RESUMO
To investigate physiologic functions and structural correlates for actin capping protein (CP), we analyzed site-directed mutations in CAP1 and CAP2, which encode the alpha and beta subunits of CP in Saccharomyces cerevisiae. Mutations in four different regions caused a loss of CP function in vivo despite the presence of mutant protein in the cells. Mutations in three regions caused a complete loss of all aspects of function, including the actin distribution, viability with sac6, and localization of CP to actin cortical patches. Mutation of the fourth region led to partial loss of only one function-formation of actin cables. Some mutations retained function and exhibited the complete wild-type phenotype, and some mutations led to a complete loss of protein and therefore loss of function. The simplest hypothesis that can explain these results is that a single biochemical property is necessary for all in vivo functions. This biochemical property is most likely binding to actin filaments, because the nonfunctional mutant CPs no longer co-localize with actin filaments in vivo and because direct binding of CP to actin filaments has been well established by studies with purified proteins in vitro. More complex hypotheses, involving the existence of additional biochemical properties important for function, cannot be excluded by this analysis.
Assuntos
Proteínas dos Microfilamentos/genética , Saccharomyces cerevisiae/genética , Proteínas de Capeamento de Actina , Fatores de Despolimerização de Actina , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Destrina , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/fisiologia , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Terminação Traducional da Cadeia Peptídica , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/fisiologia , Relação Estrutura-AtividadeRESUMO
The ADE1 gene of the yeast Pichia methanolica encodes phosphoribosyl-5-aminoimidazole-carboxylase (AIRC, EC 4.1.1.21), which is involved in purine biosynthesis. The gene was cloned by complementation of an ade2 mutation in Saccharomyces cerevisiae and a 3077 nucleotide DNA fragment was sequenced. The sequence possessed a single open reading frame, corresponding to a 543 amino acid sequence. The sequence of this putative protein has been compared to the proteins of homologous genes from S. cerevisiae, Schizosaccharomyces pombe, Escherichia coli, chicken and man. The analysis revealed remarkable homology between yeast AIRCs, while for other proteins homology was limited to defined regions.
Assuntos
Carboxiliases/genética , Genes Fúngicos , Pichia/enzimologia , Pichia/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Fúngico/genética , Humanos , Dados de Sequência Molecular , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Restriction fragments of recombinant plasmids containing a proviral sequence of Rous sarcoma virus (RSV) were Southern hybridized with double-stranded (ds) RNA isolated from the cells transformed with RSV. Hybridization data show that the major subpopulation of dsRNA molecules is homologous to the 5'-end region of the viral genome including the leader sequence. We have analysed the RNAs of RSV-transformed cells by the Northern procedure hybridizing them with the proviral fragment containing double long terminal repeats. The results demonstrate that the 14-16S RNA fraction is enriched in sequences which are homologous to the proviral end regions. We consider this RNA fraction to be homologous to the 5'-terminal region of the viral genome and (or) to its antisense strand.
Assuntos
Vírus do Sarcoma Aviário/genética , Sequência de Bases , Transformação Celular Viral , RNA de Cadeia Dupla/genética , RNA Viral/genética , Homologia de Sequência do Ácido Nucleico , Animais , Embrião de Galinha , Enzimas de Restrição do DNA , Hibridização de Ácido Nucleico , PlasmídeosRESUMO
Several methods were used for isolation of double-stranded (ds) RNA from the cytoplasm of Rous sarcoma virus-transformed chick embryo cells. The dsRNA was shown to have a high melting temperature (82.5 degrees C) in 0.16 M phosphate buffer (pH 6.8), which shifted to more than 90 degrees C after RNase treatment. The size of a single strand was approximately 1300-1600 nucleotides and RNase-resistant fragments were 50-250 nucleotides long. Double-stranded RNA formed hybrids with the labeled genomic RSV RNA RNA so that the major subpopulation of the dsRNA hybridized to 6-10% of RSV RNA and the minor subpopulation -- to 90-94% of RSV RNA. It was suggested that this large subpopulation of dsRNA was abundant in sequences homologous to proviral end fragments as judged by Southern procedure. The data are discussed by considering the analogy between retroviral proviruses and mobile genetic elements.
