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

Area of Science:

• Cell Biology
• Molecular Genetics
• Biochemistry

Background:

• Cell cycle regulation is crucial for cell proliferation.
• Glycoprotein synthesis is essential for various cellular functions, including cell signaling and structure.

Purpose of the Study:

• To characterize a temperature-sensitive mutant of Chinese hamster cells with defects in cell cycle progression and glycoprotein synthesis.
• To investigate the molecular basis of the observed defects at the non-permissive temperature.

Main Methods:

• Cell synchronization and temperature shift experiments.
• DNA synthesis analysis using [3H]-thymidine incorporation.
• Flow microfluorimetry for cell cycle analysis.
• Analysis of labeled sugar incorporation (fucose) into glycoproteins.
• Chromatographic analysis of glycoprotein structure.
• Assessment of overall protein synthesis and polysome association with the rough endoplasmic reticulum.

Main Results:

• The mutant cells arrest in the G1 phase of the cell cycle at 40°C.
• Glycoprotein synthesis, particularly fucose incorporation, is significantly reduced at 40°C in all glycoprotein classes.
• Uptake and conversion of fucose to GDP-fucose are unaffected.
• Overall protein synthesis and rough endoplasmic reticulum-associated polysomes remain normal.
• The defect appears to be in an early step of glycoprotein synthesis or its regulation.

Conclusions:

• The identified mutation causes a temperature-sensitive defect in cell cycle progression and early glycoprotein synthesis.
• The mutation is recessive, and revertants restore normal growth and glycoprotein synthesis at the non-permissive temperature.