GENETIC AND PHYSICAL ANALYSES OF THE ILVGEDA OPERON AND THE ILVB GENE OF ESCHERICHIA COLI K-12
Abstract
Genetic and physical analyses of the ilvGEDA operon and the ilvB gene of Escherichia coli K-12 were undertaken to study gene regulation. Transcription termination factor (rho) was shown not to be involved in the "downstream amplification" pattern of ilvGEDA expression. A cis-trans analysis of three ilvO mutations (ilvO mutations allow both ilvG expression and increased ilvEDA expression) demonstrated that the ilvO determinant functions in cis. A specialized transducing phage carrying a mutation that causes elevated ilva and ilvD expression under ilv-specific control was isolated to facilitate the physical analysis of this mutation. Restriction endonuclease and heteroduplex analysis of this phage, (lamda)h80dilv-2130, revealed the deletion to be 1.75 kb, from within the ilvE gene to within the ilvG gene, thus removing the ilvO determinant which is assumed to be a site of natural polarity within the ilvG gene, downstream of the ilvGEDA control region. The ilvB gene was cloned from E. coli K-12 to physically define the gene and to study its mechanisms of control. Analysis of the hybrid plasmid, pPU38, and its derivatives showed that the ilvB gene is approximately 1600 bp and that the gene is transcribed in a clockwise direction, both on pPU38 and on the E. coli K-12 chromosome as the maps are usually represented. The cya or carbon source effect on ilvB expression appeared to be that of a slight increase. Construction of the ilvB::galK fusion confirmed the location of the ilvB promoter and showed that the leuS and valS gene products affect ilvB expression at the level of transcription initiation. The fusion demonstrated that ilvB transcription initiation was normal in cpxA, cpxB strains. Neither the E. coli ilvB gene nor the E. coli ilvB::galK gene was expressed as well in Salmonella typhimurium as in E. coli K-12, indicating that some factor required for ilvB expression in E. coli k-12 is lacking in S. typhimurium. Base sequence analysis of the ilvB promoter yielded no evidence that ilvB is controlled by attenuation. A model for the positive control of expression of the ilvB gene is discussed.
Degree
Ph.D.
Subject Area
Biochemistry
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