KLS = proteinase. K-SDS-lysozyme treatment,. LS = lysozyme-SDS treatment, S = SDS treatment. (B) Dilutions of extracted samples of LdNPV spotted on nylon ...
Journal of Virological Methods, 37 (1992) 235-240 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00
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VIRMET 01316
(Short Communication)
A simple method for the extraction of baculovirus DNA D. Desmarteauxa’b,
G. Charpentier”
and M. Arellab
aUniversitP du QuPbec ri Trois-RiviPres, Dipartement de Chimie-Biologie, Trois-Rivikres. Quebec and bInstitut Armand-Frappier, Lava1 des Rapides, QuPbec, Canada (Accepted 6 December
1991)
Summary We have developed a method using lysozyme for DNA extraction from Baculoviruses using as a model Lymantria dispar nuclear polyhedrosis virus (LdNPV) obtained from infected larvae. This method proved to be quick, inexpensive and the extracted DNA was successfully used in molecular hybridization experiments. DNA extraction;
Baculovirus; Lysozyme
The genome of Baculoviruses consists of a double-stranded, covalently closed circular DNA. These viruses are attractive biological control agents in forest pest management programs and have, more recently, also been employed as expression vectors for eukaryotic and prokaryotic genes of medical and veterinary importance (Hausser et al., 1988). The expression vector methodology is based on the high level of expression of the polyhedrin gene responsible for the coding of inclusion bodies (Luckow and Summers, 1988). Several examples of mass production of proteins using baculovirus expression vectors have been reviewed (Doerfler, 1986; Luckow and Summers, 1988). Recent methods used for field analysis of the spread of viral infection and in molecular studies of baculoviruses such as molecular hybridization, the polymerase chain reaction and restriction enzyme analysis require a minimum of nucleic acid purity as well as access to the target sequences on DNA. Current procedures used for the extraction and purification of baculovirus DNA are Correspondence to: M. Arella, Institut Armand-Frappier, Qutbec, Canada, H7N 423.
531 boul. des Prairies, Lava1 des Rapides,
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based on the proteolytic digestion by proteinase K and anionic detergents, such as sodium dodecyl sulfate (SDS) or sarcosyl, followed by organic extractions (Summers and Smith, 1987). We describe the efficient release of baculovirus DNA using different SDS treatments in the presence or absence of lysozyme and proteinase K. Moribund gypsy moth (Lymantriu dispar) larvae were collected in the field from the middle of May to June in the province of Quebec (Canada), as recommended by Ode11 et al. (1979). Forty NPV-infected larvae were homogenised in 25 ml sterile distilled water and the homogenate was filtered through cheese cloth. Larval debris were removed by pelleting in a Sorvall RCSC centrifuge at 1000 x g for 1 min. Viral particles and polyhedra in the supernatants were purified by centrifugation at 99 000 x g for 3 h at 4°C in a 25-60% (w/w) linear sucrose gradient in distilled water (Harrap et al., 1977). The white pellets containing polyhedra were collected, washed twice in distilled water and sedimented at 20000 x g for 30 min. The pellet was finally resuspended in 1 ml of distilled water. The third instar larvae from a sterile insect rearing were infected per OS as described previously (Shapiro et al., 1984). Larvae were fed on 0.5-cm3 diet cubes soaked with 10 ml of the purified suspension of polyhedra to a final concentration of 1.5 x 10’ polyhedra/cm3. Ten days post-infection, groups of 3 moribund larvae each were crushed in 1.5-ml microtubes with 500 ~1 of dilute alkaline saline (DAS) buffer: 100 mM NaHC03.Na2C03, 50 mM NaCl, 2 mM Na-EDTA, pH 10.5 (Whitt and Manning, 1987) and incubated at 37°C for 30 min to solubilize the inclusion bodies. The suspension was centrifuged in an Eppendorf microfuge (table model 5415) for 15 s at 1000 x g and the supernatant transferred to a second microtube in which 100 ~1 of 1 M Tris-HCl, pH 6.8, were added to achieve a neutral pH. Viral DNA was released from the suspension of free virus using the following method: (1) The suspension was incubated for 60 min at 37°C with proteinase K (BRL, Gaithersburg, USA) at a final concentration of 200 ,ug/ml and containing 0.5% SDS. (2) The suspension was incubated for 60 min at 37°C with 200 pg/ml proteinase K, 200 pg/ml of lysozyme (E.C. 3.2.1.17 muramidase, Sigma Chemical Co., St. Louis, USA) and 0.5% SDS. (3) The suspension was kept on ice for 30 min in the presence of 200 mg/ml lysozyme and then transferred to 37°C for the next 30 min in the presence of 0.5% SDS. (4) The suspension was incubated for 60 min at 37°C in the presence of 0.5% SDS.
