Nucleotide sequence and functional analysis of the RAD1 gene of Saccharomyces cerevisiae.

The RAD1 gene of Saccharomyces cerevisiae is involved in excision repair of damaged DNA. The nucleotide sequence of the RAD1 gene presented here shows an open reading frame of 3,300 nucleotides. Two ATG codons occur in the open reading frame at positions +1 and +334, respectively. Since a deletion of about 2.7 kilobases of DNA from the 5' region of the RAD1 gene, which also deletes the +1 ATG and 11 additional codons in the RAD1 open reading frame, partially complements UV sensitivity of a rad1 delta mutant, we examined the role of the +1 ATG and +334 ATG codons in translation initiation of RAD1 protein. Mutation of the +1 ATG codon to ATC affected the complementation ability of the RAD1 gene, whereas mutation of the +334 ATG codon to ATC showed no discernible effect on RAD1 function. These results indicate that translation of RAD1 protein is initiated from the +1 ATG codon. Productive in-frame RAD1-lacZ fusions showed that the RAD1 open reading frame is expressed in yeasts. The RAD1-encoded protein contains 1,100 amino acids with a molecular weight of 126,360.     

Nucleotide sequence of the RAD10 gene of Saccharomyces cerevisiae.

The RAD10 gene is one of several genes in Saccharomyces cerevisiae required for incision of u.v.-irradiated or cross-linked DNA. We have determined the nucleotide sequence of the RAD10 gene and its flanking regions. The RAD10 nucleotide sequence presented here differs significantly from that recently reported. The RAD10 protein predicted from the nucleotide sequence contains 210 amino acids with a calculated mol. wt. of 24 310. The middle portion of the RAD10 protein, which is highly basic and also contains eight of the total of 10 tyrosine residues present in the protein, may be involved in DNA binding by ionic interactions and tyrosine intercalation between the bases of DNA. A genomic deletion of the entire RAD10 gene does not affect viability; however, the rad10 deletion mutant is highly u.v. sensitive.     

Molecular cloning, DNA structure, and RNA analysis of the arginase gene in Saccharomyces cerevisiae. A study of cis-dominant regulatory mutations.

The Saccharomyces cerevisiae gene cargA + or CAR1 , encoding arginase has been cloned by recovering function in transformed yeast cells. It was used to analyse RNA and chromosomal DNA from six strains bearing cis-dominant regulatory mutations leading to constitutive arginase synthesis. The DNA from the four cargA + O- strains in which constitutive arginase synthesis was independent of the mating-type functions showed no detectable differences with the wild- typye . The cargA + O- mutations were, therefore, small alterations, possibly single base substitutions. On the other hand, the cargA + Oh-1 and cargA + Oh-2 mutations, leading to a constitutive and mating-type dependent arginase synthesis, were identified as insertions. Their size and restriction pattern strongly suggested that they were induced by the Ty1 yeast transposable element. This was confirmed by cloning and analysis of the cargA + Oh-1 mutant gene. The concentration of arginase RNA was significantly increased in the mutants, indicating that the regulation of arginase synthesis was exerted, at least in part, at the level of RNA synthesis or stability. In the cargA + Oh-2 strain the Ty1 element was located at a distance of approximately 600 base pairs from the insertion present in the cargA + Oh-1 strain. This result suggests either a surprisingly large arginase regulatory region or an indirect influence of the Ty1 element on gene expression over long distances.     

Nucleotide sequence of the dihydrofolate reductase gene of Saccharomyces cerevisiae

The nucleotide sequence of a DNA fragment that contained the Saccharomyces cerevisiae gene DFR coding for dihydrofolate reductase (DHFR) was determined. The DHFR was encoded by a 633-bp open reading frame, which specified an Mr24264 protein. The polypeptide was significantly related to the DHFRs of chicken liver and Escherichia coli. The yeast enzyme shared 60 amino acid (aa) residues with the avian enzyme and 51 aa residues with the bacterial enzyme. DHFR was overproduced about 40-fold in S. cerevisiae when the cloned gene was present in the vector YEp24. As isolated from the Saccharomyces library, the DFR gene was not expressed in E. coli. When the gene was present on a 1.8-kb BamHI-SalI fragment subcloned into the E. coli vector, pUC18, weak expression in E. coli was observed.     

Identification of a sequence containing the positive regulatory region of Saccharomyces cerevisiae gene ENO1

A DNA fragment which contains the 5'-flanking region of the Saccharomyces cerevisiae enolase 1 gene (ENO1) and a portion of the coding sequence was cloned in a plasmid pMC1587. This fragment was fused in frame to the lacZ gene of Escherichia coli. Many mutants which deleted a portion of the 5'-flanking region of ENO1 were isolated from this ENO1-lacZ fusion plasmid by in vitro recombination. Analysis of beta-galactosidase activity of these mutants indicated that the regulatory region responsible for an efficient expression of the ENO1-lacZ fused gene resides within an 86-bp sequence located at -487 to -402 upstream from the start codon of ENO1. We found that the segment encompassing the 86-bp region worked equally well in an inverted orientation, but the tandem duplication of the sequence did not enhance the expression of the fused gene.     

Nucleotide sequence of the ADE 1 gene of the yeast Saccharomyces cerevisiae

The yeast ADE 1 gene has been cloned and sequenced. The primary structure deduced from the nucleotide sequence demonstrated that phosphoribosylaminoimidazole-succinocarboxamide synthetase is a protein with molecular weight of 34 500 D.     

A positive regulatory sequence of the Saccharomyces cerevisiae ENO1 gene

ENO1-'lacZ fusions with various lengths of the ENO1 5'-flanking region were constructed on various types of yeast plasmid vectors. The fully expressed level of beta Gal directed by ENO1-'lacZ fusions differed depending on the type of vector, but on any type of vector, beta Gal activity was not greatly influenced by the carbon source in the medium. The 86-bp DNA region of ENO1 at position -487 to -402 upstream of the initiation codon, in which we had previously delimited the positive regulatory region of ENO1 (Uemura, H., Shiba, T., Paterson, M., Jigami, Y., & Tanaka, H. (1986) Gene 45, 67-75), exerted its function without requiring precise location with respect to the TATA box. The action of the positive regulatory region was not affected by its orientation. In addition, the substitution of the UASs of PHO5, encoding repressible acid phosphatase, with the regulatory region of ENO1 changed the expression of PHO5-'lacZ gene to constitutive, irrespective of the concentration of inorganic phosphate in the medium. Furthermore, the GCR1 gene cloned in a multicopy plasmid increased the expression of the ENO1-'lacZ fused genes.