Graham S V, Wymer B, Barry J D
Wellcome Unit of Molecular Parasitology, The Anderson College, University of Glasgow, Scotland, United Kingdom.
Mol Cell Biol. 1998 Mar;18(3):1137-46. doi: 10.1128/MCB.18.3.1137.
African trypanosomes evade the mammalian host immune response by antigenic variation, the continual switching of their variant surface glycoprotein (VSG) coat. VSG is first expressed at the metacyclic stage in the tsetse fly as a preadaptation to life in the mammalian bloodstream. In the metacyclic stage, a specific subset (<28; 1 to 2%) of VSG genes, located at the telomeres of the largest trypanosome chromosomes, are activated by a system very different from that used for bloodstream VSG genes. Previously we showed that a metacyclic VSG (M-VSG) gene promoter was subject to life cycle stage-specific control of transcription initiation, a situation unique in Kinetoplastida, where all other genes are regulated, at least partly, posttranscriptionally (S. V. Graham and J. D. Barry, Mol. Cell. Biol. 15:5945-5956, 1985). However, while nuclear run-on analysis had shown that the ILTat 1.22 M-VSG gene promoter was transcriptionally silent in bloodstream trypanosomes, it was highly active when tested in bloodstream-form transient transfection. Reasoning that chromosomal context may contribute to repression of M-VSG gene expression, here we have integrated the 1.22 promoter, linked to a chloramphenicol acetyltransferase (CAT) reporter gene, back into its endogenous telomere or into a chromosomal internal position, the nontranscribed spacer region of ribosomal DNA, in both bloodstream and procyclic trypanosomes. Northern blot analysis and CAT activity assays show that in the bloodstream, the promoter is transcriptionally inactive at the telomere but highly active at the chromosome-internal position. In contrast, it is inactive in both locations in procyclic trypanosomes. Both promoter sequence and chromosomal location are implicated in life cycle stage-specific transcriptional regulation of M-VSG gene expression.
非洲锥虫通过抗原变异来逃避哺乳动物宿主的免疫反应,即不断更换其可变表面糖蛋白(VSG)外衣。VSG最初在采采蝇的循环后期阶段表达,作为对哺乳动物血液中生活的一种预适应。在循环后期阶段,位于最大锥虫染色体端粒的特定VSG基因子集(<28个;占1%至2%),由一个与用于血液中VSG基因的系统截然不同的系统激活。此前我们表明,一个循环后期VSG(M-VSG)基因启动子受到转录起始的生命周期阶段特异性控制,这在动质体目中是独特的,在动质体目中所有其他基因至少部分是在转录后受到调控(S.V.格雷厄姆和J.D.巴里,《分子细胞生物学》15:5945 - 5956,1985年)。然而,虽然核转录分析表明ILTat 1.22 M-VSG基因启动子在血液中的锥虫中转录沉默,但在血液形式的瞬时转染实验中进行测试时它却高度活跃。由于推断染色体背景可能有助于抑制M-VSG基因表达,在此我们将与氯霉素乙酰转移酶(CAT)报告基因相连的1.22启动子整合回其内源端粒或染色体内部位置,即核糖体DNA的非转录间隔区,在血液和前循环锥虫中均如此。Northern印迹分析和CAT活性测定表明,在血液中,启动子在端粒处转录无活性,但在染色体内部位置高度活跃。相反,它在前循环锥虫的这两个位置均无活性。启动子序列和染色体位置都与M-VSG基因表达的生命周期阶段特异性转录调控有关。