Eur. J. Biochem. 98, 61 -66 (1979)
Characterization of Long Guanosine-Free RNA Sequences from the Dahlemense and U2 Strains of Tobacco Mosaic Virus Brice A. KUKLA, Hubert A. GUILLEY, Gerard X. JONARD, Kenneth E. RICHARDS, and Kurt W. MUNDRY Labordtoire de Virologie, Institut de Biologie Moleculaire et Cellulaire du Centre National de la Recherche Scientifique, Strasbourg, and Institut fur Botanik, Universitat Stuttgart (Received December 27, 1978)
Four naturally occurring strains of tobacco mosaic virus, U2, Dahlemense, CV4, and the bean form of tobacco mosaic virus, were tested for the existence of long TI RNAase oligonucleotides analogous to the oligonucleotide 52 found in the common or U1 strain of tobacco mosaic virus and which makes up the 5‘ non-coding region of the RNA molecule. U2 and Dahlemense RNA were each found to contain this type of long TI RNAase oligonucleotide with chain lengths of 54 and 74-77 residues, respectively. The sequence of the two oligonucleotides was determined mostly by using 5‘-32P-labelled material in vitro and rapid polyacrylamide gel sequencing techniques. The RNA of the common or U1 strain of tobacco mosaic virus [TMV (Ul)] contains an exceptionally long nucleotide sequence devoid of guanosine residues [l - 31. This guanosine-free sequence is 69 nucleotides in length and may be easily isolated from a total T1 RNAase digest of tobacco mosaic virus RNA as part of a very long T1 oligonucleotide called 52 [2,4]. It is now known that 52 is situated immediately after the capped guanosine residue at the 5‘ extremity of the TMV RNA molecule, the 5’-terminal sequence being m7G5’ppp5’G-52.. . [5]. The sequence at the 3‘ end of Q is . . . A-C-AA-U-G [6]. Because there can be no A-U-G or G-U-G triplet to the left of this sequence, it follows that the A-U-G at the 3‘ terminus of !2i is the first potential initiaton codon in the RNA molecule and we have demonstrated elsewhere that protein synthesis does in fact start at this point, at least in cell-free proteinsynthesizing systems [7]. Thus the non-coding sequence of 68 nucleotides at the 5‘ end of TMV RNA (UI) is remarkable in that it contains no internal guanosine residues. In addition to TMV (Ul), a great many naturally occurring strains of TMV have been isolated and classified with respect to divergence of the coat protein sequence from that of the wild-type strain [8]. Evidently it would be interesting to know how the guanosine-free sequence has evolved in such strains, In an earlier study Mundry and Priess [3] show that the RNAs of the two strains most closely related to TMV (Ul), Dahlemense and U2, each contain a long T1 oligonucleotide resembling O(U1) in length (79 and 56 Abbreviation. TMV, tobacco mosaic virus.
residues respectively) and overall base composition. In this paper we describe the use of 32P-labelling techniques in vitro with polynucleotide kinase to determine the sequence of these two oligonucleotides. The RNA of the two other strains examined, CV4 and the bean form of TMV, did not possess comparable guanosine-free sequences. MATERIALS AND METHODS Preparation of 5’-32P-LabelledOligonucleotides
U2 and Dahlemense strains of TMV were propagated and purified as described by Mundry [4]. CV4 strain TMV was propagated on Cucumis sativus and purified as described by Von Wechmar and Van Regenmortel [9]. The bean form of TMV (inoculum provided by M. Zaitlin) was propagated on Phaseolus vulgaris. A preparation greatly enriched in long particles was prepared from the infected leaves as described by Whitfeld and Higgins [lo]. RNA was obtained from purified virus by phenol extraction. Total TI RNAase hydrolysis was with 1 unit of enzyme (Sankyo)/lO pg RNA at 37°C in 50 mM Tris-HCI, 10 mM MgClz, pH 7.4. After 1 h a like quantity of T1 RNAase was added and incubation was continued for another hour. The oligonucleotides were precipitated with two volumes of ethanol. The ethanol precipitate was taken up in 50 mM TrisHCI 10 mM MgC12, 15 mM 2-mercaptoethanol, pH 7.4, and the RNA was 5’-32P-labelledby treatment with polynucleotide kinase in the presence of [y-”PIATP [6]. Purification of 5’-labelled oligonucleotides was by polyacrylamide gel electrophoresis [6].
