Identification, mapping and cloning of the thymidine kinase gene of fish lymphocystis disease virus

63

Virus Research, 9 (1988) 63-12 Elsevier

VRR 00375

Identification, mapping and cloning of the thymidine kinase gene of fish lymphocystis disease virus Jiirgen Scholz, Angela R&en-Wolff, Musa Touray, Paul Schnitzler and Gholamreza Darai Institut ftir Medirinische

Virologie der Universitiit Heidelberg, Heidelberg. F.R.G.

(Accepted

for publication

18 September

1987)

Summary The thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) was identified by biochemical transformation of 3T3 TK negative (TK-) to 3T3 TK positive (TK+) cells using specific viral DNA sequences. DNA fragments of the viral genome used in this study were obtained from a defined gene library of FLDV genome containing the complete viral DNA sequences. The selection of the converted cells was carried out under the condition of the HAT selection procedure. The results of these experiments revealed that the EcoRI FLDV DNA fragment C (11.2 kbp; 0.611 to 0.718 map units) is able to transform 3T3 TK- to 3T3 TK+ cells. Additional experiments using the subclones of EcoRI DNA fragment C revealed that DNA sequences of 4.1 kbp size between the coordinates 0.669 to 0.718 of the FLDV genome possessed the ability for biochemical transformation, indicating that the TK gene locus is located in this particular region. Iridovirus; 3T3 TKtion; Transformation;

Fish lymphocystis disease, a common

cell; Recombinant plasmids; HATG medium; Restriction enzyme; DNA hybridization

transfec-

disease virus (FLDV) is the causative virus for lymphocystis chronic disease of pleuronectes (Fliigel, 1985; Darai, 1986).

Correspondence to: G. Darai, Institut fiir Medizinische heimer Feld 324, 6900 Heidelberg, F.R.G.

0168-1702/88/$03.50

DNA

0 1988 Elsevier Science Publishers

Virologie

der Universitlt

B.V. (Biomedical

Heidelberg,

Division)

Im Neuen-

64 FLDV belongs to the icosahedral cytoplasmic deoxyriboviruses and has been classified as a separate genus of the Iridoviridae family, with the proposed name Cystivirus (Willis, 1985). FLDV has many interesting biological aspects, e.g., the genome structure of FLDV was found to be circularly permuted and terminally redundant (Darai et al., 1983, 1985). The other interesting property of FLDV genome is the high level of methylation in cytosine residues (Darai et al., 1983; Wagner et al., 1985). The characterization of the FLDV genome by molecular cloning and physical mapping has been reported (Darai et al., 1985). Viral components (Darai, 1986) and some virion-associated enzymes have been determined, e.g., adenosine triphosphohydrolase (Fltigel et al., 1982; Fltigel, 1985). In addition, a protein kinase and a thymidine kinase activity were found to be associated with the FLDV (Fhigel, 1985). This supports the previous observation of Aubertin and Longchamps (1974) on the detection of a thymidine kinase activity in TK negative mouse cells after infection with frog virus 3 (FV3). FV3 is another member of the family Iridoviridae which has common properties compared to FLDV, e.g., a similar genomic structure (Goorha and Murti, 1982) and a comparable methylation pattern of cytosine residues (Willis and Granoff, 1980). The identification and cloning of the thymidine kinase gene of FLDV is the subject of this report. In order to determine whether or not the FLDV genome possesses DNA sequences expressing a gene for thymidine kinase (TK) the following strategy was used. The purified virion DNA of FLDV (Darai et al., 1983) was used for transformation of 3T3 cells with TK negative phenotype (3T3 TK-). The cell cultures were grown and propagated in Eagle’s basal medium balanced with Earle’s salt solution supplemented with 10% fetal calf serum (Darai et al., 1980) which contained 30 pg/rnl BrdUrd. The transfection assay was carried out according to the calcium phosphate technique (Stow and Wilkie, 1976) and the DNA of 3T3 TK- cells was used as carrier DNA. In this experiment 10 pg of FLDV DNA, native or cleaved with the restriction endonucleases BumHI, EcoRI, and double digested with BstEII/PstI were coprecipitated with 10 pg carrier DNA and introduced to 0.6 to 1 X lo7 3T3 TK- cells. The transfected cultures were processed under HATG (hypoxanthin, 10e4 M; aminopterin, 4 x low7 M; thymidine, 1.6 x lop5 M; glycine, 10e5 M) selection pressure (Kit et al., 1980, 1981). According to the results of this study, documented in Table 1, it was found that native FLDV DNA or DNA cleaved with EcoRI or double digested with BstEII/PstI was able to convert 3T3 TK- cells to 3T3 TK+ cells. These results are based on the DNA sequence arrangement of the viral genome as determined previously for the restriction endonucleases BarnHI, EcoRI, PstI, and BstEII (Fig. l,A; Darai et al., 1983) and led to the conclusion that the FLDV genome possesses a gene coding for thymidine kinase. This locus has to be located within the the viral DNA sequences that do not have any recognition sites for EcoRI, BstEII, and PstI. For the further mapping of the TK gene locus on the FLDV genome and to ascertain these results, the defined gene library of FLDV, which represents 100% of the DNA sequences of the viral genome (Darai et al., 1985) was screened for the ability to transform 3T3 TK- cells to 3T3 TK+ cells. This gene library, for example,

