DNA Products in In Vitro DNA Synthesis Using Bacteriophage ϕX174 Single-stranded DNA

We described product analysis of DNA synthesized in chloroplast lysate from liverwort Marchantia polymorpha L. cell suspension cultures. Characteristics of in vitro DNA synthesis by chloroplast lysate using bacteriophage ?X174 single-stranded DNA were very similar to those in the case of double-stranded calf thymus DNA reported previously. Autoradiographic analysis clearly showed the incorporation of radioactive [α-³²P]-dCTP into DNA molecules associated with bacteriophage ?X174 single-stranded template DNA, indicating conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III, double-stranded linear molecule). Experiments on the fate of [³²P]-labeled single-stranded DNA also showed a clear conversion of the single-stranded DNA to double-stranded DNA. Furthermore, patterns of sucrose density gradient centrifugations (neutral and alkaline) showed the production of two major components in in vitro DNA synthesis by chloroplast lysate. This also indicated conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III form). Our results suggest that the mechanism of chloroplast DNA replication could be the mode of strand-displacement DNA synthesis as seen in animal mitochondrial DNA synthesis.     

Cloning,expression, and PCR application of DNA polymerase from the hyperthermophilic archaeon, <Emphasis Type="Italic">Thermococcus celer</Emphasis>

The family B DNA polymerase gene was amplified from Thermococcus celer genomic DNA by using the degenerate primers and DNA walking PCR. The Tce DNA polymerase gene was cloned and sequenced. The gene contains an ORF of 2,325 bp encoding 774 amino acid residues with a calculated molecular weight of 89,788.9 kDa. The Tce DNA polymerase was purified by heat treatment and heparin column chromatography. The optimal conditions for PCR were determined. Long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tce DNA polymerases (Tce plus DNA polymerase). Tce plus DNA polymerase surpassed the PCR performance of Tce, Taq and Pfu DNA polymerases in terms of yield and efficiency.     

Intrinsically bent DNA sites in the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> third chromosome amplified domain

Bent DNA sites promote the curvature of DNA in both eukaryotic and prokaryotic chromosomes. Here, we investigate the localization and structure of intrinsically bent DNA sites in the extensively characterized Drosophila melanogaster third chromosome DAFC-66D segment (Drosophila amplicon in the follicle cells). This region contains the amplification control element ACE3, which is a replication enhancer that acts in cis to activate the major replication origin ori-β. Through both electrophoretic and in silico analysis, we have identified three major bent DNA sites in DAFC-66D. The bent DNA site (b1) is localized in the ACE3 element, whereas the other two bent DNA sites (b2 and b3) are localized in the ori-β region. Four additional bent DNA sites were identified in the intron of the S18 gene and near the TATA box of the S15, S19, and S16 genes. The identification of DNA bent sites in genomic regions previously characterized as functionally relevant for DNA amplification further supports a function for DNA bent sites in DNA replication in eukaryotes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of DNA topoisomerases involved in immediate and transient DNA relaxation induced by heat shock inEscherichia coli

The linking number of plasmid DNA in exponentially growingEscherichia coli increases immediately and transiently after heat shock. The purpose of this study was to search for DNA topoisomerases that catalyze this relaxation of DNA. Neither introduction of atopA deletion mutation nor treatment of cells with DNA gyrase inhibitors affected the DNA relaxation induced by heat shock. Thus, DNA topoisomerase I and DNA gyrase are apparently not involved in the process. However, the reaction was inhibited by nalidixic acid or by oxolinic acid in thetopA mutant and the reaction was resistant to nalidixic acid in atopA mutant carrying, in addition, thenalA26 mutation. These results are interpreted as indicating that both DNA topoisomerase I and DNA gyrase are involved in the DNA relaxation induced by heat shock.     

