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PubMed2025

Efficient site-specific integration of kilobase-length DNA fragments in plant cells via Kp03 recombinase.

Targeted insertion of large DNA sequences into plant genomes remains a major challenge in synthetic biology. Here, we evaluate the large serine recombinase Kp03 for site-specific integration of DNA fragments in rice and Arabidopsis. In transient protoplast assays, Kp03 mediates efficient insertion of donor DNA up to 27.3 kilobases (kb), with plasmid integration efficiencies reaching 99.1% for fragments up to 3.4 kb. Truncation experiments reveal that a minimal 15-bp attB sequence is necessary for integration. As a proof of concept, Kp03 successfully incorporates a 3.4-kb donor DNA into the rice genome at a locus containing this minimal attB sequence. Moreover, in rice callus, combining Kp03 with the NM-PE genome editing system to install a 26-bp attB site enables targeted integration of a 3.4-kb donor at the desired genomic locus. These findings establish Kp03 as a versatile tool for plant genome engineering, with broad applications for synthetic biology.

Oryza↗
PubMed1979

DNA fragmentation in some organs of rats and mice treated with cycasin.

Cycasin (methylazoxymethanol-beta-D-glucoside) is carcinogenic in several animal species. It produces a variety of malignant tumours, mainly in the liver of mice, and in the liver, kidney and large intestine in rats. It does not appear to be mutagenic in the Ames test, even in the presence of liver microsome fraction, and it is among those carcinogens (less than 10%) ranked as "false negatives" in this test. The ability of cycasin to damage in vivo liver, kidney, lung and colonic DNA of Wistar rats and C57BL/L mice was investigated by means of alkaline elution technique. Oral single-dose administration of cycasin, in the range of 50-400 mg/kg body weight, produced in the rat a clearly evident dose-dependent DNA fragmentation in the liver, and less marked damage to DNA from kidney and colon mucosa. In mice, the same treatment produced dose-dependent DNA damage only in the liver. DNA repair up to 18 h appeared to be incomplete both in mice and rats. Methylazoxymethanol acetate is considered to be an active form of cycasin. While in vivo methylazoxymethanol acetate caused DNA damage, in vitro it appeared inactive and required metabolic activation, possibly consisting in its hydrolysis by esterase activity, to be able to cause DNA fragmentation.

Animals↗
PubMed1995

Formation of apoptotic bodies is associated with internucleosomal DNA fragmentation during drug-induced apoptosis.

The onset of apoptosis is often coincident with internucleosomal DNA fragmentation or ladders which are considered a hallmark of the process. However, several studies have indicated that MOLT-4 human lymphoblastoid cells exposed to various agents, including VP16, display some apoptotic characteristics in the absence of either internucleosomal ladders or production of apoptotic bodies. The present study records that, in the presence of aurintricarboxylic acid (ATA), internucleosomal ladders were detected in DNA isolated from VP16-treated MOLT-4 cells; a paradoxical result in view of inhibition by ATA of nuclease activity in cell free preparations. The activity of ATA in mediating genomic fragmentation was dose- and time-dependent. Moreover, addition of ATA to VP16-treated MOLT-4 cells also resulted in production of apoptotic bodies, this effect being quantified by morphological examination and flow cytometry. Detection of ladders and apoptotic bodies after addition of ATA was not attributable to increased toxicity in cells exposed to the combined treatment relative to VP16 alone. A similar response, that is the appearance of both internucleosomal fragmentation and apoptotic bodies, occurred after exposure of MOLT-4 cells to the mitotic inhibitor podophyllotoxin. The consistent association between internucleosomal fragmentation of DNA and formation of apoptotic bodies exhibited during death of MOLT-4 cells, insofar as both characteristics are either present or absent following different agents, suggests interdependence.

Apoptosis↗
PubMed1981

Construction and characterization of three yeast-Escherichia coli shuttle vectors designed for rapid subcloning of yeast genes on small DNA fragments.

