The psi factor of yeast: a problem in inheritance.
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Genes, Fungal↗
Biomedical subjects
M F Tuite
Publications and source records attributed to M F Tuite.
The allosuppressor gene SAL4 encodes a protein important for maintaining translational fidelity in Saccharomyces cerevisiae.
Allosuppressor (sal) mutations enhance the efficiency of the yeast ochre suppressor SUQ5 and define five unlinked loci, SAL1-SAL5. A number of sal4 mutants were isolated and found to have pleiotropic, allele;specific phenotypes, including hypersensitivity in vivo to paromomycin and other antibiotics that stimulate translational errors in yeast. To examine further the nature of the SAL4 gene product, the wild type SAL4 gene was isolated by complementation of a conditional lethal allele sal4-2, and demonstrated to be a single copy gene encoding a single 1.6 kb transcript. Restriction mapping and DNA hybridisation analysis were used to demonstrate that the SAL4 gene is identical to the previously identified omnipotent suppressor gene SUP45 (SUP1). Our results implicate the SAL4 gene product as playing a major role in maintaining translational accuracy in yeast.
Anti-Bacterial Agents↗
A ribosome-associated inhibitor of in vitro nonsense suppression in [psi-] strains of yeast.
All classes of tRNA-mediated nonsense suppression are much more efficient in yeast cell-free lysates prepared from a [psi+] strain than in those prepared from an isogenic [psi-] strain. Mixed [psi+]/[psi-] lysates do not support efficient suppression. Fractionation of the [psi-] lysate demonstrated the presence of an inhibitor of in vitro suppression that is loosely associated with the 80 S ribosome. The data indicate that the inhibitor is a factor involved in the termination of translation in this simple eukaryote.
Protein Biosynthesis↗
The functions and relationships of Ty-VLP proteins in yeast reflect those of mammalian retroviral proteins.
We have identified the major structural core proteins of Ty virus-like particles (Ty-VLPs) and shown that they are generated by proteolytic cleavage of the primary translation product of TYA, p1. This precursor protein is therefore functionally similar to the gag precursor of retroviruses. Cleavage is mediated by a Ty-encoded protease located at the 5' region of TYB and is accompanied by a change in particle morphology. p1 contains sufficient information for the assembly of a pre-Ty-VLP complex, which does not require the presence of either Ty protease or reverse transcriptase. The results indicate that the requirements and pathway of Ty-VLP formation reflect the initial stages of mammalian retroviral assembly and further support the idea of a common origin for Ty elements and retroviruses.
Amino Acid Sequence↗
Genetic control of translational fidelity in yeast: molecular cloning and analysis of the allosuppressor gene SAL3.
The fidelity of translation in the yeast Saccharomyces cerevisiae is controlled by a number of gene products. We have begun a molecular analysis of such genes and here describe the cloning and analysis of one of these genes, SAL3. Mutations at this locus, and at least four other unlinked loci (designated SAL1-SAL5), increase the efficiency of the tRNA ochre suppressor SUQ5, and are thus termed allosuppressors. We have cloned the SAL3 gene from a yeast genomic library by complementation of a sal3 mutation. Integration of the cloned sequence into the yeast chromosome was used to confirm that the SAL3 gene had been cloned. SAL3 gene is present in a single copy in the yeast genome, is transcribed into a 2.3-kb polyadenylated mRNA and encodes a protein of Mr 80,000. The size of the SAL3 gene product strongly suggests that it is not a ribosomal protein.
Cloning, Molecular↗
A novel suppressor tRNA from the dimorphic fungus Candida albicans.
Unfractionated tRNAs from a number of prokaryotes and eukaryotes were examined for their ability to promote termination codon readthrough in a cell-free system isolated from Saccharomyces cerevisiae. tRNA from the dimorphic fungus Candida albicans was found to have significant UGA and UAG readthrough activity and this activity was present in tRNA extracted from both the yeast and the hyphal phase of the fungus. Unusually the efficiency of readthrough activity in vitro was not affected by the [psi] determinant. C. albicans tRNA was fractionated by one-dimensional and two-dimensional gel electrophoresis and both readthrough activities appeared to be associated with a single species of tRNA.
Candida albicans↗
mRNA-dependent yeast cell-free translation systems: theory and practice.
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Cell Fractionation↗
The effects of paromomycin on the fidelity of translation in a yeast cell-free system.
The effects of the aminoglycoside antibiotic paromomycin on the fidelity of translation of the synthetic template poly(U), and two natural mRNAs (rabbit globin mRNA and Brome Mosaic virus RNA), were examined in an mRNA-dependent cell-free system from the yeast Saccharomyces cerevisiae. At antibiotic concentrations that did not inhibit translation (100 microM) optimal mistranslation of all three templates was observed, with the effects declining at higher antibiotic concentrations. Synthesis of the opal termination read-through protein of rabbit beta-globin mRNA was induced by paromomycin, but only in lysates prepared from a [psi+] strain of yeast. The antibiotic did not induce detectable levels of either ochre or amber read-through, but did induce general misreading of Brome Mosaic virus RNA to the same degree in both [psi+] and [psi-] lysates. This misreading was enhanced by addition of the polyamine spermidine.
Animals↗
Expression of Ty-lacZ fusions in Saccharomyces cerevisiae.
We have determined the nucleotide sequence of about 520 bp spanning the 5' delta regions (Figure 1) of two Tyl elements. There is an open reading frame running out of the deltas for at least 180 nucleotides into the internal region of each element. The functional significance of these open reading frames has been tested by fusing them to a defective E.coli lacZ gene. Expression of B-galactosidase in yeast transformants containing these fusions shows that Tyl elements contain functional translation signals.
Amino Acid Sequence↗
Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.
