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Genetic mapping of regA mutants of bacteriophage T4D.

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Copyright © 1977 American Society for Microbiology Printed in U.S.A.

Genetic

Mapping of regA Mutants

of

Bacteriophage

T4D

JOHN S. WIBERG,* STEVEN L. MENDELSOHN, VIRGINIA WARNER,' CYNTHIA ALDRICH,2 AND THOMAS S. CARDILLO

Department ofRadiation Biologyand Biophysics, School ofMedicine and Dentistry, University ofRochester, Rochester, New York14642

Received for publication 22 November 1976

SP62, a mutant ofbacteriophage T4 shown by Wiberg et al. (1973) to be defectiveinregulation ofT4proteinsynthesis, wasshown by complementation

tests todefineanew gene,regA,and byintergenic mapping tolie betweengenes

43and62.ThemappinginvolvedcrossingSP62 withaquadruple ambermutant

defectiveingenes 42, 43, 62,and44,selecting allsixclasses of amber-containing recombinantscaused by single crossover events, andthen scoring the presence orabsence of SP62intheserecombinants.Inaddition, 15 new,spontaneous regA

mutants wereisolated, and13 oftheseweremapped against eachother;a total

ofeight different mutation siteswerethus defined. Most of the new mutants wereisolatedaspseudorevertants ofaleaky ambermutant ingene 62,according

to KaramandBowles (1974), whereas one was identified by virtue of the "white

ring"arounditsplaque,aphenotype possessedby all the regA mutants at high

temperature. SP62 was renamed regA1, and the new mutants were named

regA2,regA3, etc.

Aviablemutantofbacteriophage T4Dthat is

defectiveinregulationofphageprotein

synthe-sis, namely SP62, wasdescribed by Wiberget

al. (18); unpublished datawere cited to argue

that the mutant, SP62, defines a new gene,

regA, thatmapsbetweengenes 43 and62. The

present paper presents those data, which

in-volve both complementation tests and

inter-genic mapping of SP62. Also described is the

isolationandintragenic mappingofmoreregA

mutants.

MATERIALS AND METHODS

Bacteriophage and bacterial strains. Escherichia coli B and E. coli K-12 strain W3110, both nonper-missive for T4 amber (am) mutants, and E. coli CR63, permissive for T4 am mutants, have been described (8, 17). We isolatedaspontaneous mutant of E.coliBthat is resistantto 150pgof

streptomy-cinpermlandassigneditbacterialstock no. 150;it

exhibits drastically reduced ribosomal ambiguity suppression ofT4 amber mutants. Wild type T4D (T4+) and the unbackcrossed versions of the am

mu-tantswereobtained fromR.S. Edgar. SP62 has been described (18), and all studies on it in this paper

were done with the genetically purified version, SP62x3; this has been renamed regA1 (see below), andbothnames areusedinthis paper. The quadru-pleam mutant used for mapping (see Table 2) was constructed by standard crosses and identified by the spot-testprocedure describedbelow;itcontains

1Presentaddress: 20Offgrove St., E. Weymouth, MA 02189.

2Presentaddress:1857LindenSt.,E.Lansing,MI 48823.

the following mutations: amN55x5 (gene 42), amB22x5 (gene 43), amE1140 (gene 62),and amN82 (gene 44).

Growthmedia and chemicals. Most of these have been described (18). GCA medium is the

glycerol-CasaminoAcids medium of Fraser and Jerrel (5). Thermometers. All temperatures above 40°C were read on thermometers calibrated against a

thermometer certified by the National Bureau of Standardstobeaccuratewithin0.3°C.

Complementation tests. For complementation

tests (Table 1), E. coliB wasgrownat37°CinGCA medium to about 5 x 10f cells per ml and then chilled. For eachinfection,1ml ofcellswaswarmed

by shakingfor3 min at 44.8°C in a 10-ml, baffled

Erlenmeyer flask. Phage (0.1 ml) were thenadded

at atotal multiplicity of infection (MOI) of10(5 of each, whereamixtureoftwophagewasadded).At 50 min after infection, samples were diluted into broth saturated with CHCl3 (to lyse the cells) for determination ofphageproduction; platingwas on

E. coli CR63 at30°C, conditions permissive for all progeny. Bacterialsurvivorsof infection were deter-minedas colony-formers by spreading appropriate dilutionsonGCA agar,alongwithtwodropsof anti-T4 serum havinga K value of 260; theantiserum

preventskilling ofsurvivorsby phageproducedon

theplates. Controls showed that this level of antise-rum was adequate and harmless to the bacteria. Unadsorbed phage were measuredas

plaque-form-ers thatsurvive dilution through CHCl3-saturated broth; theywere sampledat 5 min afterinfection, before progenyphage appear.

Spot-test identification of mutant genotypes. Ourcurrentprocedureforspot-testidentificationof mutantgenotypes(Table 2)istheresult ofaseriesof

742

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regA MUTANTS OF 743

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modifications in this laboratory (8, 9, 17, 18) of the technique ofEdgar et al. (4). Each plaque, chosen at random from the progeny of a cross, was stabbed

TABLE 1. Complementation testsof SP62 against nearbymutantsa

Total phage pro- %of corre-Pha e duced/cellat

44.80Cb

Avg% sponding

Phg of T4+ (T4++am)

Expt 1 Expt 2 cross

T4+ 74(100) 64(100) 100

SP62 15(20) 14(22) 21

SP62 +T4+ 65(88) 83 (130) 109

T4+ + 43- 86(116) 56(88) 102

T4+ + 62- 44 (60) 38 (59) 60 T4+ + 44- 83 (112) 45 (70) 91

T4+ + 45- 68(92) 38 (59) 76

SP62 + 43- 68 (92) 44 (69) 81 80

SP62 + 62- 37 (50) 25 (39) 45 75

SP62 + 44- 40(54) 49 (77) 66 73

SP62 + 45- 40(54) 29(45) 50 66

43- 0.16 0.17

62- 0.44 0.22

44- 0.18 0.09

45- 0.13 0.14

a Procedures aredescribed in thetext.Theresults

of twoindependentexperimentsareshown. Nomore than 1.5% bacterial colony-formers survived at 5

minafterinfection; this wasmeasuredonlyin the infections bysingle phage.Unadsorbedinputphage

were measured at 5 min after all infections and

representedlessthan0.7phagepercell;thesewere

not subtracted from the values shown. Thefigures

in parentheses represent the percentage ofphage

production relative to T4+ as 100%. The 43-, 62-,

44-, and 45- mutants were amE4332, amE1165,

amN82,andamE10,respectively.

bCell titerbasedoncolony-formerspriorto infec-tion.

oncewith a sterile, wooden toothpick; a toothpick canpickup over108phage. The toothpickwasthen rinsed in two drops of medium in one well ofa sterile, plastic Linbro plate (Disposo Tray, catalog no.IS-FB-96, clear, Linbro Scientific Co., New Ha-ven,Conn.).Wemarked offan arrayof56suchholes onthe Linbro plate such that thepatternfit comfort-ablyinastandardplastic petridish(15by100mm). About 1 ,ul of each plaque suspension was then transferredwithareplica-plating devicetothe sur-face of thetopagarlayer (3 ml) seededwithinthe preceding5min with108bacteria and5 x 107helper phage where appropriate. We now use a "floating-loop replica-plating device" (J. S. Wiberg, J. Appl. Bacteriol., in press), whichwas developed both to

givegreater accuracyandreproducibility of liquid transfer andtoavoidtearing the softagarlayer. It featuresanarrayof 56thin, wire loops mountedso that theyarefreetomove upand downuponcoming intocontactwiththeagar.To identify amber

geno-typesfor Table2,agarplateswereseededasfollows:

(i)E.coli CR63 (replica); (ii) E. coli B (no ambers grow); (iii) E. coli B plus helper amN55x5 (gene

42)-only candidatescontainingtheamN55x5 muta-tion will failtogiveapositive complementationtest; (iv, v, vi) same as iii, except substitute amB22x5 (gene 43), amE1140 (gene 62),oramN82 (gene44), respectively,ashelper. A positive complementation

testappears as ashotgunpatternofmanydiscrete

plaques, similarto spot B8of Fig. 2; these plaques result from complementation, which permits phage production, and (equallynecessary)from the

subse-quent replication of resulting wild-type recombi-nants.

Thepresence orabsence of SP62inthe six recom-binantclasseswastested by spot-plate procedure2

ofWibergetal. (18), whichmeasurestheabilityof the candidatetocomplement helper SP62onE.coli B atabout450C, where SP62 doesnotgrow. Since

temperature control is very delicate for this test (18), manyreplicaswereprinted, andoneach spot-plate several control spots ofT4+ and SP62 were included; the whole plate was rejected whenever these controlsgaveunsatisfactory results.

