Acquisition and processing of a conditional dicentric chromosome in Saccharomyces cerevisiae.

Abstract

The introduction of a conditional centromere into chromosome III of Saccharomyces cerevisiae provided an opportunity to evaluate phenotypic and karyotypic consequences in cells harboring dicentric chromosomes upon entry into mitosis. A mitotic pause ensued, and monocentric derivatives of chromosome III were generated at a high frequency.The transmission of chromosomes in Saccharomyces cerevisiae can be manipulated effectively with a conditional centromere. The conditional centromere consists of a centromere that is regulated by the inducible GAL] promoter in yeast cells (6). Utilization of growth conditions that favor transcriptional induction (galactose as the carbon source) or repression (glucose as the carbon source) from the GAL] promoter results in centromere function that is switched off or on, respectively. In this paper, we describe the construction of a conditional dicentric chromosome. Observations made in a number of different organisms suggest diverse behaviors for dicentric chromosomes. These range from the extreme instability of chromosomes that undergo a breakage-fusion-bridge cycle through subsequent mitotic cell divisions (9,10) to the stable transmission of a chromosome with eight centromeres (13). With an increased understanding of the molecular structure of the centromere, this diversity of behavior may be explained.Acquisition of a dicentric chromosome results in mitotic lag. A conditional centromere was introduced into the left arm of chromosome III at the HIS4 locus, approximately 45 kilobases (kb) from CEN3 ( Fig. 1). Logarithmically growing cultures of strains containing this construction (J178-1D#7 MATa adel metl4 ura3 leu2 his3 HIS4::pBR322; URA3; GALCEN3) were switched from galactose-containing to glucose-containing medium. Cells appeared to be synchronous for mitosis after approximately 1.5 to 2 generations in the glucose-containing medium. The parent strain, J178-1D (MATa adel metl4 ura3 leu2 his3), did not exhibit a similar mitotic pause. A majority of the cells had buds that were equivalent in size to those of the mother cells. Staining with the fluorescent dye revealed that cells in the nuclear division stage were approximately three times more numerous in the experimental cultures than in control cultures raised in the galactose-containing medium.Monocentric derivatives. Figure 2 shows the electrophoretic karyotype of chromosomes after the carbon source switch. After two generations in the glucose-containing medium, a new chromosome band smaller than chromosome III was evident (Fig. 2, lane 1). This chromosome appeared when the mitotic lag was first observed. The intensity of this chromosome increased over time, while that of its wild-type counterpart decreased. It was apparent after eight generations (Fig. 2, lane 4) to the GALJ-GALJO probe, while the 370-kb chromosome III band in all lanes in Fig. 2 (WT to 6), as well as the 325-kb chromosome in lanes 1 to 6, contained sequences homologous to CEN3 (data not shown). The major rearrangement involving the dicentric chromosome i...

DNA relaxation dynamics as a probe for the intracellular environment

“…A conditional dicentric chromosome has been constructed in budding yeast by means of site-directed integration of a second copy of centromere 3 (CEN3) at the HIS4 locus (29). This extra copy of CEN3 is regulated by the GAL1 promoter (GALCEN), allowing cells to be propagated in the presence or absence of a functional dicentric chromosome.…”

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…It also facilitates techniques such as ChIP, which cannot be easily performed on the highly repetitive centromeres in other organisms. In addition, the centromere can be moved to other genomic regions, allowing the construction of artificial chromosomes and plasmids as well as tools such as conditional centromeres (Murray and Szostak 1983;Hill and Bloom 1989).…”

De Novo Kinetochore Assembly Requires the Centromeric Histone H3 Variant

“…Yeast strains used in this study are listed in Table 1 and are derived from the W303 background. cCEN yeast strains were generated by integration of pR285#7 (pGAL-CEN3-URA3; Hill and Bloom, 1989) that was digested with XhoI to direct integration to the HIS4 locus. Strains containing pGAL-UBR1-myc were made by transforming PmeI-digested pLK54#300 (Labib et al, 2000) to direct integration to the UBR1 locus.…”

“…When cells are grown in galactose media, transcription through the cCEN keeps it inactive. The addition of glucose represses transcription at the cCEN, allowing a functional kinetochore to form within 20 min (Hill and Bloom, 1989;Neff and Burke, 1992;Dewar et al, 2004;Tanaka et al, 2005).To begin analyzing the cell cycle restrictions over kinetochore assembly, we first determined whether kinetochores could assemble in G 1 cells. Kinetochore assembly at the cCEN was analyzed using ChIP.…”