62
Long Guanosine-Free Sequences from TMV U2 and Dahlemense RNA
Sequence Analysis Techniques for partial PI nuclease digestion of 5‘-32P-labelled oligonucleotide and analysis by twodimensional electrophoresis/homochromatography [l I] or two-dimensional gel electrophoresis have been described elsewhere [6]. For rapid sequencing on monodimensional gels the procedure of Donis-Keller et al. [I21 was followed except that the ‘ladder’, with cuts after all four types of nucleotide monophosphate residues, was prepared by partial formamide digestion of 5’4abelled material [13]. In order to prepare larger quantities of the long T1 oligonucleotides, as much as 20 mg U2 or Dahlemense RNA was totally digested with T I RNAase as described above. The longer oligonucleotides in the digest were preferentially precipitated with 55 ethanol [I41 and purified by polyacrylamide gel electrophoresis. Prior to electrophoresis a trace of a 5‘-32Plabelled total digest of U2 or Dahlemense RNA was added to the appropriate hydrolysate to allow the oligonucleotides of interest to be located by autoradiography of the gel. After extraction from the gel partial pancreatic RNAase digestion of oligonucleotides was at an enzyme-to-RNA ratio of 1/100000 (w/w) for 10 min at 0 ° C in 50 mM Tris-HC1, 10 mM MgC12, pH 7.5. Enzyme action was stopped by phenol extraction and the subfragments were 5’ labelled by treatment with polynucleotide kinase and [y3’P]ATP as described above. The 5’-labelled fragments were separated from one another by two-dimensional gel electrophoresis [15] with analysis by partial PI nuclease digestion and two-dimensional electrophoresis/homochromatography. RESULTS
Isolation of L’ong Guanosine Free Sequences ,from Naturally Occurring Strains of T M V The genome RNAs of the U1, U2, bean, CV4 and D strains of TMV were exhaustively digested with T1 RNAase and the oligonucleotides were 5’-32P-labelled by incubation with polynucleotide kinase in the presence of [Y-~’P]ATP(Materials and Methods). Fractionation of the digests by polyacrylamide gel electrophoresis reveals that the RNAs of strains U2 and D each contain a long TI oligonucleotide which is well separated from the mass of smaller oligonucleotides in the gel (Fig. 1). The long T1 oligonucleotide of U2, which we shall term O(U2), migrates as a discrete band somewhat faster than Q(U1) whereas the long T1 oligonucleotide of strain D, Q(D), migrates more slowly than Q(U1) and shows signs of being heterodisperse in length (Fig. 1). Neither the bean nor the CV4 strain of TMV gave rise to unusually long T1 oligonucleotides upon T1 RNAase digestion of their RNAs (Fig. 1).
a”, G”2
Fig. 1. Isolution qf long 5’-32P-lubelledoligonucleotides Jkm the R N A qf’severul strains of T M V . Autoradiogram of a 10% polyacrylamide gel staining the T1 RNAase oligonucleotides isolated from the R N A of TMV (Ul) (lanes A and F), TMV (U2) (lane B), CV4 (lane C), bean form of TMV (lane D), and TMV (D) (lane E). The oligonucleotides were 5’ labelled by treatment with polynucleotide kinase and [y-32P]ATPprior to electrophoresis
Sequence Analysis of 5’-32P-LabelledOligonucleotide by Partial PI Nuclease Digestion After purification by polyacrylamide gel electrophoresis as described above, the 5‘-labelled Q(U2) and O(D) were extracted from the gel [no effort was made to separate the Q(D) species of different electrophoretic mobilities] and digested with the non-specific nuclease P1 under conditions designed to yield a digest containing all possible intermediate stages of degradation. Sequence analysis was by the wanderingspot method [16,17]. The sequence of the first 16 residues of O(U2) was read in this manner as pA-C-A-AC-A-A-C-A-A-U-U-A-A-A-A . . . (Fig. 2A). Q(D) may be seen to begin with the sequence pU-A followed by a tract of uridine residues (Fig. 2 B), but beyond the first eight residues the spots become increasingly diffuse so that neither the exact length of the uridine tract nor the sequence following it could be determined. It will be shown below that the uridine tract of Q(D) is heterodisperse in length, which makes the wandering-spot pattern near the 5’ terminus difficult to interpret. In an effort to gain additional sequence information, partial PI nuclease digests of 5’-32P-labelled Q(U2) and Q(D) were fractionated by two-dimensional polyacrylamide gel electrophoresis [6]. Fig. 3 A shows
B. A. Kukla, H. A. Guilley, G. X. Jonard, K. E. Richards, and K. W. Mundry
63
Fig, 2. Sequenw at the 5' terminus of5'-32P-lahelledB(U 2 ) ( 4 ) a n d Q ( D ) ( B ) . Autoradiogram of a PI nuclease partial digest ot5'-32P-labelled B separated by two-dimensional electrophoresis/homochromatography
Fig. 3. T,~~o-dimensionalpolyacrylamidegel electrophorc~sisof PI partial digests qf5'-32P-labelledQ( U2) ( A ) and B ( D ) ( B ) .The first dimension was electrophoresis through a 10% polyacrylamide gel at pH 3.5 and the second dimension was electrophoresis through a 20% polyacrylamide gel at p H 8.3
64
Long Guanosine-Free Sequences from TMV U2 and Dahlemense RNA
a partial PI nuclease digest of Q(U2) fractionated in this manner. It can be seen that the two uridine residues at positions 11- 12, which were detected by two-dimensional electrophoresis/homochromatography, are separated from the next uridine in the sequence (U28) by 15 residues. Following U28 there is an A or C residue and then two more uridine residues. After this there is another long tract devoid of U. At this stage we can read the sequence pA-CA-A-C-A-A-C-A-A-U-U-A- A-A-A-N - N - N - N - N -NN-N-N-N-N-U-N-U-U . . . where N may be either A or C. Two-dimensional polyacrylamide gel electrophoresis of a partial P1 nuclease digest of Q(D) (Fig. 3 B) shows clearly that the oligo(U) tract near the 5‘ terminus is variable in length; the major component has eight uridine residues in the tract but sequences with six, seven and nine uridine residues are present as well, giving rise to wandering-spot patterns slightly displaced with respect to that of the principal species. The oligo(U) tract is followed by a sequence of about seven residues containing no uridine. Next come two uridine residues and then a very long sequence devoid of uridine (Fig. 3 B). It is immediately evident from the wandering-spot pattern that the sequence of Q(D) closely resembles that of sZ(U1) (compare to Fig.3 of [61).
sive electrophoresis of 5’-32P-labelled Q(D) through a 20 % polyacrylamide gel in the presence of urea freed the species of shortest chain length, containing six uridine residues in the oligo(U) tract, from the longer molecules. Fig. 4 B shows a monodimensional sequencing gel from which the sequence of pyrimidine and adenosine residues from positions 8-47 of this oligonucleotide can be read.
