PLASMID
2, 59-68 (1979)
Physical
Mapping of a Plasmid from Bacillus megaterium by Restriction Endonuclease Cleavage BRUCE C.CARLTON
Program
AND BARBARA J. BROWN
in Genetics and Departments of Biochemistry and Microbiology, University of Georgia, Athens, Georgia 30602
Accepted August 31, 1978 A CMdalton plasmid from Bacillus megaterium strain 216 has been physically mapped by restriction endonuclease digestion. A combination of single and double digests with seven restriction enzymes, together with a terminal labeling procedure, has produced a physical map containing 21 apparently unique cleavage sites. The data are most consistent with the view that this plasmid does not contain extensive variability in sequence.
selves (Carlton, 1976). Preliminary renaturation kinetic studies of the two smallest plasmids have likewise indicated that as much as 30% of the plasmid mass may contain sequences as complex as the fragmented, linear chromosomal DNA (L. Carreira, unpublished experiments). These data have suggested the possibility that at least some of the plasmid species may represent molecular hybrids between plasmid elements and segments of the bacterial chromosome. The discovery of site-specific restriction endonucleases, with their high specificities for sequence recognition in cleaving doublestranded DNA, (Boyer, 1971; Meselson et al., 1972))has provided a means of analyzing the molecular structures of plasmid and other DNA molecules. The analysis of fragments derived by restriction endonuclease digestion of specific DNA molecules on agarose (Sharp et al., 1973) or acrylamide (Danna and Nathans, 1971) gels, or by direct electron microscopic measurement has permitted the construction of cleavage maps (Nathans and Smith, 1975) and the determination of the unique nature of specific viral and plasmid genomes. For a complex plasmid system such as r Although these molecules do not necessarily posthat of Bacillus megaterium, the analysis sess all the properties ascribed to conventional plasmids (Novick et al., 1976), we prefer to refer to them of restriction endonuclease cleavage prodby this nomenclature until studies should prove that ucts should be a valuable approach to dethey are not. termining the uniqueness of plasmid moleThe gram-positive soil bacterium Bacillus megaterium has been shown to contain a heterogeneous group of closed-circular plasmid’ DNA molecules which can comprise a substantial fraction of the total cellular DNA (Carlton and Helinski, 1969; Henneberry and Carlton, 1973). These molecules were found to be distributed in at least nine discrete size classes, six of which comprised the vast majority of the plasmid molecule population. These six major plasmid species were shown by sedimentation velocity analysis and electron microscope measurement to have masses of 4, 6.2, 15.9, 30.9, 47, and 60 Mdaltons (md) (Carlton and Smith, 1974), and together were estimated to be present in as many as 800 molecules/cell (Carlton, 1976). The possible structural similarities among the several plasmid species and their relationship to the total genome of this organism have been of prime interest. DNA-DNA hybridization studies have suggested that substantial sequence homology exists between the total plasmid population and the chromosomal DNA sequences, as well as among three of the plasmid species them-
59
0147-619X/79/010059-10$02.00/0 Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
60
CARLTON
cules of a given size and for determining whether structural similarities occur between plasmids of different sizes. The studies described here are aimed at these two objectives, beginning with the 4-md plasmid from this organism. MATERIALS
AND METHODS
