Proc. Nati. Acad. Sci. USAVol. 83, pp. 58-62, January 1986Biochemistry

Viroid processing: A model involving the central conserved regionand hairpin I

(introns/RNA cleavage and ligation/splicing)

T. 0. DIENERPlant Virology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705

Contributed by T. 0. Diener, August 29, 1985

ABSTRACT A model is proposed for the processing ofoligomeric viroid replication intermediates into monomeric,circular progeny viroids. The model identifies a thermodynam-icafly extremely stable base-paired configuration that partiallyor completely dimeric, as well as higher, viroid oligomers canassume and postulates that this structure, which involvesstructural features common to all viroids (the central conservedregion and secondary hairpin I), is essential for precise cleavageand ligation. The model explains why recombinant plasmidscontaining tandem repeats of two or more viroid sequenceequivalents are highly infectious when inoculated into viroid-susceptible plants, why certain plasmids containing partiallyduplicated viroid-specific inserts are less infectious, and whyplasmids containing monomeric inserts are noninfectious or atbest marginally infectious. The model also accounts for the factthat vector-derived sequences on either or both sides of theviroid sequence(s) of a restriction fragment are preciselyexcised and are lacking in progeny viroids.

Detailed structural and thermodynamic studies have shownthat viroids, the smallest known agents of infectious disease(for reviews, see refs. 1 and 2), are covalently closed,single-strandedRNA molecules ofabout 270-360 nucleotidesthat can assume, because of extensive regions of intramo-lecular complementarity, a rod-like quasi-double-strandedconfiguration, jn which short base-paired regions alternatewith small internal loops (3, 4). Sequence determination ofseveral viroids and viroid isolates (5-11) has revealed certainregularities which, with the exception of one,* are commonto all viroids. Most conspicuous among these sequenceregularities is what has become known as the central con-served region of viroids. In the conventional depiction of the"native" viroid molecules (5), this region occupies a stretchof 20 nucleotides of the upper strand close to the center anda stretch of 20 nucleotides (interrupted by 3 nonconservednucleotides) directly below on the lower strand (Fig. 1).Because of its strict conservation, this central viroid regionhas long been believed to serve an essential role in viroidfunction (1, 7), but what this function might be has remainedobscure.The other striking regularity among all viroids sequenced

so far are two or three "secondary" hairpins (13, 14) that arenot present in the "native" configuration, but are transientlyformed during thermal denaturation of the viroid molecules.These hairpins, usually termed I, II, and III, are the result ofinverted repeat sequences present in all viroid primarystructures. Of particular interest to our discussion is hairpinI, which consists of a 9-nucleotide segment, in the upperstrand, 5' to and partially overlapping the central conservedregion and a complementary 9-nucleotide segment, also onthe upper strand, but on the 3' side of and partially overlap-

10=

2 9R 9L B A 9R'

FIG. 1. Putative functional regions within the native structure ofPSTV. The positions of the upper and lower portions of the centralconserved region are indicated by the bars within the rodlikestructure; the positions of sequences forming the stems of secondaryhairpins I-III are shown as boxes containing the appropriate Romannumerals, while the cross-hatched boxes indicate the presumed"pathogenicity-modulating" regions. The open boxes marked 9R',A, B, 9L, 9R, and 2 indicate sequences homologous to the "box"sequences of class I introns. A, The unique cleavage site in PSTVcDNA for BamHI. 1, Nucleotide 1 in conventional representation(5).

ping the central conserved region (Fig. 1). No specificfunctional significance ofthese inverted repeat sequences hasbeen demonstrated, but their presence in all viroids impliesthat they are essential. Also, statistically, such sequences arevery unlikely to be found in random sequences of viroidlength (12).In vivo, viroids are replicated via RNA intermediates of

opposite polarity (15-18); no DNA intermediates exist (see,for example, ref. 19). Because ofthe circularity of viroids andbecause oligomeric replication intermediates exist in infectedcells (16-18), a rolling circle-type replication mechanism hasbeen proposed (16, 18). The existence of these oligomericreplication intermediates requires that monomers are pro-duced by specific cleavage of oligomers, followed by ligationof monomers to form covalently closed-circular molecules.Conceptually, such a process resembles the cleavage-liga-tion reaction by which introns are spliced out of precursorRNAs and exons joined to form functional RNA.

