Kbp Fragments Research Articles

Prophage-like gene transfer agents promote Caulobacter crescentus survival and DNA repair during stationary phase.

Gene transfer agents (GTAs) are prophage-like entities found in many bacterial genomes that cannot propagate themselves and instead package approximately 5 to 15 kbp fragments of the host genome that can then be transferred to related recipient cells. Although suggested to facilitate horizontal gene transfer (HGT) in the wild, no clear physiological role for GTAs has been elucidated. Here, we demonstrate that the α-proteobacterium Caulobacter crescentus produces bona fide GTAs. The production of Caulobacter GTAs is tightly regulated by a newly identified transcription factor, RogA, that represses gafYZ, the direct activators of GTA synthesis. Cells lacking rogA or expressing gafYZ produce GTAs harboring approximately 8.3 kbp fragment of the genome that can, after cell lysis, be transferred into recipient cells. Notably, we find that GTAs promote the survival of Caulobacter in stationary phase and following DNA damage by providing recipient cells a template for homologous recombination-based repair. This function may be broadly conserved in other GTA-producing organisms and explain the prevalence of this unusual HGT mechanism.

High-Throughput Separation of Long DNA in Deterministic Lateral Displacement Arrays.

Length-based separation of DNA remains as relevant today as when gel electrophoresis was introduced almost 100 years ago. While new, long-read genomics technologies have revolutionised accessibility to powerful genomic data, the preparation of samples has not proceeded at the same pace, with sample preparation often constituting a considerable bottleneck, both in time and difficulty. Microfluidics holds great potential for automated, sample-to-answer analysis via the integration of preparatory and analytical steps, but for this to be fully realised, more versatile, powerful and integrable unit operations, such as separation, are essential. We demonstrate the displacement and separation of DNA with a throughput that is one to five orders of magnitude greater than other microfluidic techniques. Using a device with a small footprint (23 mm × 0.5 mm), and with feature sizes in the micrometre range, it is considerably easier to fabricate than parallelized nano-array-based approaches. We show the separation of 48.5 kbp and 166 kbp DNA strands achieving a significantly improved throughput of 760 ng/h, compared to previous work and the separation of low concentrations of 48.5 kbp DNA molecules from a massive background of sub 10 kbp fragments. We show that the extension of DNA molecules at high flow velocities, generally believed to make the length-based separation of long DNA difficult, does not place the ultimate limitation on our method. Instead, we explore the effects of polymer rotations and intermolecular interactions at extremely high DNA concentrations and postulate that these may have both negative and positive influences on the separation depending on the detailed experimental conditions.

Modeling radiation-induced cell death: role of different levels of DNA damage clustering.

Some open questions on the mechanisms underlying radiation-induced cell death were addressed by a biophysical model, focusing on DNA damage clustering and its consequences. DNA "cluster lesions" (CLs) were assumed to produce independent chromosome fragments that, if created within a micrometer-scale threshold distance (d), can lead to chromosome aberrations following mis-rejoining; in turn, certain aberrations (dicentrics, rings and large deletions) were assumed to lead to clonogenic cell death. The CL yield and d were the only adjustable parameters. The model, implemented as a Monte Carlo code called BIophysical ANalysis of Cell death and chromosome Aberrations(BIANCA), provided simulated survival curves that were directly compared with experimental data on human and hamster cells exposed to photons, protons, α-particles and heavier ions including carbon and iron. d=5μm, independent of radiation quality, and CL yields in the range ~2-20CLsGy(-1)cell(-1), depending on particle type and energy, led to good agreement between simulations and data. This supports the hypothesis of a pivotal role of DNA cluster damage at sub-micrometric scale, modulated by chromosome fragment mis-rejoining at micrometric scale. To investigate the features of such critical damage, the CL yields were compared with experimental or theoretical yields of DNA fragments of different sizes, focusing on the base-pair scale (related to the so-called local clustering), the kbp scale ("regional clustering") and the Mbp scale, corresponding to chromatin loops. Interestingly, the CL yields showed better agreement with kbp fragments rather than bp fragments or Mbp fragments; this suggests that also regional clustering, in addition to other clustering levels, may play an important role, possibly due to its relationship with nucleosome organization in the chromatin fiber.

