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Effects of ultraviolet light on transducing phage P22
VIROLOGY
18, 614-626
Effects
(1962)
of Ultraviolet ROLF
Department
Light
BENZINGER
of Biology,
The
on AND
Johns
PHILIP
Hopkins
Accepted
Transducing
P22
E. HARTMAN
University,
August
Phage
Baltimore
18, Maryland
S, 1962
The effects of ultraviolet irradiation on suspensions of P22 particles are examined. Evidence is presented which indicates that a single ultraviolet hit on an abortively transducing element enables it to form a complete transduction, i.e., to undergo recombination with the chromosome of recipient bacteria. Thus, abortive transductions are rapidly inactivated while the number of complete transductions increases. During the conversion of abortive transductions to complete transductions, jointly transduced markers become less tightly linked. The rate of linkage disruption is proportional to the genetic map distance between linked markers and allows an estimate to be made for the physical size of the histidine (his) region of the Salmonella chromosome. The rates of inactivation of complete transductions are proportional to the sizes of the genetic defects in the recipients, allowing a second estimate of the size of the his region to be made. The eight-gene his region comprises an amount of DNA equivalent to approximately 8 X 10“ molecular weight, i.e., one-fifth that contained in a normal P22 phage particle. The coding ratio for the his region is about three nucleotide pairs per amino acid in the eight proteins whose structure it controls.
an initial increase at low ultraviolet doses, and inactivation at higher doses. Ozeki (personal communication, 1956) observed that the abortive transduction activity of P22 for the athC-5 recipient decreased exponentially with ultraviolet dose. The inactivation rate of complete transduction (after the initial rise) was considerably less than that of abortive transduction. Further observations on the ultraviolet inactivation kinetics of abortive and complete transduction, the inactivation of linkage, and attempts to characterize the nature of the process causing the initial rise in complete transductions will be reported below. These will be used to draw inferences concerning the molecular size and structure of genetic regions carried by transducing particles in P22 lysates.
INTRODUCTION
The action of ultraviolet irradiation on complete transduction by P22 has been investigated by Garen and Zinder (1955). They noted an increased number of transductions at low ultraviolet doses, although phage infectivity had been reduced to as little as 1O-4 of the zero time value. An increase in transduction frequency following ultraviolet irradiation of free phage has also been observed for coliphage h (Arber, 1958), for Pl (Luria et aZ., 1960; Wilson, 1960)) for Pseudomonas aeruginosa phage (Holloway and Monk, 1959, 1961), and for Proteus mirabilis phage (Coetzee and Sacks, 1960). The increased transduction frequency is not specifically induced by ultraviolet alone; nitrous acid produces the same effect with P22 (Adye, 1962). The lytic ability of P22 (Garen and Zinder, 1955; Prell, 1961) and the number of cells doubly lysogenized by x phage (Kellenberger et al., 1959) also show 1 Present Biochemie,
address: Max-Planck Munich 15, Germany.
Institut
MATERIALS
AND
METHODS
Bacterial strains. All strains are from the collection of Dr. M. Demerec, Brookhaven National Laboratory, and are derived
fiir 614
from S. t~yphimurium strains LT2 or LT7. Methods for obtaining most of these mutants have been reported elsewhere (Hartman et al., 1960b). Salmonella typhimurium 0 form 13 and S. gallinarum were donated by Dr. N. D. Zinder. Escherichia coli strain B was used as T2 phage indicator. Phages. PLT22 (P22) and T2 phage suspensions were prepared, assayed, and stored as detailed previously by Hartman (1956). The H5 (VW) mutant has been described by Zinder (1958). Media. Difco nutrient broth enriched with 0.2% dextrose was normally used as a complete medium. T2 phage was assayed on the Difco tryptone bottom Iayer agar of Hershey and Rotman (1949) but without citrate. Minimal medium, enriched minimal medium, and enriched minimal supplemented with L-histidinol were prepared as described by Hartman et al. (1960b). Calcium pantothenate and adenine were added to enriched minimal medium to permit visual detection of athC-5+ abortive transductions as detailed by Ozeki (1956). To enlarge the colonies from abortive transduction to his+, minimal medium was supplemented with a final concentration of 80 pg L-glutamic acid per milliliter or with a pool of amino acids (20 pg/ml each) lacking histidine. Ultraviolet irradiation. A 15-watt General Electric “germicidal” lamp was used in all studies. After allowing the lamp to warm up for 30 minutes, no more than 10 ml of phage in saline at a concentration below lOlo plaque-forming units per milliliter were irradiated in a flat-bottomed lOO-mm petri dish (which was continually agitated mechanically). At this phage concentration, only 5% of the incident 253.7rnp light was absorbed, as measured in t,he Beckman DU spectrophotometer. To prevent photoreactivation, yellow illumination was utilized for all operations during and after irradiation and the plates were incubated in the dark for at Ieast 12 hours. Biological calibration by means of T2 phage inactivation and reference to the standard curve of Latarjet et al. (1953) gave a dose rate of 13.8 ergs/mm2/sec at 60
cm. In this calibration, six experiments with five p0int.s per curve to 1O-4 survival gave close agreement. Physical calibration with a General Electric watt.meter gave a value of 12.5 ergs/mm2/sec at 60 cm. The inactivation of the biological activities studied will be represented graphically by plotting the logarithm of survivors on the ordinate against ultraviolet dose on the abscissa. In order to compare the results of different experiments, the inactivation of phage infectivity is used as a standard. Thus, inactivation rates are expressed relative to the phage infectivity slope which = 1, by definition. Transduction tests. Recipient bacteria were prepared as follows: log or stationary phase nutrient broth cultures were centrifuged at 3000 g for 15 minutes and the pellet was resuspended in saline. When clearly visible his+ abortives were desired, the pellet was washed t,hree times with distilled water to reduce residual growth of the recipients on t,he glutamic acid medium. This treatment has no effect on the viable count (C. Kirchner, personal communication). After 10 minutes at 37” were allowed for phage adsorption, 0.05 to 0.3 ml samples were spread evenly on thick agar plates which had been dried for at least 24 hours at 37”. These plat.es were incubated at 37” for periods ranging from 18 to 60 hours. Complete and abort,ive transductions generally required about 48 hours to develop on minimal medium and 18 hours on glutamic acid medium. Joint transduct’ions (“donor type t,ransductional clones,” Hartman, 1956) were clearly visible after 60 and 48 hours on the respective media. Complete t,ransduction and athC-5+ abortive transduction assays varied *20% in replicate platings. Abortive transductions to his+ were counted under a Bausch and Lomb “Stereozoom” microscope at, a magnification of 30x, employing 10 x wide-field eyepieces. At least 100 abortive colonies were counted in a known area no smaller than one-fiftieth of the plat’e. The count obt,ained was then used to calculate the total number of abort.ive transductions on the plate. Assays were carried out, either in du-
616
BENZINGER
AND HARTMAN
plicate or in triplicate; the precision of the method in replicate counts was -1150%. For each inactivation curve of transduction, the titers of unirradiated phage and recipient bacteria were determined. The multiplicity of infection was generally kept below 1 and seldom was above 5. Each experiment was accompanied by a control with uninfected bacteria to determine the number of spontaneous reversions to wild type in the culture.
tested (Fig. lb). Figures la and lb also show a net increase in recovery of transductional clones at low dosesof ultraviolet light. Some features of this phenomenon are described in the following section. Table 1 depicts abortive and complete transduction inactivation rat’es for a number of auxotrophs. The rates were calculated from the exponential portions of the inactivation curves to 1% survival isee Figs. la and lb). Abortive transduction generally was more sensitive to ultraviolet than was RESULTS complete transduction. For the his region, the rate of abortive transduction inactivaThe slopes of the curves for inactivation tion was not significantly dependent, on the of phage infectivity obtained in different experiments at distances of 42, 60, or 80 recipient used. In contrast, t’he rate of incm varied 1+ 10%. They extrapolated to a activation of complete transduction devalue of 3 * 1 at zero ultraviolet dose pended on the recipient employed. Transduction to wild t,ype of multisite mutants (e.g., Fig. la, lb, 2a, and 5a) as previously noted by Prell (1960). Preadsorption of was more sensitive when the size of the phage to bacteria at 37” before dilution and genetic defect in the recipient was greater. plating by the standard agar layer tech- Complete transduction of single-site mutanique did not significantly influence the tions in different bacterial gene regions exhibited similar inactivation rates, approxrate of inactivation. (Table Garen and Zinder (1955) and Prell imately one-fifth that of infectivity 1). When complete transductions were as(1961) found an increased number of lytic responses after infection of sensitive bac- sayed on t,he enriched minimal or on amino teria by lightly irradiated P22; at higher acid pool media, all inactivation rates were lowered by a constant amount of about 0.04. doses the lytic activity was inactivated exponentially. These results were confirmed. The host-killing activity differed for the The Increase in Transduction Frequencies at Low Doses of Ultraviolet Light same phage preparation used at different multiplicities, however. Occasional fluctuSeveral experimental approaches were ations in the inCal rise of transduction at attempted in an effort to influence the initial low ultraviolet doses (see Figs. 1 and 5) rise in the number of complete transducmight be due to the killing effects of lightly tions, noted at low dosesof ultraviolet light. irradiated phages. Since the killer effect was By irradiation at 42, 60, and 80 cm, it was less pronounced when lysogenic recipients found that the increase was not dependent and P22 H5 transducing phage were used, on the light intensity. P22 phage, previously some of the studies on his+ abortive trans- host,-modified on either S. typhimurium 0 duction inactivation were carried out using form 13 or S. gallinarum (Garen and Zinder. this system. 1955) still exhibited the rise when irradiated and used to transduce LT2 strains. The rise Inactivation of Transduction by Ultraviostill occurred when P22-lysogenic recipients let Light were used (Fig. lb). When the recipient Typical experiments, including inactivabacteria were irradiated and transduced tion curves of phage infectivity as well as with unirradiated phage, both donor and of abortive and complete transductions, are wild-type transductions were inactivated at the same rate as the colony-forming units shown in Figs. la and lb. The inactivation (Benzinger, 1961). Storage of the phage at of abortive transduction followed one-hit 4” did not alter the ultraviolet light effects (m = 1) kinetics both for the athC-5 marker (Fig. la) and for the his recipients (Benzinger, 1961).
