Study of genes involved in leucine transamination in {\it Escherichia coli\/}

Date of Completion

January 1990

Keywords

Biology, Molecular|Biology, Genetics|Biology, Microbiology

Degree

Ph.D.

Abstract

A Tn5 induced presumptive tyrB mutation isolated in an ilvE strain of Escherichia coli K-12 was shown to map in the leu operon. In spite of the presence of an active tyrB gene, this mutant was unable to grow on 2-KIC, the immediate leucine precursor. Other ilvE leu operon double mutants constructed had a similar phenotype as the presumptive ilvE tyrB mutant. Accumulation of 2-KIV in these strains interferes with transamination of 2-KIC to form leucine by the tyrB encoded transaminase, transaminase D.^ The Tn5 insertion in this strain was cloned in vivo using a mini-Mu vector, Mu d114042. A novel way was developed to isolate in vivo deletion derivatives of these plasmids which carried either one or the other end of Tn5 and the adjacent chromosomal DNA. These derivatives were useful in sequencing the adjacent chromosomal region. Sequencing revealed that the Tn5 was inserted in the leu leader region.^ Mu d114042 plasmids which complemented this leuL::Tn5 insertion and thus carried the entire leu operon were isolated and a fragment carrying the leu operon was subcloned. This subclone did not complement the leu::Tn5 insertion mutation in one strain, but complemented a leuD mutant. The same fragment complemented the leu::Tn5 strain when present in another plasmid. Results show that DNA topology and the level of plasmid supercoiling plays a role in leu operon expression.^ Results show that DNA topology and the level of plasmid supercoiling plays a role in leu operon expression.^ A mini (1.8 kb) Kan$\sp{\rm r}$ derivative of the transposon $\gamma\delta$ (Tn1000), called M$\gamma\delta$-1 was constructed by cloning the Tn5 kan gene and the res region required for transposition in between 38 bp of the $\delta$ end of $\gamma\delta$. This element is useful for plasmid mutagenesis, allele replacement and DNA sequencing, and transposes as well as wild type $\gamma\delta$.^ A plasmid containing a contraselectable marker (thyA) and, adjacent to it, a $\gamma\delta$ derived mini transposon ($\delta$-kan-$\delta$) was constructed for obtaining nested deletions in vivo. Nested deletions for sequencing DNA adjacent to thyA would be easily obtainable using this plasmid by selecting for Tmp$\sp{\rm r}$ colonies. ^

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