Institut für Botanik, Fachbereich Biologie, Universität Hannover, Herrenhäuser Str. 2, 3000, Hannover, F. R. G..
Plant Mol Biol. 1985 Jul;4(4):241-5. doi: 10.1007/BF02418242.
cDNA clones were isolated for a chloroplast protein, the mRNA of which is induced to maximum levels within 2-4 h after onset of illumination in five day old, etiolated pea seedlings.The cDNA library was constructed from poly(A)(+)-mRNA which was isolated from 4 h illuminated seedlings. The extremely short induction period of the early light induced protein(ELIP)-mRNA established the basis of our screening procedure. Colony hybridization experiments were performed with(32)P-labelled cDNA probes, synthesized from RNA of seedlings which had been exposed to different programs of illumination. Plasmid DNAs were isolated from colonies showing strong hybridization signals exclusively with cDNA corresponding to the 4 h-mRNA. Hybrid released translation of preselected plasmids p 17/C2 and p17/C4 revealed a peptide of Mr 24 000. After posttranslational importin vitro, the processed product of Mr 17 000 appears in the chloroplast. Using these clones, the expression of the ELIP-mRNA was investigated by DOT-hybridization. The ELIP-mRNA reaches maximum levels within 2-4 hours after onset of illumination. Our results correspond precisely to thein vivo characteristics and indicate positive identification of the sought clones.
cDNA 克隆被分离出来的叶绿体蛋白，其 mRNA 是诱导最大水平在 2-4 小时后，光照开始在五天大，黄化豌豆幼苗。cDNA 文库构建的多（A）（+）-mRNA 是从 4 小时光照苗中分离出来的。早期光诱导蛋白（ELIP）-mRNA 的极短诱导期为我们的筛选程序奠定了基础。用（32）P 标记的 cDNA 探针进行菌落杂交实验，这些 cDNA 探针是从暴露于不同光照程序的幼苗的 RNA 中合成的。仅与对应于 4 小时-mRNA 的 cDNA 显示出强杂交信号的菌落中分离出质粒 DNA。从预选择的质粒 p17/C2 和 p17/C4 中释放的杂交翻译揭示了 Mr24000 的肽。在体外进行翻译后，Mr17000 的加工产物出现在叶绿体中。使用这些克隆，通过 DOT 杂交研究了 ELIP-mRNA 的表达。ELIP-mRNA 在光照开始后 2-4 小时内达到最大水平。我们的结果与体内特征完全一致，表明所寻找的克隆得到了准确的鉴定。
