scholarly journals Recombinant pseudorabies virus DNase exhibits a RecBCD-like catalytic function

1998 ◽  
Vol 330 (1) ◽  
pp. 55-59 ◽  
Author(s):  
Chien-Yun HSIANG ◽  
Tin-Yun HO ◽  
Chin-Hui HSIANG ◽  
Tien-Jye CHANG
Keyword(s):  
Pseudorabies Virus ◽  
Nuclease Activity ◽  
Alkaline Ph ◽  
E Coli ◽  
Catalytic Function ◽  
Double Stranded Dna ◽  
Recombinant Pseudorabies Virus ◽  
Absolute Requirement ◽  
Na Ions

The pseudorabies virus (PRV) DNase gene has previously been mapped within the PRV genome. To characterize further the enzymic properties of PRV DNase, this enzyme was expressed in Escherichia coli with the use of a pET expression vector. The protein was purified to homogeneity and assayed for nuclease activity in vitro. Recombinant PRV DNase exhibited an alkaline pH preference and an absolute requirement for Mg2+ ions that could not be replaced by Ca2+ and Na+ ions. Further studies showed that PRV DNase exhibited endonuclease, 5ʹ-exonuclease and 3ʹ-exonuclease activities in both single-stranded and double-stranded DNA. This activity occurred randomly and no significant base preference was demonstrated. The multiple biochemical activities of PRV DNase are similar to the activities of Neurospora crassa endo-exonuclease and E. coli RecBCD, two additional enzymes that are involved in recombination. Taken together, the similarity of action between N. crassa endo-exonuclease, E. coli RecBCD, and PRV DNase suggests that PRV DNase might have a role in the process of recombination that occurs during PRV infection.

scholarly journals Synthesis, Characterization and Biological Activity of Two New Copper (II) Complexes with N-sulfonamide Ligand

2019 ◽  
Vol 70 (11) ◽  
pp. 4060-4067
Author(s):  
Adriana Corina Hangan ◽  
Laura Gratiela Vicas ◽  
Roxana Liana Stan ◽  
Emoke Pall ◽  
Luminita Simona Oprean ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Nuclease Activity ◽  
Normal Fibroblast ◽  
Diffusion Method ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Biological Studies ◽  
Cause Of Deaths

Despite the fact that a large number of chemotherapeutic anticancer agents have been discovered, cancer still remains a great cause of deaths worldwide. The purpose of our researches is to discover a new antitumor drug. In this aim, two new Cu(II) complexes, [Cu(L)2(py)2(H2O)2](C1) and [Cu(L)2(phen)](C2) with a new ligand, N-(5-trifluoromethyl-[1,3,4]-thiadiazole-2-yl)-benzensulfonamide(HL) were synthesized. The complexes were characterized by elemental analysis, spectral and magnetic determinations. The nuclease activity studies of the complexes confirm their capacity to cleavage the DNA molecule. Both complexes have in vitro antioxidant activity (DPPH, FRAP methods), in vitro (using xanthine /xanthine oxidase system) and in vivo (using S.cerevisiae)SOD mimetic activity.The results of MTT assay on two carcinoma cell lines (HeLa and WM35) indicate that both complexes have antitumor activity, but (C2) has a superior activity compared with (C1) and with Cisplatin. On normal fibroblast (HDFa), (C1) showed toxicity comparable with Cisplatin, but (C2) showed a lower one. Bacterial assays were also performed (by the disk diffusion method) and both complexes have antibacterial activity against S. aureus, E. coli, P. aeroginosa and B. cereus. All the biological studies are in concordance and show that both complexes have biologic activity but (C2)is much more active.

scholarly journals Diversity and prevalence of colibactin- and yersiniabactin encoding mobile genetic elements in enterobacterial populations: insights into evolution and co-existence of two bacterial secondary metabolite determinants

2021 ◽  
Author(s):  
Haleluya Wami ◽  
Alexander Wallenstein ◽  
Daniel Sauer ◽  
Monika Stoll ◽  
Rudolf von Bünau ◽  
...  
Keyword(s):  
Structural Diversity ◽  
Eukaryotic Cell ◽  
Genomic Region ◽  
Dna Breaks ◽  
E Coli ◽  
Integrative And Conjugative Elements ◽  
Double Stranded Dna ◽  
Bacterial Genetic ◽  
Species Specific

1 AbstractThe bacterial genotoxin colibactin interferes with the eukaryotic cell cycle by causing double-stranded DNA breaks. It has been linked to bacterially induced colorectal cancer in humans. Colibactin is encoded by a 54-kb genomic region in Enterobacteriaceae. The colibactin genes commonly co-occur with the yersiniabactin biosynthetic determinant. Investigating the prevalence and sequence diversity of the colibactin determinant and its linkage to the yersiniabactin operon in prokaryotic genomes, we discovered mainly species-specific lineages of the colibactin determinant and classified three main structural settings of the colibactin-yersiniabactin genomic region in Enterobacteriaceae. The colibactin gene cluster has a similar but not identical evolutionary track to that of the yersiniabactin operon. Both determinants could have been acquired on several occasions and/or exchanged independently between enterobacteria by horizontal gene transfer. Integrative and conjugative elements play(ed) a central role in the evolution and structural diversity of the colibactin-yersiniabactin genomic region. Addition of an activating and regulating module (clbAR) to the biosynthesis and transport module (clbB-S) represents the most recent step in the evolution of the colibactin determinant. In a first attempt to correlate colibactin expression with individual lineages of colibactin determinants and different bacterial genetic backgrounds, we compared colibactin expression of selected enterobacterial isolates in vitro. Colibactin production in the tested Klebsiella spp. and Citrobacter koseri strains was more homogeneous and generally higher than that in most of the E. coli isolates studied. Our results improve the understanding of the diversity of colibactin determinants and its expression level, and may contribute to risk assessment of colibactin-producing enterobacteria.

scholarly journals Characterization of Argonaute nucleases from mesophilic bacteria Paenibacillus borealis and Brevibacillus laterosporus

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Huarong Dong ◽  
Fei Huang ◽  
Xiang Guo ◽  
Xiaoyi Xu ◽  
Qian Liu ◽  
...  
Keyword(s):  
Small Molecule ◽  
Dna Cleavage ◽  
Low Cost ◽  
Nuclease Activity ◽  
Nucleic Acid Detection ◽  
Mesophilic Bacteria ◽  
Brevibacillus Laterosporus ◽  
Double Stranded Dna ◽  
Small Molecule Detection

AbstractThermophilic Argonaute proteins (Agos) have been shown to utilize small DNA guides for cleaving complementary DNA in vitro, which shows great potential for nucleic acid detection. In this study, we explored mesophilic Agos for the detection of small molecule by cooperating with allosteric transcription factors (aTFs). Two Agos from mesophilic bacteria, Paenibacillus borealis (PbAgo) and Brevibacillus laterosporus (BlAgo), showed nuclease activity for single-stranded DNA at moderate temperatures (37 °C) by using 5′-phosphorylated and 5′-hydroxylated DNA guides. Both Agos perform programmable cleavage of double-stranded DNA, especially in AT-rich regions of plasmid. Furthermore, we developed a simple and low-cost p-hydroxybenzoic acid detection method based on DNA-guided DNA cleavage of Agos and the allosteric effect of HosA, which expands the potential application of small molecule detection by Agos.

scholarly journals Recombinant Pseudorabies Virus with TK/gE Gene Deletion and Flt3L Co-Expression Enhances the Innate and Adaptive Immune Response via Activating Dendritic Cells

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 691
Author(s):  
Lun Yao ◽  
Qiao Hu ◽  
Siqi Chen ◽  
Tong Zhou ◽  
Xuexiang Yu ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Neutralizing Antibodies ◽  
Pseudorabies Virus ◽  
Gene Deletion ◽  
Viral Evolution ◽  
Clinical Signs ◽  
Adaptive Immune Response ◽  
Lethal Challenge ◽  
Recombinant Pseudorabies Virus

Owing to viral evolution and recombination, emerging pseudorabies virus (PRV) strains have caused unprecedented outbreaks in swine farms even when the pigs were previously vaccinated, which might indicate that traditional vaccines were unable to provide effective protection. The development of safe and efficacious vaccines presents prospects to minimize the clinical signs and eventually eradicate the infection. In this study, we used an emerging PRV strain, HNX, as the parental strain to construct a recombinant PRV with TK/gE gene deletion and Fms-related tyrosine kinase 3 ligand (Flt3L) expression, named HNX-TK−/gE−-Flt3L. HNX-TK−/gE−-Flt3L enhanced the maturation of bone marrow derived dendritic cells (DCs) in vitro. Significantly more activated DCs were detected in HNX-TK−/gE−-Flt3L-immunized mice compared with those immunized with HNX-TK−/gE−. Subsequently, a remarkable increase of neutralizing antibodies, gB-specific IgG antibodies, and interferon-gamma (IFN-γ) was observed in mice vaccinated with HNX-TK−/gE−-Flt3L. In addition, a lower mortality and less histopathological damage were observed in HNX-TK−/gE−-Flt3L vaccinated mice with upon PRV lethal challenge infection. Taken together, our results revealed the potential of Flt3L as an ideal adjuvant that can activate DCs and enhance protective immune responses and support the further evaluation of HNX-TK−/gE−-Flt3L as a promising PRV vaccine candidate.

