A novel untranslated 'exon H' of the human choline acetyltransferase gene in placenta.
To investigate the existence of 5'-region(s) of human choline acetyltransferase (hChAT) mRNA in placenta we analyzed the presence or absence of ChAT 5'-untranslated regions (UTR) in human neuronal and non-neuronal cells. Total RNA from human spinal cord, placenta, cultured choriocarcinoma JEG-3 and neuroblastoma CHP126 and MC-IXC cells was reverse transcribed and used for polymerase chain reaction amplification (RT-PCR). We used a sense primer located in the 5'-flanking region, in the previously defined intronic sequence and an anti-sense primer located in the common coding exon 2 of the hChAT gene. An amplified product of 567 bp in size was obtained only in human placenta and in JEG-3 cells whereas it was absent in spinal cord, CHP126 and MC-IXC cells. It was designated 'H-type' of ChAT mRNA. Whereas CHP126 produced the R- and N-type of ChAT mRNAs, no transcript of the N-and R-type was detected in JEG-3 and human placenta. In addition, CHP126 and JEG-3 cells and placenta showed the expression of the M-type of ChAT mRNA. The identity of the amplified 567 bp product (H-type) was confirmed by Southern hybridization and sequencing. The nucleotide sequence of the amplified fragment in placenta revealed the existence of a previously unknown type of ChAT mRNA produced by alternative splicing. Using primer extension we further determined the transcription initiation site of the H-type hChAT mRNA in placenta. These results demonstrate the expression of a novel ChAT mRNA isoform in human placenta in addition to the M-type. These data may be possibly explained by the presence of a placenta specific promoter in the ChAT gene, which might be the proximal promoter P1.
Robert
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Laboratoire de Génétique Moléculaire des Eucaryotes, CNRS, Biologie Moléculaire et de Génie Génétique, INSERM, Institut de Chimie Biologique, Faculté de Médecine, Strasbourg, France.
Quirin-Stricker
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002795:Choline O-Acetyltransferase / Q000235:genetics*; D002822:Choriocarcinoma / Q000201:enzymology; D005091:Exons; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000201:enzymology; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013116:Spinal Cord / Q000201:enzymology; D014327:Trophoblasts / Q000201:enzymology; D014407:Tumor Cells, Cultured; D014594:Uterine Neoplasms / Q000201:enzymology
D012333:RNA, Messenger; D002795:Choline O-Acetyltransferase