68EN19TICS: LOONEY, CAMPIPELL, AND hOLMES PROC. N. A. S.

* Special U.S. Public Health Service Fellow, National Cancer Institute.t Present address: Radiobiology Laboratory, The Johns Hopkins University, School of

Hygiene and Public Health, Baltimore 5, Maryland.1 Higgins, G. M., and R. M. Anderson, Arch. Path., 12, 186 (1931).2 Davidson, J. N., and C. Waymouth, Biochem. J., 38, 375 (1944).3Novikoff, A. B., and V. R. Potter, J. Biol. Chem., 173, 223 (1948).4Price, J. M., and A. K. Laird, Cancer Research, 10, 650 (1950).6 Holmes, B. E., and L. K. Mee, Radiobiology Symposium, 220, Leige (1954), ed. A. M. Bacq

and P. Alexander (London: Butterworth Scientific Publ., 1955).6 Nygaard, 0. F., and H. P. Rusch, Cancer Research, 15, 240 (1955).7 Mirsky, A. E., in Proc. III International Congress Biochemistry, ed. C. Lie-Bacq (New York:

Academic Press, 1956), p. 349.8 Beltz, R. E., J. Van Lancker, and V. R. Potter, Cancer Research, 17, 688 (1957).9 Davidson, J. N., "Chemistry of the Liver Cell," Brit. med. Bull., 13, 77 (1957).

10 Looney, W. B., Dissertation for Ph.D. Degree, University of Cambridge, England, November,1959.

11 Simpson, W. L., Anat. Rec., 80, 173 (1941).12 Lillie, R. D., Histopathologic Technic and Practical Histochemistry (New York: Blakiston,

1954).13 Pelc, S. R., "Autoradiographic Technique," Nature, 160, 749 (1947).14 Looney, W. B., Proceedings of the IXth International Radiological Congress (Munich: Georg

Thieme, Verlag (in press).11 Ornstein, L., Lab. Invest., 1, 250 (1952).16 Patau, K., Chromosoma, 5, 341 (1952-53).17 Naora, H., Science, 114, 279 (1951)18 Mendelsohn, M. L, Dissertation for Ph.D. Degree, University of Cambridge, Cambridge,

England, 1957.19 Mendelsohn, M. L., J. Biophys. Biochem. Cytol, 4,407 (1958).20 Looney, W. B., in Faraday Society Conference on the Cell Nucleus, Ed. B. E. Holmes (London:

Butterworth Scientific Publ., 1960).21 Naora, H., J. Biophy. Biochem. Cytol., 3, 949 (1957).

THE EFFECT OF IRRADIATION ON THE REPLICATION OF DESOXY-RIBONUCLEIC ACID IN HEPATOCYTES

BY W. B. LOONEY,* R. C. CAMPBELLt AND BARBARA E. HOLMES

DEPARTMENT OF RADIOTHERAPEUTICS, UNIVERSITY OF CAMBRIDGE, ENGLAND

Communicated by Alfred E. Mirsky, March 24, 1960

By 1925 it was generally accepted that the cell nucleus was more sensitive to theeffects of irradiation than the cytoplasm.' The discovery by Muller2 in 1927 thatX rays could produce gene mutations firmly established the importance of theeffects of irradiation on the genetic material in the nucleus. Mitchell3' 4and Eulerand Hevesy5 were first to show that irradiation would produce disturbances innucleic acid metabolism.

Additional results suggesting that irradiation directly affects DNA synthesiswere reported by Holmes,6 Hevesy,7 Skipper and Mitchell,8 Pelc and Howard,9Klein and Forssberg,'0 Vermund, Barnum, Huseby, and Stenstrom," Bennett,Kelly, and Kreukel,'2 Lajtha, Oliver, and Ellis,13 Looney,14' 15 Lajtha, Kumatori,Oliver, and Ellis,'6 Holmes and Mee,17 Beltz, Van Lancker, and Potter,'8 and

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Stocken. I9 Some of the more pertinent findings were that (1) DNA was synthesizedduring interphase in the mitotic cycle and (2) irradiation given before the onset ofsynthesis is more effective than irradiation during the active synthesis of DNA.Conflicting results were found in the large number of studies carried out under awide diversity of conditions on different organs, systems, and animals. Kelly,20in reviewing the extensive amount of information on the effects of irradiation onDNA, suggested that almost all the results were consistent with the assumptionthat the results could be explained by factors such as cell death or mitotic delay.The labeling of the hepatocytes immediately after irradiation and sacrifice of the

animals at varying times after irradiation provides a means of identifying thehepatocytes synthesizing DNA at the time of irradiation. Measurement of theDNA content of the labeled nuclei microspectrophotometrically permits the deter-mination of the changes in the DNA content of individual nuclei following irradia-tion.

Materials and Methods.-Combined autoradiographic and cytochemical studieshave been carried out on irradiated and paired control rats sacrificed between17 and 261/2 hr after partial hepatectomy. X radiation was given at the rate of127 r per min for a total dose of 3,000 r. Tritiated thymidine was given immediatelyafter the termination of irradiation. The remaining lobe of the liver was directlyirradiated and the remainder of the body shielded. (See Materials and Methods,preceding paper, this issue.)Results.-The results of the combined cytochemical and autoradiographic meas-

urements on the same nucleus are illustrated by an experiment in which tritiatedthymidine and 3,000 r of X irradiation were given at 17 hr after hepatectomy andboth irradiated and paired control animals sacrificed 6 hr later. (See Fig. 1.) Allof the hepatocytes in the paired control which were synthesizing DNA at 17 hrafter hepatectomy had doubled their DNA content by 23 hr after hepatectomy.However, the mean DNA content of the labeled hepatocytes in the irradiatedanimal was only 63 per cent above the 4N value for DNA (see Table 1).

In one experiment the tritiated thymidine was injected at 17 hr after hepa-tectomy and the animal sacrificed 9 hr later (Fig. 2). It was found in the controlanimal that 14 of the hepatocytes labeled with tritiated thymidine at 17 hr after

TABLE 1CHANGE IN DNA CONTENT WITH TIME AFrER HEPATECTOMY MEASURED

MICROSPECTROPHOTOMETRICALLY IN TRITIATED THYMIDINE-LABELED HEPATOCYTESLabeled Nuclei-

MeantUnlabeled Nuclei grain

countsTime Mean perafter content nucleus

hepatectomy 25 (25Date (hr) 2 N 4 N 8 N nuclei S.D. D* nuclei)

21.7.59 (A) 15'/2-181/2 (C) 347 735 1447 975 4115 0.34 23.74(R) 364 714 1382 915 i 52 0.27 11.00

21.7.59 (B) 17-23 (C) 314 692 1223 1317 i 82 1.20 33.78(R) 377 801 1575 1291 4173 0.63 24.33

17.3.59 (A) 17-26 (C) 222 554 935 904 ±119 0.92 19.58(R) 138 322 662 640 ± 75 0.94 14.89

24.4.59 20-201/2(C) 184 389 725 641 A 77 0.75 52.75(R) 209 440 790 582 i 77 0.41 15.35

* Mean DNA content of labeled nuclei expressed as a fraction between the 4N and 8N amount of DNA.t These grain counts were made on the labeled hepatocytes before removal and measurement of the DNA con-

tent.(C) control. (R) irradiated.

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* Labelled nucleiO Unlabelled

600

5C0*

2Y 40- 0

A 0

*6C030- ^* 6i; ~~~~~~~~* 4cn2co 4c0so looo 120O 14o0 cooloooC-3

LI ~~~~~~~~~~~~~~~~~~~C200 400 6O 00901000 1200 1400 l6b0 1002000

DNA (Arbitrary units)

(Rod. dose 3000r)

* Labelled nucleiA * divided nuclei

60 [ Unlabelled nuclei

50-

' 40-~~4O 0

z30- OS0

.t20 t ; DS0

200 40 600 800 CO0 12 140 1660 18DN.A (Arbitrary units)

FIG. 1.-Plot of grain counts versus DNA content of the same hepatic nucleiof a rat given tritiated thymidine at 17 hr after hepatectomy andsacrificed 6 hr later.

hepatectomy had completed the doubling of the DNA content and divided. Atthis time the mean DNA content of the labeled hepatocytes of the irradiated animalwas 92 per cent above the 4N amount of DNA.

Estimates have been made for the time necessary for DNA replication in thelabeled hepatocyte following 3,000 r of irradiation. The estimates of the time forreplication of the DNA of an hepatocyte from the data of different experimentsfollowing irradiation are given in Table 2. A straight line can almost be fitted tothe experimentally determined value of the mean DNA content of labeled hepato-cytes of both irradiated and control animals (Fig. 3).

In one experiment tritiated thymidine was injected at 20 hr after hepatectomyand the animal sacrificed 30 min after injection. As tritiated thymidine is onlyavailable for a short time, the animal was sacrificed shortly after administration todetermine what correlation, if any, could be made between the grain counts pernucleus in the irradiated and control nuclei and the DNA content in the irradiated

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(Rod. dow 300 r)

A Labelled nucleiQ Unkbekd -

40-

c30- * 6

o 0 2c0 .0 SOO70 zCl

1002020050t1::Fti2N 4N N__

N Arbitrary

70-

* Labelled nucleiA Al divided nuclei0

Unlabelled nuclei 10

-9

*40- -8

fa7

'z 30 * -6 ,P ~~~~~~~~A 6u A A 0 5c A

* * * .oo0 0 -4!

mA

A A .32

2N |4N j 9100 200300 4M 5M M0060 t- sboi ~oo

DNA. (Arbitrary units)

Fig. 2.-Plot of grain counts versus DNA content of the same hepatic nucleiof a rat given tritiated thymidine at 17 hr after hepatectomy and sacrificed 9hr later.

DNA content -18N oepatocytes

Fractions ofDNA contentbetween 4N and8N . ,

-40/ x "-20- ~ I

I

DNA content x

4N hepatocytes . -1516 17 IS ~~~19 20 21 22 23 24 25 26 27 28

Tirns - hours otter hepatfctriny.

FIG. 3.-.Change in DNA content with time after hepatectomy measuredmicrospectrophotometrically in tritiated thymidine labeled hepatocytes. Key:* control. X * irradiated ,u.

and control nuclei. Twenty hr after hepatectomy was selected because it is thetime of most active DNA synthesis as shown by tritiated thymidine and auto-radiographic means. Twenty hr was also selected because little change in the

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TABLE 2ESTIMATIONS FOR THE TIME FOR DOUBLING OF THE DNA CONTENT OF AN HEPATOCYTE AFTER

3,000 R OF IRRADIATION

Estimated Doubling Time (hr)Time after Duration ofHepatectomy synthesis Linear Exponential Quadratic

No. Date (hr) (hr) D* model model model

1 21.7.59 15'/2-181/2 3.5 0.27 13.0 9.5 10.32 21.7.59 17-23 8 0.63 12.6 14.0 13.63 17.3.59 17-26 11 0.92 13.7 14.0 13.34 24.4.59 20-201/2 5.5 0.41 12.4 12.8 12.5

Mean 12.9 12.4 12.4* DNA content of labeled nuclei expressed as a fraction between the 4 N and 8 N content.

per cent of labeled hepatocytes was present, yet sufficient time had elapsed sincethe onset of synthesis to have a population of nuclei in all stages of synthesis be-tween a 4N and 8N amount. (See Table 1.)The two methods compare reasonably well as means for the determination of

changes in DNA immediately following irradiation with the limits of error of theprocedures. The combined method is superior to either method used independ-ently. It eliminates the weakness of the cytochemical method which is thedifficulty of detecting nuclei which are synthesizing DNA and it provides a meansfor determining relative changes in the same nucleus both autoradiographicallyand cytochemically.A normal curve with the same mean and standard deviation as the mean and

standard deviation for the log of the grain counts per nucleus of the irradiatedanimal in the 20- to 201/2-hr experiment has also been used as the model for thechanging rates of DNA synthesis following irradiation as it was used in the con-trol (Fig. 4). The following information about the log of the grain counts pernucleus for the irradiated animal of the 20- to 201/2-hr experiment, has been sub-stituted into the general equation for the normal distribution.

Mean log of grain counts =IRRADIATED U1 == 1.266.

24 []CONTROLStandard deviation = 01in

4±0.167.U

DZ16- Thus,~~~~~~~~~~~~~~~~x .25)

LOG GRAIN COUNFTS PER NUCLEUS.22 .0 ipsdo hto o ri

LL.~~~~ ~ ~ ~ ~~~~~~~onstht3h o elc

Fig. 4.Histogram of log of grain count distributionofenuclei with superimposed normal curve. ation covering that part of

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the axis within which readings fall 95 per cent of the time. Transforming thesevalues to the time scale, t = time in hr of the observation from the midpoint of thetime for replication (13 hr).Upon substitution of these values into the equation for DNA content and inte-

grating, the following theoretical values of the DNA content have been obtainedfor different times during the 13-hr period of replication.

TABLE 3THEORETICAL VALUES FOR DNA CONTENT

Time Theoretical DNA Time Theoretical DNA(hr) Content* (hr) Content-6.5 +1.0 0.5935-6.0 0.0101 +2.0 0.7018-5.0 0.0409 +3.0 0.7921-4.0 0.0889 +4.0 0.8611-3.0 0.1579 +5.0 0.9091-2.0 0.2482 +6.0 0.9398-1.0 0.3465 +6.5 0.95000 0.4750 ... ...

* Expressed as a fraction of the DNA content between 4N and 8N.

The theoretical curve for the change in the DNA content of hepatodytes, after3,000 r of irradiation, and during the process of replication is shown in Figure 5.

DNA content8N hepatocytes °

Frdctions of .80DNA contentbetween 4N and .60/8N

.40-

X

DNA content X4N hepatocytes 15 16 17 i' 19 20 21 22 23 24 25 26 27 28

Time - hours otter hepotectomy.

FIG. 5.-.Change in DNA content with time after hepatectomy measured microspectrophoto-metrically in tritiated thymidine labeled hepatocytes. Key: * *, theoretical curve forchange in DNA content between 4N and 8N after 3000 r of irradiation. X, DNA content de-termined experimentally after 3000 r of irradiation.

The values for the DNA content determined experimentally are not in as closeproximity as the control values. However, the agreement is still considered to begood. Thus, the predicted values for the DNA content obtained by integrationof the curve for the changing rate of DNA synthesis from autoradiographic resultsagree with the experimental values for DNA content determined by the cytochemi-cal measurement of the labeled hepatocytes at different times during replication.Both the autoradiographic and cytochemical results suggest that irradiation pri-marily causes a reduction in the DNA synthetic rate.

Estimates of the DNA doubling time have been made by using the exponentialequation for changing rates of synthesis rather than the linear equation as usedinitially. This has been done by assuming different times for doubling of the DNAand determining the DNA content at the end of the assumed time. Estimates

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of the DNA content at the end of 12 hr, 12.6 hr, and 13 hr have been made fromthe results of the DNA content found at different times in the four experimentsgiven in Table 4. Substitution of a doubling time of 12.6 hr into the exponentialequation for the change in DNA content during replication most closely approxi-mates the doubled amount of DNA.

TABLE 4ESTIMATIONS OF DNA DOUBLING BASED ON AN ASSUMED

EXPONENTIAL CHANGE IN DNA CONTENT DURING REPLICATIONTime after Estimated DNA Content* Based on an Assumed

Hepatectomy --- -Doubling Time of:- -'No. (hr) (a) 12 hr (b) 12.6 hr (c) 13 hr1 151/2-181/2 0.67 0.63 0.592 17-23 1.14 1.08 1.033 17-26 1.00 0.98 0.974 20-201/2 1.00 0.93 0.88

* DNA content expressed as a fraction between the 4N and 8N content.

Discussion.-It is evident from these investigations that any attempt to evolvea simple working model illustrating how irradiation affects DNA synthesis isfraught with many inherent dangers. However, the results of other investigationsand the results of this study do permit certain inferences to be made in regard tothe sequence of events resulting in disturbance in DNA synthesis by irradiation.The linear model for the change in DNA content during replication was unsatis-

factory for both the irradiated and control experiments. Two curved models wereused to directly estimate the DNA doubling times in the irradiated experiments.The results of the direct estimation of DNA doubling time using directly an ex-ponential and a simple quadratic (a parabola) equation are given in Table 2.Both the exponential and quadratic models fit reasonably well with the experimentalresults obtained cytochemically. Since the changing rate of DNA synthesis, basedon the autoradiographic results, can be expressed as an exponential function andsince the exponential model for the change in DNA content during replication canbe fitted to the experimental results obtained cytochemically, it is considered thatthe exponential model is the most satisfactory model for DNA replication inhepatocytes.One of the well-established examples of the direct effect of irradiation is the

production of mutations in Drosophila. Analysis of the results of this and otherinvestigations by the single-hit target theory has provided much useful informationabout the quantitative relationship between irradiation and gene mutations. Theuse of this theory has also been effectively utilized in the study of inactivation ofenzymes, phages, and viruses. The studies on the quantitative relationship be-tween irradiation and molecular inactivation have been reviewed by Guild.2' Thecorrelation between the radiation inactivation and estimates of molecular weightsbased on target size and the known molecular weight for 32 molecules with molec-ular weights ranging from 3 X 102 to 10 was presented. The correlation betweenthese results and the known molecular weights is within a factor of 2 or less, madeunder standardized conditions. Guild states that this empiric relationship seemsto demonstrate that a primary ionizing event anywhere in a biological molecule hasa probability of near unity of inactivating as its special function.

Puck, Morkovin, Marcus, and Cieciura22 reported that the mean lethal dose for a

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large number of normal and malignant human cells grown in tissue culture wasbetween 50 to 150 r. It was found that the survival curve for the more radio-resistant cells approximated a 2-hit type of curve. The survival curves for themore radiosensitive cells such as fibroblasts could be fitted by either a 1- or 2-hitcurve. They conclude that the radiosensitivity of these cells could be explainedon the basis of a defect resulting from primary damage localized in one or morechromosomes.

Alexander and Charlesby23 consider that the accumulation of information-of theeffects of irradiation in polymers has demonstrated that the difference betweendirect and indirect effects is much less than previously thought. Mitchell24 sug-gests, on general grounds, that the indirect effects from the diffusion of effectiveagents to the essential target do not represent the most important factdr in theproduction of biological effects of irradiation.The results of recent investigations have given a more precise picture of the

time sequence of the pre-synthetic period and the effects of irradiation on the pre-cursors of DNA in the pre-synthetic period. An increase in the desoxyribosidiccompounds has been found in the pre-synthetic period of DNA in rats followinghepatectomy by both Schneider and Brownell25 and Jaffe, Lajtha, Lascelles, Ord,and Stocken.26 The fact that thymidylate kinase is indetectable in normal ratliver had led Bollum, Anderegg, McElya, and Potter27 to suggest that the inductionof this enzyme is essential for the build-up of deoxynucleotides.The results of Jaffe et al. show that an increase in the desoxynucleoside com-

pounds occurs after irradiation and they suggest that the irradiation is affectingthe process by which the desoxynucleosides are converted to deoxynucleotides.These investigations suggest, therefore, that the low level of irradiation givenin these experiments affects the process by which thymidylic kinase is induced,rather than inhibition of the existing enzyme. Since the presence of a substrate isinvolved in the evocation of an induced enzyme, and since at 8 to 12 hr afterhepatectomy cytidine deoxynucleoside is the predominant deoxy-compound, theysuggest that a nucleoprotein complex may be the synthetic site. They suggestfurther that the disturbances found after irradiation may be due to a derangementof nucleoprotein at the site of synthesis of the enzyme.

Bloch and Godman28 have shown from cytochemical studies that the synthesisof histones occurred at the same time as the synthesis of DNA in cells preparing fordivision. Holmes29 found that nuclear "residue" protein synthesis paralleled DNAsynthesis in regenerating rat liver as determined by isotopic incorporation of labeledC-14 lysine and C-14 arginine. The incorporation of these basic amino acids intothe residual proteins indicates that the formation of the basic proteins closelyparallels DNA synthesis. Their formation may occur simultaneously with DNAsynthesis and may be an essential part of the replication process of the nucleus.Numerous studies have shown that the nucleoproteins are radiosensitive and

may be more affected by irradiation than DNA. Anderson and Fisher30 foundthat viscosity reductions can be obtained in solutions of DNAP with as little as25 r. They emphasize the fact that kilo-roentgen doses of irradiation are necessaryto produce changes in viscosity in pure DNA in solution.The preceding results and the results of this investigation would be consistent

with the hypothesis of Mitchell3" that the primary effect of irradiation is the pro-

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duction of a macromolecular lesion which involves the desoxyribonucleoproteinsystem. A mechanism of separation analogous to the opening of a "zipper" ispostulated to occur at some time during the duplication of the DNA or DNA1P.Therefore, the changes produced by one ion pair occurring at any point in a sensi-tive region could block the process of duplication.The results of this investigation have demonstrated that, in regenerating liver,

the factor of change in the cell population does not enter into the consideration of theimmediate effects of irradiation on DNA synthesis during the period under study.This was shown by two independent methods. Firstly, no significant differencewas shown between the total number of cells in the livers of the irradiated and con-trol animals. Secondly, no significant difference could be shown between the percent of the hepatocytes labeled with tritiated thymidine in the irradiated controlanimals of 14 experiments carried out between 17 and 26'/2 hr after hepatectomy.The comparison of the cumulative distribution curve for the grain counts per

nucleus of the irradiated with the paired control has given some of the more con-vincing results of this investigation with regard to the direct effects of irradiation onDNA synthesis. The reduction in the irradiated grain counts per nucleus wasproportional to the control grain counts per nucleus over a range which varied by afactor of 10 or more. These results suggest that the reduction in the rate of DNAsynthesis following irradiation is proportionate to the rate of synthesis at the timeof irradiation (Looney"4).

Tritiated thymidine was given at the end of the irradiation, and in 14 experi-ments the rate of uptake was 42.42 per cent of the paired control values. It would,therefore, appear that the reduction of the labeled thymidine incorporation im-mediately after irradiation is a measure of the direct effect of irradiation on theformation of the DNA molecule.From previous calculations it was estimated that an average of 3,000 molecules

were formed per min in the 8 hr necessary for replication in the controls.f There-fore, 4,000 X 0.42 = 1,260 molecules per min = the rate of formation of DNAimmediately after irradiation.The estimate that 3 ion pairs within the DNA of an hepatocyte inhibits the rep-

lication of one DNA molecule suggests that an extremely small number of ionizingeffects are needed to prevent the formation of a new molecule of DNA. It is ofinterest to compare the estimation of the ionizing effects necessary to prevent for-mation of a new DNA molecule with the estimate for the inactivation of a largenumber of molecules21 and the 1- and 2-hit curves attributed to primary damage inone or more chromosomes.22The sacrifice of the animals at different times after irradiation and tritiated

thymidine administration has permitted the tracing of the labeled hepatocytesafter irradiation until the DNA content is doubled. The determination of theDNA content cytochemically after irradiation is in all probability a measure of theeffect of irradiation on the entire process of DNA replication.The results of these combined autoradiographic and cytochemical experiments

indicate that after 3,000 r of irradiation the mean synthetic time is increased from1.34 X 106

8 hr to 13 hr. Therefore, = 1,720 molecules per min = average rate offa13 X 60aformation of DNA after irradiation. Therefore, 15 per cent of this reduction might

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be considered to be directly affecting the formation of DNA molecules and the re-mainder of the reduction affecting the entire biosynthetic process by which DNAis produced.

Summary and Conclusions.-Autoradiographic studies with tritiated thymidinehave shown that no significant difference exists between the number of labeledhepatocytes in the paired controls and the irradiated rats after partial hepatectomyand immediately following 3,000 r of X radiation. Quantitative autoradiographicstudies with tritiated thymidine have given results which suggest that the reductionin DNA synthesis immediately following irradiation is directly proportional to therate of synthesis at the time of irradiation.The mean time for replication of the DNA content of an hepatocyte of 8 hr in the

paired controls was increased to 13 hr in the irradiated animals. The shape of theexponential curve for the changing DNA content during the period of replicationfollowing irradiation was similar to the shape of the curve in the controls. Theresults of this investigation are, therefore, consistent with the assumption thatirradiation directly affects the biosynthetic process of DNA replication.

We would like to express our sincere appreciation to Prof. Joseph S. Mitchell,F.R.S., for his assistance and encouragement throughout this investigation, andfor making facilities available in his department for this study. We would alsolike to acknowledge the cooperation and assistance given us by the entire staff of theDepartment of Radiotherapeutics. Their willingness to discuss the various tech-nical problems concerning this study has been of great benefit. It has been possiblefor one of us (W. B. L.) to carry out this study through a Special United StatesPublic Health Service Fellowship of the National Cancer Institute.

* Present address: Radiobiology Laboratory, The Johns Hopkins University School of Hygieneand Public Health, Baltimore 5, Maryland.

t University Lecturer in Statistics, Department of Agriculture, University of Cambridge.§ The average rate of synthesis of 3,000 molecules of DNA per minute was obtained from:(1) Mw of D)NA molecule = 6,000,000(2) Time of replication = 8 hr(3) Weight of DNA in an hepatocyte = 1.34 X 10-1" gm.(4) Number of molecules 1.34 X 1061 Heilbrunn, L. V., and D. Mazia, Biological Effects of Radiation, ed. B. M. D)uggar (New York:

McGraw-Hill, 1936), chap. 18.2 Muller, H. J., Science, 66, 84 (1927).3 Mitchell, J. S., Nature, 146, 272 (1940).4 Mitchell, J. S., Brit. J. Exp. Path., 23, 285 (1942).6 Euler, H., and G. Hevesy, K. Danske Vidensk. Selsk. Bit l. Medd., 17, 1 (1942).6 Holmes, B. E., Brit. J. Radiol., 20, 450 (1947).Hevesy, G., Nature, 163, 869 (1949).

8 Skipper, H. E. and J. H. Mitchell, Cancer, 4, 363 (1951).9 Howard, A.. and S. R. Pelc, Heredity (Suppl.) 6, 261 (1953).

10 Klein, G., and A. Forssberg, Exptl. Cell Research, 6, 211 (1954).11 Vermund, H., C. P. Barnum, R. A. Huseby, and K. W. Stenstrom, Cancer Research, 13, 633

(1953).12 Bennett, E. L., L. S. Kelly, and B. Kreukel, Fed. Proc., 13, 181 (1954).13 Lajtha, L. G., R. Oliver, and F. Ellis, Radiotiology Symposium, Liege, 1954, ed. A. M.

Bacq and P. Alexander (London: Butterworth Scientific Publ., 1954), p. 216.

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14 Looney, W. B., Proceedings of the IXth International Radiological Congress (Munich: GeorgThieme Verlag, in press).

15 Looney, W. B., Faraday Society Conference on the Cell Nucleus, Cambridge, England, Aug. 31-Sept. 1, 1959, ed. B. E. Holmes (London: Butterworth Scientific Publ., 1960).

16 Lajtha, L. G., T. Kumatori, R. Oliver, and F. Ellis, Radiation Research, 8, 1 (1958).17 Holmes. B. E., and L. K. Mee, Radiobiology Symposium, Liege, 1954, ed. Z. M. Bacq and P.

Alexander, (London: Butterworth Scientific Publ., 1954), p. 220.'s Beltz, R. E., J. Van Lancker, and V. R. Potter, Cancer Research, 17, 688 (1957).19 Stocken, L. A., Radiation Research, Suppl. I, 53 (1959).20 Kelly, L. S., Progress in Biophysics and Biophysical Chemistry, vol. 8 (London: Pergamon

Press, 1957), p. 144.21 Guild, W. R., Radiatiin Research, 9, 124 (1958).22 Puck, T. T., D. Morkovin, P. I. Marcus, and S. J. Cieciura, J. Exp. Med., 106, 485 (1957).23 Alexander, P. A., and A. Charlesby, Nature, 173, 578 (1954).24 Mitchell, J. S., Studies in Radistherapeutics, (Oxford: Blackwells, 1959).25 Schneider, W. B., and L. W. Brownell, J. Nat. Cancer Inst. 18, 579 (1957).26 Jaffe, J. J., L. G. Lajtha, J. Lascelles, M. G. Ord, and L. A. Stocken, International J. Radiation

Biology I, 241 (1959).27 Bollum, F. J., J. W. Anderegg, A. B. McElya, and V. R. Potter, Cancer Research, 20, 138

(1960).28 Bloch, D. P., and G. C. Godman, J. Biophys. Biochem. Cytol., 1, 17 (1955).29 Holmes, B. E., Abstract, Fourth International Biochemical Congress, Vienna, 1958.30 Anderson, N. G., and W. D. Fisher, Faraday Society Conference on the Cell Nucleus, Aug. 31-

Sept. 1, Cambridge, England, 1959 (London: Butterworth Scientific Publ., 1960).31 Mitchell, J. S., J. Colloid Science, 11, 317 (1956).

GENE STRUCTURE AND FUNCTION IN NEUROSPORA*

BY R. P. WAGNER, CAROLYN E. SOMERSt AND ARLOA BERGQUIST

THE GENETICS LABORATORY OF THE DEPARTMENT OF ZOOLOGY, THE UNIVERSITY OF TEXAS, AUSTIN

Communicated by J. T. Patterson, March 14, 1960

The considerable effort made over the last decade and a half to analyze moreincisively the structure and function of those segments of genetic material calledgenes has led both to the discovery of some remarkable relationships betweenclosely linked segments of chromosomes, and a general obfuscation of what waspreviously a rather clear concept of genes.The discovery of pseudoalleles in Drosophila by Oliver, Green, and Lewis,'-'

which partially demolished the allele concept,4 the studies of the fine structure ofthe genetic material in phage,5 and the discovery and experimental exploitation ofthe phenonena of transformations and transduction7 in bacteria have made itevident that the event leading to recombination can occur probably anywhere alongthe length of the chromosome or genetic string inside or outside of "genes." Fur-thermore, much of this work, particularly that on pseudoalleles and transduction,has made it clear that closely linked genes, or functional segments, may have relatedor cooperative functions. This has been especially well illustrated in Salmonella byDemerec8 and others.9-11

Carrying the cis-trans test of complementation versus non-complementationover into the protocaryotes such as phage and bacteria has resulted in the recog-

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