Pseudouridine Excretion and Transfer RNA Primers for Reverse Transcriptase in Tumors of Retroviral Origin1

(CANCER RESEARCH 45,6260-6263, December 1985]

Franca Esposito, Tommaso Russo, Rosario Ammendola, Angela Duilio, Francesco Salvatore, and Filiberto Cimino2

Istituto di Scienze Biochimiche, II FacoltÃ di Medicina e Chirurgia, UniversitÃ di Napoli, Via S. Pansini 5,80131 Naples, Italy

ABSTRACT several human neoplasias (more frequently in lymphomas, leu- kemias, and hepatomas) and precociously decreases when a To evaluate the relationship between pseudouridine increase response to therapy is achieved (12). Similarly in AKR mice in biological fluids and retroviral cell transformation, we have bearing lymphomas of viral origin (AKR-MuLV3), either sponta studied the effect of retrovirus infection and/or transformation neous or transplanted, we found that pseudouridine serum levels on the rate of pseudouridine excretion by chick embryo fibro- are significantly higher than in control mice and are normal in blasts. mice bearing chemically induced lymphoid tumors (13). Further The results show that: (a) pseudouridine excretion by chick more in the AKR mice the increase of serum pseudouridine embryo fibroblasts transformed by Rous sarcoma virus is several appeared to be an early event, evident in the preneoplastic period times higher than that of normal cells; (b) this increased excretion (13) when the transforming virus is produced (14) and before precedes by many hours the appearance of the morphological histopathoiogical signs of the disease are apparent. signs of transformation and it is always present when neosyn- All these findings suggested that the overproduction of pseu thesized infectious viral particles are released into the culture douridine, rather than being a general aspect of the neoplastic medium; and (c) pseudouridine excretion was also increased in phenotype, could be related to the alteration of the metabolism cells infected by a mutant of Rous sarcoma virus (RAV-1 ) which, of specific tRNA molecules involved in retroviral replication. lacking the src gene, does not transform the cells but replicates To investigate this point we used the AKR system, the etiolog- normally. ical agent (AKR-MuLV) of which utilizes cellular tRNApra as primer To investigate if pseudouridine overproduction is related to an for its reverse transcriptase (15), and the CEF transformed by altered turnover rate of specific transfer RNA (tRNA) species RSV, which utilizes cellular tRNATlp as primer for its reverse which functions as primer of retrovirus reverse transcriptase, the transcriptase (16). concentration of non-acylated proline-accepting tRNA and non- We addressed two points, (a) Is the increased production of acylated tryptophan-accepting tRNA, primers of reverse tran pseudouridine in lymphomatous AKR mice a consequence of scriptase of murine leukemia virus and of Rous sarcoma virus, viral infection and replication or a response of normal cells to the respectively, has been measured, the former in normal and appearance of neoplastic foci? (b) Is the cellular content of the transformed AKR thymus and the latter in normal fibroblasts and specific tRNA species that acts as primer of reverse transcriptase in fibroblasts infected by Rous sarcoma virus or by its nontrans- altered in the cell transformed by a retrovirus? forming mutant. The results show that in both systems a signif The first point was studied by evaluating the excretion of icant increase of the primer tRNA species occurs in the infected pseudouridine and other nucleosides by CEF infected by RSV or transformed cells. or its defective mutant RAV-1 which, lacking the src gene, fails to transform the cells but replicates normally (17). The second point was evaluated by measuring the amino acid acceptance of INTRODUCTION several tRNA species purified from both normal and lymphoma Increased urinary levels of modified nucleosides have been tous AKR thymuses or from normal and RSV- or RAV-1-infected found in numerous tumor-bearing subjects (1-8). The types of CEF. modified nucleosides found in the urine of cancer patients and the observation that tRNA contains the highest and most varied MATERIALS AND METHODS number of modified nucleosides (up to 20% of the total number of nucleosides are posttranscriptionally modified) suggest that Animals. AKR mice were from colonies established from Jackson alterations in the structure and/or metabolism of tRNA in trans Laboratory mice by Drs. C. Gurgo and S. Bridges. Thymuses were formed cells cause the observed phenomenon. However, tumor excised under ether anesthesia by thoracotomy, cleaned of adherent cells appear to lack hypermodified tRNA species (9), whereas tissues, and quickly frozen in liquid nitrogen. the same cells show enhanced activity of the enzymes involved Cells. Primary cultures of CEF were prepared from body walls of 11- in the main tRNA modification reactions, i.e., methylation (10) day-old White Leghorn chick eggs (Lohman Tierzucht GMBH; Cuxhaven, and pseudouridylation (11). Federal Republic of Germany) and cultured in a 10-ml/100-mm plastic We have found previously that blood serum pseudouridine, dish (Falcon, Cockeysville, MD) of E199 medium (Difco Laboratories, Detroit, Ml) containing 2% tryptose phosphate broth, 1% calf serum, and the most abundant modified nucleoside in tRNA, increases in 1% chicken serum at 37Â°Cin 5% CO2. This incubation procedure was

