INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 71

Integration of Oncogenic Viruses in Mammalian Cells

CARLO M. CROCE

The Wisfar Institute of Anatomy and Biolog-v, Philadelphia, Pennsylvania

1. Introduction . . . . . . . . . . . . . . . . . . . . 11. Somatic Cell Hybridization Techniques to Map Human Genes . .

IV. Expression of the Transformed Phenotype and of Tumorigenicity in Hybrids between Normal Rodent Cells and SV40 Transformed Human Cells . . . . . . . . . . . . . . . . . . .

V. Integration of Other Oncogenic Viruses in Transformed Cells . . VI. Does the Integration of Tumor Virus DNA in the Chromosomal DNA

Occur by DNA Homology? . . . . . . . . . . . . . . VII. DNA-Mediated Cell Transformation of Mouse Teratocarcinoma

Cells . . . . . . . . . . . . . . . . . . . . . . VIIl. Conclusion . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . .

1Il. Somatic Cell Hybrids to Map Viral Integration Sites . . . . .

I 2 2

6 6

10

1 1 14 16

I. Introduction

Cells transformed by either RNA- or DNA-containing oncogenic viruses retain the genomes of the transforming viruses and, in general, express antigens, coded, at least in part, by the viral genomes (Black et af., 1963; Tegtmeyer, 1974; Lai and Nathans, 1974; Cooper and Temin, 1974). Nucleic acid hybridiza- tion techniques have been used to determine the state of the genomes of the tumor viruses such as Simian virus 40 (SV40) in transformed cells (Sambrook et al., 1968). These studies indicated that viral DNA sequences are integrated in the cellular DNA of viral-transformed cells (Sambrook et af., 1968; Croce et af., 1973, 1974). Since these early studies additional investigations have shown that all cells transformed by oncogenic DNA tumor viruses and by RNA tumor viruses such as avian and murine sarcoma and leukemia viruses contain viral DNA sequences integrated in the cellular DNA (Sambrook et al., 1968; Varmus et al., 1973; Kettner and Kelly, 1975; Botchan et al., 1976). In the case of RNA-containing viruses, DNA copies of the viral RNA are transcribed by viral RNA-dependent DNA plymerases (Temin, 1971; Temin and Baltimore, 1972) and the resulting complementary DNAs become covalently integrated in the cellular DNA (Varmus et al., 1973, 1974). Deoxyribonucleic acid, derived from mam- malian cells transformed by the RNA-containing Rous Sarcoma virus, has also

1 Copyright @ 1981 by Academic h a s , h c .

All rights of reproduction in any form reserved. ISBN 0-12-364471-2

2 CARL0 M . CROCE

been shown to contain the genome of the virus since it could be used to transfer the viral genetic information into uninfected chicken cells (this virus is of avian origin and can replicate only in certain chicken cells) (Hill and Hillova, 1972; Hill er al . , 1974; Cooper and Temin, 1974). Genomes of RNA-containing tumor viruses are present, however, not only in transformed cells, but also in normal cells (Huebner et a / . . 1970; Aaronson et af., 1969; Lowry et al., 1971; Hanafusa et a / . , 1974). These genomes appear to segregate in mammalian fash- ion (Rowe. 1972, 1973; Rowe arid Hartley, 1972; Rowe and Pincus, 1972; Rowe et d., 1972a.b) and, therefore, appear to be part of the genetic make-up of most of mammalian cells with the possible exception of those of human origin, since no bonufide human RNA tumor viruses have been discovered at the present time.

We intended to investigate the state of the viral genome in virus transformed cells in order to determine:

1. Whether the viral genomes are chromosomally integrated in transformed

2. Whether the integration of the viral genome is chromosome specific. cells.

For this purpose we used somatic cell hybridization techniques.

11. Somatic Cell Hybridization Techniques to Map Human Genes

Somatic cell hybrids between human and rodent cells have been used to map human genes to their specific chromosomes (Rudde, 1973; Croce er al., 1979). This was made possible by the finding that rodent-human hybrids, in general, segregate human chromosomes (Weiss and Green, 1967; Croce, 1976). If the expression of a human specific phenotype such as an enzyme segregates concor- dantly with a specific human chromosome in a relatively large number of inde- pendent rodent-human hybrids, it can be concluded that the gene coding for that phenotype is located on that human chromosome. In Table I we report the results of the analysis of a series of mouse-human hybrids for the expression of the human form of the enzyme P-glucuronidase (Chern and Croce, 1975). As shown in Table I , the expression of the human enzyme segregated concordantly with the retention of human chromosome 7. Therefore, the gene coding for P-glucuronidase can be mapped on this human chromosome.

111. Somatic Cell Hybrids to Map Viral Integration Sites

Sambrook et al. (1968) have determined that the DNA-containing tumor virus SV40 is integrated into the cellular DNA of transformed cells. Since human

INTEGRATION OF ONCOGENIC VIRUSES 3

TABLE I

WITH T H E PRESENCE OF CHROMOSOME 7 CONCORDANT SEGREGATION OF HUMAN ~-GLUCURONIDASE EXPRESSION

~~~~

Human chromosome 7

~~~~ ~

Human P-glucuronidase + -

15 0

0 12

cells can be transformed by SV40 (Girardi et al. , 1965) we produced somatic cell hybrids between rodent cells and SV40 transformed human cells and studied the hybrids for the expression of SV40 T antigen, which is coded by the A gene of SV40 (Black et al . , 1963) and for the presence of human chromosomes. As shown in Table I1 the first two SV40 transformed human cell lines we have studied contained SV40 integrated in the human chromosome 7. The expression of the T antigen segregated with this human chromosome and with no other human chromosome in a large number of independent hybrid clones (Table n) (Croce et al., 1973). In order to confirm this finding, we subcloned three SV40 T antigen positive hybrid clones, one of which (52-61(1) C1 5 BUdR) contained only human chromosome 7 and no other human chromosome (Table DI) (Croce and Koprowski, 1974a). We also rehybridized cells of this hybrid line that were deficient in thymidine kinase with mouse cells deficient in hypoxanthine phos- phoribosyltransferase. The subclones and the triple hybrids segregated into T antigen positive and negative cells. The expression of SV40 T antigen segregated concordantly with the presence of human chromosome 7 in the hybrid subclones and triple hybrids (Table IV) (Croce and Koprowski, 1974b). We have also produced mouse x human somatic cell hybrids with the SV40 transformed cell line GM54VA. In these hybrids the expression of SV40 T antigen segregated

TABLE I1 CONCORDANT SEGREGATION BETWEEN HUMAN CHROMOSQME

C-7 AND SV40 T ANTIGEN

All hybrids

- + c - 7

SV40 T antigen + 71

3" - 0

12

"Exceptions are three LN-SV X CI-ID hybrid clones that contain the C-7 chromosome, but are negative for T antigen.

4 C A R L 0 M. CROCE

TABLE 111 EXPREWOK OF SV40 T ANTIGEN I N MOUSE-HUMAN HYBRID SUBCLONES A N D TRIPLE HYBRIDS

Hybnd clones

Number of T antigen positive

Number of subclones subclones or or triple hybrids triple hybrids

52-58 C1 19 52-62 ( I ) CI 5 BUdR 52-62 ( I ) CI 16 BUdR 52-62 ( 1 ) C1 5 BUdR x IR

15 9 5

2 0

3 I 4

12

concordantly with the presence of human chromosome 17 (Table V) (Croce, 1977). In the line GM637 that contained two SV40 integration sites, we have assigned one of the two integration sites to human chromosome 12 (M. Shander, E. DeJesus, and C. M. Croce, in preparation). This was made possible by the finding that a Sac1 19 kb fragment containing SV40 DNA segregated concor- dantly with human chromosome 12 in mouse x human hybrids. The second integration site is not in human chromosome 12 and is not in human chromosome 8. Recently, Kucherlapati ef al. (1978) have assigned the SV40 integration site to human chromosome 8 in this cell line. From these studies it can be concluded that the gene coding for SV40 T antigen is located in the chromosomal DNA of kims-transformed cells and that this gene can be located on different chromo- somes in different SV40 transformed cells. Since we were able to recover infec- tious SV40 only from SV40 T antigen positive mouse hybrid cells following fusion with permissive African green monkey cells we concluded that the chromosome that coded for SV40 T antigen contained the entire genome of the kims (Croce et al. 1974). These results were conf i ied by experiments of

