Some properties of deoxyribonucleic acid preparations from Chilo, Sericesthis and Tipula iridescent viruses

J. Mol. Biol. (1967) 25, 425-432

Some Properties of Deoxyribonucleic Acid Preparations from Chile, S’ericesthis and Tipula Iridescent Viruses ~LLAK J.D. BELLETT

Department of Microbiology, John Curtin School of Medical Research Australian National University, Canberra, Australia A-ND

Ross B. Department

of Physical

INMAN

and Inorganic Chemistry, University of Adelaide Adelaide, South Australia

(Received 30 June 1966, and in revisedform 23 Januarzy 1967) The amount of DNA in the particles of three viruses of the iridescent virus group has been estimated, and some properties of DNA preparations from the viruses have been investigated. All the viruses contain double-stranded DNA. The DNA of Chile iridescent virus contains 28 to 29% G + C base pairs, tha,t of Se&e&his iridescent virus 31% G + C and that of Tip&a iridescent virus 32% 6: + C. The differences in base ratios were confirmed by analysis of mixed DNA bands at equilibrium in CsCl. The contents of DNA per virus particle (expressed in molecular weight units) and the molecular weights of extracted DNA were estimated to be 135 million and 130 million for Chile iridescent virus, 147 million and 134 million for Sericesthis iridescent virus and 155 million and 126 million for Tipula iridescent virus, respectively.

1. Introduction Sericesthis

iridescent

virus

and Tipula iridescent virus are indistinguishable in most

and are serologically related, although not identical (Steinhaus SsLeutenegger, 1963; Day & Mercer, 1964). Chilo iridescent virus has very similar properties (Fukaya & Nasu, 1966), but is not serologically related to SIV; (Day, personal communication). Both SIV and TIV contain DNA (Thoma,s, 1961; Day & Mercer, 1964); chemical analysis of TIV suggestedthat each.pa,rtiele contains about 150 million daltons of DNA, which is probably double stranded with about 31% G + C base pairs (Thomas, 1961; Allison & Burke, 1962). In this paper we report further studies of the nucleic acids of these viruses; they were found to be very similar, but not identical. physical

and

biological

properties

2. Materials CIV and SIV were paEudosa was kindly Virus Research Unit,, 7 Abbreviations Pespectively.

used:

and Methods

(a) Viruses of Galleria mellonella;

grown in larvae TIV extracted from Tip&a supplied by Mr C. F. Rivers of the Agricultural Research Council Cambridge, England. The viruses were purified by one or two cycles SIV,

CIV

and

TIV,

Sericesthis, 425

Chile

and

Tip&r

iridescent

viruses,

426

A.

of centrifugation in sucrose tions appeared homogeneous 2200. SIV prepared in this

J.

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density-gradients (Day & Mercer, 1964). The purified preparain the analytical ultracentrifuge, with an &‘20,w, of about way oontains no detectable host antigen (Day $ Merger, 1964). (b)

DNA

contents

of the vimses

The numbers of virus particles were estimated in the electron microscope by the spraydroplet method of Williams & Backus (1949). Portions (O-5-ml.) containing a known number of virus particles were twice extracted with 5% trichloroacetic acid at 90°C for 20 mm, and the combined extracts from each portion made up to 5 ml. The DNA contents of l-ml. samples of each extract were estimated by the diphenylamine reaction (Burton, 1956) using highly polymerized calf thymus DNA (Worthington Biochemical Corp.) as a standard. The DNA content of the virus suspension was divided by the particle count to give the amount of DNA per virus particle. (c) DNA

preparations

DNA was released from the viruses by incubation with 3% sodium dodecyl sulphate at 60°C for 2 to 3 hr in 1.25 M-SUCIOSe, 0.15 M-NaCl, 0.005 M-EDTA, 0.005 M-Tris buffer (pH 8.0). Protein and detergent were precipitated with 1 M-NaCl and removed by centrifuging at 15,000 g for 30 min. The crude DNA preparations were dialysed against SSC (0.15 M-Nacl, O-015 m-sodium citrate (pH 7)), then extracted 3 times with phenol and 5 times with ether by the rolling method, and finally dialysed against SSC. The usual precautions were taken to avoid damage by shear to the DNA. The recovery of DNA varied from about 60 to 90%, depending on the concentration of virus and the time of incubation in detergent. (d) Thermal delzaturation of DNA This was followed in a Zeiss PM& II spectrophotometer at 260 mp. Samples were heated by circulating water from a thermostatically controlled bath through the cell housing, and temperature was measured by a thermistor in one cuvette. Denaturation temperatures (T,) were used to estimate base ratios (Marmur & Doty, 1962). (e) Electron