Received 3/21/85; revised 8/1/85; accepted 8/6/85. 1This work was supported by grants of the Italian National Research Council, 3 The abbreviations used are: MuLV, murine leukemia virus; tRNA'*', nonacy- Special Project "Oncologia," Contract 84.00516, and of M.P.I., Rome, Italy. lated proline-accepting tRNA (other tRNA's are similarly designated); CEF, chick 2Towhomrequestsforreprintsshouldbeaddressed. embryo fibroblasts; RSV, Rous sarcoma virus.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1985 American Association for Cancer Research. RETROVIRUSES AND LEVELS OF PSEUDOURIDINE AND tRNA used in all experiments. Secondary cultures were seeded at 5 x 105 RESULTS cells/60-mm plastic dish in 3 ml of E199 medium containing 2% tryptose phosphate broth, 1% calf serum and 1% chicken serum and exposed to Pseudouridine Excretion by Chick Embryo Fibroblasts. The 0.5 ml of RSV, Schmidt Ruppin strain, subgroup A, or to RAV-1 for 1 h. Chromatographie profile of the nucleosides purified from the Control cells underwent the same procedure, but with 0.5 ml of medium. culture medium of CEF is shown in Chart 1. Pseudouridine, After 1 h of incubation, the medium was replaced by 3 ml of fresh medium cytidine, uridine, inosine, guanosine, and adenosine were identi and incubated as described. In two different sets of dishes (each in fied by comparing retention times and absorbance ratios quadruplicate), the media were collected every 24 h (one set at 12, 36, 60, and 84 h and the other at 24,48,72, and 96 h) and stored at -20Â°C; (A28o:A254)ofthe unknown peaks with those of pure compounds chromatographed under the same conditions. The abundance of 3 ml of fresh medium replaced the original medium. Control plates were inosine is probably due to adenosine deaminase contained in the treated in the same way for cell counting (in duplicate). Virus Production Assay. To determine RSV and RAV-1 production calf serum present in the culture medium. by infected cells, 0.5 ml of each medium collection (from 36 to 96 h) was The concentrations in culture medium of the major nucleosides used to infect secondary cultures. The appearance of the transformed at various times after RSV or RAV-1 infection were very similar phenotype indicated the presence in the sample of RSV, whereas the to those of normal CEF (data not shown) and consistent with impossibility of obtaining transformation when these secondary cultures the data reported by Uziel and Selkirk (21). A dramatic quanti were reinfected with RSV indicated the presence of RAV-1 (challenge tative difference was detectable for pseudouridine excretion. assay). Chart 2 depicts the time course of pseudouridine excretion in Nucleoside Purification and Analysis. The purification, identification, normal and infected CEF. A significant (P < 0.02) difference and quantitation of major and modified nucleosides excreted by cells in between normal and infected cells was already evident 60 h after the culture medium were performed according to the methods of Colonna RSV infection. The production of viral particles was evident at et al. (18), with modifications. Briefly culture medium samples were centrifugea to remove cells and debris; 1-ml aliquots of the supernatant 60 h after the infection, and at 72 h the difference between were deproteinized with 1 ml ice-cold acetonitrile, placed on ice for 15 pseudouridine excretion of normal and infected cells was maxi min, and centrifuged at 3000 x g for 30 min. The supernatant was mal, even though no morphological sign of the transformed treated with bacterial alkaline