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J Rine

Publications and source records attributed to J Rine.

At least 109 records · Page 6Linked to original sources
PubMed1988

Increased amounts of HMG-CoA reductase induce "karmellae": a proliferation of stacked membrane pairs surrounding the yeast nucleus.

Overproduction of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in yeast resulted in striking morphological effects on the structure of intracellular membranes. Specifically, stacks of paired membranes closely associated with the nuclear envelope were observed in strains that over-produced the HMG1 isozyme, one of two isozymes for HMG-CoA reductase in yeast. These nuclear-associated, paired membranes have been named "karmellae." In strains that overproduced the HMG1 isozyme, HMG-CoA reductase was present in the karmellar layers. At mitosis, karmellae were asymmetrically segregated: the mother cells inherited all of the karmellae and the daughter cells inherited none. A membranous structure of different morphology was occasionally found in cells that overproduced the HMG2 isozyme. These observations further establish the existence of cellular mechanisms that monitor the levels of membrane proteins and compensate for changes in these levels by inducing synthesis of particular types of membrane.

Cell Nucleus↗
PubMed1988

Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae.

Two DNA-binding factors from Saccharomyces cerevisiae have been characterized, GRFI (general regulatory factor I) and ABFI (ARS-binding factor I), that recognize specific sequences within diverse genetic elements. GRFI bound to sequences at the negative regulatory elements (silencers) of the silent mating type loci HML E and HMR E and to the upstream activating sequence (UAS) required for transcription of the MAT alpha genes. A putative conserved UAS located at genes involved in translation (RPG box) was also recognized by GRFI. In addition, GRFI bound with high affinity to sequences with the (C1-3A)-repeat region at yeast telomeres. Binding sites for GRFI with the highest affinity appeared to be of the form 5'-(A/G)(A/C)ACCCANNCA(T/C)(T/C)-3', where N is any nucleotide. ABFI-binding sites were located next to autonomously replicating sequences (ARSs) at controlling elements of the silent mating type loci HMR E, HMR I, and HML I and were associated with ARS1, ARS2, and the 2 micron plasmid ARS. Two tandem ABFI binding sites were found between the HIS3 and DED1 genes, several kilobase pairs from any ARS, indicating that ABFI-binding sites are not restricted to ARSs. The sequences recognized by ABFI showed partial dyad-symmetry and appeared to be variations of the consensus 5'-TATCATTNNNNACGA-3'. GRFI and ABFI were both abundant DNA-binding factors and did not appear to be encoded by the SIR genes, whose products are required for repression of the silent mating type loci. Together, these results indicate that both GRFI and ABFI play multiple roles within the cell.

Chromosomes↗
PubMed1988

Structural and functional conservation between yeast and human 3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limiting enzyme of sterol biosynthesis.

The pathway of sterol biosynthesis is highly conserved in all eucaryotic cells. We demonstrated structural and functional conservation of the rate-limiting enzyme of the mammalian pathway, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase), between the yeast Saccharomyces cerevisiae and humans. The amino acid sequence of the two yeast HMG-CoA reductase isozymes was deduced from DNA sequence analysis of the HMG1 and HMG2 genes. Extensive sequence similarity existed between the region of the mammalian enzyme encoding the active site and the corresponding region of the two yeast isozymes. Moreover, each of the yeast isozymes, like the mammalian enzyme, contained seven potential membrane-spanning domains in the NH2-terminal region of the protein. Expression of cDNA clones encoding either hamster or human HMG-CoA reductase rescued the viability of hmg1 hmg2 yeast cells lacking this enzyme. Thus, mammalian HMG-CoA reductase can provide sufficient catalytic function to replace both yeast isozymes in vivo. The availability of yeast cells whose growth depends on human HMG-CoA reductase may provide a microbial screen to identify new drugs that can modulate cholesterol biosynthesis.

Amino Acid Sequence↗
PubMed1987

Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae.

Mating type interconversion in Saccharomyces cerevisiae occurs by transposition of copies of the a or alpha mating type cassettes from inactive loci, HML and HMR, to an active locus, MAT. The lack of expression of the a and alpha genes at the silent loci results from repression by trans-acting regulators encoded by SIR (Silent Information Regulator) genes. In this paper we present evidence for the existence of four SIR genes. Inactivation of any of these genes leads to expression of cassettes at both HML and HMR. Unusual complementation properties are observed for a number of sir mutations. Specifically, some recessive mutations in different genes fail to complement. The correspondence between SIR1, SIR2, SIR3, SIR4 and other genes with similar roles (MAR, CMT, STE8 and STE9) is presented.

