X-ray-induced mutation in extracellular bacteriophage T4

)N IN E X T R A C E L L U L A R BAC1

GE T 4

Laboratories, Chalk River, Ont. (Canad~

dMARY

Two kinds of experiments were performed to test tes for a r ffect of rays on extracellular bacteriophage T4: ( ¢ ) the fie(tuencies , nottled ques were determined in platings of irradiated and unirradi ~pe T 4 ~ge ; and ( 2 ) the frequencies of/'1i+ reverse mutants were we measul ogenies irradiated and control T 4 rH phage. In both kinds of experim¢ quency Lhe mutant type was increased by irradiation of the extracellul e Reconaction experiments", in which artificial mixtures of ri~ and ~'ii re irrated, showed that only a minor part of the radiationrradiation-induced a t u a u o n - l n t t u c e t t i n c r e a s e Ill 7"II' 'n + f~rereracy could be accounted for by selection in favour of already-existing rn + revert a nLts. The results indicate that genes can be induced to mutate ml by exposure to ionizingg radiation [iation in the absence of any metabolic activity. A multihit mechanism for the induction of mutatiorms was indicated by the dose!2 from irradiated rli rn restponse curve for the frequency of r~F revertants in progenies phatige. The chemical changes involved are unknown.

INTRODUCTION

Extracellular virus tparticles offer the possibility of studying the effects of aa mutagenic agent on genes when these are completely isolated from any metabolic abolic system. Information from such studies m a y be basic to an understanding of the mechanism b y which the atgent produces mutations in living cells. Although mutations have been induced in extracellular virus particles byY exposure to a number of phy Lysical and chemical agents ~,~, it has not been clear whethel vhether they could be induced b y ionizing radiation. GOWEN i reported an increase in the frequency of mutants in tobacco )bacco mosaic virus after X-irradiation of free virus parti~articles, but MUNDRYs later showed howed that this increase m a y have reflected an improved ~roved efficiency in detecting mutants ants already present in the unirradiated virus population alation. 6 reported the induction of "clear" plaqu~ KAPLAN, W I N K L E R AND W7OLF-ELMAUER O L F --ELMAUER EL ue mutations in the lysogenic bacteriophage × of Serratia by extracellular X-irradiatior 1on but noted that other kindss of mutants in this virus remained rare after irradiation tiation. More recently, ARDASHNIK(:OV, SOYFER AND GOLDFARB 1 d e s c r i b e d a n i n c r e a s e i n tthe he Mutation Res., 3 (1966) 365-37. 373~

s of b a c t e r i o p h a g e T2 after extrac, El;: E N T , B L O K AND LINCKENS 12 fo

nge m u t a n t s of p h a g e q~ X I74 aft, in these l a t t e r studies, however, g m u t a n t s were sufficiently sensiti e r p r e t a t i o n of the results. e question of w h e t h e r n m t a t i o n s ionizing r a d i a t i o n , a s t u d y was un this phage to " r a p i d lysis", the soy s t e m for the detection a n d analy ;ess; f o r w a r d m u t a t i o n s from r~ to r are r e a d i l y ddee t e c t e d , e s for reverse m u t a t i o n s from r n to rn + can he Ferfor ::erformed, a n t ~tic p r o p e r t i e s of i n d u c e d rn n m t a n t s can be s t u d i e d :~. a. I n this s carried out to test for the i n d u c t i o n of f o r w a r d m u t a tions fron ations from ru to ru ~ b y exposure of e x t r a c e l l u l a r p h a g e to :onstruction e x p e r i m e n t s " , in which artificial mixture res of r~F :liMed, were used to test for selection in favour of rn + reverse I m 5rm t h a t n m t a t i o n s can, in fact, be induced in e x t r a c e l l u l a r ation.

sure of :'ase in tion of sts for de this teed in ith the ations, agenic nsitive ;al a n d iments reverse -called [e were results mizing

ERIALS

Phage T4B w i l d - t y p e phage, o b t a i n e d from Dr. A. D. HERSHEY, HEI (;old Spring H a r b o r , N , Y . ., was used in p r e l i m i n a r y tests for the i n d u c t i o n of f o r w a r d m u t a t i o n s from r + to r•• A double m u t a n t d e r i v a t i v e of this phage, containing contair an fi m u t a n t gene, r~, a n d a n / ' i i m u t a n t gene, r~, was used in tests for the iin& n d u c t i o n of reverse m u t a t i o n s from r n to rn +. The rx a n d r n n m t a n t genes in this l a t t e r pi h a g e were each d e r i v e d b y le-step m u t a t i o n from u n t r e a t e d w i l d - t y p e p h a g e a n d b o t h c o m b i n e d in the same single-step )me b y a cross b e t w e e n the singly m u t a n t p a r e n t sti strains. The p u r p o s e of the r~ genome m u t a n t was to e l i m i n a t e lysis inhibition, which would otherwise be expressed b y lZlI+ r e v e r t a n t s , a n d which m i g h t o p e r a t e to select for or against the m u t a n t t y p e in the rn reversion e x p e r i m e n tks. s . The r~x m u t a n t , rg, was chosen for use in these experineou s reversion r a t e ; the frequency of r e v e r t a n t s in m e n t s because of its low spontane( .ontaneous stocks of this m u t a n t p r o d utced c e d from small inocula is a b o u t 5 " Io-~. P h a g e stocks for the e x p e r i m e n t s were p r o d u c e d as clones from single p h a g e particles 9. Log phase cultures res of E. coli S/6 in H b r o t h were infected with a single p l a q u e from a p l a t i n g m a d e 4 h earlier a n d t h e n i n c u b a t e d u n t i l t h e y cleared. T h e titres of t h e cultures at t h e t i m e of~f infect infection ection were a b o u t 2 • Io~/ml, to p r o d u c e T4 w i l d - t y p e stocks, a n d lOS)M, to p r o d uace c e T4 r~r~ stocks. L y s a t e s from t h e infected cultures were :o min to r e m o v e u n l y s e d cells a n d large cellular debris. c e n t r i f u g e d at 4ooo g for 2o T h e l y s a t e s were n o t f u r t h e ;r r purified• (b) Bacteria E. coli S/6 b a c t e r i a , in which b o t h r n a n d rn + p h a g e can grow, was used for m u l t i p l i c a t i o n of p h a g e a nLd d for assays of t h e t o t a l t i t r e of p h a g e lysates. E. coli I I 2 - I 2 (2h) b a c t e r i a , in which dch rn + b u t n o t r n p h a g e can grow, was used for assays Mutation Res., 3 (1966) 305 373

~XTRACELLULAR BACTERIOPHAGE T

n lysates. Both of these strains ~titute of Technology, Pasadena. r use in the experiments were pr ~r Io8/ml or 5 " IO8/ml) in H broth asions were stored at 4 °.

wth of phage and bacteria and for ns, per litre distilled water, 8 g Ba chloride, I g glucose. Indole broth was used for single growth cycles of exper: nposition, per litre distilled water, is o.I g indole, no sodium c ;e the same ingredients as H broth. E H A top- and bottom-layer agar was used for plating. plat: EHA ltains, per litre distilled water, IO g Bacto agar, 13 g Bacto tr: oride, 2 g sodium citrate (dihydrate), 1. 3 g glucose. E H A top-1 ' litre distilled water, 6. 5 g Bacto agar, 3 g glucose, ar and otherw nts as the bottom-layer agar.

d from :oncenio*/ml

zge and broth, Lge. Its Lotherer agar sodium retains, ingre-

THODS

Irradiation The radiation source was a General Electric 2 MeV Me X-ray ?erated E.5 A. Phage were irradiated in a nutrient broth solution solutior (H broth) to protect them hem from m inactivation b y the indirect effect of X - r a y s ' . T h e ,survival curves of the phagee in this :his medium were exponential down to a survival of o.oi. o At survivals lower than thiss, an increase in the rate of inactivation became evident, evk suggesting that radiochemical mical products in the medium were contributing significantly sigJ to inactivation at these lower survivals 13. Phage suspensions were irradiated in baffled aluminum alun capsules which were electricall' :trically rotated to insure uniform exposure of the phage. ph~

(b) Early test for induced mutation A relatively crude tes ~st for induced mutation was used initially and replaced later b y a more refined procedure :ocedure. a r e . The early test consisted simply of determining the frequencies of r and r-motth nottled plaques in platings of irradiated and unirradiated ~diated T 4 wild-type phage. This tes est for induced mutation is crude in three respects: (_r) the spontaneous mutation rate from r ÷ to r is quite high, giving rise to a background frequency of uninduced mutants nts of lO-3 to lO-4; (2) r mutants cannot be screened from wild-type phage, thus limitin .iting the size of phage population which can be tested conveniently; (3) the sconr •ing of mottled r plaques is somewhat subjective since all grades of mottling occur. Irradiated and control :ol phage were plated with S/6 bacteria so as to produce ,roduce IOO-3OO plaques per plate. To prevent multiplicity reactivation on the plates, phagee were preadsorbed to plating lg bacteria at a constant multiplicity of o.ooi phage parti)article per bacterium. The plates tes were incubated at 3 °0 to produce m a x i m u m differentiferentiation between wild-type and ad r plaques. Mutation Res., 3 (1966) 365-373 365-37~

platings of irradiated phage were in control platings. To minimize atant plaques were identified by s

~t n l o r e

of this plates

tion ctive test for induced m u t a t i o n is /'everse ~v background of uninduced muta~ Ltained w spontaneous reverse n m t a t i o n r )rtants by plating the phage with a selee rain in ,o 4 • ~ . ,o ch only the reverse m u t a n t s will multiply and produce prodl plaqu counts end only on the presence or absence of plaques, not on o plaque To test for the induction of ru + revertants b y X-irradiati( X. :ellular ge, frequencies of rn + revertants were determined in progenie', ed and :radiated T 4 rn phage. The reason for testing the progenies p: phage r[l 4 ler than the survivors themselves was to insure t h a t latent la nduced :he radiation would be detected. Irra( Phage progenies were produced in the following way. v :ontrol phage parti ge were mixed with S/b bacteria in the ratio of IO ph eriurn. mixtures were incubated at 37 ° for IO rain, d u r i n g ' which tin n 95% iable phage adsorbed to the bacteria. Indole broth was wa then ad uspenpha s (diluting them 5-fold) to inhibit adsorption of progeny proi d cells aeration until phage growth was cellular debris. Incubation was continued with aerat ~ v l i i ~pleted. The lysates so produced were sterilized with th a few drops of chloroform centrifugati at 4ooo g for 2o rain. and freed of cells and large cellular debris b y centrifugation 'sates were determined, either The frequencies of rn + revertants in these lysat~ at appropriate dilutions the immediately or after overnight storage, b y plating them (2h) bacteria to deterII withi S/6 bacteria, to determine total titres, and with 112-12 mine the titres of rn + revertants To correct for a somewhat lower efficiency of ing of the phage with ~i2-~2 (aa) bacteria than with S/6 { bacteria, a known numplating her of T4 r1 phage particles were added to several plates in the assay of each lysate ().h). Correction factors were determined from fron the plaque counts on these withi III2:- - I 2 (~,h). dculation ttion of rn + titres. plates and applied in the calculati( --

•

)n in favour of rn + revertants (d) Control to test f o r selection A higher frequency of[ rn + revertants among progeny from irradiated r~t phage, as compared with progeny from unirradiated r~i phage, might reflect a favoured suivival and multiplication of rn + revertants already present in the unirradiated phage etion of new r~t+ mutations b y the radiation. To test this stock, rather than the induction possibility, "reconstruction experiments" were performed in which rn + revertants ge in numbers sufficient to mask any additive increase in were added to parental phage their frequency which might.t occur b y mutation, but small enough to reveal a multiection. The procedure for these experiments was identical plicative increase due to selection. to t h a t for tile refined test:, with the exception t h a t only one dose of X-rays was employed, 25 ° kR. " S t a n d aard r d experiments", in which no rii + phage were added to ions, were performed in parallel with the "reconstruction the parental phage suspensmns, experiments". 2VIutalion Res.,

.3 ( I 9 6 6 )

365

373

~XTRACELLULAR BACTERIOPHAGE T

phage for the "reconstruction" ex] phage stock with the selective hos vertant plaques from the plates int dded to the parental phage should 1 e already present.

ned in tests for the induction of r dted to be conclusive, the results are suggestive of a mutager extracellular phage. The frequencies of r plaques fr~ from platin ~ge exposed to 125 k R and 250 k R of X-rays were highher than t anirradiated phage (Table I). Only in the case of the higher i do

ere obh), and [ broth. ve been

are too X-rays ld-type fiatings t, how-

BLE I OF r MUTATIONS I N T4B W I L D - T Y P E PHAGE ated and control phage were plated with a surplus of host ba bacteria so ques per plate. The frequencies of r and r-mottled plaques from eac] ermined. ~UCTION

;e (kR)

Plaque counts Total

r Mottled 9 22 9

980o 8684

5 4

25 °

2112

2

a

O//ora

Whole

x~ D

lOO-3OO 91e were

o.153 (o.o78-o.239) 0.299 (o.196-o.439) o.521 (0.260-0.931)

I nn brackets are s h o w n the limits at the 0.05 level of probability probabilit 2 b y STEVENS test n.

ever ;r, was the difference in the frequencies of r plaques from fro platings of irradiated and control ltrol phage statistically significant (o.oi < P < 0.05). That this difference reflects the,,induction of r mutations mu by the radiation and not a pre )referential survival of already-r_ existin sting r mutants is suggested b y its origin in the frequeJ uencies of mottled, rather er than whole ole, r plaques. plaq Already-existing y-existin~ ing r mutants might existing ght app ppear in mottled plaques after aftei segregation from reverse mutants m t a n t s or from phage heterozygotes, but the frequencies uencles of both these events are exxpected to be several orders of magnitude below that of ot mottled plaques in these e',xperiments. x Induced r mutants, on the other hand, may ~y occur regularly in mottled plaques under certain conditions 2.

(b) Refined test The frequencies of rrii l f + revertants in progenies from irradiated and control rol T4 rlr~ phage were examined ~d in two series of experiments differing in the range of Xray doses to which the parental ental phage were exposed. The doses used in the first series 3S of experiments were I, 5, 25, and 125 kR; in the second series of experiments, they ,y were I25, 250, and 500 kR. The frequency of rn + revertants in progeny phage was increased b y irradiation diation of parental rn phage withi doses of X-rays in excess of 25 kR (Tables I I and III). Doses of less than 25 k R to parental phage produced no significant change in rii + revertant frequency (Table II). Mutation Res., 3 (1966) 365-373

MUTATIONS BY" LO\V DOSES OF X-RAYS e r e e x p o s e d t o a r a n g e of d o s e s of X - r a nonselective host bacteria. The frequcl ~gc p r o g e n i e s . T h e r i f t f r e q u e n c i e s in t

crmitted vertants ) a s c d on

lO 8

z4. t

BLE T

z.3z

Ill

FOR INDUCTION

OF l'II

>

Yll t MUTATIONS

BY

HIGH

DOSES O OIF X-RAYS

: p r o c e d u r e w a s t h e s a n l e as in tile p r e c e d i n g e x p e r i m e n t s (T~ ( T a b l e I 1). Ti ed o n 6 r e p l i c a t e e x p e r i m e n t s , o n l y 4 of w h i c h i n c l u d e d t h e h t i g h e s t do,'

5. [4 1.1 27.8 lo8

ncies are

o.34 I • o.947 : 3.15 I5.8

30 20 110 0 xlO0 ¢o l.z90

/

>ws0 t~ R:

7O u_60 o >-50 z

w D40 o w u_ 30 2O ,o

,

/ I00

200 X - RAY

300 DOSE

iN

400

500

kR

Fig. 1, I n c r e a s e in f r e q u e n c y of r l i ~ r e v e r t a n t s a m o n g p r o g e n y p h a g e d u e t o X - i r r a d i a t i o n oof p a r e n t a l p h a g e . T h e p o i n t s o nI t h e c u r v e a r e b a s e d o n 6 r e p l i c a t e e x p e r i m e n t s , o n l y 4 of w h i cc]h mee n t . T h e v e r t i c a l line a t e a c h p o i n t i n d i c a t e s its s t a n d a r d e r r o r i n c l u d e d t h e h i g h e s t d o s e t r e a ttm t h e l i m i t s for t h e z e r o d o s e p o iint, n t , [ : o.341, a r e t o o s m a l l t o b e s h o w n . Mutation

R e s . , 3 (1966) 365 373

XTRACELLULAR BACTERIOPHAGE

T,

ertants in progeny phage increase Fig. I). The increases in revertant 5 • lO-1°, and 20.6 • lO-l° at doses

y with per kR 5okR,

rn + revertants in progenies trom iI m control phage, were not artifact, by cross-reactivation phenomena l radiated phage contained inactive 1 he assays for rii + frequency by mt ~tion phenomena were absent or in ays was verified by plating the lysates at different dilul dilutions wiff frequencies of rn + revertants determined from these platings ltions. Neither were the increased frequencies of rn + revertants re~ i tdiated rH phage due to an accumulation of spontane( 3ontaneous revert :mally extended growth period of these phage. This possibilit determining the average burst sizes and latent period ?eriods of cells i d and control phage in one of the experiments from which th re derived. The average burst sizes of phage irradiated with o, I: X-rays were found to be respectively, 185,164, 17o an~ 155, am 70,, and ent periods were 22, 25, 24 and 35 rain. It is clear from fro these J teased multiplication nor increased time of growth of irradiated i the higher frequencies of revertants in progenies from fron these phage.

phage, ortions ortions - genes ions of n these 'e host. r at all nies of an abtigated mirradiLble I I I 5oo kR onding neither recount

(c)

Control to test for selection in favour of rii + revertants Only a small part of the radiation-induced increase increa in rii + frequency can be accounted ounted for by selection in favour of already-existin~g r l i + r e v e r t a n t s . W h e n r l i +÷ revertant 'ertants were added to the parental phage stock so as to t( raise their initial frequency tuency by 2 or 3 orders of magnitude ("reconstruction experimen~ )eriments"), irradiation of the stock increased reased the frequency of revertants in progeny phage phag( by a factor of only about ation of parental phage to which 1.5 (Table IV). The corresponding factor after irradiatior TABLE IV TESTS FOR SELECTION IN FAVOUR JR OF REVERTANTS (rli + PHAGE) IN IRRADIATED PHAGE SUSPENSION. )NS~ DURING EXPERIMENTS The p r o c e d u r e w a s t h e s a m e asS for t h e e x p e r i m e n t s of T a b l e s I I a n d I l I w i t h t he e x c e p t i o n s t ha h ait r i i + r e v e r t a n t s were a d d e d to tthe h e p a r e n t a l p h a g e s t o c k in t h e " r e c o n s t r u c t i o n e x p e r i m e n t s " , a n d t o n l y one dose of X - r a y s w a s used, 25o kR. The figures a re b a s e d on 5 r e p l i c a t e e x p e r i m e n ttss. T e s t s A a n d B differ in t h e n u m b e r of r e v e r t a n t s a d d e d to t h e p a r e n t a l p h a g e in t h e reconrecon struction experiments; the sim i l a r r e s u l t s in t h e t w o t e s t s i n d i c a t e t h a t s e l e c t i on in f a v o u r of cnilar o r e v e r t a n t s in i r r a d i a t e d p h a g e su~, s u s p e n s i o n s d u r i n g the e x p e r i m e n t s is i n d e p e n d e n t of t h e i r absolute absolute f r e q u e n c y . S u r v i v a l following i r r a d i a t i o n was a p p r o x i m a t e l y 0.5%.

Test

K i n d of experiment

~tant'freq. × io s Revertant [req. × Io 8 in pregeny phage Revertant" in parental wental phage Unirrad. Irrad. (250 kR) Irrad./Unirrad.

A

Standard Reconstruction

4.37 124

5-23 ± o.331 131 ~ 8.o8

45.6 i 1.69 239 ~ 27.6

8.78 ~ 0.878 1.82 ± o.321

B

Standard Reconstruction

3.25 137o

5.Ol ~ 0.363 1198 ± 69.7

47 .0 ± 4.44 1569 ~_ 218

9.54 ± 1.59 1.34 ~ 0.264

Mutation Res., 3 (1966) 365-373 365-37~

ted ( " s t a n d a r d e x p e r i m e n t s " ) was frequency due to the r a d i a t i o n in tl y c h a n g e d b y a i o - f o l d difference ntal phage, as between tests A an. f a v o u r of rn + phage in i r r a d i a t e d ese phage is reduced.

tely q. uction ~ber of is thus ~nsions

t h a t n m t a t i o n s can be i n d u c e d in age "1"4 S were exposure of e x t r a c e l l u l a r particles of the p h a g e to X N - r a y s . T1 ls. The :cted after m u l t i p l i c a t i o n of the i r r a d i a t e d phage in u1 n i r r a d i a a d i a t i o n on r virus lts t h u s confirm a m u t a g e n i c effect of ionizing rradi~ e t a b o l i c acti :icles, i.e. on genetic m a t e r i a l isolated from a n y m metal phage A m u l t i h i t m e c h a n i s m for the m u t a g e n i c action of X - r a y s on progeLdicated b y the dose response curve for the frequenc t u e n c y of rll + r the infrom i r r a d i a t e d ru phage (Fig. I). A t r i v i a l a l t e r n a t i v e expl , ertant ~ction in favo ,sing slope of this curve, in t e r m s of increasing selectio much )lies selec with increasing dose, can be ruled out since it im[ ~). The )eriments Aer t h a n those o b s e r v e d in the " r e c o n s t r u c t i o n ex F :action ~roduced ,'e indicates, r a t h e r , t h a t rn + reverse m u t a t i o n s were p ~vo or more r a d i a t i o n effects. :x-rays on e x t r a c e l t u l a r pllage ~hage I t m a y be asked w h e t h e r the m u t a g e n i c action of X ~articles, or indirectly, b y the Lediated directly, b y ionizations within the p h a g e pat :nslon medium. Tests for an inof m u t a g e n i c i n t e r m e d i a t e s in the suspenskn p r o dtuction uct ct m u t a g e n i c effect of X r a y s on p h a g e s u s p e n d e d in ir n u t r i e n t b r o t h have given direct + rr,e v e r t a n t s from u n i r r a d i a t e d ~tive results: ( z ) no increase in the f r e q u e n c y or r . negat u ~hage was d e t e c t e d in e x p e r i m e n t s in which n i r r a d i a t e d phage were i n c u b a t e d 7II 12 il the n u m b e r s of r~ + reverin i r:rra d i a t e d n u t r i e n t b r o t h solution; (2) no r e d u c t i o n in tl c o n c e n t r a t i o n of n u t r i e n t r a n t :s s i n d u c e d in rn p h a g e b y X - r a y s was d e t e c t e d when the b r o t :h h in the suspension m e d i u m was increased 5-fold. These results indicate t h a t }ension m e d i u m a n d t h a t s l . wtde n m t a g e n i c p r o d u c t s do not a c c u m u l a t e in the susper stable lived free radicals from thee b r e a k d o w n of w a t e r are p r o b a b l y not inw)lwxt in the m u t a g e n i c process. ich give rise to the i n d u c e d n m t a t i o n s a p p e a r to become W h i l e the lesions which fixed in the genetic m a t e r i aall of the virus at the time of irradiation, the m u t a t i o n s mtil s u b s e q u e n t replication of the genetic m a t e r i a l in host t h e m s e l v e s m a y not occur until race of the i n d u c e d m u t a t i o n s is suggested b y the a p p e a r cells. A d e l a y in the occurrence in m o t t l e d plaques containing b o t h r a n d w i l d - t y p e phage ance of i n d u c e d r m u t a n t s in particles (Table I). A n a l t e r n a t i v e e x p l a n a t i o n for this observation, t h a t m u t a t ii oo nnss tile a n d of the p h a g e D N A a n d b o t h s t r a n d s replicate in the are i n d u c e d in only one stra: ikely b y evidence from UV i n a c t i v a t i o n of gene function, host cells, is r e n d e r e d less likel, t h a t the genetic i n f o r m a t i o n1 of T 4 is t r a n s m i t t e d to p r o g e n y p h a g e b y only one s t r a n d of the phage D N A (ref. I4). .uced m u t a t i o n s does not a p p e a r to d e p e n d on m u l t i p31icitv licity The occurrence of indue r_ ~eactivation in infected hostt cells. I n d u c e d r m u t a n t s were d e t e c t e d b y p l a t i n g singlyinfected cells in which there,. was no o p p o r t u n i t y for m u l t i p l i c i t y r e a c t i v a t i o n to occur. Mutation Res., 3 ( I 9 6 6 ) 365 -373

gXTRACELLULAR BACTERIOPHAGE T i r r a d i a t i o n of e x t r a c e l l u l a r p h a g e

sidered

f t h e f i d e l i t y of t h e g e n e r e p l i c a t i ,

~y n o n -

NA.

COMBE for his h e l p f u l s u g g e s t i o n s kUNDERS for h e r a b l e a s s i s t a n c e in r

io II I2 I3

14

, these hem.

ARDASHNIKOV, S. N., V. N. SOYFER AND D. M. GOLDFARB, lInduction , s in the extrace]]ular phage T2 by 7-irradiation, Biochem. Biophys. Res. R~ Commu~ ~55-459. BAUTZ-FREESE, E., AND E. FREESE, Induction of reverse mutations m a tivation of nitrous acid-treated phage T4, Virology, 13 (I96I) 19-3o. FREESE, E., Induced and spontaneous mutations in bacteriop~hage, in XA :E (Ed.), Methodology in Basic Genetics, Holden Day, San Francisco, 1963. 1 Co!d '~ GOWEN, J. W., Mutation in Drosophila, bacteria, and viruses, viru Syrup. Quant. Biol., 9 (1941) 187-193. KAPLAN, R. W . , U. WINKLER AND H. WOLF-ELMAUI~R,Induction Induct and re, utations by irradiation of the extracellular x-phage of Serratia, Nature, Naturt 186 (196 ltraviolet irrad ~cellular KRIEG, D. R., Induced reversion of T 4 rlI mutants by ultrav phage, V{rology, 9 (1959) 215-227. LEA, D. E., Actions of Radiations on Living Cells, Cambridge University l n, 1954. tationsMUNDRY, K. W., Zur Frage des Einflusses yon Rbntgen- und UV-Strahl ~arate, Z. in& rate des Tobakmosaikvirus nach Bestrahlung reiner Pr~ipara mungs-, Vererbungslehre, 88 (I957) 115-127. STEINBERG, C. M., AND R. S. EDGAR, A critical test of a current theory of genetic recombination in bacteriophage, Genetics, 47 (1962) 187-2o8. STENT, G. S., Mating in the reproduction of bacterial virus~ viruses, Advan. Virus Res., 5 (1958) 95-149. STEVENS, W. L., Accuracy of mutation rates, J. Genet., 42 (I94I) (19 3Ol-3O 7. induction of mutations by ionizing VAN DER ENT, Cr. M., JOH. BLOK AND E. M. LINCKENS, The im radiation in bacteriophage ~bXI74 and in its purified DNA, Mutation /k Res., 2 (1965) 197-2o4. WATSON, J. D., The properties of X-ray inactivated bacteri bacteriophage, II. Inactivation by indire{ direct effects, J. Bacleriol., 63 (1952) 473-485 . evidence that only one of the two WOMACK, F,, AND N. A. BARRICELL[, Radiation-genetic evil DNA strands injected by phage T 4 transmits the genetic inforJ information to the progeny, Virology, 27 (1965) 6oo-613.

Mutation Res., 3 (1966) 365-373