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.

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 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.

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.

scholarly journals Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres.

1990 ◽  
Vol 10 (11) ◽  
pp. 5721-5727 ◽  
Author(s):  
M J Saunders ◽  
E Yeh ◽  
M Grunstein ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Base Pairs ◽  
Centromere Function ◽  
Nuclease Digestion ◽  
Dna Elements ◽  
Chromosome Iii ◽  
Sequence Requirements

Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200 base pairs of DNA. The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core and encompasses the conserved centromere DNA elements (CDE I, II, and III). Extensive mutational analysis has revealed the sequence requirements for centromere function. Mutations affecting the segregation properties of centromeres also exhibit altered chromatin structures in vivo. Thus the structure, as delineated by nuclease digestion, correlated with functional centromeres. We have determined the contribution of histone proteins to this unique structural organization. Nucleosome depletion by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation. Histone repression resulted in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic attack. Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding CEN3. These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes may be the central core of the centromere itself.

scholarly journals Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres

1990 ◽  
Vol 10 (11) ◽  
pp. 5721-5727
Author(s):  
M J Saunders ◽  
E Yeh ◽  
M Grunstein ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Base Pairs ◽  
Centromere Function ◽  
Nuclease Digestion ◽  
Dna Elements ◽  
Chromosome Iii ◽  
Sequence Requirements

Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200 base pairs of DNA. The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core and encompasses the conserved centromere DNA elements (CDE I, II, and III). Extensive mutational analysis has revealed the sequence requirements for centromere function. Mutations affecting the segregation properties of centromeres also exhibit altered chromatin structures in vivo. Thus the structure, as delineated by nuclease digestion, correlated with functional centromeres. We have determined the contribution of histone proteins to this unique structural organization. Nucleosome depletion by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation. Histone repression resulted in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic attack. Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding CEN3. These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes may be the central core of the centromere itself.

In vivo characterization of the Saccharomyces cerevisiae centromere DNA element I, a binding site for the helix-loop-helix protein CPF1

1991 ◽  
Vol 11 (7) ◽  
pp. 3545-3553
Author(s):  
R Niedenthal ◽  
R Stoll ◽  
J H Hegemann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Mutational Analysis ◽  
Synergistic Effects ◽  
Point Mutations ◽  
Artificial Chromosome ◽  
Palindromic Sequence ◽  
Base Pairs ◽  
Helix Loop Helix

The centromere DNA element I (CDEI) is an important component of Saccharomyces cerevisiae centromere DNA and carries the palindromic sequence CACRTG (R = purine) as a characteristic feature. In vivo, CDEI is bound by the helix-loop-helix protein CPF1. This article describes the in vivo analysis of all single-base-pair substitutions in CDEI in the centromere of an artificial chromosome and demonstrates the importance of the palindromic sequence for faithful chromosome segregation, supporting the notion that CPF1 binds as a dimer to this binding site. Mutational analysis of two conserved base pairs on the left and two nonconserved base pairs on the right of the CDEI palindrome revealed that these are also relevant for mitotic CEN function. Symmetrical mutations in either half-site of the palindrome affect centromere activity to a different extent, indicating nonidentical sequence requirements for binding by the CPF1 homodimer. Analysis of double point mutations in CDEI and in CDEIII, an additional centromere element, indicate synergistic effects between the DNA-protein complexes at these sites.

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 In vivo characterization of the Saccharomyces cerevisiae centromere DNA element I, a binding site for the helix-loop-helix protein CPF1.

1991 ◽  
Vol 11 (7) ◽  
pp. 3545-3553 ◽  
Author(s):  
R Niedenthal ◽  
R Stoll ◽  
J H Hegemann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Mutational Analysis ◽  
Synergistic Effects ◽  
Point Mutations ◽  
Artificial Chromosome ◽  
Palindromic Sequence ◽  
Base Pairs ◽  
Helix Loop Helix

The centromere DNA element I (CDEI) is an important component of Saccharomyces cerevisiae centromere DNA and carries the palindromic sequence CACRTG (R = purine) as a characteristic feature. In vivo, CDEI is bound by the helix-loop-helix protein CPF1. This article describes the in vivo analysis of all single-base-pair substitutions in CDEI in the centromere of an artificial chromosome and demonstrates the importance of the palindromic sequence for faithful chromosome segregation, supporting the notion that CPF1 binds as a dimer to this binding site. Mutational analysis of two conserved base pairs on the left and two nonconserved base pairs on the right of the CDEI palindrome revealed that these are also relevant for mitotic CEN function. Symmetrical mutations in either half-site of the palindrome affect centromere activity to a different extent, indicating nonidentical sequence requirements for binding by the CPF1 homodimer. Analysis of double point mutations in CDEI and in CDEIII, an additional centromere element, indicate synergistic effects between the DNA-protein complexes at these sites.

scholarly journals Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site.

1988 ◽  
Vol 8 (2) ◽  
pp. 647-654 ◽  
Author(s):  
S L Forsburg ◽  
L Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Striking Similarity ◽  
Base Pairs ◽  
Functional Elements ◽  
Upstream Activation Sequence ◽  
Regulatory Loci ◽  
Transition Mutation ◽  
Activation Sequence

We analyzed upstream activation sequence 2 (UAS2), one of two independent UAS elements in the CYC1 gene of Saccharomyces cerevisiae. Deletions and linker scanning mutations across the 87 base pairs previously defined as UAS2 showed two separate functional elements required for full activity. Region 1, from -230 to -200, contains the principal activation site and responds to the trans-acting regulatory loci HAP2 and HAP3. A portion of region 1 is homologous to two other HAP2-HAP3-responsive UASs and includes the G----A transition mutation UP1, which increases UAS2 activity. This consensus sequence TNATTGGT bears striking similarity to several CAAT box sequences of higher cells. Region 2, from -192 to -178, substantially enhances the activity of region 1, yet has little activity by itself. These regions bind distinct proteins found in crudely fractionated yeast extracts.

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