Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated host restriction as a barrier to transformation and developed a method for gene transfer into the previously untransformable, heterotrophic cyanobacterium Nostoc PCC 7121. A restriction endonuclease, designated Nsp 7121I, has been partially purified by phosphocellulose chromatography of Nostoc cell extracts. Comparisons of Nsp 7121I digests of bacteriophage lambda and plasmid DNAs with computer-generated restriction fragment profiles showed that Nsp 7121I is an isoschizomer of restriction endonucleases, such as Asu I, Nsp 7524IV, Sau 96I, and Eco 47II, that recognize the sequence GGNCC. Cleavage by Nsp 7121I within this sequence was confirmed by sequence analysis of DNA fragments cleaved at a unique Nsp 7121I site. These data further suggested that cleavage occurs after the first G (5'-G/GNCC-3') in this site to generate a three base 5' overhang. Nsp 7121I degraded all plasmids used in previous transformation attempts but modification of these DNA molecules by Eco 47II methylase effectively prevented digestion by Nsp 7121I. Plasmids premethylated by passage through Escherichia coli carrying a plasmid encoded Eco 47II methylase have now been used in an electroporation procedure to transform Nostoc PCC 7121 to neomycin resistance at frequencies as high as one transformant per 10(3) viable cells. Transformation, and stable replication within Nostoc of one of the transforming plasmids (pRL25), was confirmed by recovery of pRL25, in its original form, from transformants. Conjugal transfer of pRL25 from E. coli into Nostoc was also possible but at much lower efficiency than by electroporation. These findings establish the basis for genetic analysis of Nostoc PCC 7121, from which genes for photosynthetic electron transport have been cloned.
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PMID:Characterization of a restriction barrier and electrotransformation of the cyanobacterium Nostoc PCC 7121. 821 99

Etoposide, a topoisomerase II inhibitor used in cancer therapy, has been shown to induce apoptosis in vitro in a variety of cell types. In the present study, we have characterized the effects of etoposide on undifferentiated rat pheochromocytoma PC12 cells. Etoposide killed PC12 cells in a time- and concentration-dependent manner. 20-24 h incubation with 10 micrograms/ml etoposide induced 25-50% cell death. Hoechst 33258 staining revealed apoptotic morphology in dying cells. No evidence was found of either oligonucleosomal DNA fragmentation, as shown by agarose gel electrophoresis, or endonuclease involvement, as shown by the inability of aurintricarboxylic acid to prevent cell death. Cycloheximide and actinomycin-D were unable to prevent etoposide cytotoxicity indicating that the process is not dependent upon de novo protein or mRNA synthesis. NGF (5 ng/ml) prevented etoposide-induced PC12 cell death. These results offer an example of how the morphological features of apoptosis are not necessarily associated with oligonucleosomal DNA fragmentation or with de novo macromolecule synthesis.
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PMID:Etoposide-induced PC12 cell death: apoptotic morphology without oligonucleosomal DNA fragmentation or dependency upon de novo protein synthesis. 933 35

We have tested the specific hypothesis that the pathway of nuclear collapse in apoptosis is governed by the early attack on active chromatin at spatially restricted nuclear sites. Cell death in PC12 pheochromocytoma cells deprived of serum growth factors, in HL-60 leukemic cells treated with inhibitors of protein or RNA biosynthesis, and in U937 histiocytic lymphoma cells exposed to the cytokine tumor necrosis factor alpha showed a common mechanism in the targeting of DNA for degradation. An incorporation assay with labeled nucleotide revealed an early selective nicking in peripheral nuclear chromatin with concomitant diminution in the amount of immunoreactive lamin B protein. This was followed by a phase of more extensive cleavages, continued nuclear protein loss, chromatin collapse, and fragmentation of nuclei. The spatial restriction of early cleavages is similar to the nicking obtained by the application of exogenous DNase I to fixed nuclei of normal cells and to that obtained in the activation of the endogenous endonuclease of liver nuclei by Ca2+. These similarities suggest that, in apoptosis, activation of an endonuclease preferentially recognizing a specific chromatin configuration, such as that of active (DNase I-sensitive) genes, underlies the early spatial demarcation of cleavages.
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PMID:Preferential sites of early DNA cleavage in apoptosis and the pathway of nuclear damage. 1021 26

