C,ene, 7 (1979) 1--14 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
1
PREPARATION AND CHARACTERIZATION OF LARGE AMOUNTS OF RESTRICTION FRAGMENTS CONTAINING THE E. coff lac CONTROL
ELEMENTS (Promoter; operator; CRP binding site; RPC-5 column chromatography; restriction endonucleases EcoRI, HaeIII, HindIII, AluI; plasmids; T M curves) ffI'gPHEN C. HARDIES* and ROBERT D. WELLS University of Wisconsin, Department of Biochemistry, College of Agricultural and Life 8¢ienees, Madison, WI 53706 (U.S.A.) (Received March 19th, 1979) (Revision received May 15th, 1979) (Accepted May 22nd, 1979)
SUMMARY
Large quantities of pure DNA fragments (789, 203 and 95 bp in length) containing the £scherichia coil la¢ controlling elements (operator, promoter, CRP binding site) were prepared from appropriate recombinant plasmids. High pre~ure liquid chromatography on RPC-5 or preparative sucrose gradient centrifugation was used to fractionate the pVH51 vector from the inserts. The fragmer~ts had few, if any, nicks or depurinated sites, and the majority of the fragment ends were intact. Absorbance-temperature profiles on the fragments showed multiphasic transitions. INTRODUCTION
Previous studies in this laboratory and others (Wells et al., 1977; Jovin, 1976) have ndsed the possibility that conformational flexibility in DNA is important to its biological function. Of particular interest is the idea that the sequence in and around a D N A regulatory site generates special conformational properties which are involved in the regulatory interaction. Direct observa-
tions bearing on this hypothesis are difficult to obtain because only average properties are evident when a long D N A molecule is studied. Also, most measurements ~which are sensitive to conformation require large amounts of sam-
pie. *Present address: University of North Carolina, School of Medicine, Department of Bacteriology and Immunology, Chapel Hm, NC 27514 (U.S.A.). Abbreviations: bp, base paL,s; CRP, cAMP receptor protein; PEG, polyethylene glycol; RPC, reversed phase chromatography; TM, melting temperature.
This paper describes the p i n , cation of DNA restriction fragments from recombinant plasmids in suitable quantity to allow investigations of their conformational properties. The construction of recombinant plasmids containing three different insertions (789, 203, and 95 bp in length) of the genetic controlling elements of the £. eoff lactose operon (Hardies et aL, 1979a), and their genetic and biochemical properties were described. Fragments of more than one length were chosen so that the effect of length on the physical properties of the DNA could be determined. This paper describes the isolation of 0.2-4.0 mg of these/ac f~agments. The regulatory proteins which bind in this region, the/ac repressor (Rosenberg et al., 1977), £. co/i RNA polymerase (Burgess and Jendrisak, 1975) and CRP (Eflen et al., 1978) can also be isolated in pure form in quantity. Hence, it is now possible to initiate studies on the mechanism of gene regulation at the level of conformation in ~he protein-DNA complex. The use of RPC-5 column chromatography (Larson et al., 1979, Hsrdies and Wells, 1976, Wells et al., 1979) for the fractionation of the/ac fragments from the vector was m/fispensable in come cases. We have also developed sucrose gradient centrifu~_~ation as a preparative fractionation tool for cases where the vector is at least 20 times as large as the insert. Partial characterization of the fragments is reported. MATERIALSAND ~ETHODS
Preparation of piasmkf DNA The construction and characte~ation of all recombinant DNA plasmids were described (Hardies et al., 1979a). Originally, pRZ3 was prepared using E. ¢oli MO as a host, and pEW1, 2 and 4 using £. ¢oli C600 as a host. £. ¢oU C600 was suitable because it provided high transformation frequencies; however strain MO was more appropriate for the large scale preparation of plas. mid DNA because it gave 10--20-fold higher yields. Hence, pRWl, 2 and 4 (Hardies et al., 1979a) were transformed into strain MO. Plesmid DNA was prepared by a scaled-up version of the published procedune (Hardies et aL, 1979a) and was, in part, suggested by W. Barnes (personal communication). 15 liter cultures were grown with forced aeration in the pilot plant of this Department. Plasmid DNA was amplified by the addition of chloramphenicol at Ass0 = 0.5. The growth medium and amplification procedure were as described previously (Clewell, 1972). Typical preparations gave approx. 30 g packed cells. The cell pellets were suspended in 100 m125% sucrose, 50 mM Tris-HCl (pH 8.0). Lysis was by incubation for 10 min at 0°C with 40 mg lysozyme added in 10 ml 0.25 M Tris.HCl (pH 8.0) followed by addition of 25 ml 0.25 M EDTA and 13 mi 6% Brij 58, 3% deoxycholate. After 10 rain at 0°C the lysate was spun at 25 000 rev./min for 30 min at 4°C in a Spinco 42.1 rotor. The supernatant was made 10% in polyethylene glycol 6000 (Union Carbide Corp.) (Humphreys et al., 1975) and 0.5 M in NaCI and then centrifuged at 6000 rev./min for 15 min in a Sorvall GSA rotor. The pellet
was resuspended in 60 ml 0.I M Tris-HCl (pH 8.0), 0.01 M EDTA, 0.2 mg/ml ethidium bromide by vigorous magnetic stirring. CsCI was added on an equal weight:volume basis. The solutions were then centrifuged at 30 000 rev./min I at 20°C for 48 h in a Spinco 42.1 rotor. Resolution of linear DNA from supercoiled plasmid DNA at this concentration depends on prior steps having removed almost all of the former. After rebanding, the DNA was extracted with isopropanol, dialyzed, and extracted with phenol and dialyzed as described (Hardies etal., 1979a). The recovery of plasmid DNA was usually about 30 rag.
Restriction enzyme digestion Each of the purified plasmid DNAs (pRZ3, pRW2, pRWl or pRW4) was digested with enough of the appropriate restriction enzyme (HindII, HaeHI or EcoRI) to give complete digestion in 100 h under the reaction conditions described (Hardies et al., 1979a). These enzymes maintain full activity (within 30%) for this time period; the long digestion periods were for economy of enzyme. The reactions were analyzed by gel electrophoresis. Sodium azide !(0.02%) was included in all but the HindII reaction to inhibit microbial growth in these long incubations. This concentration of azide did not inhibit any of the restriction enzymes tested (HaeHI, HindII, EcoRI; data not shown).
,preparatiue sucrose gradients Restrict|on digests were prepared for fractionation by phenol extraction, concentration to less than 10 ml and dialysis versus 10 mM Tris.HCl (pH 7.4), ,0.1 mM EDTA. Small scale experiments (using approx. 0.1 mg DNA per tube) i ~ e r e performed with a 5.0 ml linear 5--20% sucrose gradient. The sucrose solu~ l o n s were prepared in 0.1 M NaCI, 0.1 mM EDTA, 10 mM Tris.HCl (pH 7.4), and 0.1 mg/ml ethidium bromide and the DNA in 0.2 ml was layered over the gradient. Centrifugation was at 49 000 rev./min in a Spinco SW50.1 rotor for 11"6h a t 4°C. Larger scale separations (up to 5 mg of DNA digest per tube) were ~"on 36 ml linear 3--22% sucrose gradients in the above buffer. The DNA in ! ~l.4ml was layered over the gradients. Centrifugation was at 25 000 rev./min !,~~ a spinco SW27 rotor for 36 h at 4 °C. The bands were visualized under UV ~t, r etnbved with a pipet from the top, made 2.5 M in NaCI, extracted five ~t~ witli~isoamyl alcohol, extracted once with ether, and dialyzed versus ~(} ~nM ~ , H C I (pH 7.4), 0.1 mM EDTA. !
