The long-term objective of the proposed study is to relate gene structure with protein structure and function to elucidate the molecular organization of human thrombospondin. Methodologies which have been made possible by recombinant DNA technology will be used to complement the existing thrombospondin project which utilizes a traditional protein biochemical approach. It is anticipated that this combined approach will help to identify the functions of thrombospondin in the formation, maintenance and repair of tissues. Specific focus in this proposal will be directed toward the following areas: (1) The Structure of the Thrombospondin Gene.
The specific aims of this section are (1) to determine the chromosomal location of the thrombospondin gene, (2) to clone the thrombospondin gene from human chromosome-specific libraries, (3) to determine the nucleotide sequence of the promotor region of the thrombospondin gene and (4) to determine the position of exon-intron boundaries. Determination of the location of the thrombospondin gene may suggest an association of thrombospondin with properties and disease states which have been mapped to the same chromosomal location. Structural analysis of the thrombospondin gene will permit the identification of regulatory elements and will provide another basis for identification of repeating and homologous sequences. (2) Functional Organization of the Thrombospondin Molecule. Through interactions with proteins, carbohydrates and cell surfaces, thrombospondin can participate in cell-to-cell and cell- to-matrix interactions and can modulate fibrin clot structure and dissolution.
A specific aim of the proposed study is to locate the functional domains for these interactions through the production of (1) fusion proteins which retain the functional activity of the native molecule, (2) polyclonal antibodies to the fusion proteins which inhibit specific functions of the native molecule and (3) variant forms of thrombospondin in which specific sites or structural regions have been mutated or deleted. Variant forms of thrombospondin will be expressed in cultured murine cell lines by retroviral-mediated gene transfer. (3) Production of Thrombospondin during Tissue Synthesis and Repair. Recent data on the production of thrombospondin by endothelial and smooth muscle cells in culture have led to the hypothesis that thrombospondin is involved in normal morphogenic processes and the cell's response to injury.
A specific aim of the proposed study is to test this hypothesis. The temporal expression and matrix deposition of thrombospondin in the tissues of the developing mouse embryo and in granulation tissue will be determined by in situ hybridization and immunofluorescence.
| Oquendo, P; Hundt, E; Lawler, J et al. (1989) CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes. Cell 58:95-101 |
