Contour-clamped Homogeneous Electric Field Electrophoresis Research Articles

Use of Molecular Methods in the Clinical Microbiology Laboratory

Molecular DNA methods are essential for evaluating the spread of hospital-acquired microorganisms. These methods are increasingly used in epidemiologic investigation of bacteria, viruses, mycobacteria, and yeast. Evaluation of plasmids and chromosomal DNA is done using restriction enzyme analysis and agarose gel or pulsed-field gradient gel electrophoresis. Contour-clamped homogeneous electric field electrophoresis (CHEF) is the method of choice in most situations. A knowledge of epidemiology is essential for control of spread.

Clonal spread of vancomycin-resistant Enterococcus faecium between patients in three hospitals in two states.

The DNAs of 3* vancomycin-resistant Enterococcus faecium isolates from five hospitals in three states were analyzed by contour-clamped homogeneous electric field electrophoresis and plasmid analysis. There were 22 strain types. One strain type was common to patients in three hospitals in two states. These results suggest the apparent intra- and interhospital spread of vancomycin-resistant E. faecium.

Mobilization of the penicillinase gene in Enterococcus faecalis

Enterococcus faecalis SF4855 is a beta-lactamase-producing isolate resistant to high levels of gentamicin, with determinants for these resistances on the chromosome. SF4855 transferred both determinants into E. faecalis FA2-2 and UV202 at a frequency of 10(-9) in the presence of the MLS plasmid pYN120. beta-Lactamase and gentamicin resistance probes hybridized to three locations on the chromosome of FA2-2 transconjugants on contour-clamped homogeneous electric field electrophoresis. The study results suggest mobilization of the beta-lactamase determinant.

Evidence for binding of extrachromosomal DNA sequences to nuclear matrix proteins in multidrug-resistant KB-V1 cells

Multidrug-resistant KB-V1 cells carry amplified mdrl gene sequences located in an extrachromosomal compartment (on episomes). Since episomes do not contain centromeric or telomeric sequences it is unclear whether they are able to bind to nuclear matrix proteins that may regulate episomal gene expression. Using high salt treatments followed by in situ hybridization and dot blot analyses we found evidence for direct binding of episomal DNA to nuclear matrix proteins. This binding could only be reversed after incubation with trypsin or proteinase K as determined by contour-clamped homogeneous electric field (CHEF) electrophoresis. Our findings are consistent with the concept that circular extrachromosomal DNA may not only reintegrate into nuclear DNA but may also be subject to functional control by regulatory proteins within the nuclear matrix.

Comparison of Pulsed-Field Gel Electrophoresis with Isoenzyme Profiles as a Typing System for Candida tropicalis

Candida species are important nosocomial pathogens, particularly in immunocompromised and critically ill patients. A variety of methods have been used to differentiate strains, but an optimal system has not been established. We compared methods for typing a panel of nine related isolates of Candida tropicalis from an outbreak of sternal wound infections as well as four unrelated control isolates of this species. (The genetic relationships of the nine isolates in the panel had been confirmed previously by restriction fragment analysis.) Typing was undertaken without knowledge of an isolate's origin. Karyotyping by contour-clamped homogeneous electric field (CHEF) gel electrophoresis failed to distinguish between outbreak and control isolates. However, when chromosome-sized DNA was digested with SfiI, EagI, SacII, or NaeI and the fragments were separated by CHEF electrophoresis, the outbreak isolates were readily identified. The isoenzyme profiles of the outbreak isolates were identical and were distinctly different from those of the control isolates. While both isoenzyme profiles and the modified CHEF procedure were discriminatory, the latter is recommended as a relatively convenient and reproducible technique for comparison of types of C. tropicalis.

