J. gen. ViroL (1977) , 36, I 9 5 - I 9 7 I95 Printed in Great Britain Adenine-rich R N A in t h e M y c o v i r u s of Allomyces arbuscula (Accepted 17 February I977) SUMMARY An adenine-rich fraction has been extracted from the mycovirus infecting the Phycomycete Allomyces arbuscula. This fraction which accounts for approximately 8 ~ of the total R N A is heterogeneous and contains tracts of 25 to 45 nucleotides in length. The majority of the poly(A) tracts have a mol. wt. of 1.2 × IO4 and a sedimentation value of 2 to 3 S. The mycovirus infecting the Phycomycete Allomyces arbuscula contains a three segmented double-stranded R N A genome (Khandjian, Turian & Eisen, I977). About 8 ~ of the total 3~P-labelled RNA is resistant to pancreatic RNase A in conditions where doublestranded RNA is digested, namely heat denaturation before RNase treatment, while no acid precipitable radioactivity is recovered after alkaline hydrolysis. Furthermore, resistance of this material to RNase A and T1 provides evidence for a polyadenylic acid [poly(A)] sequence. Mycelium was grown in the presence of 32PO4 and the virus was purified by high-speed centrifugation followed by sucrose and CsC1 density gradients (Khandjian et al. I977). ~2P-labelled virus R N A was obtained from purified particles by two different procedures: (a) particles were dialysed against o'o48 M-Na-phosphate buffer, p H 6.8, and lysed by addition of an equal volume of 8 M-urea containing z ~ (w/v) SDS. The lysate was applied to a column of bydroxylapatite and washed with several volumes of 0"048 M-Na-phosphate buffer. The ~2P-labelled R N A was eluted with o'48 M of the same buffer (Khandjian et al. 1977). (b) Extraction at 6o °C with phenol equilibrated with Io mM-tris-HC1, p H 8"5, containing IO mM-EDTA. Both batches of R N A were dialysed against Io mM-tris-HC1, p H 7"5, containing o'5 M-KC1 before being fractionated by affinity chromatography on a polyuridylic acid [poly(U)] agarose column, type 6 (P-L Biochemicals, Inc.). Elution of the column with buffer containing o'5 M-KC1 yielded an R N A fraction I. Bound poly(A) was then eluted with buffer containing no KC1 to give fraction II (approx. 8 ~ of total). R N A I which was not bound to the poly(U) column was identified after acrylamide gel electrophoresis as the three segmented double-stranded R N A which has previously been studied. In contrast, the poly(A) fraction II migrated ahead of tRNA and was found to be heterogeneous in size. The poly(A) tracts were estimated to be composed of 25 to 45 nucleotides with a mean value of 37, corresponding to a rnol. wt. of I'2 × Io 4 and a mean sedimentation value of 2 to 3S (Fig. 0. Nucleotides were extracted from the gel after alkali digestion of the corresponding slices at 37 °C for 18 h. The base composition was determined after thin-layer chromatography on PEI-cellulose (Merck No. 5579) by the method of East (I968). The R N A had a base composition of 84 ~ AMP, Io ~ U M P and 3 C M P + GMP. The poly(A) fraction is an integral part of the virion since it was recovered from the particles after the two different centrifugation procedures used. It is unlikely that the virus preparation was contaminated with poly(A) sequences derived from degraded host cell messenger RNAs. Downloaded from www.microbiologyresearch.org by IP: 54.166.135.204 On: Sun, 12 Jun 2016 12:05:31 Short communications I96 I I I Nmnber o~ nucleotides Mol. wt. x I 120 [ 10 3 37 S i I 76 I 25 1 I I I i 37 I 12 I I I 5 4 2"2 61 I 6 -X ~3 4 ~d -2 0 50 40 30 20 10 0 Slice nmnber Fig. I. Polyacrylamide gel electropherogram of a2P-labelled poly(A) fraction obtained by poly(U) agarose chromatography. Samples were subjected to electrophoresis for Izo rain at 5 m A per tube through a I 2 cm 3"5 ~ acrylamide-o.5 ~ agarose-composite gel (Peacock & Dingman, I968). Gels were calibrated with 4 and 5S R N A from E. coli according to Gould, Pinder & Matthews (I969) and sliced into 2 mm sections for radioactivity determination. Reovirus, the best studied prototype of double-stranded R N A viruses, contains an adenine-rich R N A molecule (Bellamy & Joklik, ~967; Shatkin & Sipe, I968). This adeninerich molecule is not linked to pieces of the double-stranded R N A genome and unlike most other R N A viruses, reovirus messengers contain no detectable adenylic acid polymers at their 3'-termini (Stoltzfus, Shatkin & Banerjee, I973; see also review by Silverstein, Christman & Acs, I976). On the other hand, poly(A) tracts have been demonstrated associated with the double-stranded R N A from Saeeharomyees eerevisiae strains carrying the killer character (Vodkin, Katterman & Fink, I974; Shalitin & Fischer, I975). The relation between the killer character and the presence of virus-like particles is well established (Herring & Bevan, I975). In the case of the mycovirus infecting A. arbuscula, the adenine-rich R N A has been identified as a free molecule by the two different extraction procedures used. This reduces but does not exclude the possibility of an artifact of preparation which might have caused the breakage of single-stranded poly(A) tails associated with one or all of the three different double-stranded R N A species. Free poly(A) sequences have been proposed as a possible inhibitor of endonuclease activity in eukaryotic cells, and in so doing can prevent the degradation of messenger R N A devoid of poly(A) tails (Levy et al. 1975). Since all viruses must perform messenger R N A synthesis (Baltimore, ~97I), one might expect that the adenine-rich molecule would play a role in the protection of this messenger. We wish to thank Dr W. E. Timberlake (Wayne State University) for helpful discussions. Laboratoire de Microbiologie Ddpartement de Biologie vdgdtale University of Geneva I2IZ Geneva 4, Switzerland Downloaded from www.microbiologyresearch.org by IP: 54.166.135.204 On: Sun, 12 Jun 2016 12:05:31 E. W. KHANDJIAN G. TURIAN S h o r t communications I97 REFERENCES BALTIMORE, D. (1970. Expression o f animal virus genomes. Bacteriological Reviews 35, 235-24I. BELLAMV, A. R. & JOKUK, W. K. 0967). Studies on the A-rich R N A of reovirus. Proceedings o/the National Academy o/Science of the United States o/America 58, I389-I395. EAST, J.L. 0968). Nucleotide composition o f ribonucleie acid by spectral analysis and thin-layer chromatography. Analytical Biochemistry z4, 4o9-418. COULD, H. J., PINDER, J. C. ~ MATTHEWS,H. R. 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(Received 7 February I 9 7 7 ) Downloaded from www.microbiologyresearch.org by IP: 54.166.135.204 On: Sun, 12 Jun 2016 12:05:31