Navarro M, Cross G A
Laboratory of Molecular Parasitology, The Rockefeller University, New York, New York 10021-6399, USA.
Mol Cell Biol. 1996 Jul;16(7):3615-25. doi: 10.1128/MCB.16.7.3615.
Changes in variant surface glycoprotein (Vsg) expression allow Trypanosoma brucei to elude the immune response. The expressed vsg is always located at the telomeric end of a polycistronic transcription unit known as an expression site (ES). Although there are many ESs, only one is active at any particular time. The mechanisms regulating ES transcription and switching are unknown. Chromosome rearrangements within or upstream of the ES have been described to occur in occasional switch events, but no changes have been consistently associated with switching. We inserted the drug resistance genes neo and ble, conferring resistance to G418 and phleomycin, respectively, 1 kb downstream of "silent" ES promoters. This demonstrated that short-range transcription could be achieved from a silent ES promoter. From one initial transformant clone, panels of independent consecutive on-off-on switch clones were generated and analyzed. The first activation of the neo-targeted ES was always associated with deletion of the upstream tandem promoter in this ES, but no further rearrangements were detected in consecutive off-on switches of this ES. On the other hand, direct analysis of ES promoters showed that deletions and duplications occurred elsewhere. Activation of a ble-tagged 300-kb chromosome could not be achieved, but phleomycin-resistant clones could be obtained. One such clone arose from recombination between three ESs. Taken together, our experiments suggest that ES switching may occur after a period of chromosomal interactivity that may or may not leave tangible evidence in the form of detectable sequence changes.
变异表面糖蛋白（Vsg）表达的变化使布氏锥虫能够逃避免疫反应。表达的vsg总是位于称为表达位点（ES）的多顺反子转录单元的端粒末端。尽管有许多ES，但在任何特定时间只有一个是活跃的。调节ES转录和转换的机制尚不清楚。已描述在偶尔的转换事件中ES内部或上游会发生染色体重排，但没有变化与转换始终相关。我们在“沉默”ES启动子下游1 kb处插入了分别赋予对G418和博来霉素抗性的耐药基因neo和ble。这表明可以从沉默的ES启动子实现短程转录。从一个初始转化体克隆中，产生并分析了一系列独立的连续开-关-开转换克隆。neo靶向ES的首次激活总是与该ES中上游串联启动子的缺失相关，但在该ES的连续关-开转换中未检测到进一步的重排。另一方面，对ES启动子的直接分析表明，缺失和重复发生在其他地方。无法实现带有ble标签的300 kb染色体的激活，但可以获得博来霉素抗性克隆。一个这样的克隆来自三个ES之间的重组。综上所述，我们的实验表明，ES转换可能在一段时间的染色体相互作用之后发生，这种相互作用可能会或可能不会以可检测的序列变化形式留下确凿的证据。
