An extensive deletion causing overproduction of yeast iso-2-cytochrome c

CYC7-H3 is a cis-dominant regulatory mutation that causes a 20-fold overproduction of yeast iso-2-cytochrome c. The CYC7-H3 mutation is an approximately 5 kb deletion with one breakpoint located in the 5' noncoding region of the CYC7 gene, approximately 200 base from the ATG initiation codon. The deletion apparently fuses a new regulatory region to the structural portion of the CYC7 locus. The CYC7-H3 deletion encompasses the RAD23 locus, which controls UV sensitivity and the ANP1 locus, which controls osmotic sensitivity. The gene cluster CYC7-RAD23-ANP1 displays striking similarity to the gene cluster CYC1-OSM1-RAD7, which controls, respectively, iso-1-cytochrome c, osmotic sensitivity and UV sensitivity. We suggest that these gene clusters are related by an ancient transpositional event.     

Deletions extending from a single Ty1 element in Saccharomyces cerevisiae.

Chromosomal rearrangements associated with one Ty1 element in the iso-1-cytochrome c (CYC1) region of Saccharomyces cerevisiae yeast cells were examined. Most of the rearrangements were deletions of the three linked genes, CYC1, OSM1, and RAD7, and resulted from recombination involving the single Ty1 element and a solo delta in the same orientation. These deletions differed by the number of Ty1 elements (zero, one, or two) remaining after deletion and by restriction site heterogeneities associated with these elements. A single Ty1 element remained at the deletion junction point much more frequently than no Ty1. Apparently the Ty1-associated delta element nearer to the solo delta was involved more often in recombination than the more distal Ty1-associated delta element. The restriction site data implicate gene conversion and suggest that site-specific recombination within the deltas, if occurring, is not the only mechanism of delta-delta recombination. Three other rearrangements bore deletions which began at the end of the Ty1 element and extended into regions not bearing Ty1 or delta sequences. Two of these deletions eliminated 7 kilobases of DNA, although they differed by an associated reciprocal translocation. The third involved a deletion of 14.7 kilobases of DNA associated with an overlapping inversion.     

Identification of regulatory regions within the Ty1 transposable element that regulate iso-2-cytochrome c production in the CYC7-H2 yeast mutant.

The CYC7-H2 mutation in the yeast Saccharomyces cerevisiae was caused by insertion of a Ty1 transposable element in front of the iso-2-cytochrome c structural gene, CYC7. The Ty1 insertion places iso-2-cytochrome c production under control of regulatory signals that are normally required for mating functions in yeast cells. We have investigated the regions of the Ty1 insertion that are responsible for the aberrant production of iso-2-cytochrome c in the CYC7-H2 mutant. Five alterations of the CYC7-H2 gene were obtained by specific restriction endonuclease cleavage of the cloned DNA and ligation of appropriate fragments. The CYC7+, CYC7-H2, and modified CYC7-H2 genes were each inserted into the yeast vector YIp5 and used to transform a cytochrome c-deficient yeast strain. Expression and regulation of each allele integrated at the CYC7 locus have been compared in vivo by determination of the amount of iso-2-cytochrome c produced. These results show that distal regions of the Ty1 element are not essential for the CYC7-H2 overproducing phenotype. In contrast, alterations in the vicinity of the proximal Ty1 junction abolish the CYC7-H2 expression and give rise to different phenotypes.     

Ty1 sequence with enhancer and mating-type-dependent regulatory activities.

Some insertion mutations in Saccharomyces cerevisiae activate the expression of adjacent structural genes. The CYC7-H2 mutation is a Ty1 insertion 5' to the iso-2-cytochrome c coding region of CYC7. The Ty1 insertion causes a 20-fold increase in CYC7 expression in a and alpha haploid cell types of S. cerevisiae. This activation is repressed in the a/alpha diploid cell type. Previous computer analysis of the CYC7-H2 Ty1 activator region identified two related sequences with homology both to mammalian enhancers and to a yeast a/alpha control site. A 112-base-pair (bp) DNA fragment encompassing one of these blocks of homology functioned as one component of the Ty1 activator. A 28-bp synthetic oligonucleotide with the wild-type homology block sequence was also functional. A single base pair mutation within the enhancer core of the synthetic 28-bp regulatory element reduced its activation ability to near background amounts. In addition, the 112-bp Ty1 fragment by itself functioned as a target for repression of adjacent gene expression in a/alpha diploid cells.     

