Structure-function integrity of the adult hippocampus depends on the transcription factor Bcl11b/Ctip2.

The dentate gyrus is one of the only two brain regions where adult neurogenesis occurs. Throughout life, cells of the neuronal stem cell niche undergo proliferation, differentiation and integration into the hippocampal neural circuitry. Ongoing adult neurogenesis is a prerequisite for the maintenance of adult hippocampal functionality. Bcl11b, a zinc finger transcription factor, is expressed by postmitotic granule cells in the developing as well as adult dentate gyrus. We previously showed a critical role of Bcl11b for hippocampal development. Whether Bcl11b is also required for adult hippocampal functions has not been investigated. Using a tetracycline-dependent inducible mouse model under the control of the forebrain-specific CaMKIIα promoter, we show here that the adult expression of Bcl11b is essential for survival, differentiation and functional integration of adult-born granule cell neurons. In addition, Bcl11b is required for survival of pre-existing mature neurons. Consequently, loss of Bcl11b expression selectively in the adult hippocampus results in impaired spatial working memory. Together, our data uncover for the first time a specific role of Bcl11b in adult hippocampal neurogenesis and function.

Functional features of EVI1 and EVI1Δ324 isoforms of MECOM gene in genome-wide transcription regulation and oncogenicity.

The MDS1 and ecotropic viral integration site 1 (EVI1) complex locus (MECOM) gene encodes several transcription factor variants including MDS1-EVI1, EVI1 and EVI1Δ324. Although MDS1-EVI1 has been associated with tumor-suppressing activity, EVI1 is a known oncogene in various cancers, whose expression is associated with poor patient survival. Although EVI1Δ324 is co-transcribed with EVI1, its activity in cancer cells is not fully understood. Previous reports described that unlike EVI1, EVI1Δ324 protein cannot transform fibroblasts because of its disrupted N-terminal zinc finger (ZNF) domain. To better understand EVI1Δ324 biology and function, we obtained genome-wide binding occupancies and expression data in ovarian cancer cells. We characterized its DNA-binding sites, binding motif and target genes. Comparative analyses with previous study show that EVI1 and EVI1Δ324 share similar transcriptional activities linked to their common C-terminus ZNF domain. They bind to an E-twenty-six family (ETS)-like motif, target to a large extent the same genes and cooperate with AP1 transcription factor. EVI1Δ324-occupied genes were 70.7% similar to EVI1-bound genes. More strikingly, EVI1 and EVI1Δ324 differentially expressed genes were 99.87% identical, indicating comparable transcriptional regulatory functions. Consistently with gene ontologies linked to these target genes, EVI1Δ324 expression in HeLa cells could enhance anchorage-independent growth, such as EVI1, showing that EVI1Δ324 expression also lead to pro-oncogenic effects. The main specific feature of EVI1 variant is its N-terminus ZNF domain that binds DNA through GATA-like motif. We found that most GATA-like EVI1 chromatin immunoprecipitation sequencing peaks are far from genes and are not involved in transcriptional regulation. These genomic regions were enriched in simple sequence repeats and displayed high meiotic recombination rates. Overall, our genomics analyses uncovered common and specific features of two major MECOM isoforms. Their influence on transcription and downstream cell proliferation was comparable. However, EVI1-specific GATA-like binding sites, from its N-terminus ZNF domain, associated with high recombination rates, suggesting possible additional oncogenic potential for EVI1 in modulating genomic stability.

F-MuLV acceleration of myelomonocytic tumorigenesis in SV40 large T antigen transgenic mice is accompanied by retroviral insertion at Fli1 and a novel locus, Fim4.

We describe here the development of a murine system for the identification of genes involved in myelomonocytic neoplasms. Transgenic C57BL/6J mice expressing SV40 early region under a myelomonocytic promoter develop histiocytic sarcomas with a latency of 167 days. We used retroviral proviral tagging to accelerate tumorigenesis and to uncover genetic changes that contribute to tumor development. Infection of transgenic mice with Friend murine leukemia virus (F-MuLV) shortened the latency of morbidity to 103 days (P< 0.001); this was associated with clonal proviral integrations in tumor DNA. As expected for F-MuLV, proviral insertions occurred at Fli1 in both transgenic and nontransgenic tumors. Four insertions were found at a novel locus, termed Fim4, on chromosome 6. This region is syntenic to human 7q32, a region that is commonly deleted in human myelodysplastic syndrome and acute myeloid leukemia. A murine BAC containing Fim4 was sequenced and analyzed, and while there was significant human-mouse homology in the area of the insertions, no candidate gene has been identified. Thus we have established a system to identify genes involved in myelomonocytic tumors, and have used it to identify Fim4, a new common site of proviral insertion. Study of this locus may provide insight into genes involved in AML-associated 7q32 deletions in humans.

The murine cysteinyl leukotriene 2 (CysLT2) receptor. cDNA and genomic cloning, alternative splicing, and in vitro characterization.

Two classes of cysteinyl leukotriene receptor, CysLT(1) and CysLT(2), have been identified and pharmacologically characterized in human tissues. Although the CysLT(1) receptor mediates the proinflammatory effects of leukotrienes in human asthma, the physiological roles of CysLT(2) receptor are not defined, and a suitable mouse model would be useful in delineating function. We report here the molecular cloning and characterization of the mouse CysLT(2) receptor (mCysLT(2)R) from heart tissue. mCysLT(2)R cDNA encodes a protein of 309 amino acids, truncated at both ends compared with the human ortholog (hCysLT(2)R). The gene resides on the central region of mouse chromosome 14 and is composed of 6 exons with the entire coding region located in the last exon. Two 5'-untranslated region splice variants were identified with the short form lacking exon 3 as the predominant transcript. Although the overall expression of mCysLT(2)R is very low, the highest expression was detected in spleen, thymus, and adrenal gland by ribonuclease protection assay, and discrete sites of expression in heart were observed by in situ hybridization. Intracellular calcium mobilization in response to cysteinyl leukotriene administration was detected in human embryonic kidney 293T cells transfected with recombinant mCysLT(2)R with a rank order of potency leukotriene C(4)(LTC(4) ) = LTD(4)>>LTE(4). [(3)H]LTD(4) binding to membranes expressing mCysLT(2)R could be effectively competed by LTC(4) and LTD(4) and only partially inhibited by LTE(4) and BAYu9773. The identification of mCysLT(2)R will be useful for establishing CysLT(2)R-deficient mice and determining novel leukotriene functions.

Nuclear membrane protein LAP2beta mediates transcriptional repression alone and together with its binding partner GCL (germ-cell-less).

LAP2beta is an integral membrane protein of the nuclear envelope involved in chromatin and nuclear architecture. Using the yeast two-hybrid system, we have cloned a novel LAP2beta-binding protein, mGCL, which contains a BTB/POZ domain and is the mouse homologue of the Drosophila germ-cell-less (GCL) protein. In Drosophila embryos, GCL was shown to be essential for germ cell formation and was localized to the nuclear envelope. Here, we show that, in mammalian cells, GCL is co-localized with LAP2beta to the nuclear envelope. Nuclear fractionation studies reveal that mGCL acts as a nuclear matrix component and not as an integral protein of the nuclear envelope. Recently, mGCL was found to interact with the DP3alpha component of the E2F transcription factor. This interaction reduced the transcriptional activity of the E2F-DP heterodimer, probably by anchoring the complex to the nuclear envelope. We demonstrate here that LAP2beta is also capable of reducing the transcriptional activity of the E2F-DP complex and that it is more potent than mGCL in doing so. Co-expression of both LAP2beta and mGCL with the E2F-DP complex resulted in a reduced transcriptional activity equal to that exerted by the pRb protein.

Retroviral integration at the Epi1 locus cooperates with Nf1 gene loss in the progression to acute myeloid leukemia.

Juvenile myelomonocytic leukemia (JMML) is a disease that occurs in young children and is associated with a high mortality rate. In most patients, JMML has a progressive course leading to death by virtue of infection, bleeding, or progression to acute myeloid leukemia (AML). As it is known that children with neurofibromatosis type 1 syndrome have a markedly increased risk of developing JMML, we have previously developed a mouse model of JMML through reconstitution of lethally irradiated mice with hematopoietic stem cells homozygous for a loss-of-function mutation in the Nf1 gene (D. L. Largaespada, C. I. Brannan, N. A. Jenkins, and N. G. Copeland, Nat. Genet. 12:137-143, 1996). In the course of these experiments, we found that all these genetically identical reconstituted mice developed a JMML-like disorder, but only a subset went on to develop more acute disease. This result strongly suggests that additional genetic lesions are responsible for disease progression to AML. Here, we describe the production of a unique tumor panel, created using the BXH-2 genetic background, for identification of these additional genetic lesions. Using this tumor panel, we have identified a locus, Epi1, which maps 30 to 40 kb downstream of the Myb gene and appears to be the most common site of somatic viral integration in BXH-2 mice. Our findings suggest that proviral integrations at Epi1 cooperate with loss of Nf1 to cause AML.

