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G R Fink

Publications and source records attributed to G R Fink.

At least 217 records · Page 12Linked to original sources
PubMed1985

A synthetic HIS4 regulatory element confers general amino acid control on the cytochrome c gene (CYC1) of yeast.

Hybrid promoters constructed from upstream sequences of the yeast HIS4 promoter and the downstream element of the yeast CYC1 promoter place iso-1-cytochrome c (CYC1) expression under the general amino acid control, typical of HIS4. HIS4 fragments that confer regulation contain at least one copy of the sequence T-G-A-C-T-C that is repeated at HIS4 and other genes subject to the general control. A 14-base-pair synthetic oligonucleotide containing a single copy of the HIS4 repeat places CYC1 under the general control. Two copies of this oligonucleotide produce a derepressed level of expression nearly equivalent to that conferred by the largest HIS4 5' noncoding fragments we examined and direct regulated expression of a set of transcripts with 5' ends typical of the CYC1 promoter. Comparison of the expression levels conferred by the short synthetic repeat and larger HIS4 5' fragments reveals additional promoter elements required for maintaining efficient gene expression under repressing growth conditions.

Amino Acids↗
PubMed1985

The relationship between the "TATA" sequence and transcription initiation sites at the HIS4 gene of Saccharomyces cerevisiae.

Transcription of the HIS4 gene begins at a single site (I) at position -60 from the ATG that begins translation. We have made linker insertions/deletions in the 5' noncoding region to identify the elements required for the specificity of transcription initiation. Although there are four sequences that begin TATA and are near the start of transcription (-170, -132, -123, and -102) only the sequence at -123 (TATA-123) is required for transcription initiation. By inserting synthetic oligonucleotides into a mutant from which TATA-123 had been deleted, we found that just TATA or TATAA does not work but that TATAAA functions almost as well as the wild-type sequence. This hexamer does not work in the opposite orientation (TTTATA). When a synthetic TATA sequence is placed upstream from the normal site, the site of initiation also moves upstream in a roughly cometric way even when TATA-123 is present. Analysis of transcripts in strains where the distance between the TATA sequence and the wild-type site of transcription initiation (I site) has been altered shows that in yeast, unlike higher cells, transcription does not initiate at a strictly defined distance from the TATA sequence. Constructions that alter the distance between the TATA and the I site or remove the I site change the pattern of transcription initiation without affecting the level of HIS4 expression. Deletions that eliminate the I site produce heterogeneous transcripts and deletions that substantially shorten the distance between TATA-123 and the I site initiate at multiple sites downstream from the I site. Thus, both the TATA and the sequences downstream from it determine the pattern of transcription initiation.

Base Sequence↗
PubMed1985

Meiotic recombination between duplicated genetic elements in Saccharomyces cerevisiae.

We have studied the meiotic recombination behavior of strains carrying two types of duplications of an 18.6-kilobase HIS4 Bam HI fragment. The first type is a direct duplication of the HIS4 Bam HI fragment in which the repeated sequences are separated by Escherichia coli plasmid sequences. The second type, a tandem duplication, has no sequences intervening between the repeated yeast DNA. The HIS4 genes in each region were marked genetically so that recombination events between the duplicated segments could be identified. Meiotic progeny of the strains carrying the duplication were analyzed genetically and biochemically to determine the types of recombination events that had occurred. Analysis of the direct vs. tandem duplication suggests that the E. coli plasmid sequences are recombinogenic in yeast when homozygous. In both types of duplications recombination between the duplicated HIS4 regions occurs at high frequency and involves predominantly interchromosomal reciprocal exchanges (equal and unequal crossovers). The striking observation is that intrachromosomal reciprocal recombination is very rare in comparison with interchromosomal reciprocal recombination. However, intrachromosomal gene conversion occurs at about the same frequency as interchromosomal gene conversion. Reciprocal recombination events between regions on the same chromatid are the most infrequent exchanges. These data suggest that intrachromosomal reciprocal exchanges are suppressed.

Chromosomes↗
PubMed1984

A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance.

