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Immunosorbent electron microscopy (ISEM) and antibody coating
G.P.Martelli
The principle of immunosorbent electron microscopy (ISEM) is the selective trapping of plant viruses on to electron microscope grids precoated with a specific antiserum. This technique has been described in a number of papers and review articles (Derrick, 1973; Milne and Luisoni, 1977; Garnsey et al., 1979; Roberts and Harrison, 1979; Russo, Martelli and Savino, 1980; Van Regenmortel, 1982; Milne and Lesemann, 1984; Hampton, Ball and De Boer, 1990) to which the reader is referred for comprehensive information.
ISEM may be combined with antibody coating (often referred to as "decoration"), a procedure whereby virus particles trapped on the microscope grid are exposed to the homologous antiserum, thus becoming visibly covered with antibody molecules.
The consensus is that ISEM is highly reliable (there are virtually no false positives), as sensitive as ELISA, fast (results can often be obtained within one or two hours) and operationally simple (it requires tools and reagents readily available in most laboratories).
Unfortunately, ISEM requires an electron microscope and accordingly is not suitable for large-scale routine testing. Specimens for ISEM, however, can readily be prepared in laboratories with no electron microscope facilities and can then be shipped for observation (even over long distances) to properly equipped institutions.
BASIC TOOLS AND REAGENTS
The following are required (see Figures 280 to 282):
Mix 3.86 ml of solution A with 6.14 ml of solution B and dilute tenfold
PREPARATION OF TISSUE EXTRACTS
Extracts may be prepared from tissues of different organs of field- or greenhouse-grown plants (leaves, roots, bark, dormant or breaking buds) (Figure 283) or vectors (insects, nematodes). Plant tissues (usually 100 to 200 ma) are ground in a mortar in the presence of carborundum powder or quartz sand and 0.3 to 0.5 ml of phosphate buffer or, especially with grapevine and stone fruits, one of the above protectants (nicotine, PVP, PEG). When a smooth paste is obtained, 0.3 to 0.5 ml of buffer are added and the sample is ground again. The slurry is transferred to a centrifuge tube and centrifuged at 1 500 to 2 000 g. The supernatant fluid is collected and used (Figure 284).
If a centrifuge is not available, tissue extracts can be further diluted with phosphate buffer to 1:15 to 1:20 with respect to tissue weight, and used as such.
Insect and nematode vectors are crushed with a glass rod on a glass slide in a droplet of buffer or protectant. A droplet of buffer is then added and the extract is ready to be used.
ANTISERUM DILUTIONS
The purpose of using an antiserum is twofold: to coat EM grids for trapping virus particles and to "decorate" virus particles by attachment of antibody molecules to the antigenic sites of the particles. Crude antisera are perfectly suitable for both uses, provided that they are properly diluted. For coating of grids, dilute antiserum to near or above its end point (usually 1:1 000 to 1 :5 000) with buffer. For decorating virus, dilute antiserum to 1:10 to 1:100 with buffer. Use of freshly diluted antisera is advisable.
PRECOATING OF EM GRIDS
In certain cases, precoating of EM grids with protein A, a bacterial wall protein that binds specifically to the basal part (Fc portion) of antibody molecules, can be advantageous. For instance, protein A allows trapping of more virus particles because of the richer antibody layer on the grid. It also allows the use of undiluted, lowtitre (1:8 to 1:16) antisera which would not be suitable after high dilution as required by ordinary ISEM.
Protein A is diluted in phosphate buffer at a final concentration of 10 to 100 ?g per ml, a drop is placed on the grid for five minutes at room temperature and the excess is rinsed off before exposure to antiserum.
ANTISERUM COATING OF EM GRIDS
Drops of diluted antiserum (1:1 000 to 1 :5 000) are placed on dental wax or other hydrophobic supports (parafilm strips, silicone-treated paper) in a plastic Petri dish containing moist filter paper (moist chamber) (Figure 285). A freshly prepared carbon-coated grid is gently placed, film-down, on top of each antiserum drop and floated for 5 to 10 minutes at room temperature. Grids are then removed with tweezers and rinsed.
RINSING THE EM GRIDS
Throughout the ISEM procedure, grids must be carefully rinsed to obtain clean preparations. Buffer rinse (Figure 286) is used after protein A precoating, antiserum coating and incubation of the grid with tissue extract. Distilled water rinse is used after second antibody coating (decoration of virus particles), before negative staining for uranyl acetate precipitates in the presence of phosphate ions, or at neutral pH.
