Optimization of differential display of prokaryotic mRNA: application to pure culture and soil microcosms.
The differential display (DD) technique, which is widely used almost exclusively for eukaryotic gene discovery, was optimized to detect differential mRNA transcription from both pure-culture and soil-derived bacterial RNA. A model system which included toluene induction of todC1 in Pseudomonas putida F1 was used to optimize the procedure. At 24-h tod induction was determined to be approximately 8 x 10(7) transcripts/microg or 0.08% of the total mRNA. The primer concentration, primer length, annealing temperature, and template, deoxynucleoside triphosphate, and MgCl2 concentrations were varied to optimize amplification of a todC1 fragment. The limit of detection of todC1 by DD was found to be 0.015 ng of total RNA template or approximately 10(3) transcripts. Once optimized, a todC1C2 gene fragment from P. putida F1 RNA was detected by using an arbitrary primer for the reverse transcriptase step in conjunction with the same arbitrary primer and a Shine-Dalgarno primer in the PCR. To verify the results, an arbitrary primer was used to detect recovery of a new salicylate-inducible naphthalene dioxygenase in Burkholderia cepacia JS150. The method was then used to detect mRNA induction in both inoculated and uninoculated toluene-induced soil microcosms. Several putative differentially expressed partial gene sequences obtained from the uninoculated microcosms were examined, and one novel fragment was found to be differentially expressed.
Fleming
J T
JT
Center for Environmental Biotechnology, The University of Tennessee, Knoxville, Tennessee 37996, USA. jtf@utk.edu
Yao
W H
WH
Sayler
G S
GS
D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D016133:Polymerase Chain Reaction; D016958:Pseudomonas putida / Q000235:genetics* / Q000254:growth & development; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D012988:Soil Microbiology; D014050:Toluene / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects*
D012329:RNA, Bacterial; D012333:RNA, Messenger; D014050:Toluene