Transcription of specific adenovirus genes in isolated nuclei by exogenous RNA polymerases.

Previous studies have shown that nuclei isolated from KB cells infected with adenovirus 2 synthesize discrete low molecular weight RNAs via an endogenous RNA polymerase III activity. The major transcript is the well characterized 5.5 S or VA-RNA which is encoded by the adenovirus genome. Other transcripts include additional low molecular weight viral RNAs and host 5 S RNA and pre-tRNA. In the present studies, partially purified class III RNA polymerases are shown to stimulate nuclear RNA synthesis when incubated with nuclei from adenovirus-infected or uninfected KB cells. These exogenous RNA polymerases stimulate predominantly the synthesis of low molecular weight RNAs. 5.5 S RNA synthesis in nuclei from virus-infected cells is stimulated 3to 5-fold by KB RNA polymerases III, and III, isolated either from uninfected or from virusinfected KB cells. Similar results were obtained with class III RNA polymerases from mouse plasmacytoma cells and from Xenopus laevis oocytes. The RNA polymerase III activity in isolated nuclei was found to be irreversibly inactivated with low concentrations of N-ethylmaleimide. All class III RNA polymerases which stimulated viral 5.5 S RNA synthesis in untreated control nuclei from virus-infected cells (above) also enhanced the synthesis of 5.5 S RNA in N-ethylmaleimide-treated nuclei. However, the degree of stimulation above the residual endogenous RNA polymerase level was much higher in Nethylmaleimide-treated nuclei (up to 30.fold). In contrast, neither RNA polymerase II from adenovirus-infected cells nor Escherichiu coli RNA polymerase selectively enhanced the transcription of the viral 5.5 S genes in isolated nuclei, although the levels of total RNA synthesis were increased in each case.

Previous studies have shown that nuclei isolated from KB cells infected with adenovirus 2 synthesize discrete low molecular weight RNAs via an endogenous RNA polymerase III activity. The major transcript is the well characterized 5.5 S or VA-RNA which is encoded by the adenovirus genome. Other transcripts include additional low molecular weight viral RNAs and host 5 S RNA and pre-tRNA. In the present studies, partially purified class III RNA polymerases are shown to stimulate nuclear RNA synthesis when incubated with nuclei from adenovirus-infected or uninfected KB cells. These exogenous RNA polymerases stimulate predominantly the synthesis of low molecular weight RNAs. 5.5 S RNA synthesis in nuclei from virus-infected cells is stimulated 3-to 5-fold by KB RNA polymerases III, and III, isolated either from uninfected or from virusinfected KB cells. Similar results were obtained with class III RNA polymerases from mouse plasmacytoma cells and from Xenopus laevis oocytes.
The RNA polymerase III activity in isolated nuclei was found to be irreversibly inactivated with low concentrations of N-ethylmaleimide.
All class III RNA polymerases which stimulated viral 5.5 S RNA synthesis in untreated control nuclei from virus-infected cells (above) also enhanced the synthesis of 5.5 S RNA in N-ethylmaleimide-treated nuclei. However, the degree of stimulation above the residual endogenous RNA polymerase level was much higher in Nethylmaleimide-treated nuclei (up to 30.fold). In contrast, neither RNA polymerase II from adenovirus-infected cells nor Escherichiu coli RNA polymerase selectively enhanced the transcription of the viral 5.5 S genes in isolated nuclei, although the levels of total RNA synthesis were increased in each case. to exogenous probes, such as bacterial RNA polymerases (2-4) and specific nucleases (5, 6). More recent studies of the transcription of Xenopus laeuis 5 S RNA genes (7,8) and murine 5 S and tRNA genes (9)   uses -Detergent-treated nuclei were employed for the studies reported here. The kinetics and the salt and divalent cation optima of the endogenous RNA polymerase reactions in these nuclei are similar to those described in previous nuclear transcription studies (14,15). In these experiments, nuclei from uninfected KB cells and from virus-infected cells were incubated in the absence and in the presence of purified RNA polymerases. The incubations were carried out under conditions optimal for the endogenous RNA polymerase III reaction and under conditions where the endogenous RNA polymerase II activity is inhibited (see "Experimental Procedures"). The level of total RNA synthesis in each nuclear preparation was increased from 2-to 3-fold by each of the exogenous class III RNA polymerases examined. The latter included both class III enzymes (III, and III,) from uninfected cells and from virus-infected cells. Moreover, the actual levels of stimulation of RNA polymerase III activity by the exogenous RNA polymerases were considerably greater since the endogenous RNA polymerase activity represents both RNA polymerase I plus III activities (data not shown, see Ref. 29;cf. Ref. 9).
The RNAs synthesized in isolated nuclei in response to exogenous KB class III RNA polymerases were analyzed by sedimentation in sucrose gradients and shown to consist primarily of low molecular weight transcripts (<lo S) (data not shown, see Ref. 29;cf. Ref. 9). To further characterize the nuclear transcripts, the isolated RNAs were subjected to electrophoresis in 12% polyacrylamide gels. Autoradiograms of the gels in which these 32P-labeled RNAs were analyzed are shown in Fig. 1. As shown in Fig. IA, the synthesis of 5 S RNA and 4.5 S RNA @RNA precursors) species is markedly stimulated by RNA polymerases III, and III,, isolated either from uninfected or from virus-infected cells. These observations are similar to those reported previously for the synthesis of 5 S and 4.5 S RNAs in mouse plasmacytoma cell nuclei (9).
An analysis of the low molecular weight RNAs synthesized in nuclei from virus-infected cells is shown in Fig. 1B. The viral RNAs synthesized by the endogenous RNA polymerase III (lane 1) include the major 5.5 S RNA and a minor 200nucleotide RNA (designated V,,,) visible just above the 5.5 S RNA (15). Other low molecular weight viral RNAs synthe-  sized by RNA polymerase III (15) are not apparent in these experiments. The low molecular weight RNAs synthesized in response to exogenous class III RNA polymerases are shown in lanes 2 to 5 in Fig. IB. The synthesis of the viral 5.5 S and V,,, RNAs, as well as the cellular 4.5 S RNAs, is stimulated by RNA polymerases III, and III, from uninfected and from virus-infected KB cells. The synthesis of some RNA species which are excluded from the 12% acrylamide gel is also stimulated.
The nature of these RNAs, apparently greater than 8 S in size, is unknown. Levels of a-amanitin (200 pg/ ml) which inhibit RNA polymerase III inhibit the synthesis of all these RNA species in the presence of exogenous RNA polymerase III (data not shown; see also below). Thus, the enhanced synthesis of the various RNAs in the presence of partially purified exogenous RNA polymerase III preparations is clearly due to an RNA polymerase III activity.
To quantitate the increased levels of RNA synthesis effected by the exogenous RNA polymerases, polyacrylamide gels similar to those shown in Fig. 1 were sliced and the radioactivity in specific RNAs was determined after solubilization.
The upper panel of Fig. 2 shows that exogenous RNA polymerase III, stimulates the synthesis of the 5 S RNA and the pre-tRNAs (the broad band between the 4 S and 5 S markers) about 3-fold relative to the level of synthesis by the endogenous activity in uninfected nuclei. The lower panel of Fig. 2 shows the RNAs synthesized in nuclei from virus-infected cells. Exogenous RNA polymerase III, stimulates the synthesis of the viral V,,, and 5.5 S RNAs about S-fold. Synthesis of host 5 S RNA and pre-tRNA is also stimulated although the relative levels of synthesis differ from those observed in nuclei from uninfected cells. Possibly this reflects a preferential  Fig. 3 (lanes 1  and 7) as did the KB class III enzymes. All these class III enzymes (lanes 3 to 6) also stimulated the synthesis of material (apparently 28 S in size) which does not enter the gel. The nature of this RNA has not been examined further.
In contrast to the results with class III enzymes and in agreement with studies described earlier, RNA polymerase II from virus-infected KB cells had no noticeable effect on the synthesis of viral 5.5 S RNA (compare lanes 1 and 8 in Fig. 9) although total RNA synthesis was increased about 2-to 3fold. These assays (analyzed in lanes 1 and 8) were performed in the absence of a-amanitin.
In addition, the effects of E. coli RNA polymerase were monitored. This enzyme markedly stimulates transcription when incubated withN-ethylmaleimide-treated nuclei. However, as shown in Fig. 9 (lane 7) a very heterodisperse array of transcripts is generated. When the gel analyzed in Fig. 9 was subjected to a shorter autoradiographic exposure, no 5.5 S RNA band was evident (data not shown). Moreover, no 5.5 S RNA band was evident when the gels were sliced and analyzed by liquid scintillation counting. DNA -As an independent measure of viral 5.5 S RNA synthesis in these reconstructed transcription systems, the in vitro transcripts have been characterized by hybridization to adenovirus DNA. In the experiment shown in Table II, RNA synthesized in N-ethylmaleimide-treated nuclei was hybridized to adenovirus DNA in the presence and absence of a nonradioactive 5.5 S RNA competitor. The total radioactivity in 5.5 S RNA was calculated as DIE, where D equals the difference between the total amount of radioactive RNA hybridized to adenovirus DNA and the amount hybridized in the presence of the 5.5 S RNA competitor, and E equals the fractional efficiency of hybridization of an 3H-labeled 5.5 S RNA internal standard. In the experiments shown in Table  II, only a very small and possibly insignificant amount of 5.5 S RNA was synthesized by the endogenous RNA polymerase. In contrast, exogenous KB class III RNA polymerases from uninfected KB cells and from X. Zuevis oocytes markedly stimulated (at least 30-to 40-fold) the synthesis of 5.5 S RNA. The stimulation is almost completely inhibited by a-amanitin at a concentration (160 pg/ml which inhibits RNA polymerase III activity about 90% (14). In addition, 5.5 S RNA sequences