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PubMed1999

In situ hybridization with polymerase chain reaction-derived single-stranded DNA probe and S1 nuclease.

A rapid and simplified protocol for in situ hybridization (ISH) with polymerase chain reaction (PCR)-derived single-stranded DNA probes and S1 nuclease revealed transcripts of bone matrix proteins on decalcified skeletal bone specimens. Mouse bone tissue was fixed with 4% paraformaldehyde, decalcified with 20% EDTA, and embedded in paraffin. Each pair of primers for reverse transcriptase -PCR was designed to amplify a 280-bp DNA fragment from the coding region of the mature protein of mouse osteonectin (ON) and a 320-bp fragment from the coding region of mouse osteopontin (OP). Initial PCR products were eluted, purified, and reamplified by unidirectional PCR in the presence of the digoxigenin (DIG)-labeled dUTP. ISH was carried out by proteinase K treatment, hybridization, and washing. The unhybridized single-stranded DNA probe was selectively removed by S1 nuclease treatment. Hybridized probes were visualized with the alkaline phosphatase-conjugated anti-DIG antibody. The transcripts of ON and OP were clearly detected on the thin sections of the decalcified bone. Because this protocol does not require cloning or in vitro transcription, reliable and stable ISH can be done in an ordinary laboratory equipped with a thermal cycler.

Animals↗

PubMed1978

S1 nuclease-specific nicking of mitochondrial DNA containing displacement loops.

Conditions are described in which the single strand-specific nuclease S1 selectively nicks mitochondrial DNA containing displacement loops without nicking supercoiled mitochondrial DNA. Using these conditions, the percentage of molecules containing displacement loops can be easily and accurately determined. This method is superior to the traditional electron microscopic examination for assessing the frequency of displacement loop-containing molecules. In addition, this method permits the determination of the relative specific activities of displacement loop and nondisplacement loop-containing mitochondrial DNA after various radioactive labeling protocols. S1 nuclease is shown to cleave the displaced strand of the displacement loop, to partially degrade the 7S-initiation strand, but not to cleave the parental template strand complementary to the 7S-initiation strand. The final product is a nicked circular molecule with at least two breaks localized within the displacement loop region in only one of the two parental strands.

Animals↗

PubMed1990

S1-nuclease enhancement of the ethidium bromide binding assay of drug-induced DNA interstrand crosslinking in human brain tumor cells.

A modification, using S1-nuclease, of a simple and sensitive fluorometric assay with ethidium bromide was developed for the measurement of cellular DNA interstrand crosslinking induced by bifunctional alkylators. Cells are lysed and treated with proteinase K and sodium dodecyl sulfate followed by extensive dialysis to yield intact high-molecular-weight DNA, free of contaminating proteins, on which the crosslink assay is then performed. The assay depends on the differential binding of ethidium bromide to single- and double-stranded DNA. Because of the higher ethidium bromide binding capacity of double-stranded DNA, the fluorescence retained after a heating/cooling cycle is directly proportional to the drug-induced cellular DNA interstrand crosslinking. We demonstrate that the sensitivity of this assay can be increased up to fourfold by including an S1-nuclease digestion step. This modified technique is simple and suited to the quantitation of low levels of DNA-interstrand crosslinking in cells.

Alkylating Agents↗

PubMed1982

Phospholipase A activity in commercial nucleases. Implications for membrane vesicle isolation.

Phospholipase A activity was detected in commercial DNAases I and II and in RNAase preparations. The amount of phospholipase correlates inversely with the degree of nuclease purification. The assessment of the level of phospholipase in commercial nucleases is important in cases where enzymatic properties other than those of DNAases and RNAases are to be investigated and when these preparations are to be used in the isolation of biological membranes.

Cell Fractionation↗

PubMed1978

The radiation-releasable cell wall nuclease of Micrococcus radiodurans. Purification and properties of the native enzyme.

