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PubMed1979

The regulatory region of MS2 phage RNA replicase cistron. III. Characterization of fragments resulting from S1 nuclease digestion.

The functionally active fragments MS2 R(-53 leads to 6) and MS2 R(-53 leads to 3) comprising the regulatory region for the replicase cistron have been isolated from MS2 RNA-coat protein complex following T1 RNase digestion. In order to obtain shorter fragments, active in coat protein binding and initiation of translation, MS2 R(-53 leads to 6) was cleaved with S1 nuclease. The results indicate that S1 nuclease attacks the most susceptible loop regions of the two hairpin helices of MSZ R(-53) leads to 6). Among the three fragments which have been isolated, only MS2 R(-35/33 leads to 6) containing the intact hairpin (b) region with initiation codon AUG is active in the coat protein binding. Functional activity exerted by another polynucleotide MS R(-17 leads to 6) supports the assumption that specific binding with the coat protein is determined by the hairpin (b) region prior to the replicase cistron.

Base Sequence↗

PubMed1979

S1 nuclease definition of highly repeated DNA sequences in the Guinea pig, Cavia porcellus.

Native DNA of the Guinea pig, Cavia porcellus, purified from liver or tissue culture cells, was heat denatured and reassociated to a Cot value of 0.01 (equivalent Cot value of 7.2 x 10(-2)). The reassociated DNA was isolated by digestion with the single-strand DNA specific enzyme S1 nuclease. Spectrophotometric and radioactivity assays demonstrated that 24% of the total DNA was resistant to S1 nuclease treatment. Zero-time reassociation indicated that approximately 3% of the DNA was inverted repeat sequences. Thus, highly repeated sequences comprised 21% of the total genome. CsCl buoyant density ultracentrifugation indicated that this fraction was composed of both main band and satellite sequences. Although actinomycin D - CsCl density gradients failed to give significant separation of the repetitive sequences, distamycin A - CsCl gradients were able to fractionate the DNA into several overlapping bands. The heterogeneity of the repetitive DNA was further demonstrated by the first derivative plots calculated from their thermal denaturation profiles. This analysis revealed six major thermalytes which indicate that there may be at least six discrete components in the repetitive DNA.

Animals↗

PubMed1980

Accessibility of expressed and non-expressed genes to a restriction nuclease.

Nuclei of mouse liver and an immunoglobulin producing myeloma were digested with HaeIII or its isoschizomer BspRI. The DNA fragment patterns after electrophoresis, blotting, and hybridization were very similar for the two types of nuclei when a probe for the non-expressed beta-globin gene was used. The constant (C) region of the kappa light chain gene, on the other hand, was more accessible to the nuclease in myeloma than on liver nuclei. In myeloma nuclei the BspRI sites were about equally sensitive, in fact the pattern resembled a partial digestion pattern of free DNA. In contrast, in liver nuclei some sites were much more protected than others. This is interpreted by assuming that this single copy non-expressed gene region is covered by nucleosomes which are preferentially located on certain DNA sequences. Restriction nuclease digestion of nuclei seems to be a promising method for the analysis of genes in their expressed and non-expressed states.

Animals↗

PubMed1981

Nuclease sensitivity of chromatin containing active genes: kinetic analyses utilizing continuous elution of digestion products from an ultrafiltration cell.

Methods have been developed to analyze the kinetics of digestion of chromatin by nucleases. Radioactively labeled nuclei were incubated with enzyme in an ultrafiltration apparatus and digestion rates of different chromatin samples were computed employing a least-squares curve fitting technique to fit the data to zero-order and/or first-order kinetic models. These methods allow detailed kinetic analyses on small amounts of chromatin. Two biological systems were studied. 1) Tetrahymena thermophila macronuclei and micronuclei were compared; these nuclei differ in their transcriptional activities. 2) Ribosomal DNA (rDNA) of Tetrahymena pyriformis, approximately 60% of which codes for rRNA, can be preferentially labeled during starvation-refeeding; its digestion kinetics relative to bulk chromatin were studied. DNase I digested 20-40% of the macromolecular DNA about 3 times faster than bulk macronuclear or micronuclear DNA, and 60-80% of ribosomal gene-containing chromatin about 5 times faster than bulk chromatin. Filter hybridization studies of the DNAase I sensitivity of tRNA, 5S RNA, and ribosomal genes yielded similar results. These data are consistent with the observation that transcribed genes are especially sensitive to attach by DNase I and suggest that activated chromatin structure as probed by extensive DNase I digestion is the same in higher and lower eucaryotes for genes transcribed by all three RNA polymerases. Digestion kinetics of micrococcal nuclease were found to depend on the digestion conditions employed. These two biological systems and the methods we have developed should facilitate analyses of the factors responsible for maintaining an active chromatin structure.

