Muthukrishnan S, Chandra G R, Maxwell E S
J Biol Chem. 1983 Feb 25;258(4):2370-5.
Starting with an enriched alpha-amylase mRNA preparation, cDNA was synthesized by reverse transcription and was cloned at the Pst I site of plasmid vector pBR322 by the "G-C tailing" procedure. Clones containing alpha-amylase cDNA sequences were identified among the recombinant clones by in vitro translation of hybrid-selected mRNA followed by immunoprecipitation of translation products with antiserum to alpha-amylase. The longest alpha-amylase cDNA clone isolated was 630 base pairs long and was characterized by digestion with restriction enzymes. Using this clone as a labeled nucleic acid hybridization probe, we observed that multiple bands of DNA with alpha-amylase sequences were present in restriction digests of barley embryo DNA. This indicates the presence of a family of barley alpha-amylase or alpha-amylase-like genes. Analysis of RNA blots with the cloned alpha-amylase hybridization probe indicated that alpha-amylase mRNA is synthesized de novo in barley aleurones after addition of gibberellic acid. The gibberellic acid-induced accumulation of alpha-amylase mRNA is blocked by cycloheximide which suggests the requirement of a newly synthesized protein factor for efficient expression of alpha-amylase gene.
从富集的α-淀粉酶mRNA制剂开始，通过逆转录合成cDNA，并通过“G-C加尾”程序克隆到质粒载体pBR322的Pst I位点。通过对杂交选择的mRNA进行体外翻译，然后用抗α-淀粉酶抗血清对翻译产物进行免疫沉淀，在重组克隆中鉴定出含有α-淀粉酶cDNA序列的克隆。分离得到的最长的α-淀粉酶cDNA克隆长630个碱基对，并通过限制性酶切进行表征。用该克隆作为标记的核酸杂交探针，我们观察到在大麦胚DNA的限制性消化物中存在多条带有α-淀粉酶序列的DNA条带。这表明存在大麦α-淀粉酶或α-淀粉酶样基因家族。用克隆的α-淀粉酶杂交探针分析RNA印迹表明，在添加赤霉素后，大麦糊粉层中从头合成α-淀粉酶mRNA。赤霉素诱导的α-淀粉酶mRNA积累被环己酰亚胺阻断，这表明高效表达α-淀粉酶基因需要新合成的蛋白质因子。
