Katakura K, Peng Y, Pithawalla R, Detke S, Chang K P
Department of Microbiology and Immunology, University of Health Sciences, Chicago Medical School, IL 60064.
Mol Biochem Parasitol. 1991 Feb;44(2):233-43. doi: 10.1016/0166-6851(91)90009-u.
Twelve independent variants were selected from five species of Leishmania for resistance to tunicamycin by exposure of cultured promastigotes to increasing concentrations of this antibiotic, an inhibitor of the microsomal N-acetylglucosamine-1-phosphate transferase in the dolichol pathway of N-glycosylation. All variants obtained from all species, as found previously with Leishmania amazonensis, contain amplified chromosomal DNA exclusively as extrachromosomal circles. These circular amplicons hybridize with amplified DNAs cloned previously from tunicamycin-resistant Leishmania amazonensis, but not with those from Leishmania resistant to other drugs. The amplicons from tunicamycin-resistant cells vary with different species in size from 30 to 70 kb, but all share a homologous region of 20 kb. Multiple independent transcripts are overexpressed from this region. Elevation of the microsomal glycosyltransferase activity is demonstrated in these variants from representative species. The results thus provide further evidence that this enzyme is overexpressed due to amplification of the gene in these cells. The consistent observation of this event in all cases studied also suggests that this is the predominant, if not the only mechanism of tunicamycin resistance in Leishmania.
从五种利什曼原虫中挑选出12个独立变体，通过将培养的前鞭毛体暴露于浓度不断增加的衣霉素（一种N-糖基化多萜醇途径中微粒体N-乙酰葡糖胺-1-磷酸转移酶的抑制剂）中来获得对衣霉素的抗性。如先前在亚马逊利什曼原虫中发现的那样，从所有物种获得的所有变体均仅包含扩增的染色体DNA作为染色体外环状DNA。这些环状扩增子与先前从抗衣霉素的亚马逊利什曼原虫中克隆的扩增DNA杂交，但不与抗其他药物的利什曼原虫的扩增DNA杂交。抗衣霉素细胞的扩增子在不同物种中的大小从30到70 kb不等，但都共享一个20 kb的同源区域。该区域有多个独立转录本过度表达。在这些来自代表性物种的变体中证实了微粒体糖基转移酶活性的升高。因此，结果提供了进一步的证据表明该酶在这些细胞中由于基因扩增而过度表达。在所有研究的案例中对该事件的一致观察也表明，这是利什曼原虫中抗衣霉素的主要机制，即便不是唯一机制。