Micrococcus radiodurans is known to possess a surface nuclease located in a mid-wall layer. Previous work showed that hydroxyl radicals, generated by sublethal doses of ionizing radiation, attack the cell wall and initiate release of the active enzyme into the external medium. The enzyme from unirradiated cells has now been purified to homogeneity by a simple 3-step process. The nuclease is a dimer of molecular weight about 260 000 and exonucleolytically degrades both DNA and RNA to 5'-mononucleotides. Single-stranded DNA is degraded at a rate about 200 times faster than double-stranded DNA. Oligonucleotides bearing a terminal 3'-phosphate are resistant to digestion. Dinucleotides must possess a 5'-terminal phosphate and a free 3'-terminal OH to be hydrolyzed. The enzyme has a pH optimum of about 9.0. A metal ion, possibly Ca2+, appears to be tightly bound to the protein. Removal of this metal with EDTA inactivates the enzyme. Simple readdition of Ca2+ does not restore activity although partial function can be recovered by renaturation from urea in the presence of this ion. The availability of pure enzyme should aid in the detection of radiation induced alterations which may be involved in the mechanism of its release from the cell wall.

Cell Wall↗

PubMed1976

Purification of sea urchin ribosomal RNA genes with a single-strand specific nuclease.

Ribosomal RNA genes (rDNA) from Lytechinus variegatus were isolated by selective heat denaturation of main band DNA followed by single-strand specific nuclease (S1) treatment to remove the single-stranded DNA. After S1 nuclease treatment the partially purified fraction contained 10% rDNA, representing a 50-fold purification. Preparative CsCl centrifugation of this fraction resulted in highly purified rDNA with an average molecular weight of 1.9 - 10(7) and no single-strand breaks. High molecular weight sea urchin DNA was refractory to selective heat denaturation. DNA with an average molecular weight of greater than or equal to 2.9 - 10(7) was only 60-80% denatured after heating 13 degrees C above the Tm, whereas, DNA with an average molecular weight of less than or equal to 1.9 - 10(7) was 98% denatured. This phenomenon appears not to be due to time, buffer, or pH, but is dependent on size.

Animals↗

PubMed1976

Peptides isolated from Enterobacter nuclease as potential polyamine binding sites.

The Enterobacter nuclease, which cleaves RNA between the 3'-phosphate group of cytidylic acid and the 5'-hydroxyl group of adenylic acid, has been shown to be affected by the polyamines, spermidine, spermine and putrescine. These substances enhance the hydrolytic activity of the enzyme against both poly(C) and yeast RNA. Sperimidine and spermine also reverse the inhibition of the enzyme by the ordered polynucleotides, apparently by removing them from the surface of the enzyme. Treatment of poly(G)-bound peptides (obtained from tryptic digests of poly(G)-bound nuclease) with an excess of spermidine resulted in the isolation of spermidine-bound peptides. Purification of these peptides through ion-exchange chromatography resulted in the isolation of three spermidine-bound peptides which consisted of 17 residues (6 amino acids), 19 residues (10 amino acids), and 12 residues (9 amino acids). The binding ratio of spermidine to peptides varied from 1:1 to 3:1.

Amino Acids↗

PubMed1983

Demonstration of an S1-nuclease sensitive site near the human beta-globin gene, and its protection by HMG 1 and 2.

An S1-nuclease sensitive site exists within supercoiled plasmids containing the 5'-flanking sequences of the human beta-globin gene. This site is located approximately 540 base pairs upstream from the start of the gene within a region of 52 alternating purine-pyrimidine residues which has the potential to adopt either cruciform structures or Z-form DNA. This site is protected from specific cleavage by S1-nuclease by the high-mobility-group chromosomal proteins HMG1 and 2, which may be specifically acting to protect short sequences of single-stranded DNA.

Base Sequence↗

PubMed1989

Molecular changes associated with induction of cell death in a human T-cell leukaemia line: putative nucleases identified as histones.

Following treatment of the human T-cell leukaemia line, CEM-C7, with the glucocorticoid, dexamethasone, a rapid decrease in viability occurred after 40 h which coincided with fragmentation of DNA in these cells. A similar pattern of DNA fragmentation was observed when these cells were gamma-irradiated or treated with cycloheximide. Distinct morphological changes occurred after treatment, indicating a form of cell death, regulated from within, termed apoptosis. A set of nuclear proteins ranging in size from 10-18 kDa appeared by 40 h following treatment with dexamethasone. Treatment of cells with gamma-irradiation or cycloheximide also produced the same protein pattern. This set of proteins, and a doublet approximately 55 kDa in size, had apparent nuclease activity which was not observed in untreated cells. However, protein microsequencing of these bands in the 10-18 kDa region revealed that they were histone proteins. These results cast doubt on a recent report which provided evidence that these proteins were induced nucleases.

Amino Acid Sequence↗

PubMed1991

Nuclease P1-mediated fluorescence postlabeling assay of AAF modified DNA model d(TACGTA) and calf-thymus DNA.

Fluorescence postlabeling assay for DNA damage combines enzymatic digestion of modified DNA to nucleoside monophosphates and fluorescence postlabeling. However, to facilitate the quantitative release of bulky adducts that are not readily obtained as mononucleotides, a different mechanism is essential. In order to test the application potential of nuclease P1-mediated fluorescence postlabeling to assay bulky adduct, d(TACGTA) was reacted with N-acetoxy-2-acetylamino-fluorene. The major product, characterized by nmr as AAF adduct of the guanine moiety at the C-8 position, was used as a DNA model. Nuclease P1 digestion of the modified oligomer excised the adduct in two forms (3:1) which were identified as AAF modified d(pGpT) and dpG respectively by cochromotography with authentic markers. Fluorescence postlabeling assay of AAF modified d(TACGTA) detected both forms of the excised adduct. The application of the overall procedure to assay AAF modified calf-thymus DNA demonstrated that the extension of fluorescence postlabeling technique from the mononucleotide to the dinucleotide version expands the scope of the assay.

Acetoxyacetylaminofluorene↗

PubMed1987

A nuclease-derived fragment of metaphase DNA and its relationship to the replicon.

When isolated Chinese hamster cells (CHO) metaphase chromosomes are treated with nuclease Bal-31, the DNA is reduced to a size class that is resistant to further degradation. This size class resembles the distribution of replicon sizes in this particular cell line in both average size and size range. Tests based on molecular weight (MW) analysis were devised to locate the origin of replication within the Bal-31 segments. The evidence indicates that replication origins are positioned at or near the center of these segments. The tests were made possible by the additional discovery that BrdU-substituted DNA is highly susceptible to Bal-31 nuclease attack while still contained in the isolated metaphase chromosome.

Animals↗

PubMed1992

Potentiation of DNA damage by inhibition of poly(ADP-ribosyl)ation: a test of the hypothesis for random nuclease action.

Poly(ADP-ribosyl)ation is a cellular response to DNA strand breaks by which a large array of proteins becomes covalently modified for a brief period during the lifetime of the DNA breaks. Inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide after many types of DNA damage leads to a marked increase in DNA strand breakage, repair replication, cytogenetic damage, mutagenesis, and cell killing. It has been hypothesized that poly(ADP-ribose) polymerase may modify potentially degradative endogenous nucleases that can reduce cellular viability. Thus, in the presence of DNA strand breakage, the polymer would bind these enzymes to inhibit their activity. When synthesis of the polymerase is inhibited, the enzymes would act randomly to produce nonspecific damage in the DNA. We tested this hypothesis by electroporating restriction enzymes into human cells containing the shuttle vector pHAZE. Restriction enzymes cleave at specific recognition sequences in the lacZ target gene of pHAZE, and mutations result from rejoining errors at the cleavage sites. If the hypothesis were correct, enzyme-treated cells cultured with 3-aminobenzamide to inhibit synthesis of poly(ADP-ribose) polymers would result in a significant increase in mutations outside the restriction enzyme sites. The spectrum of mutations observed after electroporation of PvuII (which produces blunt-end double-strand breaks) or PvuI (which produces cohesive-end double-strand breaks) was similar in untreated and 3-aminobenzamide-treated cells. Thus, our results do not support the hypothesis that the increase in damage observed when poly(ADP-ribosyl)ation is inhibited is due to a chaotic, nonspecific attack on DNA by endogenous cellular nucleases.