Two rinsing procedures can be utilized:
NEGATIVE STAINING
Negative stain can be applied with either system used for rinsing, i.e. floating grids on small drops of the staining solution for 30 seconds to one minute, or applying the stain dropwise (five drops) with a Pasteur pipette.
SUMMARY OF THE PROCEDURE
REFERENCES
Derrick, K.S. 1973. Quantitative assay for plant viruses using serologically specific electron microscopy. Virology, 56: 652-653.
Garnsey, S.M., Christie, R.G., Derrick, K.S. & Bar-Joseph, M. 1979. Detection of citrus tristeza virus. II. Light and electron microscopy of inclusions and virus particles. Proc. 8th Conf: IOCV, p. 9-16. Riverside, CA, IOCV.
Hampton, R., Ball, E. & De Boer, S., eds. 1990. Serological methods for detection and identification of viral and bacterial plant pathogens. A laboratory, manual. St Paul, MN, USA, Am. Phytopathol. Soc. Press. 389 pp.
Milne, R.G. & Lesemann, D.-E. 1984. Immunosorbent electron microscopy in plant virus studies. Methods Virol., 8: 85-101.
Milne, R.G. & Luisoni, E. 1977. Rapid immune electron microscopy of virus preparations. Methods Virol., 6: 265-281.
Roberts, I.M. & Harrison, B.D. 1979. Detection of potato leafroll and potato moptop viruses by immunosorbent electron microscopy. Ann. Appl. Biol., 93: 289-297.
Russo, M., Martelli, G.P. & Savino, V. 1980. Immunosorbent electron microscopy for detection of sap-transmissible viruses of grapevine. Proc. 7th Meet. ICVG, Niagara Falls, NY, USA, 1980, p. 251-257.
Van Regenmortel, M.H.V. 1982. Serology and immunochemistry of plant viruses. New York, Academic Press. 302 pp.
FIGURE 283 Plant organs commonly used for preparation of extracts: leaves, bark, roots, buds
FIGURE 286 Rinsing EM grids with phosphate buffer applied dropwise
FIGURE 287 EM grids being floated on drops of tenfold diluted antiserum for particle "decoration"
FIGURE 288 Rinsing EM grids with distilled water applied dropwise
FIGURE 289 Staining EM grids with uranyl acetate applied dropwise
FIGURE 290 Petri dish with EM grids ready for observation
Detection and identification of viroids
Isolation and analysis of double-stranded RNAs
Western
blot
Detection and identification of viroids
J.S. Semancik
HOST PLANTS FOR PURIFICATION AND BIOASSAY
Indicator plants used for viroid purification and bioassay commonly respond with some form of stunting reaction which may be accompanied by leaf symptoms of rugosity, epinasty, mottling and chlorotic spotting and vein browning. However, since viroid replication may occur in the absence of any discernible symptoms, all inoculated species should be extracted and analysed for viroid content. Plant species suspected of containing viroids can be analysed directly, provided that the extraction conditions necessary to obtain a quality nucleic acid preparation are determined.
In viroid transmission studies, the preferred host plants are seedlings, which in most cases are viroid-free, or vegetatively propagated plant sources made viroid-free by shoot-tip culturing. Woody plant species, such as citron and grapevine, can be inoculated by slashing the stem with a razor-blade moistened with inoculum. With more succulent species, such as tomato, a needle or fine Pasteur pipette is used to puncture the hypocotyl of a very young plant at the point where a drop of inoculum has been applied.
Major indicator hosts
TISSUE EXTRACTION AND PURIFICATION
Ultimate success in detecting viroids as discrete bands on polyacrylamide gels is dependent upon the quality of the nucleic acid preparations obtained from infected tissues. High concentrations of phenolic and acidic compounds can seriously interfere with the recovery of all nucleic acid species. Therefore, the composition of the extraction medium must be customized to the particular tissue under investigation to assure the consistency of factors such as a pH maintained at about 6.5 to 9.0, the presence of appropriate additives such as polyvinylpyrrolidone to neutralize the effects of polyphenols, and adequate concentration of anti-oxidants.
Two protocols commonly used for the extraction of a tissue are presented. The procedures differ basically in the treatment of the aqueous phase from the initial phenolextraction step (Figure 291). Concentration by ethanol precipitation is employed with tissue extracts from plants such as citron and tomato from which good nucleic acid preparations can routinely be recovered. "Trapping" of nucleic acids, including viroid RNA, on CF- 11 cellulose (Figure 292) has been customized for use with direct extraction of grapevine tissue or other tissues from which nucleic acids are difficult to recover.
Even though the primary focus of the procedures presented here is the analysis of citrus, the alternative approach indicated for grapevines should be employed if nucleic acid preparations are either difficult to obtain or of poor quality. The designation of the procedures for grapevines simply indicates the plant tissues for which the technique was developed and does not imply an exclusive application. To date, grapevine tissues have been the most challenging for recovery of nucleic acid preparations of high quality and adequate quantity for analysis of viroid content. Therefore, the information developed for this tissue may become valuable for the analysis of other species.
Unless one can demonstrate the recovery of a typical profile of host nucleic acids, it is difficult to evaluate the relative concentration or even the very presence of viroid molecules. Therefore, it is good practice to inspect the presence and relative concentration of particularly the 4S and 5S RNA components of 2 M LiCl soluble nucleic acids following electrophoresis in the native polyacrylamide gels.
Materials (Figures 292 to 297)
Extraction of citrus and most herbaceous species
1. Grind tissue in 1 ml pre-cooled EM- 1 and 3 ml phenol per
gram tissue, in an ice bath if possible.
Note: Phenol can cause severe burns; therefore, protection
for hands (disposable gloves) as well as a plastic barrier should
be employed to guard against accidents during homogenization.
2. Transfer to centrifuge tubes or bottles and centrifuge for 20 minutes at 7 000 to 12 000g.
3. A clear but pigmented aqueous layer is found over a solid interface of plant debris and a lower heavily pigmented phenol phase. The plant debris may also form a pellet below a liquid bilayer between the aqueous and phenol phases.
4. Remove aqueouslayer and add a 1/10 volume of 3 M sodium acetate, pH 5.5 and a minimum of three volumes of 95 to 100 percent ethanol. Hold at -20°C for 30 minutes or an indefinite period.
5. Centrifuge for 20 minutes at 7 000 to 12 000 g.
6. Discard supernatant and drain pellets containing nucleic acids until excess ethanol is removed.
7. Cover pellets with a minimum volume of RM [1 to 10 ml per 5 to 100 g tissue, fresh weight (FW)] and resuspend with agitation.
8. Transfer slurry to dialysis tubing.
Note: At this point the solution may appear quite turbid
and particulate, but it should clear considerably with dialysis.
9. Dialyse with rapid stirring on a magnetic stirrer at 4°C overnight against 1 litre of RM.
10. Remove sample from dialysis tubing to centrifuge tubes and add one volume of 4 M LiCI. Hold at 4°C for 4 hours or overnight.
11. Centrifuge for 20 minutes at 7 000 to 12 000g.
12. Retain supernatant containing LiCIsoluble nucleic acids (mainly DIVA, 4S and 5S RNA, dsRNAs and viroids). Discard the pellet of LiCI-insoluble nucleic acids (mainly ribosomal RNA).
13. Add a minimum of three volumes of 95 to 100 percent ethanol and hold at -20°C for 30 minutes or overnight.
14. Centrifuge for 20 minutes at 7 000to 12 000g.
15. Decant and drain ethanol from pellets and dry in vacuo.
16. Resuspend pellets in an appropriate volume of RM [100??l per 5 g tissue (FW)].
17. Store at -20 to -80°C.
Note: These preparations are sufficiently purified for
routine viroid detection procedures and infectivity tests.
However, the quality of the analysis will be markedly improved by
further processing by cellulose chromatography.
Extraction of grapevines and tissues from which it is difficult to recover nucleic acids
1. Grind tissue as indicated above, substituting EM2.
2. Centrifuge as above.
3. Note as above.
4. Remove aqueous phase from above interface and lower phenol layer.
5. Make solution to 35 percent ethanol and IX STE with stirring. Add dry CF- 11 cellulose powder [ 1 g per 5 g (FW) tissue extract]. Stir for 2 hours or overnight at room temperature.
6. Collect cellulose by centrifuging at 7 000g for 10 minutes.
7. Discard supernatant and wash cellulose pellet with a solution of 30 percent ethanol in IX STE buffer with agitation (Figure 300).
8. Collect cellulose as in Step 6.
9. Repeat washing procedure with 30 percent ethanol-STE solution two or three times until all traces of pigmented materials have been removed from the wash solution.
10. With the cellulose in 30 percent ethanolSTE, form a chromatography column and continue to wash cellulose with three to four void volumes of 30 percent ethanol-STE.
11. Elute bound nucleic acids using two to three void volumes of STE buffer, collecting the eluent in a serial manner and not as a single batch.
12. Add 10 percent volume of 3 M sodium acetate, pH 5.5, and a minimum of three volumes of ethanol. Let solution stand at -20°C for 30 minutes or longer as convenient.
13. Collect precipitated nucleic acids by centrifugation at 12 000g for 20 minutes.
14. Discard supernatant and allow pellet to drain until reasonably dry.
15. Resuspend pellet in minimum amount of RM buffer.
16. Add one volume of 4 M LiCI and let stand at 4°C for 4 hours or overnight.
17. Centrifuge at 12 000g for 20 minutes. Retain supernatant of 2 M LiCI-soluble nucleic acids.
18. Add a minimum of three volumes of ethanol to the supernatant and let stand at -20°C for 30 minutes or longer.
19. Collect precipitated nucleic acids by centrifugation at 12 000g for 20 minutes.
20. Discard supernatant, drain liquid from pellet and dry in vacuo.
21. Resuspend pellet in a minimum volume of RM buffer, usually in the range of 100 ?l per 5 to 10 g tissue (FW).
22. Store samples at -20 to -80°C prior to analysis by polyacrylamide gel electrophoresis or infectivity.
See Duran-Vila, Flores and Semancik, 1986; Semancik et al., 1975; Semancik, RiveraBustamante and Goheen, 1987.
CF-11 CELLULOSE CHROMATOGRAPHY
This technique (Figures 301 and 303) can be utilized routinely as a preparative procedure for the removal of DNA and other pigmented components of viroid-containing LiCI-soluble nucleic acid preparations. The property of selective binding of viroid RNA at specific ethanol concentrations can also be exploited in recovering viroids from tissue extracts, as was demonstrated in the "trapping" procedure presented in the previous section.
More recently, an analytical approach to CF11 cellulose chromatography has been introduced (Semancik, 1986) to characterize different viroid RNAs by serial elusion with an ethanol gradient. This procedure can be utilized to remove contaminating host RNAs from viroid preparations as well as to separate individual viroids with selective elusion by different ethanol concentrations.
Materials
Preparative chromatography
1. An aqueous sample containing nucleic acids is made to 35 percent ethanol in STE buffer.
2. Apply the solution to a CF- 11 cellulose column which has
been equilibrated with 35 percent ethanol-STE.
Note: (a) The amount of cellulose used is dependent upon
the amount of nucleic acid in the preparation. A proportion of 1
to 10 g cellulose per 5 to l 00 g (FW) extraction is usually
adequate. (b) A "trapping" procedure can also be
employed with dry cellulose added directly to the aqueous phase
from a phenol extraction made to 35 percent ethanol-STE.
3. Wash the cellulose with sufficient 30 percent ethanol-STE to remove all traces of colour from the cellulose or with a volume equivalent to at least four to six column void volumes.
4. Elute the nucleic acids retained on the column with two to four void volume equivalents of STE ((0 percent ethanol).
5. Precipitate nucleic acids with addition of 1/10 volume of 3 M sodium acetate, pH 5.5, and at least three volumes of 95 to 100 percent ethanol, and hold at -20°C for 30 minutes or longer.
6. Centrifuge for 20 minutes at 12 000g.
7. Dry pellet in vacuo and resuspend in TKM buffer (resuspension medium from extraction procedure).
Analytical chromatography
1. Nucleic acid sample from the extraction procedure or preferably a sample pre-treated on apreparative CF-11 column is made to 35 percent ethanol-STE.
2. Apply to a chromatography column containing an adequate amount of CF- 11 cellulose equilibrated with 35 percent ethanol-STE.
3. Wash column with 35 percent ethanol-STE (four to six column void volumes).
4. Elute with 25 percent ethanol-STE, collecting two to four column void volumes. Retain eluent. 5. Wash column with 25 percent ethanol-STE (four to six column void volumes).
6. Elute with 20 percent ethanol-STE, collecting two to four column void volumes. Retain eluent.
7. Continue alternating wash and elusion cycles either with a progressively reduced ethanol concentration (reduced in 5 percent increments for example) or with a decreasing linear ethanol gradient to a final elusion in STE buffer.
