DNA from two Orgyia pseudotsugata baculoviruses: Molecular weight determination by means of electron microscopy and restriction endonuclease analysis

VIROLOGY

95,

176-184 (1979)

DNA from Two Orgyia pseudotsugata Baculoviruses: Determination by Means of Electron Microscopy Endonuclease Analysis1 M. P. SCHAFER,* *National

Cancer

Institute, Agricultural

G. ROHRMANN,Y National Institutes Chemistry, Oregon Accepted

U. HEINE,*

AND G. S. BEAUDREAUt

of Health, Bethesda, Maryland State University, Coruallis, January

Molecular Weight and Restriction

20014, and TDepartment Oregon 97331

of

15, 1979

Both aqueous and urea-formamide procedures for spreading nucleic acid were employed for electron microscopic studies on the DNAs from the nucleopolyhedrosis bundle virus (NPBV) and the nucleopolyhedrosis single-rod virus (NPSV) both of which are pathogenic for Orgyia pseudotsugata. The molecular weight estimates via electron microscopy were derived by comparison of the mean length values for the double-stranded relaxed circular DNAs with that of SV40 DNA. The aqueous and urea-formamide spreading methods yielded NPSV DNA molecular weight values of 103 x lo6 and 104 x lo6 daltons, respectively, and molecular weight values of 87 x lo6 and 85 x lo6 daltons for NPBV DNA. These molecular weights were compared with molecular weight estimates from restriction endonuclease analysis, sedimentation analysis, and renaturation kinetic analysis. DNA located within the NPBV polyhedra but external to virions was characterized by restriction endonuclease analysis and examined by electron microscopy. It was determined to be composed of fragments of random size and to be of viral origin.

analysis and renaturation kinetics respectively (Rohrmann et al., 1977). Reports on Two distinct baculoviruses pathogenic for the NPBV from another laboratory indicated Orgyia pseudotsugata (the Douglas-fir a genome size of 64 x lo6 based on tussock moth) have been reported. These restriction endonuclease analysis (Miller have been termed the nucleopolyhedrosis and Dawes, 1978). The genome of the bundle virus (NPBV) and the nucleopolyNPSV was estimated to be 88.5 x lo6 based hedrosis single-rod virus (NPSV) based on on optical renaturation and 85 x lo6 by the extent of aggregation of their nucleo- restriction endonuclease analysis (Rohrmann capsids (Hughes and Addison, 1970). The and Beaudreau, 1977). two viruses are widely divergent genetically In order to further investigate the and demonstrate 1% or less nucleotide molecular weights of the two viral genomes sequence homology (Rohrmannet al., 1978). we have examined the genome size of both An insecticide containing these viruses has viruses utilizing electron microscopy, and recently been registered by the EPA for use the NPBV with two different restriction against 0. pseudotsugata (U. S. Environendonucleases. In addition, the extraviral mental Protection Agency registration No. DNA present in NPBV polyhedra was 27586-1, August 11, 1976). characterized by means of electron microsStudies have recently been undertaken to copy and its restriction endonuclease fragdetermine the size and complexity of the ment pattern. genomes of these viruses. Molecular weights of the NPBV genome of 96 x lo6 and MATERIALS AND METHODS 86 x lo6 were estimated by sedimentation Purijication of virions. Viral inclusion ’ Oregon Agricultural Experiment Station Technical bodies (polyhedra) were produced and paper No. 5134. purified as previously described (Rohrmann, * To whom requests for reprints should be sent. INTRODUCTION

1’76

0042-6822/‘79/070176-09$02.00/O Copyright All rights

0 1979 by Academic Press, of reproduction in any form

Inc. reserved.

DNA FROM TWO Orgyia pseudotsugata BACULOVIRUSES

177

FIG. la. A circular NPSV DNA duplex molecule. The DNA was spread in the presence of SV40 DNAutilizing the aqueous method for electron microscopy: (A) NPSV DNA; (B) SV40 DNA (Form II).

1977). Purity of the polyhedron suspensions was confirmed by dark-field microscopy procedure, with alkaline hydrocolloid (Martignoni, 1972). Polyhedra were suspended in distilled water and dissolved by adding 0.1 vol of 1 M Na&O,-0.5 M NaCl, at room temperature for 10 min. This solution was centrifuged at 4300 g for 5 min. The free virions in the supernatant were then pelleted at 40,000 g for 30 min. The resuspended virions were banded on 30-65% sucrose gradients by centrifugation at 60,OOOg for 2 hr at 20”.