TABLE IV POSITIVE CORRELATION BETWEEN THE PRESENCE O F S V M T ANTIGEN A N D

H U M A N CHROMOSOME 7 [pi 29 SUBCLONES OFTHREE T ANTIGEN POSITIVE MOUSE-HUMAN HYBRID CLONES A N D I N 20 TRIPLE HYBRID CLONES

D E R I V E D F R O M T H E F U S I O N O F ~ ~ - ~ ~ ( ~ ) C I ~ B U ~ R W T H M O U S E I R C E L L S

All hybrid subclones and triple hybrids Human chromosome 7

- +

SV40 T antigen + 26 0

0 23 -

TABLE V EXPRESSION OF SV40 T ANTIGEN IN HYBRIDS BETWEEN A3 TK-CHINESE HAMSTER CELLS A N D GM54VA SV40 TRANSFORMED HUMAN CELLS

Selected in HAT Counterselected in BUdR

Percentage T Number of hybrid cells Percentage T Number of hybrid cells Hybrid antigen positive containing human chromosome antigen positive containing human chromosome clones cells 17itotal analyzed cells 17/tofal analyzed

56-11 CI 1 56-11 C1 2 56-11 CI 5 56-11 CI 8 56-11 CI 9

56-11 CI 14

56-11 CI 16 56-11 CI 17 56-11 CI 18 56-1 1 CI 24 56-1 1 CI 29

56-1 I CI 11

56-11 C1 15

56-11 CI 30 56-11 C1 31

>90 80

>90 15 50

< 1 > 80 >90 < I > 90 > 90 > 90 >90 > 90 <I

15/15 10/11 12/12 9/10

13/13 15/15 13/14 15/15 13/14 12/12 12/13 12/12 11/11 11/13 12/12

ND“ < I <1 ND < l < I ND < I ND < I < I < I < I ND ND

ND 0/10 0/12 ND 0/10 0/10 ND 0/10 ND 0/15 0/10 0/11 0/10 ND ND

“Not done.

6 CARL0 M. CROCE

DNA-DNA reassociation kinetics using labeled viral DNA as a probe (Khoury and Croce, 1975). The number of copies of SV40 DNA corresponded to the number of copies of the human chromosome carrying the SV40 genome to the hybrid cells (Khoury and Croce, 1975). In this case, the parental human cell line was LN-SV in which the SV40 genome is integrated in human chromosome 7 (Khoury and Croce, 1975; Campo et al., 1978).

Recently the Southern transfer method (1975) has been applied to the study of the integration of SV40 in different transformed cells (Kettner and Kelly, 1976). Resuits of these studies indicated that the viral DNA is inserted in different sites in different SV40 transformed rodent cells (Kettner and Kelly, 1976; Botchan el al . . 1976). Study of mouse-human hybrids with LN-SV human cells by the same method confirmed that SV40 is integrated in human chromosome 7 in this cell line (Campo er al., 1978).

IV. Expression of the Transformed Phenotype and of Tumorigenicity in Hybrids between Normal Rodent Cells and SV40 Transformed Human

Cells

In order to determine whether the integration of SV40 in a human chromosome results in the expression of the transformed phenotype and in tumorigenicity, SV40 transformed human cells containing the SV40 genome integrated in either human chromosome 7 or 17 were hybridized with cells directly derived from the mouse such as peritoneal macrophages. Interestingly, all the hybrids with the human cell line (LN-SV) carrying the SV40 genome in human chromosome 7 (Figs, 1 and 2 ) retained human chromosome 7 and all the hybrids with the human cell line carrying the SV4Q genome in human chromosome 17 G54VA retained human chromosome 17 (Fig. 3) (Croce and Koprowski, 1974b, 1975; Croce, 1977) whereas all the other human chromosomes segregated in the hybrid clones. All hybrids were expressing SV40 T antigen, were transformed in v i m , and were capable of forming tumors in immunosuppressed animals such as nude mice (Croce et a / . 1975a,b: Koprowski and Croce, 1977). The presence of a single copy of the chromosome carrying the SV40 genome was sufficient for the ex- pression of the transformed phenotype and for the tumorigenicity of the hybrids (Figs. 4 and 5) (Croce et al. , 1975b). These results indicate that the chromo- somes containing the viral genomes are cancer chromosomes that make the cells transformed and tumorigenic.

