VIROLOGY
65,129-127
(1975)
Polar DNA Ejection
Mitsubishi-Kasei
Institute
of Life
in Bacteriophage
KAORU
SAIGO
Sciences,
11, Minamiooya,
Accepted
January
T7
Machida-shi,
Tokyo,
Japan
3, 1975
T7 phage particles were disrupted by treatment with 40% formamide followed by dialysis against EDTA and were analyzed using an electron microscope. About 20% of the products of this treatment were phage capsid-DNA complexes in which the genetical left-hand end of the DNA appeared to be ejected first from the distal end of the tail. INTRODUCTION
MATERIALS
AND
METHODS
Very little is known about the way in Bacterial and Phage Strains which DNA is arranged in a bacteriophage Escherchia coli, strain BBy and a wildcapsid. If the pattern of DNA arrangement type T7 phage were kindly supplied by Dr. in each particle is the same, one might Y. Sakaki. expect that DNA would be injected from one definite end upon infection. Thus, Buffers and Media investigation of the polarity of DNA injecT2 buffer contained per liter: Na,HPO,, tion might give some clues to the manner of 3.0 g; KH2POI, 1.5 g; NaCl, 4.0 g; K&SO,, the DNA arrangement and encapsulation. 5.0 g; 10-3moles; CaCl,, MgS0.e Unfortunately, such an investigation is 10-4moles. M9 buffer contained per liter: difficult to carry out, because, except for a Na,HPO,, 5.8 g; KH2POI, 3.0 g; NaCl, 5.0 few bacteriophage strains (T5: Lanni, 1.0 g; CaCl,, 10m4moles; MgCl,, 1968; SP82G: McAllister, 19701, there is no g; NH,Cl, 10m3moles; FeCl,, 10mGmoles. MSCAA memethod available to interrupt the process dium was M9 buffer supplemented with of DNA injection. The difficulty may, how0.4% casamino acids (Difco) and 0.4% gluever, be surmounted by provoking artificial cose. DNA ejection with some reagents. Recently, we (K. S. and H. Uchidal have Purification of Phage Particles developed a method by which phage partiThe lysate of T7 was obtained by lytic cles can be disrupted to form protein-DNA complexes (Saigo and Uchida, 1974). X infection of BBy cultured in MSCAA medium. After removal of cell debris by lowphage particles were converted by this speed centrifugation, T7 phage particles method to either phage capsids in which were purified by a few cycles of high- and the DNA was partially ejected from the low-speed differential centrifugation and distal end of the tail or complexes in which CsCl banding. Purified phage particles the right-hand terminus of the DNA was were stored in T2 buffer. connected to the proximal end of the tail. Escherichia coli phage T5 particles can Phage Disruption also be converted to similar complexes using this method (unpublished results). (i) Phage disruption with formamide and The results of application of this method to EDTA. After 0.16 A,,, unit of purified T7 E. coli phage T7 will be presented in this phage particles were dialyzed against 0.01 communication. M Tris-HCl buffer, pH 7.4, the dialysate 120 Copyright All rights
0 1975 by Academic Press. Inc. of reproduction in any form reserved.
DNA
EJECTION
was treated with 40(50)% formamide at pH 7.0 for 1 min at room temperature. Then, the mixture was again dialyzed against 0.1 M EDTA in 0.02 M Tris-HCl, pH 8.2, for more than 3 hr at 4”. As observed under an electron microscope, more than 80% of the input particles were converted to particles with empty heads. (ii) Phage disruption with NaCt0,. T7 phage particles were disrupted by NaClO, according to the method of Freifelder (1965). One hundred microliters of 0.01 M sodium POI, pH 7.8, containing 0.001 M sodium EDTA and 300 ~1 cf 7.4 M NaClO, solution in 0.002 M sodium EDTA, pH 7.5, were added to 100 ~1 of the phage suspension in 0.01 M Tris-HCl buffer (pH 7.4). The mixture was maintained at room temperature for 5 min and then dialyzed against Tris-HCl, pH 7.4, for more than 3 hr at 4”. Electron microscopic observation using the Kleinschmidt technique (see Electron Microscopy) revealed no association between DNA and the phage capsid and, furthermore, the existence of tails or taillike protrusions in about one-third of the phage capsids.
121
IN T7
by chilling to 0” and 90 ~1 of cold 1 M ammonium acetate was added to the reaction mixture. Specimens for electron microscopy were prepared by the methods indicated below. Electron Microscopy (i) Negatiue staining. Details of the procedure have already been described elsewhere (Saigo and Uchida, 1974). (ii) Kleinschmidt procedure. The procedure was essentially the same as that described by Davis et al. (1971), and its modification has already been described (Saigo and Uchida, 1974). The spreading solution contained 0.08 A,,, unit of DNA or disrupted phage particles, 0.01% cytochrome c and 0.5 M ammonium acetate, while the hypophase was 0.25 M ammonium acetate. In the present studies, we used pentan-l-01 in place of isopentane when DNA was stained with many1 acetate. RESULTS
Production of Phage Particles Containing Ejected DNA
T7 phage particles, like X, were disrupted when treated with formamide, followed by dialysis against buffer containing The in vitro transcription of T7 DNA by EDTA, although the precise experimental E. coli RNA polymerase was performed by conditions were somewhat different. About the procedures described by Davis and 80% of the input wild-type T7 particles were converted to empty-headed particles, Hyman (1970). A suspension of disrupted T7 phage particles (see Phage Disruption as observed under an electron microscope (i)) was dialyzed against 0.05 M Tris-HCl, after negative staining with uranyl acetate pH 7.9, containing 150 mM KC1 and 1 mM (Fig. 1). Observation by the Kleinschmidt techEDTA. The following reagents were then added to 50 /*l of the dialysate; 5 ~1 of 20 nique of the disruption products revealed mM dithiothreitol and 10 mM EDTA, 5 ~1 the presence of the following two types of of 6 mM dATP, 10 ~1 of 3 mM dCTP, phage capsid-DNA complexes; A complex dGTP and dTTP, and 10 ~1 of E. coli in which only one DNA fiber was protrudRNA-polymerase (80 pg/ml). Escherichia ing from the phage capsid, type I complex coli RNA-polymerase purified by the pro(Fig. 2) and a complex in which two DNA cedures of Chamberlin and Berg (1962) was fibers appeared to be protruding from the kindly supplied by Dr. S. Higuchi and was phage capsid, type II complex. These two stored in a liquid-nitrogen refrigerator until types of complexes together represented use. After the mixture was preincubated 20% of the input particles, while the refor 1 min at 25”, the reaction was started maining 80% were ghosts (phage particles by the addition of 10 ~1 of 0.1 M MgCl, without DNA) or morphologically intact and maintained at 34”. At 2.5 min after ad- phage particles. In the case of T7, unlike dition of MgCl,, the reaction was stopped lambda, we could detect no tail-DNA
In vitro Transcription Polymerase
with
E. coli RNA-
122
FIG. 1. Negatively fo1 .mamide-EDTA-treated de, scribed in Materials
KAORU
stained
phage preparations. T7 phage particles. and Methods.
SAIGO
The bar is 0.5 Mm. (a), Nontreated T7 phage particles; (b), About 2 x 10” particles/ml were disrupted by the procedures
DNA
EJECTION
IN T7
123
FIG. 2. Typical examples of the type I complexes. (a) About 3.5 pm of DNA appears to be ejected from the phage capsid. The arrow indicates the phage capsid. The bar is 0.3 pm. (b) Nearly the entire DNA (12.0 pm) appears to be ejected from the phage capsid. The arrow indicates the phage capsid. The bar is 0.5 km. (c) Enlarged photograph of the phage capsid-DNA connection site in the complex shown in (a). The arrow shows the short tail of T7. The bar is 0.2 pm.
complexes in which one end of the DNA was connected to the proximal end of the tail. Some Properties of the Complexes The type I complexes constituted S5% of the total phage capsid-DNA
about corn-
plexes. The lengths of DNA in the type I complexes were distributed as shown in Fig. 3. About 45% of the tvne I comnlexes seem to have DNA partially ejected, because the DNA ejected from the phage capsid was shorter than the entire length of T7-DNA (12.3 h 0.2 pm), and, under the
124
KAORU
DNA
5n.nrln. LENGTH
10
(urn)
FIG. 3 The length distribution of DNA ejected from the Fihage capsid. Phase particles treated with 50% formamide were disrupted by the procedures described in Materials and Methods: Histograms were made by measuring the length of DNA in 39 randomly selected type I complexes and 104 randomly selected phage capsid-free DNA molecules. The distribution of the former is shown by solid lines and the latter by dotted lines.
