DNase I hypersensitivity in the gamma globin gene locus of K562 cells.

We have probed the chromatin conformation of the G gamma-A gamma-delta-beta globin gene locus of K562 cells, a human hematopoietic cell line, with the enzyme pancreatic DNAse I. This enzyme preferentially digests genes in an active configuration. We have found that in K562 cells, which produce embryonic and fetal but not adult hemoglobins, both the active gamma and inactive beta genes are DNAse I sensitive. However, only the active gamma genes have DNAse I hypersensitive regions. The hypersensitive regions have been mapped to an area approximately 100 base pairs 5' to the G gamma and A gamma genes.     

Development of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection and identification of Campylobacter jejuni, Campylobacter coli and Campylobacter fetus

A cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection of cdtA, cdtB or cdtC gene of Campylobacter jejuni, Campylobacter coli or Campylobacter fetus, respectively, was developed and evaluated with 76 Campylobacter strains belonging to seven different species and 131 other bacterial strains of eight different genera. The cdtA, cdtB or cdtC gene of C. jejuni, C. coli or C. fetus, respectively, could be successfully amplified using the corresponding set of primers in a highly species-specific manner. Furthermore, the specific primer set for the cdtA, cdtB or cdtC gene of a particular species could amplify the desired gene from a mixture of DNA templates of any of two or all three species. The detection limit of C. jejuni, C. coli or C. fetus was 10-100 CFU tube(-1) by the multiplex PCR assay on the basis of the presence of the cdtA, cdtB or cdtC gene. These data indicate that the cdt gene-based multiplex PCR assay may be useful for rapid and accurate detection as well as identification of Campylobacter strains in a species-specific manner.     

Inhibition of DNAse I activity in vivo]

Immune gamma-globulins containing antibodies to bovine DNAse I inhibit activities of bovine and mouse DNAse I both in vitro and in vivo. Bovine DNAse I was used as exogenous DNAse I in the in vivo studies and was injected to mice intraperitoneally in combination with gamma-globulins. The serous fluid of mice was used as a source of endogenous DNAse I. Both in in vitro and in vivo studies immune gamma-globulins caused a practically complete inhibition of bovine DNAse I activity, whereas the activity of mouse DNAse I (endogenous) was inhibited by 60-80%. Nonimmune gamma-globulins had no inhibitory effects whatsoever.     

Functional studies of the recombinant subunits of a cytolethal distending holotoxin

Cytolethal distending toxin (CDT) is a multicomponent bacterial holotoxin that targets most eukarytotic cells causing distension and cell cycle arrest. A number of diverse pathogenic bacterial species associated with diarrhoea, chancroid, chronic hepatitis and periodontal disease produce a CDT. Synthesis of the holotoxin is directed by the expression of three genes, cdtA , cdtB and cdtC . Although the product of the CdtB gene was previously identified as a type I deoxyribonuclease, the functions of the cdtA and cdtC products have not been characterized. Using the periodontal pathogen, Actinobacillus actinomycetemcomitans , we demonstrate that the recombinant product of the CdtA gene binds to the surface of Chinese hamster ovary (CHO) cells. This protein did not induce distension or cytotoxicity when introduced into the cytosol using a lipid-based protein delivery system. Recombinant CdtB and CdtC proteins failed to bind to CHO cells. However, the delivery of either CdtB or CdtC into the cytosol resulted in the characteristic pattern of distension followed by cell death. Based on these results, it appears that the CdtA protein subunit alone is responsible for anchoring the holotoxin to the cell surface. The CdtC subunit, in concert with CdtB, contributes to the cytotoxic activities of the holotoxin. The specific mechanism of CdtC cytotoxicity is currently unknown.     

Expression of the rat aldolase B gene: a liver-specific proximal promoter and an intronic activator

The nature and location of the cis-acting DNA sequences regulating expression of the rat aldolase B gene has been investigated. Two liver-specific DNAse I hypersensitive sites were detected, one located just upstream from the cap site, the second in the middle of the first, 4.8-kbp-long, intron. A fragment of 190 bp 5' to the cap site behaved as a tissue-specific but weak core promoter: it directed a detectable reporter gene expression in the Hep G2 cells and hepatocytes, but not in fibroblasts. The tissue-specific expression was stimulated at least 16 fold when constructs contained the entire first intron. The intronic activating sequences could be ascribed to an inner 2 kbp fragment in which the downstream liver-specific DNAse I hypersensitive site was located.     

Invasive capabilities of Campylobacter jejuni strains isolated in Bahrain: molecular and phenotypic characterization

The association between putative virulence genes in Campylobacter jejuni clinical isolates, in vitro invasive capability and severity of infection is yet to be clearly described. We have characterized three virulence genes and correlated their presence with the severity of infection and in vitro invasiveness. We studied eight C. jejuni strains isolated from patients whose clinical data were scored to determine severity of infection. Cytolethal distending toxin (cdtB), invasion associated marker (iam) and Campylobacter invasion antigen (ciaB) genes were detected by PCR and INT407 cells used for invasion assays. Two strains positive for all three genes were the most invasive and isolated from patients with the most severe infection. Four strains positive for two genes and two strains negative for all the three genes were identified. The two cdtB(+ve)/ciaB(+ve) strains were more invasive than the cdtB(+ve)/iam(+ve) strains. One of the cdtB(-ve)/ciaB(-ve) strains showed invasion levels similar to cdtB(+ve)/ciaB(+ve) strains, but the second strain had a non-invasive phenotype. The findings indicate a correlation between in vitro invasive capability, and the presence of all three genes. The pattern of association between invasiveness and molecular characterization suggests that the ciaB gene confers a more invasive capability.     

Inhibition of muscle tropomyosin polymerization by DNAse I

Tropomyosin polymerization is inhibited by DNAse I, an endonuclease which also interacts with G-actin. A 1:4 molar ratio of DNAse I to adult chicken pectoralis muscle tropomyosin almost completely prevents the increased viscosity of tropomyosin under polymerizing ionic conditions. While G-actin binding to DNAse I inhibits the DNAse I hydrolysis of DNA, tropomyosin does not affect this enzymatic activity. G-actin-DNAse I interaction is also not altered by tropomyosin.     

Biochemical characteristics of functionally active actin-like protein from liver mitochondria

The actin-like protein with a molecular weight of 42 kDa was obtained from the preparation of freshly isolated mitochondria of the rat liver using the method of immobilized DNAse affinity chromatography. The inhibitory ability of the isolated protein with respect to pancreatic DNAse I was the same as that of muscular actin. The native structure of the mitochondria protein is confirmed by the data of spectral analysis and its ability to globular-fibrillar transformation with an increased ionic strength of the solution. The polymerization ability as well as a stimulating effect of the actin-like protein of mitochondria on the ATPase activity of myosin is much less pronounced as compared to actin of skeletal muscles.     

Biochemical properties and localization of the chromosomal protein IP25.

The protein IP25, which has previously been reported to accumulate in the chromatin during erythroid differentiation of Friend-virus-transformed erythroleukemia cells (FL cells), is shown to behave like histone H1 without being structurally related to it. Like H1, IP25 is not released by digestion of FL cells nuclei with DNAse I. After micrococcal digestion IP25 and H1 are differentially distributed in the nucleosome monomers and dimers. This distribution suggests an internucleosomal location for IP25 and H1. Different rates of digestion are observed between nuclei of differentiating and non-differentiating FL cells with both DNAse I and micrococcal nuclease. These differences could be due to the presence of IP25 in the chromatin of differentiating cells.     

Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.

Previous studies of fluorescence probes for labeling the monomeric actin pool have demonstrated lack of specificity. We have used quantitative analytical methods to assess the sensitivity and specificity of rhodamine DNAse I as a probe for monomeric (G) actin. The G-actin pool of attached or suspended fibroblasts was stabilized by ice-cold glycerol and MgCl2. Formaldehyde fixation was used to clamp the filamentous (F) actin pool. G- and F-actins were stained by rhodamine DNAse I and FITC-phalloidin, respectively. Confocal microscopy indicated that the G- and F-actins were spatially separate in substrate-attached cells. Flow cytometry and fluorescence spectrophotometry demonstrated low co-labeling of the separate actin pools, although measureable background binding of rhodamine DNAse I was detectable. Estimates of the extent of actin polymerization after trypsinization demonstrated reciprocal changes of monomeric and filamentous actins, consistent with the formation of a perinuclear array of F-actin. The labeling and quantitation methods were also sufficiently sensitive to detect cell type-dependent variations in actin content. Dual labeling of cells with rhodamine DNAse I and FITC-phalloidin may provide a simple and direct method to image and quantify actin rearrangement in individual cells.     

Actin in mammalian lens.

In this paper evidence is provided that one of the protein components of the water-soluble fraction of the calf lens binds specifically to deoxyribonuclease I (DNAse I). On the basis of this property, the polypeptide could be purified by applying DNAse I affinity chromatography. Concomitantly a protein of Mr55000 and a rather large amount of alpha-crystallin copurify with this polypeptide, which has a molecular weight of 42000. Highly purified 42000-Mr protein was also obtained by extraction of the water-insoluble fraction of the calf lens with 2-([tris(hydroxymethyl)methyl]amino) ethanesulfonic acid followed by gel filtration. Amino acid analyses, peptide mapping and electron microscopy show that the protein obtained from both lens fractions is identical to non-muscle actin. Furthermore the amino acid composition of the 55000-Mr protein is identical to hog stomach skeletin and very similar to calf brain desmin.     

Digestion of chromatin with deoxyribonuclease II

The action of DNAse II on DNA in chromatin was studied. The formation of acid-soluble products followed a two-phase kinetic curve. At the end of the first more rapid phase about 25% of DNA was degraded. Early in the degradation process DNA was converted into double stranded fragments, whose sizes were multiples of about 180 base pairs. As the degradation proceeded these fragments were reduced in size. After denaturation DNA from digested chromatin was resolved into discrete single stranded fractions, exact multiples of a ten-nucleotide length, forming a pattern very similar to that observed with DNAse I.     

Nuclease Digestion of Chromatin from the Eukaryotic Algae Olisthodiscus luteus,Peridinium balticum,and Crypthecodinium cohnii 1, 2

Chromatin from a uninucleate dinoflagellate, Crypthecodinium cohnii, a binucleate dinoflagellate, Peridinium balticum, and a chromophyte, Olisthodiscus luteus, was examined by nuclease digestion and the results were compared to those from vertebrates. Gel analysis of the products of staphylococcal (micrococcal) nuclease digestion revealed a DNA repeat unit of 220(±5) base pairs for O. luteus and 215(±5) for P. balticum. Limit digestion gave a core particle of 140 base pairs, revealing that these longer repeat sizes are due to longer linker regions. No repeating subunit structure was found upon electrophoresis of digests of C. cohnii nuclei. Examination of the DNA fragments produced by DNAse I digestion of nuclei isolated from P. balticum and O. luteus showed the same ladder of ten base multiples as seen in chromatin from other eukaryotes. Examination of the kinetics of digestion by DNAse II of Peridinium chromatin revealed less susceptibility when compared to DNAse I digestions while 70% of Olisthodiscus chromatin and 35% of C. cohnii chromatin was sensitive to DNAse II. These data, taken together with previous results from Euglena, indicate that while algal chromatin is similar to that of higher eukaryotes in regard to DNAse I and II action, it differs in that the linker DNA is longer. In addition, the Hl-like histone from O. luteus and P. balticum is located in the linker DNA as in higher eukaryotes.     

Nuclear architecture,intranuclear DNA distribution,and nuclease digestion

G0, G1, and mammalian cells and nuclei were shortly digested with either micrococcal nuclease or DNAse I, both before and after mild fixation, either before (G0) or after (G1) partial hepatectomy. Cells were Feulgen stained and examined by high resolution light microscopy. In metabolically active G1 nuclei, intranuclear DNA appears organized at least in two distinct domains, whereby, the highly dispersed one is large enough to be detected at the resolution of the light microscope and appears preferentially attacked by limited DNAse I digestion. The action of the enzyme is readily apparent only in the nuclei that are first digested and then fixed. Spectroscopic characterization of the same nuclei reveals that the fixation causes a sizeable removal of proteins, mostly in the soluble chromatin subfraction. Results are discussed in terms of two control levels for gene expression and for higher order DNA structure.     

In situ DNAse I sensitivity assay indicates DNA conformation differences between CHO cells and the radiation-sensitive CHO mutant IRS-20

The radiosensitive mutant cell line IRS-20, its wild type counterpart CHO and a derivative of IRS-20 with a transfected YAC clone (YAC-IRS) that restores radioresistance were tested for DNAse I sensitivity. The three cell lines were cultured under the same conditions and had a mitotic index of 2-5%. One drop of fixed cells from the three lines was always spread on the same microscopic slide. After one day of ageing, slides were exposed to DNAse I and stained with DAPI. Images from every field were captured and the intensity of blue fluorescence was measured with appropriate software. For untreated cells, the fluorescence intensity was similar for all of the cell lines. After DNAse I treatment, CHO and YAC-IRS had an intensity of 85% but IRS-20 had an intensity of 60%, when compared with the controls. DNAse I sensitivity differences between the cell lines indicate that overall conformation of chromatin might contribute to radiation sensitivity of the IRS-20 cells.     

Nuclear architecture, intranuclear DNA distribution, and nuclease digestion

G0, G1, and mammalian cells and nuclei were shortly digested with either micrococcal nuclease or DNAse I, both before and after mild fixation, either before (G0) or after (G1) partial hepatectomy. Cells were Feulgen stained and examined by high resolution light microscopy. In metabolically active G1 nuclei, intranuclear DNA appears organized at least in two distinct domains, whereby the highly dispersed one is large enough to be detected at the resolution of the light microscope and appears preferentially attacked by limited DNAse I digestion. The action of the enzyme is readily apparent only in the nuclei that are first digested and then fixed. Spectroscopic characterization of the same nuclei reveals that the fixation causes a sizeable removal of proteins, mostly in the soluble chromatin subfraction. Results are discussed in terms of two control levels for gene expression and for higher order DNA structure.