Isolation, sequence identification and expression profile of three novel genes Rab2A, Rab3A and Rab7A from Black-boned sheep (Ovis aries).

Complete coding sequences of three Black-boned sheep (Ovis aries) genes Rab2A, Rab3A and Rab7A were amplified using reverse transcription polymerase chain reaction (RT-PCR) based on the conserved sequence information of cattle or other mammals known to be highly homologous to sheep ESTs. The Black-boned sheep Rab2A gene encodes a protein of 226 amino acids which contains the conserved putative RabL2 domain and is highly homologous to the Rab2A proteins of seven other species--cattle (96%), human (83%), Sumatran orangutan (82%), rat (81%), mouse (80%), African clawed frog (72%) and zebrafish (71%). The Black-boned sheep Rab3A gene encodes a protein of 220 amino acids that contains the conserved putative Rab3 domain and is very similar to the Rab3A proteins of four species--cattle (99%), African clawed frog (99%), Western clawed frog (98%) and zebrafish (95%). And the Black-boned sheep Rab7A gene encodes a protein of 207 amino acids that contains the conserved putative Rab7 domain and has high homology with the Rab7A proteins of six other species--human (99%), dog (99%), Sumatran orangutan (99%), zebrafish (97%), rabbit (97%) and African clawed frog (96%). Analysis of the phylogenetic tree has demonstrated that the Black-boned sheep Rab2A, Rab3A and Rab7A proteins share a common ancestor and the tissue expression analysis has shown that the corresponding genes are expressed in a range of tissues including leg muscle, kidney, skin, longissimus dorsi muscle, spleen, heart and liver. Our experiment is the first to provide the primary foundation for a further insight into these three sheep genes.

Phylogenetic analysis of 16S rRNA gene sequences reveals rumen bacterial diversity in Yaks (Bos grunniens).

Six matured male Yaks (Bos grunniens) with a mean live weight of 450 +/- 23 kg (mean +/- SD), were housed indoors in metabolism cages and fed pelleted lucerne (Medicago sativum). After an adjustment period of 24 days of feeding the diet, samples of rumen content were obtained for analysis of the bacteria in the liquor. The diversity of rumen bacteria was investigated by constructing a 16S rRNA gene clone library using the general bacterial primers F27 and R1492. A total of 130 clones, comprising nearly full length sequences (approx. 1.5 kb) were sequenced and submitted to BLAST and phylogenetic analysis. Using the criterion that similarity of 97% or greater with the sequences of cultivated bacteria, 16 clones were identified as Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Ruminococcus flavefaciens, Succiniclasticum ruminis, Selenomonas ruminantium and Prevotella ruminicola, respectively. A further 10 clones shared similarity ranging from 90 to 97% with cultivated bacteria but the similarity in sequences for the remaining 104 clones were less than 90% of those of cultivated bacteria. Using a phylogenetic analysis it was found that the majority of the clones identified (63.8%) were located in the Low G + C Subdivision, with most of the remainder (35.4% of clones) located in the Cytophaga-Flexibacter-Bacteroides phylum and one clone (0.8%) was identified as a Proteobacteria. It was apparent that Yaks have a large and diverse range of bacteria in the rumen content which differ from those of cattle and other ruminants.

Pigmentation in Black-boned sheep (Ovis aries): association with polymorphism of the Tyrosinase gene.

Measurements were made in Black-boned (n = 40) and normal (n = 23) sheep (Ovis aries) from a flock in Nanping County of Yunnan Province, China, as well as a group (n = 21) of Romney Marsh sheep (O. aries) with the view to explaining the basis of the dark pigmentation occurring in the Black-boned animals. Plasma colour was significantly darker (P < 0.01) in Black-boned sheep than in their normal flock mates, which in turn had significantly darker plasma (P < 0.01) than the Romney Marsh sheep. Similar significant (P < 0.01) differences were measured for plasma tyrosinase activity and both groups of sheep from Nanping County had similar plasma concentrations of glutathione which were significantly smaller (P < 0.01) than for the Romney Marsh sheep.A partial fragment of 750 bp of exon 1 of the gene encoding tyrosinase was constructed and found to contain two silent mutation sites (G192C and C462T) but there was no effect on amino acid sequences of tyrosinase. Using restriction fragment length polymorphism analyses two allelic variants of site G192C were identified giving rise to the genotypes GG, GC and CC; the frequencies of allele G being 0.914, 0.824 and 0.286 in the Black-boned sheep, their flock mates and the Romney Marsh sheep respectively. Plasma tyrosinase activity was similar for genotypes GG and GC and for both genotypes significantly higher (P < 0.05) than for genotype CC. The sheep from Nanping County displayed only the GG and GC genotypes and had predominantly black or black and white coat colour whereas the Romney Marsh sheep were of either genotype GC or CC and exhibited only white coat colouration. It is not appears that the dark pigmentation of the Black-boned sheep arises because of polymorphisms in the exon 1 of tyrosinase gene. However, this result could explain the differences between Black-boned and Romney Marsh sheep but not for differences between Black-boned and Nanping Normal sheep. Moreover, this result has provided evidence of genetic markers in the form of polymorphisms of the tyrosinase gene which may help to find the black traits causing mutations. There would be merit in further studies using histochemical and molecular techniques to elucidate the causes of the dark pigmentation in these Black-boned sheep.