A genetic study of temperature-sensitive mutants of the Subtilis phage SP82

VIROLOGY

30, G50-660

(1966)

A Genetic

Study

of Temperature-Sensitive

of the S&i/is

Phage

EUNICE Department The

oj Chemistry, Harvard University, Public Health Research Institute

SP82’

KAHANz

Cambridge, of the City

Accepted

Mutants

July

1?1assachusetts, and Department of Trirology, of iVew York, Inc., New York 10009

8.8, 1966

Temperature-sensitive mutants of the Bacillus subtilis phage SP82 have been isolated. Forty-nine mutations were assigned to 17 separate cistrons on the basis of complementation tests and, when mapped by two-factor crosses, comprised a single linear linkage group with approximately 26% recombination between end markers. Results from three-factor crosses confirmed the order of the markers. INTRODUCTION

The Bacillus subtilis phage SP82 (Green, 1964) is one of a number of independently isolated B. subtilis phages containing 5hydroxymethyluracil in place of thymine in their DNA (Marmur et al., 1963; Pene and Marmur, 1964; Okubo et al., 1964). The DNA isolated from SP82, like that of several of the related phages, is capable of producing infected centers in competent cultures of B. subtilis. In preparation for future experiments that would investigate the role of the unusual basesin viral nucleic acids, we isolated a number of temperaturesensitive (ts) mutants of this phage. This paper describes the results of genetic experiments involving the ts mutants. We were particularly interested in establishing whether or not these genetic markers would have a circular distribution when mapped, as reported for mutations of the eoliphage T4 (Streisinger et al., 1964; Edgar and Lielausis, 1964). DNA of the latter phage resemblesthat of SP82 both in size (120 X 1 This investigation was supported in part by research grants HD-01229 and AI-04360 and in part by a Public Health Service fellowship No. 7-F2-GM-18,603-02-A2 from the National Institutes of Hea1t.h. 2 Present address: The Public Health Research Institute of the City of New York, Inc., New York 10009.

lo6 daltons) and in the presence of an unusual base completely replacing one of the four normally found. Our recombination experiments indicate that the temperaturesensitive markers we have isolated from mutagen-treated SP82 comprise a single noncircular linkage group, comparable to the genetic maps of the smaller coliphages T5 (Fattig and Lanni, 1965) and lambda (Campbell, 1961), both of which contain the usual basesin their DNA. Previous genetic studies of subtilis phage include a report on the isolation and characterization of 12 temperature-sensitive mutants of SP3, a phage containing the usual bases (Nishihara and Romig, 1964). Recombination has been reported in SPOl, a 5 - hydroxymethyluracil - containing phage (Okubo et al., 1964). One host-dependent and two plaque morphology mutants were mapped. These three markers were linked, the two outside markers exhibiting 15% recombination. MATERIALS

AND

METHODS

Bacteria and bacteriophage.The properties of bacteriophage SP82 have been described by Green (1964). The host bacterial strains were B. subtilis SB-1 (Nester et al., 1963) and B. subtilis 168 thy- ind- (Farmer and Rothman, 1965). The former strain requires histidine and tryptophan for growth; the 650

