A direct selection procedure for isolating yeast mutants with an impaired segregation of artificial minichromosomes.

The nondisjunction of artificial yeast minichromosomes (2:0 segregation events) during mitosis is accompanied by the appearance of cells containing more than one copy of the minichromosome. A mathematical simulation of this process has demonstrated that under certain conditions, a nondisjunction of the minichromosomes may result in their accumulation in a considerable portion of the cell population. An increase in the copy number of artificial minichromosomes as a result of impaired segregation has been used to develop a new experimental procedure for directly selecting yeast mutants showing an impaired segregation of artificial minichromosomes during mitosis. Four new genes, AMC1, AMC2, AMC3, and AMC4, which control the segregation of artificial minichromosomes in mitosis, have been identified (AMC3 and AMC4 are mapped to chromosome IV and VII, respectively). Mutations in the genes AMC1-AMC4 also affect the mitotic transmission of natural chromosomes. We suggest that the genes AMC1, AMC2, AMC3, and AMC4 control the segregation of natural chromosomes in yeast.

The stability of chromosomes in yeast.

CL mutants with high instability of chromosome III were UV-induced in haploid strain disomic for chromosome III. The obtained CL mutants can be divided into two groups: (1) CL2, CL3, CL7, CL11-CL13 with elevated level of spontaneous inter- and intragenic recombination and (2) CL4, CL8 in which instability of chromosome III is not accompanied by elevation of mitotic recombination frequency. CL4 and CL8 mutants also show unstable maintenance of artificial minichromosomes with different chromosomal replicators and centromeric loci. The instability of chromosome III and minichromosomes in CL4 and CL8 is determined by two nonallelic genes designated ch14 and ch18. The role of ch14 and ch18 genes in mitotic chromosome transmission is discussed.

A mutant of Saccharomyces cerevisiae with impaired maintenance of centromeric plasmids.

A mutant with unstable maintenance of hybrid plasmids containing either one of the centromeric loci CEN3, CEN6, CEN11 and ars1 or the replicator of the 2 mu plasmid has been obtained. The frequency of loss of hybrid plasmids in the mutant was up to 3.10(-1) per one generation versus 10(-2) in the original strain. The unstable maintenance of minichromosomes in the mutant is controlled by a recessive nuclear gene, named SMC for stability of minichromosomes. Loss of some minichromosomes is connected with impairment of their segregation in cell division. In diploids homozygous for smc mitotic chromosomal segregation is not affected but sporulation is impaired. The question of adequacy of usage of minichromosomes for selection of mutants with impaired function of centromeric loci is discussed.

The study of a rDNA replicator in Saccharomyces.

We have previously demonstrated that the loss of Rcp-CEN3, a centromeric plasmid containing yeast rDNA autonomously replicating sequences (ARS) is as high as around 50% per generation for most yeast strains. In this study we have attempted to elucidate mechanisms underlying the high mitotic instability of Rcp-CEN3. For this purpose a tandem duplication of a rDNA ARS was constructed in Rcp-CEN3. The new plasmid having two ARSs possesses a markedly higher mitotic stability as compared to a monoARS Rcp-CEN3. The mitotic stability of this centromere-containing plasmid which has two replicators corresponds to the calculated value for the mitotic stability of two monoARS plasmids Rcp-CEN3 in given cells. Genetic analysis has demonstrated that both plasmids having one or two ARSs are maintained in the single copy state. These results demonstrate that the mitotic instability of centromeric plasmid Rcp-CEN3 carrying a rDNA ARS is associated with the absence of stringent control of replication from the rDNA ARS. A possible mechanism of replication of the chromosomal rDNA repeats in yeast is discussed in the light of this data.