Conditional lethal mutants of bacteriophage ϵ34

VIROLOGY

36,

.51&%8

(1968)

Conditional

Lethal

Mutants

I. Genetic S. IKAWA, Department

of Serology

and Immunology,

of Bacteriophage

Map

S. TOYARIA, Znslitute

for

Accepted May

c341

of P H. UETAKE

AND

Virus

Research, Kyoto

Universily,

Kyofo,

Japan

1, 1968

Temperature-sensitive mutants of phage e31 have been isolated after treatment with nitrous acid, 5-bromouracil, or nitrosoguanidine. One hundred ninety mutants have been classified into 11 cistrons on a single circular linkage map by complementation and genetic recombination tests. INTRODUCTION

was designated simply as c34cin our previous reports (Toyama et al., 1966a, 1967). &II-l is a clear-plaque mutant capable of establishing 1Tsogeny with the help of CI mutants or c+ wild type. e34deZN21is a delet’ion mutant lacking episite region, a gene(s) for phage integration function, and a gene for the synthesis of O-antigen 34; E3”intD;16 appears to be a mutant concerned with some function necessary for integration. Media. Bacto Penassay broth (Difco) was used throughout. Nutrient agar: polypeptone (Daigo), 5 g; meat ext’ract (Mikuni), 4 g; yeast extract (Difco), 2 g; NaCI, 5 g; agar, 12 g; distilled water, 1000 ml. Nutrient soft agar was the same as nutrient agar, except that 4 g of agar was used. Both nutrient agar and nutrient soft agar described in this report could be replaced by Difco nutrient broth supplemented with 1.2%1 and 0.4% MATERIALS AND METHODS agar, respectively. TCG medium has been Bacterial strains. SAlOO is an ~l~-lyso- described (Toyama et al., 1966a). Indicator agar (Smith and Levine, 1967) in which genie derivative of Salmonella anatum strain 1 (Toya.ma et al., 1966a). SA104 is its mu- polypeptone (Daigo) replaced Bacto-tryptone was used for the detection of integratant requiring thymine and uracil. Phage strains. The wild-type cs4c (=ek34c tion deficient mutants. Phage techniques followed t)hose described in previous reports, Toyama et al., 1966) and its following derivatives were used: by Adams (1959). ~~~~1-1is a clear-plaque mutant unable to Preparation of mutagen-treated phage. All produce active immunity substance; this mutants described in this report were isolated from a ~~~~1-1by following mut’agens. 1 The outline of this work was presented at the 1. Nitrous acid : The phage were treated 14th and 15th General Meetings of the Society of Japanese Virologists in 1966 and 1967. for 30 minutes at pH 4.2 and 25” in 0.2 M For a full understanding of the processes involved in virus multiplication, morphogenesis, and/or lysogenization, the use of mutations that can occur in many different genes controlling a variety of different functions is desirable. Conditional lethal mut,ants provide the most convenient tool since they are distributed over the whole length of the phage genome (Campbell, 1961; Edgar and Lielausis, 1964; Edgar et al., 1964; Epstein et al., 1963). We have made use of temperature-sensitive mutants of phage t34in the present research, because suitable suppressor mutants, such as amber or ochre, are not yet available in group E salmonellas. The experiments described below have shown that the genetic ma,p of phage c34is circular.

