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Aneuploidy is ubiquitous in cancer, and its phenotypes are inevitably dominant and abnormal. In view of these facts we recently proposed that aneuploidy is sufficient for carcinogenesis generating cancer-specific aneusomies via a chain reaction of autocatalytic aneuploidizations. According to this hypothesis a carcinogen initiates carcinogenesis via a random aneuploidy. Aneuploidy then generates transformation stage-specific aneusomies and further random aneusomies autocatalytically, because it renders chromosome segregation and repair mechanisms error-prone. The hypothesis predicts that several specific aneusomies can cause the same cancers, because several chromosomes also cooperate in normal differentiation. Here we describe experiments on the Chinese hamster (CH) that confirm this hypothesis. ( i ) Random aneuploidy was detected before transformation in up to 90% of CH embryo cells treated with the carcinogen nitrosomethylurea (NMU). ( ii ) Several specific aneusomies were found in 70–100% of the aneuploid cells from colonies transformed with NMU in vitro and from tumors generated by NMU-transformed cells in syngeneic animals. Among the aneuploid in vitro transformed cells, 79% were trisomic for chromosome 3, and 59% were monosomic for chromosome 10, compared with 8% expected for random distribution of any aneusomy among the 12 CH chromosomes. Moreover, 52% shared both trisomy 3 and monosomy 10 compared with 0.6% expected for random distribution of any two aneusomies. Among the tumor cells, 65% were trisomic for chromosome 3, 51% were trisomic for chromosome 5, and 30% shared both trisomies. Aneuploid cells without these specific aneusomies may contain minor transformation-specific aneusomies or may be untransformed. ( iii ) Random aneusomies and structurally altered chromosomes increased with the generations of transformed cells to the point where their origins became unidentifiable in tumors. We conclude that specific aneusomies are necessary for carcinogenesis.

In their recent paper, Valind et al. test the theory that “aneuploidy automatically” destabilizes the karyotype (1). According to this theory, aneuploidy, an abnormal balance of chromosomes, destabilizes the karyotype automatically by unbalancing the cooperations of thousands of genes, especially mitosis genes: The more aneuploid the cell the more unstable is the karyotype (2⇓⇓–5).

Cancer cells and aneuploid cell lines can acquire resistance against multiple unrelated chemotherapeutic drugs that are over 3,000-fold those of normal levels and display spontaneous resistances up to 20-fold of normal levels. Two different mechanisms were proposed for this phenotype: ( i ) classical mutation of drug metabolizing genes or ( ii ) chromosome reassortments, catalyzed by cancer- and cell line-specific aneuploidy, which generate, via new gene dosage combinations, a plethora of cancer phenotypes, including drug resistance. To distinguish between these mechanisms, we have asked whether three mouse cell lines can become drug resistant, from which two or three genes have been deleted, and on which multidrug resistance is thought to depend: Mdr1a , Mdr1b , and Mrp1 . Because all three lines could acquire multidrug resistance and were aneuploid, whereas diploid mouse cells could not, we conclude that aneuploid cells become drug resistant via specific chromosome assortments, independent of putative resistance genes. We have asked further whether aneuploid drug-resistant Chinese hamster cells revert spontaneously to drug sensitivity in the absence of cytotoxic drugs at the high rates that are typical of chromosome reassortments catalyzed by aneuploidy or at the very low or zero rates (i.e., deletion) of gene mutation. We found that four drug-resistant hamster cell lines reverted to drug sensitivity at rates of about 2–3% per generation, whereas two closely related lines remained resistant under our conditions. Thus, the karyotypic instability generated by aneuploidy emerges as the common source of the various levels of drug resistance of cancer cells: minor spontaneous resistances reflect accidental chromosome assortments, the high selected resistances reflect complex specific assortments, and multidrug resistance reflects new combinations of unselected genes located on the same chromosomes as selected genes.

