Journal of Virological Methods, 19 (1988) 325-330 Elsevier
Method for rapid isolation and analysis of algal virus DNA Alan J. Howarth Department of Plan1 Pathology, University of Arizona, (Accepted
Summary A quick method has been developed for isolating viral DNA from small cultures of eukaryotic algae infected with PBCV-1 or similar viruses. The DNA preparations are virtually free of contaminating host DNA and are suitable substrates for restriction enzymes. 5 ml cultures yield 8-25 kg of viral DNA. Many plaque-purified isolates from environmental water samples can be analyzed in a day. PBCV-1; DNA extraction;
RFLP analysis; Water-borne
PBCV-1 (Van Etten et al., 1982) and similar viruses which infect specific isolates of unicellular algae were first discovered infecting symbiotic algae from Paramecium bursaria and Hydra viridis. These large polyhedral viruses are about 150-190 nm in diameter (Schuster et al., 1986), may be composed of as many as fifty or more structural proteins and contain a lipid component (Skrdla et al., 1984), and have large dsDNA genomes of about 333 kb (Girton and Van Etten, 1987). Other viruses with similar properties have been found to be common in ponds, lakes, and rivers across the United States (Van Etten et al., 1985a, b). Despite their widespread occurrence, little is known about their hosts, replication, persistence, and other factors related to their ecology. Purification of algal virus DNAs is not difficult, but has required purification of virus particles (Van Etten et al., 1983b) followed by disruption of the particles and Correspondence to: Alan J. Howarth, AZ 85721, U.S.A. 0166-0934/88/$03.50
of Plant Pathology,
isopycnic banding of DNA in CsCl-Hoechst dye gradients (Van Etten et al., 1981). Both of these purification protocols require one or more ultracentrifugation steps which are costly in terms of both time and equipment. Protocols for rapid, small-scale isolation of plasmid (Birnboim and Doly, 1979; Ish-Horowitz and Burke, 1981; Kado and Liu, 1981) and viral (Messing, 1983; Gardner and Shepherd, 1980; Xu, 1986) DNAs have been devised to facilitate inexpensive and quick screening of DNA. These methods replaced ultracentrifugation with precipitation and short high-speed centrifugation steps. I have developed a method of isolating algal virus DNA which eliminates ultracentrifugation and, thus, reduces from days to hours the time needed to prepare small amounts of sample for analysis. Cultures of Chlorefla NC64A cells were grown to a density of 5 x 10’ cells/ml in MBBM medium (Van Etten et al., 1983b) supplemented with tetracycline to 25 kg/ml. Five ml aliquots were distributed into sterile 125 ml flasks and were inoculated with a plaque-purified virus isolate using a flamed needle. Cultures were incubated for 2-3 days at room temperature with shaking (120 rpm) in continuous light. Cultures were transferred to sterile glass tubes with screw caps and centrifuged for 10 min at 6000 rpm in a Beckman JA-20 rotor. Pellets were resuspended in 200 l~_lof 50 mM Tris-HCl, pH 7.8, and the suspensions were transferred to 1.5 ml polypropylene tubes. To these suspensions were added 10 l~,l of 20% SDS and 2 p,l of proteinase K (10 mg/ml). After brief mixing the tubes were incubated for at least 10 min at 55-65°C. The solutions were extracted twice with equal volumes of phenol and twice with equal volumes of phenol/chloroform (1:l). Although incubation with proteinase K may be omitted, additional phenol extractions may be needed to remove protein. The aqueous phase contained high molecular weight DNA which was removed carefully to minimize shearing. The deproteinized DNA was precipitated by addition of 0.5 vol of 7.5 M ammonium acetate and 2.5 vol of ethanol. After chilling on ice for 10 min, samples were centrifuged for 5 min in a microfuge. Ethanol was removed and pellets were washed in 70% ethanol and then dried. DNA was dissolved in 50 l_~lof 10 mM Tris-HCl, pH 7.5, 1 mM EDTA or other suitable buffer. Restriction enzyme digests were performed according to manufacturers’ protocols. Electrophoresis was performed in 6 x 10 cm minigels of 0.7% agarose in TBE buffer (Maniatis et al., 1982) at 50 volts until the bromphenol blue marker dye reached the bottom of the gel. While attempting to precipitate algal viruses in solutions of polyethylene glycol (PEG) and NaCl, I found that neither PEG nor NaCl were necessary to obtain good yields of DNA. Although mostly composed of unlysed cells, as judged by light microscopy, the initial centrifugal pellets apparently contained large numbers of mature virions because microgram amounts of viral DNA were obtained after phenol extraction. Incubation of lysates for 1 h at 25°C with cell wall fragments, prepared by methanol extraction (Meints et al., 1984), from 2.5 x 10s NC64A cells markedly increased the yield of DNA by this procedure (data not shown). This result suggests that attachment of virions to residual cell walls is the reason that algal viruses sediment in the pellet after short low-speed centrifugations. Typical yields from minipreparations from 5 ml cultures contained 8-25 kg of
Fig. 1. Electrophoresis of algal virus DNAs prepared by a rapid technique. About 0.5 kg of EcoRIdigested DNA was loaded in each lane: lane 1) lambda; lane 2) DNA which was isolated from a PBCV1 preparation purified as described (Van Etten et al., 1983b); lane 3) PBCV-1 DNA prepared by the rapid method; lane 4-7) DNAs from four randomly-selected algal virus isolates obtained from the Santa Cruz River, Nogales, Arizona; lane 8) DNA from an isolate obtained from a surface water concentrate from Utah. Electrophoresis conditions are described in the text.
