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Bacteriophage P1-mediated generalized transduction in Escherichia coli: Structure of abortively transduced DNA
VIROLOGY
106,
30-40 (1980)
Bacteriophage
PI-Mediated Generalized Structure of Abortively
Transduction in Escherichia co/i: Transduced DNA
ROZANNE M. SANDRI’ AND HILLARD Departwnt
of Biology,
The Johns Hopkins
University,
BERGERZ
Baltimore,
Maryland
21218
Accepted May 30, 1980
DNA was extracted from [3H]thymidine-labeled Escherichia coli infected with 32P-5-bromouracil-labeled transducing particles, and analyzed in equilibrium CsCl gradients. About 75% of the 32P-labeled transducing DNA equilibrated in the heavy region of the gradient. This nonchromosomally associated DNA, which represented abortively transduced DNA, remained fully heavy up to 5 hr after infection. Sedimentation of abortively transduced DNA in neutral and in alkaline sucrose gradients showed that both strands remained intact up to 3 hr after infection. When infected recipient bacteria were lysed by a nonionic detergent procedure, about 60% of the abortively transduced DNA could be recovered in a form which sedimented 1.7 to 1.8 times faster than linear Pl phage DNA in neutral sucrose gradients. Sedimentation of abortively transduced DNA and Pl prophage DNA in neutral sucrose gradients showed faster and slower sedimenting forms of abortively transduced DNA which corresponded to supercoiled and relaxed circular prophage DNA, respectively. Agarose gel electrophoresis of abortively transduced DNA showed bands which comigrated with circular prophage DNA. Treatment of abortively transduced DNA with sodium lauryl sulfate, with heat (W’), or with Pronase converted the DNA to a form which sedimented with linear Pl phage DNA in neutral sucrose gradients, and which migrated with linear Pl transducing particle DNA in agarose gels. The data suggest that abortively transduced DNA exists as a circular DNA-protein complex.
transduced DNA has been shown to retain its fully heavy position on CsCl gradients In genetic studies of Pl and PZ2-mediated for up to 5 hr after infection (Ebel-Tsipis et generalized transduction, a majority of aZ., 1972). transductants were found to be abortive In our analysis of the fate of Pl trans(Gross and Englesberg, 1959; Hertman and Luria, 1967; Ozeki, 1956, 1959), that is, the ducing DNA after its entry into recipient transduced elements were not integrated bacteria, we found that a large percentage (85-90%) of the transduced DNA was not into recipient genomes, but were transintegrated into recipient chromosomes mitted unilinearly to daughter cells (Leder(Sandri and Berger, 1980). This “nonassoberg, 1956). Abortively transduced DNA ciated” DNA presumably represented aborfragments apparently do not replicate since the number of abortive transductants has tively transduced DNA. In the present study, we have extended been shown to remain constant with inour observations on the nonassociated transcreased growth of recipient cultures (Lederberg, 1956; Ozeki, 1959), and sensi- duced DNA and have investigated the structivity to decay of incorporated 32Pdoes not ture of the DNA by velocity sedimentation change (Hartman and Kozinski, 1962). in neutral sucrose gradients and by agarose gel electrophoresis. For these studies, the Additionally, density labeled P22 abortively abortively transduced DNA was isolated from recipient cells lysed by a nonionic de1 Address reprint requests to author at Department tergent method. We found that in the abof Human Genetics, The University of Michigan, Ann sence of agents which digest or denature Arbor, Mich 48109. * Deceased. protein, abortively transduced DNA sediINTRODUCTION
00426822!80/130030-11$02.00/O Copyright All rights
Q 1980 by Academic F’ress, Inc. of reproduction in any form reserved.
30
STRUCTURE
OF Pl ABORTIVELY
TRANSDUCED
DNA
31
0.2% Triton X-100, 0.0625 M EDTA, and 0.05 M Tris-HCl (pH 8.0). The suspension cleared in 20-30 min. The lysate was centrifuged at 30,000 g in an SS-34rotor for 15 min at 4” to sediment the chromosomal DNA. Such lysates are termed “cleared lysates.” This procedure resulted in the sedimentation of over 90% of the host chromosomal DNA while 40-60% of the nonchromosomally associated, “abortively transduced” MATERIALS AND METHODS DNA remained in the supernatant. Sucrose gradient velocity sedimentation. Materials. Bacterial and phage strains, media, and the preparation of 32P-5-bromo- Linear neutral sucrose gradients of either uracil-labeled transducing particles were 5-20% or 20-31% sucrose contained 0.03 M Tris-HCl (pH 8.0), 0.005 M EDTA, and 0.5 described in the accompanying publication (Sandri and Berger, 1980). Sodium lauryl M NaCI. Centrifugation was performed in sulfate (SDS) was purchased from BDH an SW 50.1 rotor at 32,500 rpm for 100 min Chemicals. Triton X-100 was obtained from at 20” in the case of the 5-20% sucrose graSigma Chemical Company. Agarose was dients, and at 45,000 rpm for 55 min at 20” purchased from Sea Kern (Marine Col- for the 20-31% sucrose gradients. Linear 5-20% alkaline sucrose gradients loids Ltd.). Solutions of Pronase (5 mg/ml in 0.25 M contained 1.0 M NaCl, 0.001 M EDTA, and Tris-HCl, pH 8.0) were heated routinely at 0.3 M NaOH. Centrifugation was performed 83” for 20 min prior to use. RNase (3 mg/ml in an SW 50.1 rotor at 32,500 rpm for 2 in 0.25 M Tris-HCl, pH 8.0) also was heated hr at 20”. Agarose gel electrophoresis. Agarose slab at 83” for 20 min before use. gels (0.8% agarose) were prepared and run Lysis of infected bacteria by Triton X-100. The procedure was a modification of a method in TEB buffer (0.09 M Tris, 0.09 M boric described by Kuperstoch-Portnoy et al. acid, and 0.0025 M EDTA, at pH 8.0). The (1974). Recipient bacteria were infected dimensions of the slab gels were 17 x 12.5 with 32P-BU-labeled transducing particles x 0.25 cm. Gels were run at 150 V for 4.5 hr. as described by Sandri and Berger (1980). The slabs were dried onto Whatman 3MM Samples of infected cultures were removed filter paper in a Bio-Rad slab gel dryer. Dried gels were autoradiographed for at the times indicated in each experiment, and were diluted into an equal volume of 2 weeks. TELS buffer LO.05M Tris-HCl (pH S.O>, RESULTS 0.005 M EDTA, and 0.05 M NaCl]. The infected bacteria were centrifuged at 10,000 g for 10 min at 4”, then resuspended and re- Nonchromosomally Associated DNA centrifuged twice in 10 ml of TELS buffer. In analyzing the fate of Pl transducing The bacterial pellet was resuspended in 1.0 DNA following its entry into recipient bacml of 25% sucrose in 0.05 M Tris-HCl (pH teria, we found that a large percentage of 8.0). The suspension was placed in an ice the transduced DNA was not associated bath and maintained on ice throughout the with recipient chromosomes (Sandri and following operations. After 5 min, 0.2 ml of a Berger, 1980). Figure 1 shows the CsCl grafreshly prepared lysozyme solution (10 mgl dient profile of DNA extracted from [3H]ml in 0.25 M Tris-HCl, pH 8.0) was added. thymidine-labeled recipient bacteria which After an additional 5 min, RNase was added were infected with 32P-BU-labeled transto a final concentration of 100 pg/ml. Again ducing particles for various times at 37”. after 5 min. 0.4 ml of 0.25 M EDTA (pH 8.0) About 75% of the 32Plabel was recovered was added, and the incubation was continued in the heavy region at the times measured, for 5 min. The bacteria were lysed by the in accord with our previous results (Sandri addition of 1.6 ml of a solution containing and Berger, 1980). These data also illustrate
mented and migrated in a manner which displayed the properties expected of circular DNA, while in the presence of these agents, the abortively transduced DNA cosedimented and comigrated with linear Pl DNA suggesting that abortively transduced DNA may exist as a circular DNA-protein complex.
32
SANDRI AND BERGER
6
FRACTION
NUMBER
FIG. 1. C&l equilibrium density gradient analysis of DNA extracted from bacteriainfected with =PBU-labeled transducing particles. [SHlThymidine-labeled recipient bacteria (strain AB115’7 (Plkc)) were infected with S2P-BU-labeled transducing particles, then incubated at 37”. Samples of the infected bacterial culture were lysed by the procedure described by Sandri and Berger (1980) at: (A) 0 min, (B) 30 min, (C) 180 min, and (D) 300 min after a 20-min period of adsorption of labeled transducing particles. DNA-CsCl solutions were adjusted to a refractive index of 1.3990. Centrifugation was performed in a Type 50 fixed-angle rotor at 35,000 rpm for 48 hr at 20”. Tubes were punctured at the bottom and lo-drop fractions were collected. Radioactivity was counted as described previously (Sandri and Berger, 1980). Density increased from right to left. Symbols: ?‘P (0); [3H]-thymidine (0).
that the “nonassociated” DNA had not replicated, even after 5 hr incubation, since the same percentage of 32Pcounts equilibrated in the heavy region in samples extracted at 5 hr as in samples extracted at 30 min (Figs. 1B and D). Our studies on Pl transduction showed that transductants doubled by 180 min after adsorption, and that integrated transducing DNA was replicated by 180 min (Sandri and Berger, 1980). Thus, the majority input transducing DNA component, the nonassociated, nonreplicated DNA, most likely represented abortively transduced DNA. In fact, a majority of Pl transductants are abortive (Gross and Englesberg, 1959; Hertman and Luria, 1967).
lecular weight as Pl DNA (6 x 10’) (Ikeda and Tomizawa, 1965a; Sandri and Berger, 1980). We analyzed the molecular weight of abortively transduced DNA by velocity sedimentation in neutral and alkaline sucrose gradients to determine if the abortively transduced DNA remained intact. In Fig. 2, abortively transduced DNA extracted at 30 min (panel A) and at 180 min (panel B) was analyzed on neutral sucrose gradients. The 32P-labeledDNA sedimented at approximately the position of the 3Hlabeled Pl marker DNA. The density label probably accounts for the slightly faster sedimentation of abortively transduced DNA. DNA extracted directly from BUlabeled transducing particles shows the Molecular Weight of Abortively Transduced same sedimentation pattern (Sandri and DNA Berger, 1980). An aliquot of abortively Transducing DNA extracted from puri- transduced DNA extracted at 180 min also fied transducing particles has the same mo- was run on an alkaline sucrose gradient
STRUCTURE
OF Pl ABORTIVELY
TRANSDUCED
Structure
FRACTION
NUMBER
FIG. 2. Velocity sedimentation analysis of abortively transduced DNA. Recipient bacteria (strain AB1157 (PI/cc)), infected with 92P-BU-labeled transducing particles, were lysed as described previously (Sandri and Berger, 1980). The extracted DNA was centrifuged to equilibrium in CsCl as described in the legend to Fig. 1. Fractions containing heavy, abortively transduced DNA were pooled then dialyzed to remove CsCl. (A) A 0.1-ml aliquot of abortively transduced DNA extracted at 30 min was run on a 5-20% neutral sucrose gradient. (B) A O.l-ml aliquot of abortively transduced DNA extracted at 180 min was run on a 5-20% neutral sucrose gradient. (C) A O.l-ml aliquot of abortively transduced DNA extracted at 180 min was run on a 5-20% alkaline sucrose gradient. In each case, [3H]thymidine-labeled PI DNA was added as a marker. Centrifugation was performed as described under Materials and Methods. Ten-drop fractions were collected and counted for radioactivity. Sedimentation was from right to left in these gradients and in all succeeding gradients. Symbols: 35P(0); [3H]thymidine (0).
(panel C). The abortively transduced DNA sedimented with Pl marker DNA illustrating that abortively transduced DNA remained intact up to 3 hr after entry into recipient bacteria.
DNA
of Abortively
33
Transduced DNA
Abortively transduced DNA escaped degradation by host nucleases, indicating that it was protected in some way. Ikeda and Tomizawa (1965b) reported that there was protein associated with Pl transducing DNA. Their observations suggested to us that abortively transduced DNA might circularize via attached protein, rendering the DNA resistant to nuclease attack. We analyzed the structure of abortively transduced DNA by velocity sedimentation in neutral sucrose gradients (Fig. 3). Nonlabeled recipient bacteria infected with 32PBU-labeled transducing particles were lysed by the Triton X-100 procedure (see Materials and Methods) at 60 min after adsorption of transducing particles. This procedure differs from the DNA extraction procedure used in previous experiments in that it did not utilize ionic detergent or protease. Aliquots of the “cleared” supernatant, which contained abortively transduced DNA, were run on ZO-31% neutral sucrose gradients. In the untreated sample (Fig. 3A), about 60% of the 32Pcounts were found in a peak which sedimented 1.7 to 1.8 times faster than linear Pl DNA, whose position is indicated by an arrow. A peak of slower sedimenting DNA also was observed which sedimented 1.2-1.3 times faster than linear DNA. When abortively transduced DNA was treated with SDS (panel B), or was incubated at 70” for 20 min (panel C), 32P-labeled DNA was found at the position of linear Pl DNA. Abortively transduced DNA which was incubated with RNase (panel D) showed no significant differences in the gradient profile from the untreated sample (panel A). The 32P-labeled DNA which sedimented 1.7-1.8 times faster than linear DNA may have been supercoiled. It has been shown that supercoiled X DNA sedimented 1.5 to 1.9 times faster than the linear form in neutral sucrose gradients (Bode and Kaiser, 1965), and that supercoiled P22 DNA sedimented 1.7 times faster than linear P22 DNA (Rhoades and Thomas, 1968). The slower sedimenting material may have represented relaxed, circular DNA. The data in Fig. 3B also suggest that protein may
34
SANDRI AND BERGER
have been involved in the maintenance of the DNA structures which sediment faster than linear DNA. Sedimentation of Pl Prophage DNA and Abortive1 y Transduced DNA Pl prophage exist as circular plasmids which do not integrate into bacterial chromosomes (Ikeda and Tomizawa, 1968). We have used the Pl lysogen, E. coli strain AB1157 (Plkc), as the recipient in our transduction experiments to prevent expression of the small percentage of Pl infectious phage present in the purified transducing particle preparations (Sandri and Berger, 1980). Since prophage DNA also would remain in the supernatant of cleared lysates, we used prophage DNA as a marker for the position of Pl circular DNA in neutral sucrose gradients. We infected 13H]thymidinelabeled strain AB1157 (Plkc) with 32P-BUlabeled transducing particles. Cleared lysates were prepared at 60 min after adsorption of the transducing particles. Aliquots were run on 20-31% neutral sucrose gradients (Fig. 4). In the untreated sample (Fig. 4A), two peaks were observed for both the 3H-labeled Pl prophage DNA and the 32Plabeled abortively transduced DNA. However, both peaks of abortively transduced DNA sedimented ahead of the corresponding prophage peaks. Similar profiles were seen when DNA samples were extracted at 30 and 180 min after adsorption of labeled transducing particles (Fig. 5). When samples extracted at 60 min were treated with SDS (Fig. 4B), or were heated at 70” (Fig. 4C), two peaks were observed again for the prophage DNA, illustrating that these treatments had no appreciable effect on the sedimentation profile of Pl prophage DNA. However, these treatments completely converted the abortively transduced DNA to a more homogeneous, slower sedimenting form. In the previous section, we showed that after treatment with SDS or heat, abortively transduced DNA sedimented in the position of linear Pl DNA (Figs. 3B and C). The peaks of abortively transduced DNA in Figs. 4B and C, then, probably represented linear DNA. The slower sedimenting peak
‘.,& IO
I 20 30 FRACTION
IO
20
30
NUMBER
FIG. 3. Velocity sedimentation analysis of abortively transduced DNA in cleared lysates. Nonlabeled recipient bacteria (strain AB1157 (Plkc)) were infected with SZP-BU-labeled transducing particles then incubated at 37” for 60 min. Infected bacteria were lysed by the Triton X-100 procedure and the lysates were “cleared” as described under Materials and Methods. (A) A O.l-ml aliquot of the cleared supernatant containing abortively transduced DNA was mixed with 0.1 ml 3H-labeled Pl phage DNA. (B) A O.l-ml ahquot of the cleared supernatant was mixed with 0.1 ml 3H-labeled Pl DNA and SDS (0.25%, final concentration). (C) A O.l-ml aliquot of the supernatant fraction combined with 0.1 ml 3Hlabeled Pl DNA was incubated at 70” for 20 min. (D) A O.l-ml aiiquot of the cleared supernatant was mixed with Pl marker DNA and RNase (100 pg/ml). The sample was incubated at 25” for 15 min. Samples were layered onto 20-318 neutral sucrose gradients which contained a 0.2-ml shelf of 70% sucrose at the bottom. The gradients were centrifuged as described under Materials and Methods. Fifteen-drop fractions were collected and the radioactivity was counted. Arrows indicate the position of 3H-labeled Pl DNA.
of prophage DNA sedimented about 1.07 times faster than the abortively transduced DNA treated with SDS or heat (Figs. 4B, and C). Ikeda and Tomizawa (1968) showed that open circular Pl prophage DNA sedi-
STRUCTURE
IO
20
30
OF Pl ABORTIVELY
IO FRACTION
20 NUMBER
TRANSDUCED
30
DNA
IO
20
30
FIG. 4. Velocity sedimentation analysis of abortively transduced DNA and Pl prophage DNA. [3H]Thymidine-labeled E. coli strain AB1157 (Plkc)) was infected with 3ZP-BU-labeled transducing particles. The infected bacteria were lysed at 60 min after absorption of transducing particles by the Triton X-100 method, and the lysates were “cleared” by centrifugation. (A) A O.l-ml aliquot of the cleared supernatant, which contained abortively transduced DNA and Pl prophage DNA. (B) A 0.1-ml aliquot of the cleared supernatant treated with SDS (0.25%). (C) A O.l-ml aliquot of the supernatant following incubation at 70” for 20 min. SampIes were layered onto 20-310/oneutral sucrose gradients with a 0.2ml shelf of 70% sucrose at the bottom. Centrifugation was performed as described under Materials and Methods. Fifteen-drop fractions were collected and counted for radioactivity. Symbols: 32P(0); [3H]thymidine (0).
mented about 1.09 times faster than linear Pl DNA under their conditions. The data above suggest that the slower sedimenting peak of prophage DNA represented relaxed circular prophage DNA. The faster sedimenting prophage peak sedimented 1.4 to 1.6 times faster than abortively transduced DNA (Figs. 4B, C), indicating that the prophage DNA in this peak was probably super-coiled. In the untreated samples (Figs. 4A and 5A, B), both the faster and slower sedimenting peaks of abortively transduced DNA banded ahead of the corresponding circular prophage peaks. Pl prophage DNA is 12% shorter than Pl phage DNA (Ikeda and Tomizawa, 1968;Yun and Vapnek, 1977), whereas abortively transduced DNA has the same molecular weight as Pl phage DNA (Fig. 2). The difference in sedimentation may be due, at least in part, to the size difference in prophage and abortively transduced DNA. Additionally, the protein associated with abortively transduced DNA may contribute to its faster rate of sedimentation.
Such a phenomenon has been observed in studies on complexed (protein associated) versus noncomplexed plasmid DNA (Clewell and Helinski, 1969;Kline and Helinski, 1971). Agarose Gel Electrophoresis DNA
of Circular
Circular DNA can be separated readily from linear DNA upon electrophoresis in agarose gels (Meyer et al., 1976; Mickel et al., 1977; Mickel and Bauer, 1976). Since we planned to use Pl prophage DNA as a marker, we first determined the position of relaxed circular prophage DNA and supercoiled prophage DNA relative to linear Pl phage DNA (Fig. 6). Prophage DNA was isolated as indicated in the legend to Fig. 6. Lane 1 of Fig. 6 shows the migration of 32Plabeled Pl DNA extracted from phage particles. Relaxed circular Pl prophage DNA (prophage DNA from the slower sedimenting peak in a preparative sucrose gradient-see legend to Fig. 6) was run in Lane 2. Pl phage DNA displayed roughly
36
SANDRI AND BERGER
Analysis of Abortively on Agarose Gels
Transduced
DNA
Abortively transduced DNA was isolated from nonlabeled recipient bacteria infected with 32P-BU-labeled transducing particles as outlined in the legend to Fig. 7. Cleared lysates were not prepared as in the sedimentation studies described above, instead, transduced cultures were lysed with Triton X-100 then centrifuged to equilibrium in CsCl. The heavy, nonassociated transducing DNA peak was pooled for electrophoresis studies. Aliquots were run on a 0.8% agarose slab gel (Fig. 7). Untreated abortively transFraction
Number
FIG. 5. Velocity sedimentation analysis of abortively transduced DNA and Pl prophage DNA isolated 30 and 180 min affer infection. [3H]Thymidine-labeled strainAB115’7(Plkc) wasinfectedwith3*P-BU-labeled transducing particles. The infected bacteria were lysed at (A) 30 min and (B) 180 min after adsorption of transducing particles as outlined in the legend to Fig. 4. A O.l-ml aliquot of each cleared supernatant was layered onto a 20-31% neutral sucrose gradient with a 0.2-ml shelf of 70% sucrose. Centrifugation was performed as described under Materials and Methods. Thirty-drop fractions were collected and counted for radioactivity. Symbols: 32P(0); [3H]thymidine (0).
1Btimes the mobility of prophage relaxed circular DNA. Super-coiled prophage DNA (prophage DNA from the faster sedimenting peak in a preparative sucrose gradient) was run in Lane 3. A predominant band displayed 6-times the mobility of relaxed circular DNA and about O.× the mobility of linear Pl phage DNA. A band also was observed at the position of relaxed circular DNA as well as a faint band at the linear position. Conversion to these forms may have occurred during handling of the prophage DNA. The data on the migration of Pl prophage DNA are in accord with the results of Mickel et al. (1977), who analyzed plasmid DNA molecules having a wide range of molecular weights under a variety of agarose gel electrophoresis conditions. Higher molecular weight super-coiled DNA migrated more slowly than the corresponding linear DNA in their studies.
FIG. 6. Autoradiograph of Pl prophage DNA following agarose gel electrophoresis. E. coli strain AB1157 (Plkc) was labeled with 32P (2 &i/ml) in TCG medium for 3 hr at 37”. The bacteria were lysed by the Triton X-100 method and the chromosomal DNA was sedimented. Aliquots of the supernatant fraction of the cleared lysate, containing Pl prophage DNA, were banded in 20-31% neutral sucrose gradients. The faster and slower sedimenting peaks containing supercoiled and relaxed circular prophage DNA, respectively, were pooled separately for dialysis. Samples containing 20 ~1 of the pooled fractions (about 200 cpm each) were run on a 0.8% agarose slab gel as described under Materials and Methods. In Lane 1, a 20-~1 sample (with about 2000 cpm) of 3ZP-labeled PI DNA extracted from infectious phage particles was run as a marker for the position of linear Pl DNA. Lane 2 contained an aliquot of the slower sedimenting (relaxed circular) prophage DNA. An aliquot of the faster sedimenting (supercoiled) prophage DNA was run in Lane 3. The origin is at the top of the gel.
STRUCTURE
OF Pl ABORTIVELY
FIG. 7. Autoradiograph of 32P-labeled abortively transduced DNA following agarose gel electrophoresis. Nonlabeled recipient bacteria (strain AB1157 (Plkc) were infected with 3ZP-BU-labeled transducing particles for 60 min before lysis with Triton X-100. The chromosomal DNA was not sedimented by centrifugation, but rather, the lysate was mixed with CsCl immediately and centrifuged to equilibrium. Fractions in the heavy region containing abortively transduced DNA were pooled. After dialysis, aliquots were electrophoresed on a 0.8% agarose slab gel. 3ZP-Labeled Pl prophage DNA which was used as a circular marker was isolated separately as described in the legend to Fig. 5. Pooled fractions of neutral sucrose gradients containing faster sedimenting (supercoiled) and slower sedimenting (relaxed circular) prophage DNA were combined in this experiment. 32P-BUlabeled transducing DNA which was used as a linear DNA marker was extracted from purified transducing particles as described previously (Sandri and Berger, 1980). Transducing particle DNA was run in Lane 1. Pl prophage DNA was run in Lanes 2-5. The following treatments were used: Lane 2, untreated; Lane 3, incubation with Pronase (500 pg/ml) for 30 min at 37”; Lane 4, incubation with RNase (200 pg/ml) for 30 min at 37”, and Lane 5, treatment with SDS (0.25%). Abortively transduced DNA was run in Lanes 6-12. The following treatments were used: Lane 6, untreated; Lane 7, incubation at 37” for 30 min; Lane 8, incubation with Pronase (500 pg/ml) for 30 min at 37”; Lane 9, incubation with RNase (200 pg/ml) for 30 min at 37”; Lane 10, treatment with SDS (0.25%); Lane 11, treatment with Sarkosyl (0.25%), and Lane 12, incubation for 20 min at 70”. Twenty microliters of each
TRANSDUCED
DNA
97
duced DNA was run in Lanes 6 and 7. Two bands were observed which corn&rated with relaxed circular prophage DNA and supercoiled prophage DNA markers, respectively (Lanes 2-5). A third band corn&rated with linear transducing particle DNA (Lane 1). A similar migration pattern was observed when abortively transduced DNA was incubated with RNase (Lane 9) or Sarkosyl (Lane 11). In contrast, when samples were treated with SDS (Lane 10) or were incubated at 70” (Lane 12>,only linear abortively transduced DNA was observed. In addition, abortively transduced DNA, which was incubated with Pronase for 30 min at 37” before electrophoresis, migrated predominantly as linear DNA, although circular species also were observed (Lane 8). We have noted in several experiments that incubation of abortively transduced DNA with Pronase for 60 min at 37” completely converted the DNA to the linear form (data not shown). However, after incubation times of 15-30 min, some circular species persisted. The reason for this is not clear. We also observed an additional faint band which migrated faster than linear DNA in the abortively transduced DNA samples. The identity of this band is not known. It appears to have been unaffected by any of the treatments since its intensity was about the same in all of the lanes. Finally, we noted that when Pl prophage DNA was treated with RNase, the supercoiled band appeared to be converted to the relaxed form (Lane 4). It is unlikely that the RNase preparation was contaminated with DNase activity since RNase solutions were heated at 83” for 20 min prior to use to inactivate any DNase activity. In addition, the abortively transduced DNA which sedimented and migrated like supercoiled DNA was not affected by RNase treatment (Fig. 3D and Fig. 7, Lane 9). The prophage DNA in these experiments was isolated from exponentially growing cultures of Pl lysogens so that the prophage DNA was presumably replicating as well. Although we cannot explain why RNase converted supercoiled DNA sample containing about 200 cpm was loaded onto the slab gel. The origin is at the top. A *“C dye marker (in upper right-hand corner) was used to orient the gel during autoradiography.
38
SANDRI
AND BERGER
prophage DNA to the relaxed form, it is possible the prophage DNA may contain RNA during its replication. Blair et al. (1972) have shown that supercoiled Co1 El DNA made in the presence of chloramphenicol is converted by RNase to the open circular structure. DISCUSSION
Our studies on Pl transduction showed that the nonchromosomally associated, abortively transduced DNA escaped degradation by host nucleases (Figs. 1 and 2), a fact which suggested to us that the structure was modified to a form resistant to the recBC nuclease (exonuclease V), a nuclease chiefly responsible for degradation of superinfecting linear duplex DNA (Benzinger et al., 1975; Oishi and Irbe, 19’77; Wackernagel, 1973).While circularization of the DNA would engender resistance to the recBC nuclease (Goldmark and Linn, 1972), it seemed unlikely that transducing DNA, which consists of donor bacterial DNA fragments, could circularize by recombination or annealing of homologous ends. Since the results of Ikeda and Tomizawa (1965b) showed that there was protein associated with Pl transducing DNA and that the protein was most likely associated with the ends of the DNA molecules, we investigated the possibility that abortively transduced DNA might circularize via attached protein. We analyzed abortively transduced DNA by velocity sedimentation in neutral sucrose gradients and by agarose gel electrophoresis. Those experiments showed that abortively transduced DNA species having the sedimentation and migration patterns expected of circular DNA molecules could be detected when recipient bacteria were lysed with Triton X-100. This procedure was used to prevent denaturation or dissociation of any protein which may be involved in joining the ends of the DNA molecules. In fact, treatment of abortively transduced DNA with SDS, with heat (70”), or with Pronase converted the DNA to a form which sedimented with linear Pl DNA and which migrated with linear transducing particle DNA. These treatments did not affect the sedimentation or migration of circular Pl prophage DNA.
The protein associated with transducing particle DNA has not been isolated or identified and it is not known whether it is of host or viral origin. Ikeda and Tomizawa (196513) have estimated its molecular weight to be around 5 x lo5 based on the density shift of deproteinated vs native transducing particle DNA in CsCl gradients. Circular DNA-protein complexes have been isolated from adenovirus (Robinson et al., 1973) and from Bacillus bacteriophages #29 and GA-l (Arnberg and Arwert, 1976; Ortin et al., 1971). Phage 429 and GA-1 complexes, like the abortively transduced DNA structures, were linearized by treatment with proteolytic enzymes (Arnberg and Arwert, 1976; Ortin et al., 1971), although, as we have previously discussed, the conversion of the abortively transduced DNA required a relatively long incubation with Pronase to effect complete conversion. Circular adenovirus molecules were converted to the linear form by treatment with proteases or SDS (Robinson et al., 1973), but were resistant to Sarkosyl (Rekosh et al., 1977). We observed a similar behavior with the abortively transduced DNA structures (Fig. 7). Pl transducing DNA is presumably injected into the recipient as a linear molecule since transducing DNA extracted from transducing particles by a nonproteolytic method is linear (Sandri, unpublished results). It is possible that it is the linear form which recombines with the chromosome to form stable transductants. This could occur if the linear form fails to circularize fast enough upon entry, in which case the DNA may be attacked by nucleases. The resulting fragments could have some structural property such as single-stranded ends which might stimulate their recombination with the chromosome. Two lines of evidence lend support to this hypothesis. First, in the previous paper we showed that the integrated DNA is substantially smaller than intact transducing DNA and the unincorporated portions of the transducing DNA molecules are degraded. Second, most of the 32P label becomes associated with the recipient DNA during the first hour after transduction with only a small increase in association after that (Sandri and Berger,
STRUCTURE
OF Pl ABORTIVELY
1980). Once the transducing DNA circularizes it becomes protected from nucleases and may undergo recombination with the chromosome at very low frequency, similar perhaps to the recombination frequency between the chromosome and a plasmid having a region of homology to the chromosome, as for example, homozygosis of lac-IF’lac’ bacteria. It has been estimated that the frequency of conversion from an abortive to stable transductant was as low as lop3 per generation (Ozeki and Ikeda, 1968). However, Benzinger and Hartman (1962) showed that if transducing phage are uv irradiated, the number of abortive transductants decreases while the number of stable transductants increases. These authors estimated that a single hit was sufficient to convert an abortive to a complete transductant, that is, to undergo recombination with the chromosome. Since uv repair mechanisms involve removal of DNA damage, presumably donor DNA was nicked at the site of damage. In fact, Helling (1973) has shown that the excision-repair genes uvrA+ and uvrB+ are required for most uv-stimulated transduction as is the RecA+ gene. Single-strand nicks alone probably are not sufficient for the conversion of abortive to complete transductants since nicks made by 32P decay (Hartman and Kozinski, 1962) or X-irradiation (Takabe and Hartman, 1962) failed to stimulate the formation of stable transductants. Some process following the nicking such as the generation of a single-stranded end as proposed above may be required for recombination with the chromosome. In any event, the formation of a circular DNA molecule through a protein linker can account for the stability of abortively transduced DNA. ACKNOWLEDGMENTS
The authors wish to thank Dr. Philip E. Hartman for critical reading of the manuscript and for valuable advice. We also thank Drs. Marc Rhoades and Peter Gauss for helpful suggestions. R.M.S. would alsolike to thank Dr. Robert P. Dottin, in whose laboratory this research was completed, for his gracious hospitality and helpful discussions. R.M.S. was a predoctoral trainee of the National Institutes of Health (Grant GM-57) during part of this research. This research was supported by Grants AI0861 from the National Institute of Allergy
TRANSDUCED
DNA
39
and Infectious Diseases, NP-175 from the American Cancer Society, and GB-43825 from the National Science Foundation. H.B. was recipient of a Career Development Award, National Institutes of Health. REFERENCES ARNBERG, A. C., and ARWERT, F. (1976). DNA-protein complex in circular DNA fromBacillus bacteriophage GA-l. J. Viral. 18, 783-784. BENZINGER, R., ENQUIST, L. W., and SKALKA, A. (1975). Transfection of Escherichia coli spheroplasts. V. Activity of reeBC nuclease in ret+ and ret- spheroplasts measured with different forms of bacteriophage DNA. J. Viral. 15, 861-871. BENZINGER, R., and HARTMAN, P. E. (1962). Effects of ultraviolet light on transducing phage P22. Viralogy 18, 614-626. BLAIR, D. G., SHERRATT,D. J., CLEWELL, D. G., and HELINSI(I, D. R. (1972). Isolation of super-coiled colicinogenic factor E, DNA sensitive to ribonuclease and alkali. Proc. Nat. Acad. Sci. USA 69, 25182522.
BODE, V. C., and KAISER, A. D. (1956). Changes in the structure and activity of h DNA in a superinfected immune bacterium. J. Mol. Biol. 14, 399-417. CLEWELL, D. G., and HELINSKI, D. R. (1969). Supercoiled circular DNA-protein complex inEscherichin coli: Purification and induced conversion to an open circular DNA form. Proc. Nut. Acad. Sci. GSA 62, 1159-1166. EBEL-TSIPIS, J., Fox, M. S., and BOTSTEIN,D. (1972). Generalized transduction by bacteriophage P22 in Salmonella typhimurium. II. Mechanism of integration of transducing DNA. J. Mol. Riol. 71, 449469. GOLDMARK,P. J., and LINN, S. (1972). Purification and properties of the recBC NAase of Esckerickia coli K12. J. Biol. Chem. 247, 1849-1860. GROSS,J., and ENGLESBERG,E. (1959). Determination of the order of mutational sites governing L-arabinose utilization in Escherickia coli B/r by transduction with phage Plbt. Virology 9, 314-331. HARTMAN, P. E., and KOZINSKI, A. W. (1962). Effects of P3* decay on transduction by Salmonella phage P22. Virology 17, 223-244. HELLING, R. B. (1973). Ultraviolet light-induced recombination Biochem. Biopkys. Res. Commun. 55, 752-757.
HERTMAN, I., and LURIA, S. E. (1967). Transduction studies on the role of a ret* gene in the ultraviolet induction of prophage lambda. J. Mol. Riot. 23, 117-133. IKEDA, H., and TOMIZAWA,J. I. (1965a). Transducing fragments in generalized transduction by phage PI. I. Molecular origin of the fragments. 6. Mol. Rio!. 14, 85- 109. IKEDA, H., and TOMIZAWA,J. I. (1965b). Transducing fragments in generalized transduction by phage Pl.
40
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II. Association of DNA and protein in the fragments. J. Mol. Biol. 14, 110-119. IKEDA, H., and TOMIZAWA,J. I. (1968). Prophage Pl, an extrachromosomal replication unit. Cold Spring Harbor Symp. Qua&. Biol. 33, 791-798. KLINE, B. C., and HELINSKI, D. R. (1971). F, sex factor ofEscherichia coli. Size and purification in the form of a strand-specific relations complex of supercoiled deoxyribonucleic acid and protein. Biochemistry 10,49’75-4980. KUPERSTOCH-PORTNOY, Y. M., LOVETT, M. A., and HELINSKI, D. R. (1974). Strand and site specificity of the relaxation event for the relaxation complex of the 13, antibiotic resistant plasmid R6K. Biochemistry 5484-5490. LEDERBERG, J. (1956). Linear inheritance in transductional clones. Genetics 41, 845-871.
MEYER, J. A., SANCHEZ, D., ELWELL, L. J., and FALKOW, S. (1976). Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J. Bacterial. 127, 1529- 1537. MICKEL, S., ARENA, JR., V., and BAUER, W. (1977). Physical properties and electrophoretic behavior of R-12 derived plasmid DNA. Nucleic Acids Res. 4, 1465- 1482. MICKEL, S., and BAUER, W. (1976). Isolation, by tetracycline selection, of small plasmids derived from R-factor R12 in Eschetihia coli K-12. J. Bacterial. 127, 644-655. OISHI, M., and IRBE, R. M. (19’77). Circular chromosomes and genetic transformation. In “Bacterial Transformation and Transfection” (R. Portoles, R. Lopez, and M. Espinoza, eds.), pp. 121-134. Elsevier/North-Holland, Amsterdam.
ORTIN, J., VINUELA, E., and SALAS, M. (1971). DNA-protein complex in circular DNA from phage 029. Nature
New Biol.
234, 275-277.
OZEKI, H. (1956). Abortive transduction. Carnegie Inst. Wash. Yearb. 55,302-303.
OZEKI, H. (1959). Chromosome fragments participating in transduction in Salmonella typhimurium. Genetics 44, 457- 470. OZEKI, H., and IKEDA, H. (1968). Transduction mechanisms. Annu. Rev. Genet. 2,245-278. REKOSH,D. M. K., RUSSELL,W. C., BELLES, A. J. D., and ROBINSON, A. J. (1977). Identification of a protein linked to the ends of adenovirus DNA. Cell 11,283-295. RHOADES, M., and THOMAS, C. A. (1968). The P22 bacteriophage molecule. II. Circular intracellular forms. J. Mol Biol. 37, 41-61. ROBINSON, A. J., YOUNGHUSBAND, H. B., and BELLETT, A. J. D. (1973). A circular DNA-protein complex from adenoviruses. Virology 56,54-69. SANDRI, R. M., and BERGER,H. (1980). Bacteriophage Pl-mediated generalized transduction in Escherichia coli: Fate of transduced DNA in Ret+ and RecArecipients. Virology 106, 14-29. TAKEBE, H. K., and HARTMAN, P. E. (1962). Effectsof X-Rays on transduction by Salmonella phage P22. Virology 17,295-302. WACKERNAGEL,W. (1973). Genetic transformation in E. coli: The inhibitory role of the recBC DNase. Biochem. Biophys. Res. Commun. 51,306-311. YUN, T., and VAPNEK, D. (1977). Electronmicroscopic analysis of bacteriophages Pl, PlCm, and P7: Determination of genome sizes, sequence homology, and location of antibiotic resistance determination. Virology
77, 376-385.
