Bakalara N, Kendall G, Michels P A, Opperdoes F R
International Institute of Cellular and Molecular Pathology, Research Unit for Tropical Diseases ICP/TROP 74.9, Brussels, Belgium.
EMBO J. 1991 Dec;10(12):3861-8. doi: 10.1002/j.1460-2075.1991.tb04955.x.
Regions 5' of the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene from Trypanosoma brucei were tested for their ability to promote chloramphenicol acetyl-transferase (CAT) expression on reintroduction by electroporation into the parasite. Deletion analysis mapped the gGAPDH promoter to within 403 nts of the start of translation. A transcription initiation site was mapped at around -190 nts from the ATG start codon by RNase protection and by primer extension. The higher expression of gGAPDH in bloodstream T. brucei, compared to procyclic (insect) forms, was largely attributed to differences in promoter activity. The gGAPDH promoter gave rise to relatively high CAT signals upon transfection into bloodstream T. brucei and relatively low signals in procyclic T. brucei, compared with levels resulting from transfection with the procyclic acidic repetitive protein (PARP) promoter. In addition, RNase protection data showed a higher level of gGAPDH primary transcripts in bloodstream. T. brucei. The PARP mini-exon addition region abolished transient CAT expression directed by either the gGAPDH or PARP promoters in bloodstream T. brucei implying that transplicing can be a point of stage-specific gene regulation.
对布氏锥虫糖体甘油醛-3-磷酸脱氢酶(gGAPDH)基因5'端区域进行了检测,以评估其在通过电穿孔重新导入寄生虫后促进氯霉素乙酰转移酶(CAT)表达的能力。缺失分析将gGAPDH启动子定位在翻译起始点的403个核苷酸范围内。通过核糖核酸酶保护和引物延伸,将转录起始位点定位在距ATG起始密码子约-190个核苷酸处。与前循环(昆虫)形式相比,gGAPDH在血流型布氏锥虫中的高表达主要归因于启动子活性的差异。与用前循环酸性重复蛋白(PARP)启动子转染所产生的水平相比,gGAPDH启动子在转染到血流型布氏锥虫后产生相对较高的CAT信号,而在原循环型布氏锥虫中产生相对较低的信号。此外,核糖核酸酶保护数据显示,血流型布氏锥虫中gGAPDH初级转录本水平较高。PARP小外显子添加区域消除了gGAPDH或PARP启动子在血流型布氏锥虫中指导的瞬时CAT表达,这意味着剪接可能是阶段特异性基因调控的一个点。