Onc Genes Research Articles

Complete genomic sequence and phylogenomics analysis of Agrobacterium strain AB2/73: a new Rhizobium species with a unique mega-Ti plasmid

BackgroundThe Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We used whole genome sequencing to fully characterize and comparatively analyze the complex genome of strain AB2/73, including its Ti plasmid and virulence factors.ResultsWe obtained a high-quality, full genomic sequence of AB2/73 by a combination of short-read Illumina sequencing and long-read Nanopore sequencing. The AB2/73 genome has a total size of 7,266,754 bp with 59.5% GC for which 7012 genes (6948 protein coding sequences) are predicted. Phylogenetic and comparative genomics analysis revealed that strain AB2/73 does not belong to the genus Agrobacterium, but to a new species in the genus Rhizobium, which is most related to Rhizobium tropici. In addition to the chromosome, the genome consists of 6 plasmids of which the largest two, of more than 1 Mbp, have chromid-like properties. The mega Ti plasmid is 605 kbp in size and contains two, one of which is incomplete, repABC replication units and thus appears to be a cointegrate consisting of about 175 kbp derived from an unknown Ti plasmid linked to 430 kbp from another large plasmid. In pTiAB2/73 we identified a complete set of virulence genes and two T-DNAs. Besides the previously described T-DNA we found a larger, second T-DNA containing a 6b-like onc gene and the acs gene for agrocinopine synthase. Also we identified two clusters of genes responsible for opine catabolism, including an acc-operon for agrocinopine degradation, and genes putatively involved in ridéopine catabolism. The plasmid also harbours tzs, iaaM and iaaH genes for the biosynthesis of the plant growth regulators cytokinin and auxin.ConclusionsThe comparative genomics analysis of the high quality genome of strain AB2/73 provided insight into the unusual phylogeny and genetic composition of the limited host range Agrobacterium strain AB2/73. The description of its unique genomic composition and of all the virulence determinants in pTiAB2/73 will be an invaluable tool for further studies into the special host range properties of this bacterium.

Viral-Mediated Noisy Gene Expression Reveals Biphasic E2f1 Response to MYC

Gene expression mediated by viral vectors is subject to cell-to-cell variability, which limits the accuracy of gene delivery. When coupled with single-cell measurements, however, such variability provides an efficient means to quantify signaling dynamics in mammalian cells. Here, we illustrate the utility ofthis approach by mapping the E2f1 response to MYC, serum stimulation, or both. Our results revealed an underappreciated mode of gene regulation: E2f1 expression first increased, then decreased as MYC input increased. This biphasic pattern was also reflected in other nodes of the network, including the miR-17-92 microRNA cluster and p19Arf. A mathematical model of the network successfully predicted modulation of the biphasic E2F response byserum and a CDK inhibitor. In addition to demonstrating how noise can be exploited to probe signaling dynamics, our results reveal how coordination of the MYC/RB/E2F pathway enables dynamic discrimination of aberrant and normal levels of growth stimulation.

Host range restrictions of oncogenes: myc genes transform avian but not mammalian cells and mht/raf genes transform mammalian but not avian cells.

The host range of retroviral oncogenes is naturally limited by the host range of the retroviral vector. The question of whether the transforming host range of retroviral oncogenes is also restricted by the host species has not been directly addressed. Here we have tested in avian and murine host species the transforming host range of two retroviral onc genes, myc of avian carcinoma viruses MH2 and MC29 and mht/raf of avian carcinoma virus MH2 and murine sarcoma virus MSV 3611. Virus vector-mediated host restriction was bypassed by recombining viral oncogenes with retroviral vectors that can readily infect the host to be tested. It was found that, despite high expression, transforming function of retroviral myc genes is restricted to avian cells, and that of retroviral mht/raf genes is restricted to murine cells. Since retroviral oncogenes encode the same proteins as certain cellular genes, termed protooncogenes, our data must also be relevant to the oncogene hypothesis of cancer. According to this hypothesis, cancer is caused by mutation of protooncogenes. Because protooncogenes are conserved in evolution and are presumed to have conserved functions, the oncogene hypothesis assumes no host range restriction of transforming function. For example, mutated human proto-myc is postulated to cause Burkitt lymphoma, because avian retroviruses with myc genes cause cancer in birds. But there is no evidence that known mutated protooncogenes can transform human cells. The findings reported here indicate that host range restriction appears to be one of the reasons (in addition to insufficient transcriptional activation) why known, mutated protooncogenes lack transforming function in human cells.

