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Polymorphism in sedimentation velocity and density of λ bacteriophage
J. Mol. Biol.
(1968) 32, 513-520
Polymorphism
in Sedimentation Velocity and Density of 1 Bacteriophage L. PICA AND E. CALEF
International
Laboratory
(Received 14 August
of Genetics and Biophysics Naples, Italy
(C.N. R.)
1967, and in revised form 6 November 1967)
A new genetic characteristic of h bacteriophage has been detected. This characteristic concerns the sedimentation velocity of the whole particle in a CsCl preformed gradient. The detection of this new characteristic has allowed us to verify the genetic affinity between h crypticogen and h wild. The sedimentation velocity is probably determined by the same genetic factor which causes a light-density phenotype in some /\ strains (h wild). This can be stated because of the complete linkage found between the two determinants and because both characters display phenotypic mixing. that these two phages Genetic crosses between X erg and h ref have indicated differ in two factors determining the density in buoyant equilibrium centrifugation. The contrasting effect of these two factors confers on them equal phenotype status in assays of buoyant equilibrium density sedimentation; their progeny, however, displays two new densities as well as the parental typos.
1. Introduction Following the finding of the crypticogen h phage (A erg) (Calef & Fischer-Fantuzzi, 1965), so called because it is capable of giving cryptic deletions after ultraviolet curing, we have searched for other distinctive characteristics of this phage with respect to its presumed parent, h wild. While we were measuring the sedimentation behaviour in the CsCl gradient, we found that it was possible and advantageous t’o obtain density measurements in a preformed CsCl gradient. In these gradients it is also possible to observe a previously undetected polymorphism among different h strains, consisting in the velocity with which the particles reach equilibrium in a preformed gradient,.
2. Materials and Methods (a) Media The phage crosses were made in Tryptone broth (Bacto-tryptone vitamin B1 1 x 1OmB g/l.). The phages were titrated in plates containing the same medium
Difco
lo/,, NaCl O*5y0,
plus agar
(Oxoid)
at.
1.5%.
For dilution and suspension of purified phagcs we used (Weigle, Meselson & Paigen, 1959). The caesium chloride was obtained from Merck, Darmstadt. (b) Strains Bacteria: Escherichia coli. C600, indicator strain (Appleyard, 1954). CR63, selective strain for the host range (h) mutants 1956). 33
613
suspension
(Appleyard,
medium
McGregor
(SM)
Jz Baird,
614
L. PICA
AND
E. CALEF
U173, cryptic lysogenic strain (Fischer-Fantuzzi & Calef, 1964). U279, non-permissive strain for all “eus” mutants (suppressor sensitivity) Fantuzzi & Calef, 1964).
(Fischer-
(c) Bmtetiophuger, h wild obtained by induction of K12 wild-type strain (Lederberg t Tatum, 1946). h ref (Kaiser, 1957) and the mutants of h ref: Xc1 clear-plaque selection from A ref, A 8~5 (Campbell, 1961), h 8~~6 (Campbell, 1961), /\ h cl (Calef & Licciardello, 1960). )\ b2 (Kellenberger, Zichichi & Weigle, 1961). h erg (Cdef & Fischer-Fantuzzi, 1965) and its mutant: A sw9 cl sus54 obtained by recombination between X erg and Campbell strains. h s.d. super dense from crosses between X erg and A ref. (d) Phage terminology In order to avoid possible confusion in the terminology of h strains, we should emphasize the following: the h designated as wild is the one present in the strain of E. coli K12 as it was isolated from nature and described by Lederberg & Tatum (1946). The K12 strain was a gift from J. Lederberg to L. Cavalli. We received this strain from L. Cavalli in 1962. This phage makes small turbid plaques of rather unequal diameter. Often in the literature, by h wild or X + + -I- one indicates another A temperate phage selected by Kaiser (1957). This phage given to us by A. D. Kaiser in 1956 produces large turbid plaques. This is the one we call X reference.t This strain is identical, from the point-of-view of density, plaque morphology, ability to lysogenize and origin, to the h used by Weigle et al. (1959) which they call “h ref Kaiser 1956”. We use this term (A ref) in spite of the facts mentioned by Kellenberger et al. (1961, p. 399, footnote), because the Kaiser X strains to which we apply it has been for many years the reference in X phage biology. (0) Crossing procedure The vegetative phage crosses were carried out according to Kaiser (1955). (f) Caesium chloride preformed gradient sedimentation procedure Baldwin & Shooter have extensively studied preformed CsCl gradients (1963). They have already pointed out the advantages and the limits of this method in the determination of density and molecular weight of macromolecules. These authors used this technique for the separation of X phage of diierent density in a mixture. In their paper, the time to reach the equilibrium under many conditions was studied. At the time the present experi. ments were performed, we were unaware of that paper. The gradients were made in Spinco SW39 tubes with the gradient device (described by Martin & Ames, 1961) consisting of two cylindrical wells joined by means of a small channel. One of the two wells contains 2 ml. of a solution two-third saturated with &Cl at room temperature; density = 1.627 g/ml. The other contains 2 ml. of a solution one-third saturated with CsCl; density = l-318 g/ml. The solutions were made in 0.015 M-Tris buffer, pH8. The flow of the solution was kept at 2 ml./min. Over the preformed gradient we layered 0.2 to 0.4 ml. of phage suspension, the titre of which was not less than 1 x lOi particles/ml. The runs were carried out in a Spinco preparative ultracentrifuge model L at a speed of 27,000 rev&in at a temperature of 18°C. The density values are calculated by measuring the refractive index of the solutions for sodium light at 25’C in an Abbe refractometer and the subsequent use of the empirical formula of Weigle et cd. (1959). We shall call this technique P.G.S. We have also carried out density measurement with CsCl gradients according to the standard method of buoyant equilibrium density sedimentation described by Meselson, Stahl & Vinograd (1957). We shall call this technique B.E.D.S. t Editor’s note: To avoid misunderstanding, it should be pointed out that the terminology of Drs Pica and C&f ss regards X reference and h wild type is just reversed from that of many other workers: the “X reference” of Pica t C&f is the strain termed “A wild type or X papa” by Kaiser (1957). who first described its properties.
