Grondin K, Kündig C, Roy G, Ouellette M
Centre de Recherche en Infectiologie du CHUL and Département de Biologie Médicale, Division de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada.
Nucleic Acids Res. 1998 Jul 15;26(14):3372-8. doi: 10.1093/nar/26.14.3372.
Gene amplification is frequently observed in Leishmania cells selected for drug resistance. By gene targeting we have tagged both alleles of the H locus of Leishmania tarentolae with the neomycin and hygromycin phosphotransferase genes ( neo and hyg ). Selection of these recombinant parasites for low level methotrexate resistance led to amplification of the H locus as part of linear amplicons. The availability of tags has permitted us to determine that both alleles can be amplified in the same cell and that chromosomal deletions are frequent. When methotrexate concentration was increased in subsequent selection steps, circles were observed in several mutants. We have introduced a hyg marker into linear amplicons to test whether the circles originated from linear amplicons. After selection with a high methotrexate concentration, circles with the hyg marker were observed, showing that circles can indeed be formed from linear amplicons. The tagging of H locus alleles permits appreciation of the extent of genetic rearrangements leading to amplicon formation in Leishmania cells selected for drug resistance.
在为耐药性而选择的利什曼原虫细胞中经常观察到基因扩增。通过基因靶向,我们用新霉素和潮霉素磷酸转移酶基因(neo和hyg)标记了大利什曼原虫H位点的两个等位基因。选择这些对低水平甲氨蝶呤具有抗性的重组寄生虫导致H位点作为线性扩增子的一部分发生扩增。标记的可用性使我们能够确定两个等位基因可以在同一细胞中扩增,并且染色体缺失很常见。在随后的选择步骤中增加甲氨蝶呤浓度时,在几个突变体中观察到了环状结构。我们将一个潮霉素标记引入线性扩增子中,以测试这些环状结构是否源自线性扩增子。在用高浓度甲氨蝶呤进行选择后,观察到带有潮霉素标记的环状结构,表明环状结构确实可以由线性扩增子形成。H位点等位基因的标记有助于了解在为耐药性而选择的利什曼原虫细胞中导致扩增子形成的基因重排程度。