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The ABO blood group is of great importance in blood transfusion and organ transplantation. However, the mechanisms regulating human ABOgene expression remain obscure. On the basis of DNase I–hypersensitive sites in and upstream of ABOin 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 ABOpromoter 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 Bmphenotypes. Therefore, it is plausible that deletion of the erythroid cell–specific regulatory element could down-regulate transcription in the Bmallele, 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 ABOhas a significant function in erythroid cells.

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 Bm phenotypes. Therefore, it is plausible that deletion of the erythroid cell–specific regulatory element could down-regulate transcription in the Bm 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.

Deoxyribonuclease I (DNase I) is known to be a glycoprotein, and two potential N-linked glycosylation sites (N18 and N106) are known for mammalian enzymes. In the present study, N18 and N106 were mutated in order to investigate the biological role of N-linked glycosylation in three mammalian (human, bovine, and equine) DNases I. The enzyme activities of N18Q and N106Q were lower than that of the wild type, and that of the double mutant (N18Q/N106Q) was lower than those of the single mutants, in accord with the sugar moiety contents in the three mammals. In addition, all mutant enzymes were unstable to heat, suggesting that both sites are required for heat stability. Moreover, in human and equine enzymes, the N18Q and N106Q mutant enzymes were less resistant to trypsin, while N18Q/N106Q was the most sensitive to trypsin. As for bovine DNase I, the trypsin resistance of N18Q and N106Q was similar to that of the wild type, but that of N18Q/N106Q decreased in a time-dependent manner. On the other hand, N-linked glycosylation was not related to pH sensitivity. The results of the present study suggest that N18 and N106 are both necessary for (i) full enzymatic activity, (ii) heat-stability, and (iii) trypsin resistance.

Abstract: Deoxyribonuclease I (DNase I) polymorphism has been used as a valuable marker in genetic and clinical investigations. Six codominant alleles are known for DNase I, DNASE1∗1, ∗2, ∗3, ∗4, and the recently discovered alleles ∗5 and ∗6. To detect these two new alleles, we added a new DNase I genotyping method based on both an allele-specific amplification and mismatched polymerase chain reaction (PCR). These methods were used to examine the distribution of DNase I genotypes in unrelated individuals from bloodstains of Ovambo and Turkish populations. The DNASE1∗1 allele was found to be most dominant in the Ovambos. In contrast, Turks showed the highest allele frequency for DNASE1∗2. This study is the first to demonstrate that there is a certain genetic heterogeneity in the worldwide distribution of DNase I polymorphism using the genotyping method of human DNase I polymorphism with PCR.

We purified four amphibian deoxyribonucleases I from the pancreases of one toad, two frog and one newt species, by using three different column chromatography methods in sequence. Each of the purified enzymes had a molecular mass of approx. 40kDa and an optimal pH for activity of approx. 8.0. These values were significantly greater than those for other vertebrate DNases I. The full-length cDNA encoding each amphibian DNase I was constructed from the total RNA of the pancreas by using rapid amplification of cDNA ends. Nucleotide sequence analyses revealed two structural characteristics unique to amphibian DNases I: a stretch of approx. 70 amino acids with a high cysteine content (approx. 15%) in the C-terminal region, and the insertion of a serine residue at position 205 (in a domain containing an essential Ca2+-binding site). Expression analysis of a series of mutant constructs indicated that both of these structures are essential in generating the active form of the enzyme. ‘DNase I signature sequences’, which are well conserved in other vertebrate DNases I, could not be found in any of the amphibian DNases I tested, whereas a ‘somatomedin B motif’ was identified in the Cys-rich stretches of all four. Although DNase I has so far been considered to be a secretory glycoprotein, amphibian DNase I seems to be non-glycosylated. These structural findings indicate strongly that amphibian DNases I are situated in a unique position on the phylogenetic tree of the DNase I family.

Administration of somatostatin to rats induced a transient reduction of serum levels of deoxyribonuclease I (DNase I) activity in a dose-dependent manner, followed by a substantial decrease of DNase I activity in the lower gut. Activity in the parotid gland, liver, and kidney did not change. Real-time PCR analysis of the DNase I gene transcript in ileum indicated that the decrease was due to down-regulation of gene expression. Based on these responses, rat tissues expressing DNase I could be classified into two types, somatostatin-sensitive and somatostatin-resistant, and the level of DNase I activity in the lower gut seems to be controlled by somatostatin.

