Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major.
Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major.
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塔伦托利什曼原虫实验室库存中的扩增 DNA：染色体外环在结构和功能上类似于甲氨蝶呤耐药利什曼原虫重大的倒置 H 区扩增。
DOI：
10.1128/mcb.8.12.5188-5199.1988
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发表时间：
1988
影响因子：
5.3
通讯作者：
Beverley,SM
中科院分区：
文献类型：
--
作者：
Petrillo-Peixoto,ML;Beverley,SM
我们描述了扩增的DNA的结构，发现在两个实验室股票的原生动物寄生虫利什曼原虫tarentolae。限制性酶切图谱和分子克隆显示，42个酶的区域在这些品系中扩增了8- 30倍。对消化的DNA或脉冲场电泳分离的染色体进行Southern印迹分析表明，扩增的DNA对应于H区，这是一个最初在抗甲氨蝶呤的硕大利什曼原虫（S. M.贝弗利，J. A. Coderre，D. V. Santi和R. T. Schimke，Cell 38：431-439，1984）。这两个种的DNA扩增产物之间的相似性包括：（i）广泛的交叉杂交;（ii）序列顺序的近似保守性;（iii）染色体外定位;（iv）作为环状140-组氨酸酶四聚体分子的整体倒置的头对头构型;（v）DNA序列重排的两个区域，每个区域都与反向重复的两个中心密切相关;（v）DNA序列重排的两个区域与反向重复的两个中心密切相关。（vi）与甲氨蝶呤抗性相关;和（vii）表型保守扩增，其中野生型染色体排列被保留而没有明显的修饰。我们的数据表明，扩增的DNA介导的耐药性出现在unselectedL。tarentolae，虽然压力导致显然自发扩增和维护的H区是未知的。这些和其他利什曼原虫DNA的简单结构和有限的扩增范围表明，利什曼原虫基因扩增的研究。为研究培养的哺乳动物细胞和肿瘤中的扩增提供了一个有吸引力的模型系统。我们还介绍了一种测量大的环状DNA的大小的方法，使用γ射线照射引入有限的双链断裂，然后通过脉冲场电泳对线性DNA进行大小测定。
We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasiteLeishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistantLeishmania major(S. M. Beverley, J. A. Coderre, D. V. Santi, and R. T. Schimke, Cell 38:431-439, 1984). Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-head configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselectedL. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and otherLeishmanialines suggests that the study of gene amplification inLeishmaniaspp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.