New mutation in Saccharomyces cerevisiae SRM genes and some features of their phenotypic effects

The effects of the previously identified mutations in nuclear genes SRM8, SRM12, SRM15, and SRM17 on the maintenance of chromosomes and recombinant plasmids in Saccharomyces cerevisiae cells and on cell sensitivity to ionizing radiation were studied. The srm8 mutation caused instability of chromosome maintenance in diploid cells. In yeast cells with the intact mitochondrial genome, all examined srm mutations decreased the mitotic stability of a centromeric recombinant plasmid with the chromosomal ARS element. Mutations srm12, srm15, and srm17 also decreased the mitotic stability of a centromereless plasmid containing the same ARS element, whereas the srm8 mutation did not markedly affect the maintenance of this plasmid. Mutations srm8, srm12, and srm17 were shown to increase cell sensitivity to gamma-ray irradiation. The SRM8 gene was mapped, cloned, and found to correspond to the open reading frame YJLO76w in chromosome X.     

The SRM12/ADA1 Gene Sequence Inserted into Recombinant Plasmids Makes Them More Mitotically Stable in Budding Yeast Cells

TheSRM12/ADA1 gene sequence inserted into a recombinant circular plasmid improves its maintenance in budding yeast (Saccharomyces cerevisiae) cells. Plasmid stabilization caused by the integrated SRM12 sequence does not require the SRM12 function complementing the srm12 mutation and depends on the orientation of the inserted sequence in the vector. This stabilization is mainly due to a decrease in spontaneous plasmid underreplication/copy loss rather than an increase in the fidelity of mitotic plasmid segregation.     

Genetic analysis of mitochondrial rho-mutability in Saccharomyces yeasts. VI. Quantitative characteristics of the effect of mutation srm5 on the mitochondrial stability of natural and recombinant genetic structures

The srm5 mutation diminishes the spontaneous rho- mutation rate by an order of magnitude. Frequency of rho- mutations is 500 times lower in homozygous cultures, as compared with those of normal SRM+/SRM+ diploids. The rate of spontaneous loss of extra chromosome IV is about 25 times higher in srm5 disomes, as compared with SRM+ ones. Haploid srm1 srm5 transformants loose recombinant circular minichromosomes spontaneously about 4 times more frequently than srm1SRM5 cells. The data presented suggest that general control of mitotic stability of different (mitochondrial and nuclear, nuclear as well as recombinant) genetic structures operates in Sacch. cerevisiae. Autonomously replicating sequences (ARS elements) seem to be involved in this mechanism.     

The SRM12/ADA1 gene acts as a cis-active factor when inserted into recombinant plasmids and makes them more stable in Saccharomyces

A DNA fragment containing the SRM12/ADA1 gene sequence inserted into a recombinant circular plasmid improves its maintenance in budding yeast (Saccharomyces cerevisiae) cells. Plasmid stabilization caused by the integrated SRM12 sequence does not require the SRM12 function complementing the srm12 mutation and depends on the orientation of the inserted fragment in the vector. This stabilization is mainly due to a decrease in spontaneous plasmid underreplication/copy loss rather than an increase in the fidelity of mitotic plasmid segregation.     

Participation of <Emphasis Type="Italic">SRM5/CDC28</Emphasis>, <Emphasis Type="Italic">SRM8/NET1</Emphasis>, and <Emphasis Type="Italic">SRM12/HFI1</Emphasis> genes in checkpoint control in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

About twenty genes participating in checkpoint control are known in yeast Saccharomyces cerevisiae. The involvement of SRM genes in the cell cycle arrest under the action of DNA damaging agents was studied in this work. These genes were earlier defined as genes affecting genetic stability and radiosensitivity. It was shown that mutations srm5/cdc28-srm, srm8/net1-srm, and srm12/hfi1-srm fail the cell cycle arrest in the presence of DNA damage and influence the checkpoint arrest in G0/S (srm5, srm8), G1/S (srm5, srm8, srm12), S (srm5, srm12), and G₂/M (srm5). It seems likely that genes SRM5/CDC28, SRM12/HFI1/ADA1, and SRM8/NET1 are involved in a cell response to DNA damage, and in checkpoint regulation in particular.     

