1977
Nucleic Acids Research
September Vlue4Nme9Setme197NcicAisR
Volume 4 Number 9
The nucleotide sequence surrounding the origin of DNA replication of Col E1
Deepak Bastia
Department of Human Genetics, Yale University School of Medicine, New Haven, CT 06510, USA Received 23 June 1977
SUMMARY The DNA of Col E1 replicates from a unique origin located at a distance of 17-19% of the genome length from the single Eco RI cleavage site. The nucleotide sequence about this site has been determined by a combination of RNA and DNA sequencing techniques. The principal features of the sequence are two palindromes, one of which resembles a palindrome located in the intercistronic region of 0X174. The sequence also contains stretches of purine and pyrimidine clusters of the following compositions: pAT G, pC T G, pGT G. The origin sequence demonstrates that initiation o?DNA repSi.ation takes place in an intercistronic region of Col E DNA, although the possibility that this region makes small polypeptides 313-40 residues long cannot be strictly eliminated at this time.
INTRODUCTION The colicinogenic plasmid Col E1 not only has become a useful vector in molecular cloning experiments (1), but also a favorable system for the study of DNA replication in vivo (2) and in.vitro (3). The DNA of Col E1 replicates from a unique origin located at a distance of 17-19% of the genome length from the single Eco RI cleavage site (3, 4, 5). The replication of this DNA is unidirectional and therefore the terminus of replication is located at or near the origin. The replication intennediates are partially supercoiled "Cairns forms" or B shaped structures (3, 5, 6). The replication in vivo requires DNA polymerase I and the initiation is rifampicin sensitive (2). Tomizawa (7) has shown that only the initiation in vitro of a new DNA chain complementary to the heavy strand at the origin is rifampicin sensitive, thus suggesting the involvement of an RNA primer at this step of initiation. The on-going replication does not require RNA synthesis or protein synthesis (2). Inhibition of host protein synthesis by chloramphenicol leads to continuation of plasmid replication in the CX Information Retrieval Limited 1 Falconberg Court London W1 V 5F6 England
3123
Nucleic Acids Research absence of host DNA replication and thus results in up to 100-fold amplification of the number of copies of plasmids per cell (8).
Hitherto, the structure of only one replication origin; namely, that of SV40 is known (9). Although the entire sequence of 0X174 chromosome has been deduced, the exact location of the replication origin is not known (10). In view of the lack of information regarding the replication origin of plasmid chromosomes, we have sequenced approximately 260 nucleotides of Col E spanning the chromosome from 16. 5-20% of the genome length from the Eco RI cleavage site. Electron microscopic data from three different laboratories have located the replication origin in this part of Col E1 chromosome (3,4, 5). Analysis of Col E1 DNA carrying large deletions also supports this localization (Inselburg, personal communication) of the replication origin.
MATERIATS AND METHODS DNA was prepared from JC411 (Col E1) (kindly supplied by Don Helinski) as previously described (8). Preparation of restriction fragments (19), RNA sequencing (12, 13), DNA sequencing by partial snake venom digestions (14) and by chemical cleavage method (15) are published. RESULTS Bastia and Weissman (submitted for publication) have reported a detailed restriction map of the part of Col E DNA containing the replication origin and the relaxation site which is shown in Figure 1. Oka and Takanami (11) have previously reported the Hae III and Hae II cleavage map of Col E 1. The electron microscopic localization of replication origin, translated into the restriction map, places the replication origin in the fragment Hae II-E (see Figure 1) near the Hae ITT-N - Hae Ill-H junction. In this paper I present the sequence of the fragments F1, F4, and approximately half of F9 (see Figure 1). These fragments cover approximately 16. 5-20% of the genome length from the Eco RI cleavage site. Sequence of F: The fragment of F1 Is 160 base pairs long and contains three Hinf sites and a single Hha I/Hae II site. The strategy for sequencing F1 was to trans3124
