Comparative and functional study of micro open reading frames (uORF) in the 5' untranslated regions (UTR) of genes in Saccharomyces cerevisiae.
Zhihong Zhang, Fred Dietrich
MGM, Duke University Medical Center, Box 3568 DUMC, Durham, NC, 27710, USA
Micro ORFs upstream of GCN4 and CPA1 have been studied and shown to be important in translational regulation. We have taken advantage of seven recently sequenced related fungal genomes to search by comparative methods to identify additional potential regulatory uORF. First, the start codon of orthologues of S. cerevisiae genes was identified by tblastn against the comparative species. The putative orthologous UTR sequences were pulled out to search for uORF(s). Comparison was applied to find candidates in which uORFs were found in multiple species for experimental verification. A total of 14 new uORF containing genes were found and verified in S. cerevisiae by 5' RACE and primer extension. Most of these 14 genes have only a single uORF in the 5'UTR, and the length of these uORFs ranges from 3 to 18 codons. Additional work is being conducted to attempt to elucidate the role of these uORF in S. cerevisiae. By using a luciferase reporter system and doing site-direct mutagenesis on start codon in the uORF(s), the luciferase activity and mRNA level are being measured and compared. We are interested in understanding the regulatory role of these uORFs.
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