Mouse chromosomal mapping of a murine leukemia virus integration region (Mis-1) first identified in rat thymic leukemia.

We have previously identified a region of genomic DNA which constitutes the site of frequent provirus integration in rat thymomas induced by Moloney murine leukemia virus (Lemay and Jolicoeur, Proc. Natl. Acad. Sci. USA 81:38-42, 1984). This genetic locus is now designated Mis-1 (Moloney integration site). Cellular sequences homologous to Mis-1 are present in mouse DNA. Using a series of hamster-mouse somatic cell hybrids, we mapped the Mis-1 locus to mouse chromosome 15. Frequent chromosome 15 aberrations have been described in mouse thymomas. Mis-1 represents a putative new oncogene which might be involved in the initiation or maintenance or both of these neoplasms.     

Dsi-1, a region with frequent proviral insertions in Moloney murine leukemia virus-induced rat thymomas.

Dsi-1 is a region of chromosomal DNA that underwent proviral insertion in 3 of 24 Moloney murine leukemia virus-induced rat thymomas. In one of these tumors, a provirus is also integrated adjacent to the proto-oncogene c-myc. The proviruses in Dsi-1 have been characterized and appear to be complete. The proviruses were located within a 2-kilobase region that contained four prominent DNase I-hypersensitive sites. These hypersensitive sites were observed in Moloney murine leukemia virus-induced thymomas but not in NRK cells. The region of Dsi-1 immediately 3' to the insertions cross-hybridized with human and chicken DNA, indicating that it contains highly conserved sequences. No evidence could be found for the expression of this highly conserved region. Dsi-1 was mapped to mouse chromosome 4. This location demonstrates that Dsi-1 is different from 16 of the known proto-oncogenes (c-abl, c-erbA c-erbB, c-ets-1, c-ets-2, c-fes, c-fos, c-myb, c-myc, c-raf, A-raf, c-Ha-ras, c-Ki-ras, N-ras, c-sis, and c-src) and 12 cellular regions of tumor-associated integrations in retrovirus-induced tumors (c-erbB, Fis-1, int-1, int-2, Mis-1/pvt-1, Mlvi-1, Mlvi-2, c-mos, c-myb, c-myc, Pim-1, and c-Ha-ras). Hybridization experiments indicated that Dsi-1 is probably different from five additional proto-oncogenes (c-fgr, c-fms, c-mos, neu, and c-yes) and from two additional frequent integration regions (lck and Mlvi-3).     

The Mlvi-1 locus involved in the induction of rat T-cell lymphomas and the pvt-1/Mis-1 locus are identical.

Mlvi-1 defines a locus of proviral integration in rat thymomas induced by Moloney murine leukemia virus. pvt-1/Mis-1 represents an independently identified locus which becomes rearranged either by chromosomal translocation in murine plasmacytomas or by provirus insertion in retrovirus-induced murine and rat thymic lymphomas. Although it had been claimed that pvt-1/Mis-1 and Mlvi-1 represent two different loci, we present here evidence showing that they are identical. This finding demonstrates the need for rigorous characterization of any newly identified common regions of integration in retrovirus-induced neoplasms.     

A replication-competent promoter-trap retrovirus.

A promoter-trap retrovirus has been constructed in which a promoterless polyomavirus middle T antigen gene was inserted in the U3 region of the long terminal repeat of a replication-competent Moloney murine leukemia virus. The resulting virus, designated PyT, was used to infect mouse mammary glands in situ. As expected, mammary tumors appeared in some infected animals. These tumors were found to contain PyT proviruses of the predicted structure. From one such tumor, the PyT provirus and surrounding sequences from the integration site were cloned. The provirus was found to have integrated adjacent to the promoter of a novel mouse gene (TRAP1) that was expressed at low levels in various mouse tissues. These data show that the PyT retrovirus provides a sensitive means of detecting active promoters in vivo.     

Sint1, a common integration site in SL3-3-induced T-cell lymphomas, harbors a putative proto-oncogene with homology to the septin gene family

The murine retrovirus SL3-3 is a potent inducer of T-cell lymphomas when inoculated into susceptible newborn mice. Previously, DNAs from twenty SL3-3-induced tumors were screened by PCR for provirus integration sites. Two out of 20 tumors demonstrated clonal provirus insertion into a common region. This region has now been isolated and characterized. The region, named SL3-3 integration site 1 (Sint1), maps to the distal end of mouse chromosome 11, corresponding to human chromosome 17q25, and may be identical to a mouse mammary tumor virus integration site in a T-cell lymphoma, Pad3. Two overlapping genomic lambda clones spanning about 35 kb were isolated and used as a starting point for a search for genes in the neighborhood of the virus integration sites. A genomic fragment was used as a hybridization probe to isolate a 3-kb cDNA clone, the expression of which was upregulated in one of two tumors harboring a provirus in Sint1. The cDNA clone is predicted to encode a protein which shows 97.0% identity to a human septin-like protein encoded by a gene which has been found as a fusion partner gene of MLL in an acute myeloid leukemia with a t(11;17)(q23;q25). Together these findings raise the possibility that a proto-oncogene belonging to the septin family, and located about 15 kb upstream of the provirus integration sites, is involved in murine leukemia virus-induced T-cell lymphomagenesis.     

