HETEROLOGOUS GENE EXPRESSION IN THE YEAST SACCHAROMYCES CEREVISIAE

Efficient expression in the yeast Saccharomyces cerevisiae of two heterologous genes, one prokaryotic the other eukaryotic was accomplished only after the 5' region of each gene was supplemented with appropriate yeast DNA sequences. The yeast sequences contain signals that initiate the translation and/or transcription mechanisms in yeast. Once initiation of these mechanisms occurs, "read-through" into the coding regions of the foreign DNA ensues, yielding a functional gene product.;The Herpes Simplex Virus type 1 (HSV-1) thymidine kinase (TK) gene present on a 3.2 kilobase (kb) BamH1 endonuclease restriction fragment and when cloned into the appropriate vehicle, will not initiate transcription of the TK coding region in yeast. Consequently, no functional gene product is produced. A functional HSV-1 TK gene product was produced in Saccharomyces cerevisiae when the coding region of the TK gene was placed under transcription/translational control of a yeast promoter region. Expression of the TK gene is due to the formation of a protein fusion between HSV-1 DNA sequences containing the entire coding region of the TK gene with remnants of an unidentified yeast gene. The remaining portion of the yeast gene includes a 5' noncoding region responsible for transcription initiation and 336 base pairs (bps) of a coding region. Translation initiation occurs at the first AUG into the yeast coding region and continues on into the TK coding region. The protein fusion consists of both TK gene product plus approximately 113 extraneous amino acids located at the amino terminus end derived from the yeast coding region, and a portion of the 5' noncoding region of the TK gene.;The Escherichia coli gene leuB which codes for (BETA)-isopropylmalate dehydrogenase will express a functional gene product and complement the yeast leu2 mutation, though weakly, when the gene is under the control of the E. coli leu operon promoter. Efficient complementation of the corresponding leu2 yeast mutation occurred after in vitro DNase digestion of plasmid bound leuB and the yeast HIS3 genes, followed by DNA ligation which placed the 5' end of the yeast HIS3 gene immediately adjacent to the coding region of the leuB gene. Two structurally distinct classes of plasmid were isolated consisting of varying portions of the yeast HIS3 gene which contribute DNA sequences responsible for initiation of leuB gene expression in yeast.