The expression in yeast of the Escherichia coli galK gene on CYCI :: galK fusion plasmids

Abstract

A set of gene fusions have been constructed between the transcriptional and translational initiation signals of the yeast CYC1 gene, encoding iso-l-cytochrome c, and the coding sequence of the Escherichia coli galK gene encoding galactokinase. These fusions are contained on plasmids which have both yeast and E. coli replication origins and selectable markers and, therefore, can be used to transform either yeast or E. coli cells. When galactokinase-deficient (gall⁻) yeasts were transformed with these plasmids the resulting Gal⁺ transformants were heterogeneous with respect to their galactokinase levels. The galactokinase levels in all were subject to glucose repression, characteristic of the transcriptional regulation of the CYCI gene. The fusion points for representative plasmids were determined by DNA sequence analysis, and from these data, the differential expression of the galK gene could be explained. One fusion plasmid, YRpRl, which gave the highest level of galK expression, was characterized further. As an additional demonstration that galactokinase expression from the fusion was under CYC1 transcriptional control, a cis-dominant, CYC1-linked mutation known to drastically reduce CYC1 gene transcription was introduced into YRpR1 and shown to similarly effect galK expression. The galK mRNA produced from the fused gene of YCpRl, a centromere-containing derivative of YRpR1, consisted of the mRNA leader sequence plus the first four codons of the CYC1 gene, the galK coding sequence, then the remainder of the CYCI coding sequence and the 175 nucleotide non-translated 3' sequence. As a demonstration of the usefulness of these plasmids for the selection of regulatory mutants, two mutants capable of greatly enhanced levels of galactokinase expression were isolated. Preliminary characterization of these mutations indicates that they likewise affect the expression of the chromosomal CYC1 gene.