COMPARATIVE GENETICS OF THE T-EVEN BACTERIOPHAGES

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COMPARATIVE GENETICS

OF THE T-EVEN BACTERIOPHAGES

RICHARD L. RUSSELL

Division of Biology, California Institute of Technology, Pasadena, California 91109

Manuscript received April 25, 1974 Revised copy received July 5, 1974

ABSTRACT

A system of amber mutants has been developed for each of the T-even bacteriophages T2 and T6, to complement those already available in T4. I n

T2 these mutants identify 52 genes, of which 49 are homologous with T4

genes; in T6 they identify 45 genes, of which 42 have T4 homologs, and an additional one which is homologous to a T2 gene not yet identified i n T4.

In both T2 and T6, recombination between mutants is characterized by con- siderable negative interference, which is correctable by a mapping function designed for T4. Recombinational maps of T2 and T6 constructed with these mutants have the same gene order and nearly the same gene spacings as in T4,

with the exception of the tail fiber region; T2 and T6 appear to lack a localized recombinational expansion of this region found in T4. Homologous gene prod- ucts from all three phages are in general interchangeable, with the exception of those from two apparently "co-adapted" tail fiber genes, 37 and 38. The general genetic similarity of all three phages suggests that they are anal- ogous to races of higher organisms, retaining the capacity for genetic exchange despite some clear genetic differences and some incipient isolating mecha- nisms.

extensive set of conditional lethal mutations isolated i n bacteriophage

T:fD

has been used to provide a description of its genome which is unsur-

passed in completeness

(EPSTEIN

et

al.

1963; EDGAR,

DENHARDT

and

EPSTEIN

1964; STAHL;

EDGAR

and

STEINBERG

1964; EDGAR

and

LIELAUSIS

1964; EDGAR

and

EPSTEIN

1965; WOOD

1974).

The related bacteriophages

T2

and

T6,

how-

ever, have received much less genetic attention

(SEKELY

1960; STAHL

and

,MURRAY

1966),

even though conditional lethal mutants can be isolated with

equal ease in all three phages.

I

report below the isolation and partial character-

ization of conditional lethal mutants of bacteriophages T2 and

T6.

These mutants

have proved useful for a detailed genetic comparison of the three T-even bac-

teriophages (see below) and for a study of their interactions in mixed infections

(PEES

and

DEGROOT

1970; PEES 1970; RUSSELL

and

HUSKEY

1974)

;

they have

also been used to construct phage hybrids of defined composition for a variety

of

purposes

(BECKENDORF

and

WILSON

1972; BECKENDORF,

KIM

and

LIELAUSIS

1973; BECKENDORF

1973;

KIM

and

DAVIDSON

1974).

'Research supported by Public Health Service Training Grant GM 00086 and by grants to R. S. EDGAR from the National Foundation for Infantile Paralysis (CRBS-120) and the National Science Foundation (GB 3930). It was com- pleted in 1967 and submitted as part of a Ph.D. thesis at the California Institute of Technology. Although unpublished, it came to the attention of several interested workers who have subsequently investigated some of its aspects more fully.

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968

R . L. RUSSELL

MATERIALS A N D M E T H O D S

Materials

Phages; T4D wild type, T2L wild type, T6 wild type, and all mutants of T4D were obtained from the collection of DR. ROBERT S. EDGAR. Amber mutants of T 2 with the prefix FS were obtained from DR.

FRANKLIN

W. STAHL. All other mutants of T 2 and all mutants of T6 were isolated in this study and are described below.

Bacteria: All bacterial strains were obtained from the collections of DRS. ROBERT S. EDGAR and JEAN J. WEIGLE. The E . coli strain BB from these collections proved to be permissive for most T 2 and T4 amber mutants, in contrast to the BB strain maintained at the Virus Labora- tories of the University of California at Berkeley. The origin of this difference is not clear.

Media: Hershey broth contained 8 g DIFCO Nutrient Broth (dehydrated), 5 g DIFCO Bacto Peptone, 5 g NaC1, 1 g dextrose (glucose), and 1000 ml distilled H,O, and was adjusted to pH 7.4 with 4% NaOH before distribution and autoclaving. Enriched Hershey bottom agar

(used for most plates, 30-35 ml per plate) contained 10 g DIFCO Bacto-Agar, 13 g DIFCO Bacto-Tryptone, 8 g NaC1, 2 g sodium citrate dihydrate (Na3C,H,*2H,O), 1.3 g dextrose (glu- cose) and 1000 ml H,O. Enriched Hershey top agar (used for most plates, 2 ml per plate) con- tained 6.5 g DIFCO Bacto-Agar, 13 g DIFCO Bacto-Tryptone, 8 g NaC1, 2 g sodium citrate dihydrate, 3 g dextrose (glucose), and 1000 ml H,O. Slant Medium No. 1 contained 18 g DIFCO Bacto-Agar, 8 g DIFCO-Tryptone, 1 g DIFCO Yeast Extract, 5 g NaC1, and 1000 ml H,O, and was adjusted to p H 7.4 with 4% NaOH before distribution and autoclaving.

Chemicals: 5-bromodeoxyuridine (5-BUdR) was obtained from the California Corporation for Biochemical Research (A grade). Propylene glycol monolaurate, often used as an anti-foam- ing agent, was obtained from K & K Laboratories, Inc. All other chemicals were standard reagent grades.

Antisera: The antisera used in this study were gifts from various people, as follows: anti-T2, J. J. WEIGLE; anti-T3, R. S. EDGAR and J. E. FLATGAARD; anti-T6, R. S. EDGAR.

Methods

Mnintenance of bacterial strains: Bacteria were maintained on slants of Slant Medium No. 1 ;

transfers to fresh slants were carried out every six to eight weeks, followed by one day of growth at 37" and subsequent storage in the cold (4").

Stock cullures of bacteria were made by inoculating about 30 ml of Hershey broth with a loopful of cells from a slant and aerating for a period of about 16 hr at 30'. The final concen- tration of bacteria in such cultures varies from 2-8

x

IO!)/ml, depending on the strain. Stock cultures were stored in the cold (4") and used for 7-10 days.

Plating bacteria were made by diluting a stock culture 100-fold into fresh Hershey broth, aerating for either 2.5 hr at 30" or 1.75 hr at 37", centrifuging the turbid suspension for 10 min a t 1500 X g in the cold ( 4 O ) , and resuspending the bacterial pellet in one-tenth the original volume of chilled Hershey broth. Bacteria prepared in this manner have a higher efficiency of plating than do saturated cultures, and maintain their higher efficiency for about two days if stored at 4". Two drops of this suspension were used for each plate.