Strain. B. megaterium strain 216 (megacin A and megacin C producer) was obtained from the collection of Dr. G. Ivanovics. DNA was isolated from a spontaneous derivative of this strain resistant to 100 pg/ml streptomycin (R. C. Henneberry, unpublished data). Reagents and special materials. Technical grade CsCl was purchased from Kawecki Chemical Company, New York, New York, and Seakem (ME) agarose from Marine Colloids, Inc., Rockland, Maine. Ethidium bromide, lysozyme, and RNase A were products of Sigma Chemical Company, St. Louis, Missouri. Protease K was purchased from EM Laboratories, Elmsford, New York. Sarkosyl NL30 was purchased from Geigy Chemical Company, and acrylamide reagents were obtained from BioRad Laboratories, Richmond, California. Restriction endonuclease enzymes (BamHI, Bg1II,EcoRI,HaeIII,HindIII,HpaI,HhaI, and HpaII) were purchased from either Bethesda Research Laboratories, Inc., or New England Biolabs. Polynucleotide kinase and alkaline phosphatase were products of P-L Biochemicals and Worthington Biochemical Corporation, respectively. Phage A DNA was a gift of Dr. C. R. Wilson and PM-2, #~X174 RF, and SV40 DNA standards were provided by Drs. D. Vapnek and R. Meagher. [Y~~P]ATP (sp act, 1000-3000 Ci/mmol) was a product of New England Nuclear, Boston, Massachusetts. Isolation and purijcation of closed circular DNA. Cells were streaked on Difco
nutrient agar plates and grown overnight at 37°C. These cells were used to innoculate 3- or 4-liter batch cultures which were grown in Spizizen salts medium (Spizizen, 1958)
AND BROWN
supplemented with 0.04% yeast extract and 0.4% glucose. Cultures were grown in a rotary shaker-incubator at 37°C until late log phase (ca. 1 x lo9 cells/ml), harvested by centrifugation, and washed twice with cold TES buffer (0.03 M Tris-HCl, 0.005 M EDTA, and 0.05 M NaCl, pH 8.0). Cells were resuspended in warm 10%(w/v) sucrose in TES containing 500 &ml lysozyme and incubated for 1 h at 37°C. Cells were lysed by adding 0.5 vol of 2% (w/v) sarkosyl in TES. Protease K was added to 50 &ml and the cell lysate was incubated for 45 min. at 37°C. The lysate was extracted for 1 h at room temperature on a horizontal roller apparatus with 1 vol of chloroform-isoamyl alcohol (24: 1, v/v), the layers were separated by a low-speed centrifugation, and the DNA was precipitated from the aqueous layer with 2 vol of 95% ethanol. The DNA was spooled onto a hooked glass rod and redissolved in TES buffer. The DNA solution was incubated for 1 h at 37°C with 200 &ml RNase A (preboiled 10 min) and then for 30 min at 37°C with 50 &ml protease K. The chloroform-isoamyl alcohol extraction was repeated, and the DNA reprecipitated in absolute ethanol and redissolved in TES buffer. Closed-circular DNA was isolated by preparative dye-buoyant density centrifugation in the presence of ethidium bromide as previously described (Henneberry and Carlton, 1973; Carlton and Smith, 1974). The denser banding closed-circular DNA fractions were pooled, the dye extracted with CsCl-saturated isopropanol, dialyzed against TES buffer, and concentrated by centrifugation or by dialysis against solid sucrose. The circular DNA was fractionated into discrete size classes by electrophoresis in 0.7% agarose on a circular preparative gel apparatus modified after the Southern apparatus (E.M. Southern, personal communication). DNA was electrophoresed out of the agarose and fractions were collected. Samples were scanned by running a small aliquot on a vertical 0.8%
MAPPING OF A
Bacillus
