J. Mol. Riol. (1978) 118. 127-135
A New Restriction Endonuclease from Streptomyces albus G h rt)st riction albus U. This h DNA at two ri~cognizes the
endonuclease, SalI, has been partially purified from Stre~ton~yces enzyme cleaves adenovirus-2 DNA at t,hree sites, bacteriophage sites, but dox not, cleave simian vir\~s 40 DNA or +X174 DNA. It, sequenre
and cuts at the sites indicatecl by the arrows. An endonuclease (XamI) wit,h similar specificity has also been isolated from Xanthomonas amaru~~thicoZa.
The physical dissection and characterization of DNA genomes is being greatly facilitated by the increasing number of specific enclodeoxyribonucleases which both recognize and cleave a specific sequence of base-pairs within a DNA duplex (Roberts, 1976). Their detection and purification are facilitated by a simple assay procedure using agarose slab gel electrophoresis (Sharp et al., 1973; Sugden et al., 1975) which gives a characteristic banding pattern after digestion of DNA with a specific endonuclease. Streptomyces albus Gt contains a specific endonuclease, SalI, which functions as a restriction enzyme in viva (Chater & Wilde, 1976). We report the purification and characterization of SaZI. S. albus G, obtained from J. &I. Ghuysen, was grown at 32°C in liquid medium containing per litre: 8 g nutrient broth (Difco), 340 g sucrose, 10 g MgCl,, 5 g glucose. Cells were harvested after 24 hours for the preparation of Sal1 (yield approx. 5 to 8 g/l) and after 48 hours or longer for the preparation of Sal1 + Sal11 (yield approx. 8 to 12 g/l). For growth on solid medium, we used sporulation agar (ATCC medium 5) which contained per litre: 1-Og yeast extract, 1-Og beef extract, 2.0 g tryptone, 10.0 g glucose, 15.0 g agar and FeSO, (trace). Cells (10 g wet weight) were resuspended in 10 ml of a buffer containing 0.01 M-Tris*HC!l (pH 7.5), 0.01 M-SHCH,CH,OH, 25 mg lysozyme, incubated at room temperature for 15 minutes, and then disrupted by souication. After high-speed centrifugation (100,000 g for 90 min), the supernatant was made 1-OM-NaCl and fractionated on a column (100 cm x 2.5 cm diam.) of Biogel A, 0.5 m (BioRad) which was elutecl with a buffer containing 1.0 M-NaCl, 0.01 M-Tris . HCl (pH 7.5), 0.01 M-SHCH,CH,OH. Fractions were assayed by incubation with aclenovirus-2 (Ad-2) DNA and the digest examined by agarose gel electrophoresis. Those containing endonucleolytic activity were combined and dialyzed against a buffer containing 0.01 M-potassium phosphate (pH 7*4), 0.01 M-SHCH,CH,OH, O*OOOl tS. albus solvifaciens, extensively retained the
G, originally isolated from soil by Gratis, has been reclassified as a subspecies, of S. grtieus (Pridham & Tresner, 1974). Since the former name has been used and another strain of S. griseus contains a different specific endonuclease we have name S. albw G. 127
128
.I.
K,. ARKANI).
1’.
t\.
