De novo purine nucleotide biosynthesis: cloning of human and avian cDNAs encoding the trifunctional glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide transformylase by functional complementation in E. coli.
The trifunctional enzyme encoding glycinamide ribonucleotide synthetase (GARS)-aminoimidazole ribonucleotide synthetase (AIRS)-glycinamide ribonucleotide transformylase (GART) was cloned by functional complementation of an E. coli mutant using an avian liver cDNA expression library. In E. coli, genes encoding these separate activities (purD, purM, and purN, respectively) produce three proteins. The avian cDNA, in contrast, encodes a single polypeptide with all three enzyme activities. Using the avian DNA as a probe, a cDNA encoding the complete coding sequence of the trifunctional human enzyme was also isolated and sequenced. The deduced amino acid sequence of the human and avian polyproteins show extensive sequence homologies to the bacterial purD, purM, and purN encoded proteins. Avian and human liver RNAs appear to encode both a trifunctional enzyme (G-ARS-AIRS-GART) as well as an RNA which encodes only GARS. The trifunctional protein has been implicated in the pathology of Downs Syndrome and molecular tools are now available to explore this hypothesis. Initial efforts to compare the expression of GARS-AIRS-GART between a normal fibroblast cell line and a Downs Syndrome cell line indicate that the levels of RNA are similar.
Aimi
J
J
Purdue University, Department of Biochemistry, West Lafayette, IN 47907.
Qiu
H
H
Williams
J
J
Zalkin
H
H
Dixon
J E
JE
D000217:Acyltransferases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001717:Birds / Q000235:genetics*; D019731:Carbon-Nitrogen Ligases; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004314:Down Syndrome / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases; D008025:Ligases / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D051026:Phosphoribosylglycinamide Formyltransferase; D011685:Purine Nucleotides / Q000096:biosynthesis; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D009097:Multienzyme Complexes; D011685:Purine Nucleotides; D004247:DNA; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D000217:Acyltransferases; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C022380:phosphoribosylaminoimidazole synthase; C494823:GART protein, human; C022387:phosphoribosylamine-glycine ligase