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首页> 外文期刊>Molecular and Cellular Biology >The 63-kilobase circular amplicon of tunicamycin-resistant Leishmania amazonensis contains a functional N-acetylglucosamine-1-phosphate transferase gene that can be used as a dominant selectable marker in transfection.
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The 63-kilobase circular amplicon of tunicamycin-resistant Leishmania amazonensis contains a functional N-acetylglucosamine-1-phosphate transferase gene that can be used as a dominant selectable marker in transfection.

机译：耐衣霉素的亚马逊利什曼原虫的63碱基对圆形扩增子包含一个功能性N-乙酰氨基葡萄糖-1-磷酸转移酶基因，可以用作转染中的主要选择标记。

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摘要

Tunicamycin (TM)-resistant Leishmania amazonensis has been found previously to contain amplified chromosomal DNA, existing exclusively as extrachromosomal circles of 63 kb. Fragments of this DNA cloned into plasmids were functionally analyzed by transfection of wild-type cells. A clone with a 15-kb fragment of the 63-kb circle was initially found to confer TM resistance. A library of the 15-kb fragment was then prepared and used in toto to transfect wild-type cells. The transfectants that emerged after selection were found to contain a plasmid with an insert of 4.6 kb. Evidence from deletion experiments suggests that this is the minimal transfection-effective fragment. Sequencing of the 4.6-kb DNA revealed 1.4-kb homolog of N-acetylglucosamine-1-phosphate transferase genes. The L. amazonensis gene is similar to those from two other sources in their deduced peptide sequence by 65 to 70% and in hydropathic characteristics. The L. amazonensis gene is amplified by more than 128-fold over the wild type and overproduces a major transcript of 2.4 kb in all transfectants. The endogenous copy of this gene was amplified by polymerase chain reaction from the wild type and cloned into pX-NEO, a Leishmania expression vector. Amplification of this plasmid in the transfectants by selection with G418 simultaneously made them resistant to TM. Evidence provided thus indicates that the 1.4-kb DNA is an N-acetylglucosamine-1-phosphate transferase gene whose amplification is responsible for TM resistance in Leishmania variants and transfectants.
机译：先前已发现耐抗衣霉素的亚马逊利什曼原虫（Leishmania amazonensis）含有扩增的染色体DNA，仅以63 kb的染色体外环存在。通过转染野生型细胞对克隆到质粒中的该DNA片段进行功能分析。最初发现具有63kb环的15kb片段的克隆具有TM抗性。然后制备15kb片段的文库，并用于转染野生型细胞。发现在选择后出现的转染子包含具有4.6kb插入片段的质粒。缺失实验的证据表明，这是最小的转染有效片段。 4.6-kb DNA的测序揭示了N-乙酰氨基葡糖-1-磷酸转移酶基因的1.4-kb同源物。亚马逊乳杆菌基因与其他两个来源的基因相似，其推导的肽序列具有65％至70％的亲水性。亚马逊乳杆菌基因比野生型扩增了128倍以上，并在所有转染子中过量产生了2.4 kb的主要转录本。通过聚合酶链反应从野生型扩增该基因的内源拷贝，并将其克隆到利什曼原虫表达载体pX-NEO中。通过用G418选择在转染子中扩增该质粒，同时使其对TM具有抗性。因此，提供的证据表明1.4kb DNA是N-乙酰氨基葡糖-1-磷酸转移酶基因，其扩增负责利什曼原虫变体和转染子中的TM抗性。

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