Tail-DNA connection and chromosome structure in bacteriophage T5

VIROLOGY

68, 154-165 (1975)

Tail-DNA

Connection

and Chromosome

in Bacteriophage KAORU Mitsubishi-Kasei

Institute

Structure

T5

SAIGO

of Life Sciences, II, Minamiooya,

Machida-xhi,

Tokyo, Japan

Accepted June 26, 197.5 Most T5 phage particles were converted to tail-DNA or ghost-DNA complexes by dialyzing against EDTA or heating in the presence of sodium dodecyl sulfate. In these complexes, one of the apparent DNA ends was connected to the proximal end of the tail. Using the tail (or the ghost) located at one of the DNA ends as a marker, a precise map of shearsensitive points on TBDNA was constructed. From the map positions of shear-sensitive points, the DNA end connected to the proximal end of the tail seems to be the one that is injected first into bacteria upon infection. In the disruption products obtained after formamide-EDTA treatment, two types of particles with partially ejected DNA were found. By analyzing such products it is suggested that the polarity of artificial DNA ejection through the tail is the same as that of injection upon infection, and that artificial DNA ejection through the tail is preferentiallv interrunted at or near the shear-sensitive points on the DNA. INTRODUCTION

In bacteriophage T5, the DNA is known to be injected from its one definite end upon infection (Labedan et al., 1973). Similar polar DNA injection or ejection also has been shown in a few other bacteriophages (SP82G: McAllister, 1970; lambda: Thomas, 1974; T7: Saigo, 1975a). On the other hand, by analyzing phage particles disrupted in various ways, connection of the tail to one definite end of the DNA has been found in several bacteriophages having DNA with cohesive ends (X+: Saigo and Uchida, 1974; Chattoraj and Inman, 1974; Thomas, 1974; 186, P2, P4: Chattoraj and Inman, 1974; d-80, lambda deletion and insertion mutants, Xb2b5 and Xs.d.: the present author, unpublished results). It has been suggested that lambda-DNA is connected to the proximal end of the tail within the particle (Saigo and Uchida, 1974) and is ejected through the tail beginning with the end to which the proximal end of the tail is apparently connected (Thomas, 1974). Thus, also in other bacteriophages having DNA with cohesive ends, DNA might be ejected from its tail-attached end.

In this communication, we present evidence demonstrating that when T&DNA. which does not contain cohesive ends but has a large terminal repetition (Rhoades and Rhoades, 1972), is packaged in the particle, one of the DNA ends is connected to the proximal end of the tail, and that the DNA is injected into bacteria upon infection from its tail-attached end. Furthermore, using such a specific connection of the tail to one definite end of the DNA, it has been possible to construct a more precise map of shear-sensitive points on the DNA. MATERIALS

AND

METHODS

Bacterial and phage strains. Escherichia coli, strain Ymell and a wild-type phage T5 were kindly supplied by Dr. A. Tsugita and Dr. K. Mizobuchi, respectively. Buffers and media. TB medium contains 1% bactotryptone (Difco) and 0.5% NaCl. Unless otherwise mentioned, the concentration and pH of Tris-HCl buffer are 0.01 M and 7.4, respectively. Tris/EDTA buffer consists of 0.1 M EDTA and 0.01 M Trissolution con- HCl, pH 8.2. NaCl/EDTA 154

