On the mechanism of genetic recombination in transforming Bacillus subtilis

On the mechanism of genetic recombination in transforming Bacillus subtilis

J. Mol. Biol. (1964) 9, 236-245 On the Mechanism of Genetic Recombination in Transforming Bacillus subtilis JACQUES J. PENE AND W. R. ROMIG Depart...

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J. Mol. Biol. (1964) 9, 236-245

On the Mechanism of Genetic Recombination in Transforming Bacillus subtilis JACQUES J. PENE AND

W. R.

ROMIG

Department of Bacteriology, University of California Los Angeles 24, California, U.S.A. (Received 1 July 1963, and in revised form 3 April 1964) The molecular fate of transforming DNA in competent Bacillus 8ubtilis hag been studied. The physical differentiation of transforming DNA and the genetic material of recipient bacteria was effected by employing the isotopes 15N and 32P. Competent bacteria labeled with 15N and 31p were allowed to incorporate 14N32P·labeled transforming DNA intracellularly. DNA was isolated from these recipient cells and centrifuged in cesium chloride. Analysis of drop fractions collected at equilibrium showed the presence of radioactivity in a region of density corresponding to recipient DNA. Material contribution from donor DNA to the resident DNA did not occur when genetically inert Bacillus cereus DNA was substituted for homologous DNA. It was concluded that radioactivity appearing in the unlabeled resident DNA was not the result of degradation of input (32P]DNA and subsequent incorporation by normal metabolic processes. Since single-stranded DNA of donor origin was not detected, nor was there evidence for the non-specific aggregation of donor and recipient DNA, it was concluded that the observed physical association of transforming and recipient DNA occurs as a result of genetic recombination in transformed B. subtilis.

1. Introduction Genetic characters in bacteria are transferred by conjugation, transformation, transduction and episomal infection. These methods of genetic transfer have marked differences, such as the physical state of the causal agent, the efficiency of transfer and the physiological properties of the recipient bacteria. A fraction of the bacterial population gives rise to genetic recombinants, that is, to cells capable of passing on bilaterally to their progeny some of the genetic information of the exogenotic nucleic acid. In these systems, a description ofthe mechanism of recombination on the molecular level requires, therefore, an understanding of the interactions and the relationships governing nucleic acids that are dissimilar both in structure and genetic information. The two general hypotheses (Lederberg, 1955) which have been advanced to describe genetic recombination can be stated as follows. (1) Copy-choice. The precise sequential replication of the host chromosome is altered by using the infectious DNA as a template. Some of the information contained on the exogenotic DNA is thereby transferred to the newly synthesized chromosome to form a genetic recombinant. The DNA of the recombinant is materially derived from only one parent. (2) Breakage and reunion. The recombinant structure is a physical hybrid produced by the association of DNA fragments derived largely from the host chromosome but in part from the exogenotic DNA. 236

GENETIC RECOMBINATION IN B. SUBTILIS

237

Fuerst, Jacob & Wollman (1961) carried out experiments to determine whether the destruction of the physical integrity of Hfr chromosomes labeled with 32p affects the formation of genetic recombinants at all times after transfer to F- bacteria. Hfr cells highly labeled with 32p were crossed with F- bacteria. At various times after transfer, samples of the population were frozen and destruction of the male chromosome was permitted to occur by radioactive decay. The samples were thawed at regular intervals and the number of genetic recombinants was determined. Their results showed that the number of recombinants increased as time was allowed for recombination to take place before the majority of damage to the Hfr chromosome occurred by radioactive decay. These experiments suggest that in bacterial conjugation the recombinant structure is not physically derived from both parental chromosomes. The mechanism of genetic recombination in bacteriophage has been recently examined by Meselson & Weigle (1961). The analysis of the progeny of crosses made between physically and genetically different viruses warranted the conclusion that recombination occurs by breakage of both participating chromosome units, and that the replication of the viral chromosome is not a requirement for recombination. The discovery of transformation in Bacillus subtilis (Spizizen, 1958) has made available another system in which genetic recombination can be investigated on the molecular level. The ability of this organism to grow on an inorganic source of nitrogen has allowed the physical differentiation of the genetic material of recipient cells and donor DNA. Experiments were performed to determine the fate of 14N32P_Iabeled transforming principle in recipients grown on media containing I5N and 3IP. These investigations provide evidence suggesting the direct incorporation of exogenotic DNA into the genetic material of recipient bacteria as the basis of recombination in transforming B. 8ubtilis.

2. Materials and Methods (a) Bacterial strains The diauxotroph B. subtilis 168-2 requiring leucine and indole for growth (ind-leu-) was isolated from survivors of B. subtilis 168 (ind-) spores (Burkholder & Giles, 1947) irradiated with ultraviolet light. The prototrophic B. subtilis SB19 was derived by Dr. E. Nester by transforming B. subtilis 168 with DNA from the Marburg strain of B. subtili«, B. cereus was obtained from the stock culture collection of the Department of Bacteriology of the University of California, Los Angeles.

(b) Preparation of competent bacteria (1) 15N31 Pilabeled recipients. B. subtilis 168-2 was grown overnight at 37°C with shaking in a minimal medium (Spizizen, 1958) devoid of (14N]ammonium sulfate but supplemented with 0'01% casein hydrolysate (Nutritional Biochemicals Corporation), 100/A-g of (15N]ammonium chloride (Bio-Rad Laboratories, NE-1), and 50 p.g/ml. of L-tryptophan and L-Ieucine (medium A). The bacteria were centrifuged and used to adjust 100 ml. of medium A in a 500·ml. Erlenmeyer flask to an optical density of 0·3 at 600 mp.. The suspension was incubated on a reciprocal shaker for 4 hr at 37°C. At that time the cells were collected by centrifugation and resuspended in 500 ml. of a medium similar to medium A but containing 5 p.g of L.tryptophan/ml. The suspension, in a 2·8-1. Fernbaeh flask, was incubated as above for 70 min. These cells, now maximally competent, were centrifuged and re-suspended in 20 ml. of medium A containing 10% glycerol. The culture was rapidly frozen in a dry-icc-ethanol mixture and stored at - 40°C. Under these conditions, the bacteria remain viable and competent for several days. (2) UN31P-labeled recipients. The procedure for the production of competent 14N31P_ labeled recipients is as described in section (1) with the following modifications:

238

J. J. PENE AND W. R. ROMIG

[UN]ammonium sulfate replaced [lSN]ammonium chloride; the concentration of casein hydrolysate was 0'02% during the 4·hr incubation period.

(c) Determination of competency The frequency of transformation in competent cultures was determined by the enumeration of leucine-independent colonies growing on minimal medium supplemented with 50 p.g/ml. of r.-tryptophan after exposure to SB19 DNA (5 /lgjml.) for 15 min at 37°C.

(d) Preparation of

I4N32 P-labeled

SB19 and B. cereus DNA

Homologous or transforming uN32P·labeled SB19 DNA and heterologous UN3·P_ labeled B. cereus DNA were obtained as described by Marmur & Grossman (1961). The concentration of radioactive phosphorus (NaH 2PO., Yolk Radiochemical Company) was 40 mejl. of growth medium. The specific activity of homologous and heterologous DNA obtained under these conditions was 3 X 105 ctsjmin//lg and 5'6 X 10' cts/min/p.g of purified DNA. Extraction of DNA from competent recipients following uptake of homologous or heterologous DNA was performed as above. DNA was estimated by the method described by Keck (1956) using highly polymerized salmon sperm DNA as standards (California Corporation for Biochemical Research). Radioactivity was measured by counting samples spread on aluminum planchets with a Nuclear Chicago D47 Geiger detector equipped with a "micrornil" window.

(e) De'Mity-gradient centrifugation studies (I) Analytical GaGl de1UJity-gradient centrifugation. The buoyant density of DNA from UN_ and l5N·labeled 168-2 recipients, B. cereus, and UN-labeled heat-denatured SB19 was measured from their respective positions in CsCl gradients established at 25°0 by centrifugation in an analytical cell fitted with a Kel-F centerpiece in a Spinco model E ultracentrifuge at 44,770 rev./min (144,000 g) for 18 hr (AN-D rotor). The principles and methods of carrying out these measurements have been published by Meselson, Stahl & Vinograd (1957). (2) Preparative GaGl de1l.'Jity-gradient centrifugation. DNA extracted from competent l5N_ or UN-labeled 168-2 recipients exposed to heterologous or homologous radioactive DNA was centrifuged in 4 ml, of CsCI (Harshaw Company, optical grade) at 35,000 rev. jmin (99,972 g) for 48 hr at 25°C in the SW39 rotor of the Spinco model L centrifuge. At equilibrium, drops were collected through a canula (a 2-cm, 21-gauge hypodermic needle) inserted at the bottom of the Lusteroid tube and sealed in a vertical position with a preparation of liquid rubber. Drops were collected to determine the distribution of radioactivity in the density gradient. The density at any point in tho gradient was interpolated by using the values of the refractive index of several drops measured with a Zeiss-Abbe refractometer (Weigle, Meselson & Paigen, 1959). The reproducibility of drop size with a given canula was determined by counting the radioactivity in drops of 6 M-NaCl containing 32P. The variability of drops measured in this fashion was found never to exceed 1%.

