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Characteristics of DNA molecules extracted from bacteriophages φ80 and φ80pt1
VIROLOGY
30, 29-35 (1966)
Characteristics of DNA Molecules Extracted from Bacteriophages
KOIG SATO
Department of Mic"obial Genetics, Research Institute for Microbiol Diseases, Osaka University, Osaka, Japan Accepted May 10, 1966 80 DNA prepared by a mild phenol extrllction method had a buoyant density in a CsCl gradient of 1.710 g cm- 8 and a thermal dcnuturution temperature of 90.5°. The buoyant density and thermal denaturation temperature of ¢80ptl DNA were identical to those of ¢80 DNA. ¢80 DNA had 11 guanine plus cytosine content of 53 mole per cent which was slightly higher than that of A DNA or Escherichia coli DNA. The molecular weight of ¢80 D~A estimated from the sedimentation rate was 29.3 X 106 , and that of 80pt1 DNA was 31.0 X 106 • The significance of the observed difference in molecular weight between ¢80 DNA and 80ph is discussed. INTRODUCTION
MATERIALS AND METHODS
Specialized transduction of tryptophan markers by phage ¢80 has many features similar to those of transduction of galactose genes by phage A (Matsushiro, 1963; Matsushiro ei al., 1964). The DNA molecules of ¢80 has terminal cohesive ends which are comparable to those of A (Yamagishi et al., 1965: Shinagawa ei ol., 1966). Moreover, genetic recombination between
Bacterial and phage strains. A prototrophic strain, W3102 of Escherichia coli K12 was used as host bacterium in the propagation of bacteriophages. Phage 4>80 is a normal active phage and phage ¢80pt1 is a non defective transducing phage carrying the tryptophan region with a deletion at anthranilate synthetase (E) cistron (Matsushiro et al., 1964) and a partial deletion at anthranilate phosphoribosyl transferase CD) cistron (Sata and Matsushiro, 1965). Phage AC was supplied by the courtesy of Dr. H. Ozeki. Phage A ("wild-type") was obtained from E. coli K12(A) (supplied by the courtesy of Dr. Y. Hirota) induced by ultraviolet light. Media. L-broth (Lennox, 1955) was used for the propagation of 4>80, nutrient broth for ¢80pt11 and lambda broth (polypeptone, 10 g and NaCl, 2.5 g per liter) for A and xc. For the preparation of labeled phages, Tris-glucose medium (Hershey, 1955) was
1 Present address: Research Unit in Biochemistry, Biophysics and Molecular Biology, MeMaster University, Hamilton, Ontario, Canada.
29
30
YAMAGISRI , YOSRIZAKO, AND SATO
used. T 1 medi um, which contained NaCl-0.1 M phosphate buffer (pH 7.1). T o lVIgSOi ·7 H 2 0, 6 X 10- 4 1tf; CaCb, 5 X 10- 4 prepare D NA samples for the base analysis, J11; gelatin, 0.001 % and Tris-HCI buffer phage particles were collected by precipita(pH 7.3) , 6 X 10- 3 M , was u sed as the t ion with ammonium sulfate (330 g/ ml) and suspending me dium for ph age particles. purified without the esCI centrifugation Preparation of phages . Cells of W3102 step. Extraction of DNA. T o the purified were infected , at a mu ltiplicity of infection of 1.0, with phages. After the cells were phage suspension, ethylenedi amine tetr alysed, cult ures were treated with chloroform acetate (2 X 10- 3 M ), sodium dodecyl and kept overnight in the cold. To prepare sulfate (0.2 %) , and an equal volume of labeled phage DNA, 3ZPOi (20 /olC/ml) or buffer-saturated phenol were added, and 3H -uridille (90 MC/ml ) was added to bac- DNA was gently extracted in a roller tube terial cultures in T ris -glucose medium at at 60 rpm for 30-60 minutes at 60 as dethe time of phage infection. Phage lys ates scribed by Frankel (1963). The phenolwere treated with DNase (1 J.lg/ml) and treated phage suspension was cent rifuged RN ase (1 JLg/ml) at 37° for 30 minutes and at 2000 rpm for 5 minutes, and the phenol purified by differen ti al centrifuga tion. Phage layer was pipetted off. This treatment was particles were resuspended in T 1 medium repeated 3 times. The phenol in water and purified further by centrifugation in a layer was removed by dialysis in SSC CsCI gradient, followed by dialysis in 0. 1 il1 (0.15 M NaCI-O.015 M sodium citrate).
