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Electron microscopic visualization of restriction sites on DNA molecules
Gene, 6 (1979) 319--330 319 O mseviar/North-Holland Biomedicsl Press, .4n~terdAm -- Printed in The Netherlands
ELECTRON MICROSCOPIC VISUALIZATION OF RESTRICTION SITES ON D N A MOLECULES
(Polylysine adsorption; Xdvland fd; EcoRI and HinHI; in situ digestion; DNA gap at cleavage sites)
TAKF,SHI MIWA*, MITSURU TAKANAMI** and HIDEO YAMAGISHI
Department of Biophysic& Faculty of Science, Kyoto University, Kyoto, Kyoto 606 and **Institute for Chemical Research, Kyoto University, Uj~ Kyoto 611 (Japan) (Received March 24th, 1979) (Revision received and accepted April 20th, 1979)
SUMMARY DNA molecules were adsorbed to a polylysine-treated carbon film and digested directly on the film by restriction enzymes. After washing the film with 1 M NaCl, 0.4% Kodak Photo-Flo and 9% form~mide, each cleavage site introduced was visualized as a gap under the electron microscope. By measuring the gapped positions on linear DNA molecules induced by other enzymes, a~ EeoRl site on aXdvlmolecule and three HinHI sites on an fdlP.F molecule were mapped at the positions expected from the cleavage maps, respectively. This electron-microscopic procedure may be useful for the construction o f a cleavage map. INTRODUCTION
Restriction endor, ucleases have proved extremely useful for cleaving a DNA molecule into unique fragments. Fragments produced by the enzymes have generally been ordered on the DNA molecule by analysis of partial cleavage products, but this mapping procedure requires many biochemical steps. If the cleavage sites on a DNA molecule could be visualized under an electron microscope, it would be helpful for the construction of a cleavage map. In this paper, DNA molecules were adsorbed to a polylysine-treated carbon film; ,and after digestion with restriction enzymes the cleavage sites were visualized as gaps.
*Present addrem: Laboratory of Molecular Genetics, University of 0~__ka, Medical School, Kita-ku, Osaka 530 (Japan).
820
DNA and enzymes Phage fd RFI DNA was prepared as described by Sugiura et al. (1969). Plasmid Xdvl DNAwas prepared (Matsubara et al., 1975) and generously donated by IL Matsubam. Covalently closed circles of ~,dvl DNA were convetted to open circles by X-ray irradiation at the dose corresponding to 0.5 hit. Restriction endonuclesses Eco RI, Bam HI, Hin dll and Hin Hl (is~schizomer of HaeH; see Roberts, 1978) were purified as described earlier (Takanami, 1973). One unit of enzyme activity is expressed as the activity which completely digests 1 ~g Z DNA by incubation for 60 mln at 37°C. In situ d ~ t i o n o f DNA Polylysine treatment of support filmRand adsorption of DNA to the films were performed as described by Williams (!977) with the following modifications; the films were made hydrophilic by subjecting them for 10 sec to a glow discharge at 200 millitorr air pressure; satisfactory DI~A concentrations for adsorption were in the range from I to 8 #g/m' ,.'n 100 mM Tris-HCI buffer (pH 7.6) and 10 mM EDTA; droplets of about 8/~l of DNA solution were applied on the treated fflmR and allowed to adsorb for 3 to 5 rain. After the drop was drained away, the filmed surface of the grid was rinsed twice by touching the surface of 25/d-drops of distilled water. After draining, a 5/d drop of 0.1 to 0.6 units of restriction enzyme solutions was then applied on the film. Enzyme solutions of EcoRI contained, besides the enzyme, 10 mM Tris.HCl (pH 7.6), 5 mM MgCI2 and 100 mM NaCI, while the other enzyme solutions contained only 10 mM Trk-HCI (pH 7.6) and 5 mM MgCI2. Digestion was carried out for 10 to 60 rain at 37°C in the humid chamber. After incubation, the film was rinsed for 15 sec with 0.1 ml droplet of I M NaCI and occasionally followed by rinsings with 0.4% aqueous Kodak Photo-Flo (pH 8.8) or 9% formamide in 10 mM Tris-HCI (pH 7.6) and I mM EDTA. Drops for rinsing were conveniently held on a fresh sheet of parAfllm and an inverted grid was touched to a droplet surface. To observe the DNA/enzyme complex, the ~,rocedure of rin~ngs was omitted. After the excess liquid was drained away the grid was rinsed with distilled water and stained with 5% aqueous uranyl acetate as described by Williams (1977). Electron microscopy The dried specimen grids were rotary shadowed at an angle of 6 ° with tungsten (Griffith et al., 1971). A tungsten wire of 0.5 mm in diameter and 5 cm in free length, was inltially heated with a 30.5-A current at 2-10 -6 tort, after which the applied voltage was left constant for 80 sec during rotary shadowing. Electron micrographs were obtained on a JEM 7A electron microscope, with primary magnification of 20 000 X. Length measurements were made with a Hewiett-Packard 9864A digitizer and 9810A calculator.
321 RESULTS
Length measurements in DNA molecules adsorbed to the polylysine-treated carbon film The protein monolayer film technique originated by Kleinschmidt ~ d Zahn (1959) has been demonstrated to be enormously useful to spread the DNA evenly on the electron microscope grid. However, electron microscopic studies on protein-nueleic acid interaction can be performed only with f~ee nucleic acids. To mount the protein-free DNA on the film, we have exploited the polylysine~carbon film technique (Williams, 1978). In this technique,the stretching and distribution of the long DNA polymers may be uneven in the absence of embedment in the carrier protein. To see the extent of the length variability, we compared the length measurements by the two different techniques, the polylysine technique and the protein monofilm technique and the results are summarized in Fig. 1. In this figure, the standard deviation is plotted against the square root of the mean length of the DNA according to Davis et al. (1971). The standard deviation of the length measurement employing the polylysine technique is about twice as large as that obtained when using the protein monofilm technique. The contour length is shortened by about 6% in the polylysine technique; the mean length of BamHI-digested Xdvl DNA was 2.01 + 0.15 #m by the polylysine technique (30 measurements) and 2.13 + 0.05 #m by the protein monolayer technique (18 measurements). As shown in Fig. 2a, DNA molecules tend to aggregate laterally and to distribute unevenly, when using the polylysine technique.
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322
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Fig.2. gleetron m ~ o g r s p h s of the open circuls~ xdvl DNA ( s - e ) and the £coRI.Umearized DNA ( f - j ) which were first adsorbed to polylysine f'dm (t, f), and then treated with ~.¢oR[ (b) or J~zmHI (g), respectively and followed by washings with 1 M NaCI (e, h), with 1 M NsC! and l~hoto-Flo (d, i) or with I M NaCI and 9% fornutmide (e, j). Restriction enzyme molecules were ehown by trianllles and the DNA gape by arrows. (× 34 900.)
323
Visualization o f cleavage sites. The important features of our technique include the following preparation steps; (1) adsorption of DNA to the polylysine. treated f i l m s ; ( 2 ) in situ digestion of the adsorbed DNA with restriction enzymes; (3) removal of the enzymes and conversion of the breaks into visible gaps. Fig. 2 shows the open circular Xdvl DNA(a--e) and the linear restriction fragments ( f - j ) that were observed at each preparation step; step I (a and f), step 2 (b and g) and step 3 (c--e and h--j). At step 2, Xdvl DNA is treated with EcoRI (b) and the EcoRI-fragment with BamHI (g). The globular molecules associated with the DNA must be the restriction enzyme, since such complexes were observed only when the enzyme was added. As shown in Fig. 2g, DNA strand is occasionally gapped at the site of complex formation. Rinsing with 1 M NaCI at step 3 (c and h) was useful to remove the enzyme and thus disclose the break. Successive rinsings with dilute Photo-Fie (d and i) or 9% formamide (e and j) served to enlarge the gap. Mapping the EcoRl site on BamHl-fragment o f Xdvl DNA Plasmid Xdvl DNA is the circular tandem dimer and the cleavage sites for EcoItl and BamHI are located at the symmetrical position as shown in Fig. 3& Therefore, two DNA gaps at the symmetrical position are expected in the open circular Xdvl DNA treated with EcoRI. However, under the present in situ digestion, most DNA molecules are intact and only a few molecules bear a single gap (Fig. 2c--e). Hardly any molecules bearing two gaps were observed. These frequencies were statistically predictable. It may be that the enzyme digests the molecule only partially. To show the gap is introduced at a specific site, we prepared the linear BamHI-fragments, subjected them to in situ digestion with EcoRI, rinsed in 1 M NaCI and examined the molecule bearing the gap (Fig. 4). The gap wa~
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Ban~4(0500) (0A21 Fi~ 3. Cleavage maps of the ~dvl DNAdimer (a) and the fd RF DNA (b). One of the two equivsdant BamHl cleavqe sites is designated as the zero point in xdvl m a p ; t h e Hindll site k designated as the zero in fd map. Each cleavage site is shown by an arrow. The joints betwmm xdvl monomers are shown by double bars. The map distance from the zero point is measured in the counterclockwise direction and expressed as fraction of the total length. xdvlcleavage map is constructed according to the rasults by Chow eta]. (1974), Thomas et sd. (1975) and Perricaudet and Tiollsis (1975~ fd RF cleavage map is described by Tskanami et aL (1975). Total genome length is 14 300 base pairs for x d v l (Chow et al., 1974; Fiandt et al., 1977) and 6408 base pairs for fd RF (Beck et aI., 1978).
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Fig. 4. Typical electron micrograph of linezr xdvlBamllY-frsgment bearing a single gap introduced by EcoRI digestio~ The gap is shown by an arrow. The ssmple was rinsed in 1 M NsCI. (x 77 600.)