Alleles↗
PubMed1987

Identifying mutations in duplicated functions in Saccharomyces cerevisiae: recessive mutations in HMG-CoA reductase genes.

The two yeast genes for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG1 and HMG2, each encode a functional isozyme. Although cells bearing null mutations in both genes are inviable, cells bearing a null mutation in either gene are viable. This paper describes a method of screening for recessive mutations in the HMG1 gene, the gene encoding the majority of HMG-CoA reductase activity in the cell. This method should be applicable to the isolation of mutations in other recovered in HMG1. These mutations exhibited intragenic complementation: one allele is in one complementation group and three alleles are in a second complementation group. Assays of HMG-CoA reductase activity indicated that the point mutations destroy most if not all of the activity encoded by HMG1. Intragenic complementation occurred with partial restoration of enzymatic activity. HMG1 was mapped to the left arm of chromosome XIII near SUP79, and HMG2 was mapped to the right arm of chromosome XII near SST2. A slight deleterious effect of a null mutation in either HMG-CoA reductase gene was detected by a co-cultivation experiment involving the wild-type strain and the two single mutants.

DNA Restriction Enzymes↗
PubMed1987

Replication and segregation of plasmids containing cis-acting regulatory sites of silent mating-type genes in Saccharomyces cerevisiae are controlled by the SIR genes.

In Saccharomyces cerevisiae, two cis-acting regulatory sites called E and I flank the silent mating-type gene, HMRa, and mediate SIR-dependent transcriptional repression of the a1-a2 promoters. It has been shown previously that the E and I sites have plasmid replicator (ARS) activity. We show in this report that the ARS activity of the E and I sites is governed by the SIR genotype of the cell. In wild-type cells, a plasmid carrying the E site from HMRa (HMR E) in the vector YIp5 exhibited very high mitotic stability at a copy number of approximately 25 per cell. However, in sir2, sir3, or sir4 mutants, plasmids with HMR E had the low mitotic stability characteristic of plasmids containing ARS1, a SIR-independent replicator. Elevated mitotic stability of plasmids that carry HMR E is due to a segregation mechanism provided by SIR and HMR E. In sir2 and sir4 mutants, the plasmid copy number was significantly lowered, suggesting that these gene products also participate in the replication of plasmids carrying HMR E. The phenotype of point mutations introduced at an 11-base-pair ARS consensus sequence present at HMR E indicated that this sequence is functional but not absolutely required for autonomous replication of the plasmid and that it is not required for SIR-dependent mitotic stabilization. A plasmid carrying both a centromere and HMR E exhibited reduced mitotic stability in wild-type cells. This destabilization appeared to be due to antagonism between the segregation functions provided by the centromere and by HMR E.

Base Sequence↗
PubMed1986

Saccharomyces cerevisiae contains two functional genes encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

We have isolated two genes from yeast encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase [hydroxymethylglutaryl-coenzyme A reductase (NADPH); HMG-CoA reductase; EC 1.1.1.34], the rate-limiting enzyme of sterol biosynthesis. These genes, HMG1 and HMG2, were identified by hybridization to a cDNA clone encoding hamster HMG-CoA reductase. DNA sequence analysis reveals homology between the amino acid sequence of the proteins encoded by the two yeast genes and the carboxyl-terminal half of the hamster protein. Cells containing mutant alleles of both HMG1 and HMG2 are unable to undergo spore germination and vegetative growth. However, cells containing a mutant allele of either HMG1 or HMG2 are viable but are more sensitive to compactin, a competitive inhibitor of HMG-CoA reductase, than are wild-type cells. Assays of HMG-CoA reductase activity in extracts from hmg1- and hmg2- mutants indicate that HMG1 contributes at least 83% of the activity found in wild-type cells.

Chromosome Mapping↗
PubMed1986

Precocious meiotic centromere separation of a novel yeast chromosome.

In Saccharomyces cerevisiae, a reciprocal translocation between chromosome II and a linear plasmid carrying a centromere (CEN6) has split chromosome II into two fragments: one, approximately 530 kilobase pairs (kbp) in size, has the left arm and part of the right arm of chromosome II; the other, a telocentric fragment approximately 350 kbp in size, has CEN6 and the rest of the right arm of chromosome II. A cross of this yeast strain with a strain containing a complete chromosome II exhibits a high frequency of precocious centromere separation (separation of sister chromatids during meiosis I) of the telocentric fragment. Precocious centromere separation is not due to the position of the centromere per se, since diploids that are homozygous for both fragments of chromosome II segregate the telocentric fragment with normal meiotic behavior. The precocious centromere separation described here differs from previously described examples in that pairing and synapsis of this telocentric chromosome seem to be normal. One model of how centromeres function in meiosis is that replication of the centromere is delayed until the second meiotic division. Data presented in this paper indicate that replication of the centromere is complete before the first meiotic division. The precocious separation of the centromere described here may be due to improper synapsis of sequences flanking the centromere.

