Isolation, fractionation and reconstitution of a nuclear extract capable of transcribing ribosomal DNA.

A procedure for preparing a nuclear extract that efficiently transcribes rat rDNA in vitro has been developed. This procedure, which is based on the protocol described by Dignam et al. (Nucl Acids Res 11:1475, 1983), allows the preparation of extract from large or small amounts of material and requires neither ultracentrifugation nor column chromatography. These extracts were found to be more efficient than other transcription systems. Extract prepared as described routinely synthesize 1-2 transcripts per linear template, and could synthesize upto 6 transcripts per linear template at an elongation rate of 2.1 nucleotides per second. 0.3 M NaCl extracts of nuclei contained RNA polymerase I, but did not transcribe rat rDNA in vitro, whereas extract prepared with 0.42 M NaCl did. The 0.42 M NaCl extract of nuclei was fractionated by chromatography on DEAE-Sephadex and heparin-Sepharose. Two activities were identified that were required for accurate in vitro transcription by endogenous RNA polymerase I. One of these activities was required for accurate initiation, and the second inhibited non-specific transcription. The fraction required for accurate initiation by the endogenous RNA polymerase I is that factor which directs species specific transcription, as it also directed the transcription of rat rDNA by nuclear extracts of HeLa cells. Combining that same chromatographic fraction of the 0.42 M NaCl extract with the 0.3 M NaCl extract resulted in specific transcription. These results suggest that a fraction of the RNA polymerase I molecules may exist in a complex with some, or all, of the factors required for transcription.

32P-post-labelling analysis of DNA adducts formed in the livers of animals treated with safrole, estragole and other naturally-occurring alkenylbenzenes. I. Adult female CD-1 mice.

The binding of a series of alkenylbenzenes to liver DNA of adult female CD-1 mice, isolated 24 h after i.p. administration of non-radioactive test compound (2 or 10 mg/mouse), was investigated by a modified 32P-post-labelling assay. The known hepatocarcinogens, safrole, estragole and methyleugenol, exhibited the strongest binding to mouse-liver DNA (1 adduct in 10 000 - 15 000 DNA nucleotides or 200 - 300 pmol adduct/mg DNA after administration of a 10 mg dose), while several related compounds, which have not been shown thus far to be carcinogenic in rodent bioassays, bound to mouse-liver DNA at 3 - 200x lower levels. The latter compounds included allylbenzene, anethole, myristicin, parsley apiol, dill apiol and elemicin. Eugenol did not bind. Low binding to mouse-liver DNA was also observed for the weak hepatocarcinogen, isosafrole. Two main 32P-labelled adducts, which appeared to be guanine derivatives, were detected for each of the binding chemicals on thin-layer chromatograms. The loss of safrole adducts from liver DNA was biphasic: a rapid loss during the first week (t 1/2 approximately 3 days) was followed by a much slower decline up to 20 weeks after treatment (t 1/2 approximately 2.5 months). Adducts formed by reaction of 1'-acetoxysafrole, a model ultimate carcinogen, with mouse-liver DNA in vitro were chromatographically identical to safrole-DNA adducts formed in vivo. Pretreatment with pentachlorophenol, a known inhibitor of sulphotransferases, inhibited the binding of safrole to mouse-liver DNA, providing further evidence that the metabolic activation of the allylbenzenes proceeds by the formation of 1'-hydroxy derivatives as proximate carcinogens and 1'-sulphoöxy derivatives as ultimate carcinogens.