Proc. Nat. Acad. Sci. USA Vol. 70, No. 2, pp. 503-506, February 1973

Separation of Specific Segments of Transforming DNA after Treatment with Endodeoxyribonuclease (bacterial transformation/Hemophilus species/separating markers/unique DNA segments) SOL H. GOODGAL AND ROSA GROMKOVA Microbiology Department, School of Medicine, University of Pennsylvania, Philadelphia, Pa. 19104 Communicated by Britton Chance, September 29, 1972

ABSTRACT Hemophilus parainfluenzae endodeoxy- MATERIALS AND METHODS ribonuclease was used to degrade the DNA of H. influenzae and to follow the biological activity of 14 markers associ- Bacterial Strains Used. H. infiuenzae strain Rd was origi- ated with this DNA. It was found that some H. influenzae nally obtained from Alexander and Leidy (4). Strain H. markers were completely inactivated by endodeoxyribo- influenzae super 8, derived from strain Rd carrying eight nuclease treatment, while others appeared to retain all novobiocin or almost all of their original activity. The bulk of the H. antibiotic-resistant markers [streptomycin (str), influenzae DNA was reduced to double-stranded pieces (nov), erythromycin (ery), streptovaracin (sty), spectino- of the order of 8 X 105 to 1 X 106 daltons. Velocity sedimen- mycin (spec), kanamycin (kan), viomycin (vio), and naladixic tation of the DNA in sucrose gradients disclosed that acid (nal)], was obtained by J. Bendler (5). The auxotrophic markers that retained biological activity were present in strain H. infiuenzae Bio 7 (ala-, pro-, try-, lys-, bio-, val-, DNA particles that were of the order of I X 106 daltons or larger, and indicated a close correlation between the size leu-) has been described by Michalka and Goodgal (6). of the DNA fragment and the amount of biological activity H. parainfluenzae "colony 14" was obtained from Grace retained. These data suggest that H. parainfluenzae Leidy. Its high-level streptomycin-resistant (strr) mutant endodeoxyribonuclease breaks DNA at specific sites. The was isolated from "colony 14" by Nickel and Goodgal (7). nalr marker was shown to have twice as much biological wild used in trans- activity after treatment with endodeoxyribonuclease when Bacillus subtilis strain 168 type (recipient assayed at saturating DNA concentrations. In the linear formation) was obtained from J. Marmur, and strain 168 portion of the DNA dose-response curve, the biological erythromycin-resistant (eryr) streptomycin-resistant (strr) activity of this marker was reduced 3- to 10-fold com- was obtained from N. Kallenback. pared to untreated DNA (in accord with the reduced size of its DNA). These data demonstrate a specific enrich- Media. Brain-Heart Infusion medium (BHI; Difco) was ment of the nalr marker by about 6- to 20-fold, and suggest supplemented with 10 mg/ml of hemin (Eastman Kodak) and a technique for the separation and purification of specific 2 jug/ml of nicotinamide adenine dinucleotide (NAD; Nu- segments of DNA. tritional Biochemicals) for the growth of H. infiuenzae. After the discovery by Smith and Wilcox (2) of an endo- Minimal medium was a modification by Michalka and nuclease in Hemophilus influenzae that attacks foreign DNA Goodgal (6) of the synthetic medium for H. infiuenzae but does not degrade H. influenzae DNA, we have studied developed by Talmadge and Herriott (8). H. parainfluenzae the effects of this enzyme on the biological properties of was grown in BHI obtained from Baltimore Biological DNAs in order to assess the specific role of endonuclease in Laboratories and was supplemented with 2 4g/ml of NAD. restriction of DNA during transformation. In addition, we B. subtilis strains were grown according to the procedure de- have discovered another enzyme from Hemophilus para- scribed by Young and Spizizen (9). that also has the property of attacking foreign infiuenzae Bacterial DNAs for transformation were obtained from DNAs including that of H. influenzae but not H. para- of and Herriott influenzae DNA. We would like to conclude from these bacteria according to the procedure Goodgal (10). For the preparation of DNA from B. subtilis, cells were studies and the pioneer work of Meselson and Yuan (3) that of restriction involves an attack of endonucleases on specific first washed and treated with 100 /Ag/ml lysozyme. sites in the DNA and that different enzymes recognize