TABLE 2. Genetic mappingof SP62 relative to genes 42, 43, 62, and44a

No. inexpt1 No.inexpt2 Totalno.of TotalregA+

Amber genotype by gene: withregAgeno- with regA geno- Total regA genotype (%)

type type no.

screened

42 43 62 44 + - + - + - Found Predicted"

+ - - - 22 1 28 1 52 50 2 96 100

- + + + 1 14 0 33 38 1 37 3 0

+ + - - 41 5 20 9 75 61 14 81

- - + + 13 16 10 27 66 23 43 35

+ + + - 0 5 1 6 12 1 11 8 0

- - - + 16 1 8 1 26 24 2 92 100

aE.coli CR63 was grown at

370C

in GCA medium to about5x 108 cells per ml. One milliliter was infected

at37°C with0.1mlof a mixture of SP62 and the quadruple am mutant (genes 42, 43, 62, and 44) at an MOI of

5each.At60min, the culture was sampled into chloroform-saturated broth, diluted, and then plated on E. coli CR63at30°C,conditions permissive for all progeny. Plaques of these progeny were stabbed at random andspot-tested against helper phage to identify all amber genotypes, and then some of these were tested for the presence of the SP62 mutation (see the text).

b Predicted on the assumptions (i) that SP62 lies between genes 43 and 62, and (ii) that all recombinants scoredweretheresult of single crossover events.

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Single-plaque identification ofregA+

recombi-nants. Forsingle-plaqueidentification of regA+ re-combinants (Table 3), progeny of the various

pair-wisecrossesof regA mutantswereplacedin 3 mlof

topagar(8)with about3x 108cells ofE.coliBorE.

coli K-12-W3110, plushydroxyurea (11, 18),toa

con-centrationof62mM in the topagar;thiswaspoured

onto20 mlof bottomagar, and theplatewas incu-batedat42.8°C for about18 h.Both thehigh temper-ature(18) and thehydroxyurea (11, 18)contributed

to the result that the regA mutantsproduced

vir-tually no plaques, whereas T4+ produced small plaquesatanefficiency of plating (EOP)of around

40%;this EOPwasmeasured with eachsetplated, and a correction factor was applied. To maintain temperature towithin0.1°C,wemodifiedastandard Thelco model4 incubatorsothatnow (i)afanwas

mounted atthe bottom to circulate the air down-ward throughahole(11-cmdiameter)cut outofthe metal plate mountedovertheheating coils, upward through a 6-mm gap at each side formedby false walls of cardboard that extendedtowithin 15cmof the topof the chamber, and then down the central chamber where the petri disheswereplaced; (ii)the metal plate just above the heating coils was insu-lated with cardboard tominimize radiant heating; (iii) the thermostatwasreplaced with an Athena, solid state proportional temperature controller, model52-3(which featuresaslower heatingratethe

closertothe specified temperature),whose

thermis-tor sensoris mounted in the middle of the center

shelf; (iv) the convection ports at top and bottom

were plugged to minimize heat loss. For faster warm-up,petri disheswereneverstackedmorethan

two high. To minimize evaporation (which gave

very unevenplaqueappearance),close-fitting,

plas-tic petri dish covers were used rather than the common, ridgedtype that sit about0.5 mmabove the bottom dish.

RESULTS

SP62 defines a new gene. Since SP62 maps

between markers in genes 43 and 62 (see be-low), itseemed possible that SP62mapswithin

one of these genes. Totestthis,

complementa-tion tests were performed. Wiberg et al. (18)

showedthatphage production by SP62 is rela-tivelymoresensitivethan T4+tohigh

tempera-ture. Table 1 presents measurementsofphage

production at 44.8°C in mixed infections of E.

coliB bySP62 and ambermutantsingenes43

and 62,aswellasinthenearbygenes44and 45.

At this temperature, SP62 made only about

20%as many phageasdid T4+at50 minafter

infection; no conditions are known that

com-pletely suppress SP62 phage production

with-out drastically decreasing that of T4+. In the

controlcrosses of T4+ with each of theam

mu-tants,essentially full complementation(91 and

102%) was seen with the 44- and 43-am

mu-tants, respectively; T4+ complemented the

45-and 62-am mutants somewhat less efficiently

(76 and 60%,respectively).Asafurthercontrol,

it is shown that each of the am mutants

pro-duced fewer than 0.44phagepercell.

