Vol. 174, No. 14
JOURNAL OF BACTERIOLOGY, JUlY 1992, p. 4850-4852
0021-9193/92/144850-03$02.00/0 Copyright X 1992, American Society for Microbiology
Interallelic Complementation of dnaE(Ts) Mutations SHARON K. BRYAN' AND ROBB E. MOSES2*
Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030,1 and Department of Molecular and Medical Genetics, Oregon Health Sciences University, 3181 S. W. Sam Jackson Park Road, L 103, Portland, Oregon 97201-30982 Received 6 January 1992/Accepted
5
May 1992
Some Escherichia coli dnaE(Ts) alleles will functionally complement in trans. The complementation is not due to copy number and is compatible with dimeric interaction.
containing the dnaE1026 allele for other functions including mutagenesis or DNA repair (Table 1). In addition, extracts showed temperature-sensitive DNA polymerase III holoenzyme activity. Plasmid pSB5 contained a dnaE allele which did not complement the dnaE(Ts) mutation in the parent strain but which could complement dnaE(Ts) functions in E486. Plasmid pSB5 also complemented strain E511 for growth at 43°C, although less well than strain E486, and functioned as well for complementation of temperature sensitivity as plasmid pDS4-26, which contains the authentic dnaE gene (15) in strains E486 and Hfr74. Therefore, the dnaE1026 mutation could complement the dnaE486 mutation, the dnaE74 mutation, and the dnaESll mutation. Thus, the complementation effect was general and not allele specific for the dnaE486-containing strain. The dnaE486 allele was isolated by the cloning of a partial chromosomal digest from E486 and selection for temperature-resistant phenotype following transformation into Hfr74 (dnaE74). The restriction endonuclease digest of the insert matched that of the authentic dnaE gene. We were unable to demonstrate complementation with the cloned dnaE486 gene transformed into strain E511. The DNA polymerase III holoenzyme activity in extracts reflected the apparent complementation in vivo. Holoenzyme extracts were prepared by the method of McHenry and Kornberg (10). By using a gapped template assay (10), DNA polymerase III holoenzyme prepared from strain E486 (dnaE486) containing pSB5 (dnaE1026) showed normal temperature resistance compared with that of holoenzyme prepared from wild-type strains (Fig. 2). This indicated that the complementation observed in vivo was reflected in the holoenzyme assembly which could be prepared from cells containing one dnaE(Ts) allele in the chromosome and a different dnaE(Ts) allele in trans. This observation is in support of the dimer hypothesis. The failure of the pSB5 clone to complement the temperature sensitivity of defects associated with the dnaE1026 allele indicated that the plasmid contained a temperaturesensitive dnaE allele. Likewise, the holoenzyme extract from CSM61 (a derivative of HS432 [12]) containing pSB5 showed temperature-sensitive DNA polymerase III activity. However, such a plasmid was capable of complementing several other dnaE(Ts) alleles in vivo and in vitro. The normal temperature resistance apparently was not due to copy number, or it would have been apparent in strains carrying the dnaE1026 allele. Complementation occurred only in heteroallelic tests. The mechanisms for the complementation observed might require interaction of the alpha-subunits of the DNA poly-
DNA polymerase III is the normal replicative enzyme in Escherichia coli. The holoenzyme consists of at least 13 components (8, 10). The alpha-subunit is the product of the dnaE (polC) gene and is the catalytic unit for synthesis. Conditionally defective mutations in this gene do not allow DNA replication (3). The dnaE gene has been cloned, and the alpha-subunit has been overproduced (6, 18). It has been proposed that the holoenzyme functions in the form of an asymmetric dimer. Such a structure would provide coordinate synthesis functions on the leading and lagging strands of DNA (1, 4, 5, 7-9, 16). We report here the interallelic complementation of several dnaE(Ts) alleles. The results suggest that there is alphadimeric-subunit interaction in DNA replication. Holoenzyme extracts prepared from cells carrying certain heterologous dnaE(Ts) alleles showed normal temperature resistance in a DNA polymerase III assay. Not