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G R Fink

Publications and source records attributed to G R Fink.

At least 199 records · Page 11Linked to original sources
PubMed1988

The Saccharomyces cerevisiae genome contains functional and nonfunctional copies of transposon Ty1.

Saccharomyces cerevisiae Ty elements are transposons closely related to retroviruses. The DNA sequence of a functional Ty element (TyH3) is presented. The long terminal repeat sequences are different, suggesting that TyH3 is a recombinant Ty element. A chromosomal Ty element near the LYS2 gene, Ty173, was found to be nonfunctional, even though it has no detectable insertions or deletions. The defect in Ty173 transposition is caused by a missense mutation giving rise to a Leu-to-Ile substitution in the TYB (pol) open reading frame. Several chromosomal Ty elements carry this lesion in their DNA, indicating that nonfunctional Ty elements are common in the yeast genome.

Amino Acid Sequence↗
PubMed1988

TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae.

We identified a 180-kilodalton plasma membrane protein in Saccharomyces cerevisiae required for high-affinity transport (uptake) of potassium. The gene that encodes this putative potassium transporter (TRK1) was cloned by its ability to relieve the potassium transport defect in trk1 cells. TRK1 encodes a protein 1,235 amino acids long that contains 12 potential membrane-spanning domains. Our results demonstrate the physical and functional independence of the yeast potassium and proton transport systems. TRK1 is nonessential in S. cerevisiae and maps to a locus unlinked to PMA1, the gene that encodes the plasma membrane ATPase. Haploid cells that contain a null allele of TRK1 (trk1 delta) rely on a low-affinity transporter for potassium uptake and, under certain conditions, exhibit energy-dependent loss of potassium, directly exposing the activity of a transporter responsible for the efflux of this ion.

Base Sequence↗
PubMed1987

Multiple global regulators control HIS4 transcription in yeast.

Gene expression is dependent on the interaction of DNA binding factors with distinct promoter control elements to activate RNA synthesis. The expression of the HIS4 gene in yeast is under two different control systems. One of these, general amino acid control, involves a DNA binding protein, GCN4, that stimulates transcription in response to amino acid starvation by binding to 5'-TGACTC-3' sequences in the HIS4 promoter region. A second system, the basal level control, stimulates HIS4 transcription in the absence of amino acid starvation. The basal level transcription of the HIS4 gene is under the control of two genes, BAS1 and BAS2, which are also required for the control of purine biosynthesis. In addition, BAS2 is required for the utilization of organic phosphates in the growth medium. Genetic mapping and DNA sequence analysis show that BAS2 is PHO2, a gene previously identified as a regulator of phosphate metabolism. Direct biochemical analysis shows that the BAS2 gene encodes a protein that binds to both the HIS4 and PHO5 promoters. The involvement of a single DNA binding protein in the regulation of histidine, adenine, and phosphate metabolism suggests that yeast may use a few key DNA binding proteins to coordinate the regulation of diverse metabolic pathways.

Acid Phosphatase↗
PubMed1987

KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast.

Molecular analysis of the KAR1 gene of yeast has shown that it is required for both mitosis and conjugation. The gene was originally identified by mutations that prevent nuclear fusion. By in vitro mutagenesis and gene replacement we have demonstrated that the gene is an essential cell division cycle gene. Temperature-sensitive mutant strains show defects in spindle pole body duplication and chromosome disjunction. Overproduction of the gene product blocks spindle pole body duplication, producing a cell cycle arrest phenotype similar to that of the Kar- temperature-sensitive mutations. Long, aberrant extranuclear microtubules are formed in the temperature-sensitive mutants arrested at the nonpermissive temperature as well as in kar1-1 during conjugation. These observations suggest that the KAR1 gene is required for the normal function of both the intranuclear and extranuclear microtubules.

Amino Acid Sequence↗
PubMed1987

5-Fluoroorotic acid as a selective agent in yeast molecular genetics.

5-FOA is an extremely useful reagent for the selection of Ura- cells amid a population of Ura+ cells. The selection is effective in transformation and recombination studies where loss of URA3+ is desired. A new plasmid shuffling procedure based on the 5-FOAR selection permits the recovery of conditional lethal mutations in cloned genes that encode vital functions.

Alleles↗
PubMed1987

A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector.

A set of genomic plasmid banks was constructed using the centromere-containing yeast shuttle vector YCp50. The centromere-containing vector is useful for the isolation of genes that are toxic to yeast when present in high copy number. Fourteen independent banks were prepared each with an average representation of two to three times the yeast genome. Any individual plasmid from a given bank is guaranteed to be of independent origin from plasmids obtained from each of the other banks. The banks were constructed from three different size classes of DNA fragments that resulted from varying conditions of partial digestion with Sau3A. This avoided the bias caused by differential sensitivity of sites to cleavage with Sau3A. Insert DNA is sufficiently large that most genes will be present in the set of plasmid banks at a frequency of about 0.1%.

Centromere↗
PubMed1987

Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein.

