Jul 17, 1981 - ABSTRACT Genomic DNA sequences of equine herpesvirus type 1 (EHV-1) ... DI particles and were cloned as Bgl II and EcoRI fragments into.
Proc. Natl Acad. Sci. USA Vol. 78, No. 11, pp. 6684-6688, November 1981
Biochemistry
Molecular cloning of equine herpesvirus type 1 DNA: Analysis of standard and defective viral genomes and viral sequences in oncogenically transformed cells (herpesvirus oncogenesis/defective interfering particles/integrated viral DNA)
ROBIN A. ROBINSON*, PHILIP W. TUCKERt, STEVEN A. DAUENHAUER*, AND DENNIS J. O'CALLAGHAN* Departments of *Microbiology and tBiochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216
Communicated by Bernard Roizman, July 17, 1981
ABSTRACT Genomic DNA sequences of equine herpesvirus type 1 (EHV-1) have been cloned as BamHI and EcoRI restriction fragments into the plasmid pBR322 and propagated in Escherichia coli. With the exception of two EcoRI restriction fragments that reside in the S region of the viral genome, all of the cloned fragments demonstrated the same electrophoretic mobilities, restriction cleavage sites, and blot-hybridization patterns as did the parent fragments produced by BamHI or EcoRI digestion of virion DNA. The EcoRI J fragment and the BamHI E fragment of the L-region terminus were cloned after the addition of appropriate linker oligonucleotides. Fragments originating from each of the two isomeric forms of EHV-1 DNA were contained in this library of clones. Supramolar DNA fragments present only in the DNA of defective interfering (DI) particles of EHV-1 were generated from Bgl II digestion of DNA preparations enriched for EHV-1 DI particles and were cloned as Bgl II and EcoRI fragments into the plasmid vector. The cloned viral sequences represented in this defective genome mapped to the S region of EHV-1 DNA. Blothybridization analysis of EHV-1 transformed and tumor cell DNAs with the cloned BamHI B fragment confirmed that subgenomic viral sequences are present and indicated that those sequences map to the viral genome between 0.32 and 0.43 map unit.
and demonstrate the utilization of cloned viral DNA fragments to identify EHV-1 DNA sequences in oncogenically transformed and tumor cells. MATERIALS AND METHODS Cells, Virus, and DNA. Procedures have been described for isolation of viral DNA (2, 8, 9), generation of DI particles (4, 5) and monitoring for DI particles by density analysis (4), and restriction enzyme analysis of viral DNA (1). EHV-1 DI-particle DNA was obtained from the supramolar fragment of Bgl II digests of DI-particle preparations (1). Plasmid pBR322 DNA (10) was extracted from large preparations of bacteria by the technique of Guerry et aL (11) and was banded by centrifugation in CsCl/ethidium bromide gradients (12). Cell DNA was isolated and purified from normal, transformed, and tumor cells as described (6). Construction ofRecombinant Plasmids and Bacterial Transformation. Methods for restriction enzyme digestions, separation of DNA fragments by agarose gel electrophoresis, and isolation of individual DNA fragments have been described (1, 2). EHV-1 and pBR322 (13) DNAs were digested separately to completion with BamHI or EcoRI. Restriction digests of EHV1 genomic DNA (1 ,.g) or individual EHV-1 DNA restriction fragments (0.5 ,ug) were ligated overnight at 40C to alkaline phosphatase-treated (14) pBR322 DNA (0.2 ug) with 2 units of