all of the dnaE(Ts) allele mutations tested showed this complementation. The results reported here were noted in a search for E. coli genes which would complement dnaE defects. The genetic screen was for restoration of normal temperature resistance in a strain with a dnaE(Ts) mutation (E486 with dnaE486 [13]). Obviously a wild-type dnaE gene would complement in trans. To avoid this problem, we made a library from a strain, HS432 (poLAl polBlOO dnaEl026), containing a different dnaE(Ts) allele. After a partial HindIII digestion, we obtained several candidate clones in pBR322 which restored normal temperature resistance by transformation (11) into the dnaE486 strain (growth at 43°C). One of these, pSB5, had a 7-kb chromosomal fragment which by restriction endonuclease mapping (Fig. 1) showed a pattern identical to that reported by Welch and McHenry for the dnaE gene (18). Identification of the gene as dnaE was confirmed by the maxicell technique (14) (Fig. 1). For Southern (17) analysis (data not shown) the chromosomal portions from pDS4-26 and pMWE303 (6, 18), plasmids containing the wild-type dnaE gene, were hybridized with a probe from the AvaIAvaI portion of pSB5. The hybridization indicated homology. The conclusion was that pSB5 contained the dnaE gene from strain HS432; however, that strain contains the dnaE1026 allele. It was possible that the dnaEl026 allele had reverted during cloning procedures. However, the cloned gene did not complement temperature-sensitive growth in strain HM10 (isogenic to parent strain HS432 except with apolA12 gene). Also, the cloned gene did not complement strains *
Corresponding author. 4850
NOTES
VOL. 174, 1992
A B C
D
4851
240 220
A
200
926180
66
160
.!0 140
31 *-
21
0
>120
I
c)
14 10(
Hind III BamHI Cla I
Ava I
Hind III Ava I BamHI
8
Ava I LJ
400bp FIG. 1. Polypeptides encoded by dnaE plasmids and the restriction map. All plasmids were transformed into strain CSR603 (uvrA recA phr) (14). Analysis of plasmid-encoded proteins was then done by the maxicell technique (14). Lane A, pSB5, a dnaE1026 plasmid; lane B, pSB5A1, derivative of pSB5 with a BamHI deletion as indicated by arrows; lane C, pMWE303, wild-type dnaE structural gene in pBR322 (18); lane D, pBR322. Arrow on right side of gel indicates 140-kDa position.
TABLE 1. Complementation of dnaE(Ts) Alleles with pSB5 (dnaE1026) at 43°C Strain Allele Growtha Repairb Mutagenesisc HM10 dnaE1026 No growth H202s MMSs No mutagenesis E511 dnaESII 3/60 H202R MMSR Not done E486 dnaE486 60/60 H202R MMSR Normal mutagenesis Hfr74 dnaE74 67/75 H202R MMSR Normal mutagenesis a Growth was scored on Luria broth plates with ampicillin at 32 and 43'C. Growth is the fraction of ampicillin-resistant colonies at 32'C growing at 43'C. By using ampicillin resistance as an index, all strains shared equivalent transformation and efficiency of plating at 32'C. b R, normal survival; S, survival decreased by at least 3 orders of magnitude after 30 min of incubation with 38 mM methyl methanesulfonate (MMS) or 14 mM H202 (2). c Normal, an increase of 10-fold or more mutants per 10i survivors over background mutation frequency after 140 s of UV at 1 J/m2/s or 0.2 M ethyl methanesulfonate (2).
+NEM 0
5 10 15 20 30 Incubation Time at 430C (min)
FIG. 2. DNA polymerase III holoenzyme in vitro complementation. Holoenzyme extracts were prepared from E486 +/- pSB5, CSM61 +/- pSB5, and HMS83 (pol41 polB100) (13). A, E486 (pSB5); A, E486; 0, CSM61 (pSB5); 0, CSM61; O, HMS83. The extracts were heated at 43°C, and samples were withdrawn at various times, added to the reaction mixture, and incubated for 30 min at 30°C as previously described (12). For all extracts, 100% activity was 150 to 200 pmol. Strain E486 contains the dnaE486 allele; strain CSM61 contains the dnaEl026 allele, the same allele as in pSB5; and strain HMS83 contains wild-type dnaE. N-Ethylmaleimide was present in the assay at 6 mM. All extracts showed similar N-ethylmaleimide sensitivity to E486 containing pSB5, as shown.
merase III holoenzyme. The complementation occurred at a frequency too high for DNA recombination; the frequency of temperature-resistant cells was the same as the frequency of transformed cells, thus fulfilling a requirement for function in trans.
