Vol. 18, No. 2
JOURNAL OF VIROLOGY, May 1976, p. 659-663 Copyright © 1976 American Society for Microbiology
Printed in U.SA.
Location in Bacteriophage Lambda DNA of Cleavage Sites of the Site-Specific Endonuclease from Bacillus amyloliquefaciens H1 DENNIS M. HAGGERTY AND ROBERT F. SCHLEIF* Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154 Received for publication 30 December 1975
The sites in Escherichia coli bacteriophage lambda DNA cleaved by the sitespecific endonuclease isolated from Bacillus amyloliquefaciens H (BamI) are found to be at 0.114, 0.466, 0.580, 0.713, and 0.861 lambda units. The sites were located by analysis of the products of digestion of lambda DNA and lambda-ara transducing phage DNA, and verified by double digestion with BamI and EcoRI.
Site-specific endonucleases permit the production of specific DNA fragments, thereby enabling one to perform a wide variety of experiments. Furthermore, there is considerable interest in the use of lambda as a vehicle for the amplification of foreign DNA in Escherichia coli. Production of these molecular hybrids is assisted by nucleases that cleave DNA the size of lambda only a few times. Here we map the cleavage sites of BamI-originally isolated from Bacillus amyloliquefaciens H by G. Wilson and F. Young (9)-in the lambda chromosome. The accurate mapping of these sites and their locations with respect to the EcoRI cleavage sites should assist work in the formation of hybrids as well as the study of existing transducing and hybrid phage. We also note the particular ease in mapping of some new enzymes that follows from the locations of the BamI cleavage sites relative to the EcoRI cleavage sites.
rated by electrophoresis on 0.7% agarose (Sea-Kem) in a vertical slab gel (0.3 by 14 by 17 cm). Samples were adjusted to 5% in glycerol and 0.01% in bromophenol blue and run at 35 mA for 6 to 24 h, stained for 15 min in 0.2 ug of ethidium bromide per ml, and washed with water for 15 min before examination. The DNA was visualized by illumination with a short-wavelength (260 mm) transilluminator (UV Products) and photographed on Polaroid type 107 film (using the contrast filter provided with the transilluminator) and a 2-mm glass filter (Corning; CS2-73). For this work, we have assumed that migration velocity on agarose gels is independent of base composition. Gels were calibrated using the 1,000-base-pair fragment isolated from heteroduplexes of XparaB114 and XparaC116 (2) and the fragments produced by digestion of lambda DNA with EcoRI (6). Purification of endonuclease. Purification of the endonuclease from B. amyloliquefaciens H followed that reported by Smith and Wilcox (5) for the endonuclease from Haemophilus influenzae. Bacteria were grown in Penassay broth (Bacto antibiotic medium 3, Difco) to late-logarithmic phase and collected by centrifugation. Cells were opened by MATERIALS AND METHODS grinding with 2 weights of alumina, resuspended in Phage strains. Growth and isolation of the var- buffer, and spun at 100,000 x g for 90 min. The ious phage strains used in this study have been supernatant was run through a Bio-Gel column (Adescribed previously (1, 4). Purified phage were 0.5 m, 100 to 200 mesh; Bio-Rad) and assayed. The phenol extracted, and the deproteinized DNA, at 200 fractions containing endonuclease activity were Ag/ml was dialyzed against three changes of DNA pooled and adjusted to 70% of saturation with solid buffer (10 mM Tris-hydrochloride, pH 8.0, 0.1 M ammonium sulphate; the dialyzed precipitate was KCl, and 0.1 mM EDTA). then loaded onto a phosphocellulose column (WhatDigestion with endonuclease. The digestion of man P-11). The enzyme eluted in the 0.2 to 0.5 M lambda DNA was carried out in a total volume of 50 fractions of KCl gradient. These fractions were comM1 at 37 C, containing approximately 0.2 tmg of DNA. bined, concentrated, and stored in 50% glycerol at BamI digestions were done in 6 mM Tris-hydrochlo- -10 C. ride, pH 7.4, 6 mM MgCl2, and 6 mM 2-mercaptoethanol; EcoRI digestions and the co-digestions using RESULTS AND DISCUSSION both enzymes were done in 0.1 M Tris-hydrochloLocation of cleavage sites. The structures of ride, pH 7.4, and 10 mM MgCl2. After digestion, the samples were heated to 65 C for 10 min. the ara transducing phage genomes shown in Electrophoresis. The DNA fragments were sepa- Fig. 1 have been determined by electron mi' This is publication no. 1064 from the Department of Biochemistry, Brandeis University.
