The chromosome of bacteriophage T5

J. Mol. Biol. (1972) 63, 397407

The Chromosome II.

of Bacteriophage

T5

Arrangement of the Single-stranded DNA Fragments in the T5 + and TSst(0) Chromosomes G.

S.

HAYWARD

AND

M. G. SMITH

Department of Biochemistry-f, University of Otago P.O. Box 913, Dunedin, New Zealand and Department of Cell Biology, University of Auckland Private Bag, Auckland, New Zealand (Received 6 July 1971) Denatured bacteriophage T5 DNA contains a large number of single-stranded DNA fragments which have been separated by agarose gel electrophoresis and classified as “major” or “minor” species on the basis of their relative abundances (Hayward & Smith, 1972). For further study of these fragments we have centrifuged denatured T5 DNA in CsCl density-gradients in the presence of poly(G). Gel electrophoretic analysis of fractions from these gradients shows that the 37.0 and 13.9 million major fragments of T5+ DNA and the 35.3 and 17.2 million of TBst(0) DNA are found in the high buoyant density regions. The other fragments vary in the extent of their interactions with poly(G) and a minor fragment, which has anomalous electrophoretic properties, exhibits the strongest poly(G) interaction. Agarose gel electrophoresis has also been used for analysing the results of hybridization experiments with isolated major fragments and for studying the distribution of single-strand fragments in the double-strand breakage products of sheared T5 DNA. From the results of these experiments we have proposed a model for the arrangment of major single-strand fragments in the duplex DNA molecules of T5+ and T&t(O). In our model, both molecules contain one intact strand, and 3.8 and 14.5 million fragments at opposite ends of the other strand, but T5+ contains three “primary” interruptions and TSst(0) contains only two. This difference is explained by a deletion in TBst(0) DNA which eliminates the central T5+ interruption and creates the new 17.2 million fragment of T5st(O) from the 5.1 and 13.9 million fragments of T5 +. The first break by shearing in duplex T5 DNA occurs in the intact strand at a position approximately opposite the interruption separating the 14.5 million fragment from the rest of the “fragmented” strand. We have also studied the single strands of first-step-transfer DNA and suggest that the division between f&t-step-transfer DNA and the rest of the molecule is not associated with a single-strand interruption.

1. Introduction Bacteriophage T5 possesses a double-stranded DNA molecule containing single-strand interruptions and, as a result, denatured T5 DNA consists of a number of singlestranded DNA fragments (Abelson 6 Thomas, 1966; Bujard, 1969; Jacquemin-Sablon t Present

address

of both

authors. 397

398

G. S. HAYWARD

AND

M. G. SMITH

& Richardson, 1970; Hayward & Smith, 1972). Several other unusual properties of the T5 chromosome are thought to be related to the presence of interruptions. These include: (i) a two-step DNA injection process which functionally separates the fstDNAt (8% of the molecular length) from the rest of the molecule (Lanni, 1968); (ii) an asymmetric breakage of the native DNA molecule when sheared, yielding two double-strand fragments of approximately 40 and 60% of the molecular length (Burgi, Hershey & Ingraham, 1966; Rubenstein, 1968); and (iii) an irreversible partial denaturation of native T5 DNA (Hershey, Goldberg, Burgi & Ingraham, 1963) which can be explained by the releaseof a short single-strand fragment from near one end of the duplex T5+ molecule (Bujard, 1969). There have been several attempts to map the arrangement of the single-strand fragments in the intact T5 chromosome.Abelson & Thomas (1966) used the results of sedimentation studies on denatured T5 DNA to propose a series of models, each containing six fragments and with interruptions in both strands ; but, unexpectedly, their results did not suggestany simple relationship between the fragments present in T5+ DNA and those of the heat-stable mutant TBst(0). Bujard (1969) examined partially denatured T5 + DNA in the electron microscope and presented evidence for a different model in which three interruptions divided one strand into regions of 7.9, 11.1, 41 and 40% of the molecular length. In the preceding paper, we showed that T5 DNA contains more than forty singlestrand fragments which can be resolved by agarosegel electrophoresis (Hayward & Smith, 1972). The five major fragments observed in our studies of T5+ DNA had molecular weights of 37.0,14.5, 13.9,5*1 and 3.8 million and they closely resembledthe fragments in Bujard’s model. T5st(O) DNA contained only four major fragments which had molecular weights of 35.3, 17.2, 14.5 and 3.8 million. The remaining minor fragments were thought to arise from subdivision of major fragments by occasional secondary interruptions. Many of the single-strand fragments of T5st(O) DNA appeared to correspond to fragments with similar electrophoretic properties from T5 + DNA. In this paper we provide evidence for a model of the arrangement of fragments in both the T5+ and TBst(0) DNA molecules and we describe experiments designed to test the relationship between the interruptions and the unusual features of these chromosomes.

2. Materials

and Methods

(a) Preparation Growth and purification of 32P-labelled

of bacteriophage DNA T5 phage, and isolation of 32P-labelled T&DNA were performed exactly as describedin the precedingpaper (Hayward & Smith, 1972). (b) Isolation of first-step-transfer DNA E. coli B/2 were grown to 5 x 10s cells/ml. at 37°C in peptone broth and then resuspended at 5 x loo/ml. in the MGM-Ca2+ buffer of Lanni (1961). The cells were starved at 37°C for 10 mm and then infected with 32P-labelled T5 phage at a multiplicity of 10. After 10 min the cells were diluted threefold and stirred at full speed for 5 min in a V&is blender at 4°C. The blended cells were washed twice with chilled MGM-Ca2+ buffer and resuspended in 0.1 M-!&is, 0.01 M-EDTA, 1 m&r-KCN (pH 8). Intracellular DNA from fst-infected cells was isolated by incubation with lysozyme (200 pg/ml.) for 10 mm with 3 cycles of freezing and thawing followed by a phenol extraction (Lanni, McCorquodale & Wilson, 1964). t Abbreviations

used: fstDNA, first-step-transfer

DNA; poly(G), polyriboguanylic

acid.

ARRANGEMENT

OF

(c) Preparative

T5

DNA

caesium chEoride/poly(G)

399

FRAGMENTS

density-gradients

T5 DNA samples in 0.01 M-Tris, 1 mnn-EDTA (pH 8) were mixed with poly(G) (Miles Laboratories Inc., Indiana) at a DNA : poly (G) ratio of 3 : 1 and denatured by the addition of 0.1 vol. of 1 M-NaGH. After 2 min at room temperature, the mixtures were chilled and neutralized with 0.1 vol. of sodium phosphate buffer containing 1.2 M-NaH2PG4 and 0.4 MNa2HP04. Saturated CsCl was added to bring the density at 25°C to I.705 g/cm3 and the volume to 5 ml. The samples were centrifuged for 44 hr at 36,000 rev./min and 4°C in a Spinco 50Ti rotor. Fractions were collected dropwise into vials and 32P radioactivity measured by cerenkov counting (Clausen, 1968). Fractions required for subsequent electrophoretic analysis were dialysed against 0.1 M-NaCl, 0.01 M-T&, 1 mm-EDTA (pH 8) to remove CsCl, followed by dialysis against 0.01 M-Tris, 1 mM-EDTA, 1 y. ethanol (pH 8). (d)

Shearing

and isolation

of breakage products

Native 32P-labelled T5 DNA (0.2~ml. samples at 20 pg/ml. in 0.6 M NaCI, 0.06 M-sodium citrate, pH 7) was sheared by blowing the solution through a selected O-2 ml. pipette. This process was repeated 10 times at 4°C to produce a double-stranded break in approximately 50% of the molecules. Samples of sheared DNA (5 pg) were applied to 13-ml. 5 to 20% sucrose gradients containing 2 M.NaCl, 0.1 M-Tris, 1 mu-EDTA (pH 8) and these were centrifuged at 24,000 rev./min for 16 hr at 4°C in a Spinco SW 40 rotor. IO-drop fractions were collected in vials and assayed by Cerenkov counting. Selected fractions were then dialysed against 0.01 M-Tris, 1 mM-EDTA, 1% ethanol (pH 8) and prepared for electrophoresis.

(e) AnaZyticaE gel electrophoresk Agarose autoradiography 1972).

and

agarose-polyacrylamide were performed

(f)

gel as described

Preparative

electrophoresis, in the preceding

gel

gel fractionation paper (Hayward

and 32P & Smith,

electrophoresis

Individual 3aP-labelled fragments of single-stranded T5 DNA were isolated from normal gel separations of 30 pg of denatured 32P-labelled T5 DNA in 12-mm diameter, 0.6% agarose gels. Longitudinal gel slices were not dried before autoradiography, but were placed in a Perspex holder, covered with thin plastic and autoradiographed directly. The developed film was then used as a template for locating and cutting out the desired sections of the gel slices. The gel samples were dissolved by heating at 70°C for 10 min in 6 vol. of 50% (v/v) formamide, 0.01 M-Tris, 1 mM-EDTA (pH 8). At this concentration (0.1%) the agarose formed a loose gel network which could be removed by centrifuging at 5000 g for 5 min. Recovery of DNA in the supernatant from gel slices was 80 to 90%.