phosphatase (0.75 unit/ml) in 0.1 M Tris- phenotype was detectable as yet; this latter began to appear HCI, pH 8.5, for 3 h to convert nucleotides to nucleosides. This procedure only 84 h after viral infection. eliminates small quantities of GMP that coelute with pseudouridine. The RAV-1-infected cells show amounts of pseudouridine excre ribonucleosides were purified on a phenylboronate column (Affi-Gel 601 ; tion very similar to those observed in RSV-infected cells. The Bio-Rad Laboratories, Richmond, CA) by eluting with 0.1 M formic acid time of appearance of neosynthetized viral particles in the me and then were lyophilized. These samples were resuspended (once) in dium was identical to that of RSV-infected cells; obviously no H2O, and 250-Ml aliquots were injected on a C,B-nBondapak column sign of transformation was observed in these cells. (Waters Associates, Milford, MA) using previously described high per formance liquid chromatography equipment (18). Nucleosides were tRNA Isoaccepting Species in Normal and Transformed eluted at a flow rate of 1 ml/min with 0.01 M NH4H2P04, pH 5.1, containing Cells. Because the tRNA primer assembled in the de novo- 60 ml of methanol/liter and quantitated with a Hewlett-Packard 3390 produced viral particles is of host cell origin (15, 16), we deter- integrator using deoxyguanosine as internal standard. tRNA Aminoacylation Assay. tRNA-aminoacylsynthetase was puri fied from wheat germ according to the method of Corbo ef al. (19). Deacylated tRNA from both AKR thymuses and CEF (confluent second ary cultures) was prepared as follows. Cells or tissues were homogenized in 2 volumes of 0.14 M NaCI:0.05 M sodium acetate, pH 5.1:1% sodium dodecyl sulfate, and centrifuged at 10,000 x g for 1 h; the supernatant was centrifuged at 105,000 x g for 90 min. Clear supernatant was extracted for 15 min at room temperature with an equal volume of phenol:chloroform:isoamyl alcohol (20:20:1) saturated with the homog- enization buffer. The aqueous phase was precipitated with 2.5 volumes of ice-cold ethanol at -80Â°C for 1 h, dried under vacuum, and resus pended in 10 mM MgCI2:10 HIM Tris-HCI, pH 7.5. This sample was u applied on a DE52 column (0.7 x 10 cm) equilibrated with the above- Â»Z mentioned buffer. tRNA was eluted with 1 M LiCl in the same buffer, m after the column was washed with 100 mM LiCl. After precipitation with ice-cold ethanol, tRNA was incubated at 37Â°Cfor 30 min in 100 mM Tris- 254 nm HCI:10mMEDTA, pH 9. Aminoacylation reaction was performed as described (19) using [3H]amino acids (Radiochemical Centre, Amersham, Buckinghamshire, England) at a specific activity of about 2.5 Ci/mmol, in a final volume of 40 jil, using 0.2 Aaeonmunit of tRNA, 40 MM[3H]amino acid, and 0.15 Â¿tg 280 nm of enzyme in 30 p\. RPC-5 Chromatography. Two Aaeonmunitsof tRNA were aminoacy- lated as described and applied onto a 0.7- x 80-cm column of RPC-5 10 20 30 and eluted with a linear gradient from 0.3 to 1 M NaCI in 0.01 M sodium TIME (min) acetate:0.01 M MgCI2:0.001 M EDTA:0.002 M /3-mercaptoethanol, pH Chart 1. High performanceliquid Chromatographieprofiles of nucteosktespuri fied from culture medium of CEF. Purification steps and high performance liquid 4.5. Fractions of 1 ml were collected and the radioactivity of the whole chromatography conditions are those reported under "Materials and Methods." ^, fraction was counted in 3 ml of Insta-Gel in a Packard Tri-Carb liquid pseudouridine;C, cytidine; U, uridine;/, inosine;G, guanosine;dG, deoxyguanosine scintillation system (20). (internalstandard);A. adenosine.

