Determination of genome sizes of Rickettsia spp. within the spotted fever group, using pulsed-field gel electrophoresis.

The chromosome lengths of six spotted fever group Rickettsia species (Rickettsia rickettsii, R. conorii, R. rhipicephali, R. sibirica, R. australis, and R. akari) were estimated by pulsed-field gel electrophoresis. The genome size of R. rickettsii was about 2,100 kb, but the chromosome lengths of the five other species were, surprisingly, much lower and ranged between 1,200 and 1,300 kb.     

Analysis of rice mitochondrial genome organization using pulsed-field gel electrophoresis

Most of the plant mitochondrial (mt) genomes that have been mapped are believed to be organized as master circle molecules from which sub-genomic molecules arise through homologous recombination. We have evidence to suggest that a major part of the rice mt genome is organized as independent, sub-genomic molecules or mt chromosomes, one of which has already been mapped. This study is aimed at the identification of the other molecular entities that comprise the genome. Pulsed-field gel electrophoresis of the native rice mt DNA and Southern analysis with different mt gene probes have shown that in addition to the 117 kb mt chromosome, at least four more such molecules of sizes 130 kb, 95 kb, 70 kb and 56 kb account for most of the rice mt genome. A majority of the rice mt genes that encode products involved in oxidative phosphorylation are distributed among these five chromosomes. Partial restriction map of the 95 kborf 25/cox 3 chromosome, indicating the sites for the enzymesBglII andHindIII has also been determined.     

Human ureaplasmas show diverse genome sizes by pulsed-field electrophoresis.

Contour clamped homogeneous field (CHEF) agarose gel electrophoresis (AGE), ramped to give linear separation of DNA molecules of 600-1600 kilobase pairs (kbp), was used to determine mobilities for full-sized genomic DNA of the serotype standard strains of the human genital mollicutes, Ureaplasma urealyticum relative to yeast chromosomal DNA markers. Indicated genome sizes (in kbp) were 760 for the four biotype 1 strains and 840-1140 for eleven biotype 2 strains. Other estimates were: 720 for Mycoplasma hominis, 1070 for Mycoplasma hyopneumoniae, 890 for Mycoplasma flocculare, 1180 and 1350 for Mycoplasma mycoides subsp. mycoides Y and GC1176-2, respectively, and 1650 and 1580 for Acholeplasma laidlawii B and PG 8, respectively. These data supplement previous evidence from CHEF AGE that the genomes of the Mycoplasmataceae are diverse in size with some larger than previously estimated from DNA renaturation kinetics.     

Virtual digest identification of secondary enzymes for use in pulsed-field gel electrophoresis of Staphylococcus aureus

Pulsed-field gel electrophoresis (PFGE) is currently the gold standard for methicillin-resistant Staphylococcus aureus (MRSA) typing but only one enzyme, SmaI, is currently used for restriction digest. We report the use of virtual digestion to identify enzymes for S. aureus PFGE. Two enzymes (EagI and SacII) were identified and successfully used to characterize two sets of S. aureus isolates, 12 USA300, and 14 additional MRSA isolates comprised of seven SmaI patterns. Phylogenetic analysis of patterns generated by all enzymes determined that the USA300 MRSAs are identical. In contrast, digestion with EagI or SacII resolved one to two band differences among three MRSA pattern sets that were not detected using SmaI. These results demonstrate that a second enzyme may detect differences in S. aureus isolates not detected by single enzyme digestion. However, because isolates differing by one to two bands are considered identical, such discrimination may not be clinically or epidemiologically relevant.     

Determination of Wolbachia genome size by pulsed-field gel electrophoresis

Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular.     

Sizing of the Leptospira genome by pulsed-field agarose gel electrophoresis

Pulsed-field gel electrophoresis allowed the determination of the size of the genome of Leptospira, a bacterium of the spirochete family. The three restriction enzymes, NotI (5'GC/GGCCGC), NheI (5'G/CTAGC), ApaI (5'-GGGCC/C) generated DNA fragments of suitable size. The results are compatible with a size of 5000 kb for the chromosome of both the pathogenic and the saprophytic species of Leptospira.     

Sizing of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis.

The four restriction enzymes ApaI (5'-GGGCCC), EagI (5'-CGGCCG), NaeI (5'-GCCGGC), and SmaI (5'-CCCGGG) were found to produce distributions of DNA fragment sizes useful for mapping of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis. ApaI produced 21 fragments (range, 1.6 to 305 kilobases [kb]), EagI yielded 30 fragments (0.6 to 339 kb), NaeI produced 32 fragments (2.3 to 290 kb), and SmaI yielded 16 fragments (6.0 to 377 kb). Summation of the fragment lengths in each digest yielded estimates for the size of the H. influenzae chromosome ranging from 1,834 kb.     

