We now know that ubiquitination participates not only in the proteolytic function but also in many non-proteolytic reactions with crucial roles in cell metabolisms. For example, fluorescence ubiquitination-based cell cycle indicator enabled us to examine cell division within living cells by the Ub-proteasome system. In mammalian cells, there are dozens of E2s and several hundred E3s, and both define families of proteins displaying substrate specificity. However, there are only two E1 enzymes for the entire array of downstream reactions in mammals, Uba1 and Uba6. Uba1 encodes canonical E1. Previously, introduction and expression of epitope-tagged Uba1 cDNA constructs revealed that nuclear and cytoplasmic isoforms of Uba1 translate from first and second ATG codons: E1a, localized predominantly in the nucleus, and E1b, localized in the cytoplasm, respectively. To avoid confusion in terminology, we respectively refer to these two isoforms as Uba1A, defined here as the predominantly nuclear form of Uba1, and Uba1B, defined here as the cytoplasmic form of Uba1, instead of E1a and E1b. Uba6 is required to activate the E2Use1 both in vitro and in vivo and can also activate another ubiquitin-like modifier, FAT10. To identify genes responsible for the maintenance of chromosome integrity, Tsuji and colleagues isolated 25 temperaturesensitive mutants from hamster wild-type CHO-K1 cells. Using two of these mutants, we revealed that ts defects in RNA polymerase II and a protein involved in splicing gave rise both to chromosome instability and to cell cycle arrest. Another ts CHO-K1 mutant, tsTM3, exhibits chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39uC. Complementation tests with other mutants showed that tsTM3 did not complement with the Uba1-defective ts mutant ts85 and DNA replication-defective ts mutant ts131b, suggesting that these mutants harbor the same genetic defect. From 1980 to 1990, many ts mutants of Uba1 were isolated from several cell lines: ts85 of FM3A, ts20 of CHO, ts131b of FM3A, ts20 of Balb/c 3T3, tsBN75 of BHK21, tsFS20 of FM3A, and tsFT5 of FM3A. This unusually high incidence of Uba1 mutations was discussed in terms of Uba1 as a determinant of heat tolerance of cells and the fact that the Uba1 locus is located on the X chromosome. In regard to the connection between Uba1 and human disease, a recent study identified the association of pathogenic mutations in human UBA1 with an early-onset neurodegenerative disorder involving lower motor neurons. It provided evidence that the rare missense and synonymous mutations detected in exon 15 of UBA1 are associated with Xlinked spinal muscular atrophy. In the present study, to identify the mechanism underlying the tsTM3 phenotypes, we performed sequence analysis of the Uba1 gene and investigated the relation between a wild-type isoform of Uba1 tagged with green fluorescent protein and its localization.