[Various tests for detection of extrachromosomal elements of Staphylococci].
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Acridine Orange↗
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The termini of extrachromosomal linear copia elements.
Extrachromosomal linear copia elements were isolated and cloned from cultured Drosophila melanogaster cells. Four full length copia recombinants were characterised. Each contains a full sized copy of copia bounded by its direct repeats. The sequences of the ends of the copia inserts were determined. All of the termini are identical to those of genomic copias except for a single base deletion from one end of one clone. These results suggest that the priming for initiation of copia reverse transcription differs from normal retroviral models. Southern blot analysis of uncloned full length copia circles shows that approximately 50% correspond to the predicted circularisation product of such linears. We propose that this class of circles and the linear DNAs described here are precursors to integrated genomic copia elements.
Animals↗
Characterisation of an extrachromosomal DNA element from Theileria annulata.
Extrachromosomal nucleic acid elements are found in all organisms, commonly as organelle, viral or plasmid genomes. In this paper we describe the initial characterisation of a novel 6.5-kb linear, double-stranded extrachromosomal element from Theileria annulata, and a 2.6-kb RNA species. The DNA element is present in different stages of the life cycle and in different stocks of the parasite. Northern blots of total RNA isolated from different stages of the parasite, probed with the purified element, detect three major transcripts, of 1.45, 1.05 and 0.24 kb, present in all life-cycle stages examined. The possible origin and function of this element is discussed, together with its possible use as a transfection vector for the introduction of genes into protozoan cells.
Animals↗
Growth and Survival of Genetically Manipulated Lactobacillus plantarum in Silage.
The growth and persistence of two genetically manipulated forms of Lactobacillus plantarum NCDO (National Collection of Dairy Organisms) 1193 have been monitored in grass silage. Both recombinants contained pSA3, a shuttle vector for gram-positive organisms that encodes erythromycin resistance. In one of the recombinants, pSA3 was integrated onto the chromosome, whereas in the other, a pSA3 derivative designated pM25, which contains a Clostridium thermocellum cellulase gene cloned into pSA3, was maintained as an extrachromosomal element. This extrachromosomal element is a plasmid. Rifampin-resistant mutants were selected for the recombinants and the parent strain. When applied to minisilos at a rate of 10 CFU/g of grass, both the recombinants and the parent strain proliferated to dominate the epiphytic microflora and induced an increase in the decline in pH compared with that of the noninoculated silos. The presence of extra genetic material did not appear to disadvantage the bacterium in comparison with the parent strain. The selective recovery of both strains by using rifampin and erythromycin was confirmed by Southern hybridization. Interestingly, the free plasmid (pM25) appeared more stable in silage than was expected from studies in MRS broth. The plasmid was retained by 85% of the rifampin-resistant L. plantarum colonies isolated from a day 30 silo. These data answer an important question by showing that genetically manipulated recombinants of L. plantarum can proliferate and compete with epiphytic lactic acid bacteria in silage.
Journal Article↗
[Electron microscopic research on the extrachromosomal genetic elements of Escherichia coli].
The structural organization of extrachromosomal genetic elements were studied in a subfraction obtained after centrifugation of the lysate of E. coli spheroplasts. With this method of isolation, the tertiary structure of the extrachromosomal genetic elements was preserved. The majority of DNA macromolecules were released in the form of single and connected rosettes. Typical rosettes composed of radial loops of DNA clustered around the central dense core (the diameter is about 60 nm). The mean length of the rosette loops was 1.06 +/- 0.4 micron. Both relaxed folded and supercoiled folded forms of DNA were observed on the preparation. Sometimes the rosettes were connected with large aggregates of DNA (possibly the material of bacterial chromosomes) and had the appearance of thick fibers with numerous lateral loops. Linear, cyclic and various replicative forms of DNA have also been observed. It is assumed that rosettes of the extrachromosomal elements of E. coli reflect one of the levels of organization of prokaryotic genetic material.
DNA, Bacterial↗
The origin of extrachromosomal circular copia elements.
Cloned extrachromosomal circular copia elements were studied by nucleotide sequence and restriction enzyme analysis to determine their mechanisms of formation and their possible roles in copia transposition. Rearranged circular copias containing inverted segments flanked by 5 bp sequence duplication were observed, suggesting that circular copias are capable of integrating into their own sequences. Such copia circles are analogous to similarly rearranged retrovirus circles, strengthening the relationship between the copia-like elements and retroviruses. There is marked sequence heterogeneity at the junction between the fused terminal direct repeats of seven copia circles. The junctions contain 0-15 bp inserts, the sequences of which are inconsistent with the creation of these particular molecules by reverse transcription.
