Immunochemistry, 1976, Vol. 13, pp. 985489. Pergamon Press. Printed in Great Britain
I M M U N O G E N I C D N A OF SHIGELLA SONNEI BACTERIOPHAGE L U D M I L A A. Z A M C H U K , N I N A M. M A G R A D Z E and D A V I D M. G O L D F A R B Institute of General Genetics, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. (First received 21 May 1976; in revised form 30 June 1976)
Abstract~Immunochemical and some other properties of Shigella sonnei 'Ufa' bacteriophage DNA have been studied. The DNA has been shown to possess high immunogenic activity indicating the presence of an unusual base which is neither 5-HMC* nor glucosylated 5-HMC, dihydroxypentiluracil or 5-HMU. The DNA is double-stranded, its mol. wt is 1.07.108 daltons and T,, = 85.5°C. Common base sequence have been found in 'Ufa' DNA and T4 DNA using competition DNA-DNA hybridization technique, Cross-neutralization experiments with intact 'Ufa' and T4 phages using corresponding antisera have confirmed the presence of common antigenic determinants in these phages.
INTRODUCTION Recently it has been shown (Zamchuk & Magradze, 1976) that D N A s isolated from 6 Shigella sonnei bacteriophages can be subdivided into 2 groups according to their immunochemical properties, i.e. those reacting and those not reacting with antisera to T-even phage DNA. The 'Ufa' phage, the D N A of which belongs to the latter group, has been previously studied by Chanishvili & Kapanadze (1967, 1970) with respect to several properties including particle and plaque morphology, serologic properties and host range. Based on these criteria, the authors have related 'Ufa' phage to the T-even phage group. As is known T-even bacteriophage D N A contains glucosylated or non-glucosylated 5-HMC (Wyatt & Cohen, 1953; Lehman & Pratt, 1960) which determines high immunogenic specificity of its. D N A (Levine et al., 1960; Murakami et al., 1962; Zamchuk, 1965). Antisera)o T-even phage D N A react only with D N A containing this unusual base. Hence, the inability of D N A from any source, including phages, to react with T-even D N A antisera is an indication of the absence of glycosylated or non-glucosylated 5-HMC in such DNA. Thus the conclusion that 'Ufa' phage relates to the T-even group appears to be arguable. It was, therefore, thought worthwhile to study immunogenic and certain other properties of D N A and intact particles of 'Ufa' phage in more detail.
MATERIALS A N D M E T H O D S
Bacteria E. coli B and B. subtilis 168 (ind- str-r) strains were obtained from the bacteria and phage collection of the Institute of General Genetics, U.S.S.R. Academy of Sciences; E. coli K172 rgl- su- and E. coli CR63 rgl-
su + were kindly provided by Dr. E. Goldberg (Tufts University School of Medicine, Boston, MA); Sh. sonnei strain 1188 was obtained from the Research Institute of Vaccines and Sera, Tbilisi, U.S.S.R. Phages Wild4ype T2, T4 and T6 phages and B. subtilis SP01 phage were obtained from the collection of the Institute of General Genetics; Sh. sonnei 'Ufa' phage was obtained from the Research Institute of Vaccines and Sera; phage T4 ctgtam8 flgtaml0 was provided by Dr. E. Goldberg. Preparation of phage stocks T2, T4 and T6 phage were grown on E. coli B in the standard M9 medium supplemented with 20/~g/ml e-tryptophan. Glucoseless phage T4 ctgtam8 flgtaml0 was grown on E. coli K172 rgl- su- (McNicol & Goldberg, 1973). 'Ufa' phage was grown on Sh. sonnei 1188 in the following medium: 7 g NaH2PO 4. 12H20 ; 3 g KH2POa, 1 g NH4C1, 0.5g NaC1, 0.02g CaClz, 0.025g MgSO4'7HzO and 4g of glucose per litre of water (Tikhonenko & Solovieva, 1961). Bacteria were aerated by shakirrg or bubbling (Birkadze et al., 1967). Infection was carried out at 1 to 2.10 s cell per ml with moi 0.1. The phages were concentrated and purified by either DEAE-column chromatography (Tikhonenko et al., 1963) or differential centrifugation. The final phage titers were from 1011 to 1012 pfu per ml. Phage absorption Phage absorption was measured according to the amount of unabsorbed phage following inactivation of infected bacteria with chlorophorm (Adams, 1961). Isolation of DNA Isolation of DNA from concentrated phage suspensions was carried out by phenol technique according to Goldberg (1966). No protein was detected in DNA by the Lowry technique. Hence, protein contamination was less than 1%. For immunological studies DNA (10 #g/ml) was denatured in a boiling water bath for 10 min, and immediately placed into an ice bath.
Preparation of antisera to Sh. sonnei 'Ufa' and T4 phages Rabbits were immunized intravenously with 1 ml of phage suspensions at intervals of 2-4 days, increasing a *Abbreviations used: 5-HMC, 5-hydroxymethylcytosine; 5-HMU, 5-hydroxymethyluracil; MBSA, methylated single dose from 1011 to 1012pfu. Each rabbit received bovine serum albumin; moi, multiphcity of infection; SSC, 6-9"1012 phage particles. The blood was taken a week 0.15 M NaCI, 0.015 M Na citrate. after the last injection. 985
986
LUDMILA A. ZAMCHUK, NINA M. MAGRADZE and DAVID M. GOLDFARB
Preparation of antisera to phage DNA Antisera to T2 DNA were obtained after 1-3 times a week immunization of rabbits with increasing (1012-10 ~3) phage equivalents doses of disrupted, alum-absorbed phage. Each animal received 28 mg of DNA (Zamchuk, 1965). To obtain antisera to SP01, 'Ufa' and T4 phages DNA, rabbits were immunized with MBSA-denatured DNA complexes according to Plescia (Plescia et al., 1964). Each animal received 1.2 mg of 'Ufa' DNA or 2 mg of T4 or SP01 DNA during the immunization course. The rabbits were bled a week after the last immunization.
°°t g-
~) 40 f 20
Phage neutralization with antisera This was performed as described by Adams (1961). Neutralization constants were calculated from the following equation: K = 2.3 D/t lg (Po/P), where Po is the amount of phage at 0 time, P, the amount of phage at t time, D, the final dilution of the serum, and K, the neutralization rate constant.
