J..Mol. Biol. (1975) 97, 123-125
Isolation o f a Sequence-specific Endonuclease (BamI) from
Bacillus amyloliquefaciens H A restriction endonuclease has been isolated from Bacillus amyloliquefaciens H (strain RUB500). The enzyme, BamI, cleaves adenovirus-2 DNA at three sites, phage XDNA at five sites, Xplac DNA at four sites, ~80 pt DNA at 14 sites, and ~b3T+ DNA at four sites. However, it does not cleave DNA from bacteriophage SPO2, ~b105or ~29. Our previous studies (Wilson & Young, 1972,1973) with Bacillus 8ubtilis and a number of closely related bacilli, including Bacillus amyloli~usfaciens H, demonstrated that genetic material could be exchanged between them by transformation and that they therefore comprised a genospecies {Ravin, 1963). However, the frequency of genetic exchange with heterospecific DNA is several orders of magnitude below that obtained with homospecific DNA thus suggesting the presence of restriction and modification enzymes. This hypothesis is strengthened by the recent discovery of a classical restriction and modification system between B. amyloliquefacien8 H and B. subtilis 168 (Wilson & Young, 1974; Wilson d al., 1975). Furthermore, a restriction endonuclease (BsuI) has been reported in a strain of B. s~btilis (Trautner et al., 1974). We, therefore, examined members of the B. subtilis genospecies for the presence of endonucleases to determine if they were responsible for the inefficiency of genetic exchange among heterologons strains. This report describes the purification and partial characterization of a restriction endonuclease from B. amyloliquefaciens H. B. amyloliqusfaciens H (strain RUB500) was grown in Penassay broth (Difco) to the late logaritbmlc phase of growth and harvested b y centrifugation. The cells (20 g) were disrupted b y grinding with alumina (3 times the wet weight of the cells) and suspended in 10 ml of extraction buffer (25 mM-Tris.HC1 (pH 8-0), 5 mM-2mercaptoethanol). Cell debris and alumina were removed by centrifugation (23,000 g, 60 mln at 4°C) and the supernate (20 ml) was diluted with 80 ml extraction buffer. Nucleic acids were removed by precipitation with streptomycin sulfate (1.0 ml 10% streptomycin sulfate/1500 A2so units). Solid ammonium sulfate was added to the supernate with constant stirring at 4°C. Although some activity was precipitated by 5 0 ~ saturation with (NH4)2S04, most of the activity was in the 50 to 80% precipitate. This precipitate was suspended in 10 ml of buffer I (0.01 E-sodium phosphate, p H 7.4) and applied to a bed of Sephadex G25 (5 cm × 39 cm). After ehition with buffer I, the fractions that contained endonuclease activity were pooled, and chromatographed on DEAE-cellulose (DE52, Whatman, 2.4 cm × 13.5 cm bed) by elution with a one liter linear gradient of 0 M to 0.6 ~-NaCl in buffer I. Fractions were pooled and tested for endonuclease activity b y agarose/ethidium bromide gel electrophoresis of DNA (Sharp eta/., 1973). Fractions 10 t o 2 5 that eluted between 0.05 and 0.I5 MNaC1 showed endonuclease activity (Plate I). Fractions that eluted at higher salt concentrations had considerable exonuclease activity and were discarded. Fractions 10 through 25 from the DEAE-cellulose chromatography were dialyzed twice ag~.in~t three liters of buffer I and chromatographed on phosphocellulose (Whatman P l l , 1.2 cm × 8.0 cm bed). The phosphocellulose was precycled first with 0-5 ~-NaOH 123
124
G. A. WILSON AND F. E. YOUNG
and then 0-5 ~-HC1 and equilibrated with buffer H (0.01 M-NaPO~(pH 7.4), 0.01 ~2-mercaptoethanol). The enzyme was eluted with a one liter linear gradient (0 ~ to 0.6 M-NaCI) in buffer II. Most of the proteins did. not bind to the phosphocellulose. Endonuelease, as determined b y electrophoretic analysis of DNA t h a t had been treated with samples from the column, eluted mainly between 0.31 and 0"36 MNaCl (fractions 110 to 125). As seen in Plate II, some endonuclease activity was found in all fractions between 75 and 140. I t is not known at this time whether the patterns reflect partial digestion of DNA or actually represent another enzyme with a different specificity. The pooled fractions from phosphocellulose chromatography (110 to 125) were used in the subsequent characterizations. The enzyme at this stage of purification has very little or no exonuclease activity (Table 1) and is stable for prolonged periods of time at 4°C. The enzyme has been designated B a m I according to the nomenclature proposed by Smith & Nathans (1973). TABLE 1
E,xonuclease activity of enzyme preparation8 Assay conditions No enzyme Pancreatic DNAase BamI 50 to 80% (NH4)2SO4 DEAE-cellulose pool PhosphoceUulose
Total acid-soluble radioactivity (ors/rain) 1380 547,080 30,295 5770 1640
The reaction mixture consisted of 8 ~g B. subtilis DNA radioactively labeled with [3H]thymidine, 50 pl enzyme preparation, 6 mM-Tris (pH 7.5), 6 mM-MgC12and 6 mM-2-mercaptoethanol in a final volume of 120 ~d. The reactions were incubated for 13.5 h at 37°C. Cold 5% trichloroacetic acid was added, the reaction mixture stored at 0°C for 30 rain and subsequently filtered through 0.65 Millipore membrane (DAWP). The extent of exonuclease activity is indicated by the amount of radioactivity in the filtrate in the presence and absence of BamI. DNA preparations from a variety of organisms have been used to characterize B a m I . The initial cleavage patterns, determined by agarose/ethidium bromide gel electrophoresis indicated t h a t B a m I recognized three sites in the adenovirus-2 DNA, four sites in ~p/ac DNA, four sites in ~ DNA and four sites in ¢3T + (Plate III). (Recentiy, P. W. J. Rigby has informed us that B a m I cleaves the A genome at 5 sites. His experiments demonstrated t h a t the second band contained a heterogeneous population of molecules. One large fragment was actually composed of two molecules held together b y the cohesive ends of the 2 genome. When separated, one fragment co-migrates with the fifth band illustrated in Plate III.) These patterns clearly establish t h a t B a m I is a unique enzyme and is not an isoschizomer of one of the several endonucleases t h a t have been isolated from micro-organisms t h a t cleave unmodified, double-stranded DNA at unique nueleotide sequences (Smith & Wilcox, 1970; Yoshimori, 1971; Middleton et al., 1972; Sharp et al., 1973; Takanami & Kojo, 1973; Takanami, 1973; Roberts et al., 1974 and R. Roberts, personal communication). The enzyme therefore should be useful in studies directed toward determining the nueleotide sequence of various DNA molecules. To date the enzyme has only been partially examined for its biological function. Although DNA from bacteriophages
(10-20) 2-2
(26-41)
( 4 2 - 5 7 ) ( 5 8 - 6 2 ) ( 6 3 -81)
-
2'0
1-8--
I-6--
0 ~o oa
1.4--
c
2
1"2--
1.0 --
0.8
--
0 - 6 --
0"4--
0-2-
10
20
30
40
50
I 60
I 70
80
90
I00
I10
120
Fraction no.
PLATE I. DEAE-cellulose chromatography of B a m I . The enzyme was eluted from DEAE-celhdose in 5-ml fractions (total 124) as described in the text, Absorbanee was determined at 280 n m (1 cm path) on a Gilford 2400 S spectrophotometer. Fractions were pooled according to numbers given in parentheses and assayed for nuclease activity as described in legend to Plate II. Samples from pooled fractions 63 to 81 also contained low molecular weight material t h a t migrated too far on the gel to be included.
