Autoradiographic detection and characterization of a Chinese hamster ovary cell mutant deficient in fucoproteins

Publisher Website
Google Scholar
Abstract
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°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 this defect in the mutant is due to reduction in UDP‐N‐acetylglucosamine‐glycoprotein N‐acetyl‐glucos‐aminyltransferase, 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.