JOURNAL OF VIROLOGY, Aug. 1984, p. 315-320 Vol. 51, No. 20022-538X/84/080315-06$02.00J0Copyright © 1984, American Society for Microbiology

Bacteriophage T4 bypass3l Mutations That Make Gene 31Nonessential for Bacteriophage T4 Replication: Mapping bypass3l

Mutations by UV Rescue ExperimentsA. H. DOERMANN1* AND LEE D. SIMON2

Department of Genetics, University of Washington, Seattle, Washington 98195,1 and Waksman Institute of Microbiology,Rutgers University, New Brunswick, New Jersey 089032

Received 6 February 1984/Accepted 6 April 1984

The product of gene 31 is normally required for assembly of the T4 capsid. Two mutations that each bypassthat requirement are shown to be located at separate sites in gene 23, which encodes the major structuralprotein of the capsid. A second phenotypic effect that characterizes both bypass3l mutant strains is the ability tomultiply in host-defective strains, such as hdB3-1 and groEL mutants, on which wild-type T4 is unable toassemble capsids. The genetic data indicate that both phenotypic effects are due to the bypass3l mutation.Elimination of the requirement for both the phage protein, gp3l, and the host protein, GroEL, by either of twosingle mutations in gene 23 indicates that GroEL and gp3l are normally needed to interact with gp23 in capsidassembly of wild-type T4.

In the accompanying paper, Simon and Randolph (9)describe a mutation of bacteriophage T4D, bypass31-1(byp31-1), with two phenotypic characteristics that distin-guish it from the wild type: (i) the normally required gene 31product is dispensable, i.e., the mutant has the bypass 31phenotype; and (ii) in contrast to wild-type T4, it canmultiply in the hdB3-1 (herein referred to simply as B3-1) (8)and groEL defective strains of Escherichia coli. Thus thenormal T4 host range has, with this mutation, been extendedto include such strains. In addition, another mutant strain,byp31-2, has now been isolated. The two mutant strains aresimilar: both bypass the gp3l requirement and share the B3-1host-range extension. They differ in the bypass characteris-tic, however. Whereas byp31-1 is unable to bypass the gp3lrequirement at 41°C, byp31-2 retains its bypass ability at thattemperature.The purpose of this paper is to provide a genetic analysis

of these two mutations. It relies heavily on the sensitivemapping procedure that makes use of genetic rescue of shortsegments from heavily UV-irradiated byp3l genomes. Theresults indicate that the two mutations are at geneticallyseparable sites in gene 23 and that both phenotypic charac-teristics result from a single mutation.

MATERIALS AND METHODSBacterial and phage strains. In addition to the widely used

Escherichia coli strains B and CR63, the derivative of B, B3-1, was also employed. This su- bacterium is described indetail by Simon et al. (8). All platings were incubated for 48 hat 28°C, at which temperature the wild type cannot makeplaques on B3-1, although byp3l does so with 100% efficien-cy compared with platings on B.The byp31-1 mutant of T4D used here was derived from

the byp31-1 isolate described in the accompanying paper (9).The original strain, which carried an r mutation in addition tothe bypass, was exposed to UV radiation at a dose ofapproximately 100 phage-lethal hits (PLH) and then crossed,in the amber-permissive host CR63, to the gene 23 double-amber mutant amHllamE1236 (see map in Fig. 1). The

* Corresponding author.

progeny of that cross was plated against B3-1. All phageparticles capable of initiating plaques necessarily carried thetwo gene 23 am' sites and the host-range characteristicpermitting T4 to multiply in B3-1. A plaque from theseplatings was isolated, suspended in broth, and plated againstB3-1 to eliminate any residual phage of other genotypes thatmight have been carried along in mixedly infected bacteriaduring growth of the original plaque. A plaque from thesecond plating was the progenitor of the stock used in thebyp31-1 mapping experiments.The byp3l-lamN54(31) double mutant used in one of the

UV rescue experiments was obtained by using amN54(31) asa helper phage to rescue the byp31-1 mutation from the strainisolated as described above after the latter had been irradiat-ed with about 180 PLH of UV. A plaque from a B3-1 platingwas suspended and plated. A plaque from the replatingserved as the source of the desired stock. It segregatedamN54. Its ability to form plaques on B3-1 showed that ithad both the host-range and bypass characteristics of theoriginal byp31-1 mutant.An additional set of byp3l mutants, each originating

independently of the others, was isolated and studied in thegenetics laboratory course at the University of Washing-ton. The mutant strains were selected after treatment ofT4amN54(31)amNG71(31) with ethyl methanesulfonate. Theclass experiments suggested that most of the mutations arelocated at gene 23, but at a site genetically separable fromthe site occupied by byp31-1. Some of the mutants in thatgroup show phenotypic differences from byp31-1. Onestrain, byp31-2, forms plaques on the su- hosts at 41°C aswell as at 28°C and does so with an efficiency of plating of100% compared with platings on su+ hosts. That mutant,first isolated and studied by Alan Dayton, was chosen forfurther investigation. All mutants isolated by the classshowed high efficiency of plating on the bacterial host B3-1su at 28°C, like byp31-1.The amber mutants used here originated largely from the

Caltech collection that is now being maintained by W. B.Wood. All multiple amber mutants were constructed in thislaboratory by a conventional crossing technique and identi-fying the recombinant genotypes by the method of Doer-mann and Boehner (4).

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316 DOERMANN AND SIMON

Culture and UV rescue techniques. Media and culturetechniques have been described previously (2). The generalprocedure for UV rescue has been discussed by Doermann(3).UV radiation procedure. Phage stocks to be irradiated

were diluted 100-fold or more to a titer of 4 x 109 particlesper ml. The dilution fluid was M9+ medium from which theglucose and Casamino Acids were omitted. Up to 100 ml ofthe diluted suspension was placed in a watch glass contain-ing a tiny stirring magnet. The target suspension was stirredconstantly during UV exposure. A 15-W low-pressure mer-cury vapor lamp 1 meter from the phage sample, poweredthrough a Sola constant-voltage transformer, was the sourceof UV irradiation. The number of PLH was calculatedaccording to target theory from low-dose survival curves (5),which indicated that a dose of about 3 PLH was deliveredper min of exposure. The dose rate of 3 PLH per min is notincreased either by making a greater dilution of the phagestock or by reducing the volume irradiated (i.e., thereappears to be no detectable shielding of one phage particleby another or UV absorption by the fluid in which the phageis suspended).

RESULTSMapping the byp31-1 site responsible for the extension of T4

host range to B3-1. In the progeny from a cross of an ambermutant by byp31-1, the wild type can be identified because,among the four genotypes emerging from the cross, it isunique: it is able to form plaques on B, but not on B3-1.Crosses were made between byp31-1 and strains carryingone of a set of widely distributed amber mutations. Theprogenies were plated against strain B on which only oneparent, byp31-1, and one recombinant, the wild type, areable to make plaques. These plaques were stabbed withsterile toothpicks, and transfers were made to plates seededwith B3-1 which were then incubated at 27 to 30°C. Thefrequency of stabs that failed to grow into plaques (wildtype) is a measure of recombination. The results indicatedthat the bypass mutation is located in the general vicinity ofgene 23. To map the mutation more precisely, however,required a more sensitive procedure. The method employedhere took advantage of the well-defined amber map of gene23 and the adjacent genes (Fig. 1), and it made use of geneticrescue of short segments from the genomes of UV-irradiatedparental phage (3). In the general procedure the phagecarrying the mutation to be mapped (byp31-1 in this case)was irradiated with a high dose of UV as described above.Pairwise mixtures were made with irradiated byp31-1 and avariety of amber helper strains. These mixtures were addedto exponentially growing CR63 bacteria (permissive foramber mutants) in H-broth at 30°C. The multiplicity ofinfection was approximately 5 UV-treated and 5 helperphage per bacterium. After a 10-min period for adsorption ofphage to cells, a 50-fold dilution was made and incubated at30°C for an additional 70 min. Chloroform was then added to

assure complete lysis, and platings of progeny phage weremade 10 min later.