FIG. lb. A circular NPBV DNA duplex molecule. The aqueous spreading method for electron microscopy was employed: (A) NPBV DNA; (B) SV40 DNA (Form II); (Cl SV40 DNA (Form I).

The virus band was removed and again pelleted and suspended in 0.02 M Tris, 0.15 M NaCl, 0.05% Na-azide, pH 7.4. Extraction of baculovirus DNA for electron microscopy studies. One-tenth milliliter of the resuspended viral pellet was mixed with 0.9 ml of 0.02 M Tris, pH 7.4, and 0.01 M EDTA. The solution was then made 0.5% in sodium dodecyl sulfate (SDS). Proteinase K, 200 pg/ml, was added and the solution incubated at 37” for 30 min. The DNA was purified by phenol extraction. Purified DNA was dissolved in 0.02 M Tris, pH 7.4, and 0.01 M EDTA. SV.40 marker DNA. Super-coiled SV40 DNA (Form I), was a gift from Dr. George Khoury, National Institutes of Health, Bethesda, Maryland. A limited digestion with DNase I (Sambrook and Shatkin, 1969) was used to convert approximately 40% of the supercoiled Form I DNA to the open circular duplex Form II DNA. DNase I was removed by phenol extraction. Electron microscopy (method 1). Solutions of DNA were spread for electron microscopic examination using a modification of the urea-formamide method (Westphal and Lai, 1977). The hyperphase contained 55% formamide, 2.6 M urea, 0.009 M EDTA, 0.09 M Tricine, 0.09 M NaOH, pH 8.3, and DNA (0.1 to 1.0 pg/ml). The mixture was heated at 53” for 30 set, placed in ice water, and then allowed to reach room temperature. Cytochrome c,

178

SCHAFER

50 pg/ml, was added immediately before spreading onto a deionized water hypophase. Method 2. In the standard aqueous method (Kleinschmidt and Zahn, 1959), 50 ~1 of DNA (0.1 to 1.0 @g/ml) in 0.5 M ammonium acetate, 0.001 M EDTA was mixed with cytochrome c immediately before spreading onto a 0.25 M ammmonium acetate hypophase. All DNA-protein films were adsorbed onto parlodion-coated grids, stained with uranyl acetate (Davis et al., 1971), dehydratedin 90% ethanol, and rotary-shadowed with platinum-palladium (80:20) at an angle of 5”. As an internal length standard, SV40 DNA was added to the baculovirus DNA before spreading. Micrographs were taken with a Siemens Elmiskop 1A electron microscope at an original magnification of 6000 and an accelerating voltage of 60 kV. The molecules were measured at a final magnification of 21,000 with a HewlettPackard 9810A calculator equipped with a 98648 digitizer. Miscellaneous. Extraction of baculovirus DNA and its analysis by restriction endonucleases was performed using methods and enzymes previously described (Rohrmann et al., 1978). Extraviral DNA was extracted as previously described (Rohrmann and Beaudreau, 1977). RESULTS

Baculovirus Electron

Genome Size Examined Microscopy

by

ET AL.

giving a rise of 3.8 A per base pair for SV40 DNA. All but one of the NPSV molecules spread by this method exhibited a length of 55-65 pm while all 23 of the NPBV DNA molecules ranged from 45 to 55 pm (Table 1). Regardless of which nucleic acid spreading method was employed, the NPBV DNA and NPSV DNA were consistently 25- and 30-fold, respectively, larger than the SV40 DNA standard. The molecular weights of the circular baculovirus DNAs were calculated from the ratios of the baeulovirus DNA mean length to the mean length of the SV40 DNA, using 3.4 x lo6 daltons for SV40 DNA (Reddy et al., 1978). The molecular weights of the circular NPSV DNA are 104 x lo6 (urea-formamide spreading) and 103 x lo6 (aqueous spreading) and for NPBV circular DNA 85 x lo6 (ureaformamide spreading) and 87 x lo6 (aqueous spreading) (Table 1). The urea-formamide method has the advantage over the aqueous spreading method in that it prevents random base-base interactions in regions of DNA molecules which may be partially single stranded. This permits full extension of the DNA molecule for more accurate length determination and also permits the visualization of supercoiled regions (See Fig. 2) which when spread by the aqueous method could be confused with collapsed single-stranded super-coiled DNA regions. [In addition from NPBV and NPSV was demonstrated by banding [32P]DNA in CsCl gradients containing ethidium bromide (data not shown). In one preparation 54% of the NPBV DNA was supercoiled.] A circular duplex DNA molecule of 6-8 x 10” daltons or approximately twice the size of the SV40 DNA was found in very low concentration in both baculovirus DNA preparations (Fig. 3A). The unknown DNA molecule also exhibited a supercoiled form (Fig. 3B).

