[Stable maintenance of dicentric mini-chromosomes in CHL4 mutants in yeast]. / Stabil'noe podderzhanie ditsentricheskikh mini-khromosom u mutanta drozhzhei po genu chl4.

Earlier we have identified the chl4-1 mutation in a screen for yeast mutants with increased loss of chromosome III and circular artificial minichromosome in mitosis. Mutation in the CHL4 gene leads to a 50-100-fold promotion in the rate of chromosome loss per cell division compared to the isogenic wild type strain. Detailed analysis of behaviour of the circular minichromosome marked by the CUP1 gene has shown that minichromosome nondisjunction (2:0 segregation) leading to an increase in the copy number of minichromosome in part of a cell population is the main reason of minichromosome instability in the mutant. The unique peculiarity of chl4-1 mutation is the ability of the strains carrying this mutation to stably maintain circular dicentric minichromosomes without any rearrangement during many generations. (In the wild type strains dicentric minichromosomes are extremely unstable. As a consequence of that there is a strong selection for cells harboring monocentric derivatives in a population of cells derived from a cell containing a dicentric plasmid). Introduction of the second centromere into one of the natural chromosomes (chromosomes II or III) in the chl4-1 mutant leads to the same dramatic consequences as that in the wild type strain (mitotic lag of cells harboring dicentric chromosomes and, as a result of that, selective pressure for cells harboring monocentric derivatives of dicentric chromosome). A genomic clone of CHL4 was isolated by complementation of the chl4-1 mutation. Nucleotide sequence analysis of CHL4 revealed a 1.4-kb open reading frame with a predicted 53-kDa protein sequence. Analyzing the sequence of the CHL4 protein we have found a region meeting the necessary requirements for the helix-turn-helix (HTH) structure. This region of the CHL4 protein has about 40% homology with the repressor of tryptophane operon (TrpR) of E. coli. A strain containing a null allele of CHL4 was viable under standard growth conditions, but had temperature-sensitive phenotype (conditional lethality at 34 degrees C). We suggest that the CHL4 gene product is one of the components of the segregation cell machinery.

[CHL15--a new gene controlling the replication of chromosomes in saccharomycetes yeast: cloning, physical mapping, sequencing, and sequence analysis]. / CHL15--novyi gen, kontroliruiushchii replikatsiiu khromosom u drozhzhei--sakharomitsetov: klonirovanie, fizicheskoe kartirovanie, sekvenirovanie i funktsional'nyi analiz.

We have analyzed the CHL15 gene, earlier identified in a screen for yeast mutants with increased loss of chromosome III and artificial circular and linear chromosomes in mitosis. Mutations in the CHL15 gene lead to a 100-fold increase in the rate of chromosome III loss per cell division and a 200-fold increase in the rate of marker homozygosis on this chromosome by mitotic recombination. Analysis of segregation of artificial circular minichromosome and artificially generated nonessential marker chromosome fragment indicated that sister chromatid loss (1:0 segregation) is a main reason of chromosome destabilization in the chl15-1 mutant. A genomic clone of CHL15 was isolated and used to map its physical position on chromosome XVI. Nucleotide sequence analysis of CHL15 revealed a 2.8-kb open reading frame with a 105-kD predicted protein sequence. At the N-terminal region of the protein sequences potentially able to form DNA-binding domains defined as zinc-fingers were found. The C-terminal region of the predicted protein displayed a similarity to sequence of regulatory proteins known as the helix-loop-helix (HLH) proteins. Data on partial deletion analysis suggest that the HLH domain is essential for the function of the CHL15 gene product. Analysis of the upstream untranslated region of CHL15 revealed the presence of the hexamer element, ACGCGT (an MluI restriction site) controlling both the periodic expression and coordinate regulation of the DNA synthesis genes in budding yeast. Deletion in the RAD52 gene, the product of which is involved in double-strand break/recombination repair and replication, leads to a considerable decrease in the growth rate of the chl15 mutant. We suggest that CHL15 is a new DNA synthesis gene in the yeast Saccharomyces cerevisiae.

[The causes of instability of artificial mini-chromosomes in yeasts mutant for chl genes]. / Izuchenie prichin nestabil'nosti iskusstvennykh mini-khromosom u mutantov drozhzhei po genam chl.

In mutants chl2, chl3, chl5, and chl6, which control mitotic chromosome transmission, the behaviour of the centromeric plasmids with various genes was analyzed. The main cause of chromosome instability in chl2, chl5, and chl6 is chromosome loss during cell division (1:0 segregation). The main cause of chromosome instability in chl3. is nondisjunction (2:0 segregation). According to this, the chl3 mutant, but not other chl's, cannot maintain the mini-chromosome with SUP11 gene. This gene causes cell death in high copy number. Chromosome nondisjunction in chl3 is also confirmed by the data on the mini-chromosome carrying CUP1 gene responsible for copper-resistance in yeast. The copper resistancy level in chl3 transformants is much higher than in chl5 or wild type transformants. Elevated copper resistance of chl3 transformants is caused by the transit accumulation of CUP1-carrying mini-chromosome in part of the cell population as a result of segregation mistakes upon cell divisions. Thus, the CHL3 gene is a new gene that controls the process of mitotic chromosome disjunction in yeast.

[Behavior in a cell of artificial mini-chromosomes during induced transcription of centromeric DNA]. / Povedenie v kletke iskusstvennykh mini-khromosom pri indutsirovannoi transkriptsii tsentromernoi DNK.

Hybrid yeast plasmids were constructed, containing the centromere loci CEN3 under the control of two inducible yeast promoters--GAL10 and PHO5. It was shown, that during the induction of transcription from the GAL10 promoter the decrease in mitotic stability of minichromosome is affected both by partial disruption of centromere function by transcription and by influence of galactose on the number of residual cell divisions. In two strains the activity of GAL10 promoter was considerably higher than that of the PHO5 promoter. It is proposed to use the effect of minichromosome destabilization for evaluation of the relative promoter strength.

[Extrachromosomal DNA in yeast-Saccharomyces]. / Issledovanie vnekhromosomnoi DNK u drozhzhei-Sakharomitsetov.

The recombinant plasmids containing autonomously replicating sequence (ARS) of yeast rDNA repeat are characterized by a high instability in transformed yeast cells. The instability of chimaric plasmids in yeast may result from improper replication and/or irregular mitotic segregation. To study the replication properties alone we have constructed series of hybrid plasmids containing centromeric DNA (CEN3), a selective marker (leu2) and ARS of rDNA. Each of these plasmids with the functional centromere should exhibit chromosomal i. e. regular type of mitotic segregation. The study of mitotic segregation of constructed plasmids has shown that the ARS rDNA from yeast is distinguished from other ARSs described in literature: ARS1, ARS2, ARS o-micron DNA. 1. The activation of replication of ARS rDNA is accidental, i. e. probability of ARS rDNA in the cell cycle is much less than one. 2. Some nuclear mutations as well as rho- mutation result in the increase of replicative activity of ARS rDNA. In some yeast strains the activity of ARS rDNA can reach the activity of ARS1, i. e. was close to one. The features of ARS rDNA may account for the phenomenon of amplification of rDNA genes.