CENTRIFUGATION
POLYMORPHISM
OF A
516
3. Results (a) Sedimentation and density equilibrium behaviour of X ref, X wild an.cEX erg A lysate stratified over a gradient, preformed as described in Materials and Methods, forms a well-defined phage band as early as ten minutes after the 27,000 rev./min speed is reached. The study of the kinetics of the run in Cscl preformed gradients under the conditions described has shown that after 130 minutes a well-defined separation of two bands is obtained from a mixture of h phages differing in density by 1o/oas measured by the standard method of Meselson et al. (1957). We have called this separation paraequilibrium of densities, and we consider it valid for relative measurements. The interruption of the run at 10, 25, 40, 60 and 100 minutes shows a change in position of the phage band8 as they move towards para-equilibrium. Observation of various phage mixtures has led us to the following conclusion: different phage particles reach the density equilibrium with different velocities, as expected according to Baldwin & Shooter (1963). Using a mixture of h ref and h erg, a 25-minute run shows two well-defined bands. The same tube spun for an additional 110 minutes shows only one very sharp band, as expected from the observation of B.E.D.S. After a run of 25 minutes also in a preformed gradient, the h erg and X &Ed mixture displayed only one band, which split into two bands at para-equilibrium. The X wild and X ref mixture displayed two bands after 25 minutes and two bands after 130 minutes. In each of the above cases, the two types of phages were labelled with morphological markers. The relative positions of the two types of phages in the C&l tube were determined by puncturing the tube and assaying the drops for phage types. Plate 1 shows the results of the experiments and Table 1 summarizes these results. TABLE 1 Density and sedhnentation velocity of some X strains
Density
(g/ml.)
Sedimentation velocity in C&l preformed gradient
h wild
= 1.604t
slow
h ref
= 1.608#
standard
h erg
= I.608
slow
t Calef & Fischer-Fantuzzi, 1965. $ Weigle, Mea&on & Peigen, 1959.
We have shown that different strains of h, displaying the same density under conditions of standard test of B.E.D.S. can be distinguished by the use of very short runs in preformed gradients. Conversely, other h strains having different density can be shown to move together for some time and then separate according to their density. We can conclude that our strains display two physical properties the roles of which under the set of condition8 used are distinguishable.
516
L.
PICA
AND
E. CALEP
(b) Genetics of sedimentation velocity and density The first question we wish to answer concerns the genetic determination of the distinctive behaviour of the three h strains the properties of which we have compared in the P.G.S. and B.E.D.S. techniques. This .can be formulated as follows: (a) How many factors are involved? (b) What are their roles on the physio-chemical phenomena observed? We shall consider only the simplest possibilities and we shall assume, for the time being, only two factors. On the assumption of only two factors being responsible for the behaviour reported in Table 1, only one of the three pairs h refh wild, X erg-A wild and h erg-A ref will differ in both factors. The first question we will be dealing with will be aimed at finding the pair in which the two factors are operating. Let us analyse the three possibilities: in the case X wild-A ref, does the pair differ for both factors? Since the two phages are distinguishable both by speed and density, we can explain the result simply by saying that the phenotype of h wild is determined by a slow sedimentation factor and a light density factor. The phenotype of A ref, by definition, will be determined by the two standard alleles of the factor. In line with this, we shall have that h erg compared to h ref differs only because of the slow sedimentation factor, and X wild compared to h erg differs only because of its light density. If this possibility corresponds to a real situation, the progeny of a cross of the pair X wild-h ref should give four different genotypes and also, under the conditions of the P.G.S. technique which we used, the two factors should interact very little. We do not see a simple hypothetical way for the pair h wild-h erg to differ by two factors, hence we can turn to consider the last possibility, namely, the pair which differs by two factors is X erg-h ref. In this last case, X erg should be of slow speed and heavy density, while, as usual, X ref would show standard speed and standard density. Under the set conditions which we used, the expression of the two factors interacts appreciably. The combined effect of the two contrasting factors gives rise to the pattern found, On this hypothesis and always on the assumption that the three phages differ only because of two factors, we would predict: (a) slow sedimentation and light density of X wild are due to the same factor; (b) the cross h erg and h ref yields four genotypes. Avoiding more complex possibilities where the genetic factors involved are more than two, the physical chemistry of the situation can be explained in terms of two possibilities, both requiring two factors. The first possibility we envisaged is hard to uphold to since it requires the unlikely assumption that the differences in velocity we are able to detect are without any influence on the density. Our experiments were planned to test the number of physical chemical genotypes in the progeny of both of the two critical pairs mentioned above. While the work was in progress we came across, almost accidentally, one unexpected observation indicating that the light density factor characteristic of h wild was expressed by something undergoing phenotypic mixing. This indication came from a genetic cross between the deleted prophage h cry contained in the bacterium U173 and h b2. We knew from previous work that, from a cryptic prophage, X ref can rescue the same light density factor present in h wild (Fischer-Fantuzzi & Calef, 1964) and also that X b2 could rescue from the cryptic prophage the full density complement of X ref. This genetic set-up, which allows us to perform a two-factor cross the segregation of which can be directly scored by analysis of the density of the progeny, was available