Molecular characterization of the mouse Tem1/endosialin gene regulated by cell density in vitro and expressed in normal tissues in vivo.

Human tumor endothelial marker 1/endosialin (TEM1/endosialin) was recently identified as a novel tumor endothelial cell surface marker potentially involved in angiogenesis, although no specific function for this novel gene has been assigned so far. It was reported to be expressed in tumor endothelium but not in normal endothelium with the exception of perhaps the corpus luteum. Here we describe the cDNA and genomic sequences for the mouse Tem1/endosialin homolog, the identification and characterization of its promoter region, and an extensive characterization of its expression pattern in murine and human tissues and murine cell lines in vitro. The single copy gene that was mapped to chromosome 19 is intronless and encodes a 92-kDa protein that has 77.5% overall homology to the human protein. The remarkable findings are 1) this gene is ubiquitously expressed in normal human and mouse somatic tissues and during development, and 2) its expression at the mRNA level is density-dependent and up-regulated in serum-starved cells. In vitro, its expression is limited to cells of embryonic, endothelial, and preadipocyte origin, suggesting that the wide distribution of its expression in vivo is due to the presence of vascular endothelial cells in all the tissues. The ubiquitous expression in vivo is in contrast to previously reported expression limited to corpus luteum and highly angiogenic tissues such as tumors and wound tissue.

Inhibition of myeloid differentiation by Hoxa9, Hoxb8, and Meis homeobox genes.

OBJECTIVE: The homeobox gene Hoxb8 is activated in the murine myelomonocytic cell line WEHI-3B as a result of intracisternal A particle integration. Cooperative activation between Hoxa9 and Meis1 is induced by retroviral integration in BXH2 murine myeloid leukemias and the myeloid leukemia cell line M1. The present study was conducted to examine possible Meis gene activation and cooperative DNA binding of homeobox proteins in WEHI-3B and to reveal the specific role of Hox and Meis genes in myeloid differentiation. MATERIALS AND METHODS: Northern blot analysis and reverse transcriptase polymerase chain reaction were performed to examine homeobox genes expression. Electrophoretic mobility shift assay was performed to evaluate DNA binding of homeobox proteins. Myeloid differentiation of 32Dcl3 was induced by granulocyte colony-stimulating factor. RESULTS: Meis2 was coactivated with Hoxb8 in WEHI-3B cells. DNA-protein complexes including Hox, Meis, and Pbx were observed in WEHI-3B and 32Dcl3. Expression and the DNA-binding complex of Hoxa9, Hoxb8, Meis1, and Meis2 were down-regulated during myeloid differentiation of 32Dcl3 cells. Enforced expression of Hox or Meis genes inhibited myeloid differentiation of 32Dcl3. CONCLUSION: The results indicate that Meis2 is an important Meis gene for myeloid leukemogenesis and that Hox and Meis are important genes for myeloid leukemogenesis through differentiation block.

Sequence, chromosomal location and expression analysis of the murine homologue of human RAD51L2/RAD51C.

The Rad51 protein has been shown to play a vital role in the DNA repair process. In humans, its interaction with proteins like BRCA1 and BRCA2 has provided an insight into the mechanism of how these molecules function as tumor suppressors. Several members of the Rad51-like family have been recently identified, including RAD51L2. This gene has been found to be amplified in breast tumors suggesting its role in tumor progression. Here, we describe the cloning of the murine homologue of the human RAD51L2/RAD51C gene. Sequence analysis has revealed that the murine Rad51l2 protein is 86% identical and 93% similar to its human homologue. In spite of such high sequence conservation, the murine protein lacks the first nine amino acids present in the human protein. We have cloned and confirmed the sequence of the 5' end of the murine Rad51l2 cDNA using 5' RACE technique as well as by sequencing the genomic region flanking the first exon of the murine Rad51l2 gene. Northern analysis shows that Rad51l2 is expressed in several adult tissues as well as in embryos at various developmental stages. The murine Rad51l2 gene maps to chromosome 11 and is located in the syntenic region of human chromosome 17q22-23, where the human RAD51L2 is present.

