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At least 199 records · Page 11Linked to original sources
PubMed1996

Effects on NaeI-DNA recognition of the leucine to lysine substitution that transforms restriction endonuclease NaeI to a topoisomerase: a model for restriction endonuclease evolution.

Substituting lysine for leucine at position 43 (L43K) transforms NaeI from restriction endonuclease to topoisomerase and makes NaeI hypersensitive to intercalative anticancer drugs. Here we investigated DNA recognition by Nael-L43K. Using DNA competition and gel retardation assays, NaeI-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preference for the former. Sedimentation studies showed that under native conditions NaeI-L43K, like NaeI, is a dimer. Introduction of mismatched bases into double-stranded DNA significantly increased that DNA's ability to inhibit NaeI-L43K. Wild-type NaeI showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration range studied. These results demonstrate that the L43K substitution caused a significant change in recognition specificity by NaeI and imply that NaeI-L43K's topoisomerase activity is related to its ability to bind single-stranded and distorted regions in DNA. A mechanism is proposed for the evolution of the NaeI restriction-modification system from a topoisomerase/ligase by a mutation that abolished religation activity and provided a needed change in DNA recognition.

DNA↗
PubMed2006

Methionine restriction decreases visceral fat mass and preserves insulin action in aging male Fischer 344 rats independent of energy restriction.

Reduced dietary methionine intake (0.17% methionine, MR) and calorie restriction (CR) prolong lifespan in male Fischer 344 rats. Although the mechanisms are unclear, both regimens feature lower body weight and reductions in adiposity. Reduced fat deposition in CR is linked to preservation of insulin responsiveness in older animals. These studies examine the relationship between insulin responsiveness and visceral fat in MR and test whether, despite lower food intake observed in MR animals, decreased visceral fat accretion and preservation of insulin sensitivity is not secondary to CR. Accordingly, rats pair fed (pf) control diet (0.86% methinone, CF) to match the food intake of MR for 80 weeks exhibit insulin, glucose, and leptin levels similar to control-fed animals and comparable amounts of visceral fat. Conversely, MR rats show significantly reduced visceral fat compared to CF and PF with concomitant decreases in basal insulin, glucose, and leptin, and increased adiponectin and triiodothyronine. Daily energy expenditure in MR animals significantly exceeds that of both PF and CF. In a separate cohort, insulin responses of older MR animals as measured by oral glucose challenge are similar to young animals. Longitudinal assessments of MR and CF through 112 weeks of age reveal that MR prevents age-associated increases in serum lipids. By 16 weeks, MR animals show a 40% reduction in insulin-like growth factor-1 (IGF-1) that is sustained throughout life; CF IGF-1 levels decline much later, beginning at 112 weeks. Collectively, the results indicate that MR reduces visceral fat and preserves insulin activity in aging rats independent of energy restriction.

Adipose Tissue↗
PubMed1998

Characterization of an extremely thermostable restriction enzyme, PspGI, from a Pyrococcus strain and cloning of the PspGI restriction-modification system in Escherichia coli.

An extremely thermostable restriction endonuclease, PspGI, was purified from Pyrococcus sp. strain GI-H. PspGI is an isoschizomer of EcoRII and cleaves DNA before the first C in the sequence 5' CCWGG 3' (W is A or T). PspGI digestion can be carried out at 65 to 85 degrees C. To express PspGI at high levels, the PspGI restriction-modification genes (pspGIR and pspGIM) were cloned in Escherichia coli. M.PspGI contains the conserved sequence motifs of alpha-aminomethyltransferases; therefore, it must be an N4-cytosine methylase. M.PspGI shows 53% similarity to (44% identity with) its isoschizomer, M.MvaI from Micrococcus variabilis. In a segment of 87 amino acid residues, PspGI shows significant sequence similarity to EcoRII and to regions of SsoII and StyD4I which have a closely related recognition sequence (5' CCNGG 3'). PspGI was expressed in E. coli via a T7 expression system. Recombinant PspGI was purified to near homogeneity and had a half-life of 2 h at 95 degrees C. PspGI remained active following 30 cycles of thermocycling; thus, it can be used in DNA-based diagnostic applications.

Amino Acid Sequence↗
PubMed1998

Molecular characterization of the Lactococcus lactis LlaKR2I restriction-modification system and effect of an IS982 element positioned between the restriction and modification genes.