Further Sequence Analysis by Polyacvylumide Gel Electrophoresis of Partial Digests
DISCUSSION
Additional sequence information was gained by partial enzymatic digestion of 5’-32P-labelled O(U2) and O(D) followed by polyacrylamide gel electrophoretic size fractionation of the partial digestion products. Conditions for random partial digestion after guanosine residues with T1 RNAase and after adenosine residues with U2 RNAase have been described by Donis-Keller et al. [12]. Partial pancreatic RNAase digestion was used to cleave after pyrimidines (cleavage almost exclusively limited to Y-A linkages) and formamide treatment to cleave after all four residues [13]. Fig. 4 A shows the pattern of bands obtained when portions of a sample of 5’-32P-labelled O(U2) were partially digested with each of the above enzymes and the digests were fractionated in adjacent lanes of a 20 % polyacrylamide gel. The first nine residues have been run off the gel. A partial formamide digest of 5’-32P-labelledQ(U2) was also included on the gel to provide a ‘ladder’ indicating all possible chain lengths. Since the products of enzymatic digestion are ordered by size, the gel determines the position of each adenosine and pyrimidine in the sequence. Before using the rapid monodimensional gel sequencing technique on Q(D) it was necessary to separate the components of different chain length. Exten-
Completion of the Sequence of Q( U2) and Q ( D ) The foregoing observations define the sequence of the first 32 residues of Q(U2) and the first 46 residues of Q(D). In order to complete the sequences, nonradioactive Q(U2) and Q(D) were partially digested with pancreatic RNAase; the subfragments of each were then 5‘ labelled by polynucleotide kinase treatment and separated from one another by two-dimensional polyacrylamide gel electrophoresis. Characterization of the 5’4abelled subfragments by the wandering-spot method provided enough overlaps to establish the complete sequence of both oligonucleotides (Fig.5). Note that the sequences are in fairly good accord with the pancreatic RNAase catalogues of O(U2) and Q(D) previously obtained by Mundry and Priess [3] with uniformly 32P-labelled oligonucleotide.
The exact position of the long guanosine-free sequences of Dahlemense and U2 RNAs along the RNA chain has not yet been directly determined. However, the marked resemblance in sequence between Q(D) and Q(U1) (Fig.5) leaves little doubt that the two guanosine-free sequences are functionally homologous, implying that O(D), like Q(Ul), is situated at the 5‘ extremity of the intact RNA. Consistent with this conclusion is the observation that treatment of Dahlemense virus ‘with sodium dodecyl sulfate, a treatment which, in the case of TMV (Ul), has been shown to uncoat particles in a polar fashion starting at the end of the rod which contains the 5‘ end of the RNA chain [18], uncovers the Q(D) sequence during the earliest stages of uncoating (Mundry, K., personal observations). Similar experiments with TMV (U2) show that Q(U2) is also situated at or very near the sodium-dodecyl-sulfate-labile extremity of the virus particle (Mundry, K., personal observations). Thus, although the sequence relation between Q(U2) and sZ(U1) (Fig. 5 ) is less close than between the latter and O(D), it seems reasonable to hypothesize that Q(U2) likewise forms all or part of the 5‘ non-coding region of U2 RNA. Q(U2) possesses two long sequences composed of only adenosine and cytosine (residues 13-27 and 32-48) like the A-A-C tract of Q(Ul), but much of
B. A. Kukla, H. A. Guilley, G. X. Jonard, K . E. Richards, and K. W. Mundry
65
A
B U, A
F
A A
50 Y A A Y A
1
4 Y A
1; Y 40
A 40 Y
A A Y A A
A Y A A 30 Y A
Y A Y
1
A Y
A
Y 20 Y A Y Y A A
A
20 A A A Y A A A A Y Y 10 A
Y
1; 10
A Y
Fig. 4. Monodimensional polyacrylamide gelfioctionation of'partial digests of S'--('P-labeNed O(U2) ( A ) and Q ( D ) ( B ) . Partial digestion was with formamide (F),which cuts after all four types of residue; U2 RNAase, which cuts after adenine; and pancreatic RNAase (A), which cuts after pyrimidines (mainly at Y-A linkages). Electrophoresis was through a 20% polyacrylamide gel at p H 8.3
A
U A U U U U U A C A A C A A U U A C C A A C A A C A A C A A A C A A C A A A C A A C A U UA C A A U U ACU AUU U A C A A U UAC A A U G
B
UA(U U ....l,"C
A A C A A U UAC C A A C A A C A A!
