C L E A V A G E OF B A C T E R I O P H A G E M13 D N A
H A E M O P H I L US I N F L U E N Z A E C. M.
VAN DEN HONDELAND J.
BY
ENDONUCLEASE-R
G. G. SCHOENMAKERS
Laboratory of Molecular Biology, University of Ni/megen, Nijmegen, The Netherlands (Received 12 April, 1973) ABSTRACT. The restriction enzyme from Haemophilus influenzae, endonuclease-R, has only one cleavage site on the double-stranded replicative form DNA of bacteriophage M13. Circular replicative forms are broken to yield full-length liniar M 13-DNA molecules (RF-III). The cleavage site appears to be specific as the RF-III molecules, produced by endonuclease-R, cannot be circularized by denaturation and renaturation. I. INTRODUCTION The genome of the small filamentous bacteriophage M13 consists of a covalently closed, circular DNA molecule of approximately 2 x 10 6 daltons [1]. In any attempt to sequence this DNA, it seems logical that an initial step would be the production of small unique fragments of the whole molecule. The utilization of restriction enzymes is very attractive for this purpose since these nucleases make a limited number of duplex cleavages by recognizing specific nucleotide sequences in double-stranded DNA [2, 3]. The utilization of two distinct restriction enzymes from Haemophilus for the production of specific fragments of ~X-174 replicative form DNA has already been described [4, 5]. This report describes the specific fragmentation obtained from the closed replicative form DNA (RF-I DNA) of coliphage M13 after digestion with endonuclease-R, a restriction enzyme from Haemophilus influenzae Rd [6].
II. MATERIALS AND METHODS
1. Bacteria and Phages Haemophilus influenzae Rd was obtained from Dr. H. O. Smith. The bacteria were grown in Brain Heart Infusion medium (Difco), supplemented with 2 lag of nicotine adenine nucleotide per ml and 10 lag of hemin(Sigma) per ml. For the preparation of M13 RF-I DNA the strain E. coli C89 (K12, 159F § uvs, su-) was used. Phage M13 was originally obtained from Dr. P. H. Hofschneider, Miinchen, phage T~'was a gift from Dr. D. Rabussay. 2. Preparation of(3H)-RF-I DNA E. coli C89 was grown in 100 ml of M-9 medium. At a titer of 4 x 108 cells m1-1 the culture was supplemented with 300 lagm1-1 deoxyadenosine [7], 0.5 lag ml -~ thymidine and 10 I~Ciml -~ 41 Molecular Biology Reports 1 (1973) 41-45. All Rights Reserved Copyright 9 1973 by D. Reidel Publishing Company, Dordrecht-Holland
(3H)-thymidine (specific radioactivity 27.7 Ci mmo1-1) and infected with M13 phage at a multiplicity of 20. Growth was continued for 60 min at 37 ~ The cells were then spun out, washed with 0.1 M Tris (pH 7.6), 0.1 M NaC1, 0.01 ~ EDTA and resuspended in 20 ml of the same buffer. Lysozyme (2 mg) and heat-treated RNAase (2 mg) were added and after 30 min incubation at 37 ~ lysis was fairly complete. To the viscous solution 1 ml of 20 ~ sodium dodecylsulphate was added and the incubation continued for 5 min. The solution became now water-clear. Host DNA was removed by careful addition of NaC1 to a final concentration of 1 M, and after standing for 2 hr at 4~ the D N A precipitate was spun down for 30 rain at 25000 rpm. The supernatant was carefully pipetted off and then extracted twice with one volume of redistilled buffer-saturated phenol. The aqueous phase was dialyzed against 0.01 t,l Tris (pH 7.6), 1.0 t,i NaCI, 0.001 M EDTA. The D N A was melted by addition of 1.0 M N a O H to give a pH of 11.8. After 3 min the solution was returned to pH 7.6 and then it was percolated through a nitrocellulose column (10 x 1 cm), equilibrated in 0.01 M Tris (pH 7.6), 1.0 ~ NaCI, 0.001 M EDTA. The radioactive eluate, containing the RF-I DNA was pooled and dialyzed against 0.01 M Tris (pH 7.6), 0.1 MNaCI, 0.001 M EDTA. The RF-I D N A was finally precipitated with 2 volumes of isopropanol and dissolved in 2 ml of 0.02 M Tris-HC1 (pH 7.5). (3H)-~bX174 RF-I D N A was a gift of Dr. H. Heineker, Rijswijk.
3. Enzymatic Digestions An amount (1-10 pl) of endonuclease-R at approximately 1 unit per ml [6] was added to 50 ~tl of (3H)-RF-I D N A (1000-10000 counts m i n - l ; 0.01 A26 o units) in enzyme buffer, containing 6 m u Tris-HCl (pH 7.6), 50 mM NaC1, 6 mM mercaptoethanol. The solution was made 6 mM in MgCI 2 and digested for the appropriate period at 37~ The reaction was stopped by adding 50 Ixl of 0.1 M EDTA (pH 7.6).
4. Denaturation and Renaturation An endonuclease-R digest (0.25 ml) of (3H)-M13 RF-I DNA was extracted twice with an equal volume of phenol, saturated with 0.01 M Tris (pH 7.6), 0.1 M NaC1, 0.001 ~ EDTA. The D N A was precipitated from the aqueous phase by the addition of 2 volumes of ethanol at - 2 0 ~ the precipitate was collected by centrifugation at 15000 x # for 30 min and the pellet was finally dissolved in 0.05 ml of 0.1 x SSC. A sample of D N A (25 pl) was denatured by heating for 5 min at 100~ and then rapidly chilled in ice. To anneal the denatured sample, the salt concentration was raised to 2 x SSC and the sample was incubated for 24 hr at 66 ~ in a sealed capillary tube. Thereafter it was cooled down to room temperature.
5. Counting All samples were counted in a Packard Tri-Carb scintillation counter with a toulene-based scintillation fluid containing 0 . 4 ~ Omnifluor and 25 ~ (v/v) Triton X-100. III. RESULTS A N D DISCUSSION The endonuclease-R was isolated from H. influenzae cells according to a modified procedure as given by H. O. Smith (personal communication), i.e. streptomycin sulphate precipitation of the supernatant fraction, obtained after disrupting the cells by sonication and centrifugation at 50000 rpm for 75 min, followed by ammonium sulphate precipitation (50-70 ~o) and phosphocellulose column chromatography. 42
Radioactivity of 3HlCpm)
Q
1200 -
21S 16S
1000
I!
_
Radioactivity of 3H(Cpm]
2/,S 17S 19S
1200
8S
It
I000
f l
'I I
800
_
600
-
I I I
~ ! I I
L,O0 200
I
It fl
I I
800
t ~ t
600
10
l,O0
I
i! I
I !
I
~
'I
_222222_2_222"2SS.2-~---_
O
@
Fraction
i* ll
-
t
0
10
I
t
I
t
200 -~-
20
_
~:~,o_ 30 t,O TOP
0 20
Fraction
30
~0
TOP
Fig. 1. Sedimentation analysis of the endonuclease-R digestion products of ffX174 RF-I D N A and M13 RF-I DNA. A 10 pl enzyme fraction was added to a reaction mixture (0.05 ml), containing 6 mM Tris-HCl (pH 7.6) 6 mM MgCI2, 50mM NaC1, 6 mM rnercaptoethanol and (3H)-~X174 RF-I D N A (6600 cpm; 0.01 A260 unit) or (aH)-M13 RF-I D N A (4000 cpm; 0.01 A2n0 unit). After incubation of 1 hr at 37~ the reaction was stopped by adding 50 pl of 0.1 M EDTA. The hydrolysate was layered on a 5-20 % (w/v) liniar sucrose gradient (in 1 M NaCI, 0.05 M Tris (pH 7.6), 0.003 ra EDTA) and centrifuged for 150 min at 65000 rpm and 4~ Two-drop fractions were collected in scintillation vials and the radioactivity measured as described under Methods section. (A): Digestion products of (all)- ~X174 RF-I D N A ( O Q). A control of (3H)- ~X174 R F - D N A ( 9 . . . . (3) incubated without enzyme was sedimented in a separate tube. (B): Digestion products of (aH)-M 13 RF-I D N A ( 0 0 ) . (3H)-M 13 RF-I D N A ( ( 3 - - - ( 3 ) sedimented in a separate tube as a control.
