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Two new genetic assays for noninfectious fragments of ∅X174 DNA
VIROLOGY
69, 782-785
(1976)
SHORT Two New Genetic
COMMUNICATIONS
Assays for Noninfectious
Fragments
of +X174 DNA
JERRY B. DODGSON, INGOLF F. NES, BRUCE W. PORTER, AND ROBERT D. WELLS University
of Wisconsin, Department
of Biochemistry, College of Agricultural Wisconsin 53706
and Life Sciences, Madison,
Accepted September 24, 1975 A genetic salvage assay for noninfectious 4X174 DNA fragments of the viral strandedness is described. This assay is complementary to the assay previously described for the negative strand of 4X174 RF fragments. Also, a novel DNA fragment assay was developed which depends on the formation of an infective complex made up of a DNA fragment and linear +X174 viral DNA.
Previous workers (1, 2) developed a genetic assay for fragments of the complementary’ strand of $X174 DNA. In these assays, conditionally lethal temperaturesensitive (ts) 4X174 viral DNA was annealed to noninfectious complementary DNA fragments that were wild type (wt) in the gene containing the mutation in the viral DNA. When the resultant hybrid was used to infect Escherichia coli spheroplasts, the progeny phage from these spheroplasts showed an increased frequency of the wt genotype compared to that observed when the spheroplasts were infected with ts viral-DNA vector2 alone. This increase was apparently due to the replication of the wt DNA marker inside the spheroplasts. Typical dose response curves obtained with this type of assay for the two mutants used in this study are given in Fig. 1. The fold stimulations were linearly related to the weight ratio of wt DNA fragments to ts viral DNA on this log-log plot with a slope of approximately ’ 4X174 DNA species were defined by Sinsheimer (3) and include: viral or ( + ) strand DNA, replicative forms (RF I and RF II), and complementary or C-1 strand. p Vector DNA is the term used to describe an infectious DNA (usually ts) which was used to transport a noninfectious DNA or RNA (usually wt) into the cell. 782 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
1.2, in agreement with previous data (2). The actual fold stimulations at a given weight ratio of fragment to vector DNA varied depending on the mutation in question (Fig. 1). Stimulations as low as one- to twofold over the background reversion frequency of the ts viral DNA could be detected. To extend these studies, we developed an assay for (+) strand wt DNA fragments with (-1 strand ts DNA as the vector. For these studies ts replicative form (RF) was randomly nicked and denatured to serve as the vector. Noninfectious fragments of either viral or RF DNA served as the source of wt (+I strand. Table I shows that substantial stimulations in reversion frequencies were found when these nicked ts RF preparations were annealed to either wt DNA fragments of RF or viral DNA. The use of reversion frequencies allowed the results of experiments with different spheroplast and hybrid preparations to be compared regardless of the competence of the spheroplasts in any particular experiment. While the use of wt RF fragments with denatured ts RF vector presumably allows both (+I and ( -> strand fragments to be salvaged, the experiments with viralDNA fragments, which can hybridize only to complementary strands of the ts RF, demonstrate that (+) DNA as well as (-1
SHORT
783
COMMUNICATIONS
DNA fragments can be salvaged. The differences in the magnitude of the stimulations observed with the two types of fragments may be related to differences in fragment size. Experiments with a more highly digested viral-DNA fragment sample (lo-fold increase in DNase over that used in Table I) showed a lO-20-fold decrease in response of fold stimulation to TABLE
1
