Tamiko Nakajima

MOUSE DEOXYRIBONUCLEASE I (DNASE I) : BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION, CDNA STRUCTURE AND TISSUE DISTRIBUTION

Mouse urinary deoxyribonuclease I (DNase I) resembles rat and human DNase Is in terms of its proteochemical and enzymological properties. Furthermore, mouse DNase I was demonstrated to be immunologically closer to the rat than to the human enzyme. A 1176 bp full length cDNA encoding mouse DNase I was constructed from RNA obtained from the kidney and parotid glands. The amino acid sequence up to the 45th residue from the N‐terminal of the mature enzyme was identical to that deduced from the cDNA sequence. This DNase I was distributed most densely in the parotid glands from the standpoint of both enzyme activity and gene transcript levels.

Expression of ABO blood-group genes is dependent upon an erythroid cell-specific regulatory element that is deleted in persons with the B(m) phenotype.

The ABO blood group is of great importance in blood transfusion and organ transplantation. However, the mechanisms regulating human ABO gene expression remain obscure. On the basis of DNase I-hypersensitive sites in and upstream of ABO in K562 cells, in the present study, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays indicated a novel positive regulatory element in intron 1. This element was shown to enhance ABO promoter activity in an erythroid cell-specific manner. Electrophoretic mobility-shift assays demonstrated that it bound to the tissue-restricted transcription factor GATA-1. Mutation of the GATA motifs to abrogate binding of this factor reduced the regulatory activity of the element. Therefore, GATA-1 appears to be involved in the cell-specific activity of the element. Furthermore, we found that a partial deletion in intron 1 involving the element was associated with B(m) phenotypes. Therefore, it is plausible that deletion of the erythroid cell-specific regulatory element could down-regulate transcription in the B(m) allele, leading to reduction of B-antigen expression in cells of erythroid lineage, but not in mucus-secreting cells. These results support the contention that the enhancer-like element in intron 1 of ABO has a significant function in erythroid cells.

Phenotype 2 of deoxyribonuclease I may be Used as a risk factor for gastric carcinoma

Human deoxyribonuclease I (DNase I) (EC3.1.21.1) is one of the candidate nucleases that acts in mammalian cells during apoptosis. Genetic polymorphism of DNase I has been classified into 15 phenotypes controlled by 5 autosomal codominant alleles. The purpose of this study was to determine whether DNase I polymorphism is closely related to the incidence of gastric carcinoma.

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.

Caucasian-specific allele in non-synonymous single nucleotide polymorphisms of the gene encoding deoxyribonuclease I-like 3, potentially relevant to autoimmunity, produces an inactive enzyme

BACKGROUND Deoxyribonuclease I-like 3 (DNase Il3), a member of human DNase I family, is postulated to be involved in the genesis of autoimmune diseases. In the DNase Il3 gene, 2 non-synonymous SNPs, R178H and R206C, have been identified, however relevant population data are not available. METHODS Genotyping of the SNPs was performed in healthy subjects belonging to 3 ethnic groups (n=1708), including nine different populations, using an amplification refractory mutation system and the PCR-RFLP technique. RESULTS All of the 9 populations were typed as a single genotype in R178H. All Asian and African populations exhibited only a homozygous C686 allele in R206C, whereas a heterozygote, C686/T686, was found (frequency of 3.5-15.4%) in three Caucasian populations (Turk, German and Mexican); Caucasian-specific allele T686 was identified. The substitution of Arg by Cys corresponding to R206C resulted in elimination of DNase Il3 activity. CONCLUSION A Caucasian-specific allele in SNP R206C produces an inactive form of DNase Il3. It seems plausible that levels of DNase Il3 activity in Caucasian subjects with the heterozygote in R206C are lower than those in individuals with the predominant homozygote. Our results may have clinical implications in relation to the prevalence of autoimmune diseases.

Identification of the three non‐identical subunits constituting human deoxyribonuclease II

We purified DNase II from human liver to apparent homogeneity. The N‐terminal amino acid sequences of each of three components constituting the purified mature enzyme were then separately determined by automatic Edman degradation. A combination of this chemical information and the previously reported nucleotide sequence of the cDNA encoding human DNase II [Yasuda et al. (1998) J. Biol. Chem. 273, 2610–2626] allowed detailed elucidation of the enzymes subunit structure: human DNase II was composed of three non‐identical subunits, a propeptide, proprotein and mature protein, following a signal peptide. Expression analysis of a series of deletion mutants derived from the cDNA of DNase II in COS‐7 cells suggested that although a single large precursor protein may not be necessary for proteolytic maturation, the propeptide region L17–Q46 may play an essential role in generating the active form of the enzyme.

Two novel screening methods for selecting monoclonal antibodies which specifically inhibit DNase I enzyme activity

Two novel screening methods, single radial enzyme diffusion and the DNA-cast polyacrylamide gel electrophoresis, for selecting monoclonal antibodies which detect human deoxyribonuclease I (DNase I) enzyme activity are described. The former was adopted for initial screening to select potential objective antibodies from numerous hybridoma culture supernatants, because it was easy to perform and a powerful mass-screening tool. The latter was utilized for the subsequent precise selection of the antibodies in the supernatants selected after preliminary screening by the former, because it was clearly more accurate and sensitive, although the procedure was slightly more complicated. The consecutive use of these two methods resulted in the isolation of 25 anti-human DNase I antibodies, all of which specifically inhibited the activity of human DNase I.

Mutation of the GATA site in the erythroid cell–specific regulatory element of the ABO gene in a Bm subgroup individual

The ABO blood group is important in blood transfusion. Recently, an erythroid cell–specific regulatory element has been identified in the first intron of ABO using luciferase reporter assays with K562 cells. The erythroid cell–specific regulatory activity of the element was dependent upon GATA‐1 binding. In addition, partial deletion of Intron 1 including the element was observed in genomic DNAs obtained from 111 Bm and ABm individuals, except for one, whereas the deletion was never found among 1005 individuals with the common phenotypes.

Molecular, biochemical and immunological studies of hen pancreatic deoxyribonuclease I.

Deoxyribonuclease I (DNase I) was purified from the hen pancreas to electrophoretic homogeneity using six-step column chromatography. The purified enzyme showed a molecular mass of about 33 kDa and maximum activity at pH 7.0. It required divalent cations, Mg2+ and Ca2+, for its activity and was inhibited by EDTA, EGTA and an antibody specific to the purified enzyme but not by G-actin. A 1066-bp cDNA encoding hen DNase I was constructed from the total RNA of a hen pancreas using a combination of the reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends methods, followed by sequencing. The cDNA was expressed in Escherichia coli, and the recombinant polypeptide exhibited significant enzyme activity. The mature hen DNase I protein was found to consist of 262 amino acids. In human and bovine DNase I four amino acid residues, Glu-13, Tyr-65, Val-67 and Ala-114 are involved in actin binding, whereas in the hen DNase I these positions were occupied by Asp, Phe, Ser and Phe, respectively. A survey of the DNase I distribution in 15 hen tissues showed that the pancreas had the highest levels of both DNase I enzyme activity and DNase I gene expression. The results of our phylogenetic and immunological analyses indicate that the hen DNase I is not closely related to the mammalian enzymes. This is the first report in which has been described the results of molecular, biochemical and immunological analyses on hen DNase I.