Possible connections between viroids and introns havebeen postulated by several authors (20-22), and the presenceof a nucleotide sequence in the potato spindle tuber viroid(PSTV) complement that exhibits complementarity with the5' end of small nuclear RNA U1 (as do nuclear encodedmRNA introns) (23, 24) has added plausibility to suchspeculations. Evidence indicates, however, that nuclearmRNA introns are excised in the form of lariat RNAscontaining, at the branch site, an unusual nuclease-resistantstructure with 2'-5' and 3'-5' phosphodiester bonds joined toa single residue (25). No such structures have been detectedin viroids.

Analysis of viroid nucleotide sequences also has disclosedthe presence of features characteristic of class I introns[encompassing nuclear rRNA and certain mitochondrial

Abbreviation: PSTV, potato spindle tuber viroid.*The avocado sunblotch viroid differs from all others in its thermo-dynamic and structural properties (12). Its inclusion among viroidsmay be questioned and, in this report, the term "all viroids"excludes this pathogen.

58

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mRNA and rRNA introns (26, 27)]. These features include a16-nucleotide consensus sequence and 3 pairs of short com-plementary sequences (Fig. 1, boxes 9L and 2, 9R and 9R',A and B). As compared with random sequences of equallength, occurrence of these class I intron-like features inviroids is highly significant statistically (27), but whether theyare involved in viroid cleavage and ligation is not known.Evidence from several laboratories has demonstrated that

appropriately constructed, viroid-specific cDNAs obtainedby recombinant DNA technology, as well as certain in vitroRNA transcripts derived therefrom, are infectious whenintroduced into viroid-susceptible plants (28-34). Such plantsdevelop the characteristic symptoms of viroid infection, andprogeny viroids of the proper sequence are synthesized.Evidently, the infecting cDNAs must be transcribed intoviroid-specific RNAs, from which progeny viroids are thensynthesized. Although the exact mechanisms of this processare unknown, experimental results point to specific require-ments that must be met in order for the cDNAs or their RNAtranscripts to be infectious (Table 1).Most conspicuous are the high levels of infectivity with all

multimeric, viroid-specific DNAs, regardless of polarity, andthe low levels or absence of infectivity with DNAs containingmonomeric inserts.The circularity of viroids and the presumed rolling circle-

type of replication suggest that repeat units of the viroidsequence may be needed to permit transcription of the entiresequence from a linear template with a specific initiation site(which is unlikely to be at the exact beginning ofa monomericinsert). Naturally occurring linear, monomeric viroid mole-cules, however, are as infectious as circular ones (35) and soare artificially nicked viroids, provided their 3' terminicontain 2',3' cyclic phosphates (36). Presumably, such linearmolecules are ligated in vivo by an RNA ligase and viroidreplication takes place, as usual, by transcription from theresulting circular molecules. The requirement of 2',3'-cyclicphosphate termini forRNA ligation by an enzyme from wheatgerm (37) supports this contention.

In vivo ligation by a host enzyme cannot, however, explainthe trace levels of infectivity often observed with plasmidscontaining monomeric viroid inserts (Table 1). A possibleexplanation for this phenomenon will be given below.

Table 1. Infectivities of cloned, viroid-specific cDNAs andRNA transcripts

Polarity of Infec-Nucleic acid inoculated transcript* tivityt Refs.

MonomertssDNA + - or ± 30

- - 30ds DNA in vector + - or ± 28, 30

- - or ± 28, 30ds DNA excised + + or ++ 30, 31RNA (E. colt) + + 28RNA (in vitro transcripts) + - 32, 34

- - 32, 34MultimerssDNA + +++ 30

- +++ 30ds DNA in vector + +++ 28, 30

- +++ 28, 30ds DNA excised + +++ 28, 30RNA (in vitro transcripts) + +++ 32, 34

- - 32,34

Excision of monomeric DNA inserts from their vectorsresults in greatly increased levels of infectivity (Table 1). Ashas been suggested (30, 31), it is likely that these restrictionfragments become ligated in vivo to form multimeric units ofviroid sequence equivalents, particularly when, as is the casewith all constructs reported, excision results in the formationof protruding ends, thus facilitating ligation.

