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Separation of Specific Segments of Transforming DNA After Treatment

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Separation of Specific Segments of Transforming DNA After Treatment 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.
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