Molec. gen. Genet. 164, 285-288 (1978) © by Springer-Verlag 1978
Restriction Endonuclease Mapping of pSC101 and pMB9 Robert C. Tait and Herbert W. Boyer Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143, USA
Summary. A restriction endonuclease analysis of the plasmids pSC101 and pMB9 has allowed a determination of the alterations that occurred in the tetracycline resistance locus during the construction of pMB9 from pSC101. The genes for four of the polypeptides involved in tetracycline resistance have been positioned on the restriction endonuclease map of pSC101.
the plasmid pBR345 (Bolivar et al., 1977c) joined to the EcoRI cleavage site of pSC101, pBRI45 DNA can be amplified by the addition of 170 gg/ml chloramphenicol during the logarithmic growth of cells (Clewell, 1972) to obtain 1-2 mg plasmid DNA per liter of culture. This plasmid allowed the preparation of sufficient amounts of pSC101 DNA for the isolation of specific restriction endonuclease cleavage fragments by the use of preparative polyacrylamide gel electrophoresis. The preparation of covalently closed circular pBR145 and pMB9 DNA was according to Bolivar et al., 1977a.
Enzymes
Introduction
During the construction of the plasmid pMB9, the replication properties of pMB8 were combined with a tetracycline (Tc) resistance derived from pSC101 by the use of the EcoRI* enzymatic activity (Rodriguez et al., 1976). pMB9 was subsequently used in the construction of other plasmids, including the molecular cloning vehicles pBR313 (Bolivar et al., 1977 a) and pBR322 (Bolivar et al., 1977b). Investigation of the Tc resistance generated by pSC101 and pMB9 in Escherichia coli revealed that only a portion of the resistance mechanism encoded for by pSC101 was encoded for by pMB9 (Tait and Boyer, 1978). A restriction endonuclease analysis of pSC101 and the Tc resistance locus of pMB9 has provided insight concerning the restriction endonuclease fragment rearrangement of pMB9 and allowed the localization on pSC101 of the genes for three of the proteins involved in Tc resistance.
The restriction endonucleases used were purified according to Greene et al. (1978). The reaction conditions used for the digestion of DNA were according to Bolivar et al. (1977a).
Gel Electrophoresis Agarose and polyacrylamide gel electrophoresis was performed according to Bolivar et al. (1977a). Preparative polyacrylamide gel electrophoresis was performed using 3% polyacrylamide gels with very brief staining of gels with ethidium bromide. DNA fragments were visualized with ultraviolet light and recovered from the polyacrylamide by excising and mincing regions of the gel containing desired DNA fragments. The minced gel was incubated at 65°C for 12 h with an equal volume of 0.5 M ammonium acetate, 10 mM magnesium acetate, 0.1 mM EDTA, 0.1% sodium dodecyl sulfate. Gel fragments were removed by filtration or centrifngation (Sorvall SS34 rotor, 10 krpm, 15 min, 4° C). Traces of ethidium bromide were removed by extraction with butanol, followed by the addition of 2 volumes of ethanol at - 2 0 ° C to precipitate the DNA. The precipitate was collected by centrifugation, resuspended in 50 100 ~tl of buffer and dialyzed prior to further analysis of the DNA fragments. Typical recoveries of DNA fragments of 1-3 megadaltons were 60-90%.