519

520

IKAWA,

TOYAMA,

sodium acetate-acetic acid buffer containing 0.05 M KNO2 . Survivors were about 10L4. Cont’rol phages were treated for 30 minutes in the same buffer without KNO,. There was no loss of infectivity. 2. 5-Bromouracil: An overnight culture of cells SA104 at 37” in TCG medium supplemented, per milliliter, with 50 pg of thymine and 50 pg of uracil was diluted lofold into t’he same fresh medium and grown to about 2 X lo8 cells/ml. The cells were harvested by centrifugation, washed once with physiological saline solution, and resuspended in fresh TCG medium supplemented, per milliliter, with 1 pg of thymine, 50 pg of uracil, and 9 clg of 5-bromouracil (Sigma). After 5 minutes of incubation, e34cwas added at a multiplicity of about 10, and the culture was kept at 37” for 120 minutes. After treat’ment with chloroform and centrifugation, mutants were searched for in the lysates. 3. Sitrosoguanidine (N-methyl-N’-nitro N-nitrosoguanidine) : An overnight culture of SAlOO in TCG medium was diluted lofold in the same fresh medium, grown to about 2 X lo8 cells/ml, and then infected with e34cat a multiplicity of about 5. After 5 minutes, the culture received nitrosoguanidine (Aldrich Chemical Co., Ltd.) at a final concentration of 10 pg/ml, was kept at 37” for 120 minutes, treated with chloroform, and centrifuged. The lysates were used for isolation of mutants. Isolation of mutants. Surviving phages in the mutagen-treated lysates were plated on nutrient agar plates, incubated at 25” for several hours, by which time plaques had become just visible, then shifted to 38”, and kept overnight. Small plaques were transferred with sterile glass pins onto duplicate plates seeded with SAlOO, of which one was incubated at 25” and the other at 38” overnight. Phage clones which gave rise to plaques at 25’ and not at 38” were picked, subjected to three successive single-plaque isolations, propagated, and stored in ice box (Toyama et al., 1966a). Jlutants whose reversion frequency was higher than 10e4 were discarded. Isolation of double mutants. The lysate of a cross between a pair of mutants was plated at 2?‘. Individual plaques were ran-

AND UETAKE

domly transferred with sterile glass pins to 3 plates; one was seeded with indicator cells alone, the second with cells and one parental phage (lo6 phages/plate), and the third with cells and the other parental phage. The first plate was incubated at 25’, and the other two at 38”. The spots which failed to show clearing on bot’h of the 38” plates were picked, propagated, and reconfirmed to be double ts mutants by crossing with each of the parents. Genetic crosses. An overnight culture of cells SAlOO at 37” in Penassay broth was diluted lo-fold in the same fresh medium, grown with aeration to 2 X lo8 cells/ml, and received KCN at a final concentration of S mM. The cells were mixed with an equal volume of phage mixture containing 1.4 X log particles/ml of each parental type, shaken gently at 25” by hand for 10 minutes, then diluted lo-fold into Penassay broth containing antiphage serum and 4 mM KCN. Ten minutes later, the infected cells were diluted 104-fold in Penassay broth and incubated at 25” for 240 minutes. Lysis was completed by the addition of chloroform. The lysates were assayed at 25” for total progeny and at 38” for wild-type recombinants. Recombination values are given as t’wice the percentage of ts+ recombinants, since double ts recombinants are not detected by this assay. Complementation test. (1) Spot test: Agar plates were overlayed with soft agar plus indicator cells and kept at 38” for 60 minutes before spotting. One drop of each mixture containing a pair of mutants to be tested (each at a concentration of about 10’ phages/ml) was spotted, and the plates were immediately incubated at 38”. As control, each mutant was spotted alone. Although complete clearing of spot is classed as positive complementation, only negative complementation was ta.ken as significant, because intragenic complementation is also possible (Edgar et al., 1964). In indicator cells were some experiments, seeded together with one of known ts mutants (lo6 phages/plate). After keeping plates at 38” for 30 minutes, one drop of each unknown mutant to be t’ested was spotted, and the plates were incubated at 38”. This modified spot test proved very

