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Expressional dynamics of minisatellite 33.15 tagged spermatozoal transcriptome in Bubalus bubalis.

Srivastava J, Premi S, Kumar S, Ali S - BMC Genomics (2009)

Bottom Line: Of these 33.15 tagged transcripts, only one was found to be homologous to Bovine steroid 21-hydroxylase (P-450-c21) gene.Moreover, genes corresponding to all the 33.15 tagged spermatozoal transcripts were found to be conserved across 13 other species analyzed.Our results show MASA as an important tool to capture mRNA transcript diversity tagged with minisatellites in the spermatozoa.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Genetic Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India. jayanshi@gmail.com

ABSTRACT

Background: Transcriptionally quiescent spermatozoa have been established to be a repository of mRNA coding for several functionally essential cellular proteins. This entourage of mRNA is envisaged to be involved in post-fertilization and early embryogenesis. Minisatellites tagged with mRNA transcripts have been implicated with gene organization, regulation and function. However, the organization and expression of the minisatellite tagged transcript diversity, particularly in spermatozoa, remains unclear.

Results: In the present study, we identified and characterized 12 mRNA transcripts from the spermatozoa of water buffalo Bubalus bubalis employing minisatellite associated sequence amplification (MASA) and a consensus sequence of 33.15 repeat loci. Of these 33.15 tagged transcripts, only one was found to be homologous to Bovine steroid 21-hydroxylase (P-450-c21) gene. Other ten transcripts showed significant similarity with various mRNAs or chromosomal contigs across the species. The remaining one construed to be novel since this was unreported in the database (NCBI GenBank). All these uncharacterized and known transcripts showed highest expression in testis and spermatozoa compared to that in somatic tissues and ovary. Of these 12 mRNA transcripts, 4 showed differential expression in the forebrain and hindbrain of buffalo. Moreover, genes corresponding to all the 33.15 tagged spermatozoal transcripts were found to be conserved across 13 other species analyzed.

Conclusion: Our results show MASA as an important tool to capture mRNA transcript diversity tagged with minisatellites in the spermatozoa. Comprehensive characterization of these transcripts is envisaged to augment our understanding on the genes involved in testicular functions and sustenance of a viable paternal genome during pre- and post- fertilization events and early stages of development. Prospects of this approach in genome analysis in general and comparative genomics in particular are highlighted.

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Minisatellite associated sequence amplification (MASA) performed using oligo based on the consensus of 33.15 repeat loci and cDNA from the spermatozoa of 7 different animals. Four to seven transcripts in the range of 0.15 kb to 0.5 kb were detected (A). β-actin was used as an internal control with cDNA from all the samples (B).
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Figure 1: Minisatellite associated sequence amplification (MASA) performed using oligo based on the consensus of 33.15 repeat loci and cDNA from the spermatozoa of 7 different animals. Four to seven transcripts in the range of 0.15 kb to 0.5 kb were detected (A). β-actin was used as an internal control with cDNA from all the samples (B).

Mentions: In previous study, we identified 6 different mRNA transcripts from various tissues of water buffalo using oligos based on the consensus of 33.15 repeat loci [20]. In the present study, we used this oligo (5' CACCTCTCCACCTGCC 3') for MASA and uncovered a total of 72 amplicons comprising 12 different mRNA transcripts from the spermatozoa (Figure 1). Cloning and sequencing followed by the BLAST search of these transcripts showed that 1 had no homology with the sequences in the GenBank and 10 were similar to uncharacterized BAC clones originating from cattle Bos taurus, human Homo sapiens and mouse Mus musculus. Only one mRNA transcript was found to be homologous to the Bovine steroid 21-hydroxylase (P-450-c21) gene (Table 1). Interestingly, comparative analysis of these spermatozoal mRNA transcripts showed that none was common to those identified from different somatic tissues, testis and ovary in earlier study [20]. However, cloning and sequencing of ~20 recombinant clones corresponding to each of these 72 fragments from seven different animals demonstrated their consistent presence in the spermatozoa. In order to ascertain the structural, functional and regulatory status of the MASA uncovered genes/gene fragments, we conducted a comprehensive database search for each mRNA transcript independently. The details of these homologous genes, their accession numbers with chromosomal locations (if available), species, position of our cDNA sequences and their possible functions are given in the table 1.