Not all of the dnaE(Ts) alleles tested complemented each other in trans. Specifically, the dnaE486 and the dnaESJJ
4852
J. BACTERIOL.
NOTES
alleles did not complement. Presumably, this reflects inadequate interaction of the peptides on the basis of the mutation in one or the other or both of the alleles. Our results do not require or demonstrate that both of the alpha-subunits are restored to activity. However, if alpha-subunits are required for coordinate synthesis in replication, then dimeric interaction of components of the holoenzyme is an attractive hypothesis. This work was supported by USPHS grant GM-24711.
REFERENCES 1. Alberts, B., J. Barry, P. Bedinger, T. Formosa, C. V. Jongeneel, and K. N. Kreuzer. 1983. Studies on DNA replication in the T4 bacteriophage in vitro system. Cold Spring Harbor Symp. Quant. Biol. 47:655-668. 2. Bryan, S. K., M. E. Hagensee, and R. E. Moses. 1988. DNA polymerase III is required for mutagenesis, p. 305-313. In (R. E. Moses and W. C. Summers (ed.), DNA Replication and Mutagenesis. American Society for Microbiology, Washington, D.C. 3. Gefter, N., Y. Hirota, T. Kornberg, J. Wechsler, and C. Barnoux. 1971. Analysis of DNA polymerase II and III in mutants of Eschenichia coli thermosensitive for DNA synthesis. Proc. Natl. Acad. Sci. USA 68:3152-3153. 4. Johanson, K. O., and C. S. McHenry. 1984. Adenosine 5'-O-(3Thio-triphosphate) can support the formation of an initiation complex between the DNA polymerase III holoenzyme and primed DNA. J. Biol. Chem. 259:4589-4595. 5. Maid, H., S. Maid, and A. Kornberg. 1988. DNA polymerase III holoenzyme of Escherichia coli. IV. The holoenzyme is an asymmetric dimer with twin active sites. J. Biol. Chem. 263: 6570-6578. 6. McHenry, C. S. 1982. Purification and characterization of DNA polymerase III. J. Biol. Chem. 257:2657-2663. 7. McHenry, C. S. 1985. DNA polymerase III holoenzyme of Escherichia coli: components and function of a true replicative complex. Mol. Cell. Biochem. 66:71-85.
8. McHenry, C. S. 1991. DNA polymerase III holoenzyme. J. Biol. Chem. 266:19127-19130. 9. McHenry, C. S., and K. 0. Johanson. 1984. DNA polymerase III holoenzyme of Escherichia coli: an asymmetric dimeric replicative complex containing distinguishable leading and lagging strand polymerases, p. 315-319. In U. Hubscher and S. Spadari (ed.), Proteins involved in DNA replication. Plenum Press, New York. 10. McHenry, C. S., and A. Kornberg. 1977. DNA polymerase III holoenzyme of Escherichia coli: purification and resolution into subunits. J. Biol. Chem. 252:6478-6484. 11. Morrison, D. A. 1977. Transformation of Escherichia coli: cryogenic preservation of competent cells. J. Bacteriol. 132: 349-351. 12. Niwa, O., S. K. Bryan, and R. E. Moses. 1979. Replication at restrictive temperatures in Escherichia coli containing a polCts mutation. Proc. Natl. Acad. Sci. USA 76:5572-5576. 13. Niwa, O., S. K Bryan, and R. E. Moses. 1981. Alternative pathways of DNA replication: DNA polymerase I-dependent replication. Proc. Natl. Acad. Sci. USA 78:7024-7027. 14. Sancar, A., A. M. Hack, and W. D. Rupp. 1979. Simple method for identification of plasmid-coded proteins. J. Bacteriol. 137: 692-693. 15. Shepard, D., R. W. Oberfelder, M. M. Welch, and C. S. McHenry. 1984. Determination of the precise location and orientation of the Escherichia coli dnaE gene. J. Bacteriol. 158:455-459. 16. Sinha, N. K., C. F. Morris, and B. M. Alberts. 1980. Efficient in vitro replication of double-stranded DNA templates by a purified T4 bacteriophage replication system. J. Biol. Chem. 255:
4290-4303. 17. Southern, E. 1979. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-509. 18. Welch, M., and S. S. McHenry. 1982. Cloning and identification of the product of dnaE of Eschenichia coli. J. Bacteriol. 152: 351-356.
