scholarly journals Structure and sequence of the centromeric DNA of chromosome 4 in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (1) ◽  
pp. 241-245 ◽  
Author(s):  
C Mann ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Consensus Sequence ◽  
The Other ◽  
Yeast Cells ◽  
Chromosome 4 ◽  
Mitotic Stability ◽  
Centromeric Dna ◽  
Sequence Homologies ◽  
Sequence Elements

The CEN4 sequences from chromosome 4 that impart mitotic stability to autonomously replicating (ARS) plasmids in yeast cells have been localized to a 1,755-base-pair (bp) fragment. This fragment could be cut in half to give two adjacent, nonoverlapping fragments, that each contained some mitotic stabilization sequences. One of the half-fragments worked as efficiently as the larger fragment from which it was derived, while the other half provided a much poorer degree of mitotic stabilization. Sequencing of 2,095 bp of DNA including this region revealed the presence of a centromere consensus sequence, elements I, II, and III (M. Fitzgerald-Hayes, L. Clarke, and J. Carbon, Cell 29:235-244, 1982), in the half-fragment providing high levels of mitotic stability. The poorly stabilizing half-fragment did not contain any obvious sequence homologies to other centromere sequences. Deletion analysis of the 1,755-bp fragment indicated that removal of the 14-bp element I plus 16 of the 82 bp of element II impaired mitotic stability. Removal of elements I and II eliminated the mitotic stability provided by the consensus sequence.

Structure and sequence of the centromeric DNA of chromosome 4 in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (1) ◽  
pp. 241-245
Author(s):  
C Mann ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Consensus Sequence ◽  
The Other ◽  
Yeast Cells ◽  
Chromosome 4 ◽  
Mitotic Stability ◽  
Centromeric Dna ◽  
Sequence Homologies ◽  
Sequence Elements

The CEN4 sequences from chromosome 4 that impart mitotic stability to autonomously replicating (ARS) plasmids in yeast cells have been localized to a 1,755-base-pair (bp) fragment. This fragment could be cut in half to give two adjacent, nonoverlapping fragments, that each contained some mitotic stabilization sequences. One of the half-fragments worked as efficiently as the larger fragment from which it was derived, while the other half provided a much poorer degree of mitotic stabilization. Sequencing of 2,095 bp of DNA including this region revealed the presence of a centromere consensus sequence, elements I, II, and III (M. Fitzgerald-Hayes, L. Clarke, and J. Carbon, Cell 29:235-244, 1982), in the half-fragment providing high levels of mitotic stability. The poorly stabilizing half-fragment did not contain any obvious sequence homologies to other centromere sequences. Deletion analysis of the 1,755-bp fragment indicated that removal of the 14-bp element I plus 16 of the 82 bp of element II impaired mitotic stability. Removal of elements I and II eliminated the mitotic stability provided by the consensus sequence.

scholarly journals The Unstable F-box Protein p58-Ctf13 Forms the Structural Core of the CBF3 Kinetochore Complex

1999 ◽  
Vol 145 (5) ◽  
pp. 933-950 ◽  
Author(s):  
Iain D. Russell ◽  
Adam S. Grancell ◽  
Peter K. Sorger
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Properties ◽  
Chromosome Segregation ◽  
Half Life ◽  
Degradation Pathway ◽  
The Other ◽  
Yeast Cells ◽  
Centromeric Dna ◽  
Structural And Functional Properties ◽  
F Box Protein

Kinetochores are smaller and more accessible experimentally in budding yeast than in any other eukaryote. Believing that simple and complex kinetochores have important structural and functional properties in common, we characterized the structure of CBF3, the essential centromere-binding complex that initiates kinetochore formation in Saccharomyces cerevisiae. We find that the four subunits of CBF3 are multimeric in solution: p23Skp1 and p58Ctf13 form a heterodimer, and p64Cep3 and p110Ndc10 form homodimers. Subcomplexes involving p58 and each of the other CBF3 subunits can assemble in the absence of centromeric DNA. In these subcomplexes, p58 appears to function as a structural core mediating stable interactions among other CBF3 proteins. p58 has a short half-life in yeast, being subject to ubiquitin-dependent proteolysis, but we find that it is much more stable following association with p64. We propose that p23Skp1-p58-p64 complexes constitute the primary pool of active p58 in yeast cells. These complexes can either dissociate, reexposing p58 to the degradation pathway, or can bind to p110 and centromeric DNA, forming a functional CBF3 complex in which p58 is fully protected from degradation. This pathway may constitute an editing mechanism preventing the formation of ectopic kinetochores and ensuring the fidelity of chromosome segregation.