Copyright 0 1975by Academic Press, Inc. All rights of reproduction

in any form reserved

TAIL-DNA

CONNECTION

sists of 0.02 M NaCl and 0.005 M EDTA, pH 7.4. Purification of phage particles. The T5 phage lysate was obtained by lytic infeotion of Ymell cultured in TB medium supplemented with 0.001 M CaCl,. After removal of cell debris, phage particles were purified by a few cycles of high- low-speed differential centrifugation and CsCl banding. Purified phage particles were stored in Tris-HCl buffer supplemented with 0.01 M MgCl,. Phage disruption. (i) Disruption with EDTA. Eight A,,, units of T5 phage stock solution was diluted with a nine times volume of Tris-HCl buffer and then dialyzed against Tris/EDTA buffer of 3 hr at 4”. Electron microscopic observation using Kleinschmidt procedures showed that about two-thirds of the input particles were disrupted. (ii) Disruption ulith sodium dodecyl sulfate (SDS). Eight A,,, units of T5 phage stock solution was diluted with a nine times volume of NaCl/ EDTA solution and dialyzed against an NaCl/EDTA solution at 4°C for 3 hr. After addition of 0.2% SDS to the dialysate, the mixture was heated at 60°C for 3 min and then chilled to 0°C. The SDS was removed by dialyzing for several hours at room temperature. After these treatments, almost no morphologically intact particles remained. (iii) Disruption with formamide and EDTA. Basic procedures of phage disruption with formamide and EDTA were the same as that described elsewhere (Saigo, 1975a; Saigo and Uchida, 1974). Eight tenth A,,, units of T5 phage suspension in Tris-HCl was treated with 50% formamide at room temperature for 1 min and then dialyzed against Tris/EDTA buffer at 4°C for more than 3 hr. Electron microscopic observation using negative staining showed that most of the input particles were disrupted by this treatment. Hydrod.ynamic shear breakage of DNA. Phage disruption products, whose concentration was 0.8 to 0.3 A,,, units in 0.01 M EDTA in Tris-HCl buffer or in NaCl/ EDTA solution, were mildly sheared by sucking and blowing with a needle (No. 21 gauge) 10 times at 4°C. Electron microscop>,. (i) Negative

155

IN T5

staining. The detailed procedures have already been described elsewhere (Saigo and Uchida, 1974). (ii) Kleinschmidt procedure. The detailed procedures have already been described elsewhere (Saigo and Uchida, 1974). The spreading solution consisted of 0.01%’ cytochrome c, 0.5 M ammonium acetate, and 0.08 to 0.008 A,,, units of DNA or disrupted phage particles while the hypophase was 0.25 M ammonium acetate. RESULTS

Production of ghost-DNA and tail-DNA complexes. Most of the input T5 phage particles were disrupted either by dialyzing against EDTA (see phage disruption (i)) or by heating in the presence of SDS (see phage disruption (ii)). Table 1 shows the fractions of the disrupted products observed under an electron microscope using the Kleinschmidt technique. It also shows that without EDTA or SDS treatment almost all parts of the input particles were not disrupted by the Kleinschmidt technique. When T5 phage particles were disrupted with EDTA, about 80% of the disrupted particles were scored as ghostDNA complexes in which one of the apparent DNA ends appeared to be associated with the head part of the ghost (Fig. 1). Under the present conditions, the full length of the TS-DNA was 36.7 * 0.6 pm, while the length of DNA protruding from the head was also 36.7 * 0.4 pm (Fig. 2A). Therefore, nearly the entire length of T5-DNA seemed to be protruding from the head. Another type of ghost-DNA complex was found rarely, in which ghosts appeared to attach to the DNA at points other than at DNA ends (Table 1B). Small fractions of the tail-DNA complexes (Fig. 3B, Table 1) also were recognized among the disruption products obtained after EDTA-treatment. In all tail-DNA complexes, nearly the full length of the T5DNA was apparently connected to the tail proximal end (identifiable as the end lacking fine tail fibers (Figs. 3A, and 3B)). Similar tail-DNA complexes also were produced by heating T5 phage particles in the presence of SDS (Fig. 3C, Table 1). In this case, no ghost&DNA complexes could

156

KAORU TABLE

SAIGO 1

GOMPONENTS IN DISRI’PTIONPKODI~CTS (A) Relative

distribution

of tail-containing disruption Expt A” 56 (0.30) 11 (0.06) 0 (0.00) 103 (0.55) 6 (0.03) 1 (0.01) 9 (0.05) 0 (0.00)

Morphologically intact particle Ghost (DNA-free) Isolated taild End-to-end type complex (ghost-DNA) End-to-end type complex (tail-DNA) Particle ejecting DNA from tail distal end Aggregate (ghost-DNA)’ Aggregate (tail-DNA)’ (B) Relative

distribution

products Expt B”

of disruption

Free DNA DNA attached with a tail at the end DNA attached with a ghost at the end DNA attached with a head capsid at the end DNA attached with a tail at points other than at the ends DNA attached with a ghost at points other than at the ends DNA attached with a head capsid other than at the ends Aggregated DNA associated with a tail Aggregated DNA associated with a ghost Aggregated DNA associated with a head capsid

Expt C’

