Cloning and physical mapping of Yaba monkey tumor virus DNA

VIROLOGY

143, 399-406

Cloning

(1985)

and Physical

Mapping

D. R. KILPATRICK Laboratory

of Yaba Monkey AND

Tumor

Virus DNA

H. ROTJHANDEH’

of Molecular and C~ULCBTViwlo~~, Department Southern Illinois University, Carbondale, Illinois

of Microbiology. 62901

Received October 12, 1984; accepted December 7, 19X.4 The physical map positions for the BarnHI, EcoRI, and Sal1 restriction fragments of Yaba monkey tumor pox virus DNA were determined using cloned virus DNA fragments as probes for hybridization as well as analyzing the secondary digests of larger DNA restriction fragments. Digests of EcoRI A and B fragments and Sal1 A and B fragments with i?a,mHI allowed for the orientation of most of the BamHI restriction map. These secondary digest products were confirmed and the map positions for the EcoRI fragments were established using cloned BamHI fragments. Yaba monkey tumor virus DNA was cloned using the plasmid vector pBR322. G WE Academic Press, Inc. INTRODUCTION

Poxviruses contain a linear doublestranded DNA genome which is crosslinked at the termini. The molecular weight of poxvirus DNA ranges from 85 to 185 X lo6 (Holowczak, 1982). The genomes of poxviruses have been well characterized with restriction endonucleases (Muller ef al., 197’7; Wittek et aZ., 1977; McCarron et al., 1978). The Orthopoxvirus family, in particular, has been extensively analyzed with a central conserved region being identified by the cross-hybridization of DNA among this family (Mackett and Archard, 1979). All members of this family also have the ends of their genome crosslinked by a single strand of DNA (Geshelin and Berns, 1974; Moyer and Graves, 1981). Yaba monkey tumor virus (YMTV) is an unclassified poxvirus capable of inducing benign histiocytomas in man and monkey (Bear-croft and Jamieson, 1958; Andrewes et al., 1969; Ambrus et ul., 1963; Sproul et al., 1963). Previous work with YMTV DNA showed that the T,,, value for its DNA was around 82.3” and that its bouyant density was 1.695 g/ml in CsCl. The percentage G + C ratio of YMTV DNA ’ Author addressed.

to whom requests for reprints

should be 399

was found to be 32.5 (Yohn and Gallagher, 1969). Rouhandeh et al. (1982), showed that there was less than 10% homology between the genomes of YMTV and monkeypox virus. YMTV did not hybridize to the ends of the MPV genome, but did hybridize to two adjacent pieces within the genome. The genome of YMTV was shown to be crosslinked and to have an average molecular weight of 95 X lo6 (Kilpatrick and Rouhandeh, manuscript submitted). This study reports the partial restriction map of the YMTV genome for the restriction endonucleases BumHI, EcoRI, and S&I. MATERIALS

AND

METHODS

Virus and cells. Yaba monkey tumor virus was grown in monolayers of CV-1 cells. Adsorption was allowed to continue for 2 hr at 35”. Eagle’s modified minimum essential medium (MEM) containing 2Y0 calf serum was then added. Yaba virus was harvested at 6 to 7 days postinfection. Puri&cution of virus. Yaba virus was purified from tissue culture fluid as described previously (Fenger and Rouhandeh, 1976; Kilpatrick and Rouhandeh, 1981). Isolation of virus DNA. DNA was isolated from purified Yaba virus by treatment of the virus with 1% SDS in TNE 0042-6822/85 Copynyht AII rirhls