Outbreak of Pseudomonas aeruginosa Infections in a Surgical Intensive Care Unit: Probable Transmission via Hands of a Health Care Worker

Pseudomonas aeruginosa was isolated from nine patients (16.2 isolations/1,000 patient-days) in a surgical intensive care unit during an outbreak in November 1990; this rate of isolation was three times higher than that noted previously on this unit. Three patients were infected with the same strain, as defined by identical serotypes, pyocin types, and contour-clamped homogeneous electric field (CHEF) electrophoresis patterns of digested genomic DNA. The hands of 80 health care workers were cultured, and a strain of P. aeruginosa identical to that infecting the three patients was isolated from the hands of a nurse providing care to all three. Environmental surfaces, medical devices, and ward stock supplies were cultured; none of these cultures yielded this strain. No clusters of infection with this strain or other strains of P. aeruginosa were observed after compliance with hand-washing and universal precautions was reemphasized. Thus this outbreak was linked to the carriage of P. aeruginosa on the hands of a health care worker. It could not be determined definitively whether this carriage was the source of the cluster or a consequence of it. However, the geographic and temporal clustering of carriage with an outbreak due to a strain of an apparently identical molecular type underlines the importance of routine hand washing between contacts with different patients.

Analysis of the Inversion Effect in Pulsed Field Gel Electrophoresis by a Two-Dimensional Contour-Clamped Homogeneous Electric Field System

A two-dimensional contour-clamped homogeneous electric field electrophoresis was used for the separation of the yeast Saccharomyces cerevisiae chromosomes. The role of different pulse times and field strengths was examined. The resulting complex migration curves were similar to those predicted by the bag model. They are characterized by a straight line in the region of lower molecular weight where the migration is inversely proportional to the molecular weight and a mobility inversion in the region of higher molecular weight both depending on the conditions chosen for the two directions. The quantitative analysis of the data was compared with the predictions of the bag model which is shown to be a good approximation. Using this experimental approach the separation pattern of a commercially available S. cerevisiae standard was examined under standard electrophoresis conditions, and chromosomes IV and XII were found to be inverted.

Molecular characterization of highly gentamicin-resistant Enterococcus faecalis isolates lacking high-level streptomycin resistance.

Antimicrobial susceptibilities and DNA contents were analyzed for six clinical isolates of Enterococcus faecalis that had high-level resistance to gentamicin (MIC > 2,000 micrograms/ml) but not streptomycin and were obtained from patients in diverse geographic areas. Contour-clamped homogeneous electric field electrophoresis of genomic DNA showed all isolates to be different strains. Gentamicin resistance was transferred from four isolates to plasmid-free enterococcal recipients in filter matings. Restriction enzyme analysis of transconjugants showed distinct gentamicin resistance plasmids. A probe specific for the gentamicin resistance determinant hybridized to the plasmids of four isolates and to the chromosomes of two isolates. These findings suggest that clonal dissemination is not responsible for the spread of these resistant strains, that resistance determinants occur on different plasmids as well as on the chromosome of E. faecalis, and that the genetic determinants of resistance are related.

Chromosome Size Polymorphism inLeptosphaeria maculans

Chromosome size polymorphism among nine isolates of Leptosphaeria maculans was studied by hybridization of homologous and heterologous DNA fragments to chromosomes separated by contour-clamped homogeneous electric field (CHEF) electrophoresis. Four of the fungal isolates were highly virulent and three were weakly virulent in Brassica plants; the remaining two originated from the cruciferous weed Thlaspi arvense. There was a high degree of chromosome size polymorphism, and no two isolates had the same karyotype. However, three general patterns could be distinguished by differences in size range, distribution of bands within the size ranges, and hybridization to probes. These three banding patterns corresponded to the three pathogenicity groups []

DNA probe for strain typing of Cryptococcus neoformans.

A 7-kb linear plasmid, harbored by a URA5 transformant, hybridized to all the chromosomes of Cryptococcus neoformans separated by contour-clamped homogeneous electric field electrophoresis. Its linear maintenance was determined to have been facilitated by the presence of telomere-like sequences at its free ends. Hybridization of this plasmid to AccI-digested genomic DNAs of 26 C. neoformans strains generated 21 unique DNA fingerprints. The DNA fingerprints of isolates within the same serotype were more similar to one another than to those from different serotypes. An acapsular clinical isolate, strain 602, widely used in immunological studies and previously thought to be in serotype D, showed DNA fingerprints typical of serotype A isolates. Isogenic strains of C. neoformans exhibited DNA fingerprints that were identical to one another. The DNA fingerprints were stable and reproducible in spite of repeated transfers in the laboratory on either complex (1% yeast extract, 2% Bacto Peptone, 2% glucose) or minimal (yeast nitrogen base) medium. The DNA fingerprints of isolates recovered from primary blood and cerebrospinal fluid cultures of patients for whom AIDS had been diagnosed showed that the original infection in each of these patients contained a homogeneous population of C. neoformans. The DNA fingerprints of isolates recovered from different tissues of infected mice and from patients undergoing different drug therapy regimens were also found to be very stable.

