Biomedical subjects

E J Louis

Publications and source records attributed to E J Louis.

At least 19 recordsLinked to original sources

PubMed2003

A role for the mismatch repair system during incipient speciation in Saccharomyces.

The cause of reproductive isolation between biological species is a major issue in the field of biology. Most explanations of hybrid sterility require either genetic incompatibilities between nascent species or gross physical imbalances between their chromosomes, such as rearrangements or ploidy changes. An alternative possibility is that genomes become incompatible at a molecular level, dependent on interactions between primary DNA sequences. The mismatch repair system has previously been shown to contribute to sterility in a hybrid between established yeast species by preventing successful meiotic crossing-over leading to aneuploidy. This system could also promote or reinforce the formation of new species in a similar manner, by making diverging genomes incompatible in meiosis. To test this possibility we crossed yeast strains of the same species but from diverse historical or geographic sources. We show that these crosses are partially sterile and present evidence that the mismatch repair system is largely responsible for this sterility.

Base Pair Mismatch↗

PubMed2001

SGS1 is required for telomere elongation in the absence of telomerase.

In S. cerevisiae, mutations in genes that encode telomerase components, such as the genes EST1, EST2, EST3, and TLC1, result in the loss of telomerase activity in vivo. Two telomerase-independent mechanisms can overcome the resulting senescence. Type I survival is characterized by amplification of the subtelomeric Y' elements with a short telomere repeat tract at the terminus. Type II survivors arise through the abrupt addition of long tracts of telomere repeats. Both mechanisms are dependent on RAD52 and on either RAD50 or RAD51. We show here that the telomere elongation pathway in yeast (type II) is dependent on SGS1, the yeast homolog of the gene products of Werner's (WRN) and Bloom's (BLM) syndromes. Survival in the absence of SGS1 and EST2 is dependent upon RAD52 and RAD51 but not RAD50. We propose that the RecQ family helicases are required for processing a DNA structure specific to eroding telomeres.

Cell Survival↗

PubMed2000

Exploring redundancy in the yeast genome: an improved strategy for use of the cre-loxP system.

Gene families having more than three members are a common phenomenon in the Saccharomyces cerevisiae genome. As yeast research enters the post-genome era, the development of existing deletion strategies is crucial for tackling this apparent redundancy, hence a method for performing rapid multiple gene disruptions in this organism has been developed. We constructed three replacement cassettes in which different selectable markers were placed between two loxP loci. Multiple deletions (of members of a gene family) were generated, in one strain, using sequential integration of different replacement markers (kanMX, LYS2, KlURA3 and SpHIS5). Their excision from the genome was performed simultaneously, as the final step, using a new cre recombinase vector, which carries the cycloheximide-resistance gene from Candida maltosa as a selectable marker. Our multiple gene deletion system significantly accelerates and facilitates the functional analysis process and is particularly useful for studying gene families in either laboratory or industrial yeast strains.

Blotting, Southern↗

PubMed2000

Chromosomal evolution in Saccharomyces.

The chromosomal speciation model invokes chromosomal rearrangements as the primary cause of reproductive isolation. In a heterozygous carrier, chromosomes bearing reciprocal translocations mis-segregate at meiosis, resulting in reduced fertility or complete sterility. Thus, chromosomal rearrangements act as a post-zygotic isolating mechanism. Reproductive isolation in yeast is due to post-zygotic barriers, as many species mate successfully but the hybrids are sterile. Reciprocal translocations are thought to be the main form of large-scale rearrangement since the hypothesized duplication of the whole yeast genome 10(8) years ago. To test the chromosomal speciation model in yeast, we have characterized chromosomal translocations among the genomes of six closely related species in the Saccharomyces 'sensu stricto' complex. Here we show that rearrangements have occurred between closely related species, whereas more distant ones have colinear genomes. Thus, chromosomal rearrangements are not a prerequisite for speciation in yeast and the rate of formation of translocations is not constant. These rearrangements appear to result from ectopic recombination between Ty elements or other repeated sequences.

Chromosomes, Fungal↗

PubMed2000

Minisatellite variants generated in yeast meiosis involve DNA removal during gene conversion.

Two yeast minisatellite alleles were cloned and inserted into a genetically defined interval in Saccharomyces cerevisiae. Analysis of flanking markers in combination with sequencing allowed the determination of the meiotic events that produced minisatellites with altered lengths. Tetrad analysis revealed that gene conversions, deletions, or complex combinations of both were involved in producing minisatellite variants. Similar changes were obtained following selection for nearby gene conversions or crossovers among random spores. The largest class of events involving the minisatellite was a 3:1 segregation of parental-size alleles, a class that would have been missed in all previous studies of minisatellites. Comparison of the sequences of the parental and novel alleles revealed that DNA must have been removed from the recipient array while a newly synthesized copy of donor array sequences was inserted. The length of inserted sequences did not appear to be constrained by the length of DNA that was removed. In cases where one or both sides of the insertion could be determined, the insertion endpoints were consistent with the suggestion that the event was mediated by alignment of homologous stretches of donor/recipient DNA.

Alleles↗

PubMed2000

Three new species in the Saccharomyces sensu stricto complex: Saccharomyces cariocanus, Saccharomyces kudriavzevii and Saccharomyces mikatae.

On the basis of genetic analysis, molecular karyotyping and sequence analyses of the 18S rRNA and internal transcribed spacer (ITS) region, three new Saccharomyces species are described, Saccharomyces cariocanus (with type strain NCYC 2890T), Saccharomyces kudriavzevii (with type strain NCYC 2889T) and Saccharomyces mikatae (with type strain NCYC 2888T). Genetic and molecular analyses did not confirm the previously observed conspecificity of Saccharomyces paradoxus and S. cariocanus. The latter species exhibits postzygotic isolation from representative strains from all known geographical populations of S. paradoxus: European, Far-East Asian, North American and Hawaiian.

DNA, Fungal↗

PubMed1999

Limitations of silencing at native yeast telomeres.

Silencing at native yeast telomeres, in which the subtelomeric elements are intact, is different from silencing at terminal truncations. The repression of URA3 inserted in different subtelomeric positions at several chromosome ends was investigated. Many ends exhibit very little silencing close to the telomere, while others exhibit substantial repression in limited domains. Silencing at native ends is discontinuous, with maximal repression found adjacent to the ARS consensus sequence in the subtelomeric core X element. The level of repression declines precipitously towards the centromere. Mutation of the ARS sequence or an adjacent Abf1p-binding site significantly reduces silencing. The subtelomeric Y' elements are resistant to silencing along their whole length, yet silencing can be re-established at the proximal X element. Deletion of PPR1, the transactivator of URA3, and SIR3 overexpression do not increase repression or extend spreading of silencing to the same extent as with terminally truncated ends. sir1Delta causes partial derepression at X-ACS, in contrast to the lack of effect seen at terminal truncations. orc2-1 and orc5-1 have no effect on natural silencing yet cause derepression at truncated ends. X-ACS silencing requires the proximity of the telomere and is dependent on SIR2, SIR3, SIR4 and HDF1. The structures found at native yeast telomeres appear to limit the potential of repressive chromatin.

Base Sequence↗

PubMed1999

The topoisomerase II-associated protein, Pat1p, is required for maintenance of rDNA locus stability in Saccharomyces cerevisiae.

The Pat1 protein of Saccharomyces cerevisiae was identified during a screen for proteins that interact with topoisomerase II. Previously, we have shown that pat1 delta mutants exhibit a slow-growth phenotype and an elevated frequency of both mitotic and meiotic chromosome mis-segregation. Here, we have studied the effects of deleting the PAT1 gene on chromosomal stability, with particular reference to rates of homologous recombination within the rDNA locus. This locus was analyzed because rDNA-specific hyperrecombination is known to occur in conditional top2 mutants. We show that pat1 delta strains mimic top2 mutants in displaying an elevated rate of intrachromosomal excision recombination at the rDNA locus, but not elsewhere in the genome. The elevated rate of recombination is dependent upon Rad52p, but not upon Rad51p or Rad54p. However, pat1 delta strains display additional manifestations of more general genomic instability, in that they show mild sensitivity to UV light and an increased incidence of interchromosomal recombination between heteroalleles.

Alleles↗

PubMed1998

The effect of sex on adaptation to high temperature in heterozygous and homozygous yeast.