The active fraction obtained did show restriction-like enzyme properties as it produced a rapid decrease in viscosity of Tv-DNA and of native calf thymus D N A but did not degrade homologous H. influenzae DNA. Incubation of (3H)-T7 D N A with the purified enzyme fraction produced little radioactivity in the trichloroacetic acid-soluble fraction, indicating that the fraction did not contain an exonuclease activity attacking double-stranded DNA. In order to verify that the enzyme isolated was indeed endonuclease-R (endo-R), a sample of (3H)-RF-I D N A of phage ~bX174 was incubated with an excess of the enzyme fraction and the hydrolysate was then analyzed by sedimentation on sucrose gradients (Fig. 1A). Under the conditions used, RF-I with a twisted circular form, sedimented at a rate of about 21 S and the open circular form RF-II D N A at about 16 S. Incubation of (kX174-RF D N A with the enzyme fraction resulted in a complete conversion of both RF-I and RF-II. The fragments produced sedimented at 8.0S, which corresponds to the value already reported by Edgell et al. [4]. Treatment of M13-RF D N A with endo-R under further similar conditions resulted however in the appearance of a single sharp peak with a sedimentation value slightly slower than that of RF-II DNA, namely 17S (Fig. 1B). No smaller fragments were observed even after a prolonged 4-hr period of digestion, although some radioactivity could be detected now on top of the gradient, apparently due to the presence of a slight contamination of exonuclease activity in the endo-R 43
preparation. A plausible assumption for the appearance of 17S fragments would be that such fragments might be produced by a single double-stranded cleavage of the circular replicative form molecule by endo-R, resulting in double-stranded linear form molecules. (RF-III.) That M 13-RF D N A has a single cleavage site for endo-R could be substantiated by denaturation studies. Denaturation of the digestion products of (3H)-M13 R F - I D N A by alkali-treatment, followed by analysis on alkaline sucrose gradients revealed that all radioactivity is located in a single peak sedimenting at 16S. This is equivalent to the position of linear M I 3 D N A molecules, which sediment slightly slower than the circular M13 D N A used as a reference (Fig. 2). It is known that endo-R makes duplex cleavages in double-stranded D N A by recognizing a specific hexanucleotide sequence [2]. The presence of only one site of cleavage, located on a genome of about 6000 nucleotides long, as evidenced for M13 D N A , is rather unexpected. An alternative hypothesis would be that there is more than one cleavage site present in the molecule, but once the twisted circular form R F - I D N A has been cleaved into linear R F - I I I D N A molecules, the remaining cleavage sites will not be attacked by the restriction enzyme. I f this hypothesis is correct, denaturation and renaturation of the R F - I H molecules will give rise to circularization of the linear duplexes. As is shown in Fig. 3, denaturation and renaturation of the D N A fragments obtained by digestion of (3H)-M13 R F - I D N A by endo-R, did not produce detectable circular duplex D N A molecules. The reannealed D N A still sedimented at 17 S, which is exactly the same position as has been found for the original R F - I I I molecules. F r o m these results it can be concluded that the M13 genome has only a single cleavage site for
Radioacivity of 3H(dpml
Radioactivity ofl~C(dpml . . . .
/ I
90 -
.
.
.
Circ. Lin
.
3000
/
/
;
2000
i
1000
2001-
/ 0
10
2O
30
TOP
0
Fraction Fig. 2. Alkaline sucrose gradient sedimentation of M13 RF-I DNA digested with endonuclease-R. (aH)-MI3 RF-I DNA was digested under the conditions as described in Fig. 1. The hydrolysate was denatured by the addition of one volume of 1 M NaOH~nd subsequently layered on a 10-30~ linear sucrose gradient in 0.5 ra NaOH. Centrifugation was performed for 389hr at 65000 rpm and 4 ~ Two-drop fractions were collected in scintillation virals and the radioactivity was measured as described in Section II. (aH)-M13 RF-I DNA digested with endonuclease-R (e-------O) (14C)-M13 viral DNA ( O - - - O ) added as a marker. 44
Radioactivity ofZ~C(dprnl--Radioactivity o1 3Hldpm}
Radioactivityofl~Cldpm]
-- - - - ' 0 - - - - - - -
-----'O------
1000
t
800-
9
|
30S 9,
1000 1 800
1000!-
- 800
800-
-
600
6oo-
~I " , t
l i
2oo
,~ ~
0
- 1000
I1
ii
400
|
30S
I~
600
Radioactivity of 3H(dpm)
- 600
I I
L,O0
"~ 20
Fraction
- 200 30
"~0 TOP
0
-
or;-0
10
20 Fraction
30
0o
40 0 TOP
Fig. 3. Denaturation and renaturation of M13 RF-III DNA, produced by endonuclease-R digestion of M13 RF-I DNA. The endo-R digestion of (aH)-RF-I DNA and the subsequent isolation of (aH)-RF-III DNA was performed as described in Section II. (A): (aH)-M13 RF-III DNA (4000 cpm) was centrifuged for 150 min at 65000 rpm. Thereafter two-drop fractions were collected in scintillation virals and the radioactivity was measured. (3H)-M13 RF-III DNA (O Q). (t4C)-M13 viral DNA ( O - - - O ) added as a marker. (B): A sample of MI3 RF-III DNA in 0.1 x SSC was heated for 5 min at 100~ and then rapidly chilled in ice. After raising the concentration to 2 x SSC, the sample was reannealed for 24 hr at 66~ and subsequently layered on a 5-20~ linear sucrose gradient in 1 M NaC1, 0.05 M Tris (pH 7.6), 0.003 M EDTA and centrifuged for 150 min at 65000 rpm and 4~ Reannealed (aH)-M13 RF-III DNA (e~----O). (x4C)-M13 viral DNA ( O - - - O ) added as a marker.