EFFECTS
OF
UV
ON
FIG. 1. (a) Kinetics of inactivation of phage infectivity and of abortive and complete transduct,ions (TD) to wild type of athC-5 following ultraviolet irradiation. Values on the ordinates refer to activities per milliliter of irradiated phage suspension. Phage were assayed on athC-5 by the standard dilution and agar layer-plating technique. In the transduction assays, 0.3 ml of the T2 buffer suspension of P22 phage previously grown on wildtype bacteria was mixed with 0.3 ml of a 1 X lO’/ml culture of recipient bacteria (multiplicity of infection of 2). After 6 minutes’ adsorption at 37”, O&ml aliquots were spread on duplicate enriched minimal agar plates supplemented with adenine and pantothenate (see Materials and Methods). In the exponential portion of the curve, a tenfold inactivation of infective centers was obtained for 0.93 minutes of ultraviolet treatment at 60 cm. The relative slopes of the exponential portions of the inactivation curves were: infective centers = 1, abortive transductions (TD) = 0.31, complete transductions (TD) = 0.24.
UV
(b)
DOSE
Kinetics of inactivation of phage infectivity and of abortive and complete transductions (TD) to wild type of histidine-requiring mutants, hisF42(P%?) and hisE,P.A,H,B,C,D-7~Z(P~d). Values on the ordinates refer to infective centers or transductions (TD) per milliliter of phage suspension irradiated at 60 cm. Phage were assayed on wildt,ype LT2 bacteria by the standard dilution and agar layer-plating technique. In the transduction assays, 0.1 ml of the treated T2 buffer suspension of wild-type P22 phage, previously grown on wildtype bacteria, was mixed with 0.3 ml of 2 X loo/ml T2 buffer suspension of P22-lysogenic nutrient
617
P22
IO6
105
I04
2 103
IO’
0 I
1
I
I
I
I
2
4
6
I3
IO
12
UV
DOSE
IN
IO’
MINUTES
IN MINUTES
broth-grown recipient bacteria (multiplicity = 0.5). After 5 minutes’ adsorption, O.l-ml aliquots were spread on duplicate plates of minimal medium containing histidine-free amino acid pool (see Materials and Methods). The broken line is the backextrapolate to zero ultraviolet dose of the his-@+ complete transduction curve. The asterisk at zero ultraviolet dose (on left ordinate) refers to the number of h&712+ abortive transductional clones before irradiation. The very slight shoulder in the his-@+ abortive transduction curve may be due to an inaccuracy in the zero time assays of transductional clones since it was not noted in over twenty similar experiments.
618
BENZINGER
AND
HARTMAN
TABLE EFFECT
OF DIFFERENT
RECIPIENTS COMPLETE
AND
F-48
(P22)
hisB-12 h&B-24 hisA -?3 h&E, F-135 hisB-22 hisF,A-703 (PRR) hisD ,G-6’9 hisF,A,H,B,C,D-162 hisE,F,A,H,B,C,D-712 712 (P22) hisG-208 hisF,A,H,B,C,D,G-644 hisE,F,H,B-612 hisB,C,D,G-67 leu-39 cysB-20 cyst-36
Single Single Single Single Single Single
2 3 5 (2) 4 (2) 6 (1) (1) 4 4 (1) 3 (2)
RATES
INACTIVATION
-
FLelative inactivat ion slope of corn P lete transduction
0.27 0.20 0.19 0.19 0.24 0.21
f f ZlZ * f f
59& 10% 10% 15% 25% 10%
Single site Single site Stable single site l-Gene multisite 1 -Gene multisite l-Gene multisite Multisite g-Gene multisite Multisite
0.22 0.18 0.25 0.32 0.28
f f f f f
10% 1570 5% 3% 157,
0.36 0.43 0.45
f zt f
5% 10% 10%
Multisite Multisite Multisite Multisite S-Gene a-Gene 2-Gene
0.65 0.45 0.45 0.65 0.31 f 0.36 0.30
4 (2)
6
-I
OF
TRANSDUCTIONS
site site site site site site
2 (3)
and
ULTRAVIOLET
Approximate size of genetic defect in recipient (Hartman, et al., 1960a, b; Ames and Hartman, 1962)
2 3 2 10 (10) and
1
THE
OF ABORTIVE
Iumber of inacti ation curves ana rzed for complete ransduction and, in parentheses, abortive transductions
Recipient
cys-7 leu-128 leu-21 athC-5 hisD-1 hisF-4.2
ON
multisite multisit#e multisite
0.32
zk 10% -
0.95(3), 0.70 (5), 0.94(3) 0.50*, 0.55(3) 0.60*, 0.44* 0.82 (3) 0.60* 0.57, 0.65
0.65
0.65 8%
-
a In the first column the abbreviations designate mutants requiring for growth adenine plus thiamine (ath), cysteine (cys), histidine (his), and leucine (ZezL). The capital letters designate the gene loci (Hartman et al., 196Oa, b) involved in the mutation, and the numbers in the genetic symbols are the isolation and stock numbers of the particular mutants. The inactivation slopes are based on the exponential portions of the inactivation curves and are expressed relative to the slope of the exponential portion of the inactivation curves for plaque formation, set equal to 1. At least six points were utilized in determining the slope in each experiment unless otherwise indicated. In such exceptional cases, the number of points used is indicated in parentheses following the figure for the relative inactivation slope. Each point is based on duplicate assays except where indicated by an asterisk, in which case triplicate assays were used. The collective data indicate a relative inactivation slope of abortive t,ransduction of his+ nf 0.60 f 2096.
At low multiplicities of infection (less than lo), the rise in transductional clones was independent of the multiplicity of infection. The peak recovery averaged 3.5 * l-fold in frequency over the unirradiated control for single-site mutants. Effects
of High Doses
of Ultraviolet
Light
When phages grown on his donor strains with incomplete genetic blocks are used to
infect other mutants, two of the possible recombinant classes, wild type and donor type, can be recovered. The result,s of one experiment, using the donor phage hid-56, are depicted in Fig. 2. After an initial exponential decline of both recombinant types, the inactivation rates decrease steadily. When the data of Fig. 2 are replotted using ult,raviolet dose as the abscissa and l/v’s (where S is survival) as the ordinate (Ru-
EFFECTS
OF UV ON P22
FIG. 2. (a) Kinetics of inactivation of phage infectivity and of wild-type and of donor-type transductions (TD). Values on the ordinates refer to activities per milliliter of irradiated P22 phage suspension. Phage (7.2 X lO’/ml) were assayed on hisE,F-135 bacteria by the standard dilution and agar layer-plating technique. In the transduction assays, P22-sensitive hisE, F155 bacteria were infected with a multiplicity of 2 donor phage. Large colonies on the transduction assay plates of minimal agar were scored as wild type (h&135+ his-56’) and small colonies were scored as donor type (his-135’ his-56) transductions (TD). A tenfold inactivation of infectious centers was obtained with 0.67 minutes of ultraviolet light at 42 cm. (b) Replot of the data of (a). The ordinate is l/square root of the survival of donor type and of wild-type transductions (TD).
UV
DOSE
IN
MINUTES
FIG. 3. Inactivation of linkage by ult.raviolet light. The fraction of linked transductions [donor type/(donor type + 2 X wild type) = 1 -PI is plotted on a log scale versus the dose of ultraviolet light. (a) The top curve shows data for his&f2 recipient bacteria infected with phage grown on hisH-32. The P for unirradiated phage is 0.03. The slope of the curve relative to phage infectivity inactivation rate is 0.009. The bottom curve shows dat’a for hisD-18 recipient and hi.&36 phage. The initial P is 0.5, and the relative slope of the curve is 0.16. (b) The top curve shows data for his.63 bacteria infected with phage grown on hi&-M. The initial P is 0.18. The relative slope is 0.018. The bottom curve shows data for hisD-f recipient and hisE-145 phage. The initial P is 0.41, and t,he relative slope is 0.12.
619
620
BENZINGER
.50
-
1235
134X56 l
i35xt.6.
AND
135x56 l
.
