On the triggers in antigenic variation of African trypanosomes

需要建立测量变化频率的方法，以了解什么是诱导VSG表达转换的最有效方法。为此，我们尝试通过对锥虫B的RT-PCR检测，通过表达异型VSG的有机体与表达同型VSG的有机体的群体比例来确定变异体出现的准确率。冈比亚的。因此，有必要对部分VSG基因的DNA序列进行进一步的研究。初步克隆了三个最具优势的VSG，KuTat 1.1、KuTat 1.2和KuTat 1.3。然而，由于在任何移码中终止密码子的异常出现，很难推断这些VSG基因的核苷酸序列。测定了KuTat 1.1 VSG基因的347个核苷酸序列，包括最小外显子和3 '末端序列。在锥虫mRNA的特征性结构元件mini-exon后设计了18个核苷酸的引物，确定的序列分别为5'和3'引物。在各种条件下，对这些引物进行RT-PCR，并且意外地注意到KuTat 1.2以及KuTat 1.1的PCR产物。当比较产生阳性PCR的阈值时，与异源锥虫同源结合的锥虫的极限数量为1：1000。通过数据库的同源性分析，不同的VSG之间有较高的同源性。考虑到这些事实，我们不得不重新考虑如何实现改进的方法来确定少数VSG的比例，因为他们的比例报告不超过10^<-4>。

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>.