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FIG . 1. CsCI de nsity gradient cen trifugation analysis of rjl80 and ¢80ptl DNA . Samples of DNA in 3 ml of CsCI solution at an av erage dens ity of 1.69 g cm- a were centrifuged at 33,000 rpm for 60 hours. De nsity gradien ts were determined from the refractive index me asured with fractions 8, 15, and 55. (a) 0.2 Ilg aU-hC D NA (open circl e) and 0.2 Ilg 82P _rjl80 DNA (filled circle). T he total number of fractions was 66. (b) 2/Lg 82P-Ql80pt1 DNA (ope n circl e) and 1 1lg 3H-¢80 DNA (filled circle ). The total number of fractions was 65.
31
DNA OF SO AND SOpt1
For the preparation of DNA used in the base analysis, the mixture of phage suspension and phenol was manually shaken. Biunjomt density determination, It was found that AC DNA and 4>80 DNA had common cohesive ends and that they readily formed heteropolymers (Yamagishi et al., 1965). Therefore, DNA samples in SSC were heated at 750 for 10 minutes separately and rapidly cooled to obtain linear monomers. To 0.6 ml of the above Dl\A solutions, 2.4 ml of saturated CsCl solution in SSC was added to obtain an average density of 1.69 g cnr". The amount of DNA used was from 0.4 to 4 p.g. Furthermore, to avoid the polymer re-formation in concentrated CsCI solutions, formaldehyde was added to 1.8 % (Hershey, 1964). Usually, about 1.5 ml of mineral oil was layered on the top of this solution to prevent evaporation. Samples were centrifuged at 33,000 rpm for 60 hours in a Hitachi RPS '10 swinging-bucket rotor at 100 • The size of drops collected from the tubes was controlled according to the method of Burgi and Hershey (1963). Groups of 4 drops were collected on a Toyoroshi No. 2 filter paper disk, washed in cold 5 % trichloroacetic acid and 95 % ethanol, and dried. Radioactivity was measured with a Packard Tri-Carb scintillation counter. Densities of the fractions were calculated from the refractive index measured at 250 (Schildkraut et al., 1962). Determination oj thermal deruiiuraium temperature. The thermal denaturation temperature of DNA samples was determined by the method of Marmur and Doty (1962). The concentration of DNA used was 20 p.g/mI. Base analysis. About 1 mg of Dl\A was precipitated with 2 volumes of ethanol and dried. The dried DNA fiber was hydrolyzed in 0.5 ml of 6 N HCI under CO2 in sealed tubes for 3 hours at 1000 • Hel was removed from the hydrolyzate in a vacuum desiccator over EOH and CaCb. Thereafter, the residue was dissolved in 50 f.Ll of 0.1 N HCI. A portion (10 I-d) of the hydrolyzate was used in the determination of total phosphorus, and the same amount of the hydrolyzate was used in the base analysis
i
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FIG. 2. Distribution of xc DNA (open circle) and SO DNA (filled circle) in a CsCl density gradient. The results of Fig. la were plotted on probability paper.
by paper chromatography. Total phosphorus was determined by the method of Lowry et al. (1954). Paper chromatography was carried out with Toyoroshi No. 51A paper and the ieopropanol-HCl solvent system by the method of Smith and Wyatt (1951). The recovery of bases was 95-98 %. Zone sedimentation analysis. Zone sedimentation pattern was analyzed in a linear concentration gradient of 5-20 % (wIv) sucrose in 0.1 JJ!I NaCI-O.05 JJ!I phosphate, pH 6.7. Immediately before centrifugation, DNA was incubated at 750 for 10 minutes and rapidly cooled to obtain linear monomers. A sample containing 0.1 f-Lg of DNA in 0.1 ml of sse was layered on 4.8 1111 of sucrose solution and centrifuged in a Hitachi RPS 40 swinging-bucket rotor at 100 at 30,000 rpm for 5 hours. Groups of 3 drops were collected, and their radioactivity was assayed. The molecular weight was calculated according to the following equation (Burgi and Hershey, 1963),
32
YAMAGlSHI, YOSHIZAKO, AND SATO
in which D is the distance sedimented in the sucrose gradient and ill is the molecular weight. RESULTS
Buouant Density 32P-Labeled 80 DNA and 3H-labeled reference Xc DNA were centrifuged in a
esCI gradient as described in Materials and Methods. Fractions were collected on filter paper, and their radioactivity was assayed (Fig. l a), The above results were also plotted on a probability paper to test the distributions (fig. 2). Both 80 DNA and Xc DNA showed a single banel. Xc DNA formed a peak at a position corresponding to a density of about 1.709 g cm- 3• The density of ¢8G DNA was found to be 0.0015
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Temperature (oel FIG. 3. Thermal denaturation of80, q,80pt1 , and AC DNA. The absorbance at 260mu at the elevated temperatures relative to that of native material at 25° was plotted as a function of the temperature to which DNA samples in sse were exposed. The absorbance was corrected for thermal expansion of the solutions. Arrow shows the midpoint of the helix-coil transition. (a) AC DNA (open circle) and 80 DNA (filled circle). (b) 80pt1 DNA (open circle) and 80 DNA (filled circle).
