Location of DNA ends in P2, 186, P4 and lambda bacteriophage heads

J. Mol. Bid. (1974) 87, 11-22

Location of DNA Ends in PZ, 186, P4 and Lambda Bacteriophage Heads DHFUJBA K. CHtl!rToRA.rLJt AKD Ross B. INMAN

Biophysics Laboratory and Biochemistry Department University of Wisconsin, Madison, Wis. 53706, U.S.A. (Received 21 Januay

1974)

When mature phage particles were suspended in a solution containing formaldehyde (0.07 ~-Nat, pH 9.0, 10% HCHO for 10 min at 23°C) and the mixture then spread for electron microscopy in the presence of 50% formamide and cytochrome c, the phage lysed and a high proportion of the DNA molecules were seen to be attached to phage tails. The phage tails were found to be attached at only one end of each DNA molecule and denaturation mapping showed that this end wss unique for each of the phages P2, 186, P4 and h. It is argued that in these mature phage particles one specific end of the DNA molecule is present at the head-tail attachment site.

1. Introduction The arrangement of DNA in bacteriophage heads is largely unknown. Recent experiments with lambda phage have shown that if the phage heads are not joined to the tail, micrococcal nuclease treatment of the phage heads removes the four terminal bases from the right-hand$ cohesive end of the packaged DNA but leaves the lefthand cohesive end intact. The results strongly indicate that the right-hand end of DNA is present at the head-tail attachment site, and that in this respect the arrangement of DNA is uniform in all h heads (Padmanabhan et al., 1972). Similar conclusions have been reached in the present study from an altogether different approach involving four different Escherichia coli bacteriophages. When the technique of electron microscope partial denaturation mapping (Schnos & Inman, 1970) was applied to a phage solution rather than to purified DNA, we observed that the phage lysed and a significant proportion of the DNA molecules were attached to phage tails. Furthermore, one specific DNA end was involved in tail attachment. We will present evidence that a formaldehyde-induced tail-DNA attachment took place in phage particles before they had a chance to separate and that one specific end of the DNA molecule is in close proximity to the tail in mature phage and that this arrangement

is uniform

in all phages studied

so far.

t Present address: Institute of Molecular Biology, University of Oregon, Eugene, Ore. 97403. U.S.A. $ For definition of right and left ends see the legend to Fig. 1. 11

12

D. K. CHATTORAJ

AND

R. B. INMAN

2. Materials and Methods (it) Solution8 NaCl-EDTA: 0.02 M-N&I, 0.006 M-Na,EDTA (pH 7.4). High-pH buffer containing HCHO: O-068 ~-NazC0~, 34% HCHO, 0.0107 M-NasEDTA (pH 10.0). The denaturation pH was achieved by carefully adding the desired amount of 5 N-NaGH. Similarly the pH was lowered by adding 1 N-HCl. High-pH buffer without HCHO: exactly the same sa high-pH buffer except water was added in place of HCHO. The pH of such a solution was 10.4. SSC: 0.16 M-NaCl, 0.016 M-sodium citrate (pH 7.0). (b) Bacteriophage strah

and preparation

of phage stocka

P2 Reel and P2 Rec2 phage were gifts from Dr G. Bertsni; 186~ (a gift from Dr R. L. Baldwin) was grown by thermal induction of E. coli CR34 (186~) according to Baldwin et aZ. (1966). P4 virl (Lindqvist & Six, 1971) was a gift from Dr E. Six and was grown basically according to Inman et al. (1971). h ~I857 aus &117 was grown by thermal induction of E. coli CR34 lysogen (a gift from Dr W. F. Dove). The choice of the particular phage strains was incidental and irrelevant to the present work. For brevity the phages will be referred to as P2, 186, P4 and h. All phage were purified by CaCl density-gradient centrifugation in 0.01 M-Tris*HCl (pH 7*1), 0.01 M-MgCl, (60-Ti rotor at 47,000 revs/mm overnight at 4°C). The mean density of the CsCl solutions were 1.44 for P2 and 186, 1.38 for P4 and 1.6 for X. (c) Spreading of phage solution8 and electron microecopg Density-gradient purified phage stocks were dialyzed and diluted in NaCl-EDTA = O-03) and 7 ~1 of this solution was solution to a titer of about lOlo phage/ml (0.D.280 then added to 3 ~1 of high-pH buffer (at pH values between 8.6 and 11.3 in various experiments). The mixture was usually incubated for 10 min at 23°C and then diluted with 10 ~1 of formamide and 2 ~1 of 0.1% cytochrome c solution (several variations of this procedure will be described in the text). The remaining procedure for spreadmg, electron microscopy and data computation have already been reported (S&n& & Inman, 1970). Harrison et al. (1973) have shown that X is inactivated in 0.01 M-EDTA at 38°C. In our experiments the phage were in contact with EDTA under less extreme conditions (0.006 M at 4°C) but for periods of many hours. There is therefore the possibility that some phage were broken by contact with EDTA. This will be discussed in Results (section (b)). (d) Preparation,