scholarly journals A YoeB toxin from A. tumefaciens has metal-dependent DNA cleaving activity

2019 ◽  
Author(s):  
Julia McGillick ◽  
Jessica R. Ames ◽  
Tamiko Murphy ◽  
Eswar Reddem ◽  
Christina R. Bourne
Keyword(s):  
Dna Cleavage ◽  
Potential Role ◽  
Fold Increase ◽  
Nuclease Activity ◽  
Dose Dependence ◽  
Bacterial Cells ◽  
Toxin Concentration ◽  
E Coli ◽  
Dna Cleaving

ABSTRACTToxin-antitoxin (TA) systems, including YoeB-YefM, are important mediators of bacterial physiological changes. Agrobacterium tumefaciens YoeB and YefM are similar to that from E. coli, and interact as a tight heterotetramer with a KD of 653 pM. We have verified that AtYoeB can perform both ribosome-dependent and –independent RNA cleavage. We have also characterized a newly described metal-dependent and pH-sensitive DNA cleaving ability. We note that this DNA cleaving ability is observed at toxin concentrations as low as 150 nM. The dose-dependence of in vitro ribosome-independent RNA and metal-dependent DNA cleavage is equivalent, and requires a ten-fold increase in toxin concentration as opposed to in the presence of the ribosome. The toxin concentration inside bacterial cells is unknown and according to current models, should increase upon activation of YoeB through degradation of the YefM antitoxin. The discovery of general nuclease activity by AtYoeB, and perhaps other YoeB toxins, offers an opportunity to explore the plasticity of this protein fold and its potential role in the evolution of nucleases.

scholarly journals ORF157 from the Archaeal Virus Acidianus Filamentous Virus 1 Defines a New Class of Nuclease

2010 ◽  
Vol 84 (10) ◽  
pp. 5025-5031 ◽  
Author(s):  
Adeline Goulet ◽  
Mery Pina ◽  
Peter Redder ◽  
David Prangishvili ◽  
Laura Vera ◽  
...  
Keyword(s):  
Hot Springs ◽  
Protein Structures ◽  
Three Dimensional ◽  
Nuclease Activity ◽  
Open Reading Frames ◽  
Double Stranded Dna ◽  
Substitution Mutations ◽  
Reading Frames

ABSTRACT Acidianus filamentous virus 1 (AFV1) (Lipothrixviridae) is an enveloped filamentous virus that was characterized from a crenarchaeal host. It infects Acidianus species that thrive in the acidic hot springs (>85°C and pH <3) of Yellowstone National Park, WY. The AFV1 20.8-kb, linear, double-stranded DNA genome encodes 40 putative open reading frames whose sequences generally show little similarity to other genes in the sequence databases. Because three-dimensional structures are more conserved than sequences and hence are more effective at revealing function, we set out to determine protein structures from putative AFV1 open reading frames (ORF). The crystal structure of ORF157 reveals an α+β protein with a novel fold that remotely resembles the nucleotidyltransferase topology. In vitro, AFV1-157 displays a nuclease activity on linear double-stranded DNA. Alanine substitution mutations demonstrated that E86 is essential to catalysis. AFV1-157 represents a novel class of nuclease, but its exact role in vivo remains to be determined.

scholarly journals Xylella fastidiosa Plasmid-Encoded PemK Toxin Is an Endoribonuclease

2012 ◽  
Vol 102 (1) ◽  
pp. 32-40 ◽  
Author(s):  
Min Woo Lee ◽  
Elizabeth E. Rogers ◽  
Drake C. Stenger
Keyword(s):  
Escherichia Coli ◽  
Xylella Fastidiosa ◽  
Nuclease Activity ◽  
Single Amino Acid ◽  
Double Stranded Rna ◽  
E Coli ◽  
Double Stranded Dna ◽  
Rna Targets ◽  
Direct Binding ◽  
Rapid Amplification

Stable inheritance of pXF-RIV11 in Xylella fastidiosa is conferred by the pemI/pemK toxin-antitoxin (TA) system. PemK toxin inhibits bacterial growth; PemI is the corresponding antitoxin that blocks activity of PemK by direct binding. PemK and PemI were overexpressed in Escherichia coli and activities of each were assessed. Purified PemK toxin specifically degraded single-stranded RNA but not double-stranded RNA, double-stranded DNA, or single-stranded DNA. Addition of PemI antitoxin inhibited nuclease activity of PemK toxin. Purified complexes of PemI bound to PemK exhibited minimal nuclease activity; removal of PemI antitoxin from the complex restored nuclease activity of PemK toxin. Sequencing of 5′ rapid amplification of cDNA ends products of RNA targets digested with PemK revealed a preference for cleavage between U and A residues of the sequence UACU and UACG. Nine single amino-acid substitution mutants of PemK toxin were constructed and evaluated for growth inhibition, ribonuclease activity, and PemI binding. Three PemK point-substitution mutants (R3A, G16E, and D79V) that lacked nuclease activity did not inhibit growth. All nine PemK mutants retained the ability to bind PemI. Collectively, the results indicate that the mechanism of stable inheritance conferred by pXF-RIV11 pemI/pemK is similar to that of the R100 pemI/pemK TA system of E. coli.

