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Macromolecule synthesis in temperature-sensitive mutants of methanol-utilizing Hansenula polymorpha
1Department of Agricultural Chemistry, National Taiwan University, Taipei, ROC.
Zhonghua Minguo Wei Sheng Wu Ji Mian Yi Xue Za Zhi = Chinese Journal of Microbiology and Immunology
|May 1, 1989
Summary
Researchers isolated temperature-sensitive (ts) mutants of Hansenula polymorpha. These mutants exhibit defects in DNA, RNA, or protein synthesis at restrictive temperatures, impacting cellular functions.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Methanol-utilizing yeast Hansenula polymorpha is a model organism for studying eukaryotic cell functions.
- Temperature-sensitive (ts) mutants are valuable tools for dissecting essential cellular processes.
- Understanding the molecular basis of ts phenotypes is crucial for yeast genetics and biotechnology.
Purpose of the Study:
- To isolate and characterize temperature-sensitive mutants of Hansenula polymorpha.
- To investigate the effects of temperature-sensitive mutations on cellular biosynthesis pathways (DNA, RNA, protein).
- To identify specific genes or pathways involved in temperature sensitivity.
Main Methods:
- Isolation of ts mutants using UV irradiation, Ethyl methanesulfonate (EMS), and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatments.
- Growth analysis at permissive (38°C) and restrictive (46°C) temperatures.
- Chemical composition analysis (RNA, protein content).
- Enzyme activity assays for aminoacyl-tRNA synthetases, particularly leucinyl-tRNA synthetase.
- Assessment of DNA, RNA, and protein synthesis under restrictive conditions.
Main Results:
- Several ts mutants of H. polymorpha were successfully isolated.
- Mutants NTU-AM-L2 and NTU-AM-L3 showed increased RNA content, while NTU-AM-E19 had higher protein content.
- Leucinyl-tRNA synthetase activity was prominent in both wild-type and ts strains.
- Shifting to restrictive temperature reduced total aminoacyl-tRNA synthetase activity, with a significant decrease in leucinyl-tRNA synthetase for mutant NTU-AM-E10.
- Specific mutants exhibited defects in DNA synthesis (NTU-AM-E10, NTU-AM-E20), RNA synthesis (NTU-AM-E15, NTU-AM-E20, NTU-AM-N37, NTU-AM-m5), and protein synthesis (NTU-AM-E10, NTU-AM-E20, NTU-AM-m5).
- Mutants NTU-AM-L2 and NTU-AM-L3 did not fit these classifications, suggesting distinct mechanisms of temperature sensitivity.
Conclusions:
- Temperature-sensitive mutations in H. polymorpha can lead to specific defects in DNA, RNA, or protein synthesis.
- Leucinyl-tRNA synthetase plays a critical role, as its activity is sensitive to temperature shifts.
- The isolated mutants provide a resource for further genetic and biochemical studies of essential cellular processes in H. polymorpha.
- Further investigation is needed to elucidate the precise molecular defects in mutants like NTU-AM-L2 and NTU-AM-L3.