In the first crosses to be discussed the helper phage was,in each case, a double mutant carrying two rather closelylinked ambers. The progeny phage of interest were thoserecombinants able to form plaques on the two amber-restrictive hosts, B and B3-1, which represent only a smallfraction of the progeny of the cross. Its magnitude dependsinversely on both the UV dose and on the size of the intervalspanned by the two amber mutations in the helper phage. Inthe present experiments it ranged from 10-3 to 10-5. Figure 2illustrates the types of recombinant that would be diagnosticin these experiments. In both crosses diagrammed there aretwo classes of recombinant capable of forming plaques onthe amber-restrictive host B, namely, the class that has, andthe class that has not, rescued byp31-1 together with the twoam+ sites from the irradiated parent. The former class will,and the latter class will not, be able to make plaques on thehost B3-1. In the first set of experiments reported here, theratio of plaques on B3-1 to plaques on B was determined. Ahigh ratio indicates frequent corescue of byp31-1 with theam+ loci and implies close linkage. A maximum ratio wasachieved when byp31-1 was located in the interval spannedby the two amber mutations. Table 1 gives the ratios ofplaques on B3-1 to plaques on B for two different rescueexperiments. The results indicate that the mutation enablingT4 to form plaques on E. coli strain B3-1, i.e., effecting theB3-1 host range, is probably located between amA489 andamC208 in gene 23 (Fig. 1).Are both the bypass 31 and the host-range properties caused

by a single mutation? Two UV rescue experiments were alsoperformed to test whether the mutation needed for growth inB3-1 is the same mutation that allows bypassing the normalrequirement for the product of gene 31. The first used a gene31 amber mutant (amN54) as the helper phage with UV-irradiated byp31-1. The progeny from this rescue cross wasplated on strains B and B3-1. On the assumption that bothproperties are conferred by the single mutation, byp31-1, thevast majority of progeny phage particles able to form plaqueson B will result from either of two types of rescue. One typewill rescue amN54+ from the UV-irradiated phage particles,replacing the amN54 of the helper. The other will rescuebyp31-1 into a genome still carrying amN54. There is noobvious reason why the two types of rescue should not beequally frequent, since in either case only a single site needbe rescued, and the sites are so far apart that corescueshould not occur with detectable frequency at the high UVdoses employed. If the assumption were correct that thesingle mutation byp31-1 confers both properties, then half ofthe phage particles plating on B should also be able to formplaques on B3-1.

If, alternatively, the bypass 31 phenotype were due to asecond mutation that was genetically distant from the gene23 mutation conferring B3-1 host range, three classes ofsingle rescues would be expected. One class would rescue

E 509 E 1270 A 489 E 1236E1193 Hil B17 B272 E 506 E 389 C208

I I I

GENE 22 GENE 23

E 355

GENE 24

FIG. 1. Genetic map of the amber mutations used in this study. The major mapping information for gene 23 was obtained from the work ofSarabhai et al. (7) and of Celis et al. (1). Additions to and modifications of their maps have been confirmed by three-point tests. The ambersites in gene 23 have been identified in the DNA sequence by Parker et al. (personal communication). The spatial distribution of ambers ingene 23 is based on sequence data.

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MAPPING T4byp-31 MUTATION BY UV RESCUE CROSSES

UV-hit am1 + am2+ hr UV-hit UV-hit&A Y

-- I I I IRescue-1 .-__j I IRescue-2 --- -------- -Parent-2 I I 0

am1 am2 hr+

UV-hit am3 + hr am4+ UV-hit UV-hitParent-1 x O I O _ )t

Rescue-3 . l I l lRescue-4 -Parent-3 I I;amI hr+ am4

FIG. 2. Mapping the tnutation in the bypass3l strain that is responsible for B3-1 host range (hr) by using double-amber helper phages inUV-rescue crosses. The figure diagrams two crosses: cross-I, in which the ambers of the helper phage do not span the location of the hrmutation, and cross-II, in which they do. All progeny into which the two am' alleles of the helper ambers have been rescued will makeplaques on E. coli strain B. For plaque formation on B3-1 su , not only the am' alleles, but also the hr site, must be rescued into the progenygenome. The ratio of plaques on B3-1 to plaques on B is a measure of the proximity of hr to the interval spanned by the two amber mutations.The ratio will increase as ambers are employed that are closer and closer to the hr site, i.e., in cross-I the rescue-1 class will become anincreasingly greater component of the total am' rescues. The maximum ratios will be obtained when hr is included in the interval spanned bythe two ambers, because then four crossovers will be required to effect a rescue of the two am' sites without also rescuing hr (comparerescue-3 and rescue-4 in cross-II). The position within the interval will also, of course, have some effect on the frequency of the quadruple-crossover rescue.

