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PubMed2026

CRISPR/Cas- and Argonaute-Based In Vivo Nucleic-Acid Imaging Technologies: Strategies, Challenges, and Perspectives.

Live-cell monitoring of sequence-specific nucleic acids is essential to understanding genome organization, RNA regulation, and disease progression. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) and Argonaute (Ago) systems provide programmable, guide-directed recognition of DNA or RNA and are increasingly used as platforms for in vivo bioimaging. This review summarizes the structural and mechanistic features of representative CRISPR and Ago effectors and discusses design strategies for sensitive, specific, and multiplexed imaging of genomic loci, extrachromosomal DNA, and endogenous RNA in living cells. We compare the analytical performance and limitations of CRISPR- and Ago-based imaging, with particular emphasis on the major technical and biological challenges affecting their accuracy, applicability, and reliability. Finally, this review offers insights into developing high-resolution and user-friendly bioimaging platforms for fundamental biology and future translational applications.

CRISPR↗

PubMed1984

Plasmid-mediated resistance in multiply resistant Haemophilus influenzae type b causing meningitis: molecular characterization of one strain and review of the literature.

The increasing prevalence of infections due to ampicillin-resistant strains of Haemophilus influenzae type b requires that suspected H. influenzae meningitis in children be initially treated with both ampicillin and chloramphenicol. Previously, the recognition of strains resistant to chloramphenicol but susceptible to ampicillin supported combination chemotherapy. In this study one case of meningitis due to a strain of H. influenzae resistant to ampicillin, chloramphenicol, and tetracycline was analyzed. The patient involved received intravenous trimethoprim-sulfamethoxazole, but putative resistance to this combination prompted the additional administration of intravenous moxalactam. The resistance of this organism was mediated by a conjugative 43-megadalton R plasmid; the determinants of ampicillin and chloramphenicol resistance were transferred as a single unit. However, not all of the multiply resistant transconjugants contained a detectable plasmid; DNA homology studies with R plasmids of H. influenzae confirmed that these extrachromosomal DNA sequences were associated with chromosomal DNA and that an extrachromosomal location was rare.

Ampicillin↗

PubMed1996

The growing importance of the plastid-like DNAs of the Apicomplexa.

Organisms in the phylum Apicomplexa possess, in addition to their mitochondrial genome, an extrachromosomal DNA that possesses significant similarities with the extrachromosomal genomes of plastids. To date, the majority of data on these plastid-like DNAs have been obtained from the human malarial organism, Plasmodium falciparum. In common with plastid DNAs, the plastid-like DNA of P. falciparum possesses genes for DNA-dependent RNA polymerase subunits beta and beta 1 and for organellar-like large- and small-subunits ribosomal RNAs. Both the polymerase subunit and ribosomal RNA gene sequences share a number of features with those from plastid DNAs. In addition, the ribosomal RNA genes are organised in an inverted repeat arrangement, reminiscent of plastid DNAs. Additional molecular features shared between the 2 genomes are discussed. Plastid-like DNAs have also been identified in other Plasmodium species as well as Toxoplasma gondii, Eimeria tenella, Babesia bovis and a number of Sarcocystis species. A cryptic organelle often observed in apicomplexans has been proposed as the organelle that harbours the plastid-like DNAs, but conclusive evidence for this has not yet been obtained. Although approximately 1/2 of the plastid-like DNA of P. falciparum has been sequenced to date, no function has yet been ascribed to this DNA or its putative organelle. Phylogenetic inferences based on sequence data from this DNA have indicated an evolutionary origin from photosynthetic organisms, but the true provenance of the plastid-like DNAs remains to be determined. Because of the specific nature of the plastid-like DNAs, they may prove useful as effective targets for chemotherapeutics.

Animals↗

PubMed1990

Plasmid profiles and genomic DNA restriction endonuclease patterns of 30 independent Staphylococcus lugdunensis strains.

Extrachromosomal DNA analysis and restriction endonuclease analysis of whole cellular DNA were used to characterize 30 Staphylococcus lugdunensis strains isolated from 13 different hospitals from 1977 to 1988. All the strains were susceptible to most of the antibiotics tested, including penicillin G. A single 3.2 kilobase plasmid was detected in 13 strains and one or two plasmids, ranging from 2.3 to 6.6 kilobases, were found in 7 strains. EcoRI, PstI and PvuII restriction patterns of total cellular DNA were identical for 23 isolates, indicating strong conservation of endonuclease sites in this species. One or two additional DNA bands occurred in seven isolates. Molecular markers show rather little variations between different S. lugdunensis isolates suggesting that they are closely related.

