No abstract

We investigated DNA damage caused by carcinogenic metals in a murine sarcoma virus (MuSV)-based mutagenicity assay in which mutations targeted to v-mos expression can be selected. Nickel chloride treatment of NRK cells (termed 6m2 cells) infected with MuSVts110, a retrovirus conditionally defective in viral RNA splicing and cell transformation, caused the outgrowth of transformed "revertants" with changes in the MuSVts110 RNA splicing phenotype. Cadmium and chromium treatment of 6m2 cells resulted in the selection of a second class of revertants with what appeared to be frameshift mutations allowing the translation of a readthrough gag-mos protein. In both classes of metal-induced revertants, viral gene expression was distinct from that observed in revertants arising in untreated 6m2 cultures, arguing that metal treatment did not simply enhance the rate of spontaneous reversion. In one representative nickel revertant line the operative nickel-induced mutation affecting MuSVts110 RNA splicing was a duplication of 70 bases surrounding the 3' splice site. The effect of this mutation was to direct splicing to the most downstream of the duplicated 3' sites and concomitantly relax its characteristic thermosensitivity. These data establish the mutagenic potential of nickel and provide the first example of a defined nickel-induced mutation in a mammalian gene.

We investigated whether the MuSVtsllO gag gene product (P58gag) can regulate the novel growth temperature dependence of MuSVtsllO RNA splicing. MuSVtsllO mutants with either frameshifts or deletions in the gag gene were tested for their ability to maintain the MuSVts1lO splicing phenotype. Only small decreases in splicing efficiency and no changes in the thermosensitivity of viral RNA splicing were observed in MuSVtsllO gag gene frameshift mutants. Deletions within the gag gene, however, variably decreased MuSVtsllO splicing efficiency but had no effect on its thermosensitivity. Another class of MuSVtsllO splicing mutants generated by treatment of MuSVtsllO-infected cells with NiCl2 was also examined. In these "nickel revertants," P58gag is made, but splicing of the viral transcript is nearly complete at all growth temperatures. The splicing of "tagged" viral RNA transcribed from a modified MuSVtsllO DNA introduced into nickel revertant cells remained thermosensitive, arguing against trans effects of viral gene products on splicing efficiency. These experiments indicated that neither the MuSVtsllO P58gas protein nor any other viral gene product acts in trans to regulate MuSVtsllO splicing.