8. Precipitate nucleic acids with addition of 1 /10 volume of 3 M sodium acetate, pH 5.5, and at least three volumes of 95 to 100 percent ethanol, and hold at -20°C for 30 minutes or longer.
9. Centrifuge for 20 minutes at 12 000g.
10. Dry pellet in vacuo and resuspend in RM buffer.
11. Analyse by sequential PAGE under native and denaturing conditions.
See Barber, 1966; Duran-Vila, Flores and Semancik, 19X6; Franklin, 1966; Semancik, 1986.
POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)
Optimum resolution of viroid RNA is obtained by a sequential gel electrophoresis procedure (Figure 302) involving migration of the sample into a standard gel (5 percent PAGE) (Figure 304), followed by excision of a piece of the gel, which is then placed in contact with a second, denaturing gel (dPAGE) containing 8 M urea (Figure 305). This procedure exploits the unique properties of the single-stranded closed circular structure of the viroid for the separation of a distinct band. Placement of the excised gel piece in contact with the top (Semancik and Harper, 1984) or the bottom (Schumacher, Randles and Riesner, 1983) of the denaturing gel with migration to the anode will produce similar results.
The discontinuous pH dPAGE (Rivera-Bustamante, Gin and Semancik, 1986) with the gel cast at pH 6.5 (TAE buffer) but migrated in a pH 8.3 running buffer (TBE buffer) enhances the separation between the circular and linear molecular forms of the viroid. In addition, the background of host nucleic acids is reduced, which aids in the recovery of pure viroid preparations for physical characterization and hybridization analysis.
Verification of a suspected viroid can be made by a PAGE analysis sequence involving:
Staining with ethidium bromide to visualize nucleic acid bands is necessary when gels are to be subjected to a second electrophoresis and/or when biologically active viroid is to be recovered. Sensitivity of detection can be increased with silver nitrate staining. However, this procedure renders the viroid inactivated and immobilized in the gel.
Materials
Native 5 percent PAGE
1. Assemble glass form to receive polymerization solution.
2. Mix contents of two beakers containing the following solutions in the indicated amounts or similar proportions:
Beaker 1
12.0 ml distilled water
10.0 ml Stock C
2.4 ml Stock B
Beaker 2
5.0 ml Stock A
0.48 ml ammonium persulphate
3. Fill form, place sample-well comb and let stand for 30 minutes.
4. Withdraw sample-well comb and lower spacer. Attach to chamber and fill electrode reservoirs with 1/10 dilution of Stock D.
5. Mix samples with about 1/4 volume of glycerol and load into wells with fine-tip Pasteur pipettes. Load outermost wells with mixture of tracking dyes.
6. Apply constant current, 54 mA, at 4°C for 2.5 to 3 hours or until bromophenol blue dye has migrated about 8 cm and xylene cyanol has reached about 4 cm.
7. Remove gel from the chamber and form. Soak with gentle agitation in the ethidium bromide staining solution for 10 minutes.
8. View the gel directly over a UV transillumination source. Cut horizontal strip as defined by "viroid zone" (CEV-ASV) or smaller, depending upon viroid, and transfer to denaturing gel.
Denaturing PAGE (pH 6.5)
1. Assemble glass form for polymerization solution.
2. Prepare two beakers with the following contents:
Beaker 1
14.4 g urea
7.0 ml H2O
3.0 ml Stock F (TAE, pH 6.5)
5.0 ml Stock A
Beaker 2
2.5 ml Stock B
0.5 ml ammonium persulphate
Dissolve the contents of Beaker 1 over low heat. After the urea
is dissolved, rapidly mix the contents of the two beakers.
3. Immediately fill form, leaving a flat surface with sufficient space for the excised native gel piece. Allow to stand for a minimum of 1 hour.
4. Remove lower spacer and attach to chamber. Do not add buffer or any liquid to the gel surface until immediately prior to use.
5. After section has been removed from native gel, fill electrode reservoirs and cover top surface of gel with Stock G diluted tenfold (TBE buffer, pH 8.3, 1 X).
6. Float excised section on to the top of the denaturing gel, making as close a contact as possible.
7. Add a few drops of xylene cyanol-glycerol mix next to the outer edges of the gel strip.
8. Apply constant current, 15 mA, at 24°C for about 4 hours or until the xylene cyanol tracking has migrated to within 0.5 cm of the bottom of the gel.
