scholarly journals Allosuppressors that Enhance the Efficiency of Omnipotent Suppressors in Saccharomyces cerevisiae

Genetics ◽  
1987 ◽  
Vol 115 (3) ◽  
pp. 451-460
Author(s):  
Jae Mahn Song ◽  
Susan W Liebman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Haploid Cell ◽  
Suppressor Activity ◽  
Translational Ambiguity

ABSTRACT Two recessive Mendelian-allosuppressors have been isolated and have been shown to enhance the efficiency of omnipotent suppressors thought to be translational ambiguity mutations. These allosuppressors are unlinked to each other or to the omnipotent suppressors on which they act. They also increase the efficiency of the serine-inserting UAA-suppressor, SUP16 . One allosuppressor is allelic or tightly linked to the previously isolated sal2. Another allosuppressor, called sal6, represents a new locus, unlinked to the previously isolated sal1-sal5 that enhance the efficiency of the UAA-suppressors. When present singly in the absence of suppressors or other modifiers the sal2 and sal6 mutations do not have suppressor activity. However, when sal2 and sal6 are combined together in a haploid cell they do suppress weakly. In addition sal2 becomes a weak suppressor in the presence of the [η+] modifying factor.

scholarly journals Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo.

1986 ◽  
Vol 6 (7) ◽  
pp. 2663-2673 ◽  
Author(s):  
M C Strobel ◽  
J Abelson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trna Gene ◽  
Nuclear Extract ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Specific Sequence ◽  
Made In

The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.

Isolation and transcriptional characterization of three genes which function at start, the controlling event of the Saccharomyces cerevisiae cell division cycle: CDC36, CDC37, and CDC39

1983 ◽  
Vol 3 (5) ◽  
pp. 881-891
Author(s):  
H J Breter ◽  
J Ferguson ◽  
T A Peterson ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Dna Sequences ◽  
Shuttle Vector ◽  
Control Process ◽  
Haploid Cell ◽  
Loop Analysis ◽  
Mrna Species ◽  
Coding Regions ◽  
Plasmid Library

The genes CDC36, CDC37, and CDC39, thought to function in the cell division control process in Saccharomyces cerevisiae, were isolated from a recombinant plasmid library prepared by partial digestion of S. cerevisiae genomic DNA with Sau3A and insertion into the S. cerevisiae-Escherichia coli shuttle vector YRp7. In each case, S. cerevisiae DNA sequences were identified which could complement mutant alleles of the gene in question and which could direct integration of a plasmid at the chromosomal location known to correspond to that gene. Complementing DNA segments were subcloned to remove extraneous coding regions. The coding regions corresponding to CDC36, CDC37, and CDC39 were then identified and localized by R-loop analysis. The estimated sizes of the three coding regions were 615, 1,400, and 2,700 base pairs, respectively. Transcriptional orientation of the coding regions was established by using M13 vectors to prepare strand-specific probes followed by hybridization to blots of electrophoresed S. cerevisiae mRNA. The intracellular steady-state abundance of the mRNA species corresponding to the genes was estimated by comparing hybridization signals on RNA blots to that of a previously determined standard, the cell cycle start gene CDC28. The quantities calculated for the three mRNA species were low, ranging from 1.5 +/- 1 copies per haploid cell for the CDC36 mRNA to 3.1 +/- 1.5 and 4.6 +/- 2 copies per haploid cell for the CDC37 and CDC39 mRNAs, respectively. The CDC28 mRNA had been previously estimated at 7.0 +/- 2 copies per cell.

scholarly journals Engineering of Polyploid Saccharomyces cerevisiae for Secretion of Large Amounts of Fungal Glucoamylase

2002 ◽  
Vol 68 (11) ◽  
pp. 5693-5697 ◽  
Author(s):  
Keisuke Ekino ◽  
Hiroyuki Hayashi ◽  
Masahiro Moriyama ◽  
Minoru Matsuda ◽  
Masatoshi Goto ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ploidy Level ◽  
Copy Number ◽  
Vector System ◽  
Aspergillus Awamori ◽  
Haploid Cell ◽  
Single Chromosome ◽  
Integration Vector ◽  
A Cell

ABSTRACT We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the δ-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. δ-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.

scholarly journals GPA1Val-50 mutation in the mating-factor signaling pathway in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (6) ◽  
pp. 2289-2297 ◽  
Author(s):  
I Miyajima ◽  
K Arai ◽  
K Matsumoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
G Proteins ◽  
Double Mutant ◽  
Growth Defect ◽  
Haploid Cell ◽  
Short Term ◽  
Alpha Factor ◽  
High Concentrations ◽  
Mating Factor

The GPA1 gene of Saccharomyces cerevisiae encodes a protein that is highly homologous to the alpha subunit of mammalian hetrotrimeric G proteins and is essential for haploid cell growth. A mutation of the GPA1 protein, GPA1Val-50, in which Gly-50 was replaced by valine, could complement the growth defect of a GPA1 disruption, gpal::HIS3. However, cells with gpa1::HIS3 expressing the GPA1Val-50 protein were supersensitive to alpha-factor in a short-term incubation but resumed growth after long-term incubation even after exposure to high concentrations of alpha-factor. The former phenotype associated with GPA1Val-50 is recessive, and the latter phenotype is dominant to GPA1+. The supersensitivity of GPA1Val-50 to alpha-factor was dependent on STE2 and STE4, which demonstrates that this GPA1Val-50-produced phenotype requires the mating-factor receptor and the beta subunit of the G protein. The double mutant of sst2-1 GPA1Val-50 recovered from division arrest, which suggested that SST2 is not required for recovery of the GPA1Val-50 mutant.

scholarly journals Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

1995 ◽  
Vol 15 (9) ◽  
pp. 4754-4762 ◽  
Author(s):  
M Remacha ◽  
A Jimenez-Diaz ◽  
B Bermejo ◽  
M A Rodriguez-Gabriel ◽  
E Guarinos ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Cell Extract ◽  
Suppressor Activity ◽  
Cell Extracts ◽  
Absolute Requirement ◽  
Type Pattern ◽  
Cold Sensitive ◽  
Acidic Proteins ◽  
P Proteins

Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently.

scholarly journals Pheromones and pheromone receptors are the primary determinants of mating specificity in the yeast Saccharomyces cerevisiae.

Genetics ◽  
1989 ◽  
Vol 121 (3) ◽  
pp. 463-476 ◽  
Author(s):  
A Bender ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Types ◽  
Haploid Cell ◽  
Wild Type ◽  
Cell Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
A Cells ◽  
Alpha Factor ◽  
A Cell ◽  
Specific Proteins

Abstract Saccharomyces cerevisiae has two haploid cell types, a and alpha, each of which produces a unique set of proteins that participate in the mating process. We sought to determine the minimum set of proteins that must be expressed to allow mating and to confer specificity. We show that the capacity to synthesize alpha-factor pheromone and a-factor receptor is sufficient to allow mating by mat alpha 1 mutants, mutants that normally do not express any alpha- or a-specific products. Likewise, the capacity to synthesize a-factor receptor and alpha-factor pheromone is sufficient to allow a ste2 ste6 mutants, which do not produce the normal a cell pheromone and receptor, to mate with wild-type a cells. Thus, the a-factor receptor and alpha-factor pheromone constitute the minimum set of alpha-specific proteins that must be produced to allow mating as an alpha cell. Further evidence that the pheromones and pheromone receptors are important determinants of mating specificity comes from studies with mat alpha 2 mutants, cells that simultaneously express both pheromones and both receptors. We created a series of strains that express different combinations of pheromones and receptors in a mat alpha 2 background. These constructions reveal that mat alpha 2 mutants can be made to mate as either a cells or as alpha cells by causing them to express only the pheromone and receptor set appropriate for a particular cell type. Moreover, these studies show that the inability of mat alpha 2 mutants to respond to either pheromone is a consequence of two phenomena: adaptation to an autocrine response to the pheromones they secrete and interference with response to alpha factor by the a-factor receptor.

scholarly journals Allosuppressors in yeast

1977 ◽  
Vol 30 (2) ◽  
pp. 187-205 ◽  
Author(s):  
B. S. Cox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Phenotypic Expression ◽  
Chain Termination ◽  
Class I ◽  
Temperature Sensitive ◽  
Suppressor Activity ◽  
Recessive Mutations ◽  
Suppressor Mutations ◽  
Cold Sensitive

