RESUMO
ShcA and Grb2 are crucial components in signalling by most tyrosine kinase-associated receptors. How ever, it is not clear whether Grb2 bound directly to the receptor is equivalent to Grb2 associated via ShcA. We have used signalling stimulated by the middle T-antigen (MT) of polyoma virus to address this question. The two known Grb2-binding sites from murine ShcA, 313Y and 239/240YY, could functionally replace the MT ShcA-interacting region in transformation assays using Rat2 fibroblasts. This demonstrates that signal output from membrane-bound ShcA requires only these two sequences and the ShcA-binding site in MT does not recruit other signalling molecules. Two standard Grb2-interacting sequences, either from the EGF receptor or the ShcA 313Y region, could not replace the requirement for ShcA binding to MT, indicating an enhanced role for the ShcA 239/240YY motif. Sos1 and the docking protein Gab1 are brought into the MT complex through Grb2 association and this may be more effective using the 239/240YY sequence.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas/metabolismo , Tirosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Membrana Celular/metabolismo , Receptores ErbB/metabolismo , Fibroblastos/metabolismo , Proteína Adaptadora GRB2 , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Ratos , Proteína SOS1/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Domínios de Homologia de srcRESUMO
The SIN3 gene (also known as SDI1) is a negative regulator of the yeast HO gene. Mutations in SIN3 suppress the requirement for the SWI5 activator for expression of the yeast HO gene and change the normal asymmetric pattern of HO expression in mother and daughter cells. Furthermore, the in vitro DNA-binding activity of several DNA-binding proteins is reduced in extracts prepared from sin3 mutants. We have cloned the SIN3 gene and determined that a haploid strain with a SIN3 gene disruption is viable. We determined the sequence of the SIN3 gene, which is predicted to encode a 175-kDa polypeptide with four paired amphipathic helix motifs. These motifs have been identified in the myc family of helix-loop-helix DNA-binding proteins and in the TPR family of regulatory proteins. The SIN3 transcript was mapped, and it was determined that the SIN3 transcript was absent in stationary-phase cells. Immunofluorescence microscopy with anti-SIN3 antibody demonstrated that SIN3 protein was present in nuclei. A comparison of restriction map and sequence data revealed that SIN3 is the same as regulatory genes UME4 and RPD1.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Genes Reguladores , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Conformação Proteica , RNA Mensageiro/genética , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
A DNA-binding protein has been identified from extracts of the budding yeast Saccharomyces cerevisiae which binds to sites present in the promoter regions of a number of yeast genes transcribed by RNA polymerase II, including SIN3 (also known as SDI1), SWI5, CDC9, and TOP1. This protein also binds to a site present in the enhancer for the 35S rRNA gene, which is transcribed by RNA polymerase I, and appears to be identical to the previously described REB1 protein (B. E. Morrow, S. P. Johnson, and J. R. Warner, J. Biol. Chem. 264:9061-9068, 1989). When oligonucleotides containing a REB1-binding site are placed between the CYC1 upstream activating sequence and TATA box, transcription by RNA polymerase II in vivo is substantially reduced, suggesting that REB1 acts as a repressor of RNA polymerase II transcription. The in vitro levels of the REB1 DNA-binding activity are reduced in extracts prepared from strains bearing a mutation in the SIN3 gene. A greater reduction in REB1 activity is observed if the sin3 mutant strain is grown in media containing galactose as a carbon source.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Transcrição Gênica , Sequência de Bases , Proteínas de Ligação a DNA/isolamento & purificação , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , RNA Polimerase II/metabolismo , RNA Ribossômico/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
A computerized statistical quality control system has been developed for use in the industrial hygiene chemistry laboratory. The system is practical and sufficiently flexible to allow for multiple analytes, concentrations, replicate sizes and sample types. The computerized system provides an immediate evaluation of the quality of analytical results and produces automatically simple but informative accuracy and precision quality control charts.
