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51.
52.
Furukawa T Kimura S Ishibashi T Mori Y Hashimoto J Sakaguchi K 《Plant molecular biology》2003,51(1):59-70
A novel endonuclease, a new member of the RAD2 nuclease family, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare), and designated as OsSEND-1. The open reading frame of the OsSEND-1 cDNA encoded a predicted product of 641 amino acid residues with a molecular weight of 69.9 kDa. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nuclease family proteins, especially RAD2 nuclease, but it differed markedly from FEN-1, XPG or HEX1/EXO1. The N- and I-domains in the family were highly conserved in the OsSEND-1 sequence. The protein was much smaller than XPG, but larger than HEX1/EXO1 and FEN-1. The genome sequence was composed of 14 exons, and was localized at the almost terminal region of the short arm of chromosome 8. Northern blotting and in situ hybridization analyses demonstrated preferential expression of OsSEND-1 mRNA in proliferating tissues such as meristem. The mRNA level of OsSEND-1 was induced by UV and DNA-damaging agent such as MMS or H2O2, indicating that OsSEND-1 has some roles in the repair of many types of damaged DNA. The recombinant peptide showed endonuclease activity. 相似文献
53.
Koprowski P Fikus MU Dzierzbicki P Mieczkowski P Lazowska J Ciesla Z 《Molecular genetics and genomics : MGG》2003,269(5):632-639
We reported previously that the product of DIN7, a DNA damage-inducible gene of Saccharomyces cerevisiae, belongs to the XPG family of proteins, which are involved in DNA repair and replication. This family includes the S. cerevisiae protein Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Interestingly, Din7p is the only member of the XPG family which specifically functions in mitochondria. We reported previously that overexpression of DIN7 results in a mitochondrial mutator phenotype. In the present study we wished to test the hypothesis that this phenotype is dependent on the nuclease activity of Din7p. For this purpose, we constructed two alleles, din7-D78A and din7-D173A, which encode proteins in which highly conserved aspartates important for the nuclease activity of the XPG proteins have been replaced by alanines. Here, we report that overexpression of the mutant alleles, in contrast to DIN7, fails to increase the frequency of mitochondrial petite mutants or erythromycin-resistant (Er) mutants. Also, overproduction of din7-D78Ap does not result in destabilization of poly GT tracts in mitochondrial DNA (mtDNA), the phenotype observed in cells that overexpress Din7p. We also show that petite mutants induced by enhanced synthesis of wild-type Din7p exhibit gross rearrangements of mtDNA, and that this correlates with enhanced recombination within the mitochondrial cyt b gene. These results suggest that the stability of the mitochondrial genome of S. cerevisiae is modulated by the level of the nuclease Din7p.Communicated by R. Devoret 相似文献
54.
Meng XW Fraser MJ Feller JM Ziegler JB 《Apoptosis : an international journal on programmed cell death》2000,5(3):243-254
Single-strand DNase and poly rAase, activities characteristic of endo-exonuclease, were co-activated in nuclear fractions of HL-60 cells by caspase-3. Activation was accompanied by cleavages of large soluble polypeptides (130–185 kDa) and a 65 kDa inactive chromatin-associated polypeptide related to the endo-exonuclease of Neurospora crassa as detected on immunoblots. The major products seen in vitro were a 77 kDa soluble polypeptide and an active chromatin-associated 34 kDa polypeptide. When HL-60 cells were induced to undergo apoptosis by treating with 50 M etoposide (VP-16) for 4 hours, 77 kDa and 40 kDa polypeptides accumulated in nuclear fractions. Chromatin DNA fragmentation activity was also activated in cytosol and nuclear extract either by pre-treating the cells in vivo with VP-16 or by treating the cytosol in vitro with caspase-3 or dATP and cytochrome c. Endo-exonuclease activated by caspase-3 in cytosol-derived fractions augmented chromatin DNA fragmentation activity in vitro. Endo-exonuclease is proposed to act in vivo in conjunction with the caspase-activated DNase (CAD) to degrade chromatin DNA during apoptosis of HL-60 cells. 相似文献
55.
Probing the contribution of internal cavities to the volume change of protein unfolding under pressure. 总被引:5,自引:2,他引:3 下载免费PDF全文
The structural origin of the decrease in system volume upon protein denaturation by pressure has remained a puzzle for decades. This negative volume change upon unfolding is assumed to arise globally from more intimate interactions between the polypeptide chain and water, including electrostriction of buried charges that become exposed upon unfolding, hydration of the polypeptide backbone and amino acid side chains and elimination of packing defects and internal void volumes upon unfolding of the chain. However, the relative signs and magnitudes of each of these contributing factors have not been experimentally determined. Our laboratory has probed the fundamental basis for the volume change upon unfolding of staphylococcal nuclease (Snase) using variable solution conditions and point mutants of Snase (Royer CA et al., 1993, Biochemistry 32:5222-5232; Frye KJ et al., 1996, Biochemistry 35:10234-10239). Our prior results indicate that for Snase, neither electrostriction nor polar or nonpolar hydration contributes significantly to the value of the volume change of unfolding. In the present work, we investigate the pressure induced unfolding of three point mutants of Snase in which internal cavity size is altered. The experimentally determined volume changes of unfolding for the mutants suggest that loss of internal void volume upon unfolding represents the major contributing factor to the value of the volume change of Snase unfolding. 相似文献
56.
