Scott-Craig J S, Guerinot M L, Chelm B K
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing 48824-1312.
Mol Gen Genet. 1991 Sep;228(3):356-60. doi: 10.1007/BF00260627.
DNA sequences have been isolated that are expressed at high levels in bacteroids, the differentiated form of the soybean microsymbiont, Bradyrhizobium japonicum. Random-primed cDNA was synthesized using total RNA isolated from purified B. japonicum bacteroids or from cells grown in culture. When used directly to screen bacteriophage lambda libraries, these cDNA probes produced a high background hybridization signal due to sequence similarity between B. japonicum and E. coli ribosomal DNA (rDNA) operons. To reduce this background signal, the rDNA operon of B. japonicum was cloned and the rDNA plasmid DNA used in subtractive hybridization with the cDNA probes and as a competitor in hybridization solutions. This method greatly reduced the background signal in screening of genomic libraries and thus permitted the identification of twelve unique recombinant phage which contained sequences that are expressed at higher levels in B. japonicum bacteroids than in cells grown in culture.
已经分离出在类菌体（大豆微共生体日本慢生根瘤菌的分化形式）中高水平表达的DNA序列。使用从纯化的日本慢生根瘤菌类菌体或培养生长的细胞中分离的总RNA合成随机引物cDNA。当直接用于筛选λ噬菌体文库时，由于日本慢生根瘤菌和大肠杆菌核糖体DNA（rDNA）操纵子之间的序列相似性，这些cDNA探针产生了高背景杂交信号。为了降低这种背景信号，克隆了日本慢生根瘤菌的rDNA操纵子，并将rDNA质粒DNA用于与cDNA探针的消减杂交以及作为杂交溶液中的竞争物。该方法大大降低了基因组文库筛选中的背景信号，从而使得能够鉴定出十二个独特的重组噬菌体，它们包含在日本慢生根瘤菌类菌体中比在培养生长的细胞中表达水平更高的序列。
