Effect of 5-fluorouracil on ultraviolet inactivation of virus production by murine sarcoma-leukemia virus complex carrier cells

48, 193-200

VIROLOGY

#ect

(1972)

af 5Fhorouracil urine

on Ultraviolet

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Sarcoma-Leukemia

Virus

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H. YOSHIKURA Departme& Tokyo

of Oncology, 108, bapan,l

Institute of and Institut

Medical Science, Uhersity of Tokyo, P. 0. Takanatua~ du Radium, PacuEt6 des Sciences, 91 Chsay, France

Accepted December 10, 1971 Addition of 5-fluorouracil (10-z M) to murine sarcoma virus-carrier cells enhanced sensitivity to ultraviolet inactivation not only of the virions produced, but also of the virus-producing capacity of the cells. The latter sensitization required 24 hr contact of the carrier cells with Wluorouracil, the former only 2 hr. The sensitization of the virus-producing capacity was partially reversible. On the other hand, .?+bromodeoxyuridine was unable to sensitize these functions to ultraviolet light. These facts may suggest that the function which is the target for ultraviolet inactivation of the cellular capacity to produce virions was carried bv RNA species whose turnover rate was far slower than that of viral RNA. INTRODUCTION

Ultraviolet light (UV) inactivates various cellular functions (Smith and Hanawalt, 1969). The virus-producing capacity of cells infected with RNA tumor viruses is also inactivated by UV (Bader, 1966; Yoshikura, 197’0). In this paper, the author tried to determine the target molecule for the UV inactivation of the virus-producing capacity of cells which were chronically infected with musine sarcoma-leukemia complex. MATERIALS

AND

METHODS

li?,rus and cells. The Maloney isolate of murine sarcoma-leukemia virus complex (MSV) was used; preparation and storage of the virus are described elsewhere (Yoshikura et al., 1968). The C3H2K cell line originating from the kidney tissues of a newborn C3N/He mouse was cultivated in medium consisting of 8 parts modified Eagle’s MEN (Yamaue et al., 1968), 1 part calf serum inactivated by heating at 56” for 30 min, and I part tryptose phosphate broth. Ultraviolet light (UV) irracliation. UV irra1 Present

diation was produced by a germicidal mei cury vapor lamp. The dose rate was measured by a Latarjet dosimeter. The IX dose indicated in t’he figures represents the su3 face dose. liTi inactivation of virus. UV inac-tivaI!ion of virus was performed in a 6O-mm glass petri dish containing L ml of the sample; the dish fyas constantly shaken during the irradiation. The cells cultivated in 6 glass petri dishes 17;ereexposed to 0,4 ml of the inoculum for 2 hr, and then 3.5 ml of culture medium was added. The cultures were kept in a CO, incubator. Six days later, the plates were submit,ted to plaque assay of murine leukemia virus by the XC test (Rowe et zcE.,1970). After the medium was discarded, the ceils were IX-irradiated at a dose of 680 ergs/mm? and overlaid with XC cells in an amount of IO6 cells per dish 1 medium was changed on the second day: and on the fourth day the cultures were fixed and stained for plaque eowting, Two plates were used for each point throughoai the experiments. UV inactivation of survival of LUSi,w cwriei~ cells. C3lT2K cells which had been cu!tivated

for 14-20 days after

address.

193 Copyright

@ 1972 by Academic

Press,

Inc.

XE3V infection:

and

YOSHIKURA

194

subcultured at least once were used as MSV carrier cells. The plating efficiency of the cells was 5-10 % without feeder layer. Appropriate numbers of the cells were plated in 60-mm glass petri dishes; after being settled, the cells were UV-irradiated. Colonies were counted when they became macroscopically visible. UV inactivation of focus forming centers. The MSV carrier cells were trypsinized and suspended into the medium to a concentration of lo5 cells/ml. The suspension in a volume of 1 ml was transferred to a 60-mm glass petri dish and exposed to UV; the dishes were constantly shaken during the irradiation. Then, the irradiat.ed samples were diluted appropriately and plated onto semiconfluent cultures of C3H2K cells. In some experiments, the cells were seeded into empty glasspetri dishes in appropriate amounts. After the cells had settled, the medium was removed and the cells were UV-irradiated. Then, uninfected cells were plated onto the irradiated cells in an amount of 3 X lo5 cells per dish. The medium was changed on the next day. Under these conditions, the contribution of free or cell-associated virions to the registered focus number was negligible (Yoshikura, 1970). The efficiency of focus formation was around 20 %. The number of plaques obtained by the XC test, which was carried out after counting foci, exceeded that of foci by 20 %; 17 % of the cells were infected only with leukemia virus and 83 % with sarcoma and leukemia viruses. RESULTS

