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Lethal effects of 32P decay on transfecting activity of Bacillus subtilis phage φe DNA
VIROLOGY
96,2'70-273
(1979)
Lethal Effects of 32PDecay on Transfecting Activity of Bacillus subtilis phage 4e DNA KENNETH
S. LOVEDAY’
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02199 Accepted February 22, 1979 Disintegration of 3zP present in the DNA of Bacillus subtilis phage & (a phage containing double-strand DNA) results in the loss of viability of intact phage as well as transfecting activity of isolated DNA. Only 52 of the 32Pdisintegrations per phage DNA equivalent inactivates the intact phage while nearly every disintegration inactivates the transfecting DNA. This result provides evidence for a single-strand intermediate in the transfection of B. subtilis by & DNA.
The yield of infective centers resulting since several DNA molecules would have from transfection with DNA isolated from to be taken up by a single cell to provide the the majority of Bacillus subtilis phages necessary complementary or partially comincreases with the second or third power of plementary single-strand fragments. A simiDNA concentration. This observation sug- lar description has been suggested by Porter gests that several DNA molecules must and Guild to describe transfection in S. participate in the formation of an infective pneumoniae (10). center (l-5). It has been suggested that No double-stranded transfecting DNA phage DNA is damaged either during or was detected in the competent cells (9). after entry and that several DNA mole- However, the limits of resolution in CsCl cules (taken up independently) must there- gradients (9) make it impossible to exclude fore recombine to form an intact phage double-stranded DNA below a level of 5% genome (3,640. of the total DNA taken up by the cells. The physical characterization of the +e Since transfection is an inefficient process transfecting DNA that has been taken up (the maximum efficiency observed was 1.5% by B. subtilis suggests the following se- infective centers per phage equivalent of quence of events (9). Double-stranded DNA taken up (9)), it remains possible that transfecting DNA (9), like bacterial trans- a small amount of double-stranded DNA is forming DNA (11 -I,$>, is reduced to single taken into the cells by a different mechanism strands during uptake by competent bac- and is responsible for infective center forteria. Fragments of complementary single- mation. Although this hypothesis seems unstranded phage DNA can then anneal within likely (9), independent evidence demonthe cell and be repaired to form double- strating a role for single stranded DNA as stranded structures by bacterial polym- an intermediate in transfection-mediated infective center formation is desirable. erases. Those cells in which a full-length To differentiate between infective center phage genome had been reassembled would become infective centers. This proposal ac- formation initiated by double-stranded counts for the DNA concentration de- versus single-stranded transfecting DNA, pendence of infective center formation the inactivation of transfecting DNA and of viable phage particles by 32Psuicide were of incor1 Present address: Division of Clinical Genetics, measured. The disintegration porated 3zPatoms (32Psuicide) is more effecChildren’s Hospital Medical Center, 300 Longwood tive in inactivating phages which contain Ave., Boston, Mass. 02115. 0042-6822/79/090270-04$02.00/O Copyright 0 19’79by Academic Press, Inc. All rights of reproduction in any form reserved.
270
SHORT COMMUNICATIONS
single-stranded DNA than phages containing double-stranded DNA (15-18). Each 32P disintegration causes a single-strand break in the DNA chain (16); and for single stranded DNA phages, each 32Pdisintegration is a lethal event (a = 1) (15). For doublestranded DNA phages, between Y12 and YZO of the 32P disintegrations are lethal events resulting from a recoil-induced double-strand break (18, 19). If doublestranded DNA were responsible for initiating infective centers, transfecting DNA labeled with 32P would be expected to be inactivated with an (I! = #2. If an intermediate in the process were single stranded, an inactivation rate corresponding to an (Yof about 1 would be expected. The following experiment will provide
271
support for the proposal that single-stranded DNA is an intermediate in transfection since the efficiency with which 32Pdisintegrations result in inactivation of transfecting DNA is near 1. The techniques for growing competent B. subtilis strains, for purifying competent cells on renografm gradients, for labeling $e DNA with 32P, for isolating DNA, and for performing transfection experiments have been previously described (9). To minimize external radiation damage, the phage were purified as quickly as possible and the DNA was stored at a low concentration (2 pg/ml) at 4”. Isolated $e DNA labeled with 32Prapidly lost transfecting activity as the 32P atoms disintegrated (Fig. 1A). Since the yield of infective centers is so sensitive to DNA t1
I
I
I
1
I
:
FRACTDN
OF =P
ATOMS
DECAYED
I I-c?
