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G R Fink

Publications and source records attributed to G R Fink.

At least 235 records · Page 13Linked to original sources
PubMed1982

Movement of yeast transposable elements by gene conversion.

We have constructed yeast strains in which Ty (transposon yeast) elements at the HIS4 locus are genetically marked with the yeast URA3 gene. By isolating and analyzing Ura- derivatives of these strains, we have detected a variety of Ty-mediated recombination events. In this paper, we describe events in which the DNA sequence of the Ty element at the HIS4 locus is replaced by the DNA sequence of a different Ty element. These replacements occur without alterations in the flanking DNA sequence and without chromosomal aberrations. We believe that these events result from gene conversion between the Ty element at HIS4 and a Ty element at a different site in the yeast genome. Gene conversion can occur between Ty elements that differ by large insertion and substitution mutations. These recombination events result not only in the movement of Ty sequences but also in alterations in expression of the adjacent HIS4 gene. Different Ty elements at the same site in the HIS4 regulatory region can result in His-, His+, and cold-sensitive His+ phenotypes. Several Ty elements render expression of the HIS4 gene subject to control by genes at the mating type locus.

DNA Restriction Enzymes↗
PubMed1982

Regulation of HIS4-lacZ fusions in Saccharomyces cerevisiae.

The beginning of the Saccharomyces cerevisiae HIS4 gene has been fused to the structural gene for Escherichia coli beta-galactosidase. This construction, which contains HIS4 DNA from -732 to +30 relative to the translation initiation codon, has been integrated into the yeast genome at two chromosomal locations, HIS4 and URA3. At both locations, this 762-base-pair stretch of DNA is sufficient for initiating expression of beta-galactosidase activity in S. cerevisiae and confers upon this activity the regulatory response normally found for HIS4.

Base Sequence↗
PubMed1982

Construction of yeast strains containing genetically marked transposons.

Haploid yeast cells contain approximately 35 Ty (transposon yeast) elements. To facilitate the study of these elements, we have constructed yeast strains in which one of these transposons carries a genetic marker. The elements that we have modified are Ty912 and Ty917, elements originally detected at the HIS4 locus in spontaneously occurring his4- mutants. The strain constructions took place in three stages: 1) cloning of the mutant HIS4 genes containing the Ty elements; 2) introduction of a HindIII restriction fragment containing the yeast URA3 gene into the cloned transposons; and 3) replacement of the chromosomal HIS4 gene with the modified genes constructed in vitro. These strains will be extremely useful in the study of Ty transposition and other Ty-promoted DNA rearrangements.

Chromosomes↗
PubMed1982

Yeast genetics.

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DNA Replication↗
PubMed1981

Gene conversion between duplicated genetic elements in yeast.

The mitotic recombination behaviour of a duplication of the his4 region on chromosome III in the yeast Saccharomyces cerevisiae was studied. The major recombination event between the duplicated segments is gene conversion unassociated with reciprocal recombination. The rad52-1 mutation preferentially decreases mitotic gene conversion. These results suggest that mitotic gene conversion may occur by a different pathway from that occurring in meiosis. This mitotic gene conversion may be important in yeast mating type interconversion and the maintenance of sequence homogeneity in families of repeated eukaryotic genes.

Alleles↗
PubMed1981

The yeast his4 multifunctional protein. Immunochemistry of the wild type protein and altered forms.

A procedure for rapid purification of the yeast his4 protein has been developed. A combined biochemical and immunological study of the his4 proteins from wild type and a number of mutant strains indicates that the his4 gene product is a trifunctional protein with three independent functional domains. Active fragments of the his4 protein possessing one or several activities can result from proteolysis or from genetic alteration. Such fragments can be purified to homogeneity by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Immunological experiments with antibody to the wild type protein and with antibody to genetically or proteolytically altered forms indicate that the sole product of the wild type his4 region is a 95,000-dalton protein. Furthermore, such data indicates that each mutant protein has a characteristic stability that correlates with the type of mutation and with the location of the mutation within the his4 locus. By varying the ionic strength of the extraction buffer, it is possible to find conditions under which such mutant proteins are quite stable.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

Suppressible four-base glycine and proline codons in yeast.

