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Mutation near the polyoma DNA replication origin permits productive infection of F9 embryonal carcinoma cells
Cell, Vol. 23, 809-814, March 1981, Copyright 0
1981
by MIT
Mutation near the Polyoma DNA Replication Origin Permits Productive Infection of F9 Embryonal Carcinoma Cells Frank K. Fujimura,* Prescott L. Deininger,? Theodore Friedmannt and Elwood Linney* * La Jolla Cancer Research Foundation 2945 Science Park Road La Jolla, California 92037 t Department of Pediatrics M-009 University of California, San Diego La Jolla, California 92093
Summary F9 mouse embryonal carcinoma cells are resistant to productive infection by wild-type polyoma virus. Continued passage of F9 cells initially infected with wild-type polyoma virus eventually leads to the selection of polyoma virus mutants that are capable of productive infection of undifferentiated F9 cells. Three mutants, PyFlOl, PyFll 1 and PyF441, have been- plaque-purified and examined. All three PyF mutant DNAs are altered from the wild-type sequence in the Pvu II-4 fragment that spans 67.6 to 70.2 map units on the polyoma genome. PyF441 has a single base change of A to G at 69.6 map units: PyFl 01 and PyFl 11 DNAs also contain this point mutation at 69.6 map units. In addition, PyFl 01 and PyFl 11 DNAs have exact tandem duplications of 54 and 31 bp, respectively, of sequences encompassing the point mutation, and both copies of the tandem duplication have the point mutation. Other than these changes, no difference exists in the nucleotide sequences of wildtype and PyF mutant DNAs from the Bcl I site at 65.6 map units clockwise through the origin of viral DNA replication to the Bgl I site at 72.2 map units. DNA infections of F9 cells with wild-type-mutant hybrid DNAs formed by ligation of heterologous combinations of the small and large DNA fragments generated by double digestion with the restriction enzymes Bcl I and Bgl I show that the DNA sequence changes described above are responsible for the ability of the PyF mutants to infect F9 cells. Introduction Murine teratocarcinomas are malignant tumors usually containing a wide variety of somatic cell types. These tumors also contain a characteristic cell called embryonal carcinoma (EC). The EC cell is the stem cell of teratocarcinomas and can differentiate to form the somatic cell types present in these tumors (Kleinsmith and Pierce, 1964). Studies have indicated that EC cells have many similarities to multipotent cells of early mouse embryos and that some of the cellular changes accompanying differentiation of EC cells mimic events during normal embryonic differentiation
(Pierce, 1967; Stevens, 1967; Martin, 1975; Graham, 1977; Jacob, 1978). In addition to changes in expression of cellular genes, differentiation of EC cells leads to changes in expression of some viral genes. No viral tumor(T) antigens can be detected in undifferentiated EC cells after infection by simian virus 40 (SV40; Swartzendruber and Lehman, 1975; Topp et al., 1977) or polyoma virus (Swartzendruber and Lehman, 1975). Differentiated cells arising from EC cells can allow papovavirus T antigen expression and, in the case of polyoma, can support lytic infection. Similarly, EC cells are resistant to minute virus of mouse (Miller et al., 1977) and C-type RNA tumor viruses (Peries et al., 1977; Teich et al., 1977; Gautsch, 1980), while some differentiated mouse cells are sensitive to these viruses. Viral resistance is not a general phenomenon in EC cells, as these cells will support growth of a number of other viruses (Lehman et al., 1975; Kelly and Boccara, 1976; Teich et al., 1977; Oldstone et al., 1980). The nature of the block to papovavirus infection of EC cells is not known. Swartzendruber et al. (1977) showed that SV40 and polyoma adsorption and penetration take place in EC cells. Fusion of polyomainfected EC cells with baby hamster kidney cells leads to both early and late viral gene expression (Boccara and Kelly, 1978), while fusion of SV40-infected EC cells with permissive monkey cells does not rescue expression of SV40 T antigen (Swartzendruber et al., 1977). Segal et al. (1979) examined F9 EC cells infected with SV40 and found low levels of early viral RNA that, when analyzed by the method of Berk and Sharp (1977), was unspliced. F9 cells allowed to differentiate in the presence of retinoic acid as described by Strickland and Mahdavi (1978), when infected with SV40, contain spliced early viral RNA (Segal and Khoury, 1979). Unpublished results cited by Vasseur et al. (1980) for polyoma-infected PCC4 aza EC cells indicated that the low level of viral RNA detected in these infected cells was spliced. During our investigation of the interactions of polyoma virus with EC cells, we found polyoma mutants that are capable of productive infection of F9 EC cells. This report describes the selection and characterization of these mutants. Our data suggest that the significant change affecting the ability of polyoma virus to infect F9 cells is a point mutation at 69.6 map units in a noncoding region near and to the late side of the origin of DNA replication. Tandem duplications of DNA sequences including the point mutation appear to increase the efficiency of polyoma growth in F9 cells. The nature of these nucleotide changes permitting polyoma infection of F9 cells differs from the more extensive rearrangements Katinka et al. (1980) recently reported to be present in the DNAs of polyoma virus mutants that infect another EC cell line, PCC4 aza (Vasseur et al, 1980). ’
Cell 810
gel electrophoresis (data not shown). No difference was detected between wild-type and PyF441 DNAs in the Hpa II, Pvu II or Hae Ill fragments. The only detectable difference in the Hpa II fragments of wildtype and PyFl 01 DNAs was that the PyFl 01 Hpa II-3 fragment was about 55 bp larger than the corresponding wild-type fragment. Similarly, the PyFl 11 Hpa II-3 fragment was about 30 bp larger than the wild-type Hpa II-3 fragment. Digestion with Pvu II indicated that the PyFlOl and PyFlli Pvu II-4 fragments were about 55 bp and 30 bp, respectively, larger than the wild-type Pvu II-4 fragment. Thus restriction enzyme analyses indicated that, while PyF441 DNA shows no detectable size difference from wild-type DNA, PyFl 01 and PyFl 11 DNAs contain additional nucleotides located within the Pvu II-4 fragment (see Figure
Results Selection of Polyoma Virus Mutants F9 cells were infected with wild-type polyoma at multiplicities of infection between 10 and 1000 pfu/cell, and infected cultures were passed every three days. Four days after each passage, the cells were assayed for polyoma T antigen by indirect immunofluorescence and for virus production by hemagglutination. No T antigen or virus was detected in initial passages of the infected F9 cells. In contrast, later passages did show T antigen immunofluorescence and hemagglutinating activity. The virus obtained from these later passages, when plated back onto fresh F9 cells, was able to induce polyoma T antigen within 48 hr after infection. Polyoma viruses capable of productive infection of F9 ceils were detected in later passages of all wildtype polyoma-infected F9 cultures. These mutant viruses are capable of productive infection of 3T6 cells, so they were plaque-purified and propagated on 3T6. Three plaque-purified mutants, designated PyFl 01, PyFlll and PyF441, were studied in more detail. These three PyF mutants were isolated from three independent infections of F9 cells with two separate stocks of wild-type polyoma.
2). Marker Rescue of PyF Phenotype To determine whether the alteration in the Pvu II-4 fragment of PyFlOl DNA was responsible for the ability of this mutant to infect F9 cells, we performed marker rescue experiments (Hutchinson and Edgell, 1971; Miller and Fried, 1976) by infecting F9 cells with ligated combinations of wild-type and mutant DNA fragments and then assaying for expression of polyoma T antigen by immunofluorescence. For ligation, we isolated the small and large fragments of wildtype and mutant DNAs generated by double digestion with the restriction enzymes Bcl I and Bgl I. The small fragment spans nucleotides 5046 clockwise to 101 (65.6-72.2 map units) and contains the entire Pvu II4 fragment (see Figure 2). The four combinations of wild-type and mutant small and large fragments were ligated, and the resulting DNAs were used to infect F9 cells in the presence of diethylaminoethyl (DEAE)dextran (McCutchan and Pagano, 1968). As shown in Table 1, the ability to express T antigen in F9 cells depended upon the small fragment of PyFlOi DNA. Ligated DNA lacking PyFlOl small fragment did not express polyoma T antigen after infection of F9. All four ligated combinations of small and large DNA fragments led to expression of T antigen in 3T6 cells (Table 1). To verify that the virus rescued in F9 cells was due to the altered small Bcl I/Bgl I fragment of
T Antigen Expression by PyF Mutants in F9 Cells F9 cells infected with wild-type polyoma at multiplicities of infection as high as 1000, when assayed 48 hr after infection for T antigen immunofluorescence, were consistently negative. No T antigen staining was observed after scanning numerous coverslips, each with more than lo5 cells. F9 cells infected for 48 hr with the PyF mutants exhibited T antigen immunofluorescence (data not shown). At a multiplicity of infection of 50, approximately 5-10% of F9 cells infected with either PyFl 01 or PyFl 11 and approximately 0.5 1 .O% of F9 cells infected with PyF441 were positive, by immunofluorescence, for T antigen 48 hr after infection. Analysis of PyF DNAs with Restriction Enzymes Wild-type and PyF mutant form I DNAs were digested with the restriction enzymes, Hpa II, Pvu II and Hae Ill, and the resulting DNA fragments were analyzed by Table DNA
1.
Rescue
Fragments
of PyFl
01 Phenotype
Ligated
by the Small Bcl I/Bgl
I Fragment
T Antigen
lmmunofluorescence
3T6
F9
Small
Large
No. of Cells
% T Antigen Positive
No. of Cells
% T Antigen Positive
Wild-type PyFl 01 PyFl 01 Wild-type
Wild-type PyFlOl Wild-type PyFl 01
1042 1015 1028 1040
4.3 5.3 8.8 6.8
* 2097 2106 *
0 4.5 7.0 0
* More
than
1 O5 cells
were
scanned
with no T antigen-positive
cells
seen.
Polyoma 811
Mutant
Infection
of F9
Ceils
PyFlOl DNA, virus produced from F9 infected with wild-type large fragment ligated to PyFl 01 small fragment was plated back onto fresh F9 cells. Forty-eight hours after infection, Hirt (1967) supernatants were prepared and digested with Hpa II. Gel electrophoresis showed that the Hpa II fragment pattern was identical to that of PyFl 01 DNA (data not shown). Marker rescue with PyF441 directly in F9 cells was not successful because of the low efficiency of infection. As an alternative (Katinka et al., 1980) ligated combinations of wild-type and PyF441 small and large Bcl I/Bgl I fragments were used to infect 3T6 cells in the presence of DEAE-dextran. Viruses arising from these DNA infections were used to infect F9 cells at a multiplicity of infection of 100 pfu/cell, and T antigen was assayed by indirect immunofluorescence 48 hr after infection. Virus arising in 3T6 cells from ligated DNA containing the small Bcl I/Bgl I fragment of PyF441 DNA induced 2-5% T antigen-positive nuclei in F9 cells, while virus arising from ligated DNA containing the small Bcl I/Bgl I fragment of wild-type DNA showed less than 0.1% T antigen-positive nuclei in FQ cells, indicating that the PyF441 phenotype is rescued more efficiently by the small Bcl I/Bgl I fragment.
1979, and Deininger et al., 1980, which begins at the TsA sequence near the origin of DNA replication and proceeds in the direction of early transcription; the sequences presented here are of the DNA strand with the same polarity as early mRNA.) The only difference between wild-type and PyF441 DNAs is at nucleotide 5258 (69.6 map units), where the wild-type A is changed to a G in PyF441. Both PyFl 01 and PyFl 11 DNAs also have a G at nucleotide 5258. In addition, these two mutants have exact tandem duplications of sequences surrounding the point mutation at nucleotide 5258. PyFlOl has a tandem duplication of 54 nucleotides from position 5211 to position 5264 (68.7-69.7 map units), while PyFl 11 has a tandem duplication of 31 nucleotides from position 5243 to position 5273 (69.3-69.8 map units). Both copies of the duplications in these two mutants contain the point mutation at nucleotide 5258. Other than these changes, there was no difference among the wild-type and mutant DNAs in the region sequenced. The nucleotide sequences for these DNAs from nucleotide 5201 to nucleotide 5295 (68.5-70.3 map units) are shown in Figure 1. The locations of the changes in PyF DNAs with respect to restriction maps of the polyoma virus genome are shown in Figure 2.
Nucleotide Sequences of PyF DNAs Restriction enzyme analyses and marker rescue suggested that nucleotide sequence changes within the Pvu II-4 fragments of PyF mutants were involved in allowing productive infection of FQ cells, so we sequenced wild-type and mutant DNAs from the Bcl I site through the Pvu II-4 fragment to the Bgl I site (see Figure 2). Our wild-type polyoma DNA sequence is identical to that reported by Deininger et al. (1979) for polyoma large plaque strain 3, except for a single base change of G to A at position 5180. (We used the nucleotide numbering system of Friedmann et al.,
Discussion Although polyoma and SV40 are capable of adsorbing to and penetrating EC cells (Swartzendruber et al., 1977; Boccara and Kelly, 1978), the infection process apparently is blocked at a stage before T antigen synthesis. The data of Segal et al. (1979) suggest that the block in FQ cells for SV40 infection occurs at the level of early RNA processing. Results cited by Vasseur et al. (1980) however, suggest that initiation of polyoma early transcription is blocked in PCC4 aza Pvu
Ibe Ill lb/M
z 0 1 .
: 2 1 .
hXi%GG
: 4 1 .
I
2’ 6 1 .
&UKiAGG
AAGCMMAG
tCTCTCUCC
CAGGCCTAGA iTGTTlCUC
(UGTGTGGTT
TlcCMUW
MGUAMAG
CCTCTCUCC
UGfXCliGA
UGGCCliGh . . i
1. Sequences
of Wild-type
and PyF Mutant
CUATUTTA
hUUAC
Ylld-Type
PyilOl
ATGT) .
Figure
z 8 1 .
UGTGTGGTT
(TCTCUCC
II
4/2
2 ATGTTTCUC
Pyflll
CU) 2
PyF441
DNAs
The nucleotide sequence of wild-type polyoma DNA between nucleotide positions 5201 (68.5 map units) and 5295 (70.3 map units) is shown. The changes found in the sequences of PyF mutant DNAs are the presence of a G instead of an A at position 5258 and tandem duplications, indicated by parentheses, in PyFl 01 and PyFl 1 1 DNAs. Sequences shown are of the strand of DNA having the same polarity as early mRNA. The orientation from left to right is 5’ to 3’. Arrows indicate Hae Ill and Pvu II restriction sites.
Cell 812
5000
5100
6300
5195/o
100
300
LlllIlll1
I
300 I
I
1
I ICI
I
w
3
Figure 2. Locations ative to the Physical
I
1
.
of Sequence Changes in PyF Mutant Map of the Polyoma Genome
nm. II
DNAs
Rel-
Fragment maps and the nucleotide numbering system for the polyoma genome are from Deininger et al. (1980). The polyoma genome is divided into 100 map units proceeding clockwise from the Eco RI site, nucleotide 1575 (Griffin et al., 1974). The origin of DNA replication (0s) and the directions and locations of early (E) and late (L) transcription are shown within the circular fragment maps. The expanded map in the upper part of the figure shows the region around the portion of the polyoma genome affected in the PyF mutants. The blocks represent the nucleotides that are tandemly duplicated in PyFlOl and PyFl 11 DNAs. Solid triangles (A) indicate the site of the point mutation from A to G at nucleotide 5258.
