature podocyte foot processes. In developing glomeruli of newborn mouse, antinephrin immunolocalized to the earliest slit pore regions between differentiating podocytes, sites where slit diaphragms first become visible. CONCLUSION: As a putative cell adhesion molecule of the immunoglobulin superfamily, nephrin likely participates in cell-cell interactions between podocyte foot processes and may represent a component of the slit diaphragm.
Assuntos
Células Epiteliais/química , Junções Intercelulares/química , Glomérulos Renais/citologia , Proteínas/análise , Proteínas/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Northern Blotting , Comunicação Celular/fisiologia , Clonagem Molecular , DNA Complementar , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Imunofluorescência , Expressão Gênica/fisiologia , Glomérulos Renais/crescimento & desenvolvimento , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Dados de Sequência Molecular , RNA Mensageiro/análise , Homologia de Sequência de Aminoácidos , Transcrição Gênica/fisiologiaRESUMO
Induced production of arginase (CAR1) enzyme activity and steady-state CAR1 mRNA in Saccharomyces cerevisiae requires wild-type ARG80/ARGRI and ARG81/ARGRII gene products. We demonstrate here that these gene products, along with that of the MCM1 gene, are required for the inducer-dependent USAI-A, UASI-B and UASI-C elements to function but they are not required for operation of inducer-independent CAR1 UASC1 or UASC2. Through the use of single and multiple point mutations, the CAR1 UASI-B and UASI-C elements were demonstrated to be at least 23 bp in length. Moreover, simultaneous mutation of both ends of an elements gave stronger phenotypes than mutations at either end. The center of the element was more sensitive to mutation than were the ends.
Assuntos
Arginase/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Genes Reguladores/genética , Sequências Reguladoras de Ácido Nucleico/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Deleção de Genes , Genes Reporter , Óperon Lac , Proteína 1 de Manutenção de Minicromossomo , Dados de Sequência Molecular , Mutagênese , Fenótipo , Proteínas Recombinantes de Fusão/biossíntese , Sequências Repetitivas de Ácido Nucleico , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Arginase (CAR1) gene expression in Saccharomyces cerevisiae is induced by arginine. The 5' regulatory region of CAR1 contains four separable regulatory elements--two inducer-independent upstream activation sequences (UASs) (UASC1 and UASC2), an inducer-dependent UAS (UASI), and an upstream repression sequence (URS1) which negatively regulates CAR1 and many other yeast genes. Here we demonstrate that three homologous DNA sequences originally reported to be present in the inducer-responsive UASI are in fact three exchangeable elements (UASI-A, UASI-B, and UASI-C). Although two of these elements, either the same or different ones, are required for transcriptional activation to occur, all three are required for maximal levels of induction. The elements operate in all orientations relative to one another and to the TATA sequence. All three UASI elements bind protein(s); protein binding does not require arginine or overproduction of any of the putative arginine pathway regulatory proteins. The UASI-protein complex was also observed even when extracts were derived from arg80/argRI or arg81/argRII deletion mutants. Similar sequences situated upstream of ARG5,6 and ARG3 and reported to negatively regulate their expression are able to functionally substitute for the CAR1 UASI elements and mediate reporter gene expression.