IX
14s
HARTMAN
(Figs. 3a, 3b, and 4). The rapid decrease in linkage could not be accounted for by the different target sizes or donor and wildtype transducing segments. Exceptional Behavior of his-24
In confirmation of Hartman et al. (1960b), single-site mutant hisB-24 yielded 34 times as many wild type recombinants with markers located to either side of it than did hisB-12, with which hisB-24 failed to recombine to produce wild type. This effect was independent of the distance between the markers and occurred whether .24x 32 hisB-24 was used as donor or as recipient. When hisB-24 was transduced with ultra.02 violet-irradiated phage grown on hisB-32, .02 .06 .I0 .I4 donor type recombinants were inactivated SLOPE OF LOG at the expected rate (Fig. 5b) while wild[(I-S )/INFECTIVE CENTERS] FIG. 4. Relation between the map distance be- type transductions were uniquely resistant (Fig. 5a). Mutant hisB-12 tween two linked his markers and the rate of to ultraviolet was used as recipient for the same phage disruption of linkage by ultraviolet light. The and showed normal behavior (Figs. 5a and ordinate indicates the probability of independent transduction (P) obtained with unirradiated 5b). phage. The abscissa gives values for the rate of A similar disparate ultraviolet resistance linkage disruption (obtained from plots such as of the wild-type (single) recombinant class those in Figs. 3a and 3b) divided by the rate of only was noted when hid?-211 and hisB-56 inactivation of infective centers. Thus, the values were used as donors and his-24 the recipient. on the abscissa are relative values. Each point When phages grown on hisB-24 (which is in the figure represents the results of one experiable to grow on histidinol) were used in ment with the pair of his markers indicated. Each relative slope was calculated from the best recombination experiments with histidine mutants unable to grow on histidinol, the straight line fit to at least five experimental points. In each case, the stock number of the his same anomalous behavior was observed recipient bacteria is followed by the stock numonly for t.he wild-type recombinant class. ber of the mutant bacteria upon which the phage In all cases, the disruption of linkage conwas grown. tinued uninterrupted over the entire ultraviolet dose range investigated, in contrast pert and Goodgal, 1960)) a straight line to other two-point tests where exponential is obtained for the heavier doses (Fig. 2b). linkage disruption ceased after the number The bend in the curve on a semilog plot of complete transductions had returned to is noticeable sooner when point mutants the level of unirradiated phage preparations. or small multisite mutants are used as reConversion of Abortive to Complete Transcipients (e.g., Fig. lb). duetions Disruption of Linkage The back extrapolates of the complete Exponential decreases in linkage values were observed at low doses of ultraviolet light, while the recovery of complete transductional clones remained at or above the value obtained for unirradiated phage. The rate of linkage disruption was proportional to the initial map distances between the two bacterial markers involved in the cross
transduction inact.ivation curves (e.g., Figs. la and lb) led to values (m) which averaged about 10-11. The values were quite variable in different experiments. Some representative data are shown in Table 2. The ratio of abortive to complete transductions for most unirradiated phage stocks was also about IO. Where wild type and donor type
EFFECTS
621
OF UV ON P22
b
\
12 16 20 i UV
DOSE
IN
MINUTES
0
4 UV
8 DOSE
I2 16 20 24 IN
MINUTES
FIG. 5. (a) Inactivation kinetics for phage infectivity and for wild type (I&74’ his42’ anti his-Id+ his-%+) recombinant formation, produced by infection of hi&24 and hi&-12, respectively, with P22 phage grown on hisH-32. Values on the ordinates refer to plaque-forming units (infective centers) and complete transducing particles (TD) per milliliter of irradiated phage suspension. In the exponential portion of the infective centers curve, a tenfold inactivation of infective centers was obtained for 4.7 minutes of ultraviolet treatment at 80 cm. The multiplicity of infection of hisI%24 was 0.3, that of hi&lb was 0.2. (b) Same experiment as that recorded in Fig. 5a. Inactivation kinetics for formation of donor type (his-24+ his-%? and his-12’ his-%‘) recombinant clones from hi&24 and his&l%‘, respectively.
transductions were assayed separately (Figs. 2 and 5) the back extrapolates for the wild-type transduction curves gave values much higher than 10-11. This is a reflection of the initial disruption of linkage (see Fig. 4 and Discussion).
(3) The sensitivity to ultraviolet light of a particular portion of a P22 phage genome or of bacterial (SalmoneZZa) genetic material contained in t,ransducing particles is proportional to its physical size. Thus, the two genetic materials are considered to be comparable in ultraviolet sensitivity. InDISCUSSION herent in this assumption is the proposition Some assumptions underlying several of that’ the “homology” suggested for P22 the conclusions drawn below are: (1) The phage and its host by Garen and Zinder processes of adsorption and injection by (1955) is the same in transducing particles P22 particles are extremely resistant to as it is in infectious phage particles. The base ultraviolet irradiation (Benzinger, 1961) compositions of phage and host DN4 are and thus play at most a minor role in the very similar (Zinder, 1955). PhotoreacCvainact,ivations described. (2) Transducing tion data (Benzinger, 1961) show that inparticles in P22 lysat,es contain the same fectivity, abortive transduction, complete mean amount of DNA as do P22 infective transduction, and linkage disruption all exparticles. This content,ion is supported by hibit similar photoreactive sectors. Finally, the density gradient centrifugation data of nitrous acid inactivation data (Adye, 1962) Sheppard (1962) and the Pz2 and S-ray show the same relative cross sections for inactivation data of Hartman and Kozinski inactivation as noted below. (1962) and Takebe and Hartman (1962). On the basis of the above assumptions,
622
BENZINGER TABLE
AND
2
VALUE OF m FOR ULTRAVIOLET INACTIVATION OF COMPLETE TRANSDUCTION COMPARED TO THE RATIO OF ABORTIVE TO COMPLETE TRANSDUCTION FOR UNIRRADIATED WILD TYPE P22” -7 BackRatio of xtrapolate 0 f/ abortive to Mutant and lysate complete complete ransduction 1:ransduction curve &C-b (X) P22 lysate
wild-type 1
7 9 18 12 10 37 6
hisD-I
wild-type 2
10 10
hisA-
wild-type 2
5 15
(X) P22 lysate (X) P22 lysate
hisB-22 (X) wild-type
12 10 10 10 11 10 10
-
4 12
0 In the first column the abbreviations designate mutants requiring for growth adenine plus thiamine (&C-J) (Ozeki, 1956) or histidine (hisD-I, hi&b, and hi&-22) (Hartman et al., 1960a, b). The genetic tests are indicated listing the recipient strains first and then the wild-type P22 lysate. The data are grouped according to individual lysates because the initial ratio (for unirradiated phage) of abortive to complete transducing particles varies from lysate to lysate (Hartman et al., 196Oa). The second column lists the multiplicity of hits (m = back extrapolate of a complete transduction inactivation curve to the ordinate divided by the value obtained for unirradiated phage) required for inactivation of complete transductions. The back extrapolates (cf. Fig. lb) are drawn from curvea based on at least six experimental points. The third column lists the ratio of the number of abortive transductions to the number of complete transductions as determined by duplicate assays of the unirradiated P22 lysate. P22 lysate
2
the exponential rate of inactivation of plaque formation by ultraviolet treatment of P22 suspensions is set, by definition, equal to 1.0, and all inactivation rates (assumed “target sizes”) are expressed relative to this value.
HARTMAN
Possible Conversion of Abortive ple te Transducing Elements
to Com-
Abortive transductions of histidine mutants are inactivated exponentially at a rate of about 0.65, regardless of the recipient (single-site or multisite mutants) used in the assay (Fig. lb and Table 1). This finding indicates that the entire his region is eliminat,ed as a unit from abortive transduction. It is possible that the inactivation of abortively transducing elements is due to the gaining of an ability, following ultraviolet damage, to undergo recombination with the genome of recipient bacteria; i.e., potential abortive transductions are converted to potential complete transductions. If the converting hits take place only in DNA strictly homologous with that of the host (i.e., in bacterial genes), the bacterial gene content of his+ transducing elements is at least 65% of the total DNA of the particle. For elements carrying ath+, the bacterial gene content is at least 32% (Fig. la, Table 1). The converting hits could be akin to those which stimulate recombination in other systems (Jacob and Wollman, 1955; Roman and Jacob, 1958). The exponential portions of the inactivation curves for complete transduction often extrapolate to a value of about 10 at zero ultraviolet dose (Table 2). This value coincides with the ratio of abortive to complete transductions observed when unirradiated phage is used (e.g., Fig. lb, Table 2). Furthermore, an increased number of complete transductions is found at low ultraviolet doses. These observations lead to the hypothesis that an abortive transducing element is converted by an ultraviolet hit to a complete t,ransducing segment which then is converted to an inactive segment by an additional ultraviolet hit. Theoretical kinetics of a sequenceof two first-order reactions fit the observed values found for the rise in complete transductions, both in the dark and under photoreactivating conditions (Benzinger, 1961). Inactivation of Complete Transductions and the Physical Size of the his Region The inactivation of complete transduction of the multisite mutant, his-712,
EFFECTS
OF UV ON P22
proceeds at a rate of about 0.45. In contrast, transduction of his single-site mutants is inactivated at a rate of about 0.20 (Fig. lb, Table 1). Thus, although single-site mutants are presumably altered in one, or at most a few, nucleotide pairs, sensitivity of the wild-type segment necessary for recombinant formation represents a sensitivity proportional to a considerable fraction of the sensitivity of the total phage genome. The sensitivity of complete transductions to high dosesof ultraviolet light (Figs. lb and 2a) indicates that only extremely rarely are a few nucleotide pairs involved in eliciting a recombinational event, i.e., the production of wild-type recombinants most often involves a genetic region of considerable size. A “spreading effect” of ultraviolet light damages appears unlikely in view of the quite analogous results obtained with nitrous acid treatments by Adye (1962). The approximate value of 0.20, then, may be considered to consist of a segment of mean size comprising not only the genetic material actually entering the recombinant genome, but also a region in which synapsis and recombinational events take place. The difference in the rates of inactivation of point mutants and long multisite mutants can be used to obtain an approximation of the size of the his region relative to the total gentic material carried by the transducing particle (E 1). The difference in inactivation rates for his-152, a multisite mutant encompassing 6 of the 8 histidine genes (Ames and Hartman, 1962) as opposed to that for single-site mutants is 0.43 minus 0.20, or 0.23. The parallel calculation for the 7-gene multisite mutant, his-712, is 0.45 - 0.20, or 0.25 (data in Table 1). This value is probably only an approximation of the size of the his region since, owing to ‘(loop” formation of nonhomologous regions, the segmentsrequired for successful synapsis of elements involving multisite mutations probably would be larger, on the average, than those utilized in synapsis preceding complete transduction of single-site mutants. For example, multisite mutants inhibit recombinations in adjacent regions (Hartman, 1956; Hartman et al., 1960b). This larger mean pairing region also would presumably be ultraviolet sensitive; there-