We have constructed three new subcloning plasmid vectors, pRC1, pRC2, and pRC3, derived from pKC7, which allow the rapid, single-step subcloning of yeast genes. Subcloning with these vectors utilizes a partial digestion with Sau3A to generate a quasi-random set of DNA fragments from the original plasmid. All three vectors contain a kanamycin resistance gene. Therefore, if the original cloned yeast DNA fragment is present in a vector that does not specify kanamycin resistance, the subclone pool can be propagated in Escherichia coli in the presence of kanamycin to select against parent plasmids that escaped restriction by Sau3A. Selection by complementation in yeast yields a collection of plasmids with smaller yeast DNA inserts containing the gene of interest. In the vectors pRC2 and pRC3, constructed from pRC1, the unique BamHI site is located within an intact tetracycline resistance gene, thus making it possible to screen bacterial transformants for those containing recombinant plasmid molecules. Vectors pRC2 and pRC3 also contain the yeast 2 micrometers DNA replication origin, and thus are more stable than plasmids carrying only the TRP1-associated replicator (ars1).

Cloning, Molecular↗
PubMed2004

DNA fragment transplacement in Saccharomyces cerevisiae: some genetic considerations.

The ability to make specific genomic alterations is an invaluable tool to researchers who use genetics and biochemistry to study problems in biology. We have investigated some of the parameters governing DNA fragment transplacement in two commonly used strains of Saccharomyces cerevisiae, S288C and W303-1A. These strains exhibited a marked difference in their capacity to take up plasmid DNA and utilize linear DNA fragments as substrates for transplacement. The contributions of transformation efficiency, length of homology, and alternative target site configuration were assessed. This analysis indicates that several genetic parameters are important for optimizing the efficiency of gene transplacement.

Base Sequence↗
PubMed1998

Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation.

Acute lung injury is a frequent and treatment-limiting consequence of therapy with hyperoxic gas mixtures. To determine if IL-11 is protective in oxygen toxicity, we compared the effects of 100% O2 on transgenic mice that overexpress IL-11 in the lung and transgene (-) controls. IL-11 markedly enhanced survival in 100% O2 with 100% of transgene (-) animals dying within 72-96 h and > 90% of transgene (+) animals surviving for more than 10 d. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Significant differences in copper zinc superoxide dismutase and catalase activities were not noted and the levels of total, reduced and oxidized glutathione were similar in transgene (+) and (-) animals. Glutathione reductase, glutathione peroxidase, and manganese superoxide dismutase activities were slightly higher in transgene (+) as versus (-) mice after 100% O2 exposure, and IL-11 diminished hyperoxia-induced expression of IL-1 and TNF. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals and IL-11 markedly diminished this cell death response. These studies demonstrate that IL-11 markedly diminishes hyperoxic lung injury. They also demonstrate this protection is associated with small changes in lung antioxidants, diminished hyperoxia-induced IL-1 and TNF production, and markedly suppressed hyperoxia-induced DNA fragmentation.

Animals↗
PubMed1997

[A new PCR-primer for specific amplification of human DNA fragments selected on the basis of computer analysis of the nucleotide sequences of MER1 dispersed repeats in man].

The possibility of using oligonucleotides from MER1 family repeats as PCR primers for the amplification of human genome DNA fragments was studied. The recommended oligonucleotide primers were chosen by computer analysis of the data base of the EMBL (release 37.0) nucleotide sequences. Use of one of the oligonucleotides was shown to allow specific human DNA amplification to be carried out.

Base Sequence↗
PubMedDate not supplied

Polymerase chain reaction method to detect canis materials by amplification of species-specific DNA fragment.

Rapid identification of mammal materials in feeding stuffs and food is essential for effective control of a potential source of pathogens, such as those that cause bovine spongiform encephalopathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a canis-specific mitochondrial DNA sequence in foodstuffs and food. The amplified canis-specific PCR product was a 213 base pair band from the D-loop DNA fragment of mitochondria, a high copy gene which should improve the possibility of amplifying template molecules of adequate size among the degraded DNA fragments brought about by heat denaturation. The specificity of this method was confirmed by 8 canis blood DNA samples (from different breeds of dog) and 9 noncanis animal blood DNA samples (bovine, sheep, porcine, chicken, fish, donkey, rabbit, deer, horse). This method was able to detect the presence of canis material in foodstuffs and in food mixtures even when the concentration of canis-derived meat was reduced to 0.05%. Furthermore, it did not appear to be affected by prolonged heat treatment. This method was developed for detection of canis materials in feeding stuffs, and occasionally for medical jurisprudence detection of canis-derived materials.

Animals↗
PubMed2007

Initial yields of DNA double-strand breaks and DNA Fragmentation patterns depend on linear energy transfer in tobacco BY-2 protoplasts irradiated with helium, carbon and neon ions.

