The molecular analysis of gene structure, function, and regulation depends upon the ability to correlate physiological, genetic, and structural data relating to a specific gene or set of genes. Many mutants of the yeast Saccharomyces cerevisiae have been isolated, and techniques for the manipulation and mapping of the associated genetic characteristics are routinely performed (1). Genetically defined yeast DNA sequences have been isolated in the form of viable molecular hybrids with bacteriophage X or Escherichia coli plasmids (2-5). Derivatives of the cloned his3 gene that delete DNA sequences near or in the structural gene have been isolated and physically defined (unpublished data). The demonstration by Hinnen et al. (5) that recombinant DNA containing cloned yeast genes can be used to transform yeast cells clearly expands the potential of molecular analysis considerably. These workers showed that yeast transformation occurred at low frequency and that it was usually accompanied by homologous recombination between the transforming DNA and the host chromosomal DNA.The present paper reports two additional modes of yeast transformation. In both, the transformation event occurs at high frequency and is associated with autonomous replication of the transforming DNA. Yeast vectors useful for a wide variety of genetic manipulations have been constructed by combining the three mechanistically different modes of transformation with structural information of the endogenous yeast plasmid (6) MATERIALS AND METHODS Organisms, DNAs, and Enzymes. The following strains were used: yeast-A3617C (a his3-532 gal2) (3) and D13-IA (a his3-532 trpl gal2); E. coli-trpC 9830 (7), hisB 463, SF8, C600 (rK -mK+) (2), and MB1000 (rK-mK+lac-trp-pyrF-) (D. Botstein, personal communication); phage-Xgt-Sc2601 (2), X590 (8), and Xgt-Sc4104; plasmid DNAs-pMB9-Sc2601 (3), Scpl (6), pBR322 (9), pGT2-Sc2605, pBR322-Sc2676 (unpublished data), and pMB1068 (D. Botstein, personal communication). Propagation of strains and preparation of DNAs have been described (2, 3, 6).EcoRI, E. coli DNA ligase, and deoxynucleotidyl terminal transferase were the gifts of Marj Thomas, Robert Alazard, and Tom St. John, respectively. Other restriction endonucleases were purchased from New England BioLabs and Bethesda Research Laboratories (Rockville, MD) and used as directed. Cloning procedures have been described (2, 10).Where appropriate p2,EK1 conditions, as described by the National Institutes of Health Guidelines fo Recombinant DNA Research, were used.Rapid Yeast DNA Preparations. Total yeast DNA was prepared from 5-ml cultures of cells grown to the stationary phase. Yeast cells were harvested and resuspended in 0.4 ml of 0.9 M sorbitol/50 mM potassium phosphate, pH 7.5/14 mM 2-mercaptoethanol. Lyticase (25 units) (a gift from R. Schekman) was added and spheroplast formation was allowed to proceed for 30 min at 300C. At this stage, the procedure for rapid phage DNA preparations (6)
We present the diploid genome sequence of the fungal pathogen Candida albicans. Because C. albicans has no known haploid or homozygous form, sequencing was performed as a whole-genome shotgun of the heterozygous diploid genome in strain SC5314, a clinical isolate that is the parent of strains widely used for molecular analysis. We developed computational methods to assemble a diploid genome sequence in good agreement with available physical mapping data. We provide a whole-genome description of heterozygosity in the organism. Comparative genomic analyses provide important clues about the evolution of the species and its mechanisms of pathogenesis.
We used DNA microarrays to profile gene expression patterns in the C. elegans germline and identified 1416 germline-enriched transcripts that define three groups. The sperm-enriched group contains an unusually large number of protein kinases and phosphatases. The oocyte-enriched group includes potentially new components of embryonic signaling pathways. The germline-intrinsic group, defined as genes expressed similarly in germlines making only sperm or only oocytes, contains a family of piwi-related genes that may be important for stem cell proliferation. Finally, examination of the chromosomal location of germline transcripts revealed that sperm-enriched and germline-intrinsic genes are nearly absent from the X chromosome, but oocyte-enriched genes are not.
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