T4 DNA ligase in 66 mM Tris-HCl, pH 7.5/6.6 mM MgCl2/ 10 mM dithiothreitoV0.4 M ATP (total reaction volume, 20 Al) (10). Escherichia coli strains C600SF8 and HB101 (15) were transformed with the ligated DNAs by the calcium-shock method (16). A transformation efficiency of 160,000 colonies per Ag of ligated DNA was obtained. Bacteria carrying plasmids with inserted EcoRI fragments were selected and screened for resistance to both ampicillin and tetracycline; bacteria having plasmids with inserted BamHI fragments were selected and screened for sensitivity to tetracycline and resistance to ampicillin. The terminal sequences of the EHV-1 L region were cloned by the introduction of chemically synthesized restriction enzyme recognition-site sequences (17) to the noncohesive ends of the terminal restriction fragments by the method of Maniatis et aL (18). Colony Hybridization. Bacterial colonies were screened by the replica plate method of Grunstein and Hogness (19). Briefly, the colonies were transferred to nitrocellulose discs, which were placed in denaturing solution for 10 min, neutralized, air-dried, and baked at 80'C for 2 hr in vacuo. The filters were prehybridized at 650C for 4-8 hr in a modified Denhardt's solution
The EHV41 genome is a 92-megadalton (MDal) DNA molecule comprised of L and S covalently linked regions. Only the S region contains unique sequences flanked by inverted repetitive sequences (1) which allow the genome to exist in two isomeric forms (2). Upon repeated, high-multiplicity passage of EHV-1 in animals (3) or cell culture (4, 5), defective interfering (DI) particles, whose genome is comprised of tandem repeat units originating from the S region (1, 4), emerge. One striking consequence of the interference activity ofthese DI particles is that infection of permissive hamster embryo cells with viable preparations of these defective particles results in oncogenic transformation and establishment of persistent infection (1, 6). Because DNA sequences located in the L region appear to be associated with EHV-1 oncogensis (7), we have proposed a model in which these herpesvirus DI particles mediate oncogenic transformation indirectly by tempering cytocidal viral functions and thereby allowing oncogenic DNA sequences to be dominantly expressed (6). One approach for providing ample quantities of viral DNA sequences necessary for biochemical analyses of virus-transformed cells is the insertion of viral DNA restriction enzyme fragments by recombinant DNA methods into plasmids for subsequent propagation in bacteria. In this report we describe the isolation and characteristics of cloned plasmids containing restriction fragments of EHVr1 standard and defective genomes The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. ยง1734 solely to indicate this fact.
Abbreviations: EHV-1, equine herpesvirus type 1; DI, defective interfering; Mdal, megadaltons. 6684
Biochemistry: Robinson et aL
Proc. NatL Acad. Sci. USA 78 (1981)
L
6685
S | w Us Tw
JO GPKL F 1 U2 62 'W'4.2 8.2 4.9k~~~~~~~6.2 i~~~~~~~~~7~~~I4~~~~I 8.2
EcoR:
HQI N I d62 It 62 '27' I27i 12.4
A
'6.22
23.4
4.9
IC
.7
Bgl
E MI DIQK' 2. 6.6
NF
14
6723 3
G
M
1L1J J t4 8 |
C
9.2 X.1A Y 63
431 5.1
6
G L K
t5'
F II' H II3j4 3.
A
E
61
e
J
MO
As 24.5
1
12.9 E , H J.N. D 6.2 4.3 4.03.2 7.2
BamH
F
B
G
5.4
9.8
4.6
A 13.7
iMs '3.47 9.1
ISi N Ci 12.9i+ I Be N Ca
D
.7 8&3
i
P
D
A4 *
4.4
E .
24.5
a
XbaI
M 3
'_
1 J 7t
Ci PzOM.i, L PiQi 28 i 9g6@ b 3.&334 Ca Pi L M OPaQa
I ,K 41 3.6
96
i 3.4'&3433.1 .U 2.8
Map Units I
0.0
I
I
0.1
I
I
0.2
I
I
0.3
I
I
0.4
I
I
0.5
I
I
I
Q6
(1) and then were incubated at 650C overnight in sealed plastic bags containing 106 cpm/ml of32P-labeled EHV-1 DNA or fragments (20), 20% (vol/vol) formamide, and hybridization buffer. The filters were washed extensively (1), air dried, and exposed to Kodak X-Omat XR-1 film with intensifying screens for autoradiography. Blot Hybridization with Plasmids to EHV-1 Virion DNA and to EHV-1 Transformed- and Tumor-Cell DNA. Plasmid DNA (13) 32P-labeled by nick-translation (20) was hybridized to EHV1 DNA fragments immobilized on nitrocellulose as described (1). Blot hybridizations or recombinant plasmid DNA (1-2 108 cpm/,ug) to nitrocellulose blots of restriction enzyme digests of cell DNAs were performed under conditions described elsewhere (1, 21). X
RESULTS Cloning of BamHI and EcoRI Fragments. Unfractionated BamHI and EcoRI fragments (Fig. 1) of virion DNA and individual BamHI and EcoRI fragments purified from agarose gels were ligated to BamHI- or EcoRI-digested pBR322 DNA and used to transform competent E. coli. Ampicillin- and tetracycline-resistant transformants were obtained from bacteria transformed with plasmids containing EHV-1 EcoRI fragments. Ampicillin-resistant and tetracycline-sensitive transformants were obtained from bacteria transformed with plasmids containing EHV-1 BamHI fragments. Transformants transferred to nitrocellulose filters by a replica plating method were screened for positive colonies by hybridization with 32P-labeled purified EHV-1 DNA restriction fragments. Antibiotic-resistant transformants that demonstrated positive hybridization were picked and grown in small suspension cultures to provide crude DNA lysate preparations for electrophoretic analysis. The DNA preparations from each isolated clone were digested with BamHI and EcoRI, and these digests, as well as BamHI and EcoRI digests of EHV-1 DNA, were analyzed by gel electrophoresis (Figs. 2 and 3). Upon cleavage of the recombinant DNAs with these restriction enzymes, the linear plasmid vector DNA and the BamHIor EcoRI-inserted viral DNA fragments were observed. The electrophoretic mobilities of most of the viral DNA fragments were identical to those of fractionated digests of virion DNA. However, two notable exceptions were the plasmids containing the EcoRI B and C fragments, which had molecular masses that
Il* I
I
0.7
Q8
I
I
09
4I
1.0
FIG. 1. Restriction enzyme map of the genome of EHV-1. Reproduced from ref 2 with permission from Academic Press.
2.4 and 3.1 MDal, respectively, in excess ofthe molecular observed for the same fragments in virion DNA digests (Fig. 3, lanes 3 and 4). Cloning of L-Region Terminal Sequences. Because fragments containing the internal repeat unit of the S region were cloned, it was deemed unnecessary for our purposes to isolate clones containing the S-region terminal repeat sequences. However, sequences present in the L-region terminus are not present internally as a repeat unit (2); therefore, these sequences were cloned by the addition of oligonucleotide linkers (containing restriction enzyme recognition sites) to EHV-1 Lregion terminal fragments EcoRI J and BamHI E. The dodecameric DNA molecules containing the EcoRI [d(5'-G-G-C-AA-T-T-C-G-G-3')] and the BamHI [d(5'-C-C-G-G-A-T-C-C-Gwere
masses
FIG. 2. Electrophoretic analysis of recombinant DNAs containing EHV-1 BamHI fragments. Recombinant DNAs (lanes 2-12) and standard EHV-1 DNA (lane 1) were digested to completion with BamHI, fractionated by electrophoresis through 0.9% agarose gels, stained with ethidium bromide, and visualized by UV illumination. The recombinant DNAs were isolated from positive bacterial colonies transformed by the ligation products of pBR322 and EHV-1 BamHI fragments. The heavy-staining band with a molecular mass of 2.5 MDal present in all recombinant DNAs is the linearized plasmid DNA molecule. Lanes: 2, pD01-031 (containing BamHI A fragment); 3, pD01032 (BamHl B); 4, pD01-033 (BamHI C); 5, pD01-034 (BamHI D); 6, pD01-037 (BamHI F); 7, pD01-038 (BamHI G); 8, pD01-039 (BamHI
H); 9, pD01-040 (BamHI I); 10, pD01-041 (BamHI J); 11, pD01-042 (BamHI K); 12, pD01-043 (BamHI L).
Proc. Nad Acad. Sci. USA 78 (1981)
Biochemistry: Robinson et aL
6686
1 2 34
FIG. 4. Electrophoretic analysis of recombinant DNAs containing L-region terminal sequences of EHV-1 DNA. EHV-1 DNA and recombinant DNA from plasmids prepared as described in the text were digested with EcoRP or Bamland analyzed as in Fig. 2. Lanes: 1, EcoRP digest of EHV-1 DNA; 2, BamHI digest of EHV-1 DNA; 3, EcoRP digest of pD01-150 [containing EcoRI J fragment); 4, BamHI digest of pD01-036 (BamHI E).
FIG. 3. Electrophoretic analysis of recombinant DNAs containing EHV-1 EcoRI fragments. EHV-1 DNA (lane 1) and recombinant DNA (lanes 2-13) were digested to completion with EcoRI and analyzed as described in the legend to Fig. 2. The recombinant DNAs were isolated from positive bacterial colonies transformed by the ligation products of pBR322 and EHV-1 EcoRI fragments. The 2-MDal heavy-staining band present in all of the recombinant DNAs is the linearized plasmid DNA molecule. Lanes: 2, pD01-141 (containing EcoRI A fiagment); 3, pD01-142 (EcoRI B); 4, pD01-143 (EcoRI C); 5, pD01-146 (EcoRI F); 6, pD01-147 (EcoRI G); 7, pD01-148 (EcoRI H); 8, pD01-149 (EcoRI I); 9, pD01-151 (EcoRI K); lane 10, pD01-152 (EcoRI L; 11, pD01-153 (EcoRI M); 12, pD01-155 (EcoRI N); 13, pD01-156 (EcoRI 0).
quences are shown in Fig. 4. The electrophoretic mobilities of the cloned terminal fragment inserts in agarose gels were comparable to those observed for the same fragments in restriction digests of virion DNA. Mapping of Restriction Fragments from Genomic Digests. The map positions of cloned fragments from genomic EHV-1 DNA digests were determined by blot-hybridization methods. EHV-1 DNA was digested separately with EcoRI, Xba I, Bgl II, or BamHI and was electrophoresed in agarose gels. These enzymes were chosen because restriction cleavage maps of EHV-1 DNA with these enzymes have been constructed (1, 2). The fractionated fragments were transferred to nitrocellulose filters by the method of Southern (21), and these blots were hybridized to recombinant DNAs 32P-labeled by nick-translation. Results from several of these hybridizations (Fig. 5) confirmed that the inserts of these recombinant plasmids were of viral origin and identified the viral sequences that were shared among various restriction enzyme fragments. These data were in good agreement with previous mapping studies of EcoRI, Xba I, Bgl II, and BamHI fragments of EHV-1 DNA (1, 2).
G-3')] restriction sites were phosphorylated with T4 kinase to add phosphate groups at the 5' end of the linkers. The phosphorylated linkers were blunt-end ligated to the corresponding EHV-1 DNA fragments purified from agarose gels, and then these molecules were cleaved with the appropriate restriction enzyme to produce fragments with cohesive termini that were joined to the complementary ends of pBR322 DNA. Prior to ligation to the vector DNA, restriction fragments with the newly acquired linkers were sedimented through a 10-40% sucrose gradient to remove unincorporated linker molecules that would compete for the binding sites of restriction enzymes. The results of an electrophoretic analysis of EcoRI and BamHI digests of DNA from isolated clones containing L-region terminal sepDOI-032
pDQI-031 1
2
3
A 4
1 2 3
4
A
A
13.7 _-.o.
Z2
6.2
--w
---o
5$4
B
D
ED
12 3
2 3 4
B *- C
am
46
H
a-
a-
-E
E G
4.6 4.3 = 4.1
K.
I
-
H 3.6 3.4 = 3.1
28
-
-.
4
A
4 B B.
d*
9.8
pDOI-034
pDOI-033 1
4
Q
*
FIG. 5. Blot hybridization of recombinant plasmids to EHV-1 DNA. Autoradiograms of gels containing EcoRI, Xba I, Bgll , andBam HI digests of EHV-1 DNA (designated as lanes 1, 2, 3, and 4, respectively) after hybridization with 32P-labeled recombinant DNAs as probes. The EHV-1 DNA restriction enzyme digests were transferred to nitrocellulose filters after fractionation on 0.7% agarose gels. The plasmids used for this experiment were pD01-031 (containingBamHI A frag-
ment), pD01-032 (BamHI B), pD01-033 (BamHI C), and pD01-034 (BamHI D). BamHI-digested EHV-1 DNA gave the molecular size markers shown on the left in MDal.
Biochemistry: Robinson et at
Cloning of the Defective Genome. The defective genome of EHV-1 DNA has been suggested to be comprised of several tandem repeat units of approximately 20 MDal in size (4). The viral DNA sequences represented within these repeat units have been located within the S region of the EHV-1 genome (unpublished data). It was our intention to clone a single repeat unit of the defective genome to understand fully the nature of EHV-1 DI particles. A preparation of EHV-1 DNA enriched for defective DNA as judged by the absence of certain restriction fragments in fractionated restriction digests of this DNA and by the overabundance of newly formed restriction fragments within these digests is shown in Fig. 6 (lanes 1 and 2). The Bgl II supramolar fragment, unique to defective DNA (1), was isolated from the gel and ligated to the BamHI sites of pBR322. Transformants containing the recombinant DNA were screened with 32P-labeled EHV-1 DNA. Cloned isolates were examined by electrophoretic analysis of EcoRI digests of the recombinant DNA (Fig. 6, lane 3). The linear vector DNA was observed to have a molecular mass greater than normal, and three fragments with molecular masses of 12.2, 10.4, and 3.4 M Dal were present in these digests. Each ofthe defective DNA fragments generated with EcoRI were isolated and recloned into the plasmid vector. Hybridization of 3P-labeled recombinant DNAs containing defective viral DNA to blots of standard EHV-1 DNA restriction enzyme digests revealed that the DNA sequences comprising this DI-particle species mapped only within the S region and the terminus of the L region ofthe standard EHV-1 genome. Blot Hybridization of EHV-1 Transformed- and Tumor-Cell DNAs with the BamHI B Cloned Fragment. Hamster embryo cells transformed by UV-irradiated EHV-1 (22) or live virus preparations enriched for EHV-1 DI particles (6) have been demonstrated to contain one or more copies per diploid genome of subgenomic viral sequences. The hybridization probes used in the previous reassociation kinetic studies were 32P-labeled EHV-1 genomic DNA that lacked the specificity necessary to
Proc. Natd Acad. Sci. USA 78 (1981)
6687
detect single copy viral genes in restricted cell DNAs by blothybridization analyses. Therefore, the EHV-1 restriction fragment recombinants provided a source of viral DNA for hybridization probes to fine map those viral sequences present in the transformed and tumor cells. To discern the identity of these viral sequences, initial experiments involved the hybridization of the 32P-labeled BamHI B cloned fragment (0.32 to 0.43 map units) to blots containing Bgl II digests of several EHV-1 transformed- and tumor-cell DNAs. The results of typical hybridization experiments (Fig. 7) are summarized here. The hybridization conditions used in these experiments were demonstrated to be sufficient to detect a single copy of the BamHI B fragment at the level of one copy per diploid genome (see reconstruction experiment, lane 2). Fragments sharing various degrees of homology with this viral fragment were evident within the Bgl II digests from all of these EHV-1 transformed- and tumor-cell DNAs. The detection of these viral sequences in fragments with molecular weights in excess of the probe DNA indicated the covalent linkage of viral and cellular DNA sequences. The distribution patterns of Bgl II fragments containing viral sequences differed among the transformed-cell DNAs examined in this experiment and suggested that these viral sequences represent either random integration or multiple integration at specific cellular sites by specific herpesvirus DNA sequences within the host genome. Viral sequences homologous to the BamHI B fragment were not detectable in the restriction digests of normal hamster DNA. DISCUSSION In this report, we described the construction of recombinant plasmids containing EHV-1 DNA restriction fragments whose 2
12
3 4 4
5
6
7
8
9 1011
0.
24.5
-
9.2 _ 7.3 _ 66_6 51
0 *
AM
4*
.,#
4.
so~~* -~~ ~~~~. 4o
_
4.3
24
FIG. 6. Electrophoretic analysis of recombinant plasmids containing the EHV-1 defective genome. EHV-1 DNA and recombinant DNAs were digested with BamHI or EcoRI and analyzed as described in the legend to Fig. 2. Lanes: 1, Bgl II digest of EHV-1 DNA: 2, BamHIdigested pD01-818 (containing Bgl II supramolar fragment of DI-particle-enriched infection); 3, EcoRI digest of pD01-818; 4, EcoRI digest of pD01-846 (recloned 12.2-MDal fragment from pD01-818); 5, pDO1848 (recloned 10.4-MDal fiagment from pD01-818); 6, pD01-850 (recloned 3.4-MDal fiagment from pD01-818); 7, Bgi II digest of a preparation of EHV-1 DNA enriched for DI particles (65%); 8, EcoRI digest of EHV-1 DNA.
_
FIG. 7. IntegrationpatternsofEHV-1 DNAinEHV-1 transformed hamster cells and progeny tumor cell lines. EHV-1 DNA or cell DNAs (20 ,ug) indicated were digested to completion with Bgi II. The BamHI B cloned fragment (9.8 MDal; 48.8 pg) was mixed with 20 lug of hamster embryo DNA (lane 2). Fragments were fractionated by electrophoresis in 0.7% agarose gels and transferred to nitrocellulose filters by Southern blotting (21). Virus-specific sequences were detected by hybridization with the plasmid pD1-032 (containing BamHI B fragment; 0.32-0.43 map units) and 32P-labeled by nick-translation, followed by autoradiography. EHV-1 transformed (LSEH) and tumor (LSEH-T) cells established with UV-inactivated standard virus have been described (1, 22); EHV-1 transformed (DI-5 and -10) and tumor (DI-1OT) cells established by infection with viable DI-particle preparations have been described (1, 7). Lanes: 1, Bgi II-digested EHV-1 DNA molecular mass markers in MDal; 2, BamHI-B(IX) cloned fragment with hamster embryo DNA; 3, LSEH-8; 4, LSEH-3; 5, LSEH-4; 6, LSEH-8T; 7, LSEH3T; 8, LSEH-4T; 9, DI-5; 10, DI-10; 11, DI-1OT.
6688
Biochemistry: Robinson et aLPProc. Nad Acad. Sci. USA 78 (1981)
total coding capacity exceeded 95% of the viral genome. The BamHI and EcoRI fragments of standard EHV-1 DNA were inserted at the appropriate restriction sites ofthe E. coli plasmid pBR322. Cloning of the terminal sequences from the EHV-1 L region into recombinant plasmid vectors was facilitated by the covalent linkage of specific oligonucleotide sequences to the BamHI E and EcoRI J fragments. Hybridization of each of the cloned fragments as radioactively labeled probes to blots of various restriction enzyme digests of EHV-1 DNA verified the identity of these viral DNA recombinants and confirmed the location of EHV-1 BamHI fragments. Manifestations of the nucleotide sequence heterogeneity reported earlier in the EHV1 genome resulted in variable molecular sizes for several restriction fragments (1) and may account for some of the minor size discrepancies observed in a few of our cloned fragments (e.g., EcoRI fragments B and C). Alternatively, these additional sequences may have resulted from the duplication and recombination ofplasmid DNA sequences within these recombinants. The defective genome isolated initially as a supramolar Bgl II fragment was cloned. The viral sequences constituting the target DNA insert had a molecular mass of 28.3 MDal and may represent a subunit of the tandem repeats within the EHV-1 defective genome as these DNAs were shown by blot-hybridization analysis to map in the S region and in the terminal sequences of the L region. Whether the EHV-1 DI genome originates from one or both isomers of thie standard virus is not known at present; however, further cloning of DNA from successive passages of EHV-1 DI particles and restriction enzyme mapping of these clones should answer this question. The ability to discern discrete units of integrated herpesvirus DNA sequences within EHV-1 transformed- and tumor-cell genomes was recognized in the blot-hybridization experiment using the cloned BamHI B fragment. These data confirmed the presence of subgenomic viral DNA sequences in these cell lines and supported the conclusion that integration of herpesvirus DNA is associated with oncogenic transformation. Alone, these blot hybridization results do not constitute a formal survey of all possible viral sequences and their arrangements in these cell lines; however, analyses of these cell DNAs with a collection of cloned EHV-1 restriction fragments whose sum coding capacity approximated the entire EHV-1 genome revealed the presence of a consensus, integrated viral sequence mapping from 0.32 to 0.38 that was shared among all EHV-1 transformedand tumor-cell lines (7). The covalent linkage (integration) of EHV-1 'DNA to host sequences has been corroborated by the isolation of recombinant A phage molecules that contain viral and adjoining cellular sequences (unpublished data). Further delineation of herpesvirus gene(s) that initiate and maintain the oncogenic transformed state is possible by using the cloned fragments described here as transfecting inocula in transformation experiments of hamster embryo cells.