Materials and Methods Results Preparation of Plasmid DNA The plasmid pBR145, constructed by Dr. R.L. Rodriguez, was used as the source of pSC101 DNA. This plasmid consists of
For offprints contact: R.C. Tait
Digestion of pBR145 DNA with EcoRI endonuclease generated linear pSC101 of 5.8 megadaltons and linear pBR345 of 0.78 magedaltons, which can be seen in Figure 1 a. Further digestion with HpaI converted
0026-8925/78/0164/0285/$01.00
286
R.C. Tait and H.W. Boyer: Restriction Endonuclease Mapping of pSC101 and pMB9
Fig. 1 a-f. Restriction endonuclease mapping of pSC101 and pMB9
DNA. The plasmids pBR145 and pMB9 were digested with various restriction enzymes and analyzed by electrophoresis on a 1% agarose gel. a pBR145 digested with EcoRI and HpaI. b pBR145 digested with EcoRI, HpaI and HsuI, c the 1.85 megadalton EcoRIHpaI fragment of pSC101 purified and partially digested with HincII, d pMB9 digested with EcoRI and HpaI, e pMB9 digested with EcoRI, HpaI and HsuI, f pMB9 digested with HincII, s, molecular weight standards, EcoRI digested 2 DNA with HindIII digested SV40 DNA
the linear pSC101 D N A to fragments of 4.0 and 1.85 megadaltons, also shown in Figure 1 a. Digestion of pMB9 D N A with EcoRI generated a D N A fragment of 3.2 megadaltons, while further digestion with HpaI generated D N A fragments of 1.85 and 1.4 megadaltons, shown in Figure 1 d. It was demonstrated that the 1.85 megadalton EcoRI-HpaI D N A fragments of pSC101 and pMB9 were not identical by digestion of the fragments with HsuI. As is shown in Figure 1 b, digestion with HsuI did not cause any detectable alteration in the molecular weight of the EcoRI-HpaI pSC101 D N A fragment. This is consistent with the observation that the EeoRI site and the HsuI site of pSC10I are only 30 base pairs (0.019 megadaltons) apart (Rodriguez et al., 1977). However, as is shown in Figure 1 e, digestion of pMB9 D N A with HsuI reduced the size of the 1.85 megadalton D N A fragment to 1.65 megadaltons, suggesting that the HsuI site of pMB9 is 0.2 megadaltons from either the EcoRI site or the HpaI site. The 1.85 megadalton EcoRI-HpaI pSC101 D N A fragment was purified and digested with HincII. As
Fig. 2a-d. Analysis of the EcoRI-HpaI DNA fragments of pSC101 and pMB9. The 1.85 megadalton EcoRI-HpaI fragment of pSC101 and the HincII fragments of pMB9 were recoveredfrom preparative polyacrylamidegels, further digested, and analyzed on a 5% polyacrylamide gel. s, molecular weight standards, a pMB9 digested with HincII, b EcoRI-HpaI fragment of pSC101 digested with HincII, c EcoRI-HpaI fragment of pSC101 digested with SalI, d large HincII fragment of pMB9 digested with EcoRI
shown in Figure 2b, this D N A fragment contained two HincII cleavage sites, one of which was also recognized by Sal!, shown in Figure 2c. A partial HineII digest of this D N A fragment, shown in Figure 1 c, was used to determine the positions of these sites relative to the EcoRI site of pSC101. Digestion of pMB9 D N A with HincII, shown in F i g u r e s l f and 2a, also revealed the presence of two HincII cleavage sites, but the resulting D N A fragments were of different molecular weights than those generated by digestion of pSC101 with HincII. The two HincII generated pMB9 D N A fragments were purified and further analyzed. It was found that the larger HincII fragment contained the EcoRI site of pMB9, as shown in Figure 2d, at a distance of 0.52 megadaltons from the HincII site that was also recognized by SalI. The distance from the EcoRI site to the SalI site in pSC101 was found to be 0.37 megadaltons, suggesting that during the construction of pMB9, 0.15 megadaltons of D N A was added between the EcoRI and SalI sites of pSC101, pSC101 contained a HincII site between the SalI and HpaI sites,
287
R.C. Tait and H.W. Boyer: Restriction Endonuclease Mapping of pSC101 and pMB9 pSCIOI