the B3-1 host-range mutation into an amN54 genome. An-other would rescue the 31 bypass mutation into an amN54genome. The third would rescue amN54+, producing a wild-type genome. Although the second and the third would makeplaques on B, none of the three would do so on B3-1. Thefirst would fail because the requirement for gp3l is neithersatisfied nor bypassed, whereas the second and third wouldnot grow on B3-1 because neither includes the host-rangemutation. The only type of phage that would be able to plateon B3-1 is the infrequent one that corescues the host-rangemutation together with either the 31 bypass mutation or theamN54+ site.The result of the test was that platings on B yielded 3,012

plaques, whereas an equal sample plated on B3-1 yielded1,518 plaques. This result implies either that both pheno-types are effected by a single mutation or that two tightlylinked mutations are responsible. Because the B3-1 hostrange has been shown to be due to a mutation in gene 23, thesecond mutation, if it exists, must also be in or near gene 23.The second type of rescue experiment is also designed to

test whether the two phenotypic properties of byp31-1 (i.e.,bypassing the need for gp3l and extending the T4 host rangeto include B3-1) are effected by a single mutation or by twoseparate mutations. It is more sensitive than the previouslydescribed procedure. Diagrammed in Fig. 3, it consists of aUV rescue experiment in which the irradiated phage carriesthe byp31-1 mutation(s) and, in addition, an amber mutationin gene 31 (amN54). The helper phage likewise has an

amN54 mutation, but also a gene 23 amber, which is as close

TABLE 1. Frequencies of the B3-1 host-range mutation amongrescues of gene 23 segments that include two amber sitesa

Rescue titer on B3-1/rescueHelper titer on B

Expt 1 Expt 2b

amE1193amH11 0.05camHllamB17 0.05amB17amB272 0.10amB272amE509 0.07amE509amE506 0.16 0.12amE506amE1270 0.22amE506amE389 0.32amE1270amE389 0.25amE389amA489 0.47amA489amC208 0.94 0.82amC208amE1236 0.80 0.44amE1236amE355 0.35

a The byp31-1 strain used in experiment 1 was irradiated to a dose ofapproximately 180 PLH. Exponentially growing CR63 was mixedly infectedwith a multiplicity of 5 UV-irradiated byp31-1 particles and 5 helper phagecarrying two amber mutations as indicated in the first column (see Fig. 1 forlocations of the am mutations). The progenies of the rescue crosses wereplated both on B and on B3-1. The plaque counts in each experiment werelarge, making the sampling error negligible. Control platings were included ineach experiment to determine the efficiency of plating by byp31-1 on B3-1compared with that on B. The experimental plaque counts on B3-1 werecorrected by these factors, which ranged from 0.95 to 1.07.bExperiment 2 was carried out with a different stock of byp3l-lamN54. It

was irradiated with a UV dose of approximately 200 PLH.c The values given are the corrected rescue titer on B3-1 divided by the

rescue titer on B.

Parent-1 -At-

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CROSS-Il

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318 DOERMANN AND SIMON

UV-hit am+ byp hram

UV-hit UV-hit N54 UV-hitParent-1 .. I K

I .__ __ 11

Rescue-1I ............J_Rescue-2 ------Parent-2

UV-hit

am byp+ hr+A489

amN54

ambyp hr am + UV-hit UV-hit N54 UV-hitI I CiX...... y I y8

I I..1 . - .

- - --- -

byp+ hr+ amEl 236

amN54

FIG. 3. Rationale of UV rescue experiment to test whether the two phenotypic properties of byp3l result from one or from two separablemutations. The crosses diagrammed assume that two separable mutations are responsible and that their map order is amA489, byp (themutation effecting independence of gp3l), hr (the mutation extending host range to B3-1), amE1236. To make a plaque on either B or B3-1, a