w
25 min
130 min
Cl
W + CRC
Cl
CRG
th
Its iwmlcer; h
rqferencr;
CENTRIFUGATION
POLYMORPHISM
OF h
51;
since we had some double lysogens (h cry) (h b2). From these double lysogens, lysates ‘vere obtained by ultraviolet light. The lysates were concentrated to 5 X 10” particles/ ml. and banded in C&l for the B.E.D.S. technique. Out of six different double lysogens, we obtained one which produced a lysate with four distinct phage bands (see Plate II). The photograph shows, in addition to a density marker towards the bottom of the tube, four X phage bands. These bands have the following densities: X ref, X wild, X b2, h b2 “light”. It can be seen, and the titration confirmed this, that the four bands are of roughly the same phage concentration. In other words, it appea,rs that out of two-factor crosses, we have a segregation situation mimicking two noulinked factors. Indeed, four bands would only be possible if together with a DXBqciated density marker such as b2 (Kellenberger et ul., 196lj we also had one other actor expressed as a phenotype freely associating with any genotype. This tentative t .clusion can be strengthened by analysing the progeny of the cross, where the parents arc likely to differ by only this factor. We crossed h ref h cl with h wild. The progeny of t,!ie cross was submitted to the B.E.D.S. technique and about 300 fractions were taken out of a 3-ml. tube. In each of two repetitions, the cl character was found to ?c distributed with nearly equal frequency at the tn-o parental density levels. In a,l~~arent contrast with this observation, we verified that the parental combination of visible markers is linked to the density marker after one cycle of growt’h. At this point 3 can strongly suspect phenotypic mixing. To verify further whet,her the random .,sociation of these two characters is due to recombination or phenotypic mixing, we Are able to show that the determinant of the density character is closely linked to one )^the standard chromosomal markers. To do so, we have analysed the density of four ’ and seven h recombinants taken from the progeny before the density separat#ion; 11the members of the first group had the slow sedimentation velocity and light density; all the members of the second group had the fast sedimentation velocity and he heavy density. Moreover, we have taken 12 h recombinants from one of the density .,;parations mentioned above from 12 evenly spaced density Ievels and we have oloned and centrifuged them again individually. The results showed that all were of t ie fast, heavy type. Finally, the percentage of recombinants between h and cl in ne cross was measured by analysing an unselected sample of the progeny. The value \ as found to be equal to 6%. To summarize, we have four relevant observations: (1) Apparent random association of a known chromosomal character (cl) and the -nsity character by banding by the B.E.D.S. technique the phage progeny directly ter the cross. (2) A tendency of the parental combinations to persist after cloning. (3) Strong linkage of the density character and another chromosomal charact,er 5) after cloning the recombinants. (4) Usual linkage value of the two chromosomal h and cl characters. Following the same criteria, phenotypic mixing has also been shown in the same ross with respect to the sedimentation velocity in a preformed CsCl gradient. Again the two visible bands obtained in the 25-minute run of the progeny of the cross were found, when analysed, to be equally rich in cl + and cl- phages. All this leads to the conclusion that the determinant of t,he light density of the ,J,wild is probably the same as that affecting the sedimentation value in the P.G;.S. technique. This hypothetical factor is linked to the determinant of the h character and expresses itself by a phenotype mixable with any genotype. According to the prediction made
L.
618
PICA
AND
E. CALEF
at the beginning of this paragraph, the situation found corresponds to one of the possibilities envisaged: namely, the pair X erg-X ref differs in its sedimentation properties because of two factors. To contim this, we crossed A erg au89 cl ~~854 x A ref aus& Table 2 gives, together with the score for traditional markers, the characterization of all the recombinants found for sedimentation velocity and density in the P.G.S. technique. TABLE
2
Analysis of progeny phugea from a erosa involving sedimentation and density characters No. of genotypes found
eua9 m&i Parental types
+
+
cl
SW351
-I-
-I-
+
+
88 164
+
-
+
+
+
+
-
-I-
+
+ Recomb. types
Centrifugation pattern
standard 8 standards
x erg X ref
standard heavy
h erg x 8.d.
light standard heavy standard
x wild h ref x ad. X ref
1 slow
standard
x erg
2 standard 1 untested
standard untested
X ref
1 standard
standard
X ref
2 slow
standard
1 slow
light
A&
1 slow
light
h wild
1 slow 1 standard 1 1 2 i 1
f -
slow§ standards
Density$
0 2
4
Sed. velocityt in C&l preformed gradient
slow standard standard standard
+
-I-
+
-
+
+
+
-
-
-
+
+
-
-
-
+
+
+
-i1
+
0
0
+ + +
2
t Standard sedimentation velocity is X ref sedimentation velocity. ia h wild and X erg sedimentation velocity. Slow sedimentation velocity is X ref density. $ Standard density is h wild density. Light density is about 2% greater than the density of h ref. Heavy density 5 In this experiment only one lysate for each parental type was tested for density and sedimentation velocity in a preformed gradient.
