PHYSIOLOGY AND METABOLISM OF VITAMIN D

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date4/1/797/31/05

ASJC

  • Medicine(all)