Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes

Bloom, Kerry; Carbon, John

doi:10.1016/0092-8674(82)90147-7

Cited by 310 publications

(220 citation statements)

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“…Deletion of the CBF1 gene or deletion of CDEI motifs from centromeric DNA led to similar defects in mitotic and meiotic centromere function, indicating that Cbf1p is an important component of the centromerekinetochore complex (9,10). Although the precise role of Cbf1p at the centromere is not yet understood, it is known to interact with other protein components of the kinetochore complex to form a compact nucleosome-based structure (11)(12)(13)(14)(15).…”

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Cbf1p Is Required for Chromatin Remodeling at Promoter-proximal CACGTG Motifs in Yeast

Kent

Eibert

Mellor

2004

Journal of Biological Chemistry

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Cbf1p is a basic-helix-loop-helix-zipper protein of Saccharomyces cerevisiae required for the function of centromeres and MET gene promoters, where it binds DNA via the consensus core motif CACRTG (R ‫؍‬ A or G). At MET genes Cbf1p appears to function in both activator recruitment and chromatin-remodeling. Cbf1p has been implicated in the regulation of other genes, and CACRTG motifs are common in potential gene regulatory DNA. A recent genome-wide location analysis showed that the majority of intergenic CACGTG palindromes are bound by Cbf1p. Here we tested whether all potential Cbf1p binding motifs in the yeast genome are likely to be bound by Cbf1p using chromatin immunoprecipitation. We also tested which of the motifs are actually functional by assaying for Cbf1p-dependent chromatin remodeling. We show that Cbf1p binding and activity is restricted to palindromic CACGTG motifs in promoter-proximal regions. Cbf1p does not function through CACGTG motifs that occur in promoter-distal locations within coding regions nor where CACATG motifs occur alone except at centromeres. Cbf1p can be made to function at promoter-distal CACGTG motifs by overexpression, suggesting that the concentration of Cbf1p is normally limiting for binding and is biased to gene regulatory DNA by interactions with other factors. We conclude that Cbf1p is required for normal nucleosome positioning wherever the CACGTG motif occurs in gene regulatory DNA. Cbf1p has been shown to interact with the chromatin-remodeling ATPase Isw1p. Here we show that recruitment of Isw1p by Cbf1p is likely to be general but that Isw1p is only partially required for Cbf1p-dependent chromatin structures.

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Cbf1p Is Required for Chromatin Remodeling at Promoter-proximal CACGTG Motifs in Yeast

Kent

Eibert

Mellor

2004

Journal of Biological Chemistry

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“…The molecular organization of the centromere region of the chromosome is characterized by a conserved core particle that is surrounded by distinct arrays of nucleosomal subunits (4). The centromere core DNA is organized in a 220-to 250-base-pair (bp) chromatin particle that is protected from nuclease digestion in the cell nucleus.…”

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“…Hybridization studies reveal that these flanking sequences are not repeated in the yeast genome, although they may contain very small (10-to 50-bp) regions of homology (4,10 Thomas (35).…”

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“…Transfer of DNA from agarose gels to nitrocellulose paper (BA 85; Schleicher & Schuell) was performed essentially as described by Southern (32). Hybridization analysis was performed as described by Bloom and Carbon (4). RESULTS Transcription map of regions flanking the centromere in chromosome III.…”

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“…Our findings revealed that the centromere region in yeasts is structurally distinct from the flanking chromosomal arms; however, repetitive satellite sequences that seem to be associated with the centromere in higher eucaryotes (27) are noticeably absent in this region of the yeast genome. A highly repeating array of nucleosomal subunits is characteristic of the flanking centromere region in chromosomes III and XI in yeasts (4) (Fig. 3).…”

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Tightly centromere-linked gene (SPO15) essential for meiosis in the yeast Saccharomyces cerevisiae.

Yeh¹,

Carbon²,

Bloom³

1986

Mol. Cell. Biol.

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We used DNA fragments from the centromere regions of yeast (Saccharomyces cerevisiae) chromosomes III and XI to examine the transcriptional activity within this chromosomal domain. RNA transcripts were found 200 to 300 base pairs from the 250-base-pair centromere core and lie within an ordered chromatin array. No transcripts were detected from the functional centromere region. We examined the cellular function of one of these tightly centromere-linked transcripts, (CENJl)L, by disrupting the coding sequences in vivo and analyzing the phenotype of the mutant yeast cell. Diploids heterozygous for the (CENII)L disruption sporulated at wild-type levels, and the absence of the (CENII)L gene product had no effect on the viability or mitotic growth of haploid cells. Diploids homozygous for the (CENJI)L disruption were unable to sporulate when induced by the appropriate nutritional cues. The mutant cells were competent for intragenic recombination and appeared to be blocked at the mononucleate stage. The temporal ordering of (CENII)L function with respect to the sporulation mutant spol3 suggests that the (CENJl)L gene product may be required at both the first and second meiotic cell divisions. This new sporulation gene has been termed SPOIS.The transmission of genetic material to progeny cells during the cell cycle is an essential process that proceeds with a high degree of precision in all eucaryotic organisms. This process involves the establishment of a spindle apparatus that interacts with specific regions along the eucaryotic chromosome, the centromeres, to direct the accurate segregation of identical genetic information to the daughter cells. Although yeast centromeres cannot be visualized at the morphological level, the genetic segregation properties of yeast chromosomes clearly indicate the involvement of specific chromosomal loci. The centromeric regions from five chromosomes in yeasts (CEN) [14]) and are able to confer properties of chromosome stabilization and proper Mendelian segregation to replicating DNA molecules in yeasts (10). Furthermore, when CEN3 sequences are deleted from their host chromosome in vivo, an acentric chromosome is produced which is lost from the population, presumably owing to the missing spindle attachment site (11). The centromere sequence is therefore required to stabilize the entire yeast chromosome. Chromosome stability is recovered when the centromere DNAs from chromosomes III, XI, and VI are substituted in either orientation for the CEN3 region in chromosome III (3, 11). The resulting chromosomes segregate normally through mitosis and meiosis in a manner identical to that of normal chromosome III. These results indicate that the yeast centromeres are fully functional in either orientation and are not necessarily chromosome specific.The molecular organization of the centromere region of the chromosome is characterized by a conserved core particle that is surrounded by distinct arrays of nucleosomal subunits (4). The centromere core DNA is organized in a 220-to 250-base-pair (b...

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Preparation of Protein Extracts from Yeast

Dunn

Wobbe

1993

CP Molecular Biology

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Three protocols are presented for preparing protein extracts; they differ primarily in the way the cells are broken. In the basic protocol, cells are enzymatically converted to spheroplasts, which are then lysed by a combination of osmotic shock and Dounce homogenization. A support protocol for isolating intact nuclei by differential centrifugation is also presented. An alternate protocol describes mechanical breakage of cells by vortexing in the presence of glass beads. In a second alternate protocol, growing cells are frozen immediately in liquid nitrogen and then lysed by grinding in an industrial-strength blender in the presence of liquid nitrogen.

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Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes

Cited by 310 publications

References 38 publications

Cbf1p Is Required for Chromatin Remodeling at Promoter-proximal CACGTG Motifs in Yeast

Cbf1p Is Required for Chromatin Remodeling at Promoter-proximal CACGTG Motifs in Yeast

Tightly centromere-linked gene (SPO15) essential for meiosis in the yeast Saccharomyces cerevisiae.

Preparation of Protein Extracts from Yeast

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