Genetic interaction of DED1 encoding a putative ATP-dependent RNA helicase with SRM1 encoding a mammalian RCC1 homolog in Saccharomyces cerevisiae

 The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1. In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene. The double mutant, srm1-1 ded1-21, could grow at 35° C, but not at 37° C. A revertant of srm1-1 ded1-21 that became able to grow at 37° C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1. In all the rcc1 ^- strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature. While mutated SRM1 protein was stabilized in all of the rcc1 ^- strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S. cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2. Received: 20 March 1996/Accepted: 1 July 1996     

Genetic analysis of mitochondrial rho-mutability in Saccharomyces. III. Comparative analysis of the effect of various nuclear srm mutations and disomy for chromosome IV on rho-mutagenesis

Different combinations of modifying genes which enhance the rho- mutability of haploid yeast cells are shown to be suppressible by the srm1, srm2, srm3 mutations and by the disomy for chromosome IV. The srm1 mutation leads to dramatic decrease in both the spontaneous and ethidium-bromide induced rho- mutability. Other srm mutations studied and the disomy appear to cause relatively moderate quantitative changes in the spontaneous rho- mutation rate and to have no significant effect on mutation induction by ethidium bromide. Neither additivity nor synergism was revealed by the analysis of the interaction between the srm mutations. We suggest that in Saccharomyces an efficient mechanism of the rho- mutagenesis operates which can be directly affected by the srm1 mutation and more or less modified by other srm mutations under study and by the disomy for chromosome IV.     

Genetic interaction of DED1encoding a putative ATP-dependent RNA helicase with SRM1 encoding a mammalian RCC1 homolog in Saccharomyces cerevisiae

The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1. In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene. The double mutant, srm1-1 ded1-21, could grow at 35°?C, but not at 37°?C. A revertant of srm1-1 ded1-21 that became able to grow at 37°?C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1. In all the rcc1 ^- strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature. While mutated SRM1 protein was stabilized in all of the rcc1 ^- strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S. cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2.     

A yeast mutation that stabilizes a plasmid bearing a mutated ARS1 element.

To identify the trans-acting factors involved in autonomously replicating sequence (ARS) function, we initiated a screen for Saccharomyces cerevisiae mutants capable of stabilizing a plasmid that contains a defective ARS element. The amm (altered minichromosome maintenance) mutations recovered in this screen defined at least four complementation groups. amm1, a mutation that has been studied in detail, gave rise to a 17-fold stabilization of one defective ARS1 plasmid over the level seen in wild-type cells. The mutation also affected the stability of at least one plasmid bearing a wild-type ARS element. amm1 is an allele of the previously identified TUP1 gene and exhibited the same pleiotropic phenotypes as other tup1 mutants. Plasmid maintenance was also affected in strains bearing a TUP1 gene disruption. Like the amm1 mutant, the tup1 disruption mutant exhibited ARS-specific plasmid stabilization; however, the ARS specificities of these two mutants differed. The recovery of second-site mutations that suppressed many of the tup1 phenotypes but not the increased plasmid maintenance demonstrates that the plasmid stability phenotype of tup1 mutants is not a consequence of the other defects caused by tup1.     

Genetic Collection of Meiotic Mutants of Rye <Emphasis Type="Italic">Secale cereale</Emphasis> L.