DN A melting temperature This was measured on Unicam SP-8000 spectrophotometer in a thermostated cell at 260 nm as described by Marmur & Doty (1962) and DNA was dissolved at 20 pg/ml in l x SSC (pH 7.0 _+ 0.3). G + C content This was calculated from the equation Tm= 69.3 + 0.41 x (G + C) which is valid for DNA preparations containing from 25 to 75% of G + C (Marmur & Doty, 1962). Sedimentation coefficients Sedimentation coefficients (S°20. w) for phage DNA were determined using Spinco E ultracentrifuge at 36,000 rev/ min and 20°C. DNA was dissolved in 1 x SSC at 20 #g per ml. DNA tool. wt determination This was carried out according to equations suggested by Studier (1965). Hybridization The following technique of competition DNA-DNA hybridization was used ' t o detect homologous base sequences. Membrane filters (Millipore, dia 25 mm) were soaked overnight in 2 x SSC solution. Five ml of denatured unlabeled DNA (5 #g/ml) in 6 x SSC were passed through each filter, followed by 5 ml of 6 x SSC. The filters were dried for 1 hr in vacuum at room temperature and incubated for 3 hr at 65°C. After drying, the filters were put into 1 ml of 3 x SSC containing 0.02% Ficoll (mol. wt 400,000), 0.02% polyvinylpyrollidone and 0.02% BSA (Denhardt, 1966). 3H-labeled T4 DNA (0.5#g per 0.25 ml of 3 x SSC) was heat denatured and mixed with competing unlabeled denatured DNA (3.5 #g in 0.25 ml of 3 x SSC), incubated for 6 hr at 65°C, and added to the vials with the filters. In the control only labeled denatured T4 DNA (0.5 gg in 0.5 ml of 3 x SSC) was added (direct hybridization). The incubation continued for 12-15 hr at 65°C. After that the filters were washed by 40ml of 2 x SSC on each side, dried and placed into scintillation fluid (50mg P O P O P and 4g PPO per 1 of toluene). Radioactivity of the filters was measured on Nuclear Chicago MARK-1 liquid scintillation counter. Enzymatic treatment of DNA Crystalline DNase 1 (Worthington), trypsin (Spofa, Czechoslovakia) and pronase (Calbiochem, U.S.A.) were used at 10#g/ml. The treatment with DNase was per-
0.01
0.025
0.05 ONA,
0.1 /~g
Fig. 1. C' fixation of anti-T2 DNA with homologous and heterologous denatured DNA test-antigens: O, T2; 0, T4; x, Sh. sonnei 'Ufa'. Antiserum dilution; O, 0, 1/8000; x,
1/400. formed for 1 hr in 10 -3 M MgC12 at pH 7.0. Trypsin and pronase treatment was performed for 1 hr in 0.01 M Tris-HCl buffer, pH 8.0.
Complement fixation test This was carried out according to Wasserman & Levine (1961).
RESULTS
Immunochemical characterization of Sh. sonnei 'Ufa' phage D N A Antisera to T2 and T4 did not react with 'Ufa' D N A at the dilution of 1:400 while the same sera at 1:2000-1:8000 dilution were positive in the complementation fixation test with homologous antigens (Figs. 1 a n d 2). O n the other hand, antisera of rabbits immunized with M B S A - ' U f a ' D N A complexes were highly reactive with the corresponding antigen but failed to react with T2, T4 and T6 or glucoseless T4 ctgtam8 flgtaml0 phage D N A (Fig. 3, Table 1). It follows from Fig. 3 that with anti-'Ufa' D N A serum diluted 1 : 1600 a n d 0.1 #g of homologous DNA, 85% of C' were fixed while the same serum at 1:400 did not give complement fixation with T-even phage DNA.
I O0
80
g~ ~=
4O
20 I
0.01
~
0025
=4
f
0.05 DNA,
O.I #g
Fig. 2. C' fixation of anti-T4 DNA with homologous and heterologous denatured DNA test-antigen: O, T4; O, T2; x, Sh. sonnei 'Ufa'. Antiserum dilution: O, 0, 1/2000; x,
1/400.
987
Immunogenic Properties of Phage DNA Table 1. Immunochemical specificity of sera to different phage DNA C' fixation (~o) with DNA" of phage Sh. sonnei T4 ~tgtam8 B. subtilis 'Ufa' flgtaml0 T4 SP01
Antisera to phage DNA Sh. sonnei 'Ufa' (1 : 1800)b T4 (1:1500) b B. subtilis SP01 (1:900) b
95.5 0d 9.0~
0c 0~ 17.0c
0c 98.3 0c
(Y 3n 86.0
= DNA dose, 0.1 k~g. b Serum dilution in C' fixation with homologous DNA. c Serum dilution 1:300. a Serum dilution 1 : 600.
Antibodies that appear in sera of rabbits, immunized with MBSA-'Ufa' DNA react with denatured DNA (Fig. 4) which is characteristic of all types of antibodies to DNA resulting from immunization of normal animals (Levine et al., 1960; Murakami et al., 1962; Zamchuk, 1965). Specificity of antibodies to 'Ufa' D N A was confirmed by enzymatic controls. Trypsin and pronase treatment of 'Ufa' D N A antigen had no effect on its ability to react with the antiserum whereas DNase digestion led to complete loss of antigenic activity. Therefore the following conclusions can be made on the basis of these data: (1) 'Ufa' phage DNA possesses high immunogenic activity; (2) immunogenic determinant of this D N A is immunodominant and distinct from glucosylated 5-HMC. Additional experiments indicated that 5-HMU is also absent in 'Ufa' DNA. Table 1 shows that antiserum to SP01 DNA which contains antibodies to 5-HMU does not react with 'Ufa' DNA as well as antiserum to 'Ufa' DNA is negative in the complement fixation test with SP01 DNA. Physical properties of Sh. sonnei 'Ufa' phage D N A 'Ufa' is a large phage. It contains double-stranded DNA which produces a typical A260 vs temperature curve on melting (Fig. 5). Tm calculated from this curve equals 85.5°C. Hence, it follows that the unusual base of 'Ufa' phage DNA does not disturb the interaction of D N A complementary strands. G + C content calculated from the Tm value of 'Ufa' DNA is 39.5~o which is different from the corresponding value for T2 and T4 DNAs (Table 2). The average
I00
°~°
~
I00
80
o o ;,7-
60 40
20
0.0 I
O. 025
0.05
O. I
DNA,
/.¢g
Fig. 4. C' fixation of anti-'Ufa' DNA (1 : 1200) with denatured (O) and native (O) homologous DNA.
mol. wt calculated from the S°2o,w value equals 1.07.10 s (-fable 3) and is close to the mol. wt of T4 DNA determined in our experiments (1.15. 108). In the competition D N A - D N A hybridization assay there was found that only a part of 'Ufa' DNA sequence was homologous to that of T-even phages (Table 4). The table shows that T4, T2 and 'Ufa' DNAs inhibits hybridization of T4 3H-DNA by the average 58, 53 and 28~o, respectively. Specificity of the observed 28~o inhibition is confirmed by the fact that pre-hybridization of E. coli DNA with the filterimmobilized unlabeled T4 DNA did not affect the ability of the latter to hybridize with 'Ufa' DNA (Table 4, exp. 1) and also by the insignificant inhibition of hybridization with calf thymus DNA (Table 4, exp. 3).
o 0.6E
60 o
L~
4C-
d
2C--
o.5a
d 1/400
I/8(X)
Dilution
111600
of
11/2400
serum
0.44
@J
I
80
Fig. 3. C' fixation of anti-'Ufa' DNA with homologous and heterologous denatured DNA test-antigen: @, Sh. sonnei 'Ufa'; x, T4; O, T2; /x, T6. DNA dose, 0.1/tg.
]
8 ;>
I
84
I
86
I
88
J
90
I
92
1-*
Fig. 5. Optical density vs temperature for DNA of phages T4 (@) and Sh. sonnei 'Ufa' (O).
988
LUDMILA A. ZAMCHUK. NINA M. MAGRADZE and DAVID M. GOLDFARB Table 2. Melting temperatue (T,~) and G + C content of different phages DNA G + C content taken
Phages DNA
Tm
C + C (/o) °/
T2
85.5 83.5
39.5 34.6
T4
84.0
35.8
Sh. sonnei 'Ufa'
Table 3. S"2o.w and mol. wt of 'Ufa' and T4 DNA Phages DNA Sh. sonnei 'Ufa' E. coli T4
S:2o.w
mol. wt x 106 dalton
53.0 54.0
107 115
from the literature 33.0 (Marmur & Doty, 1962) 35.8 (Marmur & Dory, 1962)
functional difference between the tail of T-even and 'Ufa' phages. DISCUSSION
Serologic properties o f Sh. sonnei 'Lfa' phage
The partial inhibition of T4 D N A x T4 3H-DNA by 'Ufa' D N A indicates the presence of common base sequences in D N A s of T4 and 'Ufa' phages which may result in common proteins in their capsides. To investigate this possibility, we have carried out crossneutralization experiments with "Ufa' and T-even phages and the corresponding antisera and determined the inactivation constants. Table 5 shows that antiserum to intact T4 phage inactivated 'Ufa' phage to the same degree as it did T2 and T6 phages. At the same time, antiserum to 'Ufa' phage did not inactivate any of the T-even phages. In addition it was demonstrated that the efficiency of adsorption of 'Ufa' and T4 phages on E. coil B was 7 and 99%, respectively. This also indicates the
The main result of the present work is the finding that Sh. sonnei 'Ufa' phage D N A possesses specific immunogenic activity which indicates the presence of an unusual base in this DNA. D N A of this phage has the mol. wt of 1.07- 108 dalton. This value is not absolute since it was determined only on the basis of S°2o, w and therefore, needs to be specified by additional techniques. We believe, however, that it is close to the true mol. wt since parallel determinations for T4 D N A have the value very close to that found by Lang (1970) who measured the contour length of T4 D N A (1.19.10 s dalton). The melting profile of 'Ufa' D N A indicates its double-stranded structure (Fig. 5); G + C content, as calculated from the T~, value, is 39.5Yo, i.e. higher than for T2 and T4 phage D N A (Table 2). lmmunochemical studies of 'Ufa' phage DNA have demonstrated that this phage is different from the T-even E. coil phages since rabbit antisera against
Table 4. Inhibition of T4 DNA × T4 H 3 DNA hybridization by homo- and heterologous DNA Competing DNA (3.5/~g/ml)
Exp.
..... Ic
Sh. sonnei 'Ufa;
T4 T2 Ka -
2
-
Sh. sonnei 'Ufa'
T4 T2 Ks -3
Sh. sonnei 'Ufa'
T4 T2 Thymus K*
Retention of ~H-DNA (counts/min) (,%)
Retention inhibition (°o)b
5005 3771 2650 3212 9856
50.7 38.2 26.9 32.5
0 24.7 47.1 35.9
5890 4362 2856 2912 9856
59.7 44.2 28.9 29.5
0 26.0 51.5 50.6
3138 2055 803 914 2844 9357
33.5 22.0 8.6 9.8 30.4
0 34.5 74.4 71.2 9.4
° K reference 3H-DNA counts (0.5 #g) taken for 100~o. b When retention inhibition was calculated, the amount of label retained in the absence of competing DNA was taken for the 100%. 'In exp. 1 the filters with immobilized, denatured, unlabeled T4 DNA were pre-incubated with 5/~g of denatured E. coli DNA for 4 hr at 65°C. Then 3H-DNA of T4 and competing DNA were added (see Materials and Methods).