Bacteria for infections, including crosses and liquid culture complementation tests, were made by diluting a stock culture 500-fold into fresh Hershey broth, aerating for either 2.5 h r at 30" or 1.75 hr a t 37", centrifuging the somewhat turbid suspension for 10 min at 1500

x

g in the cold (4"), and suspending the bacterial pellet in one-fiftieth the original volume of chilled Hershey broth. By this procedure, early logarithmic phase bacteria were obtained at a final concentration of 1-2

x

IO"m1, and could be diluted as desired.

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T-EVEN P H A G E GENETICS

969

lated culture at 25". Incubation at higher temperatures resulted in lower stock titers and often higher reversion indices, probably because of the limited restriction exerted by CR63 and other

E . coli

K

strains against T 2 (REVEL 1967). The resulting stocks were sometimes centrifuged for

10 min at 5800

x

g to rid them of large bacterial debris, and were then filtered through a Mandler candle filter to complete the cleaning. I n the other method of stock-making, about

IO5

phages were plated on each of several plates, and after 12-15 hr of incubation a t 25", 0.5 ml of chloroform was added to the surface of the confluent plate and allowed to evaporate. Five ml of Hershey broth were then added to the plate, and 2-3 hr at room temperature were allowed for the phage to diffuse from the top agar layer into the broth. The broth was then collected ( 3 4 m! could be recovered from a normal plate), and the resulting "plate stock" was centrifuged and filtered in the same way as the normal liquid culture stock described above. The Sagik effect

(SAGIK 1954) did not hamper use of T2 stocks, probably because a matter of weeks usually elapsed between the making of a stock and its use for experiments i n which its titer had to be accurately known.

Standard phage techniques (ADAMS 1959) were used for the dilution, plating and counting of phage samples, with the €allowing restrictions: (1) no more than 0.5 ml and no less than 0.05 ml of a phage sample was ever plated; ( 2 ) no plates were used later than 5 days after they had been poured (the use of older plates leads to smaller plaques and a n apparent drop i n efficiency of plating); (3) no plating bacteria were used later than 2 days after they had been prepared; and (4) no counts of less than 100 plaques were accepted.

Mutagenesis of phages was accomplished as follows. The phage to be treated was grown i n a restrictive host, to eliminate any large clones of amber mutants already present by chance. This stock was then used to infect permissive bacteria (CR63, 1-4

x

108,"l) at a multiplicity of 5, and 5BUdR was added to bring the final concentration to 10 pg/ml. (This concentration had previously been found to give a reasonably high frequency of amber mutants in T4.) The in- fected cells were then aerated vigorously until they lysed, and the progeny were plated as de- scribed below

Selection of amber mutants was carried out by a method based on a suggestion by DR. RICH- ARD

H

EPSTEIN. The phage sample plus 2 drops of normal CR63 plating bacteria were added to 2 ml of melted top agar, mixed, and poured over 2-3-day-old plates. After this initial layer had hardened, a second layer, consisting of 2 ml of melted top agar plus 2 drops of a IOO-fold dilu- tion of restrictive (B/5 or S/6/5) plating bacteria was poured over the first layer and allowed to harden. After incubation the bacteria in the upper layer gave rise to clearly separated micro- colonies which were easily distinguished from the bacterial lawn below. Over a wild-type plaque these microcolonies were missing from an area somewhat larger than the plaque. Over a plaque of an amber mutant, however, the microcolonies were present without reduction in number or size, giving the plaque a characteristic stippled appearance. With T 2 and T6, about 10% of the plaques selected for their stippled appearance contained amber mutants.

Stab testing of suspected mutants was carried out by stabbing into a stippled plaque a sterile pin which had been coated with a sterile mixture of vegetable dye and glycerine (to identify the plaque stabbed). The pin was then stabbed successively into defined positions on each of two plates, the first of which had been seeded with restrictive, and the second with permissive, bac- teria. Tests indicating the presence of an amber mutant were checked by picking the area of lysis from the permissive plate with a sterile glass capillary, transferring it to a few ml of chloroform-saturated Hershey broth, allowing a few hours for the phage to diffuse out of the agar plug, and then respotting the resulting suspension on two plates seeded. respectively, with restrictive and permissive bacteria.

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970

R . L. RUSSELL

a considerably greater number of plaques; controls and negative tests usually contained only a few scattered plaques, whereas in positive tests there was nearly confluent lysis of the bacteria. Rare intermediate tests were repeated or abandoned in favor of liquid culture complementation tests. (The numerous plaques in positive spot tests were due to wild-type recombinants formed in mixedly infected bacteria; since the bacteria were restrictive, the formation of these recom- binants depended on the ability of the two mutants to complement one another.) Spot tests be- tween mutants of the same phage worked well, whether the phage was T2, T4 or T6. Spot tests between mutants of different phages worked for T2-T4 tests and for T2-T6 tests, but considerably less well f o r T4-T6 tests. The latter could be markedly improved by a 100-fold reduction in the bacterial concentration, and this was adopted as the standard method for T4-T6 tests.

Liquid culture complementation tests were used to check the results of spot tests and to clarify situations in which these results were ambiguous. Restrictive bacteria were prepared for infection, at a final concentration osf 4

x

1018/ml. Just before use, KCN was added to give a final concentration of 0.004 M. A test was performed by mixing together equal volumes of two amber mutant phage suspensions, each at 6

x

IO~'m1, and then adding to one volume of the mixture an equal volume of bacteria. After 8 min osf adsorption at 30", anti-phage serum was added to give a final neutralization constant (k) of 1 min-1. The antiserum was allowed 5 min to inactivate unadsorbed phages, and the adsorption mixture was then diluted 40,000-fold into Hershey broth at 25O. Samples were quickly taken for measuring infective centers and residual unadsorbed phages. After 120 min of incubation, chloroform was added to ensure complete lysis. The progeny phage were then assayed, and the burst size was computed and compared to that of simultaneous controls. These controls were infections with wild-type phages a t the same total multiplicity (ca.15) used in the tests, and they were usually inserted at the beginning and at the end of a series of tests. When the tests were between mutants of different phages, the controls were mixed infections between the wild types of the same two phages. The liquid culture tests have advantage over spot tests in that the results do not depend upon the production of recombinants.

Crosses. Bacteria were prepared for infection at a final concentration of 4

x

108/ml, and just before use, KCN was added to give a final concentration of 0.004 M. A cross was performed by mixing equal volumes of two amber mutant phage suspensions, each at 6 X 108/ml, and then adding one volume of the mixture an equal volume of bacteria. Adsorption was allowed to proceed for 8 min if unadsorbed phage were to be eliminated with antiserum (occasionally) or 10 min if not (usually). Antiserum was used at a final

k

of 1 min-1 fo'r 5 min. After adsorp- tion the mixture was diluted 40,000-fold into Hershey broth at 25", incubated f o r 120-150 min, and then treated with chloroform to ensure complete lysis. A sufficiently small number of crosses (usually 20) was done with any one batch of bacteria to ensure that no bacteria had been ex- posed to KCN for more than 22 min (such exposure tends to reduce the burst sizes obtained).