MYERS
AND
Irminally labelcrl WI fragments. Atl-2 DNA (23 pg) was incubated in a reaction mix (100 ~1) containing 0 rn>l-Tris.HCl (l)H 7.9), (i mx-MgCl,, 6 mu-SHCH,CH,OH, 25 units Sal1 for 1 h at 3’7°C. Alkaline phosphatasc (10 /Lg) was added and the volume increased to 200 ~1 by adjusting to 50 mmTris.HCl (pH 8.9), 10 ~RIMgCI, and incubation continued for a further 1 h at 37°C. Following oxt,raction with phenol (4 x 200 ~1) the DNA fragments were precipitated with ethanol and recovered by centrifugation (100,000 g for 20 min). The DNA was phosphorylated in a reaction mix (100 ~1) containing 50 mM-Tris-HCI (pH 9.5), 10 mM-MgCl,, 5 m%r-dithiothreit.il, 5:/, glycerol, IO ~M-[~-~~P]A'I'P
LETTERS
TO THE TABLE
Idmtijkation
of the 5’-tlimcleotide
EDITOIZ
133
1 present
after
cleavqe
with
SalI
The f’our standard dinuclt~otidex \vtar(a prepared by phonl>horylating t,ho caorresponding dinuc*lraoside monophosphates (Collaborativ(x &searrh) with polynurleotide kinase and [y-“2P]ATP. as desrrihrd in the legend to Fig. 5. and purifying the products by clectrophoresis on DEAEc~llulost* paper in 7 o/0 (v/v) formic acitl. Oligonucleotides from the fingerprint shown in Fig. 5 w-em incubated in a reaction mix (20 ~1) containing 66 mM-glyrine-NaOH (pH 9.6), 6.6 mM-MgCl,, 3.3 mn-dithiothreitol, 5 units exonuclease I for 30 min at 37°C’. The products were analyzed by clectrophoresis on Whatman 540 paper at pH 3.5 (Barrftll, 1971). The oligonucleotide labeled pTC in Fig. 5 was unaffected by this treatment and was itlentical with the product of digestion of all longer oligonucleotides. RR rcfcrs to the r*lrc%rophoretic, mobility with respect to that of thr blue dyn xylene cyanol FF.
their mobility shifts (Sangcr pt al., 1973) and suggested a unique pentanucleotide, pT-C-G-S-C, present, at t,he 5’-end of each Au11 digestion product. The presence of all four possible hexanucleotides. pT-C-(:-A-C-N. indktes that specificity is lost at the sixt’h posit’ion. The simplest intwpretation of t’hcse result’s is that, Sal1 recognizes the hexanucleotidc ,j'-G-fi'-CJmGeA+-C-3'
3’-C-8-G-C-T-G-5 \vith the sites of cleavage indicated by the arro\vs. ‘I!his would mean that Sal1 fragpossess a 5’-tetranucleot,ide extension and hence should be a substrate for a DEA polymerase. This prediction was tested by preparing Sal1 fragments of Ad-2 DNX and incubat,ing them with avian myeloblastosis virus reverse transcriptase in the presence of one [+32P]deoxynucleoside triphosphate and three unlabeled deoxynucleoside triphosphat,es. All four possible combinations were used and t’he products wcrc analyzed by nearest-neighbor analysis. The results of these experiments are shown in Table 2 and provide contirmat8ion of the recognition sequence assigned earlier on thrx basis of mobilit’y shifts. Although Xaml must recognize this same sequence, we haw not, been able t’o obtain this enzyme sufficient’lv pure to d&ermine if the site of cleavage is identical w&h that of SalI. men&
(spec. act. 800 Ci/mmol), 10 units polynucleotide kinase and incubated for 1 h at 37°C. Unreacted 1y-32P]ATI’ was removed by pa,ssage through a Sephades G50 column run in 0.1 M-NaCl, 0.01 11.Tris.HC’l (pH 7.9), 0.001 al-Na,EDTA. Labeled Sal1 fragments were rerovered from the void volumc~ by precipitation with ethanol (2 vol.) and centrifugation (100,000 g for 20 min). The DNA was then incubated in a rear&m mix (30 ~1) containing 0.1 ar-sodium acetate (pH 5.0), 5 mM Mg(‘1,. 5 pg pancreatic DNAase for 30 min at, 37°C. Then 10 ~1 was lyophilized, redissolved in wat,or (3 ~1) and fingerprinted using elertrophoresis on c*c>llulose acetate (CA) at pH 3.5 in the In-cscnc~> of 7 If-urea for the first dimension, and homochromatography (HC) (Brownlee & Sanger, 1969) on a thin lavor of DEAE-rrllulosc (1 : 10) cluting with Homomix VI (Jay et al., 1974) for the s~(~mtl tlimt*nsron. The 5’.oligonllc~leotitles mere detrctcd by aut~oradiography and cluted from t htr thin lay
A New Restriction Endonuclease from Streptomyces albus G h rt)st riction albus U. This h DNA at two ri~cognizes the
endonuclease, SalI, has been partially purified from Stre~ton~yces enzyme cleaves adenovirus-2 DNA at t,hree sites, bacteriophage sites, but dox not, cleave simian vir\~s 40 DNA or +X174 DNA. It, sequenre
and cuts at the sites indicatecl by the arrows. An endonuclease (XamI) wit,h similar specificity has also been isolated from Xanthomonas amaru~~thicoZa.
