A method of directed random mutagenesis of the yeast chromosome shows that the iso-1-cytochrome c heme ligand His18 is essential

Abstract

A method to perform site-directed random mutagenesis directly in the yeast chromosomal DNA at the iso-1-cytochrome c-encoding gene locus ( CYCI) is described. To test the effectiveness of the random mutagenesis procedure, the heme ligand His 18 was mutated to Ala (H18A), rendering cytochrome c (Cyc) nonfunctional. Random mutagenesis was performed by transforming yeast cells with a synthetic oligodeoxyribonucleotide (oligo) that randomizes the codon for His 18. The transformed cells were then selected for reversion to a functional Cyc on selective media. Ten functional mutants were recovered, all of which had integrated the synthetic oligo. Sequencing showed that five of the recovered mutants carried the His codon, CAU, and five mutants contained the His codon, CAC. Because Arg had previously been found as a heme ligand, this mutant was produced by standard techniques and integrated into the yeast chromosome. These yeast did not produce a holo cytochrome c that was detectable by low-temperature spectroscopy. To develop a selection for nonfunctional Cyc, competent yeast (which lack the ability to synthesize tryptophan) were cotransformed with a plasmid carrying the TRP1 gene and the random oligo, and were plated on media lacking tryptophan. Of the 1200 colonies that grew, 120 tested negative for the integration of the random oligo, demonstrating that this particular selection for nonfunctional protein is not feasible. A method is thus described for directed, random mutagenesis directly in the yeast chromosome that can be used to probe structure/function relationships in Cyc. Only His can act as a heme ligand at position 18, using the functional selection described here.