microscopy

of DNA

The DNA was examined by the method of Kleinschmidt, Lang, Jacherts & Zahn (1962). Although lengths of the molecules can be estimated to within -& 3%, there is now some uncertainty in converting the lengths to molecular weights, since the ionic composition of the hypophase affects the lengths obtained (Inman, manuscript in preparation). Experiments with X Cl857 and X hi DNA under the conditions used here (hypophase O-1 M-ammonium acetate) suggested a provisional empirical mass-to-length ratio about 17% higher than the theoretical ratio of 1.92 million daltons/micron calculated for native DNA in the B configuration, assuming a molecular weight of 32 million for X DNA. Although this empirical correction gives weight-average molecular weights which agree with those obtained from sedimentation coefficients, the longest iridescent virus DNA molecules then appear to have a higher molecular weight than expected from the DNA contents of the particles. Molecular weights calculated from lengths must therefore be treated as provisional estimates only. (f) Analytical

ultracentrifugation

Sedimentation coefficients were determined under the conditions used by Burgi &s Hershey (1961) except that SSC was used as the solvent. The sedimentation coefficient was independent of DNA concentration under these conditions, as found by Burgi 85 Hershey. Molecular weights were calculated using the empirical calibration of Rubenstein, Thomas & Hershey (1961). Equilibrium oentrifugation in CsCl (Meselson, Stahl & Vinograd, 1957) was used to estimate buoyant densities of DNA preparations. Densities were calculated by t.he method of Ifft, Voet & Vinograd (1961) including correction for pressure (Hearst, Ifft & Vinograd, 1961), or from the position of the DNA band relative to a marker band of Escherichia COG DNA; the two methods gave densities which agreed to within 0.002 g cmw3. Base ratios were calculated from buoyant densities (Rolfe & Meselson, 1959).

IRIDESCENT

VIRUS

3. Results

4%

DNA4

and Discussion

(a) Base ratios The thermal denaturation curves of the DNA preparations were typical of doublestranded DNA (Fig. 1). On cooling the denatured samples to 35°C over a period of about 10 minutes, they became renat.ured with 73 to 76% efkiency as judged by the decrease in absorbance at 260 rnp, and could then be denatured agein without significant change in the T,. SIV DNA and TIV DNA also showed about 400/, irrcrea~se in absorbance on raising the pH from 11.5 to 11-S (Fig. 2) or on inoubating sx
I

I

I

(a) SiV DNA

++t+ I(c) TIV DNA

I

a Temperature FIG. FIGS

A/A0 is the ratio absorbance FIG.

arrowed

to 35°C

at room

(“cl

it-;

12

PH

1

1 and 2. Denaturation

I

9 FXG.

of iridescent

virus

DNA

of the absorbance of the solution (at 260 mp) temperature and neutral pH at the beginning

1. Thermal denaturation of (a) CIV DNA; points show the absorbance of the solution for 10 min.

(b) SIV DNB; after the fully

2,

preparations. under a given condition of the experiment.

to its

(c) TIT;’ DNA at pR 7. The denatured DNA was caoled

FIG. 2. Alkaline denaturation of (a) SIV DNA; (b) TIV DNA. The pH wa,s adjusted by adding ~1. amounts of NaOH, and nitrogen was passed through the solution while the pH and absorbance of the solution were measured at room temperature.

29

A.

428

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to 98°C for three minutes followed by chilling. All these results are consistent with the hypothesis that the DNA of the iridescent viruses is double stranded. The T, values and buoyant densities of DNA preparations from the three iridescent viruses were very similar, although the results suggested that the percentage G -+- C in SIV DNA might be greater than that in CIV DNA but less than that in TIV DNA (Table 1). The T, results were subjected to an analysis of variance and none of the differences was significant at the 5% level in an P-test. TABLE ;Z Molar base ratios oj DNA. from three iridescent viruses Virus

Property of DNA measured

T, (“‘W Buoyant

CIV

81.3

density

%G %G

+ C (from + C (from

%G

+ C (mean)

(g cme3)$ T,) density)

1.686

& 0.6

& 0.001 29.3

SIV

TIV

82.0

&

0.6

1.689

f

0.001

82.6

f

0.5

1.690

f

0.001

28.0

31.0 31.0

32.4 32.0

28.7

31.0

32.2

t Means and 95% confidence limits for the means of three experiments seven experiments with SIV DNA and TIV DNA. $ Means and ranges for two experiments with each DNA. Denatured SIV density of 1.710 g cmm3.