The ability of ion beams to kill or mutate plant cells is known to depend on the linear energy transfer (LET) of the ions, although the mechanism of damage is poorly understood. In this study, DNA double-strand breaks (DSBs) were quantified by a DNA fragment-size analysis in tobacco protoplasts irradiated with high-LET ions. Tobacco BY-2 protoplasts, as a model of single plant cells, were irradiated with helium, carbon and neon ions having different LETs and with gamma rays. After irradiation, DNA fragments were separated into sizes between 1600 and 6.6 kbp by pulsed-field gel electrophoresis. Information on DNA fragmentation was obtained by staining the gels with SYBR Green I. Initial DSB yields were found to depend on LET, and the highest relative biological effectiveness (about 1.6) was obtained at 124 and 241 keV/microm carbon ions. High-LET carbon and neon ions induced short DNA fragments more efficiently than gamma rays. These results partially explain the large biological effects caused by high-LET ions in plants.

Animals↗
PubMed1995

Separation of DNA fragments induced by ionizing irradiation using a graded-field gel electrophoresis.

A method is described that allows a separation of X-ray induced DNA fragments by graded-field gel electrophoresis. Synchronized G1 and asynchronous CHO cells were embedded in agarose and irradiated with X-ray doses ranging from 1 to 100 Gy. Following proteolysis by sarcosine and proteinase K, electrophoresis was run for 49 h using graded electric fields with stepwise increasing field strength (0.6, 1.5, 3 and 9 V/cm). Since the molecular size of DNA able to migrate decreased with increasing voltage, each voltage step led to the generation of a distinct DNA band with the largest fragments in band 1, fragments of intermediate size in bands 2 and 3 and the smallest fragments in band 4. Using yeast chromosomal DNA as a reference, the molecular weight of eluted fragments was calculated to range from 1 to 10 Mbp. It could be shown that the fragment size was not the only criterion that discriminates migrating from non-migrating DNA. DNA fragments were found to be retained in the well by an unknown factor presumably associated with DNA conformation. This retention factor increased with increasing fragment size. Graded-field gel electrophoresis also allowed the determination of the absolute number of double-stranded breaks (dsb) induced, which amounted to 11.5 x 10(-12) dsbs Gy-1 Da-1 corresponding to 37 dsbs/G1 cell.

Animals↗
PubMed2005

Restriction enzyme cleavage of fluorescently labeled DNA fragments--analysis of the method and its usage in examination of digestion completeness.

Restriction enzymes have proven to be among the most valuable tools in molecular biology. In this work, we demonstrate that the cleavage of fluorescently labeled, PCR-amplified DNA can be used as a simple and highly sensitive technique for detection of sequences present in a percentage as low as 0.6% in a DNA pool. Due to the fact that fluorescent labeling of DNA fragments enables such sensitive detection and quantification of restriction enzyme cleavage, the method was further exploited in monitoring of the enzymatic digestion completeness and in determination of factors that influence restriction enzyme effectiveness. We analyzed the activity of six restriction endonucleases; the percentage of uncleaved DNA fragments predominantly ranged between 2.0 and 2.5 and the highest value was 8.00%. We conclude that, since the enzymatic digestion completeness may not always be assured, each assay based on restriction enzyme cleavage that is intended to be used in investigations of heterogeneity in a DNA pool should be constructed so that the presence of cleaved sequences is the indication of pool nonuniformity. When the presence of uncleaved sequences indicates pool heterogeneity, the results could be misleading due to possible incompleteness of enzymatic cleavage.

Base Sequence↗
PubMed2004

Reduction of DNA fragmentation and hydroxyl radical production by hyaluronic acid and chondroitin-4-sulphate in iron plus ascorbate-induced oxidative stress in fibroblast cultures.