cells. Whatever the nature of the block or blocks to papovavirus infection of EC cells, our results, along with those of Vasseur et al. (1980) indicate that polyoma virus mutants able to overcome the block can be isolated. The three mutants, PyFl 01, PyFl 11 and PyF441, contain alterations in their DNAs within the Pvu II-4 fragment. Marker rescue shows that the DNA sequence changes in the Pvu II-4 fragments of PyF441 and PyFl 01 are responsible for the ability of these mutants to infect F9 cells. The alteration in PyF441 DNA consists of a single base pair change at 69.6 map units, indicating that a point mutation in the polyoma genome is sufficient to permit productive infection of F9 cells. Alterations of wild-type polyoma DNA sequences within the Pvu II-4 fragment (67.6-70.2 map units) apparently are necessary for generation of viral mutants that grow in EC cells. Polyoma mutants that grow in PCC4 aza EC cells have been isolated by Vasseur
et al. (1980) and two of these mutants, designated Py97 and Py204, have been characterized. Like the PyF mutants, Py97 and Py204 have DNA sequence changes around 69 map units, but these changes are more complicated than those present in the PyF mutants. Py97 deletes nucleotides 5214 to 5244, and its nucleotide 5258 is an A as in wild-type DNA (Katinka et al., 1980). Thus the DNA sequence changes in Py97 and Py204 are different from those in the PyF mutants. These differences may be significant for determining the host range of polyoma virus because Py97 and Py204 do not infect F9 cells (Vasseur et al., 1980). The function of the apparently noncoding region of polyoma DNA containing the PyF mutations is not known. This region is located near and to the late side of the postulated origin of viral DNA replication (Friedmann et al., 1979; Soeda et al., 1979) but its involvement in viral DNA replication has not been demonstrated. It has been suggested that PCC4 aza cells are defective in initiation of early transcription on wildtype polyoma DNA, and that the sequence rearrangements observed in Py97 and Py204 DNAs introduce new early promoter sites that are functional in PCC4 aza cells (Vasseur et al., 1980; Katinka et al., 1980). Although it is possible that the PyF mutations affect early transcriptional initiation in F9 cells, the effect of these mutations on other processes, such as posttranscriptional processing or initiation of viral DNA replication cannot be ruled out. Experimental
Procedures
Cell Culture and Virus Infections All cell cultures were grown in plastic tissue culture plates. We used Dulbecco’s modified Eagle’s medium (DME; 4.5 mg/ml glucose; Flow Laboratories) containing 50 units/ml penicillin, 50 pig/ml streptomycin, and either horse or calf serum. The mouse embryonal carcinoma cell line, F9 (Bernstine et al., 1973). was provided by R. Oshima and decontaminated of mycoplasma by his method (1978). F9 cells were grown in DME containing 10% horse serum. Mouse 3T6 cells were cultured in DME with 10% calf serum; polyoma virus-infected cultures of 3T6 were grown in DME with 5% calf serum. All cell cultures were at 37°C in a humidified atmosphere of 5-l 0% CO?. The wild-type polyoma virus used in this investigation is the large plaque strain designated WS by Hutchinson et al. (1978), which is identical to the wild-type strain 3 sequenced by Deininger et al. (1980). It was derived from a plaque isolate of polyoma strain LP (Eckhart, 1969; Friedmann et al., 1978). A stock of wild-type polyoma virus was obtained from W. Eckhart. We obtained all wild-type virus stocks by low multiplicity infection of 3T6. using this original virus stock or a plaque isolate of the original stock. Polyoma virus mutants selected in F9 cells were plaque purified on 3T6, and mutant stocks were prepared by amplification of plaque isolates by low multiplicity infection of 3T6. Virus infections were performed in DME buffered at pH 7 with 10 mM HEPES. After an adsorption period of 1.5-2 hr at 37OC with occasional rocking, infected cultures were fed with DME supplemented with the appropriate serum. Restriction Enzyme Digestions and Preparation of DNA Fragments Wild-type and mutant polyoma supercoiled form I DNA was purified from 3T6 cells infected at a multiplicity of infection of 1 O-20 pfu/cell for 48 hr by the selective extraction method of Hirt (1967) followed
Polyoma 813
Mutant
Infection
of F9 Cells
by equilibrium centrifugation in CsCl/ethidium bromide. Restriction endonucleases were obtained from New England Biolabs or Bethesda Research Laboratories and were used under conditions specified by the supplier. Msp I is an isoschizomer of Hpa II. Because they are identical, we refer to Msp I fragments of polyoma DNA as Hpa II fragments to be consistent with published fragment maps of polyoma DNA. Restricted DNA fragments were analyzed by electrophoresis in 1.4% agarose or 5% or 7.5% polyacrylamide (2O:i acrylamide:bisacrylamide) slab gels. Electrophoresis buffer for both agarose and acrylamide gels contained 40 mM Tris, 5mM NaOCOCH, and 1 mM EDTA (adjusted to pH 7.8 with HOCOCH,). To visualize DNA bands, gels were soaked approximately 30 min in electrophoresis buffer containing 0.5 pg/ml ethidium bromide, then transilluminated with an ultraviolet light source. For preparative fractionation of DNA fragments, desired samples were run in 1.4% agarose gels and stained with ethidium bromide. The regions of the gel containing DNA fragments were excised with a razor blade. The gel sections were solubilized in saturated KI solution (Blin et al., 1975). and DNA was separated from the agarose and KI by retention on hydroxyapatite. After elution from hydroxyapatite, the DNA fragments were concentrated by precipitation with cetyltrimethylammonium bromide (CETAB; Stehelin et al., 1976) and carrier yeast RNA. Residual CETAB was removed by several cycles of precipitation with 67% ethanol. DNA Infections For ligation, wild-type or mutant form I DNA was digested with Bgl I followed by Bcl I, and DNA fragments were purified as described above. Appropriate combinations of small and large Bgl I/Bcl I fragments ware ligated in reaction mixtures (50 pl) containing 50 mM Tris-Cl, pH 7.5, 10 mM MgCl>, 1 mM dithiothreitol, 0.1 mM ATP, 1 unit T4 DNA ligase (New England Biolabs) and a total of 0.5-l pg of DNA fragments at equimolar ratio. Reaction was at 13°C for 12-l 6 hr. After ligation, each reaction mixture was diluted with 0.4 ml of DME containing 50 mM Tris-Cl, pH 7.4, and 750 pg/ml DEAEdextran (McCutchan and Pagano, 1968). Cells to be infected with DNA were trypsinized and washed with phosphate-buffered saline, and 1 x lo5 washed cells were suspended in 0.2 ml of the ligated DNA solution. The suspension was left at room temperature for 30 min. washed once with growth medium and plated. 3T6 cells infected with DNA were plated directly onto gelatinized coverslips and were fixed in methanol and stained for T antigen immunofluorescence 3 days after infection. F9 cells were plated into 6 cm plastic dishes and were split I:20 after 2 days. Five days after infection, infected F9 cultures were split 1 :50 onto gelatinized coverslips. F9 coverslips were fixed and stained 7-8 days after infection. We assayed polyoma T antigen by indirect immunofluorescence, using rat antipolyoma tumor serum (provided by W. Eckhart) followed by fluorescein isothiocyanate-conjugated rabbit antirat IgG (Miles Laboratories). DNA Sequence Analysis DNA sequence analysis was carried out by the method of Maxam and Gilbert (1977), with modifications that have been described previously (Friedmann and Brown, 1978). End-labeled DNA fragments were prepared by incubation of either Hpa II or Bcl I cleaved DNAs with a-32P-deoxynucleotide triphosphates in the presence of reverse transcriptase. as has been previously described (Friedmann et al., 1978). Secondary cleavages were carried out by Barn HI, Bcl I, Hpa II or Hph I digestion, and fragments were separated and isolated from 5% (29:l acrylamide:bisacrylamide) polyacrylamide gel. The chemical degradation sequencing reactions were carried out as described previously and fractionated on 0.5 mm thick 20% and 8% (2O:l) gels of either 32 cm or 72 cm length. Autoradiography was carried out as described previously (Friedmann et al., 1978). Acknowledgments We are grateful to Drs. Walter Eckhart, Cole Manes and Robert Oshima for supplying materials used in this work and to Ms. Patty Saxen for help with preparation of the manuscript. This investigation was supported by grants from the National Cancer Institute and a
Cancer Research Coordinating Committee grant. F.K.F. was supported as a Leukemia Society of America fellow; P.L.D. was supported by a USPHS training grant; E.L. was supported as a Leukemia Society of America scholar. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
August
13, 1980;
revised
December
11, 1980.
