Field Inversion Gel Electrophoresis Research Articles

Molecular epidemiological analysis of quinolone-resistant Escherichia coli causing bacteremia in neutropenic patients with leukemia in Korea.

We analyzed an outbreak of Escherichia coli bacteremia in eight patients with leukemia in a hematology-oncology unit from July to September 1994. The antibiograms and genotypic patterns of the isolates were different, thus suggesting that the outbreak did not originate from a single clone. However, all the isolates were resistant to quinolones, which led us to examine the microbiological records from 1992 to 1994. The incidence of quinolone-resistant E. coli bacteremia in the hematology-oncology unit ranged from 81.8% to 94.6% during this period. We then analyzed 36 more isolates recovered from late 1994 to 1995. Field inversion gel electrophoresis patterns of these isolates were also different. Analysis of the quinolone resistance determining region in gyrA revealed that all the isolates had a double mutation in gyrA. In conclusion, quinolone-resistant E. coli could be an emerging threat to neutropenic patients with leukemia who receive a quinolone prophylactically, and attention must be paid to this trend of resistance.

Direct sputum analysis of Pseudomonas aeruginosa macrorestriction fragment genotypes in patients with cystic fibrosis.

The distribution of bacterial populations in the airways of 13 patients with cystic fibrosis who were colonized for 6-23 years with Pseudomonas aeruginosa was investigated by genotyping of bacterial chromosomes directly isolated from 21 sputa. After removal of host material from sputum by hypotonic cell lysis and repetitive washing and centrifugation steps, agarose-embedded bacterial cells were lysed, residual eukaryotic DNA separated by field inversion gel electrophoresis, and the purified bacterial chromosomes subjected to macrorestriction fragment pattern and Southern analyses. Bacterial populations consisted of a single P. aeruginosa clone in 17 sputa, of which more than one clonal variant was apparent in two SpeI fragment fingerprints. Two clones of P. aeruginosa and another species co-existed in four samples. Genomically homogeneous populations of P. aeruginosa are characteristic for chronically colonized lungs in most cases of cystic fibrosis.

The Multiple cpb Cysteine Proteinase Genes ofLeishmania mexicana Encode Isoenzymes That Differ in Their Stage Regulation and Substrate Preferences

The cpb genes of Leishmania mexicana encode stage-regulated, cathepsin L-like cysteine proteinases that are leishmanial virulence factors. Field inversion gel electrophoresis and genomic mapping indicate that there are 19 cpb genes arranged in a tandem array. Five genes from the array have been sequenced and their expression analyzed. The first two genes, cpb1 and cpb2, differ significantly from the remaining 17 copies (cpb3-cpb19) in that: 1) they are expressed predominantly in metacyclic promastigotes (the form in the insect vector which is infective to mammalian macrophages) rather than amastigotes (the form that parasitizes mammals); 2) they encode enzymes with a truncation in the COOH-terminal extension, an unusual feature of these cysteine proteinases of trypanosomatids. Transfection of cpb1 into a cpb null mutant resulted in expression of an active enzyme that was shown by immunogold labeling with anti-CPB antibodies to be targeted to large lysosomes. This demonstrates that the 100-amino acid COOH-terminal extension is not essential for the activation or activity of the enzyme or for its correct intracellular trafficking. Transfection into the cpb null mutant of different copies of cpb and analysis of the phenotype of the lines showed that individual isoenzymes differ in their substrate preferences and ability to restore the loss of virulence associated with the null mutant. Comparison of the predicted amino acid sequences of the isoenzymes implicates five residues located in the mature domain (Asn18, Asp60, Asn61, Ser64, and Tyr84) with differences in the activities of the encoded isoenzymes. The results suggest that the individual isoenzymes have distinct roles in the parasite's interaction with its host. This complexity reflects the adaptation of cathepsin L-like cysteine proteinases to diverse functions in parasitic protozoa.

Evidence for rolling circle replication of Autographa californica M nucleopolyhedrovirus genomic DNA.

Autographa californica M nucleopolyhedrovirus (AcMNPV) is a large ds DNA virus restricted to larval lepidopteran insect hosts. Using field inversion gel electrophoresis and digestion with a restriction enzyme which cuts the AcMNPV genome once, we detected multiple unit-length genome fragments from replicating viral DNA. Our data suggest that AcMNPV replicates in a head-to-tail manner via rolling circle replication.

Glucocorticoid/oxysterol-induced DNA lysis in human leukemic cells.

Both glucocorticoids and oxysterols, steroids with quite different known transduction pathways, cause the death of lymphoid cells. Dual TUNEL/propidium iodide assays on sensitive human leukemic CEM-C7 clones treated with either steroid were clearly positive by 48 h, consistent with apoptosis. Both steroids evoked two distinctive types of DNA lysis: cleavage into large fragments of several different sizes and the classic "ladders", multiples of approximately 200 base pairs. Conventional gel electrophoresis showed that a small proportion of total DNA had undergone laddering 36-48 h after treatment with glucocorticoid or 24 h after oxysterol exposure. On field inversion gel electrophoresis of cellular DNA both steroids caused an increase in an array of large DNA fragments <50 kb in size. A 50 kb fragment appeared 36 h after treatment with either steroid, but only oxysterol treatment caused a significant increase in a 300 kb fragment. Oxysterol treatment did not result in DNA fragmentation in the resistant M10R5 subclone, which retained sensitivity to glucocorticoids. We conclude that glucocorticoids and oxysterols kill these cells with similar, but not identical, patterns of DNA lysis which occur just before or concomitant with the onset of cell death.

Multi-organ characterization of mitochondrial genomic rearrangements in ad libitum and caloric restricted mice show striking somatic mitochondrial DNA rearrangements with age.

Mitochondrial DNA (mtDNA) rearrangements have been shown to accumulate with age in the post-mitotic tissues of a variety of animals and have been hypothesized to result in the age-related decline of mitochondrial bioenergetics leading to tissue and organ failure. Caloric restriction in rodents has been shown to extend life span supporting an association between bioenergetics and senescence. In the present study, we use full length mtDNA amplification by long-extension polymerase chain reaction (LX-PCR) to demonstrate that mice accumulate a wide variety of mtDNA rearrangements with age in post mitotic tissues. Similarly, using an alternative PCR strategy, we have found that 2-4 kb minicircles containing the origin of heavy-strand replication accumulate with age in heart but not brain. Analysis of mtDNA structure and conformation by Southern blots of unrestricted DNA resolved by field inversion gel electrophoresis have revealed that the brain mtDNAs of young animals contain the traditional linear, nicked, and supercoiled mtDNAs while old animals accumulate substantial levels of a slower migrating species we designate age-specific mtDNAs. In old caloric restricted animals, a wide variety of rearranged mtDNAs can be detected by LX-PCR in post mitotic tissues, but Southern blots of unrestricted DNA reveals a marked reduction in the levels of the age- specific mtDNA species. These observations confirm that mtDNA mutations accumulate with age in mice and suggest that caloric restriction impedes this progress.

Concatemeric Intermediates of Equine Herpesvirus Type 1 DNA Replication Contain Frequent Inversions of Adjacent Long Segments of the Viral Genome

In common with other α-herpesviruses, the genome of equine herpesvirus type-1 (EHV-1) comprises covalently linked long and short unique sequences of DNA, each flanked by inverted repeats. Equimolar amounts of two genomic isomers, generated by free inversion of the short segment, relative to the long segment, are packaged into EHV-1 virions. In contrast with herpes simplex virus (HSV), inversion of genomic long segments has not been described. In the current work, the structures of high molecular weight intermediates of EHV-1 DNA replication were studied by field inversion gel electrophoresis. It is shown that adjacent long segments of the viral genome are frequently inverted in concatemeric intermediates of EHV-1 DNA replication. Further, like HSV concatemers, high molecular weight intermediates of EHV-1 replication are flanked exclusively by the long segment of the viral genome. Hence, despite the fact that only two, rather than four, isomers of EHV-1 DNA are packaged into virions, the intermediates of EHV-1 DNA replication closely resemble those of herpes simplex virus type 1 in structure. These data have implications relating to the mechanisms involved in packaging of α-herpesvirus DNA.

Concerted evolution of the tandemly repeated genes encoding human U2 snRNA (the RNU2 locus) involves rapid intrachromosomal homogenization and rare interchromosomal gene conversion.

We have surveyed the tandemly repeated genes encoding U2 snRNA in a diverse panel of humans. We found only two polymorphisms within the U2 repeat unit: a SacI polymorphism (alleles SacI+ or SacI-) and a CT microsatellite polymorphism (alleles CT+ or CT-). Surprisingly, individual U2 tandem arrays are entirely SacI+ or SacI-, and entirely CT+ or CT-, although the SacI and CT alleles can occur in any combination. We also found that polymorphisms in the left and right junction regions flanking the tandem array fall into only two haplotypes (JL+ and JL-, JR+ and JR-). Most surprisingly, JL+ is always associated with JR+, and JL- with JR-. Thus individual U2 arrays do not exchange flanking markers, despite independent assortment and subsequent homogenization of the SacI and CT alleles within the U2 repeat units. We propose that the primary driving force for concerted evolution of the tandem U2 genes is intrachromosomal homogenization; interchromosomal genetic exchanges are much rarer, and reciprocal nonsister chromatid exchange apparently does not occur. Thus concerted evolution of the U2 tandem array occurs in situ along a chromosome lineage, and linkage disequilibrium between sequences flanking the U2 array may persist for long periods of time.

Downregulation of Bcr-Abl in K562 cells restores susceptibility to apoptosis: Characterization of the apoptotic death

We examined the susceptibility of a variety of human leukemic cell lines to the induction of apoptosis. K562, a chronic myelogenous leukemic cell line which expresses the bcr-abl fusion gene, was found to be extremely resistant to apoptosis, irrespective of the inducing agent. This resistance can be attributed to the deregulated Abl kinase activity of bcr-abl, as downregulation of its expression using antisense oligodeoxynucleotides targeted to the beginning of the abl sequence in this chimeric gene rendered these cells susceptible to cytotoxic drug-induced apoptosis. Examination of the morphological and biochemical features of apoptosis in K562 cells revealed the typical membrane blebbing and chromatin condensation associated with this form of cell death. In situ TdT-mediated end labeling of the DNA revealed the presence of strand breaks in the treated cells and field inversion gel electrophoresis revealed the presence of large 10-50 kb fragments. However there was an absence of oligonucleosomal DNA fragmentation, whether or not Bcr-Abl was expressed. Thus, while inhibition of expression of Bcr-Abl renders K562 cells susceptible to apoptosis, the absence of oligonucleosomal DNA fragmentation in these cells is independent of the function of this molecule.

COMPARISON OF CONSTANT VOLTAGE AND FIELD INVERSION GEL ELECTROPHORESIS METHODS FOR MEASURING DNA STRAND BREAKAGE IN FRESHWATER BIVALVES

To compare constant voltage and field inversion gel electrophoresis (FIGE) methods for detecting DNA strand breakage, two sets of DNA samples from freshwater bivalves were electrophoresed on both gel types. The first sample group contained DNA from mapleleaf mussels (Quadrula quadrula) exposed to lead; the second sample group contained DNA from Asiatic clams (Corbicula fluminea) that was passed through a syringe to physically induce strand breakage. A series of comparisons revealed that FIGE separated DNA into a greater number of bands, had less DNA in the band with the highest relative quantity of DNA, and resulted in a higher calculated average DNA strand length. The periodic inversion of the electric field on FIGE gels resulted in more complete separation of DNA fragments of different sizes, making FIGE the better of the two methods. Thorough separation of DNA fragments is a very important factor in the accurate calculation of average DNA strand lengths. Correctly determined DNA strand lengths are inte...

Structural organization of the Streptococcus pneumoniae chromosome and relatedness of penicillin-sensitive and -resistant strains in type 9V.

Fragmentation of Streptococcus pneumoniae genomic DNA with low-frequency-cleavage restriction endonucleases and separation of the fragments by field-inversion gel electrophoresis (FIGE) provides a DNA-fingerprint of a strain. This method enables us to construct a physical and genetic map of the R6 laboratory strain what will be presented. The origin of replication containing several Dna boxes was located in the dnaA region. It was of interest to compare the profiles of subclones. Two clones of strain R36A (R6 and C13) were cultivated separately for more than 15,000 generations in two laboratories. FIGE profiles differed by only one band. Another R36A descendant, isolated in 1958 by Ravin, strain Rx was of interest since it was deficient in Dpn restriction enzymes and methylases and in the hex B function. Its origin was questionable; its profile is identical to others R6 descendants, demonstrating that Rx is derived from R36A. FIGE analysis was carried out on several penicillin-resistant strains of type 9V because penicillin-resistance in this type increased recently. The profiles of a collection of a number of these resistant isolates were very similar, showing that they result from a clone. The profiles of penicillin sensitive isolates of the same type are very similar to the resistant isolates. This suggests that the 9V type has spread recently from a clone, and the resistance genes have mutated and were selected when penicillin was extensively used.

