Journal of Virological Methods, 36 0 1992 Elsevier Science Publishers
VIRMET
271
(I 992) 277-282 B.V. / All rights
reserved
/ Ol66-0934/92/$05.00
01312
Short Communication
Establishment of a genomic bank of bovine herpesvirus 1 using a novel positive selection plasmid vector L.S. Christensen State
Veterinary Institute for
and M. Boye
Virus Research,
(Accepted
6 December
Lindholm. Kalvehave, Denmark 1991)
Summary
A small positive selection cloning vector, designated pSiig1, suitable for the construction of genomic banks in E. coli is described and used for the establishment of a bank of the bovine herpesvirus 1 genome. Hybrid transformants are directly selected on agar plates containing ampicillin. The vector is based on the replicon of Rl and has a lambda PR promotor inserted upstream of the replication control genes. The vector has an uncontrolled (runaway) replication and is lethal to the host cell unless the PR promotor is brought under trans-acting control of the lambda cI repressor or runaway replication is blocked by an insertion between the PR promotor and the replicon. The vector contains a unique BgZII site between Pn and the replicon which is suitable for insertion of genomic DNA. Cloning vector; pSiig1; Genomic bank construction;
Bovine herpesvirus 1
Introduction
Plasmid vectors are widely used for the establishment of genomic banks in bacteria. Among the commonly used principles for selection of hybrid transformants are insertional inactivation of genes coding for antibiotic resistance or metabolic functions. However, using such vectors, screening for hybrids requires either replica plating on selective plates or complex agar Correspondence to: L. Siig Christensen. At present address: Institute for Medical Microbiology, Building 24-2. DK-2200 Copenhagen N, Denmark.
University
of Copenhagen,
Panum
Institute
278
media. The vector pSiig1 described in the present report provide the simplest possible selection of hybrids directly on agar plates with ampicillin (Ap). pSiig1 is based on the replicon of the resistance plasmid Rl . This replicon is comprised within a 2.5 kb region (Kollek et al., 1978; Molin et al., 1979) and is well characterized (for a review, see Nordstrom et al., 1984). Based on the knowledge of the replication control system of this replicon a series of vectors were constructed, where a DNA fragment containing the lambda Pa promotor and the temperature sensitive ~1857 allele of the repressor gene was inserted upstream of the replicon (Larsen et al., 1984). In these vectors runaway replication is induced by Pa when the repressor is inactivated at increased temperatures. In pSiig1 this principle is applied in a modified version. pSiig1 does not contain the ~1857 repressor and can only be harbored in a host strain containing the repressor. Hybrids in which runaway replication is blocked by the insert can be selected in a suitable host not containing the repressor.
Materials and Methods Bacterial strains Three derivatives of Escherichia coli K-12 were used. N4830-1 (F-, su-, his-, ilv-, galKd8, d(ch1 D-pgl), [/2, ABarn, N +, cI857dHll) (Gottesman et al., 1980) was used as host for pSiig1. C600 (F-, thr-1, leuB6, thi-1, supE44) (Bachmann, 1972) was used as host for the parental plasmid pOU57. MT102 (MC1000 hsd RK12) (M. Trier Hansen) was used as recipient in selection of hybrid transformants. Virus strain A Danish strain of bovine herpesvirus 1 was used as the source of the genomic bank. The strain DK540/89 was isolated from a nasal swab of a cow during an outbreak of rhinotracheitis. DK540/89 was categorized as type I (Cooper-type, IBR-like) according to Misra et al. (1983) by digestion of viral DNA with HilrdIII and EcoRI. Construction
of pSiig1
The parental plasmid pOU57 (Larsen et al., 1984) contains the bla gene of tn3 and a region originating from the bacteriophage lambda strain ED//4 (Dempsey and Willetts, 1976) comprising ~1857 and Pa upstream of the replicon of Rl as shown in Fig. 1. The replicon is deleted of a 0.5 kb BgnI fragment containing the constitutive promotor and the promotor-proximal part of the copB gene. pOU57 was digested with Hind111 and religated. The ligation mixture was transformed into N4830- 1, and a plasmid deleted of the 2 minor Hind111 fragments was identified.
279
!I ~ B
‘3
b.4
Basic replicon
n S
._ 4,
4
4
b 4
I I 1 S,
HC
pou57
tiEx
!
1.
Digestion with Hindl~l,re~iga~ion
t t
H
ä
4
a
'P S2
I P
S
pSiig1
P Sl
Fig. 1. Genetic and physical map of the basic replicon of plasmid RI, the parental vector ~01157, and pSiigl_ In the basic replicon copB and copA encode products that control the expression of the repA gene product, which is required for initiation of replication at ori (cf. Nordstrom et al., 1984). bla represent the /& lactamase gene mediating resistance to penicillin. b indicate the localization of promoters essential for replication or replication control. Pa is the rightward promotor of lambda being under control of the temperature sensitive repressor mutant ~1857. Restriction sites: B 1 = BarnHI, B2 = &$I, C = CM, E = EcoRI, H = HindIII, P .= f’stl, S = SalI.
Media and growth conditions
The bacteria were grown in LB medium (Bertani, 1951) and on LA plates containing 1.5% agar. Selective pressure was achieved by supplementing with Ap (Doktacillin, Astra) to a final concentration of 50 pg/ml. Strains harboring pOU57 or pSiig1 were grown at 30°C and amplification of the plasmids was achieved by elevating the temperature to 42°C and incubation 3 h. Strains containing hybrids were grown at 37°C. ~re~aratia~ of virus DNA Virus was propagated and viral DNA prepared as described (Ronshoh et al., 1987; Christensen and Normann, 1992). Ligation of DNA fragments
previously
and transformation
DNA fragments were gel-purified as described previously (Christensen, 1988) and ligated to pSiig1 with T4 DNA ligase, supplied by New England BioLabs and according to the recommendations of the manufacturer. Ligation
280
mixtures
were transformed
Preparation digests
by the procedure
described
of plasmid DNA, restriction endonuclease
by Davis et al. (1980). digestion, and analvses oj
Small scale preparation of plasmid DNA was done as described by Holmes and Quigley (1981). Restriction endonucleases were provided by New England BioLabs and used in accordance with the recommendations of the manufacturer. Digests of plasmids and viral DNA were analyzed by agarose gel electrophoresis.
Result and Discussion A plasmid deleted of the 2 minor Hind111 fragments of pOU57 was identified. The identity of this plasmid designated pSiig1 was confirmed by digestion with PstI and BgflI (not shown). The size was found to be 5.3 kb. pSiig1 could not be transformed into C600. Among different strains harboring the ~I857 repressor allele, N4830-1 was found to be the most suitable host of pSiig1. At temperatures below 30°C the PR is repressed. At these temperatures the yield of pSiig1 is comparable to the yield of pOU57 reflecting a copy number of 6-8 per chromosome equivalent (Larsen et al., 1984) or 15-20 per cell. As with pOU57 runaway replication of pSiig1 is achieved at temperatures between 39°C and 42°C providing a simple means of amplification of the vector. A genomic bank of BHV-1 was established based on a digest of BHV- 1 DNA with BgflI and religation with BglII cleaved pSiig1 as shown in Fig. 2. In spite of repeated efforts fragment 1 was not cloned, presumably because it is located at one of the termini of the genome. A variety of fragments with sizes in the range of fragment 10 was identified (not shown) indicating that this fragment exhibits hypervariability in the isolate DK540/89. All transformants analyzed were shown to contain foreign DNA in the BgflI site of pSiig1. The size of the inserts varies from 2 kb to 27 kb. Since the basic replicon of Rl has been shown to be sufficiently strong to drive the replication of the E. coli chromosome (Bird et al., 1976. Molin and Nordstrom, 1980) it might be anticipated that the size limit of foreign DNA that can be cloned with pSiig1 is set only by the method of transformation. The vector pSiig1 is suitable as cloning vector for digests with endonucleases compatible with BgflI, and the versatility might be extensitied by modulating the BgflI site with adapters. The replicon of RI including the region of the BgflI site has been sequenced (Stougaard et al., 198 1), thus providing suitable targets for priming of sequencing or amplilication by the polymerase chain reaction.
281
2 3 4 5 6 7 8 9 10 11 DK 5~40/89:BglII Lainkbda:HindIII Fig. 2. A partial genomic bank of BHV-I strain DK540/89. The lanes of the hybrids are numbered according to the designation of the insert. DK540/89. Lane 12: A Hind111 digest of lambda DNA.
Acknowledgements
A sample of the plasmid pOU57 was donated by Dr. J.E.L. Larsen, Dept. of Molecular Biology, Odense University, Denmark. The bovine herpesvirus 1 strain DK540/89 was kindly provided by Dr. W. Palfi, The National Veterinary Laboratory, Copenhagen, Denmark. The technical assistance of Margrethe Nielsen and Preben Normann is appreciated.
References B.J. (1972) Pedigrees of some mutant strains of Escherichia coli K-12. Bact. Rev. 36, 525-557. Bertani, G. (1951) Studies on lysogenesis, I: The mode of phage liberation by lysogenic Escherichiu co/i. J. Bacterial. 62, 2933300. Bird, R.E., Chandler, M. and Caro, L. (1976) Suppression of an Escherichia coli dnaA mutation by the integrated R factor R.lO0.1: change of chromosome replication origin in synchronized cultures. J. Bacterial. 126, 1215-1223. Christensen, L.S. (1988) Comparison by restriction fragment pattern analyses and molecular characterization of some European isolates of Suid herpesvirus I: A contribution to strain differentiation of European isolates. Arch. Virol. 102, 3947. Christensen, L.S. and Normann, P. (1992) A simple method for purification of herpesvirus DNA. J. Virol. Meth. In press. Davis, R.W., Botstein, D. and Roth, J.R. (1980) A manual for genetic engineering: Advanced bacterial genetics. Cold Spring Harbor Laboratory, New York. Dempsey, W.B. and Willets, N.S. (1976) Plasmid cointegrates of prophage lambda and R factor RIOO. J. Bacterial. 143, 166-176. Gottesman et al. (1980) Transcriptional antitermination by bacteriophage lambda N gene product. J. Mol. Biol. 140, 57-75. Holmes, D.S. and Quigley, M. (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114, 193-197. Bachmann,
282 Kollek, R., Oertel, W. and Goebel, W. (1978) Isolation and characterization of the minimal fragment required for autonomous replication of a copy mutant (pKN102) of the antibiotic resistance factor RI. Molec. Gen. Genet. 162, 51-58. Larsen, J.E.L., Gerdes, K.. Light, J. and Molin, S. (1984) Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promotor. Gene 28, 45-54. Misra, V., Babiuk, L.A. and Darcel. C. Le Q. (1983) Analysis of bovine herpesvirus-type I isolates by restriction endonuclease fingerprinting. Arch. Virol. 76, 341-354. Molin. S. and Nordstrom, K. (1980) Control of plasmid Rl replication: function involved in replication, copy number control, incompatibility, and switch-off of replication. J. Bacterial. 141. I I I-120. Molin, S., Stougaard, P., Uhlin. B.E., Gustafsson, P. and Nordstrom, K. (1979) Clustering of genes involved in replication. incompatibility and stable maintenance of the resistance plasmid Rldrd19. J. Bacterial. 138, 70-79. Nordstrom, K.. Molin, S. and Light, J. (1984) Control of replication of bacterial plasmids: genetics, molecular biology and physiology of the plasmid RI system. Plasmid 12, 71-90. Ronsholt, L., Christensen. L.S. and Bitsch, V. (1987) Latent herpesvirus infection in Red deer: Characterization of a specific deer herpesvirus including comparison of genomic restriction fragment patterns. Actd Vet. Stand. 28, 23-31. Stougaard. P., Molin. S., Nordstrom. K. and Hansen, F.G. (1981) The nucleotide sequence of the replication control region of the resistance plasmid Rldrd-19. Molec. Genet. Genet. 181, 1l6122.