GENETIC

STUDY

OF

SUBTILIS

latter strain requires thymine (or thymidine) and indole (or tryptophan). Growth and assay of bacteriophage. The media and techniques already described (Green, 1964) for the growth and titration of SP82 have been modified by the substitution of a supplemented minimal medium (NM) in place of H-broth. We used the medium described by Nomura et al. (1962), containing 1 mg of glucose per millilit,er in place of glycerol and supplemented with 1 mg of casamino acids (Nutritional Biochemicals Corporation, Cleveland, Ohio) per milliliter, 20 ,ug tryptophan per milliliter and 1OV M MnS04. (In those cases where the thymidine-requiring strain was grown, 10 ,ug of thymidine per milliliter was also included.) In this medium lysates were found to retain 60-30 % of their original titer after 6 months’ storage at 4”. Early in this study it was found that lysates made in veal infusion broth, in antibiotic medium 3 (Difco Laboratories, Detroit, Michigan) or in H-broth were unstable, losing up to half their titer in 1 week. To prepare high-titer phage stocks a confluently lysed standard overlay plate was flooded with 5 ml of NM and allowed to stand for 4 hours at room temperature. The fluid phase was added to 20 volumes of a growing culture of SB-1 in NM having a cell density between 2 and 3 X lO*/ml. (Estimates of the titer were obtained by measuring the optical density at 550 rnp of the culture with a Coleman Jr. Spectrometer and comparing the reading to a calibration curve.) This culture was agitated at 37” for about 1 hour, by which time clearing occurred. Such lysates had titers of 2 to 4 X lOlo plaque-forming units (PFU) per milliliter. Bacterial debris was removed by centrifugation (6000 g for 10 minutes) and the stocks were stored at 4”. Production and isolation of bacteriophage mutants. Most mutants were isolated from a single st’ock of viruses treated with hydroxylamine according to the procedure described by Freese et al. (1961). An SP82 lysate in antibiotic medium 3 containing 5 X log PFU/ml was exposed for 12 hours at 37” to 1 M hydroxylamine HCl in 0.1 M sodium phosphate at pH 6.0. The survivors

PHAGE

SP82

651

(3 X lo6 PFU/ml) were added at an input ratio (IR) of 1 PFU/cell to a culture of SB-1, in H-broth having a cell density of 106/ml. This culture was agitated at 37” for 6 hours; during this time the culture became visibly turbid and then cleared. The resulting lysate contained lOlo PFU/ml. It was assumed that multiple cycles of growth at the low temperature would permit segregation under nonselective conditions of the mutational heterozygotes that were chemically induced (Tessman, 1959). Sufficient lysate was plated on SB-1 to produce approximately 100 plaques/plate on incubation at 37”. These “master” plates, incubated at the nonselective temperature, were replica,ted by means of a wooden block covered with sterile velvet (Lederberg and Lederberg, 1952) onto plates overlaid with top agar seeded with SB-1, and the replicas were incubated at 47”. Phages that produced plaques at 37” but not at 47” were termed temperature-sensitive (ts). Ts mutants comprised 1% of the plaques on the master plate. Plaque-morphology mutants were also present in the hydroxylamine-treated lysate. These mutants formed plaques with a turbid center (tu) on plates seeded with SB-1 but formed plaques indistinguishable from the wild type on B. subtilis 168 thy- ind-. Ts mut’ants were also obtained from a lysate prepared from bacteria that had been exposed to 2-aminopurine during infection. In this experiment, SB-1 was grown in NM to a cell density of approximately 1 X 108/ml, 2-aminopurine (Sigma Chemical Company, St. Louis, Missouri) was added to final concentration of 1 mg/ml, and the culture was incubated further until the O.D. had doubled. Bacteriophage were then added at an IR of 5 and the infected culture was incubated at 37” for 2 hours. By the end of this time the culture had cleared and the lysate was screened for ts mutants by the above procedure. In this case the mutation frequency was approximately 0.1%. A third series of ts mutants was isolated from phage grown in the presence of N-methyl-N’-nitro-N-nitrosoguanidine (Al-

652

KAHAN

drich Chemical Company, Milwaukee, Wisconsin). Cultures of B. subtilis SB-1 were grown to a cell density of 2 X lO*/ml in NM and combined with 1 pg nitrosoguanidine per milliliter immediately before infection with phage (IR of 5). The burst size obtained in this culture was reduced by 65 %. Ts mutants occurred at a frequency of approximately 1%. Mutants isolated from the mutagentreated stocks are identified by a prefix letter to indicate the mutagen employed (“A” in the case of 2-aminopurine, “G” for nitrosoguanidine, and “H” for hydroxylamine) and a number. We will deal primarily with temperature-sensitive phage; however, in those cases where plaque morphology mutants were also involved, the symbols ts will identify the former class, tu the latter. Assay of genetic recombination. To 0.6 ml of minimal medium containing approximately 2 X lo8 cells of a logarithmically growing culture of SB-1, 0.004 LW KCN was added! followed 3 minutes afterward by the addition of 0.4 ml of NM containing I X log PFU of each of the two mutants between which recombination was being tested. Ten minutes later unadsorbed phage were inactivated by the addition of 0.01 ml of a lo-fold dilution of antiSPS2 rabbit serum. [This serum was obtained from rabbits that had been injected on each of 5 successive days with lOI PFU of SP82, rested for 10 days, and again injected on each of 5 successive days with 1012PFU. They were bled 6 days later and yielded a serum with an inactivating coefficient, K, of 300 units (Adams, 1959).] Ten minutes after the addition of antiserum, the suspensionswere diluted lO,OOOfold into growth tubes containing NM. Samples were plated on H-broth agar plates to establish the concentration of surviving colony formers. The concentration of infected centers (ICs) was established by plating samples on H-broth plates seeded with SB-1 and incubated at 37”. The diluted suspension of infected cells was then incubated for 2 hours at 37”. Lysis was completed by the addition per milliliter of 10 pg of crystalline egg white lysozyme (Worthington Biochemicals Corporation, Freehold,

