Isolation and structure of phage λ head-mutant DNA

J. Mol. Biol. (1972) 64, 5194540

Isolation and Structure of Phage k Head-mutant DNA R. G. WAKE, A.D.

KAISER

Department of Biochemistry, Stanford University Stanford, Calif. 94305, U.S.A.

Ross B. INMAN

Biophysics Laboratory and Biochemisty Department of Wisconsin, Madison, Wis. 53706, U.S.A.

University

(Received 2 August 1971) High molecular weight DNA accumulates in bacteria in which X is multiplying but cannot complete the formation of new phage particles due to a defect in head assembly. Accumulated h DNA has been isolated from induced mitomycin Ctreated lysogens by means of a shift in buoyant density labels from heavy to light and fractionation by density-gradient sedimentation for completely light DNA. Head formation was blocked in these lysogens by amber mutations in genes D or E, which specify the two major head proteins. The purified DNA is at least 80% A by DNA-DNA hybridization and some preparations are close to 100% I by this test. Electron microscopy and sedimentation velocity measurements on these preparations showed that they were a population of linear molecules (about 5% were circular) with lengths ranging from one to more than four monomer units. After partial denaturation, the long molecules showed a repeating h-like pattern of denatured sites. The length of the repeating unit equalled the length of DNA molecules isolated from phage particles. The distribution of denatured sites within the repeating unit ww the same as that for DNA isolated from phage particles. Thus, the long molecules are head-to-tail polymers of h DNA. These structures may be head precursor DNA, which accumulates because one of the structural proteins of the head is missing. Perhaps h DNA is normally cut into monomer units at the same time and as an integral part of head protein assembly and DNA encapsulation.

1. Introduction DNA molecules isolated from particles of bacteriophage X are about 46,000 base pairs long (Davidson & Szybalski, 1971). The ends of each molecule have projecting single strands, 12 nucleotides long, which are complementary to each other (Hershey, Burgi & Ingraham, 1963; Wu & Kaiser, 1968; Wu & Taylor, 1971). Within five minutes after infection, the ends join and most of the DNA molecules are converted intro covalently closed circles (Young & Sinsheimer, 1964; Bode & Kaiser, 1965). Lambda replicates its DNA through forms which do not have cohesive ends, and molecules with cohesive ends are not detectable until complete phage heads are formed (Dove, 1966; Mackinlay & Kaiser, 1969). 34

519

520

R. G. WAKE,

A. D. KAISER

AND

R. B. INMAN

There are indications that the structure of h DNA depends upon proper assembly of the phage head. At least seven genes, A, W, B, C, D, E and F, clustered at the left end of the phage chromosome, are known to control head synthesis and assembly (Weigle, 1966; Parkinson, 1968). Two of these genes, W and F, specify head proteins which are required for the attachment of tails (Casjens, 1971; Casjens, Hohn & Kaiser, 1972). Mutants defective in either of these genes produce normal sized, DNA-filled heads which, however, are unable to attach tails. The DNA isolated from F - or W - heads, or directly from F - or W - infected cells, is of monomer length and has cohesive ends (Casjens et al., 1972). However, mutants in any of the remaining genes, A, B, C, D or E, cannot fill heads with DNA and the h DNA in extracts of cells infected with any of these mutants lacks cohesive ends, as tested by capacity to donate genes to helper-infected recipient bacteria (Dove, 1966; Mackinlay & Kaiser, 1969). Lambda DNA accumulates in bacteria within which an iI-, B-, C-, D- or Emutant is multiplying, but it has an unusual structure. Salzman & Weissbach (1967) reported that induced lysogens of hAam or ADaml5 accumulated DNA which sedimented faster than DNA isolated from phage particles. Subsequently, the DNA which accumulates in bacteria infected with XA- has been partially purified and found to be a mixture of circular molecules of phage length and linear molecules of various lengths (Weissbach, Bartl & Salzman, 1968; Kiger & Sinsheimer, 1969; McClure, MacHattie & Gold, personal communication). Hoping to shed some light on how head assembly affects DNA structure we have studied the X DNA, which accumulates in induced lysogens for h mutants defective in genes D or E. These genes specify two proteins, which together account for 980/, of the head protein mass. The first task was to separate X DNA from the larger amount of Escherichia coli DNA present in extracts of induced lysogens. This step is particularly important because it turns out that much of the accumulated h DNA is linear and thus easily confused with host DNA. The purified intracellular X DNA was then examined by hybridization, centrifugat,ion and electron microscopy to determine the size, shape and arrangement of h monomer units.

2. Materials and Methods (a) Bacteriophage Both h&857 and @80 were used for the preparation of reference DNA, which was extracted by the method of Kaiser & Hogness (1960). Head mutant stocks XDam123 ind+ ~1857 and xEaml3 ind+c1857 and helper phage Xi434 &am21 Ram60 were gifts from L. Reichardt . 32P-labeled X was prepared by temperature induction of Js’3350 (Ac1857) grown as described previously (Bode & Kaiser, 1965). (b) Bacteria The Escherichia coli K12 strain 159T- and F + su-thy-hstrain (Kiger & Sinsheimer, 1969) were kindly provided by Dr R. L. Sinsheimer. The lysogenic derivatives, 159T(XDam123 ind+c1857) and 159T- (maml3 ind+cI857), wereisolated following infection of 159T- by the appropriate phage. The phage genotype of each lysogen was checked by complementation and recombination tests following superinfection with other head mutants as well as hJaml7, a tail mutant. (c) Btrflers

and culture

medium

TE is 0.01 M-Tris.HCl + 0.001 M-EDTA (pH 8.1); SSC is 0.15 M-NaCl + O.O15Msodium citrate (pH 7.0); TCM is 0.01 M-Tris.HCl + 0.01 M-CaCl, + 0.01 M-MgCl,

X HEAD-MUTANT

DNA

521

3 g KH,PO,, 1 g NH&I, (pH 7.1) ; KW medium, per liter: 7 g NaaHPO,, 0.6 g MgS04, 2 g glucose, 15 g Casamino acids, charcoal adsorbed. (d) Isolation

