The mechanism of extrachromosomal homologous DNA recombination in plant cells

Summary By cotransfecting plasmids carrying particular mutations in the -glucuronidase (GUS) gene into Nicotiana plumbaginifolia protoplasts and by monitoring the recombination rates using a recently developed transient assay, we were able to obtain insights into the mechanism of extrachromosomal recombination operating in plant cells. An exchange of flanking markers takes place in over 90% of the recombination events. In most of the remaining cases two consecutive, independent single crossover events occur. These events involve the same DNA substrate and lead to two successive exchanges of flanking markers, thus mimicking a presumed double crossover intermediate. A comparison of the outcome of our experiments with the predictions of two recombination models originally proposed for mammalian cells indicates that extrachromosomal recombination in plant cells is best described by the single strand annealing model. According to this model all recombination events result in an exchange of flanking markers. Our results rule out the double strand break repair model which predicts that flanking markers are exchanged in only half of all events.     

Dynamic behavior of histone H1 microinjected into HeLa cells

Histone H1 was purified from bovine thymus and radiolabeled with tritium by reductive methylation or with 125I using chloramine-T. Red blood cell-mediated microinjection was then used to introduce the labeled H1 molecules into HeLa cells synchronized in S phase. The injected H1 molecules rapidly entered HeLa nuclei, and a number of tests indicate that their association with chromatin was equivalent to that endogenous histone H1. The injected molecules copurified with HeLa cell nucleosomes, exhibited a half-life of approximately 100 h, and were hyperphosphorylated at mitosis. When injected HeLa cells were fused with mouse 3T3 fibroblasts less than 10% of the labeled H1 molecules migrated to mouse nuclei during the next 48 h. Thus, the intracellular behavior of histone H1 differs markedly from that of high mobility group proteins 1 and 2 (HMG1 and HMG2), which rapidly equilibrate between human and mouse nuclei after heterokaryon formation (Rechsteiner, M., and L. Kuehl, 1979, Cell, 16:901-908; Wu, L., M. Rechsteiner, and L. Kuehl, 1981, J. Cell Biol, 91: 488-496). Despite their slow rate of migration between nuclei, the injected H1 molecules were evenly distributed on mouse and human genomes soon after mitosis of HeLa-3T3 heterokaryons. These results suggest that although most histone H1 molecules are stably associated with interphase chromatin, they undergo extensive redistribution after mitosis.     

Fate and structure of DNA microinjected into mouse TK L cells

Co-microinjection of single linearized molecules of plasmids containing the human β-globin gene (pRK1) and the herpes simplex virus (HSV) type I thymidine kinase gene (pX1) into the mouse TK^? L cell nucleus results in covalent linkage between these (or derived) molecules within the nucleus as revealed by Southern blotting, plasmid rescue, and recovery of plasmid-derived DNA from a Charon 4A phage library of cellular DNA. The microinjected DNA is predominantly found as high molecular weight DNA as determined by Hirt fractionation. Southern blotting data and recombinants from the Charon 4A library suggest that the plasmid DNA is in the form of a head-to-tail linear concatamer of up to 80 copies. Passage of these microinjected cells in selective medium (HAT) results in coordinate amplification of both plasmids, which are maintained in an approx. 3:1 molar ratio of pRK1 to pX1-derived molecules. Hybridization in situ shows the DNA to be integrated on a translocation chromosome, t(4;4). Integration does not appear to be site-specific, since plasmid DNA from another microinjected cell line, C2B, appears on a different translocation chromosome, t(8?;14). Plasmid rescue experiments confirm a previous finding that passage of pBR322 DNA through eukaryotic cells may result in deletions of normally stable plasmid DNA upon subsequent transformation of E. coli. These deletions appear to occur in the bacteria, and originate in a 128 bp region between the Sal I and Hae II sites of pBR322.     

