|Da Mota, A - EMBRAPA|
|Van Tassell, Curtis|
|Matukamalli, Lakshmi - GEORGE MASON UNIVERSITY|
|Brito, M A - EMBRAPA|
|Martinez, M - EMBRAPA|
Submitted to: Animal Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 24, 2004
Publication Date: March 24, 2004
Citation: Da Mota, A.F., Sonstegard, T.S., Van Tassell, C.P., Shade, L.L., Matukamalli, L.K., Wood, D.L., Capuco, A.V., Brito, M.P., Connor, E.E., Martinez, M.L. 2004. Characterization of open reading frame expressed sequence tags generated from bos indicus and bos taurus mammary gland cDNA libraries. Animal Genetics 35:213-219. Interpretive Summary: Functional genomic studies of the mammary gland require the construction of sequence resources based on genes expressed from the different developmental and operational states of this gland. The mammary gland presents a unique challenge for research aimed at sequence discovery due to the amount of mRNA expression redundancy in the gland. As such, new methodologies were tested that were based on cDNA synthesis from random annealing events not those reactions conditions typically directed for annealing to the 3' end of the mRNA. This cost-effective method of making cDNA libraries was used to generate over 6,000 expressed sequence tags (EST) from healthy and infected mammary gland tissues of Brazilian Gir and Holstein cattle. This method was also found to be effective at identifying rare and/or lowly expressed bovine genes as nearly 300 new bovine genes were sequenced. In addition, the number of gene sequences derived from Bos indicus animals was increased by 40-fold. This information will be important for mapping studies utilizing single nucleotide polymorphism markers to determine alleles by breed of origin.
Technical Abstract: Sequence-based gene expression data are used to interpret results from functional genomic and proteomics studies. Even though more than 300,000 bovine expressed sequence tags (EST) are available in public databases, a more thorough and directed sampling of the expressed genome is needed to identify new transcripts and improve assembly and annotation of existing transcript sequences. Accordingly, we examined the utility of constructing cDNA libraries synthesized by arbitrarily primed RT-PCR of mRNA from tissues not well represented in the publicly available bovine EST database. A total of 33 cDNA libraries were constructed from healthy and infected mammary gland tissues of Brazilian Gir and Holstein cattle. This series of libraries was used to generate 6,481 open reading frame-expressed sequence tags (ORESTES) that assembled into 1,798 unique sequence elements of which, 1,157 did not significantly match sequence assemblies available in the Bos taurus gene index. However, a total of 264 of these 1,157 sequence elements aligned with mouse and human expressed sequences demonstrating that ORESTES is an effective resource for discovery of novel expressed sequences in cattle. Furthermore, comparison of the alignment position of bovine ORESTES-derived sequence elements to human gene reference sequences suggested that the priming events for cDNA synthesis more often occurred at the central portion of a transcript, which may have contributed to the relatively high rate of novel sequence discovery.