659
croscopy of heteroduplexes (1, 4). It is also known that BamI produces a single cleavage in
660
HAGGERTY AND SCHLEIF
the 1,000-base-pair region of ara DNA common to phages XparaB1l4 and XparaC116 (John T. Lis, Ph.D. dissertation, Brandeis Univ., Waltham, Mass., 1975). Examination of the genome structures and of the fragments produced by digestion of lambda XparaB114 and XparaCll6 DNA (Table 1) shows that fragment 0.352 lies completely to the left of the att site and fragment 0.133 lies completely to the right of the att site (Fig. 2a). This is indicated by the absence of the 0.352 fragment in the digest of XparaB114 DNA and the absence of the 0.133 fragment in the digest of XparaCll6 DNA. The fragments containing the left and right ends of the genome were determined by electrophoresis of a digestion of XparaB114 DNA with the cohesive ends annealed together (7, 8). Under these conditions, bands from fragments 0.138 and 0.114 vanished and a new band of their combined molecular mass appeared. The hybrid 080 phage was utilized to determine the fragments found in the right-hand region of the DNA, since that is the only region
x1
0 114 -T
%J.
J
A r) 1-+ U.1 It
XporaB114
0 a'x U.145
*i.
hi
containing X DNA. The right end of the genome was found to contain fragments 0.149 and 0.138, because these were the only fragments found in both the 080 and X DNA digests. This locates the 0.114 fragment at the left end and the 0.138 fragment at the right end; fragment 0.149 is tentatively placed between 0.133 and 0.138. The fragments can thus be oriented as shown in Fig. 2b. To complete the map, the fragment containTABLE 1. Size of fragments produced by BamI digestion ofphage DNA"
DNA source
Fragment size (lambda units)
0.352
x
XparaB1l4
0.138 0.138 0.138 0.138
0.149 0.149 0.149 0.149
0.133 0.133
0.114 0.114 0.114
XparaC116 0.352 Xh80dara " The following fragments not corresponding to lambda fragments were produced: XparaB114, 0.43, 0.067, and 0.019; XparaCll6, 0.131 and 0.125; Xh80dara, 0.24, 0.096, 0.070, 0.067, 0.063, and 0.030.
.L %V
0 152 V.Juc_
6 w
J. VIROL.
41--
0114-T --cog -_If
b2
0.149
.1
0133
%J.
w
%-,. I.., 11
.L
w
0.138
R
Cl
PP
,*0.0670019 w wIffm- U.100 n IAA
vL w
n 14Q V.11tv
.L W
0 nI wao
4I 0.131 a, MZ
)V. 149 I-T;7
lw
,
0 13a V.1,JW
*
0.149
2
A B IOCP'
114
XporaCI16
Xh80dara
V.1 It
A S9
lw
lw
-
C 01 B
A
D
a
V. CZ), --I
m
+ 0.138
FIG. 1. Structures ofphage chromosomes. BamI cleavage sites are indicated by arrows. Light line, lambda DNA; dashed line, 480 DNA; heavy line, E. coli DNA.
a)
0352
A
%LI w J
b)
0.114
*
0.352
C)
0.114 *
0.352
t.* 0.133
b2 ppI
* 0.114
rT
R
Cl
0.133
*
0.149
*
0,l38
0.133
*
0.149
*
0.138
FIG. 2. Relative locations of the fragments produced by digestion of lambda DNA with BamI. (a) After analysis of XparaB114 and XparaC116 DNA digestion patterns; (b) after analysis of Xh8Odara digestion pattern and the annealing of lambda cohesive ends; (c) after analysis ofannealed patterns of XparaB114, and XparaC116 DNA.