GENETIC

OF PHAGE

MAP TABLE

TEMPERATURE

1

SENSITIVITY

OF SOME

Burst size Cistron

521

84

ts MUT.4NTS Plaque-forming

ability

Mutant 25°C

37OC

39°C

25°C

30°C

35°C

3i”C

38°C

ts+ 3 13

450 390 370

150 2.7 1.5

40 2.0 1.5

+ + +

+ + +

+ -

+ -

+ -

C c II E

59 213 14 28

480 250 280 230

37 25 3.0 1.7

3.1 2.4 2.1 1.5

+ + + +

+ + + +

+ + -

+ -

-

E F G H I

45 24 1 35 6

150 330 390 470 260

3.6 1.0 1.1 1.1 0.8

1.5 1.1 2.2 0.6 0.2

+ + + + +

+ + + + +

+ + + + -

-

-

J K K

130 50 4

150 210 310

4.6 36 5.7

4.6 3.0

+ + +

+ + +

+ +

+ +

-

A B

useful for screening a large number of mutants and for isolation of double ts mutants. (2) Burst size test: The burst size under nonpermissive conditions was determined as described for the cross except that the incubation temperature after neutralization of free phage was shifted to 37” and that the durat,ion of incubation was reduced to 120 minutes. As control, cells infected with either mutant alone and those with wild-type phages were employed for comparison. RESULTS

Isolation ancl Characterization of Mutants Since preliminary experiments revea.led that the burst size of wild type is a little reduced even at 37” and the efficiency of plating decreases above about 40”, mutants which gave rise to plaques at 25” but not at 38” were isolated as described in Materials and Methods. Individual mutants were given consecutive numbers prefixed with ts, to indicate the nature of the mutant phenotype, and with a letter which indicates the particular mutagen used: (1) Mutants tsN1 through tsN61 were induced with nitrous acid; (2) mutants tsB71 through tsB73 were induced with 5-bromouracil; (3) mutants tsNGlO1 through tsNG228 were induced with nitrosoguanidine. In the present report prefixes N, B, and NG will generally

not be used and the mutants will be identified by numbers only. Plaque-forming abilities and burst sizes at various temperatures have been determined. Table 1 gives some of the results. The mutants are very heterogeneous with regard to temperature sensitivity. All mutants plate at both 25’ and 30”, while most of them do not plate at 37”. A few give rise to very minute plaques at 37”, and their efficiencies of plating remain about constant within a certain range of elevated temperature. In general, burst sizes of mutants are reduced at 37” to Ho - ,;SO of those at 25”, and are near the wild-type value at 25”, although a few mutants have small burst sizes even at 25”. Compbmextatirm. Tests At first, spot complementation tests between pairs of mutant phages were carried out in all possible combinations, and 190 ts mutants used in this experiment were classified into 11 cistrons, A through K. The distribution of various mutations is given in Table 2. There is heterogeneity in distribution of mutations. Mutants induced by nitrosoguanidine include many double ts mutants, indicating its strong mutagenicity. Although not completely unambiguous, the spot test proved to be of great value for assigning new or known cistron to mutants.

IKAWA,

522 TABLE DISTRIBUTION

TOYAMA,

2

OF Is MUTANTS Number of mutants from each isolation series

Cistron is

B

NG

A B C II E F G H I J K E-F E-I F-G F-K G-K Unclassified

50 3 1 8 8 2 8 10 3 00 22 0 0 0 0 0 0

Total mutants Number of cistrons

61 10

Total mutants

2 0 0 0 0 0 0

2 4 1 9 12 5 9 0 18 3 53 1 1 1 1 5 1

7 7 2 18 20 7 17 1 21 3 77 1 1 1 1 5 1

3 2

126 10

190 11

0 0 1 0 0 0 0

Burst-size tests were performed to confirm the results obtained from the spot. tests. Complementation index was expressed as percentage of burst size of the wild type at 37”. Some of the results are summarized in Tables 3 and 4. Complementation between different cistrons results in significantly larger burst size than those obtained with either mutant alone. On the other hand, among mutants of the same cistron, complementation index remained at the level of background. The data with ts59 in Table 3 are not unambiguous; this is probably due to the nature of the function of t’his cistron, which is regarded to be concerned with cell lysis, since in tdY-infected cells intracellular phages are synthesized even in larger number than in wild type-infected cells and some of them may be released by chloroform treatment. When assa,yed after centrifugation without chloroform treatment, free phage titers of ts.59 were very low, comparable to those of ts mutants in other cistrons. Two-Factor

Crosses

Two-factor crosses among ts mutants were performed to construct the genetic map.