Physical analysis of the CYC1-sup4 interval in Saccharomyces cerevisiae.

CYC1 and sup4 are part of a tightly linked cluster of genes on chromosome X in the yeast Saccharomyces cerevisiae. Using as probes previously cloned fragments containing the CYC1 and sup4 genes, we have identified and cloned the deoxyribonucleic acid (DNA) present between these genes in one strain of yeast. We find that the CYC1 and sup4 genes are approximately 21 kilobases apart. In the same strain, the meiotic map distance is approximately 3.7 centimorgans, for a ratio of 5.6 kilobases per centimorgan in this interval. The physical mapping has allowed unambiguous determination of the orientation of CYC1 and sup4 relative to each other, the centromere, and a nearby transfer ribonucleic acid (tRNA(2Ser)) gene. The spontaneous mutation cyc1-1 inactivates the CYC1 gene as well as the neighboring loci OSM1 and RAD7. We have determined that a cyc1-1-bearing strain lacks approximately 13 kilobases of single-copy DNA from the CYC1-sup4 region, including all of the CYC1 coding information. There is a sequence homologous to the middle-repetitive element Ty1 at or near the breakpoint of the cyc1-1 deletion. We discuss the possibility that Ty elements play a role in the formation of such large, spontaneous deletions, which occur frequently in this region of chromosome X in certain yeast strains.     

Mechanisms of human minisatellite mutation in yeast

Minisatellites are tandem repeat loci, with repeat units ranging in size from 5 bp to 100 bp. The total lengths of repeat arrays vary from about 0.5 kb to 30 kb, and excessive variability in allele length at human minisatellite loci is the result of germline-specific complex recombination events generating new length alleles. Minisatellite alleles also mutate to new lengths in somatic cells, but this occurs at a much lower rate than in the germline. Since recombination is involved in minisatellite mutation, the yeast Saccharomyces cerevisiae is a suitable model organism that has been employed to further dissect the molecular basis of mutation events at human minisatellites. These studies have shown that the mutational behaviour of a minisatellite in meiosis is not determined by the intrinsic properties of the repeat array, but are highly dependent on the position of the minisatellite in the genome. The processes for minisatellite mutation in yeast and humans are identical in the sense that mutation is indeed driven by meiotic recombination, but differ with regard to the types of structural changes that are generated by the recombination events. Tetrad analyses showed that inter-allelic transfers of repeats occur by conversion and not crossing over, and that several chromatids can be involved in successive recombination events in one meiosis, resulting in mutant alleles in several spores. It has been demonstrated that the genes SPO11 and RAD50, involved in the initiation of recombination events, are required for human minisatellite mutation in yeast meiosis. Intrinsic properties of the repeat array appear to determine the stability of human minisatellites in yeast mitosis, since mitotic mutation rates in yeast are highly variable between minisatellites. The repair genes RAD27 and DNA2 stabilise human minisatellites in yeast mitosis, while RAD5 has no effect on mitotic stability. MSH2 depresses human minisatellite frequency in meiotic cells of yeast.     

Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast

Homologous recombination is believed to play important roles in processing stalled/blocked replication forks in eukaryotes. In accordance with this, recombination is induced by replication fork barriers (RFBs) within the rDNA locus. However, the rDNA locus is a specialised region of the genome, and therefore the action of recombinases at its RFBs may be atypical. We show here for the first time that direct repeat recombination, dependent on Rad22 and Rhp51, is induced by replication fork blockage at a site-specific RFB (RTS1) within a 'typical' genomic locus in fission yeast. Importantly, when the RFB is positioned between the direct repeat, conservative gene conversion events predominate over deletion events. This is consistent with recombination occurring without breakage of the blocked fork. In the absence of the RecQ family DNA helicase Rqh1, deletion events increase dramatically, which correlates with the detection of one-sided DNA double-strand breaks at or near RTS1. These data indicate that Rqh1 acts to prevent blocked replication forks from collapsing and thereby inducing deletion events.     

