Haruo Takeshita

Chromosomal assignment of the human deoxyribonuclease I gene, DNASE1 (DNL1), to band 16p13.3 using the polymerase chain reaction

To localize the human deoxyribonuclease I (DNase I) gene, DNASE1 (DNL1), we performed a polymerase chain reaction (PCR) using DNA extracted from a panel of cloned human x rodent hybrid cell lines carrying different human chromosomes and screened for the presence of the expected PCR products. Two different sets of oligonucleotide primers specific for human DNase I cDNA sequences were used to amplify unique fragments in the human DNase I gene. Based on this work, DNL1 could be assigned to human chromosome 16. Furthermore, regional localization of the gene to 16p13.3 was performed by PCR analysis of a high-resolution mouse x human somatic cell hybrid panel that contained defined portions of human chromosome 16.

Human seminal deoxyribonuclease I (DNase I): purification, enzymological and immunological characterization and origin

Deoxyribonuclease I (DNase I) was purified from the semen of a 38-year-old male and then characterized. The catalytic properties of the purified enzyme closely resembled those of DNase I purified from the urine of this individual and the following other similarities were observed: molecular masses, iodoacetic acid inactivation kinetics, desialylated isoenzyme patterns. However, the behavior of the purified enzymes determined on several different lectin-affinity chromatography columns differed, which suggests that organ-specific glycosylation of DNase I occurs. Multiple forms of the purified seminal DNase I were demonstrated, each of which had a different pI value separated by isoelectric focusing, which is compatible with the reported existence of genetic polymorphism of seminal DNase I (Sawazaki et al., Forensic Sci Int 1992;57:39-44). Furthermore, enzymological and immunological comparisons of purified seminal and urinary and partially purified prostatic DNases I indicated that the prostate may be one of seminal enzyme source tissues.

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.

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.

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.

Deoxyribonuclease I (DNase I) typing from semen stains : Low enzyme activity in vaginal fluids does not interfere with seminal DNase I typing from mixture stains

We describe the use of deoxyribonuclease I (DNase I) polymorphism for individualization of semen in body fluid stain mixtures, as a means of providing new and more useful information to practicing forensic biologists as a genetic marker. We have already reported that human DNase I isozyme patterns from different subjects are classifiable into ten groups. Isoelectric focusing of DNase I isozymes on polyacrylamide gel (IEF-PAGE, pH 3.5 to 5) was accomplished using a 0.5 mm thick gel. Pretreatment of semen samples with neuraminidase enhanced the isozyme band resolution and sensitivity. Activity detection using the dried agarose film overlay (DAFO) procedure was reliable, sensitive and simple, with high resolution, and the phenotypes of DNase I were determined in semen stains of about 0.3 microL stored at room temperature for up to a year in most of the samples tested. The DNase I types in semen stains were correlated with the types found in the corresponding blood and urine samples, although most of the vaginal fluid samples had no typable DNase I activity. This is considerably advantageous for seminal individualization from body fluid mixture stains in criminal cases. An evaluation of DNase I typing by IEF-PAGE and DAFO was also performed on casework samples submitted to our laboratory, and the results showed that DNase I was expected to be one of the most useful individualization marker of semen in practical application.

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, DNASEI*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).

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 DNASEI*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.

The STR loci HumTPO and HumLPL : population genetic data in eight populations

Abstract The two STR systems HumTPO and HumLPL were investigated in eight human populations (Moroccans, Ovambos, Papuans, Australian aborigines, Germans, Turks, Japanese and Chinese). After electrophoresis, seven and eight alleles were identified in the HumTPO and HumLPL systems, respectively. In each population, no deviations from Hardy-Weinberg equilibrium were observed, but considerable differences in phenotype frequencies of each system were found between major ethnic groups.

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.