present conditions, less than 1% of free DNA was broken (Fig. 3). On the other hand, in the remaining 55% of the type I complexes, the length of DNA ejected was 12.3 i 0.2 pm and seemed to be identical to the full length of T7-DNA within the limits of error. This may suggest that the terminal segment of DNA associates with some sites in a phage capsid even after nearly the whole DNA has been released. In order to clarify the site of DNA protrusion on the surface of the phage capsid, the manner of connection between the apparent end of DNA and the phage capsid was investigated in 105 randomly selected complexes. Although the tail structure could not be identified in 22 complexes, possibly because of the shortness of the T7 tail (Fig. la) (Fraser and Williams, 1953), in 78 out of the remaining 83 complexes, one end of the DNA was apparently connected to the distal end of the tail (Fig. 2~). It is known that T7-DNA is completely dissociated from the phage capsid by treating the phage with NaClO, (Serwer, 1974). When T7 particles were treated with NaClO, [see Phuge Disruption (ii) ] and subsequently with formamide
SAIGO
and EDTA [see Phuge Disruption (i) ] no attachment of DNA could be detected in 100 randomly selected phage capsids. Therefore, it is unlikely that the type I complex was formed by reassociation between the phage capsid-free DNA and the DNA-free phage capsid. From these results, we conclude that in almost all of the type I complexes, DNA was ejected partially or nearly completely through the tail. In the type II complexes, the phage capsids were associated with various regions of the DNA, and the length of DNA protruding from the phage capsid appeared to be nearly equal to or somewhat shorter than the full length of T7-DNA. Since, under the conditions employed, the DNA does not appear to be fragmented, this might indicate that a part of the entire DNA is entrapped in the phage capsid at least in some fraction of the complexes. Structures similar to the type II complexes were also found by Serwer (1974) after heat disruption of T7 phage particles. Ejection
of the Left-Hand
Terminus
In order to determine which end of the DNA was ejected first from the phage capsid, RNA was synthesized by the in vitro RNA-polymerase reaction using ejected T7-DNA as a template, and the distribution of RNA bushes was examined. Since only DNA in the vicinity of the genetical left-hand end of the T7-DNA is known to be transcribed in vitro by the RNA-polymerase after a short incubation (Davis and Hyman, 1970), we can readily discriminate between the left-hand and the right-hand ends of the DNA. Under the conditions employed, about 99% of the DNA molecules were transcribed, while 16% of them still had the phage capsid at one of the two apparent ends. In 82 randomly selected complexes, the major clusters of RNA bushes were always observed to be located in the vicinity of the opposite extremity of DNA with respect to the phage capsid-attached end. Figure 4 shows typical examples having DNA of various lengths. Figure 5 shows maps of RNA bushes in five free DNA molecules, ten randomly selected type I complexes and five type I com-
DNA
EJECTION
IN T7
FIG. 4. Typical examples of the type I complexes with RNA bushes. The length of the DNA follows: (a), 10.4 pm; (b), 6.1 pm; (c), 2.9 Frn; (d), 2.1 pm; (e), 1.2 pm; (f), 0.7 pm. Open arrowheads phage capsids, while filled arrowheads indicate the clusters of RNA bushes. ‘l’he bar is 0.5 pm.
plexes with a short length of DNA ejected. The main clusters of RNA bushes always appeared to be located within the region of 2.5 pm from the left-hand end of DNA
125
ejected is as indicate the
regardless of the phage capsid attachment. Thus, it is concluded that it is the lefthand end of T7-DNA that is ejected first through the tail.