purification

and assay of P2 t&L

Phage stock was prepared by multiple-cycle growth of plaque-purified P2 am7 wip in E. coli C620 at 32°C in LB medium containing 2.5 x 10m3 a6-CaCl,, as described previously (Chattoraj & Inman, 1973). P2 tails were grown according to Lindahl (personal communication). E. coli Cl-a was grown in LB at 32°C to an o.D.~~,, = 0.2, the oells were chilled, centrifuged and resuspended in O-1 vol. LB containing 2.6 x 10V3 m-CaCl,. P2 am7 vir phage was added at a multiplicity of infection of 5 and adsorbed for 10 min at 32”C, the suspension was chilled and centrifuged. The supernatant was poured off and the pellet resuspended in 3 vol. cold LB and subsequently incubated at 32’C. After 30 min, potassium phosphate buffer (pH 6.8) was added to a final concentration of O-2 M in phosphate. After 2 h, the debris was removed by centrifugation. The supernatant (titre 6 x log tails/ml) was directly layered (2 ml per tube) on 10.6 ml of a 6% to 20% sucrose gradient in 0.01 M-triethanolamine buffer (pH 7.6) and centrifuged at 6°C in a Spinco SW41 rotor for 100 min at 40,000 revs/mm 0.5~ml fractions were collected and about 6 fractions from the middle of the tube were dialyzed against N&l-EDTA solution and gradually concentrated 20-fold by dialyzing alternately against solid polyethylene glycol and NaCl-EDTA solution. The final concentration of purified tails was 3 x log tails/ml. The purity of the tail solution was confkmed by electron microscopy on negatively stained (1 o/o uranyl acetate) samples. An essentially similar method was used to prepare the P2 heads except that P2 am6 wir was used to infect E. coEi Cl-a. The crude lyeate (9 x log heads/ml) was stored at 4°C. To

LOCATION

OF DNA

ENDS

IN PHAGE

HEADS

13

assay for tails, O-1 ml of the tail solution was mixed with O-9 ml of the head lysate and the mixture incubated for 3 h at 32°C. The mixture was diluted and plated on a sensitive lawn of E. coli C620. Contamination of complete phage particles in crude head and tail lysates was about 0.2% and 0.06%, respectively. Details of the phage and bacterial strains (gifts from Dr C. Bertani) were reported elsewhere (Lindahl, 1971).

