Widespread retinal degenerative disease mutation (rdAc) discovered among a large number of popular cat breeds.

The recent discovery of a mutational variant in the CEP290 gene (CEP290: IVS50+9T>G), conferring recessive retinal degeneration in Abyssinian and Somali (long-haired Abyssinian) cats (rdAc) prompted a survey among 41 cat breeds (846 individuals) to assess the incidence, frequency and clinical consequence of rdAc. The rdAc allele displayed widespread distribution, observed in 16/43 (37%) breeds, exhibiting a high allele frequency (∼33%) in North American and European Siamese populations. Clinical evaluations demonstrated high concordance between rdAc pathology and the CEP290 (IVS50+9T>G) homozygous genotype (P=1.1E-6), with clinical disease similar to affected Abyssinians/Somalis. This retinal degeneration has not been reported in breeds other than the Abyssinian/Somali and poses a significant health risk particularly in the Siamese breed group. Alertness of the veterinary community and the present availability of commercial diagnostic testing could synergistically enable breeders to reduce the incidence of rdAc blindness in pure-bred cat populations.

Second-generation integrated genetic linkage/radiation hybrid maps of the domestic cat (Felis catus).

We report construction of second-generation integrated genetic linkage and radiation hybrid (RH) maps in the domestic cat (Felis catus) that exhibit a high level of marker concordance and provide near-full genome coverage. A total of 864 markers, including 585 coding loci (type I markers) and 279 polymorphic microsatellite loci (type II markers), are now mapped in the cat genome. We generated the genetic linkage map utilizing a multigeneration interspecies backcross pedigree between the domestic cat and the Asian leopard cat (Prionailurus bengalensis). Eighty-one type I markers were integrated with 247 type II markers from a first-generation map to generate a map of 328 loci (320 autosomal and 8 X-linked) distributed in 47 linkage groups, with an average intermarker spacing of 8 cM. Genome coverage spans approximately 2,650 cM, allowing an estimate for the genetic length of the sex-averaged map as 3,300 cM. The 834-locus second-generation domestic cat RH map was generated from the incorporation of 579 type I and 255 type II loci. Type I markers were added using targeted selection to cover either genomic regions underrepresented in the first-generation map or to refine breakpoints in human/feline synteny. The integrated linkage and RH maps reveal approximately 110 conserved segments ordered between the human and feline genomes, and provide extensive anchored reference marker homologues that connect to the more gene dense human and mouse sequence maps, suitable for positional cloning applications.

Radiation hybrid mapping of 304 novel microsatellites in the domestic cat genome.

Effective utilization of the domestic cat as an animal model for hereditary and infectious disease requires the development and implementation of high quality gene maps incorporating microsatellites and conserved coding gene markers. Previous feline linkage and radiation hybrid maps have lacked sufficient microsatellite coverage on all chromosomes to make effective use of full genome scans. Here we report the isolation and genomic mapping of 304 novel polymorphic repeat loci in the feline genome. The new loci were mapped in the domestic cat radiation hybrid panel using an automated fluorescent TAQ-Man based assay. The addition of these 304 microsatellites brings the total number of microsatellites mapped in the feline genome to 580, and the total number of loci placed onto the RH map to 1,126. Microsatellites now span every autosome with an average spacing of roughly one polymorphic STR every five centimorgans, and full genome coverage of one marker every 2.7 megabases. These loci now provide a useful tool for undertaking full-genome scans to identify genes associated with phenotypes of interest, such as those relating to hereditary disease, coat color, patterning and morphology. These resources can also be extended to the remaining 36 species of the cat family for population genetic and evolutionary genomic analyses.

A genetic linkage map of microsatellites in the domestic cat (Felis catus).

Of the nonprimate mammalian species with developing comparative gene maps, the feline gene map (Felis catus, Order Carnivora, 2N = 38) displays the highest level of syntenic conservation with humans, with as few as 10 translocation exchanges discriminating the human and feline genome organization. To extend this model, a genetic linkage map of microsatellite loci in the feline genome has been constructed including 246 autosomal and 7 X-linked loci. Two hundred thirty-five dinucleotide (dC. dA)n. (dG. dT)n and 18 tetranucleotide repeat loci were identified and genotyped in a two-family, 108-member multigeneration interspecies backcross pedigree between the domestic cat (F. catus) and the Asian leopard cat (Prionailurus bengalensis). Two hundred twenty-nine loci were linked to at least one other marker with a lod score >/=3.0, identifying 34 linkage groups. Representative markers from each linkage group were assigned to specific cat chromosomes by somatic cell hybrid analysis, resulting in chromosomal assignments to 16 of the 19 feline chromosomes. Genome coverage spans approximately 2900 cM, and we estimate a genetic length for the sex-averaged map as 3300 cM. The map has an average intragroup intermarker spacing of 11 cM and provides a valuable resource for mapping phenotypic variation in the species and relating it to gene maps of other mammals, including human.

Genetic individualization of domestic cats using feline STR loci for forensic applications.

A group of ten short tandem repeat (STR) loci suitable for PCR typing from DNA of domestic cats is evaluated for genetic individualization using blinded samples of eight putative feline blood specimens. The ten loci were also typed in a 70 member cat pedigree to demonstrate Mendelian inheritance and independent assortment. A "match window" or measurement precision estimate was empirically established by determining the maximum gel migration difference among alleles identical by descent in different individuals of the pedigree. Hardy-Weinberg equilibrium and abundant heterozygosity was observed for each locus in cat population samples from Canada and the USA. The probabilities of two unrelated individuals matching by chance (Pm) at all ten loci was estimated as 1.35 x 10(-10). We present a conservative approach to compute, for forensic consideration, the mathematical likelihood of a chance genotypic match between DNA evidence from a crime scene and the suspect composite STR genotypes for species or populations when genotype frequency information is not available.