Avian erythroblastosis virus E26: only one (myb) of two cell-derived coding regions is necessary for oncogenicity.

The oncogene hypothesis postulates that mutated cellular genes, termed proto-onc genes, function as cancer genes because they are related to retroviral onc genes. However, in contrast to retroviral onc genes, mutated proto-onc genes from cancers are not sufficient for carcinogenesis. Therefore, it has been proposed that mutated proto-onc genes depend on other proto-onc genes for carcinogenesis. Since the oncogene of the avian leukemia virus E26 includes coding regions derived from two cellular proto-onc genes, proto-myb and proto-ets, this hybrid gene has been proposed to be a model for two-gene-carcinogenesis. Here we set out to test this proposal. For this purpose myb and ets deletion mutants of cloned E26 provirus were prepared, and the corresponding viruses, produced by transfected primary chicken embryo cells, were tested for leukemogenicity in newborn chickens. It was found that an ets deletion mutant was just as leukemogenic as the wild-type virus and that a myb deletion mutant lacked leukemogenicity completely. To eliminate the possibility that our E26 myb deletion mutant failed to be leukemogenic because it failed to replicate, the virus was titered by a quantitative polymerase chain reaction (PCR) method. By this method, E26 from the plasma of infected chickens was first allowed to reverse-transcribe viral RNA to cDNA in vitro, and then the cDNA concentration was determined from the lowest dilution that gave a positive signal after amplification of E26 cDNA by the PCR method. Virus titers of about 10(5) per ml were found for wild type and for myb and ets deletion mutants of E26. It is concluded that the ets region is not essential for carcinogenesis, and E26 derives transforming function from overexpression of its proto-myb coding region via the retroviral promoter. Thus, E26 is a single-hit carcinogen and, like all other oncogenic retroviruses, is not a model for two-gene-carcinogenesis. Viral ets probably reflects a genetic accident that transduced sequences of proto-ets together with proto-myb in generating E26.

Unmutated proto-src coding region is tumorigenic if expressed from the promoter of Rous sarcoma virus: implications for the gene-mutation hypothesis of cancer.

The transforming (onc) genes of oncogenic retroviruses share most or all of their coding sequences with normal cellular genes termed proto-onc genes. The viral genes differ from proto-onc genes in virus-specific promoters and in various point mutations and substitutions of cell-derived coding regions. In view of the structural similarities between viral oncogenes and cellular proto-onc genes, the hypothesis has been advanced that proto-onc genes become cellular cancer genes if they have suffered mutations. Indeed, point mutations and substitutions have been observed in the proto-onc genes of some cancers. However, the hypothesis has been difficult to prove because mutated proto-onc genes from tumors do not transform diploid cells. Moreover, owing to the popularity of this hypothesis, even viral oncogenes are thought to derive transforming function from mutations of this cell-derived coding region. A competing hypothesis proposes that enhanced expression from retroviral promoters is necessary and sufficient for oncogenic function of proto-onc genes. To distinguish between these hypotheses we have tested tumorigenicity of RpSV, a synthetic retrovirus with the normal proto-src coding region in a vector derived from Rous sarcoma virus (RSV). In addition, we have tested the role of RSV-specific src point mutations on the tumorigenicity of RpSV. It was found that RpSV with an unmutated proto-src coding region is tumorigenic in chickens and that tumorigenicity is enhanced by RSV-specific src point mutations. It is concluded that retroviral promoters are essential for the transforming function of viral oncogenes and that certain point mutations merely supplement their transforming function. Thus retroviral onc genes are not models for the hypothesis that mutated, but transcriptionally normal, proto-onc genes of certain tumors are cancer genes.

Novel cell lines derived from transgenic mice expressing recombinant human proteins. Transgenic hepatoma-derived cell lines.

We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human alpha 1-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of an onc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human alpha 1-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing.

Characterization of trans-immortalized hepatic cell lines established from transgenic mice

Hepato-speciflc regulatory (promoter/enhancer) DNA sequences were used for targeting the expression of onc genes, such as murine c- myc and Simian Virus 40 T Antigen, to hepatocytes of transgenic mice which subsequently developed hepatocellular carcinomas after a variable period of time (depending on the type of onc gene employed). Several trans-immortalized cell lines were established and compared with respect to the expression of adult hepatic markers and response to growth factors. Despite the morphological differences observed between trans-hepatomas, owing to the expression of the two different onc genes, all tumor-derived cell lines behaved in a comparable fashion during long-term culture displaying an adult hepatic phenotype for at least 40 passages. They differed, however, in response to epidermal growth factor. When the gene coding for human α1-antitrypsin was placed under the control of the same hepato-specific promoter/enhancer, high levels of the human recombinant protein could be harvested from the supernatants of trans-hepatoma-derived cell lines.

