Gene Expression During the Development of Bacillus subtilis Bacteriophage φ29 II. Resolution of Viral-Specific Ribonucleic Acid Molecules

JOURNALOFVIROLOGY,Jan.1973,p.87-97

Copyright( 1973AmericanSocietyforMicrobiology

Vol.11, No. 1 PrintedinU.S.A.

Gene

Expression

During the Development

of

Bacillus subtilis Bacteriophage

029

II. Resolution of

Viral-Specific

Ribonucleic Acid Molecules

D. J. LOSKUTOFF AND J. J. PENE

DepartmentofMolecular, Cellular, and Developmental Biology, University of Colorado,

Boulder,

Colorado 80302

Received forpublication8September1972

The ribonucleic acid (RNA) specified by bacteriophage 029was isolated

un-der conditions which minimized physical and enzymatic degradation, reduced

aggregation, and enriched for completed molecules. This RNA was

fraction-ated both by sedimentation through sucrose density gradients and electro-phoresisthrough polyacrylamide gelstomeasurethesize and relativeamountof

each component. Early RNA consisted of six components of molecularweight 0.75 x 106, 0.44 x 106, 0.37 x 106, 0.25 x 106, 0.09 x 106, and 0.04 x 106,

ac-counting for 35% of the coding capacity of

029

deoxyribonucleic acid (DNA).

All of these components except the one at 0.44 x 106 were detected when

in-fection occurred in the presence of chloramphenicol. Synthesis of the major

early component (0.75 x 106) ceased shortly after theonsetof viral DNA

syn-thesis. The other species of early RNAwere synthesized throughout the latent

period. Three additional components, 1.75 x 106, 0.93 x 106, and 0.07 x 106,

appear at late times. The two large RNAs may be polycistronic messenger

RNAs corresponding to the seven viral capsid proteins.

Characterization of individual species of

messengerribonucleic acid(mRNA) would

con-tribute significantly to understanding mech-anisms which affect gene expression during

phage development. The isolation and resolu-tion of intact mRNA molecules have been constrained both by technical problems

as-sociated with the purification of these

meta-bolically unstable macromolecules and by the complexity of most systemsjudged attractive for genetic analysis. Thus, previous attempts to characterize transcription during bacterio-phage development have relied primarily

upon analysis of RNA by deoxyribonucleic

acid(DNA)-RNA hybridization (4, 7), orupon

detection of specific proteins by using

stan-dard biochemical techniques (4, 10). The tech-nique of competition-hybridization is

inad-equate to detectsingle species ofmRNA since

this approach doesnot distinguish between

in-tact and fragmented molecules. Inaddition, it

lacks the sensitivity requiredtodetectchanges in single species of mRNA among complex

populations of molecules. Examination of geneexpression by direct measurement of

pro-teins is similarly limiting, since in most cases

only a few viral functions are known. Neither

appraoch yields information about the number

87

ofindividual mRNAsortheir size. In addition, information about the intramolecular organiza-tion of polycistronic mRNAs and the rates of initiation and termination of transcriptive units are difficulttoobtainwhenutilizingthese techniques. This paper summarizes the re-sults ofexperimentsemploying polyacrylamide gel electrophoresis to fractionate the mRNA molecules specified by phage k29. Their me-tabolism throughout the virallytic cycle is de-scribed.

429is aBacillus subtilisbacteriophage which contains a single molecule ofdouble-stranded DNA ofmolecular weight 11 x 101 (1,9). Over 50% ofthe viral geneticinformation is devoted to the synthesis of seven capsid polypeptides (D. J. Loskutoff, A. R. Ayers, and J. J. Pene, Bacteriol. Proc., p. 197, 1971; see also refer-ence 14). Ifhalf ofthe DNA can betranscribed into mRNA, the sum total of potential viral-specific RNA should be 5.5 x 106.

In a previous communication the transcrip-tion ofphage k29 chromosomes was examined by competition-hybridization (13). Two classes of

029-specific

RNA were defined. "Early" genes were those activated before the onset

of viral DNA replication, whereas "late" genes were those expressed at later times in

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LOSKUTOFF AND

PItNE

the viral lytic cycle, temporally at approxi-mately the same time as the onset of DNA

synthesis. Forty percent of the genome was

activated early, an additional 60% after 10

min. Although late genes were expressed in

conjunction with viral DNA replication, the

replication of

029

chromosomeswas not neces-sary for their expression. Most of the early

genome was found tobe active throughout the

viral latent period. No late transcripts and only 80% of the early ones could be detected

when infection occurred in the presence of

chloramphenicol.

Preliminary experiments described the iden-tification of four distinct andatleastthreeless

defined size classes of late viral RNA (D. J. Loskutoff, A. R. Ayers, and J. J. Pene, Bac-teriol. Proc., p. 197, 1971). This paper

de-scribes the results of further experiments to

characterize the RNA molecules specified by

p29. Nine components, accounting for over

85% of the coding capacity of029 DNA, have been identified. Their time of appearance in

the viral life cycle and the percentage ofthe

genome devoted to their synthesis is in good

agreement with the hybridization-competition datapreviouslyreported (13).

MATERIALS AND METHODS

Bacteria and bacteriophages, media, growth, and

purification of 029, isolation of viral DNA, and the

procedures for DNA-RNA hybridization were

de-scribedpreviously (13).

Labeling and purification ofRNA. RNA from uninfected and infected bacteria was labeled by a

4-min exposure to uridine-5-3H (New England Nu-clear Corp.; 25.6 Ci/mmole) as described inthe

fig-ures. The cells were treated simultaneously with

chloramphenicol (CAP; Calbiochem, Los Angeles, Calif., 250 gg/ml) to minimize normal intracellular

degradationofRNA(11).Thistreatmentwasfoundto

increasesignificantlythestability of RNA after

addi-tion ofrifamycin (a giftofCiba-GeigyCorp., Summit,

N.J.) or actinomycin D (a gift of Merck and Co.,

Rahway, N.J.). RNA to be analyzed by

polyacryla-mide gel electrophoresis was prepared by a modi-fication of thediethylpyrocarbonate(DEP) technique

(20, 22). Bacteria wereexposed tolysozyme (500,ug/ ml) for the final 30 sec ofthe labeling period, fol-lowed by the addition of sodium dodecyl sulfate

(SDS) to 1.5% and DEP (Eastman Organic Chemi-cals, Rochester, N.Y.) to 3%. The clear, viscous ly-satewas incubated for an additional 3 min at 37 C and 0.5 volume ofcold saturated NaCl was added. Thechilled solutionwascentrifugedat12,000 xgfor 10 min and the supernatant solution was filtered

through a Nalgene 0.45-gm membrane filter unit

(Nalge Co., Rochester, N.Y.) and precipitated for 4 hr at -20 C with two volumes of ethanol. After centrifugation, the pellet was resuspended in water

and dialyzed against 0.1X Loenings buffer (12) for 1 hrat 4 C. Loeningsbuffer contains 0.036mm

tris-(hydroxymethyl)aminomethane (Tris), 0.03 M

NaH2PO4, 0.001 M ethylenediaminetetraacetic acid (EDTA) and 0.2% sodium dodecyl sulfate, pH 7.7.

The final RNA preparation was free ofDNA (13) andhad a specific activity ofapproximately 100,000 countsper min per.tg. Allpreparations for gel analy-sis were stored at -70 C until used. 029RNA proc-essed and stored as described above was stable for weeks.

Sucrose gradients. Initial approximations ofthe size of 429 RNA were obtained by subjecting total infected RNAto analysis by centrifugation through 15 to 30% linear sucrose density gradients

contain-ing 0.05 M sodium acetate, 0.01 Mdisodium EDTA,

0.01 M Tris (pH 7.4), and0.2% sarkosyl (Ciba-Geigy Corp., Ardsley, N.Y.). Centrifugation was for 12 to

14 hr (or as indicated) at 4 C, in a Beckman SW41 rotor at41,000rev/min.Aftercentrifugation, the

con-tents of eachtube were pumped through aflowcell and monitored at 260 nm to locate bacterial ribo-somal and transfer RNAs; each tube was

fraction-ated into 50 fractions containing 12 drops. Under these conditions, the23S RNA moves approximately two-thirds of the waythrough the gradient.

The positions of 23, 16, and 4S RNA markers in

the gradient were used to calculate the

sedimenta-tion coefficient of 029 RNA. The amount of viral-specific RNA in each fraction was determined by

hydridization to 029 DNA (8, 13). Blank filters

in-itially were included in each hybridization reaction

to evaluate the extent of nonspecific binding (e.g., resulting from bacterial DNA in the preparation), but since the background was low and constant

throughout the gradient, this step wassubsequently

eliminated.

Polyacrylamide gel electrophoresis. RNA was

analyzed by electrophoresis through columns (0.5

cm by 10cm or 15cm) of 2.5%polyacrylamide

con-taining 0.07% N,N'-methylenebisacrylamide (East-man Kodak, Rochester, N.Y.). The gels were poly-merized for at least 12hr, and electrophoresis was

conducted for 4hrat5v/cm priortothe addition of the sample. Gel tubes were completely submerged

in electrophoresis buffer to minimizeheating. Elec-trophoresis was conducted at room temperature at 10 v/cm (constant voltage) resultingin a current of

8 to 10 ma per gel. After electrophoresis, the gels were removed from the glass tubes and scanned at 260 nm using the linear transport attachment of a

Gilford 2000 recording spectrophotometer. The gelswerethen frozen inanethanol-dry icebath and cut into 2-mm slices. The molecular weight of the

RNA in each slice was estimated by comparing its

relative position with that of 23, 16, 5, and 4S bac-terial RNAs. Under these conditions a linear

re-lationship exists between the distance traveled by

an RNA molecule and the logarithm ofits molecu-lar weight (2). Each slice was incubatedfor 6 hr in

2 ml of 2 x SSC at 65 C to elute the RNA. Viral-specific RNA was detected by DNA-RNA

hybrid-ization. Gel slices did not appear to interfere with thehybridization reaction.

RESULTS

Analysis of viral RNA by sedimentation

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029 MESSENGER RNAs

through sucrose gradients. The results of competition-hybridization experiments re-ported separately (13) indicated that gene ex-pression during

029

development occurred in a highly regulated manner and involved two distinct stages. Viral RNA was analyzed by sedimentation through 15 to 30% sucrose gra-dientsinan attempt tofurther define the tran-sition from early to late transcription. Figure 1 shows the size distribution of X29 RNA labeled at progressively late intervals in the infectious cycle. In part A, the RNA was labeledfrom 2 to 6min after infection and thus well before the onset of viral DNA replication at 10 min (9, 13). Three major components were resolved, migrating with sedimentation coefficients of 17, 12, and 9S. In addition a shoulder was present on the

9S

component with a sedimentation coefficient of approxi-mately 6S. Viral-specific RNA could not be detected in the region ofthe gradient corres-pondingto asize greaterthan23S.

Figure 1B shows the sedimentation profile ofRNA extracted5.5 to 9.5 minafter infection, a time spanning the onset ofviral DNA rep-lication. RNA greater than 23S begins to ap-pear. In addition, a 14S component becomes prominentwhile the relativeamount ofthe 17S component hasdecreased.

Figure 1C shows the profile of

029

RNA labeled from 21 to 25 min after infection, well after the onset of viral DNA replication. The first intracellular phages are detected at this time

(unpublished observation).

Data from DNA-RNA

competition-hybridization

experi-ments have shown that most ofthe sequences in early RNA were present in late RNA, but that late RNA contained unique species ab-sent from early RNA (13). Furthermore, it was previously concluded that late RNA con-tained molecules of large molecular weight since experiments measuring the stability of late RNA after addition of rifamycin showed that phage RNA synthesis continuedfor 2min after addition of the drug (13). It can be seen

fromFig. 1C that themajority ofviral-specific RNA latein

429

development

migrated

inthe region of the

gradient

corresponding

to

ma-terial

larger

than

17S,

the

largest

component foundinearlyRNA. DistinctRNA components were recognizedat 30, 26, 20, and

14S,

aswell as inthe 6to9S region. The

complexity

ofthe pattern and the lack ofresolution made it dif-ficult to determine if the 12Sand 17S compo-nents were still

synthesized

at this late time. These experiments indicate that gene

ex-pression during phage

029

developmentoccurs

via the synthesis ofat least

eight

components ranging in size from 6 to 30S. Material

larger

than30Swasalso present at the bottom of the centrifuge tube at late times after infection. An estimate of the molecular weight ofRNA molecules was obtained by comparing sedi-mentation values of the viral RNAs withRNAs of known molecular weight (2). The size dis-tributionislarge,the smallest componentbeing less than 105 daltons, whereas the largest one isapproximately 2 x 106 daltons.

Figure 2shows the result of a similar experi-ment analyzing the size distribution of

029

RNA extracted from bacteria which were in-fected in the presence of CAP. Under these conditions only 80% of the early RNA and no late RNA is detected (13). The pattern is very similar to the early pattems shown in Fig. 1 in that components were detected at 17, 12, 9, and 6S, and no material larger than 23Swasdetected.

Analysis of viral RNA by polyacryla-mide gel electrophoresis: chloramphenicol RNA. Figure 2indicated thatatleast foursize components were synthesized when infection occurred in the presence ofCAP. The limited resolution of sucrose gradients suggested the possibility that other components might be present but not detectable. CAP RNA was, therefore, subjected to analysis by electro-phoresis through polyacrylamide gels in an effort to define further its composition.

Figure 3 shows the size distribution ofRNA extracted 29 min after infection from bacteria pretreated with CAP. Five components, super-imposed on a heterogeneous background, were found. Although the characterisitcs of the

background

material varyslightlyfrom experi-ment to experiment, these five components were reproducible. Since small variations arise from cutting or counting procedures, RNA peaks which consisted of less than two pointsonthesegraphsorwhich didnot appear in all gels have been

ignored.

The molecular weights are

presented

as a fraction of 106 daltons.

The largest molecules

migrate

between the 23S and 16S ribosomal RNA and have a

molecular weight of

approximately

0.75 x 106. Littleor noviral RNAwasdetectedinthearea

of the

gel

corresponding

to sizes greater than 23S.

Viral-specific

material was also present at 0.37 x 106, 0.25 x 106, and0.09 x 10' dal-tons, withavery

prominent

component

migrat-ing between the

bromophenol

blue marker dye and 5SRNA. ThisRNAhas an estimated molecular

weight

of0.04 x 106. The five

com-ponents haveanaccumulatedmolecular

weight

ofabout 1.5 x

106,

accounting

for 27% of the

transcriptional

potential

ofthe virus

(assuming

that

only

onestrand of

029

DNA is transcribed 89

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lo1

5W n FIG.1. Fractionationof029RNAbycentrifugation through sucrose gradients. Infected bacteria were

175

_ITS

w \ exposed to CAP (250

ag/ml)

and

3H-uridine

(40

A

\Ci/ml) at the indicated times. The RNA was ex-^ 4 N %12s tracted and fractionated as described in Materials

4 and Methods.

Approximately

100 jig

of

total RNA

x 6 was layered on each 15 to 30% sucrose gradient.

_II98 Centrifugation was for 14 hr using the SW41 rotor

a 3 l at maximum

speed

at4C. The

ultraviolet-absorbing

material was monitored (arrows indicate the posi-U s \ tionof bacterial 23, 16, and 4S RNA), and each

frac-c:a t \ tion washybridized to 10jgof029DNA to localize X 2 X \ viral-specific RNA. A, 2 to 6

min

afterinfection; B,

>_ > \5.5 to 9.5 min afterinfection; C, 21 to 25 min after

\ infection.

FRACTION NUMBER

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029 MESSENGER RNAs

o

750l

600-450- 17s

300-150

0 S 10 15 20

25

30 35

Fraction Number

FIG. 2. Fractionation ofCAP RNA by

centrifuga-tion through sucrose gradients. Logarithmically growing bacteria were treated with CAP (250 gg/

ml) at -5 minand infected with 029at 0min. The

infected bacteria were labeled with 3H-uridine (40

MCi/ml) from 25 to 29 minafter infection, and the RNA was extracted by the DEP technique. Frac-tionation anddetection of viral-specificmaterialwas

thesame asthat described in thelegendtoFig.1.

and that each size class represents a distinct species ofRNAand not adegradation product of a larger class). This number is, therefore, in good agreement with the estimation ob-tained from the DNA-RNA hybridization-competition data(13).

Analysis of early and late RNA. The number of viral RNA components and their

changes

during

the

lytic cycle

were further characterized

by

examination of RNA

pre-pared

atvarious times after infection. Figure4 shows the distribution of RNA labeled from 2 to 6 min after infection. The majorityofthe radioactivity was found to be associated with the 0.75 x 106 dalton component.

Comparison

ofthisgel profile andthose inFig. 1and2 indi-cates that this is the 17S RNA component resolved by sucrose. Viral-specific RNA was also found at 0.25 x 106 and 0.09 x 106daltons. In addition, material hybridizing to

029

DNA

200,

.0

0.09

0.75

0.25

100-00

10 20 30 40 50 60 70

Fraction Number

FIG. 3. Fractionation of CAP RNA by

polyacryl-amide gel electrophoresis. Logarithmically growing bacteria were treated with CAP(250 ug/mI) 5 min

prior to infection with 029. The infected bacteria

were labeledwith 3H-uridine (40 uCi/ml) from 25 to 29 minafter infection, and the RNA was extracted by the DEP technique. Between 20 and 30 Mg of

total RNA was layeredon a15-cm, 2.5%acrylamide

gel. Electrophoresis was for2.5 hrat 10 v/cm (see Materials and Methods for details). The gel was

analyzedat 260 nm tolocalize the 23, 16, 5, and4S components(arrows) and cut into2-mm slices. The RNA from each slice washybridized to 5

Mg

of029

DNA tolocalizeviral-specific material. The molecu-lar weights ofviral-specific RNAs arepresented as afraction of 106daltons.

was detected reproducibly in the region of thegel correspondingto 0.44 x 106 and0.37 x 106 daltons. This RNA wasdifferentfrom CAP RNA in two respects, i.e., the component at 0.04 x 106 daltons was missing and a new one at 0.44 x 106 daltons was present. The RNA at 0.04 x 106 daltons was present at slightly later times but still well before the

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LOSKUTOFF AND

P:NE

0

2

I0 a.

L) 8 _

'

6-

4-0.25

0.09

00

10 20 30 40 50 60 70

80 Fraction Number

FIG.4. Fractionationof2-to 7-min RNAby poly-acrylamide gel electrophoresis. Infected bacteria

were exposed to CAP (250

gsg/ml)

and 3H-uridine (40 uCi/ml) from 2 to 6 min after infection. The RNA was extracted and fractionated by the pro-cedure outlined in Materials and Methods. The RNA from each fraction washybridized to 5

tg

of 029DNAtolocalizeviral-specificmaterial.

onset of viral DNA replication (unpublished

observation).

The accumulated molecular weight ofthe six early species is 1.94 x 106or

about 35% ofthe potential RNA.

Theprofilewasverydifferent when the RNA

was labeled from5.5 to 9.5 minafter infection (Fig. 5). Synthesisofthe componentsat0.44 x 106, 0.37 x 106 and 0.25 x 10' daltons was

stimulated relative to the 0.75 x 106 dalton component and new components at 1.75 x 106 and 0.07 x 106 daltons were detected. This interval is thusatransitionperiodcharacterized by the synthesis of new RNA components.

0-0

x

2

0. 10 .I

IM

Fraction Number

FIG. 5. Fractionation of 5.5- to 9.5-min RNA by polyacrylamide gel electrophoresis. Infected bac-teria were exposed to CAP (250 Mg/ml) and 3H-uridine (40 MCi/ml) from 5.5 to 9.5 minafter infec-tion. The RNA was extracted and fractionated by the procedure outlined in Materials and Methods. T7he RNA from each fraction was hybridized to 5 Mgof029DNAtolocalize viral-specific material.

DNA synthesis could not be detected under these conditions (CAP was added at 5.5 min), consistent with the conclusion that the transi-tion from early to late RNA occurs independ-ently of 029 DNA replication (13).

Figure 6 shows an electropherogram of 029 RNAmoleculeslabeled from 25 to 29 min after infection. Distinct components were found at

1.75 x 106 and 0.93 x 106 daltons. Viral RNA

was also located between these two compo-nents with an approximate molecular weight of 1.4 x 106 to 1.5 x 106. The component at 0.07 x 106daltons was clearly resolved at this time, whereas the major early component (0.75)

wasnotdetected.

Nine late RNA components were recognized

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V129 MESSENGER RNAs

0. .0

I2

Fraction Number

FIG. 6. Fractionation of 25- to 29-min RNA by polyacrylamide gel electrophoresis. Infected

bac-teria were exposed to CAP (250 Ag/ml) and

3H-uridine (40 gCi/ml) from 25 to 29 min after

infec-tion. The RNA was extracted and fractionated by theprocedure described in Materials and Methods. The RNA from each fraction was hybridized to 5

Mgof029DNAtolocalizeviral-specific material.

in thesegels,twomigratingslower than the 23S

RNA (1.75 x 106 and 1.4 x 106 daltons), one

between the 23S and 16S (0.93 x 106 daltons) and six moving slower than the 16S ribosomal

RNA component (0.44 x 106, 0.37 x 106,

0.25 x 106, 0.09 x 106, 0.07 x 106, and 0.04 x

106 daltons). Again, much unresolved

mate-rial was found in the middle of the gel,

mak-ing it difficult to detect the component at

0.25 orto eliminate the possibilitythat others

exist in this molecular-weight region. At these

times, the rate of synthesis of all components

relative to the one at 1.75 x 106 daltons was

decreased, resulting in the tendency of this

species to dominate the profile. Very little

labeled RNA was found in that portion of the

gel corresponding to 0.75 x 106 daltons. In

addition, some material did not enter the

gels atthese late times.

Analysis of the RNA specified in the absence of viral DNA replication. Previous experiments indicated that the transition from early to late RNA occurred independently of viral DNA replication(13). It was therefore im-portant to confirm this result directly by gel analysis of the RNA components present. In addition, it was of interest to determine the effect ofthe restrictive temperature (45 C) on the RNA profile. Accordingly, bacteria were grown at 45 C and infected with wild-type 029or

TS35,

a

k29

mutant

temperature-sensi-tive for DNA replication (9). The RNA was labeled with 3H-uridine from 25 to 28 min after infection and extracted as described in Materialsand Methods. The results are shown in Fig. 7.

It is clear that distinct components are re-solved inboth cases and that these are similar to thoseseen at late times at 37 C (see Fig. 6). The presence ofdistinct species at 45 C sup-ports the conclusion that

029

RNA is rela-tively stable (13). The RNAs extracted from wild-type-infected (Fig. 7A) and TS35-infected (Fig. 7B) bacteriaarevery similar. The absence of the 0.75 x 106 dalton component is appar-ent in both cases, and both contain the large-molecular-weight material which is character-istic oflate RNA.

Effects of CAP on late profile. Pulitzer (18) and Cascino (3) have shown that expres-sion oflate T4 genes depends both upon viral DNA replication and the products of genes 55 and 33. Continuous protein synthesis was required for the continued expression of late genes. It was interesting, therefore, to deter-mine whether late

029

RNA continues to be made in the absence of protein synthesis, or whethergene expression revertsback to a pat-tern which is characteristic of early develop-ment. These questions were answered directly by gel analysis.

CAP was added to infected bacteria at 21 min after infection, a time when late RNA is being synthesized. Radioactive uridine was then added for 4-min intervals at 21 (Fig. 8A) or 35 (Fig. 8B) min after infection, and the RNA was extracted and analyzed. The results are shown in Fig. 8. It can be seen that the RNA components present in the control (Fig. 8A) are also present after 18 minofadditional exposure to CAP. 029 transcription is thus very different from that of T4 in that expres-sion oflate

029

genes continuesevenafter pro-longed periods in the absence of protein syn-thesis.

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LOSKUTOFF AND P1NE

11

0

6

00.

0.

u

0

-o

0

500-400

300i'

10.37

0.04

200

0.93

0.25

01

0

10

20

30

40

50

60

70

80

Fraction Number

FIG. 7. Fractionation ofthe RNA specified by TS35at thenonpermissive temperature. B. subtilis60084 wasgrownat45Cto2 x 108 cells permlandinfectedwithwild-type phageormutantTS35(multiplicity of

infection = 10-15). CAP (250ug/ml) and 3H-uridine (20

ACi/mI)

wereadded at25minafterinfection. The RNA wasextracted at28min andfractionated bytheproceduredescribed in Materials and Methods. The RNAfrom eachfraction washybridized to5pg of 029DNA to localize viral-specific material.A, 029wild

type;B, TS35.

DISCUSSION

In the discussion ofthe experimentsreported

in this communication we have assumed that in vivo transcription of

029

chromosomes is asymmetric andthat the maximum amount of RNA which can be

copied

from

029

DNA

would represent half of the molecular weight of the DNA. We therefore expect, as a maxi-mumvalue, toaccountfor 5.5 x 106 daltons of RNA sequences. As shown by electrophoresis

on acrylamide gels,

early

029

RNA is

com-posedofsixcomponents: 0.75 x 106,0.44 x 106, 0.37 x 106, 0.25 x 106, 0.09 x 106, and0.04 x

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V129 MESSENGER RNAs

50 60 70 80

Froction

Number

FIG.8. Viral RNA synthesis afterprolonged exposure to CAP.Logarithmically growing bacteriawere

in-fectedwith029 at 0minandexposed toCAP(250Asg/ml)at21min. A, 3H-uridine (20 MCi/mI) added from

21 to 25 min; B, 3H-uridine addedfrom 35 to 39min. The RNA was extracted and analyzed by

electro-phoresis through 2.5%acrylamide gels. TheRNA from each fractionwas hybridizedto5 ggof 429DNA to localizeviral-specificmaterial.

106daltons.Allof theseviral-specific molecules

except the component at 0.44 were detected

when infection occurs in the presence of CAP and, therefore, probably represent

tran-scription ofviralchromosomesbythe host RNA

polymerase. Although the 029 RNA speciesat

0.04 x 106 daltons was always detected as

one of the members of CAP RNA (Fig. 3), its appearance does not coincide with that of the other species of early RNA (see Fig. 4). The

following two possibilities arise from this ob-servation. (i) The RNA at 0.04 x 106 daltons

could represent an example of delayed

tran-scription in the absence of protein synthesis.

7

6

5

4

3

2

t0

10

I-lo

._

I

0

7

6

95

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LOSKUTOFF AND P1NE

According to the interpretation, this RNA would be located distal to the promoter of one ofthe other early species and would originate by "run-through" ofthe host RNApolymerase. (ii) The RNA at 0.04 x 106 daltons could be a breakdown product of one of the larger early species. Out data, at this point, cannot sup-port either of these possibilities. The major obstacle in obtaining additional information about the origin of this species is the fact thatverylittleearly RNAismadeimmediately after infection. The sum of the molecular weight of early RNA molecules is 1.94 x 106 or 35% ofthe maximumamountofRNA which could be transcribed from 429 DNA. This value is in good agreement with the40% value obtained from competition-hybridization ex-periments (13).

A difference between CAP RNA and early RNA was the absence of the 0.44 component in the former case. We cannot conclude from this observation thataviral protein isrequired as a positive control elementfortheexpression ofthismessenger RNA since ithasbeen demon-strated that promotor distal sequences in the tryptophanoperon are notdetected inthe pres-ence of CAP (16, 17). Thus, the distinction

which has been made between "immediate early" from "delayed early" functions in bac-teriophage T4 (15) or SPOl (5, 6) may reflect an effect of CAP on promoter distal tran-scripts.

The mechanism by which a drug inhibiting protein synthesis can affect transcription is notunderstood, although it has been suggested that this antibiotic may affect the stability of the distal portion of the tryptophan operon (17). In vitro transcription of

029

DNA mole-cules by purified RNA polymerase extracted from uninfected B. subtilis gives a product which ishomologous by competition-hybridiza-tion analysis to CAP RNA isolated in vivo (unpublished observation).

Continued transcription of late

029

RNA occurred even after prolonged exposure to CAP (Fig. 8). The large-molecular-weight components were stillsynthesized 18 min after addition of CAP, and synthesis of the 0.75 x 106 early component did not resume. These observations suggest that CAP, when added before infection, may in fact restrict transcrip-tion to 30% of the genome by inhibiting the synthesis of an early viral protein. If restric-tion resulted from an uncoupling of

transcrip-10

15 20 25

30

[image:10.495.113.396.370.611.2]

Minutes After Infection

FIG. 9. Summary of029transcription.Eacharrowrepresentsasize classof029-specificmRNA. The

initia-tion andterminationpoints(heads andtails of the arrows)weredetermined both from the data in this

commu-nicationaswellasfromthehybridization-competitiondatapreviously reported(13). The numbers above each arrow arethe molecularweights of the components,expressedasafraction of 106 daltons. Thestarafter the

early speciesat 0.04 x 106 daltonsrepresentsanuncertainty about itsexact timeofappearance(see

Discus-sion).

96 J. VIROL.

1.75

I

.o

(1.40)

2

0.93

a

075

0.44 i _ __

EARLY

LATE

0.37~~~~~~~~~~

0.25 _ _ _ _ *

0C CU'

0.09 0t

0.07

4o

0.04 ___________

_I P

0

5

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029MESSENGERRNAs

tion and translation (21) orfromthe decreased

stability of operator distal portions of viral transcripts (17), the profile should revertback

to the one characteristic ofthe RNA

synthe-sized in the presence of CAP. This does not

happen.

029

late RNA is composed ofat least three andpossibly four RNA molecules. These mole-cules were detected in bacteria infected with

wild-type X29orwith029TS35,a

temperature-sensitive mutant blocked in DNA replication. It therefore appears that viral DNA

replica-tion is not a requirement for the expression

of late genes in 429. k29 late RNA contains

components of molecular weight 1.75 x 106,

0.93 x 106, and 0.07 x 106. In addition, RNA

with molecular weight ofapproximately 1.4 x

106 was often detected. We are not certain at thistime that thecomponent at 1.4 x 106

rep-resents a unique RNA species since it was

detected verylate in theinfectious cycle, after

the appearance of infective

029

particles.

Further work is in progress to determine whether or notthis RNA represents a specific

cleavage product of the larger-molecular-weight RNA. With the omission of the RNA

at 1.4 x 106 daltons, the sum of the class III RNA is 2.75 x 106daltons or50% of the total

potential RNA. This value agrees with the valuedetermined bycompetition-hybridization analysis (13). It should be noted thatsynthesis

of the majorearly component, 0.75 x 106 dal-tons, appears to cease at approximately the time in infection when late RNAbegins to be synthesized. The cessation of synthesis ofthis

molecule doesnotappeartorequire viral DNA replication since shut-off can be detected in

bacteria infected with

029

TS35atthe restric-tivetemperature.

Figure 9 is a representation of the 029

tran-scriptionprogramas weunderstand it. We have

also detected late in infection k29 RNA with

a molecular weight of 2.5 x 106 (see Fig. 7) both in bacteria infected with wild-type and

with TS35. The appearance of this large RNA

molecule after the production of intracellular

virus suggest that it may represent the failure

of RNA polymerase to terminate properly at

these late times.

ACKNOWLEDGMENTS

This workwassupported byPublic HealthServicegrant AI-08077 from the National InstituteofAllergyand Infectious

Diseases. One of us (D.J.L.) was supported by Public Health Service and National Science Foundation

trainee-shipsadministeredbytheUniversityofColorado.

LITERATURE CITED

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5.Gage, L. P., and E. P. Geiduschek. 1971. RNA synthe-sis during bacteriophage SPOl development: six classesofSPOlRNA. J. Mol. Biol. 57:279-300. 6. Gage, L. P., and E. P. Geiduschek. 1971. RNA

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8. Gillespie, D., and S. Spiegelman. 1965. Aquantitative assay forDNA-RNA hybrids with DNA immobilized on amembrane. J. Mol. Biol. 12:829-842.

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12. Loening, U. E. 1967. The fractionation ofhigh molecular weight ribonucleic acid by polyacrylamide-gel elec-trophoresis.Biochem. J.102:251-257.

13. Loskutoff, D. J., J. J. Pine, and D. P. Andrews. 1972. Gene expression during the development ofBacillus subtilis bacteriophage 029. I. Analysis of viral-spe-cific transcription by deoxyribonucleic acid-ribo-nucleic acid competition-hybridization. J. Virol. 11:78-86.

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Virology 45:567-576.

15. Milanesi, G., E. N. Brody, 0. Grau, and E. P. Geiduschek. 1970. Transcriptionofthebacteriophage T4 genome in vitro: separation of "delayed early" from "immediate early" transcription. Proc. Nat. Acad. Sci. U.S.A. 66:181-188.

16. Morse, D. E. 1971.Polarityinducedbychloramphenicol and releasebySuA. J. Mol. Biol. 55:113-118. 17. Morse, D. E. 1970. "Delayed-early" mRNA for the

tryptophan operon. An effect of chloramphenicol? Cold Spring HarborSymp. Quant. Biol. 35:495-496. 18. Pulitzer, J. F. 1970. Function ofT4gene55. I. Charac-terization of temperature-sensitive mutations in the "maturation" gene55.J. Mol.Biol. 49:473-488. 19. Reilly, B. E., and J. Spizizen. 1964. Bacteriophage

deoxy-ribonucleate infection of competent Bacillus subtilis.J.Bacteriol.89:782-790.

20. Solymosy, F., I. Fedorcsak, A. Gulyas, G. L. Farkas, and L.Ehrenberg. 1968. A newmethodbased on the useofdiethylpyrocarbonateas anuclease inhibitorfor the extractionofundergradednucleic acidfromplant tissues. Eur. J. Biochem. 5:520-527.

21. Stent, G. S. 1964. The operon: on its third anniver-sary. Science 144:816-820.

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