65 TABLE

1

TRANSFORMATION

OF 3T3 TK-

DNA a Carrier (3T3 TK-

CELLS

TO 3T3 TK+

CELLS

FLD-VIRION

No. of colonies

Treatment native sheared

)

USING

DNA.

b

0

Eco RI

native sheared BumHI BstEII+ Eco RI

FLDV

2- 5 ND 0 9-17 11-23

PstI

’ In each experiment 0.6 to 1 X 10’ cells were transfected carrier DNA. b HATG medium was used for selection. ND = not done.

(calcium

phosphate)

with 10 gg of virion

or

contains all 14 EcoRI FLDV DNA fragments which have been cloned individually into the EcoRI site of the plasmid vector pACYCl84. In these experiments 10 pg of each of the 14 recombinant plasmids were used individually for the transfection

PHYSICAL MAP OF THE GEMGME OF FlSH LYMFHOCYSTIS DISEASE VlRUS

kbP map unit

I 0

J# “/_

c7

c20 &

Fig. 1. Diagram of the restriction map of the EcoRI FLDV DNA fragment C and the map position of the individual subclones established (panel C). Physical maps of the viral genome are given as a circle (A) and linearized (B).

66 TABLE

2

TRANSFORMATION OF 3T3 TKMENTS OF THE VIRAL GENOME. DNA fragment

CELLS

a

AandB C D to M ___--__---_---_--------_-------------_---___--__--__---__--_ pACYC184

TO 3T3 TK+

CELLS

No. of colonies

USING

ECORI

DNA

FRAG-

b

0 18 to 31 0 0

a DNA fragments were obtained from the corresponding recombinant DNA fragments of the viral genome. b Average of four experiments (0.1 pM DNA/l X 10' cells).