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detected at only a single site on this fragment. The position of the gap was measured from the closest end and a histogram of the results is shown in Fig. 5. Most of the gaps were situated at 37% of the fragment length from one end. The value is very close to 35%, in agreement with the value derived from the physical map of Fig. 3 ~ Additional minor sites of the gap may represent the sites for EcoRI* activity found by Polisky et al. (1975) and Tikchonenko et al. (1978). Mapping the HinHl sites on a linear fd D N A molecule A cleavage map of the fd RF DNA is shown in Fig. 3b. There are a single cleavage site for HindII and three cleavage sites for HinHI. In this section, we mapped the binding sites of HinHI as well as their cleavage sites on a linear molecule o f fd RF DNA which had been produced by cleavage of circles with HindII. As shown in Fig. 6, we can see the complex formation with HinHI. Locations of the enzyme on the DNA molecules were measured from one end and the molecules were oriented so as to locate most of the enzymes in a close correlation for every pair of molecules as described by Giacomoni et al. (1977) (Fig. 7a). The results are shown in the form of a histogram (Fig. 7b).
Fig. 6. Complex between HinHI and a fd DNA molecule linearized with HindIl. Bound HinHI molecules are shown by triangles. (× 77 600.)
Fig. 5. Positions of EcoRI cleavage site on kdvl BamHl-fragment. (a) Graphic array of the representative fragments subjected to in sito digestion with EcoRI and rinsed in 1 M NaCI. "fine position o f the gap is shown by the vertical bar to indicate the nature of the primary data. The length of DNA molecules was normalized and the distances were measured from 'the closest end as fraction of the entire fragment length. (b) A hktogram showing the location of gaps. The arrow indicates the cleavage site derived from the physical map in Fig. 3a.
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Fif~ 7. Position of HinHI molecules on a fd RF DNA molecule linearized with H/ndIL (a) Graphic array of the representative fd RF DNAs forming s complex with HinHL The position of the enzyme is sho~m by the circle. The length of DNA molecules is normalized sad the distances of the enzyme molecules from one end of the DNA molecule are expressed Is fraction of the entire DNA lenjth. The DNA molecules ate oriented so ss to correlate the position of enzymes closdy for every pair of DNAs. (b) A hkto~am showing the location of the HinHl molecules. The arrow Indicates the cleavsge site derived from the physical map in Fig. 8b.
Although a background noise appears on the histogram, the positions of the complex peak agree well with the cleavage m a p by H l n H I (Fig. 3b) in which cleavage sites are situated at 42%, 72% and 84% respectively, of the entke molecular length from one end. To confirm the cleavage sites by showing the D N A gaps, we removed the bound enzyme from the D N A molecules. Fig. 8 shows some representative examples containing a single gap (a), two gaps (b) and three gaps (c) in a molecule. Molecules containing multiple gaps were few, probably due either to the preferential occurrences o f partial digestions or to the failure in opening the staggered ends. The gap positions on the D N A molecules were measured and the results are shown in Fig. 9. Background noise in the histogram of the complex between H i n H l and the HJndII-treated DNA is now reduced in the histogram of the cleavage gaps revealing the true peaks in the expected sites for HinHI.
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DISCUSSION The techniques of protein-free spreading of nucleic acid for electron microscopic observations have undergone a considerable improvement allowing the visualiT.ation of proteins bound to D N A (Dubochet et al., 1971; Vollenweider et al., 1975; Williams, 1977). These techniques have been used to visualize the stable complex between type I restriction enzyme E c o K and
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Fil~ 9. Position o f HinHl cleavage sites on a fd RIP D N A linearized with HindlL Same as Fig. 7 except that the HinHl-treated samples were rinsed as described in Fig. 8 and the
pcLdtion of the eleavsge gap k shown by the vertiesl bar.