Animals↗

PubMed1981

Ribosomal RNA cleavage, nuclease activation and 2-5A(ppp(A2'p)nA) in interferon-treated cells.

Ribosomal RNA (rRNA) in intact ribosomes is cleaved into discrete products on incubation of reticulocyte lysates or L-cell extracts with ppp(A2'p)3A. Cleavage of rRNA may, therefore, provide a useful assay for 2-5A (ppp)A2'p)nA; n = 2 to 4) or for the presence of a 2-5A-dependent nuclease. The results with reticulocyte lysates differed from those obtained in the L-cell-free system in that (a) a different RNA cleavage pattern was produced (with added L-cell ribosomes) and (b) cleavage was fully activated by the analogue ppp(A2'p)3A3'pCp. As might be expected from the relatively high levels of 2-5A present in interferon-treated, encephalomyocarditis virus (EMC)-infected L-cells, rRNA extracted from these cells was also cleaved. The cleavage pattern observed overlapped with that obtained on incubation of an L-cell-free system with 2-5A. Thus, not only is 2-5A present, but the 2-5A-dependent nuclease also appears to be active, in interferon-treated, EMC-infected L-cells.

Adenine Nucleotides↗

PubMed2005

DNA end-directed and processive nuclease activities of the archaeal XPF enzyme.

The XPF/Mus81 family of structure-specific nucleases cleaves branched or nicked DNA substrates and are implicated in a wide range of DNA repair and recombination processes. The structure of the crenarchaeal XPF bound to a DNA duplex has revealed a plausible mechanism for DNA binding, involving DNA distortion into upstream and downstream duplexes engaged by the two helix-hairpin-helix domains that form a dimeric structure at the C-terminus of the enzyme. A flexible linker joins these to the dimeric nuclease domain, and a C-terminal motif interacts with the sliding clamp, which is essential for the activity of the enzyme. Here, we demonstrate the importance of the downstream duplex in directing the endonuclease activity of crenarchaeal XPF, which is similar to that of Mus81-Eme1, and suggest a mechanistic basis for this control. Furthermore, our data reveal that the enzyme can digest a nicked DNA strand processively over at least 60 nt in a 3'-5' direction and can remove varied types of DNA lesions and blocked DNA termini. This in vitro activity suggests a potential role for crenarchaeal XPF in a variety of repair processes for which there are no clear pathways in archaea.

Archaeal Proteins↗

PubMed2006

Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases.

Individual zinc finger (ZF) domains that recognize DNA triplets with high specificity and affinity can be used to create designer transcription factors and nucleases that are specific for nearly any site in the genome. These domains can be treated as modular units and assembled to create a polydactyl protein that recognizes extended DNA sequences. Deter-mination of valid target sites and the subsequent design of ZF proteins (ZFPs) is error-prone and not trivial, however. As a result, the use of ZFPs have been restricted primarily to those labs with the appropriate expertise. To address these limitations, we have created a user-friendly utility called Zinc Finger Tools (ZF Tools) that can be accessed at the URL http://www.zincfingertools.org. User-supplied DNA sequences can be searched for target sites appropriate for either gene regulation or nuclease targeting. Using a database of experimentally characterized zinc finger domains, the amino acid sequence for a ZFP expected to bind to any chosen target site can be generated. A reverse engineering utility is provided to predict the binding site for a ZFP of known sequence.

Binding Sites↗

PubMed2003

Simultaneous detection of multiple single nucleotide polymorphism by single-strand-specific nuclease and PNA probe.

