Showing papers by "Bruce W. Birren published in 1996"

Disruption of the nuclear hormone receptor RORα in staggerer mice

Abstract: Homozygous staggerer (sg) mice show a characteristic severe cerebellar ataxia due to a cell-autonomous defect in the development of Purkinje cells. These cells show immature morphology, synaptic arrangement, biochemical properties and gene expression, and are reduced in numbers. In addition, sg heterozygotes show accelerated dendritic atrophy and cell loss, suggesting that sg has a role in mature Purkinje cells. Effects of this mutation on cerebellar development have been studied for 25 years, but its molecular basis has remained unknown. We have genetically mapped staggerer to an interval of 160 kilobases on mouse chromosome 9 which was found to contain the gene encoding RORalpha, a member of the nuclear hormone-receptor superfamily. Staggerer mice were found to carry a deletion within the RORalpha gene that prevents translation of the ligand-binding homology domain. We propose a model based on these results, in which RORalpha interacts with the thyroid hormone signalling pathway to induce Purkinje-cell maturation.

A bacterial artificial chromosome-based framework contig map of human chromosome 22q

Abstract: We have constructed a physical map of human chromosome 22q using bacterial artificial chromosome (BAC) clones. The map consists of 613 chromosome 22-specific BAC clones that have been localized and assembled into contigs using 452 landmarks, 346 of which were previously ordered and mapped to specific regions of the q arm of the chromosome by means of chromosome 22-specific yeast artificial chromosome clones. The BAC-based map provides immediate access to clones that are stable and convenient for direct genome analysis. The approach to rapidly developing marker-specific BAC contigs is relatively straightforward and can be extended to generate scaffold BAC contig maps of the rest of the chromosomes. These contigs will provide substrates for sequencing the entire human genome. We discuss how to efficiently close contig gaps using the end sequences of BAC clone inserts.

Molecular linkage of the mouse CD5 and CD6 genes.

Abstract: CD5 and CD6 are both lymphocyte cell surface glycoproteins that appear to play a role in T-cell activation. In humans, CD6 is a 105000/130000 Mr monomer expressed at high levels by peripheral blood T cells and medullary thymocytes, and also by a small fraction of peripheral blood B cells (Kamoun et al. 1981; Reinherz et al. 1982a, b; Morimoto et al. 1988; Endres et al. 1989; Swack et al. 1989; Cardenas et al. 1990; Aruffo et al. 1991; Swack et al. 1991). The mouse homologue has recently been shown to be a 130000Mr glycoprotein expressed on the surface of thymocytes and T cells in lymph node and spleen (Robinson et al. 1995). Monoclonal antibody (mAb) crosslinking studies have shown that CD6 ligation in combination with costimulatory signals provided by accessory cells, PMA or anti-CD2/CD3 mAbs can induce proliferation of human T cells (Walker et al. 1987; Morimoto et al. 1988; Gangemi et al. 1989; Swack et al. 1989; Bott et al. 1993; Osorio et al. 1994). CD5 is a 67000Mr monomeric glycoprotein expressed on all mature T cells, most thymocytes, and a subset of mature B cells (Ledbetter et al. 1980; Wang et al. 1980; Caligaris-Cappio et al. 1982; Hayakawa et al. 1985; Jones et al. 1986; Huang et al. 1987a). Like CD6, CD5 also seems to play an important role in T-cell activation both in humans and mice, as crosslinking of CD5 by mAbs enhances T-cell proliferation either in a monocyte-dependent manner or in response to mitogens, alloantigens, or CD3-specific mAbs (Hollander et al. 1981; Ledbetter et al. 1985; Ceuppens and Baroja 1986; Nishimura et al. 1988; Spertini et al. 1991; Vandenberghe and Ceuppens 1991; Alberola-Ila et al. 1992). Furthermore, CD5 is physically associated with the Tcr/CD3 complex on T cells (Burgess et al. 1992; Osman et al. 1992). In addition to their similar tissue distribution and costimulatory activity in T cells, CD5 and CD6 are also structurally related molecules: they both belong to the scavenger receptor cysteine rich (SRCR) protein superfamily defined by the cysteine-rich domain of the type I macrophage scavenger receptor (Freeman et al. 1990; Aruffo et al. 1991, Robinson et al. 1995; Whitney et al. 1995). This domain includes a 100 amino acid stretch with six positionally conserved cysteine residues (Freeman et al. 1990). CD5 and CD6 are particularly close homologues, since they are so far the only members of this family possessing three SRCR domains, and they additionally share two cysteine residues and 31 conserved residues in their SRCR domains that are not found in other members of the SRCR family (Aruffo et al. 1991). Furthermore, the human CD5 and CD6 genes have both been mapped to chromosome 11 (Tsuge et al. 1985; Hecht et al. 1989). In mouse, theCD5 gene has been mapped to chromosome 19 (Tada et al. 1982). Because the CD5 and CD6 genes are related and map to the same chromosome in human, the present study was undertaken to look for a possible linkage between the mouse CD5 andCD6 genes. To address this question, we performed pulse field gel electrophoresis (PFGE) experiments using DNA from the mouse CD4+ T-cell line C6VL (obtained from J. Allison, University of Berkeley, CA), which expresses both CD5 ndCD6. Cells were grown in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 1 mM sodium pyruvate, 10 mM Hepes (Applied Scientific, San Francisco, CA), penicillin (100 units/ml), streptomycin (100 μg/ml; Flow Laboratories, McLean, VA), and 10% heat-inactivated fetal calf serum (Gemini Bio Products, Calabasas, CA). Cells were washed twice and resuspended in 1 × PBS at a concentration of 2× 107 cells/ml, and mixed with an equal volume of 1.5% low-melting temperature agarose (InCert Agarose; FMC BioProducts, Rockland, ME) prepared in 20 mM Tris pH 7.5/50 mM ethylenediaminetetraacetate (EDTA). The agarose/cell plugs were then incuO. Lecomte? J. B. Bock? J. R. Parnes ( ) Department of Medicine, Division of Immunology and Rheumatology, MSLS Building, Room P306, Stanford University School of Medicine, Stanford, CA 94305-5487, USA

Correction: Disruption of the nuclear hormone receptor RORα in staggerer mice

Abstract: Nature 379, 736-739 (1996) THE labelling of Fig. 3a and b is incorrect: rostral is to the upper left and the fourth ventricle is seen as a triangular space above and to the right of the cerebellum. The RORotl cDNA sequence has been submitted to GenBank, accession number U53228.

Pulsed-field gel electrophoresis.

Abstract: Publisher Summary Pulsed-field gel electrophoresis (PFGE) of agarose gels enables the reproducible separation of large DNA fragments. In concept, PFGE is an extension of conventional electrophoresis, in which two alternating (or pulsed) electric fields are used instead of the traditional single static field. Separation occurs when these fields are oriented at an obtuse angle to one another. In a pulsed-field gel, the end of each molecule migrates in a new direction, with each change of the electric fields. The DNA molecules, thus, migrate through the agarose matrix in a zigzag motion. The tardiness of the larger molecules, in turning corners (e.g., in PFGE) or in running forward and backward [e.g., in field-inversin gel electrophoresis (FIGE)], separates them from the smaller size fragments. The effectiveness of PFGE, however, is not limited to the separation of very large DNA molecules. PFGE can improve the resolution of DNA molecules of only a few hundred bases and permits separation up to 12,000 kilobase pairs (kb). A number of models and theories have been proposed to explain some of the more complex behavior of DNA molecules in PFGE. However, biologists rarely need to consult these physical models or equations for practical PFGE applications. This chapter discusses the optimum PFG electrophoretic conditions for the separation of DNA fragments from 1 to 6000 kb.