DNA. Therefore, l-3 p,l of DNA solution was used for restriction enzyme digestions. Very little algal DNA contaminated the viral DNA preparations (Fig. l), possibly because viral restriction endonucleases may have degraded host nuclear and chloroplast DNA within hours of infection (Van Etten et al., 1984; Xia et al., 1986a, b, 1987). However, slight background of high molecular weight DNA is present in some minipreparations. Fig. 1 shows restriction enzyme digestion patterns from highly purified DNAs and from DNAs purified by this rapid procedure. Lanes 2 and 3 are, respectively, PBCV-1 DNAs isolated from a preparation of purified PBCV-1 virus (Van Etten et al., 1983b) and by this rapid method. The two lanes are virtually identical. Lanes 4-7 contained DNAs from randomly-selected plaque isolates collected from the Santa Cruz River, Arizona. These isolates were indistinguishable by plaque morphology, but they yielded three different restriction patterns. In addition, lane 8 contained DNA obtained from virus which had been adsorbed to a filter from 530 gallons of water, eluted in 850 ml, and plated from an aliquot of eluate with NC64A cells (Van Etten et al., 1983a).
To save two or three days of culturing time, I further optimized this method to purify and analyze DNA from single plaques. An agar plug containing a single plaque was cut with an alcohol-flamed cork borer, lifted into a microfuge tube with the blade of a scalpel, and covered with 200 ~1 of 50 mM Tris-HCl, pH 7.8. The plug was disrupted by repeated passage through a pipette tip attached to a Pipetman P-1000 (Gilson), the resulting slurry was centrifuged for 1 min in a microfuge and the liquid was removed to another tube. The sample was treated with SDS, proteinase K, phenol, phenol/chloroform, and ethanol as described above and further purified by passage through an Elutip-d column (Schleicher and Schuell). Typical 3 mm plaques were titered and contained about 10’ PFU, or about 3 ng of DNA. This calculation is consistent with my results that single plaques contain too little DNA for routine screening by ethidium bromide fluorescence, but enough for detection by sensitive techniques like Southern hybridization. It may be possible to manipulate the culture conditions to obtain plaques with more virions and, thus, more DNA. Simple methods not requiring ultracentrifugation were needed to efficiently screen single-plaque isolates obtained from environmental water samples. Further, genetic variation among virus isolates from the same water source has been shown (Van Etten et al., 1985b; Schuster et al., 1986), however, the extent of variation and the relative abundance of viral genotypes at particular water sources has not been surveyed. These two quick methods make it feasible to purify and analyze DNAs from many viral isolates in one day and thus facilitate such studies. Success in obtaining plaques from eluates of filtered water samples suggested that an optimized filtration method of isolating algal viruses from large quantities of water could be developed, similar to what has been done with human enteric viruses (Gerba et al., 1978) and we have developed such a method (Howarth and DeLeon, manuscript submitted).
Acknowledgements PBCV-1 and NC64A were generously provided by James Van Etten. eron, Patti Fashing-Burdette, John Voss, and Ric DeLeon collected ples. Joe Utermohlen collected water and constructively criticized the This manuscript is journal article no. 4340 of the Arizona Agricultural Station.
