This chapter describes the procedures that make use of the special properties of this biological system to isolate, clone, and characterize a specific 9-kDa CaBP cDNA. Cholecalcin, or calbindin, a vitamin D-induced calcium-binding protein (CaBP), is one of a group of intracellular proteins that bind calcium with affinity constant Ka = l06 M-1. The chick produces a single Mr 28,000 CaBP (28-kDa CaBP) whereas mammals possess both the Mr 28,000 molecule and a smaller Mr 9,000 protein (9-kDa CaBP). Five micrograms poly(A)+ RNA fraction sedimenting at 7.5 S and enriched in 9-kDa CaBP was used to synthesize the first cDNA strand. The mRNA template was then hydrolyzed and the single-stranded DNA was precipitated with ethanol. The radioactivity of the dried DNA pellet was used as a measure of total dCMP incorporation assuming that for the four nucleotides, radioactivity was incorporated in equivalent amounts. The total quantity of the synthesized first-strand cDNA of the double-strand cDNA was obtained. Plasmid DNA was prepared from the 24 colonies showing the highest signals. They were further analyzed by a hybridization translation-arrest assay. The cDNA-hybridized mRNA, isolated from rat duodenum, directs the cell-free synthesis of two proteins. A major protein comigrates with 125I-labeled purified 9-kDa CaBP whereas a minor product corresponds to a protein that is larger by 2000 Da.
ASJC Scopus subject areas
- Molecular Biology