REVERSION FREQUENCY OF NICKED ts +X174 RF ANNEALED TO wt DNA FRAGMENTS Genotype of vee- Source ofV Veight rator RF” wt DNA t.io of fragfragments to mentsb DNA ts4lD ts4lD .?sllD FIG. 1. Dose-response curve of the salvage of noninfectious fragments of wt RF by ts viral DNA vectors. All DNA samples were prepared as described (4, 5) and were characterized by analytical ultracentrifugation as well as their infectivity to spheroplasts; all viral DNAs had a specific infectivity of approximately 4 x lOI@ plaque forming unit per microgram and all RF preparations had approximately 2 x lo9 PFU/pg. All +X174 phage stocks contained the am3 mutation in the lysis gene (6); therefore 1$X174 am3 will be called wild type +X174 throughout. The temperature-sensitive mutant strains (double mutants with am3) were ts9 (0) and ts4lD (0) (7). Wild type +X174 RF was cleaved to small noninfectious DNA fragments by treatment with pancreatic DNase as described previously (2). The specific infectivity of these fragments was approximately 1 x 10” PFU/Fg DNA. Wild type fragments and ts viral vector DNA were hybridized at the given weight ratios in 2X SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.3) by incubation at 95” for 3 min followed by incubation at 65” for 2 hr. The concentration of the ts DNA vectors was kept constant at 10 pg/ml. The nucleic acid preparations were diluted to 0.1 to 1 pg/ml in 50 mM TrisHCl (pH 8.1) and assayed in E. coli K12W6 spheroplasts by the method of Guthrie and Sinsheimer (8) as modified to include protamine sulfate by Henner et al (9). The spheroplasts were lysed and were titered on E. coli HF4714 at 30” (permissive temperature for ts mutants) and 42” (nonpermissive temperature) or at 37” (for wild type phage). The reversion frequency was defined as the number of PFU/ml assayed at the nonpermissive temperature minus the number of high temperature PFU/ml due to the fragment preparation alone (usually negligible)
ts9 ts9 ts9
viral viral RF viral viral RF
0.074 0.19 0.036 0.027 0.11 0.053
Rever- Fpold sti-
,ion fre luencyC (X 109 480 630 1100 100 300 5200
mulation’ 88 120 200 7 22 400
1 1
Nicked ts 1$X174 alone
n 1#~X174viral DNA and RF were prepared as described previously (4, 5). +X174 ts RF was randomly nicked to give 75 to 90% RF II by pancreatic DNase as described UO). b Fragments of viral DNA were prepared by incubation of 60 pg/ml of 4X174 viral DNA and 1 fig/ml of pancreatic DNase in 10 mM potassium phosphate buffer (pH 8.0), 7 mM MgSO, at 30” for 6 hr. The average size of this fragment preparation measured by gel electrophoresis (11) was approximately 30 nucleotides, and its specific infectivity was 6 x lo4 PFUIpg. Fragmentation of +X174 RF was described (2 and legend to Fig. 1). c Samples were hybridized and assayed in spheroplasts as described in the legend to Fig. 1. Reversion frequency and fold stimulation are defined in the legend to Fig. 1. divided by the number of PFU/ml at the permissive temperature. In most cases reversion frequencies were relatively consistent (within a factor of approximately 2) from one spheroplast assay to another. Somewhat larger variabilities were found at 42” than at 30”. The fold stimulation is defined as the reversion frequency of the vectorqfragment hybrid minus that of the vector alone divided by the reversion frequency of the vector alone. The background reversion frequencies of viral DNAs alone were 3.9 x lo-” for ts41D and 8.0 x lo-” for ts9.
784
SHORT
COMMUNICATIONS
(+) DNA can be complemented by noninfectious fragments of (-) DNA. The relative infectivity of the linear viral strand increased with the amount of RF fragments added. The (-1 strand RF fragments hybridized with the linear viral DNA to form an infective structure perhaps due to in vivo ligation of the nick in the linear molecule to form an infectious circular molecule. This agrees with the work of Miller and Sinsheimer (13) who suggested from indirect evidence that complementary linear +X174 strands can anneal during or just prior to incubation with spheroplasts to form an infective molecule. We have also observed possible in situ hybridization of DNA fragments and complementary DNA during spheroplast incubations (unpublished results). The sensitivity of this assay was determined by the degree to which the linear DNA was free of infectious circular viral DNA. A control experiment was performed with circular viral DNA which exhibited a slight decrease in infectivity upon the annealing of RF DNA fragments. This was probably due to the inherently lower specific infectivity of doublestranded DNA relative to that of singlestranded DNA (15). The less efficient ab-