VOL. 18, 1976
BACTERIOPHAGE LAMBDA CLEAVAGE SITES
a
b
c
d
e
661
f
0.25
0.138
0.114
FIG. 3. Composite ofgels used for identification of a second 0.114 fragment in the digest of lambda DNA . DNA was annealed after digestion: (a) lambda, (b) lambda after heating, (c) AparaB114, (d) AparaB114 after heating, (e) XparaC116, and (f) XparaCll6 after heating.
ing the att site must be identified. A second fragment of size 0.114 was observed in the digest of X DNA after the cohesive ends were annealed. The annealing resulted in the loss of fragments 0.138 and 0.114 from the digest of XpcraB114 and XparaC116 DNA; however, in the digest of lambda DNA, fragment 0.138 vanished and fragment 0.114 was diminished in
intensity (Fig. 3). Therefore, band 0.114 is a doublet in the digest of lambda DNA, and this second 0.114 fragment must contain the att site. The presence of the doublet 0.114 fragment is consistent with the sizes of all the phage examined, since the B and C substitutions both eliminate the second 0.114 fragment (Fig. 2c). Verification of the cleavage sites. As the
662
J. VIROL.
HAGGERTY AND SCHLEIF
Bam I 0.114
0.861
0.713
0.580
0.466
w
A
0.445 (0.443)
O043 (0.543)
lo
I06 0.656
0.810
0.931
(0.656)
(0.8091
(0.928)
R
Eco RI FIG. 4. Cleavage-site locations in lambda DNA of BamI and EcoRI as fractions of lambda lengths. TABLE 2. Fragments produced by codigestion of five EcoRI cleavage sites in lambda are known lambda DNA with BamI and EcoRI and lie between those of BamI, further proof of the structures presented in Fig. 1 was obtained Sizes predicted Sizes predicted by co-digestion with EcoRI and BamI. Diges- Observed sizes from reported RI from adjusted RI cleavage sites cleavage sites tion of lambda DNA with both enzymes should produce a unique set of fragments whose sizes 0.329 0.331 0.33 are determined by the cleavage locations of 0.114 0.114 0.114 both enzymes. In Fig. 4 the cleavage sites of 0.097 0.096 0.096 0.077 0.077 0.077 EcoRI and BamI are presented and can be used 0.076 0.077 0.076 to predict the results of an actual co-digestion. 0.072 0.070 0.072 As can be seen in Table 2, the sizes of the frag0.067 0.069 0.067 ments observed are in excellent agreement 0.057 0.057 0.057 with the calculated sizes. 0.051 0.052 0.052 Since the BamI sites and EcoRI sites alter0.037 0.037 0.037 nate in the lambda chromosome, these two en0.023 0.021 0.023 zymes can be used to directly locate the cleavage sites of certain other site-specific endonuAlthough the accuracy of sizes determined cleases. In general, the cleavage sites for an endonuclease can be located by analysis of its using migration velocity on agarose gels has digestion pattern if it does not cleave any previ- not been extensively evaluated, we note the ously characterized fragment more than twice. following two facts: (i) there is excellent interThis approach may be extended as additional nal agreement in those experiments where predicted fragment sizes were dependent on comenzymes are isolated and their cleavage sites bined electron microscopy and gel electrophoreare mapped. After our work was completed, a cleavage sis data; and (ii) in the several cases of a slight disparity between the predicted sizes and the map of BamI on phage lambda DNA was published (3). Although most of the assigned cleav- observed sizes, the agreement can be improved by shifting the location assigned to the EcoRI age sites are close to ours, one is significantly different, the left-most site. We place this site sites. Shifting 0.445 to 0.443, 0.810 to 0.809, and at 0.114, whereas Perricaudet and Tiollais place 0.931 to 0.928, alterations that are well within this site at 0.130. This discrepancy may result the error limits determined by Thomas and from their use of acrylamide gels for size deter- Davis (6), improves the agreement of the six mination. By electron microscopy, Thomas and fragments involved. At present we see no way Davis (6) have shown the acrylamide gel sys- to determine whether these slightly shifted valtem to be unreliable for the size determination ues actually are more accurate. of the fragments produced by EcoRI digestion of ACKNOWLEDGMENTS lambda DNA; at the same time, however, the This work was supported in part by Public Health Sersizes determined by electrophoresis on agarose vice grant GM18277, Career Development Award K04-GMgels are in agreement with those from electron 38797, and training grant GM00212, all from the National of General Medical Sciences. microscopy. A check of our Bam cleavage sites Institute We thank Richard J. Roberts for supplying the B. amylowas provided by the double digestion with liquefacines H, and Donald Dean for purified EcoRI. BamI and EcoRI. Since the fragment sizes proLITERATURE CITED duced by this double digestion closely agree 1. Lis, J. T., and R. Schleif. 1975. The isolation and charwith those predicted from cleavage sites asacterization of plaque-forming arabinose transducsigned to each enzyme, we believe that the ing bacteriophage lambda. J. Mol. Biol. 95:395-407. cleavage site locations determined by our work 2. Lis, J. T., and R. Schleif. 1975. The regulatory region of are more
accurate.
