As part of a project to develop a genetic and physical map of the domestic cat major histocompatibility complex (MHC), we have constructed a domestic cat genomic DNA library in pCYPAC3.0 (Ioannou et al., Nature Genetics 6: 84, 1994) composed of 91,900 independent P1 artificial chromosomes (PACs). To determine average insert size of the feline PAC library, 35 clones were digested with Not I, which cleaves the vector at sites flanking the Bam HI cloning site and analyzes the digestion products by pulse field gel electrophoresis. The average insert size was 80,000 base pairs with a size range of 50-150 kilobases (kb). The theoretical number of genome-equivalents represented was estimated at 2.5 and the probability of finding any given gene sequence as 91%. To estimate a realistic coverage, we screened the library with 52 comparative anchor tagged sequences (CATS) using the polymerase chain reaction (PCR) (Lyons et al., Nature Genetics 15: 47, 1997) and compared the PCR products obtained from the PAC library with cat genomic DNA by agarose gel electrophoresis. The results indicated that 43 of the 52 CATS were represented in the cat genomic PAC library and, therefore, the actual probability of finding any given gene is 83%. To determine if the cat genomic PAC library would be useful for the cloning of cat genes, the library was screened with a feline MHC class I cDNA, feline MHC class II cDNAs for DRA and DRB, and a feline DPA pseudogene. A total of ten positive colonies with an average insert size of 79.9 kb were isolated, six PACs containing class I sequences, three containing DRA sequences (one of which also contained DRB sequences), and one containing DPA sequences. Restriction enzyme analysis and fingerprinting of the class I PAC clones using a nonradioactive method that gives uniform band intensities (Ota and Amemiya, Genetic Analysis 12: 173, 1996) indicates that the six class I PAC clones represent four separate contigs. For MHC class II PAC clones, fingerprinting and hybridization analyses indicated that the three DR positive clones represent two individual contigs, suggesting that two distinct DR haplotypes are represented. Sample sequencing of PAC clone ends and of random Bam HI subclones is currently underway to confirm overlapping clones, identify microsatellite markers, and determine gene and microsatellite order. This library should also prove useful for comparative mapping projects, analysis of historic recombination events, and positional cloning of disease genes in the domestic cat.
| Vazquez-Salat, Nuria; Yuhki, Naoya; Beck, Thomas et al. (2007) Gene conversion between mammalian CCR2 and CCR5 chemokine receptor genes: a potential mechanism for receptor dimerization. Genomics 90:213-24 |
| Beck, Thomas W; Menninger, Joan; Murphy, William J et al. (2005) The feline major histocompatibility complex is rearranged by an inversion with a breakpoint in the distal class I region. Immunogenetics 56:702-9 |
