Zhang Zhihong, Dietrich Fred S
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Curr Genet. 2005 Aug;48(2):77-87. doi: 10.1007/s00294-005-0001-x. Epub 2005 Sep 14.
We have taken advantage of recently sequenced hemiascomycete fungal genomes to computationally identify additional genes potentially regulated by upstream open reading frames (uORFs). Our approach is based on the observation that the structure, including the uORFs, of the post-transcriptionally uORF regulated Saccharomyces cerevisiae genes GCN4 and CPA1 is conserved in related species. Thirty-eight candidate genes for which uORFs were found in multiple species were identified and tested. We determined by 5' RACE that 15 of these 38 genes are transcribed. Most of these 15 genes have only a single uORF in their 5' UTR, and the length of these uORFs range from 3 to 24 codons. We cloned seven full-length UTR sequences into a luciferase (LUC) reporter system. Luciferase activity and mRNA level were compared between the wild-type UTR construct and a construct where the uORF start codon was mutated. The translational efficiency index (TEI) of each construct was calculated to test the possible regulatory function on translational level. We hypothesize that uORFs in the UTR of RPC11, TPK1, FOL1, WSC3, and MKK1 may have translational regulatory roles while uORFs in the 5' UTR of ECM7 and IMD4 have little effect on translation under the conditions tested.
我们利用最近测序的半子囊菌真菌基因组，通过计算来识别可能受上游开放阅读框（uORF）调控的其他基因。我们的方法基于以下观察结果：转录后受uORF调控的酿酒酵母基因GCN4和CPA1的结构（包括uORF）在相关物种中是保守的。我们鉴定并测试了38个在多个物种中发现含有uORF的候选基因。通过5' RACE我们确定这38个基因中有15个被转录。这15个基因中的大多数在其5' UTR中只有一个uORF，这些uORF的长度范围为3至24个密码子。我们将七个全长UTR序列克隆到荧光素酶（LUC）报告系统中。比较了野生型UTR构建体和uORF起始密码子发生突变的构建体之间的荧光素酶活性和mRNA水平。计算每个构建体的翻译效率指数（TEI），以测试在翻译水平上可能的调控功能。我们假设RPC11、TPK1、FOL1、WSC3和MKK1的UTR中的uORF可能具有翻译调控作用，而在测试条件下，ECM7和IMD4的5' UTR中的uORF对翻译影响很小。