Expressional dynamics of minisatellite 33.15 tagged spermatozoal transcriptome in Bubalus bubalis.

Srivastava J, Premi S, Kumar S, Ali S - BMC Genomics (2009)

Minisatellite associated sequence amplification (MASA) performed using oligo based on the consensus of 33.15 repeat loci and cDNA from the spermatozoa of 7 different animals. Four to seven transcripts in the range of 0.15 kb to 0.5 kb were detected (A). β-actin was used as an internal control with cDNA from all the samples (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713999&req=5

Figure 1: Minisatellite associated sequence amplification (MASA) performed using oligo based on the consensus of 33.15 repeat loci and cDNA from the spermatozoa of 7 different animals. Four to seven transcripts in the range of 0.15 kb to 0.5 kb were detected (A). β-actin was used as an internal control with cDNA from all the samples (B).
Mentions: In previous study, we identified 6 different mRNA transcripts from various tissues of water buffalo using oligos based on the consensus of 33.15 repeat loci [20]. In the present study, we used this oligo (5' CACCTCTCCACCTGCC 3') for MASA and uncovered a total of 72 amplicons comprising 12 different mRNA transcripts from the spermatozoa (Figure 1). Cloning and sequencing followed by the BLAST search of these transcripts showed that 1 had no homology with the sequences in the GenBank and 10 were similar to uncharacterized BAC clones originating from cattle Bos taurus, human Homo sapiens and mouse Mus musculus. Only one mRNA transcript was found to be homologous to the Bovine steroid 21-hydroxylase (P-450-c21) gene (Table 1). Interestingly, comparative analysis of these spermatozoal mRNA transcripts showed that none was common to those identified from different somatic tissues, testis and ovary in earlier study [20]. However, cloning and sequencing of ~20 recombinant clones corresponding to each of these 72 fragments from seven different animals demonstrated their consistent presence in the spermatozoa. In order to ascertain the structural, functional and regulatory status of the MASA uncovered genes/gene fragments, we conducted a comprehensive database search for each mRNA transcript independently. The details of these homologous genes, their accession numbers with chromosomal locations (if available), species, position of our cDNA sequences and their possible functions are given in the table 1.

Bottom Line: Of these 33.15 tagged transcripts, only one was found to be homologous to Bovine steroid 21-hydroxylase (P-450-c21) gene.Moreover, genes corresponding to all the 33.15 tagged spermatozoal transcripts were found to be conserved across 13 other species analyzed.Our results show MASA as an important tool to capture mRNA transcript diversity tagged with minisatellites in the spermatozoa.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Genetic Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India. jayanshi@gmail.com

ABSTRACT

Background: Transcriptionally quiescent spermatozoa have been established to be a repository of mRNA coding for several functionally essential cellular proteins. This entourage of mRNA is envisaged to be involved in post-fertilization and early embryogenesis. Minisatellites tagged with mRNA transcripts have been implicated with gene organization, regulation and function. However, the organization and expression of the minisatellite tagged transcript diversity, particularly in spermatozoa, remains unclear.

Results: In the present study, we identified and characterized 12 mRNA transcripts from the spermatozoa of water buffalo Bubalus bubalis employing minisatellite associated sequence amplification (MASA) and a consensus sequence of 33.15 repeat loci. Of these 33.15 tagged transcripts, only one was found to be homologous to Bovine steroid 21-hydroxylase (P-450-c21) gene. Other ten transcripts showed significant similarity with various mRNAs or chromosomal contigs across the species. The remaining one construed to be novel since this was unreported in the database (NCBI GenBank). All these uncharacterized and known transcripts showed highest expression in testis and spermatozoa compared to that in somatic tissues and ovary. Of these 12 mRNA transcripts, 4 showed differential expression in the forebrain and hindbrain of buffalo. Moreover, genes corresponding to all the 33.15 tagged spermatozoal transcripts were found to be conserved across 13 other species analyzed.

Conclusion: Our results show MASA as an important tool to capture mRNA transcript diversity tagged with minisatellites in the spermatozoa. Comprehensive characterization of these transcripts is envisaged to augment our understanding on the genes involved in testicular functions and sustenance of a viable paternal genome during pre- and post- fertilization events and early stages of development. Prospects of this approach in genome analysis in general and comparative genomics in particular are highlighted.

Show MeSH
Related in: MedlinePlus