Selection and analysis of galactose metabolic pathway variants of a mouse liver cell line.
To study the genetic expression and regulation of galactose-metabolizing enzymes, we mutagenized the mouse liver H2.35 cell line and selected for cell clones resistant to the toxic galactose analog, 2-deoxy-D-galactose (2-DOG). One cloned line, designated H12.10, was stably resistant to high levels of 2-DOG and was completely deficient in galactokinase activity. Galactokinase activity and growth sensitivity to 2-DOG could be restored by transfecting H12.10 cells with a plasmid containing the Escherichia coli galactokinase (galK) gene fused to a eucaryotic promoter; thus, the 2-DOG selection could be directed against transfected recombinant constructs in a liver cell line. We also found that H2.35 cells could not utilize galactose as a primary carbon source because of a deficiency in galactose-1-phosphate uridyltransferase; a variant line of H2.35 cells selected in galactose medium expressed higher levels of uridyltransferase activity. Finally, we found that in all mammalian cell lines tested, galactokinase expression was the same whether the medium contained glucose, galactose, or both sugars. These studies demonstrate differences between mammalian cells and yeast cells in the regulation of gal enzymes, and they define different schemes for obtaining altered expression of genes in the galactose metabolic pathway. The isogenic liver cell lines described here can also serve as model systems for studying galactosemias, which are inherited disorders of galactose metabolism in humans.
Zaret
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KS
Section of Biochemistry, Brown University, Providence, Rhode Island 02912.
Stevens
K A
KA
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002999:Clone Cells; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005686:Galactokinase / Q000235:genetics* / Q000378:metabolism; D005690:Galactose / Q000378:metabolism*; D005798:Genes, Bacterial; D014644:Genetic Variation; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics*; D015345:Oligonucleotide Probes; D014162:Transfection; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000235:genetics* / Q000378:metabolism
D015345:Oligonucleotide Probes; D005686:Galactokinase; D009713:Nucleotidyltransferases; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase; D005690:Galactose