Transcriptional Status of Known and Novel Genes Tagged with Consensus of 33.15 Repeat Loci Employing Minisatellite-Associated Sequence Amplification (MASA) and Real-Time PCR in Water Buffalo, Bubalus bubalis

Abstract

We conducted minisatellite-associated sequence amplification (MASA) with an oligo (5' CACCTCTCCACCTGCC 3') based on consensus of 33.15 repeat loci using cDNA from the testis, ovary, spleen, kidney, heart, liver, and lung of water buffalo Bubalus bubalis and uncovered 25 amplicons of six different sizes (1,263, 846/847, 602, 576, 487, and 324 base pairs). These fragments, cloned and sequenced, were found to represent several functional, regulatory, and structural genes. Blast search of all the 25 amplicons showed homologies with 43 transcribing genes across the species. Of these, the 846/847-bp fragment, having homology with the adenylate kinase gene, showed nucleotide changes at six identical places in the ovary and testis. The 1,263; 324; and 487-bp fragments showed homology with the secreted modular calcium binding protein (SMOC-1), leucine-rich repeat neuronal 6A (LRRN6A) mRNA, and human TTTY5 mRNA, respectively. Real-time PCR showed maximum expression of AKL, LRRN6A, and T-cell receptor gamma (TCR-gamma)-like genes in the testis, SMOC-1 in the liver, and the T-cell receptor-like (TCRL) gene in the spleen compared to those used as endogenous control. We construe that these genes have evolved from a common progenitor and conformed to various biological functions during the course of evolution. MASA approach coupled with real-time PCR has potentials to uncover accurate expression of a large number of genes within and across the species circumventing the screening of cDNA library.

Molecular Mining of Alleles in Water Buffalo Bubalus bubalis and Characterization of the TSPY1 and COL6A1 Genes

“…MASA reactions were performed using cDNA templates from different somatic and gonadal tissues of two individuals and from spermatozoa of four individuals following standard procedures [32], [34]. Representative gel pictures are shown in figure 1.…”

“…To compare the relative expression of different genes and gene fragments, SYBR green assays were conducted using Real Time PCR (Sequence Detection System, 7500, ABI) according to established protocols [34], [36]. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as an endogenous control.…”

Molecular mining of GGAA tagged transcripts and their expression in water buffalo Bubalus bubalis

“…4368814, ABI, Foster city, CA, USA). The quality of cDNA so prepared was confirmed by PCR amplification using β-actin primers (Srivastava et al, 2006).…”

“…Earlier, we used oligos based on consensus of 33.6, 33.15, GACA and GATA repeats to uncover repeat tagged mRNA transcripts from the somatic tissues, gonads and spermatozoa of B. bubalis. This led to the identification of tissue specific transcripts and in the process several candidate genes were characterized (Pathak et al, 2010;Srivastava et al, 2006Srivastava et al, , 2008Srivastava et al, , 2009. Interestingly, all the genes uncovered by MASA using different oligo primers were found to be unique as they were not reported in the database.…”

Water Buffalo Genome Science Comes of Age

“…Srivastava and colleagues 30 performed the minisatellite-associated sequence amplification with an oligo (5' CACCTCTCCACCTGCC 3'), that was designed based on consensus of 33.15 repeat loci using cDNA from the testis, ovary, spleen, kidney, heart, liver, and lung of water buffalo. The authors defined six different sizes of minisatellites with 1,263, 846/847, 602, 576, 487, and 324 bp, respectively.…”