3. Results The buoyant density in CsCl of the DNA's used in these studies is listed in Table 1. These values were not determined with the aid of reference DNA of known buoyant density. B. subtilis 168-2 was made competent in a medium containing 15N as the main nitrogen source, as described in Materials and Methods. These 15N31P_Iabeled recipients were exposed to 20 p.gfmi. of transforming DNA extracted from the prototrophic B. subtilis SB19 grown in the presence of UN and 32p. Effective contact with DNA was restricted to ten minutes at 37°C. A sample of the population was diluted and plated to determine the frequency of cells transformed to leucine-independence. The remaining bacteria were rapidly chilled to O°C, centrifuged and thoroughly

GENETIC RECOMBINATION IN B. SUBTILIS TABLE

239

I

Buoyant density of DNA's in CsClt DNA

B. B. B. B.

t

subtiii« 168·2 subtili« 168·2 subtilis cereus

Density, P2S'C (g cm- 3)

Characteristic

HN ION 14N, heat denatured

UN

1·707 1·717 1·718 1·702

These values were determined without a reference DNA of known buoyant density.

washed twice in the cold with 0'15 M·NaCI-0·05 M·EDTA (pH 8'0). The total DNA of these bacteria was extracted. Recipient 15N31P.labeled DNA and the 14N32P_ labeled donor DNA taken up by the cells were then separated by preparative cesium chloride density-gradient centrifugation. After equilibrium was attained, drop fractions were collected to determine the distribution of radioactivity in the density gradient. The results appear in Fig. 1. Two peaks of radioactivity were found, one

~

A

B

.~

1-70

....

500

o

I

E

v

~

o

10

40 Drop number

FIG. 1. Distribution ofradioactivity from uN32P-labeled B. subtilis SBI9 donor DNA in a cesium chloride density-gradient after uptake by 15N31P-labeled competent B. eubtiti» 168-2. Concentration of donor DNA added to recipients: 3 p.gjml. Specific activity of donor DNA: 3 X 105 cts/minj""g. Effective contact with donor DNA: 10 min. Total amount of DNA reo extracted from cells: 980 p.g. Specific activity of DNA re-extracted from cells: 1·2 X 10' cts/min/p.g. Transformation frequency leu - __ leu +: 1 X 10- 3. Amount of DNA centrifuged in density. gradient: 15 p.g/ml. Density at point A: 1·717. Density at point B: 1·708. - X - X - , density, P25'C (g cm- 3); -0-0-, radioactivity, 32p (ctsjmin).

240

J. J. PENE AND W. R. ROMIG

corresponding to the density of recipient 15N-Iabeled DNA and the other to the density of the UN·labeled donor DNA. Four possibilities were considered to account for the p resence of radioactivity from the "light" donor DNA into the region corresponding to the density of t he DNA from recipient cells. These can be stated as follows . (1) Precipitation by ethanol of a mixture of 15N31P_Iabeled recipient DNA and 14N32P-Iabeled donor DNA results in the formation on non-specific aggregates which do not completely separate wh en the DNA fibers are dissolved in saline and centrifuged in CsCl. (2) The radioactivity detected in recipi ent DNA is due to the degradation of input 32P-Iabeled donor DNA to smaller units which are incorporated into the rec ipient DNA by metabolic reactions. (3) Shortly after penetration, the 14N32P-Iabeled donor DNA becomes singlestranded. The transition from native or double-stranded DNA to single-st randed mol ecules is accompanied by a change in buoyant densit y (Marmur & Lane, 1960 ; Meselson & Stahl, 1958). As can be seen from t he result s in Table 1, the density of 14N-Iabeled DNA ma de p redominantl y single-stranded by heat denaturation (Marmur & Lane, 1960) is close t o the density of the 15N-Iabeled recipient DNA. (4) The material contribution from exogenotic transforming principle to the resident gen ome occurs as a result of genetic recombination. Figure 2 presents results concerning the question of the formation on no n-specific aggregates of 15N_ and 14N-Iabeled DNA by alcohol precipitation. B . subtilis 168-2 15N31P_lab eled DNA was precipitated with ethanol in the presence of 14N32P_Iabeled 900 1-72

