Posno M, Verweij W R, Dekker I C, de Waard P M, Groot G S
Biochemical Laboratory, Free University, Amsterdam, The Netherlands.
Curr Genet. 1986;11(1):25-34. doi: 10.1007/BF00389422.
We have used a variety of methods to localize the genes for ribosomal proteins S7 and S12 on Spirodela chloroplast DNA. Heterologous hybridization with a rps12 gene specific probe from Euglena has revealed the presence of rps12 homologous sequences within the inverted repeat of Spirodela chloroplast DNA on the fragment BamHI-V. In the partial nucleotide sequence of this fragment, two regions of amino acid sequence homology to Euglena S12 can be identified, separated from each other by a 542 bp intron with conserved boundary sequences. As was found for Nicotiana S12, the Spirodela S12 coding regions are for 85 amino acids homologous (79%) to E. coli S12 (starting from residue 38 to the C-terminus). Likewise, we are unable to identify the 37 5' terminal codons of rps12 in Spirodela. The functionality of the Spirodela rps12 sequence is discussed. The rps7 gene is located adjacent to rps12. Chloroplast ribosomal protein C-S11 (homologous to S7) has been detected by immunoprecipitation with both a polyspecific anti 30S serum and an anti C-S11 serum, among the in vitro translation products of mRNAs selected by Spirodela chloroplast DNA fragments BamHI-V and BamHI-P. Since in a DNA dependent E. coli cell free system, only BamHI-V appears to be capable of synthesis of C-S11, it is concluded that rps7 is located entirely within BamHI-V and is transcribed into a mRNA which extends into BamHI-P. As determined by Northern hybridization experiments, rps7 is cotranscribed with rps12; a stable transcript of approx. 1100 b is detected in total cellular Spirodela RNA with either rps12 and rps7 gene specific probes. The rps12 probe additionally detects an approx. 600 b transcript, which presumably corresponds to the excised rps12 intron RNA. Finally we have examined the expression of both rps7 and rps12 during light induced chloroplast development by Northern blotting and by immunoblotting. It is shown, that the steady-state levels of neither chloroplast ribosomal protein transcripts, nor those of the chloroplast ribosomal proteins itself, change significantly during the greening process.
我们运用了多种方法在紫萍叶绿体DNA上定位核糖体蛋白S7和S12的基因。用来自眼虫的rps12基因特异性探针进行异源杂交,揭示了紫萍叶绿体DNA反向重复序列中BamHI-V片段上存在rps12同源序列。在该片段的部分核苷酸序列中,可鉴定出与眼虫S12氨基酸序列同源的两个区域,它们被一个具有保守边界序列的542 bp内含子隔开。正如烟草S12的情况一样,紫萍S12编码区与大肠杆菌S12(从第38位残基到C端)的85个氨基酸同源(79%)。同样,我们无法鉴定紫萍中rps12的37个5'端密码子。文中讨论了紫萍rps12序列的功能。rps7基因位于rps12附近。在用多特异性抗30S血清和抗C-S11血清进行免疫沉淀时,在由紫萍叶绿体DNA片段BamHI-V和BamHI-P选择的mRNA的体外翻译产物中检测到了叶绿体核糖体蛋白C-S11(与S7同源)。由于在依赖DNA的大肠杆菌无细胞系统中,似乎只有BamHI-V能够合成C-S11,因此得出结论,rps7完全位于BamHI-V内,并转录成延伸到BamHI-P的mRNA。通过Northern杂交实验确定,rps7与rps12共转录;用rps12和rps7基因特异性探针在紫萍总细胞RNA中检测到一个约1100 b的稳定转录本。rps12探针还检测到一个约600 b的转录本,推测它对应于切除的rps12内含子RNA。最后,我们通过Northern印迹和免疫印迹检测了光诱导叶绿体发育过程中rps7和rps12的表达。结果表明,在绿化过程中,叶绿体核糖体蛋白转录本的稳态水平以及叶绿体核糖体蛋白本身的稳态水平均未发生显著变化。