Clamped Homogeneous Electric Field Gel Research Articles

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.

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.

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.

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.

The upstream sequences of the HSP82 and HSC82 genes of Saccharomyces cerevisiae: Regulatory elements and nucleosome positioning motifs

We present the upstream sequences of HSP82 and HSC82, two closely related, but differentially regulated, heat-shock genes of Saccharomyces cerevisiae. Several dozen potential regulatory elements are identified within each upstream region; interestingly, only a few are conserved between the two genes. These include a consensus heat-shock element, an upstream repressor element, and a consensus TATA element. A search for motifs known actively to position nucleosomes in vitro revealed that such sequences are three- to seven-fold enriched within each promoter; a comparable enrichment is seen near the 3' end of each transcription unit. Located approximately 1100 bp upstream of HSC82 is an open reading frame (ORF) of 255 amino acids; approximately 800 bp upstream of HSP82 is an ORF of 132 amino acids. The latter ORF contains several conserved ankyrin motifs and appears to be expressed under normal growth conditions. Finally, we show by clamped homogeneous electric field gel electrophoresis that the two genetic loci map to different chromosomes: HSP82 to chromosome XVI and HSC82 to chromosome XIII. The sequences have been deposited in the GenBank database under Accession Numbers U20323 and U20349.

Multiple Vibrio vulnificus strains in oysters as demonstrated by clamped homogeneous electric field gel electrophoresis

Clamped homogeneous electric field gel electrophoresis and a computer program for managing electrophoresis banding patterns (ELBAMAP) were used to analyze genomic DNA of 118 Vibrio vulnificus strains, isolated from three oysters by direct plating. Analysis with SfiI resulted in 60 restriction endonuclease digestion profiles (REDP), while analysis with SrfI produced 53 different REDP. Similarities between REDP ranged from 7 to 93%. Principal-component analysis showed that the strains were heterogeneous.

Molecular karyotype and chromosomal localization of genes encoding two major surface glycoproteins, gp63 and gp46/M2, hsp70, and β-tubulin in cloned strains of several Leishmania species

The molecular karyotypes of several Leishmania isolates ( Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania panamensis, Leishmania donovani, Leishmania major, Leishmania aethiopica, Leishmania tropica, Leishmania enriettii) have been analyzed by clamped homogeneous electric field (CHEF) gel electrophoresis. The chromosomal localization of genes encoding 2 major surface glycoproteins, gp63 and gp46/M2, heat shock protein 70 (hsp70), and β-tubulin was determined for cloned isolates of 8 of these Leishmania species. The chromosome size class assignment of hsp70 genes was most conserved in that all species contained a single hybridizing DNA band of approximately 1200 kb. The β-tubulin gene probe hybridized predominantly to large (1600–1750 kb) chromosome-size DNA and to 1–5 additional bands, the number of which depended on the species. The number and size of DNA bands hybridizing to gp63 or gp46/M2 gene probes were not uniformly conserved among species. In contrast to previous reports of gp63 genes being located on a single chromosome, using various CHEF gel conditions we observed a Leishmania major gp63 gene probe hybridizing to at least 2 chromosomal DNA bands in the New World species and in L. tropica. Gp46/M2 genes were located on 1 band in L. donovani, L. major, and L. aethiopica or 2 bands in L. tropica and L. amazonensis, but surprisingly, do not hybridize to any chromosomal DNA of species in the L. braziliensis complex or in L. enriettii. Whenever both genes were present in a species, gp63 and gp46/M2 genes were located on different chromosomal DNA bands.

Study of the effects on DNA of electromagnetic fields using clamped homogeneous electric field gel electrophoresis

A clamped homogeneous electric field (CHEF) elecirophoresis allowing the separation of DNA molecules in the range of 200 to 3000 kb in size was used to study the biological effects of electric and magnetic fields (EMFs). The results obtained did not show any detectable genomic damage on Saccharomyces cerevisiae.