The physical properties of the deoxyribonucleic acid from N4 coliphage

491

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95529

THE PHYSICAL PROPERTIES

OF THE DEOXYRIBONUCLEIC

ACID F R O M

N4 COLIPHAGE G. C. SCHITO*, G. R I A I . D I * * AND A. P E S C E *

Istituto di Microbiologia dell'Universitd~ di Genova*, and Sezione VI del Centro Nazionale Virus Vegetali deI Consiglio Nazionale delle Ricerche**, Genova (Italy) ( Received M a y 2oth, i966 )

SUMMARY

z. Purified p r e p a r a t i o n s of N 4 b a c t e r i o p h a g e were t r e a t e d w i t h cold phenol a n d the D N A was s u b j e c t e d to a series of p h y s i c a l a n d chemical investigations. 2. The e x t r a c t e d D N A was shown to be homogeneous b y u l t r a c e n t r i f u g a t i o n a n d c h r o m a t o g r a p h y on m a g n e s i u m p y r o p h o s p h a t e gel. 3. The size of N 4 D N A , as d e t e r m i n e d from s e d i m e n t a t i o n , v i s c o m e t r y a n d l i g h t - s c a t t e r i n g studies, was found to be in t h e range of 4o" I o 6 daltons. Since the molecular weight of N 4 b a c t e r i o p h a g e is 83" zo 6, there is p r o b a b l y o n l y one D N A molecule per virus particle. 4. T h e r m a l d e n a t u r a t i o n studies p r o v i d e d evidence for a d o u b l e - s t r a n d e d s e c o n d a r y s t r u c t u r e of N 4 DNA. The t r a n s i t i o n t e m p e r a t u r e vas ~ 7.2 corresponding to a m e a n guanine pl.us cytosine c o n t e n t of 44 moles o~ ,0' 5. The b u o y a n t d e n s i t y of n a t i v e N 4 D N A in CsCI d e n s i t y g r a d i e n t s was found to be z.7o 7 g/ml and an increase of o.or5 g/ml was o b s e r v e d for t h e r m a l l y d e n a t u r e d samples b a n d e d at the isopycnic point.

INTRODUCTION

Recent interest in the isolation a n d c h a r a c t e r i z a t i o n of new b a c t e r i a l a n d a n i m a l viruses m a y p r o b a b l y be e x p l a i n e d - - besides e x t e n d i n g our present s t r u c t u r a l , t a x o n o m i c a n d physiological k n o w l e d g e - as an a t t e m p t to secure for s t u d y a larger n u m b e r of sources of p h y s i c a l l y a n d genetically homogeneous d e o x y r i b o n u c l e i c acids 1. Following this trend, a n d as a result of a wide scanning, N 4, a b a c t e r i o p h a g e active on Escherichia coli K z'z strains, was isolated, a n d some of the biological and b i o p h y s i c a l p r o p e r t i e s of the virus particle have been r e p o r t e d 2,a. This investigation concerns the physical c h a r a c t e r i z a t i o n of the nucleic m o i e t y of N4 p h a g e a n d presents evidence t h a t this n e w l y described virus m a y be considered a v e r y convenient source of monodisperse, high-molecular-weight, d o u b l e - s t r a n d e d D N A . P a r t of the d a t a i n c l u d e d in this work was r e p o r t e d at the X I I I N a t i o n a l Congress of the I t a l i a n Society for Microbiology, Salsomaggiore, I965. ** Pap er No. 28 from Sezione VI del Centro N a z i o n a l e Virus V e g e t a l i del ('.N.I¢. ~tl the [ n s t i t u t e of I n d u s t r i a l ( ' h e m i s t r v , !Tniversity of Geno va , Genova, l t a l v .

Biochim. ]3iophys. dora, I29 (1960) 491 5~)l

492

G.C.

SCHITO, G. RIALDI, A. PESCE

MATERIALS AND METHODS

Isolation o[ N4 DNA Propagation and purification of N 4 bacteriophage have been described in the accompanying paper s. DNA was extracted from phage stocks purified by CsC1 banding employing the phenol deproteinization method described b y MANDELLAND HERSHEY4. The phage, at a concentration of o . 5 - 1 % , was dialyzed overnight against o.i M NaC1 and gently agitated with an equal volume of cold, water-saturated, alkali-neutralized, phenol. The water phase was re-extracted three times and most of the phenol was then removed by repeated washes with water-saturated ether. The extremely viscous DNA preparation was dialyzed against several changes of either I M NaC1 or standard saline-citrate buffer (o.15 M N a C l + o . o I 5 M sodium citrate, p H 7.0) (SSC). During isolation, mechanical shear was avoided as far as possible, and pipetting was performed only with pipettes whose bore was at least 2 m m wide. It was also possible to release N4 DNA by submitting the phage to repeated cycles of freezing and thawing or to dialysis at room temperature against o.I M Tris or phosphate buffer (pH 7.2), since, in these conditions, the virus slowly disrupted 5. The most satisfactory method for preparing N 4 DNA, as judged by the appearance of a sharp sedimenting boundary in the ultracentrifuge, was, however, that by phenol extraction. Approx. 25-30 mg DNA is obtainable from IOO mg of purified phage.

Analytical ultracentri/ugation Sedimentation velocities were measured in the 3o-mm path-length Kel-F cells of the Spinco Model E ultracentrifuge, using absorption optics. Films were scanned with a Beckman Analytrol densitometer, and the sedimentation coefficients were calculated from the 50 O//o concentration points of the boundaries. Values of s were corrected to the viscosity and buoyancy of water at 20 °, assuming the N4 DNA to have a partial specific volume of 0.55 ml/g (refs. 6, 7). In most experiments the solutions were centrifuged at 33 450 rev./min. Analyses were performed on DNA samples dissolved in I M NaC1 in order to improve reproducibility of the sedimentation coefficients through the stabilizing effect of the density gradient provided b y the redistribution of the salt s. Cells were filled while partially assembled, thus avoiding the shear associated with the conventional filling method 9.

Density gradient centri[ugation Equilibrium density-gradient centrifugation 10 was used to estimate the buoyant density and the base composition of N4 DNA. The DNA was suspended in CsC1 containing 0.02 M Tris buffer (pH 8.5), and the density of the solutions, as determined from their refractive indexes 11, was adjusted to a value of 1.71o g/ml. The final concentration of the DNA ranged from 0.5 to 2/~g/ml. The CsC1-DNA solutions were slowly introduced into a I 2 - m m Kel-F cell with a 1-degree negative wedge window, and the runs were performed in the Spinco Model E ultracentrifuge at 25 °, for at least 20 h at 44 77 ° rev./min. The DNA of Pseudomonas aeruginosa and of Proteus vulgaris (1.727 and 1.698 g/m1) 12, which were extracted following the proBiochim. Biophys. Acta, 129 (I960) 4 9 i - 5 o i

DNA fROM N 4 COLIPHAGE

493

cedure of MARMUR la, w e r e used as markers in order to calculate the buoyant density of native and denatured N4 DNA, respectively ~4. Tracings of the ultraviolet absorption bands were made as described for velocity sedimentation studies.

Viscometry Relative viscosities at several shear stresses were measured in a three-bulb suspended-level capillary viscometer having a flow time of 128 sec for water. Viscosities were extrapolated to zero shear and concentration. The instrument was immersed in a water bath at 25 °, constant to ± o . o i .

Light scattering The molecular weight of the phage DNA was determined by light-scattering experiments performed in a Sofica photometer with the use of a cylindrical cell and unpolarized light at a wavelength of 5460 A. Solutions of DNA in SSC buffer were clarified b y centrifugation and portions were added to clean filtered solvent in the scattering cell, in order to give a range of concentrations. The reciprocal of the molecular weight was obtained b y the method of double extrapolation in. The refractive index increment of DNA was assumed to be o.2ol ml/g (ref. 16) and concentrations were calculated from an extinction coefficient of 20 absorbance units/mg/ml at 260 m/~.

Thermal denaturation Melting experiments were performed and plotted as described by MARMUR ANn DOTYiv. DNA solutions in SSC, 15-2o/zg/ml, were heated in I.o-cm light-path, stoppered quartz cells, of a Zeiss PMQ I I spectrophotometer, equipped with thermospacers. Elevated temperatures were obtained b y circulation of a heated glycolwater mixture. Observed absorbancies were corrected for volume expansion.

RESULTS

Hydrodynamic properties and size o/N 4 DNA Traces of ultraviolet photographs of N4 DNA, sedimenting in the ultracentrifuge (Fig. 1), allowing for technical imperfections in the mechanical responsiveness of the scanning densitometer, appear to agree, within reasonable limits, with the theoretical step function expected from the migration of a boundary of a monodisperse material with a low diffusion coefficient s. This behaviour does not, by itself, demonstrate the molecular homogeneity of the extracted N 4 DNA, yet the lack of evident hydrodynamic heterogeneity, associated with chromatographic analysis, lends support to the hypothesis that the preparations are essentially monodisperse. N4 DNA, chromatographed on magnesium pyrophosphate columns, capable of separating native from denatured molecules and possibly able to fractionate according to molecular weight and base composition is, was eluted as a single peak at a phosphate buffer (pH 7.2) concentration of 0.25 M. By this criterion too, no heterogeneity was detected. Biochim. Biophys. Acta, 129 (1966) 491-5Ol

494

G. C. SCHITO, G. RIALDI, A. PESCE

- ~

Radial

distance

Fig. i. D e n s i t o m e t e r traces of u l t r a v i o l e t - a b s o r p t i o n p h o t o g r a p h s of sedimenting N 4 D N A at a concentration of 2o/~g/nll in I M NaC1. Pictures were t a k e n at 4-min intervals after reaching speed of 33 45 ° rev./min. T e m p e r a t u r e 2o°; exposure tinle 2o sec. Sedimentation proceeds f r o m left to right and the lower scale indicates radial distance in the centrifugal cell.

Self-sharpening of the sedimenting boundaries of DNA solutions has repeatedly been describedS, 19. To exclude the fact that the observed steepness of the densitometer traces may be a consequence of such an artifact, an ultracentrifugation experiment was performed in which equal weights (IO/zg) of native and partially deoxyribonuclease-digested N 4 DNA were run in the same cell, under otherwise standard conditions. Degraded DNA was readily resolved and a slower sedimenting boundary appeared. Fig. 2 shows the dependence of the median sedimentation coefficients as a function of concentration, s20,~ extrapolated to zero concentration is 4I~:O.5 S. At elevated concentrations and centrifugal forces, high molecular weight DNA manifests anomalous hydrodynamic behaviour, possibly as a consequence of rapid sedimentation of a portion of the macromolecules in these conditions 2°-2s. Although few detailed investigations have been made on N 4 DNA, no pronounced dependence of the shape of the boundaries on rotor speed has been noted. This observation may be in agreement with theoretical predictions, since the plateau effect is inversely related to molecular weight and has been described for T2 and T4 bacteriophage DNA whole molecules24, ~5, but has not been reported for T 7 DNA (refs. 8, 22). However, since the molecular weight of N 4 DNA is in the range of 4 °. Io 6 daltons (see further) the definitive demonstration of the absence of this sedimentation anomaly must await more thorough investigation. Values of the limiting viscosity number were determined on three independent preparations of N 4 DNA dissolved in SSC. A series of solutions ranging in concentration from Io to 65/zg/ml were analyzed. The estimated intrinsic viscosity, E~, Biochim. Biophys. Acta, 129 (1966) 491--5Ol

D N A FROM N 4 COLIPHAGE

495

5C

4(i

? 3C o

20

2%

~o

7%

~bo

c(mg / rnl) F i g . 2. R e c i p r o c a l of t h e s e d i m e n t a t i o n centration.

c o e f f i c i e n t s of N 4 c o l i p h a g e D N A

as a f u n c t i o n of c o n -

is 125 dl/g. It will be shown later that this value is slightly lower than that calculated from EIGNER AND DOTY'S empirical equation, which relates intrinsic viscosity to molecular weight 26. The difference may possibly be ascribed to the inaccuracy of the extrapolation of the specific viscosity to zero shear rate, when using capillary instead of rotating cylinder viscometers 23,27. As a consequence of fundamental contributions in the past few years (see EIGNER AND DOTY2e for an outstanding review), a sound theoretical basis has been given for the determination of molecular weights of linear, double-stranded D N A molecules from hydrodynamic parameters. Depending on the method of calibrat~,oa~, different empirical equations have been proposed, and, since the Watson and Crick configuration was established for N4 D N A (see further), calculations of the size ,of this macromolecule were accordingly made. The results are given in Table I, together with the appropriate substitutions used.

TABLE

I

DETERMINATION OF MOLECULAR WEIGHTS OF N 4 D N A

FROM s ° AND [~]

T h e f o l l o w i n g s u b s t i t u t i o n s w e r e u s e d : s ° = 41 S v e d b e r g u n i t s ; [~] = 125 d l / g ; N = 6 . 0 2 3 . 1o83; fl = 2 . 4 - i o ~ ; ~ = 0 . 5 5 m l / g ; ~/0 = 1 - o 1 9 " 1 o - 8 ; P = I . O l i g / m l . Equation

Re/erence

Molecular weight X IO 6

s = s = s -s = s -[~/]

0 . 0 6 3 M °'aT 0 . 0 3 4 M °.4°5 0 . 0 8 0 M °.35 0 . 0 0 2 4 M °.s43 0 . 0 8 8 2 M °.a4e ~ 6 . 9 " lO -4 M °'~°

M=

#(i

iI//

~p)

l'

(so [

2"27" 1019(1 --Vp)

]

DOTY et al. *o E I G N E R AND DOTY 26 I3URGI AND H E R S H E Y 28 R U B E N S T E I N et al. 29 STUDIER3° E I G N E R AND DOTY 26

40.2 40.6 55.2 60. 5 5 I. 2 32.5

M A N D E L K E R N AND F L O R Y 41

40.6

CROTHERS AND ZIMM 22

40.6

Biochim.

Biophys. Acta, 1 2 9 ( 1 9 6 6 ) 4 9 I - 5 O I

496

G. C. SCHITO, G. RIALDI, A. PESCI~

An analysis of the data shows that most figures are concentrated in the neighbourhood of 40" 106 daltons. Values well above this range are obtained by employin8 the equations derived from measurements on T2 DNA shear products2S, z9 and thus suffer from uncertainties related to the unknown effect of glycosylation on hydrodynamic properties 8. STUDIER'S relation 30 is undoubtedly valid, when comparable techniques of zone sedimentation are adopted, which was not the case in this work. A molecular weight of 40" I06 daltons for N 4 DNA is thus in accord with available chemical analyses of the virus particle composition 3. On the other hand, the viscosity curve 26 yields a molecular weight slightly lower than that computed from the sedimentation coefficient: this may be a consequence of the already mentioned doubtful zero shear rate extrapolation.

°6

'

'

.~

'

{ sin 2

'

'

8/2+c.10 3

g

'

16

'

'

Fig. 3. L i g h t s c a t t e r i n g of N 4 D N A in s t a n d a r d s a l i n e - c i t r a t e buffer (pH 7.o).

Light scattering The particle weight of N 4 DNA was measured by light scattering. A Zimm plot from one experiment is shown in Fig. 3. From four experiments, a mean particle weight of 41. lO 6 daltons was calculated. Extrapolation of light-scattering data to zero angle, when dealing with native DNA samples exceeding five or six million daltons, has been considered liable to underestimate the size of these molecules31, 32. More recently, reliable light-scattering experiments have been further limited to a size of not more than 3" lO6 daltonse6With this in view, the agreement of the mean molecular weight of N 4 DNA, as derived from the latter technique, with that calculated from hydrodynamic and chemical parameters, is somewhat unexpected and, at present, no clear explanation can be offered for it.

Evidence/or the double-stranded nature o / N 4 D N A DNA extracted from N 4 phage has been characterized as double-stranded by a series of commonly held criteria; the form of the melting curve, the reaction with Biochim. Biophys. Acta, 129 (1966) 491 5Ol

DNA FROM N 4 COLIPHAGE

497

E~

O tO ¢J 1.2 O)

1.2 1.1

i

~_~,

o 1.0

. . . . .

-"

Temperature F i g . 4. T h e r m a l t r a n s i t i o n s o f n a t i v e saline-citrate buffer (pH 7.0).

(O-O)

and heat-denatured

N 4 DNA

(O-Q)

in s t a n d a r d

formaldehyde, the effect of deoxyribonuclease digestion, and the change in density in CsC1 gradients upon heating and quick cooling. N4 DNA dissolved in SSC was heated as described under MATERIALS AND METHODS, and the absorbancies measured. As shown in Fig. 4, the absorbance remained essentially constant until the temperature reached 85 ° , thereafter the extinction increased until, at 8 9.5 o, a final hyperehromic increment of 4 1 % was attained. The denaturation temperature, Tin, was 87.2 °, a value corresponding to a base composition of 43.6 % guanine plus cytosine z~. When N4 DNA samples, heated for IO min in a boiling-water bath, were rapidly chilled, the increase in absorbance was 13 %, that is about 65 % of the bases were still non-specifically hydrogenbonded. Upon raising the temperature, however, these short helix regions melt out, and, on further heating, annealing conditions are provided a3. Fig. 4 shows that the melting profile of native and gradually renatured N4 DNA were very similar in the Tm region, a result to be expected assuming that a substantial proportion of the strands had resumed the specific complementary configuration. When reannealing was performed under the conditions recommended by ~/[ARMUR AND DOTY33 (in this case 4 h at 60°), probably because of the high degree of homogeneity of N 4 DNA, over 9 ° % renaturation was obtained, as judged from TABLE

II

THE EFFECT OF FORMALDEHYDE AND HEATING AT IOO ° ON THE ABSORBANCE OF N 4 D N A N 4 D N A w a s d i s s o l v e d i n O.Ol 5 M N a C 1 c o n t a i n i n g o . o o l 5 M s o d i u m c i t r a t e ( p H 7.2). S a m p l e s w e r e h e a t e d , c h i l l e d r a p i d l y , a n d t h e a b s o r b a n c e t h e n m e a s u r e d a t 2 6 0 m/z.

Treatment

E,6 o

Increment (%)

A. B. C. D. E.

o.918 o.918 1.o4o 1.325

13 44.2

1.23 °

33

I n c u b a t e d a t 37 ° f o r 6 0 m i n I n c u b a t e d a t 37 ° f o r 6 0 m i n w i t h i % f o r m a l d e h y d e Heated at ioo ° for io min H e a t e d a t IOO ° f o r IO m i n w i t h 1 % f o r m a l d e h y d e Formaldehyde (i % ) a d d e d t o s a m p l e C, i n c u b a t e d at 37 ° for 6o rain

Jgiochim. Biophys. Acta, 129 (1966) 4 9 1 - 5 O l

498

C. C. SCHITO, G. RIALDI, A. PESCE

regained hypochromicity, superimposition of the melting profile and CsC1 density gradient analysis. Native N 4 DNA does not react with formaldehyde at 37 ° (Table II). As already mentioned, heating followed by rapid cooling, produces an increment in absorbance of only 13 %. When the denaturation is performed in the presence of the aldehyde, the final increase in absorbance is 44.2 %. On the other hand, thermally denatured DNA is able to react with the formaldehyde, and an additional increase in ultraviolet absorbance is noted (Table II). This behaviour is characteristic of doublestranded DNA (refs. 34, 35). The samples of N4 DNA exhibited a hyperchromic effect of 3 1 % upon digestion for I h in a solution containing 25 #g/ml of DNA, 3/*g of pancreatic deoxyribonuclease (NBC) and o.oo3 M MgC12. This is again consistent with a doublestranded secondary structure of the virus DNA (refs. 35, 36).

1.707

1.712

1,727

Density in CsCl (9/cm3) Fig. 5. Microdensitometer tracing of N 4 coliphage D N A at equilibrium in a CsC1 density gradient. T o p curve, native N 4 D N A and reference Pseudomonas aeruginosa D N A (p = 1.727 g/ml). Lower curve, h e a t - d e n a t u r e d N 4 D N A quenched in ice w a t e r and reference P. aeruginosa DNA. Conditions of centrifugation: o.5-2/~g/ml of each D N A species; r o t o r speed 44 77 ° rev./min; t e m p e r a t u r e 25°; time 22 h.

N 4 DNA has a buoyant density of x.7o 7 g/ml, as measured by equilibrium sedimentation in CsC1 (Fig. 5), a value corresponding to a G + C content I" of 47.9 °/oHeating at IOO°, for IO min, followed by rapid cooling in liquid nitrogen, causes an increase in the density of the DNA to 1.722 g/ml, with an increment of o.oi5 density units. This is in accord with the increase expected upon denaturation of doublestranded DNA (refs. 37-39). When quenching of the heated samples was performed in ice-water, however, equilibrium sedimentation analysis revealed that a substantial Biochim. Biophys. Acta, I29 ([966) 491 5oi

DNA VROM N 4 COLIPHAGE

499

portion of the DNA renatured to a density differing little from that of native N 4 DNA (0.005 g/ml heavier), although the band of renatured material showed a noticeable broadened distribution (Fig. 5). This is taken to indicate that, owing to the molecular homogeneity of N4DNA, extensive renaturation is attained even in the short period in which reannealing conditions are provided during the chilling of the heated sample.

DISCUSSION

The study reported here was primarily aimed at the physical characterization of the nucleic acid moiety of the newly isolated N 4 bacteriophage. With this in view, most experiments were focused on the hydrodynamic behaviour of the macromolecule, in order to get some information pertaining to the size of the extracted DNA. Since, for interpretation of the data, knowledge of the secondary structure of the biopolymer was required, this point was also investigated. Previous chemical analysis of the virus particle revealed that from 47 to 50 % of the weight of the phage is accounted for by the DNA (ref. 3). Assuming a molecular weight of 83" lO 6 daltons for the intact virus, it follows that the molecular weight of N4 DNA should be approx. 39 to 41. lO 6. Calculations from the experimental hydrodynamic parameters, as summarized in Table I, yield values in accord with those expected from the particle composition. For reasons already mentioned, a molecular weight of about 40.5" lOs daltons, as derived from the equations of DOTY et al. 4o, EIGNER AND DOTY 26, MANDELKERN AND FLORY 41 a n d CROTHERS AND ZIMM 22,

is probably to be preferred for N4 DNA. In view of the ultracentrifugal and chromatographic behaviour of N 4 D~A, consistent with that of a monodisperse preparation, it is rather tempting to speculate upon the number of DNA molecules per virus particle. Indirect evidence of the type reported above, pertaining to the size of the macromolecule in solution, in association with chemical data, points to the conclusion that N 4 bacteriophage probably contains a single DNA molecule. Considering the viral source of the DNA, this hypothesis is rendered more valid by the analogy with similar conclusions drawn for T2 (refs. 29, 42), T7 (ref. 8), qSXI74 (ref. 35) and other bacteriophages 1, all of which have been shown to contain only one nucleic acid molecule per virion. Concerning the secondary structure of the phage DNA, all the experimental data thus far collected, point to the conclusion that the polymer exists in solution in a double-stranded, Watson-Crick type configuration. A definite answer to this problem, however, is expected from the chemical analysis of the DNA base content at present in progress. Preliminary results have confirmed the figure of about 45 % guanine plus cytosine established in this investigation by physicochemical methods, while the origin of the slight discrepancy observed in the G + C values obtained from thermal denaturation profiles (44 %) and CsC1 density gradients (48 %) has not yet been elucidated. In any case, the mean base composition of N4 DNA is sufficiently remote from that of the host-cell chromosome to confirm, at the molecular level, the observed impossibility for N 4 bacteriophage to establish lysogenic relationships with E. coli KI2 cells. Using the average value of 662 molecular weight units per base pair, the Biochim. Biophys. Acta, 129 (t966) 491 5oi

500

G.C.

SCHITO, G. RIALDI, A. PESCE

D N A of the virus is found to consist of about 5"10 nucleotide pairs. Assuming that during the preparation procedure the D N A retains either the B crystalline form, with an interbase separation aa of 3-46 A, or the A configuration, in which the interbase distance diminishes to 2.56 A, the overall length of the polynucleotide chain m a y be thought to vary between 15 and 21 ,u. In both cases, since the N 4 particle has a diameter of about 700 2~, the available space in the phage head cannot accommodate the D N A molecule unless this becomes highly coiled and compact. This change in conformation probably occurs in vivo during the last stages of the virus assembly, in which condensation of loose D N A fibers dispersed in the cytoplasm of the infected cell precedes the appearance of mature particles 44. It has recently been shown that most D N A molecules are members of a homologous series, in respect to sedimentation coefficients and limiting viscosity numbers 2e. Comparison of the pertinent figures relative to N 4 D N A with those plotted by EIGNER AND DOTY 26, shows that there is nothing anomalous about the hydrodynamic properties of this DNA. Thus, N4 bacteriophage seems to represent a very convenient source of monodisperse, double-stranded DNA. In addition, the size of this polymer places it in a particular position in the molecular weight range thus far described: indeed, only a few examples are known~, 2e of D N A samples in the vicinity of 40" IOe molecular weight units. While the reason for this scanty distribution in nature is obscure, the availability of a viral D N A of such size m a y be of some general interest.

ACKNOWLEDGEMENTS

The excellent technical assistance of Mr. A. TRAVERSO is

CASAGRANDE, [. CONTI

and L.

gratefully acknowledged.

}~EFERENCES I 2 3 4 5 6 7 8 9 1o II 12 13 14 15 16 17 18 19 20 21 22 23 24

I. J. :BENDET, Advan. Virus Res., IO (1963) 65. A. M. MOLINA, A. PESCE AND G. C. SCHITO, Boll. Ist. Sieroterap. Milan., 44 (1965) 329 . G. C. Sc111To, G. RIALDI AND A. PESCE, Biochim. Biophys. Acta, I29 (I966) 482. J. D. I'~IANDELL AND A. D. HERSHEY, Anal. Biochem., I (196o) 66. (;- C. SCHITO, Giorn. Microbiol. in the press. H. G. TENNENT AND G. F. VILBRANDT, J. A m . Chem. Soc., 65 (1943) 424 . J, ]2;. HEARST, J . Mol. Biol., 4 (1962) 415 . 1°. F. DAVISON AND I). FREIFELDER, dr. Mol. Biol., 5 (1962) 643. P. F. DAVISON, Proc. Natl. Acad. Sci. U.S., 45 (1959) 156o~ . ~IJe2SELSON, F. ~¥. STAItL AND J. VINCGRAD, fJfOC. Natl. Acad. Sci. U.S., 43 (1957) 581. J. ]3. IFFT, D. H. VOET AND J. ~7INOGRAD, J. Phys. Chem., 65 (1961) 1138. C. X. SCHILDKRAUT, J. ~ARMUR AND P. DOTY, J. biol. Biol., 4 (1962) 43 o, J. MARML'R, J. Mol. Biol., 3 (1961) 2o8. N. SUEOKA, J. ~I01. Biol., 3 (1961) 31. t3. H. ZIMM, J . Chem. Phys., 16 (1948) lO93. T. G. NORTHROP, l~. L. NUTTER AND I~. L. SINSHEIMER, J. A m . Chem. Soc., 75 (1953) 5134 . J. MARMUR AND P. DOTY, J. Mol. Biol., 5 (1962) lO9. ?\. ~ESCE AND G. C. SCHITO, manuscript in preparation. H. 1,~. SCHACHMAN, Ultracentri[ugation in Biochemistry, Academic Press, N e w Y o r k , 1959. J. E. HEARST ANn J. VINOGRAD, Arch. Biochem. Biophys., 92 (1961) 206. J. I~OSENBLOOM AND V. 2q. SCHUMAKER, Biochemistry, 2 (1963) 12o6. D. M. CROTHERS AND B. H. ZIMM, J. Mol. Biol., 12 (1965) 525. J. ]3. T. ATEN AND J. A. COHEN, J . Mol. Biol., 12 (1965) 537C. A. THOMAS AND K. I. BERNS, J. Mol. Biol., 3 (1961) 277.

BiochDJ~. Biophys. Acta, 129 (I966) 491-5Ol

DNA 25 26 27 28 29 3° 31 32 33 34 35 36 37 38 39 4° 41 42 43 44

VROM N 4 COLIPHAGE

501

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