Five ICR-170--induced mutations at the His4 locus in yeast are +1 G.C (G, guanine; C, cytosine) additions in DNA regions that contain multiple G.C base pairs. These mutations represents both nonsuppressible and suppressible alleles. All externally, suppressible frameshift mutations occur in glycine and proline codons to produce the four-base codons GGGU (U, uracil), GGGG, and CCCU. This implies that suppression of these four-base codons in yeast, as in bacteria, involves a four-base anticodon or its functional equivalent. Two identical four-base codons (CCCU) at widely separate regions with His4 are not suppressed equally.

Base Sequence↗
PubMed1981

Stable chromosomal integration of the entire nitrogen fixation gene cluster from Klebsiella pneumoniae in yeast.

A bacterial plasmid containing the entire nitrogen fixation (nif) gene cluster (consisting of at least 15 genes) from Klebsiella pneumoniae was used in conjunction with an Escherichia coli-yeast shuttle plasmid containing the yeast his4 gene cluster to cotransform a his4- recipient strain of Saccharomyces cerevisiae. Of 87 histidine-independent clones screened, 2 contained nif DNA. Restriction and hybridization analyses showed that two copies of the nif plasmid (46 kilobases each) are integrated in tandem in the recipient chromosome by recombination between homologous regions in the transforming plasmids. Chromosomal integration was also verified by tetrad analysis, showing that the nif DNA behaved in meiosis like a Mendelian element. During mitotic growth, one of the two copies of the nif region is frequently lost. The remaining copy of nif is stable, even after 40 generations in nonselective medium.

DNA Restriction Enzymes↗
PubMed1980

The origins of gene instability in yeast.

Two unstable mutations at the his4 locus of yeast are due to the insertion of the transposable elements Ty912 and Ty917 into the his4 regulatory region. The two transposons are related, one being derived from the other by a substitution of 4000 base pairs of DNA. Element Ty912 includes identical terminal repeats, whereas the terminal repeats of Ty917 are not identical. Transposition of Ty912 or Ty917 generates 5-base-pair duplications of the target DNA at either end of the element. Expression and reversion of a his4 gene containing Ty912 or Ty917 is controlled by three unlinked regulatory genes. The properties of these regulatory genes are similar to those described for the controlling elements in maize.

Alleles↗
PubMed1980

Insertion of the eukaryotic transposable element Ty1 creates a 5-base pair duplication.

The his4-912 mutation results from insertion of a transposable element into the 5'-non-coding region of the his4 gene of yeast. A duplication of 5 base pairs of wild-type his4 DNA flanks the inserted element. The major class of His+ revertants result from excision of most of the transposable element, leaving an inserted segment of 334 base pairs flanked by the 5-base pair repeats.

Base Sequence↗
PubMed1980

The intracellular proteinases and their inhibitors in yeast. A mutant with altered regulation of proteinase A inhibitor activity.

A mutation of yeast pai1 has been isolated which leads to altered regulation of proteinase A inhibitor activity. Under conditions of derepression, this mutant as compared with wild type has a 70% reduction in proteinase A inhibitor activity, but no change in the activities of proteinase A or B. The growth of strains carrying the pai1 mutation is sensitive to temperature and pH. The altered physiological and biochemical phenotypes of the mutant appear to be the consequences of a single mutation which segregates 2:2 at meiosis. Results obtained with this mutant indicate that proteinase A and its inhibitor are two independently synthesized polypeptide chains rather than two proteins resulting from proteolytic cleavage of a single precursor. Furthermore, proteinase A and its inhibitor appear to be regulated independently of each other. Studies on the mutant also indicate that the regulation of the proteinase A inhibitor is different from that of the proteinase B inhibitor. In wild type cells there is an excess of proteinase A inhibitor over proteinase A, whereas in the mutant cells there is a 2.5-fold excess of proteinase A over proteinase A inhibitor. This excess proteinase A does not lead to detectable alterations in overall protein degradation. However, tryptophan synthase and proteinase B inhibitor proteins which are sensitive to proteinase A attack in vitro show an enhanced loss of activity in the mutant.

