Extrachromosomal MicroDNAs and Chromosomal Microdeletions in Normal Tissues

Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, USA.
Science (Impact Factor: 33.61). 03/2012; 336(6077):82-6. DOI: 10.1126/science.1213307
Source: PubMed


We have identified tens of thousands of short extrachromosomal circular DNAs (microDNA) in mouse tissues as well as mouse and human cell lines. These microDNAs are 200 to 400 base pairs long, are derived from unique nonrepetitive sequence, and are enriched in the 5'-untranslated regions of genes, exons, and CpG islands. Chromosomal loci that are enriched sources of microDNA in the adult brain are somatically mosaic for microdeletions that appear to arise from the excision of microDNAs. Germline microdeletions identified by the "Thousand Genomes" project may also arise from the excision of microDNAs in the germline lineage. We have thus identified a previously unknown DNA entity in mammalian cells and provide evidence that their generation leaves behind deletions in different genomic loci.

Download full-text


Available from: Jack Griffith, Mar 26, 2014
  • Source
    • "Extrachromosomal DNA was prepared for visualization by electron microscopy by direct mounting as described previously (Shibata et al., 2012). For quantification, microDNAs from a defined number of cells were mounted, and 30 randomly selected images were captured from across the grid and the number of circles counted and normalized to the cell count. "
    [Show abstract] [Hide abstract]
    ABSTRACT: MicroDNAs are <400-base extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs have been found in all tissue types, including sperm. MicroDNAs arise preferentially from areas with high gene density, GC content, and exon density from promoters with activating chromatin modifications and in sperm from the 5'-UTR of full-length LINE-1 elements, but are depleted from lamin-associated heterochromatin. Analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cells defective in various DNA repair proteins reveals that homologous recombination and non-homologous end joining repair pathways are not required for microDNA production. Deletion of the MSH3 DNA mismatch repair protein results in a significant decrease in microDNA abundance, specifically from non-CpG genomic regions. Thus, microDNAs arise as part of normal cellular physiology-either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 06/2015; 11(11). DOI:10.1016/j.celrep.2015.05.020 · 8.36 Impact Factor
  • Source
    • "On the other hand, circular molecules excised from the chromosome arm have been observed as extrachromosomal closed circular (ecc) DNAs [36]–[38] or small polydisperse circular (spc) DNAs [39]. Furthermore, recent techniques have identified tens of thousands of kinds of extrachromosomal circular molecules (micro DNAs) in cells from normal mouse tissues or human cell lines [40]. Because IR/MAR sequences are scattered throughout the human genome at 50–100 kb intervals, it is very likely that some circular molecules of genomic origin contain IR/MARs, and therefore behave similarly to the IR/MAR plasmid. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The gene amplification plays a critical role in the malignant transformation of mammalian cells. The most widespread method for amplifying a target gene in cell culture is the use of methotrexate (Mtx) treatment to amplify dihydrofolate reductase (Dhfr). Whereas, we found that a plasmid bearing both a mammalian origin of replication (initiation region; IR) and a matrix attachment region (MAR) was spontaneously amplified in mammalian cells. In this study, we attempted to uncover the underlying mechanism by which the IR/MAR sequence might accelerate Mtx induced Dhfr amplification. The plasmid containing the IR/MAR was extrachromosomally amplified, and then integrated at multiple chromosomal locations within individual cells, increasing the likelihood that the plasmid might be inserted into a chromosomal environment that permits high expression and further amplification. Efficient amplification of this plasmid alleviated the genotoxicity of Mtx. Clone-based cytogenetic and sequence analysis revealed that the plasmid was amplified in a chromosomal context by breakage-fusion-bridge cycles operating either at the plasmid repeat or at the flanking fragile site activated by Mtx. This mechanism explains how a circular molecule bearing IR/MAR sequences of chromosomal origin might be amplified under replication stress, and also provides insight into gene amplification in human cancer.
    PLoS ONE 07/2014; 9(7):e103439. DOI:10.1371/journal.pone.0103439 · 3.23 Impact Factor
  • Source
    • "A possible explanation for the unstable pattern of “HGT” detected here is, as suggested earlier, the presence of extrachromosomal DNA in E. chlorotica that may encode foreign genes and/or gene fragments. Extrachromosomal DNA has been reported in a number of organisms ranging from protozoa to vertebrates (Cohen et al. 2007; Cohen and Segal 2009), and in several cases, its formation and/or disappearance is linked to specific developmental changes (Noto et al. 2003), and more recently, to distinct gene regions (Shibata et al. 2012). It is, therefore, possible that the acquisition of DNA from algal food in the sea slug differs among individuals each generation and is correlated with feeding. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The sea slug Elysia chlorotica offers a unique opportunity to study the evolution of a novel function (photosynthesis) in a complex multicellular host. Elysia chlorotica harvests plastids (absent of nuclei) from its heterokont algal prey, Vaucheria litorea. The “stolen” plastids are maintained for several months in cells of the digestive tract and are essential for animal development. The basis of long-term maintenance of photosynthesis in this sea slug was thought to be explained by extensive horizontal gene transfer (HGT) from the nucleus of the alga to the animal nucleus, followed by expression of algal genes in the gut to provide essential plastid-destined proteins. Early studies of target genes and proteins supported the HGT hypothesis, but more recent genome-wide data provide conflicting results. Here, we generated significant genome data from the E. chlorotica germ line (egg DNA) and from V. litorea to test the HGT hypothesis. Our comprehensive analyses fail to provide evidence for alga-derived HGT into the germ line of the sea slug. Polymerase chain reaction analyses of genomic DNA and cDNA from different individual E. chlorotica suggest, however, that algal nuclear genes (or gene fragments) are present in the adult slug. We suggest that these nucleic acids may derive from and/or reside in extrachromosomal DNAs that are made available to the animal through contact with the alga. These data resolve a long-standing issue and suggest that HGT is not the primary reason underlying long-term maintenance of photosynthesis in E. chlorotica. Therefore, sea slug photosynthesis is sustained in as yet unexplained ways that do not appear to endanger the animal germ line through the introduction of dozens of foreign genes.
    Molecular Biology and Evolution 05/2013; 30(8). DOI:10.1093/molbev/mst084 · 9.11 Impact Factor
Show more