fragment concentration. The background reversion frequencies of the ts vector RF DNAs alone (Table I) were similar to those of the corresponding viral DNAs (Fig. 1). This assay allows genetic salvage of (+) strand (viral DNA) fragments. It may be generally useful to determine the map position of DNA fragments generated by the action of specific nucleases on viral DNA or by its degradation in the presence of certain DNA binding proteins (to determine the position of protection). Also, it could be used to map single-stranded replication intermediates [(+) strand type1 formed in vivo. A second DNA-fragment assay that involves the complementation of noninfectious linear 1$X174DNA was also developed. This assay detects DNA fragments complementary to viral $X174 DNA (Fig. 2). Linear 4x174 viral DNA (non-specifically nicked circular DNA) is noninfectious. If fragments of (-) strand RF are hybridized to the linear (+) DNA, this could be converted to a circular structure which might be infectious. Use of conditionally lethal mutants is not required for this study since neither component is infectious at any temperature. Figure 3 shows that noninfectious linear I
LINEAR
VIRAL
DNA
FRAGMENTS
OFfiX RF
LINEAR
HYBRID
\
i
(+I STRAND FRAGMENTS
CIRCULAR HYBRlD
I
SPHEROPLAST
J ASSAY
LIGASE I NONINFECTIOUS
I
I NONINFECTIOUS
DNA REPLICATON I PROGENY
$X174
FIG. 2. Scheme of study to determine capacity of fragments of RF DNA to salvage infectivity of linear viral DNA. Noninfectious fragments of it RF were annealed to linear viral it DNA which also was noninfectious. The products of hybridization were used to infect E. coli spheroplasts. Hybrid molecules which were converted into circular DNAs in viuo could give rise to progeny phage. However, linear hybrids or the unhybridized (+) strand fragments would be noninfectious.
785
SHORT COMMUNICATIONS
mixture (161, but some preference for single-stranded molecules still occurred. A unique application of this second assay would be to determine the location of a specific nick in the viral DNA providing a source of specific RF fragments (e.g., restriction endonuclease fragments) is available.
16
ACKNOWLEDGMENTS We thank Drs. C. A. Hutchison III and M. H. Edge11 for generously providing the bacterial and phage strains and for helpful discussion. This work was supported by funds from the National Cancer Institute (CM-122751 and the National Science Foundation (GB-30528X). One of us (J. B. D.) was supported, in part, by a National Science Foundation Predoctoral Fellowship. REFERENCES
0
02
04 WEIGHT
06
08
RATIO
;=; +
1.0
12
STRAND
FIG. 3. Recovery of infectivity of linear viral DNA on annealing to complementary DNA fragments. In order to prepare linear 4X174 viral DNA, 3H-labeled circular viral DNA (wt) was partially nicked by pancreatic DNase. The reaction mixture (0.4 ml) containing 10 n&f potassium phosphate buffer (pH 7.1). 7.5 mi+4MgCl,, 60 fig/ml viral DNA (6400 3H-cpm/pg DNA), and 2.5 ng of pancreatic DNase was incubated at 30” for 20 min. The reaction was terminated by addition of EDTA (to 12.5 n&f) and the solution was heated to 100” for 2.5 min followed by cooling on ice. The DNase-nicked mixture was sedimented on a low-salt, alkaline sucrose gradient according to the procedure of Kato et al. (14). The unit-length circular and unit-length linear regions of the gradient were pooled separately and further purified on three additional low-salt, alkaline gradients. Linear @XI74 was contaminated with less than 1.7% infectious circular DNA. Both preparations were hybridized to variable amounts of fragments of wt $X174 RF DNA and assayed in spheroplasts as described in Fig. 1. The ratio of the PFU for the hybrid to the PFU of the linear (or circular) viral DNA alone is plotted versus the ratio of the concentration of the negative strand nucleic acid (half of the RF fragment concentration) to the concentration of the linear (or circular) viral DNA in the hybridization mixture. (O), linear viral DNA + RF fragments; (m), circular viral DNA + RF fragmerits.
