Harris E H, Burkhart B D, Gillham N W, Boynton J E
Department of Botany, Duke University, Durham, North Carolina 27706.
Genetics. 1989 Oct;123(2):281-92. doi: 10.1093/genetics/123.2.281.
Mutants resistant to streptomycin, spectinomycin, neamine/kanamycin and erythromycin define eight genetic loci in a linear linkage group corresponding to about 21 kb of the circular chloroplast genome of Chlamydomonas reinhardtii. With one exception, all of these mutants represent single base-pair changes in conserved regions of the genes encoding the 16S and 23S chloroplast ribosomal RNAs. Streptomycin resistance can result from changes at the bases equivalent to Escherichia coli 13, 523, and 912-915 in the 16S gene, or from mutations in the rps12 gene encoding chloroplast ribosomal protein S12. In the 912-915 region of the 16S gene, three mutations were identified that resulted in different levels of streptomycin resistance in vitro. Although the three regions of the 16S rRNA mutable to streptomycin resistance are widely separated in the primary sequence, studies by other laboratories of RNA secondary structure and protein cross-linking suggest that all three regions are involved in a common ribosomal neighborhood that interacts with ribosomal proteins S4, S5 and S12. Three different changes within a conserved region of the 16S gene, equivalent to E. coli bases 1191-1193, confer varying levels of spectinomycin resistance, while resistance to neamine and kanamycin results from mutations in the 16S gene at bases equivalent to E. coli 1408 and 1409. Five mutations in two genetically distinct erythromycin resistance loci map in the 23S rDNA of C. reinhardtii, at positions equivalent to E. coli 2057-2058 and 2611, corresponding to the rib3 and rib2 loci of yeast mitochondria respectively. Although all five mutants are highly resistant to erythromycin, they differ in levels of cross-resistance to lincomycin and clindamycin. The order and spacing of all these mutations in the physical map are entirely consistent with our genetic map of the same loci and thereby validate the zygote clone method of analysis used to generate this map. These results are discussed in comparison with other published maps of chloroplast genes based on analysis by different methods using many of the same mutants.
对链霉素、壮观霉素、新霉素/卡那霉素和红霉素具有抗性的突变体，在一个线性连锁群中定义了八个基因位点，该连锁群对应于莱茵衣藻环形叶绿体基因组中约21 kb的区域。除了一个例外，所有这些突变体均代表编码16S和23S叶绿体核糖体RNA的基因保守区域中的单碱基对变化。链霉素抗性可能源于16S基因中与大肠杆菌13、523和912 - 915位点等效的碱基变化，或者源于编码叶绿体核糖体蛋白S12的rps12基因中的突变。在16S基因的912 - 915区域，鉴定出三个突变，这些突变在体外导致了不同水平的链霉素抗性。尽管16S rRNA中对链霉素抗性可变的三个区域在一级序列中相距甚远，但其他实验室对RNA二级结构和蛋白质交联的研究表明，所有这三个区域都参与了一个与核糖体蛋白S4、S5和S12相互作用的共同核糖体邻域。16S基因保守区域内与大肠杆菌1191 - 1193位点等效的三个不同变化，赋予了不同水平的壮观霉素抗性，而对新霉素和卡那霉素的抗性则源于16S基因中与大肠杆菌1408和1409位点等效的碱基突变。两个遗传上不同的红霉素抗性位点中的五个突变位于莱茵衣藻的23S rDNA中，分别对应于大肠杆菌2057 - 2058和2611位点，分别对应于酵母线粒体的rib3和rib2位点。尽管所有五个突变体对红霉素都具有高度抗性，但它们对林可霉素和克林霉素的交叉抗性水平有所不同。物理图谱中所有这些突变的顺序和间距与我们对相同位点的遗传图谱完全一致，从而验证了用于生成此图谱的合子克隆分析方法。将这些结果与其他基于使用许多相同突变体的不同方法分析的已发表叶绿体基因图谱进行了比较讨论。
