Wilson K, Uyetake L, Boothroyd J
Department of Microbiology and Immunology, Stanford University, Stanford, California, 94305, USA.
Exp Parasitol. 1999 Mar;91(3):222-30. doi: 10.1006/expr.1998.4366.
The procyclic acidic repetitive protein (PARP or procyclin) of the parasitic protozoan Trypanosoma brucei is a developmentally regulated protein that shows extreme differences in its level of expression in different stages of the parasite's life cycle. Specifically, it is the major surface protein in the procyclic (insect) stage and, although the PARP gene is being actively transcribed in the mammalian bloodstream stage, there is no detectable PARP mRNA or protein in these cells. The 3'-untranslated region (UTR) of PARP, as well as other trypanosome genes, has the ability to confer the appropriate developmental regulation pattern onto chimeric reporter genes. To understand the mechanism of posttranscriptional regulation, selective replacement mutagenesis of the PARP mRNA 3'UTR was done to identify the cis-acting sequences involved in the down-regulation of this mRNA in bloodstream-form T. brucei. Transient transformation of constructs containing the PARP promoter and 5'UTR, the beta-glucuronidase coding region, and the selectively mutagenized or unaltered PARP 3'UTR were performed in procyclic and bloodstream T. brucei. The results of the reporter gene assays on the transformed cells indicate that there are at least two elements in the PARP 3'UTR which in bloodstream cells are involved in regulation of PARP expression and which appear to function as negative elements. In procyclic cells, there are two regions in which mutagenesis indicates positive cis-regulatory sequences, one of which has been previously defined (A. Hehl et al., 1994, Proc. Natl. Acad. Sci. USA 91, 370-374). These results indicate that multiple cis-acting elements within the PARP 3'UTR are involved in the developmental regulation of PARP expression and that regulation is controlled in a complex manner, presumably involving several cellular trans-acting factors.
寄生原生动物布氏锥虫的前循环酸性重复蛋白（PARP或前环蛋白）是一种受发育调控的蛋白，在寄生虫生命周期的不同阶段，其表达水平存在极大差异。具体而言，它是前循环（昆虫）阶段的主要表面蛋白，尽管PARP基因在哺乳动物血液阶段处于活跃转录状态，但在这些细胞中却检测不到PARP mRNA或蛋白。PARP的3'非翻译区（UTR）以及其他锥虫基因，能够将适当的发育调控模式赋予嵌合报告基因。为了解转录后调控机制，对PARP mRNA 3'UTR进行了选择性置换诱变，以鉴定参与布氏锥虫血液形式中该mRNA下调的顺式作用序列。在循环前和血液中的布氏锥虫中，对含有PARP启动子和5'UTR、β-葡萄糖醛酸酶编码区以及选择性诱变或未改变的PARP 3'UTR的构建体进行了瞬时转化。对转化细胞进行的报告基因检测结果表明，PARP 3'UTR中至少有两个元件在血液细胞中参与PARP表达的调控，且似乎起着负性元件的作用。在循环前细胞中，有两个区域诱变显示存在正性顺式调控序列，其中一个区域先前已被确定（A. Hehl等人，1994年，美国国家科学院院刊91，370 - 374）。这些结果表明，PARP 3'UTR内的多个顺式作用元件参与了PARP表达的发育调控，且调控是以复杂的方式进行的，推测涉及几种细胞反式作用因子。