Deoxyribonuclease I (DNase I) activities were measured in 14 different tissues from humans and 5 other mammals (bovine, pig, rabbit, rat, and mouse) by using the single radial enzyme diffusion (SRED) method, which is a sensitive and nonradioactive assay for nucleases. The results indicated that these species are classifiable into three groups on the basis of their different tissue distributions of DNase I. In human and pig, the pancreas showed the highest activity of DNase I; in rat and mouse, the parotid glands showed the highest activity; and in bovine and rabbit, both pancreas and parotid glands showed high activity. Therefore we designated human and pig DNase I as pancreas type, rat and mouse DNase I as parotid type, and bovine and rabbit DNase I as pancreas–parotid (or mixed) type. DNase I of the pancreas type was more sensitive to low pH than the other types. DNase I of pancreas type is secreted into the intestinal tract under neutral pH conditions, whereas the other types are secreted from the parotid gland and have to pass through the very acidic conditions in the stomach. Differences in the tissue distribution and acid sensitivity of mammalian DNases I may provide important information about their digestive function from the evolutionary perspective.

Deoxyribonuclease II (DNase II) levels in human vary depending on whether the individual has the DNASE2*H(high) allele or the DNASE2*L(low) allele. We examined the promoter activity of the 5′‐flanking region of each of these alleles by transient transfection luciferase assay. DNASE2*Hhad 5‐fold higher promoter activity than DNASE2*Lin human hepatoma HepG2 cell. Comparison of the nucleotide sequences of the proximal promoter regions revealed a G to A transition at position −75; G and A residues were assigned to DNASE2*Hand *L, respectively. Since no differences were found between the open reading frame sequences of these alleles, it is likely that the A−75G transition causes the allelic difference in the promoter activity of the gene, underlying the genetic polymorphism.

Deoxyribonuclease II (DNase II) levels in human vary depending on whether the individual has the DNASE2*H(high) allele or the DNASE2*L(low) allele. We examined the promoter activity of the 5′-flanking region of each of these alleles by transient transfection luciferase assay. DNASE2*Hhad 5-fold higher promoter activity than DNASE2*Lin human hepatoma HepG2 cell. Comparison of the nucleotide sequences of the proximal promoter regions revealed a G to A transition at position −75; G and A residues were assigned to DNASE2*Hand *L, respectively. Since no differences were found between the open reading frame sequences of these alleles, it is likely that the A−75G transition causes the allelic difference in the promoter activity of the gene, underlying the genetic polymorphism.

Using site-directed mutagenesis, we eliminated three potential N-glycosylation sites (N86, N212, and N266) of human deoxyribonuclease II (DNase II), conserved in mammalian enzymes, and a proteolytic processing site (Q46–R47), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N86and N266and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II activities, irrespective of cell line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneous attachment of a carbohydrate moiety to N86and N266, cleavage of the propeptide from the single DNase II precursor, and the inherent signal peptide might be required for subcellular sorting and proteolytic maturation of the enzyme.

A new allele, DNASE1*6,of human deoxyribonuclease I (DNase I) has been discovered by isoelectric focusing: its gene product has the most cathodic pIof the six electrophoretic variants. Results of DNA sequencing, mismatched PCR-restriction fragment length polymorphism, and transient transfection of the variant construct showed that the mutant was caused by a C–T transition at nucleotide position 1826, resulting in an Arg to Cys substitution at amino acid position 185 of the mature enzyme. The variant isoenzyme, expressed in COS-7 cells, was more labile than the other types. Instability and an increase in the pIvalue of the variant suggest that a structural alteration, perhaps due to aberrant formation of a disulfide bond, could occur in the enzyme.

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 enzyme's 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–Q46may play an essential role in generating the active form of the enzyme.