We characterized two genes, FUS1 and FUS2, which are required for fusion of Saccharomyces cerevisiae cells during conjugation. Mutations in these genes lead to an interruption of the mating process at a point just before cytoplasmic fusion; the partition dividing the mating pair remains undissolved several hours after the cells have initially formed a stable "prezygote." Fusion is only moderately impaired when the two parents together harbor one or two mutant fus genes, and it is severely compromised only when three or all four fus genes are inactivated. Cloning of FUS1 and FUS2 revealed that they share some functional homology; FUS1 on a high-copy number plasmid can partially suppress a fus2 mutant, and vice versa. FUS1 remains essentially unexpressed in vegetative cells, but is strongly induced by incubation of haploid cells with the appropriate mating pheromone. Immunofluorescence microscopy of alpha factor-induced a cells harboring a fus1-LACZ fusion showed the fusion protein to be localized at the cell surface, concentrated at one end of the cell (the shmoo tip). FUS1 maps near HIS4, and the intervening region (including BIK1, a gene required for nuclear fusion) was sequenced along with FUS1. The sequence of FUS1 revealed the presence of three copies of a hexamer (TGAAAC) conserved in the 5' noncoding regions of other pheromone-inducible genes. The deduced FUS1 protein sequence exhibits a striking concentration of serines and threonines at the amino terminus (46%; 33 of 71), followed by a 25-amino acid hydrophobic stretch and a predominantly hydrophilic carboxy terminus, which contains several potential N-glycosylation sites (Asn-X-Ser/Thr). This sequence suggests that FUS1 encodes a membrane-anchored glycoprotein with both N- and O-linked sugars.

Amino Acid Sequence↗
PubMed1986

The HTS1 gene encodes both the cytoplasmic and mitochondrial histidine tRNA synthetases of S. cerevisiae.

The gene encoding the histidine-tRNA synthetase (HTS1) has two in-frame translation start sites located 60 bp apart. One set of HTS1 transcripts (long) initiates upstream of both ATG codons, and the other set (short) initiates between the two ATG codons and therefore contains only the downstream ATG. A mutation that destroys the first AUG on the long message results in the Pet- (respiratory deficient) phenotype, but does not affect either the level of the cytoplasmic histidine-tRNA synthetase or viability. Mutations distal to the second ATG lead to loss of cytoplasmic synthetase function, lethality and respiratory deficiency. These phenotypes can be explained if the longer message were to encode the mitochondrial synthetase and the shorter message were to encode the cytoplasmic histidine-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1986

A cloned tryptophan-synthesis gene from the ascomycete Cochliobolus heterostrophus functions in Escherichia coli, yeast and Aspergillus nidulans.

A gene (TRP1) in the tryptophan biosynthetic pathway of the fungal plant pathogen Cochliobolus heterostrophus was isolated by complementation of an Escherichia coli trpF mutant which lacked phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene also complemented an E. coli trpC mutant lacking indoleglycerolphosphate synthase (IGPS) activity, a yeast trp1 mutant missing PRAI activity and an Aspergillus nidulans trpC mutant. It functioned in E. coli and A. nidulans without apparent rearrangement but in yeast only after the 5' end of the gene was deleted. The gene was subcloned on a 4.65-kb DNA fragment and the PRAI domain was localized to a 2.9-kb region. It showed homology to the A. nidulans trpC and Neurospora crassa trp-1 genes. There was one predominant transcript of C. heterostrophus TRP1, the same size (2.6-kb) as one of the two functional transcripts produced by A. nidulans trpC. The constitutive activity of the C. heterostrophus TRP1 gene was high whereas that of the A. nidulans trpC gene was low.

Aldose-Ketose Isomerases↗
PubMed1986

GCN4 protein, a positive transcription factor in yeast, binds general control promoters at all 5' TGACTC 3' sequences.

The GCN4 gene is required for the general amino acid control derepression response in yeast. GCN4 protein protects a repeated sequence motif in the 5'-untranslated region of HIS4, HIS3, ILV1, and ILV2 genes subject to general control. At low concentrations of GCN4, only certain repeats in these genes are bound. The repeats differ slightly from the 5' TGACTC 3' consensus core sequence, and the selective binding of some sites at low GCN4 concentrations is related to the relative affinity of these sites to GCN4. Using purified GCN4 protein obtained from an overproducing strain of Escherichia coli, we were able to obtain complete protection of all of the repeat elements in these four genes at high GCN4 concentrations. Analysis of the relative binding constant to the 15 repeated sequences protected by GCN4 shows that the optimal binding site for GCN4 is 5' RRTGACTC 3' followed by a short stretch of thymidines. Another protein, present mostly in yeast nuclear extracts, binds to the HIS4 promoter at a site overlapping one of the GCN4 binding sites. This protein is displaced from its binding site at high GCN4 concentrations.

Binding Sites↗
PubMed1986

Saccharomyces cerevisiae nuclear fusion requires prior activation by alpha factor.