Virus DNA packaging: the strategy used by phage λ

Phage λ, like a number of other large DNA bacterio-phages and the herpesviruses, produces concatemeric DNA during DNA replication. The concatemeric DNA is processed to produce unit-length, virion DNA by cutting at specific sites along the concatemer. DNA cutting is coordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the λ DNA packaging process is the phage-encoded enzyme terminase, which is involved in (i) recognition of the concatemeric λ DNA; (ii) initiation of packaging, which includes the introduction of staggered nicks at cosN to generate the cohesive ends of virion DNA and the binding of the prohead; (iii) DNA packaging, possibly including the ATP-driven DNA translocation; and (iv) following translocation, the cutting of the terminal cosN lo complete DNA packaging. To one side of cosN is the site cosB, which plays a role in the initiation of packaging; along with ATP, cosB stimulates the efficiency and adds fidelity to the endo-nuclease activity of terminase in cutting cosN. cosB is essential for the formation of a post-cleavage complex with terminase, complex I, that binds the prohead, forming a ternary assembly, complex II. Terminase interacts with cosN through its large subunit, gpA, and the small terminase subunit, gpNul, interacts with cosB. Packaging follows complex II formation. cosN is flanked on the other side by the site cosQ, which is needed for termination, but not initiation, of DNA packaging. cosQ is required for cutting of the second cosN, i.e. the cosN at which termination occurs. DNA packaging in λ has aspects that differ from other λ DNA transactions. Unlike the site-specific recombination system of λ, for DNA packaging the initial site-specific protein assemblage gives way to a mobile, translocating complete, and unlike the DNA replication system of λ, the same protein machinery is used for both initiation and translocation during λ DNA packaging.     

Restriction Fragment Length Polymorphism Analysis of the Mitochondrial DNA of Fusarium oxysporum f. sp. ciceris

Seven isolates of Fusarium oxysporum f. sp. ciceris, representing pathogenic races 1 , 2, 3, and 4 from India and 0, 5, and 6 from Spain, were assayed for restriction fragment length polymorphisms (RFLPs) in the mitochondrial DNA,(mt DNA). The mt DNA fraction of total fungal DNA was purified and digested with the restriction endonucleases Bam HI, Bgl II, Eco RI. Kpn I, Sac I, Sal I, Sma I, and Xho I. The mt DNA is a circular molecule of 40.5 kb. No RFLP in the mt DNA was detected among the seven races of F. o. ciceris. The identical restriction patterns of mt DNA indicates an extensive conservation in the gene composition of mt DNA without sequence variation, and suggests that mt DNA of F. o. ciceris may not be responsible for pathogenic diversity. The restriction map of mt DNA from the race 6 isolate Fo 8272 was constructed by digestion of the mt DNA with five restriction enzymes: Eco RI, Kpn I, Sac I, Sal I, and Xho I, either singly or in selected pairs.     

Single copy DNA homology in sea stars

Summary The sequence homology in the single copy DNA of sea stars has been measured. Labeled single copy DNA fromPisaster ochraceus was reannealed with excess genomic DNA fromP. brevispinus, Evasterias troschelii, Pycnopodia helianthoides, Solaster stimpsoni, andDermasterias imbricata. Reassociation reactions were performed under two criteria of salt and temperature. The extent of reassociation and thermal denaturation characteristics of hybrid single copy DNA molecules follow classical taxonomic lines.P. brevispinus DNA contains essentially all of the sequences present inP. ochraceus single copy tracer whileEvasterias andPycnopodia DNAs contain 52% and 46% of such sequences respectively. Reciprocal reassociation reactions with labeledEvasterias single copy DNA confirm the amount and fidelity of the sequence homology. There is a small definite reaction of uncertain homology betweenP. ochraceus single copy DNA andSolaster orDermasterias DNA. SimilarlySolaster DNA contains sequences homologous to approximately 18% ofDermasterias unique DNA. The thermal denaturation temperatures of heteroduplexes indicate that the generaPisaster andEvasterias diverged shortly after the divergence of the subfamilies Pycnopodiinae and Asteriinae. The twoPisaster species diverged more recently, probably in the most recent quarter of the interval since the separation of the generaPisaster andEvasterias.     

