Complementation by a Cloned Human Ubiquitin-Activating Enzyme E1 of the S-phase-arrested Mouse FM3A Cell Mutant With Thermolabile E1

Cell Struct Funct. 1992 Apr;17(2):113-22. doi: 10.1247/csf.17.113.

Abstract

A temperature-sensitive growth mutant tsFS20 isolated from mouse FM3A cells was identified as a mutant with thermolabile ubiquitin-activating enzyme E1 by transfection with a full-length cDNA encoding the human E1 enzyme and cell-cell hybridization with an authentic E1 mutant ts85 previously isolated from FM3A cells. The resulting transformants produced thermoresistant E1 activity. Upon shift-up of temperature, asynchronously growing tsFS20 cells showed multiple points of cell-cycle arrest. At the nonpermissive temperature, tsFS20 cells that had been synchronized at the G1-S-phase progressed and accumulated in the mid-S-phase, as evidenced by the absence of G2-specific cdc2 kinase activity, while ts85 mutant cells, the widely used E1 mutant, reached the G2-phase and were arrested. Thus, the E1 mutation seemed to be involved in progression in the S-phase as well as in the G2-phase in the cell cycle. Degradation of short-lived abnormal proteins in tsFS20 cells was decreased to about 50% at the nonpermissive temperature, while the block was fully restored to the wild-type level in the transformant cells. Relevance of the unusually high incidence of the temperature-sensitive E1 mutation was discussed in terms of the E1 as a determinant of heat tolerance of cells.

MeSH terms

  • Animals
  • Cell Cycle / physiology
  • Cell Line
  • Cloning, Molecular
  • DNA / genetics
  • Genetic Complementation Test
  • Hot Temperature
  • Humans
  • Ligases / chemistry
  • Ligases / genetics
  • Ligases / physiology*
  • Mice
  • Molecular Sequence Data
  • Mutation / genetics
  • Mutation / physiology*
  • Proteins / metabolism
  • RNA, Messenger / metabolism
  • S Phase / genetics
  • S Phase / physiology*
  • Ubiquitin-Activating Enzymes
  • Ubiquitin-Protein Ligases

Substances

  • Proteins
  • RNA, Messenger
  • DNA
  • Ubiquitin-Protein Ligases
  • Ligases
  • Ubiquitin-Activating Enzymes

Associated data

  • GENBANK/S47136
  • GENBANK/S47137
  • GENBANK/S47164
  • GENBANK/S47165
  • GENBANK/S47166
  • GENBANK/S47167
  • GENBANK/S47168
  • GENBANK/S51472
  • GENBANK/S72771
  • GENBANK/X56976