Vol. 30, No.3
JouRNAL OF VIROLOGY, June 1979, p. 923-928 0022-538X/79/06-0923/06$02.00/0
Physical Map of Bacteriophage BF23 DNA: Restriction Enzyme Analysist BRENDA LANGE-GUSTAFSON AND MARC RHOADES* Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 Received for publication 21 December 1978
Cleavage maps of bacteriophage BF23 DNA have been constructed for the restriction endonucleases Sall (3 fragments), BamHI (5 fragments), EcoRI (8 fragments), BailI (13 fragments), and HpaI (49 fragments, 32 of which have been ordered). The maps were determined by (i) analysis of deletion mutants, (ii) digestion with two endonucleases, (iii) digestion of isolated fragments with a second enzyme, (iv) analysis of partial digests, and (v) digestion after treatment with A exonuclease.
Bacteriophages T5 and BF23 are closely related and possess several novel properties (3). We have been engaged in an analysis of the single-chain interruptions that occur in T5 and BF23 DNAs and in a determination of the physical location of T5 genetic markers. These studies have been aided by construction ofrestriction endonuclease cleavage maps of both genomes. A number of cleavage maps of T5 are available (2, 4, 8, 10). This paper presents cleavage maps of BF23 DNA for the Sall, BamHI, EcoRI, BalI, and HpaI restriction endonucleases. The results of cleavage of BF23st(+) DNA (wild type) with the endonucleases used in this study are shown in Fig. 1. The number of fragments produced by each enzyme and their molecular weights are given in Tables 1 through 3. The cleavage maps are shown in Fig. 2. The evidence used for their construction is summarized below. The three fragments produced by cleavage of BF23 DNA with SalI were ordered by analysis of two heat-stable deletion mutants, BF23st(102) and BF23st(104). As indicated in Fig. 2, both lack 6 to 7% segments located 24 to 33% from the left end of the genome. Sall digests of both BF23st(102) and BF23st(104) DNAs were found to lack only SalI-2. Since SalI-1 is too large to be located between the left end and the st deletions, the order of the SailI fragments, from left to right, is 3-2-1. The EcoRI map of BF23 DNA was established by analysis of the st deletion mutants, use of A exonuclease to identify the terminal fragments, and combined digestion with Sall. Analysis of the st mutants revealed that the BF23st(102) deletion affects fragments 6, 7, and t Contribution no. 1013 from the Department of Biology, The Johns Hopkins University, Baltimore, Md. 920
8 and that the BF23st(104) deletion affects fragments 6 and 7. Since the BF23st(102) deletion is smaller than EcoRI-6 and extends to the left of the BF23st(104) deletion, the order of the three smallest EcoRI fragments must be 8-7-6. Prior digestion to 5% with A exonuclease resulted in loss of EcoRI fragments 1 and 3, whereas digestion to 22% resulted in the additional loss of EcoRI-2. Fragments 1 and 3 were ssigned to the left and right ends of the molecule, respectively, by combined digestion with EcoRI and SalI. EcoRI-1 and EcoRI-4 were missing from the combined digest, which contained three new fragments, representing 12.1, 10.7, and 2.9% of intact BF23 DNA. EcoRI-1 thus spans the SailI site at 11% and yields SalI-3 and the 10.7% fragment in the combined digest. Since EcoRI2 is too large to fit between fragments 1 and 8, it must occur next to fragment 3 at the right end of the DNA. EcoRI-4 must span the 47% SailI site and occur to the left of EcoRI-5 in order to satisfy the results of the combined digest. The five BamHI fragments of BF23 DNA were ordered in a manner similar to the EcoRI fragments. Prior digestion with A exonuclease resulted first in the loss of fragments 4 and 5, followed by the loss of fragments 2 and 3. Since BamHI-2 was the only fragment affected by the st deletions, the order of the central three fragments is 2-1-3. BamHI-4 and BamHI-5 were assigned to the left and right ends, respectively, by analysis of the products of a BamHI-EcoRI digest. EcoRI fragments 4 through 8 and BamHI-4 and BamHI-5 were present in the combined digest along with new fragments representing 16.7, 13.5, 5.7, and 4.6% of BF23st(+) DNA. EcoRI-1 must yield BamHI-5 and the 13.5% fragment, and EcoRI-3 yields BamHI4 and the 5.7% fragment in the combined digest.
924
J. VIROL.
NOTES
TABLE 1. Molecular weights of SalI, BamHI, and EcoRI fragments of BF23st(+) DNAa Endonuclease
U -I
IN
I m I.. I
_I
U a
-N
.M ., "1 o
'i
r.
.N E. m
-,
-'
FIG. 1. Restriction endonuclease cleavage prod-
ucts of BF23 DNA. (A) Gel 1, SalI digest of BF23st(+)
DNA; gel 2, BamHI digest ofBF23st(+) DNA; gels 3 and 4, EcoRI digest of BF23st(+) DNA. Electrophoresis was carried out in 0.5% agarose gels in gels 1, 2, and 3 and in 1.0% agarose in gel 4. (B) Gels I and 2, BalI digest of BF23st(+) DNA; gels 3 and 4, HpaI digest of BF23st(+) DNA. Electrophoresis was carried out in 0.5% agarose in gel 1, 0.7% agarose in gel 3, and 1.5% agarose in gels 2 and 4. The relative intensities of BalI band 4,5 and HpaI bands 13, 23, and 27 indicate that each contains two fragments, whereas HpaI band 25,26 contains three fragments. Both copies of HpaI fragments 13, 23, 25, and 27, which occur within the terminal repetition, have been given the same number.