fore, a fraction of the difference, 0.20, would belong not to the his region, but to outside genetic material necessary for pairing with the multisite mutation but not with the single-site mutation. Thus, 0.25 is considered a maximum estimate of the relative size of the his region. An independent estimate of the size of the his region comes from the study of the rapid initial inactivation of linkage by ultraviolet (Fig. 3). Joint transductions of two linked markers are called “donor type” recombinants. When only one of the markers carried by the donor is integrated,- a wild-type recombinant may be formed. P is defined as the probability of the separate integration of two markers, i.e.: 2 X the number of wild-type recombinants/(2 X the number of wild-type recombinants + the number of donor-type recombinants). The number of wild-t,ype recombinants is multiplied by 2 because the double-mutant recombinant is not detectable. With r’ thus defined, 1 P becomes: the number of donor-type recombinants/(the number of donor-type recombinants + 2 X the number of wildtype recombinants). 1 - P thus expresses the probability of joint integration of two markers. When ultraviolet dose is plotted against the logarithm of 1 - p for a number of different pairs of markers, points approximately fitted by a straight line are obtained (Fig. 3). The 1 - p inactivation rates are not accounted for by the difference in ultraviolet senstitvity of the two recombinant classes expected from the study of ultraviolet inactivation of transduction of singleand multisite mutants by wild-type phage. Indeed, at higher ultraviolet doses, when the number of complete transductions declines below the level obtained for unirradiated phage, the rapid disruption of linkage ceases. If the values for the slopes of the linear portions of the plots of log 1 - p against ultraviolet dose (Fig. 3) are expressed in terms relative to the phage infectivity inactivation rate and then plotted against the p values obtained in the same cross with unirradiated phage, a line extrapolating near the origin can be drawn (Fig. 4). Data of Adye (1962) on disruption of linkage by nitrous acid treatment of P22 also conform
624
BENZINGER
AND HARTMAN
to the plot shown in Fig. 4. The abscissa represents the quotient of ultraviolet hits which disrupt linkage divided by ultraviolet hits that inactivate infectivity. If P for two markers at the very opposite ends of the his region is chosen, the corresponding point on the abscissa should represent the relative size of the his region. Since the average P value for the most distant his marker pairs is 0.67 (Hartman, Stahl, and Adye, personal communication), the minimum size of the his region equals 0.17 f 0.04, as determined from Fig. 4. A critical assumption is that the genetic distance across the his region, 0.67, is a true estimate of its physical size. The validity of this assumption rests on a random distribution of crossovers along the his region and adjacent segments of the transducing fragment. Since map distances between mutants across the his region show no discontinuities (Hartman et al., 1960b) and the absolute transduction frequency for markers, including short multisite mutations, at either end of the his region is equal to that of markers located in its middle, this assumption appears justifiable. A final assumption predicates that recombinational events are of infrequent occurrence along the bacterial genes in the transducing element, i.e., double exchanges within the his region are rare. However, measurements of recombination frequencies between various pairs of his mutations located close to one another, as opposed to those further apart, suggest that recombinational events are very frequent along the element, an average of at least 1.3 events taking place within the his region (Hartman, Stahl and Adye, unpublished). Therefore, the current estimate of the size of the his region (i.e., 0.17) is considered an approximation of its minimal size. Mutant his-152 exhibits a transduction frequency with unirradiated P22 which is about 25% that for single-site mutations (Hartman et al., 1960b). For the his-712 mutant, the relative frequency is about 15%. One of these mutations (his-152) involves 6 of the 8 his genes; the other mutation involves 7 of the 8 genes (Ames and Hartman, 1962). From these reductions in transduction frequency, it is inferred that the majority (75-85 % of the class of wild-type transduc-
tional clones which ordinarily would appear) are not recovered owing to failure to replace completely the mutated region of the recipient. The transductions which do appear are formally analogous to donor-type transductions except that multiple exchanges within the his region cannot occur. The reduction in transduction frequency of 7585% thus infers a value for p of 0.75 to 0.85. The estimate for the size of a region with this p value (cf. Fig. 4) is about 0.20. The relative “target sizes” for the inactivation of complete transduction to wild type of multisite mut.ants (Table 1) suggest that the genetic defects in his-712, his-63, and his-644 do not extend very far outside of the known histidine region (Hartman et al., 1960b; Ames and Hartman, 1962). On the other hand, the large target sizes for inact,ivation of transductions of his-612, his-57, and his-203 indicate that the genetic defect in the former extends about four genes to the “left,” of the histidine region while the genetic defect,s in the latter two mutants extend about 12 genes to the “right” of the histidine region. il Coding Ratio for the his Region The above estimates for the size of the his region, as well as others discussed by Benzinger (1961)) allow us to assign an average relative value of approximately 0.20 to the his region. Eight gene loci (A-H) comprise the his region (Hartman et al., 1960a,b; Ames and Hartman, 1962). The molecular weights of some of the proteins of the histidine biosynthetic pathway have been determined by Martin and Ames (1961). They found that L-histidinol dehydrogenase (gene D) , imidazoleacetol phosphate transaminase (gene C), and phosphoribosyl ATP pyrophosphorylase (gene G) had molecular weights of roughly 75,000, 68,000, and 170,000, respectively. The latter enzyme at present is considered to be a dimer (Martin, 1962). The enzyme elicited by gene A and the presumed monomer protein elicited by gene B (imidazoleglycerol phosphate dehydrase-L-histidinol phosphate phosphatase) each have molecular weights of roughly 70,000 (Ames, personal communication). Seven of eight histidine genes are of approximately equal
EFFECTS
OF
Al
UV
ON
625
P22
If about one-fifth of these comprise the his 12,600 nucleotide region, approximately pairs code 4000 amino acids. A coding ratio of about 3 is thus obtained (cf. Ames and Hartman, 1962). This estimate is based also on the assumption that the great majority of the nucleotides of the his region are used in coding for the amino acids of the histidine biosynthetic enzymes.
Aberrant Recombinational Behavior of his24 HisB-Z.$ has been shown to exhibit ab-
f32
FIG. 6. Schematic representation of one type of molecular change in the genetic material (mutation) which might lead to expression of a mutant phenotype and, simultaneously, affect recombination in adjacent regions. In this model, the mutation at the base pair indicated results in a configuration allowing either a greatly increased or a greatly decreased tendency for intrastrand hydrogen bonding by complementary base pairing. This altered propensity for “loop” formation within single strands of the DNA molecule is suggested as critically affecting some stage in the recombination process.
sizes; the eighth genemay be slightly smaller (Ames and Hartman, 1962). It seems reasonable to assume that seven of the genes control the structural formation of seven protein monomers with an average molecular weight of 70,000 whereas the eighth gene controls a protein monomer of 30,000 molecular weight. This is an aggregate molecular weight of 5.2 X 1Oj. The average molecular weight of amino acids making up proteins is about 130; therefore, the positioning in proteins of approximat’ely 4000 amino acids appears to be determined by the DNA of the his region. The total number of nucleotide pairs in P22 is about 63,000 (Garen and Zinder, 1955).
normally high recombination frequencies with regions adjacent to it (Hartman et aE., 1960b). The ultraviolet inactivation kinetics of transduction to his+ in two-factor crosses involving h&B-24 reveal a unique resistance to ultraviolet light (Fig. 5a). These and other data pertaining to the properties of his-24 are summarized elsewhere (Hartman, 1962). The type of molecular change involved in t’he case of his-24 and a possible way in which this change leads to the observed properties of the his-24 allele are suggested in Fig. 6 and described in the legend to that, figure. The model makes two predictions, namely, that mutations affecting recombination in this manner will usually not involve “transit,ion” mutations and that the primary mutation might lead to greatly increased frequency of multisite mutations in its vicinity in later generations. The data at present are too scanty to determine whether either prediction is fulfilled. ACKNOWLEDGMENTS
genetic
We wish to thank Drs. Sol H. Goodgal and Claude S. Rupert for suggesting some of the ultraviolet irradiation experiments. The energetic technical assistance of Mr. William F. Kosch III is gratefully acknowledged. This work, in part, was supported by Research Grant E-1650 of the National Institute of Allergy and Infectious Diseases, United States Public Health Service. REFERENCES
ADYE, J. (1962). duction
Effects by Salmonella
627-632. AMES, B. N., and enzymes, and biosvnthesis.
of nitrous acid on transphage P22. Virology 18, P. E. mechanisms Molecular
HARTMAX,
control ITS “The
(1962). Genes, in histidine Basis of Neo-
626
BENZINGER
AND
plasia,” M. D. $nderson Hospital Symposium, pp. 322-345. ARBER, W. (1958). Transduction des characteres gal par le bacteriophage lambda. Arch. Sci. (Geneva) 11,259338. BENZINGER, R. (1961). Studies on the mechanism of transduction by bacteriophage of P22 of S. typkimurium. Ph.D. Thesis, The Johns Hopkins University, Baltimore, Maryland, COETZEE,J. N., and SACKS, T. G. (1960). Transduction of streptomycin resistance in Proteus mirabilis. J. Gen. Microbial. 23, 445-455. GARES, A., and ZINDER, N. D. (19551. Radiological evidence for partial genetic homology between bacteriophage and host bacteria. Virology
1,347-376.
HARTMAN, P. E. (1956). Linked loci in the control of consecutive steps in the primary pathway of histidine synthesis in Salmonella typhimurium. In “Genetic Studies with Bacteria,” pp. 35-62. Carnegie
Inst.
Wash.
Publ.
No. 612.
HARTMAN, P. E. (1962). Methodology in transduction. Symposium on Methodology in Basic Genetics (W. J. Burdette, editor), in press. HAarnrAN, P. E., and KOZINSKI, A. W. (1962). Effects of P” decay on transduction by Salmonella phage P22. Virology 17, 233-244. HARTMAN, P. E., HARTMAN, Z., and SERMA~, D. ( 1960a). Complementation mapping by abortive transduction of histidine-requiring Salmonella mutants. J. Gen. Microbial. 22, 354-368. HARTMAN, P. E., LOPER, J. C., and QERMAN, D. (1960b). Fine structure mapping by complete transduction between histidine-requiring Salmonella mutants. J. Gen. Microbial. 22, 322-353. HERSHEY, A. D., and ROTMAX, R. (1949). Genetic recombination between host-range and plaquetype mutants of bacteriophage in single bacterial cells. Genetics 34, 44-71. HOLLOWAY, B. W., and MONK, M. (1959). Transduction in Pseudomonas aeruginosa. Nature 184, 1426. HOLLOWAY, B. W., and MONK, M. (1961). Influence of ultra-violet irradiation on general transduction in Pseudomonas aeruginosa. In “Radiobiology” (P. L. T. Ilbery, ed.), Proc. 3rd Australian
Conf.
Radiobiol.,
Sydney,
Aug.