progeny phage from this cross must carry the rescued gene 23 am' site in its genome. In addition it must carry the rescued byp site because allphages carry the gene 31 mutation, amN54. Plaque formation on B3-1 su- has yet another requirement: hr must also be rescued into theprogeny genome. In cross-I, therefore, rescue-1 can make plaques on both B and B3-1, whereas rescue-2 can do so only on B. A high UV dosewas used to reduce the length of the genetic segments rescued, thereby increasing the relative frequency of rescue-2 compared with rescue-1.In cross-1I, however, with the gene 23 amber to the right of byp and hr, rescue-3 will make plaques on both bacterial hosts, whereas rescue-4will do so on neither because it cannot circumvent the need for gp3l. Because quadruple crossovers are presumably infrequent compared withdouble crossovers, they are not expected to produce a significant number of byp in cross-II without corescue of hr. Thus cross-I is diagnosticof the order assumed here, whereas cross-Il would be diagnostic if the order of byp and hr were reversed. The data from this experiment are

given in Table 2.

as practical to the byp3J-1+ location(s). To make a plaque on

the amber-restrictive host B, a progeny genome must neces-

sarily contain the am+ site rescued from the UV-treatedgene 23. Because all genotypes have amN54(31), it must alsocontain the mutation responsible for bypassing the gp3lrequirement. To make a plaque on B3-1, it must carry bothof those sites and, in addition, the site responsible for the B3-1 host range (hr). If both the bypass 31 and the host-rangeproperties are due to a single mutation, the numbers ofplaques on B and B3-1 should be equal. If they are due totwo separable mutations, the disposition of the three sites,am(23)+, byp-31, and hr, plays a decisive role. Because theorder is unknown, a priori, it is necessary to carry out rescue

crosses with two different helpers, one with the gene-23amber on one side and the second with a gene-23 amber on

the other side of the 31 bypass mutation. With the order as

shown in Fig. 3 (byp to the left of hr), the amA489 rescue isdiagnostic because one double-crossover rescue (no. 2 inFig. 3) can rescue byp alone, whereas the other (no. 1)rescues both byp and hr. If class 2 makes up a detectablefraction of the rescues, the plaque count on B will exceed tneplaque count on B3-1. If the order of byp and hr were

reversed, it would require two double-crossover rescues toprovide a genome carrying byp and amA489+ without hr.That would be too rare to be detectable and thus plaquecounts on B and on B3-1 would be statistically equal. In that

event, however, the second cross with amE1236 in thehelper phage would be diagnostic. The results of these tworescue crosses are given in Table 2. In neither cross did theplaque counts on B exceed the plaque counts on B3-1. Itmust be concluded that the two phenotypic properties aredue to a single mutation or to two mutations that are soclosely linked that this method cannot resolve them.

Additional evidence may be brought to bear on the ques-

TABLE 2. UV rescue cross to test whether the bypass3l and theB3-1 host-range phenotypes are effected by separable mutationsa

Plaque countHelper phage On Count on B3-1/

Bo3-1 On B count on B

amA489amN54 3,759 3,662 1.03bamE1236amN54 1,632 1,567 1.04b

a The rationale of this experiment is discussed in the text and in thediagrams given in Fig. 3.

b The efficiency of plating of unirradiated byp31-1 was compared on B3-1and B in these and other experiments. Seven determinations gave an averagevalue of 1.03. The individual ratios ranged from 0.95 to 1.15. The data havenot been corrected for efficiency of plating. If two separable mutations areresponsible for the two phenotypic properties of byp31-1, it should bereflected in a deviation of the experimental ratio from the control efficiency of1.03.

CROSS-I

CROSS-IlRescue-3Rescue-4Parent-3

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MAPPING T4byp-31 MUTATION BY UV RESCUE CROSSES 319

tion whether a single mutation causes both the bypass 31 andthe host-range extension phenotypes. In the class experi-ments mentioned above, 13 mutants of independent originwere selected for their ability to bypass the requirement forgp3l. Each of the 13 showed the extended host rangephenotype. That result strongly supportes the hypothesisthat a single mutation is responsible for both phenotypicproperties. A possible alternative interpretation of thosedata, that the phage strain initially mutagenized[amN54(31)amNG71(31)] carried a host-range mutation, wasruled out by UV rescue experiments.UV rescue experiments indicate that if two separate

mutations are responsible for the bypass and host-rangephenotypes, they must be closely linked. It is anticipatedthat comparison of mutant nucleotide sequences with thewild-type sequence will provide a definitive conclusion.Sequencing experiments are now under way.

