Vernet T, Fleck J, Durr A, Fritsch C, Pinck M, Hirth L
Eur J Biochem. 1982 Sep 1;126(3):489-94. doi: 10.1111/j.1432-1033.1982.tb06806.x.
A hybridization probe was used to study the regulation of expression of the gene coding for the small subunit of ribulose 1,5-bisphosphate carboxylase, during functional differentiation of protoplasts. A library of cDNA from poly(A)-containing RNA extracted from specially treated tobacco leaves was constructed in the plasmid pBR322 by blunt-end ligation. This library was screened by colony hybridization with 32P-labelled cDNA prepared from mRNA coding for the precursor of the small subunit. A positive colony was identified containing recombinant plasmids with a nucleotide sequence homologous to this mRNA. These plasmids, bound to diazobenzyloxymethylated cellulose paper, were then used as a hybridization probe. The results showed unambiguously that the small subunit was not transcribed in protoplasts but was transcribed in undifferentiated white and chlorophyll-containing green callus cultures derived from protoplasts. The discrepancy between these results and those obtained with classical techniques is discussed.
在原生质体功能分化过程中，使用杂交探针研究编码核酮糖1,5 - 二磷酸羧化酶小亚基的基因的表达调控。通过平端连接，将从经过特殊处理的烟草叶片中提取的含poly(A)RNA构建的cDNA文库克隆到质粒pBR322中。用编码小亚基前体的mRNA制备的32P标记的cDNA通过菌落杂交筛选该文库。鉴定出一个阳性菌落，其含有与该mRNA具有同源核苷酸序列的重组质粒。然后将这些与重氮苄氧基甲基化纤维素纸结合的质粒用作杂交探针。结果明确表明，小亚基在原生质体中不转录，但在源自原生质体的未分化白色和含叶绿素的绿色愈伤组织培养物中转录。讨论了这些结果与用经典技术获得的结果之间的差异。