AND

UETAKE

Crosses were also performed between ts mutants and clear-plaque mutants. For the latter crosses, ts,c+ recombinants were prepared by crossing ts,c mutants with wild type. Crosses between ts,c+ mutant’s and c mutants were carried out, in the same way as described for the crosses among ts mutant’s, and the frequency of recombination was expressed as twice the ratio of c+ plaque formers at 38” to the total progeny at 25”. Also used for crosses were integration-deficient mutants. In crosses between ts and int mutants, lysates were plated and incubated overnight at 38”. From plaque centers cells were transferred to indicator agar with wooden sticks, which were soaked in indicator culture beforehand. This modified procedure has the advantage that it facilitates the distinction between dark-green growth of int mutant carrier cell clones and light-green growth of wild-type lysogens (Smith and Levine, 1967). After overnight incubation at 37”, wild-type recombinants which give rise to light green growth were scored. For crosses between c and int mutants, three modifications were made: (1) progeny phages were plated and incubated at 29; (2) only turbid plaques were spotted on indicator agar; (3) preadsorption of indica,tor cells onto a wooden stick was not necessary, because plaques on master plates at 25” contain more sensitive cells than those at, 38”, at which temperature int mutants give rise to almost clear plaques. Recombination value between c1 and cI1 mutants was determined by crosses between ~1-1 and ~11-1 mutants. Lysates were plated and incubated overnight at 25”. cf recombinants were scored by the appearance of plaques, and recombination frequency was expressed as twice the percentage of wildtype recombinant’s. The results from these experiments are summarized in Fig. 1. Although recombination values are not strictly additive, all mutants can be arranged in one linkage group. Most of the data from crosses among very distant mutations are not included, but t’hey are consistent with the order of the mutations given in E’ig. 1. These results also indicate the circularity of the vegetative map.

GENETIC

MAP

OF PHAGE

TABLE INTERGROUP COMPLEMENTATION

523

es4

3

TESTS OF TEMPERATURE-SENSITIVE

MUTANTS

Cistron

A4

B

C

D

E

F

G

H

I

J

Mutant

3

13

59

14

28

24

1

3.5

6

130

4

16.8 7.4 19.9 19.4 38.7 15.1 12.3 37.5 1.4

11.5 15.3 21.7 24.5 23.6 14.8 15.0 1.0

14.5 14.7 27.5 26.0 0.6

9.8 49.2 21.7 0.7

11.2

2.8 3.2

K J I H G F E I) c B A

56 4 130 6 35 1 24 28 14 59 13 3

20.8 74.3 51.6 46.4 45.5 17.6 32.7 58.2 37.5 79.0 52.1 1.2

23.1 20.5 34.0 100 41.7 30.5 18.2 21.8 44.3 70.0 0.9

37.7 38.2 53.0 17.3 31.2 86.9 40.7 17.6

TABLE INTRAGROUP

COMPLEMENTATION

K 56 72 73 23 44

6.7 12.3 7.8 10.3 1.4

56 2.2

3.0

4

TESTS OF TEMPERATURE-SENSITIVE

MUTANTS

K

Cistron Mutant 50 58 4

4.8 6.8 9.4 7.1 28.7 8.6 0.2

K

44

23

73

72

56

14.1 20.0 5.6 8.6 9.1 9.7 8.7 7.9

21.2 28.0 8.7 8.5 6.9 11.4 13.1

18.8 25.9 6.4 3.0 2.4 2.1

18.8 20.0 3.1 6.9 2.1

10.0 25.8 2.8 2.2

Three-Factor Crosses A more precise determination of gene order may be made with three-factor crosses. The double ts mutants were crossed with the single ts mutants. When the single marker lies outside the segment between the markers of the double mutants, the frequency of the wild-type recombinant is comparable to that observed in two-factor cross between the single marker and the closer marker of the double mutant. On the other hand, when the single marker lies inside the segments, the frequency is far less than that observed in crosses between the single marker and either of the two markers in the double mutant. Some of these crosses are presented in Table 5. Although double crossover generating the wild-type recombinant occurs 5 to 6 times too frequently (negative interference),

