Nudeotlde sequence of the transcriptional initiation region of the yeast GAL 7 gene

The GAL7 gene of Saccharomyces cerevlsiae encodes Gal-l-P uridylyl transferase, the second enzyme of Leloir pathway for the galactose catabolism. We have determined the sequence of 1003 base pairs surrounding and upstream of the transcriptional initiation site of the GAL7 gene. The region sequenced also encompasses the 3' end of GAL10 gene. The 5 end of GAL7 mRNA was determined on the DNA sequence by the SI nucleaseand exonucleaae VII mapping, which is located 21 to 22 base pairs upstream from the translation initiating ATG codon. The primary structure of the GAL7 5' flanking region has many features common to those of multicellular eukaryotic genes. The 3' end of GAL10 mRNA was also determined by the mapping technique with the single-strand specific nucleases to be about 600 base pairs upstream from the 5' end of GAL7 mRNA. INTRODUCTION The galactose utilization system in Saccharomyces cerevisiae has been extensively studied genetically as well as biochemically: Three genes designated GAL7, GAL10 and GALl encode galactose-1-phosphate uridylyl transferase (transferase), uridine diphosphoglucose 4-epimerase (epimerase) and galactokinase respectively, which catalize the first three steps of the galactose catabolism (1). These enzymes have been purified to homogeneity (2, 3, 4). These genes, collectively referred to aa the GAL cluster genes in this paper, are tightly linked to each other near the centromere of chromosome II. The gene order is known to be centromere-GAL7-GAL10-GALl (5) . The GAL cluster genes have been cloned into \ phage (6) and the approximate location of each gene is assigned on the cloned DNA (7). It has also been shown that the transcription of each gene starts from the respective promoter (8). The expression of the GAL cluster genes is positively regulated by the genes GAL4 and GAL11, and negatively by the GAL80 gene in a coordinate fashion (9-13) at the transcriptional level (6, 7). This gene expression is also under the control of the catabolite repression which is presumably mediated by the GAL82 and GAL83 genes (14). By an analogy with the mechanism of the 6 IRL Press Limited, Oxford, England. 8555 Nucleic Acids Research prokaryotic regulation, all or at least some of these gene products are assumed to bind the GAL cluster genes at the respective 5' flanking regions (10, 11, 13). Recently the regulatory genes GAM (15-17) and GAL80 (17, 18) have been isolated, which has opened the way to purify those regulatory proteins. These knowledges prompted us to identify the signals for the controlled transcription in the GAL cluster genes. As the first step, we have determined the nucleotide sequence of the 5' flanking region of GAL7 gene. The 5' end of GAL7 mRNA as well as the 3' end of GAL10 mRNA have been assigned on the DNA sequence. These results have revealed that the 5'-flanking region of GAL7 shares many features with the corresponding region of eukaryotic genes. MATERIALS AND METHODS Strains, media and growth Saccharomyces cerevisiae strain YK3 (a_ trpl his3 GAL ) , and YK3 transformed with the plasmid pYH3003 (YK3[pYH3OO3]) were used as sources of polyadenylated RHA (polyA RNA). Yeast strains were grown on synthetic minimal media containing 0.67% yeast nitrogen base w/o amino acids (Difco) and either galactose (SGal) or glucose (SD) as the carbon source. Escherichia coli K12 strain JA221 (recAl leuB6 trpAE5 hsdR hsdM lacY) harboring the plasmid pYF1016 was grown on L-broth containing 25 ug/ml ampicillin. Plasmid DNA was prepared by the cleared-lysate procedure (19) and further purified through Bio-Gel A-150 m column fractionation followed by the ethidium bromide/CsCl equilibrium centrifugation. Recombinant plasmids The plasmid pYH3OO3 (17) was constructed by inserting the 2.3 kilobases (kb) Sail fragment containing the complete sequence of the GAL7 gene and a 3' region of the GAL10 gene from AgtSc481 (6) into an E_. coli-yeast shuttle vector, pYCl, a gift from Dr. B. Hohn via Dr. Y. Kikuchi of our laboratory at Keio University. The plasmid pYF1016 was constructed by inserting the same 2.3 kb Sail fragment into the Sail site of pBR322. DNA sequence analysis The plasmid pYF1016 was digested with HinfI, electrophoresed on a 4% polyacrylamide gel and the 1.0 kb fragment was eluted from the gel. The fragment was digested with Avail, Alul or BstNI and labeled either at the 5' end with [Y3 P]ATP and T4 polynucleotide kinase (20) or at the 3' end with an appropriate [aPJdNTP and AMV reverse transcriptase (21). The nucleotide sequence was determined by the method of Maxam and Gilbert (20). The G, A>C, C+T, C reactions were carried out and analyzed on thin (0.3 mm) 10% or 20%