agarose gel. Appropriate circular DNA samples were pooled, dialyzed, and concentrated by centrifugation. The DNA was rebanded in CsCl-ethidium bromide to ensure digestibility with restriction endonucleases. Restriction enzyme digestion. Isolated size classes of circular DNA were dialyzed against and stored in half-strength TES buffer. DNA concentration was adjusted to 410 pg/ml and 25 ~1 were used per reaction. All DNA samples were diluted with an equal volume of an appropriate 2x restriction endonuclease digestion buffer. Buffers (2x) employed were as follows: for EcoRI, 200 mM Tris (pH 7.4), 10 mM MgC&, 100 mM NaCl, 0.02% Nonidet P40 (Shell Oil Co.); HaeIII and HhaI, 100 mM Tris (pH 7.4), 10 mM MgC&; BglII and HpaII, 40 mM Tris (pH 7.4), 20 mM MgC&; HindIII, 40 mM Tris (pH 7.4), 14 mM MgC&, 120 mM NaCl; HpaI, 40 mM Tris (pH 7.4), 20 mM MgC&, 12 mM KCl; BumHI, 12 mM Tris (pH 7.5), 2 mM MgC&, 100 mM NaCl. All double digests were done sequentially, allowing the restriction enzyme requiring the lower salt concentration to react first; then appropriate adjustments were made to the reaction buffer and the second restriction enzyme was added. Samples were digested for l-2 h at 37°C; reactions were stopped with 10 ~1 of a dye-stop mix consisting of 40% sucrose in TES, 0.05% bromphenol blue, and 1.0% sodium dodecyl sulfate. Analytical gel electrophoresis. Restriction enzyme cleavage products were analyzed on vertical gels 18 cm x 16.5 cm x 3 mm thick containing 0.8% (w/v) or 1.2% (w/v) agarose poured above a bottom plug of 10% (w/v) Cyanogum 41 (Fisher Chemical). The gel and plug were made up in 1x Tris - borate electrophoresis buffer (0.09 M Tris-HCl, 0.0025 M EDTA, 0.09 M boric acid, pH 8.1). Gels were electrophoresed for 2 min at 100 V and then for 16-18 h at 30 V at room temperature. Fragments of DNA with a mass of less than 0.6 md were resolved on 6% (w/v) acrylamide gels of the same dimensions as above. Acrylamide gels were run at 120 V for 1.5 to 2.5 h.
megaterium
PLASMID
61
After electrophoresis the gels were stained with ethidium bromide and destained briefly in deionized water. The stained gels were illuminated with a shortwavelength ultraviolet light source (UltraViolet Products Inc.) and photographed with an MP-4 Polaroid camera apparatus. A 3-min exposure was used with type 55 positive-negative film (Polaroid) through a 23a gelatin filter (Kodak). The negatives were scanned on a Beckman Acta CIII recording spectrophotometer, and fragment sizes determined by comparison of their relative mobilities against standard restriction enzyme digests of phage A DNA (EcoRI and HindIII), phage PM2 DNA (HindIII), SV40 DNA (HindIII), or mixtures ofthese digests. Terminal labeling. The 4-md plasmid was linearized with EcoRI and then terminally labeled with 32P according to the method of Smith and Bimstiel(1976) with only slight modification. Approximately 2 pug(0.5 pmol) of the EcoRI-cleaved plasmid were incubated with 20 ~1 (30 pmol) of [y32P]ATP (3000 Cilmmol) for 60 min at 37°C. Thirty micrograms of Escherichiu coli carrier tRNA (Sigma) was added to the reaction mixture, which was then phenol extracted and passed over a lo-ml G-50 Sephadex column to separate the labeled plasmid from the unincorporated label. The labeled plasmid was subsequently treated with Hue111 restriction endonuclease to yield 2.7- and 1.3-md fragments, which were preparatively separated on a 4% acrylamide slab gel at 50 V for 16 h. The gel was autoradiographed as indicated below and the two fragments were cut out of the gel and freed of acrylamide by electrophoresis into a dialysis bag. The DNA was precipitated with ethanol three times prior to subsequent restriction endonuclease digestion. Each reaction mix contained 750 to 1000 total Cerenkov cpm. The partial digests necessary for autoradiograms were obtained by removing a IO-p1 sample from a SO-p1reaction mix at five time points. The five samples were pooled in one tube of dyestop mix and later applied to either a 6% (w/v) acrylamide gel or a 1.5% (w/v) agarose
62
CARLTON
14.43.5-
G s
l.l0.7* ossOH-
kl B OStE
0.30-
f iz
O.ISOJS-
AND BROWN
MAPPING OF A
Bacillus