New Jersey). The lysate was titrated on B. subtilis SB-1 at 37” and at 47”. The recombination frequencies are given as titer at 47°C x 290 titer at 37°C The factor 2 is used because recombinants that are doubly mutant are not detected by this assay but are expected to occur with the same frequency as wild-type recombinants. The criteria for acceptable crosseswere those of Doermann and Hill (1953). Complement&ion tests. The ts mutations were assigned to cistrons on the basis of pairwise complementation tests (Benzer, 1961). About lo6 PFU of each of the mutant phages to be tested was placed on a 10 X 35 mm petri dish. Three milliliters top agar containing plating bacteria (B. .subtilis SB-1) was added, and the plates were incubated at 47”. Mutational defects were considered to be in different cistrons and scored as positive for complementation in those caseswhere confluent lysis of the lawn was observed. On the other hand, where the bacterial lawn developed normally, it was believed that parental particles were mutant for the samefunction and scored as negative for complementation. In addition to these two results, occasional plates contained a few individual plaques. This result can be due either to revertants in parental phage stocks or to “leaky” phage mutants. The latter have a greatly reduced burst size at high temperatures but nevertheless form very minute plaques. This “leaky” growth may allow the occasional liberation of revertant or recombinant progeny, either of which would give rise to plaques. This result was also scored as negative for complementation. Control plates containing each mutant phage alone indicated the number of plaques to be expected due to revertants and the extent of the “leakiness” of the parental phage. Complementation was also measured quantitatively by comparing the average burst size of mixedly infected bacteria at 37” and 49”. These experiments were carried out along with recombination experiments. An additional set of growth tubes was incubated at 49”. The lysates from the two sets

GENETIC

STUDY

OF

of growth tubes were plated and incubated at 37”. The percentage of the 37” burst obtained at 49” was considered a quantitative measure of complementation. This value was corrected for the 45% reduction in burst size of the wild-type when incubated at the higher temperature. Isolation of double mutants. The progeny of a cross between a pair of mutants were plated at 37”. Randomly picked plaques were tested for complementation with each of the parental mutants. In this complementation test phages from each plaque were transferred with a toothpick to 3 plates: one seeded with plating bacteria alone, another seeded with plating bacteria and one of the parental phages (lo7 phage per plate), and the third seeded wit,h plating bacteria and the second parental phage. The plate containing no parental phage was incubated at 37”, and the other two were incubated at 47”. In those cases where clearing was absent from the stabbed region of both of the 47” plates it was suspected that the donor plaque contained double ts mutants. Failure of the suspected double ts mutant to produce wild-type recombinants in crosseswith either of the single parental ts mutants was taken to confirm the identification. Nearly all known cistrons were included in the set of double ts muta’nts. The double mutants were used for further complementation tests and for three-factor crosses. In complementation tests their use largely eliminated the problems of “leakage” and reversion. RESULTS

Properties of Mutants The burst size of various mutants at a number of different temperatures was measured. Table 1 reveals that mutants vary in their sensitivity to elevated temperatures. Some mutants exhibit a sharp drop in burst size when incubation of the infected cell is carried out above a given temperature, whereas others show a gradual decline in burst size as the temperature of incubation is raised. Those mutants with the greatest resistance to elevated temperatures produce very minute plaques on plates in-

