FISH mapping of three ammonia metabolism genes (Glul, Cps1, Glud1) in rat, and the chromosomal localization of GLUL in human and Cps1 in mouse

In recent years, the rat genes encoding glutamate dehydrogenase (GLUD; Das et al. 1993), glutamine synthetase (glutamateammonia ligase, GLUL; van de Zande et al. 1990) and carbamoylphosphate synthetase 1 (CPS; Van den Hoff et al. 1995) have been isolated. These enzymes have important functions in ammonia metabolism, and each of them is encoded by a single gene. GLUD (E.C. 1.4.1.3) catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia, using NAD+ or NADP+ as cofactor. GLUL (E.C. 6.3.1.2) catalyzes the synthesis of glutamine from glutamate, thereby hydrolyzing ATP to ADP. CPS (E.C. 6.3.4.16) is the first and rate-determining enzyme of the ornithine cycle and catalyzes the production of carbamoylphosphate from ammonia, bicarbonate and ATP. In the present communication, we mapped the position of these three genes in the rat by FISH and by somatic cell hybrids. In addition, we determined the chromosomal location of the human GLUL gene and of the mouse, Cps1 gene by FISH. For the FISH mapping of the human glutamine synthetase gene (also called glutamate-ammonia ligase, approved human gene symbol GLUL), a human cDNA (total 2738 bp in pBluescript; Van den Hoff et al. 1991) was used. As shown in Fig.1a, the gene could be unequivocally mapped to Chromosome (Chr) (HSA) 1, band q25. The three genes were mapped in the rat both with a rat-mouse somatic cell hybrid panel (Szpirer et al. 1984; Klinga Levan et al. 1993) and with FISH. For the mappings with the cell hybrid panel, cDNA probes of approximately 1000 bp from the 38 ends of the genes were used (Glul 1111 bp, van de Zande et al. 1990; Glud1 957 bp, Das et al. 1989; Cps1 883 bp, De Groot et al. 1986). The probes were labeled with radioactivity by use of a-P-CTP and the random priming method. They were subsequently hybridized to filters containing 15 mg of genomic DNA from each hybrid restricted with EcoRI or BamHI. In each case the rat hybridizing fragments could be distinguished from the mouse bands, and the genes were assigned as follows: Glud1 to rat Chr (RNO) 16, Cps1 to RNO9, and Glul to RNO13. In the rat, the FISH results corroborated and refined the findings from the somatic cell hybrid panel. Longer probes are preferred in FISH analysis, and for the regional mapping of the Glul gene with FISH, two genomic clones were used (pgGS2, 5000 bp including exons 2–6, and pgGS4, 4500 bp including exon 1; van de Zande et al. 1990). Both probes gave very clear signals at the same chromosomal location, and rat Glul could be sublocalized to RNO13q22 (Fig. 1b). The rat G-band nomenclature is according to Levan (1974); for an updated recent version of the idiogram, see RATMAP database (URL http://ratmap.gen.gu.se/ratmap/WWW Nomen/RNOIdiogrRev96new.GIF). For the mapping of rat Glud1, two clones of genomic DNA (pgGDH6d, 6700 bp containing exon 1, and pgGDH2u, 5800 bp containing exons 8–12; Das et al. 1993) were used. The results from each of the probes were the same, and Glud1 could be sublocalized to RNO16p16 (Fig. 1c). Since RNO16 is a metacentric chromosome in which both chromosome arms have very similar stainability and banding pattern except in optimal metaphases, we wanted to check our conclusion with respect to which chromosome arm carried the Glud1 gene. Sasaki and associates (1994) have published excellent pictures of FISH mapping of the Atp7b gene (Wilson Disease gene homolog), and convincingly assigned this gene to RNO16q12.3. We used the same probe (designated pWD4) in simultaneous hybridizations with the Glud1 probe and could show that the two genes were located on opposite chromosome arms (Fig. 1d), thus verifying our conclusion that Glud1 is at RNO16p16. For the FISH mapping of the rat carbamoyl-phosphate synthetase gene (Cps1), a full-length cDNA probe (cCPSf.1, 5500 bp; De Groot et al. 1986) was used. The findings corroborated the previous hybrid panel mapping, and the Cps1 gene could be sublocalized to 9q34 (Fig. 1e). Since the Cps1 gene had not been mapped in the mouse, we attempted to map it by FISH with the rat cDNA probe. This worked out well, and the mouse Cps1 gene could be assigned to mouse Chr (MMU) 1, band C3 (Fig. 1f). Comparative mapping shows that the human GLUL gene is comprised in a region spanning bands HSA 1q22–1q42 and containing 11 human genes for which there are homologous rat genes on RNO13 (Table 1). Only seven of these genes have been mapped also in the mouse, but they are all situated distally in MMU1 (spanning about 26 cM from C4bp at map position 68 to Atp1a2 at map position 94; mouse data from Mouse Genome Database, MGD). The human homolog of the rat Glud1 gene is located at HSA 10q23.3, and the mouse homolog is on MMU14. Glud1 is included in a group of three genes (also comprising Rbp3 and Sftp1) that is conserved on these chromosomes. In contrast, the human and mouse genes homologous to the Atp7b gene on the long arm of RNO16 are on HSA13 and MMU8, respectively, as is the Atp4b gene, which is also located on RNO16. Thus, it looks as if RNO16 resulted from the fusion of two chromosome segments that are on separate chromosomes in both humans and mice, and, therefore, occurred after the separation of the rat lineage from human and mouse lineages. The human homolog of Cps1 is on HSA2q33–36. In total, there are nine genes on HSA2q (spanning the segment 2q32–2q37) that have their homologs on RNO9 (Table 1). A corresponding segment in the mouse is located on MMU1 (spanning 31 cM from Slc9a2 at map position 21 to Ugt1a1 and Akp3 at position 52). We have pointed out earlier that Correspondence to: G. Levan Mammalian Genome 8, 362–364 (1997).