Combined histologic and molecular features reveal previously unappreciated subsets of lymphoma in AKXD recombinant inbred mice.

Hematopoietic neoplasms developing in AKXD recombinant inbred, NFS.V(+) and ICSBP knockout mice were assessed using morphologic, cytologic and molecular criteria that relate these disorders to human lymphoma and leukemia. Lymphoma types included precursor T-cell and B-cell lymphoblastic, small lymphocytic, splenic marginal zone, follicular, and diffuse large cell (DLCL). In addition to previously defined subtypes of DLCL composed of centroblasts or immunoblasts, two additional subtypes are defined here: lymphoblastic lymphoma like (LL) and lymphoma characterized by a histiocytic reaction (HS). DLCL(HS) were distinguished from true histiocytic lymphomas by the presence of clonal Ig gene rearrangements.

Non-Hodgkin lymphomas of mice.

Studies of lymphoid neoplasms occurring in normal or genetically engineered mice have revealed parallels and differences to non-Hodgkin lymphomas (NHL) of humans. Some mouse lymphomas have strong histologic similarities to the human NHL subsets including precursor B- and T-cell lymphoblastic, small lymphocytic, splenic marginal zone, and diffuse large-cell B-cell lymphomas (DLCL); whether molecular parallels also exist is under study. Others mouse types such as sIg+ lymphoblastic B-cell lymphoma have no histologic equivalent in human NHL even though they share molecular deregulation of BCL6 with human DLCL. Finally, Burkitt lymphoma does not appear to occur naturally in mice, but it can be induced with appropriately engineered transgenes.

Promoter function of the angiogenic inducer Cyr61gene in transgenic mice: tissue specificity, inducibility during wound healing, and role of the serum response element.

The cysteine-rich angiogenic protein 61 (Cyr61) is an extracellular matrix-associated, heparin-binding protein that mediates cell adhesion, stimulates cell migration, and enhances growth factor-induced cell proliferation. Cyr61 also promotes chondrogenic differentiation and induces neovascularization. In this study, we show that a 2-kb fragment of the Cyr61 promoter, which confers growth factor-inducible expression in cultured fibroblasts, is able to drive accurate expression of the reporter gene lacZ in transgenic mice. Thus, transgene expression was observed in the developing placenta and embryonic cardiovascular, skeletal, and central and peripheral nervous systems. The sites of transgene expression are consistent with those observed of the endogenous Cyr61 gene as determined by in situ hybridization and immunohistochemistry. The transgene expression in the cardiovascular system does not require the serum response element, a promoter sequence essential for transcriptional activation of Cyr61 by serum growth factors in cultured fibroblasts. Because the serum response element contains the CArG box, a sequence element implicated in cardiovascular-specific gene expression, the nonessential nature of this sequence for cardiovascular expression of Cyr61 is unexpected. Furthermore, the Cyr61 promoter-driven lacZ expression is inducible in granulation tissue during wound healing, as is synthesis of the endogenous Cyr61 protein, suggesting a role for Cyr61 in wound healing. Consistent with this finding, purified Cyr61 protein promotes the healing of a wounded fibroblast monolayer in culture. In addition, we mapped the mouse Cyr61 gene to the distal region of chromosome 3. Together, these results define the functional Cyr61 promoter in vivo, and suggest a role of Cyr61 in wound healing through its demonstrated angiogenic activities upon endothelial cells and its chemotactic and growth promoting activities upon fibroblasts.

Activation of the Rap1 guanine nucleotide exchange gene, CalDAG-GEF I, in BXH-2 murine myeloid leukemia.

