Iverson Stephanie, Sonnemann Kevin, Reddick Adam, McDonough Virginia
Department of Biology, Hope College P.O. Box 9000, Holland, MI 49422-9000, USA.
Biochem Biophys Res Commun. 2006 Nov 10;350(1):91-6. doi: 10.1016/j.bbrc.2006.08.196. Epub 2006 Sep 15.
The PIS1 gene encodes a key branchpoint phospholipid biosynthetic enzyme, phosphatidylinositol synthase. The PIS1 promoter contains the unusual feature of three ATG codons (ATGs1, 2, and 3) in-frame with three stop codons, located just before the authentic start codon (ATG4). Using a PIS1(promoter)-lacZ reporter expression system and site-directed mutagenesis, we investigated the role the "upstream" ATG codons play in modulation of PIS1 expression. Of the single codon changes, mutation of the first ATG (ATG1) resulted in the largest increase of the reporter gene PIS1(promoter)-lacZ expression. All combinations of altered upstream ATG codons also resulted in greater reporter expression. Reverse transcription-PCR revealed that at least some PIS1 transcripts include all AUG codons, and their synthesis is probably directed by a second TATA box upstream of the putative TATA box. These results indicate that the multiple upstream AUG codons are present in at least some PIS1 transcripts and negatively impact PIS1 expression.
PIS1基因编码一种关键的分支点磷脂生物合成酶——磷脂酰肌醇合成酶。PIS1启动子具有一个不同寻常的特征，即在真正的起始密码子（ATG4）之前有三个与三个终止密码子符合读码框的ATG密码子（ATG1、2和3）。我们使用PIS1（启动子）-lacZ报告基因表达系统和定点诱变技术，研究了“上游”ATG密码子在调节PIS1表达中所起的作用。在单个密码子变化中，第一个ATG（ATG1）的突变导致报告基因PIS1（启动子）-lacZ表达增加最多。上游ATG密码子改变的所有组合也导致报告基因表达增加。逆转录-聚合酶链反应显示，至少一些PIS1转录本包含所有AUG密码子，并且它们的合成可能由推定的TATA框上游的第二个TATA框指导。这些结果表明，多个上游AUG密码子存在于至少一些PIS1转录本中，并对PIS1表达产生负面影响。
