The molecular biology of calbindin-D₉

Calbindin-D9k is a cytosolic calcium binding protein expressed in the mammalian intestine, placenta, and uterus. The protein is probably involved in calcium transport across the intestinal and placental epithelia. The objective of this thesis was to study the structure of the human and porcine calbindin-D9k at the cDNA and genomic level. The cDNAs for the bovine, murine, and rat calbindin-D9k as well as the gene for rat calbindin-D9k had been cloned previously. The full length cDNA encoding the human and porcine calbindin-D 9k were cloned using a modified PCR (polymerase chain reaction) technique called RACE (rapid amplification of cDNAs ends) with rat and bovine cDNA sequence-derived primers for amplification. The full length of these sequences was confirmed by primer extension assay. The human cDNA includes a coding region for 79 amino acids, 57 nucleotides 5'and 159 nucleotides 3'-non-coding region, and a poly(A+) tail. Northern analysis showed that the calbindin-D 9k mRNA is expressed in human duodenum (600 nucleotides in length) but not in reproductive tissues such as placenta and uterus. The porcine clone revealed a full-length cDNA encoding calbindin-D9k , 79 amino acids, 57 nucleotides 5'and 149 nucleotides 3'non-coding region, and a poly(A+) tail. The inferred amino acid sequence of the porcine calbindin-D9k is identical to the published amino acid sequence, except for one residue. Northern analysis of porcine tissues showed a 600 nucleotide transcript in intestine, kidney, and uterus. iii The gene was found to be located on the human X-chromosome by means of PCR of hybrid DNAs from human and hamster somatic cells. The human calbindin-D9k gene was isolated from a human X-chromosome library. The genomic clone contained the complete gene as well as 5' and 3' flanking regions. The structural gene, approximately 1.3 kb of 5' flanking, and 0.5 kb of 3' flanking region were sequenced. The gene spans about 4.5 kb and consists of three exons separated by two introns. The first exon represents only non-coding sequences, while the second and third exons encode the two calcium binding domains of the protein. A partial genomic sequence from pig DNA was also cloned by the use of PCR techniques. The genomic sequences were analyzed by computer to identify consensus sequences for transcription factor binding sites. The human and porcine calbindin-D9k gene sequences revealed the presence of a putative estrogenresponsive element located at the boundary of exon I and intron A. This type of sequence in the analogous location of the rat gene has been shown to mediate estrogen regulation in the uterus. The ability of these elements to bind the estrogen receptor was investigated by gel retardation assay. No binding was detected when the human and porcine sequences were used. The inability of the human sequence to bind the estrogen receptor may explain why the gene is not expressed in human uterus (and placenta). In the case of the porcine calbindinD9k an estrogen receptor binding site could be located in the gene's 5' regulatory region. In the human gene there was no such putative region found, neither in the 5' nor 3' flanking region. iv This study is the first report on the genomic structure and expression of the human calbindin-D9k. The human gene appears to be unique in that it is the only mammalian calbindin-D 9k gene which is not expressed in the uterus and placenta. This has potential significance for fetal/maternal calcium transfer and uterine function in human.