Sensitization of MSV-Associatecl Leukemia Virions to UV Radioactive uridine added to RNA tumor virus-infected cell cultures was recovered in the virions 70 min later (Bader, 1970; Hino et al., 1971). Based upon this finding, an experiment was designed to obtain virions whose nucleic acid was partially replaced with base analogs. MSV-carrier cells were incubated for 2 hr in medium (MEM + 1% calf serum) containing either 5fluorouracil (5FUra) or 5bromodeoxyuridine (BrdUrd) at a concentration of 10v3 M. 5-FUra is known as

an analog of uracil in RNA, and not incorporated into DNA of mouse cells; BrdUrd is known as an analog of thymidine in DNA (Heidelberger, 1965). The cells were washed and incubated further for 2.5 hr in 0.5 ml of the medium free of the analogs. The medium was harvested, diluted 5-fold, irradiated with UV at various doses,and plated on the assay plates. The dose response of MSV as titrated by focus counting was bimodal; one hit at low dilutions, and two hit at higher dilutions (Hartley and Rowe, 1966; O’Connor and Fischinger, 1968; Yoshikura et al., 1968). The dose response of the leukemia virus as measured by plaque counting in the XC test was one hit throughout the dilutions. As the UV sensitivity of the leukemia virus and that of MSV are reportedly indistinguishable (Kelloff et al., 1970; Yoshikura, 1971), the comparison of the UV sensitivities of virus samples was made with plaque counting. The inactivation dose Dlo of the virus preparation obtained from 5-FUra-treated culture was about one-half of those of the virus obtained from untreated or BrdUrdtreated cultures (Fig. 1). This indicates that 5-FUra was incorporated into viral RNA, resulting in the sensitization of the virions to UV, but that BrdUrd was not. UV Inactivation of Focus-Forming Centers The author tried to identify the molecular nature of the radiation target whose inactivation resulted in the abolishment of the cellular capacity to produce virions. As MSV foci develop only by secondary infection of neighboring cells (Hartley and Rowe, 1966; Bather et al., 1968; Yoshikura et al., 196S), the UV sensitivity of the cells as focus formers is equivalent to the cellular capacity to produce infectious virions. It should be noted here that focus formation by focusforming cells also depends upon the presence and replication of helper virus. Consequently, what was measured here may be primarily the cellular capacity to replicate the leukemia virus and only secondarily the capacity for MSV replication. MSV-carrier cells were cultivated for 24 hr in medium (MEM + 1% calf serum) containing either 5-FUra or BrdUrd at a

UW IRRADIATION uv

dose

OF MSV CARRIER

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CELLS

195

carried by cell-associated Cons. The &!V sensitivity of cell survival was about twice that of the cellular capacity t,o beeomc Coeus center (Fig. 5).

MSV-carrier cells were plated in petri dishes in small numbers so as to produce countable foci. 5-FUra was added to the cultures at a final concentration of IO-” M. At intervals, the cultures were washe with phosphate-buffered saline, UV-irradiated, and overlaid with normal Incubation with S-Era for 8 hr was i eient for sensitization; incubation for 16 br sensitized

FIG. 1. Sensitization of MLV virions to UV. MSV-carrier cells were incubated for 2 hr in medium (MEM + 1% calf serum) cont.aining eit,her h-fluorouracil or bromodeoxyuridine at a concentration of lo-” M. The cells were washed and incubated further for 2.5 hr in 0.5 ml of medium free of the base analogs. The medium was harvested, diluted B-fold, irradiated with UV at various doses, and plated on the assay plates. Six days later, the plates were submitted to the XC test. 0, Virus sample obtained from untreated MEW-carrier cells. A, virus sample obtained from cells treated with bromodeoxyuridine; q , virussample obtained from cells treated with 5-fluorouracil. Two independent experiments are shown with the different size symbols.