The nucleotide sequence of the plasmid-encoded LlaKR2I restriction-modification (R-M) system of Lactococcus lactis subsp. lactis biovar diacetylactis KR2 was determined. This R-M system comprises divergently transcribed endonuclease (llaKR2IR) and methyltransferase (llaKR2IM) genes; located in the intergenic region is a copy of the insertion element IS982, whose putative transposase gene is codirectionally transcribed with llaKR2IM. The deduced sequence of the LlaKR2I endonuclease shared homology with the type II endonuclease Sau3AI and with the MutH mismatch repair protein, both of which recognize and cleave the sequence 5' GATC 3'. In addition, M. LlaKR2I displayed homology with the 5-methylcytosine methyltransferase family of proteins, exhibiting greatest identity with M. Sau3AI. Both of these proteins shared notable homology throughout their putative target recognition domains. Furthermore, subclones of the native parental lactococcal plasmid pKR223, which encode M. LlaKR2I, all remained undigested after treatment with Sau3AI despite the presence of multiple 5' GATC 3' sites. The combination of these data suggested that the specificity of the LlaKR2I R-M system was likely to be 5' GATC 3', with the cytosine residue being modified to 5-methylcytosine. The IS982 element located within the LlaKR2I R-M system contained at its extremities two 16-bp perfect inverted repeats flanked by two 7-bp direct repeats. A perfect extended promoter consensus, which represented the likely original promoter of the llaKR2IR gene, was shown to overlap the direct repeat sequence on the other side of IS982. Specific deletion of IS982 and one of these direct repeats via a PCR strategy indicated that the LlaKR2I R-M determinants do not rely on elements within IS982 for expression and that the efficiency of bacteriophage restriction was not impaired.

Amino Acid Sequence↗
PubMed1993

Restricted replication of simian immunodeficiency virus strain 239 in macrophages is determined by env but is not due to restricted entry.

Virus derived from the infectious, pathogenic, molecular clone of simian immunodeficiency virus (SIV) called SIVmac239 replicates poorly in primary rhesus monkey alveolar macrophage cultures. Variants with three to nine amino acid changes in the envelope replicate 100 to 1,000 times more efficiently in these macrophage cultures than parental SIVmac239. Early events, including virus entry into cells, were analyzed by measuring the amounts of newly synthesized viral DNA 14 to 16 h after infection of macrophages by using a quantitative polymerase chain reaction method. SIVmac239 ws found to enter macrophages with an efficiency similar to that of the macrophage-tropic derivatives. The assay indeed measured newly synthesized viral DNA since detection was inhibited by the reverse transcriptase inhibitors azidothymidine and foscarnet and by heat inactivation of the virus stock prior to infection. Furthermore, entry of SIVmac239 and macrophage-tropic variant into macrophages was inhibited by monoclonal antibody against CD4. Analysis of the time course of viral DNA accumulation showed that although initial entry of SIVmac239 into cells occurred normally, subsequent logarithmic increases in the amounts of viral DNA associated with spread of virus through the macrophage cultures was blocked. Increasing the amount of SIVmac239 incubated with macrophages increased the amount of virus entering the cell, but this could not overcome the block to replication. Thus, restricted replication of SIVmac239 in macrophages is determined by the envelope, but surprisingly it is not due to restricted virus entry.

Animals↗
PubMed2001

The neuron-restrictive silencer element-neuron-restrictive silencer factor system regulates basal and endothelin 1-inducible atrial natriuretic peptide gene expression in ventricular myocytes.

Induction of the atrial natriuretic peptide (ANP) gene is a common feature of ventricular hypertrophy. A number of cis-acting enhancer elements for several transcriptional activators have been shown to play central roles in the regulation of ANP gene expression, but much less is known about contributions made by transcriptional repressors. The neuron-restrictive silencer element (NRSE), also known as repressor element 1, mediates repression of neuronal gene expression in nonneuronal cells. We found that NRSE, which is located in the 3' untranslated region of the ANP gene, mediated repression of ANP promoter activity in ventricular myocytes and was also involved in the endothelin 1-induced increase in ANP gene transcription. The repression was conferred by a repressor protein, neuron-restrictive silencer factor (NRSF). NRSF associated with the transcriptional corepressor mSin3 and formed a complex with histone deacetylase (HDAC) in ventricular myocytes. Trichostatin A (TSA), a specific HDAC inhibitor, relieved NRSE-mediated repression of ANP promoter activity, and chromatin immunoprecipitation assays revealed the involvement of histone deacetylation in NRSE-mediated repression of ANP gene expression. Furthermore, in myocytes infected with recombinant adenovirus expressing a dominant-negative form of NRSF, the basal level of endogenous ANP gene expression was increased and a TSA-induced increase in ANP gene expression was apparently attenuated, compared with those in myocytes infected with control adenovirus. Our findings show that an NRSE-NRSF system plays a key role in the regulation of ANP gene expression by HDAC in ventricular myocytes and provide a new insight into the role of the NRSE-NRSF system outside the nervous system.

