- Email: [email protected]
Genetic exchanges within prophage φ80 correlated with exchanges between host markers on either side
VIROLOGY
45,
$23-507
(1971)
xchanges nges
within
between
Prophage Host
V. N. RYBTCHINE Institut
Pasteur,
Paris,
and
Kalinin Accepted
Four mutations of phage - h-~40. In prophage am32 - am2 - h indicating Genetic exchanges between tween bacterial markers on based on a linearly inserted am2
Markers A!'ill
April
R. SIGNER1 Institute,
teningrad,z
USSR
8, 1971
$30 were mapped in phage crosses in the order am32 crosses the same markers were mapped in the order c40 cyclic permutation as specified by the Campbell model. prophage markers were correlated with exchanges beeither side in quantitative agreement with expectations prophage.
INTRODIJCTION
MATERIALS
AXD
METIIODS
Phage strains. The origin of the Cambridge st,rain of I$% was described earlier (Signer, 1966). The h mutant was the gift of Hall. The amber mutants am2 and am32 and the clear plaque mutant ~40 were derived by the double ultraviolet, irradiation teehnique of Weigle (1953). Spot eomplementation tests according to Campbell (1961) showed ‘that am2 and am32 are in different cistrons. Bacterial strahs. indicator strains, from the collection of Dr. S. Brenner, were CA161 (Hfr II Zac,, suII+) permissive for am mutants, CAB50 (Hfr H lacochre s’u.-) nonpermissive for am mutants, and X98 (I? lac-hisara-suII+ [t~z~-!PIRldelSmR) resistani; to (p8Qh+but sensitive to +80!2. All bacterial crosses were made with lysogens from the male CA150 and the fema. (F-lac,,hre- suC+tryC-py?F-his% genetics of this region have been described elsewhere (Signer, et al., 1965; Signer, 66). The markers SUC, at&O, Iry, and py are located on the E. coli map at 24.4, 24,8, 23: and 25.4 min, respectively (Tayh~: 1970)S Media. Broth contained Baetopeptone (10 g/l), Yeast, extract (5 g/l), Na Synthetic medium contained NIL:! g/l), KIIZPOd (3 g/l), NH&l 2O (0.13 g/I), lactose (20 g1’1)~ 1 (1 mg/l), hi&dine (29 mg/ml),
Department of Biology, Masof Technology, Cambridge, of V. r\‘. Rybtchine, should be sent.
E.
Polytechnic
Prophage integration is now fairly well understood (see reviews by Signer, 196%: Gottesman and Weisberg, 1971). Campbell’s (1962) model seems t.o describe the process quite well, and many of its predictions have been verified experimentally. Most important, prophage and bacterial genes in the chromosome of a lysogen may be deleted in a single block (Franklin et aE., 1965); and cotransduction is decreased while reeombinat,ion is increased for a pair of markers spanning the inserted prophage (Rothman, 1965; Signer, 1966). Here we present support for another prediction of the model, namely that genetic exchanges within the prophage are correlated with exchanges between bacterial markers lying on either side. During the course of this work we have constructed phage and prophage recombination maps for four mutants of 480. As with phage x (Calef and Licciardello, 1960; Rothman, 1965) the two maps are related by cyclic permutation. A preliminary report of some of these findings has appeared elsewhere (Signer and Rybtchine, 1967). 1 Present address: sachusetts Institute Massachusetts. 2 present address requests for reprints
68
to whom 503
504
RYBTCHINE
AND
SIGNER
(am32, am2)-h-c40. The two am sites are ordered by results of the cross am2-c40 X am32; among selected am+ recombinants turbid plaques outnumbered clear plaques by 688 to 431, indicating that am3W is farther from ~40. The two am sites seemto be very close since the double crossover recombinants (turbid plaques) are relatively frequent. The very high frequency of recombination between h and c suggests that, like X (see Gottesman and Weisberg, 1971), 480 has a site-specific prophage integration system that can promote recombination in phage crossesat a site between h and c. The order am32-am2-h-c40 is supported by the pattern of marker rescue from lysogens carrying the defective transducing phages @O&C (Signer et al., 1965) and +80d1Zac (Beckwith and Signer, 1966). Am2+ can be rescued from neither lysogen; am32+ from the latter only; and c22+ from both (~22 is a clear-plaque mutation that does not complement ~40). The SUC and lac markers are both located relative to the prophage in a position such that the transducing phages @O&UC and $80&lac should have substitutions in the late region as in Xdg (Franklin et al., 1965), so the results are consistent with the order given by the phage crosses.
