Downey N, Donelson J E
Department of Molecular Biology Ph.D. Program, University of Iowa, Iowa City 52242, USA.
Mol Biochem Parasitol. 1999 Oct 25;104(1):25-38. doi: 10.1016/s0166-6851(99)00135-8.
A search was conducted for transcriptional promoters in Trypanosoma congolense. A promoter test plasmid was constructed utilising the luciferase coding region flanked by the intergenic regions of a T. congolense gene encoding GARP, the glutamic acid and alanine rich protein on the surface of procyclic organisms. Using this plasmid, sequences located upstream of an 18S rRNA gene were tested in transient transfection assays for their ability to promote luciferase expression. A rRNA promoter fragment of 377 bp was identified that increases luciferase activity by as much as 35,000-fold above background levels. The rRNA transcription initiation site is located 961 bp upstream of the 18S rRNA gene and immediately downstream of 6 bp imperfect repeats. The plasmid was also used to examine sequences upstream of a GARP gene cluster in two different T. congolense strains for promoter activity. In contrast to the findings of another group, we were unable to detect promoter activity upstream of these GARP genes in either strain. We conclude that the GARP gene promoter, if it exists, has less than 0.03% (1/3000) of the activity of the rRNA promoter in this luciferase-based assay.
对刚果锥虫的转录启动子进行了搜索。构建了一个启动子测试质粒，该质粒利用荧光素酶编码区，其两侧是编码GARP（前循环生物体表面富含谷氨酸和丙氨酸的蛋白质）的刚果锥虫基因的基因间区域。使用该质粒，在瞬时转染实验中测试了位于18S rRNA基因上游的序列促进荧光素酶表达的能力。鉴定出一个377 bp的rRNA启动子片段，其可使荧光素酶活性比背景水平提高多达35000倍。rRNA转录起始位点位于18S rRNA基因上游961 bp处，紧接在6 bp不完全重复序列的下游。该质粒还用于检测两种不同刚果锥虫菌株中GARP基因簇上游序列的启动子活性。与另一组的发现相反，我们在这两种菌株中均未检测到这些GARP基因上游的启动子活性。我们得出结论，在这种基于荧光素酶的检测中，GARP基因启动子（如果存在）的活性不到rRNA启动子活性的0.03%（1/3000）。