In vitro RNA transcripts from DNAs containing monomer-ic inserts of viroid sequences are noninfectious, whereasthose from multimeric inserts are highly infectious, providedthat the transcripts are of the polarity of the infectious viroid(Table 1).

All of these results indicate that more than a monomeric,viroid-specific DNA or RNA is required for expression ofinfectivity exceeding trace levels. Infectivity levels ofdimeric inserts are as high as those of trimeric or tetramericinserts, but whether less than a complete dimer would sufficeis not evident from these experiments.An important clue regarding the required extent of se-

quence duplication has come from experiments with mono-meric, double-stranded PSTV cDNAs inserted into differentvectors. Whereas plasmid pBR322 containing a monomeric,BamHI-derived ds PSTV cDNA unit, inserted into theBamHI site of the plasmid, is noninfectious (28) or onlymarginally infectious (30), the same cDNA inserted intoeither bacteriophage M13-DNA (30) or plasmids pUC9 orpSP64 (33) results in constructs with relatively high levels ofinfectivity (but not as high as those of plasmids with dimericinserts) (not shown in Table 1).

Examination of the plasmid sequences adjacent to thejunction between the vector DNA and the viroid-specificinsert reveals that insertion of the BamHI PSTV unit intoplasmid pBR322 leads to a clone consisting of the 359nucleotides of the monomeric PSTV sequence plus 6 PSTV-specific nucleotides originating from the vector, whereasinsertion into the M13, pUC9, or pSP64 vectors results inclones consisting of the 359-monomeric-PSTV nucleotidesequence plus, coincidentally, 11-PSTV-specific nucleotides(GGATCCCCGGG) originating from the vectors (30, 33).Tabler and Sanger (30) have pointed out that this difference

of five nucleotides seems to be essential for the infectivity ofthe cloned PSTV cDNA and that, interestingly, the viroidregion in question is part of the central region that is strictlyconserved in all viroids.

Clearly, far less than a complete dimer is required forinfectivity, but there seems to be a direct relationshipbetween the extent of sequence duplication and the level ofinfectivity. Whereas a 6-nucleotide duplication results in aclone with trace amounts or no infectivity, an 11-nucleotideduplication results in a clone with a substantially higher levelof infectivity, which, however, is still below that of a clonecontaining a complete duplication of the viroid sequence.Why the requirement for sequence duplication?

In analogy with known splicing and ligation mechanisms ofRNA precursors, particularly those involving nuclear pre-mRNAs, I propose a model in which complementary se-quences close to the future splice site must base pair, makingit possible for a putative "splicing" enzyme to preciselycleave the RNA into monomeric segments and to ligate theseinto the familiar covalently closed circular viroid molecules.Alternatively, this operation may proceed, as in the case ofcertain class I introns (38, 39), in the absence of protein,provided that the appropriate base pairing of complementarysequences takes place.With viroids, a complication arises as a result of their

highly stable "native" structure (4). Presumably, the nascentRNA transcript will readily assume this highly base-pairedand thermodynamically stable configuration. Hence, thedrastically different secondary structure, assumed to berequired for cleavage and ligation, requires a base-paired

*+, Polarity of infectious viroid; -, polarity of viroid complement.t-, No infectivity; ±, trace; +, low; + +, medium; + ++, high levelsof infectivity.$, with 6 or fewer nucleotide duplications.

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60 Biochemistry: Diener

configuration whose stability exceeds that of the "native"configuration in the same region. As I will show, partiallyduplicated or oligomeric viroids can indeed assume such ahighly stable configuration. My model assumes that thisconfiguration is essential for viroid cleavage and ligation. Themodel explains why a certain amount of sequence duplicationis required and assigns a defined function to the centralconserved region and to hairpin I.As shown in Fig. 2, a dimeric RNA transcript can assume

a base-paired configuration whose stability far exceeds thatof the "native" configuration in this region (about -71.1, ascompared to -38.0 kcal, calculated according to refs. 40 and41). Interestingly, this 28-base-pair long and almost uninter-rupted complementary region, consisting predominantly ofC(G base pairs, is composed of the upper portion of thecentral conserved region and of hairpin I. Hence, assumptionof this configuration is possible with all viroids. Although theidentity of nucleotides involved in formation of hairpin I isnot conserved among the various viroids, their ability to basepair is strictly maintained.