Melting studies '
Absorbance.temperature transitions (Wells et al., 1970; Burd et al., 1975) were recorded automatically with a Gflford 2000 spectrophotometer equipped with dual thermo spacers. DNA samples were dialyzed into 100 mM NaCI, 1 mM phosphate (pH 7.4), 10/~M EDTA except where noted otherwise. 0.8 ml of solution (0.5--0.7 A260) was read in a Teflon stoppered 0.4 × 1 cm cuvette. The sample was centrifuged to remove particulates and degassed with a fine stream of helium. The tern• perature was raised 1°C/15 min with a Lauda circulating water bath. Tempera-
ture was monitored with a calibrated platinum resistance thermometer positioned in the cell holder. Standard error was -+ 0.001 for an absorbance reading and less than +- 0.01°C for temperature. Readings were taken approximately every 0.025°C and a blank was recorded between each reading. The curves were smoothed graphically by drawing a line through the recordings from the blank and the sample and plotting at every 0.05 °C interval the difference between these curves in percent hyperchromicity. The individual readings did not vary from the smoothed curve by more than the rounding error of the calculation (0.1% hyperchromicity). Readings were not corrected for thermal expansion. Gel eleetrophoresis on separated strands The separated strands of the fragments were studied on a gel system modified from published procedures (Maxam and Gilbert, 1977; Szalay et al., 1977). The gel composition was 5% acrylamide (60:1 with bisacrylamide), 0.5 X Peacock's buffe~o(Peacock and Dingman, 1968), 0.03% TEMED, 0.1% ammonium persulfate. Cylindrical tube gels (0.6 X 15 cm) were run for 2 or 6 h at 200 V in 0.5 times Peacock's buffer. 0.1/~g of DNA sample was loaded in the previously described (Maxam and Gilbert, 1977) high pH solution. The behavior of DNA fragments in this sytem was similar to that described (,Maxam and Gilbert, 1977; Szalay et al., 1977). Alternatively, any pre-existing depurinated sites were first cleaved by incubating 0.1/~g DNA at 37°C for 24 h in 0.75 M NaOH, 25% glycerol, 0.5 mM EDTA ( p H ) 13.5). The sample was then diluted with an equal volume of water, mixed with bromphenol blue, and run as described above. Control studies with acid.treated fragments (0.1 M acetic acid, 10 min, 37°C) (Mitra et al., 1967) showed that this treatment was effective in cleaving depurinated sites (data not shown). RPC.5 chromatography Preparative RPC-5 column chromatography with sodium acetate gradients was done as described (Hardies and Wells, 1976). The DNA was concentrated if necessary and dialyzed into the starting sodium acetate solution for loading. In the case of the final column used to purify the 203 bp fragment from pRW1, the sample (approx. 30 mg in 135 m! 10 mM Tris-HCl, pH 6.8, 0.1 mM EDTA) was concentrated 10.fold, made approx. 0.3 M in KCI, loaded directly, and eluted at 43 °C with a 2-liter linear 0.45-0.85 M KCI gradient. The eluting solutions contained 10 mM Tris-HCl (pH 6.8) and 0.1 mM EDTA. Other materials and methods Other materials and methods were as described elsewhere: analytical buoyant density analyses (Wells and Larson, 1972), gel electrophozesis (Hardies et al., 1979a) hydroxylapatite chromatography (Bernardi, 1971), determination of base composition by UV spectral analyses (Hirschman and Felsenfeld, 1966), restriction enzymes (all prepared in this lab) (Hardies et al., 1979), RPC-5 resin (Larson et al., 1979).
RESULTS
Preparation of 789 bp lac fragment The plasmid p R Z 3 was constructed and characterized by restriction mapping as described (Hardies et al., 1979a). This plasmid consists of the 789 bp fragment, containing the lac operator and p r o m o t e r and the CRP binding site. as well as flanking regions of DNA and the 3800 bp pVH51 vector DNA. The only two HindII sites in pRZ3 DNA are at the junctions between the lac insert and the pVH51 vector. 27 mg o f pRZ3 DNA was prepared and digested with HindII as described in METHODS. Fig. I shows the gel analyses on the preparative separation of the 789 bp /ac fragment from the 3800 bp vector by column chromatography on RPC-5. As expected (Larson et al., 1979; Wells et al., 1979), the two fragments were completely separated and the smaller fragment eluted first. Fractions 72--79 were pooled and contained 4.0 mg of the 789 bp fragment.
Froction
Number
O ¢q 0 ¢q ~" (D ¢U (9 0 U} ¢0 0 0 f'- F- P- I~ O0 GO O~ O~ O~ O~ - --- ~) tO 0 ~ GO oJ m ~ ~ o ~3 p. p= p. O0 O0 O 0 0 ~
VECTOR...
Ioc 7 8 9 - . .
...................................................
Fig. 1. RFC-5 fractionation of 789 bp/ac fragment and 3800 bp pVH51 vector. The RPC-5 column (135 x 0.9 cm) was run essentially as described (Hardies and Wells, 1976) using a 24iter acetate gradient from 1.4 to 1.8 M. 27 mg of DNA digest was applied onto the column. 10-ml fractions were collected. Recovery was 90%. Portions of the fractions were analyzed on 6% polyacrylamide gels run from top to bottom. The gel marked C at the left displays the mixture which was applied onto the column.
Preparation of 203 bp lac fragment containing E c o R I ends The plasmid pRW2 (Hardies et al., 1979a) contains the 203 b p / a c HaeIII fragment which was inserted into linearized pVH51 DNA between two EcoRI half sites which were "f'flled i n " with DNA polymerase. Thus, the EcoRI sites were regenerated at the junction between the 203 bp lac fragment and the
vector. Plasmid pEW2 DNA was prepared and digested with E c o R I as described in METHODS. The two fragments were separated by sucrose gradient centrifugation. Fig. 2 shows the results of a small scale fractionation of 100/~g of the mixture on a 5 ml 5--20% sucrose gradient. The gradient profile (panel B) and the gel electrophoretic analyses on the fractions (panel A) show that the 203 bp/ac fragment was well resolved from the vector which was pelleted on the bottom of the tube. The remainder of the E c o R I digest was fractionated on preparaAO
Fraction
Number .....,q.
' I
~o o~ O l
V E C T O R ----
1o.._£c 2 0 3 - - ~ ,
m
.
0.2 BO
0.1
I0 Fraction
20 Number
Fig. 2. Separation of/o~ 903 bp and pVH51 3800 bp fragments by sucrose gradient centrifugation. A small scale sucrose gradient was used to separate the fragments as described in METHODS except that the dye was omitted. Panel B shows the absorbance at 0.54 m of the eluate, continuously monitored during withdrawal from the top by an Iseo density fractionator. The bottom of the gradient is at the right. 0.25 m | fractions were taken. Panel A shows 5% polyacrylamide gels on portions o~ selected fractions run from top to bottom. The gel marked C at left displays the starting mixture.
tive sucrose gradients in a Spinco SW27 rotor as described in METHODS. Resolution of the two fragments was comparable to that shown in Fig. 2. Recovery from the sucrose gradients was quantitative (> 98%) giving a yield of 330 #g of the 203 bp lac fragment. Preparative gradients did not show overlap of the fragment and vector bands.
C
I00 II
I10 II
A
~ 3 0 0 --~ h2~l 2 0 3 ~
tl.
B Z
1
60
0
IIIlle
(/1 (n
=E Or)
/~ I--
80
II
J
l
I
II0 90 I00 FRACTION NUMBER Fig. 3. RPC-5 fractiormtion of 203 bp ~ c fragment from 300 bp linker. The RPC-5 column (135 × 0.9 cm) was run as described in METHODS at 43°C with a KCI gradient. 10-ml fractions were taken. Panel B shows the continuous monitoring of the transmission profile at 254 nm of the column effluent. The numbers on the ve~icsl scale were arbitrarily set and do not represent true percent transmission. Panel A shows 5% polyacrylamide gels on mmzples of selected fractions. The gel marked C displays the starting mixture. Minor species originate from HaeIII cleavage of contaminants from the preceding RPC-5 chromatography step (see RESULTS).
