THE RELATION OF GROWTH TO THE LETHAL DAMAGE INDUCED BYULTRAVIOLET IRRADIATION IN ESCHERICHIA COLI'

HAZEL D. BARNER AND SEYMOUR S. COHEN

Children's Hospital of Philadelphia (Department of Pediatrics) and Department of Biochemistry of theUniversity of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Received for publication June 24, 1955

Studies in this laboratory on a thymine-requiring strain of Escherichia coli, strain 15T-(Barner and Cohen, 1954; Cohen and Barner,1954 and 1955), have revealed the lethal effect ofpreventing nuclear synthesis while cytoplasmicsyntheses and growth continue. Bacteria in whichnormal deoxyribosenucleic acid (DNA) synthesisis prevented by omitting the specific nuclear com-ponent, thymine, from the medium, rapidly andirreversibly lose the power to multiply. This lossof power to multiply, or "death," is a consequenceof continuing growth, since if other normal syn-theses are simultaneously inhibited the cells donot die. We have termed this type of killing"death by unbalanced growth."

It is possible to impose multiple deficiencies,including a requirement for exogenous thymine,by growing otherwise nonauxotrophic bacteriain the presence of sulfanilamide. Thyminelessdeath may be demonstrated in such bacteria asa consequence of specifically withholding thy-mine from the medium (Cohen and Barner, 1954,and in preparation). The replacement of thyminein the DNA of 15T- by 5-bromouracil also leadsto the loss of the power to multiply (Cohen andBarner, 1954 and in preparation). These observa-tions indicate clearly that lesions specificallyaffecting synthesis of DNA or treatments alteringthe structure of DNA are of critical importanceto cell multiplication. Such phenomena are evi-dently of chemotherapeutic interest.

Cells which die by unbalanced growth are en-larged and have considerably increased theircontent of protein and ribosenucleic acid (RNA)."

I This research was aided by a grant from theCommonwealth Fund.

2 In 15T- in the absence of thymine there is aslight synthesis of deoxyribosenucleic acid (DNA)which is proportionately far less than that ofribosenucleic acid (RNA) synthesis under thesame conditions. This DNA is marked by the pres-ence of an abnormal base, N-methylaminopurine(methyl adenine) which appears to replace thy-

In these respects such bacteria approach the en-larged or filamentous appearance of bacteriakilled by numerous other agents, such as ultra-violet radiation, penicillin, nitrogen mustards,etc. The bactericidal action of penicillin, at least,is not manifested unless the cells are permittedto metabolize actively and to grow. As a result oftreatment with ultraviolet radiation or nitrogenmustard, the patterns of nucleic acid synthesisof treated bacteria superficially resemble thepattern observed in thymineless death, and themetabolic products which accumulate followingsuch lethal treatments similarly indicate theexistence of lesions in nucleic acid metabolism.A comparison of the behavior of bacteria underseveral bactericidal conditions is sunmmarized intable 1. The bactericidal actions of these agentsshow numerous similarities which may conceiv-ably be explained by a single hypothesis, i. e., ineach case death is produced by unbalancedgrowth, as a consequence of the inability to syn-thesize an essential nuclear constituent. Fromwhat little is known of the mode of action of ultra-violet radiation and the nitrogen mustards, thisessential constituent may be deoxyribosenucleicacid (DNA), even as in thymineless death. How-ever, the lesions effected in each case may besupposed to be at different points in the meta-bolic chain leading to the synthesis of this criticalpolymer.

In order to obtain more information bearing

mine in the polymer (Dunn and Smith, 1955).Several arguments may be presented to supportthe view that the existence of the new type of DNAis not in itself the cause of death. For example,most of the DNA elaborated in the absence of thy-mine is present after 30 min of incubation in thedeficient medium. However, the cells have not yetlost the power to multiply. Indeed, addition ofthymine at this point permits the doubling of apresumably normal DNA and the synchronizationof division in all of the cells of the culture (Barnerand Cohen, 1955).

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TABLE 1Properties of bacteria under conditions producing death

Ribosenucleic Acid Deoxyrbowe Accumulated Products in Requirement forAgent Cell Size Synthesis | Acid Medium Bacterial Participation

Thymineless Enlarged Active Inhibited Uracil, orotic acid, Must metabolizedeath* hypoxanthine and grow

Ultraviolet ir- Filamentous Activet Inhibitedt Deoxyribonucleo. Subject of thisradiation tides, thymidylic paper

acidtNitrogen mus- Filamentous Active Inhibited ? ?tardi

Penicillin Filamentous Somewhat in- ? Uracil nucleotidesil Must metabolizehibited¶ and grow

* Cohen and Barner 1954.t Kelner 1953.t Kanazir 1954. Kanazir and Errera 1954.§ Herriott 1951.¶ Gale and Folkes 1953.II Park 1952.

on this hypothesis, a comparison was made instrain 15T- of the damage induced by ultravioletradiation and of thymineless death. It will beshown that these two types of death possessmany imilarities in addition to those indicatedin table 1. The attempt to demonstrate a relationof metabolism to radiation damage also revealeda number of unexpected phenomena concerningthe reversibility of ultraviolet radiation damage.A preliminary report of these results has ap-peared (Cohen and Bamer, 1954).

EXPERIMENTAL METHODS

Bacrial strains. The origin and cultivation ofE. coli, strain 15T-, strain W, and the uracil-requiring mutant Wc- have been described(Cohen, 1953; Cohen and Barner, 1955). E. colistrain B has been used in previous studies in thislaboratory.

Media. The composition of the broth mediumhas been described (Cohen, 1947) as well as thatof the basic mineral medium (Cohen andArbogast, 1950). In the latter, with glucose as acarbon source, strain 15T- and its parent strain15 had a generation time of 45 to 50 min, andthat of strain B, 55 to 60 min. Thymine at a con-centration of 2 ,ug per ml was added for growth of15T-. At 1 mg glucose per ml, this concentrationof thymine is in excess of that necessary to obtainmaximum viable counts. We have described theloss of viability of 15T- in mineral medium con-taining glucose and limiting thymine as deter-

mined by plating and colony counts on agar con-taining a thymine-rich nutrient (Barrer andCohen, 1954).

Irradiation. Cultures in the exponential phaseof multiplication were used in all experiments.These were obtained by growing cultures over-night in synthetic medium, diluting 20-fold intofresh medium to 5 X 107 per ml and incubatingwith aeration until viable count had increasedto 3 X 108. The culture was chilled, centrifuged,and washed with mineral medium. After resus-pension to a concentration of 107 viable cells perml, a 6-ml aliquot was placed in a petri dish on arotary shaker during irradiation. The radiationsource was a 15 W Sylvania germicidal lamp, andintensity was regulated by varying the distanceof the lamp from the sample. Strains 15 and15T- were irradiated at a distance of 57 cm, themore sensitive strain B at 75 cm. Immediatelyafter irradiation, a 5-fold dilution was made intothe incubation medium at 5 C. An aliquot wasremoved for determination of viable count andthe inoculated medium was incubated under theusual growth conditions, i. e., aeration at 37 C.A control unirradiated culture received identicaltreatment.

All experiments with irradiated cells were car-ried out in very considerably subdued light tominimize photoreactivation (Kelner, 1949). Thiswas particularly important since bacteria wereusually given a radiation dose permitting 10 percent survival in the dark. This dose has been

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1956JRELATION OF GROWTH TO IRRADIATION DAMAGE

20 40 60 80 100SECONDS

Figure 1. The sensitivity to ultraviolet irradia-tion of Escherichia coli strains 15 and 15T-.

found to be very extensively photoreactivable(Kelner, 1953). The inhibition of DNA synthesisin such irradiated cells is also specifically reversedbyAisible light.

Viable count. The viable count was determinedfrom the number of colonies which appearedafter incubation for 16 hr at 37 C on agar con-aining nutrient agar supplemented with 5 gNaCl per L. Each aliquot for plating was dilutedin mineral medium at S C and plated by spread-ing as quickly as possible. The radiation sensi-tivities of strains 15 and 15T- are presented infigure 1.

RESULTS

The effect of incubation on viable count of irradi-ated cultures. Strain 15T- was exposed to varyingdoses of radiation and aliquots were incubated ina glucose-containing mineral medium lackingthymine. Viable count was estimated at frequentintervals and the results of two such experimentsare presented in figure 2. Incubation produced aconsiderable restoration of the initially killedcells within 20 minutes, resulting in a 3- to5-fold increase in viable count. Upon continuedincubation, the viable count decreased at a ratecomparable with that in the control culture con-taining unirradiated celLs. The loss of viability inthe unirradiated control is the phenomenon ofthymineless death.The effect of thymine on both restoration and

the subsequent death of restored cells was thenstudied. Irradiation periods of 15 sec, which re-

4 _0 , 2

(1)1 5~~

CONTROLD

20) 40 60 90 120MINUTES

Figure 2. The restoration and death of ultra-violet-irradiated strain 15T-, as a function ofradiation dose.

8-

UNIRRADIATED.THYMINE

IRRADIATED

iTHYMINE

20 40 60 80 100 120MINUTES

Figure S. The restoration and death of ultra-violet-irradiated strain 15T-, as a function of incu-bation in the presence or absence of thymine.

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sulted in 85 to 90 per cent initial killing, wereused in subsequent experiments. A typical ex-periment is given in figure 3. Control curves arepresented in this figure for viability of the un-irradiated culture in the presence and absence ofthymine. In the irradiated culture incubatedwith glucose in the absence of thymine the pat-tern of restoration and subsequent death wascomparable to that observed in figure 2. Whenthe irradiated culture was incubated in the pres-ence of glucose and 2 ,ug of thymine, it can beseen in figure 3 that there was no apparent effectof the presence of thymine on restoration, thenumbers of viable cells having increased about5-fold in a time less than half of that essentialfor a single cell division.In the irradiated culture containing thymine,

the increased viability of most of the cells of theculture also decreased rapidly after the 20-mininterval and fell until 55 to 60 min after the be-ginning of the experiment. The viable count thenincreased at a rate characteristic of normal di-vision. The extrapolation of this latter curve ofincrease between 60 to 120 min back to 0 minintersected the ordinate at the initial number ofcells surviving irradiation. This curve then maybe taken to represent the normal division curveof the unirradiated survivors. Thus, phenomenaobserved after incubation for 1 hr may be elim-inated as significant aspects of the radiationeffects in this system.

It can be seen that irradiated cells which arerestored as a result of incubation in a liquid con-taining glucose, nitrogen, and phosphorus dieagain in that same medium whether thymine ispresent or not. The curve for the second deathmeets that of the multiplication curve of the un-irradiated survivors at about 55 min. In otherwords, the restored cells die again in a total timeafter irradiation which is approximately thatnecessary for one division.

Since the rate of this loss of viable count isthat of thymineless death, and is complete in thetime necessary for one cycle of bacterial multi-plication, it appears that the second death or lossof power to multiply may be similar to thymine-less death.Although all restored cells of this strain appear

to die in liquid medium, the existence of per-manent restoration may be inferred from thefact that curves of viable count of the characterof figures 2 and 3 may be obtained at all. For ex-

ample, if all restored cells die on continuinggrowth one would not expect to observe restora-tion; that is, cells restored in liquid mediumwould fail to give rise to colonies on plating. Sincethe viability of restored celLs is preserved on theplate, i. e., cells give rise to colonies, the condi-tions of plating may be said to prevent the ex-pression of the radiation-induced lesion, which isin some way permanently repaired. As will beindicated below, a partial permanent restorationin liquid medium does appear to occur with otherstrains of E. coli, e. g., irradiated strain B. Inaddition, irradiated strain 15T- may be pre-served in liquid medium under conditions of atemporary inhibition of growth, and a large pro-portion of the restored cells will go on to multiplywhen the inhibition is removed.The nature of the plating conditions which

permit permanent restoration on the nutrientagar plate is not yet known. If irradiated 15T- isincubated in nutrient broth, curves of restorationand death are observed comparable to those ob-tained in synthetic medium. If nutrient broth isadded to cells restored after 20 min in a syntheticmedium, death proceeds in the same manner.Although the nutritional conditions on the platedo not appear to control permanent restoration,it is conceivable that agar is providing a localenvironment which is producing this effect. Forexample, agar chelates some metals very well andmay be affecting growth in this way. Other stepswhich may be relevant to this question are thedilution of bacteria in chilled media at 5 C andthe initial incubation on the plate at room tem-perature. An analogous "agar healing" effect hasbeen described in connection with the ultravioletinduction of lysogenic bacteria (Lwoff, 1953).

In figure 3 the curve for the death of the re-stored cells extrapolates to the viable count ofthe unirradiated culture, and it may be sup-posed that the irradiated cells are losing thepower to multiply from the inception of incuba-tion after irradiation. However, this extrapola-tion may be entirely fortuitous, it having beenshown in figure 2 that after radiation doses lead-ing to less than 10 per cent survival the seconddeath curve does not extrapolate to the initialnumbers of viable cells. Also it will be seen belowthat restoration curves are similar whether me-tabolism and growth are permitted or not-aresult suggesting that the apparent restorationcurve need not be corrected for concurrent death.

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MINUTESFigure 4. The restoration and death of ultra-

violet-irradiated strain 15.

If this interpretation is correct, it would appear

that the death of restored cells occurs after an

initial lag, even as does thymineless death.Restoration and death of strain 15. Since restora-

tion and death of restored cells were independentof the presence or absence of thymine, it was ex-

pected that comparable phenomena would beobserved with nonauxotrophic bacteria. E. colistrain 15, the parent strain of 15T-, was tested as

described above. Irradiated and control cultureswere incubated at 37 C in synthetic media con-

taining glucose and aliquots were plated at fre-quent intervals. In figure 4 it can be seen thatstrain 15 on irradiation and incubation is re-

stored approximately 4-fold during 20 mi.After this period the restored cells died, and theviable count of the culture returned to the baseline of the normal division curve of the unirradi-ated survivors in a total time approximating thatnecessary for one cycle of multiplication. Subse-quent experiments on the nature of these effectscould therefore be conducted with strain 15,thereby obviating the requirement for multiplecontrol cultures containing or lacking thymine.However, initial experiments with 15T- had re-

vealed the similarity of the death of restoredcells to thymineless death. This second death

would have been very difficult to interpret ifsuch a comparison had been unavailable.Comparable phenomena of restoration and

death, although less clearly defined, have alsobeen observed for a uracil-less strain W, in thepresence of uracil, and for strain B. In the latterinstance the time over which a considerableamount of such restoration occurs can be con-siderably prolonged, and the curves may beinterpreted to indicate that a considerableamount of such restoration is permanent. Studieson DNA synthesis in irradiated cultures of strainB (Kanazir and Errera, 1954) have shown thatradiation initially inhibits synthesis of DNA.However, restoration of the synthesis ofDNA caneventually be complete with this organism. Wehave not continued our irradiation studies withstrainW or B, since a clearer picture of the deathof restored cells was possible with strain 15.

Inhibition of the death of restored cells. If thedeath of restored cells is death by unbalancedgrowth, it should be possible to inhibit this typeof death by inhibiting growth. In initial experi-ments strain 15 was grown in a complete medium,sedimented, washed, irradiated, and incubated inmedia lacking the carbon source (glucose) or thenitrogen source (NH4+). In these experimentsrestoration occurred at rates comparable to thatin complete media and, although death wasinhibited, a significant drop in viable count wasobserved. It appeared necessary to reduce theintracellular supply of carbon or nitrogen com-pounds by irradiating starved bacteria. The res-toration of irradiated starved bacteria in mediadevoid of glucose or nitrogen was not followedby a second death.

Cells of strain 15 were grown in the mineralmedium containing i of the usual glucose con-centration, i. e., 0.2 mg per ml. Growth rate wasnormal until growth ceased due to the exhaustionof glucose at a bacterial concentration approxi-mately that at which cultures were routinelyharvested. The results of an irradiation and in-cubation experiment with such glucose-starvedcells are given in figure 5. Addition of glucose tothe starved cells produced a rapid division rateafter a 40-min lag. In the absence of glucose, avery slow drift upward in cell number (10 percent) occurred over a 2-hour interval. IrradiatedcelLs were restored at a slightly higher rate inthe presence of glucose than in its absence. Inboth instances restoration continued for about

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107- - °D3 8 IRRADIATED +Ur> 4G/LUC NOGLUOS

6,//-J IRRADIATED

20 40 60 80 100 120MINUTES

Figure 5. The restoration and death of ultra-violet-irradiated strain 15 as a function of thepresence or absence of glucose.

30 min. In the presence of glucose all the restoredcells died. In the absence of glucose, the starvedirradiated cells were restored and were main-tained unchanged.

Effect of 6-methyl tryptophan. When cells weregrown normally in nondeficient media and irra-radiated, a single amino acid analogue was able toprevent death after restoration in completemedia. This effect was produced by the com-petitive antagonist for tryptophan-5-methyltryptophan-at a concentration which almostcompletely inhibited growth. Under the usual con-ditions of our experiment, such a concentrationis 2.3 X 104 M.

Strain 15 was grown under the usual condi-tions, washed, irradiated, and incubated innormal media or in media containing this ana-logue. It can be seen in figure 6 that the rate ofrestoration was essentially identical in the pres-ence or absence of the analogue. With 5-methyltryptophan present, a slightly greater total restor-ation was obtained. However, although the usualdeath of restored cells occurred in the absence of5-methyl tryptophan, in the presence of the in-hibitor the cells showed only a very low deathrate-attributable, in fact, to the slight growth ofthe bacteria which is detectable in the unirra-diated control containing 5-methyl tryptophan.

4.

2

Fl7Z) 8.

6

J

2

UNIRRADIATEDa

_|

x IRRADIATED+5MT

I *%% ,0,0I'o __ _I

I ~~~IRRADIATEDIIIII

20 40 60 80 100 120MINUTES

Figure 6. Ultraviolet-irradiated strain 15 incu-bated in media in the presence or absence of5-methyl tryptophan.

It was of interest to determine if death wouldoccur on creating conditions for normal growthin the inhibited irradiated culture. Inhibition ofnormal growth by 5-methyl tryptophan is relievedby tryptophan. When normally prepared bacteriawere irradiated and incubated simultaneously in5-methyl tryptophan and tryptophan adequatefor normal growth, the usual restoration anddeath curves were obtained as in figure 7. Ifnormal unirradiated cells were held in 5-methyltryptophan for 30 min and tryptophan was thenadded, normal growth and division occurred-al-beit with a slight decrease in division time. Ifthe irradiated cells were held in 5-methyl trypto-phan for 30 min and tryptophan was added, aconsiderable additional restoration was obtained.Following restoration the curve showed a plateau.The apparent plateau may be interpreted to sig-nify the concurrent death of some celLs and themultiplication of others. In any case it is clearthat a quantity of tryptophan capable of restoringgrowth does not provoke the precipitous deathof restored cells as had previously been observedin the uninhibited culture. It appears then that,if restored cells are only temporarily preventedfrom growth by addition of 5-methyl tryptophan,the lesion leading to death by unbalanced growth

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8ILJD.

X TRYPTOPHAN - 0 TIME* s -30.MIN.

//UNIRRADIATED

yA/

MINUTESFigure 7. Ultraviolet-irradiated strain 15 in-

cubated in media containing 5 methyl tryptophanand supplemented with tryptophan after differenttimes of inhibition with 5-methyl tryptophan.can be permanently repaired in a considerablenumber of the restored cells.The experiments with 5-methyl tryptophan

corroborate the experiments with starved cellsand indicate that, under conditions in whichnormal growth and division are inhibited, restoredcells do not die. To state the phenomenon in otherwords, the lesion residual in restored irradiatedcells is expressed only under conditions of growthand division.

The effect of temperature on restoration. Whenthe irradiated cells were incubated at tempera-tures in the range 5 to 45 C, a differential effectwas observed on restoration and on the subse-quent death of restored cells. These results arepresented in figure 8. At 25 C or below, the rateof restoration and the relative amount fellmarkedly under these conditions, and it may benoted that a 10 per cent reactivation of the ini-tially nonviable cells at 5, 15, 25 and 37 C oc-

curred at approximately 80, 40, 20, and 5 mi,respectively.On the other hand, the rate of death of restored

20 40 60 80 100 120MINUTES

Figure 8. The viability of ultraviolet-irradiatedstrain 15 incubated in glucose medium at differenttemperatures.

cells varied far less over a wide range of tem-peratures and all restored cells were killed ineach case. Since the cells restored at tempera-tures which did not support measurable divisionof controls nevertheless lost their secondarilyacquired viability at rates independent of thetemperature (15 and 45 C), factors other than asimple inhibition of division were effective underthese circumstances.

DISCUSSION

The restoration of viability to cells "killed"by ultraviolet irradiation is not a unique propertyof strain 15. The phenomenon is quite general,as an abundant literature attests. Phenomema ofspontaneous and induced cell restoration afterirradiation have been surveyed recently byseveral authors (Latarjet, 1954; Kelner et al.,1955). Restoring agents summarized in thosepapers have included temperature changes, visiblelight, catalase, metabolites, etc.The restorations observed in the present paper

appear to differ from the above in that restora-tion is revealed as a common phenomenon ofincubation under normal conditions of cultiva-tion.3 In strains other than B, these were not

' Apparent recoveries of this type had perhapsbeen observed previously (Roberts and Aldous,1949). However the experiments of these workersdid not take account of photoreactivation and aredifficult to interpret for this reason.

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seen earlier because it appears to have been anunusual practice to assay irradiated cultures peri-odically at relatively short intervals, i. e., inter-vals significantly less than a single division time.If the system were assayed at longer intervals,the phenomenon would have been missed. Inexperiments with strains 15 and W, for example,restoration is no longer apparent after one divi-sion time, since the restored cells die on continuedincubation.The phenomenon of restoration which we have

seen is relatively unaffected by a wide variety ofmetabolic conditions, and proceeds very well inthe absence of exogenous nitrogen or in thepresence of a metabolic inhibitor such as 5-methyltryptophan. These conditions may be expectedto eliminate protein synthesis. The recoveryproceeds in irradiated cells deficient with respectto an endogenous and exogenous carbon andenergy source, although it does seem slightlyinhibited under these circumstances. The tem-perature coefficient of restoration is approxi-mately 2 in the range of 5 to 37 C, and thisinformation suggests that we may be dealing witha relatively simple reaction, as in the decay of atoxic compound. Since the cells have been sub-jected to a very light dose of irradiation and arediluted prior to incubation, and since the multi-plication curve of survivors is normal in everyrespect-e. g., lack of lag, division time-it doesnot appear likely that the toxic material ispresent in the medium. It may be assumed that,if restoration reflects the decay curve of a toxicmaterial, this substance has been generatedintracellularly.The subsequent death of the restored cells has

many of the major properties of death by unbal-anced growth. Both occur under conditionsknown to result in the inhibition of deoxyribose-nucleic acid (DNA) synthesis. The rate of deathis that of thymineless death, and is complete inthe time necessary for a single cycle of multipli-cation. If metabolism and cell growth are in-

UVA. DNA -* activated DNA*

A

hibited, the secondary death of restored cells canbe completely prevented. The similarities of thistype of radiation-induced death and thyminelessdeath support the hypothesis that the initialradiation damage is localized in the nuclear DNA.The irreversible "killing" of cells restored after

low doses of ultraviolet irradiation appears torequire cellular growth under conditions in whichcytoplasmic syntheses are normal and nuclearsyntheses are incomplete. However, in speakingof this phenomenon as "death by unbalancedgrowth," it is not evident whether the decisivestep leading to the loss of the ability to multiplyis a cytoplasmic or a nuclear event. Two typesof phenomena seem possible: (1) The synthesisof cytoplasmic structures may have formed aframework within which the nucleus cannotoperate when given the thymine essential forDNA synthesis. (2) Nuclear division may haveproceeded with an incomplete complement ofnuclear structures, containing DNA, the resultingfragments being inadequate to maintain nuclearfunction and incapable of setting cell division intoplay. In either case the continuing developmentof the cell under conditions of unbalanced syn-theses leads the cell to a structural impasse fromwhich it cannot withdraw; the cell may be saidto have irreversibly lost the power to multiply.

Radiation damage in strain 15 is manifested intwo different types of damage, and their meansof repair also differ. The first type of damage isrepaired by incubation in liquid medium and notby our plating conditions. The data on this typeof repair suggest the existence of a labile sub-stance whose presence is inconsistent with con-tinuing multiplication. After the destruction ofsuch a compound, the cell may multiply if aresidual lesion is repaired. This repair of theresidual lesion cannot occur with strain 15 underconditions of growth in liqulid medium, but doesoccur under plating conditions. If it is supposedthat ultraviolet irradiation is absorbed in DNA,these phenomena may be pictured in several

DN/A repa- r DNAplating

toxic product decay ti productincubation

B. DNA -* activated DNA* decay b DN/A repair DNAincubation plating

Diagram I

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RELATION OF GROWTH TO IRRADIATION DAMAGE

ways (diagram 1). In scheme A it may be picturedthat activated DNA may decompose to form aDNA with a break in a critical bond (DN/A).In addition the decomposition produces a toxiccompound, such as an organic peroxide, whichmay inhibit critical syntheses albeit at very lowintracellular concentration. The repair in DN/Amay be perceived only after the disappearanceof the toxic agent. In scheme B the activatedDNA itself may be the toxic substance and mayhave to decay before the repair of the productDN/A becomes possible.

SUMMARY

A comparison has been made of the loss ofviability of Escherichia coli strain 15T- after lowdoses of ultraviolet irradiation and after depriva-tion of thymine. It was observed that incubationin liquid media permitted a considerable restora-tion of irradiated bacteria. The restored cells oncontinued ineubation in liquid medium underconditions of growth died in a manner similar to(tells which were peirmitted to grow in the absenceof thymine.The restoration and secondary death of re-

stored cells were independent of the presence ofthymine and were observed with nonthvmine-requiring bacteria. Inhibition of cell metabolismand growth permitted restoration but preventedthe death of restored cells. This was accomplishedb)y depletion of carbon and nitrogen sources orby treatment with 5-methyl tryptophan. Thesignificance of these results has been discussed.

REFERENCES

13ARNER, H. D. AND COHEN, S. S. 1954 The in-duction of thymine synthesis by T2 infectionof a thymine-requiring mutant of Escherichiacoli. J. Bacteriol., 68, 80-88.

BARNER, H. D. AND COHEN, S. S. 1955 Thesynchronization of cell division of a thyminerequiring mutant of Escherichia coli. Fed.Proc., 14, 177.

COHEN, S. S. 1947 Streptomycini and desoxy-ribonuclease in the study of variations in theproperties of a bacteria virus. J. Biol. Chem.,168, 511-526.

COHEN, S. S. 1953 Studies on controlling mech-anisms in the metabolism of virus-infectedbacteria. Cold Spring Harbor SymposiaQuant. Biol., 18, 221-235.

COn1EN, S. S. AND ARBOGAST, R. 1950 Chemi-

cal studies in host-virus interactions. VII.A comparison of some properties of threemutant pairs of bacterial viruses: T2r+ andT2r, T4r+ and T4r, T6r+ and T6r. J. Exptl.Med., 91, 619-636.

COHEN, S. S. AND BARNER, H. D. 1954 Studieson unbalanced growth in Escherichia coli.Proc. Nat. Acad. Sci., 40, 885-893.

COHEN, S. S. AND BARNER, H. D. 1955 Enzy-matic adaptation in a thymine-requiringstrain of Escherichia coli. J. Bacteriol., 69,59-66.

DUNN, D. B. AND SMITH, J. D. 1955 The occur-rence of a new base in the deoxyribonucleicacid of a strain of Bacterium coli. Nature,175, 336-337.

GALE, E. F., AND FOLKES, J. P. 1953 The as-similation of amino acids by bacteria. 15.Actions of antibiotics on nucleic acid and pro-tein synthesis in Staphylococcus aureus. Bio-chem. J. (London), 53, 493-498.

HERRIOTT, R. 1951 Nucleic acid synthesis inmustard gas-treated E. coli B. J. Gen.Physiol., 34, 761-764.

KANAZIR, D. 1954 Accumulation de l'acidethymidylique chez E. coli apres irradiationU.V. Biochem Biophys. Acta, 13, 587-590.

KANAZIR, D. AND ERRERA, M. 1954 Metabo-lisme des acides nucleiques chez E. coli Bapres irradiation ultraviolette. BiochemBiophys Acta, 14, 62-66.

KELNER, A. 1949 Photoreactivation of uiltr a-violet-irradiated Escherichia coli, with specialreference to the dose-reduction principle andlto ultraviolet-induced mutation. J. Bac-teriol., 58, 511-522.

KELNER, A. 1953 Growth, iespiratiotn, andnucleic acid synthesis in ultraviolet-irradiate(dand in photoreactivated Escherichia coli. J.Bacteriol., 65, 252-262.

KELNEP, A., BELLAMY, W. D., STAPLETON, G. E.,AND ZELL, M. R. 1955 Symposium on radia-tion effects on cells and bacteria. Bacteriol.Revs., 19, 22-44.

LATARJET, R. 1954 Spontanieous and induce(dcell restorations after treatments with ioniz-ing and non-ionizing radiations. ActaRadiol., 41, 84-100.

LWOFF, A. 1953 Lysogeny. Bacteriol. Revs.,17, 269-337.

PARK, J. T. 1952 Uridine-5'-pyrophosphate de-rivatives. J. Biol. Chem., 194, 877-904.

ROBERTS, R. B. AND ALDOUS, E. 1949 lRe-covery from ultraviolet irradiationi in Esch-erichia coli. J. Bacteriol., 57, 363-375.

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