|Van Tassell, Curtis - Curt|
Submitted to: American Society of Animal Science
Publication Type: Abstract Only
Publication Acceptance Date: 5/1/2002
Publication Date: 7/21/2002
Citation: Mota, A.F., Sonstegard, T.S., Van Tassell, C.P., Connor, E.E., Capuco, A.V., Brito, A.P., Machado, M.A., Martinez, M.L., Coutinho, L.L. 2002. Construction and characterization of orestes cdna libraries generated from bovine mammary gland tissues [abstract]. American Society of Animal Science.
Technical Abstract: Currently, there are approximately 200,000 expressed sequence tags (EST) available in public databases. This is approximately an order of magnitude less sequence information than that available for characterizing the transcriptomes of human and mouse. As such, additional bovine EST need to be generated to more throughly identify, annotate and classify bovine genes. This information will be essential to the interpretation of results generated from functional genomic studies. The objective of the present study was to generate 5,000 EST from the mammary gland, while maintaining a maximum discovery rate of novel EST. Based on this criteria, we chose to construct cDNA libraries using the open reading frame EST (ORESTES) method. This method uses arbitrarily primed cDNA synthesis to generate a partial profile of gene expression that can be PCR amplified, cloned, and arrayed into mini-libraries. The clones are usually sequences representing the central portion of expressed genes. In a preliminary study, primers that amplify estrogen receptor gene family members and mRNA from a pre-pubertal Holstein heifer were used to create six mini-libraries. Six 96 well plates of clones from three of these libraries were processed for sequence analysis. After sequence processing to assign quality score and trim vector sequence, 455 sequences meeting minimum GenBank submission criteria were generated. These sequences were assembly using GAP4 (Staden-Package) to assess rate of clonal redundancy and to provide consensus sequences for BLAST analysis. A total of 64 tentative consensus (TC) sequences were assembled leaving 178 singletons (ST) to generate a rate of redundancy of 47\%. However, BLAST analysis of the 242 unique sequence elements (TC and ST sequence) against GenBank nt and the Bos taurus Gene Index (BtGI) databases revealed that most of these sequences were new relative to other cattle EST. Eighty-one sequences (17\%) had no match to the nt database, and 147 (32\%) did not match BtGI. Because ORESTES produced a high rate of novel cattle sequences, this method will be exploited to generate EST from mammary gland mRNA isolated from Brazilian dairy cattle (Gir). The presence of novel sequences within these libraries will be a valuable resource for studying gene expression differences in the mammary gland of Holstein and Gir cattle.