126 0 ,
1 *
KAORU
2 n
DNA 3 4 1’
LENGTH 5 6 7 “1
(urn) 8 9 11
10 11 12 13 l4 0 “1,
cul”
SAIGO
proposed for bacteriophage lambda (Padmanabhan et al., 1972). In the case of X, it was shown that the right-hand terminus of DNA was at the tail-head attachment site (or slightly inserted into the tail) (Padmanabhan et al., 1972; Saigo and Uchida, 1974; Chattoraj and Inman, 1974; Thomas, 1974) and that the right-hand terminus of DNA was ejected first through the tail (Thomas, 1974). By analogy with lambda, we presume that the left-hand end of T7DNA is positioned at the tail-head attachment site in the T7 particle. Escherichia coli bacteriophage T5 and a Bacillus subtilis bacteriophage, SP82G, may also belong to the same category as h and T7, at least as based on the DNA arrangement in the particle. In such phages, DNA was shown by biochemical and genetic tests to be injected from its one definite end upon infection (T5: Labedan et al., 1973; SP82G: McAllister, 1970). ACKNOWLEDGMENTS
Fm. 5. Transcriptional maps of the type I complexes and free DNA molecules. The maps of five free DNA, ten randomly selected type I complexes and five type I complexes having short DNA ejected are arranged from top to bottom. They are aligned so that the cluster of RNA bushes is at the left. The position of the phage capsid is indicated by C, while that of the RNA bushes is shown by the small vertical bars on the DNA. DISCUSSION
T7 phage particles were disrupted by treating with formamide, followed by dialysis against buffer containing EDTA. Electron microscopic observations strongly suggested that T7-DNA was ejected uniquely beginning with its genetical left-hand end, although the possibility could not be completely eliminated that, in some fraction of the T7 particles, DNA was ejected first from its right-hand end and then dissociated from the phage tail. Pao and Speyer (1973) also deduced the same order of T7-DNA ejection by marker rescue experiments using X-ray-damaged T7 particles. The finding that one definite end of T7-DNA is ejected first supports the idea that the arrangement of DNA is identical in all T7 particles. A similar idea has been
I thank Dr. T. Miyake for encouragement and critical discussion and Dr. K. Yanagisawa for reading the manuscript. I am grateful to Dr. Y. Sakaki and Dr. S. Higuchi for kind gifts of T7 phage and purified RNA polymerase, respectively. REFERENCES M., and BERG, P. (19621. Deoxyribonucleic acid-directed synthesis of ribonucleic acid by an enzyme from Escherichia co/i. Proc. Nat. Acad. Sci. USA 48, 81-93. CHATTORAJ, D. K., and INMAN, R. B. (1974). Location of DNA ends in P2, 184, P4 and lambda bacteriophage heads. J. Mol. Biol. 87, 11-22. DAVIS, R. W., and HYMAN, R. W. (19701. Physical locations of the in vitro RNA initiation site and termination sites of T7M DNA. Cold Spring Harbor Symp. Quant. Biol. 35, 269-281. DAVIS, R. W., SIMON, M., and DAVIDSON, N. (1971). Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan. eds.1, Vol. 21D, pp. 413-428. Academic Press, New York. FRASER, D., and WILLIAMS, R. C. (1953). Details of frozen-dried T3 and T7 bacteriophages as shown by electron microscopy. J. Bacterial. 65, 167-170. FREIFELDER, D. (1965) A novel method for the release of bacteriophage DNA. Biochem. Biophys. Res. Commun. 18, 141-144. LABEDAN, B., CROCHET, M., LEGACLT-DEMARE, J., and STEVENS, B. J. (1973). Location of the first step CHAMBERLIN,
DNA
EJECTION
transfer fragment and single-strand interruptions in T5stO bacteriophage DNA. J. Mol. Biol. 75, 213-234. LANNI, Y. T. (1968). First-step-transfer deoxyribonucleic acid of bacteriophage T5. Bacterial. Reel. 32, 2277242. MCALLISTER, W. T. (1970). Bacteriophage infection: Which end of the SP82G genome goes in first? J. Viral. 5, 194-198. PADMANABHAN, R., Wu, R., and BODE, V. C. (19721. Arrangement of DNA in lambda bacteriophage heads. III. Localization and number of nucleotides cleaved from X DNA by micrococcal nuclease at-
IN T7
127
tack on heads. J. Mol. Biol. 69, 201-207. PAO, C.-C., and SPEYER, J. F. (1973). Order of injection of T7 bacteriophage DNA. J. Viral. 11, 1024-1026. SAIGO, K., and UCHIDA, H. (1974). Connection of the right-hand terminus of DNA to the proximal end of the tail in bacteriophage lambda. Virolog,y 61, 524-536. SERWER, P. (1974). Complexes between bacteriophage T7 capsids and T7 DNA. Virolog.v 59, 89-107. THOMAS, J. 0. (1974). Chemical linkage of the tail to the right-hand end of bacteriophage lambda DNA. J. Mol. Biol. 87, l-9.