The aqueous spreading method produced an average rise of 3.16 A per base pair for the SV40 standard DNA. This is close to the values of 2.9 to 3.0 b per base pair recently reported by Griffith (1978) for X+174 duplex DNA. Relaxed circular DNA molecules were obtained by the aqueous spreading method for both nucleopolyhedrosis viruses (Figs. la and lb). All but 3 of 39 circular NPSV DNA molecules exhibited lengths between 45-55 pm, NPBV Genome Size Estimated by Restricwhereas the NPBV was significantly tion Endow&ease Analysis smaller with 33 of 36 molecules being The molecular weight of the NPBV 40-45 ,um in length (Table 1). The nonaqueous urea-formanu ‘de spreading genome was also examined by comparing rate of restriction endomethod caused the duplex molecules to the migration nuclease fragments with DNA markers of extend 1.2 times the length of the molecules prepared by the aqueous method (Table 1) known size. Digests were made utilizing

DNA

FROM

TWO

pseudotsugata

Orgyia

TABLE

179

BACULOVIRUSES

1

DETERMINATION OF BACULOVIRUS DNA MOLECULAR WEIGHTS UTILIZING ELECTRON MICROSCOPIC NUCLEIC ACID SPREADING TECHNIQUEF Mean length (Frn f SD)

No. of molecules measured

DNA

Urea-formamide sv40 NPBV sv40 NPSV

22 23 37 17

method 2.00 f 0.17 50.00 f 1.88 1.97 * 0.14 60.34 -+ 2.52

Aqueous method 1.62 41.35 1.65 49.95

74 36 38 39

sv40 NPBV sv40 NPSV

Molecular weight (XloB)

k 2 2 f

3.4” 85 -+ 3 104 * 4 3.4” 87 lr 3

0.08 1.28 0.11 2.71

103 2 6

u Calculated from the nucleotide sequence (Reddy et al., 1978).

both Smal and Hsul. Duplicate digests and gels were done for each enzyme and a representative scan from one of these gels is shown in Fig. 4. A molecular weight of 85 + 5 x lo6 daltons was estimated for the NPBV from four digests. Characttization

of NPBV Extraviral

DNA

DNA present in NPBV polyhedra but not sedimenting with virions was examined by means of electron microscopy, buoyant density in cesium chloride density gradients, and restriction endonuclease patterns. It was found by electron microscopy to be TABLE

composed of many small fragments less than 1 pm in length as well as large doublestranded linear molecules. There were also a few large intact circular DNA duplexes comparable to baculovirus DNA. It was found to have a buoyant density (data not shown) and EcoRl restriction endonuclease pattern (Fig. 5) identical to that for NPBV DNA. DISCUSSION

Measurement of 0. pseuckkmgata baculovirus genome size by means of electron microscopy using SV40 DNA as an internal 2

SUMMARY OF Orgyia pseudotsugata BACULOVIRUS GENOME MOLECULAR WEIGHT ESTIMATES Method

NPBV”

Reference

NPSV”

Reference

Velocity sedimentation Restriction endonuclease Renaturation kinetics (uncorrected for %GC) (corrected for %GC) Electron microscopy

89 (96)c 85

Rohrmann et al., 1977 This report

110 (85)*

Rohrmann and Beaudreau, 1977

81 86 86

Rohrmann et al., 1977

101 88.5 103

This report

Rohrmann and Beaudreau, 1977 This report

u Molecular weight x lo6 dahons. * We have concluded that the restriction fragments previously reported for this virus (Rohrmann and Beaudreau, 1977) of molecular weights 7, 8.4, and 9.3 x 106 are likely to be doublets which would result in an estimated molecular weight of 110 rather than 85 x 106. c A calculation of 96 x lo6 daltons was made from sZO+,value of 57 (Rohrmann et al. 1977) using the equation of Opschoor et al. (1968), szO,w - 2.7 = 0.0152M0,‘45. A more recent equation from Reinert et al. aw, smw - 2.5 = -.019M”.4~5, gives a molecular weight of 89 x lo6 daltons.