Preparation of 203 bp lac fragment containing Haelll termini The plasmid pRWl was c o n s t r u ~ (Hardies et al., 1979) in order to prevare the 203 bp/ac HaeHI fragment which retained its HaeIH termini, instead of being changed into a 203 bp ~ e n t with £coRI termini (as described above), pRWI contains two 300 bp linkers symmetrically flanking the 203 bp /ae fragment; this 803 bp £coRI fragment was inserted into the single EcoRI site of pVH51. Hence, the only two EcoRI sites in pRWl DNA are at the junctions of the 803 bp linker-/ac-linker insert and the pVH51 vector. 25 mg of pRW1 DNA was prepared and digested with EeoRI as described in METHODS. The 803 bp linker-/ac-linker fragment was s e p ~ from the 3800 bp vector by RPC-5 chromatography (data not shown). This 803 bp fragment was dialyzed into low salt and then digested with HaeIH to give the 203 bp/a¢ fragment with HaeHI termini and the 300 bp linker fragments in 1:2 molar ratio. This mixture was chromatographed on RPC-5 at 43°C using KCI as the eluting salt at pH 6.8 (Fig. 3). These conditions were an improvement over elution with sodium acetate and permitted the separation of the 203 and 300 bp fragments. Fractions 9 2 - 9 4 contained pure/ac 203, whereas fractions 9 7 - 9 9 contained a sharp peak which was predominantly linker. Total recovery of/ac 203 was 465/~g. There was a single small second peak eluting after the major peak for eac~hmajor component: in fraction 96 for the 203 bp fragment and in fraction 103 for the linker. We do not know the significance of these peaks. Other small peaks are minor components of the starting material which originate from a small amount of vector which was not removed by the first fractionation step. Preparation of 95 bp lac fragment The 95 bp AluI fragment, co~ ~aining the/ac operator and promoter but not the entire CEP binding site, was cloned into ~ e EcoRI site of pVH51 which had been "filled in" by DNA polymerase (Hardies et al., 1979a). Thus, the 95 bp/ac fragment could be cleaved out of the recombinant plasmid pRW4 by cleavage with EcoEI. 31 mg of pRW4 DNA was prepared as described in METHODS except the PEG precipitation step was omitted. After digestion of the plasmid DNA with EcoRI, the 95 bp insert and the 3800 bp vector were separated on preparative sucrose gradients in a manner analogous to the purification of the 203 bp fragment from pEW2 (data not shown). The 95 bp fragment was contaminated with small cellular RNAs (such as tENAs) which obscured visualization of the 95 bp DNA in the sucrose gradient. These RNAs were removed by column chromatography on hydroxylapatite; recovery was excellent (~ 90%). Contamination of plasmid DNA with these cellular RNAs after the density gradient centrifugation steps was observed (as a broad band on polyacrylamide gels at the position of 80 bp) only when the PEG precipitation step was omitted. 0.2 mg of the 95 bp/ac fragment was recovered and judged to be pure by gel electrophoresis.
Assays for nicks or depurinated sites The large scale preparation of the three lac operator-promoter containing fragments (789, 203 and 95 bp in length) was performed for the purpose of carrying out spectroscopic, physical and biochemical investigations. Prior work (Chan et al., 1977) showed the marked influence of nicks in the vicinity of the lac operator on the equilibrium and rate constants for the binding of the/ac repressor. Therefore, it was important to characterize these fragments, as to nicks, depurinated sites or degraded ends, prior to embarking on other types of studies. The separated strands of the 789, 203 (with EcoRI ends), and 95 bp fragments were examined on polyacrylamide gels as described in METHODS (data not shown). In each case a pair of sharp bands appeared at the expected positions. If as few as one nick were present in each of the strands, this result could not be obtained. Thus, the fragments were not nicked or cleaved to the extent of once per strand during the isolation procedures. This extent is an upper limit of the nicking established by the fact that some intact single strands are visible at the appropriate positions on the gels. It may be that, in fact, there are no nicks. Each of the three fragments was also treated with alkali as described in METHODS under conditions known to cause nicks at depurinated sites (data not shown). No significant loss of intensity was detectable when the separated ~qtrands were examined on polyacwlamide gels. It can be concluded from these studies that neither a significant number of depurinated sites nor ribonucleotides are present in the fragments.
Assay for intactness of termini To determine if the termini of the fragments were pat~dally degraded during the isolation procedures, each of the DNAs was treated with the T4 DNA ligase to cause self.Ugation. Subsequently, each of the ligase products was treated with the appropriate restriction endonoclease, HindII for the 789 and EcoRI for the 203 and 95 bp fragments, to determine if the junctions created by the DNA ligase were susceptible to cleavage. Fig. 4 lanes g--i show that the 95 bp fragment undergoes self-ligation extremely efficiently. Thus, the majority of the ends are competent for ligation. The higher oligomers are reconverted to the 95 bp monomer by treatment with £¢oRI (lane i). The 789 and 203 bp DNAs were also self-ligated but the dimer to tetramer were the major products (Fig. 4 lanes b and e, respectively). Thus, 30--50% of the ends of these fragments may have been partially degraded. This approximation is derived by considering that a trimer has four of its six ends joined. Thus, if the trimer is the major product, 66% is an estimate of the number of ends which can be joined. The junctions created by DNA ligase could be recleaved by the appropriate restriction endonucleases (Fig. 4, lanes c, f and i) indicating that the oligomers did not arise from ligation of damaged ends. Assays similar to those described above were not performed on the 203 bp fragment with HaeHI ends.
Fig. 4. Assay for intactness of the termini of fragments. Lanes a--c 1.0% agarose gels of 789 bp fragments. Lanes d--f, 5% polyaerylamide gels of 203 bp EcoRI fragment. Lanes g-i, 5% polyacrylamide gels of 95 bp EcoRI f~agments. Gels a, d and g; untreated fragments. Gels b, e and h; fragments treated with DNA ligase. Gels c, f and i; the ligase-treated fragments which were subsequently recut with the appropriate restriction enzyme (HindH for lane c and EcoRI for lanes f and i). The band appearing across gels d--i at approx. 25% of the distance from the top is a 789 bp marker. The ligation reactions were performed essentially as described (Hardies et al., 1979) and contained 1 #g fragment and 0.5 units of T4 DNA ligase; the reactions were carried out for 18 h at 15° C for the 789 bp DNA and at 0° C for the 203 and 95 bp DNAs. The restriction enzyme recleavsge reactions were essentially as described (Hardies et al., 1979a). The band in lane b which, migrates slightly ahead of the mon, ~¢t is presumably ~ circular ligation produet. The 789 bp and 203 bp reactions are limit products. Conversely, the 95 bp reaction proceeds further with extended reaction times (data not shown).
A bsorbance-temperature transitions The ~ e r m a l denaturation curve o f t h e 789 bp f r a g m e n t in 100 mM sodium ion is shown in Fig. 5. T w e n t y d a t a points per degree are shown. T h e multiphasic nature o f the profile is apparent. This behavior is discussed b e l o w (DISCUSSION). Preliminary d a t a ( n o t shown) for t h e 203 bp fragment in 100 mM sodium ion also shows multiple, though less p r o n o u n c e d , phases. A t 10 mM sodium ion, the multiple phases for t h e 203 b p fragment converge ~oward a single s m o o t h sigmoid curve. T h e T M shifted 15.5°C for a 10-fold change in sodium ion. Detailed helix-coil transition studies on these and related fragments are the subject o f another paper (Hardies et al., 1979b). The base composition of the 7 8 9 and 203 bp fragments was d e t e r m i n e d b y three different m e t h o d s (Table I). T h e standard relationships were used t o calculate the base compositions from the T M d a t a (Marmur and D o t y , 1962), the UV spectral measurements (Hirschman and Felsenfeld, 1966) and t h e anal. ytical CsCI b u o y a n t density profiles (Szybalski and Szybalski, 1971). Each of these m e t h o d s gives the correct base composition, within error, c o m p a r e d t o the compositions from nucleotide sequence studies (Maxam and Gilbert, 1977). The standard relationships for these three m e t h o d s were all derived from mea-
11 40
30
.-e 4-" ¢,,t
E o
2O S
o Q.