VIRMET
271
(I 992) 277-282 B.V. / All rights
reserved
/ Ol66-0934/92/$05.00
01312
Short Communication
Establishment of a genomic bank of bovine herpesvirus 1 using a novel positive selection plasmid vector L.S. Christensen State
Veterinary Institute for
and M. Boye
Virus Research,
(Accepted
6 December
Lindholm. Kalvehave, Denmark 1991)
Summary
A small positive selection cloning vector, designated pSiig1, suitable for the construction of genomic banks in E. coli is described and used for the establishment of a bank of the bovine herpesvirus 1 genome. Hybrid transformants are directly selected on agar plates containing ampicillin. The vector is based on the replicon of Rl and has a lambda PR promotor inserted upstream of the replication control genes. The vector has an uncontrolled (runaway) replication and is lethal to the host cell unless the PR promotor is brought under trans-acting control of the lambda cI repressor or runaway replication is blocked by an insertion between the PR promotor and the replicon. The vector contains a unique BgZII site between Pn and the replicon which is suitable for insertion of genomic DNA. Cloning vector; pSiig1; Genomic bank construction;
Bovine herpesvirus 1
Introduction
Plasmid vectors are widely used for the establishment of genomic banks in bacteria. Among the commonly used principles for selection of hybrid transformants are insertional inactivation of genes coding for antibiotic resistance or metabolic functions. However, using such vectors, screening for hybrids requires either replica plating on selective plates or complex agar Correspondence to: L. Siig Christensen. At present address: Institute for Medical Microbiology, Building 24-2. DK-2200 Copenhagen N, Denmark.
University
of Copenhagen,
Panum
Institute
278
media. The vector pSiig1 described in the present report provide the simplest possible selection of hybrids directly on agar plates with ampicillin (Ap). pSiig1 is based on the replicon of the resistance plasmid Rl . This replicon is comprised within a 2.5 kb region (Kollek et al., 1978; Molin et al., 1979) and is well characterized (for a review, see Nordstrom et al., 1984). Based on the knowledge of the replication control system of this replicon a series of vectors were constructed, where a DNA fragment containing the lambda Pa promotor and the temperature sensitive ~1857 allele of the repressor gene was inserted upstream of the replicon (Larsen et al., 1984). In these vectors runaway replication is induced by Pa when the repressor is inactivated at increased temperatures. In pSiig1 this principle is applied in a modified version. pSiig1 does not contain the ~1857 repressor and can only be harbored in a host strain containing the repressor. Hybrids in which runaway replication is blocked by the insert can be selected in a suitable host not containing the repressor.
Materials and Methods Bacterial strains Three derivatives of Escherichia coli K-12 were used. N4830-1 (F-, su-, his-, ilv-, galKd8, d(ch1 D-pgl), [/2, ABarn, N +, cI857dHll) (Gottesman et al., 1980) was used as host for pSiig1. C600 (F-, thr-1, leuB6, thi-1, supE44) (Bachmann, 1972) was used as host for the parental plasmid pOU57. MT102 (MC1000 hsd RK12) (M. Trier Hansen) was used as recipient in selection of hybrid transformants. Virus strain A Danish strain of bovine herpesvirus 1 was used as the source of the genomic bank. The strain DK540/89 was isolated from a nasal swab of a cow during an outbreak of rhinotracheitis. DK540/89 was categorized as type I (Cooper-type, IBR-like) according to Misra et al. (1983) by digestion of viral DNA with HilrdIII and EcoRI. Construction
of pSiig1
The parental plasmid pOU57 (Larsen et al., 1984) contains the bla gene of tn3 and a region originating from the bacteriophage lambda strain ED//4 (Dempsey and Willetts, 1976) comprising ~1857 and Pa upstream of the replicon of Rl as shown in Fig. 1. The replicon is deleted of a 0.5 kb BgnI fragment containing the constitutive promotor and the promotor-proximal part of the copB gene. pOU57 was digested with Hind111 and religated. The ligation mixture was transformed into N4830- 1, and a plasmid deleted of the 2 minor Hind111 fragments was identified.
279
!I ~ B
‘3
b.4
Basic replicon
n S
._ 4,
4
4
b 4
I I 1 S,
HC
pou57
tiEx
!
1.
Digestion with Hindl~l,re~iga~ion
t t
H
ä
4
a
'P S2
I P
S
pSiig1
P Sl
Fig. 1. Genetic and physical map of the basic replicon of plasmid RI, the parental vector ~01157, and pSiigl_ In the basic replicon copB and copA encode products that control the expression of the repA gene product, which is required for initiation of replication at ori (cf. Nordstrom et al., 1984). bla represent the /& lactamase gene mediating resistance to penicillin. b indicate the localization of promoters essential for replication or replication control. Pa is the rightward promotor of lambda being under control of the temperature sensitive repressor mutant ~1857. Restriction sites: B 1 = BarnHI, B2 = &$I, C = CM, E = EcoRI, H = HindIII, P .= f’stl, S = SalI.
Media and growth conditions
The bacteria were grown in LB medium (Bertani, 1951) and on LA plates containing 1.5% agar. Selective pressure was achieved by supplementing with Ap (Doktacillin, Astra) to a final concentration of 50 pg/ml. Strains harboring pOU57 or pSiig1 were grown at 30°C and amplification of the plasmids was achieved by elevating the temperature to 42°C and incubation 3 h. Strains containing hybrids were grown at 37°C. ~re~aratia~ of virus DNA Virus was propagated and viral DNA prepared as described (Ronshoh et al., 1987; Christensen and Normann, 1992). Ligation of DNA fragments
previously
and transformation
DNA fragments were gel-purified as described previously (Christensen, 1988) and ligated to pSiig1 with T4 DNA ligase, supplied by New England BioLabs and according to the recommendations of the manufacturer. Ligation
280
mixtures
were transformed
Preparation digests
by the procedure
described
of plasmid DNA, restriction endonuclease
by Davis et al. (1980). digestion, and analvses oj
Small scale preparation of plasmid DNA was done as described by Holmes and Quigley (1981). Restriction endonucleases were provided by New England BioLabs and used in accordance with the recommendations of the manufacturer. Digests of plasmids and viral DNA were analyzed by agarose gel electrophoresis.
Result and Discussion A plasmid deleted of the 2 minor Hind111 fragments of pOU57 was identified. The identity of this plasmid designated pSiig1 was confirmed by digestion with PstI and BgflI (not shown). The size was found to be 5.3 kb. pSiig1 could not be transformed into C600. Among different strains harboring the ~I857 repressor allele, N4830-1 was found to be the most suitable host of pSiig1. At temperatures below 30°C the PR is repressed. At these temperatures the yield of pSiig1 is comparable to the yield of pOU57 reflecting a copy number of 6-8 per chromosome equivalent (Larsen et al., 1984) or 15-20 per cell. As with pOU57 runaway replication of pSiig1 is achieved at temperatures between 39°C and 42°C providing a simple means of amplification of the vector. A genomic bank of BHV-1 was established based on a digest of BHV- 1 DNA with BgflI and religation with BglII cleaved pSiig1 as shown in Fig. 2. In spite of repeated efforts fragment 1 was not cloned, presumably because it is located at one of the termini of the genome. A variety of fragments with sizes in the range of fragment 10 was identified (not shown) indicating that this fragment exhibits hypervariability in the isolate DK540/89. All transformants analyzed were shown to contain foreign DNA in the BgflI site of pSiig1. The size of the inserts varies from 2 kb to 27 kb. Since the basic replicon of Rl has been shown to be sufficiently strong to drive the replication of the E. coli chromosome (Bird et al., 1976. Molin and Nordstrom, 1980) it might be anticipated that the size limit of foreign DNA that can be cloned with pSiig1 is set only by the method of transformation. The vector pSiig1 is suitable as cloning vector for digests with endonucleases compatible with BgflI, and the versatility might be extensitied by modulating the BgflI site with adapters. The replicon of RI including the region of the BgflI site has been sequenced (Stougaard et al., 198 1), thus providing suitable targets for priming of sequencing or amplilication by the polymerase chain reaction.