TABLE 1 BASE COMPOSITION OF ¢SO DNA
Moles per cent A
T
G
c
23.0 ± 0.2
24.0 ± 0.3
27.0 ± 0.2
26.0 ± 0.2
Per cent
ere
53.0
PulPy 1.0
a The values were obtained from three independent determinations. The recoveries of bases were 95-98%. Abbreviations, A, adenine; T, thymine; 0, guanine; 0, cytosine; Pu, purine; Py, pyrimidine.
:33
DNA OF
TABLE 2 g cm- 3 higher than that of xc DNA. Since the density of the reference Xc DNA is PIWPEHTIES OF DNA EXTRACTED FROM PHAGE <,680, ¢80ptz, ),.0, ;.,dg, AND THEIR BAC'l'ERIAI, 1.7086 g cm- 3 (Hershey, 1964), the density HOST, E. coli K12 of ¢80 DNA would then be 1.710 g cm-3, Figure Ib shows band profiles of 3H-¢80 Denaturation Buoyant DNA DNA and 32P-¢80pt1 DNA in a CsCI graMole G-C% temperature density (p) source (g cm- J ) (Tm ) dient. The position of ¢80pt 1 DNA peak was found to be identical to that of ¢80 90.5 1. 710 53.0 DNA peak, indicating that both DNA .p80 (1.712)1' (91.0)· preparations had the same buoyant density, 1.710 90.5 Therefore, the presence of bacterial genome in ¢80pt1 apparently does not alter the 89.5 49.0 a 1. 7086' density of the phage D:-iA. A similar situ(89.3). (1. 7080)" ation has been observed with Xdg (Kaiser 49. ()b 1.708 b hdg and Rogness, 1960), (1. 708)"
Thermal Denaturation Temperature The thermal transition profiles of ¢80 DNA, ¢80pt1 DNA, and Xc DNA are shown in Fig. 3. Both ¢SO DNA and ¢80ptl DNA had the same T", (the midpoint of the helix-coil transition) of 90.5°. The Ton of Xc DNA used as reference was 89.5° which was slightly higher than that reported by Marmur and Doty (1962). Base Composition The result of base analysis presented in Tables 1 and 2 indicated that ¢80 DNA had a guanine plus cytosine content of 53 % which was 4% higher than that of hC DNA. The buoyant density and T", of ¢SO DNA estimated from the guanine plus cytosine content are very similar to those obtained experimentally (Table 2). The molar equalities of adenine and thymine and of guanine and cytosine observed in this experiment suggest that ¢80 DNA has the complementary double-stranded structure. No bases other than the normal four bases were found in the chromatogram. JI![ oleculor
Weight To determine the rate of sedimentation, 32P_¢80 DNA or 32P-¢80ph DNA was centrifuged in a sucrose gradient together with 3li-A DNA as reference. Figure -'1 shows the sedimentation pattern of these DNA samples. Although there was some skewness toward heavier fractions, all DNA samples sedimented as a single band. The presence of the skewness indicates that a
E. coli K12
50.1'
90.5<1 (89.8)'
1. 7101 (1. 709)"
a Reported by Ledinko (19M) to range from 48.5 to 49.4%. b Kaiser and Hogness (1960). c Gandelman et al. (1952). d Marmur and Doty (1952). 'Hershey (1964). J Schildkraut et al. (1962) . • Calculated value from mole G-C per cent using the equation, T,,, = 59.3 + 0.41(G-C) (Marrnur and Doty, 19(2). "Calculated value from mole G-C pel' cent using the equation, p = 1.550 + 0.098 (G-C) (Schildkrant el al., 19(2).
small amount of closed monomer is still present in the preparations (Yam.agishi et al., 1965). Reference A DNA sedimented 1.02 times faster than ¢80 DNA (Fig. 4a). On the other hand, ¢80pt1 DNA sedimented at the same rate as A DNA (Fig. 4b). These determinations were repeated three times using independent samples of different specific activities. Since in a preliminary experiment it was found that 32P_¢80 DNA and 3H-¢80 DNA sedimented at the same rate, radiation damage due to the presence of 32p in our DKA preparations would be negligible under the present experimental conditions, From the present data, assuming that the molecular weight of }. DNA is :31.0 X 106 (Burgi and Hershey, 1963), the molecular weight of ¢80 DNA and of ¢80pt1 DNA were estimated to be 29.3 X 106 and 31.0 X lOG,
34
YAMAGlSHI, YOSHIZAKO, AND SATO
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Fro eli on no. FIG. 4. Zone sedimentation analysis of 4>80, q,8Dpt and "DNA. Samples of D.1 ml were layered on " 4.8 ml of sucrose solution (5-20%) and centrifuged at 30,000 rpm. After 5 hours, fractions were collected on disk filter and the radioactivity was measured. (a) 0.05 J.Lg 32P-80 DNA (open circle) and 0.05 J.'g 31I-A. DNA (filled circle). The total number of fractions was 92.5. (b) 0.051'g !2P-80pt, DNA (open circle) and 0.05 J.Lg aH-x DNA (filled circle). The total number of fractions was 92.
