Molecular structure of the chicken vitamin D-induced calbindin-D28K gene reveals eleven exons, six Ca2+-binding domains, and numerous promoter regulatory elements.
The seco-steroid hormone 1,25-dihydroxyvitamin D3 is known to induce the expression of a calcium binding protein termed calbindin-D28K in a variety of target tissues. In order to comprehend the mechanism of induction we have cloned and sequenced the chicken calbindin-D28K gene. The gene spans some 18.5 kilobases (kb) of chromosomal DNA from the putative Cap site to the polyadenylation site of the 2.8 kb mRNA. It is split into 11 coding exons by 10 intervening sequences. The promoter region of this gene is markedly G + C-rich (60-80%) extending from -225 to +400. Within this region we find 70 CpG dinucleotides, four G-C boxes, and numerous known promoter regulatory signals. These putative regulatory signals include a TATA box (ATAAATA) at -30 and a CAT box (CCAAT) at -326. Ten additional variant CAT boxes are found in the upstream promoter region (-218 to -770) of this gene. Furthermore we have identified a glucocorticoid-like responsive element at -410 (TCTACACACTGTTCC) and this element overlaps a metal responsive element (TGCACTC) and a variant CAT box (CCAAAT) and juxtaposes an enhancer-like core element (AAATGGT) on its 3'-side. In addition, the calbindin-D28K promoter is composed of a variety of simple repeated sequences, some of which are components of putative regulatory signals. All splice junctions were found to conform to the GT-AG rule. A consensus sequence of the 5'-splice junction reads AG/GTAAG-TTATA. A consensus sequence of the 3'-splice site consists of two elements: a pyrimidine track (mainly T) followed by ACAG/G-T. A two-dimensional model of calbindin-D28K was constructed which projects the existence of 6 alpha-helix-loop-alpha-helix regions characteristic of calcium binding domains. The 3'-end of the gene consists of a single large (2039 base pair) uninterrupted exon, an organizational feature common to other members of the calcium binding protein gene family which include calmodulin, parvalbumin, Spec I, myosin light chains, etc. Another feature common to the gene family is the presence of the repeated sequence ATTT or TTTA located in the 3'-untranslated exons. These simple repeat sequences could be involved in regulating mRNA degradation by serving as a ribonuclease recognition signal.
Minghetti
P P
PP
Department of Biochemsitry, University of California, Riverside 92521.
Cancela
L
L
Fujisawa
Y
Y
Theofan
G
G
Norman
A W
AW
D000595:Amino Acid Sequence; D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D004274:DNA, Recombinant; D005091:Exons; D007438:Introns; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D064030:S100 Calcium Binding Protein G / Q000235:genetics*
D064026:Calbindins; D004274:DNA, Recombinant; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G; D002118:Calcium