Here we report the recurrent proviral activation of the Rap1-specific guanine nucleotide exchange factor CalDAG-GEF I (Kawasaki, H., Springett, G. M., Toki, S., Canales, J. J., Harlan, P., Blumenstiel, J. P., Chen, E. J., Bany, I. A., Mochizuki, N., Ashbacher, A., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13278-13283; Correction (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 318) gene in BXH-2 acute myeloid leukemia. We also show that CalDAG-GEF I encodes two protein isoforms, a full-length isoform (CalDAG-GEF Ia) and a C-terminally truncated isoform (CalDAG-GEF Ib). Expression of the full-length CalDAG-GEF Ia isoform in Rat2 fibroblasts enhances growth in low serum, whereas expression in Swiss 3T3 cells causes morphological transformation and increased saturation density. In FDCP1 myeloid cells, CalDAG-GEF Ia expression increases growth and saturation density in the presence of the diacylglycerol analogs phorbol 12-myristate 13-acetate (PMA), which activates CalDAG-GEF Ia exchange activity. Likewise, in 32Dcl3 myeloblast cells, CalDAG-GEF Ia expression increases cell adherence to fibronectin in response to PMA and calcium ionophore and allows higher saturation densities and prolonged growth on fibronectin-coated plates. These effects were correlated with increased Rap1, but not Ras, protein activation following PMA and calcium ionophore treatment. Our results suggest that Rap1-GTP delivers signals that favor progression through the cell cycle and morphological transformation. The identification of CalDAG-GEF I as a proto-oncogene in BXH-2 acute myeloid leukemia is the first evidence implicating Rap1 signaling in myeloid leukemia.

Functional characterization of the gene encoding RLIM, the corepressor of LIM homeodomain factors.

RLIM is a RING H2 zinc finger protein that acts as a negative coregulator for LIM homeodomain transcription factors. We have isolated genomic lambda clones that cover the entire mouse RLIM-encoding Rnf12 gene. The Rnf12 gene encompasses 20 kb and consists of at least five exons and four introns. Several transcriptional start sites within a 24-bp region were mapped around 300 nt upstream of the translational start site. Rnf12-specific mRNA can be detected in many tissues as revealed by Northern blot analysis. Transient cotransfections reveal that the proximal Rnf12 promoter can be activated in vitro by ubiquitously and more restrictively expressed transcription factors, some of which are known mediators of signal transduction pathways, e.g., mammalian Krüppel-like transcription factors, Sox and ets-related proteins, and RBP-J. We isolated a cDNA encoding human RLIM, which is highly conserved with mouse and chick RLIM. By fluorescence in situ hybridization and interspecific backcross analysis, we have localized the Rnf12 gene to the central regions of mouse and human chromosome X.

Nkx3.1, a murine homolog of Ddrosophila bagpipe, regulates epithelial ductal branching and proliferation of the prostate and palatine glands.

Nkx3.1 is a homeobox gene related to Drosophila bagpipe. Nkx3.1 is an early marker of the sclerotome and a subset of vascular smooth muscle cells, and at later stages, this gene is expressed in the prostate, palatine glands, kidney, and restricted regions of the central nervous system. In the present study, we determined the chromosomal localization of Nkx3.1 and examined the function of Nkx3. 1 in vivo by using gene targeting technique. Interestingly, Nkx3.1 mapped to the central region of the mouse chromosome 14 and was linked to Nkx2.6, a murine homolog of Drosophila tinman. Homozygous mutant mice for Nkx3.1 were viable and fertile, and the phenotype was, unexpectedly, confined to the prostate and palatine glands. The homozygous mutant mice exhibited defective branching morphogenesis of the prostate and palatine glands. Moreover, epithelial cells of the mutant prostate and palatine glands showed significant hyperplasia. No abnormalities were detected in the sclerotome, blood vessels, kidney, or brain. These results indicate that Nkx3.1 plays a critical role in epithelial branching and proliferation in the prostate and palatine glands. However, we did not observe prostate cancer in homozygous mutant mice up to 2 years of age. Therefore, involvement of NKX3.1 in carcinogenesis in men needs to be carefully determined by further investigation.

Characterization of the mouse interleukin-13 receptor alpha1 gene.

Interleukin (IL)-13 is a pleiotropic immune regulatory cytokine that shares structural and biological characteristics with IL-4. The receptor for IL-13 is comprised of the IL-4 receptor alpha (IL-4Ralpha) subunit and a low-affinity IL-13-binding subunit, IL-13Ralpha1. An additional receptor, IL-13Ralpha2, binds to IL-13 with high affinity, but lacks the cytoplasmic domain for signaling. In this study, we isolated the mouse IL-13Ralpha1 gene (Il13ra1) of approximately 56 kb that spans the entire coding region. The mouse Il13ra1 gene is composed of 11 exons, and shows striking similarity in genomic structure to the previously reported class I cytokine receptor genes. Motifs characteristic of the cytokine receptor family are similarly organized on the genome, including conserved cysteines, a WSxWS motif, and Box1, indicating closely related genetic evolution of the cytokine receptor superfamily. Alternative mRNA splicings were demonstrated to generate variant transcripts that encode soluble IL-13Ralpha1. The mouse Ill13ra1 gene was mapped to the proximal region of the mouse X chromosome, and was closely linked to the DXPas3 locus by interspecific backcross analysis. Il13ra1 mRNA was co-expressed with I14ra mRNA in mouse myeloid and natural killer cells on which IL-13 has been known to act, whereas the Il13ra2 mRNA was not detected in these cells, indicating that IL-13Ralpha1 is the major component of the IL-13 receptor complex in lymphohematopoietic cells.