4 A AC A A C - A A C A A
AAC
U U A C A Lj U UA C AJ
AAC
U e UAC A A s U AC A A U G
\ I
U
UAU,U
C
ACAACAAUUA
/
CAAC'AACAAACAACAAACAACA
ACAAUG
\
AAAACAAAAA
Fig.5. Conijilele .requences uf sLiUl) ( A ) , O i D ) ( B ) and Q(U.2) ( C ) . Sequences of O(D) and Q(U2) have been written so as to display sequence homology with sL(U1). Hyphens representing phosphodiester links have been omitted to save space
the sequence found in sZ(U1) which separates this region from the 3' terminus has been deleted (Fig. 5). sZ(U2) also differs from both sZ(U1) and Q(D) in lacking a uridine-rich sequence at its 5'-terminus. It
cannot be ruled out, however, that such a sequence exists immediately preceding sZ(U2) in the intact RNA molecule, but with a guanosine residue separating it from the sZ(U2) sequence. Fig.5 also shows that all
66
B. A. Kukla et al.: Long Guanosine-Free Sequences from TMV U2 and Dahlemense RNA
three sZ fragments have the same trinucleotide sequence A-C-A immediately preceding the final A-U-G. Dahlemense belongs to the group of TMV strains considered to be the most closely related to TMV (Ul) [8] but, nonetheless, the two strains have diverged considerably, with Dahlemense coat protein having undergone amino acid substitution in 28 of 158 positions [I91 and with overall nucleotide sequence homology falling below the level detectable by competition hybridization [20]. Thus the relatively limited difference in sequence between Q(D) and sZ(U1) may be taken as evidence that the 5' non-coding regions of the two RNAs have been subject to conservative pressures. It is interesting to note that a number of the sequence changes of Q(D) relative to sZ(U1) can be thought of as insertions or deletions of one or more units of a repeating base motif. Examples are the additional A-A-C at position 32- 35 of SZ(D) and the one to four added uridine residues in the oligo(U) tract at the 5' end of the sequence (Fig. 5). A mechanism for engendering such changes is easy to imagine if, during the course of replication of the region of RNA containing the repeating motif, the plus and minus strands of RNA occasionally slip with respect to one another so as to loop out one unit of repeat. Then either an insertion or deletion could occur, depending upon whether the looped out unit is in the plus or minus strand. We thank Prof. L. Hirth, in whose laboratory most of this work was carried out, for his interest and support. Polynucleotide kinase and [i.-32P]ATP were a gift of Dr G. Keith. This research was
supported in part by the ATP 50-77-82 of lnsrirut Nutionol dc. Iu Suntc; ef de lu Rwherchr Midicul.
REFERENCES Mandeles, S. (1968) J . Bid. Chcm. 243, 3671 -3674. Lloyd, D. A. & Mandeles, S. (1970) Biochemistry, 9, 932-938. Mundry, K. W. & Pries, M. (1971) Virology, 46, 86-97. Mundry, K . W. (1969) Mol. G m . Genet. 105, 361-377. Richards, K . F., Guillcy, H., Jonard, G. & Keith. G . (1977) Nature (Lond.) 267, 548 - 550. 6. Richards, K. E., Guilley, H., Jonard, G . & Elirth, L. (1978) Eur. J . Biochem. 54, 513-519. 7. Jonard, G., Richards, K. E., Mohier, E. & Gerlinger, P. (1978) Eur. J . Biochem. 54, 521 - 531. 8. Siegel, A. & Wildman, S. G. (1954) P/7ytopuihology, 4,277- 282. 9. Von Wechmar, M. B. & Van Regenmortel, M . H . V. (1970) S. Afr. Med. J . 44, 3 51 155. 10. Whitfeld, P. R . & Higgins, T. J. V. (1976) Virology, 71, 471 485. 11. Silberklang, M., Gillum, A. M . & RajBhandary, U . L. (1977) Nucleic Acids Res. 4, 4091 -4108. 12. Donis-Keller, H., Maxam, A. M. & Gilbert, W. (1977) Nuc,/cic Acids Res. 4, 2527-2538. 13. Simoncsits, A,, Brownlee, G. G., Brown, R. S., Rubin, J . R. & Guilley, H . (1977) Nuture (Lond.) 20,833-836. 14. Garfin, D. E. & Mandeles, S. (1975) Virology, 64, 388-3399, 15. De Waechter, R . & Fiers, W. (1972) A n d . Biochem. 49, 184197. 16. Lockard, R. E. & RajBhandary, U. L. (1976) Cell, Y, 747-760. 17. Silberklang, M., Prochiantz, A., Haenni, A. L. & RajBhandary, U . L. (1977) Eur. J . Biochem. 72. 465-478. 18. Wilson, T. M . A,, Perham, R. N., Finch, J. T. & Butler, P. J. G. (1976) FEBS Lrrr. 64, 285-289. 19. Croft, L. R. (1973) Hondhook of Protein Sequences, 3rd edn, pp, 123 - 124, Joynson-Bruvvers Limited, Eynsham Oxford. 20. Vandewalle, M. J . & Sicgel, A. (1976) Virologj.. 73, 413-418. 1. 2. 3. 4. 5.