9. Remove gel from form and stain either with ethidium bromide for additional dPAGE (pH 8.3 gel) to confirm circular and linear forms or for elusion of viroid bands for infectivity or for use as templates for cDNA probes; or with silver nitrate for maximum sensitivity of detection.
Denaturing PAGE (pH 8.3)
1. Follow the same set-up and running procedure as presented above.
2. Mix rapidly the contents of two beakers containing:
Beaker 1
14.4 g urea
7.0 ml H2O
3.0 ml Stock G (TBE buffer, pH 8.3, 10X)
5.0 ml Stock A
dissolved on low heat
Beaker 2
2.5 ml Stock B
0.5 ml ammonium persulphate
3. Stain completed gel as before with either ethidium bromide or silver nitrate.
Silver staining
1. Gel can be stained with silver directly or following ethidium bromide staining without additional treatment.
2. Soak gel at room temperature in solution of 50 percent ethanol + 10 percent acetic acid for at least 1 hour with gentle shaking. Overnight soaking can sometimes improve the background.
3. Soak gel at room temperature in solution of 10 percent ethanol + 1 percent acetic acid for 1 hour with gentle shaking.
4. Soak in solution of 12 mM AgNO3 for 1 hour with gentle shaking.
5. Rinse thoroughly (three times) with distilled water.
6. Rinse rapidly with small volume of developer solution (0.75 M KOH + 0.28 percent HCHO) and discard solution.
7. Add fresh developer solution (100 to 200 ml) and observe until bands appear, usually within 20 minutes.
8. Add excess distilled water and allow gel to expand. This process reduces the background and improves the quality of photographs.
9. Developing reaction can be stopped with 0.07 M Na2CO3.
10. Photograph gel over a light-box using Polaroid film (Figures 306 to 309).
See Igloi, 1983; Morris and Wright, 1975; Rivera-Bustamante, Gin and Semancik, 1986; Schumacher, Randles and Riesner, 1983; Semaneik and Harper. 1984.
INFECTIVITY OF NUCLEIC ACID FRACTIONS AND VIROID MOLECULES
The infectivity of a viroid-containing sample can be influenced by the quality of the preparation. In many cases, viroid transmission by highly purified preparations can be more difficult than by a more complex, less purified preparation, perhaps in part because the presence of host nucleic acids may protect the viroid molecule from inactivation. Therefore, a sample such as a 2 M LiCI-soluble fraction may be valuable to demonstrate the transmission properties and host range of suspected viroid-containing preparations.
Nevertheless, an essential proof for the detection of a viroid is the transmissibility of the putative viroid-like molecule. This can be provided by the recovery of the unique, transmissible viroid structure, the singlestranded circular RNA molecule, in highly purified form followed by transmission to a host plant.
Electro-elution of the circular forms of viroids, as detected in denaturing PAGE by ethidium bromide staining, has proved to be a highly efficient procedure for the recovery of biologically active, pure viroid.
Materials
Electro-elution
1. Prepare gel piece to be eluted within dialysis tubing or according to IBI instructions.
2. Apply about 125 V constant voltage for 30 minutes at room temperature.
3. Withdraw buffer sample containing eluted viroid, add 1/10
volume of 3 M sodium acetate, pH 5.5, plus at least three volumes
of ethanol and hold at -20°C for 30 minutes or longer.
Note: The gel piece can be checked for incomplete elusion
of viroid by restaining with ethidium bromide and viewing over a
UV transilluminator. If viroid still remains in the gel piece,
the elusion procedure can be repeated.
4. Centrifuge sample at 12 000g for 20 minutes. Pellets may be extremely small or invisible. Nevertheless, sufficient viroid to be detected by PAGE and silver staining or infectivity can be recovered many times.
5. Dry decanted centrifuge tubes in vacuo and resuspend pellets in appropriate volume of TKM buffer (RM) or desired medium.
REFERENCES
Barber, R. 1966. The chromatographic separation of ribonucleic acids. Biochim. Biophys. Acta, 114: 422424.
Brierley, P. 1953. Some experimental hosts of the chrysanthemum stunt virus. Plant Dis. Rep., 37: 343345.
Calavan, E.C., Frolich, E.F., Carpenter, J.B., Roistacher, C.N. & Christiansen, D.W. 1964. Rapid indexing for exocortis of citrus. Phytopathology, 54: 1359- 1362.
