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The positional cloning of the hypocretin receptor 2, the gene for autosomal recessive canine narcolepsy, has led to the development of a physical map spanning a large portion of canine chromosome 12 (CFA12), in a region corresponding to human chromosome 6p12-q13. More than 40 expressed sequence tags (ESTs) were used in homology search experiments, together with chromosome walking, to build both physical and radiation hybrid maps of the CFA12 13-21 region. The resulting map of bacterial artificial chromosome ends, ESTs, and microsatellite markers represents the longest continuous high-density map of the dog genome reported to date. These data further establish the dog as a system for studying disease genes of interest to human populations and highlight feasible approaches for positional cloning of disease genes in organisms where genomic resources are limited., (Copyright 2001 Academic Press.)

Early retinal degeneration (erd) is an early onset progressive retinal atrophy, a hereditary canine retinal disease phenotypically similar to human retinitis pigmentosa (RP). In previous efforts to identify the erd locus, canine homologs of genes causally associated with RP in humans, such as opsin (RHO), the beta-subunit gene for cyclic GMP phosphodiesterase (PDE6B), and RDS/peripherin, were excluded. A genome-wide screen was undertaken on canine families segregating the erd disease. Analysis of over 150 canine-specific markers has localized erd to a single linkage group comprising two previously identified canine linkage groups, 20 and 26, corresponding to canine radiation hybrid groups RH.34-a and RH.40-a. Multipoint analysis places erd in the interval between marker FH2289 (distance 23.6 cM) and FH2407 (5.9 cM) with a lod score of 12.23. Although the erd linkage group has not been assigned to an identified canine chromosome, conserved synteny of this linkage group with human 12p13-q13 suggests several candidates for erd and identifies a novel retinal degeneration locus. The rapid progress now occurring in canine genetics will expedite identification of the genes and molecular mechanisms underlying the inherited traits and diseases that make the dog a unique asset for study of mammalian traits., (Copyright 1999 Academic Press.)

A large insert canine genomic bacterial artificial chromosome (BAC) library was built from a Doberman pinscher. Approximately 166,000 clones were gridded on nine high-density hybridization filters. Insert analysis of randomly selected clones indicated a mean insert size of 155 kb and predicted 8.1 coverage of the canine genome. Two percent of the clones were nonrecombinant. Chromosomal fluorescence in situ hybridization studies of 60 BAC clones indicated no chimerism. The library was hybridized with dog PCR products representing eight genes (ADA, TNFA, GCA, MYB, HOXA, GUSB, THY1, and TOP1). The resulting positive clones were characterized and shown to be compatible with an eightfold redundant library., (Copyright 1999 Academic Press.)

We have constructed a collection of canine-rodent microcell hybrid cell lines by fusion of canine fibroblast microcell donors with immortalized rodent recipient cells. Characterization of the hybrid cell lines using a combination of fluorescence in situ hybridization and PCR analysis of canine microsatellite repeat sequences allowed selection of a panel of hybrids in which most canine chromosomes are represented. Approximately 90% of genetic markers and genes that were tested could be assigned to 1 of 31 anonymous canine chromosome groups, based on common patterns of retention in the hybrid set. Many of these putative chromosome groups have now been validated by linkage analysis. This panel of cell lines provides a tool for development of genetic, physical, and comparative maps of the canine genome.

A genetic linkage map of the canine genome has been developed by typing 150 microsatellite markers using 17 three-generation pedigrees, composed of 163 F2 individuals. One hundred and thirty-nine markers were linked to at least one other marker with a lod score > or = 3.0, identifying 30 linkage groups. The largest chromosome had 9 markers spanning 106.1 cM. The average distance between markers was 14.03 cM, and the map covers an estimated 2073 cM. Eleven markers were informative on the mapping panel, but were unlinked to any other marker. These likely represent single markers located on small, distinct canine chromosomes. This map will be the initial resource for mapping canine traits of interest and serve as a foundation for development of a comprehensive canine genetic map.

Fourteen new dinucleotide repeat polymorphisms specific for human chromosome 21 have been identified, mapped, and characterized. The average heterozygosity of all markers was 0.66. The average PIC value was 0.61. The markers were mapped by STS content mapping of YACs previously assigned to chromosome 21. The correlation of polymorphic genetic markers with substantially complete physical maps should facilitate the identification of loci of interest on chromosome 21.

A large block of simple sequence repeat (SSR) polymorphisms for the dog genome has been isolated and characterized. Screening of primary libraries by conventional hybridization methods as well as by screening of enriched marker-selected libraries led to the isolation of a large number of genomic clones that contained (CA)n repeats. The sequences of 101 clones showed that the size and complexity of (CA)n repeats in the dog genome were similar to those reported for these markers in the human genome. Detailed analysis of a representative subset of these markers revealed that most markers were moderately to highly polymorphic, with PIC values exceeding 0.70 for 33% of the markers tested. An association between higher PIC values and markers containing longer (CA)n repeats was observed in these studies, as previously noted for similar markers in the human genome. A list of primer sequences that tag each characterized marker is provided, and a comprehensive system of nomenclature for the dog genome is suggested.

Psoralen accessibility in the SV40 regulatory region has been analyzed in vivo on viral minichromosomes and extracellular virus particles using a psoralen mapping technique which we have recently described. This approach takes advantage of the fact that lambda exonuclease digestion of double-stranded DNA is inhibited by the presence of 5-methylisopsoralen monoadducts. Previous analysis of purified restriction fragments from this region irradiated in vitro revealed a region of psoralen hypersensitivity which encompassed the repeated 72-base-pair (bp) enhancers and sequences 150 bp upstream toward the late region. The studies reported here demonstrate that changes in the structure of the DNA helix due to supercoiling do not affect the pattern of psoralen accessibility in this region. Additionally, no significant difference in the precise pattern of the psoralen hypersensitive domain is observed between late nucleoprotein complexes and extracellular virus. Finally, virus particles that have been through a freeze-thaw cycle, a process which we have shown eliminates the preferential photoaddition of the regulatory region in chromatin, also exhibit the same pattern of psoralen hypersensitivity.