respectively. Therefore, the molecular weight of cP80pt1 DNA is 5.8 % higher than that of cP80 DNA. These values are comparable to those obtained by measuring the length of DNA molecules on the electron micrographs (Shinagawa ei al., 1966). DISCUSSION
Matsushiro et ol, (1964) reported that the buoyant density of ¢80pt1 particles was 0.006 g cm- 3 higher than that of ¢80 particles. These authors suggested that this difference in the buoyant density might reflect the difference in the DNA content of these phages. If we assume that the altered density of q,80ptl could result only from the increase in the DNA content of phage particles, the increase /.::,.P in the phage density corresponding to a fractional increment a of DNA can be obtained from the relation presented by Weigle et al. (1959),
/.: ,. _ p -
Fm POCl'.
1
-
F..
+ aF..
where F and F v are respectively the mass and volume fraction of DNA in ordinary 80 having a buoyant density of Po. The measured value for a is 0.058; for Po is 1.495 g cm-3 (Matsushiro et al., 1964), for ¢80 DNA the density is 1.710 g cm-3, and we assume the value 1.300 g cm- 3 for protein. Then F m and F v are found to be 0.55 and 0.48, respectively. Therefore, the value of 6p calculated from our data is 0.006 g cm- 3, which is identical to that determined by Matsushiro et al. (1964). Imamoto et al. (1965) reported that messenger RNA for the entire tryptophan operon had a sedimentation constant of 33 S, which would correspond to a molecular weight of 1.7 X 106 • The molecular weight of DNA corresponding to this messenger RNA is 3.4 X lOG. In the present study, molecular weights of ¢80 DNA and ¢80ptl DNA were found to be 29.3 X lOG and 31.0 X 10° respectively. This difference in the molecular weight (1.7 X 10°) is, therefore, equivalent to half of the tryptophan operon. According to MatsulI,
DNA OF
shiro et al. (1964) and Sato and Matsushiro (1955), E cistron is completely deleted and D cistron is partially deleted in the tryptophan genes carried by ¢80ptl' Furthermore, the possibility that a small segment of the phage genome may be replaced by the bacterial chromosome in ¢80ptl cannot be completely excluded. Therefore, further investigations have to be made to determine whether the difference in the molecular weight of DNA between ¢80 and ¢80ptl represents exactly the tryptophan region carried by ¢80ptj • ACKNOWLEDGMENTS We are indebted to Dr. H. Ozeki and members of the Department of Microbial Genetics, Research Institute for Microbial Diseases, Osaka University for their valuable criticism and encouragement during the course of this work, and we are also grateful to Dr. 1. Takahashi for his many helpful suggestions in the preparation of the manuscript. REFERENCES BURGI, E., and HEHSJ-lEY, A. D. (1963). Sedimentation rate as a measure of molecular weight of DNA. Biophsj«. J. 3, 309-321. FRANKEL, F. R. (1963). An unusual DNA extracted from bacteria infected with phage T2. Proc. Nail. Acad. Sci. U.S. 49,366-372. GANDELMAN, B., ZAMENHOF, S., and CHARGAlJ'l", E. (1952). The deoxypentose nucleic acids of three strains of Escherichia coli, Biochim, Biophys. Acta 9, 399-401. HEHSJ-lEY, A. D. (1955). An upper limit to the protein con tent of the germinal substance of bacteriophage T2. Virology 1,108-127. HERSHEY, A. D. (1964). Some idiosyncrasies of phage DNA structure. Carnegie Insi. Wash. Year Book 63, 580-592. IMAMo'l'O, F., MOHIKAWA, N., and SATO, K. (1965). On the transcription of the tryptophan operon in Escherichia coli. III. Multicistronic messenger RNA and polarity for transcription. J. Mol. BioI. 13, W9-182.
35
KAISER, A. D., and HOGNESS, D. S. (1960). The transformation of Escherichia coli with deoxyribonucleic acid isolated from bacteriophage xdg. J. Mol. Bioi. 2, 392-415. LEDINKO, N. (1964). Occurrence of 5-methyl deoxycytidylate in the DNA of phage lambda. J. Mol. Bioi. 9, 834-835. LENNOX, E. S. (1955). Transduction of linked genetic characters of the host by bacteriophage PI. Virology 1, 190-206. LOWHY, O. I-I., ROBEHTS, N. R" LEINER, K. Y., Wu, M. L., and FAHR, A. L. (1954). The quantitative histochemistry of brain. 1. Chemical methods. J. Bioi. Chern. 207, 1-17. MARMun, J., and Do-rr, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. kIol. Bioi. 5, 109-11S. MATSUSHIHO, A. (1963). Specialized transduction of tryptophan markers in Bscherichia coli K12 by bacteriophage 80 and lambda or <,681. Biochem . Biophys. Re8. Commun: 20, 727732.
