DNA sequences have been located at the fragile X site by in situ hybridization and by the mapping of breakpoints in two somatic cell hybrids that were constructed to break at the fragile site. These hybrids were found to have breakpoints in a common 5-kilobase Eco RI restriction fragment. When this fragment was used as a probe on the chromosomal DNA of normal and fragile X genotype individuals, alterations in the mobility of the sequences detected by the probe were found only in fragile X genotype DNA. These sequences were of an increased size in all fragile X individuals and varied within families, indicating that the region was unstable. This probe provides a means with which to analyze fragile X pedigrees and is a diagnostic reagent for the fragile X genotype.

The sequence of a Pst I restriction fragment was determined that demonstrate instability in fragile X syndrome pedigrees. The region of instability was localized to a trinucleotide repeat p(CCG)n. The sequence flanking this repeat were identical in normal and affected individuals. The breakpoints in two somatic cell hybrids constructed to break at the fragile site also mapped to this repeat sequence. The repeat exhibits instability both when cloned in a nonhomologous host and after amplification by the polymerase chain reaction. These results suggest variation in the trinucleotide repeat copy number as the molecular basis for the instability and possibly the fragile site. This would account for the observed properties of this region in vivo and in vitro.

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Fluorescence in situ hybridization of a tile path of DNA subclones has previously enabled the cyto-genetic definition of the minimal DNA sequence which spans the FRA16D common chromosomal fragile site, located at 16q23.2. Homozygous deletion of the FRA16D locus has been reported in adenocarcinomas of stomach, colon, lung and ovary. We have sequenced the 270 kb containing the FRA16D fragile site and the minimal homozygously deleted region in tumour cells. This sequence enabled localization of some of the tumour cell breakpoints to regions which contain AT-rich secondary structures similar to those associated with the FRA10B and FRA16B rare fragile sites. The FRA16D DNA sequence also led to the identification of an alternatively spliced gene, named FOR (fragile site FRA16D oxidoreductase), exons of which span both the fragile site and the minimal region of homozygous deletion. In addition, the complete DNA sequence of the FRA16D-containing FOR intron reveals no evidence of additional authentic transcripts. Alternatively spliced FOR transcripts (FOR I, FOR II and FOR III) encode proteins which share N-terminal WW domains and differ at their C-terminus, with FOR III having a truncated oxidoreductase domain. FRA16D-associated deletions selectively affect the FOR gene transcripts. Three out of five previously mapped translocation breakpoints in multiple myeloma are also located within the FOR gene. FOR is therefore the principle genetic target for DNA instability at 16q23.2 and perturbation of FOR function is likely to contribute to the biological consequences of DNA instability at FRA16D in cancer cells.

Crosslinking of immunoreceptor tyrosinebased activation motif (ITAM)-containing receptor complexes on a variety of cells leads to their activation through the sequential triggering of protein tyrosine kinases. Recently, DAP12 has been identified as an ITAM-bearing signaling molecule that is noncovalently associated with activating isoforms of MHC class I receptors on natural killer cells. In addition to natural killer cells, DAP12 is expressed in peripheral blood monocytes, macrophages, and dendritic cells, suggesting association with other receptors present in these cell types. In the present study, we report the molecular cloning of the myeloid DAP12-associating lectin-1 (MDL-1), a DAP12-associating membrane receptor expressed exclusively in monocytes and macrophages. MDL-1 is a type II transmembrane protein belonging to the C type lectin superfamily and contains a charged residue in the transmembrane region that enables it to pair with DAP12. Crosslinking of MDL-1͞DAP12 complexes in J774 mouse macrophage cells resulted in calcium mobilization. These findings suggest that signaling via MDL-1͞DAP12 complexes may constitute a significant activation pathway in myeloid cells.Crosslinking of immune receptors, such as the B or T cell receptor (BCR or TCR) or Fc receptors, on a variety of cells leads to their activation through the sequential activation of protein tyrosine kinases (PTKs) (1). These receptor complexes have in common that ligand binding and signal transduction occur in distinct receptor subunits. In their signal-transducing subunits (e.g., CD3 in the TCR), these complexes contain one or more immunoreceptor tyrosine-based activation motifs (ITAMs). Upon engagement of the ligand-binding subunit, the cytoplasmic ITAMs in the signaling subunits are tyrosinephosphorylated by src-family PTKs. This leads to the recruitment of syk-family PTKs, which, in turn, triggers a cascade of intracellular phosphorylation that results in cellular activation.A number of receptor complexes that recognize MHC class I molecules have been identified on natural killer (NK) cells (2-5). Although many of these receptors possess immunoreceptor tyrosine-based inhibition motifs (ITIMs) that recruit protein tyrosine phosphatases and inhibit NK cell activation, several of these MHC class I receptors lack intrinsic signaling sequences. These receptors express a charged residue in their transmembrane domain, suggesting the association with an adapter molecule that is capable of signaling. Recently, we identified DAP12, a type I glycoprotein containing an ITAM (6). DAP12 is expressed at the cell surface of NK cells noncovalently associated with the human KIR2DS receptor for HLA-C (6, 7), the mouse Ly49D and Ly49H receptors (8, 9), and the human heterodimer CD94͞NKG2C receptor complex recognizing HLA-E (10).In addition to NK cells, DAP12 is expressed in peripheral blood granulocytes, monocytes, and dendritic cells, suggesting association with other receptors present in these cell types. In the present study, we report the cloning ...

4-1BB was originally described as a cDNA expressed by activated murine T cells and subsequently demonstrated to encode a member of the tumor necrosis factor receptor family of integral membrane proteins. Recently, we identified and cloned a murine ligand for 4-1BB (mu4-1BB-L) and demonstrated it to be a member of an emerging family of ligands with structural homology to tumor necrosis factor. To characterize further the role of 4-1BB in the immune response we undertook to clone the human homologue of 4-1BB-L. However, attempts to isolate a cDNA encoding the human 4-1BB-L by cross-hybridization with the murine cDNA were unsuccessful. Therefore we first utilized cross-species hybridization to isolate a cDNA encoding human 4-1BB (hu4-1BB). A fusion protein consisting of the extracellular portion of hu4-1BB coupled to the Fc region of human immunoglobulin G1 (hu4-1BB.Fc) was then used to identify and clone a gene for human 4-1BB-L from an activated CD4+ T cell clone using a direct expression cloning strategy. Human 4-1BB-L shows 36% amino acid identity with its murine counterpart and maps to chromosome 19p13.3. Scatchard analysis demonstrated high-affinity binding of hu4-1BB.Fc to either native or recombinant human 4-1BB-L. Both monoclonal antibody to hu4-1BB and cells transfected with hu4-1BB-L induced a strong proliferative response in mitogen co-stimulated primary T cells. In contrast, ligation of 4-1BB on T cell clones enhanced activation-induced cell death when triggered by engagement of the TCR/CD3 complex.

A ligand was cloned for murine OX40, a member of the TNF receptor family, using a T cell lymphoma cDNA library. The ligand (muOX40L) is a type II membrane protein with significant identity to human gp34 (gp34), a protein whose expression on HTLV‐1‐infected human leukemic T cells is regulated by the tax gene. The predicted structures of muOX40L and gp34 are similar to, but more compact than, those of other ligands of the TNF family. Mapping of the muOX40L gene revealed tight linkage to gld, the FasL gene, on chromosome 1. gp34 maps to a homologous region in the human genome, 1q25. cDNAs for human OX40 receptor were cloned by cross‐hybridization with muOX40, and gp34 was found to bind the expressed human receptor. Lymphoid expression of muOX40L was detected on activated T cells, with higher levels found on CD4+ rather than CD8+ cells. The cell‐bound recombinant ligands are biologically active, co‐stimulating T cell proliferation and cytokine production. Strong induction of IL‐4 secretion by muOX40L suggests that this ligand may play a role in regulating immune responses. In addition, the HTLV‐1 regulation of gp34 suggests a possible connection between virally induced pathogenesis and the OX40 system.

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