the L-arabinose
operon:
its isolation
on a
1000 base-
VOL. 18, 1976
BACTERIOPHAGE LAMBDA CLEAVAGE SITES
pair fragment from DNA heteroduplexes. J. Mol. Biol. 95:409-416. 3. Perricaudet, M., and P. Tiollais. 1975. Defective bacteriophage lambda chromosome, potential vector for DNA fragments obtained after cleavage by Bacillus amyloliquefaciens endonucleases (Bam I). FEBS Lett. 56:7-lid. 4. Schleif, R., J. Greenblatt, and R. W. Davis. 1971. Dual control of arabinose genes on transducing phage Xdara. J. Mol. Biol. 59:127-150. 5. Smith, H. O., and K. W. Wilcox. 1970. A restriction enzyme from Hemophilus influenza. I. Purification and general properties. J. Mol. Biol. 51:379-391.
663
6. Thomas, M., and R. W. Davis. 1975. Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease. J. Mol. Biol. 91:315-328. 7. Wang, J. C., and N. Davidson. 1966. Thermodynamic and kinetic studies on the interconversion between the linear and circular forms of phage lambda DNA. J. Mol. Biol. 15:111-123. 8. Wang, J. C., and N. Davidson. 1966. On the probability of ring closure of lambda DNA. J. Mol. Biol. 19:469482. 9. Wilson, C. A., and F. E. Young. 1975. Isolation of a sequence-specific endonuclease (Bam I) Bacillus amyloliquefaciens H. J. Mol. Biol 97:123.
JOURNAL OF VIROLOGY, May 1976, p. 659-663 Copyright © 1976 American Society for Microbiology
Printed in U.SA.
Location in Bacteriophage Lambda DNA of Cleavage Sites of the Site-Specific Endonuclease from Bacillus amyloliquefaciens H1 DENNIS M. HAGGERTY AND ROBERT F. SCHLEIF* Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154 Received for publication 30 December 1975
The sites in Escherichia coli bacteriophage lambda DNA cleaved by the sitespecific endonuclease isolated from Bacillus amyloliquefaciens H (BamI) are found to be at 0.114, 0.466, 0.580, 0.713, and 0.861 lambda units. The sites were located by analysis of the products of digestion of lambda DNA and lambda-ara transducing phage DNA, and verified by double digestion with BamI and EcoRI.
Site-specific endonucleases permit the production of specific DNA fragments, thereby enabling one to perform a wide variety of experiments. Furthermore, there is considerable interest in the use of lambda as a vehicle for the amplification of foreign DNA in Escherichia coli. Production of these molecular hybrids is assisted by nucleases that cleave DNA the size of lambda only a few times. Here we map the cleavage sites of BamI-originally isolated from Bacillus amyloliquefaciens H by G. Wilson and F. Young (9)-in the lambda chromosome. The accurate mapping of these sites and their locations with respect to the EcoRI cleavage sites should assist work in the formation of hybrids as well as the study of existing transducing and hybrid phage. We also note the particular ease in mapping of some new enzymes that follows from the locations of the BamI cleavage sites relative to the EcoRI cleavage sites.