Mike Mathwater sammanuscript. Experiment
References Birnboim, H.C. and Daly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523. Gardner, R.C. and Shepherd, R.J. (1980) A procedure for rapid isolation and analysis of cauliflower mosaic virus DNA. Virology 106, 159-161. Gerba, C.P., Farrah, S.R., Goyal, S.M., Wallis, C. and Melnick, J.L. (1978) Concentration of enteroviruses from large volumes of tapwater, treated sewage and seawater. Appl. Environ. Microbial. 35, 540-548.
329 Girton, L.E. and Van Etten, J.L. (1987). Restriction site map of the Chlorellu virus PBCV-1 genome. Plant Molec. Biol. 9, 247-257. Ish-Horowitz, D. and Burke, J.F. (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res. 9, 2989-2998. Kado, C.I. and Liu, S.-T. (1981) Rapid procedure for detection and isolation of large and small plasmids. J. Bacterial. 145, 1365-1373. Maniatis, T., Fritsch, E.F. and Sambrook. J. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Meints, R.H., Lee, K., Burbank, D.E. and Van Etten. J.L. (1984) Infection of Chlorellu-like alga with the virus, PBCV-I: ultrastructural studies. Virology 138, 341-346. Messing, J. (1983) New Ml3 vectors for cloning. Meth. Enzymol. 101, 2&78. Schuster, A.M., Burbank, D.E., Meister. B., Skrdla. M.P.. Meints.R.H., Hattman, S., Swinton, D. and Van Etten, J.L. (1986) Characterization of viruses infecting a eukaryotic Chlorella-like green alga. Virology 150, [email protected]
Skrdla, M.P., Burbank, D.E., Xia, Y., Meints, R.H. and Van Etten, J.L. (1984) Structural proteins and lipids in a virus. PBCV-I. which replicates in a Chlorella-like alga. Virology 135, 308-315. Van Etten, J.L., Meints, R.H., Burbank, D.E., Kuczmarski, D., Cuppels, D.A. and Lane, L.C. (1981) Isolation and characterization of virus from the intracellular green alga symbiotic with Hydra viridis. Virology 113, 704-711. Van Etten, J.L., Meints. R.H., Kuczmarski, D., Burbank, D.E. and Lee, K. (1982) Viruses of symbiotic Chlorellu-like algae isolated from Paramecium bursuria and Hydra viridis. Proc. Natl. Acad. Sci. USA 79, 3867-3871. Van Etten. J.L., Burbank, D.E., Kuczmarski, D. and Meints, R.H (1983a) Virus infection of culturable Ch[orellu-like algae and development of a plaque assay. Science 219, 994-996. Van Etten, J.L., Burbank, D.E., Xia, Y. and Meints, R.H. (1983b) Growth cycle of a virus, PBCV1. that infects Chlorelln-like algae. Virology 126, 117-125. Van Etten, J.L., Burbank, D.E., Joshi, J. and Meints, R.H. (1984) DNA synthesis in a Chlorellu-like alga following infection with the virus PBCV-1. Virology, 134, 443-449. Van Etten, J.L., Burbank, D.E., Schuster, A.M. and Meints, R.H. (1985a) Lytic viruses infecting a Chlorellu-like alga. Virology 140, 135-143. Van Etten, J.L., Van Etten, C.E., Johnson, J.K. and Burbank, D.E. (1985b) A survey for viruses from fresh water that infect a eukaryotic ChloreNu-like green alga. Appl. Environ. Microbial. 49, 1326-1328. Xia, Y., Burbank, D.E., Uher, L., Rabussay, D. and Van Etten, J.L. (1986a) Restriction endonuclease activity induced by PBCV-1 virus infection of a Chlorellu-like green alga. Mol. Cell. Biol. 6, 143&1439. Xia, Y., Burbank, D.E. and Van Etten, J.L. (1986b) Restriction endonuclease activity induced by NC1A virus infection of a Chlorellu-like green alga. Nucleic Acids Res. 14, 6017-6030. Xia, Y., Burbank, D.E., Uher, L., Rabussay, D. and Van Etten, J.L. (1987) IL-3A virus infection of a Chlorellu-like green alga induces a DNA restriction endonuclease with novel sequence specificity. Nucleic Acids Res. (in press). Xu, S.-Y. (1986) A rapid method for preparing phage lambda DNA from agar plate lysates. Gene Anal. Techn. 3. 90-91.