Open reading frames (ORFs) are frequently inserted into group I self-splicing introns. These ORFs encode either maturases that are required for splicing of the intron or DNA endonucleases that promote intron mobility. A self-splicing intron in the tRNA(fMet) gene of Synechocystis PCC 6803, which has been proposed to have moved laterally within the cyanobacteria, contains an ORF that is unrelated to known intron-encoded endonucleases or maturases. Here, using an in vitro transcription-translation system, we show that this intronic ORF encodes a double-strand DNA endonuclease, I-Ssp6803I. I-Ssp6803I cleaves each strand of the intronless tRNA(fMet) gene adjacent to the anticodon triplet leaving 3 bp 3' extensions and has no activity at intron-exon boundaries. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a partially palindromic 20 bp region encompassing the entire anticodon stem and loop of the tRNA(fMet) gene. I-Ssp6803I represents a novel intron-encoded DNA endonuclease and is the first example of a chromosomally encoded group I intron endonuclease in bacteria.
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PMID:A novel group I intron-encoded endonuclease specific for the anticodon region of tRNA(fMet) genes. 1125 45

DNase gamma, which cleaves chromosomal DNA into nucleosomal units (DNA ladder formation), has been suggested to be the critical component of apoptotic machinery. Using rat pheochromocytoma PC12 cells, which are differentiated to sympathetic neurons by nerve growth factor (NGF), we investigated whether DNase gamma-like enzyme is present in neuronal cells and is involved in neuronal cell death. The nuclear auto-digestion assay for DNase catalyzing internucleosomal DNA cleavage revealed that nuclei from neuronal differentiated PC12 cells contain acidic and neutral endonucleases, while nuclei from undifferentiated PC12 cells have only acidic endonuclease. The DNA ladder formation observed in isolated nuclei from neuronal differentiated PC12 cells at neutral pH requires both Ca(2+) and Mg(2+), and is sensitive to Zn(2+). The molecular mass of the neutral endonuclease present in neuronal differentiated PC12 cell nuclei is 32000 as determined by activity gel analysis (zymography). The properties of the neuronal endonuclease present in neuronal differentiated PC12 cell nuclei were similar to those of purified DNase gamma from rat thymocytes and splenocytes. Interestingly, in neuronal differentiated PC12 cells, internucleosomal DNA fragmentation is observed following NGF deprivation, whereas undifferentiated PC12 cells fail to exhibit DNA ladder formation during cell death by serum starvation. These results suggest that the DNase gamma-like endonuclease present in neuronal differentiated PC12 cell nuclei is involved in internucleosomal DNA fragmentation during apoptosis, induced by NGF deprivation.
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PMID:Presence of DNase gamma-like endonuclease in nuclei of neuronal differentiated PC12 cells. 1464 7

We improved genetic transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, by combining electroporation with a top agar method. Transformation was also improved when a disruptant of a putative type I restriction endonuclease (tll2230) was used as recipient cells. In particular, some constructs, with which wild type has never been transformed, were successfully integrated into the tll2230-disruptant. Single-crossover recombination was detected more frequently than the double-crossover recombination. In accordance with the presence of all the homologs of pil genes in Synechocystis sp. PCC 6803, we found that T. elongatus is naturally transformable with exogenous DNA.
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PMID:Improved genetic transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1. 1498 87

Biosynthesis of transfer RNA requires processing from longer precursors at the 5'- and 3'-ends. In eukaryotes, in archaea, and in those bacteria where the 3'-terminal CCA sequence is not encoded, 3' processing is carried out by the endonuclease RNase Z, which cleaves after the discriminator nucleotide to generate a mature 3'-end ready for the addition of the CCA sequence. We have identified and cloned the gene coding for RNase Z in the cyanobacterium Synechocystis sp. PCC 6803. The gene has been expressed in Escherichia coli, and the recombinant protein was purified. The enzymatic activity of RNase Z from Synechocystis has been studied in vitro with a variety of substrates. The presence of C or CC after the discriminator nucleotide modifies the cleavage site of RNase Z so that it is displaced by one and two nucleotides to the 3'-side, respectively. The presence of the complete 3'-terminal CCA sequence in the precursor of the tRNA completely inhibits RNase Z activity. The inactive CCA-containing precursor binds to Synechocystis RNase Z with similar affinity than the mature tRNA. The properties of the enzyme described here could be related with the mechanism by which CCA is added in this organism, with the participation of two separate nucleotidyl transferases, one specific for the addition of C and another for the addition of A. This work is the first characterization of RNase Z from a cyanobacterium, and the first from an organism with two separate nucleotidyl transferases.
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PMID:Sequence-dependent cleavage site selection by RNase Z from the cyanobacterium Synechocystis sp. PCC 6803. 1605 5

