Biomedical subjects

M Kupiec

Publications and source records attributed to M Kupiec.

At least 37 records · Page 2Linked to original sources

PubMed1996

Induction of Ty recombination in yeast by cDNA and transcription: role of the RAD1 and RAD52 genes.

In the yeast Saccharomyces cerevisiae ectopic recombination has been shown to occur at high frequencies for artificially created repeats, but at relatively low frequencies for a natural family of repeated sequences, the Ty family. Little is known about the mechanism(s) that prevent recombination between repeated sequences. We have previously shown that nonreciprocal recombination (gene conversion) of a genetically marked Ty can be induced either by the presence of high levels of Ty cDNA or by transcription of the marked Ty from a GAL1 promoter. These two kinds of induction act in a synergistic manner. To further characterize these two kinds of Ty recombination, we have investigated the role played by the RAD52 and RAD1 genes. We have found that the RAD52 and RAD1 gene products are essential to carry out transcription-induced Ty conversion whereas cDNA-mediated conversion can take place in their absence.

DNA Repair Enzymes↗

PubMed1995

The role of Saccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance.

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, in Saccharomyces cerevisiae, the CDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence of CDC40 (455 amino acids) contains four copies of a beta-transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in the cdc40-1 allele) results in normal vegetative growth at 23 degrees C, and cell cycle arrest at 36 degrees C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36 degrees C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.

Amino Acid Sequence↗

PubMed1995

Recombination of Ty elements in yeast can be induced by a double-strand break.

The Ty retrotransposons are the main family of dispersed repeated sequences in the yeast Saccharomyces cerevisiae. These elements are flanked by a pair of long terminal direct repeats (LTRs). Previous experiments have shown that Ty elements recombine at low frequencies, despite the fact that they are present in 30 copies per genome. This frequency is not highly increased by treatments that cause DNA damage, such as UV irradiation. In this study, we show that it is possible to increase the recombination level of a genetically marked Ty by creating a double-strand break in it. This break is repaired by two competing mechanisms: one of them leaves a single LTR in place of the Ty, and the other is a gene conversion event in which the marked Ty is replaced by an ectopically located one. In a strain in which the marked Ty has only one LTR, the double-strand break is repaired by conversion. We have also measured the efficiency of repair and monitored the progression of the cells through the cell-cycle. We found that in the presence of a double-strand break in the marked Ty, a proportion of the cells is unable to resume growth.

Base Sequence↗

PubMed1995

The role of DNA repair genes in recombination between repeated sequences in yeast.

The presence of repeated sequences in the genome represents a potential source of karyotypic instability. Genetic control of recombination is thus important to preserve the integrity of the genome. To investigate the genetic control of recombination between repeated sequences, we have created a series of isogenic strains in which we could assess the role of genes involved in DNA repair in two types of recombination: direct repeat recombination and ectopic gene conversion. Naturally occurring (Ty elements) and artificially constructed repeats could be compared in the same cell population. We have found that direct repeat recombination and gene conversion have different genetic requirements. The role of the RAD51, RAD52, RAD54, RAD55, and RAD57 genes, which are involved in recombinational repair, was investigated. Based on the phenotypes of single and double mutants, these genes can be divided into three functional subgroups: one composed of RAD52, a second one composed of RAD51 and RAD54, and a third one that includes the RAD55 and RAD57 genes. Among seven genes involved in excision repair tested, only RAD1 and RAD10 played a role in the types of recombination studied. We did not detect a differential effect of any rad mutation on Ty elements as compared to artificially constructed repeats.

Adenosine Triphosphatases↗

PubMed1994

Transcriptional induction of Ty recombination in yeast.

Families of repeated sequences are present in the genomes of all eukaryotes. Little is known about the mechanism(s) that prevents recombination between repeated sequences. In the yeast Saccharomyces cerevisiae, recombination between homologous sequences placed at nonhomologous locations in the genome (ectopic recombination) has been shown to occur at high frequencies for artificially created repeats, but at relatively low frequencies for a natural family of repeated sequences, the Ty family. We have previously shown that a high level of Ty cDNA in the cell causes an increase in the rate of nonreciprocal recombination (gene conversion) of a marked Ty element. In the present study, we show that it is also possible to elevate the rate of recombination of a marked Ty by increasing its transcription. This induction is different from, and acts synergistically to, the one seen upon increased levels of donor Ty cDNA. We show that the induction by transcription does not require the products of the RAD50, RAD51, and RAD57 genes. In contrast, cDNA-mediated recombination is dependent on the product of the RAD51 gene but not on products of the genes RAD50 or RAD57.

DNA Repair↗

PubMed1994

Plasmid-mediated induction of recombination in yeast.

Diploid yeast cells heteroallelic at the HIS3 locus were transformed with a minichromosome (centromeric plasmid) carrying homology to the HIS3 region and containing the same two mutations as were present in the chromosomes. When a double-strand break (DSB) was introduced in the region of homology, an increase in the recombination frequency between heteroalleles (leading to His+ cells) was observed, although the plasmid was unable to donate wild-type information. This induction of recombination was dependent on the presence of homology between the plasmid sequences and the chromosomes. We show evidence for the physical involvement of the plasmid in tripartite recombination events, and we propose models that can explain the interactions between the plasmid-borne and chromosomal-borne alleles. Our results suggest that the mitotic induction of recombination by DNA damage is due to localized initiation of recombination events, and not to a general induction of recombination enzymes in the cell.

DNA Damage↗

PubMed1993

Donation: a new, facile method of gene replacement in yeast.

We describe here a new method for the introduction of non-selectable alleles into Saccharomyces cerevisiae, gene replacement by donation. This method only requires the availability of an autonomously replicating, selectable plasmid containing the allele to be introduced into yeast. The plasmid is digested at a restriction site (or sites) within this allele, and introduced into yeast by transformation. In the course of double-strand break repair, the entering plasmid donates genetic information to the chromosome, replacing the chromosomal allele in a gene conversion-like event. Gene replacement events are identified by a phenotypic screen of the transformants. When necessary, the transforming plasmid may be subsequently lost by segregation during permissive growth. We have studied several parameters affecting the utility of this protocol as a method of gene replacement. Together with our previous results, the results show gene replacement by donation to be a useful, facile method, yielding gene replacement in up to 1.5% of transformants.

Genes, Fungal↗

PubMed1992

Effect of donor copy number on the rate of gene conversion in the yeast Saccharomyces cerevisiae.

Nonreciprocal recombination (gene conversion) between homologous sequences at nonhomologous locations in the genome occurs readily in the yeast Saccharomyces cerevisiae. In order to test whether the rate of gene conversion is dependent on the number of homologous copies available in the cell to act as donors of information, the level of conversion of a defined allele was measured in strains carrying plasmids containing homologous sequences. The level of recombination was elevated in a strain carrying multiple copies of the plasmid, compared with the same strain carrying a single copy of the homologous sequences either on a plasmid or integrated in the genome. Thus, the level of conversion is proportional to the number of copies of donor sequences present in the cell. We discuss these results within the framework of currently favoured models of recombination.

Blotting, Southern↗

PubMed1992

Ectopic recombination between Ty elements in Saccharomyces cerevisiae is not induced by DNA damage.

Mitotic recombination is increased when cells are treated with a variety of physical and chemical agents that cause damage to their DNA. We show here, using Saccharomyces cerevisiae strains that carry marked Ty elements, that recombination between members of this family of retrotransposons is not increased by UV irradiation or by treatment with the radiomimetic drug methyl methanesulfonate. Both ectopic recombination and mutation events were elevated by these agents for non-Ty sequences in the same strain. We discuss possible mechanisms that can prevent the induction of recombination between Ty elements.

Blotting, Northern↗

PubMed1992

Involvement of cDNA in homologous recombination between Ty elements in Saccharomyces cerevisiae.

Strains carrying a marked Ty element (TyUra) in the LYS2 locus were transformed with plasmids bearing a differently marked Ty1 element (Ty1Neo) under the control of the GAL promoter. When these strains were grown in glucose, a low level of gene conversion events involving TyUra was detected. Upon growth on galactose an increase in the rate of gene conversion was seen. This homologous recombination is not the consequence of increased levels of transposition. When an intron-containing fragment was inserted into Ty1Neo, some of the convertants had the intron removed, implying an RNA intermediate. Mutations that affect reverse transcriptase or reverse transcription of Ty1Neo greatly reduce the induction of recombination in galactose. Thus, Ty cDNA is involved in homologous gene conversion with chromosomal copies of Ty elements. Our results have implications about the way families of repeated sequences retain homogeneity throughout evolution.

Biological Evolution↗

PubMed1989

Recombination in yeast and the recombinant DNA technology.

The development of methods to isolate eukaryotic genes, alter these genes in vitro and reintroduce them into the cell has had a major impact on the study of recombination in the yeast Saccharomyces cerevisiae. In this paper we discuss how recombinant DNA techniques have been employed in the study of recombination in yeast and the results that have been obtained in these studies.

DNA, Fungal↗

PubMed1988

Allelic and ectopic recombination between Ty elements in yeast.

Allelic and nonallelic (ectopic) recombination events were analyzed in a set of isogenic strains that carry marked Ty elements. We found that allelic recombination between Ty elements occurred at normal frequencies both in meiosis and mitosis. The marked Ty elements were involved in a large variety of different types of ectopic recombination and this variety was greater in mitosis than in meiosis. Allelic and ectopic recombination events occurred at similar frequencies in mitosis, but allelic recombination predominated in meiosis. Some of the types of ectopic mitotic recombination indicated the common occurrence of concerted recombination events. The length of homology represented by a delta element (330 bp) seemed to be sufficient for some types of mitotic and meiotic recombination.

Alleles↗

PubMed1988

Meiotic recombination between repeated transposable elements in Saccharomyces cerevisiae.