FRACTlCW
OF%
ATOMS
DECAYED
, I-e-“‘1
FIG. 1. Inactivation of 3ZP-labeled transfecting DNA. (A) Phage $e was labeled with 32P at 33 ~Ci/ml in medium containing 20 pg/ml phosphate (determined by a bioassay) (25). The labeled DNA was stored at 4” at 2 pg/ml and (5 &i/ml). The DNA was assayed for transfecting activity at 1 pg/ml using renografin purified competent cells from strain SB291 (9). The value plotted is the number of infective centers formed by labeled DNA divided by the number found using a standard of unlabeled cpe DNA (assayed at 1 pg/ml). +e DNA labeled with a*P, n ; unlabeled DNA with added 35P phosphate, 0. The labeled intact phage (6 x 10IVml) was stored at 4” and titered with competent cells as indicator, q I. (B) The relative transfecting activity of the same “P-labeled DNA as in (A), assayed at 0.33 pg/ml, is plotted (also normalized to the transfecting activity of unlabeled DNA assayed at 1 pg/ml).
SHORT COMMUNICATIONS
272
TABLE 1 EFFECT OF CONCENTRATION OF RADIOACTIVITY DURING STORAGE ON INACTIVATION OF 32P-L~~~~~~ TRANSFECTING DNA”
Storage conditions Wn-4 (1) 4 (10 &i/ml)
(2) 2 (5 &i/ml) (31 1 (2.5 @/ml) (4) Unlabeled DNA + 32P (5 $.Xml)
Concentration of DNA during transfection (&ml)
Slope of inactivation curve
Fraction surviving (e-y
Lethal hits per genome 09
1 0.33
-2.2 -2.4
0.0065 0.0043
5.0 5.5
*1 *0.33
-1.7 -1.9
0.022 0.010
3.9 4.4
0.5 0.16
-1.8 -2.0
0.017 0.011
4.1 4.6
-0.46
0.35
1.1
*1
a Phage DNA labeled with 32Pwas stored at three different concentrations, resulting in three different levels of radioactivity. Transfecting activity was assayed at two different concentrations for each DNA sample. For condition (4), unlabeled DNA was stored at 10 fig/ml and [32P]phosphoric acid was added to give a final concentration of 5 @Xml. The slopes of the inactivation curves are derived from least square plots as shown in Fig. 1. The fraction surviving was computed by dividing the extrapolated value at 100% decay by the extrapolated value at 0% decay. Lethal hits per genome is derived from the Poisson relation e-.V,where N is the number of lethal hits needed to give the observed fraction of surviving activity at infinite 32Pdecay. The asterisks designate data plotted in Fig. 1.
concentration, the activity of 32P-labeled DNA was determined at two DNA concentrations differing by a factor of 3. Similar rates of inactivation were observed at the two concentrations (Figs. 1A and B), indicating that the loss of transfecting activity was not reflecting a small change in the effective DNA concentration in the transfection assay. In contrast, loss of viability of intact phage from which the DNA had been isolated was modest. No more than half of the phage was lost when virtually all of the 32Phad disintegrated (Fig. 1A). In order to calculate the efficiency of inactivation by 32P disintegration (lethal events per genomeldisintegrations per genome), it was necessary to make a modest correction for the amount of inactivation due to external radiation. Two additional experiments were performed to estimate this contribution to the observed inactivation. First, the 32P-labeledDNA was stored at two additional concentrations, at 4 and 1 pglml, resulting in levels of radioactivity of 10 and 2.5 &i/ml. The loss of
transfecting activity during storage of DNA under these conditions was similar to that previously observed (Table 1). At the highest level of radioactivity, 10 &i/ml, a small effect of the external radiation is evident, as the rate of inactivation is slightly faster than for the other two, which are similar. Presumably the inactivation due to external radiation for DNA stored at 2.5 and 5 pCi/ml is very small. Second, an equivalent amount of 32P as phosphoric acid (5 #Z/ml) was added to unlabeled 4e DNA. Under these conditions the DNA lost a small amount of transfecting activity (Fig. lA, Table 1). The amount of inactivation due to external radiation would in this case represent about one lethal hit per genome when all of the 32Phad decayed. These observations suggest that the inactivation curves at radioactivity levels of 2.5 and 5.0 $.X/ml could include external radiation components no greater than one lethal hit per genome. These observations show that the disintegration of incorporated 32Pis far more efficient in the inactivation of transfecting
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273
COMMUNICATIONS
DNA than in the inactivation of intact phage particles. To calculate the efficiency of inactivation (a), the number of 32Patoms/ phage must be known. Based on the specific activity of phosphorous in the growth medium (1.6 $Zi/~g) and a genome size of 120 x lo6 (9), the expected number of 32P atomslphage was 7. After the phage were purified, the calculated number was 10 (based on the amount of radioactivity per plaque-forming unit). If double-stranded transfecting DNA initiated infective center formation, one would expect the same reduction in activity that was observed for intact phage particles (50% inactivation or 0.7 lethal hit/genome). However, the number of lethal hits per genome due to 32P suicide was between 3 and 4 (Table 1, using the values for the DNA stored at 2.5 and 5 &i/ml and subtracting the maximum value of 1.0 lethal hit for such damage). Since there were an average of about four 32P atoms/DNA strand, the expected number of lethal hits would be about four if one single strand of transfecting DNA were to initiate infective center formation. The calculated (Y would thus be close to 1.0. This result provides firm evidence for the proposal that the single-stranded phage DNA found in newly transfected competent cells (9) is responsible for formation of infective centers. The precise mechanism of transfection is of special interest in interpreting the results of tranfection with heteroduplex DNA made in vitro by annealing the separated complementary strands of DNAs from mutant phages. Such studies were undertaken in the search for evidence of the recognition and correction of base pair mismatches to homozygosity (5, 20-22). Their interpretation depends on the assumption that transfecting DNA remains double stranded as it is taken up by competent cells. The existence of such recognition and correction processes has been postulated to account for meiotic gene conversion (23 >and contribute to the high negative
interference observed in phage crosses (20,21). These processes have been demonstrated in other systems (21,22,24). However, it may not be possible to detect them in B. subtilis by transfection experiments using artificially constructed heteroduplex molecules. REFERENCES
2. 3. 1. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
24. 25.
FISCHBACH, K., SPATZ, H., and KLOTZ, G., Mol. Gen. Genet. 141, 121-145 (1975). FLOCK, J., and RUTBERG, L., Mol. Gen. Genet. 131, 301-311 (1974). GREEN, D. M., J. Mol. Biol. 22, 1-13. OKUBO, S., STRAUSS, B., and STODOLSKY, M., Virology 24, 552-562 (1964). SPATZ, H., and TRAUTNER, T., Mol. Gen. Genet. 109, 84-106 (1970). GREEN, D. M., J. Mol. Biol. 10, 438-451 (1964). EPSTEIN, H., J. Viral. 2, 710-715 (1968). SPATZ, H., and TRAUTNER, T., Mol. Gen. Genet. 113, 174- 190 (1971). LOVEDAY, K. S., and Fox, M. S., Virology 85, 387-403 (1978). PORTER, R., and GUILD, W., J. Viral. 25, 60-72 (1978). DAVIDOFF-ABELSON, R., and DUBNAU, D., J. Bacterial. 116, 146-153 (1973). PIECHOWSKA, M., and Fox, M. S., J. Bacterial. 108, 681-689 (1971). PIECHOWSKA, M., SOLTYK, A., and SHUGAR, D., J. Bacterial. 122, 610-622 (1975). SOLTYK, A., SHUGAR, D., and PIECHOWSKA, M., J. Bacterial. 124, 1429-1438 (1975). TESSMAN, I., Virology 7, 263-275 (1959). STENT, G. S., and FUERST, C. R., J. Gen. Physiol. 38, 441-448 (1955). DENHARDT, D. T., and SINSHEIMER, R. T., J. Mol. Biol. 12, 663-673 (1965). KRISCH, R. E., Int. J. Radiat. Biol. 25, 261-276 (1974). SONENSHEIN, A. L., Virology 42,488-495 (1970). WHITE, R. L., and Fox, M. S., Proc. Nat. Acad. Sci. USA 71, 1544-1546 (1974). WHITE, R. L., and Fox, M. S., 1Mol. Gen. Genet. 141, 163-171 (1975). WILDENBERG, J., and MESELSON, M., Proc. Nat. Acad. Sci. USA 72, 2202-2206 (1975). HOLLIDAY, R., Genet. Res. 5, 282-287 (1964). FOX, M. S., Ann. Rev. Genet. 12, 47-68 (1978). Fox, M. S., J. Mol. Biol. 6, 85-94 (1963).