989
Immunogenic Properties of Phage DNA Table 5. Constants of neutralization for different phages Phages Sh. sonnei 'Ufa'
T4 T2 T6
Antiserum to phage Sh. sonnei 'Ufa' 950.0 6.5 5.7 6.3
T4 250 1070 230 140
T2 and T4 DNA were negative in the complement fixation test with 'Ufa' phage DNA (Figs. 1 and 2). It is well known that antisera to T-even phage DNA contain antibodies to glucosylated 5-HMC which is the immunodominant antigenic determinant in this DNA (Levine et al., 1960; Murakami et al., 1962; Zamchuk, 1965). 'Ufa' phage DNA also possesses high and specific immunogenic activity. Anti-'Ufa' DNA serum was active at a 1:1600 dilution with homologous DNA and did not react at a 1:400 dilution with either glucoseless T4 DNA or wild-type T-even phage DNA containing fully or partially glucosylated 5-HMC. From this it follows that the immunogenic determinant of 'Ufa' phage DNA is an unusual base being neither 5-HMC nor glucosylated 5-HMC. Besides T-even phages, unusual bases have been found in DNA of B. subtilis phages: 5-HMU substitutes for thymine in the DNA of SP01, SP8 and SP82 phages (Romig & Brodetsky, 1961; Kallen et al., 1962; Marmur et al., 1963), uracil substitutes for thymine in the DNA of PBS1 phage (Takahashi & Martour, 1963) and dihydroxypentyluracil partially substitutes for thymine in SP15 phage (Brandon et al., 1972; Marmur et al., 1972). The unusual base in the DNA of 'Ufa' phage cannot be 5-HMU since (1) antiserum to SP01 phage DNA which contains only antibodies against 5-HMU (Braude & Zamchuk, 1971; May-Levine et al., 1967; Goldfarb & Zamchuk, 1975) does not react with 'Ufa' phage DNA, and (2) antiserum to 'Ufa' phage DNA does not react with SP01 DNA (Table 1). Nor is this base likely to be dihydroxypentyluracil since the latter sharply decreases T,, of the DNA (Brandon et al., 1972; Marmur et al., 1972). Preliminary results of chemical composition studies of 'Ufa' phage DNA have confirmed the absence of either glucosylated or glucoseless 5-HMC in this DNA. Another, yet unidentified, base is present in its place (N. I. Alexandrushkina & B. F. Vanyushin, personal communication). Structural differences between T-even and 'Ufa' DNA were also revealed in the competition hybridization assay (Table 4). It follows that the ability of 'Ufa' phage DNA to compete with the labeled T4 DNA in hybridization is much less than the ability of T4 and T2 DNA. Nevertheless, 'Ufa' DNA inhibits hybridization of T4 DNA by 28% and this is indicative of common base sequences in these DNAs. Consequently, 'Ufa' phage appears to be related to the T-even phages of E. coli though it cannot be attributed to this group. This relation was supported by the results of cross-neutralization experiments with
antisera to whole phages. The results show that in the capside of T-even phage there are common antigens with the 'Ufa' capsides. Indeed, antiserum to whole T4 phage neutralizes 'Ufa', T2 and T6 phages with about the same efficiency (Table 5). This is consistent with the results of Gachechiladze and Chanishvili (1972) who observed serological relations between T-even and 'Ufa' phages. The reason for inability of 'Ufa' phage antiserum to neutralize T-even phages is unclear and requires more detailed investigation. Acknowledgements--The authors wish to thank V. S. Mikoyan for the assistance in preparing the manuscript for publication, and N. N. Schukin and A. G. Slusarenko for provision of labeled T4 DNA and calf thymus DNA.
REFERENCES Adams M. (1959) Bacteriophages, NY. Birkadze T. V., Chirkadze I. G. & Chanishvili T. (3. (1967) Trans. Res. Inst. Vaccine Sera, Tbilisi (U.S.S.R.) 6, 73. Brandon C., Gallop P., Marmur J., Hayashi H. & Nakanishi K. (1972) Nature, New Biol. 239, 70. Braude N. & Zamchuk L. A. (1971) J. lmmun. 107, 1966. Chanishvili T. G. & Kapanadze J. S. (1967) Trans. Res. Inst. Vaccine Sera, Tbilisi (U.S.S.R.) 6, 49. Denhardt D. I. (1966) Biochem. biophys. Res. Commun. 23, 641. Gachechiladze K. K. & Chanishvili T. G. (1972) In Prohlems of Experimental and Theoretical Biology, Tbilisi, U.S.S.R. Goldberg E. (1966) Proc. natn Acad. Sei. U.S.A. 56, 1457. Goldfarb D. M. & Zamchuk L. A. (1975) Antibodies to the Nucleic Acids, Nauka, Moscow. Kallen R., Simon M. & Marmur J. (1962) J. molec. Biol. 5, 248. Kapanadze J. S. (1970) Sh. sonnei bacteriophages. Doctoral Thesis. Moscow. Lang D. (1970) J. molec. Biol. 54, 557. Lehman J. R. & Pratt E. A. (1960) J. biol. Chem. 235, 3254. Levine L., Murakami J., Van Vunakis H. & Grossman L. (1960) Proc. natn Acad Sci. U.S.A. 46, 1038. Marmur J. & Dgty P. (1962) J. molec. Biol. 5, 109. Marmur J., Greenspan C., Palecek E., Kahan F. & Mandell M. (1963) Cold Spring Harb. Symp. quant. Biol. 28, 191. Marmur J., Brandon C., Neubort S., Ehrlich M., Mandelt M. & Konviska J. (1972) Nature, New Biol. 239, 68. May-Levine F., Lacour F., Truffaut M. & May P. (1967) C.r. hedb. SOanc. Acad. Sci. Paris, ser. D, 265, 937. McNicol L. & Goldberg E. (1973) J. molec. Biol. 76, 285. Murakami W., Van Vunakis H., Lenrer H. & Levine L. (1962) J. lmmun. 89, 116. Plescia O., Braun W. & Palszuk N. (1964) Proc. natn Acad Sci. U.S.A. 52, 279. Romig W. & Brodetsky A. (1961) J. Bact. 82, 135. Studier F. (1965) J. molec. Biol. 11, 373. Takahashi J. & Marmur J. (1963) Nature, Lond. 197, 794. Tikhonenko T. I., Koudelka Y. & Borishpolez Z. N. (1963) Mikrobiologia (U.S.S.R.) 32, 723. Tikhonenko T. I. & Solovieva N. (1961) Biokhimia (U.S.S.R) 26, 794. Wasserman E. & Levine L. (1961) J. lmmun. 87, 290. Wyatt G. R. & Cohen S. S. (1953) Biochem. J. 55, 774. Zamchuk L. A. (1965) Genetika (U.S.S.R.) 3, 132. Zamchuk L. A. & Magradze N. M. (1976) Voprosi Virusologii (U.S.S.R.) 2, 161.