Duran-Vila, N., Flores, R. & Semancik, J.S. 1986. Characterization of viroid-like RNAs associated with the citrus exocortis syndrome. Virology, 150: 75-84.
Franklin, R. 1966. Purification and properties of the replicative intermediate of the RNA bacteriophage R 17. Proc. Natl. Acad. Sci. USA, 55: 1504-1511.
Igloi, G. 1983. A silver stain for the detection of nanogram amounts of tRNA following twodimensional electrophoresis. Anal. Biochem., 134: 184-188.
Morris, T.J. & Wright, N.S. 1975. Detection on polyacrylamide gel of a diagnostic nucleic acid from tissue infected with potato spindle tuber viroid. Am. Potato J., 52: 57-63.
Raymer, W.B., O'Brien, M.J. & Merriam, D. 1964. Tomato as a source of and indicator plant for the potato spindle tuber virus. Am. Potato J., 41:311-314.
Rivera-Bustamante, R. F., Gin, R. & Semancik, J.S. 1986. Enhanced resolution of circular and linear molecular forms of viroid and viroid-like RNA by electrophoresis in a discontinuous-pH system. Anal. Biochem., 156: 91-95.
Sasaki, M. & Shikata, E. 1977. Studies on the host range of the hop stunt disease in Japan. Proc. Jpn. Acad., 55: 103-108.
Schumacher, J., Randles, J.W. & Riesner, D. 1983. A two-dimensional electrophoretic technique for the detection of circular viroids and virusoids. Ana/. Biochem., 135: 288-295.
Semancik, J.S. 1986. Separation of viroid RNAs by cellulose chromatography indicating conformational distinctions. Virology, 155: 39-45.
Semancik, J.S. & Harper, K.L. 1984. Optimal conditions for cell-free synthesis of citrus exocortis viroid and the question of specificity of RNA polymerase activity. Proc. Natl. Acad. Sci. USA, 81: 4429-4433.
Semancik, J.S., Morris, T.J., Weathers, L.G., Rodorf, B.F. & Kearns, D.R. 1975. Physical properties of a minimal infectious RNA (viroid) associated with the exocortis disease. Virology, 63: 160-167.
Semancik, J.S., Rivera-Bustamante, R. & Goheen, A.C. 1987. Widespread occurrence of viroid-like RNA in grapevine. Am. J. Enol. Vitic., 38: 35-40.
Van Dorst, H.J.M. & Peters, D. 1974. Some biological observations on pale fruit, a viroid incited disease of cucumber. Neth. J. Plant Pathol., 80: 85-96.
Weathers, L.G. & Greer, F.C. Jr. 1968. Additional herbaceous hosts of the exocortis virus of citrus. Abstract of a paper accepted for presentation at the Sixtieth Annual Meeting of the American Phytopathological Society, Columbus, Ohio, 2-6 September 1968. Phytopathology, 58: 1071.
Weathers, L.G., Greer, F.C. Jr & Harjung, M.K. 1967. Transmission of exocortis virus of citrus to herbaceous plants. Plant Dis. Rep., 51: 868-871.
FIGURE 291 The procedure for tissue extraction and purification
FIGURE 292 Selection of citrus tissue for extraction
FIGURE 293 Selection of grapevine tissue for extraction
FIGURE 294 Components of extraction medium used for citrus
FIGURE 295 Components used in purification and concentration of nucleic acids
FIGURE 296 Components for resuspension of nucleic acid pellets
FIGURE 297 Components of extraction medium used for grapevines
FIGURE 298 Low-speed refrigerated centrifuge
FIGURE 299 Virtis high-speed homogenizer
FIGURE 300 Apparatus used to agitate CF-11 cellulose for "trapping" of nucleic acids
FIGURE 301 Apparatus used for preparative or analytical cellulose chromatography
FIGURE 302 Apparatus used for polyacrylamide gel electrophoresis (PAGE) and denaturing PAGE (dPAGE)
FIGURE 303 The procedure for CF-11 cellulose chromatography
FIGURE 304 The procedure for polyacrylamide gel electrophoresis (PAGE)
FIGURE 305 The procedure for denaturing polyacrylamide gel electrophoresis (dPAGE)
FIGURE 308 As for Figure 307 (with room lights off)
FIGURE 309 Silver-stained denaturing polyacrylamide gel as seen through the Polaroid viewer