Bent DNA is a structural feature of scaffold-attached regions in Drosophila melanogaster interphase nuclei

In this study the SAR DNA (scaffold attached region DNA) of some Drosophila genes was analyzed. Bent DNA regions were found to be present in all SAR DNA fragments analyzed here. Bent non-SAR DNA exhibits SAR-like properties when it is exogenously added to lithium 3,5-di-iodosalicylate-extracted Drosophila nuclear scaffolds. Thus the presence of bent regions within SAR DNA fragments might be a prerequisite for the SAR-like behavior of a DNA fragment.     

PriA participates in nascent DNA synthesis in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The question of whether discontinuous DNA replication operates only for the lagging strand or for both strands in E. coli remains unresolved. In this study, the participation of priA, B, C and rep genes in discontinuous DNA replication was examined by analyzing the size distribution of nascent DNA synthesized in wild-type, lig-7 and polA4113 genetic backgrounds. Inactivation of priA, but not priB, priC or rep, resulted in a significant increase of high molecular weight (HMW) DNA in the short pulse-labeled DNA in the wild-type lig ⁺ polA ⁺ strains. Inactivation of priA also produced a significant increase of HMW DNA in the nascent DNA synthesized in lig-7 and polA4113 strains. These results indicate that PriA is involved in the discontinuous synthesis of nascent DNA.     

Analysis of a repetitive DNA family of two closely related chironomids,Chironomus thummi thummi andCh. thummi piger (Diptera) by density-gradient centrifugation,melting analysis and restriction analysis

The DNAs of the two subspecies ofChironomus thummi, Ch. th. thummi andCh. th. piger, were investigated by CsCl density-gradient centrifugation, melting analysis and restriction analysis including Southern hybridization with AT-rich, highly repetitive DNA sequences. The melting analysis of density-fractionatedCh. th. thummi andCh. th. piger DNA has shown that thethummi DNA contains an early melting DNA fraction, which is enriched in the light fractions of the density gradient. The DNA fraction is also present inpiger DNA though in lower concentration. Restriction and Southern analysis of density fractionatedthummi andpiger DNA has revealed that there are two tandemly-repetitive DNA-sequence families that hybridize with this AT-rich, early melting DNA fraction. One sequence is characterized by anHae-III site and a basic repeat length of 130 ± 15 bp and the other by aCla-1 restriction site and a basic repeat length of 120 ± 4 bp. These sequences are present in much higher concentrations in the genome ofCh. th. thummi when compared toCh. th. piger, and are hence correlated to the higher DNA content of theCh. th. thummi genome.     

Satellite DNA specific to knob heterochromatin inCucumis metuliferus (Cucurbitaceae)

Buoyant density gradient analysis of nuclear DNA of fourCucumis species showed asymmetric profiles indicating the presence of satellite DNA sequences in the nuclear genome. A highly repeated satellite DNA sequence was isolated from the nuclear genome ofC. metuliferus under neutral CsCl gradients. The satellite DNA constitutes about 4.96% of total nuclear DNA and has 48.06% guanine plus cytosine content. The kinetic complexity of satellite DNA is 150 times smaller than T₄ phage DNA and the base sequence divergence is low.³H-labeled cRNA transcribed from satellite DNA hybridized clearly to six heterochromatic knobs of pachytene chromosomes. The knob heterochromatin can be distinguished by Giemsa C-banding of pachytene chromosomes. Restriction enzyme analysis and Southern blot hybridization indicated that the satellite DNA has a tandem arrangement and predominantly formed two bands of size 210 and 151 base pairs. Absence of knob satellite DNA ofC. metuliferus in the nuclear genomes ofC. melo, C. anguria andC. sativus showed thatC. metuliferus remains isolated within the genusCucumis.     

Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Background  

Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of γ-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair.     