References Berk, A. J. and Sharp, P. A. (1977). Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of SI endonuclease-digested hybrids. Cell 72, 721-732. Bernstine, E. G., Hooper, M. L.. Grandchamp, (1973). Alkaline phosphatase activity in mouse Acad. Sci. USA 70, 3899-3903.
S. and Ephrussi, B. teratoma. Proc. Nat.
Blin, N., Gabain. A. V. and Bujard. H. (1975). Isolation of large molecular weight DNA from agarose gels for further digestion by restriction enzymes. FEBS Letters 53, 84-86. Boccara, M. and Kelly, F. (1978). Expression of polyoma virus in heterocaryons between embryonal carcinoma cells and differentiated cells. Virology 90, 147-l 50. Deininger, P., Esty, A., LaPorte, P. and Friedmann. T. (1979). Nucleotide sequence and genetic organization of the polyoma late region: features common to the polyoma early region and SV40. Cell 18, 771-779. Deininger, P. L., Esty, A., LaPorte, P.. Hsu, H. and Friedmann, T. (1980). The nucleotide sequence and restriction enzyme sites of the polyoma genome. Nucl. Acids Res. 8, 855-860. Eckhart, W. (1969). Complementation and transformation by temperature-sensitive mutants of polyoma virus. Virology 38, 120-l 25. Freidmann, T. and Brown, D. (1978). Base-specific for DNA sequencing. Nucl. Acids Res. 5, 615-622.
reactions
Friedmann, T., LaPorte, P., and Esty, A. (1978). Nucleotide studies of polyoma DNA. J. Biol. Chem. 253, 6561-6567.
useful
sequence
Friedmann, T., Esty. A., LaPorte, P. and Deininger, P. (1979). The nucleotide sequence and genome organization of the polyoma early region: extensive nucleotide and amino acid homology with SV40. Cell 7 7. 715-724. Gautsch, function
J. W. (1980). Embryonal required for virus replication.
carcinoma stem cells lack Nature 285, 11 O-l 12.
a
Graham, C. F. (1977). Teratocarcinoma cells and normal mouse embryogenesis. In Concepts in Mammalian Embryogenesis, M. I. Sherman, ed. (Cambridge. Massachusetts: The MIT Press), pp. 315394. Griffin, physical
B. E.. Fried, M. and Cowie, A. (1974). Polyoma map. Proc. Nat. Acad. Sci. USA 77. 2077-2081.
Hirt, B. (1967). Selective extraction of polyoma mouse cell cultures. J. Mol. Biol. 26, 365-369.
DNA from
Hutchinson, C. A. and Edgell, M. H. (1971). Genetic fragments of bacteriophage +X1 74 deoxyribonucleic 181-189. Hutchinson. M. A., Hunter, T. and Eckhart, W. (1978). tion of T antigens in polyoma-infected and transformed 65-77. Jacob, F. (1978). Mouse teratocarcinoma R. Sot. Lond. B. 207, 249-270. Katinka, M., of polyoma depends on region. Cell
and mouse
DNA:
a
infected
assay for small acid. J. Virol. 8, Characterizacells. Cell 75, embryo.
Proc.
Yaniv, M., Vasseur, M. and Blangy, D. (1980). Expression early functions in mouse embryonal carcinoma cells sequence rearrangements in the beginning of the late 20. 393-399.
Kelly, F. and Boccara, M. (1976). cells to adenovirus type 2. Nature
Susceptibility 262, 409-411.
of teratocarcinoma
Cell 814
Kleinsmith. embryonal
L. J. and Pierce, G. 6. (1964). Multipotentiality carcinoma cells. Cancer Res. 24, 1544-l 552.
of single
Lehman, J. M., Klein, I. B. and Hackenberg. R. M. (1975). The response of murine teratocarcinoma cells to infection with DNA and RNA viruses. In Teratomas and Differentiation, M. I. Sherman and D. Salter, eds. (New York: Academic Press), pp. 289-301. Martin, G. R. (1975). study of embryogenesis
Teratocarcinomas and neoplasia.
as a model system Cell 5, 229-243.
Maxam, A. and Gilbert, W. (1977). A new method DNA. Proc. Nat. Acad. Sci. USA 74, 560-564.
for the
for sequencing
McCutchan, J. H. and Pagano, J. S. (1968). Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J. Nat. Cancer Inst. 4 7, 351-357. Miller, L. K. and Fried, M. (1976). Construction the polyoma genome. J. Virol. 18, 824-832.
of the genetic
map of
Miller, l?. A., Ward, D. C. and Ruddle, F. H. (1977). Embryonal carcinoma cells (and their somatic cell hybrids) are resistant to infection by the murine parvovirus MVM, which does infect other teratocarcinoma-derived cell lines. J. Cell. Physiol. 97, 393-402. Oldstone, M.B.A., Tishon, A., Dutko, F. J.. Kennedy, S.I.T., Holland, J. J. and Lampert, P. W. (1980). Does the major histocompatibility complex serve as a specific receptor for Semliki Forest virus? J. Virol. 34, 256-265. Oshima, R. (1978). Stimulation of the clonal growth and differentiation of feeder layer dependent mouse embryonal carcinoma cells by pmercaptoethanol. Differentiation 7 7, 149-l 55. Peries, J., Alves-Cardoso, E., Canivet, M., Debons-Guillemin, M. C. and Lasneret, J. (1977). Lack of multiplication of ecotropic C-type viruses in mouse teratocarcinoma primitive cells. J. Nat. Cancer Inst. .59,463-465. Pierce, concept
G. B. (1967). Teratocarcinoma: model for a developmental of cancer. Curr. Topics Dev. Biol. 2, 223-246.
Segal, S. and Khoury, G. (1979). Differentiation as a requirement for simian virus 40 gene expression in F-9 embryonal carcinoma cells. Proc. Nat. Acad. Sci. USA 76, 561 I-561 5. Segal, S.. Levine, A. J. and Khoury, spliced SV40 RNA in undifferentiated cells. Nature 280, 335-338.
G. (1979). Evidence for nonmurine teratocarcinoma stem
Soeda, E., Arrand, J.R., Smolar, N. and Griffin, B. E. (1979). Sequence from early region of polyoma virus DNA containing viral replication origin and encoding small, middle and (part of) large T antigens. Cell 17, 357-370. Stehelin, D., Guntaka, R. V., Varmus, H. E. and Bishop, J. M. (1976). Purification of DNA complementary to nucleotide sequences required for neoplastic transformation of fibroblasts by avian sarcoma viruses. J. Mol. Biol. 707, 349-365. Stevens, 31.
L. C. (1967).
The biology
of teratomas.
Adv. Morphol.
6, l-
Strickland, S. and Mahdavi, V. (1978). The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 75, 393-403. Swartzendruber. D. E., and Lehman, J. M. (1975). Neoplastic differentiation: interaction of simian virus 40 and polyoma virus with murine teratocarcinoma cells in vitro. J. Cell. Physiol. 85. 179-l 88. Swartzendruber, D. E., Friedrich, T. D. and Lehman, J. M. (1977). Resistance of teratocarcinoma stem cells to infection with simian virus 40: early events. J. Cell. Physiol. 93, 25-30. Teich, N. M., Weiss, R. A., Martin, G. R. and Lowy, D. R. (1977). Virus infection of murine teratocarcinoma stem cell lines. Cell 72, 973-982. Topp. W., Hall, J. D., Rifkin, D., Levine, A. J. and Pollack, R. (1977). The characterization of SV40-transformed cell lines derived from mouse teratocarcinoma: growth properties and differentiated characteristics. J. Cell. Physiol. 93, 269-276. Vasseur, M.. Kress, C.. Montreau. N. and Blangy, D. (1980). Isolation and characterization of polyoma virus mutants able to develop in embryonal carcinoma cells. Proc. Nat. Acad. Sci. USA 77, 10681072.