65,129-127
(1975)
Polar DNA Ejection
Mitsubishi-Kasei
Institute
of Life
in Bacteriophage
KAORU
SAIGO
Sciences,
11, Minamiooya,
Accepted
January
T7
Machida-shi,
Tokyo,
Japan
3, 1975
T7 phage particles were disrupted by treatment with 40% formamide followed by dialysis against EDTA and were analyzed using an electron microscope. About 20% of the products of this treatment were phage capsid-DNA complexes in which the genetical left-hand end of the DNA appeared to be ejected first from the distal end of the tail. INTRODUCTION
MATERIALS
AND
METHODS
Very little is known about the way in Bacterial and Phage Strains which DNA is arranged in a bacteriophage Escherchia coli, strain BBy and a wildcapsid. If the pattern of DNA arrangement type T7 phage were kindly supplied by Dr. in each particle is the same, one might Y. Sakaki. expect that DNA would be injected from one definite end upon infection. Thus, Buffers and Media investigation of the polarity of DNA injecT2 buffer contained per liter: Na,HPO,, tion might give some clues to the manner of 3.0 g; KH2POI, 1.5 g; NaCl, 4.0 g; K&SO,, the DNA arrangement and encapsulation. 5.0 g; 10-3moles; CaCl,, MgS0.e Unfortunately, such an investigation is 10-4moles. M9 buffer contained per liter: difficult to carry out, because, except for a Na,HPO,, 5.8 g; KH2POI, 3.0 g; NaCl, 5.0 few bacteriophage strains (T5: Lanni, 1.0 g; CaCl,, 10m4moles; MgCl,, 1968; SP82G: McAllister, 19701, there is no g; NH,Cl, 10m3moles; FeCl,, 10mGmoles. MSCAA memethod available to interrupt the process dium was M9 buffer supplemented with of DNA injection. The difficulty may, how0.4% casamino acids (Difco) and 0.4% gluever, be surmounted by provoking artificial cose. DNA ejection with some reagents. Recently, we (K. S. and H. Uchidal have Purification of Phage Particles developed a method by which phage partiThe lysate of T7 was obtained by lytic cles can be disrupted to form protein-DNA complexes (Saigo and Uchida, 1974). X infection of BBy cultured in MSCAA medium. After removal of cell debris by lowphage particles were converted by this speed centrifugation, T7 phage particles method to either phage capsids in which were purified by a few cycles of high- and the DNA was partially ejected from the low-speed differential centrifugation and distal end of the tail or complexes in which CsCl banding. Purified phage particles the right-hand terminus of the DNA was were stored in T2 buffer. connected to the proximal end of the tail. Escherichia coli phage T5 particles can Phage Disruption also be converted to similar complexes using this method (unpublished results). (i) Phage disruption with formamide and The results of application of this method to EDTA. After 0.16 A,,, unit of purified T7 E. coli phage T7 will be presented in this phage particles were dialyzed against 0.01 communication. M Tris-HCl buffer, pH 7.4, the dialysate 120 Copyright All rights
0 1975 by Academic Press. Inc. of reproduction in any form reserved.
DNA
EJECTION
was treated with 40(50)% formamide at pH 7.0 for 1 min at room temperature. Then, the mixture was again dialyzed against 0.1 M EDTA in 0.02 M Tris-HCl, pH 8.2, for more than 3 hr at 4”. As observed under an electron microscope, more than 80% of the input particles were converted to particles with empty heads. (ii) Phage disruption with NaCt0,. T7 phage particles were disrupted by NaClO, according to the method of Freifelder (1965). One hundred microliters of 0.01 M sodium POI, pH 7.8, containing 0.001 M sodium EDTA and 300 ~1 cf 7.4 M NaClO, solution in 0.002 M sodium EDTA, pH 7.5, were added to 100 ~1 of the phage suspension in 0.01 M Tris-HCl buffer (pH 7.4). The mixture was maintained at room temperature for 5 min and then dialyzed against Tris-HCl, pH 7.4, for more than 3 hr at 4”. Electron microscopic observation using the Kleinschmidt technique (see Electron Microscopy) revealed no association between DNA and the phage capsid and, furthermore, the existence of tails or taillike protrusions in about one-third of the phage capsids.