0 (0.00) 0 (0.00) 94 (0.39) 0 (0.00) 146 (0.61) 0 (0.00) 0 (0.00) 0 (0.00)

products having an extended Expt D’ 11 (0.09) 7 (0.06) 96 (0.77) 0 (0.00) 0 (0.00)

163 10.96) 0 (0.00) 0 (0.00) 2 (0.01) 0 (0.00) 1 (0.01) 3 (0.02) 0 (0.00)

DNA Expt E” 59 (0.32) 119 (0.65) 0 (0.00) 0 (0.00) 5 (0.03)

l(O.01)

0 (0.00)

3 (0.02)

0 (0.00)

0 (0.00) 6 (C.05) 0 (0.00)

0 (0.00) 0 (0.00) 0 (0.00)

“Phage T5 particles were disrupted by dialyzing against EDTA (see phage disruption (i)) and the relative distribution of 186 disrupted products with tails was examined. Fractions of each component are indicated in parentheses. bPhage T5 particles were disrupted by heating in the presence of SDS (see phage disruption (ii)) and the relative distribution of 240 disruption products with tails was examined. Fractions of each component are indicated in parentheses. c Phage particles (0.008 A,,, units in 0.5 M ammonium acetate) were spread on a hypophase of 0.25 M ammonium acetate and the relative distribution of 169 complexes was examined. ‘Together with DNA-free tails, “isolated tail” contains very small fractions of tail-DNA complexes in which the tail appeared to attach to the DNA at points other than at DNA ends. ‘The “aggregate” is a complex in which either a tail or a ghost attaches to the aggregated DNA. ’ Phage T5 particles were disrupted by dialyzing against EDTA (see phage disruption (i)) and the relative distribution of 124 disrupted particles having an extended DNA is indicated. g Phage T5 particles were disrupted by heating in the presence of SDS (see phage disruption (ii)) and the relative distribution of 186 disrupted particles having an extended DNA is indicated.

be found. Since no tail fibers could be detected (possibly because of separation of tail fibers from the tail tube), we could not morphologically determine whether the tail end associated with the DNA was the proximal end or not. However, the results described below (see the next section) strongly suggest that it was. From these results, it is concluded that the vicinity of one of the DNA ends is connected to the

tail proximal end in T5 phage particles, as in lambda (Saigo and Uchida, 1974). Breakage of DNA by hydrodynamic shearing forces. T5-DNA is known to be fragmented at specific points by shearing (Burgi et al., 1966) and each shear-sensitive point on the DNA is fairly well mapped (Hayward, 1974). Thus, using this character of T5-DNA, we can determine the end of the DNA to which the tail or the

TAIL-DNA

FIG. 1. A typical pm.

example of the ghost-DNA

CONNECTION

IN T5

complex. Arrowheads

ghost is connected. Figs. 2B and 4A show the distribution of the fragmented T5-DNA with the tail or the ghost at the end. In the case of the ghost-DNA cornplexes, the length of the main DNA fragments were 4.1, 6.6, 8.3, 17.5, 32.7, and

157

indicate ghosts at the DNA end. Bar. 1.0

64.1% of the full length of the T5-DNA (Fig. 2B), and DNA fragments derived from the EDTA-induced tail-DNA complexes also seemed to distribute in a similar fashion. In the case of SDS-heat induced tail-DNA complexes, the length of

KAORU

1 i

0 DNA

LENGTH

(%)

FIG. 2. (A) Histogram of the length of DNA protruding from the head part in ghost-DNA complexes. In this case, the complexes are not subjected to the procedures of shear breakage (see hydrodynamic shear breakage of DNA under Materials and Methods). Arrowhead a shows 100.0% of the total molecular length of T5-DNA (36.7 pm). (B) Length distribution of DNA protruding from the head part of the ghost-DNA complexes subjected to the procedures of shear breakage. In the present case. DNA fragments with puddlelike structure were excluded. Each arrowhead represents a peak position in the distribution and the positions of arrowheads labeled a, b, c, d, e, f, and g are 4.1, 6.6, 8.3, 17.5, 32.7. 64.1, and 100.0% of the total molecular length of T5-DNA (36.7 pm), respectively. An enlarged graph of the region from 0 to 15% is shown in Fig. 9A.

the main DNA fragments were 8.0, 18.3, 32.5, and 63.9% (Fig. 4A). Thus, except for short DNA fragments, equal to or less than 6.6%, the profiles of length distribution of the main DNA fragments in SDS-heat induced tail-DNA complexes seemed to be identical to that in the ghost&DNA complexes within the limits of error. These findings support the idea that both the tail and the ghost are associated with the same definite end of T&DNA. In the accom-

SAIGO

panying paper (Saigo, 1975b), this idea will be supported more directly by denaturation mapping of the complex. Therefore, the relative position of each shear-sensitive point and the DNA end associated with the tail (ghost) can be mapped on T5-DNA, as shown in Fig. 5. Additional shear-sensitive points may reside at the positions of 4.1 and 6.6% of the full-length DNA because the fragments of 4.1%’ and 6.67;swere found among the fragments derived from the ghost-DNA complexes. By comparing our map of shear-sensitive points (Fig. 5) to that of Labedan et al. (Fig. 7 in Labedan et al., 1973), the DNA end connected to the tail (or the ghost) is strongly suggested to be identical to the DNA end to be injected into the host bacteria first upon injection. Some other properties of sheared DNA. Fig. 4B shows the length distribution of the tail-free DNA fragments produced by shear-breakage of SDS-heat induced tailDNA complexes. Except for short DNA fragments, almost all of them could be attributable to the fragments expected from the model shown in Fig. 5. Short fragments unexplained by the model were small in weight, though large in number, and might be related to “minor nicks” on the T5-DNA (Hayward and Smith, 1972a). In about 15%’ of the DNA fragments derived from the ghost-DNA complexes, large puddlelike structures were detected either at the end or in the middle of the DNA (Fig. 6) and about 60?# of such fragments are different in length from the main DNA fragments. Since, under the present conditions for DNA visualization, it is known that the single-stranded DNA chain is usually visible as a puddlelike structure (Fuke et al., 1970), such regions found in our preparations may be attributed to either partial denaturation or from which one of the two complementary single-stranded chains is released. Hershey et al. (1963) also suggested that T5-DNA was partially denatured under an appropriate shear condition. However, for better understanding of the physical meaning of such puddlelike structures, further investigation will be required. DNA ejection. When T5 phage particles

TAIL-DNA

CONNECTION

IN T5

159

FIG. 3. (A) Negatively stained T5 particles. The arrowhead indicates the tail fiber at the distal end of the tail. Bar, 0.1 pm. (B) A part of EDTA induced tail-DNA complex. The arrowhead shows fine tail fibers at the distal end of the tail. Bar. 0.2 ym. (C) A part of SDS-heat induced tail-DKA complex. The arrowhead shows the phage tail lacking tail fibers. Bar, 0.2 pm.

were treated with formamide-EDTA (see phage disruption (iii), 9-15 and 44lo%, of the input particles were scored as phage particles partially ejecting DNA from the distal end of the tail, the type El complex (Fig. 7A), and as tail (or ghost))DNA

complexes similar to those described in the above section, respectively. Small but significant fractions of the input particles (usually l-3%) also were scored as complexes in which the head with full-head appearance is connected to one end of a

160

KAORU

i

[L ZlO I 3 z

5

1 0

10

20 30 DNA

40 50 60 LENGTH

70 80 (%)

90

100

FIG. 4. (A) Length distribution of DNA protruding from the proximal end of the tail in SDS-heat induced tail-DNA complexes subjected to the procedures of shear breakage. Each arrowhead represents. a peak position in the distribution, and the positions of arrowheads labeled a, b, c, d, and e are 8.0, 18.3, 32.5, 63.9, and 100.0% of the entire length of T5DNA, respectively. (B) Length distribution of tailfree DNA fragments found in the same preparation studied in (A).

short DNA molecule; the other end of the DNA is attached to the proximal end of the tail (type E2 complex) (Fig. 7B). In some preparations, type E2 complexes could not be detected and in place of them the phage heads having full-head appearance and ejecting only tail-free DNA were observed. The production of the type E2 complex may be interpreted as follows; in the authentic particle, the end of T5-DNA first to be ejected through the tail is connected to the proximal end of the tail; under the conditions employed, the tail is disaggregated from the head but the tail-DNA connection is still preserved. This idea is compatible with the idea described above, that the DNA end connected to the tail

SAIGO

proximal end is the one first to be injected into the bacteria upon infection. To clarify the relationship between the type El and type E2 complexes, the length distribution of the DNA ejected from the tail distal end or the phage head was compared (Figs. 8A. 8B, 9B, and 9C). The profiles of the distribution seemed to be rather discrete and under the present conditions, we could not detect any full-length DNA ejected in either case. The following peaks could be discriminated in the two cases: 1.0, 1.8, 2.5. 3.9, 6.4, 7.8 (Fig. 9B), and 15.9% (Fig. 8A) of the full-length TS-DNA in the type El complexes, and 0.7, 2.3, 3.0, 4.2, 8.4 (Fig. 9C), 20.8, and 32.4% (Fig. 8B) of the full-length T&DNA in the type E2 complexes. Considering the fact that the length of the T5 tail is about 0.2 pm (Fig. 3A) and corresponds to about 0.6%’ of the full length of T5-DNA, four of the seven peaks in the type El complexes (1.8, 2.5, 3.9, and 7.8%) are identical to four of the seven in the type E2 complexes (2.3, 3.0, 4.2, and 8.4c7r, respectively) within the difference of one tail-length (Figs. 9B and 9C). This means that the length of each peak in the El complex is identical to the corresponding peak in the type E2 complex if the length ot DNA ejected in the type El complexes is measured not from the tail distal end but from the tail proximal end. This concept together with morphological character of the type E2 complexes suggests that the discrete length distribution of ejected DNA resulted not from tail-DNA interaction but from an unknown interaction between DNA and protein in the head. Furthermore, almost all peaks with values of more than 3.9% may correspond fairly well to positions of shear-sensitive points on the DNA. That is, three of four peaks in the type El complexes, 3.9, 6.4, and 7.8% of the full-length T5-DNA, may correspond to three positions of shear-sensitive points, 4.1, 6.6, and 8.2% of the full-length DNA, respectively (Figs. 5, 7A, 9A, and 9B), while three of four peaks in the type E2 complexes, 4.2, 8.4, and 32.4%, may correspond to three positions of shear-sensitive points, 4.1, 8.2, and 32.6%’ of the full-length DNA, respectively (Figs. 5, 7B, 9A, and 9C). Thus, although some ambiguities still

TAIL-DNA

CONNECTION

161

IN T5

&&-~ :-, 4.1%

&-

66%

I @-+

8 3%

&-(

a 0 7.

@-+

'I :I

, I

~

175%

4

183%

8

:

32

7%

@

j

3 2 5%

@j

641%

@

639%

3

/ I

~

:,/

:

I 'I

!,I

1000%

I 6e.J

1000%

Q

'. : I I

I

I

: I / ,

I I

I

( ;

I 1 I

I 1

I

I ,

FIG. 5. A map of shear-sensitive points of T5-DNA. The end of the DNA associated with the proximal end of the tail is positioned at the left and positions of shear-sensitive points are shown as percentage lengths of the full length of the T5-DNA. Positions of the main shear-sensitive points are shown as the average of values deduced from shear-broken tail-DNA complexes and ghost&DNA complexes. G and T show the ghost and the tail attached to the apparent DNA fragment end. Filled and open triangles show main and additional shear-sensitive points on T5-DNA, respectively.

remain, we tentatively conclude that the shear-sensitive points on the DNA are in intimate relation to preferential interruption of DNA ejection. DISCUSSION

The results mentioned above strongly support the idea that when T5-DNA is packaged in the T5 particle, the end of the DNA first to be injected upon infection is connected to the tail proximal end. A similar tail-DNA connection has also been shown in bacteriophage lambda (Saigo and Uchida, 1974; Chattoraj and Inman, 1974; Thomas, 1974). Thus, although T5 seems to be unrelated to lambda, it appears to belong to the same category as lambda as far as the DNA arrangement in the particle is concerned. Since, in T5&DNA prepared by our methods, one definite end of the DNA was marked with the tail (or the ghost), we could construct a map of the shear-sensitive points of T5-DNA (Fig. 5), within a relative error range of 5%, using an electron microscope. Recently another map of shear-sensitive points was proposed by Hayward (1974) by using gel electrophoresis. Although in his map some ambiguities still remain, possibly because of depend-

