Parameters affecting the yield of DNA from human blood

An examination of variables affecting the yield of DNA from blood was undertaken in order to improve sample processing and to evaluate alternative methods of mailing blood samples for DNA analysis. A rapid, high-yield method was developed for the isolation of high-molecular-weight DNA from fresh and frozen blood. In addition, the following observations were made: (1) Of the anticoagulants examined, acid citrate dextrose (ACD) solution B was found to be superior to EDTA and heparin for preserving yields of DNA during incubation at room temperature. If DNA is isolated from frozen blood, high yields of undegraded DNA are achieved after incubation at 23 degrees C for 5 days with ACD solution B. (2) High yields of undegraded DNA are obtained from blood stored with ACD solution B for at least 1 day at 42 degrees C, 5 days at 0 degrees C, or 1 month at -20 degrees C. (3) Three cycles of freezing and thawing may have little if any affect on the yield of DNA. The results indicate that blood for DNA extraction may be mailed in an ambient temperature container and, in many cases, sent by first-class mail rather than by overnight delivery services.     

A non-organic and non-enzymatic extraction method gives higher yields of genomic DNA from whole-blood samples than do nine other methods tested.

We compared ten methods for extraction of DNA from whole blood. Nine methods require incubation with either enzymes or treatment of organic solvents or both. The 'Rapid Method' (RM) (Method 10) avoids the use of organic solvents (phenol/chloroform) and eliminates completely the use of proteinase K. Thus, the time and cost of DNA extraction are reduced significantly. This is accomplished by salting out and precipitation of the cellular proteins in saturated sodium chloride. This method takes less than an hour to completion, without compromising the yield or the quality of DNA. Using RM, we can make DNA from 0.1 ml of whole blood and as little as 0.5 ml of blood yields DNA sufficient to run a few Southern blots. The RM can also be applied to packed cells. The DNA is free of RNA, protein and degrading enzymes. The uncut DNA runs as a typical slow-migrating, high-molecular-weight and undegraded species in an agarose gel. The DNA is suitable for digestion by various restriction endonucleases. This procedure works equally well with fresh blood samples and with those that are stored at 4 degrees C and -70 degrees C. To our knowledge the RM reported here is the safest, fastest and most quantitative and economical method for preparation of DNA from whole blood and cells.     

Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale

Summary Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taqpolymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121°C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 10³ per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120°C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes. Offprint requests to: M. R. Fibi     

On the integrity of sperm DNA

Ejaculated rabbit spermatozoa washed with buffer prior to decondensation by Triton X-100 and dithiothreitol were good templates for DNA synthesis by Escherichia coli DNA polymerase. This result agrees with the observations of Zirkin and Chang [1977], and implies that the sperm DNA is nicked. Template activity, however, was reduced if spermatozoa were extensively washed before decondensation, and if DNase inhibitors EDTA or Na₂SO₄ were present during decondensation. Template activity was also low if decondensation was induced with DNase inhibitors thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate and dithiothreitol instead of with Triton X-100 and dithiothreitol. Calf thymus DNA was completely degraded when incubated with rabbit seminal plasma or buffer-washed spermatozoa, but much less degradation was observed if EDTA, Na₂SO₄, thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate were also present, or if spermatozoa were extensively washed with buffer. Centrifugation of spermatozoa through 2.05 M sucrose completely removed contaminating DNase, and such spermatozoa were inactive as DNA templates after decondensation. The DNA template activity of swollen rabbit sperm nuclei thus parallels the activity of a contaminating seminal plasma DNase. This suggest that the nicks in sperm DNA enabling it to act as a template for DNA synthesis were generated by the DNase during decondensation and thus are not a natural structural feature of the DNA. The presence of breaks in the DNA of decondensed buffer-washed spermatozoa (DNase contaminated) was confirmed by their incorporation of phosphate from [γ^?32 P] ATP in the presence of the enzyme polynucleotide kinase. These spermatozoa were found to contain as few as two breaks/mole of DNA, but sucrose-washed spermatozoa (DNase free) were free of breaks. The possible use of this enzymic procedure for the assessment of sperm genome damage and the evaluation of the quality of a sperm population are discussed.     

