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DNA replication during episome transfer as studied by bromodeoxyuridine photosensitization
J. Mol. Bid. (1969) 46, 365-368
DNA Replication During Episome Transfer as Studied by Bromodeoxyuridine Photosensitization In spite of extensive investigations of the location and timing of DNA replication during Escherichia wli conjugation (Curtiss, Charamella, Stallions & Mays, 1968), data so far obtained have not clearly shown whether replication occurs in the donor cell at the time of transfer (Cuzin, Buttin & Jacob, 1967) or in the recipient (Bonhoeffer & Vielmetter, 1968). Recently several laboratories have shown that only one donor strand is selectively recovered from a recipient (Cohen, Fisher, Curtiss & Adler, 1968; Rupp & Ihler, 1968; Ohki & Tomizawa, 1968). To determine which parent supplies the nucleotides for episomal DNA replication during transfer we have used one parent that requires thymine (thy-) and in the absence of thymine can incorporate 5.bromodeoxyuridine and mated it with a wild type (thy+) parent which incorporates the thymidylate it synthesizes. The DNA that contains BUdRt is photosensitive to 313 rnp light; its incativation can be assayed in the zygote by photoinactivation of &galactosidase induction (Eisenberg & Pardee, 1970). The photochemical lesions appear to be located in single genes, and are effective when BUdR is incorporated into either one of the DNA strands. If the episome is replicated in the recipient, it should be sensitized by BUdR incorporation when the recipient is the thymine auxotroph. When BUdR was present for 40 minutes during mating, 90% inactivation of enzyme formation was observed (Fig. 1). The result is consistent with a replication of the episome using BUdR supplied by the recipient during conjugation or later. When BUdR incorporation was stopped before 40 minutes by addition of thymine, enzyme synthesis after 40 minutes was less sensitive (Fig. 1). This result might suggest the episomal strand replicated slowly after an early transfer under these conditions. But alternative interpretations are possible because samples which were exposed more briefly to BUdR later spent more time in thymine-containing medium. For example, when BUdR was removed at 20 minutes the culture grew an additional 20 minutes in thymine. The episomes of these cells could have lost sensitivity during the latter period; or further mating occurring after thymine addition produced enzyme, since Dupanol did not inhibit mating completely. As a control, irradiation of recipient preloaded with BUdR for one generation before mating had no effect on /Lgalactosidase production if the BUdR was removed at the time of mating. This indicates that the effect described above depends on BUdR incorporation into the episome itself (Eisenberg & Pardee, 1970). The effect of limiting DNA synthesis was studied by withholding thymine from a thy- recipient during and after transfer. Controls showed that thymine starvation did not inhibit /Lgalactosidase formation by unmated cells. The mated cells produced less #Lgalactosidase when thymine was absent during or before mating (Table 1). Also, the number of lac+ recombinants was greatly reduced, particularly after long starvation. Restoration of thymine brought enzyme formation back more strongly than it t Abbreviation
used: BUdR,
5-bromodeoxyuridine. 366
366
R. J. EISENBERO
AND
A. B. PARDEE
Time thymine odded (min) FIQ. 1. Effect
on F-galaotoaidase
of BUdR incorpor8tion
formation
by the reoipient
before and
during mating. The recipients, kzc- straina 2340-2 -O-O8nd 2340-2 &y- -@--a-, were grown in 100 pg/ml. BUdR medium (minus glycerol) for 60 min prior to meting with F’Zuc r&rein A327. After 20 min, agitation w8a increased and Dupanol was added to reduce mating, and growth wae continued 20 min longer. Thymine (100 pg/ml.) w8a added at variow times before or after the mating period began. Samples were irradiated at 313 rnp for 2 min. Then, fl-galsctoeideee was induced with isopropyl-fl-r,-thiogalectoside; streptomycin was present. /3-g8laotosidaee formation by each irmdi8ted rxunple of zygotes is expressed 8a 8 percentage of its unirr8diated control.
TAEZLE~
Starvation for thymine before mating @w
0
Thymine added, after mating bw bw
0 10
100 86
0
23 79
10
60
None
80
/3-gal8ct offlame ** in zygot 2: % of control
None
7
Reoombinants
(x10-q Number/ml.
9.2 7.4 3.6 2.0 2.1
0.3
%
(W 80 38 28 23 3
An F’ Zoc donor (A327) and a thymine-requiring recipient (F-2340-2thy-) were g-rown on M63, Cksamino acids glycerol, and 60 pg thymine/ml. for the recipient. Cella were washed and re~uapended in minimal medium but without glycerol or thymine. A portion of the F- culture was incubated without thymine for 60 min. After mixing the culture8, thymine was added at the times indicated. After 10 min mating, Dupanol, streptomycin and isopropyl-j-D-thiogakbotoixide were added and rates of j-galaotoeidaae formation were determined. At the end of the mating period and before induction, samples were immedistely removed end diluted 1:lO into M63 medium. These diluted semples were further diluted and plated for recombinants on LSBl agar cont8ining phenylelanine end thymine.
LETTERS
TO THE
EDITOR
357
did recombinant number. Thy-mine starved cells apparently do not function well as recipients. If the donor supplies the nucleotides (used by either cell) for synthesis of the transferred DNA, then BUclR incorporation during conjugation of a thy- episome donor (F%zc+) and a thy+ recipient (F-kc-) should sensitize the injected genetic material. This was not the case, when BUclR was present from as long as 30 minutes before mating to termination of mating (Table 2). This result suggests that the donor &d not synthesize a new strand of DNA during transfer. An alternative hypothesis is that the donor synthesized a new strand during mating using thymidylate supplied by the recipient through the cytoplasmic bridge. To determine the effect of labeling the lac+ episome before transfer, thy- F’ cells were preincubated for longer periods, up to 60 minutes (one generation), in the experiment above (Table 2). Induced /Lgalactosiclase synthesis by these donors became completely sensitive to irradiation under conditions identical to those used for mating but with no recipient present (Table 2). Thus the episomes of all donor cells were completely sensitized by incorporation of BUclR into one strand. These donors were not severely inhibited in their ability to transfer the lac episome to the recipient, since without irradiation they permitted about 70% as much fi-galactosidase synthesis by their zygotes as did thymine-containing donors. TABLE 2 Effect on /3-galactosidaseformation of incorporation by the donor before and during muting
Preincubation
Rate of
plus BUdR
,Sgalectosidaseformation, (arbitrary units)
Unirradiated
W4
of BUdR
Irradiated
Ratio
______ 0 15 30 45 60 60 (Induced
F’)
22 16 15 13 16
17 12 11 4 4
0.80 0.80 0.70 0.30 0.27
350
10
0.03
A thymine-requiring F’kzc+ donor (A327/2340-2thy-) and a fi-galactosidese- recipient (F - 2340-2) were grown aa described in Table 1. The donor was grown for the times noted with 100 pg BUdR/ml. before mating. The celle were mated in the presence of BUdR, st a ratio of F’ : F- = 1 : 2 for 15 min. The cells were then irradiated for 2 min with principally 313 and 302 rnp light (Walker & Pardee, 1968). Constitutive enzyme synthesie wae mewwed over the next 46 min.
