Putative amino acid sequence of chick calcium-binding protein deduced from a complementary DNA sequence

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Vitamin D plays a crucial role in calcium homeostasis, primarily through its effects on intestinal calcium absorption.
• Calcium-binding proteins (CaBP) are essential mediators of vitamin D's action in various tissues, including the intestine.
• Understanding the genetic and molecular basis of CaBP is vital for comprehending calcium metabolism and related disorders.

Purpose of the Study:

• To isolate and sequence DNA fragments encoding chick Calcium-binding protein (CaBP).
• To characterize the mRNA structure and identify key functional regions of chick CaBP.
• To investigate the evolutionary conservation of Ca-binding sites within CaBP.

Main Methods:

• cDNA synthesis from enriched chick intestinal mRNA.
• Cloning of cDNA into pUC12 and screening using differential hybridization with vitamin D-dependent probes.
• Hybrid-arrested and hybrid-selected translation assays to confirm mRNA coding for immunoprecipitable CaBP.

Main Results:

• Two recombinant clones with high affinity for vitamin D-replete mRNA probes were identified.
• The mRNA for chick CaBP consists of a G,C-rich 5' untranslated region, a 786 bp coding sequence, and a 1250 nucleotide 3' untranslated region.
• Identified Ca-binding sites exhibit significant homology with chick calmodulin and rat intestinal CaBP, suggesting evolutionary conservation.

Conclusions:

• The study successfully isolated and sequenced chick CaBP DNA fragments, providing a molecular basis for its function.
• The deduced amino acid sequence contains five Ca-binding sites, but its size suggests post-translational modification is necessary for the native protein.
• The conserved nature of Ca-binding sites highlights their importance in protein function across species.