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A genetic map of poliovirus temperature-sensitive mutants
VIROLOGY
36, 584-596 (1968)
A Genetic
Map
of Poliovirus
Temperature-Sensitive
PETER
Mutants
D. COOPER
Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, Australia Accepted November IO, 1967 and the The conditions affecting recombination between ts mutants of poliovirus assay of tsf recombinants were standardized. The mean recombinant frequencies were then reproducible and characteristic of each pair of mutants, being between 5.2 and 31.4 times the total background reversion rates and ranging from 0.0270 t,o 0.8570. Certain genetic sequences were proved by three-factor crosses involving a mutant adapted to guanidine resistance (h-28 g), and these showed that recombination frequencies were additive. An additive linear genetic map comprising one linkage group was obtained. The g character of ts-28 g seemed to result from mutation at a single site that could be accurately located near the middle of the map. Recombination was nonrandom with time, most of the mating events occurring early in the growth cycle. INTRODUCTION
Temperature-sensitive (ts) mutants derived from a ts+ strain of poliovirus (Cooper, 1964) have been examined both by a complementation test (Cooper, 1965) and in terms of defects in physiological function (Cooper et al., 1966) and in synthesis of RNA and serum-blocking antigen (Wentworth et al., 1968). Recombination between poliovirus strains (Hi&, 1962; Ledinko, 1963) has shown the possibility of genetica,lly mapping these mutants. Much of the work described below is a technical evaluation of mapping experiments, in which ts+ recombinants were scored selectively. This work has led to a reproducible and additive genetic map of the mutants, which is also presented. As there is no published precedent for a quantitative map obtained by recombination analysis of a virus growing in animal cells, or of an RNA-containing virus growing in any cell, particular attention has been paid to possible causes of variability in recombination frequencies. MATERIALS
AND
METHODS
The parental tsf strain of poliovirus type 1 and the ts mutants have been described previously (Cooper, Virus
and cell strains.
584
1964; Cooper et al., 1966). Virus was grown and assayed as before in U cells, which were treated with 100 pg/ml of kanamycin sulphate for the 24 hours before harvest to minimize contamination with mycoplasma. All assays, viral growth, and preparations of celIs for viral growth were made with one or two large batches of pretested serum, agar, and medium components. The guanidineresistant strains ts-28 g and ts+ g were separately adapted from the guanidine-sensitive (gf) strains ts-28 and tsf, respectively, bv Dr. B. B. Wentworth, by alternate passage and cloning in the presence and absence of guanidine in a manner similar to that described by Ledinko (1963). In case the ts defect of ts-28 had been affected by the adaptation and selection of ts-28 g, the two ts loci were shown to be cIoseIy Iinked by the lack of recombinants formed in standard crosses of 28 g X 28 (mean recombination frequency = 0.003%), and by the frequencies in crosses of 20 X 28 g (mean = 0.054 %, parallel crosses of t.+20 X k-28 giving 0.049 %), and in crosses of 28g X 22 (mean = 0.249 %, va,Iues for 28 X 22 being 0.201%). Neither difference’ between k-28 and ts-28 g crosses is significant (p > 0.05) by analysis of the standard error of the
GENETIC
MAP OF POLIOVIRUS
difference. As the reversion frequencies and other characteristics of ts-28 and ts-28 g were similar, it is presumed that the ts defect had remained unchanged through the adaptation procedure. Prepamtion of mutant stocks for recombination tests. Small limited-passage stocks of mutant, were prepared with low wild-type content, and then partially purified. For this purpose, several “ministocks” were made by infecting tubes of lo5 cells with the maximum amount (50-200 PFU) of cloned mutant, stocks that would allow less than one tube in 20 to receive 1 PFU of revertant ts+. This scheme was preferred to recloning, to give t’he mutants as large an advantage in growth over revertants as possible. The ministocks were then assayed for wild-type content and for efficiency of plating at 39.3-39.7” compared with 37” (see below). Only t,hose mutants were used that gave < 1O-4 mutant plaques (< 1 mm) at restrict’ive temperatures; t’he rejection procedure on the basis of ts+ plaques (>2 mm) is given below. Three monolayers of 5 X lo6 cells were then each infected from each ministock tha,t contained the least tsf, and frozen after S hours’ growth at 36”. Several such stocks were made for each mutant, and those containing t’he least wild type were pooled; this method gave ts+ contents >{o-Koo that of cloned st’ocks for most mutants. Stocks of about 5 X lo8 PFU were the smallest suitable as perma.nent references, and mutants that failed after several attempts to give ts+ contents of <10e4 in such stocks were rejected. Smaller stocks containing up t’o 5 X 10e4 ts+ were used to map less stable mutants in a few crosses. Stocks Ivere clarified (20 minutes at 200 rpm) and recentrifuged for 90 minutes at 39,000 rpm in a Spinco 40 rotor. After standing at 4” overnight in 0.5 ml of 0.9% NaCl solution, the pellets were resuspended, trea,ted with 1 ml pH 2.5 glycine buffer (0.05 ill, Fenwick and Cooper, 1962) and neut’ralized after 3 minutes with 10 ml Eagle’s medium. This treatment, which presumably disaggregated the inoculum, was the greatest single factor in increasing reproducibilit,y of recombination; it increased titers up to 2-fold, and enhanced up to g-fold
Is MUTANTS
585
both the adsorption ability and recombination efficiencies. The virus was recentrifuged at 39,000 rpm and the pellet, after standing at 4” overnight in 1 ml Eagle’s medium plus 0.5 % calf serum, was resuspended and the concentrates pooled and reclarified. This material (5 to 10 X lo* PFU/ml) was st,ored in 0.2-ml amounts in sealed ampoules at - 45”. Standard yecombination test. The following conditions give maximum recombination frequencies, and were closely followed. Cells from semiconfluent (2- to 4-day) Roux cultures were resuspended to 2.5 X lo5 cells/ml in Eagle’s spinner medium containing 10 % of Difco tryptose phosphate broth, 7.5 % calf serum, 2.5% fetal calf serum, 1 mg/ml CaCL, and 0.77 mg/ml NaHCOs, the mixture having been previously gassed with 5 o/o CO* in air. One millilitre was added to stoppered flat-bottomed tubes of 20 mm internal diameter, and incubated for 20 hours at 37.2”, then washed twice with 2 ml of PBS. Fluid was thoroughly aspirated, and 0.08 ml of virus inoculum was added. A fresh ampoule of each special mutant stock (see above) was diluted to 1.5 X 10” PFU/ ml in Eagle’s medium. For test crosses, equal volumes of this suspension were mixed before inoculation, giving an input of 24 PFU/ cell and a measured adsorbed multiplicity of lo-15 PFU/cell of each mutant; for selfcrosses, the suspension was added undiluted (input = 48 PFU/cell), and for “controls” the suspension was diluted 1:2. The adsorption efficiencies were the same in self-crosses, controls and mixtures. Each cross was performed in duplicate and assayed separately, using 2 or 3 plates or bottles per dilution, with appropriate self-crosses and controls. Each experiment included cultures infected with tsf and t.9 g. The inoculated tubes were restoppered and shaken at 10-15’ for 4 hours (giving 4080 % virus adsorpt’ion), then 0.92 ml of Eagle’s medium (at pH 7.2 and containing 100 pg/ml of cycloheximide, 10% tryptose phosphate broth, and 5% calf serum) was added, the cultures placed in a 37.2” water bath for 20 minutes, and then chilled. The supernatant was removed for assay of unadsorbed virus, and t’he cultures washed
586
COOPER
twice. One millilitre of Eagle’s medium containing 10% tryptose phosphate broth, 5% calf serum and 0.28 mg/ml NaHC03 was then added, and the tubes frozen after a further 9 hours at 37.2”: the pH remained about 7.2 throughout. After thawing, the infectivity was stable at 4“ and pH < 7.3. Viral assay. Agar cell-suspension plaque assays involved the galactose medium (Cooper, 1961), to which was added a total of 8 % calf serum, 5 % of a 10% skim milk powder suspension, and 1 mg/ml of Difco tryptose powder. The ts defect is often associated with cystine dependence (McCahon and Cooper, unpublished data), and so leak rates were much reduced by omission of lactalbumin hydrolyzate and yeast extract at 39.3-39.7” : the recombination frequencies and tsf plating efficiencies were not affected. For assays of guanidine-resistant virus, an Analar guanidine carbonate solution adjusted to pH 7-8 with 10 N HCl was added to both agar layers to give 200 pg of guanidine carbonate per millilitre. Cystinedeficient medium was not used in presence of guanidine carbonate at 200 pg/ml level. Guanidine carbonate (10 pg/ml) was added t.o all assays of k-28 g and ts+ g recombinants to satisfy a slight guanidine dependence; this dose did not affect plaque size or number of any guanidine-sensitive (g+) strain, and g plaques at 37”-39.7” in guanidine carbonate at 200 pg/ml were the same size and number as at 10 pg/ml. In controls of every strain included in all experiments, guanidine carbonate at 200 pg/ml always reduced the plaque count of g+ strains to negligible levels (< lo-“), at 37-39.7”. All assays were diluted through 0.05 M glycine buffer, pH 2.5, to eliminate aggregation as a source of variability and to increase sensitivity. The total virus produced was assayed in petri plates (64 hours at 37”), and the ts+ virus was assayed in bottles immersed for 52 or 64 hours in a water bath at 39.3”, 39.5”, or 39.7”, all f0.05’. The temperature and time of ts+ assays were arranged to give a mutant efficiency of plating of < 10e4 and a loss of tsf plaques of <20 % in the control assays; 39.5” for 64 hours in cystine-deficient medium was usually satisfactory for all the main mutants.