Meiotic Recombination between Duplicated Genetic Elements in SACCHAROMYCES CEREVISIAE

We have studied the meiotic recombination behavior of strains carrying two types of duplications of an 18.6-kilobase HIS4 Bam HI fragment. The first type is a direct duplication of the HIS4 Bam HI fragment in which the repeated sequences are separated by Escherichia coli plasmid sequences. The second type, a tandem duplication, has no sequences intervening between the repeated yeast DNA. The HIS4 genes in each region were marked genetically so that recombination events between the duplicated segments could be identified. Meiotic progeny of the strains carrying the duplication were analyzed genetically and biochemically to determine the types of recombination events that had occurred. Analysis of the direct vs. tandem duplication suggests that the E. coli plasmid sequences are recombinogenic in yeast when homozygous. In both types of duplications recombination between the duplicated HIS4 regions occurs at high frequency and involves predominantly interchromosomal reciprocal exchanges (equal and unequal crossovers). The striking observation is that intrachromosomal reciprocal recombination is very rare in comparison with interchromosomal reciprocal recombination. However, intrachromosomal gene conversion occurs at about the same frequency as interchromosomal gene conversion. Reciprocal recombination events between regions on the same chromatid are the most infrequent exchanges. These data suggest that intrachromosomal reciprocal exchanges are suppressed.     

A genetic screen for increased loss of heterozygosity in Saccharomyces cerevisiae

Loss of heterozygosity (LOH) can be a driving force in the evolution of mitotic/somatic diploid cells, and cellular changes that increase the rate of LOH have been proposed to facilitate this process. In the yeast Saccharomyces cerevisiae, spontaneous LOH occurs by a number of mechanisms including chromosome loss and reciprocal and nonreciprocal recombination. We performed a screen in diploid yeast to identify mutants with increased rates of LOH using the collection of homozygous deletion alleles of nonessential genes. Increased LOH was quantified at three loci (MET15, SAM2, and MAT) on three different chromosomes, and the LOH events were analyzed as to whether they were reciprocal or nonreciprocal in nature. Nonreciprocal LOH was further characterized as chromosome loss or truncation, a local mutational event (gene conversion or point mutation), or break-induced replication (BIR). The 61 mutants identified could be divided into several groups, including ones that had locus-specific effects. Mutations in genes involved in DNA replication and chromatin assembly led to LOH predominantly via reciprocal recombination. In contrast, nonreciprocal LOH events with increased chromosome loss largely resulted from mutations in genes implicated in kinetochore function, sister chromatid cohesion, or relatively late steps of DNA recombination. Mutants of genes normally involved in early steps of DNA damage repair and signaling produced nonreciprocal LOH without an increased proportion of chromosome loss. Altogether, this study defines a genetic landscape for the basis of increased LOH and the processes by which it occurs.     

High-frequency gene conversion between repeated C mu sequences integrated at the chromosomal immunoglobulin mu locus in mouse hybridoma cells.

The occurrence of mitotic recombination between repeated immunoglobulin mu gene constant (C mu) region sequences stably integrated at the haploid chromosomal immunoglobulin mu locus in murine hybridoma cells was investigated. Recombination events are detected as changes in hapten-specific immunoglobulin M production. Recombination occurs with high frequency (0.5 to 0.8%) by a mechanism consistent with gene conversion. A double-strand break repair-like mechanism is suggested by the finding that repair of a 2-bp deletion mutation and a 2-bp insertion mutation occurs with parity in a donor-directed manner. The results also suggest that the gene conversion process is directional in that the 5' C mu region sequence is preferentially converted.     

Genetic and Molecular Analysis of Recombination Events in Saccharomyces Cerevisiae Occurring in the Presence of the Hyper-Recombination Mutation Hpr1

The hyper-recombination mutation hpr1 specifically increases mitotic intrachromatid crossovers, with no effect on other mitotic recombination events such as unequal sister chromatid exchange and plasmid-chromosome recombination, and no effect on meiotic recombination and a lesser effect on intrachromosomal gene conversion. The excision repair RAD1 gene is partially required for the expression on the hpr1 phenotype. The simplest hypothesis to account for some of the hpr1 stimulated recombination events is that a heteroduplex DNA intermediate and localized gene conversion are involved. hpr1 stimulated crossover events are independent of intrachromosomal gene conversion events stimulated by the hyper-gene conversion mutation hpr5. This result suggests that different intrachromosomal recombination processes are affected in each mutant strain. We propose that HPR1 may function to inhibit intrachromatid crossovers.     