the restriction enzyme o f H. influenzae Rd, which actually is cleaved resulting in linear duplex M13 D N A molecules. ACKNOWLEDGMENTS The authors are grateful to Mr. Th. Cuypers, w h o h e l p e d during the initial stages o f the work, Miss R. Matze for excellent technical assistance and Dr. R. Konings for helpful advices and suggestions. REFERENCES [1] [2] [3] [4] [5] [6] [7]
Marvin, D. A. and H o h n , B., Bacteriol. Rev. 33, 172 (1969). Kelley, T. J. and Smith, H. O., J. MoL Biol. 51, 393 (1970). Hedgpeth, J., G o o d m a n , H. M., and Boyer, H. W., Proc. Nat. Acad. Sci. 69, 3448 (1972). Edgell, M. H., Hutchison III, C. A., and Sclair, M., J. Virol. 9, 574 (1972). Middleton, J. H., Edgell, M. H., and Hutchison III, C. A., J. Virol. 10, 42 (1972). Smith, H. O. and Wilcox, K. W., J. Mol. Biol. 51, 379 (1970). Budman, D. R. and Pardee, A. B., J. BacterioL 94, 1546 (1967).
45
H A E M O P H I L US I N F L U E N Z A E C. M.
VAN DEN HONDELAND J.
BY
ENDONUCLEASE-R
G. G. SCHOENMAKERS
Laboratory of Molecular Biology, University of Ni/megen, Nijmegen, The Netherlands (Received 12 April, 1973) ABSTRACT. The restriction enzyme from Haemophilus influenzae, endonuclease-R, has only one cleavage site on the double-stranded replicative form DNA of bacteriophage M13. Circular replicative forms are broken to yield full-length liniar M 13-DNA molecules (RF-III). The cleavage site appears to be specific as the RF-III molecules, produced by endonuclease-R, cannot be circularized by denaturation and renaturation. I. INTRODUCTION The genome of the small filamentous bacteriophage M13 consists of a covalently closed, circular DNA molecule of approximately 2 x 10 6 daltons [1]. In any attempt to sequence this DNA, it seems logical that an initial step would be the production of small unique fragments of the whole molecule. The utilization of restriction enzymes is very attractive for this purpose since these nucleases make a limited number of duplex cleavages by recognizing specific nucleotide sequences in double-stranded DNA [2, 3]. The utilization of two distinct restriction enzymes from Haemophilus for the production of specific fragments of ~X-174 replicative form DNA has already been described [4, 5]. This report describes the specific fragmentation obtained from the closed replicative form DNA (RF-I DNA) of coliphage M13 after digestion with endonuclease-R, a restriction enzyme from Haemophilus influenzae Rd [6].