Irradiation of the zygotes inactivated enzyme formation after transfer increasingly as the period of BUdR incorporation into F’ increased (Table 2). But enzyme formation was partly rescued by mating: constitutive /?-galactosidase synthesis was 30% of the control value obtained with thymine-containing donors This is a much greater survival than shown by the unmated donor cells. These results could be accounted for if the donor transferred either the BUdR-containing strand or the thyminecontaining strand with equal probability; this transferred strand would then be
R. J. EISENBERG
358
AND
A. B. PARDEE
replicated in the recipient using thymidylate. Half of the episomes then would not contain BUdR and would have low sensitivity to irradiation. In contrast to the effect of thymine deprivation in the F-, enzyme formation by zygotes made with a thy- donor was unaffected by the absence of thymine. This has been observed for recombinant formation by others, although their evidence indicates a requirement of a little initial DNA synthesis in the donor for transfer of the episome (Curtiss et al., 1968). In conclusion, the simplest model to explain our results is that episomal DNA is transferred as a single strand which is replicated in the recipient. This is consistent with the small episomal “transfer” into a thymine starved recipient. It also agrees with the appearance of ultraviolet sensitivity when the recipient takes up BUdR during
mating,
as contrasted
to the decrease of sensitivity
donor is mated (in the presence of BUdR) with a F-thy+ This work was supported
by grant no. AI-04409
when the BUdR-preloaded
recipient.
from the U.S. Public Health
Program in Biochemical Sciences Moffett Laboratories Princeton University Princeton, N.J. 08640, U.S.A. Received 5 May 1969, and in revised form 4 September
Service.
ROSELYN J. EISENBERU ARTHUR B. PARDEE
1969
REFERENCES Bonhoeffer, F. & Vielmetter, W. (1968). Cold Spr. Ha& Symp. Quant. Biol. 33, 623. Cohen, A., Fisher, W. D., Curt&s, R., III. & Adler, H. I. (1968). Cold Spr. Harb. Symp. Quad. Biol. 33, 636. Curtiss, R., III., Char&m&a, L. J., Stallions, D. R. & Mays, J. A. (1968). Bact. Rev. 32, 320. Cuzin, F., Buttin, G. & Jacob, F. (1967). J. Cell Physiol. 70, Suppl. 1, 77. Eisenberg, R. & Pardee, A. B. (1970). Biochim. biophys. Acta, in the press. Ohki, M. & Tomizawa, J. (1968). Cold Spr. Harb. Symp. Quant. Bid. 33, 651. Rupp, W. D. & Ibler, G. (1968). Cold. Spr. Harb. Symp. Quant. Biol. 33, 647. Walker, J. R. & Pardee, A. B. (1968). J. Butt. 95, 123.
DNA Replication During Episome Transfer as Studied by Bromodeoxyuridine Photosensitization In spite of extensive investigations of the location and timing of DNA replication during Escherichia wli conjugation (Curtiss, Charamella, Stallions & Mays, 1968), data so far obtained have not clearly shown whether replication occurs in the donor cell at the time of transfer (Cuzin, Buttin & Jacob, 1967) or in the recipient (Bonhoeffer & Vielmetter, 1968). Recently several laboratories have shown that only one donor strand is selectively recovered from a recipient (Cohen, Fisher, Curtiss & Adler, 1968; Rupp & Ihler, 1968; Ohki & Tomizawa, 1968). To determine which parent supplies the nucleotides for episomal DNA replication during transfer we have used one parent that requires thymine (thy-) and in the absence of thymine can incorporate 5.bromodeoxyuridine and mated it with a wild type (thy+) parent which incorporates the thymidylate it synthesizes. The DNA that contains BUdRt is photosensitive to 313 rnp light; its incativation can be assayed in the zygote by photoinactivation of &galactosidase induction (Eisenberg & Pardee, 1970). The photochemical lesions appear to be located in single genes, and are effective when BUdR is incorporated into either one of the DNA strands. If the episome is replicated in the recipient, it should be sensitized by BUdR incorporation when the recipient is the thymine auxotroph. When BUdR was present for 40 minutes during mating, 90% inactivation of enzyme formation was observed (Fig. 1). The result is consistent with a replication of the episome using BUdR supplied by the recipient during conjugation or later. When BUdR incorporation was stopped before 40 minutes by addition of thymine, enzyme synthesis after 40 minutes was less sensitive (Fig. 1). This result might suggest the episomal strand replicated slowly after an early transfer under these conditions. But alternative interpretations are possible because samples which were exposed more briefly to BUdR later spent more time in thymine-containing medium. For example, when BUdR was removed at 20 minutes the culture grew an additional 20 minutes in thymine. The episomes of these cells could have lost sensitivity during the latter period; or further mating occurring after thymine addition produced enzyme, since Dupanol did not inhibit mating completely. As a control, irradiation of recipient preloaded with BUdR for one generation before mating had no effect on /Lgalactosidase production if the BUdR was removed at the time of mating. This indicates that the effect described above depends on BUdR incorporation into the episome itself (Eisenberg & Pardee, 1970). The effect of limiting DNA synthesis was studied by withholding thymine from a thy- recipient during and after transfer. Controls showed that thymine starvation did not inhibit /Lgalactosidase formation by unmated cells. The mated cells produced less #Lgalactosidase when thymine was absent during or before mating (Table 1). Also, the number of lac+ recombinants was greatly reduced, particularly after long starvation. Restoration of thymine brought enzyme formation back more strongly than it t Abbreviation
used: BUdR,
5-bromodeoxyuridine. 366
366
R. J. EISENBERO
AND
A. B. PARDEE
Time thymine odded (min) FIQ. 1. Effect
on F-galaotoaidase
of BUdR incorpor8tion
formation
by the reoipient
before and
during mating. The recipients, kzc- straina 2340-2 -O-O8nd 2340-2 &y- -@--a-, were grown in 100 pg/ml. BUdR medium (minus glycerol) for 60 min prior to meting with F’Zuc r&rein A327. After 20 min, agitation w8a increased and Dupanol was added to reduce mating, and growth wae continued 20 min longer. Thymine (100 pg/ml.) w8a added at variow times before or after the mating period began. Samples were irradiated at 313 rnp for 2 min. Then, fl-galsctoeideee was induced with isopropyl-fl-r,-thiogalectoside; streptomycin was present. /3-g8laotosidaee formation by each irmdi8ted rxunple of zygotes is expressed 8a 8 percentage of its unirr8diated control.
TAEZLE~
Starvation for thymine before mating @w
0
Thymine added, after mating bw bw
0 10
100 86
0
23 79
10
60
None
80
/3-gal8ct offlame ** in zygot 2: % of control
None
7
Reoombinants
(x10-q Number/ml.
9.2 7.4 3.6 2.0 2.1
0.3
%
(W 80 38 28 23 3
An F’ Zoc donor (A327) and a thymine-requiring recipient (F-2340-2thy-) were g-rown on M63, Cksamino acids glycerol, and 60 pg thymine/ml. for the recipient. Cella were washed and re~uapended in minimal medium but without glycerol or thymine. A portion of the F- culture was incubated without thymine for 60 min. After mixing the culture8, thymine was added at the times indicated. After 10 min mating, Dupanol, streptomycin and isopropyl-j-D-thiogakbotoixide were added and rates of j-galaotoeidaae formation were determined. At the end of the mating period and before induction, samples were immedistely removed end diluted 1:lO into M63 medium. These diluted semples were further diluted and plated for recombinants on LSBl agar cont8ining phenylelanine end thymine.
