We present here the first fully integrated, comprehensive map of the canine genome, incorporating detailed cytogenetic, radiation hybrid (RH), and meiotic information. We have mapped a collection of 266 chromosome-specific cosmid clones, each containing a microsatellite marker, to all 38 canine autosomes by fluorescence in situ hybridization (FISH). A 1500-marker RH map, comprising 1078 microsatellites, 320 dog gene markers, and 102 chromosome-specific markers, has been constructed using the RHDF5000-2 whole-genome radiation hybrid panel. Meiotic linkage analysis was performed, with at least one microsatellite marker from each dog autosome on a panel of reference families, allowing one meiotic linkage group to be anchored to all 38 dog autosomes. We present a karyotype in which each chromosome is identified by one meiotic linkage group and one or more RH groups. This updated integrated map, containing a total of 1800 markers, covers >90% of the dog genome. Positional selection of anchor clones enabled us, for the first time, to orientate nearly all of the integrated groups on each chromosome and to evaluate the extent of individual chromosome coverage in the integrated genome map. Finally, the inclusion of 320 dog genes into this integrated map enhances existing comparative mapping data between human and dog, and the 1000 mapped microsatellite markers constitute an invaluable tool with which to perform genome scanning studies on pedigrees of interest.

Saccharomyces boulardii, a yeast that was isolated from fruit in Indochina, has been used as a remedy for diarrhea since 1950 and is now a commercially available treatment throughout Europe, Africa, and South America. Though initially classified as a separate species of Saccharomyces, recent publications have shown that the genome of S. boulardii is so similar to Saccharomyces cerevisiae that the two should be classified as conspecific. This raises the question of the distinguishing molecular and phenotypic characteristics present in S. boulardii that make it perform more effectively as a probiotic organism compared to other strains of S. cerevisiae. This investigation reports some of these distinguishing characteristics including enhanced ability for pseudohyphal switching upon nitrogen limitation and increased resistance to acidic pH. However, these differences did not correlate with increased adherence to epithelial cells or transit through mouse gut. Pertinent characteristics of the S. boulardii genome such as trisomy of chromosome IX, altered copy number of a number of individual genes, and sporulation deficiency have been revealed by comparative genome hybridization using oligonucleotide-based microarrays coupled with a rigorous statistical analysis. The contributions of the different genomic and phenotypic features of S. boulardii to its probiotic nature are discussed.

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Regulatable promoters are commonly used to control the expression of, especially, essential genes in a conditional manner. Integration of such promoters upstream of an ORF using one-step PCR-mediated homologous recombination should be particularly efficient. However, integration of the original KanMX4-tetO promoter cassette (Belli et al., 1998a) into the relatively short upstream regions of many yeast genes is often problematic, presumably due to the size (3.9 kb) of the replacement cassette. We have created a new, shorter, KanMX4-tetO cassette by removing the transactivator (tTA) sequence from the original cassette. The transactivator (tTA) has been integrated into the yeast genome to create a new strain for use with the new system, which has a greatly increased efficiency of promoter substitution. With it, we have been able to create strains that could not be made with the original cassette. To increase the throughput of promoter substitutions, we have developed a new assay for testing doxycycline sensitivity, based on liquid culture using microtitre trays. Altogether, the components of this new 'tool kit' greatly increase the efficiency of systematic promoter substitutions.

We have generated a collection of yeast strains, each of which has an essential yeast gene under the control of the tetracycline-responsive, tetO, promoter. Screens using first-generation promoter-swap strains uncovered the non-specific responsiveness of the tetO 7 promoter to a known human transcription factor (hIRF-1). Non-specific regulation was not observed with the tetO 2 promoter. Reporter assays have been used to demonstrate this phenomenon. Subsequent efforts to generate a collection of tetracycline-regulatable strains have focused on the tetO 2 promoter. These strains are available to the yeast community and can be used for functional genomics studies.