$3.00

(c 1985 hy Academic Press, Inc. of rrpruducli~n in any form rcserwd

400

KILPATRICK

AND

buffer (0.01 M Tris-HCl, pH 7.4, 0.1 M NaCl, 0.001 M EDTA) and autodigested Pronase (500 pg/ml) overnight at 37”. The DNA was extracted twice with 80% phenol, twice with chloroform-n-butanol (24: l), and precipitated from the aqueous phase by the addition of 2 vol of 100% cold ethanol. The DNA was washed twice with ethanol and dissolved in TE buffer (0.5 M Tris-HC, pH 7.8 and 0.001 M EDTA). The 0D260,280ratio was shown to be 1.92, using a Gilford spectrophotometer. Restriction enzyme cleavage of DNA. Restriction endonucleases (Bethesda Research Labs) were used to digest DNA under the following conditions: EcoRI, 100 mM Tris-HCl, pH 7.2, 5 mM MgC&, 50 mM NaCl, 2 mM 2-mercaptoethanol; BamHI, 20 mM Tris-HCl, pH 8.0, 7 mM MgC&, 100 mM NaCl; SalI, 8 mM TrisHCl, pH 7.6, 6 mM MgClz, 0.2 mM NazEDTA, 150 mM NaCl. All reactions were incubated at 37” for l-2 hr. The reactions were stopped by adding an equal volume of agarose suspension (Schaffner et al., 1976) which contains 0.2% agarose in 20 mM Na-EDTA, 10 mM Tris-HCl, pH 7.6, 10% glycerol, and 0.01% bromphenol blue. Agarose gel electrophoresis. After heating the samples for 5 min at 60” and quick cooling in an ice bath, the samples were applied to a 0.7% or a 1.4% horizontal agarose gel (20 X 15 X 0.3 cm) and electrophoresed at 30 V for 12 to 16 hr. Electrophoresis was in a Tris-buffered system (Loening, 1969; 4.84 g Tris, pH 7.8, 2.82 g sodium acetate, 0.74 g EDTA, 1.8 ml acetic acid in 1 liter). After electrophoresis the gel was stained for 30 min with 1 pg/ml ethidium bromide, placed under a uv lamp and photographed through a red filter with Polaroid type 55 film. Molecular cloning of Yaba monkey tumor virus DNA. The BamHI fragments of YMTV DNA were inserted in the plasmid vector, pBR322 using procedures essentially as described in Maniatis et al. (1982). The ligated DNA was used to transform the HBlOl strain of Escherichia coli. The ligation reaction mixture was brought up to 100 ,ul with the addition of 50 ~1 of 2X ligase buffer to transformation mixture

ROUHANDEH

(20 mM CaCl and 13 mM MgCl). The cells to be transformed were then thawed in an ice-water bath. Cells (0.2 ml) were then added to the DNA sample. The cell-DNA suspension was then held at 25 min in an ice-water bath. The E. coli was then plated onto LB plates containing 20 pg/ml of ampicillin and incubated at 37” overnight. These colonies were picked and screened first on plates containing ampicillin, and then on plates containing ampicillin and tetracycline (10 fig/ml). Five hundred ampicillin-resistant, tetracycline-sensitive colonies were isolated. Isolation of plasmid DNA. Recombinant plasmid DNA was extracted from an overnight culture of E. coli using the boiling technique as described by Maniatis et al. (1982). Preparation oj’ labeled cloxed YMTV DNA probe. The extracted plasmid DNA was labeled with [32P]dCTP to a sp act of 3 to 6 X lo7 cpm/pg as described by Rigby et al. (1977). Th nick-translation system was purchased from New England Nuclear. Analysis (ti virus DNA by the SwAhern YMTV DNA was cleaved with technique. EcoRI and Sal1 restriction enzymes. The fragments were separated by electrophoresis on a 0.7% agarose gel. After electrophoresis the DNA was denatured by submerging the gel in 0.5 M NaOH, 1.5 M NaCl at room temperature for 30 min. Subsequently, the gel was neutralized in 3 M sodium acetate, pH 5.5 for 30 min and the denatured DNA fragments were then transferred to Ba 85 nitrocullulose filters (Schliecher & Schuell, Keene, N. H.) by Southern’s technique (1975). The filters were rinsed in 2X SSC, dried at 80” for 2 hr, and sealed in plastic bags. DNA-DNA hybridization, and autoradiography. The DNA hybridization was performed as described by Wahl et ul. (1979). The filters were preincubated in 70 ml of a solution containing 50% formamide, fivefold concentrated Denhardt reagent (1X reagent contains 20 mg each of bovine serum albumin, Ficoll-400, and polyvinylpyrrolidone in 100 ml), and 300 pg of heat-denatured calf thymus DNA/

YABA

MONKEY

TUMOR

ml in 0.05 M sodium phosphate, pH 6.5, and 5~ SSC. The filters were preincubated at 41” for 24 hr. The preincubation buffer was replaced by 50 ml of a solution containing the heat-denatured 32P-labeled virus DNA probe (3 to 6 X lo7 cpm) in 50% formamide, 1X Denhardt reagent, and 100 pg of heat-denatured calf thymus DNA/ ml in 5~ SSC, and 10% sodium dextran sulfate 500. The filters were then washed successively with several changes of 2X SSC and 0.5% SDS at 65” for a total of 4 hr. After drying, the filters were exposed to Kodak X-Omat AR film for 1 to ‘7 days. RESULTS

Previous studies showed that Yaba monkey tumor virus had an average molecular weight of 95.0 X lo6 when analyzed using the restriction endonucleases BumHI, EcoRI, HindIII, SalI, and XhoI (Rouhandeh et uZ., 1982; Kilpatrick and Rouhandeh, manuscript submitted). BamHI fragments D and J (14.5 and 5.1 kb, respectively) were also shown to possess crosslinks. A partial restriction map of the YMTV genome using BamHI was constructed using secondary digestions of larger restriction enzyme fragments as well as hybridization with cloned BamHI restriction fragments. Secondary di-gestion of individual frugrue&=. Restriction fragments were separated using a gel consisting of low-melting-point agarose. Individual fragments were cut out and digested with a second enzyme (BarnHI). These secondary digest fragments were separated on a 0.7% agarose gel. Adjacent BamHI fragments which were contained in a larger fragment were identified in this manner. The secondary cleavage products are listed in Table 1. EcoRI fragment A contains BamHI fragments I, K, A, L, and J, as well as several fragments which did not comigrate with any of the BamHI fragments. These fragments are at one end of the genome since BumHI fragment J has previously been shown to be crosslinked. The two molar EcoRI B fragment contains BamHI fragments D, N, F, G, and H.

VIRUS

401

DNA TABLE

1

SECONDARY CLEAVAGES OF EcoRI RESTRICTION FRAGMENTS

Second digestion l&x%-A/ BamHI EC&-B/ BnmHI &I&A/ Ba1nH1

S&-B/ BumHI

AND Sal1

Size of products (kilobases) 25.7 (BnmHI-A), 6.1 (BarnHI-I), 5.1 (BarnHI-J), 4.5 (Ban~H1-KI, 3.6 (BarnHI-L), 1.5 16.6, 14.5 (BarnHI-D), 10.6 (BarnHI-F), 8.9 (BarnHI-G), 1.9 (BarnHI-N), 1.5 12.5 (BumHI-E), 10.6 (BarnHI-F), 8.9 (BarnHI-G), 7.8 (BumHI-H), 6.8, 5.1 (BwTzHI-J), 3.0 (BarnHI-M), 1.9 (BarnHI-N) 18.1, 4.5 (BarnHI-K), 1.5

Some of these fragments are end of the genome because of of fragment D. Similarly, SulI contains BamHI fragments M. and N.

at the other the presence A fragment E, F, G, H,

14 9

267

141 123

FIG. 1. YMTV DNA was cleaved with (1) SalI/ BarnHI, (2) BamHI, and (3) EcoRI/BamHI. These double digests indicate which BnmHI fragments have EcoRI and Sal1 cleavage sites. This information was used to determine the possible origin of fragments which did not comigrate with the BamHI fragments found in the secondary digestion of individual restriction fragments. XDNA cleaved with Hind111 is shown for a molecular weight marker (X106).

402

KILPATRICK

AND

Whole YMTV DNA was cleaved with SalI/BamHI and EcoRI/BamHI to show which BamHI fragments had either Sal1 or EcoRI cleavage sites. Figure 1 shows that BamHI fragments B, E, H, and M are cleaved with EcoRI, while BamHI fragments A, B, D, I, and J are cleaved with SalI. The sizes of the DNA fragments cleaved with the three enzymes used in this study are shown in Table 2. The fact that these fragments contain at least one Sal1 or EcoRI cleavage site explains where the fragments which did not comigrate with the native BamHI fragments are generated. For example, since BamHI A and B fragments are very close in size (25.7 and 22.7 kb), it was difficult to say whether the large fragment generated from the BamHI digestion of the EcoRI A fragment was BamHI A or BamHI B. BamHI B is shown to be cleaved with EcoRI in the double digest of the whole DNA (Fig. l), therefore the large fragment generated from EcoRI A must be BamHI A. Other fragments which did not comi-

ROUHANDEH

grate with BamHI pieces were similarly identified. In order to verify these results, and to have some specific probes for hybridization, YMTV DNA was cloned using the plasmid pBR322. Molecular cloning of YMTV DNA. BamHI fragments of YMTV were inserted into the BamHI site of pBR322, thus inactivating the tetracycline gene. Five hundred ampicillin-resistant, tetracyclinesensitive colonies were isolated. Plasmid DNA was extracted from these recombinants and cleaved with BamHI to identify which viral BamHI fragments were inserted. Figure 2 shows the cloned YMTV DNA fragments. Three BamHI fragments, A, B, and C were not inserted into the vector due to their large size. The two crosslinked fragments D and J were also not inserted into the plasmid. Hybridixation using BamHI clones as probes. Recombinant plasmids containing YMTV DNA BamHI fragments were nicktranslated and hybridized to filters containing YMTV DNA cleaved with Sal1

