A high level of mixed Trypanosoma brucei infections in tsetse flies detected by three hypervariable minisatellites
A high level of mixed Trypanosoma brucei infections in tsetse flies detected by three hypervariable minisatellites
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DOI：
10.1016/s0166-6851(99)00101-2
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发表时间：
1999-08-20
影响因子：
1.5
通讯作者：
Tait, A
中科院分区：
文献类型：
--
作者：
MacLeod, A;Turner, CMR;Tait, A
在布氏锥虫的自然种群中是否以显著的频率发生遗传交换的问题是有争议的，反对高频率的论点之一是明显缺乏锥虫基因型混合物的宿主感染。在三个基因的编码区内的三个小卫星标记（MS 42，CRAM，292）已经被鉴定，并且使用感染血液的粗裂解物作为模板开发了用于检测这些位点处的变异的基于PCR的方法。使用重复序列侧翼的引物对来自寄生虫的两个克隆原种的DNA进行的初始PCR分析显示，从每个原种扩增出两个不同大小的DNA片段。对这些片段在杂交F1后代中的遗传分析表明，不同大小的片段是以孟德尔方式分离的等位基因。三个基因座中的每一个的等位基因独立分离，与它们在三条不同染色体上的定位一致。一系列的克隆分离的采采蝇的分析表明，这些位点是高度可变的，杂合性为94%，并在17个克隆分离物的样品中鉴定出12个不同的等位基因。为了确定分离株在锥虫基因型方面是否具有异质性，在肯尼亚基博科和乌干达卢加拉分离的舌蝇的未克隆样本中检查了这三个基因座的等位基因变异。一个显着比例的分离株（36%的卢加拉和47%的Kiboko）包含两个以上的等位基因在一个或多个位点，从而表明，高比例的采采蝇感染了一个以上的基因型锥虫。这是建立，明确地，两个分离株产生一系列的克隆锥虫株系，并确定每个克隆的基因型;一个分离株（927）含有7个不同的基因型，在每个位点的等位基因的可能组合的比例很高。这些结果表明，在该领域的频繁的遗传交换的可能性，他们意味着一个显着比例的哺乳动物宿主必须包含不同的锥虫基因型的混合物，他们展示了使用小卫星标记的优势，为分析的锥虫种群结构。布鲁塞。(C)1999 Elsevier Science B. V.保留所有权利。
The issue of whether genetic exchange occurs at a significant frequency in natural populations of Trypanosoma brucei is controversial and one of the arguments against a high frequency has been the apparent lack of host infections with mixtures of trypanosome genotypes. Three minisatellite markers (MS42, CRAM, 292) within the coding regions of three genes have been identified and PCR based methods developed for detecting Variation at these loci using crude lysates of infected blood as templates. Initial PCR analysis, using primers flanking the repeats, of DNA from two cloned stocks of the parasite has shown that two DNA fragments of different size were amplified from each stock. Analysis of the inheritance of these fragments into the Fl progeny of crosses demonstrated that the different size fragments were alleles that segregated in a Mendelian manner. The alleles at each of the three loci segregated independently consistent with their localisation on three different chromosomes. Analysis of a series of cloned isolates from tsetse dies showed that these loci were highly variable giving heterozygosities of 94% and the identification of 12 distinct alleles in a sample of 17 cloned isolates. In order to determine whether isolates are heterogeneous in terms of trypanosome genotype, the allelic Variation at these three loci was examined in uncloned samples from tsetse flies isolated in Kiboko, Kenya and Lugala, Uganda. A significant proportion of the isolates (36% in Lugala and 47% in Kiboko) contained more than two alleles at one or more of the loci thus demonstrating that a high proportion of tsetse flies were infected with more than one genotype of trypanosomes. This was established, unequivocally, for two isolates by generating a series of cloned trypanosome lines from each and determining the genotype of each clone; one isolate (927) contained seven different genotypes with a high proportion of the possible combinations of alleles at each locus. These results indicate the possibility of frequent genetic exchange in the field, they imply that a significant proportion of mammalian hosts must contain mixtures of different trypanosome genotypes and they demonstrate the advantages of using minisatellite markers for the analysis of the population structure of T. brucei. (C) 1999 Elsevier Science B.V. All rights reserved.