JOURNAL OF BACTERIOLOGY, JUlY 1992, p. 4850-4852
0021-9193/92/144850-03$02.00/0 Copyright X 1992, American Society for Microbiology
Interallelic Complementation of dnaE(Ts) Mutations SHARON K. BRYAN' AND ROBB E. MOSES2*
Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030,1 and Department of Molecular and Medical Genetics, Oregon Health Sciences University, 3181 S. W. Sam Jackson Park Road, L 103, Portland, Oregon 97201-30982 Received 6 January 1992/Accepted
5
May 1992
Some Escherichia coli dnaE(Ts) alleles will functionally complement in trans. The complementation is not due to copy number and is compatible with dimeric interaction.
containing the dnaE1026 allele for other functions including mutagenesis or DNA repair (Table 1). In addition, extracts showed temperature-sensitive DNA polymerase III holoenzyme activity. Plasmid pSB5 contained a dnaE allele which did not complement the dnaE(Ts) mutation in the parent strain but which could complement dnaE(Ts) functions in E486. Plasmid pSB5 also complemented strain E511 for growth at 43°C, although less well than strain E486, and functioned as well for complementation of temperature sensitivity as plasmid pDS4-26, which contains the authentic dnaE gene (15) in strains E486 and Hfr74. Therefore, the dnaE1026 mutation could complement the dnaE486 mutation, the dnaE74 mutation, and the dnaESll mutation. Thus, the complementation effect was general and not allele specific for the dnaE486-containing strain. The dnaE486 allele was isolated by the cloning of a partial chromosomal digest from E486 and selection for temperature-resistant phenotype following transformation into Hfr74 (dnaE74). The restriction endonuclease digest of the insert matched that of the authentic dnaE gene. We were unable to demonstrate complementation with the cloned dnaE486 gene transformed into strain E511. The DNA polymerase III holoenzyme activity in extracts reflected the apparent complementation in vivo. Holoenzyme extracts were prepared by the method of McHenry and Kornberg (10). By using a gapped template assay (10), DNA polymerase III holoenzyme prepared from strain E486 (dnaE486) containing pSB5 (dnaE1026) showed normal temperature resistance compared with that of holoenzyme prepared from wild-type strains (Fig. 2). This indicated that the complementation observed in vivo was reflected in the holoenzyme assembly which could be prepared from cells containing one dnaE(Ts) allele in the chromosome and a different dnaE(Ts) allele in trans. This observation is in support of the dimer hypothesis. The failure of the pSB5 clone to complement the temperature sensitivity of defects associated with the dnaE1026 allele indicated that the plasmid contained a temperaturesensitive dnaE allele. Likewise, the holoenzyme extract from CSM61 (a derivative of HS432 [12]) containing pSB5 showed temperature-sensitive DNA polymerase III activity. However, such a plasmid was capable of complementing several other dnaE(Ts) alleles in vivo and in vitro. The normal temperature resistance apparently was not due to copy number, or it would have been apparent in strains carrying the dnaE1026 allele. Complementation occurred only in heteroallelic tests. The mechanisms for the complementation observed might require interaction of the alpha-subunits of the DNA poly-
DNA polymerase III is the normal replicative enzyme in Escherichia coli. The holoenzyme consists of at least 13 components (8, 10). The alpha-subunit is the product of the dnaE (polC) gene and is the catalytic unit for synthesis. Conditionally defective mutations in this gene do not allow DNA replication (3). The dnaE gene has been cloned, and the alpha-subunit has been overproduced (6, 18). It has been proposed that the holoenzyme functions in the form of an asymmetric dimer. Such a structure would provide coordinate synthesis functions on the leading and lagging strands of DNA (1, 4, 5, 7-9, 16). We report here the interallelic complementation of several dnaE(Ts) alleles. The results suggest that there is alphadimeric-subunit interaction in DNA replication. Holoenzyme extracts prepared from cells carrying certain heterologous dnaE(Ts) alleles showed normal temperature resistance in a DNA polymerase III assay. Not all