Isolation of nuclear encoded plastid ribosomal protein cDNAs.
Isolation of nuclear encoded plastid ribosomal protein cDNAs.
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核编码质体核糖体蛋白 cDNA 的分离。
DOI：
10.1007/bf00331635
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Key,JL
中科院分区：
文献类型：
--
作者：
Gantt,JS;Key,JL
在表达载体λgt11中构建了豌豆叶片基因文库，并用从分离的豌豆叶绿体中提取的30S和50S核糖体亚基和70S核糖体蛋白制备的抗血清进行筛选。经鉴定，有6个重组噬菌体编码含有胞质核糖体蛋白抗原决定簇的融合蛋白。将含有融合蛋白的噬菌体诱导的细胞裂解物蛋白与硝酸纤维素膜结合，作为亲和基质制备单特异性抗体。然后，通过Western blotting，这些抗体被用来识别哪些胞质核糖体蛋白与融合蛋白具有共同的抗原决定簇。从产生抗原的噬菌体中插入的cDNA被用来杂交选择互补的mRNAs。将这些mRNAs的无细胞翻译产物加入豌豆叶绿体体外转运系统中，对进口蛋白进行双向凝胶电泳法分析。进口的蛋白与被鉴定为与相应重组噬菌体产生的融合蛋白具有抗原性相关的胞体核糖体蛋白一起迁移。进口蛋白质比其前体小3,500-5,500道尔顿。
A pea leaf cDNA library was constructed in the expression vector λgt11 and screened with antisera raised against proteins extracted from 30S and 50S ribosomal subunits and 70S ribosomes prepared from isolated pea chloroplasts. Six recombinant phage were identified that encoded fusion proteins containing plastid ribosomal protein antigenic determinants. Phage-induced cell lysate proteins, containing the fusion proteins, were bound to nitrocellulose membranes and used as affinity matrices to prepare monospecific antibodies. These antibodies were then used to identify by Western blotting which plastid ribosomal protein shared antigenic determinants with the fusion proteins. cDNA inserts from the antigen-producing phage were used to hybrid-select complementary mRNAs. The cell-free translation products of these mRNAs were added to a pea chloroplast in vitro transport system and imported proteins analyzed by two-dimensional gel electrophoresis. The imported proteins comigrated with the plastid ribosomal proteins that were identified as being antigenically related to the fusion proteins produced by the corresponding recombinant phage. The imported proteins were 3,500–5,500 daltons smaller than their precursors.