Identification of Autonomously Replicating Sequence (ARS) Elements in Eukaryotic Cells

Methods ◽  
1997 ◽  
Vol 13 (3) ◽  
pp. 221-233 ◽  
Author(s):  
Rosemary K. Clyne ◽  
Thomas J. Kelly
Keyword(s):  
Eukaryotic Cells ◽  
Autonomously Replicating Sequence ◽  
Ars Elements

scholarly journals Completion of Replication Map of Saccharomyces cerevisiae Chromosome III

2001 ◽  
Vol 12 (11) ◽  
pp. 3317-3327 ◽  
Author(s):  
Arkadi Poloumienko ◽  
Ann Dershowitz ◽  
Jitakshi De ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Complete Analysis ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Eukaryotic Chromosome ◽  
Ars Elements ◽  
Chromosome Iii

In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of ∼40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements onS. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in ≤10% of cells in the population and two ARS elements active in ≥90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

The replication of yeast chromosomes: lessons fromSaccharomyces cerevisiaechromosome III

1995 ◽  
Vol 73 (S1) ◽  
pp. 208-214 ◽  
Author(s):  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Replication Origins ◽  
Chromosomal Replication ◽  
Position Effects ◽  
Autonomously Replicating Sequence ◽  
Systematic Analysis ◽  
Eukaryotic Chromosome ◽  
Ars Elements ◽  
Constant Rate ◽  
Chromosome Iii

To understand how a eukaryotic chromosome is replicated, a systematic analysis of chromosome III of Saccharomyces cerevisiae has been undertaken. Replication origins are specified by autonomously replicating sequence (ARS) elements, whose sequences can be dissected using a simple plasmid assay. Only a subset of ARS elements are active as chromosomal replication origins. Replication origins are required for normal chromosome transmission, but they appear to be redundant; several origins can be deleted without affecting chromosome stability. Replication origin position has been conserved on chromosome III in diverged strains, suggesting that origin position is important for chromosome function. The inability of some ARS elements to function as chromosomal replication origins appears likely to result from chromosomal context or position effects. Replication termination occurs over broad regions between active replication origins. The position of termination can be altered by deleting origins, suggesting that no specific replication termination elements are required. Replication forks appear to move at a relatively constant rate through the chromosome. A replication pause site associated with the centromere results from the kinetochore protein complex that binds the centromere to mediate chromosome segregation. Key words: Saccharomyces cerevisiae, ARS elements, replication origins, replication termination, DNA replication intermediates.

scholarly journals Activation of Silent Replication Origins at Autonomously Replicating Sequence Elements near the HML Locus in Budding Yeast

1999 ◽  
Vol 19 (9) ◽  
pp. 6098-6109 ◽  
Author(s):  
Marija Vujcic ◽  
Charles A. Miller ◽  
David Kowalski
Keyword(s):  
Replication Origin ◽  
Budding Yeast ◽  
Transcriptional Silencing ◽  
Growth Conditions ◽  
Replication Origins ◽  
Additional Time ◽  
Autonomously Replicating Sequence ◽  
Ars Elements ◽  
Active Replication ◽  
Chromosome Iii

ABSTRACT In the budding yeast, Saccharomyces cerevisiae, replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity. Two of these ARSs comprise the transcriptional silencers E (ARS301) and I (ARS302). Another, ARS303, resides betweenHML and the CHA1 gene, and its function is not known. Here we further localized and characterized ARS303and in the process discovered a new ARS, ARS320. BothARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III. However, at their native locations, where the two ARSs are in a cluster withARS302, the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene,trans-acting mutations that abrogate transcriptional silencing at HML (sir3, orc5), orcis-acting mutations that delete the E and I silencers containing ARS elements. These results suggest that, for theHML ARS cluster (ARS303, ARS320, and ARS302), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share keycis- and trans-acting components. Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at theHML ARS cluster, as well as at ARS301, the E silencer. Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances fromHML in the chromosome. The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML, providing additional time for HML ARSs to fire as origins.

