A METHOD OF DIRECTED RANDOM MUTAGENESIS OF THE YEAST CHROMOSOME SHOWSTHAT THE ISO-1-CYTOCHROME-C HEME LIGAND HIS18 IS ESSENTIAL

A method to perform site-directed random mutagenesis directly in the y east chromosomal DNA at the iso-l-cytochrome c-encoding gene locus (CY C1) is described. To test the effectiveness of the random mutagenesis procedure, the heme ligand His(18) was mutated to Ala (H18A), renderin g cytochrome c (Cyc) nonfunctional. Random mutagenesis was performed b y transforming yeast cells with a synthetic oligodeoxyribonucleotide ( oligo) that randomizes the codon for His(18). The transformed cells we re then selected for reversion to a functional Cyc on selective media. Ten functional mutants were recovered, all of which had integrated th e synthetic oligo. Sequencing showed that five of the recovered mutant s carried the His codon, CAU, and five mutants contained the His codon , CAC. Because Arg had previously been found as a heme ligand, this mu tant was produced by standard techniques and integrated into the yeast chromosome. These yeast did not produce a hole cytochrome c that was detectable by low-temperature spectroscopy. To develop a selection for nonfunctional Cyc, competent yeast (which lack the ability to synthes ize tryptophan) were cotransformed with a plasmid carrying the TRP1 ge ne and the random oligo, and were plated on media lacking tryptophan. Of the 1200 colonies that grew, 120 tested negative for the integratio n of the random oligo, demonstrating that this particular selection fo r nonfunctional protein is not feasible. A method is thus described fo r directed, random mutagenesis directly in the yeast chromosome that c an be used to probe structure/function relationships in Cyc. Only His can act as a heme ligand at position 18, using the functional selectio n described here.