scholarly journals Subtelomeric ACS-containing Proto-silencers Act as Antisilencers in Replication Factors Mutants in Saccharomyces cerevisiae

2009 ◽  
Vol 20 (2) ◽  
pp. 631-641 ◽  
Author(s):  
Muhammad Attiq Rehman ◽  
Dongliang Wang ◽  
Genevieve Fourel ◽  
Eric Gilson ◽  
Krassimir Yankulov
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Position Effect ◽  
S Phase ◽  
Dependent Manner ◽  
Wild Type ◽  
Replication Factors ◽  
Late Stages ◽  
Subtelomeric Regions

Subtelomeric genes are either fully active or completely repressed and can switch their state about once per 20 generations. This meta-stable telomeric position effect is mediated by strong repression signals emitted by the telomere and relayed/enhanced by weaker repressor elements called proto-silencers. In addition, subtelomeric regions contain sequences with chromatin partitioning and antisilencing activities referred to as subtelomeric antisilencing regions. Using extensive mutational analysis of subtelomeric elements, we show that ARS consensus sequence (ACS)-containing proto-silencers convert to antisilencers in several replication factor mutants. We point out the significance of the B1 auxiliary sequence next to ACS in mediating these effects. In contrast, an origin-derived ACS does not convert to antisilencer in mutants and its B1 element has little bearing on silencing. These results are specific for the analyzed ACS and in addition to the effects of each mutation (relative to wild type) on global silencing. Another line of experiments shows that Mcm5p possesses antisilencing activity and is recruited to telomeres in an ACS-dependent manner. Mcm5p persists at this location at the late stages of S phase. We propose that telomeric ACS are not static proto-silencers but conduct finely tuned silencing and antisilencing activities mediated by ACS-bound factors.

scholarly journals Cell cycle-dependent chromatin dynamics at replication origins

2021 ◽  
Author(s):  
Yulong Li ◽  
Alexander J. Hartemink ◽  
David MacAlpine
Keyword(s):  
Cell Cycle ◽  
Consensus Sequence ◽  
S Phase ◽  
Micrococcal Nuclease ◽  
Dependent Manner ◽  
Replication Origins ◽  
Chromatin Dynamics ◽  
Cell Cycles ◽  
Cycle Dependent ◽  
Replication Factors

Origins of DNA replication are specified by the ordered recruitment of replication factors in a cell cycle dependent manner. The assembly of the pre-replicative complex in G1 and the pre-initiation complex prior to activation in S-phase are well characterized; however, the interplay between the assembly of these complexes and the local chromatin environment is less well understood. To investigate the dynamic changes in chromatin organization at and surrounding replication origins, we used micrococcal nuclease (MNase) to generate genome-wide chromatin occupancy profiles of nucleosomes, transcription factors and replication proteins through consecutive cell cycles in Saccharomyces cerevisiae. During each G1 phase of two consecutive cell cycles, we observed the downstream repositioning of the origin-proximal +1 nucleosome and an increase in protected DNA fragments spanning the ARS consensus sequence (ACS) indicative of pre-RC assembly. We also found that the strongest correlation between the chromatin occupancy at the ACS and origin efficiency occurred in early S-phase consistent with the rate limiting formation of the Cdc45-Mcm2-7-GINS (CMG) complex being a determinant of origin activity. Finally, we observed nucleosome disruption and disorganization emanating from replication origins and traveling with the elongating replication forks across the genome in S-phase, likely reflecting the disassembly and assembly of chromatin ahead of and behind the replication fork, respectively. These results provide insights into cell cycle-regulated chromatin dynamics and how they relate to the regulation of origin activity.

Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1.

Genetics ◽  
1993 ◽  
Vol 133 (4) ◽  
pp. 851-863 ◽  
Author(s):  
C F Lesser ◽  
C Guthrie
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Reporter Gene ◽  
Splice Site ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Intron Position ◽  
Mrna Splicing ◽  
Copper Resistance ◽  
Dependent Manner ◽  
Fusion Construct

