A genetic study of temperature-sensitive mutants of the Bacillus subtilis bacteriophage ∅29

VIROLOQY

43, 561668

A Genetic

(1971)

Study Bacillus

E. W. HAGEN, Departments

of Temperature-Sensitive subtilis

Bacteriophage

V. M. ZEECE,

AND

Mutants

of the

(~29

D. L. ANDERSON

of Genetics and Cell Biology and Microbiology, and School of Dentistry, of Minnesota, Minneapolis, Minnesota 56.455 University Accepted October 20, 1970

Temperature-sensitive mutants of the Bacillus subtilis bacteriophage $29, which grow at 37” but not at 45” were obtained by nitrous acid mutagenesis of free phage or by intracellular induction with nitrosoguanidine. Qualitative and quantitative complementation tests were used to group 32 mutants into 13 cistrons. Representative mutants from each of the functional groups were crossed with mutants of every other group. The genetic map of 429 derived from these data is linear and has a total length of 10 recombination units. Thus, 13 of the estimated 1520 genes carried by 429 have been marked. INTRODUCTION

Bacillus subtilis bacteriophage 429 is a small, virulent,, morphologically unique phage with a highly infectious DNA (Reilly, 1965). The virus has an intricate morphology consisting of at least eight distinct structural component’s (Anderson et al., 1966). Physical and electron microscopic studies on 429 DNA have revealed a linear, duplex, nonpermuted DNA of molecular weight 11 X lo6 (Anderson and Mosharrafa, 1968). Circular molecules can be produced by merely annealing phenol-extracted 629 DNA, indicating the presence of cohesive ends (Anderson and Mosharrafa, 196s). The polynucleotide chains derived from duplex 429 DNA by denaturabion are intact and can be separated into light and heavy fractions by poly UG binding (Mosharrafa et al., 1970). With the highly infectious 429 DNA, bhe kinetics of infection and the relationship of infection to DNA dilution are similar to those relationships observed in B. subtilis transformation (Reilly and Spizizen, 1965). Moreover, the biological activity of 429 DNA fragments can be evaluated using a marker rescue technique in which the wildtype allele of a temperature-sensitive 561

marker introduced by transfection is rescued by preinfection with the ts phage mutant (Tsien et al., 1970). Because of its small size, structural complexity, and the infectivity of it’s DNA for competent B. subtilis, +29 should be a valuable tool for studying a variety of biological problems. Development of hhe genet,ics of 429 was therefore needed to facilitate further analysis of this phage and its DNA. The present paper reports the use of temperature-sensitive (ts) mutants to map the 429 genome. These ts mut’ants are grouped into 13 cistrons by complementation tests. Recombination experiments show the mutat’ions to be widely distributed over a linear map. Preliminary reports on some phases of this st’udy have appeared previously (Hagen, 1969; Hagen and Anderson, 1969). MATERIALS

AND

METHODS

Phage and bacteria. B. subtilis bacteriophage 429 was obtained from Dr. B. E. Reilly, who originally isolated and characterized the virus (Reilly, 1965). The bacterial host was B. subtilis strain Asp 12A, an asporogenic, non-transformable organism derived from B. subtilis 16SM. This or-

