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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020068-09
Application #
3129592
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1987-12-01
Project End
1992-11-30
Budget Start
1990-12-01
Budget End
1992-11-30
Support Year
9
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Seattle Biomedical Research Institute
Department
Type
DUNS #
City
Seattle
State
WA
Country
United States
Zip Code
98109
Marolewski, A; Smith, J M; Benkovic, S J (1994) Cloning and characterization of a new purine biosynthetic enzyme: a non-folate glycinamide ribonucleotide transformylase from E. coli. Biochemistry 33:2531-7
Nygaard, P; Smith, J M (1993) Evidence for a novel glycinamide ribonucleotide transformylase in Escherichia coli. J Bacteriol 175:3591-7
He, B; Smith, J M; Zalkin, H (1992) Escherichia coli purB gene: cloning, nucleotide sequence, and regulation by purR. J Bacteriol 174:130-6
Andersen, P S; Smith, J M; Mygind, B (1992) Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12. Eur J Biochem 204:51-6
Meyer, E; Leonard, N J; Bhat, B et al. (1992) Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway. Biochemistry 31:5022-32
Cheng, Y S; Shen, Y; Rudolph, J et al. (1990) Glycinamide ribonucleotide synthetase from Escherichia coli: cloning, overproduction, sequencing, isolation, and characterization. Biochemistry 29:218-27
He, B; Shiau, A; Choi, K Y et al. (1990) Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction. J Bacteriol 172:4555-62
Flannigan, K A; Hennigan, S H; Vogelbacker, H H et al. (1990) Purine biosynthesis in Escherichia coli K12: structure and DNA sequence studies of the purHD locus. Mol Microbiol 4:381-92
Inglese, J; Smith, J M; Benkovic, S J (1990) Active-site mapping and site-specific mutagenesis of glycinamide ribonucleotide transformylase from Escherichia coli. Biochemistry 29:6678-87
Inglese, J; Johnson, D L; Shiau, A et al. (1990) Subcloning, characterization, and affinity labeling of Escherichia coli glycinamide ribonucleotide transformylase. Biochemistry 29:1436-43

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