The physical dissection and characterization of DNA genomes is being greatly facilitated by the increasing number of specific enclodeoxyribonucleases which both recognize and cleave a specific sequence of base-pairs within a DNA duplex (Roberts, 1976). Their detection and purification are facilitated by a simple assay procedure using agarose slab gel electrophoresis (Sharp et al., 1973; Sugden et al., 1975) which gives a characteristic banding pattern after digestion of DNA with a specific endonuclease. Streptomyces albus Gt contains a specific endonuclease, SalI, which functions as a restriction enzyme in viva (Chater & Wilde, 1976). We report the purification and characterization of SaZI. S. albus G, obtained from J. &I. Ghuysen, was grown at 32°C in liquid medium containing per litre: 8 g nutrient broth (Difco), 340 g sucrose, 10 g MgCl,, 5 g glucose. Cells were harvested after 24 hours for the preparation of Sal1 (yield approx. 5 to 8 g/l) and after 48 hours or longer for the preparation of Sal1 + Sal11 (yield approx. 8 to 12 g/l). For growth on solid medium, we used sporulation agar (ATCC medium 5) which contained per litre: 1-Og yeast extract, 1-Og beef extract, 2.0 g tryptone, 10.0 g glucose, 15.0 g agar and FeSO, (trace). Cells (10 g wet weight) were resuspended in 10 ml of a buffer containing 0.01 M-Tris*HC!l (pH 7.5), 0.01 M-SHCH,CH,OH, 25 mg lysozyme, incubated at room temperature for 15 minutes, and then disrupted by souication. After high-speed centrifugation (100,000 g for 90 min), the supernatant was made 1-OM-NaCl and fractionated on a column (100 cm x 2.5 cm diam.) of Biogel A, 0.5 m (BioRad) which was elutecl with a buffer containing 1.0 M-NaCl, 0.01 M-Tris . HCl (pH 7.5), 0.01 M-SHCH,CH,OH. Fractions were assayed by incubation with aclenovirus-2 (Ad-2) DNA and the digest examined by agarose gel electrophoresis. Those containing endonucleolytic activity were combined and dialyzed against a buffer containing 0.01 M-potassium phosphate (pH 7*4), 0.01 M-SHCH,CH,OH, O*OOOl tS. albus solvifaciens, extensively retained the
G, originally isolated from soil by Gratis, has been reclassified as a subspecies, of S. grtieus (Pridham & Tresner, 1974). Since the former name has been used and another strain of S. griseus contains a different specific endonuclease we have name S. albw G. 127
128
.I.
K,. ARKANI).
1’.
t\.
