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De novo kinetochore assembly requires the centromeric histone H3 variant.

ABSTRACT: Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of >65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly. Using a conditional centromere, we found that yeast kinetochore assembly is not temporally restricted and can occur in both G1 phase and prometaphase. We performed the first investigation of kinetochore assembly in the absence of the centromeric histone H3 variant Cse4 and found that all proteins tested depend on Cse4 to localize. Consistent with this observation, Cse4-depleted cells had severe chromosome segregation defects. We therefore propose that yeast kinetochore assembly requires both centromeric DNA specificity and centromeric chromatin.

SUBMITTER: Collins KA

PROVIDER: S-EPMC1289410 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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De novo kinetochore assembly requires the centromeric histone H3 variant.

Collins Kimberly A KA Castillo Andrea R AR Tatsutani Sean Y SY Biggins Sue S

Molecular biology of the cell 20051005 12

Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of >65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly. Using a conditional centromere, we found that yeast kinetochore assembly is not tem ...[more]

PMID: 16207811

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Project description:Lycoris species have various chromosome numbers and karyotypes, but all have a constant total number of chromosome major arms. In addition to three fundamental types, including metacentric (M-), telocentric (T-), and acrocentric (A-) chromosomes, chromosomes in various morphology and size were also observed in natural populations. Both fusion and fission translocation have been considered as main mechanisms leading to the diverse karyotypes among Lycoris species, which suggests the centromere organization playing a role in such arrangements. We detected several chromosomal structure changes in Lycoris including centric fusion, inversion, gene amplification, and segment deletion by using fluorescence in situ hybridization (FISH) probing with rDNAs. An antibody against centromere specific histone H3 (CENH3) of L. aurea (2n = 14, 8M+6T) was raised and used to obtain CENH3-associated DNA sequences of L. aurea by chromatin immunoprecipitation (ChIP) cloning method. Immunostaining with anti-CENH3 antibody could label the centromeres of M-, T-, and A-type chromosomes. Immunostaining also revealed two centromeres on one T-type chromosome and a centromere on individual mini-chromosome. Among 10,000 ChIP clones, 500 clones which showed abundant in L. aurea genome by dot-blotting analysis were FISH mapped on chromosomes to examine their cytological distribution. Five of these 500 clones could generate intense FISH signals at centromeric region on M-type but not T-type chromosomes. FISH signals of these five clones rarely appeared on A-type chromosomes. The five ChIP clones showed similarity in DNA sequences and could generate similar but not identical distribution patterns of FISH signals on individual chromosomes. Furthermore, the distinct distribution patterns of FISH signals on each chromosome generated by these five ChIP clones allow to identify individual chromosome, which is considered difficult by conventional staining approaches. Our results suggest a different organization of centromeres of the three chromosome types in Lycoris species.

Dataset Information

De novo kinetochore assembly requires the centromeric histone H3 variant.

Publications

De novo kinetochore assembly requires the centromeric histone H3 variant.

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