All plaques visible were counted; if in any plaques (< 1 mm) assays the mutant amounted to >lO% of the tsf plaques, (>2 mm) in the self crosses, all samples in that experiment were reassayed at a higher temperature. If ts+ assays differed with temperature, or if k-28 g assays differed with guanidine concentration, t.he ts+ or ts+ g recombinant titres were adjusted to the titres to be expected at 37” and 10 pg guanidine carbonate per millilitre; correction factors were within the range 0.7-1.3. All assays of one experiment were made at the same time. Calculatim of recombination frequency. The recombination frequency is given as the recombinant tsf content expressed as a percentage of the total virus (PFU/ml assayed at 37”). The recombinant tsf content is derived by subtract’ion from the total ts+ content of the ts+ content to be expected from spontaneous reversion. RESULTS Obstacles to Mapping
Poliovirus ts Mutants
Preliminary recombination tests, using the methods of Ledinko (1963), produced progeny containing four to ten times as much ts+ virus as did the self-crosses but attempts to obtain a self-consistent genetic map by such tests were unsuccessful. There were found to be four reasons for this: (1) the high and variable ts+ content of the stocks, despite frequent recloning; (2) loss of ts+ plaques or poor control of leak in the 39.5” assay of ts+ contents; (3) the presence of many double mutants; and (4) large variations between experiments (> lo-fold) in the frequencies of recombination. Factors (1) and (2) were minimized or eliminated by the procedures described in Materials and Methods. Some aspects of the assay are described in the next section; factors (3) and (4) are discussed in subsequent sect’ions. The Assay of ts+ in Presence of Excess t-s Virus Reconstruction experiments, in which 100 PFU of tsf were assayed in presence of varying doses of any main mutant, showed that no ts+ plaques were masked by excess mutant virus in a standard assay at 39.3”. The ts+
GENETIC
MAP
OF POLIOVIRUS
plaque count was still proportional to input in the presence of sufficient mutant (6 X lo5 PFU) to infect 10% of the cells; in most assays, only 1% cells were infected. The detection efficiency for t& was about lo+ for very defective mutants. Assay of any two mutants (5 X lo5 PFU of each) in one bottle gave ts+ plaque formation ehual to the sum of the mutants assayed singly: no recombination occurred in the bottle.
Singularity
Double mutants were the main cause of nonadditive recombination frequencies, and accordingly the singularity of the mutants’ defects was examined. Relevant data are given in Table 1. The main mutants are in italicsThe reversion frequencies in Table 1 are calculated from the mean wild-type content
TABLE SOME
PROPERTIES
OF POLIOVIRUS
-
-
ts+ content x 10-4
Appr0x.b reversion frequency
15
1
ts MUTANTS RELEVANT THEY CONTAINS
- I I
Character of revertants
TO THE NUMBER
OF ts DEFECTS
Character of recombinants
.g&
Plaqued size
587
Ls MUTANTS
e.0.p.
change 39.3-39.70 l/P
.dditivity of Lapdistance! l/7”
nd/nd wt/nd nd/nd nd/nd nd/nd ts/ts
nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd
Yes/Yes Yes/Yes nd/‘No Yes/No nd/nd No/No
Single Single Double Double Double Double
nd/wt
nd/wt
rid/Yes
wt/nd nd/nd wt/wt nd/nd nd/nd nd/nd nd/nd nd/nd
wt/nd nd/nd wt/wt nd/nd nd/nd nd/nd nd/nd nd/nd
Yes/No rid/No Yes/Yes Yes/Yes No/rid nd/nd nd/nd nd/nd
Prob. single Double Double Single Single Double ? ? Prob. single Prob. single Prob. single Double Single Prob. single Prob. single Prob single
Stock
Self-crossc
2 3 5 9 18 19
2
3.75 1.60 f 0.4: 2 6.46 2.2 0.8
nd 0.56 0.70 2.2 0.77 0.28
wt wt s 9 S s
20
<0.2
1.93 f
0.67
wt
wt
22 23 28 28 37 44 46 89
2.33 f 1.0 0.81 2.6 0.90 1.26 f 0.6~ 0.44 2.07 f 0.41 0.72 2.1 0.73 nd nd nd >40 100-600 >30
wt S wt wt s
nd wt wt
nd wt wt
nd nd wt
wt/s wt/s wt/wt wt/wt s/rid wt/nd wt/nd wt/nd
nd
wt
wt
wt/nd
nd/nd
nd/nd
nd/nd
nd
wt
wt
wt/nd
nd/nd
nd/nd
Yes/rid
11.5 5.2 5.23 f 1.7:
4.0 nd 1.8
wt wt wt
wt nd wt
wt/wt wt/wt wt/nd
nd/nd nd/nd nd/nd
nd/nd nd/nd wt/nd
Yes/No Yes/Yes Yes/rid
15
nd
wt
wt
wt/nd
nd/nd
nd/nd
nd/nd
nd
wt
wt
wt/nd
nd/nd
nd/nd
nd/nd
39.339.Y
onclusions on 8ingularity
.o.p. 39.3’ f is00
-
94
5-10
96
1
99 104
1-2 nd 1.5
149 150
nd
155
.lO
-
0.5:
30 3.3
>60
nd wt ts
wt nd
wt
ts
ts
ts
wt/wt wt/wt wt/s wt/wt wt/nd s + wtl s + wt nd/wt
a Abbreviations: wt = wild type; s = small; nd = not determined * Per particle per duplication X 10m4. c Mean of all values. d At restrictive temperature. e In crosses with single mutants located to left or right (Z/r) in the map,
588
COOPER
of the self crosses, using method 3 of Breeze and Subak-Sharpe (1967); in some cases the 95 % confidence limits of the mean are also given. The most stable mutants reverted at a rate of 0.5 to 1 X 1O-4 per particle per duplication. Columns 5 and 6 of Table 1 give the character of revertant plaques, as determined in 39.5” assays of the self crosses. In some cases the decrease in plaque number in duplicate bottles incubated at higher temperatures was compared with wild-type values; a decrease >3 times the decrease in tsf plating efficiency indicates that the revertants were ts. Columns 7-10 give similar data for characters of recombinants, and the last column gives final conclusions on singularity. Table 1 shows that the reversion frequencies of these mutants are useless as criteria of singularity, presumably because all mutants have been highly selected for stability. Revertant character (plaque size and change of plating efficiency with temperature) apparently are fairly useful, but are not reliable, because of the probable presence of suppressor mutations elsewhere in the defective viral gene. Partial revertants from single poliovirus ts mutant’s are in fact of frequent occurrence (McCahon, personal communication). More reliable are the recombination characteristics. As double crossovers are unlikely, the provision of additive map distances and of solely wild-type recombinants with mutants t’hat map on either side are strong evidence of singularity, or of closely linked defects. Only ts-2, -3, -28, and -104 gave such evidence, but the mutants at the ends of the map (ts-20 and k-149, see below) behaved in all other respects like single mutants. Time of incubation. Ledinko (1963) suggested that poliovirus recombination frequencies increased about 2-fold during a single growth cycle. Preliminary experiments with ts mutants indicated that any increase was likely to be smaller than that, and this question was therefore tested more rigorously. A standard recombination test was made involving twenty replicates of the cross 28 9 X 3; ten replicates were harvested
3.1 hours and the remainder 7.0 hours after infection, when the average yields (with standard deviations in brackets) were, respectively, 29.8 (3.25) and 124 (9.0) PFU/ cell. The mean recombination frequencies and standard deviations for the two samples were, respectively, 0.279 (0.053) %, and 0.419 (0.0339) 70. Thus there was a small but significant (p < 104) increase in the recombination frequency, at times corresponding to a 417 % increase in virus content. E$ect of Multiplicity
of Infection
Ledinko (1963) also showed that variations in total multiplicities between 10 and 30 PFU/cell had no effect on recombination frequency provided that the input ratio remained between 3 and 0.5. This finding was confirmed for ts mutants in a standard recombination test of 20 X 28 g, in which the proportion of each mutant could also be determined in the yield (Table 2). Growth of ts-20 and ts-28 g in mixed infection was approxima.tely proportional to the input frequency ratio, and the recombination showed no systematic variation over a wide TABLE
2
EFFECT ON RECOMBINATION FREQUENCY IN THE CROSS~OX 28 ~OFVARYINGTHEPROPORTION OF INPUT VIRUS AT .4 CONSTANT MULTIPLICITY"
-
Input virus (proportion of ts-28 g) 1.0 0.9 0.8 0.6 0.5 0.4 0.2 0.1 0 ts+
/
Total yield PFU/ml x 10’
Proportion b-28 g
3.55 4.9, 3.25 6.0, 5.35 5.3,4.5 3.75,4.2 5.2, 4.5 2.15, 2.45 2.0,2.2 3.2 2.0
1.0 0.68, 0.68 0.56, 0.54 0.50,0.50 0.32,0.44 0.26,0.34 0.26, 0.16 0.10,0.091 0
-I ts+ content” (%) 0.042 0.080,0.100 0.125,0.149 0.118,0.165 0.198, nd 0.140, 0.162 0.221,0.198 0.180,0.114 0.030 00
0 The procedure was the standard recombination test given in Materials and Methods. The total input multiplicity was 40; nd = not determined. Values are the results from duplicate tubes. b Uncorrected for spontaneous reversion.
GENETIC
MAP
OF POLIOVIRUS
589
ts MUTANTS
and ts-28 y, ts-3, and ts-149, respecbively. The tests in each experiment were made and assayed on the same occasion, as always. They illustrate the type of result that was aimed for in the assays, and the way of obtaining the corrected recombination frequencies. Figures 1 and 2 show the plaque characteristics of the recombinant progeny of several crosses; such recombinants are typical of all crosses except those giving nonadditive recombination frequencies (see below).
range of ratios. Thus input ratios do not need to be precisely equal for maximum recombination frequencies. Effect of Spontaneous Reversion to ts+ Table 1 gives the t.s+content of the special recombination stocks, together with the mean Is+ content of the self-crosses made with them. The ts+ content increased IO-fold or more in this single cycle at 37.2”, presumably due to reversion rather than selection as the growth rates of t.@ and mutants were similar. In all “control” crosses (i.e., selfcrosses at half multiplicity) t’he ts+ content did not differ from that of the corresponding self-crosses; hence small changes in multiplicity did not affect reversion rates. Spontaneous reversion contributed relatively little to the tot’al ts+ content of recombination tests except for closely linked mutants, and the procedure for correcting for this is given in Materials and Methods. In the main crosses (20 X 28,28 X 22,20 X 22, 20 x 3, 28 x 3, 28 x 149, 20 x 149) the mean recombination frequencies exceeded the reversion background by, respect)ively, 5.2-, 12.5-, 15.7-, 31.4-, 30.0-, 21.8-, and 23-fold.