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The essentialdataofTable1arethoseinthe

TABLE 3. Recombination betweenregA mutantsa

Mutantatindicatedmap site

regA1, regA2,

Map site regA15 regA14 regA9 regA5 regA11 regA 12, regA4, regA8 regA13, regA6,

regA16 regA7

A B C D E F G H

A 0.88 (2) 1.07 (2) 0.91(2) 0.93(2) 0.13(2) 0.70(4) 0.59(2)

(0.56-1.2) (0.83-1.3) (0.90-0.91) (0.87-0.98) (0.10-0.16) (0.40-0.96) (0.56-0.61) B 0.50 (2) 0.26 (2) 0.21 (2) 0.074(2) 0.33(3) 0.24(2)

(0.24-0.76) (0.20-0.31) (0.19-0.23) (0.033-0.115) (0.17-0.47) (0.13-0.35)

C 0.20 (2) 0.23 (2) 0.11(2) 0.28(11) 0.47(2)

(0.19-0.21) (0.14-0.31) (0.09-0.12) (0.17-0.47) (0.34-0.60)

D 0.29(2) 0.17(2) 0.17(2) 0.24(2)

(0.27-0.30) (0.16-0.18) (0.15-0.18) (0.23-0.24)

E 0.049(4) 0.37(2) 0.32(2)

(0.033-0.088) (0.36-0.38) (0.12-0.52)

F 0.12(4) 0.31(2)

(0.08-0.20) (0.28-0.33)

G 0.13(2)

(0.12-0.14)

aE.coli W3110 at 5 x 108 cells per ml in GCA medium was infected at 37°C with amixtureof two phage at an MOI of 5 each.Progeny phagewere sampled 60minlater. The value (not in parentheses) at theintersectionof a vertical column and ahorizontalrow represents the average percent recombination (2 x percentage of regA+recombinants) among progeny of a cross ofthe mutants definingthat column and row. The value in parentheses at its right gives the number of crosses performed. The values in parentheses below that indicate the range of values ofpercent recombination. Where several mutants atthe same site were used in various crosses, the particular mutant used isnotindicated to simplify presentation ofdata.Background T4+ revertants in the single mutant stocks corresponded to apercentrecombination value of 0.05 for regA8, and less than 0.005 for all the other mutants; these values were subtracted.Otherdetails are given in the text.

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last column, where the degree of complementa-tion of each of the am mutants by SP62 is shownrelative tothat given by T4+. It is seen

that SP62 complements all to about the same

extent-66 to 80%-as wellas does T4+. Thus,

SP62 complemented am mutants in genes 43 and 62, itsimmediate neighbors, as well as it complemented thoseingenes44and 45, which

are more distant neighbors. Comparable

re-sults were observed with another regA mutant, regA 15 (datanot shown). This argues that SP62

was inneithergene 43 nor 62, and that,

there-fore, SP62 defineda newgene;wehave already named this generegA (18). Ambermutants in

genes 43 and 62 made essentially no phage

DNA; gene 43 is the structuralgene for DNA

polymerase,whereasthe exact function of gene

62is unknown (12).

Intergenic mapping of SP62. To locate the approximate genetic map position ofSP62, it was crossed at equal multiplicity with amber

mutants in various T4 genes, starting from

gene 49 and proceeding counterclockwise to

gene 60. The progeny were plated on E. coli CR63 at 30'C for total phage, and wild-type recombinants were scored on plates at 40'C con-taining E.coliBplus5-fluorodeoxyuridine and uracil (at 33 and 100

AM,

respectively, inthe

top agar); these conditions were essentially

those of spot-plateprocedure 1ofWibergetal. (18). Under these conditions, amber mutants

didnot grow, SP62 gave tiny plaques atbest,

andwild-type recombinantsgavelarger plaques.

The results (not shown) revealed minimum

recombination frequencies in the vicinity of genes 43, 62, and 44.

To map SP62moreprecisely,atechnique was used thatisanalogoustothe secondprocedure used by Warner et al. (16) to map the dexA

gene;thisapproachavoids the scoring of

recom-binantsresultingfromdoublecrossoverevents,

thus avoiding the ambiguity often caused by

high negative interference (1-3). SP62 was

crossed atequal multiplicity withaquadruple

ambermutantdefectiveingenes42, 43,62,and 44;these four genesarelistedinclockwise order

on the T4 genetic map (13). First the progeny

were scored for thesixclasses of amber

recom-binants that can result from single crossover

events. These recombinants were then tested

for the presence orabsence of the SP62

muta-tion. The results of two such experiments are

shown in Table 2. They support a prediction that the SP62 mutation is between genes 43

and 62; 81% ofthe + +- - recombinants were

regA+, whereas 35% of the reciprocal recom-binants, - -++, were regA+. These two

val-ues should total 100%; that they total 116% is

doubtless due to statistical variation. If the values are normalized to a totalof 100%, then

70 and 30% of the + + - - and -- + + recom-binants, respectively, are regA+. This argues that the SP62 mutation is located about 70% of

the distance from am B22x5 in gene 43 to

amEl140 in gene 62 (Fig. 1). The two regA re-combinants found in the +--- and ---+ progeny, and the one regA+recombinant found

in the - + + + and + + + - progeny, were

prob-ably due to a low frequency of multiple cross-overs or insertions (2), and do not affect the

conclusion. Thetwo-factorcrossesof Fig. 1also

placeSP62about70% of the way fromam B22x5 to amE1140, thus providing independent con-firmation of the more rigorous conclusion from the experiments of Table 2. Figure 1 also shows therelative map positions of the four mutations used in thecrosses of Table 2.