DNA Restriction Enzymes↗

PubMed1981

Cellular location of Mu DNA replicas.

To ascertain the form and cellular location of the copies of bacteriophage Mu DNA synthesized during lytic development, DNA from an Escherichia coli lysogen was isolated at intervals after induction of the Mu prophage. Host chromosomes were isolated as intact, folded nucleoids, which could be digested with ribonuclease or heated in the presence of sodium dodecyl sulfate to yield intact, unfolded nucleoid DNA. Almost all of the Mu DNA in induced cells was associated with the nucleoids until shortly before cell lysis, even after unfolding of the nucleoid structure. We suggest that the replicas of Mu DNA are integrated into the host chromosomes, possibly by concerted replication-integration events, and are accumulated there until packaged shortly before cell lysis. Nucleoids also were isolated from induced lambda lysogens and from cells containing plasmid DNA. Most of the plasmid DNA sedimented independently of the unfolded nucleoid DNA, whereas 50% or more of the lambda DNA from induced lysogens cosedimented with unfolded nucleoid DNA. Possible explanations for the association of extrachromosomal DNA with nucleoid DNA are discussed.

Bacteriophage lambda↗

PubMed1994

[Nucleotide sequence of DNA isolated from protein cores of rosette-like structures (elementary chromomeres) of mouse interphase chromosomes].

Data are presented on the primary nucleotide sequence of 15 DNA fragments cloned from protein cores of rosette-like structures of mouse interphase chromosomes. According to data of a computer analysis, these DNA fragments represent protein-noncoding regions of interphase chromosomes, contain nucleotide sequences causing DNA strand bending, and contain a large amount of cis-regulatory transcriptional elements and sites closely related to sites of DNA topoisomerase II cleavage. Five clones demonstrated partial length homology with satellite DNA, short and long interspersed sequences, and extrachromosomal DNA from mouse thymocytes. Six clones showed homology with sites of initiation of replication of viruses, yeast, and mice. Six clones were found to contain consensus sequences for non-homologous recombination in somatic cells and six clones contained consensus sequences of human VTR1.1 loci.

Animals↗

PubMed1987

rRNA genes of Naegleria gruberi are carried exclusively on a 14-kilobase-pair plasmid.

An extrachromosomal DNA was discovered in Naegleria gruberi. The 3,000 to 5,000 copies per cell of this 14-kilobase-pair circular plasmid carry all the 18S, 28S, and 5.8S rRNA genes. The presence of the ribosomal DNA of an organism exclusively on a circular extrachromosomal element is without precedent, and Naegleria is only the third eucaryotic genus in which a nuclear plasmid DNA has been found.

Animals↗

PubMed1992

Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells.

This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-ChR-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-ChR-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads.

Chromosomes, Human↗

PubMed2006

Retroviral E-DNA: persistence and gene expression in nondividing immune cells.

Following retroviral infection of cells, not only is the proviral DNA integrated into the host genome, but there is also an accumulation of unintegrated extrachromosomal DNA (E-DNA), both linear and circular. Although the integrated DNA is responsible for the production of viral proteins and new viral progeny, the role of E-DNA has remained uncertain. Several reports have shown that E-DNA is transcriptionally active producing both RNA, as well as viral proteins and that circular E-DNA can persist in nondividing cells, raising questions regarding the potential consequences of this reservoir. Furthermore, integrase inhibitors, presently in clinical trials, shifts the balance of proviral DNA to the E-DNA form. This review is focused on recent work in this field with an emphasis on exploring the potential role of E-DNA in both pathogenesis of retroviral infections, especially HIV-1, and as a tool to deliver and express genes.

Animals↗

PubMed1995

An extrachromosomal fragment of telomeric DNA in wheat.