Evidence that retroviral transduction is mediated by DNA not by RNA.

Retroviral transduction of cellular nucleic acid sequences requires illegitimate RNA or DNA recombination. To test a model that postulates transduction via efficient illegitimate recombination during reverse transcription of viral and cellular RNAs, we have measured the ability of Harvey sarcoma viruses (HaSVs) with artificial 3' termini to recover a retroviral 3' terminus from helper Moloney virus (MoV) by illegitimate and homologous recombination. For this purpose, mouse NIH 3T3 cells were transformed with Harvey proviruses and then superinfected with MoV. The proviruses lacked the 3' long terminal repeat and an untranscribed region of the 5' long terminal repeat to prevent virus regeneration from input provirus. Only 0-11 focus-forming units of HaSV were generated upon MoV superinfection of 3 x 10(6) cells transformed by Harvey proviruses with MoV-unrelated termini. This low frequency is consistent with illegitimate DNA recombination via random Moloney provirus integration 3' of the transforming viral ras gene in the 10(6)-kilobase mouse genome. When portions of murine viral envelope (env) genes were attached 3' of ras, 10(2)-10(5) focus-forming units of HaSV were generated, depending on the extent of homology with env of MoV. These recombinants all contained HaSV-specific sequences 5' and MoV-specific sequences 3' of the common env homology. They were probably generated by recombination during reverse transcription rather than by recombination among either input or secondary proviruses, since (i) the yield of recombinants was reduced by a factor of 10 when the env sequence was flanked by splice signals and (ii) HaSV RNAs without retroviral 3' termini would be inadequate templates for provirus synthesis. We conclude that there is no efficient illegitimate recombination in retroviruses. In view of known precedents of illegitimate DNA recombination, the structure of known viral onc genes, and our evidence for illegitimate DNA recombination via provirus integration, we favor the DNA model of transduction over the RNA model.

Cancer genes by illegitimate recombination.

Retroviral onc genes are as yet the only proven cancer genes. They are generated by rare illegitimate recombinations between retroviruses and cellular genes, termed proto-onc genes. The claims that these proto-onc genes cause virus-free cancers upon "activation" by mechanisms that do not alter their germline structure are challenged. Instead, it is proposed that retroviral onc genes and cellular cancer genes are generated de novo by illegitimate recombinations that alter the germline structure of normal genes.

The Agrobacterium tumefaciens T-DNA gene 6b is an onc gene

In this article it is shown that the T-DNA of Agrobacterium tumefaciens contains besides the well-known cyt and aux genes another gene with an oncogenic effect in plants. The gene in question is called 6(b) and causes the formation of small tumors in plant species such as Nicotiana glauca and Kalanchoe tubiflora.

Two autonomous myc oncogenes in avian carcinoma virus OK10.

The oncogenic avian retrovirus OK10 has the genetic structure gag-delta pol-myc-delta-env. The myc sequence is transduced from a cellular gene, proto-myc, while gag, pol, and env are essential retrovirus genes. By analogy with other directly oncogenic retroviruses, the specific myc sequence of OK10 is thought to be essential for transforming function. However, unlike the specific sequences of all other transforming retroviruses that encode unique transforming proteins, the myc sequence of OK10 encodes two potential transforming proteins, p58 and p200. p200 is translated from the gag-delta pol-myc region of genomic RNA, while p58 is thought to be translated from the gag leader and the open reading frame of myc via a subgenomic mRNA. In this paper, we ask whether both myc genes of OK10 are autonomous transforming genes. By differentially inactivating the p200 myc gene of OK10 provirus in vitro and analyzing transforming function in quail embryo cells, it was found that mutants expressing only p58 transformed like wild-type OK10. Further, it was shown that p58 with and without the gag leader had transforming function and that p58 of wild-type OK10 is initiated from the gag leader. Mutants expressing only p200 were also transforming but less efficiently than mutants that express only p58. A mutant OK10 virus in which the native frameshift of retroviruses between gag and pol was deleted expressed a shortened p200 (delta p200). Although this virus expressed more delta p200 than wild-type OK10 did, it transformed cells less efficiently. It follows that OK10 expresses two autonomous transforming genes, which is unique among retroviruses with onc genes.

Genes specifically expressed at growth arrest of mammalian cells

A subtraction cDNA library enriched for RNA sequences preferentially expressed in growth-arrested cells was prepared. Six cDNA clones were identified, varying in abundance from 2% to 0.0002% of the library and in size from 0.8 to 10 kb. The corresponding mRNAs are downregulated with different kinetics upon induction of growth by serum. The kinetics of induction after serum starvation and density-dependent inhibition of two of these growth-arrest-specific (gas) genes were investigated in more detail. Two cell lines transformed by viral onc genes did not express the two gas genes. The full-length cDNA for one gene has been sequenced and the protein product preliminarily characterized by in vitro translation.

Expression of “cell-cycle-dependent” genes in regenerating rat liver

The expression of genes coding for the ATP/ADP translocase, calcyclin, ornithine decarboxylase, vimentin, proto- onc genes p53 and c-Ha- ras1 and also for two genes JE and KC with as yet unknown function was studied during regeneration of rat liver. Genes highly induced were: JE (2–8 h of regeneration), ATP/ADP translocase (8–18 h), c-Ha- ras-1 (6–48 h) and p53 (6–12 h). Vimentin and KC gene transcripts were not detectable in the first 48 h of liver regeneration, whereas ornithine decarboxylase and calcyclin gene transcripts were present at constant levels. Our findings extend the list of genes expressed at the early stages of liver regeneration.

Retroviral transduction of oncogenic sequences involves viral DNA instead of RNA.

We have studied whether the origin of retroviral onc genes, by transduction of sequences from cellular proto-onc genes, involves DNA or RNA recombination. By using altered Harvey sarcoma proviruses as models for transduction intermediates, we have investigated the mechanism of regeneration of transforming virus from truncated proviruses with only a single 5' long terminal repeat (LTR) but with a complete 5'-LTR-ras transforming gene. The Harvey proviruses were specifically altered to discriminate between virus regeneration by RNA template switching during reverse transcription, as has been postulated, and virus regeneration by DNA recombination with either helper virus or among elements of the defective provirus alone. For this purpose U3 elements of the Harvey proviral LTR, which are essential for replication but not for transcription, were deleted in vitro. Only proviral constructions with an intact or a nearly intact single LTR regenerated infectious Harvey sarcoma virus. Since all constructions transformed cells and produced identical RNAs, our results exclude a model of virus regeneration by switching of RNA templates during reverse transcription. We conclude that regeneration of infectious Harvey viruses from truncated provirus involved illegitimate recombination of cellular or cotransfected DNAs flanking the 5'-LTR-ras gene of Harvey sarcoma virus. Based on this and evidence from the literature, we propose that retroviral transduction proceeds by way of rare illegitimate recombinations between proviral and cellular DNAs.

Analysis of functional domains in the epidermal growth factor receptor using site-directed mutagenesis.

Protein tyrosine kinase activity is a central feature of one class of retroviral onc genes and of several growth factor receptors including those for epidermal growth factor receptor (EGF) (Ushiro and Cohen, 1980; Hunter and Cooper, 1985), platelet-derived growth factor (PDGF) (Yarden et al., 1986), insulin and insulin-like growth factors (IGF) (Ebina et al., 1985; Ullrich et al., 1985), colony-stimulating factor 1 (CSF1) (Sherr et al., 1985), and other ligands (neu) (Bargmann et al., 1986a; Yamamoto et al., 1986). The EGF receptor is the proto-oncogene for the retroviral oncogene erb B (Yamamoto et al., 1983; Downward et al., 1984a; Lin et al., 1984; Ullrich et al., 1984; Xu et al., 1984); the CSF1 receptor for v-fms (Sherr et al., 1985; Coussens et al., 1986); and the neu receptor for the transforming gene neu (Bargmann et al., 1986b). The sis oncogene of the simian sarcoma virus is homologous to the B chain of PDGF and presumably exerts its transforming effects through interaction with the normal PDGF receptor (Doolittle et al., 1983; Waterfield et al., 1983; Huang et al., 1984). Constitutive expression of TGF-α and of EGF, both of which bind to the EGF receptor, also results in cell transformation, presumably via activation of normal EGF receptors (Rosenthal et al., 1986; Stern et al., 1987). Constitutive activation of ligands may be analogous to gene amplification and quantitative over-expression of receptor protein as occurs in some human tumors (Thompson and Gill, 1985) because the initial ligand receptor interaction in a bimolecular event dependent on the concentration of both ligand and receptor (Gill et al., 1985).