Preparation of Purified Restriction Enzymes. Endodeoxy- specific base sequences. If the biochemical data support our ribonuclease (endo-DNase) was prepared according to the genetic and physical studies, it should be possible to attack procedure of Smith and Wilcox (2). The same method was DNA in the same way that proteolytic enzymes like chymo- used for isolation and purification of H. parainfluenzae and trypsin and trypsin attack proteins. These specific breaks H. influenzae enzymes. should be feasible since we are now able to use the enzyme from H. parainfluenzae to prepare segments of H. influenzae Transformation Assay. Transformation of H. influenzae DNA that contain biological markers with greatly increased was as described in ref. 10, with competent cells prepared specific activity. Danna and Nathans (17) and Edgell, according to the method of Goodgal and Herriott as modified Hutchinson, and Sclair (18) have also demonstrated that by Cameron (11). Unless otherwise indicated, the standard endoxyribonucleases can be used to prepare specific segments assay contained 0.2 ml of competent cells (2 X 108/ml final of DNA. density), 0.1 ml of the DNA to be assayed, and 2.7 ml of 503 Downloaded by guest on September 23, 2021 504 Biochemistry: Goodgal and Gromkova Proc. Nat. Acad. Sci. USA 70 (1973) TABLE 1. Effect of H. influenzae endo-DNase on and the mixture was incubated at 30°. Aliquots were re- transforming activity of different markers moved after various times of incubation and examined for transforming activity. Controls in these experiments con- Remaining sisted of DNA, which was treated in a similar manner, without DNA Marker activity (%) the addition of the enzyme. H. parainfluenzae Strr <1 Attempts at Fractionation of DNA. 32P-labeled DNA (100 NoVr 10 from H. treated or untreated with H. Eryr <1 MAg/ml) influenzae, B. subtilis Novr <1 parainfluenzae enzyme, was applied to two Biogel A50 columns Eryr < 1 (2.5 X 49 cm), and 52 fractions were collected from each H. influenzae Strr 100 column and examined for transforming activity and radio- activity. The same material was also applied to and eluted To 0.5 ml of DNA (25 Mg/ml) was added 51.1 (0.04 units/ml) of from methylated albumin kieselguhr columns (MAK) (12). endo-DNase from H. influenzae. The reaction mixture was in- Density Gradient Centrifugation. CsC1 gradient. H. influenzae cubated at 300. Samples were taken after 60 min and diluted with cold medium to give a final concentration of 1 ug/ml. Cells [32P]DNA (25 Mg), treated with 10-MA of H. parainfluenzae were added and transformation was assayed as indicated in enzyme for 60 min, was centrifuged in a CsCl gradient at Methods. 35,000 rpm for 48 hr at 18° in a Spinco rotor SW65. Untreated DNA was used as a control. BHI broth. After 30 min of incubation at 340, 1 Mug/ml of Sucrose Gradient. H. influenzae [32P]DNA (25 Mg), untreated pancreatic DNase was added. After an additional 5-min or treated for 60 min with 10 /Ag of enzyme, were layered on incubation, the mixture was appropriately diluted and plated for transforming activity. The plates to be used for assay of transformations to antibiotic resistance were over- 10 20 30 40 layed with antibiotics after 3 hr of incubation at 37°. The a~~~~~~~nOOnqt~ bi4 concentration of antibiotic in the overlay was designed to voal ala pro . yield a final concentration of 200 ug/ml of streptomycin, 1000o_ 15 of novobiocin or erythromycin, 8 of strepto- Ag/ml ,ug/ml 0t0n varacin, 10 ,g/ml of spectinomycin, 7 gg/ml of kanamycin, 8) 800 The for I0 and 3 Aug/ml of nalidixic acid. assay auxotrophic 0) / 'b markers was as described in ref. 6. Preparation of H. para- 0z 600 03 influenzae competent cells and transformation of H. para- I8I- .~~~~~~~00 / influenzae were as described by Nickel and Goodgal (7). For 400F .~0 transformation of B. subtilis, the preparation of competent :0 y cells and the transformation assay were essentially those used 200 by Young and Spizizen (9). L Enzyme Assay. To test the effect of endo-DNase on the 10 20 30 40 top biological activity of DNA, the DNA was diluted to a specific Sucrose Gradient Fraction concentration in Tris-Mg-mercaptoethanol buffer [6.6 mM b each of Tris buffer (pH 7.4), MgCl2, and 2-mercaptoethanol]. To 0.5 ml of this material was added 5 ,.l of purified enzyme, 800 _ ~~~~~I'd I'.