gel, or both, for resolution of digestion products. Reference markers of the undigested fragments, together with standards of a Hue111digest of 32P-labeled4X174RF DNA, were also included on the gels. At the completion of electrophoresis the gels were dried under vacuum onto a sheet of Whatman No. 1 filter paper. The dried gels were autoradiographed against DuPont Cronex 2DC X-ray film with a DuPont Cronex Hi-plus intensifying screen. The film was exposed at -70°C for 48-72 h before developing. RESULTS
Digestion patterns of the 4-md plasmid of B. megaterium produced by eight different restriction endonucleases are shown in Fig. 1 and summarized in Table 1. These enzymes produced a total of 21 unique polynucleotide fragments from the intact plasmid. Two of the enzymes (EcoRI and HueIII) cleaved the molecule only once, each yielding unit-length, linear 4-md molecules (Fig. 1A). The Bgl II enzyme produced 2 fragments, while the HhaI enzyme yielded 4 fragments. HpaI produced 5 fragments, 2 of which corn&rated on acrylamide gels. The occurrence of 2 fragments of similar size was readily evident by the greater ethidium bromide staining intensity of the 0.2-md band when the photographic negative was scanned spectrophotometrically. The Hind111 enzyme produced 5 digestion fragments. The 2 small (0.3 md) Hind111 fragments appeared to be nearly identical in size on acrylamide gels (see Fig. 1B). They were, however, clearly distinguishable in double digests. The HpaII enzyme yielded 6 fragments, 3 of which (3.2,2.6, and 2.2 md) were present in variable, generally low amounts, together with the 3 major fragments which are shown in Table 1 and Fig. 1. The 3 larger fragments
megaterium
PLASMID
63
produced by the HpaII enzyme were judged to be incomplete digestion products arising from incomplete digestion of the three sites of attack by the enzyme. Further studies revealed that all except the 2.2-md overlap fragment could be completely cleaved by extended digestion at elevated enzyme concentrations (data not shown). The partial digestion fragments were useful in establishing the fragment order A-B-C on the physical map (Fig. 2). Treatment of the 4-md plasmid with the BamHI enzyme yielded a partial digest of linear unit-length molecules and nicked, opencircular forms (Fig. 1A). A secondary digestion with other enzymes (e.g., EcoRI or HueIII) produced a smear of fragments ranging from 4 md downward. It was, therefore, concluded that the action of the BamHI enzyme was probably due to a contaminating nonspecific nuclease, leading to random single- or double-strand cleavages throughout the molecule, and that no BamHI recognition site exists on this plasmid. Ordering of the cleavage sites on the plasmid for construction of a physical map was accomplished by two approaches. The first involved sequential digestion of the intact plasmid with two (or occasionally three) restriction enzymes in order to establish overlaps between adjacent fragments. The second approach, which yields both absolute order of restriction enzyme sites as well as the distances between sites (when appropriate standards are available), was the terminal-labeling procedure of Smith and Birnstiel(l976). This procedure has the advantages that very little material is required, and the sensitivity of fragment detection is limited principally by the extent of terminal labeling. Very small fragments thus appear with the same approximate intensity as large ones, unlike the usual ethidium bromide staining procedure. In addition, only those
FIG. 1. Restriction endonuclease digestion patterns of the 4-md plasmid of Bacillus megaterium. Reactions were carried out as described under Materials and Methods. (A) Agarose gel, 1.2%. The molecular weight standards are mixed Hind111 digests of SV40 and phage A DNAs. Electrophoresis was carried out in Tris-borate buffer at 30 V for 17 h. (B) Acrylamide gel, 6%, run for 3.5 h at 100 V in Tris-borate buffer.