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Fig. 3. EcoRI* mapping of pSC101 and pMB9. Electrophoresis of DNA was on a 5% polyacrylamide gel. s, molecular weight standards, a EcoRI-HpaI fragment of pSC101 digested with EcoRI*, b EcoRI-HpaI fragment of pSC101 digested with EcoRI* and SaII, c EcoRI* digest of pMB9, d large HincII fragment of pMB9 digested with EcoRI* e small HincII fragment of pMB9 digested with EcoRI* but pMB9 contained only two HincII sites, one recognized by SalI and one by HpaI. This suggested that during the construction of pMB9 from pSC101, a D N A fragment containing this HincII site was deleted from between the SalI and HpaI sites of pSC101. Because the distance from the EcoRI site to the HpaI site is the same in both plasmids and because it has been shown that 0.15-0.18 megadaltons of D N A was added to pMB9 between the EcoRI and SalI sites of pSC101, the deletion of approximately 0.17 megadaltons of D N A including the HinclI site between the SalI site and the HpaI site of pSC101 should have been involved in the construction of pMB9. To test this hypothesis, the EcoRI-HpaI fragment of pSC101 and the HincII fragments of pMB9 were purified and digested with EcoRI* activity. The EcoRI* fragments of pSC101, shown in Figure 3a, revealed a minor D N A fragment of 0.21 megadaltons and major D N A fragments of 0.14 and approximately 0.06 megadaltons that could not be detected in either of the HincII fragments of pMB9, shown in Figures 3d and 3e. Because of the difficulties in obtaining complete EcoRI* digestions, it is probable that the 0.14 and the 0.06 megadalton D N A fragments together form the 0.21 megadalton D N A fragment that was deleted during the construction of pMB9 with the use of the EcoRI* activity.
Fig. 4. Restriction map of the Tc resistance locus of pSC101 and pMBg. The cleavage sites for a variety of restriction endonucleases have been indicated on the 1.85 megadalton EcoRI-HpaI DNA fragments from pSC101 and pMB9. EcoRI* sites are indicated by *. At least four more major EcoRI* sites occur in both plasmids between the SalI and HpaI sites. Although these sites could not be positioned precisely, digestions with the enzyme AluI suggested that linear sequence of the EcoRI* fragments is the same in the two plasmids, with the exception of the deletion of the 0.21 megadalton fragment containing the HincII site of pSC101 and the 0.17 megadalton fragment that was added to the EeoRI site of pSC101
The results of the restriction endonuclease mapping of the 1.85 megadalton EcoRI-ItpaI D N A fragments of pSC101 and pMB9 have been summarized in Figure 4. During the construction of pMB9, a 0.17 megadalton EcoRI* generated D N A fragment was added to the EcoRI site of pSC101, converting it to an EcoRI* site and generating an EcoRI site 0.17 megadaltons away from the original EcoRI site. In addition, an EcoRI* generated D N A fragment of 0.21 megadaltons containing an EcoRI* site and a HincII site was deleted from pSC101.
Discussion Previous investigation has implicated polypeptides of 34,000, 26,000, 18,000 and 14,000 daltons in the generation of Tc resistance by pSC101 (Tait and Boyer, 1978). Insertion of D N A at the EcoRI site of pSC101 has been found to cause apparent alterations in the molecular weight of the 14,000 dalton polypeptide, suggesting that the EcoRI site occurs in the carboxyterminal region of the gene for the 14,000 dalton polypeptide (Meagher et al., 1977). The deletion from pSC101 of the region from the HpaI site to the BamHI site prevented the synthesis in minicells of the polypeptides of 34,000, 26,000 and 18,000 daltons, suggesting that the genes for these polypeptides are contained within this D N A fragment (Tait and Boyer, 1978). The insertion of D N A in the B a m H I site was observed to reduce the size of the 34,000 dalton poly-