106,
30-40 (1980)
Bacteriophage
PI-Mediated Generalized Structure of Abortively
Transduction in Escherichia co/i: Transduced DNA
ROZANNE M. SANDRI’ AND HILLARD Departwnt
of Biology,
The Johns Hopkins
University,
BERGERZ
Baltimore,
Maryland
21218
Accepted May 30, 1980
DNA was extracted from [3H]thymidine-labeled Escherichia coli infected with 32P-5-bromouracil-labeled transducing particles, and analyzed in equilibrium CsCl gradients. About 75% of the 32P-labeled transducing DNA equilibrated in the heavy region of the gradient. This nonchromosomally associated DNA, which represented abortively transduced DNA, remained fully heavy up to 5 hr after infection. Sedimentation of abortively transduced DNA in neutral and in alkaline sucrose gradients showed that both strands remained intact up to 3 hr after infection. When infected recipient bacteria were lysed by a nonionic detergent procedure, about 60% of the abortively transduced DNA could be recovered in a form which sedimented 1.7 to 1.8 times faster than linear Pl phage DNA in neutral sucrose gradients. Sedimentation of abortively transduced DNA and Pl prophage DNA in neutral sucrose gradients showed faster and slower sedimenting forms of abortively transduced DNA which corresponded to supercoiled and relaxed circular prophage DNA, respectively. Agarose gel electrophoresis of abortively transduced DNA showed bands which comigrated with circular prophage DNA. Treatment of abortively transduced DNA with sodium lauryl sulfate, with heat (W’), or with Pronase converted the DNA to a form which sedimented with linear Pl phage DNA in neutral sucrose gradients, and which migrated with linear Pl transducing particle DNA in agarose gels. The data suggest that abortively transduced DNA exists as a circular DNA-protein complex.
transduced DNA has been shown to retain its fully heavy position on CsCl gradients In genetic studies of Pl and PZ2-mediated for up to 5 hr after infection (Ebel-Tsipis et generalized transduction, a majority of aZ., 1972). transductants were found to be abortive In our analysis of the fate of Pl trans(Gross and Englesberg, 1959; Hertman and Luria, 1967; Ozeki, 1956, 1959), that is, the ducing DNA after its entry into recipient transduced elements were not integrated bacteria, we found that a large percentage (85-90%) of the transduced DNA was not into recipient genomes, but were transintegrated into recipient chromosomes mitted unilinearly to daughter cells (Leder(Sandri and Berger, 1980). This “nonassoberg, 1956). Abortively transduced DNA ciated” DNA presumably represented aborfragments apparently do not replicate since the number of abortive transductants has tively transduced DNA. In the present study, we have extended been shown to remain constant with inour observations on the nonassociated transcreased growth of recipient cultures (Lederberg, 1956; Ozeki, 1959), and sensi- duced DNA and have investigated the structivity to decay of incorporated 32Pdoes not ture of the DNA by velocity sedimentation change (Hartman and Kozinski, 1962). in neutral sucrose gradients and by agarose gel electrophoresis. For these studies, the Additionally, density labeled P22 abortively abortively transduced DNA was isolated from recipient cells lysed by a nonionic de1 Address reprint requests to author at Department tergent method. We found that in the abof Human Genetics, The University of Michigan, Ann sence of agents which digest or denature Arbor, Mich 48109. * Deceased. protein, abortively transduced DNA sediINTRODUCTION
00426822!80/130030-11$02.00/O Copyright All rights
Q 1980 by Academic F’ress, Inc. of reproduction in any form reserved.
30
STRUCTURE
OF Pl ABORTIVELY
TRANSDUCED
DNA
31
0.2% Triton X-100, 0.0625 M EDTA, and 0.05 M Tris-HCl (pH 8.0). The suspension cleared in 20-30 min. The lysate was centrifuged at 30,000 g in an SS-34rotor for 15 min at 4” to sediment the chromosomal DNA. Such lysates are termed “cleared lysates.” This procedure resulted in the sedimentation of over 90% of the host chromosomal DNA while 40-60% of the nonchromosomally associated, “abortively transduced” MATERIALS AND METHODS DNA remained in the supernatant. Sucrose gradient velocity sedimentation. Materials. Bacterial and phage strains, media, and the preparation of 32P-5-bromo- Linear neutral sucrose gradients of either uracil-labeled transducing particles were 5-20% or 20-31% sucrose contained 0.03 M Tris-HCl (pH 8.0), 0.005 M EDTA, and 0.5 described in the accompanying publication (Sandri and Berger, 1980). Sodium lauryl M NaCI. Centrifugation was performed in sulfate (SDS) was purchased from BDH an SW 50.1 rotor at 32,500 rpm for 100 min Chemicals. Triton X-100 was obtained from at 20” in the case of the 5-20% sucrose graSigma Chemical Company. Agarose was dients, and at 45,000 rpm for 55 min at 20” purchased from Sea Kern (Marine Col- for the 20-31% sucrose gradients. Linear 5-20% alkaline sucrose gradients loids Ltd.). Solutions of Pronase (5 mg/ml in 0.25 M contained 1.0 M NaCl, 0.001 M EDTA, and Tris-HCl, pH 8.0) were heated routinely at 0.3 M NaOH. Centrifugation was performed 83” for 20 min prior to use. RNase (3 mg/ml in an SW 50.1 rotor at 32,500 rpm for 2 in 0.25 M Tris-HCl, pH 8.0) also was heated hr at 20”. Agarose gel electrophoresis. Agarose slab at 83” for 20 min before use. gels (0.8% agarose) were prepared and run Lysis of infected bacteria by Triton X-100. The procedure was a modification of a method in TEB buffer (0.09 M Tris, 0.09 M boric described by Kuperstoch-Portnoy et al. acid, and 0.0025 M EDTA, at pH 8.0). The (1974). Recipient bacteria were infected dimensions of the slab gels were 17 x 12.5 with 32P-BU-labeled transducing particles x 0.25 cm. Gels were run at 150 V for 4.5 hr. as described by Sandri and Berger (1980). The slabs were dried onto Whatman 3MM Samples of infected cultures were removed filter paper in a Bio-Rad slab gel dryer. Dried gels were autoradiographed for at the times indicated in each experiment, and were diluted into an equal volume of 2 weeks. TELS buffer LO.05M Tris-HCl (pH S.O>, RESULTS 0.005 M EDTA, and 0.05 M NaCl]. The infected bacteria were centrifuged at 10,000 g for 10 min at 4”, then resuspended and re- Nonchromosomally Associated DNA centrifuged twice in 10 ml of TELS buffer. In analyzing the fate of Pl transducing The bacterial pellet was resuspended in 1.0 DNA following its entry into recipient bacml of 25% sucrose in 0.05 M Tris-HCl (pH teria, we found that a large percentage of 8.0). The suspension was placed in an ice the transduced DNA was not associated bath and maintained on ice throughout the with recipient chromosomes (Sandri and following operations. After 5 min, 0.2 ml of a Berger, 1980). Figure 1 shows the CsCl grafreshly prepared lysozyme solution (10 mgl dient profile of DNA extracted from [3H]ml in 0.25 M Tris-HCl, pH 8.0) was added. thymidine-labeled recipient bacteria which After an additional 5 min, RNase was added were infected with 32P-BU-labeled transto a final concentration of 100 pg/ml. Again ducing particles for various times at 37”. after 5 min. 0.4 ml of 0.25 M EDTA (pH 8.0) About 75% of the 32Plabel was recovered was added, and the incubation was continued in the heavy region at the times measured, for 5 min. The bacteria were lysed by the in accord with our previous results (Sandri addition of 1.6 ml of a solution containing and Berger, 1980). These data also illustrate
mented and migrated in a manner which displayed the properties expected of circular DNA, while in the presence of these agents, the abortively transduced DNA cosedimented and comigrated with linear Pl DNA suggesting that abortively transduced DNA may exist as a circular DNA-protein complex.