The mutation rates of cancer cells to drug and multidrug resistance are paradoxically high, i.e., 10 −3 to 10 −6 , compared with those altering phenotypes of recessive genes in normal diploid cells of about 10 −12 . Here the hypothesis was investigated that these mutations are due to chromosome reassortments that are catalyzed by aneuploidy. Aneuploidy, an abnormal number of chromosomes, is the most common genetic abnormality of cancer cells and is known to change phenotypes (e.g., Down's syndrome). Moreover, we have shown recently that aneuploidy autocatalyzes reassortments of up to 2% per chromosome per mitosis because it unbalances spindle proteins, even centrosome numbers, via gene dosage. The hypothesis predicts that a selected phenotype is associated with multiple unselected ones, because chromosome reassortments unbalance simultaneously thousands of regulatory and structural genes. It also predicts variants of a selected phenotype based on variant reassortments. To test our hypothesis we have investigated in parallel the mutation rates of highly aneuploid and of normal diploid Chinese hamster cells to resistance against puromycin, cytosine arabinoside, colcemid, and methotrexate. The mutation rates of aneuploid cells ranged from 10 −4 to 10 −6 , but no drug-resistant mutants were obtained from diploid cells in our conditions. Further selection increased drug resistance at similar mutation rates. Mutants selected from cloned cells for resistance against one drug displayed different unselected phenotypes, e.g., polygonal or fusiform cellular morphology, flat or three-dimensional colonies, and resistances against other unrelated drugs. Thus our hypothesis offers a unifying explanation for the high mutation rates of aneuploid cancer cells and for the association of selected with unselected phenotypes, e.g., multidrug resistance. It also predicts drug-specific chromosome combinations that could become a basis for selecting alternative chemotherapy against drug-resistant cancer.

For nearly a century, cancer has been blamed on somatic mutation. But it is still unclear whether this mutation is aneuploidy, an abnormal balance of chromosomes, or gene mutation. Despite enormous efforts, the currently popular gene mutation hypothesis has failed to identify cancer-specific mutations with transforming function and cannot explain why cancer occurs only many months to decades after mutation by carcinogens and why solid cancers are aneuploid, although conventional mutation does not depend on karyotype alteration. A recent high-profile publication now claims to have solved these discrepancies with a set of three synthetic mutant genes that “suffices to convert normal human cells into tumorigenic cells.” However, we show here that even this study failed to explain why it took more than “60 population doublings” from the introduction of the first of these genes, a derivative of the tumor antigen of simian virus 40 tumor virus, to generate tumor cells, why the tumor cells were clonal although gene transfer was polyclonal, and above all, why the tumor cells were aneuploid. If aneuploidy is assumed to be the somatic mutation that causes cancer, all these results can be explained. The aneuploidy hypothesis predicts the long latent periods and the clonality on the basis of the following two-stage mechanism: stage one, a carcinogen (or mutant gene) generates aneuploidy; stage two, aneuploidy destabilizes the karyotype and thus initiates an autocatalytic karyotype evolution generating preneoplastic and eventually neoplastic karyotypes. Because the odds are very low that an abnormal karyotype will surpass the viability of a normal diploid cell, the evolution of a neoplastic cell species is slow and thus clonal, which is comparable to conventional evolution of new species.

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell lines, recently analyzed by Lengnauer et al . [Lengnauer, C., Kinzler, K. W. & Vogelstein, B. (1997) Nature (London) 386, 623–627], also corresponds exactly to their degree of aneuploidy. We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

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.

Oncogenic retroviruses carry coding sequences that are transduced from cellular protooncogenes. Natural transduction involves two nonhomologous recombinations and is thus extremely rare. Since transduction has never been reproduced experimentally, its mechanism has been studied in terms of two hypotheses: (i) the DNA model, which postulates two DNA recombinations, and (ii) the RNA model, which postulates a 5' DNA recombination and a 3' RNA recombination occurring during reverse transcription of viral and protooncogene RNA. Here we use two viral DNA constructs to test the prediction of the DNA model that the 3' DNA recombination is achieved by conventional integration of a retroviral DNA 3' of the chromosomal protooncogene coding region. For the DNA model to be viable, such recombinant viruses must be infectious without the purportedly essential polypurine tract (ppt) that precedes the 3' long terminal repeat (LTR) of all retroviruses. Our constructs consist of a ras coding region from Harvey sarcoma virus which is naturally linked at the 5' end to a retroviral LTR and artificially linked at the 3' end either directly (construct NdN) or by a cellular sequence (construct SU) to the 5' LTR of a retrovirus. Both constructs lack the ppt, and the LTR of NdN even lacks 30 nucleotides at the 5' end. Both constructs proved to be infectious, producing viruses at titers of 10(5) focus-forming units per ml. Sequence analysis proved that both viruses were colinear with input DNAs and that NdN virus lacked a ppt and the 5' 30 nucleotides of the LTR. The results indicate that DNA recombination is sufficient for retroviral transduction and that neither the ppt nor the complete LTR is essential for retrovirus replication. DNA recombination explains the following observations by others that cannot be reconciled with the RNA model: (i) experimental transduction is independent of the packaging efficiency of viral RNA, and (ii) experimental transduction may invert sequences with respect to others, as expected for DNA recombination during transfection.

