RESUMO
SUP2 (SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, intimately involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. In the present study a SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of the temperature-sensitive sup2 mutation of S. cerevisiae. The nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa, exceeding the Sup2 protein of S. cerevisiae by 6 kDa. Like the SUP2 gene product of S. cerevisiae, the Sup2 protein of P. pinus represents a fusion of a unique N-terminal part and a region homologous to EF-1 alpha. The comparison of amino acid sequences of the Sup2 proteins reveals high conservation (76%) of the C-terminal region and low conservation (36%) of the N-terminal part where, in addition, the homologous correspondence is ambiguous. Proteins related to the Sup2 of S. cerevisiae were found in P. pinus and some other yeast species by the immunoblotting technique. The relation between the evolutionary conservation of different regions of the Sup2 protein and their functional significance is discussed.
Assuntos
Proteínas Fúngicas/genética , Genes Supressores , Pichia/genética , Príons , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon/genética , DNA Fúngico/genética , Proteínas Fúngicas/análise , Immunoblotting , Dados de Sequência Molecular , Fatores de Terminação de Peptídeos , Plasmídeos , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Transformação GenéticaRESUMO
SUP2(SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. A SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of temperature-sensitive sup2 mutation of S. cerevisiae. Nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa exceeding the SUP2 protein of S. cerevisiae for 6 kDa. The SUP2 gene product of P. pinus is similar to the Sup2 protein of S. cerevisiae by its structure and includes a highly conservative (76%) C-terminal region homologus to EF-1 alpha and a lowly conservative N-terminal region. The relation between the evolutionary conservativity of different regions of the Sup2 protein and their functional significance is discussed.
Assuntos
Genes Fúngicos , Pichia/genética , Saccharomyces cerevisiae/genética , Supressão Genética , Sequência de Aminoácidos , Sequência de Bases , Códon , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The collection of overlapping lys2 deletions (five in the chromosomal and seven in the plasmid LYS2 gene) is constructed in this work. The deletions overlap the whole coding region of the gene and provide the system for intragenic recombinational mapping of lys2 mutations in one of 14 controlled regions. A portion of these regions can be correlated with the regions on the physical map of LYS2. Mutations in two regions can be easily cloned. The system constructed gives the possibility for the study of intragenic and molecular specificity of mutagenesis.
Assuntos
Genes Fúngicos , Mutação , Saccharomyces cerevisiae/genética , Alelos , Deleção Cromossômica , Cromossomos Fúngicos , Engenharia Genética , Plasmídeos , Mapeamento por RestriçãoRESUMO
Seven mutants of Saccharomyces cerevisiae deficient in production of extracellular glucoamylase have been analyzed. For each of the seven a monogenic pattern of inheriting the mutant phenotype has been observed. The mutations have been shown to map within five different genetic loci, three independent mutations affecting the STA2 locus and the other four residing in four formerly unidentified genes. As expected, the sta2 mutants recover the wild phenotype when transformed with a STA2-bearing multicopy plasmid. Such reversion has also been observed for the transformed stall mutant. Unlike the others, the sta16 mutant is unable to secrete heterologous alpha-amylase encoded by a plasmid-borne DNA fragment. All the mutants have a moderately reduced ability to secrete the invertase and acid phosphatase.
Assuntos
Sequência de Bases , Análise Mutacional de DNA , DNA Fúngico/genética , Saccharomyces cerevisiae/metabolismo , Amido/metabolismo , Genótipo , Saccharomyces cerevisiae/genéticaRESUMO
Red adenine-dependent mutants of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts have been transformed by the plasmid pYE (ADE2) 2 DNA containing ADE2 gene from Saccharomyces cerevisiae. The analysis of two P. guilliermondii Ade+-transformants has revealed the integration of pYE (ADE2)2 sequence into the recipient strain genome and partial restoration of the deficient function.
Assuntos
Genes Fúngicos , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Transformação Genética , Carboxiliases/genética , Marcadores Genéticos , Mutação , Pichia/genética , Plasmídeos , Saccharomyces cerevisiae/enzimologiaRESUMO
A novel Escherichia coli-Saccharomyces cerevisiae shuttle vector lambda MAN78 has been constructed. The vector contains phage lambda 47.1 DNA, Sacch. cerevisiae chromosomal segment with TRP1 gene and the yeast ARS1 replicator. This vector may be propagated as a phage and, similar to parental lambda 47.1, allows direct selection of large DNA inserts (15-24 kbp) in E. coli. lambda MAN78 can efficiently transform LiCl-treated yeast cells (3-5 X 10(3) transformants per 1 microgram DNA). Replication of hybrid molecules in E. coli cells does not influence the ability of the molecules to transform yeast cells and replicate in the cells.