32
SANDRI AND BERGER
6
FRACTION
NUMBER
FIG. 1. C&l equilibrium density gradient analysis of DNA extracted from bacteriainfected with =PBU-labeled transducing particles. [SHlThymidine-labeled recipient bacteria (strain AB115’7 (Plkc)) were infected with S2P-BU-labeled transducing particles, then incubated at 37”. Samples of the infected bacterial culture were lysed by the procedure described by Sandri and Berger (1980) at: (A) 0 min, (B) 30 min, (C) 180 min, and (D) 300 min after a 20-min period of adsorption of labeled transducing particles. DNA-CsCl solutions were adjusted to a refractive index of 1.3990. Centrifugation was performed in a Type 50 fixed-angle rotor at 35,000 rpm for 48 hr at 20”. Tubes were punctured at the bottom and lo-drop fractions were collected. Radioactivity was counted as described previously (Sandri and Berger, 1980). Density increased from right to left. Symbols: ?‘P (0); [3H]-thymidine (0).
that the “nonassociated” DNA had not replicated, even after 5 hr incubation, since the same percentage of 32Pcounts equilibrated in the heavy region in samples extracted at 5 hr as in samples extracted at 30 min (Figs. 1B and D). Our studies on Pl transduction showed that transductants doubled by 180 min after adsorption, and that integrated transducing DNA was replicated by 180 min (Sandri and Berger, 1980). Thus, the majority input transducing DNA component, the nonassociated, nonreplicated DNA, most likely represented abortively transduced DNA. In fact, a majority of Pl transductants are abortive (Gross and Englesberg, 1959; Hertman and Luria, 1967).
lecular weight as Pl DNA (6 x 10’) (Ikeda and Tomizawa, 1965a; Sandri and Berger, 1980). We analyzed the molecular weight of abortively transduced DNA by velocity sedimentation in neutral and alkaline sucrose gradients to determine if the abortively transduced DNA remained intact. In Fig. 2, abortively transduced DNA extracted at 30 min (panel A) and at 180 min (panel B) was analyzed on neutral sucrose gradients. The 32P-labeledDNA sedimented at approximately the position of the 3Hlabeled Pl marker DNA. The density label probably accounts for the slightly faster sedimentation of abortively transduced DNA. DNA extracted directly from BUlabeled transducing particles shows the Molecular Weight of Abortively Transduced same sedimentation pattern (Sandri and DNA Berger, 1980). An aliquot of abortively Transducing DNA extracted from puri- transduced DNA extracted at 180 min also fied transducing particles has the same mo- was run on an alkaline sucrose gradient
STRUCTURE
OF Pl ABORTIVELY
TRANSDUCED
Structure
FRACTION
NUMBER
FIG. 2. Velocity sedimentation analysis of abortively transduced DNA. Recipient bacteria (strain AB1157 (PI/cc)), infected with 92P-BU-labeled transducing particles, were lysed as described previously (Sandri and Berger, 1980). The extracted DNA was centrifuged to equilibrium in CsCl as described in the legend to Fig. 1. Fractions containing heavy, abortively transduced DNA were pooled then dialyzed to remove CsCl. (A) A 0.1-ml aliquot of abortively transduced DNA extracted at 30 min was run on a 5-20% neutral sucrose gradient. (B) A O.l-ml aliquot of abortively transduced DNA extracted at 180 min was run on a 5-20% neutral sucrose gradient. (C) A O.l-ml aliquot of abortively transduced DNA extracted at 180 min was run on a 5-20% alkaline sucrose gradient. In each case, [3H]thymidine-labeled PI DNA was added as a marker. Centrifugation was performed as described under Materials and Methods. Ten-drop fractions were collected and counted for radioactivity. Sedimentation was from right to left in these gradients and in all succeeding gradients. Symbols: 35P(0); [3H]thymidine (0).
(panel C). The abortively transduced DNA sedimented with Pl marker DNA illustrating that abortively transduced DNA remained intact up to 3 hr after entry into recipient bacteria.
DNA
of Abortively
33
Transduced DNA
Abortively transduced DNA escaped degradation by host nucleases, indicating that it was protected in some way. Ikeda and Tomizawa (1965b) reported that there was protein associated with Pl transducing DNA. Their observations suggested to us that abortively transduced DNA might circularize via attached protein, rendering the DNA resistant to nuclease attack. We analyzed the structure of abortively transduced DNA by velocity sedimentation in neutral sucrose gradients (Fig. 3). Nonlabeled recipient bacteria infected with 32PBU-labeled transducing particles were lysed by the Triton X-100 procedure (see Materials and Methods) at 60 min after adsorption of transducing particles. This procedure differs from the DNA extraction procedure used in previous experiments in that it did not utilize ionic detergent or protease. Aliquots of the “cleared” supernatant, which contained abortively transduced DNA, were run on ZO-31% neutral sucrose gradients. In the untreated sample (Fig. 3A), about 60% of the 32Pcounts were found in a peak which sedimented 1.7 to 1.8 times faster than linear Pl DNA, whose position is indicated by an arrow. A peak of slower sedimenting DNA also was observed which sedimented 1.2-1.3 times faster than linear DNA. When abortively transduced DNA was treated with SDS (panel B), or was incubated at 70” for 20 min (panel C), 32P-labeled DNA was found at the position of linear Pl DNA. Abortively transduced DNA which was incubated with RNase (panel D) showed no significant differences in the gradient profile from the untreated sample (panel A). The 32P-labeled DNA which sedimented 1.7-1.8 times faster than linear DNA may have been supercoiled. It has been shown that supercoiled X DNA sedimented 1.5 to 1.9 times faster than the linear form in neutral sucrose gradients (Bode and Kaiser, 1965), and that supercoiled P22 DNA sedimented 1.7 times faster than linear P22 DNA (Rhoades and Thomas, 1968). The slower sedimenting material may have represented relaxed, circular DNA. The data in Fig. 3B also suggest that protein may
34
SANDRI AND BERGER
have been involved in the maintenance of the DNA structures which sediment faster than linear DNA. Sedimentation of Pl Prophage DNA and Abortive1 y Transduced DNA Pl prophage exist as circular plasmids which do not integrate into bacterial chromosomes (Ikeda and Tomizawa, 1968). We have used the Pl lysogen, E. coli strain AB1157 (Plkc), as the recipient in our transduction experiments to prevent expression of the small percentage of Pl infectious phage present in the purified transducing particle preparations (Sandri and Berger, 1980). Since prophage DNA also would remain in the supernatant of cleared lysates, we used prophage DNA as a marker for the position of Pl circular DNA in neutral sucrose gradients. We infected 13H]thymidinelabeled strain AB1157 (Plkc) with 32P-BUlabeled transducing particles. Cleared lysates were prepared at 60 min after adsorption of the transducing particles. Aliquots were run on 20-31% neutral sucrose gradients (Fig. 4). In the untreated sample (Fig. 4A), two peaks were observed for both the 3H-labeled Pl prophage DNA and the 32Plabeled abortively transduced DNA. However, both peaks of abortively transduced DNA sedimented ahead of the corresponding prophage peaks. Similar profiles were seen when DNA samples were extracted at 30 and 180 min after adsorption of labeled transducing particles (Fig. 5). When samples extracted at 60 min were treated with SDS (Fig. 4B), or were heated at 70” (Fig. 4C), two peaks were observed again for the prophage DNA, illustrating that these treatments had no appreciable effect on the sedimentation profile of Pl prophage DNA. However, these treatments completely converted the abortively transduced DNA to a more homogeneous, slower sedimenting form. In the previous section, we showed that after treatment with SDS or heat, abortively transduced DNA sedimented in the position of linear Pl DNA (Figs. 3B and C). The peaks of abortively transduced DNA in Figs. 4B and C, then, probably represented linear DNA. The slower sedimenting peak
‘.,& IO
I 20 30 FRACTION
IO
20
30
NUMBER
FIG. 3. Velocity sedimentation analysis of abortively transduced DNA in cleared lysates. Nonlabeled recipient bacteria (strain AB1157 (Plkc)) were infected with SZP-BU-labeled transducing particles then incubated at 37” for 60 min. Infected bacteria were lysed by the Triton X-100 procedure and the lysates were “cleared” as described under Materials and Methods. (A) A O.l-ml aliquot of the cleared supernatant containing abortively transduced DNA was mixed with 0.1 ml 3H-labeled Pl phage DNA. (B) A O.l-ml ahquot of the cleared supernatant was mixed with 0.1 ml 3H-labeled Pl DNA and SDS (0.25%, final concentration). (C) A O.l-ml aliquot of the supernatant fraction combined with 0.1 ml 3Hlabeled Pl DNA was incubated at 70” for 20 min. (D) A O.l-ml aiiquot of the cleared supernatant was mixed with Pl marker DNA and RNase (100 pg/ml). The sample was incubated at 25” for 15 min. Samples were layered onto 20-318 neutral sucrose gradients which contained a 0.2-ml shelf of 70% sucrose at the bottom. The gradients were centrifuged as described under Materials and Methods. Fifteen-drop fractions were collected and the radioactivity was counted. Arrows indicate the position of 3H-labeled Pl DNA.
of prophage DNA sedimented about 1.07 times faster than the abortively transduced DNA treated with SDS or heat (Figs. 4B, and C). Ikeda and Tomizawa (1968) showed that open circular Pl prophage DNA sedi-
STRUCTURE
IO
20
30
OF Pl ABORTIVELY
IO FRACTION
20 NUMBER
TRANSDUCED
30
DNA
IO
20
30
FIG. 4. Velocity sedimentation analysis of abortively transduced DNA and Pl prophage DNA. [3H]Thymidine-labeled E. coli strain AB1157 (Plkc)) was infected with 3ZP-BU-labeled transducing particles. The infected bacteria were lysed at 60 min after absorption of transducing particles by the Triton X-100 method, and the lysates were “cleared” by centrifugation. (A) A O.l-ml aliquot of the cleared supernatant, which contained abortively transduced DNA and Pl prophage DNA. (B) A 0.1-ml aliquot of the cleared supernatant treated with SDS (0.25%). (C) A O.l-ml aliquot of the supernatant following incubation at 70” for 20 min. SampIes were layered onto 20-310/oneutral sucrose gradients with a 0.2ml shelf of 70% sucrose at the bottom. Centrifugation was performed as described under Materials and Methods. Fifteen-drop fractions were collected and counted for radioactivity. Symbols: 32P(0); [3H]thymidine (0).
mented about 1.09 times faster than linear Pl DNA under their conditions. The data above suggest that the slower sedimenting peak of prophage DNA represented relaxed circular prophage DNA. The faster sedimenting prophage peak sedimented 1.4 to 1.6 times faster than abortively transduced DNA (Figs. 4B, C), indicating that the prophage DNA in this peak was probably super-coiled. In the untreated samples (Figs. 4A and 5A, B), both the faster and slower sedimenting peaks of abortively transduced DNA banded ahead of the corresponding circular prophage peaks. Pl prophage DNA is 12% shorter than Pl phage DNA (Ikeda and Tomizawa, 1968;Yun and Vapnek, 1977), whereas abortively transduced DNA has the same molecular weight as Pl phage DNA (Fig. 2). The difference in sedimentation may be due, at least in part, to the size difference in prophage and abortively transduced DNA. Additionally, the protein associated with abortively transduced DNA may contribute to its faster rate of sedimentation.