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.

Oncogenic retroviruses are generated by transduction of the coding region of a protooncogene and acquire genetic changes during subsequent replication. Critical genetic events which occurred during and after transduction of rat proto-ras-1Ha into Harvey sarcoma virus were identified by evaluating the transforming activity of plausible synthetic progenitor proviruses encompassing the complete proto-ras genomic region with or without various 5' deletions. All progenitor proviruses induced phenotypic transformation of mouse NIH 3T3 cells, although with a 5- to 10-fold lower frequency than Harvey sarcoma provirus. Although no tumor formation was observed in vivo after inoculation in the absence of helper murine retrovirus, both wild-type and progenitor viruses inoculated in the presence of helper virus induced tumors in newborn BALB/c mice. No critical alterations of the p21ras coding region and no deletion of 5' genomic elements were detected in a progenitor virus encompassing the complete proto-ras genomic region that had been isolated from tumors. However, one progenitor virus that included all proto-ras exons induced tumors with a decreased latency. This virus contained a mutation in codon 12 (glycine to valine), which had apparently been selected during tumorigenesis in vivo. During the genesis of Harvey sarcoma virus, critical steps conferring transforming function are therefore transduction of coding proto-ras exons and enhancement of their transforming function by specific amino acid changes in p21ras.

Based on transfection into cells in culture or natural transduction into retroviruses, proto-ras genes seem to derive transforming function either from heterologous promoters or from point mutations. Here we ask how such different events could achieve the same results. To identify homologous regulatory elements, about 3 kilobases of rat DNA upstream of the first untranslated proto-Ha-ras exon was sequenced. Surprisingly, the sequence shares at -1858 a homology of 148 nucleotides with Harvey (Ha) sarcoma virus, 5' of viral ras, signaling possibly a second untranslated proto-Ha-ras exon. In addition the sequence contains a perfect repeat of 25 CA dinucleotides at -2655. A retroviral promoter, even from upstream of the poly(CA), conferred transforming function on proto-Ha-ras and increased transcription greater than 100-fold compared with that of unrearranged proto-ras. Point mutations were not necessary for transforming function of rat and human proto-Ha-ras genes with retroviral promoters but did enhance it greater than 10-fold. A unifying hypothesis proposes that proto-ras genes depend on high expression from heterologous promoters or enhancers for transforming function, which is modulated by ras point mutations. The hypothesis makes two testable predictions. (i) Unrearranged proto-ras genes with point mutations, which occur in some cancers, have no transforming function. Indeed, tumors with mutated proto-ras genes, even those that also lack hypothetical tumor-suppressor genes, are indistinguishable from counterparts with normal proto-ras genes. (ii) Proto-ras genes in transfected cells derive transforming function from heterologous promoters or enhancers acquired via illegitimate recombination from vector DNAs and particularly from viral helper genes that must be cotransfected for transformation of primary cells. Indeed, expression of exogenous proto-ras genes in cells transformed by transfection is as high as for viral ras genes and is much higher than in the cells of origin.

After mixed infection, up to half of related retroviruses are recombinants. During infection, retroviral RNA genomes are first converted to complementary DNA (cDNA) and then to double-stranded DNA. Thus recombination could occur during reverse transcription, by RNA template switching, or after reverse transcription, by breakage and reunion of DNA. It has not been possible to distinguish between these two potential mechanisms of recombination because both single-stranded cDNA and double-stranded proviral DNA exist in infected cells during the eclipse period. Therefore we have analyzed for recombinant molecules among cDNA products transcribed in vitro from RNA of disrupted virions. Since recombinants from allelic parents can only be distinguished from parental genomes by point mutations, we have examined the cDNAs from virions with distinct genetic structures for recombinant-specific size and sequence markers. The parents share a common internal allele that allows homology-directed recombination, but each contains specific flanking sequences. One parent is a synthetically altered Harvey murine sarcoma virus RNA that lacks a retroviral 3' terminus but carries a Moloney murine retrovirus-derived envelope gene (env) fragment 3' of its transforming ras gene. The other parent is intact Moloney virus. Using a Harvey-specific 5' primer and a Moloney-specific 3' primer, we have found recombinant cDNAs with the polymerase chain reaction, proving directly that retroviruses can recombine during reverse transcription unassisted by cellular enzymes, probably by template switching during cDNA synthesis. The recombinants that were obtained in vitro were identical with those obtained in parallel experiments in vivo.

