Mutations in the Chinese hamster ovary cell GART gene of de novo purine synthesis

Abstract

Mutations in several steps of de novo purine synthesis lead to human inborn errors of metabolism often characterized by mental retardation, hypotonia, sensorineural hearing loss, optic atrophy, and other features. In animals, the phosphoribosylglycinamide transformylase (GART) gene encodes a trifunctional protein carrying out 3 steps of de novo purine synthesis, phosphoribosylglycinamide synthase (GARS), phosphoribosylglycinamide transformylase (also abbreviated as GART), and phosphoribosylaminoimidazole synthetase (AIRS) and a smaller protein that contains only the GARS domain of GART as a functional protein. The GART gene is located on human chromosome 21 and is aberrantly regulated and overexpressed in individuals with Down syndrome (DS), and may be involved in the phenotype of DS. The GART activity of GART requires 10-formyltetrahydrofolate and has been a target for anti-cancer drugs. Thus, a considerable amount of information is available about GART, while less is known about the GARS and AIRS domains. Here we demonstrate that the amino acid residue glu75 is essential for the activity of the GARS enzyme and that the gly684 residue is essential for the activity of the AIRS enzyme by analysis of mutations in the Chinese hamster ovary (CHO-K1) cell that require purines for growth. We report the effects of these mutations on mRNA and protein content for GART and GARS. Further, we discuss the likely mechanisms by which mutations inactivating the GART protein might arise in CHO-K1 cells.

Fig. 1

The de novo purine biosynthesis pathway. Enzymes associated with pathology are underlined. The steps encoded by the GART gene are indicated. Abbreviations are: R-5-P, ribose-5-phosphate; PRPP, phosphoribosylpyrophosphate; PRA, phosphoribosylamine; GAR, phosphoribosylglycinamide; FGAR, phosphoribosylformylglycinamide; FGAM, phosphoribosylformylglycineamidine; AIR, phosphoribosylaminoimidazole; CAIR, phosphoribosylaminoimidazole- carboxylate; SAICAR, phosphoribosylsuccinylaminoimidazole; AICAR, phosphoribosylaminoimidazolecarboaxamide; FAICAR, phosphoribosylformylaminoimidazolecarboxamide; IMP, inosine monophosphate; SAMP, adenylosuccinate; XMP, xanthosine monophosphate; AMP, adenosine monophosphate; GMP, guanosine monophosphate. Pathway enzymes are: (1) PRAT, phosphoribosylamidotransferase; (2) GARS, GAR synthetase; (3) GART, GAR transformylase; (4) FGARAT, FGAR amidotransferase; (5) AIRS, AIR synthetase; (6) AIRC, AIR carboxylase; (7) SAICARS, SAICAR synthetase; (8) and (12) ADSL, adenylosuccinate lyase; (9) AICART, AICAR transformylase; (10) IMPS, IMP synthetase; SAMPS, SAMP synthetase; IMPDH, IMP dehydrogenase; GMPS, GMP synthetase. PRPS1, phosphoribosylpyrophosphate synthase 1

Fig. 2

Transfection of AdeC and AdeG (55-1) cells with wild-type and mutant cDNA constructs. The wild-type and mutant GART clones indicated were transfected into AdeC or AdeG (55-1) cells in F12 without purines and clones able to grow in the absence of purines were scored as described in Materials and Methods. Error bars indicate the standard deviation of the measurements. pT indicates transfection with the empty cloning vector.

Fig. 3

Northern blot of mRNA from wild-type and GART mutant CHO cells. Total RNAs were isolated from CHO cells, separated on 1% agarose-formaldehyde gel, transferred to nylon membrane and probed with (A) Labeled PCR fragment of GARS cDNA. (B) The membrane was stripped then reprobed with a control cyclophilin cDNA. 1 = CHO-K1; 2 = AdeC; 3 = AdeG (55-1).

Fig. 4

CHO-GART copy number in CHO mutants. Fluorescence in situ hybridization of metaphase chromosomes from CHO-K1, AdeC, and AdeG (55-1) cells. The left panel is CHO-K1, the middle panel is AdeC, and the right panel is AdeG (55-1). The probe was made from two fragments of the CHO GART gene that were purified and labeled with SpectrumOrange (Vysis Inc.).