SUMMARYFive loci have been identified inSaccharomyces cerevisiaewhose function reduces suppressor activity in strains carrying ochre super-suppressor mutations. Recessive mutations which allow an increased level of suppression occur at these loci. In such mutants, termed allosuppressors, the serine-inserting suppressorSUPQ5suppresses ochre mutations in a [psi−] background and Class I tyrosine-inserting suppressors are lethal or have a reduced viability. Mutations at two allosuppressor loci,sal3 andsal4, have a lethal interaction with one another and with the extrachromosomal determinant [psi+]. This interaction is expressed in the absence of any suppressor mutation. All the mutant alleles of one allosuppressor locussal3 are cold sensitive. One allosuppressor mutation,sal4.2, is temperature-sensitive for growth, as well as for other aspects of its phenotypic expression; namely the expression ofSUPQ5and the lethal interactions with Class I super-suppressors, with [psi+] and withsal3. At low temperature (24 °C),sal3-sal4.2 double mutants weakly suppress the ochre mutationade2.1, but do not suppresshis5.2 orlys1.1. It is argued that the site of function of the products of these loci is ribosomal and that they are involved in chain termination at UAA codons. It is inferred that the [psi+] factor or its product affects protein synthesis by interaction with the ribosome.

GPA1Val-50 mutation in the mating-factor signaling pathway in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (6) ◽  
pp. 2289-2297
Author(s):  
I Miyajima ◽  
K Arai ◽  
K Matsumoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
G Proteins ◽  
Double Mutant ◽  
Growth Defect ◽  
Haploid Cell ◽  
Short Term ◽  
Alpha Factor ◽  
High Concentrations ◽  
Mating Factor

The GPA1 gene of Saccharomyces cerevisiae encodes a protein that is highly homologous to the alpha subunit of mammalian hetrotrimeric G proteins and is essential for haploid cell growth. A mutation of the GPA1 protein, GPA1Val-50, in which Gly-50 was replaced by valine, could complement the growth defect of a GPA1 disruption, gpal::HIS3. However, cells with gpa1::HIS3 expressing the GPA1Val-50 protein were supersensitive to alpha-factor in a short-term incubation but resumed growth after long-term incubation even after exposure to high concentrations of alpha-factor. The former phenotype associated with GPA1Val-50 is recessive, and the latter phenotype is dominant to GPA1+. The supersensitivity of GPA1Val-50 to alpha-factor was dependent on STE2 and STE4, which demonstrates that this GPA1Val-50-produced phenotype requires the mating-factor receptor and the beta subunit of the G protein. The double mutant of sst2-1 GPA1Val-50 recovered from division arrest, which suggested that SST2 is not required for recovery of the GPA1Val-50 mutant.

scholarly journals Isolation and transcriptional characterization of three genes which function at start, the controlling event of the Saccharomyces cerevisiae cell division cycle: CDC36, CDC37, and CDC39.

1983 ◽  
Vol 3 (5) ◽  
pp. 881-891 ◽  
Author(s):  
H J Breter ◽  
J Ferguson ◽  
T A Peterson ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Dna Sequences ◽  
Shuttle Vector ◽  
Control Process ◽  
Haploid Cell ◽  
Loop Analysis ◽  
Mrna Species ◽  
Coding Regions ◽  
Plasmid Library

The genes CDC36, CDC37, and CDC39, thought to function in the cell division control process in Saccharomyces cerevisiae, were isolated from a recombinant plasmid library prepared by partial digestion of S. cerevisiae genomic DNA with Sau3A and insertion into the S. cerevisiae-Escherichia coli shuttle vector YRp7. In each case, S. cerevisiae DNA sequences were identified which could complement mutant alleles of the gene in question and which could direct integration of a plasmid at the chromosomal location known to correspond to that gene. Complementing DNA segments were subcloned to remove extraneous coding regions. The coding regions corresponding to CDC36, CDC37, and CDC39 were then identified and localized by R-loop analysis. The estimated sizes of the three coding regions were 615, 1,400, and 2,700 base pairs, respectively. Transcriptional orientation of the coding regions was established by using M13 vectors to prepare strand-specific probes followed by hybridization to blots of electrophoresed S. cerevisiae mRNA. The intracellular steady-state abundance of the mRNA species corresponding to the genes was estimated by comparing hybridization signals on RNA blots to that of a previously determined standard, the cell cycle start gene CDC28. The quantities calculated for the three mRNA species were low, ranging from 1.5 +/- 1 copies per haploid cell for the CDC36 mRNA to 3.1 +/- 1.5 and 4.6 +/- 2 copies per haploid cell for the CDC37 and CDC39 mRNAs, respectively. The CDC28 mRNA had been previously estimated at 7.0 +/- 2 copies per cell.

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