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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020068-04
Application #
3129588
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1982-09-01
Project End
1987-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
4
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Louisiana State University Hsc Shreveport
Department
Type
Schools of Medicine
DUNS #
City
Shreveport
State
LA
Country
United States
Zip Code
71103
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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