Pierre Thuriaux

A revised chromosome map of the fission yeast Schizosaccharomyces pombe.

SummaryThe genetic map of the nuclear genome of the fission yeast Schizosaccharomyces pombe has been extended by mitotic and meiotic mapping data. A total of 158 markers are now assigned to the three linkage groups known in this organism, and 118 of them have been located on the corresponding chromosome map. Chromosome II and III each consist of one linkage group. There is some indication that the two large fragments which define chromosome I are meiotically linked, but the linkage observed is significant at the P = 0.05 level only. The length of the map is at least 1,700 map units, corresponding to an average of about 8 kilobases per map unit. The latter figure is comparable to the one obtained for intragenic recombination in the sup3 gene (Hofer et al. 1979). The basic frequency of gene conversion as measured for 21 genes varies according to a distribution of Poisson (with a modal value of 0.6% conversion per meiosis and per gene), in sharp contrast with Saccharomyces cerevisiae (Fogel et al. 1980) and Ascobolus immersus (Nicolas 1979). This may reflect the rarity of gene or region-specific rec alleles in S. pombe and may be related to the homothallism of this organism.

Genetic analysis of antisuppressor mutants in the fission yeast Schizosaccharomyces pombe

SummaryFourteen unlinked sin genes could be mutated to recessive antisuppressor alleles preventing the expression of suppressors in the fission yeast Schizosaccharomyces pombe. cyh1 alleles, resistant to the ribosomal inhibitor cycloheximide, also have some antisuppressor effect. The genetical and physiological characterization of these mutants is consistent with the hypothesis that they affect components of the messenger RNA translation machinery such as tRNA modifying enzymes or ribosomal proteins.

Either one of the two yeast EF-1α genes is required for cell viability

SummaryTwo genes,TEF1 andTEF2, encode the protein elongation factor EF-1α in the yeastSaccharomyces cerevisiae. We have generated yeast haploid strains containing eitherTEF1 orTEF2 interrupted by insertion of a large piece of foreign DNA. Cells which contain either one functional copy of the EF-1α genes are viable. In contrast, attempts to isolate a yeast haploid strain with bothTEF1 andTEF2 inactivated have failed suggesting that the double gene disruption is a lethal event.

The origin of a centromere effect on mitotic recombination : A study in the fission yeast Schizosaccharomyces pombe.

SummarySpontaneous meiotic and mitotic rates of recombination were measured for 50 intragenic intervals in 9 genes of Schizosaccharomyces pombe. A much smaller mitotic/meiotic recombination ratio is observed for genes unlinked to the centromere (average ratio: 0.005) than for genes close to the centromere (up to 0.17). The high ratio observed for the latter genes is due to a high rate of mitotic recombination rather than to a centromeric inhibition of meiotic recombination, since there was no meiotic inhibition in the genes considered. As already reported for one pair of genes in Saccharomyces cerevisiae (Hénaut et Luzzati, 1972), cells that have recombined at one locus have a meiotic rate of coincident recombination at an unlinked locus, even if the latter is not on the same chromosome. This coincidence is much lower when intragenic recombinants are selected in the centromere-linked gene lys1. In modification of an earlier hypothesis (Hurst and Fogel, 1964), we propose that: (a) pairing between homologous chromosomes is the major rate-limiting factor in mitotic recombination; (b) mitotic pairing of homologous chromosomes is frequent, possibly occuring in all vegetative cells, but is usually restricted to the centromere region; and (c) pairing along the whole genome is restricted to a small subpopulation among the mitotically dividing cells.

Gene conversion in nonsense suppressors of Schizosaccharomyces pombe : II. Specific marker effects.

SummaryGene conversion and postmeiotic segregation patterns have been analysed at 14 mutant sites of sup3, sup8 and sup9 including 5 alleles with a strong marker effect on recombination frequencies in two-factor crosses. The total frequency of gene conversion and postmeiotic segregation tetrads is fairly constant within each gene, but may vary from one gene to another. About 97% of the conversion events are coconversions spanning the whole sup gene. Postmeiotic segregations are usually quite rare. None of the marker-effect alleles has an increased rate of hybrid DNA formation at the allele considered, as judged from the frequency of gene conversion and postmeiotic segregation in one-factor crosses. At least two of them, sup3-e and sup9-e, are associated with a high frequency of postmeiotic segregation indicating a poor repair of the corresponding base-pair mismatches. This is also observed in a two-factor cross and can account for the marker effect on recombination frequencies. The properties of a third marker effect allele, sup3-e,r10, are best explained by a higher probability of single site conversions as opposed to coconversions in two-factor crosses involving the mutant site r10.