The combination of PNA (peptide nucleic acid) and single-strand-specific nuclease have been used for detection of single nucleotide polymorphisms (SNPs). When DNA is perfectly complementary to PNA, it is protected from digestion by the nuclease. If there exists a single-base mismatch between them, however, the DNA is completely digested. These differences are visualized by using 3,3'-diethylthiadicarbocyanine (DiSc2(5)), which changes its color from blue to purple upon binding to PNA/DNA hybrids. In terms of this methodology, homozygous and heterozygous SNPs in apoE gene have been successfully analyzed. Furthermore, the multiplex SNPs are simultaneously genotyped. This technique provides a simple, straightforward, facile, and visual genetic screening, with no need for expensive and complicated equipment.

Base Sequence↗

PubMed2004

High-throughput SNP genotyping by combining exonuclease III, nuclease S1, and acridine-bearing PNA.

Recently we reported a precise and straightforward method for genotyping of single nucleotide polymorphisms (SNPs) in double-stranded DNA substrates. The substrates were consecutively treated by two enzymes (exonuclease III and nuclease S1) in the presence of peptide nucleic acid (PNA) probes, and the resultant DNA fragments were analyzed by mass spectroscopy. Here, the selectivity of DNA scission by nuclease S1 has been greatly improved by attaching an acridine to the PNA probes. Modification at the C-terminus was especially effective. The number of DNA fragments formed has been greatly decreased so that double-stranded DNA substrates can be genotyped by using the two enzymes still more easily and precisely.

Acridines↗

PubMed2002

Genes for a nuclease and a protease are involved in the drastic decrease in cellular RNA amount in fission yeast cells during nitrogen starvation.

Cellular RNA in Schizosaccharomyces pombe cells drastically decreases in amount during nitrogen starvation. Previously, we found and purified a soluble RNA-degrading enzyme whose activity drastically increased in the cells of S. pombe undergoing nitrogen starvation. The enzyme was a nuclease encoded by pnu1(+). In this study, the increase in the RNA-degrading activity and the decrease in cellular RNA level are examined in a null-mutant of pnu1(+) (pnu1Delta). During nitrogen starvation, wild-type cells show an apparent increase in RNA-degrading activity, whereas the pnu1Delta cells do not. The wild-type cells show a drastic decrease in cellular RNA amount, whereas the pnu1Delta cells show only a slight decrease. These results suggest that Pnu1 nuclease is implicated in the decrease in cellular RNA amount during nitrogen starvation, probably via the RNA-degrading activity. The increase in the RNA-degrading activity is independent of both the Wis1 stress-activated MAP kinase cascade and Tor1 signaling pathway, but it is strongly dependent on isp6(+), a gene for a possible protease, whose expression is induced during nitrogen starvation. A disruption mutant for isp6(+) (isp6Delta) is deficient in both the increase in the RNA-degrading activity and the drastic decrease in the cellular RNA amount during nitrogen starvation, which suggests that isp6(+) is involved in the RNA degradation via regulating the RNA-degrading activity of Pnu1.

Endopeptidases↗

PubMed2005

Mononuclear cells from patients recovered from cutaneous leishmaniasis respond to Leishmania major amastigote class I nuclease with a predominant Th1-like response.

The Leishmania major amastigote class I nuclease (LmaCIN) is a developmentally regulated protein that is highly expressed in the amastigote stage of L. major. This protein is homologous to the P4 nuclease of L. pifanoi, which has been shown to induce protective immune response in a murine model. To evaluate LmaCIN as a potential human vaccine candidate, cellular immune responses to recombinant LmaCIN were examined in individuals recovered from Old World cutaneous leishmaniasis. Peripheral blood mononuclear cells (PBMC) from patients recovered from L. major infection were cultured either with recombinant LmaCIN or autoclaved L. major (ALM) as control. rLmaCIN induced significant proliferation of PBMC from 90% of recovered patients. Phenotypic analysis of proliferating cells showed that CD8(+) cells were the predominant cell type proliferating in response to rLmaC1N. Screening of culture supernatants for cytokines showed that rLmaCIN induced high levels of interferon (IFN)-gamma (mean +/- s.e.m.: 1398 +/- 179 pg/ml) associated with little interleukin (IL)-10 and little or no IL-5 production. These findings show that LmaCIN is immunogenic in humans during L. major infection and that it can elicit immunological responses relevant to immunoprophylaxis of leishmaniasis.