DNA (Bracket al., 1976). In the absence of ATP, EcoK forms a stable complex with DNA without cleaving it. However, such a stable complex is not found for a type II restriction enzyme. In the present study (Figs. 6 and 7), the recognition sites for type II enzyme HinHI were mapped by examining the binding of enzyme to the DNA immobilized on the carbon film. The method of in situ complex formation may be applicable for other type II enzymes. However, a high background noise due to nonspecific adsorption of enzyme molecules is the drawback in this method. To overcome this drawback, we have developed the new method to visualize the cleavages by removing the restriction enzyme molecule from the complex and disclosing the endonucleolytic breaks on the DNA molecule. In the in situ digestion, most DNA molecules are only partially cleaved with restriction enzymes as shown for EcoRI-treated ~,dvl DNA(Fig. 2b-e) and H/nHI-treated fd RF DNA (Figs. 8 and 9). By showing the position of the DNA gaps in the form of a histogram (Figs. 5 and 9), cleavage sites can be mapped on the molecule. In the previous studies, a partial digestion was required to determine the order of restriction fragments on a genome, as reviewed by Nathans and Smith (1975). Therefore, the present method is very useful to order the restric. tion fragments on the cleavage map. Moreover, an accurate length measure-
329
ment o f each restriction fragment can be performed separately by protein monolayer method. For direct visualization o f all the cleavage sites on each entire genome DNA, we still have to perfect our technique as to obtain a complete in situ digestion. ACKNOWLEDGEMENTS
This work was supported by a grant-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science and Culture, Japan. REFERENCES
Beck, E., Sommer, R., Auerswald, E.A., Kurz, C., Zink, 13., Osterburg, G., Schaller, H., Sugimoto, K., Sugisaki, H., Okamoto, T. and Takanami, M., Nueleotide sequence of bacteriophage fd DNA, Nucleic Acids Re~, 5 (1978)4495--4503. Brack, C., Eberle, NL, Bickle, T.A. and Yuan, R., Mapping of recognition sites for the restriction endonuclease from Escherichia ¢oli K-12 on bacteriophage PM2 DNA, J. biol. Biol., 108 (1976) 583--593. C~ow, LT., Davidson, N. and Berg, D., Electron microscope study of the structures of kdv DNAs, J. Mol. Biol., 86 (1974) 69-89. Davis, l~W., Simon, M. and Davidson, N., Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, Methods Enzymol., 21D
(1971) 413-428. Duboehet, J., Ducommun, )/L, Zollinger, M. and Kellenberger, E., A new preparation method for dark-field electron microscopy of biomolecules, J. Ultrastruct. Res., 35 (1971) 147--167. Fiandt, M., Honlgman, A., Rosenvold, E.C. and Szybaiski, W., Precise measurement of the b2 deletion in coliphage larnbda, Gene, 2 (1977) 289-293. Giacomoni, P.U., Delain, E. and Le Pecq, J.B., Electron microscopy analysis of the interaction between EmcheHchia ¢oli DNA-dependent RNA polymerase and the replicative form of phage fd DNA, Eur. J. Biochem., 78 (1,977) 205--213. Griffith, J., Huberman, J.A. and Kornberg, A., Electron microscopy of DNA polymerase bound to DNA, J. Mol. Biol., 56 (1971) 209-214. Kleinschmidt, A. and Zshn, I~K., Uber Desoxyribonucleinsiiure-Molekeln in ProteinMischfilmen, Z. Naturforsch., 14b (1959) 779-779. Matsubera, K., Takagi, Y. and Mukai, T., In vitro construction of different oligomerie forms of ~.dv DNA and studies on their transforming activities, J. Virol., 16 (1975) 479-485. Nathans, D. and Smith, H.O., Restriction endonucleascs in the analysis and restrt.~turing of DNA molecules, Annu. Rev. Biochem., 44 (1975) 273--293. Perr~candet, M. and Tiollsjs, P., Defective bacteriophage lambda chromosome, potential vector for DNA fragments obtained after cleavage by Bacili~ amyiolique[acienJ endonuelease (Baml), FEBS Lett., 56 (1975) 7--11. Polisky, B., Greene, P., Graf'm, D.E., McCarthy, ILJ., Goodman, ILM. and Boyer, ELW., Specificity of substrate recognition by the E¢oRI restriction endonuelease, Prnc. Natl. Acad. 8eL USA, 72 (1975) 3319-3314. Roberts, R.J., Restriction and modification enzymes and their recognition sequences, Gene, 4 (1978) 183--193. Su&~ura, M., Okmnoto, T. and Takanami, M., Starting nucleotide sequences of RNA synthesized on the replieative form DNA of eoliphqe fd, J. Mol. Biol., 43 (1969) 299-315. Takansmi, M., Specific cleavage of coliphage fd DNA by five different restriction endonucleases from Haemophilus genus, FEBS Lett., 34 (1973) 318-322.
880
Taksnmn~ M., Okamoto, T., 8uliimoto, K. n d 8ul~bski, I-L, 8 t u d i l on baet~riophqe fd DNA, L A eleavase map of the fd gemom~ J. Idol. Biol., 95 (1976) 21--81. Thomas, M. and Davis, R.W., 8t'udim on the d u v s g e of bactmiophqe lsmbds DNA with EcoRl re~a~iefion mdonucleme, J. MoL Biol., 91 (1975)315--328. 11kehone~o, T.L, ~ o v , E.V., Zavizion, B.A. and Nm~xlitaky, B.8., EeoRl activity; emzyme modification or sctivM.ion of accompanying endonucleme? Gene, 4 (1978) 195--'212.
