Analysis of Paramecium Macronuclear DNA Using Pulsed Field Gel Electrophoresis

.We have analyzed the macronuclear DNA of Paramecium tetraurelia using orthogonal-field-altemation gel electrophoresis. The mean size of the linear macronuclear DNA molecules is approximately 450 kb. Less than 6% of the macronuclear DNA is larger than 800 kb. Using pulse times of 20, 40, 60 and 90 s we show that the macronuclear fragment containing the A type variable surface antigen gene migrates reproducibly as a 320-kb linear DNA. Over the same pulse times we describe the unusual migration of the ribosomal RNA gene (rDNA) of P. tetraurelia. At pulse times of 20 and 40 s the rDNA migrates at limit mobility (300 and 500 kb, respectively) whereas with 60- and 90-s pulse times, 2 components of rDNA are observed; 1 fraction independent of pulse time migrating at limit mobility, and a 2nd component migrating between 100-kb and 400-kb linear markers. Based upon previous electron micrographic studies of Paramecium rDNA as well as data presented here we conclude that the majority of Paramecium rDNA molecules are a circular DNA form.     

Plasmid migration using orthogonal-field-alternation gel electrophoresis.

The migration properties of a series of supercoiled plasmids ranging in size from 4 to 16 kilobases (kb) have been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE). These circular DNAs enter the gel and are well resolved. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a wide range of pulse times, from 10 to 120 seconds, as well as over a broad range of total running times, from 6 to 24 hours. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of pBR322 shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.     

High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 4. Influence of DNA topology

Pulsed-field gel electrophoresis is a powerful technique for the fractionation of linear DNA molecules with sizes above 50 kilobase pairs (kb). Here it is demonstrated that this technique is also effective for separating smaller DNAs including linear, circular, and supercoiled species. The mobilities of linear DNAs larger than 8 kb can be modulated by pulse times between 0.1 and 100 s. The mobility of supercoiled DNA molecules up to 16 kb is generally unaffected by these pulse times except that 10-s pulse times cause a small but distinct increase in the mobility. The general insensitivity of small supercoiled DNAs to pulse time presumably occurs because these species reorient so rapidly that they spend most of their time undergoing conventional electrophoresis. However, the mobilities of larger supercoiled DNAs are affected by pulse times of less than 1 s, and at 0.1 s the molecules are better resolved by pulsed electrophoresis than by ordinary electrophoresis. The mobility of 3-19 kb nicked and relaxed circular DNA molecules is also affected by pulse time but in a complex way.     

Moving pictures of DNA released upon lysis from bacteria,chloroplasts, and mitochondria

Summary Cells and organelles suspended in gelled agarose agarose were lysed with detergent and protease, stained with ethidium bromide and their DNA was observed by fluorescence microscopy. The migration of individual DNA molecules during electrophoresis on a microscope slide was recorded on video tape so that moving pictures could be analyzed. The DNA from lysed bacteria (Escherichia coli andAgrobacterium tumefaciens) appeared as a rosette of at least twenty loops of varying size, whereas that from bacterial spheroplasts (E. coli andPseudomonas aeruginosa) appeared as circular forms or rods with many fine filaments of RNA extending toward the anode. The DNA from chloroplasts of watermelon (Citrullus vulgaris) and pea (Pisum sativum) did not appear as a rosette of loops. Many or most of the chloroplast DNA molecules per lysed chloroplast were immobile in the electric field, as if in circular form hooked on agarose fibers. The amount of DNA-fluorescence per watermelon mitochondrial particle was much less than that found for either chloroplasts or bacteria. The appearance of the mitochondrial DNA during electrophoresis was that of linear molecules, no obviously circular forms were evident and no rosette structures were observed.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole - kb kilobase pairs - mtDNA mitochondrial DNA - PFGE pulsed-field gel electrophoresis     