30, 29-35 (1966)
Characteristics of DNA Molecules Extracted from Bacteriophages
KOIG SATO
Department of Mic"obial Genetics, Research Institute for Microbiol Diseases, Osaka University, Osaka, Japan Accepted May 10, 1966 80 DNA prepared by a mild phenol extrllction method had a buoyant density in a CsCl gradient of 1.710 g cm- 8 and a thermal dcnuturution temperature of 90.5°. The buoyant density and thermal denaturation temperature of ¢80ptl DNA were identical to those of ¢80 DNA. ¢80 DNA had 11 guanine plus cytosine content of 53 mole per cent which was slightly higher than that of A DNA or Escherichia coli DNA. The molecular weight of ¢80 D~A estimated from the sedimentation rate was 29.3 X 106 , and that of 80pt1 DNA was 31.0 X 106 • The significance of the observed difference in molecular weight between ¢80 DNA and 80ph is discussed. INTRODUCTION
MATERIALS AND METHODS
Specialized transduction of tryptophan markers by phage ¢80 has many features similar to those of transduction of galactose genes by phage A (Matsushiro, 1963; Matsushiro ei al., 1964). The DNA molecules of ¢80 has terminal cohesive ends which are comparable to those of A (Yamagishi et al., 1965: Shinagawa ei ol., 1966). Moreover, genetic recombination between
Bacterial and phage strains. A prototrophic strain, W3102 of Escherichia coli K12 was used as host bacterium in the propagation of bacteriophages. Phage 4>80 is a normal active phage and phage ¢80pt1 is a non defective transducing phage carrying the tryptophan region with a deletion at anthranilate synthetase (E) cistron (Matsushiro et al., 1964) and a partial deletion at anthranilate phosphoribosyl transferase CD) cistron (Sata and Matsushiro, 1965). Phage AC was supplied by the courtesy of Dr. H. Ozeki. Phage A ("wild-type") was obtained from E. coli K12(A) (supplied by the courtesy of Dr. Y. Hirota) induced by ultraviolet light. Media. L-broth (Lennox, 1955) was used for the propagation of 4>80, nutrient broth for ¢80pt11 and lambda broth (polypeptone, 10 g and NaCl, 2.5 g per liter) for A and xc. For the preparation of labeled phages, Tris-glucose medium (Hershey, 1955) was
1 Present address: Research Unit in Biochemistry, Biophysics and Molecular Biology, MeMaster University, Hamilton, Ontario, Canada.
29
30
YAMAGISRI , YOSRIZAKO, AND SATO
used. T 1 medi um, which contained NaCl-0.1 M phosphate buffer (pH 7.1). T o lVIgSOi ·7 H 2 0, 6 X 10- 4 1tf; CaCb, 5 X 10- 4 prepare D NA samples for the base analysis, J11; gelatin, 0.001 % and Tris-HCI buffer phage particles were collected by precipita(pH 7.3) , 6 X 10- 3 M , was u sed as the t ion with ammonium sulfate (330 g/ ml) and suspending me dium for ph age particles. purified without the esCI centrifugation Preparation of phages . Cells of W3102 step. Extraction of DNA. T o the purified were infected , at a mu ltiplicity of infection of 1.0, with phages. After the cells were phage suspension, ethylenedi amine tetr alysed, cult ures were treated with chloroform acetate (2 X 10- 3 M ), sodium dodecyl and kept overnight in the cold. To prepare sulfate (0.2 %) , and an equal volume of labeled phage DNA, 3ZPOi (20 /olC/ml) or buffer-saturated phenol were added, and 3H -uridille (90 MC/ml ) was added to bac- DNA was gently extracted in a roller tube terial cultures in T ris -glucose medium at at 60 rpm for 30-60 minutes at 60 as dethe time of phage infection. Phage lys ates scribed by Frankel (1963). The phenolwere treated with DNase (1 J.lg/ml) and treated phage suspension was cent rifuged RN ase (1 JLg/ml) at 37° for 30 minutes and at 2000 rpm for 5 minutes, and the phenol purified by differen ti al centrifuga tion. Phage layer was pipetted off. This treatment was particles were resuspended in T 1 medium repeated 3 times. The phenol in water and purified further by centrifugation in a layer was removed by dialysis in SSC CsCI gradient, followed by dialysis in 0. 1 il1 (0.15 M NaCI-O.015 M sodium citrate).