/
-r
/
/
I0
b
/
J •
80
" Temperature
n 85 (°C)
"
II 90
Fig. 5. Absorbance-temperature profile for 789 bp fragment in 1 0 0 mM sodium ion. Absorbance changes at 260 nm were monitored from 22°C to the temperatures shown; no change in absorbance was observed b e l o w 78°C. Other details are in METHODS. The recorded T M is 88.7°C and when correctvd for the difference in temperature between the cuvette and the temperature probe, is 86.7°C.
surements on large and polydisperse chromosomal DNAs of various base compositions. Hence, Table I demonstrates that these relationships may be extended to homogeneous restriction fragments as small as 203 bp in length. Moreover, the agreement of the base composition determined from buoyant density values with the sequence shows that the fragments probably are not heavily modified (Szybalski and Szybalski, 1971). The influence of fragment size on the breadth of peaks found in analytical buoyant density determinations is shown elsewhere (Patient et al., 1979). These determinations were not performed on the 95 bp fragment due to the relative scarcity of material and becal~.se it will not effectively band in a cesium salt gradient due to its small size (P~tient et al., 1979). DISCUSSION
Large amounts of four different lac operator-promoter containing fragments were prepared. The fragments were isolated using recombinant plas~lids as a source. The purifications described herein do not approach the limits to which the techniques can be extended. For example, we (S.M. Stirdivant, S.C. Hardies and R.D. Wells, unpublished) have recently purified 6.0 mg of the 203 bp lac fragment from one preparation by a 10-fold scale-up and by using a plasmid containing a triple lac insert (Hardies et al., 1979a).
12
TABLE I PERCENT GC OF/a¢ FRAGMENTS MEASURED BY VARIOUS METHODS Method o f % (G + C) determination
Fragment 789
Fragment 203 a
Nueleotide sequence b ITj~ d e t e ~ t i o ~ spectral determinations e Native Hyperehromie shift Analytical CsCi buoyant density f
54.6 c 56
48.7 47
51 55 53
49 52 49
aThe 203 bp fragment with EcoRI termini was studied. bThe single-stranded termini were not included in this calculation. eTwo of the bp in the 789 bp sequence are unknown (Maxam and Gilbert, 1977) and hence this value is ± 0.2 %(G + C). dT M is dei"med in the classical way. The observed T M's (86.7 and 82.6 for the 789 and 203 DNAs respectively under the conditions of Fig. 5) were corrected to 0.2 M sodium ion (Dove and Davidson, 1962) and the base composition was calculated by the standard relationship (Marmur and Dory, 1962) derived for chromosomal DNAs. The expected error is • 2%(G + C). eThe accuracy of these methods is ± 3%(G + C). fThe error from these determinations is ± 1%(G + C). The density values were 1.706 and 1.702 g/era s for the 789 and 203 D N A fragments respectively. We are grateful to J.E. L a t i n for performing these experiments.
Several experiments were done to establish the intactness and homogeneity of these fragments. The separated ~ n d s form sharp bands on polyacrylamide gels; thus, the fragments were not nicked or depurinated to the extent of once per strand. The efficiency with which the ends were ligated showed that the 95 bp fragment had nearly all ends intact whereas the 203 and 789 bp fragments had no more than half of their ends damaged. We believe that the improvement in the condition of the 95 bp fragment over that of the 203 and 789 bp fragments was due to the inclusion of 0.02% sodium azide in the lengthy restriction reactions and the inclusion of 0.1 mM EDTA in all buffers. In this work preparative RPC-5 columns were run either using sodium acetate or KCI as an eluting salt. The elution profiles for the separation of 789 bp fragment from vector and 803 bp fragment from vector showed many peaks per fragment. Attempted separations of 203 bp fragment (with EcoRI ends) from linker using sodium acetate elution from RPC-5 failed due to loss of resolution brought about by this peak splitting (data not shown). In the latter case, the same material was successfully fractionated using the KCI elution conditions (Fig. 3). This improvement in performance using KCI instead of sodium acetate was expected in the light of results obtained from analytical RPC-5 experiments (Larson et al., 1979). Peak splitting with sodium acetate has been shown to be a column artifact and not due to heterogeneity in the DNA fragments (Wells et al., 1979).
13 The absorbance-temperature profiles show a complex behavior since the entire ler, gth o f the fragments does n o t melt at the same temperature. These studies are currently being extended (Hardies et al., 1979b) by performing determinations at highe~ resolution (finer temperature control and spectroscopic resolution) and by employing other restriction fragments related to those described herein. Also, the cause of the submelting transitions is uncertain at present and is under investigation. It is anticipated that studies on the melting properties o f the sequence constituting the p r o m o t e r will be relevant to the mechanism o f RNA polymerase action. Previous w o r k (Vizard and Ansevin, 1 9 7 6 Guttman et al., 1977; Blake and Lefolev, 1978; Tachibana et al., 1978) has identified subspecies of thermal transitions (thermalites) in larger DNAs. ACKNOWLEDGEMENTS
This w o r k was supported by grants from the National Institutes of Health (CA 20279) and the National Scienc:~ Foundation (PCM 77-15033). Partial support was provided to S.C.H. by a predoctoral fellowship from the National Science F o u n d a t i o n and to R.D.W. by the J o h n Simon Guggenheim Founu dation during a portion of this work. REFERENCES
Bernardi, G., Chromatography of nucleic acids on hydroxyapatite columns. Meth. Enzymol., 21 (1971) 95--139. Blake, R.D. and Lefoley, S.G., Spectral analysis of high resolution direct-derivative meltring curves of DNA for instantaneous and total base composition, Biochim. Biophys. Acta, 518 (1978) 233--246. Burd, J.F., Wartell, R.M., Dodgson, J.B. and Wells, R.D., Transmission of stability (telestability) in DNA: physical and enzymatic studies on the duplex block polymer d(ClsAis).d(TzsGls), J. Biol. Chem., 250 (1975) 5109--5113. Burgess, R.R. and Jendrisak, J.J., A procedure for the rapid, large-scale purification of Escherichia coU DNA-dependent RNA polymerase involving polymin P precipitation and DNA-cellulose chromatography, Biochemistry, 14 (1975) 4634--4638. Chan, H.W., Dodgson, J.B. and Wells, R.D., The influence of DNA structure on the lactose operator-repressor interaction, Biochemistry, 16 (1977) 2356--2366. Clewell, D.B., Nature of ColE1 plasmid replication in Escherichia coli in the presence of chloramphenicol, J. Bacteriol., 110 (1972) 667--676. Dove, W.F. and Davidson, N., Cation effects on the denaturation of DNA, J. Mol. Biol., 5 (1962) 467--478. Eilen, E., Pampeno, C. and Krakow, J.S., Production and properties of the a ~ore derived from the cyclic AMP receptor protein of Escheriehia coli, Biochemistry~ 17 (1978) 2469--2473. Guttmann, T., Vitek, A. and Pivec, L., High resolution thermal denaturation of mammalian DNAs, Nucl. Acids Res., 4 (1977) 285--297. Hardies, S.C. and Wells, R.D., Preparative fractionation of DNA restriction fragments ~y reversed phase column chromatography, Proc. Natl. Acad. Sci. USA, 73 (1976) 3117-3121, Hardies, S.C., Patient, R.K., Klein, R.D., Ho, F., Reznikoff, W.S. and Wells, R.D., Construction and mapping of recombinant plasmids used for the p~eparation of DNA fragments containing the E. coli lactose operator and promoter, J. Biol. Chem., 254 (1979a) 5527--5534.