plasmids

harboring

the EcoRI

assay which was performed as described above. As shown in Table 2, it was found that the EcoRI FLDV DNA fragment C (11.2 kbp; 0.611-0.718 map units) was able to convert the TK negative phenotype of parental 3T3 cells to TK positive phenotype. This result is in full agreement with the data obtained from the first experiments carried out with the uncleaved or EcoRI-cut viral DNA, since the EcoRI DNA fragment C does not have any recognition sites for PstI and BstEII (Fig. l,A). To determine the exact position of the TK gene locus of FLDV the Hind111 restriction map of the EcoRI DNA fragment C was constructed as shown in Fig. l,C. Subsequently, subclones of this particular region were established in which the EcoRI FLDV DNA fragment C was cleaved partially with the restriction endonuclease Hind111 and molecularly cloned into the corresponding Hind111 or EcoRI/HindIII sites of the plasmid vector pAT153 (Twigg and Sheratt, 1980) as described previously (Darai et al., 1985). The results of this study are summarized in Table 3 and Fig. l,C. According to these data a defined gene library of the EcoRI FLDV DNA fragment C was established, containing ten recombinant plasmids (C3, C5, C7, C16, C19, C20, C22, C23, C27, and C53) that harbor overlapping DNA sequences between the coordinates 0.611 and 0.718 of the viral genome. In the final experiments the capability of these recombinant plasmids to transform 3T3 TK- cells to 3T3 TK+ cells was screened under the same procedure as described above. As shown in Table 3, it was found that only the recombinant plasmid C19, which harbours DNA sequences of the viral genome (4.1 kbp) at the coordinates 0.669 to 0.718, was able to convert the TK negative phenotype of 3T3 cells to TK positive phenotype. In contrast, it was found that no other recombinant plasmids, including C23, C27, and C53 which contain overlapping DNA sequences of this particular region (from genome coordinates 0.669 to 0.675) were able to transform the 3T3 TK- cells. This indicates that the DNA sequences of the TK gene locus of FLDV continue downstream of coordinate 0.675 (a Hind111 site). Thus the TK gene locus of FLDV spans map coordinates from 0.675 to 0.718. The presence of the complete TK gene on the last-mentioned coordinates is not possible, because two other recombinant plasmids C3 and C5 which both harbor the suspected DNA sequences at the genome coordinates 0.680 to 0.718 did not have the ability to

67 TABLE

3

TRANSFORMATION DNA FRAGMENT

OF 3T3 TK-

CELLS

TO 3T3 TK+

CELLS

USING

SUBCLONES

Plasmid

DNA fragment a (kbp) map units

No. of colonies

pyLV17-E-C

EcoRI-C (11.2) 0.611 to 0.718 0.680 to 0.718 0.669 to 0.718 0.669 to 0.675 0.641 to 0.669 0.641 to 0.675 0.621 to 0.675 0.621 to 0.669 0.611 to 0.648 0.621 to 0.641 0.611-0.648 and 0.680-0.718

21

CS c 19 C 27 c 20 C 23 c 53 c7 C 16 c 22 c 3

OF ECORI

C.

a DNA fragments were obtained from the corresponding recombinant DNA fragments of the viral genome. b Average of two experiments (0.1 pM DNA/l X 10’ cells).

b

0 37 0 0 0 0 0 0 0 0 plasmids

harboring

the EcoRI

convert the TK negative phenotype of 3T3 cells to the TK positive phenotype. Consequently, these data indicate that the FLDV TK gene locus is unambiguously located in the right part of the EcoRI DNA fragment C between the map coordinates 0.669 and 0.718 of the FLDV genome. The state of the specific DNA sequences of the TK gene of FLDV in the biochemically transformed cells was investigated by hybridization experiments, using the 32P-labeled DNA of the recombinant plasmid pC19 which was hybridized to the EcoRI-digested DNA (15 pg) of five transformed cell lines. The transformed cell lines 3T3-Tcltk+-Cl to C4 were established after biochemical transformation of 3T3 TK- cells by the recombinant plasmid pyLV17-E-C harboring the FLDV EcoRI DNA fragment C, and the transformed cell line designated 3T3-Tc19tk+ was transformed by the recombinant plasmid pC19. All transformed cell lines used in this study were cloned and established in tissue cultures by being picked from foci of colonies which had been formed after biochemical transformation of 3T3 TKcells by the individual recombinant plasmids, as described above. Hybridizations were carried out according to a modified procedure of the Southern technique (Southern, 1975) as described elsewhere (Schnitzler et al., 1987). The nick translation was performed using cu[32P]dATP and dCTP (spec. act. 6000 Ci/mmol; New England Nuclear, Dreieich, FRG) according to Rigby et al. (1977) as described previously (Darai et al., 1985). As shown in Fig. 2 (lanes 2 to 6), significant hybridization signals were detected in all five transformed cell lines tested, indicating that DNA sequences of the FLDV TK gene were integrated into the cellular DNA of the biochemically transformed cells. In contrast, no hybridization signals were found when the DNA of parental 3T3 TK- cells was analyzed under the same conditions (Fig. 2, lane 1). The size of the hybridizing fragments found by the

Fig. 2. Identification of the FLDV specific DNA sequences in the genome of 3T3 TK- cells which were converted to TK+ phenotype after transformation with the DNA sequences of the EcoRI FLDV DNA fragment C (0.611 to 0.718 map units) by Southern blot hybridization. Hybridization was performed using 32P-labeled DNA of recombinant plasmid pC19 harboring FLDV DNA sequences at the genome coordinates 0.669 to 0.718 (4.1 kbp) which was hybridized to the 15 pg celhtlar DNA cleaved with restriction endonuclease EcoBI. DNA fragments were separated electrophoretically (Sharp et al., 1983) on a 0.8% agarose slab gel and immobilized on nitrocellulose filters. (A) Ethidium bromide staining. (B) Autoradiogram of hybridization experiment of the same gel. Lane 1, DNA of 3T3 TK- cells which served as internal control; lanes 2 to 6, DNAs of five clones of 3T3 TK+ &Is established after biochemical transformation with the EcoRl FLDV DNA fragment C (3T3-Tcltk+-Cl to C4 correspond to lanes 2 to 5, respectively) or with a part of DNA sequences of this fragment (0.669 to 0.718 viral map units; 3T3-Tc19tk+, lane 6). The DNA of recombinant plasmid pC19 (0.25 I.rg = 0.05 PM) cleaved with EcoFU/HindIII (lane 7) served as internal marker and 32P-labeled phage lambda DNA digested with Hind111 (M) served as molecular weight marker. Arrows mark the position of the hybridization signals found in transformed cells and the triangle labeled with letter V indicates the position of the vector.

transformed cell lines 3T3-Tcltk+-Cl to C4 was about 10 and 4.3 kbp, and in the case of the transformed cell line 3T3-Tc19tk+ about 15 and 4.5 kbp. With respect to the fact that the transformed cells tested in this study were of clonal origin and consisted of one population of HATG resistant cells only, and the fact that DNA sequences of the FLDV TK gene possessed no EcoRI sites and therefore cannot be cleaved with the restriction endonuclease EcoRI, which was used for the digestion of the cellular DNA tested, one can assume that the specific FLDV DNA sequences have undergone a recombination in two different regions of the cellular chromosomes. However, a DNA nucleotide alteration which can lead to the appearance

69

of a new EcoFU site within the introduced FLDV DNA sequences can also be expected. The detection of the hybridization signals by the EcoRI-digested DNA of transformed cell lines 3T3-Tcltk+-Cl to C4 of a size of about 10 and 4.3 kbp is surprising and can lead to the conclusion that the EcoRI restriction pattern of chromosomal DNA of these four cell lines could be identical. The consequence of this conclusion is that the integration of FLDV TK DNA sequences into cellular DNA is not random but specific. However, the detection of hybridization signals for DNA fragments of a size of about 15 and 4.5 kbp by the DNA of transformed cell line 3T3-Tcl9tk+, which was cleaved with EcoRI and analyzed under the same conditions, contradicts this conclusion. Alternatively, the presence of chromosomal DNA fragments of about the same size but of different origin which contain the DNA sequences of FLDV TK gene is obvious, and should also be considered. The situation can be clarified when the corresponding flanking regions of this particular gene in the cellular DNA of transformed cells have been determined. To answer the question of how many copies of DNA sequences of the FLDV TK gene in the cellular chromosome were integrated, hybridization tests were carried out. The 32P-labeled DNA of the recombinant plasmid PC19 was used as hybridization probe. The intensity of hybridization signals found by the EcoRI digest of the DNA of 1 10’ cells of the transformed 3T3-Tcltk+ cell line was compared to the intensity of the hybridization signals of various concentrations (lo3 to lo8 DNA molecules) of the EcoRI/HindIII digest of the recombinant plasmid PC19 analyzed under the same conditions. The results of this study revealed that the intensity of the hybridization signals found by the DNA of the transformed cell line 3T3-Tcltk+ (Fig. 3,B, lane 8) was twice as high as the intensity of the hybridization signal found by 1 x 10’ molecules of the recombinant plasmid PC19 (Fig. 3,B, lane 2). This indicates that cellular DNA of the transformed cells tested harbor one to two copies of the DNA sequences of the FLDV TK gene. The results presented in this study demonstrate that the FLDV genome (98 kbp) contains DNA sequences located at the map coordinates 0.669 to 0.718 (4.1 kbp) which were able to convert the TK negative phenotype of 3T3 cells to the TK positive phenotype by biochemical transformation assay. Although Aubertin and Longchamps (1974) were able to detect a thymidine kinase gene activity in TKmouse cells after infection with frog virus 3, the present study is the first report which has identified, localized, and molecularly cloned the TK gene locus of FLDV, another member of the family Iridoviridae. The genomes of herpesviruses (Jamieson et al., 1974; Kit et al., 1980, 1981; Otsuka and Kit, 1984; Wagner et al., 1981; Bodemer et al., 1986) and orthopoxviruses (Bajszar et al., 1983; Boyle and Coupar, 1986; Boyle et al., 1987; Dubbs et al., 1983; Esposito and Knight, 1984; Hruby and Ball, 1982; Reyes et al., 1982; Upton and McFadden, 1986; Weir et al., 1982; Weir and Moss, 1983) have been shown to encode a viral TK gene. The further characterization of the cloned TK gene of FLDV by DNA nucleotide sequence analysis and the determination of the promoter and the deduced protein sequences will allow determination of the evolutionary relatedness and organization of the TK gene of the FLDV for the Iridoviridae in comparison to other viral or other known

70

123458789

Fig. 3. Determination of the copy number of the FLDV-specific DNA sequences integrated in the cellular DNA of 3T3-Tcltk+ cells by Southern blot hybridization test using 32P-labeled DNA of the recombinant plasmid pC19 which were hybridized to the EcoRI digest of the DNA of 1 x10’ transformed cells (lane 8). The intensity of the hybridization signals detected were compared to the intensity of the hybridization signals found by various concentrations of the DNA molecules of the ~c~~I/~~~dIII digest of pC19 (lanes 1 to 6 correspond to 10’ to lo3 DNA moiecules, respectively). The DNA fragments had been separated electrophoretically on a 0.8% agarose slab gel and immobilized on nitrocellulose filter. (A) Ethidium bromide staining (B) Autoradiogram of hybridization experiment of the same gel. The DNA of 1 x lo7 parental 3T3 TK- cleaved with EcoRI (lane 9) served as control. 3zP-labeled phage lambda DNA digested with Hind111 (lane 7) served as molecular weight marker. Arrows mark the position of the hybridization signal found in transformed cells and the triangle labeled with letter V indicates the position of the vector (lanes 1 to 6) which resulted when the r~mbinant plasmid pC19 was double digested with the restriction endonucleases EcoIU/HindIII. The black solid lines marked with the letters A and B indicate the position of viral inserts in lanes 1 to 6 (A = HindIII/EcoBI DNA fragment 0.675 to 0.718 mu and B = Hind111 DNA fragment 0.669 to 0.675 mu).

eukaryotic TK genes such as human (Bradshaw, 1983; Bradshaw and Deininger, 1984; Lin et al., 1983), murine (Lin et al., 1985), and chicken (Kwoh and Engler, 1984; Merril et al., 1984)).

This study was supported by the Deutsche Forschungsgemeinschaft, project II B 6 Da 142/2-2. The authors thank Dr. R.M. F&gel for critical moments, and thank Miss Elke Fischer for technical assistance.

