Zomerdijk J C, Ouellette M, ten Asbroek A L, Kieft R, Bommer A M, Clayton C E, Borst P
Division of Molecular Biology, Netherlands Cancer Institute, Amsterdam.
EMBO J. 1990 Sep;9(9):2791-801. doi: 10.1002/j.1460-2075.1990.tb07467.x.
The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5' ends of the major transcripts coming from the initiation region map at nucleotide sequences that do not strongly resemble rRNA transcriptional starts even though the transcripts are synthesized by an RNA polymerase highly resistant to alpha-amanitin. The cloned VSG gene 221 ES transcription initiation region promotes high CAT gene expression, when reintroduced by electroporation into T. brucei. We show that the activity of this expression site is controlled at or near transcription initiation in bloodstream trypanosomes. The 221 ES is inactivated without any sequence alteration within 1.4 kb of the transcription start site. This excludes mechanisms of promoter inactivation involving DNA rearrangements in the vicinity of the transcription start site, e.g. promoter inversion or conversion.
布氏锥虫的变异特异性表面糖蛋白（VSG）基因221作为一个60 kb表达位点（ES）的一部分进行转录。我们通过使用富含221号染色体的DNA文库和VSG基因221表达位点特异性转录本，鉴定了控制这个多基因转录单元的启动子。转录起始点通过对新生RNA的杂交和RNase保护分析来确定。来自起始区域的主要转录本的5'端定位在核苷酸序列上，这些序列与rRNA转录起始点不太相似，尽管这些转录本是由对α-鹅膏蕈碱高度抗性的RNA聚合酶合成的。当通过电穿孔重新导入布氏锥虫时，克隆的VSG基因221 ES转录起始区域促进了高CAT基因表达。我们表明，在血流型锥虫中，这个表达位点的活性在转录起始时或附近受到控制。221 ES在转录起始位点1.4 kb范围内没有任何序列改变的情况下被灭活。这排除了涉及转录起始位点附近DNA重排的启动子失活机制，例如启动子倒位或转换。