NORE1A (RASSF5) and RASSF1A are newly described Ras effectors with tumour suppressor functions. Both molecules are frequently inactivated in various cancers. In this study, we aimed to explore the potential involvement of NORE1A and RASSF1A in pheochromocytoma and abdominal paraganglioma tumorigenesis. A panel of 54 primary tumours was analysed for NORE1A and RASSF1A mRNA expression by TaqMan quantitative RT-PCR. Furthermore, NORE1A and RASSF1A promoter methylation was assessed by combined bisulphite restriction endonuclease assay and methylation-sensitive Pyrosequencing respectively. The anti-tumorigenic role of NORE1A was functionally investigated in Nore1A-transfected PC12 rat pheochromocytoma cells by fluorescent inhibition of caspase activity and soft agar assays. Significantly suppressed NORE1A and RASSF1A mRNA levels were detected in primary tumours compared with normal adrenal medulla (P<0.001). Methylation of the NORE1A promoter was not observed in primary tumours. On the other hand, 9% (5/54) of the primary tumours examined showed RASSF1A promoter methylation greater than 20% as detected by Pyrosequencing. Methylation of the RASSF1A promoter was significantly associated with malignant behaviour (P<0.05). Transient expression of Nore1a resulted in enhanced apoptosis and impaired colony formation in soft agar. Our study provides evidence that NORE1A and RASSF1A are frequently suppressed in pheochromocytoma and abdominal paraganglioma. Silencing of NORE1A contributes to the transformed phenotype in these tumours.
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PMID:The Ras effectors NORE1A and RASSF1A are frequently inactivated in pheochromocytoma and abdominal paraganglioma. 1739 81

Anabaena PCC 7120 genome contains three elements, which get excised out during late stages of heterocyst differentiation by a site-specific recombination process. The XisA protein, which excises the nifD element, shows sequence homology with the integrase family of tyrosine recombinase. The 11 bp target site of XisA CGGAGTAATCC contains a 3 bp inverted repeat. Here, we report restriction endonuclease activity of XisA by specific loss of plasmids containing single or double target sites. The pMX25 plasmid containing two target sites demonstrated endonuclease activity proportional to excision frequency. Different plasmid substrates containing one base pair mutation in the inverted repeat of the target site were monitored for endonuclease activity. Mutation of A4C retained endonuclease activity, while other modifications lost endonuclease activity. The presence of an additional copy of the target site enhanced endonuclease activity. These results suggest that the XisA protein could be an IIE type of restriction endonuclease in addition to being a recombinase.
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PMID:In vivo restriction endonuclease activity of the Anabaena PCC 7120 XisA protein in Escherichia coli. 1802 66

Synechococcus sp. PCC 7002 is an ideal model cyanobacterium for functional genomics and biotechnological applications through metabolic engineering. A gene expression system that takes advantage of its multiple, endogenous plasmids has been constructed in this cyanobacterium. The method involves the integration of foreign DNA cassettes with selectable markers into neutral sites that can be located on any of the several endogenous plasmids of this organism. We have exploited the natural transformability and powerful homologous recombination capacity of this organism by using linear DNA fragments for transformation. This approach overcomes barriers that have made the introduction and expression of foreign genes problematic in the past. Foremost among these is the natural restriction endonuclease barrier that can cleave transforming circular plasmid DNAs before they can be replicated in the cell. We describe herein the general methodology for expressing foreign and homologous genes in Synechococcus sp. PCC 7002, a comparison of several commonly used promoters, and provide examples of how this approach has successfully been used in complementation analyses and overproduction of proteins with affinity tags.
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PMID:Expression of genes in cyanobacteria: adaptation of endogenous plasmids as platforms for high-level gene expression in Synechococcus sp. PCC 7002. 2096 Jan 36


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