(g) Annealing The single-stranded fragments of T5 DNA, isolated by preparative gel electrophoresis, were self-annealed or mixed in equimolar proportions with other fragments and incubated for 3 days at 25°C in 50% (v/v) formamide, 0.01 M-Tris, 1 mu-EDTA (pH 8). The DNA concentration of the largest fragment was approximately 3 pg/m.l. in these annealing experiments. Samples of annealed mixtures were used directly for gel electrophoretic analysis.

3. Experimental (a) Fragment

distribution

Results

in caesium chloride/poly(G)

density

gradients

We have used the technique of centrifuging denatured DNA in CsClin the presence of poly(G) (Kubinski, Opara-Kubinski & Szybalski, 1966) to study the distribution of poly(G)-binding sites within the T5 chromosome and for further comparison of the properties of corresponding fragments of T5+ and T&t(O) DNA. The buoyant density profiles of T5+ and T&t(O) DNA were very similar and a typical result for T5st(O) DNA is shown in Figure 1. The large heavy band obtained in the presenceof poly(G)

400

G.

S. HAYWARD

Buoyonl

AND

M.

G.

SMITH

dens,!y (g/cm”)

FIG. 1. CsCl density-gradients of denatured T5 DNA in the presence and absence of poly(G). Centrifugation was performed at 36,000 rev./min for 44 hr in a Spinco 50-Ti rotor at 4°C as described in Materials and Methods. Densities were calibrated using the known values for doublestranded and single-stranded TS DNA in the absence of poly(G) and checked by refractive index measurements. 32P-labelled TSst(0) DNA (ss) (a) T&t(O) without poly(G). A sample (27 pg) of alkali-denatured was neutralized and then centrifuged with 12 UP: of native sap-labelled T5st(O) DNA (ds) in neutral CsCl. (b) T&t(O) with polG(G). 3ZP-labelgd TBst(0) DNA (65 pg) and 22 pg of p&(G) were treated with 0.1 M-N&OH, then neutralized and centrifuged in neutral CsCl. Twenty-five IO-drop fraotions were oollected and counted. Samples labelled 1 to 9 were further processed for electrophoretio analysis (see Plate III).

has a density of 1.744 g/cm3 and the smaller lighter band has a peak at approximately 1.725 g/cm3. The region between these two bands also contains a considerable portion of the DNA. Without poly(G) a single band was obtained at 1.718 g/cm3. We have used agarose gel electrophoresis to analyse the distribution of individual fragments within the CsCl + poly(G) d ensity-profiles. Fractions from preparative CsCl gradients of denatured T5 + and T5st(O) DNA were dialysed, treated with alkali and placed on agarose gels for electrophoretic separation. An autoradiograph of TEist(0) DNA fractionated in this manner is shown in Plate I, gels 1 to 9, and the peak densities of prominent, fragments of T5+ and T6st(O) DNA are given in Table 1. The results show that individual fragments have different buoyant densities in the presence of poly(G). The major fragments of T5+ and TBst(0) DNA can be ordered with respect to their relative poly(G)-binding properties as shown in Table 2. In T5st(O) DNA the 3.8 and 14.5 million major fragments and many of the minor fragments appear to have identical poly(G)-binding properties to those of the corresponding T5+ fragments. The 3.8 million major fragment has a buoyant density of only 1.720 g/cm3 and hence may not have any poly(G)-binding capacity. On the other hand, the 8.2 million minor fragment (arrowed in the photograph), which bands at l-756 g/cm3, presumably

401

ARRANGEMENTOFT5DNAFRAGMENTS

TABLE 1 Poly(G)-binding

properties

Mol. wt T5’

37.0 28.3 20.4 -14.5 -13.9 13.0 12.3 11.5 10.0 9.2 8.21 7.4 6.3 5.3 5.1 G 3.8 Ti 3.0 2.7 2.2 1-9 1.8 1.6 1.35 0.9

and distribution in breakuge products single-strand fragments

x 1O-B TBst(0)

Buoyant density in CsCl + poly(G) k/cm3)

35.3 y 22.3 -17,2 -14.5 13.0 12.3 11.5 9.2 &2$ 7.6 7.4 6.3 5.3 4.8 3.8 5% 3.0 2,7 2.2 l-9 1.8 1.6 1.35 0.9

1.745 1-746 1.741 1.743 1.746 1.731 1.747 1.726 1.726 1.726 1.726 1.733 1.756 1.725 1.738 1.723 (1.723)t 1.727 1.722 1.720 1.740 I.740 1.741 1.742 1-744 or 1.721 1.744 or 1.721 1.740 1.721 1.721

Location or 40%

of the larger T5

in 00% product

-

60% 60% 6’3% 40%

60% 40% 40% 40%

‘30%

(4O%H (CO%H 60%

0 (~~~)t ‘30%§ 40%

f3%§ 60%

‘30%

Mol. weights of major fragments are underlined. The density increase observed in the presence of poly(G) varies in different experiments but the relative increase is constant. t Tentative assignments. $ Variable mobility fragment (Hayward & Smith, 1972). 5 The 5.1 and 3.8 million major fragments of T5* appear to be present in the 40% breakage produot also (see text).

contains a greater concentration of poly(G)-binding sites than any other T5 fragment. The density separation reveals a new 38 million minor fragment which was not separated from the 3-8 million major fragment by direct gel electrophoresis. (b) Fragment distribution

in the double-stranded products of sheared T5 DNA

We have used our electrophoretic procedure to study the distribution of singlestrand fragments within the “4Oo/o” and “60%” breakage products of double-stranded T5 DNA (Burgi, Hershey & Ingraham, 1966; Rubenstein, 1968). Samples of double-stranded T5+ and T&t(O) DNA were sheared by pipetting in a manner sufficient to break half the molecules in the population. The sheared samples

402

G.

S. HAYWARD

AND

M.

G.

SMITH

TABLE 2 Relative

afinity

for poly(G)

T5+ TEist(0)

37.0, 35.3,

of the major fragments

from

T5 DNA

13.9 > 14.5 > 5.1 > 3.8 17.2

>

14.5

>

3.8

separated into three main bands of double-stranded DNA when sedimented through neutral sucrosegradients (Fig. 2). The fastest sedimenting band contained unbroken moleculesand the other two bands contained the breakage product,s. Electrophoretic separation of unfractioned shearedDNA after denaturation (Plate II, gels 2 and 6) revealed two new single-strand fragments with molecular weights of 28.3 and 16.0 million in T5 + and 25.5 and 16.0 million in T6st( 0). The amount of DNA at the position of the intact strand was considerably reduced, suggestingthat the two new fragments were breakage products of the intact strand. No other significant alterations in the fragment patterns could be detected. Electrophoretic separations of denatured DNA from the three main peaks in t,he sedimentation profiles of shearedT5 DNA are also shown in Plate II. The 60% breakage product of TEist(0) DNA contained the new 25.5 million piece of the intact strand and the 17.2 and 3.8 million major fragments, and the 40% breakage product contained the new 16.0 million piece and the 14.5 million major fragment. In T5 + the

Froctm

number

FIG. 2. Sedimentation of sheared T5 DNA. Native T5 DNA was sheared as described in Materials and Methods. Samples of sheared DNA (5 pg) were centrifuged through neutral sucrose gradients for 16 hr at 24,000 rev./min and 4°C in a Spinco SW 40 rotor. Forty lo-drop fractions wore collected and counted. (a) Sheared T5+ DNA, (b) sheared TSst(0) DNA. Fractions 13, 17 and 22 of (a) and 15, 19 and 23 of (b) were further processed for electrophoretic analysis (Plate II).

35.3

17 2 14 5

38

I2

3

4

5

6

7

8

9

IO

II

12

PLATE I. Gel separations of T5st(O) fragments after banding in CsCl in the presence of poly(G). Fractions from a preparative CsCl + poly(G) density-gradient of denatured 32P-labelled TBst(0) DNA were dialysed, treated with 0.1 M-Nash and electrophoresed in Tris-acetate buffer through 0.7% agarose gels (6 mm). Mol. wt values (in millions) are given for major fragments. Arrows indicate the variable T5 fragment (see text). Gels 1 to 9. Samples 1 to 9 from the gradient shown in Fig. l(b). Buoyant density increases from right to left. Gels 10 to 12. Controls of unfractionated 32P-labelled T&t(O) DNA. Various amounts of DNA (0.12, 0.3 and 0.70 pg) were applied to the gels to assist in evaluation of the autoradiograph*,.

0

5-

16 O-

I

+

2

3

T551!0)

4

5

7

6

Q

15’

PLATE 11. Distribution of fragments in the breakage products of sheared T5 USA. Peak samples of the 3 main components from neutral sucrose gradients of sheared T5 DSA wow dialysed, treated with 0.1 M-NaOH and electrophorosedinTris-phosphate-sodium dodecyl sulphato buffer through 0.6% agarose gels (12 mm). T&t(O) DNA: gel 1, unbroken molecules; gel 2, control of sheared but unfract,ionated DNA: gel 3, 60% breakage product; gel 4, 40% breakage product.. T5+ DNA: gel 5, unbroken molecules; gel 6, control of sheared but unfractionated DNA; gel 7, 60% breakage product; gel 8, 40% breakage product.

2

3

4

5

6

7

8

9

PLATE III. Isolation and annealing of major fragments of T%t(O) DNA. JIajor fragments were isolated from gel slices and annealed in approximately equimolar proportions as described in Materials and Methods. Samples containing 0.05 to 0.15 pg of DNA in SOW, formamide, 0.01 M-Tris, 1 mM-EDTA (pH 8) were then electrophoresed through 0.0% agarose gels (6 mm) in Tris-acetate buffer. Triangles (A) indicate the two positions at which renatured DNA has been found. Gel 1, control of unfractionated TBst(0) DNA in 50% formamide (not self-annealed). Gels 2 to 5, isolated major fragments after self-annealing; gel 2, 35.3 million fragment; gel 3, 17.2 million fragment; gel 4, 14.5 million fragment; gel 5, 3.8 million fragment. Gels 6 t,o 9, annealed mixtures of isolated fragments; gel 6, 35.3 and 17.2 million fragments; gel 7, 35.3 and 14.5 million fragments; gel 8, 35.3 and 3.8 million fragments; gel 9, 17.2, 14.5 and 3.8 million fragments.

x

?

-

+ I

2

3

4

5

6

7

(b) PLATE IV. Electrophvrcais of single-strantlcd fstDNA in agaroao and agarose-pulyacrylarnltll: gels. fstDSd was prepawd as described under Materials and AIethods. Whew necessary, IISA samples were denatured with alkali prior to eiectrophoresis, which was performed in Id-mm diameter gels in Tris-phosphate-sodium dodocyl sulphate buffer. (a) TEist(0) DNA in 0.6% agarose gels. Gel I, fl and Xb2b5 phage DNA (note the apparent separation of h DNA strands and of linear and circular forms of fl DNA); gel 2, T5st(O) phage DNA; gel 3, mixed T5st(O) phage DNA and T&t(O) fstDNA; gel 4, T&t(O) fstDNA. (b) T5’ DNA in 0.7% agarose-2.27; polyacrylamide gels. (~($1 1. fl phagc I)NA ; gtsl P. ‘I’5 phage DNA ; gel 3, T5 i- fst DK.4.

ARRANGEMENT

OF

T5

DNA

FRAGMENTS

403

60% breakage product contained the 28.3, 13+9,5-l and 3.8 million fragments, but the 40% breakage product contained large amounts of the 5.1 and 3-8 million fragments in addition to the expected 16-O and 14-5 million fragments. Most minor fragments are associated with only one or the other of the breakage products (Table 1). The apparent presence of 5.1 and 3-8 million fragments in both T5+ breakage products could be explained by contamination of the 40% product by further breakage of the 60% product (Rubenstein, 1968) or by the existence of a second site of breakage at an equal distance from the other end of the intact molecule. Despite these complications, our results show that the 14.5 million fragment is probably one complete DNA strand of most of the 40% breakage products. Since the 40% breakage product comes from one end of the original duplex, we conclude that the 14.5 million fragment is a terminal portion of both the T5+ and T5st(O) DNA molecules. The sedimentation profiles of sheared DNA (Fig. 2(a) and (b)) were complicated by incomplete resolution between peaks and by the appearance of minor components,. The presence of at least five minor double-stranded species in sheared T5+ DNA can be deduced from the results of gel electrophoretic analysis of each fraction in the sedimentation profile. One of these minor species sedimented between the intact molecules and the 60% breakage product, two were in the minor peak between the main breakage products and two were in minor peaks which sedimented slower than the 40% product. These results suggest that there are several additional sites which a’re susceptible to breakage by shearing. (c) Annealing experiments with isolated fragments We have used agarose gel electrophoresis as a simple assay procedure for complementarity between major fragments. When denatured DNA (T5 or X) was reannealed at low concentration and then subjected to electrophoresis, the renatured DNA was found partly trapped at the top surface of the gel and partly at the position of double-stranded molecules. Therefore, the annealed complex formed between two complementary T5 fragments would be expected to migrate near the top of the gel. We have isolated each major single-strand fragment, annealed it with the others, and examined the gel profile for changes in the positions of the bands. Renatured species should still be detected even if one of the fragments has been degraded during isolation. The results of gel separations of self-annealed fragments of T5st(O) DNA are shown in Plate III, gels 2 to 5. Most of the 35.3 million fragments and some of the 17.2 million fragments had been broken at least once during isolation and a small amount of renaturation occurred in self-annealed preparations of the 35.3, 17.2 and 14.5 million fragments. No additional renaturation occurred when the 17.2, 14.5 and 3.8 million fragments were annealed together (gel 9), but each of these fragments was converted to renatured material when mixed with the 35.3 million fragment preparation (gels 6 to 8). Experiments involving T5+ DNA gave similar results. The 14.5, 13.9, 5.1 and 3.9 million fragments all annealed with DNA from the 37.0 million fragment but not with each other. These smaller T5 + fragments also annealed with the 35.3 million fragment of TBst(0) DNA. (d) Single-strands

of jirst-step-transfer

DNA

When T5 infection occurs in the absence of protein synthesis, only 8% of the duplex DNA molecule enters the host cell. This fstDNA can be isolated from 27

404

G.

S. HAYWARD

AND

M.

G.

SMITH

partly-infected cells after removal of the phage particles containing the rest of the DNA (McCorquodale & Lanni, 1964; Lanni et al., 1964; Lanni, 1968). The barrier between fst infection and full infection of the bacterium could be a single-strand interruption, and to test this possibility we prepared fst-infected cells using parental 32P-labelled T5 phage. The DNA isolated from these cells was used directly for electrophoretic analysis in agarose and agarose-polyacrylamide gels (Plate IV). For a direct comparison of fst and viral DNA, samples of the two were mixed before denaturation and subjected to electrophoresis in the same gel (Plate IV, gel 3). The single-strands of fstDNA migrated faster than any of the known major fragments, and the molecular weight of fstDNA strands from both T5+ and T5st(O) was 3.0 million. Since the smallest major fragment has a molecular weight of 3% million, we conclude that the fstDNA is not directly associated with a single-st’rand interruption. Our measurements give t,he size of fst-DNA as 8.1 and 8*5o/o of the molecular length in T5+ and T5st(O) DNA, respectively, which is in excellent agreement with previous estimates on duplex fstDNA (McCorquodale & Lanni, 1964).

4. Arrangement

of Major

Fragments

in the T5 Chromosome

In this section we propose a model for the arrangement of major single-strand fragments in the intact duplex DNA of T5+ and T5st(O). We suggest that the T5st(O) DNA molecule differs from T5+ DNA by a deletion which removes one of the singlestrand interruptions. This model is presented in Figure 3, and the supporting evidence and reasoning are summarized below. For the present purposes we have disregarded the minor fragments, which we consider to be secondary features of the molecular structure. (1) T5+ DNA contains five major fragments with molecular weights of 37.0, 14.5, 13.9, 5.1 and 3.8 million. TBst(0) DNA contains four major fragments with molecular weights of 35.3, 17.2, 14.5 and 3.8 million (Hayward & Smith, 1972). (2) In both viruses, t’he largest fragment is an intact strand and the other strand contains all the interruptions (Bujard, 1969; Jacquemin-Sablon & Richardson, 1970; Hayward & Smith, 1972). This conclusion is supported by our annealing results, which show that the largest fragment (37-O or 35.3 million) is complementary to all the other fragments in either T5 + or T5st(O). (3) The intact strand of T5 + is basically the same DNA strand (Eor r) as the intact strand of TBst(O), because both have similar relative abundances and poly(G)binding properties, and both are complementary to the 14.5, 13.9, 5.1 and 3.8 million fragments of T5 + . (4) The 14.5 million fragments of T5+ and TSst(0) DNA have similar electrophoretic, relative abundance and poly(G)-binding properties and must have closely related or identical base sequences. Therefore, from the results of our shearing experiments, we suggest that these 14.5 million fragments are located at the same ends of both duplexes. (5) Bujard (1969) presented elect,ron microscopic evidence for a single-strand fragment of 11% of the molecular length which could be preferentially released from T5+ DNA during partial denaturation. The resulting single-stranded section was separated from the end of the molecule by a duplex region of 8% of the molecular length. We infer that our 5.1 million fragment (13% of the molecular length) is the releasable fragment and our 3-8 million fragment (10% of t#he molecular length) is the end fragment in T5+ DNA.

4o;i

ARRANGEMENTOFT5DNAFRAGMENTS

(6) The 3.8 million fragments of T5+ and T&t(O) DNA have apparently identical electrophoretic, relative abundance and poly(G)-binding properties; and because the T5+ fragment is at one end of the molecule, the T.%t(O) fragment would be expected to occupy a corresponding position in the TBst(0) duplex. (7) There are two non-terminal fragments in T5+ (5.1 and 13.9 million), but in T5st (0) there is only one (17.2 million). We believe that this difference can be explained by the deletion of a duplex segment of 3.6 million molecular weight from T5+ DNA. This deletion would encompass the interruption between the 5-l and 13.9 million fragmenta and produce a new single-strand fragment of 17.2 million molecular weight (Fig. 3).

1,

I: T5st(O)-

353

’ 38

17 2

14 5

FIG. 3. Model for the arrangement of single-strand fragments in the duplex DNA molecules of T5+ and TBst(0) bacteriophage. The moleoular weight in millions is given for each fragment. The broken lines show the prosumptive location of the st(0) deletion and the arrows indicate the preferred points for shear breakage.

5. Discussion We have presented experimental evidence for a model describing the arrangement of major fragments in the T5+ and T5st(O) chromosomes (Fig. 3). There are several features of our results and model which we wish to discuss and relate to previous models and observations of unusual properties of T5 DNA. We envisage that every T5 DNA molecule has the same basic arrangement of major fragments and primary interruptions as depicted in our model. In addition, every molecule has an unknown number of secondary interruptions the dist’ribution of which is not equivalent between individual molecules but is constant for the total population (Hayward & Smith, 1972). Previous models (Abelson & Thomas, 1966) were based on sedimentation studies which did not resolve minor components, and thus the models contained erroneous values for the number and sizes of fragments. Bujard’s study was not affected by the presence of secondary interruptions, and co.nsequently he was able to construct a better model for the basic structure of T5 + DNA. Our model for the arrangement of major fragments in T5+ DNA is similar to Bujard.‘s and, in fact, is partly based on his evidence (Bujard, 1969). An important feature of our model is the description of the difference between T5 + and T5st(O) DNA molecules. T5st(O) is the extreme example of a series of heat-stable mutants derived from T5 + . Similar mutants occur in T3, T7 and h, and in all cases increasing heat-stability is correlated with decreasing size of the DNA molecule and decreasing density of the phage particle (Rubenstein, 1968 ; Ritchie & Malcolm, 1970 ; Parkinson $ Huskey, 1971). Abelson & Thomas (1966) could not explain the difference between the fragments thought to be present in T5+ DNA and those of T5st(O) in

406

G.

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AND

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G.

SMITH

terms of a simple deletion or deletions. However, our results show that TBst(0) could be produced by a single deletion at the site of one of the single-strand interruptions in T5+ DNA. This deletion in TBst(0) appears to have affected at least one protein in the phage particle because the sodium dodecyl sulphate-polyacrylamide gel pattern of disrupted T5+ phage contains an extra protein of approximately 13,000 molecular weight, which is not present in TBst(0) phage particles (G. S. Hayward & L. Percival, unpublished observations). In the preceding paper we reported that at least one strand of h DNA and one of the T5 minor fragments had variable mobilities in different buffer systems, and we suggested that some unusual feature of their secondary structure or base composition may be responsible for this behaviour. Our present finding that the T5 variable fragment has a higher buoyant density in the presence of poly(G) than any other T5 fragment implies that the unusual property may also be associated with a strong poly(G)interaction. Further examples of anomalous mobility appear to be exhibited by the breakage products obtained when the duplex molecule is sheared. The two DNA strands of the 40% breakage product, which have apparent molecular weights of 145 and 16.0 million, may have identical real molecular weights but separate during electrophoresis, as do the strands of h DNA. In addition, the intact strand of T5st(O) DNA has a molecular weight of 35.3 million but is broken into two pieces with apparent molecular weights of 16.0 and 25.5 million (which total 415 million). Clearly one or more of these species exhibit anomalous electrophoretic properties, and further experiments are in progress to try to resolve the problem. Previous workers have suggested that the points in T5 DNA which show fragility to shear are single-strand interruptions (Abelson & Thomas, 1966; Rubenstein, 1968; Bujard, 1969) and we have obtained evidence that this is probably correct. The first breakage products of other viral DNA molecules have a Gaussian distribution of size with a peak at half-molecular weights, but in both T5+ and T5st(O) DNA the major break in the intact strand occurs acentrically at a point which is located 16.0 million molecular weight from one end of the molecule and approximately opposite the most central single-strand interruption. None of the other major fragments appears to be affected by the breakage, suggesting that no new break is introducted into the opposite strand. We cannot determine whether the break in the intact strand occurs directly opposite the interruption (as depicted in Fig. 3), or slightly to one side SOas to give double-stranded products with single-stranded ends. However, it seems certain that the preferred site of breakage is associated with the interruption. The minor points of breakage which were detected could be at secondary interruptions. Several authors have suggested that the fst-portion of the T5 DNA molecule may be terminated by a single-strand interruption which acts as a temporary barrier during DNA injection (Abelson & Thomas, 1966; Lanni, 1968 ; Bujard, 1969). This now seems unlikely, because the smallest terminal major fragment in T5 DNA has a molecular weight of 3-8 million and the single strands of fstDNA have a molecular weight of only 3-O million. The first single-strand interruption from either end of the molecule would still be inside the phage particle during fst infection. The T5 fragments exhibited a broad buoyant density profile in the presence of poly(G), which was similar to that obtained with poly(U,G) (Lanni & Szybalski, 1969; Jacquemin-Sablon & Richardson, 1970). Electrophoretic analysis of fractions from CsCl + poly(G) density-gradients showed that individual T5 fragments interact differently with poly(G) and confirmed our nrevious suggestions (Hayward & Smith,

ARRANGEMENTOFTT5DNAFRAGMENTS

4oi

1972) that a large number of the T5 fragments are common to both T5+ and T5st(O} DNA. Lanni & Szybalski (1969) found that most phage-specific RNA from T5+-, infected E. coli hybridized with DNA from the heavy regions of their CsCl + poly (U,G) density profiles, and our experiments suggest that the only major fragments present in these heavy regions were the 37.0 and 13.9 million fragments of T5+ DNA or the 35.3 and 17.2 million fragments of T5st(O) DNA. Therefore, these two frag-. ments, which represent sections of both DNA strands, probably contain most of the template regions for T5 transcription. A study of T5 transcription patterns using the isolated single-strand fragments will be published later (S. D. Hayward & M. G. Smith, manuscript in preparation). We gratefully acknowledge the advice and encouragement received from Dr P. L. Bergquist, Dr W. D. Sutton and S. D. Hayward. Dr Bergquist generously provided laboratory facilities in Auckland. We thank Drs G. B. Petersen and W. D. Sutton for a gift of poly(G). This work was supported in part by grants from the New Zealand Medical Research Council, the Golden Kiwi Medical Research Fund and the New Zealand IJniversity Grants Committee. One of us (G. S. H.) is the recipient of a postgraduate scholarship from the New Zealand University Grants Committee. REFERENCES Abelson, J. & Thomas, C. A., Jr. (1966). J. Mol. Biol. 18, 262. Bujard, H. (1969). Proc. Nat. Acod Sci., Wash. 62, 1167. Burgi, E., Hershey, A. D. & Ingraham, L. (1966). Virology, 28, 11. Clausen, T. (1968). Analyt. Biochem. 22, 70. Hayward, G. S. & Smith, M. G. (1972). J. Mol. BioZ. 63, 383. Hershey, A. D., Goldberg, E., Burgi, E. & Ingraham, L. (1963). J. Mol. BioZ. 6, 230. Jacquemin-Sablon, A. & Richardson, C. C. (1970). J. Mol. BioZ. 47, 477. Kubinski, H., Opara-Kubinski, Z. & Szybalski, W. (1966). J. Mol. BioZ. 20, 313. Lanni, Y. T. (1961). Virology, 15, 127. Lanni, Y. T. (1968). Bad. Rev. 32, 227. Lanai, Y. T., McCorquodale, D. J. & Wilson, C. M. (1964). J. Mol. BioZ. 10, 19. Lanni, Y. T. 6t Szybalski, W. (1969). Bact. Proc. 69, 192. McCorquodale, D. J. & Lanni, Y. T. (1964). J. Mol. BioZ. 10, 10. Parkinson, J. S. & Huskey, R. J. (1971). J. Mol. BioZ. 56, 369. Ritchie, D. A. & Malcolm, F. E. (1970). J. Gen. ViroZ. 9, 35. Rubenstein, I. (1968). V;roZogy, 36, 356.