Canadian Journal of Plant Pathology Use of DNA to ...

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Use of DNA to diagnose plant diseases caused by single-strand DNA plant viruses a

Steve Haber , Jane E. Polston

a b

& Julio Bird

a c

a

Research Station, Agriculture Canada , 195 Dafoe Rd., Winnipeg, MB, R3T2M9 b

Department of Plant Pathology , University of California , Riverside, CA, 95251 c

Crop Protection Division, Agriculture Experiment Station , Rio Piedras, Puerto Rico Published online: 29 Dec 2009.

To cite this article: Steve Haber , Jane E. Polston & Julio Bird (1987) Use of DNA to diagnose plant diseases caused by single-strand DNA plant viruses, Canadian Journal of Plant Pathology, 9:2, 156-161, DOI: 10.1080/07060668709501896 To link to this article: http://dx.doi.org/10.1080/07060668709501896

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CANADIAN JOURNAL OF PLANT PATHOLOGY 9: 156-161. 1987

Use of D N A to diagnose plant diseases caused by single-strand D N A plant viruses Steve Haber, Jane E. Polston, and Julio Bird Research Station, Agriculture Canada, 195 Dafoe Rd., Winnipeg, MB R3T2M9, (J.E.P) Department of Plant Pathology, University of California, Riverside CA 95251, (J.B.) Crop Protection Division, Agriculture Experiment Station, Rio Piedras, Puerto Rico Accepted for publication 1987 03 20

Downloaded by [Agriculture & Agri-Food Canada] at 08:10 05 December 2013

This paper was presented at a symposium held during the annual meeting of the Canadian Phytopathological Society, University of Saskatchewan, Saskatoon, Saskatchewan, 27-31 July 1986. Diagnosing the single-stranded DNA geminiviruses by using specific, labelled DNA from one member of the geminivirus group is one way to approach the ideal of reliable, rapid and sensitive detection of a wide range of members of a virus group, with specific identification of individual members of the group. Purified or cloned DNA from the genome of bean golden mosaic virus (BGMV) was made into probes corresponding to sequences from either one or both genome components. Low molecular weight-DNA was extracted from plants suspected of geminivirus infection, restricted, and Southern blotted to from a'fingerprint'of hybridizing sequences when probed with labelled sequences from the BGMV genome. The presence of hybridizing bands identified sequences of likely geminivirus origin, and their pattern identified the individual infecting geminivirus. Ways of adapting this diagnostic approach to other plant virus groups are discussed. Haber, S.,J.E. Polston, and J.Bird. 1987. Use of DNA to diagnose plant diseases caused by single-strand DNA plant viruses. Can. J, Plant Pathol. 9: 156-161. Le diagnostic des geminivirus à monobrin d'ADN au moyen d'ADN marqué spécifique provenant d'un membre du groupe des geminivirus est une façon de se rapprocher de l'idéal en matière de dépistage: dépisgage sûr, rapide et sensible d'un large éventail de membres d'un groupe de virus et identification particulière des individus du groupe. De l'ADN purifié ou clone du génome du virus de la mosaïque dorée du haricot (BGMV) a servi à construire des sondes correspondant à des séquences d'une ou des deux composantes du génome. De l'ADN à faible poids moléculaire, extrait de plantes soupçonnées d'infection par geminivirus, a été fragmenté puis traité par transfert de Southern, constituant une 'empreinte digitale' des séquences hybridantes en présence de séquences marquées du génome du BGMV. La présence de bandes hybridantes caractérise les séquences provenant vraisemblablement de geminivirus et leur configuration permet d'identifier le geminivirus particulier en cause. Les auteurs discutent des possibilités d'adapter cette méthode de diagnostic à d'autres groupes de phytovirus.