Chicken myeloblasts transformed by avian myeloblastosis virus (AMV) in the absence of nondefective helper virus (termed nonproducer cells) were found to release a defective virus particle (DVP) that contains avian tumor viral gag proteins but lacks envelope glycoprotein and a DNA polymerase. Nonproducer cells contain a Pr76 gag precursor protein and also a protein that is indistinguishable from the Pr180 gag-pol protein of nondefective viruses. The RNA of the DVP is 7.5 kilobases (kb) long and is 0.7 kb shorter than the 8.2-kb RNAs of the helper viruses of AMV, MAV-1 and MAV-2. Comparisons based on RNA·cDNA hybridization and mapping of RNase T1-resistant oligonucleotides indicated that DVP RNA shares with MAV RNAs nearly isogenic 5′-terminal gag and pol -related sequences of 5.3 kb and a 3′-terminal c-region of 0.7 kb that is different from that found in other avian tumor viruses. Adjacent to the c-region, DVP RNA contains a contiguous specific sequence of 1.5 kb defined by 14 specific oligonucleotides. Except for two of these oligonucleotides that map at its 5′ end, this sequence is unrelated to any sequences of nondefective avian tumor viruses of four different envelope subgroups as well as to the specific sequences of fibroblast-transforming avian acute leukemia and sarcoma viruses of four different RNA subgroups. The specific sequence of the DVP RNA is present in infectious stocks of AMV from this and other laboratories in an AMV-transformed myeloblast line from another laboratory, and it is about 70% related to nucleotide sequences of E26 virus, an independent isolate of an AMV-like virus. Preliminary experiments show DVP to be leukemogenic if fused into susceptible cells in the presence of helper virus. We conclude that DVP RNA is the leukemogenic component of infectious AMV and that its specific sequence, termed AMV , may carry genetic information for oncogenicity. Thus we have found here a transformation-specific RNA sequence, unrelated to helper virus, in a highly oncogenic virus that does not transform fibroblasts.

The RNA of myelocytoma virus MC29, a replication-defective avian acute leukemia virus, was investigated. Sedimentation and electrophoretic analyses indicated that the virus contains a distinct 28S RNA with about 5700 nucleotides. It is the smallest avian tumor virus RNA detected to date. The small size of the RNA suggests that the defectiveness of the virus is due to deletions in replicative genes. The RNA shared 3 to 5 of 30 large RNase T 1 -resistant oligonucleotides with the RNA of other avian leukosis and sarcoma viruses. Hybridization indicated that 61% of the viral RNA contains sequences in common with other avian sarcoma and leukosis viruses. At least 32% of the RNA (about 1800 nucleotides) appear to be MC29-specific and may represent the transforming information of the virus. Sequences of the conserved transforming gene of avian sarcoma viruses were not detected in MC29 RNA. It was concluded that the transforming sequences of MC29 RNA define a new class of avian tumor viral transforming genes.

The hypothesis is tested that the ras gene of Harvey sarcoma virus (Ha-SV) and the proto-ras DNAs from certain tumor cells derive transforming function from specific codons in which they differ from normal proto-ras genes. Molecularly cloned Harvey proviral vectors carrying viral ras, normal rat proto-ras, and recombinant ras genes in which the virus-specific ras codons 12 and 59 were replaced by proto-ras equivalents each transformed aneuploid mouse 3T3 cells after latent periods that ranged from 4 to 10 days. Viruses with or without virus-specific ras codons all transformed diploid rat cells in 3-5 days equally well. However, in the absence of virus replication, mutant codons were beneficial for transforming function. Deletion of non-ras regions of Ha-SV did not affect transforming function. We conclude that specific ras codons are not necessary for transforming function. Comparisons of the ras sequences of Ha-SV, BALB SV, and Rasheed SV with sequences of proto-ras genes from rat and man revealed an upstream proto-ras exon, termed exon -1. The 3' end of this exon is present in all three viruses and in a ras pseudogene of the rat. Since ras genes transform without mutation and since exon -1 is truncated in viral ras genes and all transforming proto-ras DNAs of the Harvey and the Kirsten ras family, we propose that ras genes are activated by truncation of exon -1 either via viral transduction or artificially via cloning and transfection. The proposal implies that untruncated proto-ras genes with point mutations may not be cellular cancer genes.