B. A. Kukla, H. A. Guilley, G. X. Jonard, and K. E. Richards, Laboratoire de Virologie, Institut de Biologie Moleculaire et Ccllulaire du C.N.R.S., 15 Rue Rene-Descartes, Esplanade, F-67084 Strasbourg-Cedex, France K. W. Mundry, Institut fur Botanik, Universitat Stuttgart, D-7000 Stuttgart, Federal Republic of Germany
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Characterization of Long Guanosine-Free RNA Sequences from the Dahlemense and U2 Strains of Tobacco Mosaic Virus Brice A. KUKLA, Hubert A. GUILLEY, Gerard X. JONARD, Kenneth E. RICHARDS, and Kurt W. MUNDRY Labordtoire de Virologie, Institut de Biologie Moleculaire et Cellulaire du Centre National de la Recherche Scientifique, Strasbourg, and Institut fur Botanik, Universitat Stuttgart (Received December 27, 1978)
Four naturally occurring strains of tobacco mosaic virus, U2, Dahlemense, CV4, and the bean form of tobacco mosaic virus, were tested for the existence of long TI RNAase oligonucleotides analogous to the oligonucleotide 52 found in the common or U1 strain of tobacco mosaic virus and which makes up the 5‘ non-coding region of the RNA molecule. U2 and Dahlemense RNA were each found to contain this type of long TI RNAase oligonucleotide with chain lengths of 54 and 74-77 residues, respectively. The sequence of the two oligonucleotides was determined mostly by using 5‘-32P-labelled material in vitro and rapid polyacrylamide gel sequencing techniques. The RNA of the common or U1 strain of tobacco mosaic virus [TMV (Ul)] contains an exceptionally long nucleotide sequence devoid of guanosine residues [l - 31. This guanosine-free sequence is 69 nucleotides in length and may be easily isolated from a total T1 RNAase digest of tobacco mosaic virus RNA as part of a very long T1 oligonucleotide called 52 [2,4]. It is now known that 52 is situated immediately after the capped guanosine residue at the 5‘ extremity of the TMV RNA molecule, the 5’-terminal sequence being m7G5’ppp5’G-52.. . [5]. The sequence at the 3‘ end of Q is . . . A-C-AA-U-G [6]. Because there can be no A-U-G or G-U-G triplet to the left of this sequence, it follows that the A-U-G at the 3‘ terminus of !2i is the first potential initiaton codon in the RNA molecule and we have demonstrated elsewhere that protein synthesis does in fact start at this point, at least in cell-free proteinsynthesizing systems [7]. Thus the non-coding sequence of 68 nucleotides at the 5‘ end of TMV RNA (UI) is remarkable in that it contains no internal guanosine residues. In addition to TMV (Ul), a great many naturally occurring strains of TMV have been isolated and classified with respect to divergence of the coat protein sequence from that of the wild-type strain [8]. Evidently it would be interesting to know how the guanosine-free sequence has evolved in such strains, In an earlier study Mundry and Priess [3] show that the RNAs of the two strains most closely related to TMV (Ul), Dahlemense and U2, each contain a long T1 oligonucleotide resembling O(U1) in length (79 and 56 Abbreviation. TMV, tobacco mosaic virus.
residues respectively) and overall base composition. In this paper we describe the use of 32P-labelling techniques in vitro with polynucleotide kinase to determine the sequence of these two oligonucleotides. The RNA of the two other strains examined, CV4 and the bean form of TMV, did not possess comparable guanosine-free sequences. MATERIALS AND METHODS Preparation of 5’-32P-LabelledOligonucleotides
U2 and Dahlemense strains of TMV were propagated and purified as described by Mundry [4]. CV4 strain TMV was propagated on Cucumis sativus and purified as described by Von Wechmar and Van Regenmortel [9]. The bean form of TMV (inoculum provided by M. Zaitlin) was propagated on Phaseolus vulgaris. A preparation greatly enriched in long particles was prepared from the infected leaves as described by Whitfeld and Higgins [lo]. RNA was obtained from purified virus by phenol extraction. Total TI RNAase hydrolysis was with 1 unit of enzyme (Sankyo)/lO pg RNA at 37°C in 50 mM Tris-HCI, 10 mM MgClz, pH 7.4. After 1 h a like quantity of T1 RNAase was added and incubation was continued for another hour. The oligonucleotides were precipitated with two volumes of ethanol. The ethanol precipitate was taken up in 50 mM TrisHCI 10 mM MgC12, 15 mM 2-mercaptoethanol, pH 7.4, and the RNA was 5’-32P-labelledby treatment with polynucleotide kinase in the presence of [y-”PIATP [6]. Purification of 5’-labelled oligonucleotides was by polyacrylamide gel electrophoresis [6].