The ROX3 gene encodes an essential nuclear protein involved in CYC7 gene expression in Saccharomyces cerevisiae.

The ROX3 gene was identified during a hunt for mutants with increased expression of the heme-regulated CYC7 gene, which encodes the minor species of cytochrome c in the yeast Saccharomyces cerevisiae. The rox3 mutants caused a 10-fold increase in CYC7 expression both in the presence and absence of heme, had slightly increased anaerobic expression of the heme-activated CYC1 gene, and caused decreases in the anaerobic expression of the heme-repressed ANB1 gene and the aerobic expression of its heme-induced homolog. The wild-type ROX3 gene was cloned, and the sequence indicated that it encodes a 220-amino-acid protein. This protein is essential; deletion of the coding sequence was lethal. The coding sequence for beta-galactosidase was fused to the 3' end of the ROX3 coding sequence, and the fusion product was found to be localized in the nucleus, strongly suggesting that the wild-type protein carries out a nuclear function. Mutations in the rox3 gene showed an interesting pattern of intragenic complementation. A deletion of the 5' coding region complemented a nonsense mutation at codon 128 but could not prevent the lethality of the null mutation. These results suggest that the amino-terminal domain is required for an essential function, while the carboxy-terminal domain can be supplied in trans to achieve the wild-type expression of CYC7. Finally, RNA blots demonstrated that the ROX3 mRNA was expressed at higher levels anaerobically but was not subject to heme repression. The nuclear localization and the lack of viability of null mutants suggest that the ROX3 protein is a general regulatory factor.     

Characterization of TUP1, a mediator of glucose repression in Saccharomyces cerevisiae.

The TUP1 and CYC8 (= SSN6) genes of Saccharomyces cerevisiae play a major role in glucose repression. Mutations in either TUP1 or CYC8 eliminate or reduce glucose repression of many repressible genes and induce other phenotypes, including flocculence, failure to sporulate, and sterility of MAT alpha cells. The TUP1 gene was isolated in a screen for genes that regulate mating type (V.L. MacKay, Methods Enzymol. 101:325-343, 1983). We found that a 3.5-kb restriction fragment was sufficient for complete complementation of tup1-100. The gene was further localized by insertional mutagenesis and RNA mapping. Sequence analysis of 2.9 kb of DNA including TUP1 revealed only one long open reading frame which predicts a protein of molecular weight 78,221. The predicted protein is rich in serine, threonine, and glutamine. In the carboxyl region there are six repeats of a pattern of about 43 amino acids. This same pattern of conserved residues is seen in the beta subunit of transducin and the yeast CDC4 gene product. Insertion and deletion mutants are viable, with the same range of phenotypes as for point mutants. Deletions of the 3' end of the coding region produced the same mutant phenotypes as did total deletions, suggesting that the C terminus is critical for TUP1 function. Strains with deletions in both the CYC8 and TUP1 genes are viable, with phenotypes similar to those of strains with a single deletion. A deletion mutation of TUP1 was able to suppress the snf1 mutation block on expression of the SUC2 gene encoding invertase.     

Genome rearrangement in top3 mutants of Saccharomyces cerevisiae requires a functional RAD1 excision repair gene.

Saccharomyces cerevisiae cells that are mutated at TOP3, a gene that encodes a protein homologous to bacterial type I topoisomerases, have a variety of defects, including reduced growth rate, altered gene expression, blocked sporulation, and elevated rates of mitotic recombination at several loci. The rate of ectopic recombination between two unlinked, homologous loci, SAM1 and SAM2, is sixfold higher in cells containing a top3 null mutation than in wild-type cells. Mutations in either of the two other known topoisomerase genes in S. cerevisiae, TOP1 and TOP2, do not affect the rate of recombination between the SAM genes. The top3 mutation also changes the distribution of recombination events between the SAM genes, leading to the appearance of novel deletion-insertion events in which conversion tracts extend beyond the coding sequence, replacing the DNA flanking the 3' end of one SAM gene with nonhomologous DNA flanking the 3' end of the other. The effects of the top3 null mutation on recombination are dependent on the presence of an intact RAD1 excision repair gene, because both the rate of SAM ectopic gene conversion and the conversion tract length were reduced in rad1 top3 mutant cells compared with top3 mutants. These results suggest that a RAD1-dependent function is involved in the processing of damaged DNA that results from the loss of Top3 activity, targeting such DNA for repair by recombination.     