Such a phenomenon has been observed in studies on complexed (protein associated) versus noncomplexed plasmid DNA (Clewell and Helinski, 1969;Kline and Helinski, 1971). Agarose Gel Electrophoresis DNA
of Circular
Circular DNA can be separated readily from linear DNA upon electrophoresis in agarose gels (Meyer et al., 1976; Mickel et al., 1977; Mickel and Bauer, 1976). Since we planned to use Pl prophage DNA as a marker, we first determined the position of relaxed circular prophage DNA and supercoiled prophage DNA relative to linear Pl phage DNA (Fig. 6). Prophage DNA was isolated as indicated in the legend to Fig. 6. Lane 1 of Fig. 6 shows the migration of 32Plabeled Pl DNA extracted from phage particles. Relaxed circular Pl prophage DNA (prophage DNA from the slower sedimenting peak in a preparative sucrose gradient-see legend to Fig. 6) was run in Lane 2. Pl phage DNA displayed roughly
36
SANDRI AND BERGER
Analysis of Abortively on Agarose Gels
Transduced
DNA
Abortively transduced DNA was isolated from nonlabeled recipient bacteria infected with 32P-BU-labeled transducing particles as outlined in the legend to Fig. 7. Cleared lysates were not prepared as in the sedimentation studies described above, instead, transduced cultures were lysed with Triton X-100 then centrifuged to equilibrium in CsCl. The heavy, nonassociated transducing DNA peak was pooled for electrophoresis studies. Aliquots were run on a 0.8% agarose slab gel (Fig. 7). Untreated abortively transFraction
Number
FIG. 5. Velocity sedimentation analysis of abortively transduced DNA and Pl prophage DNA isolated 30 and 180 min affer infection. [3H]Thymidine-labeled strainAB115’7(Plkc) wasinfectedwith3*P-BU-labeled transducing particles. The infected bacteria were lysed at (A) 30 min and (B) 180 min after adsorption of transducing particles as outlined in the legend to Fig. 4. A O.l-ml aliquot of each cleared supernatant was layered onto a 20-31% neutral sucrose gradient with a 0.2-ml shelf of 70% sucrose. Centrifugation was performed as described under Materials and Methods. Thirty-drop fractions were collected and counted for radioactivity. Symbols: 32P(0); [3H]thymidine (0).
1Btimes the mobility of prophage relaxed circular DNA. Super-coiled prophage DNA (prophage DNA from the faster sedimenting peak in a preparative sucrose gradient) was run in Lane 3. A predominant band displayed 6-times the mobility of relaxed circular DNA and about O.× the mobility of linear Pl phage DNA. A band also was observed at the position of relaxed circular DNA as well as a faint band at the linear position. Conversion to these forms may have occurred during handling of the prophage DNA. The data on the migration of Pl prophage DNA are in accord with the results of Mickel et al. (1977), who analyzed plasmid DNA molecules having a wide range of molecular weights under a variety of agarose gel electrophoresis conditions. Higher molecular weight super-coiled DNA migrated more slowly than the corresponding linear DNA in their studies.
FIG. 6. Autoradiograph of Pl prophage DNA following agarose gel electrophoresis. E. coli strain AB1157 (Plkc) was labeled with 32P (2 &i/ml) in TCG medium for 3 hr at 37”. The bacteria were lysed by the Triton X-100 method and the chromosomal DNA was sedimented. Aliquots of the supernatant fraction of the cleared lysate, containing Pl prophage DNA, were banded in 20-31% neutral sucrose gradients. The faster and slower sedimenting peaks containing supercoiled and relaxed circular prophage DNA, respectively, were pooled separately for dialysis. Samples containing 20 ~1 of the pooled fractions (about 200 cpm each) were run on a 0.8% agarose slab gel as described under Materials and Methods. In Lane 1, a 20-~1 sample (with about 2000 cpm) of 3ZP-labeled PI DNA extracted from infectious phage particles was run as a marker for the position of linear Pl DNA. Lane 2 contained an aliquot of the slower sedimenting (relaxed circular) prophage DNA. An aliquot of the faster sedimenting (supercoiled) prophage DNA was run in Lane 3. The origin is at the top of the gel.
STRUCTURE
OF Pl ABORTIVELY
FIG. 7. Autoradiograph of 32P-labeled abortively transduced DNA following agarose gel electrophoresis. Nonlabeled recipient bacteria (strain AB1157 (Plkc) were infected with 3ZP-BU-labeled transducing particles for 60 min before lysis with Triton X-100. The chromosomal DNA was not sedimented by centrifugation, but rather, the lysate was mixed with CsCl immediately and centrifuged to equilibrium. Fractions in the heavy region containing abortively transduced DNA were pooled. After dialysis, aliquots were electrophoresed on a 0.8% agarose slab gel. 3ZP-Labeled Pl prophage DNA which was used as a circular marker was isolated separately as described in the legend to Fig. 5. Pooled fractions of neutral sucrose gradients containing faster sedimenting (supercoiled) and slower sedimenting (relaxed circular) prophage DNA were combined in this experiment. 32P-BUlabeled transducing DNA which was used as a linear DNA marker was extracted from purified transducing particles as described previously (Sandri and Berger, 1980). Transducing particle DNA was run in Lane 1. Pl prophage DNA was run in Lanes 2-5. The following treatments were used: Lane 2, untreated; Lane 3, incubation with Pronase (500 pg/ml) for 30 min at 37”; Lane 4, incubation with RNase (200 pg/ml) for 30 min at 37”, and Lane 5, treatment with SDS (0.25%). Abortively transduced DNA was run in Lanes 6-12. The following treatments were used: Lane 6, untreated; Lane 7, incubation at 37” for 30 min; Lane 8, incubation with Pronase (500 pg/ml) for 30 min at 37”; Lane 9, incubation with RNase (200 pg/ml) for 30 min at 37”; Lane 10, treatment with SDS (0.25%); Lane 11, treatment with Sarkosyl (0.25%), and Lane 12, incubation for 20 min at 70”. Twenty microliters of each
TRANSDUCED
DNA
97
duced DNA was run in Lanes 6 and 7. Two bands were observed which corn&rated with relaxed circular prophage DNA and supercoiled prophage DNA markers, respectively (Lanes 2-5). A third band corn&rated with linear transducing particle DNA (Lane 1). A similar migration pattern was observed when abortively transduced DNA was incubated with RNase (Lane 9) or Sarkosyl (Lane 11). In contrast, when samples were treated with SDS (Lane 10) or were incubated at 70” (Lane 12>,only linear abortively transduced DNA was observed. In addition, abortively transduced DNA, which was incubated with Pronase for 30 min at 37” before electrophoresis, migrated predominantly as linear DNA, although circular species also were observed (Lane 8). We have noted in several experiments that incubation of abortively transduced DNA with Pronase for 60 min at 37” completely converted the DNA to the linear form (data not shown). However, after incubation times of 15-30 min, some circular species persisted. The reason for this is not clear. We also observed an additional faint band which migrated faster than linear DNA in the abortively transduced DNA samples. The identity of this band is not known. It appears to have been unaffected by any of the treatments since its intensity was about the same in all of the lanes. Finally, we noted that when Pl prophage DNA was treated with RNase, the supercoiled band appeared to be converted to the relaxed form (Lane 4). It is unlikely that the RNase preparation was contaminated with DNase activity since RNase solutions were heated at 83” for 20 min prior to use to inactivate any DNase activity. In addition, the abortively transduced DNA which sedimented and migrated like supercoiled DNA was not affected by RNase treatment (Fig. 3D and Fig. 7, Lane 9). The prophage DNA in these experiments was isolated from exponentially growing cultures of Pl lysogens so that the prophage DNA was presumably replicating as well. Although we cannot explain why RNase converted supercoiled DNA sample containing about 200 cpm was loaded onto the slab gel. The origin is at the top. A *“C dye marker (in upper right-hand corner) was used to orient the gel during autoradiography.
38
SANDRI
AND BERGER
prophage DNA to the relaxed form, it is possible the prophage DNA may contain RNA during its replication. Blair et al. (1972) have shown that supercoiled Co1 El DNA made in the presence of chloramphenicol is converted by RNase to the open circular structure. DISCUSSION
Our studies on Pl transduction showed that the nonchromosomally associated, abortively transduced DNA escaped degradation by host nucleases (Figs. 1 and 2), a fact which suggested to us that the structure was modified to a form resistant to the recBC nuclease (exonuclease V), a nuclease chiefly responsible for degradation of superinfecting linear duplex DNA (Benzinger et al., 1975; Oishi and Irbe, 19’77; Wackernagel, 1973).While circularization of the DNA would engender resistance to the recBC nuclease (Goldmark and Linn, 1972), it seemed unlikely that transducing DNA, which consists of donor bacterial DNA fragments, could circularize by recombination or annealing of homologous ends. Since the results of Ikeda and Tomizawa (1965b) showed that there was protein associated with Pl transducing DNA and that the protein was most likely associated with the ends of the DNA molecules, we investigated the possibility that abortively transduced DNA might circularize via attached protein. We analyzed abortively transduced DNA by velocity sedimentation in neutral sucrose gradients and by agarose gel electrophoresis. Those experiments showed that abortively transduced DNA species having the sedimentation and migration patterns expected of circular DNA molecules could be detected when recipient bacteria were lysed with Triton X-100. This procedure was used to prevent denaturation or dissociation of any protein which may be involved in joining the ends of the DNA molecules. In fact, treatment of abortively transduced DNA with SDS, with heat (70”), or with Pronase converted the DNA to a form which sedimented with linear Pl DNA and which migrated with linear transducing particle DNA. These treatments did not affect the sedimentation or migration of circular Pl prophage DNA.