Glycosaminoglycans (GAGs), components of extracellular matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. Oxidative stress is one of the most frequent causes of tissue and cell injury and the consequent lipid peroxidation is the main manifestation of free radical damage. It has been found to play an important role in the evolution of cell death. Since several reports have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) are able to inhibit lipid peroxidation during oxidative stress, We investigated the antioxidant capacity of these GAGs in reducing oxidative damage in fibroblast cultures. Free radicals production was induced by the oxidizing system employing iron (Fe2+) plus ascorbate. We evaluated cell death, membrane lipid peroxidation, DNA damage, protein oxidation, hydroxyl radical (OH*) generation and endogenous antioxidant depletion in human skin fibroblast cultures. The exposition of fibroblasts to FeSO4 and ascorbate caused inhibition of cell growth and cell death, increased OH* production determined by the aromatic trap method; furthermore it caused DNA strand breaks and protein oxidation as shown by the DNA fragments analysis and protein carbonyl content, respectively. Moreover, it enhanced lipid peroxidation evaluated by the analysis of conjugated dienes (CD) and decreased antioxidant defenses assayed by means of measurement of superoxide dismutase (SOD) and catalase (CAT) activities. When fibroblasts were treated with two different doses of HYA or C4S a protective effect, following oxidative stress induction, was shown. In fact these GAGs were able to limit cell death, reduced DNA fragmentation and protein oxidation, decreased OH* generation, inhibited lipid peroxidation and improved antioxidant defenses. Our results confirm the antioxidant activity of HYA and C4S and this could represent a useful step in the understanding of the exact role played by GAGs in living organisms.

Alkadienes↗
PubMed1989

Cooperative DNA binding by Xenopus transcription factor IIIA. Use of a 66-base pair DNA fragment containing the intragenic control region of the 5 S RNA gene to study specific and nonspecific interactions.

The interaction of Xenopus transcription factor IIIA (TFIIIA) with a synthetic 66-base pair DNA fragment containing the intragenic control region (ICR) of the 5 S RNA gene was investigated by mobility shift gel electrophoresis and DNase I protection experiments. Specific EDTA-sensitive protein-DNA complexes were observed only in the presence of high concentrations of either the ICR-containing fragment or an excess of unlabeled nonspecific DNA. This predominant complex was determined to be composed of equal molar ratios of TFIIIA and the 66-base pair ICR-containing fragment, although complexes containing two TFIIIA molecules/ICR and three TFIIIA molecules/ICR were also found. In the absence of excess unlabeled specific or nonspecific DNA, TFIIIA was observed to interact with the labeled 66-mer as an aggregate that remained at the top of the gel matrix. Although the binding affinities of TFIIIA for the 66-base pair specific DNA fragment and the 74-base pair nonspecific DNA fragment were comparable as observed by direct measurement with mobility shift gel electrophoresis, a greater than 500-fold difference was observed by competition experiments at high DNA concentrations. Furthermore, a similar difference was also detected when proteolytically cleaved TFIIIA was used in the binding reaction, which substantially reduced the protein-protein interactions. DNase I digestion patterns of the noncoding strand of the 66-mer in the absence or presence of TFIIIA demonstrated that this region of DNA is structurally equivalent to the ICR contained within the 5 S RNA gene. In addition, the mode of binding observed in the mobility shift gels is identical to that observed by direct footprint analysis. Protein-protein interactions appear, therefore, to be an intrinsic and necessary activity of TFIIIA.

Animals↗
PubMed1995

Identification of a member of the TIS11 early response gene family at the insertion point of a DNA fragment containing a gene for the T-cell receptor beta chain in an acute T-cell leukemia.

In a previous paper (Proc. Natl. Acad. Sci. USA 84, 4264, 1987) we reported that a DNA fragment containing a gene for the T-cell receptor beta chain, which had been excised from chromosome 7q35 during D beta-J beta joining, was inserted into chromosome 6p21.3 in a patient with acute T-cell leukemia. We have since screened for genes in the vicinity of the insertion point and have identified a gene that is equivalent to the murine TIS11d gene, a member of TIS11 early response gene family, that contains unique Cysteine-Histidine motifs. The human TIS11d gene consists of two exons and encodes a polypeptide of 492 amino acids. The insertion of the DNA fragment observed in this patient is located at the carboxy-terminal portion of the TIS11d protein.

Amino Acid Sequence↗
PubMed1991

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes.

A collection of 2,000 lambda phage-carrying human single-copy inserts (greater than 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two human-hamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs; 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100-150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.

Animals↗
PubMed1995

Overexpression of Bcl-XL by cytotoxic drug exposure confers resistance to ionizing radiation-induced internucleosomal DNA fragmentation.