Location of byp31-2. Comparison of UV rescue data forbyp31-2 with results of parallel experiments with UV-irradi-ated byp31-1 suggested that the former is at a separatelocation, lying more toward the center of gene 23 than thelatter. A more compelling argument for that hypothesiswas needed, however, because, for reasons not yet clear,UV rescue data from the byp31-2 experiments were not asclear cut as those with byp31-1. It had been observed thatwhen byp31-lamN54(31) was crossed with byp3l-2amN54(31)amNG71(31), approximately 0.7% of the proge-ny plaques on B su- were distinguishably larger than thoseof either parental type. It seemed possible that these mightbe double mutants, carrying both byp31-1 and byp31-2. Oneof these large plaques was used to produce a lysate that willbe referred to as large-plaque stock (LPS). It made distin-guishably larger plaques on both B su- and on B3-1 thaneither parental byp3l strain. Two observations suggestedthat the double-mutant hypothesis was worth testing. Thefirst came from comparison of LPS platings on B su-.Although at low temperature (30°C) plaques of LPS weresignificantly larger than byp3l-lamN54, similar platings athigh temperature (41°C) showed LPS plaques to be indistin-guishable from byp31-2 plaques. This suggested that thetemperature-sensitive bypassing effect of the byp31-1 muta-tion was eliminated from the double mutant phenotype at41°C, leaving expression of only the temperature-insensitivebypassing influence of byp31-2 to promote plaque formation.The second observation came from burst size comparisons

of LPS and the two parental types. If the larger plaque sizeof LPS is due to an r (rapid lysis) mutation, it would not beexpected to make a larger burst size than its r+ counterpart(6). If, however, it results from more efficient bypassing ofthe gp3l requirement, increased burst size could be thecause. Burst size measurements of LPS and both parentswere made by using B su- as the host at 30°C in H-broth.The multiplicity of infection in each case was approximately6. The following burst sizes were found: 65 for byp3l-lamN54(31); 39 for byp31-2amN54(31)amNG71(31); and 92for LPS. In single-infection experiments, the burst sizeswere 31, 18, and 44, respectively. Thus both burst sizeand temperature dependence comparisons were consistentwith the hypothesis that LPS carries both the byp31-1 andbyp31-2 mutations.

Provided LPS is the double-bypass mutant it should bepossible to separate the components and check their identitywith the original parental types used in the cross from whichit had segregated. A sequence of steps used to make that testfollows. LPS was irradiated with a UV dose of approxi-mately 200 PLH. Two rescue crosses were male with that

irradiated stock. In one, the helper was amE1270(23)-amE389(23)amN54(31)amNG71(31). That helper was select-ed to rescue preferentially any byp3l mutation located in thecentral part of gene 23 (presumably byp31-2). In the secondcross the helper was amE1236(23)amE355(24)amN54-(31)amNG71(31). It was chosen to rescue any byp3l muta-tion near the amE1236 end of gene 23 (presumably byp31-1).Since LPS also carries at least amN54 in gene 31, only byp3lrescues will make plaques on B su-. Platings at 30°C (fromboth rescue crosses) yielded small plaques (similar to thesingle bypass strains) as well as large plaques (similar toLPS).

Small-plaque suspensions from both rescue crosses wereplated on B su- to exclude any helper phage carried alongduring growth of byp3l. Plaques from those platings wereused to prepare stock lysates of the segregants fromboth crosses. The stocks derived from theamE1270(23)amE389(23) rescue cross proved to be indistin-guishable from the byp31-2 stock used in the original cross.They made similar plaques at both 30 and 41°C. Whencrossed back to byp31-lamN54(31) and to byp3l-2am-N54(31)amNG71(31), large-plaque segregants were foundonly in the former cross. The stocks originating from theamE1236(23)amE355(24) rescue cross resembled the byp3l 1parent. They made similar plaques at 30°C, but no plaquesat 41°C. When crossed to byp31-2amN54(31)-amNG71(31),large plaque formers were observed in the 0.7% range. Whencrossed to byp3l-lamN54(31), no large plaques were found.

All of the results presented in this section are thusconsistent with the interpretation that byp31-2 is located at asite genetically separable from byp31-1 and that it is locatedcloser to the center, whereas byp31-1 is nearer the amE1236end of gene 23.

