Duplicate upstream activating sequences in the promoter region of the Saccharomyces cerevisiae GAL7 gene.

Abstract

We constructed a series of deletions in the 5' noncoding region of the Saccharomyces cerevisiae GAL7 gene, fused them to the Escherichia coli gene lacZ, and introduced them into yeasts by using a multicopy vector. We then studied the effect of the deletions on P-galactosidase synthesis directed by the gene fusions in media with various carbon sources. This analysis identified a TATA box and two upstream activating sequences as necessary elements for galactose-controlled GAL7 transcription. Two upstream activating sequences exhibiting 71% homology with each other were located 255 and 168 base pairs, respectively, upstream of the GAL7 transcription start point. Each sequence consists of 21 base pairs, displaying an approximate rotational symmetry with a core consensus sequence of GAA--GCT CT -{CCG. At least one of the two sequences is required for galactose induction and also for glucose repression of the GAL7'-lac'Z gene. Analysis with host regulatory mutants Agal4 and Agal80 suggests that these sequences are the site at which the GAL4 product exerts its action to activate the GAL7 gene. We also observed that a deletion lacking both upstreamn activation sequences allowed the gene fusion to be expressed in the absence of galactose at about 10% of the fully induced level of the intact fusion. This constitutive expression depended on the presence of the TATA box of GAL7 in cis but not on a functional GAL4 gene. The level of the uncontrolled expression was decreased by increasing the distance between the TATA box and the pBR322 sequence in the vector plasmid.

Identification of ras-related, YPT family genes in Schizosaccharomyces pombe.

“…In addition to the S.cerevisiae URA3 gene retained by the original GAL7 expression vector pMT34 (Tajima et al, 1986), a S.cerevisiae LEU2 cassette was (Schmitt et al, 1986). However, neither pMT(ypt3) nor pMT(ypt1)R could rescue S. cerevisiae yptl disruptants (Table II).…”

A family of hexosephosphate mutases in Saccharomyces cerevisiae

“…Fragments of genomic DNA with sizes between 3.0-3.4 kb and 4.3-4.7 kb, respectively, corresponding to the size of the expected PGM2 gene (Oh and Hopper, 1990) were cloned into plasmid YEplacl81. Both gene li- (Struhl, 1986), transcription-termination signals (Zaret and Sherman, 1982), and putative binding sites for the GAL4 activator (West et al, 1984;Tajima et al, 1986) and MIGl repressor proteins (Nehlin et al, 1991;Griggs and Johnston, 1991) are printed in bold and are underlined. The active-site serine residues are marked by asterisks.…”

Use of yeast nuclear DNA sequences to define the mitochondrial RNA polymerase promoter in vitro.

“…3. This sequence overlaps the RNA polymerase II TATA box promoter element reported by Tajima et al (43). This sequence, -ATAAAAGCA-, is also present in the upstream region of the MEL] gene, and transcription runoff experiments demonstrated that the mitochondrial RNA polymerase selectively initiates transcription from the MELI promoter sequence.…”

“…6 and 7 in Table 3. higher levels of HIS3 gene expression (41). To test the effectiveness of these plate assays, the strains were also transformed with a YCp19 plasmid bearing the HIS3 gene with a fully functional RNA polymerase II promoter (YCp19-HIS3+) and a YCp19 plasmid bearing the HIS3 gene lacking all promoter elements (YCp19-his3-) (30,43). Previous Northern blot experiments have demonstrated that YCpl9-HIS3+ produces a strong HIS3 band corresponding to an estimated 5 to 10 mRNA molecules per cell, whereas YCpl9-his3-produces very low levels of HIS3 mRNA (28).…”