Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris.

Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neutrophil extracellular traps. Since a systematic and direct comparison of the specific activities and properties of the secretory DNase1 family members is still missing, we expressed and purified recombinant murine DNase1 (rmDNase1), DNase1-like 2 (rmDNase1L2) and DNase1-like 3 (rmDNase1L3) using Pichia pastoris. Employing different strategies for optimizing culture and purification conditions, we achieved yields of pure protein between ~3 mg/l (rmDNase1L2 and rmDNase1L3) and ~9 mg/l (rmDNase1) expression medium. Furthermore, we established a procedure for post-expressional maturation of pre-mature DNase still bound to an unprocessed tri-N-glycosylated pro-peptide of the yeast α-mating factor. We analyzed glycosylation profiles and determined specific DNase activities by the hyperchromicity assay. Additionally, we evaluated substrate specificities under various conditions at equimolar DNase isoform concentrations by lambda DNA and chromatin digestion assays in the presence and absence of heparin and monomeric skeletal muscle α-actin. Our results suggest that due to its biochemical properties mDNase1L2 can be regarded as an evolutionary intermediate isoform of mDNase1 and mDNase1L3. Consequently, our data show that the secretory DNase1 family members complement each other to achieve optimal DNA degradation and chromatinolysis under a broad spectrum of biological conditions.

Endonuclease G modulates the alternative splicing of deoxyribonuclease 1 mRNA in human CD4+ T lymphocytes and prevents the progression of apoptosis.

Apoptotic endonucleases act cooperatively to fragment DNA and ensure the irreversibility of apoptosis. However, very little is known regarding the potential regulatory links between endonucleases. Deoxyribonuclease 1 (DNase I) inactivation is caused by alternative splicing (AS) of DNase I pre-mRNA skipping exon 4, which occurs in response to EndoG overexpression in cells. The current study aimed to determine the role of EndoG in the regulation of DNase I mRNA AS and the modulation of its enzymatic activity. A strong correlation was identified between the EndoG expression levels and DNase I splice variants in human lymphocytes. EndoG overexpression in CD4+ T cells down-regulated the mRNA levels of the active full-length DNase I variant and up-regulated the levels of the non-active spliced variant, which acts in a dominant-negative fashion. DNase I AS was induced by the translocation of EndoG from mitochondria into nuclei during the development of apoptosis. The DNase I spliced variant was induced by recombinant EndoG or by incubation with EndoG-digested cellular RNA in an in vitro system with isolated cell nuclei. Using antisense DNA oligonucleotides, we identified a 72-base segment that spans the adjacent segments of exon 4 and intron 4 and appears to be responsible for the AS. DNase I-positive CD4+ T cells overexpressing EndoG demonstrated decreased progression towards bleomycin-induced apoptosis. Therefore, EndoG is an endonuclease with the unique ability to inactivate another endonuclease, DNase I, and to modulate the development of apoptosis.

High-Level Production of DNA-Specific Endonuclease AsEndI with Synonymous Codon and its Potential Utilization for Removing DNA Contamination.

Endonuclease I is a widely distributed periplasmic or extracellular enzyme. A method for the high-level production of recombinant AsEndI (endonuclease I from Aliivibrio salmonicida) in Escherichia coli with secretion expression is investigated. The coding sequence of AsEndI gene was assembled according to the E. coli codon usage bias, and AsEndI was expressed in the periplasm of E. coli TOP10 with a C-terminal 6× His-tagged fusion. The recombinant AsEndI (His-AsEndI) was purified by Ni-NTA resin with a yield of 1.29 × 107 U from 1-L LB medium. His-AsEndI could be classified into Ca2+/Mg2+-dependent nucleases and showed highest nuclease activity to dsDNA at pH 8.0 and 37 °C. His-AsEndI is highly active in a broad range of salt concentration range up to 1.0 M with optimal NaCl concentration at 0.4 M. His-AsEndI can effectively remove DNA contamination in RNA sample or in PCR reagents to the level that cannot be detected by highly sensitive nested PCR and without adverse effects on the subsequent PCR reaction. His-AsEndI can remove DNA contamination at high salt conditions, especially for the DNA that may be shielded by DNA-binding protein at low salt conditions.