In addition to assigning a definite biological function to thecentral conserved region and to hairpin I, the model explainswhy decreasing extents of sequence duplication result indecreasing levels of infectivity. Thus, the base-paired struc-ture that an RNA transcript of a monomeric, BamHI-bordered, cDNA restriction fragment with an 11-nucleotidesequence duplication can assume is less stable than thebase-paired structure possible for a complete dimer or highermultimer; and the structure possible for a transcript contain-ing only a 6-nucleotide duplication is still less stable (Fig. 2).The model is compatible with an involvement of the class Iintron-like sequences in the splicing process. Folding ofPSTV into a conformation analogous to that proposed for theTetrahymena rRNA intron (26) determines a splice site thatis close to or identical with the position envisaged in mymodel (Fig. 3). The model furthermore explains why vector-derived sequences on either or both sides of the viroidsequence(s) of a restriction fragment are precisely excisedand lacking in progeny viroids (Fig. 3).The model does not, however, predict the mechanism of

cleavage-ligation or specify the exact position of cleavage.This could take place directly across the base-paired struc-ture between positions 94 and 95 or at offset locationstheoretically anywhere from positions 93 or 94 to positions 79or 80. Most likely, however, splicing occurs within the rigidlyconserved central portion of the complex that may serve asa recognition site identical in all viroids, whereas the flank-ing, hairpin I-derived, portions may be required for addedstability. Evidently, once the monomeric sequence is ex-cised, the thermodynamic stability of the base-paired "cleav-age-ligation complex" is greatly reduced, presumably per-mitting the monomeric viroid to assume its familiar "native"configuration.The question arises why, in natural infections, or in

purified viroid preparations, the identified highly stablecleavage-ligation complex is not entered into by two mono-meric, linear, or covalently closed circular viroid molecules.Viroid aggregates that are dissociable by compounds such asformaldehyde, urea, or formamide have been describedrepeatedly (42-44), but whether these are base paired in themanner here proposed is not known. Also, the increasedstability of the cleavage-ligation complex, as compared tothat of the native configuration, may not be sufficient todisrupt base pairing of two native viroid molecules, but maybe sufficient to disrupt that of one native molecule (the first359 nucleotides of the PSTV transcript) by the nascent,unpaired duplicate portion of the transcript.The model requires that cleavage occur in the neighbor-

hood of position 94 or 95 which, prima facie, is in conflictwith several reported observations: (i) Cress et al. (28) have

Hairpin I -

Central

Conserved

Region

Hairpin I -

79 C * G

G * CC - GU * A

U *G

110

G -C

Bam Hi G

G\A

90 U

*cAA 100

* A

-l

C - G 11-nucl. repeat

C * G

C - G94 C- G 95

95 G. C 94

0.C-C 6-nuci. repeat

G . C

G - C

A AU

A G

1U%C * G

C - G

IU - AI

G - C

G - U

A - U

G - C

C - G

110 G- C

90

79

- I

FIG. 2. The highly base-paired, thermodynamically stable con-

figuration that dimeric or higher oligomeric viroids can assume.Contribution to the structure of the central conserved region and ofsecondary hairpin I (I, I') is indicated. The extent of sequenceduplication of some recombinant clones containing BamHI-bor-dered, viroid-specific inserts is shown.

observed that RNA isolated from Escherichia coli trans-formed with a recombinant plasmid containing a monomericdouble-stranded PSTV cDNA unit bordered by the uniqueHae III restriction site was marginally infectious when

