Genotyping of human deoxyribonuclease I polymorphism by the polymerase chain reaction.

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.

Molecular analysis of the third allele of human deoxyribonuclease I polymorphism.

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.

Correlation of alpha-1-antitrypsin (PI) polymorphism between blood and semen.

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.

The molecular basis for genetic polymorphism of human deoxyribonuclease I: identification of the nucleotide substitution that generates the fourth allele.

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).

Genetically polymorphic alpha-L-fucosidase (FUCA1) isozymes detected in blood plasma.

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.

Detection of deoxyribonucleases I and II (DNases I and II) activities in reproductive organs of male rabbits.

Deoxyribonucleases (DNases) I and II activities in 13 different organs and body fluids from healthy male rabbits were measured using the single radial enzyme diffusion method. We now show that testis, epididymis, ampulla, seminal vesicle, vesicular gland, prostate, and semen have both of the DNases I and II activities, whereas spermatozoa do not. DNase I activities were highest in epididymis and seminal vesicle, whereas DNase II activities were highest in epididymis and prostate among the reproductive organs. The presence of these two enzyme activities outside the digestive system suggests that they have another biological function in addition to their digestive roles.

Salivary deoxyribonuclease I polymorphism separated by polyacrylamide gel-isoelectric focusing and detected by the dried agarose film overlay method.

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.

Survey of the association of deoxyribonuclease I polymorphism with disease.

The deoxyribonuclease I (DNase I) system was studied in 120 unrelated Japanese patients with liver disease, malignant neoplasms, alimentary-canal disease and inflammatory conditions with respect to the distribution of phenotypes and gene frequencies in serum samples. In patients with alimentary-canal disease a significant deficit of the DNase I phenotype 1-2 was demonstrated, which suggests that heterozygosity may confer protection against such disease. Furthermore, a significant association between the DNase I phenotype 2 and liver disease was found. The possible involvement of these phenotypes in the response to these diseases would appear to merit further study.

A new individualization marker of semen: deoxyribonuclease I (DNase I) polymorphism.

We describe a method for obtaining specific and reproducible deoxyribonuclease I (DNase I) typing from liquid semen. Isoelectric focusing of the enzymes on polyacrylamide gel (IEF-PAGE, pH 3.5-5) was accomplished using a 0.5-mm thick gel. The separated isozymes were visualized by a new activity staining method, dried agarose film-overlay (DAFO). Pretreatment of semen samples with neuraminidase markedly enhanced the isozyme-band resolution and sensitivity. The method was simple and reliable, with high resolution and sensitivity. The DNase I types in semen samples were correlated with the types found in corresponding blood and urine samples. DNase I typing could therefore provide an additional discriminant characteristic in the forensic examination of semen.

The zymogram method for detection of ribonucleases after isoelectric focusing: analysis of multiple forms of human, bovine, and microbial enzymes.

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.

Transferrin polymorphisms in Japanese populations: north-south cline in the distribution of the TF*C2 allele.

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.

Genetic polymorphism of human glutamate oxaloacetate transaminase (GOT1) detected using isoelectric focusing and a sensitive and positive staining method.

Genetic polymorphism of glutamate oxaloacetate transaminase (GOT1) was demonstrated in human erythrocytes by isoelectric focusing in thin layer polyacrylamide gels and a sensitive and positive detection method. Using this technique, five phenotypes, GOT1 1, 2, 2-1, 3-1 and 3-2 were determined and the estimated gene frequencies of GOT1*1, GOT1*2 and GOT1*3 in the Japanese population were 0.9740, 0.0173 and 0.0087, respectively.