Repetitive Dictyostelium heat-shock promotor functions in Saccharomyces cerevisiae

1984 ◽  
Vol 4 (4) ◽  
pp. 591-598
Author(s):  
J Cappello ◽  
C Zuker ◽  
H F Lodish
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Nucleotide Sequence ◽  
Dna Sequence ◽  
Base Pair ◽  
Shock Treatment ◽  
Yeast Cells ◽  
Genomic Segment ◽  
Heat Shock Treatment ◽  
Shock Response

The Dictyostelium genome contains 40 copies of a 4.7-kilobase repetitive and apparently transposable DNA sequence (DIRS-1) and about 250 smaller elements that appear to be deletions or rearrangements of DIRS-1. Transcripts of these sequences are induced during differentiation and also by heat shock treatment of growing cells. We showed that one such cloned element, pB41.6 (2.5 kilobases) contains a nucleotide sequence identical to the Drosophila consensus heat shock promotor. To test whether this sequence might indeed control the expression of DIRS-1-related RNAs, we have cloned this genomic segment into yeast cells. In yeast cells, 41.6 directs synthesis of a 1.7-kilobase RNA that is induced at least 10-fold by heat shock. Transcription initiates at about 124 bases 3' of the putative promotor sequence and terminates within the 41.6 insert. A 381-base-pair subclone that contains the putative promotor sequence is sufficient to induce the heat shock response of 41.6 in yeast cells.

Two separate regions of the extrachromosomal ribosomal deoxyribonucleic acid of Tetrahymena thermophila enable autonomous replication of plasmids in Saccharomyces cerevisiae

1981 ◽  
Vol 1 (6) ◽  
pp. 535-543
Author(s):  
G B Kiss ◽  
A A Amin ◽  
R E Pearlman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Frequency ◽  
Deoxyribonucleic Acid ◽  
Tetrahymena Thermophila ◽  
The Other ◽  
Yeast Cells ◽  
Origin Of Replication ◽  
Autonomous Replication ◽  
Coding Regions ◽  
Dna Fragment

Plasmids containing the nontranscribed central and terminal, but not the coding, regions of the extrachromosomal ribosomal deoxyribonucleic acid (rDNA) of Tetrahymena thermophila are capable of autonomous replication in Saccharomyces cerevisiae. These plasmids transform S. cerevisiae at high frequency; transformants are unstable in the absence of selection, and plasmids identical to those used for transformation were isolated from the transformed yeast cells. One plasmid contains a 1.85-kilobase Tetrahymena DNA fragment which includes the origin of bidirectional replication of the extrachromosomal rDNA. The other region of Tetrahymena rDNA allowing autonomous replication of plasmids in S. cerevisiae is a 650-base pair, adenine plus thymine-rich segment from the rDNA terminus. Neither of these Tetrahymena fragments shares obvious sequence homology with the origin of replication of the S. cerevisiae 2-microns circle plasmid or with ars1, an S. cerevisiae chromosomal replicator.

scholarly journals Repetitive Dictyostelium heat-shock promotor functions in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (4) ◽  
pp. 591-598 ◽  
Author(s):  
J Cappello ◽  
C Zuker ◽  
H F Lodish
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Nucleotide Sequence ◽  
Dna Sequence ◽  
Base Pair ◽  
Shock Treatment ◽  
Yeast Cells ◽  
Genomic Segment ◽  
Heat Shock Treatment ◽  
Shock Response

The Dictyostelium genome contains 40 copies of a 4.7-kilobase repetitive and apparently transposable DNA sequence (DIRS-1) and about 250 smaller elements that appear to be deletions or rearrangements of DIRS-1. Transcripts of these sequences are induced during differentiation and also by heat shock treatment of growing cells. We showed that one such cloned element, pB41.6 (2.5 kilobases) contains a nucleotide sequence identical to the Drosophila consensus heat shock promotor. To test whether this sequence might indeed control the expression of DIRS-1-related RNAs, we have cloned this genomic segment into yeast cells. In yeast cells, 41.6 directs synthesis of a 1.7-kilobase RNA that is induced at least 10-fold by heat shock. Transcription initiates at about 124 bases 3' of the putative promotor sequence and terminates within the 41.6 insert. A 381-base-pair subclone that contains the putative promotor sequence is sufficient to induce the heat shock response of 41.6 in yeast cells.

Different sequence elements are required for function of the cauliflower mosaic virus polyadenylation site in Saccharomyces cerevisiae compared with in plants

1992 ◽  
Vol 12 (5) ◽  
pp. 2322-2330
Author(s):  
S Irniger ◽  
H Sanfaçon ◽  
C M Egli ◽  
G H Braus
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mosaic Virus ◽  
Cauliflower Mosaic Virus ◽  
Yeast Cells ◽  
Polyadenylation Site ◽  
Proper Function ◽  
Single Point Mutation ◽  
Dependent Manner ◽  
Sequence Element ◽  
Sequence Elements

We show that the polyadenylation site derived from the plant cauliflower mosaic virus (CaMV) is specifically functional in the yeast Saccharomyces cerevisiae. The mRNA 3' endpoints were mapped at the same position in yeast cells as in plants, and the CaMV polyadenylation site was recognized in an orientation-dependent manner. Mutational analysis of the CaMV 3'-end-formation signal revealed that multiple elements are essential for proper activity in yeast cells, including two upstream elements that are situated more than 100 and 43 to 51 nucleotides upstream of the poly(A) addition site and the sequences at or near the poly(A) addition site. A comparison of the sequence elements that are essential for proper function of the CaMV signal in yeast cells and plants showed that both organisms require a distal and a proximal upstream element but that these sequence elements are not identical in yeast cells and plants. The key element for functioning of the CaMV signal in yeast cells is the sequence TAGTATGTA, which is similar to a sequence previously proposed to act in yeast cells as a bipartite signal, namely, TAG ... TATGTA. Deletion of this sequence in the CaMV polyadenylation signal abolished 3'-end formation in yeast cells, and a single point mutation in this motif reduced the activity of the CaMV signal to below 15%. These results indicate that the bipartite sequence element acts as a signal for 3'-end formation in yeast cells but only together with other cis-acting elements.

