Submitted to: Animal Genetics International Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: June 1, 2002
Publication Date: August 11, 2002
Citation: Damota, A.F., Sonstegard, T.S., Van Tassell, C.P., Connor, E.E., Capuco, A.V., Brito, M.P., Machado, M.A., Martinez, M.L., Coutinho, L.L. 2002. Sampling gene expression from mammary tissues by est sequencing or orestes cdna libraries from holstein and gir cattle.. Animal Genetics International Conference Proceedings.
Even though more than 230,000 bovine expressed sequence tags (EST) are available in public databases, additional EST are needed. The objective of this study was to generate EST to better characterize the mammary gland transcriptome. Construction of cDNA libraries was performed using the open reading frame EST (ORESTES) method to avoid the transcript bias inherent to mammary and to maximize discovery rate of novel EST. ORESTES relies on arbitrarily primed RT-PCR to generate a partial expression profile that can be shotgun cloned. In a preliminary study, six libraries were constructed using mRNA from pre-pubertal mammary and primers that amplify the estrogen receptors. Sequencing of 576 clones generated 455 GenBank quality submissions. Sequence assembly was used to assess rate of clonal redundancy relative to each RT-PCR primer and provide tentative consensus (TC) sequences for BLAST analysis. A total of 64 TC sequences were assembled from three libraries leaving 178 singletons and an average redundancy rate of 47%. BLAST analysis of the 242 unique sequence elements revealed 81 sequences (17%) that had no match to GenBank nt and 147 (32%) were novel relative to cattle. Because ORESTES produced a high rate of novel sequences, this method will be exploited to generate mammary-derived EST from Holstein- and Gir-derived libraries. The resultant breed specific polymorphisms and novel sequences will be a valuable resource for understanding gene expression differences between breeds.