MITOCHONDRIAL GENOME CONFORMATION AMONG CW‐GROUP CHLOROPHYCEAN ALGAE1

Most green algal taxa have circular‐mapping mitochondrial genomes, whereas some have linear genome‐ or subgenomic‐sized mitochondrial DNAs (mtDNA). It is not clear, however, if the circular‐mapping genomes represent genome‐sized circular molecules, if such circular molecules and the linear forms are the predominant in vivo mtDNA structures, or if the linear forms arose only once or multiple times among extant green algal lineages. We therefore examined the DNA components detected with homologous mtDNA probes after pulsed‐field gel electrophoresis of total cellular DNA from the chlorophycean basal bodies displaced clockwise(CW)‐group taxa Chlamydomonas reinhardtii and Chlamydomonas moewusii. For C. reinhardtii, the 15.8‐kb linear mtDNA was the only DNA component detected, and there was no evidence of circular or large linear precursors of this DNA. In the case of C. moewusii, which is known to have a circular‐mapping 22.9‐kb mitochondrial genome, three DNA components were detected; these appeared to be circular (relaxed and supercoiled) and genome‐sized linear DNA molecules, the latter of which likely resulted from random double‐strand breaks in the circular forms during DNA isolation. In further studies, DNA from additional CW‐group taxa was examined using conventional gel electrophoresis and DNA‐filter blot analysis with C. reinhardtii and C. moewusii mtDNA probes. We conclude that all taxa from the “Volvox clade” (sensu Nakayama et al. 1996 of the CW‐group have genome‐ or subgenomic‐sized linear mtDNAs as their predominant mtDNA form and that these arose from a genome‐sized circular form in an ancestor that existed near the base of this clade.     

Some physicochemical properties of rice mitochondrial DNA

Summary Certain physicochemical properties of rice mitochondrial DNA (mtDNA) were determined. Certain low-molecular-weight mtDNA bands were found in addition to the major mtDNA band. Rice mtDNA appeared in the electron microscope as a collection of linear molecules with heterogeneous length in the range of 1–156 kb. The major distribution area was 60–105 kb. A small fraction (less than 5%) of rice mtDNA was found in the form of a circular molecule. Some molecules had the appearance of being supercoiled. Replication fork structures were found in both circular and linear mtDNA molecules. In one rice species, Jin Nante, 15 different circular molecules were found. Rice mtDNA was digested with different restriction enzymes. The total molecular weight of rice mtDNA was calculated to be about 300 kb according to the data of restriction enzyme digestion and electron microscopy.     

Genetic stability and DNA rearrangements associated with a 2?×?1.1-Kb perfect palindrome in Escherichia coli

We show that circular plasmids containing perfect palindromic regions of 2 × 1.1 kb can be propagated in sbcC strains of Escherichia coli, a result that is at variance with the well known observation that λ DNA cannot tolerate palindromic regions larger than 2 × 265 bp. However, a significant fraction of these palindrome-containing plasmids can be recovered from E. coli strains either as linear molecules with hairpins at their ends or as head-to-head dimers, both in a RuvC-and RusA-independent manner. Our results suggests that large palindromes may form cruciforms in E. coli. However, palindrome-associated DNA rearrangements occur by a process that does not require any known cruciform resolvase activity. Our data support a replication-dependent model for the induction of DNA rearrangements by perfect palindromes. Received: 11 May 1998 / Accepted: 24 June 1998     

