Sensitivity of phage precursor nucleic acid, synthesized in the presence of chloramphenicol, to ultraviolet irradiation

VIROLOGY

6, 55-80 (1958)

Sensitivity of Phage Precursor Nucleic in the Presence of Chloramphenicol, Irradiation’*2 JUN-ICHI Department

of Genetics,

Carnegie

Acid, Synthesized to Ultraviolet

TOMIZAWA~

Institution of Washington, New York

Accepted March

Cold Spring

Harbor,

$1, 1958

Infected bacteria, in which phage precursor nucleic acid (DNA) has been allowed to accumulate in the presence of chloramphenicol, can be irradiated with multiple phage-lethal doses of ultraviolet light (UV) without preventing formation of phage particles after subsequent removal of chloramphenicol. Considerable numbers of such particles prove to be noninfective. With a small dose of UV, phage particles and deoxyribonucleic acid (DNA) are produced more or less normally after resumption of protein synthesis, but more than half of the particles lack infectivity. The number of noninfective particles produced is roughly proportional to the amount of precursor DNA present at the time of irradiation. Irradiation of bacteria before infection does not cause the production of noninfective particles. Large doses of irradiation permit synthesis of protein and maturation of phage particles with little concomitant DNA synthesis, after removal of chloramphenirol. Ahout 99% of the particles produced under these conditions are noninfective. With such cross reactivation, and multiplicity reacpreparations, photoreactivation, tivation can be demonstrated with an efficiency appropriate t#o the dose of UV actually used. These results mean that phage precursor DNA formed in the presence of chloramphenicol presents a characteristic UV-sensitive target equivalent in all respects to that contained in finished phage particles. Since this target is presumed to be the phage chromosome, its formation in the presence of chloramphenicol seems to show that multiplying phage chromosomes are composed of DNA to the exclusion of all other known phage constituents. Irradiation of such chromosomes under the conditions described produces persistent 1 Supported in part by a grant (C2158) from the National Cancer Institute, National Institutes of Health, United States Public Health Service. 2 Isotopes were supplied by the Oak Ridge National Laboratory on allocation from the Atomic Energy Commission. 3 On leave from the Department of Biochemistry, National Institute of Health, Tokyo, Japan. 55

56

TOMIZAWA

lesions which do not prevent the subsequent formation of normal chromosomes; the lesions themselves are neither repaired nor replicated. Other experiments show that DNA synthesized at different times is incorporated into single phage particles and therefore that the phage precursor DNA destined for a given particle does not consist of a unitary structure finished in advance. Moreover, both irradiated and unirradiated DNA is found in both live and dead particles after maturation. Preliminary analysis of the manner of mixing shows that the bulk of the phage precursor DNA, like that of parental origin, consists of roughly equal parts of DNA that is (in a formal sense) resistant or sensitive, respectively, to UV damage. INTRODUCTION

Doermann (1953) found that phage genetic material multiplies in a noninfective “vegetative” form inside infected bacteria. An important role of DNA in the initiation of phage growth was suggested when Hershey and Chase (1952) showed that most of the phage DNA, but little phage protein, enters the cells at the time of infection. These results, with other supporting evidence, especially the high efficiency of transfer of parental DNA to the progeny (Kozloff, 1953; Hershey, 1957) and the instability of phage genetic material against decay of incorporated radiophosphorus (Stent, 1953; Stahl, 1956), suggest that’ the genetic material of phage particles is composed of DNA. Experiments with inhibitors showed that protein synthesis is necessary for the initiation of DNA synthesis in infected bacteria but is not necessary for its continuation (Burton, 1955; Melechen, 1955; Tomizawa and Sunakawa, 1956). Chloramphenicol, which inhibits synthesis of known phage precursor proteins (Hershey, 1957) proved especially useful for the separation of protein and DNA synthesis during phage growth. Experiments with this substance revealed that the synthesis of phage precursor DNA is independent of synthesis of other phage precursors and of protein synthesis in general (Hershey and Melechen, 1957; Hershey, 1957). The facts outlined above generate the hypothesis that genetic replication is synonymous with DNA synthesis and independent of other aspects of phage growth. A test of this hypothesis is made possible by evidence that UV produces its characteristic effects on phage particles by damaging their genetic material (Luria, 1947; Doermann et aZ., 1955; Krieg, 1957). The test takes the following form. Bacteria are infected with phage, and chloramphenicol is added 9 minutes later to stop protein synthesis. Phage precursor DNA is allowed to accumulate in the presence of the inhibitor, which is then removed. The cells are promptly irradiated

SESSITIVITY

OF

DNA

TO

UV

IRRBDISTION

57

with several phage-lethal doses of ultraviolet light and warmed to permit resumption of protein synthesis. At appropriate times thereafter, samples of the culture are lysed and phnge particles are isolated. These are enumerated in terms of Dn’A content and killing power for phagesensitive bacteria. These two measurements agree. In contrast, enumeration by plaque count shows that many of the particles are noninfective. By examining the character of the defects, their equivalence to genetic damages of previously described kinds can be recognized. By appropriate labeling with radiophosphorus, a correlation between the numbers of noninfective particles and t,he amount of previously irradiated DNA incorporated into them can be established. Noninfective phage particles containing radiation-damaged DSA can be recognized by their ability to kill bacteria and by their susceptibility to photoreactivation, multiplicity reactivation, and marker rescue (cross reactivation). In addition to these methods, we make use of a “transfer method” based on the following principle. If bacteria are infected with live radioactive phage particles, radiophosphorus is transferred to the progeny particles. The efficiency of transfer is the same in single and multiple infections. On the other hand, if bacteria are infected singly with UV-irradiated radioactive phage particles, no progeny is produced and of course no transfer is observed. However, if bacteria are mixedly infected with irradiated radioactive phage and live nonradioactive phage, radiophosphorus does appear in the progeny (Kozlofi, 1953). Under suitable conditions, the efficiency of transfer of radiophosphorus from irradiated phage particles to the progeny formed by mixed infection with live phage is the same as the efficiency of transfer of radiophosphorus from live particles to progeny. By comparing the efficiency of transfer of radiophosphorus from parents to progeny in single and multiple infection, the distribution of radiophosphorus between infective and noninfective particles can be determined (Hershey and Burgi, 1956). When combined with selective labeling of phage precursor DNA, irradiated or not, and applied to the subsequently formed phage particles, the transfer method thus yields information quite different from the other methods listed above. The application of this method shows, in short, that phage precursor DNA resembles the DNA in phage particles in still another way. Both consist of a fraction that, after irradiation, transfers lethal damages into phage particles that receive it, and of a second fraction that does not.

58

TOMIZAWA

MATERIALS

AND

METHODS

Phage T2H and h’scherichia coli strain H grown in a glucose-ammonium medium were used in experiments employing chloramphenicol. Cultural conditions were similar to those described by Hershey and Melechen (1957) unless otherwise stated. Before irradiation, the cultures were centrifuged and resuspended in fresh culture medium at 5 X lOa bacteria per milliliter. Twenty-fivemilliliter portions were exposed with rotation at room temperature in 14 cm petri dishes 53 cm distant from a 15-watt germicidal lamp (incident intensity 17 erg rnrnM2set-I, mostly 2537 A). Under these conditions there was appreciable screening by the bacteria, which was measured by comparing the sensitivity to irradiation of intracellular mature phage with extracellular phage as follows. A culture was infected with phage, sedimented, resuspended in fresh medium, allowed to develop for 21 minutes, and irradiated. Samples were diluted into cyanide broth after 0, 15, and 30 seconds’ irradiation, corresponding to 0, 5.7, and 12 “hits” to unshielded phage particles under the same conditions, and titrated after cellular lysis was complete. The effective dose to intracellular phage particles was 0, 3.8 and 7.9 hits, respectively, or 66 % of the dose to unshielded particles. This result was checked by measuring the efficiency of multiplicity reactivat’ion (see below) of phage particles irradiated intracellularly, and agrees qualitatively with previous checks in which the efficiency of photoreactivation was measured (Tomizawa and Horiuchi, unpublished). Dosage of UV to infected cells given in this paper is therefore expressed as multiples of the e-l survival dose (hits) to unshielded phage times 0.66. Synthesis of nucleic acid and its incorporation into phage particles was measured by trichloroacetic acid precipitation and Schmidt-Thannhauser fractionation of materials labeled with radiophosphorus, as described by Hershey and Melechen (1957). The isolation of phage particles from infected cultures without addition of carrier phage was effected as follows. A sample of the culture was diluted fivefold in a lysing medium (Hershey, 1957) containing indole to prevent inactivation of phage by bacterial debris. After warming 30 minutes at 36” and keeping overnight in a refrigerator, the lysate was clarified by low-speed centrifugation, and the phage were isolated by further centrifugations as usual. The DNA content of the isolated phage was measured radiochemically in

SENSITIVITY

OF

DNA

TO

UV

IRRADIATION

5“9

phage equivalent units of phosphorus (2 X lo-” pg per phage) or as optical density units (7 X lo-l2 cm2 per phage) at 260 rnp (Luria et al., 1951; Hershey, 1957). The number of bacteria-killing particles in phage preparations was measured in the usual way by reduction in colony count of a standard bacterial suspension, assuming a Poisson distribution of phsge particles over bacteria and perfect adsorption (Adams, 1950). Photoreactivation was brought about by exposing infected cells at’ 36” in broth containing 0.005 M KCK to two 15-watt General Electric blacklight lamps at a distance of 8 cm. Multiplicity reactivation and cross reactivation will be described in connection with the appropriate experiments below. The transfer method for measuring the distribution of radiophosphorus between infective and noninfective phage particles in a mixture consists of the following. Bacteria are grown to 5 X IO* cells per milliliter in glucose-ammonium medium, centrifuged, and resuspended in fresh medium at 2.5 X log cells per milliliter. Aliquots of this suspension are infected either singly (0.13 phage particles or less, infective or not, per bacterium) with the purified test phage, or mixedly with the test phage and infective, nonradioactive particles (0.5 and 4.5 per bacterium, respectively). After warming 5 minutes for adsorption of phage to bacteria, the mixtures are centrifuged to remove unadsorbed radioactivity (5% to 15%) and resuspended (time 0) in warm aerated culture medium at 5 X lo8 cells per milliliter. To achieve equal transfer from infective phage particles in single and multiple infection, the following device is resorted to (Hershey and Burgi, 1.956). To the singly infected culture described above is added, at the seventh minute, 1.5 volumes of a bacterial suspension that was multiply infected with nonradioactive phage particles according to the same time schedule as the test culture. In this way one obtains similar conditions with respect to reinfection and lysis-inhibition in all the test cultures. Samples of the cultures are then taken at appropriate intervals, lysed, fractionated, and assayed for radioactivity in phage particles as described by Hershey and Melechen (1957). The results of control measurements with irradiated and unirradiated phage particles are shown in Fig. 1. From such graphs the per cent transfer for a given preparation is computed as the difference between the interpolated 40-minute value and the average of blanks prepared by lysis at 8 to 10 minutes. (Lysis at times

60

TOMIZAWA 1

2

/

I

I

I -

50z

MIxed

infection’

50-

I

I

Smgls

mfsction

I -p

0

’

;:

f7@-

i= 40-

/-

0

40Mixed

mfection“1

z g 4 2 z

30-

-

30-

20-

-

20-

0 g

Single

IO-

Y

infection ,oap-

3%

‘O

&$

s L

2 P

I

Oo

IO

I

I

I

20

30

40

TIME

I

50

Oo

OF SAMPLING

IO

I

I

I

20

30

40

50

(min.)

1. Transfer of radiophosphorus from irradiated (left) and unirradiated (right) phage to progeny in single and mixed infections. The irradiated phage received sixteen hits of UV before infection. The results illustrate the principle of the transfer method for measuring distribution of radiophosphorus between infective and noninfective phage particles (see Materials and Methods in text). FIG.

later than 45 minutes is avoided because of a slow rise in the apparent transfer from irradiated phage particles in single infection after that time.) For all phage preparations, the transfer in mixed infection computed as above is about 40%. In single infection the transfer is 40% for infective particles and none for noninfective. For a test phage giving 41% transfer in mixed infection and 18 % transfer in single infection the radioactivity in infective phage particles is said to be 18/41 or 44 f approximately 5% of the total radioactivity in phage particles. RESULTS

Production of Noninfective Particles in Irradiated Bacteria The following experiments illustrate our principal finding, that irradiation of infected bacteria containing large amounts of phage precursor DNA causes them afterwards to produce noninfective phage particles. The demonstration is simplified by the use of chloramphenicol to prevent formation of phage particles before irradiation. Bacteria were grown for three generations (to 5 X IO8 cells per milliliter) in medium containing radiophosphorus and infected with five phage particles per bacterium at time zero. At 9 minutes, 20 pg per

SENSITIVITY

OF

DNA

TO

UV

61

IRRADIATION

milliliter of chloramphenicol was added. At 40 minutes, the cells were sedimented and resuspended in fresh medium containing radiophosphorus but lacking chloramphenicol. One portion of the culture, now at room temperature, was irradiated for 15 seconds (4 hits). A second portion was not irradiated. This schedule is described as “irradiation at 40 minutes” since cooling and anaerobiosis in the centrifuge effectively stop DNA synthesis. To both portions indole was added to final concentration 0.01 % (to minimize losses of phnge progeny by adsorption to bacteria), the cultures were warmed, and aeration resumed at the 55th minute. At this time more than 90% of the initially infected bacteria, irradiat,ed or not, could form plaques on nutrient-agar plates. During the ensuing period samples of the culture were lysed at intervals for isolation of phage particles and throughout the experiment other samples mere analyzed for total labeled DNA. The results, expressed per initially infected bacterium, are presented in Fig. 2, in which the following may be noted. I 400-

I

0 0

NoUV

A

15sec.UV

A

MINUTES FIG. 2. Production

I

AFTER

I

I

INFECTION

of noninfective phage particles after irradiation of infected bacteria containing phsge precursor DNA. Chloramphenicol (CM) present from the 9th to 55th minute. The unit of phosphorus is 2 X lo-” kg.

62

TOMIZAWA

30

60

MINUTES FIG. 3. Experiment of irradiation.

identical

AFTER

INFECTION

to that shown in Fig. 2 except

for the larger

dose

At the time of irradiation, the culture contained 140 phage equivalent units of DNA per bacterium, of which perhaps 25 units represent impurities labeled before infection. Most of the remainder is phage precursor. After irradiation DNA synthesis continues and phage particles start to accumulate, neither process being greatly affected by the irradiation. Judging by the plaque counts, however, more than half the phage particles produced by the irradiated culture are noninfective. An experiment identical to the above, except for a larger dose of irradiation (40 seconds = 11 hits), is presented in Fig. 3. The results lead to the same qualitative conclusions but differ from those of the preceding experiment in certain respects. First, only 10 % of the bacteria, after irradiation, yield infective phage particles, as judged by plating before lysis in nutrient agar. Second, the irradiated culture fails to resume Dh’A synthesis at an appreciable rate after irradiation. Third, phage particles are nevertheless produced at about 30 % of the rate seen in the unirradiated culture. Fourth, virtually no infective phage particles

SENSITIVITY

OF

DNA

TABLE 0F

ENUMERATION

NONINFECTIVE

-.

Plaque titer (per ml)

(a)

0 15

2.3 x 10” 7.2 X 10”’

0 40

3.0 x 3.4 x

10” 108

Bacteria-killing titer (b) (Per ml)

UV

IRRADIATION

63

1 PHASE

IRRADIATED uv dose (set)

TO

PARTICLES

FORMED

IN

BACTERIA* Phosphorus content (c) (units/ml)

Experiment 2.5 2.3 X 10” 1.8 1.7 X 10” Experiment 3.6 3.2 3.0 x 10’0

Optical density ratio (d) (units/ml)

oj Fig. 2 X 10” 2.7 X 10” x 10” 1.8 X IO” of Fig. 3 X 10” 3.6 X 10” X IO”’ 3.4 x 10’0

Plaques particle

per (e)

approx.

1 .o 0.4

approx.

1.0 0.01

* Phage particles are isolated as described in text and enumerated in four ways (a, b, c, d). Phosphorus (c) is assayed radiochemically and expressed in phage equivalent units, 2 X IO-” pg per phage. Optical density (d) is measured at 260 rnp and expressed in multiples of the optical cross section per phage particle, 7 X lo-l2 cm*. The ratio (e) between titer (a) and titer (b), (c), or (d) measures the fraction of t,he particles capable of forming plaques.

are produced. Under these conditions, some bacteria apparently produce noninfective phage particles exclusively. Additional evidence is required to prove that the radiochemical results presented above really measure production of noninfective phage particles. To obtain such evidence, samples of phage corresponding to the 150-minute time of Fig. 2 and t’o the 180-minute time of Fig. 3 were isolated without added carrier phage and analyzed further with results shown in Table 1. These results demonstrate clearly that phage produced in unirradiated cultures show the normal relation between infective titer, phosphorus content, optical density, and bacteria-killing titer, whereas phage produced in otherwise identical cultures exposed to CV fail to form plaques but are normal with respect to DNA content and ability to attach to and kill bacteria. The two preparations described in Table 1 contain, respectively, 60% and 99 % of noninfective particles, as expected from the corresponding kinetic measurements (Figs. 2 and 3). The phage preparations analyzed in Table 1 provided a valuable opportunity to check the precision of the transfer method for measuring the distribution of radioactivity between infective and noninfective phage particles. The preparation known to contain about 60% noninfective phage particles, tested by this method, transferred 40% of its radioactivity in mixed infection and 16% in single infection. The preparation known to contain 99 % noninfective particles likewise transferred 40% of its radioactivity in mixed infection, but virtually

64

TOMIZAWA

none in single infection. These results nicely verify the meaning of the transfer measurements and also show that the noninfective phage particles inject their DNA with normal efficiency into the bacteria to which they attach. Direct versus Indirect Particles

Action

of UV on Subsequently Formed Phage

The conclusions we wish to draw from the present experiments depend very much on the hypothesis that irradiation produces the effects described by direct action on phage precursor DNA. One support for this idea derives from the fact that unirradiated bacteria infected with a mixture of irradiated and Lmirradiated phage particles produce a phage progeny containing a few noninfective particles in which irradiated DIVA is concentrated (Hershey and Burgi, 1956). Further support is gained from the following experiments, in which we vary the time of irradiation of infected bacteria. In a first experiment we ask whether irradiation of the nucleotide precursors of DNA can cause lesions in phage particles into which the nucleotides are eventually incorporated. Unfortunately there is no straightforward test of this possibility and we resort to the following expedient. Bacteria were labeled with radiophosphorus and were subsequently allowed to grow for one generaCon in nonradioactive culture medium to rid them of considerable amounts of phage precursor phosphorus other t.han bacterial DNA (Hershey and Melechen, 1957). The bacteria were irradiated for 1 minute and then infected with 5 phage particles per bacterium. Forty minutes after infection the cells were lysed and a yield of labeled phage particles isolated. These particles were tested for the presence of radiophosphorus in noninfective particles by the transfer method. None was found. Bacteria uniformly labeled by continuous feeding of radiophosphorus until irradiation or labeled mainly in DNA precursors by short feeding for 3 minutes just before irradiation and infection gave the same results. The sensitivity of these tests depends, in part, on whether nucleotides are transferred intact from bacterial DNA to phage DNA, a possibility for which there is some support from isotope competition tests (Hershey et al., 1954). Even if this is not so, the test should not be too insensitive, since the labeled phage particles examined presumably obtained about one-third of their carbon from irradiated sources. At any rate it is clear that incorporation of irradiated

SENSITIVITY

OF

IlKA

TO

UV

IItIL~I)I.~TION

A5

bacterial constituents into phnge particles does not produce large numbers of noninfect,ive phage particles. In a eecond type of experiment* we ask n-het.her ncninfec~tive phage part~iclcs can be produced in consequence of some unspec+icd met’aholic aftereffect of irradiat,ion of infwtcd bac~teria. To test t,his possibility t)he experiment described in Fig. 2 was repeated with irrndintjion at various times, and the number of noninfe:cativc part,ic*les formed up to the 150th minute estimated (not very accurately) by subtracting infect.ive particles per bacterium from u&s of DK.4-I’ in phage per bacterium. There numbers were: for irradiat,ion at, 9 minut.es, none; at 15 minutes, 20; at 80 minut’es, 80; and at 10 minutjes (lcig. 2), 130. Thus t’he number of noninfective particles produced is roughly proportional to the amountJ of phage precursor DKA irradiated, excluding any indirect’ metabolic effects not mediat,ed by the phage precwrror DIiA &elf, and showing that radiat’ion damages do not, themselves replicate. The result for irradiation at 1) minutes again excludes any efficient production of defective phnge by irradiation of intrnbac,t,eri:ll precursors of I)XL4. l+?nally, V-Cshow by the following experimwt that irradiat ioll of phagc prwuwor DNA at !I minut,es after infc&on does prodwe a few noninfective phage particles containing mcxh, of’ fh,c phoxphorus oj the irradiated L)NA. The experiment, NXS pcrformrd in likrl mannor t’o t,hat shown in E’ig. 2 except t,hat radiophosphorus was derived solely from the parent.al phage, t.he culture was irradiated for 10 seconds at I) minutes after infection and centrifuged to rcmovc c:hlorarnphelli~ol at 50 minutes. (Ill ot,hcr experimentas it was fomld that irradiation at, 9 minutes has relatively litt,lc effect on subsequent rventjs as caornparcd 1vit.h irradiation at later times. Under t.he conditions used here the start, of I>SA synthesis is post,poned for about, 10 tninutes by the irrudiatioll, tShenproceeds at a normal rate.) I’hage particles isolated from a lysate prepared at 150 minutes showed a normal corrcspondenw bctww plaque count and optical density, illdiwting that most of t,hem wrc infective. =l11:dysis by the transfer method showed, however, that about half of the irradiat,ed DNA derived from t.he parental phngt: was cont’ained in noninfective particles. Our principal conclusion is that noninfective phage particles produced by irradiating infected bacteria are noninfective because they receive damaged structures already present in D-VA at the time of irradiation. It seems extremely unlikely that, proportionality between amount’ of precursor DKA irrudiat,ed and numbers of noninfective particles pro-

66

TOMIZAWA

duced could prevail if phage precursor DNA synthesized in the presence of chloramphenicol was subject to breakdown and resynthesis en route to phage particles. However, more rigorous evidence for this conclusion derives from a different kind of experiment reported below. l@iciency of Incorporation Particles

of Irradiated Phage Precursor DNA into Phage

The experiment described in Fig. 3 clearly suggests that DNL4 synthesized in the presence of chloramphenicol, then irradiated, is incorporated into phage particles after subsequent removal of chloramphenicol. In the following experiments we label the irradiated DNA more specifically in order to measure the efficiency of its incorporation into phage particles. The first experiment is identical to that described in Fig. 2 except that radiophosphorus was added to the culture at 7 minutes, and its assimilation was stopped 8 minutes later by adding sufficient neutral ammonium dihydrogen phosphate solution to increase the concentration of phosphate in the culture medium loo-fold. Results are presented in Fig. 4.

h

5C

3opLJgJ=q ‘in phase particles

d2

fad

PrrrrntI

-0

30

I

-

60

MINUTES

90

AFTER

120

150

180

INFECTION

FIG. 4. Efficiency of incorporation of irradiated DNA into phage particles. Radiophosphorus fed from the 7th to 15th minute. Chloramphenicol present from the 9th to 55th minute.

SENSITIVITY

OF

DNA

TO

UV

IRRADIATION

67

The results given in Fig. 4 refer to labeled DNA, computed in phage equivalent units at the specific activity of the radiophosphorus fed. Fig. 2 should be consulted for a comparison with the amounts of DNA actually present. Figure 4 shows that 27 units of DNA were labeled during the chloramphenicol period, and only 4 units more were labeled after the removal of chloramphenicol and irradiation. Of this, 23 units were recovered in phage particles, representing an efficiency of incorporat,ion of 70% (actually more, since not all phage particles produced are recovered). This is identical to the efficiency of incorporation of unirradiated DNA in the control culture. The yields of infective phage particles at 150 minutes were 60 and 190 per bacterium for the irradiatedandunirradiated cultures, respectively. Transfer measurements of the distribution of radiophosphorus between infective and noninfective particles isolated from the irradiated culture yielded the result 40:60, showing that phosphorus derived mostly from the irradiated DNA is distributed between infective and noninfective particles in t,he same manner as previously determined for total phosphorus. In a second experiment, analogous to that described in Fig. 3, radiophosphorus was fed from the 10th to the 25th minute, and irradiated for 40 seconds at “the 40th minute”. DNA labeled during the chloramphenicol period and recovered after the subsequent centrifugation to remove chloramphenicol measured 32 units per bacterium, and the amount of labeled DNA did not increase after irradiation. Of the labeled DNA available, 19 units per bacterium were recovered in phage particles following lysis at 180 minutes, representing an efficiency of incorporation of 59 %. The yield of phage particles was 60 per bacterium, of which 98 % were noninfective. Other experiments were performed in the same way except that labeled parental phage particles served as the source of radiophosphorus. Here too, efficient incorporation of the labeled, irradiated DNA into phage particles was observed. We conclude that the phosphorus of DNA synthesized in the presence of chloramphenicol, irradiated or not, is efficiently incorporated into phage particles formed after the subsequent removal of chloramphenicol. Properties of the Noninjective Particles We have already described experiments showing that noninfective particles produced in consequence of irradiation of their DNA precursors

68

TOMIZAWA

contain the normal amount of DXA, adsorb to bacteria specifically, kill them, and inject their DNA with normal efficiency. In the following experiments we apply the tests of multiplicity reactivation, cross reactivation, and photoreactivation to gain further insight into the nature of the lethal damages. Specifically, we wish to know whether the noninfecbive particles are noninfectivc kernuse they cont,ain radiation dnma,gex or because they received DKA t,hat was already imperfect, at the time of irradiation, the effect of the latter being merely to prevent the completion of yome final step in DNA synt,hesis normally occurring after removal of chloramphelGo1. The preparation of noninfect,ive particles employed in the following experiments, already described in Table 1, was produced by irradiation for 40 seconds (11 hits) of phage precursor DKA synthesized in the presence of c~hlcPrlLmphellico1.It contained only 1 % of infective particles, and the total number of particles was accurately known from several independent cstimat~es. This prep:trat,ion is referred to as the test phage below. 111 the first experirnent, we compare the efficiency of multiplicity reactivation observed with the test phage to that observed wit,h “culihrated” phage particles irradiated with known doses of ultraviolet light and so measure an effect,ive “hit nurnber” of the presumed radiat,ion damages in the test phage. E’or this purpose exponentially growing broth cultures of IX’. coli strain S were transferred to buffered saline solution at 2.5 X 10y cells per milliliter. Aliquots of this suspension were mixed with equal volumes of test phage or calibrated phage of identical concenbration calculated to give phage: bacterium ratios of 2 to 3.5 in different experiments. After 10 minutes at So, the number of product,ivc bacteria in each mixture was determined by plaque titrations. At the same time a diluted sample of cwh tube was assayed for unadsorbed, infective particles after centrifugation. Ratios of produc*t,ive bacteria to t,otal numbers of multiply infected bacteria were then computed (Curia and Dulbecco, 1949), and the results plotted as in Fig. 5. By placing the result yielded by the test phage on the curve yielded by the calibrated phage an effective hit number of 10.5 f 1.5 is obtained. This agrees with the actual dose of 40 sec*onds (11 hits) that’ the precursors of t,hc test phage had received and leads to the conclusion that, the particles are noninfective owing to the same kinds of damage that, art’ produced by irradiat,ing phage particalcs themselves.

SENSITIVITY

;

O.I-0

OF

DNA

TO

UV

IitiL4I~IATION

I

I

I

I

I

I

2

4

6

8

IO

12

DOSE OF IRRADIATION

14

(hits)

FIG. 5. Multiplicity reactivation with noninfective phage particles. Efficienq of multiplicity reactivation means fraction of bact,eria, infected with two or more noninfective particles, yielding a phage progeny. The calibrat,ion curve shows results for known doses of UV given to phage particles. The test phage was produced after irradiation of its precursors nit,h 11 phage-lethal hits. The average multiplicity of infection was 4.5 in both cases. The data are eorrertrd for infcct,ive particles present in the preparations.

In a second experiment, we confirm the above result by employing the method of genetic marker rescue (Doermann et al., 1955). For t)his purpose a sample of noninfertive particles of a host-range (h) mutant, of T2 was prepared according to the schedule of Fig. 3 (lysis at 180 minutes), yielding a test phage very similar to the one employed in the preceding experiment. A “calibrated” series of irradiated phage part,icles was also prepared, this time using the h mutant stock. Both calibrated and test phages were then mixed with unirradiated wild-type particles and the mixtures were used to infect suspensions of bacteria in buffer to obtain phage: bacterium ratios of 2 infective wild-type and 0.2 noninfective h mutant particles per bacterium. The ahilit)y of the mixedly

70

TOMIZAWA

infected cells to yield h mutant offspring was then measured in two ways: by plating before lysis on mixed-indicator (B/2 + B), and on single-indicator (B/2), strains of bacteria. Both methods of plating detect h mutant particles, but with different efficiencies owing to the phenomenon of phenotypic mixing (Streisinger, 1956). The exact meaning of the counts is doubtless in either case very complicated, but the comparison of test phage and calibrated phage is unambiguous. The results for one experiment are illustrated in Fig. 6. From several such experiments an estimated number of effective hits to the test phage of 10.5 f 1.5 was obtained. This result is in good agreement with the previous measurement and with the actual dose of UV given to the precursor DNA of the test phage. It shows that phage precursor DNA

on mixed

on single

0

2

4

6

indicator

indicator

6

DOSE OF IRRADIATION

IO

12

14

(hits)

FIG. 6. Cross reactivation of the host-range marker of noninfective phage particles. Efficiency of cross reactivation means fraction of bacteria infected with hoth infective (h+) and noninfective (h) particles yielding a progeny amoqg whirh h particles are detected. The calibration curves show the results for known doses of UV given to h mutant phage particles. The filled circles refer to h mutant particles produced after irradiation of precursors with eleven phage-lethal hits.

SENSITIVITY

OF

DSA

TO

UV

IRRSDIATION

71

formed in the presence of chloramphenicol includes phage-specific DKA associated with the h locus, and possessing the same radiation sensitivity as that similarly situated in phage particles. Photoreactivation was also demonstrated with noninfective phage particles of the kind under discussion, and the efficiency of photoreactivation was roughly appropriate to the radiation dosage used to produce them. Estimates of hit number by this method proved rabher inaccurate, however, owing to the relatively large fraction of infective particles unavoidably present in the test phages. These results, again, could hardly be accounted for if phage precursor DNA synthesized in the presence of chloramphenicol had to undergo extensive reorganization before incorporation into phage particles. We conclude, therefore, that such DXA is already functionally equivalent to the DNA in phage particles. These experiments also confirm in a particularly direct manner the expectation that UV damages phage particles by producing lesions in DNA, and show that such damages are not erased with appreciable efficiency in bacteria in which phage growth is proceeding. The Possible Bipartite

Nature

of Phage Precursor

DNA

At this point our experiments present a paradox. On the one hand we have concluded that our noninfective particles are noninfective because they contain lesions in their DNA produced by irradiation of precursor DNA. On the other hand, we do not find any marked concentration of irradiated DICTA in noninfective particles or of unirradiated DNA in infective particles under conditions in which both are produced. Before pursuing this paradox we turn to a related question and take advantage of specifically labeled phage particles to determine whether DNA synthesized in the presence of chloramphenicol consists of a “UVresistant” part and a “UV-sensitive” part in the sense of the following experiment, which is due to Hershey and Burgi (1956). Phage particles specifically labeled in DNA that had been synthesized in the presence of chloramphenicol were prepared as described in Fig. 4. and preparations containing noninfective Infective preparations, particles produced by irradiation of their precursors, were both used. These were irradiated for 40 seconds (16 hits), which is sufficient to produce a maximum effect of the kind to be described. Bacteria were then infected with an average of five particles each of the specifically labeled irradiated particles and of unlabeled, normally infective particles, to

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obtain a progeny phage that contained radioactivity derived exclusively from irradiated particles and, ultimately, from DNA synthesized in the presence of chloramphenicol. This progeny was then analyzed by the transfer method to determine the distribution of radioactivity between infective and noninfective particles. The distribution was found to be about 50:50 for both preparations. An identical result was obtained start,ing wit,h phage particles (described below) containing radiophosphorus assimilated exclusively after the end of the chloramphenicol period. These results, together wit,h those of Hershey and Rurgi (1956), have the following meaning. DNA synthesized in the presence of chloramphenicol, irradiated or not, and DNA synthesized in the absence of chloramphenicol, irradiated or not, once incorporated into finished phage particles, have the following characteristics identical to the whole DXA found in phage particles prepared without these special treatments. All of them consist of a “UV-sensitive” fraction and a “UV-resistant” fraction, of which the former does and the latter does not, transmit detectable lesions into a progeny formed by mixed infection with irradiated and unirradiated phage particles. The meaning of this fact is not at all clear, but the suggestion of a functionally differentiated DNA separable into two fractions is obvious. This suggestion is being pursued. In the meantime the above experimenk demonstrate one more resemblance betweeu the DKA synthesized in the presence of chloramphenicol and the DXA in phage particles. Incorporation of DNA Phage Particles

Synthesized

after

Irradiation

into

Noninfective

The previous experiments show clearly that the phosphorus of irradiated DNA is incorporated into both noninfective and infective particles formed subsequent to irradiation, and quantitative considerations indicate that the reverse is true, that noninfecbive particles contain unirradiated DNA. The following experiments demonstrate the latter more direct’ly, and also yield some information about the nature of the mixing process. The schedule of a typical experiment, identical to that of Fig. 2 except for labeling schedule, may be described as follows. Unlabeled bacteria are infected at time zero. Chloramphenicol is added to the culture at 9 minutes and removed by centrifugation beginning at 40 minutes. After resuspension in nutrient medium without chlorsmphenicol the culture is

SENSITIVITY

OF

DNA

TO

UV

IRRADIATION

7.3

divided and one portion is irradiated. Indole and radiophosphate are now added to both portions and phage growth is allowed to resume, both cultures being analyzed at intervals for labeled DNA and labeled phage particles. In this case the results of the assays measure phage equivalent units of phosphorus assimilated after the time of irradiation. In an experiment of this type in which the dose of irradiation was 15 seconds (4 hits), the following results were obtained. In the unirradiated culture, the rate of incorporation of radiophosphorus into D?;A increased gradually, the final amount incorporated at>150 minut,es being 150 units tot,al and 120 units in phnge parhirles. In the irradiated culture t,hese amounts were 120 units and 80 units, respect’ively. As usual about half of the particles produced by the irradiated culture were noninfective. Analysis of the progeny by the transfer method showed, as expert.ed from the results of the reverse labeling experiment (Fig. 4), that the infective and noninfective particles were about equally labeled. Thus no concentration of irradiated Dn’A in noninfertive particles or of unirradiated DNA in infective particles can he detected under these conditions. A repetition of the experiment in an identical manner, but with a larger dose of UV (40 seconds = 11 hit,s), yielded the following result. In the irradiated culture only 20 units of labeled DNA accumulated up to the 180th minute, representing only IO % of the amount labeled in the unirradiated culture. (This small amount of DNA synthesis after irrudintion was undetectable in the uniform labeling experiment, shown in J’ig. 3.) The phage particles labeled after irradiation comained about 30 % of the t)otal labeled DNA in the culture, that, is, the incorporation of DNA synthesized after irradiation into phage particles was no more efficient t,han the incorporation of D?CA labeled before irradiation (Fig. 3). As before, only 1% of the phage purt,icles formed after irradiation were infective. Transfer measurements showed that, these contained only about 2% of the radioactivity present in the particles. Most, of the radiophosphorus had entered noninfect,ive particles. Again no great concentjration of unirradiated DIVA in infective particles could be detected, though the methods do not permit an exact test, of this point. These result+ evidently imply some kind of mixing of irradiated and unirradiated DNA (and of DNA synthesized at different times) in individual phage particles. Such mixing can be interpret,ed in several ways, but these can be divided into two main classes; Ilamely, mixing in which the idemity of the admixed components is preserved, or mixing

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in which identity is lost. The hypothesis of a bipartite DNA discussed in the preceding section would be an example of the first class. The following experiment shows that identity, as recognized by presence or absence of UV lesions, is preserved after mixing. The experiment consists, in effect, in transferring labeled unirradiated DNA from noninfective particles into a new generation of particles and then testing whether or not the labeled particles of the new generation are infective. The starting phage for this experiment were t,hose described above, namely, phage particles produced in a culture subjected to chloramphenicol treatment followed by irradiation for 40 seconds followed by introduction of radiophosphorus to label unirradiated DXA, virtually all of which is contained in noninfective particles of the final yield. Bacteria were infected with an average of one such labeled, noninfective particle and five nonradioactive, normally infective particles to produce a progeny containing radioactivity derived from the noninfective particles and, ultimately, from unirradiated DKA. This progeny was then analyzed by the transfer method to determine the distribution of radioactivity between infective and noninfective particles. The result showed that 90% of the transferred radiophosphorus was present in infective particles. When the above experiment was repeated with an average multiplicity of 5 each of the specifically labeled noninfective and unlabeled infective particles, 75 % to 80% of the transferred radioactivity was found in infective particles among t,he offspring. The results described above are to be contrasted with those obtained in identical experiments performed with noninfective phage particles prepared according to the schedule of Fig. 3, containing labeled irradiated DNA. At multiplicity one, such particles (in mixed infection with live particles) transfer radioactivity of which 60% ends up in infective particles; at multiplicity five, the fraction is about 50%. These results are identical to those obta.ined with phage particles containing labeled DNA irradiated in situ, and are subject to the same types of interpretation (Hershey and Burgi, 1956). Experiments of this type are being continued and will be presented in more detail elsewhere. At present we conclude only that irradiated and unirradiated DNA can be mixed in individual phage particles and sorted out again during subsequent growth, without loss of association between the lethal effects of UV and atoms of phosphorus. In the present context the chief inference is this. DNA synthesized in the presence of chloram-

SENSITIVITY

OF

DNA

TO

UV

IRRADIATION

75

phenicol must serve as a high molecular weight precursor of the DNA in phage particles, not simply as a source of nucleotides reused for DNA synthesis after removal of chloramphenicol. Thus the experiments described above provide our best formal proof for a conclusion already reached by previous experiments reported in this paper: that DNA synthesized in the presence of chloramphenicol presents a radiationsensitive target in which a specific molecular organization is already evident. LlISCUSSION