Determination of Some Crosses with ts-28 g
Sequences from
Certain mutants could unequivocally be assigned a sequence in the genetic map by a form of three-factor cross with ts-28 g. In standard crosses of 20 X 28 g, all recombinants in excess of the ts-28 g reversion background were g+, and this finding is illustrated in Table 3, Fig. 3 and, together with the plaque characteristics of the recombinants, in Fig. 2. The same was found in crosses of ts-5, -23, and -99 with ts-28 g, and ts-5, -20, -23, and -99 are accordingly assigned arbitrary positions to the left of ts-28 in the genetic map, and the g locus or loci to the right. Crosses between ts-28 g and the remainder of the mutants either produced mean recombination frequencies of co.3 %, which
Results of Crosses Using the Standard Method Tables 3 and 4 give the results of two experiments involving ts-20 and ts-28 g, TABLE
3
RESULTS OF THE CROSS 20 X 28 aa Percentage in yield of
PFU per plate or bottle6 Cross
ts-28 g control k-28 g self ts-20 control k-20 self ts+ control ts+ g control 20 X 28 g 20 X 28 g
37”, Ill: pg/ 83, 131, 85, 132, 76, 179 153, 112,
72 113 108 89 61 134 121
37”, 200 rg/ml 75, 87, 0, 0, 0, 157 58, 83,
82 90 0 0 0 71 58
39.5”, 10 e/ml
39.5”, 200 /Lg/ml
ts+
Is+ g
22, 23 20, 21 29, 28 24, 38 76 EO-2QO 117, 143 127, 154
20, 20 29, 35 0, 0 0, 0 0, 0 150-200 19, 17 28, 22
0.0289 0.0168 0.0289 0.0280 100 100 0.090 (0.065)~ 0.121 (0.095)c
0.0256 0.0364
D The procedure was the standard recombination test given in Materials and Methods. b Plates were incubated for 64 hours at 37” with 0.1 ml of lO+ dilution: bottles were incubated for 64 hours at 39.5” with 0.1 ml of lo+ dilution. The concentrations indicate the guanidine carbonate content of the assay medium. c Recombination frequencies after correction for spontaneous reversion are given in parentheses.
590
COOPER TABLE
4
RESULTS OF THE CROSSES 18 g X 3, R8 g X 1.49 and 3 X 14Sa PFU per plate or bottle*
Percentage in yield of
Recombination frequenciesc
Cross 37",200 ,&ml 28 g control 28 g self 3 control 3 self 149 control 149 self ts+ control ts+ g control 28gX3 28gXS 28 g X 149 28 g X 149 3 x 149 3 x 149
99 95 117, 95, 80, 78, 52, ca. 131, 95, 90, 103, 66, 58,
84 113 77 76 59 200 108 144 112 105 63 65
143 120 0, 0 090 0, 0 0, 0 0, 0 ca. 200 87, 73 76, 68 57, 60 72, 45 0, 0 0, 0
19 20 2, 5 1, 3 49, 49 41, 32 64, 69 ca. 200 89, 84, 90 105, 92, 83 141, 135, 110 142, 153, 147 38, 21 36, 37
32 19 0, 0 0, 0 0, 0 0, 0 0, 0 ca. 200 106, 82, 80 85, 89, 79 92, 71, 77 68, 72, 84 0, 0 0, 0
ts+
ts+ g
0.019 0.021 0.0035 0.0019 0.062 0.047 100 100 0.367 0.387 0.643 0.665 0.231 0.295
0.032 0.020
ts+
ts+g
-
-
-
-
0.347 0.367 0.605 0.665 0.194 0.258
0.266 0.253 0.288 0.259
0 The procedure was the standard recombination test described in Materials and Methods. * Plates were incubated for 64 hours at 37” with 0.1 ml of 1W6 dilution; bottles were incubated for 64 hours at 39.5” with 0.1 ml of 1OW’dilution for controls and self crosses, or 2 X low3 dilution for the recombination tests. The concentrations indicate the guanidine carbonate contents of the assay medium. c After correction for spontaneous reversion. d Corrected for enhancement of h-28 g in guanidine.
were the same when plated in presence or absence of guanidine (ts-9, -18, -96, -22, -94, and -155), or produced mean recombination frequencies of 0.4-0.8% when assayed in absence of guanidine but 0.360.39% in presence of guanidine (k-3, -2, -104, -149). Examples of the former type are given in Fig. 3 for the crosses 28 g X 22 and 28 g X 94; the differences between the assays in presence and absence of guanidine are not significant (p > 0.3). The latter type of cross is exemplified in Table 4 and in Fig. 4 for 28 g X 3 and 28 g X 149. Thus all the recombinants in crosses of L-28 g with ts-9, -18, -96, -22, -94 and -155 appeared to be g, while in crosses of ts 28 g with k-3, -2, -104 and -149 a fixed number of recombinants was g and a variable number was gf. The recombinants in the latter type of cross were confirmed as a mixture of g and gf by isolating wild-type plaques at random from a 39.5” assay of the cross 28 g X 149. The efficiencies of plating at 39.5” of the resulting clones were all wild-type (0.5-1.5).
The unselected g marker was then scored on 42 of the clones by assay at 37” in presence (100 and 200 pg/ml) and absence of guanidine carbonate: 28 were g and 14 were gf ; none showed intermediate sensitivity, and their g or g+ behavior wa.s identical with that of k-28 g and ts+, respectively, in parallel assays. This proportion of g particles among the tsf progeny (67 %) was not significantly different from that (about 53%, see below) to be expected from the mean recombination frequencies. Thus the presence of 100 % g recombinants for k-9, -18, -96, -22, -94 and -155 placed these mutants between ts-28 and the g locus, and the presence of a mixture of gf and g recombinants placed k-3, -2, -104 and -149 to the right of the g locus. Certain other leaky or unstable mutants (ts-44, -46, -89, -150) could be assayed in crosses with h-28 g in the presence of guanidine, as their background (leak plus reversion) was then reduced to that of ts-28 g; these mutants produce g recombinants in a frequency of
GENETIC
MAP
OF POLIOVIRUS
Is MUTANTS
FIG. 1. Plaque characters of is+ and of recombinants. The crosses illustrated (all plated in O-l-ml amounts of the dilutions given) are: 3 X 2 (lo+), 3 X 104 (lo+), 3 X 149 (2 X 10V3), and 28 g X 3 (2 X contained 200 pg of guanidine carbonate per millilitre in com10-3). The bottle marked “guanidine” plete medium, and the other bottles all contained the drug at 10 pg/ml in cystine-free medium; bottles were incubated at 39.5” for 64 hours.
< 0.3 %, and so are assigned to loci between h-28 and 9. In summary, these sequences may be expressed: (5, 20, 23, 39)28 -(.3, 18, 22, 44, @, 80,94,150,155)-g(2,3,104,149),
with the order of mutants remaining undetermined.
in parentheses
Acldihity oj Map Distances and Reproducibility of the Test The sequences from the previous section were fairly accurately reflected in the mean map distances for certain triads of mutants,
that is, the map distances were additive; Fig. 3 illustrates this with the results of all standard crosses for the triad h-20 -28 -22. Similar results were obtained for the triad ts-20 -28 -9, and with several others as elaborated below. The relative frequencies of t.riads for which three-factor crosses were not available also appeared to give reliable sequences, provided that sufficient tests were made. Figure 4 illustrates this with the results of standard crosses for 28 g X 3, 28 9 X 149, and 3 X 149. The mean posi-
COOPER
FIG. 2. Plaque characters of ts+ and of recombinants obtained from the experiment of Table 3. The cross illustrated (all samples plated in O.l-ml amounts of the dilutions given) is 28 9 X 20. The bottles marked “guanidine” contained 200 rg/ml of guanidine carbonate, and all the remainder contained the drug at 10 pg/ml; bottles were incubated at 39.5” for 64 hours.
tions for the g locus were closely similar for both types of cross, and the mean map distances were additive. However, in repeated crosses of most single mutants made and assayed on different occasions the absolute frequencies varied over about a 2-fold range, and so the source of this variability was examined. The lower part of Fig. 4 shows that the variability within an experiment was less than between experiments. This is confirmed by comparing
the standard deviation of the frequencies for the ten 7-hour replicates of the cross 28 g X 3 mentioned above (8.1% of the mean) with that of ten 28 g X 3 crosses made on five different occasions (28.0% of the mean). Similar analysis of some twenty other crosses gave similar results, namely that the root mean square of the difference from the mean of duplicate tests made on the same occasion was S-10% of the mean, whereas the standard deviation of tests (ten or more)
GENETIC FREQUENCY
OF tS+ 0
0.1
MAP
RECOMBINANTS 0.2
0.3
OA
OF POLIOVIRUS (X)
0.5
0.6
289X22
Is MUTANTS
593
for the crosses 28 g X 3, 28 g X 149, and 3 X 149 (0.495%, 0.775 %, and 0.280% respectively) were taken as standards for ts+ recombinants, and 0.364% as the standard for g recombinants in the crosses 28 g X 3 and 28 g X l&9. The correction factors ranged from 0.696 to 2.22 (mean 1.18) for the ts+ recombinants, and from 0.730 to 1.37 (mean 1.00) for the g recombinants. This standardization had very little effect on the mean recombination frequencies (see Fig. 5 below), but standard deviations were reduced from about 28% to about 10% of the mean. The procedure finally adopted was to accept the mean standardized frequencies of four crosses made on two different occasions. The Genetic Map
asgx
94 I-
I 0
I
I I
f&B
FIG. 3. Unstandardized ts+ recombination frequencies from the crosses 28 g X 22, 20 X 28 g, 20 X 22, and 28 g X 94, made on several different occasions; is-28 is arbitrarily located at 0.1%. The bracketed values, g, were obtained from 39.37” assays containing guanidine carbonate (200 pg/ml). Values to the right are the mean recombination frequencies of that cross; values at A are the mean of all crosses for the triad 20-28-22 and show the additivity obtained; the map at B is derived from the values in this figure; the order of ls-22 and -94 is undetermined.