High-Resolution Hydrodynamic Chromatographic Separation of Large DNA Using Narrow, Bare Open Capillaries: A Rapid and Economical Alternative Technology to Pulsed-Field Gel Electrophoresis?

A high-resolution, rapid, and economical hydrodynamic chromatographic (HDC) method for large DNA separations in free solution was developed using narrow (5 μm diameter), bare open capillaries. Size-based separation was achieved in a chromatographic format with larger DNA molecules being eluting faster than smaller ones. Lambda DNA Mono Cut Mix was baseline-separated with the percentage resolutions generally less than 9.0% for all DNA fragments (1.5 to 48.5 kbp) tested in this work. High efficiencies were achieved for large DNA from this chromatographic technique, and the number of theoretical plates reached 3.6 × 10(5) plates for the longest (48.5 kbp) and 3.7 × 10(5) plates for the shortest (1.5 kbp) fragments. HDC parameters and performances were also discussed. The method was further applied for fractionating large DNA fragments from real-world samples (SacII digested Arabidopsis plant bacterial artificial chromosome (BAC) DNA and PmeI digested Rice BAC DNA) to demonstrate its feasibility for BAC DNA finger printing. Rapid separation of PmeI digested Rice BAC DNA covering from 0.44 to 119.041 kbp was achieved in less than 26 min. All DNA fragments of these samples were baseline separated in narrow bare open capillaries, while the smallest fragment (0.44 kbp) was missing in pulsed-field gel electrophoresis (PFGE) separation mode. It is demonstrated that narrow bare open capillary chromatography can realize a rapid separation for a wide size range of DNA mixtures that contain both small and large DNA fragments in a single run.

Development of a Multiplex PCR and PCR-RFLP Method for Serotyping of Avibacterium paragallinarum

Avibacterium (Haemophilus) paragallinarum (A. paragallinarum) is a causative agent of infectious coryza in chickens and is classified into three serovars by agglutination tests. In an effort to identify the serovars easily, PCR and PCR-RFLP were employed. As the target gene for PCR, the hypervariable region of HMTp210, which encodes the HA antigen, was used. PCR using primer sets around the hypervariable region amplified 0.8, 1.1 and 1.6 kbp fragments for serovars A, B and C, respectively. Alternatively, the 1.6 kbp fragments were amplified with another primer pair encompassing the hypervariable region and was subjected to digestion with Bgl II, which resulted in the detection of serovar-specific digestion patterns. These results indicate that PCR and PCR-RFLP using the hypervariable region of HMTp210 are alternative methods to identify the serovar of A. paragallinarum.

DNA Separation in Nanowall Array Chips

A nanowall array structure was fabricated on a quartz chip as a separation matrix of DNA fragments, and a 30 s separation was realized for a mixture of DNA fragments (48.5 and 1 kbp fragments) by applying the electric voltage. A longer DNA fragment migrates faster than a shorter one in a nanowall array chip, and it is completely different from the separation of DNA based on gel electrophoresis, nanopillar chips, and nanoparticle array chips. Although the result is similar to DNA separation by entropic trapping, it could not be fully explained by entropic trapping phenomena. Direct observation of single-DNA molecular dynamics inside a nanowall array structure indicates that both confined elongation and relaxation recoiling of a DNA molecule occur, and an elongated DNA molecule migrates faster than a recoiled DNA molecule. Numerical fitting of DNA molecular dynamics reveals that the balance between times for the transverse of a DNA molecule in the nanowall array chip and the relaxation-recoiling of a DNA molecule governs the separation of DNA.