We shall now itemize and separately discuss our principal findings: 1. The phosphorus of DNA synthesized by infected bacteria in the presence of chloramphenicol is efficiently incorporated into phage particles formed after removal of chloramphenicol, as reported by Hershey and Melechen (1957). Since chloramphenicol inhibits synthesis of phage precursor protein, one separates in this way the synthesis of phage precursor DNA and its final incorporation into phage particles. Hershey and Melechen suggested that the precursor DNA formed under these conditions might be already functionally complete at the time of synthesis, but their evidence was limited to the demonstration of normal kinetics of incorporation into phage particles. 2. Irradiation of infected bacteria containing phage precursor DNA synthesized in the presence of chloramphenicol with moderate doses of ultraviolet light does not greatly suppress the subsequent formation of phage particles and does not otherwise suppress the incorporation of the preformed DNA into them. 3. Irradiation of infected bacteria containing phage precursor DNA synthesized in the presence of chloramphenicol does, however, suppress subsequent DNA synthesis. Thus one can observe the formation of phage particles with very little concomitant formation of DNA. In experiments not described in this paper we found that DNA synthesis is much less sensitive to irradiation in infected bacteria not subjected to theactionof chloramphenicol. Thechloramphenicol-induced sensitivity persists after removal of chloramphenicol and resumption of protein synthesis. We have no explanation for this secondary effect of chloramphenicol, which, however, has no obvious connection with our principal findings. 4. A considerable fraction of the phage particles formed after irradiation of their precursor DNA proves to be noninfective. The size of this

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fraction varies in a plausible manner with the amount of precursor DNA present in the cells at the time of irradiation and with the dose of irradiation to which the cells are subjected. The quantitative relationships suggest t)hat UV owes its effects t,o direct action on the precursor DNA and that the precursor DNA is about as sensitive to radiation as the DNA in phage particles. 5. The noninfective part,icles formed after irradiation of precursor DNA synthesized in the presence of chloramphenicol resemble in important respects noninfective particles produced by direct irradiation. Both attach to bacteria, inject their DNA, and kill the cells to which they attach. Both are subject to multiplicity reactivation, photoreactivat,ion, and genetic marker rescue. Moreover, the efficiency of these processes is dependent on the dose of irradiation according to the same quantitative rules whether the exposure to radiation occurs before or after incorporation of DNA into phage particles. Since DNA is the only important phuge precursor synthesized in the presence of chloramphenicol (Hershey, 1957), the radiation-sensitive target in either infected bacteria or phage particles must be DNA. Since in either case the radiation sensitivity is about the same, lethal damages of the type circumvented by multiple infection must be virtually irreparable in infected bacteria, at, least in the absence of photoreactivating light. It follows that multiplicity reactivation bypasses, but does not erase, radiation damages. 6. Phage precursor DNA synthesized in the presence of chloramphenico1 resembles the whole DNA present in phage particles in still another way, as demonstrated by the following experiments. If labeled phage particles are irradiated and bacteria are simultaneously infected with the irradiated particles and with unirradiated, unlabeled particles, a phage progeny is produced that contains labeled DNA partly in infective particles and part.ly in noninfective particles (Hershey and Burgi, 1956). We find the same result when the irradiated particles are specifically labeled in DNA synthesized in the presence of chloramphenicol. In both cases the DNA is apparently composed of a radiation-sensitive fraction and a radiation-resistant fraction, though the literal separation of these “fractions” has not yet been achieved. 7. It is evident in all our experiments that irradiated phage precursor DNA, and DNA synthesized after irradiation, are distributed almost at random between infective and noninfective particles formed after irradiation. Thus irradiated and unirradiated DNA, which means DNA

SESSITIVITY

OF

DNA

TO

UV

IHHADIlTION

77

synthesized at different times, are somehow mixed in single particles. The nature of the mixing process is unknown, but, one wn usk whether the mixing is reversible or irreversible. The following experiment shows that it is reversible. When noninfective particles labeled itt unirradinted DNA are used to produce a mixed infect,iott with infectJive, unl:d~eled p:trticles, the libheled DNA segregates almost completely from the radiatiott damages among the offspring. When the noninfcctive partkles are labeled in irradi:tt,ed DNA, however, the expeckd association hetweett label and rttdintiott damages persists. Thus one ctt,tt say that the st’ructural units by whicah DNA passes from the st,atus of phage pcrcursor synt,hcsixed it1 the presence of chlorumphetticol to the status of DSA in phage particles are not smaller than the structurnl units by which JIKA passes from parental to offspring phage. At least’ some of these units are large (S”ltettt and ,Jcrne, 19%; Levinthal, 1956; Hershey and Burgi, 195(i), and may consist of half-molecules (Levint,hal, 19%; Meselsott and Stahl, 19.58). Thus DNA synthesized in the presence of chloramphenkol is clearly not degraded to the level of low molecular weight precursors hefore incorporation into phage particles. 8. IIoerm:mn et al. (1955) and Krieg (19.5’7)have proposed that ultra violet, light inactivates phage p:uClcs mtGnly by producittg localized damages in the genetic material of t’he p;u%iclcs. The principal evidence for this proposal comes from experiments, similar to those described in this p:Lper, demonstrating the rescue of genetic markers from irradiated phage particles during mixed infection with uttirr:tdi:~ted p:u$cles. A4ccording to the same criterk, therefore, the d:mlagcs produced hy irradiating phage precursor DNA synthesized in t*he prwencc of c*hloramphenicol are genetic damages. 9. Our pritwipal finding, that radiation d:tm:rge t)o phage precursor DNA sufficient to rettder part~icles c*ontaining it noninfective does not seriously impair bhc m:Lturatiott of phage partkles, ~~11sfor some commenb. In t,he first place it must he recalled that ;t noninfectivc p:u$cle is ottc unable to itiiti:k infection by itself; two or more noninfective particles abtaching to the same haoterium tend t,o produce successful infections (Lurk, 1947). Thus ideas serving to wcount for mult#iplicity reactivat,ion help t*o account for our results. There is an important difference, however. In multiplicity reactivat,ion damaged chromosomes cooperate to produce undamaged chromosomes, by a process currently thought to involve two mwhanisms: cooperation at metabolic levels necessary to ret up a new machinery for 1)&-A

78

TOMIZAWA

synthesis (Krieg, 1957)) and subsequent genetic recombinations by which undamaged chromosomes are produced from damaged ones (Luria and Dulbecco, 1949). In certain of our experiments, phage particles are produced containing almost exclusively damaged chromosomes. The cooperation here must be mainly at metabolic levels concerned with the complex chain of terminal processes beginning with protein synthesis and ending with the final steps of morphogenesis, all carried out without obvious flaws except for the failure to discriminate between damaged and undamaged DNA. Several explanations can be advanced for the efficient production of phage particles containing irradiated DNA. a. By literal collaboration among irradiated chromosomes, according to the classical genetic doctrine of independent action of chromosome parts. Since each irradiated chromosome in the cell contains only a few radiation damages, presumably distributed at random, a specified function would be impaired in only a small minority of chromosomes. Interchromosomal collaboration at a similar level has already been demonstrated with phage in the phenomenon of phenotypic mixing, in which two allelic determinants of host specificity are involved in the formation of single phage particles (Streisinger, 1956). Other less easily characterized examples of interchromosomsl cooperation are described by Benzer (1955), Levine (1957), and Kaiser (1957). b. Alternatively, one could suppose that the terminal steps in phage growth are controlled by DNA functions that are intrinsically resistant to irradiation. c. Finally, one could suppose that the role of DNA in the termina.1 steps of phage growth has already been performed at the time of irradiation in our experiments-by the transfer of specifications to protein synthesized before addition of chloramphenicol or to RNA, for instance. Then the hypothetical protein or RNA might be either intrinsically resistant to irradiation, or able to function cooperatively in spite of damages. A possible analogy to the phenomenon under discussion is seen in the production of noninfective particles containing DNA in which 5bromouracil is substituted for thymine (Dunn and Smith, 1954). However, in this case it remains to be ascertained whether or not the particles are noninfective as a direct result of the abnormalities in their DNA. The phenomenon here described was also seen in unpublished experiments (Tomizawa and Horiuchi) in which TZ-infected cultures were

SENSITIVITY

OF

DNA

TO

UV

IRRADIATION

79

allowed to accumulate DNA in the presence of acriflavine, irradiated, and the inhibitor removed. The production of noninfective but otherwise functional particles under these conditions was demonstrated by photoreactivation. The finding of Watanabe (1957), that synthesis of phage-specific protein is less sensitive than synthesis of DNA in bacteria subjected to ultraviolet irradiation, is also consistent with our results. In sum, all our experiments support the idea that phnge precursor DNA synthesized in the presence of chloramphenicol consists of the finished “chromosomes” of future phage particles. If this is so, phage chromosomes are evidently composed of DNA to the exclusion of all other known phage constituents and can multiply independently of other phage-precursor syntheses and independently of concomitant protein synthesis in general. ACKNOWLEDGMENT The author is deeply indebted to Dr. A. I). Hershey and criticism during the course of this investigation.

for invaluable

advice

REFERENCES

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Research (J. H. Comroe, Jr., ed.), Vol. II, pp. l-73. Year Book Publishers, Chicago. BENZER, S. (1955). Fine structure of a genetic region in bacteriophage. Proc. Natl. Acad. Sri. U. S. 41. 344-354. BURTON, K. (1955). The relation between the synthesis of deoxyribonucleic acid and the synthesis of protein in the multiplication of bacteriophage T2. Biothem. J. 61, 473-483. DOERMANN, A. H. (1953). The vegetative state in the life cycle of bacteriophage: evidence for its occurrence and it,s genet,ic characterization. Cold Spring Harbor Symposia &ant. Riol. 16, 3-11. I~OERMANN, A. H., CHASE, M., and STAHL, F. W. (1955). Genetic recombination and replication in bacteriophage. J. Cellular Comp. Physiol. 46, Suppl. 2, 51-74. DUNN, D. B., and SMITH, J. I>. (1954). Incorporation of halogenated pyrimidines into the deoxyribonucleic acids of Bacterium coli and its bacteriophages. Nature 174, 305-306. HERSHEY, A. D. (1957). Some minor components of bacteriophage T2 particles. Virology 4, 237-264. HERSHEY, A. I)., and BC‘RGI, E. (1956). Genetic significance of the transfer of nucleic acid from parental to offspring phage. Cold Spring Harbor Symposia Qucd. Riol. 21,91-101. HERSHEY, A. II)., and CNASE, M. (1952). Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36,39-56.

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HERSHEY, A. D., and MELECHEX, N. E. (1957). Synthesis of phage-precursor nucleic acid in the presence of chloramphenicol. Virology 3,207-236. HERSHEY, A. D., GAREN, A., FRASER, D. K., and HUDIS, J. D. (1954). Growth and inheritance in bacteriophage. Carnegie Institution of Washington Yearbook 63, 210-225. KAISER, A. D. (1957). Mutations in a temperate bacteriophage affecting its ability to lysogenize Escherichia coli. Virology 3, 42-61. KOZLOFF, L. M. (1953). Origin and fate of bacteriophage material. Cold Spying Harbor Sytzposia Quant. Biol. 18, 209-220. KRIEG, 1). R. (1957). A study of gene action in ultraviolet,-irradiated hacteriophage. Ijissertation. 1Jniversit.v of Rochester, Rochester, New York. LEVINE, M. (1957). Mutations in the temperate phnge P22 and lysogeny in Salnlonella. Virology 3, 22-41. LEVINTHAL, C. (1956). The mechanism of DNA replication and genetic recombination in phage. Proc. Natl. Acad. Sri. C. S. 42,394-404. LURIA, S. E. (1947). Reactivation of irradiated bacteriophage by transfer of selfreproducing units. Proc. Natl. Acad. Sci. U. S. 33, 253-264. LURIA, S. E., and I)CLBECCO, R. (1949). Genetic recombinations leading to production of active bacteriophage from ultraviolet inact,ivat,ed bacteriophage particles. Genetics 34, 93-122. LIIRIA, S. E., WILLIAXS, R., and BACKUS, R. (1951). Electron micrographic counts of bacteriophage particles. J. Bacterial. 61, 179-188. MELECHEN, N. E. (1955). Relationship of phage IINA synthesis to protein synthesis in replication of bacteriophage T2. Genetics 40, 585. MESICI,S~N, M., and STAHL, F. W. (1958). The replication of IINA in Escherichia roli. Proc. ilTatZ. .-I Chad.Sri. U.S., in press:. STAHL, F. W. (1956). The eficcts of the decay of incorporated radioactive phosphorus on the genome of bacteriophage T4. Virology 2, 206-234. STENT, G. S. (1953). Cross reactivation of genet,ic loci of T2 bacteriophage after decay of incorporated radioactive phosphorus. Proc. AVatl. Acad. Sri. U. S. 39, 1234-1241. STENT, G. S., and JERNE, N. K. (1955). The distribution of parental phosphorus at,oms among bacteriophage progeny. Proc. Natl. ilcad. Sci. U. S. 41, 704.-709. STREISINGER, G. (1956). Phenotypic mixing of host range and serological specificities in bacteriophages T2 and T4. Virology 2,38%398. TNVIIZAWA, J., and SIXAKA~A, S. (1956). The effect of chloramphenicol on deoxyribonucleic acid synthesis and the development of resistance to ult,raviolet irradiation in E. coli infected with bacteriophage T2. J. Gen. Physiol. 39, 553565. WATANABE, I. (1957). The effect of ultraviolet light on the production of bacterial virus protein. .I. Gen. Ph,ysiol. 40, 551.-531.