st.reptomycin (250 mg/ml) and agar (20 g/l) ; tryptophan and uracil (20 mg/ml each) were added where necessary. Phage crosses.CA161 was grown in broth to log per ml, centrifuged and resuspended in equal volume of 0.01 M MgCO* , and aerated for 30 min at 37”. Phage were adsorbed at total m.o.i. 5-7 for 20 min at 37”. The adsorption mixture was centrifuged and the supernatant titered for unadsorbed phage. The pellet was resuspended and diluted in broth (final dilution lo4 from the adsorption tube) and aerated at 37” for 3 hr and then chloroformed. Bacterial crosses.Male and female were grown to 2 to 3 X lo8 per ml, mixed in the ratio 1: 10, kept at 37” for 3 hr with mild aeration, centrifuged, resuspended in 0.01 M MgS04, and plated on selective agar plates, which were then incubated for about 60 hr at 32”. Recombinant colonies, puriffed once on the selective medium, were then scored for prophage genotype by picking with sterile toothpicks onto a lawn of CA161, CA150, or X 98 and irradiating with a low dose of ultraviolet light to induce the prophage. Identical results were obtained when the crosseswere done in the presence or in the absenceof anti-480 serum. RESULTS
Recombination Map fro?n Phage Crosses Correlation of Phage and Bacterial Crossovers
Four markers have been mapped in phage crosses in the order am32-am2-h-c40. The nonselective crosses in Table 1 and the selective crossesin Table 2 give the order
The remaining experimental results comprise a. detailed study of the mating of lysogens. Male and female strains both
TABLE
an22 + 640 X fh
+
Parental am2 Recombinant Single
+ h + 394$)23 + c40 529
+ h c40 180 286 am2 + + 106 ";"Ycf
Double
Order
+ am2 deduced:
1
NONSELECTIVE
am2
;; + + h c40 -
73
14 26 12 1308 h - ~40
ad
++
CROSSES X +h ~40
+ h c40350 738 am2 + + 388 am2;hc~ am2+ +h c40 + am2 + + h c40 +
:;;
265
11 21 32 11 1052
22
am2 h ~40 X +++
am;:y3;y am2 h + + + ~40 am2 + + + h ~40 am2 +h+ + ~40
Total
722 90 159 69 15 31 16 167 23 935
Frequency
2383
710
0.214
136
0.041
71
0.021
3300
CORRELATED
PROPHAGE
carried prophages that are differentially marked. As shown in Pig. 1, recombinants Were selected to carry a distal marker from the male (try+ or pyrF+) on one side of the pro&age and a proximal marker from the female (suC+) on the other. The recombinant colonies were then tested for their prophage markers. This approach has been used to show that the presence of a prophage leads to an increase in recombination bet(ween t’he selected bact,erial markers (Signer, 1966). A typical set of results is shom;n in Table 3.
Among the selected bacterial recombinants are somethat carry a recombinant
prophage, and comparison of the two crosses indicates that marker effects do not significantly distort the results. The presence of prophage recombinants indicates some sort of correlation of crossovers between prophage and
bacterial markers. To reveal the nature of this correlation further the data have been TSKLE
(a)
am2
+
+ + -f + + c40
+ x
+ h c-lo
among selected 4652
progeny:
urn+
'780 135 75 ~40
among selected urn+ progeny:
+ h c40 2095 267 +h+ t + + 235 -t + c40 95 Order
2692 deduced
am32 - h - 40
c
om
h
FIG. 1. Scheme for prophage crosses. Male lysogens are derived from CA150 (HfrH SUC try+pyrF+) and
female
lysogens
ill 041 001 000 100 101 010 110
EXCfE.4KGE;S
1 2 3 4 124
111 98 15 5.5 20
85 53 5 34 4
195 151 20 89 24
0 39 0 30 0.N 0 18 0 05
123 234 134
9 3 --- i 312
0 1 2
9 4 3
0 02 0.000b --0.006
184
496
0 9w
A (40 nm2 +) x (+ -i- h) X (~40 a7122h,) selection sz17 /7!/-B (+ t h: n Tn this
and Table
4, the genotype
notations
I.
and 0 indicate c, a?%, h, the alleles comingfrom the male and female parent, respectively. b Exchange regions are numbered as fr>iiows’ su-(i)-c-(2)-am-(3)-h-(4)-try-(~)-pyr. fitted
CROSSES
5642 Order deduced nm2 - h (b) am32 + + X + h 40
Genotypes
HOST
2
SELECTIVE Genotypes + h 40 +h+
AND
from
X
108
(F-suC+t~y--
pyrF-). Order of prophage markers is based on Tables 3 a,nd 4, for which the exchange intervals are shown below. Distances are not to scale.
to a map including
a, linearly
inserted
prophage. Table 3 shows the fit for r’he order su-c-an&-try (see Fig. 1). This order provides the best fit among possibleprophage orders, and t’he fit is in fact quite good. This can be seen in the following way: Considering only the classesyielding a recombimmt prophage, it is clear that the most frequent class must represent a single exchange. Since this is class 011 the order must be w-c (UWL,h)-ky. The order of ana and 1~can. then be determined by considering those classesin which there has been an exchange: between am and h (i.e., classes001, 101, OiOp and 110 of Table 3). Since a,ll these classes involve an am-h exchange, the most frequent one must represent a single exchange and the others triple exchanges. Thus t#hefact chat the most frequent classis 001 indicates that the order is su-c-am-h-&y (for the order c-h-ant t,he most frequent class would have been 010). The expected frequency ol‘ a given class of triple exchanges can be computed by multiplying together the singk enchange frequencies for intervals exchanges occurred, and I minus the exchange frequency for intervals exchanges did not occur (Bailey, Computation in this way (for Tablcc?
\&erc single ~-here 1951 j. :j :t,nd
506
RYBTCHINE
AND TABLE
Unselected genotype
Exchange in interval 1 2 3 4 5 123 124 125 134 135 145 234 235 245 345
1111 0111 0011 0001 0000 1011 1001 1000 1101 1100 1110 0101 0100 0110 0010 * Crosses
TABLE
84 50 10 80 59 4 21 9 3 0 6 1 0 4 0
200 168 7 65 108 2 15 7 2 1 15 7 0 24 1
her ____
Frequency
E*
0.30 0.23 0.02 0.15 0.18 0.006 0.04 0.02 0.005 0.001 0.02 0.008 0.03 0.001
42 16 22 29 44 6 6 1 0 0 3 1 0 1 2 I--
1.011
173
284 218 17 145 167 6 36 16 5 1 21 8 0 28 1 c+ (c40 (~40 (+
+Y am2 h) -I- h) am32 +)
0.31 0.26 0.03 0.20 0.20
%:-
158
on32 om2 h
Fre-
Number
-I -/---I112 / 0.28
Fre-
0.02 0.006 0.009 0.02
331
0.997
c40
-_
WC
0 x
W PY~F--
quency
/ 481 ( 0.30
ii I- =uc
c40
on32 am2 h
try
PY’~-_
FIG. 2. Recombination maps from phage (upper) and prophage (lower) crosses and their relation by the Campbell model. Distances are not to scaIe.
4; see below) gives expected values for the presumed triples which are generally in the correct order but 2- to 5-fold lower than t.he observed values, suggesting that, there is some negative interference. Map for-
0.26 0.11 0.13 0.19 0.20 0.02 0.02 0.009 0.003
Jl am?2 ml2 h c‘+c
T&%1
quency
87 36 42 63 68 8 7 3 1 0 6 2 0 3 5
45 20 20 34 24 2 1 2 1 0 3 1 0 2 3
-
Fre-
Frequency
su+ pyr+
5
1197
Recombination
Selection:
am32 Total
F*
EXCHANGES
quency
369 312 38 244 234
am2 Total
_
Region
Num-
CROSSES
622 X x X x
4
SIX-FACTOR
am32
am2
(su-c) (c-am) (am-h) (h-try) (try-pyr)
D*
331 (~40 urn2 +) (+ + h) (+ am32 +) (c40 + h)
c D E F
TOTAL
1 2 3 4 5
C*
SIGNER
Prophage
Crosses
Crosses similar to those described above but including the additional interval try-pyr (Fig. 1) are presented in Table 4 for am2 and am32. In most instances, marker effects are not sign&ant, and the few apparent exceptions have ndt been studied further.