The 16.7 and 4.6% fragments are derived from EcoRI-2. Initial analysis of the 13 BalI fragments of BF23 DNA was carried out by repetition of the procedures described above. Digestion with A exonuclease first resulted in the loss of BalI-1 and BalI-3, followed by the loss of one fragment (arbitrarily designated BalI-4) from the band that contains fragments 4 and 5. BalI-1 must be at the left end since EcoRI-1 is not cleaved by BalI (see below). BF23st(102) lacks BalI-6, whereas BF23st(104) also lacks Ball-10. The fragment orders at the left and right ends of the DNA are therefore 1-6-10 and 4-3, respectively. Size considerations and the locations of the deletions prevent any fragment other than BalI-1 from occuring between the left end and BalI-6. The remaining BalI fragments were mapped by analyses of isolated EcoRI fragments, combined EcoRI-BalI and BamHI-BalI digests, and partial BalI digests. The results of these experiments are summarized in Table 2. BalI
SalI
Fragment no.
Mol wt (x106)
1
40.9b
28.3 8.4 26.2 BamHI 2c 20.0 16.9 3 7.8 4 7.0 5 17.3 1 EcoRId 16.3 2 12.2 3 11.8 4 5 10.5 5.6 6 7 2.10 2.06 8 a Molecular weights were determined from electrophoretic mobilities relative to restriction endonuclease fragments of T5 DNA (2). b The molecular weight of Sall-1 was determined from the sum of EcoRI fragments 2, 3, and 5 plus the 2.1 x 106-dalton (2.8%) fragment in SalI-EcoRI digests. cBamHI-2 is replaced by a 14.8 x 10-dalton fragment in BF23st(102) DNA and by a 15.3 x 10-dalton frapment in BF23st(104) DNA. BF23st(102) DNA lacks EcoRI fragments 6 through 8 and contains a new fragment having a molecular weight of 4.4 x 106. BF23st(104) DNA lacks EcoRI fragments 6 and 7 and contains a new fragment having a molecular weight of 2.8 x 106. 2 3 1
digestion of a mixture of isolated EcoRI fragments 1 and 2 yielded EcoRI-1 and BalI-5 plus two new fragments. Therefore, BalI-5 must come from EcoRI-2 and occur next to BalI-4. BalI digestion of isolated EcoRI fragments 3 and 4 yielded BalI fragments 3, 11, 12, and 13 plus two new fragments, the larger of which must be derived from BalI-2. Since Ball-3 maps within EcoRI-3, BalI fragments 11 through 13, and most of BalI-2, must occur in EcoRI-4, with BalI-2 on the right of the three smaller fragments. Treatment of a BalI digest with EcoRI led to the loss of BalI-8 and five previously mapped fragments. BalI-8 thus spans the EcoRI site at 63.4% and occurs next to BalI-5. BalI-7 and BalI-9 must therefore occupy the gap between BalI-2 and BalI-8. The absolute order of fragments 11, 12, and 13 and fragments 7, 8, and 9 was determined by analysis of partial BalI digests. As shown in Table 2, nearly complete BalI digests contain fragments whose sizes are most consistent with the fragment orders shown in Fig. 2. The results of a combined BalI-BamHI digest (Table 2) provide confirmation for several
NOTES
VOL. 30, 1979
BF23(+) (77.9) 1 (18.6)
TABLE 2. Summary of evidence for Ball cleavage map of BF23 DNA BalI frgments present in:' B723(+) BamHIb BF23(+) EcoRI Partial BalI digests' EcoRI-1,2' EcoRI-3,4d (77.9) (77.9) (33.6) (24.0)
EcoRI-1(17.3) 2 (11.5)
EcoRI-1(17.3)
2
(11.5) 3(8.9) 4 (8.5) 5(8.5)
925
(9.1) 3
(9.2) 3
(17.5) 3 + (4 or 5) (16.5) 2 + 7 (10.5) = 7 + 8 + 9 (10.0) =6+ 10+ 12
4 5
5
BamHI-5(7.0)
(5.2)
(5.2)
7(4.5)
7
7
8 (3.4)
8
9 (2.6)
9
BamHI-4(7.8) (7.5) 6 (7.2)
6
(6.1) = 8 + 9
(3.96)
(3.33)
10 (1.76)
(3.33)
9
(2.57)
(1.84) 10
(3.96) = 10 + 11 + 12
(2.33) EcoRI-7(2.1)
(2.20) = 11 + 12 or 10 + 12
(2.33)
(1.65) 11 (1.54)
12 (0.75)
11 (1.17) (1.05) 12
11 (1.15) (0.98)
11
12
12
(0.61) 13 (0.28) Z 78.03
13 13 13 X, 78.70 Z 33.33 E: 76.74 l 24.00 0Numbers in parentheses indicate molecular weights (x106). Molecular weights were determined from electrophoretic mobility relative to T5st(+) EcoRI(4) and BF23st(+) HpaI fragments. Fragments not appearing in BalI digests of BF23st(+) DNA are listed as molecular weights without fragment numbers. The new in BF23st(102) and BF23st(104) equal 1.62 and 3.74 x 10E, respectively. fragments b The BalI site at 35.3% and the BamHI site at 34.7% cannot be distinguished. 'EcoRI-1 and EcoRI-2 were isolated as one band by the procedure of Bln et al. (1). Since EcoRI-1 is not cleaved by BalI, all resulting BalI framents are from EcoRI-2. d EcoRI-3 and EcoRI-4 were isolated as one band. EcoRI-3 contains mostly BalI-3 plus a fragment from BalI-4. All other fiagments are from EcoRI-4. ' Enzyme dose and time of incubation were limited such that the digests were near completion and thus likely to contain fragments that were the sum of two neighboring fragments. All detectable composite fragments are shown.