of lo+ M. The cells were UT-irradiated in suspension, diluted. appropriately, and plated on assay cultures. Incubation with 5-FUra result,ed in a marked sensitization, while incubation with BrdUrd caused little, if any, sensitization (Fig. 2). 5-FUra-induced sensitization was detectable at a concentration of lo-* M, but not at concentrations lower t.han 10-j M. The efficiency of plating as focus center was unaffected by the treatment with the base analogs. Under these conditions, neither 5-NJra nor BrdUrd was able to sensit,ize the colony formation t’o UV inactivation. The inactivation dose I& of focus-forming center was 600 ergs/mmZ, that of the infectivity of the virions 3000 ergs/mmz. Thus, it is likely that the former function was not

concentration trypsinizeci,

Frc. 2. Sensitization to OV of the celiular capacity to become focus center. MSV-carrier cells were cultivated for 24 hr ir, medium (ME&l $ 1% calf serum) containing either 5-fluoronracil or bromodeoxyuridine at a concentration of lo-” .%!!. The cells were trypsinized, UV-irradiated ?c suspension, diluted appropriately, and plated on assay cultures. Foci were counted 5 days later 0, Untreated cells; A, cells treated with bromodeoxyuridine; IJ, cells treated with 5-fiuorousaeZI. Two independent experiments are shown with different-sized symbols.

196

YOSHIKURA

the cells partially, and incubation for 24 hr extensively (Fig. 3).

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dose egdmm2

Reversibility of the 5-F U-Induced Xensitixation MSV carrier cells were cultivated in medium (MEM + 1% calf serum) with or without 1O-3RJ 5-FUra for 24 hr. One set of the cultures were assayed for UV sensitivity of focus-forming centers. The other cultures were further cultivated in the medium free of the base analog; 24 hr later, UV sensitivity of focus formation was tested. As shown in Fig. 4, A, the sensitization bs 5FUra was partially reversed by incubation of the cells in 5-FUra-free medium for 24 hr. UV sensitivity of the virions produced at each point uv

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FIG. 3. Time required for sensitization to UV of MSV-carrier cells as focus-forming centers. MSV-carrier cells were plated in petri dishes (IO3 cells per plate for irradiation at doses of 0, 100, and 200 ergs/mm%; 104 cells per plate for irradiations at doses of 300 and 400 ergs/mm2). 5Fluorouracil was added to the cultures at a final concentration of lo+ M. At intervals (0, 0 hr; 0, 3 hr ; Cl, 8 hr; A, 16 hr; A, 24 hr), the cultures were washed twice with phosphate-buffered saline, W-irradiated, and overlaid with normal cells. The inactivation curves were steeper than those obtained by irradiating the cells in suspension (Fig. 2). Two independent experiments are shown with different-sized symbols.

. .

A

FIG. 4. Reversibility of 5-fluorouracil-induced sensitization of MSV carrier cells as focus center. MSV carrier cells were incubated in medium (MEM + 1% calf serum) with or without Lfluorouracil (lo+ M) for 24 hr. One set of cultures were assayed for UV sensitivity of the cells as focus center, and also for that of the virions produced. The other set was further incubated in the medium free of 5fluorouracil for 24 hr; UV sensitivity of the focus center and that of virions were tested again. Panel A: UV sensitivity of MSV carrier cells as focus cent’er. A, MSV carrier cells treated with 5-Buorouracil for 24 hr; A, untreated control for the preceding group; 0, MSV-carrier cells treated with 5-fluorouracil for 24 hr and then incubated in 5-fluorouracil-free medium for 24 hr. 0, Untreated control for the third group. Two independent experiments are shown with the different size symbols. Panel B : UV sensitivity of virions. A., Virions produced by the cells treated with 5-fluorouracil for 24 hr; A, control for the preceding group; 0, virions produced by cells treated with 5-fluorouracil for 24 hr and then incubated in 5-fluorouracil-free medium for 24 hr; 0, control for the preceding group.

was also tested. The virions produced by such partially desensitized cells were of normal UV sensitivity (Fig. 4B). Effect of BrdUrd on the UV Xensitivity Focus-Forming Centers

of

The cells were cultivated for 4 days in the medium containing BrdUrd at a concentration of 2.5 X low5 M. The cells were tryp-

sinized, and plated in dishes. After being settled on the glass surface, the cells were