4 11.0 15.3 3.2

58

50

25.9 22.1

14.7

the results confirm the order determined by two-factor crosses. The construction of double ts mutants involving various combinations of the mutations in the 32-14 segment was very difficult because of very close linkage in most combinations. This technical difficulty was eliminated by using crosses between c+, tsx and c, tsy. Progenies of the crosses were assayed at 25” for total phages, and at 35” for ts+ recombinants. The map order can be determined from the distribution of c markers among ts+ recombinants. If a c marker appears as a major class among ts+ recombinants, the order of genes should be c-tsxtsy. The results in Table 6 show that the order is c-32-30-3-9-(47,55)-13-59-14. To confirm positions of int mutants, crosses involving c, Is, and int markers were

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GENETIC TABLE THREE-FSCTOR

-

Sene order

-

Parent 1

Measured”

arent 1

1

3-13 55-59 55-59 55-59 55-14 59-14 59-14 59-28 14-28 14-61 14-45 6145 45-5 45-5 2-5 45-5 45-24 5-35 24-35 24-35 24-l 24-l l-35 1-35 134-35 24-35 24-6 35-6 35-6 35-6 35-6 35-50 G-50 6-50 6-50 6-50 6-181 6-181 181-50 181-50 6-50 6-50 6-4 50-4 50-4 50-4 50-4 50-56 50-56

E xpected’ TABLE

0.072 0.51 0.081 3.6l 0.6l 2.82 0.62 0.8” 0.23 0.23 0.32 0.32 1.5’ 0.12 1.12 1.33 0.33 0.53 0.83 1.02 0.52 1.1’ 0.12 0.31 0.45 1.52 0.83 7.12 0.22 0.0Q2 6.22 0.64 1.83 0.62 0.41 0.31 0.24 0.43 0.53 0.084 0.21 2.53 1.02 4.53 0.1’ 0.12 0.72 0.22 0.22 -

5 (Conlimed)

a The number of experiments repeated is indicated as a superscript to the mean recombination value. b Calculated by assuming that the three mutations are located in the order shown in Fig. 1.

-

55 3 13 14 59 55 57 14 57 28 61 12 12 2 45 24 5 24 5 1 134 140 134 140 140 6 35 24 17 11 50 6 35 130 181 192 130 192 130 192 178 4 50 6 178 58 56 4 58

525

@ TABLE

ts MUT.~NTS

Percent recombination

i

OF PHAGE

5

CROSSES BETWEEN

Cross

MAP

0.002 0.5 0.007 4.7 0.2 4.4 0.7 0.2 0.02 0.05 0.2 0.07 3.0 0.02 0.6 1.5 0.05 0.1 1.5 0.2 0.8 2.1 0.03 0.05 0.03 1.5 0.1 6.2 0.01 0.008 4.9 0.07 1.5 0.1 0.05 0.02 0.02 0.3 0.5 0.004 0.5 2.7 0.13 4.9 0.02 0.02 0.6 0.02 0.03

3-55-13 3-55-59 55-13-59 55-59-14 55-59-14 55-59-14 59-14-57 59-14-28 14-57-28 14-28-61 14-61-45 61-12-45 12-45-5 45-2-5 45-2-5 45-5-24 45-5-24 5-24-35 5-24-35 24-l-35 24-1-134 24-l-140 1-134-35 l-140-35 134-140-35 24-35-6 2435-6 24-35-6 35-17-6 35-11-6 35-6-50 35-6-50 35-6-50 6-130-50 6-181-50 6-192-50 6-130-181 6-181-192 130-181-50 181-192-50 6-50-178 6-50-5 6-50-4 6-50-4 50-178-4 50-58-4 50-4-56 50-4-56 a-58-56