We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication Enhancer (RE) domain, has no inherent replication competence but is essential for ensuring maximum function of the RS domain. The nucleotide sequence of these domains reveals several conserved sequences one of which is strikingly similar to the yeast ARS consensus sequence.
Base Sequence↗
Expression in Saccharomyces cerevisiae of human interferon-alpha directed by the TRP1 5' region.
The complete 5' flanking region of the yeast TRP1 gene encoding N-(5'-phosphoribosyl)- anthranilate isomerase, a nonabundant protein, has been cloned and the nucleotide sequence data has been extended from -102 to -440. The CT block--CAAG structure common to all efficiently expressed yeast genes is altered in the 5' region of TRP1 and a sequence postulated to be involved in general amino acid regulation is absent. There are two possible TATA boxes at -224 and -262. TRP1, in common with HIS3, HIS4 and TRP5 has a region of dyad symmetry upstream of the coding sequence which may play a role in initiation of transcription. The relative efficiency of gene expression directed by the complete 5' TRP1 region was assessed by comparison with that of PGK by inserting a cDNA for a human interferon-alpha downstream of their respective 5' regions. The respective interferon yields indicate that their in vivo expression capabilities are a function of their 5' regions.
Aldose-Ketose Isomerases↗
The complete amino acid sequence of yeast phosphoglycerate kinase.
The complete amino acid sequence of yeast phosphoglycerate kinase, comprising 415 residues, was determined. The sequence of residues 1-173 was deduced mainly from nucleotide sequence analysis of a series of overlapping fragments derived from the relevant portion of a 2.95-kilobase endonuclease-HindIII-digest fragment containing the yeast phosphoglycerate kinase gene. The sequence of residues 174-415 was deduced mainly from amino acid sequence analysis of three CNBr-cleavage fragments, and from peptides derived from these fragments after digestion by a number of proteolytic enzymes. Cleavage at the two tryptophan residues with o-iodosobenzoic acid was also used to isolate fragments suitable for amino acid sequence analysis. Determination of the complete sequence now allows a detailed interpretation of the existing high-resolution X-ray-crystallographic structure. The sequence -Ile-Ile-Gly-Gly-Gly- occurs twice in distant parts of the linear sequence (residues 232-236 and 367-371). Both these regions contribute to the nucleoside phosphate-binding site. A comparison of the sequence of yeast phosphoglycerate kinase reported here with the sequences of phosphoglycerate kinase from horse muscle and human erythrocytes shows that the yeast enzyme is 64% identical with the mammalian enzymes. The yeast has strikingly fewer methionine, cysteine and tryptophan residues.
Amino Acid Sequence↗
Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae.
We have constructed a high-efficiency expression vector to direct the synthesis of heterologous polypeptides in yeast. The vector is termed a sandwich expression vector as the heterologous gene is inserted between the 5' and 3' control regions of the efficiently expressed yeast PGK gene. We have used this vector to direct the expression of three derivatives of the calf chymosin cDNA gene; preprochymosin, prochymosin and chymosin. Prochymosin is synthesised to at least 5% of total yeast-cell protein and furthermore, it can be readily activated to produce an enzyme which has milk-clotting activity.
Animals↗
In vitro nonsense suppression in [psi+] and [psi-] cell-free lysates of Saccharomyces cerevisiae.
An homologous in vitro assay for yeast nonsense suppressors was used to examine the effect of the cytoplasmically inherited genetic determinant [psi] on the efficiency of in vitro nonsense suppression. The efficiency of all three types of yeast tRNA-mediated nonsense suppressor (ochre, amber, and UGA) is much greater in cell-free lysates prepared from a sup+ [psi+] strain than in lysates prepared from an isogeneic sup+ [psi-] strain. Lysates prepared from a [psi-] strain, into which the [psi+] determinant was reintroduced by kar1-mediated cytoduction, support efficient suppression. Evidence is also presented that [psi-] lysates contain an inhibitor of in vitro nonsense suppression.
Cell-Free System↗
Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae.
The position of the yeast phosphoglycerate kinase (PGK) gene has been mapped on a 2.95kb Hind III fragment. We have determined the nucleotide sequence of the 5' flanking region and compared this sequence with those from 16 other yeast genes. PGK, like all other yeast genes has an adenine residue at position -3. It has two possible TATA boxes at positions -114 and -152 and a CAAT box at -129. In addition we have defined a structure at position -63 to -39 that is common to all yeast genes that encode an abundant RNA. This structure is a CT-rich block followed, about 10 nucleotides later, by the sequence CAAG.
Amino Acid Sequence↗
Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae.
The 5' control region of the yeast phosphoglycerate kinase gene (PGK) was fused to the coding sequence of a human interferon-alpha. This PGK-interferon fusion was then introduced into yeast on a high copy number 2mu-based plasmid vector. Strains containing this plasmid produced a PGK-interferon-alpha fusion protein as 1-2% of cell protein and the expression of interferon activity was regulated by the availability of a fermentable carbon source. The system is capable of making as much as 15 mg of human interferon-alpha per litre of batch culture.
Amino Acid Sequence↗
Sequence and structure of yeast phosphoglycerate kinase.
The structure of yeast phosphoglycerate kinase has been determined with data obtained from amino acid sequence, nucleotide sequence, and X-ray crystallographic studies. The substrate binding sites, as deduced from electron density maps, are compatible with known substrate specificity and the stereochemical requirements for the enzymic reaction. A carboxyl-imidazole interaction appears to be involved in controlling the transition between the open and closed forms of the enzyme.
Amino Acid Sequence↗
Relationship of the [psi] factor with other plasmids of Saccharomyces cerevisiae.
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DNA, Fungal↗