Characterization and polymorphism of keratin associated protein 1.4 gene in goats.

Keratin-associated proteins (KAPs) are among the main structural components of the animal fibers and form semi-rigid matrix wherein the keratin intermediate filaments (KIFs) are embedded. Variation in the KAP genes has been reported to affect the structure of KAPs and hence fiber characteristics. As no information is available on this gene in Capra hircus therefore, present work was undertaken to characterize and explore the different polymorphic variants of KAP1.4 gene at DNA level in different breeds/genetic groups of goats of Kashmir. Cashmere (Changthangi, 30 animals) and non-Cashmere (Bakerwal and Kargil goats, 20 animals each) goats formed the experimental animals for the study. Single strand conformation polymorphism technique was employed for exploring variability at gene level. On exploring the size variability in KAP1.4 gene between Ovine and Caprine, it was concluded that sheep KAP1.4 gene has a deletion of 30 nucleotides. In comparison to published nucleotide sequences of sheep, goat sequences explored are differing at positions 174, 462 and 568 and at these positions "G", "T" and "T" nucleotides are present in sheep, but are replaced by "A", "C" and "C" respectively, in goats. By SSC studies, two genotypes were observed in each genetic group and in Bakerwal goats the genotypes were designated as A1A1 (0.40) and A1A2 (0.60) and were formed by two alleles A1 (0.70) andA2 (0.30). The different SSC patterns observed in Kargil goats were designated as B1B1 (0.35) and B1B2 (0.65) genotypes with frequencies of B1 and B2 alleles as 0.675 and 0.325, respectively. Similarly, two genotypes C1C1 (0.60) and C1C2 (0.40) were observed in Changthangi goats and the frequencies of C1 and C2 alleles were 0.80 and 0.20, respectively. These alleles were later confirmed by sequencing. The sequences of these alleles are available in NCBI under Acc. No's. JN012101.1, JN012102.1, JN000317.1, JN000318.1, JQ436929 and JQ627657. It was concluded that all the alleles observed in a breed were unique to the breed. The designated A1 and A2 alleles of Bakerwal goats differ from each other at positions 245 and the nucleotides observed were "C" or "A" and at position 605 of the nucleotide sequence "T" or "C", were observed. The designated B1 and B2 alleles of Kargil goats differed from each other at positions 224, 374, 375 and 521. The nucleotides observed in two SSC pattern were C→G, A→G, G→A and T→C, respectively. The designated C1 and C2 alleles of Changthangi goats differed from each other at one position 440 with the change of "A"→"C". Only two mutations C224G and G375A in Kargil goats resulted in change of the Cysteine (C)→Serine (S) and Alanine (A)→Threonine (T), respectively. The nucleotide sequences of KAP 1.4 gene in Bakerwal, Kargil and Changthangi goats showed 99.7% similarity with each other and 96.7% with sheep and 74.4% with mice. Average guard fiber length and diameter were 81.02±0.16 mm and 67.53±0.97 µm, respectively, and average down fiber length and diameter was 48.38±0.70 mm and 13.32±0.29 µm, respectively for Changthangi goats. Average guard fiber length and diameter were 63.51±4.52 mm and 105.31±4.48 µm, respectively for Bakerwal goats and 62.60±5.03 mm and 107.18±2.30 µm, respectively for Kargil goats. The effects of the observed genotypes on Cashmere fiber diameter, Cashmere fiber length in Changthangi goats and guard fiber length and guard fiber diameters in Changthangi, Kargil and Bakerwal goats were found to be non-significant (P>0.05).The nonsignificant association between the polymorphism and fiber attributes reported herein may be due to small sample size.

Polymorphism study of growth differentiation factor 9B (GDF9B) gene and its association with reproductive traits in sheep.

GDF9B protein plays a critical role in growth and differentiation of early ovarian follicles. In Inverdale and Hanna sheep, mutations in exon-2 of GDF9B gene have been recorded to show increased ovulation rate in heterozygous condition whereas homozygotes are infertile. Present screen study was carried out to explore the presence of these reported mutations in Corriedale and Local Kashmir Valley sheep with high rate of twinning. Exon-2 of GDF9B gene was amplified and the polymorphism was explored by SSCP technique. In the process three different bandings were observed. Later on these patterns corresponded with three different allelic forms on nucleotide sequencing. Phylogenetic analysis revealed that the nucleotide sequences of alleles observed in the present study and that of a published sequence of sheep were having the same point of origin. The results were also compared with goats, large ruminants and humans. The allelic frequencies of allele A and B were 0.64 and 0.36, respectively in Corriedale sheep whereas the allelic frequencies of all the three alleles in Kashmir Valley sheep were 0.60, 0.34 and 0.06. SNP "C" of the designated genotype AC was observed to pronounce a significant effect on litter size with average litter size going up by 0.63 as compared with the nearest genotype AB wherein the litter size was 1.29±0.05. The average litter size between AA and AB genotypes did not vary significantly.

Nucleotide sequencing and DNA polymorphism studies of BMP 15 gene in Corriedale and local Kashmir valley sheep (Ovis aries).

The families of TGF-ß proteins are the most important growth factors in the ovary for growth and differentiation of early ovarian follicles. Three related oocyte-derived members of the transforming growth factor-ß superfamily, namely GDF9, BMP15 and BMPR-IB have been shown to be essential for follicular growth and ovulation. The objective of the present study was to detect the incidence of mutation in intronic portion of BMP 15 gene in Corriedale and local Kashmir valley sheep breeds. Blood samples were collected from 85 ewes and genomic DNA was extracted using the modified phenol chloroform method. The quantity and quality of extracted DNA was examined using spectrophotometry and gel electrophoresis, respectively. A fragment with the size of 356 bp was amplified using polymerase chain reaction (PCR) with a pair of specific primers. The amplified PCR products were digested with Mph11031 restriction enzyme. In the presence of mutation at this locus, the Mph11031 enzyme cannot recognize the restriction site. However, here in the absence of mutations, the enzyme recognizes one restriction site and divides the amplified fragment into two fragments of 152 and 204 bp. The 356 bp fragment was also analyzed for polymorphism by SSCP technique. The results indicated two different banding patterns AA and AB for this fragment. Later on two different allelic forms A and B were confirmed by nucleotide sequencing. The 356 bp nucleotide sequence was subjected to alignment analysis and it was observed that sequence similarity of this fragment with that of other sheep and Jining grey goat was more than 97.8%. Phylogenetic analysis revealed that both designated A and B alleles as well as published sequence of sheep form a common cluster indicating their evolutionary closeness. The origin of Jining grey goat was located some distance away from the sheep. The overall frequencies of AA and AB genotypes were 0.79 and 0.21. The breed wise frequencies were 0.78 and 0.22 in Corriedale sheep and the frequencies in Kashmir valley sheep were 0.80 and 0.20 for AA and AB genotypes, respectively. The overall allelic frequencies of A and B alleles were 0.89 and 0.11 whereas allelic frequencies Corriedale sheep was 0.89 and 0.11 and that of Kashmir valley sheep were 0.90 and 0.10.

Contemporary update of cancer control after radical prostatectomy in the UK.

Despite a significant increase of the number of radical prostatectomies (RPs) to treat organ-confined prostate cancer, there is very limited documentation of its oncological outcome in the UK. Pathological stage distribution and changes of outcome have not been audited on a consistent basis. We present the results of a multicentre review of postoperative predictive variables and prostatic-specific antigen (PSA) recurrence after RP for clinically organ-confined disease. In all, 854 patient's notes were audited for staging parameters and follow-up data obtained. Patients with neoadjuvant and adjuvant treatment as well as patients with incomplete data and follow-up were excluded. Median follow-up was 52 months for the remaining 705 patients. The median PSA was 10 ng ml(-1). A large migration towards lower PSA and stage was seen. This translated into improved PSA survival rates. Overall Kaplan-Meier PSA recurrence-free survival probability at 1, 3, 5 and 8 years was 0.83, 0.69, 0.60 and 0.48, respectively. The 5-year PSA recurrence-free survival probability for PSA ranges <4, 4.1-10, 10.1-20 and >20 ng ml(-1) was 0.82, 0.73, 0.59 and 0.20, respectively (log rank, P<0.0001). PSA recurrence-free survival probabilities for pathological Gleason grade 2-4, 5 and 6, 7 and 8-10 at 5 years were 0.84, 0.66, 0.55 and 0.21, respectively (log rank, P<0.0001). Similarly, 5-year PSA recurrence-free survival probabilities for pathological stages T2a, T2b, T3a, T3b and T4 were 0.82, 0.78, 0.48, 0.23 and 0.12, respectively (log rank, P=0.0012). Oncological outcome after RP has improved over time in the UK. PSA recurrence-free survival estimates are less optimistic compared to quoted survival figures in the literature. Survival figures based on pathological stage and Gleason grade may serve to counsel patients postoperatively and to stratify patients better for adjuvant treatment.