Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe

1988 ◽  
Vol 8 (2) ◽  
pp. 754-763
Author(s):  
B Fishel ◽  
H Amstutz ◽  
M Baum ◽  
J Carbon ◽  
L Clarke
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Dna Sequences ◽  
Centromeric Dna ◽  
Centromere Function ◽  
Leu2 Gene ◽  
Centromeric Repeat ◽  
Sequence Elements ◽  
Chromosome Iii ◽  
Chromosome Ii

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.

scholarly journals Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

1988 ◽  
Vol 8 (2) ◽  
pp. 754-763 ◽  
Author(s):  
B Fishel ◽  
H Amstutz ◽  
M Baum ◽  
J Carbon ◽  
L Clarke
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Dna Sequences ◽  
Centromeric Dna ◽  
Centromere Function ◽  
Leu2 Gene ◽  
Centromeric Repeat ◽  
Sequence Elements ◽  
Chromosome Iii ◽  
Chromosome Ii

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.

scholarly journals The centromeric K-type repeat and the central core are together sufficient to establish a functional Schizosaccharomyces pombe centromere.

1994 ◽  
Vol 5 (7) ◽  
pp. 747-761 ◽  
Author(s):  
M Baum ◽  
V K Ngan ◽  
L Clarke
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Dna Sequences ◽  
Specific Protein ◽  
Central Core ◽  
Critical Elements ◽  
Centromere Function ◽  
Centromeric Repeat ◽  
Proposed Model ◽  
Kinetochore Function

The DNA requirements for centromere function in fission yeast have been investigated using a minichromosome assay system. Critical elements of Schizosaccharomyces pombe centromeric DNA are portions of the centromeric central core and sequences within a 2.1-kilobase segment found on all three chromosomes as part of the K-type (K/K"/dg) centromeric repeat. The S. pombe centromeric central core contains DNA sequences that appear functionally redundant, and the inverted repeat motif that flanks the central core in all native fission yeast centromeres is not essential for centromere function in circular minichromosomes. Tandem copies of centromeric repeat K", in conjunction with the central core, exert an additive effect on centromere function, increasing minichromosome mitotic stability with each additional copy. Centromeric repeats B and L, however, and parts of the central core and its core-associated repeat are dispensable and cannot substitute for K-type sequences. Several specific protein binding sites have been identified within the centromeric K-type repeat, consistent with a recently proposed model for centromere/kinetochore function in S. pombe.

scholarly journals Centromeres of the fission yeast Schizosaccharomyces pombe are highly variable genetic loci.

1993 ◽  
Vol 13 (8) ◽  
pp. 4578-4587 ◽  
Author(s):  
N C Steiner ◽  
K M Hahnenberger ◽  
L Clarke
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Genetic Locus ◽  
Central Core ◽  
Structural Motif ◽  
Centromeric Dna ◽  
Repeat Structure ◽  
Food And Beverage ◽  
Chromosome Iii ◽  
Chromosome Ii

Gross variations in the structure of the centromere of Schizosaccharomyces pombe chromosome III (cen3) were apparent following characterization of this centromeric DNA in strain Sp223 and comparison of the structure with that of cen3 in three other commonly used laboratory strains. Further differences in centromere structure were revealed when the structure of the centromere of S. pombe chromosome II (cen2) was compared among common laboratory strains and when the structures of cen2 and cen3 from our laboratory strains were compared with those reported from other laboratories. Differences observed in cen3 structure include variations in the arrangement of the centromeric K repeats and an inverted orientation of the conserved centromeric central core. In addition, we have identified two laboratory strains that contain a minimal cen2 repeat structure that lacks the tandem copies of the cen2-specific block of K-L-B-J repeats characteristic of Sp223 cen2. We have also determined that certain centromeric DNA structural motifs are relatively conserved among the four laboratory strains and eight additional wild-type S. pombe strains isolated from various food and beverage sources. We conclude that in S. pombe, as in higher eukaryotes, the centromere of a particular chromosome is not a defined genetic locus but can contain significant variability. However, the basic DNA structural motif of a central core immediately flanked by inverted repeats is a common parameter of the S. pombe centromere.

