Stewart Scherer

Sterile host yeasts (SHY): A eukaryotic system of biological containment for recombinant DNA experiments

A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewers/Bakers yeast) is described. The principle of containment is sterility: the haploid host strains all contain a mating-type-non-specific sterile mutation. The hosts also contain four auxotrophic mutations suitable for selection for the various kinds of vectors used. All vectors are derivatives of pBR322 which can be selected and maintained in both yeast and Escherichia coli. The system has recently been certified at the HV2 level by the National Institutes of Health.

[49] Rapid DNA isolations for enzymatic and hybridization analysis

Publisher Summary This chapter describes the rapid DNA isolations for enzymatic and hybridization analysis. The methods described are for the isolation of DNA from bacteria, yeast, and tissue culture cells. The ability to rapidly isolate DNA from a large number of individual organisms has facilitated the characterization of the genome of the organisms. Ability to rapidly isolate DNA from organisms containing cloned DNA segments using recombinant DNA techniques has facilitated the identification of particular clones. These methods have been used for identifying the size of a particular cloned DNA fragment or for surveying the genome organization in a large number of individual organisms or strains. The general approach is to remove enzymatically or physically any rigid cell wall and then to lyse the cells with a detergent, usually SDS. Nucleases are inactivated with diethyl oxydiformate and most of the cell debris, proteins, and SDS are precipitated by addition of potassium. The DNA is then recovered by ethanol precipitation. RNA present in the preparations interferes with most restriction endonucleases, and is eliminated by ribonuclease treatment. The digested RNA does not interfere with analysis and is not removed.

Metabolic specialization associated with phenotypic switching in Candida albicans

Phase and antigenic variation are mechanisms used by microbial pathogens to stochastically change their cell surface composition. A related property, referred to as phenotypic switching, has been described for some pathogenic fungi. This phenomenon is best studied in Candida albicans, where switch phenotypes vary in morphology, physiology, and pathogenicity in experimental models. In this study, we report an application of a custom Affymetrix GeneChip representative of the entire C. albicans genome and assay the global expression profiles of white and opaque switch phenotypes of the WO-1 strain. Of 13,025 probe sets examined, 373 ORFs demonstrated a greater than twofold difference in expression level between switch phenotypes. Among these, 221 were expressed at a level higher in opaque cells than in white cells; conversely, 152 were more highly expressed in white cells. Affected genes represent functions as diverse as metabolism, adhesion, cell surface composition, stress response, signaling, mating type, and virulence. Approximately one-third of the differences between cell types are related to metabolic pathways, opaque cells expressing a transcriptional profile consistent with oxidative metabolism and white cells expressing a fermentative one. This bias was obtained regardless of carbon source, suggesting a connection between phenotypic switching and metabolic flexibility, where metabolic specialization of switch phenotypes enhances selection in relation to the nutrients available at different anatomical sites. These results extend our understanding of strategies used in microbial phase variation and pathogenesis and further characterize the unanticipated diversity of genes expressed in phenotypic switching.

Genomic evidence for a complete sexual cycle in Candida albicans

Candida albicans is a diploid fungus that has become a medically important opportunistic pathogen in immunocompromised individuals. We have sequenced the C. albicans genome to 10.4-fold coverage and performed a comparative genomic analysis between C. albicans and Saccharomyces cerevisiae with the objective of assessing whether Candida possesses a genetic repertoire that could support a complete sexual cycle. Analyzing over 500 genes important for sexual differentiation in S. cerevisiae, we find many homologues of genes that are implicated in the initiation of meiosis, chromosome recombination, and the formation of synaptonemal complexes. However, others are striking in their absence. C. albicans seems to have homologues of all of the elements of a functional pheromone response pathway involved in mating in S. cerevisiae but lacks many homologues of S. cerevisiae genes for meiosis. Other meiotic gene homologues in organisms ranging from filamentous fungi to Drosophila melanogaster and Caenorhabditis elegans were also found in the C. albicans genome, suggesting potential alternative mechanisms of genetic exchange.

Assembly of the Candida albicans genome into sixteen supercontigs aligned on the eight chromosomes

BackgroundThe 10.9× genomic sequence of Candida albicans, the most important human fungal pathogen, was published in 2004. Assembly 19 consisted of 412 supercontigs, of which 266 were a haploid set, since this fungus is diploid and contains an extensive degree of heterozygosity but lacks a complete sexual cycle. However, sequences of specific chromosomes were not determined.ResultsSupercontigs from Assembly 19 (183, representing 98.4% of the sequence) were assigned to individual chromosomes purified by pulse-field gel electrophoresis and hybridized to DNA microarrays. Nine Assembly 19 supercontigs were found to contain markers from two different chromosomes. Assembly 21 contains the sequence of each of the eight chromosomes and was determined using a synteny analysis with preliminary versions of the Candida dubliniensis genome assembly, bioinformatics, a sequence tagged site (STS) map of overlapping fosmid clones, and an optical map. The orientation and order of the contigs on each chromosome, repeat regions too large to be covered by a sequence run, such as the ribosomal DNA cluster and the major repeat sequence, and telomere placement were determined using the STS map. Sequence gaps were closed by PCR and sequencing of the products. The overall assembly was compared to an optical map; this identified some misassembled contigs and gave a size estimate for each chromosome.ConclusionAssembly 21 reveals an ancient chromosome fusion, a number of small internal duplications followed by inversions, and a subtelomeric arrangement, including a new gene family, the TLO genes. Correlations of position with relatedness of gene families imply a novel method of dispersion. The sequence of the individual chromosomes of C. albicans raises interesting biological questions about gene family creation and dispersion, subtelomere organization, and chromosome evolution.

Deletion analysis of the Saccharomyces GAL gene cluster. Transcription from three promoters.

Five deletions, constructed in vitro, have been incorporated into the Saccharomyces Cerevisiae GAL7-GAL10-GAL1 gene cluster on chromosome II. These deletions permit unambiguous assignment of genetic function to each of the previously identified transcribed regions. The GAL7 and GAL10 genes are transcribed right to left while the GAL1 gene is transcribed left to right. A deletion of the divergently transcribed region between GAL10 and GAL1 including the sequences homologous to the 5′ ends of all transcripts for both of these genes results in a GAL7+, gal10−, gal1− genotype. This result suggests that each of these three genes can be transcribed from separate promoter regions. A yeast strain, containing the rna1-1 temperature-sensitive mutation, at non-permissive temperatures accumulates a large RNA containing both GAL7 and GAL10 sequences, a small RNA containing GAL7 sequences and at least three larger RNAs containing GAL1 sequences. Deletion of DNA sequences contained within the larger GAL1 transcripts but beyond the GAL1 (galactokinase) coding sequences results in a strain able to complement gal1− mutants. This result suggests that the larger transcripts may not be obligate precursors to the stable GAL1 mRNAs.

Cloning of bacterial DNA replication genes in bacteriophage λ

SummaryRecombinant lambda phages containing the genes for dnaZ protein (the γ subunit of DNA polymerse III holoenzyme), primase (dnaG protein) and dnaC protein from Escherichi coli and Salmonella typhimurium were isolated. Each gene cloned from S. typhimurium has extensive DNA sequence homology to the corresponding E. coli gene. Clones selected by complementation of a dnaA temperature-sensitive mutant appear similar to other isolated suppressors of dnaA (Projan and Wechsler 1981). Derivatives of each cloned fragment suitable for overproduction of the protein were constructed. Of those tested, only the phage containing the E. coli dnaZ gene resulted in significant overproduction.