In each case the order indicated is su-c-am-htry-pyr. The total number of exchanges in each interval is tabulated in Table 5 for each of the sets of crosses of Table 4. Values for intervals 1, 4, and 5, which should be invariant, are in very good agreement. On the the other hand, from the values for intervals 2 and 3 it is clear that am32 is closer to c and farther from h than aqn2. Thus the prophage order appears to be su-c40-am32am2-h-try-pyr. In Table 4 the ratio of the sum of recombinants having an odd number of exchanges
CORRELATED
PROPHAGE
AND
between
su and try to recombinants having b&Teen try and pyr is 3.0 (summing over both crosses). This compares well with the value 4.1 found earlier (Signer, 1966) in somewhat different conditions. In the earlier work the value for nonlysogens was 1.6. Assuming an integrated prophage, Ohis would indicate that in lysogens about 60% of the su-try exchanges occur within the prophage. Table 5 shows that about 37 70 (0.30/0.81) of the su-try exchanges occur between c and h. If exchanges in the a-try segment are di&ributed randomly, then the distance from c to h is about three-fifths (37/60) the length of the prophage, comparable in magnitude to that in X.
an
exchange
DISCUSSION Early work by Calef and Licciardello (1960) with prophage X suggested a correlation of exchanges within the prophage and nearby bacterial exchanges. Our experiments show that exchanges within the prophage are strongly correlated with those involving bacterial markers on either side and in quantit’at’ive agreement with a model of a linearly inserted prophage. Thus these results may be added to the large body of evidence supporting the Campbell (1962) model (see Signer, 1968). Ahhough the phage order of markers is am32-am%h-c, the prophage order is su-cal?i32-am2-h-t~y-pyr. The two orders are related by cyclic permutation. This has already been shown for phage X (Calef and Licciardeho, 1960; Rothman, 1965), which is related to 480 at least to the extent of recombining with it’ (Signer, 1964). The phage and prophage maps are compared within the calnext of the Campbell model in Fig. 2. ,4sexpected, there is a close resemblance to the map of Xin that the circle opens between h and c in insertion and that all the an2 sites are located between c and h on the prophage map. We do not know how the markers described here correspond to the ones characterized by Sato et al. (1968). ACKXOWLEDGNIENT The authors whose laboratory
are grateful this work
to Dr. F. Jacob, was initiated, for
in his
KOST
;5,si’
EXCIIA~GES
hospitality and interest. Work in Paris ported by grant,s to Dr. Jacob from the Science Foundation and La Delegation % la Recherche Scientifique et Technique, postdoctoral fellowship to Ethan Signer Jane CofIin Childs Memorial Fund for Research. We thank Mrs. T. Kirpichnikova technical assistance.
was supXational G&&ale and a, from the Medical for
REFERENCES
BAILEY, N. T. J. (1951).
The estimation of linkage Beredity 5, 111-124. BECKWITH, J. It., and SIGNER: %:. R. (1966). Transposition of the Lae Region of E. toll. I. Diversion of the lac Operon and transduction of Zac by &O. J. Mol. Biol. 19, 254-265. CALEF, E., and LICCIARDELLO, G. (1960), Reeombination experiments on prophage-host relation.ships. Y&Zoy~ 5.2, 81-103. CAMPBELL, A. (1961). Sensitive mutants of baeteriophage A. ‘virology 14, 2232. CAMPBELL, A. (1962). Episomes. Advan. Chel. 1B, 101-145. FRANKLIN, S. C., DOVE, W. F., and YANOFSICY~ C. (1965). The linear insertion of a prophage into the chromosome of E. coli shown by deletion mapping. Biochem,. Biophys. Res. Common, 18, 910-23. GOTTESMAN, M. E., and WEISBERG. R. A. (1911j. Specialized recombination in bacteriophage X: the Int system. ‘LBacteriophage Xzl (A. D. Hershey, ed.), in press. ROTHMAS, J. L. (1965). Transduction studies on the relation between prophage and host chro. mosome. J. Mol. Biol. P2, 8922912. f&o, K., NISEIMUNE, Y., &TO, X, ~'IXUC;I~ I<.? MATSITSHIRO, A., IKOKUCWI, H., and OZEKI, H. (1968). Suppressor-sensitive mutants of coliphage +SO. Yirobogy 34, 637649. SIGNER, E. R. (1964). Recombination between coliphages A and $80. Virology 22, 650-2. SIGSER, E. R. (1966). Interaction of prophagea 62% the at@0 site with the chromosome of Escherichia coli. J. Mol. Biol. 15, 243-S. SIGIER, E. R. (1968). Lysogeny: The Integration Problem. Annu. Rev. Xicrobiol. 12, 457.-88. in bacteria.
SIGSEIL, E. R., arrd Run~cms~,
V. (1967). Ke-
combination between prophages of Escherichzn coli. Genefika 3, 114-121 (in Russianj. SIGPZER,E.I~.,~ECI~~~~ITH, J.R., andBnES~Erz,S. (1965). Mapping of suppressor loci in Esch.erirh;cL cozi. J. MOZ. Biol. 14, 153-66. WEIGLE, J. J. (1953). Induction of mutations in a bacterial virus. Proc. Nai. Acad. Sci. t:. S. 39, 628-33.
