A SINGLE-BASE-PAIR DELETION IN THE BETA-GLUCURONIDASE GENE ACCOUNTS FOR THE PHENOTYPE OF MURINE MUCOPOLYSACCHARIDOSIS TYPE-VII
A SINGLE-BASE-PAIR DELETION IN THE BETA-GLUCURONIDASE GENE ACCOUNTS FOR THE PHENOTYPE OF MURINE MUCOPOLYSACCHARIDOSIS TYPE-VII
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DOI：
10.1073/pnas.90.14.6567
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发表时间：
1993-07-15
影响因子：
11.1
通讯作者：
BIRKENMEIER, EH
中科院分区：
文献类型：
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作者：
SANDS, MS;BIRKENMEIER, EH
小鼠粘多糖沉积症VII型是一种由自发突变gus（mps）引起的遗传性疾病，与5号染色体上的β-葡萄糖醛酸苷酶结构基因密切相关。突变纯合子小鼠的β-葡萄糖醛酸苷酶mRNA水平降低>200倍，并且通过灵敏的荧光测定法几乎检测不到酶活性。从gus（mps）/gus（mps）小鼠和+/+小鼠的祖先品系克隆了约20 kb的含有β-葡糖醛酸糖苷酶基因Gus和>2 kb的5'和3'侧翼序列的基因组DNA。从两个Gus等位基因中的每一个产生含有长度为20-400 bp的DNA片段的限制性内切酶片段，然后通过使用非变性聚丙烯酰胺DNA测序凝胶进行比较。该方法快速鉴定了大量的限制性位点，并且灵敏度足以检测由gus（mps）等位基因中的1-bp缺失引起的限制性片段长度变化。突变体基因组片段的DNA序列分析表明，1-bp的缺失在外显子10内产生了移码突变。当转染到gus（mps）/gus（mps）成纤维细胞中时，通过寡核苷酸定点突变插入缺失的核苷酸恢复了校正的突变基因的功能。我们的结论是，移码突变，即在外显子10的497密码子处引入一个提前终止密码子，解释了与gus（mps）表型相关的分子、生化和病理异常。
Murine mucopolysaccharidosis type VII is a heritable disease caused by a spontaneous mutation, gus(mps), closely linked to the beta-glucuronidase structural gene on chromosome 5. Mice homozygous for the mutation have a >200-fold decrease in beta-glucuronidase mRNA levels and virtually no enzyme activity detectable by a sensitive fluorometric assay. Approximately 20 kb of genomic DNA containing the beta-glucuronidase gene Gus and >2 kb of 5' and 3' flanking sequences were cloned from both a gus(mps)/gus(mps) mouse and a +/+ mouse of the progenitor strain. Restriction enzyme digests containing DNA fragments 20-400 bp in length were generated from each of the two Gus alleles and then compared by using nondenaturing polyacrylamide DNA-sequencing gels. This method rapidly identified a large number of restriction sites and was sensitive enough to detect a restriction fragment length variation resulting from a 1-bp deletion in the gus(mps) allele. DNA-sequence analysis of the mutant genomic fragment showed that the 1-bp deletion created a frameshift mutation within exon 10. Insertion of the deleted nucleotide by oligonucleotide site-directed mutagenesis restored function to the corrected mutant gene when transfected into gus(mps)/gus(mps) fibroblasts. We concluded that the frameshift mutation, which introduces a premature stop codon at codon 497 in exon 10, accounts for the molecular, biochemical, and pathological abnormalities associated with the gus(mps) phenotype.