Using adaptive mutagenesis system for identification of early genes encoding de novo purine biosynthesis in methylotrophic yeast Pichia methanolica MH4

Abstract

A collection of spontaneous "Roman's mutants" (1654 mutants) for early genes of purine biosynthesis PUR1-PUR5 was obtained from 16 parental ade1(pur6) and ade2(pur7) strains of the methylotrophic yeast Pichia methanolica. Two genes, bifunctiional ADE7,4(PUR2,5) and ADES(PUR4), were identified earlier. For identification of the two remaining early genes (ADE3 and ADE8), a novel approach was used: a comparison of spectra of spontaneous Roman's mutants and relative sizes of genes (with regard to the length of polypeptides in amino acid residues). Significant correlation between relative sizes of genes and a proportion of mutants in the spectrum was shown in yeast Saccharomyces cerevisiae (according to analysis of data from the literature). Data on average polypeptide product lengths of genes encoding the first five steps of purine biosynthesis in 22 species of lower eucaryotes were obtained using the PubMed database. A correlation between the spectrum of spontaneous Roman's mutants and relative gene sizes was also shown experimentally in P. methanolica yeast. On the strength of this evidence, the gene ADE3 was identified as PUR1 and gene ADE8 as PUR3. Spontaneous mutagenesis producing Roman's mutants most probably is the result of transcription activity of early genes, which essentially agrees with experimental data on the correlation between the mutant spectrum and gene sizes. It was supposed that the adaptive mutagenesis, i.e., an enhancement of early genes transcription activity in response to stress, the accumulation of toxic metabolites (AIR or CAIR) as a result of the block of 6-7 stages of purine biosynthesis underlies the Roman's effect.