Autoradiographic detection and characterization of a Chinese hamster ovary cell mutant deficient in fucoproteins.
Autoradiography of colony replicas immobilized on filter paper was used to isolate a Chinese hamster ovary cell line deficient in incorporation of radiolabeled fucose into a trichloroacetic acid-insoluble fraction. This cell line, called 62.1, has the same growth rate at 37 degrees C as wild-type cells, but incorporates five times less fucose into acid-insoluble radioactivity. Chemical analysis of fucose bound to macromolecules also showed a fivefold reduction in the mutant. The fucoproteins of the mutant cell line differ qualitatively from those of wild-type cells as visualized by SDS gel electrophoresis fluorography; no differences were detected between total proteins as visualized by coomassie blue staining. The macromolecular sialic acid content of the mutant was somewhat higher than the wild type (20%). Studies of the synthesis of the glycoprotein of vesicular stomatitis virus in mutant and wild-type cells showed that the mutant is unable to synthesize complex-type N-linked oligosaccharides. Enzyme assays show that ths defect in the mutant is due to reduction in UDP-N-acetylglucosamine-glycoprotein N-acetyl-glucosaminyltransferase, a key enzyme in the assembly of complex glycopeptides. Hybridization studies have shown that mutant 62.1 has common mutations belonging to the same complementation group as mutant PhaR1-1. This latter mutant was previously isolated using lectin resistance by Stanley et al. (1975) and was also deficient in the above N-acetyl-glucosaminyltransferase.
D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D002472:Cell Transformation, Viral; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006023:Glycoproteins / Q000096:biosynthesis / Q000172:deficiency*; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D008562:Membrane Glycoproteins; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis; D010053:Ovary; D014316:Tritium; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000235:genetics
C028391:G protein, vesicular stomatitis virus; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C016465:fucoproteins; D014316:Tritium; D008358:Mannose