Glycoprotein synthesis in a temperature-sensitive Chinese hamster cell cycle mutant

Abstract

A temperature-sensitive mutant of Chinese hamster cells is described which has two interesting properties: (1) it is a cell cycle mutant and (2) glycoprotein synthesis appears to be affected at the at the non-permissive temerature (40degreesC). Synchronized cells shifed to 40degreesC in the beginning of their G1 phase do not incorporate [3H]-thymidine into DNA during the expected S-phase, but once DNA synthesis has been initiated ( approximately 10 hours after termination of serum starvation) a shift to 40 degrees C no longer leads to an arrest of DNA synthesis. Flow microfluorimetric analysis of DNA content/cell supports this conclusion and indicates that a majority of cells become arrested in the G1 phase of the cell cycle when a non-synchronized population of cells is transferred to 40degreesC. Apparently at all times in the cell cycle there is a drastic reduction if incorporation of labeled sugars (particularly fucose) into glycoproteins. The uptake of fucose and its conversion to GDP-fucose appears to be normal at 40degreesC. Chromatographic analysis indicates that all classes of glycoproteins are affected, and we do not find any evidence for partially completed oligosaccharides at 40 degrees C. Overall protein synthesis is not reduced at he nonpermissive temperature during the time interval under consideration and the number of polysomes attached to membranes (RER) is also normal at 40degreesC. This suggests that the defect is at an early step in the synthesis or regulation of synthesis of glycoproteins. The mutation is a recessive mutation in hybrid cells and mutagen induced revertants can be obtained which grow normally at 40degreesC and in which glycoprotein synthesis at 40 degrees C is restored to normal, wild type levels.