15-18,
1960, pp. 231-237. Butterworths, London. JACOB, F., and WOLLMAN, E. L. (1955). Etude genetique d’un bacteriophage temper+ d’Escherichia coli. III. Effet du rayonnement ultraviolet sur la recombinaison gCnCtique. Ann. inst. Pasteur g&724749. KELLEXBERGER, G., ARBER,
W., and KELLEICBERGER,
HARTMAN
E. (1959). Eigenschaften uv-bestrahlter APhagen. Z. Naturjorsch. 14b, 6W629. LATARJET, R., MORENNE, P., and BERGER, R. (1953). Un appareil simple pour le dosage des rayonnements ultraviolets Cmis par les lampes germicides. Ann. inst. Pasteur 85, 174-184. LURIA, S. E., ADAMS, J. N., and TING, R. C. (1960). Transduction of lactose-utilizing ability among strains of Escherichia coli and Shigella dysenteriae and the properties of the transducing particles. Virology 12, 348-390. MARTIN, R. G. (1962). The first enzyme in histidine biosynthesis : The nature of feedback inhibition by histidine. J. Biol. Chem. 237, in press. MARTIX, R. G., and AMES, B. N. (1961). A method for determining the sedimentation behavior of enzymes ; application to protein mixtures. J. Biol. Chem. 236, 1372-1379. OZEKI, H. (1956). Abortive transduction in purinerequiring mutants of Salmonella typhimurium. In “Genetic Studies with Bacteria,” pp. 97-106. Carnegie PRELL, H.
Inst.
Wash.
Publ.
No.
612.
H. (1960). Ueber die Mehrfachreaktivierung des Infektionsund Lysogenierungsvermoegens des Phagen P22 von Salmonella typhimurium nach UV-Bestrahlung. Arch. Mi-
krobiol. 35, 384390. PRELL, H. H. (1961).
Analyse des Lysogenisierungsvorganges im System Salmonella typhimuriumphage P22 mit Hilfe von UV-Bestrahlung. Arch. Mikrobiol. 38, 171-200. ROMAN, H., and JACOB, F. (1958). A comparison of spontaneous and ultraviolet-induced allelic recombinations with reference to the recombination of outside markers. Cold Spring Harbor Symposia Quant. Biol. 23, 155-159. RUPERT, C. S., and GOODGAL, S. H. (1960). Shape of ultraviolet inactivation curves of transforming deoxyribonucleic acid. Nature 185, 556-557. SHEPPARD, D. E. (1962). Density gradient centrifugation of bacteriophage P22. Virology 17, 212214. TAKEBE, H., and HARTYAN, P. E. (1962). Effects of X-rays on transduction by Salmonella phage P22. Virology 17,295-300. WILSON, D. E. (1960). The effects of ultraviolet light and ionizing radiation on the transduction of Escherichia coli by phage Pl. Virology 11,533-546. ZINDER, N. D. (1955). Bacterial transduction. J. Cellular Comp. Physiol. 45, Suppl. 2, 23-49. ZINDER, N. D. (1958). Lysogenization and superinfection immunity in Salmonella. Virology 5,
291-326.
18, 614-626
Effects
(1962)
of Ultraviolet ROLF
Department
Light
BENZINGER
of Biology,
The
on AND
Johns
PHILIP
Hopkins
Accepted
Transducing
P22
E. HARTMAN
University,
August
Phage
Baltimore
18, Maryland
S, 1962
The effects of ultraviolet irradiation on suspensions of P22 particles are examined. Evidence is presented which indicates that a single ultraviolet hit on an abortively transducing element enables it to form a complete transduction, i.e., to undergo recombination with the chromosome of recipient bacteria. Thus, abortive transductions are rapidly inactivated while the number of complete transductions increases. During the conversion of abortive transductions to complete transductions, jointly transduced markers become less tightly linked. The rate of linkage disruption is proportional to the genetic map distance between linked markers and allows an estimate to be made for the physical size of the histidine (his) region of the Salmonella chromosome. The rates of inactivation of complete transductions are proportional to the sizes of the genetic defects in the recipients, allowing a second estimate of the size of the his region to be made. The eight-gene his region comprises an amount of DNA equivalent to approximately 8 X 10“ molecular weight, i.e., one-fifth that contained in a normal P22 phage particle. The coding ratio for the his region is about three nucleotide pairs per amino acid in the eight proteins whose structure it controls.
an initial increase at low ultraviolet doses, and inactivation at higher doses. Ozeki (personal communication, 1956) observed that the abortive transduction activity of P22 for the athC-5 recipient decreased exponentially with ultraviolet dose. The inactivation rate of complete transduction (after the initial rise) was considerably less than that of abortive transduction. Further observations on the ultraviolet inactivation kinetics of abortive and complete transduction, the inactivation of linkage, and attempts to characterize the nature of the process causing the initial rise in complete transductions will be reported below. These will be used to draw inferences concerning the molecular size and structure of genetic regions carried by transducing particles in P22 lysates.
INTRODUCTION
The action of ultraviolet irradiation on complete transduction by P22 has been investigated by Garen and Zinder (1955). They noted an increased number of transductions at low ultraviolet doses, although phage infectivity had been reduced to as little as 1O-4 of the zero time value. An increase in transduction frequency following ultraviolet irradiation of free phage has also been observed for coliphage h (Arber, 1958), for Pl (Luria et aZ., 1960; Wilson, 1960)) for Pseudomonas aeruginosa phage (Holloway and Monk, 1959, 1961), and for Proteus mirabilis phage (Coetzee and Sacks, 1960). The increased transduction frequency is not specifically induced by ultraviolet alone; nitrous acid produces the same effect with P22 (Adye, 1962). The lytic ability of P22 (Garen and Zinder, 1955; Prell, 1961) and the number of cells doubly lysogenized by x phage (Kellenberger et al., 1959) also show 1 Present Biochemie,
address: Max-Planck Munich 15, Germany.
Institut
MATERIALS
AND
METHODS
Bacterial strains. All strains are from the collection of Dr. M. Demerec, Brookhaven National Laboratory, and are derived
fiir 614
from S. t~yphimurium strains LT2 or LT7. Methods for obtaining most of these mutants have been reported elsewhere (Hartman et al., 1960b). Salmonella typhimurium 0 form 13 and S. gallinarum were donated by Dr. N. D. Zinder. Escherichia coli strain B was used as T2 phage indicator. Phages. PLT22 (P22) and T2 phage suspensions were prepared, assayed, and stored as detailed previously by Hartman (1956). The H5 (VW) mutant has been described by Zinder (1958). Media. Difco nutrient broth enriched with 0.2% dextrose was normally used as a complete medium. T2 phage was assayed on the Difco tryptone bottom Iayer agar of Hershey and Rotman (1949) but without citrate. Minimal medium, enriched minimal medium, and enriched minimal supplemented with L-histidinol were prepared as described by Hartman et al. (1960b). Calcium pantothenate and adenine were added to enriched minimal medium to permit visual detection of athC-5+ abortive transductions as detailed by Ozeki (1956). To enlarge the colonies from abortive transduction to his+, minimal medium was supplemented with a final concentration of 80 pg L-glutamic acid per milliliter or with a pool of amino acids (20 pg/ml each) lacking histidine. Ultraviolet irradiation. A 15-watt General Electric “germicidal” lamp was used in all studies. After allowing the lamp to warm up for 30 minutes, no more than 10 ml of phage in saline at a concentration below lOlo plaque-forming units per milliliter were irradiated in a flat-bottomed lOO-mm petri dish (which was continually agitated mechanically). At this phage concentration, only 5% of the incident 253.7rnp light was absorbed, as measured in t,he Beckman DU spectrophotometer. To prevent photoreactivation, yellow illumination was utilized for all operations during and after irradiation and the plates were incubated in the dark for at Ieast 12 hours. Biological calibration by means of T2 phage inactivation and reference to the standard curve of Latarjet et al. (1953) gave a dose rate of 13.8 ergs/mm2/sec at 60
cm. In this calibration, six experiments with five p0int.s per curve to 1O-4 survival gave close agreement. Physical calibration with a General Electric watt.meter gave a value of 12.5 ergs/mm2/sec at 60 cm. The inactivation of the biological activities studied will be represented graphically by plotting the logarithm of survivors on the ordinate against ultraviolet dose on the abscissa. In order to compare the results of different experiments, the inactivation of phage infectivity is used as a standard. Thus, inactivation rates are expressed relative to the phage infectivity slope which = 1, by definition. Transduction tests. Recipient bacteria were prepared as follows: log or stationary phase nutrient broth cultures were centrifuged at 3000 g for 15 minutes and the pellet was resuspended in saline. When clearly visible his+ abortives were desired, the pellet was washed t,hree times with distilled water to reduce residual growth of the recipients on t,he glutamic acid medium. This treatment has no effect on the viable count (C. Kirchner, personal communication). After 10 minutes at 37” were allowed for phage adsorption, 0.05 to 0.3 ml samples were spread evenly on thick agar plates which had been dried for at least 24 hours at 37”. These plat.es were incubated at 37” for periods ranging from 18 to 60 hours. Complete and abort,ive transductions generally required about 48 hours to develop on minimal medium and 18 hours on glutamic acid medium. Joint transduct’ions (“donor type t,ransductional clones,” Hartman, 1956) were clearly visible after 60 and 48 hours on the respective media. Complete t,ransduction and athC-5+ abortive transduction assays varied *20% in replicate platings. Abortive transductions to his+ were counted under a Bausch and Lomb “Stereozoom” microscope at, a magnification of 30x, employing 10 x wide-field eyepieces. At least 100 abortive colonies were counted in a known area no smaller than one-fiftieth of the plat’e. The count obt,ained was then used to calculate the total number of abort.ive transductions on the plate. Assays were carried out, either in du-
616
BENZINGER
AND HARTMAN
plicate or in triplicate; the precision of the method in replicate counts was -1150%. For each inactivation curve of transduction, the titers of unirradiated phage and recipient bacteria were determined. The multiplicity of infection was generally kept below 1 and seldom was above 5. Each experiment was accompanied by a control with uninfected bacteria to determine the number of spontaneous reversions to wild type in the culture.