64
CARLTON AND BROWN TABLE 1 RESTRICTION
ENDONUCLEASE
FRAGMENT
OF THE
4-md
PATTERNS PLASMID
GENERATED
OF Bacillus
BY SINGLE
AND DOUBLE
DIGESTS
megaterium”
Enzymes for double digests Enzymes for single digest
EcoRI
HaeIII
BglII A B
2.4 (2.4 +
2, 59-68 (1979)
Physical
Mapping of a Plasmid from Bacillus megaterium by Restriction Endonuclease Cleavage BRUCE C.CARLTON
Program
AND BARBARA J. BROWN
in Genetics and Departments of Biochemistry and Microbiology, University of Georgia, Athens, Georgia 30602
Accepted August 31, 1978 A CMdalton plasmid from Bacillus megaterium strain 216 has been physically mapped by restriction endonuclease digestion. A combination of single and double digests with seven restriction enzymes, together with a terminal labeling procedure, has produced a physical map containing 21 apparently unique cleavage sites. The data are most consistent with the view that this plasmid does not contain extensive variability in sequence.
selves (Carlton, 1976). Preliminary renaturation kinetic studies of the two smallest plasmids have likewise indicated that as much as 30% of the plasmid mass may contain sequences as complex as the fragmented, linear chromosomal DNA (L. Carreira, unpublished experiments). These data have suggested the possibility that at least some of the plasmid species may represent molecular hybrids between plasmid elements and segments of the bacterial chromosome. The discovery of site-specific restriction endonucleases, with their high specificities for sequence recognition in cleaving doublestranded DNA, (Boyer, 1971; Meselson et al., 1972))has provided a means of analyzing the molecular structures of plasmid and other DNA molecules. The analysis of fragments derived by restriction endonuclease digestion of specific DNA molecules on agarose (Sharp et al., 1973) or acrylamide (Danna and Nathans, 1971) gels, or by direct electron microscopic measurement has permitted the construction of cleavage maps (Nathans and Smith, 1975) and the determination of the unique nature of specific viral and plasmid genomes. For a complex plasmid system such as r Although these molecules do not necessarily posthat of Bacillus megaterium, the analysis sess all the properties ascribed to conventional plasmids (Novick et al., 1976), we prefer to refer to them of restriction endonuclease cleavage prodby this nomenclature until studies should prove that ucts should be a valuable approach to dethey are not. termining the uniqueness of plasmid moleThe gram-positive soil bacterium Bacillus megaterium has been shown to contain a heterogeneous group of closed-circular plasmid’ DNA molecules which can comprise a substantial fraction of the total cellular DNA (Carlton and Helinski, 1969; Henneberry and Carlton, 1973). These molecules were found to be distributed in at least nine discrete size classes, six of which comprised the vast majority of the plasmid molecule population. These six major plasmid species were shown by sedimentation velocity analysis and electron microscope measurement to have masses of 4, 6.2, 15.9, 30.9, 47, and 60 Mdaltons (md) (Carlton and Smith, 1974), and together were estimated to be present in as many as 800 molecules/cell (Carlton, 1976). The possible structural similarities among the several plasmid species and their relationship to the total genome of this organism have been of prime interest. DNA-DNA hybridization studies have suggested that substantial sequence homology exists between the total plasmid population and the chromosomal DNA sequences, as well as among three of the plasmid species them-
59
0147-619X/79/010059-10$02.00/0 Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
60
CARLTON
cules of a given size and for determining whether structural similarities occur between plasmids of different sizes. The studies described here are aimed at these two objectives, beginning with the 4-md plasmid from this organism. MATERIALS
AND METHODS