1981
by MIT
Mutation near the Polyoma DNA Replication Origin Permits Productive Infection of F9 Embryonal Carcinoma Cells Frank K. Fujimura,* Prescott L. Deininger,? Theodore Friedmannt and Elwood Linney* * La Jolla Cancer Research Foundation 2945 Science Park Road La Jolla, California 92037 t Department of Pediatrics M-009 University of California, San Diego La Jolla, California 92093
Summary F9 mouse embryonal carcinoma cells are resistant to productive infection by wild-type polyoma virus. Continued passage of F9 cells initially infected with wild-type polyoma virus eventually leads to the selection of polyoma virus mutants that are capable of productive infection of undifferentiated F9 cells. Three mutants, PyFlOl, PyFll 1 and PyF441, have been- plaque-purified and examined. All three PyF mutant DNAs are altered from the wild-type sequence in the Pvu II-4 fragment that spans 67.6 to 70.2 map units on the polyoma genome. PyF441 has a single base change of A to G at 69.6 map units: PyFl 01 and PyFl 11 DNAs also contain this point mutation at 69.6 map units. In addition, PyFl 01 and PyFl 11 DNAs have exact tandem duplications of 54 and 31 bp, respectively, of sequences encompassing the point mutation, and both copies of the tandem duplication have the point mutation. Other than these changes, no difference exists in the nucleotide sequences of wildtype and PyF mutant DNAs from the Bcl I site at 65.6 map units clockwise through the origin of viral DNA replication to the Bgl I site at 72.2 map units. DNA infections of F9 cells with wild-type-mutant hybrid DNAs formed by ligation of heterologous combinations of the small and large DNA fragments generated by double digestion with the restriction enzymes Bcl I and Bgl I show that the DNA sequence changes described above are responsible for the ability of the PyF mutants to infect F9 cells. Introduction Murine teratocarcinomas are malignant tumors usually containing a wide variety of somatic cell types. These tumors also contain a characteristic cell called embryonal carcinoma (EC). The EC cell is the stem cell of teratocarcinomas and can differentiate to form the somatic cell types present in these tumors (Kleinsmith and Pierce, 1964). Studies have indicated that EC cells have many similarities to multipotent cells of early mouse embryos and that some of the cellular changes accompanying differentiation of EC cells mimic events during normal embryonic differentiation
(Pierce, 1967; Stevens, 1967; Martin, 1975; Graham, 1977; Jacob, 1978). In addition to changes in expression of cellular genes, differentiation of EC cells leads to changes in expression of some viral genes. No viral tumor(T) antigens can be detected in undifferentiated EC cells after infection by simian virus 40 (SV40; Swartzendruber and Lehman, 1975; Topp et al., 1977) or polyoma virus (Swartzendruber and Lehman, 1975). Differentiated cells arising from EC cells can allow papovavirus T antigen expression and, in the case of polyoma, can support lytic infection. Similarly, EC cells are resistant to minute virus of mouse (Miller et al., 1977) and C-type RNA tumor viruses (Peries et al., 1977; Teich et al., 1977; Gautsch, 1980), while some differentiated mouse cells are sensitive to these viruses. Viral resistance is not a general phenomenon in EC cells, as these cells will support growth of a number of other viruses (Lehman et al., 1975; Kelly and Boccara, 1976; Teich et al., 1977; Oldstone et al., 1980). The nature of the block to papovavirus infection of EC cells is not known. Swartzendruber et al. (1977) showed that SV40 and polyoma adsorption and penetration take place in EC cells. Fusion of polyomainfected EC cells with baby hamster kidney cells leads to both early and late viral gene expression (Boccara and Kelly, 1978), while fusion of SV40-infected EC cells with permissive monkey cells does not rescue expression of SV40 T antigen (Swartzendruber et al., 1977). Segal et al. (1979) examined F9 EC cells infected with SV40 and found low levels of early viral RNA that, when analyzed by the method of Berk and Sharp (1977), was unspliced. F9 cells allowed to differentiate in the presence of retinoic acid as described by Strickland and Mahdavi (1978), when infected with SV40, contain spliced early viral RNA (Segal and Khoury, 1979). Unpublished results cited by Vasseur et al. (1980) for polyoma-infected PCC4 aza EC cells indicated that the low level of viral RNA detected in these infected cells was spliced. During our investigation of the interactions of polyoma virus with EC cells, we found polyoma mutants that are capable of productive infection of F9 EC cells. This report describes the selection and characterization of these mutants. Our data suggest that the significant change affecting the ability of polyoma virus to infect F9 cells is a point mutation at 69.6 map units in a noncoding region near and to the late side of the origin of DNA replication. Tandem duplications of DNA sequences including the point mutation appear to increase the efficiency of polyoma growth in F9 cells. The nature of these nucleotide changes permitting polyoma infection of F9 cells differs from the more extensive rearrangements Katinka et al. (1980) recently reported to be present in the DNAs of polyoma virus mutants that infect another EC cell line, PCC4 aza (Vasseur et al, 1980). ’
Cell 810
gel electrophoresis (data not shown). No difference was detected between wild-type and PyF441 DNAs in the Hpa II, Pvu II or Hae Ill fragments. The only detectable difference in the Hpa II fragments of wildtype and PyFl 01 DNAs was that the PyFl 01 Hpa II-3 fragment was about 55 bp larger than the corresponding wild-type fragment. Similarly, the PyFl 11 Hpa II-3 fragment was about 30 bp larger than the wild-type Hpa II-3 fragment. Digestion with Pvu II indicated that the PyFlOl and PyFlli Pvu II-4 fragments were about 55 bp and 30 bp, respectively, larger than the wild-type Pvu II-4 fragment. Thus restriction enzyme analyses indicated that, while PyF441 DNA shows no detectable size difference from wild-type DNA, PyFl 01 and PyFl 11 DNAs contain additional nucleotides located within the Pvu II-4 fragment (see Figure
Results Selection of Polyoma Virus Mutants F9 cells were infected with wild-type polyoma at multiplicities of infection between 10 and 1000 pfu/cell, and infected cultures were passed every three days. Four days after each passage, the cells were assayed for polyoma T antigen by indirect immunofluorescence and for virus production by hemagglutination. No T antigen or virus was detected in initial passages of the infected F9 cells. In contrast, later passages did show T antigen immunofluorescence and hemagglutinating activity. The virus obtained from these later passages, when plated back onto fresh F9 cells, was able to induce polyoma T antigen within 48 hr after infection. Polyoma viruses capable of productive infection of F9 ceils were detected in later passages of all wildtype polyoma-infected F9 cultures. These mutant viruses are capable of productive infection of 3T6 cells, so they were plaque-purified and propagated on 3T6. Three plaque-purified mutants, designated PyFl 01, PyFlll and PyF441, were studied in more detail. These three PyF mutants were isolated from three independent infections of F9 cells with two separate stocks of wild-type polyoma.