Reduced DNASE1 gene expression in thyroid autoimmunity.

The enzyme DNASE1 plays an important role in the hydrolysis of double-stranded DNA and might be related to autoimmunity. Therefore, the authors hypothesized that patients with autoimmune thyroid disease show a reduced expression of the DNASE1 gene. DNASE1 mRNA was quantitatively analyzed in 20 patients (10 with Hashimoto's thyroiditis and 10 with Graves' disease) and 20 age- and sex-matched healthy controls by real-time reverse transcription PCR in a lightcycler using SYBR-Green-format. For relative quantification, the mRNA ratio of the DNASE1 gene to the house keeping gene ß2-MICROGLOBULIN was used. The house keeping gene was proved not to be regulated by autoimmune thyroid disease. The interassay coefficient of variation for patients and controls was 22.2% and 15.6%, respectively, suggesting good reproducibility of measurements. The mean expression of the DNASE1 mRNA in patients was 0.52±0.22 (range 0.18-0.99) and in controls 0.95±0.22 (0.66-1.43). The expression level of the DNASE1 gene was strongly decreased in patients, amounting only 54.7% of that in controls (p<0.001). The lowered expression level in patients was not related to age or sex. This study demonstrated for the first time a downregulation of the DNASE1 mRNA expression in patients with autoimmune thyroid disease. This might result in degrading less DNA from dying cells, thereby promoting the development of thyroid autoimmunity.

Dissection of the molecular basis for hypervirulence of an in vivo-selected phenotype of the widely disseminated M1T1 strain of group A Streptococcus bacteria.

Group A streptococci (GAS) may engage different sets of virulence strategies, depending on the site of infection and host context. We previously isolated 2 phenotypic variants of a globally disseminated M1T1 GAS clone: a virulent wild-type (WT) strain, characterized by a SpeB(+)/SpeA(-)/Sda1(low) phenotype, and a hypervirulent animal-passaged (AP) strain, better adapted to survive in vivo, with a SpeB(-)/SpeA(+)/Sda1(high) phenotype. This AP strain arises in vivo due to the selection of bacteria with mutations in covS, the sensor part of a key 2-component regulatory system, CovR/S. To determine whether covS mutations explain the hypervirulence of the AP strain, we deleted covS from WT bacteria (DeltaCovS) and were able to simulate the hypervirulence and gene expression phenotype of naturally selected AP bacteria. Correction of the covS mutation in AP bacteria reverted them back to the WT phenotype. Our data confirm that covS plays a direct role in regulating GAS virulence.

Gln222Arg (A2317G) polymorphism in the deoxyribonuclease I gene exhibits ethnic and functional differences.

BACKGROUND: The single nucleotide polymorphism (SNP) at deoxyribonuclease I (DNase I) in exon 8 (A2317G: Gln222Arg) has been shown to be associated with several diseases. METHODS: The allele frequency of the DNASE1 polymorphism in Chinese (Shenyang and Guangzhou in China), Uygurs (Urumqi), Tamils (Sri Lanka), and Tibetans (Katmandu in Nepal) was investigated, and the results were compared with those of other studies. RESULTS: This study revealed that DNASE1*1 is more common in Africans and DNASE1*2 is more common in Caucasians. Expression vectors of DNASE1*1 and DNASE1*2 were constructed and compared to the enzyme properties secreted into a medium from transfected COS-7 cells. The activity of the type-2 enzyme was significantly higher than that of the type-1 enzyme. In addition, the type-1 enzyme was heat-labile when compared to the type-2 enzyme. Moreover, the optimum pH of the DNase I type-2 enzyme was more acidic than that of DNase I type-1. CONCLUSIONS: This study revealed that the distribution of Gln222Arg in the DNASE1 gene is different among ethnic groups and that the DNASE1 polymorphism appears to affect the specific activity, heat sensitivity, and optimum pH of the DNase I enzyme.

Murine serum deoxyribonuclease 1 (Dnase1) activity partly originates from the liver.

