Randolph-Anderson B L, Boynton J E, Gillham N W, Huang C, Liu X Q
Department of Botany, Duke University, Durham, NC 27708, USA.
Mol Gen Genet. 1995 May 10;247(3):295-305. doi: 10.1007/BF00293197.
The ribosomal protein gene rps4 was cloned and sequenced from the chloroplast genome of Chlamydomonas reinhardtii. The N-terminal 213 amino acid residues of the S4 protein are encoded in the single-copy region (SCR) of the genome, while the C-terminal 44 amino acid residues are encoded in the inverted repeat (IR). The deduced 257 amino acid sequence of C. reinhardtii S4 is considerably longer (by 51-59 residues) than S4 proteins of other photosynthetic species and Escherichia coli, due to the presence of two internal insertions and a C-terminal extension. A short conserved C-terminal motif found in all other S4 proteins examined is missing from the C. reinhardtii protein. In E. coli, mutations in the S4 protein suppress the streptomycin-dependent (sd) phenotype of mutations in the S12 protein. Because we have been unable to identify similar S4 mutations among suppressors of an sd mutation in C. reinhardtii S12 obtained using UV mutagenesis, we made site-directed mutations [Arg68 (CGT) to Leu (CTG and CTT)] in the wild-type rps4 gene equivalent to an E. coli Gln53 to Leu ribosomal ambiguity mutation (ram), which suppresses the sd phenotype and decreases translational accuracy. These mutants were tested for their ability to transform the sd S12 mutation of C. reinhardtii to streptomycin independence. The streptomycin-independent isolates obtained by biolistic transformation all possessed the original sd mutation in rps12, but none had the expected donor Leu68 mutations in rps4. Instead, six of 15 contained a Gln73 (CAA) to Pro (CCA) mutation five amino acids downstream from the predicted mutant codon, irrespective of rps4 donor DNA. Two others contained six- and ten-amino acid, in-frame insertions at S4 positions 90 and 92 that appear to have been induced by the biolistic process itself. Eight streptomycin-independent isolates analyzed had wild-type rps4 genes and may possess mutations identical to previously isolated suppressors of sd that define at least two additional chloroplast loci. Cloned rps4 genes from streptomycin-independent isolates containing the Gln73 to Pro mutation and the 6-amino acid insertion in r-protein S4 transform the sd strain to streptomycin independence.
核糖体蛋白基因rps4是从莱茵衣藻的叶绿体基因组中克隆并测序的。S4蛋白的N端213个氨基酸残基由基因组的单拷贝区域（SCR）编码，而C端44个氨基酸残基由反向重复序列（IR）编码。莱茵衣藻S4推导的257个氨基酸序列比其他光合物种和大肠杆菌的S4蛋白长得多（长51 - 59个残基），这是由于存在两个内部插入片段和一个C端延伸。在所有其他检测的S4蛋白中发现的一个短的保守C端基序在莱茵衣藻蛋白中缺失。在大肠杆菌中，S4蛋白的突变会抑制S12蛋白突变的链霉素依赖（sd）表型。由于我们在使用紫外线诱变获得的莱茵衣藻S12的sd突变的抑制子中未能鉴定出类似的S4突变，我们在野生型rps4基因中进行了定点突变[将Arg68（CGT）突变为Leu（CTG和CTT）]，这相当于大肠杆菌中Gln53到Leu的核糖体模糊突变（ram），该突变抑制sd表型并降低翻译准确性。测试了这些突变体将莱茵衣藻的sd S12突变转化为链霉素非依赖型的能力。通过生物弹道转化获得的链霉素非依赖型分离株在rps12中都具有原始的sd突变，但在rps4中没有预期的供体Leu68突变。相反，15个中有6个在预测的突变密码子下游五个氨基酸处含有Gln73（CAA）到Pro（CCA）的突变，与rps4供体DNA无关。另外两个在S4的90和92位含有6个和10个氨基酸的框内插入，这似乎是由生物弹道过程本身诱导的。分析的8个链霉素非依赖型分离株具有野生型rps4基因，可能具有与先前分离的sd抑制子相同的突变，这些抑制子定义了至少两个额外的叶绿体基因座。从含有Gln73到Pro突变以及r蛋白S4中6个氨基酸插入的链霉素非依赖型分离株中克隆的rps4基因将sd菌株转化为链霉素非依赖型。
