Many inborn errors of metabolism involve aberrations in either the basic biosynthetic pathway or the salvage and interconversion pathways for purine nucleotides. Understanding the mechanism of regulation of purine metabolism may ultimately aid in the control and treatment of such inborn errors of purine metabolism as gout, Lesch-Nyhan and severe combine immunodeficiency. Using the bacterium Escherichia coli K12, the purpose of this study is to investigate the nature of the regulation of this major biosynthetic pathway. We plan to extend our previous studies with pur-lac fusion strains to more fully exploit these strains for the isolation of regulatory mutations. Our proposed research will focus on DNA sequence studies of the control regions of the various pur loci for the comparison between the wild type and cis-acting regulatory mutations to determine the essential regulatory control features. Plasmids from the Clarke and Carbon collection will serve as the source of DNA for the DNA sequencing of the purA, purB, purE, purC and purJHD loci. In vitro pur-lac fusions will be constructed on a plasmid before transfer to a att+ bacteriophage for the isolation of regulatory mutations. The cis-acting regulatory mutations isolated on the bacteriophage will be recovered and sequenced. A method for the specific isolation of trans-acting regulatory mutations is proposed. The previously isolated trans-acting class of regulatory mutations will be genetically characterized for the purF (purR), purB (ARM::Tn10) and guaBA (guaR) loci and subcloned for DNA sequencing. A systematic search will be made for other transacting loci for these structural genes. These regulatory mutations will be transferred to other pur-lac fusion strains to determine how many different pur loci each regulatory locus controls.
Showing the most recent 10 out of 26 publications