Benzamides↗

PubMed1984

Configuration of beta-globin messenger RNA in rabbit reticulocytes. Identification of sites exposed to endogenous and exogenous nucleases.

Masked and exposed sites in rabbit beta-globin messenger RNA were identified through S1 nuclease mapping of RNase T1 cleavage sites. Sites exposed to this enzyme were compared in deproteinized polysomal RNA and in mRNA in its native configuration in reticulocyte extracts. The analysis showed that most of the 3' non-coding region is well accessible to the enzyme, both in deproteinized RNA and in the cell extract. A possible protecting function for the poly(A) sequence is suggested by the fact that molecules with very short poly(A) segments were cleaved preferentially in this region. The G residues in the 5' non-coding region were inaccessible to RNase T1. A highly sensitive site adjacent to the initiation AUG codon was evident in the deproteinized RNA. This site was far less accessible to the enzyme in the mRNA associated with ribosomes in the cell extract. The first 150 nucleotides in the coding region showed very little susceptibility to digestion by the enzyme, in deproteinized RNA as well as in the cell extracts. Preparations of untreated mRNA showed the occurrence of truncated molecules, apparently generated by cleavage by endogenous nucleases. These cleavages were most prevalent in the two non-coding regions. They occurred at sites containing A-U sequences in the 3' non-coding region, and at sites with different sequences in the 5' non-coding region. Incubation of cell extracts at 37 degrees C did not cause any increase in these endogenous cleavages. It is suggested that they may have been generated in the intact cells, possibly as part of the mRNA degradation process in maturing reticulocytes.

Animals↗

PubMed1985

Conservation of major nuclease S1-sensitive sites in the non-conserved spacer region of ribosomal DNA in Drosophila species.

We have analysed nuclease S1-sensitive sites in cloned ribosomal DNA repeats from Drosophila melanogaster, D. hydei and D. virilis. All species contain major S1-sensitive sites in the spacer near the region of transcription termination, albeit with somewhat different positions and sensitivities. The same sites are also sensitive to the single-strand specificity of Bal31 nuclease at neutral pH. Additional major sites exist at each end of the intervening sequence within the 28 S gene of non-transcribed intervening-sequence-positive ribosomal DNA units of D. hydei. Only minor sites, however, were detected in the Pol I promoter regions. This is in contrast to Pol II transcribed genes, where S1 hypersensitivity becomes apparent at the 5' ends during gene expression. We have sequenced and mapped the S1 sites in the D. hydei spacer. They consist mainly of alternating A and T nucleotides that could form small cruciform structures. Cross-hybridization at low stringencies between the relevant S1-sensitive spacer regions of the three species indicates that the sites lie within very divergent sequences. We discuss the potential functional significance of S1 sites in rDNA spacers and intervening sequences, and the manner in which they might be maintained during rDNA sequence divergence.

Animals↗

PubMed1988

Molecular cloning and sequence of the concatemer junction from vaccinia virus replicative DNA. Viral nuclease cleavage sites in cruciform structures.

The concatemer junction from replicative forms of vaccinia virus DNA was cloned into plasmid vectors and shown to be a precise duplex copy of the viral terminal hairpin structure, with each strand corresponding to one of the alternative sequence isomers. The plasmids were relaxed circles with extruded cruciforms representing two copies of the vaccinia telomere hairpin structure. Head-to-head dimers containing two copies of the vaccinia virus concatemer junction were observed to contain only one set of stem-loop structures per molecule, suggesting that the initial formation of a small cruciform, and not branch migration, was the rate-limiting step in cruciform formation. The plasmids containing the concatemer junction were converted into nicked circular, linear and cross-linked linear molecules by a nuclease isolated from vaccinia virions. The region-specific cleavage near the border of the hairpin loop and the formation of DNA cross-links in some of the molecules is consistent with the nuclease acting as a nicking-closing enzyme that participates in the resolution of mature termini from replicative concatemer intermediates.