2). Marker Rescue of PyF Phenotype To determine whether the alteration in the Pvu II-4 fragment of PyFlOl DNA was responsible for the ability of this mutant to infect F9 cells, we performed marker rescue experiments (Hutchinson and Edgell, 1971; Miller and Fried, 1976) by infecting F9 cells with ligated combinations of wild-type and mutant DNA fragments and then assaying for expression of polyoma T antigen by immunofluorescence. For ligation, we isolated the small and large fragments of wildtype and mutant DNAs generated by double digestion with the restriction enzymes Bcl I and Bgl I. The small fragment spans nucleotides 5046 clockwise to 101 (65.6-72.2 map units) and contains the entire Pvu II4 fragment (see Figure 2). The four combinations of wild-type and mutant small and large fragments were ligated, and the resulting DNAs were used to infect F9 cells in the presence of diethylaminoethyl (DEAE)dextran (McCutchan and Pagano, 1968). As shown in Table 1, the ability to express T antigen in F9 cells depended upon the small fragment of PyFlOi DNA. Ligated DNA lacking PyFlOl small fragment did not express polyoma T antigen after infection of F9. All four ligated combinations of small and large DNA fragments led to expression of T antigen in 3T6 cells (Table 1). To verify that the virus rescued in F9 cells was due to the altered small Bcl I/Bgl I fragment of
T Antigen Expression by PyF Mutants in F9 Cells F9 cells infected with wild-type polyoma at multiplicities of infection as high as 1000, when assayed 48 hr after infection for T antigen immunofluorescence, were consistently negative. No T antigen staining was observed after scanning numerous coverslips, each with more than lo5 cells. F9 cells infected for 48 hr with the PyF mutants exhibited T antigen immunofluorescence (data not shown). At a multiplicity of infection of 50, approximately 5-10% of F9 cells infected with either PyFl 01 or PyFl 11 and approximately 0.5 1 .O% of F9 cells infected with PyF441 were positive, by immunofluorescence, for T antigen 48 hr after infection. Analysis of PyF DNAs with Restriction Enzymes Wild-type and PyF mutant form I DNAs were digested with the restriction enzymes, Hpa II, Pvu II and Hae Ill, and the resulting DNA fragments were analyzed by Table DNA
1.
Rescue
Fragments
of PyFl
01 Phenotype
Ligated
by the Small Bcl I/Bgl
I Fragment
T Antigen
lmmunofluorescence
3T6
F9
Small
Large
No. of Cells
% T Antigen Positive
No. of Cells
% T Antigen Positive
Wild-type PyFl 01 PyFl 01 Wild-type
Wild-type PyFlOl Wild-type PyFl 01
1042 1015 1028 1040
4.3 5.3 8.8 6.8
* 2097 2106 *
0 4.5 7.0 0
* More
than
1 O5 cells
were
scanned
with no T antigen-positive
cells
seen.
Polyoma 811
Mutant
Infection
of F9
Ceils
PyFlOl DNA, virus produced from F9 infected with wild-type large fragment ligated to PyFl 01 small fragment was plated back onto fresh F9 cells. Forty-eight hours after infection, Hirt (1967) supernatants were prepared and digested with Hpa II. Gel electrophoresis showed that the Hpa II fragment pattern was identical to that of PyFl 01 DNA (data not shown). Marker rescue with PyF441 directly in F9 cells was not successful because of the low efficiency of infection. As an alternative (Katinka et al., 1980) ligated combinations of wild-type and PyF441 small and large Bcl I/Bgl I fragments were used to infect 3T6 cells in the presence of DEAE-dextran. Viruses arising from these DNA infections were used to infect F9 cells at a multiplicity of infection of 100 pfu/cell, and T antigen was assayed by indirect immunofluorescence 48 hr after infection. Virus arising in 3T6 cells from ligated DNA containing the small Bcl I/Bgl I fragment of PyF441 DNA induced 2-5% T antigen-positive nuclei in F9 cells, while virus arising from ligated DNA containing the small Bcl I/Bgl I fragment of wild-type DNA showed less than 0.1% T antigen-positive nuclei in FQ cells, indicating that the PyF441 phenotype is rescued more efficiently by the small Bcl I/Bgl I fragment.
1979, and Deininger et al., 1980, which begins at the TsA sequence near the origin of DNA replication and proceeds in the direction of early transcription; the sequences presented here are of the DNA strand with the same polarity as early mRNA.) The only difference between wild-type and PyF441 DNAs is at nucleotide 5258 (69.6 map units), where the wild-type A is changed to a G in PyF441. Both PyFl 01 and PyFl 11 DNAs also have a G at nucleotide 5258. In addition, these two mutants have exact tandem duplications of sequences surrounding the point mutation at nucleotide 5258. PyFlOl has a tandem duplication of 54 nucleotides from position 5211 to position 5264 (68.7-69.7 map units), while PyFl 11 has a tandem duplication of 31 nucleotides from position 5243 to position 5273 (69.3-69.8 map units). Both copies of the duplications in these two mutants contain the point mutation at nucleotide 5258. Other than these changes, there was no difference among the wild-type and mutant DNAs in the region sequenced. The nucleotide sequences for these DNAs from nucleotide 5201 to nucleotide 5295 (68.5-70.3 map units) are shown in Figure 1. The locations of the changes in PyF DNAs with respect to restriction maps of the polyoma virus genome are shown in Figure 2.
Nucleotide Sequences of PyF DNAs Restriction enzyme analyses and marker rescue suggested that nucleotide sequence changes within the Pvu II-4 fragments of PyF mutants were involved in allowing productive infection of FQ cells, so we sequenced wild-type and mutant DNAs from the Bcl I site through the Pvu II-4 fragment to the Bgl I site (see Figure 2). Our wild-type polyoma DNA sequence is identical to that reported by Deininger et al. (1979) for polyoma large plaque strain 3, except for a single base change of G to A at position 5180. (We used the nucleotide numbering system of Friedmann et al.,
Discussion Although polyoma and SV40 are capable of adsorbing to and penetrating EC cells (Swartzendruber et al., 1977; Boccara and Kelly, 1978), the infection process apparently is blocked at a stage before T antigen synthesis. The data of Segal et al. (1979) suggest that the block in FQ cells for SV40 infection occurs at the level of early RNA processing. Results cited by Vasseur et al. (1980) however, suggest that initiation of polyoma early transcription is blocked in PCC4 aza Pvu
Ibe Ill lb/M
z 0 1 .
: 2 1 .
hXi%GG
: 4 1 .
I
2’ 6 1 .
&UKiAGG
AAGCMMAG
tCTCTCUCC
CAGGCCTAGA iTGTTlCUC
(UGTGTGGTT
TlcCMUW
MGUAMAG
CCTCTCUCC
UGfXCliGA
UGGCCliGh . . i
1. Sequences
of Wild-type
and PyF Mutant
CUATUTTA
hUUAC
Ylld-Type
PyilOl
ATGT) .
Figure
z 8 1 .
UGTGTGGTT
(TCTCUCC
II
4/2
2 ATGTTTCUC
Pyflll
CU) 2
PyF441
DNAs
The nucleotide sequence of wild-type polyoma DNA between nucleotide positions 5201 (68.5 map units) and 5295 (70.3 map units) is shown. The changes found in the sequences of PyF mutant DNAs are the presence of a G instead of an A at position 5258 and tandem duplications, indicated by parentheses, in PyFl 01 and PyFl 1 1 DNAs. Sequences shown are of the strand of DNA having the same polarity as early mRNA. The orientation from left to right is 5’ to 3’. Arrows indicate Hae Ill and Pvu II restriction sites.
Cell 812
5000
5100
6300
5195/o
100
300
LlllIlll1
I
300 I
I
1
I ICI
I
w
3
Figure 2. Locations ative to the Physical
I
1
.
of Sequence Changes in PyF Mutant Map of the Polyoma Genome
nm. II
DNAs
Rel-
Fragment maps and the nucleotide numbering system for the polyoma genome are from Deininger et al. (1980). The polyoma genome is divided into 100 map units proceeding clockwise from the Eco RI site, nucleotide 1575 (Griffin et al., 1974). The origin of DNA replication (0s) and the directions and locations of early (E) and late (L) transcription are shown within the circular fragment maps. The expanded map in the upper part of the figure shows the region around the portion of the polyoma genome affected in the PyF mutants. The blocks represent the nucleotides that are tandemly duplicated in PyFlOl and PyFl 11 DNAs. Solid triangles (A) indicate the site of the point mutation from A to G at nucleotide 5258.
cells. Whatever the nature of the block or blocks to papovavirus infection of EC cells, our results, along with those of Vasseur et al. (1980) indicate that polyoma virus mutants able to overcome the block can be isolated. The three mutants, PyFl 01, PyFl 11 and PyF441, contain alterations in their DNAs within the Pvu II-4 fragment. Marker rescue shows that the DNA sequence changes in the Pvu II-4 fragments of PyF441 and PyFl 01 are responsible for the ability of these mutants to infect F9 cells. The alteration in PyF441 DNA consists of a single base pair change at 69.6 map units, indicating that a point mutation in the polyoma genome is sufficient to permit productive infection of F9 cells. Alterations of wild-type polyoma DNA sequences within the Pvu II-4 fragment (67.6-70.2 map units) apparently are necessary for generation of viral mutants that grow in EC cells. Polyoma mutants that grow in PCC4 aza EC cells have been isolated by Vasseur
et al. (1980) and two of these mutants, designated Py97 and Py204, have been characterized. Like the PyF mutants, Py97 and Py204 have DNA sequence changes around 69 map units, but these changes are more complicated than those present in the PyF mutants. Py97 deletes nucleotides 5214 to 5244, and its nucleotide 5258 is an A as in wild-type DNA (Katinka et al., 1980). Thus the DNA sequence changes in Py97 and Py204 are different from those in the PyF mutants. These differences may be significant for determining the host range of polyoma virus because Py97 and Py204 do not infect F9 cells (Vasseur et al., 1980). The function of the apparently noncoding region of polyoma DNA containing the PyF mutations is not known. This region is located near and to the late side of the postulated origin of viral DNA replication (Friedmann et al., 1979; Soeda et al., 1979) but its involvement in viral DNA replication has not been demonstrated. It has been suggested that PCC4 aza cells are defective in initiation of early transcription on wildtype polyoma DNA, and that the sequence rearrangements observed in Py97 and Py204 DNAs introduce new early promoter sites that are functional in PCC4 aza cells (Vasseur et al., 1980; Katinka et al., 1980). Although it is possible that the PyF mutations affect early transcriptional initiation in F9 cells, the effect of these mutations on other processes, such as posttranscriptional processing or initiation of viral DNA replication cannot be ruled out. Experimental
Procedures
Cell Culture and Virus Infections All cell cultures were grown in plastic tissue culture plates. We used Dulbecco’s modified Eagle’s medium (DME; 4.5 mg/ml glucose; Flow Laboratories) containing 50 units/ml penicillin, 50 pig/ml streptomycin, and either horse or calf serum. The mouse embryonal carcinoma cell line, F9 (Bernstine et al., 1973). was provided by R. Oshima and decontaminated of mycoplasma by his method (1978). F9 cells were grown in DME containing 10% horse serum. Mouse 3T6 cells were cultured in DME with 10% calf serum; polyoma virus-infected cultures of 3T6 were grown in DME with 5% calf serum. All cell cultures were at 37°C in a humidified atmosphere of 5-l 0% CO?. The wild-type polyoma virus used in this investigation is the large plaque strain designated WS by Hutchinson et al. (1978), which is identical to the wild-type strain 3 sequenced by Deininger et al. (1980). It was derived from a plaque isolate of polyoma strain LP (Eckhart, 1969; Friedmann et al., 1978). A stock of wild-type polyoma virus was obtained from W. Eckhart. We obtained all wild-type virus stocks by low multiplicity infection of 3T6. using this original virus stock or a plaque isolate of the original stock. Polyoma virus mutants selected in F9 cells were plaque purified on 3T6, and mutant stocks were prepared by amplification of plaque isolates by low multiplicity infection of 3T6. Virus infections were performed in DME buffered at pH 7 with 10 mM HEPES. After an adsorption period of 1.5-2 hr at 37OC with occasional rocking, infected cultures were fed with DME supplemented with the appropriate serum. Restriction Enzyme Digestions and Preparation of DNA Fragments Wild-type and mutant polyoma supercoiled form I DNA was purified from 3T6 cells infected at a multiplicity of infection of 1 O-20 pfu/cell for 48 hr by the selective extraction method of Hirt (1967) followed
Polyoma 813
Mutant
Infection
of F9 Cells
by equilibrium centrifugation in CsCl/ethidium bromide. Restriction endonucleases were obtained from New England Biolabs or Bethesda Research Laboratories and were used under conditions specified by the supplier. Msp I is an isoschizomer of Hpa II. Because they are identical, we refer to Msp I fragments of polyoma DNA as Hpa II fragments to be consistent with published fragment maps of polyoma DNA. Restricted DNA fragments were analyzed by electrophoresis in 1.4% agarose or 5% or 7.5% polyacrylamide (2O:i acrylamide:bisacrylamide) slab gels. Electrophoresis buffer for both agarose and acrylamide gels contained 40 mM Tris, 5mM NaOCOCH, and 1 mM EDTA (adjusted to pH 7.8 with HOCOCH,). To visualize DNA bands, gels were soaked approximately 30 min in electrophoresis buffer containing 0.5 pg/ml ethidium bromide, then transilluminated with an ultraviolet light source. For preparative fractionation of DNA fragments, desired samples were run in 1.4% agarose gels and stained with ethidium bromide. The regions of the gel containing DNA fragments were excised with a razor blade. The gel sections were solubilized in saturated KI solution (Blin et al., 1975). and DNA was separated from the agarose and KI by retention on hydroxyapatite. After elution from hydroxyapatite, the DNA fragments were concentrated by precipitation with cetyltrimethylammonium bromide (CETAB; Stehelin et al., 1976) and carrier yeast RNA. Residual CETAB was removed by several cycles of precipitation with 67% ethanol. DNA Infections For ligation, wild-type or mutant form I DNA was digested with Bgl I followed by Bcl I, and DNA fragments were purified as described above. Appropriate combinations of small and large Bgl I/Bcl I fragments ware ligated in reaction mixtures (50 pl) containing 50 mM Tris-Cl, pH 7.5, 10 mM MgCl>, 1 mM dithiothreitol, 0.1 mM ATP, 1 unit T4 DNA ligase (New England Biolabs) and a total of 0.5-l pg of DNA fragments at equimolar ratio. Reaction was at 13°C for 12-l 6 hr. After ligation, each reaction mixture was diluted with 0.4 ml of DME containing 50 mM Tris-Cl, pH 7.4, and 750 pg/ml DEAEdextran (McCutchan and Pagano, 1968). Cells to be infected with DNA were trypsinized and washed with phosphate-buffered saline, and 1 x lo5 washed cells were suspended in 0.2 ml of the ligated DNA solution. The suspension was left at room temperature for 30 min. washed once with growth medium and plated. 3T6 cells infected with DNA were plated directly onto gelatinized coverslips and were fixed in methanol and stained for T antigen immunofluorescence 3 days after infection. F9 cells were plated into 6 cm plastic dishes and were split I:20 after 2 days. Five days after infection, infected F9 cultures were split 1 :50 onto gelatinized coverslips. F9 coverslips were fixed and stained 7-8 days after infection. We assayed polyoma T antigen by indirect immunofluorescence, using rat antipolyoma tumor serum (provided by W. Eckhart) followed by fluorescein isothiocyanate-conjugated rabbit antirat IgG (Miles Laboratories). DNA Sequence Analysis DNA sequence analysis was carried out by the method of Maxam and Gilbert (1977), with modifications that have been described previously (Friedmann and Brown, 1978). End-labeled DNA fragments were prepared by incubation of either Hpa II or Bcl I cleaved DNAs with a-32P-deoxynucleotide triphosphates in the presence of reverse transcriptase. as has been previously described (Friedmann et al., 1978). Secondary cleavages were carried out by Barn HI, Bcl I, Hpa II or Hph I digestion, and fragments were separated and isolated from 5% (29:l acrylamide:bisacrylamide) polyacrylamide gel. The chemical degradation sequencing reactions were carried out as described previously and fractionated on 0.5 mm thick 20% and 8% (2O:l) gels of either 32 cm or 72 cm length. Autoradiography was carried out as described previously (Friedmann et al., 1978). Acknowledgments We are grateful to Drs. Walter Eckhart, Cole Manes and Robert Oshima for supplying materials used in this work and to Ms. Patty Saxen for help with preparation of the manuscript. This investigation was supported by grants from the National Cancer Institute and a
Cancer Research Coordinating Committee grant. F.K.F. was supported as a Leukemia Society of America fellow; P.L.D. was supported by a USPHS training grant; E.L. was supported as a Leukemia Society of America scholar. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
August
13, 1980;
revised
December
11, 1980.