II. MATERIALS AND METHODS
1. Bacteria and Phages Haemophilus influenzae Rd was obtained from Dr. H. O. Smith. The bacteria were grown in Brain Heart Infusion medium (Difco), supplemented with 2 lag of nicotine adenine nucleotide per ml and 10 lag of hemin(Sigma) per ml. For the preparation of M13 RF-I DNA the strain E. coli C89 (K12, 159F § uvs, su-) was used. Phage M13 was originally obtained from Dr. P. H. Hofschneider, Miinchen, phage T~'was a gift from Dr. D. Rabussay. 2. Preparation of(3H)-RF-I DNA E. coli C89 was grown in 100 ml of M-9 medium. At a titer of 4 x 108 cells m1-1 the culture was supplemented with 300 lagm1-1 deoxyadenosine [7], 0.5 lag ml -~ thymidine and 10 I~Ciml -~ 41 Molecular Biology Reports 1 (1973) 41-45. All Rights Reserved Copyright 9 1973 by D. Reidel Publishing Company, Dordrecht-Holland
(3H)-thymidine (specific radioactivity 27.7 Ci mmo1-1) and infected with M13 phage at a multiplicity of 20. Growth was continued for 60 min at 37 ~ The cells were then spun out, washed with 0.1 M Tris (pH 7.6), 0.1 M NaC1, 0.01 ~ EDTA and resuspended in 20 ml of the same buffer. Lysozyme (2 mg) and heat-treated RNAase (2 mg) were added and after 30 min incubation at 37 ~ lysis was fairly complete. To the viscous solution 1 ml of 20 ~ sodium dodecylsulphate was added and the incubation continued for 5 min. The solution became now water-clear. Host DNA was removed by careful addition of NaC1 to a final concentration of 1 M, and after standing for 2 hr at 4~ the D N A precipitate was spun down for 30 rain at 25000 rpm. The supernatant was carefully pipetted off and then extracted twice with one volume of redistilled buffer-saturated phenol. The aqueous phase was dialyzed against 0.01 t,l Tris (pH 7.6), 1.0 t,i NaCI, 0.001 M EDTA. The D N A was melted by addition of 1.0 M N a O H to give a pH of 11.8. After 3 min the solution was returned to pH 7.6 and then it was percolated through a nitrocellulose column (10 x 1 cm), equilibrated in 0.01 M Tris (pH 7.6), 1.0 ~ NaCI, 0.001 M EDTA. The radioactive eluate, containing the RF-I DNA was pooled and dialyzed against 0.01 M Tris (pH 7.6), 0.1 MNaCI, 0.001 M EDTA. The RF-I D N A was finally precipitated with 2 volumes of isopropanol and dissolved in 2 ml of 0.02 M Tris-HC1 (pH 7.5). (3H)-~bX174 RF-I D N A was a gift of Dr. H. Heineker, Rijswijk.
3. Enzymatic Digestions An amount (1-10 pl) of endonuclease-R at approximately 1 unit per ml [6] was added to 50 ~tl of (3H)-RF-I D N A (1000-10000 counts m i n - l ; 0.01 A26 o units) in enzyme buffer, containing 6 m u Tris-HCl (pH 7.6), 50 mM NaC1, 6 mM mercaptoethanol. The solution was made 6 mM in MgCI 2 and digested for the appropriate period at 37~ The reaction was stopped by adding 50 Ixl of 0.1 M EDTA (pH 7.6).
4. Denaturation and Renaturation An endonuclease-R digest (0.25 ml) of (3H)-M13 RF-I DNA was extracted twice with an equal volume of phenol, saturated with 0.01 M Tris (pH 7.6), 0.1 M NaC1, 0.001 ~ EDTA. The D N A was precipitated from the aqueous phase by the addition of 2 volumes of ethanol at - 2 0 ~ the precipitate was collected by centrifugation at 15000 x # for 30 min and the pellet was finally dissolved in 0.05 ml of 0.1 x SSC. A sample of D N A (25 pl) was denatured by heating for 5 min at 100~ and then rapidly chilled in ice. To anneal the denatured sample, the salt concentration was raised to 2 x SSC and the sample was incubated for 24 hr at 66 ~ in a sealed capillary tube. Thereafter it was cooled down to room temperature.
5. Counting All samples were counted in a Packard Tri-Carb scintillation counter with a toulene-based scintillation fluid containing 0 . 4 ~ Omnifluor and 25 ~ (v/v) Triton X-100. III. RESULTS A N D DISCUSSION The endonuclease-R was isolated from H. influenzae cells according to a modified procedure as given by H. O. Smith (personal communication), i.e. streptomycin sulphate precipitation of the supernatant fraction, obtained after disrupting the cells by sonication and centrifugation at 50000 rpm for 75 min, followed by ammonium sulphate precipitation (50-70 ~o) and phosphocellulose column chromatography. 42
Radioactivity of 3HlCpm)
Q
1200 -
21S 16S
1000
I!