Nucleic Acids Research IHha K
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THE DIRECTION OF REPLICATION
Fig. 1
Restriction map of Col E1
cribe it with E. coli RNA polymerase in the presence of a- 32P rNTP s (one labeled precursor in a given reaction) and digest the labeled RNA transcript with T1 RNase. The T 1 oligonucleotides were sequenced following the procedure of Brownlee and Sanger (12) and Weissman and Radding (13). The T1 oligonucleotides were then overlapped by: (a) obtaining base specific chemical degradation patterns of end labeled subfragments of F 1; a nd (b) pa rtial snake venom phosphodiesterase digestion patterns of 53 end labeled subfragments of F1. This strategy obviated the need for the laborious procedure of overlapping T1 oligonucleotide by the classical partial T1digestion procedure. The T 1 oligonucleotides of F 1were fractionated in the first dimension by electrophoresis on a cellogel strip and then transferred to a thin layer plate containing 9 parts of cellulose to 1 part of DEAE cellulose. The plates were homochromatographed, using the homomixture B (13). A repres entative T1 oligonucleotide homochromatogram is presented in Figure 2. The sequences of the T1 oligonucleotides were determined by secondary digestion of the spots eluted from the homochromatograms with pancreatic and U2 RNases and the nearest neighbors of the secondary products were determined by alkaline hydrolysis. The details of this procedure have been described before (13). The results of the analysis of T1 oligonucleotides are presented in Table 1 and Table 2. The complete pancreatic RNase products of the transcript of F 1were also analyzed and the data (not shown) was consistent with the sequence of F1 presented in Figure 6A. 3125
Nucleic Acids Research
6
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Partial snake venom digestions of 5' end labeled subfragments of F The subfragments of F1 were labeled at one of the two 5' phosphoryl ends (with y- P ATP and polynucleotide kinase) by labeling the appropriate parental fragment containing the subfragment at both the 5' ends followed by recleaving with an appropriate restriction enzyme or by separating the two strands of the subfragment labeled at both 5' ends, by twodimensional homochromatography as previously described (14), The fragments asymmetrically labeled at one of the two 5' ends were subjected to partial hydrolysis with snake venom phosphodiesterase and the partial di3126
Nucleic Acids Research TABLE 1
Oligonucleotides in extensive T1 RNase digest of transcripts of the DNA fragment F1(a) Products of extensive pancreatic RNase digestion (c) OligoSequence of nucleotide [c- 32PI labeled precursor product (d) tide number (b) 1 2
3 4 5 6 8
CTP
AAAAAC AC U AC C,U AC,G,C,U
AU,C
ATP
GTP
C,AC, AAAAAC C,AC C,U U,G
AAAAAC, G C,G AC U
U, C,AC, AG AC, AAC, U
AG AG C C U
9 10
AU,AC,C AAC,AC G,C,U C,U,G AC,U
11 13
AAU U
AAU, C
15
AU,AC
16 17 18 131 181 201 19
-
AAAAG -
AC AU U
AU, C, U AU, C, U AU,U
AG U
AAU
AU,C,G
C,U,G
U,C
AU,C,U,G
CAUCUG(U) UCACUG(A)
AU,AAG AAAAG U,AAG
AAG
AC AU
UUACCG (U) AUAAG (G)
C,G
U,G
AC
AC, C,AG
G,U,C
C,U,G
AG
C,U C,U AC,C
AAAAG AAG (G) U U AG AC,C,AG
U,G AC
AC,C
AC
23
U,G
24 25
AC G,AG G U,G G C,G AC G,U AAG AG AC, AG
26 27 30 124
231 241 261 281
ACUCACACAAAAACG (G) CAUUUCACACCCGG CAUUUUUCUCCUUACG (C) CCUUAUCCCCUG (A) UUAUCCACAG (A) UAACACAG (U) CUUUUCCG (C) CUUCCUCG (C) CUCACUG (A) AUAACUG (U) AAUCAG (G) AUUCUG (U)
-
AC
UTP
G,C,U
U,G G G
C, AAG AG C,G
AC, C AU, AG U,G C,U,G C,G C U, AG AAG AG AC,AG
C
AU G C G
AG,AC
G
AAAAG (C) UAAG (G) UUUUG (U) UACUG (A) CACAG (A)
UACCG(C) ACUCGC UCCAGC CUACGC
CUCG(G) (C) ACCG (A) CACG (U) AUG (C) UAG (A) (C)
UG(G) (U) (A) (C) CUG(C)UCG(G) CG(A) (U) (C) (G) CCG (C) UCAG (U) ACUG (C) AAG (C)
AG(C) (A) (G) CAG(C)ACG(A) (U)
a) See Figure 1. b) Number refers to the oligonucleotide in Figure 2. c) The underlined products are present in more than one mole.Where appropriate,the products were analyzed by alkaline hydrolysis to determine the nearest neighbors. d) Letters in parenthesis indicate nearest neighbors. Sequences were deduced from results shown in Tables 1 and 2 and in Figures.