A procedure developed originally for selective extraction of viral (extrachromosomal) DNA from virus-infected mammalian cells was applied to cell nuclei isolated from uninfected wheat embryos. The resulting nuclear extrachromosomal DNA (exDNA) was enriched for telomere-type sequences by isopycnic centrifugation and inserted into the Sma I site of pUC119. A cloned DNA fragment (241 bp) was found to consist primarily of tandemly repeated heptamere units of the same sequence (5'-CCCTAAA-3') that is known to predominate in telomeric DNA of Arabidopsis thaliana. Hybridization experiments indicate that extrachromosomal telomeric repeats are abundant in resting embryos and disappear rapidly during germination.

Arabidopsis↗

PubMed1984

Circular extrachromosomal copia-like transposable elements in Drosophila tissue culture cells.

We find that in the circular extrachromosomal DNA from Drosophila tissue culture cells the transposable elements copia, 412, 297, and mdg 1 are present in variable amounts. There is no detectable circular DNA homologous to B104 . From the relationship between the intra- and extrachromosomal forms it appears that the amount of different circular elements is not related to the amount of the respective chromosomal elements.

Animals↗

PubMed1992

Range of action and genetic bacteriocin codification of Pseudomonas aeruginosa isolated from three different ecological niches.

Strains of Pseudomonas aeruginosa isolated from sediments in wells showed a greater bacteriocinogenic activity in those isolated from river and clinical specimens. More than half of all the strains examined had extrachromosomal DNA. Plasmid DNA was extracted from all the strains and in only 24/120, all from different origins, was the curing achieved; all these strains coded their bacteriocins in the chromosomal DNA.

Bacteriocins↗

PubMed1991

Inverse relationship between human papillomavirus (HPV) type 16 early gene expression and cell differentiation in nude mouse epithelial cysts and tumors induced by HPV-positive human cell lines.

Two human papillomavirus type 16 (HPV 16)-immortalized human keratinocyte cell lines (HPK) were shown to have retained the ability for differentiation after subcutaneous injection into nude mice. These properties were maintained even at late passage. HPK cells gave rise to transiently growing cysts which exhibited an epitheliumlike architecture. Moreover, differentiation-specific markers such as cytokeratin 10, involucrin, and filaggrin were shown to be expressed in an ordered succession. RNA-RNA in situ hybridization revealed heterogeneous and low levels of HPV 16 E6-E7 RNA in the basal layer of the cysts. In contrast, in progressively growing tumors induced by HPK cells containing an activated ras oncogene (EJ-ras) or in tumors induced by the cervical carcinoma cell line CaSki, high levels of E6-E7-specific RNA could be detected. Irrespective of the growth potential of these cell lines in nude mice, viral transcription was always more evident in the basal layer and in proliferatively active cells rather than in differentiated cells. This contrasts with viral gene expression in HPV 16 positive low-grade cervical dysplasia, in which abundant viral transcriptional activity was mapped to the upper third of the epithelium. It is suggested that the physical state of the viral DNA, i.e., integrated viral DNA in the cell lines as opposed to extrachromosomal DNA in low-grade cervical dysplasia, may influence viral gene regulation.

Animals↗

PubMed1986

[Isolation and properties of recombinant DNA from a plasmid and filamentous phage].

In the course of studying extrachromosomal DNA with composite replicons, a hybrid has been constructed by the in vitro recombination of the filamentous phage M13mp2 DNA (RF) and plasmid pUR222 (ApR). Both parental DNAs contain a fragment of lac-operon (ca. 800 bp), which includes the distal end of lacI gene, lacPO segments, and the lacZ gene proximal region coding for 145 N-terminal amino acid residues of beta-galactosidase and thus providing for alpha-complementation, the effect being cancelled with a polynucleotide insertion at the unique EcoRI site in the lacZ gene segment. E. coli BMH71-18 cells were transformed with the ligated mixture of EcoRI restricts of both DNAs. A phage-like nucleoprotein was isolated from colourless plaques (on the Xgal- and IPTG-supplemented medium); its deproteinization yielded a DNA which contains the ApR-determinant and, according to PAGE, structurally specific staining, restriction analysis, sequencing by the Sanger procedure, and electron microscopy data, is a linear double-stranded molecule comprising the phage and plasmid genomes in an equimolar ratio. Since the hybrid DNA does not display the alpha-complementation effect, both bacterial inserts are in the opposite orientation. Transformation of both phage (F+) and plasmid (F-) hosts with the hybrid DNA led to cultures which, after precipitation of the nucleoprotein from the extracellular medium and deproteinization, afforded the same composite DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Mapping↗

PubMed1974

Some properties of the PBP1 transduction system in Bacillus pumilus.

