scholarly journals Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (7) ◽  
pp. 2443-2451 ◽  
Author(s):  
A Percival-Smith ◽  
J Segall
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intergenic Region ◽  
Mutational Analysis ◽  
Fusion Gene ◽  
Lacz Fusion ◽  
Regulatory Sequences ◽  
Wild Type ◽  
Base Pairs ◽  
Differential Hybridization ◽  
Dna Library

A differential hybridization screen of a genomic yeast DNA library previously identified 14 genes of Saccharomyces cerevisiae that are expressed preferentially during sporulation. Three of these sporulation-specific genes, SPS1, SPS2, and SPS3, have been shown to be closely linked. A mutational analysis has demonstrated that expression of the SPS1 gene, but not the SPS2 gene, is essential for the completion of sporulation. A diploid MATa/MAT alpha strain homozygous for a disruption of the SPS1 gene failed to form asci when subjected to sporulation conditions. The 3' end of the transcript encoded by the SPS1 gene was found to map only 185 base pairs from the 5' end of the SPS2 gene. The SPS1-SPS2 intergenic region was shown to contain all of the regulatory sequences necessary for the sporulation-specific activation of the SPS2 gene as assessed by expression of a translational SPS2-lacZ fusion gene present on a replicating, centromere-containing plasmid. The fusion gene was found to be expressed at the same time during sporulation as the chromosomal wild-type SPS2 gene.

Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharomyces cerevisiae

1986 ◽  
Vol 6 (7) ◽  
pp. 2443-2451
Author(s):  
A Percival-Smith ◽  
J Segall
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intergenic Region ◽  
Mutational Analysis ◽  
Fusion Gene ◽  
Lacz Fusion ◽  
Regulatory Sequences ◽  
Wild Type ◽  
Base Pairs ◽  
Differential Hybridization ◽  
Dna Library

A differential hybridization screen of a genomic yeast DNA library previously identified 14 genes of Saccharomyces cerevisiae that are expressed preferentially during sporulation. Three of these sporulation-specific genes, SPS1, SPS2, and SPS3, have been shown to be closely linked. A mutational analysis has demonstrated that expression of the SPS1 gene, but not the SPS2 gene, is essential for the completion of sporulation. A diploid MATa/MAT alpha strain homozygous for a disruption of the SPS1 gene failed to form asci when subjected to sporulation conditions. The 3' end of the transcript encoded by the SPS1 gene was found to map only 185 base pairs from the 5' end of the SPS2 gene. The SPS1-SPS2 intergenic region was shown to contain all of the regulatory sequences necessary for the sporulation-specific activation of the SPS2 gene as assessed by expression of a translational SPS2-lacZ fusion gene present on a replicating, centromere-containing plasmid. The fusion gene was found to be expressed at the same time during sporulation as the chromosomal wild-type SPS2 gene.

Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae

1984 ◽  
Vol 4 (10) ◽  
pp. 1985-1998
Author(s):  
R R Yocum ◽  
S Hanley ◽  
R West ◽  
M Ptashne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Elements ◽  
Lacz Fusion ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Yeast Plasmid ◽  
Coding Sequence ◽  
Lacz Fusions ◽  
Base Pair Fragment

We present the DNA sequence of a 914-base pair fragment from Saccharomyces cerevisiae that contains the GAL1-GAL10 divergent promoter, 140 base pairs of GAL10 coding sequence, and 87 base pairs of GAL1 coding sequence. From this fragment, we constructed four pairs of GAL1-lacZ and GAL10-lacZ fusions on various types of yeast plasmid vectors. On each type of vector, the fused genes were induced by galactose and repressed by glucose. The response of a GAL1-lacZ fusion to gal4 and gal80 regulatory mutations was similar to the response of intact chromosomal GAL1 and GAL10 genes. A set of deletions that removed various portions of the GAL10 regulatory sequences from a GAL10-CYC1-lacZ fusion was constructed in vitro. These deletions defined a relatively guanine-cytosine-rich region of 45 base pairs that contained sequences necessary for full-strength galactose induction and an adjacent guanine-cytosine rich 55 base pairs that contained sequences sufficient for weak induction.

scholarly journals Elements involved in oxygen regulation of the Saccharomyces cerevisiae CYC7 gene.

