scholarly journals Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA

2011 ◽  
Vol 108 (11) ◽  
pp. 2535-2543 ◽  
Author(s):  
Jennifer Tullman ◽  
Gurkan Guntas ◽  
Matthew Dumont ◽  
Marc Ostermeier
Keyword(s):  
Plasmid Dna ◽  
S1 Nuclease ◽  
Supercoiled Form ◽  
Nuclease Digestion

scholarly journals Improved downstream process for the production of plasmid DNA for gene therapy.

2005 ◽  
Vol 52 (3) ◽  
pp. 703-711 ◽  
Author(s):  
Jochen Urthaler ◽  
Wolfgang Buchinger ◽  
Roman Necina
Keyword(s):  
Gene Therapy ◽  
Plasmid Dna ◽  
Viral Vectors ◽  
Continuous System ◽  
Size Exclusion ◽  
High Yield ◽  
Downstream Process ◽  
Naked Dna ◽  
Supercoiled Form ◽  
Exclusion Chromatography

Gene therapy and genetic vaccines promise to revolutionize the treatment of inherited and acquired diseases. Since viral vectors are generally associated with numerous disadvantages when applied to humans, the administration of naked DNA, or DNA packed into lipo- or polyplexes emerge as viable alternatives. To satisfy the increasing demand for pharmaceutical grade plasmids we developed a novel economic downstream process which overcomes the bottlenecks of common lab-scale techniques and meets all regulatory requirements. After cell lysis by an in-house developed gentle, automated continuous system the sequence of hydrophobic interaction, anion exchange and size exclusion chromatography guarantees the separation of impurities as well as undesired plasmid isoforms. After the consecutive chromatography steps, adjustment of concentration and final filtration are carried out. The final process was proven to be generally applicable and can be used from early clinical phases to market-supply. It is scaleable and free of animal-derived substances, detergents (except lysis) and organic solvents. The process delivers high-purity plasmid DNA of homogeneities up to 98% supercoiled form at a high yield in any desired final buffer.

scholarly journals Isolation of Developmentally Regulated Genes fromToxoplasma gondii by a Gene Trap with the Positive and Negative Selectable Marker Hypoxanthine-Xanthine-Guanine Phosphoribosyltransferase

1998 ◽  
Vol 18 (2) ◽  
pp. 807-814 ◽  
Author(s):  
Laura J. Knoll ◽  
John C. Boothroyd
Keyword(s):  
Antimicrobial Agents ◽  
Selectable Marker ◽  
Insertion Site ◽  
Gene Trap ◽  
Specific Gene ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
S1 Nuclease ◽  
Nuclease Digestion

ABSTRACT Within its intermediate host, Toxoplasma gondiiswitches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT). T. gondii was transfected with promoterlessHXGPRT and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing HXGPRT. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using Dolichos biflorus agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had Dolichos immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the Toxoplasmafamily of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive P36 gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.

Detection of Non-B-DNA Secondary Structures by S1 Nuclease Digestion

1998 ◽  
Vol 75 (6) ◽  
pp. 762 ◽  
Author(s):  
Marcel.li del Olmo ◽  
Agustin Aranda ◽  
Jose E. Perez-Ortin ◽  
Vicente Tordera
Keyword(s):  
Secondary Structures ◽  
S1 Nuclease ◽  
Nuclease Digestion ◽  
Dna Secondary Structures

scholarly journals First Report of an OXA-48- and CTX-M-213-ProducingKluyveraSpecies Clone Recovered from Patients Admitted in a University Hospital in Madrid, Spain

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Marta Hernández-García ◽  
Ricardo León-Sampedro ◽  
Blanca Pérez-Viso ◽  
María Isabel Morosini ◽  
Nieves López-Fresneña ◽  
...  
Keyword(s):  
Genomic Analysis ◽  
Rflp Analysis ◽  
Site Directed Mutagenesis ◽  
University Hospital ◽  
The Novel ◽  
Illumina Hiseq ◽  
S1 Nuclease ◽  
Content Type ◽  
Nuclease Digestion ◽  
Chromogenic Agar

ABSTRACTEnterobacteralesspecies other thanKlebsiella pneumoniaealso contribute to OXA-48 carbapenemase endemicity. We studied the emergence of an OXA-48-producingKluyveraspecies clone, which expresses the novel CTX-M-213 enzyme, colonizing patients in our hospital. Rectal swabs from patients admitted in four wards (March 2014 to March 2016; R-GNOSIS project) were seeded onto Chromo ID-ESBL) and Chrom-CARB/OXA-48 chromogenic agar plates. Carbapenemases and extended-spectrum β-lactamases (ESBLs) were characterized (PCR, sequencing, cloning, and site-directed mutagenesis), and antibiotic susceptibility was determined. Clonal relatedness was established (XbaI pulsed-field gel electrophoresis [XbaI-PFGE]), and plasmid content was studied (transformation, S1 nuclease digestion-PFGE, SB-hybridization, restriction fragment length polymorphism [RFLP] analysis [DraI and HpaI], and PCR [incompatibility group andrepA,traU, andparA genes]). Whole-genome sequencing (WGS) (Illumina HiSeq-2500) and further bioinformatics analysis of plasmids (PLACNET and plasmidSPAdes) were performed. Patients' charts were reviewed. Six unrelated patients (median age, 75 years [range, 59 to 81 years]; 4/6 male patients) colonized with OXA-48-producingKluyveraspecies isolates (>95% similarity of the PFGE pattern) were identified. Nosocomial acquisition was demonstrated. In two patients, OXA-48-producingKluyveraspecies isolates coexisted with OXA-48-producingRaoultella ornithinolytica,K. pneumoniae, andEscherichia coli. TheblaOXA-48gene was located on an ∼60-kb IncL plasmid related to IncL/M-pOXA-48a and the novelblaCTX-M-213gene in a conserved chromosomal region ofKluyveraspecies isolates. CTX-M-213, different from CTX-M-13 (K56E) but conferring a similar β-lactam resistance profile, was identified. Genomic analysis also revealed a 177-kb IncF plasmid (class I integron harboringsul1andaadA2) and an 8-kb IncQ plasmid (IS4-blaFOX-8). We describe the firstblaOXA-48plasmid inKluyveraspp. and the novel chromosomal CTX-M-213 enzyme and highlight further nosocomial dissemination ofblaOXA-48through clonal lineages or plasmids related to IncL/M-pOXA-48a.

A Synthetic Block Copolymer Regulates S1 Nuclease Fragmentation of Supercoiled Plasmid DNA

2005 ◽  
Vol 117 (23) ◽  
pp. 3610-3614 ◽  
Author(s):  
Kensuke Osada ◽  
Yuichi Yamasaki ◽  
Satoshi Katayose ◽  
Kazunori Kataoka
Keyword(s):  
Block Copolymer ◽  
Plasmid Dna ◽  
S1 Nuclease ◽  
Supercoiled Plasmid

scholarly journals Single-strand nuclease action on heat-denatured spermiogenic chromatin.

1976 ◽  
Vol 24 (8) ◽  
pp. 901-907 ◽  
Author(s):  
J D Hunter ◽  
A J Bodner ◽  
F T Hatch ◽  
R L Balhorn ◽  
J A Mazrimas ◽  
...  
Keyword(s):  
Thermal Denaturation ◽  
Deoxyribonucleic Acid ◽  
Elevated Temperatures ◽  
Chromatin Condensation ◽  
Heat Denaturation ◽  
S1 Nuclease ◽  
Nuclease Digestion ◽  
Aldehyde Groups ◽  
Phosphate Groups