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Vector Sequences

7 4

Cleavage-Ligation

110 79

9R

Box 2

Class I Consensus

FIG. 3. A schematic diagram illustrating the putative cleavage-ligation complex (nucleotides 79 to 110) that oligomeric viroids can assume(see Fig. 2) and the folding of the remainder of a monomeric PSTV unit (bottom) in analogy with the folding of class I introns (26, 27).Cleavage-ligation at the center of the base-paired complex (solid line) or at any one of several off-set positions (broken line) results in the excisionof a circular, monomeric PSTV molecule (bottom) from the vector-derived and remaining viroid sequences (top). With multimeric viroids,complex formation and excision ofmonomers may occur repeatedly until exhaustion of longer-than-unit-length viroid sequences in the molecule.Because oligomeric viroid complements (minus strands) can assume a "cleavage-ligation" complex involving the same region (nucleotides 79'to 110'), which is almost as stable as that of Fig. 2, the model is equally applicable to the processing of minus strands.

inoculated into tomato plants; (it) Tabler and Sanger (30) havereported that M13 ds DNA containing a monomeric unit ofdouble-stranded PSTV cDNA bordered by the unique Ava IIrestriction site and M13 single-stranded DNA containing anidentical, but single-stranded insert, were marginally infec-tious; (iHO) Kikuchi et al. (45) have shown that natural linearPSTV molecules are predominantly nicked at one of twopositions (181/182 or 348/349), neither of which correspondto the position postulated in the model; and (iv) Robertson etal. (46) have reported that, in an in vitro reaction, a smallportion (1-5%) of tandemly duplicated PSTV was "sponta-neously" cleaved somewhere between positions 250 and 270.

Interestingly, in contrast to the trace infectivity levelsobserved with plasmids containing monomeric, viroid-spe-cific inserts [(i) and (ii) above], monomeric in vitro RNAtranscripts containing vector-derived sequences at either endare completely noninfectious (32, 34). These observationssuggest that the infectivity of the plasmids with monomericinserts is the result of in vivo events during which a modifiedDNA or RNA transcript is produced that can be ligated intoa viable monomeric circle. Such events, for example, mightconsist of in vivo recombination of plasmid DNAs or dupli-cation of viroid sequences by mechanisms similar to thosepostulated by Keese and Symons (47) involving discontinu-ous transcription by a "jumping" RNA polymerase. What-

ever the mechanisms involved, at best, marginal levels ofinfectivity are reached, indicating that these events areinfrequent and clearly distinct from those occurring afterinoculation with cDNAs containing oligomeric viroid copies.

In the case of the natural linear PSTV molecules [see (iii)above], the authors left open the possibility that one or bothof the specific nicks may be artifacts. They presentedconvincing evidence, however, against the possibility thatthese molecules originated during viroid purification (45) butthese linear molecules (which were present in trace amountsonly) might have originated by in vivo nicking of circularmolecules. It is conceivable that the two predominant sites ofnicking could be more exposed than other sites in viroid-hostprotein complexes (46) and, therefore, be more susceptible tonicking by host nucleases.

Similar considerations may apply to the spontaneous invitro cleavage of viroid dimers [see (iv) above]. In view of thesmall portion of dimer cleaved, the particular cleavagereaction demonstrated may not represent an event that is partof the in vivo replication cycle of viroids.

In conclusion, no compelling reason exists to postulate invivo cleavage of viroids at positions different from thoseenvisaged by the model presented. Results with viroid cDNAmutants (33) are compatible with the proposed model-i.e.,with an in vivo cleavage-ligation site within the upper portion

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of the central conserved region. The infectivity of plasmidswith head-to-tail dimeric, BamHI-bordered, PSTV cDNAinserts was substantially affected only when HSO3-inducedcytidine to uridine transitions were present at positions 92and 103 of the second monomer (that is, within the centralregion presumably involved in the cleavage-ligation com-plex), but not when cytidine to uridine transitions werepresent in one or two other portions of either monomer (33).