Animals↗
Stable propagation of 'selfish' genetic elements.
Extrachromosomal or chromosomally integrated genetic elements are common among prokaryotic and eukaryotic cells. These elements exhibit a variety of 'selfish' strategies to ensure their replication and propagation during the growth of their host cells. To establish long-term persistence, they have to moderate the degree of selfishness so as not to imperil the fitness of their hosts. Earlier genetic and biochemical studies together with more recent cell biological investigations have revealed details of the partitioning mechanisms employed by low copy bacterial plasmids. At least some bacterial chromosomes also appear to rely on similar mechanisms for their own segregation. The 2 mm plasmid of Saccharomyces cerevisiae and related yeast plasmids provide models for optimized eukaryotic selfish DNA elements. Selfish DNA elements exploit the genetic endowments of their hosts without imposing an undue metabolic burden on them. The partitioning systems of these plasmids appear to make use of a molecular trick by which the plasmids feed into the segregation pathway established for the host chromosomes.
Cell Cycle Proteins↗
Preferential inclusion of extrachromosomal genetic elements in yeast meiotic spores.
During meiosis and sporulation in the yeast Saccharomyces cerevisiae, extrachromosomal traits are efficiently transmitted to haploid spores. Although the pattern of inheritance of chromosomal traits reflects the mechanism of regular chromosomal segregation in meiosis, it is not known what processes are reflected by the efficient inheritance of extrachromosomal traits. Because extrachromosomal genetic elements in yeast are present in multiple copies, perpetuation of an extrachromosomal trait could occur by the passive envelopment of a subset of copies or by an active sequestering of all or a subset of copies within the four spores. We show that only subsets of the four extrachromosomal nucleic acids commonly found in yeast are transmitted through meiosis--55% of mitochondrial DNA copies, 82% of the 2-micron DNA plasmids, and about 70% of the L and M double-stranded RNAs. However, electron micrographs of serial sections through yeast asci indicate that the four spore enclose only 30% of the total ascus material. Thus these extrachromosomal elements are preferentially included within the spores, indicating that their inheritance is not a random process. Transmission of mitochondrial DNA can be accounted for by the observed enclosure of 52% of the mitochondrial volume within the spores. The high transmission frequencies of the double-stranded RNAs (which exist as virus-like particles in the cytoplasm) and 2-micron DNA must indicate that either these nucleic acids are actively recruited from the cytoplasm by some mechanism or they are associated in some way with the nucleus during meiosis.
DNA, Mitochondrial↗
[Extrachromosomal genetic elements of Clostridium botulinum. I. Plasmids of toxigenic and atoxigenic strains of Clostridium botulinum type A].
Plasmid profiles in fifteen toxigenic and six nontoxigenic strains of Clostridium botulinum type A were investigated. The electrophoretic patterns of nucleic acids from the bacterial lysates were shown to vary in relation to the mode of bacterial cells lysis and the stage of microbial population growth. Two toxigenic strains harbour a unique 3.9 MD plasmid, five strains have a plasmid of about 6 Md and seven other strains carry an 11.5 Md plasmid. The nontoxigenic derivatives of two toxigenic strains harbouring a 6 Md plasmid have lost this extrachromosomal element. The analysis of the derivatives has not indicated the 6 Md plasmid to be connected with bacteriocin production or antibiotic resistance in Clostridium botulinum. Nontoxigenic variants of three other strains possess an 11.5 Md plasmid similar to their toxigenic parents' one. One of the studied toxigenic strains harboured no plasmids, as well as its nontoxigenic derivative. The functions of all studied plasmids remain unknown. Physical maps of the 11.5 and 6 Md plasmids were constructed on the basis of restriction analysis. The obtained results can be used for identification and differentiation of Clostridium botulinum strains and in further molecular biological experiments with this organism.
Botulinum Toxins↗
[Extrachromosomal genetic elements of Clostridium botulinum. II. Isolation and analysis of DNA from bacteriophages of Clostridium botulinum types C and A].
The DNAs of bacteriophage c-st, known to realize the lysogenic conversion of toxinogenicity among C. botulinum types C and D strains, and the nucleic acid of a virulent mutant of bacteriophage CB propagated in type A C. botulinum cells were purified and examined. Heterogeneity of phage c-st preparations was observed during purification, manifesting by formation of several bands in isopiknic CsCl gradient during centrifuging. An extra nucleic acid fraction was detected in some DNA preparations of phage c-st; the origin of this fraction is discussed. Plasmid extrachromosomal elements were for the first time found in the cells of nontoxigenic type C C. botulinum A02 strain, known as the indicator for c-st phage. The sensitivity of phage c-st DNA to 25 restriction endonucleases was examined. Analysis of the results of restriction analysis of c-st and CB phage DNAs and plasmid nucleic acids, revealed earlier in type A C. botulinum strains, disclosed several DNA modification enzymes with different recognition sites in type C C. botulinum. At least two of these activities are not found in type A strains. According to restriction analysis, total size of phage c-st DNA is about 160 kbp and of phage CB DNA 35 kbp. Individual EcoRI and HindIII restricts of phage c-st DNA, containing the initial site of botulinum toxin CI gene, were recognized by radioisotope labeled oligonucleotide probe Enzyme immunoassay revealed slight expression of the N-terminal region of bntc I gene in E. coli recombinant variants. These data can be used in further investigation of C. botulinum genetics.
Bacteriophages↗
Genetic basis of the biodegradation of salicylate in Pseudomonas.
The genetic basis of the biodegradation of salicylate in Pseudomonas putida R1 has been studied. This strain utilizes the meta pathway for oxidizing salicylate through formation of catechol and 2-hydroxymuconic semialdehyde. The enzymes of the meta pathway are induced by salicylate but not by catechol, and the genes specifying these enzymes are clustered. The gene cluster can be eliminated from some salicylate-positive cells by treatment with mitomycin C and appears to exist inside the cell as an extrachromosomal element. This extrachromosomal gene cluster, termed the SAL plasmid, can be transferred by conjugation from P. putida R1 to a variety of other Pseudomonas species.
Aldehyde-Lyases↗
Chromosomal destabilization during gene amplification.
Acentric extrachromosomal elements, such as submicroscopic autonomously replicating circular molecules (episomes) and double minute chromosomes, are common early, and in some cases initial, intermediates of gene amplification in many drug-resistant and tumor cell lines. In order to gain a more complete understanding of the amplification process, we investigated the molecular mechanisms by which such extrachromosomal elements are generated and we traced the fate of these amplification intermediates over time. The model system consists of a Chinese hamster cell line (L46) created by gene transfer in which the initial amplification product was shown previously to be an unstable extrachromosomal element containing an inverted duplication spanning more than 160 kilobases (J. C. Ruiz and G. M. Wahl, Mol. Cell. Biol. 8:4302-4313, 1988). In this study, we show that these molecules were formed by a process involving chromosomal deletion. Fluorescence in situ hybridization was performed at multiple time points on cells with amplified sequences. These studies reveal that the extrachromosomal molecules rapidly integrate into chromosomes, often near or at telomeres, and once integrated, the amplified sequences are themselves unstable. These data provide a molecular and cytogenetic chronology for gene amplification in this model system; an early event involves deletion to generate extrachromosomal elements, and subsequent integration of these elements precipitates a cascade of chromosome instability.
Animals↗
Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.
Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25-50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.
DNA, Intergenic↗
Physical mapping of the Bacillus thuringiensis subsp. kurstaki and alesti chromosomes.
Two strains of the well-known insect pathogen and biopesticide, Bacillus thuringiensis (Bt), belonging to subspecies alesti (strain Bt5) and kurstaki (strain Bt213), were chosen for genetic characterization. The two strains belong to different serotypes and are currently classified into different subspecies, although their insecticidal activity is similar. Physical maps were constructed of Bt alesti and Bt kurstaki using Pulsed Field Gel Electrophoreses (PFGE), and the map positions of several genes were determined. The 5.5 Mb combined genetic and physical chromosome maps of the two strains were found to be indistinguishable, and the only differences detected between the strains were of extrachromosomal origin. A cryIA toxin gene probe hybridised to a chromosome fragment and to two extrachromosomal elements in both strains, migrating as 100 kb and 350 kb, respectively. In addition a cry hybridizing extrachromosomal element migrating as 80 kb was present only in Bt alesti. Both strains were also found to contain sequences hybridizing to an enterotoxin (hbla) gene probe. Such sequences were positioned on the 350 kb extrachromosomal element, as well as on the chromosome.
Bacillus thuringiensis↗
Characterization of a linear extrachromosomal DNA element (pBL1) isolated after interspecific mating between Streptomyces bambergiensis and S. lividans.
