Pogue G P, Koul S, Lee N S, Dwyer D M, Nakhasi H L
Division of Hematologic Products, CBER, Food and Drug Administration, Bethesda, MD 20892, USA.
Parasitol Res. 1995;81(4):282-90. doi: 10.1007/BF00931531.
Arbitrary primed polymerase chain reactions (AP-PCR) were used to amplify different polymorphic genomic DNA fragments from various Old World Leishmania species. Using four 10-mer AP primers, geographic isolates of L. donovani and various Old World species of Leishmania could be readily distinguished from one another by the pattern of amplified DNA products. Our studies confirmed two important characteristics of AP-PCR: its abilities to amplify a consistent pattern of DNA fragments from the genomes of different isolates of a single species and to identify genetic polymorphisms between the species isolates. We selected three polymorphic DNA fragments that differentiate L. donovani geographic isolates for further analysis. Sequence analysis of the clones derived from these three polymorphic fragments revealed eight unique sequences. Six of eight unique clones hybridized to distinct RNAs upon Northern-blot analysis. Three of these six clones hybridized to RNAs expressed differentially in in vitro grown L. donovani pro- and "amastigotes." One of the differentially expressed clones, LdE-6-1, exhibited restriction length polymorphisms that distinguished L. donovani from L. tropica and L. major. Comparative Northern blotting revealed that LdE-6-1 was differentially expressed in some members of the L. donovani species complex but not in L. major or L. tropica. These results demonstrate that AP-PCR can be used to generate products reflecting particular genes in organisms with low-complexity genomes.
任意引物聚合酶链反应（AP-PCR）被用于扩增来自不同旧世界利什曼原虫物种的不同多态性基因组DNA片段。使用四种10聚体AP引物，杜氏利什曼原虫的地理分离株和各种旧世界利什曼原虫物种可以通过扩增的DNA产物模式轻松区分开来。我们的研究证实了AP-PCR的两个重要特征：它能够从单个物种的不同分离株基因组中扩增出一致的DNA片段模式，以及识别物种分离株之间的遗传多态性。我们选择了三个区分杜氏利什曼原虫地理分离株的多态性DNA片段进行进一步分析。对源自这三个多态性片段的克隆进行序列分析，揭示了八个独特序列。在Northern印迹分析中，八个独特克隆中的六个与不同的RNA杂交。这六个克隆中的三个与在体外培养的杜氏利什曼原虫前鞭毛体和“无鞭毛体”中差异表达的RNA杂交。其中一个差异表达的克隆LdE-6-1表现出限制性长度多态性，可将杜氏利什曼原虫与热带利什曼原虫和硕大利什曼原虫区分开来。比较Northern印迹显示，LdE-6-1在杜氏利什曼原虫物种复合体的一些成员中差异表达，但在硕大利什曼原虫或热带利什曼原虫中不表达。这些结果表明，AP-PCR可用于在低复杂性基因组的生物体中生成反映特定基因的产物。