Mapping and Gene Conversion Studies with the Structural Gene for Iso-1-Cytochrome C in Yeast

We have investigated the order of the four genes cyc1, rad7, SUP4, and cdc8 which form a tightly linked cluster on the right arm of chromosome X in the yeast Saccharomyces cerevisiae. Crossing over and coconversion data from tetrad analysis established the gene order to be centromere-cyc1-rad7-SUP4. Also cdc8 appeared to be distal to SUP4 on the basis of crossovers that were associated with conversion of SUP4. The frequencies of recombination and the occurrence of coconversions suggest that these four genes are contiguous or at least nearly so. Gene-conversion frequencies for several cyc1 alleles were studied, including cyc1-1, a deletion of the whole gene that extends into the rad7 locus. The cyc1-1 deletion was found to be capable of conversion, though at a frequency some fivefold less than the other alleles studied, and both 3:1 and 1:3 events were detected. In general 1:3 and 3:1 conversion events were equally frequent at all loci studied, and approximately 50% of conversions were accompanied by reciprocal recombination for flanking markers. The orientation of the cyc1 gene could not be clearly deduced from the behavior of the distal marker SUP4 in wild-type recombinants that arose from diploids heteroallelic for cyc1 mutations.     

A yeast centromere acts in cis to inhibit meiotic gene conversion of adjacent sequences

The centromere of chromosome III (CEN3) of yeast has been examined for its ability to inhibit meiotic recombination in adjacent sequences. The effect of the centromere was investigated when it was adjacent to both of the recombining sequences (homozygous) or adjacent to only one of the two recombining DNA segments (hemizygous). When homozygous, CEN3 exerts a bidirectional repression of crossing over and a strong inhibition of gene conversion. This suggests that CEN3 reduces the frequency of crossing over by interfering with the initiation of proximal recombination events. When hemizygous, CEN3 impairs the ability of adjacent sequences to act as the recipient of genetic information during gene conversion. These results support the idea that the initiating event in yeast meiotic recombination involves the recipient molecule.     

Analysis of the Mechanism for Reversion of a Disrupted Gene

A positive selection system for intrachromosomal recombination in Saccharomyces cerevisiae has been developed. This was achieved by integration of a plasmid containing an internal fragment of the HIS3 gene into its chromosomal location. This resulted in two copies of the HIS3 gene one with a terminal deletion at the 3' end and the other with a terminal deletion at the 5' end. Reversion of the gene disruption could be brought about by plasmid excision, unequal sister chromatid exchange or sister chromatid conversion. The purpose of this study was to define the mechanisms involved in reversion of the gene disruption. The frequency of plasmid excision could be determined by placing a yeast sequence bearing an origin of replication onto the plasmid that was subsequently integrated into the yeast genome. Unequal sister chromatid exchange and conversion could be distinguished by determining the nature of the reciprocal product by Southern blotting. The results indicate that reversion might occur mainly by conversion between sister chromatids. This is because the frequency of plasmid excision was about two orders of magnitude lower than the overall frequency of reversion and no reciprocal product indicative of sister chromatid exchange was found. The findings of this presentation suggest that conversion might be an important mechanism for recombination of sister chromatids and possibly for repair of damaged DNA in S or G2.     

Genome structure of Halobacterium halobium: plasmid dynamics in gas vacuole deficient mutants

Halobacterium halobium contains two gas vacuole protein genes that are located in plasmid pHH1 (p-vac) and in the chromosomal DNA (c-vac). The mutation frequency for these genes is different: the constitutively expressed p-vac gene is mutated with a frequency of 10(-2), while the chromosomal gene expressed in the stationary phase of growth is mutated with a frequency of 10(-5). The difference in the mutation susceptibility is due to the dynamics of plasmid pHH1. p-vac gene mutations are caused (i) by the integration of an insertion element or (ii) by a deletion event encompassing the p-vac gene region. In contrast, c-vac mutants analyzed to date incurred neither insertion elements nor deletions. Deletion events within pHH1 occur at high frequencies during the development of a H. halobium culture. The investigation of the fusion regions resulting from deletion events indicates that insertion elements are involved. The analysis of pHH1 deletion variants led to a 4 kilobase pair DNA region containing the origin of replication of the pHH1 plasmid.