180

SCHAFER

FIG. 2. A circular, partially supercoiled NPSV DNA duplex molecule. The urea-formamide spreading method for electron microscopy was used: (R) NPSV DNA relaxed region; (S) NPSV DNA supercoiled region; (SS) SV40 DNA (supercoiled).

length standard showed that the mean molecular weights of the DNA from NPBV and NPSV were 86 x lo6 and 103 x lo6 daltons, respectively. The standard deviation of the contour lengths of DNA circles was very small (about +2 pm) which

ET AL.

suggests that the viral genomes are nearly homogeneous. These molecular weights from electron microscopic studies were strongly supported by previous molecular weight determinations using velocity sedimentation, restriction endonuclease analysis, and renaturation kinetic studies (Table 2). One of the objectives of these studies was to determine if the data obtained from reassociation kinetics required correction for GC content as recommended by Seidler and Mandel (1971). Clearly, the molecular weights that have not been corrected for GC content are in close agreement with the electron microscopic studies (see Table 2) and fall within the standard deviation. Therefore we conclude in our system that the %GC does not have the effect on DNA renaturation kinetics as reported for other organisms. The use of restriction endonucleases for molecular weight determination of large genomes presents two difficulties: (1) multiple fragments which migrate close together in an unresolved band on the gel can cause errors, especially in the high molecular weight region of the gel; (2) the size of the high molecular weight components (>6 x lo6 daltons) on the gel cannot be accurately determined because migration in that region is not linear. Extensive enzymatic studies with a variety of restriction enzymes would have to be undertaken in order to clarify such ambiguities. Our earlier estimate of 85 x lo6 for NPSV genome based on restriction endonuclease analysis appears to be too low. A reexamination of the densitometer tracing of the endonuclease fragments leads us to conclude that the ‘7, 8.4, and 9.3 x 106-dalton fragments are doublets (see Fig. 5, Rohrmann and Beaudreau, 1977). This results in a molecular weight of 110 x lo6 daltons which is closer to the value derived by electron microscopy (103 2 6 x 106). A similar misinterpretation of restriction endonuclease fragments may have resulted in the molecular weight of 64 x lo6 Miller and Dawes (1978) reported for the NPBV. Velocity sedimentation analysis of DNA from NPBV in an analytical ultracentrifuge (Carnegie, 1970; Rohrmann et al., 1977) gave a molecular weight for the viral genome within the standard deviation of

DNA FROM TWO Orgyia pseudotsugatu BACULOVIRUSES

FIG. 3. (A) A circular DNA duplex molecule of 6-8 x lo6 daltons found in low concentration in the baculovirus DNA preparations. (B) The supercoiled form of the 6-8 x 106-dalton DNA duplex molecule.

D

RELATIVE

DISTANCE

MIGRATED

-

FIG. 4. Scans of restriction endonuclease fragments of NPBV DNA and two sets of DNA markers. Numbers indicate molecular weight x 106. The DNA markers were EcoRl digests of A phage DNA (Thomas and Davis, 1975) and Hue111 digest of PM-2 DNA (Shaw et al., 1976). The molecular weights of the DNA fragments of the NPBV DNA was estimated by scanning the photographs of agarose gel electrophoresis profiles with an ORTEC Model 4310 densitometer and comparing the NPSV scans to those of marker DNA fragments. The asterisks indicate two fragments with the same molecular weight. The following scans are present: (A) EcoRl digest of A DNA, (B) SmaI digest of NPBV DNA, (C) Hsul digest of NPBV DNA, (D) Hue111 digest of PM-2 phage DNA.

181

182

FIG. 5. Restriction endonuclease extraviral DNA. Left gel: untreated Right gel: extraviral DNA digested

SCHAFER

digest of NPBV extraviral DNA. with EcoRl.

the values calculated from lengths of the DNA molecules (Table 2). The relationship between sZO,Wand molecular weight appears to be best expressed by the equation of

ET

AL.