_
Radioactivity of 3H(Cpm]
2/,S 17S 19S
1200
8S
It
I000
f l
'I I
800
_
600
-
I I I
~ ! I I
L,O0 200
I
It fl
I I
800
t ~ t
600
10
l,O0
I
i! I
I !
I
~
'I
_222222_2_222"2SS.2-~---_
O
@
Fraction
i* ll
-
t
0
10
I
t
I
t
200 -~-
20
_
~:~,o_ 30 t,O TOP
0 20
Fraction
30
~0
TOP
Fig. 1. Sedimentation analysis of the endonuclease-R digestion products of ffX174 RF-I D N A and M13 RF-I DNA. A 10 pl enzyme fraction was added to a reaction mixture (0.05 ml), containing 6 mM Tris-HCl (pH 7.6) 6 mM MgCI2, 50mM NaC1, 6 mM rnercaptoethanol and (3H)-~X174 RF-I D N A (6600 cpm; 0.01 A260 unit) or (aH)-M13 RF-I D N A (4000 cpm; 0.01 A2n0 unit). After incubation of 1 hr at 37~ the reaction was stopped by adding 50 pl of 0.1 M EDTA. The hydrolysate was layered on a 5-20 % (w/v) liniar sucrose gradient (in 1 M NaCI, 0.05 M Tris (pH 7.6), 0.003 ra EDTA) and centrifuged for 150 min at 65000 rpm and 4~ Two-drop fractions were collected in scintillation vials and the radioactivity measured as described under Methods section. (A): Digestion products of (all)- ~X174 RF-I D N A ( O Q). A control of (3H)- ~X174 R F - D N A ( 9 . . . . (3) incubated without enzyme was sedimented in a separate tube. (B): Digestion products of (aH)-M 13 RF-I D N A ( 0 0 ) . (3H)-M 13 RF-I D N A ( ( 3 - - - ( 3 ) sedimented in a separate tube as a control.
The active fraction obtained did show restriction-like enzyme properties as it produced a rapid decrease in viscosity of Tv-DNA and of native calf thymus D N A but did not degrade homologous H. influenzae DNA. Incubation of (3H)-T7 D N A with the purified enzyme fraction produced little radioactivity in the trichloroacetic acid-soluble fraction, indicating that the fraction did not contain an exonuclease activity attacking double-stranded DNA. In order to verify that the enzyme isolated was indeed endonuclease-R (endo-R), a sample of (3H)-RF-I D N A of phage ~bX174 was incubated with an excess of the enzyme fraction and the hydrolysate was then analyzed by sedimentation on sucrose gradients (Fig. 1A). Under the conditions used, RF-I with a twisted circular form, sedimented at a rate of about 21 S and the open circular form RF-II D N A at about 16 S. Incubation of (kX174-RF D N A with the enzyme fraction resulted in a complete conversion of both RF-I and RF-II. The fragments produced sedimented at 8.0S, which corresponds to the value already reported by Edgell et al. [4]. Treatment of M13-RF D N A with endo-R under further similar conditions resulted however in the appearance of a single sharp peak with a sedimentation value slightly slower than that of RF-II DNA, namely 17S (Fig. 1B). No smaller fragments were observed even after a prolonged 4-hr period of digestion, although some radioactivity could be detected now on top of the gradient, apparently due to the presence of a slight contamination of exonuclease activity in the endo-R 43
preparation. A plausible assumption for the appearance of 17S fragments would be that such fragments might be produced by a single double-stranded cleavage of the circular replicative form molecule by endo-R, resulting in double-stranded linear form molecules. (RF-III.) That M 13-RF D N A has a single cleavage site for endo-R could be substantiated by denaturation studies. Denaturation of the digestion products of (3H)-M13 R F - I D N A by alkali-treatment, followed by analysis on alkaline sucrose gradients revealed that all radioactivity is located in a single peak sedimenting at 16S. This is equivalent to the position of linear M I 3 D N A molecules, which sediment slightly slower than the circular M13 D N A used as a reference (Fig. 2). It is known that endo-R makes duplex cleavages in double-stranded D N A by recognizing a specific hexanucleotide sequence [2]. The presence of only one site of cleavage, located on a genome of about 6000 nucleotides long, as evidenced for M13 D N A , is rather unexpected. An alternative hypothesis would be that there is more than one cleavage site present in the molecule, but once the twisted circular form R F - I D N A has been cleaved into linear R F - I I I D N A molecules, the remaining cleavage sites will not be attacked by the restriction enzyme. I f this hypothesis is correct, denaturation and renaturation of the R F - I H molecules will give rise to circularization of the linear duplexes. As is shown in Fig. 3, denaturation and renaturation of the D N A fragments obtained by digestion of (3H)-M13 R F - I D N A by endo-R, did not produce detectable circular duplex D N A molecules. The reannealed D N A still sedimented at 17 S, which is exactly the same position as has been found for the original R F - I I I molecules. F r o m these results it can be concluded that the M13 genome has only a single cleavage site for
Radioacivity of 3H(dpml
Radioactivity ofl~C(dpml . . . .