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FIG . 1. CsCI de nsity gradient cen trifugation analysis of rjl80 and ¢80ptl DNA . Samples of DNA in 3 ml of CsCI solution at an av erage dens ity of 1.69 g cm- a were centrifuged at 33,000 rpm for 60 hours. De nsity gradien ts were determined from the refractive index me asured with fractions 8, 15, and 55. (a) 0.2 Ilg aU-hC D NA (open circl e) and 0.2 Ilg 82P _rjl80 DNA (filled circle). T he total number of fractions was 66. (b) 2/Lg 82P-Ql80pt1 DNA (ope n circl e) and 1 1lg 3H-¢80 DNA (filled circle ). The total number of fractions was 65.
31
DNA OF SO AND SOpt1
For the preparation of DNA used in the base analysis, the mixture of phage suspension and phenol was manually shaken. Biunjomt density determination, It was found that AC DNA and 4>80 DNA had common cohesive ends and that they readily formed heteropolymers (Yamagishi et al., 1965). Therefore, DNA samples in SSC were heated at 750 for 10 minutes separately and rapidly cooled to obtain linear monomers. To 0.6 ml of the above Dl\A solutions, 2.4 ml of saturated CsCl solution in SSC was added to obtain an average density of 1.69 g cnr". The amount of DNA used was from 0.4 to 4 p.g. Furthermore, to avoid the polymer re-formation in concentrated CsCI solutions, formaldehyde was added to 1.8 % (Hershey, 1964). Usually, about 1.5 ml of mineral oil was layered on the top of this solution to prevent evaporation. Samples were centrifuged at 33,000 rpm for 60 hours in a Hitachi RPS '10 swinging-bucket rotor at 100 • The size of drops collected from the tubes was controlled according to the method of Burgi and Hershey (1963). Groups of 4 drops were collected on a Toyoroshi No. 2 filter paper disk, washed in cold 5 % trichloroacetic acid and 95 % ethanol, and dried. Radioactivity was measured with a Packard Tri-Carb scintillation counter. Densities of the fractions were calculated from the refractive index measured at 250 (Schildkraut et al., 1962). Determination oj thermal deruiiuraium temperature. The thermal denaturation temperature of DNA samples was determined by the method of Marmur and Doty (1962). The concentration of DNA used was 20 p.g/mI. Base analysis. About 1 mg of Dl\A was precipitated with 2 volumes of ethanol and dried. The dried DNA fiber was hydrolyzed in 0.5 ml of 6 N HCI under CO2 in sealed tubes for 3 hours at 1000 • Hel was removed from the hydrolyzate in a vacuum desiccator over EOH and CaCb. Thereafter, the residue was dissolved in 50 f.Ll of 0.1 N HCI. A portion (10 I-d) of the hydrolyzate was used in the determination of total phosphorus, and the same amount of the hydrolyzate was used in the base analysis
i
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Density(g cm- 3 }
FIG. 2. Distribution of xc DNA (open circle) and SO DNA (filled circle) in a CsCl density gradient. The results of Fig. la were plotted on probability paper.
by paper chromatography. Total phosphorus was determined by the method of Lowry et al. (1954). Paper chromatography was carried out with Toyoroshi No. 51A paper and the ieopropanol-HCl solvent system by the method of Smith and Wyatt (1951). The recovery of bases was 95-98 %. Zone sedimentation analysis. Zone sedimentation pattern was analyzed in a linear concentration gradient of 5-20 % (wIv) sucrose in 0.1 JJ!I NaCI-O.05 JJ!I phosphate, pH 6.7. Immediately before centrifugation, DNA was incubated at 750 for 10 minutes and rapidly cooled to obtain linear monomers. A sample containing 0.1 f-Lg of DNA in 0.1 ml of sse was layered on 4.8 1111 of sucrose solution and centrifuged in a Hitachi RPS 40 swinging-bucket rotor at 100 at 30,000 rpm for 5 hours. Groups of 3 drops were collected, and their radioactivity was assayed. The molecular weight was calculated according to the following equation (Burgi and Hershey, 1963),
32
YAMAGlSHI, YOSHIZAKO, AND SATO
in which D is the distance sedimented in the sucrose gradient and ill is the molecular weight. RESULTS
Buouant Density 32P-Labeled 80 DNA and 3H-labeled reference Xc DNA were centrifuged in a
esCI gradient as described in Materials and Methods. Fractions were collected on filter paper, and their radioactivity was assayed (Fig. l a), The above results were also plotted on a probability paper to test the distributions (fig. 2). Both 80 DNA and Xc DNA showed a single banel. Xc DNA formed a peak at a position corresponding to a density of about 1.709 g cm- 3• The density of ¢8G DNA was found to be 0.0015
•• 140
(0)
140
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EI.30
1.30
0
E 0
to
to
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65
90
95
100
80
85
90
ioo
95
Temperature (oel FIG. 3. Thermal denaturation of
TABLE 1 BASE COMPOSITION OF ¢SO DNA
Moles per cent A
T
G
c
23.0 ± 0.2
24.0 ± 0.3
27.0 ± 0.2
26.0 ± 0.2
Per cent
ere
53.0
PulPy 1.0
a The values were obtained from three independent determinations. The recoveries of bases were 95-98%. Abbreviations, A, adenine; T, thymine; 0, guanine; 0, cytosine; Pu, purine; Py, pyrimidine.