288
R.C. Tait and H.W. Boyer: Restriction Endonuclease Mapping of pSC101 and pMB9
References
E~S!U~l ]l i HIal HIn~l,Hin~llUHi]¢ll Hi~cllHinl¢ll HincllI
Hinlc II
O. of Rep.
0
I
2
~
4
5
5.8
MegadaJtons
Fig. 5. Map of the Tc resistance locus of pSCI0I. The genes for four polypeptides involved in Tc resistance have been positioned on the restriction map of pSC101. The approximate origin of replication as determined by Cabetlo, Timmis and Cohen (1976) is indicated by 0. of Rep
peptide (Rambach and Hogness, 1977). Minicells containing pMB9 synthesized the 34,000 and 18,000 dalton polypeptides but not the 26,000 dalton polypeptide (Tait and Boyer, 1978). In addition, during the construction of pBR322 from pMB9, 0.6 megadaltons of DNA was deleted from the HpaI site of pMB9 towards the SalI site (Bolivar et al., 1977b). Minicells containing pBR322 synthesize both the 34,000 and the 18,000 dalton polypeptides (Covarrubias et al., 1978). Combined, these results indicate that the gene for the 34,000 dalton polypeptide is interrupted by the BamHI site, with the gene for the 18,000 dalton polypeptide adjacent to the gene for the 34,000 dalton polypeptide. The gene for the 26,000 dalton polypeptide or information necessary for its expression should then exist in the 0.21 megadalton DNA fragment that was deleted during the construction of pMB9. Using a molecular weight average of 110 daltons per amino acid residue, the approximate sizes of these genes have been calculated and the positions of the genes indicated on the restriction map of pSC101 shown in Figure 5. Acknowledgements. This work was supported by a grant from the National Science Foundation (PCM7510468). H.W.B. is an Investigator for the Howard Hughes Medical Institute.
Bolivar, F., Rodriguez, R.L., Betlach, M.C., Boyer, H.W.: Construction and characterization of new cloning vehicles : I. Ampicillin resistant derivatives of the plasmid pMB9. Gene 2, 75-93 (1977a) Bolivar, F., R0driguez, R.L., Greene, P.J., Betlach, M.C., Heyneker, H.L., Boyer, H.W., Crosa, J.H., Falkow, S.: Construction and characterization of new cloning vehicles: II. A multipurpose cloning system. Gene 2, 95-113 (1977b) Bolivar, F., Betlach, M.C., Heyneker, H.L., Shine, J., Rodriguez, R.L., Boyer, H.W.: Origin of replication of pBR345 plasmid DNA. Proc. nat. Acad. Sci. (Wash.) 74, 5265-5269 (1977c) Cabello, F., Timmis, K., Cohen, S.N.: Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons. Nature (Lond.) 259, 285-290 (1976) Clewell, D.B. : Nature of Cole 1 plasmid replication in the presence of chloramphenicol. J. Bact. 110, 667 676 (1972) Covarrubias, A.A., Tait, R.C., Bolivar, F.G., Boyer, H.W., to be submitted to Molec. gen. Genet. Greene, P.J., Heyneker, H.L., Bolivar, F., Rodriguez, R.L., Betlach, M.C., Corvarrubias, A.A., Backman, K., Russel, D.J., Tait, R., Boyer, H.W.: A general method for the purification of restriction enzymes. Submitted to Nuc. Acids Res. Meagher, R.B., Tait, R.C., Betlach, M., Boyer, H.W.: Protein expression in E. coli minicells by recombinant plasmids. Cell 10, 521-536 (1977) Rambach, A., Hogness, D.S. : Translation of Drosophila melanogaster sequences in Escheriehia eoli. Proc. nat. Acad. Sci. (Wash.) 74, 5041-5045 (1977) Rodriguez, R.L., Bolivar, F., Goodman, H.M., Boyer, H.W., Betlach, M.C. : Construction and characterization of cloning vehicles. In: Molecular mechanisms in the control of gene expression (eds. D.P. Nierlich, W.J. Rutter and C.F. Fox.), pp. 471-478. New York: Academic Press 1976 Rodriguez, R.L., Tait, R.C., Shine, J., Bolivar, F., Heyneker, H., Betlach, M., Boyer, H.W. : Characterization of tetracycline and ampicillin resistant plasmid cloning vehicles. In: Molecular cloning of recombinant DNA (eds. W.A. Scott and R. Werner), pp. 73-84. New York: Academic Press 1977 Tait, R.C., Boyer, H.W.: On the nature of tetracycline resistance controlled by the plasmid pSC101. Cell 13, 73-81 (1978)
Communicated by W. Arber Received May 17, 1978
Restriction Endonuclease Mapping of pSC101 and pMB9 Robert C. Tait and Herbert W. Boyer Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143, USA
Summary. A restriction endonuclease analysis of the plasmids pSC101 and pMB9 has allowed a determination of the alterations that occurred in the tetracycline resistance locus during the construction of pMB9 from pSC101. The genes for four of the polypeptides involved in tetracycline resistance have been positioned on the restriction endonuclease map of pSC101.