Mitochondrial plasmids do not determine host range in crucifer‐infecting strains of Fusarium oxysporum

The role of mitochondrial plasmids in determining host range in Fusarium oxysporum was tested by using protoplast fusion and fluorescence‐assisted cell sorting to produce hybrid cells between two strains (ATCC 9990 and ATCC 16601) of the fungus which differ in host specialization to cabbage and radish. Three nuclear restriction fragment length polymorphism‐defined loci and mitochondrial chromosomal RFLP markers were identified which distinguish the parental strains. In addition, parents were differentiated by mitochondrial plasmids, one unique to each strain and previously correlated to host specialization. In single‐spored cultures derived from cell fusions (‘fusion cultures’), only parental combinations of the three nuclear genetic markers were detected. However, non‐parental combinations of nuclear and cytoplasmic markers were recovered. One mitochondrial plasmid type was not found in fusion cultures; the other plasmid type was detected in association with both parental mitochondrial chromosome types. Several fusion cultures lacked detectable plasmid sequences of either type. Chromosome‐sized DNAs from parent strains and fusion cultures were separated using contour‐clamped homogeneous electric field (CHEF) electrophoresis. Electrophoretic karyotypes of the parent strains differed greatly, with eight and 11 chromosome‐sized bands detected for ATCC 9990 and ATCC 16601, respectively. Electrophoretic karyotypes of fusion cultures generally were indistinguishable from one or the other parental types. Host specialization was not correlated with mitochondrial plasmid type, but rather with nuclear genotype.

Use of contour-clamped homogeneous electric field (CHEF) electrophoresis to type methicillin-resistant Staphylococcus aureus.

Strains of methicillin-resistant Staphylococcus aureus (MRSA) from Australia and the UK were compared by digesting their chromosomal DNA with the low-frequency-cutting restriction enzyme SmaI and separating the restriction fragment length polymorphisms (RFLPs) by contour-clamped homogeneous electric field (CHEF) electrophoresis. The numbers of restriction fragments produced were in the range 14-17 and the sizes of the bands were 7-700 kb. Generally, the results confirmed previous conclusions based on antimicrobial resistance and plasmid profiles. The earlier MRSA isolates were different from more recent isolates, and the epidemic MRSA from eastern Australia (EA MRSA) was the same as the epidemic MRSA (EMRSA) found in London hospitals. However, contrary to previous results, the EA MRSA did not constitute a homogeneous group. The results showed that comparison of RFLPs by CHEF electrophoresis is a useful technique for studying the epidemiology of MRSA.

Physical map of the chromosome of Neisseria gonorrhoeae FA1090 with locations of genetic markers, including opa and pil genes

A physical map of the chromosome of Neisseria gonorrhoeae FA1090 has been constructed. Digestion of strain FA1090 DNA with NheI, SpeI, BglII, or PacI resulted in a limited number of fragments that were resolved by contour-clamped homogeneous electric field electrophoresis. The estimated genome size was 2,219 kb. To construct the map, probes corresponding to single-copy chromosomal sequences were used in Southern blots of digested DNA separated on pulsed-field gels, to determine how the fragments from different digests overlapped. Some of the probes represented identified gonococcal genes, whereas others were anonymous cloned fragments of strain FA1090 DNA. By using this approach, a macrorestriction map of the strain FA1090 chromosome was assembled, and the locations of various genetic markers on the map were determined. Once the map was completed, the repeated gene families encoding Opa and pilin proteins were mapped. The 11 opa loci of strain FA1090 were distributed over approximately 60% of the chromosome. The pil loci were more clustered and were located in two regions separated by approximately one-fourth of the chromosome.