tested (Fig. lb). Figures la and lb also show a net increase in recovery of transductional clones at low dosesof ultraviolet light. Some features of this phenomenon are described in the following section. Table 1 depicts abortive and complete transduction inactivation rat’es for a number of auxotrophs. The rates were calculated from the exponential portions of the inactivation curves to 1% survival isee Figs. la and lb). Abortive transduction generally was more sensitive to ultraviolet than was RESULTS complete transduction. For the his region, the rate of abortive transduction inactivaThe slopes of the curves for inactivation tion was not significantly dependent, on the of phage infectivity obtained in different experiments at distances of 42, 60, or 80 recipient used. In contrast, t’he rate of incm varied 1+ 10%. They extrapolated to a activation of complete transduction devalue of 3 * 1 at zero ultraviolet dose pended on the recipient employed. Transduction to wild t,ype of multisite mutants (e.g., Fig. la, lb, 2a, and 5a) as previously noted by Prell (1960). Preadsorption of was more sensitive when the size of the phage to bacteria at 37” before dilution and genetic defect in the recipient was greater. plating by the standard agar layer tech- Complete transduction of single-site mutanique did not significantly influence the tions in different bacterial gene regions exhibited similar inactivation rates, approxrate of inactivation. (Table Garen and Zinder (1955) and Prell imately one-fifth that of infectivity 1). When complete transductions were as(1961) found an increased number of lytic responses after infection of sensitive bac- sayed on t,he enriched minimal or on amino teria by lightly irradiated P22; at higher acid pool media, all inactivation rates were lowered by a constant amount of about 0.04. doses the lytic activity was inactivated exponentially. These results were confirmed. The host-killing activity differed for the The Increase in Transduction Frequencies at Low Doses of Ultraviolet Light same phage preparation used at different multiplicities, however. Occasional fluctuSeveral experimental approaches were ations in the inCal rise of transduction at attempted in an effort to influence the initial low ultraviolet doses (see Figs. 1 and 5) rise in the number of complete transducmight be due to the killing effects of lightly tions, noted at low dosesof ultraviolet light. irradiated phages. Since the killer effect was By irradiation at 42, 60, and 80 cm, it was less pronounced when lysogenic recipients found that the increase was not dependent and P22 H5 transducing phage were used, on the light intensity. P22 phage, previously some of the studies on his+ abortive trans- host,-modified on either S. typhimurium 0 duction inactivation were carried out using form 13 or S. gallinarum (Garen and Zinder. this system. 1955) still exhibited the rise when irradiated and used to transduce LT2 strains. The rise Inactivation of Transduction by Ultraviostill occurred when P22-lysogenic recipients let Light were used (Fig. lb). When the recipient Typical experiments, including inactivabacteria were irradiated and transduced tion curves of phage infectivity as well as with unirradiated phage, both donor and of abortive and complete transductions, are wild-type transductions were inactivated at the same rate as the colony-forming units shown in Figs. la and lb. The inactivation (Benzinger, 1961). Storage of the phage at of abortive transduction followed one-hit 4” did not alter the ultraviolet light effects (m = 1) kinetics both for the athC-5 marker (Fig. la) and for the his recipients (Benzinger, 1961).
EFFECTS
OF
UV
ON
FIG. 1. (a) Kinetics of inactivation of phage infectivity and of abortive and complete transduct,ions (TD) to wild type of athC-5 following ultraviolet irradiation. Values on the ordinates refer to activities per milliliter of irradiated phage suspension. Phage were assayed on athC-5 by the standard dilution and agar layer-plating technique. In the transduction assays, 0.3 ml of the T2 buffer suspension of P22 phage previously grown on wildtype bacteria was mixed with 0.3 ml of a 1 X lO’/ml culture of recipient bacteria (multiplicity of infection of 2). After 6 minutes’ adsorption at 37”, O&ml aliquots were spread on duplicate enriched minimal agar plates supplemented with adenine and pantothenate (see Materials and Methods). In the exponential portion of the curve, a tenfold inactivation of infective centers was obtained for 0.93 minutes of ultraviolet treatment at 60 cm. The relative slopes of the exponential portions of the inactivation curves were: infective centers = 1, abortive transductions (TD) = 0.31, complete transductions (TD) = 0.24.
UV
(b)
DOSE
Kinetics of inactivation of phage infectivity and of abortive and complete transductions (TD) to wild type of histidine-requiring mutants, hisF42(P%?) and hisE,P.A,H,B,C,D-7~Z(P~d). Values on the ordinates refer to infective centers or transductions (TD) per milliliter of phage suspension irradiated at 60 cm. Phage were assayed on wildt,ype LT2 bacteria by the standard dilution and agar layer-plating technique. In the transduction assays, 0.1 ml of the treated T2 buffer suspension of wild-type P22 phage, previously grown on wildtype bacteria, was mixed with 0.3 ml of 2 X loo/ml T2 buffer suspension of P22-lysogenic nutrient
617
P22
IO6
105
I04
2 103
IO’
0 I
1
I
I
I
I
2
4
6
I3
IO
12
UV
DOSE
IN
IO’
MINUTES
IN MINUTES
broth-grown recipient bacteria (multiplicity = 0.5). After 5 minutes’ adsorption, O.l-ml aliquots were spread on duplicate plates of minimal medium containing histidine-free amino acid pool (see Materials and Methods). The broken line is the backextrapolate to zero ultraviolet dose of the his-@+ complete transduction curve. The asterisk at zero ultraviolet dose (on left ordinate) refers to the number of h&712+ abortive transductional clones before irradiation. The very slight shoulder in the his-@+ abortive transduction curve may be due to an inaccuracy in the zero time assays of transductional clones since it was not noted in over twenty similar experiments.
618
BENZINGER
AND
HARTMAN
TABLE EFFECT
OF DIFFERENT
RECIPIENTS COMPLETE
AND
F-48
(P22)
hisB-12 h&B-24 hisA -?3 h&E, F-135 hisB-22 hisF,A-703 (PRR) hisD ,G-6’9 hisF,A,H,B,C,D-162 hisE,F,A,H,B,C,D-712 712 (P22) hisG-208 hisF,A,H,B,C,D,G-644 hisE,F,H,B-612 hisB,C,D,G-67 leu-39 cysB-20 cyst-36
Single Single Single Single Single Single
2 3 5 (2) 4 (2) 6 (1) (1) 4 4 (1) 3 (2)
RATES
INACTIVATION
-
FLelative inactivat ion slope of corn P lete transduction
0.27 0.20 0.19 0.19 0.24 0.21
f f ZlZ * f f
59& 10% 10% 15% 25% 10%
Single site Single site Stable single site l-Gene multisite 1 -Gene multisite l-Gene multisite Multisite g-Gene multisite Multisite
0.22 0.18 0.25 0.32 0.28
f f f f f
10% 1570 5% 3% 157,
0.36 0.43 0.45
f zt f
5% 10% 10%
Multisite Multisite Multisite Multisite S-Gene a-Gene 2-Gene
0.65 0.45 0.45 0.65 0.31 f 0.36 0.30
4 (2)
6
-I
OF
TRANSDUCTIONS
site site site site site site
2 (3)
and
ULTRAVIOLET
Approximate size of genetic defect in recipient (Hartman, et al., 1960a, b; Ames and Hartman, 1962)
2 3 2 10 (10) and
1
THE
OF ABORTIVE
Iumber of inacti ation curves ana rzed for complete ransduction and, in parentheses, abortive transductions
Recipient
cys-7 leu-128 leu-21 athC-5 hisD-1 hisF-4.2
ON
multisite multisit#e multisite
0.32
zk 10% -
0.95(3), 0.70 (5), 0.94(3) 0.50*, 0.55(3) 0.60*, 0.44* 0.82 (3) 0.60* 0.57, 0.65
0.65
0.65 8%
-
a In the first column the abbreviations designate mutants requiring for growth adenine plus thiamine (ath), cysteine (cys), histidine (his), and leucine (ZezL). The capital letters designate the gene loci (Hartman et al., 196Oa, b) involved in the mutation, and the numbers in the genetic symbols are the isolation and stock numbers of the particular mutants. The inactivation slopes are based on the exponential portions of the inactivation curves and are expressed relative to the slope of the exponential portion of the inactivation curves for plaque formation, set equal to 1. At least six points were utilized in determining the slope in each experiment unless otherwise indicated. In such exceptional cases, the number of points used is indicated in parentheses following the figure for the relative inactivation slope. Each point is based on duplicate assays except where indicated by an asterisk, in which case triplicate assays were used. The collective data indicate a relative inactivation slope of abortive t,ransduction of his+ nf 0.60 f 2096.
At low multiplicities of infection (less than lo), the rise in transductional clones was independent of the multiplicity of infection. The peak recovery averaged 3.5 * l-fold in frequency over the unirradiated control for single-site mutants. Effects
of High Doses
of Ultraviolet
Light
When phages grown on his donor strains with incomplete genetic blocks are used to
infect other mutants, two of the possible recombinant classes, wild type and donor type, can be recovered. The result,s of one experiment, using the donor phage hid-56, are depicted in Fig. 2. After an initial exponential decline of both recombinant types, the inactivation rates decrease steadily. When the data of Fig. 2 are replotted using ult,raviolet dose as the abscissa and l/v’s (where S is survival) as the ordinate (Ru-
EFFECTS
OF UV ON P22
FIG. 2. (a) Kinetics of inactivation of phage infectivity and of wild-type and of donor-type transductions (TD). Values on the ordinates refer to activities per milliliter of irradiated P22 phage suspension. Phage (7.2 X lO’/ml) were assayed on hisE,F-135 bacteria by the standard dilution and agar layer-plating technique. In the transduction assays, P22-sensitive hisE, F155 bacteria were infected with a multiplicity of 2 donor phage. Large colonies on the transduction assay plates of minimal agar were scored as wild type (h&135+ his-56’) and small colonies were scored as donor type (his-135’ his-56) transductions (TD). A tenfold inactivation of infectious centers was obtained with 0.67 minutes of ultraviolet light at 42 cm. (b) Replot of the data of (a). The ordinate is l/square root of the survival of donor type and of wild-type transductions (TD).