the plasmid pBR345 (Bolivar et al., 1977c) joined to the EcoRI cleavage site of pSC101, pBRI45 DNA can be amplified by the addition of 170 gg/ml chloramphenicol during the logarithmic growth of cells (Clewell, 1972) to obtain 1-2 mg plasmid DNA per liter of culture. This plasmid allowed the preparation of sufficient amounts of pSC101 DNA for the isolation of specific restriction endonuclease cleavage fragments by the use of preparative polyacrylamide gel electrophoresis. The preparation of covalently closed circular pBR145 and pMB9 DNA was according to Bolivar et al., 1977a.
Enzymes
Introduction
During the construction of the plasmid pMB9, the replication properties of pMB8 were combined with a tetracycline (Tc) resistance derived from pSC101 by the use of the EcoRI* enzymatic activity (Rodriguez et al., 1976). pMB9 was subsequently used in the construction of other plasmids, including the molecular cloning vehicles pBR313 (Bolivar et al., 1977 a) and pBR322 (Bolivar et al., 1977b). Investigation of the Tc resistance generated by pSC101 and pMB9 in Escherichia coli revealed that only a portion of the resistance mechanism encoded for by pSC101 was encoded for by pMB9 (Tait and Boyer, 1978). A restriction endonuclease analysis of pSC101 and the Tc resistance locus of pMB9 has provided insight concerning the restriction endonuclease fragment rearrangement of pMB9 and allowed the localization on pSC101 of the genes for three of the proteins involved in Tc resistance.
The restriction endonucleases used were purified according to Greene et al. (1978). The reaction conditions used for the digestion of DNA were according to Bolivar et al. (1977a).
Gel Electrophoresis Agarose and polyacrylamide gel electrophoresis was performed according to Bolivar et al. (1977a). Preparative polyacrylamide gel electrophoresis was performed using 3% polyacrylamide gels with very brief staining of gels with ethidium bromide. DNA fragments were visualized with ultraviolet light and recovered from the polyacrylamide by excising and mincing regions of the gel containing desired DNA fragments. The minced gel was incubated at 65°C for 12 h with an equal volume of 0.5 M ammonium acetate, 10 mM magnesium acetate, 0.1 mM EDTA, 0.1% sodium dodecyl sulfate. Gel fragments were removed by filtration or centrifngation (Sorvall SS34 rotor, 10 krpm, 15 min, 4° C). Traces of ethidium bromide were removed by extraction with butanol, followed by the addition of 2 volumes of ethanol at - 2 0 ° C to precipitate the DNA. The precipitate was collected by centrifugation, resuspended in 50 100 ~tl of buffer and dialyzed prior to further analysis of the DNA fragments. Typical recoveries of DNA fragments of 1-3 megadaltons were 60-90%.
