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Purification of supercoiled DNA of plasmid Col E1 by RPC-5 chromatography
AN.41
YTICAI.
114, 235-243
BIOCHEMISTRY
IPurification
(1981)
of Supercoiled RPC-5
DNA
of Plasmid
Division,
Oak
Ridge
by
Chromatography’,2
AUDREY N. BEST, DAVID P. ALLISON, Biology
Col El
National
Received
Laboratory. October
AND Oak
G. DAVID NOVELLI Ridge,
Tennessee
37830
16, 1980
Col El DNA can be purified to a high degree by RPC-5 chromatography of a partially purified cell lysate with a very shallow linear NaCl gradient at pH 7.8. Electron micrographs demonstrated that the purest fractions were composed of 93% supercoiled (form I) DNA and 7% open circular (form II) DNA. The actual chromatography can be accomplished in 13- 14 h and is designed for the production of several milligrams of plasmid DNA.
The DNA of the plasmid Co1 El has proved to be a useful vehicle for genetic, biochemical and biophysical studies ( l-3). Procedures bary somewhat in detail for the isolation of the plasmid DNA as the supercoiled closed circular duplex (form I) from Escherichia co/i carrier cells, with subsequent purification to remove contaminants (2,4,5). However, in most procedures the final purification step involves one or two 40h centrifugations by the CsClkethidium bromide buoyant density technique (2,4~-6). Ideally, the resulting Co1 El plasmid DNA preparation will be composed of form I DNA. The other plasmid forms [open circular duplex DNA (form II) and linear duplex DNA (form III)] should be present only as very minor components, and cellular RNAs, chromosomal DNA, and proteins should be removed. Whether the presence of these impurities can be tolerated depends on the intended experimental use of the plasmid DNA. To obtain ;1 preparation with a high con’ Research sponsored by the Office of Health and Environmental Research. U. S. Department of Energy, under Contract IV-7405-eng-26 with the Union Carbide Corporation. ’ The U. S. government’s right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper, for governmental purposes, is acknowledged.
centration of form I Co1 El DNA, we have employed standard isolation and preliminary purification procedures. Avoiding the timeconsumivg buoyant density centrifugations, we have substituted a 13- to 14-h chromatographic procedure using the RPC-5 system (7) to remove residual contaminants such as oligoribonucleotides, form III DNA, chromosomal fragments, and most of form II DNA to give a Co1 E 1 plasmid DNA preparation consisting of 93% form I and 7% form II DNA. This chromatographic procedure should be useful in the preparation of milligram quantities of form I plasmid DNA. MATERIALS
AND METHODS
Bacteria. E. coli strain JC4 11 (thy-, met-, leu-, his-, arg-) (Co1 El), obtained from Dr. D. M. Crothers (Department of Chemistry, Yale University, New Haven, Corm.), was used for the production of the Co1 El plasmid. Reagents. Buffer A was 10 mM Triss HCl/l mM EDTA (pH 7.8); Buffer B, 10 mM Tris-HCl/SO mM NaCl/2 mM EDTA (pH 8); Buffer C, 10 mM Tris-HCl/O.l mM EDTA (pH 7.8); Buffer D, 40 mM TrisHCl/5 mM sodium acetate/l mM EDTA (pH 7.8). Brij 58 (Atlas Chemical Indus-
235 0003-2697/X
l/100235-09$02.00/O
236
BEST,
ALLISON,
tries), and sodium deoxycholate and lysozyme (Sigma) were used for cell lysis. Adogen 464 (Ashland Chemical Co.) and Plaskon CTFE 2300 powder (Allied Chemical Co.) were prepared for RPC-5 chromatography by method C of Pearson et al. (7). Agarose powder, low electroendosmosis grade, was manufactured by Marine Colloids, Inc. The restriction endonucleases HaeII, HindHI, and PstI were from Bethesda Research Labs., Inc., and Eco RI was from Sigma. Lambda phage DNA was purchased from Sigma. T7 phage DNA was the gift of Dr. A, L. Stevens, Biology Division. All organic solvents and other chemicals were of reagent grade. Production of Co1 El plasmid DNA. The Co1 El-bearing strain of E. cofi JC411 was grown in 1- to 3-liter cultures on M-9 medium containing 0.4% glucose, 0.5% Casamino Acids (Difco), and 3 pg thymine/ml (Sigma) under conditions similar to those described by Clewell (8) and Herschman and Helinski (9). When the cell concentration reached 2-3 X 10’ cells/ml, 300 pg chloramphenicol/ml (Sigma) was added permitting amplification of the Co1 El plasmid DNA over a period of 16-20 h. A “cleared lysate” was produced from the harvested cells, following the Brij lysis procedure of Clewell and Helinski ( 10). The clear supernatant of the Brij lysate, designated as BL I, was digested first with RNase A (Worthington) and RNase T, (Sankyo Co., Japan), then with proteinase K (E. Merck, Darmstadt, West Germany), in a procedure similar to that described by Zasloff et al. ( 11). The digested lysate was extracted twice with a mixture consisting of 1 vol water-saturated phenol (pH 8) and 0.5 vol chloroform/isoamyl alcohol mixture, 24: 1. The combined aqueous fractions were reextracted once with one volume of chloroform/ isoamyl alcohol, then three times with ether prior to the precipitation of nucleic acid material by alcohol at -20°C. The precipitate was dissolved in a minimum volume of Buffer B and dialyzed exhaustively in this
AND
NOVELL1
buffer. This preparation, designated BL II, was used for the RPC-5 chromatography experiments. RPC-5 chromatography. The Adogen / Plaskon powder mixture was equilibrated with starting NaCl/buffer mixture and packed under pressure at the desired temperature in either of two Glenco 3202 jacketed columns, 0.6 X 30 and 0.6 X 100 cm with final column heights of 20 and 90 cm. Unless otherwise specified, linear gradients (total volume 1 liter) of 0.6-0.7, 0.62-0.72, and 0.68-0.78 M NaCl in Buffer A were used for chromatography at 28-30,37, and 50°C respectively. Theoretically, the NaCl concentration should increase in 0.1 mM increments per milliliter of influent. A Milton Roy minipump was used to adjust the column flow rate between 0.6 and 0.8 ml/min, and the column eluant was monitored by a Chromatronix dual-channel absorbance detector, Model 230, at 254 and 280 nm. Collection volumes were 2-4 ml for the DNA portion of the eluate. A Zeiss PMQ II spectrophotometer was used to measure the absorbance at 260 and 280 nm of all collection tubes containing ultraviolet-absorbing material. When the absorbance values permitted, selected collections were monitored for various forms of Co1 El plasmid DNA by agarose gel electrophoresis before the fractions were pooled. Dialysis with Buffer C preceded the concentration by freeze-drying of the pooled fractions. The residues were dissolved in minimal volumes of Buffer A, and several levels of DNA of comparable concentration, usually 0. l-l .O gg, from each pooled fraction were analyzed to determine the purity, i.e., the proportion of form I Co1 El DNA in relation to forms II and III and chromosomal DNA fragments. Gel electrophoresis. Horizontal agarose slab gels, 13 cm wide X 22 cm long, were prepared by use of the electrophoresis apparatus (Aquebogue, Model 850) initially described by McDonell et al. (12). Agarose gels of 1% in Buffer D were used to estimate the relative amounts of Co1 El plasmid
237