121
IN T7
by chilling to 0” and 90 ~1 of cold 1 M ammonium acetate was added to the reaction mixture. Specimens for electron microscopy were prepared by the methods indicated below. Electron Microscopy (i) Negatiue staining. Details of the procedure have already been described elsewhere (Saigo and Uchida, 1974). (ii) Kleinschmidt procedure. The procedure was essentially the same as that described by Davis et al. (1971), and its modification has already been described (Saigo and Uchida, 1974). The spreading solution contained 0.08 A,,, unit of DNA or disrupted phage particles, 0.01% cytochrome c and 0.5 M ammonium acetate, while the hypophase was 0.25 M ammonium acetate. In the present studies, we used pentan-l-01 in place of isopentane when DNA was stained with many1 acetate. RESULTS
Production of Phage Particles Containing Ejected DNA
T7 phage particles, like X, were disrupted when treated with formamide, followed by dialysis against buffer containing The in vitro transcription of T7 DNA by EDTA, although the precise experimental E. coli RNA polymerase was performed by conditions were somewhat different. About the procedures described by Davis and 80% of the input wild-type T7 particles were converted to empty-headed particles, Hyman (1970). A suspension of disrupted T7 phage particles (see Phage Disruption as observed under an electron microscope (i)) was dialyzed against 0.05 M Tris-HCl, after negative staining with uranyl acetate pH 7.9, containing 150 mM KC1 and 1 mM (Fig. 1). Observation by the Kleinschmidt techEDTA. The following reagents were then added to 50 /*l of the dialysate; 5 ~1 of 20 nique of the disruption products revealed mM dithiothreitol and 10 mM EDTA, 5 ~1 the presence of the following two types of of 6 mM dATP, 10 ~1 of 3 mM dCTP, phage capsid-DNA complexes; A complex dGTP and dTTP, and 10 ~1 of E. coli in which only one DNA fiber was protrudRNA-polymerase (80 pg/ml). Escherichia ing from the phage capsid, type I complex coli RNA-polymerase purified by the pro(Fig. 2) and a complex in which two DNA cedures of Chamberlin and Berg (1962) was fibers appeared to be protruding from the kindly supplied by Dr. S. Higuchi and was phage capsid, type II complex. These two stored in a liquid-nitrogen refrigerator until types of complexes together represented use. After the mixture was preincubated 20% of the input particles, while the refor 1 min at 25”, the reaction was started maining 80% were ghosts (phage particles by the addition of 10 ~1 of 0.1 M MgCl, without DNA) or morphologically intact and maintained at 34”. At 2.5 min after ad- phage particles. In the case of T7, unlike dition of MgCl,, the reaction was stopped lambda, we could detect no tail-DNA
In vitro Transcription Polymerase
with
E. coli RNA-
122
FIG. 1. Negatively fo1 .mamide-EDTA-treated de, scribed in Materials
KAORU
stained
phage preparations. T7 phage particles. and Methods.
SAIGO
The bar is 0.5 Mm. (a), Nontreated T7 phage particles; (b), About 2 x 10” particles/ml were disrupted by the procedures
DNA
EJECTION
IN T7
123
FIG. 2. Typical examples of the type I complexes. (a) About 3.5 pm of DNA appears to be ejected from the phage capsid. The arrow indicates the phage capsid. The bar is 0.3 pm. (b) Nearly the entire DNA (12.0 pm) appears to be ejected from the phage capsid. The arrow indicates the phage capsid. The bar is 0.5 km. (c) Enlarged photograph of the phage capsid-DNA connection site in the complex shown in (a). The arrow shows the short tail of T7. The bar is 0.2 pm.
complexes in which one end of the DNA was connected to the proximal end of the tail. Some Properties of the Complexes The type I complexes constituted S5% of the total phage capsid-DNA
about corn-
plexes. The lengths of DNA in the type I complexes were distributed as shown in Fig. 3. About 45% of the tvne I comnlexes seem to have DNA partially ejected, because the DNA ejected from the phage capsid was shorter than the entire length of T7-DNA (12.3 h 0.2 pm), and, under the
124
KAORU
DNA
5n.nrln. LENGTH
10
(urn)
FIG. 3 The length distribution of DNA ejected from the Fihage capsid. Phase particles treated with 50% formamide were disrupted by the procedures described in Materials and Methods: Histograms were made by measuring the length of DNA in 39 randomly selected type I complexes and 104 randomly selected phage capsid-free DNA molecules. The distribution of the former is shown by solid lines and the latter by dotted lines.