SUBTILIS

PHAGE

653

SP82

cubated at 47”. These plaques, however, could be distinguished easily from the much larger recombinant plaques. Note that H38, H166, and H180, which are located TABLE TER~PERATURE

1

SENSITIVITY MLJTANTP

OF

Average

Mutant

H48 A4 Hl6G H38 H180 A3 HI1 H385 H189 H2

burst

SOME

TS

sizeb

34°C

38°C

42°C

46°C

48°C

223 224 83 101 112 60 121 143 131 38

230 lG7 61 37 108 70 G8 132 164 42

252 25 25 7 35 16 10 6 54 <1

12 10 3
<1
a SB-I. was infected at 37” in the presence of 0.004 M KCN at an IR of about 5. Unadsorbed phage were neutralized with antiserum. IC’s were diluted lo- to &fold and incubated at the desired temperature for 2 hours, whereupon lysozyme was added and the lysates were assayed at 37”. b The average burst size of wild-type SP82 was found to be reduced by 45yo between the temperatures of 37” and 49” (see Table 3). TABLE ASSIGNMENT CISTROKS Cistron 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1G 17

2

OF TX MLJT~TIONS BY COMPLEMENT~TION

TO DIFFERENT SPOT TESTS

Mutations H177, H176, G145 G55, G108, HI0 H20 H48, H173, G79, G4, G152, H38, G39, H50, HG3, H166, Cl09 G95 A4, G121, G87 G48, G104, H385 A3 H56, G99, G7, H189, H18 H2, G132 HI, Hll, G29, H8 H175, G76, G82 H108, H15, G112 H24 H201

H326, H180,

G119 G75

RESULTS Mutations H177 x G55 H177 x H108 G55 X H20-H326 G55 X H56 G55 X G112 G119 X H38 H48 X H56 H38 X G109 H38 X G95 H38 X A4 H38 x H385 H38 X H56 G109 x G95 G109 x A4 G109 x H385 G95 X A4 G95 x H385 G95 x A3 G95 x G112 A4 X H385 A4 X A3 A4 X H56 A4 X G112 H385 X A3 H385 X H56 A3 X H56 H56 X H2 H56 X HI H56 x G112 Hl X H2 Hl x G112 H175 x G112 G112 X H24 H24 X H177-H201 H177 X H176 H20-H326 X H48 H48 X G119 H56 X H18 G112 x H108 Wild type (WT) H177 H55 H20-H326 H48 G119 H38 G109 G95 A4 H385 A3 H56 H2 Hl H175 G112 H24 (1 No significance is attached and Table 1 inasmuch as burst

TABLE OF COMPLEMENTATION Cistrons

1x2 1 x 2 x 2 x 2 x 4x5 4 x 5X6 5x7 5X8 5x9 5 x 6X7 6X8 6X9 7X8 7x9 7 x 7 x 8X9 8 X 8 X 8 X 9 x 9 x 10 x 11 x 11 x 11 x 12 x 12 x 14 x 15 X 16 X 1x1 3-4 x 4x4 11 x 15 x -

15 34 11 15 11

11

10 15 10 11 15 10 11 11 12 13 15 13 15 15 16 1-17 4 11 15

1 2 3-4 4 4 5 6 7 8 9 10 11 12 13 14 15 16

3 BURST

SIZE

TESTS

Burst

size=

37°C

49°C

250 183 196 155 275 250 302 335 112 140 128 174 314 138 200 160 247 110 710 420 110 187 410 172 125 108 114 210 387 136 228 188 250 205 80 610 345 106 340 600 196 79 256 570 220 69 640 104 170 62 210 82 56 117 385 330 235