RPC-5CHROMATOGRAPHYOFColElPLASMlDDNA DNA and chromosomal fragments and to establish the: presence of RNA contaminants at various sltages of purification. In general the electrophoresis procedure followed that of Sharp et al. (13). We found that a constant current of 25 mA (l&20 h) was satisfactory for the detection of 445 S RNA in preparation BL I and oligoribonucleotides in BL II. The various forms of Co1 El DNA were resolved in greater detail at 40 mA ( 18- 20 h). When necessary, comparisons were made with 445 S RNA and rRNA preparations. After electrophoresis, DNA and RNA were detected by staining with ethidium bromide (Sigma) 1 pg/ml in Buffer D, then visualized and photographed during shortwave ultraviolet illumination (13). Restriction endonuclease cleavage of Co1 El DNA. EcoRI, HaeII, and PstI restriction enzymes were incubated in separate reactions with purified Co1 El DNA for 18-20 h at 37°C and the reaction conditions were as described by the manufacturer. The amount of DNA, gel strength, and current were adjusted according to the size of fragments generated by the restriction endonucleases. Marker fragments, generated by EcoRI or Hind111 cleavage of phage X DNA, were used to estimate the size of the restriction enzyme-generated Co1 El DNA fragments ( 14). Electron microscopy. DNA samples were prepared for electron microscopy by the aqueous protein film technique (15). The spreading solutions contained 10 ~1 of DNA (4-5 pg/ml in Buffer A), 85 ~1 of 0.5 M ammonium acetate/l mM EDTA (pH 7.8) and 5 ~1 of cytochrome c (2 mg/ml, Calbiochem.). The hypophase was 0.25 M ammonium acetate (pH 7.5) in a 90-mm circular plastic petri dish. The spreading solution (30 ,ul) was allowed to flow down a clean glass slide that had been stored in 0.25 M ammonium acetate (pH 7.5) onto the hypophase. A. parlodion film supported on a 200-mesh csopper grid was touched to the protein film at about one grid width from the slide; rinsed by being touched to 0.25 M
ammonium acetate (pH 7.5), 50% ethanol, 95% ethanol; and finally stained with uranyl acetate. The grids were rotary shadowed with platinum at a 6” angle, and micrographs were taken at random at a magnification of 2000 on a Siemens Elmiskop IA. RESULTS Co1 El DNA Purification
Isolation
and Preliminary
Visualization of the DNA and RNA present in the plasmid-containing lysates (BL I) after gel electrophoresis indicated that form I Co1 El DNA was the predominant DNA, although small amounts of form II Co1 El DNA and E. coli chromosomal DNA were present. After enzyme digestions and phenol extraction, the amount of form II DNA in most BL II preparations appeared to increase to about one-third of the amount of form I. This transition can be expected since random insertion of small RNA segments into one of the plasmid DNA strands occurs during Co1 El replication in the presence of chloramphenicol and up to 50% of the form I DNA can be converted to form II by alkali or RNase A treatment (16). Approximately 95% of the Az6,, units in our BL I preparations consisted of 4-5 S RNA ( 17) which had to be eliminated along with cellular proteins before RPC-5 chromatography could proceed. Residual nondialyzable, low-molecular-weight oligoribonucleotides present in BL II preparations were easily removed by RPC-5 chromatography. RPC-5
Chromatography
In a preliminary experiment (Fig. 1) the oligoribonucleotide fraction (peak 1) of the BL II preparations was excluded almost immediately from a 20-cm RPC-5 column at 37°C with a linear gradient of 0.55-0.925 M NaCl in 250 ml Buffer A. The DNAs were eluted as a single tailing peak between 0.65 and 0.7 M NaCl. Most of form I plasmid
238
BEST,
ALLISON,
AND
NOVELL1
b’ffect of Temperature Chromatography
2a
2b
2.0 -
4
s 0 l.Oc%
0 260 GRADIENT (ml) FIG. I. Separation of various components of a BL II preparation by RPC-5 chromatography. The column load was I ml of BL II (39.5 AzhOunits) followed by 10 ml of starting buffer. The absorbance peaks are identified as follows: peak I (oligoribonucleotides), 7.5-45 ml; peak 2a (main DNA fraction), 80-90 ml; peak 2b (tailing portion of DNA), 91-105 ml. Column conditions are described under Results. The distribution of the various forms of DNA after electrophoresis of two levels of peaks 2a and 2b (0.5 and I.0 bg) is shown above the profile. Gel bands are identified in Fig. 2
DNA emerged in the main portion of the peak (2a), whereas the chromosomal DNA was concentrated in the last tailing portion (2b). The main DNA fraction (2a) contained approximately 80% of the DNA eluted from the column, and analysis by gel electrophoresis indicated that: the concentration of form I DNA was enriched to three to four times the amount of form II; form III was present in trace amounts; and chromosomal DNA was not present. Portions of the 2a fraction were rechromatographed on RPC-5 columns to test the effect of temperature on the separation of forms I, II, and III plasmid DNA with shallow gradients of 0.1 mM increments as described under Methods. Longer columns (90 cm) were used in these experiments because they provided better separation of the three forms of plasmid DNA at 37°C by retarding the emergence of form III.
on RPC-5
DNA was eluted from RPC-5 columns equilibrated at 28830°C and 37°C in the concentration ranges of 0.62-0.63 and 0.660.67 M NaCl, respectively. Although the absorbance profile indicated only one DNA peak, there was enrichment of the three forms in different portions of the DNA fraction. Forms I and II eluted before form III. Although both forms II and III were found in the early collection tubes where the Ale0 values were higher, they were still eluting in the final tailing portion that contained little or no form I DNA. Improved separation of form I DNA from forms II and III was noted with the lower column temperatures. It was necessary to increase the NaCl concentration to elute DNA from RPC-5 columns at 50°C. A series of multiple peaks emerged between 0.71 and 0.73 M NaCI. The gel analysis showed no real separation or concentration of the three forms in any particular peak. In contrast to RPC-5 chromatography at lower temperatures, there was a positional shift, and forms II and III emerged in the first peak. Forms I and II were spread out over the entire profile, so there was no apparent possibility of further enrichment of form I DNA at this temperature. Since an NaCl gradient of 0.1 mM/ml at column temperatures of 28-30°C provided improvement in the separation of Co1 El form I DNA from forms II and III, the gradient concentration was decreased to 0.05 mM/ml increments. A portion of BL II containing the oligoribonucleotide fraction and fragmented chromosomal DNA as well as the three forms of plasmid DNA was applied to the 90-cm RPC-5 column. The elution profile of the DNAs is shown in Fig. 2. Gel electrophoresis analysis of selected collection tubes indicated that forms I and II were eluted in approximately the same proportions until the absorbance peak started to tail off. Gel patterns of the distribution of
RPC-5
CHROMATOGRAPHY
OF Col El PLASMID
3c --II TEL --I BL
ABCDEF
G
-0.64 -0.62 g 300
SbO
5do GRADIENT (ml)
6bO
0.60 =
FIG. 2. Absorbance profile of Col El DNA with RPC5 chromatography. The column load was 65 A200 units of BL II in I.65 ml on a 0.6 X 90-cm RPC-5 column (25.5.ml resin bed) at 3O’C: gradient, I liter 0.6W.65 M NaCl in Buffer A; flow rate. 35 ml/h at 180 psi; fraction collection volume, 2 ml. The oligoribonucleotide fraction (not depicted) was completely eluted in the first 100 ml of gradient. The distribution of the DNAs in BL II and the pooled fractions A-G is shown above the absorbance profile. Gel bands of form I, III, II, and chromosomal DNA fragments (C) are indicated. Samples of DNA (I pg except for fraction A which was 0.2 pg) were electrophoresed in I ‘% agarose gels for I8 h at 40 mA. Chromatography was discontinued temporarily after about I4 h (indicated by I). then resumed until the gradient volume was exhausted.