aspects of the BalI cleavage map. Cleavage of BF23st(+) DNA with HpaI results in 49 fragmnents, the largest 32 of which have been ordered. Because of the complexity of the HpaI map, only the general strategy used to order the fragments is presented. However, all of the critical results are summarized in Tables 3 and 4. Approximate fragment locations were determined by digestion of isolated BamHI fragments (Table 3), analysis of the st deletion mutants, and prior digestion with A exonuclease. BF23st(102) DNA lacks fragments 6, 12, 14, and 22, whereas BF23st(104) DNA lacks fragments 6 and 12. The A exonuclease experiments revealed that HpaI-21 and HpaI-28 are terminally located, followed by fragments 13, 23, 25 and 27.
Further digestion led to the loss of fragments 5 and 18. The order of fragments 13, 23, 25, and 27, which are present in two copies because of the terminal repetition in BF23 DNA (5), has been determined by an analysis of T5-BF23 hybrid genomes and will be described in another publication. HpaI fragments 32, 34, 37, and 38 also occur within the terminal repetition, but remain unordered. Many of the HpaI fragments were further localized within each BamHI fragment by analyses of combined digests with each of the four other endonucleases. Except for fragments 13 through 17, the 20 largest HpaI fragments could be localized with some precision in this manner. HpaI-1, for example, is in BamHI-1 and must
926
J. VIROL.
NOTES
TABLE 3. Summary of evidence for HpaI cleavage map of BF23 DNA HpaI fragments present in:a
BF23(+)b BF23(+) BamHI BamHI-1 (77.9)
(77.9)
(26.2)
1(8.0)
1
1
2(6.5) 3 (6.0)
2 3
4(5.2) 5(4.9)
4
6(4.2)
6 7c
BamHI-2 (20.0)
BamHI-3 (16.9)
BamHI-4 (7.8)
BamHI-5 (7.0)
BF23(+) EcoRI
(77.9)
BF23(+) BalI (77.9) (77.
1 2
2
(7.9)
3
(5.35)
7(3.4)
4
4
5 (4.60) 6 7
6 7
7
d
(3.4) 8 (3.3) 9 (3.04)
8 gd
(3.1) (3.04)e
9e
(2.9)
(2.83)f 10 (2.67) 11 (2.63)
10
10
10
(2.61) (2.52)
(2.52)
(2.45) (2.41) 12 (2.19) 13 (2.01)
12 13
12
13
(1.94)
13
13
12 13
(1.94)
(1.94) (1.82)
(1.80)
14 (1.72)
14
15 (1.60)
15
16 (1.54)
16 17
17 (1.51)
14
14
15 16
17
15
(1.63) 15
16
(1.57) 16
17
17
18
18
(1.50) 18 (1.38)
18
(1.38)'
18'
(1.33) 19 (1.29) 20 (1.24) 21 (1.21)
19
19 20 21
22 (1.17) 23 (1.02) 24 (0.86) 25 (0.75)
22 23 24 25
26 (0.75)
26
27 (0.52)
27
20
20
21
21
22
19 20 21
(1.20) 22
22
23
23
23
23
25
25
24 25
24 25
26
26
24 26
(0.63) 27
27
27
(0.63) 27
(0.46) 28 28 28 28 28(0.39) 29 29 29 29 29 (0.35) 30 30 30 30 30 (0.27) 31 31 31 31 31 (0.24) 2 7.33 h X 76.15 X 76.12 X 25.83 X 19.98 X 16.74 6.07h 72.73h 75.05O a Numbers in parentheses are molecular weights (x 106). Molecular weights for fragments 1 through 31 were determined from their electrophoretic mobilities relative to TS HpaI fragments (2). Fragments not appearing in HpaI digests of BF23st(+) are listed as molecular weights without fragment numbers. SalI-HpaI digests lack HpaI-1 and HpaI-7. Two new products appear (molecular weights, 7.3 x 106 and 1.87 x 106).
VOL. 30, 1979
927
NOTES
TABLE 3-Continued The molecular weights of the following fragments, which were not mapped because of their small size, were determined from their electrophoretic mobilities relative to OX174 Taq fragments (6) on polyacrylamide gels and are: fragment 32, 0.22 x 106; fragment 33, 0.19 x 106; fragment 34, 0.14 x 106; fragment 35, 0.136 106; fragment 36, 0.12 x 106; fament 37, 0.11 106; fragment 38, 0.10 x 106; fragment 39, 0.08 x 106; fragment 40, 0.06 x 106; and fragment 41, 0.02 x 106. Fragments 32, 34, 37, and 38 appear to be present in two copies per genome. The new fragments in BF23st(102) and BF23st(104) equal 3.8 x 106 and 1.2 x 106, respectively. 'The BamHI site at 9.0% and the HpaI site at 8.9% cannot be distinguished. d This fragment appears to be a doublet. 'HpaI-9 must be in BamHI-1 since it is cleaved by both BalI and EcoRI. f This fragment appears to be a triplet. HpaI-18 must be in BamHI-5 as indicated by A exonuclease experiments. h These values are approximate since fragments smaller than HpaI-31 were not identified. 'Determined, when possible, by using molecular weights of BalI products from isolated HpaI fragments (see Table 4). b
x
x
cr
IT
2
0
BamHZI
s
2
1
I
3
EcoRi '
'
2
WI
'
I
.