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 Stabilizing Effects of Interruptions on Trinucleotide Repeat Expansions in Saccharomyces cerevisiae

2000 ◽  
Vol 20 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Michael L. Rolfsmeier ◽  
Robert S. Lahue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Trinucleotide Repeat ◽  
Primary Factor ◽  
Yeast Cells ◽  
Repeat Expansions ◽  
Yeast Genetic ◽  
Hairpin Formation ◽  
Lagging Strand ◽  
Genetic Assay

ABSTRACT In most trinucleotide repeat (TNR) diseases, the primary factor determining the likelihood of expansions is the length of the TNR. In some diseases, however, stable alleles contain one to three base pair substitutions that interrupt the TNR tract. The unexpected stability of these alleles compared to the frequent expansions of perfect TNRs suggested that interruptions somehow block expansions and that expansions occur only upon loss of at least one interruption. The work in this study uses a yeast genetic assay to examine the mechanism of stabilization conferred by two interruptions of a 25-repeat tract. Expansion rates are reduced up to 90-fold compared to an uninterrupted allele. Stabilization is greatest when the interruption is replicated early on the lagging strand, relative to the rest of the TNR. Although expansions are infrequent, they are often polar, gaining new DNA within the largest available stretch of perfect repeats. Surprisingly, interruptions are always retained and sometimes even duplicated, suggesting that expansion in yeast cells can proceed without loss of the interruption. These findings support a stabilization model in which interruptions contribute in cis to reduce hairpin formation during TNR replication and thus inhibit expansion rates.

scholarly journals Isolation and characterization of the centromere from chromosome V (CEN5) of Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (1) ◽  
pp. 86-91 ◽  
Author(s):  
G T Maine ◽  
R T Surosky ◽  
B K Tye
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Base Pair ◽  
Mapping Technique ◽  
Chromosome Loss ◽  
Tetrad Analysis ◽  
Autonomously Replicating Sequence ◽  
Centromeric Dna ◽  
Isolation And Characterization

We have cloned a functional centromeric DNA sequence from Saccharomyces cerevisiae. Using the 2 mu chromosome-loss mapping technique and meiotic tetrad analysis, we have identified this DNA sequence as the centromere of chromosome V (CEN5). The CEN5 sequence has been localized on an 1,100-base-pair BamHI-BglII restriction fragment. Plasmids containing CEN5 and an autonomously replicating sequence are mitotically stable in S. cerevisiae and segregate in a Mendelian fashion during meiosis.

scholarly journals Different sequence elements are required for function of the cauliflower mosaic virus polyadenylation site in Saccharomyces cerevisiae compared with in plants.

1992 ◽  
Vol 12 (5) ◽  
pp. 2322-2330 ◽  
Author(s):  
S Irniger ◽  
H Sanfaçon ◽  
C M Egli ◽  
G H Braus
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mosaic Virus ◽  
Cauliflower Mosaic Virus ◽  
Yeast Cells ◽  
Polyadenylation Site ◽  
Proper Function ◽  
Single Point Mutation ◽  
Dependent Manner ◽  
Sequence Element ◽  
Sequence Elements

We show that the polyadenylation site derived from the plant cauliflower mosaic virus (CaMV) is specifically functional in the yeast Saccharomyces cerevisiae. The mRNA 3' endpoints were mapped at the same position in yeast cells as in plants, and the CaMV polyadenylation site was recognized in an orientation-dependent manner. Mutational analysis of the CaMV 3'-end-formation signal revealed that multiple elements are essential for proper activity in yeast cells, including two upstream elements that are situated more than 100 and 43 to 51 nucleotides upstream of the poly(A) addition site and the sequences at or near the poly(A) addition site. A comparison of the sequence elements that are essential for proper function of the CaMV signal in yeast cells and plants showed that both organisms require a distal and a proximal upstream element but that these sequence elements are not identical in yeast cells and plants. The key element for functioning of the CaMV signal in yeast cells is the sequence TAGTATGTA, which is similar to a sequence previously proposed to act in yeast cells as a bipartite signal, namely, TAG ... TATGTA. Deletion of this sequence in the CaMV polyadenylation signal abolished 3'-end formation in yeast cells, and a single point mutation in this motif reduced the activity of the CaMV signal to below 15%. These results indicate that the bipartite sequence element acts as a signal for 3'-end formation in yeast cells but only together with other cis-acting elements.

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