通过限制性消化聚合酶链反应克隆具有聚（A）尾的大肠杆菌信使核糖核酸
[Cloning E. coli mRNAs with poly (A) tails by restriction digest polymerase chain reaction].
作者信息
Hu Zi-You, Ma Wen-Li, Song Yan-Bin, Zhang Bao, Wu Qing-Hua, Guo Qiu-Ye, Peng Yi-Fei, Zheng Wen-Ling
机构信息
Institute of Molecular Biology, First Military Medical University, Guangzhou 510515, China.
出版信息
Di Yi Jun Yi Da Xue Xue Bao. 2002 Mar;22(3):203-5.
OBJECTIVE
To investigate the polyadenylation at the 3' terminal of the mRNAs in E.coli.
METHODS
mRNAs of E.coli was enriched from total RNA with oligo (dT)-cellulose, and reverse transcription was performed using oligo(dT)18 as primer prior to synthesis of double strands cDNA which was digested with Sau 3A I to produce multiple gene fragments that were then ligated with adapters. Restriction digest-polymerase chain reaction (RD-PCR) was employed to divide the fragments into 10 groups using 10 different combinations of the 4 primers, and the products were cloned into T-vectors.
RESULTS
More than 100 gene fragments were cloned, 30 of which were sequenced.
CONCLUSION
Polyadenylation of E.coli mRNA is not a biochemical curiosity, and very likely, it is a general attribute of the mRNAs of bacteria.
目的
研究大肠杆菌中mRNA 3'末端的多聚腺苷酸化情况。
方法
用寡聚（dT）纤维素从总RNA中富集大肠杆菌的mRNA，以寡聚（dT）18为引物进行反转录，然后合成双链cDNA，用Sau 3A I消化产生多个基因片段，再与接头连接。采用限制性消化-聚合酶链反应（RD-PCR），用4种引物的10种不同组合将片段分为10组，产物克隆到T载体中。
结果
克隆了100多个基因片段，其中30个进行了测序。
结论
大肠杆菌mRNA的多聚腺苷酸化并非生化上的奇闻，很可能是细菌mRNA的普遍特性。
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