562

HAGEN,

ZEECE, AND ANDERSON

ganism, also supplied by Dr. Reilly, was used in preference to strain 168M because it gave a higher efficiency of plating and larger, more distinct plaques. Media. Difco Antibiotic Medium No. 3 (PB) was used for phage infections in broth. Difco Tryptose Blood Agar Base (TBAB) was utilized for bacterial subcultures and as the base layer for phage assays. Using standard techniques (Adams, 1959), 2-day-old plates were overlaid with a soft agar described by Reilly and Spizizen (1965). A phage diluent consisting of 0.02 M Tris-HCl (pH 7), 0.1 M NaCl and 0.01 M iL3gS04 was used for short-term storage of mutant phages. Growth of phage stocks. Mutant phages were grown by a method modified from that used by Kahan (1966). Sufficient mutant phage (about 106) were plated to confluently lyse t,he host in an agar overlay. After incubation at 37”, the plate was flooded wit,h 5 ml of phage diluent, then allowed to remain at room temperature for about 4 hr. The buffer was collected, centrifuged at 1000 g for 20 min and filtered through a 0.45 p M&pore membrane. These phage suspensions were concentrated about 15 fold by centrifugation for 3.54 hr at 54,500 g in a Beckman Spinco rotor No. 30. The resuhing phage pellets were resuspended overnight at 4” in 2 ml of a solution containing 10% (w/v) dimethyl sulfoxide and 0.5 % (w/v) glucose (Yehle and Doi, 1965). These suspensions were subdivided and st’ored at -20”. Preparation of antiserum. Antiserum was prepared by injecting rabbits with 2 X 1012 phage per milliliter via standard techniques as described by Adams (1959). Repeated booster injections yielded antiserum with K values ranging from 2500 to 4300. Induction of phage mutations. Temperature-sensitive (ts) mutants of 429 were induced by mutagenizing the wild-type virus with nitrous acid or nitrosoguanidine. The nitrous acid procedure was taken from that described by Bautz-Freese and Freese (1961). Phage suspensions treated v&h nitrous acid for 3040 min yielded the ts mutants used in the present study. Samples were titered and assayed for mutants as described below.

Nitrosoguanidine mutagenesis was based upon the procedure used by Ikawa et al. (1968) in their work with the E. coli phage c34.Five minutes after cells were infected at a multiplicity of 5, nitrosoguanidine (Aldrich Chemical Company, Milwaukee, Wisconsin) was added to give a final concentration of 10 pg/ml. The cells were incubated at 37” for 2 additional hours, treated with 10 pg/ml of lysozyme, then centrifuged at 1000 g to remove debris. All mutants were isolated from one phage lysate produced in the presence of nitrosoguanidine. Isolation of phage mutants. Mutagenized phage were plated on 3-day-old TBAB plates to obtain well isolated plaques. After incubation at 37”, t,hese master plates were replicated in duplicate (Lederberg and Lederberg, 1952) ; one set of replica plates was incubated at the restrictive temperature of 47” and the other set at 37”. Plaques present only at the permissive temperature were stabbed with sterile toothpicks to t,ransfer the phage to a second set’ of duplicate overlay plates to verify temperature sensitivity. Presumpt#ive phage mutants growing only at 37” were subjected to three successive single-plaque isolations. Mutants possessing a significant degree of temperature sensitivity at this point were grown according to t’he previously described procedure. Phage from the total of 32 mutant stock lysates used in our experiments differed by at least a factor of lo6 in plating efficiency at 37” and 45”. Mutants indiced by nitrous acid were designated NA and assigned numbers from 1 to 199 corresponding to the order of their isolation. Nitrosoguanidine-induced mutations were designat’ed NG and given numbers ranging from 200 to 225. Some of the mutants carry plaque morphology alterations in addition to temperature-sensitivity markers. Complementation tests. The method used for qualitative or spot complementation tests was a modification of the procedure described by Kahan (1966). The two phage mut’ants to be tested were mixed prior to their addition to 10 X 35 mm petri dishes at a concentration of 2 X lo6 phage per dish. Two ml of overlay agar containing plating bacteria were added and mixed

GENETICS

563

OF PHAGE 629

with the phage. As controls, mutants were also plated singly at a concentration of lo6 phage/dish and incubated at 47” with the test plates. Burst-size complementation methods used here were also modified from those used by Kahan (1966). Bacterial cells (2 X 108/ml) were infected at a multiplicity of 5 of each mutant per cell. After adsorption for 10 min at 37”, antiserum (K value of 1000) was added to remove unadsorbed phage. Ten minutes later, the infected cells were diluted by a factor of lo* into growth medium. Cells were sampled, plated, and incubated at 37” immediately after dilution to assay for infectious centers. After a 2-hr incubation at the restrictive temperature (45’), the infected culture was lysed by the addition of 10 pg/ml of lysozyme. Tot’al progeny were assayed at 37”. Burst size complementation results were expressed as the ratio of the burst size of mixedly infected bacteria to the wild-type burst size at the restrictive temperature, multiplied by 100. Controls for each mutant and the wild-type were prepared using the same procedure. Burst sizes of the individual mutants at various temperatures were determined as above, except that t’he 2-hr incubation period was carried out at 37”, 43”, 45”, or 47”. Recombination. Methods for crosses were derived from those used by Fattig and Lanni (1965) in work with coliphage T5. Cells of strain 12A grown in PB to a concentration of 3.3 X 108/ml were concentrated lo-fold by centrifugation and resuspension of the pellet in the minimal growth medium (without glucose) of Anagnostopoulos and Spizizen (1961). Afber incubation in this medium for 15 min at 37”, the starved bacteria were mixedly infected with the two mutants (multiplicity of 5 of each mutant per cell), chilled in ice water, and kept at 4” for 30 mm while adsorption took place. The infected cells were then warmed by incubat,ion (at 37”) for about 3 mm, antiserum was added, and incubation was continued for an additional 10 min. The cells were then diluted lOOOfold into growth medium and incubated for 2 hr at 37”. Finally, 20 pg/ml lysozyme