CANCER RESEARCH VOL. 45 DECEMBER 1985 6261

4 1.25

zÂ° 9 x 0.75 oc =

Ã» (A 13

0.25

36 48 60 72 84 96 o- 80 90 100 110 TIME (hrs) FRACTION Chart 2. Pseudouridine conÅ“ntration in culture medium of normal (â€¢),RSV- Charts. RPC-5 Chromatographie profiles of 3H-labeted tRNA"â„¢of normal (â€¢) infected (A), or RAV-1 -infected (CU)CEFat various times after seeding of secondary and lymphomatous AKR thymus (â€¢).Conditions are reported under 'Materials and cultures. Closed arrows, presence of infectious virus in the medium (RSV or RAV- Methods." 1); open arrows, presence of morphological signs of transformation (for RSV- mfected cells). Pseudouridine was quantitated as described under "Materials and Methods." showeda singlepeak,with identicalChromatographieproperties (datanot shown).

Table 1 Amino acid acceptance of tRNA purified from normal and lymphomatous AKR thymus and from normal and RSV-transformed CEF DISCUSSION pmol of charged tRNA/Aao m units of total tRNA" Our previous studies suggested that pseudouridine can be AKR CEF considered a marker of viral cell transformation. In fact the Normal Lymphomatous Normal RSV RAV-1 increased pseudouridine serum levels in mice with lymphomas tRNA"â„¢ 8.75 34.30 105.00 74.20 96.20 of viral origin before the appearance of the neoplastic phenotype tRNATrp 29.65 24.75 21.45 33.30 44.80 and the absence of this increase in a murine chemically induced tRNATy* 6.15 5.10 5.25 5.70 7.80 lymphoma supported the hypothesis that pseudouridine overpro Â°Charged tRNA amount was calculated on the basis of amino acid specific duction is not a general phenomenon of neoplasias but a specific activity. consequence of one of the steps of retrovirus-cell interaction (13). This hypothesis is strongly supported by the present report mined its concentration in two experimental systems for which of an overproduction of pseudouridine in RSV-infected CEF many the tRNA primer for reverse transcriptase is known (tRNA^0 for hours before the appearance of the transformed phenotype. AKR-MuLV and tRNATrpfor RSV). As reported in Table 1, proline Moreover increased pseudouridine excretion was evident also in acceptance by tRNA from lymphomatous AKR thymus is 4-fold RAV-1 -infected CEF, in which the mutant virus does not trans higher than that of normal (2-month-old mice) AKR thymus tRNA, form the cells but replicates normally, therefore causing virus whereas tRNATrp and tRNATyr were unchanged. To exclude that particle production. The two experimental systems, i.e., AKR the observed increase could be due to the presence in trans mouse strain and CEF transformed by RSV, have similar prop formed cells of "new" tRNA''â„¢species with higher affinity for the erties. In both cases an oncogenic retrovirus is the etiological specific aminoacylsynthetase, reverse phase Chromatographie agent of cell transformation, and both viruses use a cellular tRNA analysis on an RPC-5 column of acylated tRNA was performed as primer for reverse transcriptase. The tRNA primers for MuLV (see Chart 3). The close similarity in the retention volumes of and RSV have been identified as tRNA^ and tRNATrp, respec tRNAPrÂ°peaks between normal and lymphomatous AKR thymus tively, and completely sequenced (15,16). Incidentally they both suggests an increase of normally present tRNA species in trans contain in loop IV an extra pseudouridine residue instead of the formed tissue. normally present ribothymidine (15, 16); this means that their Similarly in CEF a 2-fold increase of the specific tRNA primer, pseudouridine content is four and three residues per molecule, tRNATrp, was observed in RSV-transformed cells (see Table 1). respectively. Such an increase was also present in RAV-1-infected CEF. Also We found a significant increase of the concentration of the in this case the Chromatographie elution profiles on RPC-5 two specific primers, tRNApro in lymphomatous AKR thymus and excluded the presence of a "new" tRNATrp species because they tRNATrp in RSV- or RAV-1-infected CEF. The finding that also in

