Complementation by a cloned human ubiquitin-activating enzyme E1 of the S-phase-arrested mouse FM3A cell mutant with thermolabile E1.
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.
Ayusawa
D
D
Laboratory of Mutagenesis, National Institute of Genetics, Mishima, Japan.
Kaneda
S
S
Itoh
Y
Y
Yasuda
H
H
Murakami
Y
Y
Sugasawa
K
K
Hanaoka
F
F
Seno
T
T
D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005816:Genetic Complementation Test; D006358:Hot Temperature; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics / Q000502:physiology*; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016196:S Phase / Q000235:genetics / Q000502:physiology*; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases
D011506:Proteins; D012333:RNA, Messenger; D004247:DNA; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes