Pulsed-field Gel Electrophoresis Techniques Research Articles

Heavy ion-induced DNA double-strand breaks with yeast as a model system.

Cells of diploid yeast, Saccharomyces cerevisiae, were exposed to a variety of energetic heavy ions (provided by the UNILAC facility at the Gesellschaft für Schwerionenforschung, GSI), 241Am alpha-particles and 80-keV x-rays after which they were assessed for DNA double-strand breaks (DSB) using either the neutral sedimentation or the pulsed-field gel electrophoresis (PFGE) technique. Both yielded comparable results. The DSB production cross-sections are compared with inactivation studies performed for the same cells under identical conditions. The measurements show that with lighter ions DSB induction cross-sections increase with linear energy transfer (LET), but the situation is less clear with the heavier ions. A close parallelism was found between DSB induction and cell inactivation in these yeast cells.

Fused pyrazine mono-N-oxides as bioreductive drugs. III. Characterization of RB 90740 in vitro and in vivo.

RB 90740 is the lead compound in a series of aromatic mono-N-oxide bioreductive drugs. The compound shows considerably greater toxicity towards hypoxic verses aerobic mammalian cells in vitro. The differential in concentration required to give the same level of cell killing under these conditions ranges from 3.5 in a human bronchio-alveolar tumor cell line up to 120 in a rodent cell line defective in the repair of DNA strand breaks. The ability of RB 90740 to cause DNA strand breaks under hypoxic conditions was confirmed by alkaline sucrose gradient and pulsed field gel electrophoresis techniques. Despite these properties demonstrated in vitro, RB 90740 was shown not to be cytotoxic to hypoxic cells in experimental murine tumors in vivo. This may be due, in part, to the level of hypoxia (< 0.02% O2) necessary to produce toxicity in vitro.

Molecular characterization of Yersinia enterocolitica by pulsed-field gel electrophoresis and hybridization of DNA fragments to ail and pYV probes.

Sixty strains of Yersinia enterocolitica from five serogroups (O:3; O:9; O:8; O:5; and O:5,27) and eight non-Y. enterocolitica strains, recovered from diverse sources (humans, animals, food, and the environment) in Europe, Argentina, and the United States, were examined by the pulsed-field gel electrophoresis (PFGE) technique of contour clamped homogeneous electric field electrophoresis (CHEF) by using NotI and XbaI as restriction enzymes. NotI and XbaI generated 36 and 33 restriction endonuclease digestion profiles (REDP), respectively. By combining the results of both enzymes, 42 unique genomic groups were differentiated. DNA fragments were transferred to nylon membranes and hybridized with digoxigenin-labelled oligonucleotide probes to the ail gene and virulence plasmid to determine hybridization patterns and the potential virulence of the strains. The strains were tested for the presence of the plasmid by PFGE-CHEF and phenotypic characteristics encoded for by the virulence plasmid. Thirty of the 60 Y. enterocolitica strains tested harbored the virulence plasmid. The specificity of the ail and pYV probes was 100% when tested with 68 Yersinia strains and 19 different non-Yersinia strains. Sixteen selected Y. enterocolitica strains were tested for their virulence by lethality in iron- and desferrioxamine-sensitized mice. No correlation between REDP and the virulence of the strains was observed. The observed REDP and the hybridization patterns were very homogeneous within a serogroup and independent of the source of isolation. In addition, PFGE-CHEF was shown to be valuable in identifying and confirming serogroups. Principal component analysis of Dice similarity indices from REDP was an excellent tool for determining genetic relatedness among strains.

Conservation of gene arrangement and an unusual organization of rRNA genes in the linear chromosomes of the Lyme disease spirochaetes Borrelia burgdorferi, B. garinii and B. afzelii

Physical maps of the chromosomes of the Lyme disease spirochaetes Borrelia garinii and Borrelia afzelii have been elucidated for the enzymes CspI, SgrAI, I-CeuI, SmaI, EagI, BssHII, MluI and ApaI by two-dimensional pulsed-field gel electrophoresis techniques. The maps contain 42 sites for B. garinii and 32 for B. afzelii. The mapping studies showed that the two chromosomes are linear DNA molecules of 953 and 948 kbp, respectively. A comparison of the physical maps of B. garinii and B. afzelii and the published map of the other Lyme disease spirochaete, Borrelia burgdorferi [Davidson, B.E., MacDougall, J. & Saint Girons, I. (1992) J Bacteriol 174, 3766-3774] revealed that the three chromosomes have few endonuclease sites in common, apart from a cluster in rrl (encoding 23S rRNA) and rrs (encoding 16S rRNA). Cloned borrelial genes were used as specific hybridization probes to construct genetic maps, using the physical maps as a basis. The resulting maps contain 41 genetic loci for B. burgdorferi, 39 for B. garinii, and 33 for B. afzelii. In contrast to the physical maps, the three genetic maps are closely related, with no detectable differences in gene order along the entire length of the chromosome. It is concluded that the chromosomes of these three borrelial species have undergone no major rearrangements, deletions or insertions during their evolution from a common ancestor. Detailed mapping of the region of the B. garinii and B. afzelii chromosomes that encodes rRNA revealed that each chromosome contains one copy of rrs separated by 5 kbp from two copies each of rrl and rrf (encoding 5S rRNA). (ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of mutants of Rhizobium meliloti unable to synthesize poly-β-hydroxybutyrate

The isolation and characterization of four mutants of Rhizobium meliloti unable to synthesize poly-β-hydroxybutyrate (PHB) are reported. The mutants were independently obtained via Tn5 transposon mutagenesis and exhibited physiological and cytomorphological characteristics similar to those of the parental strain, as well as overlapping DNA profiles. These were assessed at both the plasmid and total genome level, using for the latter the sensitive technique of pulsed-field gel electrophoresis in a contour-clamped homogeneous electric field. With respect to the parental PHB+ strain, the loss of PHB-synthesizing ability in the four mutants was demonstrated by gas chromatography, transmission electron microscopy, and enzymatic tests. Localization studies of Tn5 insertion showed that the PHB− phenotype had, in all mutants, a transposon insertion in the same region, although not in the same position. The symbiotic traits (nodule-inducing ability on Medicago sativa and acetylene-reducing activity of nodules) of the mutants did not differ significantly from those of the parental R. meliloti.Key words: Rhizobium meliloti, poly-β-hydroxybutyrate (PHB), PHB synthase.

Bacterial genome mapping by two-dimensional pulsed-field gel electrophoresis (2D-PFGE).

Macrorestriction mapping is often the first step toward a thorough physical and genetic characterization of a bacterial genome. The problem of deducing the order of partially or completely digested macrorestriction fragments to yield a physical genome map may readily be solved by applying two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. These powerful methods are quick and technically easy to perform; specifically, they are independent of DNA probes and should therefore be applicable to any bacterial species irrespective of its prior genetic characterization. In this article, detailed step-by-step protocols are given to set up, run, and evaluate 2D pulsed-field gels. Two basic methods are described: partial/complete 2D gels of one restriction enzyme and complete/complete 2D gels of two different restriction enzymes. Other topics include preparation of bacterial genomic DNA, screening for suitable rare-cutting restriction enzymes and determination of optimal running conditions. Accompanied by many notes, these protocols are meant to offer the novice a sound and rapid access to these important methods.

Damage Induced in Episomal EBV DNA in Raji Cells by Antitumor Drugs as Measured by Pulsed Field Gel Electrophoresis

The studies described below were carried out to analyze the damage induced by DNA active drugs to episomal (Epstein-Barr virus, EBV) DNA in the Raji Burkitt′s lymphoma cell line. This work: (i) applies pulsed-field gel electrophoresis (PFGE) techniques to quantify DNA damage on a large (≍180 kbp), circular target, (ii) investigates the DNA strand-scission behavior of different classes of drugs on the EBV episome, and (iii) compares EBV episomal damage to that generated in genomic DNA in the Raji cell line. Cells were treated with ionizing radiation to induce random strand scission, and the migration of topological forms of EBV was measured using PFGE. DNA damage induced in the episome by DNA active drugs was then assayed. Three drugs, acting by different types of DNA interactive mechanisms, were used: bleomycin, an intercalative DNA strand-scission agent; and amsacrine (mAMSA) and teniposide (VM26), intercalative and nonintercalative topoisomerase II active drugs, respectively. Rad equivalency of damage was determined by comparing the drug-induced change in percentage of Forms I and III to that generated by ionizing radiation. Additionally, single- and double-strand scission induced in genomic (total cellular) DNA by X-rays, bleomycin, amsacrine, and teniposide were assayed by high-sensitivity alkaline and neutral filter elution techniques. We demonstrate that pulsed-field gel electrophoresis is a useful technique for measuring form conversion in large episomal DNA. While all three drugs effect both episomal and genomic DNA strand scission, bleomycin appears to preferentially damage the EBV episome. The topoisomerase II active drugs mAMSA and VM26 show no evidence of episome-directed damage in this system and, in fact, damage genomic DNA at somewhat higher rates.

Analysis of Schizosaccharomyces pombe mitochondrial DNA replication by two dimensional gel electrophoresis.

The entire mitochondrial genome of Schizosaccharomyces pombe ura4-294h- was analyzed by the 2D pulsed field gel electrophoresis technique developed by Brewer and Fangman. The genome consists of multimers with an average size of 100 kb and analysis of the overlapping restriction fragments of the complete mitochondrial DNA (mtDNA) genome resulted in simple Y 2D gel patterns. Large single-stranded DNA molecules or double-stranded DNA molecules containing large or numerous single-stranded regions were found in the S. pombe mtDNA preparation. The replication of mtDNA monomers was found to occur in either direction. On the basis of these results, a replication mechanism for S. pombe mtDNA that is most consistent with a rolling circle model is suggested.

Measurement of DNA Double-Strand Breaks in Mammalian Cells by Pulsed-Field Gel Electrophoresis: A New Approach Using Rarely Cutting Restriction Enzymes

A new method is presented for measuring DNA double-strand breaks (DSBs) which combines the technique of pulsed-field gel electrophoresis (PFGE) with the idea of velocity sedimentation. Purified DNA samples were treated with restriction enzymes, which results in DNA of sizes which can be separated by PFGE. After electrophoresis, unirradiated DNA shows a size distribution (obtained with the help of a specially developed software program) similar to that obtained with the sedimentation technique; with X irradiation, this distribution is shifted to lower molecular weight with increasing dose. The rate of DSB induction was calculated by comparing the curves obtained experimentally with theoretical distributions (based on the assumption that breaks are formed according to Poisson statistics). The method was tested by measuring X-ray-induced DSBs in P3 (derived from human epithelial teratoma) cells. The induction of DSBs was found to be linear with dose and a rate of 5.4 x 10(-3)/Mbp/Gy was obtained.

Molecular characterization of a recurring complex chromosomal translocation in two human extragonadal germ cell tumors

The molecular characterization of a recurring complex chromosomal translocation involving 6p21, 6p22, 6q23, and 11q13 in two independent but similar extragonadal human germ cell tumors was initiated using fluorescence in situ hybridization (FISH) and pulse field gel electrophoresis (PFGE) techniques. By using a series of specific probes from the 11q13 region, the translocation breakpoint in this chromosomal band could be located within a long-range restriction enzyme map in between the markers D11S457 and D11S546. In addition, aberrantly hybridizing restriction fragments were revealed by PFGE in both tumors, indicating that the breakpoint region must be located within a distance of at maximum 200 kilobase pairs (kbp) from the nearest DNA marker (D11S546).

Physical map of the extremely thermophilic bacterium Thermus thermophilus HB27 chromosome

The physical map of the chromosome of Thermus thermophilus HB27 was constructed using three restriction enzymes; EcoRI, SspI and MunI by applying pulsed-field gel electrophoresis techniques. Although the genome size of 1.82 Mb was almost the same as that (1.74 Mb) reported for T. thermophilus HB8 [Borges, K.M., and P.L. Bergquist. (1993) J. Bacteriol. 175, 103-110], the MunI cleavage maps were different. A 240 kb plasmid was detected in HB27, and its physical map was also constructed. In addition, several genes were located on the chromosomal physical map.