ence of apparent molecular weight on base sequences, the distribution of shear-sensitive points as a whole seems to be identical to ours. In disruption products obtained after formamide-EDTA treatment, particles were found in which DNA was ejected either from the phage head with full-head appearance or from the distal end of the tail. Analysis of the length distribution of ejected DNA in these complexes suggested that in vitro DNA ejection was interrupted preferentially at or near the shear-sensitive points on the DNA by an unknown mechanism present in the head. And the length of DNA ejected through the tail was shorter by about one tail-length than the distance from the tail-attached end to the corresponding shear-sensitive point. Interruption of DNA ejection is also known upon infection of host bacteria by T5 phage (Lanni, 1968). That is, at first only the “8%” portion of the total molecular length (FST-DNA) is injected into bacteria and, several minutes later, the rest of the DNA is transferred into the host cell (Lanni, 1968). The most reliable value for the length of the FST-DNA segment seems to be the value given by Hayward and Smith (1972b) using gel electrophoresis. They

162

KAOHU

SAIGO

FIG. 6. Examples of puddlelike structures found in DPiA fragments after shear treatments. indicate the puddlelike structures residing either at the end or in the middle of the DNA fragments.

gave estimates of 3,000,OOO and 3,800,OOO for the molecular weight (as a singlestranded chain) of the FST-DNA segment and of one of the main single-stranded chains corresponding to the 8.2-17.9% fragment on our map, respectively. (The “3,800,OOO daltons fragment” was assigned to the 8.2P17.9Ya fragment on our map according to reasoning given by Bujard and Hendrickson (1973) and by considering the

Arrowheads Bar, 0.3 pm.

intimate relation between the shear-sensitive points and the single-strand breaks (Hayward, 1974; Saigo, 1975b)). Thus, the length of the FST-DNA segment was calculated to be 7.7% of the total molecular length of T5-DNA and seemed to be shorter by about one tail-length (0.6% of the total molecular length of T5-DNA) than the length of the O-8.2% DNA segment. Since a main shear-sensitive point is lo-

TAIL-DNA

CONNECTION

IN T5

163

FIG. 7. (A) An example of phage particles ejecting DhrA from the distal end of the tail (type El complex). Arrowhead shows tail fibers at the tail distal end. Bar, 0.3 pm. (B) Three examples of complexes in which the tail is disaggregated from the head but connected to the head with full-head appearance (type E2 complex). Open and filled arrowheads show the phage heads with full-head appearance and the tail proximal ends, respectively. Bar, 0.2 pm.

164

KAORI:

SAIGO

I I

LL5 0

E w

Lz W’O m I 3 1 z .j “i!

mO I: 3 z 5

“I i

0

IIIIL All 10

20 30 76 DNA LENGTH

0

5 DNA

( “1. )

FIG. 8. Length distribution of DiXA ejecting from the distal end of the tail in type El complexes (A) and from the head in type E2 complexes (B). Each arrowhead represents a peak position in the distribution. The positions of arrowheads labeled a, b. and c in (A) while arroware 3.7, 7.4, and 15.9?, respectively, heads labeled a, b, c, and d in (B) are 4.1, 8.4, 20.8, and 32.4%,, respectively. Enlarged profiles of the length distribution of DNA in the region from 0 to 154 are shown in Figs. 9B and 9C.

cated at 8.2% from the tail-attached end of the DNA, ejection of DNA upon infection, as in in vitro DNA ejection, might be interrupted at a shear-sensitive point by a similar mechanism present in the head. The position of each shear-sensitive point on the DNA seems to be quite identical to that of the corresponding single-strand break (Saigo, 197513).Thus, it might be suggested that the single-strand break on the DNA generally serves as a kind of mechanical stop in the ejection process. Using isolated T5 receptors, Zarybnicky et al. (1973) investigated the process of DNA ejection and concluded that the whole phage DNA should be expelled once the tail core has been opened. Apparently.