the various DNAs in seven pooled fractions, A-G, are represented in Fig. 2. Since all samples with the exception of fraction A were the same DNA concentration, visual inspection revealed the relative proportions of the various DNA forms in each fraction with some degree of accuracy. The composition of fractions A-E, eluted between 0.6 16 and 0.62 M NaCl, could be estimated as 8090% form I and 20-10% form II. Neither form III nor chromosomal DNA were visible in the 1-pg samples. The reported lower limit of sensitivity for detection of the DNA/ ethidium bromide complex is about 0.05 pg (I 3), and it can be assumed that the amount of these two forms is less than 5% of the total DNA. The main peak, fractions A-E,
DNA
239
accounted for about 67% of the recovered DNA. In the tailing portion of the DNA peak, represented by fractions F and G, there was more form II than form I, and form III and chromosomal DNA were also eluted. Comparison of fraction F with fraction G suggests that form III began emerging at a somewhat lower NaCl concentration than the chromosomal fragments. The purification of form I (fractions A-E) was complete when the concentration of the NaCl gradient became 0.62 M; the total time required to achieve this portion of the chromatography was 13 h. At least 90% of the column load of 65 Az,,,units of BL II could be recovered from the combined oligoribonucleotide fraction and total DNA eluted from the column. No additional DNA was eluted from the column with 1.0 M NaCl in Buffer A. The composition of the total AZbo units of recovered material was as follows: oligoribonucleotide fraction, 30%; DNA fractions A-E, 47%; and fractions F and G, 23%. Decreasing the gradient concentrations below 0.05 mM/ml increments was not particularly effective in the isolation of form I from form II DNA. The DNA elution was spread out over a larger volume in proportion to the decrease in concentration.
Electron Micrographs
of RPC-S Fractions
Electron micrographs of the pooled Co1 El DNA fractions A-G resulting from RPC-5 chromatography of BL II (Fig. 2) were scored for the distribution of forms I, II, and III and E. co/i chromosomal fragments. The results are presented in Table 1 along with the AzbO units recovered in each fraction. The purest fractions, A--E, which were estimated by agarose gel electrophoresis (Fig. 2) to contain 80-90% form I DNA, were shown by electron microscopy to be composed individually of 86-95% form I DNA. Form II was the only other form of DNA present in these fractions. Fraction C had appeared in several gel electrophoresis
240
BEST,
ALLISON,
AND
TABLE ELECTRON
MICROKOPIC
ANALYSIS AFTER RPC-5
NOVELL1
I
OF POOLED FRACTIONS CHROMATOGRAPHY
Co1 El DNA Recovered DNA (%) 1.83 16.38 12.35 21.41 14.93 8.45 24.65
Pooled fraction A B C D E F
G B-E mixture’ BL II
Form
I
Form
OF Co1 El
DNA
E. coli chromosomal fragments” (%)
(%)
II
Form
III
87 95 86 95 94 57 30
13 5 14 5 6 40 54
0 0 0 0 0 3 8
0 0 0 0 0 0 8
92 86
8
0
0
11
2
I
’ Fragmented chromosomal contaminants were of variable electrophoresis as phage T7 DNA, 25 X lo6 M,. * This mixture consisted of equal volumes of fractions B-E was tested for reactivity with restriction endonucleases.
experiments to be of equal purity with B, D, and E. Therefore, the value of 86% form I in this fraction was surprising. However, a composite mixture of equal volumes of fractions B-E was found to contain 92% form I. An electron micrograph of this mixture (Fig. 3) shows the predominantly superhelical nature of the purified preparation. The amount of form I DNA in fractions A-E represented 93% of the total ASGounits recovered in this portion of the eluate. Linear form III, 4.2 X lo6 M, (18), visible in fractions F and G in the gel represented in Fig. 2, was present in measurable amounts as shown by electron micrographic analysis. Fraction F showed a loss in form I relative to form II DNA which appeared to be of the same magnitude in both the gel and electron micrographs. The last fraction, G, showed almost twice as much form II as form I in the electron micrographs; the pattern in the gel indicated form II to be present in a larger amount. Chromosomal fragments, collected only in fraction G, were visible in the electron micrographs. It is difficult to correlate the gel pattern
size; the major
portion
showed
the same mobility
(25, 18. 34, and 23% of the respective
fractions)
in and
of the starting material, BL II, with the results from electron micrographic analysis, as the proportion of form I relative to form II DNA appears to be lower in the gel (Fig. 2). However, electrophoresis with vertical agarose gels (not represented here) gave a pattern which was in better agreement with the results in Table 1. In the final analysis, we can state that RPC-5 chromatography eliminated the oligoribonucleotides, form III, and chromosomal fragments from 65 A260 units of the BL II preparation, giving a purified preparation of 27.6 AZbo units or 1.38 mg of Co1 El DNA composed of 93% form I and 7% form II DNA. Cleavage of Co1 El DNA Endonucleases
with Restriction
Co1 El DNA has a single cleavage site for restriction endonuclease EcoRI, two sites for PstI, and six sites for HaeII (19-22). The purified Co1 El DNA (mixture B-E, 92% form I and 8% form II DNA, Table 1) obtained from RPC-5 chromatography was
RPC-5
: . .
‘
< -.
CHROMATOGRAPHY
‘...’ ;.. :‘I ..:
OF
Col El
PLASMID
DNA
241
.‘, .,
FIG. 3. Eclectron micrograph of Col El mixture circles are Indicated. Magnification, 14.000X.
cleaved into fragments of the expected number and size. We can conclude from these results that the suitability of the Col El
of fractions
B-E
(see Table
1). Two
form
II open
DNA as a substrate for these restriction endonucleases was not affected by the chromatographic process.
242
BEST,
ALLISON.
