Nicking activity on pBR322 DNA of ribosome inactivating proteins from Phytolacca dioica L. leaves

2005 ◽  
Vol 386 (4) ◽  
pp. 307-317 ◽  
Author(s):  
Serena Aceto ◽  
Antimo Di Maro ◽  
Barbara Conforto ◽  
Gesualdo G. Siniscalco ◽  
Augusto Parente ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Mung Bean Nuclease ◽  
Ribosome Inactivating Proteins ◽  
Dna Molecule ◽  
Pbr322 Dna ◽  
Low Salt ◽  
Control Plasmid ◽  
Ampicillin Resistance Gene ◽  
Transforming Activity ◽  
Linear Molecules

Abstract Ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves are rRNA-N-glycosidases, as well as adenine polynucleotide glycosylases. Here we report that some of them cleave supercoiled pBR322 dsDNA, generating relaxed and linear molecules. PD-L1, the glycosylated major form isolated from the winter leaves of adult P. dioica plants, produces both free 3′-OH and 5′-P termini randomly distributed along the DNA molecule, as suggested by labelling experiments with [α-32P]dCTP and [γ-32P]dATP. Moreover, when the reaction is carried out under low-salt conditions, cleavage is observed mainly at a specific site, located downstream of the ampicillin resistance gene (close to position 3200), ending with the deletion of a fragment of approximately 70 nucleotides. This cleavage pattern is similar to that obtained under the same conditions with mung bean nuclease, a single-strand endonuclease. Furthermore, pBR322 DNA treated with PD-L1 shows reduced transforming activity with E. coli HB101 competent cells in comparison to untreated control plasmid DNA.

Electron microscopic assays of restriction endonuclease cutting, exonuclease digestion, and hybridization

1984 ◽  
Vol 42 ◽  
pp. 686-687
Author(s):  
Mark Hannibal ◽  
Jacob Varkey ◽  
Michael Beer
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Restriction Endonuclease ◽  
Double Helix ◽  
Test Material ◽  
Electron Microscopic ◽  
Exonuclease Iii ◽  
Dna Molecule ◽  
Linear Molecules ◽  
Exonuclease Digestion

Workman and Langmore have recently proposed a procedure for isolating particular chromatin fragments. The method requires restriction endonuclease cutting of the chromatin and a probe, their digestion with two exonucleases which leave complimentary single strand termini and low temperature hybridization of these. We here report simple electron microscopic monitoring of the four reactions involved.Our test material was ϕX-174 RF DNA which is cut once by restriction endonuclease Xho I. The conversion of circles to linear molecules was followed in Kleinschmidt spreads. Plate I shows a circular and a linear DNA molecule. The rate of cutting is shown in Figure 1.After completion of the endonuclease cutting, one portion of the DNA was treated with exonuclease III, an enzyme known to digest the 3' terminals of double helical DNA. Aliquots when examined in the electron microscope reveal a decreasing length of double helix and increasing bushes at the ends.

scholarly journals Ribosome-inactivating proteins depurinate poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase and have transforming activity for 3T3 fibroblasts

FEBS Letters ◽  
2003 ◽  
Vol 538 (1-3) ◽  
pp. 178-182 ◽  
Author(s):  
Luigi Barbieri ◽  
Maurizio Brigotti ◽  
Paolo Perocco ◽  
Domenica Carnicelli ◽  
Marialibera Ciani ◽  
...  
Keyword(s):  
Ribosome Inactivating Proteins ◽  
3T3 Fibroblasts ◽  
Transforming Activity

Photocleavage of Plasmid pBR322 DNA by Some Anionic Porphyrins

1998 ◽  
Vol 02 (04) ◽  
pp. 337-343 ◽  
Author(s):  
SHAMPA R. CHATTERJEE ◽  
T. S. SRIVASTAVA ◽  
J. P. KAMAT ◽  
T. P. A. DEVASAGAYAM
Keyword(s):  
Lipoic Acid ◽  
Plasmid Dna ◽  
Dna Cleavage ◽  
Irradiation Time ◽  
Water Soluble ◽  
Plasmid Pbr322 ◽  
Limited Extent ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Pbr322 Dna

The water-soluble porphyrins meso-tetrakis[4-(carboxymethyleneoxy)phenyl]porphyrin ( H 2 T4CPP ), meso-tetrakis[3-(carboxymethyleneoxy)phenyl]porphyrin ( H 2 T3CPP ) and meso-tetrakis[3,4-bis(carboxymethyleneoxy)phenyl]porphyrin ( H 2 T3 , 4BCPP ) cleave plasmid pBR322 DNA to single-strand breaks (SSBs) in the presence of molecular oxygen and visible light. These porphyrins induced SSBs in DNA as a function of irradiation time as well as porphyrin concentration. Under similar conditions (10 μM or more), H 2 T3CPP showed more SSBs in DNA than the porphyrins H 2 T 4CPP and H 2 T 3,4 BCPP . The DNA cleavage was more in D 2 O -based buffer than in H 2 O buffer. In addition, this DNA cleavage was inhibited by the presence of sodium azide and lipoic acid, which are potent quenchers of singlet oxygen (1 O 2). These observations suggest the involvement of 1 O 2 in photocleavage of DNA. Further, the DNA cleavage, to a limited extent, was also inhibited by tert-butanol and mannitol, both quenchers of hydroxyl radical (· OH ), suggesting the involvement of · OH in photocleavage of DNA. Thus both 1 O 2 and · OH are involved in photocleavage of plasmid DNA by these porphyrins.

scholarly journals Differences in intracellular DNA ligation after microinjection and transfection.

