We propose to characterize, at the DNA sequence level, the genetic damage done by metal carcinogens (e.g., compounds of nickel, chromium and cadmium) known to induce respiratory and bladder cancers as well as birth defects. Our approach will be to study stably inherited, metal compound-induced mutations that affect the expression of the v-mos gene in NRK cells infected by the MuSVts110 retrovirus (6m2 cells). In 6m2 cells, expression of the MuSVts110 v-mos region is restricted to growth temperatures of 33degrees C and below by a conditionally defective viral RNA splicing event. Hence, 6m2 cells appear morphologically transformed if grown at 33degrees C and normal if shifted to 39degrees C. Previously, we have shown that treatment of 6m2 cells with nickel, chromium, or cadmium salts causes the outgrowth of """"""""revertant"""""""" cells that have stably acquired the ability to maintain the transformed state at 39degrees C (1,2,3). In the chromium- and cadmium-induced revertants, apparent frameshift mutations in the MuSVts110 viral DNA allow the expression of a gag-mos fusion protein from the unspliced viral RNA. In the nickel revertants, splicing of the MuSVts110 viral RNA has become essentially growth-temperature-independent as a result of what seem to be base substitution mutations. The central objective of this proposal will be to determine precisely the DNA damage done by metal carcinogens in the various classes of 6m2 revertants outlined above. Specifically, we will test whether the metal-induced lesion is in the viral DNA or in some cellular gene whose product can exert a compensatory effect on the conditional defect in MuSVts110 RNA splicing. This will be accomplished by transfection of cellular DNA from the revertants into NIH3T3 cells to determine whether the """"""""altered splicing phenotype"""""""" or """"""""frameshifted phenotype"""""""" tracks with the integrated MuSVts110 viral DNA. Cis- vs. trans-acting mutations with respect to the viral DNA will also be tested using a """"""""tagged"""""""" MuSVts110 viral DNA with a normal thermosensitive splicing phenotype whose transcripts can be distinguished from """"""""native"""""""" MuSVts110 RNA. Mutated viral DNA in the metal revertants will then be amplified by the """"""""PCR"""""""" technique and directly sequenced. To determine whether a particular mutation or set of mutations is neutral or can confer either the altered splicing phenotype or the synthesis of a gag- mos protein from an unspliced viral RNA, chimeric viral DNAs containing mutated regions (amplified by PCR and then subcloned) substituted for the cognate region of native MuSVts110 DNA will be introduced into cells and tested for either altered growth-temperature dependence of splicing or the ability to support the translation of an elongated gag-mos protein from the unspliced transcript.
