Razin S, Nur I, Glaser G
Department of Membrane and Ultrastructure Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Isr J Med Sci. 1987 Jun;23(6):678-82.
Extrachromosomal DNA, constituting plasmids or replicative forms of viruses, has been detected in a variety of spiroplasmas, particularly in Spiroplasma citri. Only a few of the S. citri plasmids were characterized by restriction enzyme mapping, and essentially nothing is known on functions encoded by the plasmids. Our studies revealed in S. citri (R8A2) an 8.0-kbp plasmid that differed from previously described plasmids in its restriction map. It was also clonable in pBR322. The plasmid, named pRA1, was found in large quantities as free plasmid in S. citri (R8A2) subclones of low passage level. In subclones of higher passage levels, free plasmid was replaced by plasmid sequences integrated into the spiroplasma chromosome, as revealed by Southern hybridization blots of digested spiroplasmal DNA with nick-translated pRA1 or its recombinant as probes. Significant quantities of integrated plasmid sequences were also observed in S. kunkelii and in Spiroplasma sp. P40. Small quantities of free and/or integrated plasmid DNA were detected in some spiroplasmas serologically and genotypically remote from S. citri. Chromosome-integrated pRA1 sequences were cloned into the Escherichia coli plasmids pUC13 and M13. Hybridization tests and restriction maps of these clones indicated that the integrated plasmid sequences consisted of small repetitive sequences inserted into specific sites on the spiroplasma chromosome. Despite the large number of the inserts they do not appear to affect significantly gene expression in the spiroplasma. Due to the abundance of free and integrated pRA1 in S. citri, nick-translated pRA1 was effective as a DNA probe in detecting small numbers of S. citri in infected periwinkle plants and leafhoppers.
构成质粒或病毒复制形式的染色体外DNA已在多种螺旋体中被检测到，尤其是在柑橘螺旋体中。柑橘螺旋体中只有少数质粒通过限制性酶切图谱进行了表征，而关于这些质粒所编码的功能基本上一无所知。我们的研究在柑橘螺旋体（R8A2）中发现了一个8.0千碱基对的质粒，其限制性图谱与先前描述的质粒不同。它也可克隆到pBR322中。这个名为pRA1的质粒在低传代水平的柑橘螺旋体（R8A2）亚克隆中大量以游离质粒形式存在。在较高传代水平的亚克隆中，如用缺口平移法标记的pRA1或其重组体作为探针，对消化后的螺旋体DNA进行Southern杂交印迹分析所显示的，游离质粒被整合到螺旋体染色体中的质粒序列所取代。在库氏螺旋体和螺旋体属P40中也观察到了大量整合的质粒序列。在一些血清学和基因型上与柑橘螺旋体相距较远的螺旋体中检测到了少量游离和/或整合的质粒DNA。染色体整合的pRA1序列被克隆到大肠杆菌质粒pUC13和M13中。这些克隆的杂交试验和限制性图谱表明，整合的质粒序列由插入螺旋体染色体特定位点的小重复序列组成。尽管有大量的插入片段，但它们似乎并未对螺旋体中的基因表达产生显著影响。由于柑橘螺旋体中游离和整合的pRA1含量丰富，缺口平移法标记的pRA1作为DNA探针可有效检测受感染长春花植物和叶蝉中少量的柑橘螺旋体。