Cloning, sequencing and expression of the gene encoding the extracellular neutral protease, vibriolysin, of Vibrio proteolyticus.

The gene (nprV), encoding the extracellular neutral protease, vibriolysin (NprV), of the Gram- marine microorganism, Vibrio proteolyticus, was isolated from a V. proteolyticus DNA library constructed in Escherichia coli. The recombinant E. coli produced a protease that co-migrated with purified neutral protease from V. proteolyticus on non-denaturing polyacrylamide gels, and that demonstrated enzymatic specificity towards the neutral protease substrate N-[3-(2-furyl)acryloyl]-L-alanylphenylalanine amide. The nucleotide (nt) sequence of the cloned nprV gene revealed an open reading frame encoding 609 amino acids (aa) including a putative signal peptide sequence followed by a long 'pro' sequence consisting of 172 aa. The N-terminal aa sequence of NprV purified from cultures of V. proteolyticus, identified the beginning of the mature protein within the aa sequence deduced from the nt sequence. Comparative analysis of mature NprV to the sequences of the neutral proteases from Bacillus thermoproteolyticus (thermolysin) and Bacillus stearothermophilus identified extensive regions of conserved aa homology, particularly with respect to active-site residues, zinc-binding residues, and calcium-binding sites. NprV was overproduced in Bacillus subtilis by placing the DNA encoding the 'pro' and mature enzyme downstream from a Bacillus promoter and signal sequence.

Detection of bovine alpha s1-casein genomic variants using the allele-specific polymerase chain reaction.

A method was developed to distinguish between genotypic variants B and C of bovine alpha s1-casein, using the allele-specific polymerase chain reaction (ASPCR). The alpha s1-casein genotype determined for 17 Jersey cows by the ASPCR method was confirmed by typing the alpha s1-casein milk proteins on isoelectric focusing gels. Using the ASPCR method described, rapid analysis of the alpha s1-casein genotype of bulls is now possible. In addition, kappa-casein genotypes can be determined from the same PCR reaction.

External cephalic version with ruptured membranes and adequate amniotic fluid volume.

The use of external cephalic version in the patient with ruptured membranes is examined and the first successful reported case is described. No difficulties or complications were encountered. We conclude that use of this procedure in the presence of ruptured membranes has been unjustly slighted and that external version may offer an important alternative to the patient with ruptured membranes, adequate amniotic fluid, and malpresentation. We caution that this should only be attempted under continuous monitoring, in patients with adequate amniotic fluid, and in facilities where immediate cesarean section is available. We believe that a larger trial is warranted, and we encourage that this be done.

Identification of a distal regulatory element in the 5' flanking region of the bovine prolactin gene.

The 5'-flanking region of the bovine prolactin gene was cloned and sequenced. The expression of chimeric gene constructs containing 5'-flanking DNA fragments from the prolactin gene joined to a reporter gene encoding human growth hormone (hGH) was examined using transiently transfected rat pituitary cells. Prolactin nucleotide sequences located at position -1213 to -925 enhance the basal level of expression of growth hormone by 5-fold and function in a position- and orientation-independent fashion. In addition to increasing the basal level of growth hormone expression, this enhancer element also responds to induction by epidermal growth factor. The nucleotide sequence of the bovine prolactin gene enhancer element is highly similar to an enhancer element located approximately -1.5 kb from the rat prolactin transcription initiation site. Deletion analysis of the enhancer region shows that sequences -1124 to -985 are necessary and sufficient for enhancer activity.

Exclusive presence of lactose-sensitive fimbriae on a typical strain (WVU45) of Actinomyces naeslundii.

Lactose-sensitive fimbriae were identified as the only fimbriae present on Actinomyces naeslundii WVU45 (ATCC 12104). A single antigen reactive with antiserum against WVU45 cells was detected by cross immunoelectrophoresis of isolated fimbriae, and a monospecific antiserum against this antigen reacted with all fimbriae observed on the bacterial surface by immunoelectron microscopy. Moreover, the loss of one cell surface antigen by a spontaneous mutant of A. naeslundii WVU45 (WVU45M), isolated by its failure to react with a monospecific antibody against the fimbriae, was associated with the loss of all fimbriae. The functional involvement of the fimbriae in lactose-sensitive bacterial adherence was demonstrated by the ability of WVU45, but not WVU45M, cells to agglutinate neuraminidase-treated erythrocytes and by the lactose-sensitive hemagglutinating activity of immune complexes formed with isolated fimbriae and monospecific antibody. Bacterial agglutination assays with different monospecific antibodies revealed an antigenic similarity between the fimbriae of A. naeslundii WVU45 and the lactose-sensitive fimbriae (type 2) of Actinomyces viscosus T14V. In contrast, cross-reactivity was not observed between the WVU45 fimbriae and type 1 fimbriae, the structures involved in lactose-resistant adherence of strain T14V to saliva-treated hydroxyapatite. Functional differences between the fimbriae of A. naeslundii and A. viscosus strains may be correlated with well-established differences in the in vivo distribution of these organisms: namely, the preference of typical A. naeslundii for epithelial surfaces and of A. viscosus for tooth surfaces.