Genetic collection of meiotic mutants of winter rye Secale cereale L. (2n = 14) was created. Mutations were detected in inbred F₂ generations after self-fertilization of the F₁ hybrids, obtained by individual crossing of rye plants (cultivar Vyatka) or weedy rye with plants from autofertile lines. The mutations cause partial or complete plant sterility and are maintained in collection in a heterozygous state. Genetic analysis accompanied by cytogenetic study of meiosis has revealed six mutation types. (1) Nonallelic asynaptic mutations sy1 and sy9 caused the formation of only axial chromosome elements in prophase and anaphase. The synaptonemal complexes (SCs) were absent, the formation of the chromosome “bouquet” was impaired, and all chromosomes were univalent in meiotic metaphase I in 96.8% (sy1) and 67% (sy2) of cells. (2) Weak asynaptic mutation sy3, which hindered complete termination of synapsis in prophase I. Subterminal asynaptic segments were always observed in the SC, and at least one pair of univalents was present in metaphase I, but the number of cells with 14 univalents did not exceed 2%. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19, which caused partially nonhomologous synapsis: change in pairing partners and fold-back chromosome synapsis in prophase I. In metaphase I, the number of univalents varied and multivalents were observed. (4) Mutation mei6, which causes the formation of ultrastructural protrusions on the lateral SC elements, gaps and branching of these elements. (5) Allelic mutations mei8 and mei8-10, which caused irregular chromatin condensation along chromosomes in prophase I, sticking and fragmentation of chromosomes in metaphase I. (6) Allelic mutations mei5 and mei10, which caused chromosome hypercondensation, defects of the division spindle formation, and random arrest of cells at different meiotic stages. However, these mutations did not affect the formation of microspore envelopes even around the cells, whose development was blocked at prophase I. Analysis of cytological pictures of meiosis in double rye mutants reveled epistatic interaction in the mutation series sy9 > sy1 > sy3 > sy19, which reflects the order of switching these genes in the course of meiosis. The expression of genes sy2 and sy19 was shown to be controlled by modifier genes. Most meiotic mutations found in rye have analogs in other plant species.     

Cloning and mapping of the RAD50 gene of Saccharomyces cerevisiae

Summary The RAD50 gene was cloned as a 4.8 kb fragment in the 2 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 -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 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 tow different strains carrying rad50-1 showed the mutation to map next to pet2 on chromosome XIV, and not on the right arm of chromsome IV, as previously published.     

Genetic analysis of mitochondrial rho-mutability in Saccharomyces. IV. Relation between spontaneous rho-mutability and mitotic stability in disomic Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae the disomy for chromosome XIV resembles the previously described disomy for chromosome IV in that it leads to a significant decrease in spontaneous rho- mutability. The nuclear srm1 mutation, reducing spontaneous rho- mutability, diminishes significantly the mitotic disome stability. So, the mechanisms of spontaneous rho- mutagenesis and mitotic disome stability seem to compete for the function affected by the srm1 mutation.     

Chromosome stability in saccharomycete yeasts

Mutants with high instability of chromosome III designated Chl+ (chromosome loss) were obtained after irradiation with UV the Z4221-3c1 haploid disomic for chromosome III. The Chl+ mutants can be divided into two classes: 1) CL2, CL3, CL7, CL9, CL11, CL12, CL13 with elevated level of spontaneous inter- and intragenic recombination; 2) CL4, CL8 which unstable maintenance of chromosome III not accompanied with elevation of mitotic recombination frequency. The CL4 and CL8 mutants also reveal, in contrast to other mutants, unstable maintenance of artificial mini-chromosomes with chromosomal replicator ARS1 and centromeric loci CEN3, CEN4, CEN5, CEN6, CEN11. Substitution of ARS1 for other yeast replicators (ARS2, ARS of 2 micron plasmid) leads to no stabilization of mini-chromosomes in mutants. The noncentromeric plasmids containing homologous replicator (or replicators) from Candida maltosa are maintained with the same frequency both in wild type and in mutants. So, the stability of mini-chromosomes in CL4 and CL8 is not connected with uneffective replication of these chromosomes. Instability of chromosome III and mini-chromosomes in CL4 and CL8 is controlled by two nonallelic genes designated chl14 and chl18. We suppose that these genes control the process of centromere interaction with mitotic spindle microtubules.     

A quantitative assay to measure chromosome stability in Schizosaccharomyces pombe

Summary The fidelity of mitotic chromosome transmission in Schizosaccharomyces pombe was estimated quantitatively by using cycloheximide resistance as a means to select cells that had undergone chromosome loss or nondisjunction. We aimed to investigate the connection between recombination and mitotic chromosome stability. A number of mutants defective in mitotic recombination such as cdc17-L16, rec59-72, and rec50-25 were tested and in these an approximately ten fold elevation of mitotic haploidization rate was found compared with controls. Our data suggest that recombination is important in controlling the maintenance of chromosomes during mitosis.     