Effects of Applied Voltage and Fence Configurations on DNA Separation Based on Electrophoresis Using Nanostructured Fence Matrix

We have demonstrated effective biomolecule separation methods based on size exclusion chromatography (SEC) type separation using nanostructured channels constituted with a flat fence matrix and a convex fence matrix. The performances of biomolecule separations were compared between different applied voltages and between the flat and convex fence matrices. The flat fence matrix has wide streets along the electrophoresis direction and narrow lanes perpendicular to the streets. In the SEC type separation using the flat fence matrix, the narrow lanes were designed for the passages for smaller molecules to detour. The specific feature of the convex fence matrix is that the lane width is changed at the halfway of the lane length. It was found from the result of the separation of λ-Hin d III digest (125-23 130 bp) that the separation resolution of the long DNA was highest for the lowest voltage of 20 V. In contrast, the result of the separation of φX 174-Hae III digest (72-1 353 bp) showed that the separation resolution of the short DNA was highest for the highest voltage of 100 V. In addition, it is interesting to note that the convex fence matrix enabled us to separate the peaks of 4, 6 and 9 kbp fragments from the peak of 23 kbp, while those peaks overlapped each other for the flat fence matrix.

A novel genetic marker to differentiate feline herpesvirus type 1 field isolates

Five recent field isolates of feline herpesvirus type 1 (FHV-1) were compared by digestion with a restriction endonuclease, SalI or MluI. The SalI digestion showed a potentially useful difference in one isolate 00-035 that had an approximately 3.0 kbp fragment instead of a 2.6 kbp fragment in the other strains. After cloning the 3.0 and 2.6 kbp fragments, the nucleotide sequences were analyzed. The result showed that the 3.0 kbp fragment of 00-035 included a complete open reading frame of the herpes simplex virus 1 (HSV-1) homologue of the UL17 gene and a 5'-part of UL16 gene and that only one nucleotide substitution was found in the 5'-region of UL17 gene where the SalI site of the 2.6 kbp fragment locates. Based on these nucleotide sequences, two PCR primers were designed to amplify the region around the SalI site in the UL17 gene and the PCR was carried out using 78 field isolates from various parts of Japan. The SalI digestion of the PCR products revealed an interesting profile in that the genotype without the SalI site in UL17 gene was dominant in Tottori and Yamagata prefectures (69% and 75%, respectively) but minor in the other regions of Japan (0-10%). These results suggest that the SalI digestion method described in the present study can be used as a genetic marker to differentiate some FHV-1 field isolates and this is the first report that showed different distributions of FHV-1 genotypes using the novel genetic marker.

Mechanisms of cell death induced by 5-fluoro-2'-deoxyuridine (FUdR)--necrosis or apoptosis after treated with FUdR.

5-Fluoro-2'-deoxyuridine (FUdR) inhibits thymidylate synthase (TS). The inhibition of TS causes an imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools which subsequently induced cell death. We investigated the molecular mechanisms of cell death after treated with FUdR in F28-7 strain (which induced necrosis) and F28-7-A strain (mutant of F28-7 strain which induced apoptosis). After treated with FUdR, we observed different size of DNA fragmentations. F28-7 strain induced DNA cleavaged into 100-200 kbp fragments and F28-7-A strain induced DNA cleavaged into oligonucleosomal sized fragments. In F28-7 strain, FUdR induced the increased mRNA level of c-jun, c-fos and c-myc genes, caspase-3 like protease activity and the changes of mitochondrial membrane potential which were greater and earlier than those of F28-7-A strain. On the other hand, F28-7-A strain induced release of cytochrome c from mitochondrial, but not F28-7 strain. Furthermore, caspase-5 inhibitor was strongly inhibited the cell death of F28-7 strain. We suggest that it is concerned with intensity of intracellular signals in the cell death of F28-7 strain and F28-7-A strain.

Simulation of DNA Damage after Proton and Low LET Irradiation

A module for proton track structure simulation in liquid water was implemented in the biophysical model PARTRAC. Simulated tracks of energy deposition events from the radiation under investigation were superimposed on a higher-order DNA target model describing the whole genome inside a human cell. The parameters controlling DNA damage from direct and indirect effects were adapted to agree with yields and pathway contributions derived from gamma ray irradiation experiments. Single and double strand break (DSB) induction was simulated for irradiations by protons, photons and electrons over a wide range of initial energies. The relative biological effectiveness for DSB induction after proton irradiation was found to rise from 1.2 at 5 keV.micron-1 to about 2.5 at 70 keV.micron-1. About half of this growth resulted from an increased production of DSB clusters associated with small (< 10 kbp) fragments.