sorption of double-stranded molecules was somewhat alleviated by the use of protamine sulfate in the spheroplast incubation
1. WEISBEEK, P. J., and VAN DE POL, J. H., Biochim. Biophys. Acta 224, 32b338 (1970). 2. HUTCHISON, C. A., III, and EDGELL, M. H., J. Viral. 8, 181-189 (1971). 3. SINSHEIMER, R. L., in “Progress in Nucleic Acid
Research and Molecular Biology” (J. N. Davidson and W. E. Cohn, eds.), Vol. 8, pp. 115 169, Academic Press, New York, 1968. 4. PAGANO, J. S., and HLJTCHISON, C. A., III. in “Methods in Virology” (K. Maramorosch, and H. Koprowski, eds.), Vol. 5, pp. 79-123, Academic Press, New York, 1971. 5. RUSH, M. G., and WARNER, R. C., J. Biol. Chem. 245, 2704-2708 (1970). 6. HUTCHIS~N, C. A., III, and SINBHEIMER, R. L., J. Mol. Biol. 18, 429-447 (1966). 7. BENBOW, R. M., HUTCHISON, C. A., III, FABRICANT, J. D., and SINSHEIMER, R. L., J. Viral. 7, 549-558 (1971). 8. GUTHRIE, G. D., and SINSHEIMER, R. L., B&him. Biophys. Actu 72, 290-297 (1963). 9. HENNER, W. D., KLEBER, I., and BENZINGER, R., J. Viral. 12, 741-747 (1973). 10. JANSZ, H. S., BAAS, P. D., POUWELS, P. H., VAN BRUGGEN, E. L. J., and OLDENZIEL, H., J. Mol. Biol. 32, 159168 (1968). 11. BURD, J. G., and WELU, R. D., J. Biol. Chem.
249, 7094-7101(1974). 12. BURTON, A., and SINSHEIMER, R. L., J. Mol. Biol. 14, 327-347 (1965). 13. MILLER, L. K., and SINSHEIMER, R. L., J. Virol.
14, 1503-1514(1974). 14. KATO, A. C., BARTOK, K., FRASER, M. J., and DENHARIYP, D. T., Biochim. Biophys. Acta 308, 6&78 (1973). 15. SINSHEIMER, R. L., LAWRENCE, M., and NAGLER, C., J. Mol. Biol. 14, 348-360 (1965). 16. BENZINGER, R., KLEBER, I., and HUSKEY, R., J. Viral. 7, 646650 (1971).
69, 782-785
(1976)
SHORT Two New Genetic
COMMUNICATIONS
Assays for Noninfectious
Fragments
of +X174 DNA
JERRY B. DODGSON, INGOLF F. NES, BRUCE W. PORTER, AND ROBERT D. WELLS University
of Wisconsin, Department
of Biochemistry, College of Agricultural Wisconsin 53706
and Life Sciences, Madison,
Accepted September 24, 1975 A genetic salvage assay for noninfectious 4X174 DNA fragments of the viral strandedness is described. This assay is complementary to the assay previously described for the negative strand of 4X174 RF fragments. Also, a novel DNA fragment assay was developed which depends on the formation of an infective complex made up of a DNA fragment and linear +X174 viral DNA.
Previous workers (1, 2) developed a genetic assay for fragments of the complementary’ strand of $X174 DNA. In these assays, conditionally lethal temperaturesensitive (ts) 4X174 viral DNA was annealed to noninfectious complementary DNA fragments that were wild type (wt) in the gene containing the mutation in the viral DNA. When the resultant hybrid was used to infect Escherichia coli spheroplasts, the progeny phage from these spheroplasts showed an increased frequency of the wt genotype compared to that observed when the spheroplasts were infected with ts viral-DNA vector2 alone. This increase was apparently due to the replication of the wt DNA marker inside the spheroplasts. Typical dose response curves obtained with this type of assay for the two mutants used in this study are given in Fig. 1. The fold stimulations were linearly related to the weight ratio of wt DNA fragments to ts viral DNA on this log-log plot with a slope of approximately ’ 4X174 DNA species were defined by Sinsheimer (3) and include: viral or ( + ) strand DNA, replicative forms (RF I and RF II), and complementary or C-1 strand. p Vector DNA is the term used to describe an infectious DNA (usually ts) which was used to transport a noninfectious DNA or RNA (usually wt) into the cell. 782 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
1.2, in agreement with previous data (2). The actual fold stimulations at a given weight ratio of fragment to vector DNA varied depending on the mutation in question (Fig. 1). Stimulations as low as one- to twofold over the background reversion frequency of the ts viral DNA could be detected. To extend these studies, we developed an assay for (+) strand wt DNA fragments with (-1 strand ts DNA as the vector. For these studies ts replicative form (RF) was randomly nicked and denatured to serve as the vector. Noninfectious fragments of either viral or RF DNA served as the source of wt (+I strand. Table I shows that substantial stimulations in reversion frequencies were found when these nicked ts RF preparations were annealed to either wt DNA fragments of RF or viral DNA. The use of reversion frequencies allowed the results of experiments with different spheroplast and hybrid preparations to be compared regardless of the competence of the spheroplasts in any particular experiment. While the use of wt RF fragments with denatured ts RF vector presumably allows both (+I and ( -> strand fragments to be salvaged, the experiments with viralDNA fragments, which can hybridize only to complementary strands of the ts RF, demonstrate that (+) DNA as well as (-1
SHORT
783
COMMUNICATIONS
DNA fragments can be salvaged. The differences in the magnitude of the stimulations observed with the two types of fragments may be related to differences in fragment size. Experiments with a more highly digested viral-DNA fragment sample (lo-fold increase in DNase over that used in Table I) showed a lO-20-fold decrease in response of fold stimulation to TABLE
1
REVERSION FREQUENCY OF NICKED ts +X174 RF ANNEALED TO wt DNA FRAGMENTS Genotype of vee- Source ofV Veight rator RF” wt DNA t.io of fragfragments to mentsb DNA ts4lD ts4lD .?sllD FIG. 1. Dose-response curve of the salvage of noninfectious fragments of wt RF by ts viral DNA vectors. All DNA samples were prepared as described (4, 5) and were characterized by analytical ultracentrifugation as well as their infectivity to spheroplasts; all viral DNAs had a specific infectivity of approximately 4 x lOI@ plaque forming unit per microgram and all RF preparations had approximately 2 x lo9 PFU/pg. All +X174 phage stocks contained the am3 mutation in the lysis gene (6); therefore 1$X174 am3 will be called wild type +X174 throughout. The temperature-sensitive mutant strains (double mutants with am3) were ts9 (0) and ts4lD (0) (7). Wild type +X174 RF was cleaved to small noninfectious DNA fragments by treatment with pancreatic DNase as described previously (2). The specific infectivity of these fragments was approximately 1 x 10” PFU/Fg DNA. Wild type fragments and ts viral vector DNA were hybridized at the given weight ratios in 2X SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.3) by incubation at 95” for 3 min followed by incubation at 65” for 2 hr. The concentration of the ts DNA vectors was kept constant at 10 pg/ml. The nucleic acid preparations were diluted to 0.1 to 1 pg/ml in 50 mM TrisHCl (pH 8.1) and assayed in E. coli K12W6 spheroplasts by the method of Guthrie and Sinsheimer (8) as modified to include protamine sulfate by Henner et al (9). The spheroplasts were lysed and were titered on E. coli HF4714 at 30” (permissive temperature for ts mutants) and 42” (nonpermissive temperature) or at 37” (for wild type phage). The reversion frequency was defined as the number of PFU/ml assayed at the nonpermissive temperature minus the number of high temperature PFU/ml due to the fragment preparation alone (usually negligible)
ts9 ts9 ts9
viral viral RF viral viral RF
0.074 0.19 0.036 0.027 0.11 0.053
Rever- Fpold sti-
,ion fre luencyC (X 109 480 630 1100 100 300 5200
mulation’ 88 120 200 7 22 400
1 1
Nicked ts 1$X174 alone
n 1#~X174viral DNA and RF were prepared as described previously (4, 5). +X174 ts RF was randomly nicked to give 75 to 90% RF II by pancreatic DNase as described UO). b Fragments of viral DNA were prepared by incubation of 60 pg/ml of 4X174 viral DNA and 1 fig/ml of pancreatic DNase in 10 mM potassium phosphate buffer (pH 8.0), 7 mM MgSO, at 30” for 6 hr. The average size of this fragment preparation measured by gel electrophoresis (11) was approximately 30 nucleotides, and its specific infectivity was 6 x lo4 PFUIpg. Fragmentation of +X174 RF was described (2 and legend to Fig. 1). c Samples were hybridized and assayed in spheroplasts as described in the legend to Fig. 1. Reversion frequency and fold stimulation are defined in the legend to Fig. 1. divided by the number of PFU/ml at the permissive temperature. In most cases reversion frequencies were relatively consistent (within a factor of approximately 2) from one spheroplast assay to another. Somewhat larger variabilities were found at 42” than at 30”. The fold stimulation is defined as the reversion frequency of the vectorqfragment hybrid minus that of the vector alone divided by the reversion frequency of the vector alone. The background reversion frequencies of viral DNAs alone were 3.9 x lo-” for ts41D and 8.0 x lo-” for ts9.