Meng XW Fraser MJ Ireland CM Feller JM Ziegler JB 《Apoptosis : an international journal on programmed cell death》1998,3(6):395-405
Since mitochondrial factors have been implicated in apoptosis, experiments were designed to assess whether or not the potent mitochondrial nuclease could be one of these factors. Nuclei isolated by two different methods were found to contain mitochondrial nuclease in masked form. This nuclease was released by treatment with the non-ionic detergent NP-40 and rendered trypsin-sensitive. It was not removed appreciably from the nuclei by washing and sedimentation of the nuclei through a sucrose cushion. Levels of the mitochondrial nuclease were followed during drug-induced apoptosis. Time courses of apoptosis in cultures of HL-60 cells were monitored by flow cytometry of propidium iodide-stained cells and by agarose gel electrophoresis of extracted DNA. Changes in the inner mitochondrial transmembrane potential were monitored by flow cytometry of chloromethyl-X-Rosamine-stained cells. Apoptosis was induced by treatment with either the chemotherapeutic agent etoposide (VP-16 at 10 M) over an 8 h period or with the anti-rheumatic agent hydroxychloroquine (HCQ at 0.28 mM) over a 24 h period. These two drugs likely act in different pathways of apoptosis. VP-16 caused loss of the mitochondrial transmembrane potential 1.0–1.5 h before apoptosis was detected. On the other hand, treatment with HCQ caused these processes to occur in parallel possibly indicating that the mitochondrial changes are secondary events. No losses of masked mitochondrial nuclease were detected with either drug treatment during the course of apoptosis. HL-60 mitochondrial DNA was also not degraded during apoptosis induced by either agent. These observations likely explain why the mitochondrial DNA is not degraded and make it unlikely that mitochondrial nuclease plays any role in vivo in chromatin DNA fragmentation. 相似文献
57.
We have applied atomic force microscopy (AFM) to the measurement of BAL 31 nuclease activities. BAL 31 nuclease, a species
of exonuclease, is used to remove unwanted sequences from the termini of DNA before cloning. For cutting out only the appropriate
sequences, it is important to know the nuclease properties, such as digestion speed and the distribution of the lengths of
the digested DNA. AFM was used to obtain accurate measurements on the lengths of DNA fragments before and after BAL 31 nuclease
digestion. We analyzed 4 DNAs with known number of base pairs (288, 778, 1818, and 3162 base pairs) for correlating the contour
length measured by AFM with the number of base pairs under the deposition conditions used. We used this calibration for analyzing
DNA degradation by BAL 31 nuclease from the AFM measurement of contour lengths of digested DNAs. In addition, the distribution
of digested DNA could be analyzed in more detail by AFM than by electrophoresis, because digested DNA were measured as a population
by electrophoresis, but were measured individually by AFM. These results show that AFM will be a useful new technique for
measuring nuclease activities.
Received: 8 August 1997 / Accepted: 10 September 1997 相似文献
58.
Aims: The ability to transform Vibrio spp. is limited by the extracellular nuclease that their cells secrete. The reported transformation efficiency of this organism is 102 –105 transformants per microgram DNA. We tried different buffers and conditions, aiming to elevate its transformation efficiency.
Methods and Results: MgCl2 and sucrose are often included in the washing and/or electroporation buffers to stabilize the cell membrane. However, Mg2+ is required for production and activity of the extracellular nuclease. A simple electroporation buffer lacking Mg2+ was found to increase transformation efficiency dramatically, to levels 50-fold more than the buffers containing Mg2+ . To maintain the stability of the cell membranes, Mg2+ was replaced with high concentrations of sucrose, from 272 to 408 mmol l−1 . With the new buffers, the transformation efficiency of Vibrio parahaemolyticus was increased to 2·2 × 106 transformants per microgram DNA.
Conclusions: Mg2+ in the buffer adversely affected transformation of V. parahaemolyticus by electroporation. The cell membranes of vibrio can be stabilized by high concentration of sucrose when Mg2+ is absent.
Significance and Impact of the Study: A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg2+ can be used for other nuclease-producing organisms. 相似文献
Methods and Results: MgCl
Conclusions: Mg
Significance and Impact of the Study: A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg
59.
Defining the molecular basis of the DNA sequence selectivity of polyamine binding is central to understanding polyamine-dependent gene expression. We have studied, by selective NMR experiments, the variation of spermine mobility and conformation in the presence of G-quadruplexes formed by sequences of the purine-rich strand of the c-Myc promoter, nuclease hypersensitivity element III1 (NHE III1). All the NHE quadruplexes restrict spermine mobility and induce a spermine conformational change but the most effective immobilisation occurs when all five G-tracts of the NHE III1 are present. This suggests structure within the nucleotides flanking the G-quadruplex has a role in immobilising spermine. 相似文献
60.