Fig. 1. (A) DNA extracted from LdNPV using the different treatments described in the manuscript and separated on agarose. The arrow is pointing at LdNPV DNA. M = molecular weight markers, KS = proteinase K-SDS treatment, KLS = proteinase K-SDS-lysozyme treatment, LS = lysozyme-SDS treatment, S = SDS treatment. (B) Dilutions of extracted samples of LdNPV spotted on nylon membranes and hybridized using a polyhedrin gene probe. Positive and negative controls are at the bottom of the membrane.
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M
KS
KLS
LS
S
Ld NPV DNA
PERCENTA GE OF RXED DNA
loo.M I31.60
-
to.oD 3.16
-
1.66 0.31 o.to
-
0.03
-
om
-
CONT ROL @I CON-fROL@
)(s
KLS
LS
S
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Each DNA suspension was then extracted with phenol, phenol-chloroform and chloroform-isoamyl alcohol (24:1, v/v) and precipitated with ethanol. Finally, DNA was lyophylised and resuspended in sterile distilled water. An aliquot of the extracted DNA was electrophoresed on a 0.8% agarose gel for 45 min at 50 V. The gel was then stained for 15 min in ethidium bromide and observed with an U.V. transilluminator at 254 nm (Fig. 1A). The residual volumes of crude DNA was completed to 600 ~1 with distilled water. Semi-logarithmic dilutions were prepared from each DNA extract, the samples were then denatured at 100°C and immobilised on nylon membranes pretreated with 2 x SSC (sodium salt citrate; 0.3 M NaCl, 0.03 M Na-citrate, pH 7.0) using a slot-blot manifold system (Schleicher & Schuell Inc., Keene, U.S.A.) (Maniatis et al., 1982). Membranes were then baked under vacuum at 80°C for 60 min. Prehybridization and hybridization were carried out as described by Maniatis et al. (1982). Hybridization was carried out for 16 h at 42°C using lo6 CPM of a [cr-32P]dCTP-labelled LdNPV polyhedrin gene prepared by nicktranslation (Smith et al., 1988). Membranes were washed twice in 500 ml of 1 x SSC and twice in 500 ml of 0.1 x SSC and 0.1% SDS (w/v) at 55°C for 30 min. X-Omat AR Kodak films were exposed overnight to the dried membranes and developed in a Kodak X-Omat M20 film processor. The experiments allowed comparison of the efticiency of proteinase K-SDS (KS), proteinase K-lysozyme-SDS (KLS), lysozyme-SDS (LS) and 0.5% SDS (S) in releasing LdNPV DNA from a common pool of 3 infected larvae (Fig. 1A). The figure shows that lysozyme-SDS (LS) and 0.5% SDS (S) seem to release much more DNA than the conventional method (KS). This difference is also apparent when lysozyme is added to proteinase K-SDS (KLS). Nevertheless, the samples treated with proteinase K (KS and KLS) appear to be much more pure than the others, which showed evidence of partial degradation. Hybridization of labelled LdNPV polyhedrin gene, used as probe, to semilog dilutions of viral DNA samples treated with KS, KLS, LS and S (Fig. 1B) show that the signal obtained for the highest dilution of the lysozyme-treated samples (LS, lane 7) is very similar to those obtained for a lo-fold dilution of the samples treated with KS, KLS and S. It is well known that lysozyme (mucopeptide N-acetyl muramoyl hydrolase) hydrolyzes bonds between ZV-acetyl muramic acid and N-acetyl-b-D-glucosamine in the polysaccharide backbone of peptidoglycans. Lysozyme is currently used in bacterial cell walls lysis and in the extraction of nucleic acids (Haas and Dawding, 1975). It is interesting to note that lysozyme and a group of peptide or small protein antibiotics were recently found to be active in immune and hemostatic systems of insects and are important for the protection of the insect from foreign organisms (Law and Wells, 1989). The greater efficiency of lysozyme over proteinase K in releasing LdNPV DNA could be explained by two hypotheses: (1) lysozyme hydrolyses well target proteins linked to nucleic acids of LdNPV (deoxynucleoproteins) very