of head-mutant

O-5 g NaCl,

DNA

As far as possible all steps were carried out under sterile conditions. The appropriate 159T lysogen was grown overnight at 30°C in KW medium supplemented with 20 pg thymine/ml. The generation time under these conditions is 80 to 90 min. The culture was diluted 1 in 50 into fresh medium and allowed to grow to IO9 cells/ml. (absorbance at 590 nm, A,,,, of O-70) at which time it was diluted into 4 vol. of warmed KW medium containing 5bromouracil, such that the final concentration of the latter was 40 pg/ml. (bromouracil: thymine weight ratio 10: 1). All cultures growing in the presence of bromouracil were protected from fluorescent light. When A5a0 reached 0.80, the culture was mixed with mitomycin C @al concentration 20 pg/ml., prepared as described by Young & Sinsheimer, 1967b) and kept in the dark at 3O“C for 10 min without shaking. After cooling to 0°C the cells were centrifuged, washed with an equal volume of ice-cold KW medium and resuspended in the same volume of KW medium containing thymine (10 rg/ml.) and [methyZ-3H]thymine (10 &i/ml.). Temperature induction was effected by warming at 44°C for 7.5 min (for 200 ml. vol.) and the culture was then incubated in the dark at 37°C with shaking. After 40 min (lysis commences at 45 to 50 min) NaN, was added to a final to 0°C. The cells were centrifuged concentration of 0.02 M and the culture cooled rapidly and, for a 200-ml. culture, suspended in a mixture of 8 ml. of 25% sucrose in 0.04 MTris.HCl (pH 8.0) + 0.8 ml. of 0.2 M-NaN, + 1.6 ml. of 0.2 M-EDTA (pH 8.1) at OOC. Lysozyme (0.8 ml. of 6.4 mg/ml. in 0.25 n/r-Tris*HCl, pH 7.4) was added and the mixture held at 0°C for 20 min. Then 10.4 ml. of cold 0.7% sodium sarcosinate in TE buffer was added. The mixture cleared rapidly and after 5 min at O”C, it was heated at 70°C for 10 min to inactivate nucleases. After cooling to room temperature, 2.0 ml. pronase (1 mg/ml., predigested and heated according to Young $ Sinsheimer, 1967b) was added and the mixture incubated at 37°C for 2 hr. The total volume was made up to approximately 27 ml. with TE buffer, and 25.0 to 25.5 g of the very viscous lysate mixed with 31.8 g CsCl. After an initial gentle mixing it was allowed to stand overnight in the refrigerator. After low-speed centrifugation a small amount of brownish solid was removed from the top. The clear solution, density 1.725 g/ml., was then dispensed by means of a wide bore pipette into six polyallomer tubes (5 ml. in each), overlaid with mineral oil and centrifuged in a Beckman SW40 rotor at 15’C. After 20 to 24 hr at 30,000 rev./min the speed was lowered to 15,000 rev./min for a further 3 days. Fractions were collected by allowing the solution to drip slowly (0.5 to 1 hr/tube) through a wide bore needle (0.7 mm internal diameter) inserted at the bottom. After measuring the acid-precipitable radioactivity throughout the region of the light DNA, appropriate fractions were pooled and dialyzed against 4 x 500 ml. lots of TE buffer-O.1 M-NaCl. Solid CsCl was added to give a density of 1.725 g/ml. in half the volume of the first CsCl centrifugation step and the material was refractionated as before. The final pooled fractions were dialyzed against SSC, concentrated to 1.0 ml. by dialysis against 20% polyethylene glycol (Carbowax 6000) in SSC and then finally dialyzed against several changes of SSC. The final volume of 1 to 2 ml. had an Azao = 0.8 to 1.6. was 1.9. A 23OlA23Cl of all preparations Two variations on this procedure were tried in attempting to reduce the exposure of the DNA to shear forces. In one variation, used for preparations DII, DIV, EII and EIII, the second CsCl density gradient fractionation and final dialysis concentration steps were eliminated. In a second variation, used for preparations DV and EIV, the HH and LH species were removed from the first CsCl density gradient tube. To the remaining solut,ion in the tube, which still contained the LL species, more CsCl solution was added. The mixture was centrifuged again and collected through a large (l-mm) hole. The dialysis concentration step was also omitted. (e) Analytical

cesium

chloride

density-gradient

centrifugation

A volume of 0.505 ml. of the sample (in TE buffer) was mixed with 0.696 g of CsCI, loaded into a centrifuge cell (Kel-F centerpiece, 12 mm, 4”) and centrifuged for 16 hr at 44,000 rev./min and 25°C. Photographs were taken and scanned with a Joyce-Loebl microdensitometer.

522

R. G. WAKE,

A. D. KAISER (f) DNA-DNA

AND

R. B. INMAN

hybridization

The method of Warnaar & Cohen (1966) was used except that the input DNA samples were denatured by heating for 7 min at 100°C in 0.5 M-KOH (Tomizawa & Ogawa, 1968). Using a final volume of 0.2 ml. and 9 pg DNA on a 6-mm filter (Schleicher & Schuell type B6), the number of counts bound (after subtraction of the blank) was directly proportional to the input counts up to a total input of at least 0.12 pg for both A-X and E. coliE. coli DNA hybridizations. 3H-labeled X DNA was kindly provided by G. Ordal and aHlabeled E. coli DNA by Dr Stanley Cohen. hc18.57 DNA for loading on to the filters was prepared as described in section (a), E. coli DNA was purified from the non-lysogenic strain 159T- by the method of Marmur (1961). The input DNA was measured by precipitating portions of the alkali-denatured sample, containing 50 pg calf thymus DNA carrier, with 2 vol. ice-cold 10% trichloroacetic acid and collecting after 30 min at 0°C on 2.4.cm Whatman GF/C filters. They were washed with ice-cold 0.2 M-HCl, then alcohol, and dried before counting. It was found that trichloroacetic acid precipitated 70% of the radioactivity, which could be detected by spotting the input sample directly on to membrane filters for /\, E. coli or a purified head-mutant DNA. Because trichloroacetic acid does not precipitate all of the radioactivity, the extent of hybridization of total DNA may be only 70% of that obtained relative to trichloroacetic acid-precipitable counts. To obtain the most reliable comparison of radioactivity in input DNA samples, the samples were counted under exactly the same conditions of quenching which precipitation and washing give. Using the above procedure, the extents of hybridization for the four possible combinations in various experiments were as follows :

Input

DNA

DNA

A E. coli E. cozi h t Relative

Per cent hybridized?

on filter

h E. coli x E. coli to trichloroacetic

60-75 25-35 <2 2-4

acid-precipitable

input counts.

The small amount of E. coZi DNA binding to h DNA filter has been ignored in the analysis outlined below. The percentage radioactivity in a head-mutant preparation present as h and E. coli DNA has been obtained by comparing the extent of hybridization to h DNA and to E. coli DNA bound to filters after correcting for the hybridization efficiencies of X DNA from phago particles and E. coli DNA, passing through all steps in parallel with the unknowns. A single batch of DNA filters was used in each comparison. (g) Infectivity

assay for

h DNA

Helper-infected bacteria were prepared by the procedure of Kaiser & Inman (1965) employing Yanofsky’s strain 9605 which is su + h sensitive as recipient and himm4a4 QamZl Ram60 as the helper. In some cases the cells were frozen under the conditions described by Bode & Kaiser (1965) and stored in liquid nitrogen until use. Assays were carried out as described by Kaiser & Inman (1965). The indicators for immA and imma” were C600 (immaO), C600 (imm434) (h%mmso a@‘) and W3101 (imm434) (hi80 imm”), respectively. Specific infectivities for hc1857 DNA were generally in the range 1 to 3 x lo9 plaques/A,,, unit. (h) Sedimentation

velocity

in sucrose gradients

This was carried out in a manner similar to that described by Radding & Kaiser (1963). Linear 5 to 20% sucrose gradients (25.5 ml. total vol.) were prepared in 1 in. x 3 in. polyallomer tubes by means of a Beckman gradient former. In all cases at neutral pH,

h READ-MUTANT

DNA

523

TE buffer was used and in some cases N&l was present at a final concentration of 1.0 M. Alkaline gradients contained 0.3 M-NaCH + 0.7 M-NaCl + O+OOl M-EDTA. A volume of plastic l*O-ml. pipette 0.5 ml. of the solution to be analyzed was layered from a wide-bore directly on top of the gradient. Samples were centrifuged in the Beckman SW251 rotor at 5°C. Radioactivity in the gradient fractions was measured after precipitation of portions with trichloroacetic acid as described in section (f). Infectivity was measured on sampies diluted sufficiently to ensure a linear response between input DNA and number of plaques. (i) Examination Preparation of Huberman (1968) t’ion technique has dialyzed into 0.02

of DNA

by electron microscopy

native DNA followed the aqueous spreading technique described by using a small, plastic spreading trough. The high pH partial denaturaalready been described (Inman & Schnos, 1970). The DNA samples were M-NaCl, 0.005 M-sodium EDTA, at pH 7.5 before partial denaturation.