Centromere↗
PubMed1986

A position effect on the expression of a tRNA gene mediated by the SIR genes in Saccharomyces cerevisiae.

The SIR genes of Saccharomyces cerevisiae are responsible for the position-dependent regulation of the a and alpha mating-type genes. Previous work by others has shown that the products of the SIR genes prevent the accumulation of stable transcripts of the a and alpha genes at HML and HMR. Results of this study establish that this regulation is a region-specific effect rather than a gene-specific effect since expression of a tRNA gene placed at HMR is repressed by the products of the SIR genes.

DNA Restriction Enzymes↗
PubMed1985

Regulated expression of endonuclease EcoRI in Saccharomyces cerevisiae: nuclear entry and biological consequences.

In an investigation to determine how proteins are localized within the nucleus of a cell, we demonstrate that the restriction endonuclease EcoRI is able to enter and function within the nucleus of Saccharomyces cerevisiae when this prokaryotic protein is synthesized in vivo. The EcoRI endonuclease was produced in yeast under the transcriptional control of a regulated yeast promoter by ligating a DNA fragment containing only coding sequences for the endonuclease to the promoter element of the yeast GAL1 gene (the structural gene for galactokinase, EC 2.7.1.6). Yeast cells harboring a plasmid containing this promoter-gene fusion are able to grow under conditions that repress transcription from the GAL1 promoter. However, under inducing conditions, these yeast cells are unable to grow. Moreover, rad52 mutants, which are deficient in the repair of double-strand breaks, are more sensitive to the presence of the promoter-gene fusion plasmid than are wild-type cells. We demonstrate that the EcoRI endonuclease activity is present in lysates prepared from yeast transformants grown under conditions that induce transcription of GAL1, but this activity is not detectable in cells grown under conditions that repress transcription from the promoter. Furthermore, analysis of yeast chromosomal DNA shows that the endonuclease enters the yeast nucleus and cleaves DNA specifically at EcoRI recognition sites.

Biological Transport↗
PubMed1984

Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step.

Asparagine-linked glycosylation is a form of covalent modification that distinguishes proteins that are either membrane bound or are in cellular compartments topologically outside of the cell from those proteins that remain soluble in the cytoplasm. This type of glycosylation occurs stepwise, with core oligosaccharide added in the endoplasmic reticulum and subsequent modifications occurring in the golgi. We used tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharide, to select for mutants that are resistant to this antibiotic. Genetic, biochemical, and physiological experiments led to the following conclusions. The synthesis of N-linked oligosaccharide is an essential function in cells. In contrast to mammalian cells, yeast cells do not transport tunicamycin by a glucosamine transport function. We identified a gene, ALG7, that is probably the structural gene for UDP-N-acetylglucosamine-1-P transferase, the enzyme inhibited by tunicamycin. Dominant mutations in this gene result in increased activity of the transferase and loss of the ability of the cell to sporulate. In addition, we identified another gene, TUN1, in which recessive mutations result in resistance to tunicamycin. The ALG7 and TUN1 genes both map on chromosome VII.

Asparagine↗
PubMed1983

Targeted selection of recombinant clones through gene dosage effects.

The availability of multicopy plasmid vectors for the yeast Saccharomyces cerevisiae allows the selective amplification of individual segments of the genome. Increased dosage of particular genes results in overproduction of specific gene products and thereby confers resistance to certain metabolic inhibitors. Advantage was taken of this fact to isolate recombinant clones that increase the activities of the enzymes UDP-N-acetylglucosamine-1-P transferase and 3-hydroxy-3-methylglutaryl-CoA reductase.

Cloning, Molecular↗
PubMed1981

Homology and non-homology at the yeast mating type locus.

Four mutations of the alpha mating type locus of Saccharomyces cerevisiae have been analysed to determine their relationship to the a mating type locus. Mat alpha+ recombinations are produced by mat alpha 2-/MAT but not by mat alpha 1-/MATa diploids. MAT alpha and MATa thus contain regions of homology (coding for at least part of MAT alpha 2) and regions of non-homology (coding for at least part of MAT alpha 1)-the genetic determinant for cell type is larger than the non-homologous sequence seen by DNA-DNA heteroduplexes and genetic analysis. The segment transposed in mating type interconversion includes both types of sequence.

Base Sequence↗