1987 ◽  
Vol 7 (6) ◽  
pp. 2212-2220 ◽  
Author(s):  
R S Zitomer ◽  
J W Sellers ◽  
D W McCarter ◽  
G A Hastings ◽  
P Wick ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutational Analysis ◽  
Direct Repeat ◽  
Regulatory Sequences ◽  
Oxygen Regulation ◽  
Base Pairs ◽  
Protein Coding ◽  
C Protein ◽  
Intact Gene ◽  
Positive Site

The CYC7 gene of Saccharomyces cerevisiae encodes the minor species, iso-2, of the cytochrome c protein. Its expression is governed by two regulatory sequences upstream from the gene: a positive site which stimulates transcription 240 base pairs 5' from the protein-coding sequence (-240) and a negative site which inhibits transcription at -300. In this study, the nature of the positive site and its relationship to the negative site has been investigated. Expression of the CYC7 gene is weakly inducible by oxygen. This effect was greatly enhanced by the semidominant CYP1-16 mutation in the trans-acting gene CYP1. The weak oxygen regulation in wild-type cells and the enhanced induction in CYP1-16 mutants were found to be mediated through the positive site. A mutational analysis of this site implicated at least part of a tandem, direct repeat of 9 base pairs as essential for the functioning of this site. The relationship between the positive and negative sites was investigated by comparing the expression of the intact gene with that of derivatives lacking either one or the other site. The expression of the gene containing only the negative site was actually stimulated anaerobically, while the gene containing the positive site alone, although having higher expression aerobically than anaerobically, had higher anaerobic expression than did the intact gene. Thus, it appeared that the combination of the positive and negative sites suppressed anaerobic expression. A model which attempts to explain these properties of the two sites and account for the regulation of the expression of the intact gene is presented.

scholarly journals Suppressors of a gpa1 mutation cause sterility in Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 119 (4) ◽  
pp. 797-804
Author(s):  
I Miyajima ◽  
N Nakayama ◽  
M Nakafuku ◽  
Y Kaziro ◽  
K Arai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Gene ◽  
Lacz Fusion ◽  
Wild Type ◽  
Cell Type ◽  
Recessive Mutations ◽  
Cycle Arrest ◽  
Alpha Factor ◽  
A Cell ◽  
Mating Factor

Abstract The Saccharomyces cerevisiae GPA1 gene encodes a protein highly homologous to the alpha subunit of mammalian G proteins and is essential for haploid cell growth. We have selected 77 mutants able to suppress the lethality resulting from disruption of GPA1 (gpa1::HIS3). Two strains bearing either of two recessive mutations, sgp1 and sgp2, in combination with the disruption mutation, showed a cell type nonspecific sterile phenotype, yet expressed the major alpha-factor gene (MF alpha 1) as judged by the ability to express a MF alpha 1-lacZ fusion gene. The sgp1 mutation was closely linked to gpa1::HIS3 and probably occurred at the GPA1 locus. The sgp2 mutation was not linked to GPA1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11 and ste12). sgp2 GPA1 cells showed a fertile phenotype, indicating that the mating defect caused by sgp2 is associated with the loss of GPA1 function. While expression of a FUS1-lacZ fusion gene was induced in wild-type cells by the addition of alpha-factor, mutants bearing sgp1 or sgp2 as well as gpa1::HIS3 constitutively expressed FUS1-lacZ. These observations suggest that GPA1 (SGP1) and SGP2 are involved in mating factor-mediated signal transduction, which causes both cell cycle arrest in the late G1 phase and induction of genes necessary for mating such as FUS1.

