Seven genetic diseases have been identified to date that involve alterations in purine metabolism. These include Lesch-Nyhan disease, severe combined immunodeficiency and most recently discovered, a defect in adenylosuccinate lyase that results in autism. Understanding the regulation of purine metabolism will aid in comprehending the basis of these diseases. The object of this proposed project is to understand the regulation of the de novo purine biosynthetic pathway of Escherichia coli K12 on a molecular basis. The regulation of de novo purine biosynthesis has three components: (1) the promoter-control region where the regulatory protein(s) bind; (2) the trans-acting regulatory protein(s); and (3) the effector molecule(s). The DNA sequence of the purJHD and purB loci will be determined to complete the sequence of the all known pur loci. The control regions and associated promoters will be identified by primer extension studies and examined for the presence of the homologies and two regions of imperfect dyad symmetry we have identified in the other loci. We will determine the role of these regions of dyad symmetry in pur loci regulation by in vitro saturation mutagenesis of selected control regions, i.e., purF, purA and guaBA loci. After characterization of the control region mutations by Sanger dideoxy sequencing, in vitro created pur-lac fusions will be employed to assess the effect on the expression of individual genes. Mutations that result in decreased expression will be used in a selection scheme to specifically identify trans-acting regulatory loci. This approach will distinguish between authentic trans-acting regulatory mutations and effector molecule level mutations which are normally genetically indistinguishable from true regulatory mutations. After isolation and identification, the specific trans-acting regulatory mutations will be mapped with linked transposons and standard recombinant DNA techniques will be used to clone and sequence the corresponding locus. The regulation of the different loci of the pathway will be tested under defined conditions in standard genetic background to determined the relationship between the different control region homologies and regulatory responses.
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