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herpes simplex virus. Virology 31 : 120-128 - Mercola KE, Stang HO, Browne J, Salser W, Cline MJ (1980) Insertion of a new gene of viral origin into bone marrow cells of mice. <strong>Science</strong> 208: 1033-1035 - Nunberg JH, Kaufman RJ, Chang ACY, Cohen SN, Schimke RT (1980) Structure and genomic organization of the mouse dihydrofolate reductase gene. CeIl19:355-364 - Salser W, Tong BO, Stang HO, Browne JK, Mercola K, Bar-Eli M, Cline MJ (to be published) Gene therapy techniques: Use of drug resistance selections in intact animals to insert human globin genes into bone-marrow cells of living mice. - Wigler M, Pellicer A, Silverstein S, Axel R (1978) Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell 14:725-731 170
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 Organization of the Methotrexate Resistant Mouse L5178YR Dihydrofolate Reductase Gene and Transformation of Human HCT-8 Cells by This Gene D. I. Scheer, s. Srimatkandada, B. A. Kamen, S. Dube, andJ. R. Bertino A. Introduction Gene amplification has been recently shown to play a pivotal role in the development of resistance to the folate analogue, methotrexate (MTX), in several cell culture systems (Schimke et al. 1978). Stepwise increases in the concentration of MTX supplied to the medium in which the murine lymphoblastoid line L5178Y was cultivated resulted in the development of 100,000-fold elevation in resistance (Dolnick et al. 1979). The resistant line, L5178YR, was able to grow in the presence of millimolar concentrations of MTX, and it was shown that these ceHs had a 300-fold elevation in the activity of the target enzyme, dihydrofolate reductase (DHFR). Moreover, the increase in DHFR activity could be correlated with a proportional increase in DHFR-specific mRNA (300-fold) as weH as a 300 fold-increase in DHFR gene copies. To clarify the mechanism of the amplification event as weH as gain insight into the regulation of the DHFR gene an analysis has been made of the DNA sequence organization of the DHFR gene. Further, DNA-mediated gene transfer has been employed to transform a human colon carcinoma line (HCT-8) with DHFR genes from the L5178YR line. B. Methods and Materials I. Cell Culture Mouse L5178Y and L5178YR lines were maintained in Fischer's medium with 10% horse serum. Human colon carcinoma, HCf -8, was maintained in RPMI medium containing 20% fetal calf serum. 11. Isolation of DNAs Essentially as described by Blin and Stafford (1976) 111. 1ransformation Essentially as described by Graham and van der Eb (1973) with minor modifications. IV. Enzymology Essentially as described by Hayman et al (1978) for DHFR and by Lomax and Greenberg (1967) for TMP synthetase, with modifications by Dolnick and Cheng (1977) and Kamen et al. (1976) for the cofactor binding studies. V. Southern Blot Hybridization Southern blot hybridization was done essentially as described by Southern (1975) with modifications by Wahl et al. (1979). Cloned DHFR cDNA probes were kindly provided by Dr. R. Schimke (See Chang etal.1978). C. Results and Discussion Data obtained from our laboratory as weIl as that of Schimke (Nunberg et al. 1980) suggests that the murine DHFR gene is interrupted by at least five intervening sequences. Figure 1 depicts a Southern blot of amplified L5178YR DNA which has been cleaved by several enzymes, transferred to DBM paper, and hybridized with the c10ned DHFR cDNA probes (pDHFR 21 and 26). Since none ofthe indicated enzymes c1eave within the structural gene, the patterns of fragmentation result from sites provided by the intervening sequences. From limited and complete Pstl digestion 171
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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
Page 131 and 132: Highrisk Standard risk Table 1. Cli
Page 133 and 134: Cumulo/ive comp/el9 remission 1.0 .
