Demonstration, by somatic cell genetics, of coordinate regulation of genes for two enzymes of purine synthesis assigned to human chromosome 21.

Abstract

A method for determining coordinate genetic regulation is proposed for mammalian cells. The method involves (i) isolation of a set of mutants defective in the relevant pathway; (ii) complementation analysis of these mutants to determine dominance and to categorize the mutants into various different complementation groups; (iii) determination of the biochemical blocks in the mutants; (iv) identification of individual mutants that fail to complement the members ofat least two distinct complementation groups that complement each other, such mutants being said to show coordinate regulation of the affected functions; (v) biochemical and reversion analysis ofthe relevant cell types to confirm the basis for the observed coordinate regulation; (vi) assignment ofthe individual genes to particular human chromosomes; (vii) mapping of the genes to determine contiguity on the genome; and (viii) examination of the structure of the relevant gene products. This method has allowed the demonstration of coordinate regulation between the gene coding for phosphoribosylglycineamide synthetase [5-phosphoribosylamine:glycine ligase (ADP-forming), EC 6.3.4.13], defective in our Ade-C mutants, and the gene coding for phosphoribosylaminoimidazole synthetase [5'-phosphoribosylformylglycinamidine cyclo-ligase (ADP-forming), EC 6.3.3.1], defective in our Ade-G mutants. Moreover, both genes can be assigned to human chromosome 21. Because at least two genes for purine biosynthesis have now been assigned to chromosome 21, and because patients with trisomy 21 (Down syndrome) show increased levels of serum purines, it may be that cells ofthese patients overproduce purines and that this overproduction may be relevant to the pathology of the syndrome.

Measurement of Glycinamide Ribonucleotide Synthetase Activity in Intact Human Fibroblasts and Chinese Hamster Ovary Cells

“…This low value is due mainly to the low NH4CI and high ribose 5'-phosphate con centrations used. The second group of papers [Bartley and Epstein, 1980;Patterson et al, 1981;Bradley et al, 1982] based their assays on the method developed by Oates [1976]. While these conditions were similar to those reported here, there are two notable excep tions.…”

Potential Interaction Between the GARS-AIRS-GART Gene and CP2/LBP-1c/LSF Transcription Factor in Down Syndrome-related Alzheimer Disease

“…The human GARS-AIRS-GART gene is an important candidate gene in this context, as it localizes to chromosome 21q22.1 within the Down syndrome critical region ( Fig. 1A) (Patterson et al 1981;Hard et al 1986), subserves a critical biochemical function in terms of de novo purine biosynthesis ( Fig. 1B) (Aimi et al 1990), and is (Epstein 2001).…”

No Evidence for Mutations that Deregulate GARS–AIRS–GART Protein Levels in Children with Down Syndrome

“…Protein samples were dissolved in reducing Laemmli buffer containing [62.5 mM Tris-Cl (pH 6.8), 100 mM b-mercaptoethanol, 2% SDS, 0.1% bromophenol blue and 10% glycerol] [23] by boiling for 10 min. Equal amounts (10 lg) of protein were resolved by 10% SDS-PAGE at 20 mA constant current for 2 hours followed by wet transfer to nitrocellulose membrane (NC; Schleicher & Schuell, Keene, N.H.) at 110 volts (constant voltage) for 2 2 1/ hours at 4°C. The blots were placed in Subsequently blots were incubated with 1:5000 dilution of Horse Radish Peroxidase-conjugated secondary goat-antimouse antibody (IgG) [Bangalore Genei, India] for 2 hours at RT.…”

“…Human glycinamide ribonucleotide synthetase (GARS)-aminoimidazole ribonucleotide synthetase (AIRS)-glycinamide ribonucleotide transformylase (GART) is an important candidate gene in studies of DS-related MR by virtue of localization to chromosome 21q22.1 [2,3], over-expression in fetal postmortem DS cerebellum [4] and biochemical function in de novo purine biosynthesis [5]. GARS-AIRS-GART spans *38 kb with 22 exons [6] and the cDNA has been cloned by functional complementation in Escherichia coli [7].…”