Abstract: In addition to the three polymorphic sites responsible for protein polymorphism, a new polymorphic site has been identified in intron 7 of the human deoxyribonuclease I (DNase I) gene. Three phenotypes were observed on single-strand conformational polymorphism analysis of a 266-bp polymerase chain reaction-amplified fragment containing exon 7 and part of intron 7 of the human DNase I gene. DNA sequencing analysis demonstrated that a C-G substitution occurred at position 1978 in intron 7. This substitution was confirmed by restriction fragment length polymorphism analysis, since a new Msp1 site is created by the substitution. Population and family studies showed that the inheritance of the genotypes for DNase I C1978G polymorphism is controlled by two codominant alleles, tentatively designated DNASE1*1978C and *1978G. The gene frequencies in a Japanese population were significantly different from those in a Caucasian (German) population. The C1978G polymorphism is in linkage disequilibrium with the common DNase I protein phenotypes 1, 1–2, and 2.

The main isozyme patterns of desialylated blood plasma or serum a-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-a-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 FUCA11* and FUCA12* 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 FUCA11* gene product in plasma has about 1.4 times the activity of FUCA12*.

Affinity chromatography on immobilized ribonuclease (RNase) inhibitor was developed for purification of mammalian RNase. Human placental RNase inhibitor was conjugated to CNBr-activated Sepharose in the presence of dithiothreitol. About 80% of the immobilized RNase inhibitor was capable of binding bovine pancreatic RNase A. The bound RNase A was eluted with 3MNaCl at pH 5.0. Two 25-kDa and 18-kDa RNases, which were obtained from human liver using a cellulose phosphate column, were bound to the immobilized RNase inhibitor and recovered in a pure and active form after treatment of the resin withp-hydroxymercuribenzoate. These enzymes were considered to be nonsecretory-type RNases with different sugar contents.

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.

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).

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).

DNase I from rabbit urine was purified approx. 3600-fold to apparent homogeneity with a 41% yield by affinity chromatography utilizing DNA–cellulose; the purity of the final preparation was assessed by SDS/PAGE, lack of contamination by other nucleases and production of a monospecific antibody against the enzyme. Although the proteochemical and enzymological properties of the purified enzyme resembled those of other mammalian DNases I, the enzymic activity of rabbit DNase I was less efficiently inhibited by monomeric actin than was that of human DNase I, probably due to substitution of an amino acid residue involved in actin binding (Tyr-65 to Phe). The effects of specific antibodies to human, rabbit and rat DNases I on the activities of the corresponding purified enzymes revealed that human DNase I lies between the rat and rabbit enzymes with regard to its immunological properties. An 1158 bp full-length cDNA encoding rabbit DNase I was constructed from the total RNA of rabbit pancreas using a combination of reverse transcriptase-PCR and rapid amplification of cDNA ends, followed by sequencing. This identified a 17- or 21-amino-acid signal sequence, with the mature enzyme containing 260 amino acids and a single N-glycosylation site at Asn-18. The amino acid sequence deduced from the cDNA sequence exactly matched that determined proteochemically from the purified enzyme up to residue 20. A systematic survey of DNase I distribution as measured by both enzymic activity and DNase I gene transcripts in 12 rabbit tissues showed the pancreas and parotid gland to produce equivalent levels, higher than those in other tissues. Enzymic activity and DNase I gene expression levels in each tissue correlated well. The results of phylogenetic and sequence identity analysis, immunological properties and tissue-distribution patterns of DNase I indicated a closer relationship between the rabbit and human enzymes than for other mammalian DNases I. Furthermore, differences between the enzymic activities expressed in mammalian parotid gland and pancreas suggest that the distribution of DNase I in mammalian tissue is species-specific.

Recently obtained information on the cDNA encoding human deoxyribonuclease II (DNase II) (T. Yasudaet al.,1998, J. Biol. Chem. 273, 2610–2616) has made it possible to demonstrate the precise position of the the human DNase II gene (DNASE2) on human chromosomes. Two different sets of oligonucleotide primers specific for human DNase II cDNA sequences were used to amplify unique DNA fragments in the human DNase II gene from a panel of human × rodent hybrid cell lines carrying different human chromosomes. Based on this analysis,DNASE2was assigned to human chromosome 19. Furthermore, regional localization of the gene to 19p13.2–p13.1 was achieved by fluorescencein situhybridization analysis using a full-length cDNA probe corresponding to the entire open reading frame.