3127
Nucleic Acids Research TABLE 2 Products of extensive U2 RNass. digest of oligonucleotides l'isted in Table 1 Product (b) Sequence of Oligo[a- PI labeled precursor nucleotide product number(a) CTP UTP GTP (c) 1.0 1 CG .2 0.66 CUCA(C) 2 .22 CCCG .19 .05 0 UUUCA(C) 0 CA CA(G) 4 0 0 0 UCCCCUG .18 .12 CCUUA 5 .22 CA 1.1 .23 UCCA(C) .41 UUA 6 .82 .8 CA UG .45 .45 10 CUG .48 .49 11 UCA .05 13 UUCUG 15 .12 .14 .13 UCUG .23 .25 UUA(C) .5 .5 UCA+CCG
a) See footnote (b) of Table 1. b) Numbers refer to the relative mobility with respect to the xylene cyanol tracking dye. c) Letters in parenthesis indicate nearest neighbors. Products were analyzed by (i) digestion with alkali; (ii) re-electrophoresis on DEAE paper, pH 1.7, where appropriate.
gests were resolved into a trail of spots by electrophoresis on Cellogel in the first dimension followed by homochromatography in the second dimension. The trails generated by partial digestion (referred to hereafter as SVP trails) of various subfragments of F1 were used to overlap the T products (representative SVP trails are shown in Figure 3).
The subfragments of F1 were also asymmetrically labeled at one of the two 5' phosphoryl ends as described above and subjected to base specific chemical degradation procedure described by Maxam and Gilbert (15). The products were fractionated in 20% acrylamide-urea slab gels and representative gel patterns are shown in Figures 4 and 5. The T oligonucleotides of F were ordered and overlapped using the sequence derived from snake venom trails and the Maxam-Gilbert patterns. Sequence of F4: The fragment F4 (Hae III-N, Figure 1) is 43 base pairs long and its sequence was derived entirely by DNA sequencing methods: (a) by examining 3128
Nucleic Acids Research
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Nucleic Acids Research
September Vlue4Nme9Setme197NcicAisR
Volume 4 Number 9
The nucleotide sequence surrounding the origin of DNA replication of Col E1
Deepak Bastia
Department of Human Genetics, Yale University School of Medicine, New Haven, CT 06510, USA Received 23 June 1977
SUMMARY The DNA of Col E1 replicates from a unique origin located at a distance of 17-19% of the genome length from the single Eco RI cleavage site. The nucleotide sequence about this site has been determined by a combination of RNA and DNA sequencing techniques. The principal features of the sequence are two palindromes, one of which resembles a palindrome located in the intercistronic region of 0X174. The sequence also contains stretches of purine and pyrimidine clusters of the following compositions: pAT G, pC T G, pGT G. The origin sequence demonstrates that initiation o?DNA repSi.ation takes place in an intercistronic region of Col E DNA, although the possibility that this region makes small polypeptides 313-40 residues long cannot be strictly eliminated at this time.
INTRODUCTION The colicinogenic plasmid Col E1 not only has become a useful vector in molecular cloning experiments (1), but also a favorable system for the study of DNA replication in vivo (2) and in.vitro (3). The DNA of Col E1 replicates from a unique origin located at a distance of 17-19% of the genome length from the single Eco RI cleavage site (3, 4, 5). The replication of this DNA is unidirectional and therefore the terminus of replication is located at or near the origin. The replication intennediates are partially supercoiled "Cairns forms" or B shaped structures (3, 5, 6). The replication in vivo requires DNA polymerase I and the initiation is rifampicin sensitive (2). Tomizawa (7) has shown that only the initiation in vitro of a new DNA chain complementary to the heavy strand at the origin is rifampicin sensitive, thus suggesting the involvement of an RNA primer at this step of initiation. The on-going replication does not require RNA synthesis or protein synthesis (2). Inhibition of host protein synthesis by chloramphenicol leads to continuation of plasmid replication in the CX Information Retrieval Limited 1 Falconberg Court London W1 V 5F6 England
3123
Nucleic Acids Research absence of host DNA replication and thus results in up to 100-fold amplification of the number of copies of plasmids per cell (8).
Hitherto, the structure of only one replication origin; namely, that of SV40 is known (9). Although the entire sequence of 0X174 chromosome has been deduced, the exact location of the replication origin is not known (10). In view of the lack of information regarding the replication origin of plasmid chromosomes, we have sequenced approximately 260 nucleotides of Col E spanning the chromosome from 16. 5-20% of the genome length from the Eco RI cleavage site. Electron microscopic data from three different laboratories have located the replication origin in this part of Col E1 chromosome (3,4, 5). Analysis of Col E1 DNA carrying large deletions also supports this localization (Inselburg, personal communication) of the replication origin.
