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Haematology and Blood Transfusion Vol. 26 <strong>Modern</strong> Trends in Human Leukemia IV Edited by Neth, Gallo, Graf, Mannweiler, Winkler © Springer-Verlag Berlin Heidelberg 1981 Proteolytic Processing of A vian and Simian Sarcoma and Leukemia Viral Proteins B. Konze-Thomas and K. von der Helm A. Introduction Viral proteins of avian and mammalian leukemia and sarcoma virus es are synthesized in their host cells primarily as precursors. They are successively processed to sm aller proteins, which then can be assembled into virus particles (Vogt et al. 1975; Areement et al. 1976; Okasinsky and Velieer 1976; Eisenman and Vogt 1978). This processing has been found to be caused by a virus-specific proteolytic enzyme. The protease p15 is eoded by the genome of leukemia/sareoma viruses; it is part of the internal viral eore, the group-specifie antigen (gag) protein, and c1eaves its own gag precursor (von der Helm 1977). This preeursor c1eavage ean be demonstrated in vitro with purified p 15 from AMV and gag preeursor bound to an antibody (direeted against gag proteins) and Staph. aureus protein A (Dittmar and Moelling 1978). Using this system we studied whether protein preeursors with gag sequences of other related or unrelated oncornaviruses could be c1eaved by the avian gag protein p15. This paper deseribes the c1eavage of a 110 k dalton fusion protein of the replication-defeetive avian aeute leukemia virus MC 29 (Langlois et al. 1967; Bister et al. 1977) and a gag protein preeursor pr65 of Simian sareoma virus (SSV) (Wolfe et al. 1971; Deinhardt et al. 1978) by avian p15 into diserete smaller proteins. Since protease p15 evidently c1eaves off the gag sequenees of the fusion protein gag-x (whereas the x sequences may represent the putative "one"-gene sequences), we diseuss how p15 eould be used as a tool for mapping transformation proteins. B. Results and Discussion To determine the optimal eonditions for c1eavage of IgG-bound pr76 by the p 15 protease, the immunoeomplex containing 35S-methionine-Iabeled pr76 was ineubated for different periods of time and different salt eoneentrations in presenee of p15 protease (B. Konze Thomas and K. von der Helm, to be published). Protease p15 was isolated from virions by eonventional methods (von der Helm and Konze-Thomas 1980; B. Konze-Thomas and K. von der Helm, to be published). Figure 1 shows that during 30 min of ineubation in 0.15 M NaCI proeessing of pr76 and pr180 eommeneed and is eompleted after 6 h. In presenee of higher salt eoncentration (i.e., 1 M NaCl) in vitro proeessing is already eompleted after 2 h of ineubation. Evidenee for the eompletion of the proeessing is the disappearanee of the polypeptide pr32 whieh is an intermediate preeursor to p19 (Vogt et al. 1975; von der Helm 1977). Cleavage of other non-gag-containing polypeptides (i.e., gp85) or further c1eavage of the proeessed gag proteins (p27, ete.) has never been observed, even with very long ineubation periods (not shown). J. C1eavage of Me 29 Protein 110 k by pIS The defeetive avian aeute leukemia virus MC 29 has among other defeets adeletion of the p15 gene (Bister et al. 1977; Mellon et al. 1978). A 110 k dalton protein, the only virus-specifie polypeptide synthesized in infeeted and transformed nonprodueer eells is not proeessed. It is a fusion protein of an ineomplete gag gene (p19 and p27) and unknown 409
pr 180 - - - P 12/15 - A B c D E F Fig. 1. In vitro cleavage of pr76 by pIS. RSV~infected chicken fibroblasts were labeled fOT 1 h with 35S-methionine, lysed, and immunoprecipitated by anti-p27 serum and protein A sepharose (B. Konze-Thomas and K. von der Helm, to be published) (A), untreatedcontrol orincubatedin 0.15 MNaCI at 37°C with 0.7 ug of protease pIS for 112 h (B), 2 h (C), 6 h (D) or for 2 h in 0.5 MNaCI (E) or 1 MNaCi (F). After incubation the sampies were analysed by 12.5% SDS-PAGE and autoradiography sequences coding for a putative transformation protein (Dittmar and Moelling 1978). We used the pIS protease as a tool for restrictive protein cleavage in vitro and tried to cleave off the gag sequences in order to determine the size of the non-gag sequences of the 110 k dalton polypeptide. From a 35S-methionine-Iabeled lysate of nonproducer cells, the 110 k d protein had been immunoprecipitated by aserum directed against p27 structural protein of avian leukemia/sarcoma virus (B. Konze-Thomas and K. von der Helm, 1980, to be published). After incubation of the immunocomplex with pIS the 110 k dalton pro tein had been cleaved into proteins of the molecular weight 75 k, 55 k, 32 k and 25/24 k dalton (Fig. 2). A comparison of these proteins by a V8 protease digestion analysis suggested that all of them derived from the 110 k dalton protein (not shown). By this analysis the 75 k d polypeptide appears to be an intermediate precursor to the 55 k d product. The 25/24 and 32 k d polypeptides react stronglywith anti-gag serum, the 55 k d polypeptide, veryweakly. We assume thatthe24/25 kdand 32k dpolypeptides are gag-related proteins, while the 55 k d polypeptide contains no gag sequences and may represent the putative "one" part of the fusion protein. 11. Immunoprecipitation of Intracellular SSV -Specific Proteins Simian sarcoma virus was originally isolated from a spontaneous sarcoma of a woolly monkey (Wolfe et a1. 1971). Similar to the Me 29 virus, this virus transforms eells and is replication-defective. Isolates of this virus always contain excess helper virus (SSAV) (Wolfe et a1. 1971). Cells infected solely with SSV do not produce virus. We immunoprecipitated 35S-methionine-Iabeled lysates of several SSV nonproducer or SSV (SSAV) producer cells with serum against the' SSAV viral gag 410
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Haematology and Blood Transfusion 2
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Dr. Rolf Neth, Universitäts-Kinder
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Participants of the Meeting Aaronso
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Kurth, R, Friedrich-Miescher-Labora
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Wilsede Scholarship Holders (Grante
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Preface Gut ist eine Lehrart, wo ma
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Personal and scientific discussion
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Acknowledgement We should like to t
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Wilsede, June 21 1978 Memorial Trib
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limiting benign lymphoproliferative
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this most unlikely, however (for re
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longer subject to the same control
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Bregula U, Wiener F, Harris H (1971
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fresh lymph node biopsies from ten
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Fig. 3. Binucleate mitosis in an ob
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y Fig. 4. Schematic diagram of post
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than that among patients with Stage
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Tests of binding affinity, agglutin
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N Engl J Med 297: 963-968 - Green I
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Clinical Aspects
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"ring chromosome". "Mar" is marker,
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191 chromosomally abnormal patients
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tumors of both African and non-Afri
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Haematology and Blood Transfusion V
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Retrospective group 26/93 evolved l
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Treatment
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advantage in terms of duration of f
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leukaemia. An analysis of 168 patie
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11. Treatment Program Chemotherapy
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efractory congestive heart failure
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period (Sequence II) was gene rally
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children with a multiple drug proto
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DAYS 1 5 10 15 20 25 30 35 , I I I
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Biologically Drug Outcome fit" sens
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may counterbalance the potential be
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Ht (0--0) 50 40 30 20 10 0 Platelet
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10 7 ,-----------------------------
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11. Haematological and Biochemical
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0\ 0\ INTERFERON ... 10 3 rl E "'
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("pre-B" phenotype) has been descri
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human bone marrow cells exhibit the
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10 9.,0 MBKCC .. .,4 B prot:oool 1'
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demann W, Büehner T, Arlin Z, Gee
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-...l 00 a-tLDRENS CAt«:ER S TU>Y
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prognostic characteristics under in
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significantly higher in HR} (14.5%,
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normal or elevated immunoglobulin l
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References Andreeff M, Darzynkiewic
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R GROUP 1 ( 1970 - 1971 ) 17 PTS. R
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Table 3. Influence of initial featu
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LIJ (f) a.. < -1 LIJ Q: u. 0 >- I-
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me nt indicated that additional the
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42:2123-2134 - Chessells JM, Cornbl
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Baum et al. 1979). This study was b
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1. 0 '--'1--" 0.9 e: o .- VI VI E C
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e: o e: 1. 0 ,.--...-- 1'"'\ ......
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In conclusion, we can state that th
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Haematology and Blood Transfnsion V
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INDUCTION VCR + PRED + ASNASE (wk 3
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SCHED.INT 1 MTX • ! Adria MTX •
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~ looh---------ooooc>----o---c>----
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Consoll- Irra .tlon t) .tlon Indu
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Highrisk Standard risk Table 1. Cli
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Cumulo/ive comp/el9 remission 1.0 .