Genome Analysis of Different Haloferax mediterranei Strains Using Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) has been used to analyze the different genomic elements of the halophilic archaeon Haloferax mediterranei strains characterized by numerical taxonomy (M2a/b, M4, M6, M10 and M14) and to compare them with those of the type collection microorganism, ATCC 33500 (strain R-4), whose characterization had been previously reported. Both PFGE and DNA/DNA hybridization experiments have shown that M14 does not belong to this species. The rest of the strains contained the same four replicons described for Hf. mediterranei ATCC 33500 (chromosome, pHM500, pHM300 and pHM100) except for M10, which contained an additional replicon of approximately 66 Kb (pHM50). The size and topology of all the elements have been established, their BamHI and PacI restriction patterns characterized, and as a result, a high level of polymorphism observed. Chromosomal physical maps of M2a, M2b, M4, and M10 were constructed using PacI . Comparative genomic analysis allowed us to establish three subgroups for these strains. The potential applicability of PFGE techniques for archaeal taxonomy and the understanding of the evolution of genomic lineages is discussed.

A NheI macrorestriction map of the Neisseria meningitidis B1940 genome.

A macrorestriction map of the Neisseria meningitidis strain B1940 genome was constructed by two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. Digestion of the genomic DNA with the restriction endonuclease NheI revealed 15 fragments between 10 kb and 450 kb. The sum of the fragments and resolution of the linearized chromosome yielded a total genome size of about 2.3 Mbp. By overlapping methylation with the AluI-methylase six NheI recognition sites could be blocked. Fragments were ordered by partial/complete 2D-PFGE of genomic DNA with and without prior AluI methylation, respectively. All nine AluI-methylase/NheI and 14 NheI restriction sites could be mapped on a single circular chromosome. This map will serve as a useful tool for further genetic analysis of meningococci and exemplifies the power of non-radioactive 2D-PFGE techniques to construct large physical genome maps with a single restriction enzyme.

Pulsed-field gel electrophoresis analysis of higher-order chromatin structures of Zea mays. Highly methylated DNA in the 50 kb chromatin structure.

We have investigated the presence of higher-order chromatin structures in different maize tissues. Taking advantage of the pulsed-field gel electrophoresis technique to analyse large DNA fragments from intact nuclei and cells, we have determined the size distribution of the high-molecular-weight DNA fragments obtained from chromatin degradation by endogenous nucleases in isolated nuclei. Chromatin digestion leads to the appearance of stable DNA fragments of about 50 kb in all the tissues examined, suggesting the folding of DNA in higher-order chromatin domain structures. It has been reported that such chromatin domains are formed by loops of the 30 nm fibers anchored to the nuclear matrix by a complex set of proteins, including DNA topoisomerase II. Treatment of maize protoplasts with the calcium ionophore A23187 and the antitumour drug VM-26, which specifically inhibit the religation of the cleaved DNA in the topoisomerase II reaction, also produces the 50 kb structure. Analysis of the DNA contained in the 50 kb chromatin structure shows a higher degree of methylation than in bulk maize chromosomal DNA. The role of methylated DNA in the chromatin folding is discussed.

Genomic restriction map of the extremely thermophilic bacterium Thermus thermophilus HB8

A physical map of the chromosome of the extremely thermophilic eubacterium Thermus thermophilus HB8 has been constructed by using pulsed-field gel electrophoresis techniques. A total of 26 cleavage sites for the rarely cutting restriction endonucleases HpaI, MunI, and NdeI were located on the genome. On the basis of the sizes of the restriction fragments generated, the genome size was estimated to be 1.74 Mbp, which is significantly smaller than the chromosomes of Escherichia coli and other mesophiles. Partial digestion experiments revealed the order of the six HpaI bands on the chromosome. Hybridization of isolated restriction fragments to pulsed-field gel-separated restriction digestions confirmed the deduced order of the HpaI fragments and allowed ordering and alignment of the NdeI and MunI fragments. In addition, 16 genes or gene clusters cloned from several different Thermus strains were located on the T. thermophilus HB8 chromosomal map by hybridization of gene probes to pulsed-field gel-resolved restriction digestions.