784
SHORT
COMMUNICATIONS
(+) DNA can be complemented by noninfectious fragments of (-) DNA. The relative infectivity of the linear viral strand increased with the amount of RF fragments added. The (-1 strand RF fragments hybridized with the linear viral DNA to form an infective structure perhaps due to in vivo ligation of the nick in the linear molecule to form an infectious circular molecule. This agrees with the work of Miller and Sinsheimer (13) who suggested from indirect evidence that complementary linear +X174 strands can anneal during or just prior to incubation with spheroplasts to form an infective molecule. We have also observed possible in situ hybridization of DNA fragments and complementary DNA during spheroplast incubations (unpublished results). The sensitivity of this assay was determined by the degree to which the linear DNA was free of infectious circular viral DNA. A control experiment was performed with circular viral DNA which exhibited a slight decrease in infectivity upon the annealing of RF DNA fragments. This was probably due to the inherently lower specific infectivity of doublestranded DNA relative to that of singlestranded DNA (15). The less efficient ab-
fragment concentration. The background reversion frequencies of the ts vector RF DNAs alone (Table I) were similar to those of the corresponding viral DNAs (Fig. 1). This assay allows genetic salvage of (+) strand (viral DNA) fragments. It may be generally useful to determine the map position of DNA fragments generated by the action of specific nucleases on viral DNA or by its degradation in the presence of certain DNA binding proteins (to determine the position of protection). Also, it could be used to map single-stranded replication intermediates [(+) strand type1 formed in vivo. A second DNA-fragment assay that involves the complementation of noninfectious linear 1$X174DNA was also developed. This assay detects DNA fragments complementary to viral $X174 DNA (Fig. 2). Linear 4x174 viral DNA (non-specifically nicked circular DNA) is noninfectious. If fragments of (-) strand RF are hybridized to the linear (+) DNA, this could be converted to a circular structure which might be infectious. Use of conditionally lethal mutants is not required for this study since neither component is infectious at any temperature. Figure 3 shows that noninfectious linear I
LINEAR
VIRAL
DNA
FRAGMENTS
OFfiX RF
LINEAR
HYBRID
\
i
(+I STRAND FRAGMENTS
CIRCULAR HYBRlD
I
SPHEROPLAST
J ASSAY
LIGASE I NONINFECTIOUS
I
I NONINFECTIOUS
DNA REPLICATON I PROGENY
$X174
FIG. 2. Scheme of study to determine capacity of fragments of RF DNA to salvage infectivity of linear viral DNA. Noninfectious fragments of it RF were annealed to linear viral it DNA which also was noninfectious. The products of hybridization were used to infect E. coli spheroplasts. Hybrid molecules which were converted into circular DNAs in viuo could give rise to progeny phage. However, linear hybrids or the unhybridized (+) strand fragments would be noninfectious.
785
SHORT COMMUNICATIONS
mixture (161, but some preference for single-stranded molecules still occurred. A unique application of this second assay would be to determine the location of a specific nick in the viral DNA providing a source of specific RF fragments (e.g., restriction endonuclease fragments) is available.
