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We recently completely elucidated the molecular basis of genetic polymorphism in human deoxyribonuclease I and found it to be controlled by four codominant alleles, DNASE1*1, *2, *3 and *4. In this paper we describe a novel DNase I-genotyping system that could be used directly on DNA samples using the polymerase chain reaction (PCR) based on the three nucleotide substitutions underlying the protein polymorphism. The system consists of three independent reactions. Since the substitutions neither suppress nor create any known enzyme recognition site in the DNase I gene, two separate mismatched PCR followed by XhoI digestion methods were introduced to discriminate between the DNASE1*1 (or *3) and the DNASE1*2 (or *4) alleles, and to detect the DNASE1*4 allele. An amplification refractory mutation system was employed to detect DNASE1*3. A 100% correlation was found between the results of this genotyping method and those obtained by phenotyping using conventional isoelectric focusing. The high sensitivity and specificity of this genotyping method allows us to survey DNase I-polymorphism in small DNA samples.

The correlation between isoprotein types of alpha-1-antitrypsin (PI) in blood and semen samples from the same individual was determined in 48 Japanese men by the combined technique of isoelectric focusing with immobilized pH gradients and immunoblotting. Five common PI types (M1, M1M2, M1M3, M2 and M2M3) were detected in the blood plasma samples. However, PI-specific bands in semen migrated more cathodally than those in plasma into a pI region of approximately 5.05, and about 17% of the semen samples could not be phenotyped: the rest were easily phenotyped and their PI types were found to correlate with the type found in the corresponding blood samples. PI typing could therefore provide an additional discriminant characteristic for the forensic examination of individualization from semen samples.

In addition to the three alleles commonly responsible for the protein polymorphism of human deoxyribonuclease I, a mutation encoded by a fourth allele, DNASE1*4, was detected by isoelectric focusing. All 8 exons covering the entire open reading frame of the human DNase I gene were amplified by the polymerase chain reaction and subjected to direct sequencing. Only one nucleotide substitution, a C-to-G transition (CAG-->GAG), in the codon for amino acid 9 of the mature enzyme was found. This substitution resulted in the replacement of Gln with Glu (Q9E).

The main isozyme patterns of desialylated blood plasma or serum alpha-L-fucosidase (FUCA) were found to be almost identical to those of semen, urine, placental extracts, and leukocyte lysates, when detected by polyacrylamide gel isoelectric focusing, and activity staining using the fluorogenic substrate 4-methylumbelliferyl-alpha-L-fucopyranoside. Three phenotypes (1, 2-1, and 2) determined from plasma samples were identical to the phenotypes from urine and leukocyte lysates from the same individuals. A population study of plasma samples collected from 485 Japanese individuals indicated that the frequencies of the FUCA1*1 and FUCA1*2 alleles were 0.7505 and 0.2495, respectively. The mean plasma enzyme activities (+/- SD) of the three phenotypes were 318.8 +/- 116.7 nmol/ml per h for type 1, 268.0 +/- 108.3 nmol/ml per h for type 2-1, and 233.2 +/- 84.4 nmol/ml per h for type 2. The mean activities of types 1 and 2 suggest that, on average, the FUCA1*1 gene product in plasma has about 1.4 times the activity of FUCA1*2.

Isoelectric focusing of whole saliva samples on polyacrylamide gels (pH 3.5-5), followed by dried agarose film overlay detection, was employed to determine the type of salivary deoxyribonuclease I (DNase I). Since this detection method had not only a high sensitivity, but also a high band resolution, it was possible to determine DNase I types from saliva samples of 2-5 microL. Pretreatment of saliva samples with neuraminidase simplified the isozyme pattern and enhanced the sensitivity. The DNase I types in all 30 saliva samples showed a good correlation with the types found in the corresponding blood, semen, and urine samples. This preliminary trial indicates that DNase I typing from saliva samples is a new and promising method for individualization of casework samples in the field of forensic biology.

A zymogram method for detection of in situ ribonuclease (RNase) activity, combined with isoelectric focusing in a thin layer of polyacrylamide gel (IEF-PAGE), has been developed. After incubation with a dried agarose film containing substrate RNA, ethidium bromide, and an appropriate reaction buffer, which was placed tightly on the top of the focused gel, sharp and distinct dark bands corresponding to RNase isoenzymes on a fluorescent background appeared under uv light. Addition of urea to the IEF-PAGE gel at a final concentration of 4.8 M permitted optimal focusing of the RNases. This method had not only a high sensitivity of less than 0.1 ng purified RNase A, but also a high band resolution compared with the immunostaining method. It was also useful for analysis of purified enzymes, including bovine pancreatic RNases and two types of human urine RNase as mammalian enzymes, and RNases T1 and T2 as microbial enzymes, as well as for detection of RNases present in crude tissue extracts, resulting in more detailed elucidation of the multiplicity of these enzymes.

Allele frequencies for human transferrin (TF) subtypes were determined using serum samples from Japanese subjects living in Fukui prefecture, Japan, and compared with other Japanese populations using isoelectric focusing (IEF) and immunoblotting. The application of IEF revealed considerable heterogeneity in the TF system, enhancing its potential value for anthropologic and genetic studies. So far, TF subtypes of about 27,000 Japanese individuals from 35 population groups have been analyzed to evaluate the degree of genetic variation at the TF locus. Possible geographic and biologic factors are discussed.