Molecular cloning and expression of cDNA for murine galactocerebrosidase and mutation analysis of the twitcher mouse, a model of Krabbe's disease
Molecular cloning and expression of cDNA for murine galactocerebrosidase and mutation analysis of the twitcher mouse, a model of Krabbe's disease
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DOI：
10.1046/j.1471-4159.1996.66031118.x
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发表时间：
1996-03-01
影响因子：
4.7
通讯作者：
Okada, S
中科院分区：
文献类型：
--
作者：
Sakai, N;Inui, K;Okada, S
根据与人半乳糖脑苷酶cDNA的同源性，从小鼠睾丸cDNA文库中分离到鼠半乳糖脑苷酶cDNA，用PCR方法克隆其5′端。它有2278个核苷酸序列，包括一个2004个核苷酸的开放阅读框，编码668个氨基酸残基。人与鼠氨基酸序列的一致性很高，估计为84%。对抽动鼠肝脏的cDNA进行测序，发现密码子339 （TGG—> TGA）处存在无义突变。半乳糖脑苷酶mRNA最丰富的条带为3.6 kb，在抽搐小鼠中未检测到。这表明我们所鉴定的cDNA （2278 bp）代表了一个由交替多聚(a)信号产生的小物种，并且大多数mRNA具有更长的3'-未翻译区。基因组分析显示，该突变在抽动鼠中为纯合子，在携带者中为杂合子，但在正常小鼠中不存在。将正常小鼠的半乳糖脑苷酶cDNA转染至COS1细胞后，酶活性升高，而将突变的半乳糖脑苷酶cDNA转染至COS1细胞后，酶活性升高。我们得出结论，这种突变导致抽搐小鼠半乳糖脑苷酶缺乏。
The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5' end. It has a 2,278-nucleotide sequence including a 2,004-nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG --> TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6-kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3'-untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.