Contrasting Patterns of rDNA Homogenization within the Zygosaccharomyces rouxii Species Complex

PubMed Central

Chand Dakal, Tikam; Giudici, Paolo; Solieri, Lisa

2016-01-01

Arrays of repetitive ribosomal DNA (rDNA) sequences are generally expected to evolve as a coherent family, where repeats within such a family are more similar to each other than to orthologs in related species. The continuous homogenization of repeats within individual genomes is a recombination process termed concerted evolution. Here, we investigated the extent and the direction of concerted evolution in 43 yeast strains of the Zygosaccharomyces rouxii species complex (Z. rouxii, Z. sapae, Z. mellis), by analyzing two portions of the 35S rDNA cistron, namely the D1/D2 domains at the 5’ end of the 26S rRNA gene and the segment including the internal transcribed spacers (ITS) 1 and 2 (ITS regions). We demonstrate that intra-genomic rDNA sequence variation is unusually frequent in this clade and that rDNA arrays in single genomes consist of an intermixing of Z. rouxii, Z. sapae and Z. mellis-like sequences, putatively evolved by reticulate evolutionary events that involved repeated hybridization between lineages. The levels and distribution of sequence polymorphisms vary across rDNA repeats in different individuals, reflecting four patterns of rDNA evolution: I) rDNA repeats that are homogeneous within a genome but are chimeras derived from two parental lineages via recombination: Z. rouxii in the ITS region and Z. sapae in the D1/D2 region; II) intra-genomic rDNA repeats that retain polymorphisms only in ITS regions; III) rDNA repeats that vary only in their D1/D2 domains; IV) heterogeneous rDNA arrays that have both polymorphic ITS and D1/D2 regions. We argue that an ongoing process of homogenization following allodiplodization or incomplete lineage sorting gave rise to divergent evolutionary trajectories in different strains, depending upon temporal, structural and functional constraints. We discuss the consequences of these findings for Zygosaccharomyces species delineation and, more in general, for yeast barcoding. PMID:27501051

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA.

PubMed Central

Khan, A S; Repaske, R; Garon, C F; Chan, H W; Rowe, W P; Martin, M A

1982-01-01

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA. Images PMID:6281459

Heterochromatic siRNAs and DDM1 Independently Silence Aberrant 5S rDNA Transcripts in Arabidopsis

PubMed Central

Blevins, Todd; Pontes, Olga; Pikaard, Craig S.; Meins, Frederick

2009-01-01

5S ribosomal RNA gene repeats are arranged in heterochromatic arrays (5S rDNA) situated near the centromeres of Arabidopsis chromosomes. The chromatin remodeling factor DDM1 is known to maintain 5S rDNA methylation patterns while silencing transcription through 5S rDNA intergenic spacers (IGS). We mapped small-interfering RNAs (siRNA) to a composite 5S rDNA repeat, revealing a high density of siRNAs matching silenced IGS transcripts. IGS transcript repression requires proteins of the heterochromatic siRNA pathway, including RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent. DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats. We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays. PMID:19529764

Environmental stress induces trinucleotide repeat mutagenesis in human cells

PubMed Central

Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A.; Yotnda, Patricia; Wilson, John H.

2015-01-01

The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)—the cause of multiple human diseases—have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential. PMID:25775519

Environmental stress induces trinucleotide repeat mutagenesis in human cells.

PubMed

Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A; Yotnda, Patricia; Wilson, John H

2015-03-24

The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)-the cause of multiple human diseases-have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential.

Molecular characterization of the canine mitochondrial DNA control region for forensic applications.

PubMed

Eichmann, Cordula; Parson, Walther

2007-09-01

The canine mitochondrial DNA (mtDNA) control region of 133 dogs living in the area around Innsbruck, Austria was sequenced. A total of 40 polymorphic sites were observed in the first hypervariable segment and 15 in the second, which resulted in the differentiation of 40 distinct haplotypes. We observed five nucleotide positions that were highly polymorphic within different haplogroups, and they represent good candidates for mtDNA screening. We found five point heteroplasmic positions; all located in HVS-I and a polythymine region in HVS-II, the latter often being associated with length heteroplasmy. In contrast to human mtDNA, the canine control region contains a hypervariable 10 nucleotide repeat region, which is located between the two hypervariable regions. In our population sample, we observed eight different repeat types, which we characterized by direct sequencing and fragment length analysis. The discrimination power of the canine mtDNA control region was 0.93, not taking the polymorphic repeat region into consideration.

Coordinated DNA dynamics during the human telomerase catalytic cycle

NASA Astrophysics Data System (ADS)

Parks, Joseph W.; Stone, Michael D.

2014-06-01

The human telomerase reverse transcriptase (hTERT) utilizes a template within the integral RNA subunit (hTR) to direct extension of telomeres. Telomerase exhibits repeat addition processivity (RAP) and must therefore translocate the nascent DNA product into a new RNA:DNA hybrid register to prime each round of telomere repeat synthesis. Here, we use single-molecule FRET and nuclease protection assays to monitor telomere DNA structure and dynamics during the telomerase catalytic cycle. DNA translocation during RAP proceeds through a previously uncharacterized kinetic substep during which the 3‧-end of the DNA substrate base pairs downstream within the hTR template. The rate constant for DNA primer realignment reveals this step is not rate limiting for RAP, suggesting a second slow conformational change repositions the RNA:DNA hybrid into the telomerase active site and drives the extrusion of the 5‧-end of the DNA primer out of the enzyme complex.

Heteroplasmy and evidence for recombination in the mitochondrial control region of the flatfish Platichthys flesus.

PubMed

Hoarau, Galice; Holla, Suzanne; Lescasse, Rachel; Stam, Wytze T; Olsen, Jeanine L

2002-12-01

The general assumption that mitochondrial DNA (mtDNA) does not undergo recombination has been challenged recently in invertebrates. Here we present the first direct evidence for recombination in the mtDNA of a vertebrate, the flounder Platichthys flesus. The control region in the mtDNA of this flatfish is characterized by the presence of a variable number of tandem repeats and a high level of heteroplasmy. Two types of repeats were recognized, differing by two C-T point mutations. Most individuals carry a pure "C" or a pure "T" array, but one individual showed a compound "CT" array. Such a compound array is evidence for recombination in the mtDNA control region from the flounder.

Slipped-strand mispairing at noncontiguous repeats in Poecilia reticulata: a model for minisatellite birth.

PubMed Central

Taylor, J S; Breden, F

2000-01-01

The standard slipped-strand mispairing (SSM) model for the formation of variable number tandem repeats (VNTRs) proposes that a few tandem repeats, produced by chance mutations, provide the "raw material" for VNTR expansion. However, this model is unlikely to explain the formation of VNTRs with long motifs (e.g., minisatellites), because the likelihood of a tandem repeat forming by chance decreases rapidly as the length of the repeat motif increases. Phylogenetic reconstruction of the birth of a mitochondrial (mt) DNA minisatellite in guppies suggests that VNTRs with long motifs can form as a consequence of SSM at noncontiguous repeats. VNTRs formed in this manner have motifs longer than the noncontiguous repeat originally formed by chance and are flanked by one unit of the original, noncontiguous repeat. SSM at noncontiguous repeats can therefore explain the birth of VNTRs with long motifs and the "imperfect" or "short direct" repeats frequently observed adjacent to both mtDNA and nuclear VNTRs. PMID:10880490

Selective recognition of N4-methylcytosine in DNA by engineered transcription-activator-like effectors.

PubMed

Rathi, Preeti; Maurer, Sara; Summerer, Daniel

2018-06-05

The epigenetic DNA nucleobases 5-methylcytosine (5mC) and N 4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'. © 2018 The Author(s).

Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability

PubMed Central

Reddy, Kaalak; Schmidt, Monika H.M.; Geist, Jaimie M.; Thakkar, Neha P.; Panigrahi, Gagan B.; Wang, Yuh-Hwa; Pearson, Christopher E.

2014-01-01

R-loops, transcriptionally-induced RNA:DNA hybrids, occurring at repeat tracts (CTG)n, (CAG)n, (CGG)n, (CCG)n and (GAA)n, are associated with diseases including myotonic dystrophy, Huntington's disease, fragile X and Friedreich's ataxia. Many of these repeats are bidirectionally transcribed, allowing for single- and double-R-loop configurations, where either or both DNA strands may be RNA-bound. R-loops can trigger repeat instability at (CTG)·(CAG) repeats, but the mechanism of this is unclear. We demonstrate R-loop-mediated instability through processing of R-loops by HeLa and human neuron-like cell extracts. Double-R-loops induced greater instability than single-R-loops. Pre-treatment with RNase H only partially suppressed instability, supporting a model in which R-loops directly generate instability by aberrant processing, or via slipped-DNA formation upon RNA removal and its subsequent aberrant processing. Slipped-DNAs were observed to form following removal of the RNA from R-loops. Since transcriptionally-induced R-loops can occur in the absence of DNA replication, R-loop processing may be a source of repeat instability in the brain. Double-R-loop formation and processing to instability was extended to the expanded C9orf72 (GGGGCC)·(GGCCCC) repeats, known to cause amyotrophic lateral sclerosis and frontotemporal dementia, providing the first suggestion through which these repeats may become unstable. These findings provide a mechanistic basis for R-loop-mediated instability at disease-associated repeats. PMID:25147206

High-throughput STR analysis for DNA database using direct PCR.

PubMed

Sim, Jeong Eun; Park, Su Jeong; Lee, Han Chul; Kim, Se-Yong; Kim, Jong Yeol; Lee, Seung Hwan

2013-07-01

Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high-throughput and cost-effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter-loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to <3%. In conclusion, the newly developed direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time- and cost-saving manner. © 2013 American Academy of Forensic Sciences Published 2013. This article is a U.S. Government work and is in the public domain in the U.S.A.

Identification of presumed ancestral DNA sequences of phaseolin in Phaseolus vulgaris.

PubMed Central

Kami, J; Velásquez, V B; Debouck, D G; Gepts, P

1995-01-01

Common bean (Phaseolus vulgaris) consists of two major geographic gene pools, one distributed in Mexico, Central America, and Colombia and the other in the southern Andes (southern Peru, Bolivia, and Argentina). Amplification and sequencing of members of the multigene family coding for phaseolin, the major seed storage protein of the common bean, provide evidence for accumulation of tandem direct repeats in both introns and exons during evolution of the multigene family in this species. The presumed ancestral phaseolin sequences, without tandem repeats, were found in recently discovered but nearly extinct wild common bean populations of Ecuador and northern Peru that are intermediate between the two major gene pools of the species based on geographical and molecular arguments. Our results illustrate the usefulness of tandem direct repeats in establishing the polarity of DNA sequence divergence and therefore in proposing phylogenies. Images Fig. 1 Fig. 3 PMID:7862642

Structures of ω repressors bound to direct and inverted DNA repeats explain modulation of transcription

PubMed Central

Weihofen, Wilhelm Andreas; Cicek, Aslan; Pratto, Florencia; Alonso, Juan Carlos; Saenger, Wolfram

2006-01-01

Repressor ω regulates transcription of genes required for copy number control, accurate segregation and stable maintenance of inc18 plasmids hosted by Gram-positive bacteria. ω belongs to homodimeric ribbon-helix-helix (RHH2) repressors typified by a central, antiparallel β-sheet for DNA major groove binding. Homodimeric ω2 binds cooperatively to promotors with 7 to 10 consecutive non-palindromic DNA heptad repeats (5′-A/TATCACA/T-3′, symbolized by →) in palindromic inverted, converging (→←) or diverging (←→) orientation and also, unique to ω2 and contrasting other RHH2 repressors, to non-palindromic direct (→→) repeats. Here we investigate with crystal structures how ω2 binds specifically to heptads in minimal operators with (→→) and (→←) repeats. Since the pseudo-2-fold axis relating the monomers in ω2 passes the central C–G base pair of each heptad with ∼0.3 Å downstream offset, the separation between the pseudo-2-fold axes is exactly 7 bp in (→→), ∼0.6 Å shorter in (→←) but would be ∼0.6 Å longer in (←→). These variations grade interactions between adjacent ω2 and explain modulations in cooperative binding affinity of ω2 to operators with different heptad orientations. PMID:16528102

Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system

NASA Technical Reports Server (NTRS)

Dar, M. E.; Jorgensen, T. J.

1995-01-01

Using the radiomimetic drug, bleomycin, we have determined the mutagenic potential of DNA strand breaks in the shuttle vector pZ189 in human fibroblasts. The bleomycin treatment conditions used produce strand breaks with 3'-phosphoglycolate termini as > 95% of the detectable dose-dependent lesions. Breaks with this end group represent 50% of the strand break damage produced by ionizing radiation. We report that such strand breaks are mutagenic lesions. The type of mutation produced is largely determined by the type of strand break on the plasmid (i.e. single versus double). Mutagenesis studies with purified DNA forms showed that nicked plasmids (i.e. those containing single-strand breaks) predominantly produce base substitutions, the majority of which are multiples, which presumably originate from error-prone polymerase activity at strand break sites. In contrast, repair of linear plasmids (i.e. those containing double-strand breaks) mainly results in deletions at short direct repeat sequences, indicating the involvement of illegitimate recombination. The data characterize the nature of mutations produced by single- and double-strand breaks in human cells, and suggests that deletions at direct repeats may be a 'signature' mutation for the processing of DNA double-strand breaks.

Mitochondrial genome maintenance: roles for nuclear nonhomologous end-joining proteins in Saccharomyces cerevisiae.

PubMed

Kalifa, Lidza; Quintana, Daniel F; Schiraldi, Laura K; Phadnis, Naina; Coles, Garry L; Sia, Rey A; Sia, Elaine A

2012-03-01

Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat-mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial double-strand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria.

Quantitative analysis of TALE-DNA interactions suggests polarity effects.

PubMed

Meckler, Joshua F; Bhakta, Mital S; Kim, Moon-Soo; Ovadia, Robert; Habrian, Chris H; Zykovich, Artem; Yu, Abigail; Lockwood, Sarah H; Morbitzer, Robert; Elsäesser, Janett; Lahaye, Thomas; Segal, David J; Baldwin, Enoch P

2013-04-01

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN > NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

Genome-Wide Stochastic Adaptive DNA Amplification at Direct and Inverted DNA Repeats in the Parasite Leishmania

PubMed Central

Plourde, Marie; Gingras, Hélène; Roy, Gaétan; Lapointe, Andréanne; Leprohon, Philippe; Papadopoulou, Barbara; Corbeil, Jacques; Ouellette, Marc

2014-01-01

Gene amplification of specific loci has been described in all kingdoms of life. In the protozoan parasite Leishmania, the product of amplification is usually part of extrachromosomal circular or linear amplicons that are formed at the level of direct or inverted repeated sequences. A bioinformatics screen revealed that repeated sequences are widely distributed in the Leishmania genome and the repeats are chromosome-specific, conserved among species, and generally present in low copy number. Using sensitive PCR assays, we provide evidence that the Leishmania genome is continuously being rearranged at the level of these repeated sequences, which serve as a functional platform for constitutive and stochastic amplification (and deletion) of genomic segments in the population. This process is adaptive as the copy number of advantageous extrachromosomal circular or linear elements increases upon selective pressure and is reversible when selection is removed. We also provide mechanistic insights on the formation of circular and linear amplicons through RAD51 recombinase-dependent and -independent mechanisms, respectively. The whole genome of Leishmania is thus stochastically rearranged at the level of repeated sequences, and the selection of parasite subpopulations with changes in the copy number of specific loci is used as a strategy to respond to a changing environment. PMID:24844805

Transcription arrest by a G quadruplex forming-trinucleotide repeat sequence from the human c-myb gene.

PubMed

Broxson, Christopher; Beckett, Joshua; Tornaletti, Silvia

2011-05-17

Non canonical DNA structures correspond to genomic regions particularly susceptible to genetic instability. The transcription process facilitates formation of these structures and plays a major role in generating the instability associated with these genomic sites. However, little is known about how non canonical structures are processed when encountered by an elongating RNA polymerase. Here we have studied the behavior of T7 RNA polymerase (T7RNAP) when encountering a G quadruplex forming-(GGA)(4) repeat located in the human c-myb proto-oncogene. To make direct correlations between formation of the structure and effects on transcription, we have taken advantage of the ability of the T7 polymerase to transcribe single-stranded substrates and of G4 DNA to form in single-stranded G-rich sequences in the presence of potassium ions. Under physiological KCl concentrations, we found that T7 RNAP transcription was arrested at two sites that mapped to the c-myb (GGA)(4) repeat sequence. The extent of arrest did not change with time, indicating that the c-myb repeat represented an absolute block and not a transient pause to T7 RNAP. Consistent with G4 DNA formation, arrest was not observed in the absence of KCl or in the presence of LiCl. Furthermore, mutations in the c-myb (GGA)(4) repeat, expected to prevent transition to G4, also eliminated the transcription block. We show T7 RNAP arrest at the c-myb repeat in double-stranded DNA under conditions mimicking the cellular concentration of biomolecules and potassium ions, suggesting that the G4 structure formed in the c-myb repeat may represent a transcription roadblock in vivo. Our results support a mechanism of transcription-coupled DNA repair initiated by arrest of transcription at G4 structures.

Length Variation in Mitochondrial DNA of the Minnow Cyprinella Spiloptera

PubMed Central

Broughton, R. E.; Dowling, T. E.

1994-01-01

Length differences in animal mitochondrial DNA (mtDNA) are common, frequently due to variation in copy number of direct tandem duplications. While such duplications appear to form without great difficulty in some taxonomic groups, they appear to be relatively short-lived, as typical duplication products are geographically restricted within species and infrequently shared among species. To better understand such length variation, we have studied a tandem and direct duplication of approximately 260 bp in the control region of the cyprinid fish, Cyprinella spiloptera. Restriction site analysis of 38 individuals was used to characterize population structure and the distribution of variation in repeat copy number. This revealed two length variants, including individuals with two or three copies of the repeat, and little geographic structure among populations. No standard length (single copy) genomes were found and heteroplasmy, a common feature of length variation in other taxa, was absent. Nucleotide sequence of tandem duplications and flanking regions localized duplication junctions in the phenylalanine tRNA and near the origin of replication. The locations of these junctions and the stability of folded repeat copies support the hypothesized importance of secondary structures in models of duplication formation. PMID:8001785

Divergence, differential methylation and interspersion of melon satellite DNA sequences.

PubMed Central

Shmookler Reis, R; Timmis, J N; Ingle, J

1981-01-01

Melon (Cucumis melo) satellite DNA consists of two components, Q and S, each with a buoyant density in CsCl of 1.707 g/ml, but differing by 9 degrees C in "melting" temperature. These physical properties appear to be in contradiction, since both depend on G + C content. In order to resolve this anomaly, base compositions were directly determined for isolated fractions. the low-"melting" component S contains 41.8% G + C, with 6% of C present as 5-methylcytosine, whereas Q DNA contains 54% G + C, with 41% of C methylated. Analyses of restriction site loss agreed well with the direct determinations of methylation and divergence, and indicated some clustering of methylated sites in Q DNA. Analysis of restricted main-band DNA by hydridization with RNA complementary to Q satellite DNA ("Southern transfer") showed satellite Q tandem arrays interspersed in DNA of main-band density. Sequence divergence and extent of methylation did not appear to depend on whether a repeat array was present as satellite or interspersed in main-band DNA. Hydridization in situ indicated considerable heterogeneity in the genomic proportion of the Q-DNA sequences in melon fruit nuclei, implying over- and under-representation consistent with extensive unequal recombination in satellite Q tandem arrays. The cucumber, Cucumis sativus, contains less than 8% as much Q-homologous DNA per genome as the melon, suggesting rapid evolutionary gain or loss of these tandem repeat sequences. Images Fig. 2. PLATE 1 Fig. 4. Fig. 10. PMID:6172117

Structural basis of DNA sequence recognition by the response regulator PhoP in Mycobacterium tuberculosis.

PubMed

He, Xiaoyuan; Wang, Liqin; Wang, Shuishu

2016-04-15

The transcriptional regulator PhoP is an essential virulence factor in Mycobacterium tuberculosis, and it presents a target for the development of new anti-tuberculosis drugs and attenuated tuberculosis vaccine strains. PhoP binds to DNA as a highly cooperative dimer by recognizing direct repeats of 7-bp motifs with a 4-bp spacer. To elucidate the PhoP-DNA binding mechanism, we determined the crystal structure of the PhoP-DNA complex. The structure revealed a tandem PhoP dimer that bound to the direct repeat. The surprising tandem arrangement of the receiver domains allowed the four domains of the PhoP dimer to form a compact structure, accounting for the strict requirement of a 4-bp spacer and the highly cooperative binding of the dimer. The PhoP-DNA interactions exclusively involved the effector domain. The sequence-recognition helix made contact with the bases of the 7-bp motif in the major groove, and the wing interacted with the adjacent minor groove. The structure provides a starting point for the elucidation of the mechanism by which PhoP regulates the virulence of M. tuberculosis and guides the design of screening platforms for PhoP inhibitors.

Phylogeographic patterns of Armillaria ostoyae in the western United States

Treesearch

J. W. Hanna; N. B. Klopfenstein; M. -S. Kim; G. I. McDonald; J. A. Moore

2007-01-01

Nuclear ribosomal DNA regions (i.e. large subunit, internal transcribed spacer, 5.8S and intergenic spacer) were sequenced using a direct-polymerase chain reaction method from Armillaria ostoyae genets collected from the western USA. Many of the A. ostoyae genets contained heterogeneity among rDNA repeats, indicating intragenomic variation and likely intraspecific...

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.

PubMed

Blochlinger, K; Diggelmann, H

1984-12-01

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells.

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.

PubMed Central

Blochlinger, K; Diggelmann, H

1984-01-01

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells. Images PMID:6098829

Clustered regularly interspaced short palindromic repeats (CRISPRs) for the genotyping of bacterial pathogens.

PubMed

Grissa, Ibtissem; Vergnaud, Gilles; Pourcel, Christine

2009-01-01

Clustered regularly interspaced short palindromic repeats (CRISPRs) are DNA sequences composed of a succession of repeats (23- to 47-bp long) separated by unique sequences called spacers. Polymorphism can be observed in different strains of a species and may be used for genotyping. We describe protocols and bioinformatics tools that allow the identification of CRISPRs from sequenced genomes, their comparison, and their component determination (the direct repeats and the spacers). A schematic representation of the spacer organization can be produced, allowing an easy comparison between strains.

Identification of apple cultivars on the basis of simple sequence repeat markers.

PubMed

Liu, G S; Zhang, Y G; Tao, R; Fang, J G; Dai, H Y

2014-09-12

DNA markers are useful tools that play an important role in plant cultivar identification. They are usually based on polymerase chain reaction (PCR) and include simple sequence repeats (SSRs), inter-simple sequence repeats, and random amplified polymorphic DNA. However, DNA markers were not used effectively in the complete identification of plant cultivars because of the lack of known DNA fingerprints. Recently, a novel approach called the cultivar identification diagram (CID) strategy was developed to facilitate the use of DNA markers for separate plant individuals. The CID was designed whereby a polymorphic maker was generated from each PCR that directly allowed for cultivar sample separation at each step. Therefore, it could be used to identify cultivars and varieties easily with fewer primers. In this study, 60 apple cultivars, including a few main cultivars in fields and varieties from descendants (Fuji x Telamon) were examined. Of the 20 pairs of SSR primers screened, 8 pairs gave reproducible, polymorphic DNA amplification patterns. The banding patterns obtained from these 8 primers were used to construct a CID map. Each cultivar or variety in this study was distinguished from the others completely, indicating that this method can be used for efficient cultivar identification. The result contributed to studies on germplasm resources and the seedling industry in fruit trees.

In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

PubMed

Paca-Uccaralertkun, S; Zhao, L J; Adya, N; Cross, J V; Cullen, B R; Boros, I M; Giam, C Z

1994-01-01

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.

Flexible DNA binding of the BTB/POZ-domain protein FBI-1.

PubMed

Pessler, Frank; Hernandez, Nouria

2003-08-01

POZ-domain transcription factors are characterized by the presence of a protein-protein interaction domain called the POZ or BTB domain at their N terminus and zinc fingers at their C terminus. Despite the large number of POZ-domain transcription factors that have been identified to date and the significant insights that have been gained into their cellular functions, relatively little is known about their DNA binding properties. FBI-1 is a BTB/POZ-domain protein that has been shown to modulate HIV-1 Tat trans-activation and to repress transcription of some cellular genes. We have used various viral and cellular FBI-1 binding sites to characterize the interaction of a POZ-domain protein with DNA in detail. We find that FBI-1 binds to inverted sequence repeats downstream of the HIV-1 transcription start site. Remarkably, it binds efficiently to probes carrying these repeats in various orientations and spacings with no particular rotational alignment, indicating that its interaction with DNA is highly flexible. Indeed, FBI-1 binding sites in the adenovirus 2 major late promoter, the c-fos gene, and the c-myc P1 and P2 promoters reveal variously spaced direct, inverted, and everted sequence repeats with the consensus sequence G(A/G)GGG(T/C)(C/T)(T/C)(C/T) for each repeat.

DNA topoisomerase 1α promotes transcriptional silencing of transposable elements through DNA methylation and histone lysine 9 dimethylation in Arabidopsis.

PubMed

Dinh, Thanh Theresa; Gao, Lei; Liu, Xigang; Li, Dongming; Li, Shengben; Zhao, Yuanyuan; O'Leary, Michael; Le, Brandon; Schmitz, Robert J; Manavella, Pablo A; Manavella, Pablo; Li, Shaofang; Weigel, Detlef; Pontes, Olga; Ecker, Joseph R; Chen, Xuemei

2014-07-01

RNA-directed DNA methylation (RdDM) and histone H3 lysine 9 dimethylation (H3K9me2) are related transcriptional silencing mechanisms that target transposable elements (TEs) and repeats to maintain genome stability in plants. RdDM is mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti-cancer properties that targets DNA topoisomerase 1α (TOP1α) was able to de-repress LUCL by reducing its DNA methylation and H3K9me2 levels. Further studies with Arabidopsis top1α mutants showed that TOP1α silences endogenous RdDM loci by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at TEs and repeats. This study assigned a new role in epigenetic silencing to an enzyme that affects DNA topology.

Msh2-Msh3 Interferes with Okazaki Fragment Processing to Promote Trinucleotide Repeat Expansions

PubMed Central

Kantartzis, Athena; Williams, Gregory M.; Balakrishnan, Lata; Roberts, Rick L.; Surtees, Jennifer A.; Bambara, Robert A.

2012-01-01

Summary Trinucleotide repeat (TNR) expansions are the underlying cause of more than forty neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington’s disease. Although genetic evidence has attributed the cause of these diseases to errors in DNA replication and/or repair, clear molecular mechanisms have not been described. We have focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. We further provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA Ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging strand DNA replication. PMID:22938864

Msh2-Msh3 interferes with Okazaki fragment processing to promote trinucleotide repeat expansions.

PubMed

Kantartzis, Athena; Williams, Gregory M; Balakrishnan, Lata; Roberts, Rick L; Surtees, Jennifer A; Bambara, Robert A

2012-08-30

Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease. Although genetic evidence points to errors in DNA replication and/or repair as the cause of these diseases, clear molecular mechanisms have not been described. Here, we focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. Furthermore, we provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging-strand DNA replication. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Absence of MutSbeta leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks

PubMed Central

Slean, Meghan M.; Panigrahi, Gagan B.; Castel, Arturo López; Pearson, August B.; Tomkinson, Alan E.; Pearson, Christopher E.

2016-01-01

Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats. PMID:27155933

Direct single-stranded DNA binding by Teb1 mediates the recruitment of Tetrahymena thermophila telomerase to telomeres.

PubMed

Upton, Heather E; Hong, Kyungah; Collins, Kathleen

2014-11-15

The eukaryotic reverse transcriptase telomerase copies its internal RNA template to synthesize telomeric DNA repeats at chromosome ends in balance with sequence loss during cell proliferation. Previous work has established several factors involved in telomerase recruitment to telomeres in yeast and mammalian cells; however, it remains unclear what determines the association of telomerase with telomeres in other organisms. Here we investigate the cell cycle dependence of telomere binding by each of the seven Tetrahymena thermophila telomerase holoenzyme proteins TERT, p65, Teb1, p50, p75, p45, and p19. We observed coordinate cell cycle-regulated recruitment and release of all of the subunits, including the telomeric-repeat DNA-binding subunit Teb1. Using domain truncation and mutagenesis approaches, we investigated which subunits govern the interaction of telomerase holoenzyme with telomeres. Our results show that Teb1 is critical for telomere interaction of other holoenzyme subunits and demonstrate that high-affinity Teb1 DNA-binding activity is necessary and sufficient for cell cycle-regulated telomere association. Overall, these and additional findings indicate that in the ciliate Tetrahymena, telomerase recruitment to telomeres requires direct binding to single-stranded DNA, unlike the indirect DNA recognition through telomere-bound proteins essential in yeast and mammalian cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

SV40 host-substituted variants: a new look at the monkey DNA inserts and recombinant junctions.

PubMed

Singer, Maxine; Winocour, Ernest

2011-04-10

The available monkey genomic data banks were examined in order to determine the chromosomal locations of the host DNA inserts in 8 host-substituted SV40 variant DNAs. Five of the 8 variants contained more than one linked monkey DNA insert per tandem repeat unit and in all cases but one, the 19 monkey DNA inserts in the 8 variants mapped to different locations in the monkey genome. The 50 parental DNAs (32 monkey and 18 SV40 DNA segments) which spanned the crossover and flanking regions that participated in monkey/monkey and monkey/SV40 recombinations were characterized by substantial levels of microhomology of up to 8 nucleotides in length; the parental DNAs also exhibited direct and inverted repeats at or adjacent to the crossover sequences. We discuss how the host-substituted SV40 variants arose and the nature of the recombination mechanisms involved. Copyright © 2011 Elsevier Inc. All rights reserved.

High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase.

PubMed

Romi, Erez; Baran, Nava; Gantman, Marina; Shmoish, Michael; Min, Bosun; Collins, Kathleen; Manor, Haim

2007-05-22

Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in functional enzyme complexes. Our assays reveal contact of the single-stranded DNA adjacent to the primer-template hybrid and tTERT residue W187 at the periphery of the N-terminal domain. This contact was detected in complexes with three different registers of template in the active site, suggesting that it is maintained throughout synthesis of a complete telomeric repeat. Substitution of nearby residue Q168, but not W187, alters the K(m) for primer elongation, implying that it plays a role in the DNA recognition. These findings are the first to directly demonstrate the physical location of TERT-DNA contacts in catalytically active telomerase and to identify amino acid determinants of DNA binding affinity. Our data also suggest a movement of the TERT active site relative to the template-adjacent single-stranded DNA binding site within a cycle of repeat synthesis.

Identification of TTAGGG-binding proteins in Neurospora crassa, a fungus with vertebrate-like telomere repeats.

PubMed

Casas-Vila, Núria; Scheibe, Marion; Freiwald, Anja; Kappei, Dennis; Butter, Falk

2015-11-17

To date, telomere research in fungi has mainly focused on Saccharomyces cerevisiae and Schizosaccharomyces pombe, despite the fact that both yeasts have degenerated telomeric repeats in contrast to the canonical TTAGGG motif found in vertebrates and also several other fungi. Using label-free quantitative proteomics, we here investigate the telosome of Neurospora crassa, a fungus with canonical telomeric repeats. We show that at least six of the candidates detected in our screen are direct TTAGGG-repeat binding proteins. While three of the direct interactors (NCU03416 [ncTbf1], NCU01991 [ncTbf2] and NCU02182 [ncTay1]) feature the known myb/homeobox DNA interaction domain also found in the vertebrate telomeric factors, we additionally show that a zinc-finger protein (NCU07846) and two proteins without any annotated DNA-binding domain (NCU02644 and NCU05718) are also direct double-strand TTAGGG binders. We further find two single-strand binders (NCU02404 [ncGbp2] and NCU07735 [ncTcg1]). By quantitative label-free interactomics we identify TTAGGG-binding proteins in Neurospora crassa, suggesting candidates for telomeric factors that are supported by phylogenomic comparison with yeast species. Intriguingly, homologs in yeast species with degenerated telomeric repeats are also TTAGGG-binding proteins, e.g. in S. cerevisiae Tbf1 recognizes the TTAGGG motif found in its subtelomeres. However, there is also a subset of proteins that is not conserved. While a rudimentary core TTAGGG-recognition machinery may be conserved across yeast species, our data suggests Neurospora as an emerging model organism with unique features.

CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats.

PubMed

Bland, Charles; Ramsey, Teresa L; Sabree, Fareedah; Lowe, Micheal; Brown, Kyndall; Kyrpides, Nikos C; Hugenholtz, Philip

2007-06-18

Clustered Regularly Interspaced Palindromic Repeats (CRISPRs) are a novel type of direct repeat found in a wide range of bacteria and archaea. CRISPRs are beginning to attract attention because of their proposed mechanism; that is, defending their hosts against invading extrachromosomal elements such as viruses. Existing repeat detection tools do a poor job of identifying CRISPRs due to the presence of unique spacer sequences separating the repeats. In this study, a new tool, CRT, is introduced that rapidly and accurately identifies CRISPRs in large DNA strings, such as genomes and metagenomes. CRT was compared to CRISPR detection tools, Patscan and Pilercr. In terms of correctness, CRT was shown to be very reliable, demonstrating significant improvements over Patscan for measures precision, recall and quality. When compared to Pilercr, CRT showed improved performance for recall and quality. In terms of speed, CRT proved to be a huge improvement over Patscan. Both CRT and Pilercr were comparable in speed, however CRT was faster for genomes containing large numbers of repeats. In this paper a new tool was introduced for the automatic detection of CRISPR elements. This tool, CRT, showed some important improvements over current techniques for CRISPR identification. CRT's approach to detecting repetitive sequences is straightforward. It uses a simple sequential scan of a DNA sequence and detects repeats directly without any major conversion or preprocessing of the input. This leads to a program that is easy to describe and understand; yet it is very accurate, fast and memory efficient, being O(n) in space and O(nm/l) in time.

CRISPR Recognition Tool (CRT): a tool for automatic detection ofclustered regularly interspaced palindromic repeats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bland, Charles; Ramsey, Teresa L.; Sabree, Fareedah

Clustered Regularly Interspaced Palindromic Repeats (CRISPRs) are a novel type of direct repeat found in a wide range of bacteria and archaea. CRISPRs are beginning to attract attention because of their proposed mechanism; that is, defending their hosts against invading extrachromosomal elements such as viruses. Existing repeat detection tools do a poor job of identifying CRISPRs due to the presence of unique spacer sequences separating the repeats. In this study, a new tool, CRT, is introduced that rapidly and accurately identifies CRISPRs in large DNA strings, such as genomes and metagenomes. CRT was compared to CRISPR detection tools, Patscan andmore » Pilercr. In terms of correctness, CRT was shown to be very reliable, demonstrating significant improvements over Patscan for measures precision, recall and quality. When compared to Pilercr, CRT showed improved performance for recall and quality. In terms of speed, CRT also demonstrated superior performance, especially for genomes containing large numbers of repeats. In this paper a new tool was introduced for the automatic detection of CRISPR elements. This tool, CRT, was shown to be a significant improvement over the current techniques for CRISPR identification. CRT's approach to detecting repetitive sequences is straightforward. It uses a simple sequential scan of a DNA sequence and detects repeats directly without any major conversion or preprocessing of the input. This leads to a program that is easy to describe and understand; yet it is very accurate, fast and memory efficient, being O(n) in space and O(nm/l) in time.« less

Translocation and gross deletion breakpoints in human inherited disease and cancer II: Potential involvement of repetitive sequence elements in secondary structure formation between DNA ends.

PubMed

Chuzhanova, Nadia; Abeysinghe, Shaun S; Krawczak, Michael; Cooper, David N

2003-09-01

Translocations and gross deletions are responsible for a significant proportion of both cancer and inherited disease. Although such gene rearrangements are nonuniformly distributed in the human genome, the underlying mutational mechanisms remain unclear. We have studied the potential involvement of various types of repetitive sequence elements in the formation of secondary structure intermediates between the single-stranded DNA ends that recombine during rearrangements. Complexity analysis was used to assess the potential of these ends to form secondary structures, the maximum decrease in complexity consequent to a gross rearrangement being used as an indicator of the type of repeat and the specific DNA ends involved. A total of 175 pairs of deletion/translocation breakpoint junction sequences available from the Gross Rearrangement Breakpoint Database [GRaBD; www.uwcm.ac.uk/uwcm/mg/grabd/grabd.html] were analyzed. Potential secondary structure was noted between the 5' flanking sequence of the first breakpoint and the 3' flanking sequence of the second breakpoint in 49% of rearrangements and between the 5' flanking sequence of the second breakpoint and the 3' flanking sequence of the first breakpoint in 36% of rearrangements. Inverted repeats, inversions of inverted repeats, and symmetric elements were found in association with gross rearrangements at approximately the same frequency. However, inverted repeats and inversions of inverted repeats accounted for the vast majority (83%) of deletions plus small insertions, symmetric elements for one-half of all antigen receptor-mediated translocations, while direct repeats appear only to be involved in mediating simple deletions. These findings extend our understanding of illegitimate recombination by highlighting the importance of secondary structure formation between single-stranded DNA ends at breakpoint junctions. Copyright 2003 Wiley-Liss, Inc.

DIRECT BINDING OF GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE TO TELOMERIC DNA PROTECTS TELOMERES AGAINST CHEMOTHERAPY-INDUCED RAPID DEGRADATION

PubMed Central

Demarse, Neil A.; Ponnusamy, Suriyan; Spicer, Eleanor K.; Apohan, Elif; Baatz, John E.; Ogretmen, Besim; Davies, Christopher

2009-01-01

GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is a glycolytic enzyme that displays several non-glycolytic activities, including the maintenance and/or protection of telomeres. In this study, we determined the molecular mechanism and biological role of the interaction between GAPDH and human telomeric DNA. Using gel shift assays, we show that recombinant GAPDH binds directly with high affinity (Kd = 45 nM) to a single-stranded oligonucleotide comprising three telomeric DNA repeats and that nucleotides T1, G5 and G6 of the TTAGGG repeat are essential for binding. The stoichiometry of the interaction is 2:1 (DNA: GAPDH), and GAPDH appears to form a high-molecular weight complex when bound to the oligonucleotide. Mutation of Asp32 and Cys149, which are localized to the NAD-binding site and the active site center of GAPDH, respectively, produced mutants that almost completely lost their telomere-binding functions both in vitro and in situ (in A549 human lung cancer cells). Treatment of A549 cells with the chemotherapeutic agents gemcitabine and doxorubicin resulted in increased nuclear localization of expressed wild-type GAPDH, where it protected telomeres against rapid degradation, concomitant with increased resistance to the growth inhibitory effects of these drugs. The non-DNA-binding mutants of GAPDH also localized to the nucleus when expressed in A549 cells, but did not confer any significant protection of telomeres against chemotherapy-induced degradation or growth inhibition, and this occurred without the involvement of caspase activation or apoptosis regulation. Overall, these data demonstrate that GAPDH binds telomeric DNA directly in vitro and may have a biological role in the protection of telomeres against rapid degradation in response to chemotherapeutic agents in A549 human lung cancer cells. PMID:19800890

Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells

NASA Technical Reports Server (NTRS)

Dar, M. E.; Winters, T. A.; Jorgensen, T. J.

1997-01-01

Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

DDM1 represses noncoding RNA expression and RNA-directed DNA methylation in heterochromatin.

PubMed

Tan, Feng; Lu, Yue; Jiang, Wei; Zhao, Yu; Wu, Tian; Zhang, Ruoyu; Zhou, Dao-Xiu

2018-05-24

Cytosine methylation of DNA, which occurs at CG, CHG, and CHH (H=A, C, or T) sequences in plants, is a hallmark for epigenetic repression of repetitive sequences. The chromatin remodeling factor DECREASE IN DNA METHYLATION1 (DDM1) is essential for DNA methylation, especially at CG and CHG sequences. However, its potential role in RNA-directed DNA methylation (RdDM) and in chromatin function is not completely understood in rice (Oryza sativa). In this work, we used high-throughput approaches to study the function of rice DDM1 (OsDDM1) in RdDM and the expression of non-coding RNA (ncRNA). We show that loss of function of OsDDM1 results in ectopic CHH methylation of transposable elements and repeats. The ectopic CHH methylation was dependent on rice DOMAINS REARRANGED METHYLTRANSFERASE2 (OsDRM2), a DNA methyltransferase involved in RdDM. Mutations in OsDDM1 lead to decreases of histone H3K9me2 and increases in the levels of heterochromatic small RNA (sRNA) and long noncoding RNA (lncRNA). In particular, OsDDM1 was found to be essential to repress transcription of the two repetitive sequences, Centromeric Retrotransposons of Rice1 (CRR1) and the dominant centromeric CentO repeats. These results suggest that OsDDM1 antagonizes RdDM at heterochromatin and represses tissue-specific expression of ncRNA from repetitive sequences in the rice genome. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

PubMed

Warmerdam, Daniël O; van den Berg, Jeroen; Medema, René H

2016-03-22

rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Multiple bidirectional initiations and terminations of transcription in the Marek's disease virus long repeat regions.

PubMed Central

Chen, X B; Velicer, L F

1991-01-01

Marek's disease is an oncogenic disease of chickens caused by a herpesvirus, Marek's disease virus (MDV). Serial in vitro passage of pathogenic MDV results in amplification of a 132-bp direct repeat in the MDV genome's TRL and IRL repeat regions and loss of tumorigenicity. This led to the hypothesis that upon such expansion, one or more tumor-inducing genes fail to be expressed. In this report a group of cDNAs mapping in the expanded regions were isolated from a pathogenic MDV strain in which the 132-bp direct repeat number was found to range between one and seven. Partial cDNA sequencing and S1 nuclease protection analysis revealed that the corresponding transcripts are either initiated or terminated within or near the expanded regions at multiple sites in both rightward and leftward directions. Furthermore, each 132-bp repeat contains one TATA box and two polyadenylation consensus sequences in each direction. These RNAs contain a partial copy or one or more full copies of the 132-bp direct repeat at either their 5' or 3' end. Northern (RNA) blot analysis showed that the majority of transcripts are 1.8 kb in size, while the minor species range in size from 0.67 to 3.1 kb. Together, these data raise the possibility that the 132-bp direct repeat, and indirectly its copy number, may be involved in the regulation of transcriptional initiation and termination and therefore in the generation of four groups of transcripts from the TRL and IRL, although this remains to be demonstrated. Images PMID:1850022

R-loops: targets for nuclease cleavage and repeat instability.

PubMed

Freudenreich, Catherine H

2018-01-11

R-loops form when transcribed RNA remains bound to its DNA template to form a stable RNA:DNA hybrid. Stable R-loops form when the RNA is purine-rich, and are further stabilized by DNA secondary structures on the non-template strand. Interestingly, many expandable and disease-causing repeat sequences form stable R-loops, and R-loops can contribute to repeat instability. Repeat expansions are responsible for multiple neurodegenerative diseases, including Huntington's disease, myotonic dystrophy, and several types of ataxias. Recently, it was found that R-loops at an expanded CAG/CTG repeat tract cause DNA breaks as well as repeat instability (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Two factors were identified as causing R-loop-dependent breaks at CAG/CTG tracts: deamination of cytosines and the MutLγ (Mlh1-Mlh3) endonuclease, defining two new mechanisms for how R-loops can generate DNA breaks (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Following R-loop-dependent nicking, base excision repair resulted in repeat instability. These results have implications for human repeat expansion diseases and provide a paradigm for how RNA:DNA hybrids can cause genome instability at structure-forming DNA sequences. This perspective summarizes mechanisms of R-loop-induced fragility at G-rich repeats and new links between DNA breaks and repeat instability.

Short Tandem Repeat DNA Internet Database

National Institute of Standards and Technology Data Gateway

SRD 130 Short Tandem Repeat DNA Internet Database (Web, free access)   Short Tandem Repeat DNA Internet Database is intended to benefit research and application of short tandem repeat DNA markers for human identity testing. Facts and sequence information on each STR system, population data, commonly used multiplex STR systems, PCR primers and conditions, and a review of various technologies for analysis of STR alleles have been included.

Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase.

PubMed

Fu, Changlin; Donovan, William P; Shikapwashya-Hasser, Olga; Ye, Xudong; Cole, Robert H

2014-01-01

Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17-30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50 °C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90-95%.

Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

PubMed Central

Fu, Changlin; Donovan, William P.; Shikapwashya-Hasser, Olga; Ye, Xudong; Cole, Robert H.

2014-01-01

Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17–30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50°C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90–95%. PMID:25551825

DNA looping by FokI: the impact of synapse geometry on loop topology at varied site orientations

PubMed Central

Rusling, David A.; Laurens, Niels; Pernstich, Christian; Wuite, Gijs J. L.; Halford, Stephen E.

2012-01-01

Most restriction endonucleases, including FokI, interact with two copies of their recognition sequence before cutting DNA. On DNA with two sites they act in cis looping out the intervening DNA. While many restriction enzymes operate symmetrically at palindromic sites, FokI acts asymmetrically at a non-palindromic site. The directionality of its sequence means that two FokI sites can be bridged in either parallel or anti-parallel alignments. Here we show by biochemical and single-molecule biophysical methods that FokI aligns two recognition sites on separate DNA molecules in parallel and that the parallel arrangement holds for sites in the same DNA regardless of whether they are in inverted or repeated orientations. The parallel arrangement dictates the topology of the loop trapped between sites in cis: the loop from inverted sites has a simple 180° bend, while that with repeated sites has a convoluted 360° turn. The ability of FokI to act at asymmetric sites thus enabled us to identify the synapse geometry for sites in trans and in cis, which in turn revealed the relationship between synapse geometry and loop topology. PMID:22362745

Target Site Recognition by a Diversity-Generating Retroelement

PubMed Central

Guo, Huatao; Tse, Longping V.; Nieh, Angela W.; Czornyj, Elizabeth; Williams, Steven; Oukil, Sabrina; Liu, Vincent B.; Miller, Jeff F.

2011-01-01

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification. PMID:22194701

Stabilization of perfect and imperfect tandem repeats by single-strand DNA exonucleases

PubMed Central

Feschenko, Vladimir V.; Rajman, Luis A.; Lovett, Susan T.

2003-01-01

Rearrangements between tandemly repeated DNA sequences are a common source of genetic instability. Such rearrangements underlie several human genetic diseases. In many organisms, the mismatch-repair (MMR) system functions to stabilize repeats when the repeat unit is short or when sequence imperfections are present between the repeats. We show here that the action of single-stranded DNA (ssDNA) exonucleases plays an additional, important role in stabilizing tandem repeats, independent of their role in MMR. For perfect repeats of ≈100 bp in Escherichia coli that are not susceptible to MMR, exonuclease (Exo)-I, ExoX, and RecJ exonuclease redundantly inhibit deletion. Our data suggest that >90% of potential deletion events are avoided by the combined action of these three exonucleases. Imperfect tandem repeats, less prone to rearrangements, are stabilized by both the MMR-pathway and ssDNA-specific exonucleases. For 100-bp repeats containing four mispairs, ExoI alone aborts most deletion events, even in the presence of a functional MMR system. By genetic analysis, we show that the inhibitory effect of ssDNA exonucleases on deletion formation is independent of the MutS and UvrD proteins. Exonuclease degradation of DNA displaced during the deletion process may abort slipped misalignment. Exonuclease action is therefore a significant force in genetic stabilization of many forms of repetitive DNA. PMID:12538867

The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mak, Amanda Nga-Sze; Bradley, Philip; Cernadas, Raul A.