71 References Aubertin, A. and Longchamps, M. (1974) Thymidine kinase induction in FV 3-infected mouse cells. Virology 58,111-118. Bajszar, G., Wittek, R., Weir, J.P. and Moss, B. (1983) Vaccinia virus th~dine kinase and nei~bou~ng genes; mRNAs and polypeptides of wild-type virus and putative non-sense mutants. J. Virol. 45, 62-72. Bodemer, W., Niller, H.H., Nitsche, N., Scholl, B. and Fleckenstein, B. (1986) Orgauisation of the thymidylate synthase gene of herpes saimiri. J. Viral. 60, 114-123. Boyle, D.B. and Coupar, B.E.H. (1986) Identification and cloning of the fowlpox virus thymidine kinase gene using vaccinia virus. J. Gen. Viral. 67, 1591-1600. Bradshaw, H.D., Jr. (1983) Molecular cloning and cell cycle-specific regulation of a functional human thymidine kinase gene. Proc. Natl. Acad. Sci. USA 80, 5.588-5591. Bradshaw, H.D., Jr. and Deiuinger, P.L. (1984) Human thymidine kinase gene: molecular cloning and nucleotide sequence of a cDNA expressible in mammalian cells. Mol. Cell. Biol. 4, 2316-2320. Darai, G., Matz, B., Fhigel, R., Grafe, A., Gelderblom, H. and Delius, H. (1980) An adenovirus from Tupaia (Tree Shrew): growth of the virus, characte~atioR of viral DNA, and transforming ability. Virology 104,122-138. Darai, G., Anders, K., Koch, H.G., Delius, H., Gelderblom, H., Samalecos, C. and Fhigel, R.M. (1983) Analysis of the genome of fish lymphocystis disease virus isolated directly from epidermal tumours of pleuronectes. Virology 126, 466-479. Darai, G., Delius, II., Clarke, J., Apfel, H., Schnittler, P. and Fltlgel, R.M. (1985) Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus. Virology 146, 292-301. Darai, G. (1986) Molecular biology of fish lymphocystis disease virus. In: Pathology in Marine Aquaculture (Vivares, C.P., Bonami, J.R. and Jaspers, E., eds.), Special Publication No. 9, pp. 261-280. European Aquac~ture Society, Bredene, Belgium. Dubbs, D.R., Otsuka, H. and Kit, S. (1983) Mapping thymidme kinase-deficient mutants of vaccinia virus by marker rescue with hybrid plasmid DNAs containing portions of the HindHI-J fragment of virus DNA, Virology 126, 408-411. Esposito, J.J. and Knight, J.C. (1984) Nucleotide sequence of the thymidine kinase gene region of monkeypox and variola viruses. Virology 1359, 561-562. Ftigel, R.M., Darai, G. and Gelderblom, H. (1982) Viral proteins and adenosine t~phosphate phosphohydrolase activity of fish lymphocystis disease virus. Virology 122,48-X. FliIgel, R.M. (1985) Lymphocystis dsease virus. In: Iridoviridae (Willis, D.B., ed.), Current Topics in Microbiology and Immunology 116, pp. 133-150. Springer-Verlag, Berlin. Goorha, R. and Murti, K.G. (1982) The genome of frog virus 3, an animal DNA virus, is circularly permuted and terminally redundant. Proc. Natl. Acad. Sci. USA 79, 248-252. Hruby, D.E. and Ball, L.A. (1982) Mapping and identification of the vaccinia virus thymidine kinase gene. J. Viral. 2, 403-404. Hruby, D.E., Maki, R.A., Miller, D.B. and Ball, L,A. (1983) Fine structure analysis and nucleotide sequence of vaccinia virus thymidine kinase gene. Proc. Natl. Acad. Sci. USA 80, 3411-3415. Inglis, M.M. and Darby, G. (1987) Analysis of the role of cysteine 171 residue in the activity of herpes simplex virus type 1 thymidine kinase by oligonucleotide-directed mutagenesis. J. Gen. Virol. 68, 39-46. Jar&son, A.T. and Subak-Sharpe, J.H. (1974) Bi~be~cal studies on the herpes simplex virus-specified d~x~y~~ne kinase activity. J. Gen. Virol. 24,482-492. Kit, S., Otsuka, H., Qavi, H., Dubbs, D.R. and Trkula, D. (1980) Biochemical transformation of mouse cells by a purified fragment of marmoset herpesvirus DNA. Intervirology 13, 110-121. Kit, S., Qavi, H., Hazen, M., Trkula, D. and Otsuka, H. (1981) Biochemical transformation of LM(TK- ) cells by hybrid plasmids containing the coding region of the herpes simplex virus type 1 thymidine kinase gene. Virology 113, 452-464. Kwoh, T.J. and Engler, J.A. (1984) The nucleotide sequence of the chicken thymidine kinase gene and relations~p of its predicted polypeptide to that of the vaccinia virus thy~dine kinase. Nucl. Acids Res. 12, 3959-3971.

