The transforming gene of Moloney murine sarcoma virus

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.     

Orthotopic bone induction at sites of Moloney murine sarcoma virus inoculation in mice.

Moloney murine sarcoma virus (M-MSV) induces at the site of inoculation in newborn and adult mice and rats various types of sarcoma, including osteosarcoma. The induced tumours are fatal in newborn, whereas sarcomas developed in adult animals regress spontaneously. The regression is mediated mainly by a cellular response. We have now demonstrated that the presence of sarcomas induced by M-MSV is a powerful stimulus for periosteal osteogenesis around tumour masses. Orthotopically induced osteogenesis by M-MSV may serve as a model for local regulation of bone growth and for cell differentiation studies, and may help explain the aetiology of some human bone disorders.     

Non-function of a Moloney murine leukaemia virus regulatory sequence in F9 embryonal carcinoma cells

Moloney murine leukaemia virus (M-MuLV) infection of embryonal carcinoma (EC) cells results in the integration of proviral DNA into the host cell genome, but not in virus production. One suggested explanation for the lack of viral gene expression in EC cells has been methylation of the integrated viral DNA. However, subsequent reports indicated that integration of the M-MuLV DNA occurs soon after infection, but that viral DNA methylation occurs considerably later. Nevertheless, viral gene expression is not observed even at early times. One possible explanation is that certain M-MuLV regulatory sequences do not function in EC cells. We now present evidence which supports this hypothesis.     

Activation of SV40 genome by 72-base pair tandem repeats of Moloney sarcoma virus

The simian virus 40 (SV40) 72-base pair (bp) tandem-repeated sequences have a crucial role as an activator element in viral gene expression. We replaced the SV40 72-bp repeat with a 72-bp repeat derived from the long terminal repeat (LTR) of cloned Moloney murine sarcoma virus (MSV) DNA. Although there is no detectable sequence homology to SV40, the MSV repeats can substitute functionally for the SV40 repeats and generate a viable virus in monkey kidney cells.     

Mature murine B lymphocytes immortalized by Kirsten sarcoma virus

Clonal, antigen-specific, functionally responsive cell populations have proved critical for the analysis of the activation and regulation of lymphocytes. Such studies with B lymphocytes, the precursors of antibody-secreting cells, are hampered by the difficulty in generating phenotypically mature, antigen-reactive lines from defined cell populations. One method is to use acutely transforming retroviruses, which can transform B-lineage lymphocytes in vitro. However, Abelson murine leukaemia virus (A-MuLV) infection of murine bone marrow cells in vitro yields mostly immature B-cell lines, and infection of murine bone marrow cells with murine sarcoma viruses carrying ras related genes produces only immature lymphoid cell lines. Retroviruses which contain ras can immortalize nonlymphoid cells without causing loss of mature phenotypic characteristics. We used ras-containing Kirsten sarcoma virus (KiSV) pseudotyped with an amphotropic MuLV helper virus, to infect a purified population of mature, hapten-binding murine splenic B lymphocytes, aiming to generate mature B-cell lines to use as models for the study of B-cell growth and differentiation physiology. Immortalized B-cell lines which retain the same mature phenotype as the starting population, including hapten-specific binding, were produced. This is the first demonstration of a method for immortalizing selected antigen-binding B lymphocytes, and the first example of immortalization of mature B cells in vitro with an acutely transforming ras-containing retrovirus.     

Interferon inhibits transformation by murine sarcoma viruses before integration of provirus

Neoplastic transformation by C-type retroviruses requires synthesis of a DNA copy (the provirus) of the RNA genome and its integration into the host cell DNA. We have previously shown that interferon (IFN) can stably prevent transformation of murine fibroblasts by the Kirsten strain of murine sarcoma virus (KiMSV), a murine leukaemia virus (MLV). A series of cell clones (IFN clones), isolated in the presence of IFN (10(4) U ml-1) from cultures of NIH-3T3 cells which had been treated with IFN, and then infected with KiMSV (KiMLV) in conditions where every cell was infected, were shown to be phenotypically untransformed. These untransformed cells did not produce virus or contain rescuable KiMSV. However, cells isolated using an identical procedure, but in the absence of IFN, were uniformly transformed and all produced KiMSV (KiMLV) or contained rescuable KiMSV. It was concluded that IFN either prevents synthesis or integration of the provirus, or else that in the presence of IFN the provirus is integrated such that it is not expressed. We now show that five representative clones contain no detectable KiMSV proviral DNA, and also that the initial stages of infection by KiMSV (KiMLV) are inhibited by IFN treatment. IFN seems to act before integration, preventing either the synthesis or the integration of proviral DNA.     