I M M U N O G E N I C D N A OF SHIGELLA SONNEI BACTERIOPHAGE L U D M I L A A. Z A M C H U K , N I N A M. M A G R A D Z E and D A V I D M. G O L D F A R B Institute of General Genetics, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. (First received 21 May 1976; in revised form 30 June 1976)
Abstract~Immunochemical and some other properties of Shigella sonnei 'Ufa' bacteriophage DNA have been studied. The DNA has been shown to possess high immunogenic activity indicating the presence of an unusual base which is neither 5-HMC* nor glucosylated 5-HMC, dihydroxypentiluracil or 5-HMU. The DNA is double-stranded, its mol. wt is 1.07.108 daltons and T,, = 85.5°C. Common base sequence have been found in 'Ufa' DNA and T4 DNA using competition DNA-DNA hybridization technique, Cross-neutralization experiments with intact 'Ufa' and T4 phages using corresponding antisera have confirmed the presence of common antigenic determinants in these phages.
INTRODUCTION Recently it has been shown (Zamchuk & Magradze, 1976) that D N A s isolated from 6 Shigella sonnei bacteriophages can be subdivided into 2 groups according to their immunochemical properties, i.e. those reacting and those not reacting with antisera to T-even phage DNA. The 'Ufa' phage, the D N A of which belongs to the latter group, has been previously studied by Chanishvili & Kapanadze (1967, 1970) with respect to several properties including particle and plaque morphology, serologic properties and host range. Based on these criteria, the authors have related 'Ufa' phage to the T-even phage group. As is known T-even bacteriophage D N A contains glucosylated or non-glucosylated 5-HMC (Wyatt & Cohen, 1953; Lehman & Pratt, 1960) which determines high immunogenic specificity of its. D N A (Levine et al., 1960; Murakami et al., 1962; Zamchuk, 1965). Antisera)o T-even phage D N A react only with D N A containing this unusual base. Hence, the inability of D N A from any source, including phages, to react with T-even D N A antisera is an indication of the absence of glycosylated or non-glucosylated 5-HMC in such DNA. Thus the conclusion that 'Ufa' phage relates to the T-even group appears to be arguable. It was, therefore, thought worthwhile to study immunogenic and certain other properties of D N A and intact particles of 'Ufa' phage in more detail.
MATERIALS A N D M E T H O D S
Bacteria E. coli B and B. subtilis 168 (ind- str-r) strains were obtained from the bacteria and phage collection of the Institute of General Genetics, U.S.S.R. Academy of Sciences; E. coli K172 rgl- su- and E. coli CR63 rgl-
su + were kindly provided by Dr. E. Goldberg (Tufts University School of Medicine, Boston, MA); Sh. sonnei strain 1188 was obtained from the Research Institute of Vaccines and Sera, Tbilisi, U.S.S.R. Phages Wild4ype T2, T4 and T6 phages and B. subtilis SP01 phage were obtained from the collection of the Institute of General Genetics; Sh. sonnei 'Ufa' phage was obtained from the Research Institute of Vaccines and Sera; phage T4 ctgtam8 flgtaml0 was provided by Dr. E. Goldberg. Preparation of phage stocks T2, T4 and T6 phage were grown on E. coli B in the standard M9 medium supplemented with 20/~g/ml e-tryptophan. Glucoseless phage T4 ctgtam8 flgtaml0 was grown on E. coli K172 rgl- su- (McNicol & Goldberg, 1973). 'Ufa' phage was grown on Sh. sonnei 1188 in the following medium: 7 g NaH2PO 4. 12H20 ; 3 g KH2POa, 1 g NH4C1, 0.5g NaC1, 0.02g CaClz, 0.025g MgSO4'7HzO and 4g of glucose per litre of water (Tikhonenko & Solovieva, 1961). Bacteria were aerated by shakirrg or bubbling (Birkadze et al., 1967). Infection was carried out at 1 to 2.10 s cell per ml with moi 0.1. The phages were concentrated and purified by either DEAE-column chromatography (Tikhonenko et al., 1963) or differential centrifugation. The final phage titers were from 1011 to 1012 pfu per ml. Phage absorption Phage absorption was measured according to the amount of unabsorbed phage following inactivation of infected bacteria with chlorophorm (Adams, 1961). Isolation of DNA Isolation of DNA from concentrated phage suspensions was carried out by phenol technique according to Goldberg (1966). No protein was detected in DNA by the Lowry technique. Hence, protein contamination was less than 1%. For immunological studies DNA (10 #g/ml) was denatured in a boiling water bath for 10 min, and immediately placed into an ice bath.
Preparation of antisera to Sh. sonnei 'Ufa' and T4 phages Rabbits were immunized intravenously with 1 ml of phage suspensions at intervals of 2-4 days, increasing a *Abbreviations used: 5-HMC, 5-hydroxymethylcytosine; 5-HMU, 5-hydroxymethyluracil; MBSA, methylated single dose from 1011 to 1012pfu. Each rabbit received bovine serum albumin; moi, multiphcity of infection; SSC, 6-9"1012 phage particles. The blood was taken a week 0.15 M NaCI, 0.015 M Na citrate. after the last injection. 985
986
LUDMILA A. ZAMCHUK, NINA M. MAGRADZE and DAVID M. GOLDFARB
Preparation of antisera to phage DNA Antisera to T2 DNA were obtained after 1-3 times a week immunization of rabbits with increasing (1012-10 ~3) phage equivalents doses of disrupted, alum-absorbed phage. Each animal received 28 mg of DNA (Zamchuk, 1965). To obtain antisera to SP01, 'Ufa' and T4 phages DNA, rabbits were immunized with MBSA-denatured DNA complexes according to Plescia (Plescia et al., 1964). Each animal received 1.2 mg of 'Ufa' DNA or 2 mg of T4 or SP01 DNA during the immunization course. The rabbits were bled a week after the last immunization.
°°t g-
~) 40 f 20
Phage neutralization with antisera This was performed as described by Adams (1961). Neutralization constants were calculated from the following equation: K = 2.3 D/t lg (Po/P), where Po is the amount of phage at 0 time, P, the amount of phage at t time, D, the final dilution of the serum, and K, the neutralization rate constant.
