Characterization of ß-casein gene in Indian riverine buffalo.

The study aimed at characterization of buffalo ß-casein gene and its promoter by PCR-SSCP analysis. Complete ß-casein exon VII region analysis revealed two SSCP band patterns, with pattern-I representing predominant allele B (85%) present in homozygous (genotype BB) condition and pattern-II representing a rare allele A1 present in heterozygous condition (genotype A1B). Sequencing of two patterns revealed three nucleotide substitutions at codon 68, 151 and 193 of exon VII. The cDNA sequence of buffalo ß-casein gene indicated three further nucleotide substitutions between allele A1 and B at codon 10, 39, and 41. Analysis of ß-casein proximal promoter region (-350 upstream to +32) revealed four SSCP band patterns. These SSCP patterns corresponded to nucleotide substitutions at seven locations within 382 bp 5' UTR region of ß-casein gene. Haplotype analysis suggested pattern-I of exon VII (wild type) was associated with three types of promoters and pattern-II of exon VII (rare type) corresponded to one exclusive type of promoter. The study suggested two haplotypes of exon VII and four haplotypes of promoter for buffalo ß-casein.

A reporter promoter assay confirmed the role of a distal promoter NOBOX binding element in enhancing expression of GDF9 gene in buffalo oocytes.

Growth differentiation factor 9 is primarily expressed in oocytes and plays a vital role in oocyte cumulus crosstalk. Earlier studies with buffalo oocytes revealed differential expression of this gene under different media stimulation conditions which, in turn, are correlated with the blastocyst yield. In this study, different germ cell specific cis elements including a NOBOX binding elements (NBE) and several E-boxes were identified at the 5' upstream region of buffalo GDF9 gene and their potential role in GDF9 expression was investigated. Transfecting oocytes with GDF9 promoter deletion constructs harbouring the NBE reporter gene revealed a 33% increase in GFP as well as the luciferase signal signifying its role in stimulating the minimal promoter activity of GDF9 in buffalo oocytes. Site directed mutation of core binding nucleotides at NBE at 1.8 kb upstream to TSS further confirmed its role for enhancing the basal transcriptional activity of GDF9 promoter in buffalo oocytes. Current work will provide important leads for understanding the role of GDF9 in oocytes competence and designing a more physiological IVF protocol in case of buffalo.

Heterologous expression and characterization of Indian Sahiwal cattle (Bos indicus) alpha inhibin.

Inhibin is a non-steroidal glycoprotein hormone of gonadal origin with major action as negative feedback control of the production of FSH by the anterior pituitary gland. The physiological role of inhibin has led to the development of inhibin immunogens for fertility enhancement in farm animals. It is envisaged that a reduction of endogenous inhibin secretion would increase FSH concentrations and thus offers a potential for increasing the number of ovulatory follicles in the ovary. The present work was carried out to produce recombinant bovine (Indian Sahiwal Cattle; Bos indicus) alpha inhibin (bINH-α) in E. coli by optimizing its expression and purification in biologically active form and to study its immunological characterization. A bacterial protein expression vector system based on the phage T(5) promoter was used. The bINH-α encoding gene was successfully cloned and expressed in E. coli and the purified recombinant bINH-α was characterized. Recombinant bINH-α (25 µg mL(-1)) immunized guinea pigs had a significant increase in litter size compared to the control group. These results indicate a role for recombinant bINH-α as a fecundity vaccine to enhance the ovulation rate and litter size in animals.

Polymorphism of fecundity genes (FecB, FecX, and FecG) in the Indian Bonpala sheep.

The present study was designed for screening polymorphism of known fecundity genes in prolific Indian Bonpala sheep. Employing tetra-primer amplification refractory mutation system PCR, 11-point mutations of BMP1B, BMP15, and GDF9 genes of 97 Bonpala ewes were genotyped. The FecB locus of the BMPR1B gene and two loci (G1 and G4) of GDF9 gene were found to be polymorphic. In FecB locus, three genotypes, namely, wild type (Fec++, 0.02), heterozygous (FecB+, 0.23), and mutant (FecBB, 0.75) were detected. At G1 locus of GDF9 gene, three genotypes, namely, wild type (GG, 0.89), heterozygous (GA, 0.10), and mutant (AA, 0.01) were detected. At G4 locus of GDF9 gene, three genotypes, namely, wild type (AA, 0.01), heterozygous (AG, 0.14), and mutant (GG, 0.85) were detected. Statistically no significant correlation of polymorphism of FecB, G1, and G4 loci and litter size was found in this breed. All five loci of BMP15 and three loci of GDF 9 genes were monomorphic. This study reports Bonpala sheep as the first sheep breed where concurrent polymorphism at three important loci (FecB, G1, and G4) of two different fecundity genes (BMPR1B and GDF9) has been found.

