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.
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.
复制标题
抗衣霉素利什曼原虫的 63 KB 环状扩增子含有功能性 N-乙酰氨基葡萄糖-1-磷酸转移酶基因，可用作转染中的显性选择标记。
DOI：
10.1128/mcb.12.9.4112-4122.1992
复制
发表时间：
1992
影响因子：
5.3
通讯作者：
Chang,KP
中科院分区：
文献类型：
--
作者：
Liu,X;Chang,KP
先前已发现抗衣霉素 (TM) 亚马逊利什曼原虫含有扩增的染色体 DNA，仅以 63 kb 的染色体外环形式存在。通过转染野生型细胞对克隆到质粒中的 DNA 片段进行功能分析。最初发现具有 63 kb 环的 15 kb 片段的克隆具有 TM 抗性。然后制备 15 kb 片段的文库并用于全部转染野生型细胞。发现选择后出现的转染子含有带有4.6 kb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-乙酰葡萄糖-胺-1-磷酸转移酶基因的 1.4-kb 同源物。 TheL。 amazonensisgene 与其他两个来源的基因在推导的肽序列和亲水特性方面相似 65% 至 70%。 L.amazonensis 基因比野生型扩增超过 128 倍，并在所有转染子中过量产生 2.4 kb 的主要转录物。该基因的内源拷贝通过聚合酶链式反应从野生型中扩增并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，同时使它们对 TM 产生抗性。因此提供的证据表明1.4-kb DNA是N-乙酰葡糖胺-1-磷酸转移酶基因，其扩增导致利什曼原虫变体和转染子中的TM抗性。
Tunicamycin (TM)-resistantLeishmania amazonensishas 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 a 1.4-kb homolog ofN-acetylglucos-amine-1-phosphate transferase genes. TheL. amazonensisgene is similar to those from two other sources in their deduced peptide sequence by 65 to 70% and in hydropathic characteristics. TheL.amazonensisgene 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, aLeishmaniaexpression 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 anN-acetylglucosamine-1-phosphate transferase gene whose amplification is responsible for TM resistance inLeishmaniavariants and transfectants.