Isolation of new regA mutants. We isolated

a number of new regA mutants by the tech-nique of Karam and Bowles (11). This tech-niquedepends on two observations: (i) amE1140

inT4gene 62isleakyon anam-restrictive host

because of high ribosomalambiguity, but isnot

quite ableto make a plaque; (ii) addition ofa

regA mutation suppresses amE1140, permit-ting formation of a tiny plaque, presumably because the low level of gene62protein is now increased. Six independent stocks (A through F)of amE1140 wereprepared, one from each of

six separate plaques on E. coli CR63. These unmutagenized stocks were plated at various

gene42 amN55x5

.. 9.7

gene43 gene regA gene62 gene44 amB22x5 amEll4Ox3 amN82x3

14.3 4.8

SP62

9.0 3.8

FIG. 1. Relative map positions of SP62 and the amber mutants used in the multifactor crosses of Table2.Percent recombination (=2xpercent T4+)

is indicated between markers and represents the

av-erageoftwoindependent experiments;variationfrom

the average was less than 13%ofthe values given. The arrow indicates the position ofSP62 derived from the data of Table 2, i.e. 70%ofthe distancefrom amB22x5toamE114Ox3. Toone mlofE. coli CR63 (5 x108 cells per ml) inGCA at37°C was added 0.2

mlofphageinGCA(MOI of eachphage,5); progeny phage weresampled60 minlater. Totalphage pro-duction (150to200phage percell) was determined

onplatesat30°Cseeded with E. coli CR63. For the

am x am crosses, T4+recombinants werescoredas thosephage givingplaquesonE. coliBat30°C.For the am x SP62 crosses, T4+ recombinants were

scored as thoseplating at 43°C on E. coli B plus

hydroxyurea (see text).

I..

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746 WIBERG ET AL.

dilutionson E. coli B at 30and40'C, andtiny

plaqueswere selected. These plaques were

di-luted and replated, and single plaques were

stabbedtomake stocksonCR63at30'C. Those thatfailedtogrow onbacterial stockno. 150,an

am-restrictive, streptomycin-resistant (strr)

strain, were retained for further testing; the

strrstraineliminates the ribosomal ambiguity

on which suppression by the regA mutation

depends (11). Of these, mostfailed to

comple-mentSP62 (regA 1) in liquid culture on E. coli

B at44.50Candwerejudgedto containan regA

mutation. The gene 62 amber mutation was

then bredout ofall by crossing with T4+. The resulting regA single mutants were given the

followingdesignations, where the letter in

pa-renthesesisthe amE1140 stockinwhicha

mu-tant arose and the temperature is that of the

plate on which itwas found: regA2 (A, 300C);

regA 6,regA7 (A,400C);regA 3(B, 300C);regA 8,

regA9 (B,

400C);

regA10 (C,

400C);

regA11 (D,

40C);regA4 (E, 30C); regA14(E, 40C); regA 5

(F, 300C) regA 12, regA13, regA15 (F, 400C).

Comparisonof thisdata with themappingdata

of Table 3 shows thatregA 2, regA 6, andregA7

are at site G, and all arose from the same

plaque isolate ofamE1140; also, regA12 and

regA13 mapped atsite F and arose from a

dif-ferentplaqueisolate. The rest of the mutations

aroseindependentlyof these and of each other.

Although mutants that arose in a common

plaque isolatewereprobably identical,wegave

themseparatenamesbecausethey maynot be

identical; one may be an ambermutation,

an-othermay be an ochre at the same codon; or,

they could be mutant at different, but very close, sites indistinguishable in our

experi-ments.

One other mutant, regA 16, was detected

solelyonthe basis of its white halo (see below

andFig. 2). It was apparentlyarare

contami-nant in the same T4+ stock from which SP62

(regA 1) arose. This and the fact thatregA1 and

regA16 map atthesame site (Table3)suggests

thattheyareidentical.