Treponema pallidum subsp. pallidum has a single, circular chromosome with a size of approximately 900 kilobase pairs

The genome size and chromosome conformation of Treponema pallidum subsp. pallidum, Nichols strain, were determined by contour-clamped homogeneous electric field electrophoresis, a pulsed-field gel electrophoresis technique. Digestion of T. pallidum subsp. pallidum DNA with the restriction endonucleases NotI and SpeI produced 12 and 26 fragments, respectively. Summation of the physical lengths of the fragments produced by NotI and SpeI cleavage yielded average sizes of 900 and 913 kbp, respectively, for the genome of T. pallidum subsp. pallidum. Contour-clamped homogeneous electric field electrophoresis of T. pallidum subsp. pallidum DNA exposed to 4 krads of gamma irradiation resolved a single band of 800 to 1,000 kbp; treatment of the DNA with 16 krads of gamma irradiation resulted in the production of smaller fragments, whereas untreated DNA did not migrate into the gels. The gamma irradiation results indicate that T. pallidum subsp. pallidum has a single, circular chromosome that was linearized at a dosage of 4 krads of gamma irradiation. The size estimate provided by restriction endonuclease digestion with NotI and SpeI shows that the genome of T. pallidum subsp. pallidum, at approximately 900 kbp, is considerably smaller than the 13,700-kbp genome size calculated from renaturation kinetics.

Physical map of the Myxococcus xanthus chromosome

The genome of Myxococcus xanthus, which is 9,454 kbp, is one of the largest bacterial genomes. The organization of the DNA and the distribution of genes encoding social and developmental behaviors were examined by using pulsed field gel electrophoresis. Intact genomic DNA was digested with AseI into 16 restriction fragments, which were separated by contour-clamped homogeneous electric field electrophoresis, purified, and radiolabeled. Each AseI fragment was hybridized to SpeI-digested DNA and to an M. xanthus genomic library contained in yeast artificial chromosomes. Some SpeI restriction fragments and yeast artificial chromosome clones contained AseI sites and hybridized with two different AseI restriction fragments, providing evidence for the juxtaposition of these AseI restriction fragments in the chromosome. The deduced AseI physical map is circular, suggesting that this bacterium contains a single, circular chromosome. Transposable elements shown by transduction to be in or near genes of interest were located on specific AseI restriction fragments by restriction analysis and Southern hybridization. Most AseI restriction fragments contained genes involved in social and developmental behaviors.

SfiI and NotI polymorphisms in Theileria stocks detected by pulsed field gel electrophoresis

DNAs of Theileria parva parva, T. p. lawrencei, T. p. bovis and Theileria mutans stocks, from Kenya, Uganda, Zanzibar and Zimbabwe were digested with either SfiI or NotI and analysed using contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE). The SfiI-digested T. parva genomic DNA resolved into approximately 30 fragments while the NotI digestion produced between 4–7 bands. The summation of the sizes of SfiI fragments gave an estimate of 9–10×10 6 base pairs for the size of the T. parva genome. Heterogeneity within T. p. parva Muguga, Pemba/Mnarani and Mariakani stocks was detected. All the T. parva stocks analysed showed SfiI and NotI restriction fragment length polymorphisms (RFLP). Hybridisation of 5 SfiI-digested T. parva DNAs with a Plasmodium berghei telomeric repeat probe suggested that most of the polymorphisms and heterogeneity occurred in the telomeric or sub-telomeric regions of the genome. The recognition by the Plasmodium telomeric probe of 7–8 strongly hybridising SfiI bands indicates that the T. parva genome may possess at least 4 chromosomes. The T. mutans genome was cut frequently with the above enzymes resulting in large numbers of fragments predominantly below 50 kb, thus suggesting either a much higher G+C content than T. parva or the presence of highly reiterated G+C-rich regions.

Use of yeast artificial chromosome clones for mapping and walking within human chromosome segment 18q21.3.

