Physical and genetic map of the Spiroplasma kunkelii CR2-3x chromosome
Physical and genetic map of the Spiroplasma kunkelii CR2-3x chromosome
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DOI：
10.1139/w06-044
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发表时间：
2006-09-01
影响因子：
2.8
通讯作者：
Davis, Robert E.
中科院分区：
文献类型：
--
作者：
Dally, Ellen L.;Barros, Thereza S. L.;Davis, Robert E.
昆克螺原体（软体纲）是一种典型的螺旋状、无壁细菌，可引起玉米矮化病。结合限制酶分析、脉冲场凝胶电泳 (PFGE) 和 Southern 杂交分析，构建了 S. kunkelii CR2-3x 染色体的物理和遗传图谱。图谱上限制性片段的顺序是通过使用 I-CeuI、AscI、ApaI、EagI、SmaI、BssHII、BglI 和 SalI 对相互核酸内切酶双消化进行分析来确定的；在二维脉冲场电泳凝胶上鉴定相邻片段。染色体的大小估计为 1550 kb。设计寡核苷酸对以引发聚合酶链式反应 (PCR) 中 26 个 S. kunkelii 基因序列的扩增。使用 PCR 扩增子作为探针，通过对 PFGE 中分离的染色体片段进行 Southern 杂交分析，将 27 个 S. kunkelii 推定单拷贝基因的位置定位在图谱上。确定了单个核糖体 RNA 操纵子的核苷酸序列，并将其位置映射到带有 SalI、SmaI、EagI 和 I-CeuI 识别位点的染色体片段。
Spiroplasma kunkelii (class Mollicutes) is the characteristically helical, wall-less bacterium that causes corn stunt disease. A combination of restriction enzyme analysis, pulsed-field gel electrophoresis (PFGE), and Southern hybridization analysis was used to construct a physical and genetic map of the S. kunkelii CR2-3x chromosome. The order of restriction fragments on the map was determined by analyses of reciprocal endonuclease double digests employing I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI, and SalI; adjacent fragments were identified on two-dimensional pulsed-field electrophoresis gels. The size of the chromosome was estimated at 1550 kb. Oligonucleotide pairs were designed to prime the amplification of 26 S. kunkelii gene sequences in the polymerase chain reaction (PCR). Using PCR amplicons as probes, the locations of 27 S. kunkelii putative single-copy genes were positioned on the map by Southern hybridization analyses of chromosomal fragments separated in PFGE. The nucleotide sequence of the single ribosomal RNA operon was determined and its location mapped to a chromosomal segment bearing recognition sites for SalI, SmaI, EagI, and I-CeuI.