UV IRRADIATION

OF MSV CARRIER

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FIG. 5. Sensitization by bromodeoxyuridine. Panel at left: MSV-carrier cells were cultivated in medium containing bromodeoxyuridine (2.5 X 10-S M) for 4 days. The cells were trypsinized and plated in @&mm glass petri dishes. Aft,er being setled on the glass surface, the cells were UT-irradiated. Open symbols: control; filled symbols: bromodeoxyuridine-treated ceils. Three independent, experiments are shown. I’anel at right: The cultures prepared in the same manner as described above were assayed for fiV sensitivity of the cells as focus-forming centers. Open symbols: untreated control; fdled symbols: bromodeoxguridine-treated cells. Four independent experiments are shown.

UV-irradiated, A4 set of cultures for focus center assay were overlaid with normal cells. he inactivation dose I&, of the cellular survival was about 140 ergs/mm2 for the control, and 80 ergs/mm2 for the BrdUrdtreated cells (Fig. 5, left). On the other hand, the UV sensit,ivit,ies of these cells as focusforming centers were almost identical for both control and BrdUrd treated cells (Fig. 5, right) D

in the loss of virus-producing capacity resided in an RXA specieswith a Lurnover rate far slower than that of viral RNA. BrdUrd, an analog of thymidine in DNA, on the other hand, was unable to sensitize the virus-producing capacity under conditiona where the cellular survival was sensitized LO UV. On the other hand, Bader (1966) re.ported that virus production by chick cells NA which had incorporated BrdUrd into prior to infeet,ion with Rous sarcoma virus~ DISCUSSION (RSV) was more susceptible to UV than tha-t 5-Pluorouracil sensitized to inactivation by untreated cells. He suggested th by UV not only virions but also the capacity continuous participation of cellular of carrier cells to produce virions. Sensitiza- was required for growth of RSV. Since this tion of virus-producing capacity required suggestion was based upon t,he virus yield 24-hr contact of the carrier cells with 5-FUra, per culture, the UV sensitivity of virus prothat of virions only 2 hr. Incubation of the duction may have been influenced by that of sensitized carrier cells in medium free of the cell multiplication; consequently, the es~5-FUra for 24 hr partially reversed the sensi- hancement of the latter by BrdGrd may tization; the virions produced by such only have resulted in that of the former. Morepartially desensitized cells were of normal over, the replication of the RNA tumor vi,” UV sensitivity. These facts suggest that the ruses is reportedly dependent upon the ceil function whose inactivation by UV resulted cycle (Temin, 1967; Hobom-Schnegg eZ al,,

198

YOSHIKURA

1970; Yoshikura, 1970); the disturbance of the cell cycle by BrdUrd may thus have resulted in a delay of virus replication. As the UV sensitivity of the virus-producing capacity gradually decreasedwith the lapse of time after infection (Bader, 1966; Yoshikura, 1970), the delay of the cell cycle by BrdUrd may quite well have resulted in the apparent sensitization of the virus-producing capacity of the infected cells. The discovery of DNA polymerase in the virions of RNA tumor viruses (Temin and Mizutani, 1970; Baltimore, 1970; Spiegelman et al., 1970; Rokutanda et aH.,1970), and the lack or extremely low levels of this enzyme in noninfectious virions (Hanafusa and Hanafusa, 1971; Robinson and Robinson, 1971) have suggested t,hat after infection viral RNA is transcribed into DNA (provirus) which may inversely serve as template in the synthesis of viral RNA. Experiments with DNA inhibitors (Temin, 1964; Vigier, 1964; Bader, 1964; Yoshikura, 1967; Nakata and Bader, 1968; Buck and Bather, 1969) and inhibition of virus replicaCon by actinomycin D, a specific inhibitor of DNAdependent RNA synthesis (Temin, 1963; Vigier, 1964; Vigier and Gold& 1964; Bases and King, 1967; Duesberg and Robinson, 1967; Yoshikura, 1967) may support this view. The participation of the newly formed DNA in the cellular transformation by RSV has been clearly demonstrated photobiologically (Boettiger and Temin, 1970; Balduzzi and Morgan, 1970). The lack of photosensitization of the virusproducing capacity by BrdUrd in my experiments is apparently inconsistent with these data, which suggest the DNA template as critical for the replication of viral RNA. Indeed, MSV carrier cells used in my experiments already contained the viral DNA, but the viral DNA, once formed in the infected cells, would be expected to replicate in pace with the replication of host cell DNA. Therefore, the capacity to produce virions should have been sensitized by BrdUrd under conditions where the cellular survival was sensitized. One possible explanation for this contradiction is to supposethat the viral DNA synthesis is such that the incorporation of BrdUrd or BrdUrd-induced sensitization was somehow efficient in the early phases of the