AND
NOVELL1
quaternary ammonium columns of the RPC category. The secondary and tertiary stucThe usefulnessof a procedure for the iso- tures are important factors in the order of lation of a plasmid DNA in the most desired tRNA elution, and it appears from our work form, usually as a supercoiled duplex, is de- that this may also be true for DNA. The gel pendent upon many factors which have been in Fig. 2 showed that superhelical form I discussed in several reviews (2,4). We have DNA eluted at the lowest salt concentration addressed ourselves to the problem of ob- accompanied by a small amount of open cirtaining sufficient quantities of form I Co1 El cular form II DNA. During the preliminary DNA, perhaps 5-20 mg or more, for testing purification, the use of RNase caused the the experimental parameters of the chro- removal of small RNA segments from the matographic separation of restriction endo- form I DNA, resulting in the formation of nuclease-cleaved fragments. RPC-5 columns form II DNA. If the RNA is inserted ranhave been used to separate large amounts domly in either DNA strand, as reported by of restriction endonuclease-cleaved frag- some investigators, form 11 DNA would be ments by several investigators (23-25). heterogeneous since the single-stranded gaps We have not investigated the merits of in the open circular DNA would be at varvarious procedures in the initial stages of ious locations and possibly of different isolation or preliminary purification. Our lengths (2,16,27). The structural alteration main objective was to perform initial puri- due to the opening of the superhelix and the fications to remove as much 4-5 S RNA as existence of single-strand DNA in the nicked possible, eliminate cellular proteins and section are probably important factors in the DNA, and delete RNA sequences which elution pattern of form II DNA. Linear form have been reported to be inserted in Co1 El III DNA and chromosomal fragments were DNA synthesized during chloramphenicoleluted by higher salt concentrations. Base stimulated amplification of the plasmid copy composition, size, and structural change are number (16). The resulting preparation, BL important factors in the chromatography of II, was the test material for developing an these linear DNAs, as demonstrated by RPC-5 chromatographic technique that RPC-5 chromatography of restriction eneliminated an estimated 78% of form II donuclease-generated fragments (25). DNA as well as all of form III and chroThe capacity of the 0.6 X 90-cm RPC-5 mosomal DNA fragments. About 16% of columns for plasmid purification has not form I DNA from BL II was eluted in the been determined; we have not attempted a last fractions containing the above-men- column load of BL II in excessof the amount tioned contaminants. used for the experiment in Fig. 2. Wells et al. (25) after separating restriction endoAlthough electrophoresis on horizontal agarose slabs was a valuable tool in esti- nuclease-generated fragments from several mating the amount of each component pres- plasmids on RPC-5 columns, have suggested ent at various stagesof purification, electron that the upper ratio limit for these fragments micrographs gave conclusive evidence of the may be 0.5 mg/ml resin bed. In general, they recommend working in the range of I- 10 mg superhelical nature of our final preparation. The purified Col El DNA obtained by RPC- of DNA/30-ml resin bed. It is difficult to 5 chromatography was a satisfactory sub- correlate the column capacity for the resostrate for three restriction endonucleases lution of linear DNA fragments of varying base pair size with that for form I or II Co1 which have been reported to cleave Co1 El El DNA. Our column load of 65 AZhOunits DNA (EcoRI, PstI, and HaeII). of BL II (70% DNA material) on a 25.5-ml Kelmers et al. (26) have discussedthe facresin bed is far below the upper limits of tors regulating the elution of tRNAs from DISCUSSION
RPC-5
CHROMATOGRAPHY
Wells et al. If necessary, it should be possible to expand column capacity by increasing the amount of quaternary ammonium compound (Adogen 464) bound to the hydrophobic support material (Plaskon CTFE 2300 powder) and/or extending the length of the column. The time saved by use of RPC-5 chromatography instead of the usual one or two 40-h buoyant density centrifugations is significant. In a recent publication El-Gewely and Helling reported that they reduced the centrifugation time to 18 h by use of a vertical rotor (28). In our anticipated scale-up of the production of Co1 El DNA, it will be necessary to determine the maximum yield of the plasmid by investigatmg various parameters such as time of addit.ion of chloramphenicol to actively growing cultures, optima1 growth conditions for the E. coli carrier cells, etc. Other phases in the preliminary purification might be modified. and improvements in the postchromatographic concentration could be introduced. ACIKNOWLEDGMENTS The authors express their appreciation to the following members of the Biology Division for advice and encouragement in the preparation of this work: Drs. R. K. Fujimura, S. K. Niyogi. and S. Mitra; Ms. B. C. Roop; and Ms. C. E. Snyder.
REFERENCES 1. Helinski, D. R. (1975) in Comparative Microbiology (Hasegawa, T., ed.), Vol. I, pp. 1977215, Science C’ouncil of Japan, Tokyo. 2. Staudenbauer, W. L. (1978) Curr. Top. Microbial. Itnmunol. 83, 933155. 3. Tomizawa, J.-I. (1978) in DNA Synthesis: Present and Future (Molineaux. I.. and Kohiyama, M., eds.). pp. 7977826. Plenum, New York. 4. Kahn. M., Kolter, R.. Thomas. C., Figurski, D., Meyer. R., Remaut, E., and Helinski. D. R. (1979) in Methods in Enzymology (Wu, R.. ed.), Vol. 68, pp. 2688280, Academic Press, New York.
OF Col El
PLASMID
DNA
243
5. Bastia, D. (1978) J. Mol. Biol. 124, 601-639. 6. Bauer, W.. and Vinograd. J. (1968) J. Mol. Biol. 33, 141-171. 7. Pearson, R. L., Weiss, J. F., and Kelmers, A. D. (1971) Biochim. Biophys. Acta 228, 770-774. 8. Clewell. D. B. (1972) J. Bacferiol. 110, 667-676. 9. Herschman, H. R., and Helinski. D. R. (1967) J. Bacterial. 94, 69 1-699. IO. Clewell, D. B., and Helinski, D. R. (1970) Biu chemistry 9, 4428-4440. Il. Zasloff, M., Cinder, G. D., and Felsenfeld, G. (1978) Nucleic Acids Res. 5, I 139-l 152. 12. McDonell, M. W., Simon, M. N., and Studier, F. W. (1977) J. Mol. Biol. 110, 119-146. 13. Sharp, P. A., Sugden, B., and Sambrook, J. ( 1973) Biochemistry 12, 3055-3063. 14. Helling, R. B., Goodman, H. M., and Boyer. H. W. (I 974) J. Virol. 14, 1235-I 244. 15. Ferguson, J., and Davis, R. W. (1978) in Advanced Techniques in Biological Electron Microscopy (Koehler, J. K., ed.), Vol. 2, pp. 123-167, Springer-Verlag. New York. 16. Blair, D. G.. Sherratt, D. J., Clewell, D. B., and Helinski, D. R. (1972) Proc. Nat. Acad. Sci. USA 69, 25 1882522. 17. Clewell. D. B.. and Evenchik, B. G. (1973) J. Mol. Biol. 75, 503-513. 18. Bazaral. M., and Helinski, D. R. (1968) J. Mol. Biol. 36, 1855194. 19. Tomizawa, J., Sakakibara, Y., and Kakefuda, T. (1974) Proc. Nat. Acad. Sri. USA 71. 22602264. 20. Selker, E., Brown, K., and Yanofsky. C. (1977) J. Bacterial. 129, 388-394. 21. Oka, A., and Takanami, M. (1976) Nature (Londoni 264, 1933196. 22. Inselburg, J., and Ware, P. ( 1977) J. Bacterial. 132, 321-331. 23. Landy. A., Foeller. C., Reszelbach, R., and Dudock, B. (I 976) Nucleic Acids Res. 3, 2575-2592. 24. Eshaghpour, H., and Crothers, D. M. (1978) Nucleic Acids Res. 5, 13-2 1. 25. Wells, R. D., Hardies, S. C., Horn, G. T., Klein, B.. Larson, J. E., Neuendorf, S. K., Panayotatos, N.. Patient, R. K., and Selsing, E. (1980) in Methods in Enzymology. 65, 327-347. 26. Kelmers, A. D.. Weeren, H. 0.. Weiss, J. F.. Pearson, R. L., Stulberg, M. P.. and Novelli, G. D. (I 97 1) in Methods in Enzymology (Moldave, K., and Grossman L. eds.), Vol. 20, pp. 9934, Academic Press, New York. 27. Sugino, Y., Tomizawa, J., and Kakefuda, T. (I 975) Nature (London) 253, 652-654. 28. El-Gewely, M. R.. and Helling, R. B. (I 980) Anal. Biochem. 102, 423-428.