72 Lit-r. P.F., Zhao, S.Y. and Ruddle, F.H. (1983) Genomic cloning and preliminary characterization of the human thymidine kinase gene. Proc. Natl. Acad. Sci. USA 80, 6528-6532. Lin, P.F., Lieberman, H.B., Yeh, D.B., Yu, T., Zhao, S.Y. and Ruddle, F.H. (1985) Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences. Mol. Cell. Biol. 5, 2149-3156. Merril, G.F., Harland, R.M., Grondine, M. and M&night, L. (1984) Genetic and physical analysis of the chicken tk gene. Mol. Cell. Biol. 4, 1769-1776. Otsuka, H. and Kit, S. (1984) Nucleotide sequence of the marmoset herpesvirus thymidine kinase gene and predicted ammo acid sequence of thymidine kinase polypeptide. Virology 135, 316-330. Reyes, G.R., Jeang, K. and Hayward, G.S. (1982) Transfection with the isolated herpes simplex virus thymidine kinase genes. Minimal size of the active fragments from HSV-1 and HSV-2. J. Gen. Virol. 62,191-206. Rigby, P.W.J., Dieckmann, M., Rhodes, C. and Berg, P. (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 114, 237-256. Schnitzler, P., Soltau, J., Fischer, M., Reisner, H., Scholz, J., Delius, H. and Darai, G. (1987) Molecular cloning and physical mapping of the genome of insect iridescent virus type 6; further evidence for circular permutation of the viral genome. Virology 160, 66-74. Sharp, P.A., Sugden, B. and Sambrook, J. (1983) Detection of two restriction endonuclease activities in Haemophilus paruinfluenrae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12, 3055-3061. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. Stow, N.D. and Wilkie, N.M. (1976) An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA. J. Gen. Virol. 33, 447-458. Twigs, A. and Sheratt, D. (1980) Trans-complementable copy-number mutants of plasmid Co1 El. Nature (London) 283, 216-218. Upton, C. and McFadden, G. (1986) Identification and nucleotide sequence of the thymidine kinase gene of shope fibroma virus. J. Gen. Virol. 60, 920-927. Wagner, M.J., Sharp, J.A. and Summers, W.C. (1981) Nucleotide sequence of the thymidine kinase gene of herpes simplex type 1. Proc. Natl. Acad. Sci. USA 78, 1441-1445. Wagner, H., Simon, D., Werner, E., Gelderblom, H., Darai, G. and Fltigel, R.M. (1985) Methylation pattern of DNA of fish lymphocystis disease virus. J. Virol. 53, 1005-1007. Weir, J.P., Bajsxar, G. and Moss, B. (1982) Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA. Proc. Natl. Acad. Sci. USA 79, 1210-1214. Weir, J.P. and Moss, B. (1983) Nucleotide sequence of the vaccinia virus thymidine kinase gene and the nature of spontaneous frameshift mutants. J. Virol. 46, 530-537. Willis, D.B. (1985) Iridoviridae, Current Topics in Microbiology and Immunology 116, Springer-Verlag, Berlin. Willis, D.B. and Granoff, A. (1980) Frog virus 3 DNA is heavily methylated at CpG sequences. Virology 107, 250-257. (Manuscript

received

22 July 1987; revision

received

15 September

1987)