CANCER RESEARCH VOL. 45 DECEMBER 1985 6262

RAV-1-infected CEF there is an increase of tRNATrp indicates REFERENCES that the phenomenon is not linked to the transformed phenotype, 1. Borek, E., and Sharma,O. K. New applicationsof urinary nucleoside markers. because RAV-1 lacks the src gene. The identity of the elution Recent Results Cancer Res., 84: 301-316, 1983. profiles of 3H-labeled tRNApro from normal and lymphomatous 2. Gehrke, C. W., Kuo, K. C., Waalkes, T. P., and Borek, E. Pattern of urinary thymus as well as of 3H-labeled tRNATrp from normal and RSV- excretion of modified nucleosides.Cancer Res., 39:1150-1153,1979. 3. Heldman, D. A., Grever, M. R., and Trewyn, R. W. Differential excretion of infected cells seems to rule out the possibility that the increased modified nucleosidesin adult acute leukemia. Blood, 67: 291-296,1983. concentration of these tRNA species could be due to the ap 4. Salvatore, F., Colonna,A., Costanze, F., Russo, T., Esposito, F., and Cimino, pearance of "new" isoaccepting tRNA, as described in other F. Modified nucleosides in body fluids of tumor-bearing patients. Recent Results Cancer Res., 84: 360-377,1983. systems (22, 23). It is worth noting that the proportion between 5. Speer, J., Gehrke, C. W., Kuo, K. C., Waalkes, T. P., and Borek, E. tRNA the two isoaccepting species of tRNApro seems to be slightly breakdown products as markers for cancer. Cancer (Phila.),44: 2120-2123, 1979. changed in transformed cells. 6. Tormey, D. C., Waalkes,T. P., and Gehrke, C. W. Biological markers in breast carcinoma. Clinical correlation with pseudouridine, N2,N2,dimethylguanosine During the assembling of neosynthesized viral particles, the and 1-methylinosine.J. Surg. Oncol., 14: 267-273, 1980. tRNA primer is the only component not coded for by the viral 7. Tormey, D. C., Waalkes, T. P., Hamann,D., Gehrke, C. W., Zumwalt, R. W., genome and it must be supplied by the host cell (24). This could Snyder, J., and Hansen,H. Biologicalmarker in breast carcinoma. I. Incidence of abnormalitiesof CEA, HCG,three polyaminesand three minor nucleosides. indicate that a more active synthesis of a specific tRNA species Cancer (Phila.),35: 1095-1100,1975. occurs in the host cell because a consistent aliquot of the primer 8. Trewyn, R. W., Glaser, R., Kelly, D. R., Jakson, D. G., Graham, W. P., and Speicher,C. E. Elevatednucleosideexcretion by patients with nasopharyngeal molecules is entrapped in neosynthetized viral particles. Specific carcinoma. Cancer (Phila.),49: 2513-2517,1982. activation of the transcription of particular tRNA species may 9. Russo, T., Salvatore, F., and Cimino, F. Determination of pseudouridine in tRNA and in acid soluble tissue extracts by high performance liquid chroma also be speculated. tography. J. Chromatogr., 296: 387-393, 1984. The latter hypothesis is supported by the observation that 10. Salvatore, F., Izzo, P., Traboni, C., Esposito, F., and Cimino, F. Studies of cultured fibroblasts contain much more tRNA^0 than do thymo- transfer RNA methylation in cell transformation. In: E. Usdin, R. T. Borchardt, and C. R. Creveling (eds.), The Biochemistry of S-Adenosylmethionineand cytes (see Table 1). This is probably due to the preponderant Related Compounds, pp. 389-397. London: MacMillan, 1982. translation of proline codon-rich collagen mRNA, which requires 11. Fujimura, S., and Shimizu, M. Enhancedactivity of tRNA pseudouridine syn- an elevated number of adaptor molecules. Furthermore in the thetase in Yoshida ascites sarcoma. Biochem. Biophys. Res. Commun., 79: RSV-transformed cells a decrease of tRNA''â„¢is observed, and it 763-768,1977. 12. Salvatore, F., Russo, T., Colonna, A., Cimino, L., Mazzacca, G., and Cimino, F. Pseudouridine determination in blood serum as tumor marker. Cancer seems to be related to the decrease of the collagen mRNA Detect. Prev., 6: 531-536,1983. transcription (25). 13. Russo, T., Colonna, A., Salvatore, F., Cimino, F., Bridges, S., and Gurgo, C. We are now attempting to identify and clone the tRNA""â„¢and Serum pseudouridine as a biochemical marker in the development of AKR tRNATrpgenes to study the mechanisms that control their expres mouse lymphoma. Cancer Res., 44: 2567-2570,1984. 14. Cloyd, M. W., Hatley, J. W., and Rowe, W. P. Lymphomagenicityof recombi sion in the two experimental systems. As yet no mechanism is nant mink cell focus-inducing murine leukemia viruses. J. Exp. Med., 757: known for the regulation of the tRNA gene expression in eukar- 542-552,1980. 