Mutations at the URA3 locus of Saccharomyces cerevisiae can be obtained by a positive selection. Wild-type strains of yeast (or ura3 mutant strains containing a plasmid-borne URA3+ gene) are unable to grow on medium containing the pyrimidine analog 5-fluoro-orotic acid, whereas ura3- mutants grow normally. This selection, based on the loss of orotidine-5'-phosphate decarboxylase activity seems applicable to a variety of eucaryotic and procaryotic cells.

Carboxy-Lyases↗
PubMed1984

The SPT3 gene is required for normal transcription of Ty elements in S. cerevisiae.

The transposable Ty elements consist of a central core, epsilon, flanked by direct repeats called deltas. In wild-type strains Ty transcripts initiate in one delta and terminate in the other. Insertion mutations caused by Ty elements have a wide variety of phenotypes, ranging from inhibition of gene expression to constitutive gene expression. Mutations in the SPT3 gene suppress these effects of Ty and delta insertion mutations on adjacent genes. In spt3 null mutants the Ty transcription pattern for the entire ensemble of Ty elements is changed. The delta-delta transcripts are absent and initiation begins at a position 800 bp into the epsilon region. In these spt3 strains, transcription that initiates in solo deltas and proceeds into adjacent structural genes is also abolished. The requirement of SPT3 for normal transcription from delta can explain the ability of spt3 mutations to suppress the mutations caused by Ty and delta insertions. In SPT3 strains transcription from the delta into adjacent sequences interferes with normal expression of those sequences, whereas in spt3 strains the aberrant transcript is not made. spt3 mutations also lead to defects in diploid formation and sporulation, suggesting that SPT3 is important for the expression of genes in addition to Ty elements.

Alleles↗
PubMed1984

Ty-mediated gene expression of the LYS2 and HIS4 genes of Saccharomyces cerevisiae is controlled by the same SPT genes.

Five Ty insertion mutations were isolated at the LYS2 locus of Saccharomyces cerevisiae. Genetic and physical analyses show that four Ty insertions are in the 5' noncoding region of LYS2 and one is within the structural gene. Three of these Ty elements have been cloned and characterized. The Ty mutations differ from each other in restriction pattern, phenotypic effects on LYS2, reversion frequency, and the nature of reversion events. Spt2 and spt3 mutations, known to suppress Ty insertions and their solo delta derivatives at HIS4, can also suppress at least one of the Ty insertions (Ty61) at LYS2 and can also suppress the Lys- phenotype of a solo delta derivative of another Ty insertion (Ty128) at LYS2. These results demonstrate that spt mutations can suppress Ty and delta mutations at both HIS4 and LYS2, suggesting that they are general for their effects on Ty and delta elements.

Cloning, Molecular↗
PubMed1984

Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae.

We have identified mutations in seven unlinked genes (SPT genes) that affect the phenotypes of Ty and delta insertion mutations in the 5' noncoding region of the HIS4 gene of S. cerevisiae. Spt mutants were selected for suppression of his4-912 delta, a solo delta derivative of Ty912. Other Ty and delta insertions at HIS4 are suppressed by mutations in some but not all of the SPT genes. Only spt4 suppresses a non-Ty insertion at HIS4. In addition to their effects on Ty and delta insertions, mutations in several SPT genes show defects in general cellular functions--mating, DNA repair and growth.

Alleles↗
PubMed1984

Effects of Ty insertions on HIS4 transcription in Saccharomyces cerevisiae.

Insertion of two different Ty elements into the Saccharomyces cerevisiae HIS4 regulatory region eliminates transcription of HIS4. Transcription can be restored by genetic rearrangements involving the Ty element inserted at HIS4. Several deletions, an inversion, a translocation, and a gene conversion are capable of restoring HIS4 transcription. Some of the rearrangements result in new transcriptional initiation sites. One type of revertant of his4-912 results from recombination between the delta elements flanking the Ty element, leaving a solo delta in place of the complete Ty. Strains carrying a Ty912 delta at HIS4 are His- at 23 degrees C. Unlinked suppressors (SPT) lead to suppression of this His- phenotype and increase levels of the normal HIS4 transcript. These suppressor genes affect not only the amount of transcription from the normal HIS4 initiation site, but also that from new initiation sites within Ty sequences adjacent to HIS4.

Alleles↗
PubMed1984

Positive regulatory interactions of the HIS4 gene of Saccharomyces cerevisiae.

The role of cis- and trans-acting elements in the expression of HIS4 has been examined by using HIS4-lacZ fusions in which lacZ expression is dependent upon the HIS4 5' noncoding region. The cis-acting sequences involved in regulation were defined by studying the effects of the wild-type and various deletions and their revertants on regulation via the general control of amino acid biosynthesis. The role of trans-acting genes was analyzed by studying the regulation of the HIS4-lacZ fusions in strains carrying mutations in the GCN (AAS) or GCD (TRA) genes and in strains carrying the GCN genes on high-copy-number plasmids. These studies have led to the following conclusions. (i) HIS4 is positively regulated by the general control. (ii) At least one copy of the 5'TGACTC3' repeat at -136 is required in cis for this regulation. (iii) Both the GCN4 gene and at least one copy of the repeated sequence are required for expression at the repressed level. (iv) The open reading frames in the 5' noncoding region are not required in either cis or trans for the regulation of HIS4.

Chromosome Deletion↗
PubMed1983

The histidine tRNA genes of yeast.

Yeast has at least seven nuclear histidine tRNA genes although there is a single tRNAHis. We have sequenced three of the histidine tRNA genes. The genes have identical coding sequences and the DNA anti-codon sequence GTG corresponds to the GUG anti-codon in tRNAHis. None of the three yeast histidine tRNA genes has an intervening sequence. Two of the three genes contain repeated DNA elements in the region adjacent to the 5' end of the histidine tRNA gene. One of the elements, sigma, is 18 base pairs (bp) from the 5' end of each of these genes, sigma elements are highly conserved and flanked by 5-bp repeats. The other element, delta, is at variable distances from the tRNA gene; one is 439 bp from a histidine tRNA gene and the other is 52 bp from a histidine tRNA gene. These solo delta elements are quite divergent when compared with delta s associated with transposon yeast elements and are not flanked by 5-bp repeats.

Base Sequence↗
PubMed1983

Structure and expression of two aminoacyl-tRNA synthetase genes from Saccharomyces cerevisiae.

We have isolated the structural genes for methionyl-tRNA synthetase (L-methionine:tRNAMet ligase, EC 6.1.1.10) and isoleucyl-tRNA synthetase (L-isoleucine:tRNAIle ligase, EC 6.1.1.5) from the yeast Saccharomyces cerevisiae by transformation. Both genes exist in a single copy in the yeast genome and show no homology to either yeast mitochondrial or Escherichia coli DNA as judged by Southern analysis. The region corresponding to the DNA segment altered in mes1 or ils1-1 mutants was defined by transformation of synthetase mutants with subclones of the respective gene. Although the isolated fragment containing the methionyl-tRNA synthetase gene complements the mes1 mutation in yeast, it does not complement the analogous metG mutation in E. coli. A single transcript of about 3100 nucleotides codes for the isoleucyl-tRNA synthetase. This transcript is about 2-fold elevated in strains starved for isoleucine, histidine, or arginine as compared to wild type. It is regulated by the general control of amino acid biosynthesis, which affects several biosynthetic pathways. Two polyadenylated transcripts are homologous to the methionyl-tRNA synthetase gene. The major MES1 transcript is not affected by the general control or starvation for several different amino acids, including methionine.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Repeated DNA sequences upstream from HIS1 also occur at several other co-regulated genes in Saccharomyces cerevisiae.

The HIS1 gene of Saccharomyces cerevisiae encodes ATP phosphoribosyltransferase, the first enzyme in the pathway of histidine biosynthesis. We have cloned this gene by complementation of a his1 auxotroph. The HIS1 coding region was localized within the cloned segment by assay of subcloned fragments for their ability to complement a his1 auxotroph. We determined the DNA sequence of the HIS1 region defined by this complementation test. S1 nuclease and exonuclease VII mapping of the 5' and 3' termini of HIS1 mRNA reveal considerable heterogeneity at both ends of the transcript, especially the 5' end which displays 13 different termini that span a 110-base pair region. Northern analysis shows that derepression of HIS1 enzyme activity under conditions of amino acid deprivation can be accounted for by an increase in the steady state level of HIS1 mRNA. There are no large differences between the relative levels of HIS1 mRNA molecules with different 5' termini in repressed and derepressed cells. In the DNA sequence upstream from the 5' termini of HIS1 mRNA we have found four closely related copies of a 9-base pair sequence. This sequence is also repeated in the 5' noncoding regions of HIS4, HIS3, and TRP5. Closely related sequences are not found flanking a number of other yeast genes, suggesting that the repeated sequence plays a role in the regulation of amino acid biosynthetic genes subject to the general amino acid control.

ATP Phosphoribosyltransferase↗
PubMed1983

A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast.

We have made deletions of the HIS4 5' noncoding region in vitro and inserted these deletions into the yeast genome by transformation. Deletions that extend from -588 to -235 have no detectable effects on either promoter or regulatory functions. Deletions that extend to -138 affect promoter function, but are still regulated by the general control of amino acid biosynthesis. A deletion that extends to -136 cannot derepress HIS4 mRNA in response to the general control. This deletion removes all copies of the sequence 5'-TGACTC-3', which appears at positions -194, -182 and -138 in strains without the deletion. The importance of at least one copy of this repeat for regulation of HIS4 is shown by the reappearance of this sequence in revertants of the -136 deletion that have regained the regulatory response. The fact that deletion of this sequence leads to the inability to derepress suggests that HIS4 is under positive control.

Base Sequence↗
PubMed1983

Positive regulation in the general amino acid control of Saccharomyces cerevisiae.

Starvation of yeast for a single amino acid leads to derepression of enzymes in many different amino acid biosynthetic pathways. This general control is regulated by several transacting genes. Mutations in the TRA3 gene result in constitutive derepression, whereas mutations in AAS genes lead to the inability to derepress. We have isolated aas mutations as suppressors of the tra3-1 mutation. Some of these suppressors are alleles of AAS2 and others define a heretofore unidentified gene, AAS3. We have studied the regulatory behavior of strains containing both aas and tra3 mutations and strains containing the cloned AAS genes in high copy number. Either aas1- or aas2- in combination with tra3- has the Tra- phenotype, whereas aas3- in combination with tra3- has the Aas- phenotype. These interactions suggest that the AAS1 and AAS2 products act indirectly to bring about derepression by disabling the repressive effect of TRA3, whereas the AAS3 product functions more directly and is required even in the absence of the TRA3 function. When present in high copy number, the AAS3 gene complements mutations in AAS1 and AAS2, whereas AAS1 and AAS2 only complement their cognate mutations. Taken together these data suggest that AAS1 and AAS2 are negative regulators of TRA3, which in turn is a negative regulator of AAS3. AAS3 is a positive regulator, which is required for the general control response. This model of negative and positive interactions is formally identical to those proposed for the regulation of the galactose and phosphatase systems in yeast.

Amino Acids↗
PubMed1982

The nucleotide sequence of the HIS4 region of yeast.

We have determined the nucleotide sequence of the yeast HIS4 gene and its 5' and 3' flanking sequences. The protein chain has a calculated Mr-value of 87 935. Th 5' end of the HIS4 transcript maps at a position 63 bp upstream from the site of initiation of protein synthesis. The 3' end of the HIS4 transcript maps at a position approx. 118 bp after the UAG termination codon. There is no evidence for intervening sequences within the transcription unit. Functional sub-regions within the HIS4 coding frame have been identified by determining the sequence changes for various his4 mutations.

Base Sequence↗
PubMed1982

sigma, a repetitive element found adjacent to tRNA genes of yeast.

sigma is a DNA element of about 340 base pairs (bp) that is repeated many times in the yeast genome. The element has 8-bp inverted repeats at its ends and is flanked by 5-bp direct repeats. The 5-bp repeats are different for each sigma and have no homology with the ends of the sigma sequence. sigma is located 16 or 18 bp from the 5' end of several tRNA genes. Southern analysis of different yeast strains shows that the pattern of hybridization is different even for closely related strains.

Base Composition↗