RESUMO
The yeast genome has been shown to contain a significant number of gene families with more than three members. In order to study these families it is often necessary to generate strains carrying deletions of all members of the family, which can require a wide range of auxotrophic markers. To facilitate such studies, we have generated yeast strains containing deletions of a selection of nutritional marker genes (ade2, ade4, ade8, met3 and met14). We have also cloned the corresponding cognate genes, allowing their use in PCR-based gene disruptions. Two new pRS family Saccharomyces cerevisiae-Escherichia coli shuttle vectors containing ADE8 (one low-copy, pRS4110, and one high-copy, pRS4210) have been produced for use in conjunction with the new strains. A system for easier synthetic lethal screening using one of these new markers is also presented. The ADE8 and HIS3 genes have been cloned together on a high-copy vector (pRS4213), providing a plasmid for red-white colour screening in the ade2 Delta 0 ade8 Delta 0 strains we have generated. In contrast to some conventional systems, this plasmid allows for screening using gene libraries constructed in URA3 plasmids.
Assuntos
Vetores Genéticos/genética , Plasmídeos/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Clonagem Molecular , Primers do DNA , Deleção de Genes , Genes Fúngicos/fisiologia , Marcadores Genéticos/genética , Genoma Fúngico , Dados de Sequência Molecular , Plasmídeos/química , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
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.
Assuntos
Genoma Fúngico , Família Multigênica/genética , Saccharomyces cerevisiae/genética , Proteínas Virais , Southern Blotting , Cromossomos Fúngicos/genética , DNA Fúngico/genética , DNA Recombinante/genética , Eletroforese em Gel de Campo Pulsado , Deleção de Genes , Genes Fúngicos/genética , Marcadores Genéticos , Integrases/genética , Integrases/metabolismo , Plasmídeos , Recombinação Genética , Saccharomyces cerevisiae/citologiaRESUMO
A physical map of a 14.5 kb region close to the centromere on the left arm of chromosome IV of Saccharomyces cerevisiae is presented. This map has been constructed by restriction analysis of a clone from a YCp50 genomic library and by use of pre-existing and new sequence data from this region. The map reveals the following gene order (reading from the most centromere-distal to the most centromere-proximal locus): USO1/INT1-MBP1-PSA1-SLC1-YLA1 and defines the size of the open reading frames and intergenic regions.