96,2'70-273
(1979)
Lethal Effects of 32PDecay on Transfecting Activity of Bacillus subtilis phage 4e DNA KENNETH
S. LOVEDAY’
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02199 Accepted February 22, 1979 Disintegration of 3zP present in the DNA of Bacillus subtilis phage & (a phage containing double-strand DNA) results in the loss of viability of intact phage as well as transfecting activity of isolated DNA. Only 52 of the 32Pdisintegrations per phage DNA equivalent inactivates the intact phage while nearly every disintegration inactivates the transfecting DNA. This result provides evidence for a single-strand intermediate in the transfection of B. subtilis by & DNA.
The yield of infective centers resulting since several DNA molecules would have from transfection with DNA isolated from to be taken up by a single cell to provide the the majority of Bacillus subtilis phages necessary complementary or partially comincreases with the second or third power of plementary single-strand fragments. A simiDNA concentration. This observation sug- lar description has been suggested by Porter gests that several DNA molecules must and Guild to describe transfection in S. participate in the formation of an infective pneumoniae (10). center (l-5). It has been suggested that No double-stranded transfecting DNA phage DNA is damaged either during or was detected in the competent cells (9). after entry and that several DNA mole- However, the limits of resolution in CsCl cules (taken up independently) must there- gradients (9) make it impossible to exclude fore recombine to form an intact phage double-stranded DNA below a level of 5% genome (3,640. of the total DNA taken up by the cells. The physical characterization of the +e Since transfection is an inefficient process transfecting DNA that has been taken up (the maximum efficiency observed was 1.5% by B. subtilis suggests the following se- infective centers per phage equivalent of quence of events (9). Double-stranded DNA taken up (9)), it remains possible that transfecting DNA (9), like bacterial trans- a small amount of double-stranded DNA is forming DNA (11 -I,$>, is reduced to single taken into the cells by a different mechanism strands during uptake by competent bac- and is responsible for infective center forteria. Fragments of complementary single- mation. Although this hypothesis seems unstranded phage DNA can then anneal within likely (9), independent evidence demonthe cell and be repaired to form double- strating a role for single stranded DNA as stranded structures by bacterial polym- an intermediate in transfection-mediated infective center formation is desirable. erases. Those cells in which a full-length To differentiate between infective center phage genome had been reassembled would become infective centers. This proposal ac- formation initiated by double-stranded counts for the DNA concentration de- versus single-stranded transfecting DNA, pendence of infective center formation the inactivation of transfecting DNA and of viable phage particles by 32Psuicide were of incor1 Present address: Division of Clinical Genetics, measured. The disintegration porated 3zPatoms (32Psuicide) is more effecChildren’s Hospital Medical Center, 300 Longwood tive in inactivating phages which contain Ave., Boston, Mass. 02115. 0042-6822/79/090270-04$02.00/O Copyright 0 19’79by Academic Press, Inc. All rights of reproduction in any form reserved.
270
SHORT COMMUNICATIONS
single-stranded DNA than phages containing double-stranded DNA (15-18). Each 32P disintegration causes a single-strand break in the DNA chain (16); and for single stranded DNA phages, each 32Pdisintegration is a lethal event (a = 1) (15). For doublestranded DNA phages, between Y12 and YZO of the 32P disintegrations are lethal events resulting from a recoil-induced double-strand break (18, 19). If doublestranded DNA were responsible for initiating infective centers, transfecting DNA labeled with 32P would be expected to be inactivated with an (I! = #2. If an intermediate in the process were single stranded, an inactivation rate corresponding to an (Yof about 1 would be expected. The following experiment will provide
271
support for the proposal that single-stranded DNA is an intermediate in transfection since the efficiency with which 32Pdisintegrations result in inactivation of transfecting DNA is near 1. The techniques for growing competent B. subtilis strains, for purifying competent cells on renografm gradients, for labeling $e DNA with 32P, for isolating DNA, and for performing transfection experiments have been previously described (9). To minimize external radiation damage, the phage were purified as quickly as possible and the DNA was stored at a low concentration (2 pg/ml) at 4”. Isolated $e DNA labeled with 32Prapidly lost transfecting activity as the 32P atoms disintegrated (Fig. 1A). Since the yield of infective centers is so sensitive to DNA t1
I
I
I
1
I
:
FRACTDN
OF =P
ATOMS
DECAYED
I I-c?