DN A melting temperature This was measured on Unicam SP-8000 spectrophotometer in a thermostated cell at 260 nm as described by Marmur & Doty (1962) and DNA was dissolved at 20 pg/ml in l x SSC (pH 7.0 _+ 0.3). G + C content This was calculated from the equation Tm= 69.3 + 0.41 x (G + C) which is valid for DNA preparations containing from 25 to 75% of G + C (Marmur & Doty, 1962). Sedimentation coefficients Sedimentation coefficients (S°20. w) for phage DNA were determined using Spinco E ultracentrifuge at 36,000 rev/ min and 20°C. DNA was dissolved in 1 x SSC at 20 #g per ml. DNA tool. wt determination This was carried out according to equations suggested by Studier (1965). Hybridization The following technique of competition DNA-DNA hybridization was used ' t o detect homologous base sequences. Membrane filters (Millipore, dia 25 mm) were soaked overnight in 2 x SSC solution. Five ml of denatured unlabeled DNA (5 #g/ml) in 6 x SSC were passed through each filter, followed by 5 ml of 6 x SSC. The filters were dried for 1 hr in vacuum at room temperature and incubated for 3 hr at 65°C. After drying, the filters were put into 1 ml of 3 x SSC containing 0.02% Ficoll (mol. wt 400,000), 0.02% polyvinylpyrollidone and 0.02% BSA (Denhardt, 1966). 3H-labeled T4 DNA (0.5#g per 0.25 ml of 3 x SSC) was heat denatured and mixed with competing unlabeled denatured DNA (3.5 #g in 0.25 ml of 3 x SSC), incubated for 6 hr at 65°C, and added to the vials with the filters. In the control only labeled denatured T4 DNA (0.5 gg in 0.5 ml of 3 x SSC) was added (direct hybridization). The incubation continued for 12-15 hr at 65°C. After that the filters were washed by 40ml of 2 x SSC on each side, dried and placed into scintillation fluid (50mg P O P O P and 4g PPO per 1 of toluene). Radioactivity of the filters was measured on Nuclear Chicago MARK-1 liquid scintillation counter. Enzymatic treatment of DNA Crystalline DNase 1 (Worthington), trypsin (Spofa, Czechoslovakia) and pronase (Calbiochem, U.S.A.) were used at 10#g/ml. The treatment with DNase was per-
0.01
0.025
0.05 ONA,
0.1 /~g
Fig. 1. C' fixation of anti-T2 DNA with homologous and heterologous denatured DNA test-antigens: O, T2; 0, T4; x, Sh. sonnei 'Ufa'. Antiserum dilution; O, 0, 1/8000; x,
1/400. formed for 1 hr in 10 -3 M MgC12 at pH 7.0. Trypsin and pronase treatment was performed for 1 hr in 0.01 M Tris-HCl buffer, pH 8.0.
Complement fixation test This was carried out according to Wasserman & Levine (1961).
RESULTS
Immunochemical characterization of Sh. sonnei 'Ufa' phage D N A Antisera to T2 and T4 did not react with 'Ufa' D N A at the dilution of 1:400 while the same sera at 1:2000-1:8000 dilution were positive in the complementation fixation test with homologous antigens (Figs. 1 a n d 2). O n the other hand, antisera of rabbits immunized with M B S A - ' U f a ' D N A complexes were highly reactive with the corresponding antigen but failed to react with T2, T4 and T6 or glucoseless T4 ctgtam8 flgtaml0 phage D N A (Fig. 3, Table 1). It follows from Fig. 3 that with anti-'Ufa' D N A serum diluted 1 : 1600 a n d 0.1 #g of homologous DNA, 85% of C' were fixed while the same serum at 1:400 did not give complement fixation with T-even phage DNA.
I O0
80
g~ ~=
4O
20 I
0.01
~
0025
=4
f
0.05 DNA,
O.I #g
Fig. 2. C' fixation of anti-T4 DNA with homologous and heterologous denatured DNA test-antigen: O, T4; O, T2; x, Sh. sonnei 'Ufa'. Antiserum dilution: O, 0, 1/2000; x,
1/400.
987
Immunogenic Properties of Phage DNA Table 1. Immunochemical specificity of sera to different phage DNA C' fixation (~o) with DNA" of phage Sh. sonnei T4 ~tgtam8 B. subtilis 'Ufa' flgtaml0 T4 SP01
Antisera to phage DNA Sh. sonnei 'Ufa' (1 : 1800)b T4 (1:1500) b B. subtilis SP01 (1:900) b
95.5 0d 9.0~
0c 0~ 17.0c
0c 98.3 0c
(Y 3n 86.0
= DNA dose, 0.1 k~g. b Serum dilution in C' fixation with homologous DNA. c Serum dilution 1:300. a Serum dilution 1 : 600.
Antibodies that appear in sera of rabbits, immunized with MBSA-'Ufa' DNA react with denatured DNA (Fig. 4) which is characteristic of all types of antibodies to DNA resulting from immunization of normal animals (Levine et al., 1960; Murakami et al., 1962; Zamchuk, 1965). Specificity of antibodies to 'Ufa' D N A was confirmed by enzymatic controls. Trypsin and pronase treatment of 'Ufa' D N A antigen had no effect on its ability to react with the antiserum whereas DNase digestion led to complete loss of antigenic activity. Therefore the following conclusions can be made on the basis of these data: (1) 'Ufa' phage DNA possesses high immunogenic activity; (2) immunogenic determinant of this D N A is immunodominant and distinct from glucosylated 5-HMC. Additional experiments indicated that 5-HMU is also absent in 'Ufa' DNA. Table 1 shows that antiserum to SP01 DNA which contains antibodies to 5-HMU does not react with 'Ufa' DNA as well as antiserum to 'Ufa' DNA is negative in the complement fixation test with SP01 DNA. Physical properties of Sh. sonnei 'Ufa' phage D N A 'Ufa' is a large phage. It contains double-stranded DNA which produces a typical A260 vs temperature curve on melting (Fig. 5). Tm calculated from this curve equals 85.5°C. Hence, it follows that the unusual base of 'Ufa' phage DNA does not disturb the interaction of D N A complementary strands. G + C content calculated from the Tm value of 'Ufa' DNA is 39.5~o which is different from the corresponding value for T2 and T4 DNAs (Table 2). The average
I00
°~°
~
I00
80
o o ;,7-
60 40
20
0.0 I
O. 025
0.05
O. I
DNA,
/.¢g
Fig. 4. C' fixation of anti-'Ufa' DNA (1 : 1200) with denatured (O) and native (O) homologous DNA.
mol. wt calculated from the S°2o,w value equals 1.07.10 s (-fable 3) and is close to the mol. wt of T4 DNA determined in our experiments (1.15. 108). In the competition D N A - D N A hybridization assay there was found that only a part of 'Ufa' DNA sequence was homologous to that of T-even phages (Table 4). The table shows that T4, T2 and 'Ufa' DNAs inhibits hybridization of T4 3H-DNA by the average 58, 53 and 28~o, respectively. Specificity of the observed 28~o inhibition is confirmed by the fact that pre-hybridization of E. coli DNA with the filterimmobilized unlabeled T4 DNA did not affect the ability of the latter to hybridize with 'Ufa' DNA (Table 4, exp. 1) and also by the insignificant inhibition of hybridization with calf thymus DNA (Table 4, exp. 3).
o 0.6E
60 o
L~
4C-
d
2C--
o.5a
d 1/400
I/8(X)
Dilution
111600
of
11/2400
serum
0.44
@J
I
80
Fig. 3. C' fixation of anti-'Ufa' DNA with homologous and heterologous denatured DNA test-antigen: @, Sh. sonnei 'Ufa'; x, T4; O, T2; /x, T6. DNA dose, 0.1/tg.
]
8 ;>
I
84
I
86
I
88
J
90
I
92
1-*
Fig. 5. Optical density vs temperature for DNA of phages T4 (@) and Sh. sonnei 'Ufa' (O).
988
LUDMILA A. ZAMCHUK. NINA M. MAGRADZE and DAVID M. GOLDFARB Table 2. Melting temperatue (T,~) and G + C content of different phages DNA G + C content taken
Phages DNA
Tm
C + C (/o) °/
T2
85.5 83.5
39.5 34.6
T4
84.0
35.8
Sh. sonnei 'Ufa'
Table 3. S"2o.w and mol. wt of 'Ufa' and T4 DNA Phages DNA Sh. sonnei 'Ufa' E. coli T4
S:2o.w
mol. wt x 106 dalton
53.0 54.0
107 115
from the literature 33.0 (Marmur & Doty, 1962) 35.8 (Marmur & Dory, 1962)
functional difference between the tail of T-even and 'Ufa' phages. DISCUSSION
Serologic properties o f Sh. sonnei 'Lfa' phage
The partial inhibition of T4 D N A x T4 3H-DNA by 'Ufa' D N A indicates the presence of common base sequences in D N A s of T4 and 'Ufa' phages which may result in common proteins in their capsides. To investigate this possibility, we have carried out crossneutralization experiments with "Ufa' and T-even phages and the corresponding antisera and determined the inactivation constants. Table 5 shows that antiserum to intact T4 phage inactivated 'Ufa' phage to the same degree as it did T2 and T6 phages. At the same time, antiserum to 'Ufa' phage did not inactivate any of the T-even phages. In addition it was demonstrated that the efficiency of adsorption of 'Ufa' and T4 phages on E. coil B was 7 and 99%, respectively. This also indicates the
The main result of the present work is the finding that Sh. sonnei 'Ufa' phage D N A possesses specific immunogenic activity which indicates the presence of an unusual base in this DNA. D N A of this phage has the mol. wt of 1.07- 108 dalton. This value is not absolute since it was determined only on the basis of S°2o, w and therefore, needs to be specified by additional techniques. We believe, however, that it is close to the true mol. wt since parallel determinations for T4 D N A have the value very close to that found by Lang (1970) who measured the contour length of T4 D N A (1.19.10 s dalton). The melting profile of 'Ufa' D N A indicates its double-stranded structure (Fig. 5); G + C content, as calculated from the T~, value, is 39.5Yo, i.e. higher than for T2 and T4 phage D N A (Table 2). lmmunochemical studies of 'Ufa' phage DNA have demonstrated that this phage is different from the T-even E. coil phages since rabbit antisera against
Table 4. Inhibition of T4 DNA × T4 H 3 DNA hybridization by homo- and heterologous DNA Competing DNA (3.5/~g/ml)
Exp.
..... Ic
Sh. sonnei 'Ufa;
T4 T2 Ka -
2
-
Sh. sonnei 'Ufa'
T4 T2 Ks -3
Sh. sonnei 'Ufa'
T4 T2 Thymus K*
Retention of ~H-DNA (counts/min) (,%)
Retention inhibition (°o)b
5005 3771 2650 3212 9856
50.7 38.2 26.9 32.5
0 24.7 47.1 35.9
5890 4362 2856 2912 9856
59.7 44.2 28.9 29.5
0 26.0 51.5 50.6
3138 2055 803 914 2844 9357
33.5 22.0 8.6 9.8 30.4
0 34.5 74.4 71.2 9.4
° K reference 3H-DNA counts (0.5 #g) taken for 100~o. b When retention inhibition was calculated, the amount of label retained in the absence of competing DNA was taken for the 100%. 'In exp. 1 the filters with immobilized, denatured, unlabeled T4 DNA were pre-incubated with 5/~g of denatured E. coli DNA for 4 hr at 65°C. Then 3H-DNA of T4 and competing DNA were added (see Materials and Methods).