3. Results (a) Isolation

of h DNA

from

induced lysogens by shift in density label

Following either infection of E. coli with h or induction of a h lysogen, host DNA synthesis continues (Joyner, Isaacs, Echols & Sly, 1966). However, host DNA synthesis can be inhibited preferentially by exposing the bacteria before induction to mitomycin C (Young & Sinsheimer, 1967a,b; Kiger & Sinsheimer, 1969). We have used this preferential inhibition to label A DNA with density and radioactivit)y markers. Density shift procedures have also been developed independently by Weissbath et aE. (1968) and Carter, Shaw & Smith (1969). I n our experiments lysogens were grown in the presence of a density label (bromouracil + thymine, 10 : 1 by weight) until all DNA had been shifted away from the light region. The bacteria were treated with mitomycin C then induced by warming to 44°C. Immediately before induction bromouracil + thymine was replaced by [3H]thymine. Finally, LL DNA was isolated, which, if host DNA synthesis had been completely inhibited, should contain only phage DNA. When incorporation of [3H]thymine into acid-precipitable material was measured after induction of 159T- (XDam123 ind+ ~1857) and mock induction of non-lysogenic 159T-, 8-fold more incorporation was observed in the lysogen over a period of 35 minutes. This degree of inhibition is similar to that reported by Kiger & Sinsheimer (1969). Figure 1 shows the density spectra of DNA extracted before and after thermal induction of a lysogen, treated with mitomycin C and bromouracil as described. Growth in bromouracil + thymine medium is seen in Figure l(a) to shift all DNA away from the LL region and into the position of hybrid (LH) and heavy (HH) species. Following induction (Fig. l(b)) a substantial LL peak, accounting for more than 20% of the DNA, appears. A comparable experiment with an homologous but non-lysogenic strain showed the same pattern before induction, but no LL peak after mock induction. Attempts to reduce the proportion of LH material present at the time of induction by longer growth in bromouracil were fruitless, because in bromouracil + thymine medium the rate of growth decreased shortly after the cell concentration reached about 10g/ml. Two successive preparative CsCl density gradient steps were used to isolate the LL DNA. Replicating h DNA has been reported to be very shear-sensitive (Smith & Skalka, 1966; Salzmann & Weissbach, 1967 ; Kiger & Sinsheimer, 1969), therefore precautions were taken to minimize shear forces. Deproteinization with phenol was avoided and a lysozyme-detergent-pronase lysate was fractionated directly in a

524

R. G. WAKE,

A. D.

KAISER

AND

R. B. INMAN

FIG. 1. Analytical CsCl density-gradient patterns showing the DNA species present before and after shifting a culture of 159T- (kDam123 ind+ ~1857) from a bromouracil to thymine medium, with mitomycin C treatment and thermal induction at the time of the shift (see Materials and Methods for details). (a) Before induction, (b) 40 min after induction, (c) D- DNA obtained by fractionating out the LL species in (b) in two successive preparative gradients (see Fig. 2). The lysates in (a) and (b) were treated with ribonuclease and dialyzed to remove low mol. wt u.v.-absorbing material before analysis.

CsCl gradient. The material is unavoidably subjected to some shear forces during collection of fractions from the bottom of the tubes following centrifugation, but this was minimized by slow drop collection through a large-bore needle. Some 75% of the acid-precipitable radioactivity present in the original lysate remained in the CsCl solution after an initial low speed centrifugation to remove a small amount of solid material. Figure 2 shows that symmetrical peaks of radioactive DNA are obtained in the first and second preparative CsCl gradients. Hybrid DNA should have banded about ten fractions below light DNA, and in other experiments in which all of the fractions from the gradient were assayed, no peak of radioactivity was found elsewhere in the gradient. Essentially complete recovery of radioactivity (80 to 100%) was obtained from these gradients. An analytical buoyant-density spectrum of the product of the second preparative gradient centrifugation is displayed in Figure l(c). No DNA, labeled or unlabeled, is detectable in the LH or HH regions of the gradient. This LL DNA was then shown to be isopycnic, as determined by preparative density-gradient sedimentation with unlabeled, thymine-containing DNA isolated from Xc1857 phage particles. The effectiveness of growth in bromouracil + thymine medium in shifting DNA away from the LLregion of the gradient, and of the purification procedure, can be judged by comparing frames (a) and (c) of Figure 1 which were derived from equal amounts of extract.

A HEAD-MUTANT 6000,

1

1

2nd

CsCl

..--

626

DNA _

.---~

..-~--~-

gradient

r

2000'1000;

i$ 3 Fraction

no

FIG. 2. Preparative CsCl density-gradient fractionation of a lysozyme-detergent-pronase lysate prepared from 159T- (hDamlBSind+ ~1857) at 40 min, following a shift from bromouracil to [3H]thymine medium, with mitomycin C treatment and thermal induction accompanying tbe shift (see Materials and Methods for details). Two successive centrifugations were carried out and lo-$. samples of fractions in the region of the LL species were precipitated with trichloroacetic acid and counted. The fractions collected and pooled in each case are indicated by a bar.

TABLE 1 DNA-DNA

Preparation

hybridization

tests of head-mutant

Percentage radioactivity binding to E. coli DNA A DNA

DNA Percentage? radioactivity as A DNA

DI DIIj DIII DIV DV DVI

78

6

80 101 103 69

12

78-94 75 80-88

32

68-69

EI EII$ EIII EIV

100 96 92 101

3 5

97-100 95-96

t Figures give upper and lower limits. $ DNA preparations DII and EII were purified from a common lysate as described in the text. The percentage of radioactivity as X DNA in DII DNA was estimated directly from the ratio of lrC counts binding to A and E. co& filters, aft,er taking into account t,he different efficiencies of h--A and E. cc&E. coli hybridization.