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10° 8 = 6 ö 4 Non T Cell .~ :ö
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% SURVIVAL 100 ~ ~II 0 80 60 40 - -
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a) Percentage of donor cell mitoses
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Table 5. Results of bone marrow tra
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...... w N Table 7. Clinical result
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agent had to be added and that in t
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C. Chronic Myelogenous Leukemia The
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Patients with a suitable donor rece
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No. Recurrent Leukaemia Other death
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followed by dialysis against isoton
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HS, HL- Heme >_ 40 0 HS I ;:. =====
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knowing at present. These genes may
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esistance to antifolates. In Vitro
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a DNA probe specific for the gene t
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translocation between chromosomes 9
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Table 1. Subdivision of 1827 Myelo-
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NORMAL CELL ®
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some change of the primary type, in
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F. Transformation of Mouse Bone Mar
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were carried out with 3H-MTX in the
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the lysozyme cDNA prepared from ovi
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et al. 1976). The BJAB cellline in
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dalton bands corresponding to light
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1979). The EBV-carrying cell lines
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producer lines (DAUDI and P3HR-1),
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G, Giovanella B, Westman A, Stehlin
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Dc;bt CT + i J.I9 Raji; 1. i .. 100
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c. Discussion During infectious mon
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C. Nonepisomal Viral DNA We have us
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A 260 3.0 A B 1670 70 N2 2.0 c: E .
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vivo. Nature 260:302-306 - Kirk JTO
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Fig. 1. a Polyacrylamide gelelectro
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1979). The lymphocyte subpopulation
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lysin 0 antigens are found, and mix
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Pope JH (1978) Long-term T cell-med
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Haematology and Blood Transfnsion V
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Charaeteristie Morphologie maturati
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after TPA-induction (Table 3). The
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PA (1978) Reactivity of a rabbit an
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Blood Monocyte. B lood Monocytes 1-
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and nonhistone proteins can serve a
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The findings of the evaluation of s
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Fig. 3. Tumor grawth of L 428 cells
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Cell biological and Immunological A
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consideration. Sanel (1973) obtaine
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endotoxin serum, were used. A parti
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monocytic leukemia cell line in cul
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B. Isolation, Characterization and
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don al hemopathies generally displa
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nitors. Blood Cells 6:595-607 - Min
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\I) ....I ....I IU Jl 200 r 100 90
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their haemopoietic cells' ability t
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normal normal ALL ALL ALL persons p
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64.5 { V)
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References Biermann E, Neth R, Gros
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ehambers, the growth pattern observ
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References Anderssen LC, 10kinen M,
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REH CELL UNE TO CALLb~Ö ~q~ V.M. c
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the macrophage series in which the
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Table 1. Production of factor depen
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Table 1. Characteristics of the adh
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Fig. 3. A human marrow culture at 6
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300 250 • HYPERPROLIFERATIVE PATT
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Fig. 9. Nonadherent population from
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The cobblestone areas were often no
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L (1976) Induction of colony format
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B. Materials and Methods J. Tissue
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Fig. 2. Ultrastructural appearance
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Table 1. Monoclonal antibody reacti
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al. 1979; Janossy et al. 1979) is e
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to which this is an exact replica o
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inger JL (1976) Distribution of la-
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I region antigens were found to be
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have been reported to be present on
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V. FunctionalAssays Assays for PHA
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Functional studies of UCHT1 separat
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40 ,...... (5 L.. C o u 30 .9 ~ .~
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with immunohistochemieal methods (L
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can be dissected by cloning. Utiliz
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Table 1. Origin and marker profile
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complete identity of gp 100 from no
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Table 1. Reaction of single anti se
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media. Following 24-h incubation, c
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nonleukemic eells (Greaves 1979). L
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Fig. 2. Cytocentrifuged smears of b
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Modification of amino-groups of hum
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disturb this balance in the directi
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The majority of human blood lymphoc
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of the EBV infected B cells. Howeve
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and immunoglobulins. J Immunol 120:
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" K 10 • j ~ ALL RNlL AL wtfh les
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tion akuter Leukämiezellen in "spo
Page 367 and 368: D. Results and Discussion J. Acute
Page 369 and 370: selectively responsive to different
Page 371 and 372: Haematology and Blood Transfusion V
Page 373 and 374: l1J ~ 100 ~ Q. ::) l1J Z 0 ~ z >- .