3. Results (a) Attachment of phage tails to DNA when P2, 186, P4 and h phage are spread from high-pH buffer containing HCHO Many of the following observations were essentially similar for the four phage types studied and mention of any particular phage type will be made only when necessary. We first note that efkient visualization of native and partially denatured DNA can be effected in high-pH buffer by spreading phage rather than purified DNA. Thus, isolation and purification of phage DNA, and damage to DNA caused thereby, can be avoided. A very small number of phage particles (7 x 10’) are required in one experiment. Recently CSmez & Lang (1972) and Mayer et al. (1973) have also used intact phages for partial denaturation mapping of DNA following a different procedure from ours. Under the electron microscope the DNA molecules released from the phage were almost always full-length and linear. Many of the molecules were seen to be attached to phage tails. There was only one tail connected to each DNA molecule and the attachment was invariably at a DNA-end (Plates I to III and Table 1). The frequency of tail-DNA attachment was variable and depended on the spreading conditions (Table 1). DNA molecules with or without tails were found to be of identical length within experimental error. Tail attachment of DNA other than at the end was not observed (Table 1). However, rarely (4 out of 622 molecules) the tails attached to DNA at one end, were also attached to a second point on the same DNA through their distal end (Plate III). Free tails and empty heads, or parts of heads, could also be seen in these solutions but mature phage particles were not observed. (b) Attachment of phage tails to unique DNA ends On partial denaturation in the presence of HCHO (pH 11.0, 50% formamide (Table 1) or pH 8*6,80% formamide, Table 2, line (c)) DNA molecules were still seen to be attached to the tails. The degree of denaturation was chosen so that the two ends of the DNA molecules could be unambiguously assigned. At comparable degrees of denaturation the maps of molecules attached to tails were indistinguishable from those without a tail and from purified DNA (Fig. 1). Thus, the presence of phage proteins in the spreading solution and the attachment of a tail to the DNA does not interfere with the partial denaturation mapping of DNA. On the basis of the denaturation maps of P2, 186, P4 and X DNA it can be unambiguously concluded that tails are attached at unique DNA ends. The results in Figure 1 indicate that tail attachment is at the left ends of P2 and 186 and at the right ends of P4 and )r DNA, as defined by denaturation mapping. As will be shown later, the attachment of tails to DNA can only be demonstrated when intact phages are lysed in the presence of HCHO or after preincubation in HCHO (free DNA and tails do not combine). The presence of free DNA or tail-less heads in the phage suspension decreases the frequency of observed tail-DNA

p2

p2 186 186 186 p4 P4 A A

33

(4

lb) 6) (d) (e) (f) h3) @I (9 (j)

Q-9 11.0 Il.0 9.0 11.0 11.2 9.0 11.0 9.0 Il.0

pH of high-pH buffer containing HCHO 212 94 119 100 118 250 108 180 202 100

Total no. of molecules counted /

74 76 74 16 92 100 22 70 18 92

Free DNA

of t&l-DNA

82

78

84

26

At undefined DNA end?

0

0

8 0

24 26

Left end of DNAS

8

30

0 0

0 0

Right end of DNAS

Observed tail position (as y0 of total molecules examined)

association

t As observed in undenatured molecules. $ As deduced from partial denatumtion mapping.

0 6.2 4.1 0 10.2 58.0 0 11.9 0 5.3

Average degree of DNA denaturation (%)

Speci$city

TAEZE 1

0 0 0 0 0 0 0 0 0 0

Tail attached but not at DNA end

.

186

186

186

h h

P4

(b)

(c)

(d)

(4 m

k)

11.5

11.0

225

76

89 90

209 200

5.3


10.4 11.0

100

100

60

8.6

66

204

8.6

6

90

Free DNA

204

346

,

10.4


10.4

Total no. of molecules counted

m.sociuticm

4

94

4

Undefined DNA endl

10

24

0

0

0

0

0

Right end of DNA$

34

Left end of DNA3

Observed tail position (as y0 of total molecules examined)

in tail-DNA

2

0

7 0

0

0

0

6

Tail attached but not at a DNA end

.

Special treatment

were usually

exposed

High-pH buffer without HCHO Phage fhst &reds, then spread as in (a) Same as (b) but spread with 80% formamide Same as (b) but spread with 90% formamide Same a8 (a) The phage was first suspended in pH 9.0 buffer with HCHO for 10 min, diluted (1 : 50) in NaClEDTA and then spread at the indicated pH in the presence of HCHO Same a8 (f)

t As observed in undenatured molecules. $ As deduced from partial denaturation mapping. 3 1% HCHO, pH 7.0, 0.12 M-Na+, 2 h, 23°C. but the ends of the molecules T[ About 48% of the molecules were still attached to head or head fragments and formed “rosettes”, and could be defined by denaturation mapping (a still higher proportion (88%) of rosettes were observed in (b)).

