Effect of x-irradiation on the physicochemical properties of deoxyribonucleic acid

ANCHIVEs

OF

BIOCHEMISTRY

ASD

BIOPHYSICS

Effect of X-Irradiation

61.5422

117,

on the

Physicochemical

Deoxyribonucleic HELES

(1966)

Properties

of

Acid

HARRINGTOS

With the assistance of Marlene Ricanati Departments

of Radiology

and Biochemistry, Received

WesfeTn Reserve C’niversity, March

Cleveland,

Ohio

7, 1966

Intact lymphoblasts and isolated DNA were irradiated under conditions known to reduce the priming act,ivity of DNA for Escherichia coli DNA-polymerase in order to detect structural alterations responsible for the decrease in biological activity. Irradiation of intact lymphoblaets with lOCKI R caused single-strand breaks in the DNA as evidenced by a great,er reduction in the viscosity of the DNA subsequentl) extracted and heat-denatured as compared with t,hat observed in DPr’A extracted from control cells and denatured. Irradiation of DNA in dilute solution with 1000 1L was more effective in producing alterations in t,he DNA, as evidenced by a reduct,ion in the viscosity of native DNA, a reduction in the ?‘,,‘ and sedimentat.ion rate, and an increased sensitivity to alkaline phosphatase and a nuclease preparation from E. co/i. It, has been proposed that enough single breaks are produced on opposite strands b) the in vitro radiation to cause horizontal cleavage. Iodouracil substitution in the l>NA increased the irradiation effect when intact cells were irradiated but not when the DNA was irradiated in dilute solution.

The effects of x-irradiation on the physicochemical properties of DKA have been st’udied for many years; the alt’erations observed include a decreased molecular weight (l-6)) deamination and depurination (7)) denaturation (S-la), and cross-linking (13-15). Evidence for the occurrence of single-strand breaks in x-irradiated DNA has also recently been accumulating (16-15). In general, very large doses of x-irradiation have been found necessary to cause detectable structural alterations in the DNA. In contrast, we have previously reported doses of 1000 R and below to produce a marked decrease in the priming activity of DNA for the DKA- and RKA-polymerases of Escherichia coli (19). In order to invest,igat!e the structural alterations responsible for the decreased biological activity observed after low doses of x-irradiation, we have studied the physicochemical nature of DSA irradiated under the same conditions shown to produce the decrease in priming Mivity. Cnder t,hese conditions WC have 615

observed changes in the viscosity, sedimentation rate, t’hermal transition, and sensitivity to hydrolyt,ic enzymes, indicating the occurrence of single- and doublestrand breaks, perhaps accompanied by small regions of denaturation. In addit,ion, it was found that substitution of the DSA thymine with iodouracil increased the sensitivity of DSA to x-irradiation if the DNA were irradiated in viuo (in the intact cell), but not if irradiated in z&o (in dilute solution). Preliminary reports of t,hesc results have appeared previously (20-22). MATERIALS

A?il)

METH01)S

1. DXA. Deoxyribonucleic acid was prepared from the leukemic lymphoblast, L5178Y, either by the method of Kay et al. (23), but using 0.15 M NaCl - 0.015 M sodium citrate instead of distilled water as a solvent, or by the met’hod of Marmur (24). Calf thymus DNA was prepared by the method of Hurst (25) and Bacillus subtilis I)NA by the method of Young and Hpizizen (26). The preparations cont,ained 8-13 mg RN9 and 6-13 mg protein per 100 mg I)P\‘A. Ribonllcleic arid

616

HARRINGTON

was determined by the method of Barnum et al. (27), protein by the method of Lowry et al. (28), and DNA by the method of Burton (29). Deoxyribonucleic acid substituted with iodouracil (IU) was isolated from lymphoblasts grown in the presence of 12SI-iododeoxyuridine (IUDR) (5.64 PM) and fluorodeoxyuridine (FUDR) (0.175 PM) for 36 hours. IUDR was synthesized in collaboration with Dr. W. H. Prusoff according to the method described by Prusoff (30) using 1261obtained from Oak Ridge National Laboratories. FUDR was the generous gift of Hoffman-LaRoche, Inc. The degree of IU substitution in the DNA was determined either by density gradient centrifugation in CsCl or by determination of the 1*61 content by scintillation counting. Results obtained with the two methods were in good agreement and showed that the IU substitution in different DNA preparations ranged from 20 to 60% of the thymine content. Deoxyribonucleic acid labeled with 3H, 14C, or 32P was prepared from lymphoblasts grown in the presence of 0.25 pC per milliliter 3H-thymidine (Schwarz Bioresearch, Inc.), 0.08 PC per milliliter 14C-adenine (Schwarz Bioresearch, Inc.), or l-3 PC per milliliter a2P 04 (Oak Ridge National Laboratories). 2. Irradiation. Irradiation was carried out at the rat,e of 73 R per minute (250 kV, 15 mA, filtered through 1.5 mm Cu). For in vivo (in the intact cell) irradiation studies, cells were irradiated in the growth medium (19) and were then immediately centrifuged and frozen, the DNA being extracted at a later time. For in vitro (in dilute solution) irradiation studies, DNA was dissolved in 0.01 M phosphate buffer, pH 7, to a final concentration of 3&100 pg per milliliter. Control cells or control solutions of DNA were treated similarly except for the irradiation. For irradiation in the presence of 02 or NP, the vessels containing the DNA solution were evacuated for 5 minutes and flushed 5 minutes three times. After the final flushing the vessels were stoppered and irradiated. 3. Viscosity. Viscosity was measured on solutions containing 33 rg DNA per milliliter 0.01 M phosphate buffer. Reduced specific viscosities were determined in Cannon-Manning (WC) semi-micro viscometers with flow times of approximately 10 minutes and gradients of 550-700 set-1, and suspended in a water bath regulated at 25 f 0.01”. Intrinsic viscosity [v] was determined in a Cannon-Ubbelohde 4-bulb viscometer which allowed correction for the shear rate. 4. Thermal transition. Thermal transitions were carried out in a Beckman DU spectrophotometer fitted with a circulating water jacket. Deoxyribonucleic acid was dissolved in 0.01 M phosphate buffer, pH 7. Control and irradiated DNA solutions were heated simultaneously. 6. Sedimentation velocity. The sedimentation

rates were determined in a Spinco model E centrifuge fitted with ultraviolet optics, at a speed of 50,740 rpm, using cells with an aluminum centerpiece and a light path of 30 mm and DNA at a concentration of 20 rg per milliliter. The solvent used was either 0.01 M phosphate buffer or 0.15 M NaCl. 6. Hydrolysis by alkaline phosphatase. Chromatographically pure alkaline phosphatase (EC.3.1.3.1) was obtained from Worthington Biochemical Corporation. Fifty pg a2P-DNA (10e105 cpm per microgram P) was incubated with 18 units (as defined in the Worthington catalogue) of the enzyme in a solution containing 0.0625 M Tris buffer, pH 9, at 37” for 1 hour. The total incubation volume was 1.6 ml. The reaction was terminated by the addition of 15 mg bovine serum albumin and 0.05 ml of 12 N HClOd. The solutions were diluted with cold 0.6 N HCIOl and were centrifuged at 35,000g for 10 minutes at 4”. To the supernatant fluid was added 10 mg of activated charcoal, and the solutions were shaken intermittently for 1 hour and were then centrifuged. The supernatant fluid was filtered through Whatman No. 1 filter paper, and aliquots were counted by liquid scintillation and corrected for quenching. The percentage hydrolysis was calculated by comparing the radioactivity in the charcoal filtrate with the total radioactivity in the added DNA. When 10 pg DNase I (EC.3.1.4.5)-digested z2P-DNA was incubated for 1 hour with 7 units of alkaline phosphatase, 25.7’% of the added a2P was found in the charcoal filtrate. 7. Sensitivity to a nuclease preparation from E. coli. An enzyme preparation was isolated from E. coli according to the method of Lehman (31). After chromatography on DEAE-cellulose the preparation contained both exonuclease I and a nuclease capable of hydrolyzing double-stranded DNA. Five pg of labeled DNA was incubated with approximately 200 units, as defined by Lehman and Nussbaum (32), of the enzyme in an incubation medium consisting of 0.06 M glycine, pH 9.2, and 0.008 M MgC12, in a total volume of 0.25 ml for 30 minutes at 37” The reaction was terminated by addition of albumin and HCIOa as described above, the solutions were centrifuged, and the radioactivity in the acid-soluble supernatant fluid was determined. Two hundred units of the nuclease preparation converted 95-1009& of heatdenatured DNA and 12-25yo of native DNA to an acid-soluble form during the incubation period. RESULTS

1. E$ect of Irradiation

on Xpecif;c Viscosity

In vivo irradiation. Table I shows the reduced specific viscosities of DNA preparations from (1) control, (2) irradiated,

STRUCTCRAL

CHANGES

IN

X-II~1~.~DIATEI~

case of the in civo irradiation, heatming the DNA reduced t,he viscosity of the irradiated DiY;h IO a great)er extent than that, of the nonirradiated DNX. Since the viscosity of native DXA$ ~1s reduced by in vitro iI.radiation before heat denaturation ~vhilc :t reduction in viscosity aft,er in ZV%Oirr:uliat’ion lvas apparent only aft,er heat denaturation, the in vik.0 irradiation appeared IO be more effecbive in altering t)he DK:\. Thcb in vitm irradiation also reduced the vi+ cosit,y of the K-substituted DK-A. but llot to a greater extent than in the case of IIW unsubstituted DNA, i.e., IV-substitut ioIl did not potentiate the in vitro irradiation effect as it did in the case of Gl. hvo irratli at,ion. If the reduced specific viscosities Hertz corrected for the shear rate, the effecl of irradiation on dilute soWions of DS:\ hc-

(3) Ikubstituted, and (4) irradiated, IGsubstituted lymphoblasts. The irradiation of nonsubstituted cells did not cause a significsnt~ decerease in the viscosity of the DS,2 subsequently extracted, but the viscosity of DKA extracted from irradiated ICsubstituted cells TV~S 40% lower than that rxtrwctc~d from nonirradiated IUsubstituted cells. Heating the DPl;A subquent to its extraction decreased its viscosity in all (‘LIES, but t)he decrease mas greater in the wsc of the Dn’h ext’racted from irradiated cells than from non-irradiated ills, whefiler 01’ not the DNL4 confained It’. In uitro irradiation. When dilute solut’ions of unsubstituted lymphoblast DXA were irradiated Jvit’h 500-1000 R, the reduced specific visc>osit,y of the DNA shomed a significant decrease (Table II). As in the TABLE OF DNA

61 T

I )?;A

I

TREATED WITH IUDR AND 1000 I< DNA was extracted from control, irradiat,ed, W-substituted, and IU-subst,ituted-irradiated cells. Viscosity was determined before (native) and after (heat denatured) heating in a boiling water bath for 10 minrttes and rapidly cooling. Molecular weights were estimated from a chart given by Eigner and Doty (34). ‘C’MYJHITY

ISOLATED

FROM L 5178 Y CELLS

Reduced specific viscosity

Treatment

Kative

Control

15.4 14.5 16.3b 9.76

1000 R

IFslthstituted W-substituted

+ 1000 R

u Standard deviation. b, c Menu values which

differ

from

each other

f i zk *

Heat denatured

3.8” 3.6 5.4 1.G

at the 5% confidence

TSBLE EFFECT

30 pg DNA/ml trol samples were

before

and after

f rt rt f

0.Q 2.1 1.24 1.90

level.

DNA of the irradiated

OF LYMPHOBLAST

viscosities

Control 500 R 1000 R X-substituted IU-substituted IU-substituted

Native

+ 500 R + 1000 R

and con.

heating. Reduced specific viscosity

Treatment

-4 1 2 5 3 .5 1.x

II

OF in Vitro IRRADIATION ON THE VISCOSITY 0.01 M PO1 buffer was irradiated and the specific

determined

5.@ 3.4c 4.8 2.5

Approximate molecular weight of heated DNA (X UP)

22.4~~ d 17.3c 14.4d 14.2 13.1 10.8

Heat denatured

zk 3.8” f 2.1 zk 2.3 f

1.1

n Standard deviat,ion. b Estimat,ed from a chart given by Eigner and Doty (34). ~1d, e Mean values which differ from each other at the 5y0 confidence

8.8C~d 3.3d 2.oc 4.7” 2.7 2.1”

level.

It 2.10” + 0.49 * 0.15 f

0.39

Approximate molecule weight of denatured DNA* (X lOmE) 6.4

2.4 1.4 3.5 2.0 1.4

618

HARRINGTON TABLE

III

COMPARISON OF REDUCED SPECIFIC VISCOSITY

EFFECT

DNA AFTER in Viva in Vitro Reduced specific viscosity (IJ~~,~)was determined in Cannon-Manning semi-micro viscometers, and intrinsic viscosity [TJ] in a CannonUbbelohde 4-bulb viscometer. AND

INTRINSIC IRRADIATION

VISCOSITY

0~ AND

Calf Thymus DNA

%P/C

bll

Lymphoblast

Control

lp,“ti,T‘,

% ;;:b”; 1000 R

Control

26.0 32.1

14.5 12.9

44 60

21.9 31.3

DNA

60

70

80

60

ON

in

EFFECT

?$ decrease in specific viscosity after 1000 R

33 50 67 100 250 500 750 1000

30.6 36.5 31.5 35.0, 24.8 16.9 19.8 11.9 9.2

taining 1 X 1W3M mercaptoethanol. The reduction in specific viscosity observed when solutions of DNA in 0.01 M phosphate buffer were irradiated was similar whether the irradiation was carried out under an atmosphere of air, oxygen, or nitrogen. 2. Thermal Transition Irradiation of intact cells did not alter the midpoint temperature of the thermal transition (T,) of unsubstituted DNA, but the T, of IU-substituted DNA ex-

80

70

TEMPERATURE,

FIG. 1. Effect of irradiation

RADIATION

Cont. DNA during irradiation (&ml)

16 -

came even more pronounced, since the viscosity of the in vitro irradiated DNA was not as greatly affected by the shear rate as was control DNA or DNA irradiated in viva (Table III). The intrinsic viscosity of DNA was not reduced by irradiation of intact cells. If solutions containing more than 100 pg DNA per milliliter were irradiated, less effect on specific viscosity was observed (Table IV). The reduction in specific viscosity was not observed when solutions of DNA were irradiated in tissue culture medium, in 0.15 M NaCl-0.015 M sodium citrate, or in 0.01 M phosphate buffer con-

TABLE IV DNA CONCENTRATION

All solutions were diluted to 33 pg DNA/ml 0.01 M PO1 buffer before running viscosity determinations. Calf thymus DNA was used in these experiments.

% !OOO.R Red- by tn VIYO tmn 1OOOR

18.4 33.2

OF Vitro

‘C

on the thermal transition of DNA. a. Irradiation of intact cells. (---) DNA extracted from control cells (T, = 67.3). (- - -) DNA extracted from of intact cells containing IU-substituted cells given 1000 R (T, = 67.3). b. Irradiation DNA. (---) IU-substituted DNA extracted from control cells (T, = 68”). (---) IU-substituted DNA extracted from cells given 1000 R (T,, = 65”). c. Irradiation of dilute solutions of DNA. (--) control DNA (T, = 69.0”). (---) DNA given 1000 R in dilute solution (T, = 67.5”).

STRUCTURAL

CHANGES

tracted from irradiated cells was decreased by 2-3.5”. The T, of unsubstituted DSA irradiat,ed in dilute solution was decreased the initial rise in absorption by 0.713, occurring 5” lower. One set of curves is shown in Fig. 1. The increase in hyperchromic*ity seen in the case of DNA extracted from irradiat)ed cells (Part a, Icig. I ) was not observed in subsequent expcrinicnts. 3. Sedinwztation Rate Irradiation of intact cells did not significantly affect the sedimentation rate of the DXA subsequentSly extracted, while irradiation of DNA in dilute solution appeared to cause a decrease, especially in the wse of B. subtilis DNA. The comparatively large decrease in sedimentat,ion rate observed when irradiated calf thvmus DNX was denat,ured indicates the presence of single-strand breaks in the irradiat’ed DXA (Table \‘). J. Hydrolysis by Alkaline Phosphatase Breaks in t,he phosphodiester chains of DNA1 Avould be expected to increase the TABLE

06

Sedimentation velocit,ies were run in 30 mm cells with DNA at a concentration of 20 rg/ml in 0.01 II phosphate buffer or 0.15 M NaCl saml,le

L5178 Y Control 1000 R, in Z,i/,O 1000 R, in ~~i!ro B. subtilis Control 1000 R, in llitro Calf

s8. VJ sm.to 0.01~phosphate buffer O.lhlNaCl

25.4 (f5.4)" 22.06 (f2.2) 17.1" (zt3.2)

31.8 27.4 24.0

32.1 19.6

thyms

Control Denatured 1000 R, in &TO Denat rlred after 1000 R, in c,ilro

~X-II~1:A1)IATEI)

(il!)

IJNA

amount of inorganic phosphanb groups sensitive to alkaline phosphatase. Irrxtliation of intact cells or dilute sohnions of DiYA wit’h 1000 R did not appear to have a significant effect on t,he extent, of suhsequent hydrolysis. A tlefinim increase in the amount of phosphate liberated by t,htt PIIzyme was apparent, however, when dilut,o solutions of t,hc DNA were irradiat,ed with 5000 or 10,000 Ii (Table VI). llcc~rt cl\,idence has indicdated that alkaline phosph:itase may not liberate internal monocstcrficti phosphat,e from double-stranded DNA (33). Therefore, the method used probably (lid not, detect- single-strand breaks, but onlv the double-strand breaks caused by irra~l~at,ion. The number of double-strand breaks in DSA irradiated witjh 1000 l( wer(l rvidentlp I101 suffic+nt t0 (‘:111sC3 (It~lCX~tilhlt~

32P-11NA samples were incubated with slkaliue phosphatase for 1 hollr, and arid-soluble, charcoal-nonadsorbable 32P was determined. D?;A sample

G; IIydrolysis

V

EFFECT 0~ S-IRRADIATION SEDIMENTATION RATE

DS.4

IN

17.4 7.3 12.4 4.5

n 8t)alrdard deviation. b Sot significantly different from the control vallle (0.5 > p > 0.4). c Ijiffers from the control mean at a confidence level of (0.1 > p > 0.05).

Cant rol 1000 R in Vi,‘0 1000 R in G/W 5000 R in vitro 10,000 I< in Z'iltY~

0.08 0.10 0.09 0.21" 0.5@

It 0.06” i 0.06 f 0.K zk 0.15

(LMean valltes which differ from mean at the lyc confidence level D Stantlard deviation. TABLE

the control

\‘I1

~IYI)HOLYSIS OF DNA HY .1. NCCI,E.\SE Pru?.:Paxa~rloN Fltoxf E. coli Labeled I)!XA samples were incubated 30 mirl with the Itucleasc preparation and acid-solllt)le radioactivity was determiued. I)TA sample (:ontrol 1000 R ire aim 1000 It in vitro 5000 R in vitro 10,000 R in Glro

‘;

Hydrolysis

li.9 17.6 25.W 44.P

f

fi.fi”

f i

4i.8”

f

a.ti 10.1 11.4

a Meal1 values which differ from mean at the 1% confidence level. b Standard deviation.

the 0011tro1

620

HARRINGTON

TIME (HOURS)

FIG. 2. Hydrolysis of native, denatured and irradiated DNA by a nuclease preparation from E. co&. Labeled DNA samples were incubated with the enzyme preparation for various intervals, and the acid-soluble radioactivity determined. (-) Native DNA; (--) denatured DNA; (----) DNA irradiated with 5000 R in dilute solution.

increase in hydrolyzable 32P043-, one break per DNA molecule being calculated to cause an increase of 5-50 cpm in acid-soluble radioactivity.

5. Hydrolysis by an E. coli

whether the alteration consisted of singlestranded regions hydrolyzable by exonuclease I, or an increase in available binding sites per nucleic acid molecule for a contaminating nuclease, or both.

Nuclease Preparatim Irradiation of dilute solutions of DNA with lOOO-10,000 R increased its sensitivity to a 30-minute hydrolysis by a nuclease preparation containing both exonuclease I and another nuclease capable of hydrolyzing double-stranded DNA (Table VII). When pure, exonuclease I hydrolyzes only singlestranded DNA beginning at a 3’-OH end (31). With our preparation, the initial rate of hydrolysis of DNA given 5000 R in dilute solution was twice as fast as that of the control DNA (Fig. 2), but since both control and irradiated samples were almost completely hydrolyzed in 4 hours, the presence of another nuclease in the preparation was indicated. Hydrolysis of DNA by this enzyme preparation was also increased by a limited pretreatment of control DNA with DNase I, DNase II (EC.3.1.4.6), and ultrasound. Thus, although the results indicate that the irradiation produced an alteration in the DNA, it cannot be certain

DISCUSSION

Irradiation of L 5178 Y lymphoblasts with 1000 R produced an alteration in the DNA as evidenced by a greater reduction of viscosity upon heat denaturation than in the case of DNA extracted from nonirradiated cells. It therefore appears that the in vivo irradiation produced single-strand breaks in the phosphodiester chain since such breaks would be expected to affect viscosity only when the hydrogen bonds of the double helix are disrupted. The change in viscosity indicates that 1000 R in viva produced approximately one single-strand break per DNA molecule. The singlestrand breaks could result from direct or indirect action of the radiation or from increased nuclease action. Other properties of unsubstituted DKA extracted from

irradiated

cells did not

differ

signifi-

cantly from the control. Although more extensively degraded DNA could have been lost during the isolation procedure,

STRUCTURAL

CHANGES

the extracted Dr\‘,4 was of interest since it was shown to have a decreased biological activity (19). When dilute solutions of DNA were irradiated, a decrease in the viscosity was apparent even before heat denaturation. Other changes which we have observed in the in vitro irradiated DNA include a lowered II’nl, a decrease in sedimentation rate, and an increased sensitivity to hydrolysis of phosphomonester bonds by alkaline phosphatase and a nuclease preparation from E. coli. The greater effect of the in cit~o irradiation could be due to the relative lack of protective substances, the dilute nat,ure of the DNA, and the absence of enzymic repair. The increased sensitivity t,o alkaline phosphatase after in vitro irradiat,ion indicates that t,he number of breaks in the phosphodiester chain had increased. Collyns et al. (11) have also observed that the sensitivity to hydrolysis bv prostate phosphomonoesterase increased &cr x-irradiation of DiYA in dilute solution. If single-strand breaks occur on opposite strands :md within several nucleotides of each other, cleavage of the double helix would hc expected (16, 35). The decrease in viscosit,y and sedimentation rate indicates that the in vitro irradiation of 1000 R produc*ed 1-2 double-strand breaks and 4-G single-st,rand breaks per DNA molecule. The decrease in Z’, could be due to the lowered molecular weight of the irradiated DNA1 (36) or to a weakening of hydrogen hood.:: caausedby t#he single-strand breaks, or both. hs discussed previously, the increased sensit,ivity to the E. coli nuclease preparat,ion cdtl be due eit,her to the presence of single-st’randed regions or to breaks in the phosphodiester chain. Irrtversiblc alterations present in DKh after ia vitro irradiation may be mediated by the OH. radical, since the irradiat,ion r#ect was prevented by mercaptoethanol but was not, drcareased in an atmosphere of nitrogen or increased in an atmosphere of oxygen. The incarcased effect observed when dilut,e solut,ions were irradiated could be clue to the increase in the percentage of DSA molerules affect’ed by a given number of activated water molecules or to the pre\-ent,ion of aggregation of damaged

IN X-IRRATjIATED

DNA

6'21

molecules. The influence of protective substances, 02, and the concenbrat,ion of the DNA solution on the radiation effect has been studied previously by many workers and has been discussed by Butler (3). Erikson and Szybalski (37) have shown that cells containing IU-substituted DNA are sensitized to x-irradiation with respect to their capacity for further replication. In the present studies, IF-substitut,ion increased the alteration in DKA when intact cells were irradiat’ed but, not when DXA was irradiated in dilute solution. The results are therefore in agreement, with recent evidence relating halogenated pyrimidine substitution to prevention of repair of damaged DNA (3S-41). Deoxyribonucleic acid irradiated under the conditions used in these experiments has been shown to have a marked decrease in priming activity for RS.\- and DN& polymernses of E. coli (19). However, Ihc presence of single- and double-strand break:: in the irradiated DSX apparently cLannot entirely account for the decrease in priming activity (32). Although single-stranded :rutl partially denatured DKA act+ similarly to irradiated DSA in the polymcrasr system (42), little evidence has been foulId in t’he present work that an irradiation of 1000 R produces denat,urntion of the 11SA4. It is possible that several types of altjemtjions occur in irradi:&cd DS=i :mtl that the combined effects acacaountfor thck c~hnngc~in priming a(~1ivity.

This investigation was snpport rd 1)~ (JOIII r:~t W31-109.eng-78 of the I-.9. Atomic Allergy COIW mission with bf:estern Reserve lTnivrrsitv ad Iy research grant HI>-00669 from the N:l.tion:+l Institute of Child Iledth urd Htmx~u J)evelopmer~t~, 1J.S. Plthlic ITealth Service. We :rre indebted to Doris Roberts for determinations of sedimelltation rate, to I,llthcr Lindner for s~)me c#f the I?‘. coli ilrlclense determinations, :ctrd III (:hxrlw Belle for valu:~hlc techl\ic:tl assist;nlw

622

HARRINGTON

3. BUTLER, G. C., Can. J. Res. 27B, 972 (1949). 4. TAYLOR, B., GREENSTEIN, J. P., AND HOLLAENDER, A., Arch. Biochem. 16, 19 (1948). 5. KOENIG, V. L., AND PERRINGS, J. D., Arch. Biochem. Biophys. 44, 443 (1953). 6. SCHOLES, G., AND WEISS, J. J., Biochem J. 66, 65 (1954). 7. SCHOLES, G., AND WEISS, J. J., Nature 166, 640 (1950). 8. Cox, R. A., OVEREND, W. G., PEACOCKE, A. R., AND WILSON, S., Proc. Roy. SOC. (London) Ser. B 149, 511 (1958). 9. PEACOCKE, A. R., AND PRESTON, B. N., Proc. Roy. Sot. (London), Ser. B 163, 105 (1960). 10. HAGEN, U., AND WILD, R., Strahlentherapie 134, 18 (1964). 11. COLLYNS, B., OKADA, S., SCHOLES, G., WEISS, J. J., AND WHEELER, C. M., Radiation Res. 26, 526 (1965). 12. SARKAR, M., Nature 196, 269 (1962). 13. LETT, J. T., STACEY, K. A., AND ALEXANDER, P., Radiation Res. 14, 349 (1961). 14. SHUGAR, D., AND BARANOW’SKA, J., Nature 186, 33 (1960). 15. GUDA, H. E., FRAJOLA, W. J., AND LESSLER, M. A., Science 137, 607 (1962). 16. HAGEN, U., Strahlentherapie 134, 428 (1964). 17. DAVISON, D. F., FREIFELDER, O., AND HOLLOWAY, B., J. Mol. Biol. 8, 1 (1964). 18. SUMMERS, W., AND SZYBALSKI, W., Radiation Res. 26, 76 (1965). 19. HARRINGTON, H., Proc. Natl. Acad. Sci., U.S. 61, 59 (1964). 20. HARRINGTON, H., Federation Proc. 22, 582 (1963). 21. HARRINGTON, H., .~ND RICANATI, M., Radiation Res. 19, 188 (1963). 22. HARRINGTON, H., AND LINDNER, L., Biophysics Sot. Abstr. p. 42 (1965).

23. KAY, E. R. M., SIMMONS, N. S., AND DOUNCE, A. L., J. Am. Chem. Sot. 74, 1724 (1952). 24. MARMCR, J., J. Mol. Biol. 3, 208 (1961). 25. HURST, R. 0.. Can. J. Biockm. Physiol. 36, 1115 (1958). 26. YOUNG, F., AND SPIZIZEN, J., J. Bacterial. 81, 823 (1961). 27. BARNUM, C. P., NASH, C. W., JENNINGS, E., NYGAhRD, O., .~ND VERMUND, H., Arch. Biochem. 26, 376 (1950). 28. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1961). 29. BURTON, K., Biochem. J. 62, 315 (1956). 30. PRUSOFF, W. H., Biochim. Biophys. Acta 32, 295 (1959). 31. LEHMAN, I., J. Biol. Chem. 236, 1479 (1960). 32. LEHM.4~, I., AND NUSSBAUM, A. L., J. Biol. Chem. 239, 2628 (1964). 33. RICHARDS, 0. C., AND BOYER, P. D., J. Mol. Biol. 11, 327 (1965). 34. EIGNER, J., AND DOTY, P., J. Mol. Biol. 12, 549 (1965). 35. THOMAS, C. A., J. Am. Chem. Sot. 73, 1861 (1955). 36. CROTHERS, D. M., KALLENBACH, N. R., AND ZIMM, B. H., J. Mol. Biol. 11, 802 (1965). 37. ERIKSON, R. L., AND SZYBALSKI, W., Cancer Res. 23, 122 (1963). 38. HOWARD-FLANDERS, P., BOYCE, R. P., AND THERIOT, L., Nature 196, 51 (1962). 39. HOWARD-FLANDERS, P., BOYCE, R. P., SIMPSON, E., AND THERIOT, L., Proc. Natl. Acad. Sci., U.S. 48, 2109 (1962). 40. SAURBIER, W., Virology 16, 465 (1961). 41. STAHL, F. W., CRASEMAN, J. M., OKUN, L., Fox, E., AND LAIRD, C., Virology 13, 98 (1961). 42. HARRINGTON, H., J. Mol. Biol. 16,152 (1966).