Field Inversion Gel Research Articles

Erythromycin-resistant group A streptococci in schoolchildren in Pittsburgh.

Resistance to erythromycin has been very uncommon among group A streptococci in the United States. As part of a longitudinal study, we obtained surveillance throat cultures twice monthly and with each new respiratory tract illness from children in kindergarten through grade 8 at one school in Pittsburgh. Screening for resistance to erythromycin and clindamycin was initially accomplished with use of the Kirby-Bauer disk-diffusion test. The minimal inhibitory concentration of resistant isolates was determined by the E test. A double disk-diffusion test was used to characterize the resistance phenotype, and the polymerase-chain-reaction assay was used to identify the resistance gene. The molecular relatedness of strains was determined by field-inversion gel electrophoresis. A total of 1794 throat cultures were obtained from 100 children between October 2000 and May 2001, of which 318 cultures (18 percent) from 60 of the children were positive for group A streptococci. Forty-eight percent of these isolates (153 of 318) were resistant to erythromycin. None were resistant to clindamycin. Results of the double disk-diffusion test indicated the presence of the M phenotype of erythromycin resistance. Molecular typing indicated that the outbreak was due to a single strain of group A streptococci. Of 100 randomly selected isolates of group A streptococci obtained from the community between April and June 2001, 38 were resistant to erythromycin. In January 2001, during a longitudinal study of schoolchildren, we detected the emergence of erythromycin resistance in pharyngeal isolates of group A streptococci. This clonal outbreak also affected the wider community.

A reappraisal of the role of Zn2+ in TGF-beta1-induced apoptosis in primary hepatocytes.

There is now a wealth of information regarding the apoptotic mode of cell death and its importance in toxicological studies in many mammalian organs including the liver. In this study, we investigated the modulatory effects of the heavy metal Zn2+ on transforming growth factor-beta1 (TGF-beta1)-induced apoptosis in primary rat hepatocytes. Apoptosis induced by TGF-beta1 (1 ng/ml) in hepatocytes was accompanied by nuclear condensation as assessed morphologically by staining with Hoechst 33258 and DNA cleavage as detected biochemically by in situ end-labeling, field inversion and conventional gel electrophoresis. Pretreatment with 100 micromol/L Zn2+ abrogated the nuclear condensation, in situ end-labeling, and DNA laddering in TGF-beta1-treated hepatocytes. Surprisingly, Zn2+ did not inhibit the formation of high-molecular-weight DNA fragments (30-50 kbp to 250-300 kbp). These data provide evidence that Zn2+ exerts its effects on the endonucleases that act downstream in the execution phase of TGF-beta1-induced apoptosis in hepatocytes.

Maintenance of replicative intermediates in ganciclovir-treated human cytomegalovirus-infected retinal glia.

To characterize the molecular structure of the human cytomegalovirus (HCMV) DNA maintained in cultures of human retinal glia following ganciclovir treatment and to determine the biological activity of the DNA. Cultures of human retinal glia were established, infected with HCMV, treated with ganciclovir, and embedded in agarose, and the viral DNA was analyzed by field inversion gel electrophoresis. The HCMV DNA was found to persist in cultures of infected, ganciclovir-treated retinal glial cells in the form of replicative intermediates. After removal of ganciclovir, processed forms of DNA in the 500-to 1000-kilobase range were found as well as 230-kb unit length genome. Infectious virus was recovered after termination of ganciclovir treatment. The data are consistent with the concept that ganciclovir's virostatic nature permits maintenance of HCMV DNA in retinal glia in a biologically active form that is capable of replication after removal of the drug.

Physical linkage of the genes for platelet membrane glycoproteins IIb and IIIa.

The fibrinogen receptor on human platelets is a prototypic member of the integrin family and is composed of subunit glycoproteins IIb (gpIIb) and IIIa (gpIIIa) in a 1:1 stoichiometric ratio. We have isolated cDNA clones for gpIIb and gpIIIa and localized both genes to chromosome 17. In the current study, several approaches were used to localize and map the genes for gpIIb and gpIIIa. A preliminary evaluation of subchromosomal localization was performed by using a panel of mouse-human somatic cell hybrids that contain different amounts of the long arm of human chromosome 17. Southern hybridization to the DNA of these hybrids shows that both genes map near the thymidine kinase gene. In situ hybridization to intact human chromosomes localized both genes to the 17q21-22 region. To better define the physical distance between the two genes, we examined the genomic hybridization pattern of each cDNA probe to high molecular weight restriction fragments separated by pulsed-field gel electrophoresis. Serial hybridizations of the same filter have allowed construction of long-range Mlu I and Sfi I restriction maps spanning more than 500 kilobases. Finally, nonoverlapping portions of the cDNAs for both gpIIb and gpIIIa were used to probe Sfi I digests of genomic DNA separated by field-inversion gels. This confirmed that the genes are physically linked within the same 260-kilobase Sfi I fragment and suggests that the gene for gpIIb is located on the 3' side of the gene for gpIIIa. These results suggest that coordinate expression of gpIIb and gpIIIa may depend on physical proximity.

5S rRNA genes in Pisum: Sequence, long range and chromosomal organization

We have employed a combination of techniques to examine the organization of pea 5S rRNA genes. These include the analysis of length variant interspersion patterns in cosmid clones, sequence analysis, Southern analysis of both conventional gels and field inversion gels and in situ hybridization. From these analyses we conclude that the 5S rRNA genes of pea are arranged in three major tandem arrays which are represented by three large EcoRI fragments and that these correspond to the three sites of in situ hybridization in the haploid pea complement.

Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family.

A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows one to estimate the number of hybridizing genes contained in each large DNA fragment. We have also developed a novel method to increase the separation, resolution and hybridization signal in the second dimension by condensing the bands from the first dimension into spots. As an example, we have applied these techniques to determine the organization of the murine T-cell receptor gamma locus. The murine gamma gene family was found to be contained on two DNA fragments encompassing 195 kilobases of DNA. The two-dimensional gel electrophoresis method is particularly useful in the analysis of the organization of multigenic families where single copy probes are not readily available, and should extend the potential usefulness of field inversion gel electrophoresis in gene mapping.