526

R. G. WAKE,

A. D. KAISER

(b) Hybridization

AND

R. B. INMAN

tests of radiochemical

purity

The head-mutant DNA preparations were examined for their content of h and E. coli DNA by DNA-DNA hybridization, with results summarized in Table 1. The preparations were hybridized separately to A and E. coli DNA immobilized on filters. In all preparations, except DI, the radioactivity hybridizing t.o h DNA plus that hybridizing to E. coli DNA sums to 90 to 100°& This shows that the hybridizat’ion tests give valid measures of X and E. coli DNA sequences. On this basis contamination of the head-mutant DNApreparations by E. coli DNA ranged from 3 to 32%, with 10% or less as typical. The presence of some E. coli DNA in all preparations is most likely the result of residual host DNA symhesis after mitomycin C treatment. In particular, it might arise from the semi-conservative replication of hybrid (LH) E. coZi DNA. A reverse type of density-shift regime was also tried, leading to the isolation of bromouracilsubstituted head-mutant DNA. After growth in the presence of thymine alone, a culture of 159T- (Dam123 ind+ ~1857) was temperature induced and incubated in bromouracil + thymine medium for 40 minutes before collecting the cells for preparation of a lysate. The HH species, which accumulates to a considerable extent in a lysogen but to a barely detectable level in a non-lysogen, was obtained by densitygradient fractionation. However, this preparation, called DVI, was the least pure of all. (c) Sedimentation

properties

of head-mutant

DNA

The most striking feature of the sedimentation profiles at neutral pH has been the presence of significant quantities of material sedimenting faster than linear X DNA of monomer length. To mark the sedimentation of linear monomers a very small amount of @SO DNA was added. In control experiments this DNA was found to co-sediment with mature hcI857 DNA. The @SO marker DNA was then detected by its infectivity. Figure 3 shows the sedimentation of E- DNA at two different ionic strengths. The two profiles are identical, as was the behavior at a lo-fold higher DNA concentration at low ionic strength. Some radioactivity sediments more slowly, but most of it scdiments faster than linear monomer h DNA. Approximately 50% of the input. sediments faster than 1.3 times the marker and there is an appreciable quantity of material in the region of 1.5 to 2.0. The identity of the profiles at low and high ionic strength shows that the fast sedimenting DNA is neither largely single-stranded nor in the form of twisted, covalently closed duplex circles, because the sediment,ation behavior of these structures is markedly dependent on ionic strength (Bode & Kaiser, 1965; Young & Sinsheimer, 1967b). To investigate the possibility that the fast sedimentation was due to aggregation of h DNA through its cohesive ends (Hershey et al., 1963), the effect of heat,ing in SSC at 70°C for 10 minutes and quenching t.o O”C, conditions known to dissociate cohcsivc ends, was examined. The result, showninpanel (c) of Figure 3, is a virtually identical sedimentation profile. Thus there is no evidence for aggregation due to non-covalent joining of cohesive ends. The sedimentation of DNA preparation DI is shown in Figure 4. Again, an appreciable fraction of the radioactivity sediments ahead of the marker and up to at least 1.5 times its rat,e. In this experiment 3.5 pg of DNA were applied to the gradient. DNA preparation DIII had a similar sedimentation profile, which was unchanged over the range of 0.7 to 7 IJ-gDNA applied and, as in the case of E - DNA, heating and fast cooling had no apparent effect on the sedimentation profile.

h HEAD-MUTANT

IO Fraction

DNA

20 no.

527

30

FIa. 3. Sedimentation of E- DNA in neutral sucrose gradients. (a) Sedimentation in TE buffer, (b) sedimentation in TE buffer-l M-NaCl. In these cases, 0.50 ml. of a mixture containing sH-labeled EI DNA (1.3 pg/ml.) and @SODNA (0.015 pg/ml., heated at 70°C and cooled) in the appropriate solvent was layered on top of a linear 5 to 20% sucrose gradient and centrifuged in a Beckman SW25.1 rotor at 5’C and 25,000 rev./min for 5 hr. Fractions were collected and 0.50-ml. samples precipitated with trichloroacetic acid and counted. Portions suitwith helper-infected bacteria. The recoveries of ably diluted were assayed for immsO activity input radioactivity for (a) and (b) were 102 and 83%, respectively. -O-O--, Radioactivity; -@-•--, iwwn@ activity in arbitrary units. (c) Effect of heating on the sedimentation of E- DNA. EI DNA (17 pg/ml. in SSC) was heated at 70°C for 10 min, cooled rapidly to 0°C and diluted with TE buffer to 2.8 pg/ml. @80 DNA (0.03 pg/ml.) was added and 0.50 ml. of the mixture sedimented as in (a). 3H ratios for corresponding fractions of the unheated and heated mixtures, normalized to a mean of 1.0, were calculated for fractions 10 to 29. The down-pointing arrows at the top of each panel idicate the expected positions of molecules sedimenting 1.0, 1.5 or 2.2 times faster than linear monomers.

Comparison of Figures 3 and 4 suggests that a larger fraction of the radioactivit’y sediments ahead of the marker in EI than in DI DNA. To test whether or not this reflects a real difference between the two head-mutant DNA’s, cultures of the 159Tlysogens were induced, and allowed to accumulate DNA in the presence of different radioactive isotopes, [3H]thymine for E- and [14C]thymine for D-. The two cultures were mixed together, extracted, and fractionated. They were carried through one rather than the usual two CsCl density gradient steps. Figure 5 shows that the E- DNA does have a slighly higher average sedimentation

828

R. G. WAKE, 1500

A. D. I

KAISER

AND II-5

R. B. INMAN IO

I

t

Fraction no.

FIG. 4. Sedimentation velocity of D- DNA in a neutral sucrose gradient. 0.5 ml. of a mixture of 3H-labeled DI DNA (7 pg/ml.) and 080 DNA (0.025 pg/ml., heated at 70°C and cooled) in TE buffer was layered on top of a linear 5 to 20% sucrose gradient in TE buffer and centrifuged in a Beckman SW 25.1 rotor at 5°C and 25,000 rev./min for 5 hr. Fractions were collected and O.lO-ml. samples precipitated with trichloroacetic acid and counted. Recovery of input radioactivity was 93%. Samples of suitable dilutions of the fractions were assayed for immsc and imna” (shown in Fig. 10) activity. -O-O-, Radioactivity; -e-e-, imms”.

coefficient that the D- DATA though the difference is not great. There is a great difference, however, between the sedimentation of the mixed DNA and that of the preparations which had been through two preparative CsCl steps, shown in Figures 3 and 4. There is more fast sedimenting DNA; for example, 60% of EII DNA (Fig. 5) sedimented at more than 1.5 times the rate of the marker, whereas 30% of EI DNA (Fig. 3) did so. One possible explanation for the higher proportion of fast sedimenting DNA in the mixed preparation is its fractionation through only one CsCl density gradient and thus less opportunity for shear breakage. This possibility was borne out in subsequent preparations (DIV, DV, EIII and EIV of Table 1) where the purification procedure was varied, as described in Materials and Methods, to reduce exposure of the DNA to shear forces. The sedimentation profiles of these DNA preparations showed amounts of fast sedimenting materials comparable to that in Figure 5. The high sedimentation rate at neutral pH of the DNA isolated from induced headmutant lysogens could be due either to a more compact configuration, or to a higher molecular weight than linear DNA of unit length. The presence of significant quantities of covalently closed, twisted circles has already been ruled out by the identical behavior at high and low salt, at least in the case of Ed DNA. Single strands longer than monomer length could arise only from molecules of higher molecular weight than mature DNA. To investigate the length of single strands in head-mutant DNA, the same mixture of E- and D- DNA which was analyzed in Figure 5 at neutral pH was sedimented in alkali. This was done as soon as the preparation was completed in order to avoid the accumulation of single-strand breaks resulting

Fraction

no

FIG 5. Sedimentation velocity in neutral sucrose of D - and E - DNA isolated together from a common lysozyme-detergent-pronase lysate. The appropriate D- and E- lysogens of 159Twere taken separately through the density shift, mitomycin C, thermal induction procedure outlined in Materials and Methods. In the case of the D- lysogen, [14C]thymine (2.5 rCi/ml.) replaced the usual [3H]thymine (10 @i/ml.) used with the E- lysogen, following induction. Only one CsCl density-gradient fractionation step was used in the isolation of the mixed X species. For sedimentation, 0.50ml. of purified DII plus EII DNA (8 pg/ml.) plus @SO DNA (0.075 pg/ml., heated at 70°C and cooled) was layered on top of a linear 5 to 20%, sucrose gradient in TE buffer and centrifuged as in Figure 4. Fractions were collected and O-04-ml. samples precipitated with trichloroacetic acid for radioactivity counting. The recoveries of input radioactivity were 94 and 97% for 3H and 14C, respectively. Suitable portions of the appropriateby diluted fractions were assayed for imnz*O activity. -O-O-, 3H (E-); --A-A--, r4C (D-); -@--•---, inamss in arbitrary units.

Fraction

no.

FIU. 6. Sedimentation velocity of D- and E- DNA in an alkaline sucrose gradient. A mixture of 3H-labeled EII and r4C-labeled DII DNA’s (see the legend to Fig. 5) at a total concentration of 5 pg/ml. and 3ZP-labeled X DNA (0.4 pg/ml.) was allowed to stand in 0.2 MNaOH, 0.35 M-NaCl, 0.001 M-EDTA at room temperature for 20 min. After cooling to O”C, 0.50 ml. was layered on top of a linear 5 to 20% sucrose gradient in 0.3 ix-NaOH, 0.7 M-NaCl, 0.001 MEDTA and centrifuged in a Beckman SW25.1 rotor at 5°C and 23,000 rev./min for 8.5 hr. Fractions were collected and precipitated with triohloroaoetio acid for counting. Recoveries of input 3H, ‘% and saP were 105, 98 and 104%, respectively. -O-O-, 3H (E-); -- A-A--, “C (D-); -.-.-, =P.

530

R. G. WAKE,

A. D. KAISER

AND

R. B. INMAN

from 3H decay. The result, shown in Figure 6, is that approximately 60% of both radioactive DNA’s sediment ahead of the marker for strands of monomer length. In the case of the E- DNA, which carries the 3H label, approximately 30% moves with a rate > 1.5 times the marker. Hybridization analysis, Table 1, indicated that at least 95% of the 3H-labeled DNA is X DNA. This experiment therefore confirms the existence of molecules with covalently continuous lengths greater than one h unit. (d) Structure

and length distribution

of head-mutant

DNA

To determine the size and shape of the phage DNA which accumulates during multiplication of a head-defective hmutant, DNA purified as described above was spread in a film of cytochrome c, according to Kleinschmidt’s procedure (see Materials and Methods section (i)), shadowed, and examined in the electronmicroscope. Allmolecules which were sufficiently spread to be traceable were photographed andmeasured. A total of 67 molecules from DNA preparation DIV and 75 from preparation EIII were photographed and measured. Of the 67 molecules of DIV, 64 were linear and. three were circular. The three circles had lengths 0.94, 1.01 and 1.97 X units. Of the 75 molecules of EIII examined, 71 were linear and four were circular. The four circles had lengths 1.03, 1.05, 1.14 and 2.04. The distribution of lengths of all molecules measured is given in Figure 7. Most, of the molecules are greater than monomer length and some of the molecules are greater than tetramer in length. 20

,

,

,

,

,

,

/

,

I

I

I

I

1

1

g 15-

1

I

E-DNA

D-DNA

.'

2

6 0

I.0

2.0

3.0

I.0

4.0>40 Length-X

20

3.0

40

unlls

FIQ. 7. Length histograms are shown for 67 DNA molecules from DIV DNA and 75 from EIII DNA. The lengths were measured on electron miorocrgraphs and are expressed relative to tho length of DNA molecules isolated from hcI857 phage particles, spread under the same conditions.

There are two arguments that most, if not all, of the long DNA molecules in these preparations are X DNA and not contaminating E. coli DNA. One argument is based on an examination of the partial denaturation profile of individual molecules and this experiment will be described in the following section. The second argument is based on a comparison of the total mass of X DNA to the total mass of long molecules. In preparing DIV and EIII DNA, [14C]thymine was added to the culture medium along with bromouracil, before induction, to label E. coli and prophage DNA. Then at the time of induction only [3H]thymine, to label newly synthesized DNA, was present. The distribution of 14C and 3H between the density species in the preparative C&l gradient, and the distribution of u.v.-absorbing material from an analytical C&l

X HEAD-MUTANT

DNA

531

gradient,weremeasured. Thisinformationin conjunction with the 14C/3H ratios of the unfractionated lysates and the final products, permitted calculation of the degree of contamination of the purified material by host DNA. To calculate the maximum possible contamination it was assumed that all of the 14Cwas in host DNA. On this basis it was found that DIV DNA contained less than 26% by mass, host DNA an.d the EIII DNA less than 30% host DNA. By contrast, 46% of the mass of DIV DNA was in molecules of more than twice monomer length and 52% of the mass of EIII DKA was in molecules of more than twice monomer length. On this basis, molecules longer than dimers make up at least 20 to 46% of the DNA mass in preparation DIV and 22 to 52% of the DNA mass in preparation EIII. (e) IdentiJcation

and location of h monomer units in head-mutant DNA

To determine whether there are individual DNA molecules which contain more than one monomer unit of A DNA we have made use of the characteristic denaturation map of h DNA. It has been demonstrated that denaturation at a temperature or pII below the T, or pH, of the entire molecule occurs in unique zones (Inman, 1967 ; Inman & Schnos, 1970). These zones are probablythesegments most rich in A*T base pairs. The positions of the first three zones should give an unambiguous identification of the position and orientation of h DNA monomers in a polymeric molecule. Both E- and D- DNA were examined by denaturation mapping. EIII DNA was refractionated, after one CsCl density gradient step, by preparative sedimentation velocity through a neutral sucrose gradient. Fractions corresponding to molecules averaging twice unit length were used for denaturation mapping. DV DNA was not refractionated on a sucrose gradient, but was examined directly after two CsCl density gradient steps. However, analytical sucrose-gradient sedimentation of D DNA revealed that the bulk of the radioactivity sedimented faster than mature h DNA. In fact the profile was quite similar to that shown in Figure 3. Both E- and D- DNA samples were prepared for denaturation mapping at pH 11.1. To test the hypothesis that these molecules are linear polymers of X DNA monomers joined head to tail, a gauge was constructed showing the expected position of denatured zones in three X DNA monomers joined head to tail. (The data used to construct the gauge are given in Fig. 8(c) .) The gauge was laid against the denaturation map of each of the molecules and was slipped to the right or left, and reversed if necessary until, by eye, an optimal matching was obtained between the positions of denatured zones in the molecule and the positions of expected zones on the gauge. It was usually possible to obtain a good linear correspondence between denatured segments on the gauge and those on the molecule. In the case of DV DNA, 39 partially denatured molecules were photographed. Forty-nine per cent of these molecules had a repeated h-like sequence. These 19 molecules are listed in Figure 8(a). A further eight (20%) were circular, having X DNA length and a h-like denaturation map; the remaining molecules consisted of ten (26%,) with a non-h-like map (presumed to be E. coli) and two (5%) with maps too short for unambiguous identification. In the case of EIII DNA, a total of 72 molecules were photographed and 51 (71”/;,) exhibited denaturation maps consistent with a repeated X sequence. Only the first 31 of these 51 molecules are depicted in Figure 8(b). Five molecules (7 %) were found to be simple circular structures with a h-like denaturation pattern (circles were selected while scanning the sample and are therefore actually present in lower proportions tha.n

Length

( pm)

250

30.0

(b)

5.0

lcm

15.0

20.0

35.0

40.0

450

50-O

I”

Length

(pm)

FIG. 8. (a) Denaturation maps of DV DNA at pH 11.1. Each horizontal line represents a DNA molecule and the black rectangles denote the size and location of denatured sites. Maps have been plotted with a direction and an alignment giving the best visual fit of the denaturation pattern with the X control shown in (0). (b) Denaturation maps of EIII DNA at pH 11.1. Only the first 31 of 51 molecules examined are shown. (c) Denaturation of computed head-to-tail trimers of Xc1857 DNA. Each horizontal line represents the expected map resulting from the head-to-tail union of 3 real monomeric X denaturation maps (pH 11.1). The left half of a X denaturation map is defined as the G+C richer (or less denatured) half of the molecule. According to the scale used in this Figure, mature ends of X DNA should be situated at 0.0, 17.5, 35.0 and 52.5 pm.

X HEAD-MUTANT

DNA

533

7 %). The remaining molecules consisted of six (8%) which were too short to allow for unambiguous mapping and ten (14%) which had denaturation maps unlike a repeated X-like sequence, and which we assume to be E. coli DNA. The interval between repeats in the pattern of denatured sites in the DV DNA molecules was found to be the same as the average length of partially denatured monomer circles in the same preparation, 18.4 +0*9 microns. The repeat interval in the EIII DNA was 17.5 pm based on the nominal microscope magnification calibration, whereas X DNA is usually 18.9 pm long at the degree of denaturation employed. However, the microscope calibration was subsequently found to be inaccurate yielding lengths that were about 15% too small. In view of the possibility of length discrepanc,y we tested this DNA for deletion or insertion relative to normal h : DNA lengths from hEam13cI857 (the phage carried by the strain from which EIII DNA was generated) and from hEcI857 were measured with phage P4 DNA as an internal standard. Both DNA’s were of similar length and equal to 17.60 *0*3 pm and 17.58 f0.3 pm, respectively. We conclude that the repeat interval in EIII DNA is the normal h monomer length, but cannot assert this to an accuracy better than 15%. To permit comparison of the D- and E- DNA maps with each other, and with the maps of wild-type X at different degrees of denaturation (Inman & SchnGs, 1970), all three have been scaled down in Figure 8 to a monomer length of 17.5 pm, the length of undenatured X DNA under our spreading conditions. All but the six shortest maps in Figure S(a) and (b) show at least two repeated sequences. The exceptions are the last three in (a) and the last three in (b). The longest molecules, of which the first molecule in (b) is an example, are consistent with three repeated segments. Another comparison of the pattern of denatured sites in the long molecules with that of DNA isolated from phage particles was made. In Figure 9, the frequency distribution of denatured sites, as a function of distance from the visually deduced positions of the left and right ends of a A DNA monomer, can be compared with the corresponding frequency distribution for DNA isolated from phage particles. Figure 9(a) and (b) are histograms based on all molecules in DV and EIII DNA exhibiting a X-like denaturation pattern. The structures were artificially opened up at the position(s) corresponding to mature ends, as judged by their individual denaturation pattern, then each of the resulting linear fragments was aligned either at 0.0 or at 17.5 ,um on the denaturation map of the mature molecule. The resulting histograms are quite similar to that for h DNA isolated from phage particles presented in Figure 9(c). The differences in peak heights between D- , E- and f DNA, apparent in Figure 9, indicate that the three preparations were not denatured to the same extent. We believe this is due to slight sample to sample variation in temperature or pH (however, all were adjusted to pH 11.1) rather than to differences in ease of denaturation, because similar variation has been observed between replicate experiments with the same DNA. In spite of the differences in height, the similarity of position and shape of the peaks, evident by comparison of Figure 9(a), (b) and (c), is strong and direct evidence that some 49 and 71%, for D and E DNA respectively, of the long intracellular molecules are in fact h genomes joined head to tail by covalent linkage. (A select,ed sucrose gradient fraction of EIII DNA was examined microscopically. Since the 14C13Hratio in that fraction was the same as that of the material applied to the gradient, the purity of the fraction is a good estimate of the purity of the entire EIII: preparation.)

534

R. G. WAKE,

0.60

A. D. KAISER

AND

R. B. INMAN

-

(b) g i,oo2 ,” z 0+30‘ij 5

0,60-

I .ooI-

0.60

(c)

-

2.5

5.0

7.5 Length

IO.0

125

15.0

17.5

(pm)

FIG. 9. Histograms representing the frequency of denatured sites at each posit.ion on t.he ,%DNA molecule. All three are weight averaged (Inmen, 1967). broken at positions (a) DV DNA. Maps of the 19 molecules shown in Fig. 8 (a) were artificially corresponding to the mature ends (17.5 pm and 35.0 pm). The histogram was then generated by alignment of the artificial left and right ends at 0.0 pm and 17.5 pm respectively. (b) XIII DNA. All 51 molecules analyzed are represented. The maps were artificially broken as described in (a). (c) + DNA. DNA isolated from phage particles of /\~I857 are represented.

Although denatured sites in lI- and E- DNA were usually at correct positions, in some cases they were longer than expected. For instance the top map in Figure 8(b) shows two sites centered at 13 and27 pm, whicharelocated at correct positions but are much too large. Moreover, large single-stranded regions were often observed at the

,I HEAD-MUTANT

DNA

535

ends of molecules ; see for instance molecules 10, 11, 12 and 13 in Figure 8(b). These single-stranded regions have been recorded as part of the denaturation maps, but they may not be due to high pH denaturation because single-stranded regions were also observed in these molecules before partial denaturation, but under conditions which would resolve single-stranded material. Thus many of the large single-stranded regions at ends of molecules, and perhaps some of these within molecules, are not strictly part of the denaturation map. The real ends of the long molecules seem to bear no fixed relationship to the positions deduced for the joined cohesive ends. There is a tendency for the ends of the D- DNA molecules to cluster around the origin of replication. Ten among nineteen molecules terminate in the 13 to 15 pm interval and the replication origin has been mapped at 14.3 pm (Schn& & Inman, 1970). However, more molecules are needed to establish the significance of this point. On the other hand, in the selected population of E- DNA molecules examined the ends occur at approximately random positions, as if they had been derived by random breakage of longer structures. (f) Infectivity

of head-mutant

DNA

Free h DNA can donate phage genes to recipient bacteria which have been infected with a helper phage and that donation is dependent upon the presence of cohesive ends on the donor DNA (Kaiser & Wu, 1968). Under optimum conditions, the efficiency can be one gene donated per hundred molecules of mature h DNA. DNA with donor activity can also be extracted from bacteria which have beeninfected with wildtype X, but only during the second half of the latent period when complete heads are formed. On the other hand, bacteria infected with X mutants defective in head genes A, B, C, D or E do not produce DNA with donor activity (Dove, 1966; Mackinlay tSt Kaiser, 1969). The absence of donor activity in the h DNA which accumulates in these infected cells suggests that the DNA lacks cohesive ends. However, the DNA extracts tested in those experiments had not been fractionated and consequently they contained large amounts of bacterial DNA, which may have influenced activity measurements. The availability of purified D- and E- DNA permits assay of donating activity without possible competitive inhibition by bacterial DNA and also permits a determination of the donating activity per unit mass of h DNA. In Table 2 the specific donating activities of several purified h D- and h E- DNA preparations are summarized. In all the cases the donating activity was low. However, D- DNA appears to TABLE

XpeciJic donor activity DNA Preparation DI DIII DVI EI

2

of head-mutant

DN,4

Donor activity 0.6 x 1O-3 0.6- 3 x 10-3t 2x10-3 2x10-5

Donor activity is expressed as the fraction of the activity of an equivalent mass of DNA extracted from Xc1857 phage particles. t The lower value in this range was obtained after the preparation had been stored for 2 months. 35

536

R. G. WAKE,

A. r). KArsm

3.02-0 -i“

before heat iA after heat

AND

2.0

I.0

+

+

0

0

I.0 -

o

0

R. B. INMAN

cl

O. ‘“0”

o”

oI IO

I 20 Fraction

I 30

no.

FIG. 10. A comparison of the effect of heating and cooling on the sedimentation of the immA aotivity of D- DNA in neutral sucrose. 3H-labeled DI DNA was mixed with @SODNA and sedimented through a linear 6 to 20% suerose gradient in TE buffer. This is part of the experiment described in Fig. 4. Another sample was prepared by first heating DI DNA (41 pg/ml. in SSC) at 70°C for 10 min and cooling rapidly to 0°C. The sample was diluted immediately to 7 pg/ml. with TE buffer mixed with @SODNA and sedimented under identical conditions. Fractions were collected and assayed for immh and immsO activity, and counted for radioactivity. In the top panel a comparison of the specific immh activity in fractions 14 to 29 of the unheated DNA is made. In the lower panel E comparison of the immr activities in corresponding fractions from the unheated and heated samples is made. The peak of immso activity was used to define the position (1.0) of linear, monomer molecules.

have a significantly higher activity (lo- 3, than E - DNA (2 x 10 - 5). The same small but definite activity of DNA from D - , in comparison with E - and A - , has also been observed in unfkactionated extracts prepared at various times following induction of lysogenic bacteria. For example, at 40 minutes after thermal induction, the extracts of su- lysogens of Dam15 or Dam123 had a tenfold higher activity than extracts of induced Aam or Earn4 lysogens. The activity of the induced D - lysogens was 10 - 3 to 1O-2 the activity of the extract of an induced am+ lysogen. The activity of D- DNA cannot be accounted for by the presence of sus+ revertants, because the frequency of revertants is too low. Upon induction of 159T(h Dam123 ind+cI857) fewer than one am+ in lo7 was observed. Furthermore, the donating activity has two properties which distinguish it from mature h DNA. In the first place, as shown in Figure 10, much of the donor activity of D- DNA sediments faster than mature DNA of monomer length, even after heating and quenching under conditions which would dissociate molecules held together by hydrogen-bonded cohesive ends. Reference to Figure 4 shows that approximately 50% of the radioactivity (3H) in this preparation sediments faster than monomers. Secondly, DDNA is not able to form monomer circles by cohesion as shown in Table 3. Circles do not have donor activity for helper-infected bacteria (Kaiser & Inman, 1965). This behavior is an inherent property of the D- DNA and is not due to the presence of some factor inhibiting circularization, because admixed @SODNA does form circles.

h HEAD-MUTANT

TABLE

DNA

637

3

Donor activity D-h DNA

080 DNA (admixed)

Experiment

(a)

Before annealing After annealing Annealed, reheated

1.0 0.65 0.91

1.0 0.06 1.1

Experiment

(b)

Before annealing After annealing Annealed, reheated

1.0 0.85 o-94

1.0 0.18 0.98

Monomer h DNA (parallel run)

1.0 0.08 0.91

In experiment (a), unfractionated DI DNA was used. In experiment (b), that fraction of DII DNA sedimenting as h linear monomers obtained by fractionation on a sucrose gradient was used. Annealing was carried out by heating samples in TE buffer-O.5 M-NaCl to 76°C for 10 min and allowing to cool to room temperature over a period of approximately 16 hr. Reheating was achieved by holding samples at 75°C for 10 min and cooling rapidly to 0°C. In the mixture of D- h DNA and @SODNA the D- DNA was followed by immA donor activity and the 4180 DNA by immss donor aotivity.

Similar differences in behavior were also observed when unpurified extracts prepared from W3101 (X D-) and W3101 (h+) were compared in the same way (Veldhuisen & Kaiser, unpublished observations).

4. Discussion Even though bacterial DNA synthesis continues while X multiplies, intracellular ,I DNA can be separated from E. coli DNA in extracts of induced lysogens. A combination of mitomycin C treatment before induction, with a shift from heavy to light density label at the time of induction followed by density fractionation, yields light radioactive DNA which is at least 80 to 95% X DNA by DNA-DNA hybridization tests. Some preparations of both D- and E- DNA contained more than 98% of their radioactive label in X DNA. Because radioactivity bound to DNA filters was measured in the hybridization tests, contamination by non-radioactive E. coli DNA would not have been detected. Light (LL) non-radioactive bacterial DNA should, however, have been detected in the analytical density-gradient sedimentation which measure;9 all DNA molecules by their ultraviolet absorbance. The absence of detectable LL DNA in Figure l(a) indicates that the amount of LL host DNA synthesized before induction is less than 5% of the amount of LL h DNA which has accumulated by 40 minutes after induction. Electron microscope studies of partially denatured DNA provides an independent measure of the fraction of the purified DNA which has the h base sequence. Among the molecules long enough to have a recognizable pattern of denatured sites, 71 o/oof the mass of DV DNA was h-like, as was 82% of the EIII DNA. Thus the density shift procedure can yield h DNA which is both radiochemically and chemically almost pure. Lambda DNA, isolated in this way from induced lysogens for head-defective mutants D- or E-, is a collection of covalently joined linear aggregates of X DNA monomers joined head to tail. Evidence for this proposition can be summarized as follows. Electron microscopic examination of undenatured DNA shows it to be a population of linear molecules of variable length. Among 142 molecules examined,