scholarly journals Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (10) ◽  
pp. 1985-1998 ◽  
Author(s):  
R R Yocum ◽  
S Hanley ◽  
R West ◽  
M Ptashne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Elements ◽  
Lacz Fusion ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Yeast Plasmid ◽  
Coding Sequence ◽  
Lacz Fusions ◽  
Base Pair Fragment

We present the DNA sequence of a 914-base pair fragment from Saccharomyces cerevisiae that contains the GAL1-GAL10 divergent promoter, 140 base pairs of GAL10 coding sequence, and 87 base pairs of GAL1 coding sequence. From this fragment, we constructed four pairs of GAL1-lacZ and GAL10-lacZ fusions on various types of yeast plasmid vectors. On each type of vector, the fused genes were induced by galactose and repressed by glucose. The response of a GAL1-lacZ fusion to gal4 and gal80 regulatory mutations was similar to the response of intact chromosomal GAL1 and GAL10 genes. A set of deletions that removed various portions of the GAL10 regulatory sequences from a GAL10-CYC1-lacZ fusion was constructed in vitro. These deletions defined a relatively guanine-cytosine-rich region of 45 base pairs that contained sequences necessary for full-strength galactose induction and an adjacent guanine-cytosine rich 55 base pairs that contained sequences sufficient for weak induction.

Elements involved in oxygen regulation of the Saccharomyces cerevisiae CYC7 gene

1987 ◽  
Vol 7 (6) ◽  
pp. 2212-2220
Author(s):  
R S Zitomer ◽  
J W Sellers ◽  
D W McCarter ◽  
G A Hastings ◽  
P Wick ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutational Analysis ◽  
Direct Repeat ◽  
Regulatory Sequences ◽  
Oxygen Regulation ◽  
Base Pairs ◽  
Protein Coding ◽  
C Protein ◽  
Intact Gene ◽  
Positive Site

The CYC7 gene of Saccharomyces cerevisiae encodes the minor species, iso-2, of the cytochrome c protein. Its expression is governed by two regulatory sequences upstream from the gene: a positive site which stimulates transcription 240 base pairs 5' from the protein-coding sequence (-240) and a negative site which inhibits transcription at -300. In this study, the nature of the positive site and its relationship to the negative site has been investigated. Expression of the CYC7 gene is weakly inducible by oxygen. This effect was greatly enhanced by the semidominant CYP1-16 mutation in the trans-acting gene CYP1. The weak oxygen regulation in wild-type cells and the enhanced induction in CYP1-16 mutants were found to be mediated through the positive site. A mutational analysis of this site implicated at least part of a tandem, direct repeat of 9 base pairs as essential for the functioning of this site. The relationship between the positive and negative sites was investigated by comparing the expression of the intact gene with that of derivatives lacking either one or the other site. The expression of the gene containing only the negative site was actually stimulated anaerobically, while the gene containing the positive site alone, although having higher expression aerobically than anaerobically, had higher anaerobic expression than did the intact gene. Thus, it appeared that the combination of the positive and negative sites suppressed anaerobic expression. A model which attempts to explain these properties of the two sites and account for the regulation of the expression of the intact gene is presented.

Meiotic expression of human ornithine transcarbamylase in the testes of transgenic mice

1988 ◽  
Vol 8 (4) ◽  
pp. 1821-1825
Author(s):  
K A Kelley ◽  
J W Chamberlain ◽  
J A Nolan ◽  
A L Horwich ◽  
F Kalousek ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fusion Gene ◽  
Regulatory Region ◽  
Transgenic Animals ◽  
Ornithine Transcarbamylase ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Protein Coding ◽  
Wide Range ◽  
Flanking Sequences

In an attempt to use mouse metallothionein-I (mMT-I) regulatory sequences to direct expression of human ornithine transcarbamylase in the liver of transgenic animals, fusion genes joining either 1.6 kilobases or 185 base pairs of the mMT-I regulatory region to the human ornithine transcarbamylase protein-coding sequence were used to produce transgenic mice. In mice carrying the fusion gene with 1.6 kilobases of the mMT-I 5'-flanking sequences, transgene expression was observed in a wide range of tissues, but, unexpectedly, expression in liver was never observed. Surprisingly, in mice carrying the fusion gene regulated by only 185 base pairs of the mMT-I 5'-flanking sequences, the transgene was expressed exclusively in male germ cells during the tetraploid, pachytene stage of meiosis.