was added and incubation was continued for 15 min. The lysate was plated at 37” to enumerate t’otal progeny, and at 45’ to measure recombinants. The recombination frequency is expressed as twice the plaque count at 45” divided by the plaque count at 37”. RESULTS

Burst Sizes The burst size of wild-type 429 on B. subtilis 12A decreased as the temperature was increased. There was virtually no difference in the efficiency of plating of the wild type at 37” and 45”, but at 47” the plating efficiency was about 83 % of that at 37”. The growth of plating bacteria was normal at 45” but slightly impaired at 47”. Burst sizes of representative ts mutants at 37”, 43”, 45”, and 47” are presented in Table 1. A wide variation in burst sizes among the mutants was observed at 37”, and most were considerably lower than the wild-type burst. Burst sizes are quite low at 43” and almost zero at 45”. For this reason and those cited above, all experiments, with the exception of our early TABLE 1 TEMPERATURE SENSITIVITY OF REPRESENTATIVE Is MUTANTS Cistron 1 3 4 5 5 6 8 10 10 11 12 13

Mutant w.t. 224 204 219 156 123 110 116 142 150 225 221 218

Burst sizea at 37

43

45

47

220 30 66 110 230 115 170 80 120 80 78 90 87

220 9 14 77 13 10 10 3 5 4 5 0

200 2 1 1 3 3 4 1 6 0 4 3 0

120 0 0 0 1 0 0 0 0 0 2 0 0

0 Bacteria were infected at a multiplicity of 5, and 629 antiserum was added 10 min later to remove unadsorbed phage. The infected cells were then diluted lO,OOO-fold into PB, incubated for 2 hr at the appropriate temperature, and lysed with lysozyme.

564

HAGEN,

ZEECE,

AND

TABLE

-

-

224

Mutant:

-224 205 204 219 35 216 110 158 138 116 136 142 225 221 218

0.61’

-

_

-

I

‘

-

2

INTERCISTRONIC BURST SIZE COMPLEMENTATION -

1

Cistrons

ANDERSON

2

3

205

204

4 219 --

-

a Burst size measurements the numbers of experiments

-

7

110 ___-

138

-

8

9

-

10

11

12

142 --__

225

221

13

116 ~__c-

136

9.52 17.912 28.38 31.06 35.34 8.022 il.410 35.1* 23.02 :26.7lQ 32.1’0 13.82 8.0244 2.7320 0.83l’

2.83 4.12 4.52 10.25* 14.51

10.4” 18.44 26.6% 18.44 27.312

26.82 34.32 10.4% 44.84 27.812

24.P 12.7a 34.08 20.02 32.72 20.32 34.12 46.5” 29.23 27.Q6

39.72 56.42 75.5e 41.71 23.86

0.85’

4.16l’

4.0”

28.3lo

32.6r0

38.0’

30.16

63.14

0.972

3.652 11.34 23.22 7.61a 0.406 6.58 1.0210 6.01’8 0.82’

13.12 25.03 18.41° 60.6* 3.02

22.1” 26.5* 19.22 24.Sa 11.54 1.96 6

16.72 l.Ba 23.41a 14.5% 73.02 73.02 0.702

-

30.42 /50.02 2.04 1.05 21 L9.72 1!8.54 0.91 43 ;3.6O 1.62 3

28

OF ts MUTANTP

-

are expressed as percent of the wild-type used to compute the average values.