45,
$23-507
(1971)
xchanges nges
within
between
Prophage Host
V. N. RYBTCHINE Institut
Pasteur,
Paris,
and
Kalinin Accepted
Four mutations of phage - h-~40. In prophage am32 - am2 - h indicating Genetic exchanges between tween bacterial markers on based on a linearly inserted am2
Markers A!'ill
April
R. SIGNER1 Institute,
teningrad,z
USSR
8, 1971
$30 were mapped in phage crosses in the order am32 crosses the same markers were mapped in the order c40 cyclic permutation as specified by the Campbell model. prophage markers were correlated with exchanges beeither side in quantitative agreement with expectations prophage.
INTRODIJCTION
MATERIALS
AXD
METIIODS
Phage strains. The origin of the Cambridge st,rain of I$% was described earlier (Signer, 1966). The h mutant was the gift of Hall. The amber mutants am2 and am32 and the clear plaque mutant ~40 were derived by the double ultraviolet, irradiation teehnique of Weigle (1953). Spot eomplementation tests according to Campbell (1961) showed ‘that am2 and am32 are in different cistrons. Bacterial strahs. indicator strains, from the collection of Dr. S. Brenner, were CA161 (Hfr II Zac,, suII+) permissive for am mutants, CAB50 (Hfr H lacochre s’u.-) nonpermissive for am mutants, and X98 (I? lac-hisara-suII+ [t~z~-!PIRldelSmR) resistani; to (p8Qh+but sensitive to +80!2. All bacterial crosses were made with lysogens from the male CA150 and the fema. (F-lac,,hre- suC+tryC-py?F-his% genetics of this region have been described elsewhere (Signer, et al., 1965; Signer, 66). The markers SUC, at&O, Iry, and py are located on the E. coli map at 24.4, 24,8, 23: and 25.4 min, respectively (Tayh~: 1970)S Media. Broth contained Baetopeptone (10 g/l), Yeast, extract (5 g/l), Na Synthetic medium contained NIL:! g/l), KIIZPOd (3 g/l), NH&l 2O (0.13 g/I), lactose (20 g1’1)~ 1 (1 mg/l), hi&dine (29 mg/ml),
Department of Biology, Masof Technology, Cambridge, of V. r\‘. Rybtchine, should be sent.
E.
Polytechnic
Prophage integration is now fairly well understood (see reviews by Signer, 196%: Gottesman and Weisberg, 1971). Campbell’s (1962) model seems t.o describe the process quite well, and many of its predictions have been verified experimentally. Most important, prophage and bacterial genes in the chromosome of a lysogen may be deleted in a single block (Franklin et aE., 1965); and cotransduction is decreased while reeombinat,ion is increased for a pair of markers spanning the inserted prophage (Rothman, 1965; Signer, 1966). Here we present support for another prediction of the model, namely that genetic exchanges within the prophage are correlated with exchanges between bacterial markers lying on either side. During the course of this work we have constructed phage and prophage recombination maps for four mutants of 480. As with phage x (Calef and Licciardello, 1960; Rothman, 1965) the two maps are related by cyclic permutation. A preliminary report of some of these findings has appeared elsewhere (Signer and Rybtchine, 1967). 1 Present address: sachusetts Institute Massachusetts. 2 present address requests for reprints
68
to whom 503
504
RYBTCHINE
AND
SIGNER
(am32, am2)-h-c40. The two am sites are ordered by results of the cross am2-c40 X am32; among selected am+ recombinants turbid plaques outnumbered clear plaques by 688 to 431, indicating that am3W is farther from ~40. The two am sites seemto be very close since the double crossover recombinants (turbid plaques) are relatively frequent. The very high frequency of recombination between h and c suggests that, like X (see Gottesman and Weisberg, 1971), 480 has a site-specific prophage integration system that can promote recombination in phage crossesat a site between h and c. The order am32-am2-h-c40 is supported by the pattern of marker rescue from lysogens carrying the defective transducing phages @O&C (Signer et al., 1965) and +80d1Zac (Beckwith and Signer, 1966). Am2+ can be rescued from neither lysogen; am32+ from the latter only; and c22+ from both (~22 is a clear-plaque mutation that does not complement ~40). The SUC and lac markers are both located relative to the prophage in a position such that the transducing phages @O&UC and $80&lac should have substitutions in the late region as in Xdg (Franklin et al., 1965), so the results are consistent with the order given by the phage crosses.