of the dnaE(Ts) allele mutations tested showed this complementation. The results reported here were noted in a search for E. coli genes which would complement dnaE defects. The genetic screen was for restoration of normal temperature resistance in a strain with a dnaE(Ts) mutation (E486 with dnaE486 [13]). Obviously a wild-type dnaE gene would complement in trans. To avoid this problem, we made a library from a strain, HS432 (poLAl polBlOO dnaEl026), containing a different dnaE(Ts) allele. After a partial HindIII digestion, we obtained several candidate clones in pBR322 which restored normal temperature resistance by transformation (11) into the dnaE486 strain (growth at 43°C). One of these, pSB5, had a 7-kb chromosomal fragment which by restriction endonuclease mapping (Fig. 1) showed a pattern identical to that reported by Welch and McHenry for the dnaE gene (18). Identification of the gene as dnaE was confirmed by the maxicell technique (14) (Fig. 1). For Southern (17) analysis (data not shown) the chromosomal portions from pDS4-26 and pMWE303 (6, 18), plasmids containing the wild-type dnaE gene, were hybridized with a probe from the AvaIAvaI portion of pSB5. The hybridization indicated homology. The conclusion was that pSB5 contained the dnaE gene from strain HS432; however, that strain contains the dnaE1026 allele. It was possible that the dnaEl026 allele had reverted during cloning procedures. However, the cloned gene did not complement temperature-sensitive growth in strain HM10 (isogenic to parent strain HS432 except with apolA12 gene). Also, the cloned gene did not complement strains *
Corresponding author. 4850
NOTES
VOL. 174, 1992
A B C
D
4851
240 220
A
200
926180
66
160
.!0 140
31 *-
21
0
>120
I
c)
14 10(
Hind III BamHI Cla I
Ava I
Hind III Ava I BamHI
8
Ava I LJ
400bp FIG. 1. Polypeptides encoded by dnaE plasmids and the restriction map. All plasmids were transformed into strain CSR603 (uvrA recA phr) (14). Analysis of plasmid-encoded proteins was then done by the maxicell technique (14). Lane A, pSB5, a dnaE1026 plasmid; lane B, pSB5A1, derivative of pSB5 with a BamHI deletion as indicated by arrows; lane C, pMWE303, wild-type dnaE structural gene in pBR322 (18); lane D, pBR322. Arrow on right side of gel indicates 140-kDa position.
TABLE 1. Complementation of dnaE(Ts) Alleles with pSB5 (dnaE1026) at 43°C Strain Allele Growtha Repairb Mutagenesisc HM10 dnaE1026 No growth H202s MMSs No mutagenesis E511 dnaESII 3/60 H202R MMSR Not done E486 dnaE486 60/60 H202R MMSR Normal mutagenesis Hfr74 dnaE74 67/75 H202R MMSR Normal mutagenesis a Growth was scored on Luria broth plates with ampicillin at 32 and 43'C. Growth is the fraction of ampicillin-resistant colonies at 32'C growing at 43'C. By using ampicillin resistance as an index, all strains shared equivalent transformation and efficiency of plating at 32'C. b R, normal survival; S, survival decreased by at least 3 orders of magnitude after 30 min of incubation with 38 mM methyl methanesulfonate (MMS) or 14 mM H202 (2). c Normal, an increase of 10-fold or more mutants per 10i survivors over background mutation frequency after 140 s of UV at 1 J/m2/s or 0.2 M ethyl methanesulfonate (2).
+NEM 0
5 10 15 20 30 Incubation Time at 430C (min)
FIG. 2. DNA polymerase III holoenzyme in vitro complementation. Holoenzyme extracts were prepared from E486 +/- pSB5, CSM61 +/- pSB5, and HMS83 (pol41 polB100) (13). A, E486 (pSB5); A, E486; 0, CSM61 (pSB5); 0, CSM61; O, HMS83. The extracts were heated at 43°C, and samples were withdrawn at various times, added to the reaction mixture, and incubated for 30 min at 30°C as previously described (12). For all extracts, 100% activity was 150 to 200 pmol. Strain E486 contains the dnaE486 allele; strain CSM61 contains the dnaEl026 allele, the same allele as in pSB5; and strain HMS83 contains wild-type dnaE. N-Ethylmaleimide was present in the assay at 6 mM. All extracts showed similar N-ethylmaleimide sensitivity to E486 containing pSB5, as shown.
merase III holoenzyme. The complementation occurred at a frequency too high for DNA recombination; the frequency of temperature-resistant cells was the same as the frequency of transformed cells, thus fulfilling a requirement for function in trans.
Not all of the dnaE(Ts) alleles tested complemented each other in trans. Specifically, the dnaE486 and the dnaESJJ
4852
J. BACTERIOL.
NOTES
alleles did not complement. Presumably, this reflects inadequate interaction of the peptides on the basis of the mutation in one or the other or both of the alleles. Our results do not require or demonstrate that both of the alpha-subunits are restored to activity. However, if alpha-subunits are required for coordinate synthesis in replication, then dimeric interaction of components of the holoenzyme is an attractive hypothesis. This work was supported by USPHS grant GM-24711.
REFERENCES 1. Alberts, B., J. Barry, P. Bedinger, T. Formosa, C. V. Jongeneel, and K. N. Kreuzer. 1983. Studies on DNA replication in the T4 bacteriophage in vitro system. Cold Spring Harbor Symp. Quant. Biol. 47:655-668. 2. Bryan, S. K., M. E. Hagensee, and R. E. Moses. 1988. DNA polymerase III is required for mutagenesis, p. 305-313. In (R. E. Moses and W. C. Summers (ed.), DNA Replication and Mutagenesis. American Society for Microbiology, Washington, D.C. 3. Gefter, N., Y. Hirota, T. Kornberg, J. Wechsler, and C. Barnoux. 1971. Analysis of DNA polymerase II and III in mutants of Eschenichia coli thermosensitive for DNA synthesis. Proc. Natl. Acad. Sci. USA 68:3152-3153. 4. Johanson, K. O., and C. S. McHenry. 1984. Adenosine 5'-O-(3Thio-triphosphate) can support the formation of an initiation complex between the DNA polymerase III holoenzyme and primed DNA. J. Biol. Chem. 259:4589-4595. 5. Maid, H., S. Maid, and A. Kornberg. 1988. DNA polymerase III holoenzyme of Escherichia coli. IV. The holoenzyme is an asymmetric dimer with twin active sites. J. Biol. Chem. 263: 6570-6578. 6. McHenry, C. S. 1982. Purification and characterization of DNA polymerase III. J. Biol. Chem. 257:2657-2663. 7. McHenry, C. S. 1985. DNA polymerase III holoenzyme of Escherichia coli: components and function of a true replicative complex. Mol. Cell. Biochem. 66:71-85.
8. McHenry, C. S. 1991. DNA polymerase III holoenzyme. J. Biol. Chem. 266:19127-19130. 9. McHenry, C. S., and K. 0. Johanson. 1984. DNA polymerase III holoenzyme of Escherichia coli: an asymmetric dimeric replicative complex containing distinguishable leading and lagging strand polymerases, p. 315-319. In U. Hubscher and S. Spadari (ed.), Proteins involved in DNA replication. Plenum Press, New York. 10. McHenry, C. S., and A. Kornberg. 1977. DNA polymerase III holoenzyme of Escherichia coli: purification and resolution into subunits. J. Biol. Chem. 252:6478-6484. 11. Morrison, D. A. 1977. Transformation of Escherichia coli: cryogenic preservation of competent cells. J. Bacteriol. 132: 349-351. 12. Niwa, O., S. K. Bryan, and R. E. Moses. 1979. Replication at restrictive temperatures in Escherichia coli containing a polCts mutation. Proc. Natl. Acad. Sci. USA 76:5572-5576. 13. Niwa, O., S. K Bryan, and R. E. Moses. 1981. Alternative pathways of DNA replication: DNA polymerase I-dependent replication. Proc. Natl. Acad. Sci. USA 78:7024-7027. 14. Sancar, A., A. M. Hack, and W. D. Rupp. 1979. Simple method for identification of plasmid-coded proteins. J. Bacteriol. 137: 692-693. 15. Shepard, D., R. W. Oberfelder, M. M. Welch, and C. S. McHenry. 1984. Determination of the precise location and orientation of the Escherichia coli dnaE gene. J. Bacteriol. 158:455-459. 16. Sinha, N. K., C. F. Morris, and B. M. Alberts. 1980. Efficient in vitro replication of double-stranded DNA templates by a purified T4 bacteriophage replication system. J. Biol. Chem. 255:
4290-4303. 17. Southern, E. 1979. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-509. 18. Welch, M., and S. S. McHenry. 1982. Cloning and identification of the product of dnaE of Eschenichia coli. J. Bacteriol. 152: 351-356.
