Enzymatic digestion of DNA gamma-irradiated in aqueous solution separation of the digests by ion-exchange chromatography.

Aqueous solutions of DNA were gamma-irradiated in the presence and absence of oxygen and enzymatically hydrolysed by the combined action of pancreatic deoxyribonuclease (DNase I), snake-venom phosphodiesterase (PDE I), spleen phosphodiesterase (PDE II) and alkaline phosphatase. In contrast to unirradiated DNA, which is fully hydrolysed to nucleosides by these enzymes, gamma-irradiated DNA yields a series of oligonucleotides. Their isolation might enalbe the future identification of the chemical nature of DNA lesions.     

Molecular, biochemical and immunological analyses of canine pancreatic DNase I

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.     

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.     

Single-cell cDNA-PCR: removal of contaminating genomic DNA from total RNA using immobilized DNase I.

A procedure utilizing immobilized DNase I that allows the efficient amplification of cDNA by PCR from a single cell in the absence of contaminating genomic DNA is described. DNase I treated, total RNA derived from single cells was reverse transcribed into cDNA followed by PCR using beta-actin and c-fos specific primers that recognize different exons of the respective genes. Amplification products corresponding to cDNA, but not to genomic sequences, were detected after treatment with immobilized DNase I in samples previously shown to be contaminated with genomic DNA. This method allows the efficient removal of DNA contaminating total RNA derived from a single cell.     

Susceptibility of mammalian deoxyribonucleases I (DNases I) to proteolysis by proteases and its relationships to tissue distribution: biochemical and molecular analysis of equine DNase I

Equine (Equus caballus) deoxyribonuclease I (DNase I) was purified from the parotid gland, and its 1295-bp cDNA was cloned. The mature equine DNase I protein consisted of 260 amino acid residues. The enzymatic properties and structural aspects of the equine enzyme were closely similar to those of other mammalian DNases I. Mammalian DNases I are classified into three types--pancreatic, parotid and pancreatic-parotid-based on their tissue distribution; as equine DNase I showed the highest activity in the parotid gland, it was confirmed to be of the parotid-type. Comparison of the susceptibility of mammalian DNases I to proteolysis by proteases demonstrated a marked correlation between tissue distribution and sensitivity/resistance to proteolysis; pancreatic-type DNase I shared properties of resistance to proteolysis by trypsin and chymotrypsin, whereas parotid-type DNase I did not. In contrast, pancreatic-parotid-type DNase I exhibited resistance to proteolysis by pepsin, whereas the other enzyme types did not. However, site-directed mutagenesis analysis revealed that only a single amino acid substitution could not account for acquisition of proteolysis resistance in the mammalian DNase I family during the course of molecular evolution. These properties are compatible with adaptation of mammalian DNases I for maintaining their activity in vivo.     

Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.     

Carp hepatopancreatic DNase I: biochemical,molecular, and immunological properties

A survey of DNase I in nine different carp tissues showed that the hepatopancreas has the highest levels of both DNase I enzyme activity and gene expression. Carp hepatopancreatic DNase I was purified 17,000-fold, with a yield of 29%, to electrophoretic homogeneity using three-step column chromatography. The purified enzyme activity was inhibited completely by 20 mM EDTA and a specific anti-carp DNase I antibody and slightly by G-actin. Histochemical analysis using this antibody revealed the strongest immunoreactivity in the cytoplasm of pancreatic tissue, but not in that of hepatic tissue in the carp hepatopancreas. A 995-bp cDNA encoding carp DNase I was constructed from total RNA from carp hepatopancreas. The mature carp DNase I protein comprises 260 amino acids, the same number as the human enzyme, however, the carp enzyme has an insertion of Ser59 and a deletion of Ala225 in comparison with the human enzyme. These alterations have no influence on the enzyme activity and stability. Three amino acid residues, Tyr65, Val67, and Ala114, of human DNase I are involved in actin binding, whereas those of carp DNase I are shifted to Tyr66, Val68, and Phe115, respectively, by the insertion of Ser59: the decrease in affinity to actin is due to one amino acid substitution, Ala114Phe. The results of our phylogenetic and immunological analyses indicate that carp DNase I is not closely related to the mammalian, avian or amphibian enzymes, and forms a relatively tight piscine cluster with the tilapia enzyme.     

Estrogen induces tissue specific changes in the chromatin conformation of the vitellogenin genes in Xenopus.

Nuclei from male Xenopus liver were digested extensively with DNase I and the residual amount of the four vitellogenin genes measured by hybridization with a moderate excess of vitellogenin cDNA. The saturation value was about twofold lower in chromatin isolated from liver cells of estrogen treated than from untreated males or from erythrocytes. Analyzing the disappearance of several defined restriction fragments specific for the A1 and A2 vitellogenin genes, after limited digestion with DNase I, suggested that the entire A1 and A2 vitellogenin genes are about twofold more sensitive to DNase I in chromatin of hepatocytes isolated from estrogen treated than from untreated males. Using the same assay no change in the DNase I sensitivity of the two vitellogenin genes in erythrocyte chromatin was observed. Analysis of the beta 1-globin and an albumin gene demonstrated that the DNase I sensitivity of these genes in both cell types is not altered by estrogen. All these data indicate that estrogen stimulation results in an increased DNase I sensitivity specific for the vitellogenin genes in hepatocytes.     

Differential regulation of PDE5 expression in left and right ventricles of feline hypertrophy models

Background

Though long known to affect smooth muscle biology, recent studies indicate that phosphodiesterase 5 (PDE5) is also expressed in myocardium. Recognizing that the regulation of PDE5 in hypertrophy is not well understood, we assessed the response of PDE5 expression and the level of cGMP-dependent kinase I (cGKI) in the left and right ventricles of feline hypertrophy models.