Sizing and mapping of the genome of Campylobacter coli strain UA417R using pulsed-field gel electrophoresis.

Agarose-immobilized chromosomal DNA from the nalidixic-acid-resistant Campylobacter coli strain UA417 and its streptomycin-resistant (StrR) derivative, UA417R, were digested with the restriction enzymes SalI (GTCGAC) and SmaI (CCCGGG). The sizes of the resulting fragments were determined using pulsed-field gel electrophoresis. The two genomes showed similar restriction patterns of seven and 13 fragments for the two respective enzymes and the total genome size was determined to be approx. 1.7 Mb. Analysis of partial digestion fragments, as well as Southern-blot hybridization, were used to construct a physical map of the C. coli UA417R genome. Natural transformation studies using DNA fragments extracted from UA417R, as well as the erythromycin-resistant (EryR) C. coli strain UA585, were used to locate the StrR and EryR resistance markers on the genomic map.     

Determination of microbial genome sizes by two-dimensional denaturing gradient gel electrophoresis.

In two-dimensional denaturing gradient gel electrophoresis, DNA is digested with a restriction endonuclease and the resulting DNA fragments are separated as a function of size by conventional agarose gel electrophoresis. Following this first dimension electrophoresis, the fragment distribution is placed at the top of a denaturing gradient slab gel and electrophoresis is carried out parallel to the gradient direction. This second dimension separation is a complex function of the base sequence of each fragment. Analysis of the DNA fragment distribution as a function of fragment size allows the DNA size to be calculated. This method has been applied to calculate three microbial genome sizes: Mycoplasma capricolum, 724 kb; Acholeplasma laidlawii, 1646 kb; and Hemophilus influenzae, 1833 kb.     

New approaches in genome analysis by pulsed-field gel electrophoresis: application to the analysis of Pseudomonas species

A general method for the evaluation of macrorestriction fragment patterns is presented and its applicability to the taxonomy of bacteria is demonstrated for 32 Pseudomonas species. Strains were differentiated at the species and subspecies level by genome size and macrorestriction fragment fingerprints of the chromosome that had been separated on pulsed-field gels. The relatedness of bacteria was ascertained from the similarity of AsnI, DraI, SpeI, SspI or XbaI fragment patterns. In general, the dendrograms calculated from the genome fingerprints corresponded with the phylogenetic classification obtained from phenotypic marker or nucleic acid hybridization analysis, but several exceptions were noted. The techniques and algorithms presented herein are generally applicable to the genome analysis of bacteria, lower eukaryotes, and DNA fragments cloned in yeast artificial chromosomes.     

Analysis of the genome of the gram-negative moderate halophiles Halomonas and Chromohalobacter by using pulsed-field gel electrophoresis

The genomes of 11 moderately halophilic bacteria belonging to the genera Halomonas and Chromohalobacter have been analyzed by restriction endonuclease digestion and pulsed-field gel electrophoresis (PFGE). By using the infrequently cutting restriction endonucleases SpeI and SwaI, highly characteristic fingerprintings were obtained for each of the isolates studied. On the basis of the lengths of the SpeI and SwaI fragments, separated by PFGE, the genome size of the 11 strains studied was estimated. The genome size for 8 Halomonas strains ranged from 1450 to 2830 kb, whereas for the 3 Chromohalobacter strains studied it ranged from 1770 to 2295 kb. Finally, we show that macrorestriction fingerprints could be a useful tool to elucidate the taxonomic position of bacteria belonging to the Halomonas–Deleya complex. Received: October 13, 1997 / Accepted: May 12, 1998     

Analysis of rice (Oryza sativa L.) genome using pulsed-field gel electrophoresis and rare-cutting restriction endonucleases

TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.     

Estimation of circular DNA size using gamma-irradiation and pulsed-field gel electrophoresis

A method is described for estimating the size of large circular DNAs found within complex chromosomal DNA preparations. DNAs are treated with low levels of gamma-irradiation, sufficient to introduce a single double-stranded break per circle, and the resulting linear DNA is sized by pulsed-field electrophoresis and blot hybridization. The method is fast, reproducible, and very conveniently applied to the agarose-enclosed chromosomal DNA preparations commonly used in pulsed field electrophoresis.     

Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macro-restriction fragments

AIMS: To examine whether pulsed-field gel electrophoresis (PFGE) of DNA macro-restriction fragments could provide better discrimination among the different biotypes previously described within the species Staphylococcus aureus than the traditional biochemical approach. METHODS AND RESULTS: Seventy three Staph. aureus strains from various sources (human, animal or food origin) and belonging to eight biotypes, including the poultry-like biotype, tentatively designated as an 'abattoir' biotype, were genotyped by PFGE after SmaI digestion of DNA. The PFGE patterns were compared using the average linkage matching method (UPGMA) with the Dice coefficient. A total of 61 PFGE patterns were observed, showing between 31 and 100% similarity. In most cases, strains with the same biotype were grouped specifically into one, two or three separate sub-clusters. Strains from the 'abattoir' biotype were clustered in one separate sub-cluster. CONCLUSIONS: The PFGE typing is useful to distinguish the traditional biotypes of Staph. aureus and has a more discriminatory power than the biochemical typing. SIGNIFICANCE AND IMPACT OF THE STUDY: The PFGE typing confirms the 'abattoir' biotype as a separate group on a genetic level and is well suited to investigate modes of staphylococcal contamination of food.     

Restriction analysis of the Streptomyces glaucescens genome by agarose gel electrophoresis

A method for the analysis of total DNA of Streptomyces glaucescens is described. The relevant steps are (a) extraction and purification of DNA, (b) restriction of DNA samples with type II restriction enzymes, (c) one dimensional separation of restriction fragments by agarose gel electrophoresis. A typical banding pattern was obtained for each wild type strain, independant of growth conditions or age of the culture. Mutant strains exhibited in most cases the same banding pattern as the parent wild type strain. Only in one specific mutant class a fragment of about 9 megadalton was missing.     

Fine analysis of the Pneumocystis carinii f. sp. carinii genome by two-dimensional pulsed-field gel electrophoresis

Pneumocystis carinii is a general designation for a group of unusual unicellular fungal parasites responsible of pneumopathy in animal hosts. Divided into several subgroups termed the 'special forms', P. carinii is prone to an extensive karyotype variation. In previous studies, the nuclear genome of these organisms has been considered to be haploid and a set of 16 chromosomes has been assigned to P. carinii f. sp. carinii, a special form known to infect rats. We report the analysis of the genome of an isolate representative of the karyotype 1 of this special form, using two-dimensional pulsed-field gel electrophoresis procedures. The 'karyotype and restriction display' (KARD) fingerprints indicated the presence of 17 different chromosomes. The haploid genome size was estimated to be 8.4 Mbp. Some homologous chromosomes were distinguished on the basis of a single restriction fragment length polymorphism, which raises the possibility of a diploid nucleus. A restriction map of the chromosome 15, characterized by two homologues with a size difference of 7 kb, was constructed. Hybridization data indicated that insertion/deletion events may have occurred within subtelomeric regions which carry genes encoding the major surface glycoprotein (MSG) of Pneumocystis.     

Differentiation between human and ovine isolates of Bordetellaparapertussis using pulsed-field gel electrophoresis

Abstract The genetic relatedness of 18 human and 29 ovine isolates of Bordetella parapertussis was examined by macrorestriction digestion of DNA with the rarely cutting enzyme Xba I and resolution by pulsed-field gel electrophoresis. There was clear separation of human and ovine isolates and variation within host types. The human isolates were separated into three types as were the 24 Scottish ovine isolates. Species-specific bands were observed with the human isolates at 114, 134, 166, 213, 346 and 372 kb. No species-specific bands were found in the B. parapertussis ovine isolates. Isolates of B. parapertussis recovered from sheep in New Zealand gave a further two DNA banding patterns which were clearly different from the Scottish ovine and the human isolates. These results indicate that human and ovine isolates of B. parapertussis are genetically distinct and that variation exists within isolates from the same host species. Pulsed-field gel electrbphoresis therefore appears to be a powerful discriminatory tool for the classification of B. parapertussis .     

Differentiation of Erwinia amylovora strains by pulsed-field gel electrophoresis.

Erwinia amylovora strains, isolated from several host plants in various geographic regions during different years, were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the DNA from lysed, agar-embedded cells with rare-cutting restriction enzymes. The banding patterns obtained with enzyme XbaI digests revealed significant differences among strains from different areas. North American strains E9 and Ea-Rb, a Rubus strain, were highly divergent from other E. amylovora strains. French strains were different from central European and English strains. E. amylovora strains from central Europe and New Zealand had identical PFGE patters, as had strains from Egypt, Greece, and Turkey. PFGE of genomic DNA from American and English strains gave rise to dissimilar patterns. Patterns of some American strains resembled those from strains isolated in other parts of the world. The restriction fragment length polymorphisms observed by PFGE analysis can be used to group strains and may give hints about the course of distribution of the plant disease. From the sizes of the restriction fragments obtained, a molecular mass of approximately 4.5 Mb was calculated for the genome of E. amylovora.     

Development of a modified DNA extraction method for pulsed-field gel electrophoresis analysis of Staphylococcus aureus and enterococci without using lysostaphin

A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to clinical isolates of Staphylococcus aureus and enterococci to reduce the cost of using lysostaphin. This protocol reduces the expenses of PFGE typing of S. aureus and enterococci as it removes the use of lysostaphin during the spheroplast formation from these bacteria.