Page 375 and 376: Table 2. Growth inhibition of S49 e
Page 377 and 378: Reverse Infectious ASC/2X1Q6 transc
Page 381 and 382: munized animals to 1316 tumor cells
Page 383 and 384: Haematology and Blood Transfnsion V
Page 385 and 386: Fig. 3. Expression of retroviral gp
Page 387 and 388: pg70. J Exp Med 143: 151-166 - McGr
Page 389 and 390: Table 1. Expression of tumor antige
Page 391 and 392: Virological and Molecularbiological
Page 393 and 394: Table 1. Oncogenic properties of re
Page 395 and 396: also Fig. 1). It followed that the
Page 397 and 398: whether this sequence is related to
Page 399 and 400: zed in infected cells argue that th
Page 401 and 402: a specific viral transforming prote
Page 403 and 404: detect viral RNA as the only charac
Page 405 and 406: Eisen H (1980) Myeloproliferate vir
Page 407 and 408: p,60- -34K - 1 2 3 4 Fig. 1. In vit
Page 409 and 410: .. p - • a'T ... I .t'l Fig. 4. D
Page 413 and 414: Fig. 2. Photomicrographs of NY68 in
Page 415 and 416: DEAE (o(lJmn 'liI GI -2 2 o e s 1
Page 417: vity was deterrnined. For endogenou
Page 421 and 422: 85,- 66- -27 - -66 40- 27- -1'9 19-
Page 425 and 426: = 0.5 o E Q. o ILI 0.4 (J) « ILI .
Page 427 and 428: Table 1. Oncogenic potential of avi
Page 429 and 430: 100 III GI c: o "8 80 > w
Page 431 and 432: Expt. No. Infecting virus Proportio
Page 433 and 434: Haematology and Blood 'fransfusion
Page 435 and 436: sequences, provided its gag portion
Page 437 and 438: 77:3009-3013 - Hu SSF, Moscoviei C,
Page 439 and 440: RI 2.2 md and Hind III 2.6 md leuke
Page 443 and 444: RELATIONSHIP OF ENDOGENOUS AND EXOG
Page 445 and 446: E ........ E Cl. U E :::l .... Q) (
Page 447 and 448: References Astrin S (1978) Endogeno
Page 449 and 450: the same animal were examined (e.g.
Page 451 and 452: I) 'IQ a \0 - CL -- Fig. 2. Restrie
Page 453 and 454: ~ ..................... ~ Cell 3' 5
Page 455 and 456: a :Sa,C! + . ~ 111 i' ( " '~ .J. b
Page 457 and 458: Table 1 Sampie Tissue TS fragment"
Page 459 and 460: a b Sac I / rep ._-_.-_ K J nl rep
Page 461 and 462: Haematology and Blood 'fiansfusion
Page 463 and 464: Osteopetrosis and osteosarcomas occ
Page 465 and 466: Lymphoma cellline Table 1. T and B
Page 467 and 468: Table 2. In vivo growth and oneogen
Page 469 and 470:
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Table 1. Transforming aetivity of c
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Table 3. Transformation aetivity of
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- Shoemaker C, Goff S, Gilboa E, Pa
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indieate a shifting of target eell
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Table 2. Expression of Jl and Sourc
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~ i ,..1---------,,':::::::::::::::
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fragment of pBR322 DNA, a 3.0-kbp S
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Mak TW (1979) Proc Natl Acad Sci US
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Table 1. Transformation of 3T3 cell
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inserts in the genome of rapidly tr
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pulations, it seemed likely that th
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contains antigens which react with
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le of reacting in sensitive radioim
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gic approach. In: Hiatt HH, Watson
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12 Eco R [ Fig. 1. Hybridization pa
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10 - '1~ )( -~ S:! E A U I o 1 2
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Haematology and Blood 'fransfusion
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cultures. These cells did not conta
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indication of malignant T-cells in
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Fig. 3. Thin-section electron micro
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Table 3. Lack of detectable related
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J. HTLV Was Present in the Primary
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specific signals into nonspecific s
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Table 2. Distribution of HTLV-relat
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u 10 ~ 20 ~ :c 30 a 40 o Fig. 1. Ki
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SSV TrS-gp labeled effectively with
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60 HFF/SSV cDNA CELL RNAs: 0 HF/SSV
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Antisera a Table 1. Immunofluoresce
Page 539 and 540:
Haematology and Blood 'ftansfusion
Page 541 and 542:
y competition RIA yielded virtually
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SiS'V' gp70: ANTIGEN, ANTI BODY, IM
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type-C virus p30 antigen in cells f
Page 547 and 548:
c. Results The fractionated whole-b
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induced antibodies as well as exper
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- Fig. la-f. Retravirus particles p
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eplication of infecting murine leuk
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immunodeficiency, multic10nal lymph
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Bovine leukemia --, proteins of 499
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Human T- cell - -, characterization
Page 563 and 564:
RNA -, of tumor virus 439 Rous sarc
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Haematology andBlood Transfusion Su
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