Mutation detection using fluorescent enzyme mismatch cleavage with T4 endonuclease VII.

Mutation detection techniques are often limited by sensitivity, ease of use and short fragment lengths. Enzyme mismatch cleavage (EMC) is a technique capable of rapidly scanning 1 kbp fragments of DNA for mutations. It relies on the ability of a bacteriophage resolvase enzyme, T4 endonuclease VII, to cleave DNA at single base pair mismatches and small heteroduplex loops. Originally the process was performed using radioactively labeled DNA and the results analysed after denaturing polyacrylamide gel electrophoresis and autoradiography. However, access to systems capable of detecting fluorescent species migrating through a gel and the widespread availability of fluorescently tagged primers have greatly improved upon the original technique. A number of mutations were detected using fluorescent EMC and the results compared to performing the technique using radiolabeled DNA. Fluorescent EMC detected the presence, position and number of mutations in DNA fragments as large as 1 kbp. The fluorescent method was found to have advantages over the original method in its ease of use, increase in signal-to-noise ratio and the ability to multiplex samples by labeling DNA fragments with different fluorophores. This improvement on an already established method provides a sensitive, robust technique for mutation detection.

De novo methylation and co-suppression induced by a cytoplasmically replicating plant RNA virus.

The relationship between co-suppression and DNA methylation was explored in transgenic plants showing inducible co-suppression following infection with a cytoplasmically replicating RNA virus. Induction resulted in a loss of transgene mRNA and resistance to further infection, factors typical of post-transcriptional gene silencing. In infected plants, de novo methylation of the transgene appeared to precede the onset of resistance and only occurred in plants where the outcome was co-suppression. The methylation was limited to sequences homologous to the viral RNA and occurred at both symmetric and non-symmetric sites on the DNA. Although methylation is predicted to occur in mitotic cells, the virus was found not to access the meristem. A diffusible sequence-specific signal may account for the epigenetic changes in those tissues.

Association between DNA cleavage during apoptosis and regions of chromatin replication.

We have addressed the association between the site of DNA cleavage during apoptosis and DNA replication. DNA double strand breaks were introduced into chromatin containing pulse labeled nascent DNA by the induction of apoptosis or autocleavage of isolated nuclei. The location of these breaks in relation to nascent DNA were revealed by Bal31 exonuclease digestion at the cut sites. Our data show that Bal31 accessible cut sites are directly linked to regions enriched in nascent DNA. We suggest that these regions coincide with the termini of replication domains, possibly linked by strong DNA-matrix interactions with biophysically defined topological structures of 0.5-1.3 Mbp in size. The 50 kbp fragments that are commonly observed as products of apoptosis are also enriched in nascent DNA within internal regions but not at their termini. It is proposed that these fragments contain a subset of replicon DNA that is excised during apoptosis through recognition of their weak attachment to the nuclear matrix within the replication domain.

Association of Ceramide Accumulation with Photodynamic Treatment‐Induced Cell Death

Ceramide, a stress-induced second messenger, has been associated with apoptosis in several malignant and non-malignant cell lines. We have shown that photodynamic treatment (PDT), using the phthalocyanine photosensitizer Pc 4 (HOSiPcOSi[CH3]2[CH2]3N[CH3]2), causes increased ceramide generation and subsequent induction of apoptosis in L5178Y-R (LY-R) mouse lymphoma cells. To test further if ceramide generation accompanies photocytotoxicity, we treated various cell lines with a PDT dose producing a 99-99.9% loss of clonogenicity. Like LY-R cells, human leukemia (U937) cells underwent rapid DNA fragmentation initiating within 1 h after PDT. Similarly, Chinese hamster ovary (CHO) cells showed rapid DNA laddering, beginning 1 h following the treatment. In contrast, mouse radiation-induced fibrosarcoma (RIF-1) cells showed no apoptosis within 24 h post-PDT, as judged by the absence of 50 kbp and oligonucleosome size DNA fragments, as well as no annexin V binding to cells with preserved membrane integrity. Using the same doses of PDT, we observed a time-dependent ceramide accumulation in all three cell lines. While a significant increase in ceramide levels was reached within 1 and 10 min in U937 and CHO cells, respectively, elevated ceramide production was measured only after 30 min in RIF-1 cells. In addition, exogenous N-acetyl-sphingosine was able to mimic PDT-induced apoptosis in U937 and CHO cells. We suggest that ceramide accumulation is associated with PDT-induced apoptosis and photocytotoxicity.