YTICAI.
114, 235-243
BIOCHEMISTRY
IPurification
(1981)
of Supercoiled RPC-5
DNA
of Plasmid
Division,
Oak
Ridge
by
Chromatography’,2
AUDREY N. BEST, DAVID P. ALLISON, Biology
Col El
National
Received
Laboratory. October
AND Oak
G. DAVID NOVELLI Ridge,
Tennessee
37830
16, 1980
Col El DNA can be purified to a high degree by RPC-5 chromatography of a partially purified cell lysate with a very shallow linear NaCl gradient at pH 7.8. Electron micrographs demonstrated that the purest fractions were composed of 93% supercoiled (form I) DNA and 7% open circular (form II) DNA. The actual chromatography can be accomplished in 13- 14 h and is designed for the production of several milligrams of plasmid DNA.
The DNA of the plasmid Co1 El has proved to be a useful vehicle for genetic, biochemical and biophysical studies ( l-3). Procedures bary somewhat in detail for the isolation of the plasmid DNA as the supercoiled closed circular duplex (form I) from Escherichia co/i carrier cells, with subsequent purification to remove contaminants (2,4,5). However, in most procedures the final purification step involves one or two 40h centrifugations by the CsClkethidium bromide buoyant density technique (2,4~-6). Ideally, the resulting Co1 El plasmid DNA preparation will be composed of form I DNA. The other plasmid forms [open circular duplex DNA (form II) and linear duplex DNA (form III)] should be present only as very minor components, and cellular RNAs, chromosomal DNA, and proteins should be removed. Whether the presence of these impurities can be tolerated depends on the intended experimental use of the plasmid DNA. To obtain ;1 preparation with a high con’ Research sponsored by the Office of Health and Environmental Research. U. S. Department of Energy, under Contract IV-7405-eng-26 with the Union Carbide Corporation. ’ The U. S. government’s right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper, for governmental purposes, is acknowledged.
centration of form I Co1 El DNA, we have employed standard isolation and preliminary purification procedures. Avoiding the timeconsumivg buoyant density centrifugations, we have substituted a 13- to 14-h chromatographic procedure using the RPC-5 system (7) to remove residual contaminants such as oligoribonucleotides, form III DNA, chromosomal fragments, and most of form II DNA to give a Co1 E 1 plasmid DNA preparation consisting of 93% form I and 7% form II DNA. This chromatographic procedure should be useful in the preparation of milligram quantities of form I plasmid DNA. MATERIALS
AND METHODS
Bacteria. E. coli strain JC4 11 (thy-, met-, leu-, his-, arg-) (Co1 El), obtained from Dr. D. M. Crothers (Department of Chemistry, Yale University, New Haven, Corm.), was used for the production of the Co1 El plasmid. Reagents. Buffer A was 10 mM Triss HCl/l mM EDTA (pH 7.8); Buffer B, 10 mM Tris-HCl/SO mM NaCl/2 mM EDTA (pH 8); Buffer C, 10 mM Tris-HCl/O.l mM EDTA (pH 7.8); Buffer D, 40 mM TrisHCl/5 mM sodium acetate/l mM EDTA (pH 7.8). Brij 58 (Atlas Chemical Indus-
235 0003-2697/X
l/100235-09$02.00/O
236
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ALLISON,
tries), and sodium deoxycholate and lysozyme (Sigma) were used for cell lysis. Adogen 464 (Ashland Chemical Co.) and Plaskon CTFE 2300 powder (Allied Chemical Co.) were prepared for RPC-5 chromatography by method C of Pearson et al. (7). Agarose powder, low electroendosmosis grade, was manufactured by Marine Colloids, Inc. The restriction endonucleases HaeII, HindHI, and PstI were from Bethesda Research Labs., Inc., and Eco RI was from Sigma. Lambda phage DNA was purchased from Sigma. T7 phage DNA was the gift of Dr. A, L. Stevens, Biology Division. All organic solvents and other chemicals were of reagent grade. Production of Co1 El plasmid DNA. The Co1 El-bearing strain of E. cofi JC411 was grown in 1- to 3-liter cultures on M-9 medium containing 0.4% glucose, 0.5% Casamino Acids (Difco), and 3 pg thymine/ml (Sigma) under conditions similar to those described by Clewell (8) and Herschman and Helinski (9). When the cell concentration reached 2-3 X 10’ cells/ml, 300 pg chloramphenicol/ml (Sigma) was added permitting amplification of the Co1 El plasmid DNA over a period of 16-20 h. A “cleared lysate” was produced from the harvested cells, following the Brij lysis procedure of Clewell and Helinski ( 10). The clear supernatant of the Brij lysate, designated as BL I, was digested first with RNase A (Worthington) and RNase T, (Sankyo Co., Japan), then with proteinase K (E. Merck, Darmstadt, West Germany), in a procedure similar to that described by Zasloff et al. ( 11). The digested lysate was extracted twice with a mixture consisting of 1 vol water-saturated phenol (pH 8) and 0.5 vol chloroform/isoamyl alcohol mixture, 24: 1. The combined aqueous fractions were reextracted once with one volume of chloroform/ isoamyl alcohol, then three times with ether prior to the precipitation of nucleic acid material by alcohol at -20°C. The precipitate was dissolved in a minimum volume of Buffer B and dialyzed exhaustively in this
AND
NOVELL1
buffer. This preparation, designated BL II, was used for the RPC-5 chromatography experiments. RPC-5 chromatography. The Adogen / Plaskon powder mixture was equilibrated with starting NaCl/buffer mixture and packed under pressure at the desired temperature in either of two Glenco 3202 jacketed columns, 0.6 X 30 and 0.6 X 100 cm with final column heights of 20 and 90 cm. Unless otherwise specified, linear gradients (total volume 1 liter) of 0.6-0.7, 0.62-0.72, and 0.68-0.78 M NaCl in Buffer A were used for chromatography at 28-30,37, and 50°C respectively. Theoretically, the NaCl concentration should increase in 0.1 mM increments per milliliter of influent. A Milton Roy minipump was used to adjust the column flow rate between 0.6 and 0.8 ml/min, and the column eluant was monitored by a Chromatronix dual-channel absorbance detector, Model 230, at 254 and 280 nm. Collection volumes were 2-4 ml for the DNA portion of the eluate. A Zeiss PMQ II spectrophotometer was used to measure the absorbance at 260 and 280 nm of all collection tubes containing ultraviolet-absorbing material. When the absorbance values permitted, selected collections were monitored for various forms of Co1 El plasmid DNA by agarose gel electrophoresis before the fractions were pooled. Dialysis with Buffer C preceded the concentration by freeze-drying of the pooled fractions. The residues were dissolved in minimal volumes of Buffer A, and several levels of DNA of comparable concentration, usually 0. l-l .O gg, from each pooled fraction were analyzed to determine the purity, i.e., the proportion of form I Co1 El DNA in relation to forms II and III and chromosomal DNA fragments. Gel electrophoresis. Horizontal agarose slab gels, 13 cm wide X 22 cm long, were prepared by use of the electrophoresis apparatus (Aquebogue, Model 850) initially described by McDonell et al. (12). Agarose gels of 1% in Buffer D were used to estimate the relative amounts of Co1 El plasmid
237