.., I

"" ~"N

800 700

)C

600

1·71

c:

E

'E

v

500 ';;;-

.!J

.oJ

u

v

!!l

400

e,

170

Drop number

FlO. 2. Distribution of radioactivity from 14N32P·labeled B. subtil i« 8B I9 DNA in a cesium chloride d en sit y-gradient following precipitation by ethanol in the presenc e of 15NJlP_labeled B. subtilis 168· 2 DNA. Mixture of DNA precipitated: [15N31P]B. subt ilis 168 -2 DNA: 1000,..g. [14N32P]B. subt ilis 8B1 9 DNA : 8·4,..g. The mixture was precipitat ed by addit ion o f :2 vol. et hano l. The DNA fibers were dissolved in 0·15 !>i-NaC!. The amount of re-precipitated DNA centrifug ed in CsCl was 20 p.g/m!. - X - X - , density, P2'· C (g cm- 3 } ; - 0-0-, radioactivity, 32p (ct s/ mi n}.

GENETIC RECOMBINATION IN B. SU BT I LIS

241

SB19 DNA. The fibers were collected, dissolved in 0·15 M-NI1Cl and centrifuged in CsCl as above. The distribution of radioactivity in the density-gradient was determined. As can be seen from Fig. 2, the radioactivity falls at a position corresponding to l4N-labeled DNA. We conclude, therefore, that under the conditions of our experiments the formation on non-specific aggregates cannot account for the incorporation of radioactive material found in the DNA with density corresponding to that of the recipient bacteria. To test the second hypothesis, experiments were performed to determine if radioactivity could be detected in lSN-labeled recipient DNA when competent bacteria were exposed to heterologous 14N32P-labeledDNA incapable of genetic recombination

"73

x______

!lsN B. sublllls

t

x

DNA

114N B.cereus

+

"70

DNA

x

'"I E u ~

~

~

so o

10

20

30

40

Drop number

FIG. 3. Distribution of radioactivity from HN3'P-labeled B. cereus DNA in a cesium chloride density gradient after uptake by competent 15N31P_labeled B. 8ubtili8 168·2. Concentration of donor DNA added to recipients: 20 p.gjml. Specific activity of donor DNA: 5·6 X 10 4 cts/min/p.g. Effective contact with donor DNA: 10 min. Total amount of DNA reextracted from cells: 520 p.g. Specific activity of DNA re-extracted from cells: 2 X 10 1 ctsjminjp.g. Transformation frequency leu - --+ leu +: (Olt. Amount of DNA centrifuged in density gradient: 25p.g/ml. - X - X - , density, p,sOc (g cm- 3 ) ; -0-0-, radioactivity, aop (ets/min).

with the resident genome. Competent bacteria are capable of intracellular incorporation of heterologous DNA which does not give rise to recombinants (Schaeffer, 1961). The buoyant density ofthe heterologous DNA was chosen so that it could be separated from the recipient DNA by density-gradient centrifugation. B. subtilis 168-2 15N31P_labeled recipients were exposed to 14N32P-Iabeled DNA of density 1·702 extracted from B. cereus under conditions similar to those of the experiment in Fig. 1. The results are presented in Fig. 3. The total DNA of these bacteria was extracted and a fraction centrifuged in CsCI as described above. Drops were collected to determine the position of radioactive material in the density-gradient. The distribution of radioactivity is restricted to the region corresponding to B. cereus DNA only.

t The frequency of transformation of leu to a IlaInple of the population was 5 X 10- 4. 1&

--+

leu +

6S

determined by addition of SB19 DNA

242

J. J. PENE AND W. R. ROMIG

The time of contact with DNA (10 minutes) represents approximately one-seventh of the generation time of B. subtilis recipients growing under these conditions. For this reason, and since heterologous DNA is not incorporated in the recipient genome during that time, we conclude that re-utilization of donor DNA cannot, in all probability, account for the presence of the radioactivity in the DNA of recipient bacteria. With homologous and heterologous DNA, it has generally been found that the specific activity of the re-extracted DNA is a function of the competency of the recipient bacteria. In order to test whether intracellular donor DNA becomes single stranded and forms bands at a region in the density-gradient corresponding to the [15N]DNA of the recipient bacteria, 14N32P-transforming SBl9 DNA was added to 14N31P-Iabeled

14N native DNA

"72

.., I

~

300

1·71

E v

200

~

.s

~ ..., v

U

~ <>. ),70

o

o

20

50

60

168-2 recipients. The DNA re-extracted from these cells was centrifuged in CsCI as above. The results appear in Fig. 4. Radioactivity appears only at a point in the gradient of density 1·707 corresponding to 14N-labeled double- stranded DNA.