Accurate and sensitive diagnosis is often more critical for plant diseases caused by viruses than for those caused by other types of plant pathogen. No broad-spectrum 'virucides1 are available at present that could be seen as the counterparts to the widespread use of broad-spectrum fungicides against fungal plant pathogens. The control measures against plant viruses that are available aim at a) eradicating sources of inoculum, b) stopping or slowing the spread of virus by controlling vectors, or c) incorporating in plants resistance or tolerance to the virus, rather than using chemical agents with broad-spectrum efficacy to cure plants after the infection has started. Diagnosis is thus especially critical for plant diseases caused by viruses and must try to meet several — on occasion conflicting — aims. Most of all, diagnosis must be reliable. Ideally this should be true not only in the sense of avoiding false negative and false positive results, but also in establishing the activity of infectious agents rather than the simple physical presence of particles or their components.

Diagnosis should be sensitive. Viral inoculum present at levels lower than those that can be detected by a given method could nonetheless still be the source of a disease epidemic, or the continuing cause of insidious yield losses, especially in vegetatively propagated plants such as potato and fruits. Diagnosis should be rapid. Control measures may need to be applied quickly and at the earliest possible stage in the disease cycle to be effective. Plant viruses were first described by their transmission as infectious agents from plant to plant by grafts, insect vectors or mechanical means, and these methods can be extremely useful in disease diagnosis. They are, however, timeconsuming and especially in slow-growing plants one may have to wait for as long as years for appropriate results (Gibbs & Harrison 1976). Finally, diagnosis should be specific. Because different strains of a virus may differ widely in host range or virulence, it is often desirable to know the type and strain of the infecting virus. Some diagnostic approaches, such as the enzyme-linked 156


HABER ET AL.: SYMPOSIUM/BIOTECHNOLOGY

immunosorbent assay (ELISA) in its commonly used 'double antibody sandwich'form, achieve this specificity, at the expense of failing to detect altogether viruses that are similar, but not identical, to the virus under test (van Regenmortel 1983). Diagnostic approaches based on the transmis sion of the virus to a range of healthy hosts with subsequent observation of ensuing symptoms are reliable in the sense of rarely yielding false positives, and sensitive in those cases where it is known how to achieve the transmission. They require, at the very least, several days to yield results, and usually transmission to a range of hosts by a range of vector taxa to determine the specific type of the infecting virus. Approaches which detect virus particles themselves, or their protein or nucleic acid constituents, are usually much more rapid, may be highly sensitive, and do not presuppose knowledge of how to transmit the virus. They are inherently more prone to the risk of yielding false positives, however, since detecting the (sometimes spurious) presence of virus-related material does not prove the 'diagnosed1 virus is causing the disease. An ideal approach to diagnosis would sensitively detect a virus-specific product of the infection process. Biotechnology, in the form of manipula tions of complementary DNA (cDNA) made from viral genes, makes it possible to produce in pure form, virutally unlimited quantities of nucleic acids with highly specific affinities for plant virus genomes, or the products or intermediates of their infections. The application of such an approach to the diagnosis of geminiviruses illustrates how advances in basic understanding of the structure of one member of a virus group have served the practical aim of detecting or confirming infection by other geminiviruses. The geminivirus model Geminiviruses are far from being 'typical1 plant viruses: they are the only ones employing singlestranded (ss) DNA as their genetic material. Partly because of this, they offer a convenient model to illustrate the application of cDN A technology and restriction mapping to plant virus identification. In the majority of geminivirus (and suspected geminivirus-) infections, the infectious particles may often be difficult to transmit mechanically, propagate, and purify, making conventional diagnostic approaches inappropirate for routine use, while geminivirus genomes and genome products, as DNA, are amenable to precise and sensitive manipulation — and therefore reliable and sensitive detection.