Reinert et al. (1971) derived from studies with homogeneous, native DNA. Other equations by Opshoor (1968), Freifelder (19’70), Rubenstein (1961), and Eigner and Doty (1965) produced molecular weight values ranging from 90 to 111 x lo6 daltons. Sedimentation analysis in sucrose gradients also presents a number of problems. Overloading can lead to artifacts which can significantly alter the molecular weight estimates (Mosmann and Hudson, 1973). In the past cosedimentation with marker DNA in density gradients has been hampered because the size of the marker DNA was not precisely known (e.g., Summers and Anderson, 1973). Often the distribution of DNAs in the gradient is broad and may result in errors in measurement. The presence of extraviral DNA occluded in polyhedra was previously reported for the NPSV (Rohrmann and Beaudreau, 1977). It was concluded that this DNA was of viral origin because it had the same buoyant density as the NPSV DNA. NPBV extraviral DNA was identified as being of viral origin by its restriction endonuclease pattern and it appears to be composed of DNA fragments covering a wide range of sizes. This indicates that a certain amount of partially replicated or degraded viral DNA was trapped as the polyhedra crystallize. The presence of a low level of a 6-8 x lo6 dalton supercoiled DNA in the baculovirus DNA preparations (Fig. 4) may indicate the presence of another virus associated with the polyhedra. This could represent an endogenous insect virus present in the nucleus which becomes occluded in the polyhedra as they crystallize. Our studies on the two 0. pseudotsugata baculoviruses by means of restriction enzyme analysis, sedimentation velocity, renaturation kinetics, and electron microscopy utilizing two different spreading techniques is one of the most complete investigations to determine the genome size of a large virus. The genome size of the NPSV of 103 x lo6 is the largest baculovirus genome determined by means of electron microscopy. This expands the range of baculovirus genome sizes determined by electron microscopy to include values of 58-103 x lo6 daltons (Bud and Kelly,

DNA FROM TWO Orgyia

pseudotsugata

1977; Brown et al., 1977; Burgess, 1977; Tweeten et al., 1977). Our studies also indicate that supercoiled DNA genomes in excess of 100 x lo6 daltons occur. ACKNOWLEDGMENTS We are grateful to the following from the National Institutes of Health: Benjamin Elliott, Jr., for technical assistance, Douglas Jones for photography, and Stella Gregory for typing assistance. The assistance of Dr. M. Martignoni USDA Forestry Sciences Laboratory, Corvallis, Oregon, in supplying Orgyia pseudotsugata larvae and for his advice is also gratefully acknowledged. We also very much appreciated the assistance with the restriction endonuclease analysis by Dr. R. McParland, Department of Biochemistry and Biophysics, Oregon State University. This project was supported in part by Postdoctoral Grant 1 F32 ES05110-01 from NIH (G.R.). REFERENCES BROWN, D. A., BUD, H. M., and KELLY, D. C. (1977). Biophysical properties of the structural components of a granulosis virus isolated from the cabbage white butteriIy(Pieris brassicae). Virology 81, 317-32’7. BUD, H. M., and KELLY, D. C. (1977). The DNA contained by nuclear polyhedrosis viruses isolated from fourSpodoptera spp. (Lepidoptera, Noctuidae); Genome size and configuration assessed by electron microscopy. J. Gen. Virol. 37, 135-143. BURGESS, S. (1977). Molecular Weights of Lepidopteran baculovirus DNAs: Derivation by electron microscopy. J. Gen. Virol. 37, 501-510. CARNEGIE, J. W., Ph.D. Thesis, Oregon State University (1970). DAVIS, R. W., SIMON, M. M., and DAVIDSON, N. (1971). Electron microscopic heteroduplex methods for mapping regions of base sequence homology, p. 413-428. In “Methods in Enzymology” (L. Grossman and K. Moldare, eds.), Vol. 21, Part D. Academic Press, New York. EIGNER, J., and DOTY, P. (1965). The native, denatured and renatured states of deoxyribonucleic acid. J. Mol. Biol.12, 549-580. FREIFELDER, D. (1970). Molecular weights of coliphages and coliphage DNA. IV. Molecular weights of DNA from bacteriophages T4, T5 and T7 and the general problem of determination of M. J. Mol. Biol.

54, 567-577.

GRIFFITH, J. D. (1978). DNA Structure: Evidence from electron microscopy. Science 201, 525-527. HUGHES, K. M., and ADDISON, R. B. (1970). TWO nuclear polyhedrosis viruses of the Douglas-fir tussock moth. J. Znvertebr. Pathol. 16, 196-204.