Promiscuous patching of broken chromosomes in mammalian cells with extrachromosomal DNA

To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we stably transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. Cells were then electroporated with a plasmid expressing endonuclease I-SceI to induce a DSB, and clones that had lost tk function were selected. In a previous study of DSB-induced tk-deficient clones, we found that ~8% of recovered tk mutations involved the capture of one or more DNA fragments at the DSB site. Almost half of the DNA capture events involved the I-SceI expression plasmid, and several events involved retrotransposable elements. To learn whether only certain DNA sequences or motifs are efficiently captured, in the current work we electroporated an I-SceI expression plasmid along with HaeIII fragments of X174 genomic DNA. We report that 18 out of 132 tk-deficient clones recovered had captured DNA fragments, and 14 DNA capture events involved one or more fragments of X174 DNA. Microhomology existed at most junctions between X174 DNA and genomic sequences. Our work suggests that virtually any extrachromosomal DNA molecule may be recruited for the patching of DSBs in a mammalian genome.     

Cationic lipid-mediated intracellular delivery of antibodies into live cells

The ability to introduce antibodies to live cells opens new insights to a wide range of applications, such as protein intracellular trafficking studies, protein interference studies with blocking antibodies, and live immunolabeling or protein phosphorylation states studies. Apart from single-chain format variable (scFv) antibodies, DNA transfection of eukaryotic cells is rarely used to produce antibodies in situ, mainly due to inappropriate folding of the antibody in the cytoplasm. Thus, the development of dedicated carriers is needed since antibodies, which are large, unable to cross the plasma membrane and effective release of the antibody in the cytoplasm need to be overcome. We studied these two crucial steps using a dedicated delivery reagent in live cells and compared the results with immunocytochemistry experiments in fixed cells.     

Formation of extrachromosomal circular DNA in HeLa cells by nonhomologous recombination.

Extrachromosomal circular DNA (eccDNA) generated from chromosomal DNA is found in all mammalian cells and increases with cell stress or aging. Studies of eccDNA structure and mode of formation provide insight into mechanisms of instability of the mammalian genome. Previous studies have suggested that eccDNA is generated through a process involving recombination between repetitive sequences. However, we observed that approximately one half of the small eccDNA fragments cloned from HeLa S3 cells were composed entirely of nonrepetitive or low-copy DNA sequences. We analyzed four of these fragments by polymerase chain reaction and nucleotide sequencing and found that they were complete eccDNAs. We then screened a human genomic library with the eccDNAs to isolate the complementary chromosomal sequences. Comparing the recombination junctions within the eccDNAs with the chromosomal sequences from which they were derived revealed that nonhomologous recombination was involved in their formation. One of the eccDNAs was composed of two separate sequences from different parts of the genome. These results suggest that rejoining of ends of fragmented DNA is responsible for the generation of a substantial portion of the eccDNAs found in HeLa S3 cells.     

Genetic transformation of somatic cells. III. An analysis of the status of the plasmid nucleotide sequences in the extrachromosomal DNA of transformant clone cells and the rescue of extrachromosomal molecules of the plasmid DNA

Extrachromosomal DNAs from TK+ transformant clones of A238 Chinese hamster cells isolated after the treatment with plasmid pST826 containing thymidine kinase gene (TK-gene) of Herpes simplex virus (HSV1) and 1.8 kb insert of human satellite III DNA (HSIII) were studied by hybridization technique. In two TK+-clones (2T301 and 2T16) large quantities of rearranged plasmid DNA molecules were found. Electron microscopy show in clone 2T301 the presence of circular DNAs with average length being 4.64 +/- 0.27 kb. These molecules were rescued by retransformation into E. coli and analysed by restriction mapping and hybridization. All of them contain deletions spanning the entire TK gene of HSV1 and pBR325 sequences situated just downstream from the ORI of replication. The origin of extra-replicating circular DNA in 2T301 clone is discussed.     

Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.     