Bacteriophage PBP1 is a flagella-specific virus that performs generalized transduction in strains of Bacillus pumilus. PBP1 is morphologically and serologically distinct from two other flagella-specific phages, PBS1 and SP-15, which perform generalized transduction in certain Bacillus species. The DNA extracted from PBP1 particles has a buoyant density of 1.690 g/cm(3) in cesium chloride gradients, a melting temperature of 86.1 C, and a sedimentation velocity of 47S in neutral sucrose gradients. Assuming the molecule is a linear duplex, PBP1 DNA has a molecular weight of approximately 76 x 10(6). In two strains of B. pumilus which are sensitive to both PBP1 and PBS1, co-transducible genetic markers are more tightly linked by PBS1 transduction than by PBP1 transduction. The size of the fragment of bacterial DNA carried by PBP1-transducing particles, inferred from transduction studies and sedimentation analysis of viral DNA, suggests that PBP1 may be useful for genetic studies of extrachromosomal DNA elements present in two strains of B. pumilus. Genetic exchange of chromosomally located genes between the plasmid(+) and plasmid(-)B. pumilus strains NRS 576 and NRRL B-3275 has been demonstrated by PBP1 transduction.

Bacillus↗

PubMed2004

Genomic DNA is captured and amplified during double-strand break (DSB) repair in human cells.

Genomic stability is maintained by the surveillance and repair of DNA damage. Here, we describe a mechanism whereby repair of extrachromosomal DNA double-strand breaks (DSBs) in human cells can be accompanied by capture of genomic DNA fragments. The availability of the human genome sequence enabled us to characterize these inserts in cells from a normal individual and from a patient with ataxia telangiectasia (AT), deficient for the damage response kinase ATM and prone to genomic instability. We find AT cells exhibit insertions of human chromosomal DNA fragments in excess of 17 kb during DSB repair, whereas we detected no such genomic inserts in normal cells. However, the presence of simian virus 40 (SV40), used to transform these cell lines, resulted in capture of genomic DNA associated with sites of viral integration in both cell types. The genomic instability at sites of SV40 integration was exported to other sites of DNA damage, and acquisition of the viral origin of replication resulted in gene amplification through autonomous replication of the plasmid harbouring the repaired extrachromosomal DSB. Should this same phenomenon apply to the repair of chromosomal DSBs, genome rearrangements made possible via this DSB insertional repair pose risks to genomic integrity, and may contribute to tumorigenic progression.

DNA↗

PubMed1990

Extrachromosomal HIV-1 DNA in persistently infected U937 cells.

Persistent human immunodeficiency virus type 1 (HIV-1) infection of U937 monocytic cells resulted in the accumulation of novel forms of extrachromosomal viral DNA. These DNA species are larger than the genome size of HIV-1 and persist indefinitely. The extrachromosomal viral DNA species (E-DNA) were shown to be structurally stable by subcloning of infected cell lines and restriction fragment analysis. Similar E-DNA structures were observed in independent infections. Persistently infected monocytic cells had low levels of viral antigens, reflecting the low levels of viral RNA that were detected. These results support a role for E-DNA in persistent HIV-1 infection of monocytic cells.

Cell Line↗

PubMedDate not supplied

Spiroplasma species share common DNA sequences among their viruses, plasmids and genomes.

Alkaline-Southern-blot analyses showed that a spiroplasma plasmid, pRA1, obtained from Spiroplasma citri (Maroc-R8A2), contained DNA sequences that were homologous to spiroplasma type 3 viruses (SV3) obtained from S. citri (Maroc-R8A2), S. citri (608) and S. mirum (SMCA). In addition, pRA1 and SV3(608) DNA shared common, but not necessarily related, sequences with extrachromosomal DNA derived from 11 Spiroplasma species or strains. Furthermore, SV3(608) had DNA homology with the chromosome from 6 distinct spiroplasmas but not with chromosomal DNA from eight other Spiroplasma species or strains. The biological function of these common sequences is unknown.

Bacteriophages↗