Cell inactivation by heavy charged particles.

The inactivation of cells resulting in lethal or aberrant effects by charged particles is of growing interest. Charged particles at extremely high LET are capable of completely eliminating cell-type and cell-line differences in repair capacity. It is still not clear however whether the repair systems are inactivated, or merely that heavy-ion lesions are less repairable. Studies correlating the particle inactivation dose of radioresistant cells with intact DNA analyzed with pulse field gel electrophoresis and other techniques may be useful, but more experiments are also needed to assess the fidelity of repair. For particle irradiations between 40-100 keV/microns there is however evidence for particle-induced activation of specific genes in mammalian cells, and certain repair processes in bacteria. New data are available on the inactivation of developmental processes in several systems including seeds, and cells of the nematode C. elegans. Future experimental and theoretical modeling research emphasis should focus on exploring particle-induced inactivation of endpoints assessing functionality and not just lethality, and on analyzing molecular damage and genetic effects arising in damaged but non-inactivated survivors. The discrete nature of selective types of particle damage as a function of radiation quality indicates the value of accelerated ions as probes of normal and aberrant biological processes. Information obtained from molecular analyses of damage and repair must however be integrated into the context of cellular and tissue functions of the organism.

Distribution of legionellae in a hospital water system: prevalence of immunologically and genetically related Legionella pneumophila serogroup 6 isolates

A hospital warm water system was monitored for the presence and distribution of legionellae. Subtyping of ten selected Legionella pneumophila isolates, originating from four different sites in the system by using serogroup specific antisera in an indirect immunofluorescence test, revealed that nine of the ten isolates belonged to serogroup 6, while the remaining one was serogroup 10. Two monoclonal antibodies (mAbs) specific for a subgroup of serogroup 6 strains were further used for characterization. None of the strains reacted with these mAbs. Genome analysis by elaborating NotI profiles using the pulsed field gel electrophoresis (PFGE) technique revealed that nearly all serogroup 6 isolates derived from different sites, including a new building connected by a ring pipe, were identical according to restriction fragment patterns. The patterns were distinguishable from those of the two L. pneumophila serogroup 6 reference strains, and from that of the L. pneumophila serogroup 10 isolate. These data argue for a relatively homogeneous L. pneumophila serogroup 6 population in the entire water system.

Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells.

This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-ChR-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-ChR-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads.

Effect of 2-deoxy-D-glucose on DNA double strand break repair, cell survival and energy metabolism in euoxic Ehrlich ascites tumour cells.

Effects of 2-deoxy-D-glucose (2-DG) on DNA double strand break (dsb) repair, cell survival and on the energy metabolism were investigated in exponentially growing Ehrlich ascites tumour (EAT) cells. Cells in suspension were exposed to 40 Gy of X-rays and allowed to repair (up to 4 h) with or without 2-DG at 37 degrees C. DNA dsb rejoining was measured by means of clamped homogeneous electric field (CHEF), a pulsed field gel electrophoresis technique. The fraction of activity released (FAR) during electrophoresis (DNA associated 14C-thymidine) was used as a parameter to determine the number of dsb present in the DNA. Biphasic kinetics for dsb repair were observed. The presence of 2-DG significantly inhibited the slow component of dsb repair. The presence of 2-DG also enhanced radiation-induced cell killing. ATP content of cells was measured by a bioluminescence method. ATP content in exponentially growing cells was about 4 pg per cell. The level of ATP was reduced by 50% in presence of 2-DG (C2-DG/CG = 1.0).

Analysis of large DNA from soybean (Glycine max L. Merr.) by pulsed-field gel electrophoresis.

The technique of pulsed-field gel electrophoresis (PFE) has been used to study chromosomal regions and entire genomes of several organisms. Techniques are presented for the isolation of high molecular weight DNA from embedded soybean protoplasts and the conditions for separating large DNA fragments using PFE. Digestion was detected by Southern hybridization using single copy nodulin clones. These data are being used to generate a physical map of the nodulin region(s) of the soybean genome.