15. Harada, F., Peters, G. G., and Dahlberg,J. E. The primer tRNA for Moloney yotic cells (26). murine leukemia virus DNA synthesis. J. Biol. Chem., 254: 10979-10985, The data presented here suggest that the increased produc 1979. 16. Harada, F., Sawyer, R. C., and Dahlberg, J. E. A primer ribonucleic acid for tion of pseudouridine could reflect the increased turnover of the initiation of in vitro Rous sarcoma virus deoxyribonucleic acid synthesis. J. specific tRNA the synthesis of which is enhanced in the cell as Biol. Chem., 250: 3487-3497,1975. 17. Rubin, H., and Vogt, P. K. An avian leukosis virus associated with stocks of a consequence of retroviral replication. Any increase of modified Rous sarcoma virus. Virology, 77: 184-194, 1962. nucleosides other than pseudouridine (e.g., 1-methyladenosine, 18. Colonna, A., Russo, T., Esposito, F., Salvatore, F., and Cimino, F. Determi 1-methylguanosine, 2-rnethylguanosine, etc.) cannot be detected nation of pseudouridineand other nucleosidesin human blood serum by high performanceliquid chromatography.Anal. Biochem., 730: 19-26, 1983. because they are present in tRNATrp in amounts 3-fold lower 19. Corbo, L., Ciliberto, G., Traboni, C., SantamarÃa,R.,Cimino, F., Cortese, R., and Salvatore, F. Functional assay of tRNA molecules transcribed from a than is pseudouridine and are therefore below the minimal de purified gene. NucleicAcids Res., 70: 7363-7372,1982. tection limit of the high performance liquid chromatography pro 20. Pearson,R. L., Weiss,J. F.,and Keimers,A. D. Improvedseparationof transfer cedure (18) used (25 pmol of nucleoside). Moreover these com RNAs on polychlorotrifluoroethylene-supportedreversed-phasechromatogra phy columns. Biochim. Biophys. Acta, 228: 770-774,1979. pounds are also excreted as modified bases because they 21. Uziel,M., and Selkirk, J. K. Pyrimidinenucleoside,pseudouridineand modified possess an A/-glycosidic bond that is subject to hydrolysis, nucleoside excretion by growing and resting fibroblasts. J. Cell. Physiol., 99: 217-222,1979. whereas the C-glycosidic bond present in pseudouridine is highly 22. Marini, M., and Mushinsky,J. F. Transfer ribonucleicacids from eleven immu- stable (27) and thus only this nucleoside is a true end product. noglobulin secreting mouse plasmacytomas. Constant and variable Chroma tographie profiles compared with the myeloma protein sequences. Biochim. The same applies to modified nucleosides in the blood serum of Biophys. Acta, 562: 252-270,1979. cancer patients (12) and of AKR lymphomatous mice (13). How 23. Nishimura,S. Modified nucleosidesin tRNA. In: P. R. Shimmel,D. Soil, and J. N. Abelson (eds.), Transfer RNA: Structure, Properties and Recognition, pp. ever, in our previous studies (4) on human lymphomas we found 59-79. Cold Spring Harbor, NY: Cold Spring Harbor Laboratories, 1979. that increased pseudouridine urinary concentration was accom 24. Gilboa,E., Mitra, S. W., Goff, S., and Baltimore, D. A detailed modelof reverse panied by an increase of other modified nucleosides, such as 1- transcription and tests of crucial aspects. Cell, 78: 93-100,1979. 25. Adams, S. L., Sobel, M. E., Howard, B. H., Olden, K., Yamada, K. M., de methyladenosine, 2-methylguanosine, 1-methylguanosine, and Crombrugghe,B., and Pastan, I. Levelsof translatablemRNAs for cell surface 7-methylguanosine. Similar results have also been reported by protein, collagen precursors, and two membrane proteins are altered in Rous sarcoma virus-transformed chick embryo fibroblasts. Proc. Nati. Acad. Sci. other authors (2, 5, 8). USA, 74: 3399-3403,1977. The evaluation of pseudouridine as an etiological marker for 26. Ciliberto,G., Castagnoli,L., and Cortese, R. Transcription by RNA polymerase III. Curr. Top. Dev. Biol., 78: 59-88, 1983. neoplasias where retroviruses are implicated is a promising area 27. Chambers, R. W. The chemistry of pseudouridine. Progr. Nucleic Acid Res. for further experimental studies. Mol. Btol., 5:349-375,1966.

CANCER RESEARCH VOL. 45 DECEMBER 1985 6263

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1985 American Association for Cancer Research. Pseudouridine Excretion and Transfer RNA Primers for Reverse Transcriptase in Tumors of Retroviral Origin

Franca Esposito, Tommaso Russo, Rosario Ammendola, et al.

Cancer Res 1985;45:6260-6263.

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