GENETIC AND PHYSICAL ANALYSES OF THE ILVBN OPERON OF ESCHERICHIA COLI K-12 (CLONING, SEQUENCING)
Abstract
Genetic and physical analysis of the ilvBN operon of Escherichia coli K-12 was undertaken to study its regulation. This operon, located at 81.5 minutes on the Escherichia coli K-12 chromosome, codes for the large and small subunits of acetohydroxy acid synthase I. The ilvB and ilvN genes were physically separated and cloned onto individual plasmids. The ilvN protein was found to be associated with a 1.6 kb ilvBN fragment. The ilvN protein was produced in vivo in a maxicell system and in vitro. The size of the ilvN protein is approximately 11 kda. Deletions that extend into the carboxy terminal of the ilvN gene were generated by nuclease Bal31 digestion. These deletions were cloned and tested for the ability to complement mutations generated by insertion of the (OMEGA) fragment into either the ilvB or ilvN gene. ilvN deletion plasmids did complement the ilvB::(OMEGA) mutation, although the activity of acetohydroxy acid synthase I was very low. Plasmids carrying the ilvN gene controlled presumably by a plasmid promoter were able to complement the ilvN::(OMEGA) mutation. Thus, the ilvN gene can act in trans. Sequence analysis revealed the extent of each ilvN deletion. The nucleotide sequence of the ilvN and the distal third of the ilvB genes was determined. A nucleotide sequence typical of a rho-independent terminator was found just distal to the ilvBN operon.
Degree
Ph.D.
Subject Area
Molecular biology
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