Materials and Methods Results Preparation of Plasmid DNA The plasmid pBR145, constructed by Dr. R.L. Rodriguez, was used as the source of pSC101 DNA. This plasmid consists of
For offprints contact: R.C. Tait
Digestion of pBR145 DNA with EcoRI endonuclease generated linear pSC101 of 5.8 megadaltons and linear pBR345 of 0.78 magedaltons, which can be seen in Figure 1 a. Further digestion with HpaI converted
0026-8925/78/0164/0285/$01.00
286
R.C. Tait and H.W. Boyer: Restriction Endonuclease Mapping of pSC101 and pMB9
Fig. 1 a-f. Restriction endonuclease mapping of pSC101 and pMB9
DNA. The plasmids pBR145 and pMB9 were digested with various restriction enzymes and analyzed by electrophoresis on a 1% agarose gel. a pBR145 digested with EcoRI and HpaI. b pBR145 digested with EcoRI, HpaI and HsuI, c the 1.85 megadalton EcoRIHpaI fragment of pSC101 purified and partially digested with HincII, d pMB9 digested with EcoRI and HpaI, e pMB9 digested with EcoRI, HpaI and HsuI, f pMB9 digested with HincII, s, molecular weight standards, EcoRI digested 2 DNA with HindIII digested SV40 DNA
the linear pSC101 D N A to fragments of 4.0 and 1.85 megadaltons, also shown in Figure 1 a. Digestion of pMB9 D N A with EcoRI generated a D N A fragment of 3.2 megadaltons, while further digestion with HpaI generated D N A fragments of 1.85 and 1.4 megadaltons, shown in Figure 1 d. It was demonstrated that the 1.85 megadalton EcoRI-HpaI D N A fragments of pSC101 and pMB9 were not identical by digestion of the fragments with HsuI. As is shown in Figure 1 b, digestion with HsuI did not cause any detectable alteration in the molecular weight of the EcoRI-HpaI pSC101 D N A fragment. This is consistent with the observation that the EeoRI site and the HsuI site of pSC10I are only 30 base pairs (0.019 megadaltons) apart (Rodriguez et al., 1977). However, as is shown in Figure 1 e, digestion of pMB9 D N A with HsuI reduced the size of the 1.85 megadalton D N A fragment to 1.65 megadaltons, suggesting that the HsuI site of pMB9 is 0.2 megadaltons from either the EcoRI site or the HpaI site. The 1.85 megadalton EcoRI-HpaI pSC101 D N A fragment was purified and digested with HincII. As
Fig. 2a-d. Analysis of the EcoRI-HpaI DNA fragments of pSC101 and pMB9. The 1.85 megadalton EcoRI-HpaI fragment of pSC101 and the HincII fragments of pMB9 were recoveredfrom preparative polyacrylamidegels, further digested, and analyzed on a 5% polyacrylamide gel. s, molecular weight standards, a pMB9 digested with HincII, b EcoRI-HpaI fragment of pSC101 digested with HincII, c EcoRI-HpaI fragment of pSC101 digested with SalI, d large HincII fragment of pMB9 digested with EcoRI
shown in Figure 2b, this D N A fragment contained two HincII cleavage sites, one of which was also recognized by Sal!, shown in Figure 2c. A partial HineII digest of this D N A fragment, shown in Figure 1 c, was used to determine the positions of these sites relative to the EcoRI site of pSC101. Digestion of pMB9 D N A with HincII, shown in F i g u r e s l f and 2a, also revealed the presence of two HincII cleavage sites, but the resulting D N A fragments were of different molecular weights than those generated by digestion of pSC101 with HincII. The two HincII generated pMB9 D N A fragments were purified and further analyzed. It was found that the larger HincII fragment contained the EcoRI site of pMB9, as shown in Figure 2d, at a distance of 0.52 megadaltons from the HincII site that was also recognized by SalI. The distance from the EcoRI site to the SalI site in pSC101 was found to be 0.37 megadaltons, suggesting that during the construction of pMB9, 0.15 megadaltons of D N A was added between the EcoRI and SalI sites of pSC101, pSC101 contained a HincII site between the SalI and HpaI sites,
287
R.C. Tait and H.W. Boyer: Restriction Endonuclease Mapping of pSC101 and pMB9 pSCIOI