2012-02-10

DNA recognition by TAL effectors is mediated by tandem repeats, each 33 to 35 residues in length, that specify nucleotides via unique repeat-variable diresidues (RVDs). The crystal structure of PthXo1 bound to its DNA target was determined by high-throughput computational structure prediction and validated by heavy-atom derivatization. Each repeat forms a left-handed, two-helix bundle that presents an RVD-containing loop to the DNA. The repeats self-associate to form a right-handed superhelix wrapped around the DNA major groove. The first RVD residue forms a stabilizing contact with the protein backbone, while the second makes a base-specific contact to the DNA sense strand.more » Two degenerate amino-terminal repeats also interact with the DNA. Containing several RVDs and noncanonical associations, the structure illustrates the basis of TAL effector-DNA recognition.« less

Discovery of Escherichia coli CRISPR sequences in an undergraduate laboratory.

PubMed

Militello, Kevin T; Lazatin, Justine C

2017-05-01

Clustered regularly interspaced short palindromic repeats (CRISPRs) represent a novel type of adaptive immune system found in eubacteria and archaebacteria. CRISPRs have recently generated a lot of attention due to their unique ability to catalog foreign nucleic acids, their ability to destroy foreign nucleic acids in a mechanism that shares some similarity to RNA interference, and the ability to utilize reconstituted CRISPR systems for genome editing in numerous organisms. In order to introduce CRISPR biology into an undergraduate upper-level laboratory, a five-week set of exercises was designed to allow students to examine the CRISPR status of uncharacterized Escherichia coli strains and to allow the discovery of new repeats and spacers. Students started the project by isolating genomic DNA from E. coli and amplifying the iap CRISPR locus using the polymerase chain reaction (PCR). The PCR products were analyzed by Sanger DNA sequencing, and the sequences were examined for the presence of CRISPR repeat sequences. The regions between the repeats, the spacers, were extracted and analyzed with BLASTN searches. Overall, CRISPR loci were sequenced from several previously uncharacterized E. coli strains and one E. coli K-12 strain. Sanger DNA sequencing resulted in the discovery of 36 spacer sequences and their corresponding surrounding repeat sequences. Five of the spacers were homologous to foreign (non-E. coli) DNA. Assessment of the laboratory indicates that improvements were made in the ability of students to answer questions relating to the structure and function of CRISPRs. Future directions of the laboratory are presented and discussed. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(3):262-269, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.

Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase

PubMed Central

Schawalder, James; Paric, Enesa; Neff, Norma F

2003-01-01

Background Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci. Results Chromatin immunoprecipitation of BLM complexes recovered telomere and ribosomal DNA repeats. The N-terminus of BLM, required for NB localization, is the same as the telomere association domain of BLM. The C-terminus is required for ribosomal DNA localization. BLM localizes primarily to the non-transcribed spacer region of the ribosomal DNA repeat where replication forks initiate. Bloom syndrome cells expressing the deletion alleles lacking the ribosomal DNA and telomere association domains have altered cell cycle populations with increased S or G2/M cells relative to normal. Conclusion These results identify telomere and ribosomal DNA repeated sequence elements as chromosomal targets for the BLM DNA helicase during the S/G2 phase of the cell cycle. BLM is localized in nuclear bodies when it associates with telomeric repeats in both telomerase positive and negative cells. The BLM DNA helicase participates in genomic stability at ribosomal DNA repeats and telomeres. PMID:14577841

Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double-strand break in the genome.

PubMed

Odom, Obed W; Baek, Kwang-Hyun; Dani, Radhika N; Herrin, David L

2008-03-01

Certain group I introns insert into intronless DNA via an endonuclease that creates a double-strand break (DSB). There are two models for intron homing in phage: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR). The Cr.psbA4 intron homes efficiently from a plasmid into the chloroplast psbA gene in Chlamydomonas, but little is known about the mechanism. Analysis of co-transformants selected using a spectinomycin-resistant 16S gene (16S(spec)) provided evidence for both pathways. We also examined the consequences of the donor DNA having only one-sided or no homology with the psbA gene. When there was no homology with the donor DNA, deletions of up to 5 kb involving direct repeats that flank the psbA gene were obtained. Remarkably, repeats as short as 15 bp were used for this repair, which is consistent with the single-strand annealing (SSA) pathway. When the donor had one-sided homology, the DSB in most co-transformants was repaired using two DNAs, the donor and the 16S(spec) plasmid, which, coincidentally, contained a region that is repeated upstream of psbA. DSB repair using two separate DNAs provides further evidence for the SDSA pathway. These data show that the chloroplast can repair a DSB using short dispersed repeats located proximally, distally, or even on separate molecules relative to the DSB. They also provide a rationale for the extensive repertoire of repeated sequences in this genome.

Expanded CAG/CTG Repeat DNA Induces a Checkpoint Response That Impacts Cell Proliferation in Saccharomyces cerevisiae

PubMed Central

Sundararajan, Rangapriya; Freudenreich, Catherine H.

2011-01-01

Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases. PMID:21437275

Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

PubMed Central

Bergquist, Helen; Rocha, Cristina S. J.; Álvarez-Asencio, Rubén; Nguyen, Chi-Hung; Rutland, Mark. W.; Smith, C. I. Edvard; Good, Liam; Nielsen, Peter E.; Zain, Rula

2016-01-01

Expansion of (GAA)n repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich’s ataxia. (GAA)n expansions form non-canonical structures, including intramolecular triplex (H-DNA), and R-loops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)n expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)n repeats of different lengths (short: n=9, medium: n=75 or long: n=115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA)4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT)5-PNA did not. We present evidence that (GAA)4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT)5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression. PMID:27846236

Molecular Dynamics Simulations of DNA-Free and DNA-Bound TAL Effectors

PubMed Central

Wan, Hua; Hu, Jian-ping; Li, Kang-shun; Tian, Xu-hong; Chang, Shan

2013-01-01

TAL (transcriptional activator-like) effectors (TALEs) are DNA-binding proteins, containing a modular central domain that recognizes specific DNA sequences. Recently, the crystallographic studies of TALEs revealed the structure of DNA-recognition domain. In this article, molecular dynamics (MD) simulations are employed to study two crystal structures of an 11.5-repeat TALE, in the presence and absence of DNA, respectively. The simulated results indicate that the specific binding of RVDs (repeat-variable diresidues) with DNA leads to the markedly reduced fluctuations of tandem repeats, especially at the two ends. In the DNA-bound TALE system, the base-specific interaction is formed mainly by the residue at position 13 within a TAL repeat. Tandem repeats with weak RVDs are unfavorable for the TALE-DNA binding. These observations are consistent with experimental studies. By using principal component analysis (PCA), the dominant motions are open-close movements between the two ends of the superhelical structure in both DNA-free and DNA-bound TALE systems. The open-close movements are found to be critical for the recognition and binding of TALE-DNA based on the analysis of free energy landscape (FEL). The conformational analysis of DNA indicates that the 5′ end of DNA target sequence has more remarkable structural deformability than the other sites. Meanwhile, the conformational change of DNA is likely associated with the specific interaction of TALE-DNA. We further suggest that the arrangement of N-terminal repeats with strong RVDs may help in the design of efficient TALEs. This study provides some new insights into the understanding of the TALE-DNA recognition mechanism. PMID:24130757

Variable-Number Tandem Repeats That Are Useful in Genotyping Isolates of Salmonella enterica subsp. enterica Serovars Typhimurium and Newport▿

PubMed Central

Witonski, D. ; Stefanova, R.; Ranganathan, A.; Schutze, G. E.; Eisenach, K. D.; Cave, M. D.

2006-01-01

The genome of Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 was analyzed for direct repeats, and 54 sequences containing variable-number tandem repeat loci were identified. Ten primer pairs that anneal upstream and downstream of each selected locus were designed and used to amplify PCR targets in isolates of S. enterica serovars Typhimurium and Newport. Four of the 10 loci did not show polymorphism in the length of products. Six loci were selected for analysis. Isolates of S. enterica serovars Typhimurium and Newport that were related to specific outbreaks and showed identical pulsed-field gel electrophoresis patterns were indistinguishable by the length of the six variable-number tandem repeats. Isolates that differed in their pulsed-field gel electrophoresis patterns showed polymorphism in variable-number tandem repeat profiles. Length of the products was confirmed by DNA sequence analysis. Only 2 of the 10 loci contained exact integers of the direct repeat. Eight loci contained partial copies. The partial copies were maintained at the ends of the variable-number tandem repeat loci in all isolates. In spite of having partial copies that were maintained in all isolates, the number of direct repeats at a locus was polymorphic. Six variable-number tandem repeat loci were useful in distinguishing isolates of S. enterica serovars Typhimurium and Newport that had different pulsed-field gel electrophoresis patterns and in identifying outbreak-associated cases that shared a common pulsed-field gel pattern. PMID:16943354

Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage*

PubMed Central

Acevedo, Julyana; Yan, Shan; Michael, W. Matthew

2016-01-01

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes. PMID:27129245

Damage-induced ectopic recombination in the yeast Saccharomyces cerevisiae.

PubMed

Kupiec, M; Steinlauf, R

1997-06-09

Mitotic recombination in the yeast Saccharomyces cerevisiae is induced when cells are irradiated with UV or X-rays, reflecting the efficient repair of damage by recombinational repair mechanisms. We have used multiply marked haploid strains that allow the simultaneous detection of several types of ectopic recombination events. We show that inter-chromosomal ectopic conversion of lys2 heteroalleles and, to a lesser extent, direct repeat recombination (DRR) between non-tandem repeats, are increased by DNA-damaging agents; in contrast, ectopic recombination of the naturally occurring Ty element is not induced. We have tested several hypotheses that could explain the preferential lack of induction of Ty recombination by DNA-damaging agents. We have found that the lack of induction cannot be explained by a cell cycle control or by an effect of the mating-type genes. We also found no role for the flanking long terminal repeats (LTRs) of the Ty in preventing the induction. Ectopic conversion, DRR, and forward mutation of artificial repeats show different kinetics of induction at various positions of the cell cycle, reflecting different mechanisms of recombination. We discuss the mechanistic and evolutionary aspects of these results.

Direct observation of TALE protein dynamics reveals a two-state search mechanism

PubMed Central

Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M.

2015-01-01

Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins for which the fundamental mechanisms governing the search process are not fully understood. Here we use single-molecule techniques to directly observe TALE search dynamics along DNA templates. We find that TALE proteins are capable of rapid diffusion along DNA using a combination of sliding and hopping behaviour, which suggests that the TALE search process is governed in part by facilitated diffusion. We also observe that TALE proteins exhibit two distinct modes of action during the search process—a search state and a recognition state—facilitated by different subdomains in monomeric TALE proteins. Using TALE truncation mutants, we further demonstrate that the N-terminal region of TALEs is required for the initial non-specific binding and subsequent rapid search along DNA, whereas the central repeat domain is required for transitioning into the site-specific recognition state. PMID:26027871

Direct observation of TALE protein dynamics reveals a two-state search mechanism.

PubMed

Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M

2015-06-01

Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins for which the fundamental mechanisms governing the search process are not fully understood. Here we use single-molecule techniques to directly observe TALE search dynamics along DNA templates. We find that TALE proteins are capable of rapid diffusion along DNA using a combination of sliding and hopping behaviour, which suggests that the TALE search process is governed in part by facilitated diffusion. We also observe that TALE proteins exhibit two distinct modes of action during the search process-a search state and a recognition state-facilitated by different subdomains in monomeric TALE proteins. Using TALE truncation mutants, we further demonstrate that the N-terminal region of TALEs is required for the initial non-specific binding and subsequent rapid search along DNA, whereas the central repeat domain is required for transitioning into the site-specific recognition state.

MMS Exposure Promotes Increased MtDNA Mutagenesis in the Presence of Replication-Defective Disease-Associated DNA Polymerase γ Variants

PubMed Central

Stumpf, Jeffrey D.; Copeland, William C.

2014-01-01

Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but suppresses MMS-induced mutagenesis. These results suggest a novel mechanism wherein mutations that lead to hypermutation by DNA base-damaging agents and associate with mitochondrial disease may contribute to previously unexplained phenomena, such as the wide variation of age of disease onset and acquired mitochondrial toxicities. PMID:25340760

MMS exposure promotes increased MtDNA mutagenesis in the presence of replication-defective disease-associated DNA polymerase γ variants.

PubMed

Stumpf, Jeffrey D; Copeland, William C

2014-10-01

Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but suppresses MMS-induced mutagenesis. These results suggest a novel mechanism wherein mutations that lead to hypermutation by DNA base-damaging agents and associate with mitochondrial disease may contribute to previously unexplained phenomena, such as the wide variation of age of disease onset and acquired mitochondrial toxicities.

Integrating a high-force optical trap with gold nanoposts and a robust gold-DNA bond.

PubMed

Paik, D Hern; Seol, Yeonee; Halsey, Wayne A; Perkins, Thomas T

2009-08-01

Gold-thiol chemistry is widely used in nanotechnology but has not been exploited in optical-trapping experiments due to laser-induced ablation of gold. We circumvented this problem by using an array of gold nanoposts (r = 50-250 nm, h approximately 20 nm) that allowed for quantitative optical-trapping assays without direct irradiation of the gold. DNA was covalently attached to the gold via dithiol phosphoramidite (DTPA). By using three DTPAs, the gold-DNA bond was not cleaved in the presence of excess thiolated compounds. This chemical robustness allowed us to reduce nonspecific sticking by passivating the unreacted gold with methoxy-(polyethylene glycol)-thiol. We routinely achieved single beads anchored to the nanoposts by single DNA molecules. We measured DNA's elasticity and its overstretching transition, demonstrating moderate- and high-force optical-trapping assays using gold-thiol chemistry. Force spectroscopy measurements were consistent with the rupture of the strepavidin-biotin bond between the bead and the DNA. This implied that the DNA remained anchored to the surface due to the strong gold-thiol bond. Consistent with this conclusion, we repeatedly reattached the trapped bead to the same individual DNA molecule. Thus, surface conjugation of biomolecules onto an array of gold nanostructures by chemically and mechanically robust bonds provides a unique way to carry out spatially controlled, repeatable measurements of single molecules.

Analysis of the genome of fish lymphocystis disease virus isolated directly from epidermal tumours of pleuronectes.

PubMed

Darai, G; Anders, K; Koch, H G; Delius, H; Gelderblom, H; Samalecos, C; Flügel, R M

1983-04-30

Virions of fish lymphocystis disease virus (FLDV), a member of the iridovirus family, were isolated directly from lymphocystis disease lesions of individual flatfishes and purified by sucrose and subsequent cesium chloride gradient centrifugation to homogeneity as judged by electron microscopy. The isolated FLDV DNAs appear to be heterogeneous in size. Contour length measurements of 43 DNA molecules gave an average length of 49 +/- 23 microns, corresponding to 93 +/- 44 X 10(6) D. Molecular weight estimations of FLDV DNA by restriction enzyme analysis resulted in only 64.8 X 10(6) D indicating an excess length of the DNA of about 50%. FLDV DNA was sensitive to lambda 5'-exonuclease and to E. coli 3'-exonuclease III without preference of any one terminal DNA restriction fragment. Denaturation and reannealing experiments of FLDV DNA resulted in the formation of circular DNA molecules of 34.25 microns contour length (= 65.22 X 10(6) D). This result suggests that FLDV DNA contains directly repeated sequences at both ends and that it is terminally redundant. FLDV DNA is methylated in cytosine. FLDV DNA did not hybridize with frog virus DNA indicating that the two iridoviruses are not closely related to each other. Restriction enzyme analysis and Southern blot hybridizations revealed that FLDV isolates can be classified into two different strains: FLDV strain 1 occurs in flounders and plaice, whereas strain 2 is usually found in lesions of dabs.

Morphology and genome organization of the virus PSV of the hyperthermophilic archaeal genera Pyrobaculum and Thermoproteus: a novel virus family, the Globuloviridae.

PubMed

Häring, Monika; Peng, Xu; Brügger, Kim; Rachel, Reinhard; Stetter, Karl O; Garrett, Roger A; Prangishvili, David

2004-06-01

A novel virus, termed Pyrobaculum spherical virus (PSV), is described that infects anaerobic hyperthermophilic archaea of the genera Pyrobaculum and Thermoproteus. Spherical enveloped virions, about 100 nm in diameter, contain a major multimeric 33-kDa protein and host-derived lipids. A viral envelope encases a superhelical nucleoprotein core containing linear double-stranded DNA. The PSV infection cycle does not cause lysis of host cells. The viral genome was sequenced and contains 28337 bp. The genome is unique for known archaeal viruses in that none of the genes, including that encoding the major structural protein, show any significant sequence matches to genes in public sequence databases. Exceptionally for an archaeal double-stranded DNA virus, almost all the recognizable genes are located on one DNA strand. The ends of the genome consist of 190-bp inverted repeats that contain multiple copies of short direct repeats. The two DNA strands are probably covalently linked at their termini. On the basis of the unusual morphological and genomic properties of this DNA virus, we propose to assign PSV to a new viral family, the Globuloviridae.

Formation of Linear Amplicons with Inverted Duplications in Leishmania Requires the MRE11 Nuclease

PubMed Central

Laffitte, Marie-Claude N.; Genois, Marie-Michelle; Mukherjee, Angana; Légaré, Danielle; Masson, Jean-Yves; Ouellette, Marc

2014-01-01

Extrachromosomal DNA amplification is frequent in the protozoan parasite Leishmania selected for drug resistance. The extrachromosomal amplified DNA is either circular or linear, and is formed at the level of direct or inverted homologous repeated sequences that abound in the Leishmania genome. The RAD51 recombinase plays an important role in circular amplicons formation, but the mechanism by which linear amplicons are formed is unknown. We hypothesized that the Leishmania infantum DNA repair protein MRE11 is required for linear amplicons following rearrangements at the level of inverted repeats. The purified LiMRE11 protein showed both DNA binding and exonuclease activities. Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents. The MRE11−/− parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity. These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment. PMID:25474106

Rolling Circle Amplification For Spatially Directed Synthesis Of A Solid Phase Anchored Single-Stranded DNA Molecule

NASA Astrophysics Data System (ADS)

Reiß, Edda; Hölzel, Ralph; von Nickisch-Rosenegk, Markus; Bier, Frank F.

2006-09-01

In this article the usefulness of the enzyme phi29 DNA polymerase and the principle of rolling circle amplification (RCA) for creating single-stranded DNA (ssDNA) nanostructures is described. Currently we are working on the spatial orientation of a growing ssDNA molecule during its RCA-based synthesis by the application of a hydrodynamic force. Starting at an immobilized primer at single molecule level, the aim is to construct a nanostructure of known location and orientation, providing multiple repeating binding sites that can be addressed via complementary base-pairing. Proof-of-principle experiments demonstrate the potential of the enzymatic reaction. ssDNA molecules of more than 20 μm length were created at an immobilized primer and detected by means of fluorescence microscopy.

Characterization of a cDNA encoding a protein involved in formation of the skeleton during development of the sea urchin Lytechinus pictus.

PubMed

Livingston, B T; Shaw, R; Bailey, A; Wilt, F

1991-12-01

In order to investigate the role of proteins in the formation of mineralized tissues during development, we have isolated a cDNA that encodes a protein that is a component of the organic matrix of the skeletal spicule of the sea urchin, Lytechinus pictus. The expression of the RNA encoding this protein is regulated over development and is localized to the descendents of the micromere lineage. Comparison of the sequence of this cDNA to homologous cDNAs from other species of urchin reveal that the protein is basic and contains three conserved structural motifs: a signal peptide, a proline-rich region, and an unusual region composed of a series of direct repeats. Studies on the protein encoded by this cDNA confirm the predicted reading frame deduced from the nucleotide sequence and show that the protein is secreted and not glycosylated. Comparison of the amino acid sequence to databases reveal that the repeat domain is similar to proteins that form a unique beta-spiral supersecondary structure.

Replication intermediates of the linear mitochondrial DNA of Candida parapsilosis suggest a common recombination based mechanism for yeast mitochondria.

PubMed

Gerhold, Joachim M; Sedman, Tiina; Visacka, Katarina; Slezakova, Judita; Tomaska, Lubomir; Nosek, Jozef; Sedman, Juhan

2014-08-15

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria*

PubMed Central

Gerhold, Joachim M.; Sedman, Tiina; Visacka, Katarina; Slezakova, Judita; Tomaska, Lubomir; Nosek, Jozef; Sedman, Juhan

2014-01-01

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. PMID:24951592

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae.

PubMed

Galli, Alvaro; Cervelli, Tiziana; Schiestl, Robert H

2003-05-01

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.

High Satisfaction and Low Distress in Breast Cancer Patients One Year after BRCA-Mutation Testing without Prior Face-to-Face Genetic Counseling.

PubMed

Sie, Aisha S; Spruijt, Liesbeth; van Zelst-Stams, Wendy A G; Mensenkamp, Arjen R; Ligtenberg, Marjolijn J L; Brunner, Han G; Prins, Judith B; Hoogerbrugge, Nicoline

2016-06-01

According to standard practice following referral to clinical genetics, most high risk breast cancer (BC) patients in many countries receive face-to-face genetic counseling prior to BRCA-mutation testing (DNA-intake). We evaluated a novel format by prospective study: replacing the intake consultation with telephone, written and digital information sent home. Face-to-face counseling then followed BRCA-mutation testing (DNA-direct). One year after BRCA-result disclosure, 108 participants returned long-term follow-up questionnaires, of whom 59 (55 %) had previously chosen DNA-direct (intervention) versus DNA-intake (standard practice i.e., control: 45 %). Questionnaires assessed satisfaction and psychological distress. All participants were satisfied and 85 % of DNA-direct participants would choose this procedure again; 10 % would prefer DNA-intake and 5 % were undecided. In repeated measurements ANOVA, general distress (GHQ-12, p = 0.01) and BC-specific distress (IES-bc, p = 0.03) were lower in DNA-direct than DNA-intake at all time measurements. Heredity-specific distress (IES-her) did not differ significantly between groups. Multivariate regression analyses showed that choice of procedure did not significantly contribute to either general or heredity-specific distress. BC-specific distress (after BC diagnosis) did contribute to both general and heredity-specific distress. This suggests that higher distress scores reflected BC experience, rather than the type of genetic diagnostic procedure. In conclusion, the large majority of BC patients that used DNA-direct reported high satisfaction without increased distress both in the short term, and 1 year after conclusion of genetic testing.

Twisting Right to Left: A…A Mismatch in a CAG Trinucleotide Repeat Overexpansion Provokes Left-Handed Z-DNA Conformation

PubMed Central

2015-01-01

Conformational polymorphism of DNA is a major causative factor behind several incurable trinucleotide repeat expansion disorders that arise from overexpansion of trinucleotide repeats located in coding/non-coding regions of specific genes. Hairpin DNA structures that are formed due to overexpansion of CAG repeat lead to Huntington’s disorder and spinocerebellar ataxias. Nonetheless, DNA hairpin stem structure that generally embraces B-form with canonical base pairs is poorly understood in the context of periodic noncanonical A…A mismatch as found in CAG repeat overexpansion. Molecular dynamics simulations on DNA hairpin stems containing A…A mismatches in a CAG repeat overexpansion show that A…A dictates local Z-form irrespective of starting glycosyl conformation, in sharp contrast to canonical DNA duplex. Transition from B-to-Z is due to the mechanistic effect that originates from its pronounced nonisostericity with flanking canonical base pairs facilitated by base extrusion, backbone and/or base flipping. Based on these structural insights we envisage that such an unusual DNA structure of the CAG hairpin stem may have a role in disease pathogenesis. As this is the first study that delineates the influence of a single A…A mismatch in reversing DNA helicity, it would further have an impact on understanding DNA mismatch repair. PMID:25876062

Large Polyglutamine Repeats Cause Muscle Degeneration in SCA17 Mice

PubMed Central

Huang, Shanshan; Yang, Su; Guo, Jifeng; Yan, Sen; Gaertig, Marta A.; Li, Shihua; Li, Xiao-Jiang

2015-01-01

SUMMARY In polyglutamine (polyQ) diseases, large polyQ repeats cause juvenile cases with different symptoms than adult-onset patients, who carry smaller expanded polyQ repeats. The mechanisms behind the differential pathology mediated by different polyQ repeat lengths remain unknown. By studying knock-in mouse models of spinal cerebellar ataxia-17 (SCA17), we found that a large polyQ (105 glutamines) in the TATA box-binding protein (TBP) preferentially causes muscle degeneration and reduces the expression of muscle-specific genes. Direct expression of TBP with different polyQ repeats in mouse muscle revealed that muscle degeneration is mediated only by the large polyQ repeats. Different polyQ repeats differentially alter TBP’s interaction with neuronal and muscle-specific transcription factors. As a result, the large polyQ repeat decreases the association of MyoD with TBP and DNA promoters. Our findings suggest that specific alterations in protein interactions by large polyQ repeats may account for the unique pathology in juvenile polyQ diseases. PMID:26387956

Role of Replication and CpG Methylation in Fragile X Syndrome CGG Deletions in Primate Cells

PubMed Central

Nichol Edamura, Kerrie; Leonard, Michelle R.; Pearson, Christopher E.

2005-01-01

Instability of the fragile X CGG repeat involves both maternally derived expansions and deletions in the gametes of full-mutation males. It has also been suggested that the absence of aberrant CpG methylation may enhance repeat deletions through an unknown process. The effect of CGG tract length, DNA replication direction, location of replication initiation, and CpG methylation upon CGG stability were investigated using an SV40 primate replication system. Replication-dependant deletions with 53 CGG repeats were observed when replication was initiated proximal to the repeat, with CGG as the lagging-strand template. When we initiated replication further from the repeat, while maintaining CGG as the lagging-strand template or using CCG as the lagging-strand template, significant instability was not observed. CpG methylation of the unstable template stabilized the repeat, decreasing both the frequency and the magnitude of deletion events. Furthermore, CpG methylation slowed the efficiency of replication for all templates. Interestingly, replication forks displayed no evidence of a block at the CGG repeat tract, regardless of replication direction or CpG methylation status. Templates with 20 CGG repeats were stable under all circumstances. These results reveal that CGG deletions occur during replication and are sensitive to replication-fork dynamics, tract length, and CpG methylation. PMID:15625623

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats

PubMed Central

de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

2015-01-01

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. PMID:26481363

All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus.

PubMed Central

Klobutcher, L A; Swanton, M T; Donini, P; Prescott, D M

1981-01-01

In hypotrichous ciliates, all of the macronuclear DNA is in the form of low molecular weight molecules with an average size of approximately 2200 base pairs. Total macronuclear DNA from four hypotrichs has been shown to have inverted terminal repeats by direct sequence analysis. In Oxytricha nova, Oxytricha sp., and Stylonychia pustulata, this terminal sequence may be written as 5'-C4A4C4A4C4 ... 3'-G4T4G4T4G4T4G4T4G4 ... In Euplotes aediculatus, the sequences is similar but differs in the lengths of the duplex region (28 base pairs) and of the putative 3' extension (14 base pairs). Also in Euplotes, a second common sequence of 5 base pairs (A-A-C-T-T-T-T-G-A-A) occurs internal to the terminal repeat and a 17-base-pair heterogeneous region: 5'-C4A4C4A4C4A4C4(X)17T-T-G-A-A ... 3'-G2T4G4T4G4T4G4T4G4T4G4(X)17A-A-C-T-T ... The length of the terminal repeat sequence for O. nova was confirmed in cloned macronuclear DNA molecules. Images PMID:6265931

Structure of the highly repeated, long interspersed DNA family (LINE or L1Rn) of the rat.

PubMed Central

D'Ambrosio, E; Waitzkin, S D; Witney, F R; Salemme, A; Furano, A V

1986-01-01

We present the DNA sequence of a 6.7-kilobase member of the rat long interspersed repeated DNA family (LINE or L1Rn). This member (LINE 3) is flanked by a perfect 14-base-pair (bp) direct repeat and is a full-length, or close-to-full-length, member of this family. LINE 3 contains an approximately 100-bp A-rich right end, a number of long (greater than 400-bp) open reading frames, and a ca. 200-bp G + C-rich (ca. 60%) cluster near each terminus. Comparison of the LINE 3 sequence with the sequence of about one-half of another member, which we also present, as well as restriction enzyme analysis of the genomic copies of this family, indicates that in length and overall structure LINE 3 is quite typical of the 40,000 or so other genomic members of this family which would account for as much as 10% of the rat genome. Therefore, the rat LINE family is relatively homogeneous, which contrasts with the heterogeneous LINE families in primates and mice. Transcripts corresponding to the entire LINE sequence are abundant in the nuclear RNA of rat liver. The characteristics of the rat LINE family are discussed with respect to the possible function and evolution of this family of DNA sequences. Images PMID:3023845

Interaction of the putative tyrosine recombinases RipX (UU145), XerC (UU222), and CodV (UU529) of Ureaplasma parvum serovar 3 with specific DNA

PubMed Central

Zimmerman, Carl-Ulrich R; Rosengarten, Renate; Spergser, Joachim

2013-01-01

Phase variation of two loci (‘mba locus’ and ‘UU172 phase-variable element’) in Ureaplasma parvum serovar 3 has been suggested as result of site-specific DNA inversion occurring at short inverted repeats. Three potential tyrosine recombinases (RipX, XerC, and CodV encoded by the genes UU145, UU222, and UU529) have been annotated in the genome of U. parvum serovar 3, which could be mediators in the proposed recombination event. We document that only orthologs of the gene xerC are present in all strains that show phase variation in the two loci. We demonstrate in vitro binding of recombinant maltose-binding protein fusions of XerC to the inverted repeats of the phase-variable loci, of RipX to a direct repeat that flanks a 20-kbp region, which has been proposed as putative pathogenicity island, and of CodV to a putative dif site. Co-transformation of the model organism Mycoplasma pneumoniae M129 with both the ‘mba locus’ and the recombinase gene xerC behind an active promoter region resulted in DNA inversion in the ‘mba locus’. Results suggest that XerC of U. parvum serovar 3 is a mediator in the proposed DNA inversion event of the two phase-variable loci. PMID:23305333

Spectroscopic insights into quadruplexes of five-repeat telomere DNA sequences upon G-block damage.

PubMed

Dvořáková, Zuzana; Vorlíčková, Michaela; Renčiuk, Daniel

2017-11-01

The DNA lesions, resulting from oxidative damage, were shown to destabilize human telomere four-repeat quadruplex and to alter its structure. Long telomere DNA, as a repetitive sequence, offers, however, other mechanisms of dealing with the lesion: extrusion of the damaged repeat into loop or shifting the quadruplex position by one repeat. Using circular dichroism and UV absorption spectroscopy and polyacrylamide electrophoresis, we studied consequences of lesions at different positions of the model five-repeat human telomere DNA sequences on the structure and stability of their quadruplexes in sodium and in potassium. The repeats affected by lesion are preferentially positioned as terminal overhangs of the core quadruplex structurally similar to the four-repeat one. Forced affecting of the inner repeats leads to presence of variety of more parallel folds in potassium. In sodium the designed models form mixture of two dominant antiparallel quadruplexes whose population varies with the position of the affected repeat. The shapes of quadruplex CD spectra, namely the height of dominant peaks, significantly correlate with melting temperatures. Lesion in one guanine tract of a more than four repeats long human telomere DNA sequence may cause re-positioning of its quadruplex arrangement associated with a shift of the structure to less common quadruplex conformations. The type of the quadruplex depends on the loop position and external conditions. The telomere DNA quadruplexes are quite resistant to the effect of point mutations due to the telomere DNA repetitive nature, although their structure and, consequently, function might be altered. Copyright © 2017. Published by Elsevier B.V.

DNA Replication Dynamics of the GGGGCC Repeat of the C9orf72 Gene.

PubMed

Thys, Ryan Griffin; Wang, Yuh-Hwa

2015-11-27

DNA has the ability to form a variety of secondary structures in addition to the normal B-form DNA, including hairpins and quadruplexes. These structures are implicated in a number of neurological diseases and cancer. Expansion of a GGGGCC repeat located at C9orf72 is associated with familial amyotrophic lateral sclerosis and frontotemporal dementia. This repeat expands from two to 24 copies in normal individuals to several hundreds or thousands of repeats in individuals with the disease. Biochemical studies have demonstrated that as little as four repeats have the ability to form a stable DNA secondary structure known as a G-quadruplex. Quadruplex structures have the ability to disrupt normal DNA processes such as DNA replication and transcription. Here we examine the role of GGGGCC repeat length and orientation on DNA replication using an SV40 replication system in human cells. Replication through GGGGCC repeats leads to a decrease in overall replication efficiency and an increase in instability in a length-dependent manner. Both repeat expansions and contractions are observed, and replication orientation is found to influence the propensity for expansions or contractions. The presence of replication stress, such as low-dose aphidicolin, diminishes replication efficiency but has no effect on instability. Two-dimensional gel electrophoresis analysis demonstrates a replication stall with as few as 20 GGGGCC repeats. These results suggest that replication of the GGGGCC repeat at C9orf72 is perturbed by the presence of expanded repeats, which has the potential to result in further expansion, leading to disease. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Deciphering the Role of Alternative Non-Homologous End Joining (Alt-NHEJ) DNA Repair in Breast Cancer

DTIC Science & Technology

2017-12-01

homology-directed repair genes. Here we report that PolQ inhibition can be used to increase the efficiency of CRISPR targeting. Function-Structure...aberrancies. • Chromosomal fusions. • CRISPR : Clustered Regularly Interspaced Short Palindromic Repeats. • DNA ligase 3 and 4 • Ku80 5 • Mouse...corresponding junctions. To introduce the cleavages, I expressed the CRISPR Cas9 nuclease and two different guide RNAs in PolQ proficient and PolQ

The Chromatin Remodeler Isw1 Prevents CAG Repeat Expansions During Transcription in Saccharomyces cerevisiae

PubMed Central

Koch, Melissa R.; House, Nealia C. M.; Cosetta, Casey M.; Jong, Robyn M.; Salomon, Christelle G.; Joyce, Cailin E.; Philips, Elliot A.; Su, Xiaofeng A.; Freudenreich, Catherine H.

2018-01-01

CAG/CTG trinucleotide repeats are unstable sequences that are difficult to replicate, repair, and transcribe due to their structure-forming nature. CAG repeats strongly position nucleosomes; however, little is known about the chromatin remodeling needed to prevent repeat instability. In a Saccharomyces cerevisiae model system with CAG repeats carried on a YAC, we discovered that the chromatin remodeler Isw1 is required to prevent CAG repeat expansions during transcription. CAG repeat expansions in the absence of Isw1 were dependent on both transcription-coupled repair (TCR) and base-excision repair (BER). Furthermore, isw1∆ mutants are sensitive to methyl methanesulfonate (MMS) and exhibit synergistic MMS sensitivity when combined with BER or TCR pathway mutants. We conclude that CAG expansions in the isw1∆ mutant occur during a transcription-coupled excision repair process that involves both TCR and BER pathways. We observed increased RNA polymerase II (RNAPII) occupancy at the CAG repeat when transcription of the repeat was induced, but RNAPII binding did not change in isw1∆ mutants, ruling out a role for Isw1 remodeling in RNAPII progression. However, nucleosome occupancy over a transcribed CAG tract was altered in isw1∆ mutants. Based on the known role of Isw1 in the reestablishment of nucleosomal spacing after transcription, we suggest that a defect in this function allows DNA structures to form within repetitive DNA tracts, resulting in inappropriate excision repair and repeat-length changes. These results establish a new function for Isw1 in directly maintaining the chromatin structure at the CAG repeat, thereby limiting expansions that can occur during transcription-coupled excision repair. PMID:29305386

The Neandertal genome and ancient DNA authenticity

PubMed Central

Green, Richard E; Briggs, Adrian W; Krause, Johannes; Prüfer, Kay; Burbano, Hernán A; Siebauer, Michael; Lachmann, Michael; Pääbo, Svante

2009-01-01

Recent advances in high-thoughput DNA sequencing have made genome-scale analyses of genomes of extinct organisms possible. With these new opportunities come new difficulties in assessing the authenticity of the DNA sequences retrieved. We discuss how these difficulties can be addressed, particularly with regard to analyses of the Neandertal genome. We argue that only direct assays of DNA sequence positions in which Neandertals differ from all contemporary humans can serve as a reliable means to estimate human contamination. Indirect measures, such as the extent of DNA fragmentation, nucleotide misincorporations, or comparison of derived allele frequencies in different fragment size classes, are unreliable. Fortunately, interim approaches based on mtDNA differences between Neandertals and current humans, detection of male contamination through Y chromosomal sequences, and repeated sequencing from the same fossil to detect autosomal contamination allow initial large-scale sequencing of Neandertal genomes. This will result in the discovery of fixed differences in the nuclear genome between Neandertals and current humans that can serve as future direct assays for contamination. For analyses of other fossil hominins, which may become possible in the future, we suggest a similar ‘boot-strap' approach in which interim approaches are applied until sufficient data for more definitive direct assays are acquired. PMID:19661919

Overstretching of B-DNA with various pulling protocols: Appearance of structural polymorphism and S-DNA

NASA Astrophysics Data System (ADS)

Garai, Ashok; Mogurampelly, Santosh; Bag, Saientan; Maiti, Prabal K.

2017-12-01

We report a structural polymorphism of the S-DNA when a canonical B-DNA is stretched under different pulling protocols and provide a fundamental molecular understanding of the DNA stretching mechanism. Extensive all atom molecular dynamics simulations reveal a clear formation of S-DNA when the B-DNA is stretched along the 3' directions of the opposite strands (OS3) and is characterized by the changes in the number of H-bonds, entropy, and free energy. Stretching along the 5' directions of the opposite strands (OS5) leads to force induced melting form of the DNA. Interestingly, stretching along the opposite ends of the same strand leads to a coexistence of both the S- and melted M-DNA structures. We also do the structural characterization of the S-DNA by calculating various helical parameters. We find that the S-DNA has a twist of ˜10° which corresponds to a helical repeat length of ˜36 base pairs in close agreement with the previous experimental results. Moreover, we find that the free energy barrier between the canonical and overstretched states of DNA is higher for the same termini pulling protocol in comparison to all other protocols considered in this work. Overall, our observations not only reconcile with the available experimental results qualitatively but also enhance the understanding of different overstretched DNA structures.

Molecular architecture of classical cytological landmarks: Centromeres and telomeres

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyne, J.

1994-11-01

Both the human telomere repeat and the pericentromeric repeat sequence (GGAAT)n were isolated based on evolutionary conservation. Their isolation was based on the premise that chromosomal features as structurally and functionally important as telomeres and centromeres should be highly conserved. Both sequences were isolated by high stringency screening of a human repetitive DNA library with rodent repetitive DNA. The pHuR library (plasmid Human Repeat) used for this project was enriched for repetitive DNA by using a modification of the standard DNA library preparation method. Usually DNA for a library is cut with restriction enzymes, packaged, infected, and the library ismore » screened. A problem with this approach is that many tandem repeats don`t have any (or many) common restriction sites. Therefore, many of the repeat sequences will not be represented in the library because they are not restricted to a viable length for the vector used. To prepare the pHuR library, human DNA was mechanically sheared to a small size. These relatively short DNA fragments were denatured and then renatured to C{sub o}t 50. Theoretically only repetitive DNA sequences should renature under C{sub o}t 50 conditions. The single-stranded regions were digested using S1 nuclease, leaving the double-stranded, renatured repeat sequences.« less

Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis

PubMed Central

Henriksson-Peltola, Petri; Sehlén, Wilhelmina; Haggård-Ljungquist, Elisabeth

2007-01-01

Bacteriophages P2, P2 Hy dis and WΦ are very similar but heteroimmune Escherichia coli phages. The structural genes show over 96% identity, but the repressors show between 43 and 63% identities. Furthermore, the operators, which contain two directly repeated sequences, vary in sequence, length, location relative to the promoter and spacing between the direct repeats. We have compared the in vivo effects of the wild type and mutated operators on gene expression with the complexes formed between the repressors and their wild type or mutated operators using electrophoretic mobility shift assay (EMSA), and real-time kinetics of the protein–DNA interactions using surface plasmon resonance (SPR) analysis. Using EMSA, the repressors formed different protein–DNA complexes, and only WΦ was significantly affected by point mutations. However, SPR analysis showed a reduced association rate constant and an increased dissociation rate constant for P2 and WΦ operator mutants. The association rate constants of P2 Hy dis was too fast to be determined. The P2 Hy dis dissociation response curves were shown to be triphasic, while both P2 and WΦ C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages. PMID:17412705

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats.

PubMed

de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

2015-11-16

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream regulatory elements.

PubMed

Fisher, R P; Topper, J N; Clayton, D A

1987-07-17

Selective transcription of human mitochondrial DNA requires a transcription factor (mtTF) in addition to an essentially nonselective RNA polymerase. Partially purified mtTF is able to sequester promoter-containing DNA in preinitiation complexes in the absence of mitochondrial RNA polymerase, suggesting a DNA-binding mechanism for factor activity. Functional domains, required for positive transcriptional regulation by mtTF, are identified within both major promoters of human mtDNA through transcription of mutant promoter templates in a reconstituted in vitro system. These domains are essentially coextensive with DNA sequences protected from nuclease digestion by mtTF-binding. Comparison of the sequences of the two mtTF-responsive elements reveals significant homology only when one sequence is inverted; the binding sites are in opposite orientations with respect to the predominant direction of transcription. Thus mtTF may function bidirectionally, requiring additional protein-DNA interactions to dictate transcriptional polarity. The mtTF-responsive elements are arrayed as direct repeats, separated by approximately 80 bp within the displacement-loop region of human mitochondrial DNA; this arrangement may reflect duplication of an ancestral bidirectional promoter, giving rise to separate, unidirectional promoters for each strand.

Direct Interaction between the WD40 Repeat Protein WDR-23 and SKN-1/Nrf Inhibits Binding to Target DNA

PubMed Central

Leung, Chi K.; Hasegawa, Koichi; Wang, Ying; Deonarine, Andrew; Tang, Lanlan; Miwa, Johji

2014-01-01

SKN-1/Nrf transcription factors activate cytoprotective genes in response to reactive small molecules and strongly influence stress resistance, longevity, and development. The molecular mechanisms of SKN-1/Nrf regulation are poorly defined. We previously identified the WD40 repeat protein WDR-23 as a repressor of Caenorhabditis elegans SKN-1 that functions with a ubiquitin ligase to presumably target the factor for degradation. However, SKN-1 activity and nuclear accumulation are not always correlated, suggesting that there could be additional regulatory mechanisms. Here, we integrate forward genetics and biochemistry to gain insights into how WDR-23 interacts with and regulates SKN-1. We provide evidence that WDR-23 preferentially regulates one of three SKN-1 variants through a direct interaction that is required for normal stress resistance and development. Homology modeling predicts that WDR-23 folds into a β-propeller, and we identify the top of this structure and four motifs at the termini of SKN-1c as essential for the interaction. Two of these SKN-1 motifs are highly conserved in human Nrf1 and Nrf2 and two directly interact with target DNA. Lastly, we demonstrate that WDR-23 can block the ability of SKN-1c to interact with DNA sequences of target promoters identifying a new mechanism of regulation that is independent of the ubiquitin proteasome system, which can become occupied with damaged proteins during stress. PMID:24912676

Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein-Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis.

PubMed

Barreales, Eva G; Vicente, Cláudia M; de Pedro, Antonio; Santos-Aberturas, Javier; Aparicio, Jesús F

2018-05-15

The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, pimM A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimR SARP appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding. IMPORTANCE Here, we have shown that a modulation of the production of the antifungal pimaricin in Streptomyces natalensis can be accomplished via promoter engineering of the PAS-LuxR transcriptional activator pimM The expression of this gene is controlled by the Streptomyces antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator PimR, which binds a series of heptameric direct repeats in its promoter region. The structure and importance of such repeats in protein binding, transcriptional activation, and polyene production have been investigated. These findings should provide important clues to understand the regulatory machinery that modulates antibiotic biosynthesis in Streptomyces and open new possibilities for the manipulation of metabolite production. The presence of PimR orthologues encoded by gene clusters for different secondary metabolites and the conservation of their operators suggest that the improvements observed in the activation of pimaricin biosynthesis by Streptomyces natalensis could be extrapolated to the production of different compounds by other species. Copyright © 2018 Barreales et al.

Probing Human Telomeric DNA and RNA Topology and Ligand Binding in a Cellular Model by Using Responsive Fluorescent Nucleoside Probes.

PubMed

Manna, Sudeshna; Panse, Cornelia H; Sontakke, Vyankat A; Sangamesh, Sarangamath; Srivatsan, Seergazhi G

2017-08-17

The development of biophysical systems that enable an understanding of the structure and ligand-binding properties of G-quadruplex (GQ)-forming nucleic acid sequences in cells or models that mimic the cellular environment would be highly beneficial in advancing GQ-directed therapeutic strategies. Herein, the establishment of a biophysical platform to investigate the structure and recognition properties of human telomeric (H-Telo) DNA and RNA repeats in a cell-like confined environment by using conformation-sensitive fluorescent nucleoside probes and a widely used cellular model, bis(2-ethylhexyl) sodium sulfosuccinate reverse micelles (RMs), is described. The 2'-deoxy and ribonucleoside probes, composed of a 5-benzofuran uracil base analogue, faithfully report the aqueous micellar core through changes in their fluorescence properties. The nucleoside probes incorporated into different loops of H-Telo DNA and RNA oligonucleotide repeats are minimally perturbing and photophysically signal the formation of respective GQ structures in both aqueous buffer and RMs. Furthermore, these sensors enable a direct comparison of the binding affinity of a ligand to H-Telo DNA and RNA GQ structures in the bulk and confined environment of RMs. These results demonstrate that this combination of a GQ nucleoside probe and easy-to-handle RMs could provide new opportunities to study and devise screening-compatible assays in a cell-like environment to discover GQ binders of clinical potential. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Repeated sequence sets in mitochondrial DNA molecules of root knot nematodes (Meloidogyne): nucleotide sequences, genome location and potential for host-race identification.

PubMed Central

Okimoto, R; Chamberlin, H M; Macfarlane, J L; Wolstenholme, D R

1991-01-01

Within a 7 kb segment of the mtDNA molecule of the root knot nematode, Meloidogyne javanica, that lacks standard mitochondrial genes, are three sets of strictly tandemly arranged, direct repeat sequences: approximately 36 copies of a 102 ntp sequence that contains a TaqI site; 11 copies of a 63 ntp sequence, and 5 copies of an 8 ntp sequence. The 7 kb repeat-containing segment is bounded by putative tRNAasp and tRNAf-met genes and the arrangement of sequences within this segment is: the tRNAasp gene; a unique 1,528 ntp segment that contains two highly stable hairpin-forming sequences; the 102 ntp repeat set; the 8 ntp repeat set; a unique 1,068 ntp segment; the 63 ntp repeat set; and the tRNAf-met gene. The nucleotide sequences of the 102 ntp copies and the 63 ntp copies have been conserved among the species examined. Data from Southern hybridization experiments indicate that 102 ntp and 63 ntp repeats occur in the mtDNAs of three, two and two races of M.incognita, M.hapla and M.arenaria, respectively. Nucleotide sequences of the M.incognita Race-3 102 ntp repeat were found to be either identical or highly similar to those of the M.javanica 102 ntp repeat. Differences in migration distance and number of 102 ntp repeat-containing bands seen in Southern hybridization autoradiographs of restriction-digested mtDNAs of M.javanica and the different host races of M.incognita, M.hapla and M.arenaria are sufficient to distinguish the different host races of each species. Images PMID:2027769

Revisiting the TALE repeat.