6

THREE-FACTOR CROSSES HETWEEN c ts MUTANTS Multiplicity

cross

Recombinant ratio

Gene order

Parent 1 Parent 2 Pa;ent PaY2cnt $SJ c,

-

c+, Cf, c+, c+, Cf, c+, c+, Cf, Cf, Cf, c+, c+, Cf,

13 3 3 32 3 13 3 13 14 3 14 14 13

c, c, c, c, c, c, c, c, c, c, c, c, c,

47 30

Q 30 13 3 55 55 59 59 3 55 59

4.2 3.8 5.4 2.8 5.5 5.5 4.7 5.6 3.0 5.5 5.6 5.5 6.4

4.2 3.8 6.1 3.5 5.5 5.5 4.9 5.9 3.0 5.4 5.7 5.6 4.9

0.81 0.73 0.04 0.02 0.20 0.72 0.12 0.86 0.64 0.03 0.85 0.66 0.2-l

13-47-c 3-30-c Q-3-c 30-32-c 13-3-c 13-3-c 55-3-c 13-55-c 14-59-c 59-3-c 14-3-c 14-55-c 59-13-c

also carried out. As seen in Table 7, the order is ts3-cI-int-ts56. The results described above taken as a whole give conclusive evidence for the circularity of the genetic map. DISCUSSION

All the results obtained from complementation and recombination tests can be summarized into a map in which distances are expressed as mean values obtained from two-factor crosses between nearestneighbor mutants. Such a map is shown in Fig. 2. It gives clear indication of the circularity of the vegetative map. Circular vegetative maps have been described in phages T2, T4, and PK (Baylor et al., 1965; Streisinger et al., 1964; Edgar and Lielausis, 1964; Rutberg et al., 1967). In addition to genetic evidences, T2 and T4 DNA molecules have been shown physically to be circularly permuted and terminally repetitious (Thomas, 1963; Thomas and

526

IKAWA,

TOYAMA, TABLE

THREE-FACTOR

AND

UETAKE

7

CROSSES INVOLVING c, ts, AND int MARKERS

Cross

Recombinant ratio int+ c’ ts+

Multiplicity Parent 2

Parent 1

Parent 2

(G&t+c+ ts+) + (int+ Cf ts)

