Clamped Homogeneous Electric Field Research Articles

Rapid Protocol for Characterizing Klebsiella pneumoniae Isolates by Pulsed Field Gel Electrophoresis (PFGE) in Contour Clamped Homogeneous Electric Field (CHEF) Minigels

Klebsiella pneumoniae strains may act as opportunistic pathogens infecting susceptible patients, or as hypervirulent germs infecting healthy people, or as multidrug-resistant organisms due to extended-spectrum beta-lactamase (ESBL)-production provoking infection outbreaks in hospital settings and communities. Consequently, it is a priority to encourage the development of simple, rapid and economical methods for the molecular subtyping of the outbreak strains and then to facilitate the infection control. Pulsed field gel electrophoresis (PFGE) remains a good choice for K. pneumoniae subtyping and for the laboratory confirmation of outbreaks. We report the implementation of a rapid protocol for characterizing K. pneumoniae isolates by contour clamped homogeneous electric field (CHEF) in mini gels in a total analysis time of less than 12 h. This approach is based on the preparation of K. pneumoniae DNA of PFGE-quality by a single incubation step of immobilized bacteria for 2 h with a nonenzymatic solution containing 4 M urea and nonionic detergents with further XbaI DNA fingerprint resolution in CHEF mini gels in 5.5 h. The DNA suitable for PFGE obtained by this procedure was approximately 91% of the total DNA in the plugs. This procedure enabled the reproducible subtyping of 26 isolates of K. pneumoniae and three of K. oxytoca. The XbaI DNA fingerprint analysis in CHEF mini gels was able to discriminate the seven isolates of K. pneumoniae that were epidemiologically related and the nineteen that were unrelated. The nonenzymatic DNA plugs preparation reported in this paper is a cost-effective alternative for K. pneumoniae PFGE subtyping in laboratories where the technique is already implemented.

Sgs1 RecQ Helicase Inhibits Survival of Saccharomyces cerevisiae Cells Lacking Telomerase and Homologous Recombination

In yeast telomerase mutants, the Sgs1 RecQ helicase slows the rate of senescence and also facilitates the appearance of certain types of survivors of critical telomere shortening via mechanisms dependent on Rad52-dependent homologous recombination (HR). Here we describe a third function for Sgs1 in telomerase-deficient cells, inhibition of survivors that grow independent of Rad52. Unlike tlc1 rad52 double mutants, which do not form survivors of telomere dysfunction, tlc1 rad52 sgs1 triple mutants readily generated survivors. After emerging from growth crisis, the triple mutants progressively lost telomeric and subtelomeric sequences, yet grew for more than 1 year. Analysis of cloned chromosome termini and of copy number changes of loci genome-wide using tiling arrays revealed terminal deletions extending up to 57 kb, as well as changes in Ty retrotransposon copy numbers. Amplification of the remaining terminal sequences generated large palindromes at some chromosome termini. Sgs1 helicase activity but not checkpoint function was essential for inhibiting the appearance of the survivors, and the continued absence of Sgs1 was required for the growth of the established survivors. Thus, in addition to facilitating the maintenance of telomere repeat sequences via HR-dependent mechanisms, a RecQ helicase can prevent the adoption of HR-independent mechanisms that stabilize chromosome termini without the use of natural telomere sequences. This provides a novel mechanism by which RecQ helicases may help maintain genome integrity and thus prevent age-related diseases and cancer.

Value of morphotyping for the characterization of Candida albicans clinical isolates

Until recently, morphotyping, a method evaluating fringe and surface characteristics of streak colonies grown on malt agar, has been recommended as a simple and unexpensive typing method for Candida albicans isolates. The discriminatory power and reproducibility of Hunter's modified scheme of Phongpaichit's morphotyping has been evaluated on 28 C. albicans isolates recovered from the oral cavity of asymptomatic human immunodeficiency virus-positive subjects, and compared to two molecular typing methods: randomly amplified polymorphic DNA (RAPD) fingerprinting, and contour clamped homogeneous electric field (CHEF) electrophoretic karyotyping. Morphological features of streak colonies allowed to distinguish 11 different morphotypes while RAPD fingerprinting yielded 25 different patterns and CHEF electrophoresis recognized 9 karyotypes. The discriminatory power calculated with the formula of Hunter and Gaston was 0.780 for morphotyping, 0.984 for RAPD fingerprinting, and 0.630 for karyotyping. Reproducibility was tested using 43 serial isolates from 15 subjects (2 to 6 isolates per subject) and by repeating the test after one year storage of the isolates. While genetic methods generally recognized a single type for all serial isolates from each of the subjects studied, morphotyping detected strain variations in five subjects in the absence of genetic confirmation. Poor reproducibility was demonstrated repeating morphotyping after one year storage of the isolates since differences in at least one character were detected in 92.9% of the strains.

Kinetic properties of DNA migration under clamped homogeneous electric field conditions: DNA size, migration velocities and reorientation time determined in a single clamped homogeneous electric field run

By analyzing DNA migrations per pulse at different pulse times we determined the reorientation times and migration velocities, during and after DNA reorientation of molecules separated under different clamped homogeneous electric field (CHEF) electrophoretic conditions. We obtained functions relating these parameters and DNA migration per pulse with running variables and DNA size. A statistical procedure that validates the predictions of these functions was also proposed using identical data for function fitness and validation. A method is proposed to estimate DNA size, reorientation time and migration velocities using the distance migrated by a DNA molecule in a single CHEF experiment.

Comparison of DNA migrations in two clamped homogeneous electric field chambers of different sizes: Relation between sample thickness and electrophoresis time

We present here a method to compare the mathematical descriptions of DNA migration per pulse as a function of pulse time. It is based on obtaining robust estimates and variances of DNA reorientation time, migration velocities during and after DNA reorientation; and on the statistical comparisons of these estimates. We demonstrated an equal description for the migration per pulse of each DNA molecule separated under identical conditions in clamped homogeneous electric field (CHEF) and miniCHEF chambers. However, miniCHEF resolved the patterns in shorter times, because it uses thinner samples. The relationship between sample thickness and CHEF run time is also presented.

Molecular Typing to Demonstrate Transmission of Gram-Negative Bacteria in the NICU † 1417

Objectives: To study spread of clones of aerobic gram-negative rods (GNR) in the NICU and the influence of antibiotic resistance on transmission. To evaluate the use of contour clamped homogeneous electric field (CHEF) gel electrophoresis in demonstrating spread of GNR in non-epidemic situations. Setting: 45 bed unit with care by two medical teams covering rooms 1&3 (team A) and 2&4 (team B). Ampicillin and gentamicin are used routinely for empiric treatment of sepsis.Methods: A prospective surveillance study. Sequential stool/rectal swab cultures were obtained on admission, weekly, and on discharge from 1/1-3/31/97. GNR were isolated on selective media, tested for gentamicin susceptibility, and typed by CHEF. Staff hand pool cultures were obtained in the last week of the study. Results: 239 infants had 561 cultures during the study period. 317(56%) cultures had GNR and 145(45%) cultures had>1 GNR for a total of 484 isolates; percentage positive increased linearly with age. 417(86%) isolates were gentamicin susceptible (GS) and 67(14%) were gentamicin non-susceptible (GNS). The species identified were:Klebsiella 184(38%), E.coli 151(31%),Enterobacter 74(15%), Citrobacter 46(10%),Pseudomonas 14(3%), Serratia 12(2%), others 3(1%). All 5 hand pools grew ≥1 GNR and 3 isolates were identical to strain types from colonized infants. Data for E. coli are presented.

Fusion between protoplasts of Pichia stipitis and isolated filamentous fungi nuclei

Nuclei were isolated from mycelium of the filamentous fungi Fusarium moniliforme and Trichoderma reesei, and fused with protoplasts of the xylose-fermenting yeast Pichia stipitis using PEG as the fusogenic agent. Hybrids which were morphologically yeastlike and able to hydrolyze xylan showed a similar chromosomal pattern to the parental yeast when clamped homogeneous electric field (CHEF) electrophoresis was done. These hybrids were isolated and their xylanase activity determined quantitatively. Xylanase activity in the parental fungal species was maximum at 96 h growth; it reached 796 nkat ml −1 in the culture medium for F. moniliforme and 898 nkat ml −1 at 120 h growth for T. reesei. Xylanase activity in one of the hybrids reached 350 nkat ml −1 in 9 h. P. stipitis itself showed no xylanase activity.

Human histocompatibility leukocyte antigen (HLA)-G molecules inhibit NKAT3 expressing natural killer cells : Munz, C., Holmes, N., King, A., Loke, Y.W., Colonna, M., Schild, H., Rammensee, H.-G. Department of Immunology, Institute for Cell Biology, Auf der Morgenstelle 15, 72076 Tubingen, Germany [Journal of Experimental Medicine 185/3, 385–391 (1997)

The class I region of the human leukocyte antigen (HLA) complex includes genes encoding the classical transplantation antigens (HLA-A, -B, -C), at least three nonclassical class I genes (HLA-E, -F, and -G), and many class I pseudogenes (including HLA-7.5p). We have used probes from DNA within or flanking the HLA -A, -F, -G, and −7.5p genes to construct a physical linkage map that places the HLA-F, -G, and −7.5p loci in order with respect to HLA-A. The map was constructed using clamped homogeneous electric field pulsed-field gel electrophoresis. DNA was isolated from LCL 721 (A1:B8, A2:B5), a human Epstein-Barr virus-transformed lymphoblastoid cell line (LCL), and from two γ-irradiation-induced mutants of LCL 721 lacking complementary class I haplotypes. The physical linkage data place HLA-G closest to HLA-A and place HLA-7.5p between HLA-G and HLA-F. The map constructed supports a maximum distance of 490 kilobases between HLA-A and HLA-F. Human Immunology 31, 180–185 (1991).

Physical and genetic maps of the human herpesvirus 7 strain SB genome.

Human herpesvirus 7 (HHV-7) is a close relative of human herpesvirus 6A (HHV-6A) and human herpesvirus 6B (HHV-6B) based on limited biologic and genetic data. In this work we describe physical and genetic maps for HHV-7 strain SB [HHV-7(SB)], which was obtained from the saliva of a healthy adult. The HHV-7(SB) genome length is approximately 144 kb by clamped homogeneous electric field gel electrophoresis and approximately 135 kb by summation of restriction endonuclease fragments. We constructed plasmid clones and PCR amplimers that span the HHV-7 genome, except for the genomic termini, and determined the maps of the restriction endonuclease cleavage sites for BamHI, PstI, and SacI. The HHV-7(SB) genome is composed of a single unique region of approximately 122 kb bounded at each end by a 6 kb direct repeat. Homologs to thirty-five herpesvirus genes were identified. The highest similarity was with the HHV-6 genes, with an average amino acid identity of 50%, followed by the human cytomegalovirus counterpart. The genomic and genetic maps indicated that the HHV-7 and HHV-6 genomes are colinear. There was no sequence variation in a segment of the gene encoding the DNA polymerase-associated factor homolog among six HHV-7 isolates, while the corresponding segment of the HHV-6A and HHV-6B counterparts differed by 4.6%. These data support previous observations that the closest genetic relatives of HHV-7 are betaherpesviruses.

Mucin-like glycoprotein genes are closely linked to members of the trans-sialidase super-family at multiple sites in the Trypanosoma cruzi genome

In Trypanosoma cruzi a cell surface enzyme with trans-sialidase (TS) activity has been implicated as an important factor in establishing infection. The enzyme is encoded by genes belonging to a large super-family which on the basis of sequence has been subdivided into 4 groups. TS mediates the transfer of sialic acid residues from host glycoconjugates to acceptor molecules on the parasite surface. To study the organisation of the TS genes we isolated several distinct cosmids from a library constructed with DNA from the T. cruzi X10.6 clone. In these cosmids, the TS genes (group I) were present either as single copies or as a direct tandem repeat. A common feature of the cosmids was the presence of a related group III gene located 10–12kb downstream of the TS gene(s) and arranged in the same orientation. In several of the cosmids we also identified a mucin-like glycoprotein gene located between the group I and group III genes. The mucin-like genes are part of a large polymorphic family and contour clamped homogeneous electric field electrophoresis (CHEFE) analysis showed that they were linked to members of the TS super-family at multiple sites in the X10.6 genome. Screening of a second cosmid library made with DNA from the CL-Brener clone confirmed this multiple linkage suggesting that it is a common feature of the species. This genetic organisation may have important functional significance since the mucin-like glycoproteins are the major cell surface acceptors of sialic acid.

Molecular karyotype of the phytopathogenic fungusSclerotinia sclerotiorum

Molecular techniques have been used to characterize different field isolates of Sclerotinia sclerotiorum, an ubiquitous phytopathogen. Chromosomal DNA resolved by pulsed-field gel electrophoresis (PFGE) revealed that S. sclerotiorum contains at least 16 chromosomes ranging from 1.5 Mb to 4.0 Mb. The size of the haploid genome was estimated to be 43.5 Mb. Six field isolates with different levels of virulence on sunflower germlings or green beans were differentiated by random amplification of polymorphic DNA (RAPD), and analysed by clamped homogeneous electric field electrophoresis. This analysis revealed few chromosome-length polymorphisms among these strains. Chromosomal DNA hybridization indicated that the endopolygalacturonase-encoding pg1 gene is localized on the smallest chromosome of all the strains, whereas the ribosomal DNA mapped to different-sized chromosomes. The less-aggressive strain was characterized by the presence of a supernumary small band, presumably consisting of dsRNA. In contrast to numerous other phytopathogenic fungi, this study reveals a strong karyotypic stability among the strains of S. sclerotiorum which may be preserved by the sexual mode of reproduction of this species

5S rRNA genes in tribe Phaseoleae: array size, number, and dynamics

The organization of 5S rRNA genes in plants belonging to tribe Phaseoleae was investigated by clamped homogeneous electric field gel electrophoresis and Southern blot hybridization. Representatives of subtribe Glycininae included the diploid species Neonotonia wightii and Teramnus labialis, as well as three soybean accessions: an elite Glycine max (L.) Merr. cultivar (BSR101), an unadapted G. max introduction (PI 437.654), and a wild Glycine soja (PI 468.916). A cultivar of Phaseolus vulgaris (kidney bean), a member of subtribe Phaseolinae, was also examined. We determined the number of 5S rDNA arrays and estimated the size and copy number of the repeat unit for each array. The three soybean accessions all have a single 5S locus, with a repeat unit size of ~345 bp and a copy number ranging from about 600 in 'BSR101' to about 4600 in the unadapted soybean introduction. The size of the 5S gene cluster in 'BSR101' is the same in roots, shoots, and trifoliate leaves. Given that the genus Glycine probably has an allotetraploid origin, our data strongly suggest that one of the two progenitor 5S loci has been lost during diploidization of soybean. Neonotonia wightii, the diploid species most closely related to soybean, also has a single locus but has a repeat unit of 520 bp and a copy number of about 1300. The more distantly related species T. labialis and P. vulgaris exhibited a more complex arrangement of 5S rRNA genes, having at least three arrays, each comprising a few hundred copies of a distinct repeat unit. Although each array in P. vulgaris exhibits a high degree of homogeneity with regard to the sequence of the repeat unit, heterogeneity in array size (copy number) was evident when individual plants were compared. A cis-dependent molecular drive process, such as unequal crossing-over, could account for both the homogenization of repeat units within individual arrays and the observed variation in copy number among individuals. Key words : pulsed-field gel electrophoresis, rRNA genes, soybean, tandem arrays.

Fast Procedure To Distinguish Circular and Linear DNA Molecules in Pulsed Field Gel Electrophoresis

Abstract We describe here a fast procedure to identify, by Pulsed Field Gel Electrophoresis (PFGE), circular DNA molecules, displaying shift mobility dependent on ethidium bromide (EtBr) concentration. Several identical DNA samples were coelectrophoresed in a multilane gel with different EtBr concentrations in each lane. Samples were run in a Contour Clamped Homogeneous Electric Field (CHEF) or in a small CHEF (miniCHEF) equipment for 15 or 4 h, respectively. Circular DNA molecules from the protozoan parasite Entamoeba histolytica were detected in a single running. The circles were isolated from the gels and then their structure was confirmed by transmission electron microscopy. By hybridization it was shown that the circles obtained corresponded to the ribosomal DNA episome.

Dose-rate effect on radiation-induced DNA double-strand breaks in the human fibroblast HF19 cell line.

We measured DNA double-strand breaks (dsbs) immediately after exposure of a non-transformed human fibroblast cell line (HF19) to gamma-rays (0-40 Gy) at four dose-rates (10, 1, 0.1, and 0.01 Gy/min) at 37 degree C using clamped homogeneous electric field (CHEF) gel electrophoresis. The shape of the dose-response curves, which could be approximated by a straight line over the range 0-20 Gy for irradiation at 4 degree C, became curvilinear when irradiation was carried out at 37 degree C at 10, 1, 0.1, and 0.01 Gy/min and reached a plateau at 10 Gy after irradiation at 0.01 Gy/min. We present a mathematical analysis that predicts the results of irradiation at 37 degree C from dsb induction and repair data obtained at 4 degree C, followed by incubation for repair at 37 degree C. The model assumes that the rate of dsb rejoining changes continuously with repair time and that it is independent of dose and dose-rate in the range 10-40 Gy. The model also assumes a linear induction of dsb with dose at 4 degree C and dsb induction is independent of dose-rate and of temperature during irradiation. Independent measurements of dsb induction at 4 degree C and of repair rate accurately predict the dsb levels after irradiation at 37 degree C, during which both phenomena occur simultaneously.

Physical mapping and expression of gene families encoding the N-acetyl D-galactosamine adherence lectin of Entamoeba histolytica.

Adherence of the enteric protozoan parasite Entamoeba histolytica is mediated by an N-acetyl D-galactosamine (GalNAc)-specific lectin, a heterodimer of heavy (170 kDa) and light (35/31 kDa) subunits. The gene families encoding the lectin subunits were characterized using clamped homogeneous electric field (CHEF) gel electrophoresis in the strain HM1:IMSS. The heavy subunit was shown to be encoded by a family of five hgl genes, which were physically mapped to five distinct HindIII restriction fragments. The light subunit was shown to be encoded by a family of lgl genes located at six loci in the genome. Heavy and light subunit genes did not appear to be linked. Partial sequences of new members of the hgl and lgl gene families were obtained. Several different strains of E. histolytica were found to contain multiple hgl loci in their genomes. Expression of hgl and lgl genes in HM1:IMSS trophozoites was examined under different growth conditions using the reverse transcription-polymerase chain reaction (RT-PCR). mRNA transcripts were detected from three hgl genes and three lgl genes, with no significant differences between cultured amoebae and amoebae from liver abscesses. The complexity of GalNAc lectin gene expression observed suggests distinct biological functions for the products of the individual genes during pathogenesis.

Primary characterization of a herpesvirus agent associated with Kaposi's sarcomae

Detection of novel DNA sequences in Kaposi's sarcoma (KS) and AIDS-related body cavity-based, non-Hodgkin's lymphomas suggests that these neoplasms are caused by a previously unidentified human herpesvirus. We have characterized this agent using a continuously infected B-lymphocyte cell line derived from an AIDS-related lymphoma and a genomic library made from a KS lesion. In this cell line, the agent has a large episomal genome with an electrophoretic mobility similar to that of 270-kb linear DNA markers during clamped homogeneous electric field gel electrophoresis. A 20.7-kb region of the genome has been completely sequenced, and within this region, 17 partial and complete open reading frames are present; all except one have sequence and positional homology to known gammaherpesvirus genes, including the major capsid protein and thymidine kinase genes. Phylogenetic analyses using both single genes and combined gene sets demonstrated that the agent is a gamma-2 herpesvirus (genus Rhadinovirus) and is the first member of this genus known to infect humans. Evidence for transient viral transmission from infected to uninfected cells is presented, but replication-competent virions have not been identified in infected cell lines. Sera from patients with KS have specific antibodies directed against antigens of infected cell lines, and these antibodies are generally absent in sera from patients with AIDS without KS. These studies define the agent as a new human herpesvirus provisionally assigned the descriptive name KS-associated herpesvirus; its formal designation is likely to be human herpesvirus 8.

Carboplatin- and cisplatin-induced potentiation of moderate-dose radiation cytotoxicity in human lung cancer cell lines.

The interaction between moderate-dose radiation and cisplatin or carboplatin was studied in a cisplatin-sensitive (GLC4) and -resistant (GLC4-CDDP) human small-cell lung cancer cell line. Cellular toxicity was analysed under oxic conditions with the microculture tetrazolium assay. For the platinum and radiation toxicity with the clinically relevant dose ranges applied, this assay was used to obtain information on cell survival after the treatments. Apart from effects on cell survival effects on DNA were also investigated. Configurational DNA changes could be induced by platinum drugs and thereby these drugs might change the frequency of DNA double-strand breaks (dsbs). DNA fragmentation assayed with the clamped homogeneous electric field (CHEF) technique was used as a measure for dsbs in DNA. The radiosensitising effect of the platinum drugs was expressed as enhancement ratio (ER) calculated directly from survival levels of the initial slope of the curve. The highest ER for cisplatin in GLC4 was 1.39 and in GLC4-CDDP 1.38. These were all at 75% cell survival. Carboplatin showed increased enhancement with prolonged incubation up to 1.21 in GLC4 and was equally effective as cisplatin in GLC4-CDDP. According to isobologram analysis, prolonged incubation with both platinum drugs showed at least additivity with radiation for both cell lines at clinically achievable doses. GLC4-CDDP showed cross-resistance to radiation. The radiosensitising capacity of both lung cancer cell lines was not dependent on their platinum sensitivity. The formation of dsbs in DNA directly after radiation was not influenced by pretreatment of either drug in the sensitive or in the resistant cell line. Drug treatment resulted in decreased DNA extractability in control as well as in irradiated cells. Modest enhancement ratio for radiosensitisation by platinum drugs cannot be explained on the level of dsb formation in DNA in both cell lines. Interaction of radiation with the clinically less toxic carboplatin can be improved by prolonged low-dose carboplatin exposure before irradiation and is as potent as cisplatin in the resistant lung cancer cell line. This suggests an advantage in combining radiation and carboplatin in lung cancer patients.

Cloning and assignment of linkage group loci to a karyotypic map of the filamentous fungusAspergillus flavus

The purpose of this study was to develop a karyotypic map of A. flavus and assign parasexually defined linkage groups to chromosomes. Seven A. flavus chromosomal bands, ranging in size from 2.3 to 7.0 megabases were separated by clamped homogeneous electric field gel electrophoresis. Polyethylene glycol-mediated transformation of A. flavus with a wildtype cosmid genomic library was used to complement seven strains having auxotrophic mutations in genes mapped to six of the eight linkage groups. DNA from cosmids complementing each of these seven mapped auxotrophic genes was used to make linkage group-specific probes. Hybridization of these probes to chromosomal separations allowed us to assign six linkage groups (I, II, IV, VI, VII, VIII) to five chromosomal bands. In addition, five previously cloned but unmapped genes were located on this karyotypic map and thus assigned to their corresponding linkage groups, i.e., benA, rDNA, pyrG, aflR and adhl to groups I, II, II, VII, and IV or VIII, respectively. This karyotypic map will provide linkage group mapping of most cloned genes without parasexual analysis.

A Video Technique for the Quantification of DNA in Gels Stained with Ethidium Bromide

We have developed a method for the comparison of quantitative differences between nucleic acid samples run on agarose gels. It is useful for the analysis of digested genomic DNA and RNA preparations, as well as for clamped homogeneous electric field gel analysis of damaged chromosomal DNA. The technique utilizes a standard color charge-coupled device video camera, a microcomputer, and commercially available software. While not as elaborate as other image analysis systems, our method is suitable for many purposes and can be set up for a relatively low cost. In principle, any system capable of recording 24-bit color scans and measuring pixel color intensity could be used.

Apoptosis without decrease of cell DNA content

Apoptosis of human B cells and murine T and B cells was analyzed by DNA agarose gel electrophoresis, clamped homogeneous electric field, measurement of cell DNA content by flow cytometry, transmission electron microscopy and by UV microscopy. Apoptosis was induced by etoposide (an inhibitor of topoisomerase II), by the calcium ionophore ionomycin or by cross-linking of membrane immunoglobulins (Ig) with anti-Ig-antibodies. Two types of apoptosis could be defined. Apoptosis resulting in small DNA fragments (180–200 base pairs and multiples thereof) was associated with a typical ‘ladder’ in agarose gel electrophoresis and a decrease in cell DNA content assessed by flow cytometry. Conversely apoptosis with large DNA fragments (100–150 kilobase pairs) was only demonstrated by clamped homogeneous electric field but was not associated with decreased cell DNA content or the observation of DNA ladders. Nuclear condensation without fragmentation was more frequent when apoptosis generated large DNA fragments. The type of apoptosis appears to be an intrinsic property of each cell type.