PubMed

Deng, Dong; Yan, Chuangye; Wu, Jianping; Pan, Xiaojing; Yan, Nieng

2014-04-01

Transcription activator-like (TAL) effectors specifically bind to double stranded (ds) DNA through a central domain of tandem repeats. Each TAL effector (TALE) repeat comprises 33-35 amino acids and recognizes one specific DNA base through a highly variable residue at a fixed position in the repeat. Structural studies have revealed the molecular basis of DNA recognition by TALE repeats. Examination of the overall structure reveals that the basic building block of TALE protein, namely a helical hairpin, is one-helix shifted from the previously defined TALE motif. Here we wish to suggest a structure-based re-demarcation of the TALE repeat which starts with the residues that bind to the DNA backbone phosphate and concludes with the base-recognition hyper-variable residue. This new numbering system is consistent with the α-solenoid superfamily to which TALE belongs, and reflects the structural integrity of TAL effectors. In addition, it confers integral number of TALE repeats that matches the number of bound DNA bases. We then present fifteen crystal structures of engineered dHax3 variants in complex with target DNA molecules, which elucidate the structural basis for the recognition of bases adenine (A) and guanine (G) by reported or uncharacterized TALE codes. Finally, we analyzed the sequence-structure correlation of the amino acid residues within a TALE repeat. The structural analyses reported here may advance the mechanistic understanding of TALE proteins and facilitate the design of TALEN with improved affinity and specificity.

Molecular and chromosomal evidence for allopolyploidy in soybean.

PubMed

Gill, Navdeep; Findley, Seth; Walling, Jason G; Hans, Christian; Ma, Jianxin; Doyle, Jeff; Stacey, Gary; Jackson, Scott A

2009-11-01

Recent studies have documented that the soybean (Glycine max) genome has undergone two rounds of large-scale genome and/or segmental duplication. To shed light on the timing and nature of these duplication events, we characterized and analyzed two subfamilies of high-copy centromeric satellite repeats, CentGm-1 and CentGm-2, using a combination of computational and molecular cytogenetic approaches. These two subfamilies of satellite repeats mark distinct subsets of soybean centromeres and, in at least one case, a pair of homologs, suggesting their origins from an allopolyploid event. The satellite monomers of each subfamily are arranged in large tandem arrays, and intermingled monomers of the two subfamilies were not detected by fluorescence in situ hybridization on extended DNA fibers nor at the sequence level. This indicates that there has been little recombination and homogenization of satellite DNA between these two sets of centromeres. These satellite repeats are also present in Glycine soja, the proposed wild progenitor of soybean, but could not be detected in any other relatives of soybean examined in this study, suggesting the rapid divergence of the centromeric satellite DNA within the Glycine genus. Together, these observations provide direct evidence, at molecular and chromosomal levels, in support of the hypothesis that the soybean genome has experienced a recent allopolyploidization event.

Linkage map of the fragments of herpesvirus papio DNA.

PubMed Central

Lee, Y S; Tanaka, A; Lau, R Y; Nonoyama, M; Rabin, H

1981-01-01

Herpesvirus papio (HVP), an Epstein-Barr-like virus, causes lymphoblastoid disease in baboons. The physical map of HVP DNA was constructed for the fragments produced by cleavage of HVP DNA with restriction endonucleases EcoRI, HindIII, SalI, and PvuI, which produced 12, 12, 10, and 4 fragments, respectively. The total molecular size of HVP DNA was calculated as close to 110 megadaltons. The following methods were used for construction of the map; (i) fragments near the ends of HVP DNA were identified by treating viral DNA with lambda exonuclease before restriction enzyme digestion; (ii) fragments containing nucleotide sequences in common with fragments from the second enzyme digest of HVP DNA were examined by Southern blot hybridization; and (iii) the location of some fragments was determined by isolating individual fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. Terminal heterogeneity and internal repeats were found to be unique features of HVP DNA molecule. One to five repeats of 0.8 megadaltons were found at both terminal ends. Although the repeats of both ends shared a certain degree of homology, it was not determined whether they were identical repeats. The internal repeat sequence of HVP DNA was found in the EcoRI-C region, which extended from 8.4 to 23 megadaltons from the left end of the molecule. The average number of the repeats was calculated to be seven, and the molecular size was determined to be 1.8 megadaltons. Similar unique features have been reported in EBV DNA (D. Given and E. Kieff, J. Virol. 28:524-542, 1978). Images PMID:6261015

Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Jamy C.

Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) thatmore » binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in euchromatin. Remarkably, human euchromatin and fly heterochromatin share similar features; such as repeated DNA content, intron lengths and open reading frame sizes. Human cells likely stabilize their DNA content via mechanisms and factors similar to those in Drosophila heterochromatin. Furthermore, my thesis work raises implications for H3K9me and chromatin functions in complex-DNA genome stability, repeated DNA homogenization by molecular drive, and in genome reorganization through evolution.« less

Complex structure of knob DNA on maize chromosome 9. Retrotransposon invasion into heterochromatin.

PubMed Central

Ananiev, E V; Phillips, R L; Rines, H W

1998-01-01

The recovery of maize (Zea mays L.) chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses enables us to analyze the structure and composition of specific regions, such as knobs, of individual maize chromosomes. A DNA hybridization blot panel of eight individual maize chromosome addition lines revealed that 180-bp repeats found in knobs are present in each of these maize chromosomes, but the copy number varies from approximately 100 to 25, 000. Cosmid clones with knob DNA segments were isolated from a genomic library of an oat-maize chromosome 9 addition line with the help of the 180-bp knob-associated repeated DNA sequence used as a probe. Cloned knob DNA segments revealed a complex organization in which blocks of tandemly arranged 180-bp repeating units are interrupted by insertions of other repeated DNA sequences, mostly represented by individual full size copies of retrotransposable elements. There is an obvious preference for the integration of retrotransposable elements into certain sites (hot spots) of the 180-bp repeat. Sequence microheterogeneity including point mutations and duplications was found in copies of 180-bp repeats. The 180-bp repeats within an array all had the same polarity. Restriction maps constructed for 23 cloned knob DNA fragments revealed the positions of polymorphic sites and sites of integration of insertion elements. Discovery of the interspersion of retrotransposable elements among blocks of tandem repeats in maize and some other organisms suggests that this pattern may be basic to heterochromatin organization for eukaryotes. PMID:9691055

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PubMed

Nakano, Kazuma; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Ashimine, Noriko; Ohki, Shun; Shinzato, Misuzu; Minami, Maiko; Nakanishi, Tetsuhiro; Teruya, Kuniko; Satou, Kazuhito; Hirano, Takashi

2017-07-01

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II's sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

Measurement of plasma cell-free DNA concentrations in dogs with sepsis, trauma, and neoplasia.

PubMed

Letendre, Jo-Annie; Goggs, Robert

2017-05-01

To determine if cell-free DNA (cfDNA) was identifiable in canine plasma, to evaluate 3 techniques for the measurement of plasma cfDNA concentrations in dogs presented to an emergency service, and to compare the plasma cfDNA concentrations of healthy dogs to those with sepsis, trauma, and neoplasia. Retrospective study of banked canine plasma samples collected between May 2014 and December 2014. Dogs presented to the emergency service of a university veterinary teaching hospital. Plasma cfDNA was measured on residual plasma samples obtained from 15 dogs with sepsis, 15 dogs with moderate-severe trauma, 15 dogs diagnosed with a sarcoma. Plasma cfDNA was also measured in 15 healthy dogs. None. Assay linearity, repeatability, and reproducibility were evaluated. Quantification of cfDNA was performed in duplicate on diluted citrated plasma and following DNA purification using 2 fluorescence assays (SYBR-Gold; Quant-iT) and by ultraviolet absorbance spectroscopy. Fluorescence intensities (FIs) were converted to cfDNA concentrations using standard curves. Median FI values and cfDNA concentrations were compared to healthy controls using the Kruskal-Wallis test, with adjustment for multiple comparisons. Alpha was set at 0.05. Both assays had excellent linearity, and acceptable repeatability and reproducibility. Compared to controls, plasma cfDNA concentrations were significantly increased in dogs with sepsis or moderate-severe trauma with both assays (P ≤ 0.003). Dogs with neoplasia had significantly increased cfDNA concentrations with the Quant-iT assay only (P = 0.003). When measurements were performed on purified DNA, only dogs with moderate-severe trauma had significantly increased cfDNA concentrations (P < 0.001; SYBR-Gold assay). cfDNA can be readily identified in canine plasma using 2 fluorescence assays. DNA extraction offers no advantage over direct measurement. Compared to healthy controls, dogs with sepsis or moderate-severe trauma have significantly increased plasma cfDNA concentrations. © Veterinary Emergency and Critical Care Society 2017.

Topological diversity of chromatin fibers: Interplay between nucleosome repeat length, DNA linking number and the level of transcription

PubMed Central

Norouzi, Davood; Katebi, Ataur; Cui, Feng; Zhurkin, Victor B.

2016-01-01

The spatial organization of nucleosomes in 30-nm fibers remains unknown in detail. To tackle this problem, we analyzed all stereochemically possible configurations of two-start chromatin fibers with DNA linkers L = 10–70 bp (nucleosome repeat length NRL = 157–217 bp). In our model, the energy of a fiber is a sum of the elastic energy of the linker DNA, steric repulsion, electrostatics, and the H4 tail-acidic patch interaction between two stacked nucleosomes. We found two families of energetically feasible conformations of the fibers—one observed earlier, and the other novel. The fibers from the two families are characterized by different DNA linking numbers—that is, they are topologically different. Remarkably, the optimal geometry of a fiber and its topology depend on the linker length: the fibers with linkers L = 10n and 10n + 5 bp have DNA linking numbers per nucleosome ΔLk ≈ −1.5 and −1.0, respectively. In other words, the level of DNA supercoiling is directly related to the length of the inter-nucleosome linker in the chromatin fiber (and therefore, to NRL). We hypothesize that this topological polymorphism of chromatin fibers may play a role in the process of transcription, which is known to generate different levels of DNA supercoiling upstream and downstream from RNA polymerase. A genome-wide analysis of the NRL distribution in active and silent yeast genes yielded results consistent with this assumption. PMID:28133628

Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species.

PubMed

Kuipers, A G J; Kamstra, S A; de Jeu, M J; Visser, R G F

2002-01-01

Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68-127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed.

Small tandemly repeated DNA sequences of higher plants likely originate from a tRNA gene ancestor.

PubMed Central

Benslimane, A A; Dron, M; Hartmann, C; Rode, A

1986-01-01

Several monomers (177 bp) of a tandemly arranged repetitive nuclear DNA sequence of Brassica oleracea have been cloned and sequenced. They share up to 95% homology between one another and up to 80% with other satellite DNA sequences of Cruciferae, suggesting a common ancestor. Both strands of these monomers show more than 50% homology with many tRNA genes; the best homologies have been obtained with Lys and His yeast mitochondrial tRNA genes (respectively 64% and 60%). These results suggest that small tandemly repeated DNA sequences of plants may have evolved from a tRNA gene ancestor. These tandem repeats have probably arisen via a process involving reverse transcription of polymerase III RNA intermediates, as is the case for interspersed DNA sequences of mammalians. A model is proposed to explain the formation of such small tandemly repeated DNA sequences. Images PMID:3774553

Excess single-stranded DNA inhibits meiotic double-strand break repair.

PubMed

Johnson, Rebecca; Borde, Valérie; Neale, Matthew J; Bishop-Bailey, Anna; North, Matthew; Harris, Sheila; Nicolas, Alain; Goldman, Alastair S H

2007-11-01

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.

Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair

PubMed Central

Bishop-Bailey, Anna; North, Matthew; Harris, Sheila; Nicolas, Alain; Goldman, Alastair S. H

2007-01-01

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Δ cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Δ cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins. PMID:18081428

The highly conserved MraZ protein is a transcriptional regulator in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eraso, Jesus M.; Markillie, Lye Meng; Mitchell, Hugh D.

2014-05-05

The mraZ and mraW genes are highly conserved in bacteria, both in sequence and location at the head of the division and cell wall (dcw) gene cluster. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin, and MraW is known to methylate ribosomal RNA, mraZ and mraW null mutants have no detectable growth phenotype in any species tested to date, hampering progress in understanding their physiological role. Here we show that overproduction of Escherichia coli MraZ perturbs cell division and the cell envelope, is more lethal at high levels or in minimal growth medium,more » and that MraW antagonizes these effects. MraZGFP localizes to the nucleoid, suggesting that it binds DNA. Indeed, purified MraZ directly binds a region upstream from its own promoter containing three direct repeats to regulate its own expression and that of downstream cell division and cell wall genes. MraZ-LacZ fusions are repressed by excess MraZ but not when DNA binding by MraZ is inhibited. RNAseq analysis indicates that MraZ is a global transcriptional regulator with numerous targets in addition to dcw genes. One of these targets, mioC, is directly bound by MraZ in a region with three direct repeats.« less

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts.

PubMed

Bernstein, Diana L; Le Lay, John E; Ruano, Elena G; Kaestner, Klaus H

2015-05-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator-like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics.

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts

PubMed Central

Bernstein, Diana L.; Le Lay, John E.; Ruano, Elena G.; Kaestner, Klaus H.

2015-01-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator–like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics. PMID:25866970

Alu repeats: A source for the genesis of primate microsatellites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arcot, S.S.; Batzer, M.A.; Wang, Zhenyuan

1995-09-01

As a result of their abundance, relatively uniform distribution, and high degree of polymorphism, microsatellites and minisatellites have become valuable tools in genetic mapping, forensic identity testing, and population studies. In recent years, a number of microsatellite repeats have been found to be associated with Alu interspersed repeated DNA elements. The association of an Alu element with a microsatellite repeat could result from the integration of an Alu element within a preexisting microsatellite repeat. Alternatively, Alu elements could have a direct role in the origin of microsatellite repeats. Errors introduced during reverse transcription of the primary transcript derived from anmore » Alu {open_quotes}master{close_quote} gene or the accumulation of random mutations in the middle A-rich regions and oligo(dA)-rich tails of Alu elements after insertion and subsequent expansion and contraction of these sequences could result in the genesis of a microsatellite repeat. We have tested these hypotheses by a direct evolutionary comparison of the sequences of some recent Alu elements that are found only in humans and are absent from nonhuman primates, as well as some older Alu elements that are present at orthologous positions in a number of nonhuman primates. The origin of {open_quotes}young{close_quotes} Alu insertions, absence of sequences that resemble microsatellite repeats at the orthologous loci in chimpanzees, and the gradual expansion of microsatellite repeats in some old Alu repeats at orthologous positions within the genomes of a number of nonhuman primates suggest that Alu elements are a source for the genesis of primate microsatellite repeats. 48 refs., 5 figs., 3 tabs.« less

Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly.

PubMed

Reyon, Deepak; Maeder, Morgan L; Khayter, Cyd; Tsai, Shengdar Q; Foley, Jonathan E; Sander, Jeffry D; Joung, J Keith

2013-07-01

Customized DNA-binding domains made using transcription activator-like effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in various organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or downregulate expression of endogenous genes in human cells and plants. This unit describes a detailed protocol for the recently described fast ligation-based automatable solid-phase high-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multichannel pipet. Using the automated approach, a single researcher can construct up to 96 DNA fragments encoding TALE repeat arrays of various lengths in a single day, and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in a week or less. Plasmids required for FLASH are available by request from the Joung lab (http://eGenome.org). This unit also describes improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) web server (http://ZiFiT.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high throughput. © 2013 by John Wiley & Sons, Inc.

Engineering Customized TALE Nucleases (TALENs) and TALE Transcription Factors by Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) Assembly

PubMed Central

Reyon, Deepak; Maeder, Morgan L.; Khayter, Cyd; Tsai, Shengdar Q.; Foley, Jonathan E.; Sander, Jeffry D.; Joung, J. Keith

2013-01-01

Customized DNA-binding domains made using Transcription Activator-Like Effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in multiple different organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or down-regulate expression of endogenous genes in human cells and plants. Here we describe a detailed protocol for practicing the recently described Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multi-channel pipet. With the automated version of FLASH, a single researcher can construct up to 96 DNA fragments encoding various length TALE repeat arrays in one day and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in one week or less. Plas-mids required to practice FLASH are available by request from the Joung Lab (http://www.jounglab.org/). We also describe here improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) webserver (http://ZiFiTBeta.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high-throughput. PMID:23821439

Duplication in DNA Sequences

NASA Astrophysics Data System (ADS)

Ito, Masami; Kari, Lila; Kincaid, Zachary; Seki, Shinnosuke

The duplication and repeat-deletion operations are the basis of a formal language theoretic model of errors that can occur during DNA replication. During DNA replication, subsequences of a strand of DNA may be copied several times (resulting in duplications) or skipped (resulting in repeat-deletions). As formal language operations, iterated duplication and repeat-deletion of words and languages have been well studied in the literature. However, little is known about single-step duplications and repeat-deletions. In this paper, we investigate several properties of these operations, including closure properties of language families in the Chomsky hierarchy and equations involving these operations. We also make progress toward a characterization of regular languages that are generated by duplicating a regular language.

Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation.

PubMed Central

Garcia, J A; Harrich, D; Soultanakis, E; Wu, F; Mitsuyasu, R; Gaynor, R B

1989-01-01

The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted this stem and loop mutation relative to the HIV LTR mRNA start site resulted in even larger decreases in tat-activation. This suggests that multiple determinants, including protein binding, the loop sequence, and RNA or DNA secondary structure, are important in tat-activation and suggests that tat may interact with cellular proteins binding to DNA to increase HIV gene expression. Images PMID:2721501

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription

PubMed Central

Stefanovsky, Victor Y.; Tremblay, Michel G.; Lindsay, Helen; Robinson, Mark D.

2017-01-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes. PMID:28715449

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

PubMed

Herdman, Chelsea; Mars, Jean-Clement; Stefanovsky, Victor Y; Tremblay, Michel G; Sabourin-Felix, Marianne; Lindsay, Helen; Robinson, Mark D; Moss, Tom

2017-07-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.

TAL effector-DNA specificity.

PubMed

Scholze, Heidi; Boch, Jens

2010-01-01

TAL effectors are important virulence factors of bacterial plant pathogenic Xanthomonas, which infect a wide variety of plants including valuable crops like pepper, rice, and citrus. TAL proteins are translocated via the bacterial type III secretion system into host cells and induce transcription of plant genes by binding to target gene promoters. Members of the TAL effector family differ mainly in their central domain of tandemly arranged repeats of typically 34 amino acids each with hypervariable di-amino acids at positions 12 and 13. We recently showed that target DNA-recognition specificity of TAL effectors is encoded in a modular and clearly predictable mode. The repeats of TAL effectors feature a surprising one repeat-to-one-bp correlation with different repeat types exhibiting a different DNA base pair specificity. Accordingly, we predicted DNA specificities of TAL effectors and generated artificial TAL proteins with novel DNA recognition specificities. We describe here novel artificial TALs and discuss implications for the DNA recognition specificity. The unique TAL-DNA binding domain allows design of proteins with potentially any given DNA recognition specificity enabling many uses for biotechnology.

Mitochondrial Genome Maintenance: Roles for Nuclear Nonhomologous End-Joining Proteins in Saccharomyces cerevisiae

PubMed Central

Kalifa, Lidza; Quintana, Daniel F.; Schiraldi, Laura K.; Phadnis, Naina; Coles, Garry L.; Sia, Rey A.; Sia, Elaine A.

2012-01-01

Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat–mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial double-strand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria. PMID:22214610

Influence of long-term repeated prescribed burning on mycelial communities of ectomycorrhizal fungi.

PubMed

Bastias, Brigitte A; Xu, Zhihong; Cairney, John W G

2006-01-01

To demonstrate the efficacy of direct DNA extraction from hyphal ingrowth bags for community profiling of ectomycorrhizal (ECM) mycelia in soil, we applied the method to investigate the influence of long-term repeated prescribed burning on an ECM fungal community. DNA was extracted from hyphal ingrowth bags buried in forest plots that received different prescribed burning treatments for 30 yr, and denaturing gradient gel electrophoresis (DGGE) profiles of partial fungal rDNA internal transcribed spacer (ITS) regions were compared. Restriction fragment length polymorphism (RFLP) and sequence analyses were also used to compare clone assemblages between the treatments. The majority of sequences derived from the ingrowth bags were apparently those of ECM fungi. DGGE profiles for biennially burned plots were significantly different from those of quadrennially burned and unburned control plots. Analysis of clone assemblages indicated that this reflected altered ECM fungal community composition. The results indicate that hyphal ingrowth bags represent a useful method for investigation of ECM mycelial communities, and that frequent long-term prescribed burning can influence below-ground ECM fungal communities.

Functional interrogation of non-coding DNA through CRISPR genome editing

PubMed Central

Canver, Matthew C.; Bauer, Daniel E.; Orkin, Stuart H.

2017-01-01

Methodologies to interrogate non-coding regions have lagged behind coding regions despite comprising the vast majority of the genome. However, the rapid evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has provided a multitude of novel techniques for laboratory investigation including significant contributions to the toolbox for studying non-coding DNA. CRISPR-mediated loss-of-function strategies rely on direct disruption of the underlying sequence or repression of transcription without modifying the targeted DNA sequence. CRISPR-mediated gain-of-function approaches similarly benefit from methods to alter the targeted sequence through integration of customized sequence into the genome as well as methods to activate transcription. Here we review CRISPR-based loss- and gain-of-function techniques for the interrogation of non-coding DNA. PMID:28288828

Preferential Nucleosome Assembly at DNA Triplet Repeats from the Myotonic Dystrophy Gene

NASA Astrophysics Data System (ADS)

Wang, Yuh-Hwa; Amirhaeri, Sorour; Kang, Seongman; Wells, Robert D.; Griffith, Jack D.

1994-07-01

The expansion of CTG repeats in DNA occurs in or near genes involved in several human diseases, including myotonic dystrophy and Huntington's disease. Nucleosomes, the basic structural element of chromosomes, consist of 146 base pairs of DNA coiled about an octamer of histone proteins and mediate general transcriptional repression. Electron microscopy was used to examine in vitro the nucleosome assembly of DNA containing repeating CTG triplets. The efficiency of nucleosome formation increased with expanded triplet blocks, suggesting that such blocks may repress transcription through the creation of stable nucleosomes.

Medium-sized tandem repeats represent an abundant component of the Drosophila virilis genome.

PubMed

Abdurashitov, Murat A; Gonchar, Danila A; Chernukhin, Valery A; Tomilov, Victor N; Tomilova, Julia E; Schostak, Natalia G; Zatsepina, Olga G; Zelentsova, Elena S; Evgen'ev, Michael B; Degtyarev, Sergey K H

2013-11-09

Previously, we developed a simple method for carrying out a restriction enzyme analysis of eukaryotic DNA in silico, based on the known DNA sequences of the genomes. This method allows the user to calculate lengths of all DNA fragments that are formed after a whole genome is digested at the theoretical recognition sites of a given restriction enzyme. A comparison of the observed peaks in distribution diagrams with the results from DNA cleavage using several restriction enzymes performed in vitro have shown good correspondence between the theoretical and experimental data in several cases. Here, we applied this approach to the annotated genome of Drosophila virilis which is extremely rich in various repeats. Here we explored the combined approach to perform the restriction analysis of D. virilis DNA. This approach enabled to reveal three abundant medium-sized tandem repeats within the D. virilis genome. While the 225 bp repeats were revealed previously in intergenic non-transcribed spacers between ribosomal genes of D. virilis, two other families comprised of 154 bp and 172 bp repeats were not described. Tandem Repeats Finder search demonstrated that 154 bp and 172 bp units are organized in multiple clusters in the genome of D. virilis. Characteristically, only 154 bp repeats derived from Helitron transposon are transcribed. Using in silico digestion in combination with conventional restriction analysis and sequencing of repeated DNA fragments enabled us to isolate and characterize three highly abundant families of medium-sized repeats present in the D. virilis genome. These repeats comprise a significant portion of the genome and may have important roles in genome function and structural integrity. Therefore, we demonstrated an approach which makes possible to investigate in detail the gross arrangement and expression of medium-sized repeats basing on sequencing data even in the case of incompletely assembled and/or annotated genomes.

[Direct human DNA damage by unfavorable environmental and climatic factors].

PubMed

Doroshtuk, N A; Postnov, A Iu; Doroshtuk, A D; Khasanova, E B; Konovalova, N V; Khesuani, Iu D; Osiaeva, M K; Rodnenkov, O V; Chazova, I E

2014-01-01

To study the impact of simulated climatic conditions of the 2010 summer in Moscow on the telomere repeats of chromosomes in human blood cells. The climatic conditions of July-August 2010 in Moscow were simulated at the Medical Technical Complex, Institute of Biomedical Problems, Russian Academy of Sciences. The relative length of the telomeric repeats of blood cell chromosomes from 6 apparently healthy volunteers was measured by quantitative real-time polymerase chain reaction. These conditions were ascertained to lead to a statistically significant decline in the length of telomere repeats in the terminal portions of chromosomes by 15%. Environmental changes and abnormal temperature rises may result in oxidative stress accompanied by telomere shortening, which can be, in turn, a factor of premature aging.

Identification and nucleotide sequence analysis of the repetitive DNA element in the genome of fish lymphocystis disease virus.

PubMed

Schnitzler, P; Delius, H; Scholz, J; Touray, M; Orth, E; Darai, G

1987-12-01

The genome of the fish lymphocystis disease virus (FLDV) was screened for the existence of repetitive DNA sequences using a defined and complete gene library of the viral genome (98 kbp) by DNA-DNA hybridization, heteroduplex analysis, and restriction fine mapping. A repetitive DNA sequence was detected at the coordinates 0.034 to 0.057 and 0.718 to 0.736 map units (m.u.) of the FLDV genome. The first region (0.034 to 0.057 m.u.) corresponds to the 5' terminus of the EcoRI FLDV DNA fragment B (0.034 to 0.165 m.u.) and the second region (0.718 to 0.736 m.u.) is identical to the EcoRI DNA fragment M of the viral genome. The DNA nucleotide sequence of the EcoRI FLDV DNA fragment M was determined. This analysis revealed the presence of many short direct and inverted repetitions, e.g., a 18-mer direct repetition (TTTAAAATTTAATTAA) that started at nucleotide positions 812 and 942 and a 14-mer inverted repeat (TTAAATTTAAATTT) at nucleotide positions 820 and 959. Only short open reading frames were detected within this region. The DNA repetitions are discussed as sequences that play a possible regulatory role for virus replication. Furthermore, hybridization experiments revealed that the repetitive DNA sequences are conserved in the genome of different strains of fish lymphocystis disease virus isolated from two species of Pleuronectidae (flounder and dab).

Structure of chromatin and the linking number of DNA.

PubMed Central

Worcel, A; Strogatz, S; Riley, D

1981-01-01

Recent observations suggest that the basic supranucleosomal structure of chromatin is a zigzag helical ribbon with a repeat unit made of two nucleosomes connected by a relaxed spacer DNA. A remarkable feature of one particular ribbon is that it solves the apparent paradox between the number of DNA turns per nucleosome and the total linking number of a nucleosome-containing closed circular DNA molecule. We show here that the repeat unit of the proposed structure, which contains two nucleosomes with -1 3/4 DNA turns per nucleosome and one spacer crossover per repeat, contributes -2 to the linking number of closed circular DNA. Space-filling models show that the cylindrical 250-A chromatin fiber can be generated by twisting the ribbon. Images PMID:6940168

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

PubMed

Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

2014-03-06

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

NASA Astrophysics Data System (ADS)

Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

2014-03-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Controlled Nucleation and Growth of DNA Tile Arrays within Prescribed DNA Origami Frames and Their Dynamics

PubMed Central

2015-01-01

Controlled nucleation of nanoscale building blocks by geometrically defined seeds implanted in DNA nanoscaffolds represents a unique strategy to study and understand the dynamic processes of molecular self-assembly. Here we utilize a two-dimensional DNA origami frame with a hollow interior and selectively positioned DNA hybridization seeds to control the self-assembly of DNA tile building blocks, where the small DNA tiles are directed to fill the interior of the frame through prescribed sticky end interactions. This design facilitates the construction of DNA origami/array hybrids that adopt the overall shape and dimensions of the origami frame, forming a 2D array in the core consisting of a large number of simple repeating DNA tiles. The formation of the origami/array hybrid was characterized with atomic force microscopy, and the nucleation dynamics were monitored by serial AFM scanning and fluorescence spectroscopy, which revealed faster kinetics of growth within the frame as compared to growth without the presence of a frame. Our study provides insight into the fundamental behavior of DNA-based self-assembling systems. PMID:24575893

Upstream mononucleotide A-repeats play a cis-regulatory role in mammals through the DICER1 and Ago proteins.

PubMed

Aporntewan, Chatchawit; Pin-on, Piyapat; Chaiyaratana, Nachol; Pongpanich, Monnat; Boonyaratanakornkit, Viroj; Mutirangura, Apiwat

2013-10-01

A-repeats are the simplest form of tandem repeats and are found ubiquitously throughout genomes. These mononucleotide repeats have been widely believed to be non-functional 'junk' DNA. However, studies in yeasts suggest that A-repeats play crucial biological functions, and their role in humans remains largely unknown. Here, we showed a non-random pattern of distribution of sense A- and T-repeats within 20 kb around transcription start sites (TSSs) in the human genome. Different distributions of these repeats are observed upstream and downstream of TSSs. Sense A-repeats are enriched upstream, whereas sense T-repeats are enriched downstream of TSSs. This enrichment directly correlates with repeat size. Genes with different functions contain different lengths of repeats. In humans, tissue-specific genes are enriched for short repeats of <10 bp, whereas housekeeping genes are enriched for long repeats of ≥10 bp. We demonstrated that DICER1 and Argonaute proteins are required for the cis-regulatory role of A-repeats. Moreover, in the presence of a synthetic polymer that mimics an A-repeat, protein binding to A-repeats was blocked, resulting in a dramatic change in the expression of genes containing upstream A-repeats. Our findings suggest a length-dependent cis-regulatory function of A-repeats and that Argonaute proteins serve as trans-acting factors, binding to A-repeats.

Analysis of Duck Hepatitis B Virus Reverse Transcription Indicates a Common Mechanism for the Two Template Switches during Plus-Strand DNA Synthesis

PubMed Central

Havert, Michael B.; Ji, Lin; Loeb, Daniel D.

2002-01-01

The synthesis of the hepadnavirus relaxed circular DNA genome requires two template switches, primer translocation and circularization, during plus-strand DNA synthesis. Repeated sequences serve as donor and acceptor templates for these template switches, with direct repeat 1 (DR1) and DR2 for primer translocation and 5′r and 3′r for circularization. These donor and acceptor sequences are at, or near, the ends of the minus-strand DNA. Analysis of plus-strand DNA synthesis of duck hepatitis B virus (DHBV) has indicated that there are at least three other cis-acting sequences that make contributions during the synthesis of relaxed circular DNA. These sequences, 5E, M, and 3E, are located near the 5′ end, the middle, and the 3′ end of minus-strand DNA, respectively. The mechanism by which these sequences contribute to the synthesis of plus-strand DNA was unclear. Our aim was to better understand the mechanism by which 5E and M act. We localized the DHBV 5E element to a short sequence of approximately 30 nucleotides that is 100 nucleotides 3′ of DR2 on minus-strand DNA. We found that the new 5E mutants were partially defective for primer translocation/utilization at DR2. They were also invariably defective for circularization. In addition, examination of several new DHBV M variants indicated that they too were defective for primer translocation/utilization and circularization. Thus, this analysis indicated that 5E and M play roles in both primer translocation/utilization and circularization. In conjunction with earlier findings that 3E functions in both template switches, our findings indicate that the processes of primer translocation and circularization share a common underlying mechanism. PMID:11861843

Disease-associated repeat instability and mismatch repair.

PubMed

Schmidt, Monika H M; Pearson, Christopher E

2016-02-01

Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential. Copyright © 2015 Elsevier B.V. All rights reserved.

E-motif formed by extrahelical cytosine bases in DNA homoduplexes of trinucleotide and hexanucleotide repeats

PubMed Central

Pan, Feng; Zhang, Yuan; Man, Viet Hoang; Roland, Christopher

2018-01-01

Abstract Atypical DNA secondary structures play an important role in expandable trinucleotide repeat (TR) and hexanucleotide repeat (HR) diseases. The cytosine mismatches in C-rich homoduplexes and hairpin stems are weakly bonded; experiments show that for certain sequences these may flip out of the helix core, forming an unusual structure termed an ‘e-motif’. We have performed molecular dynamics simulations of C-rich TR and HR DNA homoduplexes in order to characterize the conformations, stability and dynamics of formation of the e-motif, where the mismatched cytosines symmetrically flip out in the minor groove, pointing their base moieties towards the 5′-direction in each strand. TRs have two non-equivalent reading frames, (GCC)n and (CCG)n; while HRs have three: (CCCGGC)n, (CGGCCC)n, (CCCCGG)n. We define three types of pseudo basepair steps related to the mismatches and show that the e-motif is only stable in (GCC)n and (CCCGGC)n homoduplexes due to the favorable stacking of pseudo GpC steps (whose nature depends on whether TRs or HRs are involved) and the formation of hydrogen bonds between the mismatched cytosine at position i and the cytosine (TRs) or guanine (HRs) at position i − 2 along the same strand. We also characterize the extended e-motif, where all mismatched cytosines are extruded, their extra-helical stacking additionally stabilizing the homoduplexes. PMID:29190385

Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

Treesearch

M.N. lslam-Faridi; C.D. Nelson; S.P. DiFazio; L.E. Gunter; G.A. Tuskan

2009-01-01

The 185-285 rDNA and 55 rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 185-285 rDNA sites and one 55 rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis-type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones...

Genetic variation and DNA fingerprinting of durian types in Malaysia using simple sequence repeat (SSR) markers.

PubMed

Siew, Ging Yang; Ng, Wei Lun; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Tan, Soon Guan; Yeap, Swee Keong

2018-01-01

Durian ( Durio zibethinus ) is one of the most popular tropical fruits in Asia. To date, 126 durian types have been registered with the Department of Agriculture in Malaysia based on phenotypic characteristics. Classification based on morphology is convenient, easy, and fast but it suffers from phenotypic plasticity as a direct result of environmental factors and age. To overcome the limitation of morphological classification, there is a need to carry out genetic characterization of the various durian types. Such data is important for the evaluation and management of durian genetic resources in producing countries. In this study, simple sequence repeat (SSR) markers were used to study the genetic variation in 27 durian types from the germplasm collection of Universiti Putra Malaysia. Based on DNA sequences deposited in Genbank, seven pairs of primers were successfully designed to amplify SSR regions in the durian DNA samples. High levels of variation among the 27 durian types were observed (expected heterozygosity, H E  = 0.35). The DNA fingerprinting power of SSR markers revealed by the combined probability of identity (PI) of all loci was 2.3×10 -3 . Unique DNA fingerprints were generated for 21 out of 27 durian types using five polymorphic SSR markers (the other two SSR markers were monomorphic). We further tested the utility of these markers by evaluating the clonal status of shared durian types from different germplasm collection sites, and found that some were not clones. The findings in this preliminary study not only shows the feasibility of using SSR markers for DNA fingerprinting of durian types, but also challenges the current classification of durian types, e.g., on whether the different types should be called "clones", "varieties", or "cultivars". Such matters have a direct impact on the regulation and management of durian genetic resources in the region.

Genetic variation and DNA fingerprinting of durian types in Malaysia using simple sequence repeat (SSR) markers

PubMed Central

Siew, Ging Yang; Tan, Sheau Wei; Tan, Soon Guan; Yeap, Swee Keong

2018-01-01

Durian (Durio zibethinus) is one of the most popular tropical fruits in Asia. To date, 126 durian types have been registered with the Department of Agriculture in Malaysia based on phenotypic characteristics. Classification based on morphology is convenient, easy, and fast but it suffers from phenotypic plasticity as a direct result of environmental factors and age. To overcome the limitation of morphological classification, there is a need to carry out genetic characterization of the various durian types. Such data is important for the evaluation and management of durian genetic resources in producing countries. In this study, simple sequence repeat (SSR) markers were used to study the genetic variation in 27 durian types from the germplasm collection of Universiti Putra Malaysia. Based on DNA sequences deposited in Genbank, seven pairs of primers were successfully designed to amplify SSR regions in the durian DNA samples. High levels of variation among the 27 durian types were observed (expected heterozygosity, HE = 0.35). The DNA fingerprinting power of SSR markers revealed by the combined probability of identity (PI) of all loci was 2.3×10−3. Unique DNA fingerprints were generated for 21 out of 27 durian types using five polymorphic SSR markers (the other two SSR markers were monomorphic). We further tested the utility of these markers by evaluating the clonal status of shared durian types from different germplasm collection sites, and found that some were not clones. The findings in this preliminary study not only shows the feasibility of using SSR markers for DNA fingerprinting of durian types, but also challenges the current classification of durian types, e.g., on whether the different types should be called “clones”, “varieties”, or “cultivars”. Such matters have a direct impact on the regulation and management of durian genetic resources in the region. PMID:29511604

Novel 5.712 kb mitochondrial DNA deletion in a patient with Pearson syndrome: a case report.

PubMed

Park, Joonhong; Ryu, Hyejin; Jang, Woori; Chae, Hyojin; Kim, Myungshin; Kim, Yonggoo; Kim, Jiyeon; Lee, Jae Wook; Chung, Nack-Gyun; Cho, Bin; Suh, Byung Kyu

2015-05-01

Pearson marrow‑pancreas syndrome (PS) is a progressive multi‑organ disorder caused by deletions and duplications of mitochondrial DNA (mtDNA). PS is often fatal in infancy, and the majority of patients with PS succumb to the disease before reaching three‑years‑of‑age, due to septicemia, metabolic acidosis or hepatocellular insufficiency. The present report describes the case of a four‑month‑old infant with severe normocytic normochromic anemia, vacuolization of hematopoietic precursors and metabolic acidosis. After extensive clinical investigation, the patient was diagnosed with PS, which was confirmed by molecular analysis of mtDNA. The molecular analysis detected a novel large‑scale (5.712 kb) deletion spanning nucleotides 8,011 to 13,722 of mtDNA, which lacked direct repeats at the deletion boundaries. The present report is, to the best of our knowledge, the first case reported in South Korea.

A septal chromosome segregator protein evolved into a conjugative DNA-translocator protein

PubMed Central

Sepulveda, Edgardo; Vogelmann, Jutta

2011-01-01

Streptomycetes, Gram-positive soil bacteria well known for the production of antibiotics feature a unique conjugative DNA transfer system. In contrast to classical conjugation which is characterized by the secretion of a pilot protein covalently linked to a single-stranded DNA molecule, in Streptomyces a double-stranded DNA molecule is translocated during conjugative transfer. This transfer involves a single plasmid encoded protein, TraB. A detailed biochemical and biophysical characterization of TraB, revealed a close relationship to FtsK, mediating chromosome segregation during bacterial cell division. TraB translocates plasmid DNA by recognizing 8-bp direct repeats located in a specific plasmid region clt. Similar sequences accidentally also occur on chromosomes and have been shown to be bound by TraB. We suggest that TraB mobilizes chromosomal genes by the interaction with these chromosomal clt-like sequences not relying on the integration of the conjugative plasmid into the chromosome. PMID:22479692

DNA replication stress restricts ribosomal DNA copy number.

PubMed

Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

Sequences in the intergenic spacer influence RNA Pol I transcription from the human rRNA promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, W.M.; Sylvester, J.E.

1994-09-01

In most eucaryotic species, ribosomal genes are tandemly repeated about 100-5000 times per haploid genome. The 43 Kb human rDNA repeat consists of a 13 Kb coding region for the 18S, 5.8S, 28S ribosomal RNAs (rRNAs) and transcribed spacers separated by a 30 Kb intergenic spacer. For species such as frog, mouse and rat, sequences in the intergenic spacer other than the gene promoter have been shown to modulate transcription of the ribosomal gene. These sequences are spacer promoters, enhancers and the terminator for spacer transcription. We are addressing whether the human ribosomal gene promoter is similarly influenced. In-vitro transcriptionmore » run-off assays have revealed that the 4.5 kb region (CBE), directly upstream of the gene promoter, has cis-stimulation and trans-competition properties. This suggests that the CBE fragment contains an enhancer(s) for ribosomal gene transcription. Further experiments have shown that a fragment ({approximately}1.6 kb) within the CBE fragment also has trans-competition function. Deletion subclones of this region are being tested to delineate the exact sequences responsible for these modulating activities. Previous sequence analysis and functional studies have revealed that CBE contains regions of DNA capable of adopting alternative structures such as bent DNA, Z-DNA, and triple-stranded DNA. Whether these structures are required for modulating transcription remains to be determined as does the specific DNA-protein interaction involved.« less

Performance testing of a semi-automatic card punch system, using direct STR profiling of DNA from blood samples on FTA™ cards.

PubMed

Ogden, Samantha J; Horton, Jeffrey K; Stubbs, Simon L; Tatnell, Peter J

2015-01-01

The 1.2 mm Electric Coring Tool (e-Core™) was developed to increase the throughput of FTA(™) sample collection cards used during forensic workflows and is similar to a 1.2 mm Harris manual micro-punch for sampling dried blood spots. Direct short tandem repeat (STR) DNA profiling was used to compare samples taken by the e-Core tool with those taken by the manual micro-punch. The performance of the e-Core device was evaluated using a commercially available PowerPlex™ 18D STR System. In addition, an analysis was performed that investigated the potential carryover of DNA via the e-Core punch from one FTA disc to another. This contamination study was carried out using Applied Biosystems AmpflSTR™ Identifiler™ Direct PCR Amplification kits. The e-Core instrument does not contaminate FTA discs when a cleaning punch is used following excision of discs containing samples and generates STR profiles that are comparable to those generated by the manual micro-punch. © 2014 American Academy of Forensic Sciences.

Insights on genome size evolution from a miniature inverted repeat transposon driving a satellite DNA.

PubMed

Scalvenzi, Thibault; Pollet, Nicolas

2014-12-01

The genome size in eukaryotes does not correlate well with the number of genes they contain. We can observe this so-called C-value paradox in amphibian species. By analyzing an amphibian genome we asked how repetitive DNA can impact genome size and architecture. We describe here our discovery of a Tc1/mariner miniature inverted-repeat transposon family present in Xenopus frogs. These transposons named miDNA4 are unique since they contain a satellite DNA motif. We found that miDNA4 measured 331 bp, contained 25 bp long inverted terminal repeat sequences and a sequence motif of 119 bp present as a unique copy or as an array of 2-47 copies. We characterized the structure, dynamics, impact and evolution of the miDNA4 family and its satellite DNA in Xenopus frog genomes. This led us to propose a model for the evolution of these two repeated sequences and how they can synergize to increase genome size. Copyright © 2014 Elsevier Inc. All rights reserved.

Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna.

PubMed

Volkov, Roman A; Panchuk, Irina I; Borisjuk, Nikolai V; Hosiawa-Baranska, Marta; Maluszynska, Jolanta; Hemleben, Vera

2017-01-23

Polyploid hybrids represent a rich natural resource to study molecular evolution of plant genes and genomes. Here, we applied a combination of karyological and molecular methods to investigate chromosomal structure, molecular organization and evolution of ribosomal DNA (rDNA) in nightshade, Atropa belladonna (fam. Solanaceae), one of the oldest known allohexaploids among flowering plants. Because of their abundance and specific molecular organization (evolutionarily conserved coding regions linked to variable intergenic spacers, IGS), 45S and 5S rDNA are widely used in plant taxonomic and evolutionary studies. Molecular cloning and nucleotide sequencing of A. belladonna 45S rDNA repeats revealed a general structure characteristic of other Solanaceae species, and a very high sequence similarity of two length variants, with the only difference in number of short IGS subrepeats. These results combined with the detection of three pairs of 45S rDNA loci on separate chromosomes, presumably inherited from both tetraploid and diploid ancestor species, example intensive sequence homogenization that led to substitution/elimination of rDNA repeats of one parent. Chromosome silver-staining revealed that only four out of six 45S rDNA sites are frequently transcriptionally active, demonstrating nucleolar dominance. For 5S rDNA, three size variants of repeats were detected, with the major class represented by repeats containing all functional IGS elements required for transcription, the intermediate size repeats containing partially deleted IGS sequences, and the short 5S repeats containing severe defects both in the IGS and coding sequences. While shorter variants demonstrate increased rate of based substitution, probably in their transition into pseudogenes, the functional 5S rDNA variants are nearly identical at the sequence level, pointing to their origin from a single parental species. Localization of the 5S rDNA genes on two chromosome pairs further supports uniparental inheritance from the tetraploid progenitor. The obtained molecular, cytogenetic and phylogenetic data demonstrate complex evolutionary dynamics of rDNA loci in allohexaploid species of Atropa belladonna. The high level of sequence unification revealed in 45S and 5S rDNA loci of this ancient hybrid species have been seemingly achieved by different molecular mechanisms.

Molecular structure and chromosome distribution of three repetitive DNA families in Anemone hortensis L. (Ranunculaceae).

PubMed

Mlinarec, Jelena; Chester, Mike; Siljak-Yakovlev, Sonja; Papes, Drazena; Leitch, Andrew R; Besendorfer, Visnja

2009-01-01

The structure, abundance and location of repetitive DNA sequences on chromosomes can characterize the nature of higher plant genomes. Here we report on three new repeat DNA families isolated from Anemone hortensis L.; (i) AhTR1, a family of satellite DNA (stDNA) composed of a 554-561 bp long EcoRV monomer; (ii) AhTR2, a stDNA family composed of a 743 bp long HindIII monomer and; (iii) AhDR, a repeat family composed of a 945 bp long HindIII fragment that exhibits some sequence similarity to Ty3/gypsy-like retroelements. Fluorescence in-situ hybridization (FISH) to metaphase chromosomes of A. hortensis (2n = 16) revealed that both AhTR1 and AhTR2 sequences co-localized with DAPI-positive AT-rich heterochromatic regions. AhTR1 sequences occur at intercalary DAPI bands while AhTR2 sequences occur at 8-10 terminally located heterochromatic blocks. In contrast AhDR sequences are dispersed over all chromosomes as expected of a Ty3/gypsy-like element. AhTR2 and AhTR1 repeat families include polyA- and polyT-tracks, AT/TA-motifs and a pentanucleotide sequence (CAAAA) that may have consequences for chromatin packing and sequence homogeneity. AhTR2 repeats also contain TTTAGGG motifs and degenerate variants. We suggest that they arose by interspersion of telomeric repeats with subtelomeric repeats, before hybrid unit(s) amplified through the heterochromatic domain. The three repetitive DNA families together occupy approximately 10% of the A. hortensis genome. Comparative analyses of eight Anemone species revealed that the divergence of the A. hortensis genome was accompanied by considerable modification and/or amplification of repeats.

Comparative genomics and repetitive sequence divergence in the species of diploid Nicotiana section Alatae.

PubMed

Lim, K Yoong; Kovarik, Ales; Matyasek, Roman; Chase, Mark W; Knapp, Sandra; McCarthy, Elizabeth; Clarkson, James J; Leitch, Andrew R

2006-12-01

Combining phylogenetic reconstructions of species relationships with comparative genomic approaches is a powerful way to decipher evolutionary events associated with genome divergence. Here, we reconstruct the history of karyotype and tandem repeat evolution in species of diploid Nicotiana section Alatae. By analysis of plastid DNA, we resolved two clades with high bootstrap support, one containing N. alata, N. langsdorffii, N. forgetiana and N. bonariensis (called the n = 9 group) and another containing N. plumbaginifolia and N. longiflora (called the n = 10 group). Despite little plastid DNA sequence divergence, we observed, via fluorescent in situ hybridization, substantial chromosomal repatterning, including altered chromosome numbers, structure and distribution of repeats. Effort was focussed on 35S and 5S nuclear ribosomal DNA (rDNA) and the HRS60 satellite family of tandem repeats comprising the elements HRS60, NP3R and NP4R. We compared divergence of these repeats in diploids and polyploids of Nicotiana. There are dramatic shifts in the distribution of the satellite repeats and complete replacement of intergenic spacers (IGSs) of 35S rDNA associated with divergence of the species in section Alatae. We suggest that sequence homogenization has replaced HRS60 family repeats at sub-telomeric regions, but that this process may not occur, or occurs more slowly, when the repeats are found at intercalary locations. Sequence homogenization acts more rapidly (at least two orders of magnitude) on 35S rDNA than 5S rDNA and sub-telomeric satellite sequences. This rapid rate of divergence is analogous to that found in polyploid species, and is therefore, in plants, not only associated with polyploidy.

High-Resolution Whole-Genome Sequencing Reveals That Specific Chromatin Domains from Most Human Chromosomes Associate with Nucleoli

PubMed Central

van Koningsbruggen, Silvana; Gierliński, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J.; Ariyurek, Yavuz; den Dunnen, Johan T.

2010-01-01

The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope. PMID:20826608

High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

PubMed

van Koningsbruggen, Silvana; Gierlinski, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J; Ariyurek, Yavuz; den Dunnen, Johan T; Lamond, Angus I

2010-11-01

The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

Coexistence of minicircular and a highly rearranged mtDNA molecule suggests that recombination shapes mitochondrial genome organization.

PubMed

Mao, Meng; Austin, Andrew D; Johnson, Norman F; Dowton, Mark

2014-03-01

Recombination has been proposed as a possible mechanism to explain mitochondrial (mt) gene rearrangements, although the issue of whether mtDNA recombination occurs in animals has been controversial. In this study, we sequenced the entire mt genome of the megaspilid wasp Conostigmus sp., which possessed a highly rearranged mt genome. The sequence of the A+T-rich region contained a number of different types of repeats, similar to those reported previously in the nematode Meloidogyne javanica, in which recombination was discovered. In Conostigmus, we detected the end products of recombination: a range of minicircles. However, using isolated (cloned) fragments of the A+T-rich region, we established that some of these minicircles were found to be polymerase chain reaction (PCR) artifacts. It appears that regions with repeats are prone to PCR template switching or PCR jumping. Nevertheless, there is strong evidence that one minicircle is real, as amplification primers that straddle the putative breakpoint junction produce a single strong amplicon from genomic DNA but not from the cloned A+T-rich region. The results provide support for the direct link between recombination and mt gene rearrangement. Furthermore, we developed a model of recombination which is important for our understanding of mtDNA evolution.

A Dynamic Tandem Repeat in Monocotyledons Inferred from a Comparative Analysis of Chloroplast Genomes in Melanthiaceae.

PubMed

Do, Hoang Dang Khoa; Kim, Joo-Hwan

2017-01-01

Chloroplast genomes (cpDNA) are highly valuable resources for evolutionary studies of angiosperms, since they are highly conserved, are small in size, and play critical roles in plants. Slipped-strand mispairing (SSM) was assumed to be a mechanism for generating repeat units in cpDNA. However, research on the employment of different small repeated sequences through SSM events, which may induce the accumulation of distinct types of repeats within the same region in cpDNA, has not been documented. Here, we sequenced two chloroplast genomes from the endemic species Heloniopsis tubiflora (Korea) and Xerophyllum tenax (USA) to cover the gap between molecular data and explore "hot spots" for genomic events in Melanthiaceae. Comparative analysis of 23 complete cpDNA sequences revealed that there were different stages of deletion in the rps16 region across the Melanthiaceae. Based on the partial or complete loss of rps16 gene in cpDNA, we have firstly reported potential molecular markers for recognizing two sections ( Veratrum and Fuscoveratrum ) of Veratrum . Melathiaceae exhibits a significant change in the junction between large single copy and inverted repeat regions, ranging from trnH_GUG to a part of rps3 . Our results show an accumulation of tandem repeats in the rpl23-ycf2 regions of cpDNAs. Small conserved sequences exist and flank tandem repeats in further observation of this region across most of the examined taxa of Liliales. Therefore, we propose three scenarios in which different small repeated sequences were used during SSM events to generate newly distinct types of repeats. Occasionally, prior to the SSM process, point mutation event and double strand break repair occurred and induced the formation of initial repeat units which are indispensable in the SSM process. SSM may have likely occurred more frequently for short repeats than for long repeat sequences in tribe Parideae (Melanthiaceae, Liliales). Collectively, these findings add new evidence of dynamic results from SSM in chloroplast genomes which can be useful for further evolutionary studies in angiosperms. Additionally, genomics events in cpDNA are potential resources for mining molecular markers in Liliales.

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

PubMed

Lakshmikumaran, M S; D'Ambrosio, E; Laimins, L A; Lin, D T; Furano, A V

1985-09-01

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this.

Functional interrogation of non-coding DNA through CRISPR genome editing.

PubMed

Canver, Matthew C; Bauer, Daniel E; Orkin, Stuart H

2017-05-15

Methodologies to interrogate non-coding regions have lagged behind coding regions despite comprising the vast majority of the genome. However, the rapid evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has provided a multitude of novel techniques for laboratory investigation including significant contributions to the toolbox for studying non-coding DNA. CRISPR-mediated loss-of-function strategies rely on direct disruption of the underlying sequence or repression of transcription without modifying the targeted DNA sequence. CRISPR-mediated gain-of-function approaches similarly benefit from methods to alter the targeted sequence through integration of customized sequence into the genome as well as methods to activate transcription. Here we review CRISPR-based loss- and gain-of-function techniques for the interrogation of non-coding DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

Directed evolution of an RNA enzyme

NASA Technical Reports Server (NTRS)

Beaudry, Amber A.; Joyce, Gerald F.

1992-01-01

An in vitro evolution procedures was used to obtain RNA enzymes with a particular catalytic function. A population of 10 exp 13 variants of the Tetrahymena ribozyme, a group I ribozyme that catalyzes sequence-specific cleavage of RNA via a phosphoester transfer mechanism, was generated. This enzyme has a limited ability to cleave DNA under conditions of high temperature or high MgCl2 concentration, or both. A selection constraint was imposed on the population of ribozyme variants such that only those individuals that carried out DNA cleavage under physiologic conditions were amplified to produce 'progeny' ribozymes. Mutations were introduced during amplification to maintain heterogeneity in the population. This process was repeated for ten successive generations, resulting in enhanced (100 times) DNA cleavage activity.

A polymorphism in the MSH3 mismatch repair gene is associated with the levels of somatic instability of the expanded CTG repeat in the blood DNA of myotonic dystrophy type 1 patients.

PubMed

Morales, Fernando; Vásquez, Melissa; Santamaría, Carolina; Cuenca, Patricia; Corrales, Eyleen; Monckton, Darren G

2016-04-01

Somatic mosaicism of the expanded CTG repeat in myotonic dystrophy type 1 is age-dependent, tissue-specific and expansion-biased, contributing toward the tissue-specificity and progressive nature of the symptoms. Previously, using regression modelling of repeat instability we showed that variation in the rate of somatic expansion in blood DNA contributes toward variation in age of onset, directly implicating somatic expansion in the disease pathway. Here, we confirm these results using a larger more genetically homogenous Costa Rican DM1 cohort (p<0.001). Interestingly, we also provide evidence that supports subtle sex-dependent differences in repeat length-dependent age at onset and somatic mutational dynamics. Previously, we demonstrated that variation in the rate of somatic expansion was a heritable quantitative trait. Given the important role that DNA mismatch repair genes play in mediating expansions in mouse models, we tested for modifier gene effects with 13 DNA mismatch gene polymorphisms (one each in MSH2, PMS2, MSH6 and MLH1; and nine in MSH3). After correcting for allele length and age effects, we identified three polymorphisms in MSH3 that were associated with variation in somatic instability: Rs26279 (p=0.003); Rs1677658 (p=0.009); and Rs10168 (p=0.031). However, only the association with Rs26279 remained significant after multiple testing correction. Although we revealed a statistically significant association between Rs26279 and somatic instability, we did not detect an association with the age at onset. Individuals with the A/A genotype for Rs26279 tended to show a greater propensity to expand the CTG repeat than other genotypes. Interestingly, this SNP results in an amino acid change in the critical ATPase domain of MSH3 and is potentially functionally dimorphic. These data suggest that MSH3 is a key player in generating somatic variation in DM1 patients and further highlight MSH3 as a potential therapeutic target. Copyright © 2016 Elsevier B.V. All rights reserved.

Laser mass spectrometry for DNA fingerprinting for forensic applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, C.H.; Tang, K.; Taranenko, N.I.

The application of DNA fingerprinting has become very broad in forensic analysis, patient identification, diagnostic medicine, and wildlife poaching, since every individual`s DNA structure is identical within all tissues of their body. DNA fingerprinting was initiated by the use of restriction fragment length polymorphisms (RFLP). In 1987, Nakamura et al. found that a variable number of tandem repeats (VNTR) often occurred in the alleles. The probability of different individuals having the same number of tandem repeats in several different alleles is very low. Thus, the identification of VNTR from genomic DNA became a very reliable method for identification of individuals.more » DNA fingerprinting is a reliable tool for forensic analysis. In DNA fingerprinting, knowledge of the sequence of tandem repeats and restriction endonuclease sites can provide the basis for identification. The major steps for conventional DNA fingerprinting include (1) specimen processing (2) amplification of selected DNA segments by PCR, and (3) gel electrophoresis to do the final DNA analysis. In this work we propose to use laser desorption mass spectrometry for fast DNA fingerprinting. The process and advantages are discussed.« less

Structural and biophysical properties of h-FANCI ARM repeat protein.

PubMed

Siddiqui, Mohd Quadir; Choudhary, Rajan Kumar; Thapa, Pankaj; Kulkarni, Neha; Rajpurohit, Yogendra S; Misra, Hari S; Gadewal, Nikhil; Kumar, Satish; Hasan, Syed K; Varma, Ashok K

2017-11-01

Fanconi anemia complementation groups - I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein-protein and protein-DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.

Sunflower centromeres consist of a centromere-specific LINE and a chromosome-specific tandem repeat.

PubMed

Nagaki, Kiyotaka; Tanaka, Keisuke; Yamaji, Naoki; Kobayashi, Hisato; Murata, Minoru

2015-01-01

The kinetochore is a protein complex including kinetochore-specific proteins that plays a role in chromatid segregation during mitosis and meiosis. The complex associates with centromeric DNA sequences that are usually species-specific. In plant species, tandem repeats including satellite DNA sequences and retrotransposons have been reported as centromeric DNA sequences. In this study on sunflowers, a cDNA-encoding centromere-specific histone H3 (CENH3) was isolated from a cDNA pool from a seedling, and an antibody was raised against a peptide synthesized from the deduced cDNA. The antibody specifically recognized the sunflower CENH3 (HaCENH3) and showed centromeric signals by immunostaining and immunohistochemical staining analysis. The antibody was also applied in chromatin immunoprecipitation (ChIP)-Seq to isolate centromeric DNA sequences and two different types of repetitive DNA sequences were identified. One was a long interspersed nuclear element (LINE)-like sequence, which showed centromere-specific signals on almost all chromosomes in sunflowers. This is the first report of a centromeric LINE sequence, suggesting possible centromere targeting ability. Another type of identified repetitive DNA was a tandem repeat sequence with a 187-bp unit that was found only on a pair of chromosomes. The HaCENH3 content of the tandem repeats was estimated to be much higher than that of the LINE, which implies centromere evolution from LINE-based centromeres to more stable tandem-repeat-based centromeres. In addition, the epigenetic status of the sunflower centromeres was investigated by immunohistochemical staining and ChIP, and it was found that centromeres were heterochromatic.

Evidence of birth-and-death evolution of 5S rRNA gene in Channa species (Teleostei, Perciformes).

PubMed

Barman, Anindya Sundar; Singh, Mamta; Singh, Rajeev Kumar; Lal, Kuldeep Kumar

2016-12-01

In higher eukaryotes, minor rDNA family codes for 5S rRNA that is arranged in tandem arrays and comprises of a highly conserved 120 bp long coding sequence with a variable non-transcribed spacer (NTS). Initially the 5S rDNA repeats are considered to be evolved by the process of concerted evolution. But some recent reports, including teleost fishes suggested that evolution of 5S rDNA repeat does not fit into the concerted evolution model and evolution of 5S rDNA family may be explained by a birth-and-death evolution model. In order to study the mode of evolution of 5S rDNA repeats in Perciformes fish species, nucleotide sequence and molecular organization of five species of genus Channa were analyzed in the present study. Molecular analyses revealed several variants of 5S rDNA repeats (four types of NTS) and networks created by a neighbor net algorithm for each type of sequences (I, II, III and IV) did not show a clear clustering in species specific manner. The stable secondary structure is predicted and upstream and downstream conserved regulatory elements were characterized. Sequence analyses also shown the presence of two putative pseudogenes in Channa marulius. Present study supported that 5S rDNA repeats in genus Channa were evolved under the process of birth-and-death.

MutSβ and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells

PubMed Central

Gannon, Anne-Marie M.; Frizzell, Aisling; Healy, Evan; Lahue, Robert S.

2012-01-01

Trinucleotide repeat (TNR) expansions cause at least 17 heritable neurological diseases, including Huntington’s disease. Expansions are thought to arise from abnormal processing of TNR DNA by specific trans-acting proteins. For example, the DNA repair complex MutSβ (MSH2–MSH3 heterodimer) is required in mice for on-going expansions of long, disease-causing alleles. A distinctive feature of TNR expansions is a threshold effect, a narrow range of repeat units (∼30–40 in humans) at which mutation frequency rises dramatically and disease can initiate. The goal of this study was to identify factors that promote expansion of threshold-length CTG•CAG repeats in a human astrocytic cell line. siRNA knockdown of the MutSβ subunits MSH2 or MSH3 impeded expansions of threshold-length repeats, while knockdown of the MutSα subunit MSH6 had no effect. Chromatin immunoprecipitation experiments indicated that MutSβ, but not MutSα, was enriched at the TNR. These findings imply a direct role for MutSβ in promoting expansion of threshold-length CTG•CAG tracts. We identified the class II deacetylase HDAC5 as a novel promoting factor for expansions, joining the class I deacetylase HDAC3 that was previously identified. Double knockdowns were consistent with the possibility that MutSβ, HDAC3 and HDAC5 act through a common pathway to promote expansions of threshold-length TNRs. PMID:22941650

Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering.

PubMed

Moyer, Tyler C; Holland, Andrew J

2015-01-01

The ability to rapidly and specifically modify the genome of mammalian cells has been a long-term goal of biomedical researchers. Recently, the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system from bacteria has been exploited for genome engineering in human cells. The CRISPR system directs the RNA-guided Cas9 nuclease to a specific genomic locus to induce a DNA double-strand break that may be subsequently repaired by homology-directed repair using an exogenous DNA repair template. Here we describe a protocol using CRISPR/Cas9 to achieve bi-allelic insertion of a point mutation in human cells. Using this method, homozygous clonal cell lines can be constructed in 5-6 weeks. This method can also be adapted to insert larger DNA elements, such as fluorescent proteins and degrons, at defined genomic locations. CRISPR/Cas9 genome engineering offers exciting applications in both basic science and translational research. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of non-CG genomic hypomethylation associated with gamma-ray-induced suppression of CMT3 transcription in Arabidopsis thaliana.

PubMed

Kim, Ji Eun; Lee, Min Hee; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin-Hong

2013-12-01

Ionizing radiation causes various epigenetic changes, as well as a variety of DNA lesions such as strand breaks, cross-links, oxidative damages, etc., in genomes. However, radiation-induced epigenetic changes have rarely been substantiated in plant genomes. The current study investigates whether DNA methylation of Arabidopsis thaliana genome is altered by gamma rays. We found that genomic DNA methylation decreased in wild-type plants with increasing doses of gamma rays (5, 50 and 200 Gy). Irradiation with 200 Gy significantly increased the expression of transcriptionally inactive centromeric 180-bp (CEN) and transcriptionally silent information (TSI) repeats. This increase suggested that there was a substantial release of transcriptional gene silencing by gamma rays, probably by induction of DNA hypomethylation. High expression of the DNA demethylase ROS1 and low expression of the DNA methyltransferase CMT3 supported this hypothesis. Moreover, Southern blot analysis following digestion of genomic DNA with methylation-sensitive enzymes revealed that the DNA hypomethylation occured preferentially at CHG or CHH sites rather than CG sites, depending on the radiation dose. Unlike CEN and TSI repeats, the number of Ta3, AtSN1 and FWA repeats decreased in transcription but increased in non-CG methylation. In addition, the cmt3-11 mutant showed neither DNA hypomethylation nor transcriptional activation of silenced repeats upon gamma irradiation. Furthermore, profiles of genome-wide transcriptomes in response to gamma rays differed between the wild-type and cmt3-11 mutant. These results suggest that gamma irradiation induced DNA hypomethylation preferentially at non-CG sites of transcriptionally inactive repeats in a locus-specific manner, which depends on CMT3 activity.

Foldback intercoil DNA and the mechanism of DNA transposition.

PubMed

Kim, Byung-Dong

2014-09-01

Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNA at as sharp as 180° and the intertwining of two double helixes within each other's major groove to form an intercoil with a diameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion and inversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated by the direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition of DNA transposons involves the pairing of terminal inverted repeats and 5-7-bp tandem target duplication. FBI DNA configuration effectively explains simple as well as replicative transposition, along with the involvement of an enhancer element. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologous end-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.

Nature of frequent deletions in CEBPA.

PubMed

Fuchs, Ota; Kostecka, Arnost; Provaznikova, Dana; Krasna, Blazena; Brezinova, Jana; Filkukova, Jitka; Kotlin, Roman; Kouba, Michal; Kobylka, Petr; Neuwirtova, Radana; Jonasova, Anna; Caniga, Miroslav; Schwarz, Jiri; Markova, Jana; Maaloufova, Jacqueline; Sponerova, Dana; Novakova, Ludmila; Cermak, Jaroslav

2009-01-01

C/EBPalpha (CCAAT/enhancer binding protein alpha) belongs to the family of leucine zipper transcription factors and is necessary for transcriptional control of granulocyte, adipocyte and hepatocyte differentiation, glucose metabolism and lung development. C/EBPalpha is encoded by an intronless gene. CEBPA mutations cause a myeloid differentiation block and were detected in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), multiple myeloma and non-Hodgkin's lymphoma (NHL) patients. In this study we identified in 41 individuals from 824 screened individuals (290 AML patients, 382 MDS patients, 56 NHL patients and 96 healthy individuals) a single class of 23 deletions in CEBPA gene which involved a direct repeat of at least 2 bp. These mutations are characterised by the loss of one of two same repeats at the ends of deleted sequence. Three most frequent repeats included in these deletions in CEBPA gene are CGCGAG (493-498_865-870), GCCAAGCAGC (508-517_907-916) and GG (486-487_885-886), all according to GenBank accession no. NM_004364.2. A mechanism for deletion formation between two repetitive sequences can be recombination events in the repair process. Double-stranded cut in DNA can initiate these recombination events of adjacent DNA sequences.

The Repeat Expansion Diseases: the dark side of DNA repair?

PubMed Central

Zhao, Xiao-Nan; Usdin, Karen

2015-01-01

DNA repair normally protects the genome against mutations that threaten genome integrity and thus cell viability. However, growing evidence suggests that in the case of the Repeat Expansion Diseases, disorders that result from an increase in the size of a disease-specific microsatellite, the disease-causing mutation is actually the result of aberrant DNA repair. A variety of proteins from different DNA repair pathways have thus far been implicated in this process. This review will summarize recent findings from patients and from mouse models of these diseases that shed light on how these pathways may interact to cause repeat expansion. PMID:26002199

Analysis of the giant genomes of Fritillaria (Liliaceae) indicates that a lack of DNA removal characterizes extreme expansions in genome size.

PubMed

Kelly, Laura J; Renny-Byfield, Simon; Pellicer, Jaume; Macas, Jiří; Novák, Petr; Neumann, Pavel; Lysak, Martin A; Day, Peter D; Berger, Madeleine; Fay, Michael F; Nichols, Richard A; Leitch, Andrew R; Leitch, Ilia J

2015-10-01

Plants exhibit an extraordinary range of genome sizes, varying by > 2000-fold between the smallest and largest recorded values. In the absence of polyploidy, changes in the amount of repetitive DNA (transposable elements and tandem repeats) are primarily responsible for genome size differences between species. However, there is ongoing debate regarding the relative importance of amplification of repetitive DNA versus its deletion in governing genome size. Using data from 454 sequencing, we analysed the most repetitive fraction of some of the largest known genomes for diploid plant species, from members of Fritillaria. We revealed that genomic expansion has not resulted from the recent massive amplification of just a handful of repeat families, as shown in species with smaller genomes. Instead, the bulk of these immense genomes is composed of highly heterogeneous, relatively low-abundance repeat-derived DNA, supporting a scenario where amplified repeats continually accumulate due to infrequent DNA removal. Our results indicate that a lack of deletion and low turnover of repetitive DNA are major contributors to the evolution of extremely large genomes and show that their size cannot simply be accounted for by the activity of a small number of high-abundance repeat families. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

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

PubMed Central

Komosa, Martin; Root, Heather; Meyn, M. Stephen

2015-01-01

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. PMID:25662602

The organization of repeating units in mitochondrial DNA from yeast petite mutants.

PubMed

Bos, J L; Heyting, C; Van der Horst, G; Borst, P

1980-04-01

We have reinvestigated the linkage orientation of repeating units in mtDNAs of yeast ρ(-) petite mutants containing an inverted duplication. All five petite mtDNAs studied contain a continuous segment of wild-type mtDNA, part of which is duplicated and present in inverted form in the repeat. We show by restriction enzyme analysis that the non-duplicated segments between the inverted duplications are present in random orientation in all five petite mtDNAs. There is no segregation of sub-types with unique orientation. We attribute this to the high rate of intramolecular recombination between the inverted duplications. The results provide additional evidence for the high rate of recombination of yeast mtDNA even in haploid ρ(-) petite cells.We conclude that only two types of stable sequence organization exist in petite mtDNA: petites without an inverted duplication have repeats linked in straight head-to-tail arrangement (abcabc); petites with an inverted duplication have repeats in which the non-duplicated segments are present in random orientation.

High-throughput analysis of the satellitome illuminates satellite DNA evolution

NASA Astrophysics Data System (ADS)

Ruiz-Ruano, Francisco J.; López-León, María Dolores; Cabrero, Josefa; Camacho, Juan Pedro M.

2016-07-01

Satellite DNA (satDNA) is a major component yet the great unknown of eukaryote genomes and clearly underrepresented in genome sequencing projects. Here we show the high-throughput analysis of satellite DNA content in the migratory locust by means of the bioinformatic analysis of Illumina reads with the RepeatExplorer and RepeatMasker programs. This unveiled 62 satDNA families and we propose the term “satellitome” for the whole collection of different satDNA families in a genome. The finding that satDNAs were present in many contigs of the migratory locust draft genome indicates that they show many genomic locations invisible by fluorescent in situ hybridization (FISH). The cytological pattern of five satellites showing common descent (belonging to the SF3 superfamily) suggests that non-clustered satDNAs can become into clustered through local amplification at any of the many genomic loci resulting from previous dissemination of short satDNA arrays. The fact that all kinds of satDNA (micro- mini- and satellites) can show the non-clustered and clustered states suggests that all these elements are mostly similar, except for repeat length. Finally, the presence of VNTRs in bacteria, showing similar properties to non-clustered satDNAs in eukaryotes, suggests that this kind of tandem repeats show common properties in all living beings.

Mapping Simple Repeated DNA Sequences in Heterochromatin of Drosophila Melanogaster

PubMed Central

Lohe, A. R.; Hilliker, A. J.; Roberts, P. A.

1993-01-01

Heterochromatin in Drosophila has unusual genetic, cytological and molecular properties. Highly repeated DNA sequences (satellites) are the principal component of heterochromatin. Using probes from cloned satellites, we have constructed a chromosome map of 10 highly repeated, simple DNA sequences in heterochromatin of mitotic chromosomes of Drosophila melanogaster. Despite extensive sequence homology among some satellites, chromosomal locations could be distinguished by stringent in situ hybridizations for each satellite. Only two of the localizations previously determined using gradient-purified bulk satellite probes are correct. Eight new satellite localizations are presented, providing a megabase-level chromosome map of one-quarter of the genome. Five major satellites each exhibit a multichromosome distribution, and five minor satellites hybridize to single sites on the Y chromosome. Satellites closely related in sequence are often located near one another on the same chromosome. About 80% of Y chromosome DNA is composed of nine simple repeated sequences, in particular (AAGAC)(n) (8 Mb), (AAGAG)(n) (7 Mb) and (AATAT)(n) (6 Mb). Similarly, more than 70% of the DNA in chromosome 2 heterochromatin is composed of five simple repeated sequences. We have also generated a high resolution map of satellites in chromosome 2 heterochromatin, using a series of translocation chromosomes whose breakpoints in heterochromatin were ordered by N-banding. Finally, staining and banding patterns of heterochromatic regions are correlated with the locations of specific repeated DNA sequences. The basis for the cytochemical heterogeneity in banding appears to depend exclusively on the different satellite DNAs present in heterochromatin. PMID:8375654

[Polymorphic loci and polymorphism analysis of short tandem repeats within XNP gene].

PubMed

Liu, Qi-Ji; Gong, Yao-Qin; Guo, Chen-Hong; Chen, Bing-Xi; Li, Jiang-Xia; Guo, Yi-Shou

2002-01-01

To select polymorphic short tandem repeat markers within X-linked nuclear protein (XNP) gene, genomic clones which contain XNP gene were recognized by homologous analysis with XNP cDNA. By comparing the cDNA with genomic DNA, non-exonic sequences were identified, and short tandem repeats were selected from non-exonic sequences by using BCM search Launcher. Polymorphisms of the short tandem repeats in Chinese population were evaluated by PCR amplification and PAGE. Five short tandem repeats were identified from XNP gene, two of which were polymorphic. Four and 11 alleles were observed in Chinese population for XNPSTR1 and XNPSTR4, respectively. Heterozygosities were 47% for XNPSTR1 and 70% for XNPSTR4. XNPSTR1 and XNPSTR4 localized within 3' end and intron 10, respectively. Two polymorphic short tandem repeats have been identified within XNP gene and will be useful for linkage analysis and gene diagnosis of XNP gene.

DNA replication stress restricts ribosomal DNA copy number

PubMed Central

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

PubMed Central

Lakshmikumaran, M S; D'Ambrosio, E; Laimins, L A; Lin, D T; Furano, A V

1985-01-01

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this. Images PMID:3016521

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

PubMed Central

de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

2014-01-01

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. PMID:24792163

Primary analysis of repeat elements of the Asian seabass (Lates calcarifer) transcriptome and genome

PubMed Central

Kuznetsova, Inna S.; Thevasagayam, Natascha M.; Sridatta, Prakki S. R.; Komissarov, Aleksey S.; Saju, Jolly M.; Ngoh, Si Y.; Jiang, Junhui; Shen, Xueyan; Orbán, László

2014-01-01

As part of our Asian seabass genome project, we are generating an inventory of repeat elements in the genome and transcriptome. The karyotype showed a diploid number of 2n = 24 chromosomes with a variable number of B-chromosomes. The transcriptome and genome of Asian seabass were searched for repetitive elements with experimental and bioinformatics tools. Six different types of repeats constituting 8–14% of the genome were characterized. Repetitive elements were clustered in the pericentromeric heterochromatin of all chromosomes, but some of them were preferentially accumulated in pretelomeric and pericentromeric regions of several chromosomes pairs and have chromosomes specific arrangement. From the dispersed class of fish-specific non-LTR retrotransposon elements Rex1 and MAUI-like repeats were analyzed. They were wide-spread both in the genome and transcriptome, accumulated on the pericentromeric and peritelomeric areas of all chromosomes. Every analyzed repeat was represented in the Asian seabass transcriptome, some showed differential expression between the gonads. The other group of repeats analyzed belongs to the rRNA multigene family. FISH signal for 5S rDNA was located on a single pair of chromosomes, whereas that for 18S rDNA was found on two pairs. A BAC-derived contig containing rDNA was sequenced and assembled into a scaffold containing incomplete fragments of 18S rDNA. Their assembly and chromosomal position revealed that this part of Asian seabass genome is extremely rich in repeats containing evolutionarily conserved and novel sequences. In summary, transcriptome assemblies and cDNA data are suitable for the identification of repetitive DNA from unknown genomes and for comparative investigation of conserved elements between teleosts and other vertebrates. PMID:25120555

Fasting-induced apoptosis in rat liver is blocked by cycloheximide.

PubMed

Tessitore, L; Tomasi, C; Greco, M

1999-08-01

The effect of cycloheximide (CH) on the fasting-induced changes of rat liver cell and protein turnover has been investigated. Late starvation phase (3-4-day-fasting period) was characterised by a decrease in liver weight and protein and DNA content. The loss of DNA was not related to liver cell necrosis but due not only to depression of cell proliferation as shown by the drop in the labelling index but also induction of apoptosis. This type of apoptosis was documented by the increase in the apoptotic index (cells labelled by TUNEL) and transglutaminase activity as well as by DNA fragmentation. The liver cells of fasted rats appeared smaller as shown by the higher cell density and DNA/protein ratio than in controls. Females were more resistant to fasting-induced apoptosis than males. A single dose of CH, a drug primary known as inhibitor of protein synthesis, induced or enhanced apoptosis in fed and 2-days fasted male rats, respectively, without any sign of cell necrosis. On the contrary, the administration of repeated doses of CH blocked apoptosis induced by fasting. CH "froze" protein and DNA content as well as apoptotic process at the level of 2 days-fasted rats. While fasting-induced liver protein loss resulted from a marked reduction in protein synthesis with a slight decrease in degradation, repeated treatment with CH virtually blocked protein loss by abolishing protein catabolism. These data suggest a direct relationship between the catabolic side of protein turnover and the apoptotic process.

DNA Data Visualization (DDV): Software for Generating Web-Based Interfaces Supporting Navigation and Analysis of DNA Sequence Data of Entire Genomes.

PubMed

Neugebauer, Tomasz; Bordeleau, Eric; Burrus, Vincent; Brzezinski, Ryszard

2015-01-01

Data visualization methods are necessary during the exploration and analysis activities of an increasingly data-intensive scientific process. There are few existing visualization methods for raw nucleotide sequences of a whole genome or chromosome. Software for data visualization should allow the researchers to create accessible data visualization interfaces that can be exported and shared with others on the web. Herein, novel software developed for generating DNA data visualization interfaces is described. The software converts DNA data sets into images that are further processed as multi-scale images to be accessed through a web-based interface that supports zooming, panning and sequence fragment selection. Nucleotide composition frequencies and GC skew of a selected sequence segment can be obtained through the interface. The software was used to generate DNA data visualization of human and bacterial chromosomes. Examples of visually detectable features such as short and long direct repeats, long terminal repeats, mobile genetic elements, heterochromatic segments in microbial and human chromosomes, are presented. The software and its source code are available for download and further development. The visualization interfaces generated with the software allow for the immediate identification and observation of several types of sequence patterns in genomes of various sizes and origins. The visualization interfaces generated with the software are readily accessible through a web browser. This software is a useful research and teaching tool for genetics and structural genomics.

Activation of both acfA and acfD transcription by Vibrio cholerae ToxT requires binding to two centrally located DNA sites in an inverted repeat conformation.

PubMed

Withey, Jeffrey H; DiRita, Victor J

2005-05-01

The Gram-negative bacterium Vibrio cholerae is the infectious agent responsible for the disease Asiatic cholera. The genes required for V. cholerae virulence, such as those encoding the cholera toxin (CT) and toxin-coregulated pilus (TCP), are controlled by a cascade of transcriptional activators. Ultimately, the direct transcriptional activator of the majority of V. cholerae virulence genes is the AraC/XylS family member ToxT protein, the expression of which is activated by the ToxR and TcpP proteins. Previous studies have identified the DNA sites to which ToxT binds upstream of the ctx operon, encoding CT, and the tcpA operon, encoding, among other products, the major subunit of the TCP. These known ToxT binding sites are seemingly dissimilar in sequence other than being A/T rich. Further results suggested that ctx and tcpA each has a pair of ToxT binding sites arranged in a direct repeat orientation upstream of the core promoter elements. In this work, using both transcriptional lacZ fusions and in vitro copper-phenanthroline footprinting experiments, we have identified the ToxT binding sites between the divergently transcribed acfA and acfD genes, which encode components of the accessory colonization factor required for efficient intestinal colonization by V. cholerae. Our results indicate that ToxT binds to a pair of DNA sites between acfA and acfD in an inverted repeat orientation. Moreover, a mutational analysis of the ToxT binding sites indicates that both binding sites are required by ToxT for transcriptional activation of both acfA and acfD. Using copper-phenanthroline footprinting to assess the occupancy of ToxT on DNA having mutations in one of these binding sites, we found that protection by ToxT of the unaltered binding site was not affected, whereas protection by ToxT of the mutant binding site was significantly reduced in the region of the mutations. The results of further footprinting experiments using DNA templates having +5 bp and +10 bp insertions between the two ToxT binding sites indicate that both binding sites are occupied by ToxT regardless of their positions relative to each other. Based on these results, we propose that ToxT binds independently to two DNA sites between acfA and acfD to activate transcription of both genes.

Tandemly repeated sequences in mtDNA control region of whitefish, Coregonus lavaretus.

PubMed

Brzuzan, P

2000-06-01

Length variation of the mitochondrial DNA control region was observed with PCR amplification of a sample of 138 whitefish (Coregonus lavaretus). Nucleotide sequences of representative PCR products showed that the variation was due to the presence of an approximately 100-bp motif tandemly repeated two, three, or five times in the region between the conserved sequence block-3 (CSB-3) and the gene for phenylalanine tRNA. This is the first report on the tandem array composed of long repeat units in mitochondrial DNA of salmonids.

Dynamics and biological relevance of DNA demethylation in Arabidopsis antibacterial defense.

PubMed

Yu, Agnès; Lepère, Gersende; Jay, Florence; Wang, Jingyu; Bapaume, Laure; Wang, Yu; Abraham, Anne-Laure; Penterman, Jon; Fischer, Robert L; Voinnet, Olivier; Navarro, Lionel

2013-02-05

DNA methylation is an epigenetic mark that silences transposable elements (TEs) and repeats. Whereas the establishment and maintenance of DNA methylation are relatively well understood, little is known about their dynamics and biological relevance in plant and animal innate immunity. Here, we show that some TEs are demethylated and transcriptionally reactivated during antibacterial defense in Arabidopsis. This effect is correlated with the down-regulation of key transcriptional gene silencing factors and is partly dependent on an active demethylation process. DNA demethylation restricts multiplication and vascular propagation of the bacterial pathogen Pseudomonas syringae in leaves and, accordingly, some immune-response genes, containing repeats in their promoter regions, are negatively regulated by DNA methylation. This study provides evidence that DNA demethylation is part of a plant-induced immune response, potentially acting to prime transcriptional activation of some defense genes linked to TEs/repeats.

Regulation of DNA methylation patterns by CK2-mediated phosphorylation of Dnmt3a.

PubMed

Deplus, Rachel; Blanchon, Loïc; Rajavelu, Arumugam; Boukaba, Abdelhalim; Defrance, Matthieu; Luciani, Judith; Rothé, Françoise; Dedeurwaerder, Sarah; Denis, Hélène; Brinkman, Arie B; Simmer, Femke; Müller, Fabian; Bertin, Benjamin; Berdasco, Maria; Putmans, Pascale; Calonne, Emilie; Litchfield, David W; de Launoit, Yvan; Jurkowski, Tomasz P; Stunnenberg, Hendrik G; Bock, Christoph; Sotiriou, Christos; Fraga, Mario F; Esteller, Manel; Jeltsch, Albert; Fuks, François

2014-08-07

DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Fragile DNA Motifs Trigger Mutagenesis at Distant Chromosomal Loci in Saccharomyces cerevisiae

PubMed Central

Saini, Natalie; Zhang, Yu; Nishida, Yuri; Sheng, Ziwei; Choudhury, Shilpa; Mieczkowski, Piotr; Lobachev, Kirill S.

2013-01-01

DNA sequences capable of adopting non-canonical secondary structures have been associated with gross-chromosomal rearrangements in humans and model organisms. Previously, we have shown that long inverted repeats that form hairpin and cruciform structures and triplex-forming GAA/TTC repeats induce the formation of double-strand breaks which trigger genome instability in yeast. In this study, we demonstrate that breakage at both inverted repeats and GAA/TTC repeats is augmented by defects in DNA replication. Increased fragility is associated with increased mutation levels in the reporter genes located as far as 8 kb from both sides of the repeats. The increase in mutations was dependent on the presence of inverted or GAA/TTC repeats and activity of the translesion polymerase Polζ. Mutagenesis induced by inverted repeats also required Sae2 which opens hairpin-capped breaks and initiates end resection. The amount of breakage at the repeats is an important determinant of mutations as a perfect palindromic sequence with inherently increased fragility was also found to elevate mutation rates even in replication-proficient strains. We hypothesize that the underlying mechanism for mutagenesis induced by fragile motifs involves the formation of long single-stranded regions in the broken chromosome, invasion of the undamaged sister chromatid for repair, and faulty DNA synthesis employing Polζ. These data demonstrate that repeat-mediated breaks pose a dual threat to eukaryotic genome integrity by inducing chromosomal aberrations as well as mutations in flanking genes. PMID:23785298

Alu repeated DNAs are differentially methylated in primate germ cells.

PubMed Central

Rubin, C M; VandeVoort, C A; Teplitz, R L; Schmid, C W

1994-01-01

A significant fraction of Alu repeats in human sperm DNA, previously found to be unmethylated, is nearly completely methylated in DNA from many somatic tissues. A similar fraction of unmethylated Alus is observed here in sperm DNA from rhesus monkey. However, Alus are almost completely methylated at the restriction sites tested in monkey follicular oocyte DNA. The Alu methylation patterns in mature male and female monkey germ cells are consistent with Alu methylation in human germ cell tumors. Alu sequences are hypomethylated in seminoma DNAs and more methylated in a human ovarian dysgerminoma. These results contrast with methylation patterns reported for germ cell single-copy, CpG island, satellite, and L1 sequences. The function of Alu repeats is not known, but differential methylation of Alu repeats in the male and female germ lines suggests that they may serve as markers for genomic imprinting or in maintaining differences in male and female meiosis. Images PMID:7800508

Length and sequence heterogeneity in 5S rDNA of Populus deltoides.

PubMed

Negi, Madan S; Rajagopal, Jyothi; Chauhan, Neeti; Cronn, Richard; Lakshmikumaran, Malathi

2002-12-01

The 5S rRNA genes and their associated non-transcribed spacer (NTS) regions are present as repeat units arranged in tandem arrays in plant genomes. Length heterogeneity in 5S rDNA repeats was previously identified in Populus deltoides and was also observed in the present study. Primers were designed to amplify the 5S rDNA NTS variants from the P. deltoides genome. The PCR-amplified products from the two accessions of P. deltoides (G3 and G48) suggested the presence of length heterogeneity of 5S rDNA units within and among accessions, and the size of the spacers ranged from 385 to 434 bp. Sequence analysis of the non-transcribed spacer (NTS) revealed two distinct classes of 5S rDNA within both accessions: class 1, which contained GAA trinucleotide microsatellite repeats, and class 2, which lacked the repeats. The class 1 spacer shows length variation owing to the microsatellite, with two clones exhibiting 10 GAA repeat units and one clone exhibiting 16 such repeat units. However, distance analysis shows that class 1 spacer sequences are highly similar inter se, yielding nucleotide diversity (pi) estimates that are less than 0.15% of those obtained for class 2 spacers (pi = 0.0183 vs. 0.1433, respectively). The presence of microsatellite in the NTS region leading to variation in spacer length is reported and discussed for the first time in P. deltoides.

Intramolecular transposition by a synthetic IS50 (Tn5) derivative

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomcsanyi, T.; Phadnis, S.H.; Berg, D.E.

1990-11-01

We report the formation of deletions and inversions by intramolecular transposition of Tn5-derived mobile elements. The synthetic transposons used contained the IS50 O and I end segments and the transposase gene, a contraselectable gene encoding sucrose sensitivity (sacB), antibiotic resistance genes, and a plasmid replication origin. Both deletions and inversions were associated with loss of a 300-bp segment that is designated the vector because it is outside of the transposon. Deletions were severalfold more frequent than inversions, perhaps reflecting constraints on DNA twisting or abortive transposition. Restriction and DNA sequence analyses showed that both types of rearrangements extended from onemore » transposon end to many different sites in target DNA. In the case of inversions, transposition generated 9-bp direct repeats of target sequences.« less

Caffeine inhibits homology-directed repair of I-SceI-induced DNA double-strand breaks.

PubMed

Wang, Huichen; Boecker, Wilfried; Wang, Hongyan; Wang, Xiang; Guan, Jun; Thompson, Larry H; Nickoloff, Jac A; Iliakis, George

2004-01-22

We recently reported that two Chinese hamster mutants deficient in the RAD51 paralogs XRCC2 and XRCC3 show reduced radiosensitization after treatment with caffeine, thus implicating homology-directed repair (HDR) of DNA double-strand breaks (DSBs) in the mechanism of caffeine radiosensitization. Here, we investigate directly the effect of caffeine on HDR initiated by DSBs induced by a rare cutting endonuclease (I-SceI) into one of two direct DNA repeats. The results demonstrate a strong inhibition by caffeine of HDR in wild-type cells, and a substantial reduction of this effect in HDR-deficient XRCC3 mutant cells. Inhibition of HDR and cell radiosensitization to killing shows similar dependence on caffeine concentration suggesting a cause-effect relationship between these effects. UCN-01, a kinase inhibitor that effectively abrogates checkpoint activation in irradiated cells, has only a small effect on HDR, indicating that similar to radiosensitization, inhibition of checkpoint signaling is not sufficient for HDR inhibition. Recombination events occurring during treatment with caffeine are characterized by rearrangements reminiscent to those previously reported for the XRCC3 mutant, and immunofluorescence microscopy demonstrates significantly reduced formation of IR-specific RAD51 foci after caffeine treatment. In summary, our results identify inhibition of HDR as a significant contributor to caffeine radiosensitization.

Horizontal gene transfer from Agrobacterium to plants.

PubMed

Matveeva, Tatiana V; Lutova, Ludmila A

2014-01-01

Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named "cellular T-DNA" (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rubinson, Emily H.; Prakasha Gowda, A.S.; Spratt, Thomas E.

DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation,more » providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.« less

DNA Fingerprint Analysis of Three Short Tandem Repeat (STR) Loci for Biochemistry and Forensic Science Laboratory Courses

ERIC Educational Resources Information Center

McNamara-Schroeder, Kathleen; Olonan, Cheryl; Chu, Simon; Montoya, Maria C.; Alviri, Mahta; Ginty, Shannon; Love, John J.

2006-01-01

We have devised and implemented a DNA fingerprinting module for an upper division undergraduate laboratory based on the amplification and analysis of three of the 13 short tandem repeat loci that are required by the Federal Bureau of Investigation Combined DNA Index System (FBI CODIS) data base. Students first collect human epithelial (cheek)…

Determining orientation and direction of DNA sequences

DOEpatents

Goodwin, Edwin H.; Meyne, Julianne

2000-01-01

Determining orientation and direction of DNA sequences. A method by which fluorescence in situ hybridization can be made strand specific is described. Cell cultures are grown in a medium containing a halogenated nucleotide. The analog is partially incorporated in one DNA strand of each chromatid. This substitution takes place in opposite strands of the two sister chromatids. After staining with the fluorescent DNA-binding dye Hoechst 33258, cells are exposed to long-wavelength ultraviolet light which results in numerous strand nicks. These nicks enable the substituted strand to be denatured and solubilized by heat, treatment with high or low pH aqueous solutions, or by immersing the strands in 2.times.SSC (0.3M NaCl+0.03M sodium citrate), to name three procedures. It is unnecessary to enzymatically digest the strands using Exo III or another exonuclease in order to excise and solubilize nucleotides starting at the sites of the nicks. The denaturing/solubilizing process removes most of the substituted strand while leaving the prereplication strand largely intact. Hybridization of a single-stranded probe of a tandem repeat arranged in a head-to-tail orientation will result in hybridization only to the chromatid with the complementary strand present.

Localization of Action of the Is50-Encoded Transposase Protein

PubMed Central

Phadnis, Suhas H.; Sasakawa, Chihiro; Berg, Douglas E.

1986-01-01

The movement of the bacterial insertion sequence IS50 and of composite elements containing direct terminal repeats of IS50 involves the two ends of IS50, designated O (outside) and I (inside), which are weakly matched in DNA sequence, and an IS50 encoded protein, transposase, which recognizes the O and I ends and acts preferentially in cis. Previous data had suggested that, initially, transposase interacts preferentially with the O end sequence and then, in a second step, with either an O or an I end. To better understand the cis action of transposase and how IS50 ends are selected, we generated a series of composite transposons which contain direct repeats of IS50 elements. In each transposon, one IS50 element encoded transposase (tnp +), and the other contained a null (tnp-) allele. In each of the five sets of composite transposons studied, the transposon for which the tnp+ IS50 element contained its O end was more active than a complementary transposon for which the tnp - IS50 element contained its O end. This pattern of O end use suggests models in which the cis action of transposase and its choice of ends is determined by protein tracking along DNA molecules. PMID:3007274

Polymerase chain reaction amplification of DNA from aged blood stains: quantitative evaluation of the "suitability for purpose" of four filter papers as archival media.

PubMed

Kline, Margaret C; Duewer, David L; Redman, Janette W; Butler, John M; Boyer, David A

2002-04-15

In collaboration with the Armed Forces Institute of Pathology's Department of Defense DNA Registry, the National Institute of Standards and Technology recently evaluated the performance of a short tandem repeat multiplex with dried whole blood stains on four different commercially available identification card matrixes. DNA from 70 stains that had been stored for 19 months at ambient temperature was extracted or directly amplified and then processed using routine methods. All four storage media provided fully typeable (qualitatively identical) samples. After standardization, the average among-locus fluorescence intensity (electropherographic peak height or area) provided a suitable metric for quantitative analysis of the relative amounts of amplifiable DNA in an archived sample. The amounts of DNA in Chelex extracts from stains on two untreated high-purity cotton linter pulp papers and a paper treated with a DNA-binding coating were essentially identical. Average intensities for the aqueous extracts from a paper treated with a DNA-releasing coating were somewhat lower but also somewhat less variable than for the Chelex extracts. Average intensities of directly amplified punches of the DNA-binding paper were much larger but somewhat more variable than the Chelex extracts. Approximately 25% of the observed variation among the intensity measurements is shared among the four media and thus can be attributed to intrinsic variation in white blood count among the donors. All of the evaluated media adequately "bank" forensically useful DNA in well-dried whole blood stains for at least 19 months at ambient temperature.