Intragenic mapping of

regA

mutants.Many

F 1

- t

8 3O

}1

*

v

v* *

2

w

J-15 0 s <

T4+

reqAI

FIG. 2. Whitehalo phenotype of regAmutants.ForallbutF, phage were dilutedto109lmlindilution broth (0.9%nutrientbroth[Difco]and 0.5%NaCl)andprinted, with the floating-loop printer, onto plates seeded with0.15mlofE. coli Batabout2 x109cells per mlinGCA medium by using modified (9) GCA bottom and top agar. Theplates were then placed at the temperatures indicated for the periods indicated. All but Dwere

photographed under dark-field illumination, on a Bactronic colony counter, model C110, New Brunswick

ScientificCo.;D wasphotographed with direct lighting from behind,on alight boxdesigned for viewing

X-ray films. (A) T4+ and all the regA mutants, incubated at 42.8°C for 21 h; regA3 contains a 'junk"

temperature-sensitive (ts) mutation in an unknown gene, whereas this ts mutation has been removed in

regA3xl. The names of the mutants have been abbreviated fromregAl,etc.to1,etc.,andT4+ istermed+.In B through E, the phageare inthe sameorderasshown here. (B) Same as A, except incubatedat 43.9°C for21h.

(C)SameasA, except incubatedat38°C for20 h. (D)Same petri dishas inA, but lightis directlybehind dish. (E) SameasA, except incubatedat44.6°C for21 h.(F)Single plaques ofT4+andregAl incubatedat

43.5'Cfor21 h.

J.

-IM.

-V

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regA MUTANTS 747

pairwisecrosses oftheregAmutantswere

per-formed in an effort to construct an intragenic

map. The results are shown in Table 3. The

most important conclusion from the table is that themutantsfellintoeight distinct sites,A

throughH. SitesFand Gweredefined by four and five mutants, respectively. Initially these

mutants at sites Fand Gwereassignedtotheir

respective sites because each failed to

recom-bine withoneother(tester) mutant inthesite.

However it seemed possible that ifthe tester

mutation were a small deletion, other (point)

mutants atthatsite might define twoor more

close but different sites within the deletion

re-gion. Thus, within eachsite, all possible

pair-wise crosses were performed; no recombinants

were detected, which argues that themutants atsitesFand Grepresenteitherall point

muta-tions or (less likely) all deletions.regA mutant

R9 of Karam and Bowles (11) is included in

Table3andwasseen to map at siteG.Mutants

regA3 and regA10 were notincludedinTable3

becausetheyweretooleakyto map.

We tried to assign an unambiguous, linear

mapordertothesitesbutwereunabletodoso; the data of Table 3 were simply not precise

enough, and we did not find conditions that eliminate thevariation. The primaryfactor in

these large variations in recombination fre-quencies for replicate crosses is undoubtedly the fact thatnocompletely selective,restrictive

conditions for regA mutants are known. For

example, theplatingconditions usedtorepress

growth of regA mutants decreased the effi-ciency ofplatingof T4+toabout 40%; this was

measuredineachexperimentand corrected for

(see above). Other factors that maycontribute

to the variation are the following. (i) Perhaps

the "wild" recombinants differ in their EOP, depending on the cross, because of

unrecog-nized "junk" mutations; we did, in fact, find

thatregA3, as originally isolated, containeda

temperature-sensitive mutation in someother

gene (Fig. 2B). (ii) These same plating

condi-tions aredifficulttoreproduce precisely, partly

because thehydroxyurea sensitivity and

tem-perature sensitivity among the regA mutants

differ slightly; sometimes duplicate plates in

thesameincubatordiffer somewhatin

appear-ance. (iii)It ispossible thatinthissmall gene

there aresignificant recombinationalhot spots

orsite-specific effects that disturbalinear rela-tionship betweenphysical distanceand

recom-bination frequency.

We did not succeed in orienting the most

separated regA mutants with respect to genes 43 and 62, primarily because the regA gene is

apparentlysosmall. We find that the distance

between the closest known gene 43 amber

(amE4306) and the three known gene 62

am-bers (which map within 0.3% recombination frequency [RF] of each other) is about 11%

RF, and the regA mutants (about 1% apart)

map roughly in the middle of this gap. Thus,

any differences among the regA mutants in

their RFs with the gene 62 or43 ambersare not

convincing,especially considering the problems

of reproducibility in scoring regA+

recombi-nants among many regA mutants (just

dis-cussed).

White halo phenotype of regA mutants. We

observed that regA1 produced a white halo around the centralzoneof clearinginbacterial lawns, both on spot plates and as single plaques, whereas T4+ didnot, under the same

plating conditions (Fig. 2). Allof the regA

mu-tantsexhibited the white haloto somedegree,

and high temperature appeared to be

neces-sary:theywere seen at 43.9and 42.8°C (Fig. 2A

and B), but not at 38 (Fig. 20) or 30°C (not

shown), even ifthe plates were incubated for several days. The white haloappearedto

repre-sentlargerbacterial microcolonies thaninthe

phage-free areas of the bacterial lawn, as

judged byexaminationwithamicroscope. Also supporting this view is the observation that,

withlight directly behind the plaque(Fig. 2D), ascontrasted with dark-field illumination (Fig. 2A, B, C, E,F),the white halo appearsdarker than the adjoining bacterial lawn.

Itis notclear why the white halooccurs,but

ithasprovided useful confirmationof the usual

spot-test identifications ofthe regA genotype.

Occasionally, the white halois not seenaround

regA zones athigh temperature; this sporadic

ficklenessmaybecaused byvariations in ageof inoculating bacteria, dryness ofagar, duration ofincubation of the seededplate,etc.Forall the halos shown (Fig.2),theapplied phagewere in abroth medium; substitution of GCA medium had no effect on development of the halo. We

were tempted to view the white halo as

diag-nosticforregA mutants and weresupportedin

thisview by the discovery of regA16 solely on

thebasis ofitswhitehalo(seeabove).However,

we recently found that ifthe temperature is

raised to 44.6°C, where the regA mutants are

essentially dead, T4+ now produces a white

halo (Fig. 2E). The significanceof this halo is

discussed below.

DISCUSSION

The fact that the phenotype of SP62 is very

differentfromthat ofmutantsintheadjoining

genes, 43and 62, already suggestedthatSP62

defines a new gene, but thecomplementation

experiments of Table 1 constitute direct

evi-VOL. 22, 1977

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748 WIBERG ET AL.

dence for thatconclusion. Our location ofSP62 between genes 43 and62 (Table 2) documents

our preliminary mapping report (18). Karam

andBowles located theirregA mutant R9 also between genes 43 and62, by using adifferent approach fromours (11).

Untilnow itseemed possible that SP62was a

special kind of missensemutantand that there-fore theregA phenotype might be the result ofa

partial loss, or subtle modification, of regA function. However, our observation that the

mutants define eight different sites, spread

over a distance ofover 1% recombination

fre-quency, suggests that no subtlety is involved

and that the phenotype is due to total loss of

regA function.

The whitehalo seen on petridishes

contain-ing regA mutants is clearly due to faster

growth ofnearbybacterial cells. One

explana-tion may be the release of some factor from

regA-infectedcellsthat promotes thegrowthof

uninfected cells. We are awareofonlyone

pre-vious report ofa white halo phenotype forT4

mutants. Halletal. (7)showed thatT4 mutants

defective in dihydrofolate reductase and, to a

lesserextent, thymidylate synthetase, produce a bright white halo, but only under special

platingconditions. The key factorintheir

plat-ingconditions is apparently auracil-requiring bacterial host. Hallsuggested thatasignificant

amountof dUMP accumulates incells infected

by thesemutantsand, uponlysis,isreleasedas

deoxyuridine or uracil (6). The genefor dihy-drofolate reductase was originally named wh

(for whitehalo) (7)buthas sincebeenrenamed

frd (10, 14);tdisthegene forthymidylate

syn-thetase (14). Inthiscontext, it isworthnoting

that regA1underproducesthymidylate

synthe-tase at

370C

(18);perhaps highertemperatures

cause greater underproduction. For this to be

anexplanation of the whitehalo,wewould also

have to propose that, athightemperature, E.

coliBdevelopsatleastapartialdependenceon

exogenous products (e.g., dUMP) thatmay

ac-cumulate when thymidylate synthetase

activ-ity is low. We have no way of knowing at this

point whetherthe white halo seen withT4+,at yethigher temperatures, iscausedbythe same

mechanism aswith regA mutants.

In preliminary experiments, we recently

found that extracts of E. coli B cells infected

with most ofthe regA mutants lack a protein

havingamolecular weight oflessthan 12,000,

when displayed on sodium dodecyl

sulfate-polyacrylamide gels (Landry, Cardillo, and

Wiberg, unpublisheddata). Thus, regAmaybe

the structural gene for this protein. If so, we

may be able to detect nonsense fragments (if

someofthesemutants areindeednonsense

mu-tants) and order them bysize. This should aid

in constructing a rigorous map of the regA gene. Further,if nonsense-suppressor strains of

E. coliconvert someof thenonsensefragments

toafull sizeprotein, this willprovethatthese

mutants are nonsense mutants.

ACKNOWLEDGMENTS

Our thanks toDwightHall forvaluable discussion about thewhite halo phenotype.

This paper is based on workperformed undercontract with the U.S. Energy Research and Development Adminis-tration at the University of Rochester Biomedical and Envi-ronmental research project and has been assigned report no.UR-3490-1034. This work wasalso supported by Public Health Service grant GM-21999 from the National Institute ofGeneral Medical Sciences.

LITERATURE CITED

1. Berger, H., and A. J.Warren. 1969. Effects of deletion mutations onhigh negativeinterference in T4D bac-teriophage. Genetics 63:1-5.

2. Broker, J. R., and I. R. Lehman. 1971. Branched DNA molecules: intermediates in T4 recombination. J. Mol. Biol. 60:131-149.

3. Chase, M.,and A. H.Doermann. 1958. High negative interference over short segments of the genetic struc-ture ofbacteriophage T4. Genetics 43:332-353. 4. Edgar, R. S., G. H. Denhardt, and R. H. Epstein. 1964.

A comparative genetic study of conditional lethal mutations ofbacteriophage T4D. Genetics 49:635-648.

5. Fraser,D., and E. A. Jerrel. 1953. Amino acid composi-tionof T3bacteriophage.J.Biol. Chem. 205:291-295. 6. Hall, D.H.1967. Mutantsofbacteriophage T4 unable to induce dihydrofolate reductase activity. Proc. Natl. Acad. Sci. U.S.A. 58:584-591.

7. Hall, D. H., I. Tessman, and 0. Karlstrom. 1967. Link-age of genescontrolling a series of steps in pyrimidine biosynthesis.Virology31:442-448.

8. Hercules, K., J. L. Munro, S. Mendelsohn, and J. S.

Wiberg.1971.Mutantsinanonessential gene of bac-teriophage T4 which aredefective in the degradation of Escherichia coli deoxyribonucleic acid. J. Virol. 7:95-105.

9. Hercules, K., and J. S. Wiberg. 1971. Specific suppres-sion of mutationsingenes46and47bydas, a new class of mutations in bacteriophage T4D. J. Virol. 8:603-612.

10. Johnson, J. R., and D. H. Hall. 1973. Isolation and characterization of mutants of bacteriophage T4 re-sistant to folateanalogs. Virology 53:413-426. 11. Karam, J. D., and M. G. Bowles. 1974. Mutation to

overproduction of bacteriophage T4 gene products. J. Virol.13:428-438.

12. Morris, C. F., N. K. Sinha, and B. M. Alberts. 1975. Reconstruction ofbacteriophageT4DNAreplication apparatus from purified components: rolling circle replication following de novo chain initiation on a

single-strandedcircular DNAtemplate. Proc. Natl. Acad. Sci. U.S.A. 72:4800-4804.

13. Mosig, G. 1976. Linkage map of bacteriophage T4, p. 664-676. In G. D. Fasman (ed.), Handbook of bio-chemistry and molecular biology, 3rd ed., Nucleic acids, vol.II.CRCPress,Cleveland,Ohio. 14. Simon, E. H., and I. Tessman. 1963.

Thymidine-requir-J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

(8)

regA 749

ing mutants ofphage T4. Proc. Natl. Acad. Sci. U.S.A. 50:526-532.

15. Tessman,I., and D. B. Greenberg. 1972. Ribonucleotide reductase genes ofphage T4: map location of the

thioredoxingenenrdC. Virology49:337-338. 16. Warner,H.R., D. P.Snustad, J.F.Koerner, and J.D.

Childs.1972.Identification and genetic characteriza-tionofmutantsofbacteriophage T4defectiveinthe

abilitytoinduceexonuclease A. J.Virol. 9:399-407. 17. Wiberg, J. S. 1966. Mutants of bacteriophage T4 unable

tocausebreakdown of host DNA. Proc. Natl. Acad.

Sci. U.S.A. 55:614-621.

18. Wiberg,J.S., S. Mendelsohn, V. Warner, K.Hercules,

C. Aldrich, and J. Munro. 1973. SP62,aviable

mu-tantofbacteriophageT4Ddefective inregulationof phageenzymesynthesis. J. Virol. 12:775-792.

VOL. 22, 1977

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Figure

TABLE 2. Genetic mapping of SP62 relative to genes 42, 43, 62, and 44a
TABLE 3. Recombination between regA mutantsa
FIG.1.phagefromerageamB22x5ambertheonam(5TheisTablescoredhydroxyureathoseductiontheml indicated x plates Relative map positions of SP62 and the mutants used in the multifactor crosses of 2
FIG. 2.photographedScientific43.5'Cthroughdish.topregA3xl.temperature-sensitive(0.9%raywith(C) White halo phenotype ofregA mutants

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