made on different occasions was 24-28 % of the mean. This difference depended more on the day-to-day variations in the sensitivity of the assay system than on assay errors or variations in the recombination test, since absolute frequencies often varied 2-fold in assays of the same test repeated on different occasions, while the relative frequencies of different crosses assayed together stayed the same. Accordingly, the day-to-day variations were minimized by normalizing each test cross in terms of standard crosses made and assayed on the same occasion. The mean recombination frequencies shown in Fig. 2
The mean standardized values for the crosses giving additive recombination frequencies enabled a genetic map of the main mutants (ts-20, 28, 3, 2, 104, 149) to be constructed (Fig. 5). The standardized and unstandardized values were closely similar (the differences were all within the 95 % confidence limits of the means of the unstandardized values). In all individual experiments the standardized or unstandardized frequencies between the main mutants showed the same map sequence. The 95 % confidence limits given in Fig. 5 for t’he means of some self-crosses show that the variability from spontaneous reversion was not large. This background determined the smallest detectable frequencies, which were 0.02-0.05 YCfor most crosses. The limit for g crosses with ts-28 g was 0.01%. The positions for g given by ts-28 g crosses with ts+ and with four ts mutants were closely similar. In addition, ts+ and tsf g plaques in assays of these crosses were exactly alike in size (see Fig. 1) and no tsf progeny of intermediate guanidine sensitivity were found in the isolates from the cross 28 g X 149 described above. Hence the g character of ts-28 g is probably determined by a single locus, or by several closely linked loci between ts-22 and ts-3. Figure 5 also shows the mean standardized frequencies for certain unstable mutants that
594
COOPER FREQUENCY
OF tS+
RECOMBINANTS
(%)
2agx3
3x
149
A I 3
apt.1
apt.
2
2ag x 3 2ag x 149 3x149
1‘.
‘22 l
:: :49
. 3x149
FIG. 4. Unstandardized ts+ recombination frequencies from the crosses 28 g X 5, 28 g X 149 and 3 X 149; k-88 is arbitrarily located at 0.1%. The squares on the lines for crosses involving h-28 g represent the frequencies for ts+ g recombinants, determined by 39.3-7” assays containing guanidine carbonate (200 pg/ml). The values at A are the mean of the crosses given for the triad 38-S-149 and show the additivity obtained; the inferred map locations of the mutants are also given. The lower part of the figure gives the results of paired crosses in two illustrative experiments made on separate occasions (see text).
could only be crossed with ts-28 g. Some of these (ts-44, -46, -89, -150) could only be assayed in presence of guanidine carbonate (200 pg/ml). This device could be used only for mutants with loci between 28 and g; they fall into two groups, one near k-28 and the other near k-22. The frequencies in crosses between some main mutants and t-s-5, -9, -19, -22, -23, -S7, and -99 were nonadditive, that is, the sum of the frequencies between these mutants and two main mutants was x-J$o of the frequency between the main mutants themselves. Other evidence indicates that most of these mutants contain multiple ts defects. Two of the double mutants (b-9 and -22) gave additive frequencies and ts+ plaques with main mutants to their left in the map, but not to their right; thus only their major defects are located in Fig. 5, and the minor ones lie to the right. In certain other crosses involving double mutants (28 + 99,20 X 23, 20 X 5, I.43 X 37), the recombinants were all wild type, and so these crosses probably reflect the true map positions of the major defects of ts-5, -23, -37 and -99. Other crosses involving these double mutants yielded mixed recombinants that all or mainly produced Is plaques at 39.3-5”.
The relative positions in the map of loci within the groups (5, 23, 99), (46, 18, 96, 9, 44, 150), and (22, 155, 89, 94) are undetermined. DISCUSSION
This work shows that one can select a group of poliovirus ts mutants between which the recombination frequencies are all additive. The selfconsistent nature of the genetic map obtained, in particular from the three-factor crosses, dispels any doubt that the enhanced ts+ contents of the progeny result from genetic recombination. The reproducibility and error of the method are satisfactory: the coefficients of variation of the unstandardized frequencies are similar to those of phage T4D (Edgar, 1958), and after standardization resemble those of other phage crosses. Poliovirus strains giving additive map distances also behaved as single mutants by all other criteria, while those that did not almost all presented other evidence of being double mutants. It was particularly important to resolve this question of singularity, as so large a portion (9/15) of the apparently
useful
mut’ants
turned
out
to
have more than one ts defect. Only 4 of the
GENETIC
MAP OF POLIOVIRUS
FREQUENCY c I
-
OF ts+
c
595
ts MUTANTS
RECOMBINANTS
(2) B.8
0.9
1.0
ts: arg
x ts+B
2rgx3 28gx149 3x149 23gx104 3x104 104x149
q q q q fg
2x104
q q q q
2oxarg
•J
2rgxa 2x149 3x2
q 20x3 q aogx22 pJ 20x22 q 20x9 q 2rgx9 q 20x149
-I-
Eil
= -
FIG. 5. Mean tsf recombination frequencies from those crosses showing additive map distances; the width of the stippled boxes shows the discrepancies in additivity. The values in the squares are the number of crosses made. The upper of two frequencies for a particular cross represents the standardized, and the lower the unstandardized, frequency (see text); a single frequency is the standardized one, except that no standards were available in the tests made for the cross 20 X $2. The symbol n represents the frequency of ts+ g recombinants in crosses with ts-28 g, from which the locus for guanidine resistance (g) is derived. The map at A (L-28 being arbitrarily fixed at 0.1%) represents the relative positions of those mutants showing additivity, together with others (ts-46, -96, -44, -150, -94, -155, -89) whose positions have only been determined in crosses with k-28 g, and with the loci of the major defects of some double mutants (k-5, -23, -99, -18, -37). The recombination frequencies from crosses of ts-28 g with ts-46, 44, 150, and 89 (not circled) could only be determined by assay in 200 pg of guanidine per millilitre; frequencies from crosses of ts-28 g with all other mutants (circled) were determined by assay in presence and absence of guanidine. The lengths of the horizontal bars at the positions of some main mutants represent the 95% confidence limits for the means of their self crosses. The relative position of loci within a bracketed group is uncertain. 15 (ts-2, -3, 428 and -104) could be shown to have all the criteria of single mutants. The proportion identified as double mutants
(ts-5, -9, -18, -19, -22, -2S, -37, -58, -99) was within the limits to be expected from their frequency of isolation (Cooper et al., 1966). The remainder (ts-20, -44, -46, -89, -94, -96, -149, -150 and -155) were probably single
mutants, but were either at the extremes of the map or could only be mapped by means of crosses with ts-28 g. The finding of only a 50% increase in recombinant frequency between 3.1 and 7 hours after infection, times representing, respectively, a minority and the large majority of RNA replicative events, indi-
596
COOPER
&es that the mating events are not necessarily linked with replicative events. Their markedly nonrandom occurrence with time suggests that multiple rounds of mating do not occur. In fact’, most mating events occurred less than 3 hours after infection, when RNA replication had barely begun, and so progeny strands did not as a rule participate in mating. Since certain sequences (e.g., 20-28-g-S) are proved by three-factor crosses and the recombination frequencies are additive, it follows that each parental strand contributes equally and that the recombination should be reciprocal. However, doubly defective recombinants were not sought, and results have been given only in terms of the recombination frequencies yielding ts+. The frequencies found (up to 0.85 %) were similar to those of Hirst (1962) and Ledinko (1963). There is a close correspondence between the genetic map given above and the physiological defects of the ts mutants (Cooper et al., 1966; Wentworth et al., 1968). Particularly interesting is the fact that the g locus lies in the region concerned with structural (virion) proteins. These relations will be considered in detail elsewhere (Cooper, 1968). REFERENCES BREEZE, D. C., and SUBAK-SHARPE, H. (1967). The mutability of small-plaque-forming encephalomyocarditis virus. J. Gen. ViiroZ. 1, 81-88.
COOPER, P. D. (1961). An improved agar cellsuspension plaque assay for poliovirus: some factors affecting efficiency of plating. Virology 13, 153-157. COOPER, P. D. (1964). The mutation of poliovirus by 5-fluorouracil. Virology 22, 186-192. COOPER, P. D. (1965). Rescue of one phenotype in mixed infections with heat-defective mutants of type 1 pohovirus. Virology 25, 431-438. COOPER, P. D. (1968). The genetic analysis of poliovirus. In preparation. COOPER, P. D., JOHNSON, R. T., and GARWES, D. J. (1966). Physiological characterization of heat-defective (temperature-sensitive) poliovirus mutants: preliminary classification. Virology 30,638-649. EDGAR, R. S. (1958). Mapping experiments with rI1 and h mutants of bacteriophage T4D. Virology 6, 215-225. FENWICK, XI. L., and COOPER, P. D. (1962). Early interactions between poliovirus and ERK cells: some observations on the nature and significance of the rejected particles. Virology 18, 212-223. HIRST, G. K. (1962). Genetic recombination with Newcastle disease virus, poliovirus and influenza. Cold Spring Harbor Symp. Quant. BioZ. 27,303-308. LEDINKO, N. (1963). Genetic recombination with poliovirus type 1. Studies of crosses between a normal horse serum-resistant mutant and several guanidine-resistant mutants of the same strain. Virology 20, 107-119. WENTWORTH, B. B., MCCAHON, D., and COOPER, P. D. (1968). Production of infectious RNA and serum-blocking antigen by poliovirus temperature-sensitive mutants. J. Gen. ViroZ. 2, 297307.