The p53-inducible gene EI24/PIG8 localizes to human chromosome 11q23 and the proximal region of mouse chromosome 9.

Activation of the p53 tumor suppressor leads to either a cell cycle arrest or to apoptosis and the factors that influence these responses are poorly understood. It is clear, however, that p53 regulates these processes by inducing a series of downstream target genes. One recently identified p53-target gene, EI24 (alias PIG8), induces apoptosis when ectopically expressed. To better understand the biological properties of EI24 and its potential relevance to disease, in particular cancer, we determined the chromosomal location and pattern of gene expression of EI24. EI24 is widely expressed in adult tissues and throughout mouse embryogenesis. The genomic locus of EI24 was mapped to the proximal region of mouse chromosome 9 and human chromosome 11q23-->q24, a region frequently altered in human cancers. These results suggest that EI24 may play an important role in the p53 tumor suppressor pathway.

Cloning of the murine Drm gene (Cktsf1b1) and characterization of its oncogene suppressible promoter.

The Drm gene, first identified in rat cells in our laboratory, appears to play a significant role in early embryo patterning and limb bud development. We have now isolated mouse Drm (mDrm) cDNA as well as genomic DNA clones and have mapped the Drm gene (Cktsf1b1) to murine chromosome 2. Cktsf1b1 is regulated in a tissue specific fashion and is expressed only in nontransformed mouse cells or primary fibroblasts in culture, but not in established transformed or tumor-derived mouse cell lines. The major transcription start sites map to within 69 bp upstream of the initiating ATG. A promoter was contained in the -214 to +1 bp 5' flanking region, and promoter/reporter constructs showed 10-fold higher activity than control in REF-1 (rat), A31 (mouse) and CHO (hamster) cells. The region contains a TATA sequence and multiple potential transcription factor binding sites. Promoter activity was dose-dependently inhibited by cotransfection with either ras or mos oncogenes, but oncogene inhibition was reversed and the overall activity increased when cells were treated with the MAP kinase kinase (MKK) inhibitor PD98059. An NF-1 and Yi-like site, identified in the minimal promoter region, showed different mobility shift patterns when normal and transformed cell nuclear extracts are compared. Mutation of the NF-1 site reduced Cktsf1b1 promoter activity 25%, while mutation of the Yi-like site destroyed all the activity. Our results indicate that the expression of Cktsf1b1, a gene associated with early development and cell transformation, is sensitive to MKK levels and may be regulated via multiple transcription factor complexes.

Detection of integrated murine leukemia viruses in a mouse model of acute myeloid leukemia by fluorescence in situ hybridization combined with tyramide signal amplification.

This study was undertaken to develop a reliable method to enumerate and map somatically acquired, clonal, murine leukemia virus (MuLV) proviral insertions in acute myeloid leukemia (AML) cells from the BXH-2 mouse strain. This was achieved by using fluorescence in situ hybridization combined with tyramide signal amplification (FISH-TSA) and an 8.8 kilobase pair (kb) full-length ecotropic MuLV or 2.0 kb MuLV envelope (env) gene probe. Two-color FISH was utilized combining chromosome-specific probes for regions near the telomere and/or centromere and the MuLV probes. The technique reliably detected germline and somatically acquired, tumor-specific, MuLV proviruses in BXH-2 AML cell lines. It was possible to readily verify homozygous insertions at endogenous ecotropic MuLV loci, Emv1 (chromosome 5), Emv2 (chromosome 8) and a BXH-2 strain-specific locus (chromosome 11). This strategy also verified the presence of molecularly cloned proviral insertions within the mouse Nf1 gene and another locus on distal chromosome 11, as well as on chromosome 7 and chromosome 9 in BXH-2 AML cell line B117. The technique was also used to detect several new tumor-specific, proviral insertions in BXH-2 AML cell lines.