Biomedical subjects
Publications and source records attributed to D Ayusawa.
The human gene for ubiquitin-activating enzyme E1 (UBE1) was localized by a direct mapping system that combined fluorescence in situ hybridization with replicated R-bands on prometaphase chromosomes. The fluorescent signals were localized to Xp11.3----p11.23. Simple procedures for the detection of R-bands are also described.
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.
Synchronization of cells to various phases of the cell cycle in mammalian cells is crucial to analyze cell cycle progression and many other cellular functions. To date, various methods have been developed, such as mitotic selection, use of cell-cycle mutants, use of drugs which inhibit DNA replication (nucleoside analogue, excess thymidine, hydroxyurea, and aphidicolin), elutriation, deprivation of nutrients (amino acid, and serum), and new promising drugs of protein kinase inhibitors. Although above methods have both advantage and disadvantage, a practical and satisfactory method can be chosen from these method if taking into account goals of experiments and cell types to use. In near future, more powerful and reliable ones will be discovered since our understanding of the cell cycle has been increasing quite abruptly.
We studied whether homologous recombination activity in mammalian cells could be induced by thymidylate stress (thymidylate deprivation). In vitro recombination activity in cell extracts was measured with pSV2neo-derived plasmids. When prior to the preparation of extracts, mouse FM3A cells were grown in 5-fluorodeoxyuridine (FdUrd), an inducer of thymidylate stress, the homologous recombination activity was significantly induced, as judged from an increase in the number of neomycin-resistant bacterial colonies. Maximum induction was observed in cells treated with 1 microM FUdR for 16 h. However, 3-8 h of treatment of FM3A cells with the drug followed by an additional 8-16-h incubation in its absence was sufficient to induce the recombination activity while slightly reducing their growth rates. These results indicate that thymidylate stress induces homologous recombination activity in mammalian cells as observed in Escherichia coli and in yeast.
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The complete nucleotide sequence of the human thymidylate synthase (TS) gene was determined. The biologically active unit spans about 16 kilobase pairs (kbp) and is composed of seven exons and six introns. The promoter region and the major transcriptional start sites were located within about 400 base pairs (bp) and 160-180 bp, respectively, upstream from the ATG initiation codon. A minigene consisting of a 5'-flanking sequence of about 4 kbp, a coding sequence of about 1.7 kpb was sufficient for cell cycle-dependent expression of TS when introduced into a TS-deficient mutant of rodent cells. The 5'-flanking region does not contain a "TATA," "CAAT," or the consensus sequence for Sp1 binding (GC box), whereas intron 1 contains three GC boxes. The gene is G + C-rich and the CpG-rich region extends from 5'-flanking region to intron 1. These sequence characteristics were compared with those of the mouse TS gene and other cell cycle-regulated genes.
The human thymidylate synthase (TS) gene was regionally assigned to chromosome band 18p11.32 by nonisotopic in situ hybridization using biotinylated cDNA (1.1 kb insert) and genomic DNA (6.8 kb insert) probes of the human gene. There have been two provisional assignments for the TS gene to 18pter-q12 and 18q21-qter. The present result confirmed the first of these and further localized the TS gene to the telomeric region of the short arm of chromosome 18. The TS gene appears to be a novel telomeric anchor point for the construction of both physical and genetic linkage maps of human chromosome 18.
