HUMAN CHOLINE-ACETYLTRANSFERASE GENE - LOCALIZATION OF ALTERNATIVE FIRST EXONS

Two overlapping cosmids containing the 5' end of human choline acetylt ransferase (ChAT) gene have been cloned. Using heterologous probes, we localized two alternative first exons homologous to rodent ChAT exons R and M (Misawa et al.: J Biol Chem 267:20392-20399, 1992), The seque nce of rodent exon N was not conserved in the human gene, Northern blo t analysis of mRNA purified from the human neuroepithelioma cell lines LA-N2 and MC-I-XC revealed that both exons R and M were transcribed i n mRNA species of 6.0 and 2.5 kb, Only the 6-kb species was detected w ith both R- and M-specific probes in the neuroepithelioma cell line CH P126. Reverse transcription-polymerase chain reaction (RT-PCR) analysi s suggested that the major mRNA species in MC-I-XC and CHP126 cells co ntained the proximal part of exon M spliced to exon 1, which contains the alternative ACG initiation codon. RT-PCR also allowed the characte rization of a mRNA species containing exon R spliced to exon 1, but no species containing both exon R and the distal part of exon M could be detected. RT-PCR was also used to evidence an alternative exon (tenta tively numbered exon 8) in the coding sequence. (C) 1995 Wiley-Liss, I nc.