14 Hardies, S.C., Hillen, W., Goodman, T.C. and Wells, ILD., High resolution thermal denatur ation analyses of small sequenced DNA restriction fragments containing £. coff lactose genetic control loci, J. BioL Chem,, (1979b) in press. Himehnum, $,~. and Felsenfeld, G., Determln~tion of DNA composition and concentration by spectral analysis, J. Mol. Biol., 16 (1966) 347--358. Humphreys, GO., Willshaw, G.A., and Anderson, E.8., A simple method for the preparation of large quantities of pure plasmid DNA, Bioehim Biophys. Acte, 383 (1975) 457--463. Jovin, TXI., Recognition mechanisms of DNA-specifie enzymes, .annu. Rev. Bioehem., 45 (1976} 889--920. Lamon, J.E., Hardies, S.C., Patient, R.K. and Wells, R.D., Factors influencing fraetionation of DNA restriction fragments by RPC-§ column ~hromatography, J. Biol. Chem., (19/9) in the press. Mannur, J. and Dory, P., DetenninJion of the base composition of deoxyribonucleic acid from its thermal denaturation temperature, J. Mol. Biol., 5 (1962) 109--118. M~r~m, AM. and Gilbert, W., A new method for sequencing DNA, Proe. Natl. Acad. Sei. USA, 74 (1977) 560--664, and personal communication. Mitra, A., Rekhard, P., Inman, R.B., Bertseh, L.L. and Kornberg, A., Enzymic synthesis of deoxyribonucleic acid, XXIL Replication of a circular s i n g l ~ e d DNA template by DNA polymerase of Eseherichia eoli, J. Mol. Biol., 24 (1967) 429-447. Patient, R.K., Hazdies, S.C., Larson, J~E., Inman, R.B., Maquat, L.E. and Wells, R.D., Influence of A-T content on the fractionation of DNA restriction fragments by RPC-5 column chromatography, J. Biol. Chem., 254 (1979) 5548--5554. Peacock, A.C. and Dingman, C.W., Molacular weight estimation and separation of ribonueleie acid by eleetrophoresis in agaroee-acrylamide composite gels, Biochemistry, 7 (1968) 668--674. Rosenberg, JJd., Kallai, O.B., Kopka, M.L., Diekerson, R.F~ and l~ias, A.D., Lac repressor puri/'~ation without inactivation of DNA binding activity, Nncl. Acids, ReL, 4 (1977) §67--572. Szalay, A.A., Grohmann, K. and Sinsheimer, R.L., Separation of the complementary strands of DNA fragments in polyacgyhunide gels, Nuel. Acids Res., 4 (1977) 1569-1578. 8zybalski, W. and 8zybalski, E.H., Equilibrium density gradient eentrifugatlon, in Procedures in Nucleic Acid Research, Vol. 9, G.L. Centoni and D.R. Davies (gds.), Harper and Row, New York, 1971, pp. $U--$54. Tachibana, H., Wada, d,, Gotoh, O and Takanami, M., Location of the cooperative melting regions in bacteriophage fd DNA, Bioehim. Biophys. Aeta, 517 (1978) 319-328.
Vizard, D.L. and Ansevin, A.T., High resolution thermal denaturation of DNA: thermalites of bacteriophqe DNA, Bioehemktry, 15 (1976) 741-750. Wells, R.D., Larson, J.E., Grant, R.C., 8hortle, B.E. and Cantor, C.R., Phyaleoehemieal studies on poly0eoxyribonueleotides containing defined repeating nueleotide sequences, J. Mol. Biol., 54 (1970) 465-497. Wells, R.D. and Larson, J.E., Buoyant density studies on natural synthetic DNAs in neutral and alkaline solutions, J. Biol. Chem., 247 (1972) 3405--3409. Wells, R.D., Blakesley, R.W., Burd, J.F., Chan, H.W., Dodgson, J.B., Hardies, S.C., Horn, G.T., Jensen, K.F., Larson, J.E., Nes, LF., 8elsing, E. and Wartell, R.M., The role of DNA structure in genetic regulation, CRC Crit. Rev. Bioehem., 4 (1977) 305-340.
Wells, R.D., Hardies, S.C., Horn, G.T., Klein, B., Lanon, J.E., Neuendorf, 8.K., Panayotatos, N., Patient, R.K. and Seking, E., RPC-5 eolunm chromatography f,~ *,he isolation of DNA fragments, Meth. Enzymol., (1979) in the press. Communicated by H.O. Smith.
1
PREPARATION AND CHARACTERIZATION OF LARGE AMOUNTS OF RESTRICTION FRAGMENTS CONTAINING THE E. coff lac CONTROL
ELEMENTS (Promoter; operator; CRP binding site; RPC-5 column chromatography; restriction endonucleases EcoRI, HaeIII, HindIII, AluI; plasmids; T M curves) ffI'gPHEN C. HARDIES* and ROBERT D. WELLS University of Wisconsin, Department of Biochemistry, College of Agricultural and Life 8¢ienees, Madison, WI 53706 (U.S.A.) (Received March 19th, 1979) (Revision received May 15th, 1979) (Accepted May 22nd, 1979)
SUMMARY
Large quantities of pure DNA fragments (789, 203 and 95 bp in length) containing the £scherichia coil la¢ controlling elements (operator, promoter, CRP binding site) were prepared from appropriate recombinant plasmids. High pre~ure liquid chromatography on RPC-5 or preparative sucrose gradient centrifugation was used to fractionate the pVH51 vector from the inserts. The fragmer~ts had few, if any, nicks or depurinated sites, and the majority of the fragment ends were intact. Absorbance-temperature profiles on the fragments showed multiphasic transitions. INTRODUCTION
Previous studies in this laboratory and others (Wells et al., 1977; Jovin, 1976) have ndsed the possibility that conformational flexibility in DNA is important to its biological function. Of particular interest is the idea that the sequence in and around a D N A regulatory site generates special conformational properties which are involved in the regulatory interaction. Direct observa-
tions bearing on this hypothesis are difficult to obtain because only average properties are evident when a long D N A molecule is studied. Also, most measurements ~which are sensitive to conformation require large amounts of sam-
pie. *Present address: University of North Carolina, School of Medicine, Department of Bacteriology and Immunology, Chapel Hm, NC 27514 (U.S.A.). Abbreviations: bp, base paL,s; CRP, cAMP receptor protein; PEG, polyethylene glycol; RPC, reversed phase chromatography; TM, melting temperature.
This paper describes the p i n , cation of DNA restriction fragments from recombinant plasmids in suitable quantity to allow investigations of their conformational properties. The construction of recombinant plasmids containing three different insertions (789, 203, and 95 bp in length) of the genetic controlling elements of the £. eoff lactose operon (Hardies et aL, 1979a), and their genetic and biochemical properties were described. Fragments of more than one length were chosen so that the effect of length on the physical properties of the DNA could be determined. This paper describes the isolation of 0.2-4.0 mg of these/ac f~agments. The regulatory proteins which bind in this region, the/ac repressor (Rosenberg et al., 1977), £. co/i RNA polymerase (Burgess and Jendrisak, 1975) and CRP (Eflen et al., 1978) can also be isolated in pure form in quantity. Hence, it is now possible to initiate studies on the mechanism of gene regulation at the level of conformation in ~he protein-DNA complex. The use of RPC-5 column chromatography (Larson et al., 1979, Hsrdies and Wells, 1976, Wells et al., 1979) for the fractionation of the/ac fragments from the vector was m/fispensable in come cases. We have also developed sucrose gradient centrifu~_~ation as a preparative fractionation tool for cases where the vector is at least 20 times as large as the insert. Partial characterization of the fragments is reported. MATERIALSAND ~ETHODS
Preparation of piasmkf DNA The construction and characte~ation of all recombinant DNA plasmids were described (Hardies et al., 1979a). Originally, pRZ3 was prepared using E. ¢oli MO as a host, and pEW1, 2 and 4 using £. ¢oli C600 as a host. £. ¢oU C600 was suitable because it provided high transformation frequencies; however strain MO was more appropriate for the large scale preparation of plas. mid DNA because it gave 10--20-fold higher yields. Hence, pRWl, 2 and 4 (Hardies et al., 1979a) were transformed into strain MO. Plesmid DNA was prepared by a scaled-up version of the published procedune (Hardies et aL, 1979a) and was, in part, suggested by W. Barnes (personal communication). 15 liter cultures were grown with forced aeration in the pilot plant of this Department. Plasmid DNA was amplified by the addition of chloramphenicol at Ass0 = 0.5. The growth medium and amplification procedure were as described previously (Clewell, 1972). Typical preparations gave approx. 30 g packed cells. The cell pellets were suspended in 100 m125% sucrose, 50 mM Tris-HCl (pH 8.0). Lysis was by incubation for 10 min at 0°C with 40 mg lysozyme added in 10 ml 0.25 M Tris.HCl (pH 8.0) followed by addition of 25 ml 0.25 M EDTA and 13 mi 6% Brij 58, 3% deoxycholate. After 10 rain at 0°C the lysate was spun at 25 000 rev./min for 30 min at 4°C in a Spinco 42.1 rotor. The supernatant was made 10% in polyethylene glycol 6000 (Union Carbide Corp.) (Humphreys et al., 1975) and 0.5 M in NaCI and then centrifuged at 6000 rev./min for 15 min in a Sorvall GSA rotor. The pellet
was resuspended in 60 ml 0.I M Tris-HCl (pH 8.0), 0.01 M EDTA, 0.2 mg/ml ethidium bromide by vigorous magnetic stirring. CsCI was added on an equal weight:volume basis. The solutions were then centrifuged at 30 000 rev./min I at 20°C for 48 h in a Spinco 42.1 rotor. Resolution of linear DNA from supercoiled plasmid DNA at this concentration depends on prior steps having removed almost all of the former. After rebanding, the DNA was extracted with isopropanol, dialyzed, and extracted with phenol and dialyzed as described (Hardies etal., 1979a). The recovery of plasmid DNA was usually about 30 rag.