TABLE 2. Effect of H. parainfluenzae endo-DNase on 600 OfI'-\ transforming activity of different markers of H. influenzae DNA I ' '\ . 400- 04 Marker Remaining activity (%) 1 i Streptomycin <1 200k Erythromycin <1 ;*m*S~~~~~I Novobiocin 75 Kanamycin <1 10 20 30 40 top Streptovaracin <1 Sucrose Gradient Fraction Viomycin <1 FIG. 1. Separation of biological activity in a sucrose density Nalidixic acid 200 gradient. Biological activity was determined by the standard Spectinomycin <1 transformation assay. 10 ug of DNA treated (a) or untreated (b) Alanine 60 with endo-DNase was layered onto the top of a 5-20% sucrose Proline 2 gradient and centrifuged for 3 hr at 35,000 rpm in a Spinco SW39 Tryptophan 2 rotor. Fractions were collected and analyzed for biological activi- Biotin 14 ties. The biological activities of the auxotrophic markers in the Lysine <1 untreated DNA (b) showed the same peak locations and distribu- Valine 200 tion as the markers indicated. Transformants (a) X (2 X 10-3), (b) X (2 X 10-4). *- - I*, 32P; A, nal; 0--O, nov; Conditions are the same as in Table 1. X X, str; A- *, pro. Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Transforming DNA 505

the top of a linear 5-20% sucrose gradient, (0.3 M NaCl, TABLE 3. Effect of H. parainfluenzae endo-DNase on pH 7.6). Samples were then centrifuged at 39,000 rpm for the biological activity of H. influenzae DNA 4 hr at 180 in a Spinco rotor SW41. No. of transformants at RESULTS Saturating concen- Limiting concen- It has been observed that treatment of DNAs with endo- trations of DNA trations of DNA DNase from H. influenzae or H. parainfluenzae resulted in a Nal Nov Nal Nov limited digestion of foreign DNAs, but these enzymes did not attack DNA from the same organism (1, 2). The size of seg- Treated 2908 1092 85 25 ments produced on the average was similar to that found by Untreated 1689 640 840 248 Smith and Wilcox (2), about 8 X 105 to 1 X 106 daltons. In analyzing the biological activity of transforming DNAs after Conditions are the same as those for Table 1, except that the treatment with H. influenzae endo-DNase, we found that DNA was diluted to 1 ng/ml for measurement of limiting concen- some of the activity of the novr marker of H. parainfluenzae trations of DNA. DNA was maintained, while all the activity of the strr and eryr markers was destroyed. These results are shown in The size of the DNA for individual markers is sufficiently Table 1. In addition, the species specificity of the enzyme distinct to indicate that the data represent a limit digest. toward foreign B. subtilis markers is shown as well as the total Further enzyme treatment or incubation for longer periods refractory behavior of the homologous DNA from H. in- of time do not increase the activity of most endo-DNase fluenzae. preparations. Endo-DNase preparations contaminated with In order to improve evaluation of the marker specificity of exonuclease activity do give additional inactivation with endo-DNase, the enzyme was prepared from H. parainfluenzae time, but this affect is also noted with homologous DNA. and used to treat the DNA of H. influenzae that contained The position of the nalr marker was approximately that of a 14 markers. From the data presented in Table 2 it is apparent DNA molecule of 8 X 106 daltons, while the molecular weight that markers show a considerable variability in their response of the novr marker was close to 4 X 106. The major band of the to the action of II. parainfluenzae endo-DNase. Some of the DNA was at a position corresponding to 8 X 105 to 1 X 106 markers retained all or almost all of their original activity, and daltons, in agreement with the results found by Smith and others were completely inactivated. Wilcox (2). A CsCJ density gradient analysis of the products of the In view of the amount of DNA present in the region of the digestion of H. influenzae DNA by H. parainfluenzae endo- nalr marker, it was anticipated that the specific activity of this DNase demonstrated a broader band width for the endo- DNA might be greater than that of the bulk DNA. When DNase-treated material, which is characteristic of the DNA treated with endo-DNase and untreated DNA were reduced size of the DNA, and corroborated the finding that compared for their transforming activities under conditions of the enzyme completely destroyed the strr marker but not the saturating and