DISCUSSIONThe experiments reported here establish several facts

about the bypass3l mutations: (i) they are located in gene 23,(ii) they occur at two (or more) genetically separable sites inthat gene, and (iii) two phenotypic consequences appear toresult from such mutations, namely, bypassing the require-ment for gp3l as well as extension of host range to cells thatlack normal groEL function. It is also shown that a doublebypass3l mutant strain is more effective at bypassing therequirement for gp3l than either single mutant. On the B su-host the burst size of the double mutant is nearly as large asthe sum of the burst sizes of the two component singlemutants. Plaques of the double mutant are larger than thoseof either single mutant on either B su- or on B3-1 su-.Apparently the two mutations act additively.The accompanying paper (9) proposes the hypothesis that

the T4 protein, gp3l, and the host protein, GroEL, are bothrequired to carry out a single function in T4 capsid assembly.The suggested function is one that is essential for properpolymerization of gp23, the major structural protein of theT4 capsid. The evidence presented here indicates that bothhost range extension to the capsid-assembly defective strainB3-1 and bypass3l activity of the mutants are due to a singlemutation. This fact, together with the mapping data whichclearly locate the two byp3l mutations in gene 23, givesstrong support to the hypothesis.The fact that the two bypass3l mutations have additive

effects on burst size (presumably on the efficiency of capsidproduction) in the absence of gp3l is difficult to interpret inmore than a general way. Evidently the conformation ofgp23 is more amenable to effective polymerization when twosites in gene 23 are altered by bypass3l mutations. Attempts

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320 DOERMANN AND SIMON

to sequence the byp3l mutants of gene 23 are now under wayin the hope that comparing the mutant sequences with wild-type gp23 sequence may yield some insight. It may be addedthat all mutations that appear to be located at the byp31-1site have, to date, proved to be equally temperature sensitivein their bypassing activity. The mutations that appear to belocated at or near the byp31-2 site, however, vary in tem-perature sensitivity, implying different amino acid substitu-tions in that part of gp23.

ACKNOWLEDGMENTSThe experiments reported here were carried out with the assist-

ance of Linde Boehner, whose expert help is gratefully acknowl-edged. The 1981 class in Genetics 461 at the University of Washing-ton isolated and made preliminary studies on 13 mutants, includingbyp31-2.

This work was supported by Public Health Service grantsGM13280 to A.H.D. and GM27314 to L.D.S. from the NationalInstitutes of Health.

LITERATURE CITED

1. Celis, J. E., J. D. Smith, and S. Brenner. 1973. Correlationbetween genetic and translational maps of gene 23 in bacterio-

phage T4. Nature (London) New Biol. 241:130-132.2. Chase, M. C., and A. H. Doermann. 1958. High negative interfer-

ence over short segments of the genetic structure of bacterio-phage T4. Genetics 43:332-353.

3. Doermann, A. H. 1983. Mapping of mutations and construction ofmulti-mutant genomes, p. 302-311. In C. K. Mathews, E. M.Kutter, G. Mosig, and P. Berget (ed.), Bacteriophage T4. Ameri-can Society for Microbiology, Washington, D.C.

4. Doermann, A. H., and L. Boehner. 1970. Identification of com-plex genotypes in bacteriophage T4. I. Methods. Genetics66:551-567.

5. Harm, W. 1980. Biological effects of ultraviolet radiation. Cam-bridge University Press, Cambridge.

6. Hershey, A. D. 1946. Mutation of bacteriophage with respect totype of plaque. Genetics 31:620-640.

7. Sarabhai, A. S., A. 0. W. Stretton, S. Brenner, and A. Bolle.1964. Colinearity of the gene with the polypeptide chain. Nature(London) 201:13-17.

8. Simon, L. D., J. M. McLaughlin, D. Snover, J. Ou, C. Grisham,and M. Loeb. 1975. E. coli membrane lipid alteration affecting T4morphogenesis. Nature (London) 256:379-383.

9. Simon, L. D., and B. Randolph. 1984. Bacteriophage T4 bypass3lmutations that make gene 31 nonessential for bacteriophage T4replication: isolation and characterization. J. Virol. 51:321-328.

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