Most explanations for the evolutionary maintenance of sex depend on the assumption that sex produces variation by recombining parental haplotypes in the offspring. Therefore, meiosis is expected to be useful only in heterozygotes. We tested this assumption by competing sexual strains of yeast against constitutive asexuals in a hot (37 degrees C) culture for 500 generations, in either heterozygous or homozygous genetic backgrounds. We found that there was an initial cost of sex for all the sexual strains, which was indicated by a sharp increase in the proportion of asexuals after the induction of sex. The cost was larger in the heterozygotes than in the homozygotes, probably because of recombinational load. However, in two of the three heterozygote backgrounds, after the initial success of the asexuals, the remaining sexuals eventually drove them out of the population. These two heterozygotes also suffered the largest initial cost of sex. In the other heterozygote and in the three homozygote backgrounds it appeared to be a matter of chance whether sexuals or asexuals won. The average relative fitness increased in all the strains, but the increase was largest in the two strains that showed both the clearest advantage and the largest cost of sex. We conclude that these results are consistent with the traditional view that sex has a short-term cost but a long-term benefit.

Adaptation, Biological↗

PubMed1998

Mutation of yeast Ku genes disrupts the subnuclear organization of telomeres.

The mammalian Ku70 and Ku86 proteins form a heterodimer that binds to the ends of double-stranded DNA in vitro and is required for repair of radiation-induced strand breaks and V(D)J recombination [1,2]. Deletion of the Saccharomyces cerevisiae genes HDF1 and HDF2--encoding yKu70p and yKu80p, respectively--enhances radiation sensitivity in a rad52 background [3,4]. In addition to repair defects, the length of the TG-rich repeat on yeast telomere ends shortens dramatically [5,6]. We have shown previously that in yeast interphase nuclei, telomeres are clustered in a limited number of foci near the nuclear periphery [7], but the elements that mediate this localization remained unknown. We report here that deletion of the genes encoding yKu70p or its partner yKu80p altered the positioning of telomeric DNA in the yeast nucleus. These are the first mutants shown to affect the subnuclear localization of telomeres. Strains deficient for either yKu70p or yKu80p lost telomeric silencing, although they maintained repression at the silent mating-type loci. In addition, the telomere-associated silencing factors Sir3p and Sir4p and the TG-repeat-binding protein Rap1p lost their punctate pattern of staining and became dispersed throughout the nucleoplasm. Our results implicate the yeast Ku proteins directly in aspects of telomere organization, which in turn affects the repression of telomere-proximal genes.

Animals↗

PubMed1998

Minisatellite origins in yeast and humans.

Variable numbers of tandem repeats are valuable markers in genetic studies. The arrays of interest are simple microsatellites, containing repetitions of 1-5 nucleotides, and minisatellites, with multiple iterations of approximately 10 to 100 bp. Microsatellite origins can be explained by replication errors in regions fortuitously containing two or more adjacent short repeats. Microsatellite variation arises by replication errors in the absence of mismatch correction (R. Parsons et al., 1993, Cell 75: 1227-1236; M. Strand et al., 1993, Nature 365: 274-276). Variation in the size of minisatellites is thought to involve homologous recombination processes, including gene conversions (J. Buard and G. Vergnaud, 1994, EMBO J. 13: 3203-3210; A. J. Jeffreys et al., 1994, Nature Genet. 6: 136-145) and possibly unequal exchanges among repeats. However, the origins of minisatellites are less obvious. The probability of finding a direct tandem repeat of minisatellite size by chance alone is very low [<4-(10 to 100)]. Here we report the finding of short direct repeats of 5 to 10 bp flanking many yeast and human minisatellites that may be involved in their origins through replication slippage or unequal crossings over.

Base Sequence↗

PubMed1997

The nucleotide sequence of Saccharomyces cerevisiae chromosome XII.

The yeast Saccharomyces cerevisiae is the pre-eminent organism for the study of basic functions of eukaryotic cells. All of the genes of this simple eukaryotic cell have recently been revealed by an international collaborative effort to determine the complete DNA sequence of its nuclear genome. Here we describe some of the features of chromosome XII.

Base Sequence↗

PubMed1997

Chromosome ends: all the same under their caps.

The recent characterisation of subtelomeric regions from a variety of organisms from yeast to man has led to the realisation that all chromosome ends are similar in structure although maintenance of the terminus varies. The mosaic of repeats and proteins associated with telomeres has an architectural role which divides the genome into two domains, allowing for the adaptive use of the region as well as the evolution of non-telomerase-mediated telomere maintenance.

Adaptation, Biological↗

PubMed1997

Sequence comparison of human and yeast telomeres identifies structurally distinct subtelomeric domains.

We have sequenced and compared DNA from the ends of three human chromosomes: 4p, 16p and 22q. In all cases the pro-terminal regions are subdivided by degenerate (TTAGGG)n repeats into distal and proximal sub-domains with entirely different patterns of homology to other chromosome ends. The distal regions contain numerous, short (<2 kb) segments of interrupted homology to many other human telomeric regions. The proximal regions show much longer (approximately 10-40 kb) uninterrupted homology to a few chromosome ends. A comparison of all yeast subtelomeric regions indicates that they too are subdivided by degenerate TTAGGG repeats into distal and proximal sub-domains with similarly different patterns of identity to other non-homologous chromosome ends. Sequence comparisons indicate that the distal and proximal sub-domains do not interact with each other and that they interact quite differently with the corresponding regions on other, non-homologous, chromosomes. These findings suggest that the degenerate TTAGGG repeats identify a previously unrecognized, evolutionarily conserved boundary between remarkably different subtelomeric domains.

Base Sequence↗

PubMed1997

Saccharomyces cerevisiae telomeres. A review.

Recent work has yielded considerable information concerning the structure and function of telomeres and their associated sequences in the budding yeast Saccharomyces cerevisiae. The structure and maintenance of telomeres depends not only on the RNA template and the catalytic subunit of telomerase, but on a number of other proteins. These include proteins involved in assessing DNA damage and cell cycle regulation. There are also non-telomerase mediated processes involved in the normal maintenance of telomeres. In addition to proteins involved in telomere maintenance, there are a number of other proteins involved in the chromatin structure of the region. Many of these proteins have roles in silencing, ageing, segregation and nuclear architecture. The structure of the subtelomeric regions has been well characterized and consists of a mosaic of repeats found in variable copy numbers and locations. Amidst the variable mosaic elements there are small conserved sequences found at all ends that may have functional roles. Recent work shows that the subtelomeric repeats can rescue chromosome ends when telomerase fails, buffer subtelomerically located genes against transcriptional silencing, and protect the genome from deleterious rearrangements due to ectopic recombination. Thus the telomeres of yeast have a variety of roles in the life of the yeast cell beyond the protection of the ends and overcoming the end replication problem associated with linear molecules.

Base Sequence↗

PubMed1996

Pat1: a topoisomerase II-associated protein required for faithful chromosome transmission in Saccharomyces cerevisiae.

Saccharomyces cerevisiae top2 mutants deficient in topoisomerase II activity are defective in chromosome segregation during both mitotic and meiotic cell divisions. To identify proteins that act in concert with topoisomerase II during chromosome segregation in S.cerevisiae, we have used a two-hybrid cloning approach. We report the isolation of the PAT1 gene (for protein associated with topoisomerase II), which encodes a novel 90 kDa proline- and glutamine-rich protein that interacts with a highly conserved, leucine-rich region of topoisomerase II in vivo. Strains lacking Pat1p exhibit a slow growth rate and a phenotype reminiscent of conditional top2 mutants grown at the semi-permissive temperature; most notably, a reduced fidelity of chromosome segregation during both mitosis and meiosis. These findings indicate that the PAT1 gene is necessary for accurate chromosome transmission during cell division in eukaryotic cells and suggest that the interaction of Pat1p and topoisomerase II is an important component of this function.

Binding Sites↗

PubMed1996

Life with 6000 genes.

The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.

Amino Acid Sequence↗

PubMed1996

The mismatch repair system contributes to meiotic sterility in an interspecific yeast hybrid.

The mismatch repair system is the major barrier to genetic recombination during interspecific sexual conjugation in prokaryotes. The existence of this anti-recombination activity has implications for theories of evolution and the isolation of species. To determine if this phenomenon occurs in eukaryotes, the effect of a deficiency of mismatch repair on the meiotic sterility of an interspecific hybrid of Saccharomyces cerevisiae and the closely related species Saccharomyces paradoxus was examined. The results demonstrate that the rare viable spores from these hybrids have high frequencies of aneuploidy and low frequencies of genetic exchange. Hybrids lacking mismatch repair genes PMS1 or MSH2 display increased meiotic recombination, decreased chromosome non-disjunction and improved spore viability. These observations are consistent with the proposal that the mismatch repair system is an element of the genetic barrier between eukaryotic species. We suggest that an anti-recombination activity during meiosis contributes towards the establishment of post-zygotic species barriers.

Aneuploidy↗