Yasuda T, Nadano D, Takeshita H, Tenjo E, Kishi K
Department of Legal Medicine, Fukui Medical School, Japan.
Ann Hum Genet. 1995 Apr;59(2):139-47. doi: 10.1111/j.1469-1809.1995.tb00737.x.
In addition to common phenotypes 1, 1-2 and 2 of human deoxyribonuclease I (DNase I), phenotypes 1-3 and 2-3, encoded by a third allele DNASE1*3, have been found by means of isoelectric focusing. The main objective of this study was to identify the mutation site(s) underlying phenotype 3. All eight exons covering the entire open reading frame of the human DNase I structural gene were amplified by the polymerase chain reaction (PCR) and subjected to direct DNA sequencing. When the entire 780-bp coding region and exon/intron junctions of the DNase I gene of two individuals with phenotypes 1-3 and 2-3 were sequenced, only one nucleotide substitution, a C-G transition (CCC-->GCC), in the codon for amino acid 132 of the mature enzyme located in exon VI was found that resulted in the replacement of proline with alanine (P132A). The mutation was confirmed by allele-specific amplification of genomic DNA. The replacement of the amino acid residue may reduce the hydrophobicity of the enzyme and thus increase the pI value of the type-3 isozyme compared with that of type 1, as increasing the hydrophobicity of a protein is known to decrease its pI value. The specific PCR-amplifications of exons and alleles developed in this study may provide a new tool suitable for rapid screening of DNase I variants.
除了人类脱氧核糖核酸酶I（DNase I）常见的1型、1 - 2型和2型表型外，通过等电聚焦法还发现了由第三个等位基因DNASE1*3编码的1 - 3型和2 - 3型表型。本研究的主要目的是确定表型3背后的突变位点。通过聚合酶链反应（PCR）扩增覆盖人类DNase I结构基因整个开放阅读框的所有八个外显子，并进行直接DNA测序。对两名具有1 - 3型和2 - 3型表型个体的DNase I基因的整个780 bp编码区及外显子/内含子连接区进行测序时，仅在外显子VI中成熟酶第132位氨基酸的密码子处发现一个核苷酸替换，即C - G转换（CCC→GCC），导致脯氨酸被丙氨酸取代（P132A）。通过基因组DNA的等位基因特异性扩增证实了该突变。与1型相比，氨基酸残基的替换可能会降低酶的疏水性，从而提高3型同工酶的等电点（pI）值，因为已知增加蛋白质的疏水性会降低其pI值。本研究中开发的外显子和等位基因的特异性PCR扩增可能提供一种适用于快速筛查DNase I变体的新工具。