s"UI
I.11 f 11 IN I
Bali
b9
9 a7,9gS I I
2
!A P C
I
I
#A
0
3
5 I "4 a
wIdid
i 212 1(1741(23e 2t2,2T2 2IT.4 I MI 2a .11, HpaZL3~la 7Ialli 1a2 I, I I&AIRA1 1UI 11
25232l7 111
2
o0,CwAuCee .4t,* Idt Mm
Id
44
S
C:
M
0
**t Oj"4
*SS
'4I.sbb 444o4
II I Del9tions
6
lb
2b
4
^b
4
*
^
4
160
X SF23 st(+) lngith FIG. 2. Restriction endonuclease cleavage maps of BF23st(+) DNA. The numbers above the lines are fragment numbers; the numbers below the lines are the coordinates of sites expressed as percentages of the wild-type length from the left end of the molecule. In the HpaI map, fragments occurring within the 8.4% terminal repetition have been given the same number. Fragments in parentheses have not been ordered. The locations of the st deletions were determined from heteroduplex analysis (unpublished data).
928
NOTES
TABLE 4. BalI cleavage products of isolated HpaI fragments in BF23 DNA a BF23(+)m Hpal fragBalI products ment
2 (6.5) 3 (6.0) 5 (4.9) 6 (4.2)
(3.46) (2.89) (2.99) (2.91) (2.88) (1.97) (2.55) (1.63) 7 (3.4)-8 (3.3) HpaI-7 (3.4) (3.13) 9 (3.04) (1.61) (1.33) 10 (2.67)-11 (2.63) (1.55) (1.49) (1.11) (0.74) a Numbers in parentheses indicate molecular weights (x106). Molecular weights were determined from electrophoretic mobilities relative to T5(+) HpaI fragments (2). Fragments were isolated by the procedure of Blin et al. (1).
span the Sall site at 47%, but not the EcoRI site at 50%. Similarly, HpaI-2 is in BamHI-3 and must span the BalI site at 78% in order to avoid cleavage by the other enzymes. Exact locations for HpaI fragments 2, 3, and 5 through 11 were determined by digestion of the isolated fragments with BalI (Table 4). This information in turn allowed several of the smaller fragments to be mapped on the basis of size. Within BamHI1, for example, the only gap large enough to accomodate HpaI-16 occurs between fragments 8 and 9. Similar logic dictated the placement of HpaI fragments 17, 26, and 29 through 31. HpaI fragments 15, 20, and 24 were tentatively mapped by analysis of T5-BF23 hybrid genomes (unpublished data). The order of HpaI fragments 22 and 14 fits best with the fact that neither is cut by BalI. A comparison of the common restriction maps of BF23 and T5 reveals that 30 out of 84 cleavage sites have been conserved between the two ge-
J. VIROL.
nomes. Based on this value, two methods of estimating sequence homology (7, 9) both indicate that T5 and BF23 have undergone approximately 15% divergence in base sequence. This research was supported by grant PCM 76-24036 from the National Science Foundation. LITERATURE CITED 1. Blin, N., A. von Gabain, and H. Bujard. 1975. Isolation of large molecular weight DNA from agarose gels for further digestion by restriction enzymes. FEBS Lett. 53:84-6. 2. Hamlett, N. V., B. Lange-Gustafson, and M. Rhoades. 1977. Physical map of the bacteriophage T5 genome based on the cleavage products of the restriction endonucleases SalI, SmaI, BamI, and HpaI. J. Virol. 24: 249-260. 3. McCorquodale, D. J. 1975. The T-odd bacteriophages. Crit. Rev. Microbiol. 4:104-159. 4. Rhoades, M. 1975. Cleavage of T5 DNA by the Escherichia coli RI restriction endonuclease. Virology 64: 170-179. 5. Rhoades, M., and B. Lange-Gustafson. 1979. Physical map of bacteriophage BF23 DNA: terminal redundancy and localization of single-chain interruptions. J. Virol. 30:777-786. 6. Sato, S., C. A. Hutchison, and J. I. Harris. 1977. A thermostable sequence-specific endonuclease from Thermus aquaticus. Proc. Natl. Acad. Sci. U.S.A. 74: 542-546. 7. Schumperli, D., R. Lagadec, and H. K. Miiller. 1977. DNA sequence homology estimation by combinational analysis of endonuclease restriction data. J. Gen. Virol. 38:161-166. 8. Tchernov, A. P., N. P. Kouzmin, and I. Fodor. 1978. Physical mapping of cleavage sites recognized by restriction endonucleases on the genome of bacteriophage T5. Gene 3:293-302. 9. Upholt, W. B. 1977. Estimation of DNA sequence divergence from comparison of restriction endonuclease digests. Nucleic Acids Res. 4:1257-1265. 10. von Gabain, A., G. S. Hayward, and H. Bujard. 1976. Physical mapping of the HmndIII, EcoRI, Sal, and Sma restriction endonucleases cleavage fragments from bacteriophage T5 DNA. Mol. Gen. Genet. 143:279-290.