Progeny phage were plated at various dilutions on permissive and restrictive bacteria. The frequency of recombination was calculated by multiplying the titer on the restrictive strain by 2 (to compensate for the unscored double mutant recombinants), dividing by the titer on the permissive strain, and multiplying by a correction factor f o r the inherent differences, if any, between the two strains of efCiciency of plating. This correction factor was determined sep-

arately for each batch of plating bacteria by plating on them 0.5 ml of a large volume stock

of wild-type phage at a titer of 1.2

x

1 0 3 / ~ l ; it was often quite significant, usually being, for example, about 0.7 with T 2 on CR63 and S/6/5.

Often a given cross was repeated a number of times. In general the agreement between the various values for the frequency of recombination was good, and an average value was taken. Occasionally, however, one of or even sometimes two of a series of crosses seemed out of line with the rest of the series. In these cases, a n average of the remainder of the values was taken, and the exclusion of some values from the average was noted.

RESULTS

Mutant isolation:

From stocks of

T2L

and T6 grown in the presence of

5-

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T-EVEN P H A G E GENETICS

971

by a mixed-lawn screening technique described above. The isolated mutants

occurred after mutagenesis at frequencies of approximately 0.5

%

;

in general

they were not backcrossed to wild type before subsequent testing and 12% of

them turned out to harbor more than one amber mutation. Thirty-five additional

mutants of

T2

were obtained from the collection of

F. W.

STAHL,

described else-

where

(

STAHL

and

MURRAY

1966)

;

these were given the prefix

FS

to distinguish

them from mutants isolated in this study.

Complementation testing:

On the assumption that T2 and T 6 genes might

have homologs in T4, the T2 and T6 mutants were first tested in complementa-

tion spot tests with a series of T4 amber mutants. These tests, originally designed

for complementation testing

of

T4 mutants, proved satisfactory for testing T2

mutants as well; for T6 mutants, however, the initial testing procedure had to be

modified by a reduction in the bacterial concentration to achieve satisfactory

results. With the appropriate testing procedure, each new T 2 and T6 mutant

(with a few exceptions

to

be described below) could be unambigously assigned,

either to a gene with a T 4 homolog. o r to a T2

o r

T6 gene for which a T4 homolog

was not yet known. The testing procedure was sequential; each new mutant was

first tested against a set of seven multiple amber mutants of T4 which, between

them, contained mutations in 24 T4 genes.

If

it failed to complement one of

these, it was tested next against the T 4 single mutant constituents of the un-

complemented multiple;

if

it complemented all the multiple mutants, it was

subsequently tested against a set of single and double mutants which, between

them, contained mutations in the remaining 46 identified genes of T4.

By this procedure 144 of the 152 T2 mutants, and 129 of the 146 T6 mutants

were assigned to identified genes. The remaining mutants were tested against

each other in all possible combinations; they proved to identify a total of 5 new

genes, of which one was found in both

T2

and T 6 , 2 were found in

T2

alone, and

2 were found in T6 alone. All new mutants assigned to a given gene were tested

against each other in all possible combinations, and with the exception (2)

noted below, all such tests were negative, as expected.

A

summary of these gene

assignments is presented in Table 1.

During the spot testing, occasional exceptional results were observed; these

were of the following types.

1)

M u l t i p l e mutants.

A

total

of

17

T2

mutants and 18 T6 mutants showed

noncomplementation with more than a single T4 tester mutant. These appeared,

by subsequent complementation testing with single mutants of T2, T4 and T6, to

harbor multiple amber mutations in different genes. In T2 there were a total of

15 apparent double mutants and 2 apparent triples, In T2,

11 of the apparent

double mutants were tested by backcrossing to T2 wild type; in all cases the

expected single mutant constituents were isolated. The high proportion of mul-

tiple mutants is probably a consequence of the relatively heavy mutagenesis

hpplied, but it should also be pointed out that the multiple mutants appeared at a

frequency much higher than expected by assuming independent origins for the

multiple mutations.

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9 72

R. L. RUSSELI,

TABLE 1

Gene locations of T2 and T6 amber mutations

Gene T 1 nlolanl5 TG mutants

e

1 153,99a,166a 57

15

2 163 64 FS109 50 FS48

4

FS114 53 72

5

[38,12~,1~,117b,132b,166b] [FSIS] 6 I06,160,FS63,30a,l54a

7 14,27,77 8 37,140 9 98c

10

11

12 60,FS13 13

14 69,93,167,FS62 16 FS15

17 16,98a 18 117a 20 108 21 111,124 22 99b

23 94,104,113,138,157,148a

25

11

26 28,56

51

FS82,148b 27 156,FS57,30b 29 7,65,81,FS29 48

54 95,FS66 30

FS5

63 43,54,FS51,152a 32 8,51,145

33 96,13O,FS4%,164a

34 12,67,73,74,75,78,79,135,FS4.01,25a,l10a 35 FS6

36 21,24,120,FS23 3 7 123,129,FS4,25b 38 35,91,125,FSf% 52 [32] [89,5a]

39 [33,71,84,114,70a]

[5b]

56

13,61,64,87,1 O12,133,165,FS2,FS 18,154b 58 100

41 82,86,137,FS99,98b 42 3,6,68,136,FS85,76a,i 10b

43 83,141,146,151,162,FS115,76b,105a,147a,164b

44 FS8

85

67,114

92

78,97

22,106

[ 10,58,64,109,13 1,124b,142b] [43,127] [9,27,29,50,51 ,59,60i,82,24bl [I321 80

28 102,96a 14,79,117

18,39,13 7,144,24c,l11 a

63,84,73a 7,54,94,34a,99b 17,21,71,138 6,124a 87,130,42b,124c 93,112 119 57 73b 20,118,31b

[4,8,40,1G~4,31b,52a,99a,lI

Ib]

[I431 98,91a

[ 11 3,116,1251 [44a]

13,88,121,128,134,141,26a,135b 53,75,1015,15a

45,91b

33,83,9O,107,11O1,146,15b,42a 3 7,62,26 b

3,126,24a

23,72,115a [65,96b] [I223 25,66,89,95,99c,1408b

44b

16,108,129,31a

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T-EVEN P H A G E GENETICS

TABLE I-Continued

9

73

Gene TL mutant? ‘I’G mutants

46 FS98 133

46 31,59,115,122,155,FS32,70b,132a,152b,lO5b 32,76,136,52b,135a

47 4,80,92,FS7,FS45 21,61,60,101

55 17,15QFS56,147b

49 2,FS87 115b

26,36,109 1.36,47,56,103,14~a,142a

10~,159,FS10,117c FS78,FS43

123 86

Mutants are listed with the genes to which their defects were assigned by spot testing. T2 mutants with prefix FS are from the set isolated by DR. F. W. STAHL. Multiple mutants are listed once for each defect, with suffixes a, b, and (where necessary) c to distinguish their different defects. Within a gene, brackets are used to separate groups f o r which the spot tests suggested either unusually high intragenic recombination or some degree of intragenic complementation.

against one another in all possible combinations; the

T2 mutants fell into two

clear complementation groups, and both

T6 mutants fell into one of these two

groups. The two groups apparently correspond to genes

37

and

38,

and the in-

ability of the T2 and T6 mutants in them to complement either gene

37

or

gene

38

mutants of T9 apparently results from a specific gene product incompatibility,

as

previously reported by STAHL

and

MURRAY

(1966). This point is discussed in

more detail below

3 ) Weak positiue tests due to exclusion:

Some of the apparently positive spot

tests results were considerably weaker than usual; this was observed usually in

tests involving mutants

of

T4 with those of T2 or

T6

(i.e., not usually

in T2-

T2, T4-T4, T6-T6, or T2-T6 tests), and only when the T4 mutants came from

the “early regions” of the T4 genetic map. Such results are easily interpreted as

the result

of partial exclusion; the spot tests depend for positive results on the

production of a sizeable number

of

wild-type recombinants in mixed infections

between complementing phages. I n T4-T2 or T4-T6 mixed infections, partial

exclusion hampers the production of these recombinants, particularly those which

contain wild-type alleles from the strongly excluded T2 or T6 early regions (see

the accompanying paper,

RUSSELL

and

HUSKEY

1974). Thus tests involving T4

early gene mutants with mutants of

T2 and T6 produce fewer than normal wild-

type recombinants (i.e., only a weakly positive result) even when full comple-

mentation has occurred. (Similar results observed by STAHL

and

MURRAY

[

19661

can be explained in the same way). Besides the ones listed above, no other types

of exceptional results were observed.

In total, the new mutants served

to

identify 52 genes in T2 and 45 genes in T6.

Most

of

these genes are homologous

to

T4 genes by complementation, and the

success of the spot testing procedure further argues that most of the genes are,

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9 744

R. L. RUSSELL

phages which must contain a particular combination

of

T2

and T4 genes. Appar-

ently any such combination of genes (whether from T2, T4

o r

T6) is compatible

with viability, except for the case of genes 37 and 38, discussed in more detail

below.

Mapping

Gene order:

The success of the spot testing procedure also argues that the

genomes of T2 and T6 are likely to be organized very much like that of T4. To

investigate this conclusion further, genetic maps of T2 and T6 were constructed,

using one representative mutant from each mapped gene. I n each case the

working hypothesis was that the desired map would strongly resemble that of T4

in its gene order; consequently each mutant was crossed first with its three

expected nearest neighbors on either side, and subsequently, if necessary, with

more distantly linked mutants. Data from these crosses are presented in Figures

1

and 2. In each case the gene order in the figure is that

of

T4, and in both T2 and

T6 the data are consistent with the same order. However the T2 and T6 data do

not establish this as the unique order

for

the map as a whole; rather the data

divide the mapping genes into

a

set of well mapped groups (in T2, for example,

the well-mapped groups are genes 57-30, genes 32-39, and genes 56-49). Within

each of these groups

a

unique order is established, but the ordering of the groups

with respect to one another is much less clear from these data, because the groups

are only distantly linked. Fortunately, the DNA-hybrid studies

of

KIM

and

DAVIDSON

(1974)

show unequivocally that these groups must have the same

57 I 2 50 64 53 5 5' 6 7 8 12 (101 14 16 17 20 21 23 25 26

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-134- C133-185---rC136-138-163-250-

-142 = : 112 134 -23 7 153

-

134-

-66- 130- 105 194 z -OM 236-

-156 ~ 148- 176 100- -24 0 275-

23 25 26 51 27 29 54 30 63 32 33 34 35 36 37 38 52 52' 39 39' 56 ~173-94-49~82-ao-126cr154rr~7-25~17-152-91-75-66-14 I u 1 6 ~ 2 4 - 1 3 ~ 4 ~ 3 4 r

C l 582-31-4 0-35 1-196- 171-164----rC170-26 1-

-24 -3 313 = 306

-

24 -5 24 I 222-

~193-ai-rci53-~0-133-15-85-261-163-3~4-

-23-20-444 = : 191 177 2 394-

-142- 40 I -41 6 5-12 205- 471-

39 39' 56 58 41 42 43 44 45 46 47 55 49 57 I

' * 4 5 ~ 3 1 4 ~ 3 3 ~ 1 4 3 ~ 1 ~ 9 ~ l 1 ~ 1 2 ~ 7 5 ~' ~ 8 ~ 1 1 ~ ~ 1 0 ~ 1 6 ~ ~ ~ ~

-324-136- 84-1-33 147-234-491 --377- 175.---CI43---r--94-172-

-3 3 9

- -163 22 0 252-

-404- 187 z =

152--274 : = 258- 21 6

(9)

T-EVEN P H A G E GENETICS

975

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13.9 :I 9.4 29.1-

C-27.3 16.3 21 0 14.7

-

43.7

-

FIGURE 2.-Recombination data from T6 crosses. Presented as in Figure 1. The average number of determinations per number in the figure is 2.0.

relative orientations in all three phages, and hence the gene orders of

T2

and

T6

appear identical to that of

T4.

2)

Gene

spacing:

To

construct genetic maps from the data of Figures

1

and

2

it was necessary

to

devise a relationship between map distance and measured

recombination frequency. Several such relationships, called mapping functions,

have been devised for

T4

(STAHL,

EDGAR

and STEINBERG

1964)

to deal with the

extensive negative interference seen in

T4

crosses. Similar negative interference

was observed in

T2

and

T6

crosses, as shown in Figure

3;

in the absence of nega-

tive interference the data should have scattered about the drawn lines. In a n

attempt to circumvent this negative interference, an average

of

the two best

T4

mapping functions, the Four-Parameter Switch Function and the Four-Param-

100 I l l I I I I , I

(A)

60

-

40

-

30 -

20 -

15-

10

8 -

6 -

-

4 -

4 6 8 IO 15 20 30 40 60 80 100

PERCENTAGE OF RECOMBINATION, SUMMED FOR SHORTEST INTERVALS

(10)

976

R . L. RUSSELL

FIGURE 4.-Better additivity of T2 and T6 computed map distances. (A) T2, ( B ) T6. All distances are expressed as fractions of the total map length, as computed from recombination percentages using the T4 mapping function described in the text. Presentation is analogous with that of Figure 3, and in both cases the fit to the drawn line is better than in Figure 3.

eter Modified Bernstein Function

(

STAHL,

EDGAR

and STEINBERG

1964)

,

were

applied to recombination data from T2 and T6. This attempt was partially suc-

cessful for

T2

and more so for T6, as shown in Figure 4, and the resulting map

distances were used to construct the T2 and T6 maps shown in Figures 5 and 6.

