Feb 1, 1982 - For exam- ple, the transforming gene of the avian sarcoma viruses v-src, ... oncogene may contain two functional domains, v-erbA and v-erbB ...
The EMBO Journal, Vol. 1 No. 2, pp.237- 242, 1982
Molecular cloning and characterization of the chicken DNA locus related to the oncogene erbB of avian erythroblastosis virus
A.Sergeantl*, S.Saule, D.Leprince, A.Begue, C.Rommens, and D.Stehelin U 186 INSERM, Oncologie Moleculaire, Institut Pasteur, 15, Rue Camille Guerin, 59019 Lille Cedex, France Communicated by W.Keller Received on 1 February 1982
Chicken cell DNA contains sequences which are homologous to the avian erythroblastosis virus oncogene v-erb. These cellular sequences (c-erb) have been isolated from a library of chicken cell DNA fragments generated by partial digestion with AluI and HaeII and shown to be shared by at least two loci in the chicken DNA. One of them, denoted c-erbB, contains 1.8 kilobase pairs of chicken DNA homologous to the 3' part of the v-erb oncogene (v-erbB). Restriction mapping studies show that the c-erbB DNA sequences homologous to v-erbB are distributed among six EcoRI fragments located in a single genomic region. Heteroduplexes between v-erbB in viral RNA and cloned c-erbB DNA show that the chicken DNA sequences homologous to v-erbB are interrupted by 11 DNA sequences not present in the v-erb oncogene. We conclude from our data that the c-erbB locus might represent the cellular progenitor for the v-erbB domain of the v-erb oncogene.
Key words: viral oncogene/cellular oncogene/DNA cloning/ restriction analysis/heteroduplex mapping Introduction Recent progress in retroviral oncology supports the theory that some cancers could be due to DNA rearrangements that bring cellular genes under the control of highly active transcription promoters (Klein, 1981). Moreover, it has been shown that the retroviral oncogenes, v-onc, are homologous to "normal" genes of their host cells, c-onc, which may or may not be interrupted by intervening sequences (Stehelin et al., 1976, Roussel et al., 1979, Goff et al., 1980, Jones et al., 1980, Francini et al., 1981, Shalloway et al., 1981). For example, the transforming gene of the avian sarcoma viruses v-src, which contains an uninterrupted coding sequence of 1590 bp (Czemilofsky et al., 1980b), is homologous to chicken DNA sequences interrupted by five or six intervening sequences (Shalloway et al., 1981), whereas the cellular gene c-mos, homologous to the transforming gene of Moloney sarcoma virus v-mos, is colinear with the viral sequences (Jones et al., 1980). Experimental data suggest that the retroviral oncogenes represent cellular genes that play essential roles in cell growth and/or differentiation (Beug et al., 1979, Stehelin et al., 1980). If such genes evade cell regulation and are continuously expressed at a high level their products might change the growth and/or affect the differentiation programme of the cell and lead directly or indirectly to malignant 1Present address: German Cancer Research Center, Institute of Cell and Tumor Biology, Im Neuenheimer Feld 280, D-6900 Heidelberg, FRG. *To whom reprint requests should be sent.
IRL Press Limited, Oxford, England. 0261-4189/82/0102-0237$2.00/0
transformation. This occurs in retrovirus-transformed cells where the cell-derived viral oncogenes, stably integrated in the host genome, are continuously transcribed under the control of a viral promoter, at a level 30-100 times higher than their cellular progenitors (Shank et al., 1978, Czerlinofsky et at., 1980a). This also occurs in some avian leukosis virus (ALV) induced lymphomas where the "non-transforming" ALV integrates immediately upstream from the cellular counterpart c-myc of the viral oncogene v-myc, providing this cellular gene with a promoter (present in the viral large terminal repeat) and increasing its transcription by a factor of 30 to 100 (Hayward et al., 1981). Finally, a constructed vector consisting of the c-mos gene flanked by viral sequences necessary for replication, integration, and transcription induced a transformed phenotype in target cells into which it was transfected (Oskarsson et al., 1980). That the cellular progenitors of viral oncogenes are important for cell growth regulation and differentiation is particularly well documented by biological and biochemical studies on defective avian leukaemia viruses (DLV). These viruses contain oncogenes called v-erb, v-myc, and v-myb (Roussel et al., 1979) which transform and block the differentiation of specific haematopoietic target cells in vitro (Graf and Beug, 1978; Graf et al., 1978; Beug et al., 1979). Again these viral oncogenes seem to be derived from normal cellular genes that could code for specific proteins involved in the control of haematopoietic differentiation (Graf and Beug, 1978). Avian erythroblastosis virus (AEV) is a DLV that induces erythroleukaemia with a short period of latency and transforms both avian fibroblasts and avian erythroblasts in culture (Graf et al., 1976). The AEV viral oncogene (v-erb), is homologous to avian and mammalian chromosomal DNA sequences (c-erb) (Saule et al., 1981). In the AEV RNA, v-erb is flanked by sequences isogenic to the ALV gag and env genes (Lai et al., 1979; Saule et al., 1981; Vennstrom et al., 1980) (Figure 1) and, in all AEV-transformed cells tested, it is transcribed into two mRNAs with lengths of 6 and 3.2 kb. The 6-kb RNA species is a genomic-size mRNA whereas the 3.2-kb species, which contains -2 kb from the 3' half of v-erb, might be generated by splicing of the 6-kb RNA (Figure 1) (Sheiness et al., 1981; Saule et al., 1981). These two mRNAs probably encode two different proteins. The 6-kb RNA codes for a p75 gag-erb fusion polyprotein (Hayman et al., 1979), while in vitro translation studies suggest that the 3.2-kb mRNA could code for a p40 erb protein, unrelated to the p75 protein (Figure 1) (Lai et al., 1980; Pawson and Martin, 1980). Accordingly, it has been proposed that the AEV oncogene may contain two functional domains, v-erbA and v-erbB, acquired from two different cellular progenitors (Sheiness et al., 1981; Saule et al., 1981), Thus, v-erbA can be defined as the domain of v-erb coding for the erb part of p75, i.e., -1 kb of the 5' part of v-erb. The v-erbB domain is defined by the sequences present in the 3.2-kb mRNA species, i.e., -2 kb of the 3' half of v-erb (Figure 1). This paper describes our attempts to characterize the chicken DNA counterpart of v-erb. In chicken DNA frac237
A.Sergeant et al.
