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This work was supported in part by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 29.
Kahle, P., Hoppe-Seyler, P. & Kroger, H. (1971) Bioclrini. Biophys. Acta 240, 384-391
Kerr, S. J. & Borek, E. (1972) Ado. Enzymol. 36,l-27
Leboy, P. S. (1970) Ann. N. Y. Acad. Sci. 171, 895-900
Nishimura, S. (1971) Praced. Nucleic Acid Res. 2, 544564
Sharma, 0.K.,Kerr, S. J., Lipshitz-Wiesner, R. &Borek, E. (1971) Fed. Proc. Am. Fed.Soc.Exp.
Bid. 30, 167-176
Purification of Methionine-Accepting Transfer Ribonucleic Acid from
Scenedesmus obliquiis D3
DAVID S. JONES and FRANCIS T. JAY
Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX,
U.K.
Previously we have described the isolation and prelimiiiary characterization of tRNA
from the green alga Scenedesmus obliquus D3 (Jones & Jay, 1975). This material has
been fractionated by several different ion-exchange chromatographic methods in an
attempt to isolate and identify the various species of methione-accepting tRNA. On
both DEAE-cellulose and DEAE-Sephadex the methionine-accepting t RNA is separated into one major and one minor peak. However, as these peaks occur within a region
of high absorbance
the enrichment of methionine-accepting t RNA is poor.
Fractionation of crude tRNA on arginine-agarose (Jay & Jones, 1973) also gave one
major and one minor peak of methionine-accepting activity, but in this case the major
peak is located in an area of low absorbance and the purification is 14-fold. By using a
crude enzyme preparation from Escherichia coli the formyl-accepting activity of
methionyl-tRNA in the peaks was determined, which showed that the latter part of the
major peak is formylated to a low extent (24%) and the minor peak is formylated to a
much higher extent (69 %).
Chromatography of the crude tRNA on BD-cellulose) (benzoylated DEAE-cellulose)
gave two major peaks of methionine-accepting activity. Here methionyl-tRNA,
contained in all areas of the first peak, is formylated to a low extent (22%) whereas
only the methionyl-tRNA in the faster-running part of the second peak is formylated
and this to a high extent (69 %).
From these latter two fractionation methods it appears that there are at least three
different species of methionine-accepting tRNA. Two are formylatable, one to a small
extent, and one to a much greater extent and the third is a non-formylatable species.
In E. coli two formylatable species of methionyl-tRNAP" have been separated on
BD-cellulose (Petrissant & Favre, 1972)and one has been shown to be a modified form of
the other (Shugart et al., 1969; Petrissant & Favre, 1972). The two species of tRNAPe'
which have been found here are, on the contrary, thought to be different entities. The
methionine derivative of one is formylated only poorly with the E. coli enzyme, a feature
found with other eukaryotic initiator tRNA species (Ecarot & Cedergren, 1974), whereas
the other is formylated to a high extent which is typical of prokaryotic initiator tRNA
species.
By isolating the major peak from arginine-agarose chromatography and re-running
it on BD-cellulose, the poorly formylated species of tRNAMCt
has been obtained with a
high degree of purity. Nucleoside analysis of this material by the method of Randerath
et a[. (1972) shows it to be lacking in ribothymidine, which is a feature of eukaryotic
initiator tRNA species (see Barrel1 & Clark, 1974). It is concluded therefore that this
purified species of tRNAMe'isolated from S. obliqirus is the cytoplasmic initiator tRNA.
The second species of tRNAMe',which is formylated more efficiently, may be the initiator
tRNA from the chloroplast.
Vol. 3
910
BIOCHEMICAL SOCIETY TRANSACTIONS
Barrell, B. G . & Clark, B. F. C. (eds.) (1974) Handbook of Nucleic Acid Sequences, JoynsonBruvvers, Oxford
Ecarot, B. & Cedergren, R. J. (1974) Biochim. Eioplrys. Acta340, 13g139
Jay, F. T. &Jones, D. S. (1973) Prep. Biocheni. 3, 517-523
Jones, D. S. &Jay, F. T. (1975) Biochem. SOC.Trans. 3,660-661
Petrissant, G . & Favre, A. (1972) FEBSLett. 23, 191-194
Randerath, E., Yu, C. T. & Randerath, K. (1972) Anal. Biocliem. 48, 172-198
Shugart, L. Chastain, B. & Novelli, D. G. (1969) Biochem. Biopliys. Res. Corninnuti.37,305-312
Polyacrylamide-Gel Electrophoresis of Messenger Ribonucleic Acid
Extracted from Cells Infected with Pig Herpes Virus 1
J. KEITH VASS and WILLIAM S. STEVELY
Dcpurtment of’Biochemistry, University of Glusgow, Glusgow GI 2 S Q Q ,
U.K.
We have used polyacrylamide-gel electrophoresis to examine thc polyadenylated polyribosomal R N A extracted from HeLa cells infected with pig herpes virus I (pseudorabies
virus). This method was used to examine the effect of cycloheximide on viral messenger
R N A synthesis. Rakusanova et al. (1971) have stated that only 25 % of the part of the
viral genome normally transcribed before D N A synthesis gives rise t o transcripts in
cells treated with cycloheximide from the time of infection. This finding has been
confirmed by Rakusanova e f ul. (I 972), Ben-Porat et al. (1 974) and Kozak & Roizman
(1974); the last of these groups of workers used human herpes virus 1 (herpes simplex
virus type 1). In the above studies DNA-RNA hybridization was used to analyse the
polyribosomal RNA. We decided to attempt to find differences in particular species of
R N A which could be detected by using polyacrylamide-gel electrophoresis.
Polyadenylated R N A was isolated from polyribosomes, by using phenol and chloroform, and purified by using poly(U)-Sepharose chromatography. HeLa cells, either
mock-infected or infected with 20 plaque-forming units/cell of virus were used. [3H]Uridine was added at various times after infection and the cells were harvested either
1 or 2 h later. In some cases cycloheximide was present from the time of infection.
We have found two peaks of R N A on polyacrylamide-gel electrophoresis, which are
synthesized 2 h after injection, and are still not present 5 h after infection when cycloheximide is present from the time of infection. These species may belong to the early
virus-messenger-RNA class proposed by Kaplan (1973). The appearance of this class
is thought to require prior synthesis of immediate-early m R N A and protein. In herpes
simplex virus type 1 it has been suggested by Honess & Roizman (1974) that a similar
situation occurs with three sequential classes of proteins. The latter two d o not appear
without the prior synthesis of the class of proteins appearing immediately before the
class in question. The effects of cycloheximide on the synthesis of a t least two species
of polyribosomal RNA, noted in our experiments, suggest the theory that there is a
requirement for early protein synthesis before some later genes are transcribed into
viable messenger RNA.
This work was supported by the Medical Research Council.
Ben-Porat, T., Jean, J. H. & Kaplan, A. S. (1974) Virology 59, 524-531
Honess, R. W. & Roizrnan, B. (1974) J . Virol. 14, 8-19
Kaplan, A. S. (1973) Cancer Rcs. 33, 1393-1398
Kozak, M. & Roizrnan, B. (1974) Proc. Narl. Acad. Sci. U.S.A. 71,4322-4326
Rakusanova, T., Ben-Porat, T., Hirneno, M. & Kaplan, A. S. (1971) Virology 46,877-889
Rakusanova, T., Ben-Porat, T. & Kaplan, A. S. (1972) Virology 49, 537-548
1975