Temperature sensitive allosuppressor mutants of the fission yeast S. pombe influence cell cycle control over mitosis

SummaryA collection of Schizosaccharomyces pombe mutants has been obtained which restore activity to a nonsense suppressing tRNA sup3–5 whose suppressing function has been inactivated by second site mutations within the sup3–5 gene. These mutants were screened for those that were temperature sensitive in suppressing the opal nonsense allele ade6-704. Some of these map within or close to sup3 itself and others define two allosuppressor genes sal2 and sal3. The temperature sensitive mutants fail to efficiently suppress any other opal nonsense alleles although one mutant, sup3–5, r57, rr2, weakly does so at the low temperature. sal2 and sal3 mutants have a pleiotropic effect on the cell cycle causing a transient or complete blockage of mitosis. This blockage and the allosuppressor phenotypes are both eliminated by the presence of wee mutations in wee1 or cdc2. Mutants in sal2 are allelic with cdc25, a gene required for successful completion of mitosis. It is suggested that sal3 and cdc25 influence the mechanism that links the growth rate of the cell with the initiation of mitosis. Mutants in these genes may disturb tRNA biosynthesis or protein synthesis and this disruption may have an effect on both nonsense suppression and the growth rate control over mitosis.

Direct selection of mutants influencing gene conversion in the yeast Schizosaccharomyces pombe

SummaryIn Schizosaccharomyces pombe, a suppressor-active mutation at the anticodon site of the tRNASerUCAgene sup3 leads to opal (UGA)-specific suppression. Second-site mutations (rX) in sup3 inactivate the suppressor. The sup3-UGA, rX double mutants are genetically unstable in meiotic selfings, due to the intergenic transfer of information between sup3 and the unlinked genes sup9 and sup12 (Hofer et al. 1979; Munz and Leupold 1981; Munz et al. 1982). These three genes have considerable sequence homology over about 200 base pairs (Hottinger et al. 1982).Mutants showing a decrease or an increase of the meiotic instability at sup3 have been selected. One mutation (rec3-8) increases both the genetic instability and the frequency of intragenic recombination in sup3 by one order of magnitude. It has no effect on the stability of the nonsense alleles arg1-230 (UAA), ade6-704 and ura1-61 (UGA) or on the frequency of crossing-over between sup3 and the closely linked gene cdc8.The existence of a common genetic control over intragenic recombination and genetic instability at sup3 provides a direct way of selecting for rec mutants in homothallic haploid strains of S. pombe carrying a suppressor-inactive allele of sup3. It also supports the hypothesis that the instability of mutant alleles of this gene is due to chromosome mispairing at meiosis allowing sup3 to pair with sup9 or sup12 and then to undergo recombination by gene conversion restoring the suppressor-active allele sup3-UGA from the suppressor-inactive allele sup3-UGA,rX. Two mutations (rec2-5 and rec5-11) have no effect on intragenic recombination, but considerably reduce the meiotic instability of the sup3-UGA,rX alleles. They may suppress illegitimate pairing between sup3 and sup9 or sup12.

Organisation of the complex locus trp1 in the fission yeast Schizosaccharomyces pombe

Summary65 trp1− alleles of Schizosaccharomyces pombe have been analysed for their interallelic complementation pattern, suppressibility by nonsense suppressors and position of the corresponding mutation site on an intragenic map of the trp1 locus. In addition to the three complementation classes previously described (Schweingruher and Dietrich 1973) as defective in phosphoribosylanthranilate isomerase (trp1A), indole glycerolphosphate synthetase (trp1B) and anthranilate synthetase (trp1C), two new complementation classes, trp1BC and trp1ABC, were found. The former is represented by a single allele which can only complement trp1A mutants. The latter is represented by six alleles which fail to complement tester mutants of the trp1A, trp1B or trp1C class. Classes trp1A, trp1B and trp1C correspond to mutations in three nonoverlapping regions mapping in the order trp1A, trp1B and trp1C All the alleles of the trp1BC and trp1ABC classes correspond to mutations in the trp1C region. Nonsense alleles of the opal (UGA) or ochre (UAA) type were found in the trp1A (2 alleles out of 17) and trp1ABC (5 alleles out of 6) classes only. These data indicate that the trp1 locus is transcribed as a single messenger RNA with transcription starting from the trp1C region. This messenger is probably translated in a single, multifunctional polypeptide, or at most in two polypeptides coded for by the trp1B-trp1C and by the trp1A regions. In addition, the polar effect of nonsense mutations in the trp1C region is cancelled by rare spontaneous mutations occuring at or very near the trp1 locus, which may act by creating an internal single for the initiation of transcription and/or translation.