scholarly journals Studies on sex-organ development. Changes in chromatin structure during spermatogenesis in maturing rooster testis as demonstrated by the initiation pattern of ribonucleic acid synthesis in vitro

1978 ◽  
Vol 170 (2) ◽  
pp. 203-210 ◽  
Author(s):  
C Mezquita ◽  
C S Teng
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Acid Synthesis ◽  
Chromatin Interaction ◽  
E Coli ◽  
Double Stranded Dna ◽  
The Stability ◽  
Kinetics Of

To probe the structural change in the genome of the differentiating germ cell of the maturing rooster testis, the chromatin from nuclei at various stages of differentiation were transcribed with prokaryotic RNA polymerase from Escherichia coli or with eukaryotic RNA polymerase II from wheat germ. The transcription was performed under conditions of blockage of RNA chain reinitiation in vitro with rifampicin or rifampicin AF/013. With the E. coli enzyme, the changes in (1) the titration curve for the enzyme-chromatin interaction, (2) the number of initiation sites, (3) the rate of elongation of RNA chains, and (4) the kinetics of the formation of stable initiation complexes revealed the unmasking of DNA in elongated spermatids and the masking of DNA in spermatozoa. In both cases the stability of the DNA duplex in the initiation region for RNA synthesis greatly increased. In contrast with the E. coli enzyme, the wheat-germ RNA polymerase II was relatively inefficient at transcribing chromatin of elongated spermatids. Such behaviour can be predicted if unmasked double-stranded DNA is present in elongated spermatids.

scholarly journals Direct purification of CRISPR/Cas ribonucleoprotein from E. coli in a single step

2018 ◽  
Author(s):  
Siyu Lin ◽  
Jie Qiao ◽  
Lixin Ma ◽  
Yi Liu
Keyword(s):  
Large Scale ◽  
Low Cost ◽  
Cost Effective ◽  
Nuclease Activity ◽  
Single Step ◽  
Simple Method ◽  
E Coli ◽  
Purification System

AbstractCRISPR/Cas ribonucleoprotein (RNP) complexes have been recently used as promising biological tools with plenty of applications, however, there are by far no efficient methods to prepare them at large scale and low cost. Here, we present a simple method to directly produce and purify Cas RNP, including the widely used Cas9 and Cas12a nuclease, from E.coli in a single step using an ultra-high-affinity CL7/Im7 purification system. The prepared Cas RNP shows high stability, solid nuclease activity in vitro, and profound genome editing efficiency in vivo. Our method is convenient, cost-effective, and applicable to prepare other CRISPR associated nucleases.

scholarly journals Isolation, characterization and possible mode of action of antiseminalplasmin, a new protein that inhibits the antimicrobial activity of seminalplasmin

1985 ◽  
Vol 227 (2) ◽  
pp. 609-619 ◽  
Author(s):  
V N Rao ◽  
P M Bhargava
Keyword(s):  
Seminal Plasma ◽  
Rna Synthesis ◽  
Sodium Dodecyl ◽  
Alkaline Ph ◽  
E Coli ◽  
Ph Values ◽  
Acidic Peptide ◽  
Transcription In Vitro ◽  
New Protein

The isolation from bovine seminal plasma and purification of a new protein called ‘antiseminalplasmin’, which reverses the inhibition of the growth of, and RNA synthesis in, Escherichia coli by seminalplasmin (another protein of bovine seminal plasma), is described. Antiseminalplasmin, a weakly acidic protein, has a minimum Mr of about 39 000 and appears to consist of three acidic peptide chains that move close to each other on electrophoresis on cellulose acetate strips or on sodium dodecyl sulphate/18%-(w/v)-polyacrylamide gels. Antiseminalplasmin has a tendency to oligomerize at slightly alkaline pH values; it does not bind to seminalplasmin or to DNA, and does not reverse the inhibition by seminalplasmin of transcription in vitro by purified E. coli RNA polymerase. It appears that antiseminalplasmin may act by binding to the cell surface and preventing the entry of seminalplasmin into the cells. By itself, antiseminalplasmin has no effect on the growth of E. coli.

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