281
2 3 4 5 6 7 8 9 10 11 DK 5~40/89:BglII Lainkbda:HindIII Fig. 2. A partial genomic bank of BHV-I strain DK540/89. The lanes of the hybrids are numbered according to the designation of the insert. DK540/89. Lane 12: A Hind111 digest of lambda DNA.
Acknowledgements
A sample of the plasmid pOU57 was donated by Dr. J.E.L. Larsen, Dept. of Molecular Biology, Odense University, Denmark. The bovine herpesvirus 1 strain DK540/89 was kindly provided by Dr. W. Palfi, The National Veterinary Laboratory, Copenhagen, Denmark. The technical assistance of Margrethe Nielsen and Preben Normann is appreciated.
References B.J. (1972) Pedigrees of some mutant strains of Escherichia coli K-12. Bact. Rev. 36, 525-557. Bertani, G. (1951) Studies on lysogenesis, I: The mode of phage liberation by lysogenic Escherichiu co/i. J. Bacterial. 62, 2933300. Bird, R.E., Chandler, M. and Caro, L. (1976) Suppression of an Escherichia coli dnaA mutation by the integrated R factor R.lO0.1: change of chromosome replication origin in synchronized cultures. J. Bacterial. 126, 1215-1223. Christensen, L.S. (1988) Comparison by restriction fragment pattern analyses and molecular characterization of some European isolates of Suid herpesvirus I: A contribution to strain differentiation of European isolates. Arch. Virol. 102, 3947. Christensen, L.S. and Normann, P. (1992) A simple method for purification of herpesvirus DNA. J. Virol. Meth. In press. Davis, R.W., Botstein, D. and Roth, J.R. (1980) A manual for genetic engineering: Advanced bacterial genetics. Cold Spring Harbor Laboratory, New York. Dempsey, W.B. and Willets, N.S. (1976) Plasmid cointegrates of prophage lambda and R factor RIOO. J. Bacterial. 143, 166-176. Gottesman et al. (1980) Transcriptional antitermination by bacteriophage lambda N gene product. J. Mol. Biol. 140, 57-75. Holmes, D.S. and Quigley, M. (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114, 193-197. Bachmann,
282 Kollek, R., Oertel, W. and Goebel, W. (1978) Isolation and characterization of the minimal fragment required for autonomous replication of a copy mutant (pKN102) of the antibiotic resistance factor RI. Molec. Gen. Genet. 162, 51-58. Larsen, J.E.L., Gerdes, K.. Light, J. and Molin, S. (1984) Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promotor. Gene 28, 45-54. Misra, V., Babiuk, L.A. and Darcel. C. Le Q. (1983) Analysis of bovine herpesvirus-type I isolates by restriction endonuclease fingerprinting. Arch. Virol. 76, 341-354. Molin. S. and Nordstrom, K. (1980) Control of plasmid Rl replication: function involved in replication, copy number control, incompatibility, and switch-off of replication. J. Bacterial. 141. I I I-120. Molin, S., Stougaard, P., Uhlin. B.E., Gustafsson, P. and Nordstrom, K. (1979) Clustering of genes involved in replication. incompatibility and stable maintenance of the resistance plasmid Rldrd19. J. Bacterial. 138, 70-79. Nordstrom, K.. Molin, S. and Light, J. (1984) Control of replication of bacterial plasmids: genetics, molecular biology and physiology of the plasmid RI system. Plasmid 12, 71-90. Ronsholt, L., Christensen. L.S. and Bitsch, V. (1987) Latent herpesvirus infection in Red deer: Characterization of a specific deer herpesvirus including comparison of genomic restriction fragment patterns. Actd Vet. Stand. 28, 23-31. Stougaard. P., Molin. S., Nordstrom. K. and Hansen, F.G. (1981) The nucleotide sequence of the replication control region of the resistance plasmid Rldrd-19. Molec. Genet. Genet. 181, 1l6122.
