To develop a physical map of the domestic cat major histocompatibility complex (MHC), we constructed two large insert domestic cat genomic DNA libraries. The first large-insert library was constructed using DNA from a normal male fibroblast cell line and is composed of 91,900 independent P1 artificial chromosomes (PACs) with an average insert size of 80,000 bp. A second library was constructed from DNA prepared from peripheral blood mononuclear cells of a male domestic cat using improved techniques and consists of 234,349 bacterial artificial chromosomes (BACs) with an average insert size of 137 kb and a 10.6- fold redundancy. State-of-the-art technologies are being applied to BAC/PAC contig mapping and large scale sequence analysis of the feline MHC. BAC/PAC/plasmid DNAs are purified using 96-well plate formatted mini-preparations. To generate megabase size contig maps of large insert clones, Hind III digested BAC/PAC DNAs are fingerprinted by electrophoresis in 1% agarose, stained, and imaged using a Hitachi Fluorimager. Gel images are imported into IMAGE V3.1 which extracts normalized band positions from each clone and sends the data to Fingerprinting Contigs (FPC) developed by C. Soderlund. FPC is an integrated program for assembly of a minimal tiling path of clones using an algorithm to cluster clones into contigs based on their probability of coincidence scores. The gels are transfered to nylon membranes so that genes can be mapped to specific BAC/PAC clones by hybridization. To expand our coverage of the 400 kb feline MHC class II region which we have cloned from the PAC library we screened the BAC library with probes for DRA, DRB, DMB, and unique sequences from the 5? and 3? ends of DOB6B1 which contains the interval between DOB-LMP7. To expand our coverage of the feline MHC class I region, the PAC library was screened by cross-species hybridization with 41 human expressed sequence tag and sequence tagged site markers (probe density ~ 1/60 kb). Using this approach, we were able to asssemble the six original feline class I region PAC clones and 48/53 feline PAC clone into 18 contigs spanning an estimated 1.8 Mb of the domestic cat genome. - comparative mapping, bacterial artificial chromosomes, P1 artificial chromosomes, molecular evolution, feline genetics, - Neither Human Subjects nor Human Tissues
| 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 |