RPC-5CHROMATOGRAPHYOFColElPLASMlDDNA DNA and chromosomal fragments and to establish the: presence of RNA contaminants at various sltages of purification. In general the electrophoresis procedure followed that of Sharp et al. (13). We found that a constant current of 25 mA (l&20 h) was satisfactory for the detection of 445 S RNA in preparation BL I and oligoribonucleotides in BL II. The various forms of Co1 El DNA were resolved in greater detail at 40 mA ( 18- 20 h). When necessary, comparisons were made with 445 S RNA and rRNA preparations. After electrophoresis, DNA and RNA were detected by staining with ethidium bromide (Sigma) 1 pg/ml in Buffer D, then visualized and photographed during shortwave ultraviolet illumination (13). Restriction endonuclease cleavage of Co1 El DNA. EcoRI, HaeII, and PstI restriction enzymes were incubated in separate reactions with purified Co1 El DNA for 18-20 h at 37°C and the reaction conditions were as described by the manufacturer. The amount of DNA, gel strength, and current were adjusted according to the size of fragments generated by the restriction endonucleases. Marker fragments, generated by EcoRI or Hind111 cleavage of phage X DNA, were used to estimate the size of the restriction enzyme-generated Co1 El DNA fragments ( 14). Electron microscopy. DNA samples were prepared for electron microscopy by the aqueous protein film technique (15). The spreading solutions contained 10 ~1 of DNA (4-5 pg/ml in Buffer A), 85 ~1 of 0.5 M ammonium acetate/l mM EDTA (pH 7.8) and 5 ~1 of cytochrome c (2 mg/ml, Calbiochem.). The hypophase was 0.25 M ammonium acetate (pH 7.5) in a 90-mm circular plastic petri dish. The spreading solution (30 ,ul) was allowed to flow down a clean glass slide that had been stored in 0.25 M ammonium acetate (pH 7.5) onto the hypophase. A. parlodion film supported on a 200-mesh csopper grid was touched to the protein film at about one grid width from the slide; rinsed by being touched to 0.25 M
ammonium acetate (pH 7.5), 50% ethanol, 95% ethanol; and finally stained with uranyl acetate. The grids were rotary shadowed with platinum at a 6” angle, and micrographs were taken at random at a magnification of 2000 on a Siemens Elmiskop IA. RESULTS Co1 El DNA Purification
Isolation
and Preliminary
Visualization of the DNA and RNA present in the plasmid-containing lysates (BL I) after gel electrophoresis indicated that form I Co1 El DNA was the predominant DNA, although small amounts of form II Co1 El DNA and E. coli chromosomal DNA were present. After enzyme digestions and phenol extraction, the amount of form II DNA in most BL II preparations appeared to increase to about one-third of the amount of form I. This transition can be expected since random insertion of small RNA segments into one of the plasmid DNA strands occurs during Co1 El replication in the presence of chloramphenicol and up to 50% of the form I DNA can be converted to form II by alkali or RNase A treatment (16). Approximately 95% of the Az6,, units in our BL I preparations consisted of 4-5 S RNA ( 17) which had to be eliminated along with cellular proteins before RPC-5 chromatography could proceed. Residual nondialyzable, low-molecular-weight oligoribonucleotides present in BL II preparations were easily removed by RPC-5 chromatography. RPC-5
Chromatography
In a preliminary experiment (Fig. 1) the oligoribonucleotide fraction (peak 1) of the BL II preparations was excluded almost immediately from a 20-cm RPC-5 column at 37°C with a linear gradient of 0.55-0.925 M NaCl in 250 ml Buffer A. The DNAs were eluted as a single tailing peak between 0.65 and 0.7 M NaCl. Most of form I plasmid
238
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b’ffect of Temperature Chromatography
2a
2b
2.0 -
4
s 0 l.Oc%
0 260 GRADIENT (ml) FIG. I. Separation of various components of a BL II preparation by RPC-5 chromatography. The column load was I ml of BL II (39.5 AzhOunits) followed by 10 ml of starting buffer. The absorbance peaks are identified as follows: peak I (oligoribonucleotides), 7.5-45 ml; peak 2a (main DNA fraction), 80-90 ml; peak 2b (tailing portion of DNA), 91-105 ml. Column conditions are described under Results. The distribution of the various forms of DNA after electrophoresis of two levels of peaks 2a and 2b (0.5 and I.0 bg) is shown above the profile. Gel bands are identified in Fig. 2
DNA emerged in the main portion of the peak (2a), whereas the chromosomal DNA was concentrated in the last tailing portion (2b). The main DNA fraction (2a) contained approximately 80% of the DNA eluted from the column, and analysis by gel electrophoresis indicated that: the concentration of form I DNA was enriched to three to four times the amount of form II; form III was present in trace amounts; and chromosomal DNA was not present. Portions of the 2a fraction were rechromatographed on RPC-5 columns to test the effect of temperature on the separation of forms I, II, and III plasmid DNA with shallow gradients of 0.1 mM increments as described under Methods. Longer columns (90 cm) were used in these experiments because they provided better separation of the three forms of plasmid DNA at 37°C by retarding the emergence of form III.
on RPC-5
DNA was eluted from RPC-5 columns equilibrated at 28830°C and 37°C in the concentration ranges of 0.62-0.63 and 0.660.67 M NaCl, respectively. Although the absorbance profile indicated only one DNA peak, there was enrichment of the three forms in different portions of the DNA fraction. Forms I and II eluted before form III. Although both forms II and III were found in the early collection tubes where the Ale0 values were higher, they were still eluting in the final tailing portion that contained little or no form I DNA. Improved separation of form I DNA from forms II and III was noted with the lower column temperatures. It was necessary to increase the NaCl concentration to elute DNA from RPC-5 columns at 50°C. A series of multiple peaks emerged between 0.71 and 0.73 M NaCI. The gel analysis showed no real separation or concentration of the three forms in any particular peak. In contrast to RPC-5 chromatography at lower temperatures, there was a positional shift, and forms II and III emerged in the first peak. Forms I and II were spread out over the entire profile, so there was no apparent possibility of further enrichment of form I DNA at this temperature. Since an NaCl gradient of 0.1 mM/ml at column temperatures of 28-30°C provided improvement in the separation of Co1 El form I DNA from forms II and III, the gradient concentration was decreased to 0.05 mM/ml increments. A portion of BL II containing the oligoribonucleotide fraction and fragmented chromosomal DNA as well as the three forms of plasmid DNA was applied to the 90-cm RPC-5 column. The elution profile of the DNAs is shown in Fig. 2. Gel electrophoresis analysis of selected collection tubes indicated that forms I and II were eluted in approximately the same proportions until the absorbance peak started to tail off. Gel patterns of the distribution of
RPC-5
CHROMATOGRAPHY
OF Col El PLASMID
3c --II TEL --I BL
ABCDEF
G
-0.64 -0.62 g 300
SbO
5do GRADIENT (ml)
6bO
0.60 =
FIG. 2. Absorbance profile of Col El DNA with RPC5 chromatography. The column load was 65 A200 units of BL II in I.65 ml on a 0.6 X 90-cm RPC-5 column (25.5.ml resin bed) at 3O’C: gradient, I liter 0.6W.65 M NaCl in Buffer A; flow rate. 35 ml/h at 180 psi; fraction collection volume, 2 ml. The oligoribonucleotide fraction (not depicted) was completely eluted in the first 100 ml of gradient. The distribution of the DNAs in BL II and the pooled fractions A-G is shown above the absorbance profile. Gel bands of form I, III, II, and chromosomal DNA fragments (C) are indicated. Samples of DNA (I pg except for fraction A which was 0.2 pg) were electrophoresed in I ‘% agarose gels for I8 h at 40 mA. Chromatography was discontinued temporarily after about I4 h (indicated by I). then resumed until the gradient volume was exhausted.
