Correlated physical and genetic map of the Bradyrhizobium japonicum 110 genome
Correlated physical and genetic map of the Bradyrhizobium japonicum 110 genome
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日本慢生根瘤菌 110 基因组的相关物理和遗传图谱
DOI：
10.1128/jb.175.3.613-622.1993
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发表时间：
1993
影响因子：
3.2
通讯作者：
M. Gottfert
中科院分区：
文献类型：
--
作者：
Christoph Kundig;H. Hennecke;M. Gottfert
我们描述了79个已知基因的汇编，其中63个基因被放置在染色体的相关物理和遗传图谱上。基因组DNA用PacI、PmeI和Swai酶进行限制性内切酶鉴定，分别产生2、5和9个片段。通过Southern杂交实验建立了部分片段的连锁关系。对于探针，我们使用了分离的标记片段，这些片段要么是由PmeI产生的，要么是由Swii产生的。通过基因定向突变将基因定位在单个限制性片段上。这种方法的原理是将上述三种限制性内切酶的识别位点引入或非常接近所需的基因位点。与野生型DNA相比，限制性突变体DNA的脉冲场凝胶电泳会导致片断模式的改变。这使我们能够识别重叠的片段，并确定任何选定的基因位点的确切位置。这项技术仅受到片段大小估计的准确性的限制。在连接了所有的限制性片段后，我们得出结论：日本血吸虫基因组由一个约8,700 kb长的单条环形染色体组成。与结瘤和共生固氮直接相关的基因聚集在一个约380 kb的染色体片段中。
We describe a compilation of 79 known genes of Bradyrhizobium japonicum 110, 63 of which were placed on a correlated physical and genetic map of the chromosome. Genomic DNA was restricted with enzymes PacI, PmeI, and SwaI, which yielded two, five, and nine fragments, respectively. Linkage of some of the fragments was established by performing Southern blot hybridization experiments. For probes we used isolated, labelled fragments that were produced either by PmeI or by SwaI. Genes were mapped on individual restriction fragments by performing gene-directed mutagenesis. The principle of this method was to introduce recognition sites for all three restriction enzymes mentioned above into or very near the desired gene loci. Pulsed-field gel electrophoresis of restricted mutant DNA then resulted in an altered fragment pattern compared with wild-type DNA. This allowed us to identify overlapping fragments and to determine the exact position of any selected gene locus. The technique was limited only by the accuracy of the fragment size estimates. After linkage of all of the restriction fragments we concluded that the B. japonicum genome consists of a single, circular chromosome that is approximately 8,700 kb long. Genes directly concerned with nodulation and symbiotic nitrogen fixation are clustered in a chromosomal section that is about 380 kb long.