JouRNAL OF VIROLOGY, June 1979, p. 923-928 0022-538X/79/06-0923/06$02.00/0
Physical Map of Bacteriophage BF23 DNA: Restriction Enzyme Analysist BRENDA LANGE-GUSTAFSON AND MARC RHOADES* Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 Received for publication 21 December 1978
Cleavage maps of bacteriophage BF23 DNA have been constructed for the restriction endonucleases Sall (3 fragments), BamHI (5 fragments), EcoRI (8 fragments), BailI (13 fragments), and HpaI (49 fragments, 32 of which have been ordered). The maps were determined by (i) analysis of deletion mutants, (ii) digestion with two endonucleases, (iii) digestion of isolated fragments with a second enzyme, (iv) analysis of partial digests, and (v) digestion after treatment with A exonuclease.
Bacteriophages T5 and BF23 are closely related and possess several novel properties (3). We have been engaged in an analysis of the single-chain interruptions that occur in T5 and BF23 DNAs and in a determination of the physical location of T5 genetic markers. These studies have been aided by construction ofrestriction endonuclease cleavage maps of both genomes. A number of cleavage maps of T5 are available (2, 4, 8, 10). This paper presents cleavage maps of BF23 DNA for the Sall, BamHI, EcoRI, BalI, and HpaI restriction endonucleases. The results of cleavage of BF23st(+) DNA (wild type) with the endonucleases used in this study are shown in Fig. 1. The number of fragments produced by each enzyme and their molecular weights are given in Tables 1 through 3. The cleavage maps are shown in Fig. 2. The evidence used for their construction is summarized below. The three fragments produced by cleavage of BF23 DNA with SalI were ordered by analysis of two heat-stable deletion mutants, BF23st(102) and BF23st(104). As indicated in Fig. 2, both lack 6 to 7% segments located 24 to 33% from the left end of the genome. Sall digests of both BF23st(102) and BF23st(104) DNAs were found to lack only SalI-2. Since SalI-1 is too large to be located between the left end and the st deletions, the order of the SailI fragments, from left to right, is 3-2-1. The EcoRI map of BF23 DNA was established by analysis of the st deletion mutants, use of A exonuclease to identify the terminal fragments, and combined digestion with Sall. Analysis of the st mutants revealed that the BF23st(102) deletion affects fragments 6, 7, and t Contribution no. 1013 from the Department of Biology, The Johns Hopkins University, Baltimore, Md. 920
8 and that the BF23st(104) deletion affects fragments 6 and 7. Since the BF23st(102) deletion is smaller than EcoRI-6 and extends to the left of the BF23st(104) deletion, the order of the three smallest EcoRI fragments must be 8-7-6. Prior digestion to 5% with A exonuclease resulted in loss of EcoRI fragments 1 and 3, whereas digestion to 22% resulted in the additional loss of EcoRI-2. Fragments 1 and 3 were ssigned to the left and right ends of the molecule, respectively, by combined digestion with EcoRI and SalI. EcoRI-1 and EcoRI-4 were missing from the combined digest, which contained three new fragments, representing 12.1, 10.7, and 2.9% of intact BF23 DNA. EcoRI-1 thus spans the SailI site at 11% and yields SalI-3 and the 10.7% fragment in the combined digest. Since EcoRI2 is too large to fit between fragments 1 and 8, it must occur next to fragment 3 at the right end of the DNA. EcoRI-4 must span the 47% SailI site and occur to the left of EcoRI-5 in order to satisfy the results of the combined digest. The five BamHI fragments of BF23 DNA were ordered in a manner similar to the EcoRI fragments. Prior digestion with A exonuclease resulted first in the loss of fragments 4 and 5, followed by the loss of fragments 2 and 3. Since BamHI-2 was the only fragment affected by the st deletions, the order of the central three fragments is 2-1-3. BamHI-4 and BamHI-5 were assigned to the left and right ends, respectively, by analysis of the products of a BamHI-EcoRI digest. EcoRI fragments 4 through 8 and BamHI-4 and BamHI-5 were present in the combined digest along with new fragments representing 16.7, 13.5, 5.7, and 4.6% of BF23st(+) DNA. EcoRI-1 must yield BamHI-5 and the 13.5% fragment, and EcoRI-3 yields BamHI4 and the 5.7% fragment in the combined digest.
924
J. VIROL.
NOTES
TABLE 1. Molecular weights of SalI, BamHI, and EcoRI fragments of BF23st(+) DNAa Endonuclease
U -I
IN
I m I.. I
_I
U a
-N
.M ., "1 o
'i
r.
.N E. m
-,
-'
FIG. 1. Restriction endonuclease cleavage prod-
ucts of BF23 DNA. (A) Gel 1, SalI digest of BF23st(+)
DNA; gel 2, BamHI digest ofBF23st(+) DNA; gels 3 and 4, EcoRI digest of BF23st(+) DNA. Electrophoresis was carried out in 0.5% agarose gels in gels 1, 2, and 3 and in 1.0% agarose in gel 4. (B) Gels I and 2, BalI digest of BF23st(+) DNA; gels 3 and 4, HpaI digest of BF23st(+) DNA. Electrophoresis was carried out in 0.5% agarose in gel 1, 0.7% agarose in gel 3, and 1.5% agarose in gels 2 and 4. The relative intensities of BalI band 4,5 and HpaI bands 13, 23, and 27 indicate that each contains two fragments, whereas HpaI band 25,26 contains three fragments. Both copies of HpaI fragments 13, 23, 25, and 27, which occur within the terminal repetition, have been given the same number.
