Cloning and characterization of rat dentin matrix protein 1 (DMP1) gene and its 5'-upstream region.
Rat dentin matrix protein 1 (DMP1) is a highly acidic 58-kDa phosphoprotein, and DMP1 was the first gene to be cloned from the mineralized dentin matrix. It exists as a highly phosphorylated protein with a pI of 3 in the dentin matrix and, in that state, might have an important role in the mineralization process. The spatio-temporal distribution during development indicates that the expression of this gene is tightly regulated in the odontoblasts. It is now known that DMP1 is not unique to dentin but is present in other mineralized tissues like long bone, calvaria, and ameloblasts. To study the transcriptional regulation and the function of DMP1 in these tissues, a genomic clone with a functional promoter, introns, and exons was isolated. Sequence analysis showed that the rat DMP1 gene is comprised of six exons and five introns and spans approximately 13 kilobases (kb). Exon 1 contains the 5'-untranslated sequences. Exon 2 encodes a total of 18 amino acids including the 16 amino acids of the signal sequence. Exons 3-5 encode 16, 11, and 15 amino acids, respectively. Exon 6 contains 1.3 kb of the coding sequence with the RGD domain, stop codon, and the 3'-untranslated region (1.1 kb). We have mapped two transcription start sites within the DMP1 promoter that are 280 and 321 base pairs, respectively, from the ATG start codon. The location of functional elements within the 5'-upstream DMP1 DNA fragment was determined by cloning it into a luciferase reporter gene. Transient transfection and luciferase assays revealed that the 3 kb fragment has the ability to drive the luciferase gene. However, this promoter activity was restricted to MC3T3-E1 cells (an osteoblast cell lineage). The promoter was silent in Chinese hamster ovary cells (an epithelial cell lineage), indicating the necessity of tissue-specific factors to drive the transcription.
Thotakura
S R
SR
Department of Oral Biology, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Karthikeyan
N
N
Smith
T
T
Liu
K
K
George
A
A
D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004742:Enhancer Elements, Genetic; D005091:Exons; D016326:Extracellular Matrix Proteins; D005786:Gene Expression Regulation; D007438:Introns; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection
D020121:5' Untranslated Regions; C081635:Dmp1 protein, rat; D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins