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Cloning the trpR gene.

W Roeder, R L Somerville

Molecular & General Genetics : MGG

|November 1, 1979

Summary

Researchers mapped the Escherichia coli trpR gene using specialized phages and restriction enzymes. Increased gene dosage did not affect tryptophan biosynthesis regulation, suggesting complex control mechanisms.

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Area of Science:

Microbiology
Molecular Biology
Genetics

Background:

The Escherichia coli deoD gene, encoding purine nucleoside phosphorylase, can be inactivated by prophage lambda insertion.
Deletions in the deo-thr region of the E. coli chromosome, including the trpR gene, can be isolated from lambda lysogens.

Purpose of the Study:

To physically map the Escherichia coli trpR gene within the deoD-trpR region.
To investigate the effect of increased gene dosage on tryptophan biosynthesis regulation.

Main Methods:

Isolation of specialized transducing phages carrying bacterial DNA segments.
Restriction enzyme analysis (BamHI, SalI, PvuI) to map cleavage sites.
Cloning of the trpR gene into the pBR322 plasmid.
Analysis of gene expression under conditions of increased gene dosage.

Main Results:

The trpR gene was localized to a 950 base pair BamHI-PvuI DNA segment.
A functional trpR gene was cloned on a 1250 base pair BamHI fragment.
A SalI restriction site was identified within the trpR gene.
Increased trpR gene dosage did not lead to tryptophan auxotrophy, altered sensitivity to DL-5-methyl-tryptophan, or super-repression of tryptophan biosynthetic enzymes.

Conclusions:

The physical location and a functional clone of the E. coli trpR gene were established.
The regulation of tryptophan biosynthesis is not solely dependent on repressor protein concentration, indicating complex regulatory mechanisms.

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