DNA-mediated restoration of phenylalanine hydroxylase gene expression in enzyme-deficient derivatives of enzyme-constitutive mouse cell hybrids.
Phenylalanine hydroxylase (PH) gene expression is not extinguished in hybrids between PH- mouse A9 cells, or its neomycin-resistant derivative A9Neo-3, and PH+ mouse erythroleukemia (MEL) cells, PHC-3A, in contrast to its extinction in hybrids between A9Neo-3 and PH+ rat hepatoma cells, FT-2. Two different types of 6-thioguanine (TG) -resistant derivatives of these A9 X PHC-3A hybrids (LP), are generated in regard to PH gene expression. In regular growth medium supplemented with 10(-4) M TG (Tyr+/TG), TGr derivatives, all of which continue to express PH, occur with high frequency (approximately equal to 10(-3). In contrast, in tyrosine-deficient selective medium, supplemented with 10(-4) M TG (Tyr-/TG), no actively growing colonies are observed. Nevertheless, small colonies containing quiescent cells can be rescued by supplementing the medium with tyrosine. The rescued TGr clones do not express any detectable level of PH. Biochemical, hybridization, and cybridization analyses of one such rescued clone, LPTG-3, showed that these cells lack the regulatory factor capable of activating PH gene in PH- MEL cells. The PH- phenotype of LPTG-3 cells can be converted to the PH+ phenotype by transfection with restriction enzyme-digested or -undigested PHC-3A or mouse liver DNA. Therefore, these cells could be used to clone a fragment of DNA involved in PH gene regulation through DNA-mediated gene transfer methods.
D000818:Animals; D002460:Cell Line; D002999:Clone Cells; D004247:DNA / Q000235:genetics*; D004351:Drug Resistance; D005796:Genes; D006822:Hybrid Cells / Q000201:enzymology*; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000235:genetics; D051379:Mice; D009154:Mutation; D009355:Neomycin / Q000633:toxicity; D010651:Phenylalanine Hydroxylase / Q000172:deficiency / Q000235:genetics*; D013866:Thioguanine / Q000494:pharmacology
D007527:Isoenzymes; D004247:DNA; D007770:L-Lactate Dehydrogenase; D010651:Phenylalanine Hydroxylase; D013866:Thioguanine; D009355:Neomycin