Multiple mechanisms generate extrachromosomal circular DNA in Chinese hamster ovary cells.

Seven cloned small circular DNA molecules from CHO cells were sequenced and examined for the presence of homologies to each other and to a number of other functional sequences present in transposable elements, retroviruses, mammalian repeat sequences, and introns. The sequences of the CHO cell circular DNA molecules did not reveal common structural features that could explain their presence in the circular DNA population. A gene bank was constructed for CHO chromosomal DNA and sequences homologous to two of the seven small circular DNA molecules were isolated and sequenced. The nucleotide sequences present at the junction of circular and chromosomal DNA suggest that a recombination process involving homologous pairing may have been involved in the generation of one, but not the other, of the two circular DNA molecules.     

A rapid method for the purification of extrachromosomal DNA from eukaryotic cells.

A simple and efficient procedure to purify the low molecular weight extrachromosomal DNA from eukaryotic cells is described. Gentle lysis of cells with urea and sodium dodecyl sulfate in 0.24 M phosphate buffer (pH 6.8) is followed by the removal of high molecular weight bulk DNA by centrifugation. Protein and RNA are removed from the supernatant by hydroxyapatite chromatography in urea/phosphate buffer. Urea is then removed with 0.15 M phosphate buffer and the extrachromosomal DNA, virtually free from protein and RNA, is finally eluted in 0.5 M phosphate buffer. The procedure allows the recovery of about 99% simian virus 40 (SV40) DNA from infected monkey kidney cells in the extrachromosomal fraction. In normal mouse, monkey, andhuman cells, approximately 1% of total cell DNA appears to be extrachromosomal.     

Visualization and quantitative analysis of extrachromosomal telomere-repeat DNA in individual human cells by Halo-FISH

Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain <5 ECTR DNA molecules/nucleus. ECTR DNA in ALT cells exhibit striking cell-to-cell variations in number (<20 to >300), range widely in length (<1 to >200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but <4% in U2OS cells. In addition to its use in ALT cell analysis, Halo-FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells.     

Tracking molecular particles in live cells using fuzzy rule-based system.

Recent development of detection techniques of molecular particles in live cells has stimulated interest in developing the new powerful techniques to track the molecular particles in live cells. One special type of cellular microscopy images is about the formation and transportation of clathrin-coated pits and vesicles. Clathrin-coated pits are very important in studying the behavior of proteins and lipids in live cells. To answer the question, whether there exist "hot spots" for the formation of Clathrin-coated pits or the pits and arrays formed randomly on the plasma membrane, it is necessary to track many hundreds of individual pits dynamically in live-cell microscope movies to capture and monitor how pits and vesicles were formed. Therefore, a motion correspondence algorithm based on fuzzy rule-based system is proposed to resolve the problem of ambiguous association encountered in these dynamic, live-cell images of clathrin assemblies. Results show that this method can accurately track most of the particles in the high volume images.     

Toxicity and extrachromosomal DNA in strains of the cyanobacterium Microcystis aeruginosa

Abstract Correlations were sought between toxicity and the presence of plasmids in toxic and non-toxic strains of Microcystis aeruginosa . Plasmids were present in toxic and non-toxic cultures. Cultivation of the toxic Microcystis PCC7820 in the presence of novobiocin did not influence toxicity, although extrachromosomal DNA was no longer detected after novobiocin treatment. The data indicate that it is unlikely that plasmids are involved in the toxicity of Microcystis PCC7820.     

Tracking bio‐molecules in live cells using quantum dots

Single particle tracking (SPT) techniques were developed to explore bio‐molecules dynamics in live cells at single molecule sensitivity and nanometer spatial resolution. Recent developments in quantum dots (Qdots) surface coating and bio‐conjugation schemes have made them most suitable probes for live cell applications. Here we review recent advancements in using quantum dots as SPT probes for live cell experiments. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha–beta–PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA–PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha–alpha and alpha–PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha–alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)₂.