186

(a)

Average degree of DNA denaturation (%)

pH of high-pH buffer

Effect of forntaldehyde

TABLE

D. K.

16

CHATTORAJ

AND

R. B. INMAN

P2 DNA t t t t

. I I I II

,I I” . . . .

II. I I

c

II I .,I I,

P4 DNA I -i -l----d I

A DNA

Length ( pm)

FIG. 1. Examples of partially denatured DNA molecules attached to phege tails. Eech horizontal line represents a DNA molecule and black rectangles show the size and poaition of denatured sites. The short vertical arrows mark the point of attachment of tails to DNA. In each case the maps lmve been normalized to the average length of the particular native DNA. The spreading conditions were: (1) P2, P4 and X phage suspensions were adjusted to 10% HCIIO and pH Il.0 (in high-pH buffer) and left for 10 min at 23”C, formamide was then added to a final concentration of 60%; (2) 186 phage ww suspended in 10% HCHO at pH 8.6 (in high-pH buffer) snd left for 10 min at 23’C, formamide was then added to give a f?nel concentration of 80% and the solution left a further 10 min et 23°C before spreading. In all casesthe right half of the map is deAned as the A + T richer h&lf.

attachment. Since X phage were often stored in EDTA-containing solutions (see Materials and Methods, section (b)) and were not scored for such contamination, the frequencies shown in Tables 1 and 2 could be underestimated. This was indicated by the following experiment. A X phage solution was banded in CsCl and stored 9 months at 6°C in O-01 M-Tris (pH 7-O), 0.01 M-MgCI, and spread in the usual manner at pH 9.0; in this experiment only 2% of the DNA molecules were found attached to tails. The same phage suspension, when rebanded in CsCl and spread within a day, yielded 82% of the molecules attached to tails. Thue, the purity of the phage sus-

LOCATION

OF DNA

ENDS

IN

PHAGE

HEADS

17

pension can be quite significant and this has not been systematically recorded in the present experiments. The frequency of tail attachment decreases as the pH is increased (Tables 1 and 2) and is presumably in part due to denaturation of tail protein. At pH 11.0 and above, attached X tails start to lose their characteristic shape and eventually can be observed only as ill-defined “puddles” at the right end of X DNA molecules (Plate II). If these puddles are scored as tails then the frequency of tail attachment at pH 11.0 is 66% rather than 10% (Table 2, line (f)). Attached P2, 186 and P4 tails (actually tai1 tubes) appear to be somewhat more resistant to alkali. However, at pH 11.2, 186 tails can no longer be observed; presumably, again because of protein denaturation. That the observed structures at the end of the DNA molecules were, in fact,, phage tails was confirmed by their length and diameter. In the case of P2, 186 and P4 the sheath was usually contracted and in a few cases the sheath was missing, suggesting that DNA was actually attached to the tail tube. The contracted sheath could be anywhere along the tail tube and in a few instances could be found threaded along the DNA molecule at random positions (Plate III). Empty heads could also be seen in the background (Plate III) and sometimes were still attached to the tail (Plate I). At pH Il.0 recognizable phage heads were no longer seen. We next attempted to determine if the DNA was attached exactly to the end of the tail or was, in fact, inserted some distance within the tail. In the latter case, t,he length of exposed DNA would appear shorter than free DNA molecules and the difference in length would represent the amount of DNA present within the tail. In t.he case of P2, the length difference could not be greater than 176 since the P2 tail is about 1% of P2 DNA length (I nman et al., 1971). The experimental error involved in length measurements in the present experiments precludes a direct test of the above question in the case of P2. However, an approximate estimate was obtained with P4 whose tail is the same size as the P2 tail, but whose DNA is only one-third as long (Inman et al., 1971). The length ratio of P4 DNA molecules with and without tails was measured in fields where both occurred together. The mean ratio was found to be 1.005 & 0.018. Thus, we could not demonstrate a significant difference between both types of P4 DNA. If P4 DNA was fully inserted within the tail, a length difference of about 3% would be expected. We conclude, therefore, that the amount of DNA (if any) inserted within a P4 tail is less than can be debected by our measurements (<0*06 pm). (c) Tests of three models for tail-DNA