Restriction enzyme digestion Each of the purified plasmid DNAs (pRZ3, pRW2, pRWl or pRW4) was digested with enough of the appropriate restriction enzyme (HindII, HaeHI or EcoRI) to give complete digestion in 100 h under the reaction conditions described (Hardies et al., 1979a). These enzymes maintain full activity (within 30%) for this time period; the long digestion periods were for economy of enzyme. The reactions were analyzed by gel electrophoresis. Sodium azide !(0.02%) was included in all but the HindII reaction to inhibit microbial growth in these long incubations. This concentration of azide did not inhibit any of the restriction enzymes tested (HaeHI, HindII, EcoRI; data not shown).
,preparatiue sucrose gradients Restrict|on digests were prepared for fractionation by phenol extraction, concentration to less than 10 ml and dialysis versus 10 mM Tris.HCl (pH 7.4), ,0.1 mM EDTA. Small scale experiments (using approx. 0.1 mg DNA per tube) i ~ e r e performed with a 5.0 ml linear 5--20% sucrose gradient. The sucrose solu~ l o n s were prepared in 0.1 M NaCI, 0.1 mM EDTA, 10 mM Tris.HCl (pH 7.4), and 0.1 mg/ml ethidium bromide and the DNA in 0.2 ml was layered over the gradient. Centrifugation was at 49 000 rev./min in a Spinco SW50.1 rotor for 11"6h a t 4°C. Larger scale separations (up to 5 mg of DNA digest per tube) were ~"on 36 ml linear 3--22% sucrose gradients in the above buffer. The DNA in ! ~l.4ml was layered over the gradients. Centrifugation was at 25 000 rev./min !,~~ a spinco SW27 rotor for 36 h at 4 °C. The bands were visualized under UV ~t, r etnbved with a pipet from the top, made 2.5 M in NaCI, extracted five ~t~ witli~isoamyl alcohol, extracted once with ether, and dialyzed versus ~(} ~nM ~ , H C I (pH 7.4), 0.1 mM EDTA. !
Melting studies '
Absorbance.temperature transitions (Wells et al., 1970; Burd et al., 1975) were recorded automatically with a Gflford 2000 spectrophotometer equipped with dual thermo spacers. DNA samples were dialyzed into 100 mM NaCI, 1 mM phosphate (pH 7.4), 10/~M EDTA except where noted otherwise. 0.8 ml of solution (0.5--0.7 A260) was read in a Teflon stoppered 0.4 × 1 cm cuvette. The sample was centrifuged to remove particulates and degassed with a fine stream of helium. The tern• perature was raised 1°C/15 min with a Lauda circulating water bath. Tempera-
ture was monitored with a calibrated platinum resistance thermometer positioned in the cell holder. Standard error was -+ 0.001 for an absorbance reading and less than +- 0.01°C for temperature. Readings were taken approximately every 0.025°C and a blank was recorded between each reading. The curves were smoothed graphically by drawing a line through the recordings from the blank and the sample and plotting at every 0.05 °C interval the difference between these curves in percent hyperchromicity. The individual readings did not vary from the smoothed curve by more than the rounding error of the calculation (0.1% hyperchromicity). Readings were not corrected for thermal expansion. Gel eleetrophoresis on separated strands The separated strands of the fragments were studied on a gel system modified from published procedures (Maxam and Gilbert, 1977; Szalay et al., 1977). The gel composition was 5% acrylamide (60:1 with bisacrylamide), 0.5 X Peacock's buffe~o(Peacock and Dingman, 1968), 0.03% TEMED, 0.1% ammonium persulfate. Cylindrical tube gels (0.6 X 15 cm) were run for 2 or 6 h at 200 V in 0.5 times Peacock's buffer. 0.1/~g of DNA sample was loaded in the previously described (Maxam and Gilbert, 1977) high pH solution. The behavior of DNA fragments in this sytem was similar to that described (,Maxam and Gilbert, 1977; Szalay et al., 1977). Alternatively, any pre-existing depurinated sites were first cleaved by incubating 0.1/~g DNA at 37°C for 24 h in 0.75 M NaOH, 25% glycerol, 0.5 mM EDTA ( p H ) 13.5). The sample was then diluted with an equal volume of water, mixed with bromphenol blue, and run as described above. Control studies with acid.treated fragments (0.1 M acetic acid, 10 min, 37°C) (Mitra et al., 1967) showed that this treatment was effective in cleaving depurinated sites (data not shown). RPC.5 chromatography Preparative RPC-5 column chromatography with sodium acetate gradients was done as described (Hardies and Wells, 1976). The DNA was concentrated if necessary and dialyzed into the starting sodium acetate solution for loading. In the case of the final column used to purify the 203 bp fragment from pRW1, the sample (approx. 30 mg in 135 m! 10 mM Tris-HCl, pH 6.8, 0.1 mM EDTA) was concentrated 10.fold, made approx. 0.3 M in KCI, loaded directly, and eluted at 43 °C with a 2-liter linear 0.45-0.85 M KCI gradient. The eluting solutions contained 10 mM Tris-HCl (pH 6.8) and 0.1 mM EDTA. Other materials and methods Other materials and methods were as described elsewhere: analytical buoyant density analyses (Wells and Larson, 1972), gel electrophozesis (Hardies et al., 1979a) hydroxylapatite chromatography (Bernardi, 1971), determination of base composition by UV spectral analyses (Hirschman and Felsenfeld, 1966), restriction enzymes (all prepared in this lab) (Hardies et al., 1979), RPC-5 resin (Larson et al., 1979).
RESULTS
Preparation of 789 bp lac fragment The plasmid p R Z 3 was constructed and characterized by restriction mapping as described (Hardies et al., 1979a). This plasmid consists of the 789 bp fragment, containing the lac operator and p r o m o t e r and the CRP binding site. as well as flanking regions of DNA and the 3800 bp pVH51 vector DNA. The only two HindII sites in pRZ3 DNA are at the junctions between the lac insert and the pVH51 vector. 27 mg o f pRZ3 DNA was prepared and digested with HindII as described in METHODS. Fig. I shows the gel analyses on the preparative separation of the 789 bp /ac fragment from the 3800 bp vector by column chromatography on RPC-5. As expected (Larson et al., 1979; Wells et al., 1979), the two fragments were completely separated and the smaller fragment eluted first. Fractions 72--79 were pooled and contained 4.0 mg of the 789 bp fragment.
Froction
Number
O ¢q 0 ¢q ~" (D ¢U (9 0 U} ¢0 0 0 f'- F- P- I~ O0 GO O~ O~ O~ O~ - --- ~) tO 0 ~ GO oJ m ~ ~ o ~3 p. p= p. O0 O0 O 0 0 ~
VECTOR...
Ioc 7 8 9 - . .
...................................................
Fig. 1. RFC-5 fractionation of 789 bp/ac fragment and 3800 bp pVH51 vector. The RPC-5 column (135 x 0.9 cm) was run essentially as described (Hardies and Wells, 1976) using a 24iter acetate gradient from 1.4 to 1.8 M. 27 mg of DNA digest was applied onto the column. 10-ml fractions were collected. Recovery was 90%. Portions of the fractions were analyzed on 6% polyacrylamide gels run from top to bottom. The gel marked C at the left displays the mixture which was applied onto the column.
