Construction and partial characterization of a Corynebacterium pseudotuberculosis bacterial artificial chromosome library through genomic survey sequencing

Corynebacterium pseudotuberculosis is a gram-positive bacterium that causes caseous lymphadenitis in sheep and goats. However, despite the economic losses caused by caseous lymphadenitis, there is little information about the molecular mechanisms of pathogenesis of this bacterium. Genomic libraries constructed in bacterial artificial chromosome (BAC) vectors have become the method of choice for clone development in high-throughput genomic-sequencing projects. Large-insert DNA libraries are useful for isolation and characterization of important genomic regions and genes. In order to identify targets that might be useful for genome sequencing, we constructed a C. pseudotuberculosis BAC library in the vector pBeloBAC11. This library contains about 18,000 BAC clones, with inserts ranging in size from 25 to 120 kb, theoretically representing a 390-fold coverage of the C. pseudotuberculosis genome (estimated to be 2.5-3.1 Mb). Many genomic survey sequences (GSSs) with homology to C. diphtheriae, C. glutamicum, C. efficiens, and C. jeikeium proteins were observed within a sample of 215 sequenced clones, confirming their close phylogenetic relationship. Computer analyses of GSSs did not detect chimeric, deleted, or rearranged BAC clones, showing that this library has low redundancy. This GSSs collection is now available for further genetic and physical analysis of the C. pseudotuberculosis genome. The GSS strategy that we used to develop our library proved to be efficient for the identification of genes and will be an important tool for mapping, assembly, comparative, and functional genomic studies in a C. pseudotuberculosis genome sequencing project that will begin this year.

Assignments of the tyrosinase related protein-1 and -2 genes to human chromosome bands 9p23 and 13q32.1 by in situ hybridization

To determine the precise chromosomal localization of tyrosine related protein-1 and -2 (TRP-1 and TRP-2) genes by fluorescence in situ hybridization, we used DNAs isolated from human bacterial artificial chromosome clones. They contain genomic sequences with approximately 120 kb inserts for TRP-1 and TRP-2. The TRP-1 and TRP-2 genes were assigned to human chromosome bands 9p23 and 13q32.1, respectively. These results confirmed the previously mapped location for the TRP-1 gene and more precisely located the TRP-2 gene, which had previously been mapped to chromosome 13q31-q32.