with

CIV

DNS

and

DNA

had

a buoyant

To test the possible differences between the base ratios of DNA from the three viruses, mixtures of DNA preparations from different viruses were centrifuged to equilibrium in CsCl. After 24 hours at 44,770 rev./min, mixtures of CIV DNA with either SIV DNA or TIV DNA gave two partly overlapping bands (Fig. 3), the distances between the modes of these bands being consistent with the direction and approximate magnitude of the differences suggested by the previous experiments (Table 1). lktixtures of SIV DNA with TIV DNA could not be resolved at 44,770 rev&in, but aft.er three days at 31,410 rev./mm, the composite band was non-Gaussian (Fig. 3), although either DNA alone gave a Gaussian band under these conditions. The composite band was analysed by the method of Ageno & Frontali (1963) into two Qaussian components with an estimated difference in mean buoyant density of about 1.6 x 10-3gcm-3. (b) Estimates of DNA

contents of the viruses and molecular weights of extracted DNA

Mean values for the amount of DNA per virus particle for several samples of each of the iridescent viruses are shown in Table 2: the results are also expressed in molecula8r weight units for comparison with the molecular weights of DNA preparations in Table 3. The values for CIV and SIV did not differ significantly from each other at the 5% level in a t-test. A value of 174 million daltons per particle was obtained for TIV-significantly higher than the values for CIV and SIV. However, the DNA

1RIDESCENT

VIRUS

DNA

429

A

(b) CIVi-TI\,

+------Rotor

3%. 3. Miorophotometer tracings of ultraviolet preparittions from (a) CIV f SIV; (b) CIV + density-gradients. (a) and (b) were photographed (E) after 3 days at 31,410 rev./min.

centre

absorption photographs of mixtures of DNA (c) SIV + TIV at equilibrium in CsCl 24 hr centrifugatiort at 44,77@ rev./mhn;

TIV; after

contents of TIV reported by Thomas (1961) and by Allison & Burke (1962) do not differ significantly from our estimates for CIV and SIV, and it seems likely that our experiments have overestimated the DNA content of TIV for some reason. The best estimate of the DNA content of TIV was therefore taken as 155 million daltons (Table 2), the mean of our estimate and those of Thomas (1961) and Allison & Burke (1962). DNA preparations from the iridescent viruses had sedimentation coefficients of about 62 S, corresponding to molecular weights of about 130 million dakons (Table 3). The preparations appeared polydisperse in the ultracentrifuge and in their distributions of molecular lengths in the electron microscope (Fig. 4), suggesting t,hat the DNA bad been damaged during extraction in spite of the precautions taken t’o minimize shear. No circular molecules were seen. Both CIV and SIV DNA preparations had number-average lengths of about 55 p andlength-average lengths of about 60 p, but the longest molecules were about 80 p in each case. The length-average lengths correspond to weight-average molecular weights of about 130 million on the empirical calibration used here, in good agreement with values obtained from sedimentation experiments

430

A.

3.

D.

BELLETT

AND

Virus

of DNA

Particles/ml. ( x lo-=)

Sample

INMAN

per particle

for three iridescent

DNA/ml. (g x 1w

DXA/particle (g x 1016)

268 251 257

2.63 f 0.70 2.06 & 0.44 2.06 f 0.18

1 1

2.08 1.03 0.94 0.98

490 233 240 252

2.36 & 0.56 2.26 2.55 & + 0.38 0.42

-)

2.57 & 0.20

i

1

1.24 1.11 1.29

364 329 357 -

2.94 & 0.61 2.96 -& 0.23

1

2 3

0.27

-

1 2 3

SIV SIV SIV SIV

2 3 4

TIV TIV TIV

1.02

Thomas (1961) Allison & Burke (1962)

viruses

Mean DNA/particle (daltons x 1O-6)

1.22 1.25

CIV CIV CIV

TIV TIV

of the amount

B.

2

TABLE Estimation

R.

135 & 14

147 -& II

1741

2.78 -& 0.55 2.30

151 1155 & 19

139J

The particle count and DNA content of each sample were estimated three or four times as described in Materials and Methods. Variances of the ratios of DNA/particle were calculated as var R = (var N var l/D) + (J@ var l/D) + (l/D2 var N), where R is the ratio, N the numerator and D the denominator (D. Vere-Jones, personal communication). The values given in column 5 are the means and 95% confidence limits for the means of individual samples; in column 6 the sample values have been combined to give over-all means and confidence limits for each virus. The limits for TIV in column 6 were calculated assuming that the values obtained by different authors represent random samples from a hypothetical population.

