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USNRDL-TR-65512 June 1963

IPRIMER ACTIVITY OF THYMUS DNA FRACTIONATED BYL :C 'ECTEOLA COLUMN CHROMATOGRAPHY

CD by4l:CW. D. Skidmore

R. K. Main< C/: L. J. Cole

0

SU.S. NAVAL RADIOLOGICALDEFENSE LABORATORY

yi!4SAN FRANCISCO 24, CALI FORNIADDC

Ji ~L2LTISIA E

EXPERIMENTAL PATHOLOGY BRANCHL. J. Cole, Head

BIOLOGICAL AND MEDICAL SCIENCES DIVISIONE. L. Alpen, Head

ADMINISTRATIVE INFORMATION

This work was accomplished under the Bureau of Medicineand Surgery Task MR005.08-1200, Suhtask 7, Technical Ob-jective BR 03800. as described in the U. S. Naval Radiologi-cal Defense Laboratory Annual Report to the Bureau of Medi-

cine and Surgery (OPNAV FORM 3910-1) of 31 December1962, and is listed in the U. S. Naval Radiological DefenseLaboratory Technical Program Summary for Fiscal Years1963-1965 of 1 November 1962 under Program A3, Prob-lem 1 entitled *Fundamental Studies in Radiobiology."This study was supported through funds provided by theBureau of Medicine and Surgery.

AVAILABILITY (F COPIES

Requests for additional copies by agencies or activitiesof the Department of Defense. their contractors certified toDDC (formerly ASTIA). and other government agencies oractivities should be directed to the Defense DocumentationCenter for Scientific and Technical Information. CameronStation, Alexandria, Virginia.

....... ...... 0.0 ....... ........................ .......... ....................

Evgeno P. Cooper E.S. Roth, CAPT USNScientific Director Commanding Officer and Director

12ND NRDL P1 (3-63)

ABSTRACT

The DEA-primer activity of Ecteola fractionated calf- and rat-

thymus DNIA samples was determined by a DNA-polymerase assay system. All

DNA samples assayed, heated or unheated, and fractionated or unfraction-

ated, showed some degree of DNA-primer activity.

The chromatographic profiles of heated DNA were different from

those of unheated DNA. Phenol and p-aminosalicylate, used in the prepa-

ration uf DiA, did not alter the patterns of MNA obtained by Ecteola

chromatoGraphy. Sephadex chromatography was found to be a rapid and

effect've method to deionize DNA solutions.

DNA-primer activity was increased by an anmonium hydroxide gradient.

At 40, treatment of DNA by phenol and p-aminosalicylate, Ecteola column

chromatoGraphy with a salt Cradient, Sephadex column chromatography with

deionized distilled water to desalt DNA, and !yophilization did not alter

DNA-primer activity.

The results indicate that DNA-primer activity, Mr se, is not

specifically associated with one particular fraction of DNA.

i

SLNARY

The Problem:

The unique property of DNA to act as a primer for its own synthesis

by the DNA-polymerase enzyme system is well established. In connection

with the general question of the role of DNA-primer in cell division in

vivo, the question arises whether there exists in rapidly dividing tissues

a specific fraction of DNA with primer activity. The problem was to

separate thymus DNA into fractions that demonstrated higher and lower

DNA-primer activities than did the original starting material.

The Findings

Calf- and rat-thymus DNA were found to be heterogeneous and could

be fractionated into separate chromatographic peaks by Ecteola column

chromatography.

The chromatographic profiles of heated DNA were different from those

of unheated DNA. Phenol and p-aminosalicylate, used in the preparation

of DNA, did not alter the patterns of DNA obtained by Ecteola chromatog-

raphy. Sephadex chromatography was found to be a rapid and effective

method to deionize DNA solutions.

DNA-primer activity was increased by an ammonium hydroxide gradient.

At 4, treatment of DNA by phenol and p-aminosalicylate, Ecteola column

chromatography with a salt gradient, Sephadex column chromatography with

ii

deionized distilled water to desalt DNA, and lyophilization did not alter

DNA-primer activity.

Primer assay of the DNA fractions indicated that primer DNA

was not separated from nonprimer DNA.

IN'TRODUCTIOT

The cJlasoicai. worJ of' KornbcrL-, et a? -, demontrated a requirement

i'or DIIA as primer in the enzymatic cynthczis of' DMAP b-, the rpJ.yuerase

system. Later, a miarked increa:ze in pri*minLj activity oi' DIA after

hcat~nj i~oso,e d' . -otdvr~ unheated DIIA. samrp? as haie "been

dif'ferentiated al~zc by characterl z-tl c eIhrornatcrahic ratterns 4s5

More reccntl,-, an enzoae-' rid-,ccd .ncrcce *n r- reactivity of var'.o..o

Isnati~e" DNA samr-co in tLe 2. col'A D.1-j.o..ynrerase system has .)een

reporteci

In th:s connect~or., thru qi.ect.'on arisoes whazer DIA can be fractior-

eted into pr:.rior and noniprrucr DIIA, ar.C. %:Letl~er a:2. MfA molec-- es have

primn.L civityv. Therc-lore, iwe inet~tdthe DIA-rxruzmer activity

associated u..t, Ectec~la co. inn chir.orto.,raph_'c fract-ons of' unrheated

versusn heated caJ i*- and rat-thy=:s. DIA samp,'es. In order to accomplish

ti.1s, DIA was repared *..% a phcno? eztract.cn neot,-od, 'ract-*onated v~th

an Ecteola colvmn, desulted v..tl _e-.e:,yaphilized, and assayed

for DII-primer activity rAn a I-ol.-merase sy-stem.

I

METHODS AU MATERIALS

Preparation of DNA samples. Worthington Calf-tymus DNA (67 mag)

was dissolved in deionized distilled water (93.3 ml) by stirring with a

magnetic stirrer for 22 hours at 40 . Tris-phosphate buffer (6.7 ml;

0.5 M, pH 7.3) was then added and a portion(50ma) was used for treatment

with phenol to determine if there would be an effect of phenol on DNA-

primer activity of DNA thus isolated. This portion was mixed with an

equal volume of 9O5 w/w freshly-distilled phenol. The resulting so-

lution was stirred for one hour at 40 and centrifuged at 20,000 rpm in

a Spinco Model. L centrifuge with a swinging bucket head (SW-25.2) for

30 min at 00. The upper aqueous layer was removed with the aid of a

tube-cutter and mixed with an eq ,al volume of 2-ethoxy ethanol (etl l

cellosolve, mono-ethyl ether of ethylene glycol) at 0 to precipitate

DNA in a fibrous form. The fibers were wrapped around a tglass rod in a

manner in which most of the mother liquor was squeezed out of the fibers,

removed from the flash, and dissolved in deionLzed distilled water (25

ml). EnouGh tris-phosphate buffer (0.5 M, pH 7.3) was then added to

effect a dilute D1A solution (in 0.03 M buffer) such that the A2 - 0 was

appro.:imately equal to the calf-thymis DNA aliquot not trcatea i -th

phenol.

Rat-thymus DNA was prepared by the method of IKirby 7 and aLso by a

simplified modification of it. The thymus [ ands c 5-, 2- ,ek old

female Sprague-Dawley rats were removed and homoen1zed at 0° in sucrose-

2

tris-ch-Joride buffer (sucrose, 0.2 M; tris, ii mM; pH 7.7) tnd centrifuged

(105,000 : g, 1 hr, 0). The resultant supernatant fluid, containing

D1-polymerase, was stored at -196°. The insoluble nucleoprotein pel-

let obta:ned (12 ) war c-orcd at -20 ° bcfoi'c etiac!n C the DMA. It

was homogenized with 6% wv soditum p-amdnosalicylate (15 vol) and then

stirred with 90% w, w pheno! (15 vol) for one hour at 4. The mixture

was centrifuged and the DNA was recovered as described above for the

D A treated with phenol. At this point a modification of the method

was introduced, in that the DNA was recovered without further treatment.

The DKA fibers obtained in cach case were dissolved in deionized water

(100 i.l) at , ° . Buffer wias added, the A260 was determined, and the

appropriate dilution made as above.

A port:on of each of the above samples was heated in a boiling water

'ath (-00) for 7 run and cooled quiclJy. The A2 60 of each sample was

determined in tr's-phosphate buffer (0.03 14, pH 7.3), and the DNA samples

were stored at -200 for Jater use.

Ecteola column chromatoiraphy. The method used was an extensive4

nodification of that of Rosenhranz and Bendich . A linear Cradient was

oet up *n the cold room (4') where two Graduated cylinders (250 ml) were

connected at their base so that the fluid levels in each cylinder re-

mined equal durinC clut-on. The elutinC solvent in one cylinder was

mixed with a magnetic stirrer before being conducted from the base of

that container to the column by polyethylene tubing.. Ecteola cellulose

anion c::chsngcr(type 20, 0.4 meq N, ,), purchased from Brown Co., Berlin,

3

N. H., was successively washed with water, 1 N NaOH, water, and buffer

(0.05 M Na1, 0.00. M phosphate, pH 7.0) before use. The coltumn (1.0 g

Ecteola, i. cm x 6.5 cm) was equ:ipped with a constant level siphon to

prevent the column from going dry when the eluting solvent was ex-

hausted. The amount of DNA sample (about 4 mg in 6.0 ml) put on the

column was determined in terms oi DNA-units (A260 x vol). The flow-rate

was about 0.5 ml per an and the fractions (60 drops per tube, about

S to 7 ml) were collected with an automatic fraction collector. The

method of !*ncar gradient eluton consisted of two steps: I) A salt

gradient from 0.05 M NaCl to 2.0 M NaC1 (50 ml each, buffered with

0.001 M phosphate, 1.1. 7.C); c.ni ii) .L. IH4 0H L-aicnt to

2.0 N I'IlOH in 2.0 14 NaCl (2.50 ml each). The volume and A2 O of the

fractions collected were measured before storing them at -20o. The

same column was used repeatedly by washinL the Ecteola with . N NaOH,

water, and buffer before applying a new sample.

Sephadex column chromatoLraphy. The general methods used were

modifications of those of other worhers c,9 Sephadex C2 (G-25, carse)

was purchased from Pharmacia, Uppsala, Swedenpand was washed rep~eated? y

with deionized distilled water to remove "fincs". T6c d1'.crent columns

were prepared in a sirllar manner: one (2.5 cm x 40 cii) was prepared

with 30 g and, the other (3.5 cr., 30 cm) with O U quantities. They

were eqipped with constant ! eve: siphons to prey ent the Sephade.: from

goinC dry when the clutin solvent was exhausted. The fractions eluted

from the Ecteola cc uxins and saved at -20 ° were thawed and put on the

4

Sephadex column in the cold room (4) for chromatography in order to

separate the DNA from NaCl and NH,OH. Deionized distilled water was

used as the eluting solvent. The flow-rate was 1-2 ml per min and

the fraction size collected with an automatic fraction collector was

6 -7 m per tube. The A20 x vol, electrical resistance, and pH of the

fractions were determined at 25*. Appropriate tubes selected for

Iyophilization were combined and the determinations were made as above,

in order to prepare the DNA for primer assay. The samples thus pre-

pared were stored at -20* for later use. The Sephadex columns were

used repeated:ly after thorough e ut:-'on of retained NaC2. and I 4 OH with

an excess of water.

DIIA-prmer assay. The incorporation of (311) thymidIne into newly

sm-nthcsized DIA was determined, in triplicate, by the r.ethod of Walwich

and Maitn .e assay mixture(total volume of 500 4l) contained:

(31!) th.-iydine (0 01, specific activity 3.00 c per r.mole); deoxynucleo.

si.de ''-monophosphates of adenine, cytosine, and -uanine (50 j! each);

i.c12 (6.0 z iI); KCl (45 m1M); tris-ATP (6.0 ILM); crude DIIA-polymerase

supernatant lu.d (250 pi, 2.0 iV protein) and varyin] aaotunts of DNA,

as DIIA. primer, per 150 jil tris-p-hosphate buffer (0.03 M, p1 7 .3). The

m!xture was incubated at 37o for 1 hour for the assay.

i 1Determinatlon of DRA. An indole method was used to determine

the amount of DNA assayed for DNA-primer activity.

5

RESULTS

Ecteola column chromatography. Calf- and rat-thymus DNA, prepared

as described above, were chromatographed with an Ecteola column (Figs.

3-4). The recovery of DNA-units from the column was 92-107 l. Except

for one fraction (tube nos. 1-2 of Figs. 2 and 3) identified as phenol,

and another (tube nos. 4-7 of Figs. 2 and 3) identified as p-amino-

salicylate, the elution patterns of untreated ca]f-thyrnus DNA and that

treated with phenol and p-amiLnosalicylate were sinlar. The chromatog-

raphic profiles of heated DIA were different from those of unheated

DIV, (compare Figs. IA-4A with FLgs. IB-4B), conforming to the observa-

5tions of Roserkranz and Bendich 5.

DNA prepared by the unmodified Kirby' procedure versus the modi-

fied method yielded different chromatographic patterns (compare Figs.

3 and 4). In addition, fractions obtained by the unmodified method'

contained no phenol or p-ailnosa icylate and the amount of the DNA

fraction eluted in the salt gradient was considerably reduced.

Deionization by Sephade;: column chromatography. The major DNA

fractions from the Ecteola column were deionized by Sephade;, chroma-

tography. The recovery of DA-units varied between 8U4-!0: . Typical

e:amples with the two different sizes of coc!uzins arc zhown in Fig. 5.

The concentration of VaCl found in the DNA fractions was c]L,- 0.3

mM as determined by electrical resistance of the solutions. In ad-

dition, the neutral pH of the DNA fractions demonstrated the absence

of N'4OH(Pg. .).

6

10

9 NoCI8 2.0

7/16--

4 - OA 1.0

" 0 2 X .

E 0 5 0 15 20 25 30 35 40 45 50 70 80

SI NoC

z 8 . . .. . 2.0 M

0 6r

4 1.0

3

2

0 00

0 5 10 15 20 25 30 35 40 45 50 60TUBE NUMBER

Fi.0 11 Eecteola column chroratogr:Lphic pattern of 67 DNA -unlts (A2 60x vol) of unheated calf-thymus DNA. The elution pattern in area I wasthe result of a linear salt grudient (0.C5 M NaCI to 2.0 h NaCl) at pH7 0, and in area II from a linear hN40H gradient (0.0 to 2.0 N H4OH)with constant salt concentration (2.0 M NaCl). Total recovery was 92 ,.Other details are oresented in the methods and materials section.Fire 1B Same legend as in Fir. 11 except that the calf-thvmus DNA washeated (1000, 7 min) prior to chromatograph3" and 81 DNA,-units were puton the column. Total recovery was 94%.

7

10 I9 _ __

8 INoCI 2.0

6 I

4LLJ 1.0S3

0 2

0 0 _

0 . 0 '

00 5 0 15 20 25 30 35 40 45 6 D0

W1 /9 z4 5

4 / 0

0 -- ,00

0 5 10 15 20 25 30 5 40 45 70

TUBE NUMBER

Fie. 2A Ecteola column chromatogriphic pattern of 67 DNA-units ofunheated calf-thymus DNA which had been previously treated by a modifiedphenol extraction method. The elution pattern was obtained as describedin the legend to Fig. 1. Tube nos. 1-2 and 4-6 represent phenol andp-axninosalicyvlate rospectively. Total recovery was Q5%.Fig. 2B Same legend as in Fig. 2A except that the calf-thymus DNAsample was heated prior to chromatograph;. and 78 DNA-units were put onthe column. Total recovery was 100%.

8

15

14

13 ]12

109

8 I NOCI 2.0

7 I

6

4 - 101 3

>2 /

05o 1 0 15 oE 0 10 15 20 25 30 35 40 45 70 z018 ,

,I6 In"z

"15

12

I0

98 1_____ NoCl I

4O 10

12

100.

7

0 10 15 20 2,5 30 5 40 45 65

TUBE NUMBER

Fig. 3A Ecteola column chromatographic pattern of 136 DNA-units ofunheated rat-thymus DNA obtained by a modified phenol extraction method.The elution pattern was obtained as described in the legend to Fig. 1.Tube nos. 1-3 and 4-7 represent phenol and p-aminosalicylate respective-ly. Total recovery was 107%.Fig. 3B Same legend as in Fig. 3A except that the rat-thymus DNA samplewas heated prior to chromatography and 77 DNA-units were put on thecolumn. Total recovery was 100%.

9

10

8 I I _ NaC.

/20

5 7

0 5 10 15 20 25 30 35 40 45 50

TUB3.UMBE

297

0 28 -I '~

UJ 10

0 8- .0

- 0 5 . 15 20eoa c25m 30oatgpi 35ter 40 5 50-niso4 1.0

2

2I

0 5 10 15 20 25 30 35 40 45 500TUBE NUMBER

Fig. 4iA Ecteola column chromnatographic pattern of 65 DNA-units ofunheated rat-thymus DNA obtained by the unmodified phenol method. 7

The elution pattern was obtained as described in the legend of Fig. 1.Fig. 4B Same legend as in Fig. 4A except that the rat-thymus DNA sam-ple was heated prior to chromatography and 76 DNA-units mere put onthe column.

10

E 10 10 E10 10

Wj9 9~ LU9~098 pH- 8 3 8 pH-.. 8

> 7 "---- 7 >7 .7R6 6 R6 -6 0

5 mMN0Ct o5 55S Z mM NoCI 24

4 ('44 4 4)

3 3 E 3 3Ez 2 2 <2 2

o 00 0- .

< 0 5 10 15 20 4 0 10 15 20 25TUBE NUMBER TUBE NUMBER

Fig. 5A Sephadex column (30 g G-25, 2.5 cm x 40 cm) chromatographicelution curves of an Ecteola column fraction of unheated rat-thymus DNAsample (tube nos. 20-23 of Fig. 3A, 28.6 DNA-units) at 40. The curveswere obtained by using deionized distilled water as eluting solvent andtesting the fractions for A260, volume, electrical resistance, and pHas indicated in the Methods section. The recovery was 98%.Fig. 5B Same legend as Fig. 5A except that the sample chromatographedwas heated rat-thymus DNA (tube nos. 21-26 of Fig. 3B, 45 DNA-units)and a different column was used (60 g G-25, 3.5 cm x 30 cm). Therecovery was 102%.

11

Primer assay of unfractionated DNA. In the assay, primer activity

was linear in the range of 10-30 g for unheated and heated DNA (Fig 6).

This linearity was observed also with DNA isolated either with or

without phenol and p-aminosalicylate. Thus, the use of these reagents

for preparation of DNA did not increase the primer activity of DNA.

The values for unheated rat-thymus DNA were found to fall on a straight-

line plot different from that of calf thymus DNA. The activities of

heated DA samples showed marked increases (about 10 fold) above the

corresponding activities of unheated samples.

Primer assay of fractionated DNA. In all cases, primer activity

of the isolated DRA Ecteola fractions increased with increasing I'TH4 OH

concentrations used for e3utLng the DNA fractions (for example, un-

heated calf-thymus DNA, tube nos. 44-46 and 39-43 of Table I). In

parallel with this observation was the decrease in hyperchromic shift

of the respective fractions (Table I). Primer activity of the last

fraction of unheated DNA eluted with NH4 OH approached that of the

heated DNA (Table I and Figure 6). Primer activ.:ty values for calf-

thymus DNA isolated with and without phenol were approximatei the

same (for example, unheated calf-thimus DNA, tube nos. 6-10 and 7-. of

Table I), while that of unheated DNA eluted by the NaCI gradient did

not change appreciably from the activity of the original DIA samples

before fractionation (Table I and Figure 6).

12

50 T

0

.40

S20

CL U-CT _

0 10 20 30 40 50 60 70 80 90 100

/ig DNA

Fig. 6 The effect of various amounts of thymus DNA on the incorpora-tion of ( 3H) thymidine into DNA as a measure of DNA-primer activ'ty ina DNA-polymerase assay system. The method of assay was performed asindicated in the methods section. The abbreviations are: U-CT andH-CT for unheated and heated calf-thymus DNA, U-CT' and H-CT' forunheated and heated calf-thymus DNA which had been previously treatedwith phenol, U-RT and H-RT for unheated and heated rat-thymus DNA ob-tained by a modified phenol extraction procedure as indicated in themethods section.

13

TABLE I

DNA-PRIMER ACTIVITY OF FRACTIONATED, DEIONIZED, AND LYOPHILIZEDTHYMUS DNA

SA4PLE DNA RECOVERED FOR ASSAY

Source Condition Ecteola Before After Hyperchromic DNA DNA-primer

Fraction 11eatinU Heating Shift Assayed Activity

tube ,' A2 A26 % i count/min

x 10 -3

8-30 0.100 0.130 30 10.0 8.7

Unheated 17-20 0.300 0.130 30 9.0 15.4

Calf 44-48 0.]25 0.140 12 1O.4 102

Thymus Heated 17-21 0.125 0.135 8 9.5 105

22-28 0.115 0.130 9 9.5 111

7-9 0.105 0.14o 33 10.5 8.6

Calf Unheated 16-18 0.110 0.135 23 9.1 20.5

Th)Mu 39-43 0.105 0.130 24 10.5 89.2

After 23-27 0.125 0.14o0 32 9.7 96.0

Phenol Heated

28-35 0.095 0.110 16 11.0 102

8-11 0.1.00 0.135 35 10.2 12.5

Rat Unheated 20-23 0.105 0.130 24 8.0 19.6

Thymus 29-33 0.10 o.140 17 9.0 79.0

Hleated 21-26 0.120 0.130 8 8.5 91.6

Control blank 0.0 0.0 0 0.0 4.7

Background 0.02

14

DISCUSSION

The phenol method7 was modified to permit the assay of DNA as

soon as possible in the isolation procedure in order to minimize the

degradation or denaturation of DNA through subsequent methodological

manipulations such as RNase treatment and repeated precipitations.

The Ecteola column removed two impurities (phenol and p-aminosalicylate)

from the DNA, thus isolated. Other criteria for degree of purity such

as protein content of the DNA were not investiLated. The unfractionated

DNA samples assayed did contain these reagent impurities (phenol and

p-mminosalicylate), but tneir presence did not affect the assay of

unheated DIA for primer activity.

The &cadient to 2 M NH4OH was selected in order to elute the DNA

q';antitatively without the use of NaOH (cf.12). Gradient elution was

preferred to stepwise elution to minimize the peaks associated with

5,6,13changes in eluting solvents . However, the use of 1H4 OH gradient

(in 2 M NaCl) produced an increase in primer activity of the DNA eluted by

increasing concentrations of NH4OH. It is evident that the exposure

of DNA to ITH4 0H increases its primer activity and thus introduces an

artifact.

Worh from this Laboratory10 has shown the importance of controlling

cation concentration in the DNA-polymerase assay medium; therefore, it

was necessary to remove salt from the DNA fractions obtained from

chromatography before the assay. The use of Sephadex to deionize the

15

DNA fractions followed up by lyophilization to eliminate water did not

complicate the interpretation of the data. It has also been reported14

that drying and rehydrating DNA does not uncoil its helical structure1 5

irreversibly.

If primer activity could be fractionated into one characteristic

fraction, then it would be expected that the total activity of the

unfractionated sample could be accounted for in such an active fraction

This was not found to be true in the present st d. If the DNA could

be fractionated into subfractions with different levels of primer

activity it would seem logical to anticipate that the DNA recovered

from the salt fraction would show different activity than the un-

fractionated DNA. However, the res Its from the salt Gradient, show

that this fraction had the same level of primer activity as did the

unfractionated material. It is concluded, therefore, that under these

conditions of DNA fractionation, primer activity vas not concentrated

in a particular DNA fraction.

16

REFERENCES

1. I. R. Lehman, M. J. Bessman, E. S. Sims and A. Kornberg, J. Biol.

Chem., 233 (1958) 163.

2. I. R. Lelman, Ann. N. Y. Acad. Sci., 81 (1959) 745.

3. F. J. Bollun, J. Biol. Chem., 234 (1959) 2733.

4. H. S. Rosen):ranz and A. Bendich, J. Am. Chem. Soc., 81 (1059) 6255.

5. S. Kit, Nature, 184 (1959) 36.

6. C. C. Richardson, F. Sample, C. Schildkraut, I. R. Lclman and

A. Kornber;, Federation Froc., 22 (1963) 349.

7. K. S. Kirby, Biochim. Biophys. Acta, 36 (1959) 117.

. P. Flodin, J. Chromato[:., 5 (1961) 103.

g. G. R. Shepherd and D. F. letersen, J. ChromatoG., 9 (1962) 445.

]O. E. R. Walwich and R. K. Main, Biochin. Biophys. Acta, 61 (1962)

C7.

11. K. KecI-, Arch. Biochem. Biophys., 63 (1956) 446.

'2. H. M. Ke'lr, B. finnie and R. M. G. Gme1lie, Biochem. J., 82 (1962)

3-3. D. N. Ward and J. D. Putch, Makromol. Chem., 55 (1962) 121.

34. M. Falk, K. A. Hartman, Jr. and R. C. Lord, J. Am. Chem. Soc.,

85 (1963) 391.

35. J. D. Watson and F. H. C. Crick, Nature, 171 (1953) 737.

17

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2u1 DEVENSE DOCUMI'NTAIION CENTERI DIRECTOR. ARMED FORCES RADIOEIIOLOGY RESEARCH IN4STITUTE

AEC ACTIVITIES AND OTHERS

I AEC SCIENTIFIC REPRESENTATIVE, FRANCEI AEC SCIENTIFIC REPRIESENTATIVE. JAPANI AIROJLT GINFRAL. AZUJSA5 ARGOiNE. CANCER RESfARCH HOS.PITAL10 ARHONNE N.ATIONAL LAKjORATORY2 AtnfMIC R0M11 CASUALTY COM4MISSION4I ATOMIC ENER01Y COMMIr.SION* WASHINGTON4 ATOMIC ENER(.Y OF CANADA. LIMITED3 ATOMI(S, INTERNATIONAL2 BATTELLE MEMORIAL INSTITUTE1 BORDIP. CHEMICAL COMPANY4 PROOUI4AVfN NATIONAL LABO0RATORYI CPIICAFo PATkNT GRORIP IACII COLORADO STATE tiNIVERSITYI COLUN'UA UNIVERSITY IROSSI)I COMITIEk ON THE EFFECTS OF ATOMIC RADIATION3 DtFENCE RESFARCH MEMDfR1 DIRECTOR. 0IVIT.ION OF BIOLO(AY AND MEDICINE IAECI2 DUJ PONT COMPANY. AIKENI DlIP PONT COMPANY WILMINGtrOh1 EDGERTON. GERNfSHAUStN AND CiRItR, IN(.9 CIOLETAI E!K.ENTOP. GjERMFSMAUS1Il AND GRIER. INC. LAS VEGAS2 GENERAL DYNAMICS. FORT WORTH2 GEN4ERAL ELECTRIC COMPANY. CINCINNATIa GENERAl tLECTRIC COMPANY. RICHLAND)I GENERAL ELFCTRIC COMIPANY, ST PETERSAURGMI CTfNERAL SCIENTIFIC CORPORATION

I tJGMES AIRCRAFT COMPANY, CULVER CITYI IOWA STATE UNIVERSITYI OLLS ATOMIC POWER LABORATORY2 LOS ALAMOS SCIEF4TIFIC LABORATORY ILISRARY)1 LOVELACE FOUNDATIONI 11ARTIN-MARIETTA CORPORATION1 RASSACIIJSETTS INSTITUTE Of' TECHNOLOGYI MOU0tND LABORATORY

20

1 NASA. AMES RESEARCH CENTER, MOFFETT FIELD2 NASAo SCIENTIFIC AND TECHNICAL INFORMATION FACILITY1 NATIONAL ACADEMY OF SCIENCES1 NATIONAL BUREAU OF STANDARDS ITAYLOR)1 NATIONAL CANCER INSTITUTE1 NATIONAL LEAD COMPANY OF OHIO1 NATIONAL LIBRARY OF MEDICINE1 NEW JERSEY STATE DEPARTMENT OF HEALTH1 NEW YORK OPERATIONS OFFICE(AECI1 NEW YORK UNIVERSITY (EISENBUDII OFFICE OF ASSISTANT GENERAL GENERAL COUNCEL FOR PATENTS2 PHILLIPS PETROLEUM COMPANY4 PRATT AND WHITNEY AIRCRAFT DIVISION1 PUBLIC HEALTH SERVICE. LAS VEGAS1 PUBLIC HEALTH SERVICE. MONTGOMERY2 PUBLIC HEALTH SERVICE. WASHINGTON1 RESEARCH ANALYSIS CORPORATIONI REYNOLDS ELECTRICAL AND ENGINEERING COMPANY. INC1 SANDIA CORPORATION, ALBUQUERQUE1 UNION CARBIDE NUCLEAR COMPANY IORGDPI5 UNION CARBIDE NUCLEAR COMPANY (ORNL)1 UNION CARBIDE NUCLEAR COMPANY (PADUCAH PLANT)1 UNITED NUCLEAR CORPORATION (NDA)1 UNIVERSITY OF CALIFORNIA# DAVIS1 UNIVERSITY OF CALIFORNIA. SAN FRANCISCO1 UNIVERSITY OF CALIFORNIA, LOS ANGELES3 U. OF CALIFORNIA LAWRENCE RADIATION LAB, BERKELEY2 U. OF CALIFORNIA LAWRENCE RADIATION LAB, LIVERMORE1 UNIVERSITY OF CHICAGO RADIATION LABORATORYI UNIVERSITY OF HAWAIII UNIVERSITY OF PUERTO RICO1 UNIVERSITY OF ROCHESTER IATOMIC ENERGY PROJECTI1 UNIVERSITY OF TENNESSEE (UTA)I UNIVERSITY OF UTAH1 UNIVERSITY OF WASHINGTON (DONALDSON$1 US GEOLOGICAL SURVEY* DENVER1 US GEOLOGICAL SURVEY, MENLO PARK1 US GEOLOGICAL SURVEY. NAVAL WEAPONS PLANT1 US GEOLOGICAL SURVEY, WASHINGTON1 US WEATHER BUREAU. WASHINGTON1 WAYNE STATE UNIVERSITY1 WESTERN RESERVE UNIVERSITY (FRIEDELLI1 WESTINGHOUSE ELECTRIC CORPORATION (RAHILLYI1 WESTINGHOuSE ELECTRIC CORPORATION (NASA$

25 TECHNICAL INFORMATION EXTENSION. OAK RIOGE

USNRDL

40 USNRDL9 TECHNICAL INFORMATION DIVISION

DISTRIBUTION DATE: 8 August 1963

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