V. Integration of Other Oncogenic Viruses in Transformed Cells

Evidence that polyoma virus, another small DNA tumor virus (Soeda et id., 1979; Novak et al . , 1980), certain adenoviruses (Doerfler et al., 1974), and

INTEGRATION OF ONCOGENIC VIRUSES 7

FIG. 1. Karyotype of a hybrid cell between a mouse peritoneal macrophage (MPM) and an LN-SV SV40 transformed human cell. This hybrid has retained three copies of human chromosome 7 and one copy of human chromosome 17.

FIG. 2. Metaphase plate of an MPM X LN-SV hybrid that contains only chromosome 7 and no other human chromosome.

8 CARLO M. CROCE

FIG. 3. Karjatype of an MPMxGM54VA hybrid cell. Human chromosome 17 is the only human chromosome present in the hybrid.

FIG. 4. Karyotype of a tumorigenic MPM x LN-SV hybrid clone. Human chromosome 7 is the only human chromosome present in the hybrid.

INTEGRATION OF ONCOGENIC VIRUSES 9

FIG. 5 . Tumor formed by the injection of an MPM X LN-SV hybrid into a nude mouse.

RNA tumor viruses (Varmus ef al. 1973, 1974) are also integrated in the cellular genome of viral transformed cells has been obtained. It is, however, not known where the chromosomal location of these viral genomes is or whether the integra- tion occurs at specific sites or at random. The fact that the viral genomes are located in different restriction enzyme fragments in virus transformed cells does not necessarily mean that the integration occurs at random. It is possible, in fact, that homologous DNA sequences that are repeated on different chromosomes and/or on the same chromosome are the sites of integration.

Conclusive information on this subject will be derived by studies of the nuc- leotide sequences of cloned viral integration sites. In the case of Epstein-Ban (EB) virus, a herpes virus that has been considered

as the causative agent of Burkitt lymphoma, a neoplastic condition observed in certain areas of Africa (Epstein ef af., 1964), disagreement exists on whether the viral DNA or segments of it are integrated in the cellular DNA of the malignant cells. Klein and his associates reported some experiments that suggested that the genome of the virus is not covalently integrated in the cellular DNA but it is associated with the chromosomal DNA by alkaline labile bonds in Burkitt lym- phoma cells (Adams et al., 1973). Other investigators have reported that the expression of the EB viral induced nuclear antigen (EBNA) (Reedman and Klein, 1973), is associated with chromosome 14 in hybrids between mouse cells and Burkitt lymphoma cells (Yamamato et al . , 1978). We have also attempted to

10 CARL0 M. CROCE

locate the genome of EB virus in Burkitt lymphoma cells using hybrids between mouse fibroblasts and human Burkitt lymphoma cells (Glaser er al., 1978). The results of this analysis indicated that the expression of EBNA and the presence of viral DNA sequences are not associated with any human chromosomes in these hybrids (Glaser et al., 1978). These results suggest that some of the viral DNA is an episomal state in Burkitt lymphoma cells. It is possible, however, that EB viral DNA sequences are also integrated in the chromosomal DNA of these malignant cells. It will become feasible to resolve this question when the DNA fragments of the entire EBV genome will be cloned in plasmid or phage cloning vectors. The use of these cloned DNA probes to analyze the DNA of Burkitt lymphoma cells containing a very limited number of EB viral genome equiva- lents as part of it should answer this question.