Ageratum, Croton and Malvastrum harbour geminiviruses : evidence through PCR amplification

Ageratum conyzoides, Croton bonpladianum and Malvastrum coromandelianum are common weeds found around agricultural fields. In several cases these were found to exhibit vein yellowing and yellow mosaic symptoms. Using degenerate primers specific for whitefly-transmitted geminiviruses (WTGs), and total DNA isolated from such infected plants (exhibiting the above symptoms) as a template, 1.2kbp fragments were amplified and were shown to have homology to DNA-A of Indian tomato leaf curl virus (ITLCV) by Southern hybridization. In control experiments the same primers failed to amplify any DNA fragments from the total DNA isolated from healthy plants (no symptoms as above). These results show that Ageratum, Croton and Malvastrum harbour geminivirus(es).

Heat-induced apoptosis and p53 in cultured mammalian cells

Heat-induced apoptosis was studied in M10 and MOLT-4 cells by determining nuclear morphological changes, decrease in cell size, DNA degradation into fragments of about 30 kbp, and the appearance of internucleosomal DNA fragments (DNA ladders). Morphological changes in the nucleus were detected within 30 min after heat-treatment at 44 C in M10 cells, but much later (5 h) in MOLT-4 cells. In M10 cells, 30 kbp-DNA fragments were observed even at the end of the heat-treatment and decreased 10 min later, while the DNA ladder increased at 10-30 min after heat treatment. DNA fragments of 30 kbp appeared in MOLT-4 cells at 1 h after the heat-treatment and apparently accumulated for up to 24 h. Heat-treatment increased p53 protein in MOLT-4 cells but not in M10 cells. Analysis of the DNA sequence of the p53 gene revealed that M10 cells have a heterozygous mutation in codon 173 of exon 5. These results suggest that apoptosis is induced by hyperthermia in a cell-line dependent manner, that the formation of 30 kbpDNA fragments is a very early event in apoptosis, that DNA fragmentation into a DNA ladder occurs via the 30 kbp fragments, and that apoptosis in heat-treated M10 cells may be independent of p53.

The induction of partial resistance to photodynamic therapy by the protooncogene BCL-2.

Photodynamic therapy (PDT) is an efficient inducer of apoptosis, an active form of cell death that can be inhibited by the BCL-2 oncoprotein. The ability of BCL-2 to modulate PDT-induced apoptosis and overall cell killing has been studied in a pair of Chinese hamster ovary cell lines that differ from one another by a transfected human BCL-2 gene in one of them (Bissonnette et al, Nature 359, 552-554, 1992). Cells were exposed to the phthalocyanine photosensitizer Pc 4 and various fluences of red light. Pc 4 uptake was identical in the two cell lines. The parental cells displayed a high incidence of apoptosis after PDT, whereas at each fluence there was a much lower incidence of apoptosis in the BCL-2-expressing cells. Apoptosis was monitored by (a) observation of 50 kbp and oligonucleosome-size DNA fragments by gel electrophoresis, (b) flow cytometry of cells labeled with fluorescently tagged dUTP by terminal deoxynucleotidyl transferase and (c) fluorescence microscopy of acridine orange-stained cells. The time course of apoptosis varied with the PDT dose, suggesting that only after moderately high doses (> 99% loss of clonogenicity) was there a relatively synchronous and rapid entry of many cells into apoptosis. At PDT doses reducing cell survival by 90 or 99%, significant increases in apoptotic cells were found in the population after 6-12 h. Clonogenic assays showed that BCL-2 protein inhibited not only apoptosis but overall cell killing as well, effecting a two-fold resistance at the 10% survival level. Thus, BCL-2-expressing cells may be relatively resistant to PDT.