The Abelson protein is required for initiation and maintenance for transformation in murine pre-B cells.

Abelson murine leukemia virus (Ab-MLV) is a replication defective retrovirus that carries the onc gene abl. The virus induces a rapid thymus-independent lymphoma in vivo under most conditions and transforms cells related to pre-B lymphocytes in vitro (reviewed in Rosenberg and Witte, 1988). The Abelson protein, the sole product of the virus, is a protein tyrosine kinase and expression of an enzymatically active Abelson protein is required for transformation (Rosenberg and Witte, 1988). Recent analyses of temperature sensitive (ts) mutants of Ab-MLV have indicated that expression of an active Abelson protein is also required for maintenance of NIH 3T3 cell transformation (Engelman and Rosenberg, 1987; Kipreos et al., 1987; Takemori et al., 1987). In addition, Abelson protein expression appears to play a role in maintaining the relatively undifferentiated state of some Ab-MLV-transformed lymphoid cells (Takemori et al., 1987).

Cancer genes generated by rare chromosomal rearrangements rather than activation of oncogenes.

The 20 known transforming onc genes of retroviruses are defined by sequences that are transduced from cellular genes, termed proto-oncogenes or cellular oncogenes. Based on these sequences, viral onc genes have been postulated to be transduced cellular cancer genes and proto-onc genes have been postulated to be latent cancer genes that can be activated from within the cell to cause virus-negative tumors. The hypothesis is popular because it promises direct access to cellular cancer genes. However, the existence of latent cancer genes presents a paradox since such genes are clearly undesirable. The hypothesis predicts (i) that viral onc genes and proto-onc genes are isogenic, (ii) that expression of proto-onc genes induces tumors, (iii) that activated proto-onc genes transform diploid cells upon transfection, like viral onc genes, and (iv) that diploid tumors exist that differ from normal cells only in transcriptionally or mutationally activated proto-onc genes. As yet, none of these predictions is confirmed. Moreover, the probability of spontaneous transformation in vivo is at least 10(9) times lower than predicted from the mechanisms thought to activate proto-onc genes. Therefore the hypothesis, that proto-onc genes are latent cellular oncogenes, appears to be an overinterpretation of sequence homology to structural and functional homology with viral onc genes. Here it is proposed that only rare truncations and illegitimate recombinations that alter the germline configuration of cellular genes, generate viral and possibly cellular cancer genes. The clonal chromosome abnormalities that are consistently found in tumor cells are microscopic evidence for rearrangements that may generate cancer genes. The clonality indicates that the tumors are initiated with, and possibly by, these abnormalities as predicted by Boveri in 1914 (Zur Frage der Entstehung maligner Tumoren, Jena, Fischer).

Cytogenetic evaluation of human endothelial cell cultures.

Cytogenetic evaluation of serially subcultivated human endothelial cells revealed significant differences between cultures derived from fetal umbilical cords and cultures derived from various vessel sites in adults. A rapid increase in the prevalence of polyploid cells, to levels of 100% in many cases, was detected in human umbilical vein endothelial cell cultures but not in endothelial cell cultures from adult vessels. Because the development of polyploidy has been viewed as one signpost of in vitro senescence, it may be that these in vitro observations of high levels of polyploidy are a reflection of the fact that umbilical tissue is at the end of its in vivo developmental lifespan when studied. Consistent karyotypic alterations also were observed in two clones from adult human abdominal aorta, even though these cultures exhibited low percentages of polyploid cells. Cultures of one clone exhibited a trisomy of chromosome 11, on which there are at least three onc gene loci, and a deletion of chromosome 13 through band q14. A loss of band 13q14 is a prezygotic chromosomal lesion known to predispose to retinoblastoma. In the other clone, two cell populations were observed, and each displayed a chromosomal abnormality. A trisomy of the long arm of chromosome 2 was noted in one cell population via a marker chromosome involving 2 and 14. The other cell population exhibited an abnormality of chromosome 2. Neither of these karyotypic alterations was detected in the parent culture from which the clones were derived. The results reported in this study have both practical and theoretical implications. The high incidence of polyploidy in serially cultivated umbilical cultures as well as the occurrence of chromosomal changes in umbilical and aortic cultures testify to the need for cytogenetic monitoring of cell cultures even though they are derived from presumably normal tissue. Cytogenetic changes in the endothelium may be important in atherogenesis and other pathologic states. The conversion of diploid endothelial cells into polyploid endothelial cells may provide a convenient model cell system for studying mechanisms of the development of polyploidy in cells and their relationship to in vitro senescence.

