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.

scholarly journals Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (1) ◽  
pp. 68-75 ◽  
Author(s):  
A Gaudet ◽  
M Fitzgerald-Hayes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Composition ◽  
Consensus Sequence ◽  
Fold Increase ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Core Element ◽  
Sequence Elements ◽  
Dna Elements ◽  
Chromosome Iii

Centromere DNA from 11 of the 16 chromosomes of the yeast Saccharomyces cerevisiae have been analyzed and reveal three sequence elements common to each centromere, referred to as conserved centromere DNA elements (CDE). The adenine-plus-thymine (A + T)-rich central core element, CDE II, is flanked by two short conserved sequences, CDE I (8 base pairs [bp]) and CDE III (25 bp). Although no consensus sequence exists among the different CDE II regions, they do have three common features of sequence organization. First, the CDE II regions are similar in length, ranging from 78 to 86 bp measured from CDE I to the left boundary of CDE III. Second, the base composition is always greater than 90% A + T. Finally, the A and T residues in these segments are often arranged in runs of A and runs of T residues, sometimes with six or seven bases in a stretch. We constructed insertion, deletion, and replacement mutations in the CDE II region of the centromere from chromosome III, CEN3, designed to investigate the length and sequence requirements for function of the CDE II region of the centromere. We analyzed the effect of these altered centromeres on plasmid and chromosome segregation in S. cerevisiae. Our results show that increasing the length of CDE II from 84 to 154 bp causes a 100-fold increase in chromosome nondisjunction. Deletion mutations removing segments of the A + T-rich CDE II DNA also cause aberrant segregation. In some cases partial function could be restored by replacing the deleted DNA with fragments whose primary sequence or base composition is very different from that of the wild-type CDE II DNA. In addition, we found that identical mutations introduced into different positions in CDE II have very similar effects.

scholarly journals The Anatomy of a Hypoxic Operator in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 150 (4) ◽  
pp. 1429-1441 ◽  
Author(s):  
Jutta Deckert ◽  
Ana Maria Rodriguez Torres ◽  
Soo Myung Hwang ◽  
Alexander J Kastaniotis ◽  
Richard S Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Number Of Genes ◽  
Repression Complex ◽  
Single Base Pair ◽  
Sequence Requirements

Abstract Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes.

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 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 Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (2) ◽  
pp. 530-538 ◽  
Author(s):  
J McGrew ◽  
B Diehl ◽  
M Fitzgerald-Hayes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Dna Sequences ◽  
Base Pair ◽  
Site Directed Mutagenesis ◽  
Chromosome Stability ◽  
Functional Requirements ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Centromere Function

In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.

Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (2) ◽  
pp. 530-538
Author(s):  
J McGrew ◽  
B Diehl ◽  
M Fitzgerald-Hayes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Dna Sequences ◽  
Base Pair ◽  
Site Directed Mutagenesis ◽  
Chromosome Stability ◽  
Functional Requirements ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Centromere Function

In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.

Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae

1987 ◽  
Vol 7 (1) ◽  
pp. 68-75
Author(s):  
A Gaudet ◽  
M Fitzgerald-Hayes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Composition ◽  
Consensus Sequence ◽  
Fold Increase ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Core Element ◽  
Sequence Elements ◽  
Dna Elements ◽  
Chromosome Iii

Centromere DNA from 11 of the 16 chromosomes of the yeast Saccharomyces cerevisiae have been analyzed and reveal three sequence elements common to each centromere, referred to as conserved centromere DNA elements (CDE). The adenine-plus-thymine (A + T)-rich central core element, CDE II, is flanked by two short conserved sequences, CDE I (8 base pairs [bp]) and CDE III (25 bp). Although no consensus sequence exists among the different CDE II regions, they do have three common features of sequence organization. First, the CDE II regions are similar in length, ranging from 78 to 86 bp measured from CDE I to the left boundary of CDE III. Second, the base composition is always greater than 90% A + T. Finally, the A and T residues in these segments are often arranged in runs of A and runs of T residues, sometimes with six or seven bases in a stretch. We constructed insertion, deletion, and replacement mutations in the CDE II region of the centromere from chromosome III, CEN3, designed to investigate the length and sequence requirements for function of the CDE II region of the centromere. We analyzed the effect of these altered centromeres on plasmid and chromosome segregation in S. cerevisiae. Our results show that increasing the length of CDE II from 84 to 154 bp causes a 100-fold increase in chromosome nondisjunction. Deletion mutations removing segments of the A + T-rich CDE II DNA also cause aberrant segregation. In some cases partial function could be restored by replacing the deleted DNA with fragments whose primary sequence or base composition is very different from that of the wild-type CDE II DNA. In addition, we found that identical mutations introduced into different positions in CDE II have very similar effects.

scholarly journals hsp26 of Saccharomyces cerevisiae is related to the superfamily of small heat shock proteins but is without a demonstrable function.

1989 ◽  
Vol 9 (11) ◽  
pp. 5265-5271 ◽  
Author(s):  
R E Susek ◽  
S L Lindquist
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Mutational Analysis ◽  
Small Heat Shock Protein ◽  
Major Effect ◽  
Selfish Dna ◽  
Dna Elements ◽  
Cloned Gene ◽  
Shock Proteins

Analysis of the cloned gene confirms that hsp26 of Saccharomyces cerevisiae is a member of the small heat shock protein superfamily. Previous mutational analysis failed to demonstrate any function for the protein. Further experiments presented here demonstrate that hsp26 has no obvious regulatory role and no major effect on thermotolerance. It is possible that the small heat shock protein genes originated as primitive viral or selfish DNA elements.

DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (9) ◽  
pp. 6350-6360
Author(s):  
F Houman ◽  
C Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Essential Gene ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Nonsense Mutations ◽  
Carboxy Terminal

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.

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