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Methylation of adenovirus 2 (Ad 2) late RNA was studied. RNA was double-labeled with [3H-methyl]-methionine and [14C]-uridine 15–20 h postinfection. Nuclear RNA (rRNA) and cytoplasmic RNA (mRNA) was extracted, and fractionated into polyA(+) and (−) molecules using poly(U)-Sepharose. Ad 2 specific RNA was purified by 2 cycles of hybridization to and elution from Ad 2 DNA immobilized on filters. The Ad 2 polyA(+) and (−) nRNA and mRNA fractions had the same 3 H 14 C ratios, and were estimated to contain a minimum of 1.4 methylated nucleotides per 1000 bases. Viral RNA was digested with RNase T2 and chromatographed on DEAE-Sephadex in 7 M urea at pH 7.6. All four Ad RNA fractions contained methylated constituents consistent with: (1) two classes of methylated “capped” 5′-termini with general structures m7 GpppNmpNp and m7 GpppNmpNmpNp; (2) internal base methylations; (3) minor amounts of internal ribose 2′-0-methylations. Two classes of 5′-termini have previously been reported for animal cell mRNA, but not for mRNA from a variety of viruses. Internal methylations may be unique to RNA molecules transcribed in the nucleus, since they have not been found in RNA from cytoplasmic viruses. No gross differences were observed in the DEAE-Sephadex elution profiles of the methylated constituents of the four types of Ad 2 RNA. These results suggest that the majority of methylation events occur in the nucleus, and raise the possibility that Ad 2 methylated late nRNA may differ significantly from SV40 late nRNA (Lavi, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci. USA 72 , 2012–2016).

Conidia of A. niger aggregated into clumps, and then germinated to form masses of mycelia (pellets) when grown in agitated, submerged, liquid cultures. Cyclic AMP and 5′-GMP promoted the aggregation of conidia, while adenosine, AMP, ADP, dibutyryl cyclic AMP, cyclic GMP, guanosine, GDP, GTP and theophylline were ineffective. ATP and 2′, 3′-cyclic AMP elicited a weak response. Conidia in the center of clumps did not germinate. The increase in “adhesiveness” of conidia by cyclic AMP and GMP may have implications in the regulation of germination, as well as the eventual growth and development of hyphae.

A soluble Ad2 DNA synthesizing complex was prepared from Ad2-infected KB cell nuclei and purified by exclusion chromatography on a BioGel A-50m column. The purified complex was able to synthesize DNA from all regions of the virus genome, as indicated by EcoRI restriction endonuclease analysis of in vitro labeled DNA. Experiments were performed to identify Ad2-induced early polypeptides present in the complex. Ad2-infected and mock-infected cells were labeled with [35S]methionine 7–10 h postinfection, then incubated for 8 h to allow the 35S-labeled early polypeptides to become associated with the complex. The polypeptides in the purified complex and each of the cell fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The major components of the purified complex were the 73K DNA binding phosphoprotein and 11K, two adenovirus 2-induced early polypeptides. The 11K has a preferred nuclear location. Small quantities of other Ad2-induced early proteins, 21K, 15K, and possibly 8.3K were also associated with the complex.

Aspergillus niger accumulated citric acid in the medium under certain conditions. Cyclic AMP concentrations of the order of 10−6M and higher caused an increase in the rate of citrate synthesis. Adenosine, ATP, and cyclic GMP at 10−3M also stimulated, but were ineffective at 10−4M. 5′-AMP had no effect while 5′-GMP and guanosine inhibited slightly. ADP showed a 42% inhibition. Theophylline enhanced the cyclic AMP effect. It is proposed that citric acid accumulation by Aspergillus niger may result from abnormal cyclic AMP metabolism.