Methodology/Principal Findings

Using a cDNA library of feline aortic smooth muscle cells, we identified and cloned PDE5 cDNA for the first time in this species. The sequence shares 98% identity with its human orthologue at the amino acid level. E. coli expression of the cloned allele allowed selection of antibodies with appropriate specificity, facilitating the analysis of PDE5 expression in feline models created by selective proximal aortic (Ao) or pulmonary artery (PA) banding that resulted in hypertrophy of the left ventricle (LV) and right ventricle (RV), respectively. We demonstrated that PDE5 expression responded differentially with a decreased expression in the LV and an increased expression in the RV in the Ao-banded model. Similarly, in the PA-banded model, LV showed reduced expression while the RV expression was unaltered. In addition, the expression of cGKI was significantly decreased in the RV of Ao-banded group, correlating inversely with the increase in PDE5 expression.

Conclusions/Significance

The differential regulation of PDE5 and cGKI expression suggests that the mechanisms involved in hypertrophy could be different in RV vs. LV. Reciprocal PDE5 and cGKI expression in the RV of Ao-banded model suggests functional significance for PDE5 up-regulation.     

Cloning,sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: purification and characterization of the recombinant enzyme

The bovine pancreatic (bp-) DNase I gene has been cloned from bp-cDNA and expressed in E. coli. A polynucleotide sequence of 1295 base pairs was deduced from clones of the cDNA. The sequence showed an open reading frame which can be translated as a 282-amino acid polypeptide, including a hydrophobic signal peptide and the polypeptide of bp-DNase I. An expression plasmid was constructed by inserting into the vector pET-15b, a cDNA fragment coding for bp-DNase I ligated with a hexanucleotide coding for Met–Ala at the 5′-end. The plasmid was transformed into E. coli strain DH5α and the active recombinant bovine (rb-) DNase I was produced after induction of protein synthesis. From the induced culture medium, rb-DNase I was purified by chromatography on a Mono Q column. The purified rb-DNase I showed a molecular mass of 29 kDa and had the same specific activity as bp-DNase I. The NH₂-terminus of rb-DNase I was Ala, not Met, and at position 19, corresponding to the carbohydrate attachment site of bp-DNase I, Asn was not glycosylated.     

The cloning, genomic structure, localization, and expression of human deoxyribonuclease IIbeta

Acidic endonuclease activity is present in all cells in the body and much of this can be attributed to the previously cloned and ubiquitously expressed deoxyribonuclease II (DNase II). Database analysis revealed the existence of expressed sequence tags and genomic segments coding for a protein with considerable homology to DNase II. This report describes the cloning of this cDNA, which we term deoxyribonuclease IIbeta (DNase IIbeta) and comparison of its expression to that of the originally cloned DNase II (now termed DNase IIalpha). The cDNA encodes a 357 amino acid protein. This protein exhibits extensive homology to DNase IIalpha including an amino-terminal signal peptide and a conserved active site, and has many of the regions of identity that are conserved in homologs in other mammals as well as C. elegans and Drosophila. The gene encoding DNase IIbeta has identical splice sites to DNase IIalpha. Human DNase IIbeta is highly expressed in the salivary gland, and at low levels in trachea, lung, prostate, lymph node, and testis, whereas DNase IIalpha is ubiquitously expressed in all tissues. The expression pattern of human DNase IIbeta suggests that it may function primarily as a secreted enzyme. Human saliva was found to contain DNase IIalpha, but after immunodepletion, considerable acid-active endonuclease remained which we presume is DNase IIbeta. We have localized the gene for human DNase IIbeta to chromosome 1p22.3 adjacent (and in opposing orientation) to the human uricase pseudogene. Interestingly, murine DNase IIbeta is highly expressed in the liver. Uricase is also highly expressed in mouse but not human liver and this may explain the difference in expression patterns between human and mouse DNase IIbeta.     

Chromatin structure of active and inactive human X chromosomes

Nuclei from a variety of human cell lines and tissues were digested with gradually increasing levels of DNase I. The DNA was then purified, treated with restriction enzymes and subjected to Southern blot hybridization using a cloned cDNA probe to 3-phosphoglycerate kinase (PGK) a housekeeping enzyme. At relatively high levels of DNase I, a specific, slightly sensitive site in chromatin sequences encoding PGK was observed in all of the cell types examined. This slightly sensitive site resides on the active X-chromosome since cell lines with increased numbers of inactive X-chromosomes do not show an increase in the region of chromatin which is sensitive. Except for this restricted region of enhanced sensitivity on the active X-chromosome, the data suggest that, for PGK encoding sequences, chromatin configurations on the active and inactive X-chromosomes are similar.     

Rapid measurement of deoxyribonuclease I activity with the use of microchip electrophoresis based on DNA degradation

Deoxyribonuclease I (DNase I) activity in serum has been shown to be a novel diagnostic marker for the early detection of acute myocardial infarction (AMI). However, the conventional method to measure DNase I activity is time-consuming. In the current study, to develop a rapid assay method for DNase I activity for clinical purposes, a microchip electrophoresis device was used to measure DNase I activity. Because DNase I is an endonuclease that degrades double-stranded DNA endo-nucleolytically to produce oligonucleotides, degradation of the DNA standard caused by DNase I action was detected using microchip electrophoresis. We detected DNase I activity within 10 min. This is the first study to apply microchip electrophoresis for the detection of DNase I activity; furthermore, it seems plausible that reduction of analysis time for DNase I activity could make this novel assay method using microchip electrophoresis applicable in clinical use.