538

R. G. WAKE,

A. D. KAISER

AND

R. B. INMAN

seven (or 5%) were circular, the rest were linear. A wide distribution of lengths was observed microscopically, with about half the molecules two or more times monomer length and some at least tetramer in length. Examination of the molecules after partial denaturation showed a repeating pattern of denatured sites with a repeat distance equal to the length of monomer DNA isolated from phage particles. Microscopy permits examination of a limited number of molecules, but sedimentation velocity experiments describe the entire population. The sedimentation velocity distribution of purified D- and E- DNA preparations at neutral pH shows a large fraction of material sedimenting 15 to 2.0 times the rate of linear /\ DNA of monomer length. Two facts weigh against the fast-sedimenting material being an artifact of drop collection caused by viscous E. coli DNA in the lower part of the gradient, a phenomenon described by Kiger & Sinsheimer (1969). First, the amount of E. coli DNA in the preparations examined here is very low because it was separated away in a previous buoyant density step. Second, the sedimentation profile is found to be constant over a IO-fold range of DNA concentration, Failure to observe a dependence on concentration can be explained by heterogeneity in length, such that the actual DNA concentration within any region of the gradient is too low for significant DNA-DNA interaction (Burgi & Hershey, 1963). Assuming, on the basis of the electron microscopical results, that all of the molecules are linear duplexes, then the molecular weight should be approximately proportional to the 2*5t power of the sedimentation velocity. Thus, sedimentation at 1.5 to 2.0 times the linear monomer rate implies lengths of 2.8 to 5.7 times monomer. For preparation EII, then, as shown in Figure 5, more than half the molecules were longer than dimers. Sedimentation of the same DNA at alkaline pH revealed more than 50% of the polynucleotide mass sedimenting faster than monomer length single strands ; substantial amounts sedimented faster than 1.5 times monomer. In preparation EII for example, 30% of the single strands did so. On the basis of Studier’s equation for the relation between sedimentation velocity and mass at alkaline pH, linear molecules sedimenting at 1.5 times faster than monomer would be 2.7 times as long (Studier, 1965). Thus both the sedimentation and the electron microscopical experiments show that most molecules are greater than monomer length and that many are longer than dimers. The proteins specified by genes D and E have been identified as the major structural components of the completed phage head. The E protein with a molecular weight of 38,000 accounts for 75% of the protein mass of the head (Casjens, Hohn & Kaiser 1970; Buchwald, Steed-Glaister & Siminovitch 1970). The D protein has a molecular weight of about 12,000 and makes up about 24% of the head protein mass (Casjens level et al., 1970; Murialdo & Siminovitch, 1971). Alow, andapproximatelyequivalent, of enzyme activity which generates cohesive ends, in vitro, from added circular X DNA can be detected in crude extracts of induced D- and E- lysogens (Wang & Kaiser, unpublished data). Therefore it seems likely that D and E proteins are required for the formation of cohesive-ended monomers, not because either of these proteins is the enzyme which cuts replicative DNA into linear monomers, but because these proteins are essential parts of the head structure. t Taken as the average of the values determined, in one case, for the condition of zone sodimentation through sucrose gradients (Burgi & Hershey, 1963) and, in the other case, for boundary sedimentation in the analytical oentrifuge (Crothers & Zimm, 1965).

,I HEAD-MUTANT

DNA

539

Why should D and E protein be needed to form h DNA monomers Z This question can be answered in two ways, depending on the mechanism one assumes for head assembly. If head precursor DNA is assumed to be cut into linear monomers before a monomer is encapsulated by head proteins, then, in the absence of head protein, DNA monomers might rejoin and accumulate as polymers. An argument against this mechanism arises from the data of Skalka, Poonian & Bartl (1971); for they find head-to-tail polymers of h DNA in bacteria infected with wild-type X where head assembly should be proceeding normally. An alternative explanation is more attractive. If head precursor DNA is cut into monomer units as an integral part of the process by which the head proteins assemble, then the absence of one of the structural proteins would prevent cutting and cause head precursor DNA to accumulate. This hypothesis might also explain why large or small head variants of phage T4 contain correspondingly large or small molecules of DNA (Mosig, 1968). Mrs T. Masuda provided invaluable assistance in these experiments. This work was supported by a grant from the National Institutes of Health (AI-04509) to one of us (A. D. K.) and by grants from the National Institutes of Health, American Cancer Society and the University of Wisconsin Graduate School to another author (R. B. I .). The other author (R. G. W.) is a Fulbright Research Scholar on leave from the Department of Biochemistry, University of Sydney, Australia. REFERENCES Bode, V. & Kaiser, A. (1965). J. Mol. Biol. 14, 399. Buchwald, M., Steed-Glaister, P. & Siminovitch, L. (1970). l’i’irology, 42, 375. Burgi, E. & Hershey, A. (1963). Biophys. J. 3, 309. Carter, B., Shaw, B. & Smith, M. (1969). Biochim. biophys. Acta, 195, 494. Casjens, S. (1971). In The Bacteriophage Lambda, ed. by A. D. Hershey, p. 725. New York: Cold Spring Harbor Laboratory. Casjens, S., Hohn, T. & Kaiser, A. (1970). l’irology, 42, 496. Casjens, S., Hohn, T. & Kaiser, A. (1972). J. Mol. BioZ. 64, 551. Crothers, D. & Zimm, B. (1965). J. Mol. BioZ. 12, 525. Davidson, N. & Szybalski, W. (1971). In The Bacteriophage Lambda, ed. by A. D. Hershey, p. 45. New York: Cold Spring Harbor Laboratory. Dove, W. (1966). J. Mol. BioZ. 19, 187. Hershey, A., Burgi, E. & Ingraham, L. (1963). Proc. Nat. Acad. Sci., Wash. 49, 748. Huberman, J. (1968). Cold Spr. Harb. Symp. Quant. BioZ. 33, 509. Inman, R. (1967) J. Mol. BioZ. 28, 103. Inman, R. & Schniis, M. (1970). J. MOE. BioZ. 49, 93. Joyner, A., Isaacs, L., Echols, H. & Sly, W. (1966). J. Mol. BioZ. 19, 174. Kaiser, A. & Hogness, D. (1960). J. Mol. BioZ. 2, 392. Kaiser, A. & Inman, R. (1965). J. Mol. BioZ. 13, 78. Kaiser, A. & Wu, R. (1968). Cold Spr. Harb. Symp. Qwmt. BioZ. 33, 729. Kiger, J. & Sinsheimer, R. (1969). J. Mol. BioZ. 40, 467. Mackinlay, A. & Kaiser, A. (1969). J. Mol. BioZ. 39, 679. Marmur, J. (1961). J. Mol. BioZ. 3, 208. Mosig, G. (1968). Genetics, 59, 137. Murialdo, H. & Siminovitch, L. (1971). In The Bacteriophage Lambda, ed. by A. D. Hershey, p. 711. New York: Cold Spring Harbor Laboratory. Parkinson, J. (1968). Genetics, 59, 311. Ra,dding, C. & Kaiser, A. (1963). J. Mol. BioZ. 7, 225. Salzman, L. & Weissbach, A. (1967). J. Mol. BioZ. 28, 53. Schnos, M. & Inman, R. (1970). J. Mol. BioZ. 51, 61. Skalka, A., Poonian, M. & Barth P. (1972). J. Mol. BioZ. 64, 541.

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G. WAKE,

A. D.

KAISER

AND

R. B. INMAN

Smith, M. & Skalka, A. (1966). J. Gem. PhysioZ. 49, 127. Studier, W. (1965). J. Mol. BioZ. 11, 373. Tomizawa, J. & Ogawa, T. (1968). CoZd Sp. Hurb. Symp. Q,uant. BioZ. 33, 533. Warnaar, S. & Cohen, J. (1966). Biochem. Biophys. l&s. Comm. 24, 554. Weigle, J. J. (1966). Proc. Nat. Acad. Sk., Wash. 55, 1462. Weissbach, A., Bartl, P. & Salzman, L. (1968). Cold Spr. Harb. Symp. Quant. Biol. Wu, R. & Kaiser, A. (1968). J. Mol. BioZ. 35, 523. Wu, R. & Taylor, E. (1971). J. Mol. BioZ. 57, 491. Young, E. & Sinsheimer, R. (1964). J. Mol. BioZ. 10, 562. Young, E. & Sinsheimer, R. (1967a). J. Mol. BioZ. 30, 147. Young, E. & Sinsheimer, R. (1967b). J. Mol. BioZ. 30, 165.

33,525.