Vollenweider, ELJ., ~Qgo, J.M. and Ko/ler, Th~ A routine me~od for protein-free spmsdi~ of doulde- and singlwstrsnded nucleic acid molecules, Pro~ NatL Acad. 8ci. USA, 72 (1976) 83---87. ILC., Use of polylydne for adsorption of nucleic aeicls m d enzymes to electron mierow~pe specimen films, M . M~L Acad. 8eL USA, 74 (1977) 2311--2315. Yamsgiehi, IL, Inokuehi, H, and Ozeki, IL, Excision snd duplication of s u r ~ m s d u c i n g fragments carried by bacteriophsge @80, J. Virol., 18(1976) 1016--1023. Communicated by K. MaUmbam.
ELECTRON MICROSCOPIC VISUALIZATION OF RESTRICTION SITES ON D N A MOLECULES
(Polylysine adsorption; Xdvland fd; EcoRI and HinHI; in situ digestion; DNA gap at cleavage sites)
TAKF,SHI MIWA*, MITSURU TAKANAMI** and HIDEO YAMAGISHI
Department of Biophysic& Faculty of Science, Kyoto University, Kyoto, Kyoto 606 and **Institute for Chemical Research, Kyoto University, Uj~ Kyoto 611 (Japan) (Received March 24th, 1979) (Revision received and accepted April 20th, 1979)
SUMMARY DNA molecules were adsorbed to a polylysine-treated carbon film and digested directly on the film by restriction enzymes. After washing the film with 1 M NaCl, 0.4% Kodak Photo-Flo and 9% form~mide, each cleavage site introduced was visualized as a gap under the electron microscope. By measuring the gapped positions on linear DNA molecules induced by other enzymes, a~ EeoRl site on aXdvlmolecule and three HinHI sites on an fdlP.F molecule were mapped at the positions expected from the cleavage maps, respectively. This electron-microscopic procedure may be useful for the construction o f a cleavage map. INTRODUCTION
Restriction endor, ucleases have proved extremely useful for cleaving a DNA molecule into unique fragments. Fragments produced by the enzymes have generally been ordered on the DNA molecule by analysis of partial cleavage products, but this mapping procedure requires many biochemical steps. If the cleavage sites on a DNA molecule could be visualized under an electron microscope, it would be helpful for the construction of a cleavage map. In this paper, DNA molecules were adsorbed to a polylysine-treated carbon film; ,and after digestion with restriction enzymes the cleavage sites were visualized as gaps.
*Present addrem: Laboratory of Molecular Genetics, University of 0~__ka, Medical School, Kita-ku, Osaka 530 (Japan).
820
DNA and enzymes Phage fd RFI DNA was prepared as described by Sugiura et al. (1969). Plasmid Xdvl DNAwas prepared (Matsubara et al., 1975) and generously donated by IL Matsubam. Covalently closed circles of ~,dvl DNA were convetted to open circles by X-ray irradiation at the dose corresponding to 0.5 hit. Restriction endonuclesses Eco RI, Bam HI, Hin dll and Hin Hl (is~schizomer of HaeH; see Roberts, 1978) were purified as described earlier (Takanami, 1973). One unit of enzyme activity is expressed as the activity which completely digests 1 ~g Z DNA by incubation for 60 mln at 37°C. In situ d ~ t i o n o f DNA Polylysine treatment of support filmRand adsorption of DNA to the films were performed as described by Williams (!977) with the following modifications; the films were made hydrophilic by subjecting them for 10 sec to a glow discharge at 200 millitorr air pressure; satisfactory DI~A concentrations for adsorption were in the range from I to 8 #g/m' ,.'n 100 mM Tris-HCI buffer (pH 7.6) and 10 mM EDTA; droplets of about 8/~l of DNA solution were applied on the treated fflmR and allowed to adsorb for 3 to 5 rain. After the drop was drained away, the filmed surface of the grid was rinsed twice by touching the surface of 25/d-drops of distilled water. After draining, a 5/d drop of 0.1 to 0.6 units of restriction enzyme solutions was then applied on the film. Enzyme solutions of EcoRI contained, besides the enzyme, 10 mM Tris.HCl (pH 7.6), 5 mM MgCI2 and 100 mM NaCI, while the other enzyme solutions contained only 10 mM Trk-HCI (pH 7.6) and 5 mM MgCI2. Digestion was carried out for 10 to 60 rain at 37°C in the humid chamber. After incubation, the film was rinsed for 15 sec with 0.1 ml droplet of I M NaCI and occasionally followed by rinsings with 0.4% aqueous Kodak Photo-Flo (pH 8.8) or 9% formamide in 10 mM Tris-HCI (pH 7.6) and I mM EDTA. Drops for rinsing were conveniently held on a fresh sheet of parAfllm and an inverted grid was touched to a droplet surface. To observe the DNA/enzyme complex, the ~,rocedure of rin~ngs was omitted. After the excess liquid was drained away the grid was rinsed with distilled water and stained with 5% aqueous uranyl acetate as described by Williams (1977). Electron microscopy The dried specimen grids were rotary shadowed at an angle of 6 ° with tungsten (Griffith et al., 1971). A tungsten wire of 0.5 mm in diameter and 5 cm in free length, was inltially heated with a 30.5-A current at 2-10 -6 tort, after which the applied voltage was left constant for 80 sec during rotary shadowing. Electron micrographs were obtained on a JEM 7A electron microscope, with primary magnification of 20 000 X. Length measurements were made with a Hewiett-Packard 9864A digitizer and 9810A calculator.