UV
DOSE
IN
MINUTES
FIG. 3. Inactivation of linkage by ult.raviolet light. The fraction of linked transductions [donor type/(donor type + 2 X wild type) = 1 -PI is plotted on a log scale versus the dose of ultraviolet light. (a) The top curve shows data for his&f2 recipient bacteria infected with phage grown on hisH-32. The P for unirradiated phage is 0.03. The slope of the curve relative to phage infectivity inactivation rate is 0.009. The bottom curve shows dat’a for hisD-18 recipient and hi.&36 phage. The initial P is 0.5, and the relative slope of the curve is 0.16. (b) The top curve shows data for his.63 bacteria infected with phage grown on hi&-M. The initial P is 0.18. The relative slope is 0.018. The bottom curve shows data for hisD-f recipient and hisE-145 phage. The initial P is 0.41, and t,he relative slope is 0.12.
619
620
BENZINGER
.50
-
1235
134X56 l
i35xt.6.
AND
135x56 l
.
IX
14s
HARTMAN
(Figs. 3a, 3b, and 4). The rapid decrease in linkage could not be accounted for by the different target sizes or donor and wildtype transducing segments. Exceptional Behavior of his-24
In confirmation of Hartman et al. (1960b), single-site mutant hisB-24 yielded 34 times as many wild type recombinants with markers located to either side of it than did hisB-12, with which hisB-24 failed to recombine to produce wild type. This effect was independent of the distance between the markers and occurred whether .24x 32 hisB-24 was used as donor or as recipient. When hisB-24 was transduced with ultra.02 violet-irradiated phage grown on hisB-32, .02 .06 .I0 .I4 donor type recombinants were inactivated SLOPE OF LOG at the expected rate (Fig. 5b) while wild[(I-S )/INFECTIVE CENTERS] FIG. 4. Relation between the map distance be- type transductions were uniquely resistant (Fig. 5a). Mutant hisB-12 tween two linked his markers and the rate of to ultraviolet was used as recipient for the same phage disruption of linkage by ultraviolet light. The and showed normal behavior (Figs. 5a and ordinate indicates the probability of independent transduction (P) obtained with unirradiated 5b). phage. The abscissa gives values for the rate of A similar disparate ultraviolet resistance linkage disruption (obtained from plots such as of the wild-type (single) recombinant class those in Figs. 3a and 3b) divided by the rate of only was noted when hid?-211 and hisB-56 inactivation of infective centers. Thus, the values were used as donors and his-24 the recipient. on the abscissa are relative values. Each point When phages grown on hisB-24 (which is in the figure represents the results of one experiable to grow on histidinol) were used in ment with the pair of his markers indicated. Each relative slope was calculated from the best recombination experiments with histidine mutants unable to grow on histidinol, the straight line fit to at least five experimental points. In each case, the stock number of the his same anomalous behavior was observed recipient bacteria is followed by the stock numonly for t.he wild-type recombinant class. ber of the mutant bacteria upon which the phage In all cases, the disruption of linkage conwas grown. tinued uninterrupted over the entire ultraviolet dose range investigated, in contrast pert and Goodgal, 1960)) a straight line to other two-point tests where exponential is obtained for the heavier doses (Fig. 2b). linkage disruption ceased after the number The bend in the curve on a semilog plot of complete transductions had returned to is noticeable sooner when point mutants the level of unirradiated phage preparations. or small multisite mutants are used as reConversion of Abortive to Complete Transcipients (e.g., Fig. lb). duetions Disruption of Linkage The back extrapolates of the complete Exponential decreases in linkage values were observed at low doses of ultraviolet light, while the recovery of complete transductional clones remained at or above the value obtained for unirradiated phage. The rate of linkage disruption was proportional to the initial map distances between the two bacterial markers involved in the cross
transduction inact.ivation curves (e.g., Figs. la and lb) led to values (m) which averaged about 10-11. The values were quite variable in different experiments. Some representative data are shown in Table 2. The ratio of abortive to complete transductions for most unirradiated phage stocks was also about IO. Where wild type and donor type
EFFECTS
621
OF UV ON P22
b
\
12 16 20 i UV
DOSE
IN
MINUTES
0
4 UV
8 DOSE
I2 16 20 24 IN
MINUTES
FIG. 5. (a) Inactivation kinetics for phage infectivity and for wild type (I&74’ his42’ anti his-Id+ his-%+) recombinant formation, produced by infection of hi&24 and hi&-12, respectively, with P22 phage grown on hisH-32. Values on the ordinates refer to plaque-forming units (infective centers) and complete transducing particles (TD) per milliliter of irradiated phage suspension. In the exponential portion of the infective centers curve, a tenfold inactivation of infective centers was obtained for 4.7 minutes of ultraviolet treatment at 80 cm. The multiplicity of infection of hisI%24 was 0.3, that of hi&lb was 0.2. (b) Same experiment as that recorded in Fig. 5a. Inactivation kinetics for formation of donor type (his-24+ his-%? and his-12’ his-%‘) recombinant clones from hi&24 and his&l%‘, respectively.
transductions were assayed separately (Figs. 2 and 5) the back extrapolates for the wild-type transduction curves gave values much higher than 10-11. This is a reflection of the initial disruption of linkage (see Fig. 4 and Discussion).
(3) The sensitivity to ultraviolet light of a particular portion of a P22 phage genome or of bacterial (SalmoneZZa) genetic material contained in t,ransducing particles is proportional to its physical size. Thus, the two genetic materials are considered to be comparable in ultraviolet sensitivity. InDISCUSSION herent in this assumption is the proposition Some assumptions underlying several of that’ the “homology” suggested for P22 the conclusions drawn below are: (1) The phage and its host by Garen and Zinder processes of adsorption and injection by (1955) is the same in transducing particles P22 particles are extremely resistant to as it is in infectious phage particles. The base ultraviolet irradiation (Benzinger, 1961) compositions of phage and host DN4 are and thus play at most a minor role in the very similar (Zinder, 1955). PhotoreacCvainact,ivations described. (2) Transducing tion data (Benzinger, 1961) show that inparticles in P22 lysat,es contain the same fectivity, abortive transduction, complete mean amount of DNA as do P22 infective transduction, and linkage disruption all exparticles. This content,ion is supported by hibit similar photoreactive sectors. Finally, the density gradient centrifugation data of nitrous acid inactivation data (Adye, 1962) Sheppard (1962) and the Pz2 and S-ray show the same relative cross sections for inactivation data of Hartman and Kozinski inactivation as noted below. (1962) and Takebe and Hartman (1962). On the basis of the above assumptions,
622
BENZINGER TABLE
AND
2
VALUE OF m FOR ULTRAVIOLET INACTIVATION OF COMPLETE TRANSDUCTION COMPARED TO THE RATIO OF ABORTIVE TO COMPLETE TRANSDUCTION FOR UNIRRADIATED WILD TYPE P22” -7 BackRatio of xtrapolate 0 f/ abortive to Mutant and lysate complete complete ransduction 1:ransduction curve &C-b (X) P22 lysate
wild-type 1
7 9 18 12 10 37 6
hisD-I
wild-type 2
10 10
hisA-
wild-type 2
5 15
(X) P22 lysate (X) P22 lysate
hisB-22 (X) wild-type
12 10 10 10 11 10 10
-
4 12
0 In the first column the abbreviations designate mutants requiring for growth adenine plus thiamine (&C-J) (Ozeki, 1956) or histidine (hisD-I, hi&b, and hi&-22) (Hartman et al., 1960a, b). The genetic tests are indicated listing the recipient strains first and then the wild-type P22 lysate. The data are grouped according to individual lysates because the initial ratio (for unirradiated phage) of abortive to complete transducing particles varies from lysate to lysate (Hartman et al., 196Oa). The second column lists the multiplicity of hits (m = back extrapolate of a complete transduction inactivation curve to the ordinate divided by the value obtained for unirradiated phage) required for inactivation of complete transductions. The back extrapolates (cf. Fig. lb) are drawn from curvea based on at least six experimental points. The third column lists the ratio of the number of abortive transductions to the number of complete transductions as determined by duplicate assays of the unirradiated P22 lysate. P22 lysate
2
the exponential rate of inactivation of plaque formation by ultraviolet treatment of P22 suspensions is set, by definition, equal to 1.0, and all inactivation rates (assumed “target sizes”) are expressed relative to this value.