st.reptomycin (250 mg/ml) and agar (20 g/l) ; tryptophan and uracil (20 mg/ml each) were added where necessary. Phage crosses.CA161 was grown in broth to log per ml, centrifuged and resuspended in equal volume of 0.01 M MgCO* , and aerated for 30 min at 37”. Phage were adsorbed at total m.o.i. 5-7 for 20 min at 37”. The adsorption mixture was centrifuged and the supernatant titered for unadsorbed phage. The pellet was resuspended and diluted in broth (final dilution lo4 from the adsorption tube) and aerated at 37” for 3 hr and then chloroformed. Bacterial crosses.Male and female were grown to 2 to 3 X lo8 per ml, mixed in the ratio 1: 10, kept at 37” for 3 hr with mild aeration, centrifuged, resuspended in 0.01 M MgS04, and plated on selective agar plates, which were then incubated for about 60 hr at 32”. Recombinant colonies, puriffed once on the selective medium, were then scored for prophage genotype by picking with sterile toothpicks onto a lawn of CA161, CA150, or X 98 and irradiating with a low dose of ultraviolet light to induce the prophage. Identical results were obtained when the crosseswere done in the presence or in the absenceof anti-480 serum. RESULTS
Recombination Map fro?n Phage Crosses Correlation of Phage and Bacterial Crossovers
Four markers have been mapped in phage crosses in the order am32-am2-h-c40. The nonselective crosses in Table 1 and the selective crossesin Table 2 give the order
The remaining experimental results comprise a. detailed study of the mating of lysogens. Male and female strains both
TABLE
an22 + 640 X fh
+
Parental am2 Recombinant Single
+ h + 394$)23 + c40 529
+ h c40 180 286 am2 + + 106 ";"Ycf
Double
Order
+ am2 deduced:
1
NONSELECTIVE
am2
;; + + h c40 -
73
14 26 12 1308 h - ~40
ad
++
CROSSES X +h ~40
+ h c40350 738 am2 + + 388 am2;hc~ am2+ +h c40 + am2 + + h c40 +
:;;
265
11 21 32 11 1052
22
am2 h ~40 X +++
am;:y3;y am2 h + + + ~40 am2 + + + h ~40 am2 +h+ + ~40
Total
722 90 159 69 15 31 16 167 23 935
Frequency
2383
710
0.214
136
0.041
71
0.021
3300
CORRELATED
PROPHAGE
carried prophages that are differentially marked. As shown in Pig. 1, recombinants Were selected to carry a distal marker from the male (try+ or pyrF+) on one side of the pro&age and a proximal marker from the female (suC+) on the other. The recombinant colonies were then tested for their prophage markers. This approach has been used to show that the presence of a prophage leads to an increase in recombination bet(ween t’he selected bact,erial markers (Signer, 1966). A typical set of results is shom;n in Table 3.
Among the selected bacterial recombinants are somethat carry a recombinant
prophage, and comparison of the two crosses indicates that marker effects do not significantly distort the results. The presence of prophage recombinants indicates some sort of correlation of crossovers between prophage and
bacterial markers. To reveal the nature of this correlation further the data have been TSKLE
(a)
am2
+
+ + -f + + c40
+ x
+ h c-lo
among selected 4652
progeny:
urn+
'780 135 75 ~40
among selected urn+ progeny:
+ h c40 2095 267 +h+ t + + 235 -t + c40 95 Order
2692 deduced
am32 - h - 40
c
om
h
FIG. 1. Scheme for prophage crosses. Male lysogens are derived from CA150 (HfrH SUC try+pyrF+) and
female
lysogens
ill 041 001 000 100 101 010 110
EXCfE.4KGE;S
1 2 3 4 124
111 98 15 5.5 20
85 53 5 34 4
195 151 20 89 24
0 39 0 30 0.N 0 18 0 05
123 234 134
9 3 --- i 312
0 1 2
9 4 3
0 02 0.000b --0.006
184
496
0 9w
A (40 nm2 +) x (+ -i- h) X (~40 a7122h,) selection sz17 /7!/-B (+ t h: n Tn this
and Table
4, the genotype
notations
I.
and 0 indicate c, a?%, h, the alleles comingfrom the male and female parent, respectively. b Exchange regions are numbered as fr>iiows’ su-(i)-c-(2)-am-(3)-h-(4)-try-(~)-pyr. fitted
CROSSES
5642 Order deduced nm2 - h (b) am32 + + X + h 40
Genotypes
HOST
2
SELECTIVE Genotypes + h 40 +h+
AND
from
X
108
(F-suC+t~y--
pyrF-). Order of prophage markers is based on Tables 3 a,nd 4, for which the exchange intervals are shown below. Distances are not to scale.