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Fig. 3. EcoRI* mapping of pSC101 and pMB9. Electrophoresis of DNA was on a 5% polyacrylamide gel. s, molecular weight standards, a EcoRI-HpaI fragment of pSC101 digested with EcoRI*, b EcoRI-HpaI fragment of pSC101 digested with EcoRI* and SaII, c EcoRI* digest of pMB9, d large HincII fragment of pMB9 digested with EcoRI* e small HincII fragment of pMB9 digested with EcoRI* but pMB9 contained only two HincII sites, one recognized by SalI and one by HpaI. This suggested that during the construction of pMB9 from pSC101, a D N A fragment containing this HincII site was deleted from between the SalI and HpaI sites of pSC101. Because the distance from the EcoRI site to the HpaI site is the same in both plasmids and because it has been shown that 0.15-0.18 megadaltons of D N A was added to pMB9 between the EcoRI and SalI sites of pSC101, the deletion of approximately 0.17 megadaltons of D N A including the HinclI site between the SalI site and the HpaI site of pSC101 should have been involved in the construction of pMB9. To test this hypothesis, the EcoRI-HpaI fragment of pSC101 and the HincII fragments of pMB9 were purified and digested with EcoRI* activity. The EcoRI* fragments of pSC101, shown in Figure 3a, revealed a minor D N A fragment of 0.21 megadaltons and major D N A fragments of 0.14 and approximately 0.06 megadaltons that could not be detected in either of the HincII fragments of pMB9, shown in Figures 3d and 3e. Because of the difficulties in obtaining complete EcoRI* digestions, it is probable that the 0.14 and the 0.06 megadalton D N A fragments together form the 0.21 megadalton D N A fragment that was deleted during the construction of pMB9 with the use of the EcoRI* activity.
Fig. 4. Restriction map of the Tc resistance locus of pSC101 and pMBg. The cleavage sites for a variety of restriction endonucleases have been indicated on the 1.85 megadalton EcoRI-HpaI DNA fragments from pSC101 and pMB9. EcoRI* sites are indicated by *. At least four more major EcoRI* sites occur in both plasmids between the SalI and HpaI sites. Although these sites could not be positioned precisely, digestions with the enzyme AluI suggested that linear sequence of the EcoRI* fragments is the same in the two plasmids, with the exception of the deletion of the 0.21 megadalton fragment containing the HincII site of pSC101 and the 0.17 megadalton fragment that was added to the EeoRI site of pSC101
The results of the restriction endonuclease mapping of the 1.85 megadalton EcoRI-ItpaI D N A fragments of pSC101 and pMB9 have been summarized in Figure 4. During the construction of pMB9, a 0.17 megadalton EcoRI* generated D N A fragment was added to the EcoRI site of pSC101, converting it to an EcoRI* site and generating an EcoRI site 0.17 megadaltons away from the original EcoRI site. In addition, an EcoRI* generated D N A fragment of 0.21 megadaltons containing an EcoRI* site and a HincII site was deleted from pSC101.
Discussion Previous investigation has implicated polypeptides of 34,000, 26,000, 18,000 and 14,000 daltons in the generation of Tc resistance by pSC101 (Tait and Boyer, 1978). Insertion of D N A at the EcoRI site of pSC101 has been found to cause apparent alterations in the molecular weight of the 14,000 dalton polypeptide, suggesting that the EcoRI site occurs in the carboxyterminal region of the gene for the 14,000 dalton polypeptide (Meagher et al., 1977). The deletion from pSC101 of the region from the HpaI site to the BamHI site prevented the synthesis in minicells of the polypeptides of 34,000, 26,000 and 18,000 daltons, suggesting that the genes for these polypeptides are contained within this D N A fragment (Tait and Boyer, 1978). The insertion of D N A in the B a m H I site was observed to reduce the size of the 34,000 dalton poly-