the various DNAs in seven pooled fractions, A-G, are represented in Fig. 2. Since all samples with the exception of fraction A were the same DNA concentration, visual inspection revealed the relative proportions of the various DNA forms in each fraction with some degree of accuracy. The composition of fractions A-E, eluted between 0.6 16 and 0.62 M NaCl, could be estimated as 8090% form I and 20-10% form II. Neither form III nor chromosomal DNA were visible in the 1-pg samples. The reported lower limit of sensitivity for detection of the DNA/ ethidium bromide complex is about 0.05 pg (I 3), and it can be assumed that the amount of these two forms is less than 5% of the total DNA. The main peak, fractions A-E,
DNA
239
accounted for about 67% of the recovered DNA. In the tailing portion of the DNA peak, represented by fractions F and G, there was more form II than form I, and form III and chromosomal DNA were also eluted. Comparison of fraction F with fraction G suggests that form III began emerging at a somewhat lower NaCl concentration than the chromosomal fragments. The purification of form I (fractions A-E) was complete when the concentration of the NaCl gradient became 0.62 M; the total time required to achieve this portion of the chromatography was 13 h. At least 90% of the column load of 65 Az,,,units of BL II could be recovered from the combined oligoribonucleotide fraction and total DNA eluted from the column. No additional DNA was eluted from the column with 1.0 M NaCl in Buffer A. The composition of the total AZbo units of recovered material was as follows: oligoribonucleotide fraction, 30%; DNA fractions A-E, 47%; and fractions F and G, 23%. Decreasing the gradient concentrations below 0.05 mM/ml increments was not particularly effective in the isolation of form I from form II DNA. The DNA elution was spread out over a larger volume in proportion to the decrease in concentration.
Electron Micrographs
of RPC-S Fractions
Electron micrographs of the pooled Co1 El DNA fractions A-G resulting from RPC-5 chromatography of BL II (Fig. 2) were scored for the distribution of forms I, II, and III and E. co/i chromosomal fragments. The results are presented in Table 1 along with the AzbO units recovered in each fraction. The purest fractions, A--E, which were estimated by agarose gel electrophoresis (Fig. 2) to contain 80-90% form I DNA, were shown by electron microscopy to be composed individually of 86-95% form I DNA. Form II was the only other form of DNA present in these fractions. Fraction C had appeared in several gel electrophoresis
240
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TABLE ELECTRON
MICROKOPIC
ANALYSIS AFTER RPC-5
NOVELL1
I
OF POOLED FRACTIONS CHROMATOGRAPHY
Co1 El DNA Recovered DNA (%) 1.83 16.38 12.35 21.41 14.93 8.45 24.65
Pooled fraction A B C D E F
G B-E mixture’ BL II
Form
I
Form
OF Co1 El
DNA
E. coli chromosomal fragments” (%)
(%)
II
Form
III
87 95 86 95 94 57 30
13 5 14 5 6 40 54
0 0 0 0 0 3 8
0 0 0 0 0 0 8
92 86
8
0
0
11
2
I
’ Fragmented chromosomal contaminants were of variable electrophoresis as phage T7 DNA, 25 X lo6 M,. * This mixture consisted of equal volumes of fractions B-E was tested for reactivity with restriction endonucleases.
experiments to be of equal purity with B, D, and E. Therefore, the value of 86% form I in this fraction was surprising. However, a composite mixture of equal volumes of fractions B-E was found to contain 92% form I. An electron micrograph of this mixture (Fig. 3) shows the predominantly superhelical nature of the purified preparation. The amount of form I DNA in fractions A-E represented 93% of the total ASGounits recovered in this portion of the eluate. Linear form III, 4.2 X lo6 M, (18), visible in fractions F and G in the gel represented in Fig. 2, was present in measurable amounts as shown by electron micrographic analysis. Fraction F showed a loss in form I relative to form II DNA which appeared to be of the same magnitude in both the gel and electron micrographs. The last fraction, G, showed almost twice as much form II as form I in the electron micrographs; the pattern in the gel indicated form II to be present in a larger amount. Chromosomal fragments, collected only in fraction G, were visible in the electron micrographs. It is difficult to correlate the gel pattern
size; the major
portion
showed
the same mobility
(25, 18. 34, and 23% of the respective
fractions)
in and
of the starting material, BL II, with the results from electron micrographic analysis, as the proportion of form I relative to form II DNA appears to be lower in the gel (Fig. 2). However, electrophoresis with vertical agarose gels (not represented here) gave a pattern which was in better agreement with the results in Table 1. In the final analysis, we can state that RPC-5 chromatography eliminated the oligoribonucleotides, form III, and chromosomal fragments from 65 A260 units of the BL II preparation, giving a purified preparation of 27.6 AZbo units or 1.38 mg of Co1 El DNA composed of 93% form I and 7% form II DNA. Cleavage of Co1 El DNA Endonucleases
with Restriction
Co1 El DNA has a single cleavage site for restriction endonuclease EcoRI, two sites for PstI, and six sites for HaeII (19-22). The purified Co1 El DNA (mixture B-E, 92% form I and 8% form II DNA, Table 1) obtained from RPC-5 chromatography was
RPC-5
: . .
‘
< -.
CHROMATOGRAPHY
‘...’ ;.. :‘I ..:
OF
Col El
PLASMID
DNA
241
.‘, .,
FIG. 3. Eclectron micrograph of Col El mixture circles are Indicated. Magnification, 14.000X.
cleaved into fragments of the expected number and size. We can conclude from these results that the suitability of the Col El
of fractions
B-E
(see Table
1). Two
form
II open
DNA as a substrate for these restriction endonucleases was not affected by the chromatographic process.