157

Thus it was sensible to first characterize as fully as possible the DNA of a few model geminiviruses, such as bean golden mosaic virus (BGMV), that could be propagated and purified relatively easily (Goodman 1977, Goodman et al. 1980, Haber et al. 1981, Ikegami et al. 1981, Haber et al. 1983). Geminivirus genomes of ssDNA replicate via double-standard (ds) DNA intermediates or putative replicative forms (RF) (Ikegami et al. 1981). When a geminivirus infects a cell, the dsDNA intermediate is made from the viral ssDN A; the dsDNA then serves as a template from which new molecules of viral ssDN A are 'peeled ' off to be assembled into a new generation of geminivirus particles. The precise mechanism by which this happens is not known, but detection of geminivirus-specific dsDNA from a (suspectedly) infected cell or tissue and failure to detect it from the healthy control constitutes reasonable evidence of geminivirus infection. The methods used to obtain RF preparations from tissues infected by one geminivirus (e.g. BGMV) can be used to extract analogous RFs from tissues infected by other related geminiviruses. Sequences related to those of BGMV can be detected by complementary hybridization with the labelled probes made from BGMV DNA. A probe might be labelled with a radioisotope, or any other chemical group that can be incorporated into the molecule without impairing its function and that enables it to also be sensitively and specifically detected. An alternative to the traditional radiolabelling has become available in which cytosine or deoxy-cytosine residues of nucleic acids are labelled with biotin; the biotin does not interfere with normal nucleic acid processes but can be subsequently specifically and sensitively detected because it strongly binds the egg-white protein, avidin, which has been previously labelled with an enzyme or fluorescing dye (Langer et al. 1981). The RFs produced by different (suspected) geminivirus infections are distinguished by the sets of fragments they yield when treated with restriction enzymes. The fragments are electrophoretically separated according to size, transferred to a solid support (Southern 1975) and hybridized to a labelled probe made from BGMV DNA. The patterns of fragments obtained with given sets of restriction enzymes constitute a 'fingerprint' to identify a particular geminivirus DNA, because the restriction patterns are sensitive to changes in even a very small proportion of the sequence (Hull 1980). In order for the RF sequence of related (suspected) geminiviruses to be detected, they need not be identical with those of the probe of labelled

158

CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 9, 1987


DNA made from the known geminivirus. The conditions of hybridization of labelled probe to blotted DNA can be adjusted so that those DNA sequences only approximately (40% or more) homologous to the probe DNA will be detected (Howley et al. 1979). Yet even if the RF sequence of the suspected geminivirus being diagnosed is only slightly different from that of the probing sequence (e.g. by 1%), the chances are good that one or more of the specific restriction sites of a set of restriction enzymes with different recognition sites will be altered, resulting in a different set of fingerprints. 'Fingerprint' diagnosis of geminiviruses Similarities in symptoms, and transmission of the disease agent by the whitefly Bemisia tabaci had prompted Bird and others to suggest that a group of rugose mosaic plant diseases were caused by a group of related but distinct viruses related to the agent of golden mosaic of bean (Bird 1956, Bird & Maramorosch 1978). With the characterization of BGMV, it became possible to test Bird's hypothesis, even though most of the agents of the rugose mosaics were refractory to conventional virus purification and characterization. Crude extracts enriched for putative geminivirus-RF were analysed by the fingerprinting approach described above, at first using probes prepared from DNA obtained from purified BGMV particles and later with cloned BGMV DNA. The results (Fig. 1, Table 1) confirmed the presence of distinct sets of restriction fragments specifically hybridizing to BGMV DNA sequences. Moreover, the total size of the fragments, and the respective sums of sizes of fragments hybridizing to DNA from each of the two components of BGMV DNA, conformed to the model established for BGMV (Haber 1983, Haber et al. 1983). In addition, identical fingerprints were found for the isolates from Macroptilium lathyroides, the original source of BGMV, and, unexpectedly, from Malvastrum coromandelianum, a malvaceous weed not thought to be a host of BGMV. Short of obtaining their complete nucleotide sequences, the characterization of geminivirus isolates by analysing their restriction fragment fingerprints offers the best information for diagnostic and comparative purposes. Sensitive serologically-based methods such as ELISA, though having the advantage of being more rapid and inexpensive, could not distinguish between BGMV, Boerhavea mosaic, Euphorbia mosaic, Jatropha mosaic, and Rhynchosia mosaic, and failed to detect viral antigen in field isolates of Rhynchosia mosaic, Sida mosaic, and BGMV in M. coromandelianum (Fig. 1). Although ELISA is

II

Hpa Malvastrum Mer re m a

Jatropha

Rhyncosia

Boerhavea

Sida

v

/

Euphorbia \

\

I

/

Macroptilium

/

/

BGMV Mtt/Haelll Standards

—2527

—1623

$

' 341 311/309 245 214 169 158

>H"

