Isolation of a yeast centromere and construction of functional small circular chromosomes

Abstract

The centromeric DNA (CEN3) from yeast chromosome III has been isolated on a 1.6 kilobase-pair segment of DNA located near the centromere-linked CDC10 locus of Saccharomyces cerevisiae. When present on a plasmid carrying a yeast chromosomal replicator, CEN3 enables that plasmid to function as a chromosome both mitotically and meiotically. Minichromosomes containing CEN3 are stable in mitosis and segregate as ordinary yeast chromosomes in the first and second meiotic divisions.

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

“…The diploid was homozygous for both leu2 and ura3 so the plasmid (LEU2 URA3) could be followed in the cross. The results we obtained were very similar to those obtained for other S. cerevisiae centromeres (4,9,25) (Table 3). The plasmid segregates 2+:20 ca.…”

“…Most ARS-containing plasmids .n S. cerevisiae are unstable mitotically (26,27). Cloning of a centromeric DNA sequence into an ARScontaining plasmid results in the stable transmission of the plasmid to progeny during mitosis and meiosis (4,9,10,21,25 Table 2. Restfiction enzyme sites shown are: R, EcoRl; P, PstI; Bg, BgII; H3, HindIII; S, SalI; Bm, BamHI; Xb, XbaI; Hp, HpaI.…”

“…Plasmids containing a centromeric sequence and an autonomously replicating sequence (ARS) behave as minichromosomes which are mitotically stable and segregate 2+ :20 during meiosis (4,9,10,21,25). The centromere maintains the copy number of the plasmid at approximately one in a haploid cell (4).…”

“…The centromere maintains the copy number of the plasmid at approximately one in a haploid cell (4). Hsiao and Carbon (13) and Stinchcomb et al (25) have described a general procedure for the cloning of functional centromeric DNA, exploiting the property of mitotic stability conferred on plasmids by centromeres.…”

The centromere‐binding factor Cbf1p from Candida albicans complements the methionine auxotrophic phenotype of Saccharomyces cerevisiae

“…The centromere consists of specific DNA sequences that form a complex with centromere-specific proteins. Yeast centromeres have been characterized from Saccharomyces cerevisiae (Clarke and Carbon, 1980), Schizosaccharomyces pombe (Clarke et al, 1986), Saccharomyces uvarum (Hubermann et al, 1986), Kluyveromyces lactis (Heus et al, 1990), Kluyveromyces marxianus (Iborra and Ball, 1994), Candida maltosa (Ohkuma et al, 1995) and Candida glabrata (Kitada et al, 1997). Except in Sz.…”