qualitat,ive complementation tests, conducted using 45” as the restrictive perature.

were tem-

Complementation

The ts mutants were tentatively assigned to cist#rons on the basis of qualitative complement&ion behavior. Initial results generally indicated groups of mutants needing additional analysis with the more discriminating burst size complementation methods. Because intragenic complementation has been shown to occur between ts phage mutants (Edgar et al., 1964), only those tests showing a complete lack of complementation (absence of plaques) were considered valid indications that two mutations were located within the same cistron. Table 2 consists of a summary of the intercistronic burst size complementation data. Values ranged from as low as 4% of the wild-type burst in adjacent cistrons to as high as 75 % in widely separated functional groups. Almost all values were considerably higher than controls representing bursts of each of the ts mutants alone. A more definitive assignment of mutant,s to cistrons based on quantitative or burst size complementation tests at the restrictive

control.

218 .

_

-

Superscripts

TABLE

represent

3

ASSIGNMENT OF MUTANTS TO CISTRONS BASED ON COMPLEMENTATION TESTS Cistron 1 2 3 4 5

6 7 8 9 10 11 12 13

Mutant NG 224 NG 205 NG 204 NG 219 NA 35, NA 123, NA 144, NA 151, NA 154, NA 155, NA 157, NA 161, NG 216, NG 220, NG 223 NA 110, NA 158 NA 138 NA 116, NA 139 NA 136, NA 81, NA 114, NA 129 NA 142, NA 150, NG 222 NG 225 NG 221 NG 218

temperature is shown in Table 3. The 32 mutants were arranged in 13 different functional groups, with 5 cistrons containing more than one independently isolated mutation. Intracistronic complementation data for cistrons 8, 9, and 10 are shown in Table 4. It presents in greater detail the complementation values of mutants recombining witbin

GENETICS

OF PHAGE TABLE

INTRACISTRONIC

Mutant 116 8 i 139 136 129 g I 81 (114

4

COMPLEMENTATION

IN

CISTRONS

8, 9, AND

lo” 10

9

8

Cistron

.i65

429

116

139

136

129

81

114

222

150

142

0.274

0.436 2.62

7.66 5.42

3.14 1.544

2.456 2.744

10.479 3.164

3.436 -

15.9’ 1.372

9.606 5.586

1.084

0.864 0.902

0.634 1.566 1.742

3.24 2.514 1.964

2.32’ 3.024 1.522

1.9(i4 1.14 1 .632 3.564

8.946 2.864 2.744 8.034

1.32

1.732 1.014

1.406 2.116 0.602

1.284

1222 lO( 150 (142 a Burst size measurements are expressed as per cent of the wild-type the number of experiments used to compute the average values.

a relatively short genetic distance. In ambiguous cases, cistronic assignments were based on complementation data in conjunction with corresponding recombinat,ion data. Recombination To first establish that exclusion of the parental mutants did not occur in mixedly infected bacteria, we examined the progeny of single cells infected wihh two different plaque morphology mutants, and noted the ratio of t,he parental markers. Results of several experiments indicated that about 80 % of the single cells were doubly infected. The percentage did not differ significantly when cells were infected in the presence of cyanide. The use of cyanide to facilitate mixed infections in recombination experiments caused a not)iceable reduction in both the number of recombinants and the total progeny. Therefore, a starvat,ion-cold infection treat’ment was subst,ituted to slow metabolism and allow equal adsorpt,ion of the two ts mutants. Results indicated no adverse effects on the wild-type burst size or on the burst size of cells doubly infected with ts mutants. Adsorption of phage was essentially complete after 30 min of cold treatment. There was little effect on observed recombination values in test experiments even when infection with the two phage mutants was separated by an interval of 5-15 min. A summary of the intercistronic recom-

control.