The 16.7 and 4.6% fragments are derived from EcoRI-2. Initial analysis of the 13 BalI fragments of BF23 DNA was carried out by repetition of the procedures described above. Digestion with A exonuclease first resulted in the loss of BalI-1 and BalI-3, followed by the loss of one fragment (arbitrarily designated BalI-4) from the band that contains fragments 4 and 5. BalI-1 must be at the left end since EcoRI-1 is not cleaved by BalI (see below). BF23st(102) lacks BalI-6, whereas BF23st(104) also lacks Ball-10. The fragment orders at the left and right ends of the DNA are therefore 1-6-10 and 4-3, respectively. Size considerations and the locations of the deletions prevent any fragment other than BalI-1 from occuring between the left end and BalI-6. The remaining BalI fragments were mapped by analyses of isolated EcoRI fragments, combined EcoRI-BalI and BamHI-BalI digests, and partial BalI digests. The results of these experiments are summarized in Table 2. BalI
SalI
Fragment no.
Mol wt (x106)
1
40.9b
28.3 8.4 26.2 BamHI 2c 20.0 16.9 3 7.8 4 7.0 5 17.3 1 EcoRId 16.3 2 12.2 3 11.8 4 5 10.5 5.6 6 7 2.10 2.06 8 a Molecular weights were determined from electrophoretic mobilities relative to restriction endonuclease fragments of T5 DNA (2). b The molecular weight of Sall-1 was determined from the sum of EcoRI fragments 2, 3, and 5 plus the 2.1 x 106-dalton (2.8%) fragment in SalI-EcoRI digests. cBamHI-2 is replaced by a 14.8 x 10-dalton fragment in BF23st(102) DNA and by a 15.3 x 10-dalton frapment in BF23st(104) DNA. BF23st(102) DNA lacks EcoRI fragments 6 through 8 and contains a new fragment having a molecular weight of 4.4 x 106. BF23st(104) DNA lacks EcoRI fragments 6 and 7 and contains a new fragment having a molecular weight of 2.8 x 106. 2 3 1
digestion of a mixture of isolated EcoRI fragments 1 and 2 yielded EcoRI-1 and BalI-5 plus two new fragments. Therefore, BalI-5 must come from EcoRI-2 and occur next to BalI-4. BalI digestion of isolated EcoRI fragments 3 and 4 yielded BalI fragments 3, 11, 12, and 13 plus two new fragments, the larger of which must be derived from BalI-2. Since Ball-3 maps within EcoRI-3, BalI fragments 11 through 13, and most of BalI-2, must occur in EcoRI-4, with BalI-2 on the right of the three smaller fragments. Treatment of a BalI digest with EcoRI led to the loss of BalI-8 and five previously mapped fragments. BalI-8 thus spans the EcoRI site at 63.4% and occurs next to BalI-5. BalI-7 and BalI-9 must therefore occupy the gap between BalI-2 and BalI-8. The absolute order of fragments 11, 12, and 13 and fragments 7, 8, and 9 was determined by analysis of partial BalI digests. As shown in Table 2, nearly complete BalI digests contain fragments whose sizes are most consistent with the fragment orders shown in Fig. 2. The results of a combined BalI-BamHI digest (Table 2) provide confirmation for several
NOTES
VOL. 30, 1979
BF23(+) (77.9) 1 (18.6)
TABLE 2. Summary of evidence for Ball cleavage map of BF23 DNA BalI frgments present in:' B723(+) BamHIb BF23(+) EcoRI Partial BalI digests' EcoRI-1,2' EcoRI-3,4d (77.9) (77.9) (33.6) (24.0)
EcoRI-1(17.3) 2 (11.5)
EcoRI-1(17.3)
2
(11.5) 3(8.9) 4 (8.5) 5(8.5)
925
(9.1) 3
(9.2) 3
(17.5) 3 + (4 or 5) (16.5) 2 + 7 (10.5) = 7 + 8 + 9 (10.0) =6+ 10+ 12
4 5
5
BamHI-5(7.0)
(5.2)
(5.2)
7(4.5)
7
7
8 (3.4)
8
9 (2.6)
9
BamHI-4(7.8) (7.5) 6 (7.2)
6
(6.1) = 8 + 9
(3.96)
(3.33)
10 (1.76)
(3.33)
9
(2.57)
(1.84) 10
(3.96) = 10 + 11 + 12
(2.33) EcoRI-7(2.1)
(2.20) = 11 + 12 or 10 + 12
(2.33)
(1.65) 11 (1.54)
12 (0.75)
11 (1.17) (1.05) 12
11 (1.15) (0.98)
11
12
12
(0.61) 13 (0.28) Z 78.03
13 13 13 X, 78.70 Z 33.33 E: 76.74 l 24.00 0Numbers in parentheses indicate molecular weights (x106). Molecular weights were determined from electrophoretic mobility relative to T5st(+) EcoRI(4) and BF23st(+) HpaI fragments. Fragments not appearing in BalI digests of BF23st(+) DNA are listed as molecular weights without fragment numbers. The new in BF23st(102) and BF23st(104) equal 1.62 and 3.74 x 10E, respectively. fragments b The BalI site at 35.3% and the BamHI site at 34.7% cannot be distinguished. 'EcoRI-1 and EcoRI-2 were isolated as one band by the procedure of Bln et al. (1). Since EcoRI-1 is not cleaved by BalI, all resulting BalI framents are from EcoRI-2. d EcoRI-3 and EcoRI-4 were isolated as one band. EcoRI-3 contains mostly BalI-3 plus a fragment from BalI-4. All other fiagments are from EcoRI-4. ' Enzyme dose and time of incubation were limited such that the digests were near completion and thus likely to contain fragments that were the sum of two neighboring fragments. All detectable composite fragments are shown.