321 RESULTS
Length measurements in DNA molecules adsorbed to the polylysine-treated carbon film The protein monolayer film technique originated by Kleinschmidt ~ d Zahn (1959) has been demonstrated to be enormously useful to spread the DNA evenly on the electron microscope grid. However, electron microscopic studies on protein-nueleic acid interaction can be performed only with f~ee nucleic acids. To mount the protein-free DNA on the film, we have exploited the polylysine~carbon film technique (Williams, 1978). In this technique,the stretching and distribution of the long DNA polymers may be uneven in the absence of embedment in the carrier protein. To see the extent of the length variability, we compared the length measurements by the two different techniques, the polylysine technique and the protein monofilm technique and the results are summarized in Fig. 1. In this figure, the standard deviation is plotted against the square root of the mean length of the DNA according to Davis et al. (1971). The standard deviation of the length measurement employing the polylysine technique is about twice as large as that obtained when using the protein monofilm technique. The contour length is shortened by about 6% in the polylysine technique; the mean length of BamHI-digested Xdvl DNA was 2.01 + 0.15 #m by the polylysine technique (30 measurements) and 2.13 + 0.05 #m by the protein monolayer technique (18 measurements). As shown in Fig. 2a, DNA molecules tend to aggregate laterally and to distribute unevenly, when using the polylysine technique.
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322
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Fig.2. gleetron m ~ o g r s p h s of the open circuls~ xdvl DNA ( s - e ) and the £coRI.Umearized DNA ( f - j ) which were first adsorbed to polylysine f'dm (t, f), and then treated with ~.¢oR[ (b) or J~zmHI (g), respectively and followed by washings with 1 M NaCI (e, h), with 1 M NsC! and l~hoto-Flo (d, i) or with I M NaCI and 9% fornutmide (e, j). Restriction enzyme molecules were ehown by trianllles and the DNA gape by arrows. (× 34 900.)
323
Visualization o f cleavage sites. The important features of our technique include the following preparation steps; (1) adsorption of DNA to the polylysine. treated f i l m s ; ( 2 ) in situ digestion of the adsorbed DNA with restriction enzymes; (3) removal of the enzymes and conversion of the breaks into visible gaps. Fig. 2 shows the open circular Xdvl DNA(a--e) and the linear restriction fragments ( f - j ) that were observed at each preparation step; step I (a and f), step 2 (b and g) and step 3 (c--e and h--j). At step 2, Xdvl DNA is treated with EcoRI (b) and the EcoRI-fragment with BamHI (g). The globular molecules associated with the DNA must be the restriction enzyme, since such complexes were observed only when the enzyme was added. As shown in Fig. 2g, DNA strand is occasionally gapped at the site of complex formation. Rinsing with 1 M NaCI at step 3 (c and h) was useful to remove the enzyme and thus disclose the break. Successive rinsings with dilute Photo-Fie (d and i) or 9% formamide (e and j) served to enlarge the gap. Mapping the EcoRl site on BamHl-fragment o f Xdvl DNA Plasmid Xdvl DNA is the circular tandem dimer and the cleavage sites for EcoItl and BamHI are located at the symmetrical position as shown in Fig. 3& Therefore, two DNA gaps at the symmetrical position are expected in the open circular Xdvl DNA treated with EcoRI. However, under the present in situ digestion, most DNA molecules are intact and only a few molecules bear a single gap (Fig. 2c--e). Hardly any molecules bearing two gaps were observed. These frequencies were statistically predictable. It may be that the enzyme digests the molecule only partially. To show the gap is introduced at a specific site, we prepared the linear BamHI-fragments, subjected them to in situ digestion with EcoRI, rinsed in 1 M NaCI and examined the molecule bearing the gap (Fig. 4). The gap wa~
(a)
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(b)
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Ban~4(0500) (0A21 Fi~ 3. Cleavage maps of the ~dvl DNAdimer (a) and the fd RF DNA (b). One of the two equivsdant BamHl cleavqe sites is designated as the zero point in xdvl m a p ; t h e Hindll site k designated as the zero in fd map. Each cleavage site is shown by an arrow. The joints betwmm xdvl monomers are shown by double bars. The map distance from the zero point is measured in the counterclockwise direction and expressed as fraction of the total length. xdvlcleavage map is constructed according to the rasults by Chow eta]. (1974), Thomas et sd. (1975) and Perricaudet and Tiollsis (1975~ fd RF cleavage map is described by Tskanami et aL (1975). Total genome length is 14 300 base pairs for x d v l (Chow et al., 1974; Fiandt et al., 1977) and 6408 base pairs for fd RF (Beck et aI., 1978).