LETTERS
TO THE
EDITOR
357
did recombinant number. Thy-mine starved cells apparently do not function well as recipients. If the donor supplies the nucleotides (used by either cell) for synthesis of the transferred DNA, then BUclR incorporation during conjugation of a thy- episome donor (F%zc+) and a thy+ recipient (F-kc-) should sensitize the injected genetic material. This was not the case, when BUclR was present from as long as 30 minutes before mating to termination of mating (Table 2). This result suggests that the donor &d not synthesize a new strand of DNA during transfer. An alternative hypothesis is that the donor synthesized a new strand during mating using thymidylate supplied by the recipient through the cytoplasmic bridge. To determine the effect of labeling the lac+ episome before transfer, thy- F’ cells were preincubated for longer periods, up to 60 minutes (one generation), in the experiment above (Table 2). Induced /Lgalactosiclase synthesis by these donors became completely sensitive to irradiation under conditions identical to those used for mating but with no recipient present (Table 2). Thus the episomes of all donor cells were completely sensitized by incorporation of BUclR into one strand. These donors were not severely inhibited in their ability to transfer the lac episome to the recipient, since without irradiation they permitted about 70% as much fi-galactosidase synthesis by their zygotes as did thymine-containing donors. TABLE 2 Effect on /3-galactosidaseformation of incorporation by the donor before and during muting
Preincubation
Rate of
plus BUdR
,Sgalectosidaseformation, (arbitrary units)
Unirradiated
W4
of BUdR
Irradiated
Ratio
______ 0 15 30 45 60 60 (Induced
F’)
22 16 15 13 16
17 12 11 4 4
0.80 0.80 0.70 0.30 0.27
350
10
0.03
A thymine-requiring F’kzc+ donor (A327/2340-2thy-) and a fi-galactosidese- recipient (F - 2340-2) were grown aa described in Table 1. The donor was grown for the times noted with 100 pg BUdR/ml. before mating. The celle were mated in the presence of BUdR, st a ratio of F’ : F- = 1 : 2 for 15 min. The cells were then irradiated for 2 min with principally 313 and 302 rnp light (Walker & Pardee, 1968). Constitutive enzyme synthesie wae mewwed over the next 46 min.
Irradiation of the zygotes inactivated enzyme formation after transfer increasingly as the period of BUdR incorporation into F’ increased (Table 2). But enzyme formation was partly rescued by mating: constitutive /?-galactosidase synthesis was 30% of the control value obtained with thymine-containing donors This is a much greater survival than shown by the unmated donor cells. These results could be accounted for if the donor transferred either the BUdR-containing strand or the thyminecontaining strand with equal probability; this transferred strand would then be
R. J. EISENBERG
358
AND
A. B. PARDEE
replicated in the recipient using thymidylate. Half of the episomes then would not contain BUdR and would have low sensitivity to irradiation. In contrast to the effect of thymine deprivation in the F-, enzyme formation by zygotes made with a thy- donor was unaffected by the absence of thymine. This has been observed for recombinant formation by others, although their evidence indicates a requirement of a little initial DNA synthesis in the donor for transfer of the episome (Curtiss et al., 1968). In conclusion, the simplest model to explain our results is that episomal DNA is transferred as a single strand which is replicated in the recipient. This is consistent with the small episomal “transfer” into a thymine starved recipient. It also agrees with the appearance of ultraviolet sensitivity when the recipient takes up BUdR during
mating,
as contrasted
to the decrease of sensitivity
donor is mated (in the presence of BUdR) with a F-thy+ This work was supported
by grant no. AI-04409
when the BUdR-preloaded
recipient.
from the U.S. Public Health
Program in Biochemical Sciences Moffett Laboratories Princeton University Princeton, N.J. 08640, U.S.A. Received 5 May 1969, and in revised form 4 September
Service.
ROSELYN J. EISENBERU ARTHUR B. PARDEE
1969
REFERENCES Bonhoeffer, F. & Vielmetter, W. (1968). Cold Spr. Ha& Symp. Quant. Biol. 33, 623. Cohen, A., Fisher, W. D., Curt&s, R., III. & Adler, H. I. (1968). Cold Spr. Harb. Symp. Quad. Biol. 33, 636. Curtiss, R., III., Char&m&a, L. J., Stallions, D. R. & Mays, J. A. (1968). Bact. Rev. 32, 320. Cuzin, F., Buttin, G. & Jacob, F. (1967). J. Cell Physiol. 70, Suppl. 1, 77. Eisenberg, R. & Pardee, A. B. (1970). Biochim. biophys. Acta, in the press. Ohki, M. & Tomizawa, J. (1968). Cold Spr. Harb. Symp. Quant. Bid. 33, 651. Rupp, W. D. & Ibler, G. (1968). Cold. Spr. Harb. Symp. Quant. Biol. 33, 647. Walker, J. R. & Pardee, A. B. (1968). J. Butt. 95, 123.