MATERIATS AND METHODS DNA was prepared from JC411 (Col E1) (kindly supplied by Don Helinski) as previously described (8). Preparation of restriction fragments (19), RNA sequencing (12, 13), DNA sequencing by partial snake venom digestions (14) and by chemical cleavage method (15) are published. RESULTS Bastia and Weissman (submitted for publication) have reported a detailed restriction map of the part of Col E DNA containing the replication origin and the relaxation site which is shown in Figure 1. Oka and Takanami (11) have previously reported the Hae III and Hae II cleavage map of Col E 1. The electron microscopic localization of replication origin, translated into the restriction map, places the replication origin in the fragment Hae II-E (see Figure 1) near the Hae ITT-N - Hae Ill-H junction. In this paper I present the sequence of the fragments F1, F4, and approximately half of F9 (see Figure 1). These fragments cover approximately 16. 5-20% of the genome length from the Eco RI cleavage site. Sequence of F: The fragment of F1 Is 160 base pairs long and contains three Hinf sites and a single Hha I/Hae II site. The strategy for sequencing F1 was to trans3124
Nucleic Acids Research IHha K
a*M H H(4%
Flo
M NooN* EI EoenI H pollJ-14 -HpoRK 20-% Hpo]M L
H
.1±.
Fe
HHpox
Ho!
NpsUmeso
Fl
Fe
"poll
F F4 Ft MPGDU pHeNnHasE
THE DIRECTION OF REPLICATION
Fig. 1
Restriction map of Col E1
cribe it with E. coli RNA polymerase in the presence of a- 32P rNTP s (one labeled precursor in a given reaction) and digest the labeled RNA transcript with T1 RNase. The T 1 oligonucleotides were sequenced following the procedure of Brownlee and Sanger (12) and Weissman and Radding (13). The T1 oligonucleotides were then overlapped by: (a) obtaining base specific chemical degradation patterns of end labeled subfragments of F 1; a nd (b) pa rtial snake venom phosphodiesterase digestion patterns of 53 end labeled subfragments of F1. This strategy obviated the need for the laborious procedure of overlapping T1 oligonucleotide by the classical partial T1digestion procedure. The T 1 oligonucleotides of F 1were fractionated in the first dimension by electrophoresis on a cellogel strip and then transferred to a thin layer plate containing 9 parts of cellulose to 1 part of DEAE cellulose. The plates were homochromatographed, using the homomixture B (13). A repres entative T1 oligonucleotide homochromatogram is presented in Figure 2. The sequences of the T1 oligonucleotides were determined by secondary digestion of the spots eluted from the homochromatograms with pancreatic and U2 RNases and the nearest neighbors of the secondary products were determined by alkaline hydrolysis. The details of this procedure have been described before (13). The results of the analysis of T1 oligonucleotides are presented in Table 1 and Table 2. The complete pancreatic RNase products of the transcript of F 1were also analyzed and the data (not shown) was consistent with the sequence of F1 presented in Figure 6A. 3125
Nucleic Acids Research
6
12Z
15
16'// 17:
13
...
i'
131
s I
.
._
:^g, *.
........
.. _
t
.:i... ..
