Temperature-sensitive variants of Saccharomyces cerevisiae iso-1-cytochrome c produced by random mutagenesis of codons 43 to 54.
In vitro random mutagenesis within the CYC1 gene from the yeast Saccharomyces cerevisiae was used to produce a library of mutants encompassing codons 43 to 54 of iso-1-cytochrome c. This region consists of an evolutionarily conserved structure within an evolutionarily diverse sequence. The library, on a low-copy-number yeast shuttle phagemid, was introduced into a yeast strain lacking cytochrome c. The ability of transformants harboring a functional cytochrome c to grow on the non-fermentable carbon source glycerol at 30 degrees C and 37 degrees C was used to determine the phenotype of nearly 1000 transformants. Approximately 90% of the missense mutants present in the library give rise to the wild-type phenotype, 7% result in the temperature-sensitive (Cycts) phenotype, and 3% give rise to the non-functional (Cyc-) phenotype. Phagemids from 20 Cycts and 30 Cyc- transformants were subjected to DNA sequence analysis. All the mutations occur within the targeted region. One-third of the mutants from Cyc- transformants and all the mutants from Cycts transformants are missense mutants. The remaining mutants from Cyc- transformants are nonsense or frame-shift mutants. Missense mutations within the codons for Gly45, Tyr46, Thr49, Asn52 or Ile53 alone are sufficient to produce temperature-sensitive behavior both in vivo and in the variant proteins. The deduced amino acid substitutions correlate remarkably well with side-chain dynamics, secondary structure and tertiary structure of the wild-type protein.
Wang
X H
XH
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
Pielak
G J
GJ
D001483:Base Sequence; D003062:Codon; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D045304:Cytochromes c; D004247:DNA; D003854:Deoxyribonucleotides; D015723:Gene Library; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014170:Transformation, Genetic
C014059:CYC1 protein, S cerevisiae; D003062:Codon; D003574:Cytochrome c Group; D003854:Deoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D004247:DNA