VI. Does the Integration of Tumor Virus DNA in the Chromosomal DNA Occur by DNA Homology?

We have started to address the question of whether the integration of SV40 and polyoma virus DNA in the chromosomal DNA occurs by DNA homology. We chose one somatic cell hybrid clone between thymidine kinase-deficient mouse cells (LM-TK) and GM637 SV40 transformed human cells that contain two SV40 genomes, one of which is integrated in human chromosome 12. Southern blotting analysis of the hybrid cellular DNA following restriction with the en- zyme SacI indicated that the human parental cells and some of the hybrids contained SV40 in a 19 kb DNA fragment (M. Shander and C. M. Croce, unpublished results). T antigen positive hybrid cells that contained the Sac1 19 kb DNA fragment hybridizing with SV40 DNA were counterselected in medium containing high concentrations of 5-bromodeoxyuridine and clones that were T antigen positive and thymidine kinase-deficient were obtained. Such T antigen positive thymidine kinase deficient mouse-human hybrids were then transformed with recombinant plasmids containing the gene for herpes simplex type 1 thymidine kinase and the entire SV40 genome (Fig. 6) (Linnenbach et al., 1980) and transformants were selected in HAT selective medium (Littlefield, 1964) in which only thymidine kinase positive cells can grow. Transformants were ob- tained and we are presently determining whether the exogenous recombinant SV40 molecules integrate where the endogenous SV40 DNA is located (on chromosome 12). We will restrict the transformant cellular DNA with SacI and other restriction enzymes and we will establish whether the SacI 19 kb restric- tion fragment disappears in the transformants.

The use of a thymidine kinase vector could be applied to the study of integra- tion of other oncogenic viruses in order to establish conclusively whether integra- tion of the viral genome in the cellular genome occurs by DNA homology.

INTEGRATION OF ONCOGENIC VIRUSES 11

BamHI

;IN OF

C 6 I3.2Kb

pBR322

FIG. 6. Restriction enzyme map of the plasmid C6 containing the entire genome of SV40 and the 3.4 kb fragment of herpes simplex virus type 1 carrying the gene for thymidine kinase into the plasmid pBR322.

VII. DNA-Mediated Cell Transformation of Mouse Teratocarcinoma Cells

Mouse teratocarcinoma stem cells do not express SV40 or polyoma virus T antigen following viral infection (Swartzengruber and Lehman, 1975). On the contrary, infection of differentiated cells with these viruses results in the expres- sion of the viral (tumor) T antigens (Swartzengruber et al., 1977; Seegal and Khoury, 1979; Seegal et al., 1979). Apparently, the block does not occur at the lev- el of penetration and viral uncoating in the cellular nuclei (Swartzengruber er al., 1977; Seegal and Khoury, 1979; Seegal et af . , 1979) but it involves either the tran- scription of the viral genes or the processing of viral-specific mRNAs. In order to determine the molecular basis of the lack of expression of SV40 T antigen in mouse teratocarcinoma cells we transformed TK-F9 mouse teratocarcinoma cells with a C6 plasmid vector carrying the gene for herpes simplex virus thymidine kinase and the entire SV40 genome (Fig. 6) (Linnenbach et a l . , 1980) in the plasmid pBR322 by the DNA-mediated gene transfer approach. As shown in Fig. 7 the transfor- mants selected in HAT medium expressed herpes simplex virus thymidine kinase.

12 CARL0 M . CROCE

FIG. 7 . Starch gel electrophoretic separation of thymidine kinase gene products. knzyme migra- tion is visualized hy the binding of titrated thymidine monophosphate product to DE-81 paper overlaid on electrophoretically separated thymidine kinase enzymes. Enzymes in lanes 2 and 5 are from FY stem cell transformants 13-1 and 12-1; lane 3 contains murine thymidine kinase derived from MC57G cells; lane 4 contains HSV-I thymidine kinase derived from 41-1-1 cells. Lane 1 contains HSV-I thymidine kinase derived from an F9 stem cell transformant not described in this article.