present conditions, less than 1% of free DNA was broken (Fig. 3). On the other hand, in the remaining 55% of the type I complexes, the length of DNA ejected was 12.3 i 0.2 pm and seemed to be identical to the full length of T7-DNA within the limits of error. This may suggest that the terminal segment of DNA associates with some sites in a phage capsid even after nearly the whole DNA has been released. In order to clarify the site of DNA protrusion on the surface of the phage capsid, the manner of connection between the apparent end of DNA and the phage capsid was investigated in 105 randomly selected complexes. Although the tail structure could not be identified in 22 complexes, possibly because of the shortness of the T7 tail (Fig. la) (Fraser and Williams, 1953), in 78 out of the remaining 83 complexes, one end of the DNA was apparently connected to the distal end of the tail (Fig. 2~). It is known that T7-DNA is completely dissociated from the phage capsid by treating the phage with NaClO, (Serwer, 1974). When T7 particles were treated with NaClO, [see Phuge Disruption (ii) ] and subsequently with formamide
SAIGO
and EDTA [see Phuge Disruption (i) ] no attachment of DNA could be detected in 100 randomly selected phage capsids. Therefore, it is unlikely that the type I complex was formed by reassociation between the phage capsid-free DNA and the DNA-free phage capsid. From these results, we conclude that in almost all of the type I complexes, DNA was ejected partially or nearly completely through the tail. In the type II complexes, the phage capsids were associated with various regions of the DNA, and the length of DNA protruding from the phage capsid appeared to be nearly equal to or somewhat shorter than the full length of T7-DNA. Since, under the conditions employed, the DNA does not appear to be fragmented, this might indicate that a part of the entire DNA is entrapped in the phage capsid at least in some fraction of the complexes. Structures similar to the type II complexes were also found by Serwer (1974) after heat disruption of T7 phage particles. Ejection
of the Left-Hand
Terminus
In order to determine which end of the DNA was ejected first from the phage capsid, RNA was synthesized by the in vitro RNA-polymerase reaction using ejected T7-DNA as a template, and the distribution of RNA bushes was examined. Since only DNA in the vicinity of the genetical left-hand end of the T7-DNA is known to be transcribed in vitro by the RNA-polymerase after a short incubation (Davis and Hyman, 1970), we can readily discriminate between the left-hand and the right-hand ends of the DNA. Under the conditions employed, about 99% of the DNA molecules were transcribed, while 16% of them still had the phage capsid at one of the two apparent ends. In 82 randomly selected complexes, the major clusters of RNA bushes were always observed to be located in the vicinity of the opposite extremity of DNA with respect to the phage capsid-attached end. Figure 4 shows typical examples having DNA of various lengths. Figure 5 shows maps of RNA bushes in five free DNA molecules, ten randomly selected type I complexes and five type I com-
DNA
EJECTION
IN T7
FIG. 4. Typical examples of the type I complexes with RNA bushes. The length of the DNA follows: (a), 10.4 pm; (b), 6.1 pm; (c), 2.9 Frn; (d), 2.1 pm; (e), 1.2 pm; (f), 0.7 pm. Open arrowheads phage capsids, while filled arrowheads indicate the clusters of RNA bushes. ‘l’he bar is 0.5 pm.
plexes with a short length of DNA ejected. The main clusters of RNA bushes always appeared to be located within the region of 2.5 pm from the left-hand end of DNA
125
ejected is as indicate the
regardless of the phage capsid attachment. Thus, it is concluded that it is the lefthand end of T7-DNA that is ejected first through the tail.