62
Long Guanosine-Free Sequences from TMV U2 and Dahlemense RNA
Sequence Analysis Techniques for partial PI nuclease digestion of 5‘-32P-labelled oligonucleotide and analysis by twodimensional electrophoresis/homochromatography [l I] or two-dimensional gel electrophoresis have been described elsewhere [6]. For rapid sequencing on monodimensional gels the procedure of Donis-Keller et al. [I21 was followed except that the ‘ladder’, with cuts after all four types of nucleotide monophosphate residues, was prepared by partial formamide digestion of 5’4abelled material [13]. In order to prepare larger quantities of the long T1 oligonucleotides, as much as 20 mg U2 or Dahlemense RNA was totally digested with T I RNAase as described above. The longer oligonucleotides in the digest were preferentially precipitated with 55 ethanol [I41 and purified by polyacrylamide gel electrophoresis. Prior to electrophoresis a trace of a 5‘-32Plabelled total digest of U2 or Dahlemense RNA was added to the appropriate hydrolysate to allow the oligonucleotides of interest to be located by autoradiography of the gel. After extraction from the gel partial pancreatic RNAase digestion of oligonucleotides was at an enzyme-to-RNA ratio of 1/100000 (w/w) for 10 min at 0 ° C in 50 mM Tris-HC1, 10 mM MgC12, pH 7.5. Enzyme action was stopped by phenol extraction and the subfragments were 5’ labelled by treatment with polynucleotide kinase and [y3’P]ATP as described above. The 5’-labelled fragments were separated from one another by two-dimensional gel electrophoresis [15] with analysis by partial PI nuclease digestion and two-dimensional electrophoresis/homochromatography. RESULTS
Isolation of L’ong Guanosine Free Sequences ,from Naturally Occurring Strains of T M V The genome RNAs of the U1, U2, bean, CV4 and D strains of TMV were exhaustively digested with T1 RNAase and the oligonucleotides were 5’-32P-labelled by incubation with polynucleotide kinase in the presence of [Y-~’P]ATP(Materials and Methods). Fractionation of the digests by polyacrylamide gel electrophoresis reveals that the RNAs of strains U2 and D each contain a long TI oligonucleotide which is well separated from the mass of smaller oligonucleotides in the gel (Fig. 1). The long T1 oligonucleotide of U2, which we shall term O(U2), migrates as a discrete band somewhat faster than Q(U1) whereas the long T1 oligonucleotide of strain D, Q(D), migrates more slowly than Q(U1) and shows signs of being heterodisperse in length (Fig. 1). Neither the bean nor the CV4 strain of TMV gave rise to unusually long T1 oligonucleotides upon T1 RNAase digestion of their RNAs (Fig. 1).
a”, G”2
Fig. 1. Isolution qf long 5’-32P-lubelledoligonucleotides Jkm the R N A qf’severul strains of T M V . Autoradiogram of a 10% polyacrylamide gel staining the T1 RNAase oligonucleotides isolated from the R N A of TMV (Ul) (lanes A and F), TMV (U2) (lane B), CV4 (lane C), bean form of TMV (lane D), and TMV (D) (lane E). The oligonucleotides were 5’ labelled by treatment with polynucleotide kinase and [y-32P]ATPprior to electrophoresis
Sequence Analysis of 5’-32P-LabelledOligonucleotide by Partial PI Nuclease Digestion After purification by polyacrylamide gel electrophoresis as described above, the 5‘-labelled Q(U2) and O(D) were extracted from the gel [no effort was made to separate the Q(D) species of different electrophoretic mobilities] and digested with the non-specific nuclease P1 under conditions designed to yield a digest containing all possible intermediate stages of degradation. Sequence analysis was by the wanderingspot method [16,17]. The sequence of the first 16 residues of O(U2) was read in this manner as pA-C-A-AC-A-A-C-A-A-U-U-A-A-A-A . . . (Fig. 2A). Q(D) may be seen to begin with the sequence pU-A followed by a tract of uridine residues (Fig. 2 B), but beyond the first eight residues the spots become increasingly diffuse so that neither the exact length of the uridine tract nor the sequence following it could be determined. It will be shown below that the uridine tract of Q(D) is heterodisperse in length, which makes the wandering-spot pattern near the 5’ terminus difficult to interpret. In an effort to gain additional sequence information, partial PI nuclease digests of 5’-32P-labelled Q(U2) and Q(D) were fractionated by two-dimensional polyacrylamide gel electrophoresis [6]. Fig. 3 A shows
B. A. Kukla, H. A. Guilley, G. X. Jonard, K. E. Richards, and K. W. Mundry
63
Fig, 2. Sequenw at the 5' terminus of5'-32P-lahelledB(U 2 ) ( 4 ) a n d Q ( D ) ( B ) . Autoradiogram of a PI nuclease partial digest ot5'-32P-labelled B separated by two-dimensional electrophoresis/homochromatography
Fig. 3. T,~~o-dimensionalpolyacrylamidegel electrophorc~sisof PI partial digests qf5'-32P-labelledQ( U2) ( A ) and B ( D ) ( B ) .The first dimension was electrophoresis through a 10% polyacrylamide gel at pH 3.5 and the second dimension was electrophoresis through a 20% polyacrylamide gel at p H 8.3
64
Long Guanosine-Free Sequences from TMV U2 and Dahlemense RNA
a partial PI nuclease digest of Q(U2) fractionated in this manner. It can be seen that the two uridine residues at positions 11- 12, which were detected by two-dimensional electrophoresis/homochromatography, are separated from the next uridine in the sequence (U28) by 15 residues. Following U28 there is an A or C residue and then two more uridine residues. After this there is another long tract devoid of U. At this stage we can read the sequence pA-CA-A-C-A-A-C-A-A-U-U-A- A-A-A-N - N - N - N - N -NN-N-N-N-N-U-N-U-U . . . where N may be either A or C. Two-dimensional polyacrylamide gel electrophoresis of a partial P1 nuclease digest of Q(D) (Fig. 3 B) shows clearly that the oligo(U) tract near the 5‘ terminus is variable in length; the major component has eight uridine residues in the tract but sequences with six, seven and nine uridine residues are present as well, giving rise to wandering-spot patterns slightly displaced with respect to that of the principal species. The oligo(U) tract is followed by a sequence of about seven residues containing no uridine. Next come two uridine residues and then a very long sequence devoid of uridine (Fig. 3 B). It is immediately evident from the wandering-spot pattern that the sequence of Q(D) closely resembles that of sZ(U1) (compare to Fig.3 of [61).
sive electrophoresis of 5’-32P-labelled Q(D) through a 20 % polyacrylamide gel in the presence of urea freed the species of shortest chain length, containing six uridine residues in the oligo(U) tract, from the longer molecules. Fig. 4 B shows a monodimensional sequencing gel from which the sequence of pyrimidine and adenosine residues from positions 8-47 of this oligonucleotide can be read.