Chromosomal DNA replication initiates at the same origins in meiosis and mitosis

1994 ◽  
Vol 14 (5) ◽  
pp. 3524-3534
Author(s):  
I Collins ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
S Phase ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Ars Elements ◽  
Chromosome Iii

Autonomously replicating sequence (ARS) elements are identified by their ability to promote high-frequency transformation and extrachromosomal replication of plasmids in the yeast Saccharomyces cerevisiae. Six of the 14 ARS elements present in a 200-kb region of Saccharomyces cerevisiae chromosome III are mitotic chromosomal replication origins. The unexpected observation that eight ARS elements do not function at detectable levels as chromosomal replication origins during mitotic growth suggested that these ARS elements may function as chromosomal origins during premeiotic S phase. Two-dimensional agarose gel electrophoresis was used to map premeiotic replication origins in a 100-kb segment of chromosome III between HML and CEN3. The pattern of origin usage in premeiotic S phase was identical to that in mitotic S phase, with the possible exception of ARS308, which is an inefficient mitotic origin associated with CEN3. CEN3 was found to replicate during premeiotic S phase, demonstrating that the failure of sister chromatids to disjoin during the meiosis I division is not due to unreplicated centromeres. No origins were found in the DNA fragments without ARS function. Thus, in both mitosis and meiosis, chromosomal replication origins are coincident with ARS elements but not all ARS elements have chromosomal origin function. The efficiency of origin use and the patterns of replication termination are similar in meiosis and in mitosis. DNA replication termination occurs over a broad distance between active origins.

scholarly journals Chromosomal DNA replication initiates at the same origins in meiosis and mitosis.

1994 ◽  
Vol 14 (5) ◽  
pp. 3524-3534 ◽  
Author(s):  
I Collins ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
S Phase ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Ars Elements ◽  
Chromosome Iii

Autonomously replicating sequence (ARS) elements are identified by their ability to promote high-frequency transformation and extrachromosomal replication of plasmids in the yeast Saccharomyces cerevisiae. Six of the 14 ARS elements present in a 200-kb region of Saccharomyces cerevisiae chromosome III are mitotic chromosomal replication origins. The unexpected observation that eight ARS elements do not function at detectable levels as chromosomal replication origins during mitotic growth suggested that these ARS elements may function as chromosomal origins during premeiotic S phase. Two-dimensional agarose gel electrophoresis was used to map premeiotic replication origins in a 100-kb segment of chromosome III between HML and CEN3. The pattern of origin usage in premeiotic S phase was identical to that in mitotic S phase, with the possible exception of ARS308, which is an inefficient mitotic origin associated with CEN3. CEN3 was found to replicate during premeiotic S phase, demonstrating that the failure of sister chromatids to disjoin during the meiosis I division is not due to unreplicated centromeres. No origins were found in the DNA fragments without ARS function. Thus, in both mitosis and meiosis, chromosomal replication origins are coincident with ARS elements but not all ARS elements have chromosomal origin function. The efficiency of origin use and the patterns of replication termination are similar in meiosis and in mitosis. DNA replication termination occurs over a broad distance between active origins.

scholarly journals Analysis of Chromosome III Replicators Reveals an Unusual Structure for the ARS318 Silencer Origin and a Conserved WTW Sequence within the Origin Recognition Complex Binding Site

2008 ◽  
Vol 28 (16) ◽  
pp. 5071-5081 ◽  
Author(s):  
FuJung Chang ◽  
James F. Theis ◽  
Jeremy Miller ◽  
Conrad A. Nieduszynski ◽  
Carol S. Newlon ◽  
...  
Keyword(s):  
Binding Site ◽  
Origin Recognition Complex ◽  
Consensus Sequence ◽  
Sensu Stricto ◽  
Unusual Structure ◽  
Multiple Sequence ◽  
Autonomously Replicating Sequence ◽  
Ars Elements ◽  
Chromosome Iii ◽  
Origin Recognition

ABSTRACT Saccharomyces cerevisiae chromosome III encodes 11 autonomously replicating sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to the ACS that positions the ORC binding site only 6 bp away from an Abf1p binding site. Although a largely inactive replicator on the chromosome, ARS318 becomes active if the nearby HMR-E silencer is deleted. We also performed a multiple sequence alignment of confirmed replicators on chromosomes III, VI, and VII. This analysis revealed a highly conserved WTW motif 17 to 19 bp from the ACS that is functionally important and is apparent in the 228 phylogenetically conserved ARS elements among the six sensu stricto Saccharomyces species.

