Yasushi Kaneko

A Simple Method of DNA Extraction and STR Typing from Urine Samples Using a Commercially Available DNA/RNA Extraction Kit*

We devised a simple DNA extraction procedure suitable for STR typing of urine sample. Use of a commercially available DNA/RNA extraction kit equipped with a silica-gel-based membrane made it possible to omit the recovery of urinary nucleated cells by sedimentation before the extraction. Successful genotyping of the TH01, HumTPO and multiplex STRs was achieved using aliquots of urine as small as 100 microL. Furthermore, application of this DNA extraction procedure to frozen urine samples provided STR allele results comparable to results obtained from fresh samples. Therefore, this extraction procedure is considered to be effective for STR typing of urine samples in both the frozen and aqueous state. Furthermore, addition of sodium azide to fresh urine samples prolonged their storage duration even at room temperature.

Molecular, biochemical and immunological analyses of porcine pancreatic DNase I.

Deoxyribonuclease I (DNase I) was purified 26500-fold in 39% yield from porcine pancreas to electrophoretic homogeneity using three-step column chromatography. The purified enzyme was inhibited by an antibody specific to the purified enzyme but not by G-actin. A 1303 bp cDNA encoding porcine DNase I was constructed from total RNA from porcine small intestine using a rapid amplification of cDNA ends method, followed by sequencing. Mature porcine DNase I protein was found to consist of 262 amino acids. Unlike all other mammalian DNase I enzymes that are inhibited by G-actin, porcine DNase I has H65 and S114 instead of Y65 and A114, which presumably results in the lack of inhibition. Porcine DNase I was more sensitive to low pH than rat or bovine enzymes. Compared with their primary structures, the amino acid at position 110 was N in porcine enzyme, but S in rat and bovine enzymes. A porcine mutant enzyme in which N was substituted by S alone at position 110 (N110S) became resistant to low pH to a similar extent as the rat and bovine enzymes.

Transcriptional regulation of the human ABO histo-blood group genes is dependent on the N box upstream of the proximal promoter.

BACKGROUND:  Our previous studies of the transcriptional regulation of the human ABO gene indicated that negative regulatory elements are present in the sequence just upstream from the proximal promoter. The role of the −275 to −118 region in regulation of ABO gene transcription is further characterized.

Use of Human Recombinant DNase I Expressed in COS-7 Cells as an Immunogen to Produce a Specific Anti-DNase I Antibody

To obtain human deoxyribonuclease I (DNase I) as an immunogen, we have developed a procedure that is more useful and effective than the conventional procedure, which uses human urine as a starting material. In the new procedure, we culture COS-7 cells transfected with expression vector carrying human DNase I cDNA, and then purify the enzyme from the culture medium. The enzyme can be easily isolated to apparent homogeneity by passage through only three chromatography columns. The rabbit antiserum that we used against the recombinant DNase I was not inferior to that used against DNase I from human urine, in terms of both its ability to discriminate DNase I phenotypes and its ability to neutralize enzyme activity. Therefore, our procedure may be useful for producing an antibody specific for human DNase I.

Abrupt pubertal elevation of DNase I gene expression in human pituitary glands of both sexes

Deoxyribonuclease I (DNase I) was confirmed to be expressed in the human pituitary gland, particularly the anterior lobe, at levels comparable to those in the pancreas. The DNase I activity and the amount of gene transcript present in the pituitary glands of individuals aged from 1 month to 89 years was significantly age‐dependent, with an abrupt elevation after the neonatal and prepubertal periods irrespective of gender, followed by a gradual age‐dependent decline in males and a marked reduction in females in their postreproductive period. This DNase I age dependence in the pituitary gland was not present in the pancreas and serum. These observations suggest that tissue‐specific up‐regulation of DNase I gene expression in the pituitary gland occur, possibly at the onset of puberty.

DNase I Is Present in the Chief Cells of Human and Rat Stomachs

The distribution of deoxyribonuclease I (DNase I) in human and rat stomachs was examined by biochemical, molecular biological and immunohistochemical techniques. By the use of monoclonal anti-human DNase I and polyclonal anti-rat DNase I antibodies, we determined that strong immunoreactivity was present in the cytoplasm of chief cells of the human fundus and the rat pars glandularis, respectively. High DNase I enzyme activity was detected in tissue homogenates of both human fundus and rat pars glandularis. The presence of DNase I-specific mRNA was verified by reverse transcriptase-polymerase chain reaction analysis of the total RNAs extracted from human and rat stomachs. Immunoelectron microscopy revealed gold particles localized in the chief cells, with most labelling in exocrine secretory granules. These results show that the chief cells of human and rat stomach produce DNase I. This is the first report to demonstrate that secretion of DNase I is controlled by the chief cells in human and rat stomachs.

Usefulness of deoxyribonuclease I (DNase I) polymorphism for individualization from small aged urine stains

We devised a procedure that combines a simple extraction method, isoelectric focusing and activity staining using the dried agarose film overlay method, for deoxyribonuclease I (DNase I) typing from aged urine stains. DNase I types were determined without difficulty from urine stains kept at room temperature for 3 months or more in all of the samples tested. The amounts of urine stains required for typing after 3 months of storage were estimated to be equivalent to 60-120 microl of liquid urine. Therefore, considering that useful PCR-based DNA typing has not yet been developed for urine stains, DNase I polymorphism could be considered the first biochemical marker found to be well suited for individualization from small aged urine stains.

Production and characterization of murine monoclonal anti-human DNase II antibodies, and their use for immunoaffinity purification of DNase II from human liver and urine

Four murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.

Japanese population data for two STR loci, HumTPO and HumLPL

The two short tandem repeat (STR) systems, HumTPO and HumLPL, were investigated in blood samples obtained from approximately 800 unrelated Japanese individuals living in seven geographically different areas of Japan. Neither deviation from a Hardy-Weinberg equilibrium nor significant difference between the allele distributions was found among the seven Japanese populations in the two STR systems. These findings indicate that there is a general uniformity for both the STR loci in the Japanese population.

Production of murine monoclonal anti-human DNase II antibodies, and their use for immunoaffinity purification of DNase II from human liver and urine

Murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion and DNA-cast polyacrylamide gel electrophoresis were very useful screening methods for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.