FRACTlCW
OF%
ATOMS
DECAYED
, I-e-“‘1
FIG. 1. Inactivation of 3ZP-labeled transfecting DNA. (A) Phage $e was labeled with 32P at 33 ~Ci/ml in medium containing 20 pg/ml phosphate (determined by a bioassay) (25). The labeled DNA was stored at 4” at 2 pg/ml and (5 &i/ml). The DNA was assayed for transfecting activity at 1 pg/ml using renografin purified competent cells from strain SB291 (9). The value plotted is the number of infective centers formed by labeled DNA divided by the number found using a standard of unlabeled cpe DNA (assayed at 1 pg/ml). +e DNA labeled with a*P, n ; unlabeled DNA with added 35P phosphate, 0. The labeled intact phage (6 x 10IVml) was stored at 4” and titered with competent cells as indicator, q I. (B) The relative transfecting activity of the same “P-labeled DNA as in (A), assayed at 0.33 pg/ml, is plotted (also normalized to the transfecting activity of unlabeled DNA assayed at 1 pg/ml).
SHORT COMMUNICATIONS
272
TABLE 1 EFFECT OF CONCENTRATION OF RADIOACTIVITY DURING STORAGE ON INACTIVATION OF 32P-L~~~~~~ TRANSFECTING DNA”
Storage conditions Wn-4 (1) 4 (10 &i/ml)
(2) 2 (5 &i/ml) (31 1 (2.5 @/ml) (4) Unlabeled DNA + 32P (5 $.Xml)
Concentration of DNA during transfection (&ml)
Slope of inactivation curve
Fraction surviving (e-y
Lethal hits per genome 09
1 0.33
-2.2 -2.4
0.0065 0.0043
5.0 5.5
*1 *0.33
-1.7 -1.9
0.022 0.010
3.9 4.4
0.5 0.16
-1.8 -2.0
0.017 0.011
4.1 4.6
-0.46
0.35
1.1
*1
a Phage DNA labeled with 32Pwas stored at three different concentrations, resulting in three different levels of radioactivity. Transfecting activity was assayed at two different concentrations for each DNA sample. For condition (4), unlabeled DNA was stored at 10 fig/ml and [32P]phosphoric acid was added to give a final concentration of 5 @Xml. The slopes of the inactivation curves are derived from least square plots as shown in Fig. 1. The fraction surviving was computed by dividing the extrapolated value at 100% decay by the extrapolated value at 0% decay. Lethal hits per genome is derived from the Poisson relation e-.V,where N is the number of lethal hits needed to give the observed fraction of surviving activity at infinite 32Pdecay. The asterisks designate data plotted in Fig. 1.
concentration, the activity of 32P-labeled DNA was determined at two DNA concentrations differing by a factor of 3. Similar rates of inactivation were observed at the two concentrations (Figs. 1A and B), indicating that the loss of transfecting activity was not reflecting a small change in the effective DNA concentration in the transfection assay. In contrast, loss of viability of intact phage from which the DNA had been isolated was modest. No more than half of the phage was lost when virtually all of the 32Phad disintegrated (Fig. 1A). In order to calculate the efficiency of inactivation by 32P disintegration (lethal events per genomeldisintegrations per genome), it was necessary to make a modest correction for the amount of inactivation due to external radiation. Two additional experiments were performed to estimate this contribution to the observed inactivation. First, the 32P-labeledDNA was stored at two additional concentrations, at 4 and 1 pglml, resulting in levels of radioactivity of 10 and 2.5 &i/ml. The loss of
transfecting activity during storage of DNA under these conditions was similar to that previously observed (Table 1). At the highest level of radioactivity, 10 &i/ml, a small effect of the external radiation is evident, as the rate of inactivation is slightly faster than for the other two, which are similar. Presumably the inactivation due to external radiation for DNA stored at 2.5 and 5 pCi/ml is very small. Second, an equivalent amount of 32P as phosphoric acid (5 #Z/ml) was added to unlabeled 4e DNA. Under these conditions the DNA lost a small amount of transfecting activity (Fig. lA, Table 1). The amount of inactivation due to external radiation would in this case represent about one lethal hit per genome when all of the 32Phad decayed. These observations suggest that the inactivation curves at radioactivity levels of 2.5 and 5.0 $.X/ml could include external radiation components no greater than one lethal hit per genome. These observations show that the disintegration of incorporated 32Pis far more efficient in the inactivation of transfecting
