Biomedical subjects

D I Cohen

Publications and source records attributed to D I Cohen.

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PubMedDate not supplied

Isolation of a cDNA clone corresponding to an X-linked gene family (XLR) closely linked to the murine immunodeficiency disorder xid.

The striking number of human and murine immunodeficiency disorders which map to the X chromosome suggests that genes localized on this chromosome must have important roles in lymphocyte development. At least seven distinct disorders in the human and two in the mouse disrupt lymphocyte maturation, particularly that of B cells, at characteristic stages. As functional genes mapping to the X chromosome in one mammal are found on the X chromosome in all other mammals, the same genes regulating lymphocyte development are expected to be found on the X chromosome in mouse and man. Investigations into the possible mechanisms of these X-linked disorders have been hampered by the lack of molecular probes for the genes or gene products affected; because of this, and the possibility of correlating one or more of the several hundred B- or T-cell-specific genes with a specific mutation, we surveyed 15 different B- and T-cell-specific cDNA clones for localization to the X chromosome. We report here the characterization of one of these murine cDNA clones, which hybridizes with a large, X-linked gene family, designated XLR (X-linked, lymphocyte-regulated). We show that the XLR gene family is closely linked to the X-linked immunodeficiency described in the CBA/N mouse strain (xid), by restriction fragment length polymorphism (RFLP) analysis of DNA from mice congeneic for xid. This finding, together with data on the expression of the XLR locus in B cells, indicates that this gene family either includes the locus defined by the xid mutation or is adjacent to it in a gene complex which may be important in lymphocyte differentiation.

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PubMedDate not supplied

Expression of an X-linked gene family (XLR) in late-stage B cells and its alteration by the xid mutation.

One of the most extensively studied X-linked immunodeficiency disorder is the xid mutation of the mouse strain CBA/N. This mutation may involve a maturational defect as xid animals are unable to raise antibodies to soluble polysaccharide antigens, a function normally attributed to late-stage B cells. Moreover, studies using monoclonal antibodies have defined a B-cell surface antigen (BLA-2 or 14G8) that is expressed on most or all immature B lymphocytes, but not on a subpopulation of mature splenic B lymphocytes; this late-stage, 14G8 antigen-negative splenic B-cell subpopulation is apparently absent from mice bearing the xid defect. In the accompanying paper we describe the isolation of a cDNA clone recognizing a family of genes on the X chromosome, at least some of whose members are closely linked to the xid trait. We report here that this gene family, XLR, is transcribed in certain B- and T-cell lineage tumours, but not in macrophage tumours, or liver or kidney cells. We show that it is transcribed principally in late-stage, 14G8-negative B-cell tumours and plasmacytomas, but not in immature B-cell or pre-B-cell tumours. We are able to detect transcription in all of 12 plasmacytomas (secretory B-cell tumours) derived from mice with normal X chromosomes, but not in three plasmacytomas carrying the xid mutation. These data, combined with the restriction fragment length polymorphism analysis linking the XLR gene family to the xid mutation, suggests that the xid defect occurs within a member of this gene family.

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PubMedDate not supplied

Highly specific separation of heterogeneous cell populations by lectin-coated beads: application for the isolation of inner medullary collecting duct cells.

Conditions for the highly specific selection of a cell type by the use of lectin-coated magnetic beads are reported for the isolation of inner medullary collecting duct (IMCD) cells from a heterogeneous inner medullary cell suspension, containing both single cells and tubular fragments of variable size. The lectin Dolichos Biflorus Agglutinin (DBA), which binds in rat inner medulla exclusively to IMCD cells, was coupled via the avidin-biotin system to beads. By isolating DBA-bead-IMCD cells in a magnetic field (positive selection) from a suspension containing about 50% IMCD, a fraction of 98 +/- 1% purity was obtained; recovery of cells was up to 90%. Suspensions negative on reverse-transcriptase polymerase chain reaction for vimentin as a marker of contaminating interstitial and vascular cells could be received by repeating this procedure and additional trypsinization. On the other hand, it was possible to reduce the portion of IMCD cells in the suspension by one isolation step to 1.5 +/- 0.9% (negative selection). Performing this step twice resulted in virtually pure suspensions. No significant effects of this isolation technique on cell viability, growth characteristics, and biochemical parameters were observed. Therefore, this method appears to be a powerful tool for the highly specific separation of heterogeneous cell populations.

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