RhyP nat

TT" TT T

HT

-ff

TCrap mech

ELISA Figure 1. Patterns of Hpa II restriction fragments, "finger prints", of Puerto Rican (suspected) geminivirus isolates hybridized with probe made from BGMV DNA. Note the identical patterns of BGMV, Macroptilium isolate and Malvastrum isolate. Analysis of ELISA results was by Tschebycheff's inequality: readings were considered positive if they were higher than the mean plus four standard deviations of the reading for the uninfected bean (Phaseolus vulgaris L. cv Top Crop) controls (A405 = 0.J04). Samples were designated: +■ for 0.250 > A405 > 0.104; ++for 0.400 > A405 > 0.250; +++ for A405 > 0.400

sensitive in detecting BGMV and related geminivir uses at the nanogram range (Franklin & Goodman 1982), specific hybridization of DNA to a labelled probe is clearly more sensitive still. Cloned DNA fragments homologous to the probe could be detected in quantities as small as 1 pg (Haber 1983). An additional advantage of hybridization fingerprint analysis over serologically-based methods is the lesser concern about falsely positive


159

Table 1. Hpa II restriction fragment fingerprints of suspected geminivirus îolates from Puerto Rico Macroptilium lath vroides

BGMV "Urbana'

I* 1250 1080 (150) (130)


Jatropha gossypifolia

Euphorbia prunifolia

II

I

II

I

II

I

II

I

1600

1250 1080 (150) (130)

1600

1200 1100

1550

1600 1020

1100

1300

850 730

820 465

700 300

2610

Boerhavea coccinea

2600

700 300

820

620

330 210

2610

2600

2920

2370

2620

2680

5210

5210

5290

5300

Ma/vastrum coromandelianum

Merremia quinquefolia

Rhynchosia minima

Sida car pini folia

l

11

1

II

1

II

1

1250 1080 (150) (130)

1600

1170

2650

1300

1290

700 300

950 360

1240 1050

2600

2480

2610 5210

II 940 660 550

800

2650 5130

2100 4900

510

880 600

2800

2770

2585

2690 5275

II 980 750 400 280 2130 4900

*Fragments exclusively or preferentially hybridizing to probes made from one or the other of the two components of the BGMV genome are listed for each isolate under "I" and "II". Numbers in parentheses indicate fragments not visualized in Southern blot fingerprinting but known from genome mapping to belong to component I.

results. The positive result in serologically-based methods is a single effect: a precipitin line for Ouchterlony double diffusion, a color change for ELIS A, or the number of particles per viewing field for immune-specific electron microscopy (ISEM). The positive result itself thus offers no way of distinguishing a specific, true result from a nonspecific, false result, and must be controlled by the simultaneous use of known healthy checks. Hybridization fingerprints, by contrast, as specific arrays of relatively sharp zones of fixed raidolabel, would be difficult to conceive of as arising nonspecifically. If at all possible, however, appropriate controls should be included as a precaution. The contribution of biotechnology The motivation to use cDN A in viral diagnosis is simple and powerful: identify one member of a plant virus group that is the easiest to obtain, study and manipulate. Then obtain its genes in pure form, label them, and use them to specifically and sensitively find related sequences, presumably derived from infection by a related virus, in tissues suspected of being infected. Using bacteriophage M13 that had been altered to include a gene for /3-galactosidase (Messing et al. 1981), clones of restriction fragments covering the entire genome of BGMV were prepared (Haber et al. 1983, Howarth et al. 1985), making it possible to produce limitless quantities of pure BGMV DNA

