Stability of the human dystrophin transcript in muscle.
The human dystrophin gene has 79 exons spanning >2300 kb making it the largest known gene. In previous studies we showed that approximately 16 h are required to transcribe the gene in myogenic cultures [Tennyson, C.N., Klamut, H.J. and Worton, R.G. (1995) Nature Genet. 9, 184-190]. To estimate the half-life of the dystrophin mRNA, the decay of the transcript was monitored by quantitative RT-PCR in cultured human fetal myotubes following exposure to actinomycin D. Results from this analysis indicated that the half-life of the dystrophin mRNA is 15.6 +/- 2.8 h in these cultures. Transcript accumulation profiles were predicted using a mathematical model which incorporated the measured half-life. The modeled accumulation profiles were consistent with observed profiles supporting the half-life measured using actinomycin D. The kinetic model was then used to predict the relative amount of nascent and mature dystrophin transcript at steady state. Measurements by quantitative RT-PCR indicated that in adult skeletal muscle tissue the concentration of mature dystrophin mRNA is 5-10 molecules per nucleus, demonstrating, as expected, that it is a low abundance transcript. Furthermore the ratio of nascent to mature dystrophin transcript indicated that dystrophin synthesis may not be at steady state in the adult skeletal muscle we tested. Alternatively, the kinetics of transcript production in skeletal muscle tissue may be different from those observed in cultured fetal myogenic cells.
Tennyson
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Molecular and Medical Genetics Department, University of Toronto, Ontario, Canada.
Shi
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Worton
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D000328:Adult; D001483:Base Sequence; D002999:Clone Cells; D003609:Dactinomycin / Q000494:pharmacology; D016189:Dystrophin / Q000235:genetics*; D005333:Fetus / Q000166:cytology; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D016189:Dystrophin; D012333:RNA, Messenger; D003609:Dactinomycin