c ts3

c+ i&N16

c ts3

c+ delNZ1

4.5 6.4 5.3 4.9 4.5 4.5

4.8 6.4 4.7 4.9 4.5 4.5

0.87 0.75 0.92 0.93 0.83 0.07

Parent 1

c is56

c+ AN16

Gene order

intNWc-3 intN16-c-3 deZN21-c-3 deENZl-c-3 deZN21-c-3 56-intN16-c

,4lthough the presence of terminal repetitious nucleotide sequences has not been settled as yet, the circularity of t,he map and the above physical findings are consistent with each other (Toyama et al., unpublished results). It has been shown that DT\‘A molecules from many temperat,e phages such as X, 480, 21, 186, 424, and 434 are of linear duplex structure with cohesive ends which make them able to form either circles or linear multimers (Hershey and Burgi, 1965; Yamagishi et al., 1965; Baldwin et al., 1966). Yet, the genetic map of X is not circular. In contrast, Rutberg et al. (1967) have shown that the genetic map of temperat’e Bacillus phage, PK, is circular, and Thomas (1967) has reported that DNA molecules liberated Gi from phage P22 are both circularly per1% muted and terminally repetitious. Phages x FIG. 2. Genetic map of phage 9. The extents of and ~$80 are classified as specialized transdelet,ions ill mutants delN21 and i&N16 are esducing phages (Morse et al., 1956; Rlatsutimated from recombination frequencies with shiro, 1963), and phages P22 (Zinder and tsN23 or c1, but that in intN16 is only tentative, Lederberg, 1952) and c34 as generalized as its characteristics in detail have not been transducing phages. These result’s, together clarified as yet. with our above experiments, favor the notion Rubenst’ein, 1964; MaeHattie et al., 1967). that DNA molecules of generalized transWith these in mind preliminary studies have ducing phages are circularly permuted with been undertaken to see whether this also respect to base sequences. Ikeda and Tomizawa (1965) reported that holds for &DNA molecules. Preliminary retransducing particles of phage Pl cont’ain a sults to date have shown that the E~~-DNA fragment of bacterial chromosome only. Almolecules extracted from phage particles are though the mechanism by which transducing circularly permuted in regard to base separt’icles are formed has not been definitely quences, and have no cohesive ends such as settled, it would be tempting to assume that observed in ot’her temperate phages as the production of transducing fragment of terminal complementary single-stranded nucleot’ide sequences (Hershey et al., 1963; bacterial chromosome is mediated by the Yama.gishi et al., 1965; Baldwin et al., 1966). same process as that involved in fragmenta-

GENETIC

MAP

tion of concatena,ted DNA into headful length of mature phage DNA. This interpretation is also compatible with the fact that E~~-DNA molecules extracted from vegetative pool of infected cells are larger than that of mature phage DNA. These concatemers are cut into headful length of DNA by the process involving head formation during the course of maturation (Toyama et al., 1966b, and unpublished results). Some of the results of complementation test,s are complicated and require comment. Mutant ts4 in cistron K shows relatively low values in most crosses, whereas mutant ts3 in cist,ron A and mutant ts13 in cistron B show high values (Table 3). Cistrons A and B direct the synthesis of proteins necessary for phage DNA replication, whereas others, C through cistron K, control the synthesis of structural components (Ikawa et al., 1966, and unpublished results). There remains a possibility that abnormal protein subunits of mutant types can be assembled into phage architectures together with subunits synthesized by nonmutant genes, resulting in the production of malformed structures. As for the location of cistron 034 which controls the synthesis of somatic antigen O-34, it should be noted that cistron 034 locates very close to a region required for prophage integration, since mutant delN21 lacks cist,ron 034, too. This favors the explanation that characters conferred by lysogenie conversion are under the control of bacterial genes which have become permanent#ly incorporated into a viral genome (Luria et al., 1958; Uetake and Hagiwara, 1961). ACKNOWLEDGMENT The authors are deeply indebted to Dr. S. E. Luria, Massachusetts Institute of Technology, for criticism and help in preparing the manuscript. This investigation was supported in part by grants from the Jane Coffin Childs Memorial Fund for Medical Research, U. S. Army Research and Development Group (Far East), Waksman Foundation, Japan, and Ministry of Education, Japan. REFERENCES ADAMS, M. H. (Interscience),

(1959). “Bacteriophages.” New York.

Wiley

OF PHAGE

,%I

527

BALDWIN, R. L., BARRAND, P., FRITSCH, A., GOLDTHWAIT, D. A., and JACOB, F. (1966). Cohesive sites on the deoxyribonucleic acids from several temperate coliphages. J. Mol. Biol. 17, 343-357. BAYLOR, M. B., HESSLER, A. Y., and BAIRD, J. P. (1965). The circular linkage map of bacteriophage TZH. Genetics 51, 351-361. CAMPBELL, A. (1961). Sensitive mutants of bacteriophage X. Virology 14, 22-32. EDGAR, R. S., and LIELAUSIS, I. (1964). Temperature-sensitive mutants of bacteriophage T4: their isolation and genetic characterization. Genetics

