Expression analysis of cloned chromosomal segments of Escherichia coli.
The novel transcription system of bacteriophage T7 was used to express Escherichia coli genes preferentially with a new low-copy-number plasmid vector, pFN476, to minimize toxic gene effects. Selected E. coli chromosomal fragments from an ordered genomic library (Y. Kohara, K. Ikiyama, and K. Isono, Cell 50:495-508, 1987) were recloned into this vector, and their genes were preferentially expressed in vivo utilizing its T7 promoter. The protein products were analyzed by two-dimensional gel electrophoresis. By using DNA sequence information, the gel migration was predicted for the protein products of open reading frames from these segments, and this information was used to identify gene products visualized as spots on two-dimensional gels. Even in the absence of DNA sequence information, this approach offers the opportunity to identify all gene products of E. coli and map their genes to within 10 kb on the E. coli genome; with sequence information, this approach can produce a definitive expression map of the E. coli genome.
Sankar
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P
Department of Microbiology and Immunology, University of Michigan, Ann Arbor 48109-0620.
Hutton
M E
ME
VanBogelen
R A
RA
Clark
R L
RL
Neidhardt
F C
FC
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014764:Viral Proteins
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases