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PubMed2004

Detection and traceability of genetically modified organisms in the food production chain.

Both labelling and traceability of genetically modified organisms are current issues that are considered in trade and regulation. Currently, labelling of genetically modified foods containing detectable transgenic material is required by EU legislation. A proposed package of legislation would extend this labelling to foods without any traces of transgenics. These new legislations would also impose labelling and a traceability system based on documentation throughout the food and feed manufacture system. The regulatory issues of risk analysis and labelling are currently harmonised by Codex Alimentarius. The implementation and maintenance of the regulations necessitates sampling protocols and analytical methodologies that allow for accurate determination of the content of genetically modified organisms within a food and feed sample. Current methodologies for the analysis of genetically modified organisms are focused on either one of two targets, the transgenic DNA inserted- or the novel protein(s) expressed- in a genetically modified product. For most DNA-based detection methods, the polymerase chain reaction is employed. Items that need consideration in the use of DNA-based detection methods include the specificity, sensitivity, matrix effects, internal reference DNA, availability of external reference materials, hemizygosity versus homozygosity, extrachromosomal DNA, and international harmonisation. For most protein-based methods, enzyme-linked immunosorbent assays with antibodies binding the novel protein are employed. Consideration should be given to the selection of the antigen bound by the antibody, accuracy, validation, and matrix effects. Currently, validation of detection methods for analysis of genetically modified organisms is taking place. In addition, new methodologies are developed, including the use of microarrays, mass spectrometry, and surface plasmon resonance. Challenges for GMO detection include the detection of transgenic material in materials with varying chromosome numbers. The existing and proposed regulatory EU requirements for traceability of genetically modified products fit within a broader tendency towards traceability of foods in general and, commercially, towards products that can be distinguished from each other. Traceability systems document the history of a product and may serve the purpose of both marketing and health protection. In this framework, segregation and identity preservation systems allow for the separation of genetically modified and non-modified products from "farm to fork". Implementation of these systems comes with specific technical requirements for each particular step of the food processing chain. In addition, the feasibility of traceability systems depends on a number of factors, including unique identifiers for each genetically modified product, detection methods, permissible levels of contamination, and financial costs. In conclusion, progress has been achieved in the field of sampling, detection, and traceability of genetically modified products, while some issues remain to be solved. For success, much will depend on the threshold level for adventitious contamination set by legislation.

Animals↗

PubMed2026

Characterisation of whole-blood extrachromosomal circular DNAs in Kawasaki disease complicated by coronary artery aneurysm.

BACKGROUND: Extrachromosomal circular DNAs are critical regulators of stress responses, immunity, and inflammation pathways. However, their role and underlying mechanisms in Kawasaki disease complicated by coronary artery aneurysm remain poorly understood. METHODS: Whole blood samples from six children with Kawasaki disease, including three with coronary artery aneurysm group and three without coronary artery aneurysm (control group), were subjected to extrachromosomal circular DNA sequencing. Putative extrachromosomal circular DNAs were identified using Circle-Map. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on genes associated with upregulated extrachromosomal circular DNAs, and an exploratory immune-signature profiling was performed using the single-sample gene set enrichment analysis algorithm. RESULTS: We identified 4,790 differential extrachromosomal circular DNAs between the coronary artery aneurysm and control groups, of which 4,789 were upregulated in the CAA group. The extrachromosomal circular DNAs in the coronary artery aneurysm group were more concentrated and shorter in length than those in the control group. Upregulated extrachromosomal circular DNAs were mainly derived from autosomes, particularly chromosomes 3, 4, and 7. Functional enrichment analysis indicated that genes associated with upregulated extrachromosomal circular DNAs were mainly involved in response to stimulus-related terms and pathways, including chemokine signalling, cGMP-PKG signalling, and calcium signalling. Immune-signature analysis suggested that the coronary artery aneurysm group exhibited more specific immune responses, whereas the control group tended towards broader immune regulatory processes. CONCLUSIONS: Coronary artery aneurysm and control groups exhibited distinct extrachromosomal circular DNA profiles. These findings suggest that extrachromosomal circular DNA alterations are associated with coronary artery aneurysm in Kawasaki disease and may provide new insights into the molecular features underlying this condition.

Coronary artery aneurysm↗

PubMed1992

Targeted alterations of the Caenorhabditis elegans genome by transgene instructed DNA double strand break repair following Tc1 excision.

Excision of a Tc1 transposon of Caenorhabditis elegans is thought to leave a DNA double strand break. We report here that sequence polymorphisms in a transgenic DNA template are copied into the corresponding chromosomal gene upon excision of Tc1 from the chromosome. This shows that the double strand DNA break resulting from Tc1 excision is repaired with the extrachromosomal DNA as template and that sequences flanking the break can be replaced by sequences from the transgene. Transgene instructed break repair provides a method for the targeted introduction of precise alterations into the Caenorhabditis elegans genome.

Animals↗

PubMed1989

Involvement of a Spiroplasma citri plasmid in the erythromycin-resistance transfer.

An erythromycin-resistant strain (M4 Er-1) was selected from Spiroplasma citri M4+. The transfer by transformation of the erythromycin-resistance character to the erythromycin-sensitive S. citri strain R8A2+ was studied. Transfer became effective and reproducible when cells were treated with alkali cations plus polyethylene glycol. Comparison of the efficiency of transformation of the erythromycin-sensitive strain S. citri R8A2+ by total and extrachromosomal DNA purified from the erythromycin-resistant strain M4 Er-1 showed that the plasmid pM42 was able to transfer the erythromycin-resistance. pM42 was mapped with restriction endonucleases and found to be related to the pMH1 plasmid previously isolated from S. citri MH. Hybridization analysis of DNA from sensitive and resistant strains has shown that a sequence from pM42, analogous to a sequence from pMH1, was integrated at a specific locus in the chromosome of the erythromycin-resistant cells, i.e., of the transformed R8A2 cells and of the spontaneous mutant M4 Er-1 strain.

DNA↗

PubMed2004

snRNPs are present in the karyosome capsule in the weevil germinal vesicle.

Within the oocyte nucleus of the apple blossom weevil, Anthonomus pomorum (Insecta, Coleoptera) highly condensed and transcriptionaly inactive chromosomes form the karyosome. During its formation, within the nucleoplasm numerous, variably sized spherical inclusions termed nuclear bodies occur. As oogenesis progresses, the karyosome is gradually surrounded by a prominent sheath, the karyosome capsule. The function and molecular composition of both the nuclear bodies and the karyosome capsule are largely unknown. Using cytochemical methods we demonstrate that DNA is confined to the karyosome and there is no extrachromosomal DNA accumulations within the nucleoplasm. In addition, none of the oocyte nucleus subdomains contain argyrophilic proteins. Our immunoEM study revealed that in contrast to similar structures in germinal vesicles in other insect species, the nuclear bodies of A. pomorum do not cross-react with antibodies recognising small nuclear ribonucleoproteins, coilin or the splicing factor SC-35. Unexpectedly, we found that as the karyosome capsule develops, mature small nuclear RNAs and proteins containing the Sm epitope associate with the capsule material. We suggest that the karyosome capsule is a storage site for small nuclear ribonucleoprotein particles, which may be used during early embryonic development.

Animals↗

PubMed1999

Bacterial plasmids: replication of extrachromosomal genetic elements encoding resistance to antimicrobial compounds.

Plasmids are self-replicating extrachromosomal DNA molecules found in Gram-negative and Gram-positive bacteria as well as in some yeast and other fungi. Although most of them are covalently closed circular double-stranded DNA molecules, recently linear plasmids have been isolated from different bacteria. In general, plasmids are not essential for the survival of bacteria, but they may nevertheless encode a wide variety of genetic determinants, which permit their bacterial hosts to survive better in an adverse environment or to compete better with other microorganisms occupying the same ecological niche. The medical importance of plasmids that encode for antibiotic resistance, as well as specific virulence traits has been well documented and demonstrated the important role these bacterial genetic elements play in nature. Although they encode specific molecules required for initiation of their replication, plasmids rely on host-encoded factors for their replication. Plasmid replication initiates in a predetermined cis-site called ori and can proceed either by a rolling circle or a theta replication mechanism. Some of the plasmid-encoded elements required for their replication, such antisense RNA molecules and DNA repeated sequences located close to ori, determine plasmid attributes like copy number and incompatibility.

Bacteria↗

PubMed1972

Isolation of minicircular deoxyribonucleic acids from wild strains of Escherichia coli and their relationship to other bacterial plasmids.

Supercoiled minicircular deoxyribonucleic acid (DNA) molecules with molecular weights of 1.8 x 10(6) and 2.3 x 10(6) have been isolated from two wild strains of Escherichia coli. DNA-DNA hybridization experiments indicate that these DNA molecules share extended homologies with the minicircular DNA of E. coli 15. The DNA of the colicinogenic factor E1 (ColE1) also hybridizes to a large extent with minicircular DNA of E. coli 15. In contrast, no hybridization could be detected with various large extrachromosomal DNA elements such as the colicinogenic factor V (ColV), the beta-hemolytic factor (Hly), or the P1-like DNA of E. coli 15. Two different insertion DNA species of E. coli integrated into lambdadg-DNA (lambdadg UP(in) 128, lambdadg UP(in) 308) do not show any annealing with minicircular DNA of E. coli 15.

Centrifugation, Density Gradient↗

PubMed1975

Plasmid interactions in Staphylococcus aureus: nonadditivity of compatible plasmid DNA pools.

Six different Staphylococcus aureus plasmids have been examined for compatibility relationships and their intracellular DNA pools have been measured singly and in various combinations. All six were mutually compatible, but contrary to expectation, their intracellular DNA pools were not additive; instead, there appeared to be a maximum level of extrachromosomal DNA that could be supported by the cell, and the plasmids studied approached this level individually as well as in varous combinations. One exception was encountered: a plasmid encoding kanamycin/neomycin resistance was present in a small but constant number of copies regardless of the presence of other plasmids.

DNA, Bacterial↗

PubMed1984

Mobile genetic elements in animal cells and their biological significance.

Mobile genetic elements were discovered by McClintock while analysing unstable mutations in maize. The structural and functional studies of such elements became possible after their cloning, first from the genome of Drosophila melanogaster. In particular, Ilyin et al. demonstrated the varying location of the described elements in D. melanogaster chromosomes, thus providing the first evidence of their mobility. Mobile elements comprise a significant part of the genetic material in D. melanogaster (not less than 10%). Several classes of mobile elements do exist. Mobile dispersed genetic elements (mdg elements) are among the best characterized ones. Mdg elements are represented in the genome by dozens of families, each consisting of 10-150 copies. They are very similar structurally to proviruses of endogenous retroviruses. In particular, the both contain long terminal repeats (LTRs). The nucleotide sequences of LTRs and their flanking sequences of several mdg elements were determined. Their analysis suggested that RNA reverse transcription should be involved in the mdg amplification. It has been found that putative transposition intermediates, i.e. extrachromosomal DNA copies of mdg elements, are synthesized by reverse transcriptase in D. melanogaster culture cells. Another type of mobile genes is represented by P factor and similar elements. P factor seems to encode 'transposase' participating in direct excision and insertion of P elements themselves as well as of other mobile genes (mdg and fold-back elements). Besides these 'active transposons' which encode the enzyme machinery for transposition, a number of other sequences which may be transposed are present in the genome. RNAs synthesized on such elements can serve as a template for reverse transcriptase, and the DNA formed can then be inserted at new sites of the genome. Among such sequences are the so-called short ubiquitous repeats: B1 and B2 in mouse genome and Alu in human genome. We found that, at least in several cases, B-type sequences were located at the 3' end of mRNA. Short repetitive sequences were also detected at the 3' end of certain mRNAs of D. melanogaster. Usually the transpositions of mobile genes occur very rarely. However, under certain conditions, for example, in hybrid dysgenesis, they become more frequent. The strain with a mutation in cut locus was obtained in hybrid dysgenesis. This mutation depends on an insertion of mdg4 at the cut locus. Genetic instability in this strain is maintained for a long time. 'Transposition bursts' were found to occur in some germ cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

A comparative study of male meiosis in Drosophila melanogaster and D. virilis.

Male meiosis in D. melanogaster cytologically follows the usual pattern, whereas in D. melanogaster and in D. virilis oocytes the chromosomes clump into a karyosphere at early meiotic prophase and remain so up to metaphase I. Male meiosis in D. virilis spermatocytes has an intermediate character: a part of the chromatin clumps together in a karyosphere at early prophase, whereas the other part of the chromatin remains diffuse all through prophase. At the end of prophase, the diffuse chromatin becomes integrated into the karyosphere before metaphase I. During the meiotic divisions the chromosomes have the same clumped aspect as those in Drosophila oocytes and thus differ strikingly from the dividing chromosomes in D. melanogaster spermatocytes. In D. virilis spermatocytes the nucleolus exhibits changes during the meiotic prophase that may be related to synthetical activities. The DNA specific staining with the fluorochrome DAPI reveals the existence of extrachromosomal DNA in the later prophase. Other striking differences in meiotic events between the two Drosophila species concern the centrioles and spermiogenesis.

Animals↗

PubMed1993

Epidemiological markers of coagulase-negative staphylococci.

Several different epidemiological typing methods have been used in studies of coagulase-negative staphylococci; these include biotyping, antibiotic susceptibility pattern analysis, serological typing, phage typing, slime production detection, protein profile analysis, immunoblot fingerprinting and DNA typing. The most frequently used tests are antibiotic susceptibility and extrachromosomal DNA banding patterns. Comparison of chromosomal DNA restriction patterns is facilitated when the fragments are separated by pulsed-field electrophoresis. Typing variations occur quite frequently even in isolates of demonstrated clinical significance. Caution should therefore be exercised in denying the clinical relevance of coagulase-negative staphylococci, even when successive isolates show distinct typing differences.

Bacteriological Techniques↗

PubMed1979

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.

Cell Cycle↗

PubMed1982

[Genetic control of antibiotic resistance and the R plasmid properties of clinical E. coli strains].

The electrophoretic analysis of the plasmid DNA of multiresistant clinical E. coli strains revealed the presence of extrachromosomal DNA in 97% of the strains. The plasmid nature of resistance was established in 88-100% of E. coli strains resistant to chloramphenicol, ampicillin, streptomycin or kanamycin and in 67% of the strains resistant to tetracyclin. 24 out of 40 strains contained conjugative R-plasmids in which the determinants of resistance to kanamycin, ampicillin and chloramphenicol were transferred most frequently (82-93%), while the determinants of resistance to tetracyclin and streptomycin were transferred less frequently (45% and 35%, respectively). Nonconjugative R-plasmids were detected in 15 out of 40 antibiotic-resistant strains; in these plasmids the determinants of resistance to streptomycin (65%) and tetracyclin (22%) were transferred most frequently.

Anti-Bacterial Agents↗

PubMed1980

Studies on the biological role of dna methylation; IV. Mode of methylation of DNA in E. coli cells.

Two pairs of restriction enzyme isoschizomers were used to study in vivo methylation of E. coli and extrachromosomal DNA. By use of the restriction enzymes MboI (which cleaves only the unmethylated GATC sequence) and its isoschizomer Sau3A (indifferent to methylated adenine at this sequence), we found that all the GATC sites in E. coli and in extrachromosomal DNAs are symmetrically methylated on both strands. The calculated number of GATC sites in E. coli DNA can account for all its m6Ade residues. Foreign DNA, like mouse mtDNA, which is not methylated at GATC sites became fully methylated at these sequences when introduced by transfection into E. coli cells. This experiment provides the first evidence for the operation of a de novo methylation mechanism for E. coli methylases not involved in restriction modification. When the two restriction enzyme isoschizomers, EcoRII and ApyI, were used to analyze the methylation pattern of CCTAGG sequences in E. coli C and phi X174 DNA, it was found that all these sites are methylated. The number of CCTAGG sites in E. coli C DNA does not account for all m5Cyt residues.

Base Sequence↗

PubMed1982

Comparative restriction endonuclease maps of proviral DNA of the primate type C simian sarcoma-associated virus and gibbon ape leukemia virus group.