182 55 45 84 110 31 83 41 19 48 19 70 47 52 23 43 45 33 162 162 26 60 146 35 40 66 100 84 160 27 43 29 74 39
47°C -3YC

to the differences in burst size for given mutants described size was found to be sensitive to the age of the medium. 654

x

WT37”C WT47”C

x 100

131 54 41 97 72 22 49 22 30 61 27 72 27 68 21 48 33 54 41 69 42 58 64 37 58 110 158 72 74 36 34 28 53 34 <2.2 0.28 0.68 <1.6 1.1 0.53 2.5 <0.71 0.41 0.45 <2.5 0.12 <1.6 0.64 <2.9 3.2 1.3 <3.1 0.70 0.23 <5.4 0.48 in this table

a The although

H177 G55 1120 G119 H38 G109 G95 A4 H385 A3 II56 H2 Hl 11175 G112 H24 H201

-

H177

superscript in most

_-___-_____-~~

6.87 4.1” -

H20

9.92 7.51 4.g3 -

G119

1.1’ 0.402 -

1.02 -

2.3l 1.12 0.512

12l 7.11

G95

TABLE

4

1.41 0.721 0.371 -

2.0’ 1.82 1.51 0.571 0.17” -

10’ 7.01

H385

A3

H.56

is based.

Data

2.3l 1.41 0.86> 0.732 0.46” 0.303 -

82 .__

4.5l 4.41 2.6:’

2.52 0.713

5.23 3.9’ 3.46 2.22

4.0’ 4.2l

16l 7.71 7.6O 6.6’ 6.g3

G112

the same

3.F

within

141 8.91 6.2l

H175

2.5l

7.62 6.22 3.31

9.32

Hl ~____

1’S MUTANTS

for mutants

1.22 2.6’ 1.6l 1 .5” -

4.2l 3.41 2.7l

10’ 9.1’

BETWEEN

122 121 6.9”

CROSSES

0.751 0.372 0.422 -

shown

INTEKCISTRONIC A4

8.0’

FOR

on which the average value using mutants listed.

8.4l

G109

11.P 8.51

H38

PERCENTAGEP

denotes the number of crosses cases experiments were conducted

3.3” -

G.55

RECOMBINATION

cistron

5.91 5.6l 4.8” 2.4l 1.71 -

16’ 13” lo5 8.1’ 7.83 5.8’ 8.5l 5.6l 6.62

H24

are pooled,

6.33 7.9” 5.41 3.12 2.v 1.6& -

8.8” 5.81

7.5’

15l 132 12’

H201 ___-

iii N

ii

ii

E s 2

2

z

z

z

3 I?

656

KAHAN

in the same cistron, do not have the same temperature-sensitivity pattern. With few exceptions the mutants form plaques at 37” that resemble wild-type plaques in size. Complementation Mutations were initially assigned to cistrons on the basis of the results obtained from complementation spot tests. The 49 ts mutations were assigned to 17 cistrons (Table 2). Eleven cistrons were found to contain more than one independently produced mutation. In most cases, recombination could be demonstrated between the mutations assigned to the same cistron. These sites, therefore, are clearly different. In other cases, in which the mutants were isolated from separate stocks, recombination experiments were not always performed, and the sites will be considered different until shown to be otherwise. In the case of the H-series, multiple mutations were found in two cistrons between which recombination could not be demonstrated. It was decided to include data from only one member of each of these two groups (H56, H8). Since ts mutations are known to undergo intragenic complementation (Edgar et al., 1964), only negative spot tests could be considered meaningful. As a further check, mutations from adjacent cistrons as well as some mutations in the same cistron and

2

I 3.3 H 177 HI76 6145

I I 655 GlO8 HI0

I

8

9

IO

G109 77

G95

A4 6121

648 6104

A3

4

5

6-10

II.0

I

2.3

I

FIG. 1. Linkage map of the between adjacent cistrons. The the cistron are listed below.

ts

If an exclusion reaction were rapidly established by the first infecting phage, the recombination frequency observed would be spuriously low. To test the possibility of exclusion, SB-1 was infected with equal input ratios of plaque-morphology mutant tu 1 and temperature-sensitive mutant ts H137. The infected culture was diluted in NM and distributed at a concentration of 0.1 IC per tube, to many small tubes. In one such experiment 98.5% of the bacteria were killed, 26 single bursts were examined, and 24 were found to yield both mutants in roughly equal numbers.