Spontaneous mutants of mouse FM3A cells (AC1, AC2, and AC3), highly resistant to aphidicolin (3000-, 2500-, and 300-fold increase in resistance, respectively), were isolated by multistep selection. The DNA synthesizing activity in permeabilized cells of all three mutants was substantially resistant to aphidicolin, like that in intact cells. The DNA polymerase activity in nuclear extracts in AC1 and AC3, but not AC2, was resistant to aphidicolin. Partially purified DNA polymerase alpha from AC3, but not from AC1 or AC2, showed resistance to aphidicolin. The apparent Ki value for aphidicolin of AC3 polymerase alpha was three to four times that of the enzyme from the parent cells, but the apparent Km values of the enzyme for dCTP and dTTP were normal. All the mutants showed cross-resistance to both arabinofuranosyladenine and arabinofuranosylcytosine. The AC3 mutant had expanded deoxyribonucleoside triphosphate pools. On two-dimensional polyacrylamide gel electrophoresis, AC1 gave a new protein (mol wt 40 kDa). The aphidicolin-resistance trait was reversible in AC2, unlike in AC1 and AC3. These results show that in mammalian cells there are at least two mechanisms of aphidicolin-resistance that involve an altered DNA polymerase alpha that is resistant to aphidicolin and simultaneous expansion of the four DNA-precursor pools.
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We isolated three adenine auxotrophic mutants (Ade1, Ade2 and Ade3) of mouse FM3A cells deficient in 5-aminoimidazole-4-carboxamide ribotide transformylase (EC 2.1.2.3) activity. Ade1 and Ade3 but not Ade2 also lacked inosinicase (EC 3.5.4.10) activity. While Ade2 and Ade3 complemented each other, Ade1 complemented neither Ade2 nor Ade3, suggesting that two complementation groups exist in these mutants. We introduced human genes into the Ade2 and Ade3 cells by chromosome-mediated gene transfer. All the transformants tested were found to produce the human transformylase and inosinicase, and identical DNA bands containing human Alu sequences were detected in the transformants of Ade2 and Ade3. These mutants seem to have arisen by mutation in the same gene or adjacent genes, since only human chromosome 2 was capable of rescuing the genetic defects in all these mutants.
A long L1 repetitive sequence (3.6 kilobase pairs) was found in the third intron of the human thymidylate synthase gene. This L1 family sequence is unique in that it possesses the longest open reading frame (1.7 kilobase pairs) of all L1 family members identified in sequences associated with specific genes that have been cloned thus far. Furthermore, the amino acid sequence deduced from the open reading frame of the L1 sequence was found to be highly homologous (90%) to that encoded by a known human teratocarcinoma L1 RNA species, and to contain several blocks of sequences homologous to ones in RNA-dependent DNA polymerases of various origins.
Two genomic DNA fragments partially encoding human thymidylate synthase (TS) [EC 2.1.1.45] were previously cloned in lambda phage from the mouse cell transformant, but had no transforming activity on mouse TS-negative mutant cells. In this study, an additional genomic DNA for human TS was cloned and demonstrated to have the transforming activity in combination with one of the two previously cloned DNAs and to produce human TS mRNA. The two transforming genomic DNAs overlapped and covered a region of 23 kb in total. Using fragments from one of these DNAs, the structure of the 1.2-kb region around the ATG initiator codon of the TS gene was analyzed in relation to regulatory sequences of the gene. Sequence determination demonstrated the presence of an unusual inverted repeat consisting of a triple tandem repeat of a 28-bp sequence and an inverted sequence of the same length. These sequences can form three possible, stable, stem-loop structures, which may be interconvertible. Based on S1 nuclease mapping data and a line of circumstantial evidence, we deduced two major mRNA cap sites within the inverted sequence. Comparison of the human and mouse sequences upstream from the ATG initiator codon revealed many significant blocks of sequence homology, especially in the regions around the deduced cap sites.