attachment

Several alternatives can be envisaged to explain the attachment of DNA to tails, First, during lysis of phage the entire DNA could be released through the tail; one specific end emerges first and for some reason, the trailing end of the DNA molecule remains attached either to the distal or proximal end of the tail. In experiments on osmotically shocked X phage (Caro, 196.Q the DNA appeared to eject from the tail. Second, the DNA might be released from the head but remain attached to the tail due to prior binding between a DNA end and the proximal end of the tail. In the case of X it is known that four terminal nucleotides of the right cohesive end are vulnerable to nuclease attack of heads containing DNA and it has been concluded that in h the DNA appears to be packed with its right-hand end exposed at the tail attachment site (Padmanabhan et al., 1972). Third, the phage might lyse to yield free DNA and tails which then combine to produce the observed specific tail-DNA attachment. 2

18

D. K.

CHATTORAJ

AND

R. B. INMAN

The following variations of the spreading conditions were made in order to understand which, if any, of the above models might explain tail attachment. (i) Spreading in high-pa bufSer in the absence of HCHO We first examined the effect of excluding HCHO from the spreading solution (the high-pH buffer was made without HCHO). Under these conditions, full-length free DNA molecules were observed and the number of molecules attached to tails was greatly reduced (Table 2, lines (a) and (e)) from that observed previously when HCHO was present (Table 1). This experiment suggests that HCHO greatly facilitates the tail-DNA association but may not be essential for the binding. When tails were attached to DNA they were often found at positions other than at ends (Table 2; Plate IV(a)); such was never the case when phage lysis occurred in the presence of HCHO (Tablo 1; Plates I to III). In addition, empty heads could be seen attached at random positions in about 11 y0 of the DNA molecules. Clearly, in some, the DNA was incompletely released from the head and it was apparent that DNA could come out of the head and/or the tail (Plate IV(a)). (ii) Spreading in the absence of HCHO but after pre-incubation in HCHO In order to check further on the effect of HCHO, the phage particles were first incubated in the presence of 1% HCHO (2 h, 23”C, 0.12 M-Na+, pH 7.0) diluted (1 : 50) in NaCl-EDTS and then spread in the absence of HCHO in high-pH buffer. 94% of the DNA molecules were now attached to the tail at one end (Table 2, line (b)). When these molecules were partially denatured in the presence of 80% formamide, again only one specific end of the DNA was seen to be attached to the tail (Table 2, line (c)). Pre-incubation with HCHO thus dramatically facilitates the tail-DNA association. A high proportion of the molecules (88o/o) retained the head, which could be anywhere along the DNA but usually was separated from the tail (Plate IV(b)). DNA molecules were often entangled around the head and were in most cases incompletely released. We therefore conclude that in the presence of HCHO a unique DNA end is cross-linked to the proximal end of the tail and this is then followed by release of DNA from the phage head. (iii) Attempts to attach puri$ed tails to DNA Purified preparations of P2 tails and P2 DNA, each dialyzed against NaCl-EDTA, were mixed together (3 x 10Btails/ml and DNA of o.D.,~~ = 0.03) and put in high-pH buffer containing HCHO at pH 9.0 for 10 min and then spread for electron microscopy. Out of 250 DNA molecules counted, none showed attachment to the tail at the ends. In eight molecules the tails were lying across the DNA at positions other than at ends. Sucrose gradient purification of the tails was necessary; otherwise the DNA molecules were found to be degraded when mixed with the crude lysate of the tail preparation. Under similar spreading conditions, intact phage yielded a high proportion of DNA-tail attachments (Table 1). In the above experiment HCHO was present at the time DNA and tails were mixed. It is possible that HCHO could stabilize the DNA-tail attachment once the union has been made but may, in fact, interfere with the act of attachment. For example, if attachment requires the presence of a single-stranded cohesive end (or a portion of such an end) then HCHO might prevent attachment. However, if HCHO was added after the attachment it would stabilize the connection by formation of

PLATE I. Electron micrograph showing many examples of /\ twtt to phage tails; X phage in 0.07 wNa+, pH 9.0, 10% HCHO, pwtrin monolayer technique. The two arrows show DNA-tail still prrscnt~: in these cases it appears that the DNA is connected tail.