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Fig. 4. Typical electron micrograph of linezr xdvlBamllY-frsgment bearing a single gap introduced by EcoRI digestio~ The gap is shown by an arrow. The ssmple was rinsed in 1 M NsCI. (x 77 600.)
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325
detected at only a single site on this fragment. The position of the gap was measured from the closest end and a histogram of the results is shown in Fig. 5. Most of the gaps were situated at 37% of the fragment length from one end. The value is very close to 35%, in agreement with the value derived from the physical map of Fig. 3 ~ Additional minor sites of the gap may represent the sites for EcoRI* activity found by Polisky et al. (1975) and Tikchonenko et al. (1978). Mapping the HinHl sites on a linear fd D N A molecule A cleavage map of the fd RF DNA is shown in Fig. 3b. There are a single cleavage site for HindII and three cleavage sites for HinHI. In this section, we mapped the binding sites of HinHI as well as their cleavage sites on a linear molecule o f fd RF DNA which had been produced by cleavage of circles with HindII. As shown in Fig. 6, we can see the complex formation with HinHI. Locations of the enzyme on the DNA molecules were measured from one end and the molecules were oriented so as to locate most of the enzymes in a close correlation for every pair of molecules as described by Giacomoni et al. (1977) (Fig. 7a). The results are shown in the form of a histogram (Fig. 7b).
Fig. 6. Complex between HinHI and a fd DNA molecule linearized with HindIl. Bound HinHI molecules are shown by triangles. (× 77 600.)
Fig. 5. Positions of EcoRI cleavage site on kdvl BamHl-fragment. (a) Graphic array of the representative fragments subjected to in sito digestion with EcoRI and rinsed in 1 M NaCI. "fine position o f the gap is shown by the vertical bar to indicate the nature of the primary data. The length of DNA molecules was normalized and the distances were measured from 'the closest end as fraction of the entire fragment length. (b) A hktogram showing the location of gaps. The arrow indicates the cleavage site derived from the physical map in Fig. 3a.
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Although a background noise appears on the histogram, the positions of the complex peak agree well with the cleavage m a p by H l n H I (Fig. 3b) in which cleavage sites are situated at 42%, 72% and 84% respectively, of the entke molecular length from one end. To confirm the cleavage sites by showing the D N A gaps, we removed the bound enzyme from the D N A molecules. Fig. 8 shows some representative examples containing a single gap (a), two gaps (b) and three gaps (c) in a molecule. Molecules containing multiple gaps were few, probably due either to the preferential occurrences o f partial digestions or to the failure in opening the staggered ends. The gap positions on the D N A molecules were measured and the results are shown in Fig. 9. Background noise in the histogram of the complex between H i n H l and the HJndII-treated DNA is now reduced in the histogram of the cleavage gaps revealing the true peaks in the expected sites for HinHI.
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DISCUSSION The techniques of protein-free spreading of nucleic acid for electron microscopic observations have undergone a considerable improvement allowing the visualiT.ation of proteins bound to D N A (Dubochet et al., 1971; Vollenweider et al., 1975; Williams, 1977). These techniques have been used to visualize the stable complex between type I restriction enzyme E c o K and
328
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Fil~ 9. Position o f HinHl cleavage sites on a fd RIP D N A linearized with HindlL Same as Fig. 7 except that the HinHl-treated samples were rinsed as described in Fig. 8 and the
pcLdtion of the eleavsge gap k shown by the vertiesl bar.