SHORT
273
COMMUNICATIONS
DNA than in the inactivation of intact phage particles. To calculate the efficiency of inactivation (a), the number of 32Patoms/ phage must be known. Based on the specific activity of phosphorous in the growth medium (1.6 $Zi/~g) and a genome size of 120 x lo6 (9), the expected number of 32P atomslphage was 7. After the phage were purified, the calculated number was 10 (based on the amount of radioactivity per plaque-forming unit). If double-stranded transfecting DNA initiated infective center formation, one would expect the same reduction in activity that was observed for intact phage particles (50% inactivation or 0.7 lethal hit/genome). However, the number of lethal hits per genome due to 32P suicide was between 3 and 4 (Table 1, using the values for the DNA stored at 2.5 and 5 &i/ml and subtracting the maximum value of 1.0 lethal hit for such damage). Since there were an average of about four 32P atoms/DNA strand, the expected number of lethal hits would be about four if one single strand of transfecting DNA were to initiate infective center formation. The calculated (Y would thus be close to 1.0. This result provides firm evidence for the proposal that the single-stranded phage DNA found in newly transfected competent cells (9) is responsible for formation of infective centers. The precise mechanism of transfection is of special interest in interpreting the results of tranfection with heteroduplex DNA made in vitro by annealing the separated complementary strands of DNAs from mutant phages. Such studies were undertaken in the search for evidence of the recognition and correction of base pair mismatches to homozygosity (5, 20-22). Their interpretation depends on the assumption that transfecting DNA remains double stranded as it is taken up by competent cells. The existence of such recognition and correction processes has been postulated to account for meiotic gene conversion (23 >and contribute to the high negative
interference observed in phage crosses (20,21). These processes have been demonstrated in other systems (21,22,24). However, it may not be possible to detect them in B. subtilis by transfection experiments using artificially constructed heteroduplex molecules. REFERENCES
2. 3. 1. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
24. 25.
FISCHBACH, K., SPATZ, H., and KLOTZ, G., Mol. Gen. Genet. 141, 121-145 (1975). FLOCK, J., and RUTBERG, L., Mol. Gen. Genet. 131, 301-311 (1974). GREEN, D. M., J. Mol. Biol. 22, 1-13. OKUBO, S., STRAUSS, B., and STODOLSKY, M., Virology 24, 552-562 (1964). SPATZ, H., and TRAUTNER, T., Mol. Gen. Genet. 109, 84-106 (1970). GREEN, D. M., J. Mol. Biol. 10, 438-451 (1964). EPSTEIN, H., J. Viral. 2, 710-715 (1968). SPATZ, H., and TRAUTNER, T., Mol. Gen. Genet. 113, 174- 190 (1971). LOVEDAY, K. S., and Fox, M. S., Virology 85, 387-403 (1978). PORTER, R., and GUILD, W., J. Viral. 25, 60-72 (1978). DAVIDOFF-ABELSON, R., and DUBNAU, D., J. Bacterial. 116, 146-153 (1973). PIECHOWSKA, M., and Fox, M. S., J. Bacterial. 108, 681-689 (1971). PIECHOWSKA, M., SOLTYK, A., and SHUGAR, D., J. Bacterial. 122, 610-622 (1975). SOLTYK, A., SHUGAR, D., and PIECHOWSKA, M., J. Bacterial. 124, 1429-1438 (1975). TESSMAN, I., Virology 7, 263-275 (1959). STENT, G. S., and FUERST, C. R., J. Gen. Physiol. 38, 441-448 (1955). DENHARDT, D. T., and SINSHEIMER, R. T., J. Mol. Biol. 12, 663-673 (1965). KRISCH, R. E., Int. J. Radiat. Biol. 25, 261-276 (1974). SONENSHEIN, A. L., Virology 42,488-495 (1970). WHITE, R. L., and Fox, M. S., Proc. Nat. Acad. Sci. USA 71, 1544-1546 (1974). WHITE, R. L., and Fox, M. S., 1Mol. Gen. Genet. 141, 163-171 (1975). WILDENBERG, J., and MESELSON, M., Proc. Nat. Acad. Sci. USA 72, 2202-2206 (1975). HOLLIDAY, R., Genet. Res. 5, 282-287 (1964). FOX, M. S., Ann. Rev. Genet. 12, 47-68 (1978). Fox, M. S., J. Mol. Biol. 6, 85-94 (1963).