Superscripts

represent

bination data is present’ed in Table 5. Twofactor crosses were performed in which a representative mutant from each functional group was crossed with a mutant from every other group. Representatives from cistrons containing several mutants were selected primarily for stability and absence of recombination anomolies sometimes observed. Values obtained from crosses using mutants within the same functional group were pooled, unless otherwise indicated. Intercistronic recombinabion percentages varied from as low as 0.2% between two adjacent cistrons to almost 10% for crosses involving mutants in functional groups located at opposite ends of Dhe map. Recombination frequencies varied by as much as a factor of 2 from day t’o day In addition, it was apparent that individual characteristics of mutants affected their recombination behavior. Some mutants consistently gave lower recombination frequencies, while others consistently produced higher recombination values than expected. Recombination percent)ages obtained from some intracistronic crosses are shown in Table 6. Recombinat’ion values from crosses within functional groups generally ranged from less than 0.01% to approximately 0.2 %. The Genetic Map The provisional genetic map of phage 429 (Fig. 1) is based on the complementation and recombination data just described.

566

HAGEN,

ZEECE,

AND

TABLE

-

-

-

Cistrons 1

2

4

3

Mutants :224 20.5

204

01.605 0.734 0.541”

224 205 204

INTERCISTRONIC

219

5

RECOMBINATION

5

6

OF ts MUT.ANTS~

7

219 - 35 ___

216

110 ____

158

138 ~_____

1 .326 1.9g4 1 .55 2.914 01.416 0.5g3 0.603

2.81 1.98 1.21 1.74 0.07

1.92 3.87z 0.992 0.553 0.802

3.34 2.4P 2.i’34 1.294 0.234 0.72= 0.003

2.332 1.744 1.156 1.52” 0.7g8 1.124 0.1g3 0.242

35 216 110 158 138 116 136 142 225 221 -

ANDERSON

( 8 116

j 9 136

2.79” 2.6g2 0.10” 0.754 1.49% 2.332 O.4421.922 1.252 2.564 0.97* 2.122 3.154 1.12” 3.314 0.562 2.742 0.2ga

( 10 i 11 1 12 142 ____

22.5

1.763 2.032 3.44” 6.802 2.32% 2.10g 3.7g4 2.75= 3.75a 4.272 1.56 5.46% 4.573 9.622 1.66’ 2.942 3.834 2.55% 0.6gz 1.39* 0.834 1.67* 1.127

221 _____ 8.34 5.054 5.724 6.0g4 1.652 15.02 8.8g2 2.342 8.414 2.20% 2.364 3.126 0.606

( 13 218 6.253 7.121 2.523 7.04 3.272 3.70a 7.382 5.35* 3.642 2.472 4.922 5.272 1.644

I I I ! I /2.88a

-

a Values presented are recombination percentages. Superscripts represent the number of experiments used to compute the average values. TABLE

6

INTRBCISTRONIC

RECOMBINATION

Cistron

Mutants crossed

5

6 8 9

10

35 35 35 35 35 216 216 216 216 216 110 116 136 136 136 222 150

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

216 123 157 151 153 156 154 223 161 144 158 139 129 81 114 142 142

OF ts MUTANTS

Recombination percentagea 0.072 0.113 0.232 0.033 0.204 0.1g2 0.196 0.1910 0.09’ 0.052 0.0034 0.012 0.022 0.032 0.172 0.035 0.053

a The superscripts represent the number of experiments used to compute the average values. The map consists of a single linear linkage group. Markers ordered according to increasing values, based on crosses among neighboring cistrons, also gave progressively increasing values when mapped directly.

In ordering the Is mutants, heavier emphasis was placed on the validity of small recombination values such as those obtained from crosses of adjacent and other neighboring cistrons. Three-factor crosses will be required to confirm and substantiate ts marker order. No linkage between end markers was observed. The total length of the genetic map, obtained by summing recombination frequencies between adjacent cistrons, is about 10 recombination unit#s. Nine cist)rons are located on t’he left half of the map and four on the right half. A definite clustering of cistrons occurs just t,o the left of the cent’er of t’he map. DISCUSSION Temperature-sensitive mut’ants of phage $29 were categorized into 13 functional groups by qualitative and quantitat)ive burst size complementation studies. Recombinat,ion data obtained from crosses among representative mutants from each of the cistrons yielded a genetic map with a t,otal length of about 10 recombination units. With a few exceptions, the complementation and recombination data were in good general agreement. The map order