Transformation of a methionine auxotrophic mutant of Mucor circinelloides by direct cloning of the corresponding wild type gene

Summary A transformation system has been developed for Mucor circinelloides, by direct cloning of a wild-type methionine gene that complements the auxotrophic mutation. The marker gene isolated was associated with an autonomous replication sequence (ARS) functional in this zygomycete. Southern hybridisation analyses of transformants showed sequence homology both with vector DNA and with Mucor wild-type DNA. The transformation frequency (up to 6000 per g DNA) and the mitotic instability of the transformed cells were studied. The hybridisation pattern of undigested DNA from the transformants suggests that the inserts contain a novel autonomous replication element for this filamentous fungus.     

Genetic analysis of the mitotic transmission of minichromosomes

The fidelity of the mitotic transmission of minichromosomes in S. cerevisiae is monitored by a novel visual assay that allows one to detect changes in plasmid copy number in individual mitotic divisions. This assay is used to investigate the mitotic transmission of a plasmid containing a putative yeast origin of replication (ARS 1) and a centromere (CEN3). The rate of improper segregation for the minichromosome is 200-fold higher than observed for a normal chromosome. However, the replication of the minichromosome is stringently controlled; it overreplicates less than once per one thousand mitotic divisions. We also use this assay to isolate and characterize mutations in ARS 1 and CEN3. The mutations in ARS 1 define a new domain required for its optimal activity, and the mutations in CEN3 suggest that the integrity of element II is not essential for centromere function. Finally, the phenotypes of the mutations in ARS 1 and CEN3 are consistent with their function in replication and segregation, respectively.     

Replication analysis of plasmid DNAs injected into Drosophila embryos

From a shotgun collection of DNA fragments, isolated from Drosophila melanogaster, we selected sequences that function as autonomously replicating sequences (ARS) in the yeast Saccharomyces cerevisiae. To investigate the replicative potential of such sequences in Drosophila, five of these ARS elements and also the Adh gene of D. melanogaster, which has been described earlier to have ARS function in yeast, were microinjected into developing Drosophila eggs and analysed after reisolation from first instar larvae. As an assay for DNA replication, we determined the sensitivity of recovered plasmid DNA to restriction enzymes that discriminate between adenine methylation and nonmethylation. within the limits of detection our results show that none of the plasmids replicated two or more rounds. However, a fraction of all injected plasmid DNAs, including vector DNA, seems to replicate once. The same result was obtained for a DNA sequence from mouse that had been reported to have replication origin function in mouse tissue culture cells. We excluded the possibility that methylation of the plasmids is the reason for their inability to replicate. These results demonstrate that homologous and heterologous DNA sequences that drive replication of plasmids in cells of other species are not sufficient to fulfil this function in Drosophila embryos.by J.A. Huberman     

Nuclear suppression of a mitochondrial RNA splice defect: nucleotide sequence and disruption of the MRS3 gene

Summary A mitochondrial RNA splice defect in the first intron of the COB gene (bI1) can be suppressed by a dominant nuclear mutation SUP-101. Starting with a gene bank of yeast nuclear DNA from a SUP-101 suppressor strain cloned in the YEp13 plasmid, we have isolated a recombinant plasmid which exerts a suppressor activity similar to the SUP-101 allele. The N3(2) insert of this plasmid contains an open reading frame (ORF) of 1014 bp which is transcribed to a 12 S RNA. Deletion of the 5 end of this ORF and its upstream sequences abolishes the suppressor activity. The N3(2) insert thus carries a functional gene (called MRS3) which can suppress a mitochondrial splice defect. The chromosomal equivalent of the cloned gene has been mapped to chromosome 10. Disruption of this chromosomal gene has no phenotypic effect on wild-type cells.     

Mutations that increase the mitotic stability of minichromosomes in yeast: characterization of RAR1

Summary In an attempt to identify proteins involved in the initiation of DNA replication, we have isolated a series of Saccharomyces cerevisiae mutants in which the function of putative replication origins is affected. The phenotype of these Rar^- (regulation of autonomous replication) mutants is to increase the mitotic stability of plasmids whose replication is dependent on weak ARS elements. These mutations are generally recessive and complementation analysis shows that mutations in several genes may improve the ability of weak ARS elements to function. One mutation (rar1-1) also confers temperature-sensitive growth, and thus an essential gene is affected. We have determined the DNA sequence of the RAR1 gene, which reveals an open reading frame for a 48.5 kDa protein. The RAR1 gene is linked to rna1 on chromosome XIII.