Isolation of Bradyrhizobium japonicum DNA sequences that are transcribed at high levels in bacteroids.
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.
Scott-Craig
J S
JS
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing 48824-1312.
Guerinot
M L
ML
Chelm
B K
BK
D015139:Blotting, Southern; D004247:DNA / Q000235:genetics; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization; D009876:Operon; D010957:Plasmids; D015183:Restriction Mapping; D012231:Rhizobium / Q000235:genetics* / Q000254:growth & development; D014158:Transcription, Genetic
D015342:DNA Probes; D004269:DNA, Bacterial; D004247:DNA