4. Discussion Mter uptake by competent cells of B. subtilis, a fraction of the donor DNA was found to have become physically associated with the DNA of the recipient bacteria. Several possibilities which could account for this observation were considered; these are discussed below.

GENETIC RECOMBINATION IN B. SU BT I LIS

243

(1) The failure to separate macromolecular U{Jgregates of donor and recipient DNA by GsGI centrifugation

The reconstruction experiments performed by mixing labeled and unlabeled DNA, precipitating the mixture with ethanol, and centrifuging the re-dis solved fibers in CsCI gave no evidence of non-specific aggregation of this type. Thi s possibility was therefore considered inadequate to explain our experimental results. (2) The degradation of donor DNA into smaller units which are incorporated into resident DNA by metabolic processes

This possibility was first considered unlikely because the effective contact between recipient bacteria and [32P]DNA was limited to only one-seventh of the generation time of the bacteria (ten minutes). Therefore the postulated degradation and subsequent re-utilization of donor DNA would also have to be fairly rapid. However, the quantity of radioactive donor DNA that can be re-extracted from recipient bacteria under the experimental conditions employed does not seem compatible with the assumption that input DNA is rapidly degraded following fixation by the cel1. The results of the reconstruction experiment (presented in Fig. 3) designed to test this point are likewise incompatible with our second possibility. In these experiments it was assumed that the penetration of recipient bacteria by heterologous, genetically inert B. cereus DNA is similar to the uptake of homologous transforming DNA by B. subtilis. This assumption is based on the finding that the amount of firmly bound radioactive donor DNA that can be extracted from recipient bacteria is directly related to the competency of the population used as recipients. Such a relationship would not be expected if the re-extracted DNA was merely associated non-specifically with the bacteria. This observation is similar to that of Lerman & Tolmach (1957), who found that Escherichia coli DNA is fixed by competent Pneumococcus in a DNaseinsensitive state and that it is retained by them for at least several cell divisions. The assumption that heterologous DNA penetrates competent B. subtilis is further strengthened by the observation that DNA from phage SP3, when added to competent recipients, initiates production of mature phage particles (Romig, 1962). Since it was assumed that the heterologous B. cereus DNA was actually incorporated by the recipient B. subtilis, and since radioactive material was neither transferred from this DNA to the recipient DNA, nor was it genetically functional, it was coneluded that our second possibility was unlikely. It cannot be excluded, however, that intracellular nuc1eases of B. 8ubtilis are ineffective against B . cereus DNA, yet can degrade excess intracellular B. subtili« donor DNA that fails to be integrated into the genome of the recipient bacteria. (3) After penetration of recipient bacteria, donor DNA becomes single-stranded

Our failure, in the experiments reported in Fig. 4, to detect a second peak of radioactivity corresponding to single-stranded DNA was interpreted to mean that donor DNA incorporated by B. subtilis does not become single-stranded. It is possible that small quantities of radioactive single-stranded DNA would not be separated from an excess of unlabeled recipient DNA in this experiment. Our results indicate, however, that if any DNA becomes single-stranded after it is incorporated by recipient B. 8ubtilis, the amount is small.

244

J. J. PENE AND W. R. ROMIG

These results differ from those of Lacks (1962), who used almost identical procedures (density-gradient centrifugation in CsCI) to demonstrate single-stranded DNA in extracts from Pneumococcus that were exposed for an eighth of a generation to 32P_Iabeled DNA. In his experiments, about one-half of the radioactive material re-extracted from the bacterial recipients formed bands at a position characteristic of heat-denatured DNA. (4) The material contribution from exoqenotic transforming principle to the resident genome occurs as a result of genetic recombination