TABLE

2

SIZES OF YMTV DNA FRAGMENTS PRODUCED BY RESTRICTION ENDONUCLEASES’

Fragment A B C D E F G H I J K L M N Total

size (kb) 25.7 22.7 17.4 14.5” 12.1 10.6 8.9 7.8 6.1 5.1’ 4.5 3.6 3.0 1.9 149.0

EcoRI

Sal1

BarnHI Mb 1 1
size (kb) 33.3 22.7 11.5 9.0 8.4 3.9 3.4 3.3 1.5

J’& 2 1 2 1 1 1
141.8

a Monkeypox virus and XDNA cleaved with Hind111 were used as standards. ‘Molar ratio as calculated from densitometer tracings. ‘Fragments which possess crosslinks.

size (kb) 45.4 28.7 14.3 13.1 9.4 8.3

147.9

M, 1 2 1 1
YABA 12345678

9

MONKEY 10

TUMOR

11

FIG. 2. Recombinant plasmids were analyzed by cleaving the extracted plasmid DNA and running samples with native YMTV DNA cleaved with Ba?nHI on a 0.7% agarose gel. (1) Fragment E, (2) F, (3) G, (4) H, (5) I, (6) YMTV DNA (7) J, (8) K (9) L, (10) M, and (11) N. (Note: Vector DNA migrates closely with fragment K in lane 8).

and EcoRI. This information was used to support the results of the secondary cleavage of Sal1 A and B, as well as EcoRI A and B, fragments with BamHI. Figure 3 shows the autoradiograms of this hybridization experiment. Fragments E, F, G, H, J, and M hybridized to the Sal1 A fragment. This supports the results obtained from the secondary digest of Sal1 A with BamHI. Cloned fragments E, J, and M also hybridized to the EcoRI C fragment indicating their close proximity to each other. The hybridization of the remaining cloned fragments agrees with the results shown in Table 1. The results of the hybridization are summarized in Table 3. Partial restriction map of the YMTV genome. The data collected so far could be used to construct a partial restriction map for the YMTV genome. EcoRI A contains the BamHI J fragment, so all of these fragments are at one end of the genome. Since the other J fragment was cloned and did not hybridize to the EcoRI

VIRUS

403

DNA

A fragment, this must be the J fragment which is crosslinked. EcoRI B contains the BamHI D fragment, so all fragments associated with the D fragment must be at the other end of the genome, due to presence of a crosslink in D. The presence of fragments which did not comigrate with BamHI fragments was explained by the previous double digests of the whole DNA (Fig. 1). The physical map locations for the BamHI fragments were established using the data collected in these experiments, knowing that fragments D and J represent the termini since they are crosslinked. The positions of the EcoRI fragments were possible due to the hybridization information and comparing this to the BamHI map. EcoRI fragments A through D were accordingly positioned. EcoRI fragments E and F, which are submolar, were positioned according to the location of the BamHI C fragment which is also submolar (approximate map units 0.6 to 0.7). The sum of E and F (9.4 and 8.3 kb)

5

,

6

7 -d

a

6 * BI

a -

*

FIG. 3. Hybridization of cloned YMTV DNA to individual restriction endonuclease fragments. The following cloned BamHI fragments were nick-translated and hybridized to YMTV DNA: (1) E, (2) F, (3) G, (4) H, (5) I, (6) J, (7) K, and (8) M. So11 fragments were transferred to nitrocellulose filters and hybridized to the individual probes. The autoradiograms of these filters are shown in column a. The ethidium bromide-stained DNA cleaved with Sal1 is in b. Column c is YMTV DNA cleaved with EcoRI and d is the autoradiogram of this DNA transferred to filters and hybridized to individual cloned fragments.

404

KILPATRICK TABLE

3

HYBRIDIZATION OF CLONED BamHI TO YMTV DNA

Cloned fragment E F G H I J K M

AND

FRAGMENTS

EcoRI and Sal1 fragments that hybridized to a specific cloned fragment EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI

C, B, B, D, A, C, A, C,

Sol I SoI1 Sal1 Sal1 S&I Sal1 Sol1 Sol1

A A A A C A B A

is approximately equal to the size of the BumHI C fragment (17.4 kb). The Sal1 fragments A, B, and C were positioned in agreement with the hybridization data. The remaining Sal1 fragments could not be positioned with the current information. Figure 4 shows the physical map locations for the BarnHI, EcoRI, and Sal1 restriction fragments. DISCUSSION

The partial restriction map of Yaba monkey tumor virus DNA was established using the secondary digests of larger restriction fragments and the hybridization of BarnHI cloned fragments to EcoRI and Sal1 restriction fragments. The areas where ambiguity still exists within the map are indicated in Fig. 4. Fragments N and F may switch positions and therefore the map may read D, N, F or D, F, N. The reason for this uncertainty and the regions containing the BamHI fragments M and E, as well as the EcoRI fragments E and F is that there were no cut sites in these fragments for the enzymes used. BamHI fragments N and F as well as M and E were always resolved together in the secondary digests. EcoRI fragments E and F were positioned since they represent the submolar region of the genome corresponding to the BumHI C fragment. The location of the BumHI C fragment was determined since relatively larger

ROUHANDEH

areas of the genome can be accounted for by just the EcoRI A and B secondary digests. These fragments contain the two crosslinks and therefore represent either end of the genome. Large areas on either end of the genome are accounted for in this manner. The hybridization of individual cloned BamHI fragments to the larger EcoRI and SaZI fragments also supports the result of the secondary cleavage of individual restriction fragments. The location of the BamHI C fragment must be toward the middle of the genome or at least not at its very termini. This is also supported by the finding that an original isolate of YMTV, which has the C fragment in one molar quantities has the same crosslinked termini (BarnHI fragment D and J) as does the strain which possesses the submolar C fragment. The deletion, therefore, is interior to the Barv~H1 D and J fragments. This deletion occurred as a result of continued propagation in cell culture. This is a common occurrence among poxviruses (Holowczak, 1982; Moss et ub, 1981). The original isolate of YMTV, isolated from tumors and stored frozen for 14 years, also developed this same deletion upon continued propagation. Yaba monkey tumor virus (YMTV) is one of the few tumor viruses which causes tumors in its natural host, in this case, monkeys. YMTV has shown the ability to cause tumors in man as well. This unclassified poxvirus has significant distinctions

FIG. 4. The physical map locations for BurnHI, EooRI, and S&I were determined using the information obtained from the hybridization of BamHI cloned fragments to EcoRI and Sal1 fragments. Secondary digests of larger EcoRI and Sal1 fragments with BamHI were used to identify the positions for most of the BnmHI fragments. The black dots indicate fragments whose position may change relative to one another. The broken lines indicate areas of the Sal1 map which could not be identified by hybridization.

YABA

MONKEY

TUMOR

from other viruses of the poxvirus group. In particular, the length of the growth cycle and the proteins synthesized during infection, have been previously shown to be very different from monkeypox virus (Vafai and Rouhandeh, 1982; Rouhandeh et al, 1982). The amount of DNA homology between this virus and monkeypox virus is also less than lo%, although YMTV did hybridize to two adjacent Hind111 restriction enzyme fragments (L and M) which lie in a central conserved region (as described by Mackett and Archard, 1979) of the monkeypox virus genome (Rouhandeh et al., 1982). For these reasons the analysis of the genome, as far as its structural organization, is of interest. The molecular cloning of most of the BamHI fragments for YMTV and the restriction analysis of the genome will further aid this analysis. This information will also aid the investigation of the in vitro cell transformation system which has been established using inactivated Yaba monkey tumor virus (Rouhandeh and Vafai, 1982). The three largest fragments and the crosslinked termini were not expected to be cloned using this shotgun approach. Previous work involving the cloning of vaccinia virus DNA into pBR322 also did not clone the largest DNA fragment of vaccinia (Hind111 A) and had to be subcloned by its digestion with another enzyme which was then cloned into a vector (Belle Isle et al., 1981). ACKNOWLEDGMENT This work was supported in part by the Office of Research and Development at Southern Illinois University, Carbondale, Illinois. REFERENCES

AMBRUS,J. L., PELTZ, E. T., GRACE, J. T., and OU’ENS, G. (1963). A virus induced tumor in primates. National Cancer Institute Monograph 10, pp. 447 458. AXDREWES, C. H., ALLISON, A. C., ARMSTRONG, J. A., BEARCROFT, G., NIVEN, J. S. F., and PEREIRA, H. S. (1969). A virus disease of monkeys causing large superficial growth. Acta Unionis intemationaliz

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BEARCROFT,W. C. G., and JAMIESON, M. F. (1958). An outbreak of subcutaneous tumors in rhesus monkeys. Nature (London) 182, 195-196. BELLE ISLE, H., VENKATESAN, S., and MOSS, B. (1981). Cell-free translation of early and late mRNAs selected by hybridization to cloned DNA fragments derived from the left 14 million to 72 million Daltons of the vaccinia virus genome. Virology 112, 306-317. FENCER,T., and ROUHANDEH, H. (1976). Proteins of Yaba monkey tumor virus, I. Structural proteins. J. Viral. 18, 757-764. GESHELIN,P., and BERNS, K. I. (1974). Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA. J. Mol. Biol. 88, 785-796. HOLOWCZAK, J. A. (1982). “Current Topics in Microbiology and Immunology,” Vol. 97. Springer-Verlag, New York. KILPATRICK, D., and ROUHANIIEH, H. (1981). The polypeptides of monkeypox virus. I. Analysis of the polypeptide synthesis of MPV by SDS-PAGE and by two-dimensional electrophoresis. ViroZq~~~~ 110, 455-465. LOENING, U. E. (1969). The determination of the molecular weight of ribonucleic acid by polyacrylamide gel electrophoresis. Biomerl. J. 113, 131-138. MACKETT, M., and ARCHARD, L. C. (1979). Conservation and variation in Orthopoxvirus genome structure. J. Gm. Viral. 45, 683-701. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, d. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. MCCARRON, R. J., CABRERA, C. V., ESTABAN, M., MCALLISTER, W. T., and HOLOWCZAK, J. A. (1978). Structure of vaccinia DNA: Analysis of the viral genome by restriction endoculeases. Virology 86, 88-101. Moss, B., WINTERS, E., and COOPER, J. A. (1981). Deletion of a 9,000-base-pair segment of the vacinnia virus genome that encodes nonessential polypeptides. J. Viral. 40, 387.-395. MOYER, W. R., and GRAVES, L. R. (1981). The mechanism of cytoplasmic orthopoxvirus DNA rcplication. Cell 27, 391-401. MULLER, H. K., WITTEK, R., SCHAE‘FNER, W., S~H~J%TPERLI, D., MENNA, A., and WYLER, R. (1977). Comparison of five poxvirus genomes by analysis with restriction endonucleases HindIII, BnrrrHI, and &‘coRI. J. Gen. Viral. 38, 135-147. RIGBY, P. W. J., DIECKMANN, M., RHODES, C., and BERG, P. (1977). Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237-251. ROUHAN~EH, H., and VAF~I, A. (1982). A novel cell

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transformation with DNA-containing cytoplasmic Yaba tumor poxvirus. Virology 120,77-92. ROUHANDEH, H., KILPATRICK, D., and VAFAI, A. (1982). The molecular biology of Yaba tumor pox virus: Analysis of lipids, proteins, and DNA. J. Gen. ViroL 62, 207-218. SCHAFFNER, W., GROSS, K., TELFORD, J., and BRINSTIEL, M. L. (1976). Molecular analysis of the histone gene cluster of Psammechinus miliarua II. The arrangement of the five histonecoding and spacer sequences. Cell 8,471-4’78. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. SPROUL, E. E., METZGAR, R. S., and GRACE, J. T., JR. (1963). The pathogenesis of Yaba virus-induced histiocytomas in primates. Cancer Rea 23, 671675.

ROUHANDEH WAHL, G. M., STERN, M., and STARK, G. R. (1979). Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl paper and rapid hybridization by using dextran sulfate. Proc. Natl. Acad. Sci. l% S. A. 76, 3683-3687. VAFAI, A., and ROUHANDEH, H. (1982). Analysis of Yaba tumor poxvirus-induced proteins in infected cells by two dimensional gel electrophoresis. Virology 120, 65-76. WITTEK, R., MENNA, A., SCHUMPERLI, D., STOFFEL, S., MULLER, H. K., and WYLER, R. (1977). Hind111 and Sat1 restriction sites mapped on rabbitpox virus and viccinia virus DNA. J. Viral. 23, 669678.

YOHN, D. S., and GAI,LAGHER, J. F. (1969). Some physical properties of Yaba poxvirus deoxyribonucleic acid. J. Viral. 3, 114-118.