MOLECULAR ANALYSIS OF THE 3RD ALLELE OF HUMAN DEOXYRIBONUCLEASE-I POLYMORPHISM

In addition to common phenotypes 1, 1-2 and 2 of human deoxyribonuclea se I (DNase I), phenotypes 1-3 and 2-3, encoded by a third allele DNAS E13, have been found by means of isoelectric focusing. The main objec tive of this study was to identify the mutation site(s) underlying phe notype 3. All eight exons covering the entire open reading frame of th e human DNase I structural gene were amplified by the polymerase chain reaction (PCR) and subjected to direct DNA sequencing. When the entir e 780-bp coding region and exon/intron junctions of the DNase I gene o f 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 hydrophobic ity 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 p rotein is known to decrease its pI value. The specific PCR-amplificati ons of exons and alleles developed in this study may provide a new too l suitable for rapid screening of DNase I variants.