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KLEINSCHMIDT, A. K., and ZAHN, R. K. (1959). Deoxyribonucleic acid molecules in protein-mixed film. 2. Naturforsch. B14, 770-779. MARTIGNONI, M. E. (1972). A rapid method for the identification of nucleopolyhedron types. J. Znvertebr. Pathol. 19, 281-283. MILLER, L. K., and DAWES, K. P. (1978). Restriction endonuclease analysis for the identification of baculovirus pesticides. Appl. Environ. Microbial. 35, 411-421. MOSMANN, T. R., and HUDSON, J. B. (1973). Some properties of the genome of murine cytomegalovirus (MCV). Virology 54, 135-149. OPSCHOOR,A., POUWELS, P. H., KNIJNENBURG, and ATEN, J. B. T. (1968). Viscosity and sedimentation of circular native deoxyribonucleic acid. J. Mol. Biol. 37, 13-20. REDDY, V. B., THIMMAPPAYA, B. DAHR., R., SUBRAMANIAN, K. N., ZAIN, B. S., PAN, J., GHOSH, P. K., CELMA, M. L., and WEISSMAN, S. M. (1978). The genome of Simian Virus 40. Science 200, 494502. REINERT, K. E., STRASSBURGER, J., and TRIEBEL, H. (1971). Molecular weight and hydrodynamic properties of homogeneous native DNA derived from diffusion, sedimentation and viscosity measurements on polydisperse samples. Biopolymers 10, 285-307. ROHRMANN, G. F. (1977). Characterization of N-Polyhedrin of two Baculovirus strains pathogenic for Orgyiapseudotsugata. Biochemistry 16,1631-1634. ROHRMANN, G. F., and BEAUDREAU, G. S. (1977). Characterization of DNA from polyhedral inclusion bodies of the nucleopolyhedrosis single-rod virus pathogenic for Orgyia pseudotsugata. Virology 83, 474-478. ROHRMANN, G. F., CARNEGIE, J. W., MARTIGNONI, M. E., and BEAUDREAU, G. S. (1977). Characterization of the genome of the nucleopolyhedrosis bundle virus pathogenic forOrgyiapseudotsugata. Virology 80, 421-425. ROHRMANN, G. F., MCPARLAND, R. H., MARTIGNONI, M. E., and BEAUDREAU, G. S. (1978). Genetic relatedness of two nucleopolyhedrosis viruses pathogenic for Orgyia pseudotsugata. Virology 84, 213-217. RUBENSTEIN, I., THOMAS, C. A., JR., and HERSHEY, A. D. (1961). The molecular weights of T2 bacteriophage DNA and its fist and second breakage products. Proc. Nat. Acad. Sci. USA 47,1113-1122. SAMBROOK, J., and SHATKIN, A. J. (1969). Polynucleotide ligase activity in cells infected with Simian virus 40, polyoma virus, or vaccinia virus. J. Viral. 4, 719-726. SEIDLER, R. J., and MANDEL, M. (1971). Quantitative aspects of deoxyribonucleic acid renaturation: Base composition, state of chromosome replication, and polynucleotide homologies. J. Bacterial. 106, 608-614.

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SHAW, B. R., HERMAN, T. M., KOVACIC, R. T., BEAUDREAU, G. S., and VAN HOLDE, K. E. (1976). Analysis of subunit organization in chicken erythrocyte chromatin. Proc. Nat. Acad. Sci. USA 73, 505409. SUMMERS, M. D., and ANDERSON, D. L. (1973). Characterization of nuclear polyhedrosis virus DNAs. J. Viral. 12, 1336-1346. THOMAS, M., and DAVIS, R. W. (1975). Studies on

ET AL,. the cleavage of bacteriophage lambda DNA with EcoRl restriction endonuclease. J. Mol. Biol. 91, 315-328. TWEETEN, K. A., BULLA, L. A., and CONSIGLI, R. A. (19’7’7). Supercoiled circular DNA of an insect granulosis virus. Proc. Nat. Acad. Sci. USA 74, 3574-3573. WESTPHAL, H., and LAI, S. P. (1977). Quantitative electron microscopy of early adenovirus RNA. J. Mol. Sol. 116, 525-548.