Advanced mRNA differential display: isolation of a new differentially regulated myosin heavy chain-encoding gene in amphibian limb regeneration.
In an effort to make mRNA differential display more amenable as a molecular screen, we have optimized the technology for the isotopic and non-isotopic detection of differentially regulated mRNAs. The number of amplification rounds in the displays was significantly reduced, resulting in the semi-quantitative detection of expression patterns of both low- and high-abundance transcripts. Moreover, we extended the method beyond the display of mRNAs by introducing a direct sequencing approach for the fast molecular analysis of isolated cDNAs. Applying this improved technique to the regenerating amphibian limb system, we have identified cDNA PCR products with a temporal difference in expression. This differential regulation was confirmed by Northern analysis, and DNA sequencing uncovered a novel newt differentiation-specific transcript encoding a skeletal myosin heavy chain (MHC).
Simon
H G
HG
Department of Molecular Genetics and Cell Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA. Simon#m#_Hans-Georg@msmail.bms.com
Oppenheimer
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics*; D017931:DNA Primers; D005552:Forelimb; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D051957:MSX1 Transcription Factor; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000235:genetics*; D009673:Notophthalmus viridescens; D012333:RNA, Messenger; D012038:Regeneration / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013698:Templates, Genetic; D014157:Transcription Factors; D014158:Transcription, Genetic
D017931:DNA Primers; D018398:Homeodomain Proteins; D051957:MSX1 Transcription Factor; D012333:RNA, Messenger; D014157:Transcription Factors; D018995:Myosin Heavy Chains