Characterization ofPerina nudaNucleopolyhedrovirus (PenuNPV) Polyhedrin Gene

ThePerina nudanucleopolyhedrovirus (PenuNPV) polyhedrin gene was located inEcoRI-G (6.3 kilobase pairs; kbp) andPstI-G (4.3 kbp) fragments of its genomic DNA. A portion of 1333 nucleotides (nt) containing this gene was sequenced. An open reading frame of 735 nt encoded a 245-amino-acid-long polyhedrin. A conserved TAAG motif which is associated with transcriptional start sites was identified 51 nt upstream of the translation initiation codon of PenuNPV polyhedrin gene. A putative polyadenylation signal, AATAAA, was found 116 nt downstream of the termination codon (TAA). Comparison of the amino acid sequences of PenuNPV polyhedrin with those of other NPVs showed that PenuNPV polyhedrin was most closely related toOrgyia pseudotsugatamultiple NPV(OpMNPV) polyhedrin.

Short exposures to tunicamycin induce apoptosis in SV40-transformed but not in normal human fibroblasts.

When SV40-transformed fibroblasts (line 90VAVI) were exposed to tunicamycin, an inhibitor of N-linked glycosylation, an extensive cell death occured compared with untransformed fibroblasts. A considerable cell loss was obtained within 24 h after tunicamycin addition, and after 72 h there were hardly any virus-transformed cells alive. A 2-h pulse treatment with tunicamycin was found to be almost as effective as a continuous 48-h treatment in killing the cells. Even such a short exposure as 7 min resulted in a drastically decreased cell viability (54%). The morphology of the dying tunicamycin-treated 90VAVI cells suggested that they were undergoing apoptosis. This was also supported by the appearance of nuclear condensation, as assayed by propidium iodide uptake, which was detectable within 2 h after tunicamycin addition. Furthermore, analysis of DNA from tunicamycin-treated 90VAVI cells by field inversion gel electrophoresis revealed DNA degradation into 50 kbp fragments within 2 h, and conventional agarose gel electrophoresis showed 'DNA laddering', indicating internucleosomal DNA cleavage, detectable after 36 h. Together with the finding that tunicamycin within seconds caused an elevation of [Ca2+]i, a well documented early feature of apoptosis in many experimental systems, these results strongly suggest that tunicamycin-induced cell death in 90VAVI is due to apoptosis. The short tunicamycin exposure required to trigger cell death in 90VAVI indicates that the apoptotic process is irreversibly induced soon after its addition. It seems unlikely that the pool of one or several specific N-linked glycoproteins could be depleted during such a short period. Instead the overall accumulation of unglycosylated proteins in ER might contribute to the apoptotic response in 90VAVI. Tunicamycin also killed and induced DNA degradation in the breast cancer cell line MDA-231.

Multi-step DNA cleavage in rat liver nuclei is inhibited by thiol reactive agents

DNA fragmentation in isolated rat liver nuclei is a Mg 2+-dependent, multi-step process which is potentiated by Ca 2+ and cleaves the DNA into ≥700, 200–300 and 30–50 kilobase pair (kbp) fragments, prior to internucleosomal cleavage by Ca 2+/Mg 2+-dependent endonuclease(s). We now show that Cd 2+, Hg 2+, dichloroisocoumarin (DCI, a serine protease inhibitor) and N-ethylmaleimide (NEM) block both Mg 2+ and Ca 2+/Mg 2+-dependent processes. Inhibition of DNA cleavage produced an increase in the size of the DNA fragments, from mono-/oligonucleosomes to 30–50, 200–300, ≥700 kbp and finally to intact DNA. NEM and DCI inhibition was blocked by dithiothreitol, and it is proposed that a critical thiol(s) is involved in the DNA cleavage reactions which are a feature of the apoptotic process.