The genome of the defective avian tumor virus MH2 was identified as a RNA of 5.7 kilobases by its presence in different MH2-helper virus complexes and its absence from pure helper virus, by its unique fingerprint pattern of RNase T1-resistant (T1) oligonucleotides that differed from those of two helper virus RNAs, and by its structural analogy to the RNA of MC29, another avian acute leukemia virus. Two sets of sequences were distinguished in MH2 RNA: 66% hybridized with DNA complementary to helper-independent avian tumor viruses, termed group-specific, and 34% were specific. The percentage of specific sequences is considered a minimal estimate because the MH2 RNA used was about 30% contaminated by helper virus RNA. No sequences related to the transforming src gene of avian sarcoma viruses were found in MH2. MH2 shared three large T1 oligonucleotides with MC29, two of which could also be isolated from a RNase A- and T1-resistant hybrid formed between MH2 RNA and MC29 specific cDNA. These oligonucleotides belong to a group of six that define the specific segment of MC29 RNA described previously. The group-specific sequences of MH2 and MC29 RNA shared only the two smallest out of about 20 T1 oligonucleotides associated with MH2 RNA. It is concluded that the specific sequences of MH2 and MC29 are related, and it is proposed that they are necessary for, or identical with, the onc genes of these viruses. These sequences would define a related class of transforming genes in avian tumor viruses that differs from the src genes of avian sarcoma viruses.

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.

The RNA of defective avian acute leukemia virus OK10 was isolated from a defective virus particle, released by OK10-transformed nonproducer avian fibroblasts, as a 60S complex consisting of 8.6-kilobase subunits. Oligonucleotide fingerprinting and RNA·cDNA hybridization identified two sets of sequences in OK10 RNA: group-specific sequences, which are related to all nondefective members of the avian tumor virus group, and a sequence closely related to the subgroup-specific sequences ( mcv ) of the myelocytomatosis virus (MC29) subgroup of avian acute leukemia viruses. Hence, OK10 is classified as a member of the MC29 subgroup of avian tumor viruses, in agreement with classification based on its oncogenic spectrum. The group-specific sequences of OK10 RNA include partial (Δ) pol and env genes, a c -region, and, unlike those of all other members of the MC29 subgroup, a complete gag gene. Oligonucleotide mapping revealed 5′- gag -Δ pol - mcv -Δ env - c -3′ as the order of the subgroup-specific and group-specific elements of OK10 RNA. The genetic unit gag -Δ pol - mcv , measuring ≈6.4 kilobases, codes for the nonstructural, presumably transforming, 200,000-dalton OK10-specific protein and also includes the gag gene coding for the internal virion proteins. Because gag is the only intact virion gene shared in addition to regulatory RNA sequences between OK10 and nondefective avian tumor viruses, it is concluded that the gag gene is sufficient for the formation of a defective virus particle. Comparisons among the RNAs and gene products of different viruses of the MC29 subgroup show that they share 5′-terminal gag -related and internal mcv sequences but differ from each other in intervening gag -, pol -, and mcv -related sequences. It follows that the probable transforming genes and their protein products have two essential domains, one consisting of conserved 5′ gag -related and the other of 3′ mcv -related sequence elements. In the light of this and previous knowledge we can now distinguish two designs among five different transforming onc genes of avian tumor viruses: onc genes with coding sequences unrelated to virion genes, like those of Rous sarcoma virus and avian myeloblastosis virus, and onc genes with coding sequences that are hybrids of virion genes and specific sequences, like those of the MC29 subgroup viruses, of avian erythroblastosis virus, and of Fujinami sarcoma virus.