A Simple Method for the Efficient Isolation of Genomic DNA from Lactobacilli Isolated from Traditional Indian Fermented Milk (dahi).

A simple, inexpensive and effective genomic DNA isolation procedure for Lactobacillus isolates from traditional Indian fermented milk (dahi) is described. A total of 269 Lactobacillus isolates from fermented milk collected from four places in North and west India were tested for lysis by an initial weakening of the Gram positive cell wall with Ampicillin followed by Lysozyme treatment. The average genomic DNA yield was ~50 µg/ml log phase culture. Quality and repeatability of the method was found to be adequate for subsequent molecular applications. The quality of the genomic DNA isolated by this method was verified by restriction digestion and polymerase chain reaction (PCR). No inhibition was observed in subsequent PCR amplification and restriction digestion. The presented method is rapid, cheap and useful for routine DNA isolation from gram positive bacteria such as Lactobacillus.

Polymorphism of BMPR1B, BMP15 and GDF9 fecundity genes in prolific Garole sheep.

Mutation studies in different prolific sheep breeds have shown that the transforming growth factor beta super family ligands viz. the growth differentiation factor 9 (GDF9/FecG), bone morphogenetic protein 15 (BMP15/FecX) and associated type I receptors, bone morphogenetic protein receptor (BMPR1B/FecB), are major determinant of ovulation rate and consequent increase in litter size. The Garole sheep is a highly prolific sheep breed of India. Characterization of fecundity genes in these animals could substantially improvise the breeding programme in these animals as well as other sheep breeds of the region. The present study was therefore designed with the objective of polymorphism study of fecundity genes in these prolific microsheep. A total of 11 point mutations were detected by polymerase chain reaction (PCR)-based method. A competitive technique called tetra-primer amplification refractory mutation system-PCR was adapted to type a total of ten points of two ovine fecundity genes (GDF9 and BMP15). The FecB locus of the BMPR1B gene and G1 locus of GDF9 gene were found to be polymorphic. In FecB locus, two genotypes, wild type (FecB(+)) and mutant (FecBB), were detected with allele frequencies of 0.39 and 0.61, respectively. At G1 locus, two genotypes, mutant (A) and wild types (G) were detected with allele frequencies of 0.18 and 0.82, respectively. This study reports Garole sheep as the fourth sheep breed after Belclare/Cambridge, Lacaune and Small-tailed Han sheep, where coexisting polymorphism has been found in two different fecundity genes (BMPRIB and GDF9 genes).

Expression stability of two housekeeping genes (18S rRNA and G3PDH) during in vitro maturation of follicular oocytes in buffalo (Bubalus bubalis).

The present study was undertaken to evaluate the expression stability of two housekeeping genes (HKGs), 18S rRNA and G3PDH during in vitro maturation (IVM) of oocytes in buffalo, which qualifies their use as internal controls for valid qRT-PCR estimation of other oocyte transcripts. A semi quantitative RT-PCR system was used with optimised qRT-PCR parameters at exponential PCR cycle for evaluation of temporal expression pattern of these genes over 24 h of IVM. 18S rRNA was found more stable in its expression pattern than G3PDH.

Characterization of the luteinizing hormone beta (LH-beta) subunit gene in the Indian river buffalo (Bubalus bubalis).

The cDNA sequence of leuteinizing hormone (LH) beta subunit was determined to understand the molecular basis for silent oestrus behavior and poor response to superovulation in buffalo. The LH-beta cDNA contains an open reading frame 426 bp long. The deduced sequence of the LH-beta is 141 amino acids in length. The amino acid sequences of the Indian river buffalo LH-beta subunit showed overall similarity to those of other mammals. The nucleotide sequence variability of LH-beta was studied in more than approximately 350 Indian buffaloes covering five different breeds. The results of the sequence analysis showed that the buffalo LH-beta gene is not highly conserved and non-synonymous mutations are not rare, at least in the samples collected randomly from five different breeds and buffalo populations. A total of seven different variants were obtained. In spite of its crucial role in reproduction, variation of the LH-beta gene was found present in this species. The polymorphisms found were unique in the Indian river buffalo population.