Instability of expanded CAG/CAA repeats in spinocerebellar ataxia type 17.

PubMed

Gao, Rui; Matsuura, Tohru; Coolbaugh, Mary; Zühlke, Christine; Nakamura, Koichiro; Rasmussen, Astrid; Siciliano, Michael J; Ashizawa, Tetsuo; Lin, Xi

2008-02-01

Trinucleotide repeat expansions are dynamic mutations causing many neurological disorders, and their instability is influenced by multiple factors. Repeat configuration seems particularly important, and pure repeats are thought to be more unstable than interrupted repeats. But direct evidence is still lacking. Here, we presented strong support for this hypothesis from our studies on spinocerebellar ataxia type 17 (SCA17). SCA17 is a typical polyglutamine disease caused by CAG repeat expansion in TBP (TATA binding protein), and is unique in that the pure expanded polyglutamine tract is coded by either a simple configuration with long stretches of pure CAGs or a complex configuration containing CAA interruptions. By small pool PCR (SP-PCR) analysis of blood DNA from SCA17 patients of distinct racial backgrounds, we quantitatively assessed the instability of these two types of expanded alleles coding similar length of polyglutamine expansion. Mutation frequency in patients harboring pure CAG repeats is 2-3 folds of those with CAA interruptions. Interestingly, the pure CAG repeats showed both expansion and deletion while the interrupted repeats exhibited mostly deletion at a significantly lower frequency. These data strongly suggest that repeat configuration is a critical determinant for instability, and CAA interruptions might serve as a limiting element for further expansion of CAG repeats in SCA17 locus, suggesting a molecular basis for lack of anticipation in SCA17 families with interrupted CAG expansion.

Molecular mechanism of DNA association with single-stranded DNA binding protein

PubMed Central

Maffeo, Christopher

2017-01-01

Abstract During DNA replication, the single-stranded DNA binding protein (SSB) wraps single-stranded DNA (ssDNA) with high affinity to protect it from degradation and prevent secondary structure formation. Although SSB binds ssDNA tightly, it can be repositioned along ssDNA to follow the advancement of the replication fork. Using all-atom molecular dynamics simulations, we characterized the molecular mechanism of ssDNA association with SSB. Placed in solution, ssDNA–SSB assemblies were observed to change their structure spontaneously; such structural changes were suppressed in the crystallographic environment. Repeat simulations of the SSB–ssDNA complex under mechanical tension revealed a multitude of possible pathways for ssDNA to come off SSB punctuated by prolonged arrests at reproducible sites at the SSB surface. Ensemble simulations of spontaneous association of short ssDNA fragments with SSB detailed a three-dimensional map of local affinity to DNA; the equilibrium amount of ssDNA bound to SSB was found to depend on the electrolyte concentration but not on the presence of the acidic tips of the SSB tails. Spontaneous formation of ssDNA bulges and their diffusive motion along SSB surface was directly observed in multiple 10-µs-long simulations. Such reptation-like motion was confined by DNA binding to high-affinity spots, suggesting a two-step mechanism for SSB diffusion. PMID:29059392

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

PubMed

de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

2014-06-01

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Understanding the recognition mechanisms of Zα domain of human editing enzyme ADAR1 (hZα(ADAR1)) and various Z-DNAs from molecular dynamics simulation.

PubMed

Wang, Qianqian; Li, Lanlan; Wang, Xiaoting; Liu, Huanxiang; Yao, Xiaojun

2014-11-01

The Z-DNA-binding domain of human double-stranded RNA adenosine deaminase I (hZαADAR1) can specifically recognize the left-handed Z-DNA which preferentially occurs at alternating purine-pyrimidine repeats, especially the CG-repeats. The interactions of hZαADAR1 and Z-DNAs in different sequence contexts can affect many important biological functions including gene regulation and chromatin remodeling. Therefore it is of great necessity to fully understand their recognition mechanisms. However, most existing studies are aimed at the standard CG-repeat Z-DNA rather than the non-CG-repeats, and whether the molecular basis of hZαADAR1 binding to various Z-DNAs are identical or not is still unclear on the atomic level. Here, based on the recently determined crystal structures of three representative non-CG-repeat Z-DNAs (d(CACGTG)2, d(CGTACG)2 and d(CGGCCG)2) in complex with hZαADAR1, 40 ns molecular dynamics simulation together with binding free energy calculation were performed for each system. For comparison, the standard CG-repeat Z-DNA (d(CGCGCG)2) complexed with hZαADAR1 was also simulated. The consistent results demonstrate that nonpolar interaction is the driving force during the protein-DNA binding process, and that polar interaction mainly from helix α3 also provides important contributions. Five common hot-spot residues were identified, namely Lys169, Lys170, Asn173, Arg174 and Tyr177. Hydrogen bond analysis coupled with surface charge distribution further reveal the interfacial information between hZαADAR1 and Z-DNA in detail. All of the analysis illustrate that four complexes share the common key features and the similar binding modes irrespective of Z-DNA sequences, suggesting that Z-DNA recognition by hZαADAR1 is conformation-specific rather than sequence-specific. Additionally, by analyzing the conformational changes of hZαADAR1, we found that the binding of Z-DNA could effectively stabilize hZαADAR1 protein. Our study can provide some valuable information for better understanding the binding mechanism between hZαADAR1 or even other Z-DNA-binding protein and Z-DNA.

C9orf72 nucleotide repeat structures initiate molecular cascades of disease.

PubMed

Haeusler, Aaron R; Donnelly, Christopher J; Periz, Goran; Simko, Eric A J; Shaw, Patrick G; Kim, Min-Sik; Maragakis, Nicholas J; Troncoso, Juan C; Pandey, Akhilesh; Sattler, Rita; Rothstein, Jeffrey D; Wang, Jiou

2014-03-13

A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat-length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Specifically, nucleolin, an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases.

LPA gene: interaction between the apolipoprotein(a) size ('kringle IV' repeat) polymorphism and a pentanucleotide repeat polymorphism influences Lp(a) lipoprotein level.

PubMed

Røsby, O; Berg, K

2000-01-01

In order to search for factors influencing the Lp(a) lipoprotein level, we have examined the apolipoprotein(a) (apo(a)) size polymorphism as well as a pentanucleotide (TTTTA) repeat polymorphism in the 5' control region of the LPA gene. Lp(a) lipoprotein levels were compared between individuals with different genotypes as defined by pulsed field gel electrophoresis of DNA plugs, and PCR of DNA samples followed by polyacrylamide gel electrophoresis. DNA plugs and DNA were prepared from blood samples collected from blood donors. Twenty-seven different K IV repeat alleles were observed in the 71 women and 92 men from which apo(a) size polymorphism results were obtained. Alleles encoding 26-32 Kringle IV repeats were the most frequent. Alleles encoding seven to 11 TTTTA repeats were detected in the 84 women and 122 men included in the pentanucleotide polymorphism study, and homozygosity for eight TTTTA repeats was the most common genotype. The eight TTTTA repeat allele occurred with almost any apo(a) allele. An inverse relationship between number of K IV repeats and Lp(a) concentration was confirmed. The contributions of the apo(a) size polymorphism and the pentanucleotide repeat polymorphism to the interindividual variance of Lp(a) lipoprotein concentrations were 9.7 and 3.5%, respectively (type IV sum of squares). Nineteen per cent of the variance in Lp(a) lipoprotein level appeared to be the result of the multiplication product (interaction) between the apo(a) size polymorphism and the pentanucleotide repeat polymorphism. The contribution of the apo(a) size polymorphism alone to the variation in Lp(a) lipoprotein level was lower than previously reported. However, the multiplicative interaction effect between the K IV repeat polymorphism and the pentanucleotide repeat polymorphism may be an important factor explaining the variation in Lp(a) lipoprotein levels among the populations.

DNA motifs determining the accuracy of repeat duplication during CRISPR adaptation in Haloarcula hispanica

PubMed Central

Wang, Rui; Li, Ming; Gong, Luyao; Hu, Songnian; Xiang, Hua

2016-01-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) acquire new spacers to generate adaptive immunity in prokaryotes. During spacer integration, the leader-preceded repeat is always accurately duplicated, leading to speculations of a repeat-length ruler. Here in Haloarcula hispanica, we demonstrate that the accurate duplication of its 30-bp repeat requires two conserved mid-repeat motifs, AACCC and GTGGG. The AACCC motif was essential and needed to be ∼10 bp downstream from the leader-repeat junction site, where duplication consistently started. Interestingly, repeat duplication terminated sequence-independently and usually with a specific distance from the GTGGG motif, which seemingly served as an anchor site for a molecular ruler. Accordingly, altering the spacing between the two motifs led to an aberrant duplication size (29, 31, 32 or 33 bp). We propose the adaptation complex may recognize these mid-repeat elements to enable measuring the repeat DNA for spacer integration. PMID:27085805

Ctf4 Is a Hub in the Eukaryotic Replisome that Links Multiple CIP-Box Proteins to the CMG Helicase.

PubMed

Villa, Fabrizio; Simon, Aline C; Ortiz Bazan, Maria Angeles; Kilkenny, Mairi L; Wirthensohn, David; Wightman, Mel; Matak-Vinkovíc, Dijana; Pellegrini, Luca; Labib, Karim

2016-08-04

Replisome assembly at eukaryotic replication forks connects the DNA helicase to DNA polymerases and many other factors. The helicase binds the leading-strand polymerase directly, but is connected to the Pol α lagging-strand polymerase by the trimeric adaptor Ctf4. Here, we identify new Ctf4 partners in addition to Pol α and helicase, all of which contain a "Ctf4-interacting-peptide" or CIP-box. Crystallographic analysis classifies CIP-boxes into two related groups that target different sites on Ctf4. Mutations in the CIP-box motifs of the Dna2 nuclease or the rDNA-associated protein Tof2 do not perturb DNA synthesis genome-wide, but instead lead to a dramatic shortening of chromosome 12 that contains the large array of rDNA repeats. Our data reveal unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome. Most strikingly, Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

DNA mismatch repair complex MutSβ promotes GAA·TTC repeat expansion in human cells.

PubMed

Halabi, Anasheh; Ditch, Scott; Wang, Jeffrey; Grabczyk, Ed

2012-08-24

While DNA repair has been implicated in CAG·CTG repeat expansion, its role in the GAA·TTC expansion of Friedreich ataxia (FRDA) is less clear. We have developed a human cellular model that recapitulates the DNA repeat expansion found in FRDA patient tissues. In this model, GAA·TTC repeats expand incrementally and continuously. We have previously shown that the expansion rate is linked to transcription within the repeats. Our working hypothesis is that structures formed within the GAA·TTC repeat during transcription attract DNA repair enzymes that then facilitate the expansion process. MutSβ, a heterodimer of MSH2 and MSH3, is known to have a role in CAG·CTG repeat expansion. We now show that shRNA knockdown of either MSH2 or MSH3 slowed GAA·TTC expansion in our system. We further characterized the role of MutSβ in GAA·TTC expansion using a functional assay in primary FRDA patient-derived fibroblasts. These fibroblasts have no known propensity for instability in their native state. Ectopic expression of MSH2 and MSH3 induced GAA·TTC repeat expansion in the native FXN gene. MSH2 is central to mismatch repair and its absence or reduction causes a predisposition to cancer. Thus, despite its essential role in GAA·TTC expansion, MSH2 is not an attractive therapeutic target. The absence or reduction of MSH3 is not strongly associated with cancer predisposition. Accordingly, MSH3 has been suggested as a therapeutic target for CAG·CTG repeat expansion disorders. Our results suggest that MSH3 may also serve as a therapeutic target to slow the expansion of GAA·TTC repeats in the future.

DNA Mismatch Repair Complex MutSβ Promotes GAA·TTC Repeat Expansion in Human Cells*

PubMed Central

Halabi, Anasheh; Ditch, Scott; Wang, Jeffrey; Grabczyk, Ed

2012-01-01

While DNA repair has been implicated in CAG·CTG repeat expansion, its role in the GAA·TTC expansion of Friedreich ataxia (FRDA) is less clear. We have developed a human cellular model that recapitulates the DNA repeat expansion found in FRDA patient tissues. In this model, GAA·TTC repeats expand incrementally and continuously. We have previously shown that the expansion rate is linked to transcription within the repeats. Our working hypothesis is that structures formed within the GAA·TTC repeat during transcription attract DNA repair enzymes that then facilitate the expansion process. MutSβ, a heterodimer of MSH2 and MSH3, is known to have a role in CAG·CTG repeat expansion. We now show that shRNA knockdown of either MSH2 or MSH3 slowed GAA·TTC expansion in our system. We further characterized the role of MutSβ in GAA·TTC expansion using a functional assay in primary FRDA patient-derived fibroblasts. These fibroblasts have no known propensity for instability in their native state. Ectopic expression of MSH2 and MSH3 induced GAA·TTC repeat expansion in the native FXN gene. MSH2 is central to mismatch repair and its absence or reduction causes a predisposition to cancer. Thus, despite its essential role in GAA·TTC expansion, MSH2 is not an attractive therapeutic target. The absence or reduction of MSH3 is not strongly associated with cancer predisposition. Accordingly, MSH3 has been suggested as a therapeutic target for CAG·CTG repeat expansion disorders. Our results suggest that MSH3 may also serve as a therapeutic target to slow the expansion of GAA·TTC repeats in the future. PMID:22787155

NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING

PubMed Central

Zhang, Lu; Mo, Jinyao; Swanson, Karen V.; Wen, Haitao; Petrucelli, Alex; Gregory, Sean M.; Zhang, Zhigang; Schneider, Monika; Jiang, Yan; Fitzgerald, Katherine A.; Ouyang, Songying; Liu, Zhi-Jie; Damania, Blossom A; Shu, Hong-Bing; Duncan, Joseph A.; Ting, Jenny P-Y.

2014-01-01

SUMMARY Stimulator of interferon genes (STING, also named MITA, MYPS or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich repeat containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP) and DNA viruses. NLRC3 associated with both STING and TBK1, and impeded STING-TBK1 interaction and downstream type I interferon production. Using purified recombinant proteins NLRC3 was found to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3−/− mice exhibited enhanced innate immunity, reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus and c-di-GMP PMID:24560620

Long repeating (TTAGGG)n single stranded DNA self-condenses into compact beaded filaments stabilized by G-quadruplex formation.

PubMed

Kar, Anirban; Jones, Nathan; Arat, N Özlem; Fishel, Richard; Griffith, Jack

2018-04-19

Conformations adopted by long stretches of single stranded DNA (ssDNA) are of central interest in understanding the architecture of replication forks, R loops, and other structures generated during DNA metabolism in vivo. This is particularly so if the ssDNA consists of short nucleotide repeats. Such studies have been hampered by the lack of defined substrates greater than ~150 nt, and the absence of high-resolution biophysical approaches. Here we describe the generation of very long ssDNA consisting of the mammalian telomeric repeat (5'-TTAGGG-3')n as well as the interrogation of its structure by electron microscopy (EM) and single molecule magnetic tweezers (smMT). This repeat is of particular interest as it contains a run of 3 contiguous guanine residues capable of forming G quartets as ssDNA. Fluorescent-dye exclusion assays confirmed that this G-strand ssDNA forms ubiquitous G-quadruplex folds. EM revealed thick bead-like filaments that condensed the DNA ~12 fold. The bead-like structures were 5 nm and 8 nm in diameter and linked by thin filaments. The G-strand ssDNA displayed initial stability to smMT force extension that ultimately released in steps that were multiples ~28 nm at forces between 6-12 pN; well below the >20 pN required to unravel G-quadruplexes. Most smMT steps were consistent with the disruption of the beads seen by EM. Binding by RAD51 distinctively altered the force extension properties of the G-strand ssDNA, suggesting a stochastic G-quadruplex-dependent condensation model that is discussed. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

STRBase: a short tandem repeat DNA database for the human identity testing community

PubMed Central

Ruitberg, Christian M.; Reeder, Dennis J.; Butler, John M.

2001-01-01

The National Institute of Standards and Technology (NIST) has compiled and maintained a Short Tandem Repeat DNA Internet Database (http://www.cstl.nist.gov/biotech/strbase/) since 1997 commonly referred to as STRBase. This database is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. STRBase consolidates and organizes the abundant literature on this subject to facilitate on-going efforts in DNA typing. Observed alleles and annotated sequence for each STR locus are described along with a review of STR analysis technologies. Additionally, commercially available STR multiplex kits are described, published polymerase chain reaction (PCR) primer sequences are reported, and validation studies conducted by a number of forensic laboratories are listed. To supplement the technical information, addresses for scientists and hyperlinks to organizations working in this area are available, along with the comprehensive reference list of over 1300 publications on STRs used for DNA typing purposes. PMID:11125125

Grasshopper, a long terminal repeat (LTR) retroelement in the phytopathogenic fungus Magnaporthe grisea.

PubMed

Dobinson, K F; Harris, R E; Hamer, J E

1993-01-01

The fungal phytopathogen Magnaporthe grisea parasitizes a wide variety of gramineous hosts. In the course of investigating the genetic relationship between pathogen genotype and host specificity we identified a retroelement that is present in some strains of M. grisea that infect finger millet and goosegrass (members of the plant genus Eleusine). The element, designated grasshopper (grh), is present in multiple copies and dispersed throughout the genome. DNA sequence analysis showed that grasshopper contains 198 base pair direct, long terminal repeats (LTRs) with features characteristic of retroviral and retrotransposon LTRs. Within the element we identified an open reading frame with sequences homologous to the reverse transcriptase, RNaseH, and integrase domains of retroelement pol genes. Comparison of the open reading frame with sequences from other retroelements showed that grh is related to the gypsy family of retrotransposons. Comparisons of the distribution of the grasshopper element with other dispersed repeated DNA sequences in M. grisea indicated that grasshopper was present in a broadly dispersed subgroup of Eleusine pathogens, suggesting that the element was acquired subsequent to the evolution of this host-specific form. We present arguments that the amplification of different retroelements within populations of M. grisea is a consequence of the clonal organization of the fungal populations.

Comparative Chloroplast Genomes of Pinaceae: Insights into the Mechanism of Diversified Genomic Organizations

PubMed Central

Wu, Chung-Shien; Lin, Ching-Ping; Hsu, Chi-Yao; Wang, Rui-Jiang; Chaw, Shu-Miaw

2011-01-01

Abstract Pinaceae, the largest family of conifers, has diversified organizations of chloroplast genomes (cpDNAs) with the two typical inverted repeats (IRs) highly reduced. To unravel the mechanism of this genomic diversification, we examined the cpDNA organizations from 53 species of the ten Pinaceous genera, including those of Larix decidua (122,474 bp), Picea morrisonicola (124,168 bp), and Pseudotsuga wilsoniana (122,513 bp), which were firstly elucidated. The results uncovered four distinct cpDNA forms (A−C and P) that are due to rearrangements of two ∼20 and ∼21 kb specific fragments. The C form was documented for the first time and the A form might be the most ancestral one. In addition, only the individuals of Ps. macrocarpa and Ps. wilsoniana were detected to have isomeric cpDNA forms. Three types (types 1−3) of Pinaceae-specific repeats situated nearby the rearranged fragments were found to be syntenic. We hypothesize that type 1 (949 ± 343 bp) and type 3 (608 ± 73 bp) repeats are substrates for homologous recombination (HR), whereas type 2 repeats are likely inactive for HR because of their relatively short sizes (151 ± 30 bp). Conversions among the four distinct forms may be achieved by HR and mediated by type 1 or 3 repeats, thus resulting in increased diversity of cpDNA organizations. We propose that in the Pinaceae cpDNAs, the reduced IRs have lost HR activity, then decreasing the diversity of cpDNA organizations, but the specific repeats that the evolution endowed Pinaceae complement the reduced IRs and increase the diversity of cpDNA organizations. PMID:21402866

Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.

PubMed

Pilotte, Nils; Papaiakovou, Marina; Grant, Jessica R; Bierwert, Lou Ann; Llewellyn, Stacey; McCarthy, James S; Williams, Steven A

2016-03-01

The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world's most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.

Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats

PubMed Central

Villahermosa, Desirée; Christensen, Olaf; Knapp, Karen; Fleck, Oliver

2017-01-01

Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1), which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade+ reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe. Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2FEN1. Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes. PMID:28341698

Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats.

PubMed

Villahermosa, Desirée; Christensen, Olaf; Knapp, Karen; Fleck, Oliver

2017-05-05

Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1), which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade + reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2 FEN1 Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe , but contributes to DNA repeat stability in MMR-independent processes. Copyright © 2017 Villahermosa et al.

Repetitive sequence analysis and karyotyping reveals centromere-associated DNA sequences in radish (Raphanus sativus L.).

PubMed

He, Qunyan; Cai, Zexi; Hu, Tianhua; Liu, Huijun; Bao, Chonglai; Mao, Weihai; Jin, Weiwei

2015-04-18

Radish (Raphanus sativus L., 2n = 2x = 18) is a major root vegetable crop especially in eastern Asia. Radish root contains various nutritions which play an important role in strengthening immunity. Repetitive elements are primary components of the genomic sequence and the most important factors in genome size variations in higher eukaryotes. To date, studies about repetitive elements of radish are still limited. To better understand genome structure of radish, we undertook a study to evaluate the proportion of repetitive elements and their distribution in radish. We conducted genome-wide characterization of repetitive elements in radish with low coverage genome sequencing followed by similarity-based cluster analysis. Results showed that about 31% of the genome was composed of repetitive sequences. Satellite repeats were the most dominating elements of the genome. The distribution pattern of three satellite repeat sequences (CL1, CL25, and CL43) on radish chromosomes was characterized using fluorescence in situ hybridization (FISH). CL1 was predominantly located at the centromeric region of all chromosomes, CL25 located at the subtelomeric region, and CL43 was a telomeric satellite. FISH signals of two satellite repeats, CL1 and CL25, together with 5S rDNA and 45S rDNA, provide useful cytogenetic markers to identify each individual somatic metaphase chromosome. The centromere-specific histone H3 (CENH3) has been used as a marker to identify centromere DNA sequences. One putative CENH3 (RsCENH3) was characterized and cloned from radish. Its deduced amino acid sequence shares high similarities to those of the CENH3s in Brassica species. An antibody against B. rapa CENH3, specifically stained radish centromeres. Immunostaining and chromatin immunoprecipitation (ChIP) tests with anti-BrCENH3 antibody demonstrated that both the centromere-specific retrotransposon (CR-Radish) and satellite repeat (CL1) are directly associated with RsCENH3 in radish. Proportions of repetitive elements in radish were estimated and satellite repeats were the most dominating elements. Fine karyotyping analysis was established which allow us to easily identify each individual somatic metaphase chromosome. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of satellite and centromere-specific retrotransposon at centromeres. Our study provides a valuable basis for future genomic studies in radish.

Population-scale whole genome sequencing identifies 271 highly polymorphic short tandem repeats from Japanese population.

PubMed

Hirata, Satoshi; Kojima, Kaname; Misawa, Kazuharu; Gervais, Olivier; Kawai, Yosuke; Nagasaki, Masao

2018-05-01

Forensic DNA typing is widely used to identify missing persons and plays a central role in forensic profiling. DNA typing usually uses capillary electrophoresis fragment analysis of PCR amplification products to detect the length of short tandem repeat (STR) markers. Here, we analyzed whole genome data from 1,070 Japanese individuals generated using massively parallel short-read sequencing of 162 paired-end bases. We have analyzed 843,473 STR loci with two to six basepair repeat units and cataloged highly polymorphic STR loci in the Japanese population. To evaluate the performance of the cataloged STR loci, we compared 23 STR loci, widely used in forensic DNA typing, with capillary electrophoresis based STR genotyping results in the Japanese population. Seventeen loci had high correlations and high call rates. The other six loci had low call rates or low correlations due to either the limitations of short-read sequencing technology, the bioinformatics tool used, or the complexity of repeat patterns. With these analyses, we have also purified the suitable 218 STR loci with four basepair repeat units and 53 loci with five basepair repeat units both for short read sequencing and PCR based technologies, which would be candidates to the actual forensic DNA typing in Japanese population.

DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases

PubMed Central

Bettencourt, Conceição; Hensman‐Moss, Davina; Flower, Michael; Wiethoff, Sarah; Brice, Alexis; Goizet, Cyril; Stevanin, Giovanni; Koutsis, Georgios; Karadima, Georgia; Panas, Marios; Yescas‐Gómez, Petra; García‐Velázquez, Lizbeth Esmeralda; Alonso‐Vilatela, María Elisa; Lima, Manuela; Raposo, Mafalda; Traynor, Bryan; Sweeney, Mary; Wood, Nicholas; Giunti, Paola; Durr, Alexandra; Holmans, Peter; Houlden, Henry; Tabrizi, Sarah J.

2016-01-01

Objective The polyglutamine diseases, including Huntington's disease (HD) and multiple spinocerebellar ataxias (SCAs), are among the commonest hereditary neurodegenerative diseases. They are caused by expanded CAG tracts, encoding glutamine, in different genes. Longer CAG repeat tracts are associated with earlier ages at onset, but this does not account for all of the difference, and the existence of additional genetic modifying factors has been suggested in these diseases. A recent genome‐wide association study (GWAS) in HD found association between age at onset and genetic variants in DNA repair pathways, and we therefore tested whether the modifying effects of variants in DNA repair genes have wider effects in the polyglutamine diseases. Methods We assembled an independent cohort of 1,462 subjects with HD and polyglutamine SCAs, and genotyped single‐nucleotide polymorphisms (SNPs) selected from the most significant hits in the HD study. Results In the analysis of DNA repair genes as a group, we found the most significant association with age at onset when grouping all polyglutamine diseases (HD+SCAs; p = 1.43 × 10–5). In individual SNP analysis, we found significant associations for rs3512 in FAN1 with HD+SCAs (p = 1.52 × 10–5) and all SCAs (p = 2.22 × 10–4) and rs1805323 in PMS2 with HD+SCAs (p = 3.14 × 10–5), all in the same direction as in the HD GWAS. Interpretation We show that DNA repair genes significantly modify age at onset in HD and SCAs, suggesting a common pathogenic mechanism, which could operate through the observed somatic expansion of repeats that can be modulated by genetic manipulation of DNA repair in disease models. This offers novel therapeutic opportunities in multiple diseases. Ann Neurol 2016;79:983–990 PMID:27044000

Evolutionary conservation of sequence and secondary structures inCRISPR repeats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kunin, Victor; Sorek, Rotem; Hugenholtz, Philip

Clustered Regularly Interspaced Palindromic Repeats (CRISPRs) are a novel class of direct repeats, separated by unique spacer sequences of similar length, that are present in {approx}40% of bacterial and all archaeal genomes analyzed to date. More than 40 gene families, called CRISPR-associated sequences (CAS), appear in conjunction with these repeats and are thought to be involved in the propagation and functioning of CRISPRs. It has been proposed that the CRISPR/CAS system samples, maintains a record of, and inactivates invasive DNA that the cell has encountered, and therefore constitutes a prokaryotic analog of an immune system. Here we analyze CRISPR repeatsmore » identified in 195 microbial genomes and show that they can be organized into multiple clusters based on sequence similarity. All individual repeats in any given cluster were inferred to form characteristic RNA secondary structure, ranging from non-existent to pronounced. Stable secondary structures included G:U base pairs and exhibited multiple compensatory base changes in the stem region, indicating evolutionary conservation and functional importance. We also show that the repeat-based classification corresponds to, and expands upon, a previously reported CAS gene-based classification including specific relationships between CRISPR and CAS subtypes.« less

Molecular Cytogenetic Analysis of Deschampsia antarctica Desv. (Poaceae), Maritime Antarctic.

PubMed

Amosova, Alexandra V; Bolsheva, Nadezhda L; Samatadze, Tatiana E; Twardovska, Maryana O; Zoshchuk, Svyatoslav A; Andreev, Igor O; Badaeva, Ekaterina D; Kunakh, Viktor A; Muravenko, Olga V

2015-01-01

Deschampsia antarctica Desv. (Poaceae) (2n = 26) is one of the two vascular plants adapted to the harshest environment of the Antarctic. Although the species is a valuable model for study of environmental stress tolerance in plants, its karyotype is still poorly investigated. We firstly conducted a comprehensive molecular cytogenetic analysis of D. antarctica collected on four islands of the Maritime Antarctic. D. antarctica karyotypes were studied by Giemsa C- and DAPI/C-banding, Ag-NOR staining, multicolour fluorescence in situ hybridization with repeated DNA probes (pTa71, pTa794, telomere repeats, pSc119.2, pAs1) and the GAA simple sequence repeat probe. We also performed sequential rapid in situ hybridization with genomic DNA of D. caespitosa. Two chromosome pairs bearing transcriptionally active 45S rDNA loci and five pairs with 5S rDNA sites were detected. A weak intercalary site of telomere repeats was revealed on the largest chromosome in addition to telomere hybridization signals at terminal positions. This fact confirms indirectly the hypothesis that chromosome fusion might have been the cause of the unusual for cereals chromosome number in this species. Based on patterns of distribution of the examined molecular cytogenetic markers, all chromosomes in karyotypes were identified, and chromosome idiograms of D. antarctica were constructed. B chromosomes were found in most karyotypes of plants from Darboux Island. A mixoploid plant with mainly triploid cells bearing a Robertsonian rearrangement was detected among typical diploid specimens from Great Jalour Island. The karyotype variability found in D. antarctica is probably an expression of genome instability induced by environmental stress factors. The differences in C-banding patterns and in chromosome distribution of rDNA loci as well as homologous highly repeated DNA sequences detected between genomes of D. antarctica and its related species D. caespitosa indicate that genome reorganization involving coding and noncoding repeated DNA sequences had occurred during the divergence of these species.

Molecular Cytogenetic Analysis of Deschampsia antarctica Desv. (Poaceae), Maritime Antarctic

PubMed Central

Amosova, Alexandra V.; Bolsheva, Nadezhda L.; Samatadze, Tatiana E.; Twardovska, Maryana O.; Zoshchuk, Svyatoslav A.; Andreev, Igor O.; Badaeva, Ekaterina D.; Kunakh, Viktor A.; Muravenko, Olga V.

2015-01-01

Deschampsia antarctica Desv. (Poaceae) (2n = 26) is one of the two vascular plants adapted to the harshest environment of the Antarctic. Although the species is a valuable model for study of environmental stress tolerance in plants, its karyotype is still poorly investigated. We firstly conducted a comprehensive molecular cytogenetic analysis of D. antarctica collected on four islands of the Maritime Antarctic. D. antarctica karyotypes were studied by Giemsa C- and DAPI/C-banding, Ag-NOR staining, multicolour fluorescence in situ hybridization with repeated DNA probes (pTa71, pTa794, telomere repeats, pSc119.2, pAs1) and the GAA simple sequence repeat probe. We also performed sequential rapid in situ hybridization with genomic DNA of D. caespitosa. Two chromosome pairs bearing transcriptionally active 45S rDNA loci and five pairs with 5S rDNA sites were detected. A weak intercalary site of telomere repeats was revealed on the largest chromosome in addition to telomere hybridization signals at terminal positions. This fact confirms indirectly the hypothesis that chromosome fusion might have been the cause of the unusual for cereals chromosome number in this species. Based on patterns of distribution of the examined molecular cytogenetic markers, all chromosomes in karyotypes were identified, and chromosome idiograms of D. antarctica were constructed. B chromosomes were found in most karyotypes of plants from Darboux Island. A mixoploid plant with mainly triploid cells bearing a Robertsonian rearrangement was detected among typical diploid specimens from Great Jalour Island. The karyotype variability found in D. antarctica is probably an expression of genome instability induced by environmental stress factors. The differences in C-banding patterns and in chromosome distribution of rDNA loci as well as homologous highly repeated DNA sequences detected between genomes of D. antarctica and its related species D. caespitosa indicate that genome reorganization involving coding and noncoding repeated DNA sequences had occurred during the divergence of these species. PMID:26394331

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae).

PubMed

Begum, Rabeya; Zakrzewski, Falk; Menzel, Gerhard; Weber, Beatrice; Alam, Sheikh Shamimul; Schmidt, Thomas

2013-07-01

The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100-500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S-5·8S-25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

Characterization of species-specific repeated DNA sequences from B. nigra.

PubMed

Gupta, V; Lakshmisita, G; Shaila, M S; Jagannathan, V; Lakshmikumaran, M S

1992-07-01

The construction and characterization of two genome-specific recombinant DNA clones from B. nigra are described. Southern analysis showed that the two clones belong to a dispersed repeat family. They differ from each other in their length, distribution and sequence, though the average GC content is nearly the same (45%). These B genome-specific repeats have been used to analyse the phylogenetic relationships between cultivated and wild species of the family Brassicaceae.

Concurrent beneficial (vitamin D production) and hazardous (cutaneous DNA damage) impact of repeated low-level summer sunlight exposures.

PubMed

Felton, S J; Cooke, M S; Kift, R; Berry, J L; Webb, A R; Lam, P M W; de Gruijl, F R; Vail, A; Rhodes, L E

2016-12-01

The concurrent impact of repeated low-level summer sunlight exposures on vitamin D production and cutaneous DNA damage, potentially leading to mutagenesis and skin cancer, is unknown. This is an experimental study (i) to determine the dual impact of repeated low-level sunlight exposures on vitamin D status and DNA damage/repair (via both skin and urinary biomarkers) in light-skinned adults; and (ii) to compare outcomes following the same exposures in brown-skinned adults. Ten white (phototype II) and six South Asian volunteers (phototype V), aged 23-59 years, received 6 weeks' simulated summer sunlight exposures (95% ultraviolet A/5% ultraviolet B, 1·3 standard erythemal doses three times weekly) wearing summer clothing exposing ~35% body surface area. Assessments made were circulating 25-hydroxyvitamin D [25(OH)D], immunohistochemistry for cyclobutane pyrimidine dimer (CPD)-positive nuclei and urinary biomarkers of direct and oxidative (8-oxo-deoxyguanosine) DNA damage. Serum 25(OH)D rose from mean 36·5 ± 13·0 to 54·3 ± 10·5 nmol L -1 (14·6 ± 5·2 to 21·7 ± 4·2 ng mL -1 ) in phototype II vs. 17·2 ± 6·3 to 25·5 ± 9·5 nmol L -1 (6·9 ± 2·5 to 10·2 ± 3·8 ng mL -1 ) in phototype V (P < 0·05). Phototype II skin showed CPD-positive nuclei immediately postcourse, mean 44% (range 27-84) cleared after 24 h, contrasting with minimal DNA damage and full clearance in phototype V (P < 0·001). The findings did not differ from those following single ultraviolet radiation (UVR) exposure. Urinary CPDs remained below the detection threshold in both groups; 8-oxo-deoxyguanosine was higher in phototype II than V (P = 0·002), but was unaffected by UVR. Low-dose summer sunlight exposures confer vitamin D sufficiency in light-skinned people concurrently with low-level, nonaccumulating DNA damage. The same exposures produce minimal DNA damage but less vitamin D in brown-skinned people. This informs tailoring of sun-exposure policies. © 2016 The Authors. British Journal of Dermatology published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.

Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

PubMed Central

North, Justin A.; Šimon, Marek; Ferdinand, Michelle B.; Shoffner, Matthew A.; Picking, Jonathan W.; Howard, Cecil J.; Mooney, Alex M.; van Noort, John; Poirier, Michael G.; Ottesen, Jennifer J.

2014-01-01

Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA–histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure. PMID:24561803

Roles for the Rad27 Flap Endonuclease in Mitochondrial Mutagenesis and Double-Strand Break Repair in Saccharomyces cerevisiae.

PubMed

Nagarajan, Prabha; Prevost, Christopher T; Stein, Alexis; Kasimer, Rachel; Kalifa, Lidza; Sia, Elaine A

2017-06-01

The structure-specific nuclease, Rad27p/FEN1, plays a crucial role in DNA repair and replication mechanisms in the nucleus. Genetic assays using the rad27-∆ mutant have shown altered rates of DNA recombination, microsatellite instability, and point mutation in mitochondria. In this study, we examined the role of Rad27p in mitochondrial mutagenesis and double-strand break (DSB) repair in Saccharomyces cerevisiae Our findings show that Rad27p is essential for efficient mitochondrial DSB repair by a pathway that generates deletions at a region flanked by direct repeat sequences. Mutant analysis suggests that both exonuclease and endonuclease activities of Rad27p are required for its role in mitochondrial DSB repair. In addition, we found that the nuclease activities of Rad27p are required for the prevention of mitochondrial DNA (mtDNA) point mutations, and in the generation of spontaneous mtDNA rearrangements. Overall, our findings underscore the importance of Rad27p in the maintenance of mtDNA, and demonstrate that it participates in multiple DNA repair pathways in mitochondria, unlinked to nuclear phenotypes. Copyright © 2017 by the Genetics Society of America.

Two different size classes of 5S rDNA units coexisting in the same tandem array in the razor clam Ensis macha: is this region suitable for phylogeographic studies?

PubMed

Fernández-Tajes, Juan; Méndez, Josefina

2009-12-01

For a study of 5S ribosomal genes (rDNA) in the razor clam Ensis macha, the 5S rDNA region was amplified and sequenced. Two variants, so-called type I or short repeat (approximately 430 bp) and type II or long repeat (approximately 735 bp), appeared to be the main components of the 5S rDNA of this species. Their spacers differed markedly, both in length and nucleotide composition. The organization of the two variants was investigated by amplifying the genomic DNA with primers based on the sequence of the type I and type II spacers. PCR amplification products with primers EMLbF and EMSbR showed that the long and short repeats are associated within the same tandem array, suggesting an intermixed arrangement of both spacers. Nevertheless, amplifications carried out with inverse primers EMSinvF/R and EMLinvF/R revealed that some short and long repeats are contiguous in the same tandem array. This is the first report of the coexistence of two variable spacers in the same tandem array in bivalve mollusks.

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution.

PubMed

Melters, Daniël P; Bradnam, Keith R; Young, Hugh A; Telis, Natalie; May, Michael R; Ruby, J Graham; Sebra, Robert; Peluso, Paul; Eid, John; Rank, David; Garcia, José Fernando; DeRisi, Joseph L; Smith, Timothy; Tobias, Christian; Ross-Ibarra, Jeffrey; Korf, Ian; Chan, Simon W L

2013-01-30

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data. Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution. While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes.

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution

PubMed Central

2013-01-01

Background Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data. Results Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution. Conclusions While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes. PMID:23363705

In vivo genome editing as a potential treatment strategy for inherited retinal dystrophies.

PubMed

Yanik, Mert; Müller, Brigitte; Song, Fei; Gall, Jacqueline; Wagner, Franziska; Wende, Wolfgang; Lorenz, Birgit; Stieger, Knut

2017-01-01

In vivo genome editing represents an emerging field in the treatment of monogenic disorders, as it may constitute a solution to the current hurdles in classic gene addition therapy, which are the low levels and limited duration of transgene expression. Following the introduction of a double strand break (DSB) at the mutational site by highly specific endonucleases, such as TALENs (transcription activator like effector nucleases) or RNA based nucleases (clustered regulatory interspaced short palindromic repeats - CRISPR-Cas), the cell's own DNA repair machinery restores integrity to the DNA strand and corrects the mutant sequence, thus allowing the cell to produce protein levels as needed. The DNA repair happens either through the error prone non-homologous end-joining (NHEJ) pathway or with high fidelity through homology directed repair (HDR) in the presence of a DNA donor template. A third pathway called microhomology mediated endjoining (MMEJ) has been recently discovered. In this review, the authors focus on the different DNA repair mechanisms, the current state of the art tools for genome editing and the particularities of the retina and photoreceptors with regard to in vivo therapeutic approaches. Finally, current attempts in the field of retinal in vivo genome editing are discussed and future directions of research identified. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

High-throughput single-molecule telomere characterization.

PubMed

McCaffrey, Jennifer; Young, Eleanor; Lassahn, Katy; Sibert, Justin; Pastor, Steven; Riethman, Harold; Xiao, Ming

2017-11-01

We have developed a novel method that enables global subtelomere and haplotype-resolved analysis of telomere lengths at the single-molecule level. An in vitro CRISPR/Cas9 RNA-directed nickase system directs the specific labeling of human (TTAGGG)n DNA tracts in genomes that have also been barcoded using a separate nickase enzyme that recognizes a 7-bp motif genome-wide. High-throughput imaging and analysis of large DNA single molecules from genomes labeled in this fashion using a nanochannel array system permits mapping through subtelomere repeat element (SRE) regions to unique chromosomal DNA while simultaneously measuring the (TTAGGG)n tract length at the end of each large telomere-terminal DNA segment. The methodology also permits subtelomere and haplotype-resolved analyses of SRE organization and variation, providing a window into the population dynamics and potential functions of these complex and structurally variant telomere-adjacent DNA regions. At its current stage of development, the assay can be used to identify and characterize telomere length distributions of 30-35 discrete telomeres simultaneously and accurately. The assay's utility is demonstrated using early versus late passage and senescent human diploid fibroblasts, documenting the anticipated telomere attrition on a global telomere-by-telomere basis as well as identifying subtelomere-specific biases for critically short telomeres. Similarly, we present the first global single-telomere-resolved analyses of two cancer cell lines. © 2017 McCaffrey et al.; Published by Cold Spring Harbor Laboratory Press.

GENETIC DIVERSITY OF TYPHA LATIFOLIA (TYPHACEAE) AND THE IMPACT OF POLLUTANTS EXAMINED WITH TANDEM-REPETITIVE DNA PROBES

EPA Science Inventory

Genetic diversity at variable-number-tandem-repeat (VNTR) loci was examined in the common cattail, Typha latifolia (Typhaceae), using three synthetic DNA probes composed of tandemly repeated "core" sequences (GACA, GATA, and GCAC). The principal objectives of this investigation w...

Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1*

PubMed Central

Musayev, Faik N.; Zarate-Perez, Francisco; Bishop, Clayton; Burgner, John W.; Escalante, Carlos R.

2015-01-01

Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains. PMID:26370092

Inhibition of colorectal cancer genomic copy number alterations and chromosomal fragile site tumor suppressor FHIT and WWOX deletions by DNA mismatch repair

PubMed Central

Gelincik, Ozkan; Blecua, Pedro; Edelmann, Winfried; Kucherlapati, Raju; Zhou, Kathy; Jasin, Maria; Gümüş, Zeynep H.; Lipkin, Steven M.

2017-01-01

Homologous recombination (HR) enables precise DNA repair after DNA double strand breaks (DSBs) using identical sequence templates, whereas homeologous recombination (HeR) uses only partially homologous sequences. Homeologous recombination introduces mutations through gene conversion and genomic deletions through single-strand annealing (SSA). DNA mismatch repair (MMR) inhibits HeR, but the roles of mammalian MMR MutL homologues (MLH1, PMS2 and MLH3) proteins in HeR suppression are poorly characterized. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) carrying Mlh1, Pms2, and Mlh3 mutations have higher HeR rates, by using 7,863 uniquely mapping paired direct repeat sequences (DRs) in the mouse genome as endogenous gene conversion and SSA reporters. Additionally, when DSBs are induced by gamma-radiation, Mlh1, Pms2 and Mlh3 mutant MEFs have higher DR copy number alterations (CNAs), including DR CNA hotspots previously identified in mouse MMR-deficient colorectal cancer (dMMR CRC). Analysis of The Cancer Genome Atlas CRC data revealed that dMMR CRCs have higher genome-wide DR HeR rates than MMR proficient CRCs, and that dMMR CRCs have deletion hotspots in tumor suppressors FHIT/WWOX at chromosomal fragile sites FRA3B and FRA16D (which have elevated DSB rates) flanked by paired homologous DRs and inverted repeats (IR). Overall, these data provide novel insights into the MMR-dependent HeR inhibition mechanism and its role in tumor suppression. PMID:29069730

A New Protein Architecture for Processing Alkylation Damaged DNA: The Crystal Structure of DNA Glycosylase AlkD

PubMed Central

Rubinson, Emily H.; Metz, Audrey H.; O'Quin, Jami; Eichman, Brandt F.

2013-01-01

Summary DNA glycosylases safeguard the genome by locating and excising chemically modified bases from DNA. AlkD is a recently discovered bacterial DNA glycosylase that removes positively charged methylpurines from DNA, and was predicted to adopt a protein fold distinct from other DNA repair proteins. The crystal structure of Bacillus cereus AlkD presented here shows that the protein is composed exclusively of helical HEAT-like repeats, which form a solenoid perfectly shaped to accommodate a DNA duplex on the concave surface. Structural analysis of the variant HEAT repeats in AlkD provides a rationale for how this protein scaffolding motif has been modified to bind DNA. We report 7mG excision and DNA binding activities of AlkD mutants, along with a comparison of alkylpurine DNA glycosylase structures. Together, these data provide important insight into the requirements for alkylation repair within DNA and suggest that AlkD utilizes a novel strategy to manipulate DNA in its search for alkylpurine bases. PMID:18585735

PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair

PubMed Central

Pluciennik, Anna; Dzantiev, Leonid; Iyer, Ravi R.; Constantin, Nicoleta; Kadyrov, Farid A.; Modrich, Paul

2010-01-01

MutLα (MLH1–PMS2) is a latent endonuclease that is activated in a mismatch-, MutSα-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLα activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLα function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLα incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion. PMID:20713735

C9orf72 Nucleotide Repeat Structures Initiate Molecular Cascades of Disease

PubMed Central

Haeusler, Aaron R.; Donnelly, Christopher J.; Periz, Goran; Simko, Eric A.J.; Shaw, Patrick G.; Kim, Min-Sik; Maragakis, Nicholas J.; Troncoso, Juan C.; Pandey, Akhilesh; Sattler, Rita; Rothstein, Jeffrey D.; Wang, Jiou

2014-01-01

Summary A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformationdependent manner. Specifically, nucleolin (NCL), an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases. PMID:24598541

Non-radioactive detection of trinucleotide repeat size variability.

PubMed

Tomé, Stéphanie; Nicole, Annie; Gomes-Pereira, Mario; Gourdon, Genevieve

2014-03-06

Many human diseases are associated with the abnormal expansion of unstable trinucleotide repeat sequences. The mechanisms of trinucleotide repeat size mutation have not been fully dissected, and their understanding must be grounded on the detailed analysis of repeat size distributions in human tissues and animal models. Small-pool PCR (SP-PCR) is a robust, highly sensitive and efficient PCR-based approach to assess the levels of repeat size variation, providing both quantitative and qualitative data. The method relies on the amplification of a very low number of DNA molecules, through sucessive dilution of a stock genomic DNA solution. Radioactive Southern blot hybridization is sensitive enough to detect SP-PCR products derived from single template molecules, separated by agarose gel electrophoresis and transferred onto DNA membranes. We describe a variation of the detection method that uses digoxigenin-labelled locked nucleic acid probes. This protocol keeps the sensitivity of the original method, while eliminating the health risks associated with the manipulation of radiolabelled probes, and the burden associated with their regulation, manipulation and waste disposal.