The most striking feature of the

T2

and T6 maps is their general similarity to

[hat of

T4 (shown in Figure 7). Gene spacings are, in general, very similar in all

three phages, with a few exceptions. By far the most notable of these exceptions

is

the relative shrinking

of

genes 34 and

35

in T2. and, to a lesser extent, in T6.

A

somewhat smaller shrinkage is seen between genes 23 and 25 in T2. Some

newly identified genes of

T2 and T6, denoted by arrows in the figures, fall,

within the already well mapped regions, and the T2 and T6 maps, like that of T4,

contain extensive “silent regions” in which conditional lethal mutants have not

yet been found.

The tail fiber region; a site

of

genetic divergence:

That the tail fiber region

(genes 34-38) might be one of relative divergence for the three T-even phages

was suggested by two lines of evidence:

(1) the anomalous spot test behavior

of

gene 37 and 38 mutants, and

(2) the relative genetic shrinkage of genes 34 and

35 in T 2 and T6. To investigate this possibility further, several types of experi-

ments were performed.

(11)

T-EVEN PHAGE GENETICS

977

FIGURE 5.-Genetic map of T2. Distances separating genes have been computed using the

T4 mapping function described in the text, with a final correction to provide circular additivity. The symbol (10) denotes the new T2 gene identified by T 2 am 10, and the symbol (gtl) denotes the mapped position of an a-glucosyl transferase deficient mutant of T2 kindly provided by H. R. REVEL and mapped both by RUSSELL (unpublished results) and by

MOLHOLT

and DE GROOT

(1969). Because slight fluctuations in recombination frequency strongly influence the sizes of the unmapped gaps in the map, their true sizes are necessarily somewhat uncertain.

(see the accompanying paper,

RUSSELL

and

HUSKEY

1974).

The results, pre-

sented in Table

2, can be interpreted as follows.

(12)

978

R . L. RUSSELL

5s

-

39

FIGURE 6.-Genetic map of T6. Presentation analogous to that of Figure 5. The new gene identified by T6 am 103 is apparently the gene for a-glucosyl transferase, as described in the DISCUSSION.

Secondly, from tests involving the wild type of the three phages (Table 2b), it

is clear that the burst sizes of

T2

and T6 are somewhat lower than that

of

T4, and

that the T4-T6 mixed infection is characterized by a marked “depressor effect”

(see the accompanying paper, RUSSELL

and

HUSKEY

1974) which confounds

analysis of T4-T6 mutant tests.

Thirdly, tests between the wild type of one phage and mutants of another

(Table 2c) reveal evidence for the partial exclusion which is the subject

of

the

accompanying paper (Russell and Huskey 1974). In T2-T4 infections, for ex-

ample, mixed infections between T2 wild type and T4 mutants give much lower

burst sizes than those between

T4 wild type and T2 mutants. This asymmetry

(13)

T-EVEN P H A G E GENETICS

9 79

-24

FIGURE 7.-Genetic map of T4, from WOOD (1974)

T6 excludes T2, and both suggestions are confirmed in the accompanying paper

(RUSSELL

and

HUSKEY

1974).

Fourthly, tests between mutants of different phages (Table 2c) reveal a num-

ber of instances of lowered complementation. Some

of

these appear to be further

examples

of

the polarity effects described above, but one particularly strong

effect, involving genes

37

and

38

of

T2 and T4, is novel.

In

effect, the gene

37

and gene

38

mutants of T2 cannot be distinguished from one another on the basis

of their complementation properties with

T4

mutants, and

vice

versa;

neither

mutant complements either mutant from the other phage.* Such a result might

have been due to a strong polarity effect involving genes

37

and 38, but there is

no

evidence f o r such a n effect when either T2

o r

T4 is considered separately. The

(14)

980

34

+

R. L. R U S S E L L

34 0.4 0 . 0 0 . 1 0 . 5 8 . 1 0.1 6 . 1 3.4 7.5 7 . 1

100 74 142 122 1 2 1 1 3 2

TABLE 2

Complementation between tail fiber mutants

T2 T4 T6

T4

+ 34 35 36 37 38

38 183 1 4 1 1 2 9 105 118 0 1

371121 1 0 5 106 61 O.O+

t C ) T2 T6 T6

+ 34 35 36 37 3a

37 1 4 1 4 7 . 7 1.6 0.0 p.ol 38 19 20 1 7 4 . 6 l o . y T4 36 1 4 1 2 11 0.0 12

Each value is the burst size (total phage yield per infected bacterium) of a mixed infection of strain B/5 with the two indicated phages, at a multiplicity of at least five each; the numbers 34-38 indicate amber mutants in the corresponding genes of the indicated phage. The mutants used were (in gene order) T2 ambers 135, FS6, 24, 123, and 35; T4 ambers B25, B252, El, N52, C290; and T6 ambers 15, 26, and 126. All experiments were done in one batch to avoid possible bacterial variation. For ease of comprehension the table is divided into three parts, and some values necessarily occur in more than one part. The boxes denote the results which demonstrate the unusual complementation behavior of T2 and T4 genes 37 and 38.

result might also have been due

to

a celective inactivation

of

T2 genes 37 and 38

in mixed infection with T4, but the tests between T4 gene 37 or gene 38 mutants

and wild type show no evidence of such inactivation. The only remaining inter-

pretation appears to be that genes 37 and 38 are a pair of “coadapted” genes whose

products must interact rather closely during tail fiber assembly; when both

products are drawn from the same phage (T2 or T4) the interaction is successful,

but when the two products are €rom different phages (one from T2, and the

other from T4), it is not. This interpretation has also been arrived at by

STAHL

and

MURRAY

(1966) on the basis of very similar results. With respect to this in-

terpretation, it is encouraging to note that

in

vitro

studies of tail fiber assembly

(KING

and WOOD

1969) show that the products of genes 37 and 38 are both

required

for

the initial step in assembly

of

the distal half-fiber

of T4.

Although the gene 37 and 38 products

of

T2 are apparently incompatible with

(15)

T-EVEN PHAGE GENETICS

981

compatibility is quite limited. Whether the

T6

products of these genes ,in turn,

are compatible with those of T4 cannot be determined from Table 2, because of

the

T+T6

depressor effect. However, the spot tests suggest that they are not, since

T6 gene 37 mutants (like

T2

gene 37 and gene 38 mutants) were anomalous in

spot tests against gene 37 and gene 38 mutants

of

T4. In short, then, the T2 and

T6

products

of

these genes have limited compatibility with one another but none

with those

of

T4.

2)

Recombinational variation in genes 34 and

35:

Another set of experiments

was performed to investigate further the apparent discrepancies in the relative

genetic sizes

of

genes 34 and 35.

