PHYSICAL STUDIES OF SIX LAMBDA-DILV PHAGES AND FORMATION AND ANALYSIS OF ILVGEDA OPERON LEADER REGION MUTATIONS
Abstract
Six (lamda)dilv phages contained six different gal-type inserts derived from an (TURN)35 kb region of the E. coli chromosome around 84.5 min. Analysis of the E. coli-lambda right arm junction in these phages revealed four unique secondary (lamda) attachment sites downstream from the ilvC gene. (lamda)dilv58 was found to carry an intact, normally controlled ilvGEDA operon. Phages lacking the ilv control region still exhibited the noncoordinate response that is independent of attenuation control. A derivative of (lamda)dilv58, (lamda)dilv-lac11, which carried an ilv-lac fusion operon controlled by the ilv control region was constructed. Two spontaneous derivatives of (lamda)dilv-lac11 with high level, constitutive expression of the fusion operon ((lamda)dilv-lac12 and (lamda)dilv-lac13) were isolated. (lamda)dilv-lac12 was found to have a 1597 bp deletion of the ilv leader region terminator and part of the ilvG gene, whereas (lamda)dilv-lac13 carried a 1869 bp deletion of the terminator and all of the ilvG gene. The deletion of the terminator in both mutants demonstrates that this structure must be causing the premature termination of RNA transcripts in vivo for the ilv-lac operon carried by (lamda)dilv-lac11. The loss of the terminator and the region of polarity in the ilvG gene resulted in very high (beta)-glactosidase activities with these mutant phages. There was, however, a low grade derepression of (beta)-glactosidase activity reminiscent of the non-coordinate response of the ilv operon. Three spontaneous mutant phages with low level, constitutive expression of the ilv-lac fusion operon were also isolated. Conditions for optimal formation of D-loops in the ilv leader region of plasmid pPU24 are described. S1 nuclease mutagenesis of D-loop containing pPU24 DNA was unsuccessful. Deletion mutations were formed in the ilv leader region of plasmid pPU119 by digestion of linear pPU119 DNA with Bal-31 exonuclease. Six mutant plasmids with small deletions (15 - 37 bp) in the protector structure of the leader region were isolated. One of the mutant plasmids was subjected to DNA sequence analysis and was found to contain a 15 bp deletion which removed three valine codons from the peptide coding region of the leader region transcript. This plasmid may not possess valine-specific control of ilvG expression.
Degree
Ph.D.
Subject Area
Microbiology
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