The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris.

PubMed

van Keulen, H; Gutell, R R; Campbell, S R; Erlandsen, S L; Jarroll, E L

1992-10-01

The total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined. The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp. The spacer contains long direct repeats and is heterogeneous in size. The LSU-rRNA of G. muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli. The LSU-rRNA has a size comparable to the 23S rRNA of E. coli but shows structural features typical for eukaryotes. Some variable regions are typically small and account for the overall smaller size of this rRNA. The structure of the G. muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.

Distribution and sequence homogeneity of an abundant satellite DNA in the beetle, Tenebrio molitor.

PubMed Central

Davis, C A; Wyatt, G R

1989-01-01

The mealworm beetle, Tenebrio molitor, contains an unusually abundant and homogeneous satellite DNA which constitutes up to 60% of its genome. The satellite DNA is shown to be present in all of the chromosomes by in situ hybridization. 18 dimers of the repeat unit were cloned and sequenced. The consensus sequence is 142 nt long and lacks any internal repeat structure. Monomers of the sequence are very similar, showing on average a 2% divergence from the calculated consensus. Variant nucleotides are scattered randomly throughout the sequence although some variants are more common than others. Neighboring repeat units are no more alike than randomly chosen ones. The results suggest that some mechanism, perhaps gene conversion, is acting to maintain the homogeneity of the satellite DNA despite its abundance and distribution on all of the chromosomes. Images PMID:2762148

The structures of non-CG-repeat Z-DNAs co-crystallized with the Z-DNA-binding domain, hZ alpha(ADAR1).

PubMed

Ha, Sung Chul; Choi, Jongkeun; Hwang, Hye-Yeon; Rich, Alexander; Kim, Yang-Gyun; Kim, Kyeong Kyu

2009-02-01

The Z-DNA conformation preferentially occurs at alternating purine-pyrimidine repeats, and is specifically recognized by Z alpha domains identified in several Z-DNA-binding proteins. The binding of Z alpha to foreign or chromosomal DNA in various sequence contexts is known to influence various biological functions, including the DNA-mediated innate immune response and transcriptional modulation of gene expression. For these reasons, understanding its binding mode and the conformational diversity of Z alpha bound Z-DNAs is of considerable importance. However, structural studies of Z alpha bound Z-DNA have been mostly limited to standard CG-repeat DNAs. Here, we have solved the crystal structures of three representative non-CG repeat DNAs, d(CACGTG)(2), d(CGTACG)(2) and d(CGGCCG)(2) complexed to hZ alpha(ADAR1) and compared those structures with that of hZ alpha(ADAR1)/d(CGCGCG)(2) and the Z alpha-free Z-DNAs. hZ alpha(ADAR1) bound to each of the three Z-DNAs showed a well conserved binding mode with very limited structural deviation irrespective of the DNA sequence, although varying numbers of residues were in contact with Z-DNA. Z-DNAs display less structural alterations in the Z alpha-bound state than in their free form, thereby suggesting that conformational diversities of Z-DNAs are restrained by the binding pocket of Z alpha. These data suggest that Z-DNAs are recognized by Z alpha through common conformational features regardless of the sequence and structural alterations.

HYBRIDIZATION PROPERTIES OF DNA SEQUENCES DIRECTING THE SYNTHESIS OF MESSENGER RNA AND HETEROGENEOUS NUCLEAR RNA

PubMed Central

Greenberg, Jay R.; Perry, Robert P.

1971-01-01

The relationship of the DNA sequences from which polyribosomal messenger RNA (mRNA) and heterogeneous nuclear RNA (NRNA) of mouse L cells are transcribed was investigated by means of hybridization kinetics and thermal denaturation of the hybrids. Hybridization was performed in formamide solutions at DNA excess. Under these conditions most of the hybridizing mRNA and NRNA react at values of Dot (DNA concentration multiplied by time) expected for RNA transcribed from the nonrepeated or rarely repeated fraction of the genome. However, a fraction of both mRNA and NRNA hybridize at values of Dot about 10,000 times lower, and therefore must be transcribed from highly redundant DNA sequences. The fraction of NRNA hybridizing to highly repeated sequences is about 1.7 times greater than the corresponding fraction of mRNA. The hybrids formed by the rapidly reacting fractions of both NRNA and mRNA melt over a narrow temperature range with a midpoint about 11°C below that of native L cell DNA. This indicates that these hybrids consist of partially complementary sequences with approximately 11% mismatching of bases. Hybrids formed by the slowly reacting fraction of NRNA melt within 4°–6°C of native DNA, indicating very little, if any, mismatching of bases. Hybrids of the slowly reacting components of mRNA, formed under conditions of sufficiently low RNA input, have a high thermal stability, similar to that observed for hybrids of the slowly reacting NRNA component. However, when higher inputs of mRNA are used, hybrids are formed which have a strikingly lower thermal stability. This observation can be explained by assuming that there is sufficient similarity among the relatively rare DNA sequences coding for mRNA so that under hybridization conditions, in which these DNA sequences are not truly in excess, reversible hybrids exhibiting a considerable amount of mispairing are formed. The fact that a comparable phenomenon has not been observed for NRNA may mean that there is less similarity among the relatively rare DNA sequences coding for NRNA than there is among the rare sequences coding for mRNA. PMID:4999767

Filter forensics: microbiota recovery from residential HVAC filters.

PubMed

Maestre, Juan P; Jennings, Wiley; Wylie, Dennis; Horner, Sharon D; Siegel, Jeffrey; Kinney, Kerry A

2018-01-30

Establishing reliable methods for assessing the microbiome within the built environment is critical for understanding the impact of biological exposures on human health. High-throughput DNA sequencing of dust samples provides valuable insights into the microbiome present in human-occupied spaces. However, the effect that different sampling methods have on the microbial community recovered from dust samples is not well understood across sample types. Heating, ventilation, and air conditioning (HVAC) filters hold promise as long-term, spatially integrated, high volume samplers to characterize the airborne microbiome in homes and other climate-controlled spaces. In this study, the effect that dust recovery method (i.e., cut and elution, swabbing, or vacuuming) has on the microbial community structure, membership, and repeatability inferred by Illumina sequencing was evaluated. The results indicate that vacuum samples captured higher quantities of total, bacterial, and fungal DNA than swab or cut samples. Repeated swab and vacuum samples collected from the same filter were less variable than cut samples with respect to both quantitative DNA recovery and bacterial community structure. Vacuum samples captured substantially greater bacterial diversity than the other methods, whereas fungal diversity was similar across all three methods. Vacuum and swab samples of HVAC filter dust were repeatable and generally superior to cut samples. Nevertheless, the contribution of environmental and human sources to the bacterial and fungal communities recovered via each sampling method was generally consistent across the methods investigated. Dust recovery methodologies have been shown to affect the recovery, repeatability, structure, and membership of microbial communities recovered from dust samples in the built environment. The results of this study are directly applicable to indoor microbiota studies utilizing the filter forensics approach. More broadly, this study provides a better understanding of the microbial community variability attributable to sampling methodology and helps inform interpretation of data collected from other types of dust samples collected from indoor environments.

Mitochondrial genome of the tomato clownfish Amphiprion frenatus (Pomacentridae, Amphiprioninae).

PubMed

Ye, Le; Hu, Jing; Wu, Kaichang; Wang, Yu; Li, Jianlong

2016-01-01

The complete mitochondrial (mt) genome of the tomato clownfish Amphiprion frenatus was obtained in this study. The circular mtDNA molecule was 16,774 bp in size and the overall nucleotide composition of the H-strand was 29.72% A, 25.81% T, 15.38% G and 29.09% C, with an A + T bias. The complete mitogenome encoded 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a control region (D-loop), with the gene arrangement and translation direction basically identical to other typical vertebrate mitogenomes. The D-loop included termination associated sequence (TAS), central conserved domain (CCD) and conserved sequence block (CSB), and was composed of 6 complete continuity tandem repeat units and an imperfect tandem repeat unit.

DNA Replication Origins and Fork Progression at Mammalian Telomeres

PubMed Central

Higa, Mitsunori; Fujita, Masatoshi; Yoshida, Kazumasa

2017-01-01

Telomeres are essential chromosomal regions that prevent critical shortening of linear chromosomes and genomic instability in eukaryotic cells. The bulk of telomeric DNA is replicated by semi-conservative DNA replication in the same way as the rest of the genome. However, recent findings revealed that replication of telomeric repeats is a potential cause of chromosomal instability, because DNA replication through telomeres is challenged by the repetitive telomeric sequences and specific structures that hamper the replication fork. In this review, we summarize current understanding of the mechanisms by which telomeres are faithfully and safely replicated in mammalian cells. Various telomere-associated proteins ensure efficient telomere replication at different steps, such as licensing of replication origins, passage of replication forks, proper fork restart after replication stress, and dissolution of post-replicative structures. In particular, shelterin proteins have central roles in the control of telomere replication. Through physical interactions, accessory proteins are recruited to maintain telomere integrity during DNA replication. Dormant replication origins and/or homology-directed repair may rescue inappropriate fork stalling or collapse that can cause defects in telomere structure and functions. PMID:28350373

Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System.

PubMed

Cooper, Lauren A; Stringer, Anne M; Wade, Joseph T

2018-04-17

In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo , for the type I-E system of Escherichia coli Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5' end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing. IMPORTANCE Many bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61-nucleotide (nt) CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease, which destroys the invading DNA molecule and promotes acquisition of new immunity elements. We made the first in vivo measurements of Cascade binding to DNA targets. Thus, we show that Cascade binding to DNA is highly promiscuous; endogenous E. coli crRNAs can direct Cascade binding to >100 chromosomal locations. In contrast, we show that targeted degradation and acquisition of new immunity elements require highly specific association of Cascade with DNA, limiting CRISPR-Cas function to the appropriate targets. Copyright © 2018 Cooper et al.

Sequences characterization of microsatellite DNA sequences in Pacific abalone ( Haliotis discus hannai)

NASA Astrophysics Data System (ADS)

Li, Qi; Akihiro, Kijima

2007-01-01

The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the microsatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber (1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats (13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (<20 repeats) were most abundant, accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatellite isolation in other abalone species.

Structural analysis of the rDNA intergenic spacer of Brassica nigra: evolutionary divergence of the spacers of the three diploid Brassica species.

PubMed

Bhatia, S; Singh Negi, M; Lakshmikumaran, M

1996-11-01

EcoRI restriction of the B. nigra rDNA recombinants, isolated from a lambda genomic library, showed that the 3.9-kb fragment corresponded to the Intergenic Spacer (IGS), which was sequenced and found to be 3,928 bp in size. Sequence and dot-matrix analyses showed that the organization of the B. nigra rDNA IGS was typical of most rDNA spacers, consisting of a central repetitive region and flanking unique sequences on either side. The repetitive region was composed of two repeat families-RF 'A' and RF 'B.' The B. nigra RF 'A' consisted of a tandem array of three full-length copies of a 106-bp sequence element. RF 'B' was composed of 66 tandemly repeated elements. Each 'B' element was only 21-bp in size and this is the smallest repeat unit identified in plant rDNA to date. The putative transcription initiation site (TIS) was identified as nucleotide position 3,110. Based on the sequence analysis it was suggested that the present organization of the repeat families was generated by successive cycles of deletions and amplifications and was being maintained by homogenization processes such as gene conversion and crossing-over.A detailed comparison of the rDNA IGS sequences of the three diploid Brassica species-namely, B. nigra, B. campestris, and B. oleracea-was carried out. First, comparisons revealed that B. campestris and B. oleracea were close to each other as the repeat families in both showed high sequence homology between each other. Second, the repeat elements in both the species were organized in an interspersed manner. Third, a 52-bp sequence, present just downstream of the repeats in B. campestris, was found to be identical to the B. oleracea repeats, thereby suggesting a common progenitor. On the other hand, in B. nigra no interspersion pattern of organization of repeats was observed. Further, the B. nigra RF 'A' was identified as distinct from the repeat families of B. campestris and B. oleracea. Based on this analysis, it was suggested that during speciation B. campestris and B. oleracea evolved in one lineage whereas B. nigra diverged into a separate lineage. The comparative analysis of the IGS helped in identifying not only conserved ancestral sequence motifs of possible functional significance such as promoters and enhancers, but also sequences which showed variation between the three diploid species and were therefore identified as species-specific sequences.

APE1 incision activity at abasic sites in tandem repeat sequences.

PubMed

Li, Mengxia; Völker, Jens; Breslauer, Kenneth J; Wilson, David M

2014-05-29

Repetitive DNA sequences, such as those present in microsatellites and minisatellites, telomeres, and trinucleotide repeats (linked to fragile X syndrome, Huntington disease, etc.), account for nearly 30% of the human genome. These domains exhibit enhanced susceptibility to oxidative attack to yield base modifications, strand breaks, and abasic sites; have a propensity to adopt non-canonical DNA forms modulated by the positions of the lesions; and, when not properly processed, can contribute to genome instability that underlies aging and disease development. Knowledge on the repair efficiencies of DNA damage within such repetitive sequences is therefore crucial for understanding the impact of such domains on genomic integrity. In the present study, using strategically designed oligonucleotide substrates, we determined the ability of human apurinic/apyrimidinic endonuclease 1 (APE1) to cleave at apurinic/apyrimidinic (AP) sites in a collection of tandem DNA repeat landscapes involving telomeric and CAG/CTG repeat sequences. Our studies reveal the differential influence of domain sequence, conformation, and AP site location/relative positioning on the efficiency of APE1 binding and strand incision. Intriguingly, our data demonstrate that APE1 endonuclease efficiency correlates with the thermodynamic stability of the DNA substrate. We discuss how these results have both predictive and mechanistic consequences for understanding the success and failure of repair protein activity associated with such oxidatively sensitive, conformationally plastic/dynamic repetitive DNA domains. Published by Elsevier Ltd.

A novel species-specific tandem repeat DNA family from Sinapis arvensis: detection of telomere-like sequences.

PubMed

Kapila, R; Das, S; Srivastava, P S; Lakshmikumaran, M

1996-08-01

DNA sequences representing a tandemly repeated DNA family of the Sinapis arvensis genome were cloned and characterized. The 700-bp tandem repeat family is represented by two clones, pSA35 and pSA52, which are 697 and 709 bp in length, respectively. Dot matrix analysis of the sequences indicates the presence of repeated elements within each monomeric unit. Sequence analysis of the repetitive region of clones pSA35 and pSA52 shows that there are several copies of a 7-bp repeat element organized in tandem. The consensus sequence of this repeat element is 5'-TTTAGGG-3'. These elements are highly mutated and the difference in length between the two clones is due to different copy numbers of these elements. The repetitive region of clone pSA35 has 26 copies of the element TTTAGGG, whereas clone pSA52 has 28 copies. The repetitive region in both clones is flanked on either side by inverted repeats that may be footprints of a transposition event. Sequence comparison indicates that the element TTTAGGG is identical to telomeric repeats present in Arabidopsis, maize, tomato, and other plants. However, Bal31 digestion kinetics indicates non-telomeric localization of the 700-bp tandem repeats. The clones represent a novel repeat family as (i) they contain telomere-like motifs as subrepeats within each unit; and (ii) they do not hybridize to related crucifers and are species-specific in nature.

Association between polymorphisms at promoters of XRCC5 and XRCC6 genes and risk of breast cancer.

PubMed

Rajaei, Mehrdad; Saadat, Iraj; Omidvari, Shahpour; Saadat, Mostafa

2014-04-01

Variation in DNA repair genes is one of the mechanisms that may lead to variation in DNA repair capacity. Ku, a heterodimeric DNA-binding complex, is directly involved in repair of DNA double-strand breaks. Ku consists of two subunits, Ku70 and Ku80, which are encoded by the XRCC6 and XRCC5 genes, respectively. In the present study, we investigated whether common genetic variant in variable number of tandem repeats (VNTR) XRCC5 and T-991C XRCC6 was associated with an altered risk of breast cancer. The present study included 407 females with breast cancer and 395 age frequency-matched controls which were randomly selected from the healthy female blood donors. The XRCC5 and XRCC6 polymorphisms were determined using PCR-based methods. For XRCC5 polymorphism, in comparison with the 1R/1R genotype, the 0R/0R genotype increased breast cancer risk (OR 9.55, 95%CI 1.19-76.64, P = 0.034). The 1R/3R genotype compared with 1R/1R genotype decreased the risk of breast cancer (Fisher's exact test P = 0.015). There was no association between T-991C polymorphism of XRCC6 and breast cancer risk. Mean of age at diagnosis of breast cancer for 0, 1, 2, 3, and >4 repeat in XRCC5 were 39.2, 41.9, 44.3, 45.8, and 47.3 years, respectively. The Kaplan-Meier survival analysis revealed that the number of repeat was associated with age at diagnosis of breast cancer (log rank statistic = 13.90, df = 4, P = 0.008). The findings of the present study revealed that either breast cancer risk or age at diagnosis of breast cancer was associated with the VNTR polymorphism at promoter region of XRCC5.

GENETIC VARIATION IN RED RASPBERRIES (RUBUS IDAEUS L.; ROSACEAE) FROM SITES DIFFERING IN ORGANIC POLLUTANTS COMPARED WITH SYNTHETIC TANDEM REPEAT DNA PROBES

EPA Science Inventory

Two synthetic tandem repetitive DNA probes were used to compare genetic variation at variable-number-tandem-repeat (VNTR) loci among Rubus idaeus L. var. strigosus (Michx.) Maxim. (Rosaceae) individuals sampled at eight sites contaminated by pollutants (N = 39) and eight adjacent...

Repetitive DNA loci and their modulation by the non-canonical nucleic acid structures R-loops and G-quadruplexes

PubMed Central

Hall, Amanda C.; Ostrowski, Lauren A.; Mekhail, Karim

2017-01-01

ABSTRACT Cells have evolved intricate mechanisms to maintain genome stability despite allowing mutational changes to drive evolutionary adaptation. Repetitive DNA sequences, which represent the bulk of most genomes, are a major threat to genome stability often driving chromosome rearrangements and disease. The major source of repetitive DNA sequences and thus the most vulnerable constituents of the genome are the rDNA (rDNA) repeats, telomeres, and transposable elements. Maintaining the stability of these loci is critical to overall cellular fitness and lifespan. Therefore, cells have evolved mechanisms to regulate rDNA copy number, telomere length and transposon activity, as well as DNA repair at these loci. In addition, non-canonical structure-forming DNA motifs can also modulate the function of these repetitive DNA loci by impacting their transcription, replication, and stability. Here, we discuss key mechanisms that maintain rDNA repeats, telomeres, and transposons in yeast and human before highlighting emerging roles for non-canonical DNA structures at these repetitive loci. PMID:28406751

Low abundance of microsatellite repeats in the genome of the Brown-headed Cowbird (Molothrus ater)

USGS Publications Warehouse

Longmire, Jonathan L.; Hahn, D.C.; Roach, J.L.

1999-01-01

A cosmid library made from brown-headed cowbird (Molothrus ater) DNA was examined for representation of 17 distinct microsatellite motifs including all possible mono-, di-, and trinucleotide microsatellites, and the tetranucleotide repeat (GATA)n. The overall density of microsatellites within cowbird DNA was found to be one repeat per 89 kb and the frequency of the most abundant motif, (AGC)n, was once every 382 kb. The abundance of microsatellites within the cowbird genome is estimated to be reduced approximately 15-fold compared to humans. The reduced frequency of microsatellites seen in this study is consistent with previous observations indicating reduced numbers of microsatellites and other interspersed repeats in avian DNA. In addition to providing new information concerning the abundance of microsatellites within an avian genome, these results provide useful insights for selecting cloning strategies that might be used in the development of locus-specific microsatellite markers for avian studies.

Local and systemic effect of transfection-reagent formulated DNA vectors on equine melanoma.

PubMed

Mählmann, Kathrin; Feige, Karsten; Juhls, Christiane; Endmann, Anne; Schuberth, Hans-Joachim; Oswald, Detlef; Hellige, Mareu; Doherr, Marcus; Cavalleri, Jessika-M V

2015-05-14

Equine melanoma has a high incidence in grey horses. Xenogenic DNA vaccination may represent a promising therapeutic approach against equine melanoma as it successfully induced an immunological response in other species suffering from melanoma and in healthy horses. In a clinical study, twenty-seven, grey, melanoma-bearing, horses were assigned to three groups (n = 9) and vaccinated on days 1, 22, and 78 with DNA vectors encoding for equine (eq) IL-12 and IL-18 alone or in combination with either human glycoprotein (hgp) 100 or human tyrosinase (htyr). Horses were vaccinated intramuscularly, and one selected melanoma was locally treated by intradermal peritumoral injection. Prior to each injection and on day 120, the sizes of up to nine melanoma lesions per horse were measured by caliper and ultrasound. Specific serum antibodies against hgp100 and htyr were measured using cell based flow-cytometric assays. An Analysis of Variance (ANOVA) for repeated measurements was performed to identify statistically significant influences on the relative tumor volume. For post-hoc testing a Tukey-Kramer Multiple-Comparison Test was performed to compare the relative volumes on the different examination days. An ANOVA for repeated measurements was performed to analyse changes in body temperature over time. A one-way ANOVA was used to evaluate differences in body temperature between the groups. A p-value < 0.05 was considered significant for all statistical tests applied. In all groups, the relative tumor volume decreased significantly to 79.1 ± 26.91% by day 120 (p < 0.0001, Tukey-Kramer Multiple-Comparison Test). Affiliation to treatment group, local treatment and examination modality had no significant influence on the results (ANOVA for repeated measurements). Neither a cellular nor a humoral immune response directed against htyr or hgp100 was detected. Horses had an increased body temperature on the day after vaccination. This is the first clinical report on a systemic effect against equine melanoma following treatment with DNA vectors encoding eqIL12 and eqIL18 and formulated with a transfection reagent. Addition of DNA vectors encoding hgp100 respectively htyr did not potentiate this effect.

Local and systemic effect of transfection-reagent formulated DNA vectors on equine melanoma.

PubMed

Mählmann, Kathrin; Feige, Karsten; Juhls, Christiane; Endmann, Anne; Schuberth, Hans-Joachim; Oswald, Detlef; Hellige, Maren; Doherr, Marcus; Cavalleri, Jessika-M V

2015-06-11

Equine melanoma has a high incidence in grey horses. Xenogenic DNA vaccination may represent a promising therapeutic approach against equine melanoma as it successfully induced an immunological response in other species suffering from melanoma and in healthy horses. In a clinical study, twenty-seven, grey, melanoma-bearing, horses were assigned to three groups (n = 9) and vaccinated on days 1, 22, and 78 with DNA vectors encoding for equine (eq) IL-12 and IL-18 alone or in combination with either human glycoprotein (hgp) 100 or human tyrosinase (htyr). Horses were vaccinated intramuscularly, and one selected melanoma was locally treated by intradermal peritumoral injection. Prior to each injection and on day 120, the sizes of up to nine melanoma lesions per horse were measured by caliper and ultrasound. Specific serum antibodies against hgp100 and htyr were measured using cell based flow-cytometric assays. An Analysis of Variance (ANOVA) for repeated measurements was performed to identify statistically significant influences on the relative tumor volume. For post-hoc testing a Tukey-Kramer Multiple-Comparison Test was performed to compare the relative volumes on the different examination days. An ANOVA for repeated measurements was performed to analyse changes in body temperature over time. A one-way ANOVA was used to evaluate differences in body temperature between the groups. A p-value < 0.05 was considered significant for all statistical tests applied. In all groups, the relative tumor volume decreased significantly to 79.1 ± 26.91% by day 120 (p < 0.0001, Tukey-Kramer Multiple-Comparison Test). Affiliation to treatment group, local treatment and examination modality had no significant influence on the results (ANOVA for repeated measurements). Neither a cellular nor a humoral immune response directed against htyr or hgp100 was detected. Horses had an increased body temperature on the day after vaccination. This is the first clinical report on a systemic effect against equine melanoma following treatment with DNA vectors encoding eqIL12 and eqIL18 and formulated with a transfection reagent. Addition of DNA vectors encoding hgp100 respectively htyr did not potentiate this effect.

Two synthetic Sp1-binding sites functionally substitute for the 21-base-pair repeat region to activate simian virus 40 growth in CV-1 cells.

PubMed Central

Lednicky, J; Folk, W R

1992-01-01

The 21-bp repeat region of simian virus 40 (SV40) activates viral transcription and DNA replication and contains binding sites for many cellular proteins, including Sp1, LSF, ETF, Ap2, Ap4, GT-1B, H16, and p53, and for the SV40 large tumor antigen. We have attempted to reduce the complexity of this region while maintaining its growth-promoting capacity. Deletion of the 21-bp repeat region from the SV40 genome delays the expression of viral early proteins and DNA replication and reduces virus production in CV-1 cells. Replacement of the 21-bp repeat region with two copies of DNA sequence motifs bound with high affinities by Sp1 promotes SV40 growth in CV-1 cells to nearly wild-type levels, but substitution by motifs bound less avidly by Sp1 or bound by other activator proteins does not restore growth. This indicates that Sp1 or a protein with similar sequence specificity is primarily responsible for the function of the 21-bp repeat region. We speculate about how Sp1 activates both SV40 transcription and DNA replication. Images PMID:1328672

Isolation of human simple repeat loci by hybridization selection.

PubMed

Armour, J A; Neumann, R; Gobert, S; Jeffreys, A J

1994-04-01

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING.

PubMed

Zhang, Lu; Mo, Jinyao; Swanson, Karen V; Wen, Haitao; Petrucelli, Alex; Gregory, Sean M; Zhang, Zhigang; Schneider, Monika; Jiang, Yan; Fitzgerald, Katherine A; Ouyang, Songying; Liu, Zhi-Jie; Damania, Blossom; Shu, Hong-Bing; Duncan, Joseph A; Ting, Jenny P-Y

2014-03-20

Stimulator of interferon genes (STING, also named MITA, MYPS, or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich-repeat-containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP), and DNA viruses. NLRC3 associated with both STING and TBK1 and impeded STING-TBK1 interaction and downstream type I interferon production. By using purified recombinant proteins, we found NLRC3 to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3(-/-) mice exhibited enhanced innate immunity and reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus, and c-di-GMP. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparative genomics of pyridoxal 5′-phosphate-dependent transcription factor regulons in Bacteria

PubMed Central

Suvorova, Inna A.

2016-01-01

The MocR-subfamily transcription factors (MocR-TFs) characterized by the GntR-family DNA-binding domain and aminotransferase-like sensory domain are broadly distributed among certain lineages of Bacteria. Characterized MocR-TFs bind pyridoxal 5′-phosphate (PLP) and control transcription of genes involved in PLP, gamma aminobutyric acid (GABA) and taurine metabolism via binding specific DNA operator sites. To identify putative target genes and DNA binding motifs of MocR-TFs, we performed comparative genomics analysis of over 250 bacterial genomes. The reconstructed regulons for 825 MocR-TFs comprise structural genes from over 200 protein families involved in diverse biological processes. Using the genome context and metabolic subsystem analysis we tentatively assigned functional roles for 38 out of 86 orthologous groups of studied regulators. Most of these MocR-TF regulons are involved in PLP metabolism, as well as utilization of GABA, taurine and ectoine. The remaining studied MocR-TF regulators presumably control genes encoding enzymes involved in reduction/oxidation processes, various transporters and PLP-dependent enzymes, for example aminotransferases. Predicted DNA binding motifs of MocR-TFs are generally similar in each orthologous group and are characterized by two to four repeated sequences. Identified motifs were classified according to their structures. Motifs with direct and/or inverted repeat symmetry constitute the majority of inferred DNA motifs, suggesting preferable TF dimerization in head-to-tail or head-to-head configuration. The obtained genomic collection of in silico reconstructed MocR-TF motifs and regulons in Bacteria provides a basis for future experimental characterization of molecular mechanisms for various regulators in this family. PMID:28348826

[Variability of nuclear 18S-25S rDNA of Gentiana lutea L. in nature and in tissue culture in vitro].

PubMed

Mel'nyk, V M; Spiridonova, K V; Andrieiev, I O; Strashniuk, N M; Kunakh, V A

2004-01-01

18S-25S rDNA sequence in genomes of G. lutea plants from different natural populations and from tissue culture has been studied with blot-hybridization method. It was shown that ribosomal repeats are represented by the variants which differ for their size and for the presence of additional HindIII restriction site. Genome of individual plant usually possesses several variants of DNA repeats. Interpopulation variability according to their quantitative ratio and to the presence of some of them has been shown. Modifications of the range of rDNA repeats not exceeding intraspecific variability were observed in callus tissues in comparison with the plants of initial population. Non-randomness of genome modifications in the course of cell adaptation to in vitro conditions makes it possible to some extent to forecast these modifications in tissue culture.

The DL1 repeats in the genome of Diphyllobothrium latum.

PubMed

Usmanova, Nadezhda M; Kazakov, Vasiliy I

2010-07-01

Diphyllobothrium latum is a widespread intestinal parasite, which has a great clinical relevance, but there are no sequences of its nuclear genome. In this paper, a repetitive element in the D. latum genome is firstly described. The adult D. latum was obtained in the result of expulsion from intestinum of a patient suffering from diphyllobothriasis. Genomic DNA was isolated from several proglottids of this individual. PstI restriction products of D. latum genomic DNA were sequenced. Polymerase chain reaction (PCR) amplification of these products using genomic DNA and selected primers was carried out. Thereby a cluster of a repetitive element, called DL1, was discovered. For precise identification of a beginning and an end of the repeat, a product of PCR amplification of D. latum genomic DNA with one specific primer was sequenced. In discussion, several evidences that DL1 repeat is a member of the SINE family of retroposons were adduced.

Engineering DNA Backbone Interactions Results in TALE Scaffolds with Enhanced 5-Methylcytosine Selectivity.

PubMed

Rathi, Preeti; Witte, Anna; Summerer, Daniel

2017-11-08

Transcription activator-like effectors (TALEs) are DNA major-groove binding proteins widely used for genome targeting. TALEs contain an N-terminal region (NTR) and a central repeat domain (CRD). Repeats of the CRD selectively recognize each one DNA nucleobase, offering programmability. Moreover, repeats with selectivity for 5-methylcytosine (5mC) and its oxidized derivatives can be designed for analytical applications. However, both TALE domains also nonspecifically interact with DNA phosphates via basic amino acids. To enhance the 5mC selectivity of TALEs, we aimed to decrease the nonselective binding energy of TALEs. We substituted basic amino acids with alanine in the NTR and identified TALE mutants with increased selectivity. We then analysed conserved, DNA phosphate-binding KQ diresidues in CRD repeats and identified further improved mutants. Combination of mutations in the NTR and CRD was highly synergetic and resulted in TALE scaffolds with up to 4.3-fold increased selectivity in genomic 5mC analysis via affinity enrichment. Moreover, transcriptional activation in HEK293T cells by a TALE-VP64 construct based on this scaffold design exhibited a 3.5-fold increased 5mC selectivity. This provides perspectives for improved 5mC analysis and for the 5mC-conditional control of TALE-based editing constructs in vivo.

Centromeric and non-centromeric satellite DNA organisation differs in holocentric Rhynchospora species.

PubMed

Ribeiro, Tiago; Marques, André; Novák, Petr; Schubert, Veit; Vanzela, André L L; Macas, Jiri; Houben, Andreas; Pedrosa-Harand, Andrea

2017-03-01

Satellite DNA repeats (or satDNA) are fast-evolving sequences usually associated with condensed heterochromatin. To test whether the chromosomal organisation of centromeric and non-centromeric satDNA differs in species with holocentric chromosomes, we identified and characterised the major satDNA families in the holocentric Cyperaceae species Rhynchospora ciliata (2n = 10), R. globosa (2n = 50) and R. tenuis (2n = 2x = 4 and 2n = 4x = 8). While conserved centromeric repeats (present in R. ciliata and R. tenuis) revealed linear signals at both chromatids, non-centromeric, species-specific satDNAs formed distinct clusters along the chromosomes. Colocalisation of both repeat types resulted in a ladder-like hybridisation pattern at mitotic chromosomes. In interphase, the centromeric satDNA was dispersed while non-centromeric satDNA clustered and partly colocalised to chromocentres. Despite the banding-like hybridisation patterns of the clustered satDNA, the identification of chromosome pairs was impaired due to the irregular hybridisation patterns of the homologues in R. tenuis and R. ciliata. These differences are probably caused by restricted or impaired meiotic recombination as reported for R. tenuis, or alternatively by complex chromosome rearrangements or unequal condensation of homologous metaphase chromosomes. Thus, holocentricity influences the chromosomal organisation leading to differences in the distribution patterns and condensation dynamics of centromeric and non-centromeric satDNA.

Condensin loaded onto the replication fork barrier site in the rRNA gene repeats during S phase in a FOB1-dependent fashion to prevent contraction of a long repetitive array in Saccharomyces cerevisiae.

PubMed

Johzuka, Katsuki; Terasawa, Masahiro; Ogawa, Hideyuki; Ogawa, Tomoko; Horiuchi, Takashi

2006-03-01

An average of 200 copies of the rRNA gene (rDNA) is clustered in a long tandem array in Saccharomyces cerevisiae. FOB1 is known to be required for expansion/contraction of the repeats by stimulating recombination, thereby contributing to the maintenance of the average copy number. In Deltafob1 cells, the repeats are still maintained without any fluctuation in the copy number, suggesting that another, unknown system acts to prevent repeat contraction. Here, we show that condensin acts together with FOB1 in a functionally complemented fashion to maintain the long tandem repeats. Six condensin mutants possessing severely contracted rDNA repeats were isolated in Deltafob1 cells but not in FOB1+ cells. We also found that the condensin complex associated with the nontranscribed spacer region of rDNA with a major peak coincided with the replication fork barrier (RFB) site in a FOB1-dependent fashion. Surprisingly, condensin association with the RFB site was established during S phase and was maintained until anaphase. These results indicate that FOB1 plays a novel role in preventing repeat contraction by regulating condensin association and suggest a link between replication termination and chromosome condensation and segregation.

Complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera, and comparative analyses with other grass genomes

PubMed Central

Saski, Christopher; Lee, Seung-Bum; Fjellheim, Siri; Guda, Chittibabu; Jansen, Robert K.; Luo, Hong; Tomkins, Jeffrey; Rognli, Odd Arne; Clarke, Jihong Liu

2009-01-01

Comparisons of complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera to six published grass chloroplast genomes reveal that gene content and order are similar but two microstructural changes have occurred. First, the expansion of the IR at the SSC/IRa boundary that duplicates a portion of the 5′ end of ndhH is restricted to the three genera of the subfamily Pooideae (Agrostis, Hordeum and Triticum). Second, a 6 bp deletion in ndhK is shared by Agrostis, Hordeum, Oryza and Triticum, and this event supports the sister relationship between the subfamilies Erhartoideae and Pooideae. Repeat analysis identified 19–37 direct and inverted repeats 30 bp or longer with a sequence identity of at least 90%. Seventeen of the 26 shared repeats are found in all the grass chloroplast genomes examined and are located in the same genes or intergenic spacer (IGS) regions. Examination of simple sequence repeats (SSRs) identified 16–21 potential polymorphic SSRs. Five IGS regions have 100% sequence identity among Zea mays, Saccharum officinarum and Sorghum bicolor, whereas no spacer regions were identical among Oryza sativa, Triticum aestivum, H. vulgare and A. stolonifera despite their close phylogenetic relationship. Alignment of EST sequences and DNA coding sequences identified six C–U conversions in both Sorghum bicolor and H. vulgare but only one in A. stolonifera. Phylogenetic trees based on DNA sequences of 61 protein-coding genes of 38 taxa using both maximum parsimony and likelihood methods provide moderate support for a sister relationship between the subfamilies Erhartoideae and Pooideae. PMID:17534593

Changing practice: red blood cell typing by molecular methods for patients with sickle cell disease.

PubMed

Casas, Jessica; Friedman, David F; Jackson, Tannoa; Vege, Sunitha; Westhoff, Connie M; Chou, Stella T

2015-06-01

Extended red blood cell (RBC) antigen matching is recommended to limit alloimmunization in patients with sickle cell disease (SCD). DNA-based testing to predict blood group phenotypes has enhanced availability of antigen-negative donor units and improved typing of transfused patients, but replacement of routine serologic typing for non-ABO antigens with molecular typing for patients has not been reported. This study compared the historical RBC antigen phenotypes obtained by hemagglutination methods with genotype predictions in 494 patients with SCD. For discrepant results, repeat serologic testing was performed and/or investigated by gene sequencing for silent or variant alleles. Seventy-one typing discrepancies were identified among 6360 antigen comparisons (1.1%). New specimens for repeat serologic testing were obtained for 66 discrepancies and retyping agreed with the genotype in 64 cases. One repeat Jk(b-) serologic phenotype, predicted Jk(b+) by genotype, was found by direct sequencing of JK to be a silenced allele, and one N typing discrepancy remains under investigation. Fifteen false-negative serologic results were associated with alleles encoding weak antigens or single-dose Fy(b) expression. DNA-based RBC typing provided improved accuracy and expanded information on RBC antigens compared to hemagglutination methods, leading to its implementation as the primary method for extended RBC typing for patients with SCD at our institution. © 2015 AABB.

Rif1 acts through Protein Phosphatase 1 but independent of replication timing to suppress telomere extension in budding yeast.

PubMed

Kedziora, Sylwia; Gali, Vamsi K; Wilson, Rosemary H C; Clark, Kate R M; Nieduszynski, Conrad A; Hiraga, Shin-Ichiro; Donaldson, Anne D

2018-05-04

The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast Saccharomyces cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore, we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant defective for PP1 interaction causes a long-telomere phenotype, similar to that of rif1Δ cells. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1-PP1 interaction is known to cause precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if late replication is forced through deletion of nearby replication origins, indicating that Rif1 can control telomere length independent of replication timing. Moreover we find that, even at a de novo telomere created after DNA synthesis during a mitotic block, Rif1-PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.

Repeated sampling of genes from a single cell - implications for gravitropism research

NASA Astrophysics Data System (ADS)

Scherp, P.; Hasenstein, K. H.

The need for repeated but independent extractions of mRNA from single cells and plant tissues prompted the development of Solid Phase Gene Extraction (SPGE, patent pending). Oligo dT18 coated glass needles hybridize during a 2 to 3 min sampling time with the poly A+ mRNA. The needle is withdrawn and can be used directly for RT-PCR. Because of the small probe size, no cytoplasm is lost and repeated sampling of the same cell is possible. SPGE of Chara rhizoids and internodal cells showed fluctuations of type and quantity of mRNA in specific areas of the cytoplasm of rhizoids and time-dependent gene expression in internodal cells as a function of light/dark intervals. Despite extensive cytoplasmic streaming, mRNA-samples taken in the vicinity of the nucleus revealed a higher variability than the distal ends of the cell. In rhizoids, the mRNA/cDNA varied between the different zones of cytoplasm. In Arabidopsis, we isolated cDNA species from root tips, shoots and leaves and determined their sequences. Growth studies on SPGE-sampled individuals showed that after a short recovery period, all sampled plants resumed growth with normal growth rates and graviresponse. The data indicate that SPGE is a powerful method to study gene expression in single cells and in tissues of higher plants with high spatial and temporal resolution. Supported by NASA: NAG 2-1423

Sequences spanning the leader-repeat junction mediate CRISPR adaptation to phage in Streptococcus thermophilus

PubMed Central

Wei, Yunzhou; Chesne, Megan T.; Terns, Rebecca M.; Terns, Michael P.

2015-01-01

CRISPR-Cas systems are RNA-based immune systems that protect prokaryotes from invaders such as phages and plasmids. In adaptation, the initial phase of the immune response, short foreign DNA fragments are captured and integrated into host CRISPR loci to provide heritable defense against encountered foreign nucleic acids. Each CRISPR contains a ∼100–500 bp leader element that typically includes a transcription promoter, followed by an array of captured ∼35 bp sequences (spacers) sandwiched between copies of an identical ∼35 bp direct repeat sequence. New spacers are added immediately downstream of the leader. Here, we have analyzed adaptation to phage infection in Streptococcus thermophilus at the CRISPR1 locus to identify cis-acting elements essential for the process. We show that the leader and a single repeat of the CRISPR locus are sufficient for adaptation in this system. Moreover, we identified a leader sequence element capable of stimulating adaptation at a dormant repeat. We found that sequences within 10 bp of the site of integration, in both the leader and repeat of the CRISPR, are required for the process. Our results indicate that information at the CRISPR leader-repeat junction is critical for adaptation in this Type II-A system and likely other CRISPR-Cas systems. PMID:25589547

Rat L (long interspersed repeated DNA) elements contain guanine-rich homopurine sequences that induce unpairing of contiguous duplex DNA.

PubMed Central

Usdin, K; Furano, A V

1988-01-01

The L family (long interspersed repeated DNA) of mobile genetic elements is a persistent feature of the mammalian genome. In rats, this family contains approximately equal to 40,000 members and accounts for approximately equal to 10% of the haploid genome. We demonstrate here that the guanine-rich homopurine stretches located at the right end of L-DNA induce oligonucleotide uptake by contiguous duplex DNA. The uptake is dependent on negative supercoiling and the length of the homopurine stretch and occurs even when the L-DNA homopurine stretches are introduced into a different DNA environment. The bound oligomer primes DNA synthesis when DNA polymerase and deoxyribonucleoside triphosphates are added, resulting in a faithful copy of the template to which the oligonucleotide had bound. The implications of this property of the L-DNA guanine-rich homopurine stretches in the amplification, recombination, and dispersal of L elements is discussed. Images PMID:2837766

Strand invasion structures in the inverted repeat of Candida albicans mitochondrial DNA reveal a role for homologous recombination in replication.

PubMed

Gerhold, Joachim M; Aun, Anu; Sedman, Tiina; Jõers, Priit; Sedman, Juhan

2010-09-24

Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study wild-type mtDNA maintenance in yeast. Copyright © 2010 Elsevier Inc. All rights reserved.

Repetitive part of the banana (Musa acuminata) genome investigated by low-depth 454 sequencing.

PubMed

Hribová, Eva; Neumann, Pavel; Matsumoto, Takashi; Roux, Nicolas; Macas, Jirí; Dolezel, Jaroslav

2010-09-16

Bananas and plantains (Musa spp.) are grown in more than a hundred tropical and subtropical countries and provide staple food for hundreds of millions of people. They are seed-sterile crops propagated clonally and this makes them vulnerable to a rapid spread of devastating diseases and at the same time hampers breeding improved cultivars. Although the socio-economic importance of bananas and plantains cannot be overestimated, they remain outside the focus of major research programs. This slows down the study of nuclear genome and the development of molecular tools to facilitate banana improvement. In this work, we report on the first thorough characterization of the repeat component of the banana (M. acuminata cv. 'Calcutta 4') genome. Analysis of almost 100 Mb of sequence data (0.15× genome coverage) permitted partial sequence reconstruction and characterization of repetitive DNA, making up about 30% of the genome. The results showed that the banana repeats are predominantly made of various types of Ty1/copia and Ty3/gypsy retroelements representing 16 and 7% of the genome respectively. On the other hand, DNA transposons were found to be rare. In addition to new families of transposable elements, two new satellite repeats were discovered and found useful as cytogenetic markers. To help in banana sequence annotation, a specific Musa repeat database was created, and its utility was demonstrated by analyzing the repeat composition of 62 genomic BAC clones. A low-depth 454 sequencing of banana nuclear genome provided the largest amount of DNA sequence data available until now for Musa and permitted reconstruction of most of the major types of DNA repeats. The information obtained in this study improves the knowledge of the long-range organization of banana chromosomes, and provides sequence resources needed for repeat masking and annotation during the Musa genome sequencing project. It also provides sequence data for isolation of DNA markers to be used in genetic diversity studies and in marker-assisted selection.

Repetitive part of the banana (Musa acuminata) genome investigated by low-depth 454 sequencing

PubMed Central

2010-01-01

Background Bananas and plantains (Musa spp.) are grown in more than a hundred tropical and subtropical countries and provide staple food for hundreds of millions of people. They are seed-sterile crops propagated clonally and this makes them vulnerable to a rapid spread of devastating diseases and at the same time hampers breeding improved cultivars. Although the socio-economic importance of bananas and plantains cannot be overestimated, they remain outside the focus of major research programs. This slows down the study of nuclear genome and the development of molecular tools to facilitate banana improvement. Results In this work, we report on the first thorough characterization of the repeat component of the banana (M. acuminata cv. 'Calcutta 4') genome. Analysis of almost 100 Mb of sequence data (0.15× genome coverage) permitted partial sequence reconstruction and characterization of repetitive DNA, making up about 30% of the genome. The results showed that the banana repeats are predominantly made of various types of Ty1/copia and Ty3/gypsy retroelements representing 16 and 7% of the genome respectively. On the other hand, DNA transposons were found to be rare. In addition to new families of transposable elements, two new satellite repeats were discovered and found useful as cytogenetic markers. To help in banana sequence annotation, a specific Musa repeat database was created, and its utility was demonstrated by analyzing the repeat composition of 62 genomic BAC clones. Conclusion A low-depth 454 sequencing of banana nuclear genome provided the largest amount of DNA sequence data available until now for Musa and permitted reconstruction of most of the major types of DNA repeats. The information obtained in this study improves the knowledge of the long-range organization of banana chromosomes, and provides sequence resources needed for repeat masking and annotation during the Musa genome sequencing project. It also provides sequence data for isolation of DNA markers to be used in genetic diversity studies and in marker-assisted selection. PMID:20846365

Ordered mapping of 3 alphoid DNA subsets on human chromosome 22

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antonacci, R.; Baldini, A.; Archidiacono, N.

1994-09-01

Alpha satellite DNA consists of tandemly repeated monomers of 171 bp clustered in the centromeric region of primate chromosomes. Sequence divergence between subsets located in different human chromosomes is usually high enough to ensure chromosome-specific hybridization. Alphoid probes specific for almost every human chromosome have been reported. A single chromosome can carry different subsets of alphoid DNA and some alphoid subsets can be shared by different chromosomes. We report the physical order of three alphoid DNA subsets on human chromosome 22 determined by a combination of low and high resolution cytological mapping methods. Results visually demonstrate the presence of threemore » distinct alphoid DNA domains at the centromeric region of chromosome 22. We have measured the interphase distances between the three probes in three-color FISH experiments. Statistical analysis of the results indicated the order of the subsets. Two color experiments on prometaphase chromosomes established the order of the three domains relative to the arms of chromosome 22 and confirmed the results obtained using interphase mapping. This demonstrates the applicability of interphase mapping for alpha satellite DNA orderering. However, in our experiments, interphase mapping did not provide any information about the relationship between extremities of the repeat arrays. This information was gained from extended chromatin hybridization. The extremities of two of the repeat arrays were seen to be almost overlapping whereas the third repeat array was clearly separated from the other two. Our data show the value of extended chromatin hybridization as a complement of other cytological techniques for high resolution mapping of repetitive DNA sequences.« less

An annotated genetic map of loblolly pine based on microsatellite and cDNA markers

USDA-ARS?s Scientific Manuscript database

Previous loblolly pine (Pinus taeda L.) genetic linkage maps have been based on a variety of DNA polymorphisms, such as AFLPs, RAPDs, RFLPs, and ESTPs, but only a few SSRs (simple sequence repeats), also known as simple tandem repeats or microsatellites, have been mapped in P. taeda. The objective o...