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EDGAR, R. S., DENHARDT, G. H., and EPSTEIN, R. H. (1964). A comparative genetic study of conditional lethal mutations of bacteriophage T4D. Genelics 49, 636648. EPSTEIN, R. H., BOLLE, A., STEINBERG, C. M., KELLENBERGER, E., BOY DE LA TOUR, E., CHEVALLEY, R., EDGAR, R. S., SUSMAN, M., DENHARDT, G. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375-394. HERSHEY, A. D., and BURGI, E. (1965). Complementary structure of interacting sites at the ends of lambda DNA molecules. Proc. Natl. Acad Sci. U.S.53,32&328. HERSHEY, A. D., BURGI, E., and INGRAHAM, L. (1963). Cohesion of DNA molecules isolated from phage lambda. Proc. Natl. Acad. Sci. U.S. 49, 748-755. IKAWA, S., TOYAMA, S., and UETAKE, H. (1966). Temperature sensitive early mutants of Salmonella phage @. Ann. Rept. Insf. Virus Res., Kyoto Univ. 9, 132-137. IKEDA, H., and TOMIZAWA, J. (1965). Transducing fragments in generalized transduction by phage PI. I. Molecular origin of the fragments. J. Mol. Biol. 14, 85-119. LURIA, S. E., FRASER, D. K., ADAMS, J. N., and BURROUS, J. W. (1958). Lysogenization, transduction, and genetic recombination in bacteria. Cold Spring Harbor Symp. Quant. Biol. 23, 7182. MACHATTIE, L. A., RITCHIE, D. A., THOMAS, C. A., JR., and RICHARDSON, C. C. (1967). Terminal repetition in permuted T2 bacteriophage DNA molecules. J. Mol. Biol. 23, 355-363. MATSUSHIRO, A. (1963). Specialized transduction of tryptophan markers in Escherichia coli K12 bacteriophage $80. Virology 19, 47&482. MORSE M. L., LEDERBERG, E. M., and LEDERBERG, J. (1956). Transduction in Escherichia coli K12. Genetics 41, 142-E%. RUTBERG, L., SANDSTR%M, E., and NILSSON, K. (1967). Circular chromosomal map of a temperate Bacillus phage. Virology 32, 103-108.

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SMITH, H. O., and LEVINE, M. (1967). A phage P22 gene controlling integration of prophage. Virology 31, 207-216. STREISINGER, G., EDGAR, R. S., and DENHARDT, G. H. (1964). Chromosome structure in phage T4. I. Circularity of the linkage map. Proc. N&Z. Acad. Sci. U.S. 51, 775779. THOMAS, C. A., JR. (1963). The arrangements of nucleotide sequences in T2 and T5 DNA molecules. Cold Spring Harbor Symp. Quant. Biol. 28, 395-396. THOMAS, C. A., JR. (1967). The rule of the ring. J. Cell. PhysioE 70, Suppl. 1,13-34. THOMAS, C. A., JR., and RUBEKSTEIN, I. (1964). The arrangements of nucleotide sequences in T2 and T5 bacteriophage DNA molecules. Biophys. J. 4, 93-106. TOYAMA, S., IKAWA, S., and UETAKE, H. (1966a). Ribonucleic acid metabolism in bacterial cells destined to lysogeny. Virology 30, 193-203.

AND

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TOYAMA, S., IKAWA, S., and UETAKE, H. (1966b). Deoxyribonucleic acid synthesis of Salmonella phage ss4kc. Ann. Rept. Inst. Virus Res., Kyoto Univ. 9, 129-132. TOYAMA, S., IKAWA, S., and UETAKE, H. (1967). Regulation of the synthesis of phage messenger RNA in temperate phage-infected cells. Virology 33,231-238. UETAKE, H., and HAGIWARA, S. (1961). Genetic cooperation between unrelated phages. Virology 13,500-506. YAMAGISHI, H., NAKAMURA, K., and OZEKI, H. (1965). Cohesion occurring between DNA molecules of temperate phage 480 and lambda or $81. Biochem. Biophys. Res. Commun. 20, 727732. ZINDER, N. D., and LEDERBERG, J. (1952). Genetic exchange in Salmonella. J. Bacterial. 64, 679699.