Cupolillo E, Grimaldi Júnior G, Momen H, Beverley S M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Mol Biochem Parasitol. 1995 Jul;73(1-2):145-55. doi: 10.1016/0166-6851(95)00108-d.
In the New World, Leishmania of the Viannia subgenus cause both cutaneous and mucocutaneous disease. These parasites show considerable intra-species genetic diversity and variation, which complicates taxonomic classification and epidemiology. We have used the variability of the transcribed noncoding regions between the small and large subunit rRNA genes to examine relationships in this group. In a method termed intergenic region typing (IRT), PCR amplification products were obtained for the rapidly evolving 1-1.2-kb internal transcribed spacers (ITS) between the SSU and LSU rRNAs, from 50 parasites isolated from different hosts and geographic areas. Amplified DNAs were digested with 10 different enzymes, and fragment patterns compared after acrylamide gel electrophoresis. High levels of intra- and inter-specific variation were observed, and quantitative similarity comparisons were used to associate different lineages. A complex evolutionary tree was obtained. Some species formed tight clusters (L. equatorensis, L. panamensis, L. guyanensis, L. shawi), while L. braziliensis was highly polymorphic and L. naiffi showed intraspecific distances comparable to the largest obtained within all Viannia. L. colombiensis, L. equatorensis and L. lainsoni clearly represent distinct lineages. Good agreement was obtained with molecular trees based upon isoenzyme or mini-exon repeat sequence comparisons. Overall, IRT appears to be a superb method for epidemiological and taxonomic studies of Leishmania, being sensitive, rapid and quantitative while simultaneously revealing considerable molecular diversity. IRT could also be applied to other nonconserved intergenic regions, including those separating protein-coding genes.
在新大陆，维安亚亚属的利什曼原虫可引发皮肤和黏膜皮肤疾病。这些寄生虫表现出相当大的种内遗传多样性和变异，这使得分类学分类和流行病学变得复杂。我们利用小亚基和大亚基rRNA基因之间转录非编码区的变异性来研究该类群中的关系。在一种称为基因间区域分型（IRT）的方法中，从50种分离自不同宿主和地理区域的寄生虫中，获得了小亚基和大亚基rRNA之间快速进化的1 - 1.2 kb内部转录间隔区（ITS）的PCR扩增产物。扩增的DNA用10种不同的酶进行消化，并在丙烯酰胺凝胶电泳后比较片段模式。观察到种内和种间的高度变异，并使用定量相似性比较来关联不同的谱系。得到了一棵复杂的进化树。一些物种形成紧密的聚类（赤道利什曼原虫、巴拿马利什曼原虫、圭亚那利什曼原虫、肖氏利什曼原虫），而巴西利什曼原虫具有高度多态性，奈菲利什曼原虫的种内距离与所有维安亚中获得的最大距离相当。哥伦比亚利什曼原虫、赤道利什曼原虫和莱因索尼利什曼原虫明显代表不同的谱系。与基于同工酶或小外显子重复序列比较的分子树取得了良好的一致性。总体而言，IRT似乎是一种用于利什曼原虫流行病学和分类学研究的极佳方法，它灵敏、快速且定量，同时揭示了相当大的分子多样性。IRT也可应用于其他非保守的基因间区域，包括那些分隔蛋白质编码基因的区域。
