Nepokroeff C M, Adachi K, Yan C, Porter J W
Eur J Biochem. 1984 Apr 16;140(2):441-5. doi: 10.1111/j.1432-1033.1984.tb08122.x.
Clones, containing DNA complementary (cDNA) to rat liver fatty acid synthetase mRNA, were constructed and identified. cDNA of these clones was then used as a probe to quantify mRNA. The cDNA was synthesized to partially purified rat liver fatty acid synthetase mRNA. Double-stranded cDNA was then prepared and inserted into the PstI site of pBR322 using oligo(dG) X oligo(dC) tailing. Initial selection of the clones was by differential colony hybridization employing [32P]cDNA synthesized from poly(A)-rich mRNA, enriched and non-enriched in fatty acid synthetase mRNA, as probes. Plasmids, containing specific sequences complementary to the fatty acid synthetase mRNA, were identified by hybrid-arrest translation. Cloned cDNA inserts ranged from 300 to 1400 base pairs. Cloned cDNA was employed to probe for mRNA in hybridizations via the dot-blot method. These studies demonstrated an increase in fatty acid synthetase mRNA during dietary induction, which suggests that regulation may involve changes in transcription or changes in post-transcriptional processing of the mRNA.
构建并鉴定了含有与大鼠肝脏脂肪酸合成酶mRNA互补的DNA（cDNA）的克隆。然后将这些克隆的cDNA用作探针来定量mRNA。cDNA是由部分纯化的大鼠肝脏脂肪酸合成酶mRNA合成的。接着制备双链cDNA，并使用寡聚（dG）X寡聚（dC）加尾法将其插入pBR322的PstI位点。最初通过差异菌落杂交筛选克隆，使用从富含多聚腺苷酸（poly(A)）的mRNA合成的[32P]cDNA作为探针，该mRNA在脂肪酸合成酶mRNA中富集或未富集。通过杂交抑制翻译鉴定含有与脂肪酸合成酶mRNA互补的特定序列的质粒。克隆的cDNA插入片段范围为300至1400个碱基对。通过点杂交法，使用克隆的cDNA作为探针在杂交中检测mRNA。这些研究表明，在饮食诱导过程中脂肪酸合成酶mRNA增加，这表明调节可能涉及转录变化或mRNA转录后加工的变化。