[ facinrj p. 124
PLATE II. Assay of phosphoeellulose column. Samples (20 /~1) from the phosphoeellulose column fractions (5 ml) were assayed for nuelease activity in reaction m i x t u r e (50/~1 total vol.) containing 2/~g A plac DNA, 6 mM.Tris.HCl (pH 7.4), 6 m~z-MgC12 a n d 6 mM-2-mercaptoethanol. After incubation for 2 h a t 3 7 ° 0 , t h e reaction was stopped b y t h e addition of 5/~l 100 mM-EDTA, a n d 15/~l 6 0 % sucrose, 1% bromphenol blue. Samples wez~ loaded on a 0-7% • garose gel containing 0.5 /~g e t h i d i u m bromide/ml. Electrophorosis was carried o u t a t 150 V (75 mA) for 3 h in a n EC-470 vertical gel a p p a r a t u s a t 80°C. Gels were p h o t o g r a p h e d u n d e r u.v. illumination using polaroid P N / 5 5 film a n d a Vivitar red no. 25A filter (see Sharp el al., 1973). The n u m b e r s ~bove the pockets indicate the fraction number. The entire gradient consisted of 20~ fractions. At thi~ stage of purification the yield from 10 g of cells is approximately 1,000,000 units. One unit is sufficient to cleave one/~g of A DNA in 60 min.
PLATE III. Agarose gel electrophoresis of various DNAs after digestion with B a m I endonuelease. Approximately 2 ~g of D N A from purified bacteriophage were digested for 3 h a t 37°C with B a m I endonuelease (purified t h r o u g h phosphoeellulose step). Reactions were stopped b y the addition of 100 mr~-EDTA. Conditions for digestions are described in legend to Plate I L Preparations were electrephoresed on a Blair Craft 20 cm × 20 em slab gel a p p a r a t u s for 6 h a t 60 mA. Gel was composed of 1.2% agarose containing 0.5 ~g ethidium bromide/ml. The gel was photographed on K o d a k Tri-X P a n Profe3sional film 4164 using a Vivitar red no. 25A filter under u.v. illumina. rich. (1,2) A wild type DNA; (3,4) ~ plac D N A ; (5,6) ~105 DNA; (7,8) ~3T + DNA; (9,10) S P 0 2 D N A ; (11,12) Ad-2 D N A ; (13,14) ~80 p t DNA. The even n u m b e r e d samples are digested with BamI. The molecular weight of D N A from bacteriophages ~105 a n d SPO2 are b o t h 26-3 × 108 (Chow et el., 1972).
L E T T E R S TO T H E E D I T O R
125
4105 and S P 0 2 is cleaved with EcoRI (Wilson et al., 1974), B a m I does not appear to cleave these viral genomes nor I ) N A from bacteriophage ~29. D N A from m u t a n t s of bacteriophage S P 0 2 t h a t can be propagated in both B. 8ubtilis and B. amylolique. faciens H strains, however, is cleaved b y B a m I only when these viruses are propagated on B. subtitis (unpublished data). Therefore, B. amyloliquefaviens H m u s t also have a modification system t h a t protects its I ) N A from its own restriction system. We previously reported the construction of genetic intergenoC~s between B. suStili8 and B. amyloliquefaciens (Wilson & Young, 1972). The intergenotes have a t least one foreign gene from B. amy~o~iquefaciens t h a t was introduced into the genome of B. 8ubtilis b y transformation. These foreign genes coding for resistance to rifampin are therefore small segments of D N A t h a t retain the foreign base sequence b u t lack the modification provided b y B. amy~oliquefaciens. We have shown t h a t these foreign genes are restricted in vitro b y B a m I and rapidly lose the ability to transform B. subtili8 (Wilson & Young, manuscript in preparation). This enzyme, the first to be isolated from a heterologous m e m b e r of the B. ~abtilis genospecies, therefore m a y play a significant role in restricting foreign I)NA. I t should also prove useful as a tool for selectively fragmenting D N A in vitro for sequence analyses. The authors wish to thank Dr Peter W. J. Rigby for access to his experiments demonstrating that BamI recognizes five sites in the h genome. Gratitude is expressed to Drs Carel l~Iulder and Richard J. Roberts for their gifts of adenovirus-2 DNA, Dr M. Oishi for bacteriophage ¢80 pt, and H. W. Baney for his teclmical assistance. We also thank Drs H. O. Smith and R. J. Roberts who provided us with m a n y of the techniques. This research was supported by grant VC.27-J from the American Cancer Society. Department of Microbiology University of Rochester School of Medicine and Dentistry Rochester, N.Y. 14642, U.S.A.
GARY A. WILSON FI~Am~ E. Y o u n g
Received 9 April 1975, and in revised form 2 June 1975 REFERENCES Chow, L. T., Boice, L. B. & Davidson, N. (1972). J. Mel. Biol. 68, 391-400. Middleton, J. H., Edgell, M. H. & Hutchison, C. A., I I I (1972). J. Virel. 1D, 42-50. Ravin, A. W. (1963). Amer. Natur. 97, 307-318. Roberts, R. J., Breitmeyer, J. B., Tabachnik, N. F. & Meyers, P. A. (1974). J. Mol. Biol. 91, 121-123. Sharp, P. A., Sugden, B. & Sambrook, J. (1973). Biochemistry, 12, 3055-3063. Smith, H. O. & Nathans, :D. (1973). J. Mol. Biol. 81,419-423. Smith, H. O. & ~Vilcox, K. W. (1970). J. Mol. Biol. 51, 379-391. Takanami, M. (1973). F E B S Letters, 34, 318-322. Takanami, M. & Kojo, H. (1973). -~EBS Lettzrs, 29, 267-270. Trautner, T. A., Pawlek, B., Bron, S. & Anagnospopoulos, C. (1974). Mol. Gen. Genst. 131, 181-191. Wilson, G. A. & Young, F. E. (1972). J. Bacteriol. 111,705-716. Wilson, G. h. & Young, F. E. (1973). Bacterial Transformation (Archer, L. J., ed.), pp. 269-292. Academic Press, New York. Wilson, G. A. & Young, F. E. (1974). Abstracts, 2nd European Meeting on Transformation and Trana]ection in Micro.organisms, Cracow, Poland, 141. Wilson, G. A., Williams, M. T., Baney, H. W. & Young, F. E. (1974). J. Virol. 14, 10131016. Yoshimori, R. N. (1971). Ph.D. Thesis. University of California, San Francisco, U.S.A.
Isolation o f a Sequence-specific Endonuclease (BamI) from
Bacillus amyloliquefaciens H A restriction endonuclease has been isolated from Bacillus amyloliquefaciens H (strain RUB500). The enzyme, BamI, cleaves adenovirus-2 DNA at three sites, phage XDNA at five sites, Xplac DNA at four sites, ~80 pt DNA at 14 sites, and ~b3T+ DNA at four sites. However, it does not cleave DNA from bacteriophage SPO2, ~b105or ~29. Our previous studies (Wilson & Young, 1972,1973) with Bacillus 8ubtilis and a number of closely related bacilli, including Bacillus amyloli~usfaciens H, demonstrated that genetic material could be exchanged between them by transformation and that they therefore comprised a genospecies {Ravin, 1963). However, the frequency of genetic exchange with heterospecific DNA is several orders of magnitude below that obtained with homospecific DNA thus suggesting the presence of restriction and modification enzymes. This hypothesis is strengthened by the recent discovery of a classical restriction and modification system between B. amyloliquefacien8 H and B. subtilis 168 (Wilson & Young, 1974; Wilson d al., 1975). Furthermore, a restriction endonuclease (BsuI) has been reported in a strain of B. s~btilis (Trautner et al., 1974). We, therefore, examined members of the B. subtilis genospecies for the presence of endonucleases to determine if they were responsible for the inefficiency of genetic exchange among heterologons strains. This report describes the purification and partial characterization of a restriction endonuclease from B. amyloliquefaciens H. B. amyloliqusfaciens H (strain RUB500) was grown in Penassay broth (Difco) to the late logaritbmlc phase of growth and harvested b y centrifugation. The cells (20 g) were disrupted b y grinding with alumina (3 times the wet weight of the cells) and suspended in 10 ml of extraction buffer (25 mM-Tris.HC1 (pH 8-0), 5 mM-2mercaptoethanol). Cell debris and alumina were removed by centrifugation (23,000 g, 60 mln at 4°C) and the supernate (20 ml) was diluted with 80 ml extraction buffer. Nucleic acids were removed by precipitation with streptomycin sulfate (1.0 ml 10% streptomycin sulfate/1500 A2so units). Solid ammonium sulfate was added to the supernate with constant stirring at 4°C. Although some activity was precipitated by 5 0 ~ saturation with (NH4)2S04, most of the activity was in the 50 to 80% precipitate. This precipitate was suspended in 10 ml of buffer I (0.01 E-sodium phosphate, p H 7.4) and applied to a bed of Sephadex G25 (5 cm × 39 cm). After ehition with buffer I, the fractions that contained endonuclease activity were pooled, and chromatographed on DEAE-cellulose (DE52, Whatman, 2.4 cm × 13.5 cm bed) by elution with a one liter linear gradient