CENTRIFUGATION
POLYMORPHISM
OF X
619
The cross is effected by considerable selective advantage of some types; the parent h ref, in particular, has a strong selective advantage over X erg. The analysis of the cross shows that among the progeny two non-parental densities appear. We started with h ref and /\ erg, both with a density of 1.508 g/ml. and we obtained phages with the density of X wild (l-504 g/ml.) and phages with a density roughly of 1.520 g/ml. This novel phage was called super dense (s.d.). To gain some insight into the physical basis of this new phenotype, one h s.d. cl stock was crossed to h ref sus5 and banded by the B.E.D.S. technique. We found that the cl progeny phages can be enriched by successive runs to sedimentation equilibrium of the lower band. This result is interpreted by saying that the character s.d. is not subject to phenotypic mixing; hence it is likely to be due to an increased DNA/ protein ratio. If we apply the classification criterion of the sedimentation to our recombinants, we then have four sedimentation patterns: h ref, X wild, h erg and h s.d. We interpret this in the following way: there are two factors affecting densities, one (fr,) is associated with the DNA and does not affect density by means of a genetic product; the other (fp) affects some protein of the phage envelope. The first factor distinguishes the DNA of h erg and h s.d. from the DNA of both h wild and h ref, which we assume to be alike. The second distinguishes the protein of h wild and X erg from the protein of h ref and X s.d. The fp factor is responsible for slow velocity of sedimentation in preformed gradients. Following our hypothesis, the equal density of X erg and X Tef is due to the interaction of two contrasting phenotypes. This interpretation is summarized in Table 3. According to this interpretation, one can analyse the remaining information contained in Table 2 in order to obtain a map location for the two hypothetical factors. TABLE 3
Inderpretative scheme of sedimentation and density polymorphism in, X Genetic constitution f* h wild
-
+
x ref
+
+
h erg
-
-
+
-
h s.d.
Behwiour on preformed gradient
low velocity, light density standard velocity, standard density low velocity, standard density etmnd&rd velocity, high density
f,,, Protein-related factor. fD, DNA-related factor; modiiiccltion in the sense of an increase in DNA content.
In computing the recombination percentage at any given interval we avoided the inclusion of phages which did not have any mating opportunity by ignoring the non-crossover types and considering only single and multiple crossovers. The results of the computation can be expressed with the maps of Fig. 1, which are the best fit to a linear order both in respect to f, and fu.
520
L. PICA 4
\
h\ \
\ sus9 \ \
\
E.
CALEF
‘y----4~ \
\ SW5 \
\
I ’\
I I ’ I
\
I I
‘xjwi-
I FIG.
AND
1. Location
\ “\ \
i \ SUf54 \
i, a
of the factors
f,, and fr, on t,he genetic map of X.
4. Discussion We have described a method which allows quick separation of whole phage particles according to their sedimentation velocity and/or density in preformed CsCl gradients. This method is convenient for density separation, as pointed out by Baldwin & Shooter (1963), because of the time factor and also because it allows detection of difference in sedimentation velocity. It should be noted that optimal conditions to detect sedimentation polymorphism for any two phages of the same species are those where the phages display equal or very similar densities in buoyant equilibrium density sedimentation. It was already suspected that the density of bacteriophage h can be affected by factors subject to phenotypic mixing (Kellenberger et al., 1961). We have collected evidence for one such factor present in the h wild and showed that it confers on h particles a density lighter than that of h ref. In each case so far examined, the light density appears associated with a slow sedimenting phenotype. This second characteristic, as well as the first, appears to be subject t’o phenotypic mixing. We think that these two characters are determined by a single genetic factor. By combined use of sedimentation velocity and buoyant equilibrium density we were able to distinguish four true-breeding patterns of sediment8ation characteristics. The four t,ypes (A wild, h ref, X erg and X s.d.) arose from a cross where t,he parents were distinguishable only through their sedimentation velocity. We interpreted this result as due to two genetic factors, which we call f, and f,,. The factor f, gives phenotypic mixing and can be identified because of the allele present in t.he strain h wild. This work BIAI.
was carried
out under
the Euratom-C.N.R.-C.N.E.N.
contract
no. 012-61-12
REFERENCES Appleyard, R. K. (1954). Genetics, 39, 429. Appleyard, R. K., McGregor, J. F. & Baird, K. M. (1956). ‘ViroEogy, 2, 565. Baldwin, R. L. & Shooter, E. M. (1963). Conference on the UZtracentr&ge, p. 143. New York: Academic Press. Calef, E. & Fischer-Fantuzzi, L. (1965). Atti del XIII Congress0 Naz. di Microbiologia, Parma-Salsomaggiore, vol. 3, p. 227. Calef, E. & Licciardello, G. (1960). Pirology, 12, 81. Campbell, A. (1961). F’irology, 14, 22. Fischer-Fantuzzi, L. & Calef, E. (1964). I’irology, 23, 209. Kaiser, A. D. (1955). 77iroZogy, 1, 424. Kaiser, A. D. (1957). Virology, 3, 42. Kellenberger, G., Zichichi, M. L. & Weigle, J. (1961). J. Mol. BioZ. 3, 399. Lederberg, J. & Tatum, E. L. (1946). Cold A‘@+. Harb. Symp. @ant. BioZ. 11, 113. Martin, R. G. & Ames, B. N. (1961). J. BioZ. Chem. 236, 1372. Meselson, M., Stahl, F. W. & Vinograd, J. (1957). Proc. Nat. Acad. Sci., Wash. 43, 581. Weigle, J., Meselson, M. & Paigen, K. (1959). J. Mol. BioZ. 1, 379.