Streptomyces bambergiensis S712 harbours a giant linear plasmid PSB1 of 640 kb. After mating with the plasmidless S. lividans strain TK64, conjugants carrying a smaller extrachromosomal DNA element, pBL1, were identified. pBL1 is a 43-kb linear DNA molecule bound to a protein which protects it from attack by both 3'- and 5'-exonucleases. The absence of this protein drastically reduces the transforming efficiency of pBL1. pBL1 shares homology with linear plasmids and chromosomal DNA from S. bambergiensis strains.
Conjugation, Genetic↗
Detection of same sized 4.3 Kb extrachromosomal DNA elements in weakly beta-haemolytic human intestinal spirochaetes and Serpulina pilosicoli of swine origin.
Agarose gel electrophoresis of total DNA from Italian strains of weakly beta-haemolytic human intestinal spirochaetes (w beta HIS) and porcine Serpulina pilosicoli reference strain P43/6/78 showed an extrachromosomal band having the same size and migrating at 4.3 Kb. The same results were observed after agarose gel electrophoresis of DNA obtained from the supernatant fluids of the spirochaetal cultures analysed. Swine Serpulina hyodysenteriae reference strain P18A was comparatively analysed and a 6.5 Kb extrachromosomal DNA element was found, as expected. Furthermore, S. hyodysenteriae reference strain P18A differed from all the other spirochaetes tested and had a higher number of flagella (8-12) at each cell end and was strongly beta-haemolytic. To the best of our knowledge, this is the first report on the detection of a band of extrachromosomal DNA having the same size in w beta HIS and S. pilosicoli from swine origin.
Animals↗
Effective generation of transgenic mice by Bovine papillomavirus type 1 based self-replicating plasmid that is maintained as extrachromosomal genetic element in three generations of animals.
The objective of our study was to analyze the efficiency and the properties of the inheritance of the Bovine papillomavirus type 1 (BPV1) replicator-based plasmid used as vector system for generation of transgenic animals. Previously, we have characterized a series of self-replicating plasmid vectors containing all viral factors necessary and sufficient for stable extrachromosomal replication of the BPV1 genome in the tissue culture system. We also demonstrated that the designed replicating vector system has a considerable benefit in the transgene expression, if compared to the regular expression vector. The vector, which showed the highest stability and maintenance function in the tissue culture was chosen for generation of the transgenic mice by pronuclear injections of the circular supercoiled plasmid. This method resulted in successful production of transgenic animals. Transmission efficiency of the vectors into the F(1) generation of animals varied between 0 and 48%, whereas transmission into the F(2) generation was uniformly near 50%. The maintenance of the vector-plasmids in the F(2) generation of transgenic animals as extrachromosomal genetic element was demonstrated by rescue of the plasmid into the Escherichia coli.
Animals↗
Genome of Azotobacter vinelandii: counting of chromosomes by utilizing copy number of a selectable genetic marker.
Studies utilizing several physical, biochemical and spectroscopic methods have suggested that Azotobacter vinelandii contains multiple copies (40-80) of its chromosome per cell, whereas genetic analysis indicated that these cells function like haploid cells. To further verify if A. vinelandii indeed contains 40-80 copies of its chromosome per cell, we have developed an 'in vivo chromosome counting' technique. The basic principle of this technique is to introduce the same genetic marker on the chromosome and on an extrachromosomal element of known copy number into the bacterium. The copy number of the chromosome can be determined by comparing the intensity of the hybridization signal generated by the DNA fragment carrying the chromosomal marker with that of the extrachromosomal marker when the total DNA isolated from this strain is hybridized with a probe made of the same genetic marker DNA. To do this we used an A. vinelandii BG102 strain which carries a kanamycin resistance marker gene integrated into the nifY locus on its chromosome(s). The plasmids pRK293 and pKT230, which can replicate in A. vinelandii and carry the kanamycin resistance gene (similar to the one present on the chromosome of A. vinelandii BG102), served as the extrachromosomal elements with known copy number. Southern blotting and hybridization analysis of the total DNA, isolated from A. vinelandii BG102 containing these plasmids, with a probe made of the kanamycin resistance gene clearly indicated that the copy number of A. vinelandii chromosome is slightly lower than the copy number of the low-copy plasmid pRK293 and about 21-fold lower than the copy number of the high copy plasmid pKT230. We believe that this 'in vivo chromosome counting' technique can be used for determination of the copy number of the chromosome in other cells with appropriate modifications in the nature of the extrachromosomal element and the genetic marker.
Azotobacter vinelandii↗