编码人酸性鞘磷脂酶的cDNA克隆的分离:发生交替处理的转录本。
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Quintern LE, Schuchman EH, Levran O, Suchi M, Ferlinz K, Reinke H, Sandhoff K, Desnick RJ
编码人酸性鞘磷脂酶的cDNA克隆的分离:发生交替处理的转录本。
EMBO J. 1989 9月8日(9):2469-73。
- PubMed ID
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2555181 (PubMed视图］
- 摘要
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从人尿液中纯化了酸性鞘磷脂酶(sphingomyelin phosphodiesterase, EC 3.1.4.12)，并对12个胰蛋白酶肽进行了微测序(128个残基)。根据密码子冗余度最小的区域，合成了4种寡核苷酸混合物，其中寡核苷酸混合物1 (20mer;256 mix)用于从人成纤维细胞cDNA文库中筛选3 X 10(6)个独立的重组子。推测阳性克隆92个，用寡核苷酸混合物2-4进行Southern杂交分析。这些研究揭示了两组克隆;第一组(80个克隆;插入约1.2 - 1.6 kb)与寡核苷酸混合物1-4杂交，而组II(12个克隆;插入物范围约1.2至1.4 kb)与寡核苷酸混合物杂交1-3。有几个组II的无性系比组I的插入体更大，但没有与寡核苷酸混合物4杂交。对人胎盘cDNA文库进行了450 bp的I组片段的筛选，也分离出I组和II组克隆。 Representative clones from group I (pASM-1) and group II (pASM-2) were sequenced. pASM-1 contained a 1879 bp insert which was colinear with 96 microsequenced amino acids, while the pASM-2 1382 bp insert was colinear with 78 microsequenced residues. Notably, pASM-2 did not have an internal 172 bp sequence encoding 57 amino acid residues, but had instead an in-frame 40 bp sequence encoding 13 amino acids which was not present in pASM-1. These findings demonstrate the presence of two distinct acid sphingomyelinase transcripts in human fibroblasts and placenta and suggest the occurrence of alternative processing of the mRNA encoding this lysosomal hydrolase.