Reduction of serum DNASE1 (DNase I) activity is supposed to aggravate anti-nuclear autoimmunity, i.e. Systemic Lupus Erythematosus (SLE) in man and mice. To evaluate the etiology of this reduction, more information is needed about the source(s) and regulation of serum DNASE1. In this work we used male C57BL/6 wild-type (WT) mice to verify that serum Dnase1 activity partly depends on hepatic Dnase1 gene expression. Thus serum and liver Dnase1 activity showed a parallel oscillatory course during 24h, which was accompanied by a phase-shifted fluctuation of the hepatic Dnase1 mRNA content. Performing native PAGE zymography (NPZ) we detected a presumably premature non-sialylated and a mature sialylated hepatic Dnase1 isoform, which both show a parallel circadian fluctuation, indicating continuous secretion of Dnase1. The sialylated form was also detectable in serum. By immunostaining the hepatocytes were identified as the source of hepatic Dnase1 gene expression. After 70% hepatectomy, the serum Dnase1 activity increased markedly due to the occurrence of ischemic hepatocellular necrosis in the vicinity of the surgical suture. Similarly, hepatocellular necrosis induced by injection of streptolysin-O (SLO) into the liver led to a rapid parallel increase of Dnase1 and of aspartate- and alanine aminotransferase (AST/ALT) in serum. Subsequent to hepatectomy, Dnase1 gene expression was up-regulated in the regenerating liver most likely leading to an enhanced serum Dnase1 level until complete regeneration. These data demonstrate that serum Dnase1 at least partly originates from the liver and hint to the possibility that natural as well as pathological hepatic conditions influence its activity.

Wide-ranging genomic effects of plasticisers and related compounds.

UNLABELLED: The effects of four compounds, bis(2-ethylhexyl)phthalate (BEHP); diisodecylphthalate (DIP); 4-n-octylphenol (OP); 4-chloro-3-methylphenol (CMP), on gene expression (steady-state mRNA levels) across the whole human genome were studied in human TE671 cells. Effects were studied using the Affymetrics GeneChip Human Genome U133 Plus 2.0, HG-U133 Plus 2.0 arrays, The array analyses the expression of 47,000 transcripts and variants, including approximately 38,500 well characterised. All four compounds exerted statistically significant actions, affecting between 4 and 6.5% of all genes. Each compound had its own expression signature. In most instances where there was an effect, steady-state mRNA levels were decreased, although not always. CMP treatment caused most increases in mRNA levels. A mixture of DIP and CMP caused fewer changes in mRNA levels than either of the individual compounds. CONCLUSIONS: These plasticisers affected the steady-state mRNA levels of many human genes. Exposure to these compounds over many years has the potential to influence human health.

Effects of salt on the kinetics and thermodynamic stability of endonuclease I from Vibrio salmonicida and Vibrio cholerae.

Adaptation to extreme environments affects the stability and catalytic efficiency of enzymes, often endowing them with great industrial potential. We compared the environmental adaptation of the secreted endonuclease I from the cold-adapted marine fish pathogen Vibrio salmonicida (VsEndA) and the human pathogen Vibrio cholerae (VcEndA). Kinetic analysis showed that VsEndA displayed unique halotolerance. It retained a considerable amount of activity from low concentrations to at least 0.6 m NaCl, and was adapted to work at higher salt concentrations than VcEndA by maintaining a low K(m) value and increasing k(cat). In differential scanning calorimetry, salt stabilized both enzymes, but the effect on the calorimetric enthalpy and cooperativity of unfolding was larger for VsEndA, indicating salt dependence. Mutation of DNA binding site residues (VsEndA, Q69N and K71N; VcEndA, N69Q and N71K) affected the kinetic parameters. The VsEndA Q69N mutation also increased the T(m) value, whereas other mutations affected mainly DeltaH(cal). The determined crystal structure of VcEndA N69Q revealed the loss of one hydrogen bond present in native VcEndA, but also the formation of a new hydrogen bond involving residue 69 that could possibly explain the similar T(m) values for native and N69Q-mutated VcEndA. Structural analysis suggested that the stability, catalytic efficiency and salt tolerance of EndA were controlled by small changes in the hydrogen bonding networks and surface electrostatic potential. Our results indicate that endonuclease I adaptation is closely coupled to the conditions of the habitats of natural Vibrio, with VsEndA displaying a remarkable salt tolerance unique amongst the endonucleases characterized so far.