ABUNDANT PLASMID-LIKE DNA IN VARIOUS MEMBERS OF THE ORDERS SIPHONOCLADALES AND CLADOPHORALES (CHLOROPHYTA)1

In all seven genera belonging to the order Siphonocladales and in two genera of the Cladophorales (Chlorophyta) that were examined, extracts of total DNA contained abundant low molecular weight (LMW) molecules. This DNA typically ranged in size from 1.5 to 3.0 kb among the different genera studied. The complexity of the populations of molecules found in different genera also varied with regard to sizes and numbers of bands evident in electrophoresis gels. Our data suggest that the LMW DNA is ubiquitous among these algae since it occurred in each of our six isolates of Ernodesmis verticillata (Kützing) Børgesen collected in both the Caribbean and the Gulf of California. The LMW DNA from Ernodesmis and Ventricaria ventricosa (J. Agardh) Olsen et J. West was denser than high molecular weight (HMW) DNA in CsCl/bisbenzimide gradients. Ultrastructurally, the LMW DNA molecules were linear, averaging 0.65 μm (Ernodesmis) and 1.0 μm (Ventricaria) in length. Approximately 2%–5% of the former possessed an apparent loop or lariat at one end, as visualized in the electron microscope after rotary shadowing. The LMW DNA molecules appeared to be predominantly double-stranded DNA based upon staining with acridine orange. The 2.2-kb DNA from Ernodesmis has features typical of plasmids in that it rapidly reannealed after heat denaturation, and it was selectively enriched in an alkaline-lysis extraction procedure. In denaturing gels, this DNA migrated at >4.0 kb, indicating that the molecules may actually be single-stranded DNA species with intramolecular base pairing, each containing a long inverted repeat folded back on itself in a hairpin conformation in the native state. Digestion with mung bean nuclease revealed that additional single-stranded regions may occur in many of the LMW molecules, in addition to the hairpin loop. Although the endonuclease DNase I rapidly digested the LMW DNA, neither exonuclease III nor lambda exonuclease digested it. This suggests that the ends of these DNA molecules are protected. Collectively, the data indicate that members of the Siphonocladales and Cladophorales contain linear plasmid-like DNA molecules that appear to have a novel combination of features.     

Low molecular weight DNA in the heteromeric macronuclei of two cyrtophorid ciliates

Summary— The size range of the native DNA molecules in the heteromeric macronuclei of two cyrtophorid ciliates (Trithigmostoma cucullulus, Chilodonella uncinata) was mainly investigated by using agarose gel electrophoresis. Numerous bands superimposed on a continuous spectrum of molecular sizes between about 0.35 kb and 30 kb were resolved by conventional electrophoresis. Species-specific banding patterns indicate a variation between species in the copy number of individual DNA fragments. A slight intra-specific variability of banding patterns can exist. Electrophoretic distributions for two strains of T cucullulus were indeed found to differ by at least one more intense band (‘overamplified’ sequences?). Fractionation by contour-clamped homogeneous electric field (CHEF) gel electrophoresis revealed that the size continum of macronuclear DNA molecules does not extend beyond 60–70 kb. The average size was estimated to be around 4 kb. Unresolved DNA fraction (> 1000 kb) accounted for less than 10% of the mass of cellular DNA entering CHEF gels. Macronuclear ribosomal DNA of each cyrtophorid species was identified by Southern hybridization with a Tetrahymena rDNA probe. The hybridization signal was observed on a single band of low molecular weight DNA. The corresponding size was close to 14.5 kb in Trithigmostoma and 15.5 kb in Chilodonella, which is about twice the size of monomeric rDNA in hypotrichous ciliates. We showed that S1 nuclease resistant duplexes wit half the length of the native rDNA can be formed by rapid renaturation of heat-denatured molecules and hybridized with native rDNA. This strongly suggests that the nucleotide sequence of this rDNA is a large palindrome. Unlike the hypotrichs, macronuclear rDNA in cyrtophorids should be organized into palindromic dimers as in Tetrahymena species.     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.     

共生菌Hebeloma sp.的线形质粒DNA的分子克隆及测序的研究

共生担子菌滑菇 H ebeloma circinas携带两个线形的染色体外 DNA分子 .全部的菌丝 DNA经蛋白酶 K处理后 ,通过琼脂凝胶电泳观察到 :在染色体 DNA旁有两个大小不等的 DNA带 ,命名为 p HCl和 p HC2 ,其分子量分别为 1 0 .3kb和 9.1 kb.用核酸外切酶处理 p HC DNA,确认其 5′端被保护 .对 p HC2用不同的内切酶处理并确定其内切酶图谱 ,对其 3.2 kb H ind 片段进行克隆 ,亚克隆和测序 .结果表明 :其片段有两个开读框 ( open reading frames) ,携带类似于病毒 B型的DNA和 RNA多聚酶基因编码 .p HC2为该菌携带的一个典型的线形质粒 ,这也是首次在共生担子菌中发现的线形质粒 .     

Genes coding for respiratory complexes map on all three chromosomes of the Paracoccus denitrificans genome

The genome of Paracoccus denitrificans (strain Pd1222) consists of three distinct DNA molecules when separated by standard pulsed-field gel electrophoresis with apparent molecular sizes of approximately 2, 1.1, and 0.64 Mb. When the separated chromosomes are digested by restriction enzymes and sizes of resulting fragments are summed up, the three chromosomes are composed of 1.83, 1.16, and 0.67 Mb. Since their migration behavior relative to size standards is largely independent of electrophoresis conditions, at least the two smaller chromosomes most likely represent linear molecules. The size analysis presented here allows an unequivocal distinction between groups of different strains of P. denitrificans and of Thiosphaera pantotropha, confirming an earlier cytochrome c analysis. When the genome was analyzed with different probes coding for respiratory enzymes, essential genes were found spread over all three chromosomes without any obvious clustering on any of the three forms. Received: 10 August 1997 / Accepted: 10 November 1997     

Plasmid recombination intermediates generated in a Saccharomyces cerevisiae cell-free recombination system.

We have developed an assay utilizing Saccharomyces cerevisiae cell extracts to catalyze recombination in vitro between homologous plasmids containing different mutant alleles of the tet gene. Electrophoretic analysis of product DNA indicated that a number of novel DNA species were formed during the reaction. These species migrated through agarose gels as distinct bands with decreased electrophoretic mobility compared with the substrate DNA. The DNA from each individual band was purified and shown to be enriched 5- to 100-fold for tetracycline-resistant recombinants by using a transformation assay. The structure of the DNA molecules present in these bands was determined by electron microscopy. Recombination between circular substrates appeared to involve the formation and processing of figure-eight molecules, while recombination between circular and linear substrates involved the formation of molecules in which a circular monomer had a monomer-length linear tail attached at a region of homology.     

Extrachromosomal circular DNAs in Drosophila melanogaster: Comparison between embryos and Kc0% cells

We established the size distribution of extrachromosomal covalently closed circular DNA molecules from embryos of various Drosophila melanogaster strains and from Kc0% tissue culture cells. In embryos, more than 80% of the circular DNA molecules are smaller than 2.5 kb and all the distributions show a peak of molecules of between 200 and 400 bp. The Kc0% cell distribution differs mainly from that of embryos in that 48% of the molecules have a size between 4 and 8 kb. Correlating with this, circular molecules homologous to copia, 412 and 297 were detected only in Kc0% cells. The three tandemly repeated families containing the 5S genes, the histone genes and the 240 bp repeat of the ribosomal DNA intergenic spacer, which had previously been identified in circular DNAs from embryos, were also found in cultured cells. A fourth tandemly repeated family corresponding to the 1.688 g/cm³ satellite DNA was detected, both in embryos and Kc0% cells. It consists of circular multimeric molecules containing multiple copies of the 359 bp repeated unit. No circular DNA molecules homologous to the actin genes, the type I ribosomal DNA insertion, or the F and I transposable elements were found in embryos or Kc0% cells. Thus it appears that the extrachromosomal circular DNA molecules from embryos and from tissue culture cells differ mainly in the presence of circular copies of the copia-like transposable elements.     

Circular mitochondrial DNA molecules from petite mutants of Saccharomyces cerevisiae: resolution by polyacrylamide gel electrophoresis

Mitochondrial DNA (mtDNA) from petite strain K45 ofSaccharomyces cerevisiae contains about 7% circular DNA molecules which comprise a simple oligomeric series based on a monomeric size of 1.7 kilobase pairs. Electrophoresis of K45 mtDNA on a polyacrylamide-agarose slab gel fractionates the mtDNA into a major band (containing linear DNA) and several faster running minor bands each containing particular size class of circular DNA molecules. From study of mtDNA from K45 and two other simple petites it was found that the mobility of circles is inversely proportional to the logarithm of the circle size. Polyacrylamide gel electrophoresis thus permits the separation of circular mtDNA from the linear mtDNA of simple petites, and physically resolves circles of different size from one another.     