To

determine whether these discrepancies might

seriously result from fortuitously different mutant distributions within these

genes, maximum and minimum estimates

f o r

the sizes

of

genes 34 and 35 were

made f o r each phage. As Table

3

shows, the maximum estimates for both T2 and

T6

are well below the minimum estimates for T4, and the genetic size discrep-

ancy is therefore real. Such a genetic discrepancy might result from a true

physical size discrepancy, o r it might result from the presence of local factors

which alter

the

probability

of

genetic exchange per nucleotide pair.

A

true

physical size discrepancy between the genes would imply a similar size discrep-

ancy between their products, and would make the demonstrated interchange-

ability

of

these products difficult to understand. It would also imply that genetic

TABLE 3

T h e genetic lengths of genes 34 and 35

T 2 m a x T 6 m d x Gene Estimate Phage map length TL/T4 TA/T4 T4 nnn T 4 min

Percentage

of total ~ __-

T2 3.4

T6 4.6

T2 3.0

Minimum T4 6.5 0.46 0.12

T6 0.8

Maximum T4 9.9 0.34 0.46

__- 0.52 0.71

34 -

T2 1.9

T6 2.2

T2 -

Minimum T4 4.7

T6 -

Maximum T4 9.2 0.21 0.24

_-___ 0.40 0.47

~ _ _ _ _ _

35

- -

Minimum estimates were obtained by taking the two most separated markers within a gene, measuring the frequency of recombination bstween them, and converting this frequency to a percentage of the total map length with the T4 mapping function described in the text

Maximum estimates were obtained in the same way, using the two markers lying closest to the gene but outside it an either end.

(16)

982

R. L. RUSSELL

exchange between phages within genes 34 and 35 might be severely limited. The

presence of localized recombination-enhancing factors, on the other hand, would

be consistent with similar-sized gene products and with considerable genetic

exchange between the two phages in these genes.

To test these points, T2 and T4, which showed the greatest apparent discrep-

ancy, were selected, and their gene 34 mutants were crossed in all possible com-

binations. The results are shown in Table 4. Clearly there is considerable genetic

exchange between the phages within gene 34, suggesting considerable genetic

homology there. T o test this homology further, an attempt was made to order the

T2 and T4 mutants on a common map of gene 34. Because of the complications of

T2-T4 crosses, such a map could not be constructed by two-factor crosses alone.

However, three-factor crosses could be performed by using the known host range

difference between T2 and T4 as

a

convenient outside marker; Table

5

presents

the evidence which localizes this host range difference to the 37-gene 38 end of

the tail fiber gene cluster. Table 6 shows how the host range difference has been

used to order T2 and T4 mutants with respect to one another on a common map

of gene 39. That such

a

map can be constructed attests to the homology between

T 2 and T4 in gene 34; the intermingling of T2 and T4 mutants in the map

provides further evidence that homologous regions of gene 34 have quite different

TABLE 4

Genetic exchange within gene 34

T3 T4

AhlBER AIIIBER AhlBEK AMBER AMBER AMBER AMBER AMBER AMBER ARlRER AhIBER 12 73 135 7 0 74. 75 W25 R265 N58 B25S A455

AMBER 11.7 A455 B258 AMBER 9.7 AMBER 8.7 AMBER 8.3 AMBER 4.4 T4 N58

B265 B25

75 74

AMBER 15.1 T2 79

AMBER 135 20.7 AMBER 73 16.8 AMBER

12

AMBER 24.8 AMBER 21

.o

5.3 2.2 0.4 5.8 5.3 5.4 5.5 1.4 0.8 4.5 3.9 01.2 5.2 5.0 6.6 6.2 1.3 3.2 1.3 0.9 4.0 3.9 2.1 2.9

5.7 1.3 301.9 29.5 28.4 17.4 1.1 2.9 32.3 26.3 23.9 3.1 8.4 18.5 13.4 6.2 12.7 10.4 6.4 13.0 1.3

(17)

T - E V E N P H A G E GENETICS

TABLE 5

Localization of host range determinants

983

Cross

Percentage of wild-type progeny with host range genotype of

T2 T4 T6

T2 Wild type

x

T4 Wild type

T2 Wild type

x

T4 Amber in 34 T2 Wild type

x

T4 Amber in 35

T2 Wild type

x

T4 Amber in 36 T2 Wild type

x

T4 Amber in 37 T2 Wild type

x

T4 Amber in 38

T2 Amber in 36

x

T4 Amber in 37 T2 Amber in 36

x

T4 Amber in 38 T2 Amber in 37

x

T4 Amber in 36 T2 Amber in 38

x

T4 Amber in 36 T2 Amber in 37

x

T4 Amber in 38 T2 Amber in 38

x

T4 Amber in 37

10 56 84 92 97 1 0 95 99 2 0 la0 0

T2 Amber in 35

x

T6 Amber in 37 T2 Amber in 36

x

T6 Amber in 37

T2 Amber in 37

x

T6 Amber in 35 T2 Amber in 38

x

T6 Amber in 35

1 0 100

2

1

T4 Amber in 35

x

T6 Amber in 37

T44 Amber in 36

x

T6 Amber in 37 T4 Amber in 37

x

T6 Amber in 35 T4 Amber in 38

x

T6 Amber in 35

9.0 44 16 8 3 0 5 1 98 100 0 100 0 0 98 99

100 0

100 0

0 100

1 99

Progeny phages were plated on B/5 ta select wild types, and host range genotypes were deter- mined by stabs to plates seeded with the appropriate indicator strains (resistant strains used were B/2, S/4, and S/6). Occasional stabs growing on both indicators were assumed to be due to heterozygotes and were included in the table as having both host ranges. 100 plaques were stabbed for each determination. The wild-type progeny of the crosses between T2 and T4 gene 37 and gene 38 mutants were quite rare (0.1-0.3%), as might have been expected from the incompati- bilities described in the text; consequently it is not certain that they represent the products of single exchange events between T2 and T4. Wild-type progeny from crosses of gene 37 and gene

38 mutants of T2 and T6 or T4 and T6 grew so poorly that their host ranges could not be determined.

These results localize the host range d e t e h a n t s to the gene 37-gene 38 end of the tail fiber gene cluster, but because of the gene 37-gene 38 product incompatibility, they cannot localize them further.

probabilities of genetic exchange per nucleotide pair in the two phages. From the

anomalously large genetic sizes of genes 34 and 35 in T4, it seems likely that T4

harbors local factors which selectively enhance recombination in this part of its

genome.