of 0 M to 0.6 ~-NaCl in buffer I. Fractions were pooled and tested for endonuclease activity b y agarose/ethidium bromide gel electrophoresis of DNA (Sharp eta/., 1973). Fractions 10 t o 2 5 that eluted between 0.05 and 0.I5 MNaC1 showed endonuclease activity (Plate I). Fractions that eluted at higher salt concentrations had considerable exonuclease activity and were discarded. Fractions 10 through 25 from the DEAE-cellulose chromatography were dialyzed twice ag~.in~t three liters of buffer I and chromatographed on phosphocellulose (Whatman P l l , 1.2 cm × 8.0 cm bed). The phosphocellulose was precycled first with 0-5 ~-NaOH 123
124
G. A. WILSON AND F. E. YOUNG
and then 0-5 ~-HC1 and equilibrated with buffer H (0.01 M-NaPO~(pH 7.4), 0.01 ~2-mercaptoethanol). The enzyme was eluted with a one liter linear gradient (0 ~ to 0.6 M-NaCI) in buffer II. Most of the proteins did. not bind to the phosphocellulose. Endonuelease, as determined b y electrophoretic analysis of DNA t h a t had been treated with samples from the column, eluted mainly between 0.31 and 0"36 MNaCl (fractions 110 to 125). As seen in Plate II, some endonuclease activity was found in all fractions between 75 and 140. I t is not known at this time whether the patterns reflect partial digestion of DNA or actually represent another enzyme with a different specificity. The pooled fractions from phosphocellulose chromatography (110 to 125) were used in the subsequent characterizations. The enzyme at this stage of purification has very little or no exonuclease activity (Table 1) and is stable for prolonged periods of time at 4°C. The enzyme has been designated B a m I according to the nomenclature proposed by Smith & Nathans (1973). TABLE 1
E,xonuclease activity of enzyme preparation8 Assay conditions No enzyme Pancreatic DNAase BamI 50 to 80% (NH4)2SO4 DEAE-cellulose pool PhosphoceUulose
Total acid-soluble radioactivity (ors/rain) 1380 547,080 30,295 5770 1640
The reaction mixture consisted of 8 ~g B. subtilis DNA radioactively labeled with [3H]thymidine, 50 pl enzyme preparation, 6 mM-Tris (pH 7.5), 6 mM-MgC12and 6 mM-2-mercaptoethanol in a final volume of 120 ~d. The reactions were incubated for 13.5 h at 37°C. Cold 5% trichloroacetic acid was added, the reaction mixture stored at 0°C for 30 rain and subsequently filtered through 0.65 Millipore membrane (DAWP). The extent of exonuclease activity is indicated by the amount of radioactivity in the filtrate in the presence and absence of BamI. DNA preparations from a variety of organisms have been used to characterize B a m I . The initial cleavage patterns, determined by agarose/ethidium bromide gel electrophoresis indicated t h a t B a m I recognized three sites in the adenovirus-2 DNA, four sites in ~p/ac DNA, four sites in ~ DNA and four sites in ¢3T + (Plate III). (Recentiy, P. W. J. Rigby has informed us that B a m I cleaves the A genome at 5 sites. His experiments demonstrated t h a t the second band contained a heterogeneous population of molecules. One large fragment was actually composed of two molecules held together b y the cohesive ends of the 2 genome. When separated, one fragment co-migrates with the fifth band illustrated in Plate III.) These patterns clearly establish t h a t B a m I is a unique enzyme and is not an isoschizomer of one of the several endonucleases t h a t have been isolated from micro-organisms t h a t cleave unmodified, double-stranded DNA at unique nueleotide sequences (Smith & Wilcox, 1970; Yoshimori, 1971; Middleton et al., 1972; Sharp et al., 1973; Takanami & Kojo, 1973; Takanami, 1973; Roberts et al., 1974 and R. Roberts, personal communication). The enzyme therefore should be useful in studies directed toward determining the nueleotide sequence of various DNA molecules. To date the enzyme has only been partially examined for its biological function. Although DNA from bacteriophages
(10-20) 2-2
(26-41)
( 4 2 - 5 7 ) ( 5 8 - 6 2 ) ( 6 3 -81)
-
2'0
1-8--
I-6--
0 ~o oa
1.4--
c
2
1"2--
1.0 --
0.8
--
0 - 6 --
0"4--
0-2-
10
20
30
40
50
I 60
I 70
80
90
I00
I10
120
Fraction no.
PLATE I. DEAE-cellulose chromatography of B a m I . The enzyme was eluted from DEAE-celhdose in 5-ml fractions (total 124) as described in the text, Absorbanee was determined at 280 n m (1 cm path) on a Gilford 2400 S spectrophotometer. Fractions were pooled according to numbers given in parentheses and assayed for nuclease activity as described in legend to Plate II. Samples from pooled fractions 63 to 81 also contained low molecular weight material t h a t migrated too far on the gel to be included.
[ facinrj p. 124
PLATE II. Assay of phosphoeellulose column. Samples (20 /~1) from the phosphoeellulose column fractions (5 ml) were assayed for nuelease activity in reaction m i x t u r e (50/~1 total vol.) containing 2/~g A plac DNA, 6 mM.Tris.HCl (pH 7.4), 6 m~z-MgC12 a n d 6 mM-2-mercaptoethanol. After incubation for 2 h a t 3 7 ° 0 , t h e reaction was stopped b y t h e addition of 5/~l 100 mM-EDTA, a n d 15/~l 6 0 % sucrose, 1% bromphenol blue. Samples wez~ loaded on a 0-7% • garose gel containing 0.5 /~g e t h i d i u m bromide/ml. Electrophorosis was carried o u t a t 150 V (75 mA) for 3 h in a n EC-470 vertical gel a p p a r a t u s a t 80°C. Gels were p h o t o g r a p h e d u n d e r u.v. illumination using polaroid P N / 5 5 film a n d a Vivitar red no. 25A filter (see Sharp el al., 1973). The n u m b e r s ~bove the pockets indicate the fraction number. The entire gradient consisted of 20~ fractions. At thi~ stage of purification the yield from 10 g of cells is approximately 1,000,000 units. One unit is sufficient to cleave one/~g of A DNA in 60 min.
PLATE III. Agarose gel electrophoresis of various DNAs after digestion with B a m I endonuelease. Approximately 2 ~g of D N A from purified bacteriophage were digested for 3 h a t 37°C with B a m I endonuelease (purified t h r o u g h phosphoeellulose step). Reactions were stopped b y the addition of 100 mr~-EDTA. Conditions for digestions are described in legend to Plate I L Preparations were electrephoresed on a Blair Craft 20 cm × 20 em slab gel a p p a r a t u s for 6 h a t 60 mA. Gel was composed of 1.2% agarose containing 0.5 ~g ethidium bromide/ml. The gel was photographed on K o d a k Tri-X P a n Profe3sional film 4164 using a Vivitar red no. 25A filter under u.v. illumina. rich. (1,2) A wild type DNA; (3,4) ~ plac D N A ; (5,6) ~105 DNA; (7,8) ~3T + DNA; (9,10) S P 0 2 D N A ; (11,12) Ad-2 D N A ; (13,14) ~80 p t DNA. The even n u m b e r e d samples are digested with BamI. The molecular weight of D N A from bacteriophages ~105 a n d SPO2 are b o t h 26-3 × 108 (Chow et el., 1972).
L E T T E R S TO T H E E D I T O R
125
4105 and S P 0 2 is cleaved with EcoRI (Wilson et al., 1974), B a m I does not appear to cleave these viral genomes nor I ) N A from bacteriophage ~29. D N A from m u t a n t s of bacteriophage S P 0 2 t h a t can be propagated in both B. 8ubtilis and B. amylolique. faciens H strains, however, is cleaved b y B a m I only when these viruses are propagated on B. subtitis (unpublished data). Therefore, B. amyloliquefaviens H m u s t also have a modification system t h a t protects its I ) N A from its own restriction system. We previously reported the construction of genetic intergenoC~s between B. suStili8 and B. amyloliquefaciens (Wilson & Young, 1972). The intergenotes have a t least one foreign gene from B. amy~o~iquefaciens t h a t was introduced into the genome of B. 8ubtilis b y transformation. These foreign genes coding for resistance to rifampin are therefore small segments of D N A t h a t retain the foreign base sequence b u t lack the modification provided b y B. amy~oliquefaciens. We have shown t h a t these foreign genes are restricted in vitro b y B a m I and rapidly lose the ability to transform B. subtili8 (Wilson & Young, manuscript in preparation). This enzyme, the first to be isolated from a heterologous m e m b e r of the B. ~abtilis genospecies, therefore m a y play a significant role in restricting foreign I)NA. I t should also prove useful as a tool for selectively fragmenting D N A in vitro for sequence analyses. The authors wish to thank Dr Peter W. J. Rigby for access to his experiments demonstrating that BamI recognizes five sites in the h genome. Gratitude is expressed to Drs Carel l~Iulder and Richard J. Roberts for their gifts of adenovirus-2 DNA, Dr M. Oishi for bacteriophage ¢80 pt, and H. W. Baney for his teclmical assistance. We also thank Drs H. O. Smith and R. J. Roberts who provided us with m a n y of the techniques. This research was supported by grant VC.27-J from the American Cancer Society. Department of Microbiology University of Rochester School of Medicine and Dentistry Rochester, N.Y. 14642, U.S.A.
GARY A. WILSON FI~Am~ E. Y o u n g
Received 9 April 1975, and in revised form 2 June 1975 REFERENCES Chow, L. T., Boice, L. B. & Davidson, N. (1972). J. Mel. Biol. 68, 391-400. Middleton, J. H., Edgell, M. H. & Hutchison, C. A., I I I (1972). J. Virel. 1D, 42-50. Ravin, A. W. (1963). Amer. Natur. 97, 307-318. Roberts, R. J., Breitmeyer, J. B., Tabachnik, N. F. & Meyers, P. A. (1974). J. Mol. Biol. 91, 121-123. Sharp, P. A., Sugden, B. & Sambrook, J. (1973). Biochemistry, 12, 3055-3063. Smith, H. O. & Nathans, :D. (1973). J. Mol. Biol. 81,419-423. Smith, H. O. & ~Vilcox, K. W. (1970). J. Mol. Biol. 51, 379-391. Takanami, M. (1973). F E B S Letters, 34, 318-322. Takanami, M. & Kojo, H. (1973). -~EBS Lettzrs, 29, 267-270. Trautner, T. A., Pawlek, B., Bron, S. & Anagnospopoulos, C. (1974). Mol. Gen. Genst. 131, 181-191. Wilson, G. A. & Young, F. E. (1972). J. Bacteriol. 111,705-716. Wilson, G. h. & Young, F. E. (1973). Bacterial Transformation (Archer, L. J., ed.), pp. 269-292. Academic Press, New York. Wilson, G. A. & Young, F. E. (1974). Abstracts, 2nd European Meeting on Transformation and Trana]ection in Micro.organisms, Cracow, Poland, 141. Wilson, G. A., Williams, M. T., Baney, H. W. & Young, F. E. (1974). J. Virol. 14, 10131016. Yoshimori, R. N. (1971). Ph.D. Thesis. University of California, San Francisco, U.S.A.