Strain. B. megaterium strain 216 (megacin A and megacin C producer) was obtained from the collection of Dr. G. Ivanovics. DNA was isolated from a spontaneous derivative of this strain resistant to 100 pg/ml streptomycin (R. C. Henneberry, unpublished data). Reagents and special materials. Technical grade CsCl was purchased from Kawecki Chemical Company, New York, New York, and Seakem (ME) agarose from Marine Colloids, Inc., Rockland, Maine. Ethidium bromide, lysozyme, and RNase A were products of Sigma Chemical Company, St. Louis, Missouri. Protease K was purchased from EM Laboratories, Elmsford, New York. Sarkosyl NL30 was purchased from Geigy Chemical Company, and acrylamide reagents were obtained from BioRad Laboratories, Richmond, California. Restriction endonuclease enzymes (BamHI, Bg1II,EcoRI,HaeIII,HindIII,HpaI,HhaI, and HpaII) were purchased from either Bethesda Research Laboratories, Inc., or New England Biolabs. Polynucleotide kinase and alkaline phosphatase were products of P-L Biochemicals and Worthington Biochemical Corporation, respectively. Phage A DNA was a gift of Dr. C. R. Wilson and PM-2, #~X174 RF, and SV40 DNA standards were provided by Drs. D. Vapnek and R. Meagher. [Y~~P]ATP (sp act, 1000-3000 Ci/mmol) was a product of New England Nuclear, Boston, Massachusetts. Isolation and purijcation of closed circular DNA. Cells were streaked on Difco
nutrient agar plates and grown overnight at 37°C. These cells were used to innoculate 3- or 4-liter batch cultures which were grown in Spizizen salts medium (Spizizen, 1958)
AND BROWN
supplemented with 0.04% yeast extract and 0.4% glucose. Cultures were grown in a rotary shaker-incubator at 37°C until late log phase (ca. 1 x lo9 cells/ml), harvested by centrifugation, and washed twice with cold TES buffer (0.03 M Tris-HCl, 0.005 M EDTA, and 0.05 M NaCl, pH 8.0). Cells were resuspended in warm 10%(w/v) sucrose in TES containing 500 &ml lysozyme and incubated for 1 h at 37°C. Cells were lysed by adding 0.5 vol of 2% (w/v) sarkosyl in TES. Protease K was added to 50 &ml and the cell lysate was incubated for 45 min. at 37°C. The lysate was extracted for 1 h at room temperature on a horizontal roller apparatus with 1 vol of chloroform-isoamyl alcohol (24: 1, v/v), the layers were separated by a low-speed centrifugation, and the DNA was precipitated from the aqueous layer with 2 vol of 95% ethanol. The DNA was spooled onto a hooked glass rod and redissolved in TES buffer. The DNA solution was incubated for 1 h at 37°C with 200 &ml RNase A (preboiled 10 min) and then for 30 min at 37°C with 50 &ml protease K. The chloroform-isoamyl alcohol extraction was repeated, and the DNA reprecipitated in absolute ethanol and redissolved in TES buffer. Closed-circular DNA was isolated by preparative dye-buoyant density centrifugation in the presence of ethidium bromide as previously described (Henneberry and Carlton, 1973; Carlton and Smith, 1974). The denser banding closed-circular DNA fractions were pooled, the dye extracted with CsCl-saturated isopropanol, dialyzed against TES buffer, and concentrated by centrifugation or by dialysis against solid sucrose. The circular DNA was fractionated into discrete size classes by electrophoresis in 0.7% agarose on a circular preparative gel apparatus modified after the Southern apparatus (E.M. Southern, personal communication). DNA was electrophoresed out of the agarose and fractions were collected. Samples were scanned by running a small aliquot on a vertical 0.8%
MAPPING OF A
Bacillus
