Cécile Fairhead

New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using ‘split-marker’ recombination

New tools are needed for speedy and systematic study of the numerous genes revealed by the sequence of the yeast genome. We have developed a novel transformation strategy, based on ‘split‐marker’ recombination, which allows generation of chromosomal deletions and direct gene cloning. For this purpose, pairs of yeast vectors have been constructed which offer a number of advantages for large‐scale applications such as one‐step cloning of target sequence homologs and combinatorial use. Gene deletions or gap‐repair clonings are obtained by cotransformation of yeast by a pair of recombinant plasmids. Gap‐repair vectors are based on the URA3 marker. Deletion vectors include the URA3, LYS2 and kanMX selection markers flanked by I‐SceI sites, which allow their subsequent elimination from the transformant without the need for counter‐selection. The application of the ‘split‐marker’ vectors to the analysis of a few open reading frames of chromosome XI is described.

Distance from the chromosome end determines the efficiency of double strand break repair in subtelomeres of haploid yeast.

Double strand break (DSB) repair plays an important role in chromosome evolution. We have investigated the fate of DSBs as a function of their location along the yeast chromosome XI, in a system where no conventional homologous recombination can occur. We report that the relative frequency of non-homologous endjoining (NHEJ), which is the exclusive mode of DSB repair in the internal chromosomal portion, decreases gradually towards the telomere, keeping the absolute frequency nearly constant, and that other repair mechanisms, which generally involve the loss of the distal chromosomal fragment, appear in subtelomeric regions. Distance of the DSB from chromosome ends plays a critical role in the global frequency of these repair mechanisms. Direct telomere additions are rare, and other events such as break-induced replication, plasmid incorporation, and gene conversion, involve acquisition of heterologous sequences. Therefore, in subtelomeric regions, cell survival to DSBs is higher and alternative modes of repair allow new genomic combinations to be generated. Furthermore, subtelomeric rearrangements depend on the recombination process, which, unexpectedly, also promotes the joining of heterologous sequences. Finally, we report that the Rad52 protein increases the efficiency of NHEJ.

Consequences of unique double-stranded breaks in yeast chromosomes: death or homozygosis

We have developed a system in which a unique double-stranded break (DSB) can be introduced into a yeast chromosome during mitotic growth. The recognition site for the endonuclease I-SceI was inserted at different places in the yeast genome in haploid and diploid cells expressing this endonuclease. Induction of the break in haploids results in cell death if no intact copy of the cleaved region is present in the cell. If such a copy is provided on a plasmid, as an ectopic gene duplication, or on a homologous chromosome, the break can be repaired. Repair results in two identical copies in the genome of the locus which has been cut. We call this phenomenon homozygotization by reference to diploids heterozygous for the cut site in which repair leads to homozygosis at this site. We have compared the efficiencies of repair in the various topological situations examined, and conclude that some mechanism must search for regions of homology to both sides of the DSB and that repair is successful only if the homologies are provided by the same template molecule.

Genomic polymorphism in the population of Candida glabrata: Gene copy-number variation and chromosomal translocations

The genomic sequence of the type strain of the opportunist human pathogen Candida glabrata (CBS138, ATCC 2001) is available since 2004. This allows the analysis of genomic structure of other strains by comparative genomic hybridization. We present here the molecular analysis of a collection of 183 C. glabrata strains isolated from patients hospitalized in France and around the world. We show that the mechanisms of microevolution within this asexual species include rare reciprocal chromosomal translocations and recombination within tandem arrays of repeated genes, and that these account for the frequent size heterogeneity between chromosomes across strains. Gene tandems often encode cell wall proteins suggesting a possible role in adaptation to the environment.

`Mass-murder' of ORFs from three regions of chromosome XI from Saccharomyces cerevisiae

The complete sequence of the yeast Saccharomyces cerevisiae reveals the presence of many new genes, many of which are without homologs in databases. Characterisation of these genes by novel methods includes systematic deletion followed by phenotypic analysis of mutant strains. We have developed a hierarchical strategy for such a functional analysis of genes, in which the primary phenotypic screening is performed on groups of contiguous genes which are then reinvestigated down to the single gene level. This strategy is applied to the whole chromosome XI as part of EUROFAN (the EUROpean Functional ANalysis) program, and we present here our results on a group of 22 genes from this chromosome. This sample is representative of the results that are emerging for the whole chromosome. Out of the 22 genes deleted, three were shown to be essential, and another three genes confer a mutant growth phenotype to cells when deleted. All phenotypes have been complemented. These figures are in accordance with the previously published fraction of lethal and growth-defective deletions of single genes. We have found no synthetic phenotypes resulting from a combination of deleted genes and have always been able to attribute a mutant phenotype to a single gene.

Mass-murder deletion of 19 ORFs from Saccharomyces cerevisiae chromosome XI

Nineteen open reading frames (ORFs) in the left arm of chromosome XI of the yeast Saccharomyces cerevisiae were inactivated. This was done by producing single-gene or contiguous-gene deletions in haploid and diploid strains. Four deletions are lethal to the corresponding haploid strains, and two result in a failure to grow on a rich glycerol medium. Complementation experiments showed that five of the six identified phenotypes were due to deletion of a single gene (ORFs YKL173w, YKL172w, YKL165c, YKL154w are essential, and YKL160w is required for growth on glycerol medium). One of the phenotypes observed on glycerol medium was not suppressed by the corresponding deleted genes. None of the other deletions, covering 13 ORFs in all, gave rise to any obvious phenotype when the cells were grown at three different temperatures on rich glycerol or glucose medium or on minimal synthetic medium.