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.