Page 135 and 136: 10° 8 = 6 ö 4 Non T Cell .~ :ö
Page 137 and 138: Haematology and Blood Transfusion V
Page 139 and 140: % SURVIVAL 100 ~ ~II 0 80 60 40 - -
Page 141 and 142: a) Percentage of donor cell mitoses
Page 143 and 144: Table 5. Results of bone marrow tra
Page 145 and 146: ...... w N Table 7. Clinical result
Page 147 and 148: agent had to be added and that in t
Page 151 and 152: C. Chronic Myelogenous Leukemia The
Page 153 and 154: Patients with a suitable donor rece
Page 155 and 156: No. Recurrent Leukaemia Other death
Page 157 and 158: followed by dialysis against isoton
Page 161 and 162: HS, HL- Heme >_ 40 0 HS I ;:. =====
Page 165 and 166: knowing at present. These genes may
Page 167 and 168: esistance to antifolates. In Vitro
Page 169 and 170: a DNA probe specific for the gene t
Page 171 and 172: translocation between chromosomes 9
Page 173 and 174: Table 1. Subdivision of 1827 Myelo-
Page 175 and 176: NORMAL CELL ®
Page 177 and 178: some change of the primary type, in
Page 181: F. Transformation of Mouse Bone Mar
Page 185 and 186: were carried out with 3H-MTX in the
Page 189 and 190: the lysozyme cDNA prepared from ovi
Page 193 and 194: et al. 1976). The BJAB cellline in
Page 195 and 196: dalton bands corresponding to light
Page 197 and 198: 1979). The EBV-carrying cell lines
Page 199 and 200: producer lines (DAUDI and P3HR-1),
Page 201 and 202: G, Giovanella B, Westman A, Stehlin
Page 205 and 206: Dc;bt CT + i J.I9 Raji; 1. i .. 100
Page 207 and 208: c. Discussion During infectious mon
Page 211 and 212: C. Nonepisomal Viral DNA We have us
Page 213 and 214: A 260 3.0 A B 1670 70 N2 2.0 c: E .
Page 215 and 216: vivo. Nature 260:302-306 - Kirk JTO
Page 217 and 218: Fig. 1. a Polyacrylamide gelelectro
Page 221 and 222: 1979). The lymphocyte subpopulation
Page 223 and 224: lysin 0 antigens are found, and mix
Page 225 and 226: Pope JH (1978) Long-term T cell-med
Page 227 and 228: Haematology and Blood Transfnsion V
Page 229 and 230: Charaeteristie Morphologie maturati
Page 231 and 232: 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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Page 307 and 308:
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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
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D. Results and Discussion J. Acute
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selectively responsive to different
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l1J ~ 100 ~ Q. ::) l1J Z 0 ~ z >- .
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Table 2. Growth inhibition of S49 e
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Reverse Infectious ASC/2X1Q6 transc
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munized animals to 1316 tumor cells
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Haematology and Blood Transfnsion V
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Fig. 3. Expression of retroviral gp
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pg70. J Exp Med 143: 151-166 - McGr
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Table 1. Expression of tumor antige
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Virological and Molecularbiological
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Table 1. Oncogenic properties of re
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also Fig. 1). It followed that the
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whether this sequence is related to
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zed in infected cells argue that th
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a specific viral transforming prote
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detect viral RNA as the only charac
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Eisen H (1980) Myeloproliferate vir
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p,60- -34K - 1 2 3 4 Fig. 1. In vit
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.. p - • a'T ... I .t'l Fig. 4. D
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Fig. 2. Photomicrographs of NY68 in
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DEAE (o(lJmn 'liI GI -2 2 o e s 1
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vity was deterrnined. For endogenou
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pr 180 - - - P 12/15 - A B c D E F
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85,- 66- -27 - -66 40- 27- -1'9 19-
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= 0.5 o E Q. o ILI 0.4 (J) « ILI .
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Table 1. Oncogenic potential of avi
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100 III GI c: o "8 80 > w
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Expt. No. Infecting virus Proportio
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Haematology and Blood 'fransfusion
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sequences, provided its gag portion
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77:3009-3013 - Hu SSF, Moscoviei C,
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RI 2.2 md and Hind III 2.6 md leuke
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RELATIONSHIP OF ENDOGENOUS AND EXOG
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E ........ E Cl. U E :::l .... Q) (
Page 447 and 448:
References Astrin S (1978) Endogeno
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the same animal were examined (e.g.
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I) 'IQ a \0 - CL -- Fig. 2. Restrie
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~ ..................... ~ Cell 3' 5
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a :Sa,C! + . ~ 111 i' ( " '~ .J. b
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Table 1 Sampie Tissue TS fragment"
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a b Sac I / rep ._-_.-_ K J nl rep
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Haematology and Blood 'fiansfusion
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Osteopetrosis and osteosarcomas occ
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Lymphoma cellline Table 1. T and B
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Table 2. In vivo growth and oneogen
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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
Page 481 and 482:
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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
Page 519 and 520:
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
Page 525 and 526:
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
Page 537 and 538:
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
Page 561 and 562:
Human T- cell - -, characterization
Page 563 and 564:
RNA -, of tumor virus 439 Rous sarc
Page 565 and 566:
Haematology andBlood Transfusion Su
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