989
Immunogenic Properties of Phage DNA Table 5. Constants of neutralization for different phages Phages Sh. sonnei 'Ufa'
T4 T2 T6
Antiserum to phage Sh. sonnei 'Ufa' 950.0 6.5 5.7 6.3
T4 250 1070 230 140
T2 and T4 DNA were negative in the complement fixation test with 'Ufa' phage DNA (Figs. 1 and 2). It is well known that antisera to T-even phage DNA contain antibodies to glucosylated 5-HMC which is the immunodominant antigenic determinant in this DNA (Levine et al., 1960; Murakami et al., 1962; Zamchuk, 1965). 'Ufa' phage DNA also possesses high and specific immunogenic activity. Anti-'Ufa' DNA serum was active at a 1:1600 dilution with homologous DNA and did not react at a 1:400 dilution with either glucoseless T4 DNA or wild-type T-even phage DNA containing fully or partially glucosylated 5-HMC. From this it follows that the immunogenic determinant of 'Ufa' phage DNA is an unusual base being neither 5-HMC nor glucosylated 5-HMC. Besides T-even phages, unusual bases have been found in DNA of B. subtilis phages: 5-HMU substitutes for thymine in the DNA of SP01, SP8 and SP82 phages (Romig & Brodetsky, 1961; Kallen et al., 1962; Marmur et al., 1963), uracil substitutes for thymine in the DNA of PBS1 phage (Takahashi & Martour, 1963) and dihydroxypentyluracil partially substitutes for thymine in SP15 phage (Brandon et al., 1972; Marmur et al., 1972). The unusual base in the DNA of 'Ufa' phage cannot be 5-HMU since (1) antiserum to SP01 phage DNA which contains only antibodies against 5-HMU (Braude & Zamchuk, 1971; May-Levine et al., 1967; Goldfarb & Zamchuk, 1975) does not react with 'Ufa' phage DNA, and (2) antiserum to 'Ufa' phage DNA does not react with SP01 DNA (Table 1). Nor is this base likely to be dihydroxypentyluracil since the latter sharply decreases T,, of the DNA (Brandon et al., 1972; Marmur et al., 1972). Preliminary results of chemical composition studies of 'Ufa' phage DNA have confirmed the absence of either glucosylated or glucoseless 5-HMC in this DNA. Another, yet unidentified, base is present in its place (N. I. Alexandrushkina & B. F. Vanyushin, personal communication). Structural differences between T-even and 'Ufa' DNA were also revealed in the competition hybridization assay (Table 4). It follows that the ability of 'Ufa' phage DNA to compete with the labeled T4 DNA in hybridization is much less than the ability of T4 and T2 DNA. Nevertheless, 'Ufa' DNA inhibits hybridization of T4 DNA by 28% and this is indicative of common base sequences in these DNAs. Consequently, 'Ufa' phage appears to be related to the T-even phages of E. coli though it cannot be attributed to this group. This relation was supported by the results of cross-neutralization experiments with
antisera to whole phages. The results show that in the capside of T-even phage there are common antigens with the 'Ufa' capsides. Indeed, antiserum to whole T4 phage neutralizes 'Ufa', T2 and T6 phages with about the same efficiency (Table 5). This is consistent with the results of Gachechiladze and Chanishvili (1972) who observed serological relations between T-even and 'Ufa' phages. The reason for inability of 'Ufa' phage antiserum to neutralize T-even phages is unclear and requires more detailed investigation. Acknowledgements--The authors wish to thank V. S. Mikoyan for the assistance in preparing the manuscript for publication, and N. N. Schukin and A. G. Slusarenko for provision of labeled T4 DNA and calf thymus DNA.
REFERENCES Adams M. (1959) Bacteriophages, NY. Birkadze T. V., Chirkadze I. G. & Chanishvili T. (3. (1967) Trans. Res. Inst. Vaccine Sera, Tbilisi (U.S.S.R.) 6, 73. Brandon C., Gallop P., Marmur J., Hayashi H. & Nakanishi K. (1972) Nature, New Biol. 239, 70. Braude N. & Zamchuk L. A. (1971) J. lmmun. 107, 1966. Chanishvili T. G. & Kapanadze J. S. (1967) Trans. Res. Inst. Vaccine Sera, Tbilisi (U.S.S.R.) 6, 49. Denhardt D. I. (1966) Biochem. biophys. Res. Commun. 23, 641. Gachechiladze K. K. & Chanishvili T. G. (1972) In Prohlems of Experimental and Theoretical Biology, Tbilisi, U.S.S.R. Goldberg E. (1966) Proc. natn Acad. Sei. U.S.A. 56, 1457. Goldfarb D. M. & Zamchuk L. A. (1975) Antibodies to the Nucleic Acids, Nauka, Moscow. Kallen R., Simon M. & Marmur J. (1962) J. molec. Biol. 5, 248. Kapanadze J. S. (1970) Sh. sonnei bacteriophages. Doctoral Thesis. Moscow. Lang D. (1970) J. molec. Biol. 54, 557. Lehman J. R. & Pratt E. A. (1960) J. biol. Chem. 235, 3254. Levine L., Murakami J., Van Vunakis H. & Grossman L. (1960) Proc. natn Acad Sci. U.S.A. 46, 1038. Marmur J. & Dgty P. (1962) J. molec. Biol. 5, 109. Marmur J., Greenspan C., Palecek E., Kahan F. & Mandell M. (1963) Cold Spring Harb. Symp. quant. Biol. 28, 191. Marmur J., Brandon C., Neubort S., Ehrlich M., Mandelt M. & Konviska J. (1972) Nature, New Biol. 239, 68. May-Levine F., Lacour F., Truffaut M. & May P. (1967) C.r. hedb. SOanc. Acad. Sci. Paris, ser. D, 265, 937. McNicol L. & Goldberg E. (1973) J. molec. Biol. 76, 285. Murakami W., Van Vunakis H., Lenrer H. & Levine L. (1962) J. lmmun. 89, 116. Plescia O., Braun W. & Palszuk N. (1964) Proc. natn Acad Sci. U.S.A. 52, 279. Romig W. & Brodetsky A. (1961) J. Bact. 82, 135. Studier F. (1965) J. molec. Biol. 11, 373. Takahashi J. & Marmur J. (1963) Nature, Lond. 197, 794. Tikhonenko T. I., Koudelka Y. & Borishpolez Z. N. (1963) Mikrobiologia (U.S.S.R.) 32, 723. Tikhonenko T. I. & Solovieva N. (1961) Biokhimia (U.S.S.R) 26, 794. Wasserman E. & Levine L. (1961) J. lmmun. 87, 290. Wyatt G. R. & Cohen S. S. (1953) Biochem. J. 55, 774. Zamchuk L. A. (1965) Genetika (U.S.S.R.) 3, 132. Zamchuk L. A. & Magradze N. M. (1976) Voprosi Virusologii (U.S.S.R.) 2, 161.
