The objectives of this grant continuation are: 1) To define the regulatory sequences in the gene of the bovine 9-kilodalton vitamin D-dependent calcium-binding protein (9-kD CaBP), 2) To sequence the human 9-kD CaBP gene and determine its tissue specific expression and, 3) To determine the amino-acid sequences that are critical to the binding of calcium (Ca) in the 9-kD CaBP and the related 28-kD Ca binding protein. We hypothesize that: 1) Because the synthesis of CaBP is under transcriptional control, it is likely that 1,25(OH)2D3-regulated promoter or enhancer elements are present proximal (5') to the transcription start site or in the gene itself; 2) The structure of the 9-kD CaBP gene is conserved through evolution, suggesting an important functional role for certain substructures in the gene; 3) The tissue specific expression of human 9-kD CaBP is similar to that found in other species; 4) Ca binding by the protein is mediated by critical residues or regions in the protein.
The specific aims of our proposal are as follows: 1. a) To sequence the bovine 9-kD CaBP gene and, b) to identify promoter and/or enhancer motifs adjacent to or in the gene of the 9-kD CaBP that are regulated by 1,25(OH)2D3. 2. a) To isolate and determine the sequence of the human 9- kD CaBP gene. b) Examine various tissues for the expression of the mRNA for this protein. 3. a) To examine the Ca and fluorescent lanthanide binding properties of full-length recombinant 28-kD CaBP and its mutants and to b) to express full-length 9-kD CaBP and its mutants in order to examine the Ca and fluorescent lanthanide binding properties of the proteins. Our methods will include the sequencing of genomic clones specific for the bovine and human 9-kD CaBP, construction and testing of reporter plasmids with appropriate putative promoter regions of the gene(s) and the synthesis and expression of appropriate plasmids containing the cDNAs for the 9-kD and 28-kD CaBP. The expressed products will be tested for Ca and lanthanide binding. An understanding of the mechanism(s) governing the synthesis of CaBP (structure of the 1,25(OH)2D3-regulated promoter and its function in the presence or absence of receptor) and the stereochemistry of Ca2+ binding by the protein which binds calcium are important in our understanding of calcium transport in man and experimental animals.
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