Hypoxia induces upregulation of the deoxyribonuclease I gene in the human pancreatic cancer cell line QGP-1.

We have previously demonstrated that ischemia caused by acute myocardial infarction induces an abrupt increase of serum deoxyribonuclease I (DNase I) activity. In this study, we examined whether hypoxia can affect the levels of DNase I activity and/or its transcripts in vitro. We first exposed the human pancreatic cancer cell line QGP-1, which is the first documented DNase-I-producing cell line, to hypoxia (2% O2), and found that this induced a significant increase in both the activity and transcripts of DNase I. This response was mediated by increased transcription only from exon 1a of the two alternative transcription-initiating exons utilized simultaneously in the human DNase I gene (DNASE1); exposure of QGP-1 cells to hypoxia for 24 h resulted in a 15-fold increase of DNASE1 transcripts starting from exon 1a compared with the expression level under normoxic conditions. Promoter, electrophoretic mobility shift, and chromatin immunoprecipitation assays with QGP-1 cells exposed to hypoxia or normoxia showed that the region just upstream from exon 1a was involved in this response in a hypoxia-induced factor-1-independent, but at least in a Sp1 transcription factor-dependent manner possibly through enhanced binding of Sp1 protein to the promoter. These results indicate that DNASE1 expression is upregulated by hypoxia in the cells.

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 human DNAS1L2 gene and the molecular mechanism for its transcriptional activation induced by inflammatory cytokines.

DNAS1L2, a member of the DNase I-like endonuclease family, is the only divalent cation-dependent acid DNase so far identified in mammals. The presence of a proline-rich domain (PRD) is its unique feature among family members. We found that a novel transcript encoding a short product, DNAS1L2-S, is expressed in peripheral blood leukocytes. Although DNAS1L2-S lacks the PRD, its enzymatic properties are apparently the same as those of the previously identified long form, DNAS1L2-L. Sequence analysis reveals that DNAS1L2 consists of seven exons. The exon/intron boundaries agree with the GT/AG rule with one exception: GC replaces GT at the 5' splice site in the sixth intron. TNF-alpha and IL-1beta are found to be potent inducers of DNAS1L2 expression in keratinocytes. They induce DNAS1L2 activation via the NF-kappaB pathway, and an NF-kappaB binding site located within the 5' flanking region is identified as the cis-responsive element.

Changing the enzymatic activity of T7 endonuclease by mutations at the beta-bridge site: alteration of substrate specificity profile and metal ion requirements by mutation distant from the catalytic domain.

Phage-encoded resolvase T7 endonuclease I is a structure-specific endonuclease. The enzyme acts on a broad spectrum of substrates with a variety of DNA structures. The enzyme is a dimer with two separated catalytic domains connected by an elongated beta-sheet bridge. The activities of enzymes with mutations in the beta-bridge segment were studied. Mutations that did not affect catalytic domain folding and function but did alter the relative positions of these domains retained catalytic activity but with altered specificity and metal ion dependence. Our results suggest that the enzyme recognizes its substrates by DNA conformation exclusion and offer a simple explanation for the broad substrate specificity of phage resolvase.

Changes in the topological expression of markers of differentiation and apoptosis in defined stages of human cervical dysplasia and carcinoma.

OBJECTIVE: We compared the capacity of cells in normal cervical epithelium, progressive stages of CIN, and invasive carcinoma to proliferate, differentiate, and undergo apoptosis. METHODS: We investigated 30 conizations showing regular squamous epithelium of the ectocervix, all stages of cervical preinvasive neoplastic lesions (CIN I to III), or invasive carcinoma. The expression of the cell proliferation and differentiation marker Ki67 and Mad-1, respectively, and of the apoptosis-related proteins bcl-2, active caspase-3, and DNase I was analyzed on paraffin sections by immunohistochemistry. The expression of DNase I or -like enzymes was also analyzed at the level of their gene transcripts by in situ hybridization. In addition, apoptotic events were identified by in situ end labeling of fragmented DNA (ISEL). RESULTS: Expression of Ki67 was restricted to suprabasal cells in normal cervical epithelium but increased with CIN severity and invasive carcinoma. ISEL demonstrated apoptosis in superficial layers of normal, CIN I, and CIN II epithelium, whereas in CIS (CIN III) and invasive carcinoma, ISEL-positive cells were additionally observed at varying epithelial locations. Bcl-2 immunostaining remained restricted to the basal layer of all preneoplastic and neoplastic stages. Active caspase-3 was present in the suprabasal layer and extended to all upper layers in normal epithelium and slightly decreased with increasing dysplasia. In invasive carcinoma it was restricted to few scattered cells. The differentiation marker Mad-1 extended from the spinous to the superficial layer in regular epithelium, but gradually shifted to more superficial layers with increasing CIN grade and invasive carcinoma. A similar topological change was observed for DNase I with increasing CIN grade. In CIS and invasive carcinoma, DNase I immunopositive cells were solely interspersed within neoplastic cells. In contrast, DNase I specific mRNA was present in all epithelial layers in CIN III and neoplasia, suggesting a translational block of the expression of DNase I or -like enzymes. CONCLUSION: Our data indicate that the elevated proliferation observed with increasing CIN severity and carcinoma was not paralleled by a similar increase in cell elimination. Most of the dysplastic and neoplastic cervical epithelial cells appeared incapable of entering terminal differentiation and complete it by apoptosis, possibly due to their failure to express or activate apoptosis executing enzymes.

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.

Induction of a DNA nickase in the presence of its target site stimulates adaptive mutation in Escherichia coli.

Adaptive mutation to Lac(+) in Escherichia coli strain FC40 depends on recombination functions and is enhanced by the expression of conjugal functions. To test the hypothesis that the conjugal function that is important for adaptive mutation is the production of a single-strand nick at the conjugal origin, we supplied an exogenous nicking enzyme, the gene II protein (gIIp) of bacteriophage f1, and placed its target sequence near the lac allele. When both gIIp and its target site were present, adaptive mutation was stimulated three- to fourfold. Like normal adaptive mutations, gIIp-induced mutations were recA(+) and ruvC(+) dependent and were mainly single-base deletions in runs of iterated bases. In addition, gIIp with its target site could substitute for conjugal functions in adaptive mutation. These results support the hypothesis that nicking at the conjugal origin initiates the recombination that produces adaptive mutations in this strain of E. coli, and they suggest that nicking may be the only conjugal function required for adaptive mutation.

Induction of apoptosis in the lung but not in the liver of rats receiving intra-tracheal instillations of chromium(VI).

Several studies have shown that hexavalent chromium [Cr(VI)] induces apoptosis in a variety of in vitro test systems. We instilled intra-tracheally either saline or sodium dichromate (0.25 mg/kg body weight), for three consecutive days, to Sprague-Dawley rats. TUNEL analyses showed a marked increase of the apoptotic index in both bronchial epithelium and lung parenchyma of Cr(VI)-treated rats, but no effect was detected in their liver. In parallel, the expression of 13 out of 18 apoptosis-related genes, evaluated by cDNA array analysis, was significantly enhanced in rat lung. The overexpressed genes included c-Jun N-terminal kinases 1, 2 and 3, bcl-x, bcl-2-associated death promoter and bcl-2-related ovarian killer protein, caspases 1, 3 and 6, DNase I precursor, DNA topoisomerases I and II alpha, and poly(ADP-ribose) polymerase. The enhancement of p53 expression in the lung was borderline to statistical significance. Expressions of bcl-2, bax-alpha, mdm2 and DNA topoisomerase IIB were not enhanced to a significant extent in lung. No induction of gene expression was observed in rat liver. RT-PCR analyses confirmed that Cr(VI) enhances the expression of c-Jun N-terminal kinase 1, caspase 6, and DNase I precursor but not that of bcl-2 in lung, while none of these genes was overexpressed in the liver of Cr(VI)-treated rats. The lack of stimulation of apoptosis in the liver parallels the failure of Cr(VI) to produce genotoxic damage, as we previously observed under identical experimental conditions. These negative findings may be ascribed to reduction of Cr(VI) to Cr(III) when traveling from the respiratory tract to the liver. On the other hand, induction of apoptosis in the respiratory tract parallels the occurrence of genotoxic effects and oxidative DNA damage produced by Cr(VI) in the same tissue. As previously shown in another laboratory, Cr(VI) did not induce lung tumors after 30 months of administration of the same daily dose. Therefore, apoptosis is likely to provide a protective mechanism at a post-genotoxic stage of Cr(VI) carcinogenesis.

Identification of an endonuclease secreted by human B lymphoblastic IM9 cells.

We have identified a Mg(2+)-dependent endonuclease from IM9 cell lysates and culture medium using DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) nuclease assay system. This particular endonuclease activity was not detectable in conventional DNA-SDS-polyacrylamide gel electrophoresis assay system which is similar to the method originally described by Rosenthal and Lacks (A.L. Rosenthal and S.A. Lacks, Anal. Biochem. 80 (1977) 76-90). Experimental results clearly demonstrated that the endonuclease activity was not derived from the fetal calf serum in which the cells were grown, but synthesized in the cell and secreted into the culture medium by IM9 cells. Biosynthesis and subsequent release of the endonuclease into the culture medium were significantly decreased by pretreatment of the cells with actinomycin D. Using supercoiled plasmid DNA as a substrate, the endonuclease activity was determined with the enzyme isolated from the cell culture medium by native-PAGE electroelution. The endonuclease, with Mg2+ alone, was able to catalyze the conversion of the plasmid into linear DNA followed by further degradation. This is the first report demonstrating that a distinct Mg(2+)-dependent endonuclease is secreted by a human immune cell line.

DNase I primary transcript is alternatively spliced in both normal and apoptotic cells: no evidence of up-regulation in apoptosis.

The reverse transcriptase polymerase chain reaction (RT-PCR) was used to elucidate the expression of DNase I and its possible involvement in apoptotic genome degradation. Multiple PCR products were obtained from cDNAs of different rat and human cells. The subsequent cloning and sequence analysis of seven PCR products revealed that only one of them had the expected size (639 bp) and sequence identity to that of rat DNase I cDNA. The other six PCR products were characterized by either sequence insertions or deletions. To establish the origin of this molecular diversity, a genomic fragment of the rat DNase I gene was also isolated, subcloned, and sequenced. Sequence comparison of six cDNAs with the rat genomic DNA revealed that they, indeed, resulted from inclusion of introns or deletion of exons. Southern hybridizations of the RT-PCR products from a variety of mammalian cell lines, using the major DNase I cDNA fragment as a probe, showed that in some cells as many as 20 alternatively spliced transcripts could be detected. This complexity of splice variants was widespread, and cell-specific profiles differed by the relative concentration of each transcript. None of these spliced transcripts maintained an open reading frame containing an intact catalytic site, suggesting that they do not encode any functional proteins. These complicated alternative splicing events might, however, significantly contribute to the regulation of DNase I expression. There was no apparent increase of the major DNase I transcript after induction of apoptosis in the cell lines studied. Apoptotic cells appeared to have similar normal/alternative transcript ratios as the control cells, suggesting that DNase I may not be the endonuclease involved in DNA degradation during apoptosis.

Rapid induction of Fas antigen mRNA expression in vivo by cycloheximide.

The effect of administration into rats of cycloheximide on the expression of genes, such as tissue transglutaminase, testosterone-repressed prostate message-2, Fas antigen, bcl-2, DNase I, and poly(ADP-ribose) polymerase, which were believed to be involved in the mechanism of apoptosis, was studied. While the effect of cycloheximide on the expression of genes other than Fas antigen was modest, only the expression of Fas antigen was elevated rapidly in most of the organs examined. A possible direct effect of cycloheximide on cells per se to induce Fas antigen mRNA expression was demonstrated by the tissue culture study using L929 fibroblast cells, although the magnitude of the induction detected in vitro was small compared with that in vivo. This induction of Fas antigen mRNA by cycloheximide is a first report on the modulation of Fas antigen mRNA expression in vivo.