Further Sequence Analysis by Polyacvylumide Gel Electrophoresis of Partial Digests
DISCUSSION
Additional sequence information was gained by partial enzymatic digestion of 5’-32P-labelled O(U2) and O(D) followed by polyacrylamide gel electrophoretic size fractionation of the partial digestion products. Conditions for random partial digestion after guanosine residues with T1 RNAase and after adenosine residues with U2 RNAase have been described by Donis-Keller et al. [12]. Partial pancreatic RNAase digestion was used to cleave after pyrimidines (cleavage almost exclusively limited to Y-A linkages) and formamide treatment to cleave after all four residues [13]. Fig. 4 A shows the pattern of bands obtained when portions of a sample of 5’-32P-labelled O(U2) were partially digested with each of the above enzymes and the digests were fractionated in adjacent lanes of a 20 % polyacrylamide gel. The first nine residues have been run off the gel. A partial formamide digest of 5’-32P-labelledQ(U2) was also included on the gel to provide a ‘ladder’ indicating all possible chain lengths. Since the products of enzymatic digestion are ordered by size, the gel determines the position of each adenosine and pyrimidine in the sequence. Before using the rapid monodimensional gel sequencing technique on Q(D) it was necessary to separate the components of different chain length. Exten-
Completion of the Sequence of Q( U2) and Q ( D ) The foregoing observations define the sequence of the first 32 residues of Q(U2) and the first 46 residues of Q(D). In order to complete the sequences, nonradioactive Q(U2) and Q(D) were partially digested with pancreatic RNAase; the subfragments of each were then 5‘ labelled by polynucleotide kinase treatment and separated from one another by two-dimensional polyacrylamide gel electrophoresis. Characterization of the 5’4abelled subfragments by the wandering-spot method provided enough overlaps to establish the complete sequence of both oligonucleotides (Fig.5). Note that the sequences are in fairly good accord with the pancreatic RNAase catalogues of O(U2) and Q(D) previously obtained by Mundry and Priess [3] with uniformly 32P-labelled oligonucleotide.
The exact position of the long guanosine-free sequences of Dahlemense and U2 RNAs along the RNA chain has not yet been directly determined. However, the marked resemblance in sequence between Q(D) and Q(U1) (Fig.5) leaves little doubt that the two guanosine-free sequences are functionally homologous, implying that O(D), like Q(Ul), is situated at the 5‘ extremity of the intact RNA. Consistent with this conclusion is the observation that treatment of Dahlemense virus ‘with sodium dodecyl sulfate, a treatment which, in the case of TMV (Ul), has been shown to uncoat particles in a polar fashion starting at the end of the rod which contains the 5‘ end of the RNA chain [18], uncovers the Q(D) sequence during the earliest stages of uncoating (Mundry, K., personal observations). Similar experiments with TMV (U2) show that Q(U2) is also situated at or very near the sodium-dodecyl-sulfate-labile extremity of the virus particle (Mundry, K., personal observations). Thus, although the sequence relation between Q(U2) and sZ(U1) (Fig. 5 ) is less close than between the latter and O(D), it seems reasonable to hypothesize that Q(U2) likewise forms all or part of the 5‘ non-coding region of U2 RNA. Q(U2) possesses two long sequences composed of only adenosine and cytosine (residues 13-27 and 32-48) like the A-A-C tract of Q(Ul), but much of
B. A. Kukla, H. A. Guilley, G. X. Jonard, K . E. Richards, and K. W. Mundry
65
A
B U, A
F
A A
50 Y A A Y A
1
4 Y A
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A 40 Y
A A Y A A
A Y A A 30 Y A
Y A Y
1
A Y
A
Y 20 Y A Y Y A A
A
20 A A A Y A A A A Y Y 10 A
Y
1; 10
A Y
Fig. 4. Monodimensional polyacrylamide gelfioctionation of'partial digests of S'--('P-labeNed O(U2) ( A ) and Q ( D ) ( B ) . Partial digestion was with formamide (F),which cuts after all four types of residue; U2 RNAase, which cuts after adenine; and pancreatic RNAase (A), which cuts after pyrimidines (mainly at Y-A linkages). Electrophoresis was through a 20% polyacrylamide gel at p H 8.3
A
U A U U U U U A C A A C A A U U A C C A A C A A C A A C A A A C A A C A A A C A A C A U UA C A A U U ACU AUU U A C A A U UAC A A U G
B
UA(U U ....l,"C
A A C A A U UAC C A A C A A C A A!