Southern blotting analysis of the Hirt supernatant (low-molecular-weight DNA) and pellet (chromosomal DNA) DNA indicated that clone 12-1 contained a single C6 plasmid integration site in a 15 kb XbuI DNA fragment (Linnenbach er a/., 1980) (Fig. 8). Clone 13-1, on the contrary, has lost the SV40 DNA sequences. As shown in Fig. 9 the cellular DNA of clone 12-1 was cleaved with various restriction enzymes and the DNA fragments were separated by agarose gel electrophoresis. The agarose gel was then cut in three pieces that were hybridized to three different probes, respectively: pBR322 DNA, HSV-I TK DNA, and SV40 DNA, As shown in Fig. 9, all three probes hybridized with the same 15 kb Xbul DNA fragment. This experiment indicates that all the three DNA components of the C6 plasmid are integrated in the same DNA fragment. Since the 4.2 kb pBR322 DNA fragment was not present in the clone 12-1 DNA that was cut with BumHI we can conclude that the C6 plasmid DNA was integrated through pBR322. Interestingly, clone 12- 1 teratocarcinoma stem cells

INTEGRATION OF ONCOGENIC VIRUSES 13

FIG. 8. Hybridization of 3T-labeled SV40 DNA to 13-1 and 12-1 cellular DNA after restriction endonuclease digestion and transfer from agarose gel. Hirt supernatant (S) and pellet (P) DNA (10 &lane) from 12-1 and 13-1 stem cells were cleaved with XbaI (lanes 1-4) and KpnI (lanes 5-8) , electrophoresed in a 0.7% agarose slab gel, denatured, transferred to a nitrocellulose sheet, and hybridized to 32P-labeled SV40 DNA. BumHI cleaved and similarly treated C6 DNA (lane 10, 12.5 pg; lane 1 1, 6.25 pg) was included as a marker.

did not express SV40 T antigen (Fig. 10). On the contrary, clone 12-1 cells that were induced to differentiate into endodermal cells by retinoic acid were found to express SV40 T antigen (Fig. 10). The 12-1 stem cells were also found to be negative for the expression of SV40 tumor-specific transplantation antigen (TSTA). Expression of SV40 TSTA was detected in the retinoic acid-induced differentiated cells (Linnenbach et al., 1980). We have counterselected clone 12- 1 cells in medium containing high concentrations of bromodeoxyuridine (BUdR). Most of the counterselected clones were found to have lost HSV-TK activity and did not express SV40 T antigen after exposure to retinoic acid. The minority of the counterselected clones, however, have retained the expression of SV40 T antigen following exposure to retinoic acid. At present, we are trans- forming one of these clones with the C6 plasmid and we will-establish whether the C6 plasmid molecules integrate at random or whether they integrate preferen- tially in the endogenous C6 plasmid.

14 CARL0 M . CROCE

FIG. 9. Hybridization of "P-labeled pBR322 DNA (a). HSV-1 TK DNA (b), and SV40 DNA (c) to Xbul and BarnHl cleaved 12-1 cellular DNA. Hin pellet DNA (10 @/lane) from 12-1 cells was cleaved with Xbul and BomHI resmction endonucleases and applied to an agarose slab gel as indicated above,each lane in this figure. After electrophoresis, the gel was cut into three parts and the DNA in each gel was denatured, transferred to nitrocellulose sheets, and each of the three nitrocel- lulose sheets was hybridized to a different 'T-labeled probe: (a) hybridized to 32P-labeled pBR322 DNA: (b) hybridized to "P-labeled HSV-I TK DNA; (c) hybridized to 32P-labeled SV40 DNA. C6 DNA BnmHl cleaved C6 DNA, and BamM cleaved SV40 DNA are included as markers.

VIII. Conclusion

It is clear from results presented that the genomes of oncogenic viruses can integrate in different sites in the same or different chromosomes in different viral-transformed cells. It is not clear, however, whether the viral genomes integrate into specific sites or at random in the mammalian cell genome. Nuc- leotide sequencing of the cellular DNA sequences flanking the viral integration sites and the use of recombinant plasmid DNAs containing a selectable gene (thymidine kinase) and the tumor virus genome should result in a better under- standing of the molecular mechanism of the integration of oncogenic viruses in the cellular genome.