(1968) 32, 513-520
Polymorphism
in Sedimentation Velocity and Density of 1 Bacteriophage L. PICA AND E. CALEF
International
Laboratory
(Received 14 August
of Genetics and Biophysics Naples, Italy
(C.N. R.)
1967, and in revised form 6 November 1967)
A new genetic characteristic of h bacteriophage has been detected. This characteristic concerns the sedimentation velocity of the whole particle in a CsCl preformed gradient. The detection of this new characteristic has allowed us to verify the genetic affinity between h crypticogen and h wild. The sedimentation velocity is probably determined by the same genetic factor which causes a light-density phenotype in some /\ strains (h wild). This can be stated because of the complete linkage found between the two determinants and because both characters display phenotypic mixing. that these two phages Genetic crosses between X erg and h ref have indicated differ in two factors determining the density in buoyant equilibrium centrifugation. The contrasting effect of these two factors confers on them equal phenotype status in assays of buoyant equilibrium density sedimentation; their progeny, however, displays two new densities as well as the parental typos.
1. Introduction Following the finding of the crypticogen h phage (A erg) (Calef & Fischer-Fantuzzi, 1965), so called because it is capable of giving cryptic deletions after ultraviolet curing, we have searched for other distinctive characteristics of this phage with respect to its presumed parent, h wild. While we were measuring the sedimentation behaviour in the CsCl gradient, we found that it was possible and advantageous t’o obtain density measurements in a preformed CsCl gradient. In these gradients it is also possible to observe a previously undetected polymorphism among different h strains, consisting in the velocity with which the particles reach equilibrium in a preformed gradient,.
2. Materials and Methods (a) Media The phage crosses were made in Tryptone broth (Bacto-tryptone vitamin B1 1 x 1OmB g/l.). The phages were titrated in plates containing the same medium
Difco
lo/,, NaCl O*5y0,
plus agar
(Oxoid)
at.
1.5%.
For dilution and suspension of purified phagcs we used (Weigle, Meselson & Paigen, 1959). The caesium chloride was obtained from Merck, Darmstadt. (b) Strains Bacteria: Escherichia coli. C600, indicator strain (Appleyard, 1954). CR63, selective strain for the host range (h) mutants 1956). 33
613
suspension
(Appleyard,
medium
McGregor
(SM)
Jz Baird,
614
L. PICA
AND
E. CALEF
U173, cryptic lysogenic strain (Fischer-Fantuzzi & Calef, 1964). U279, non-permissive strain for all “eus” mutants (suppressor sensitivity) Fantuzzi & Calef, 1964).
(Fischer-
(c) Bmtetiophuger, h wild obtained by induction of K12 wild-type strain (Lederberg t Tatum, 1946). h ref (Kaiser, 1957) and the mutants of h ref: Xc1 clear-plaque selection from A ref, A 8~5 (Campbell, 1961), h 8~~6 (Campbell, 1961), /\ h cl (Calef & Licciardello, 1960). )\ b2 (Kellenberger, Zichichi & Weigle, 1961). h erg (Cdef & Fischer-Fantuzzi, 1965) and its mutant: A sw9 cl sus54 obtained by recombination between X erg and Campbell strains. h s.d. super dense from crosses between X erg and A ref. (d) Phage terminology In order to avoid possible confusion in the terminology of h strains, we should emphasize the following: the h designated as wild is the one present in the strain of E. coli K12 as it was isolated from nature and described by Lederberg & Tatum (1946). The K12 strain was a gift from J. Lederberg to L. Cavalli. We received this strain from L. Cavalli in 1962. This phage makes small turbid plaques of rather unequal diameter. Often in the literature, by h wild or X + + -I- one indicates another A temperate phage selected by Kaiser (1957). This phage given to us by A. D. Kaiser in 1956 produces large turbid plaques. This is the one we call X reference.t This strain is identical, from the point-of-view of density, plaque morphology, ability to lysogenize and origin, to the h used by Weigle et al. (1959) which they call “h ref Kaiser 1956”. We use this term (A ref) in spite of the facts mentioned by Kellenberger et al. (1961, p. 399, footnote), because the Kaiser X strains to which we apply it has been for many years the reference in X phage biology. (0) Crossing procedure The vegetative phage crosses were carried out according to Kaiser (1955). (f) Caesium chloride preformed gradient sedimentation procedure Baldwin & Shooter have extensively studied preformed CsCl gradients (1963). They have already pointed out the advantages and the limits of this method in the determination of density and molecular weight of macromolecules. These authors used this technique for the separation of X phage of diierent density in a mixture. In their paper, the time to reach the equilibrium under many conditions was studied. At the time the present experi. ments were performed, we were unaware of that paper. The gradients were made in Spinco SW39 tubes with the gradient device (described by Martin & Ames, 1961) consisting of two cylindrical wells joined by means of a small channel. One of the two wells contains 2 ml. of a solution two-third saturated with &Cl at room temperature; density = 1.627 g/ml. The other contains 2 ml. of a solution one-third saturated with CsCl; density = l-318 g/ml. The solutions were made in 0.015 M-Tris buffer, pH8. The flow of the solution was kept at 2 ml./min. Over the preformed gradient we layered 0.2 to 0.4 ml. of phage suspension, the titre of which was not less than 1 x lOi particles/ml. The runs were carried out in a Spinco preparative ultracentrifuge model L at a speed of 27,000 rev&in at a temperature of 18°C. The density values are calculated by measuring the refractive index of the solutions for sodium light at 25’C in an Abbe refractometer and the subsequent use of the empirical formula of Weigle et cd. (1959). We shall call this technique P.G.S. We have also carried out density measurement with CsCl gradients according to the standard method of buoyant equilibrium density sedimentation described by Meselson, Stahl & Vinograd (1957). We shall call this technique B.E.D.S. t Editor’s note: To avoid misunderstanding, it should be pointed out that the terminology of Drs Pica and C&f ss regards X reference and h wild type is just reversed from that of many other workers: the “X reference” of Pica t C&f is the strain termed “A wild type or X papa” by Kaiser (1957). who first described its properties.