Trans-acting regulatory mutations that alter transcription of Saccharomyces cerevisiae histone genes

1987 ◽  
Vol 7 (12) ◽  
pp. 4204-4210
Author(s):  
M A Osley ◽  
D Lycan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Fusion Gene ◽  
Chromosome Replication ◽  
Lacz Fusion ◽  
Histone Genes ◽  
Promoter Elements ◽  
Regulatory Mutations ◽  
Cycle Dependent ◽  
Beta Galactosidase

Using a Saccharomyces cerevisiae strain containing an integrated copy of an H2A-lacZ fusion gene, we screened for mutants which overexpressed beta-galactosidase as a way to identify genes which regulate transcription of the histone genes. Five recessive mutants with this phenotype were shown to contain altered regulatory genes because they had lost repression of HTA1 transcription which occurs upon inhibition of chromosome replication (D. E. Lycan, M. A. Osley, and L. Hereford, Mol. Cell. Biol. 7:614-621, 1987). Periodic transcription was affected in the mutants as well, since the HTA1 gene was transcribed during the G1 and G2 phases of the cell cycle, periods in the cell cycle when this gene is normally not expressed. A similar loss of cell cycle-dependent transcription was noted for two of the three remaining histone loci, while the HO and CDC9 genes continued to be expressed periodically. Using isolated promoter elements inserted into a heterologous cycl-lacZ fusion gene, we demonstrated that the mutations fell in genes which acted through a negative site in the TRT1 H2A-H2B promoter.

Differential effectiveness of yeast cytochrome c oxidase subunit genes results from differences in expression not function

1987 ◽  
Vol 7 (10) ◽  
pp. 3520-3526
Author(s):  
C E Trueblood ◽  
R O Poyton
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Fusion Gene ◽  
Single Copy ◽  
Chimeric Gene ◽  
Lacz Fusion ◽  
Null Alleles ◽  
Activity Levels ◽  
Wild Type ◽  
Oxidase Subunit

In Saccharomyces cerevisiae, COX5a and COX5b encode two distinct forms of cytochrome c oxidase subunit V, Va and Vb, respectively. To determine the relative contribution of COX5a and COX5b to cytochrome c oxidase function, we have disrupted each gene. Cytochrome c oxidase activity levels and respiration rates of strains carrying null alleles of COX5a or COX5b or both indicate that some form of subunit V is required for cytochrome c oxidase function and that COX5a is much more effective than COX5b in providing this function. Wild-type respiration is supported by a single copy of either COX5a or COX5ab (a constructed chimeric gene sharing 5' sequences with COX5a). In contrast, multiple copies of COX5b or COX5ba (a chimeric gene with 5' sequences from COX5b) are required to support wild-type respiration. These results suggest that the decreased effectiveness of COX5b is due to inefficiency in gene expression rather than to any deficiency in the gene product, Vb. This conclusion is supported by two observations: (i) a COX5a-lacZ fusion gene produces more beta-galactosidase than a COX5b-lacZ fusion gene, and (ii) the COX5a transcript is significantly more abundant than the COX5b transcript or the COXsba transcript. We conclude that COX5a is expressed more efficiently than COX5b and that, although mature subunits Va and Vb are only 67% homologous, they do not differ significantly in their ability to assemble and function as subunits of the holoenzyme.

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

1988 ◽  
Vol 8 (6) ◽  
pp. 2523-2535
Author(s):  
J H Hegemann ◽  
J H Shero ◽  
G Cottarel ◽  
P Philippsen ◽  
P Hieter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Vector System ◽  
Chromosome Loss ◽  
Wild Type ◽  
Sequence Elements ◽  
Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

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