15

10 L E NG T H

(% )

FIG. 9. Length distribution of DNA in the region from 0 to 15% of the total molecular length of T5-DXA. The histogram was constructed by the procedure described by Smith and Sadler (19’71) assuming that the error range of every point was 0.X of the full length of the T&DNA. (A) Length distribution of DNA fragments with ghosts at the end (enlargement of Fig. 2B). Arrowheads labeled a, b, and c are 4.1, 6.6, and 8.37, respectively. (B) Length distribution of DNA ejected from the distal end of the tail in type El complexes enlargement of Fig. 8A). Arrowheads labeled a, b, c, d, e, and fare 1.0, 1.8. 2.5, 3.9, 6.4 and 7.8% respectively. (C) Length distribution of DNA ejecting from the head in type E2 complexes (enlargement of Fig. 8B). Arrowheads labeled a. b. c, d, andeshow0.7, 2.:1,3.0, 4.2. and8.47, respectively.

their results contradict ours. Although the cause of the apparent discrepancy is unknown, further experiments will be required to determine conclusively whether the single-strand break on the DNA is directly related to the mechanism of interruption of the DNA ejection process or not. ACKNOWLEDGMENTS I thank Dr. T. Miyake for encouragement and critical discussions and Dr. K. Yanagisawa for reading the manuscript.

TAIL-DNA

CONNECTION

REFERENCES BUJARD, H., and HENDRICKSON, H. E. (1973). Structure and function of the genome of coliphage T5. I. The physical structure of the chromosome of T5+. Eur. J. Biochem. 33, 517-528. BURGI. E., HERSHEY, A. D., and INGRAHAM, L. (1966). Preferred breakage points in T5 DNA molecules subjected to shear. Virology 28, 11-14. CHATTOHAJ. D. K., and INMAN, R. B. (1974). Location of DNA ends in P2, 184, P4 and lambda bacteriophage heads. J. Molec. Biol. 87, 11-22. FUKE, M., WADA. A., and TOMIZAWA, J. (1970). Denaturation at the right-hand end of the DNA molecule of lambda bacteriophage. J. Molec. Biol. 51, 2555266. HAYWARD, G. S. (1974). Unique double-stranded fragments of bacteriophage T5 DNA resulting from preferential shear-induced breakage at nicks. Proc. Nat. Acad. Sci. USA, 71, 2108-2112. HAYWARD. G. S.. and SMITH, M. G.. (1972a). The chromosome of bacteriophage T5. I. Analysis of the single-stranded DNA fragments by agarose gel electrophoresis. J. Molec. Biol. 63, 383-395. HAYWARD, G. S., and SMITH, M. G. (1972b). The chromosome of bacteriophage T5. II. Arrangement of the single-stranded DNA fragments in the T5+ and TSst(o) chromosomes. J. Molec. Biol. 63, 397407. HERSHEY, A. D., GOLDBERG, E., BURGI, E., and INGRAHAM, L. (1963). Local denaturation of DNA by shearing forces and by heat. J. Molec. Biol. 6, 230-243.

IN T5

165

LABEDAN, B., CROCHET, M., LEGAULT-DEMARE, J., and STEVENS, B. J. (1973). Location of the first step transfer fragment and single-strand interruptions in T5stO bacteriophage DNA. J. Molec. Biol. 75, 213-234. LANNI, Y. T. (1968). First-step-transfer deoxyribonucleic acid of bacteriophage T5. Bocteriol. Rev. 32, 227-242. MCALLISTER, W. T. (1970). Bacteriophage infection: Which end of the SP82G genome goes in first? J. Viral. 5, 194-198. RHOADES, M., and RHOADES, E. A. (1972). Terminal repetition in the DNA of bacteriophage T5. J. Molec. Biol. 69, 1877200. SAIGO. K. (1975a). Polar DNA ejection in bacteriophage T7. Virology 65, 120-127. SAIGO, K. (1975b). Denaturation mapping and chromosome structure in bacteriophage T5, Virology 68. SAIGO, K., and UCHIDA, H. (1974). Connection of the right-hand terminus of DNA to the proximal end of the tail in bacteriophage lambda. Virology 61, 524-536. SMITH, T. F., and SADLER, J. R. (1971). The nature of lactose operator constitutive mutations. J. Molec. Biol. 59, 273-305. THOMAS, J. 0. (1974). Chemical linkage of the tail to the right-hand end of bacteriophage lambda DNA. J. Molec. Biol. 87, l-9. ZARYBNICKY, V., ZARYBNICKA, A., and FRANK, H. (1973). Infection process of T5 phages. I. Ejection of T5 DNA on isolated T5 receptors. Virology 54, 318-329.