36, 584-596 (1968)
A Genetic
Map
of Poliovirus
Temperature-Sensitive
PETER
Mutants
D. COOPER
Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, Australia Accepted November IO, 1967 and the The conditions affecting recombination between ts mutants of poliovirus assay of tsf recombinants were standardized. The mean recombinant frequencies were then reproducible and characteristic of each pair of mutants, being between 5.2 and 31.4 times the total background reversion rates and ranging from 0.0270 t,o 0.8570. Certain genetic sequences were proved by three-factor crosses involving a mutant adapted to guanidine resistance (h-28 g), and these showed that recombination frequencies were additive. An additive linear genetic map comprising one linkage group was obtained. The g character of ts-28 g seemed to result from mutation at a single site that could be accurately located near the middle of the map. Recombination was nonrandom with time, most of the mating events occurring early in the growth cycle. INTRODUCTION
Temperature-sensitive (ts) mutants derived from a ts+ strain of poliovirus (Cooper, 1964) have been examined both by a complementation test (Cooper, 1965) and in terms of defects in physiological function (Cooper et al., 1966) and in synthesis of RNA and serum-blocking antigen (Wentworth et al., 1968). Recombination between poliovirus strains (Hi&, 1962; Ledinko, 1963) has shown the possibility of genetica,lly mapping these mutants. Much of the work described below is a technical evaluation of mapping experiments, in which ts+ recombinants were scored selectively. This work has led to a reproducible and additive genetic map of the mutants, which is also presented. As there is no published precedent for a quantitative map obtained by recombination analysis of a virus growing in animal cells, or of an RNA-containing virus growing in any cell, particular attention has been paid to possible causes of variability in recombination frequencies. MATERIALS
AND
METHODS
The parental tsf strain of poliovirus type 1 and the ts mutants have been described previously (Cooper, Virus
and cell strains.
584
1964; Cooper et al., 1966). Virus was grown and assayed as before in U cells, which were treated with 100 pg/ml of kanamycin sulphate for the 24 hours before harvest to minimize contamination with mycoplasma. All assays, viral growth, and preparations of celIs for viral growth were made with one or two large batches of pretested serum, agar, and medium components. The guanidineresistant strains ts-28 g and ts+ g were separately adapted from the guanidine-sensitive (gf) strains ts-28 and tsf, respectively, bv Dr. B. B. Wentworth, by alternate passage and cloning in the presence and absence of guanidine in a manner similar to that described by Ledinko (1963). In case the ts defect of ts-28 had been affected by the adaptation and selection of ts-28 g, the two ts loci were shown to be cIoseIy Iinked by the lack of recombinants formed in standard crosses of 28 g X 28 (mean recombination frequency = 0.003%), and by the frequencies in crosses of 20 X 28 g (mean = 0.054 %, parallel crosses of t.+20 X k-28 giving 0.049 %), and in crosses of 28g X 22 (mean = 0.249 %, va,Iues for 28 X 22 being 0.201%). Neither difference’ between k-28 and ts-28 g crosses is significant (p > 0.05) by analysis of the standard error of the
GENETIC
MAP OF POLIOVIRUS
difference. As the reversion frequencies and other characteristics of ts-28 and ts-28 g were similar, it is presumed that the ts defect had remained unchanged through the adaptation procedure. Prepamtion of mutant stocks for recombination tests. Small limited-passage stocks of mutant, were prepared with low wild-type content, and then partially purified. For this purpose, several “ministocks” were made by infecting tubes of lo5 cells with the maximum amount (50-200 PFU) of cloned mutant, stocks that would allow less than one tube in 20 to receive 1 PFU of revertant ts+. This scheme was preferred to recloning, to give t’he mutants as large an advantage in growth over revertants as possible. The ministocks were then assayed for wild-type content and for efficiency of plating at 39.3-39.7” compared with 37” (see below). Only t,hose mutants were used that gave < 1O-4 mutant plaques (< 1 mm) at restrict’ive temperatures; t’he rejection procedure on the basis of ts+ plaques (>2 mm) is given below. Three monolayers of 5 X lo6 cells were then each infected from each ministock tha,t contained the least tsf, and frozen after S hours’ growth at 36”. Several such stocks were made for each mutant, and those containing t’he least wild type were pooled; this method gave ts+ contents >{o-Koo that of cloned st’ocks for most mutants. Stocks of about 5 X lo8 PFU were the smallest suitable as perma.nent references, and mutants that failed after several attempts to give ts+ contents of <10e4 in such stocks were rejected. Smaller stocks containing up t’o 5 X 10e4 ts+ were used to map less stable mutants in a few crosses. Stocks Ivere clarified (20 minutes at 200 rpm) and recentrifuged for 90 minutes at 39,000 rpm in a Spinco 40 rotor. After standing at 4” overnight in 0.5 ml of 0.9% NaCl solution, the pellets were resuspended, trea,ted with 1 ml pH 2.5 glycine buffer (0.05 ill, Fenwick and Cooper, 1962) and neut’ralized after 3 minutes with 10 ml Eagle’s medium. This treatment, which presumably disaggregated the inoculum, was the greatest single factor in increasing reproducibilit,y of recombination; it increased titers up to 2-fold, and enhanced up to g-fold
Is MUTANTS
585
both the adsorption ability and recombination efficiencies. The virus was recentrifuged at 39,000 rpm and the pellet, after standing at 4” overnight in 1 ml Eagle’s medium plus 0.5 % calf serum, was resuspended and the concentrates pooled and reclarified. This material (5 to 10 X lo* PFU/ml) was st,ored in 0.2-ml amounts in sealed ampoules at - 45”. Standard yecombination test. The following conditions give maximum recombination frequencies, and were closely followed. Cells from semiconfluent (2- to 4-day) Roux cultures were resuspended to 2.5 X lo5 cells/ml in Eagle’s spinner medium containing 10 % of Difco tryptose phosphate broth, 7.5 % calf serum, 2.5% fetal calf serum, 1 mg/ml CaCL, and 0.77 mg/ml NaHCOs, the mixture having been previously gassed with 5 o/o CO* in air. One millilitre was added to stoppered flat-bottomed tubes of 20 mm internal diameter, and incubated for 20 hours at 37.2”, then washed twice with 2 ml of PBS. Fluid was thoroughly aspirated, and 0.08 ml of virus inoculum was added. A fresh ampoule of each special mutant stock (see above) was diluted to 1.5 X 10” PFU/ ml in Eagle’s medium. For test crosses, equal volumes of this suspension were mixed before inoculation, giving an input of 24 PFU/ cell and a measured adsorbed multiplicity of lo-15 PFU/cell of each mutant; for selfcrosses, the suspension was added undiluted (input = 48 PFU/cell), and for “controls” the suspension was diluted 1:2. The adsorption efficiencies were the same in self-crosses, controls and mixtures. Each cross was performed in duplicate and assayed separately, using 2 or 3 plates or bottles per dilution, with appropriate self-crosses and controls. Each experiment included cultures infected with tsf and t.9 g. The inoculated tubes were restoppered and shaken at 10-15’ for 4 hours (giving 4080 % virus adsorpt’ion), then 0.92 ml of Eagle’s medium (at pH 7.2 and containing 100 pg/ml of cycloheximide, 10% tryptose phosphate broth, and 5% calf serum) was added, the cultures placed in a 37.2” water bath for 20 minutes, and then chilled. The supernatant was removed for assay of unadsorbed virus, and t’he cultures washed
586
COOPER
twice. One millilitre of Eagle’s medium containing 10% tryptose phosphate broth, 5% calf serum and 0.28 mg/ml NaHC03 was then added, and the tubes frozen after a further 9 hours at 37.2”: the pH remained about 7.2 throughout. After thawing, the infectivity was stable at 4“ and pH < 7.3. Viral assay. Agar cell-suspension plaque assays involved the galactose medium (Cooper, 1961), to which was added a total of 8 % calf serum, 5 % of a 10% skim milk powder suspension, and 1 mg/ml of Difco tryptose powder. The ts defect is often associated with cystine dependence (McCahon and Cooper, unpublished data), and so leak rates were much reduced by omission of lactalbumin hydrolyzate and yeast extract at 39.3-39.7” : the recombination frequencies and tsf plating efficiencies were not affected. For assays of guanidine-resistant virus, an Analar guanidine carbonate solution adjusted to pH 7-8 with 10 N HCl was added to both agar layers to give 200 pg of guanidine carbonate per millilitre. Cystinedeficient medium was not used in presence of guanidine carbonate at 200 pg/ml level. Guanidine carbonate (10 pg/ml) was added t.o all assays of k-28 g and ts+ g recombinants to satisfy a slight guanidine dependence; this dose did not affect plaque size or number of any guanidine-sensitive (g+) strain, and g plaques at 37”-39.7” in guanidine carbonate at 200 pg/ml were the same size and number as at 10 pg/ml. In controls of every strain included in all experiments, guanidine carbonate at 200 pg/ml always reduced the plaque count of g+ strains to negligible levels (< lo-“), at 37-39.7”. All assays were diluted through 0.05 M glycine buffer, pH 2.5, to eliminate aggregation as a source of variability and to increase sensitivity. The total virus produced was assayed in petri plates (64 hours at 37”), and the ts+ virus was assayed in bottles immersed for 52 or 64 hours in a water bath at 39.3”, 39.5”, or 39.7”, all f0.05’. The temperature and time of ts+ assays were arranged to give a mutant efficiency of plating of < 10e4 and a loss of tsf plaques of <20 % in the control assays; 39.5” for 64 hours in cystine-deficient medium was usually satisfactory for all the main mutants.
All plaques visible were counted; if in any plaques (< 1 mm) assays the mutant amounted to >lO% of the tsf plaques, (>2 mm) in the self crosses, all samples in that experiment were reassayed at a higher temperature. If ts+ assays differed with temperature, or if k-28 g assays differed with guanidine concentration, t.he ts+ or ts+ g recombinant titres were adjusted to the titres to be expected at 37” and 10 pg guanidine carbonate per millilitre; correction factors were within the range 0.7-1.3. All assays of one experiment were made at the same time. Calculatim of recombination frequency. The recombination frequency is given as the recombinant tsf content expressed as a percentage of the total virus (PFU/ml assayed at 37”). The recombinant tsf content is derived by subtract’ion from the total ts+ content of the ts+ content to be expected from spontaneous reversion. RESULTS Obstacles to Mapping
Poliovirus ts Mutants
Preliminary recombination tests, using the methods of Ledinko (1963), produced progeny containing four to ten times as much ts+ virus as did the self-crosses but attempts to obtain a self-consistent genetic map by such tests were unsuccessful. There were found to be four reasons for this: (1) the high and variable ts+ content of the stocks, despite frequent recloning; (2) loss of ts+ plaques or poor control of leak in the 39.5” assay of ts+ contents; (3) the presence of many double mutants; and (4) large variations between experiments (> lo-fold) in the frequencies of recombination. Factors (1) and (2) were minimized or eliminated by the procedures described in Materials and Methods. Some aspects of the assay are described in the next section; factors (3) and (4) are discussed in subsequent sect’ions. The Assay of ts+ in Presence of Excess t-s Virus Reconstruction experiments, in which 100 PFU of tsf were assayed in presence of varying doses of any main mutant, showed that no ts+ plaques were masked by excess mutant virus in a standard assay at 39.3”. The ts+
GENETIC
MAP
OF POLIOVIRUS
plaque count was still proportional to input in the presence of sufficient mutant (6 X lo5 PFU) to infect 10% of the cells; in most assays, only 1% cells were infected. The detection efficiency for t& was about lo+ for very defective mutants. Assay of any two mutants (5 X lo5 PFU of each) in one bottle gave ts+ plaque formation ehual to the sum of the mutants assayed singly: no recombination occurred in the bottle.
Singularity
Double mutants were the main cause of nonadditive recombination frequencies, and accordingly the singularity of the mutants’ defects was examined. Relevant data are given in Table 1. The main mutants are in italicsThe reversion frequencies in Table 1 are calculated from the mean wild-type content
TABLE SOME
PROPERTIES
OF POLIOVIRUS
-
-
ts+ content x 10-4
Appr0x.b reversion frequency
15
1
ts MUTANTS RELEVANT THEY CONTAINS
- I I
Character of revertants
TO THE NUMBER
OF ts DEFECTS
Character of recombinants
.g&
Plaqued size
587
Ls MUTANTS
e.0.p.
change 39.3-39.70 l/P
.dditivity of Lapdistance! l/7”
nd/nd wt/nd nd/nd nd/nd nd/nd ts/ts
nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd
Yes/Yes Yes/Yes nd/‘No Yes/No nd/nd No/No
Single Single Double Double Double Double
nd/wt
nd/wt
rid/Yes
wt/nd nd/nd wt/wt nd/nd nd/nd nd/nd nd/nd nd/nd
wt/nd nd/nd wt/wt nd/nd nd/nd nd/nd nd/nd nd/nd
Yes/No rid/No Yes/Yes Yes/Yes No/rid nd/nd nd/nd nd/nd
Prob. single Double Double Single Single Double ? ? Prob. single Prob. single Prob. single Double Single Prob. single Prob. single Prob single
Stock
Self-crossc
2 3 5 9 18 19
2
3.75 1.60 f 0.4: 2 6.46 2.2 0.8
nd 0.56 0.70 2.2 0.77 0.28
wt wt s 9 S s
20
<0.2
1.93 f
0.67
wt
wt
22 23 28 28 37 44 46 89
2.33 f 1.0 0.81 2.6 0.90 1.26 f 0.6~ 0.44 2.07 f 0.41 0.72 2.1 0.73 nd nd nd >40 100-600 >30
wt S wt wt s
nd wt wt
nd wt wt
nd nd wt
wt/s wt/s wt/wt wt/wt s/rid wt/nd wt/nd wt/nd
nd
wt
wt
wt/nd
nd/nd
nd/nd
nd/nd
nd
wt
wt
wt/nd
nd/nd
nd/nd
Yes/rid
11.5 5.2 5.23 f 1.7:
4.0 nd 1.8
wt wt wt
wt nd wt
wt/wt wt/wt wt/nd
nd/nd nd/nd nd/nd
nd/nd nd/nd wt/nd
Yes/No Yes/Yes Yes/rid
15
nd
wt
wt
wt/nd
nd/nd
nd/nd
nd/nd
nd
wt
wt
wt/nd
nd/nd
nd/nd
nd/nd
39.339.Y
onclusions on 8ingularity
.o.p. 39.3’ f is00
-
94
5-10
96
1
99 104
1-2 nd 1.5
149 150
nd
155
.lO
-
0.5:
30 3.3
>60
nd wt ts
wt nd
wt
ts
ts
ts
wt/wt wt/wt wt/s wt/wt wt/nd s + wtl s + wt nd/wt
a Abbreviations: wt = wild type; s = small; nd = not determined * Per particle per duplication X 10m4. c Mean of all values. d At restrictive temperature. e In crosses with single mutants located to left or right (Z/r) in the map,
588
COOPER
of the self crosses, using method 3 of Breeze and Subak-Sharpe (1967); in some cases the 95 % confidence limits of the mean are also given. The most stable mutants reverted at a rate of 0.5 to 1 X 1O-4 per particle per duplication. Columns 5 and 6 of Table 1 give the character of revertant plaques, as determined in 39.5” assays of the self crosses. In some cases the decrease in plaque number in duplicate bottles incubated at higher temperatures was compared with wild-type values; a decrease >3 times the decrease in tsf plating efficiency indicates that the revertants were ts. Columns 7-10 give similar data for characters of recombinants, and the last column gives final conclusions on singularity. Table 1 shows that the reversion frequencies of these mutants are useless as criteria of singularity, presumably because all mutants have been highly selected for stability. Revertant character (plaque size and change of plating efficiency with temperature) apparently are fairly useful, but are not reliable, because of the probable presence of suppressor mutations elsewhere in the defective viral gene. Partial revertants from single poliovirus ts mutant’s are in fact of frequent occurrence (McCahon, personal communication). More reliable are the recombination characteristics. As double crossovers are unlikely, the provision of additive map distances and of solely wild-type recombinants with mutants t’hat map on either side are strong evidence of singularity, or of closely linked defects. Only ts-2, -3, -28, and -104 gave such evidence, but the mutants at the ends of the map (ts-20 and k-149, see below) behaved in all other respects like single mutants. Time of incubation. Ledinko (1963) suggested that poliovirus recombination frequencies increased about 2-fold during a single growth cycle. Preliminary experiments with ts mutants indicated that any increase was likely to be smaller than that, and this question was therefore tested more rigorously. A standard recombination test was made involving twenty replicates of the cross 28 9 X 3; ten replicates were harvested
3.1 hours and the remainder 7.0 hours after infection, when the average yields (with standard deviations in brackets) were, respectively, 29.8 (3.25) and 124 (9.0) PFU/ cell. The mean recombination frequencies and standard deviations for the two samples were, respectively, 0.279 (0.053) %, and 0.419 (0.0339) 70. Thus there was a small but significant (p < 104) increase in the recombination frequency, at times corresponding to a 417 % increase in virus content. E$ect of Multiplicity
of Infection
Ledinko (1963) also showed that variations in total multiplicities between 10 and 30 PFU/cell had no effect on recombination frequency provided that the input ratio remained between 3 and 0.5. This finding was confirmed for ts mutants in a standard recombination test of 20 X 28 g, in which the proportion of each mutant could also be determined in the yield (Table 2). Growth of ts-20 and ts-28 g in mixed infection was approxima.tely proportional to the input frequency ratio, and the recombination showed no systematic variation over a wide TABLE
2
EFFECT ON RECOMBINATION FREQUENCY IN THE CROSS~OX 28 ~OFVARYINGTHEPROPORTION OF INPUT VIRUS AT .4 CONSTANT MULTIPLICITY"
-
Input virus (proportion of ts-28 g) 1.0 0.9 0.8 0.6 0.5 0.4 0.2 0.1 0 ts+
/
Total yield PFU/ml x 10’
Proportion b-28 g
3.55 4.9, 3.25 6.0, 5.35 5.3,4.5 3.75,4.2 5.2, 4.5 2.15, 2.45 2.0,2.2 3.2 2.0
1.0 0.68, 0.68 0.56, 0.54 0.50,0.50 0.32,0.44 0.26,0.34 0.26, 0.16 0.10,0.091 0
-I ts+ content” (%) 0.042 0.080,0.100 0.125,0.149 0.118,0.165 0.198, nd 0.140, 0.162 0.221,0.198 0.180,0.114 0.030 00
0 The procedure was the standard recombination test given in Materials and Methods. The total input multiplicity was 40; nd = not determined. Values are the results from duplicate tubes. b Uncorrected for spontaneous reversion.
GENETIC
MAP
OF POLIOVIRUS
589
ts MUTANTS
and ts-28 y, ts-3, and ts-149, respecbively. The tests in each experiment were made and assayed on the same occasion, as always. They illustrate the type of result that was aimed for in the assays, and the way of obtaining the corrected recombination frequencies. Figures 1 and 2 show the plaque characteristics of the recombinant progeny of several crosses; such recombinants are typical of all crosses except those giving nonadditive recombination frequencies (see below).
range of ratios. Thus input ratios do not need to be precisely equal for maximum recombination frequencies. Effect of Spontaneous Reversion to ts+ Table 1 gives the t.s+content of the special recombination stocks, together with the mean Is+ content of the self-crosses made with them. The ts+ content increased IO-fold or more in this single cycle at 37.2”, presumably due to reversion rather than selection as the growth rates of t.@ and mutants were similar. In all “control” crosses (i.e., selfcrosses at half multiplicity) t’he ts+ content did not differ from that of the corresponding self-crosses; hence small changes in multiplicity did not affect reversion rates. Spontaneous reversion contributed relatively little to the tot’al ts+ content of recombination tests except for closely linked mutants, and the procedure for correcting for this is given in Materials and Methods. In the main crosses (20 X 28,28 X 22,20 X 22, 20 x 3, 28 x 3, 28 x 149, 20 x 149) the mean recombination frequencies exceeded the reversion background by, respect)ively, 5.2-, 12.5-, 15.7-, 31.4-, 30.0-, 21.8-, and 23-fold.
Determination of Some Crosses with ts-28 g
Sequences from
Certain mutants could unequivocally be assigned a sequence in the genetic map by a form of three-factor cross with ts-28 g. In standard crosses of 20 X 28 g, all recombinants in excess of the ts-28 g reversion background were g+, and this finding is illustrated in Table 3, Fig. 3 and, together with the plaque characteristics of the recombinants, in Fig. 2. The same was found in crosses of ts-5, -23, and -99 with ts-28 g, and ts-5, -20, -23, and -99 are accordingly assigned arbitrary positions to the left of ts-28 in the genetic map, and the g locus or loci to the right. Crosses between ts-28 g and the remainder of the mutants either produced mean recombination frequencies of co.3 %, which
Results of Crosses Using the Standard Method Tables 3 and 4 give the results of two experiments involving ts-20 and ts-28 g, TABLE
3
RESULTS OF THE CROSS 20 X 28 aa Percentage in yield of
PFU per plate or bottle6 Cross
ts-28 g control k-28 g self ts-20 control k-20 self ts+ control ts+ g control 20 X 28 g 20 X 28 g
37”, Ill: pg/ 83, 131, 85, 132, 76, 179 153, 112,
72 113 108 89 61 134 121
37”, 200 rg/ml 75, 87, 0, 0, 0, 157 58, 83,
82 90 0 0 0 71 58
39.5”, 10 e/ml
39.5”, 200 /Lg/ml
ts+
Is+ g
22, 23 20, 21 29, 28 24, 38 76 EO-2QO 117, 143 127, 154
20, 20 29, 35 0, 0 0, 0 0, 0 150-200 19, 17 28, 22
0.0289 0.0168 0.0289 0.0280 100 100 0.090 (0.065)~ 0.121 (0.095)c
0.0256 0.0364
D The procedure was the standard recombination test given in Materials and Methods. b Plates were incubated for 64 hours at 37” with 0.1 ml of lO+ dilution: bottles were incubated for 64 hours at 39.5” with 0.1 ml of lo+ dilution. The concentrations indicate the guanidine carbonate content of the assay medium. c Recombination frequencies after correction for spontaneous reversion are given in parentheses.
590
COOPER TABLE
4
RESULTS OF THE CROSSES 18 g X 3, R8 g X 1.49 and 3 X 14Sa PFU per plate or bottle*
Percentage in yield of
Recombination frequenciesc
Cross 37",200 ,&ml 28 g control 28 g self 3 control 3 self 149 control 149 self ts+ control ts+ g control 28gX3 28gXS 28 g X 149 28 g X 149 3 x 149 3 x 149
99 95 117, 95, 80, 78, 52, ca. 131, 95, 90, 103, 66, 58,
84 113 77 76 59 200 108 144 112 105 63 65
143 120 0, 0 090 0, 0 0, 0 0, 0 ca. 200 87, 73 76, 68 57, 60 72, 45 0, 0 0, 0
19 20 2, 5 1, 3 49, 49 41, 32 64, 69 ca. 200 89, 84, 90 105, 92, 83 141, 135, 110 142, 153, 147 38, 21 36, 37
32 19 0, 0 0, 0 0, 0 0, 0 0, 0 ca. 200 106, 82, 80 85, 89, 79 92, 71, 77 68, 72, 84 0, 0 0, 0
ts+
ts+ g
0.019 0.021 0.0035 0.0019 0.062 0.047 100 100 0.367 0.387 0.643 0.665 0.231 0.295
0.032 0.020
ts+
ts+g
-
-
-
-
0.347 0.367 0.605 0.665 0.194 0.258
0.266 0.253 0.288 0.259
0 The procedure was the standard recombination test described in Materials and Methods. * Plates were incubated for 64 hours at 37” with 0.1 ml of 1W6 dilution; bottles were incubated for 64 hours at 39.5” with 0.1 ml of 1OW’dilution for controls and self crosses, or 2 X low3 dilution for the recombination tests. The concentrations indicate the guanidine carbonate contents of the assay medium. c After correction for spontaneous reversion. d Corrected for enhancement of h-28 g in guanidine.
were the same when plated in presence or absence of guanidine (ts-9, -18, -96, -22, -94, and -155), or produced mean recombination frequencies of 0.4-0.8% when assayed in absence of guanidine but 0.360.39% in presence of guanidine (k-3, -2, -104, -149). Examples of the former type are given in Fig. 3 for the crosses 28 g X 22 and 28 g X 94; the differences between the assays in presence and absence of guanidine are not significant (p > 0.3). The latter type of cross is exemplified in Table 4 and in Fig. 4 for 28 g X 3 and 28 g X 149. Thus all the recombinants in crosses of L-28 g with ts-9, -18, -96, -22, -94 and -155 appeared to be g, while in crosses of ts 28 g with k-3, -2, -104 and -149 a fixed number of recombinants was g and a variable number was gf. The recombinants in the latter type of cross were confirmed as a mixture of g and gf by isolating wild-type plaques at random from a 39.5” assay of the cross 28 g X 149. The efficiencies of plating at 39.5” of the resulting clones were all wild-type (0.5-1.5).
The unselected g marker was then scored on 42 of the clones by assay at 37” in presence (100 and 200 pg/ml) and absence of guanidine carbonate: 28 were g and 14 were gf ; none showed intermediate sensitivity, and their g or g+ behavior wa.s identical with that of k-28 g and ts+, respectively, in parallel assays. This proportion of g particles among the tsf progeny (67 %) was not significantly different from that (about 53%, see below) to be expected from the mean recombination frequencies. Thus the presence of 100 % g recombinants for k-9, -18, -96, -22, -94 and -155 placed these mutants between ts-28 and the g locus, and the presence of a mixture of gf and g recombinants placed k-3, -2, -104 and -149 to the right of the g locus. Certain other leaky or unstable mutants (ts-44, -46, -89, -150) could be assayed in crosses with h-28 g in the presence of guanidine, as their background (leak plus reversion) was then reduced to that of ts-28 g; these mutants produce g recombinants in a frequency of
GENETIC
MAP
OF POLIOVIRUS
Is MUTANTS
FIG. 1. Plaque characters of is+ and of recombinants. The crosses illustrated (all plated in O-l-ml amounts of the dilutions given) are: 3 X 2 (lo+), 3 X 104 (lo+), 3 X 149 (2 X 10V3), and 28 g X 3 (2 X contained 200 pg of guanidine carbonate per millilitre in com10-3). The bottle marked “guanidine” plete medium, and the other bottles all contained the drug at 10 pg/ml in cystine-free medium; bottles were incubated at 39.5” for 64 hours.
< 0.3 %, and so are assigned to loci between h-28 and 9. In summary, these sequences may be expressed: (5, 20, 23, 39)28 -(.3, 18, 22, 44, @, 80,94,150,155)-g(2,3,104,149),
with the order of mutants remaining undetermined.
in parentheses
Acldihity oj Map Distances and Reproducibility of the Test The sequences from the previous section were fairly accurately reflected in the mean map distances for certain triads of mutants,
that is, the map distances were additive; Fig. 3 illustrates this with the results of all standard crosses for the triad h-20 -28 -22. Similar results were obtained for the triad ts-20 -28 -9, and with several others as elaborated below. The relative frequencies of t.riads for which three-factor crosses were not available also appeared to give reliable sequences, provided that sufficient tests were made. Figure 4 illustrates this with the results of standard crosses for 28 g X 3, 28 9 X 149, and 3 X 149. The mean posi-
COOPER
FIG. 2. Plaque characters of ts+ and of recombinants obtained from the experiment of Table 3. The cross illustrated (all samples plated in O.l-ml amounts of the dilutions given) is 28 9 X 20. The bottles marked “guanidine” contained 200 rg/ml of guanidine carbonate, and all the remainder contained the drug at 10 pg/ml; bottles were incubated at 39.5” for 64 hours.
tions for the g locus were closely similar for both types of cross, and the mean map distances were additive. However, in repeated crosses of most single mutants made and assayed on different occasions the absolute frequencies varied over about a 2-fold range, and so the source of this variability was examined. The lower part of Fig. 4 shows that the variability within an experiment was less than between experiments. This is confirmed by comparing
the standard deviation of the frequencies for the ten 7-hour replicates of the cross 28 g X 3 mentioned above (8.1% of the mean) with that of ten 28 g X 3 crosses made on five different occasions (28.0% of the mean). Similar analysis of some twenty other crosses gave similar results, namely that the root mean square of the difference from the mean of duplicate tests made on the same occasion was S-10% of the mean, whereas the standard deviation of tests (ten or more)
GENETIC FREQUENCY
OF tS+ 0
0.1
MAP
RECOMBINANTS 0.2
0.3
OA
OF POLIOVIRUS (X)
0.5
0.6
289X22
Is MUTANTS
593
for the crosses 28 g X 3, 28 g X 149, and 3 X 149 (0.495%, 0.775 %, and 0.280% respectively) were taken as standards for ts+ recombinants, and 0.364% as the standard for g recombinants in the crosses 28 g X 3 and 28 g X l&9. The correction factors ranged from 0.696 to 2.22 (mean 1.18) for the ts+ recombinants, and from 0.730 to 1.37 (mean 1.00) for the g recombinants. This standardization had very little effect on the mean recombination frequencies (see Fig. 5 below), but standard deviations were reduced from about 28% to about 10% of the mean. The procedure finally adopted was to accept the mean standardized frequencies of four crosses made on two different occasions. The Genetic Map
asgx
94 I-
I 0
I
I I
f&B
FIG. 3. Unstandardized ts+ recombination frequencies from the crosses 28 g X 22, 20 X 28 g, 20 X 22, and 28 g X 94, made on several different occasions; is-28 is arbitrarily located at 0.1%. The bracketed values, g, were obtained from 39.37” assays containing guanidine carbonate (200 pg/ml). Values to the right are the mean recombination frequencies of that cross; values at A are the mean of all crosses for the triad 20-28-22 and show the additivity obtained; the map at B is derived from the values in this figure; the order of ls-22 and -94 is undetermined.