Alternative cleavage-ligation sites may nevertheless exist.Thus, anRNA transcript ofa recombinant plasmid containinga monomeric double-stranded PSTV cDNA unit bordered bythe unique Hae III restriction site [as in (t0 above] can assumea base-paired structure similar to the one shown in Fig. 2, butinvolving hairpin III. Because the Hae III site is at the exactcenter of this structure (presumably defining the cleav-age-ligation site), the four-nucleotide sequence duplicationmay be marginally sufficient for its formation.Undoubtedly, this model represents a gross oversimpli-

fication ofreality. For example, it does not consider the likelypossibility that viroid replication takes place in specificviroid-host protein complexes. Association of viroids withspecific proteins has been demonstrated (48) and, in suchcomplexes, the viroid RNA may assume a secondary struc-ture that greatly differs from that believed to exist in solution.Although the model cannot be more than a first approxima-tion towards an explanation of what undoubtedly is a verycomplex phenomenon, it fulfills the requirement of a usefulhypothesis in being readily confirmable or refutable byappropriate experimentation.

Note Added in Proof. Additional evidence favoring the upper portionof the central conserved region as the cleavage-ligation site of viroidoligomers has appeared (49, 50), and the configuration illustrated inFig. 2 has been considered as one of three alternative structurespossibly involved (50).

I thank Robert A. Owens, Rosemarie W. Hammond, and MyronK. Brakke for valuable suggestions and critical reading of themanuscript.

1. Diener, T. 0. (1983) Adv. Virus Res. 28, 241-283.2. Sanger, H. L. (1984) in The Microbe 1984: Part I Viruses, eds.

Mahy, B. W. J. & Pattison, J. R. (Cambridge Univ. Press,Cambridge, U.K.), pp. 281-334.

3. Sanger, H. L., Klotz, G., Riesner, D., Gross, H. J. &Kleinschmidt, A. K. (1976) Proc. Natl. Acad. Sci. USA 73,3852-3856.

4. Riesner, D., Steger, G., Schumacher, J., Gross, H. J.,Randles, J. W. & Sanger, H. L. (1983) Biophys. Struct. Mech.9, 145-170.

5. Gross, H. J., Domdey, H., Lossow, C., Jank, P., Raba, M.,Alberty, H. & Sanger, H. L. (1978) Nature (London) 273,203-208.

6. Haseloff, J. & Symons, R. H. (1981) Nucleic Acids Res. 9,2741-2752.

7. Visvader, J. E., Gould, A. R., Bruening, G. E. & Symons,R. H. (1982) FEBS Lett. 137, 288-292.

8. Haseloff, J., Mohamed, N. A. & Symons, R. H. (1982) Nature(London) 299, 316-321.

9. Ohno, T., Takamatsu, N., Meshi, T. & Okada, Y. (1983)Nucleic Acids Res. 11, 509-518.

10. Kiefer, M. C., Owens, R. A. & Diener, T. 0. (1983) Proc.Natl. Acad. Sci. USA 80, 6234-6238.

11. Visvader, J. E. & Symons, R. H. (1985) Nucleic Acids Res.13, 2907-2920.

12. Steger, G., Hofmann, H., Fortsch, J., Gross, H. J., Randles,J. W., Sanger, H. L. & Riesner, D. (1984) J. Biomol. Struct.Dyn. 2, 543-571.

13. Riesner, D., Henco, K., Rokohl, U., Klotz, G., Kleinschmidt,

A. K., Gross, H. J., Domdey, H. & Sanger, H. L. (1979) J.Mol. Biol. 133, 85-115.

14. Henco, K., Sanger, H. L. & Riesner, D. (1979) Nucleic AcidsRes. 6, 3041-3059.

15. Grill, L. K. & Semancik, J. S. (1980) Nature (London) 283,399-400.

16. Branch, A. D., Robertson, H. D. & Dickson, E. (1981) Proc.Nati. Acad. Sci. USA 78, 6381-6385.

17. Rohde, W. & Sanger, H. L. (1981) Biosci. Rep. 1, 327-336.18. Owens, R. A. & Diener, T. 0. (1982) Proc. Natl. Acad. Sci.

USA 79, 113-117.19. Hadidi, A., Cress, D. E. & Diener, T. 0. (1981) Proc. Nati.