242
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quaternary ammonium columns of the RPC category. The secondary and tertiary stucThe usefulnessof a procedure for the iso- tures are important factors in the order of lation of a plasmid DNA in the most desired tRNA elution, and it appears from our work form, usually as a supercoiled duplex, is de- that this may also be true for DNA. The gel pendent upon many factors which have been in Fig. 2 showed that superhelical form I discussed in several reviews (2,4). We have DNA eluted at the lowest salt concentration addressed ourselves to the problem of ob- accompanied by a small amount of open cirtaining sufficient quantities of form I Co1 El cular form II DNA. During the preliminary DNA, perhaps 5-20 mg or more, for testing purification, the use of RNase caused the the experimental parameters of the chro- removal of small RNA segments from the matographic separation of restriction endo- form I DNA, resulting in the formation of nuclease-cleaved fragments. RPC-5 columns form II DNA. If the RNA is inserted ranhave been used to separate large amounts domly in either DNA strand, as reported by of restriction endonuclease-cleaved frag- some investigators, form 11 DNA would be ments by several investigators (23-25). heterogeneous since the single-stranded gaps We have not investigated the merits of in the open circular DNA would be at varvarious procedures in the initial stages of ious locations and possibly of different isolation or preliminary purification. Our lengths (2,16,27). The structural alteration main objective was to perform initial puri- due to the opening of the superhelix and the fications to remove as much 4-5 S RNA as existence of single-strand DNA in the nicked possible, eliminate cellular proteins and section are probably important factors in the DNA, and delete RNA sequences which elution pattern of form II DNA. Linear form have been reported to be inserted in Co1 El III DNA and chromosomal fragments were DNA synthesized during chloramphenicoleluted by higher salt concentrations. Base stimulated amplification of the plasmid copy composition, size, and structural change are number (16). The resulting preparation, BL important factors in the chromatography of II, was the test material for developing an these linear DNAs, as demonstrated by RPC-5 chromatographic technique that RPC-5 chromatography of restriction eneliminated an estimated 78% of form II donuclease-generated fragments (25). DNA as well as all of form III and chroThe capacity of the 0.6 X 90-cm RPC-5 mosomal DNA fragments. About 16% of columns for plasmid purification has not form I DNA from BL II was eluted in the been determined; we have not attempted a last fractions containing the above-men- column load of BL II in excessof the amount tioned contaminants. used for the experiment in Fig. 2. Wells et al. (25) after separating restriction endoAlthough electrophoresis on horizontal agarose slabs was a valuable tool in esti- nuclease-generated fragments from several mating the amount of each component pres- plasmids on RPC-5 columns, have suggested ent at various stagesof purification, electron that the upper ratio limit for these fragments micrographs gave conclusive evidence of the may be 0.5 mg/ml resin bed. In general, they recommend working in the range of I- 10 mg superhelical nature of our final preparation. The purified Col El DNA obtained by RPC- of DNA/30-ml resin bed. It is difficult to 5 chromatography was a satisfactory sub- correlate the column capacity for the resostrate for three restriction endonucleases lution of linear DNA fragments of varying base pair size with that for form I or II Co1 which have been reported to cleave Co1 El El DNA. Our column load of 65 AZhOunits DNA (EcoRI, PstI, and HaeII). of BL II (70% DNA material) on a 25.5-ml Kelmers et al. (26) have discussedthe facresin bed is far below the upper limits of tors regulating the elution of tRNAs from DISCUSSION
RPC-5
CHROMATOGRAPHY
Wells et al. If necessary, it should be possible to expand column capacity by increasing the amount of quaternary ammonium compound (Adogen 464) bound to the hydrophobic support material (Plaskon CTFE 2300 powder) and/or extending the length of the column. The time saved by use of RPC-5 chromatography instead of the usual one or two 40-h buoyant density centrifugations is significant. In a recent publication El-Gewely and Helling reported that they reduced the centrifugation time to 18 h by use of a vertical rotor (28). In our anticipated scale-up of the production of Co1 El DNA, it will be necessary to determine the maximum yield of the plasmid by investigatmg various parameters such as time of addit.ion of chloramphenicol to actively growing cultures, optima1 growth conditions for the E. coli carrier cells, etc. Other phases in the preliminary purification might be modified. and improvements in the postchromatographic concentration could be introduced. ACIKNOWLEDGMENTS The authors express their appreciation to the following members of the Biology Division for advice and encouragement in the preparation of this work: Drs. R. K. Fujimura, S. K. Niyogi. and S. Mitra; Ms. B. C. Roop; and Ms. C. E. Snyder.
REFERENCES 1. Helinski, D. R. (1975) in Comparative Microbiology (Hasegawa, T., ed.), Vol. I, pp. 1977215, Science C’ouncil of Japan, Tokyo. 2. Staudenbauer, W. L. (1978) Curr. Top. Microbial. Itnmunol. 83, 933155. 3. Tomizawa, J.-I. (1978) in DNA Synthesis: Present and Future (Molineaux. I.. and Kohiyama, M., eds.). pp. 7977826. Plenum, New York. 4. Kahn. M., Kolter, R.. Thomas. C., Figurski, D., Meyer. R., Remaut, E., and Helinski. D. R. (1979) in Methods in Enzymology (Wu, R.. ed.), Vol. 68, pp. 2688280, Academic Press, New York.
OF Col El
PLASMID
DNA
243
5. Bastia, D. (1978) J. Mol. Biol. 124, 601-639. 6. Bauer, W.. and Vinograd. J. (1968) J. Mol. Biol. 33, 141-171. 7. Pearson, R. L., Weiss, J. F., and Kelmers, A. D. (1971) Biochim. Biophys. Acta 228, 770-774. 8. Clewell. D. B. (1972) J. Bacferiol. 110, 667-676. 9. Herschman, H. R., and Helinski. D. R. (1967) J. Bacterial. 94, 69 1-699. IO. Clewell, D. B., and Helinski, D. R. (1970) Biu chemistry 9, 4428-4440. Il. Zasloff, M., Cinder, G. D., and Felsenfeld, G. (1978) Nucleic Acids Res. 5, I 139-l 152. 12. McDonell, M. W., Simon, M. N., and Studier, F. W. (1977) J. Mol. Biol. 110, 119-146. 13. Sharp, P. A., Sugden, B., and Sambrook, J. ( 1973) Biochemistry 12, 3055-3063. 14. Helling, R. B., Goodman, H. M., and Boyer. H. W. (I 974) J. Virol. 14, 1235-I 244. 15. Ferguson, J., and Davis, R. W. (1978) in Advanced Techniques in Biological Electron Microscopy (Koehler, J. K., ed.), Vol. 2, pp. 123-167, Springer-Verlag. New York. 16. Blair, D. G.. Sherratt, D. J., Clewell, D. B., and Helinski, D. R. (1972) Proc. Nat. Acad. Sci. USA 69, 25 1882522. 17. Clewell. D. B.. and Evenchik, B. G. (1973) J. Mol. Biol. 75, 503-513. 18. Bazaral. M., and Helinski, D. R. (1968) J. Mol. Biol. 36, 1855194. 19. Tomizawa, J., Sakakibara, Y., and Kakefuda, T. (1974) Proc. Nat. Acad. Sri. USA 71. 22602264. 20. Selker, E., Brown, K., and Yanofsky. C. (1977) J. Bacterial. 129, 388-394. 21. Oka, A., and Takanami, M. (1976) Nature (Londoni 264, 1933196. 22. Inselburg, J., and Ware, P. ( 1977) J. Bacterial. 132, 321-331. 23. Landy. A., Foeller. C., Reszelbach, R., and Dudock, B. (I 976) Nucleic Acids Res. 3, 2575-2592. 24. Eshaghpour, H., and Crothers, D. M. (1978) Nucleic Acids Res. 5, 13-2 1. 25. Wells, R. D., Hardies, S. C., Horn, G. T., Klein, B.. Larson, J. E., Neuendorf, S. K., Panayotatos, N.. Patient, R. K., and Selsing, E. (1980) in Methods in Enzymology. 65, 327-347. 26. Kelmers, A. D.. Weeren, H. 0.. Weiss, J. F.. Pearson, R. L., Stulberg, M. P.. and Novelli, G. D. (I 97 1) in Methods in Enzymology (Moldave, K., and Grossman L. eds.), Vol. 20, pp. 9934, Academic Press, New York. 27. Sugino, Y., Tomizawa, J., and Kakefuda, T. (I 975) Nature (London) 253, 652-654. 28. El-Gewely, M. R.. and Helling, R. B. (I 980) Anal. Biochem. 102, 423-428.