16
ACKNOWLEDGMENTS We thank Drs. C. A. Hutchison III and M. H. Edge11 for generously providing the bacterial and phage strains and for helpful discussion. This work was supported by funds from the National Cancer Institute (CM-122751 and the National Science Foundation (GB-30528X). One of us (J. B. D.) was supported, in part, by a National Science Foundation Predoctoral Fellowship. REFERENCES
0
02
04 WEIGHT
06
08
RATIO
;=; +
1.0
12
STRAND
FIG. 3. Recovery of infectivity of linear viral DNA on annealing to complementary DNA fragments. In order to prepare linear 4X174 viral DNA, 3H-labeled circular viral DNA (wt) was partially nicked by pancreatic DNase. The reaction mixture (0.4 ml) containing 10 n&f potassium phosphate buffer (pH 7.1). 7.5 mi+4MgCl,, 60 fig/ml viral DNA (6400 3H-cpm/pg DNA), and 2.5 ng of pancreatic DNase was incubated at 30” for 20 min. The reaction was terminated by addition of EDTA (to 12.5 n&f) and the solution was heated to 100” for 2.5 min followed by cooling on ice. The DNase-nicked mixture was sedimented on a low-salt, alkaline sucrose gradient according to the procedure of Kato et al. (14). The unit-length circular and unit-length linear regions of the gradient were pooled separately and further purified on three additional low-salt, alkaline gradients. Linear @XI74 was contaminated with less than 1.7% infectious circular DNA. Both preparations were hybridized to variable amounts of fragments of wt $X174 RF DNA and assayed in spheroplasts as described in Fig. 1. The ratio of the PFU for the hybrid to the PFU of the linear (or circular) viral DNA alone is plotted versus the ratio of the concentration of the negative strand nucleic acid (half of the RF fragment concentration) to the concentration of the linear (or circular) viral DNA in the hybridization mixture. (O), linear viral DNA + RF fragments; (m), circular viral DNA + RF fragmerits.
sorption of double-stranded molecules was somewhat alleviated by the use of protamine sulfate in the spheroplast incubation
1. WEISBEEK, P. J., and VAN DE POL, J. H., Biochim. Biophys. Acta 224, 32b338 (1970). 2. HUTCHISON, C. A., III, and EDGELL, M. H., J. Viral. 8, 181-189 (1971). 3. SINSHEIMER, R. L., in “Progress in Nucleic Acid
Research and Molecular Biology” (J. N. Davidson and W. E. Cohn, eds.), Vol. 8, pp. 115 169, Academic Press, New York, 1968. 4. PAGANO, J. S., and HLJTCHISON, C. A., III. in “Methods in Virology” (K. Maramorosch, and H. Koprowski, eds.), Vol. 5, pp. 79-123, Academic Press, New York, 1971. 5. RUSH, M. G., and WARNER, R. C., J. Biol. Chem. 245, 2704-2708 (1970). 6. HUTCHIS~N, C. A., III, and SINBHEIMER, R. L., J. Mol. Biol. 18, 429-447 (1966). 7. BENBOW, R. M., HUTCHISON, C. A., III, FABRICANT, J. D., and SINSHEIMER, R. L., J. Viral. 7, 549-558 (1971). 8. GUTHRIE, G. D., and SINSHEIMER, R. L., B&him. Biophys. Actu 72, 290-297 (1963). 9. HENNER, W. D., KLEBER, I., and BENZINGER, R., J. Viral. 12, 741-747 (1973). 10. JANSZ, H. S., BAAS, P. D., POUWELS, P. H., VAN BRUGGEN, E. L. J., and OLDENZIEL, H., J. Mol. Biol. 32, 159168 (1968). 11. BURD, J. G., and WELU, R. D., J. Biol. Chem.
249, 7094-7101(1974). 12. BURTON, A., and SINSHEIMER, R. L., J. Mol. Biol. 14, 327-347 (1965). 13. MILLER, L. K., and SINSHEIMER, R. L., J. Virol.
14, 1503-1514(1974). 14. KATO, A. C., BARTOK, K., FRASER, M. J., and DENHARIYP, D. T., Biochim. Biophys. Acta 308, 6&78 (1973). 15. SINSHEIMER, R. L., LAWRENCE, M., and NAGLER, C., J. Mol. Biol. 14, 348-360 (1965). 16. BENZINGER, R., KLEBER, I., and HUSKEY, R., J. Viral. 7, 646650 (1971).