AMPA Neurotoxicity in Cultured Cerebellar Granule Neurons: Mode of Cell Death

Various forms of cell death induced by the glutamate receptor agonist, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), were analyzed by determining the capacity of cultured cerebellar granule cells to metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into formazan, by measuring the leakage of lactate dehydrogenase (LDH), by using confocal microscopy to visualize propidium iodide staining of apoptotic nuclei, and by using field inversion gel electrophoresis (FIGE) for the detection of AMPA-produced cleavage of DNA into high molecular-weight fragments (50 kbp). All these measures indicated that stimulation of AMPA receptors may be involved in the neurotoxic effects of glutamate, and that AMPA-induced neurotoxicity in cerebellar granule cells display morphologically distinct features of both necrotic and apoptotic modes of cell death. In agreement with previous observations, a blockade of AMPA receptor desensitization was necessary to unmask AMPA-induced functional responses in cultured cerebellar granule neurons in vitro. Microfluorimetric measurements of free cytoplasmic calcium concentrations ([Ca2+]i) in single cerebellar neurons revealed that AMPA neurotoxicity was accompanied by a pronounced elevation of [Ca2+]i. Our current results add further evidence to the notion that glutamate-induced neurotoxicity in cerebellar granule cells is mediated not only through NMDA receptors but also through a direct activation of AMPA receptor-regulated cation channels.

Molecular typing of Streptococcus pneumoniae strains isolated in Romania.

The high incidence of pneumococcal infections and the increasing proportion of Streptococcus pneumoniae strains resistant to penicillin and other antibiotics are the major reason for epidemiological surveillance. A number of phenotypic and genotypic methods are used for this purpose. The aim of this study was to compare four of the genotyping methods that have been proposed for epidemiological surveillance with capsule serotyping, which is the method for epidemiological investigation currently used in clinical laboratories in Romania. The four genotyping methods evaluated were: polymerase chain reaction ribotyping (PCR ribotyping)3 in conjunction with restriction analysis of PCR ribotyping products; ribotyping1; pulsed-field gel electrophoresis (PFGE), specifically, field inversion gel electrophoresis (FIGE)4, 5, and BOX fingerprinting2.

Direct excision of 50 kb pair DNA fragments from megabase-sized fragments produced during apoptotic cleavage of genomic DNA

The DNA of U937 cells exposed to two different apoptotic stimuli, namely the cocktail H 2O 2/3-aminobenzamide (3AB) and etoposide, was analyzed using field inversion gel electrophoresis (FIGE) as well as programmable, autonomously controlled electrode electrophoresis (PACE). The results obtained indicate that FIGE is not appropriate for sizing apoptotic DNA fragments. PACE appears to be more accurate and reliable and the results obtained with this technique strongly suggest that the 50 kb DNA fragments are directly excised from Mb-sized DNA fragments without the intermediate cleavage of 200–300 kb products.

Detection of early and late stage apoptosis with field inversion gel electrophoresis.

Detection of early and late stage apoptosis with field inversion gel electrophoresis.

Subtyping Listeria monocytogenes isolates genetically related to the Swiss epidemic clone.

Macrorestriction analysis by pulsed-field gel electrophoresis was used to assess the diversity of strains within the epidemic-associated electrophoretic type 1 (ET1) clone of Listeria monocytogenes. For this purpose, a total of 144 isolates from Switzerland shown by multilocus enzyme electrophoresis to belong to the ET1 were examined. These isolates were subtyped by macrorestriction analysis using the enzymes ApaI and SmaI and field inversion gel electrophoresis. Among these 144 isolates, 45 were isolated in human listeriosis cases of the postepidemic period of 1988 to 1993 and 44 were isolated in animal listeriosis cases of the same period. Forty-seven isolates were from the epidemic period of 1983 to 1987, and eight additional isolates were from cattle from two different farms. Twenty-nine different subtypes could be identified among the 144 isolates tested. Five major subtypes were found more frequently than the others during the postepidemic period, both in humans and in animals. Two of these subtypes had been previously implicated in outbreaks of listeriosis, thus suggesting that particular pulsed-field gel electrophoresis subtypes may be frequently associated with disease in humans and animals. Two of these frequent subtypes were also suspected to be related to small clusters of listeriosis cases during the postepidemic period. The results obtained by typing epidemiologically related isolates from different animals within the same farms and from different body sites of a given patient confirmed the potential of macrorestriction analysis for epidemiological studies restricted to short periods of time and to small number of isolates. The analysis of 47 isolates related to the Swiss listeriosis epidemic period of 1983 to 1987 and the use of Southern blotting and hybridization experiments show that the interpretation of relatedness between isolates presenting slightly different macrorestriction patterns may be more complex than commonly accepted. In such cases, careful interpretation of the potential molecular mechanisms leading to the differences observed between patterns is necessary.

Isolation and characterization of mitochondrial nucleoids from the yeast Pichia jadinii.

Mitochondrial (mt) nucleoids were isolated with a high degree of purity from the yeast Pichia jadinii, in which the mitochondrial DNA (mtDNA) is linear. Field-inversion gel electrophoresis (FIGE) revealed that significant amounts of mtDNA could be isolated intact, as linear molecules of 41 kbp, from the isolated mt-nucleoids. Fifteen different proteins were detected in the mt-nucleoid fraction and, eight of these proteins bound to DNA. The patterns of mt-nucleoid proteins and of the DNA-binding proteins after gel electrophoresis in the presence of SDS were somewhat different from those of such proteins from Saccharomyces cerevisiae. The corresponding proteins isolated from the mt-nucleoids of four other species of yeast in the genera Pichia and Williopsis also differed from one another in terms of electrophoretic mobility in the presence of SDS. In immunoblotting experiments, antibodies that had been raised against the 67-kDa protein of mt-nucleoids from S.cerevisiae and the YMN-1 monoclonal antibody that is specific for a 48-kDa protein in the mt-nucleoids from S. cerevisiae did not recognize any proteins in the mt-nucleoids from Pichia jadinii and four other species of yeast. The results suggest the considerable diversity of the proteins in the mt-nucleoids of yeasts.

Organization of the Mouse Cardiac Natriuretic Peptide Locus Encoding BNP and ANP

The genes encoding the mouse atrial natriuretic peptide and B-type natriuretic peptide were previously shown to be physically linked on mouse chromosome 4 (Steinhelper ME, 1993, Structure, expression, and genomic mapping of the mouse natriuretic peptide type-B gene. Circ Res 72: 984–992). In the present study the spatial relationship and orientation of the mouse atrial natriuretic peptide and B-type natriuretic peptide transcription units were identified and a physical map of the mouse cardiac natriuretic peptide locus was obtained. To this end, genomic clones encoding atrial natriuretic peptide and B-type natriuretic peptide were isolated from a mouse genomic library in bacteriophage P1. Three independent clones encoding atrial natriuretic peptide were isolated and two of these also encode B-type natriuretic peptide. Both transcripts were shown to arise from the same DNA strand, with B-type natriuretic peptide encoded approximately 15 kb 5′-of atrial natriuretic peptide based on field inversion gel electrophoresis of fragments amplified with specific oligonucleotides. This finding was confirmed by isolation of subclones comprising the entire locus and by blot hybridization analysis of mouse genomic DNA. The results show that the genes encoding the two natriuretic peptides expressed predominantly in mammalian cardiac myocytes are organized in tandem on mouse chromosome 4. This information provides a physical framework for investigating mechanisms that regulate transcription of the cardiac natriuretic peptide locus.

Field inversion gel electrophoresis on Pseudomonas cepacia strains isolated from cystic fibrosis patients.

Genome fingerprinting by field inversion gel electrophoresis (FIGE) was utilized to typify 129 isolates of Pseudomonas cepacia (Pc) from 59 patients with cystic fibrosis (CF) and from environmental cultures in the CF ward. The aim of this study was to assess whether a segregation policy avoided colonization of CF patients by nosocomial strains and contamination of the environment by colonized individuals, whether or not an 'epidemic strain' was present in the ward and whether cross-colonization occurred in CF individuals subjected to prolonged close contact. The Pc strains of each patient remained unchanged over time; 78% of the genome finger printings (GFP) were individual, whereas the others gave rise to 9 GFP groups. A spirometer was probably contaminated by a newly colonized patient. Adequate sanitary measures and avoidance of excessive promiscuity are helpful for limiting but are unable to eliminate Pc transmission in the CF ward. Direct or indirect transmission, however seems, more frequent in CF patients in contacts outside the hospital.

A cleavage-site-directed inhibitor of interleukin-1β-converting enzyme-like proteases inhibits apoptosis in primary cultures of rat hepatocytes

Apoptosis induced in primary hepatocytes by transforming growth factor beta1 and staurosporine produced chromatin condensation, DNA cleavage is detected by in situ end-labelling, field inversion and conventional gel electrophoresis, and cell detachment. These effects were abolished by benzyloxycarbonyl-valinylalanylaspartylfluoromethyl ketone, a cleavage-site-directed inhibitor of interleukin-1beta-converting enzyme-like proteases, and this finding suggests that these enzymes are involved in liver apoptosis.