RecBCD enzyme is a bipolar DNA helicase

Escherichia coli RecBCD is a heterotrimeric helicase/nuclease that catalyses a complex reaction in which double-strand breaks in DNA are processed for repair by homologous recombination. For some time it has been clear that the RecB subunit possesses a 3' --> 5' DNA helicase activity, which was thought to drive DNA translocation and unwinding in the RecBCD holoenzyme. Here we show that purified RecD protein is also a DNA helicase, but one that possesses a 5' --> 3' polarity. We also show that the RecB and RecD helicases are both active in intact RecBCD, because the enzyme remains capable of processive DNA unwinding when either of these subunits is inactivated by mutation. These findings point to a bipolar translocation model for RecBCD in which the two DNA helicases are complementary, travelling with opposite polarities, but in the same direction, on each strand of the antiparallel DNA duplex. This bipolar motor organization helps to explain various biochemical properties of RecBCD, notably its exceptionally high speed and processivity, and offers a mechanistic insight into aspects of RecBCD function.     

Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication

Early tumorigenesis is associated with the engagement of the DNA-damage checkpoint response (DDR). Cell proliferation and transformation induced by oncogene activation are restrained by cellular senescence. It is unclear whether DDR activation and oncogene-induced senescence (OIS) are causally linked. Here we show that senescence, triggered by the expression of an activated oncogene (H-RasV12) in normal human cells, is a consequence of the activation of a robust DDR. Experimental inactivation of DDR abrogates OIS and promotes cell transformation. DDR and OIS are established after a hyper-replicative phase occurring immediately after oncogene expression. Senescent cells arrest with partly replicated DNA and with DNA replication origins having fired multiple times. In vivo DNA labelling and molecular DNA combing reveal that oncogene activation leads to augmented numbers of active replicons and to alterations in DNA replication fork progression. We also show that oncogene expression does not trigger a DDR in the absence of DNA replication. Last, we show that oncogene activation is associated with DDR activation in a mouse model in vivo. We propose that OIS results from the enforcement of a DDR triggered by oncogene-induced DNA hyper-replication.     

Specific monoclonal IgM is a potent adjuvant in murine malaria vaccination

Recent experiments in the murine system have indicated that the passive acquisition by offspring of maternal anti-malarial IgG antibodies while conferring some degree of immunity against a primary infection, paradoxically prevents the generation of acquired immunity through vaccination. Therefore, in view of earlier findings concerning the competitive effects of specific IgM and IgG antibodies, we investigated whether specific monoclonal IgM antibodies could be used to potentiate the response to a blood-stage murine malaria vaccine. We now report that small amounts of purified monoclonal anti-parasite IgM can specifically potentiate both priming and memory cell generation in response to vaccination as evidenced by survival after infection, and that the magnitude of this effect is greater than that found with a more conventional nonspecific adjuvant (Bordetella pertussis). Additionally, in offspring of immune mothers, where vaccination is ineffective for up to 8 weeks due to the presence of maternal IgG, we have found that IgM when administered with the vaccine can completely overcome this inhibition by its adjuvant effect.     

Spi-1 is a putative oncogene in virally induced murine erythroleukaemias

Retroviral insertional mutagenesis has been proposed as an efficient mechanism to turn on or to increase the expression of oncogenes in several avian or mammal models. Integration site studies of avian leukosis virus, murine leukaemia and murine mammary tumour viruses led to the coleutification of highly conserved genes whose expression is induced or increased during leukaemogenesis, probably through enhancer elements present in the retroviral long terminal repeats. This is reminiscent of the activation of cellular proto-oncogenes or putative oncogenes in numerous human tumours and leukaemias as a result of chromosomal translocations or DNA rearrangements. Here we report the characterization of a new putative oncogene isolated from a murine erythroleukaemia induced by the acute leukaemogenic retrovirus spleen focus forming virus (SFFV). An important and unusual feature of this genomic locus Spi-1 (for SFFV proviral integration) is that rearrangements due to SFFV integration were found in 95% of the erythroid tumours studied. A 4.0-kilobase messenger RNA was detected in rearranged tumours. No Spi-1 rearrangement was detected in other virally induced myeloid, lymphoid or erythroid tumours tested.