We conclude that genetic recombination in B. subtilis occurs by a mechanism involving the physical association of donor and resident DNAt. It is probable that the sizes of the incorporated pieces of homologous donor DNA are small, since the density of the DNA extracted from recipient bacteria is not measurably affected. It is likewise probable that the structures participating in genetic recombination remain double stranded, since we have been unable to detect single-stranded DNA of donor origin. Our conclusions concerning the mechanism of recombination are in agreement with those of Fox & Hotchkiss (1960). They reported that in Pneumococcus the biological activity of donor DNA disappears immediately after uptake, and recovery of function and integration of genetic markers occur approximately six minutes later. During that time, little or no DNA synthesis occurs. In addition, these investigators showed that donor DNA re-extraeted from bacteria six minutes or more after uptake was not physically or biologically damaged. Fox (1962) has since shown that the rate of inactivation of biological activity by 32P_decay of donor DNA re-extracted following uptake by competent cells remains identical to that of the original DNA used to transform the bacteria. The efficiency of phosphorus disintegrations in causing structural damage to donor DNA would be expected to increase if a transition from a double-stranded to a single-stranded structure occurred after uptake. These results are not in agreement with those obtained by Lacks (1962), who studied the fate of 32P-Iabeled donor DNA in Pneumococcus. Mter uptake, the distribution of radioactive material into nucleic acid components was determined by fractionation on methylated albumin-coated kieselguhr columns described by Mandell & Hershey (1960). Lacks concluded that, immediately after uptake, double-stranded 32P-Iabeled donor DNA was degraded, as evidenced by incorporation of radioactive material into fractions corresponding to low molecular weight compounds, single-stranded DNA and eventually into the resident genome. The causes for the differences between these sets of observations are not known, but might result from differences in the experimental methods or bacterial recipients used. For example, in contrast to its effect on Pneumococcus (Ephrussi-Taylor, 1958), chloramphenicol does not interfere with the formation of irreversibly transformed cells following uptake of DNA by competent B. subtilis (Pene, unpublished results). This observation could reflect a difference in the mechanism of integration of genetic markers in these two bacterial species. In B. 8ubtilis the nature of the physical association of donor and recipient DNA in the recombinant genome has not as yet been resolved. This association probably occurs by breakage of the resident genome and insertion of a fragment derived from the donor DNA, but could conceivably result from the formation of a permanent

t After these investigations were initiated, Lorkiewicz, Opara-Kubinska & Szybalski (1961) reported similar results with 5-bromouracil-labeled cella of B. tJ'Ubtili8.

GENETIC RECOMBINATION IN B. SUBTILIS

245

partial diploid without the substitution of the resident allele. A detailed description of the mechanisms of genetic recombination should become possible when cell-free systems are developed in which the components co-operating in the formation of recombinants can be isolated and studied. This work was supported by Contract NONR-233(67) from the Office of Naval Research. One of the authors (J. J. P.) is a U.S. Public Health Service Predoctoral Fellow. REFERENCES Burkholder, P. R & Giles, N. H. (1947). Amer, J. Bot. 34, 345. Ephrussi-Taylor, H. (1958). In Recent Progress in Microbiology, ed. by G. Tunevall, pp. 51 to 68. Springfield, Illinois: C. C. Thomas. Fox, M. S. (1962). Proc. Nat. Acad. s«, Wash. 48, 1043. Fox, M. S. & Hotchkiss, R D. (1960). Nature, 187, 1002. Fuerst, C. R, Jacob, F. & Wollman, E. L. (1961). In Sexuality and the Genetics of Bacteria, pp. 244 to 246. New York: Academic Press. Keck, K. (1956). Arch. Biochem. Biophys. 63, 446. Lacks, S. (1962). J. Mol. Biol. 5, Il9. Lederberg, J. (1955). J. Cell. Compo Physiol. 45, 75. Lerman, L. S. & Tolmach, L. J. (1957). Biochim. biophys. Acta, 26, 68. Lorkiewicz, Z., Opara-Kubinska, Z. & Szybalski, W. (l9Bl). Fed. Proc. 20, 360. Mandell, J. D. & Hershey, A. D. (1960). Analyt. Biochem. I,' 66. Marmur, J. & Grossman, L. (1961). Proe. Nat. Acad. Sci., Wash. 47,778. Marmur, J. & Lane, D. (1960). Proc, Nat. Acad. Sci., Wash. 46, 453. Meselson, M. & Stahl, F. W. (l958). Proc. Nat. Acad. Sci., Wash. 44, 671. Meselson, M., Stahl. F. W. & Vinograd, J. (1957). Proe, Nat. Acad. s«; Wash. 43,581. Meselson, M. & Weigle, J. (1961). Proc; Nat. Acad. Sci., Wash. 47,857. Romig, W. R (1962). Virology, 16, 452. Schaeffer, P. (1961). Thesis. Faeulta des Sciences de I'Universite de Paris. Spizizen, J. (l958). Proc, Nat. Acad, Sci., Wash. 44, 1072. Weigle, J., Meselson, M. & Paigen, K. (1959). J. Mol. Biol. I, 379.