scholarly journals Budding yeast centromeric DNA and A+T rich bacterial DNA can function as centromeres in the fission yeast Schizosaccharomyces pombe

2019 ◽  
Author(s):  
Anne C Barbosa ◽  
Zhengyao Xu ◽  
Kazhal Karari ◽  
Silke Hauf ◽  
William RA Brown
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Dna Sequence ◽  
Budding Yeast ◽  
Bacterial Dna ◽  
Centromeric Dna ◽  
Serine Recombinases ◽  
Centromere Function ◽  
And Function

Eukaryotic centromeric DNA is famously variable in evolution but currently, this cannot be reconciled with the conservation of eukaryotic centromere function. It seems likely that centromeric DNA from different organisms contains conserved functionally important features but the identity of these features is unknown. The point centromeres of the budding yeast Saccharomyces cerevisiae and the regional centromeres of the fission yeast Schizosaccharomyces pombe are separated by 350 million years of evolution and are canonical examples of the paradoxical relationship1 between centromeric DNA sequence and function. We have established a centromere-replacement strategy in Schizosaccharomyces pombe in order to resolve this paradox experimentally. Centromere-replacement shows that an A+T rich bacterial DNA sequence has weak centromere function and that elements of the Saccharomyces cerevisiae centromere embedded in short sequences from the non-centromeric S. pombe wee1 gene function almost as well as native S. pombe centromeric DNA. These observations demonstrate that determinants of centromere function are held in common by the budding and fission yeasts and that A+T rich DNA is both necessary and sufficient for function in S. pombe. Given the evolutionary distance between these yeasts, it is likely that A+T rich DNA has centromere function in a wide variety of eukaryotes. Centromere-replacement uses unidirectional serine recombinases that work well in many organisms2 3 and our experimental strategy should allow this idea to be tested in other eukaryotes.

Centromeres of the fission yeast Schizosaccharomyces pombe are highly variable genetic loci

1993 ◽  
Vol 13 (8) ◽  
pp. 4578-4587
Author(s):  
N C Steiner ◽  
K M Hahnenberger ◽  
L Clarke
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Genetic Locus ◽  
Central Core ◽  
Structural Motif ◽  
Centromeric Dna ◽  
Repeat Structure ◽  
Food And Beverage ◽  
Chromosome Iii ◽  
Chromosome Ii

Gross variations in the structure of the centromere of Schizosaccharomyces pombe chromosome III (cen3) were apparent following characterization of this centromeric DNA in strain Sp223 and comparison of the structure with that of cen3 in three other commonly used laboratory strains. Further differences in centromere structure were revealed when the structure of the centromere of S. pombe chromosome II (cen2) was compared among common laboratory strains and when the structures of cen2 and cen3 from our laboratory strains were compared with those reported from other laboratories. Differences observed in cen3 structure include variations in the arrangement of the centromeric K repeats and an inverted orientation of the conserved centromeric central core. In addition, we have identified two laboratory strains that contain a minimal cen2 repeat structure that lacks the tandem copies of the cen2-specific block of K-L-B-J repeats characteristic of Sp223 cen2. We have also determined that certain centromeric DNA structural motifs are relatively conserved among the four laboratory strains and eight additional wild-type S. pombe strains isolated from various food and beverage sources. We conclude that in S. pombe, as in higher eukaryotes, the centromere of a particular chromosome is not a defined genetic locus but can contain significant variability. However, the basic DNA structural motif of a central core immediately flanked by inverted repeats is a common parameter of the S. pombe centromere.

scholarly journals Analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

1986 ◽  
Vol 83 (21) ◽  
pp. 8253-8257 ◽  
Author(s):  
L. Clarke ◽  
H. Amstutz ◽  
B. Fishel ◽  
J. Carbon
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Centromeric Dna

Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe

1986 ◽  
Vol 6 (10) ◽  
pp. 3523-3530
Author(s):  
R Booher ◽  
D Beach
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Cell Cycle Control ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Atp Binding Site ◽  
Leu2 Gene ◽  
Cdc2 Gene ◽  
A Site

The cdc2+ gene of Schizosaccharomyces pombe is homologous to the CDC28 gene of Saccharomyces cerevisiae. Both genes share limited homology with vertebrate protein kinases and have protein kinase activity. cdc2+ has been subjected to mutagenesis in vitro. A null allele of the gene, constructed by insertion of the S. cerevisiae LEU2 gene into a site within the gene, has a phenotype similar to that of many temperature-sensitive alleles of cdc2. Mutations within the predicted ATP-binding site and in a region which may be a site of phosphorylation result in loss of cdc2+ activity. A single substitution of Gly-146 to Asp-146 has been identified in cdc2-1w, a dominant activated allele of the gene. The four introns within the cdc2+ gene have been deleted. The resulting gene not only functions in fission yeast but also rescues cdc28(Ts) strains of S. cerevisiae, a property which is not shared by the genomic cdc2+ gene.

scholarly journals Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences.

1990 ◽  
Vol 10 (5) ◽  
pp. 1863-1872 ◽  
Author(s):  
L Clarke ◽  
M P Baum
Keyword(s):  
Dna Sequences ◽  
Inverted Repeat ◽  
Repeated Sequence ◽  
Repeated Sequences ◽  
Central Core ◽  
Repeated Dna ◽  
Inverted Repeat Region ◽  
Repeated Dna Sequences ◽  
Centromere Function ◽  
Chromosome Ii

A circular minichromosome carrying functional centromere sequences (cen2) from Schizosaccharomyces pombe chromosome II behaves as a stable, independent genetic linkage group in S. pombe. The cen2 region was found to be organized into four large tandemly repeated sequence units which span over 80 kilobase pairs (kb) of untranscribed DNA. Two of these units occurred in a 31-kb inverted repeat that flanked a 7-kb central core of nonhomology. The inverted repeat region had centromere function, but neither the central core alone nor one arm of the inverted repeat was functional. Deletion of a portion of the repeated sequences that flank the central core had no effect on mitotic segregation functions or on meiotic segregation of a minichromosome to two of the four haploid progeny, but drastically impaired centromere-mediated maintenance of sister chromatid attachment in meiosis I. This requirement for centromere-specific repeated sequences could not be satisfied by introduction of random DNA sequences. These observations suggest a function for the heterochromatic repeated DNA sequences found in the centromere regions of higher eucaryotes.

scholarly journals Sequential de novo centromere formation and inactivation on a chromosomal fragment in maize

2015 ◽  
Vol 112 (11) ◽  
pp. E1263-E1271 ◽  
Author(s):  
Yalin Liu ◽  
Handong Su ◽  
Junling Pang ◽  
Zhi Gao ◽  
Xiu-Jie Wang ◽  
...  
Keyword(s):  
Dna Sequences ◽  
De Novo ◽  
B Chromosome ◽  
Chromosome 9 ◽  
Centromeric Dna ◽  
Repeat Sequences ◽  
Centromeric Repeat ◽  
And Function ◽  
Centromere Assembly ◽  
Derivatives Of

The ability of centromeres to alternate between active and inactive states indicates significant epigenetic aspects controlling centromere assembly and function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In such derivatives of TB-9Sb, we found a de novo centromere on chromosome derivative 3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. The functional B centromere in progenitor telo2-2 is deleted from derivative 3-3, but some B-repeat sequences remain. The de novo centromere of derivative 3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on each chromosome. Our results suggest that de novo centromere initiation is quite common and can persist on chromosomal fragments without a canonical centromere. However, we hypothesize that when de novo centromeres are initiated in opposition to a larger normal centromere, they are cleared from the chromosome by inactivation, thus maintaining karyotype integrity.

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