We have developed a protocol for efficient fusion of spheroplasts of the same mating type. Nuclear fusion in this whole-cell system is also efficient and closely parallels nuclear fusion in heterosexual mating of intact cells. In the spheroplast fusion system, nuclear fusion is dependent on both the KAR1 gene and prior exposure to alpha factor. The major products of nuclear fusion in the spheroplast fusion assay were true diploids that were homozygous at the mating-type locus. An additional 10% of the products were cells of ploidy greater than diploid. The dependence of nuclear fusion on alpha factor treatment could not be replaced by synchronization in G1 by mutations in CDC28 and CDC35 or by prior arrest in stationary phase. These data suggest that nuclear fusion is not a constitutive function of the nucleus, but rather is specifically induced by mating hormone.

Cell Nucleus↗
PubMed1986

Saccharomyces cerevisiae SPT3 gene is required for transposition and transpositional recombination of chromosomal Ty elements.

Mutations in the Saccharomyces cerevisiae SPT3 gene have dramatic effects on the expression of Ty elements and genes adjacent to the element. The SPT3 gene is essential for Ty transposition, because transposition of chromosomal Ty elements ceased when the SPT3 gene was replaced with the frameshift mutation spt3-101. Presumably, the elimination of transposition was due to the effect of the SPT3 gene product on Ty transcription; the transcripts of chromosomal Ty elements were largely abolished in the spt3-101 strain (F. Winston, K. J. Durbin, and G. R. Fink, Cell 39:675-682, 1984). Ty transcription in an spt3-101 strain could be reestablished by introduction of the pGTyH3 plasmid, in which transcription of the Ty element TyH3 is under the control of the GAL1 promoter; these plasmid-derived Ty transcripts were SPT3-independent. Ty transposition resumed after galactose induction in spt3-101 strains containing the pGTyH3 plasmid. In spt3 mutants nearly all of the resulting transposition events derived from pGTyH3 plasmids and not from chromosomal elements.

Chromosome Mapping↗
PubMed1985

Ty element transposition: reverse transcriptase and virus-like particles.

We have found reverse transcriptase activity and virus-like particles only in yeast cells that contain a galactose-promoted Ty element induced on galactose. The cofractionation of reverse transcriptase, genomic-length Ty RNA, and a Ty-specified protein antigen in a particulate fraction and the ability of this complex to synthesize specifically a product that is homologous to the entire Ty suggest that reverse transcription of Ty RNA takes place in the particle. The absence of appreciable levels of reverse transcriptase and particles in uninduced cells despite the presence of at least 35 copies of chromosomal Ty elements suggest that some of these elements may be defective. The numerous virus-like particles visible in thin sections of Ty transposition-induced cells appear not to be infectious. These particles resemble the intracisternal A-type particles of the mouse and copia particles of Drosophila. The results support the idea that Ty elements and retroviruses share a common origin.

Antigens, Fungal↗
PubMed1985

Ty elements transpose through an RNA intermediate.

We have followed Ty transposition with a donor Ty element, TyH3, whose expression is under the control of the GAL1 promoter. Sequence analysis reveals dramatic structural differences in TyH3 before and after transposition. If the donor TyH3 is marked with an intron-containing fragment, the intron is correctly spliced out of the Ty during transposition, suggesting that the Ty RNA is the intermediate for transposition. Furthermore, the pattern of sequence inheritance in progeny Ty insertions derived from the marked Ty follows the predictions of the model of retroviral reverse transcription. Comparison of marked Ty elements before and after movement shows that transposition is highly mutagenic to the Ty element. These results demonstrate that during transposition, Ty sequence information flows from DNA to RNA to DNA.

Base Sequence↗
PubMed1985

The histidine permease gene (HIP1) of Saccharomyces cerevisiae.

The histidine-specific permease gene (HIP1) of Saccharomyces cerevisiae has been mapped, cloned, and sequenced. The HIP1 gene maps to the right arm of chromosome VII, approx. 11 cM distal to the ADE3 gene. The gene was isolated as an 8.6-kb BamHI-Sau3A fragment by complementation of the histidine-specific permease deficiency in recipient yeast cells. We sequenced a 2.4-kb subfragment of this BamHI-Sau3A fragment containing the HIP1 gene and identified a 1596-bp open reading frame (ORF). We confirmed the assignment of the 1596-bp ORF as the HIP1 coding sequence by sequencing a hip1 nonsense mutation. Analysis of the amino acid (aa) sequence of the HIP1 gene reveals several hydrophobic stretches, but shows no obvious N-terminal signal peptide. We have constructed a deletion of the HIP1 gene in vitro and replaced the wild-type copy of the gene with this deletion. The hip1 deletion mutant can grow when it is supplemented with 30 mM histidine, 50 times the amount required for the growth of HIP1 cells. Revertants of this deletion mutant able to grow on a normal level of histidine arise by mutation in unlinked genes. Both these observations suggest that there are additional, low-affinity pathways for histidine uptake.

ATP-Binding Cassette Transporters↗