Base Sequence↗

PubMed1988

Analysis in vivo of the bacteriophage P22 headful nuclease.

Bacteriophage P22 packages its double-stranded DNA chromosomes from concatemeric replicating DNA in a processive, sequential fashion. According to this model, during the initial packaging event in such a series the packaging apparatus recognizes a nucleotide sequence, called pac, on the DNA, and then condenses DNA within the coat protein shell unidirectionally (rightward) from that point. DNA ends are generated near the pac site before or during the condensation reaction. The right end of the mature chromosome is created by a cut made in the DNA by the "headful nuclease" after a complete chromosome is condensed within the phage head. Subsequent packaging events on that concatemeric DNA begin at the end generated by the headful cut of the previous event and proceed in the same direction as the previous event. We report here accurate measurements of the P22 chromosome length (43,400( +/- 750) base-pairs, where the uncertainty is the range in observed lengths), genome length (41,830( +/- 315) base-pairs, where the uncertainty represents the accuracy with which the length is known), the terminal redundancy (1600( +/- 750) base-pairs or 3.8( +/- 1.8)%, where the uncertainty is the observed range) and the imprecision in the headful measuring device ( +/- 750 base-pairs or +/- 1.7%). In addition, we present evidence for a weak nucleotide sequence specificity in the headful nuclease. These findings lend further support to, and extend our understanding of, the sequential series model of P22 DNA packaging.

DNA, Viral↗

PubMed1989

Solution hybridization-nuclease protection assays for sensitive detection of differentially spliced substance P- and neurokinin A-encoding messenger ribonucleic acids.

In this chapter we discussed methods that can be used for the sensitive detection and quantitation of differentially or alternatively spliced mRNAs as well as mRNAs of low abundance. Although mechanisms responsible for splicing (and differential splicing in particular) have not been fully determined, many RNAs derived from a variety of genes have been observed to undergo the process. The impact of splicing with regard to the expanded potential of gene expression emphasizes the usefulness of the solution hybridization-nuclease digestion technique described here, compared to Northern blot analysis. The use of radiolabeled cRNA(s) provides for an assay of both high specificity and high sensitivity. While end-labeled cDNA probes can be used, they do not have the sensitivity inherent in the assay performed with uniformly radiolabeled cRNAs. If multiple mRNAs are derived from a single gene as a result of differential or alternative precursor RNA splicing, however, the results with a cRNA probe may initially appear to be quite complicated, and end-labeled cDNAs may yield more easily interpretable results. Nonetheless, both types of probes are useful in the context of gene expression analysis, and it is clear that for routine purposes of quantitation cRNA probes in solution hybridization-nuclease protection assays are clearly more desirable than RNA blot analyses due to their truly quantitative nature as well as ease of assay.

Autoradiography↗

PubMed1980

Secondary structure of mouse and rabbit alpha- and beta-globin mRNAs: differential accessibility of alpha and beta initiator AUG codons towards nucleases.

The nucleotide sequence from the 5' terminus inward of one third of mouse alpha- and beta maj-globin messenger RNAs has been established. In addition, using 5' 32P end-labeled mRNAs as substrates and S1 and T1 nucleases as probes for single-stranded regions, the secondary structures of mouse and rabbit alpha- and beta-globin mRNAs have been analyzed. Our results indicate that the AUG initiator codon in both mouse and rabbit beta-globin mRNA is quite susceptible to cleavage with S1 and T1 nucleases, suggesting that it resides in a single-stranded exposed region. In contrast, the initiator AUG in the alpha-globin mRNA of both species is inaccessible to cleavage, indicating that it is either buried by tertiary structure or is base-paired. Since the rate of initiation of protein synthesis with beta-globin mRNA in rabbit reticulocyte is 30--40% faster than for alpha-globin mRNA, these results imply a possible correlation between the differential rates of initiation with these two mRNAs and the accessibility of the respective AUG initiator codons.

Animals↗