Fungal Proteins↗
PubMed1980

Genetic events associated with an insertion mutation in yeast.

The his4-912 mutation shares similar genetic properties with mutations promoted by procaryotic insertion elements. This mutation lacks all three his4 functions. Many different classes of His+ revertants have been obtained from his4-912. The most frequent class of His+ revertants results from a site mutation which confers a cold-sensitive His- phenotype. Other classes of revertants contain translocations (one between chromosomes I and III and the other between chromosomes III and XII), a transposition of the his4 region to chromosome VIII, and an inversion of most of the left arm of chromosome III. Another class contains deletions which extend from his4-912 into the his4 region. In each of these classes of revertants, the his4 region is closely linked to the chromosomal aberration. Many of these revertants contain additional changes in chromosome structure (duplication, deletion and aneuploidy) that are unrelated to the reversion of his4-912 to His4+.

Chromosome Aberrations↗
PubMed1980

DNA rearrangements associated with a transposable element in yeast.

The his4-912 mutation results from insertion of a 6200 bp transposable element into the his4 gene of yeast. In order to clone the his4-912 mutation, the plasmid pBR322 was integrated into the his4 gene by means of yeast transformation, and then the vector sequences and the his4-912 insertion element were excised as a single restriction fragment. This his 4-912 insertion element is homologous to Ty1, a family of repetitive yeast DNA sequences. His+ revertants derived from the his4-912 mutant carry a number of chromosomal aberrations including deletions, translocations, a transposition and an inversion. The majority of His+ revertants result from deletions which have both endpoints within the element and which leave behind only 300 bp of the insertion element. Other derivatives of the his4-912 mutant carry deletions which have one endpoint in the insertion element and one endpoint in the his4 coding sequence. In two His+ revertants carrying reciprocal translocations, the chromosome III translocation breakpoints occur within the his4-912 insertion element. A His+ revertant carrying an inversion of most of the left arm of chromosome III may be an intermediate in transposition of the his4-912 insertion element to a new site on chromosome III.

Base Sequence↗
PubMed1980

Frameshift suppression Saccharomyces cerevisiae. II. Genetic properties of group II suppressors.

Suppressors of ICR-induced mutations that exhibit behavior similar to bacterial frameshift suppressors have been identified in the yeast Saccharomyces cerevisiae. The yeast suppressors have been divided into two groups. One of these groups (Group II: SUF1, SUF3, SUF4, SUF5 and SUF6) appears to include a set of informational suppressors in which the vehicle of suppression is glycyl-tRNA. Some of the genetic properties of Group II suppressors are described in this communication.--Corevertants of the Group II frameshift mutations his4-519 and leu2-3 have been characterized to determine the spectrum of reversion events induced by the frameshift mutagen ICR-170. Seventy-three ICR-induced corevertants were analyzed. With the exception of one cerevertant, which carried an allele of SUF1, all carried alleles of SUF3 or SUF5. SUF1, SUF3, SUF4 and SUF6 were represented among spontaneous and UV-induced corevertants. In the course of these experiments one of the suppressors was mapped. SUF5, the probable structural gene for tRNAGLY1, is located between ade2 and ade9 on chromosome XV.--SUF1, SUF4 and SUF6 have novel properties and comprise a distinct subset of suppressors. Although these suppressors show no genetic linkage to each other, they share several common features including lethality in haploid pairwise combinations, reduced tRNAGLY3 isoacceptor activity and increased efficiency of suppression in strains carrying the cytoplasmically inherited [PSI] element. In addition, strains carrying SUF1, SUF4 or SUF6 are phenotypically unstable and give rise to mitotic Suf+ segments at high frequency. These segregants invariably contain a linked, second-site mutation that maps in or adjacent to the suppressor gene itself. Strains carrying any of these suppressors also give rise to mitotic segregants that exhibit enhanced efficiency of suppression; mutations responsible for this phenotype map at two loci, upf1 and upf2. These genes show no genetic linkage to any of the group II suppressors.--Methods that permit positive selection have been devised in order to examine large numbers of variants. The importance of these interacting mutants is underscored by their potential utility in studying suppressor function at the molecular level.