rated by electrophoresis on 0.7% agarose (Sea-Kem) in a vertical slab gel (0.3 by 14 by 17 cm). Samples were adjusted to 5% in glycerol and 0.01% in bromophenol blue and run at 35 mA for 6 to 24 h, stained for 15 min in 0.2 ug of ethidium bromide per ml, and washed with water for 15 min before examination. The DNA was visualized by illumination with a short-wavelength (260 mm) transilluminator (UV Products) and photographed on Polaroid type 107 film (using the contrast filter provided with the transilluminator) and a 2-mm glass filter (Corning; CS2-73). For this work, we have assumed that migration velocity on agarose gels is independent of base composition. Gels were calibrated using the 1,000-base-pair fragment isolated from heteroduplexes of XparaB114 and XparaC116 (2) and the fragments produced by digestion of lambda DNA with EcoRI (6). Purification of endonuclease. Purification of the endonuclease from B. amyloliquefaciens H followed that reported by Smith and Wilcox (5) for the endonuclease from Haemophilus influenzae. Bacteria were grown in Penassay broth (Bacto antibiotic medium 3, Difco) to late-logarithmic phase and collected by centrifugation. Cells were opened by MATERIALS AND METHODS grinding with 2 weights of alumina, resuspended in Phage strains. Growth and isolation of the var- buffer, and spun at 100,000 x g for 90 min. The ious phage strains used in this study have been supernatant was run through a Bio-Gel column (Adescribed previously (1, 4). Purified phage were 0.5 m, 100 to 200 mesh; Bio-Rad) and assayed. The phenol extracted, and the deproteinized DNA, at 200 fractions containing endonuclease activity were Ag/ml was dialyzed against three changes of DNA pooled and adjusted to 70% of saturation with solid buffer (10 mM Tris-hydrochloride, pH 8.0, 0.1 M ammonium sulphate; the dialyzed precipitate was KCl, and 0.1 mM EDTA). then loaded onto a phosphocellulose column (WhatDigestion with endonuclease. The digestion of man P-11). The enzyme eluted in the 0.2 to 0.5 M lambda DNA was carried out in a total volume of 50 fractions of KCl gradient. These fractions were comM1 at 37 C, containing approximately 0.2 tmg of DNA. bined, concentrated, and stored in 50% glycerol at BamI digestions were done in 6 mM Tris-hydrochlo- -10 C. ride, pH 7.4, 6 mM MgCl2, and 6 mM 2-mercaptoethanol; EcoRI digestions and the co-digestions using RESULTS AND DISCUSSION both enzymes were done in 0.1 M Tris-hydrochloLocation of cleavage sites. The structures of ride, pH 7.4, and 10 mM MgCl2. After digestion, the samples were heated to 65 C for 10 min. the ara transducing phage genomes shown in Electrophoresis. The DNA fragments were sepa- Fig. 1 have been determined by electron mi' This is publication no. 1064 from the Department of Biochemistry, Brandeis University.
659
croscopy of heteroduplexes (1, 4). It is also known that BamI produces a single cleavage in
660
HAGGERTY AND SCHLEIF
the 1,000-base-pair region of ara DNA common to phages XparaB1l4 and XparaC116 (John T. Lis, Ph.D. dissertation, Brandeis Univ., Waltham, Mass., 1975). Examination of the genome structures and of the fragments produced by digestion of lambda XparaB114 and XparaCll6 DNA (Table 1) shows that fragment 0.352 lies completely to the left of the att site and fragment 0.133 lies completely to the right of the att site (Fig. 2a). This is indicated by the absence of the 0.352 fragment in the digest of XparaB114 DNA and the absence of the 0.133 fragment in the digest of XparaCll6 DNA. The fragments containing the left and right ends of the genome were determined by electrophoresis of a digestion of XparaB114 DNA with the cohesive ends annealed together (7, 8). Under these conditions, bands from fragments 0.138 and 0.114 vanished and a new band of their combined molecular mass appeared. The hybrid 080 phage was utilized to determine the fragments found in the right-hand region of the DNA, since that is the only region
x1
0 114 -T
%J.
J
A r) 1-+ U.1 It
XporaB114
0 a'x U.145
*i.
hi
containing X DNA. The right end of the genome was found to contain fragments 0.149 and 0.138, because these were the only fragments found in both the 080 and X DNA digests. This locates the 0.114 fragment at the left end and the 0.138 fragment at the right end; fragment 0.149 is tentatively placed between 0.133 and 0.138. The fragments can thus be oriented as shown in Fig. 2b. To complete the map, the fragment containTABLE 1. Size of fragments produced by BamI digestion ofphage DNA"
DNA source
Fragment size (lambda units)
0.352
x
XparaB1l4
0.138 0.138 0.138 0.138
0.149 0.149 0.149 0.149
0.133 0.133
0.114 0.114 0.114
XparaC116 0.352 Xh80dara " The following fragments not corresponding to lambda fragments were produced: XparaB114, 0.43, 0.067, and 0.019; XparaCll6, 0.131 and 0.125; Xh80dara, 0.24, 0.096, 0.070, 0.067, 0.063, and 0.030.
.L %V
0 152 V.Juc_
6 w
J. VIROL.
41--
0114-T --cog -_If
b2
0.149
.1
0133
%J.
w
%-,. I.., 11
.L
w
0.138
R
Cl
PP
,*0.0670019 w wIffm- U.100 n IAA
vL w
n 14Q V.11tv
.L W
0 nI wao
4I 0.131 a, MZ
)V. 149 I-T;7
lw
,
0 13a V.1,JW
*
0.149
2
A B IOCP'
114
XporaCI16
Xh80dara
V.1 It
A S9
lw
lw
-
C 01 B
A
D
a
V. CZ), --I
m
+ 0.138
FIG. 1. Structures ofphage chromosomes. BamI cleavage sites are indicated by arrows. Light line, lambda DNA; dashed line, 480 DNA; heavy line, E. coli DNA.
a)
0352
A
%LI w J
b)
0.114
*
0.352
C)
0.114 *
0.352
t.* 0.133
b2 ppI
* 0.114
rT
R
Cl
0.133
*
0.149
*
0,l38
0.133
*
0.149
*
0.138
FIG. 2. Relative locations of the fragments produced by digestion of lambda DNA with BamI. (a) After analysis of XparaB114 and XparaC116 DNA digestion patterns; (b) after analysis of Xh8Odara digestion pattern and the annealing of lambda cohesive ends; (c) after analysis ofannealed patterns of XparaB114, and XparaC116 DNA.
VOL. 18, 1976
BACTERIOPHAGE LAMBDA CLEAVAGE SITES
a
b
c
d
e
661
f
0.25
0.138
0.114
FIG. 3. Composite ofgels used for identification of a second 0.114 fragment in the digest of lambda DNA . DNA was annealed after digestion: (a) lambda, (b) lambda after heating, (c) AparaB114, (d) AparaB114 after heating, (e) XparaC116, and (f) XparaCll6 after heating.
ing the att site must be identified. A second fragment of size 0.114 was observed in the digest of X DNA after the cohesive ends were annealed. The annealing resulted in the loss of fragments 0.138 and 0.114 from the digest of XpcraB114 and XparaC116 DNA; however, in the digest of lambda DNA, fragment 0.138 vanished and fragment 0.114 was diminished in
intensity (Fig. 3). Therefore, band 0.114 is a doublet in the digest of lambda DNA, and this second 0.114 fragment must contain the att site. The presence of the doublet 0.114 fragment is consistent with the sizes of all the phage examined, since the B and C substitutions both eliminate the second 0.114 fragment (Fig. 2c). Verification of the cleavage sites. As the