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well and efficiently releases DNA without the risk of coprecipitation with unbound proteins during the extraction step, (2) lysozyme is not inhibited or partially inhibited by serpins (serine protease inhibitors) that have been shown to be present in insect tissues (Kanost et al., 1989). The possibility cannot be excluded that serpins, whose function(s) in insects are unknown (Law and Wells, 1989) are responsible for a partial inhibition of proteinase K, which is neither involved nor naturally regulated in the hemostatic systems of insects as is the case for lysozyme. The results demonstrate (Fig. lA,B) that the difference between the quantities of LdNPV DNA, obtained when releasing methods using proteinase K (K) and lysozyme (L) are employed, involves a loss of viral DNA during the extraction steps. Inefficient hydrolysis of bonds between nucleoproteins and viral DNA would cause their coprecipitation in the organic phase during the protein extraction step. Lysozyme appears to be equally efficient in extracting the nucleic acids of other baculoviruses (Artogeiu rupae GV and Bombyx mori NPV: data not shown) under the conditions described in this report. Separate steps using lysozyme only for viral DNA release, followed by SDS treatment to relax protein-protein interactions before extraction gave the optimal results in hybridization experiments (Fig. 1B). DNA extraction methods using lysozyme are more efficient and cheaper than those employing proteinase K in the pre-treatment of large numbers of specimens and could be appropriate for studies concerning the spreading of baculovirus infection in larval populations by DNA hybridization.
References Doerfler, W. (1986) Expression of the Autographa californicu nuclear polyhedrosis virus genome in insect cells: homologous viral and heterologous vertebrate genes - The baculovirus vector system. Curr. Topics Microbial. Immunol. 131, 51-68. Haas, M.J. and Dawding, J.E. (1975) Aminoglycoside-modifying enzymes. In: J.H. Hash (Ed), Methods in Enzymology, Vol. 43. Academic Press, New York, 621 pp. Harrap, K.C., Payne, C.C. and Robertson, J.S. (1977) The properties of three baculoviruses from closely related hosts. Virology 79, 14-3 1. Hausser, C., Fusswinkel, H., Li, J., Oelhg, C., Kunze, R., Muller-Neuman, M., Heinlein, M., Starlinger, P. and Doerfler, W. (1988) Overproduction of the protein encoded by the maize transposable element AC in insect cells by a baculovirus vector. Mol. Gen. Genet. 214, 373-378. Kanost, M.R., Prasad, S.V. and Wells, M.A. (1989) Primary structure of a member of the serpin superfamily of proteinase inhibitors from an insect Munduca sexta. J. Biol. Chem. 264,965-972. Law, J.H. and Wells, M.A. (1989) Insects as biochemical models. J. Biol. Chem. 264, 16335516338. Luckow, V.A. and Summers, M.D. (1988) Trends in the development of baculovirus expression vectors. Biotechnology 6, 47-55. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 545 pp. Matthews, J.A. and Kricka, L.J. (1988) Analytical strategies for the use of DNA probes. Anal. Biochem. 169, I-25. Odell, T.M., Butt, C.A. and Bridgeforth, A.W. (1979) Lymantriu dispar. In: P. Singh and R.F. Moore (Eds), Handbook of Insect Rearing, Vol. II. pp. 355-367. Shapiro, M., Robertson, J.L., Injac, M.G., Katagiri, K. and Bell, R.A. (1984) Comparative infectivities of gypsy moth (Lepidoptera: Lymantriidae) by chitinase. J. Econ. Entomol. 77, 153-
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156. Smith, I.R.L., Van Beek, N.A.M., Podgwaite, J.D. and Wood, H.A. (1988) Physical map and polyhedrin gene sequence of Lymantria dispar nuclear polyhedrosis virus. Gene 71, 97-105. Summers, M.D. and Smith, G.E. (1987) A manual of methods for Baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station. (Ed), Bulletin No. 1555, A and M University, pp. 56. Whitt, M.A. and Manning, J.S. (1987) Role of chelating agents, monovalent anion and cation in the dissociation of Autograph calfornicu nuclear polyhedrosis virus occlusion body matrix by zinc chloride. J. Invertebr. Pathol. 49. 61-69.
