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

Abstract

Repeat sequences are involved in regulation of gene expression both at the transcriptional and translational level. In the mammalian genomes, tri- and tetranucleotide repeats like ATA, AATA, GGAA and GAAA have been associated with diseases. In silico analysis of (GGAA)₅ distribution across the species showed maximum number of this repeat in the mouse transcriptome compared to that in other species. Following this, we conducted minisatellite associated sequence amplification (MASA) to explore the buffalo's transcriptome using cDNA from different tissues and an oligo based on (GGAA)₅ repeats. MASA uncovered twenty six mRNA transcripts showing homology to known genes in the database. qPCR studies showed the highest expression of twelve transcripts in the spleen. A transcript, pLRC107 with its partial sequence of 203 nucleotides showed sequence variation at several positions in spleen as compared to other four tissues examined. Transcript pLRC100 was found to represent the partial coding sequence of Bos taurus HECT {(homologous to E6-associated protein (UBE3A) carboxyl-terminus domain) and RCC1 (CHC1)-like domain (RLD) 1}, mRNA. We ascertained full length coding sequence of HECT gene and localized the same on buffalo chromosome 10 employing FISH. This gene was found to be conserved across the species. Another gene LRP8 uncovered in the process showed copy number variation between buffalo males (4-9) and females (34-54). The MASA approach enabled us to identify several genes in Bubalus bubalis without screening an entire cDNA library. The highest expression of 12 mRNA transcripts in spleen suggests their likely involvement with immuno transaction. A comprehensive knowledge of the repeat tagged transcriptomes is envisaged to help in understanding their significance in genome organization and evolution forming rich basis of functional and comparative genomics.