CENTRIFUGATION
POLYMORPHISM
OF A
516
3. Results (a) Sedimentation and density equilibrium behaviour of X ref, X wild an.cEX erg A lysate stratified over a gradient, preformed as described in Materials and Methods, forms a well-defined phage band as early as ten minutes after the 27,000 rev./min speed is reached. The study of the kinetics of the run in Cscl preformed gradients under the conditions described has shown that after 130 minutes a well-defined separation of two bands is obtained from a mixture of h phages differing in density by 1o/oas measured by the standard method of Meselson et al. (1957). We have called this separation paraequilibrium of densities, and we consider it valid for relative measurements. The interruption of the run at 10, 25, 40, 60 and 100 minutes shows a change in position of the phage band8 as they move towards para-equilibrium. Observation of various phage mixtures has led us to the following conclusion: different phage particles reach the density equilibrium with different velocities, as expected according to Baldwin & Shooter (1963). Using a mixture of h ref and h erg, a 25-minute run shows two well-defined bands. The same tube spun for an additional 110 minutes shows only one very sharp band, as expected from the observation of B.E.D.S. After a run of 25 minutes also in a preformed gradient, the h erg and X &Ed mixture displayed only one band, which split into two bands at para-equilibrium. The X wild and X ref mixture displayed two bands after 25 minutes and two bands after 130 minutes. In each of the above cases, the two types of phages were labelled with morphological markers. The relative positions of the two types of phages in the C&l tube were determined by puncturing the tube and assaying the drops for phage types. Plate 1 shows the results of the experiments and Table 1 summarizes these results. TABLE 1 Density and sedhnentation velocity of some X strains
Density
(g/ml.)
Sedimentation velocity in C&l preformed gradient
h wild
= 1.604t
slow
h ref
= 1.608#
standard
h erg
= I.608
slow
t Calef & Fischer-Fantuzzi, 1965. $ Weigle, Mea&on & Peigen, 1959.
We have shown that different strains of h, displaying the same density under conditions of standard test of B.E.D.S. can be distinguished by the use of very short runs in preformed gradients. Conversely, other h strains having different density can be shown to move together for some time and then separate according to their density. We can conclude that our strains display two physical properties the roles of which under the set of condition8 used are distinguishable.
516
L.
PICA
AND
E. CALEP
(b) Genetics of sedimentation velocity and density The first question we wish to answer concerns the genetic determination of the distinctive behaviour of the three h strains the properties of which we have compared in the P.G.S. and B.E.D.S. techniques. This .can be formulated as follows: (a) How many factors are involved? (b) What are their roles on the physio-chemical phenomena observed? We shall consider only the simplest possibilities and we shall assume, for the time being, only two factors. On the assumption of only two factors being responsible for the behaviour reported in Table 1, only one of the three pairs h refh wild, X erg-A wild and h erg-A ref will differ in both factors. The first question we will be dealing with will be aimed at finding the pair in which the two factors are operating. Let us analyse the three possibilities: in the case X wild-A ref, does the pair differ for both factors? Since the two phages are distinguishable both by speed and density, we can explain the result simply by saying that the phenotype of h wild is determined by a slow sedimentation factor and a light density factor. The phenotype of A ref, by definition, will be determined by the two standard alleles of the factor. In line with this, we shall have that h erg compared to h ref differs only because of the slow sedimentation factor, and X wild compared to h erg differs only because of its light density. If this possibility corresponds to a real situation, the progeny of a cross of the pair X wild-h ref should give four different genotypes and also, under the conditions of the P.G.S. technique which we used, the two factors should interact very little. We do not see a simple hypothetical way for the pair h wild-h erg to differ by two factors, hence we can turn to consider the last possibility, namely, the pair which differs by two factors is X erg-h ref. In this last case, X erg should be of slow speed and heavy density, while, as usual, X ref would show standard speed and standard density. Under the set conditions which we used, the expression of the two factors interacts appreciably. The combined effect of the two contrasting factors gives rise to the pattern found, On this hypothesis and always on the assumption that the three phages differ only because of two factors, we would predict: (a) slow sedimentation and light density of X wild are due to the same factor; (b) the cross h erg and h ref yields four genotypes. Avoiding more complex possibilities where the genetic factors involved are more than two, the physical chemistry of the situation can be explained in terms of two possibilities, both requiring two factors. The first possibility we envisaged is hard to uphold to since it requires the unlikely assumption that the differences in velocity we are able to detect are without any influence on the density. Our experiments were planned to test the number of physical chemical genotypes in the progeny of both of the two critical pairs mentioned above. While the work was in progress we came across, almost accidentally, one unexpected observation indicating that the light density factor characteristic of h wild was expressed by something undergoing phenotypic mixing. This indication came from a genetic cross between the deleted prophage h cry contained in the bacterium U173 and h b2. We knew from previous work that, from a cryptic prophage, X ref can rescue the same light density factor present in h wild (Fischer-Fantuzzi & Calef, 1964) and also that X b2 could rescue from the cryptic prophage the full density complement of X ref. This genetic set-up, which allows us to perform a two-factor cross the segregation of which can be directly scored by analysis of the density of the progeny, was available