3' Untranslated Regions↗
PubMed1991

Inhibition of cytolytic lymphocytes by homologous restriction factor. Lack of species restriction.

Homologous restriction factor (HRF) has been shown to inhibit complement-mediated lysis in a species-restrictive manner. Human HRF is able to block lysis by human complement but not by complement from other species. HRF has also been found in the membrane of lymphokine-activated killer (LAK) cells. When this HRF is inserted into sheep erythrocyte membranes, it is able to protect the erythrocyte from LAK cell lysis. In this report, we show that while HRF can inhibit human complement but not rat complement-mediated hemolysis, it is able to inhibit LAK cell lysis by both human and rat LAK cells. HRF is therefore a more general protective protein than has been previously thought.

Animals↗
PubMed1987

Inhibition of natural killer-cell mediated cytolysis with monoclonal antibodies to restricted and non-restricted epitopes of the leucocyte common antigen.

Three monoclonal antibodies recognizing different epitopes of the leucocyte common molecule, CMRF-11 (against the restricted or B-220 leucocyte common molecule), CMRF-12 and CMRF-26 [each against a different epitope on the non-restricted or T200 leucocyte common (CD45) molecule], were tested for their effects on lymphocyte cytotoxicity. The individual monoclonal antibodies inhibited human natural killer cell-mediated cytolysis (NK-CMC) weakly, but a mixture of CMRF-11 + 12 + 26 antibodies inhibited cytolysis more consistently and to a greater extent. This mixture did not inhibit cytotoxic T lymphocytes derived from secondary mixed lymphocyte cultures. The CMRF-11 + 12 + 26 mix was shown to inhibit a post-conjugate formation stage of lysis at the effector cell level. Inhibition of NK-CMC of a wide range of target cells, including the T-cell lines Jurkat, HSB2 and Molt 4, was demonstrated.

Antibodies, Monoclonal↗
PubMed1989

[A study on restriction of the areas working in speech function--application of multivariate analysis of aphasia test data and of lesions on CT images, by composing a "restricted-aphasia-lesion-map"].

Computed tomography (CT) gave a large contribution to morphological analysis of the aphasia syndromes. The aphasia problems are, however, still controversial and have many unresolved issues, even from a morphological point of view. In most cases with cerebral vascular lesion causing speech dysfunction, we are able to define an extent of density change on CT images. But its extent seems to be often not consistent with the area supposed to be deranged in functional work-up of speech. CT appearance of some areas may be little changed or normal when those areas are to be in poor condition in their functional maintenance. This may be due to such a vascular supply which permits brain tissues to be sustained but which does not work for their function to be normally performed. From 642 cases with cerebral vascular lesion we reviewed CT images of 382 cases: 171 cases having a lesion in the left hemisphere and 46 of them showing aphasia syndrome; 151, a lesion in the right hemisphere alone and 3, aphasic; and 60 cases having lesions on both sides and 7 showing aphasia syndrome. First, we determined anatomically the extent of CT change in 102 cases, all right-handed, with a lesion in the left hemisphere. The extents were piled and made up to a "non-aphasia-lesion-map". This map was used for eliminating those areas as not working in speech function. The other map of the areas was made out of CT images of aphasia cases (33 cases including one agraphia). The latter map was put upon the "non-aphasia-lesion-map", so that the very area working in speech function could be more restricted. The third map, "restricted-aphasia-lesion-map" demonstrated those areas related to speech function: (1) the region limited around the three rami in the Sylvian fissure (Broca A); (2) the inferior frontal gyrus and the posterior aspect of the middle frontal gyrus (Broca B); (3) the inferior aspect of the cortex and subcortex of the Reil's island; (4) most of the middle temporal gyrus, the posterior third of the superior temporal gyrus, a large portion of the supramarginal gyrus and a part of the limbic system; (5) a large portion of the limbic system; (6) the anterior aspect of the superior frontal gyrus; and (7) the corpus callosum. The Standard Language Test for Aphasia (SLTA) was applied for 33 cases of aphasia.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1993

Pretransplant detection of human minor histocompatibility antigen-specific naive and memory interleukin-2-secreting T cells within class I major histocompatibility complex (MHC)-restricted CD8+ and class II MHC-restricted CD4+ T-cell subsets.