Identification of Yersinia enterocolitica using a random genomic DNA microarray chip

Aims: To fabricate a DNA chip containing random fragments of genomic DNA of Yersinia enterocolitica and to verify its diagnostic ability. Methods and Results: A DNA microarray chip was fabricated using randomly fragmented DNA of Y. enterocolitica. Chips were hybridized with genomic DNA extracted from other Y. enterocolitica strains, other Yersinia spp. and bacteria in different genera. Genomic DNA extracted from Y. enterocolitica showed a significantly higher hybridization rate compared with DNA of other Yersinia spp. or bacterial genera, thereby distinguishing it from other bacteria. Conclusions: A DNA chip containing randomly fragmented genomic DNA from Y. enterocolitica can detect Y. enterocolitica and clearly distinguish it from other Yersinia spp. and bacteria in different genera. Significance and Impact of the Study: A microarray chip containing randomly fragmented genomic DNA of Y. enterocolitica was fabricated without sequence information, and its diagnostic ability to identify Y. enterocolitica was verified.     

Conditions and mutations affecting the maintenance and replication of plasmid DNA in Escherichia coli 15

Summary An Escherichia coli 15 strain has been constructed which contains, in addition to the plasmids inherent to E. coli 15 (P 1-like DNA and minicircular DNA), the colicinogenic factor E₁ (Col E₁). Whereas the P 1-like DNA of E. coli 15 is unaffected by the uptake of the colicin plasmid, the number of copies of minicircular DNA of E. coli 15 decreases and an equivalent amount of Col E₁ DNA becomes established in the cell. The ratio between these two small plasmids is dependent on the growth temperature. The mode of replication of minicircular DNA and Col E₁ DNA is very similar, but is different in various respects from that of the P 1-like plasmid: 1. Both small plasmids continue to replicate in the presence of chloramphenicol, whereas the replication of P 1-like DNA stops like the chromosomal DNA. 2. Rifampicin inhibits the synthesis of both small plasmids rather rapidly. The replication of P 1-like DNA continues during the remaining replication cycle of the chromosome in the presence of rifampicin. 3. The replication of Col E₁ DNA and of the minicircular DNA still proceeds at elevated temperatures (45–50°C), whereas little or no incorporation of ³H-thymidine into P 1-like DNA is observed at these temperatures. 4. Mutants have been obtained, which show altered properties in the maintenance and replication of the plasmids without being affected in the replication of the chromosomal DNA. In all these mutants the replication and (or) maintenance of the minicircular DNA of E. coli 15 and Col E₁ DNA is affected in the same way, but not that of the P 1-like plasmid.     

DNA lesions that block DNA replication are responsible for the dnaA induction caused by DNA damage

Summary The initiation protein DnaA of Escherichia coli regulates its own expression autogenously by binding to a 9 by consensus sequence, the dnaA box, between the promoters dnaAP1 and dnaAP2. In this study, we analysed dnaA regulation in relation to DNA damage and found dnaA expression to be inducible by DNA lesions that inhibit DNA replication. On the other hand, coding DNA lesions were not able to induce dnaA expression. These results suggest that an additional regulatory mechanism is involved in dnaA gene expression and that DnaA protein may play a role in cellular responses to DNA damage. Furthermore, they strongly suggest that in response to DNA replication inhibition by DNA damage, and enhanced (re)initiation capacity is induced by oriC.     

DNA reassociation kinetics and hybridization in different angiosperms

Reassociation kinetics ofDaucus carota andPetroselinum crispum (Apiaceae), andDatura innoxia (Solanaceae) are presented. Hybridization of³H-labelled DNA of two carrot cultivars indicate strong qualitative homologies of DNA sequences; nevertheless, certain quantitative differences in some Cotregions seem to exist. However, homologous sequences ofDaucus DNA with DNA ofDatura, and, suprisingly, even with DNA ofPetroselinum are very restricted: between 8% in the repeated regions and ca. 7–9% in the unique regions.     