Acad. Sci. USA 78, 6932-6935.20. Roberts, R. J. (1978) Nature (London) 274, 530.21. Crick, F. (1979) Science 204, 264-271.22. Diener, T. 0. (1979) Science 205, 859-866.23. Diener, T. 0. (1981) Proc. Natl. Acad. Sci. USA 78,

5014-5015.24. Gross, H. J., Krupp, G., Domdey, H., Raba, M., Alberty, H.,

Lossow, C. H., Ramm, K. & Sanger, H. L. (1982) Eur. J.Biochem. 121, 249-257.

25. Konarska, M. M., Grabowski, P. J., Padgett, R. A. & Sharp,P. A. (1985) Nature (London) 313, 552-557.

26. Dinter-Gottlieb, G. (1986) in The Viroids, ed. Diener, T. 0.(Plenum, New York), in press.

27. Diener, T. 0. & Hadidi, A. (1986) Microbiologica, in press.28. Cress, D. E., Kiefer, M. C. & Owens, R. A. (1983) Nucleic

Acids Res. 11, 6821-6835.29. Ohno, T., Ishikawa, M., Takamatsu, N., Meshi, T., Okada,

Y., Sano, T. & Shikata, E. (1983) Proc. Jpn. Acad. Ser. B 59,251-254.

30. Tabler, M. & Sanger, H. L. (1984) EMBO J. 3, 3055-3062.31. Meshi, T., Ishikawa, M., Ohno, T., Okada, Y., Sano, T.,

Ueda, I. & Shikata, E. (1984) J. Biochem. 95, 1521-1524.32. Ishikawa, M., Meshi, T., Ohno, T., Okada, Y., Sano, T.,

Ueda, I. & Shikata, E. (1984) Mol. Gen. Genet. 196, 421-428.33. Owens, R. A., Hammond, R. W., Gardner, R. C., Kiefer,

M. C., Thompson, S. M. & Cress, D. E. (1986) Plant Mol.Biol., in press.

34. Tabler, M. & Sanger, H. L. (1985) EMBO J. 4, 2191-2198.35. Owens, R. A., Erbe, E., Hadidi, A., Steere, R. L. & Diener,

T. 0. (1977) Proc. Natl. Acad. Sci. USA 74, 3859-3863.36. Hashimoto, T., Suzuki, K. & Uchida, T. (1985) J. Gen. Virol.

66, 1545-1551.37. Konarska, M., Filipowica, W., Domdey, H. & Gross, H. J.

(1981) Nature (London) 293, 112-116.38. Kruger, K., Grabowski, P. J., Zaug, A. J., Sands, J., Gott-

schling, D. E. & Cech, T. R. (1982) Cell 31, 147-157.39. van der Horst, G. & Tabak, H. F. (1985) Cell 40, 759-766.40. Tinoco, I., Borer, P. N., Dengler, B., Levine, M. D.,

Uhlenbeck, 0. C., Crothers, D. M. & Gralla, J. (1973) NatureNew Biology (London) 246, 40-41.

41. Salser, W. (1977) Cold Spring Harbor Symp. Quant. Biol. 42,985-1002.

42. Diener, T. 0. (1971) Virology 45, 411-428.43. Sogo, J. M., Koller, T. & Diener, T. 0. (1973) Virology 55,

70-80.44. Singh, R. P., Michniewicz, J. J. & Narang, S. A. (1974) Can.

J. Biochem. 52, 809-812.45. Kikuchi, Y., Kazimierz, T., Filipowicz, W., Sanger, H. L. &

Gross, H. J. (1982) Nucleic Acids Res. 10, 7521-7529.46. Robertson, H. D., Rosen, D. L. & Branch, A. D. (1985)

Virology 142, 441-447.47. Keese, P. & Symons, R. H. (1985) Proc. Natl. Acad. Sci. USA

82, 4582-4586.48. Wolff, P., Gilz, R., Schumacher, J. & Riesner, D. (1985)

Nucleic Acids Res. 13, 355-367.49. Meshi, T., Ishikawa, M., Watanabe, Y., Yamaya, J., Okada,

Y., Sano, T. & Shikata, E. (1985) Mol. Gen. Genet. 200,199-206.

50. Visvader, J. E., Forster, A. C. & Symons, R. H. (1985)Nucleic Acids Res. 13, 5843-5856.

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