126 0 ,
1 *
KAORU
2 n
DNA 3 4 1’
LENGTH 5 6 7 “1
(urn) 8 9 11
10 11 12 13 l4 0 “1,
cul”
SAIGO
proposed for bacteriophage lambda (Padmanabhan et al., 1972). In the case of X, it was shown that the right-hand terminus of DNA was at the tail-head attachment site (or slightly inserted into the tail) (Padmanabhan et al., 1972; Saigo and Uchida, 1974; Chattoraj and Inman, 1974; Thomas, 1974) and that the right-hand terminus of DNA was ejected first through the tail (Thomas, 1974). By analogy with lambda, we presume that the left-hand end of T7DNA is positioned at the tail-head attachment site in the T7 particle. Escherichia coli bacteriophage T5 and a Bacillus subtilis bacteriophage, SP82G, may also belong to the same category as h and T7, at least as based on the DNA arrangement in the particle. In such phages, DNA was shown by biochemical and genetic tests to be injected from its one definite end upon infection (T5: Labedan et al., 1973; SP82G: McAllister, 1970). ACKNOWLEDGMENTS
Fm. 5. Transcriptional maps of the type I complexes and free DNA molecules. The maps of five free DNA, ten randomly selected type I complexes and five type I complexes having short DNA ejected are arranged from top to bottom. They are aligned so that the cluster of RNA bushes is at the left. The position of the phage capsid is indicated by C, while that of the RNA bushes is shown by the small vertical bars on the DNA. DISCUSSION
T7 phage particles were disrupted by treating with formamide, followed by dialysis against buffer containing EDTA. Electron microscopic observations strongly suggested that T7-DNA was ejected uniquely beginning with its genetical left-hand end, although the possibility could not be completely eliminated that, in some fraction of the T7 particles, DNA was ejected first from its right-hand end and then dissociated from the phage tail. Pao and Speyer (1973) also deduced the same order of T7-DNA ejection by marker rescue experiments using X-ray-damaged T7 particles. The finding that one definite end of T7-DNA is ejected first supports the idea that the arrangement of DNA is identical in all T7 particles. A similar idea has been
I thank Dr. T. Miyake for encouragement and critical discussion and Dr. K. Yanagisawa for reading the manuscript. I am grateful to Dr. Y. Sakaki and Dr. S. Higuchi for kind gifts of T7 phage and purified RNA polymerase, respectively. REFERENCES M., and BERG, P. (19621. Deoxyribonucleic acid-directed synthesis of ribonucleic acid by an enzyme from Escherichia co/i. Proc. Nat. Acad. Sci. USA 48, 81-93. CHATTORAJ, D. K., and INMAN, R. B. (1974). Location of DNA ends in P2, 184, P4 and lambda bacteriophage heads. J. Mol. Biol. 87, 11-22. DAVIS, R. W., and HYMAN, R. W. (19701. Physical locations of the in vitro RNA initiation site and termination sites of T7M DNA. Cold Spring Harbor Symp. Quant. Biol. 35, 269-281. DAVIS, R. W., SIMON, M., and DAVIDSON, N. (1971). Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan. eds.1, Vol. 21D, pp. 413-428. Academic Press, New York. FRASER, D., and WILLIAMS, R. C. (1953). Details of frozen-dried T3 and T7 bacteriophages as shown by electron microscopy. J. Bacterial. 65, 167-170. FREIFELDER, D. (1965) A novel method for the release of bacteriophage DNA. Biochem. Biophys. Res. Commun. 18, 141-144. LABEDAN, B., CROCHET, M., LEGACLT-DEMARE, J., and STEVENS, B. J. (1973). Location of the first step CHAMBERLIN,
DNA
EJECTION
transfer fragment and single-strand interruptions in T5stO bacteriophage DNA. J. Mol. Biol. 75, 213-234. LANNI, Y. T. (1968). First-step-transfer deoxyribonucleic acid of bacteriophage T5. Bacterial. Reel. 32, 2277242. MCALLISTER, W. T. (1970). Bacteriophage infection: Which end of the SP82G genome goes in first? J. Viral. 5, 194-198. PADMANABHAN, R., Wu, R., and BODE, V. C. (19721. Arrangement of DNA in lambda bacteriophage heads. III. Localization and number of nucleotides cleaved from X DNA by micrococcal nuclease at-
IN T7
127
tack on heads. J. Mol. Biol. 69, 201-207. PAO, C.-C., and SPEYER, J. F. (1973). Order of injection of T7 bacteriophage DNA. J. Viral. 11, 1024-1026. SAIGO, K., and UCHIDA, H. (1974). Connection of the right-hand terminus of DNA to the proximal end of the tail in bacteriophage lambda. Virolog,y 61, 524-536. SERWER, P. (1974). Complexes between bacteriophage T7 capsids and T7 DNA. Virolog.v 59, 89-107. THOMAS, J. 0. (1974). Chemical linkage of the tail to the right-hand end of bacteriophage lambda DNA. J. Mol. Biol. 87, l-9.
