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Cloning and characterization of an Escherichia coli gene encoding a mannose-specific phosphotransferase involved in mannose catabolism.
The Escherichia coli mannose PTS was cloned using a mannose-negative E. coli strain as a host. Restriction fragments containing the mannose PTS genes were ligated into pBR322 and mapped. A 1.5-kilobase (kb) fragment which contained the mannose PTS genes and their flanking sequences was then subcloned into the Bluescript II KS+ plasmid pJN3, which carries the thiostrepton resistance gene (tsr) and the lacZ gene. Clones obtained were analyzed by Western blotting and high performance liquid chromatography. The gene (mnp) encoding the mannose-specific enzyme (E. coli alpha-d-mannose: beta-d-fructose-6-phosphate phosphotransferase) was identified by molecular cloning and complementation analysis. The mnp gene, which was located on the 1.5-kb fragment, was assigned to the 730-bp EcoRI fragment. The mnp gene consisted of a single open reading frame encoding a protein of 553 amino acid residues (Mr 58,599). The 5' end of the mRNA from the mnp gene was mapped to nucleotide (nt) +23 by the S1 nuclease protection technique, and the 3' end was mapped to nt +342 by the primer extension technique. Northern analysis using RNA from mannose-grown cells suggested that mnp mRNA accumulated to a higher level than other mRNAs. A comparison of the deduced amino acid sequence of the mnp protein with those of the phosphoenolpyruvate-dependent mannose-specific E. coli mannose PTS proteins demonstrated that the mnp protein has the most homology with the E. coli protein encoded by the mannose PTS gene mapped at nt +45.Rio de Janeiro – American missionary Alan and Brazilian flight attendant María Deane Campbell have been reunited after almost three decades of separation.
In a reunion that had the blessing of the Associated Press, the couple from Hawaii and Brazil reunited after be359ba680
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