infection, but not in the later phases. Another is that photosensitization of the viral DNA may have been concealed by the induction of indigenous murine leukemia viruses by BrdUrd (IClement et al., 1971; Lowy et al., 1971; Aaronson et al., 1971). The nature of the target RNA is obscure. Bader (1966) suggested that much of the basic cellular machinery for virus production (e.g., ribosomes, tRNA, activating enzymes) remains intact after UV irradiation, as the cellular capacity to produce vesicular stomatitis virus remained unaffected even after UV irradiation at doses 6-7 times higher than the dose required for inactivating the virus-producing capacity of the cells already infected with RSV. The same consideration may be applied here. But cellular RNA species, especially messenger RNA, participating in virus replication are quite possibly different for RNA tumor viruses and nontumor viruses; in the former the role of the cellular RNA may be more important than in t.he latter. Therefore we cannot exclude the possibility that the target molecule was. cellular. Biswal and Benyesh-Melmck (1969, 1970) and Biswal et al. (1970) have found two nuclear RNA fractions in MSV carrier cells which are complementary to viral RNA; one of the two is double stranded. A similar phenomenon has been reported by Van Griensven et al. (1970). Double-stranded RNA has been isolated from various cells, including normal cells (Montagnier, 1968; Kimball and Duesberg, 1971) ; homology between such RNA and cellular DNA has been detected (Hare1 and Montagnier, 1971). RNA-dependent RNA polymerase has been isolated from both normal and leukemic cells (Watanabe and Haruna, 1969, and personal communication; Watson and Beaudreau, 1969). If the target molecule is not cellular, the following model can be proposed for the replication of viral RNA. What is coded by proviral DNA is a “minus” strand of the RNA, which is converted into a doublestranded form by synthesis of complementary “plus” strands. If this model is applied, the RNA which is the target of UV inactivation of the virus-producing capacity may be the “minus” strand of the RNA replicating unit, as the “minus” strand of RNA phages

‘lib’

is reportedly 1967).

IRRADIATION

stable (Weissmann

OF

and Ochoa,

The author thanks Professor T. Yamamoto of this department for his encouragement, Drs. T. Odaka. alld N. Yamaguchi of this institute for critical reading of the manuscript, and Dr. R. Latarjet, who kindly offered me a UV dosimeter as a gift. A preliminary part of this experiment was performed in Dr. Latarjet’s laboratory in Institut du Radium, Orsay, France, during the tenure of & Research Training Fellowship awarded by the International Bgencg for R,esearch on Cancer. The base analogs .&fiuorouracil and bromodeoxyuridine were supplied by Kyowa Hakko Co. Ltd. and TakedaYakuhin Co. Ltd., respectively.

S. A., Toa~o, G. J., and SCOLSICK, E. M. (1971). Induction of murine C-type viruses from clonal lines of virus-free BhLB/3T3 cells.

AARONSON,

f%%?Tkce

174,

CARRIER

15’i-159.

J, P. (1964). The role of deoxyribonucleic acid in the synt,hesis of R,ous sarcoma virus. Virology 22, 462468. BADER, J. P. (1966). n’letaholic requirements for infection by Rous sarcoma virus. II. The part,icipation of cellular DNS. ‘Virology 29, 452461. BADE~; J. P. (1970). Synthesis of RNA of RXAcontaining tnmor viruses. I. The interval between synthesis and envelopment. Virology 40, 491404. BALDOZZI, I’., and MORGAS, H. R. (1570). Mechanism of oncogenie transformation by Rous sa,rcoma virus. I. Intracellular inactivation of cell-transforming abilit,y of Rous sarcoma virus by 5-bromodeoxyuridine and light. J. V?:rol. 5,470477. BALTIMORE, D. (1970). RNA-dependent DSA polymerase in virions of RNA tumor viruses. lY;ature (London). 226, 1209-1211. Baa~s, R, E., and KING, A. P. (1967). Inhibition of Rauscher murine leukemia virus growth in aifro by actinomycin D. Virology 32, 175-183. BATPIER, R., LEONAIZO, 9., and YANG, J. (1968). Characterist’ics in the in vitro assay of murine sarcoma virus (Moloney) and virus-infected cells. J. Wat. Cancer Inst. 40, .551-560. Brswa~, N., and BEX~ESH-MEL~I~IC, M. (1969). Complementary nuclear RNA’s of murine sarcoma-leukemia virus complex in transformed cells. Proc. Xat. Acad. Sci. C. S. 64, 1372-1379. Brsw.~~, N., and BESYESH-MELNICK, M. (1970). Characterization of t,he complementary nuclear RNA of murine sarcoma-leukemia virus. Viralogy 42, 1.064-1072.