:33
DNA OF
TABLE 2 g cm- 3 higher than that of xc DNA. Since the density of the reference Xc DNA is PIWPEHTIES OF DNA EXTRACTED FROM PHAGE <,680, ¢80ptz, ),.0, ;.,dg, AND THEIR BAC'l'ERIAI, 1.7086 g cm- 3 (Hershey, 1964), the density HOST, E. coli K12 of ¢80 DNA would then be 1.710 g cm-3, Figure Ib shows band profiles of 3H-¢80 Denaturation Buoyant DNA DNA and 32P-¢80pt1 DNA in a CsCI graMole G-C% temperature density (p) source (g cm- J ) (Tm ) dient. The position of ¢80pt 1 DNA peak was found to be identical to that of ¢80 90.5 1. 710 53.0 DNA peak, indicating that both DNA .p80 (1.712)1' (91.0)· preparations had the same buoyant density, 1.710 90.5 Therefore, the presence of bacterial genome in ¢80pt1 apparently does not alter the 89.5 49.0 a 1. 7086' density of the phage D:-iA. A similar situ(89.3). (1. 7080)" ation has been observed with Xdg (Kaiser 49. ()b 1.708 b hdg and Rogness, 1960), (1. 708)"
Thermal Denaturation Temperature The thermal transition profiles of ¢80 DNA, ¢80pt1 DNA, and Xc DNA are shown in Fig. 3. Both ¢SO DNA and ¢80ptl DNA had the same T", (the midpoint of the helix-coil transition) of 90.5°. The Ton of Xc DNA used as reference was 89.5° which was slightly higher than that reported by Marmur and Doty (1962). Base Composition The result of base analysis presented in Tables 1 and 2 indicated that ¢80 DNA had a guanine plus cytosine content of 53 % which was 4% higher than that of hC DNA. The buoyant density and T", of ¢SO DNA estimated from the guanine plus cytosine content are very similar to those obtained experimentally (Table 2). The molar equalities of adenine and thymine and of guanine and cytosine observed in this experiment suggest that ¢80 DNA has the complementary double-stranded structure. No bases other than the normal four bases were found in the chromatogram. JI![ oleculor
Weight To determine the rate of sedimentation, 32P_¢80 DNA or 32P-¢80ph DNA was centrifuged in a sucrose gradient together with 3li-A DNA as reference. Figure -'1 shows the sedimentation pattern of these DNA samples. Although there was some skewness toward heavier fractions, all DNA samples sedimented as a single band. The presence of the skewness indicates that a
E. coli K12
50.1'
90.5<1 (89.8)'
1. 7101 (1. 709)"
a Reported by Ledinko (19M) to range from 48.5 to 49.4%. b Kaiser and Hogness (1960). c Gandelman et al. (1952). d Marmur and Doty (1952). 'Hershey (1964). J Schildkraut et al. (1962) . • Calculated value from mole G-C per cent using the equation, T,,, = 59.3 + 0.41(G-C) (Marrnur and Doty, 19(2). "Calculated value from mole G-C pel' cent using the equation, p = 1.550 + 0.098 (G-C) (Schildkrant el al., 19(2).
small amount of closed monomer is still present in the preparations (Yam.agishi et al., 1965). Reference A DNA sedimented 1.02 times faster than ¢80 DNA (Fig. 4a). On the other hand, ¢80pt1 DNA sedimented at the same rate as A DNA (Fig. 4b). These determinations were repeated three times using independent samples of different specific activities. Since in a preliminary experiment it was found that 32P_¢80 DNA and 3H-¢80 DNA sedimented at the same rate, radiation damage due to the presence of 32p in our DKA preparations would be negligible under the present experimental conditions, From the present data, assuming that the molecular weight of }. DNA is :31.0 X 106 (Burgi and Hershey, 1963), the molecular weight of ¢80 DNA and of ¢80pt1 DNA were estimated to be 29.3 X 106 and 31.0 X lOG,
34
YAMAGlSHI, YOSHIZAKO, AND SATO
(0)
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"30
35
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25
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40
Fro eli on no. FIG. 4. Zone sedimentation analysis of 4>80, q,8Dpt and "DNA. Samples of D.1 ml were layered on " 4.8 ml of sucrose solution (5-20%) and centrifuged at 30,000 rpm. After 5 hours, fractions were collected on disk filter and the radioactivity was measured. (a) 0.05 J.Lg 32P-
respectively. Therefore, the molecular weight of cP80pt1 DNA is 5.8 % higher than that of cP80 DNA. These values are comparable to those obtained by measuring the length of DNA molecules on the electron micrographs (Shinagawa ei al., 1966). DISCUSSION
Matsushiro et ol, (1964) reported that the buoyant density of ¢80pt1 particles was 0.006 g cm- 3 higher than that of ¢80 particles. These authors suggested that this difference in the buoyant density might reflect the difference in the DNA content of these phages. If we assume that the altered density of q,80ptl could result only from the increase in the DNA content of phage particles, the increase /.::,.P in the phage density corresponding to a fractional increment a of DNA can be obtained from the relation presented by Weigle et al. (1959),
/.: ,. _ p -
Fm POCl'.