Concerted DNA rearrangements in Moloney murine leukemia virus-induced thymomas: a potential synergistic relationship in oncogenesis.

Rat thymic lymphomas induced by Moloney murine leukemia virus carry DNA rearrangements due to provirus integration in at least five independent cellular DNA domains (Mlvi-1, Mlvi-2, Mlvi-3, RMoInt-1, and c-myc). We had previously shown that rearrangements in more than one of these domains could occur in the same tumor. In this report we extend these findings by showing that, with one exception, tumors containing provirus insertions in Mlvi-1 always contained provirus insertions in a second locus, Mlvi-2. To determine whether both events occurred in the same population of tumor cells, we examined the clonal nature of these tumors by taking advantage of allelic polymorphisms that occur naturally in both Mlvi-1 and Mlvi-2. Tumors with provirus insertions in both Mlvi-1 and Mlvi-2 arising in rats heterozygous at one of these loci were identified. DNA from these tumors was analyzed by restriction endonuclease digestion and hybridization to DNA probes derived from both Mlvi-1 and Mlvi-2. Thus, we determined the clonal nature of three thymomas and showed that in these tumors both insertion events occurred in the same population of tumor cells. The concomitant appearance of provirus insertions in Mlvi-1 and Mlvi-2 suggests a synergism of these two events that may be important in tumor induction and progression.     

Identification of a new common provirus integration site in gross passage A murine leukemia virus-induced mouse thymoma DNA.

The Gross passage A murine leukemia virus (MuLV) induced T-cell leukemia of clonal (or oligoclonal) origin in inoculated mice. To study the role of the integrated proviruses in these tumor cells, we cloned several newly integrated proviruses (with their flanking cellular sequences) from a single tumor in procaryotic vectors. With each of the five clones obtained, a probe was prepared from the cellular sequences flanking the provirus. With one such probe (SS8), we screened several Gross passage A MuLV-induced SIM.S mouse tumor DNAs and found that, in 11 of 40 tumors, a provirus was integrated into a common region designated Gin-1. A 26-kilobase-pair sequence of Gin-1 was cloned from two lambda libraries, and a restriction map was derived. All proviruses were integrated as a cluster in the same orientation within a 5-kilobase-pair region of Gin-1, and most of them had a recombinant structure of the mink cell focus-forming virus type. The frequency of Gin-1 occupancy by provirus was much lower in thymoma induced by other strains of MuLV in other mouse strains. Using somatic-cell hybrid DNAs, we mapped Gin-1 on mouse chromosome 19. Gin-1 was not homologous to 16 known oncogenes and was distinct from the other common regions for provirus integration previously described. Therefore, Gin-1 appears to represent a new common provirus integration region. The integration of a provirus within Gin-1 might be an important event leading to T-cell transformation, and the Gin-1 region might harbor sequences which are involved in tumor development.     

The integration sites of endogenous and exogenous Moloney murine leukemia virus

Specific cDNA probes of Moloney and AKR murine leukemia viruses have been prepared to characterize the proviral integration sites of these viruses in the genomes of Balb/Mo and Balb/c mice. The genetically transmitted Moloney provirus of Balb/Mo mice was detected in a characteristic Eco RI DNA fragment of 16 x 10(6) daltons. No fragment of this size was detected in tissue DNAs from Balb/c mice infected as newborns with Moloney virus. We conclude that a viral integration site, occupied in preimplantation mouse embryos, is not necessarily occupied when virus infects cells in post-natal animals. Balb/Mo and Balb/c mice do carry the AkR structural gene in an Eco RI DNA fragment of 12 x 10(6) daltons. Further restriction analysis of this fragment indicated that both mouse lines carry one AKR-type provirus. Leukemogenesis in Balb/Mo and newborn infected Balb/c mice is accompanied by reintegration of Moloney viral sequences in new chromosomal sites of tumor tissues. Part of the reintegrated Moloney viral sequences are of subgenomic size. The AKR viral sequences, however, are not found in new sites. Further restriction analysis revealed that the development of Moloney virus-induced leukemia in Balb/Mo mice does not lead to detectable structural alteration of the genetically transmitted Moloney and AKR structural genes. Possible mechanisms of the reintegration process are also discussed.     

Identification of a common helper provirus integration site in Abelson murine leukemia virus-induced lymphoma DNA.

Abelson murine leukemia virus induces oligoclonal pre-B lymphoma in mice. The expression of the v-abl oncogene in target cells does not appear to be sufficient for tumor induction in several mouse strains, and additional genetic events are thought to be required. We postulated that the helper Moloney murine leukemia virus might induce these events, and its potential role as an insertional mutagen was assessed by the search of a common helper provirus integration site in Abelson murine leukemia virus lymphomas. Molecular cloning of cellular sequences adjacent to Moloney proviruses enabled us to identify a cellular region, designated Ahi-1, which was found occupied by the helper proviruses in 16% of Abelson pre-B-cell lymphomas. All proviruses for which the precise integration site within Ahi-1 could be mapped were found to be in the same orientation. Ahi-1 has been mapped to mouse chromosome 10 and represents a new common proviral integration site. These data suggest that the helper virus contributes to the induction of secondary genetic events which may be important for the development of Abelson murine leukemia virus-induced pre-B-cell lymphoma.     