GENETICS 1

2 .60

3 .54

4 .41

1.2

205

204

219

6 .60

I

I 224

5

35 216

7

.2

1 I/

8 .56

158 138 110

9

I.281 I

II 116

567

OF PHAGE $29

136

10 .83

1

11 1.12

12

1 .60

13

2.8

I 142

225

221

218

FIG. 1. The genetic map of phage 629. Cistron numbers appear above the solid line; mutants mapped are listed immediately below. Recombination percentages for crosses between adjacent cistrons are shown on the solid line. Distances are proportionate.

of the cistrons was consistent, even though recombination values do not always show good additivity. This lack of additivity is undoubtedly due, as in T4 phage (Edgar and Lielausis, 1964), to the large coefficient of variation in recombination values and to interference effects. Alore detailed complementation and recombination analyses in 429 might reveal the existence of another functional group within the fifth cistron. Three-factor crosses will be required to confirm the map order, part’icularly in segments where many mutations are clustered within a short genetic distance, and to order mutants within functional groups. The ts map of 429 consists of a single linear linkage group. No genetic evidence linking the end markers was found, as mutants at progressively increasing map distances also gave proportionately greater values when mapped directly. The possibility that only a small portion of the genome was marked with &mutants is not likely, since ts markers have been shown to be widely distributed over the genomes of other phages such as X and T4 (Campbell, 1961; Edgar and Lielausis, 1964). Thus, the genetic data agree with the physical studies indicating that 429 contains a linear, nonpermuted DNA molecule (Anderson and Mosharrafa, 1968). In this respect, $29 resembles the larger B. subtilis phage SP82 (Green, 1966b) and coliphages, such as T3 and T7 (Ritchie et al., 1967) and T5 (Thomas and Rubenstein, 1964). These phages all differ from the coliphages T2 and T4, which

have circular genetic maps (Baylor et al., 1965; Edgar et al., 1964; Streisinger et al., 1964) and chemically permuted DNA molecules (Thomas and MacHattie, 1967). The DNA molecule of 429 has a molecular weight of only 11 X lo6 daltons, and thus can probably code for only an estimated 15-20 proteins. We have mapped ts mutants in 13 of these functional groups, thereby placing markers over a large portion of the genome. Phage 429 can perhaps be compared with coliphage T7, which has recently been mapped by Studier (1969). The T7 DNA molecule has a molecular weight of 26 X lo6 daltons, an estimated ability to code for 25-30 proteins, and a genome of 19 cistrons as mapped by amber mutants. The intricate morphology of 429 coupled with its small size suggests that synthesis and assembly of Dhe complex structure probably requires most of the genome. Functional analysis of the ts mutants is currently in progress. Suppressor-sensitive mutants have been isolated and should confirm and expand the ts genetic map, as well as aid in functional analysis. In additional studies, the physical and biological properties of DNA fragments are being investigated using transfecbion and a recombination marker rescue system similar to that developed for the B. subtilis-SP82 phage system (Green, 1966a, b). This genetic analysis should be an important adjunct to studies which will hopefully allow us to correlate the physical, chemical, and biological properties of the 429 DNA molecule.