The mammalian protooncogene homologue of the avian v-ets sequence from the E26 retrovirus consists of two sequentially distinct domains located on different chromosomes. Using somatic cell hybrid panels, we have mapped the mammalian homologue of the 5' v-ets-domain to chromosome 11 (ETS1) in man, to chromosome 9 (Ets-1) in mouse, and to chromosome D1 (ETS1) in the domestic cat. The mammalian homologue of the 3' v-ets domain was similarly mapped to human chromosome 21 (ETS2), to mouse chromosome 16 (Ets-2), and to feline chromosome C2 (ETS2). Both protooncogenes fell in syntenic groups of homologous linked loci that were conserved among the three species. The occurrence of two distinct functional protooncogenes and their conservation of linkage positions in the three mammalian orders indicate that these two genes have been separate since before the evolutionary divergence of mammals.

Proviral DNA transcribed from the RNA of Moloney murine sarcoma virus was isolated from newly infected cells. Three forms of viral DNA were observed: (i) a linear double-stranded form of 3.4 X 10(6) daltons which constituted the major viral DNA species in the cell, and is thought to be a complete transcript (monomer) of viral RNA; (ii) a fast-sedimenting viral DNA bigger than the monomeric unit which can be either integrated provirls or concatamers; and (iii) covalently closed circles of monomer size representing 5% or less of the total viral DNA in the cell. The linear viral DNA was tested for its susceptibility to restriction endonucleases by electrophoretic analysis of the digestion products and their identification by hybridization with viral RNA or cDNA probes. The linear DNA is not cleaved by endonucleases EcoRI and BamHI. It is cleaved into two fragments by endonucleases HindIII and Hae II, and into three fragments by restriction endonuclease HincII. The fragments of the viral DNA added up to approximately 3.4 X 10(6) daltons; this and the uniform size of the linear DNA indicated that the viral DNA has unique ends and a complexity of 3.4 X 10(6) daltons. The different cleavage fragments were ordered with respect to each other and the 3' end of the viral RNA. It was observed that fragments from both ends of the linear DNA can be hybridized to sequence(s) at the 3' end of murine sarcoma virus RNA; this result suggested the possibility that a short redundant sequence exists at both termini of the genome.

AIDS is an acquired immunodeficiency syndrome defined by a severe depletion of T cells and over 20 conventional degenerative and neoplastic diseases. In the U.S. and Europe, AIDS correlates to 95% with risk factors, such as about 8 years of promiscuous male homosexuality, intravenous drug use, or hemophilia. Since AIDS also correlates with antibody to a retrovirus, confirmed in about 40% of American cases, it has been hypothesized that this virus causes AIDS by killing T cells. Consequently, the virus was termed human immunodeficiency virus (HIV), and antibody to HIV became part of the definition of AIDS. The hypothesis that HIV causes AIDS is examined in terms of Koch's postulates and epidemiological, biochemical, genetic, and evolutionary conditions of viral pathology. HIV does not fulfill Koch's postulates: (i) free virus is not detectable in most cases of AIDS; (ii) virus can only be isolated by reactivating virus in vitro from a few latently infected lymphocytes among millions of uninfected ones; (iii) pure HIV does not cause AIDS upon experimental infection of chimpanzees or accidental infection of healthy humans. Further, HIV violates classical conditions of viral pathology. (i) Epidemiological surveys indicate that the annual incidence of AIDS among antibody-positive persons varies from nearly 0 to over 10%, depending critically on nonviral risk factors. (ii) HIV is expressed in less than or equal to 1 of every 10(4) T cells it supposedly kills in AIDS, whereas about 5% of all T cells are regenerated during the 2 days it takes the virus to infect a cell. (iii) If HIV were the cause of AIDS, it would be the first virus to cause a disease only after the onset of antiviral immunity, as detected by a positive "AIDS test." (iv) AIDS follows the onset of antiviral immunity only after long and unpredictable asymptomatic intervals averaging 8 years, although HIV replicates within 1 to 2 days and induces immunity within 1 to 2 months. (v) HIV supposedly causes AIDS by killing T cells, although retroviruses can only replicate in viable cells. In fact, infected T cells grown in culture continue to divide. (vi) HIV is isogenic with all other retroviruses and does not express a late, AIDS-specific gene. (vii) If HIV were to cause AIDS, it would have a paradoxical, country-specific pathology, causing over 90% Pneumocystis pneumonia and Kaposi sarcoma in the U.S. but over 90% slim disease, fever, and diarrhea in Africa.(viii) It is highly improbable that within the last few years two viruses (HIV-1 and HIV-2) that are only 40% sequence-related would have evolved that could both cause the newly defined syndrome AIDS. Also, viruses are improbable that kill their only natural host with efficiencies of 50-100%, as is claimed for HIVs. It is concluded that HIV is not sufficient for AIDS and that it may not even be necessary for AIDS because its activity is just as low in symptomatic carriers as in asymptomatic carriers. The correlation between antibody to HIV and AIDS does not prove causation, because otherwise indistinguishable diseases are now set apart only on the basis of this antibody. I propose that AIDS is not a contagious syndrome caused by one conventional virus or microbe. No such virus or microbe would require almost a decade to cause primary disease, nor could it cause the diverse collection of AIDS diseases. Neither would its host range be as selective as that of AIDS, nor could it survive if it were as inefficiently transmitted as AIDS. Since AIDS is defined by new combinations of conventional diseases, it may be caused by new combinations of conventional pathogens, including acute viral or microbial infections and chronic drug use and malnutrition. The long and unpredictable intervals between infection with HIV and AIDS would then reflect the thresholds for these pathogenic factors to cause AIDS diseases, instead of an unlikely mechanism of HIV pathogenesis.