199

CELLS

BISWAL,

?“., &icCa~s, B., and BENYESR&~,(1970). Replication of murine sarcomaleukemia virus complex. In “The Biology of Oncogenic Viruses” (L. G. Silvestri, ed.), pp. 221-231. Nort,h-Holland Publ., Amsterdam BOETTIGER, D., and TEY:N, H. M. (1970). Lighi. inactivation of focus formation by chick embryo fibroblasts infect,ed with avian sarcoma virus ix]. the presence of 5.bromodeoxyLlridrlie. Nai.~~~~~~ 228, 622-624. Buts, B. &I., and B.\\THER, II. (1969). Ihequiiements for RNA and DNA synt,hesis in thr trawlsformation of mouse embryo cells by murine sarcoma virus (Maloney). J. Gen. “Jirol. 4, 457460. D~Es~EE~:, P. II., and ROBXXSON, W. S. (1967). Inhibition of mouse leukemia virus (MLV) replication by actinomyein I). T’iioiogv 31, 742-746 HSNAFUSA, H.: and H.~x.ux~.~, T. (19711. Noninfectious R,SV deficient’ in DNA polqmerase. Virology 43, 313316. HAREL, L., andi%NTAGNIER,Ii. (1971).Hon&ogy of double stranded RNA from rat liver cells with the cellular genome. Sature Neli; Biol. 229, 106-108 I HARTLEY~ J. Iv., and ROWE, w. P. (1966). Produetion of altered cell foci in tissue culture by defective Maloney sarcoma virus particles. Pt,oc. Xat. Acad. Sci. C:. S. 55, 780-786. HEIDELBERGER, C. (1965). Fluorinated pyrimi-. dines. Progr. Nucl. Acid Res. Mol. Biol 4. I-50. HINO, S.. Yos~lr~~~a, R., ancl Ysua~orno, T. (1971). The dependency of leukemia virus production on physiological condition of t$he cells. I’roc. Jap. Cancer Ass. (30th Amu. ilfeel ). p. 47. XICK,

ACKXOWLEDCXENTS

BADEB,

MSV

M.

H~BoM-S~EPI‘EGG, B., ROISMON, SON, W. S. (1970). Replication

virus 85-93.

in

synchronized

H.,

and

of ~tous J. Gen.

cells.

I~onr.xSBT-conx~

Vivob.

7,

A.\BOXSON, S. A.,and GILDE:N,R, I' Inaetivat,ion of murine sarcoma and viruses by ultraviolet irradiation:. 42, 1133-1135, KIMBALL: P. C.? and DCES~ERG, P. I-1. (19X! Virus interference by cellular double-stranded ribonucleic acid. J. Viral. 7, 697-T%.

KELLOFF,G.,

(1970). leukemia Virology

KLEx~sNT,

$7.; ~'ICOLSOS,

NI.

O.,

HUEBNEU,

1:. J.