568

HAGEN,

ZEECE,

ACKNOWLEDGMENTS This investigation was supported in part by grant AI-08088 from the Public Health Service, and grants GB-6695 and GB-15207 from the National Science Foundation. One of us (EWH) was the recipient of support from U.S.P.H.S. training grants 5 TOl-AI 00090-08 and 5 TOl-DE 00179-03. REFERENCES M. (1959). “Bacteriophages.” Wiley (Int,erscience), New York. ANAGNOSTOPOULOS, C., and SPIZIZEN, J. (1961). Requirements for transformation in Bacillus subtilis. J. Bacterial. 81, 741-746. ANDERSON, D. L., and MOSH~IRR~F.~, E. T. (1968). Physical and biological properties of $29 DNA. 1. viroz. 2, 1185-1190. ANDERSON, D. L., HICKM.~N, D. D., and REILLY, B. E. (1966). Structure of BaciZZus subtilis bacteriophage +29 and the length of $29 deoxyribonucleic acid. J. Bacterial. 91, 2081-2089. BAUTZ-FREESE, E., and FREESE, E. (1961). Induction of reverse mutations and cross reactivation of nitrous acid-treated phage T4. Vivirology 13, 19-30. BAYLOR, M. B., HESSLER, A. Y., and BAIRD, J. P. (1965). The circular linkage map of bacteriophage T2H. Genetics 51,351-361. CAMPBELL, A. (1961). Sensitive mutants of bacteriophage X. Virology 14, 22-32. EDG.%R, R. S., and LIEL~USIS, I. (1964). Temperature-sensitive mutants of bacteriophage T4D : Their isolation and genetic characterization. Genetics 49, 649-662. EDGAR, R. S., DENHARDT, G. H., and EPSTEN, R. H. (1964). A comparative genetic study of conditional lethal mutations of bacteriophage T4D. Genetics 49, 635-648. FATTIG, W. D., and LSNNI, F. (1965). Mapping of temperature-sensitive mutants of bacteriophage T5. Genetics 51, 157-166. GREEN, D. M. (1966a). Intracellular inactivation of infective SP82 bacteriophage DNA. J. Mol. Biol. 22, 1-13. GREEN, D. M. (1966b). Physical and genetic characterization of sheared infective SP82 bacteriophage DNA. J. Mol. Biol. 22, 15-22. HAGEN, E. W. (1969). A genetic study of temperature-sensitive mutants of the Bacillus subtilis AD.IMS,

AND

ANDERSON

bacteriophage 429. M.S. Thesis, University of Minnesota, Minneapolis. HAGEN, E. W., and ANDERSON, D. L. (1969). Genetic recombination in Bacillus subtilis bacteriophageQ29. Bacterial. Proc. 1969,191. IK.4w.4, S., TOYAMA, S., and UET~KE, H. (1968). Conditional lethal mutants of bacteriophage es4. I. Genetic map of tz4. Virology 35, 519-528. KBHAN, EUNICE. (1966). A genetic study of temperature-sensitive mutants of the Subtilis phage SP 82. Virology 36, 517-528. LEDERLIERG, J., and LEDERBERG, E. M. (1952). Replica plating and indirect selection of bacterial mutants. J. Bacterial. 63, 399-406. MOSH~RR.~F~, E. T., SCHACHTELE, C. F., and ANDERSON, D. L. (1970). Isolation of the complementary strands of bacteriophage $29 DNA. Bacterial. Proc. 1970, 175-176. REILLY, B. E. (1965). A study of the bacteriophages of Bacillus subtilis and their infectious nucleic acids. Ph.D. dissertation. Department of Microbiology, Western Reserve University, Cleveland. REILLY, B. E., and SPIZIZEN, J. (1965). Bacteriophage deoxyribonucleate infection of BaciZZus subtilis. J. Bacterial. 89, 782-790. RITCHIE, D. A., THOMAS, C. A., JR., MACHATTIE, L. A., and WENSINK, P. C. (1967). Terminal repetition in non-permuted T3 and T7 bacteriophage DNA molecules. J. Mol. Biol. 23,365-376. STREISINGER, G. R., EDGAR, R. S., ~~~DENH.~RDT, G. H. (1964). Chromosome structure in phage T4. I. Circularity of the linkage map. Proc. Xat. Acad. Sci. U. S. 51, 775-779. STUDIER, F. W. (1969). The genetics and physiology of bacteriophage T7. Virology 39,562-574. THOMAS, C. A., JR., and MACHSTTIE, L. A. (1967). The anatomy of viral DNA molecules. Annu. Rev. Biochem. 36, 485-518. THOM~ C. A., JR., and REBENSTEIN, I. (1964). The arrangements of nucleotide sequence in T2 and T5 bacteriophage DNA molecules. Biophys. J. 4, 93-106. TSIEN, H. C., REILLY, B. E., and ANDERSON, D. L. (1970). Gene transfer by bacteriophage+29 DNA fragments. Bacterial. Proc. 1970, 202. YEHLE, C. O., and DOI, R. H. (1965). Stabilization of Bacillus subtilis phage with dimethylsulfoxide. Can. J. Microbial. 11, 745-746.