Avian carcinoma virus MH2 has been grouped together with MC29, CMII, and OK10, because all of these viruses share a transformation-specific sequence termed myc. A 5.2-kilobase (kb) DNA provirus of MH2 has been molecularly cloned. The complete genetic structure of MH2 is 5'-delta gag(1.9-kb)-mht(1.2-kb)-myc(1.3-kb)-delta env(?) and noncoding c-region (0.2-kb)-3'. delta gag, delta env, and c are genetic elements shared with nondefective retroviruses, whereas mht is a unique, possibly MH2 transformation-specific, sequence. Hybridizations with normal chicken DNA and cloned chicken c-myc DNA indicate that the mht sequence probably derives from a normal cellular gene that is distinct from the c-myc gene. The genetic structure of MH2 suggests that the delta gag and mht sequences function as a hybrid gene that encodes the p100 putative transforming protein. The myc sequence of MH2 appears to encode a second transforming function. Therefore, it seems that MH2 contains two genes with possible oncogenic function, whereas MC29, CMII, and OK10 each carries a single hybrid delta gag-myc transforming gene. It is remarkable that, despite these fundamental differences in their primary structures and mechanisms of gene expression, MH2 and MC29 have very similar oncogenic properties.

The RNA of a replication-defective (rd) mutant, isolated from stocks of nondefective (nd) Schmidt-Ruppin Rous sarcoma virus of subgroup A (SR-A) and termed SR-N8, was compared to the RNAs of SR-A, of a transformation-defective derivative of SR-A (td SR-A) and of rd Bryan Rous sarcoma virus, RSV (minus). The molecular mass of the 30-40S species of SR-N8 RNA was estimated to be 21% (congruent to 7.5 to 8 times 10-5 daltons) smaller than that of SR-A by (i) electrophoresis in polyacrylamide gels and (ii) analyses of RNA complexity based on RNase T1-resistant oligonucleotides. ST-N8 shares probably all (=14) of its large RNase T1-resistant oligonucleotides with the RNA of SR-A as judged from the chromatographic distribution and the RNase A-resistant fragments obtained from RNase T1-resistant oligonucleotides. However, SR-N8 RNA lacked six large oligonucleotides which were present in the RNAs of SR-A and td SR-A. Conversely, the RNAs of SR-A, and of SR-N8 contained two oligonucleotides not found in td SR-A. The RNA of SR-N8 was found to differ from that of RSV (minus) in its electrophoretic mobility and its fingerprint pattern. It is concluded that the RNA of SR-N8 was generated by a deletion of SR-A. The extent of this deletion is compatible with the notion that the genetic information for the large viral envelope glycoprotein (molecular mass = 70,000-85,000 daltons) has been lost from the RNA of SR-A to yield SR-N8 RNA. From a comparison of td and rd deletion mutants, it appears that loss of different functions corresponds to the absence of different oligonucleotides in their RNA.