sequences. These sequences in turn were radiolabelled by nick translation (Maniatis et al. 1975), which made them sensitive probes for picking out BGMV-related and thus geminivirus-specific sequences in a blot of DNA restriction fragments prepared from an infected specimen (Fig. 1). Better diagnosis: better disease control The types of diagnosis described above exploit the use of biotechnology to provide a highly specific pure probe. First applications of these technolo gies, however, are neither cheap nor easy. The advantage which assures their increasingly important role in diagnosing plant viral diseases in the future is that once the clones are initially established, their routine use is simple and relatively cheap, and, most importantly, they allow the study of hitherto inaccessible problems. Using hybridization fingerprints with the cDNA technique, weds the sensitivity of specifically hybridizing radiolabel to the discriminating power of restriction analysis, indicating the presence of viral genome in a given sample. When the fingerprint analysis is applied to the diagnosis of suspected geminivirus infections, the material being detected — restriction fragments of the RF of BGMV DNA (the intermediate of replication) — shows that the virus has actually infected the tissue. The fingerprint analysis is sensitive. Cloned DNA fingerprints homologous to radiolabelled probe made from cloned BGMV DNA could be


160

CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 9, 1987

detected in quanitities as small as 1 pg. While not as rapid, in a conventional sense, as ELISA, requiring from one to two weeks from the collection of the tissue specimen to the development of an autoradiographic fingerprint, it yields information that can greatly reduce the time needed for virus characterization. The fingerprints reveal not only that a geminivirus has infected a given tissue specimen, but also precisely diagnose which one. Thus the need for extended host-range studies, if they can be carried out at all, is greatly reduced. It is this combination of precise identification by a definitive set of fingerprints, with the ability to detect a range of related viruses using cloned probes (made from only one or a few members of the group), that is the true power of the fingerprint analysis: specificity of identification with generality of detection. This has special benefits for the plant virus ecologist and epidemiologist. With finger print analysis, it becomes possible to determine precisely and fairly rapidly from which reservoir hosts inoculum is acquired, and to which other hosts a vector transmits the virus. Unanticipated hosts may be found whose ecology may make them important reservoirs of inoculum. Distinct plant virus infections that induce highly similar or indistinguishable symptoms in several common hosts may often be present in different individual plants or different plant species in the same field. It would be a tedious affair to attempt for each case to establish and identify the distinctions based on a series of studies of host range, symptomatology and transmission. Extending the application to all plant virus groups Most groups of plant viruses are not like geminiviruses: they have genomes of ssRNA and would not appear so well suited as the geminivir uses for detection and diagnosis by the fingerprint ing approach. Nonetheless, advances in manipulat ing RN A hold out the promise that the principles of the fingerprinting approach may soon be applied to all plant virus groups. The two tools that make the fingerprint analysis possible for geminiviruses with their ssDNA genomes and dsDNA-replicative forms, a) restriction endonucleases which cut dsDNA molecules at specific sites, and b) the production of limitless quantities of pure probe by recombinant DNA cloning, do not at first appear to have direct counterparts for use with RN A. There are no 'RN A restriction enzymes'. An alternative way to obtain a similar effect exists, however, by use of the enzyme Ribonuclease H, which specifically digests RNADNA hybrids. Synthetic DNA oligomers four to