We have measured the frequency of meiotic recombination between marked Ty elements in the Saccharomyces cerevisiae genome. These recombination events were usually nonreciprocal (gene conversions) and sometimes involved nonhomologous chromosomes. The frequency of ectopic gene conversion among Ty elements appeared lower than expected on the basis of previous studies of recombination between artificially constructed repeats. The conversion events involved either a subset of the total Ty elements in the genome or the conversion tract was restricted to a small region of the Ty element. In addition, the observed conversion events were very infrequently associated with reciprocal exchange.

DNA↗

PubMed1986

The RAD50 gene of Saccharomyces cerevisiae is not essential for vegetative growth.

The gene RAD50 was located by the ability of subclones to restore the Rad+ phenotype following transformation of a rad50-1 mutant. Disruption of the gene was achieved by directed integration of a plasmid carrying a fragment internal to RAD50. Haploids with the disrupted gene are viable and do not differ in growth rate or plating efficiency from isogenic rad50-1 or Rad+ strains.

Cloning, Molecular↗

PubMed1986

Regulation of the RAD6 gene of Saccharomyces cerevisiae in the mitotic cell cycle and in meiosis.

The regulation of the RAD6 gene at the mRNA level was investigated. The level of steady state RAD6 mRNA increases once every cell cycle, at late S/early G2. This stage is the one at which rad6 mutants arrest, as do wild-type cells exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS), or cdc40 cells exposed to the restrictive temperature. This appears to be a repair-specific stage in the cell cycle. RAD6 mRNA levels increase when cells are treated with MMS, but this increase seems to be due to the arrest of the cells by MMS at the repair-specific stage; cells arrested at the same stage by HU or by the cdc40 lesion also show high levels of RAD6 mRNA. A much smaller increase in the level of RAD6 transcripts is seen following UV irradiation. During meiosis, RAD6 mRNA is more abundant before commitment to recombination. The differential increase of RAD6 mRNA during the S/G2 repair-specific stage of the cell cycle relates the RAD6 function to the normally occurring radioresistance found at this stage.

Genes, Fungal↗

PubMed1986

DNA-repair characterization of cdc40-1, a cell-cycle mutant of Saccharomyces cerevisiae.

The cell-cycle specific mutation cdc40-1, which has been previously shown to be sensitive to MMS at the restrictive temperature, was further characterized as a DNA-repair-deficient mutation. cdc40-1 mutants shown only slight sensitivity to UV irradiation. Double mutant studies shown that rad6-l is epistatic to cdc40-1 with respect to sensitivity to UV irradiation and MMS. rad50-1 is epistatic to cdc40-1 with respect to MMS sensitivity in G1 stationary cells, but not in logarithmic cultures. An additive effect is seen between cdc40-1 and rad50-1 with respect to UV irradiation. cdc40-1 mutants are defective in UV-induced mutagenesis at the restrictive temperature. UV-induced levels of recombination are normal at both temperatures, while MMS-induced recombination is enhanced at the restrictive temperature.

Alleles↗

PubMed1985

Cloning and mapping of CDC40, a Saccharomyces cerevisiae gene with a role in DNA repair.

The cdc40 mutation has been previously shown to be a heat-sensitive cell-division-cycle mutation. At the restrictive temperature, cdc40 cells arrest at the end of DNA replication, but retain sensitivity to hydroxyurea (Kassir and Simchen 1978). The mutation has also been shown to affect commitment to meiotic recombination and its realization. Here we show that mutant cells are extremely sensitive to Methyl-Methane Sulfonate (MMS) when the treatment is carried out at restrictive temperature. Incubation at 37 degrees C prior to, or after MMS treatment at 23 degrees C, does not result in lower survival. It is concluded that the CDC40 gene product has a role in DNA repair, possibly holding together or protecting the DNA during the early stages of repair. The CDC40 gene was cloned on a 2.65 kb DNA fragment. A 2 mu plasmid carrying the gene was integrated and mapped to chromosome IV, between trp4 and ade8, by the method of marker loss. Conventional tetrad analysis has shown cdc40 to map 1.7 cM from trp4.

Chromosome Mapping↗

PubMed1984

Cloning and mapping of the RAD50 gene of Saccharomyces cerevisiae.

The RAD50 gene was cloned as a 4.8 kb fragment in the 2 mu derived plasmid pFL1. The gene resides in a 3.9 kb segment that was subcloned into the plasmid YRp7. The cloned gene complements the deficiency caused by the rad50-1 mutation with respect to gamma-rays, MMS resistance and UV-induced mitotic recombination. Restoration of the Rad+ phenotype occurs when the cloned gene is on a freely replicating multiple-copy plasmid or in the integrated form. Mapping of the cloned gene following integration of the 2 mu plasmid, and of the subclone in plasmid YRp7, showed it to be located on the left arm of chromosome XIV. Tetrad analysis of various crosses involving two different strains carrying rad50-1 showed the mutation to map next to pet2 on chromosome XIV, and not on the right arm of chromosome IV, as previously published.

Cloning, Molecular↗