nonsaturating concentrations of DNA, the nalr or novr markers (Table 2). The absence of a shift in the results shown in Table 3 were obtained. These data clearly densities of nalr and novr markers suggests that no major show that the nalr and novr markers of enzymatically digested change in the average G-C content had occurred in the DNA have transformed at higher efficiencies than the same product, and demonstrated that the product was still double- markers in untreated DNA under saturating conditions of stranded. DNA, but that at limiting DNA concentrations they were Since it is known that transformation is strongly dependent less efficient, indicating that the specific transforming activity upon size in the range of 1 to 10 X 106 daltons, various tech- of these DNA segments had been reduced by the enzyme. niques, including gel-filtration, methylated albumin kieselguhr Since there was a 10-fold decrease in activity of the nalr (MAK) column chromatography, and sucrose sedimentation marker in the linear portion of the DNA dose-response curve, velocity gradients were used to fractionate DNA according to and DNA saturation resulted in a 2-fold higher efficiency of size. After treatment with endo-DNase, H. influenzae DNA transformation, one can conclude that there was at least a marked with nalr, novr, strr, ala+, vai+, bio+, pro+, and try+ 20-fold specific enrichment of the nalr marker. was fractionated and tested for its remaining biological In order to demonstrate that there was a real increase in activity. It was found that the best separations were obtained the specific activity of markers preserved after endo-DNase with the sucrose gradient procedure. The results of sedi- treatment, it was necessary to show that there was no large mentation in a sucrose gradient are given in Fig. 1, and indi- change in the competing ability of the treated DNA. cate specific locations for each marker that retained biological In the case of small double-stranded molecules produced by activity. From a comparison of the amount of activity re- sonication, the competing ability of the molecules for trans- tained in the DNA treated with endo-DNase, as shown in formation is not greatly reduced for an equal mass of DNA Table 1, and the size of the marker activity after sedimenta- (13, 16). DNA reduced in size by treatment with endo-DNase tion, one finds excellent correlation between the size of the also shows effective competition in the transformation assay. DNA fragment and the biological activity retained. Peak Table 4 shows the results of an experiment in which a test positions of markers in the gradient (indicated by arrows in DNA SKCDN (streptomycinr, kanamycinr, novobiocinr, Fig. 1) are in the same order as the amount of biological ac- dalacinr, and nalidixic acidr) at 0.2 qg/ml was mixed with tivity for each marker in the bulk preparation after endo- strr marker DNA before and after treatment with endo- DNase treatment. With untreated DNA, all the markers DNase. The size of most of the treated DNA was reduced to sediment to the same position. Three of them are shown in 1 to 0.8 X 106 daltons. The strr DNA with or without treat- Fig. lb. ment with endo-DNase reduced the biological activity of the Downloaded by guest on September 23, 2021 506 Biochemistry: Goodgal and Gromkova Proc. Nat. Acad. Sci. USA 70 (1973) TABLE 4. Competing ability of endo-DNase-treated DNA

Transformations nov dal nal Enzyme Test DNA jig added No. Efficiency No. Efficiency No. Efficiency + nov dal nal 0.2 140 0.25 204 0.20 275 0.22 - nov dal nal 0.2 111 0.20 172 0.17 186 0.19 + nov dal nal 1.0 382 0.69 674 0.68 788 0.62 - nov dat nal 1.0 416 0.76 704 0.71 812 0.82 str-transformants + None 1 - None 550 The nov efficiency is calculated on the basis of 550 colonies = 1.0 without treatment; dal, 990 colonies = 1; nal, 1265 colonies = 1. strr marked DNA (0.5 ml; 50 Mug/ml) was treated with 10 Ml of H. parainfluenzae endo-DNase (0.08 units/ml); the control received 10 Ml of diluted Tris-Mg-mercaptoethanol buffer. After 2 hr of incubation, an aliquot was added to 30 ml of BHI broth to yield the concentra- tion indicated in the table. The test DNA was added just before the addition of 0.1 ml of competent H. influenzae cells. The test of the transformation assay was performed as indicated under Methods. All plating dilutions were 10-3. Competing DNA: 1.25 gg of strr- marked DNA.