six nucleotides long of known sequence can be hybridized to ssRNA. In those regions where the RNA sequence complements those of the hybridized DNA oligomers, Ribonuclease H cutting through the four to six base-pair region of RNA-DNA hybrid yields specific fragments of the original ssRN A molecule in much the same manner as a restriction enzyme acting on dsDNA (DonisKelleretal. 1981). It is already a common practice to obtain the sequence information of ssRNA molecules by making complementary DNA (cDNA) from an ssRNA template and then cloning this cDNA in ways similar to those available for any other DNA species. An alternative to the cDNA cloning of RNA molecules may soon be available which is simpler, faster and therefore more accessible for purposes such as making probes for diagnosis of plant viral sequences. The replicase enzyme of the ssRNA bacterial virus Q/3 can produce a 100 000fold incease in an ssRNA during a 10-minute reaction. Unfortuntely, this replicase does not copy most RNAs, because it is highly sensitive for its own, Q/3-RNA, template. By inserting a 'foreign' RNA into a location on Q/3-RNA where it does not appear to alter the three-dimensional structure of Q/3-RN A, the Q/3 replicase can be made to replicate the recombinant Q/3-cum-foreign-RNA as it would a normal Q/3-RNA (Miele et al. 1983). The effect would be similar to that of recombinant DNA cloning and might also be used to provide a ready source of probe molecules. Note added in proof. It has recently been shown that a shortened form of Tetrahymena ribosomal RNA intervening sequence acts as an RNA restriction enzyme and can be made by site-specific mutagenesis to cut at a variety of 4-base sequences (Zaug et al. 1986). Bird, J. 1956. Studies of symptoms and host range of whiteflytransmitted diseases from Puerto Rico. Ph.D. thesis, University of Minnesota, Bird, J., and K. Maramorosch. 1978. Viruses and virus diseases associated with whiteflies. Adv. Virus Res. 22: 55-110. Donis-Keller, H., K.S. Browning, and J.M. Clark. 1981. Sequence heterosis in satellite tobacco necrosis virus RNA. Virology 110: 43-47. Franklin, K.M., and R.M. Goodman. 1982. Bean golden mosaic virus antigen and viral specific sequences in Phaseolus vulgaris determined by ELISA and DNA-DNA hybridiza tion. Phytopathology 72: 964 (Abstr.). Gibbs, A., and B.D. Harrison. 1976. Plant virology: the principles. John Wiley, New York. Goodman, R.M. 1977b. Single-stranded DNA genome in a whitefly-transmitted plant virus. Virology 83: 171-179. Goodman, R.M., T.L. Shock, S. Haber, K.S. Browning, and G.R. Bowers, Jr. 1980. The composition of bean golden mosaic virus and its single-stranded DNA genome. Virology 106: 168-172.



Haber, S. 1983. The genome of bean golden mosaic virus: Restriction map and use as a probe to sensitively diagnose geminivirus infection. Ph.D. thesis, University of Illinois. Haber, S., M. Ikegami, N.B. Bajet, and R.M. Goodman. 1981. Evidence for a divided genome in bean golden mosaic virus, a geminivirus. Nature (Lond.) 289: 324-326. Haber, S., A.J. Howarth, and R.M. Goodman. 1983. Restriction map and Southern analysis of the two components of the bean golden mosaic virus genome. Virology 129:469-473. Howarth, A.J., J. Caton, and R.M. Goodman. 1985. Nucteotide sequence of bean golden mosaic virus and a model for gene regulation in geminiviruses. Proc. Nat. Acad. Sci. U.S.A. 82: 3572-3576. Howley, M.P., M.A. Israel, M.F. Law, and M.A.J. Martin. 1979. A rapid method for detecting and mapping homologies between heterologous DNAs. J. Biol. Chem. 245: 4876-4883. Hull, R. 1980. Structure of the cauliflower mosaic virus genome: restriction endonuclease mapping of thirty-three isolates. Virology 100: 76-90. Ikegami, M.,S. Haber, and R.M. Goodman. 1981. Isolation and characterization of virus-specific double-stranded DNA from tissues infected by bean golden mosaic virus. Proc. Nat. Acad. Sci. U.S.A. 78: 4102-4106.

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Langer, P.R., A.A. Waldrop, and D.C. Ward. 1981. Enzymatic synthesis of biotin-labeled polynucleotides: Novel nucleic acid affinity probes. Proc. Nat. Acad. Sci. U.S.A. 78: 6633-6637. Maniatis, T., A. Jeffrey, and D.G. Kleid. 1975. Nucleotide sequence of the rightward operator of phage lambda. Proc. Nat. Acad. Sci. U.S.A. 72: 1184-1188. Messing, J., R. Créa, and P.H. Seeburg. 1981. The bacteriophage M13 as a vehicle for cloning and sequencing. Nucl. Acids Res. 9: 103-109. Miele, E.A., D.R. Mills, and F.R. Kramer. 1983. Autocatalytic recombination of a recombinant RNA. J. Mol. Biol. 171: 281-288. Southern, E.M. 1975. Detection of specific sequences among DNA sequences separated by gel electrophoresis. J. Mol. Biol. 93: 503-509. Van Regenmortel, M.H.V. 1983. Serology and immunochemistry of plant viruses. Academic Press, New York. Zaug, A., M. Been, and T. Cech. 1986. Tetrahymena ribozyme acts like an RNA restriction enzyme. Nature (Lond.) 324: 420-432.