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae)

PubMed Central

Begum, Rabeya; Zakrzewski, Falk; Menzel, Gerhard; Weber, Beatrice; Alam, Sheikh Shamimul; Schmidt, Thomas

2013-01-01

Background and Aims The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. Methods A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100–500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Key Results Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S–5·8S–25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. Conclusions The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species. PMID:23666888

PERF: an exhaustive algorithm for ultra-fast and efficient identification of microsatellites from large DNA sequences.

PubMed

Avvaru, Akshay Kumar; Sowpati, Divya Tej; Mishra, Rakesh Kumar

2018-03-15

Microsatellites or Simple Sequence Repeats (SSRs) are short tandem repeats of DNA motifs present in all genomes. They have long been used for a variety of purposes in the areas of population genetics, genotyping, marker-assisted selection and forensics. Numerous studies have highlighted their functional roles in genome organization and gene regulation. Though several tools are currently available to identify SSRs from genomic sequences, they have significant limitations. We present a novel algorithm called PERF for extremely fast and comprehensive identification of microsatellites from DNA sequences of any size. PERF is several fold faster than existing algorithms and uses up to 5-fold lesser memory. It provides a clean and flexible command-line interface to change the default settings, and produces output in an easily-parseable tab-separated format. In addition, PERF generates an interactive and stand-alone HTML report with charts and tables for easy downstream analysis. PERF is implemented in the Python programming language. It is freely available on PyPI under the package name perf_ssr, and can be installed directly using pip or easy_install. The documentation of PERF is available at https://github.com/rkmlab/perf. The source code of PERF is deposited in GitHub at https://github.com/rkmlab/perf under an MIT license. tej@ccmb.res.in. Supplementary data are available at Bioinformatics online.

Functional centromeres in Astragalus sinicus include a compact centromere-specific histone H3 and a 20-bp tandem repeat.

PubMed

Tek, Ahmet L; Kashihara, Kazunari; Murata, Minoru; Nagaki, Kiyotaka

2011-11-01

The centromere plays an essential role for proper chromosome segregation during cell division and usually harbors long arrays of tandem repeated satellite DNA sequences. Although this function is conserved among eukaryotes, the sequences of centromeric DNA repeats are variable. Most of our understanding of functional centromeres, which are defined by localization of a centromere-specific histone H3 (CENH3) protein, comes from model organisms. The components of the functional centromere in legumes are poorly known. The genus Astragalus is a member of the legumes and bears the largest numbers of species among angiosperms. Therefore, we studied the components of centromeres in Astragalus sinicus. We identified the CenH3 homolog of A. sinicus, AsCenH3 that is the most compact in size among higher eukaryotes. A CENH3-based assay revealed the functional centromeric DNA sequences from A. sinicus, called CentAs. The CentAs repeat is localized in A. sinicus centromeres, and comprises an AT-rich tandem repeat with a monomer size of 20 nucleotides.

A novel strategy for highly efficient isolation and analysis of circulating tumor-specific cell-free DNA from lung cancer patients using a reusable conducting polymer nanostructure.

PubMed

Lee, HyungJae; Jeon, SeungHyun; Seo, Jin-Suck; Goh, Sung-Ho; Han, Ji-Youn; Cho, Youngnam

2016-09-01

We have developed a reusable nanostructured polypyrrole nanochip and demonstrated its use in the electric field-mediated recovery of circulating cell-free DNA (cfDNA) from the plasma of lung cancer patients. Although cfDNA has been recognized and widely studied as a versatile and promising biomarker for the diagnosis and prognosis of cancers, the lack of efficient strategies to directly isolate cfDNA from the plasma has become a great hindrance to its potential clinical use. As a proof-of-concept study, we demonstrated a technique for the rapid and efficient isolation of cfDNA with high yield and purity. In particular, the synergistic effects of the electro-activity and the nanostructured features of the polypyrrole polymer enabled repeated retrieval of cfDNA using a single platform. Moreover, polypyrrole nanochip facilitated the amplification of tumor-specific DNA fragments from the plasma samples of patients with lung cancer characterized by mutations in exons 21 of the epidermal growth factor receptor gene (EGFR). Overall, the proposed polypyrrole nanochip has enormous potential for industrial and clinical applications with significantly enhanced efficiency in the recovery of tumor-associated circulating cfDNA. This may ultimately contribute to more robust and reliable evaluation of gene mutations in peripheral blood. Copyright © 2016 Elsevier Ltd. All rights reserved.

Annotation, submission and screening of repetitive elements in Repbase: RepbaseSubmitter and Censor.

PubMed

Kohany, Oleksiy; Gentles, Andrew J; Hankus, Lukasz; Jurka, Jerzy

2006-10-25

Repbase is a reference database of eukaryotic repetitive DNA, which includes prototypic sequences of repeats and basic information described in annotations. Updating and maintenance of the database requires specialized tools, which we have created and made available for use with Repbase, and which may be useful as a template for other curated databases. We describe the software tools RepbaseSubmitter and Censor, which are designed to facilitate updating and screening the content of Repbase. RepbaseSubmitter is a java-based interface for formatting and annotating Repbase entries. It eliminates many common formatting errors, and automates actions such as calculation of sequence lengths and composition, thus facilitating curation of Repbase sequences. In addition, it has several features for predicting protein coding regions in sequences; searching and including Pubmed references in Repbase entries; and searching the NCBI taxonomy database for correct inclusion of species information and taxonomic position. Censor is a tool to rapidly identify repetitive elements by comparison to known repeats. It uses WU-BLAST for speed and sensitivity, and can conduct DNA-DNA, DNA-protein, or translated DNA-translated DNA searches of genomic sequence. Defragmented output includes a map of repeats present in the query sequence, with the options to report masked query sequence(s), repeat sequences found in the query, and alignments. Censor and RepbaseSubmitter are available as both web-based services and downloadable versions. They can be found at http://www.girinst.org/repbase/submission.html (RepbaseSubmitter) and http://www.girinst.org/censor/index.php (Censor).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yauk, C.L.; Quinn, J.S.

The authors used multi-locus DNA fingerprinting to examine families of herring gulls (Larus argentatus) from a genotoxically contaminated site (Hamilton Harbour) and from a pristine location (Kent Island, Bay of Fundy) to show significant differences in mutation rates between the locations. Overall the authors identified 17 mutant bands from 15 individuals of the 35 examined from Hamilton Harbour, and 7 mutant fragments from 7 individuals, of the 43 examined from Kent Island; a mutation frequency of 0.429 per nestling for Hamilton Harbour and 0.163 for Kent Island. The total number of individuals with mutant bands was significantly higher at Hamiltonmore » Harbour than at Kent Island (X{sup 2}=6.734; df = 1; P < 0.01). Ongoing analysis of other less contaminated sites also reveals lower mutation rates than those seen in Hamilton Harbour. With multi-locus DNA fingerprinting many regions of the genome can be surveyed simultaneously. The tandemly repeated arrays of nucleotides examined with DNA fingerprinting are known to have elevated rates of mutation. Furthermore, the mutations seen with DNA fingerprinting are predominantly heritable. Other biomarkers currently used in situ are not able to monitor direct and heritable DNA mutation, or measure biological endpoints that frequently result in spontaneous abortion creating difficulty in observing significantly elevated levels in viable offspring. The authors suggest that multilocus DNA fingerprinting can be used as a biomarker to identify potentially heritable risks before the onset of other types of ecological damage. This approach provides a direct measure of mutation in situ and in vivo in a vertebrate species under ambient conditions.« less

G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV

PubMed Central

Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P.; Robertson, Erle S.; Schildkraut, Carl L.; Verma, Subhash C.

2016-01-01

Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. PMID:26837574

Smoking, but not malnutrition, influences promoter-specific DNA methylation of the proopiomelanocortin gene in patients with and without anorexia nervosa.

PubMed

Ehrlich, Stefan; Walton, Esther; Roffman, Joshua L; Weiss, Deike; Puls, Imke; Doehler, Nico; Burghardt, Roland; Lehmkuhl, Ulrike; Hillemacher, Thomas; Muschler, Marc; Frieling, Helge

2012-03-01

Our pilot study evaluates the impact of environmental factors, such as nutrition and smoking status, on epigenetic patterns in a disease-associated gene. We measured the effects of malnutrition and cigarette smoking on proopiomelanocortin (POMC) promoter-specific DNA methylation in female patients with and without anorexia nervosa (AN). POMC and its derived peptides (alpha melanocyte stimulating hormone and adrenocorticotropic hormone) are implicated in stress and feeding response. Promoter-specific DNA methylation of the POMC gene was determined in peripheral blood mononuclear cells of 54 healthy female control subjects, 40 underweight patients with AN, and 21 weight-restored patients with AN using bisulfite sequencing. Malnutrition was characterized by plasma leptin. POMC promoter-specific DNA methylation was not affected by diagnosis or nutritional status but significantly negatively associated with cigarette smoking. Although malnutrition may be expected to reduce DNA methylation through its effects on one-carbon metabolism, our negative results are in line with several in vitro and clinical studies that did not show a direct relation between gene-specific DNA methylation and folate levels. In contrast, smoking has been repeatedly reported to alter DNA methylation of specific genes and should be controlled for in future epigenetic studies.

Effect of dactyloscopic powders on DNA profiling from enhanced fingerprints: results from an experimental study.

PubMed

Tozzo, Pamela; Giuliodori, Alice; Rodriguez, Daniele; Caenazzo, Luciana

2014-03-01

We conducted a study on the effect of fingerprint enhancement methods on subsequent short tandem repeat profiling. First, we performed a study typing blood traces deposited on 5 different surfaces, treated with 8 types of dactyloscopic powders. Three different DNA extraction methods were used. Subsequently, we analyzed latent fingerprints on the same 5 surfaces enhanced with the 8 different powders used in the first part of the study. This study has demonstrated that DNA profiling can be performed on fingerprints left on different substrates, and the substrate will affect the amount of DNA that can be recovered for DNA typing. In the first phase of the study, a profile was obtained in 92% of the 120 samples analyzed; in the second part, in 55% of the 80 samples analyzed, we obtained a profile complete in 32.5% of the cases. From the results obtained, it seems that the powders used in latent fingerprints enhancement, rather than having a direct inhibitory effect on extraction and amplification of DNA, may cause partial degradation of DNA, reducing the efficiency of amplification reaction. It should not be forgotten that these results were obtained under laboratory conditions, and in real caseworks, there may still be different problems involved.

Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers

PubMed Central

Udugama, Maheshi; Sanij, Elaine; Voon, Hsiao P. J.; Son, Jinbae; Hii, Linda; Henson, Jeremy D.; Chan, F. Lyn; Chang, Fiona T. M.; Liu, Yumei; Pearson, Richard B.; Kalitsis, Paul; Mann, Jeffrey R.; Collas, Philippe; Hannan, Ross D.; Wong, Lee H.

2018-01-01

ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers. PMID:29669917

Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beloglazova, Natalia; Petit, Pierre; Flick, Robert

Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two boundmore » metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities.« less

DNA methylation polymorphism in a set of elite rice cultivars and its possible contribution to inter-cultivar differential gene expression.

PubMed

Wang, Yongming; Lin, Xiuyun; Dong, Bo; Wang, Yingdian; Liu, Bao

2004-01-01

RAPD (randomly amplified polymorphic DNA) and ISSR (inter-simple sequence repeat) fingerprinting on HpaII/MspI-digested genomic DNA of nine elite japonica rice cultivars implies inter-cultivar DNA methylation polymorphism. Using both DNA fragments isolated from RAPD or ISSR gels and selected low-copy sequences as probes, methylation-sensitive Southern blot analysis confirms the existence of extensive DNA methylation polymorphism in both genes and DNA repeats among the rice cultivars. The cultivar-specific methylation patterns are stably maintained, and can be used as reliable molecular markers. Transcriptional analysis of four selected sequences (RdRP, AC9, HSP90 and MMR) on leaves and roots from normal and 5-azacytidine-treated seedlings of three representative cultivars shows an association between the transcriptional activity of one of the genes, the mismatch repair (MMR) gene, and its CG methylation patterns.

Biological sequence compression algorithms.

PubMed

Matsumoto, T; Sadakane, K; Imai, H

2000-01-01

Today, more and more DNA sequences are becoming available. The information about DNA sequences are stored in molecular biology databases. The size and importance of these databases will be bigger and bigger in the future, therefore this information must be stored or communicated efficiently. Furthermore, sequence compression can be used to define similarities between biological sequences. The standard compression algorithms such as gzip or compress cannot compress DNA sequences, but only expand them in size. On the other hand, CTW (Context Tree Weighting Method) can compress DNA sequences less than two bits per symbol. These algorithms do not use special structures of biological sequences. Two characteristic structures of DNA sequences are known. One is called palindromes or reverse complements and the other structure is approximate repeats. Several specific algorithms for DNA sequences that use these structures can compress them less than two bits per symbol. In this paper, we improve the CTW so that characteristic structures of DNA sequences are available. Before encoding the next symbol, the algorithm searches an approximate repeat and palindrome using hash and dynamic programming. If there is a palindrome or an approximate repeat with enough length then our algorithm represents it with length and distance. By using this preprocessing, a new program achieves a little higher compression ratio than that of existing DNA-oriented compression algorithms. We also describe new compression algorithm for protein sequences.

The fission yeast CENP-B protein Abp1 prevents pervasive transcription of repetitive DNA elements.

PubMed

Daulny, Anne; Mejía-Ramírez, Eva; Reina, Oscar; Rosado-Lugo, Jesus; Aguilar-Arnal, Lorena; Auer, Herbert; Zaratiegui, Mikel; Azorin, Fernando

2016-10-01

It is well established that eukaryotic genomes are pervasively transcribed producing cryptic unstable transcripts (CUTs). However, the mechanisms regulating pervasive transcription are not well understood. Here, we report that the fission yeast CENP-B homolog Abp1 plays an important role in preventing pervasive transcription. We show that loss of abp1 results in the accumulation of CUTs, which are targeted for degradation by the exosome pathway. These CUTs originate from different types of genomic features, but the highest increase corresponds to Tf2 retrotransposons and rDNA repeats, where they map along the entire elements. In the absence of abp1, increased RNAPII-Ser5P occupancy is observed throughout the Tf2 coding region and, unexpectedly, RNAPII-Ser5P is enriched at rDNA repeats. Loss of abp1 also results in Tf2 derepression and increased nucleolus size. Altogether these results suggest that Abp1 prevents pervasive RNAPII transcription of repetitive DNA elements (i.e., Tf2 and rDNA repeats) from internal cryptic sites. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing.

PubMed

Saveliev, Alexander; Everett, Christopher; Sharpe, Tammy; Webster, Zoë; Festenstein, Richard

2003-04-24

Gene repression is crucial to the maintenance of differentiated cell types in multicellular organisms, whereas aberrant silencing can lead to disease. The organization of DNA into chromatin and heterochromatin is implicated in gene silencing. In chromatin, DNA wraps around histones, creating nucleosomes. Further condensation of chromatin, associated with large blocks of repetitive DNA sequences, is known as heterochromatin. Position effect variegation (PEV) occurs when a gene is located abnormally close to heterochromatin, silencing the affected gene in a proportion of cells. Here we show that the relatively short triplet-repeat expansions found in myotonic dystrophy and Friedreich's ataxia confer variegation of expression on a linked transgene in mice. Silencing was correlated with a decrease in promoter accessibility and was enhanced by the classical PEV modifier heterochromatin protein 1 (HP1). Notably, triplet-repeat-associated variegation was not restricted to classical heterochromatic regions but occurred irrespective of chromosomal location. Because the phenomenon described here shares important features with PEV, the mechanisms underlying heterochromatin-mediated silencing might have a role in gene regulation at many sites throughout the mammalian genome and modulate the extent of gene silencing and hence severity in several triplet-repeat diseases.

[Tissue-specific Changes in the Polymorphism of Simple Repeats in DNA of the Offspring of Different Sex Born from Irradiated Male or Female Mice].

PubMed

Lomaeva, M G; Fomenko, L A; Vasil'eva, G V; Bezlepkin, V G

2016-01-01

Evidence is presented indicating the differences in the polymorphism of microsatellite (MCS) repeats in DNA of somatic tissues in the offspring of BALB/c mice of different sex born from preconceptionally irradiated males or females. Brother-sister groups of the offspring born by non-irradiated parental pairs were compared with the offspring obtained after the irradiation of one parent in the same pairs. The number of MCS repeats in DNA of somatic tissues of the offspring from irradiated males or females was compared by a polymerase chain reaction using an arbitrary primer. It was found that changes in the polymorphism of the number of MCS repeats in the offspring from the males irradiated at a dose of 2 Gy was insignificant as compared with the offspring from control animals. In the offspring born by the females irradiated at a dose of 2 Gy (which does not impair the reproductive capacity), a statistically significant increase in the polymorphism was observed. Changes in the polymorphism were different in the offspring of different sex. A higher level of polymorphism was revealed in the female offspring born from the females of the F0 generation after their irradiation at a dose of 2 Gy. The increase in the polymorphism of the number of MCS repeats in DNA was more pronounced in postmitotic tissues compared with proliferating tissues.

Crosstalk between MSH2-MSH3 and polβ promotes trinucleotide repeat expansion during base excision repair.

PubMed

Lai, Yanhao; Budworth, Helen; Beaver, Jill M; Chan, Nelson L S; Zhang, Zunzhen; McMurray, Cynthia T; Liu, Yuan

2016-08-22

Studies in knockout mice provide evidence that MSH2-MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2-MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2-MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion.

Crosstalk between MSH2–MSH3 and polβ promotes trinucleotide repeat expansion during base excision repair

PubMed Central

Lai, Yanhao; Budworth, Helen; Beaver, Jill M.; Chan, Nelson L. S.; Zhang, Zunzhen; McMurray, Cynthia T.; Liu, Yuan

2016-01-01

Studies in knockout mice provide evidence that MSH2–MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2–MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2–MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion. PMID:27546332

The repeating nucleotide sequence in the repetitive mitochondrial DNA from a "low-density" petite mutant of yeast.

PubMed Central

Van Kreijl, C F; Bos, J L

1977-01-01

The repeating nucleotide sequence of 68 base pairs in the mtDNA from an ethidium-induced cytoplasmic petite mutant of yeast has been determined. For sequence analysis specifically primed and terminated RNA copies, obtained by in vitro transcription of the separated strands, were use. The sequence consists of 66 consecutive AT base pairs flanked by two GC pairs and comprises nearly all of the mutant mitochondrial genome. The sequence, moreover, also represents the first part of wild-type mtDNA sequence so far. Images PMID:198740

DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases.

PubMed

Bettencourt, Conceição; Hensman-Moss, Davina; Flower, Michael; Wiethoff, Sarah; Brice, Alexis; Goizet, Cyril; Stevanin, Giovanni; Koutsis, Georgios; Karadima, Georgia; Panas, Marios; Yescas-Gómez, Petra; García-Velázquez, Lizbeth Esmeralda; Alonso-Vilatela, María Elisa; Lima, Manuela; Raposo, Mafalda; Traynor, Bryan; Sweeney, Mary; Wood, Nicholas; Giunti, Paola; Durr, Alexandra; Holmans, Peter; Houlden, Henry; Tabrizi, Sarah J; Jones, Lesley

2016-06-01

The polyglutamine diseases, including Huntington's disease (HD) and multiple spinocerebellar ataxias (SCAs), are among the commonest hereditary neurodegenerative diseases. They are caused by expanded CAG tracts, encoding glutamine, in different genes. Longer CAG repeat tracts are associated with earlier ages at onset, but this does not account for all of the difference, and the existence of additional genetic modifying factors has been suggested in these diseases. A recent genome-wide association study (GWAS) in HD found association between age at onset and genetic variants in DNA repair pathways, and we therefore tested whether the modifying effects of variants in DNA repair genes have wider effects in the polyglutamine diseases. We assembled an independent cohort of 1,462 subjects with HD and polyglutamine SCAs, and genotyped single-nucleotide polymorphisms (SNPs) selected from the most significant hits in the HD study. In the analysis of DNA repair genes as a group, we found the most significant association with age at onset when grouping all polyglutamine diseases (HD+SCAs; p = 1.43 × 10(-5) ). In individual SNP analysis, we found significant associations for rs3512 in FAN1 with HD+SCAs (p = 1.52 × 10(-5) ) and all SCAs (p = 2.22 × 10(-4) ) and rs1805323 in PMS2 with HD+SCAs (p = 3.14 × 10(-5) ), all in the same direction as in the HD GWAS. We show that DNA repair genes significantly modify age at onset in HD and SCAs, suggesting a common pathogenic mechanism, which could operate through the observed somatic expansion of repeats that can be modulated by genetic manipulation of DNA repair in disease models. This offers novel therapeutic opportunities in multiple diseases. Ann Neurol 2016;79:983-990. © 2016 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.

DNA CTG triplet repeats involved in dynamic mutations of neurologically related gene sequences form stable duplexes

NASA Technical Reports Server (NTRS)

Smith, G. K.; Jie, J.; Fox, G. E.; Gao, X.

1995-01-01

DNA triplet repeats, 5'-d(CTG)n and 5'-d(CAG)n, are present in genes which have been implicated in several neurodegenerative disorders. To investigate possible stable structures formed by these repeating sequences, we have examined d(CTG)n, d(CAG)n and d(CTG).d(CAG)n (n = 2 and 3) using NMR and UV optical spectroscopy. These studies reveal that single stranded (CTG)n (n > 2) forms stable, antiparallel helical duplexes, while the single stranded (CAG)n requires at least three repeating units to form a duplex. NMR and UV melting experiments show that the Tm increases in the order of [(CAG)3]2 < [(CTG)3]2 << (CAG)3.(CTG)3. The (CTG)3 duplex is stable and exhibits similar NMR spectra in solutions containing 0.1-4 M NaCl and at a pH range from 4.6 to 8.8. The (CTG)3 duplex, which contains multiple-T.T mismatches, displays many NMR spectral characteristics similar to those of B-form DNA. However, unique NOE and 1H-31P coupling patterns associated with the repetitive T.T mismatches in the CTG repeats are discerned. These results, in conjunction with recent in vitro studies suggest that longer CTG repeats may form hairpin structures, which can potentially cause interruption in replication, leading to dynamic expansion or deletion of triplet repeats.

Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.

PubMed Central

Pietras, D F; Bennett, K L; Siracusa, L D; Woodworth-Gutai, M; Chapman, V M; Gross, K W; Kane-Haas, C; Hastie, N D

1983-01-01

We report the construction of a small library of recombinant plasmids containing Mus musculus repetitive DNA inserts. The repetitive cloned fraction was derived from denatured genomic DNA by reassociation to a Cot value at which repetitive, but not unique, sequences have reannealed followed by exhaustive S1 nuclease treatment to degrade single stranded DNA. Initial characterizations of this library by colony filter hybridizations have led to the identification of a previously undetected M. musculus minor satellite as well as to clones containing M. musculus major satellite sequences. This new satellite is repeated 10-20 times less than the major satellite in the M. musculus genome. It has a repeat length of 130 nucleotides compared with the M. musculus major satellite with a repeat length of 234 nucleotides. Sequence analysis of the minor satellite has shown that it has a 29 base pair region with extensive homology to one of the major satellite repeating subunits. We also show by in situ hybridization that this minor satellite sequence is located at the centromeres and possibly the arms of at least half the M musculus chromosomes. Sequences related to the minor satellite have been found in the DNA of a related Mus species, Mus spretus, and may represent the major satellite of that species. Images PMID:6314268

Universal strategies for the DNA-encoding of libraries of small molecules using the chemical ligation of oligonucleotide tags

PubMed Central

Litovchick, Alexander; Clark, Matthew A; Keefe, Anthony D

2014-01-01

The affinity-mediated selection of large libraries of DNA-encoded small molecules is increasingly being used to initiate drug discovery programs. We present universal methods for the encoding of such libraries using the chemical ligation of oligonucleotides. These methods may be used to record the chemical history of individual library members during combinatorial synthesis processes. We demonstrate three different chemical ligation methods as examples of information recording processes (writing) for such libraries and two different cDNA-generation methods as examples of information retrieval processes (reading) from such libraries. The example writing methods include uncatalyzed and Cu(I)-catalyzed alkyne-azide cycloadditions and a novel photochemical thymidine-psoralen cycloaddition. The first reading method “relay primer-dependent bypass” utilizes a relay primer that hybridizes across a chemical ligation junction embedded in a fixed-sequence and is extended at its 3′-terminus prior to ligation to adjacent oligonucleotides. The second reading method “repeat-dependent bypass” utilizes chemical ligation junctions that are flanked by repeated sequences. The upstream repeat is copied prior to a rearrangement event during which the 3′-terminus of the cDNA hybridizes to the downstream repeat and polymerization continues. In principle these reading methods may be used with any ligation chemistry and offer universal strategies for the encoding (writing) and interpretation (reading) of DNA-encoded chemical libraries. PMID:25483841

Homology-dependent repair is involved in 45S rDNA loss in plant CAF-1 mutants

PubMed Central

Muchová, Veronika; Amiard, Simon; Mozgová, Iva; Dvořáčková, Martina; Gallego, Maria E; White, Charles; Fajkus, Jiří

2015-01-01

Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability. PMID:25359579

Loop-mediated isothermal amplification assay for detection and discrimination of Toxocara canis and Toxocara cati eggs directly from sand samples.

PubMed

Macuhova, K; Kumagai, T; Akao, N; Ohta, N

2010-12-01

We developed a novel and simple method, using loop-mediated isothermal amplification (LAMP), for the detection and discrimination of Toxocara canis and Toxocara cati eggs. The new method employs 4 steps: (1) concentration of Toxocara eggs in a small amount of sand; (2) dissolution of the proteinaceous membrane of eggs and simultaneously separation of them from the sand using NaClO treatment; (3) extraction of DNA using NaOH treatment; and (4) detection of T. canis / T. cati DNA using a LAMP assay. All these steps are fast, easy to perform, and do not require expensive equipment or reagents. The novel method was tested both experimentally and in a field study. In the laboratory, we could reliably detect as few as 3 T. canis eggs in artificially contaminated sand, if the experiment was repeated twice. In the field trial, we were able to detect T. cati DNA from 4 natural sandpits having moderate to heavy contamination, although not in a single lightly contaminated sandpit. All of the examined sandpits were found to be contaminated with eggs of T. cati, but none appeared to contain T. canis. Our new method could extract DNA from T. canis and T. cati eggs directly from sand samples as well as detect and distinguish these 2 species in a few easy steps, with markedly reduced time and expense.

ATP hydrolysis provides functions that promote rejection of pairings between different copies of long repeated sequences

PubMed Central

Danilowicz, Claudia; Hermans, Laura; Coljee, Vincent; Prévost, Chantal

2017-01-01

Abstract During DNA recombination and repair, RecA family proteins must promote rapid joining of homologous DNA. Repeated sequences with >100 base pair lengths occupy more than 1% of bacterial genomes; however, commitment to strand exchange was believed to occur after testing ∼20–30 bp. If that were true, pairings between different copies of long repeated sequences would usually become irreversible. Our experiments reveal that in the presence of ATP hydrolysis even 75 bp sequence-matched strand exchange products remain quite reversible. Experiments also indicate that when ATP hydrolysis is present, flanking heterologous dsDNA regions increase the reversibility of sequence matched strand exchange products with lengths up to ∼75 bp. Results of molecular dynamics simulations provide insight into how ATP hydrolysis destabilizes strand exchange products. These results inspired a model that shows how pairings between long repeated sequences could be efficiently rejected even though most homologous pairings form irreversible products. PMID:28854739

Diversity of the Arabidopsis mitochondrial genome occurs via nuclear-controlled recombination activity.

PubMed

Arrieta-Montiel, Maria P; Shedge, Vikas; Davila, Jaime; Christensen, Alan C; Mackenzie, Sally A

2009-12-01

The plant mitochondrial genome is recombinogenic, with DNA exchange activity controlled to a large extent by nuclear gene products. One nuclear gene, MSH1, appears to participate in suppressing recombination in Arabidopsis at every repeated sequence ranging in size from 108 to 556 bp. Present in a wide range of plant species, these mitochondrial repeats display evidence of successful asymmetric DNA exchange in Arabidopsis when MSH1 is disrupted. Recombination frequency appears to be influenced by repeat sequence homology and size, with larger size repeats corresponding to increased DNA exchange activity. The extensive mitochondrial genomic reorganization of the msh1 mutant produced altered mitochondrial transcription patterns. Comparison of mitochondrial genomes from the Arabidopsis ecotypes C24, Col-0, and Ler suggests that MSH1 activity accounts for most or all of the polymorphisms distinguishing these genomes, producing ecotype-specific stoichiometric changes in each line. Our observations suggest that MSH1 participates in mitochondrial genome evolution by influencing the lineage-specific pattern of mitochondrial genetic variation in higher plants.

Plasmid P1 replication: negative control by repeated DNA sequences.

PubMed Central

Chattoraj, D; Cordes, K; Abeles, A

1984-01-01

The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pieces of the incA fragment that each contain only three repeats destabilize P1 plasmids efficiently. This result makes it unlikely that incA specifies a regulatory product. Our in vivo results suggest that the repeating DNA sequence itself negatively controls replication by titrating a P1-determined protein, RepA, that is essential for replication. Consistent with this hypothesis is the observation that the RepA protein binds to the incA fragment in vitro. Images PMID:6387706

The NnCenH3 protein and centromeric DNA sequence profiles of Nelumbo nucifera Gaertn. (sacred lotus) reveal the DNA structures and dynamics of centromeres in basal eudicots.

PubMed

Zhu, Zhixuan; Gui, Songtao; Jin, Jing; Yi, Rong; Wu, Zhihua; Qian, Qian; Ding, Yi

2016-09-01

Centromeres on eukaryotic chromosomes consist of large arrays of DNA repeats that undergo very rapid evolution. Nelumbo nucifera Gaertn. (sacred lotus) is a phylogenetic relict and an aquatic perennial basal eudicot. Studies concerning the centromeres of this basal eudicot species could provide ancient evolutionary perspectives. In this study, we characterized the centromeric marker protein NnCenH3 (sacred lotus centromere-specific histone H3 variant), and used a chromatin immunoprecipitation (ChIP)-based technique to recover the NnCenH3 nucleosome-associated sequences of sacred lotus. The properties of the centromere-binding protein and DNA sequences revealed notable divergence between sacred lotus and other flowering plants, including the following factors: (i) an NnCenH3 alternative splicing variant comprising only a partial centromere-targeting domain, (ii) active genes with low transcription levels in the NnCenH3 nucleosomal regions, and (iii) the prevalence of the Ty1/copia class of long terminal repeat (LTR) retrotransposons in the centromeres of sacred lotus chromosomes. In addition, the dynamic natures of the centromeric region showed that some of the centromeric repeat DNA sequences originated from telomeric repeats, and a pair of centromeres on the dicentric chromosome 1 was inactive in the metaphase cells of sacred lotus. Our characterization of the properties of centromeric DNA structure within the sacred lotus genome describes a centromeric profile in ancient basal eudicots and might provide evidence of the origins and evolution of centromeres. Furthermore, the identification of centromeric DNA sequences is of great significance for the assembly of the sacred lotus genome. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Structural analysis of two length variants of the rDNA intergenic spacer from Eruca sativa.

PubMed

Lakshmikumaran, M; Negi, M S

1994-03-01

Restriction enzyme analysis of the rRNA genes of Eruca sativa indicated the presence of many length variants within a single plant and also between different cultivars which is unusual for most crucifers studied so far. Two length variants of the rDNA intergenic spacer (IGS) from a single individual E. sativa (cv. Itsa) plant were cloned and characterized. The complete nucleotide sequences of both the variants (3 kb and 4 kb) were determined. The intergenic spacer contains three families of tandemly repeated DNA sequences denoted as A, B and C. However, the long (4 kb) variant shows the presence of an additional repeat, denoted as D, which is a duplication of a 224 bp sequence just upstream of the putative transcription initiation site. Repeat units belonging to the three different families (A, B and C) were in the size range of 22 to 30 bp. Such short repeat elements are present in the IGS of most of the crucifers analysed so far. Sequence analysis of the variants (3 kb and 4 kb) revealed that the length heterogeneity of the spacer is located at three different regions and is due to the varying copy numbers of repeat units belonging to families A and B. Length variation of the spacer is also due to the presence of a large duplication (D repeats) in the 4 kb variant which is absent in the 3 kb variant. The putative transcription initiation site was identified by comparisons with the rDNA sequences from other plant species.

Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification.

PubMed

Li, Lixin; Piatek, Marek J; Atef, Ahmed; Piatek, Agnieszka; Wibowo, Anjar; Fang, Xiaoyun; Sabir, J S M; Zhu, Jian-Kang; Mahfouz, Magdy M

2012-03-01

Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants.

Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

DOEpatents

Studier, F. William

1995-04-18

Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient.

Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

DOEpatents

Studier, F.W.

1995-04-18

Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient. 2 figs.

A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction.

PubMed

Han, Wenyuan; Li, Yingjun; Deng, Ling; Feng, Mingxia; Peng, Wenfang; Hallstrøm, Søren; Zhang, Jing; Peng, Nan; Liang, Yun Xiang; White, Malcolm F; She, Qunxin

2017-02-28

The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr-α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr-α from S. islandicus and characterized it in vitro. Cmr-α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5΄-tag of crRNA and the 3΄-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr-2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr-α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Developmental validation of a Cannabis sativa STR multiplex system for forensic analysis.

PubMed

Howard, Christopher; Gilmore, Simon; Robertson, James; Peakall, Rod

2008-09-01

A developmental validation study based on recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM) was conducted on a multiplex system of 10 Cannabis sativa short tandem repeat loci. Amplification of the loci in four multiplex reactions was tested across DNA from dried root, stem, and leaf sources, and DNA from fresh, frozen, and dried leaf tissue with a template DNA range of 10.0-0.01 ng. The loci were amplified and scored consistently for all DNA sources when DNA template was in the range of 10.0-1.0 ng. Some allelic dropout and PCR failure occurred in reactions with lower template DNA amounts. Overall, amplification was best using 10.0 ng of template DNA from dried leaf tissue indicating that this is the optimal source material. Cross species amplification was observed in Humulus lupulus for three loci but there was no allelic overlap. This is the first study following SWGDAM validation guidelines to validate short tandem repeat markers for forensic use in plants.

Nuclease-mediated genome editing: At the front-line of functional genomics technology.

PubMed

Sakuma, Tetsushi; Woltjen, Knut

2014-01-01

Genome editing with engineered endonucleases is rapidly becoming a staple method in developmental biology studies. Engineered nucleases permit random or designed genomic modification at precise loci through the stimulation of endogenous double-strand break repair. Homology-directed repair following targeted DNA damage is mediated by co-introduction of a custom repair template, allowing the derivation of knock-out and knock-in alleles in animal models previously refractory to classic gene targeting procedures. Currently there are three main types of customizable site-specific nucleases delineated by the source mechanism of DNA binding that guides nuclease activity to a genomic target: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Among these genome engineering tools, characteristics such as the ease of design and construction, mechanism of inducing DNA damage, and DNA sequence specificity all differ, making their application complementary. By understanding the advantages and disadvantages of each method, one may make the best choice for their particular purpose. © 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.

Evidence for horizontal transfer of mitochondrial DNA to the plastid genome in a bamboo genus.

PubMed

Ma, Peng-Fei; Zhang, Yu-Xiao; Guo, Zhen-Hua; Li, De-Zhu

2015-06-23

In flowering plants, three genomes (nuclear, mitochondrial, and plastid) coexist and intracellular horizontal transfer of DNA is prevalent, especially from the plastid to the mitochondrion genome. However, the plastid genomes are generally conserved in evolution and have long been considered immune to foreign DNA. Recently, the opposite direction of DNA transfer from the mitochondrial to the plastid genome has been reported in two eudicot lineages. Here we sequenced 6 plastid genomes of bamboos, three of which are neotropical woody species and three are herbaceous ones. Several unusual features were found, including the duplication of trnT-GGU and loss of one copy of rps19 due to contraction of inverted repeats (IRs). The most intriguing was the ~2.7 kb insertion in the plastid IR regions in the three herbaceous bamboos. Furthermore, the insertion was documented to be horizontally transferred from the mitochondrial to the plastid genome. Our study provided evidence of the mitochondrial-to-plastid DNA transfer in the monocots, demonstrating again that this rare event does occur in other angiosperm lineages. However, the mechanism underlying the transfer remains obscure, and more studies in other plants may elucidate it in the future.

Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae.

PubMed

Kaniak-Golik, Aneta; Kuberska, Renata; Dzierzbicki, Piotr; Sledziewska-Gojska, Ewa

2017-01-01

Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27 activity in mitochondria.

Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae

PubMed Central

Dzierzbicki, Piotr

2017-01-01

Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27 activity in mitochondria. PMID:28678842

Site directed recombination

DOEpatents

Jurka, Jerzy W.

1997-01-01

Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

An examination of the origin and evolution of additional tandem repeats in the mitochondrial DNA control region of Japanese sika deer (Cervus Nippon).

PubMed

Ba, Hengxing; Wu, Lang; Liu, Zongyue; Li, Chunyi

2016-01-01

Tandem repeat units are only detected in the left domain of the mitochondrial DNA control region in sika deer. Previous studies showed that Japanese sika deer have more tandem repeat units than its cousins from the Asian continent and Taiwan, which often have only three repeat units. To determine the origin and evolution of these additional repeat units in Japanese sika deer, we obtained the sequence of repeat units from an expanded dataset of the control region from all sika deer lineages. The functional constraint is inferred to act on the first repeat unit because this repeat has the least sequence divergence in comparison to the other units. Based on slipped-strand mispairing mechanisms, the illegitimate elongation model could account for the addition or deletion of these additional repeat units in the Japanese sika deer population. We also report that these additional repeat units could be occurring in the internal positions of tandem repeat regions, possibly via coupling with a homogenization mechanism within and among these lineages. Moreover, the increased number of repeat units in the Japanese sika deer population could reflect a balance between mutation and selection, as well as genetic drift.

ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1.

PubMed

You, Zhongsheng; Chahwan, Charly; Bailis, Julie; Hunter, Tony; Russell, Paul

2005-07-01

ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Here we show that two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-beta. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. We conclude that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. We propose that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage.

The RTR Complex Partner RMI2 and the DNA Helicase RTEL1 Are Both Independently Involved in Preserving the Stability of 45S rDNA Repeats in Arabidopsis thaliana

PubMed Central

Knoll, Alexander; Puchta, Holger

2016-01-01

The stability of repetitive sequences in complex eukaryotic genomes is safeguarded by factors suppressing homologues recombination. Prominent in this is the role of the RTR complex. In plants, it consists of the RecQ helicase RECQ4A, the topoisomerase TOP3α and RMI1. Like mammals, but not yeast, plants harbor an additional complex partner, RMI2. Here, we demonstrate that, in Arabidopsis thaliana, RMI2 is involved in the repair of aberrant replication intermediates in root meristems as well as in intrastrand crosslink repair. In both instances, RMI2 is involved independently of the DNA helicase RTEL1. Surprisingly, simultaneous loss of RMI2 and RTEL1 leads to loss of male fertility. As both the RTR complex and RTEL1 are involved in suppression of homologous recombination (HR), we tested the efficiency of HR in the double mutant rmi2-2 rtel1-1 and found a synergistic enhancement (80-fold). Searching for natural target sequences we found that RTEL1 is required for stabilizing 45S rDNA repeats. In the double mutant with rmi2-2 the number of 45S rDNA repeats is further decreased sustaining independent roles of both factors in this process. Thus, loss of suppression of HR does not only lead to a destabilization of rDNA repeats but might be especially deleterious for tissues undergoing multiple cell divisions such as the male germline. PMID:27760121

The RTR Complex Partner RMI2 and the DNA Helicase RTEL1 Are Both Independently Involved in Preserving the Stability of 45S rDNA Repeats in Arabidopsis thaliana.

PubMed

Röhrig, Sarah; Schröpfer, Susan; Knoll, Alexander; Puchta, Holger

2016-10-01

The stability of repetitive sequences in complex eukaryotic genomes is safeguarded by factors suppressing homologues recombination. Prominent in this is the role of the RTR complex. In plants, it consists of the RecQ helicase RECQ4A, the topoisomerase TOP3α and RMI1. Like mammals, but not yeast, plants harbor an additional complex partner, RMI2. Here, we demonstrate that, in Arabidopsis thaliana, RMI2 is involved in the repair of aberrant replication intermediates in root meristems as well as in intrastrand crosslink repair. In both instances, RMI2 is involved independently of the DNA helicase RTEL1. Surprisingly, simultaneous loss of RMI2 and RTEL1 leads to loss of male fertility. As both the RTR complex and RTEL1 are involved in suppression of homologous recombination (HR), we tested the efficiency of HR in the double mutant rmi2-2 rtel1-1 and found a synergistic enhancement (80-fold). Searching for natural target sequences we found that RTEL1 is required for stabilizing 45S rDNA repeats. In the double mutant with rmi2-2 the number of 45S rDNA repeats is further decreased sustaining independent roles of both factors in this process. Thus, loss of suppression of HR does not only lead to a destabilization of rDNA repeats but might be especially deleterious for tissues undergoing multiple cell divisions such as the male germline.

Effect of CpG dinucleotides within IgH switch region repeats on immunoglobulin class switch recombination.

PubMed

Zhang, Zheng Z; Hsieh, Chih-Lin; Okitsu, Cindy Yen; Han, Li; Yu, Kefei; Lieber, Michael R

2015-08-01

Immunoglobulin (Ig) heavy chains undergo class switch recombination (CSR) to change the heavy chain isotype from IgM to IgG, A or E. The switch regions are several kilobases long, repetitive, and G-rich on the nontemplate strand. They are also relatively depleted of CpG (also called CG) sites for unknown reasons. Here we use synthetic switch regions at the IgH switch alpha (Sα) locus to test the effect of CpG sites and to try to understand why the IgH switch sequences evolved to be relatively depleted of CpG. We find that even just two CpG sites within an 80 bp synthetic switch repeat iterated 15 times (total switch region length of 1200 bp containing 30 CpG sites) are sufficient to dramatically reduce both Ig CSR and transcription through the switch region from the upstream Iα sterile transcript promoter, which is the promoter that directs transcripts through the Sα region. De novo DNA methylation occurs at the four CpG sites in and around the Iα promoter when each 80 bp Iα switch repeat contains the two CpG sites. Thus, a relatively low density of CpG sites within the switch repeats can induce upstream CpG methylation at the IgH alpha locus, and cause a substantial decrease in transcription from the sterile transcript promoter. This effect is likely the reason that switch regions evolved to contain very few CpG sites. We discuss these findings as they relate to DNA methylation and to Ig CSR. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Comparative genomics and evolutionary analysis of CRISPR loci in acetic acid bacteria].

PubMed

Xia, Kai; Liang, Xin-le; Li, Yu-dong

2015-12-01

The clustered regularly interspaced short palindromic repeat (CRISPR) is a widespread adaptive immunity system that exists in most archaea and many bacteria against foreign DNA, such as phages, viruses and plasmids. In general, CRISPR system consists of direct repeat, leader, spacer and CRISPR-associated sequences. Acetic acid bacteria (AAB) play an important role in industrial fermentation of vinegar and bioelectrochemistry. To investigate the polymorphism and evolution pattern of CRISPR loci in acetic acid bacteria, bioinformatic analyses were performed on 48 species from three main genera (Acetobacter, Gluconacetobacter and Gluconobacter) with whole genome sequences available from the NCBI database. The results showed that the CRISPR system existed in 32 species of the 48 strains studied. Most of the CRISPR-Cas system in AAB belonged to type I CRISPR-Cas system (subtype E and C), but type II CRISPR-Cas system which contain cas9 gene was only found in the genus Acetobacter and Gluconacetobacter. The repeat sequences of some CRISPR were highly conserved among species from different genera, and the leader sequences of some CRISPR possessed conservative motif, which was associated with regulated promoters. Moreover, phylogenetic analysis of cas1 demonstrated that they were suitable for classification of species. The conservation of cas1 genes was associated with that of repeat sequences among different strains, suggesting they were subjected to similar functional constraints. Moreover, the number of spacer was positively correlated with the number of prophages and insertion sequences, indicating the acetic acid bacteria were continually invaded by new foreign DNA. The comparative analysis of CRISR loci in acetic acid bacteria provided the basis for investigating the molecular mechanism of different acetic acid tolerance and genome stability in acetic acid bacteria.

The substrate binding interface of alkylpurine DNA glycosylase AlkD.

PubMed

Mullins, Elwood A; Rubinson, Emily H; Eichman, Brandt F

2014-01-01

Tandem helical repeats have emerged as an important DNA binding architecture. DNA glycosylase AlkD, which excises N3- and N7-alkylated nucleobases, uses repeating helical motifs to bind duplex DNA and to selectively pause at non-Watson-Crick base pairs. Remodeling of the DNA backbone promotes nucleotide flipping of the lesion and the complementary base into the solvent and toward the protein surface, respectively. The important features of this new DNA binding architecture that allow AlkD to distinguish between damaged and normal DNA without contacting the lesion are poorly understood. Here, we show through extensive mutational analysis that DNA binding and N3-methyladenine (3mA) and N7-methylguanine (7mG) excision are dependent upon each residue lining the DNA binding interface. Disrupting electrostatic or hydrophobic interactions with the DNA backbone substantially reduced binding affinity and catalytic activity. These results demonstrate that residues seemingly only involved in general DNA binding are important for catalytic activity and imply that base excision is driven by binding energy provided by the entire substrate interface of this novel DNA binding architecture. Copyright © 2013 Elsevier B.V. All rights reserved.

Application of FTA sample collection and DNA purification system on the determination of CTG trinucleotide repeat size by PCR-based Southern blotting.

PubMed

Hsiao, K M; Lin, H M; Pan, H; Li, T C; Chen, S S; Jou, S B; Chiu, Y L; Wu, M F; Lin, C C; Li, S Y

1999-01-01

Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein kinase gene. The clinical severity of this disease correlates with the length of the CTG trinucleotide repeats. Determination of the CTG repeat length has been primarily relied on by Southern blot analysis of restriction enzyme-digested genomic DNA. The development of PCR-based Southern blotting methodology provides a much more sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the use of PCR on the diagnosis of DM. A modified PCR protocol to amplify different lengths of CTG repeat region using various concentrations of 7deaza-dGTP has been reported (1). Here we describe a procedure including sample collection, DNA purification, and PCR analysis of CTG repeat length without using 7-deaza-dGTP. This protocol is very sensitive and convenient because only a small number of nucleate cells are needed for detection of CTG expansion. Therefore, it could be very useful in clinical and prenatal diagnosis as well as in prevalence study of DM.

Tandem repeats of the 5' non-transcribed spacer of Tetrahymena rDNA function as high copy number autonomous replicons in the macronucleus but do not prevent rRNA gene dosage regulation.