7

4.8

H20

Recombination

6

3 4.1

some mutations in widely separated cistrons were tested for complementation by the burst size method. The results of the burst size measurements are given in Table 3. Bacteria mixedly infected with phage carrying mutations in different cistrons as determined by spot tests yielded a burst size from 20% to 100% of that obtained at low temperature. Bacteria mixedly infected with phages carrying mutations within the same cistron yielded less than 2% of the number of phage obtained at low temperature. The complementation tests were consistent with mapping by recombination in that no pair of noncomplementing mutants was found to enclose a site complementary to either.

H48 HI73 679

I H36 639 H50

i?52

;16s36

K6

;:85°

II 1 1.5 I H56 G99 $9

12

13

I.711

I4 2.6

I I H2 6132

HI HII 629 H8

\

I5 2.2

1

16 1.7

I

I

I

H 175 G76 G82

HI08 HI5 6112

H24

mutations of subtilis phage SP82 showing recombination cistron number is indicated above the line, and mutations

17

1 I.6 HZ01

percentages located within

GENETIC TABLE

STUDY

5

RECOWBIN~4TIOx ISTRACISTRONIC

PERCENTAGES FOR CROSSES BETWEEN MUTANTS

Cistron

Cross

4

5 11

12 13

14

15

H177 H48 H48 H48 H38 H38 H56 H56 H56 H5G G7 H2 HI HI H8 H175 G86 G82 H108

x x x x X X X X X X X X X x X X X X x

OF

SOME

TS

Per cent recombination

H17G G79 G4 G119 H50 H180 G7 H189 G99 H18 H189 G132 G29 H8 G29 G86 G82 H175 G112

0.40 0.12 0.12 0.81 0.13 0.13 0.22 0.042 0.13 0.48 0.043 0.072 0.050 0.20 0.13 0.34 0.30
While most experiments were conducted using exponentially growing B. subtilis SB-1 as host, recombination experiments were also carried out both on B. subtilis 168 thy- try- and competent SB-1 bacteria. Recombination frequencies in the latter two situations were found to be within approximately 10 % of the values obtained under standard conditions. These differences are not considered significant since the frequency of recombination of a given pair of mutants

SUBZ’1LIS

PHAGE

crossed in a single host strain often varies as much as 10 %. The data obtained from two-factor intercistronic crosses involving ts mutants are summarized in Table 4. Although additivity is not good, markers can be arranged in order of increasing recombinational distances. A map constructed from the twofactor crosses is presented in Fig. 1. The distances given in the map were obtained from crosses involving adjacent cistrons. The map is linear and has a total length (summing smallest adjacent intervals) of nearly 26 recombination units. The largest unmarked distance in the map covers 4.8 units. Most of the mutations and cistrons appear to be located in the central portion of the right half of the map. Few mutations and cistrons fell within the left half of the map. The largest intracistronic distance was 0.81 recombination unit (Table 5), while the average was approximately 0.2 recombination unit. From existing data it is possible to order the mutations within some cistrons and with respect to adjacent cistrons (Fig. 2). In the most densely populated map regions (between H3S and H56), sites in different cistrons were found to be as close as 0.17 recombination unit. The order of the 7 cistrons located within this 2.3 unit span is not certain. Thwe-Factor Crosses The double ts mutants, constructed from pairs of single ts mutants, were crossedwith single ts mutants. The results of these three-

I

, H2

H8

t

,

56

t

G29

1

I

HI

H48 L

I

.I3

05

.90 -

I H38

6119 .81

I

I 1.0

I

-1.5

b.

a. FIG. 2. Recombination in the case of cistron

5

4 I

13

12

657

SP82

percentages within 13 (a) and 14 (b).

cistrons

with

respect

to a muta.tion

in an adjacent

cistron

658

KAHAN TABLE RECOMBINATION

PERCENTAGES

Double mutants

Hl 77

G55

3.51 (4.1)

H20

(2; -

H177-H24

H48

H38

A4

3.52 (4.4) -

2.7l 13.3)

0.951 (2.5)

1.31

H38-H15 H24-H48

CROSSES

TS

BETWEEN

Single mutants

-

HI-H15 H20-H326

6

OBTAISED IN THREE-FACTOR MUTANTS

(1.6) ii::; 0.17’ (0.43)

