SHORT COMMUNICATIONS 2 4 9

BBA 93332

Inactivation of transforming DNA by tritium disintegrations in v/vo

The exact mechanism by which tritium incorporated into intracelinlar macro- molecules kills ceils has not yet been ascertained. The efficiency of killing by tritiated thymidine is much higher than that produced by a tritLated amino acid 1,2. Pn~soN 2 concluded from this that about two thirds of the damage is due to chemical changes in the thymidine, and consequently in the DNA, and that the remainder of the damage is radiation induced. Other investigators TM, however, have provided theoretical arguments and experimental evidence that viability is lost solely because of ionizing radiation, This interpretation suggests that the radiation resulting from tritium in protein damages the DNA and that incorporated trltiated amino acids kill less efficiently than incorporated tritLated thymidine because of their greater distance from the DNA.

We lhsve pexformed a comparative study by directly assaying the biological activity of transforming DNA isolated from a culture of Bacillus subtilis which has incorporated tritiated thymidlne, histidine or leucine and has undergone suicide to varying degrees. Such an analysis assays the number of copies of a biologically active gene and therefore measures the direct effect on the DNA.

Cultures of all strains were made competent as previously described 5 and stored in tubes at --88 ° . "[he same batches of competent cells were used throughout this study. Because the quantity of DNA extracted varied from day to day, a constant amount of carrier, tryptophan-requiring cells which bad never been exposed to tritium was added to the experimental cells prior to lysls. The carrier cells, SB zo2, were of the genotype ~p- , lea +, his+,/by+ (~:b = tryptophan; lea = leucine; his = histidine; t,~y = thymine), whereas the experimental cells were always le~ ÷. The DNA extracted from the mixture of experimental (either heavily Labeled with tritium or unlabeled) and carrier cells was added in appropriate dilutions to two sets of co~a- petent cells: (I) SB 202 to uss~y the number of biologically active tr;b + genes following tritium decay and (2) the same strain as used in the tritium suicide tc v.ssay the num- ber of X + genes in the carr~_'er DR'A, where X is thymidine, histidine or leucine. The control nonradioactive cells were run concomitantly with the cell~ tha~ were expend to the ]etbel decays of tritium. The number of copies of a gone, present o~y in the carrier DNA, permits normalization of the number of biologically active l ~ + genes extracted from the experimental cells. The loss of biological activity of the ~rp+ genes is determined from the formula:

x + genes in r~onradioactive DNA lep + genes in radioactive DNA ~oo = % lass X + genes in radioactive DNA X try+ genes in nom'ad{oactive DNA ×

Note: DNA is that extracted from the mixture of carrier cells and experimental cells, the latter being radio-ctive or nonradioactive. Transforming activity wa s lost in DIgA extracted from ceils that had undergone

either [aH]thymidine or [~H]anino acid suicide. The loss of transforming ac~vity plotted on a logarithmic scale demonstrated single-hit kinetics. The decrease in viable cells also followed single-hit kinetics. The reciprocal of disint./cell required to reduce survival or transforming activity to 37 % was used to calculate the ££ficiency of inac- tivation by the tritiated compound. Regardless of tlle ti~tiated cmi;paund used, the

Biocbim. Biophys. Acre, i66 (1968) z49--25o

z5o SHORT COIdMUIffICATIOI~S

TABLE I EFFICIgN'C[ES OF KILLING AND ISACTIVA'fION OF 3"RANSFORMING DNA BY ~t~IA~D CO~OUNDB The procedure for all experiments was ~s follows. The auxotropha were grown to late logarithmic phase, washed, labeled for a generation in minimal medium e containing flluco~e and the tritiated compound, The cells were washed by centriffugatloB, resuspended in minimal medium containing 5 o/, g vcerol, d 5tributed into tubes frozen in a dry ice-ethanol bath, and stored at --88 ~. A f~nd'culture, treated identically was exposed to the nonradio.active coml~ound during the la.b~!- ng period. These nonradioactive cultures suffered no loss in vlablhty dunng the ~terage person. At interval~, individual tubes of labeled and control ce s were thawed and • ~ample plated. To the remainder of the cells, 5' xo* ceils of SB ~o2 were added, and DNA wae extracted from mixtures of experimental and carrier cells by the method of Tnozas, Bmms ANy K~L'/L The extracted DNA was essentially free off contamin~nte (< 3n/roll. The biological activity of the DXqA was then estimated as described in the text. Plate counts were nxade on alrpropri~tely sup- plemented sear.

[ltf]Thymidfnt [DH]Hiaidine [sH]Leuci~e

Strain (genotype} WB 829 T (t/~y-I SB 197 (his-) SB 27z (l~u-) ,uC/ml added 31.~ 21.6 31.6 pg/ml added o.48 i.o o.83 Disint./cell per day" 64 169 43 ~ Efficiency of killing, % o.3o o,12 0.Io Efficiency o f DNA inact ivation, % 0.o42 o,o16 o.013 Efficiency of killing 7 .~ 7.5 7,7 Efficiency ol gene inactivation

* Trichloroac~tic acid-precipitable material after cells were susiaended itt glycerol.

loss of biological activity of extracted DNA was proportional to the loss of survival (Table I). The inactivation of a single gene actually represents an assayfor theinteg- r i ty of the DNA molecule because partially degraded DNA loses biological activity s,

The fact tha t [~H]thymidine and Jail]amino acids yield parallel results with regard to the two tmrameters tested (viability loss and gene inactivation) suggests tha t the basic cause of death is independent of the macromolecule which contains the isotope. The da ta therefore support the theory ~ tha t death from tr i t ium results from

ionizing radiation inactivation of the genome. This work was supported by National Science Foundation Grant (GB 595;).

The senior author held Public Heal th Service predoetoral fellowship 5-Fx-GM-z84zz

Departments o[ Microbiology and Genetics, CHARLOTTJ~ McCARTHY" University o[ Washier#on. EUGEN~ W. NESTER

Seattle, Wash, 98z05 (U.S,A.)

t M. RA¢IIMEL~I¢ AND A. El. I>AFCDEE, Bioahim. Biep~ys. Aaa, 68 (1963) 62, z S. p~IiSON, B$¢,phy$, J , 3 i19fi3) tea, 3 S. ApELGOT ASD E. LATARJ~T. Biochim. Biopky$, Acts, 55 (I962) 4 °. 4 A. L. KneE, Radlation Re$,, 24 (I9631 398. 5 C. McCaxTJtX" ,~ND E. W. Ig~S~'~R, J. Bacteriol,, 94 (1967) 13I. 6 J, Svtztzg~, Proc. Null Acad. SoL U.S,, 44 (19581 lO72. 7 C, A. THOMAS, K, I. BERNS A~D T. J. ]{BLLY, irl G, L, CANTONI AND D. R. DAVIES, Procedures

in N~wlei~ Acid Reseamh, Harper and Row. Iqew York, 1966, p, 335. 8 W. F. BOOMER, J. Gen. Pl~ysiel,, 49 (1966} ~33, 9 S, P~RSON, Radiation Res., 33 (I9681 66,

Received March zsth, r968

Addendu~n: p~l~souP reached a similar conclusion by ~tadying DNA degradation by tritium in Esc~erichia coli (Received May z?th, 1968)-

• Presem 0ddre~s; Research Division, National Jewish Hospital, DenveL Coin,, U.S.A,

BioeMm. Biopkys, Aau, 160 (1068) 249-250