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Fig. 1. Schematic representation of AEV replication cycle and molecular cloning. 1: The preparation of cDNA,e,,, specific for v-erb has been extensively described (Saule et al., 1981). 3: The circular AEV proviral DNA has been cloned in Xgt.WES.B. by Vennstrom et al. (1980), and then recloned in the EcoRI site of PBR 313 (pAEV-l 1). The 1.8-kbp Sacl and the 2.2-kbp PstI AEV DNA fragments are respectively 7-kbp and 8-kbp long when the total plasmid DNA is digested by Sacl or Pstl.
Fig. 2. Hybridization of chicken fibroblast EcoRI DNA fragments with
tionated after EcoRI digestion, several distinct fragments could be detected with radioactive probes specific to v-erb. Six of these EcoRl fragments are located in one locus, c-erbB, spanning >21 kilobase pairs (kbp). Heteroduplex studies showed that the c-erbB locus contained 1.8 kbp of DNA homologous to v-erbB, interrupted by 11 regions not homologous to v-erb. The 20-kbp fragment containing sequences homologous to v-erbA is not reported here.
hybridizing to EcoRI fragments of the endogenous chicken virus RAV-O. Indeed, a cDNA of the gag, pol, and env genes of an ALV (cDNArep), (Saule et al., 1982) only detected a 5-kbp EcoRI DNA fragment. Thus, the numerous bands revealed by the E1 probe probably reflect the presence, in the distal 5' part of v-erb, of sequences repeated in the chicken genome. Therefore, from the results described above, it appeared that 3 kbp of the v-erb sequences span >44 kbp in the cell DNA, indicating a split structure for c-erb. Isolation of recombinant phages containing c-erb To probe the organization of the c-erb sequences we screened a library of chicken cell DNA generated by partial digestion with AluI and HaeIII, using a cDNAAEv probe (Dodgson et al., 1979). We selected several phages hybridizing with cDNAAEv, and screened them for the presence of EcoRI DNA fragments identical in size and genetic content to those found in the EcoRI digest of chicken chromosomal DNA and described above. After digestion of the cloned DNAs with EcoRI, the DNA fragments were separated by agarose gel electrophoresis, transferred to nitrocellulose and hybridized with different radiolabelled probes. As shown in Figures 3A and 3B, we isolated six different overlapping clones (NO, 3CB, 3CA, JK, 13i, lOA) containing only six of the chromosomal EcoRI fragments detected by the E2 and E3 probes. The order of the EcoRI DNA fragments in each clone is presented in Figure 3C, and part of the strategy employed to establish such maps is shown in Figure 3B and Figure 2. None of the DNA fragments hybridized with cDNArep (not shown). Probes E2 and E3 hybridized to a 12-kbp fragment of
Results c-erb is discontinuous in chicken DNA As shown previously, cDNAAEv anneals to DNA isolated from normal chicken cells with a Cot ½/ value indicating that there is' 1 or 2 full complements of the v-erb oncogene per haploid genome (Saule et al., 1981). This agrees with the presence of most eukaryotic genes in non-repetitive DNA. We first determined the restriction pattern of the c-erb sequences homologous to v-erb by hybridizing an EcoRI digest of chicken chromosomal DNA with different 32P-labelled proviral DNA probes. These probes, described in Figure 1, were called E1, E2, and E3 according to their polarity on the AEV proviral DNA. As shown in Figure 2, the E3 probe, containing 0.9 kbp from the 3' part of v-erbB, hybridized to EcoRI fragments of 0.5, 1.3, 1.9, 3.1, 5, and 12 kbp. The E2probe containing 1.4 kbp in the middle of v-erb (end of erbA + start of erbB) detected only EcoRI fragments of 4.5, 5.0, 12, and 20 kbp. The several EcoRI fragments detected by the E1 probe cannot be explained by the presence of sequences homologous to the gag, env, and LTR sequences of ALV 238
radiolabelled v-erb and c-erb probes. Chicken fibroblast HMW DNA was
digested with EcoRI. The size-separated EcoRI DNA fragments were transferred to nitrocellulose and hybridized to the following probes: lane 1, El probe; lane 2, E2 probe; lane 3, E3 probe; lane 4, cDNAP; lane 5, NO 2.3-kbp DNA fragment as probe (NO4); lane 6, 13i 4.3-kbp DNA fragment as
probe; lane 7, 13i l.9-kbp DNA fragment as probe.
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The celldar oncogene c-erb related to avian erythroblastosis virus
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