一种从人血凝块中提取基因组DNA的方法

介绍了一种新的从人血凝块中提取基因组DNA的方法,用该方法提取的DNA成功地应用于PCR和限制性酶切等后续实验中。基本过程为首先机械粉碎,然后高盐高EDTA溶液处理,含蛋白酶K和SDS的消化液变换温度消化,苯酚氯仿抽提。     

Intragenomic DNA sequence homologies in the chicken and other members of the classAves: DNA re-association under reduced stringency conditions

Summary We have investigated the intragenomic DNA sequence homologies of twelve species of birds representing five orders, and emphasizing Galliformes. This study differs in two important ways from the classical approaches taken in constructing and evaluating phylogenies based on DNA sequence similarities. Comparisons are made on the basis of sequence homologieswithin genomes of related birds, rather than between genomes. DNA is reassociated at 50°C in 0.5M phosphate buffer; these conditions allow formation and detection of duplexes containing more mismatch than would normally be permitted using more stringent conditions, affording an opportunity to observe more ancient sequence homologies. Thermal stability profiles of DNA duplexes formed under these conditions are the basis of comparison; three general patterns were observed. This approach emphasizes differences in sequence composition between genomes while the more traditional method of intergenomic tracer DNA hybridization at higher stringency emphasizes sequence similarities.No correlation was found between taxonomic position and intragenomic sequence composition, either within or between lineages. The thermal stability profiles of DNA duplexes formed within avian genomes did not reflect the biological similarities inferred from morphology, karyotype, and studies of interspecific hybridization. While all of the differences observed could have occurred over geological time, it was surprising that the genomes of the domestic chicken and the Red Jungle Fowl (Gallus gallus) differ in their sequence compositions. It appears that amplification/reduction events and/or positional changes occur rather often during evolution of a lineage.Abbreviations SDS sodium dodecyl sulphate - PB equimolar sodium phosphate buffer pH 6.8 - Cot concentration of DNA in moles of nucleotide per liter times the incubation time in seconds - Equiv. or Equivalent Cot Cot corrected for the monovalent cation concentration effect on re-association rate - HAP hydroxylapatite - Te_1/2 temperature at which one-half the DNA has eluted from HAP - SSC 0.15M sodium chloride-0.015M sodium citrate     

一种改良的植物DNA提取方法

植物组织中含有大量多糖、多酚、酯类等次生代谢产物, 要从中提取高质量的DNA比较困难。针对这一情况, 该文提出一种改良CTAB植物DNA提取方法(mCTAB), 并以10种常见植物为实验材料, 与4种常用的植物DNA提取试剂盒作对比。结果表明, mCTAB法提取的DNA产率高且质量好, PCR扩增成功率也较高, 而提取成本显著低于DNA提取试剂盒, 可有效用于植物DNA条形码等研究的植物DNA提取。     

Technical note: Bone DNA extraction and purification using silica-coated paramagnetic beads

The goal of this study was to develop a simple method to improve DNA recovery from challenging bone samples. To this end, an optimized procedure was developed that combined the demineralization and DNA extraction into a single step, followed by DNA purification using an automated silica-coated paramagnetic bead procedure. This method replaced a previous silica-membrane-based procedure, which was able to recover sufficient DNA to obtain full autosomal and Y chromosome STR profiles from greater than 90% of the samples, including samples greater than 20 years old. The development process began with the evaluation of buffer and demineralization systems to determine the best reagent combination. During the developmental process, we observed that the addition of EDTA and DTT affected silica-based DNA purification methods by raising the pH of the digest buffer. The protocols with buffer ATL, PK, EDTA, and DTT followed by lowering the pH with sodium acetate just before purification resulted in the best yields. The method reduced the extraction volume from 10 to 1.5 ml and used commercially available reagents already being utilized in forensic DNA casework. Because of the simplicity and small volume needed for the procedure, many steps where contamination could be introduced have been eliminated or minimized. This study demonstrated a new method of recovering DNA from bone samples capable of extracting trace quantities of DNA, removing potential inhibitors, and minimizing the potential for exogenous DNA contamination.     