1
-
F..
+ aF..
where F and F v are respectively the mass and volume fraction of DNA in ordinary 80 having a buoyant density of Po. The measured value for a is 0.058; for Po is 1.495 g cm-3 (Matsushiro et al., 1964), for ¢80 DNA the density is 1.710 g cm-3, and we assume the value 1.300 g cm- 3 for protein. Then F m and F v are found to be 0.55 and 0.48, respectively. Therefore, the value of 6p calculated from our data is 0.006 g cm- 3, which is identical to that determined by Matsushiro et al. (1964). Imamoto et al. (1965) reported that messenger RNA for the entire tryptophan operon had a sedimentation constant of 33 S, which would correspond to a molecular weight of 1.7 X 106 • The molecular weight of DNA corresponding to this messenger RNA is 3.4 X lOG. In the present study, molecular weights of ¢80 DNA and ¢80ptl DNA were found to be 29.3 X lOG and 31.0 X 10° respectively. This difference in the molecular weight (1.7 X 10°) is, therefore, equivalent to half of the tryptophan operon. According to MatsulI,
DNA OF
shiro et al. (1964) and Sato and Matsushiro (1955), E cistron is completely deleted and D cistron is partially deleted in the tryptophan genes carried by ¢80ptl' Furthermore, the possibility that a small segment of the phage genome may be replaced by the bacterial chromosome in ¢80ptl cannot be completely excluded. Therefore, further investigations have to be made to determine whether the difference in the molecular weight of DNA between ¢80 and ¢80ptl represents exactly the tryptophan region carried by ¢80ptj • ACKNOWLEDGMENTS We are indebted to Dr. H. Ozeki and members of the Department of Microbial Genetics, Research Institute for Microbial Diseases, Osaka University for their valuable criticism and encouragement during the course of this work, and we are also grateful to Dr. 1. Takahashi for his many helpful suggestions in the preparation of the manuscript. REFERENCES BURGI, E., and HEHSJ-lEY, A. D. (1963). Sedimentation rate as a measure of molecular weight of DNA. Biophsj«. J. 3, 309-321. FRANKEL, F. R. (1963). An unusual DNA extracted from bacteria infected with phage T2. Proc. Nail. Acad. Sci. U.S. 49,366-372. GANDELMAN, B., ZAMENHOF, S., and CHARGAlJ'l", E. (1952). The deoxypentose nucleic acids of three strains of Escherichia coli, Biochim, Biophys. Acta 9, 399-401. HEHSJ-lEY, A. D. (1955). An upper limit to the protein con tent of the germinal substance of bacteriophage T2. Virology 1,108-127. HERSHEY, A. D. (1964). Some idiosyncrasies of phage DNA structure. Carnegie Insi. Wash. Year Book 63, 580-592. IMAMo'l'O, F., MOHIKAWA, N., and SATO, K. (1965). On the transcription of the tryptophan operon in Escherichia coli. III. Multicistronic messenger RNA and polarity for transcription. J. Mol. BioI. 13, W9-182.
35
KAISER, A. D., and HOGNESS, D. S. (1960). The transformation of Escherichia coli with deoxyribonucleic acid isolated from bacteriophage xdg. J. Mol. Bioi. 2, 392-415. LEDINKO, N. (1964). Occurrence of 5-methyl deoxycytidylate in the DNA of phage lambda. J. Mol. Bioi. 9, 834-835. LENNOX, E. S. (1955). Transduction of linked genetic characters of the host by bacteriophage PI. Virology 1, 190-206. LOWHY, O. I-I., ROBEHTS, N. R" LEINER, K. Y., Wu, M. L., and FAHR, A. L. (1954). The quantitative histochemistry of brain. 1. Chemical methods. J. Bioi. Chern. 207, 1-17. MARMun, J., and Do-rr, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. kIol. Bioi. 5, 109-11S. MATSUSHIHO, A. (1963). Specialized transduction of tryptophan markers in Bscherichia coli K12 by bacteriophage 80 and lambda or <,681. Biochem . Biophys. Re8. Commun: 20, 727732.