nov and dal markers. In agreement with the experiments in transforming activity of the DNA to the size of the DNA which sonicated DNA was used, the untreated DNA was segments (13, 15). The competing ability of endo-DNase- slightly more effective than the treated DNA. treated DNA was not greatly reduced, and is explained in part by the ability of smaller segments of DNA to be ab- DISCUSSION sorbed to competent cells (13). Taken as a whole, these data demonstrate that there was a 6- to 20-fold enrichment of the Endo-DNase can selectively inactivate genetic markers; nalr segment, as compared to untreated DNA. this inactivation is achieved by digestion of the DNA to sizes that reduce the capacity of the transforming DNA to be We thank Mrs. Helga Ponce-de-Leon for her excellent technical assistance. This work was supported by Grant Al-04557-10 from integrated into the recipient chromosome (13). Furthermore, the United States Public Health Service. the production of DNA particles of specific sizes can lead to effective separation and purification of genetic markers. The 1. Gromkova, R. & Goodgal, S. H. (1972) J. Bacteriol. 109, 987-992. selectivity of the enzyme relates to the presence of sites on the 2. Smith, H. 0. & Wilcox, K. W. (1970) J. Mol. Biol. 51, substrate on which the enzyme acts (14). The sedimentation 379-391. of limit digests of DNAs treated with endo-DNase in a 3. Meselson, M. & Yuan, R. (1968) Nature 217, 1110-1114. sucrose gradient demonstrate a reduction in size of the 4. Alexander, H. E. & Leidy, G. (1953) J. Exp. Med. 97, DNA and that the biologically active markers have specific 17-31. 5. Catlin, B. W., Bendler, J. W. & Goodgal, S. H. (1972) J. locations in the sucrose gradient. Indeed, the marker survival Gen. Microbiol. 70, 411-422. was directly related to marker sizes. The natr marker was 6. Michalka, J. & Goodgal, S. H. (1969) J. Mol. Biol. 45, reduced to about 8 to 10 X 106 daltons, and retained con- 407-421. siderably more activity than the pro marker, which was 7. Nickel, L. & Goodgal, S. H. (1964) J. Bacteriol. 88, 1538- reduced to a size of 1 106 daltons. The fact that after endo- 1544. X 8. Talmadge, M. B. & Herriott, R. M. (1960) Biochem. Biophys. DNase treatment the biological activity could be correlated Res. Commun. 2, 203-206. closely with the size of fragments produced, and that these 9. Young, F. E. & Spizizen, J. (1961) J. Bacteriol. 81, 823- fragments are in some cases clearly nonoverlapping-e.g., 829. nov and pro-suggests that the segments produced are unique, 10. Goodgal, S. H. & Herriott, R. M. (1961) J. Gen. Physiol. 44, 1201-1227. and lend additional support to the experiments of Kelly and 11. Barnhart, B. J. & Herriott, R. M. (1963) Biochim. Biophys. Smith (14) that show that endo-DNase activity attacks Acta 76, 25-39. specific sequences of the DNA. The availability of endo-DNase 12. Seuoka, N. & Cheng, T. (1962) J. Mol. Biol. 4, 161-172. with different specificities provides a powerful tool for prepa- 13. Gottfried, T. D. (1967) Thesis, University of Pennsylvania. DNA to on struc- 14. Kelly, T. J., Jr. & Smith, H. 0. (1970) J. Mol. Biol. 51, ration of small segments amenable studies 393-409. ture and sequence. Assayed under conditions of DNA satura- 15. Litt, J., Marmur, J., Ephrussi-Taylor, H. & Doty, P. tion, endo-DNase-treated DNA showed a doubling in the (1958) Proc. Nat. Acad. Sci. USA 44, 144-152. absolute number of nalr transformants; when assayed in the 16. Randolph, M. L. & Setlow, J. K. (1972) J. Bacteriol. 111, linear portion of the dose-response curve, its activity was 186-191. 17. Danna, K. & Nathans, D. (1971) Proc. Nat. Acad. Sci. 0.1 that of the untreated marker. These results suggest USA 68, 2913-2917. that the intrinsic transforming ability of the natr segment was 18. Edgell, M. H., Hutchinson, C. A., III & Sclair, M. (1972) reduced, and are in good agreement with data that relate the J. Virol. 9, 574-582. Downloaded by guest on September 23, 2021