aspects of the BalI cleavage map. Cleavage of BF23st(+) DNA with HpaI results in 49 fragmnents, the largest 32 of which have been ordered. Because of the complexity of the HpaI map, only the general strategy used to order the fragments is presented. However, all of the critical results are summarized in Tables 3 and 4. Approximate fragment locations were determined by digestion of isolated BamHI fragments (Table 3), analysis of the st deletion mutants, and prior digestion with A exonuclease. BF23st(102) DNA lacks fragments 6, 12, 14, and 22, whereas BF23st(104) DNA lacks fragments 6 and 12. The A exonuclease experiments revealed that HpaI-21 and HpaI-28 are terminally located, followed by fragments 13, 23, 25 and 27.
Further digestion led to the loss of fragments 5 and 18. The order of fragments 13, 23, 25, and 27, which are present in two copies because of the terminal repetition in BF23 DNA (5), has been determined by an analysis of T5-BF23 hybrid genomes and will be described in another publication. HpaI fragments 32, 34, 37, and 38 also occur within the terminal repetition, but remain unordered. Many of the HpaI fragments were further localized within each BamHI fragment by analyses of combined digests with each of the four other endonucleases. Except for fragments 13 through 17, the 20 largest HpaI fragments could be localized with some precision in this manner. HpaI-1, for example, is in BamHI-1 and must
926
J. VIROL.
NOTES
TABLE 3. Summary of evidence for HpaI cleavage map of BF23 DNA HpaI fragments present in:a
BF23(+)b BF23(+) BamHI BamHI-1 (77.9)
(77.9)
(26.2)
1(8.0)
1
1
2(6.5) 3 (6.0)
2 3
4(5.2) 5(4.9)
4
6(4.2)
6 7c
BamHI-2 (20.0)
BamHI-3 (16.9)
BamHI-4 (7.8)
BamHI-5 (7.0)
BF23(+) EcoRI
(77.9)
BF23(+) BalI (77.9) (77.
1 2
2
(7.9)
3
(5.35)
7(3.4)
4
4
5 (4.60) 6 7
6 7
7
d
(3.4) 8 (3.3) 9 (3.04)
8 gd
(3.1) (3.04)e
9e
(2.9)
(2.83)f 10 (2.67) 11 (2.63)
10
10
10
(2.61) (2.52)
(2.52)
(2.45) (2.41) 12 (2.19) 13 (2.01)
12 13
12
13
(1.94)
13
13
12 13
(1.94)
(1.94) (1.82)
(1.80)
14 (1.72)
14
15 (1.60)
15
16 (1.54)
16 17
17 (1.51)
14
14
15 16
17
15
(1.63) 15
16
(1.57) 16
17
17
18
18
(1.50) 18 (1.38)
18
(1.38)'
18'
(1.33) 19 (1.29) 20 (1.24) 21 (1.21)
19
19 20 21
22 (1.17) 23 (1.02) 24 (0.86) 25 (0.75)
22 23 24 25
26 (0.75)
26
27 (0.52)
27
20
20
21
21
22
19 20 21
(1.20) 22
22
23
23
23
23
25
25
24 25
24 25
26
26
24 26
(0.63) 27
27
27
(0.63) 27
(0.46) 28 28 28 28 28(0.39) 29 29 29 29 29 (0.35) 30 30 30 30 30 (0.27) 31 31 31 31 31 (0.24) 2 7.33 h X 76.15 X 76.12 X 25.83 X 19.98 X 16.74 6.07h 72.73h 75.05O a Numbers in parentheses are molecular weights (x 106). Molecular weights for fragments 1 through 31 were determined from their electrophoretic mobilities relative to TS HpaI fragments (2). Fragments not appearing in HpaI digests of BF23st(+) are listed as molecular weights without fragment numbers. SalI-HpaI digests lack HpaI-1 and HpaI-7. Two new products appear (molecular weights, 7.3 x 106 and 1.87 x 106).
VOL. 30, 1979
927
NOTES
TABLE 3-Continued The molecular weights of the following fragments, which were not mapped because of their small size, were determined from their electrophoretic mobilities relative to OX174 Taq fragments (6) on polyacrylamide gels and are: fragment 32, 0.22 x 106; fragment 33, 0.19 x 106; fragment 34, 0.14 x 106; fragment 35, 0.136 106; fragment 36, 0.12 x 106; fament 37, 0.11 106; fragment 38, 0.10 x 106; fragment 39, 0.08 x 106; fragment 40, 0.06 x 106; and fragment 41, 0.02 x 106. Fragments 32, 34, 37, and 38 appear to be present in two copies per genome. The new fragments in BF23st(102) and BF23st(104) equal 3.8 x 106 and 1.2 x 106, respectively. 'The BamHI site at 9.0% and the HpaI site at 8.9% cannot be distinguished. d This fragment appears to be a doublet. 'HpaI-9 must be in BamHI-1 since it is cleaved by both BalI and EcoRI. f This fragment appears to be a triplet. HpaI-18 must be in BamHI-5 as indicated by A exonuclease experiments. h These values are approximate since fragments smaller than HpaI-31 were not identified. 'Determined, when possible, by using molecular weights of BalI products from isolated HpaI fragments (see Table 4). b
x
x
cr
IT
2
0
BamHZI
s
2
1
I
3
EcoRi '
'
2
WI
'
I
.
