High-density sampling of a bacterial operon using mRNA differential display.
We have implemented a simplified high throughput approach to differential display in order to identify transcriptionally regulated genes in bacteria. In contrast with the few previous applications of differential display to prokaryotes, we use a large number of primers which allows for a high-density sampling of the mRNA population and the identification of many differentially amplified DNA fragments. From the overlap of these short sequences, long contiguous sequences that encode several genes can be assembled. The multiplicity of sampling provides a strong indication that the genes identified are indeed differentially regulated. As a test case, we looked for the genes involved in the degradation of 2,4-dinitrophenol (2,4-DNP) in a Rhodococcus erythropolis strain, HL PM-1. In this experiment a long polycistronic mRNA was sampled repeatedly. The induction of these genes by 2,4-DNP was confirmed by dot blot analysis and two of them were confirmed to be involved in the degradation of 2,4-DNP. This work shows that mRNA differential display is an important tool for the identification of metabolic genes in prokaryotes.
Walters
D M
DM
Central Research and Development, E. I. DuPont de Nemours Co., Wilmington, DE 19800, USA.
Russ
R
R
Knackmuss
H J
HJ
Rouvière
P E
PE
D019297:2,4-Dinitrophenol / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics*; D010851:Picrates / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012240:Rhodococcus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010851:Picrates; D012333:RNA, Messenger; C005858:picric acid; D019297:2,4-Dinitrophenol