The aim of this study was to compare the sensitivity of chromatin from representative cellular stages of spermiogenesis to a single-strandeded nuclease after heat denaturation. Thermal denaturation of chromatin was assayed in situ in fixed round, elongating and elongated spermatids and in testicular sperm from mice. Production of single-stranded deoxyribonucleic acid (DNA) at elevated temperatures was monitored by digesting chromatin with endonuclease specific for single-stranded DNA (S1 nuclease), staining the residual DNA with gallocyanin-chrome alum (GAC) and measuring the stain content by absorption cytophotometry. Changes in GCA staining were minimal over the temperature range of 22-90 degrees C in each cell type not exposed to nuclease. Staining of undigested cells decreased progressively with advancing cell maturity. Nuclease had no effect on the GCA content of round spermatids below 60 degrees C, but above this temperature there was a progressive decrease in GCA-stainable chromatin. Both round and elongating spermatid stages showed a significantly greater sensitivity to nuclease digestion than did more mature stages; sperm showed no effects of nuclease action below 80 degrees C. Progressive chromatin condensation and a concomitant decrease in the number of available DNA phosphate groups during spermiogenic cell maturation may be responsible for the observed decline in sensitivity to nuclease and decreased GCA staining. Thermal denaturation of round spermatids labeled with 3H-thymidine produced no change in autoradiographic mean nuclear grain counts, indicating no loss of thymidine-labeled DNA from the slides during denaturation. When round spermatids and sperm were hydrolyzed with hot tricholoroacetic acid before staining, both nuclear GCA content and autoradiograph grain count were partially reduced, indicating incomplete DNA removal. Almost complete loss of Feulgen-stainable material occurred in these cells and may be due to depurination and elimination of Feulgren-reactant aldehyde groups.

scholarly journals Non-intertwined strands of plasmid DNA contradict the Watson and Crick model of DNA structure

F1000Research ◽  
2020 ◽  
Vol 8 ◽  
pp. 356
Author(s):  
Pawan Kumar
Keyword(s):  
Electrophoretic Mobility ◽  
Plasmid Dna ◽  
Dna Structure ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
S1 Nuclease ◽  
Circular Dna ◽  
Exonuclease I ◽  
Alternative Structures ◽  
Native Plasmid

According to Watson and Crick (W/C) model of DNA structure, a DNA molecule consists of two antiparallel polynucleotide chains, intertwined with each other. Although W/C model is accepted widely, some researchers have raised questions against it and proposed alternative structures for DNA. In the present study, we examined W/C model using plasmid DNA. It was hypothesized that two strands of plasmid will remain intertwined (and not separate from each other) under denaturing conditions if it follows W/C model. To test this, plasmid DNA was denatured using sodium hydroxide (NaOH) and analyzed by agarose gel electrophoresis. It was observed that addition of 0.5 N NaOH to pUC19 or pBR322 plasmids resulted in a new form of DNA having higher electrophoretic mobility in agarose gel. Higher electrophoretic mobility DNA (HmDNA) in NaOH-denatured pUC19 was digestible with S1 nuclease, but not with HindIII and ‘exonuclease I + alkaline phosphatase’. These results demonstrated that HmDNA is single-stranded circular DNA, formed due to separation of two strands of NaOH-denatured plasmid. Single-stranded and circular nature of HmDNA was corroborated by its comparable electrophoretic mobility with purified top and bottom strands of plasmid DNA. Next, we examined whether HmDNA can re-anneal into the native plasmid. Interestingly, when subjected to renaturing conditions, HmDNA from NaOH-denatured pUC19 re-annealed to form native pUC19 plasmid, which was digestible with HindIII and induced ampicillin resistance in Escherichia coli. These findings demonstrated the reversible separation of two strands of plasmid DNA and contradicted the W/C model of DNA structure.

A Synthetic Block Copolymer Regulates S1 Nuclease Fragmentation of Supercoiled Plasmid DNA

2005 ◽  
Vol 44 (23) ◽  
pp. 3544-3548 ◽  
Author(s):  
Kensuke Osada ◽  
Yuichi Yamasaki ◽  
Satoshi Katayose ◽  
Kazunori Kataoka
Keyword(s):  
Block Copolymer ◽  
Plasmid Dna ◽  
S1 Nuclease ◽  
Supercoiled Plasmid
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