Analysis of Variance↗

PubMed1991

The repair of double-strand breaks and S1 nuclease-sensitive sites can be monitored chromosome-specifically in Saccharomyces cerevisiae using pulse-field gel electrophoresis.

Repair under non-growth conditions of DNA double-stranded breaks (DSBs) and S1 nuclease-sensitive sites (SSSs; e.g. DNA damage which is processed by in vitro treatment with S1 nuclease to DSBs) induced by [60Co]-gamma-rays (200 Gy; anoxic conditions) was monitored in a diploid repair-competent strain of Saccharomyces cerevisiae. We used pulsed-field gel electrophoresis (PFGE), which allows the separation of chromosome-sized yeast DNA molecules, to determine the number of DSBs and SSSs in individual chromosome species of yeast. Our results indicate that SSSs which have been regarded as clusters of base damage in opposite DNA strands are repaired efficiently in a repair-proficient diploid strain of yeast. The time course of SSS repair is comparable to the one of DSB repair, indicating similarities in the molecular mechanism. Both types of repair kinetics are different for different chromosome species.

Anaerobiosis↗

PubMed1993

Characterization of the nucM gene coding for a nuclease of the phytopathogenic bacteria Erwinia chrysanthemi.

The gene nucM encoding a nuclease was cloned from a genomic library of Erwinia chrysanthemi. The nucM gene was subcloned, and mutagenized by insertion of a uidA-KanR cartridge. This mutation was introduced by recombination into the Erwinia chrysanthemi chromosome. The nucM mutant lost NucM activity when tested on a DNA plate after 24 hours, but still possessed secondary weak nuclease activity. The nucleotide sequence of nucM was determined. It presents a 798 bp open reading frame, coding for a 266-amino-acid protein, with a predicted molecular mass of 29,910 Da. The deduced NucM protein shows 59% sequence identity with the DNase I precursor from Vibrio cholerae. It contains a typical leader sequence. Experiments of cell fractionation showed that NucM is periplasmic in E. chrysanthemi. The transcription start has been determined by S1 mapping. The -10 and -35 regions do not show homology with consensus sequence of the promoters recognized by sigma 70. In fact, the promoter seems to be dependent on the sigma 70, but the first transcription nucleotide is unusually far from the -10 region. nucM seems to be expressed constitutively.

Amino Acid Sequence↗

PubMed1994

DNA-binding proteins as site-specific nucleases.

DNA-binding proteins can be converted into site-specific nucleases by linking them to the chemical nuclease 1,10-phenanthroline-copper. This can be readily accomplished by converting a minor groove-proximal amino acid to a cysteine residue using site-directed mutagenesis and then chemically modifying the sulphydryl group with 5-iodoacetamido-1,10-phenanthroline-copper. These chimeric scission reagents can be used as rare cutters to analyse chromosomal DNA, to test predictions based on high-resolution nuclear magnetic resonance and X-ray crystal structures, and to locate binding sites of proteins within genomes.

Catalysis↗

PubMed1975

Investigation of the repetitive sequences in calf DNA by cleavage with restriction nucleases.