w
25 min
130 min
Cl
W + CRC
Cl
CRG
th
Its iwmlcer; h
rqferencr;
CENTRIFUGATION
POLYMORPHISM
OF h
51;
since we had some double lysogens (h cry) (h b2). From these double lysogens, lysates ‘vere obtained by ultraviolet light. The lysates were concentrated to 5 X 10” particles/ ml. and banded in C&l for the B.E.D.S. technique. Out of six different double lysogens, we obtained one which produced a lysate with four distinct phage bands (see Plate II). The photograph shows, in addition to a density marker towards the bottom of the tube, four X phage bands. These bands have the following densities: X ref, X wild, X b2, h b2 “light”. It can be seen, and the titration confirmed this, that the four bands are of roughly the same phage concentration. In other words, it appea,rs that out of two-factor crosses, we have a segregation situation mimicking two noulinked factors. Indeed, four bands would only be possible if together with a DXBqciated density marker such as b2 (Kellenberger et ul., 196lj we also had one other actor expressed as a phenotype freely associating with any genotype. This tentative t .clusion can be strengthened by analysing the progeny of the cross, where the parents arc likely to differ by only this factor. We crossed h ref h cl with h wild. The progeny of t,!ie cross was submitted to the B.E.D.S. technique and about 300 fractions were taken out of a 3-ml. tube. In each of two repetitions, the cl character was found to ?c distributed with nearly equal frequency at the tn-o parental density levels. In a,l~~arent contrast with this observation, we verified that the parental combination of visible markers is linked to the density marker after one cycle of growt’h. At this point 3 can strongly suspect phenotypic mixing. To verify further whet,her the random .,sociation of these two characters is due to recombination or phenotypic mixing, we Are able to show that the determinant of the density character is closely linked to one )^the standard chromosomal markers. To do so, we have analysed the density of four ’ and seven h recombinants taken from the progeny before the density separat#ion; 11the members of the first group had the slow sedimentation velocity and light density; all the members of the second group had the fast sedimentation velocity and he heavy density. Moreover, we have taken 12 h recombinants from one of the density .,;parations mentioned above from 12 evenly spaced density Ievels and we have oloned and centrifuged them again individually. The results showed that all were of t ie fast, heavy type. Finally, the percentage of recombinants between h and cl in ne cross was measured by analysing an unselected sample of the progeny. The value \ as found to be equal to 6%. To summarize, we have four relevant observations: (1) Apparent random association of a known chromosomal character (cl) and the -nsity character by banding by the B.E.D.S. technique the phage progeny directly ter the cross. (2) A tendency of the parental combinations to persist after cloning. (3) Strong linkage of the density character and another chromosomal charact,er 5) after cloning the recombinants. (4) Usual linkage value of the two chromosomal h and cl characters. Following the same criteria, phenotypic mixing has also been shown in the same ross with respect to the sedimentation velocity in a preformed CsCl gradient. Again the two visible bands obtained in the 25-minute run of the progeny of the cross were found, when analysed, to be equally rich in cl + and cl- phages. All this leads to the conclusion that the determinant of t,he light density of the ,J,wild is probably the same as that affecting the sedimentation value in the P.G;.S. technique. This hypothetical factor is linked to the determinant of the h character and expresses itself by a phenotype mixable with any genotype. According to the prediction made
L.
618
PICA
AND
E. CALEF
at the beginning of this paragraph, the situation found corresponds to one of the possibilities envisaged: namely, the pair X erg-X ref differs in its sedimentation properties because of two factors. To contim this, we crossed A erg au89 cl ~~854 x A ref aus& Table 2 gives, together with the score for traditional markers, the characterization of all the recombinants found for sedimentation velocity and density in the P.G.S. technique. TABLE
2
Analysis of progeny phugea from a erosa involving sedimentation and density characters No. of genotypes found
eua9 m&i Parental types
+
+
cl
SW351
-I-
-I-
+
+
88 164
+
-
+
+
+
+
-
-I-
+
+ Recomb. types
Centrifugation pattern
standard 8 standards
x erg X ref
standard heavy
h erg x 8.d.
light standard heavy standard
x wild h ref x ad. X ref
1 slow
standard
x erg
2 standard 1 untested
standard untested
X ref
1 standard
standard
X ref
2 slow
standard
1 slow
light
A&
1 slow
light
h wild
1 slow 1 standard 1 1 2 i 1
f -
slow§ standards
Density$
0 2
4
Sed. velocityt in C&l preformed gradient
slow standard standard standard
+
-I-
+
-
+
+
+
-
-
-
+
+
-
-
-
+
+
+
-i1
+
0
0
+ + +
2
t Standard sedimentation velocity is X ref sedimentation velocity. ia h wild and X erg sedimentation velocity. Slow sedimentation velocity is X ref density. $ Standard density is h wild density. Light density is about 2% greater than the density of h ref. Heavy density 5 In this experiment only one lysate for each parental type was tested for density and sedimentation velocity in a preformed gradient.