made on different occasions was 24-28 % of the mean. This difference depended more on the day-to-day variations in the sensitivity of the assay system than on assay errors or variations in the recombination test, since absolute frequencies often varied 2-fold in assays of the same test repeated on different occasions, while the relative frequencies of different crosses assayed together stayed the same. Accordingly, the day-to-day variations were minimized by normalizing each test cross in terms of standard crosses made and assayed on the same occasion. The mean recombination frequencies shown in Fig. 2
The mean standardized values for the crosses giving additive recombination frequencies enabled a genetic map of the main mutants (ts-20, 28, 3, 2, 104, 149) to be constructed (Fig. 5). The standardized and unstandardized values were closely similar (the differences were all within the 95 % confidence limits of the means of the unstandardized values). In all individual experiments the standardized or unstandardized frequencies between the main mutants showed the same map sequence. The 95 % confidence limits given in Fig. 5 for t’he means of some self-crosses show that the variability from spontaneous reversion was not large. This background determined the smallest detectable frequencies, which were 0.02-0.05 YCfor most crosses. The limit for g crosses with ts-28 g was 0.01%. The positions for g given by ts-28 g crosses with ts+ and with four ts mutants were closely similar. In addition, ts+ and tsf g plaques in assays of these crosses were exactly alike in size (see Fig. 1) and no tsf progeny of intermediate guanidine sensitivity were found in the isolates from the cross 28 g X 149 described above. Hence the g character of ts-28 g is probably determined by a single locus, or by several closely linked loci between ts-22 and ts-3. Figure 5 also shows the mean standardized frequencies for certain unstable mutants that
594
COOPER FREQUENCY
OF tS+
RECOMBINANTS
(%)
2agx3
3x
149
A I 3
apt.1
apt.
2
2ag x 3 2ag x 149 3x149
1‘.
‘22 l
:: :49
. 3x149
FIG. 4. Unstandardized ts+ recombination frequencies from the crosses 28 g X 5, 28 g X 149 and 3 X 149; k-88 is arbitrarily located at 0.1%. The squares on the lines for crosses involving h-28 g represent the frequencies for ts+ g recombinants, determined by 39.3-7” assays containing guanidine carbonate (200 pg/ml). The values at A are the mean of the crosses given for the triad 38-S-149 and show the additivity obtained; the inferred map locations of the mutants are also given. The lower part of the figure gives the results of paired crosses in two illustrative experiments made on separate occasions (see text).
could only be crossed with ts-28 g. Some of these (ts-44, -46, -89, -150) could only be assayed in presence of guanidine carbonate (200 pg/ml). This device could be used only for mutants with loci between 28 and g; they fall into two groups, one near k-28 and the other near k-22. The frequencies in crosses between some main mutants and t-s-5, -9, -19, -22, -23, -S7, and -99 were nonadditive, that is, the sum of the frequencies between these mutants and two main mutants was x-J$o of the frequency between the main mutants themselves. Other evidence indicates that most of these mutants contain multiple ts defects. Two of the double mutants (b-9 and -22) gave additive frequencies and ts+ plaques with main mutants to their left in the map, but not to their right; thus only their major defects are located in Fig. 5, and the minor ones lie to the right. In certain other crosses involving double mutants (28 + 99,20 X 23, 20 X 5, I.43 X 37), the recombinants were all wild type, and so these crosses probably reflect the true map positions of the major defects of ts-5, -23, -37 and -99. Other crosses involving these double mutants yielded mixed recombinants that all or mainly produced Is plaques at 39.3-5”.
The relative positions in the map of loci within the groups (5, 23, 99), (46, 18, 96, 9, 44, 150), and (22, 155, 89, 94) are undetermined. DISCUSSION
This work shows that one can select a group of poliovirus ts mutants between which the recombination frequencies are all additive. The selfconsistent nature of the genetic map obtained, in particular from the three-factor crosses, dispels any doubt that the enhanced ts+ contents of the progeny result from genetic recombination. The reproducibility and error of the method are satisfactory: the coefficients of variation of the unstandardized frequencies are similar to those of phage T4D (Edgar, 1958), and after standardization resemble those of other phage crosses. Poliovirus strains giving additive map distances also behaved as single mutants by all other criteria, while those that did not almost all presented other evidence of being double mutants. It was particularly important to resolve this question of singularity, as so large a portion (9/15) of the apparently
useful
mut’ants
turned
out
to
have more than one ts defect. Only 4 of the
GENETIC
MAP OF POLIOVIRUS
FREQUENCY c I
-
OF ts+
c
595
ts MUTANTS
RECOMBINANTS
(2) B.8
0.9
1.0
ts: arg
x ts+B
2rgx3 28gx149 3x149 23gx104 3x104 104x149
q q q q fg
2x104
q q q q
2oxarg
•J
2rgxa 2x149 3x2
q 20x3 q aogx22 pJ 20x22 q 20x9 q 2rgx9 q 20x149
-I-
Eil
= -
FIG. 5. Mean tsf recombination frequencies from those crosses showing additive map distances; the width of the stippled boxes shows the discrepancies in additivity. The values in the squares are the number of crosses made. The upper of two frequencies for a particular cross represents the standardized, and the lower the unstandardized, frequency (see text); a single frequency is the standardized one, except that no standards were available in the tests made for the cross 20 X $2. The symbol n represents the frequency of ts+ g recombinants in crosses with ts-28 g, from which the locus for guanidine resistance (g) is derived. The map at A (L-28 being arbitrarily fixed at 0.1%) represents the relative positions of those mutants showing additivity, together with others (ts-46, -96, -44, -150, -94, -155, -89) whose positions have only been determined in crosses with k-28 g, and with the loci of the major defects of some double mutants (k-5, -23, -99, -18, -37). The recombination frequencies from crosses of ts-28 g with ts-46, 44, 150, and 89 (not circled) could only be determined by assay in 200 pg of guanidine per millilitre; frequencies from crosses of ts-28 g with all other mutants (circled) were determined by assay in presence and absence of guanidine. The lengths of the horizontal bars at the positions of some main mutants represent the 95% confidence limits for the means of their self crosses. The relative position of loci within a bracketed group is uncertain. 15 (ts-2, -3, 428 and -104) could be shown to have all the criteria of single mutants. The proportion identified as double mutants
(ts-5, -9, -18, -19, -22, -2S, -37, -58, -99) was within the limits to be expected from their frequency of isolation (Cooper et al., 1966). The remainder (ts-20, -44, -46, -89, -94, -96, -149, -150 and -155) were probably single
mutants, but were either at the extremes of the map or could only be mapped by means of crosses with ts-28 g. The finding of only a 50% increase in recombinant frequency between 3.1 and 7 hours after infection, times representing, respectively, a minority and the large majority of RNA replicative events, indi-
596
COOPER
&es that the mating events are not necessarily linked with replicative events. Their markedly nonrandom occurrence with time suggests that multiple rounds of mating do not occur. In fact’, most mating events occurred less than 3 hours after infection, when RNA replication had barely begun, and so progeny strands did not as a rule participate in mating. Since certain sequences (e.g., 20-28-g-S) are proved by three-factor crosses and the recombination frequencies are additive, it follows that each parental strand contributes equally and that the recombination should be reciprocal. However, doubly defective recombinants were not sought, and results have been given only in terms of the recombination frequencies yielding ts+. The frequencies found (up to 0.85 %) were similar to those of Hirst (1962) and Ledinko (1963). There is a close correspondence between the genetic map given above and the physiological defects of the ts mutants (Cooper et al., 1966; Wentworth et al., 1968). Particularly interesting is the fact that the g locus lies in the region concerned with structural (virion) proteins. These relations will be considered in detail elsewhere (Cooper, 1968). REFERENCES BREEZE, D. C., and SUBAK-SHARPE, H. (1967). The mutability of small-plaque-forming encephalomyocarditis virus. J. Gen. ViiroZ. 1, 81-88.
COOPER, P. D. (1961). An improved agar cellsuspension plaque assay for poliovirus: some factors affecting efficiency of plating. Virology 13, 153-157. COOPER, P. D. (1964). The mutation of poliovirus by 5-fluorouracil. Virology 22, 186-192. COOPER, P. D. (1965). Rescue of one phenotype in mixed infections with heat-defective mutants of type 1 pohovirus. Virology 25, 431-438. COOPER, P. D. (1968). The genetic analysis of poliovirus. In preparation. COOPER, P. D., JOHNSON, R. T., and GARWES, D. J. (1966). Physiological characterization of heat-defective (temperature-sensitive) poliovirus mutants: preliminary classification. Virology 30,638-649. EDGAR, R. S. (1958). Mapping experiments with rI1 and h mutants of bacteriophage T4D. Virology 6, 215-225. FENWICK, XI. L., and COOPER, P. D. (1962). Early interactions between poliovirus and ERK cells: some observations on the nature and significance of the rejected particles. Virology 18, 212-223. HIRST, G. K. (1962). Genetic recombination with Newcastle disease virus, poliovirus and influenza. Cold Spring Harbor Symp. Quant. BioZ. 27,303-308. LEDINKO, N. (1963). Genetic recombination with poliovirus type 1. Studies of crosses between a normal horse serum-resistant mutant and several guanidine-resistant mutants of the same strain. Virology 20, 107-119. WENTWORTH, B. B., MCCAHON, D., and COOPER, P. D. (1968). Production of infectious RNA and serum-blocking antigen by poliovirus temperature-sensitive mutants. J. Gen. ViroZ. 2, 297307.