agarose gel. Appropriate circular DNA samples were pooled, dialyzed, and concentrated by centrifugation. The DNA was rebanded in CsCl-ethidium bromide to ensure digestibility with restriction endonucleases. Restriction enzyme digestion. Isolated size classes of circular DNA were dialyzed against and stored in half-strength TES buffer. DNA concentration was adjusted to 410 pg/ml and 25 ~1 were used per reaction. All DNA samples were diluted with an equal volume of an appropriate 2x restriction endonuclease digestion buffer. Buffers (2x) employed were as follows: for EcoRI, 200 mM Tris (pH 7.4), 10 mM MgC&, 100 mM NaCl, 0.02% Nonidet P40 (Shell Oil Co.); HaeIII and HhaI, 100 mM Tris (pH 7.4), 10 mM MgC&; BglII and HpaII, 40 mM Tris (pH 7.4), 20 mM MgC&; HindIII, 40 mM Tris (pH 7.4), 14 mM MgC&, 120 mM NaCl; HpaI, 40 mM Tris (pH 7.4), 20 mM MgC&, 12 mM KCl; BumHI, 12 mM Tris (pH 7.5), 2 mM MgC&, 100 mM NaCl. All double digests were done sequentially, allowing the restriction enzyme requiring the lower salt concentration to react first; then appropriate adjustments were made to the reaction buffer and the second restriction enzyme was added. Samples were digested for l-2 h at 37°C; reactions were stopped with 10 ~1 of a dye-stop mix consisting of 40% sucrose in TES, 0.05% bromphenol blue, and 1.0% sodium dodecyl sulfate. Analytical gel electrophoresis. Restriction enzyme cleavage products were analyzed on vertical gels 18 cm x 16.5 cm x 3 mm thick containing 0.8% (w/v) or 1.2% (w/v) agarose poured above a bottom plug of 10% (w/v) Cyanogum 41 (Fisher Chemical). The gel and plug were made up in 1x Tris - borate electrophoresis buffer (0.09 M Tris-HCl, 0.0025 M EDTA, 0.09 M boric acid, pH 8.1). Gels were electrophoresed for 2 min at 100 V and then for 16-18 h at 30 V at room temperature. Fragments of DNA with a mass of less than 0.6 md were resolved on 6% (w/v) acrylamide gels of the same dimensions as above. Acrylamide gels were run at 120 V for 1.5 to 2.5 h.
megaterium
PLASMID
61
After electrophoresis the gels were stained with ethidium bromide and destained briefly in deionized water. The stained gels were illuminated with a shortwavelength ultraviolet light source (UltraViolet Products Inc.) and photographed with an MP-4 Polaroid camera apparatus. A 3-min exposure was used with type 55 positive-negative film (Polaroid) through a 23a gelatin filter (Kodak). The negatives were scanned on a Beckman Acta CIII recording spectrophotometer, and fragment sizes determined by comparison of their relative mobilities against standard restriction enzyme digests of phage A DNA (EcoRI and HindIII), phage PM2 DNA (HindIII), SV40 DNA (HindIII), or mixtures ofthese digests. Terminal labeling. The 4-md plasmid was linearized with EcoRI and then terminally labeled with 32P according to the method of Smith and Bimstiel(1976) with only slight modification. Approximately 2 pug(0.5 pmol) of the EcoRI-cleaved plasmid were incubated with 20 ~1 (30 pmol) of [y32P]ATP (3000 Cilmmol) for 60 min at 37°C. Thirty micrograms of Escherichiu coli carrier tRNA (Sigma) was added to the reaction mixture, which was then phenol extracted and passed over a lo-ml G-50 Sephadex column to separate the labeled plasmid from the unincorporated label. The labeled plasmid was subsequently treated with Hue111 restriction endonuclease to yield 2.7- and 1.3-md fragments, which were preparatively separated on a 4% acrylamide slab gel at 50 V for 16 h. The gel was autoradiographed as indicated below and the two fragments were cut out of the gel and freed of acrylamide by electrophoresis into a dialysis bag. The DNA was precipitated with ethanol three times prior to subsequent restriction endonuclease digestion. Each reaction mix contained 750 to 1000 total Cerenkov cpm. The partial digests necessary for autoradiograms were obtained by removing a IO-p1 sample from a SO-p1reaction mix at five time points. The five samples were pooled in one tube of dyestop mix and later applied to either a 6% (w/v) acrylamide gel or a 1.5% (w/v) agarose
62
CARLTON
14.43.5-
G s
l.l0.7* ossOH-
kl B OStE
0.30-
f iz
O.ISOJS-
AND BROWN
MAPPING OF A
Bacillus
gel, or both, for resolution of digestion products. Reference markers of the undigested fragments, together with standards of a Hue111digest of 32P-labeled4X174RF DNA, were also included on the gels. At the completion of electrophoresis the gels were dried under vacuum onto a sheet of Whatman No. 1 filter paper. The dried gels were autoradiographed against DuPont Cronex 2DC X-ray film with a DuPont Cronex Hi-plus intensifying screen. The film was exposed at -70°C for 48-72 h before developing. RESULTS
Digestion patterns of the 4-md plasmid of B. megaterium produced by eight different restriction endonucleases are shown in Fig. 1 and summarized in Table 1. These enzymes produced a total of 21 unique polynucleotide fragments from the intact plasmid. Two of the enzymes (EcoRI and HueIII) cleaved the molecule only once, each yielding unit-length, linear 4-md molecules (Fig. 1A). The Bgl II enzyme produced 2 fragments, while the HhaI enzyme yielded 4 fragments. HpaI produced 5 fragments, 2 of which corn&rated on acrylamide gels. The occurrence of 2 fragments of similar size was readily evident by the greater ethidium bromide staining intensity of the 0.2-md band when the photographic negative was scanned spectrophotometrically. The Hind111 enzyme produced 5 digestion fragments. The 2 small (0.3 md) Hind111 fragments appeared to be nearly identical in size on acrylamide gels (see Fig. 1B). They were, however, clearly distinguishable in double digests. The HpaII enzyme yielded 6 fragments, 3 of which (3.2,2.6, and 2.2 md) were present in variable, generally low amounts, together with the 3 major fragments which are shown in Table 1 and Fig. 1. The 3 larger fragments
megaterium
PLASMID
63
produced by the HpaII enzyme were judged to be incomplete digestion products arising from incomplete digestion of the three sites of attack by the enzyme. Further studies revealed that all except the 2.2-md overlap fragment could be completely cleaved by extended digestion at elevated enzyme concentrations (data not shown). The partial digestion fragments were useful in establishing the fragment order A-B-C on the physical map (Fig. 2). Treatment of the 4-md plasmid with the BamHI enzyme yielded a partial digest of linear unit-length molecules and nicked, opencircular forms (Fig. 1A). A secondary digestion with other enzymes (e.g., EcoRI or HueIII) produced a smear of fragments ranging from 4 md downward. It was, therefore, concluded that the action of the BamHI enzyme was probably due to a contaminating nonspecific nuclease, leading to random single- or double-strand cleavages throughout the molecule, and that no BamHI recognition site exists on this plasmid. Ordering of the cleavage sites on the plasmid for construction of a physical map was accomplished by two approaches. The first involved sequential digestion of the intact plasmid with two (or occasionally three) restriction enzymes in order to establish overlaps between adjacent fragments. The second approach, which yields both absolute order of restriction enzyme sites as well as the distances between sites (when appropriate standards are available), was the terminal-labeling procedure of Smith and Birnstiel(l976). This procedure has the advantages that very little material is required, and the sensitivity of fragment detection is limited principally by the extent of terminal labeling. Very small fragments thus appear with the same approximate intensity as large ones, unlike the usual ethidium bromide staining procedure. In addition, only those
FIG. 1. Restriction endonuclease digestion patterns of the 4-md plasmid of Bacillus megaterium. Reactions were carried out as described under Materials and Methods. (A) Agarose gel, 1.2%. The molecular weight standards are mixed Hind111 digests of SV40 and phage A DNAs. Electrophoresis was carried out in Tris-borate buffer at 30 V for 17 h. (B) Acrylamide gel, 6%, run for 3.5 h at 100 V in Tris-borate buffer.
64
CARLTON AND BROWN TABLE 1 RESTRICTION
ENDONUCLEASE
FRAGMENT
OF THE
4-md
PATTERNS PLASMID
GENERATED
OF Bacillus
BY SINGLE
AND DOUBLE
DIGESTS
megaterium”
Enzymes for double digests Enzymes for single digest
EcoRI
HaeIII
BglII A B
2.4 (2.4 +