Preservation of RNA and DNA from mammal samples under field conditions

Ecological and conservation genetics require sampling of organisms in the wild. Appropriate preservation of the collected samples, usually by cryostorage, is key to the quality of the genetic data obtained. Nevertheless, cryopreservation in the field to ensure RNA and DNA stability is not always possible. We compared several nucleic acid preservation solutions appropriate for field sampling and tested them on rat (Rattus rattus) blood, ear and tail tip, liver, brain and muscle. We compared the efficacy of a nucleic acid preservation (NAP) buffer for DNA preservation against 95% ethanol and Longmire buffer, and for RNA preservation against RNAlater (Qiagen) and Longmire buffer, under simulated field conditions. For DNA, the NAP buffer was slightly better than cryopreservation or 95% ethanol, but high molecular weight DNA was preserved in all conditions. The NAP buffer preserved RNA as well as RNAlater. Liver yielded the best RNA and DNA quantity and quality; thus, liver should be the tissue preferentially collected from euthanized animals. We also show that DNA persists in nonpreserved muscle tissue for at least 1 week at ambient temperature, although degradation is noticeable in a matter of hours. When cryopreservation is not possible, the NAP buffer is an economical alternative for RNA preservation at ambient temperature for at least 2 months and DNA preservation for at least 10 months.     

DNA degradation in fish: Practical solutions and guidelines to improve DNA preservation for genomic research

The more demanding requirements of DNA preservation for genomic research can be difficult to meet when field conditions limit the methodological approaches that can be used or cause samples to be stored in suboptimal conditions. Such limitations may increase rates of DNA degradation, potentially rendering samples unusable for applications such as genome‐wide sequencing. Nonetheless, little is known about the impact of suboptimal sampling conditions. We evaluated the performance of two widely used preservation solutions (1. DESS: 20% DMSO, 0.25 M EDTA, NaCl saturated solution, and 2. Ethanol >99.5%) under a range of storage conditions over a three‐month period (sampling at 1 day, 1 week, 2 weeks, 1 month, and 3 months) to provide practical guidelines for DNA preservation. DNA degradation was quantified as the reduction in average DNA fragment size over time (DNA fragmentation) because the size distribution of DNA segments plays a key role in generating genomic datasets. Tissues were collected from a marine teleost species, the Australasian snapper, Chrysophrys auratus. We found that the storage solution has a strong effect on DNA preservation. In DESS, DNA was only moderately degraded after three months of storage while DNA stored in ethanol showed high levels of DNA degradation already within 24 hr, making samples unsuitable for next‐generation sequencing. Here, we conclude that DESS was the most promising solution when storing samples for genomic applications. We recognize that the best preservation protocol is highly dependent on the organism, tissue type, and study design. We highly recommend performing similar experiments before beginning a study. This study highlights the importance of testing sample preservation protocols and provides both practical and economical advice to improve DNA preservation when sampling for genome‐wide applications.     

DNA modification by oxovanadium(IV) complexes of salen derivatives

Oxovanadium(IV) complexes of hydroxysalen derivatives have been prepared and tested as DNA reactive agents. The nuclease activity has been investigated under oxidative or reducing conditions, on the basis of the various oxidation states of vanadium: V^III, V^IV and V^V. In the absence of an activating agent, none of the compounds tested was able to induce cleavage of DNA, whereas in the presence of mercaptopropionic acid (MPA) or Oxone the four complexes induced DNA modifications. Under both conditions, the para-hydroxy complex was found to be the most active compound. Reaction of these salen complexes with DNA occurs essentially at guanine residues and is more efficient in the presence of Oxone than under reducing conditions. The extent of Oxone-mediated DNA oxidation by the four vanadyl complexes was clearly superior to VOSO₄ and was observed without piperidine treatment. EPR studies provided information on the reactive metal-oxo species involved under each conditions and a mechanism of reaction with DNA is discussed.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0529-0Abbreviations BPE buffer bis-phosphate EDTA buffer - DMPO 5,5-dimethylpyrroline N-oxide - DMS dimethyl sulfate - HFS hyperfine structure - Lin linear - MPA 3-mercaptopropionic acid - Nck nicked - salen (salicylidene)ethylenediamine - Sc supercoiled - TBE buffer tris-borate EDTA buffer - Tris tris(hydroxymethyl)aminomethane     