4 A AC A A C - A A C A A
AAC
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AAC
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\ I
U
UAU,U
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Fig.5. Conijilele .requences uf sLiUl) ( A ) , O i D ) ( B ) and Q(U.2) ( C ) . Sequences of O(D) and Q(U2) have been written so as to display sequence homology with sL(U1). Hyphens representing phosphodiester links have been omitted to save space
the sequence found in sZ(U1) which separates this region from the 3' terminus has been deleted (Fig. 5). sZ(U2) also differs from both sZ(U1) and Q(D) in lacking a uridine-rich sequence at its 5'-terminus. It
cannot be ruled out, however, that such a sequence exists immediately preceding sZ(U2) in the intact RNA molecule, but with a guanosine residue separating it from the sZ(U2) sequence. Fig.5 also shows that all
66
B. A. Kukla et al.: Long Guanosine-Free Sequences from TMV U2 and Dahlemense RNA
three sZ fragments have the same trinucleotide sequence A-C-A immediately preceding the final A-U-G. Dahlemense belongs to the group of TMV strains considered to be the most closely related to TMV (Ul) [8] but, nonetheless, the two strains have diverged considerably, with Dahlemense coat protein having undergone amino acid substitution in 28 of 158 positions [I91 and with overall nucleotide sequence homology falling below the level detectable by competition hybridization [20]. Thus the relatively limited difference in sequence between Q(D) and sZ(U1) may be taken as evidence that the 5' non-coding regions of the two RNAs have been subject to conservative pressures. It is interesting to note that a number of the sequence changes of Q(D) relative to sZ(U1) can be thought of as insertions or deletions of one or more units of a repeating base motif. Examples are the additional A-A-C at position 32- 35 of SZ(D) and the one to four added uridine residues in the oligo(U) tract at the 5' end of the sequence (Fig. 5). A mechanism for engendering such changes is easy to imagine if, during the course of replication of the region of RNA containing the repeating motif, the plus and minus strands of RNA occasionally slip with respect to one another so as to loop out one unit of repeat. Then either an insertion or deletion could occur, depending upon whether the looped out unit is in the plus or minus strand. We thank Prof. L. Hirth, in whose laboratory most of this work was carried out, for his interest and support. Polynucleotide kinase and [i.-32P]ATP were a gift of Dr G. Keith. This research was
supported in part by the ATP 50-77-82 of lnsrirut Nutionol dc. Iu Suntc; ef de lu Rwherchr Midicul.
REFERENCES Mandeles, S. (1968) J . Bid. Chcm. 243, 3671 -3674. Lloyd, D. A. & Mandeles, S. (1970) Biochemistry, 9, 932-938. Mundry, K. W. & Pries, M. (1971) Virology, 46, 86-97. Mundry, K . W. (1969) Mol. G m . Genet. 105, 361-377. Richards, K . F., Guillcy, H., Jonard, G. & Keith. G . (1977) Nature (Lond.) 267, 548 - 550. 6. Richards, K. E., Guilley, H., Jonard, G . & Elirth, L. (1978) Eur. J . Biochem. 54, 513-519. 7. Jonard, G., Richards, K. E., Mohier, E. & Gerlinger, P. (1978) Eur. J . Biochem. 54, 521 - 531. 8. Siegel, A. & Wildman, S. G. (1954) P/7ytopuihology, 4,277- 282. 9. Von Wechmar, M. B. & Van Regenmortel, M . H . V. (1970) S. Afr. Med. J . 44, 3 51 155. 10. Whitfeld, P. R . & Higgins, T. J. V. (1976) Virology, 71, 471 485. 11. Silberklang, M., Gillum, A. M . & RajBhandary, U . L. (1977) Nucleic Acids Res. 4, 4091 -4108. 12. Donis-Keller, H., Maxam, A. M. & Gilbert, W. (1977) Nuc,/cic Acids Res. 4, 2527-2538. 13. Simoncsits, A,, Brownlee, G. G., Brown, R. S., Rubin, J . R. & Guilley, H . (1977) Nuture (Lond.) 20,833-836. 14. Garfin, D. E. & Mandeles, S. (1975) Virology, 64, 388-3399, 15. De Waechter, R . & Fiers, W. (1972) A n d . Biochem. 49, 184197. 16. Lockard, R. E. & RajBhandary, U. L. (1976) Cell, Y, 747-760. 17. Silberklang, M., Prochiantz, A., Haenni, A. L. & RajBhandary, U . L. (1977) Eur. J . Biochem. 72. 465-478. 18. Wilson, T. M . A,, Perham, R. N., Finch, J. T. & Butler, P. J. G. (1976) FEBS Lrrr. 64, 285-289. 19. Croft, L. R. (1973) Hondhook of Protein Sequences, 3rd edn, pp, 123 - 124, Joynson-Bruvvers Limited, Eynsham Oxford. 20. Vandewalle, M. J . & Sicgel, A. (1976) Virologj.. 73, 413-418. 1. 2. 3. 4. 5.
B. A. Kukla, H. A. Guilley, G. X. Jonard, and K. E. Richards, Laboratoire de Virologie, Institut de Biologie Moleculaire et Ccllulaire du C.N.R.S., 15 Rue Rene-Descartes, Esplanade, F-67084 Strasbourg-Cedex, France K. W. Mundry, Institut fur Botanik, Universitat Stuttgart, D-7000 Stuttgart, Federal Republic of Germany
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