3

TABLE

Molecular weightsof DNA

from three iridescent

Property of DNA measured

Number-average Length-average Molecular Molecular

length length

weight weight

Mean molecular DNA/particle

Figures

(from (from

62 54 * 5 58 & 4

(p)$ (p) S) x length)

10m6 x 10m6§

weight x 1Om6 x 10e6 (chemical estimation)

shown

Virus SIV

CIV

&J,wt

are means

and 95%

11

viruses

TIV

63 i: 5 56 & 5 60 & 4

61

129

133 + 21

126

131 f 9

135 -J= 9

130

134 147 f 11

135 & 14

confidence

limits

126 155 & 191

for the means.

f Five preparations of SIV DNA and one each of CIV DNA and TIV DNA were examined. $ The numbers of molecules measured were 43 and 41 respectively. 3 Weight-average molecular weights calculated from length-average lengths, including an approximate correction for the effect of ionic composition of the hypophase on apparent lengths (see text). Ij The amount of DNA/particle expressed in daltons for comparison with the molecular weights of extracted DNA (from Table 2). 7 Includes the results of Thomas (1961) and Allison & Burke (1962) (see Table 2).

IBIDESCENT

VIRUS

431

DNA

1 (cd CIV DNA l-l

“0 2

,(b) SIV DNA 8.

Length (p) FTC. 4. Frequencies DNA. The monolayers

of molecular were spread

lengths in electron micrographs on & hypophase of 0.1 M-aImIIOlliUm

of (a) CIV DNA acetate.

ax!

(b) SlV

(Table 3). However, the longest molecules correspond to a moleoular weight of 176 million on this scale, rather higher than expected from the DXA contents of the viruses. If correction for the effect of salt concentration on molecular length is ignored aad the DRA is assumed to have the mass-to-length ratio expected for the B configuration, then the iridescent virus DNA preparations would have weight’-average molecular weights of about 115 million, the longest molecules corresponding to a molecular weight of about 150 million. The weight-average molecular weight of DNA extracted from the iridescent viruses is in each case close to, but slightly lower than, the value expected if the DNA were present in the virus particles as a single molecule (Table 3). In view of the difficuities of extracting the DNA and of estimating its molecular weight, it seems reasonable to propose the hypothesis that each iridescent virus particle contains a single molecule of DNA. However, it has not been possible to establish this hypothesis conclusively.

REFERENCES

Ageno, &I. & Frontali, C.

(1963). Nature, 198, 1294. Allison, 9. C. & Burke, D. C. (1962). J. Gen. Microbial. 27, 181. Burgi, E. & Hershey, A. D. (1961). ,T. Mol. Biol. 3, 458. Burton, K. (1956). Biochem. J. 62, 315. Day, lbf. F. & Newer, E. H. (1964). Au&-al. J. Biol. Xci. 17, 892. R&aye, X & Nasu, S. (1966). Applied Entomology and Zoology, I, GY. IrIearst, J. E., Et, J. B. & Vinograd, J. (1961). Proc. &Nat. Acad. Xc?:., +$/a&. If%, J. B., Voet, D. H. & Vinograd, J. (1961). J. Ploys. Chem 65, 1138.

47,

1015.

432

A. J. 33. BELLETT

AND

hz. B. HNn;iAN

Kleinschmidt, A. K., Lang, D., Jaeherts, D. & Zahn, R. K. (1962). Biochim. biophys. acto, 6X, 857. Marmur, J. & Doty, I?. (1962). J. MoZ. Biol. 5, 109. Meselson, M., Stahl, F. W. & Vinograd, J. (1957). Proc. Nat. Acad. Sci., Wash. 43, 581. Rolfe, R. & Meselson, M. (1959). Proc. Nut. Acad. Sci., Wash. 45, 1039. Rubenstein, I., Thomas, C. A., Jr., & Hershey, A. D. (1961). Proc. Nat. Acad. Sci., Wash. 47, 1113. Steinhaus, E. A. & Leutenegger, R. (1963). J. Insect Path. 5, 266. Thomas, 12. S. (1961). vvirology, 14, 240. Williams, R. C. & Backus, R. C. (1949). J. Amer. Chem. Sot. 71, 4052.