The MF alpha 1 gene of Saccharomyces cerevisiae: genetic mapping and mutational analysis of promoter elements.
The activity and cell-type specificity of the promoter of the MF alpha 1 gene of Saccharomyces cerevisiae were examined by measuring expression of an MF alpha 1-SUC2 gene fusion in MATa, MAT alpha, and MATa/MAT alpha cells. A high level of invertase activity was observed only in MAT alpha cells. Weak expression occurred in MATa cells when the hybrid gene was carried on a multicopy plasmid or on a centromere-containing plasmid, but not when the hybrid gene was integrated at the normal MF alpha 1 locus. Analysis of a set of 5'-deletions of the promoter region of the MF alpha 1-SUC2 gene on the multicopy plasmid indicated that sequences from -354 to -274 upstream of the translational start site were required for high level expression in MAT alpha cells. Smaller internal deletions and insertions within the promoter region of the MF alpha 1-SUC2 gene were inserted into the genome at the normal MF alpha 1 locus. These mutations further delineated four promoter domains important for expression: (1) two 26 bp elements (-365 to -340 and -312 to -287) with imperfect dyad symmetry; (2) a 40 bp segment (-264 to -226) that lies about 120 bp upstream of the TATA box; and (3) the TATA box itself (-128 to -122). The transcriptional start sites of the normal MF alpha 1 promoter and of a mutant lacking the TATA box were determined. The MF alpha 1 locus was mapped to the left arm of chromosome XVI, about 22 cM centromere-proximal to the PEP4 gene.
Flessel
M C
MC
Graduate Group in Microbiology, University of California, Berkeley 94720.
Brake
A J
AJ
Thorner
J
J
D001483:Base Sequence; D015152:Blotting, Northern; D002874:Chromosome Mapping; D004252:DNA Mutational Analysis; D004274:DNA, Recombinant / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000032:analysis / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000032:analysis; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D043324:beta-Fructofuranosidase
D004274:DNA, Recombinant; D012331:RNA, Fungal; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase