Gene Expression and Stability of mRNA Affected by DNA-Arrested Synthesis in Gene 59, 46, and 47 Mutants of Bacteriophage T4

0022-538X/78/0027-0791$02.00/0

CopyrightO1978 AmericanSocietyforMicrobiology Printed in U.S.A.

Gene

Expression

and

Stability

of-mRNA

Affected

by

DNA-Arrested

Synthesis

in

Gene 59, 46,

and 47 Mutants

of

Bacteriophage

T4

JEN-LEIHWUtANDYUN-CHI YEH*

DepartmentofBiochemistry, Universityof ArkansasforMedicalSciences,LittleRock,Arkansas 72201

Received for publication 13 March 1978

Theeffect ofbacteriophage T4gene 59mutations(DNA-arrested synthesis)on kinetics of DNA synthesis, gene expression, and stability of mRNA has been studied. WhenEscherichia coli B was infected by a T4 gene 59 mutant, DNA synthesisproceededtoincreaselinearlyafterinitiation,butstartedtodecreaseat 8 min and was completely arrested at 12 min at 370C. At various incubation

temperatures(20to420C),the initialratesand times ofarrestof DNAsynthesis

were different, but the total amount of DNA synthesized was constant. This result supports the hypothesis that function of gene 59 is required for the conversion of 63S DNA moleculesto otherreplicative intermediates (39). The abnormality in protein synthesis caused bygene 59mutation ismanifested by(i) adelayed shutoff in the expression ofearly proteins(gene 43, 46,39, 52, 63, 42-45, andsomeunidentified proteins), (ii) areducedrateoflategeneexpression (gene

34, 37, 18, 20, 23, wac, 24, 22, 38, and 19), and (iii) an absence ofcleavage of

certainlate proteins (23,24,IPII and22to23*,24*,IPIII*, and smallfragments).

Itappearsthat therewas noeffectonthe expression ofgene 33, 55, and32bya

mutation in gene 59. Results obtained from an addition of rifampin at the

prereplicativecycle after infection indicated that mRNA fromgenes 43,rIIA,46,

39, 52, and63are more stable in T4amC5 (gene 59) than in wild-type-infected

cells. mRNA remainedfunctional longerin mutant-infected cells, and thismay explain theprolonged synthesis of certain early proteins. Thegeneexpression of other DNA arrested mutants-those in genes 46and 47-showeda pattern of abnormal protein synthesis similartothat found ingene 59mutant-infectedcells,

except morelate proteinsaresynthesized. Thegeneexpression intermsofphage

DNAstructureisdiscussed.

The temporal sequenceofbacteriophage T4 gene expressionduring thedevelopment of the T4 phage in its host appears to be precisely

regulated(9,11, 21,32).Forexample, expression ofprereplicative cistrons,suchasimmediate and

delayed earlygenes, is controlled bytwo inter-related mechanisms. The early genes are ex-pressed immediatelyafter infectionasthe host RNApolymeraserecognizes theirpromoterloci,

whereas the delayed early genes are expressed after the read-through of the immediate early gene termination sites, which is caused by the

appearance of a T4-specific antiterminator

and/orthe newinitiationofquasi-latepromoters (6, 11,20).

Wehave studied the role ofgene59in DNA synthesis and repair. We have shown thatthe

mutation ofgene 59 causes arrested DNA

syn-thesis, premature release of replicative DNA t Presentaddress:InstituteofZoology, AcademiaSinica,

Taipei, Taiwan.

from the replicative complex, little or no

con-catemerformation,slow repair ofprogenyDNA

fragments, and defective repair of UV and al-kylating agent-damaged DNA (35, 36, 39-41). Genes 46 and 47 also belong to the DNA-ar-rested synthesis (DA) gene class (9, 27). The effect that abnormal DNA synthesis, resulting from mutations ofgenesin the DA gene class, hasonthegeneexpression of bothearlyand late proteins hasnotbeeninvestigated.Thestudy of

gene 59is, inpart,important because it has been

mappedbetweengenes 32and 33, theformer of

which codes for DNA-bindingprotein and the

latter of which controls late gene expression.

Whetheror notthe gene 59mutationaffectsthe

expression ofthese genesisnotknown and re-quiresfurtherstudy.

Inthispaper, we present data onthe kinetics

of DNAsynthesisatvarioustemperatures, the

abnormal regulation of early and late gene

expression, cleavageoflate proteins,and

stabil-791

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ityof mRNA in gene 59 mutant-infected cells. Protein synthesis in gene 59 mutant-infected cells has also been compared with that of gene

46and 47 mutant-infected cells.

MATERIALS AND METHODS

Bacteria. Escherichia coli K strain CR63 (30),

which carriesansuI gene, wasusedasapermissive host forpreparingstocks of T4 ambermutants.E. coli

Bstrain 021carryingansu- genewasobtained from

I. Tessmanand used in14C-labeledamino acid labeling of T4 proteins for sodium dodecyl sulfate (SDS)

slab-gelelectrophoresis.

E. coli B strain Tr201, a low-thymine-requiring strain obtained from G. R.Greenberg,wasusedasa

nonpermissive hostfor T4 amber mutants in

[3H]-thymine incorporation experiments (B. Dale, Ph.D.

thesis,UniversityofMichigan,AnnArbor, 1968).

Bacteriophage. Bacteriophage T4D wasused as wild-type phage in all of the experiments. amC5, an ambermutant in gene 59, wasobtained from R. S.

Edgar.Itwaspurifiedbyback-crossingfive times with

wild-typeT4D. dar mutantswereisolated from our

laboratory (36).All other T4mutantsused herewere

obtainedthroughG. R.Greenberg.

Media.M9medium(16)wasmodified and used for

growingE.coli B021 in"4C-labeledamino acid

label-ingexperiments. One liter of M9 medium contained:

7gofNa2HPO4-2H2O,3gofKH2PO4 (anhydride),0.5

gofNaCl,1gofNH4Cl,24.7mg ofMgSO4,14.7mg of

CaCl2,and4gofglucose.

Chemicals.A"4C-labeled amino acid mixturewas

purchased from New England Nuclear Corp. (25

mCi/matom). Acrylamide, N,N'-bismethylene

acryl-amide, and No-Screen medical X-ray film (Kodak)

werepurchasedfrom Eastman Chemical Co.SDSwas

obtained from BDH Chemicals Ltd. Other chemicals werestandard reagentgradeproducts.

Measurement ofkinetics of DNA synthesis.

The procedure of measuring the kinetics of DNA

synthesishas been describedpreviously (40).

Preparation of'C-labeledT4phage proteins

for SDS slab-gel electrophoresis. The overnight

culture of E. coli B021 wasdiluted50-fold with M9

medium and grownto acelldensityof5 x 108 cells perml withvigorousaerationat37°C,then

D,L-tryp-tophanwasadded to20,tg/ml, and the culturewas

infectedwithpurifiedT4phage particlesata

multi-plicityof infection(MOI)of10.Attheindicatedtime,

inFig.2and3, 1.5-mlportions of infected cellswere

exposedtothe"C-labeledamino acidmixture(0.5uCi

[2 ,ug]/ml, algal protein hydrolysate, New England

Nuclear Corp.)for various timeintervals. Incorpora-tion of 14C-labeled amino acids was terminated by

pouringthe incubation mixture into three volumes of

chilled 5% Casamino Acids(Difco).The infected cells

werecollectedbycentrifugation,washed with50 mM

Tris-hydrochloride (pH 6.8), and then centrifuged

again.The washedcellpelletwasresuspendedin0.5

ml ofsamplebuffer, which contained0.0625M

Tris-hydrochloride (pH6.8),2%SDS, 10%glycerol,5%

2-mercaptoethanol, and 0.01% bromophenol blue.The

phage proteins were completely dissociated and

de-naturedbyimmersingthe14C-labeledsampleinboiling

waterfor 1.5 min. The samples were loaded on the slab gel for immediate electrophoresis or stored at -20°C. Typically, 30,000 cpm in less than 15

pl

of each sample were loaded.

SDSslab-gel electrophoresisand

autoradiog-raphy. The"C-labeledproteinsampleswererun on

theverticalslab-gelsystemdescribedbyStudier (29)

byusingthe SDSgeland buffer system ofLaemmli

(18) with slight modifications.No-Screen medical X-rayfilm was exposed to the dried slab-gel by direct contactfor various periods of time(normally4 days). The identification of T4 gene products on the autora-diogram wasaccomplishedby comparison with other published data (21, 31, 38). Therefore, most of the band identificationsaretentative oronlyprobable.

RESULTS

Kinetics of DNA synthesis at different

temperatures. The synthesis of new viral

DNA, which is controlled by the phageitself, is initiatedapproximately5minafterinfection and

continues through the rest of the replicative

cycle (9). The arrest of DNA synthesis in cells

infected with amber mutants of gene 59 begins

at8min after infection at 370C and ends at 12

min(39,41).

Phage DNAsynthesiswasmeasured at

tem-peratures between20 and420Cby adding

[3H]-thymine at3min after infection. Radioactivity

in acid-insoluble material obtained at various temperatures was counted (Fig. 1). The time of phage DNA initiation and rate of phage DNA

synthesiswere studied as a function of

temper-ature;highertemperature resulted inearlier

ini-tiation and higherrateofDNA synthesis. The

initiation of phage DNA synthesis occurred 3

min afterinfectionat420C,6minat300C,and

15min at200C. After initiation,DNAsynthesis

proceededatdifferentrates at different

temper-atures. In T4amC5-infected cells, the time of

arrest of DNA synthesis, as well as the total

amountof DNA synthesis,varies with the

tem-perature, but the total amount of

[3H]thymine

incorporation(about104cpmper 5 x107infected

cells or 0.5

jig

ofDNA per 5 x107infected cells)

remainsconstantinspite oftemperature

varia-tion. The phage DNA synthesized, as

deter-minedbydiphenylamine,wasequivalentto10-8

,ugofphageDNAsynthesizedperinfectedcells.

Thisamount is equivalent to approxiinately50

phage equivalents ofDNA synthesized for one phage-infected bacterium, when the molecular weightof the T4 DNA molecule is taken to be 123x 106daltons (17). These results show that DNA replication of T4 gene 59 mutants has normalinitiation of DNAsynthesis, proceeds to somedefinitestage, andthenceases.

Effect of gene 59 mutation on T4 gene expression. Theeffect ofgene 59mutation on the expressionofphageT4 proteinswas moni-J. VIROL.

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PROTEIN SYNTHESIS IN T4 DA MUTANTS

7

6-5

4

3

II..

II II

I, II I, I,

,i

i

11 /

1I /

II f

II

I

I,

I /

II/

~/

/

1 1 / I I

II

AA

jI

Id

LI0

_2 V6

-w 7V

_RMI .2= -a

*_

_ . . . .

20 40 60 80 100

TIME AFTER INFECTION (MIN)

FIG. 1. KineticsofDNAsynthesis in T4D- and T4amC5 (gene 59)-infected cellsatvarioustemperatures.A cultureof E. coli B Tr201,ataconcentrationof5x108cellsperml,wasinfected with T4DorT4amC5(MOI of 5) withaerationatdifferenttemperatures. /3H]thymine (20 uCi [3

lig]/ml)

wasaddedat3min (42, 37, 30, and

25QC)

or6min(20°C) after infection.Samples of0.1mlwerewithdrawnatvariousintervalstomeasure radioactivity in the acid-insoluble fractionasdescribedin thetext. Symbols: T4D: O---5 420C;0---0, 37°C; 0---0, 300°C; A---A, 250°C; V---V,20°C; T4amC5: 42°C; @-O, 37°C; 0-O, 30°C;

A- A,25°C;V-V,200C.

tored by autoradiography of SDS

polyacryl-amide slab gels after electrophoresis of

'4C-la-beled amino acid-labeled viral proteins. This

procedure provides more specific information

than that of RNA-DNA hybridization, which

can only distinguish a few classes of mRNA

expressed.Either T4DorT4amC5-infectedcells

werepulse labeled with "4C-labeledaminoacids

atthetimeindicated at370C, andthe

incorpo-rationratesweredetermined. Therateof

incor-poration of '4C-labeled amino acids gradually

increased afterinfection, reaching themaximum

rate at 10 to 12 min postinfection, and then

decreased(Table 1). The incorporationratesin

both T4D- andT4amC5-infected cellswere

al-most identicalup to30 minpostinfection. The

14C-labeled

amino acid-labeledproteinsfrom

in-fectedcells werethensubjectedtoSDSslab-gel

electrophoresis after complete lysis in 2% SDS.

Identicalamounts of "C-labeledproteins were

layered onto each gel slot for electrophoresis.

The differences inintensity of each protein on

autoradiogramsshould reflect the differencein

relativerateor amountofprotein synthesized.

0

/

C,)

-J J w

0

x

IL)

1-lo

O

X

a-w z

I

x LL. 0

z

0

0

a-0 z

A

A,

K

/Z

VOL. 27, 793

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794 WU AND YEH

TABLE 1. Rateof"C-labeledamino acid incorporation in T4D- orT4amC5-infected E. coli

B021cellsa

"C-labeledaminoacid incorporation Labelingtime(min (cpm/7.5 x107cells)

postinfection)

T4D T4amC5

2-4 34,800 36,500

4-6 35,800 40,000

6-8 40,900 40,500

8-10 41,300 40,600

10-12 41,700 41,600

12-14 44,500 41,200

14-16 40,600 39,000

17-20 38,700 38,800

22-25 28,700 27,700

27-30 24,000 21,350

aE. coli

B021

cultures (5x

108 cells

perml) were grownin M9medium and infectedat37°C with phage

atanMOI of10.At the time indicated, the T4-infected cellswereexposed to a'4C-labeledamino acid mixture

(0.5jLCi [2

Itg]/ml).

Under this condition the

incorpo-ration of'4C-labeledaminoacids remainedlinear for atleast4min.The infectedcells were washed free of

'4C-labeledamino acidsby centrifugation and lysed in

0.1ml of sample buffer by heating at 100°C for2min. A10-gldportion ofeach lysate was placed onto round Whatman ffiterpaper(no. 3, 2.5-cm diameter) to de-termine the radioactivity in trichloroacetic acid-insol-uble fraction. The same sampleswere subjected to

SDS slab-gel electrophoresis (Fig. 2). Note that the last threepulsesarefor3min, whereas the rest are for

2min.

The effect of the gene 59 mutation on gene expression of early and late proteins and the conversion ofgeneproducts has been examined and isshown inFig.2.The expressionofmostof the immediateearly and delayed early proteins, whicharesynthesized before theonsetof DNA synthesis, ceased at 10to 12 minpostinfection

at

370C

in wild-type T4D-infected cells.

How-ever, in T4amC5-infected cells, the shutoff of

earlyprotein synthesiswasdelayed, and synthe-sisofsomeof theseproteinswasprolongedeven until30minpostinfection.These early proteins includedgenes43, 46,39, 52,63,42-45,andsome other unidentified phage proteins. However, otherearly proteins were not affected, suchas rIIA, 32-44,rIIB, and IPIII. In wild type, the late proteinswereexpressedat 8min postinfec-tionunderourconditions, asjudgedby autora-diogram (Fig. 2). All of the late proteins were

synthesized in T4amC5-infected cells, but the rateofsynthesis ofsomelateproteinswaslower (e.g., geneproteins 34, 37, 18, 20, 23*, wac, 24,

22, 38,and 19)whencompared with that of

wild-type-infectedcells.Therateofsynthesis of other lateproteins in T4amC5-infected cells was ap-parently thesame asthat ofwild-type-infected

cells; e.g., gene proteins 7, 10, 8, and 35. Four head-associatedphageproteins,suchasthoseof

genes22, 23,24,andIPIII,arecleavedtosmaller

polypeptides, namely, 23*,24*, andIPIII*, dur-ing morphogenesis (18). Gene 22 product is

*0 0 0* o * o * 0 0 0 0 0 0

--__- - _-_,34 -7

43 - _ -

-riA ____ - _ _ _ 10

46 o3

39 -~~;5;*_=:z=23

F- o2

t; 52 __ _52

D 63

0 32-44 *w-83

42j_s__P_;*;_*

-4 38

~# wj

PITu

2IM

4m

-

p-a-CmsmmaOnam asmsdui'wsa 'i e, 19

2 4 .4 6 68 8 10 10 12 12 14 114- 16 117 20j22 25 27-30

TIME AFTER INFECTION (MIN)

FIG. 2. Autoradiogram ofSDS slab-gel after electrophoresis ofproteinpulse labeled with "C-labeled

aminoacidsatthetimeindicatedafter infectionofE. coli B021 byT4DorT4amC5. See text for detailed

information.Geneproductsof earlyand lategenesareindicatedonboth sidesontheplate. Symbols:@, T4D;

0,T4amC5.

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PROTEIN

cleavedtoformanunidentifiedproduct(13,18). The conversion of 23 to 23* is very inefficient and that of IPIII to IPIII* did not occur in T4amC5-infectedcells (Fig. 2).The conversion

of24to24*was notresolved under these

exper-imental conditions. The available evidence

sug-geststhatthe proteolytic cleavagesof

head-as-sociatedphage proteinsareintimatelylinkedto the headassemblyprocess(19).Thecleavageof theproducts ofgenes23,24,IPHI,and22to23*, 24*, IPIII*, and small fragments is blocked by mutation inanyof theheadgenes20,21,22,23, 24,and31.Onorato and Showe havegiven evi-dence that proteolytic activity resides in the

gene 21 protein (22). However, T4-specific

pro-teolytic activities arefound at8 to 10min and are maximal at 18 min after infection (2, 23). The expressions of the proteolytic enzyme(s)

maybe reducedby thegene 59 mutation, and, in consequence, the proteolytic cleavage of a number of proteins isreduced bygene 59 mu-tation. Another possible reasonfor the lack of head protein cleavage may be the abnornal DNA synthesis in this mutant, since complete

headassembly normallydependsonDNA

pack-aging events (19). Since theearlygenes 33and 55controlthe expression of lategenes(3, 24, 28)

and since muchlate proteinismadebyagene 59ambermutant(Fig. 2),it appearsthat expres-sion ofgenes 33and55isnot

seriously

affected (ifatall) byamutation ingene 59.

StabilityofearlymRNA's ingene 59 mu-tant-infected celis. Some immediate early

genes(genes39and52)anddelayedearlygenes

(genes 43, 46, and 42-45) in amC5 (gene

59)-infected cells showedprolonged expression(Fig.

2). Possible reasonsfor this

delay

in shutoff of

earlygenes are anincreasedrateof

early

mRNA

synthesis,anincreased

stability

of

early

mRNA (or slowerrateof mRNA

degradation),

orboth. Thesealternativescanbedistinguished

by

treat-mentoftheinfected cells with

rifampin.

Rifam-pin bindsto the

fl-subunit

of theE. coli RNA

polymeraseandtherebyinhibits the initiation of

transcription(12).Ifthegene 59mutation affects

early RNA synthesis, the gene expression of mutant-infected cells after rifampin treatment at the early stages ofinfection should be the

same asthewild-type-infectedcells because the

effect of gene 59 function of gene expression

occurs after 10 min ofinfection. On the other

hand, if theearly messenger of mutant-infected cells remainsfunctionallongerthan that of wild-type-infectedcells,theearlygene shutoff in

mu-tant-infected cellswill beprolonged evenafter

rifampintreatment.

E. coli B021 cells were infected byT4D or

T4amC5 at37°C (MOI, 10), and rifampin was

addedtoafinalconcentrationof400,ug/mlat 4

mm afterinfectiontoinhibit earlymRNA

syn-thesis.Thecapacityofrifampin-treated cellsto

synthesize earlyproteinswasmeasured by

pulse-labeling

with '4C-labeled amino acids followed

by

SDS

slab-gel

electrophoresis(Fig. 3). In the

rifampin-treated cells, the same amount of mRNAwassynthesizedinT4D- and T4amC5-infectedcells,asindicatedbythe fact that both

T4D and T4amC5 have the same pattern of geneexpressionforthefirst10minafterphage

infection.

However,

someearly proteinssuchas gene proteins 43, rIIA, 46, 39, 52, and 63 were

synthesizedmoreinT4amC5- than in

wild-type-infectedcells. Thisisconsistent with the greater

stabilityofthe mRNA ofthose genes inT4amC5

infection thaninT4D. The messengers of other

earlygenes such as (32-44-rIIB) and IPIII

ap-parentlyhave the same stability in T4D- and

T4amC5-infectedcells,asindicated by the

iden-ticalintensityoftheproteinbandsinthe

auto-radiograms (Fig. 3).Since theeffect of the gene

59 mutation on the expression of some early

genes is due to this effect on the stability of mRNArather than on RNA synthesis, this in-dicates that gene 59 may act on phage gene expression by affecting some nuclease activity related to the degradation of these early

mRNA's. This gene 59-related nucleaseactivity appearstobespecific for gene 43, rIIA, 46, 39,

52, and 63 mRNA's, but not those for

(32-44-rIIB)andIPIII.Thisspecificity of mRNA

deg-radation may beduetodifferent nucleases acting on these two groups of early mRNA's. Other possible reasons for the delay in shutoff are presentedinDiscussion.

Temporal relationship between

expres-sion of gene 59 protein and its effect on

gene expressionin T4phageinfection. Nor-mal initiation of DNA synthesis in T4amC5-infected cells occurs, but the rate of DNA

syn-thesisbeginstodecreaseat 8min andcompletely

arrests at12min(41). Since the gene59

mutant-infected cells showdelayed shutoff of early genes andexpressionoflategenes,thequestioncanbe

asked: atwhattime after phage infection does

gene 59begintoexert its effectonphage gene expression?Thisquestion was examined by

add-ingrifampinatvarious times after T4Dinfection

and labeling phage proteins with "C-labeled

amino acid at the latelytic cycle (12 to 18min

afterinfection).Incontrasttotheabove

experi-ment(Fig. 3),wefocusedonlate gene expression. When rifampin was added up to 6 min after wild-type T4Dinfectiontoblock further

initia-tionof mRNAsynthesis,theT4D-infectedcells

only synthesized prereplicative gene products butshowednolategeneexpression. Thisresult 795

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796 WU AND YEH

S 0 0 0 0 0 0 0 * 0 * 0 0 0 * 0

F

43-D)

r31IA _

0

o 46

X 39

Z 52-- -- -

-C 63 f _ _

-- ab --

--a__-_

32-44-rIB

--

42-45-PIE-

-b_

.'1'1

'M

qw

vw

ww

--q q

C Sa__n___

_t~..__S a_ .. ....

amm

= -0

4 - 7

17

--10 110 13

1

13 16 116 19

TIME AFTER INFECTION (MIN)

FIG. 3. Decay ofprotein-synthesizing activity in T4D- and T4amC5-infected cells after rifampintreatment.

E.coli B021 culturewasgrown in M9 mediumto5x 108 cells per mlat37C andinfectedwithphagesatan

MOIof10.At4minafter infection, rifampinwasaddedtoafinalconcentrationof 400,ug/ml.At the indicated

time after infection, "C-labeled amino acids were added to 0.5 ,uCi (2 Lg)/ml of infected cultures. The

conditionsforelectrophoresis and autoradiographyaredescribed in thetext.Symbols:@, T4D;0, T4amC5.

is incontrast toT4amC5-infected cells without rifampintreatment, eventhough thephage pro-teinswerelabeledatthe latestageafter infection (datanotshown). Although early gene expres-sionwasaffected as early as4 min, this result shows thatthegene 59productdidnotexertits effect onlate geneexpression until 6 minafter

infection. However, whenrifampin was added at

8minafter infection,thepattern ofphage pro-teinsynthesiswasthe same asT4amC5-infected cells without rifampin treatment, showing de-layed shutoffofearlyproteins and reduced

syn-thesisof lateproteins.

Ten minutes after T4D infection, more late proteins were gradually synthesized than in T4amC5-infected cells withoutrifampin

treat-ment. The conclusion from this experiment is

that gene 59 starts to exert its effect on gene

expression at 8 min after infection. The time

that gene 59 function starts to act on phage

expression coincides with the arrest of DNA synthesis(41).

Comparison of gene expression ofgene

59 mutantswithother DNAarrestmutants

(genes 46 and 47). The gene expression of T4amC5wascomparedwith other DAmutants:

T4amN13O (gene 46) andT4amA456 (gene 47). Early gene expression was the same as in T4amC5 (gene 59)-, T4aml3O (gene 46)-,

T4amC5-amN13O (gene 59-46)-,

T4aml3O-amA456 (gene 46-47)-, and

T4amC5-amN13O-amA456 (gene59-46-47)-infected cells (datanot

shown). All of these DA mutant-infected cells

showed delayed shutoff of early genes when

compared with wild-type infected cells.

How-ever, some differences were observed in late

protein synthesis between gene 59 mutant and

gene46, 47mutant-infectedcells. Inalatestage

afterphage infection(20 to 22.5 min), somelate

protein products such as 34, 37, and 23* were N ....

19 - 22 |22-25125 28

r..Nomw

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PROTEIN SYNTHESIS IN T4 DA MUTANTS

synthesized more in T4amN130- and

T4amN130-amA456-infected cells than in

, amN130- and

T4amC5-amN130-amA456-infected cells.

Effect of dar mutation on gene

expres-sion ofgene59 mutant. Wehave isolated dar

mutants (DA restoration) whichare extragenic suppressors of thegene 59mutant (36). These

mutationssuppressthe defect in DNA synthesis

of DAmutantsofgenes46, 47, and 59. In

addi-tion, progenyformationby gene59 mutants is

restored byadar mutation. We examined the

effect ofadar mutationonthegeneexpression

of thegene59mutantby constructingadouble

mutant,T4darl-amC5 (dar-gene 59) (datanot

shown). We found in T4darl-amC5-infected

cellsthat the shutoff of knownearlygenessuch

as43, 46, 39, and 42-45wasdelayedupto18min

afterinfection, but the timeof shutoffwasstill

earlier than that of T4amC5-infectedcells.

Although the temporal expression of late

pro-teins indarl-amC5-infectedcells wassimilarto

that in T4darl-infected cells, most of the late

proteins and the conversion of head proteins (23

to 23*, 24 to 24*, and IPIII to IPIII*) were

reduced. These results indicated thatalthough

T4darl restored the arrested DNA synthesis

andprogenyformation ofgene59 mutation toa

nornal level, the gene expression of

T4darl-amC5wasnotascompleteasthat of theT4darl

control. The burst size of T4darl-amC5 was

increasedto200+ 10,ascomparedto25± 5 in

T4amC5-infectedcells.Wehavealsocompared

gene expression of T4darl with that of T4D

(37).

Expression ofgene32protein (DNA

bind-ing)ingene59mutant-infectedcelis.In

dar-amC5 (dar-gene 59) double mutant-infected

cells,wefoundoverproduction ofgene32protein

(DNA binding) as compared with amC5 (37).

Gene 32proteinis known to be involved in DNA

replication, repair, and recombination (1, 41), and gene 32 is contiguous to gene 59 in the

geneticmap.However,in thepresentstudythe

expressionofgene32proteiningene59

mutant-infected cells wasnormal (datanotshown),

in-dicatinglack ofapolareffect.

DISCUSSION

Atvarious incubationtemperatures,thetotal

DNAsynthesized in thegene59mutant-infected

cells was equivalent to about 50 phage DNA

units per infected bacterium. This constant

amountof DNAsynthesis, occurringatvarious

initial replication rates, indicates that mutant

DNA replication proceeds to some fixed point

and then stops. The arrest time of DNA

repli-cation in thismutantis thesame asthat for the

appearanceof defectivejoiningofprogenyDNA

andprematurerelease ofreplicativeDNA from

cativecomplex (41). Thissuggeststhatgene 59 isinvolved in the conversion of early replicative intermediatesto late ones,which is character-ized by theformnationof concatenated molecules

frommonomers of DNA.

We have also shown in this paper that a mutation ingene 59 has a pleiotropic effect on T4 phage gene expression, causing delayed shutoff of earlygenes,reduced expressionof late

genes,andblockage of head-associated protein

conversion. In addition,someearly mRNA's re-mained functional longer in agene 59 mutant than inwild-type-infectedcells. This result

sug-geststhatsome RNaseactivity involvedin the

degradation ofearly mRNA is affected bygene 59mutation.

Some possible explanations for delayed shut-off ofsome immediate and delayed earlygenes

are asfollows. (i) The early mRNA'sare

contin-uously synthesized in gene 59 mutant-infected cells, and the pattern of phageproteinsynthesis issimplyduetothecompetitionbetween early andlate mRNA's for the translationapparatus. Whenrifampinis addedattheearlystageafter phage infection toblock the initiation of tran-scription, the early protein synthesis inthe mu-tantstill continues longer than in the wild-type (Fig. 3). This modelassumes the same rate of mRNA synthesis, but says that the gene 59

mutantmakes it longer period of time. Another

variation ofthis model says that the gene 59

mutant could make early mRNA faster from

timezero.(ii) Thegene 59product isresponsible

foramodification (suchasmethylation) ofthe

existingearly messengers,renderingthem non-translatable. This functioncannoteasily explain

therequirement for the gene 59function in T4 DNAreplication and DNA repair (30). (iii) By mutation ingene 59,thestabilityofsome early

gene messengersisincreased (Fig. 3). Thus,gene

59 product may contribute an RNase activity which isresponsible forthe degradation of some

early messengers at a certain time after infec-tion. This kindofactivity is consistent with the

pleiotropic effects of this gene mutation. (iv) There is a greater initial rate of synthesis of

early mRNA or delayedshutoff of early tran-scription; ifribosomes are normally saturated

with mRNA, this extra mRNA willhave little

effectonproteinsynthesis until mRNAdecays belowsaturating levels. Thenprotein synthesis

should lastlongerinthe gene 59 mutant case (its

higher level would take longer to decay below saturation), withoutinvokinganychangeinthe

inherent decay rate. (v) Some translational

shutoff ofearly protein synthesismay be

dimin-797 VOL. 27,1978

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ished by the gene 59 mutant state. This could indirectly extend the lives of the affected mRNA by protecting themwith ribosomes, asseems to be the casewith regA translationalcontrol (14,

34).

The control of the shutoff of earlygenes by

gene 59is different from that inmutantsT4SP62

(33, 34)andT4R9(14),whichcanprolong early

gene expression up to 60 min compared to 12

minforthe gene 59 mutant. BothT4SP62 and

R9are mutated in regA gene, which maps

be-tweengenes 43and62intheT4genome(14, 33).

The abnormalities ofearly gene expression of SP62 and R9mutants can be detected only in DNA negative (DO) or maturation defective

(MD)states.MutationsinDOorMD genescan

cause a delayin early geneshutoff ofabout 12

min (14). Thus, it was thought that the first

stage

(Si)

was that controlled by DO or MD

genes, and the secondstage (S2) wasthat

con-trolled by regA (34). The delayed shutoff of early genesin DO orMD mutantsmay be due to lack ofa late protein that isrequired either for the switch of translation from earlyto late

messengers orthedegradation ofearly

messen-gers(4, 34), but itcouldequallybe duedirectly tolackofcompetition from late mRNA,which

isnotmadein DOorMD infections. Although

the late proteins are expressed in the gene 59

mutant, the shutoff ofearly proteinsis still

de-layed for 12min (Fig. 2 and 3). The shutoffof

earlymessengersynthesisisdelayed by addition ofchloramphenicol to block early protein syn-thesis, indicating that some early proteins are involved in the shutoff mechanism (5).Atleast

the gene 59 function, an early gene function,

may also participate in the shutoffmechanism for early proteins either directly or indirectly.

Thismaybeanexceptiontothepostulated

first-stageshutoff mechanism forearlyprotein(s) that

issolely controlled bylateprotein(s) (5). It has been shownbyusing rifampinthat the

delayed shutoff ofearly proteins by the T4R9 mutation has a correlation with increased

sta-bility (14, 33). Theincreased

stability

could be asecondaryresult of continuedselection of those mRNA by ribosomes (14, 15, 34). The T4R9 mutationprolongedtheexpressionofsomeearly proteins, such asrIIA, (32-44-rIB), and42-45,

butnot43, 39,53, and IPIII (14). Onthe other

hand,T4amC5-infected cells showedprolonged expressionofearlygenes,suchas43, 46,39, 52,

63, and 42-45, but not rIIA, (32-44-rIB), and

IPIII (Fig.2). Itappears that gene59andregA

express their effects through different

mecha-nisms to act on early gene shutoff: e.g., the

specificities of gene 59 and regA functions are

different withregardtodegradationof theearly

messengers.

In DO or MD mutant-infected cells, no late

genes areexpressed (3,28, 34), whereas the gene

59 mutant can express some late proteins. The

difference inlategeneexpressionbetween gene

59mutants andDOor MD mutants isprobably

due to the fact that gene 59 mutant-infected cells have normal initiation of DNA synthesis for3 min (35) and the assumption that it has normal phage modificationof host RNA

polym-erase. The progeny DNA molecules that are

synthesized before arrest canbe transcribed for some lategene expression. However, thissmall

amount of lateprotein synthesisby the gene 59

mutant canbe eliminatedby the introduction of

aDOorMDmutation (datanotshown). These results indicate that replicating DNA can be used for lategeneexpressionandparentalDNA isrequired forearlygene expression. T4 parental DNA canbe used as a template for late gene expression whenatriplemutant,whichis defec-tive in DNApolymerase (gene43), DNAligase

(gene30), and exonuclease (gene 46) is used to

infectanonpermissivehost (8, 38). This require-ment ofa multiple mutationfor replication-in-dependent expression of lategenes tends to

sup-port the concept that normally onlythe newly

replicated phage DNA, which is different from parental, iscompetent forlate geneexpression

(7, 25). The competent DNA contains gaps or

breaks, whichare essential forthe initiationof lategeneexpression. Soon after completearrest ofDNAsynthesis ingene 59, 46,and47 mutant-infected cells, the expression of late genes is affected (datanotshown). This effect indicates

thatgene 59, 46,and47functionsmaybe directly

orindirectly involved in the creation ofnicks or

gaps necessary for late gene expression, since

some progenyDNAissynthesized before DNA

arrest, andmodification of host RNA

polymer-aseis assumedtobe normal. Furtherstudies on abnormalities in the regulation of protein syn-thesis and the structure ofDNA molecules in gene59-infected cellsmayprovide better

under-standing of itsbiochemical function.

ACKNOWLEDGMENT

Thisinvestigationwassupported byPublicHealth Service research grant GM18012 from the National Institute of Gen-eralMedical Sciences.

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