J . Mol. Biol. (1966) 19, 174-186

DNA Replication and Messenger RNA Productionafter Induction of Wild-type A Bacteriophage and A Mutants

A NNE JOY~ER, L . N. ISAACS, H . ECHOLS

Department of Biochemistry, University of Wisconsinp!adison, Wisconsin , U.S.A.

.AND

W. S. SLY

Department of Medicine, Washington UniversitySt. Louis, Missouri, U.S.A .

(Received 8 March 1966, and in revisedform 20 April 1966)

Hybridization techniques have been used to study phage DNA synthesis andmRNA levels after induction of wild -t ype .\ and a number of.\ mutants. Mutantsof .\ defective in t he N, 0 and P cistrons are blocked in D NA replication andproduce a very low level of .\ mRNA late in the induct ion period. Mutantsdefective in the Q cistron sho w no rmal D NA repl icat ion, bu t produce muchlower levels of A mRNA than a normal phage. The mRNA results suggest thatt he pleiotropic defect in synthesis of late proteins shown by N, 0, P and Qmut ant s result s from an insufficient number ofmRNA copies for the region of the.\ genome containing the m aturation functions. The A TIl mutant, wh ich hasbeen sh own previously to pro duce gre atly elevated levels of .\ nuclease, wasfound t o be blo cked in DNA replication. Th ere does not , however, appearto be a general correlation between failure of DNA replication and the hyper­nuclease phenotype, because t he DNA-defective .O cist ron mutant has beenfound previously to be normal in nuclease product ion .

The turn-on of A-specific nucleic acid production in normal A developmentafter mitomycin and tempera t ure induction has been com pared . There is a20· to 30-minute lag after addition of mitomycin before .\ DNA and mRNAlevels show an inc rease; in the case of temperature induction, the turn-on of.\ nucleic acid production occurs much earlier. The effect of A development aftertemperature induction on host DNA and mRNA production was also studied;no strong inhibition was found.

1. IntroductionThe genome of phage A may be divided into five functional classes: (1) "late"eistrons (A-J and R) concerned with the production of phage st ru ct ural proteins andother aspects of ph age maturation (Harris, Mount, Fuerst & Siminovitch, 1966;Dove, 1966) ; (2) "early" eist rons concerned 'wi t h DNA repli cation and control oflate cist rons ; (3) " repression" cistrons (C1and probabl y Czl and Cm) concerned withthe repression and subsequent lysogeny of A when it infects a non-lysogenic cell(Kaiser, 1957; Bode & K aiser , 1965) ; (4) a " lysogeny region" defined by the b2

deletion (Kellenberger , Zichichi & Weigle, 1961) which apparently includes at leastthe region physically necessary as a prerequisite for insertion of the prophage (Camp­bell, 1965); (5) "cohesive site s" whi ch allow the A DNA to form circles (Hershey,

174

NUCLEIC ACID PRODUCTION FOLLOWING A INDUCTION 175

Burgi & Ingraham,1963; Ris & Chandler, 1963). The experiments presented in thispaper deal primarily with the identification and function of the "early" cistronsdefined above.

The concept of "early" and "late" genetic functions was introduced by Jacob,Fuerst & Wollman (1957), based upon their characterization of defective ,\ mutantswhich has formed a model for subsequent studies of phage development. The "late"mutants were found to replicate their genome after induction as judged by geneticmarker rescue experiments, while the "early" mutants did not show genome replica­tion. Subsequent work with the much more extensive set of suppressor-sensitiveconditional lethal (sus) mutants isolated and characterized by Campbell (1961) hasconfirmed and extended this concept. Campbell has classified his sus mutants into the18 cistrons A to R shown in Fig. 1. The sus mutants of ,\ are similar to the ambermutants of phage T4 (Epstein et al., 1963) in that they are sensitive to the same setof suppressors, and possess a similar usefulness in studies of the physiology of phagedevelopment. The sus mutants are able to lysogenize the non-permissive suppressor­negative (su -) host, resulting in defective lysogens, and it is as induced defectivelysogens that they have been principally studied.

R A

F

FIG.!. Location of the BUB mutations (Campbell, 1961; Franklin, Dove & Yanofsky, 1965),Tll (Eisen et al., 1966) and other genetic markers on the genetic map of A. Only the order of themarkers is shown. The order of K and L has not yet been determined. The vegetative map is linear,running from A to R; the prophage map is linear, running from Cm to J. The map is representedhere as a circle because the physical state of the genome during DNA replication and active mRNAproduction is not known; it may well be a circle (Young & Sinsheimer, 1964).

As an approach to understanding the mechanism of regulation of ,\ development,we were interested in the relationship between the production of'\ DNA and AmRNAand the synthesis of late phage proteins. Of particular interest in this regard is thepleiotropic defect intail antigen and lysozyme production found in the case of mutantsdefective in the N, 0, P or Q cistrons (Dove, 1966), even though J appears to be thestructural gene for the tail antigen protein (Dove, 1966) and R is the structural genefor lysozyme (Campbell & del Campillo-Campbell, 1963). Using hybridization tech­niques, we have examined ,\ DNA synthesis and .\ mRNA levels in induced defectivelysogens of the sus mutants N, 0, P and Q. We have also studied ,\ DNA synthesisand AmRNA level after induction of defective lysogens of '\dg A-J and TIL We haveused '\dg A-J, which has lost all of the functions from A through J (shown in Fig. 1)

176 A. JOYNER, L. N. ISAACS, H. ECHOLS AND W. S. SLY

by deletion, to see whether any genes in this region are required for ADNA replicationor the initiation of.\ mRNA production. We considered Tll of interest because of thehyperproduction of A nuclease after induction (Radding, 1~64a), which suggested apossible regulatory mutation. The phenotypes of the mutants which were the primaryobjects of study are summarized in Table 1.

TilLE 1

Phenotype of Amutants

Mutant Nuclease Tail antigen Lysis Marker multiplication

NoPQRA-JTIl

+++ ++ + +

n.a, + +hyper n.a, n.a.

Nuclease data for N, 0, P and TIl are from Radding (1964a,b); nuclease data for Q and Rarefrom Dove (unpublished work). Tail antigen data are from Dove (1966). Marker multiplicationexperiments are those of Brooks (1965). The inference that failure to lyse is associated with afailure to produce normal levels of phage lysozyme has been confirmed by direct lysozyme assaysin the case ofN, 0, P and Q (Dove, 1966; Protass & Korn, 1966). The designation n.a, means thatthe data were not available.

In addition, we have studied the time course of A-specific nucleic acid productionduring normal A development after mitomycin induction and after temperatureinduction of a temperature-sensitive 01 mutant, and we have examined the effect ofAdevelopment on host nucleic acid synthesis.

2. Materials and Methods(a) Strains and media

(i) Bacteria and phage

The non-permissive BU- strain W3350 and the defective lysogens W3350 (>' BUB N 5a)W3350 (>' BUB 0 29) and W3350 (>' TIl) were obtained from O. Radding. ,\ BUB P aD,'\ BUB R 5,,\ BUB Q21 and ,\ BUB Q73 were obtained as lysogens in the BU + host 0600 from A. Oampbelland were transferred to W3350. >'dg A-J was obtained from J. Adler and was studied asa defective lysogen of the epimerase-negative strain W4122, because the ,\dg A-J used isdeleted for the kinase and transferase mutations of W3350. The designation A-J meansthat the deletion includes K, L, M; that is, all of the genetic region from A to J of Fig. 1.The BU - strain W5274-0 and its lysogenic derivative W5274 (,\ BUB R 01857) were obtainedfrom W. Dove (Dove, 1966) as were strains P22, P30 and P32 lysogenic for the defective,\ phages d 22, daD and da2 (Jacob et al., 1957). Strain ABIl57 and its recombination-defec­tive (Rec-) derivative AB2463 were obtained from P. Howard-Flanders.

(ii) Media

The growth medium used was a mineral salts medium (Kornberg, Zimmerman, Kornberg& Josse, 1959), with 0·25% Difco Casamino acids and 0'2% and 0·25% glycerol for theRNA and DNA experiments, respectively. Thiamine (2 fLgfml.) was added for the growthof 0600 strains and thymidine (50 fLgfml.) for growth of W5274 strains.

NUCLEIC ACID PRODUCTION FOLLOWING .\ INDUCTION 177

(b) DNA experiments(i) DNA isolation

In experiments involving uninduced cells, cultures were grown to a titer of 4 X 108

oells/ml. and then pulse-labeled with [3H]thymidine. In heat induction experiments, thecells were grown at 30°C to 4 X 108 cells/ml., transferred to 42°C for 10 min and thenremoved to 37°C for further incubation and pulse-labeling. For mitomycin inductionexperiments, mitomycin C was added at 1 f'g/ml. after the cultures had reached a titer of4 X 108 cells/rnl., and [3H]thymidine was added at intervals following mitomycin addition.For pulse-labeling, 0·1 me of [methyZ-SH]thymidine (New England Nuclear Corporation)of specific activity 27'7 me/mg was added to 250-ml. cultures in the heat-induction experi­ments and 0·2 me was added to 500-ml. cultures in all other experiments. In the heatinductions, the pulse was terminated after 2 min by pouring the culture over 0·4 vol.frozen medium lacking the carbon source. In all other experiments, the pulse was termi­nated after 2·5 min by swirling the culture rapidly in an ice-salt mixture.

After sedimentation the cells were washed with 0·1 vol. 0·15 M-NaCI, 0·1 M-EDTA(pH 8,0) (saline-EDTA) and resuspended in saline-EDTA containing 1·2mg/ml.lysozymeat an optical density 30 times that of the original culture. After incubation at 37°C for,Wmin, sodium dodecyl sulfate was added to a final concentration of 0'05% and the lysatem.cubated at 60°C for 10 min with slow swirling. The lysate was then cooled to 45°C and100 f'g/ml. pronase (Calbiochem) added. Pronase digestion was continued for 22 to 24 hrat 45°C and was followed by a 30-min incubation at 37°C with 50 f'g/ml. ribonuclease(previously heated at 80°C for 10 min to inactivate DNase). The remainder of the isolationwas adapted from Marmur's DNA isolation (Marmur, 1961) and consisted of one 30-minchloroform extraction followed by ethanol precipitation and collection of the DNAthreads onto a glass rod. The DNA was then dissolved in 0·1 X SSC, during a period ofseveral days at 2 to 4°C. SSC is standard saline-citrate, which is 0·15 M-NaCI and 0·015 M­sodium citrate (pH 7). The resulting viscous solution was made 2 X SSC and the level of.\ or E ..coli DNA present was determined by the DNA agar column method of Bolton &McCarthy (1962). DNA labeled and prepared in this way from the non-thymine-requiringW3350 strains typically had a specific activity of approximately 1500 cts/minlf'g; fromthe thymine-requiring W5274 a specific activity of approximately 5000 cts/min/f'g wasobtained.

Purified ADNA used in the preparation of the DNA-agar or for DNA-RNA hybridiza­tions was prepared by phenol extraction of phage concentrates which had been purifiedby CsCI density-gradient centrifugation (Kaiser & Hogness, 1960). The [3H]A DNA usedin the experiments of Table 2 was prepared in the same way and had a specific activity of6800 cts/minff'g. The E. coli Kl2 DNA used in the preparation of E. coli DNA-agar wasobtained from General Biochemicals. The E. coli DNA used in the DNA-RNA hybridiza­tions was either prepared as described by Marmur (1961), except for a subsequent phenolextraction, or obtained from General Biochemicals. Hybridization results were the samefor both DNA sources.

(ii) DNA hybridization

Preparation and assay of the DNA-agars, shearing and denaturing of the [3H]DNAand preparation of the hybridization mixtures were as described by McCarthy & Bolton(1963). The DNA-agar was sieved 3 times through a wire screen into 200 ml. 2 X SSCat 60°C, and then the granules washed with 500 ml. 2 X SSC at 60°C. For the hybridizations,0·2 to 0·5 g of agar containing 25 or 50 f'g of ADNA or E. coli DNA was combined withan equal volume of 2 X SSC containing 0·5 or 1·0 f'g sheared, denatured [3H]DNA. Theratio of the amount of DNA in the agar to the [3H]DNA was always 50.

After incubation at 60°C for 15 to 16 hr, the hybridization mixtures were transferredto a jacketed column heated to 60°C and washed with fourteen 10-ml. portions of 2 X SSCover a period of about 90 min. The agar was then washed with eight 10-ml. portions of0·01 X SSC at 75°C. 100 f'g bovine serum albumin and 1 ml. 55% trichloroacetic acid wereadded to each of the fractions. The precipitates were collected on Millipore filters, driedand counted in a liquid-scintillation counter. The fraction of the radioactivity which

12

178 A. JOYNER, L. N. ISAACS, H. ECHOLS AND W. S. SLY

appeared in the samples at increased temperature and reduced salt concentration wastaken as the fraction of the [3H]DNA which formed a specific hybrid with the DNA inthe agar. Fraction 14 was used as background for the subsequent fractions and was alwaysonly slightly above or equal to counter background.

50 p,g of unsheared, denatured ADNA was added along with the [3H]DNA in hybridiza­tions where the fraction of E. coli DNA in the [3H]DNA was being measured. This wasdone to prevent the one-third of A DNA which is homologous with "E. coli DNA (Cowie &McCarthy, 1963) from forming a hybrid with the E. coli DNA in the agar. That suchcompetition is effective is shown in Table 2.

TABLE 2

Effect of competing unlabeled ): DNA on eH].\ DNA hybridization

E. coli DNASheared DNA Competing unlabeled, Cts/minin

in agar [3H]A DNA Unlabeled E. coli DNA unsheared ADNA hybrid(,.g) (,.g) (,.g)

(,.g) (%)

50 1 0 0 1150 1 0 5 850 1 0 50 350 0·5 0·5 50 3

0 1 0 50 2

Procedures for hybridization are described in the text. 3H·labeled E. coli DNA prepared asdescribed in Materials and Methods and hybridized in the presence of competing ADNA typicallygives approximately 50% hybrid. (See for example the uninduced result in Table 4.) The "back­ground" result obtained with no DNA in the agar (line 5) has not been subtracted from thenumbers given in the first four lines. Therefore, the competition effect is greater than that indicatedfrom the uncompensated data.

(c) mRNA experiments

In all experiments, [3H]RNA was obtained by pulse-labeling for 2 min with [3H]uridineand subsequent extraction with hot phenol (Scherrer & Darnell, 1962). The specific activityof the [3H]RNA was typically 1500 to 2000 cts/min/",g. Growth of cells and inductionwere as described for the DNA experiments. The level of A·specific mRNA or E. coli­specific mRNA was taken as that fraction of the [3H]RNA which formed a ribonuclease­resistant hybrid with denatured A DNA or E. coli DNA, respectively, as detected by thenitrocellulose membrane filter technique of Nygaard & Hall (1963). The methods used forRNA preparation and hybridization have been described in detail previously (Sly, Echols& Adler, 1965), except that phenol was added to the hybridization mixture in the presentexperiments (Isaacs, Echols & Sly, 1965). Phenol has been found to inhibit any residualRNase activity (Hill & Echols, 1966). The [3H]RNA prepared by the hot phenol techniquewas shown to be greater than 99% alkali-labile, as had been found previously (Sly et al.,1965).

(d) Incorporation of [3H]thymidine and [3H]uridine

W5274 and W5274 (Asus R Cr857) were grown at 30°C in the presence of thymidine(50 ,.g/ml.) to 4 X 108 cells/ml, The procedure for heat induction was then carried out asdescribed in section (b). At intervals following the transfer of the cultures to 37°C, 5-ml.portions were removed to flasks containing either 2,.c [methyl.3H]thymidine of specificactivity 27'7 mc/mg, or 12·5 fJ-c [3H]uridine of specific activity 5 mc/mg. The flasks wereincubated at 37°C for 2 min and then 0·5-ml. portions were removed to tubes containing0·5 ml. cold 10% trichloroacetic acid. After standing in ice for at least 30 min, the trichloro­acetic acid-insoluble material was collected onto Millipore membrane filtera and washed

NUCLEIC ACID PRODUCTION FOLLOWING A INDUCTION 179

with 20 ml. cold 5% trichloroacetic acid and 5 ml, cold 5% trichloroacetic acid contam­ing 2 mg/ml. thymidine or uridine. The radioactivity retained on the dried filters wascounted in a liquid-scintillation counter. The background correction was the small amountof radioactivity retained after filtration of cells which had been added to trichloroaceticacid prior to the addition of [3H]thymidine or [3H]uridine. The [3H]uridine incorporationwas rendered greater than 96% acid-soluble by alkali treatment (0,5 M-KOH for 15 hr at37°0), showing that the radioactive material was incorporated nearly exclusively into RNA.

3. Results(a) Increase of ADNA and AmRNA associatedwith normal

development after induction

The time course of the production of A DNA and mRNA following mitomycininduction of W3350 (A +) is shown in Fig. 2. There is a delay of 20 to 30 minutes afteraddition of mitomycin before the level of A-specific nucleic acid synthesis rises abovethe uninduced level of 2% for DNA and 0'1 %for RNA. A similar lag in AmRNA riseafter mitomycin induction was observed previously (Sly et al., 1965) with anotherstrain. After the delay, the rate of A DNA synthesis and the AmRNA level increaserapidly, and each reaches a high level by 50 to 60 minutes after addition of mitomycin.Lysis of W3350 (A+) begins at approximately 70 minutes after addition of mitomycin.

\o...--------------itoo;;;:---,

.£

<CZcr:: 4OJ0>

.3c::OJ

~ 2~

o

e

\0Minutes after mitomycin induction

c:

c:

~~~

.3<CZ

io 0OJ0>

~OJ

5 ::'OJ

<L

FIG. 2. The points on the DNA curve (-e-e-) represent the A-specific fraction of the DNAwhich is labeled during a 2·5-min exposure to [3H]thymidine, as determined by DNA-DNAhybridization on an agar column. The points on the mRNA curve (-0-0-) represent theA-specific fraction of the RNA labeled during a 2-min pulse with [3H]uridine as determined byRNase-resistant DNA-RNA hybrid collected on a membrane filter. Details of the nucleic acidpreparation and hybridization are given in Materials and Methods. The A DNA and A mRNAlevels through 20 min are indistinguishable from the uninduced level of 2% for DNA and 0·1 %for RNA. The no DNA "background" of 1·5 to 2% for the DNA-DNA hybridizations has notbeen subtracted from the numbers in the Figure. The no DNA "background" of 0·01 to 0·04%for the DNA-RNA hybridizations is negligible.

The extended lag in A nucleic acid synthesis after mitomycin treatment was notobserved after heat induction of W5274 (A sus R °1857), in which phage developmentshould be similar to that of A+. A vigorous rate of A DNA synthesis (26% hybrid)and a high level of AmRNA (6% hybrid) were found as early as two minutes after

180 A. JOYNER, L. N. ISAACS, H. ECHOLS AND W. S. SLY

transfer to 37°0 of a culture induced by a ten-minute treatment at 42°0 (see Table 4).This suggests that the two modes of induction are quite different in mechanism.Temperature induction presumably inactivates the repressor directly, .\ nucleic acidsynthesis begins immediately, and the time required for phage development and lysisof an R+ lysogen (40 minutes) is close to that observed in an infection experiment.In the case of mitomycin treatment, the delay in the start of mtcleio acid synthesissuggests that mitomycin induction does not result from a direct effect on either re­pressor or DNA; it seems likely that the induction process is an indirect one correlatedrather with repair of mitomycin damage to DNA. It should be noted that the timefrom the onset of nucleic acid synthesis until lysis at approximately 70 minutes is againclose to the 40-minute latent period found in infection, where .\ nucleic acid synthesisstarts very quickly (Sly et al., 1965; Joyner, unpublished work). The delayed lysisfound after ultraviolet induction is also consistent with a need for repair processesbefore the phage genome can be turned on. Mitomycin and ultraviolet lesions areprobably repaired by similar mechanisms (Boyce & Howard-Flanders, 1964).

(b) .\ DNA synthesis and messenger RNA level after inductionof defective lysogens

r DNA was prepared after pulse-labeling from 50 to 52·5 minutes and RNA afterpulse-labeling from 60 to 62 minutes following the addition of mitomycin to culturesof defective lysogens ofN, 0, P, Q, R, A-J and TIL The levels of.\ DNA and .\mRNAfound in these preparations are listed in Table 3. Sus R is defective only in lysozymeproduction; since its failure to lyse makes it a better control than .\+ for the otherdefectives which do not lyse, we have used the sus R levels of.\ DNA and mRNA aswild-type levels. The sus R levels were found to be similar to those of .\+ •

Sus N, 0 and P are apparently unable to undergo autonomous DNA replication,since the levels of ADNA found are the same as, or slightly less than, those found foran uninduced lysogen. Although the messenger RNA levels for N, 0 and P are low,they are distinctly higher than those found for any of the uninduced lysogens, showingthat an increase in .\ mRNA level is induced to some extent in the probable absenceof DNA replication in these defectives. Both of the sus Q mutants tested were foundto replicate DNA at close to the normal rate. The AmRNA level found after inductionof the sus Q lysogens, however, was only one-fourth to one-third that of sus R. DNAprepared at 40 minutes after induction of the sus Q21 lysogen gave 19% hybrid;therefore it is unlikely that the low mRNA level reflects delayed DNA replication.The level of .\ mRNA late in .\ development has also been shown to be low afterinfection by sus N, P and Q mutants (Skalka, Echols & Butler, unpublished work).

Tll was found to be defective in A DNA replication, but the .\ mRNA levelafter induction was clearly much higher than in the case of the other DNA-nega­tive mutants N, °and P. The .\ DNA and mRNA results with Adg A-J show thatnone of the A-J cistrons is necessary for the initiation of A DNA synthesis andsuggest that DNA replication and mRNA production by the remainder of thegenome occur normally. Our DNA replication results agree with the geneticexperiments of Brooks (1965), who found that sus N, 0 and P did not show genomemultiplication after induction, whereas sus Q, sus R and four of five .\dg's testeddid show genome multiplication.

The DNA-defective mutants, N, 0 and P, all show a pleiotropic defect in tail antigenand lysozyme production, both late functions. This suggests that A obeys the T4

NUCLEIC ACID PRODUCTION FOLLOWING ,\ INDUCTION 181

"rule" that DNA synthesis is required in order to produce substantial amounts oflate proteins (Epstein et al., 1963). The behavior of several mutants of Jacob et al.(1957) has previously been a reason for thinking that A. does not require DNA replica­tion for the normal expression of late functions; these A. defectives produced tailantigen or lysozyme or both, although genetic marker rescue experiments indicatedthat genome replication did not occur. To try to clear up the discrepancy, we measuredDNA replication by the hybridization technique for lysogens of the presumablyreplication-defective phages d22,d30 and d32. Experiments identical to those in Table 3

TABLE 3

A. DNA and mRNA levels following induction of defective lysogens

Prophage

R5

N53

0 29

PeoQ21

Q73

A-JTll,\ +, uninduced

Cts/min in ,\ DNA(%)

23<1<1<1

181410

<1<1

Cts/min in,\ RNA(%)

90·30·50·723230·1

[3H]Uridine WII8 added at 60 min after mitomycin induction to label RNA and [3H]thymidineat 50 min to label DNA. Nucleic acid preparation and hybridization were as described in Materialsand Methods. The percentage cts/min in ,\ DNA or ARNA is the percentage of the purified pulse­labeled [3H]DNA or [3H]RNA which forms a specific hybrid with ADNA. With no XDNA em­bedded in the agar, 1·5 to 2% of the [3H]DNA cts/min remained. With no ADNA in the DNA-RNAhybridization solution, 0'01 to 0·04% of the [3H]RNA cts/min remained on the filter after RNasetreatment. The no DNA "background" has been subtracted from the DNA-DNA hybridizationvalues in the Table; the no DNA "background" is negligible in the DNA-RNA hybridizationexperiments. To show that the [3H]DNA or [3H]RNA preparations were all capable of formingspecific hybrid even when there was little or no hybridization with ADNA, control hybridizationswere carried out with E. coli DNA in each ease. [3H]DNA from SUB N, BUB 0 and BUB P lysogensgave 53 to 59% hybrid; [3H]DNA from BUB Q and BUB R lysogens gave 30 to 39% hybrid, reflectingthe fact that ,\ DNA was being synthesized. [3H]RNA preparations typically gave hybrid figureswith E. coli DNA of approximately 16%. The maximum variability in E. coli DNA hybridizationamong the 22 RNA preparations used to provide the data of Table 3 was ±25% of this figure;the maximum variability in ADNA hybridization in repeat RNA preparations from the same strainwas also approximately ±25%.

gave 11, 33 and 26%, respectively, as the fraction of the [3H]DNA hybridizing witha A. DNA column. Apparently the marker rescue technique as a measure of genomereplication was not a satisfactory measure of DNA replication, perhaps because thesephages are somehow defective in recombination. Since these defective phage do showDNA replication, it seems likely that the behavior of A. in the relationship betweenDNA replication and normal expression of late functions is probably like that of T4,as suggested by the results with N, 0 and P.

182 A. JOYNER, L. N. ISAACS, H. ECHOLS AND W. S. SLY

(c) Host DNA and mRNA production during.\ development

Experiments were undertaken to determine the extent to which E. coli DNA andmRNA continue to be synthesized during autonomous growth of .\. This has beenrather a difficult point to establish, because the similarity of the base compositions of.\ DNA and E. coli DNA (Kaiser & Hogness, 1960) complicate the identification ofthe nucleic acids synthesized during development. A method utilizing homologymakes possible such identification. However, since approximately one-third of the.\ genome is homologous with the E. coli genome (Cowie & McCarthy, 1963), in orderto measure E. coli DNA synthesis it is necessary to modify the standard DNA-DNAhybridization procedure by adding competing unlabeled .\ DNA to the hybridizationmixture to prevent hybrid formation between.\ [3H]DNA in the labeled DNA prepara­tion and the E. coli DNA contained in the agar (see Materials and Methods).

A lysogen of a heat-inducible strain of .\ was chosen for the experiments on hostnucleic acid production, because the level of uninduced or surviving cells after heatinduction is 10- 4, so that the contribution of uninduced cells to the labeling of thenucleic acids is not significant. [3H]RNA and [3H]DNA were prepared at severaltime points during a 30-minute period after heat induction and transfer to 37°C ofstrain W5274 (.\ sus R C1857). Lysis of an R+ lysogen occurs at approximately 40minutes after transfer to 37°C. As shown in Table 4, the fraction of E. coli DNA andmRNA in the preparations remained high during phage development, showing thateven at late times after induction a substantial proportion of the nucleic acids synthe­sized are those ofthe host cell. Since the hybridization percentages are a measure onlyoflevels of host and phage nucleic acids relative to one another, the total rate of DNA

TABLE 4

Effect of.\ development on host DNA and mRNA production

Percentage cts/min in:Percentage of non-lysogenic

Strain Time DNA mRNA 3H incorporation

>. E. coli >. E. coli Thymidine Uridine

W5274 (>' BU8 R C1857) 2 26 6 26 180 3610 6 24 200 6620 33 27 9 23 160 62

"30 43 17 8 19 200 73

" uninduced (30°C) 3 46W5274 (0) 20 0·03 22

The times given are minutes following transfer of the induced culture to 37°C after a Hl-mininduction period at 42°C. The cultures were pulse-labeled for 2 min, beginning at the times desig­nated. The figures given in the>. and E. coli DNA and the>. and E. coli mRNA columns are thepercentage of purified [3H]DNA or [3H]RNA hybridizing with the appropriate DNA. This is a.measure of the relative levels of>. and E. coli DNA or mRNA. To estimate the effect of phagedevelopment on total RNA and DNA synthesis during a 2·min pulse label, the percentage 3Hincorporation into trichloroacetic acid-insoluble material for each time point was calculatedusing the radioactive material (cts/min) incorporated by "induced" non-lysogenic W5274 a.teach time as 100%. Details of the incorporation and hybridization techniques are described inMaterials and Methods.

NUCLEIC ACID PRODUCTION FOLLOWING A INDUCTION 183

and RNA synthesis during a pulse-labeling period was also estimated by determiningthe amount of acid-insoluble radioactive material resulting from two-minute pulses of[methyl-3H]thymidine and [3H]uridine (Table 4). The uri dine incorporation rate ofthe non-lysogenic strain was found to be greater than that of the lysogenic strain;however, the two rates are not greatly dissimilar. Thymidine incorporation by thelysogenic strain was approximately twofold greater than that by the non-lysogenicstrain. The incorporation and hybridization results for DNA taken together suggestthat phage DNA synthesis occurs in addition to a relatively normal rate of host DNAsynthesis, rather than replacing host synthesis as in the case of T4 infection. Com­bining incorporation and hybridization results for RNA, we find that, although therate of host RNA synthesis may be somewhat reduced during ,\ development aftertemperature induction, there is certainly nothing resembling the complete turn-offof host mRNA synthesis found after T4 infection (Volkin & Astrachan, 1956; Nomura,Hall & Spiegelman, 1960).

Although the experiments above do not prove that the host DNA synthesis observedis the normal chromosomal replication, the high rate and extended duration of hostDNA synthesis would seem to make it unlikely that only repair synthesis in theneighborhood of the ,\ "excision" site is involved. To test further the possibility thatthe host DNA synthesis observed might be repair, DNA synthesis was also studiedafter heat induction of a lysogen of a Bee: strain (AB2463). This strain is defectivein repair DNA synthesis after ultraviolet irradiation (Howard-Flanders & Theriot,1966), as judged by inability to incorporate radioactive thymidine into DNA fora period of at least 150 minutes after irradiation. As in the previous experiments,the total rate of DNA synthesis was estimated by thymidine incorporation forboth the Ree: strain AB2463 and its Ree: parental strain AB1l57, both lysogenicfor ,\ sus R Cr857; the fraction of the DNA specific to the host was measured by hybri­dization of [3H]DNA labeled 20 to 22 minutes after heat induction. The phage yieldfrom the two lysogens after heat induction was approximately the same (60 to 70phage/cell); the fraction of the DNA specific to the host as judged by hybridizationwas also approximately the same (31% hybrid from the Bee: and 28% hybrid fromthe Rec+); thymidine incorporation of a pulse-label by the Rec" was approximately20% greater than for the Rec: at 10 and 20 minutes after heat induction, but thisdifference in incorporation rate was also found before heat induction. These resultstherefore support the view that the host DNA synthesis observed after heat inductionis mainly chromosomal replication rather than repair-type synthesis.

4. Discussion

Two mutants which now would seem to be defective in a normally present specificregulatory function are sus Q and TIL Sus Q mutants appear to replicate their DNAnormally; however, they show a pleiotropic defect in the late functions tail antigenand lysozyme (Table 1), which cannot be explained by a gene dosage effect due toinsufficient copies of the phage genome. The low level of'\ mRNA after sus Q inductionrelative to sus R (Table 3) suggests that the basis ofthis defectiveness in late functionsis the failure to produce normal levels of mRNA for the late region of the genome.The Q product may have a specific role in turning on or enhancing mRNA productionfor the region of the Agenome which contains the cistrons for late proteins, or it maysimply enhance ,\ mRNA synthesis for the entire genome. The latter model would

184 A. JOYNER, L. N. ISAACS, H. ECHOLS AND W. S. SLY

probably allow for normal DNA replication in the absence of the Qproduct, becausefewer protein copies are likely to be required for DNA replication than for phagematuration functions.

The TIl mutant is characterized phenotypically by over-production of A nucleaseand defectiveness in A DNA synthesis and late function (Table 1). Although in thecase of T4 over-production of early enzymes because of failure to turn off earlyenzyme synthesis appears to be correlated with either an enzymic .block or an ultra­violet block in DNA replication (Wiberg, Dirksen, Epstein, Luria & Buchanan, 1962),there does not appear to be a general correlation in the case of Abetween the hyper­nuclease phenotype and a block in DNA replication. The DNA defective BUB 0 2 9

(Table 3) has been reported by Radding to be normal in nuclease production (Radding,1964a). Eisen et al. (1966) have shown that among a number of defective Amutantswhich appear to be blocked in DNA replication, only mutations mapping near theTIl site show the hypernuclease phenotype. ,\ TIl therefore apparently lacks a specificregulatory function.

The high level of'\ mRNA after TIl induction relative to the other DNA-negativemutants N, °and P (Table 3) suggests that the basis of its hypernuclease phenotypemay be over-production, at times late in normal ,\ development, of mRNA tran­scribed from the early cistrons, perhaps because of failure to turn offmRNAproductionfor some of the early region of the genome. In this connection it should be noted(Radding, 1964a) that at least the major over-production of nuclease after TIlinduction occurs after nuclease activity ceases to increase in control induction ofa A+ strain. It is not obvious why TIl is defective in DNA replication, however, if itsonly defect is failure to turn off early genes. ,\ TIl may be a polar mutant; and infact probably only a polar mutant would create a defective phage, since the turningoff of early genes may well be non-essential.

The failure of BUB N, °and P to produce substantial amounts of late proteins per­haps indicates the necessity for DNA synthesis for the turning-on of the late genesof the ,\ genome. However, the failure to find these proteins at appreciable levelscould also be a gene dosage effect, and the data we have do not distinguish betweenthe two alternatives. Experiments in progress (Skalka, Echols & Butler, unpublishedwork) to try to identify the region of the Agenome represented by the AmRNA pro­duced by the N, 0, P, Q and TIl mutants should give a clearer delineation of theregulatory role in ,\ development of DNA synthesis, the Q cistron and the regulatoryfunction affected by the TIl mutation.

The similarity between mutations of A and T4 which exert a pleiotropic effect onlate function is quite striking. In addition to the requirement already noted forphage DNA synthesis in ,\ and T4 in order to get normal levels of late proteins,cistron 33 in phage T4 (Epstein et al., 1963) appears to be identical in phenotypeto cistron Q in A.

In addition to providing a convenient mechanism for temporal control of'\ develop­ment, the requirement for DNA replication and Q function for adequate expressionof the late cistrons is of interest in consideration of the action of Arepression functions.Previous experiments on repression of'\ mRNA when a superinfecting phage infectsa lysogenic cell (Isaacs et al., 1965) showed that the 01 product appears to controlall A mRNA production and suggested that the site of action of the 01 repressor waswithin the Orimmunity region. It is now apparent that the 0 1 product may need torepress only one or more of the early N, 0, P and Q cistrons in order to block normal

NUCLEIC ACID PRODUCTION FOLLOWING ,\ INDUCTION 185

expression of the late cistrons. Such a simple model requiring only 0I repression ofearly cistrons for indirect control of the entire" genome is, however, probably notadequate to explain events leading to lysogeny after infection of a non-lysogenic cell.If, as is probably the case, Orrepression does not occur immediately after infection ofa non-lysogenic cell (Kaiser, 1957), then it is likely that expression of late functionsmust be inhibited if lysogeny is to proceed efficiently. This could occur through atransient early inhibition of DNA synthesis which limits the number of gene copies,or through a specific repression of late functions. This inhibition of late functions maybe the role of the On and Om products.

We thank Luis Soto-Krebs, Dorothy Rollefson, Richard Case, Linda Pilarski andKen Parejko for assistance in various phases of this work, and Charles Hill for the purified[3H],\ DNA. We are grateful to Charles Radding, Allan Campbell, Julius Adler and PaulHoward-Flanders for bacterial strains and phage stocks, and we acknowledge particularindebtedness to William Dove for much helpful discussion as well as the gift of strains.This research was supported by U.S. Public Health Service research grants GM 08407-05and GM Il905-02 and U.S. Public Health Service training grant 2T 1 GM 236 BCH.One of us (L. N. 1.) is a U.S. Public Health Service Predoctoral Fellow; another (W. S.) isa Faculty Research Associate of the American Cancer Society (grant no. PRA-16).

REFERENCES

Bode, V. C. & Kaiser A. D. (1965). Virology, 25, Ill.Bolton, E. T. & McCarthy, B. J. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1390.Boyce, R. P. & Howard-Flanders, P. (1964). Z. Vererbungsl. 95,345.Brooks, K. (1965). Virology, 26, 489.Campbell, A. (1961). Virology, 14, 22.Campbell, A. (1965). Virology, 27, 340.Campbell, A. & del Campillo-Campbell, A. (1963). J. Bact. 85, 1202.Cowie, D. B. & McCarthy, B. J. (1963). Pmc. Nat. Acad. Sci., Wash. 50,537.Dove, W. F. (1966). J. Mol. Biol. 19, 187.Epstein, R. H., Bolle, A., Steinberg, C. M., Kellenberger, E., Boy de 10. Tour, E., Chevalley,

R., Edgar, R. S., Susman, M., Denhardt, G. H. & Lielausis, A. (1963). Cold Spr,Harb. Symp. Quant. Biol. 28, 375.

Franklin, N. C., Dove, W. F. & Yanofsky, C. (1965). Biochem, Biophys. Res. Comm. 18, 910Eisen, H., Fuerst, C., Siminovitch, L., Thomas, R., Lambert, L., Pereira do. Silva, L. &

Jacob, F. (1966). Virology, in the press.Harris, A., Mount, J., Fuerst, C. & Siminovitch, L. (1966). Virology, in the press.Hershey, A. D., Burgi, E. & Ingraham, L. (1963). Proc, Nat. Acad. Sci., Wash. 49, 748.Hill, C. W. & Echols, H. (1966). J. Mol. Biol. 19, 38.Howard-Flanders, P. & Theriot, L. (1966). Genetics, in the press.Isaacs L. N., Echols, H. & Sly, W. S. (1965). J. Mol. Biol. 13, 963.Jacob, F., Fuerst, C. & Wollman, E. (1957). Ann. Inst. Pasteur, 93, 724.Kaiser, A. D. (1957). Virology, 3, 42.Kaiser, A. D. & Hogness, D. S. (1960). J. Mol. Biol. 2, 392.Kellenberger, G., Zichichi, M. L. & Weigle, J. J. (1961). J. Mol. Biol. 3, 399.Kornberg, A., Zimmerman, S. G., Kornberg, S. R. & Josse, J. (1959). Proc, Nat. A cad.

Sci., Wash. 48,1390.McCarthy, B. J. & Bolton, E. T. (1963). Proc, Nat. Acad. Sci., Wash. 50, 156.Marmur, J. (1961). J. Mol. Biol. 3, 208.Nomura, M., Hall, B. D. & Spiegelman, S. (1960). J. Mol. Biol. 2, 306.Nygaard, A. D. & Hall, B. D. (1963). Biochem. Biophys. Res. Comm. 12, 98.Protass, J. & Korn, D. (1966). Proc. Nat. Acad. Sci., Wash. 55, 1089.Radding, C. M. (1964a). Proc. Nat. Acad. Sci., Wash. 52,965.Radding, C. M. (1964b). Biochem. Biophys. Res. Comm. 15, 8.Ris, H. & Chandler, B. (1963). Cold Spr, Harb. Symp. Quant. Riol. 28, 1.

186 A. JOYNER, L. N. ISAACS, H. ECHOLS AND W. S. SLY

Scherrer, K. & Darnell, J. E. (1962). Biochem. Biophys. Res. Gomm. 7, 486.Sly, W. S., Echols, R. & Adler, J. (1965). Proc, Nat. Acad. Sci., Wash. 53,378.Volkin, E. & Astrachan, L. (1956). Virology, 2, 149.Wiberg, S., Dirksen, M. L., Epstein, R. R., Luria, S. E. & Buchanan, J. lVL (1962). Proc.

Nat. Acad. Sci., Wash. 48,293.Young, E. T. & Sinsheimer, R. L. (1964). J. Mol. Biol. 10, 562.