Recent studies have shown that host-reactive interleukin-2 (IL-2)-secreting donor T lymphocytes (TI) are critically involved in the development of acute graft-versus-host disease (GVHD) after allogeneic HLA-identical sibling bone marrow transplantation (BMT). To further characterize the responding TI, we determined the frequency of pretransplant IL-2-secreting TI-precursors (TI-p) between eight HLA-A, -B, -C, -DR, and -DQ-identical sibling donor-host pairs in both the graft-versus-host (GVH) and the host-versus-graft (HVG) direction. High frequencies of pretransplant host-reactive donor TI-p (1/18,000 to 1/49,000) were detectable in five patients with grade II acute GVHD. Donor-reactive host TI-p (1/3,700 to 1/31,000) were observed in previously in vivo primed (n = 5) and unprimed (n = 1) patients. In two pairs tested after previous in vivo priming, pretransplant donor-reactive host TI-p were highly enriched within the CD45RO+ memory T-cell subset. Previously unprimed host-reactive donor TI-p occurred in almost equal frequencies within CD45RO+ and CD45RO- T cells. Both CD4+ and CD8+ T-cell subsets contributed in comparable frequencies to host- and donor-reactive TI-p. Recognition of minor histocompatibility (mH) antigens by CD8+ TI-p appeared to be class I major histocompatibility complex (MHC)-restricted, whereas CD4+ TI-p operated in a class II (HLA-DR) MHC-restricted fashion. Even between oligonucleotide-defined HLA-DPB1-disparate sibling donor-host pairs (n = 3), either responding T-cell subset was found to recognize cellularly defined mH antigens. These data indicate that various T-cell subsets contribute to host- and donor-reactive IL-2-secreting TI in allogeneic sibling BMT.

Bone Marrow Transplantation↗
PubMed2004

Restricting restriction.

Systems biology is a new, fashionable and well-funded discipline, which to quote from a recent review aims to 'examine the structure and dynamics of cellular and organismal function, rather than the characteristics of isolated parts of a cell or organism em leader ' (Kitano, H. (2002) Science 295:1662-1664). Systems biology will do this by profiting from the vast amounts of biological information that are available in the genomics era and make extensive use of computer modelling. But: 'many breakthroughs in experimental devices, advanced software and analytical methods are required before the achievements of system biology can live up to their much-touted potential'. This edition of Molecular Microbiology contains a paper that is the product of traditional experimental biology but which could serve as a test case for systems biology. The paper shows how bacteria integrate such disparate subsystems as DNA restriction, homologous recombination and regulated proteolysis to protect their chromosomes from degradation. When systems biology can predict this level of choreography, it will be a mature discipline.

Binding Sites↗
PubMed1996

Restriction barrier composed of an extracellular nuclease and restriction endonuclease in the unicellular cyanobacterium Microcystis sp.

The unicellular cyanobacterium Microcystis aeruginosa K-81 has two types of restriction barrier, an extracellular nuclease and sequence-specific endonucleases. The nuclease was detected in the culture supernatant and it was easily released from the cells by washing with water or buffer containing Triton X-100. This nuclease was identified as a polypeptide of about 28 kDa that digested covalently closed circular and linear double-stranded DNAs, including chromosomal DNA from M. aeruginosa K-81. Among another 13 Microcystis strains examined, 3 produced an extracellular nuclease. Furthermore, M. aeruginosa K-81 contained two sequence-specific endonucleases, MaeK81I and MaeK81II, which were isoschizomers of SplI and Sau96I, respectively.

Bacterial Proteins↗
PubMed1975

Restriction and modification in B. subtilis. Purification and general properties of a restriction endonuclease from strain R.

All Bacillus subtilis R-type strains showing the phenomena of restriction and modification contain an endonuclease that inactivates in vitro the biological activity of a variety of DNAs lacking R-specific modification, such as transfecting SPPI, SPO2 and phi105 DNA, and transforming B. subtilis 168-type DNA. The corresponding DNAs carrying R-specific modification are resistant to the enzyme. The enzyme has been purified approximately 400-fold and is essentially free from contaminating double strand-directed unspecific exo- or endonuclease activity. Only Mg2+ is required as cofactor. The substrate DNAs are cleaved at specific sites. The double-stranded fragments produced from SPP1 DNA (molecular weight 2.5 x 10(7)) have an average molecular weight of about 3 x 10(5).

Adenosine Triphosphate↗
PubMed2006

Calorie restriction and late-onset calorie restriction extend lifespan but do not alter protein storage in female grasshoppers.

Calorie restriction (CR) and late-onset CR enhance longevity in many organisms. Resource allocation theory suggests that longevity is enhanced by increasing somatic storage, at the expense of current reproduction. Phytophagous insects accumulate amino acids as hemolymph storage proteins for major developmental events. We hypothesized that protein storage is involved in life extension from CR. In a longitudinal experiment, we tested whether CR altered protein storage in female grasshoppers. Individuals on CR (60 or 70% of ad libitum) or late-onset CR had at least 60% greater longevity than ad libitum individuals. Age at first oviposition, dry mass of the first clutch, or lifetime fecundity were not affected by CR, but CR did increase the number of clutches produced. Most important, females on life-extending CR and late-onset CR did not differ in the concentration of hemolymph storage of proteins in comparison to ad libitum females. Protein storage changed with time in all groups, demonstrating sufficient sensitivity in our methods. Previous experiments have shown that severe CR ( approximately 30% of ad libitum) can reduce hemolymph storage. Therefore, the reduction in intake needed to extend lifespan is not sufficient to reduce protein storage in the hemolymph. These results do not support the hypothesis that protein storage is involved in life extension from CR.

Animals↗
PubMed1992

Cloning and sequence analysis of the StsI restriction-modification gene: presence of homology to FokI restriction-modification enzymes.

StsI endonuclease (R.StsI), a type IIs restriction endonuclease found in Streptococcus sanguis 54, recognizes the same sequence as FokI but cleaves at different positions. A DNA fragment that carried the genes for R.StsI and StsI methylase (M.StsI) was cloned from the chromosomal DNA of S.sanguis 54, and its nucleotide sequence was analyzed. The endonuclease gene was 1,806 bp long, corresponding to a protein of 602 amino acid residues (M(r) = 68,388), and the methylase gene was 1,959 bp long, corresponding to a protein of 653 amino acid residues (M(r) = 76,064). The assignment of the endonuclease gene was confirmed by analysis of the N-terminal amino acid sequence. Genes for the two proteins were in a tail-to-tail orientation, separated by a 131-nucleotide intercistronic region. The predicted amino acid sequences between the StsI system and the FokI system showed a 49% identity between the methylases and a 30% identity between the endonucleases. The sequence comparison of M.StsI with various methylases showed that the N-terminal half of M.StsI matches M.NIaIII, and the C-terminal half matches adenine methylases that recognize GATC and GATATC.

Amino Acid Sequence↗
PubMed1994

Genotyping of Staphylococcus epidermidis by small-fragment restriction endonuclease analysis and pulsed-field gel electrophoresis of genomic restriction fragments.

Small-fragment restriction endonuclease analysis (SF-REA) was established as a typing tool for Staphylococcus epidermidis. A total of 60 isolates comprising 48 epidemiologically nonrelated strains and 12 putatively linked isolates from 7 patients in 2 wards were analyzed. Nonrelated isolates were characterized by unique fingerprints when DNA was cleaved with EcoRI or ClaI, electrophoretically separated in a polyacrylamide gel, and silver stained. Three blood culture isolates from one patient in an intensive care unit, 4 isolates obtained from a child over a span of 2 weeks, and 5 isolates from 5 newborns in the same ward were grouped into 3 DNA pattern types, indicating identity of sequential isolates from 2 patients and nosocomial transmission of one Staphylococcus epidermidis strain between 5 babies. Results from pulsed-field gel electrophoresis of SmaI and SacII DNA digests and conventional marker systems such as antibiogram and plasmid profile were in accordance with these interpretations, whereas slight variation was observed in the biotypes of several strains. From the results of this study, we conclude that SF-REA is a precise and efficient method for the genotypic characterization of Staphylococcus epidermidis strains that can be used as a rapid and reliable typing tool.

Adult↗
PubMed1987

Restriction of single-stranded M13 DNA using synthetic oligonucleotides: the structural requirement of restriction enzymes.

A targeted ss (single stranded) DNA cleavage technique is reported which involves the use of synthetic oligomers complementary to the ss M13 DNA polylinker. BamHI, SmaI, and KpnI restriction enzymes were tested with a partial duplex DNA formed from ss M13 DNA and a nested series of fragments derived from a synthetic 21-mer which were complementary to the polylinker region. These enzymes require up to two flanking nucleotides in addition to the hexameric recognition site for efficient cleavage. This technique could be useful for effecting unique cleavages of DNA with enzymes which generally give a large number of fragments and for strategies of ss DNA manipulation.

Base Sequence↗