Drosophila scaffold-attached regions bind nuclear scaffolds and can function as ARS elements in both budding and fission yeasts

1990 ◽  
Vol 10 (10) ◽  
pp. 5442-5454
Author(s):  
B Amati ◽  
S M Gasser
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fission Yeast ◽  
Genomic Dna ◽  
Yeast Species ◽  
Specific Interactions ◽  
Autonomously Replicating Sequence ◽  
Autonomous Replication ◽  
Ars Elements ◽  
Sequence Elements

Histone-depleted nuclei maintain sequence-specific interactions with genomic DNA at sites known as scaffold attachment regions (SARs) or matrix attachment regions. We have previously shown that in Saccharomyces cerevisiae, autonomously replicating sequence elements bind the nuclear scaffold. Here, we extend these observations to the fission yeast Schizosaccharomyces pombe. In addition, we show that four SARs previously mapped in the genomic DNA of Drosophila melanogaster bind in vitro to nuclear scaffolds from both yeast species. In view of these results, we have assayed the ability of the Drosophila SARs to promote autonomous replication of plasmids in the two yeast species. Two of the Drosophila SARs have autonomously replicating sequence activity in budding yeast, and three function in fission yeast, while four flanking non-SAR sequences are totally inactive in both.

scholarly journals Drosophila scaffold-attached regions bind nuclear scaffolds and can function as ARS elements in both budding and fission yeasts.

1990 ◽  
Vol 10 (10) ◽  
pp. 5442-5454 ◽  
Author(s):  
B Amati ◽  
S M Gasser
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fission Yeast ◽  
Genomic Dna ◽  
Yeast Species ◽  
Specific Interactions ◽  
Autonomously Replicating Sequence ◽  
Autonomous Replication ◽  
Ars Elements ◽  
Sequence Elements

Histone-depleted nuclei maintain sequence-specific interactions with genomic DNA at sites known as scaffold attachment regions (SARs) or matrix attachment regions. We have previously shown that in Saccharomyces cerevisiae, autonomously replicating sequence elements bind the nuclear scaffold. Here, we extend these observations to the fission yeast Schizosaccharomyces pombe. In addition, we show that four SARs previously mapped in the genomic DNA of Drosophila melanogaster bind in vitro to nuclear scaffolds from both yeast species. In view of these results, we have assayed the ability of the Drosophila SARs to promote autonomous replication of plasmids in the two yeast species. Two of the Drosophila SARs have autonomously replicating sequence activity in budding yeast, and three function in fission yeast, while four flanking non-SAR sequences are totally inactive in both.

High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

1992 ◽  
Vol 50 (1) ◽  
pp. 512-513
Author(s):  
J. Jakana ◽  
M.F. Schmid ◽  
P. Matsudaira ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Binding Proteins ◽  
Actin Binding ◽  
Eukaryotic Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Actin Bundle ◽  
Actin Bundles ◽  
3 Dimensional ◽  
Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

Structure of clathrin cages and coats in ice

1986 ◽  
Vol 44 ◽  
pp. 94-97
Author(s):  
G.P.A. Vigers ◽  
R.A. Crowther ◽  
B.M.F. Pearse
Keyword(s):  
Electron Microscopy ◽  
Heavy Chain ◽  
Outer Part ◽  
Coated Vesicles ◽  
Eukaryotic Cells ◽  
Coat Proteins ◽  
Terminal Domain ◽  
Clathrin Heavy Chain ◽  
Membrane Components

Clathrin forms the polyhedral cage of coated vesicles, which mediate the transfer of selected membrane components within eukaryotic cells. Clathrin cages and coated vesicles have been extensively studied by electron microscopy of negatively stained preparations and shadowed specimens. From these studies the gross morphology of the outer part of the polyhedral coat has been established and some features of the packing of clathrin trimers into the coat have also been described. However these previous studies have not revealed any internal details about the position of the terminal domain of the clathrin heavy chain, the location of the 100kd-50kd accessory coat proteins or the interactions of the coat with the enclosed membrane.

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