Hum Genet (1985) 70:109-115 © Springer-Verlag 1985 Direct assignment of orosomucoid to human chromosome 9 and 2HS-glycoprotein to chromosome 3 using human fetal liver x rat hepatoma hybrids Diane Wilson Cox 1 and Uta Francke 2 Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8 and Departments of Paediatrics, Genetics and Medical Biophysics, University of Toronto, Toronto, Canada 2Departments of Human Genetics and Pediatrics, Yale University School of Medicine, New Haven, CT, USA Summary. The production of plasma proteins has been moni- tored in somatic cell hybrids between a rat hepatoma cell line (7777) and human fetal liver cells. Production of 14 plasma proteins was assayed in concentrated serum-free culture supernatants by electroimmunoassay, a2HS-glycoprotein (AHSG) was produced by 10 of 19 hybrids; concordancy for presence or absence of protein production was 100% for human chromosome 3. Orosomucoid (ORM) was produced in 8 of 19 hybrids, with a concordancy for presence or absence of protein of 94.7% with human chromosome 9. The chromo- some location for genes for these two proteins, previously assigned by linkage studies, is confirmed by direct assignment. These studies have also suggested possible chromosomal assignments for loci for cq-antichymotrypsin and C1 esterase inhibitor. Other genes for proteins which could not be as- signed to specific chromosomes using these hybrids were: complement C3, ceruloplasmin, hemopexin, inter-a-trypsin inhibitor, prealbumin, retinol-binding protein, transferrin and apolipoproteins CII, B, and sinking-pre-beta [Lp(a)]. the occurrence of chromosome rearrangements, make karyo- type analysis difficult. Second, plasma protein production is not constitutive, and the relevant gene must be activated, which may not occur in all hybrid cells containing that struc- tural gene. Complete concordance between a single chromo- some and protein production therefore cannot be expected. Finally, the amount of protein secreted by the hybrids may be considerably lower than in the parental hepatoma cell line. We have used hybrids obtained from fusion of Morris rat hepatoma 7777 cells and cultured primary cells from a human fetal liver. This hybrid series XXII has previously been shown to produce human al-antitrypsin in the presence of chromo- some 14 (Pearson et al. 1983). Using immunologic assays, we have tested for the presence, in concentrated supernatant, of 14 human plasma proteins. For two plasma proteins, a chro- mosome assignment could be made. We have directly con- firmed the assignment of the orosomucoid (or cq-acid glyco- protein) gene (ORM) to chromosome 9 and the a2HS-glyco- protein gene (AHSG) to chromosome 3. Introduction Most plasma proteins are produced only in hepatocytes or their derivatives, therefore chromosomal assignment of genes for plasma proteins has not been possible with standard mouse/human fibroblast hybrids. However human plasma proteins have been produced by hybrids between hepatoma cell lines and leukocytes (Darlington et al. 1974), mouse RAG cells (Turner and Turner 1980), amniocytes (Darlington et al. 1982a), fibroblasts (Wray and Sutton 1982; Pearson et al. 1983), adult hepatocytes (Szpirer et al. 1980), lymphoblasts (Darlington et al. 1982b), and fetal hepatocytes (Pearson et al. 1983). These previous studies have indicated that the hybrids may (1) continue to express proteins already express- ed in the hepatoma parent, (2) turn off expression of such proteins, or (3) express a protein not originally produced in either of the parental cell lines. Several problems are encountered in using hepatoma hybrids for gene mapping. First, heteroploidy of the rodent hepatoma cell lines with large numbers of chromosomes, and Offprint requests to: D. W. Cox, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8 and Departments of Paediatrics, Genetics and Medical Biophysics, University of Toronto, Toronto, Canada Materials and methods Cell lines As reported previously (Pearson et al. 1983), rat hepatoma line 7777-14b-aza was derived from a Morris "minimal devia- tion" hepatocarcinoma originally generated by chemical carci- nogenesis and carried by serial transplants in Fisher rats (Mor- ris and Wagner 1968). Tumor cells were adapted to culture and were selected using 104M 8-azaguanine (AG). Clone 7777-14b-aza, which was AG resistant and HAT (hypoxan- thine-aminopterin-thymidine)-sensitive, was used for further studies. The karyotype was originally near diploid but re- arrangements have occurred during continuous culture for 15 months. Cell strain HFL 101 (human fetal liver cells) was de- rived from the liver of an apparently normal female fetus aborted at 20 weeks of gestation. Cell hybrids Series XXII hybrids were produced by fusion of rat hepatoma 7777-14b-aza with HFL 101 cells as described previously (Pearson et al. 1983). Chromosome analysis was carried out on trypsin-Giemsa-banded chromosomes as described pre- viously (Francke and Oliver 1978). The karyotype was assess-