1984 ◽  
Vol 4 (2) ◽  
pp. 240-246 ◽  
Author(s):  
J J Kopchick ◽  
D W Stacey
Keyword(s):  
Long Terminal Repeat ◽  
Plasmid Dna ◽  
Terminal Repeat ◽  
Viral Rna ◽  
Dna Molecule ◽  
Repeat Sequences ◽  
Dna Ligation ◽  
Avian Cell ◽  
Pbr322 Plasmid ◽  
Avian Sarcoma

An uninterrupted avian sarcoma viral genome terminated by viral long terminal repeat sequences was cloned into a pBR322 plasmid. After introduction into a cultured avian cell, transcription of either the circular plasmid molecule or one linearized within the pBR322 sequences could initiate and terminate at long terminal repeat sequences, yielding full-sized viral RNA. A plasmid DNA molecule linearized by cleavage within the viral pol gene, on the other hand, would have to undergo ligation to yield full-sized viral RNA. Microinjection of each of these three types of DNA into the nuclei of quail cells promoted the release of similar virus titers, indicating that the plasmid DNA cleaved within the viral pol gene had been efficiently and accurately ligated. When plasmid DNA was transfected into quail cells, circular and pBR322-cleaved molecules directed the synthesis of similar virus titers, indicating that they were similarly taken up and utilized by the cells. Compared with these results, plasmid DNA cleaved within the pol gene was reduced in activity over 95% after transfection. This reduction did not result from inefficient ligation but from the generation of mutations (of limited size) during ligation of the transfected molecules. Mutations were not observed after microinjection even into the cytoplasm. Consistent with these findings, transfected DNA termini were found to be joined regardless of their structure, whereas ligation after microinjection required that single-stranded protruding DNA termini be complementary.

scholarly journals STUDIES ON THE CHEMISTRY OF THE TRANSFORMING ACTIVITY

1953 ◽  
Vol 98 (4) ◽  
pp. 373-397 ◽  
Author(s):  
Stephen Zamenhof ◽  
Hattie E. Alexander ◽  
Grace Leidy
Keyword(s):  
Ionic Strength ◽  
Quantitative Study ◽  
Minimal Amount ◽  
Amino Groups ◽  
Active Molecule ◽  
Dna Molecule ◽  
Order Of Magnitude ◽  
Transforming Activity ◽  
Lag Period ◽  
Low Ionic Strength

The transforming principles of Hemophilus influenzae have been purified by a new method including fractional extraction. The active molecule behaves in these extractions like the bulk of the DNA preparation. The minimal amount of DNA necessary for transformation appeared to be of the same order of magnitude as the amount of DNA in a single cell. Quantitative study has been made of the resistance of transforming activity to various agents. When subjected to heat, the temperature at which the activity starts to decrease corresponds rather closely to the temperature at which the viscosity of the bulk of the DNA preparations starts to decrease. Similar correspondence was found when the transforming principle was subjected to pH changes. This is further evidence that the behavior of the active molecules is similar to the behavior of the average DNA molecule of the preparation. The activity is reduced by exposure to low ionic strength and by dehydration. Desoxyribonuclease in concentrations less than 10–4 γ/cc. is able to destroy the activity; a lag period during which the activity but not the viscosity decreases has been observed. NaNO2 at pH 5.3, HCHO and 10–5 M Fe++ reduce or destroy the activity; the importance of intact amino groups in the DNA molecule for the activity is discussed. Several protein-denaturing, sterilizing, and mutagenic agents have been found to have no effect on the transforming activity.

scholarly journals Rosmarinus officinalis Extract Inhibits Human Melanoma Cell Growth

2009 ◽  
Vol 4 (12) ◽  
pp. 1934578X0900401 ◽  
Author(s):  
Alessandra Russo ◽  
Laura Lombardo ◽  
Nicolas Troncoso ◽  
Juan Garbarino ◽  
Venera Cardile
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Plasmid Dna ◽  
Dna Cleavage ◽  
Human Melanoma ◽  
Rosmarinus Officinalis ◽  
Carnosic Acid ◽  
Human Melanoma Cell ◽  
Pbr322 Dna ◽  
Melanoma Cell Lines

Rosmarinus officinalis L. is receiving increasing attention due to its anti-inflammatory and antioxidative constituents. Our recent studies showed that R. officinalis extract, containing 31.7 % of carnosic acid, was able to counteract the deleterious effects of UV-R, by protecting plasmid DNA from hydroxyl radicals generated by UV-A. In this work, we evaluated the effects of this extract on pBR322 DNA cleavage induced by nitric oxide, and the growth inhibitory activity against two human melanoma cell lines, M14 and A375. The extract showed a protective effect on plasmid DNA damage, and at concentrations of 10-80 μg/mL was able to reduce significantly (p<0.001) the growth (MTT assay) of both melanoma cell lines. In addition, our results indicate that apoptotic cell demise is induced in M14 and A375 cells. No statistically significant increase in LDH release was observed in melanoma cells, correlated to a fragmentation of genomic DNA, determined by COMET assay.

Loss of transforming activity of plasmid DNA (pBR322) in E. coli caused by singlet molecular oxygen

FEBS Letters ◽  
1987 ◽  
Vol 211 (1) ◽  
pp. 49-52 ◽  
Author(s):  
Heribert Wefers ◽  
Dietrich Schulte-Frohlinde ◽  
Helmut Sies
Keyword(s):  
Molecular Oxygen ◽  
Plasmid Dna ◽  
Singlet Molecular Oxygen ◽  
E Coli ◽  
Transforming Activity
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