The genetics of RNA polymerases in yeasts

Conclusion and perspectivesOver the past few years, substantial progress has been made in the genetic characterization of yeast RNA polymerases. Most of their genes have been identified and their complete cloning and sequencing is within reach. Several conclusions can be drawn at this stage. First, the two large subunits are remarkably conserved in all cellular organisms, and are thus likely to define a “minimal” RNA polymerase carrying out the main catalytic aspects of the polymerizing reaction. Highly conserved domains have been identified and are being analyzed using conditional mutants. Second, with the exception of B32, the small subunits tested so far are essential for cellular growth and are directly involved in the transcription process as judged from the inhibition of transcription by corresponding antibodies, and from the properties of the conditional or leaky mutants so far available. They are thus genuine components of the transcription machinery even if their precise functions remain to be identified. Their amino acid sequences are unrelated to those of bacterial proteins involved in transcription. Finally, genetic and biochemical data concur in showing that certain subunits, such as B32, are “dispensable” and thus fulfill an accessory role in transcription.In spite of these advances, we are still far from being able to translate our knowledge of the primary sequence of the various subunits into functional terms: conditional or leaky mutants have been obtained for several subunits, but their biochemical characterization has been slow, and most appear to be defective in the accumulation of the protein rather than in its catalytic activity. Moreover, the spatial organization of eukaryotic RNA polymerases remains largely unknown, although a first electron microscopy characterization of enzyme A has been obtained at 2.5 nm resolution (Schultz et al. in preparation). The study of intragenic, or extragenic, suppressors of conditional, or even non-conditional, mutants may guide structural studies by defining sites that directly interact within or between distinct subunits (Nonet and Young 1989). Extragenic suppressors may also identify other components of the transcription machinery such as the enzyme-specific transcription factors, which are meanwhile being analyzed by direct cloning procedures (Eisenmann et al. 1989; Hahn et al. 1989; Horikoshi et al. 1989; Schmidt et al. 1989; Cavallini et al. 1989). finally, we still do not know if, and how, the synthesis of the three RNA polymerases is coordinatedly or differentially regulated in response to changes in the overall cellular growth rate.

2 Yeast RNA Polymerase Subunits and Genes

OVERVIEW Yeast RNA polymerases A(I), B(II), and C(III) are organized around a common core of subunits related to the bacterial core enzyme (β′ βα 2 ) and share a set of five small subunits (ABC27, ABC23, ABC14.5, ABC10α, and ABC10β). All these subunits are essential for growth. In addition, each enzyme contains a variable number of enzyme-specific subunits, some of which are not strictly required for growth. Most subunit genes have been cloned, sequenced, and mutagenized to produce null alleles and, for several of them, conditional mutants. A functional map of RNA polymerase active site, taken in a broad sense, has begun to emerge from a combined genetic and biochemical analysis of the large subunits. GENERAL PROPERTIES OF YEAST NUCLEAR RNA POLYMERASES The budding yeast Saccharomyces cerevisiae , with its biochemically and genetically well-characterized transcription apparatus, is currently the most suitable experimental model for a comprehensive study of eukaryotic RNA polymerases (see Sentenac 1985; Gabrielsen and Sentenac 1991 and references therein; Young 1991; Thuriaux and Sentenac 1992). Yeast RNA polymerases are typically eukaryotic in their subunit complexity, and there is a fair degree of functional equivalence between the transcription machinery of yeasts and of metazoan eukaryotes (see, e.g., Struhl 1989; Sawadogo and Sentenac 1990; and other chapters in this volume). We assume, therefore, that what is learned of the yeast enzymes may largely be extrapolated to other eukaryotes. Several chapters of this volume cover important structural and functional features of the RNA polymerases of higher eukaryotes and of associated general transcription factors (Corden...