Bovine and rodent tamm-horsfall protein (THP) genes: cloning, structural analysis, and promoter identification.
We have isolated bovine and rodent cDNA and genomic clones encoding the kidney-specific Tamm-Horsfall protein (THP). In both species the gene contains 11 exons, the first of which is noncoding. Exon/intron junctions were analyzed and all were shown to follow the AG/GT rule. A kidney-specific DNase I hypersensitive site was mapped onto a rodent genomic fragment for which the sequence is highly conserved in three species (rat, cow, and human) over a stretch of 350 base pairs. Primer extension and RNase protection analysis identified a transcription start site at the 3' end of this conserved region. A TATA box is located at 32 nucleotides upstream of the start site in the bovine gene and 34 nucleotides upstream in the rodent gene. An inverted CCAAT motif occurs at 65 and 66 nucleotides upstream of the start site in the bovine and rodent genes, respectively. Other highly conserved regions were noted in this 350 bp region and these are likely to be binding sites for transcription factors. A functional assay based on an in vitro transcription system confirmed that the conserved region is an RNA Pol II promoter. The in vitro system accurately initiated transcription from the in vivo start site and was highly sensitive to inhibition by alpha-amanitin at a concentration of 2.5 micrograms/ml. These studies set the stage for the further definition of cis-acting sequences and trans-factors regulating expression of the THP gene, a model for kidney-specific gene expression.
Yu
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Department of Medicine, Beth Israel Hospital, Boston, MA 02215.
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Sukhatme
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D003851:Deoxyribonucleases / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000235:genetics*; D011257:Pregnancy Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D058949:Uromodulin
D018076:DNA, Complementary; D009088:Mucoproteins; D011257:Pregnancy Proteins; C548612:UMOD protein, human; C548614:Umod protein, rat; D058949:Uromodulin; D012313:RNA; D003851:Deoxyribonucleases