DNA molecules attached at one 10 min at 23°C and spread by the attachments with head proteins to the proximal end of the phage

PLATE II. Electron 23W. Tails were still their normal structure were only seen at the

micrograph of X phage DNA partially tlcnatured at pH 1 I-O for 10 min at seen attachecl to DNA at the right end (see the inset) hut most often low either partially or completely (arrow). Such distorted structures (“pudtllcs”) right end of X DNA molecules.

1’1~vm: IV. (a) Phagt i X6 spread in the abscnor of HCHC) The tail tube can oftrn te swn at internal positions ulonp DNA can corm out of both heat1 and the tail. (b) Phage 186 first fixed at neutral pH (in lc;c, HCHO spread without HCHO as in (a). Tails can only he seen at appears to CO~H oat of the head in all eases.

(0.07 M-Ka -, pH 10.4, I IJ min at 23°C). the DNA molwx~le and it appcturs that. for 2 h at 23”C, 0.12 M-Na+) and then thP end of tho molecules and the DNA

LOCATION

OF DNA

ENDS

IN

PHAGE

HEADS

19

cross-links. In order to check on this point isolated P2 tails and P2 DNA were mixed together as above, and incubated overnight in the absence of HCHO (in NaCl-EDTA solution) at 4°C and then spread under the standard spreading conditions with HCHO present (Table 1, line (a)). In another experiment the tail and the DNA solutions were also incubated at higher ionic strength (0.1 M-N&I, 0.01 M-Na,EDTA, pH 8.0). In both cases, the original DNA solution was first heated to 75°C for 5 minutes and then rapidly chilled. (This treatment should open up any previously formed circles (Wang, 1967).) The linearity of the molecules was confirmed by electron microscopy. Thus, in these experiments the DNA ends were accessible for binding to the tails at least during the initial stages of incubation. After incubation for two days at 4°C the samples were spread in high-pH buffer containing HCHO. No attachment was found when 200 DNA molecules were counted but about 17% of the molecules were converted to circles, implying that the ends of at least some of the DNA molecules were intact. A similar experiment was also carried out with separated left halves of P2 DNA (Geisselsoder et al., 1973) to ensure that the left cohesive end was available for attachment throughout the incubation with tails. Again, no significant tail attachment could be demonstrated. A final attempt to force tail-DNA attachment was made at higher concentrations of DNA. We chose conditions that would lead to intermolecular cohesion between h DNA (Hershey et at., 1963). Under these conditions we again could not obtain evidence for tail-DNA attachment (the incubation was carried out in 0.6 M-Na + , DNA of O.D.,,o = 0.2, 1.5 x log tails/ml for 15 days at 5°C) when either whole P2 molecules or the left halves of P2 were used.

4. Discussion (a) Major experimental results The following experimental facts have been presented. (1) When phages P2, 186, P4 and h are spread from high-pH buffer containing HCHO, the phages lyse and phage tails (tail tubes in the case of P2, 186 and P4) are observed to be attached at specific ends of DNA molecules. Tails are attached at the left-hand ends of P2 and 186 DNA and at the right-hand ends of P4 and h DNA molecules, as defined by denaturation mapping. (2) When the phage are spread from a similar buffer, but with HCHO omitted, then the phage again lyse but now there is very little specific attachment of phage tails to DNA ends. (3) Prior incubation of phage with HCHO restores tail-DNA attachment when phage are lysed and spread in the absence of HCHO. (4) Specific tail-DNA attachment cannot be demonstrated when purified tails and purified DNA are incubated together under a variety of conditions (including the presence and absence of HCHO). (b) Con&.&on In view of our lack of success in demonstrating specific attachment between free DNA and tails it appears that attachment requires lysis from intact phage. The data presented above are consistent with the notion that in mature phage a specific end of the DNA molecule is in close proximity with the head-tail junction. We further conclude that lysis in the presence of HCHO (or in the absence of HCHO, but with prior incubation with HCHO) occurs with concomitant HCHO-mediated protein-