DISCUSSION

The amber mutants of

T2 and T6 described above have provided a useful tool

with which to extend previous genetic work

(SEKELP

1960;

STAHL

and

MURRAY

(18)

9844

R . L. RUSSELL

TABLE 6

Each value in the table is the percentage of wild-type recombinants having the T2 host range genotype in a cross between the two indicated mutants. Since progeny of a T2-T4 cross selected to contain the gene 34 of T 2 are only 58% T2 in host range, this is the maximum figure expected in the table. Values close to this maximum suggest that the T 2 mutant lies to the gene 33 side of the T4 mutant, while values close to zero suggest the opposite ordering. Intermediate values are likely to occur when the T2 and T4 mutants are close to one another; in this case a definitive order cannot be deduced. The map indicates with respect to each other as best fits the T2 mapping data of Table 4.

and

T6

are homologous with known T4 genes, and gene orders, as well as most

gene spacings, are apparently identical in all three phages. With a few excep-

tions, gene products

of one phage are interchangeable with the homologous

products of the others, and almost any combination of mixed genes derived by

recombination between different phages is compatible with viability. Such close

genetic similarity supports previous investigations on the comparative particle

morphology

(BRENNER

et

al.

1959), serological cross reactivity

(ADAMS

1952),

and

DNA

base sequence homology

(SCHILDKRAUT

et

al.

1962;

COWIE,

AVERY

and

CHAMPE

1971) of these three phages.

It also agrees fully with exhaustive

electron-microscopic studies of

DNA-DNA

hybrids subsequently carried out by

KIM

and

DAVIDSON

(

1974).

The limited number of genetic differences among these phages are of interest,

partly because they can serve as useful genetic markers, and partly because their

nature may suggest how the phages have come to differ. The most striking dif-

ferences occur within the region of the genome controlling tail fiber formation.

The already known host range differences between

T2, T4 and T6 have been

shown to map to the tail fiber genes

37 and

38;

this evidence clearly suggests that

the distal portion of the tail fiber controlled by these genes

is

responsible for the

bacterial adsorption specificity of the phage, a conclusion also arrived at by a

study of host range mutants in

T4

(BECKENDORF,

unpublished results, quoted in

(19)

T-EVEN P H A G E GENETICS

985

The products of genes 37 and 38 apparently interact closely in forming the

distal half-fiber; when both products are drawn from the same phage the inter-

action is successful, and

a

functional half-fiber is produced in good yield. When

the products come from different phages, however, the interaction is much less

successful, ranging from

a

marginal success in the case of

T2

and T6 to a n abso-

lute failure in the case of T2 and T4. It appears, then, that genes 37 and 38 are

a ‘Lco-adapted” pair, showing much greater diversity from phage

to

phage than

does the rest of the genome. This point is discussed at greater length by STAHL

and MURRAY

(1 966).

The diversity among T2, T4 and T6 in these genes might have been an artifact

of

their selection, since they were initially distinguished from one another (and

thereby deemed worthy of beng separately kept and studied) by these very dif-

ferences in adsorption specificity.

If

so.

however, one would expect that three T-

even phages drawn at random from wild populations would show

less

diversity

than do T2, T4 and T6.

A

preliminary test of this notion has been made by exam-

ining T-even phages drawn from nature (Long Island sewage treatment plant

inlets)

;

among thirty-one such phages,

a t

least ten distinct adsorption specificities

could be detected, suggesting that genetic diversity

of

host range is common

among natural populations of T-even phages as well (RUSSELL

1974).

Rationalizing such diversity is not difficult. Because of the T-even phages’

absolute dependence on their bacterial hosts and because of the apparent ease

with which the bacterial surface can be altered by mutation so as to resist these

phages, the adsorption phase

of

the phage life cycle is subject to considerable

selective pressures. The various bacterial derivatives resistant to one or another

of

the T-even phages can be viewed as different ecological niches which are poten-

tially exploitable by the T-even “species’’ as a whole. In order to exploit these

niches, however, the T-even species must exhibit sufficient diversity of adsorption

specificity so that bacterial mutants resistant to

all

of the T-even phages will be

very unlikely to arise. The differences in genes 37 and 38 of the tail fiber region

can be viewed as the result of this selection for adsorption diversity. The absence

of

comparable differences elsewhere in the genome may indicate less intense

selection for diversity in other aspects of the phage life cycle.

The detailed structure of genes 37 and 38 has subsequently been investigated

more fully by

BECKENDORF,

KIM and LIELAUSIS

(1973), using both genetic

crosses and electron microscopy

of

DNA

hybrids. They have shown that these

genes, in contrast

to

other regions

of

the genome, are mostly heterologous between

T 2 and T4, and that the functional differences within these genes map to hetero-

logous regions. The extent of the heterologous regions suggests that they may

have arisen by insertion of heterologous

DNA,

rather than by successive small

mutations. Interestingly, KIM and DAVIDSON

(1974) have found that T2 and T6,

whose gene 37 and gene 38 products are at least partially compatible, also show

extensive

DNA

heterology in these genes.

(20)

986

R. L. RUSSELL

by

MOSIG

(1968, 1970) and a direct comparison of gene product sizes

(KING

and

LAEMMLI

1971;

WARD

and

DICKSON

1971) suggest that their actual physical

sizes are much smaller. Thus T 4 apparently possesses localized factors enhancing

recombination in this region. T2 apparently lacks these fectors, since the genetic

size of T2 gene 34 is consistent with the physical, as opposed to the genetic size

of gene 34 in

T4.

This interpretation has been explored further and confirmed by

RECKENDORF

and

WILSON

(1972), who have shown that the enhanced genetic

size of gene 34 in T4 is due

to

a recombination gradient in which the probability

of recombination per nucleotide pair increases dramatically in the distal part of

the gene nearest gene 35. Additional experiments by

RAMIG

(1974) suggest that

this gradient results from a discontinuity near the junction of genes 34 and 35;

the gradient present in T4 can be eliminated by substituting this junctional

region (and only this region) with the homologous region from T2. The evo-

lutionary and functional significances

of

such a localized recombination enhance-

ment are unclear.

Other detected genetic differences among these T-even phages are much less

notable than those in the tail fiber region; slight differences in apparent gene

spacing have been found elsewhere in the genome, and reduced burst sizes sug-

gesting imperfect gene product compatibilities have sometimes been noted in

liquid culture complementation tests between mutants of different phages. In

both cases, however, the effects are relatively minor.

Some known differences among the phages have not shown up in this genetic

analysis. In particular the absence of a “collar” in T2

(EISERLING

and

BOY

DE LA

TOUR

1965), and the known internal protein diversity of these phages

(HOWARD,

WOLIN

and

CHAMPE

1972) have not produced detectable effects either on genetic

exchange between the phages or on the interchangeability of their gene products.

Perhaps even more striking, the multiple short regions

of

non-homology detected

by

KIM

and

DAVIDSON

(1974) have not been detected genetically, and must there-

fore have, at most, only slight effects on the interchangeability of gene products

between phages.

The new genes found in T2 and T6, but not in T4, may be related to some of

the known differences among the phages. In particular, the gene found in both

T2 and T6 is almost undoubtedly the gene for the enzyme a-glucosyl transferase,

needed for attachment of glucose in a-linkage to the hydroxymethylcytosine resi-

due of T-even phage

DNA.