Calf thymus DNA was digested with the restriction nucleases from Escherichia coli carrying resistance transfer factor I, Haemophilus influenzae Rd and Bacillus subtilis X5 (EcoRI, Hind II, and Bsu, respectively) and submitted to polyacrylamide gel electrophoresis. About 10% of the DNA migrated as discrete fragments in 8, 16, and 30 bands, respectively, superimposed upon a continuous distribution of various size DNA fragments. The fragments within the bands are repeated 2000 to 160000 times in the haploid genome. Their sizes range from about 10 to a few thousand nucleotide pairs. About 5% of the DNA in EcorRI and Hind II digests migrated in a band at the position of undigested DNA, probably due to the resistance of long stretches of DNA against these nucleases. Calf DNA fragments obtained with EcoRI and Hind II were isolated by preparative gel electrophoresis. DNA from the bands showed the behaviour of repetitive DNA in renaturation experiments. An EcoRI fragment 1300-nucleotide-pairs long, which represents 6% of the calf genome and occurs 130000 times, is tandemly repeated (derived from the satellite of 1.714 g/cm3, see below). Another EcoRI fragment of 970 nucleotide paris, which represents 0.5% of the calf genome and is derived from the DNA of 1.710 g/cm3 seems to be structurally related to the foregoing fragment since it shows a similar Hind II and Bsu cleavage pattern. It alternates with a 1550-nucleotide-pairs-long EcoRI fragment. In another series of experiments total calf DNA was separated into main-band and satellite fractions by density-gradient centrifugation and chromatography in the presence of a base-specific dye. Purified fractions were characterized by analytical ultracentrifugation and by Hind II and EcorRI digestions. From the cleavage patterns of purified fractions an assignment of the bands found with total calf DNA to satellite fractions was possible. Most fragments were derived from the components of density 1.709 and 1.710 g/cm3. The 1.714-g/cm3 satellite was cleaved into a 1300-nucleotide-pairs-ling EcorRI fragment and two Hind II fragments of 1100 and 180 nucleotide pairs. The satellites of 1.723 g/cm3 and 1.705 g/cm3 were not cleaved by either Hind II or EcoRI DNAase. On digestion of main band DNA with Bsu a 160-nucleotide-pairs-long fragment was obtained which was also observed, at a frequency of about 160000, in the Bsu digest of EcoRI fractions from total calf DNA.

Animals↗

PubMed1975

Hepatic nucleases. 1. Methods for the specific determination and characterization in rat liver.

With a view to the study of the subcellular localization of nucleases, methods ensuring the homogenates. The ribonuclease activity of rat liver is due to the three enzymes with different pH optimun. For acid ribonuclease (pH optimun 5.3), it is possible to avoid interference from the other ribonucleases by performing the incubation at pH 5. Neutral ribonuclease (pH optimum 7.6) is differentiated by relying on its sensitivity to the natural inhibitor from the supernatant of liver homogenate. Comparison of activities before and after pretreatment at 50 degrees C in acid medium permits the specific measurement of alkaline ribonuclease (pH optimum 8.8). The optimal conditions for the determination in liver homogenates of two deoxyribonucleases and of an enzyme acting on polyriboadenylate are also described. The activity of these various nucleases is compared and some of their properties are investigated.

Animals↗

PubMed1982

Partial P1 nuclease digestion as a probe of tRNA structure.

Partial P1 nuclease digestion of 5'-32P-labeled tRNAs, followed by polyacrylamide gel electrophoresis and autoradiography, results in a series of bands of unequal intensities. Comparison of the digestion profiles of two conformers of yeast tRNA3Leu shows that P1 nuclease is sensitive to structural features of its substrate. Examination of the digestion pattern of yeast tRNAPhe in light of its known three-dimensional conformation shows that the enzyme's activity reflects the primary, secondary and tertiary levels of tRNA structure.

Base Sequence↗

PubMed1988

Nuclease hypersensitivity in the beta-globin gene region of K562 cells.

We have investigated chromatin structure in the beta-globin gene region of the K562 human erythroleukemic cell line by using S1 and DNase I nuclease sensitivity assays. Despite the lack of beta-globin gene expression in these cells, we find nuclease-hypersensitive sites to these enzymes in its 5' and 3' flanking regions in K562 chromatin. This result is in contrast to previous reports in which no hypersensitive sites were found in the immediate vicinity of this gene. In the 3' region, one major hypersensitive site at 0.9 kpb 3' and three minor hypersensitive sites at 0.7 kbp, 0.5 kbp 3' and 0.2 kbp 5' of the polyadenylation site were observed; these sites are very similar to those found in fetal liver and adult bone marrow cells in which the beta-globin gene is expressed. We find hypersensitive sites to both enzymes in the 5' region of the beta-globin gene: a major site 0.8 kbp 5' to the cap site, and two minor sites 1.2 and 1.5 kbp 5' to the cap site. The -0.8 kbp site is also present in plasmids containing the beta-globin gene. Our results suggest that the lack of beta-globin gene expression may be related to the lack of hypersensitivity sites in the immediate (150 bp) 5' flanking region of the beta-globin gene, as occurs in other active globin genes.

Adult↗