Biomedical subjects
Publications and source records attributed to K C Robbins.
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Polyadenylated RNAs of certain human tumour cell lines are shown to contain transcripts related to the cell-derived transforming onc genes of molecularly cloned primate, murine or avian transforming retrovirus genomes. Thus, analogues of retroviral transforming genes are both present and frequently expressed in human neoplastic cells.
The sequence of the transforming region of simian sarcoma virus (SSV) has been determined by using molecularly cloned viral DNA. This region encompassed the 1.0-kilobase pair woolly monkey cell-derived insertion sequence, v-sis, and flanking simian sarcoma-associated viral (SSAV) sequences. A 675-nucleotide-long open reading frame commenced 19 nucleotides within the SSAV sequences to the left of the v-sis helper viral junction and terminated within v-sis itself. Possible promoter and acceptor splice signals were detected in helper viral sequences upstream from this open reading frame, and potential polyadenylylation sites were identified downstream both within v-sis and in helper viral sequences beyond v-sis. The recombinational event that led to the generation of SSV occurred in the middle of two functional codons, indicating that SSAV provided the regulatory elements for transcription as well as the initiation codon for translation of SSV cell-derived transforming sequences.
The primate cell-derived transforming gene (v-sis) of simian sarcoma virus (SSV) is represented as a single copy marker within cellular DNAs of mammalian species including human. The human analogue of v-sis can be distinguished from its rodent counterparts by Southern blotting analysis of EcoRI-restricted DNAs. By testing for the presence of the human v-sis-related fragment, c-sis (human), in somatic cell hybrids possessing varying numbers of human chromosome, as well as in segregants of such hybrids, it was possible to assign c-sis to human chromosome 22.
The presence of dominant transforming genes in human tumor cell lines has been investigated. High molecular weight DNAs isolated from cell lines established from carcinomas and sarcomas of various organs as well as from a glioblastoma and two melanomas were utilized to transfect NIH/3T3 mouse fibroblasts. The DNAs of T24 and A2182, two cell lines derived from a bladder and a lung carcinoma, respectively, and of HT-1080, a cell line established from a fibrosarcoma, were able to transform recipient NIH/3T3 cells. First-cycle transformants exhibited anchorage-independent growth and were tumorigenic in athymic and immunocompetent mice. Moreover, they contained human DNA sequences and were able to transmit their malignant phenotype in additional cycles of transfection. Southern blot analysis of T24-derived transformants showed that a single fragment of human DNA specifically cosegregated with the malignant phenotype, suggesting that it contained the T24 oncogene. Therefore, these human sequences were molecularly cloned with lambda Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated lambda T24-15A, was shown to contain a 15-kilobase-pair EcoRI insert of human cellular DNA. lambda T24-15A DNA (either intact or EcoRI digested) transformed NIH/3T3 fibroblasts with a specific activity of 20,000 focus-forming units per pmol of cloned DNA. Our results indicate that we have molecularly cloned a biologically active oncogene present in T24 human bladder carcinoma cells.
We sought to identify the species of origin of the cell-derived (sis) sequences of simian sarcoma virus. A molecular clone comprised of sis DNA detected related nucleotide sequences at low copy numbers in normal cellular DNAs of species as diverse as humans and quail. The extent of hybridization and degree of base-pair matching with sis DNA were greatest with New World primate DNAs. The thermal denaturation curve midpoints of hybrids formed between sis and woolly monkey DNAs were indistinguishable from homologous sis DNA hybrids, establishing the woolly monkey (Lagothrix spp.) as the source of sis sequences. In comparative studies, sis was shown to be more conserved among mammalian species than unique-sequence woolly monkey cellular DNA. There was no detectable homology between sis and the cell-derived sequences of other fibroblast-transforming retroviruses. These findings indicate that sis is likely to be a unique onc gene among transforming retroviruses.
The integrated form of simian sarcoma virus (SSV) was molecularly cloned in the Charon 16A strain of bacteriophage lambda. In transfection analysis, the recombinant viral DNAs demonstrated the ability to transform cells in tissue culture at high efficiency. Such transformants possessed typical SSV morphology, expressed simian sarcoma associated virus (SSAV) gag gene products in the absence of virus release, and released SSV after superinfection with a type C helper virus. A physical map of the 5.8-kilobase-pair (kbp) recombinant viral DNA clone, deduced from restriction endonuclease analysis, revealed a 5.1-kbp SSV genome containing 0.55-kbp-long terminal repeats flanked by 0.45 and 0.25 kbp of contiguous host cell sequences. By R-loop analysis, the viral DNA molecule contained two regions of homology to SSAV, separated by a 1.0-kbp nonhomologous region. This SSV-specific sequence was shown to be uniquely represented within the normal cellular DNA of diverse mammalian species, including human. Our results demonstrate that this primate transforming retrovirus arose in nature by recombination of a type C helper virus and a host cellular gene.
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The sequence of the transforming region of the Moloney murine sarcoma virus genome has been determined by using molecularly cloned viral DNA. This region, 3.6 to 5.8 kilobase pairs from the left end of the molecule, contains the entire cellular insertion (src) sequence as well as helper viral sequences including the large terminal repeat (LTR). On the viral RNA strand, a long (1224 bases) open reading frame commenced to the left of the src-helper virus junction and terminated at a point 58 nucleotides into helper viral sequences to the right of src. Possible promoter and acceptor splice signals were detected in helper viral sequences upstream from this open reading frame. On the antiviral RNA strand, several promoter-like sequences, including one within the src region itself, were identified. However, no open reading frame downstream from these promoters was detected in the antiviral RNA strand. The LTR was found to contain promoter-like sequences as well as LTR was found to contain promoter-like sequences as well as mRNA capping and polyadenylylation signals. In addition, it possessed an 11-base inverted terminal repeat at each end. Thus, the structure of the Moloney murine sarcoma virus genome with an LTR at each end resembles that of prokaryotic transposable elements.
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A cleavage map of the Moloney murine sarcoma viral DNA was constructed and compared with that of a spontaneously occurring deletion mutant. By restriction enzyme analysis, it was shown that a region encompassing over 40% of the viral information was not essential for transformation or rescue of the deletion mutant. The transforming region was further localised by analysis of the transforming activity in tissue culture of isolated restriction fragments of linear duoble-stranded sarcoma viral DNA. In each case, DNA fragments that retained transforming activity preserved the cell-derived insertion sequences of the viral genome. Moreover, such transformants invariably expressed RNA specific to this region. By these two approaches, it was possible to demonstrate that the transforming region of the viral genome begins very near or within the cell-derived insertion sequences. Thus, the transforming gene of this mammalian sarcoma virus originates from within the mouse cell genome.
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Type-C RNA viruses isolated from wild mice are causative of naturally occurring neoplasia and neurologic diseases. Biochemical and immunologic characterization of this virus group revealed that amphotropic viruses isolated from wild mice trapped in separate geographical areas are indistinguishable, whereas amphotropic and ecotropic viruses naturally infecting the same animal are env gene variants. Molecular hybridization studies established that neither host range variant is endogenous to the Mus musculus genome, although each demonstrates partial nucleotide sequence homology. Wild mouse type-C viruses exhibited much closer molecular and antigenic relatedness to the exogenous virus subgroup (Friend-, Moloney-, and Rauscher-MuLV) than to prototype endogenous viruses isolated from laboratory mouse strains. The evidence indicates that exogenous mouse type-C viruses have been maintained in nature over a long period of evolution as a separate virus group, causative of tumors in mice by a mechanism solely involving their transmission as infectious agents.
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Investigations aimed at defining cellular functions required for expression of transformation by mammalian sarcoma viruses have led to the isolation of a class of revertants that contain biologically active feline sarcoma virus, yet possess in vitro and in vivo properties of normal cells. The block to expression of the transformed state in these cellular revertants was spontaneously reversible at low frequency. Moreover, infection with certain helper viruses reversed the block at very high efficiency. Helper virus complementation was shown not to be a direct effect of helper virus functions expressed in the initially infected revertant cell. Rather, the helper virus acted indirectly by rescuing sarcoma virus and allowing it to infect and transform another cell within the revertant population. Using biochemical and immunologic techniques, it was possible to demonstrate a specific and very marked reduction in transcriptional and translational products of the sarcoma viral genome in the revertant cells. Findings that the reversal of this block was associated with reacquisition of the transformed phenotype, together with other evidence, suggest that reversion results from cellular transcriptional regulation of the integrated sarcoma virus genome. Reversion in this virus transformation system provides a model for oncogenesis resulting from derepression of cellular genes that possess malignant potential.