Chireux M A, Le Van Thai A, Weber M J
Laboratoire de Biologie Moléculaire Eucaryote, Centre National de la Recherche Scientifique, Toulouse, France.
J Neurosci Res. 1995 Mar 1;40(4):427-38. doi: 10.1002/jnr.490400402.
Two overlapping cosmids containing the 5' end of human choline acetyltransferase (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 sequence of rodent exon N was not conserved in the human gene. Northern blot 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 in mRNA species of 6.0 and 2.5 kb. Only the 6-kb species was detected with both R- and M-specific probes in the neuroepithelioma cell line CHP126. Reverse transcription-polymerase chain reaction (RT-PCR) analysis suggested that the major mRNA species in MC-I-XC and CHP126 cells contained the proximal part of exon M spliced to exon 1, which contains the alternative ACG initiation codon. RT-PCR also allowed the characterization 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 (tentatively numbered exon 8) in the coding sequence.
已克隆出两个包含人胆碱乙酰转移酶（ChAT）基因5'端的重叠黏粒。利用异源探针，我们定位了与啮齿动物ChAT外显子R和M同源的两个选择性第一外显子（三泽等人：《生物化学杂志》267:20392 - 20399，1992年）。啮齿动物外显子N的序列在人类基因中未保守存在。对从人神经上皮瘤细胞系LA - N2和MC - I - XC纯化的mRNA进行Northern印迹分析显示，外显子R和M均转录于6.0 kb和2.5 kb的mRNA种类中。在神经上皮瘤细胞系CHP126中，用R特异性和M特异性探针均仅检测到6 kb的种类。逆转录 - 聚合酶链反应（RT - PCR）分析表明，MC - I - XC和CHP126细胞中的主要mRNA种类包含与外显子1剪接的外显子M的近端部分，外显子1含有选择性ACG起始密码子。RT - PCR还能够鉴定出一种包含与外显子1剪接的外显子R的mRNA种类，但未检测到同时包含外显子R和外显子M远端部分的种类。RT - PCR还用于证明编码序列中的一个选择性外显子（暂编号为外显子8）。