MYERS
AND
Irminally labelcrl WI fragments. Atl-2 DNA (23 pg) was incubated in a reaction mix (100 ~1) containing 0 rn>l-Tris.HCl (l)H 7.9), (i mx-MgCl,, 6 mu-SHCH,CH,OH, 25 units Sal1 for 1 h at 3’7°C. Alkaline phosphatasc (10 /Lg) was added and the volume increased to 200 ~1 by adjusting to 50 mmTris.HCl (pH 8.9), 10 ~RIMgCI, and incubation continued for a further 1 h at 37°C. Following oxt,raction with phenol (4 x 200 ~1) the DNA fragments were precipitated with ethanol and recovered by centrifugation (100,000 g for 20 min). The DNA was phosphorylated in a reaction mix (100 ~1) containing 50 mM-Tris-HCI (pH 9.5), 10 mM-MgCl,, 5 m%r-dithiothreit.il, 5:/, glycerol, IO ~M-[~-~~P]A'I'P
LETTERS
TO THE TABLE
Idmtijkation
of the 5’-tlimcleotide
EDITOIZ
133
1 present
after
cleavqe
with
SalI
The f’our standard dinuclt~otidex \vtar(a prepared by phonl>horylating t,ho caorresponding dinuc*lraoside monophosphates (Collaborativ(x &searrh) with polynurleotide kinase and [y-“2P]ATP. as desrrihrd in the legend to Fig. 5. and purifying the products by clectrophoresis on DEAEc~llulost* paper in 7 o/0 (v/v) formic acitl. Oligonucleotides from the fingerprint shown in Fig. 5 w-em incubated in a reaction mix (20 ~1) containing 66 mM-glyrine-NaOH (pH 9.6), 6.6 mM-MgCl,, 3.3 mn-dithiothreitol, 5 units exonuclease I for 30 min at 37°C’. The products were analyzed by clectrophoresis on Whatman 540 paper at pH 3.5 (Barrftll, 1971). The oligonucleotide labeled pTC in Fig. 5 was unaffected by this treatment and was itlentical with the product of digestion of all longer oligonucleotides. RR rcfcrs to the r*lrc%rophoretic, mobility with respect to that of thr blue dyn xylene cyanol FF.
their mobility shifts (Sangcr pt al., 1973) and suggested a unique pentanucleotide, pT-C-G-S-C, present, at t,he 5’-end of each Au11 digestion product. The presence of all four possible hexanucleotides. pT-C-(:-A-C-N. indktes that specificity is lost at the sixt’h posit’ion. The simplest intwpretation of t’hcse result’s is that, Sal1 recognizes the hexanucleotidc ,j'-G-fi'-CJmGeA+-C-3'
3’-C-8-G-C-T-G-5 \vith the sites of cleavage indicated by the arro\vs. ‘I!his would mean that Sal1 fragpossess a 5’-tetranucleot,ide extension and hence should be a substrate for a DEA polymerase. This prediction was tested by preparing Sal1 fragments of Ad-2 DNX and incubat,ing them with avian myeloblastosis virus reverse transcriptase in the presence of one [+32P]deoxynucleoside triphosphate and three unlabeled deoxynucleoside triphosphat,es. All four possible combinations were used and t’he products wcrc analyzed by nearest-neighbor analysis. The results of these experiments are shown in Table 2 and provide contirmat8ion of the recognition sequence assigned earlier on thrx basis of mobilit’y shifts. Although Xaml must recognize this same sequence, we haw not, been able t’o obtain this enzyme sufficient’lv pure to d&ermine if the site of cleavage is identical w&h that of SalI. men&
(spec. act. 800 Ci/mmol), 10 units polynucleotide kinase and incubated for 1 h at 37°C. Unreacted 1y-32P]ATI’ was removed by pa,ssage through a Sephades G50 column run in 0.1 M-NaCl, 0.01 11.Tris.HC’l (pH 7.9), 0.001 al-Na,EDTA. Labeled Sal1 fragments were rerovered from the void volumc~ by precipitation with ethanol (2 vol.) and centrifugation (100,000 g for 20 min). The DNA was then incubated in a rear&m mix (30 ~1) containing 0.1 ar-sodium acetate (pH 5.0), 5 mM Mg(‘1,. 5 pg pancreatic DNAase for 30 min at, 37°C. Then 10 ~1 was lyophilized, redissolved in wat,or (3 ~1) and fingerprinted using elertrophoresis on c*c>llulose acetate (CA) at pH 3.5 in the In-cscnc~> of 7 If-urea for the first dimension, and homochromatography (HC) (Brownlee & Sanger, 1969) on a thin lavor of DEAE-rrllulosc (1 : 10) cluting with Homomix VI (Jay et al., 1974) for the s~(~mtl tlimt*nsron. The 5’.oligonllc~leotitles mere detrctcd by aut~oradiography and cluted from t htr thin lay