Preparation of 203 bp lac fragment containing E c o R I ends The plasmid pRW2 (Hardies et al., 1979a) contains the 203 b p / a c HaeIII fragment which was inserted into linearized pVH51 DNA between two EcoRI half sites which were "f'flled i n " with DNA polymerase. Thus, the EcoRI sites were regenerated at the junction between the 203 bp lac fragment and the
vector. Plasmid pEW2 DNA was prepared and digested with E c o R I as described in METHODS. The two fragments were separated by sucrose gradient centrifugation. Fig. 2 shows the results of a small scale fractionation of 100/~g of the mixture on a 5 ml 5--20% sucrose gradient. The gradient profile (panel B) and the gel electrophoretic analyses on the fractions (panel A) show that the 203 bp/ac fragment was well resolved from the vector which was pelleted on the bottom of the tube. The remainder of the E c o R I digest was fractionated on preparaAO
Fraction
Number .....,q.
' I
~o o~ O l
V E C T O R ----
1o.._£c 2 0 3 - - ~ ,
m
.
0.2 BO
0.1
I0 Fraction
20 Number
Fig. 2. Separation of/o~ 903 bp and pVH51 3800 bp fragments by sucrose gradient centrifugation. A small scale sucrose gradient was used to separate the fragments as described in METHODS except that the dye was omitted. Panel B shows the absorbance at 0.54 m of the eluate, continuously monitored during withdrawal from the top by an Iseo density fractionator. The bottom of the gradient is at the right. 0.25 m | fractions were taken. Panel A shows 5% polyacrylamide gels on portions o~ selected fractions run from top to bottom. The gel marked C at left displays the starting mixture.
tive sucrose gradients in a Spinco SW27 rotor as described in METHODS. Resolution of the two fragments was comparable to that shown in Fig. 2. Recovery from the sucrose gradients was quantitative (> 98%) giving a yield of 330 #g of the 203 bp lac fragment. Preparative gradients did not show overlap of the fragment and vector bands.
C
I00 II
I10 II
A
~ 3 0 0 --~ h2~l 2 0 3 ~
tl.
B Z
1
60
0
IIIlle
(/1 (n
=E Or)
/~ I--
80
II
J
l
I
II0 90 I00 FRACTION NUMBER Fig. 3. RPC-5 fractiormtion of 203 bp ~ c fragment from 300 bp linker. The RPC-5 column (135 × 0.9 cm) was run as described in METHODS at 43°C with a KCI gradient. 10-ml fractions were taken. Panel B shows the continuous monitoring of the transmission profile at 254 nm of the column effluent. The numbers on the ve~icsl scale were arbitrarily set and do not represent true percent transmission. Panel A shows 5% polyacrylamide gels on mmzples of selected fractions. The gel marked C displays the starting mixture. Minor species originate from HaeIII cleavage of contaminants from the preceding RPC-5 chromatography step (see RESULTS).
Preparation of 203 bp lac fragment containing Haelll termini The plasmid pRWl was c o n s t r u ~ (Hardies et al., 1979) in order to prevare the 203 bp/ac HaeHI fragment which retained its HaeIH termini, instead of being changed into a 203 bp ~ e n t with £coRI termini (as described above), pRWI contains two 300 bp linkers symmetrically flanking the 203 bp /ae fragment; this 803 bp £coRI fragment was inserted into the single EcoRI site of pVH51. Hence, the only two EcoRI sites in pRWl DNA are at the junctions of the 803 bp linker-/ac-linker insert and the pVH51 vector. 25 mg of pRW1 DNA was prepared and digested with EeoRI as described in METHODS. The 803 bp linker-/ac-linker fragment was s e p ~ from the 3800 bp vector by RPC-5 chromatography (data not shown). This 803 bp fragment was dialyzed into low salt and then digested with HaeIH to give the 203 bp/a¢ fragment with HaeHI termini and the 300 bp linker fragments in 1:2 molar ratio. This mixture was chromatographed on RPC-5 at 43°C using KCI as the eluting salt at pH 6.8 (Fig. 3). These conditions were an improvement over elution with sodium acetate and permitted the separation of the 203 and 300 bp fragments. Fractions 9 2 - 9 4 contained pure/ac 203, whereas fractions 9 7 - 9 9 contained a sharp peak which was predominantly linker. Total recovery of/ac 203 was 465/~g. There was a single small second peak eluting after the major peak for eac~hmajor component: in fraction 96 for the 203 bp fragment and in fraction 103 for the linker. We do not know the significance of these peaks. Other small peaks are minor components of the starting material which originate from a small amount of vector which was not removed by the first fractionation step. Preparation of 95 bp lac fragment The 95 bp AluI fragment, co~ ~aining the/ac operator and promoter but not the entire CEP binding site, was cloned into ~ e EcoRI site of pVH51 which had been "filled in" by DNA polymerase (Hardies et al., 1979a). Thus, the 95 bp/ac fragment could be cleaved out of the recombinant plasmid pRW4 by cleavage with EcoEI. 31 mg of pRW4 DNA was prepared as described in METHODS except the PEG precipitation step was omitted. After digestion of the plasmid DNA with EcoRI, the 95 bp insert and the 3800 bp vector were separated on preparative sucrose gradients in a manner analogous to the purification of the 203 bp fragment from pEW2 (data not shown). The 95 bp fragment was contaminated with small cellular RNAs (such as tENAs) which obscured visualization of the 95 bp DNA in the sucrose gradient. These RNAs were removed by column chromatography on hydroxylapatite; recovery was excellent (~ 90%). Contamination of plasmid DNA with these cellular RNAs after the density gradient centrifugation steps was observed (as a broad band on polyacrylamide gels at the position of 80 bp) only when the PEG precipitation step was omitted. 0.2 mg of the 95 bp/ac fragment was recovered and judged to be pure by gel electrophoresis.
Assays for nicks or depurinated sites The large scale preparation of the three lac operator-promoter containing fragments (789, 203 and 95 bp in length) was performed for the purpose of carrying out spectroscopic, physical and biochemical investigations. Prior work (Chan et al., 1977) showed the marked influence of nicks in the vicinity of the lac operator on the equilibrium and rate constants for the binding of the/ac repressor. Therefore, it was important to characterize these fragments, as to nicks, depurinated sites or degraded ends, prior to embarking on other types of studies. The separated strands of the 789, 203 (with EcoRI ends), and 95 bp fragments were examined on polyacrylamide gels as described in METHODS (data not shown). In each case a pair of sharp bands appeared at the expected positions. If as few as one nick were present in each of the strands, this result could not be obtained. Thus, the fragments were not nicked or cleaved to the extent of once per strand during the isolation procedures. This extent is an upper limit of the nicking established by the fact that some intact single strands are visible at the appropriate positions on the gels. It may be that, in fact, there are no nicks. Each of the three fragments was also treated with alkali as described in METHODS under conditions known to cause nicks at depurinated sites (data not shown). No significant loss of intensity was detectable when the separated ~qtrands were examined on polyacwlamide gels. It can be concluded from these studies that neither a significant number of depurinated sites nor ribonucleotides are present in the fragments.
Assay for intactness of termini To determine if the termini of the fragments were pat~dally degraded during the isolation procedures, each of the DNAs was treated with the T4 DNA ligase to cause self.Ugation. Subsequently, each of the ligase products was treated with the appropriate restriction endonoclease, HindII for the 789 and EcoRI for the 203 and 95 bp fragments, to determine if the junctions created by the DNA ligase were susceptible to cleavage. Fig. 4 lanes g--i show that the 95 bp fragment undergoes self-ligation extremely efficiently. Thus, the majority of the ends are competent for ligation. The higher oligomers are reconverted to the 95 bp monomer by treatment with £¢oRI (lane i). The 789 and 203 bp DNAs were also self-ligated but the dimer to tetramer were the major products (Fig. 4 lanes b and e, respectively). Thus, 30--50% of the ends of these fragments may have been partially degraded. This approximation is derived by considering that a trimer has four of its six ends joined. Thus, if the trimer is the major product, 66% is an estimate of the number of ends which can be joined. The junctions created by DNA ligase could be recleaved by the appropriate restriction endonucleases (Fig. 4, lanes c, f and i) indicating that the oligomers did not arise from ligation of damaged ends. Assays similar to those described above were not performed on the 203 bp fragment with HaeHI ends.