The protein associated with transducing particle DNA has not been isolated or identified and it is not known whether it is of host or viral origin. Ikeda and Tomizawa (196513) have estimated its molecular weight to be around 5 x lo5 based on the density shift of deproteinated vs native transducing particle DNA in CsCl gradients. Circular DNA-protein complexes have been isolated from adenovirus (Robinson et al., 1973) and from Bacillus bacteriophages #29 and GA-l (Arnberg and Arwert, 1976; Ortin et al., 1971). Phage 429 and GA-1 complexes, like the abortively transduced DNA structures, were linearized by treatment with proteolytic enzymes (Arnberg and Arwert, 1976; Ortin et al., 1971), although, as we have previously discussed, the conversion of the abortively transduced DNA required a relatively long incubation with Pronase to effect complete conversion. Circular adenovirus molecules were converted to the linear form by treatment with proteases or SDS (Robinson et al., 1973), but were resistant to Sarkosyl (Rekosh et al., 1977). We observed a similar behavior with the abortively transduced DNA structures (Fig. 7). Pl transducing DNA is presumably injected into the recipient as a linear molecule since transducing DNA extracted from transducing particles by a nonproteolytic method is linear (Sandri, unpublished results). It is possible that it is the linear form which recombines with the chromosome to form stable transductants. This could occur if the linear form fails to circularize fast enough upon entry, in which case the DNA may be attacked by nucleases. The resulting fragments could have some structural property such as single-stranded ends which might stimulate their recombination with the chromosome. Two lines of evidence lend support to this hypothesis. First, in the previous paper we showed that the integrated DNA is substantially smaller than intact transducing DNA and the unincorporated portions of the transducing DNA molecules are degraded. Second, most of the 32P label becomes associated with the recipient DNA during the first hour after transduction with only a small increase in association after that (Sandri and Berger,
STRUCTURE
OF Pl ABORTIVELY
1980). Once the transducing DNA circularizes it becomes protected from nucleases and may undergo recombination with the chromosome at very low frequency, similar perhaps to the recombination frequency between the chromosome and a plasmid having a region of homology to the chromosome, as for example, homozygosis of lac-IF’lac’ bacteria. It has been estimated that the frequency of conversion from an abortive to stable transductant was as low as lop3 per generation (Ozeki and Ikeda, 1968). However, Benzinger and Hartman (1962) showed that if transducing phage are uv irradiated, the number of abortive transductants decreases while the number of stable transductants increases. These authors estimated that a single hit was sufficient to convert an abortive to a complete transductant, that is, to undergo recombination with the chromosome. Since uv repair mechanisms involve removal of DNA damage, presumably donor DNA was nicked at the site of damage. In fact, Helling (1973) has shown that the excision-repair genes uvrA+ and uvrB+ are required for most uv-stimulated transduction as is the RecA+ gene. Single-strand nicks alone probably are not sufficient for the conversion of abortive to complete transductants since nicks made by 32P decay (Hartman and Kozinski, 1962) or X-irradiation (Takabe and Hartman, 1962) failed to stimulate the formation of stable transductants. Some process following the nicking such as the generation of a single-stranded end as proposed above may be required for recombination with the chromosome. In any event, the formation of a circular DNA molecule through a protein linker can account for the stability of abortively transduced DNA. ACKNOWLEDGMENTS
The authors wish to thank Dr. Philip E. Hartman for critical reading of the manuscript and for valuable advice. We also thank Drs. Marc Rhoades and Peter Gauss for helpful suggestions. R.M.S. would alsolike to thank Dr. Robert P. Dottin, in whose laboratory this research was completed, for his gracious hospitality and helpful discussions. R.M.S. was a predoctoral trainee of the National Institutes of Health (Grant GM-57) during part of this research. This research was supported by Grants AI0861 from the National Institute of Allergy
TRANSDUCED
DNA
39
and Infectious Diseases, NP-175 from the American Cancer Society, and GB-43825 from the National Science Foundation. H.B. was recipient of a Career Development Award, National Institutes of Health. REFERENCES ARNBERG, A. C., and ARWERT, F. (1976). DNA-protein complex in circular DNA fromBacillus bacteriophage GA-l. J. Viral. 18, 783-784. BENZINGER, R., ENQUIST, L. W., and SKALKA, A. (1975). Transfection of Escherichia coli spheroplasts. V. Activity of reeBC nuclease in ret+ and ret- spheroplasts measured with different forms of bacteriophage DNA. J. Viral. 15, 861-871. BENZINGER, R., and HARTMAN, P. E. (1962). Effects of ultraviolet light on transducing phage P22. Viralogy 18, 614-626. BLAIR, D. G., SHERRATT,D. J., CLEWELL, D. G., and HELINSI(I, D. R. (1972). Isolation of super-coiled colicinogenic factor E, DNA sensitive to ribonuclease and alkali. Proc. Nat. Acad. Sci. USA 69, 25182522.
BODE, V. C., and KAISER, A. D. (1956). Changes in the structure and activity of h DNA in a superinfected immune bacterium. J. Mol. Biol. 14, 399-417. CLEWELL, D. G., and HELINSKI, D. R. (1969). Supercoiled circular DNA-protein complex inEscherichin coli: Purification and induced conversion to an open circular DNA form. Proc. Nut. Acad. Sci. GSA 62, 1159-1166. EBEL-TSIPIS, J., Fox, M. S., and BOTSTEIN,D. (1972). Generalized transduction by bacteriophage P22 in Salmonella typhimurium. II. Mechanism of integration of transducing DNA. J. Mol. Riol. 71, 449469. GOLDMARK,P. J., and LINN, S. (1972). Purification and properties of the recBC NAase of Esckerickia coli K12. J. Biol. Chem. 247, 1849-1860. GROSS,J., and ENGLESBERG,E. (1959). Determination of the order of mutational sites governing L-arabinose utilization in Escherickia coli B/r by transduction with phage Plbt. Virology 9, 314-331. HARTMAN, P. E., and KOZINSKI, A. W. (1962). Effects of P3* decay on transduction by Salmonella phage P22. Virology 17, 223-244. HELLING, R. B. (1973). Ultraviolet light-induced recombination Biochem. Biopkys. Res. Commun. 55, 752-757.
HERTMAN, I., and LURIA, S. E. (1967). Transduction studies on the role of a ret* gene in the ultraviolet induction of prophage lambda. J. Mol. Riot. 23, 117-133. IKEDA, H., and TOMIZAWA,J. I. (1965a). Transducing fragments in generalized transduction by phage PI. I. Molecular origin of the fragments. 6. Mol. Rio!. 14, 85- 109. IKEDA, H., and TOMIZAWA,J. I. (1965b). Transducing fragments in generalized transduction by phage Pl.
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II. Association of DNA and protein in the fragments. J. Mol. Biol. 14, 110-119. IKEDA, H., and TOMIZAWA,J. I. (1968). Prophage Pl, an extrachromosomal replication unit. Cold Spring Harbor Symp. Qua&. Biol. 33, 791-798. KLINE, B. C., and HELINSKI, D. R. (1971). F, sex factor ofEscherichia coli. Size and purification in the form of a strand-specific relations complex of supercoiled deoxyribonucleic acid and protein. Biochemistry 10,49’75-4980. KUPERSTOCH-PORTNOY, Y. M., LOVETT, M. A., and HELINSKI, D. R. (1974). Strand and site specificity of the relaxation event for the relaxation complex of the 13, antibiotic resistant plasmid R6K. Biochemistry 5484-5490. LEDERBERG, J. (1956). Linear inheritance in transductional clones. Genetics 41, 845-871.
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ORTIN, J., VINUELA, E., and SALAS, M. (1971). DNA-protein complex in circular DNA from phage 029. Nature
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OZEKI, H. (1956). Abortive transduction. Carnegie Inst. Wash. Yearb. 55,302-303.
OZEKI, H. (1959). Chromosome fragments participating in transduction in Salmonella typhimurium. Genetics 44, 457- 470. OZEKI, H., and IKEDA, H. (1968). Transduction mechanisms. Annu. Rev. Genet. 2,245-278. REKOSH,D. M. K., RUSSELL,W. C., BELLES, A. J. D., and ROBINSON, A. J. (1977). Identification of a protein linked to the ends of adenovirus DNA. Cell 11,283-295. RHOADES, M., and THOMAS, C. A. (1968). The P22 bacteriophage molecule. II. Circular intracellular forms. J. Mol Biol. 37, 41-61. ROBINSON, A. J., YOUNGHUSBAND, H. B., and BELLETT, A. J. D. (1973). A circular DNA-protein complex from adenoviruses. Virology 56,54-69. SANDRI, R. M., and BERGER,H. (1980). Bacteriophage Pl-mediated generalized transduction in Escherichia coli: Fate of transduced DNA in Ret+ and RecArecipients. Virology 106, 14-29. TAKEBE, H. K., and HARTMAN, P. E. (1962). Effectsof X-Rays on transduction by Salmonella phage P22. Virology 17,295-302. WACKERNAGEL,W. (1973). Genetic transformation in E. coli: The inhibitory role of the recBC DNase. Biochem. Biophys. Res. Commun. 51,306-311. YUN, T., and VAPNEK, D. (1977). Electronmicroscopic analysis of bacteriophages Pl, PlCm, and P7: Determination of genome sizes, sequence homology, and location of antibiotic resistance determination. Virology
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