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.     

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.     

Frequent disruption of the Nf1 gene by a novel murine AIDS virus-related provirus in BXH-2 murine myeloid lymphomas.

Evi-2, a common site of viral integration in BXH-2 myeloid lymphomas, is located within a large intron of the Nf1 tumor suppressor gene. Viral integration at Evi-2 appears to induce disease by disrupting normal Nf1 expression. During our attempts to characterize the nature of the proviruses located at Evi-2, we found that approximately half of the proviruses were defective nonecotropic proviruses (A. M. Buchberg, H. G. Bedigian, N. A. Jenkins, and N. G. Copeland, Mol. Cell. Biol. 10:4658-4666, 1990). This was surprising, since most proviruses characterized at other BXH-2 common integration sites are full-length ecotropic viruses. In the studies described here, we found that this defective provirus carries two large deletions, one in pol and one in env, and is structurally related to another murine retrovirus, the murine AIDS retrovirus. By using oligonucleotide probes specific for this defective provirus, designated MRV, we showed that MRV-related proviruses are carried as endogenous germ line proviruses in most inbred strains. In addition, we identified the endogenous MRV provirus that gives rise to the defective proviruses identified at Evi-2. We present a model that accounts for the positive selection of MRV proviruses at Evi-2, which may allow selective identification of common viral integration sites harboring tumor suppressor genes.     

Moloney murine leukemia virus-induced myeloid tumors in adult BALB/c mice: requirement of c-myb activation but lack of v-abl involvement.

BALB/c mice treated with pristane and Abelson virus have been used as an animal model system for the rapid induction of plasmacytomas. Myelomonocytic tumors with helper Moloney murine leukemia virus clonally inserted into the c-myb locus were observed in about 10% of pristane-primed BALB/c mice infected with Abelson virus. However, v-abl was absent in almost all of those tumors. Since Moloney virus is thought to induce mostly T-cell lymphomas, we have carried out studies to investigate this alteration of disease specificity and to determine whether v-abl played an obligatory role in the development of these tumors. We found that, whereas lymphomas developed late (greater than 3 months) in both pristane-primed and unprinted control mice, the myelomonocytic tumors arose at a high frequency, within 3 months, but only in pristane-treated mice. Clonal Moloney virus insertion was again found in each of the seven myelomonocytic tumors examined. Northern blot analyses and S1 mapping studies revealed the presence of virally promoted chimeric mRNAs that lack the three 5'-most myb coding exons. Hence it appears that the requirement for the v-abl gene product in tumor induction is not obligatory. Our results also indicate that tumor-specific alteration at the 5' end of the myb gene plays an important role in the development of these tumors.     

Activation of the c-myb locus is insufficient for the rapid induction of disseminated avian B-cell lymphoma.

We have previously reported that infection of 9- to 13-day-old chicken embryos with RAV-1 results in rapid development of a novel B-cell lymphoma in which proviral insertion has activated expression of the c-myb gene (E. Pizer and E. H. Humphries, J. Virol. 63:1630-1640, 1989). The biological properties of these B-cell lymphomas are distinct from those associated with the B-cell lymphomas that develop following avian leukosis virus proviral insertion within the c-myc locus. In an extension of this study, more than 200 chickens, infected as 10- to 11-day-old embryos, were examined for development of lymphomas that possess disrupted c-myb loci. Fourteen percent developed disseminated B-cell lymphoma. In the majority of these tumors, the RAV-1 provirus had inserted between the first and second exons that code for p75c-myb. However, insertions between the second and third exons and between the third and fourth exons were also detected. In situ analysis of myb protein expression in tumor tissue revealed morphological features suggesting that the tumor originates in the bursa. Within the bursa, the lymphoma appeared to spread from follicle to follicle without compromising the structural integrity of the organ. Tumor masses in liver demonstrated heterogeneous levels of myb protein suggestive of biologically distinct subpopulations. In contrast to the morbidity data, immunohistological analysis of bursae from 4- to 6-week-old chickens at risk of developing lymphomas bearing altered c-myb loci revealed lesions expressing elevated levels of myb in 16 of 19 birds. The activated myb lymphoma displayed very poor capacity to proliferate outside its original host. Only 1 of 33 in vivo transfers of tumor to recipient hosts established a transplantable tumor. None of the primary tumor tissue nor the transplantable tumor exhibited the capacity for in vitro proliferation. Similar experimental manipulation has yielded in vitro lines established from avian B-cell lymphomas expressing elevated levels of c-myc or v-rel. The dependence on embryonic infection for development of activated-myb lymphoma suggests a requirement for a specific target cell in which c-myb is activated by proviral insertion. It is likely, moreover, that continued tumor development requires elevated expression of myb proteins within a specific cell population in a restricted stage of differentiation.     

An E mu-v-abl transgene elicits plasmacytomas in concert with an activated myc gene.

To clarify how the v-abl oncogene of Abelson murine leukemia virus contributes to lymphoid tumorigenesis, we introduced the gene linked to an immunoglobulin heavy chain enhancer (E mu) into the mouse germline. Although lymphoid development was not detectably affected in young E mu-v-abl mice, three transgenic lines shared a high predisposition to develop clonal plasmacytomas that secreted IgA or IgG. The unexpected absence of pre-B lymphomas suggests that Abelson virus generates such tumors by infecting an early lymphoid progenitor cell that has not yet activated the heavy chain enhancer. Most plasmacytomas bore a rearranged c-myc gene, apparently as a result of spontaneous translocation to the Igh locus. Moreover, progeny of a cross with analogous E mu-myc mice rapidly developed oligoclonal plasmacytomas. Thus, the collusion of v-abl with c-myc is stage specific, efficiently transforming plasma cells but not pre-B cells or B cells.     

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).     

New sites of proviral integration associated with murine promonocytic leukemias and evidence for alternate modes of c-myb activation.

Murine promonocytic leukemias involving insertional mutagenesis of the c-myb locus can be induced by replication-competent retroviruses. In previously studied promonocytic leukemic cells induced by Moloney murine leukemia virus (called MML), the provirus has been invariably integrated upstream of exons 3 or 4 and the leukemic cells expressed aberrant RNAs with fused virus-myb sequences. Furthermore, Myb expressed by these cells has been shown to be truncated by 47 or 71 amino acids. The present report examines the mechanisms of myb activation in leukemias induced by two other retroviruses, amphotropic virus 4070A and Friend strain FB29 (the leukemias are called AMPH-ML and FB-ML, respectively). This study revealed two additional c-myb proviral insertion sites in these promonocytic leukemias. One FB-ML had a proviral integration in exon 9, and expressed a C-terminally truncated Myb protein of 47 kDa similar to that previously demonstrated to be expressed in the myelomonocytic cell lines NFS60 and VFL-2. However, a sequence of reverse-transcribed and amplified RNA from this leukemia demonstrated that the truncation involved a loss of 248 amino acids compared with a loss of 240 amino acids in the myelomonocytic cell lines. Another leukemia had a provirus integrated in the 5' end of c-myb upstream of exon 2 (in the first intron) and produced a Myb protein that was indistinguishable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from normal Myb. This latter leukemia (FB-ML R1-4-10) expressed Myb with the smallest N-terminal truncation observed so far in promonocytic leukemias; translation begins at an ATG within c-myb exon 2, leading to loss of only 20 amino acids from the N terminus. Unlike the proteins produced in Moloney murine leukemia virus-induced promonocytic leukemias (MML) that have larger truncations, this protein has an intact DNA binding region and does not contain N-terminal amino acids encoded by gag. However, this protein is similar to all N-terminally truncated Mybs so far studied, in that the truncation resulted in deletion of a casein kinase II phosphorylation site which has been proposed to be involved in regulation of DNA binding.     

The mouse c-abl locus: molecular cloning and characterization

The mouse c-abl gene, part of the sequence of which was captured in Moloney murine leukemia virus to generate the transforming gene (v-abl) of the Abelson murine leukemia virus, has been isolated and characterized. The c-abl locus spans 40 kb in the mouse genome with the v-abl homologies distributed in no less than ten clusters along 25 kb of the cloned DNA. Partial sequence of the v-abl homologous regions indicates that v-abl derived from c-abl mainly by splicing of multiple exons of the c-abl gene. The c-abl sequences can be subdivided into two regions: a tyrosine kinase coding sequence distributed among eight small clusters on the 5' end of the gene and a C-terminal portion consisting of one small and one large cluster, which are needed neither for the tyrosine kinase activity nor for the transforming ability of v-abl. Apparent exon/intron boundaries in the homologous kinase-coding regions of c-abl and c-src are at different locations.     

Polymorphism in the neurofibromin gene,Nf1, is associated with antagonistic selection on wing size and development time in Drosophila melanogaster

In many invertebrates, body size shows genetically based clines, with size increasing in colder climates. Large body size is typically associated with prolonged development times. We consider variation in the CNS‐specific gene neurofibromin 1 (Nf1) and its association with body size and development time. We identified two major Nf1 haplotypes in natural populations, Nf1‐insertion‐A and Nf1‐deletion‐G. These haplotypes are characterized by a 45‐base insertion/deletion (INDEL) in Nf1 intron 2 and an A/G synonymous substitution (locus L17277). Linkage disequilibrium (LD) between the INDEL and adjacent sites is high but appears to be restricted within the Nf1 gene interval. In Australia, the frequency of the Nf1‐insertion‐A haplotype increases with latitude where wing size is larger, independent of the chromosomal inversion In(3R)Payne. Unexpectedly, the Nf1‐insertion‐A haplotype is negatively associated with wing size. We found that the Nf1‐insertion‐A haplotype is enriched in females with shorter development time. This suggests that the Nf1 haplotype cline may be driven by selection for development time rather than size; females from southern (higher latitude) D. melanogaster populations maintain a rapid development time despite being relatively larger, and the higher incidence of Nf1‐insertion‐A in Southern Australia may contribute to this pattern, whereas the effects of the Nf1 haplotypes on size may be countered by other loci with antagonistic effects on size and development time. Our results point to the potential complexity involved in identifying selection on genetic variants exhibiting pleiotropic effects when studies are based on spatial patterns or association studies.     

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.