In vitro expression of chloroplast genes in lysates of higher plant chloroplasts.
In vitro expression of chloroplast genes in lysates of higher plant chloroplasts.
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高等植物叶绿体裂解物中叶绿体基因的体外表达。
DOI：
10.1073/pnas.82.12.3983
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发表时间：
1985
影响因子：
11.1
通讯作者：
Zaitlin,D
中科院分区：
文献类型：
--
作者：
Bard,J;Bourque,DP;Hildebrand,M;Zaitlin,D
从分离的叶绿体裂解物中制备出依赖于 DNA 的体外转录-翻译系统。这些裂解物在转录和翻译保真度以及对给定模板 DNA 的响应效率方面与大肠杆菌的裂解物相当。当烟草叶绿体 DNA 用作叶绿体裂解物（烟草或菠菜）或大肠杆菌裂解物的模板时，NaDodSO4 凝胶分析显示相似的多肽模式，与使用大肠杆菌 DNA 获得的模式不同。含有叶绿体 DNA 的重组质粒中的基因也在这些体外系统中表达。脱氧核糖核酸。 RNA杂交实验表明转录本是由大部分叶绿体基因组合成的。使用叶绿体 DNA 或含有大亚基基因的烟草叶绿体 DNA 克隆片段作为模板，在叶绿体裂解物中观察到新合成的二磷酸核酮糖羧化酶/加氧物大亚基和大亚基基因 (rbcL) 的转录物。结果表明叶绿体基因的差异表达在体外发生。通过在该同源系统中使用克隆的叶绿体 DNA 模板，可以识别和绘制叶绿体蛋白的结构基因。
A DNA-dependent in vitro-coupled transcription-translation system has been prepared from lysates of isolated chloroplasts. These lysates are comparable to those of Escherichia coli in transcriptional and translational fidelity and efficiency in response to a given template DNA. When Nicotiana tabacum chloroplast DNA is used as template with chloroplast lysates (N. tabacum or spinach) or E. coli lysates, NaDodSO4 gel analysis reveals similar polypeptide patterns that are distinct from the patterns obtained with E. coli DNA. Genes in recombinant plasmids containing chloroplast DNA are also expressed in these in vitro systems. DNA . RNA hybridization experiments show that transcripts are synthesized from most of the chloroplast genome. Newly synthesized large subunit of ribulosebisphosphate carboxylase/oxygenate and a transcript of the large subunit gene (rbcL) are observed in chloroplast lysates using as template chloroplast DNA or cloned fragments of tobacco chloroplast DNA that contain the large subunit gene. Results suggest that differential expression of chloroplast genes occurs in vitro. By using cloned chloroplast DNA templates in this homologous system, it is possible to identify and map structural genes for chloroplast proteins.