to a map including
a, linearly
inserted
prophage. Table 3 shows the fit for r’he order su-c-an&-try (see Fig. 1). This order provides the best fit among possibleprophage orders, and t’he fit is in fact quite good. This can be seen in the following way: Considering only the classesyielding a recombimmt prophage, it is clear that the most frequent class must represent a single exchange. Since this is class 011 the order must be w-c (UWL,h)-ky. The order of ana and 1~can. then be determined by considering those classesin which there has been an exchange: between am and h (i.e., classes001, 101, OiOp and 110 of Table 3). Since a,ll these classes involve an am-h exchange, the most frequent one must represent a single exchange and the others triple exchanges. Thus t#hefact chat the most frequent classis 001 indicates that the order is su-c-am-h-&y (for the order c-h-ant t,he most frequent class would have been 010). The expected frequency ol‘ a given class of triple exchanges can be computed by multiplying together the singk enchange frequencies for intervals exchanges occurred, and I minus the exchange frequency for intervals exchanges did not occur (Bailey, Computation in this way (for Tablcc?
\&erc single ~-here 1951 j. :j :t,nd
506
RYBTCHINE
AND TABLE
Unselected genotype
Exchange in interval 1 2 3 4 5 123 124 125 134 135 145 234 235 245 345
1111 0111 0011 0001 0000 1011 1001 1000 1101 1100 1110 0101 0100 0110 0010 * Crosses
TABLE
84 50 10 80 59 4 21 9 3 0 6 1 0 4 0
200 168 7 65 108 2 15 7 2 1 15 7 0 24 1
her ____
Frequency
E*
0.30 0.23 0.02 0.15 0.18 0.006 0.04 0.02 0.005 0.001 0.02 0.008 0.03 0.001
42 16 22 29 44 6 6 1 0 0 3 1 0 1 2 I--
1.011
173
284 218 17 145 167 6 36 16 5 1 21 8 0 28 1 c+ (c40 (~40 (+
+Y am2 h) -I- h) am32 +)
0.31 0.26 0.03 0.20 0.20
%:-
158
on32 om2 h
Fre-
Number
-I -/---I112 / 0.28
Fre-
0.02 0.006 0.009 0.02
331
0.997
c40
-_
WC
0 x
W PY~F--
quency
/ 481 ( 0.30
ii I- =uc
c40
on32 am2 h
try
PY’~-_
FIG. 2. Recombination maps from phage (upper) and prophage (lower) crosses and their relation by the Campbell model. Distances are not to scaIe.
4; see below) gives expected values for the presumed triples which are generally in the correct order but 2- to 5-fold lower than t.he observed values, suggesting that, there is some negative interference. Map for-
0.26 0.11 0.13 0.19 0.20 0.02 0.02 0.009 0.003
Jl am?2 ml2 h c‘+c
T&%1
quency
87 36 42 63 68 8 7 3 1 0 6 2 0 3 5
45 20 20 34 24 2 1 2 1 0 3 1 0 2 3
-
Fre-
Frequency
su+ pyr+
5
1197
Recombination
Selection:
am32 Total
F*
EXCHANGES
quency
369 312 38 244 234
am2 Total
_
Region
Num-
CROSSES
622 X x X x
4
SIX-FACTOR
am32
am2
(su-c) (c-am) (am-h) (h-try) (try-pyr)
D*
331 (~40 urn2 +) (+ + h) (+ am32 +) (c40 + h)
c D E F
TOTAL
1 2 3 4 5
C*
SIGNER
Prophage
Crosses
Crosses similar to those described above but including the additional interval try-pyr (Fig. 1) are presented in Table 4 for am2 and am32. In most instances, marker effects are not sign&ant, and the few apparent exceptions have ndt been studied further.
