Determination of the half-life of the murine β4-galactosyltransferase-1 mRNA in somatic cells using the tetracycline-controlled transcriptional regulation system

摘要

The glycosyltransferases are recognized as a functional family of an estimated 300 distinct, intracellular, membrane-bound enzymes that are positioned along the secretory pathway and participate coordinately in the biosynthesis of the carbohydrate moieties on glycoconjugates. The full-length cDNA sequence for many of these proteins is now available yet little is known about the transcriptional or translational regulation of a given transcript or its decay rate in the cell. These issues are made more complex by the observations that transcription of a glycosyltransferase gene in different cells/tissues results in mRNAs with significantly different structures. For example, transcription of the murine β4-galactosyoltransferase-1 gene in somatic cells yields two transcripts of 3.9 and 4.1 kb. In contrast transcription of this gene in developing male germ cells results in transcripts of 2.9 and 3.1 kb which are distinguished from their somatic cell counterparts primarily by the deletion of ～1.7 kb of sequence in the 3'-untranslated region (UTR). With the long range goal of determining the role that the 3'-UTR serves in mRNA decay we have taken advantage of a recently developed methodology, the Tet-Off system, to determine the half-life of the mRNA encoding β4-galactosyltransferase-1 in the murine NIH 3T3 somatic cell line. We show that the β4-galactosyltransferase-1 mRNA has a half-life of ～84 min (range of 82-85 min) in 3T3 cells and that substitution of the galactosyltransferase coding sequence with the coding sequence of luciferase does not significantly alter the decay rate (～87 min; range of 84-91 min). This latter observation suggests that theβ4-galactosyltransferase-1 coding sequence does not contain functional elements that affect the intrinsic stability of this mRNA.