On the triggers in antigenic variation of African trypanosomes

点击翻译按钮获取中文摘要

It is required to establish the method to measure variation frequency to know what is the most effective to induce switching of VSG expression. For the purpose, we tried to determine precise rate of variant appearance by the population ratio of oraganisms expressing heterotype VSG to homotype applying RT-PCR for Trypanosoma b. gambiense. In advance it is necessary to clarify DNA sequence of some VSG genes. Three of the most dominant VSGs, KuTat 1.1, KuTat 1.2 and KuTat 1.3 had benn preliminarily cloned. It was, though, difficult to conclude the nucleotide sequences of those VSG genes for the unusual appearance of termination codons in any frame shift. Including mini-exon and following 3'terminal sequence, 347 nuculeotide sequence was determined for KuTat 1.1 VSG gene. Primers of 18 nuculeotides were designed after mini-exon, which is characteristic construct element of trypanosome mRNA, and the determined sequences as 5' and 3' primer respectively. Under various conditions RT-PCR was run on those primers and PCR products were unexpcctedly noticed for KuTat 1.2 as well as KuTat 1.1. When the threashold to give positive PCR was compared, limiting number of trypanosomes in homologous combination to hetrologous was 1 : 1000. The homology analysis through the data base gave rather high sequence homology between different VSGs. Considering those facts, we have to rethink to realize the improved way to determine the ratio of the minority with VSG because their ratios reported are not more than 10^<-4>.