662
J. VIROL.
HAGGERTY AND SCHLEIF
Bam I 0.114
0.861
0.713
0.580
0.466
w
A
0.445 (0.443)
O043 (0.543)
lo
I06 0.656
0.810
0.931
(0.656)
(0.8091
(0.928)
R
Eco RI FIG. 4. Cleavage-site locations in lambda DNA of BamI and EcoRI as fractions of lambda lengths. TABLE 2. Fragments produced by codigestion of five EcoRI cleavage sites in lambda are known lambda DNA with BamI and EcoRI and lie between those of BamI, further proof of the structures presented in Fig. 1 was obtained Sizes predicted Sizes predicted by co-digestion with EcoRI and BamI. Diges- Observed sizes from reported RI from adjusted RI cleavage sites cleavage sites tion of lambda DNA with both enzymes should produce a unique set of fragments whose sizes 0.329 0.331 0.33 are determined by the cleavage locations of 0.114 0.114 0.114 both enzymes. In Fig. 4 the cleavage sites of 0.097 0.096 0.096 0.077 0.077 0.077 EcoRI and BamI are presented and can be used 0.076 0.077 0.076 to predict the results of an actual co-digestion. 0.072 0.070 0.072 As can be seen in Table 2, the sizes of the frag0.067 0.069 0.067 ments observed are in excellent agreement 0.057 0.057 0.057 with the calculated sizes. 0.051 0.052 0.052 Since the BamI sites and EcoRI sites alter0.037 0.037 0.037 nate in the lambda chromosome, these two en0.023 0.021 0.023 zymes can be used to directly locate the cleavage sites of certain other site-specific endonuAlthough the accuracy of sizes determined cleases. In general, the cleavage sites for an endonuclease can be located by analysis of its using migration velocity on agarose gels has digestion pattern if it does not cleave any previ- not been extensively evaluated, we note the ously characterized fragment more than twice. following two facts: (i) there is excellent interThis approach may be extended as additional nal agreement in those experiments where predicted fragment sizes were dependent on comenzymes are isolated and their cleavage sites bined electron microscopy and gel electrophoreare mapped. After our work was completed, a cleavage sis data; and (ii) in the several cases of a slight disparity between the predicted sizes and the map of BamI on phage lambda DNA was published (3). Although most of the assigned cleav- observed sizes, the agreement can be improved by shifting the location assigned to the EcoRI age sites are close to ours, one is significantly different, the left-most site. We place this site sites. Shifting 0.445 to 0.443, 0.810 to 0.809, and at 0.114, whereas Perricaudet and Tiollais place 0.931 to 0.928, alterations that are well within this site at 0.130. This discrepancy may result the error limits determined by Thomas and from their use of acrylamide gels for size deter- Davis (6), improves the agreement of the six mination. By electron microscopy, Thomas and fragments involved. At present we see no way Davis (6) have shown the acrylamide gel sys- to determine whether these slightly shifted valtem to be unreliable for the size determination ues actually are more accurate. of the fragments produced by EcoRI digestion of ACKNOWLEDGMENTS lambda DNA; at the same time, however, the This work was supported in part by Public Health Sersizes determined by electrophoresis on agarose vice grant GM18277, Career Development Award K04-GMgels are in agreement with those from electron 38797, and training grant GM00212, all from the National of General Medical Sciences. microscopy. A check of our Bam cleavage sites Institute We thank Richard J. Roberts for supplying the B. amylowas provided by the double digestion with liquefacines H, and Donald Dean for purified EcoRI. BamI and EcoRI. Since the fragment sizes proLITERATURE CITED duced by this double digestion closely agree 1. Lis, J. T., and R. Schleif. 1975. The isolation and charwith those predicted from cleavage sites asacterization of plaque-forming arabinose transducsigned to each enzyme, we believe that the ing bacteriophage lambda. J. Mol. Biol. 95:395-407. cleavage site locations determined by our work 2. Lis, J. T., and R. Schleif. 1975. The regulatory region of are more
accurate.
the L-arabinose
operon:
its isolation
on a
1000 base-
VOL. 18, 1976
BACTERIOPHAGE LAMBDA CLEAVAGE SITES
pair fragment from DNA heteroduplexes. J. Mol. Biol. 95:409-416. 3. Perricaudet, M., and P. Tiollais. 1975. Defective bacteriophage lambda chromosome, potential vector for DNA fragments obtained after cleavage by Bacillus amyloliquefaciens endonucleases (Bam I). FEBS Lett. 56:7-lid. 4. Schleif, R., J. Greenblatt, and R. W. Davis. 1971. Dual control of arabinose genes on transducing phage Xdara. J. Mol. Biol. 59:127-150. 5. Smith, H. O., and K. W. Wilcox. 1970. A restriction enzyme from Hemophilus influenza. I. Purification and general properties. J. Mol. Biol. 51:379-391.
663
6. Thomas, M., and R. W. Davis. 1975. Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease. J. Mol. Biol. 91:315-328. 7. Wang, J. C., and N. Davidson. 1966. Thermodynamic and kinetic studies on the interconversion between the linear and circular forms of phage lambda DNA. J. Mol. Biol. 15:111-123. 8. Wang, J. C., and N. Davidson. 1966. On the probability of ring closure of lambda DNA. J. Mol. Biol. 19:469482. 9. Wilson, C. A., and F. E. Young. 1975. Isolation of a sequence-specific endonuclease (Bam I) Bacillus amyloliquefaciens H. J. Mol. Biol 97:123.
