Some physical properties of bacteriophage R17 and its ribonucleic acid

J. Mol. Biol. (1964) 8, 496-507

Some Physical Properties of Bacteriophage R17 and its Ribonucleic Acid RAYMOND

F.

GESTELAND

The Biological Laboratories, Harvard University, Cambridqe, Massachusetts, U.S.A. AND HELGA BOEDTKER

Department of Chemistry, Harvard University, Cambridge, Massachusetts, U.S.A. (Received 26 November 1963) The molecular weight of bacteriophage R 17 in 0·15 M-NaCI + 0·015 M-sodiulll citrate (S20,W = 79 to 80 s) was measured by light scattering and found to be 3'6:1: 0·3 million. The molecular weight (by light scattering) of RNA prepared from RI7 phage wail measured at salt concentrations between 0·1 and 0·01 M-Na+-, and found to be 1·1 ± 0·1 million, independent of the counterion concentration. Evidence that the one million molecular weight represents a single polynucleotide chain and not an aggregate of subunits was obtained from measurements of the sedimentation coefficient as a function of time of heating at 70 and 90°C. Brief exposure to 70°C had little effect on the sedimentation coefficient even though the helical regions were melted when the RNA was exposed to this temperature; at 90°C the slow decrease in sedimentation coefficient is consistent with random degradation, the first-order rate constant for bond scission being 2·9 x 10- 1 sec-to The solution conformation of bacteriophage RNA is more compact than that of other ribonucleic acids studied to date. However, there is no evidence that this tighter packing reflects an unusual secondary structure. Comparison of the relative absorbance-temperature profiles and reactivity with formaldehyde of R 17 RNA with tobacco mosaic virus and E. coli ribosomal RNA reveal only slight differences; these are consistent with the guanine-cytosine content of the three different RNA's.

1. Introduction For many years the general belief existed that the genetic component of all bacterial viruses was DNA. This impression was shattered when Loeb & Zinder (1961) showed that the spherical phage f2, active on Escherichia coli, did not contain DNA, but instead contained RNA. Since this discovery, a number of RNA phages have been found (Davis, Strauss & Sinsheimer, 1961; Paranchych & Graham, 1962). So far, all these phages are very small, having diameters of 200 to 260 A, and appear to belong to a common group which multiplies only on male (F+ or Hfr) bacteria. About 30% of their mass is RNA, the remainder is protein, most of which, if not all, forms a shell surrounding the RNA core. The small size of these viruses implies that the viral genetic component codes, at most, for only a few proteins. This provides a strong incentive for intensive work on these viruses; they have the further advantage that they multiply in E. coli, which is well characterized and easy to grow. The correct determination of the molecular weights of these viruses and of their RNA components is thus of more than routine importance. So far, two molecular weight determinations have been reported. 496

PROPERTIES OF PHAGE R 17 AND ITS RNA

497

Strauss & Sinsheimer (1963) showed that phage MS2 has a molecular weight of 3;6 million and that its RNA component has a molecular weight of 1·1 million. A slightly higher molecular weight for phage R17 is given by Kaesberg and coworkers (Enger, Stubbs, Mitra & Kaesberg, 1963). They measured a molecular weight of 4·2 million for the intact virus. Their value for the RNA (mol. wt. 1 x 106 ) , however, is very close to the Strauss & Sinsheimer value (Mitra, Enger & Kaesberg, 1963). A more striking discrepancy exists in the reported configurational properties of the RNA. Strauss & Sinsheimer concluded that MS2 RNA has an unusual secondary structure reflected in its tendency to form dimers and trimers on lowering the counterion concentration, and in its unusually compact and stable helical form. The Kaesberg group, however, concluded that R17 RNA behaves normally in low salt concentrations and has a secondary structure indistinguishable from that reported for other types of RNA. Here we report the results of light.scattering and sedimentation measurements on R17 and its RNA. The RNA measurements were made over a range of salt concentration, and the heat stability ofthe RNA was tested by measuring the sedimentation coefficient after the RNA had been exposed to an elevated temperature for various times. The secondary structure of R17 RNA was examined by comparing the breadth and midpoint of the helix-coil transition and the reaction with formaldehyde of this RNA with other well-characterized RNA samples.

2. Materials and Methods (a) Preparation of bacteriophage R17

Bacteriophage R17, originally supplied by W. Paranchych and A. F. Graham, was grown and assayed on Hfr, methionine-requiring strain of E. coli K12 (from A. Garen). Both nutrient broth and defined media were used to grow the bacteria. The former contained per liter: 10 g Bactotryptone (Difco), I g yeast extract (Difeo), 8 g Na.Cl, 2 m-moles CaCl z (added after autoclaving). The defined medium contained per liter: 5 g Casamino acids (Difeo], 5 g NaCI, I g NH.CI, 4 ml, glycerol, 0·1 m-moles NazHPO•• 10 JLmoles FeCl a, 1 m-mole MgSO., 1·21 g tris (Sigma 121),2 m-moles CaCl z (added after autoclaving). The pH was adjusted to 7·4. To prepare the bacteriophage, flasks containing 31. sterile medium were inoculated- with Hfrj and grown with vigorous aeration at 37°C to a concentration of 4 x 10 8 bacteriajml. as determined in a Petroff-Hausser counting chamber, and then infected with bacteriophage at a multiplicity of about ten. Aeration was continued for 3 hr to allow phage release. Titers of I to 4 x 10 13 phage/ml, in nutrient broth and 1 to 2 X 1012 phage/ml, in the defined medium were obtained. The lysate was cooled, made 2·5 M in ammonium sulfate and allowed to stand overnight at 4°C. The resultant precipitate was sedimented by centrifugation for 25 min at 5,500 rev.jrnin (5,000 g), suspended in SSe,t clarified by low-speed centrifugation, and then precipitated with one-third vol. of cold methyl alcohol for 2'5 hr at - 10°C. The precipitate was collected by centrifugation for 25 min at 5,500 rev.rmin, suspended in SSC and clarified by low-speed centrifugation. The solution was then centrifuged for 45 min in a Spinco model L ultracentrifuge at 40,000 rev.fmin (105,000 g). The translucent pellet was suspended in SSC and clarified again. An additional purification step was carried out on some preparations. The bacteriophage was banded in CsCI by centrifuging the stock solution in CsCI (n 25 = 1'374) in the Spinco SW39 rotor for 16 hr at 39,000 rev./min. Fractions were isolated by collecting drops from the bottom of the tube. The more \lense of the two visible bands was found to contain the viable phage. The CsCI was removed by overnight dialysis against sse. Purified stock solutions were stored at 4°C.

t Abbreviations used: SSC = standard saline-citrate (0,15 M-NaCI plus 0·015 M-sodium citrate, pH 7); TMV = tobacco mosaic virus.

498

R. F. GESTELAND AND H. BOEDTKER

(b) Preparation of El7 ENA 5 ml. of a 1% suspension of bacteriophage was exposed to 2% sodium dodecyl sulfate for 15 min at room temperature. The solution was then chilled and subsequent operations were carried out at 4°C. After adding 50 mg of bentonite (fractionated according to Fraenkel-Conrat, Singer & Tsugita, 1961), RNA was purified by shaking with an equal volume of SSC·saturated distilled phenol for 10 min. The two layers were separated by centrifugation; the aqueous layer was pipetted off, and the phenol layer was again extracted with 1 ml. of SSC. The combined aqueous layers were shaken two more times with 0·5 and 0·25 vol. phenol for 5 and 3 min respectively. The RNA was then precipitated from the aqueous phase by adding 2 vol. cold ethanol, and resuspended in 0·1 IIlM-EDTA, pH 8·0. The solution was made 0'5% with potassium acetate and the RNA was repreeipitated with 2 vol. ethanol. After a third ethanol precipitation, the RNA was dissolved in 0·1 IllM·EDTA and stored at - 20°0. In all later preparations, the RNA solutions were dialysed for 12 hr against 10 mM·EDTA and for 24 hr against 0·1 IIlM·EDTA before being stored at - 20°C.

(c) Physical measurements Sedimentation coefficients were measured in a Spinco model E ultracentrifuge. Bacteriophage concentrations were between 2 and 3 mgjml, for schlieren analysis. Bacteriophage concentrations between 120 and 140 p.g/mI. and RNA concentrations between 30 and 40 p.g/mI. were used for ultraviolet absorption analysis. The sedimentation coefficients obtained were corrected to those in water at 20°C in the usual manner. Light-scattering measurements were made at 25°C using a wavelength of 436 mp. in a slightly modified Brice-Speiser photometer, the description and calibration of which have been described previously (Brice, Hawler & Speiser, 1950; Doty & Steiner, 1950). Solvents were clarified by filtering through an ultrafine sintered glass filter, or a Millipore type HA filter, under partial vacuum. Solutions of bacteriophage were clarified by centrifuging for 45 min at 15,000 rev./min (20,000 g) in the Spinco model L ultracentrifuge. RNA solutions were clarified by filtering through Millipore type HA filters. Only solutions which were dust-free when viewed at low angles with an intense white beam were measured. Concentrations of both R17 bacteriophage and of R17 RNA were determined by measuring the relative absorbancy in Lcm cells in a Beckman DU spectrophotometer. The specific absorption of the bacteriophage in SSC at 260 mp. was 7·66/mg/ml. as determined by measuring the dry weight of a dialysed sample dried to constant weight in a vacuum at 100°C. The specific absorption of R17 RNA in 0·1 M·NaCI was assumed to be 22·5/mg/ml. at 258 mp., a value previously determined for TMV RNA (Simmons, personal communication). Relative absorbance versus temperature measurements were made in a Beckman DU spectrophotometer equipped with a thermostatically controlled cell housing. Results were corrected for the thermal expansion of water.

3. Properties of R17 bacteriophage The purity of the bacteriophage preparation was established by examination of the sedimentation pattern in the analytical ultracentrifuge. In all cases a single sharp component was observed (Plate I) in sse having an S20,W of 79 to 80 s measured at 130 p.g/ml. Light-scattering measurements were made of R17 phage in sse, both as a function of concentration and angle, and found to be almost independent of both as shown in Table 1 and Fig. 1. From the average value of Ke/ R o Doty & Steiner (1950), the weight.average molecular weight is calculated to be 3·60 million. In these calculations we have determined concentrations using our measured absorption coefficient of 7·66/mg/ml. at 260 mp.. This molecular weight agrees precisely with the value reported by Strauss & Sinsheimer (1963). Possible errors in absolute calibration of the photometer, the concentration dependence of the

PLATE

1. Sedimentation pattern of purified R17.

sse

buffer; 20 0 e; 8 min interval between exposures; 35,600 rev./min.

PROPERTIES OF PHAGE R17 AND ITS RNA

499

refractive index of the virus (dn/dc), and the concentration determination, each add several per cent uncertainty to the calculated molecular weight. Our molecular weight value thus has a probable uncertainty of ± 0·3 million. TABLE 1

Light.scattering results on R17 bacteriophage Sample

Concentration (I'gjml.)

KteiR.

Mol. wt.

(x lOS)

(x IO- S )

A

18·5 37·4 50·5 62·6

0·274 0·277 0·279 0·278

B

93·3 152

0·284 0·277

C

48·0

0·279 0·278

Average

3·60

t K = 6·55 X 10- 7 , calculated using dn/de = 0·182 determined by Strauss & Sinsheimer (1963) and corrected for the specific absorption determined by us.

0'30 -e

9 >(

~I~

0'28

0'26 0

•

. 0')

0'2

0'3

0'4

O'S

•

• 0'6

0'7

0'8

sin 2 (0/2)

FIG. 1. Light-scattering angular envelope of R17 bacteriophage in SSC (18·5/Lgjml.).

4. Properties of R17 RNA (a) Sedimentation velocity

Sedimentation coefficients were measured on all preparations of RNA. At first we had some difficulty in reproducibly, obtaining undegraded samples, and even those which were initially intact and homogeneous were quite unstable. These problems were largely resolved when we used freshly prepared' unfrozen virus, added bentonite prior to phenol extraction, and then dialysed the RNA solutions against EDTA. The resultant solutions were quite stable (see below) and the RNA sedimented with more than 90% of the material as a single sharp component as shown in Fig. 2. Sedimentation coefficients in several solvents are given in Table 2. These values are in good agreement with those obtained for Rl7 RNA by Mitra et al. (1963) who reported a value of 26·2 s in 0·1 M and 18·4 s in 0·01 M-potassium phosphate buffer (1 : 1). The sedimentation coefficients are somewhat lower than the values of 31 s in 0·2 M·NaCI and 27 sin 0·02 M-NaCI (at 5°C) reported by Strauss & Sinsheimer (1963)

500

R. F. GESTELAND AND H. BOEDTKER

-

FIG. 2. Microdel1Jlitometer traeing of ul traviolet picture of R 17 RNA se di me nting in 0·1 IC·NaCI , 0'01 M·aodium ac etate, pH 5, at 42,040 rev.fmin , for 28 min.

TABLE

2

Sedimentation coefficients of R17 RNA Solvent

RNA preparat ion

8 ••,.,

0·2 M.NaCl, 0·005 M-tI'i.s, pH 7

27 27

27·2 s ] 26·0

0·1 M-NaCl, 0·01 M-sodium a cetate, pH 5

4 14 14 23

26·0 25'5 26·2 25·0

0·1 M·sodium phosphate (I: I) , pH 6·8

l4 23

25·9 24·9

0·05 M-NaCl, 0·005 M.EDTA, pH 7

23

23·1

0·02 M-NaCl, 0·005 M-tri.s. pH 7

27 27

21·8t 20·4,

0·01 M.NaCl, 0·001 M-EDTA, pH 7

23

16'5

t Measured

at 7°C. All others a t 20·C .

501

PROPERTIES OF PHAGE R 17 AND ITS RNA

for MS2 RNA. This difference cannot be due to a temperature effect on the sedimentation coefficient since we find values of 27 sand 22 s respectively when the sedimentation coefficients of R17 are measured at 7°C. (b) Molecular weight

Light-scattering measurements of R17 RNA were made as a function of concentration and angle at pH 5·0 in 0·1 M-NaCI, 0·01 M-Bodium. acetate, and at pH 7·0 in solutions of NaCl and EDTA at several ionic strengths. The results shown in Table 3 TABLE

3

Light-scattering results on R17 RNA Preparation

Solvent

Concentration (fLg(ml.)

Kt e(R o ( x 10')

Mol. wt. (x 10- 6 )

14

0·1 M-NaCI 0-01 sr-sodium acetate pH 5·0

80 112 186

0'96 0·94 1·02

1·03

183

0-1 M-NaCI 0·01 M-sodium acetate pH 5·0

71 79 123

1-01 1·00 1·01

O'!lH

190

23

23

23

0·1 M·NaCl 0·01 M·EDTA pH 7·0 0·01 M·NaCI 0-001 M·EDTA pH 7·0

t Calculations made assuming dnjde

R~

(A)

30·1 57·485·3

{)'98

0-99 1·01

1·04

196

41-8 76·11 102·7

0-96 1·01 1·05

1·12

4-85

= 0·194_

place the molecular weight of R17 RNA at 1·04 ± 0·07 million. This is a weightaverage molecular weight and would equal the actual molecular weight of the RNA only if the preparation were 100% homogeneous. Preparation 23 contained less than 10% material sedimenting more slowly than 26 s and even this degraded material has sufficiently high molecular weight to make /1, contribution to the weight average of the whole sample. Therefore, we estimate the actual molecular weight of the 26 s component to be at most 5% higher, or 1·09 million. Comparison of the results obtained in 0·1 and 0·01 M-NaCI, given in Table 3 and Fig. 3, shows that the molecular weight is independent of counterion concentration as suggested by Kaesberg's group. Figure 3 also shows that the angular envelopes are linear over the angular range measured at all three sodium ion concentrations, as one would predict for randomly coiled polymer chains (Doty & Steiner, 1950). Lower salt concentrations were not used since high positive viral coefficients resulting from electrostatic repulsions make extrapolation to zero concentration unreliable. Figure 4 shows that there is an increase in the viral coefficient when the salt concentration is reduced from 0·1 to 0'01 M. The independence of molecular weight on counterion concentration reported above is contrary to results we originally obtained on samples not dialysed against EDTA.

R. F. GESTELAND AND H. BOEDTKER

502

2·0

1·2

o

0'1

0'2

0'3

0'4

O'S

0'6

0'7

0'8

sin! <0/2) FIG. 3. Light-scattering angular envelopes of R 17 RNA (23) at various salt concentrations. 0= 0·01 M-NaCl+ 0·001 M-EDTA, RNA concentration = 103 p.gjml.; Ct =0,05 M·NaCI + 0·005 MEDTA, RNA concentration = 99,.gjml.; • = 0·10 M-NaCI+O·OI M-EDTA, RNA concentration = 93p.gjml.

1·0

~Ici'

0'95

0'90

o

20

40

60

80

100

Concn \fL9/mU

FIG. 4. Light scattering of R17 RNA as a function of concentration. 0 = 0·01 M-NaCI + 0-001 MEDTA; • = 0·10 M-NaCI+O'OI M-EDTA.

PROPERTIES OF PHAGE R17 AND ITS RNA

503

Apparent molecular weights of 1·4 million were obtained in 0·02 lIf-NaCl. Some preparations gave even higher molecular weights when measured in 0·1 M-NaCl, and the downward curvature of the scattering envelopes reflected a high degree of polydispersity, suggesting a non-specific aggregation. The possibility that this was being caused by heavy-metal contaminants was considered and tested . The relative absorbance at 260 mJ.L was measured at 20°C as a function of Na+ concentration for both a dialysed and non-dialysed preparation of RNA. The results shown in Fig. 5 indicate that a dialysable factor is suppressing the hyperchromic change on lowering the Na+ ion concentration. This is consistent with the suggestion that heavy-metal ions are present before dialysis.

--._--_._~--~----:

10.4

10-3

o 10-2

• 10- 1

•

Molar eonen of Noel

FIG. 5. Relative absorbance of R17 RNA at 260 mp' as a function of NaCI concentration relative to that in 1 mM-MgH. 0 == before dialysis, 1500-fold dilution into appropriate solution. • == after dialysis against EDTA (see Methods), 250-fold dilution into appropriate solution.

(c) Thermal stability

To show that the one million molecular weight obtained for R17 RNA represents a single polynucleotide and not an aggregate of subunits held together by secondary valence forces , dilute (30 to 100 J.Lg/ml.) RNA solutions in 0·1 M-sodium phosphate buffer (1: 1) were incubated at 70°C for various times, rapidly cooled, and the sedimentation coefficient determined. Since 70°C is well above the denaturation temperature in this solvent (see below), any hydrogen-bonded subunits should dissociate. Figure 6 shows that more than half of the R17 RNA molecules are resistant to degradation after initial heating to 70°C although the mean sedimentation coefficient decreases slowly from its initial value of 24·9 s to 23·3 S , 22·7 sand 17·3 s after 60, 150 and 300 minutes of incubation respectively. The degradation appeared to be random since no discrete slower component is formed . To test this further, we mea sured th e degradation of R17 RNA in SSC at 90°C. These conditions were chosen since they are identical to those previously used to establish the random thermal breakage of the phospho-ester bonds of TMV RNA (Moller & Boedtker, 1962). The first-order rate constant for bond scission of R17 RNA was 2·9 x 10- 7 sec:", compared to 3·3 x 10- 7 sec"? obtained for TMV RNA.

504

R. F. GESTELAND AND H . B O E D T K E R

Iqo

o

75 50 c

·30

;:; 25 c

1/ V C

0

0

V

II

.e ., 0

"ii

a::

x-F IG. 6. Sed imentation diagrams of Rl7 RNA as a function of t im e of heating at 70°C in a·l x -sod iu m phosphat e (I : I ). Tracings from four different ultracen trifuge runs w ith time in terval ch osen t o co r respond roughly t o the ca lcula te d S •• ,w (see results) .

(d) S econdary structure Th e stability of the secondary structure of R17 RNA was studied by measuring th e relative ab sorbance-temperature profile and the rea ctivit y with formaldehyde. . Figure 7 shows a typical heating curve for R17 RNA together with results obtained for E . coli rib osomal RNA and TMV RNA. The breadth of t he helix-coil transition is similar for all three types of RNA, suggest ing that they ha ve similar secondary structures. The midpoint of the relative absorbance- t emperatur e pr ofile, TID' is roughly proportional to t he mole fraction of possible guanine-cytosine bonds (see Table 4), a relation observed pr eviously (Spirin, 1961 ; Fresco, 1963) for a variety of RNA samples. Further evidence of the similarity of R17 RNA to other RNA samples studied pr eviously is obtained from the rate of reaction with formaldehyde. RNA in 0·1 Msodium phosphate buffer (I : I) at 45°C was made 2'75% with formaldehyde, an d the rea cti on was followed by measuring the relative abs orbance at 270 mJ-L. The first- order rate constant s obtained for the three different RNA's are listed in Table 4. It can be seen that there is a correlation between the guanine-cytosine content of the RNA and the rate of reaction, the rate increasing as the guanine-cytosine content decreases, At the end of the reaction all samples had reacted to a comparable extent, indicating t hat t here are no unusually stable, form ald ehyde-resistant base pairs in any of the three sa mples. R elative absorbance-temperature profiles of R 17 RNA were measured in several solvents , but in view of the effect of dialysis on hyperchromicity, only those measured on EDTA.dialysed samples are considered reliable. In 0·3 M-NaCl, 0·03 M-sodium citrate, T m was 63°C; in 0·1 M-NaCl, 0·01 M-EDTA, T m was 59·SoC; and in 0·01 M Na Cl, 0'001 i\l-EDTA , it was 41°C.

PROPERTIES OF PHAGE R17 AND ITS RNA

505

1'00

0-95

0'90

0'80

0·75 90 70 Temp.{°C) FIG. 7. Relative absorbance-temperature profiles of different RNA samples in 0·1 M-sodium phosphate buffer (1: 1). • = TMV RNA, () '= E. coli ribosomal RNA, 0 '= R17 RNA

10

TABLE

4

Comparison of secondary structure of RNA's Source

TMV E. coli ribosomes R17

Mole fraction guanine plus cytosine

Trot

0·41 0·50 0·52

51 54 58

(oG)

kt(sec- 1 } x 10' for formaldehyde reaction 6'1 5'1 4'4

t Measured in 0'1 M-sodium phosphate (1: 1), pH 7·0.

5. Discussion The molecular weight (by light scattering) of R17 bacteriophage of 3·6 million agrees with the value of3·6 million reported by Strauss & Sinsheimer (1963) for MS2 bacteriophage, and suggests that these phages have approximately the same mass. A greater discrepancy exists between our light-scattering results and the molecular weight (by sedimentation diffusion) of 4·2 million reported for R17 (Enger et al., 1963). Since light. scattering results, if wrong, tend to give too high a molecular weight, it would be unusual if the results were in error by being too low. An additional suggestion of the

506

R. F. GESTELAND AND H. BOEDTKER

correctness of the molecular weight (by light scattering) can be obtained from the RNA composition of bacteriophage and its molecular weight. From the molecular weight of R17 bacteriophage measured here (3'6 million) and of the RNA prepared from it (1-1 million), we calculate that t he RNA content of the virus is 31%' assuming one molecule of RNA per particle. This agrees with the RNA content measured by Enger et al. (1963) for this bacteriophage (31'7 %) and by Strauss & Sinsheimer (1963) for MS2 phage (31'5 %) . The fact that most of the one million molecular weight RNA remains intact after short exposure to 70°C and degrades randomly at 90°C strongly suggests that it consists of a single covalently bonded polynucleotide chain. There seems little reason to doubt, therefore , that all the RNA of an R17 bacteriophage is contained in a single molecule. The conformation and secondary structure of bacteriophage were reported as unusual by Strauss & Sinsheimer, and as typical of other RNA's by the Kaesberg group. We find ourselves in agreement with part of both of these opposing statements. Thus we agree with Strauss & Sinsheimer that this RNA is an unusually compact molecule. Even though our values for the sedimentation coefficient are lower than those obtained for MS2 RNA, it still is impressive that phage RNA sediments with a sedimentation coefficient of 26 s in solvents in which TMV RNA having twice the molecular weight also sediments at about 26 s (Boedtker, 1960). This difference makes it clear that it is unwise to estimate RNA molecular weights from sedimentation data alone. In addition, we too find that the radius of gyration of this RNA is unusually low. For example, it is somewhat less than 200 A in 0·1 M-salt while that of pure 16 s E. coli ribosomal RNA having approximately half the molecular weight of R17 RNA is 260 A (Kurland, 1960). Thus it seems clear that bacteriophage RNA is able to form hydrodynamically more compact conformations than other RNA's studied thus far. This could be explained by contaminating metal ions , by the relatively high guanine-cytosine content, or as the result of the size of the helical regions which are, of course, a reflection of specific base sequences. Although R17 RNA, like MS2 RNA, forms unusually compact conformations in solution, there is no evidence that these are the result of an unusual secondary structure. In agreement with the results obtained by the Kaesberg group, we find the stability of the helical regions of R17 comparable to what would be pr edicted for an RNA having this particular guanine-cytosine content. Finally, we have confirmed Kaesberg's observations thatR17 RNA behaves normally in low salt concentrations. We found a significant decrease in the sedimentation coefficient but no increase in the molecular weight by light scattering as the counterion concentration is reduced. These results are in clear contradiction to those reported by Strauss & Sinsheimer. It is possible that this difference could be explained by the fact that we are studying RNA isolated from a different strain of bacteriophage. All the other physical and chemical measurements, however, indicate that the strains are very similar. The RNA content of the virus is identical, the base compositions are indistinguishable, and the molecular weight by light scattering in high salt concentrations is the same. This suggests that there was some facto r present in the preparations of MS2 RNA which caused Strauss & Sinsheimer to obtain anomalous results in low salt concentrations. Since we also obtained such anomalous results on R17 preparations which were not dial ysed against EDTA, we suspect that the unusual properties reported for MS2 bacteriophage RNA were the result of small amounts of heav y-metal ions.

PROPERTIES OF PHAGE R17 AND ITS RNA

507

We wish to thank Professor J. D. Watson for his helpful suggestions and criticism. We are indebted to Mr. John Richardson for his help in working out details of the virus purification, and to Mrs. S. Michener for help in the virus preparations. Finally, we wish to thank Miss Sigrid Stumpp for making some of the sedimentation measurements. This research was supported by National Institutes of Health grants GM 1l023-07, C-2107, GM 09541·02 and a grant from the Union Carbide Corporation to one of us (R. F. G.). REFERENCES Boedtker, H. (1960). J. Mol. Biol. 2, 171. Brice, B. A., Hawler, M. & Speiser, R. (1950). J. Optical Soc. Amer. 40, 768. Davis, J. E., Strauss,J. H., Jr. & Sinsheimer, R. L. (1961). Science, 134, 1427. Doty, P. & Steiner, R. F. (1950). J. Chem. Phys. 18, 1211. Enger, M. D., Stubbs, E. A., Mitra, S. & Kaesberg, P. (1963). Proc. Nat. Acad. Sci., Wash. 49,857. Fraenkel·Conrat, H., Singer, B. & Tsugita, A. (1961). Virology, 14, 54. Fresco, J. R. (1963). In Informational Macromolecules, ed, by H. J. Vogel, V. Bryson & J. O. Lampen. New York and London: Academic Press. Kurland, C. (1960). Ph.D. Thesis, Harvard University, Cambridge, Mass., U.S.A. Loeb, T. & Zinder, N. D. (1961). Proc. Nat. Acad. Sei., Wash. 47, 282. Mitra, S., Enger, M. D. & Kaesberg, P. (1963). Proe. Nat. Acad. Sci., Wash. 50, 68. Moller, W. & Boedtker, H. (1962). Acides Ribonucleique« et Pobrphosphates, Structure, Synthese et Fonctions, no. 106, Editions du Centre National de la Recherche Scientifique, Paris VII. Paranchyeh, W. & Graham, A. F. (1962). J. Cell. Compo Physiol. 60, 199. Spirin, A. S. (1961). Biokhimiya, 26, 511. Strauss, J. H., Jr. & Sinsheimer, R. L. (1963). J. Mol. Biol. 7, 43.