Acquired resistance to diverse chemotherapeutic agents has been associated with overexpression of the P-glycoprotein. We have selected human U-937 cells for clones resistant to the cytotoxic agents doxorubicin (U-A20) and vincristine (U-V20). The results demonstrate that P-glycoprotein-positive U-A20 and U-V20 cells exhibit resistance to inducers of internucleosomal DNA fragmentation. Although parental U-937 cells responded to ionizing radiation with the DNA laddering characteristic of physiological cell death, the drug-resistant lines were insensitive to this effect. The U-A20 and U-V20 clones were also resistant to endonucleolytic DNA cleavage associated with exposure to tumor necrosis factor or ceramide. Previous work has demonstrated that physiological cell death is inhibited by overexpression of the Bcl-2 protein. However, analysis of Bcl-2 revealed similar levels in the parental and drug-resistant cells. In contrast, we show that U-A20 and U-V20 cells overexpress the Bcl-2-related protein, Bcl-xL. Moreover, studies with a U-937 cell line transfected with a Bcl-XL expression vector confirm resistance to ionizing radiation-induced DNA fragmentation and cell killing. These findings suggest that, unlike Bcl-2, Bcl-XL may be constitutively overexpressed as a result of selection for cytotoxic drug resistance and that Bcl-XL participates in an acquired form of multimodality resistance to chemotherapeutic agents and radiation.

Base Sequence↗
PubMed1995

Probing the structure of long single-stranded DNA fragments with neocarzinostatin chromophore. Extension of the base-catalyzed bulge-specific reaction.

The base-catalyzed (bc) thiol-independent cleavage reaction of neocarzinostatin chromophore (NCS chrom) has been characterized with long single-stranded (ss) DNA in order to use this reaction as a selective probe for the tertiary structure of naturally occurring ss nucleic acids. The ss circular phi chi 174 phage and M13mp18 phage DNAs (approximately 5000 and 7500 bases, respectively) were shown to be bc NCS chrom reaction substrates, exhibiting the expected pH dependence. The ss DNA fragments (150-450 bases) were cleaved at six major sites; the lesions occurred at T-rich non-double-stranded sequences, as predicted from comparison with the minimal energy secondary structures. These sites exhibited the expected pH and drug: DNA ratio dependence shown to be required for this reaction. Optimization of the shortest sequence, which gave the highest cleavage yield, identified the minimal sequence requirements for the site (19-mer of the sequence 3'TACTGAGTCTCCTTTTGTA5', attacked residue in bold). Folding pattern analysis predicted that the oligonucleotide contained a two-base bulge at the cleavage site; this result was consistent with the observation that removing features which destabilize the bulged structure increased the cleavage yield. Furthermore, the derived 19-mer was shown to generate maximal amounts of the final drug product of the bc DNA cleavage reaction. Reaction of an RNA 339-mer containing the same sequence as one of the long ss DNA fragments showed it not to be a substrate for the bc reaction, while similar results were obtained for the RNA analog of shorter oligodeoxyribonucleotides identified in this and earlier studies. Through a combination of thermodynamic and kinetic assays, the observed difference in reactivity was shown to be the result of the low binding of the cleaving species to RNA.

Bacteriophage phi X 174↗
PubMed2003

Chromosome DNA fragmentation and excretion caused by defective prophage gene expression in the early-exponential-phase culture of Bacillus subtilis.

Bacillus subtilis 168 and its major autolysin mutant, AN8, were shown to excrete two size classes of DNA when cultured in Luria-Bertani medium. Pulsed-field gel electrophoresis of DNA harvested from the cell surface demonstrated the presence of 13-kb-long and circa 50-kb-long strands. Restriction digestion of both sizes of DNA resulted in a smearing pattern, as observed by agarose gel electrophoresis. Shotgun sequencing of DNase I partial digests of 50-kb DNA fragments revealed that the strands originate from various sites on the chromosome. SDS-PAGE analysis of cell surface fractions and culture supernatants demonstrated the presence of several proteins that were thought to be associated with the DNA. Of these, three major proteins were identified, i.e., XkdG, XkdK, and XkdM, by tandem mass spectrometry, all of which were proteins of a defective prophage PBSX residing in the Bacillus subtilis chromosome. Disruption of these PBSX genes resulted in a reduction of 13-kb fragment generation and excretion and also a great reduction of 50-kb fragment excretion. Electron microscopy showed that a few mature phages and numerous membrane vesicle-like particles existed in the cell surface fractions of strain 168. The present findings suggest that the spontaneous generation and excretion of chromosome DNA fragments in Bacillus subtilis are both closely related to the expression of defective prophage genes.

Bacillus Phages↗