s"UI
I.11 f 11 IN I
Bali
b9
9 a7,9gS I I
2
!A P C
I
I
#A
0
3
5 I "4 a
wIdid
i 212 1(1741(23e 2t2,2T2 2IT.4 I MI 2a .11, HpaZL3~la 7Ialli 1a2 I, I I&AIRA1 1UI 11
25232l7 111
2
o0,CwAuCee .4t,* Idt Mm
Id
44
S
C:
M
0
**t Oj"4
*SS
'4I.sbb 444o4
II I Del9tions
6
lb
2b
4
^b
4
*
^
4
160
X SF23 st(+) lngith FIG. 2. Restriction endonuclease cleavage maps of BF23st(+) DNA. The numbers above the lines are fragment numbers; the numbers below the lines are the coordinates of sites expressed as percentages of the wild-type length from the left end of the molecule. In the HpaI map, fragments occurring within the 8.4% terminal repetition have been given the same number. Fragments in parentheses have not been ordered. The locations of the st deletions were determined from heteroduplex analysis (unpublished data).
928
NOTES
TABLE 4. BalI cleavage products of isolated HpaI fragments in BF23 DNA a BF23(+)m Hpal fragBalI products ment
2 (6.5) 3 (6.0) 5 (4.9) 6 (4.2)
(3.46) (2.89) (2.99) (2.91) (2.88) (1.97) (2.55) (1.63) 7 (3.4)-8 (3.3) HpaI-7 (3.4) (3.13) 9 (3.04) (1.61) (1.33) 10 (2.67)-11 (2.63) (1.55) (1.49) (1.11) (0.74) a Numbers in parentheses indicate molecular weights (x106). Molecular weights were determined from electrophoretic mobilities relative to T5(+) HpaI fragments (2). Fragments were isolated by the procedure of Blin et al. (1).
span the Sall site at 47%, but not the EcoRI site at 50%. Similarly, HpaI-2 is in BamHI-3 and must span the BalI site at 78% in order to avoid cleavage by the other enzymes. Exact locations for HpaI fragments 2, 3, and 5 through 11 were determined by digestion of the isolated fragments with BalI (Table 4). This information in turn allowed several of the smaller fragments to be mapped on the basis of size. Within BamHI1, for example, the only gap large enough to accomodate HpaI-16 occurs between fragments 8 and 9. Similar logic dictated the placement of HpaI fragments 17, 26, and 29 through 31. HpaI fragments 15, 20, and 24 were tentatively mapped by analysis of T5-BF23 hybrid genomes (unpublished data). The order of HpaI fragments 22 and 14 fits best with the fact that neither is cut by BalI. A comparison of the common restriction maps of BF23 and T5 reveals that 30 out of 84 cleavage sites have been conserved between the two ge-
J. VIROL.
nomes. Based on this value, two methods of estimating sequence homology (7, 9) both indicate that T5 and BF23 have undergone approximately 15% divergence in base sequence. This research was supported by grant PCM 76-24036 from the National Science Foundation. LITERATURE CITED 1. Blin, N., A. von Gabain, and H. Bujard. 1975. Isolation of large molecular weight DNA from agarose gels for further digestion by restriction enzymes. FEBS Lett. 53:84-6. 2. Hamlett, N. V., B. Lange-Gustafson, and M. Rhoades. 1977. Physical map of the bacteriophage T5 genome based on the cleavage products of the restriction endonucleases SalI, SmaI, BamI, and HpaI. J. Virol. 24: 249-260. 3. McCorquodale, D. J. 1975. The T-odd bacteriophages. Crit. Rev. Microbiol. 4:104-159. 4. Rhoades, M. 1975. Cleavage of T5 DNA by the Escherichia coli RI restriction endonuclease. Virology 64: 170-179. 5. Rhoades, M., and B. Lange-Gustafson. 1979. Physical map of bacteriophage BF23 DNA: terminal redundancy and localization of single-chain interruptions. J. Virol. 30:777-786. 6. Sato, S., C. A. Hutchison, and J. I. Harris. 1977. A thermostable sequence-specific endonuclease from Thermus aquaticus. Proc. Natl. Acad. Sci. U.S.A. 74: 542-546. 7. Schumperli, D., R. Lagadec, and H. K. Miiller. 1977. DNA sequence homology estimation by combinational analysis of endonuclease restriction data. J. Gen. Virol. 38:161-166. 8. Tchernov, A. P., N. P. Kouzmin, and I. Fodor. 1978. Physical mapping of cleavage sites recognized by restriction endonucleases on the genome of bacteriophage T5. Gene 3:293-302. 9. Upholt, W. B. 1977. Estimation of DNA sequence divergence from comparison of restriction endonuclease digests. Nucleic Acids Res. 4:1257-1265. 10. von Gabain, A., G. S. Hayward, and H. Bujard. 1976. Physical mapping of the HmndIII, EcoRI, Sal, and Sma restriction endonucleases cleavage fragments from bacteriophage T5 DNA. Mol. Gen. Genet. 143:279-290.