288
R.C. Tait and H.W. Boyer: Restriction Endonuclease Mapping of pSC101 and pMB9
References
E~S!U~l ]l i HIal HIn~l,Hin~llUHi]¢ll Hi~cllHinl¢ll HincllI
Hinlc II
O. of Rep.
0
I
2
~
4
5
5.8
MegadaJtons
Fig. 5. Map of the Tc resistance locus of pSCI0I. The genes for four polypeptides involved in Tc resistance have been positioned on the restriction map of pSC101. The approximate origin of replication as determined by Cabetlo, Timmis and Cohen (1976) is indicated by 0. of Rep
peptide (Rambach and Hogness, 1977). Minicells containing pMB9 synthesized the 34,000 and 18,000 dalton polypeptides but not the 26,000 dalton polypeptide (Tait and Boyer, 1978). In addition, during the construction of pBR322 from pMB9, 0.6 megadaltons of DNA was deleted from the HpaI site of pMB9 towards the SalI site (Bolivar et al., 1977b). Minicells containing pBR322 synthesize both the 34,000 and the 18,000 dalton polypeptides (Covarrubias et al., 1978). Combined, these results indicate that the gene for the 34,000 dalton polypeptide is interrupted by the BamHI site, with the gene for the 18,000 dalton polypeptide adjacent to the gene for the 34,000 dalton polypeptide. The gene for the 26,000 dalton polypeptide or information necessary for its expression should then exist in the 0.21 megadalton DNA fragment that was deleted during the construction of pMB9. Using a molecular weight average of 110 daltons per amino acid residue, the approximate sizes of these genes have been calculated and the positions of the genes indicated on the restriction map of pSC101 shown in Figure 5. Acknowledgements. This work was supported by a grant from the National Science Foundation (PCM7510468). H.W.B. is an Investigator for the Howard Hughes Medical Institute.
Bolivar, F., Rodriguez, R.L., Betlach, M.C., Boyer, H.W.: Construction and characterization of new cloning vehicles : I. Ampicillin resistant derivatives of the plasmid pMB9. Gene 2, 75-93 (1977a) Bolivar, F., R0driguez, R.L., Greene, P.J., Betlach, M.C., Heyneker, H.L., Boyer, H.W., Crosa, J.H., Falkow, S.: Construction and characterization of new cloning vehicles: II. A multipurpose cloning system. Gene 2, 95-113 (1977b) Bolivar, F., Betlach, M.C., Heyneker, H.L., Shine, J., Rodriguez, R.L., Boyer, H.W.: Origin of replication of pBR345 plasmid DNA. Proc. nat. Acad. Sci. (Wash.) 74, 5265-5269 (1977c) Cabello, F., Timmis, K., Cohen, S.N.: Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons. Nature (Lond.) 259, 285-290 (1976) Clewell, D.B. : Nature of Cole 1 plasmid replication in the presence of chloramphenicol. J. Bact. 110, 667 676 (1972) Covarrubias, A.A., Tait, R.C., Bolivar, F.G., Boyer, H.W., to be submitted to Molec. gen. Genet. Greene, P.J., Heyneker, H.L., Bolivar, F., Rodriguez, R.L., Betlach, M.C., Corvarrubias, A.A., Backman, K., Russel, D.J., Tait, R., Boyer, H.W.: A general method for the purification of restriction enzymes. Submitted to Nuc. Acids Res. Meagher, R.B., Tait, R.C., Betlach, M., Boyer, H.W.: Protein expression in E. coli minicells by recombinant plasmids. Cell 10, 521-536 (1977) Rambach, A., Hogness, D.S. : Translation of Drosophila melanogaster sequences in Escheriehia eoli. Proc. nat. Acad. Sci. (Wash.) 74, 5041-5045 (1977) Rodriguez, R.L., Bolivar, F., Goodman, H.M., Boyer, H.W., Betlach, M.C. : Construction and characterization of cloning vehicles. In: Molecular mechanisms in the control of gene expression (eds. D.P. Nierlich, W.J. Rutter and C.F. Fox.), pp. 471-478. New York: Academic Press 1976 Rodriguez, R.L., Tait, R.C., Shine, J., Bolivar, F., Heyneker, H., Betlach, M., Boyer, H.W. : Characterization of tetracycline and ampicillin resistant plasmid cloning vehicles. In: Molecular cloning of recombinant DNA (eds. W.A. Scott and R. Werner), pp. 73-84. New York: Academic Press 1977 Tait, R.C., Boyer, H.W.: On the nature of tetracycline resistance controlled by the plasmid pSC101. Cell 13, 73-81 (1978)
Communicated by W. Arber Received May 17, 1978