Tumorigenesis by mouse mammary tumor virus: Evidence for a common region for provirus integration in mammary tumors

We have prepared specific probes for unique-sequence cellular DNA adjacent to each of the newly integrated proviruses in tumors induced by mouse mammary tumor virus (MMTV). The use of such probes to screen a large number of independent mammary tumors in the BR6 strain of mouse has indicated that in at least 17 out of the 40 tumors examined so far, an MMTV provirus has integrated into a common chromosomal domain. A 10 kb Eco RI fragment of single copy DNA from this region has been isolated and partially characterized by restriction enzyme mapping. Of the proviruses located within this fragment in different tumors, all but one are complete, in the same orientation, and clustered within about 3 kb of cellular DNA. These findings are consistent with an insertional mutagenesis model for tumorigenesis by MMTV, in which the integration of a provirus in a particular region of cellular DNA may activate a neighboring oncogene. The region we describe here appears to be different from that reported for mammary tumors in the C3H strain of mouse.     

The Myb and Ahi-1 genes are physically very closely linked on mouse Chromosome 10

Ahi-1 has previously been identified as a common helper provirus integration site on mouse Chromosome (Chr) 10 in 16% of Abelson pre-B-cell lymphomas and shown to be closely linked to the Myb protooncogene. By using long-range restriction mapping, we have mapped the Myb and Ahi-1 regions within a 120-kbp DNA fragment. The Ahi-1 region is located approximately 35 kbp downstream of the Myb gene. A further comfirmation of this finding was obtained by screening a mouse YAC library. The three positive clones obtained contained both the Myb and Ahi-1 gene sequences. To test whether provirus integration in the Ahi-1 region enhances the expression of Myb by a cis-acting mechanism, we have also examined Myb gene expression in A-MuLV-induced pre-B-lymphomas. Our data have revealed that there is no clear evidence for such activation in the tumors we have tested, indicating that provirus insertion in the Ahi-1 region is activating a novel gene, apparently involved in tumor formation.     

Genetic mapping of a cellular DNA region involved in induction of thymic lymphomas (Mlvi-1) to mouse chromosome 15.

Mlvi-1 defines a genetic locus representing a common domain for proviral DNA integration in Moloney murine leukemia virus-induced rat thymic lymphomas. Cellular sequences homologous to Mlvi-1 are present in mouse DNA, and we have used hamster-mouse somatic cell hybrids to chromosomally map Mlvi-1 in the mouse genome. Results demonstrated that Mlvi-1 maps to mouse chromosome 15 and that it is distinct from the Mlvi-2 integration region and from the cellular oncogenes c-myc and c-sis, which also map to this chromosome. Therefore, Mlvi-1 may contain novel sequences involved in the establishment and maintenance of virus-induced murine tumors, many of which contain abnormalities of chromosome 15.     

Isolation of an integrated provirus of Moloney murine leukemia virus with long terminal repeats in inverted orientation: integration utilizing two U3 sequences.

We have previously described the construction of a mutant of Moloney murine leukemia virus, in594-2, which carries a 2-base-pair insertion in the U5 region of the genome and is partially defective in forming the integrated proviral DNA. We have now recovered a cloned copy of an unusual provirus from rat cells infected with this mutant. The viral genome is flanked by long terminal repeats in inverted orientation, with U3 sequences joined to cellular DNA at both of the outer edges. In addition, the provirus is a recombinant, containing a segment of a VL30 element in inverted orientation in place of the Moloney murine leukemia virus env region. The recovery of this provirus indicates that two U3 regions can be used for viral integration and suggests that there may be no absolute requirement in the reaction for those U5 sequences outside the 13-base-pair inverted repeats.     

Novel integration sites at the distal 3' end of the c-myb locus in retrovirus-induced promonocytic leukemias.

In BALB/c nu/nu and sublethally irradiated DBA/2 mice, promonocytic leukemia was induced by intravenous inoculation of Friend murine leukemia virus (F-MuLV) strain C57 in conjunction with intraperitoneal injection of pristane. These tumors appear to be identical morphologically to previously reported ones induced by other MuLVs, such as Moloney, amphotropic 4070A, and F-MuLV FB29, which most commonly have provirus integrations in the 5' end of the c-myb locus. Interestingly, 2 of the 16 F-MuLV-induced tumors had viruses integrated in the distal 3' end of c-myb. To determine the precise locations of these integrations, it was necessary to clone sequences encoding the 3' c-myb exons and to prepare a physical map of this region. Exons 10 to 15 were positioned on the map, and it was found that the proviruses in the aforementioned tumors were located within narrow region in the beginning of the large (greater than 11 kb) intron 14. The predicted protein product encoded by the affected alleles is truncated by 38 amino acids. This represents a novel virus integration site which is most likely associated with oncogenic activation of the c-myb gene during leukemogenesis.