[Cloning and gene expression of the leukocytosis (lymphocytosis) stimulating factor of Bordatella pertussis in Escherichia coli by bringing the PT genes close to the lactose promoter of plasmid pUC19].
The 4.7 Kb EcoRI-fragment of phase I B. pertussis 475 (serovar 1.2.3.) chromosome carrying all five genes of the pertussis toxin (PT) operon was cloned on plasmid pUC19 in E. coli. The resulting hybrid plasmid pRH119 contained the PT operon in the same orientation of transcription as the lac promoter of plasmid pUC19. Nevertheless, the expression of the PT operon was not observed even after induction with isopropyl thio-beta-D-galactopyranoside (IPTG), which suggested either the inability of the PT operon to work in E. coli, or the presence of a transcription terminator between the lac promoter and the PT operon in plasmid pRH119. The expression was determined by the incubation of the clones harboring plasmid pRH119 with antiserum to PT and their subsequent in situ treatment with 125I-labeled protein A. Three deletion variants of plasmid pRH119 were constructed with the aim of approaching the PT genes to the lac promoter: pRH121 (the 0.45 Kb KpnI-fragment deleted), pRH122 (the 0.95 Kb SalGI-fragment deleted) and pRH123 (the 1.35 Kb XbaI-fragment deleted). In all these cases different levels of expression were observed (but only in the presence of IPTG). Site KpnI in the 4.7 Kb fragment was found to be localized in the -57 b. p. region in relation to the PT promoter, i. e. to lie, seemingly, in the promoter zone; for this reason, the expression of the PT genes in plasmid pRH121 proved the existence of a transcription terminator between the lac promoter and the PT operon in plasmid pRH119.(ABSTRACT TRUNCATED AT 250 WORDS)
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D001886:Bordetella pertussis / Q000235:genetics*; D002872:Chromosome Deletion; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D007785:Lactose / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic
D001426:Bacterial Proteins; D007785:Lactose
Klonirovanie i ékspressiia genov leĭkotsitoz (limfotsitoz)-stimuliruiushchego faktora Bordetella pertussis v Escherichia coli putem priblizheniia genov k laktoznomu promotoru plazmidy pUC19.