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4 a
2
Fig. 2 Ti fingerprint of
aP32GTP labeled RNA trnscript of F1
Partial snake venom digestions of 5' end labeled subfragments of F The subfragments of F1 were labeled at one of the two 5' phosphoryl ends (with y- P ATP and polynucleotide kinase) by labeling the appropriate parental fragment containing the subfragment at both the 5' ends followed by recleaving with an appropriate restriction enzyme or by separating the two strands of the subfragment labeled at both 5' ends, by twodimensional homochromatography as previously described (14), The fragments asymmetrically labeled at one of the two 5' ends were subjected to partial hydrolysis with snake venom phosphodiesterase and the partial di3126
Nucleic Acids Research TABLE 1
Oligonucleotides in extensive T1 RNase digest of transcripts of the DNA fragment F1(a) Products of extensive pancreatic RNase digestion (c) OligoSequence of nucleotide [c- 32PI labeled precursor product (d) tide number (b) 1 2
3 4 5 6 8
CTP
AAAAAC AC U AC C,U AC,G,C,U
AU,C
ATP
GTP
C,AC, AAAAAC C,AC C,U U,G
AAAAAC, G C,G AC U
U, C,AC, AG AC, AAC, U
AG AG C C U
9 10
AU,AC,C AAC,AC G,C,U C,U,G AC,U
11 13
AAU U
AAU, C
15
AU,AC
16 17 18 131 181 201 19
-
AAAAG -
AC AU U
AU, C, U AU, C, U AU,U
AG U
AAU
AU,C,G
C,U,G
U,C
AU,C,U,G
CAUCUG(U) UCACUG(A)
AU,AAG AAAAG U,AAG
AAG
AC AU
UUACCG (U) AUAAG (G)
C,G
U,G
AC
AC, C,AG
G,U,C
C,U,G
AG
C,U C,U AC,C
AAAAG AAG (G) U U AG AC,C,AG
U,G AC
AC,C
AC
23
U,G
24 25
AC G,AG G U,G G C,G AC G,U AAG AG AC, AG
26 27 30 124
231 241 261 281
ACUCACACAAAAACG (G) CAUUUCACACCCGG CAUUUUUCUCCUUACG (C) CCUUAUCCCCUG (A) UUAUCCACAG (A) UAACACAG (U) CUUUUCCG (C) CUUCCUCG (C) CUCACUG (A) AUAACUG (U) AAUCAG (G) AUUCUG (U)
-
AC
UTP
G,C,U
U,G G G
C, AAG AG C,G
AC, C AU, AG U,G C,U,G C,G C U, AG AAG AG AC,AG
C
AU G C G
AG,AC
G
AAAAG (C) UAAG (G) UUUUG (U) UACUG (A) CACAG (A)
UACCG(C) ACUCGC UCCAGC CUACGC
CUCG(G) (C) ACCG (A) CACG (U) AUG (C) UAG (A) (C)
UG(G) (U) (A) (C) CUG(C)UCG(G) CG(A) (U) (C) (G) CCG (C) UCAG (U) ACUG (C) AAG (C)
AG(C) (A) (G) CAG(C)ACG(A) (U)
a) See Figure 1. b) Number refers to the oligonucleotide in Figure 2. c) The underlined products are present in more than one mole.Where appropriate,the products were analyzed by alkaline hydrolysis to determine the nearest neighbors. d) Letters in parenthesis indicate nearest neighbors. Sequences were deduced from results shown in Tables 1 and 2 and in Figures.
3127
Nucleic Acids Research TABLE 2 Products of extensive U2 RNass. digest of oligonucleotides l'isted in Table 1 Product (b) Sequence of Oligo[a- PI labeled precursor nucleotide product number(a) CTP UTP GTP (c) 1.0 1 CG .2 0.66 CUCA(C) 2 .22 CCCG .19 .05 0 UUUCA(C) 0 CA CA(G) 4 0 0 0 UCCCCUG .18 .12 CCUUA 5 .22 CA 1.1 .23 UCCA(C) .41 UUA 6 .82 .8 CA UG .45 .45 10 CUG .48 .49 11 UCA .05 13 UUCUG 15 .12 .14 .13 UCUG .23 .25 UUA(C) .5 .5 UCA+CCG
a) See footnote (b) of Table 1. b) Numbers refer to the relative mobility with respect to the xylene cyanol tracking dye. c) Letters in parenthesis indicate nearest neighbors. Products were analyzed by (i) digestion with alkali; (ii) re-electrophoresis on DEAE paper, pH 1.7, where appropriate.
gests were resolved into a trail of spots by electrophoresis on Cellogel in the first dimension followed by homochromatography in the second dimension. The trails generated by partial digestion (referred to hereafter as SVP trails) of various subfragments of F1 were used to overlap the T products (representative SVP trails are shown in Figure 3).
The subfragments of F1 were also asymmetrically labeled at one of the two 5' phosphoryl ends as described above and subjected to base specific chemical degradation procedure described by Maxam and Gilbert (15). The products were fractionated in 20% acrylamide-urea slab gels and representative gel patterns are shown in Figures 4 and 5. The T oligonucleotides of F were ordered and overlapped using the sequence derived from snake venom trails and the Maxam-Gilbert patterns. Sequence of F4: The fragment F4 (Hae III-N, Figure 1) is 43 base pairs long and its sequence was derived entirely by DNA sequencing methods: (a) by examining 3128
Nucleic Acids Research
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Nucleic Acids Research
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