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The mechanism of cytotoxic action of 5-fluorodeoxyuridine (FdUrd) in mouse FM3A cells was investigated. We observed the FdUrd-induced imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools and subsequent double strand breaks in mature DNA, accompanied by cell death. The imbalance of dNTP pools was maximal at 8 h after 1 microM FdUrd treatment; a depletion of dTTP and dGTP pools and an increase in the dATP pool were observed. The addition of FdUrd in culture medium induced strand breaks in DNA, giving rise to a 90 S peak by alkaline sucrose gradient sedimentation. The loss of cell viability and colony-forming ability occurred at about 10 h. DNA double strand breaks as measured by the neutral elution method were also observed in FdUrd-treated cells about 10 h after the addition. These results lead us to propose that DNA double strand breaks play an important role in the mechanism of FdUrd-mediated cell death. A comparison of the ratio of single and double strand breaks induced by FdUrd to that observed following radiation suggested that FdUrd produced double strand breaks exclusively. Cycloheximide inhibited both the production of DNA double strand breaks and the FdUrd-induced cell death. An activity that can induce DNA double strand breaks was detected in the lysate of FdUrd-treated FM3A cells but not in the untreated cells. This suggests that FdUrd induces the cellular DNA double strand breaking activity. The FdUrd-induced DNA strand breaks and cell death appear to occur in the S phase. Our results indicate that imbalance of the dNTP pools is a trigger for double strand DNA break and cell death.
A triple tandem repeat (TTR) consisting of 90 nucleotides exists immediately upstream of the ATG initiator codon in human thymidylate synthase (TS) cDNA (pcHTS-1). To investigate the role of the TTR in the expression of the TS cDNA, we used pcHTS-1 to construct mutant cDNA clones in which part of the TTR was deleted or an additional element was inserted. The mutant cDNA plasmid was introduced into murine TS-negative mutant cells and the relative translation efficiencies of the mutant cDNAs were determined by measuring the transient expression of TS activity and the amount of TS mRNA transcribed. The translation efficiency in transient expression of the mutants was increased by deletions covering all the first two repeated elements, and the part of the third closest to the ATG initiator codon, but was not affected by deletions of only parts of the first two repeated elements at the 5' end. The translation efficiency was also not affected by insertion of an additional repeated element into the TTR. These results suggest that the first two repeated elements at the 5' end both have inhibitory effects on translation of the TS mRNA, probably due to the unique structural feature of this element.
Murine mammary carcinoma FM3A cells, deficient in cytosol thymidine (dThd) kinase (TK) activity and transformed by the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) TK gene (designated FM3A TK-/HSV-1 TK+ and FM3A TK-/HSV-2 TK+, respectively) proved extremely sensitive to the cytostatic action of the potent antiherpetic drugs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU). The fact that FM3A TK-/HSV-2 TK+ cells were 5-fold more sensitive to the cytostatic action of BVDU and IVDU but incorporated [125I]IVDU to a 20-fold lower extent into their DNA than did FM3A TK-/HSV-1 TK+ cells led us to conclude that incorporation of these compounds into DNA of HSV TK gene-transformed cell lines is not directly related to their cytostatic action. In attempts to unravel the mechanism of the cytostatic effects of BVDU and IVDU on HSV TK gene-transformed FM3A cells, both compounds were submitted to an intensive biochemical study. Thymidylate synthase was identified as the principal target enzyme for the cytostatic action of BVDU and IVDU since (i) both compounds were far more inhibitory to 2(1)-deoxyuridine (dUrd) than to dThd incorporation into HSV TK gene-transformed FM3A cell DNA, (ii) the cytostatic action of BVDU and IVDU was more readily reversed by dThd than by dUrd, (iii) both compounds strongly inhibited the metabolic pathway leading to the incorporation of 2'-deoxycytidine (dCyd) into DNA thymidylate, (iv) BVDU and IVDU strongly inhibited tritium release from [5-3H]dCyd and [5-3H]dUrd in intact HSV TK gene-transformed FM3A cells, and (v) [125I]IVDU accumulated intracellularly as its 5'-monophosphate to concentration levels considerably higher than those required to inhibit partially purified thymidylate synthase. The inhibitory effects mentioned under (i) to (iv) were not observed with the parental FM3A/0 and FM3A/TK- cells; they were more pronounced for FM3A TK-/HSV-2 TK+ cells than for FM3A TK-/HSV-1 TK+ cells, which correlates with the differential cytostatic effects of BVDU and IVDU on these cells.