Earlier electrophoretic analyses have shown that the 60-70S RNA of avian sarcoma viruses contains a characteristic subunit, termed class a subunit, which has a lower electrophoretic mobility than class b subunit found in transformation-defective derivatives of sarcoma viruses and in avian leukosis viruses. We have compared the RNAs of three nondefective avian sarcoma viruses, B77 and Prague and Schmidt-Ruppin strains of Rous sarcoma virus, with those of their transformation-defective ( td ) derivatives, td B77, td PR-C, and td SR-A, respectively, to determine the chemical basis for the difference between class a and b subunits. It was found by “fingerprinting” that ( 1 ) all (about 20-25) large T1 RNase-resistant oligonucleotides present in class b subunits of transformation-defective viruses have homologous counterparts in the class a subunits of corresponding nondefective sarcoma viruses and that ( 2 ) class a subunits contain a few (one or two) additional oligonucleotides that are not present in class b . By contrast the oligonucleotide fingerprints of avian tumor viruses of different strains and subgroups were very different. Cross hybridization of classes a and b RNA of sarcoma virus B77 with DNA transcribed from a corresponding transformation-defective virus td B77 showed that the two RNAs share at least 60% and differ by about 10% of their sequences. It is suggested that the structural relationship of class a and b subunits of corresponding viruses may be expressed as a = b + x , and that all the oligonucleotides present only in RNAs of sarcoma viruses but not in transformation-defective viruses of the corresponding strains are part of sequence(s) x . The possibility that x represents genetic information directly or indirectly involved in transformation of fibroblasts is discussed.

The 60–70S RNAs of several transforming and nontransforming avian tumor viruses have different electrophoretic mobilities. The RNA of transforming viruses contains two electrophoretically separable subunit classes: a and b . The relative concentrations of these subunits vary with the virus strain. Avian leukosis viruses and nontransforming derivatives of a sarcoma virus lack subunits of class a . It is suggested that the presence of the class a subunit is related to the transforming ability for fibroblasts of the virus.

The DNA polymerase of the Prague strain of Rous sarcoma virus of subgroup C and of the Schmidt-Ruppin strain of subgroup A has been solubilized. DNA polymerase purified by sucrose gradient sedimentation and chromatography on DEAE-cellulose represented less than 2% of the soluble [ 14 C]protein of the virus. The enzyme was separated from 90% of the viral glycoprotein; it is probably different from the viral group-specific antigen. The sedimentation coefficient ( s 20, w ) of the soluble DNA polymerase was 8 S before, and 6 S after, incubation with pancreatic RNase. The molecular weight of the 8S DNA polymerase was estimated to be about 170,000, and that of the 6S DNA polymerase to be about 110,000. Purified DNA polymerase had a high activity with 60-70S viral RNA or salmon DNA as template, but it had a low activity with heat-dissociated 60-70S RNA, influenza virus RNA, or the RNA of tobacco mosaic virus as template. Neither the 8S nor the 6S DNA polymerase had endogenous template activity. The DNA-dependent and the RNA-dependent DNA polymerase activities of the Prague strain coincided in sucrose gradients, both in the 8S and the 6S form. It is concluded that the RNA-dependent and the DNA-dependent DNA polymerase activities of the avian tumor viruses are probably due to the same enzyme.

Rous sarcoma virus harvested from cells at intervals of 3 min has the same density, sedimentation coefficient, and DNA polymerase as virus harvested at hourly intervals. The RNA of the Prague strain-C consists of a minor class of 60-70S RNA, a major class 30-40S RNA, and a 4-12S class of RNA present at variable concentration. The RNA of the Schmidt-Ruppin strain-A contains more 60-70S than 30-40S RNA. Upon incubation of virus harvested at 3-min intervals at 40° in cell growth medium or Tris-saline, most of the 30-40S RNA is converted to 60-70S RNA. The electrophoretic mobility of the 30-40S RNA of the Rous virus harvested at 3-min intervals is lower than that of the 30-40S subunits of completely dissociated 60-70S RNA; after heating, their mobilities are identical. Heating also releases some small RNAs from 30-40S RNA of virus harvested at 3-min intervals, but five times more 4S RNA is released if the 30-40S RNA is allowed to convert to 60-70S in the virus. The template activity for Rous virus DNA polymerase of the 30-40S RNA of Rous virus harvested at 3-min intervals is about five times lower than that of 60-70S RNA. It is suggested that association of 30-40S RNAs with some RNAs of the 4-12S class may take place simultaneously with their conversion to 60-70S RNA.