Tools for evaluating ubiquitin (UbC) gene expression: characterization of the rat UbC promoter and use of an unique 3' mRNA sequence.
UbC is one of three members of the ubiquitin gene family. We have cloned the rat UbC promoter and used primer extension analysis to map the UbC site of transcription initiation to 63 bp upstream of the putative first intron. We used a rat UbC promoter-luciferase reporter minigene to transfect H9c2 cardiomyocytes, HepG2 hepatocytes, CaCo2 colon cells, NIH3T3 fibroblasts or L6 myocytes and found the rat UbC promoter has constitutive activity. We also showed that dexamethasone stimulated the UbC promoter in L6 myocytes. Finally, we showed that a UbC-specific sequence at the 3' end of the rat UbC mRNA transcript can be used to selectively and quantitatively measure UbC: (1) mRNA using a RNase protection assay, and (2) transcription using a nuclear run-off assay to measure the rate of transcription of the UbC gene. These findings will be useful in studying the regulation of the UbC gene.
Renal Division, Emory University, Atlanta, Georgia, 30322, USA.
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D020413:3' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003907:Dexamethasone / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D014452:Ubiquitins / Q000235:genetics*
D020413:3' Untranslated Regions; D007527:Isoenzymes; D012333:RNA, Messenger; D014452:Ubiquitins; D003907:Dexamethasone