References Berk, A. J. and Sharp, P. A. (1977). Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of SI endonuclease-digested hybrids. Cell 72, 721-732. Bernstine, E. G., Hooper, M. L.. Grandchamp, (1973). Alkaline phosphatase activity in mouse Acad. Sci. USA 70, 3899-3903.
S. and Ephrussi, B. teratoma. Proc. Nat.
Blin, N., Gabain. A. V. and Bujard. H. (1975). Isolation of large molecular weight DNA from agarose gels for further digestion by restriction enzymes. FEBS Letters 53, 84-86. Boccara, M. and Kelly, F. (1978). Expression of polyoma virus in heterocaryons between embryonal carcinoma cells and differentiated cells. Virology 90, 147-l 50. Deininger, P., Esty, A., LaPorte, P. and Friedmann. T. (1979). Nucleotide sequence and genetic organization of the polyoma late region: features common to the polyoma early region and SV40. Cell 18, 771-779. Deininger, P. L., Esty, A., LaPorte, P.. Hsu, H. and Friedmann, T. (1980). The nucleotide sequence and restriction enzyme sites of the polyoma genome. Nucl. Acids Res. 8, 855-860. Eckhart, W. (1969). Complementation and transformation by temperature-sensitive mutants of polyoma virus. Virology 38, 120-l 25. Freidmann, T. and Brown, D. (1978). Base-specific for DNA sequencing. Nucl. Acids Res. 5, 615-622.
reactions
Friedmann, T., LaPorte, P., and Esty, A. (1978). Nucleotide studies of polyoma DNA. J. Biol. Chem. 253, 6561-6567.
useful
sequence
Friedmann, T., Esty. A., LaPorte, P. and Deininger, P. (1979). The nucleotide sequence and genome organization of the polyoma early region: extensive nucleotide and amino acid homology with SV40. Cell 7 7. 715-724. Gautsch, function
J. W. (1980). Embryonal required for virus replication.
carcinoma stem cells lack Nature 285, 11 O-l 12.
a
Graham, C. F. (1977). Teratocarcinoma cells and normal mouse embryogenesis. In Concepts in Mammalian Embryogenesis, M. I. Sherman, ed. (Cambridge. Massachusetts: The MIT Press), pp. 315394. Griffin, physical
B. E.. Fried, M. and Cowie, A. (1974). Polyoma map. Proc. Nat. Acad. Sci. USA 77. 2077-2081.
Hirt, B. (1967). Selective extraction of polyoma mouse cell cultures. J. Mol. Biol. 26, 365-369.
DNA from
Hutchinson, C. A. and Edgell, M. H. (1971). Genetic fragments of bacteriophage +X1 74 deoxyribonucleic 181-189. Hutchinson. M. A., Hunter, T. and Eckhart, W. (1978). tion of T antigens in polyoma-infected and transformed 65-77. Jacob, F. (1978). Mouse teratocarcinoma R. Sot. Lond. B. 207, 249-270. Katinka, M., of polyoma depends on region. Cell
and mouse
DNA:
a
infected
assay for small acid. J. Virol. 8, Characterizacells. Cell 75, embryo.
Proc.
Yaniv, M., Vasseur, M. and Blangy, D. (1980). Expression early functions in mouse embryonal carcinoma cells sequence rearrangements in the beginning of the late 20. 393-399.
Kelly, F. and Boccara, M. (1976). cells to adenovirus type 2. Nature
Susceptibility 262, 409-411.
of teratocarcinoma
Cell 814
Kleinsmith. embryonal
L. J. and Pierce, G. 6. (1964). Multipotentiality carcinoma cells. Cancer Res. 24, 1544-l 552.
of single
Lehman, J. M., Klein, I. B. and Hackenberg. R. M. (1975). The response of murine teratocarcinoma cells to infection with DNA and RNA viruses. In Teratomas and Differentiation, M. I. Sherman and D. Salter, eds. (New York: Academic Press), pp. 289-301. Martin, G. R. (1975). study of embryogenesis
Teratocarcinomas and neoplasia.
as a model system Cell 5, 229-243.
Maxam, A. and Gilbert, W. (1977). A new method DNA. Proc. Nat. Acad. Sci. USA 74, 560-564.
for the
for sequencing
McCutchan, J. H. and Pagano, J. S. (1968). Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J. Nat. Cancer Inst. 4 7, 351-357. Miller, L. K. and Fried, M. (1976). Construction the polyoma genome. J. Virol. 18, 824-832.
of the genetic
map of
Miller, l?. A., Ward, D. C. and Ruddle, F. H. (1977). Embryonal carcinoma cells (and their somatic cell hybrids) are resistant to infection by the murine parvovirus MVM, which does infect other teratocarcinoma-derived cell lines. J. Cell. Physiol. 97, 393-402. Oldstone, M.B.A., Tishon, A., Dutko, F. J.. Kennedy, S.I.T., Holland, J. J. and Lampert, P. W. (1980). Does the major histocompatibility complex serve as a specific receptor for Semliki Forest virus? J. Virol. 34, 256-265. Oshima, R. (1978). Stimulation of the clonal growth and differentiation of feeder layer dependent mouse embryonal carcinoma cells by pmercaptoethanol. Differentiation 7 7, 149-l 55. Peries, J., Alves-Cardoso, E., Canivet, M., Debons-Guillemin, M. C. and Lasneret, J. (1977). Lack of multiplication of ecotropic C-type viruses in mouse teratocarcinoma primitive cells. J. Nat. Cancer Inst. .59,463-465. Pierce, concept
G. B. (1967). Teratocarcinoma: model for a developmental of cancer. Curr. Topics Dev. Biol. 2, 223-246.
Segal, S. and Khoury, G. (1979). Differentiation as a requirement for simian virus 40 gene expression in F-9 embryonal carcinoma cells. Proc. Nat. Acad. Sci. USA 76, 561 I-561 5. Segal, S.. Levine, A. J. and Khoury, spliced SV40 RNA in undifferentiated cells. Nature 280, 335-338.
G. (1979). Evidence for nonmurine teratocarcinoma stem
Soeda, E., Arrand, J.R., Smolar, N. and Griffin, B. E. (1979). Sequence from early region of polyoma virus DNA containing viral replication origin and encoding small, middle and (part of) large T antigens. Cell 17, 357-370. Stehelin, D., Guntaka, R. V., Varmus, H. E. and Bishop, J. M. (1976). Purification of DNA complementary to nucleotide sequences required for neoplastic transformation of fibroblasts by avian sarcoma viruses. J. Mol. Biol. 707, 349-365. Stevens, 31.
L. C. (1967).
The biology
of teratomas.
Adv. Morphol.
6, l-
Strickland, S. and Mahdavi, V. (1978). The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 75, 393-403. Swartzendruber. D. E., and Lehman, J. M. (1975). Neoplastic differentiation: interaction of simian virus 40 and polyoma virus with murine teratocarcinoma cells in vitro. J. Cell. Physiol. 85. 179-l 88. Swartzendruber, D. E., Friedrich, T. D. and Lehman, J. M. (1977). Resistance of teratocarcinoma stem cells to infection with simian virus 40: early events. J. Cell. Physiol. 93, 25-30. Teich, N. M., Weiss, R. A., Martin, G. R. and Lowy, D. R. (1977). Virus infection of murine teratocarcinoma stem cell lines. Cell 72, 973-982. Topp. W., Hall, J. D., Rifkin, D., Levine, A. J. and Pollack, R. (1977). The characterization of SV40-transformed cell lines derived from mouse teratocarcinoma: growth properties and differentiated characteristics. J. Cell. Physiol. 93, 269-276. Vasseur, M.. Kress, C.. Montreau. N. and Blangy, D. (1980). Isolation and characterization of polyoma virus mutants able to develop in embryonal carcinoma cells. Proc. Nat. Acad. Sci. USA 77, 10681072.