Yeast Molecular Biology. General molecular biology of S. cerevisiae and the genome sequence. 2. Applications of molecular biology in yeast research of potential relevance to fermentation industry.
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‘S. cerevisiae — brewers' yeast — was the first eukaryote to have its genome fully sequenced. Nature published a special supplement — The Yeast Genome Directory — in 1997. This united the research effort and brought together chromosome, physical and genetic maps, a duplication chart and a gazetteer. We are pleased to make all this material freely available below in PDF format.
The sequence itself is available via a searchable online database hosted by the Munich Information Centre for Protein Sequences at mips.gsf.de/proj/yeast/.’
S. cerevisiae can be stably maintained as either heterothallic or homothallic strains. Both heterothallic and homothallic diploid strains sporulate under conditions of nutrient deficiency. During sporulation, the diploid cell undergoes meiosis yielding four progeny haploid cells, which become encapsulated as spores. The percent sporulation varies with the particular strain, ranging from no or little sporulation to nearly 100%. Many laboratory strains sporulate to over 50%. The majority of asci contains four haploid ascospores.
Up until recently (BG) this has been the strategy to clone a gene for a definable phenotype, where you don’t know the gene(s) involved for example a biosynthesis gene for ornithine (in arg metabolism).
There are a number of vectors that are available for cloning, making mutations, shuffling genes, expressing recombinant protein and shuffling between yeast and E. coli.
All yeast vectors contain markers that allow selection of transformants containing the desired plasmid. The most commonly used yeast markers include URA3, HIS3, LEU2, TRP1 and LYS2, which complement specific auxotrophic mutations in yeast. Also, the URA3, HIS3, LEU2 and TRP1 yeast markers can complement specific E. coli auxotrophic mutations.
The URA3 and LYS2 yeast genes have an additional advantage because both positive and negative selections are possible.
YIp. Yeast integrative plasmid. Low-frequency integration into chromosomal loci by homologous recombination. They don’t have yeast replication origins. One single copy is integrated which is then stably maintained.
YRp. Yeast replicating plasmid. ARS elements maintain replication, but do not segregate well.
YEp. Yeast episomal plasmid. Derived from s. cerevisiae 2mm plasmids (has an ori). 50-100 copies/cell.
YCp. Yeast centromeric plasmid. Has a yeast centromere. Get stable segregation, 1-3 copies/cell.
The initial step in the molecular characterization of eukaryotic genomes generally requires cloning of large chromosomal fragments, which is usually carried out by digestion with restriction endonucleases and ligation to specially developed cloning vectors. Usually 200 to 800 kb fragments are cloned as Yeast Artificial Chromosomes (YACs), and 100-200 kb fragments are cloned as Bacterial Artificial Chromosomes (BACs or PACs). The importance of YAC technology has been heightened by the recently developed methods for transferring YACs to cultured cells and to the germline of experimental animals.
With the availability of sequence, genes can be putatively be identified by computer analysis either of the nucleotide sequence or translated sequence. Genes/proteins of known function are used to search the sequence which will find homologous genes/proteins with regions of homology and tell you the degree of sequence homology. When you have identified your putative biosynthesis gene. Can then clone it by PCR, designing primers based on the published sequence. Disrupt the gene sequence with a selectable marker and replace the WT gene with the mutated gene. Lethal mutations can only be maintained as diploids.
One of the most important and widely used methods to characterize yeast genes is gene disruption. The complete disruption of a gene unambiguously reveals its function and can be helpful for generating additional mutations. Several methods can be used to produce deletions and null mutations.
The one-step gene disruption procedure is usually preferred because of its simplicity. This procedure is based on the use of a linear fragment of DNA containing a selectable marker flanked by 5' and 3' homologous regions. The free ends of the fragment, prepared by digestion with restriction endonucleases, are recombinogenic, resulting in the integration of URA3 marker and the loss of wild-type YFG1 allele.