INTEGRATION OF ONCOGENIC VIRUSES 15

FIG. 10. Indirect immunofluorescence of SV40 T antigen in transformant 12-1 cells before (a) and after (b) induction of differentiation with 0.1 I.LM retinoic acid.

16 CARL0 M. CROCE

REFERENCES

Aaronson, S. A., Hartley, J. W., andTodaro, G. J. (1969). Proc. Narl. Acud. Sci. U.S.A. 64,87-94. Adams, A.. Lindahl, T., and Klein, G. (1973). Proc. Natl. Acad. Sci. U.S.A. 70, 2888-2892. Black. P. H . Rowe. W . P. , Turner. H. C. . and Huebner, R. J. (1963). Proc. Narl. Acad. Sc,i.

Botchan. M . , Tapp. W , and Sambrook. J. (1976). Cell 9, 269-287. Campo. M . S . . Cameron. I . R.. and Rogers, M. E. (1978). Cell 15, 1411-1426. Chem. C. J . . and Croce. C. M. (1975). Am. J . Human Gener. 28, 350-358. Cooper. G. M . , andTemin, H. M. (1974). CnldSpringHarborS?mp. Quanr. B i d . 39, 1027-1032. Croce. C. M. (1976). Proc. Narl. Acad. Sci. U.S.A. 73, 3248-3252. Croce. C. M. (1977). Proc. Natl. Acad. Sri. U.S.A. 74, 315-318. Croce, C. M . , and Koprowski. H. (1974a). J. &xp. Med. 139, 1350-1353. Croce, C. M.. and Koprowski, H. (1974b). J . Exp. Med. 140, 1221-1224. Croce. C. M.. and Koprowski. H. (1975). Proc. Narl. Acad. Sri. U.S.A. 72, 1658-1660. Croce, C. M . . Girardi, A. J., and Koprowski, H. (1973). Proc. Narl. Acad. Sci. U.S.A. 70,

Croce. C. M.. Huebner, K., Girardi, A. J . . and Koprowski, H. (1974). Virology 60, 276-281. Croce, C . M . , Aden. D., and Koprowski, H. (1975a). Proc. Narl. Acad. Sci. U.S.A. 72. 1397-1400. Croce. C. M.. Aden. D.. and Koprowski. H. (1975b). Science 190, 1200-1202. Croce, C. M . . Shander. M.. Martinis, J . . Cicurel. L.. D’Ancona, G. G. , Dolby. T . W. , and

Doerfler. W . . Burger. H.. Ortin. J . . Fanning, E., Brown. D. T., Westphal. M.. Winterhoff. V. .

Epstein. M . A, , Achong. B. G., and Ban. Y . M. (1964). Lancer 1, 702-703. Cirardi. A . J . Jensen. F.. and Koprowski. H. (1965). J. Cell. Physiol. 65, 69-84. Gla$er. R . . Nanoyama, M , Hamper. B., and Croce. C. M . (1978). J . Cell. PhTsiol. 96. 316-326. Hanafusa. H.. Hayward. W . S., Chen, S . H.. and Hanafusa. T. (1974). Cold Spring Harbor Symp.

Hill. M. . and Hillova. S. (1972). Nurure (London) New Biol. 237, 35-39. Hill. M. . Hillova. S. . Dantchev, D.. Mariage. R. , and Goubin, G. (1974). Cold Spring Harbor

Huebner. R . J . , Keiloff. G . J . . Sanna. P. S.. Lane. W . T.. and Turner. H. C. (1970). P r w . Nor/.

Kettner. G . . and Kelly, T . J . (1975). Proc. Narl. Acad. Sci. U . S . A . 73, 1102-1106. Khoury, G . . and Croce. C. M. (1975). Cell 6. 535-542. Koprowski. H.. and Croce, C. M. (1977). Proc. Natl. Acud. Sci. U.S.A. 76, 1142-146. Kucherlapati, R . . Hwang, S. P., Shimuzu, N., McDougall, J . K., and Botchan, M. R. (1978). Proc.

Lai. C. J . , and Nathans. D. (1974). ColdSpring Harbor SJmp. Quant. B i d . 39, 53-60. Littlefield. J. W . (1964). Science 145, 709-710. Linnenbaeh. A.. Huebner, K.. and Croce, C. M. (1980). Proc. Nut!. Acad. Sci. U.S.A. 77,4875-4879. Low).. D. R . . Rowe, W . P.. Teich. N.. and Hanley, J . W . (19711. Science 174, 155-156. NOVA. V . . Dilworth. S. M . , and Griffin. B. E. (1980). Proc. Narl. Acad. S r i . U . S . A . 77, 3278-

Reednian. B M . . and Klein. G. (1973). Inr. J . Cuncer 1 1 , 499-520. Rowe. W’. P. (1972). J. E.rp. Med. 136, 1272-1285. Rowe. W. P. (1973). Cancer Res. 33, 3061-3068. Rowe. 6‘. P.. and Hanley. J . W. (1972). J. &.rp. Med. 136. 1286-1301, Rowe. W . P.. and Pincus. T . (1972). J Exp. Med. 135. 429-436. R o w . I”. P.. Hanley. J . W.. and Brenner. T . (1972a). Science 178, 860-862.

C‘.S.A. 50. 1148-1 156.

3617-3620.

Koprowski, H. (1979). Proc. Nut/. Acad. Sci. U.S.A. 76, 3416-3419.

Weiss. 8. . and Schick. J. ( 1974). Cold Spring Harbor Synip. Quant. B i d . 39. 505-522.

Quunr Biol. 39. 1139-1 144.

Swtp. Quanr. B i d 34. 1015-1026.

.4cud. SCC. U.S .A . 67. 366-376.

Narl Arad. Sci . U .S .A . 75.4460- 4464.

3282.

INTEGRATION OF ONCOGENIC VIRUSES 17

Rowe, W. P., Lowry, D. R., Teich, N., and Hartley, J. W. (1972b). Proc. Natl. Acad. Sci. U.S.A.

Ruddle, F. H. (1973). Nature (London) 242, 758-760. Sambrook, J . , Westphal, H., Srinivasan, S., and Dulbecco. R. (1968). Proc. Narl. Acad. Sci. U.S.A.

Southern, E. (1975). J. Mol. Biol. 98, 503-517. Seegal, S. , and Khoury, G. (1979). Proc. Natl. Acad. Sci. U.S.A. 76,5611-5615. Seegal, S . , Levine, A. S., and Khoury, G. (1979). Nafure (London) 280, 335-339. Soeda. F., Arrand, J. R., Smolar, N., and Griffin, B. E. (1979). Cell 17, 357-370. Swartzengruber, D. E., and Lehman, J. M. (1975). J . Cell. Physiol. 85, 179-185. Swartzengruber, D. E., Frederick, T. D., and Lehman, J. M. (1977). J . Cell. Physiol. 93,25-30. Tegtrneyer, P. (1974). Cold Spring Harbor Symp. Quant. Biol. 39. 9-15. Temin, H . M. (1971). Annu. Rev. Microbiol. 25, 609-648. Temin, H. M.. and Baltimore, D. (1972). I n “Advances in Virus Research” (R. M. Smith and M.

Varmus, H. E., Vogt, P. K.. and Bishop, J. M. (1973). Proc. Natl. Acad. Sci. U.S.A. 70, 3067-

Varmus, H. E., Guntaka, R. V., Deng. C. T . , and Bishop, J. M. (1974). Cold Spring Harbor S y t p .

Weiss, M . E.,andGreen, H. (1967). Proc. Natf.Acad. Sci. U.S.A. 38, 1104-1111. Yamarnoto, K.. Mizuno, F., Matsuo, T., Tanaka, A., Nonoyama, M., and Osato. T. (1978). Proc.

69, 1033-1035.

60, 1288-1295.

A. Lauffler, eds.), Vol. 17, p. 129. Academic Press, New York.

307 1 .

Quant. Biol. 39,982-996.

Natl. Acad. Sci. U . S . A . 75, 5155-5159.