Extrachromosomal DNA was purified from canine thymus cells acutely infected with different strains of infectious primate type C viruses of the woolly monkey (simian) sarcoma helper virus and gibbon ape leukemia virus group. All DNA preparations contained linear proviral molecules of 9.1 to 9.2 kilobases, at least some of which represent complete infectious proviral DNA. Cells infected with a replication-defective fibroblast-transforming sarcoma virus and its helper, a replication-competent nontransforming helper virus, also contained a 6.6- to 6.7-kilobase DNA. These proviral DNA molecules were digested with different restriction endonucleases, and the resultant fragments were oriented to the viral RNA by a combination of partial digestions, codigestion with more than one endonuclease, digestion of integrated proviral DNA, and hybridization with 3'- and 5'-specific viral probes. The 3'- and 5'-specific probes each hybridized to fragments from both ends of proviral DNA, indicating that, in common with those of other retroviruses, these proviruses contain a large terminal redundancy at both ends, each of which consists of sequences derived from both the 3' and 5' regions of the viral RNA. The proviral sequences are organized 3',5'-unique-3',5'. Four restriction enzymes (KpnI, SmaI, PstI, and SstI) recognized sites within the large terminal redundancies, and these sites were conserved within all the isolates tested. This suggests that both the 3' and 5' ends of the genomic RNA of these viruses are extremely closely related. In contrast, the restriction sites within the unique portion of the provirus were not strongly conserved within this group of viruses, even though they were related along most of their genomes. Whereas the 5' 60 to 70% of the RNA of these viruses was more closely related by liquid hybridization experiments than was the 3' 30 to 40%, restriction sites within this region were not preferentially conserved, suggesting that small sequence differences or point mutations or both exist throughout the entire unique portion of the genome among these viruses.

Animals↗

PubMed1988

Molecular cloning and characterization of the DNA of two papillomaviruses from monkeys.

Benign and malignant lesions from monkeys were analyzed for the presence of papillomavirus (PV) DNA. By hybridization with different PV DNA probes under conditions of lowered stringency, two tumors were found to contain PV-specific DNA sequences: (1) a cutaneous papilloma from a Colobus monkey; and, (2) a lymph node metastasis of a squamous cell carcinoma of the penis from a Rhesus monkey. Analysis of the DNA of the papilloma from the Colobus monkey indicated the presence of extrachromosomal DNA whereas analysis of DNA from the Rhesus tumor suggested the presence of integrated viral DNA. The physical size (7.8 and 8.1 kb), colinear alignment to HPV-5, and cross-hybridization with other PV types under low stringency indicate that the two genomic DNA clones represent new PV types that have been tentatively designated as Rhesus papillomavirus type 1 (RhPV 1) and Colobus guereza papillomavirus type 2 (CgPV 2). A putative viral-host DNA junction fragment was also isolated from the Rhesus genomic library. Nucleotide sequences very closely related to RhPV 1 were observed by in situ hybridization in a laryngeal carcinoma from the Colobus guereza monkey. This report communicates the finding of novel papillomaviruses associated with a benign cutaneous tumor and genital and laryngeal malignancies in non-human primates which may have significance as a putative system for the study of papillomavirus-induced genital and laryngeal malignancies in humans.

Animals↗

PubMed1991

Replication intermediates as substrates for DNA rearrangements.

A model for the formation of DNA rearrangements in somatic cells is presented. Two double-strand breaks at the junctions between unreplicated DNA and newly replicated DNA generate four double-stranded DNA molecules that can recombine to form tandem duplications, inversions, deletions and extrachromosomal DNA circles.

Chromosome Deletion↗

PubMed1997

Transforming DNA integrates at multiple sites in the dimorphic fungal pathogen Blastomyces dermatitidis.

Blastomyces dermatitidis is a primary fungal pathogen of man and other mammals, but like many other human fungal pathogens, relatively little is known about the factors that account for its virulence and pathogenicity. We developed a transformation system to facilitate molecular genetic studies of putative virulence factors from B. dermatitidis. Transformation of the multinucleate yeasts was achieved by electroporation of DNAs containing a dominant selectable marker, hygromycin B (HygB) resistance. Southern analysis showed that transforming DNA invariably integrated ectopically into the chromosome. No evidence was found for extrachromosomal DNA. The HygB resistance could be expressed by either a 375-bp promoter fragment of the B. dermatitidis WI-1 gene encoding adhesin or an Aspergillus gpdA promoter placed 5' of the E. coli hph gene. Primer extension analysis showed that for plasmids containing the WI-1 promoter, transcription of the hph gene initiated within the 375-bp WI-1 promoter fragment. The combination of gene transfer and two promoters capable of independent transcription will allow us to restore or augment gene expression in appropriate strains and test an influence on virulence. Molecular genetic manipulation of B. dermatitidis represents a major advance in our ability to investigate the pathogenesis of blastomycosis and other similar fungal diseases.

Animals↗