The evidence is (a) that T2 mutants wholly o r par-

tially defective in a-glucosyl transferase (T2gt mutants) map to the region

between genes 47 and 55

(MOLHOLT

and

DE

GROOT

1969;

RUSSELL,

unpublished

results) as do T2 and T6 mutants of the new gene, and (b) that T 2 and T6

mutants in this new gene, unlike those in any other gene, form perfectly normal

plaques on

E.

coli

strains which lack amber suppressors but instead contain

mutations rendering them tolerant of T-even phages with non-glucosylated

DNA.

An homologous gene for a a-glucosyl transferase exists in T4

(HOSODA

1967;

(21)

T-EVEN P H A G E GENETICS

987

from being lethal. Consequently, amber mutants in the T4 gene have not been

recovered as conditional lethals.

The significance of the other new genes is much less clear, but one of them

(identified by T2 amlO) maps in a region suggesting that it may be involved in

the “collar” difference between T2 and the other two phages.

The results described above suggest that

T2,

T4, and T6 are related to one

another in the same way as are races of higher organisms; genetic exchange

between them is considerable, but there are clear genetic differences. Co-adapted

gene pairs have been found, and the genetic diversity they represent is easily

rationalized as providing efficient exploitation of diverse ecological niches. The

accompanying paper describes what may be a n incipient isolating mechanism

by which genetic exchange between these phage races is beginning to be limited.

I am deeply indebted to R. S. EDGAR for exemplary guidance during the course of this work, and to F. W. STAHL for sharing both information and mutants. J. J. WEIGLE, R. J.

HUSKEY,

J. A. KING, and J. E. FLATGAARD provided many helpful discussions, and MRS. LAURA YOUNG provided able technical assistance during some of the later phases of the work. Copious amounts of materials were provided by BERTHA JONES, JEANETTE NAVEST, and IRENE VON HARTMANN,

to whom I am most grateful.

LITERATURE CITED

A~rlMs, M.

H.,

1952 Classification of bacterial viruses: characteristics of the T 5 species and 1959 Bac-

of the

T5

species and of the T2, C16 species. J. Bacteriol. 64: 387-396.

feriophages, Appendix. Interscience Publishers, New York. BECKENDORF, S. K., 1973

Biol. 73: 37-53.

BECKENDORF, S. K. and J. H. WILSON, 1972 34. Virology 50: 315-321.

BECKENDORF, S. K., J. S. KIM and I. LIELAUSIS, 1973 and 38. J. Mol. Biol. 73: 17-35.

___

,

Structure of the distal half of the bacteriophage T4 tail fiber. J. Mol.

A recombination gradient i n bacteriophage T4 gene

Structure of bacteriophage T 4 genes 37

BRENNER, S., G. STREISINGER, R. W. HORNE, S. P. CHAMPE,

L.

BARNETT, S. BENZER and M. W.

REES, 1959

COWIE, D. B., R. J. AVERY and S. P. CHAMPE, 1971 DNA homology among the T-even bacterio- phages. Virology 45: 30-37.

EDGAR, R. S. and R. H. EPSTEIN, 1965 EDGAR, R. S. and I. LIELAUSIS, 1964

Structural components of bacteriophage. J. Mol. Biol. 1 : 281-292.

The genetics of bacterial virus. Sci. Am. 212: 70-78. Temperature-sensitive mutants of bacteriophage T4D: their isolation and genetic characterization. Genetics 49: 649-662.

ditional lethal mutations of bacteriophage T4D. Genetics 49 : 635-648.

EISERLING, F. A. and E. BOY DE LA TOUR, 1965 Capsomeres and other structures observed on

some bacteriophages. Path. Microbiol. 28: 175-180.

EPSTEIN, R.

H.,

A. BOLLE, C. M. STEINBERG, E. KELLENBERGER, E. BOY DE LA TOUR, R. CHEVAL-

LEY, R. S . EDGAR, M. SUSMAN, G. H. DENHARDT and A. LIELAUSIS, 1963 Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28: 375-392.

Isolation and preliminary characterization of T4 mutants with nsn-glucsylated DNA. Biochem. Biophys. Res. Comm. 28: 179-184.

EDGAR, R. S., G. H. DENHARDT and R. H. EPSTEIN, 1964 A comparative genetic study of con-

(22)

988

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Figure

TABLE 1 Gene locations of T2 and T6 amber mutations

TABLE 1

Gene locations of T2 and T6 amber mutations p.6
TABLE I-Continued

TABLE I-Continued

p.7
FIGURE 1 the average percentage calculated percentages defects in the genes at the ends centage is the average by the permissive and restrictive strains used, as described in T2 am -Recombination data from T2 crosses

FIGURE 1

the average percentage calculated percentages defects in the genes at the ends centage is the average by the permissive and restrictive strains used, as described in T2 am -Recombination data from T2 crosses p.8
FIGURE - 2.-Recombination data from T6 crosses. Presented as in Figure 1. The average number of determinations per number in the figure is 2.0

FIGURE -

2.-Recombination data from T6 crosses. Presented as in Figure 1. The average number of determinations per number in the figure is 2.0 p.9
FIGURE 3.-Non-additivity lying between the same two mutations. The drawn line, among the shortest intervals used is indicated by the histogram is mutations; the abscissa ordinate of each point the line expected if observed and summed percentages were equal

FIGURE 3.-Non-additivity

lying between the same two mutations. The drawn line, among the shortest intervals used is indicated by the histogram is mutations; the abscissa ordinate of each point the line expected if observed and summed percentages were equal p.9
FIGURE 4.-Better percentages that distances are expressed as fractions additivity of T2 and T6 computed map distances

FIGURE 4.-Better

percentages that distances are expressed as fractions additivity of T2 and T6 computed map distances p.10
FIGURE of an a-glucosyl transferase deficient mutant H. T4 the mapped position (1969)

FIGURE of

an a-glucosyl transferase deficient mutant H. T4 the mapped position (1969) p.11
FIGURE 6.-Genetic identified by T6 am map of T6. Presentation analogous to that of Figure 5

FIGURE 6.-Genetic

identified by T6 am map of T6. Presentation analogous to that of Figure 5 p.12
FIGURE 7.-Genetic map of T4, from WOOD (1974)

FIGURE 7.-Genetic

map of T4, from WOOD (1974) p.13
TABLE 2 Complementation between tail fiber mutants

TABLE 2

Complementation between tail fiber mutants p.14
TABLE 3

TABLE 3

p.15
TABLE 4

TABLE 4

p.16
TABLE 5 Localization of host range determinants

TABLE 5

Localization of host range determinants p.17
TABLE 6 genotype in a cross between the Each value in the table is the percentage of two indicated mutants

TABLE 6

genotype in a cross between the Each value in the table is the percentage of two indicated mutants p.18

References