4.16 (3.9) 2.91 (0.59)

-

H56

2.21 (2.9) 0.161 (0.056) 0.341 (0.10)

0.42l (0.17)

H2

Hl

1.0’ (0.71)

-

,::i; 0.56l (0.13)

(6.2)

-

H15

H24 0.79’

(1.2) 6.01 0.171 (0.11) 0.36’ (0.21)

i:::; ,::i; 0.312 (0.08)

-

The superscript denotes the number of crosses on which the average value shown is based. The number in parentheses denotes the expected value based on data obtained from two-factor crosses. Where the single marker lies outside the two present on the doubly marked parent, the expected percentage frequency of double recombination, R, is calculated by the formula x/2 X y/2 X 4 X 1OP = R, where r and y are the percentage frequencies of single crossovers. When the single marker lies outside the segment between the double markers, the calculated probability is equal to that found in the two-factor crosses between the single marker and the closest of the markers in the doubly mutant strain.

factor crosses are summarized in Table 6. size, and the presence of an unusual base It was observed that the wild-type recombi- as a major component of the DNA, data nants occurred with a frequency which was obtained here were evaluated in terms of a either comparable to or far less than that possible additional similarity to T4, namely observed between the single marker and circularity of the genetic map. Data from the closest of the two markers carried by two-factor crossessuggest that the map of the doubly marked parent. The former SP82 is, in fact, linear with approximately result corresponds to what is expected if 26 % recombination between the end markthe single marker is located outside the ers. The average intragenic distances obsegment flanked by the two mutations served were approximately 0.2 unit, a size carried by the doubly marked parent, and that is in agreement with the comparatively the latter result to the situation where the small size of the total map (the genetic map single marker is located between the two of T4 is at least 500 recombination units). markers carried by the double ts parent. If the average intracistronic distance is However, in most cases where the single between 0.2 and 0.3 unit and if cistrons are markers were located between the two contiguous, the data suggest that the whole markers and two recombinational events genome may comprise from 90 to 130 ciswere necessary to produce wild-type re- trons. This number is reasonable considering combinants, negative interference was ob- similar estimates for T4 (Epstein et al., 1963) served, since the frequency of such recombi- and an estimate of 50-100 cistrons in the nants was higher than the product of the case of T5 (Fattig and Lanni, 1965), a two single events. phage with approximately two-thirds the These results confirm the order established amount of DNA of T4. It could be argued by the two-factor crosses. that the map is actually circular and that all the known markers are distributed DISCUSSION along one portion of the circle. This is Since SP82 is similar to T4 with respect to unlikely for two reasons. First, it is most several properties such as virulence, DNA unlikely that all presumably random muta-

GENETIC

STUDY

OF

tions would be confined to one-half of the genome; second, a rough calculation suggests that the known map comprises a number of cistrons that is commensurate with the DNA content of the phage. The cistrons that were located were found to be distributed unevenly over the map. Only 4 cistrons fell on the left half of the map, while 8 cistrons were located in the central portion of the right half of the map. Evidence that will be published separately indicates that the first three cistrons on the left side of the map are involved in the synthesis of DNA. An enzymatic system responsible for the introduction of HMU instead of thymine has been described (Kahan et al., 1964). It seemslikely that the genescontrolling these “early” enzymes and others responsible for DNA synthesis are located in this region. It was reported for T4 that few of the mutations obtained under nonselective conditions mapped in the region containing most of the cistrons responsible for “early” enzyme synthesis (Edgar and Lielausis, 1964). This could be due to the location of a number of cistrons in this region that specify the synthesis of enzymes that are already present in the host. Examples of such “redundant” funct,ions described in the coliphage system are dihydrofolate reductase (Mathews and Cohen, 1963), thymidylate synthetase (Cohen, 1961), and ribonucleoside diphosphate reductase (Cohen and Barner, 1962). Mutations in such a “redundant” cistron would not necessarily be lethal. ACKNOWLEDGMENTS The author wishes to thank Leonard Colarusso, Milica Stevanovic, and Kimberlie Cameron for their capable technical assistance. REFERENCES

ADAMS, M. H. (1959).

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