Schuchman E H, Suchi M, Takahashi T, Sandhoff K, Desnick R J
Division of Medical and Molecular Genetics, Mount Sinai School of Medicine, New York, New York 10029.
J Biol Chem. 1991 May 5;266(13):8531-9.
Two types of partial cDNAs encoding human acid sphingomyelinase (EC 3.1.4.12; ASM) were recently isolated from fibroblast and placental cDNA libraries (Quintern, L. E., Schuchman, E.H., Levran, O., Suchi. M., Ferlinz, K., Reinke, H., Sandhoff, K., and Desnick, R. J. (1989) EMBO J. 8, 2469-2473). The cDNA inserts had identical sequences with the exception of an internal region; type 1 cDNAs (representing approximately 90% of the ASM cDNAs isolated) had 172 in-frame base pairs (bp), which were replaced in the type 2 cDNAs by a 40-bp in-frame sequence. Northern hybridization and RNase protection studies indicated that both type 1 and 2 transcripts were approximately 2.5 kilobases; therefore, efforts were directed to isolate full-length type 1 and 2 cDNAs by screening human placental, testis, hepatoma, and retinal cDNA libraries. In addition to type 1 and 2 cDNAs, a new type of ASM cDNA (type 3), which did not contain the type 1- or 2-specific regions, was isolated and sequenced. The full-length type 1 and the reconstructed full-length type 2 and 3 cDNAs were transiently expressed in COS-1 cells. Only the full-length type 1 transcript encoded catalytically active human ASM, demonstrating its functional integrity. The 2347-bp full-length type 1 placental cDNA (pASM-1FL) had an 87-bp 5'-untranslated region, an 1890-bp open reading frame encoding 629 amino acids, and a 370-bp 3'-untranslated sequence. The predicted location of the signal peptide cleavage site was after alanine 46. Two base differences were identified in codons 322 and 506 and shown to be polymorphisms with the common alleles having frequencies of 0.6 and 0.7, respectively. To determine the genomic organization of the type 1, 2, and 3 sequences, a 1665-bp genomic region containing both the unique type 1 (172 bp) and type 2 (40 bp) sequences was amplified by the polymerase chain reaction and sequenced. The 172-bp sequence was exonic, flanked by 5'- and 3'-intronic sequences of 1052 and 229 bp, respectively. The 40-bp type 2 sequence was intronic, occurring at the 5' end of the 1052-bp intron due to the use of a cryptic 5' donor splice site, which deleted the entire 172-bp exon and both flanking intronic sequences. The type 3 cDNA resulted from an alternative splicing event, which excised the 172-bp exon. These studies demonstrate the occurrence of alternatively splicing of the ASM transcript, but the existence of only one functional mRNA.
最近从成纤维细胞和胎盘cDNA文库中分离出两种编码人酸性鞘磷脂酶（EC 3.1.4.12；ASM）的部分cDNA（昆特恩，L.E.，舒克曼，E.H.，莱夫兰，O.，苏奇，M.，费林兹，K.，赖因克，H.，桑德霍夫，K.，和德尼克，R.J.（1989年）《欧洲分子生物学组织杂志》8，2469 - 2473）。cDNA插入片段除内部区域外具有相同序列；1型cDNA（约占分离出的ASM cDNA的90%）有172个框内碱基对（bp），在2型cDNA中被一个40 bp的框内序列取代。Northern杂交和核糖核酸酶保护研究表明1型和2型转录本均约为2.5千碱基；因此，致力于通过筛选人胎盘、睾丸、肝癌和视网膜cDNA文库来分离全长1型和2型cDNA。除了1型和2型cDNA外，还分离并测序了一种新型的ASM cDNA（3型），它不包含1型或2型特异性区域。全长1型以及重构的全长2型和3型cDNA在COS - 1细胞中瞬时表达。只有全长1型转录本编码具有催化活性的人ASM，证明了其功能完整性。2347 bp的全长1型胎盘cDNA（pASM - 1FL）有一个87 bp的5'非翻译区、一个编码629个氨基酸的1890 bp开放阅读框和一个370 bp的3'非翻译序列。信号肽切割位点的预测位置在丙氨酸46之后。在密码子322和506中鉴定出两个碱基差异，并显示为多态性，常见等位基因的频率分别为0.6和0.7。为了确定1型、2型和3型序列的基因组结构，通过聚合酶链反应扩增并测序了一个包含独特的1型（172 bp）和2型（40 bp）序列的1665 bp基因组区域。172 bp序列是外显子，两侧分别是1052 bp和229 bp的5'和3'内含子序列。40 bp的2型序列是内含子序列，由于使用了一个隐蔽的5'供体剪接位点，出现在1052 bp内含子的5'端，该位点删除了整个172 bp外显子及其两侧的内含子序列。3型cDNA是由一个选择性剪接事件产生的，该事件切除了172 bp外显子。这些研究证明了ASM转录本存在选择性剪接，但仅存在一种功能性mRNA。
