Translation of an atypical human cDNA requires fidelity of apurine-pyrimidine repeat region and recoding.
Translation of an atypical human cDNA requires fidelity of apurine-pyrimidine repeat region and recoding.
复制标题
非典型人类 cDNA 的翻译需要保真度的脱嘌呤-嘧啶重复区域和重新编码。
DOI：
10.1016/j.gene.2008.02.006
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发表时间：
2008
期刊：
影响因子：
3.5
通讯作者：
Lindsey,JSuzanne
中科院分区：
文献类型：
--
作者：
Petty,AaronP;Dick,CharlesL;Lindsey,JSuzanne
迁移诱导基因-7（Migration inducing gene-7，Mig-7）蛋白表达的获得或丧失功能研究表明其引起侵袭性肿瘤细胞侵袭和肿瘤细胞血管样结构的形成。此外，Mig-7的表达显然是癌和滋养层细胞特异性的。Mig-7是一个非典型基因的例子，它在诱导、翻译和明显的癌特异性表达方面是独特的。然而，这种主要的整合膜蛋白的研究受到阻碍，因为需要检测Mig-7蛋白的克隆和表达技术。由于编码区具有终止密码子、重复序列和二级结构，我们推测基因工程E。当生产含有Mig-7序列的表达质粒时，需要大肠杆菌来维持嘌呤-嘧啶重复序列和阅读框的数目。用E.大肠杆菌基因工程缺乏重组和重排能力，阻止了重复区的延伸。由于序列中存在多个终止密码子，因此测试了从三个不同阅读框ATG位点开始的三种不同构建体在人癌细胞系中的蛋白质产生。约23 kD的Mig-7蛋白由Mig-7 cDNA产生，其在Kozak共有序列中的ATG下游含有多个终止密码子。计算机模拟分析表明，多个Mig-7 mRNA二级结构可能会导致多个终止密码子的移码、通读和/或重新编码。实验结果支持这些翻译事件中的一个或多个发生。在这份报告中，我们详细的要求克隆和表达这种新的，非典型的，人类基因。这些技术可用于表达这种独特的蛋白质，以供进一步研究。
Gain or loss of Migration inducting gene-7 (Mig-7) protein expression functional studies suggest it causes aggressive tumor cell invasion and tumor cell vessel-like structure formation. In addition, Mig-7 expression is apparently carcinoma and trophoblast cell-specific. Mig-7 is an example of an atypical gene that is unique in its induction, translation and apparent carcinoma-specific expression. However, studies of this predominantly integral membrane protein are hampered because of the cloning and expression techniques required for detection of Mig-7 protein. Because the encoding region possesses stop codons, repeat sequences and secondary structure, we hypothesized that genetically engineered E. coli are required to maintain the number of purine-pyrimidine repeats and reading frame when producing expression plasmids containing the Mig-7 sequence. Cloning Mig-7 sequence using E. coli genetically engineered to lack recombination and rearrangement capabilities prevented extension of the repeat region. Because of multiple stop codons in the sequence, three different constructs starting from three different reading frame ATG sites were tested for protein production in a human carcinoma cell line. Mig-7 protein of ~23 kD is produced from Mig-7 cDNA that contains multiple stop codons downstream from the ATG in a Kozak consensus sequence. In silico analyses imply that multiple Mig-7 mRNA secondary structures may cause frameshifting, read-through, and/or recoding of the multiple stop codons. Experimental results support that one or more of these translational events take place. In this report, we detail requirements for cloning and expression of this novel, atypical, human gene. These techniques can be used to express this unique protein for further studies.