Fig. 4. Assay for intactness of the termini of fragments. Lanes a--c 1.0% agarose gels of 789 bp fragments. Lanes d--f, 5% polyaerylamide gels of 203 bp EcoRI fragment. Lanes g-i, 5% polyacrylamide gels of 95 bp EcoRI f~agments. Gels a, d and g; untreated fragments. Gels b, e and h; fragments treated with DNA ligase. Gels c, f and i; the ligase-treated fragments which were subsequently recut with the appropriate restriction enzyme (HindH for lane c and EcoRI for lanes f and i). The band appearing across gels d--i at approx. 25% of the distance from the top is a 789 bp marker. The ligation reactions were performed essentially as described (Hardies et al., 1979) and contained 1 #g fragment and 0.5 units of T4 DNA ligase; the reactions were carried out for 18 h at 15° C for the 789 bp DNA and at 0° C for the 203 and 95 bp DNAs. The restriction enzyme recleavsge reactions were essentially as described (Hardies et al., 1979a). The band in lane b which, migrates slightly ahead of the mon, ~¢t is presumably ~ circular ligation produet. The 789 bp and 203 bp reactions are limit products. Conversely, the 95 bp reaction proceeds further with extended reaction times (data not shown).
A bsorbance-temperature transitions The ~ e r m a l denaturation curve o f t h e 789 bp f r a g m e n t in 100 mM sodium ion is shown in Fig. 5. T w e n t y d a t a points per degree are shown. T h e multiphasic nature o f the profile is apparent. This behavior is discussed b e l o w (DISCUSSION). Preliminary d a t a ( n o t shown) for t h e 203 bp fragment in 100 mM sodium ion also shows multiple, though less p r o n o u n c e d , phases. A t 10 mM sodium ion, the multiple phases for t h e 203 b p fragment converge ~oward a single s m o o t h sigmoid curve. T h e T M shifted 15.5°C for a 10-fold change in sodium ion. Detailed helix-coil transition studies on these and related fragments are the subject o f another paper (Hardies et al., 1979b). The base composition of the 7 8 9 and 203 bp fragments was d e t e r m i n e d b y three different m e t h o d s (Table I). T h e standard relationships were used t o calculate the base compositions from the T M d a t a (Marmur and D o t y , 1962), the UV spectral measurements (Hirschman and Felsenfeld, 1966) and t h e anal. ytical CsCI b u o y a n t density profiles (Szybalski and Szybalski, 1971). Each of these m e t h o d s gives the correct base composition, within error, c o m p a r e d t o the compositions from nucleotide sequence studies (Maxam and Gilbert, 1977). The standard relationships for these three m e t h o d s were all derived from mea-
11 40
30
.-e 4-" ¢,,t
E o
2O S
o Q.
/
-r
/
/
I0
b
/
J •
80
" Temperature
n 85 (°C)
"
II 90
Fig. 5. Absorbance-temperature profile for 789 bp fragment in 1 0 0 mM sodium ion. Absorbance changes at 260 nm were monitored from 22°C to the temperatures shown; no change in absorbance was observed b e l o w 78°C. Other details are in METHODS. The recorded T M is 88.7°C and when correctvd for the difference in temperature between the cuvette and the temperature probe, is 86.7°C.
surements on large and polydisperse chromosomal DNAs of various base compositions. Hence, Table I demonstrates that these relationships may be extended to homogeneous restriction fragments as small as 203 bp in length. Moreover, the agreement of the base composition determined from buoyant density values with the sequence shows that the fragments probably are not heavily modified (Szybalski and Szybalski, 1971). The influence of fragment size on the breadth of peaks found in analytical buoyant density determinations is shown elsewhere (Patient et al., 1979). These determinations were not performed on the 95 bp fragment due to the relative scarcity of material and becal~.se it will not effectively band in a cesium salt gradient due to its small size (P~tient et al., 1979). DISCUSSION
Large amounts of four different lac operator-promoter containing fragments were prepared. The fragments were isolated using recombinant plas~lids as a source. The purifications described herein do not approach the limits to which the techniques can be extended. For example, we (S.M. Stirdivant, S.C. Hardies and R.D. Wells, unpublished) have recently purified 6.0 mg of the 203 bp lac fragment from one preparation by a 10-fold scale-up and by using a plasmid containing a triple lac insert (Hardies et al., 1979a).
12
TABLE I PERCENT GC OF/a¢ FRAGMENTS MEASURED BY VARIOUS METHODS Method o f % (G + C) determination
Fragment 789
Fragment 203 a
Nueleotide sequence b ITj~ d e t e ~ t i o ~ spectral determinations e Native Hyperehromie shift Analytical CsCi buoyant density f
54.6 c 56
48.7 47
51 55 53
49 52 49
aThe 203 bp fragment with EcoRI termini was studied. bThe single-stranded termini were not included in this calculation. eTwo of the bp in the 789 bp sequence are unknown (Maxam and Gilbert, 1977) and hence this value is ± 0.2 %(G + C). dT M is dei"med in the classical way. The observed T M's (86.7 and 82.6 for the 789 and 203 DNAs respectively under the conditions of Fig. 5) were corrected to 0.2 M sodium ion (Dove and Davidson, 1962) and the base composition was calculated by the standard relationship (Marmur and Dory, 1962) derived for chromosomal DNAs. The expected error is • 2%(G + C). eThe accuracy of these methods is ± 3%(G + C). fThe error from these determinations is ± 1%(G + C). The density values were 1.706 and 1.702 g/era s for the 789 and 203 D N A fragments respectively. We are grateful to J.E. L a t i n for performing these experiments.
Several experiments were done to establish the intactness and homogeneity of these fragments. The separated ~ n d s form sharp bands on polyacrylamide gels; thus, the fragments were not nicked or depurinated to the extent of once per strand. The efficiency with which the ends were ligated showed that the 95 bp fragment had nearly all ends intact whereas the 203 and 789 bp fragments had no more than half of their ends damaged. We believe that the improvement in the condition of the 95 bp fragment over that of the 203 and 789 bp fragments was due to the inclusion of 0.02% sodium azide in the lengthy restriction reactions and the inclusion of 0.1 mM EDTA in all buffers. In this work preparative RPC-5 columns were run either using sodium acetate or KCI as an eluting salt. The elution profiles for the separation of 789 bp fragment from vector and 803 bp fragment from vector showed many peaks per fragment. Attempted separations of 203 bp fragment (with EcoRI ends) from linker using sodium acetate elution from RPC-5 failed due to loss of resolution brought about by this peak splitting (data not shown). In the latter case, the same material was successfully fractionated using the KCI elution conditions (Fig. 3). This improvement in performance using KCI instead of sodium acetate was expected in the light of results obtained from analytical RPC-5 experiments (Larson et al., 1979). Peak splitting with sodium acetate has been shown to be a column artifact and not due to heterogeneity in the DNA fragments (Wells et al., 1979).
13 The absorbance-temperature profiles show a complex behavior since the entire ler, gth o f the fragments does n o t melt at the same temperature. These studies are currently being extended (Hardies et al., 1979b) by performing determinations at highe~ resolution (finer temperature control and spectroscopic resolution) and by employing other restriction fragments related to those described herein. Also, the cause of the submelting transitions is uncertain at present and is under investigation. It is anticipated that studies on the melting properties o f the sequence constituting the p r o m o t e r will be relevant to the mechanism o f RNA polymerase action. Previous w o r k (Vizard and Ansevin, 1 9 7 6 Guttman et al., 1977; Blake and Lefolev, 1978; Tachibana et al., 1978) has identified subspecies of thermal transitions (thermalites) in larger DNAs. ACKNOWLEDGEMENTS
This w o r k was supported by grants from the National Institutes of Health (CA 20279) and the National Scienc:~ Foundation (PCM 77-15033). Partial support was provided to S.C.H. by a predoctoral fellowship from the National Science F o u n d a t i o n and to R.D.W. by the J o h n Simon Guggenheim Founu dation during a portion of this work. REFERENCES
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