Well-characterized large genomic clones obtained from yeast artificial chromosome (YAC) libraries provide the framework to localize genes and approach genetic disease. We developed universally applicable approaches to establish authenticity, localize and orient internal genes, map restriction sites, and rescue the distal ends of large human genomic DNA inserts. We selected human chromosome segment 18q21.3 as a model system. Molecular cloning of this segment was initiated by characterizing three plasminogen activator inhibitor type 2 (PAI-2) clones [290, 180, and 60 kilobases (kb)] isolated from a YAC library. Comparison of YAC and bacteriophage lambda genomic DNA clones confirmed the fidelity of the PAI-2 locus. Detailed rare cutting restriction maps were generated by ramped contour-clamped homogeneous electric field electrophoresis. The PAI-2 locus was located and oriented within the YACs, which span a distance 70 kb 5' to 220 kb 3' of PAI-2. Moreover, both left and right ends of the YAC genomic DNA inserts were rescued by amplifying circularized cloning sites with an inverted form of the polymerase chain reaction. These unique terminal genomic DNA fragments were used to rescreen the YAC library and isolate overlapping clones that extend the map. These approaches will enable neighboring loci to be definitively linked and establish the feasibility of using YAC technology to clone and map chromosomal segments.

Cloned Bacillus subtilis chromosomal DNA mediates tetracycline resistance when present in multiple copies

Plasmid pCIS7, containing 11.5 kilobases (kb) of Bacillus subtilis DNA, was isolated from a Tn917 transposon insertion in tetracycline-sensitive B. subtilis KS162. When integrated into the chromosome of B. subtilis 168, this plasmid conferred tetracycline resistance upon reiteration of the plasmid DNA sequences in the chromosome. Deletions and subclones of pCIS7 were constructed and introduced into an Escherichia coli in vitro transcription-translation system. A 72-kilodalton protein was localized to a 3.1-kb PstI-EcoRI fragment of the plasmid. Amplification of the 3.1-kb PstI-EcoRI fragment was required for expression of tetracycline resistance in B. subtilis 168. By hybridization to previously characterized clones, the 11.5-kb fragment was localized to the origin region of the chromosome. Through contour-clamped homogeneous electric field electrophoresis, this cluster of clones was shown to reside on a 200-kb NotI fragment bridging SfiI fragments of 150 and 250 kb and was oriented with respect to the purA and guaA loci, developing an accurate physical map of the region surrounding the origin of replication.

Electrophoretic analysis of Histoplasma capsulatum chromosomal DNA.

Seven chromosome-sized DNA molecules in the Downs strain of Histoplasma capsulatum were resolved by using chromosome-specific DNA probes in blot hybridizations of contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE) agarose gels. The sizes of the chromosomal DNA bands extended from that of the largest Saccharomyces cerevisiae chromosome to beyond that of the Schizosaccharomyces pombe chromosomes. Under our experimental conditions, the order of the five largest DNA bands was inverted in the FIGE gel relative to the CHEF gel, demonstrating a characteristic of FIGE whereby large DNA molecules may have greater rather than lesser mobility with increasing size. Comparison of the Downs strain with other H. capsulatum strains by CHEF and FIGE analysis revealed considerable variability in band mobility. The resolution of seven chromosome-sized DNA molecules in the Downs strain provides a minimum estimate of the chromosome number.

APPLICATION OF PULSED FIELD CHROMOSOME ELECTROPHORESIS IN THE STUDY OF CHROMOSOMEXIIIAND THE ELECTROPHORETIC KARYOTYPE OF INDUSTRIAL STRAINS OFSACCHAROMYCESYEASTS

Pulsed field chromosome electrophoresis is a powerful new technique in yeast genetics which permits the resolution of intact yeast chromosomes in an agarose gel matrix. We utilized contour-clamped homogeneous electric field electrophoresis (CHEF) to survey representative strains of Saccharomyces yeasts from the brewing, baking, distilling, sake and wine industries for their electrophoretic karyotypes. All of the strains tested were found to have a unique chromosomal profile, indicating the potential of this technology for “fingerprinting” prototrophic strains of Saccharomyces yeasts. By employing an ILV2 gene probe specific for chromosome XIII, we determined that all of the industrial strains of Saccharomyces yeasts possessed a chromosome XIII which migrated in an identical fashion to chromosome XIII from a reference haploid strain of Saccharomyces cerevisiae. While one lager yeast strain, Saccharomyces carlsbergensis M244, was found to contain two alleles of ILV2 when digested genomic DNA was probed with ILV2, the presence of a novel independently migrating chromosome XIII could not be detected. A homeologous chromosome XIII in this yeast will therefore have to be determined by genetic analysis. Pulsed field chromosome electrophoresis is concluded to be a technology with immediate application to Quality Control and Research and Development programs in industries using Saccharomyces yeasts.