HARTMAN
Possible Conversion of Abortive ple te Transducing Elements
to Com-
Abortive transductions of histidine mutants are inactivated exponentially at a rate of about 0.65, regardless of the recipient (single-site or multisite mutants) used in the assay (Fig. lb and Table 1). This finding indicates that the entire his region is eliminat,ed as a unit from abortive transduction. It is possible that the inactivation of abortively transducing elements is due to the gaining of an ability, following ultraviolet damage, to undergo recombination with the genome of recipient bacteria; i.e., potential abortive transductions are converted to potential complete transductions. If the converting hits take place only in DNA strictly homologous with that of the host (i.e., in bacterial genes), the bacterial gene content of his+ transducing elements is at least 65% of the total DNA of the particle. For elements carrying ath+, the bacterial gene content is at least 32% (Fig. la, Table 1). The converting hits could be akin to those which stimulate recombination in other systems (Jacob and Wollman, 1955; Roman and Jacob, 1958). The exponential portions of the inactivation curves for complete transduction often extrapolate to a value of about 10 at zero ultraviolet dose (Table 2). This value coincides with the ratio of abortive to complete transductions observed when unirradiated phage is used (e.g., Fig. lb, Table 2). Furthermore, an increased number of complete transductions is found at low ultraviolet doses. These observations lead to the hypothesis that an abortive transducing element is converted by an ultraviolet hit to a complete t,ransducing segment which then is converted to an inactive segment by an additional ultraviolet hit. Theoretical kinetics of a sequenceof two first-order reactions fit the observed values found for the rise in complete transductions, both in the dark and under photoreactivating conditions (Benzinger, 1961). Inactivation of Complete Transductions and the Physical Size of the his Region The inactivation of complete transduction of the multisite mutant, his-712,
EFFECTS
OF UV ON P22
proceeds at a rate of about 0.45. In contrast, transduction of his single-site mutants is inactivated at a rate of about 0.20 (Fig. lb, Table 1). Thus, although single-site mutants are presumably altered in one, or at most a few, nucleotide pairs, sensitivity of the wild-type segment necessary for recombinant formation represents a sensitivity proportional to a considerable fraction of the sensitivity of the total phage genome. The sensitivity of complete transductions to high dosesof ultraviolet light (Figs. lb and 2a) indicates that only extremely rarely are a few nucleotide pairs involved in eliciting a recombinational event, i.e., the production of wild-type recombinants most often involves a genetic region of considerable size. A “spreading effect” of ultraviolet light damages appears unlikely in view of the quite analogous results obtained with nitrous acid treatments by Adye (1962). The approximate value of 0.20, then, may be considered to consist of a segment of mean size comprising not only the genetic material actually entering the recombinant genome, but also a region in which synapsis and recombinational events take place. The difference in the rates of inactivation of point mutants and long multisite mutants can be used to obtain an approximation of the size of the his region relative to the total gentic material carried by the transducing particle (E 1). The difference in inactivation rates for his-152, a multisite mutant encompassing 6 of the 8 histidine genes (Ames and Hartman, 1962) as opposed to that for single-site mutants is 0.43 minus 0.20, or 0.23. The parallel calculation for the 7-gene multisite mutant, his-712, is 0.45 - 0.20, or 0.25 (data in Table 1). This value is probably only an approximation of the size of the his region since, owing to ‘(loop” formation of nonhomologous regions, the segmentsrequired for successful synapsis of elements involving multisite mutations probably would be larger, on the average, than those utilized in synapsis preceding complete transduction of single-site mutants. For example, multisite mutants inhibit recombinations in adjacent regions (Hartman, 1956; Hartman et al., 1960b). This larger mean pairing region also would presumably be ultraviolet sensitive; there-
fore, a fraction of the difference, 0.20, would belong not to the his region, but to outside genetic material necessary for pairing with the multisite mutation but not with the single-site mutation. Thus, 0.25 is considered a maximum estimate of the relative size of the his region. An independent estimate of the size of the his region comes from the study of the rapid initial inactivation of linkage by ultraviolet (Fig. 3). Joint transductions of two linked markers are called “donor type” recombinants. When only one of the markers carried by the donor is integrated,- a wild-type recombinant may be formed. P is defined as the probability of the separate integration of two markers, i.e.: 2 X the number of wild-type recombinants/(2 X the number of wild-type recombinants + the number of donor-type recombinants). The number of wild-t,ype recombinants is multiplied by 2 because the double-mutant recombinant is not detectable. With r’ thus defined, 1 P becomes: the number of donor-type recombinants/(the number of donor-type recombinants + 2 X the number of wildtype recombinants). 1 - P thus expresses the probability of joint integration of two markers. When ultraviolet dose is plotted against the logarithm of 1 - p for a number of different pairs of markers, points approximately fitted by a straight line are obtained (Fig. 3). The 1 - p inactivation rates are not accounted for by the difference in ultraviolet senstitvity of the two recombinant classes expected from the study of ultraviolet inactivation of transduction of singleand multisite mutants by wild-type phage. Indeed, at higher ultraviolet doses, when the number of complete transductions declines below the level obtained for unirradiated phage, the rapid disruption of linkage ceases. If the values for the slopes of the linear portions of the plots of log 1 - p against ultraviolet dose (Fig. 3) are expressed in terms relative to the phage infectivity inactivation rate and then plotted against the p values obtained in the same cross with unirradiated phage, a line extrapolating near the origin can be drawn (Fig. 4). Data of Adye (1962) on disruption of linkage by nitrous acid treatment of P22 also conform
624
BENZINGER
AND HARTMAN
to the plot shown in Fig. 4. The abscissa represents the quotient of ultraviolet hits which disrupt linkage divided by ultraviolet hits that inactivate infectivity. If P for two markers at the very opposite ends of the his region is chosen, the corresponding point on the abscissa should represent the relative size of the his region. Since the average P value for the most distant his marker pairs is 0.67 (Hartman, Stahl, and Adye, personal communication), the minimum size of the his region equals 0.17 f 0.04, as determined from Fig. 4. A critical assumption is that the genetic distance across the his region, 0.67, is a true estimate of its physical size. The validity of this assumption rests on a random distribution of crossovers along the his region and adjacent segments of the transducing fragment. Since map distances between mutants across the his region show no discontinuities (Hartman et al., 1960b) and the absolute transduction frequency for markers, including short multisite mutations, at either end of the his region is equal to that of markers located in its middle, this assumption appears justifiable. A final assumption predicates that recombinational events are of infrequent occurrence along the bacterial genes in the transducing element, i.e., double exchanges within the his region are rare. However, measurements of recombination frequencies between various pairs of his mutations located close to one another, as opposed to those further apart, suggest that recombinational events are very frequent along the element, an average of at least 1.3 events taking place within the his region (Hartman, Stahl and Adye, unpublished). Therefore, the current estimate of the size of the his region (i.e., 0.17) is considered an approximation of its minimal size. Mutant his-152 exhibits a transduction frequency with unirradiated P22 which is about 25% that for single-site mutations (Hartman et al., 1960b). For the his-712 mutant, the relative frequency is about 15%. One of these mutations (his-152) involves 6 of the 8 his genes; the other mutation involves 7 of the 8 genes (Ames and Hartman, 1962). From these reductions in transduction frequency, it is inferred that the majority (75-85 % of the class of wild-type transduc-
tional clones which ordinarily would appear) are not recovered owing to failure to replace completely the mutated region of the recipient. The transductions which do appear are formally analogous to donor-type transductions except that multiple exchanges within the his region cannot occur. The reduction in transduction frequency of 7585% thus infers a value for p of 0.75 to 0.85. The estimate for the size of a region with this p value (cf. Fig. 4) is about 0.20. The relative “target sizes” for the inactivation of complete transduction to wild type of multisite mut.ants (Table 1) suggest that the genetic defects in his-712, his-63, and his-644 do not extend very far outside of the known histidine region (Hartman et al., 1960b; Ames and Hartman, 1962). On the other hand, the large target sizes for inact,ivation of transductions of his-612, his-57, and his-203 indicate that the genetic defect in the former extends about four genes to the “left,” of the histidine region while the genetic defect,s in the latter two mutants extend about 12 genes to the “right” of the histidine region. il Coding Ratio for the his Region The above estimates for the size of the his region, as well as others discussed by Benzinger (1961)) allow us to assign an average relative value of approximately 0.20 to the his region. Eight gene loci (A-H) comprise the his region (Hartman et al., 1960a,b; Ames and Hartman, 1962). The molecular weights of some of the proteins of the histidine biosynthetic pathway have been determined by Martin and Ames (1961). They found that L-histidinol dehydrogenase (gene D) , imidazoleacetol phosphate transaminase (gene C), and phosphoribosyl ATP pyrophosphorylase (gene G) had molecular weights of roughly 75,000, 68,000, and 170,000, respectively. The latter enzyme at present is considered to be a dimer (Martin, 1962). The enzyme elicited by gene A and the presumed monomer protein elicited by gene B (imidazoleglycerol phosphate dehydrase-L-histidinol phosphate phosphatase) each have molecular weights of roughly 70,000 (Ames, personal communication). Seven of eight histidine genes are of approximately equal
EFFECTS
OF
Al
UV
ON
625
P22
If about one-fifth of these comprise the his 12,600 nucleotide region, approximately pairs code 4000 amino acids. A coding ratio of about 3 is thus obtained (cf. Ames and Hartman, 1962). This estimate is based also on the assumption that the great majority of the nucleotides of the his region are used in coding for the amino acids of the histidine biosynthetic enzymes.
Aberrant Recombinational Behavior of his24 HisB-Z.$ has been shown to exhibit ab-
f32
FIG. 6. Schematic representation of one type of molecular change in the genetic material (mutation) which might lead to expression of a mutant phenotype and, simultaneously, affect recombination in adjacent regions. In this model, the mutation at the base pair indicated results in a configuration allowing either a greatly increased or a greatly decreased tendency for intrastrand hydrogen bonding by complementary base pairing. This altered propensity for “loop” formation within single strands of the DNA molecule is suggested as critically affecting some stage in the recombination process.
sizes; the eighth genemay be slightly smaller (Ames and Hartman, 1962). It seems reasonable to assume that seven of the genes control the structural formation of seven protein monomers with an average molecular weight of 70,000 whereas the eighth gene controls a protein monomer of 30,000 molecular weight. This is an aggregate molecular weight of 5.2 X 1Oj. The average molecular weight of amino acids making up proteins is about 130; therefore, the positioning in proteins of approximat’ely 4000 amino acids appears to be determined by the DNA of the his region. The total number of nucleotide pairs in P22 is about 63,000 (Garen and Zinder, 1955).
normally high recombination frequencies with regions adjacent to it (Hartman et aE., 1960b). The ultraviolet inactivation kinetics of transduction to his+ in two-factor crosses involving h&B-24 reveal a unique resistance to ultraviolet light (Fig. 5a). These and other data pertaining to the properties of his-24 are summarized elsewhere (Hartman, 1962). The type of molecular change involved in t’he case of his-24 and a possible way in which this change leads to the observed properties of the his-24 allele are suggested in Fig. 6 and described in the legend to that, figure. The model makes two predictions, namely, that mutations affecting recombination in this manner will usually not involve “transit,ion” mutations and that the primary mutation might lead to greatly increased frequency of multisite mutations in its vicinity in later generations. The data at present are too scanty to determine whether either prediction is fulfilled. ACKNOWLEDGMENTS
genetic
We wish to thank Drs. Sol H. Goodgal and Claude S. Rupert for suggesting some of the ultraviolet irradiation experiments. The energetic technical assistance of Mr. William F. Kosch III is gratefully acknowledged. This work, in part, was supported by Research Grant E-1650 of the National Institute of Allergy and Infectious Diseases, United States Public Health Service. REFERENCES
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627-632. AMES, B. N., and enzymes, and biosvnthesis.
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