Chromosome Mapping↗
PubMed1980

Frameshift suppression in Saccharomyces cerevisiae. III. Isolation and genetic properties of group III suppressors.

Suppressors of ICR-induced mutations that exhibit behavior similar to bacterial frameshift suppressors have been identified in the yeast Saccharomyces cerevisiae. The yeast suppressors have been divided into two groups. Previous evidence indicated that suppressors of one group (Group II: SUF1, SUF3, SUF4, SUF5 and SUF6) represent mutations in the structural genes for glycyl-tRNA's. Suppressors of the other group (Group III: SUF2 and SUF7) were less well characterized. Although they suppressed some ICR-revertible mutations, they failed to suppress Group II frameshift mutations. This communication provides a more thorough characterization of the Group III suppressors and describes the isolation and properties of four new suppressors in that group (SUF8, SUF9, SUF10 and suf11).--In our original study, Group III suppressors were isolated as revertants of the Group III mutations his4-712 and his4-713. All suppressors obtained as ICR-induced revertants of these mutations mapped at the SUF2 locus near the centromere of chromosome III. Suppressors mapping at other loci were obtained in this study by analyzing spontaneous and UV-induced revertants of the Group III mutations. SUF2 and SUF10 suppress both Group III his4 mutations, whereas SUF7, SUF8, SUF9 and suf11 suppress his4-713, but not his4-712. All of the suppressors except suf11 are dominant in diploids homozygous for his4-713. The suppressors fail to suppress representative UAA, UAG and UGA nonsense mutations.--SUF9 is linked to the centromere of chromosome VI, and SUF10 is linked to the centromere of chromosome XIV. A triploid mapping procedure was used to determine the chromosome locations of SUF7 and SUF8. Subsequent standard crosses revealed linkage of SUF7 to cdc5 on chromosome Xiii and linkage of SUF8 to cdc12 and pet3 on chromosome VIII.

Chromosome Mapping↗
PubMed1979

The product of the his4 gene cluster in Saccharomyces cerevisiae. A trifunctional polypeptide.

The his4 region of yeast encodes the information for the third (phosphoribosyl-AMP cyclohydrolase), second (phosphoribosyl-ATP pyrophosphohydrolase), and tenth (histidinol dehydrogenase) steps in the histidine biosynthetic pathway. These three activities co-purify with a single protein which has a subunit molecular weight of 95,000 (95,000 protein), as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Extracts of yeast strains which carry nonsense or deletion mutations in various portions of the his4 region, purified in parallel by affinity chromatography on AMP-agarose columns, were examined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis slabs. All such mutant extracts examined were found to lack the 95,000 protein found in a strain carrying a wild type his4 allele. The presence of a protease inhibitor, phenylmethylsulfonyl fluoride, during the purification of the trifunctional enzyme prevented the degradation of the 95,000 protein to polypeptides of lower molecular weight. Monospecific antibody prepared against the 95,000 protein removed all three of the activities specified by his4 from solution; active 95,000 protein was recovered in the resuspended immunoprecipitates. All this evidence shows that the product of the his4 region is a trifunctional, 95,000-dalton protein. Preliminary evidence from two-dimensional gel electrophoresis, NH2-terminal analysis, and gel filtration column chromatography indicates that the native trifunctional enzyme is a dimer of identical 95,000-dalton subunits.

Adenosine Monophosphate↗