CENTRIFUGATION
POLYMORPHISM
OF X
619
The cross is effected by considerable selective advantage of some types; the parent h ref, in particular, has a strong selective advantage over X erg. The analysis of the cross shows that among the progeny two non-parental densities appear. We started with h ref and /\ erg, both with a density of 1.508 g/ml. and we obtained phages with the density of X wild (l-504 g/ml.) and phages with a density roughly of 1.520 g/ml. This novel phage was called super dense (s.d.). To gain some insight into the physical basis of this new phenotype, one h s.d. cl stock was crossed to h ref sus5 and banded by the B.E.D.S. technique. We found that the cl progeny phages can be enriched by successive runs to sedimentation equilibrium of the lower band. This result is interpreted by saying that the character s.d. is not subject to phenotypic mixing; hence it is likely to be due to an increased DNA/ protein ratio. If we apply the classification criterion of the sedimentation to our recombinants, we then have four sedimentation patterns: h ref, X wild, h erg and h s.d. We interpret this in the following way: there are two factors affecting densities, one (fr,) is associated with the DNA and does not affect density by means of a genetic product; the other (fp) affects some protein of the phage envelope. The first factor distinguishes the DNA of h erg and h s.d. from the DNA of both h wild and h ref, which we assume to be alike. The second distinguishes the protein of h wild and X erg from the protein of h ref and X s.d. The fp factor is responsible for slow velocity of sedimentation in preformed gradients. Following our hypothesis, the equal density of X erg and X Tef is due to the interaction of two contrasting phenotypes. This interpretation is summarized in Table 3. According to this interpretation, one can analyse the remaining information contained in Table 2 in order to obtain a map location for the two hypothetical factors. TABLE 3
Inderpretative scheme of sedimentation and density polymorphism in, X Genetic constitution f* h wild
-
+
x ref
+
+
h erg
-
-
+
-
h s.d.
Behwiour on preformed gradient
low velocity, light density standard velocity, standard density low velocity, standard density etmnd&rd velocity, high density
f,,, Protein-related factor. fD, DNA-related factor; modiiiccltion in the sense of an increase in DNA content.
In computing the recombination percentage at any given interval we avoided the inclusion of phages which did not have any mating opportunity by ignoring the non-crossover types and considering only single and multiple crossovers. The results of the computation can be expressed with the maps of Fig. 1, which are the best fit to a linear order both in respect to f, and fu.
520
L. PICA 4
\
h\ \
\ sus9 \ \
\
E.
CALEF
‘y----4~ \
\ SW5 \
\
I ’\
I I ’ I
\
I I
‘xjwi-
I FIG.
AND
1. Location
\ “\ \
i \ SUf54 \
i, a
of the factors
f,, and fr, on t,he genetic map of X.
4. Discussion We have described a method which allows quick separation of whole phage particles according to their sedimentation velocity and/or density in preformed CsCl gradients. This method is convenient for density separation, as pointed out by Baldwin & Shooter (1963), because of the time factor and also because it allows detection of difference in sedimentation velocity. It should be noted that optimal conditions to detect sedimentation polymorphism for any two phages of the same species are those where the phages display equal or very similar densities in buoyant equilibrium density sedimentation. It was already suspected that the density of bacteriophage h can be affected by factors subject to phenotypic mixing (Kellenberger et al., 1961). We have collected evidence for one such factor present in the h wild and showed that it confers on h particles a density lighter than that of h ref. In each case so far examined, the light density appears associated with a slow sedimenting phenotype. This second characteristic, as well as the first, appears to be subject t’o phenotypic mixing. We think that these two characters are determined by a single genetic factor. By combined use of sedimentation velocity and buoyant equilibrium density we were able to distinguish four true-breeding patterns of sediment8ation characteristics. The four t,ypes (A wild, h ref, X erg and X s.d.) arose from a cross where t,he parents were distinguishable only through their sedimentation velocity. We interpreted this result as due to two genetic factors, which we call f, and f,,. The factor f, gives phenotypic mixing and can be identified because of the allele present in t.he strain h wild. This work BIAI.
was carried
out under
the Euratom-C.N.R.-C.N.E.N.
contract
no. 012-61-12
REFERENCES Appleyard, R. K. (1954). Genetics, 39, 429. Appleyard, R. K., McGregor, J. F. & Baird, K. M. (1956). ‘ViroEogy, 2, 565. Baldwin, R. L. & Shooter, E. M. (1963). Conference on the UZtracentr&ge, p. 143. New York: Academic Press. Calef, E. & Fischer-Fantuzzi, L. (1965). Atti del XIII Congress0 Naz. di Microbiologia, Parma-Salsomaggiore, vol. 3, p. 227. Calef, E. & Licciardello, G. (1960). Pirology, 12, 81. Campbell, A. (1961). F’irology, 14, 22. Fischer-Fantuzzi, L. & Calef, E. (1964). I’irology, 23, 209. Kaiser, A. D. (1955). 77iroZogy, 1, 424. Kaiser, A. D. (1957). Virology, 3, 42. Kellenberger, G., Zichichi, M. L. & Weigle, J. (1961). J. Mol. BioZ. 3, 399. Lederberg, J. & Tatum, E. L. (1946). Cold A‘@+. Harb. Symp. @ant. BioZ. 11, 113. Martin, R. G. & Ames, B. N. (1961). J. BioZ. Chem. 236, 1372. Meselson, M., Stahl, F. W. & Vinograd, J. (1957). Proc. Nat. Acad. Sci., Wash. 43, 581. Weigle, J., Meselson, M. & Paigen, K. (1959). J. Mol. BioZ. 1, 379.