Abstract We have developed a new reporter gene fusion to monitor mRNA splicing in yeast. An intron-containing fragment from the Saccharomyces cerevisiae ACT1 gene has been fused to CUP1, the yeast metallothionein homolog. CUP1 is a nonessential gene that allows cells to grow in the presence of copper in a dosage-dependent manner. By inserting previously characterized intron mutations into the fusion construct, we have established that the efficiency of splicing correlates with the level of copper resistance of these strains. A highly sensitive assay for 5' splice site usage was designed by engineering an ACT1-CUP1 construct with duplicated 5' splice sites; mutations were introduced into the upstream splice site in order to evaluate the roles of these highly conserved nucleotides in intron recognition. Almost all mutations in the intron portion of the 5' consensus sequence abolish recognition of the mutated site, while mutations in the exon portion of the consensus sequence have variable affects on cleavage at the mutated site. Interestingly, mutations at intron position 4 demonstrate that this nucleotide plays a role in 5' splice site recognition other than by base pairing with U1 snRNA. The use of CUP1 as a reporter gene may be generally applicable for monitoring cellular processes in yeast.

scholarly journals Cell-Cycle—Dependent Chromatin Dynamics at Replication Origins

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1998
Author(s):  
Yulong Li ◽  
Alexander J. Hartemink ◽  
David M. MacAlpine
Keyword(s):  
Cell Cycle ◽  
Consensus Sequence ◽  
S Phase ◽  
Micrococcal Nuclease ◽  
Dependent Manner ◽  
Replication Origins ◽  
Chromatin Dynamics ◽  
Cell Cycles ◽  
Cycle Dependent ◽  
Replication Factors

Origins of DNA replication are specified by the ordered recruitment of replication factors in a cell-cycle—dependent manner. The assembly of the pre-replicative complex in G1 and the pre-initiation complex prior to activation in S phase are well characterized; however, the interplay between the assembly of these complexes and the local chromatin environment is less well understood. To investigate the dynamic changes in chromatin organization at and surrounding replication origins, we used micrococcal nuclease (MNase) to generate genome-wide chromatin occupancy profiles of nucleosomes, transcription factors, and replication proteins through consecutive cell cycles in Saccharomyces cerevisiae. During each G1 phase of two consecutive cell cycles, we observed the downstream repositioning of the origin-proximal +1 nucleosome and an increase in protected DNA fragments spanning the ARS consensus sequence (ACS) indicative of pre-RC assembly. We also found that the strongest correlation between chromatin occupancy at the ACS and origin efficiency occurred in early S phase, consistent with the rate-limiting formation of the Cdc45–Mcm2-7–GINS (CMG) complex being a determinant of origin activity. Finally, we observed nucleosome disruption and disorganization emanating from replication origins and traveling with the elongating replication forks across the genome in S phase, likely reflecting the disassembly and assembly of chromatin ahead of and behind the replication fork, respectively. These results provide insights into cell-cycle–regulated chromatin dynamics and how they relate to the regulation of origin activity.

scholarly journals Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions

2017 ◽  
Author(s):  
Antoine Hocher ◽  
Myriam Ruault ◽  
Petra Kaferle ◽  
Marc Descrimes ◽  
Mickael Garnier ◽  
...  
Keyword(s):  
Meta Analysis ◽  
Position Effect ◽  
Histone Mark ◽  
Eukaryotic Genome ◽  
Wild Type ◽  
Histone Marks ◽  
The Core ◽  
Defective Mutant ◽  
H3k79 Methylation ◽  
Subtelomeric Regions

AbstractThe eukaryotic genome is divided into chromosomal domains of heterochromatin and euchromatin. Transcriptionally silent heterochromatin is found at subtelomeric regions, leading to the telomeric position effect (TPE) in yeast, fly and man. Heterochromatin generally initiates and spreads from defined loci, and diverse mechanisms prevent the ectopic spread of heterochromatin into euchromatin. Here, we overexpressed the silencing factor Sir3 at various levels in yeast, and found that Sir3 spreading into Extended Silent Domains (ESD) eventually reached saturation at subtelomeres. We observed that Sir3 spreading into ESDs covered zone associated with specific histone marks in wild-type cells and stopped at zones of histone mark transitions including H3K79 tri-methylation levels. The conserved enzyme Dot1 deposits H3K79 methylation, and we found that it is essential for viability upon overexpression of Sir3, but not of a spreading-defective mutant Sir3A2Q. These data suggest that H3K79 methylation actively blocks Sir3 spreading. Lastly, our meta-analysis uncovers previously uncharacterized discrete subtelomeric domains associated with specific chromatin features offering a new viewpoint on how to separate subtelomeres from the core chromosome.

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.

Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity

1991 ◽  
Vol 11 (7) ◽  
pp. 3537-3544 ◽  
Author(s):  
M Ziman ◽  
J M O'Brien ◽  
L A Ouellette ◽  
W R Church ◽  
D I Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Mutational Analysis ◽  
Mutant Gene ◽  
Biochemical Properties ◽  
Wild Type ◽  
Mutant Proteins ◽  
Dominant Lethal ◽  
Gtp Binding ◽  
Multiple Buds

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

Yeast intragenic transcriptional control: activation and repression sites within the coding region of the Saccharomyces cerevisiae LPD1 gene

1994 ◽  
Vol 14 (1) ◽  
pp. 214-225
Author(s):  
D A Sinclair ◽  
G D Kornfeld ◽  
I W Dawes
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Rna Polymerase Ii ◽  
Mutational Analysis ◽  
Lacz Fusion ◽  
Dependent Manner ◽  
Coding Region ◽  
Coding Sequence

Though widely recognized in higher eukaryotes, the regulation of Saccharomyces cerevisiae genes transcribed by RNA polymerase II by proteins that bind within the coding sequence remains largely speculative. We have shown for the LPD1 gene, encoding lipoamide dehydrogenase, that the coding sequence between +13 and +469 activated gene expression of an LPD1::lacZ fusion by up to sixfold in the presence of the upstream promoter. This downstream region, inserted upstream of a promoterless CYC1::lacZ fusion, activated gene expression in a carbon source-dependent manner by a factor of 15 to 111, independent of orientation. Deletion and mutational analysis identified two downstream activation sites (DAS1 and DAS2) and two downstream repressor sites (DRS1 and DRS2) that influence the rate of LPD1 transcription rather than mRNA degradation or translation. Activation from the DAS1 region (positions +137 to +191), encompassing a CDEI-like element, is twofold under derepressive conditions. Activation from DAS2 (+291 to +296), a CRE-like motif, is 12-fold for both repressed and derepressed states. DRS1, a pair of adjacent and opposing ABF1 sites (+288 to +313), is responsible for a 1.3- to 2-fold repression of transcription, depending on the carbon source. DRS1 requires the concerted action of DRS2 (a RAP1 motif at position +406) for repression of transcription only when the gene is induced. Gel mobility shift analysis and in vitro footprinting have shown that proteins bind in vitro to these downstream elements.

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.

scholarly journals Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity.

1991 ◽  
Vol 11 (7) ◽  
pp. 3537-3544 ◽  
Author(s):  
M Ziman ◽  
J M O'Brien ◽  
L A Ouellette ◽  
W R Church ◽  
D I Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Mutational Analysis ◽  
Mutant Gene ◽  
Biochemical Properties ◽  
Wild Type ◽  
Mutant Proteins ◽  
Dominant Lethal ◽  
Gtp Binding ◽  
Multiple Buds

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

scholarly journals Thecdr2+Gene Encodes a Regulator of G2/M Progression and Cytokinesis inSchizosaccharomyces pombe

1998 ◽  
Vol 9 (12) ◽  
pp. 3399-3415 ◽  
Author(s):  
Connie S. Breeding ◽  
James Hudson ◽  
Mohan K. Balasubramanian ◽  
Sean M. Hemmingsen ◽  
Paul G. Young ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Cell Size ◽  
Size Control ◽  
S Phase ◽  
Nutrient Deprivation ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
M Cell ◽  
Mitotic Control

Schizosaccharomyces pombe cells respond to nutrient deprivation by altering G2/M cell size control. The G2/M transition is controlled by activation of the cyclin-dependent kinase Cdc2p. Cdc2p activation is regulated both positively and negatively. cdr2+was identified in a screen for regulators of mitotic control during nutrient deprivation. We have cloned cdr2+and have found that it encodes a putative serine-threonine protein kinase that is related to Saccharomyces cerevisiae Gin4p and S. pombe Cdr1p/Nim1p.cdr2+is not essential for viability, but cells lacking cdr2+are elongated relative to wild-type cells, spending a longer period of time in G2. Because of this property, upon nitrogen deprivationcdr2+mutants do not arrest in G1, but rather undergo another round of S phase and arrest in G2from which they are able to enter a state of quiescence. Genetic evidence suggests thatcdr2+acts as a mitotic inducer, functioning through wee1+, and is also important for the completion of cytokinesis at 36°C. Defects in cytokinesis are also generated by the overproduction of Cdr2p, but these defects are independent of wee1+, suggesting thatcdr2+encodes a second activity involved in cytokinesis.

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