Studies on the structure and expression of Escherichia coli pyrC, pyrD and pyrF using the cloned genes

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Abstract
The E. coli pyrC, pyrD and pyrF genes were cloned on multicopy plasmids derived from pBR322 and analyzed by means of restriction endonucleases. The pyrC gene is destroyed by cutting with the restriction endonuclease BamHI, the entire pyrD gene can be isolated on a 1300 base pairs DNA fragment generated by EcoRI cleavage and cutting with EcoRI removes the promoter and probably the translational start site from the pyrF gene. More details on the restriction maps are presented. The presence of a pyr gene in multiple copies on a plasmid does not significantly interfere with the activity of the chromosomal pyr genes. Using the minicell technique, the polypeptides encoded by the 3 cloned pyr genes were identified. The relative molecular masses for the pyrC-encoded and pyrD-encoded polypeptides are 38,000-40,000 and 36,000-38,000, respectively. In their native form, dihydroorotase and dihydroorotate oxidase appear to be dimeric proteins. The minicell experiments positively identified a protein chain of MW 23,000-24,000 as being a subunit of OMP decarboxylase encoded by pyrF. The coding frame for this polypeptide seems to be expressed as the 1st gene in the operon with the coding frame for another protein chain of MW 13,000-14,000. Since the native OMP decarboxylase during sedimentation and gel filtration behaves as a protein of MW 45,000 .+-. 4000, this latter polypeptide (MW 13,000-14,000) is hardly a component of the enzyme. Pyr-lac+ operon fusions were constructed by the Mu d1 procedure. By integrating an F''lac episome into the lac part of the fusions and determining the direction of chromosomal transfer from the resultant Hfr strains, the direction of pyrC transcription was found to be counter-clockwise, while pyrD and pyrF were transcribed in a clockwise direction.