The MN1-TEL fusion protein, encoded by the translocation (12;22)(p13;q11) in myeloid leukemia, is a transcription factor with transforming activity.

The Tel gene (or ETV6) is the target of the translocation (12;22)(p13;q11) in myeloid leukemia. TEL is a member of the ETS family of transcription factors and contains the pointed protein interaction (PNT) domain and an ETS DNA binding domain (DBD). By contrast to other chimeric proteins that contain TEL's PNT domain, such as TEL-platelet-derived growth factor beta receptor in t(5;12)(q33;p13), MN1-TEL contains the DBD of TEL. The N-terminal MN1 moiety is rich in proline residues and contains two polyglutamine stretches, suggesting that MN1-TEL may act as a deregulated transcription factor. We now show that MN1-TEL type I, unlike TEL and MN1, transforms NIH 3T3 cells. The transforming potential depends on both N-terminal MN1 sequences and a functional TEL DBD. Furthermore, we demonstrate that MN1 has transcription activity and that MN1-TEL acts as a chimeric transcription factor on the Moloney sarcoma virus long terminal repeat and a synthetic promoter containing TEL binding sites. The transactivating capacity of MN1-TEL depended on both the DBD of TEL and sequences in MN1. MN1-TEL contributes to leukemogenesis by a mechanism distinct from that of other chimeric proteins containing TEL.

Cloning and characterization of MN1, a gene from chromosome 22q11, which is disrupted by a balanced translocation in a meningioma.

We have isolated a gene, called MN1, which resides on chromosome 22 and which was found to be disrupted by a balanced translocation (4;22) in meningioma 32. The MN1 gene spans about 70 kb and consists of at least two large exons of approximately 4.7 kb and 2.8 kb. The MN1 cDNA codes for a protein of 1319 amino acids when the first methionine in the open reading frame is used. The MN1 cDNA contains two CAG repeats, one of which codes for a string of 28 glutamines. The t(4;22) disrupts the 5'-exon within the open reading frame. In meningioma 32 no expression of the MN1 mRNA is observed. These results suggest that inactivation of the MN1 gene in this tumour may contribute to its pathogenesis.

Plasmid rescue from transgenic mouse DNA using LacI repressor protein conjugated to magnetic beads.

A method for the efficient rescue of lac operator containing plasmids from transgenic mouse genomic DNA is described. The method is based on the high affinity of the LacI repressor protein for the lac operator sequence. Using the LacI repressor protein conjugated to magnetic beads, more than 95% of plasmid sequences could be purified from restriction enzyme digested genomic DNA. After circularization, the plasmids were introduced into Escherichia coli by means of electroporation. Since the plasmid was cloned into a bacteriophage lambda vector, the efficiency of plasmid rescue could easily be compared with in vitro packaging. Our results indicate that plasmid rescue is about 25 times more efficient. Application of this method should be especially useful with transgenic mouse models harboring LacZ plasmid shuttle vectors for studying spontaneous or induced mutations in vivo.

Expression of the neurofibromatosis type 2 gene in human tissues.

The neurofibromatosis Type 2 tumor suppressor gene is implicated in the hereditary tumor syndrome NF2, hallmarked by bilateral vestibular schwannomas, meningiomas, and ocular non-neoplastic features. The gene product has characteristics of a membrane cytoskeleton-linking protein but the mechanism of tumor suppression by the NF2 protein remains to be elucidated. The NF2 gene is widely expressed in mouse and rat tissues. In humans, most of the expression data have accumulated through Northern blot analysis, RT-PCR and, more recently, Western blot analysis, providing information on whole tissues and organs rather than on specific cell types. We report here an extensive survey of NF2 gene expression in human tissues using a combination of mRNA in situ hybridization (mRNA ISH) and immunohistochemistry (IH) with a panel of monoclonal antibodies (MAbs) supplemented by tissue immunoprecipitation experiments with affinity-purified polyclonal antibodies. Expression was observed in many different cell types, most of which appear functionally normal in individuals affected by NF2. Surprisingly, expression could not be consistently documented in Schwann cells and arachnoidal cells by IH or by mRNA ISH in formalin-fixed tissue. However, consistent immunostaining of Schwann cells was seen in frozen sections. (J Histochem Cytochem 47:1471-1479, 1999)

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins.

Genome scanning of breast cancers by two-dimensional DNA typing.

We have recently used two-dimensional DNA typing to detect genetic alterations in breast tumours. This method, which is based on size separation in neutral gels and sequence separation in denaturing gradient gels followed by hybridisation analysis with mini- and microsatellite core probes, allows the simultaneous analysis of hundreds of allelic fragments in a very short time. Here we demonstrate the potency of this method for total genome scanning of the tumour genome by analysing a small series of breast cancers. Comparison of tumour and normal DNA from ten breast cancer patients, using two-dimensional DNA typing with four core probes, revealed a considerable number of genomic alterations. In contrast, with Southern blot analysis only a few alterations were observed using the same probes. Most of the changes observed (74%) were deletions (absence of spots in the tumour) while 20% corresponded to amplifications (spots of higher intensity in the tumour) and 5% were new spots (gains). About 10% of the genomic changes detected appeared to occur in the tumours of more than one patient.