DNA (Bracket al., 1976). In the absence of ATP, EcoK forms a stable complex with DNA without cleaving it. However, such a stable complex is not found for a type II restriction enzyme. In the present study (Figs. 6 and 7), the recognition sites for type II enzyme HinHI were mapped by examining the binding of enzyme to the DNA immobilized on the carbon film. The method of in situ complex formation may be applicable for other type II enzymes. However, a high background noise due to nonspecific adsorption of enzyme molecules is the drawback in this method. To overcome this drawback, we have developed the new method to visualize the cleavages by removing the restriction enzyme molecule from the complex and disclosing the endonucleolytic breaks on the DNA molecule. In the in situ digestion, most DNA molecules are only partially cleaved with restriction enzymes as shown for EcoRI-treated ~,dvl DNA(Fig. 2b-e) and H/nHI-treated fd RF DNA (Figs. 8 and 9). By showing the position of the DNA gaps in the form of a histogram (Figs. 5 and 9), cleavage sites can be mapped on the molecule. In the previous studies, a partial digestion was required to determine the order of restriction fragments on a genome, as reviewed by Nathans and Smith (1975). Therefore, the present method is very useful to order the restric. tion fragments on the cleavage map. Moreover, an accurate length measure-
329
ment o f each restriction fragment can be performed separately by protein monolayer method. For direct visualization o f all the cleavage sites on each entire genome DNA, we still have to perfect our technique as to obtain a complete in situ digestion. ACKNOWLEDGEMENTS
This work was supported by a grant-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science and Culture, Japan. REFERENCES
Beck, E., Sommer, R., Auerswald, E.A., Kurz, C., Zink, 13., Osterburg, G., Schaller, H., Sugimoto, K., Sugisaki, H., Okamoto, T. and Takanami, M., Nueleotide sequence of bacteriophage fd DNA, Nucleic Acids Re~, 5 (1978)4495--4503. Brack, C., Eberle, NL, Bickle, T.A. and Yuan, R., Mapping of recognition sites for the restriction endonuclease from Escherichia ¢oli K-12 on bacteriophage PM2 DNA, J. biol. Biol., 108 (1976) 583--593. C~ow, LT., Davidson, N. and Berg, D., Electron microscope study of the structures of kdv DNAs, J. Mol. Biol., 86 (1974) 69-89. Davis, l~W., Simon, M. and Davidson, N., Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, Methods Enzymol., 21D
(1971) 413-428. Duboehet, J., Ducommun, )/L, Zollinger, M. and Kellenberger, E., A new preparation method for dark-field electron microscopy of biomolecules, J. Ultrastruct. Res., 35 (1971) 147--167. Fiandt, M., Honlgman, A., Rosenvold, E.C. and Szybaiski, W., Precise measurement of the b2 deletion in coliphage larnbda, Gene, 2 (1977) 289-293. Giacomoni, P.U., Delain, E. and Le Pecq, J.B., Electron microscopy analysis of the interaction between EmcheHchia ¢oli DNA-dependent RNA polymerase and the replicative form of phage fd DNA, Eur. J. Biochem., 78 (1,977) 205--213. Griffith, J., Huberman, J.A. and Kornberg, A., Electron microscopy of DNA polymerase bound to DNA, J. Mol. Biol., 56 (1971) 209-214. Kleinschmidt, A. and Zshn, I~K., Uber Desoxyribonucleinsiiure-Molekeln in ProteinMischfilmen, Z. Naturforsch., 14b (1959) 779-779. Matsubera, K., Takagi, Y. and Mukai, T., In vitro construction of different oligomerie forms of ~.dv DNA and studies on their transforming activities, J. Virol., 16 (1975) 479-485. Nathans, D. and Smith, H.O., Restriction endonucleascs in the analysis and restrt.~turing of DNA molecules, Annu. Rev. Biochem., 44 (1975) 273--293. Perr~candet, M. and Tiollsjs, P., Defective bacteriophage lambda chromosome, potential vector for DNA fragments obtained after cleavage by Bacili~ amyiolique[acienJ endonuelease (Baml), FEBS Lett., 56 (1975) 7--11. Polisky, B., Greene, P., Graf'm, D.E., McCarthy, ILJ., Goodman, ILM. and Boyer, ELW., Specificity of substrate recognition by the E¢oRI restriction endonuelease, Prnc. Natl. Acad. 8eL USA, 72 (1975) 3319-3314. Roberts, R.J., Restriction and modification enzymes and their recognition sequences, Gene, 4 (1978) 183--193. Su&~ura, M., Okmnoto, T. and Takanami, M., Starting nucleotide sequences of RNA synthesized on the replieative form DNA of eoliphqe fd, J. Mol. Biol., 43 (1969) 299-315. Takansmi, M., Specific cleavage of coliphage fd DNA by five different restriction endonucleases from Haemophilus genus, FEBS Lett., 34 (1973) 318-322.
880
Taksnmn~ M., Okamoto, T., 8uliimoto, K. n d 8ul~bski, I-L, 8 t u d i l on baet~riophqe fd DNA, L A eleavase map of the fd gemom~ J. Idol. Biol., 95 (1976) 21--81. Thomas, M. and Davis, R.W., 8t'udim on the d u v s g e of bactmiophqe lsmbds DNA with EcoRl re~a~iefion mdonucleme, J. MoL Biol., 91 (1975)315--328. 11kehone~o, T.L, ~ o v , E.V., Zavizion, B.A. and Nm~xlitaky, B.8., EeoRl activity; emzyme modification or sctivM.ion of accompanying endonucleme? Gene, 4 (1978) 195--'212.
Vollenweider, ELJ., ~Qgo, J.M. and Ko/ler, Th~ A routine me~od for protein-free spmsdi~ of doulde- and singlwstrsnded nucleic acid molecules, Pro~ NatL Acad. 8ci. USA, 72 (1976) 83---87. ILC., Use of polylydne for adsorption of nucleic aeicls m d enzymes to electron mierow~pe specimen films, M . M~L Acad. 8eL USA, 74 (1977) 2311--2315. Yamsgiehi, IL, Inokuehi, H, and Ozeki, IL, Excision snd duplication of s u r ~ m s d u c i n g fragments carried by bacteriophsge @80, J. Virol., 18(1976) 1016--1023. Communicated by K. MaUmbam.