20

D. K.

CHATTORAJ

AND

R. B. INMAN

DNA cross-linking which then binds the specific DNA end to the proximal end of the tail. Accordingly the left end of P2 and 186 DNA and the right ends of P4 and h should be situated very close or just within the tail at the head-tail junction. The left and right DNA ends are defined in this investigation by denaturation mapping and it is known in the case of X and P2 that the denaturation and genetic maps are congruent (Davidson t Szybalski, 1971; Chattoraj & Inman, 1972). Very similar results and conclusions are also presented in the accompanying paper (Thomas, 1974) where a different cross-linking reagent (N-cyclohexyl-W-/3-(4 methylmorpholinium) ethylcarbodiimide) has been used to demonstrate that X tails attach to the right ends of X DNA molecules, The above conclusions are also supported by the following observation. Sometimes DNA-tail units retain the phage head, which can be found attached at random to the DNA. In about 5% of these cases the head is still attached to the tail and if HCHO is present the DNA is always found to be connected at the head-tail junction (arrows in Plate I). In the absence of HCHO the DNA could be attached to the distal end of the tail (in this case, however, there is a much lower frequency of DNAtail attachment). Again the implication is that, in the presence of HCHO, DNA is only released from the head whereas in the absence of HCHO, DNA can also emerge from the tail. It is shown in the accompanying paper (Thomas, 1974) that lysis in the absence of HCHO results in partial ejection of DNA through the tail and that under these conditions it is the right end of h DNA which is first ejected. Whether or not there are pre-existing bonds between the DNA end and tail in mature phage can not be determined from the present investigation. If such bonds are present they must be weak because HCHO cross-linking is necessary to demonstrate significant tail-DNA attachment. We were not able to demonstrate any significant length difference in P4 DNA with or without attached tails. According to the explanation given above for tail attachment, this means that the amount of DNA, if any, intruding into the tail in mature phage is less than can be detected by our length measurements (<0*06 pm). In X, only four terminal bases of the right-hand cohesive end are exposed in tail-less heads at the head-tail attachment site (Padmanabhan et al., 1972). If the structure of the head is unaltered in complete phage particles, it is likely that these four bases could be involved in the attachment to the tail in the present experiments. In partiahy denatured X DNA, tails are in fact occasionally seen to be attached to a single DNA strand at the right end. However, at the degree of denaturation used, this is an infrequent event; more often there is no single-strand dissociation at the right end of the molecule (Fig. 1). In any event, our conclusions are consistent with the findings of Padmanabhan et al. (1972) but are in conflict with a report by Sharp et al. (1971) who concluded that the left and right ends of h were injected with equal frequency. The interpretation that has been given above for DNA-tail attachment involves a direct linkage either between a DNA end and the proximal end of the tail or by insertion of DNA within the tail. Another possibility that we can not exclude is that the DNA-tail joint also involves a small amount of head protein that may remain attached to the proximal end of the tail after lysis. We have concluded that the observed specific tail-DNA attachment that follows phage lysis in the presence of HCHO results from a phage structure in which a specific DNA end is in close proximity to the proximal end of the phage tail (or perhaps inserted slightly into the tail). If this is the case then it is reasonable to