DNA–DNA sequence homology recognition: physical mechanisms and open questions

First, we summarize recent experimental facts on homologous DNA pairing in vitro and discuss possible mechanisms of DNA–DNA sequence recognition. Then, we overview the mechanism of DNA–DNA recognition based on complementarity of DNA charge patterns. The theory predicts the recognition energy up to 10 k_BT for close parallel homologous DNA fragments of gene‐relevant lengths. We argue why this estimate cannot be directly applied to pairing of homologous DNA loci in experiments on yeast chromosomes. Namely, DNA–DNA distances assessed from experiments are much larger than those typically used in theory. Finally, we suggest some experiments that could help to judge whether short‐range electrostatic forces indeed govern DNA pairing. This viewpoint paper introduces recently developed theoretical concepts to molecular biologists, with a hope to generate a junction between theory and future experiments on DNA recognition. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of <Emphasis Type="Italic">target</Emphasis> nuclear DNA from faeces reduces technical issues associated with the assumptions of low-quality and quantity template

Faecal material has increasingly become an important non-invasive source of DNA for wildlife population genetics. However, DNA from faecal sources can have issues associated with quantity (low-template and/or low target-to-total DNA ratio) and quality (degradation and/or low DNA-to-inhibitor ratio). A number of studies utilizing faecal material assume and compensate for the above properties with minimal characterization of quantity or quality of target DNA, which can unnecessarily increase the risk of downstream technical problems. Here, we present a protocol which quantifies faecal DNA using a two step approach: (1) estimating total DNA concentration using a Picogreen^TM fluorescence assay and (2) estimating target nuclear DNA concentration by comparing amplification products of field samples at suspected concentrations to those of control DNA at known concentrations. We applied this protocol to faecal material collected in the field from two species: woodland caribou (Rangifer tarandus) and swift fox (Vulpes velox). Total DNA estimates ranged from 6.5 ng/μl to 28.6 ng/μl (X = 16.2 ng/μl) for the caribou extracts and 1.0–26.1 ng/μl (X = 7.5 ng/μl) for the swift fox extracts. Our results showed high concordance between total and target DNA estimates from woodland caribou faecal extracts, with only 10% of the samples showing relatively lower target-to-total DNA ratios. In contrast, DNA extracts from swift fox scat exhibited low target DNA yields, with only 38% (19 of 50) of the samples showing comparative target DNA amplification of at least 0.1 ng. With this information, we were able to estimate the amount of target DNA entered into PCR amplifications, and identify samples having target DNA below a lower threshold of 0.2 ng and requiring modification to genotyping protocols such as multiple tube amplification. Our results here also show that this approach can easily be adapted to other species where faeces are the primary source of DNA template.     

Integrated method for single-cell DNA extraction, PCR amplification, and sequencing of ribosomal DNA from harmful dinoflagellates Cochlodinium polykrikoides and Alexandrium catenella

A simplified technique was developed for DNA sequence-based diagnosis of harmful dinoflagellate species. This protocol integrates procedures for DNA extraction and polymerase chain reaction (PCR) amplification into a single tube. DNA sequencing reactions were performed directly, using unpurified PCR products as the DNA template for subsequent sequencing reactions. PCR reactions using DNA extracted from single cells of Cocodinium polykrikoides and Alexandrium catenella successfully amplified the target ribosomal DNA regions. DNA sequencing of the unpurified PCR products showed that DNA sequences corresponded to the expected locus of ribosomal DNA regions of both A. catenella and C. polykrikoides (each zero genetic distance and 100% sequence similarity). Using the protocol described in this article, there was little DNA loss during the purification step, and the technique was found to be rapid and inexpensive. This protocol clearly resolves the taxonomic ambiguities of closely related algal species (such as Alexandrium and Cochlodinium), and it constitutes a significant breakthrough for the molecular analysis of nonculturable dinoflagellate species.     

Effect of chloramphenicol and the recB gene product on DNA metabolism in Escherichia coli K12 strains defective in DNA ligase

Summary We have examined DNA strand breakage, DNA degradation, and the rate of DNA synthesis in lig and lig-recB strains of Escherichia coli K12 incubated in the presence and absence of 3 g/ml chloramphenicol. Substantial DNA strand breakage and DNA degradation is observed in the lig strain upon growth at 40°C; however, such strand breakage and DNA degradation is not observed in the lig-recB strain. Incubation of the lig strain at 40°C in the presence of 3 g/ml chloramphenicol reduces the amount of DNA strand breakage and DNA degradation to the level observed in the lig-recB strain. Together, these results demonstrate that exonuclease V (the recBC gene product) is responsible for the increased DNA degradation associated with DNA ligase deficiency.