(1971). Rescue OS the genome of focus forming virus from rat non-prodlrctive iines by 5’Sature Xex Bioi. 234, bromodeoxyuridine. 12-14. Lowu, D. I?.: Rowe,, W. I’., Tmca, N., a.nd H.&RTLEY, J. W. (1971). Murine leitkemia. virus: High frequency activation irk &ro by Godode-

200

YOSHIKURA

oxyuridine and 5-bromodeoxyuridine. Science 174, 155-156. MONTAGNIER, L. (1968). Presence d’un acid ribonucleique en double chaine dans des cellules animales. C. R. Acad. Xci. 267, 1417-1420. NAKATA, Y., and BADER, J. P. (1968). Transformation by murine sarcoma virus: Fixation (deoxyribonucleic acid synthesis) and development. J. vi’irol. 2, 1255-1261. O’CONNOR, T. E., and FISCHINGER, P. J. (1968). Titration patterns of a murine sarcoma-leukemia virus complex; evidence for existence of competent sarcoma virions. Science 159, 325329. ROBINSON, W. S., and ROBINSON, H. L. (1971). DNA polymerase in defective Rous sarcoma virus. Virology 44,457462. ROKUTANDA, M., ROKUTANDA, H., GREEN, M., FUJINAGA, K., RAY, R. K., and GURGO, C. (1970). Formation of viral RNA-DNA hybrid molecules by the DNA polymerase of sarcomaleukemia viruses. Nature (London) 227, 10261028. ROWE, W. P., PUGH, W. E., and HARTLEY, J. W. (1970). Plaque assay technique for murine leukemia viruses. Virology 42, 1136-1139. SMITH, K. C., and HANAWALT, P. C. (1969). “Molecular Photobiology.” Academic Press, New York. SPIEGELMAN, S., BURNY, A., DAS, M. R., KEYDAR, J., SCHLOM, J., TRAVNICEK, M., and WATSON, K. (1970). DNA-directed DNA polymerase activity in oncogenic RNA viruses. Nature (London) 227, 1029-1031. TEMIN, H. M. (1963). The effects of actinomycin D on growth of Rous sarcoma virus in vitro. Virology 20, 577-582. TEMIN, H. M. (1964).

The participation of DNA in Rous sarcoma virus production. Vi’irology 23,

486494. TEMIN,

H. M. (1967). Studies on carcinogenesis by avian sarcoma viruses. V. Requirement for new DNA synthesis and for cell division. J. Cell.

Physiol. TEMIN,

69,

53-64.

H. M., and MIZUTANI,

S. (1970). RNA-

dependent DNA polymerase in virions of Rous sarcoma virus. Nature (London) 226, 1211-1213. VAN GRIENSVEN, L. J. L. D., BAUDELAIRE, M. F., P&RI&S, J., EMANOIL-RAVICOVITCH, R., and Boiron, M. (1970). Studies on the biosynthesis of murine leukemia virus RNA. I. Some properties of Rauscher leukemia virus isolated from interferon treated JLSV5 cells. In “The Biology of Oncogenic Viruses” (L. G. Silvestri, ed.), pp. 145-153. North-Holland Publ., Amsterdam. VIGIER, P. (1964). Investigations on the replication of Rous sarcoma virus. Nat. Cancer Inst. Monogr. 17,407419. VIGIER, P., and GOLD&:, A. (1964). Effects of actinomyein D and mitomycin C on the development of Rous sarcoma virus. Virology 23, 511519. WATANABE, I., and HARUNA, I. (1969). Selfreplicating RNA in leukemic cells. Acta Haematel. Jap. 32, 593-602. WATSON, K. K., and BEAUDREAU, G. S. (1969). Isolation of and RNA-dependent RNA polymerase from virus infected myeloblasts. Biothem. Biophys. Res. Commun. 37,925-932. WEISSMANN, C., and OCHOA, S. (1967). Replication of phage RNA. Progr. Nucl. Acid Res. Mol. Biol. 6,353-399. YAMANE, I., MATSUYA,

Autoclavable mammalian 127,33&336.

Y., and JINBO, K. (1968). powdered culture medium for cells. Proc. Sot. Exp. Biol. Med.

H. (1967). Possible requirement of DNA synthesis for the growth of Friend leukemia virus. Jap. J. Med. Sci. Biol. 20,237-242. YOSHIKURA, H. (1970). Radiological studies on the chronological relation between murine sarcoma virus infection and cell cycle. J. Gen. ViTiroZ. 8, 113-120. YOSHIKURA, H. (1971). Ultraviolet inactivation of murine leukemia and sarcoma viruses. Int. J. Cancer 7,131-140. YOSHIKURA, H., HIROKAWA, Y., IKAWA, Y., and SUGANO, H. (1968). Transformation of a mouse cell line by murine sarcoma virus (Maloney). Int. J. Cancer 3,743-750.

YOSHIKURA,