LOCATION

OF DNA

ENDS

IN

PHAGE

HEADS

31

assume that it is the same DNA end that first enters the cell upon infection. Our results may, therefore, indicate that the right ends of h and P4 and the left ends of P2 and 186 first enter the infected cell. This would further indicate that the order of entry of phage genes in A and P2 is quite different because the physical and genetic right ends of X DNA are closest to “early” or control functions whereas in P2 the left physical and genetic ends correspond to “late” or structural functions (Lindahl, 1974; Chattoraj & Inman, 1972 and Chattoraj & Inman, unpublished results). However, the order of injection of h and P2 genes may be quite irrelevant because these DNAs form circles soon after infection. From the work of Little & Gottesman (1971), it appears that in h a specific cohesive end is a requirement for correct phage particle assembly and although defective particles are assembled using DNA with only one cohesive end, only particles with the right cohesive end are infectious when assayed as intact phage. This finding is consistent with the present report and that of Padmanabhan et al. (1972). Recent results also stress the role of cohesive ends in head morphogenesis. It is believed that X DNA matures from concatemers (Wake et al., 1972; Skalka et al., 1972). One of the head genes of X (gene A) appears to specify an enzyme that cleaves DNA concatemers into monomers with cohesive ends (Wang 6 Kaiser, 1973) and it is suggested that binding of the A protein at the cleavage site of the polymeric DNA is one of the initial steps in head assembly but that cleavage does not occur until the entire head is formed (Kaiser & Masuda, 1973). The formation of the correct cohesive end at the head-tail attachment site might, therefore, be one of the terminal steps in head morphogenesis. Possibly the specific DNA end which is in close proximity to the tail in a mature phage particle is a result of an orderly assembly process rather than a pre-requisite for successful infection. We wish to thank MS Selma Sachs for her technical assistance. We also thank Drs R. L. Baldwin, A. D. Kaiser, and H. B. Younghusband for many stimulating discussions and criticisms of the manuscript. The research was supported by grants from the United States Public Health Service, National Institutes of Health, the American Cancer Society and the Graduate School, University of Wisconsin.

REFERENCES Baldwin, Bid.

R. L., Barrand,

P., Fritsch,

A., Goldthwait,

D. A. & Jacob, F. (1966). J. Mol.

17, 343-357.

Caro, L. G. (1965). Virology, 25, 226-236. Chattoraj, D. K. & Inman, R. B. (1972). J. Mol. Biol. 66, 423-434. Chattoraj, D. K. BEInman, R. B. (1973). F+oZogy, 55, 174-182. Davidson, N. & Szybalski, W. (1971). In The Bacteriophage Lambda (Hershey, A. D., ed.), pp. 45-82, Cold Spring Harbor Laboratory, New York. Geisselsoder, J., Mandel, M., Calendar, R. & Chattoraj, D. K. (1973). J. Mol. Biol. 77, 405-415.

G6mez, B. & Lang, D. (1972). J. Mol. Biol. 70, 239-251. Harrison, D. P., Brown, D. T. & Bode, V. C. (1973). J. Mol. Biol. 79, 437-449. Hershey, A. D., Burgi, E. & Ingraham, L. (1963). Proc. Nat. Aeud. Sci., U.S.A. 49,748-755. Inman, R. B., Schnos, M., Simon, L. D., Six, E. W. & Walker, D. H. (1971). Virology, 44, 67-72.

Kaiser, D. & Masuda, T. (1973). PTOC. Nat. Ad. Lindahl, G. (1971). Virology, 46, 620-633.

Sci.,

U.S.A.

70, 260-264.

22

D. K.

CHATTORAJ

AND

R. B. INMAN

G. (1974). Mol. Gen. Genet. 128, 249-260. Lindqvist, B. H. & Six, E. W. (1971). ViroEogy, 48, l-7. Little, J. W. & Gottesman, M. (1971). In The Bacteriophage Lambda (Hershey, ed.), pp. 371-394, Cold Spring Harbor Laboratory, New York. Mayer, F., Lotz, W. & Lang, D. (1973). J. Birol. 11, 946-952. Padmanabhan, R., Wu, R. & Bode, V. C. (1972). J. Mol. Biol. 69, 201-207. Schnbs, M. & Inman, R. B. (1970). J. Mol. BioZ. 57, 61-73. Sharp, J. D., Donta, S. & Freifelder, D. (1971). Virology, 43, 176-184. Skalka, A., Poonian, M. & Bartl, P. (1972). J. Mol. BioZ. 64, 541-560. Thomas, J. 0. (1974). J. Mol. BioZ. 87, l-9. Wake, R. G., Kaiser, A. D. & Inman, R. B. (1972). J. MOE. BioZ. 64, 519-540. Wang, J. C. (1967). J. Mol. BioZ. 28, 403-411. Wang, J. C. & Kaiser, A. D. (1973). Nature New BioZ. 241, 16-17.

Lindahl,

A. D.,