Cancer genes: rare recombinants instead of activated oncogenes (a review).

The 20 known transforming (onc) genes of retroviruses are defined by sequences that are transduced from cellular genes termed protooncogenes or cellular oncogenes. Based on these sequences, viral onc genes have been postulated to be transduced cellular cancer genes, and proto-onc genes have been postulated to be latent cancer genes that can be activated from within the cell to cause virus-negative tumors. The hypothesis is popular because it promises direct access to cellular cancer genes. However, the existence of latent cancer genes presents a paradox, since such genes are clearly undesirable. The hypothesis predicts that viral onc genes and proto-onc genes are isogenic; that expression of proto-onc genes induces tumors; that activated proto-onc genes transform diploid cells upon transfection, like viral onc genes; and that diploid tumors exist. As yet, none of these predictions is confirmed. Instead: Structural comparisons between viral onc genes, essential retroviral genes, and proto-onc genes show that all viral onc genes are indeed new genes, rather than transduced cellular cancer genes. They are recombinants put together from truncated viral and truncated proto-onc genes. Proto-onc genes are frequently expressed in normal cells. To date, not one activated proto-onc gene has been isolated that transforms diploid cells. Above all, no diploid tumors with activated proto-onc genes have been found. Moreover, the probability of spontaneous transformation in vivo is at least 10(9) times lower than predicted from the mechanisms thought to activate proto-onc genes. Therefore, the hypothesis that proto-onc genes are latent cellular oncogenes appears to be an overinterpretation of sequence homology to structural and functional homology with viral onc genes. Here it is proposed that only rare truncations and illegitimate recombinations that alter the germ-line configuration of cellular genes generate viral and possibly cellular cancer genes. The clonal chromosome abnormalities that are consistently found in tumor cells are microscopic evidence for rearrangements that may generate cancer genes. The clonality indicates that the tumors are initiated with, and possibly by, these abnormalities, as predicted by Boveri in 1914.

Latent cellular oncogenes: the paradox dissolves.

The 20 known transforming onc genes of retroviruses are defined by sequences which are transduced from cellular genes, termed proto-onc genes. Based on these sequences, viral onc genes have been postulated to be transduced cellular cancer genes and proto-onc genes have been postulated to be latent cancer genes that can be activated from within the cell, to cause virus-negative tumours. The hypothesis is popular because it promises direct access to cellular cancer genes. However the existence of latent cancer genes presents a paradox since such genes would be most undesirable for eukaryotes. The hypothesis predicts (i) that viral onc genes and proto-onc genes are isogenic, (ii) that expression of proto-onc genes induces tumours, (iii) that activated proto-onc genes transform diploid cells upon transfection, like viral onc genes, and (iv) it predicts diploid tumours. As yet, none of these predictions is confirmed. Instead: (i) Structural comparisons between viral onc genes, essential retroviral genes, and the proto-onc genes show that all viral onc genes are indeed new genes, rather than transduced cellular cancer genes. They are genetic hybrids put together from truncated viral and truncated proto-onc genes. (ii) Proto-onc genes are frequently expressed in normal cells. (iii) To date, not one activated proto-onc gene has been isolated that transforms diploid cells. (iv) Above all, no diploid tumours with activated proto-onc genes have been found. Moreover the probability of spontaneous transformation in vivo is at least 10(9) times lower than predicted from the mechanisms thought to activate proto-onc genes. Therefore the hypothesis, that proto-onc genes are latent cellular oncogenes, appears to be an over-interpretation of sequence homology to structural and functional homology with viral onc genes. Here is is proposed that only rare truncations and recombinations, that alter the germline configuration of cellular genes, generate viral and possibly cellular cancer genes. The clonal chromosome abnormalities that are consistently found in tumour cells are microscopic evidence for rearrangements that may generate cancer genes. The clonality indicates that the tumours are initiated with, and possibly by these abnormalities as predicted by Boveri in 1914.