Relationship between DNA damage,DNA repair,metabolic state and cell lethality

Summary Induction of unrepairable DNA damage, accumulation of misrepaired DNA damage, and generation of imbalances in competing biochemical and/or metabolic processes have been proposed to explain the relationship between radiation-induced DNA damage and cell lethality. Theoretically, the temperature dependence of the critical DNA repair process(es) should be 1) either independent of or identical to the temperature dependence of cell killing if the first two hypotheses are correct, and 2) different if the third hypothesis is correct. To test this, exponentially growing rat 9L brain tumor cells were left at 37°C or equilibrated for 3–14 h at 20°C before irradiation. Cells were irradiated and allowed to repair at either 20°C or 37°C. Alternatively, the cells were irradiated at one of these temperatures and immediately shifted to the other temperature for repair. DNA damage was assessed by the alkaline elution technique; cell kill was assessed by a clonogenic assay. 9L cells maintained at 20°C or 37°C sustained the same amount of DNA damage as measured by alkaline elution. DNA repair instantaneously assumed the rate characteristic of the postirradiation temperature. For 9L cells equilibrated, irradiated, and repaired at 20°C, the half-time of the fast phase of the DNA repair decreased by a factor of 2 and the half-time of the slow phase decreased by a factor of 5 over that measured in cells incubated, irradiated and repaired at 37°C. Although the rate of DNA repair decreased substantially at 20°C, the survival of 9L cells that were equilibrated and irradiated at 20°C was greater (p <10^–4) than those incubated and irradiated at 37°C, when assayed by an immediate plating protocol. In addition, the survival of 9L cells equilibrated and irradiated at 20°C and then shifted to 37°C immediately after irradiation was greater (p <10^–2) than that obtained with any other delayed plating protocol. Thus, the temperature dependence of the DNA repair processes measured by alkaline elution was different from the temperature dependence of cell killing measured either by an immediate or delayed plating protocol. These data support the hypothesis that many irradiated 9L tumor cells die because of imbalances in sets of competing biochemical and/or metabolic processes.Presented at the 81st Annual Meeting of the American Association for Cancer Research, May 23–26, 1990 in Washington, DC     

Quick DNA preparation for direct PCR in marine invertebrates and fishes

A modified protocol was developed for extracting DNA and direct PCR from tissues of marine invertebrates and finfishes. Protocol represented combination of classical lysis using a modified buffer system comprising KCl, Tris buffer and MgCl₂. The DNA obtained was quantified and tested by mtDNA-PCR and RAPD. Test results indicated usefulness of this method in studies involving screening of large numbers of samples, such as evolutionary, forensic and population studies.     

The extracellular nuclease of Serratia marcescens: studies on the activity in vitro and effect on transforming DNA in a groundwater aquifer microcosm

A quantitative endonuclease assay, which relies on the introduction of single and double strand breaks into supercoiled plasmid DNA, was used to study the activity of the extracellular nuclease of Serratia marcescens SM6 in buffer and in groundwater. The parallel enzyme concentration-dependent production of relaxed and linear plasmid molecules suggests that the nuclease produces single and double strand breaks in duplex DNA. Bovine serum albumin stimulated the nuclease activity towards DNA and RNA and increased the stability of the enzyme against thermal inactivation. The DNase activity at 4 °C and 50 °C was almost half of that at the optimum temperature (37 °C). The nuclease was active in groundwater, although the specific activity was lower than in buffer. In a groundwater aquifer microcosm, mineral-adsorbed transforming DNA was substantially less accessible to the nuclease than was dissolved DNA. The data suggest that the extracellular nuclease of Serratia marcescens may contribute to DNA turnover in the environment and that adsorption of DNA to minerals provides protection against the nuclease.Abbreviations GW groundwater GWA groundwater aquifer     

Microwave-assisted digestion combined with silica-based spin column for DNA isolation from human bones

A protocol for the extraction of DNA from ancient skeletal material was developed. Bone specimen samples (powder or slice), buffer, pretreatment, and extraction methodologies were compared to investigate the best conditions yielding the highest concentration of DNA. The degree of extract contamination by polymerase chain reaction (PCR) inhibitors was compared as well. Pretreatment was carried out using agitation in an incubator shaker and microwave digestion. Subsequently, DNA from bones was isolated by the classical organic phenol–chloroform extraction and silica-based spin columns. Decalcification buffer for total demineralization was required as well as lysis buffer for cell lysis to obtain DNA, whereas microwave-assisted digestion proved to be very rapid, with an incubation time of 2 min instead of 24 h at an incubator shaker without using lysis buffer. The correction of isolated DNA was detected using real-time PCR with melt curve analysis, which was 82.8 ± 0.2 °C for highly repetitive α-satellite gene region specific for human chromosome 17 (locus D17Z1). Consequently, microwave-based DNA digestion followed by silica column yielded a high-purity DNA with a concentration of 19.40 ng/μl and proved to be a superior alternative to the phenol–chloroform method, presenting an environmentally friendly and efficient technique for DNA extraction.     

Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood

Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~ 250 μl volumes, at − 80 °C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at − 80 °C, unless a cryopreservative is present. Our “small volume” approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.     

Effects of high temperatures on the photosynthetic systems in spinach: Oxygen-evolving activities,fluorescence characteristics and the denaturation process

Activities of oxygen evolution, fluorescence F_v (a variable part of chlorophyll fluorescence) values, and amounts of the 33 kDa protein remaining bound to the thylakoids in intact spinach chloroplasts were measured during and after high-temperature treatment. The following results were obtained. (1) Both the F_v value and the flash-induced oxygen evolution measured by an oxygen electrode were decreased at high temperatures, but they showed partial recovery when the samples were cooled down and incubated at 25°C for 5 min after high-temperature treatment. (2) Oxygen evolution was more sensitive to high temperatures than the F_v value, and the decrease in the F_v/F_m ratio at high temperatures rather corresponded to that in the oxygen evolution measured at 25°C after high-temperature treatment. (3) Photoinactivation of PS II was very rapid at high temperatures, and this seems to be a cause of the difference between the F_v values and the oxygen-evolving activities at high temperatures. (4) At around 40°C, the manganese-stabilizing 33 kDa protein of PS II was supposed to be released from the PS II core complexes during heat treatment and to rebind to the complexes when the samples were cooled down to 25°C. (5) At higher temperatures, the charge separation reaction of PS II was inactivated, and the PS II complexes became less fluorescent, which was recovered partially at 25°C. (6) Increases in the F_v value due to a large decrease in the electron flow from Q_A to Q_B became prominent after high-temperature treatment at around 50°C. This was the main cause of the discrepancy between the F_v values and the oxygen-evolving activities measured at 25°C. Relationship between the process of heat inactivation of PS II reaction center complexes and the fluorescence levels is discussed.     

Molecular Typing of Cyst‐Forming Nematodes Globodera pallida and G. rostochiensis,Using Real‐Time PCR and Evaluation of Five Methods for Template Preparation

Globodera pallida and G. rostochiensis are two cyst‐forming nematodes known to infest potato crops, causing severe economic losses worldwide. In this study, a real‐time TaqMan PCR assay was developed and optimized for the simultaneous detection of G. pallida and G. rostochiensis. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Four different DNA extraction methods and one rapid crude template‐preparation procedure were compared in terms of extraction purity, efficiency for PCR applications, utility and cost. Extraction methods A and B included two commercially available kits that utilize silica columns and magnetic beads, respectively. Method C was based on DNA isolation using Chelex resin, and method D was a standard chemistry in‐house protocol. Procedure E included the direct use of crude mixture composed of disrupted cysts in Tris–EDTA buffer. The multiplex TaqMan PCR assay successfully discriminated the two nematode species from all reference cyst samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional (Co) PCR tests, the overall Dsp and Dse were lower and estimated at 94 and 87% for G. pallida, and 97 and 88% for G. rostochiensis, respectively. Spectrophotometric results showed that DNA extraction methods A, B and C yielded the purest DNA and gave the lowest mean C_t values as well as the most consistent results in Co PCR. Alternative crude preparation method E resulted in statistically similar and C_t values consistent with those obtained with methods A to C when tested by TaqMan PCR. The developed assay, using crude template‐preparation E, allows the simple, accurate and cost‐effective testing of a large number of cyst samples and can be applied in surveys and certification schemes.     

DNA base composition heterogeneity in some agarophytes (Gracilariales,Rhodophyta) from Mexico and the Philippines

Estimates of nuclear DNA base composition by determination of thermal denaturation temperatures (T_m) indicate guanine + cytosine (G + C) levels of 35.4–46.8% for ten species of the Gracilariaceae, representing the generaGracilaria andHydropuntia. T_m values were found to be reproducible with variation among most samples and replicates of less than 1 °C and 2 mol%. Interspecific variation in G + C values was less than 11.4% amongGracilaria species. Calculation of intragenomic base pair composition distribution based on mid-resolution thermal denaturation (A 1 °C/min with 4s interval H and dT logging) indicated an inverse relationship between maximum similarity values and taxonomic rank. Intraspecific (population level) maximum similarity (homology) values were estimated to range from 79–90% inGracilaria tikvahiae (4 isolates). Interspecific values of 46–69% were found in 13 species ofGracilaria. Nucleotide distribution similarity values for the Gracilariaceae are compared with previous information for genome organization and complexity, genome size and karyotype patterns.Author for correspondence     

DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.