In each case the order indicated is su-c-am-htry-pyr. The total number of exchanges in each interval is tabulated in Table 5 for each of the sets of crosses of Table 4. Values for intervals 1, 4, and 5, which should be invariant, are in very good agreement. On the the other hand, from the values for intervals 2 and 3 it is clear that am32 is closer to c and farther from h than aqn2. Thus the prophage order appears to be su-c40-am32am2-h-try-pyr. In Table 4 the ratio of the sum of recombinants having an odd number of exchanges
CORRELATED
PROPHAGE
AND
between
su and try to recombinants having b&Teen try and pyr is 3.0 (summing over both crosses). This compares well with the value 4.1 found earlier (Signer, 1966) in somewhat different conditions. In the earlier work the value for nonlysogens was 1.6. Assuming an integrated prophage, Ohis would indicate that in lysogens about 60% of the su-try exchanges occur within the prophage. Table 5 shows that about 37 70 (0.30/0.81) of the su-try exchanges occur between c and h. If exchanges in the a-try segment are di&ributed randomly, then the distance from c to h is about three-fifths (37/60) the length of the prophage, comparable in magnitude to that in X.
an
exchange
DISCUSSION Early work by Calef and Licciardello (1960) with prophage X suggested a correlation of exchanges within the prophage and nearby bacterial exchanges. Our experiments show that exchanges within the prophage are strongly correlated with those involving bacterial markers on either side and in quantit’at’ive agreement with a model of a linearly inserted prophage. Thus these results may be added to the large body of evidence supporting the Campbell (1962) model (see Signer, 1968). Ahhough the phage order of markers is am32-am%h-c, the prophage order is su-cal?i32-am2-h-t~y-pyr. The two orders are related by cyclic permutation. This has already been shown for phage X (Calef and Licciardeho, 1960; Rothman, 1965), which is related to 480 at least to the extent of recombining with it’ (Signer, 1964). The phage and prophage maps are compared within the calnext of the Campbell model in Fig. 2. ,4sexpected, there is a close resemblance to the map of Xin that the circle opens between h and c in insertion and that all the an2 sites are located between c and h on the prophage map. We do not know how the markers described here correspond to the ones characterized by Sato et al. (1968). ACKXOWLEDGNIENT The authors whose laboratory
are grateful this work
to Dr. F. Jacob, was initiated, for
in his
KOST
;5,si’
EXCIIA~GES
hospitality and interest. Work in Paris ported by grant,s to Dr. Jacob from the Science Foundation and La Delegation % la Recherche Scientifique et Technique, postdoctoral fellowship to Ethan Signer Jane CofIin Childs Memorial Fund for Research. We thank Mrs. T. Kirpichnikova technical assistance.
was supXational G&&ale and a, from the Medical for
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The estimation of linkage Beredity 5, 111-124. BECKWITH, J. It., and SIGNER: %:. R. (1966). Transposition of the Lae Region of E. toll. I. Diversion of the lac Operon and transduction of Zac by &O. J. Mol. Biol. 19, 254-265. CALEF, E., and LICCIARDELLO, G. (1960), Reeombination experiments on prophage-host relation.ships. Y&Zoy~ 5.2, 81-103. CAMPBELL, A. (1961). Sensitive mutants of baeteriophage A. ‘virology 14, 2232. CAMPBELL, A. (1962). Episomes. Advan. Chel. 1B, 101-145. FRANKLIN, S. C., DOVE, W. F., and YANOFSICY~ C. (1965). The linear insertion of a prophage into the chromosome of E. coli shown by deletion mapping. Biochem,. Biophys. Res. Common, 18, 910-23. GOTTESMAN, M. E., and WEISBERG. R. A. (1911j. Specialized recombination in bacteriophage X: the Int system. ‘LBacteriophage Xzl (A. D. Hershey, ed.), in press. ROTHMAS, J. L. (1965). Transduction studies on the relation between prophage and host chro. mosome. J. Mol. Biol. P2, 8922912. f&o, K., NISEIMUNE, Y., &TO, X, ~'IXUC;I~ I<.? MATSITSHIRO, A., IKOKUCWI, H., and OZEKI, H. (1968). Suppressor-sensitive mutants of coliphage +SO. Yirobogy 34, 637649. SIGNER, E. R. (1964). Recombination between coliphages A and $80. Virology 22, 650-2. SIGSER, E. R. (1966). Interaction of prophagea 62% the at@0 site with the chromosome of Escherichia coli. J. Mol. Biol. 15, 243-S. SIGIER, E. R. (1968). Lysogeny: The Integration Problem. Annu. Rev. Xicrobiol. 12, 457.-88. in bacteria.
SIGSEIL, E. R., arrd Run~cms~,
V. (1967). Ke-
combination between prophages of Escherichzn coli. Genefika 3, 114-121 (in Russianj. SIGPZER,E.I~.,~ECI~~~~ITH, J.R., andBnES~Erz,S. (1965). Mapping of suppressor loci in Esch.erirh;cL cozi. J. MOZ. Biol. 14, 153-66. WEIGLE, J. J. (1953). Induction of mutations in a bacterial virus. Proc. Nai. Acad. Sci. t:. S. 39, 628-33.