/ I
90 -
.
.
.
Circ. Lin
.
3000
/
/
;
2000
i
1000
2001-
/ 0
10
2O
30
TOP
0
Fraction Fig. 2. Alkaline sucrose gradient sedimentation of M13 RF-I DNA digested with endonuclease-R. (aH)-MI3 RF-I DNA was digested under the conditions as described in Fig. 1. The hydrolysate was denatured by the addition of one volume of 1 M NaOH~nd subsequently layered on a 10-30~ linear sucrose gradient in 0.5 ra NaOH. Centrifugation was performed for 389hr at 65000 rpm and 4 ~ Two-drop fractions were collected in scintillation virals and the radioactivity was measured as described in Section II. (aH)-M13 RF-I DNA digested with endonuclease-R (e-------O) (14C)-M13 viral DNA ( O - - - O ) added as a marker. 44
Radioactivity ofZ~C(dprnl--Radioactivity o1 3Hldpm}
Radioactivityofl~Cldpm]
-- - - - ' 0 - - - - - - -
-----'O------
1000
t
800-
9
|
30S 9,
1000 1 800
1000!-
- 800
800-
-
600
6oo-
~I " , t
l i
2oo
,~ ~
0
- 1000
I1
ii
400
|
30S
I~
600
Radioactivity of 3H(dpm)
- 600
I I
L,O0
"~ 20
Fraction
- 200 30
"~0 TOP
0
-
or;-0
10
20 Fraction
30
0o
40 0 TOP
Fig. 3. Denaturation and renaturation of M13 RF-III DNA, produced by endonuclease-R digestion of M13 RF-I DNA. The endo-R digestion of (aH)-RF-I DNA and the subsequent isolation of (aH)-RF-III DNA was performed as described in Section II. (A): (aH)-M13 RF-III DNA (4000 cpm) was centrifuged for 150 min at 65000 rpm. Thereafter two-drop fractions were collected in scintillation virals and the radioactivity was measured. (3H)-M13 RF-III DNA (O Q). (t4C)-M13 viral DNA ( O - - - O ) added as a marker. (B): A sample of MI3 RF-III DNA in 0.1 x SSC was heated for 5 min at 100~ and then rapidly chilled in ice. After raising the concentration to 2 x SSC, the sample was reannealed for 24 hr at 66~ and subsequently layered on a 5-20~ linear sucrose gradient in 1 M NaC1, 0.05 M Tris (pH 7.6), 0.003 M EDTA and centrifuged for 150 min at 65000 rpm and 4~ Reannealed (aH)-M13 RF-III DNA (e~----O). (x4C)-M13 viral DNA ( O - - - O ) added as a marker.
the restriction enzyme o f H. influenzae Rd, which actually is cleaved resulting in linear duplex M13 D N A molecules. ACKNOWLEDGMENTS The authors are grateful to Mr. Th. Cuypers, w h o h e l p e d during the initial stages o f the work, Miss R. Matze for excellent technical assistance and Dr. R. Konings for helpful advices and suggestions. REFERENCES [1] [2] [3] [4] [5] [6] [7]
Marvin, D. A. and H o h n , B., Bacteriol. Rev. 33, 172 (1969). Kelley, T. J. and Smith, H. O., J. MoL Biol. 51, 393 (1970). Hedgpeth, J., G o o d m a n , H. M., and Boyer, H. W., Proc. Nat. Acad. Sci. 69, 3448 (1972). Edgell, M. H., Hutchison III, C. A., and Sclair, M., J. Virol. 9, 574 (1972). Middleton, J. H., Edgell, M. H., and Hutchison III, C. A., J. Virol. 10, 42 (1972). Smith, H. O. and Wilcox, K. W., J. Mol. Biol. 51, 379 (1970). Budman, D. R. and Pardee, A. B., J. BacterioL 94, 1546 (1967).
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