The microbiology of butyrate formation in the human colon

A physical map of the Enterococcus faecium ATCC19434 chromosome was constructed by NotI, I-CeuI and Sse8387I. The chromosome was a circular DNA of 2600 kb in size, and contained six rRNA operons (rrn). The locations and orientations of the six rrn operons and 24 different determinants were mapped. Genomes of three additional E. faecium strains were also analyzed by I-CeuI digestion, and the genome sizes were found to vary from 2550 to 2995 kb. We further investigated the genome sizes and number of rrn operons in four E. faecalis, one E. avium, and one E. durans strains. The genome sizes were larger than E. faecium: 3000-3250 kb in E. faecalis, 3445 kb in E. avium, and 3070 kb in E. durans. E. avium and E. durans contained six rrn operons as in E. faecium, but all the E. faecalis strains possessed four rrn operons.     

DNA analysis of temperate bacteriophage Aa/23 isolated from Actinobacillus actinomycetemcomitans

The DNA of the temperate bacteriophage Aaφ23 isolated from the oral bacterium Actinobacillus actinomycetemcomitans was examined structurally both in the phage head and in the prophage. The DNA in phage particles comprises 44 kb linear molecules with a terminal redundancy of 1.6 kb, which represent circular permutations. Thus, DNA is packaged into phage heads by the headful mechanism. The Aaφ23 prophage is integrated into the host chromosome. Received: 15 September 1997 / Accepted: 10 December 1997     

Diversity of Vicia faba circular mtDNA in whole plants and suspension cultures

Summary A comparative analysis of the Vicia faba mitochondrial genome in whole plants and in longterm suspension culture has been conducted. Restriction fragment patterns of the mtDNA isolated from these two sources were notably different. Electronmicroscopic analysis also revealed significant differences. Large circular mtDNA patterns shifted from a 37–80 kb subpopulation, which was predominant in whole plants, to 18–34 kb subpopulations although in both classes notable quantities of circular molecules of 80 to 120 kb and more were also found. Both in whole plant and suspension culture cells very large circular DNAs were observed. Some of them had lengths nearly 290 kb and could be considered as evidence of the existence of master chromosomes. The minicircular DNA population was also altered. In the suspension culture we observed a notable increase of percentage of minicircles with sizes near 1 kb. Simultaneously, the percentage of minicircles with sizes near 3.5–10 kb significantly increased in suspension culture cells. In addition, a new peak (10–12 kb) of minicircles appeared. Copy number alterations for some sequences homologous to CCC1A, CCC1B and CCC2 (Negruk et al. 1982, 1985) were shown. Southern hybridization revealed the existence of a family of minicircles having sizes 1.4–2 kb with predominance of CCC1A, CCC1B and CCC2. The copy numbers of CCC1B and some minor minicircles was changed in the suspension culture when compared with the whole plants.     

Size and Structure of Replicating Mitochondrial DNA in Cultured Tobacco Cells

The BY-2 tobacco cell line was used to study the size and structure of replicating mitochondrial DNA (mtDNA). Approximately 70 to 90% of the newly synthesized mtDNA did not migrate during pulsed-field gel electrophoresis. Moving pictures of the fluorescently labeled molecules showed that most of the immobile well-bound DNA was in structures larger than the size of the BY-2 mitochondrial genome of ~270 kb. Most of the structures appeared as complex forms with multiple DNA fibers. The sizes of the circular molecules that were also observed ranged continuously from ~20 to 560 kb without prominent size classes. Pulse-chase and mung bean nuclease experiments showed that the well-bound DNA contained single-stranded regions and was converted to linear molecules of between 50 and 150 kb. MtDNA replication in plants may be initiated by recombination events that create branched structures of multigenomic concatemers that are then processed to 50- to 150-kb subgenomic fragments.