This work was supported by National Science Foundation Grant PCM-78-22700, National Institutes of Health Grants AI-02032 and S507-RR05386, and an American Cancer Society and Institutional Grant. 1. O'Callaghan, D. J., Henry, B. E., Wharton, J. H., Dauenhauer, S. A., Vance, R. B., Staczek, J., Atherton, S. A. & Robinson, R. A. (1981) in Herpesvirus DNA: Recent Studies on the Internal Organization and Replication of the Viral Genome, ed. Becker, Y. (Nijhoff, The Hague, Netherlands), in press. 2. Henry, B. E., Robinson, R. A., Dauenhauer, S. A., Atherton, S. S., Hayward, G. S. & O'Callaghan, D. J. (1981) Virology, in press. 3. Campbell, D. E., Kemp, M. C., Perdue, M. L., Randall, C. C. & Gentry, G. A. (1976) Virology 69, 737-750. 4. Henry, B. E., Newcomb, W. W. & O'Callaghan, D. J. (1979) Virology 92, 495-506. 5. Henry, B. E., Newcomb, W. W. & O'Callaghan, D. J. (1980) J. Gen. Virol 47, 343-353. 6. Robinson, R. A., Vance, R. B. & O'Callaghan, D. J. (1980)J. Virol 34, 204-219. 7. Robinson, R. A. & O'Callaghan, D. J. (1981) in Herpesvirus DNA: Recent Studies on the Internal Organization and Replication of the Viral Genome. ed. Becker, Y. (Nijhoff, The Hague, Netherlands) in press. 8. O'Callaghan, D. J., Allen, G. P. & Randall, C. C. (1978) in Equine Infectious Diseases IV, eds. Bryans, J. T. & Gerber, H. (Veterinary Publishers, Princeton, NJ), pp. 1-32. 9. Perdue, M. L., Kemp, M. C., Randall, C. C. & O'Callaghan, D. J. (1974) Virology 59, 201-216. 10. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. D., Heyneker, H. L., Boyer, H. W., Crosa, J. H., & Falkow, S. (1977) Gene 2, 95-113. 11. Guerry, P., LeBlanc, D. J. & Falkow, S. (1973)J. Bacteriot 116, 1064-1066. 12. Clewell, D. B. & Helinski, D. R. (1970) Biochemistry 9, 4428-4440. 13. Meagher, R. B., Tait, R. C., Betlach, M. & Boyer, H. W. (1977) Cell 10, 521-536. 14. Ulrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rutter, W. J. & Goodman, H. M. (1977) Science 196, 1313-1319. 15. Cameron, J. R., Panasenko, S. M., Lehman, I. R. & Davis, R. W. (1975) Proc. Nati Acad. Sci. USA 72, 3416-3420. 16. Mandel, M. & Higa, A. (1970)J. Mol Biol 53, 159-162. 17. Scheller, R. H., Dickerson, R. E., Boyer, H. W., Riggs, A. D. & Itakura, K. (1977) Science 196, 177-180. 18. Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O'Connell, C., Quon, D., Sin, G. K. & Efstratiadis, A. (1978) Cell 15, 687-701. 19. Grunstein, M. & Hogness, D. (1975) Proc. Natl Acad. Sci. USA 72, 3961-3965. 20. Rigby, P. W. S., Dieckman, M., Rhodes, C. & Berg, P. (1977)J. Mol Biol 113, 237-251. 21. Southern, E. M. (1975) J. Mol Biol 98, 503-517. 22. Robinson, R. A., Henry, B. E., Duff, R. C. & O'Callaghan, D.
J. (1980) Virology 101, 335-362.