PubMed Central

Pan, W J; Blackburn, E H

1995-01-01

The rRNA genes in the somatic macronucleus of Tetrahymena thermophila are normally on 21 kb linear palindromic molecules (rDNA). We examined the effect on rRNA gene dosage of transforming T.thermophila macronuclei with plasmid constructs containing a pair of tandemly repeated rDNA replication origin regions unlinked to the rRNA gene. A significant proportion of the plasmid sequences were maintained as high copy circular molecules, eventually consisting solely of tandem arrays of origin regions. As reported previously for cells transformed by a construct in which the same tandem rDNA origins were linked to the rRNA gene [Yu, G.-L. and Blackburn, E. H. (1990) Mol. Cell. Biol., 10, 2070-2080], origin sequences recombined to form linear molecules bearing several tandem repeats of the origin region, as well as rRNA genes. The total number of rDNA origin sequences eventually exceeded rRNA gene copies by approximately 20- to 40-fold and the number of circular replicons carrying only rDNA origin sequences exceeded rRNA gene copies by 2- to 3-fold. However, the rRNA gene dosage was unchanged. Hence, simply monitoring the total number of rDNA origin regions is not sufficient to regulate rRNA gene copy number. Images PMID:7784211

A novel activity of HMG domains: promotion of the triple-stranded complex formation between DNA containing (GGA/TCC)11 and d(GGA)11 oligonucleotides.

PubMed Central

Suda, T; Mishima, Y; Takayanagi, K; Asakura, H; Odani, S; Kominami, R

1996-01-01

The high mobility group protein (HMG)-box is a DNA-binding domain found in many proteins that bind preferentially to DNA of irregular structures in a sequence-independent manner and can bend the DNA. We show here that GST-fusion proteins of HMG domains from HMG1 and HMG2 promote a triple-stranded complex formation between DNA containing the (GGA/TCC)11 repeat and oligonucleotides of d(GGA)11 probably due to G:G base pairing. The activity is to reduce association time and requirements of Mg2+ and oligonucleotide concentrations. The HMG box of SRY, the protein determining male-sex differentiation, also has the activity, suggesting that it is not restricted to the HMG-box domains derived from HMG1/2 but is common to those from other members of the HMG-box family of proteins. Interestingly, the box-AB and box-B of HMG1 bend DNA containing the repeat, but SRY fails to bend in a circularization assay. The difference suggests that the two activities of association-promotion and DNA bending are distinct. These results suggest that the HMG-box domain has a novel activity of promoting the association between GGA repeats which might be involved in higher-order architecture of chromatin. PMID:8972860

[Length and structure of telomeric DNA in three species of Baikal gastropods (Caenogastropoda: Hydrobioidea: Benedictiidae)].

PubMed

Koroleva, A G; Evtushenko, E V; Maximova, N V; Vershinin, A V; Sintnikova, T Y; Kirilchik, S V

2015-03-01

The structure of telomeric repeat (TTAGGG)n was determined and the length of telomeric DNA (tDNA) was measured in three species of gastropods from the family Benedictiidae that are endemic to Lake Baikal. Fluorescence in situ hybridization (FISH) confirmed the localization of a telomeric repeat at the chromosome ends. The sizes of tDNA in "giant" eurybathic, psammo-pelobiontic species Benedictia fragilis and shallow water litho-psammobiontic species B. baicalensis with medium shell sizes were similar (16 ± 2.9 and 15 ± 2.1 kb, respectively), but they had a greater length than that of the shallow water spongio-litobiontic species Kobeltocochlea martensiana with small shells (10.5 ± 1.5 kb). We discuss tendencies in age-related changes in tDNA length in snails and a possible mechanism for maintaining tDNA size in ontogeny.

Asymmetric Epigenetic Modification and Elimination of rDNA Sequences by Polyploidization in Wheat[W

PubMed Central

Guo, Xiang

2014-01-01

rRNA genes consist of long tandem repeats clustered on chromosomes, and their products are important functional components of the ribosome. In common wheat (Triticum aestivum), rDNA loci from the A and D genomes were largely lost during the evolutionary process. This biased DNA elimination may be related to asymmetric transcription and epigenetic modifications caused by the polyploid formation. Here, we observed both sets of parental nucleolus organizing regions (NORs) were expressed after hybridization, but asymmetric silencing of one parental NOR was immediately induced by chromosome doubling, and reversing the ploidy status could not reactivate silenced NORs. Furthermore, increased CHG and CHH DNA methylation on promoters was accompanied by asymmetric silencing of NORs. Enrichment of H3K27me3 and H3K9me2 modifications was also observed to be a direct response to increased DNA methylation and transcriptional inactivation of NOR loci. Both A and D genome NOR loci with these modifications started to disappear in the S4 generation and were completely eliminated by the S7 generation in synthetic tetraploid wheat. Our results indicated that asymmetric epigenetic modification and elimination of rDNA sequences between different donor genomes may lead to stable allopolyploid wheat with increased differentiation and diversity. PMID:25415973

Asymmetric epigenetic modification and elimination of rDNA sequences by polyploidization in wheat.

PubMed

Guo, Xiang; Han, Fangpu

2014-11-01

rRNA genes consist of long tandem repeats clustered on chromosomes, and their products are important functional components of the ribosome. In common wheat (Triticum aestivum), rDNA loci from the A and D genomes were largely lost during the evolutionary process. This biased DNA elimination may be related to asymmetric transcription and epigenetic modifications caused by the polyploid formation. Here, we observed both sets of parental nucleolus organizing regions (NORs) were expressed after hybridization, but asymmetric silencing of one parental NOR was immediately induced by chromosome doubling, and reversing the ploidy status could not reactivate silenced NORs. Furthermore, increased CHG and CHH DNA methylation on promoters was accompanied by asymmetric silencing of NORs. Enrichment of H3K27me3 and H3K9me2 modifications was also observed to be a direct response to increased DNA methylation and transcriptional inactivation of NOR loci. Both A and D genome NOR loci with these modifications started to disappear in the S4 generation and were completely eliminated by the S7 generation in synthetic tetraploid wheat. Our results indicated that asymmetric epigenetic modification and elimination of rDNA sequences between different donor genomes may lead to stable allopolyploid wheat with increased differentiation and diversity. © 2014 American Society of Plant Biologists. All rights reserved.

[DNA prints instead of plantar prints in neonatal identification].

PubMed

Rodríguez-Alarcón Gómez, J; Martińez de Pancorbo Gómez, M; Santillana Ferrer, L; Castro Espido, A; Melchor Maros, J C; Linares Uribe, M A; Fernández-Llebrez del Rey, L; Aranguren Dúo, G

1996-06-22

To check the possible usefulness in studying DNA in dried blood spots taken on filter paper blotters for newborn identification. It set out to establish: 1. The validity of the method for analysis; 2. The validity of all stored samples (such as those kept in clinical records); 3. Guarantee of non-intrusion in the genetic code; 4. Acceptable price and execution time. Forty (40) anonymous 13-year-old samples of 20 subjects (2 per subject) were studied. DNA was extracted using Chelex resin and the STR ("small tandem repeat") of microsatellite DNA was studies using the "polimerase chain reaction method" (PCR). Three non coding DNA loci (CSF1PO, TPOX and THO1) were analyzed by Multiplex amplification. It was possible to type 39 samples, making it possible to match the 20 cases (one by exclusion). The complete procedure yielded the results within 24 hours in all cases. The estimated final cost was found to be a fifth of that conventional maternity/paternity tests. The study carried out made matching possible in all 20 cases (directly in 19 cases). It was not necessary to study DNA coding areas. The validity of the method for analyzing samples stored for 13 years without any special care was also demonstrated. The technic was fast, producing the results within 24 hours, and at reasonable cost.

Genetic analysis of 7 medieval skeletons from Aragonese Pyrenees

PubMed Central

Núńez, Carolina; Sosa, Cecilia; Baeta, Miriam; Geppert, Maria; Turnbough, Meredith; Phillips, Nicole; Casalod, Yolanda; Bolea, Miguel; Roby, Rhonda; Budowle, Bruce; Martínez-Jarreta, Begońa

2011-01-01

Aim To perform a genetic characterization of 7 skeletons from medieval age found in a burial site in the Aragonese Pyrenees. Methods Allele frequencies of autosomal short tandem repeats (STR) loci were determined by 3 different STR systems. Mitochondrial DNA (mtDNA) and Y-chromosome haplogroups were determined by sequencing of the hypervariable segment 1 of mtDNA and typing of phylogenetic Y chromosome single nucleotide polymorphisms (Y-SNP) markers, respectively. Possible familial relationships were also investigated. Results Complete or partial STR profiles were obtained in 3 of the 7 samples. Mitochondrial DNA haplogroup was determined in 6 samples, with 5 of them corresponding to the haplogroup H and 1 to the haplogroup U5a. Y-chromosome haplogroup was determined in 2 samples, corresponding to the haplogroup R. In one of them, the sub-branch R1b1b2 was determined. mtDNA sequences indicated that some of the individuals could be maternally related, while STR profiles indicated no direct family relationships. Conclusions Despite the antiquity of the samples and great difficulty that genetic analyses entail, the combined use of autosomal STR markers, Y-chromosome informative SNPs, and mtDNA sequences allowed us to genotype a group of skeletons from the medieval age. PMID:21674829

Structure of the Response Regulator PhoP from Mycobacterium tuberculosis Reveals a Dimer Through the Receiver Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Menon; S Wang

The PhoP protein from Mycobacterium tuberculosis is a response regulator of the OmpR/PhoB subfamily, whose structure consists of an N-terminal receiver domain and a C-terminal DNA-binding domain. How the DNA-binding activities are regulated by phosphorylation of the receiver domain remains unclear due to a lack of structural information on the full-length proteins. Here we report the crystal structure of the full-length PhoP of M. tuberculosis. Unlike other known structures of full-length proteins of the same subfamily, PhoP forms a dimer through its receiver domain with the dimer interface involving {alpha}4-{beta}5-{alpha}5, a common interface for activated receiver domain dimers. However, themore » switch residues, Thr99 and Tyr118, are in a conformation resembling those of nonactivated receiver domains. The Tyr118 side chain is involved in the dimer interface interactions. The receiver domain is tethered to the DNA-binding domain through a flexible linker and does not impose structural constraints on the DNA-binding domain. This structure suggests that phosphorylation likely facilitates/stabilizes receiver domain dimerization, bringing the DNA-binding domains to close proximity, thereby increasing their binding affinity for direct repeat DNA sequences.« less

Selfish DNA in protein-coding genes of Rickettsia.

PubMed

Ogata, H; Audic, S; Barbe, V; Artiguenave, F; Fournier, P E; Raoult, D; Claverie, J M

2000-10-13

Rickettsia conorii, the aetiological agent of Mediterranean spotted fever, is an intracellular bacterium transmitted by ticks. Preliminary analyses of the nearly complete genome sequence of R. conorii have revealed 44 occurrences of a previously undescribed palindromic repeat (150 base pairs long) throughout the genome. Unexpectedly, this repeat was found inserted in-frame within 19 different R. conorii open reading frames likely to encode functional proteins. We found the same repeat in proteins of other Rickettsia species. The finding of a mobile element inserted in many unrelated genes suggests the potential role of selfish DNA in the creation of new protein sequences.

Translocation and deletion breakpoints in cancer genomes are associated with potential non-B DNA-forming sequences.

PubMed

Bacolla, Albino; Tainer, John A; Vasquez, Karen M; Cooper, David N

2016-07-08

Gross chromosomal rearrangements (including translocations, deletions, insertions and duplications) are a hallmark of cancer genomes and often create oncogenic fusion genes. An obligate step in the generation of such gross rearrangements is the formation of DNA double-strand breaks (DSBs). Since the genomic distribution of rearrangement breakpoints is non-random, intrinsic cellular factors may predispose certain genomic regions to breakage. Notably, certain DNA sequences with the potential to fold into secondary structures [potential non-B DNA structures (PONDS); e.g. triplexes, quadruplexes, hairpin/cruciforms, Z-DNA and single-stranded looped-out structures with implications in DNA replication and transcription] can stimulate the formation of DNA DSBs. Here, we tested the postulate that these DNA sequences might be found at, or in close proximity to, rearrangement breakpoints. By analyzing the distribution of PONDS-forming sequences within ±500 bases of 19 947 translocation and 46 365 sequence-characterized deletion breakpoints in cancer genomes, we find significant association between PONDS-forming repeats and cancer breakpoints. Specifically, (AT)n, (GAA)n and (GAAA)n constitute the most frequent repeats at translocation breakpoints, whereas A-tracts occur preferentially at deletion breakpoints. Translocation breakpoints near PONDS-forming repeats also recur in different individuals and patient tumor samples. Hence, PONDS-forming sequences represent an intrinsic risk factor for genomic rearrangements in cancer genomes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

DNA double-strand breaks and micronuclei in human blood lymphocytes after repeated whole body exposures to 7T Magnetic Resonance Imaging.

PubMed

Fatahi, Mahsa; Reddig, Annika; Vijayalaxmi; Friebe, Björn; Hartig, Roland; Prihoda, Thomas J; Ricke, Jens; Roggenbuck, Dirk; Reinhold, Dirk; Speck, Oliver

2016-06-01

To examine the extent of genetic damage, assessed from deoxyribonucleic acid (DNA) double-strand breaks (DSBs) and micronuclei (MN) in peripheral blood mononuclear cells obtained from individuals repeatedly exposed to 7T Magnetic Resonance Imaging (MRI). The study protocol was approved by the local ethics committee. Informed consent was obtained from 22 healthy, non-smoking, non-alcoholic male individuals, who had never undergone radio-/chemo-therapy, scintigraphy, and had not undergone X-ray examination one year prior blood withdrawal. Eleven participants were repeatedly exposed to 7T and 3T MRI while working with/around scanners or frequently participating as 7T and lower field MRI research subjects (mean age 34±7years). The other half was never exposed to 7T or lower field MRI and served as controls (mean age 33±9years). The damage in lymphocytes was assessed using anti-γH2AX immunofluorescence staining of DNA DSBs and by quantification of MN. Isolated cells were further exposed in vitro to 7T MRI either alone or in the presence of the DNA damaging drug etoposide, to determine if there is any additional combined effect. The kinetics of DNA damage repair were examined. The mean base-level of γH2AX foci/cell and incidence of MN between repeatedly exposed and control group were not significantly different (P=0.618 and P=0.535, respectively). The additional in vitro exposure of cells to 7T MRI had no significant impact on MN frequencies and γH2AX foci at 1, 20 and 72h after exposure. Frequently repeated 7T MRI exposure did not result in a detectable increase in genotoxicity indices and alterations of DNA repair kinetics. Copyright © 2016 Elsevier Inc. All rights reserved.

Variation in the genomic locations and sequence conservation of STAR elements among staphylococcal species provides insight into DNA repeat evolution

PubMed Central

2012-01-01

Background Staphylococcus aureus Repeat (STAR) elements are a type of interspersed intergenic direct repeat. In this study the conservation and variation in these elements was explored by bioinformatic analyses of published staphylococcal genome sequences and through sequencing of specific STAR element loci from a large set of S. aureus isolates. Results Using bioinformatic analyses, we found that the STAR elements were located in different genomic loci within each staphylococcal species. There was no correlation between the number of STAR elements in each genome and the evolutionary relatedness of staphylococcal species, however higher levels of repeats were observed in both S. aureus and S. lugdunensis compared to other staphylococcal species. Unexpectedly, sequencing of the internal spacer sequences of individual repeat elements from multiple isolates showed conservation at the sequence level within deep evolutionary lineages of S. aureus. Whilst individual STAR element loci were demonstrated to expand and contract, the sequences associated with each locus were stable and distinct from one another. Conclusions The high degree of lineage and locus-specific conservation of these intergenic repeat regions suggests that STAR elements are maintained due to selective or molecular forces with some of these elements having an important role in cell physiology. The high prevalence in two of the more virulent staphylococcal species is indicative of a potential role for STAR elements in pathogenesis. PMID:23020678

DNABIT Compress - Genome compression algorithm.

PubMed

Rajarajeswari, Pothuraju; Apparao, Allam

2011-01-22

Data compression is concerned with how information is organized in data. Efficient storage means removal of redundancy from the data being stored in the DNA molecule. Data compression algorithms remove redundancy and are used to understand biologically important molecules. We present a compression algorithm, "DNABIT Compress" for DNA sequences based on a novel algorithm of assigning binary bits for smaller segments of DNA bases to compress both repetitive and non repetitive DNA sequence. Our proposed algorithm achieves the best compression ratio for DNA sequences for larger genome. Significantly better compression results show that "DNABIT Compress" algorithm is the best among the remaining compression algorithms. While achieving the best compression ratios for DNA sequences (Genomes),our new DNABIT Compress algorithm significantly improves the running time of all previous DNA compression programs. Assigning binary bits (Unique BIT CODE) for (Exact Repeats, Reverse Repeats) fragments of DNA sequence is also a unique concept introduced in this algorithm for the first time in DNA compression. This proposed new algorithm could achieve the best compression ratio as much as 1.58 bits/bases where the existing best methods could not achieve a ratio less than 1.72 bits/bases.

Histological analysis and ancient DNA amplification of human bone remains found in caius iulius polybius house in pompeii.

PubMed

Cipollaro, M; Di Bernado, G; Forte, A; Galano, G; De Masi, L; Galderisi, U; Guarino, F M; Angelini, F; Cascino, A

1999-09-01

Thirteen skeletons found in the Caius Iulius Polybius house, which has been the object of intensive study since its discovery in Pompeii 250 years ago, have provided an opportunity to study either bone diagenesis by histological investigation or ancient DNA by polymerase chain reaction analysis. DNA analysis was done by amplifying both X- and Y-chromosomes amelogenin loci and Y-specific alphoid repeat locus. The von Willebrand factor (vWF) microsatellite locus on chromosome 12 was also analyzed for personal identification in two individuals showing alleles with 10/11 and 12/12 TCTA repeats, respectively. Technical problems were the scarcity of DNA content from osteocytes, DNA molecule fragmentation, microbial contamination which change bone structure, contaminating human DNA which results from mishandling, and frequent presence of Taq DNA polymerase inhibiting molecules like polyphenols and heavy metals. The results suggest that the remains contain endogenous human DNA that can be amplified and analyzed. The amplifiability of DNA corresponds to the bone preservation and dynamics of the burial conditions subsequent to the 79 A.D. eruption.

The DNA Triangle and Its Application to Learning Meiosis

ERIC Educational Resources Information Center

Wright, L. Kate; Catavero, Christina M.; Newman, Dina L.

2017-01-01

Although instruction on meiosis is repeated many times during the undergraduate curriculum, many students show poor comprehension even as upper-level biology majors. We propose that the difficulty lies in the complexity of understanding DNA, which we explain through a new model, the DNA triangle. The "DNA triangle" integrates three…

Cloning and functional characterization of the Xenopus orthologue of the Treacher Collins syndrome (TCOF1) gene product.

PubMed

Gonzales, Bianca; Yang, Hushan; Henning, Dale; Valdez, Benigno C

2005-10-10

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development caused by mutations in the TCOF1 gene, which encodes the nucleolar phosphoprotein treacle. We previously reported a function for mammalian treacle in ribosomal DNA gene transcription by its interaction with upstream binding factor. As an initial step in the development of a TCS model for frog the cDNA that encodes the Xenopus laevis treacle was cloned. Although the derived amino acid sequence shows a poor homology with its mammalian orthologues, Xenopus treacle has 11 highly homologous direct repeats near the center of the protein molecule similar to those present in its human, dog and mouse orthologues. Comparison of their amino acid compositions indicates conservation of predominant specific amino acid residues. Antisense-mediated down-regulation of treacle expression in X. laevis oocytes resulted in inhibition of rDNA gene transcription. The results suggest evolutionary conservation of the function of treacle in ribosomal RNA biogenesis in higher eukaryotes.

Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system.

PubMed

Belhaj, Khaoula; Chaparro-Garcia, Angela; Kamoun, Sophien; Nekrasov, Vladimir

2013-10-11

Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.

Microhomology-mediated end joining induces hypermutagenesis at breakpoint junctions

PubMed Central

Li, Fuyang; Villarreal, Diana; Shim, Jae Hoon; Myung, Kyungjae; Shim, Eun Yong; Lee, Sang Eun

2017-01-01

Microhomology (MH) flanking a DNA double-strand break (DSB) drives chromosomal rearrangements but its role in mutagenesis has not yet been analyzed. Here we determined the mutation frequency of a URA3 reporter gene placed at multiple locations distal to a DSB, which is flanked by different sizes (15-, 18-, or 203-bp) of direct repeat sequences for efficient repair in budding yeast. Induction of a DSB accumulates mutations in the reporter gene situated up to 14-kb distal to the 15-bp MH, but more modestly to those carrying 18- and 203-bp or no homology. Increased mutagenesis in MH-mediated end joining (MMEJ) appears coupled to its slower repair kinetics and the extensive resection occurring at flanking DNA. Chromosomal translocations via MMEJ also elevate mutagenesis of the flanking DNA sequences 7.1 kb distal to the breakpoint junction as compared to those without MH. The results suggest that MMEJ could destabilize genomes by triggering structural alterations and increasing mutation burden. PMID:28419093

Microchip-based cell lysis and DNA extraction from sperm cells for application to forensic analysis.

PubMed

Bienvenue, Joan M; Duncalf, Natalie; Marchiarullo, Daniel; Ferrance, Jerome P; Landers, James P

2006-03-01

The current backlog of casework is among the most significant challenges facing crime laboratories at this time. While the development of next-generation microchip-based technology for expedited forensic casework analysis offers one solution to this problem, this will require the adaptation of manual, large-volume, benchtop chemistry to small volume microfluidic devices. Analysis of evidentiary materials from rape kits where semen or sperm cells are commonly found represents a unique set of challenges for on-chip cell lysis and DNA extraction that must be addressed for successful application. The work presented here details the development of a microdevice capable of DNA extraction directly from sperm cells for application to the analysis of sexual assault evidence. A variety of chemical lysing agents are assessed for inclusion in the extraction protocol and a method for DNA purification from sperm cells is described. Suitability of the extracted DNA for short tandem repeat (STR) analysis is assessed and genetic profiles shown. Finally, on-chip cell lysis methods are evaluated, with results from fluorescence visualization of cell rupture and DNA extraction from an integrated cell lysis and purification with subsequent STR amplification presented. A method for on-chip cell lysis and DNA purification is described, with considerations toward inclusion in an integrated microdevice capable of both differential cell sorting and DNA extraction. The results of this work demonstrate the feasibility of incorporating microchip-based cell lysis and DNA extraction into forensic casework analysis.

Chromosomal structures and repetitive sequences divergence in Cucumis species revealed by comparative cytogenetic mapping.

PubMed

Zhang, Yunxia; Cheng, Chunyan; Li, Ji; Yang, Shuqiong; Wang, Yunzhu; Li, Ziang; Chen, Jinfeng; Lou, Qunfeng

2015-09-25

Differentiation and copy number of repetitive sequences affect directly chromosome structure which contributes to reproductive isolation and speciation. Comparative cytogenetic mapping has been verified an efficient tool to elucidate the differentiation and distribution of repetitive sequences in genome. In present study, the distinct chromosomal structures of five Cucumis species were revealed through genomic in situ hybridization (GISH) technique and comparative cytogenetic mapping of major satellite repeats. Chromosome structures of five Cucumis species were investigated using GISH and comparative mapping of specific satellites. Southern hybridization was employed to study the proliferation of satellites, whose structural characteristics were helpful for analyzing chromosome evolution. Preferential distribution of repetitive DNAs at the subtelomeric regions was found in C. sativus, C hystrix and C. metuliferus, while majority was positioned at the pericentromeric heterochromatin regions in C. melo and C. anguria. Further, comparative GISH (cGISH) through using genomic DNA of other species as probes revealed high homology of repeats between C. sativus and C. hystrix. Specific satellites including 45S rDNA, Type I/II, Type III, Type IV, CentM and telomeric repeat were then comparatively mapped in these species. Type I/II and Type IV produced bright signals at the subtelomeric regions of C. sativus and C. hystrix simultaneously, which might explain the significance of their amplification in the divergence of Cucumis subgenus from the ancient ancestor. Unique positioning of Type III and CentM only at the centromeric domains of C. sativus and C. melo, respectively, combining with unique southern bands, revealed rapid evolutionary patterns of centromeric DNA in Cucumis. Obvious interstitial telomeric repeats were observed in chromosomes 1 and 2 of C. sativus, which might provide evidence of the fusion hypothesis of chromosome evolution from x = 12 to x = 7 in Cucumis species. Besides, the significant correlation was found between gene density along chromosome and GISH band intensity in C. sativus and C. melo. In summary, comparative cytogenetic mapping of major satellites and GISH revealed the distinct differentiation of chromosome structure during species formation. The evolution of repetitive sequences was the main force for the divergence of Cucumis species from common ancestor.

The Chromodomain of Tf1 Integrase Promotes Binding to cDNA and Mediates Target Site Selection▿ †

PubMed Central

Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D.; Levin, Henry L.

2009-01-01

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDΔ, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDΔ caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting. PMID:19109383

The chromodomain of Tf1 integrase promotes binding to cDNA and mediates target site selection.

PubMed

Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D; Levin, Henry L

2009-03-01

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDDelta, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDDelta caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting.

Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa

PubMed Central

Honda, Shinji; Bicocca, Vincent T.; Gessaman, Jordan D.; Rountree, Michael R.; Yokoyama, Ayumi; Yu, Eun Y.; Selker, Jeanne M. L.; Selker, Eric U.

2016-01-01

DNA methylation, heterochromatin protein 1 (HP1), histone H3 lysine 9 (H3K9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. Prior work showed that H3K9 methylation directs DNA methylation and histone deacetylation via HP1 in Neurospora crassa and that the histone deacetylase complex HCHC is required for proper DNA methylation. The complex consists of the chromodomain proteins HP1 and chromodomain protein 2 (CDP-2), the histone deacetylase HDA-1, and the AT-hook motif protein CDP-2/HDA-1–associated protein (CHAP). We show that the complex is required for proper chromosome segregation, dissect its function, and characterize interactions among its components. Our analyses revealed the existence of an HP1-based DNA methylation pathway independent of its chromodomain. The pathway partially depends on CHAP but not on the CDP-2 chromodomain. CDP-2 serves as a bridge between the recognition of H3K9 trimethylation (H3K9me3) by HP1 and the histone deacetylase activity of HDA-1. CHAP is also critical for HDA-1 localization to heterochromatin. Specifically, the CHAP zinc finger interacts directly with the HDA-1 argonaute-binding protein 2 (Arb2) domain, and the CHAP AT-hook motifs recognize heterochromatic regions by binding to AT-rich DNA. Our data shed light on the interrelationships among the prototypical heterochromatic features and support a model in which dual recognition by the HP1 chromodomain and the CHAP AT-hooks are required for proper heterochromatin formation. PMID:27681634

Seamless editing of the chloroplast genome in plants.

PubMed

Martin Avila, Elena; Gisby, Martin F; Day, Anil

2016-07-29

Gene editing technologies enable the precise insertion of favourable mutations and performance enhancing trait genes into chromosomes whilst excluding all excess DNA from modified genomes. The technology gives rise to a new class of biotech crops which is likely to have widespread applications in agriculture. Despite progress in the nucleus, the seamless insertions of point mutations and non-selectable foreign genes into the organelle genomes of crops have not been described. The chloroplast genome is an attractive target to improve photosynthesis and crop performance. Current chloroplast genome engineering technologies for introducing point mutations into native chloroplast genes leave DNA scars, such as the target sites for recombination enzymes. Seamless editing methods to modify chloroplast genes need to address reversal of site-directed point mutations by template mediated repair with the vast excess of wild type chloroplast genomes that are present early in the transformation process. Using tobacco, we developed an efficient two-step method to edit a chloroplast gene by replacing the wild type sequence with a transient intermediate. This was resolved to the final edited gene by recombination between imperfect direct repeats. Six out of 11 transplastomic plants isolated contained the desired intermediate and at the second step this was resolved to the edited chloroplast gene in five of six plants tested. Maintenance of a single base deletion mutation in an imperfect direct repeat of the native chloroplast rbcL gene showed the limited influence of biased repair back to the wild type sequence. The deletion caused a frameshift, which replaced the five C-terminal amino acids of the Rubisco large subunit with 16 alternative residues resulting in a ~30-fold reduction in its accumulation. We monitored the process in vivo by engineering an overlapping gusA gene downstream of the edited rbcL gene. Translational coupling between the overlapping rbcL and gusA genes resulted in relatively high GUS accumulation (~0.5 % of leaf protein). Editing chloroplast genomes using transient imperfect direct repeats provides an efficient method for introducing point mutations into chloroplast genes. Moreover, we describe the first synthetic operon allowing expression of a downstream overlapping gene by translational coupling in chloroplasts. Overlapping genes provide a new mechanism for co-ordinating the translation of foreign proteins in chloroplasts.

An annotated genetic map of loblolly pine based on microsatellite and cDNA markers

Treesearch

Craig S. Echt; Surya Saha; Konstantin V. Krutovsky; Kokulapalan Wimalanathan; John E. Erpelding; Chun Liang; C Dana Nelson

2011-01-01

Previous loblolly pine (Pinus taeda L.) genetic linkage maps have been based on a variety of DNA polymorphisms, such as AFLPs, RAPDs, RFLPs, and ESTPs, but only a few SSRs (simple sequence repeats), also known as simple tandem repeats or microsatellites, have been mapped in P. taeda. The objective of this study was to integrate a large set of SSR markers from a variety...

Repeated Nrf2 stimulation using sulforaphane protects fibroblasts from ionizing radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathew, Sherin T.; Bergström, Petra; Hammarsten, Ola, E-mail: ola.hammarsten@clinchem.gu.se

2014-05-01

Most of the cytotoxicity induced by ionizing radiation is mediated by radical-induced DNA double-strand breaks. Cellular protection from free radicals can be stimulated several fold by sulforaphane-mediated activation of the transcription factor Nrf2 that regulates more than 50 genes involved in the detoxification of reactive substances and radicals. Here, we report that repeated sulforaphane treatment increases radioresistance in primary human skin fibroblasts. Cells were either treated with sulforaphane for four hours once or with four-hour treatments repeatedly for three consecutive days prior to radiation exposure. Fibroblasts exposed to repeated-sulforaphane treatment showed a more pronounced dose-dependent induction of Nrf2-regulated mRNA andmore » reduced amount of radiation-induced free radicals compared with cells treated once with sulforaphane. In addition, radiation- induced DNA double-strand breaks measured by gamma-H2AX foci were attenuated following repeated sulforaphane treatment. As a result, cellular protection from ionizing radiation measured by the 5-ethynyl-2′-deoxyuridine (EdU) assay was increased, specifically in cells exposed to repeated sulforaphane treatment. Sulforaphane treatment was unable to protect Nrf2 knockout mouse embryonic fibroblasts, indicating that the sulforaphane-induced radioprotection was Nrf2-dependent. Moreover, radioprotection by repeated sulforaphane treatment was dose-dependent with an optimal effect at 10 uM, whereas both lower and higher concentrations resulted in lower levels of radioprotection. Our data indicate that the Nrf2 system can be trained to provide further protection from radical damage. - Highlights: • Repeated treatment with sulforaphane protects fibroblasts from ionizing radiation • Repeated sulforaphane treatment attenuates radiation induced ROS and DNA damage • Sulforaphane mediated protection is Nrf2 dependent.« less

The 2.1-kb inverted repeat DNA sequences flank the mat2,3 silent region in two species of Schizosaccharomyces and are involved in epigenetic silencing in Schizosaccharomyces pombe.

PubMed Central

Singh, Gurjeet; Klar, Amar J S

2002-01-01

The mat2,3 region of the fission yeast Schizosaccharomyces pombe exhibits a phenomenon of transcriptional silencing. This region is flanked by two identical DNA sequence elements, 2.1 kb in length, present in inverted orientation: IRL on the left and IRR on the right of the silent region. The repeats do not encode any ORF. The inverted repeat DNA region is also present in a newly identified related species, which we named S. kambucha. Interestingly, the left and right repeats share perfect identity within a species, but show approximately 2% bases interspecies variation. Deletion of IRL results in variegated expression of markers inserted in the silent region, while deletion of the IRR causes their derepression. When deletions of these repeats were genetically combined with mutations in different trans-acting genes previously shown to cause a partial defect in silencing, only mutations in clr1 and clr3 showed additive defects in silencing with the deletion of IRL. The rate of mat1 switching is also affected by deletion of repeats. The IRL or IRR deletion did not cause significant derepression of the mat2 or mat3 loci. These results implicate repeats for maintaining full repression of the mat2,3 region, for efficient mat1 switching, and further support the notion that multiple pathways cooperate to silence the mat2,3 domain. PMID:12399374

Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways

PubMed Central

Yu, Weishi; McIntosh, Carl; Lister, Ryan; Zhu, Iris; Han, Yixing; Ren, Jianke; Landsman, David; Lee, Eunice; Briones, Victorino; Terashima, Minoru; Leighty, Robert; Ecker, Joseph R.

2014-01-01

Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells−/− (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation. PMID:25170028

Forensic DNA Profiling and Database

PubMed Central

Panneerchelvam, S.; Norazmi, M.N.

2003-01-01

The incredible power of DNA technology as an identification tool had brought a tremendous change in crimnal justice . DNA data base is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. This article discusses the essential steps in compilation of COmbined DNA Index System (CODIS) on validated polymerase chain amplified STRs and their use in crime detection. PMID:23386793

Examples of kinship analysis where Profiler Plus™ was not discriminatory enough for the identification of victims using DNA identification.

PubMed

Hartman, D; Benton, L; Morenos, L; Beyer, J; Spiden, M; Stock, A

2011-02-25

The identification of the victims of the 2009 Victorian bushfires disaster, as in other mass disasters, relied on a number of scientific disciplines - including DNA analysis. As part of the DVI response, DNA analysis was performed to assist in the identification of victims through kinship (familial matching to relatives) or direct (self source of sample) matching of DNA profiles. The majority of the DNA identifications made (82%) were achieved through kinship matching of familial reference samples to post mortem (PM) samples obtained from the victims. Although each location affected by the bushfires could be treated as a mini-disaster (having a small closed-set of victims), with many such sites spread over vast areas, DNA analysis requires that the short tandem repeat (STR) system used be able to afford enough discrimination between all the DVI cases to assign a match. This publication highlights that although a 9-loci multiplex was sufficient for a DVI of this nature, there were instances that brought to light the short comings of using a 9-loci multiplex for kinship matching--particularly where multiple family members are victims. Moreso it serves to reinforce the recommendation that a minimum of 12 autosomal STR markers (plus Amelogenin) be used for DNA identification of victims which relies heavily on kinship matching. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV.

PubMed

Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P; Robertson, Erle S; Schildkraut, Carl L; Verma, Subhash C

2016-05-05

Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

PubMed

Modell, Joshua W; Jiang, Wenyan; Marraffini, Luciano A

2017-04-06

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.

TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.

PubMed

Lin, Jiangguo; Countryman, Preston; Buncher, Noah; Kaur, Parminder; E, Longjiang; Zhang, Yiyun; Gibson, Greg; You, Changjiang; Watkins, Simon C; Piehler, Jacob; Opresko, Patricia L; Kad, Neil M; Wang, Hong

2014-02-01

Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1's 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (∼ 9-17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ∼ 2.8-3.6 κ(B)T greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This 'tag-team proofreading' represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.

Novel adsorbent for DNA adsorption: Fe(3+)-attached sporopollenin particles embedded composite cryogels.

PubMed

Ceylan, Şeyda; Odabaşı, Mehmet

2013-12-01

The aim of this study is to prepare supermacroporous cryogels embedded with Fe(3+)-attached sporopollenin particles (Fe(3++)-ASPs) having large surface area for high DNA adsorption capacity. Supermacroporous poly(2-hydroxyethyl methacrylate) (PHEMA)-based monolithic cryogel column embedded with Fe3+(+)-ASPs was prepared by radical cryo-copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N,N´-methylene- bis-acrylamide (MBAAm) as cross-linker directly in a plastic syringe for DNA adsorption studies. Firstly, Fe3+(+) ions were attached to the sporopollenin particles (SPs), then the supermacroporous PHEMA cryogel with embedded Fe(3++)-ASPs was produced by free radical polymerization using N,N,N´, N´-Tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) as initiator/activator pair in an ice bath. Optimum conditions of adsorption experiments were performed at pH 6.0 (0.02 M Tris buffer containing 0.2 M NaCl), with flow rate of 0.5 mL/min, and at 5°C. The maximum amount of DNA adsorption from aqueous solution was very high (109 mg/g SPs) with initial concentration of 3 mg/mL. It was observed that DNA could be repeatedly adsorbed and desorbed with this composite cryogel without significant loss of adsorption capacity. As a result, higher amounts of DNA adsorbed these composite cryogels are expected to be good candidate for achieving higher removal of anti-DNA antibodies from systemic lupus erythematosus (SLE) patients plasma.

Infrared fluorescent automated detection of thirteen short tandem repeat polymorphisms and one gender-determining system of the CODIS core system.

PubMed

Ricci, U; Sani, I; Guarducci, S; Biondi, C; Pelagatti, S; Lazzerini, V; Brusaferri, A; Lapini, M; Andreucci, E; Giunti, L; Giovannucci Uzielli, M L

2000-11-01

We used an infrared (IR) automated fluorescence monolaser sequencer for the analysis of 13 autosomal short tandem repeat (STR) systems (TPOX, D3S1358, FGA, CSF1PO, D5S818, D7S820, D8S1179, TH01, vWA, D13S317, D16S359, D18S51, D21S11) and the X-Y homologous gene amelogenin system. These two systems represent the core of the combined DNA index systems (CODIS). Four independent multiplex reactions, based on the polymerase chain reaction (PCR) technique and on the direct labeling of the forward primer of every primer pair, with a new molecule (IRDye800), were set up, permitting the exact characterization of the alleles by comparison with ladders of specific sequenced alleles. This is the first report of the whole analysis of the STRs of the CODIS core using an IR automated DNA sequencer. The protocol was used to solve paternity/maternity tests and for population studies. The electrophoretic system also proved useful for the correct typing of those loci differing in size by only 2 bp. A sensibility study demonstrated that the test can detect an average of 10 pg of undegraded human DNA. We also performed a preliminary study analyzing some forensic samples and mixed stains, which suggested the usefulness of using this analytical system for human identification as well as for forensic purposes.

Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter.

PubMed

Sheridan, P L; Schorpp, M; Voz, M L; Jones, K A

1995-03-03

We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.

A novel method for sex determination by detecting the number of X chromosomes.

PubMed

Nakanishi, Hiroaki; Shojo, Hideki; Ohmori, Takeshi; Hara, Masaaki; Takada, Aya; Adachi, Noboru; Saito, Kazuyuki

2015-01-01

A novel method for sex determination, based on the detection of the number of X chromosomes, was established. Current methods, based on the detection of the Y chromosome, can directly identify an unknown sample as male, but female gender is determined indirectly, by not detecting the Y chromosome. Thus, a direct determination of female gender is important because the quality (e.g., fragmentation and amelogenin-Y null allele) of the Y chromosome DNA may lead to a false result. Thus, we developed a novel sex determination method by analyzing the number of X chromosomes using a copy number variation (CNV) detection technique (the comparative Ct method). In this study, we designed a primer set using the amelogenin-X gene without the CNV region as the target to determine the X chromosome copy number, to exclude the influence of the CNV region from the comparative Ct value. The number of X chromosomes was determined statistically using the CopyCaller software with real-time PCR. All DNA samples from participants (20 males, 20 females) were evaluated correctly using this method with 1-ng template DNA. A minimum of 0.2-ng template DNA was found to be necessary for accurate sex determination with this method. When using ultraviolet-irradiated template DNA, as mock forensic samples, the sex of the samples could not be determined by short tandem repeat (STR) analysis but was correctly determined using our method. Thus, we successfully developed a method of sex determination based on the number of X chromosomes. Our novel method will be useful in forensic practice for sex determination.

[Structural organization of 5S ribosomal DNA of Rosa rugosa].

PubMed

Tynkevych, Iu O; Volkov, R A

2014-01-01

In order to clarify molecular organization of the genomic region encoding 5S rRNA in diploid species Rosa rugosa several 5S rDNA repeated units were cloned and sequenced. Analysis of the obtained sequences revealed that only one length variant of 5S rDNA repeated units, which contains intact promoter elements in the intergenic spacer region (IGS) and appears to be transcriptionally active is present in the genome. Additionally, a limited number of 5S rDNA pseudogenes lacking a portion of coding sequence and the complete IGS was detected. A high level of sequence similarity (from 93.7 to 97.5%) between the IGS of major 5S rDNA variants of East Asian R. rugosa and North American R. nitida was found indicating comparatively recent divergence of these species.

Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).

PubMed Central

Ishiai, M; Wada, C; Kawasaki, Y; Yura, T

1994-01-01

Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication. Images PMID:8170998

Tissue- and age-specific DNA replication patterns at the CTG/CAG-expanded human myotonic dystrophy type 1 locus.

PubMed

Cleary, John D; Tomé, Stéphanie; López Castel, Arturo; Panigrahi, Gagan B; Foiry, Laurent; Hagerman, Katharine A; Sroka, Hana; Chitayat, David; Gourdon, Geneviève; Pearson, Christopher E

2010-09-01

Myotonic dystrophy, caused by DM1 CTG/CAG repeat expansions, shows varying instability levels between tissues and across ages within patients. We determined DNA replication profiles at the DM1 locus in patient fibroblasts and tissues from DM1 transgenic mice of various ages showing different instability. In patient cells, the repeat is flanked by two replication origins demarcated by CTCF sites, with replication diminished at the expansion. In mice, the expansion replicated from only the downstream origin (CAG as lagging template). In testes from mice of three different ages, replication toward the repeat paused at the earliest age and was relieved at later ages-coinciding with increased instability. Brain, pancreas and thymus replication varied with CpG methylation at DM1 CTCF sites. CTCF sites between progressing forks and repeats reduced replication depending on chromatin. Thus, varying replication progression may affect tissue- and age-specific repeat instability.

The self primer of the long terminal repeat retrotransposon Tf1 is not removed during reverse transcription.

PubMed

Atwood-Moore, Angela; Yan, Kenneth; Judson, Robert L; Levin, Henry L

2006-08-01

The long terminal repeat retrotransposon Tf1 of Schizosaccharomyces pombe uses a unique mechanism of self priming to initiate reverse transcription. Instead of using a tRNA, Tf1 primes minus-strand synthesis with an 11-nucleotide RNA removed from the 5' end of its own transcript. We tested whether the self primer of Tf1 was similar to tRNA primers in being removed from the cDNA by RNase H. Our analysis of Tf1 cDNA extracted from virus-like particles revealed the surprising observation that the dominant species of cDNA retained the self primer. This suggests that integration of the cDNA relies on mechanisms other than reverse transcription to remove the primer.

Somatic instability of the expanded allele of IT-15 from patients with Huntington disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stine, O.C.; Pleasant, N.; Ross, C.A.

1994-09-01

Huntington`s disease (HD) is an inherited neurodegenerative disorder caused by an expanded trinucleotide repeat in the gene IT-15. Although the expanded allele of IT-15 is unstable during gametogenesis, particularly, spermatogenesis, it is not clear if there is somatic stability. There are two reports of stability and one of instability. In order to test whether somatic instability occurs in the expansions found in HD, we have compared amplified genomic DNA isolated from either blood or distinct regions of autopsied brains of persons with Huntington disease. We find that somatic variation occurs in at least two ways. First, in cases with longermore » repeats (n > 47), the cerebellum often (8 of 9 cases) has a smaller number of repeats (2 to 10 less) than other tested regions of the brain. The larger the expanded allele, the larger the reduction in size of the repeat in the cerebellum (r=0.94, p<0.0001, df=12). Second, regardless of the repeat size, the number of amplification products from genomic DNA isolated from the cerebellum is smaller than that from genomic DNA from other forebrain regions such as the dorsal parietal cortex. As the length of the expanded allele increases, the number of amplification products increase in either tissue (r=0.86, p<0.001, df=12). Therefore our data demonstrates somatic instability especially for longer repeats.« less

Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903.

PubMed Central

Grindley, N D; Joyce, C M

1980-01-01

The kanamycin resistance transposon Tn903 consists of a unique region of about 1000 base pairs bounded by a pair of 1050-base-pair inverted repeat sequences. Each repeat contains two Pvu II endonuclease cleavage sites separated by 520 base pairs. We have constructed derivatives of Tn903 in which this 520-base-pair fragment is deleted from one or both repeats. Those derivatives that lack both 520-base-pair fragments cannot transpose, whereas those that lack just one remain transposition proficient. One such transposable derivative, Tn903 delta I, has been selected for further study. We have determined the sequence of the intact inverted repeat. The 18 base pairs at each end are identical and inverted relative to one another, a structure characteristic of insertion sequences. Additional experiments indicate that a single inverted repeat from Tn903 can, in fact, transpose; we propose that this element be called IS903. To correlate the DNA sequence with genetic activities, we have created mutations by inserting a 10-base-pair DNA fragment at several sites within the intact repeat of Tn903 delta 1, and we have examined the effect of such insertions on transposability. The results suggest that IS903 encodes a 307-amino-acid polypeptide (a "transposase") that is absolutely required for transposition of IS903 or Tn903. Images PMID:6261245

Novel variants of the 5S rRNA genes in Eruca sativa.

PubMed

Singh, K; Bhatia, S; Lakshmikumaran, M

1994-02-01

The 5S ribosomal RNA (rRNA) genes of Eruca sativa were cloned and characterized. They are organized into clusters of tandemly repeated units. Each repeat unit consists of a 119-bp coding region followed by a noncoding spacer region that separates it from the coding region of the next repeat unit. Our study reports novel gene variants of the 5S rRNA genes in plants. Two families of the 5S rDNA, the 0.5-kb size family and the 1-kb size family, coexist in the E. sativa genome. The 0.5-kb size family consists of the 5S rRNA genes (S4) that have coding regions similar to those of other reported plant 5S rDNA sequences, whereas the 1-kb size family consists of the 5S rRNA gene variants (S1) that exist as 1-kb BamHI tandem repeats. S1 is made up of two variant units (V1 and V2) of 5S rDNA where the BamHI site between the two units is mutated. Sequence heterogeneity among S4, V1, and V2 units exists throughout the sequence and is not limited to the noncoding spacer region only. The coding regions of V1 and V2 show approximately 20% dissimilarity to the coding regions of S4 and other reported plant 5S rDNA sequences. Such a large variation in the coding regions of the 5S rDNA units within the same plant species has been observed for the first time. Restriction site variation is observed between the two size classes of 5S rDNA in E. sativa.(ABSTRACT TRUNCATED AT 250 WORDS)

Satellite DNA Modulates Gene Expression in the Beetle Tribolium castaneum after Heat Stress

PubMed Central

Feliciello, Isidoro; Akrap, Ivana; Ugarković, Đurđica

2015-01-01

Non-coding repetitive DNAs have been proposed to perform a gene regulatory role, however for tandemly repeated satellite DNA no such role was defined until now. Here we provide the first evidence for a role of satellite DNA in the modulation of gene expression under specific environmental conditions. The major satellite DNA TCAST1 in the beetle Tribolium castaneum is preferentially located within pericentromeric heterochromatin but is also dispersed as single repeats or short arrays in the vicinity of protein-coding genes within euchromatin. Our results show enhanced suppression of activity of TCAST1-associated genes and slower recovery of their activity after long-term heat stress relative to the same genes without associated TCAST1 satellite DNA elements. The level of gene suppression is not influenced by the distance of TCAST1 elements from the associated genes up to 40 kb from the genes’ transcription start sites, but it does depend on the copy number of TCAST1 repeats within an element, being stronger for the higher number of copies. The enhanced gene suppression correlates with the enrichment of the repressive histone marks H3K9me2/3 at dispersed TCAST1 elements and their flanking regions as well as with increased expression of TCAST1 satellite DNA. The results reveal transient, RNAi based heterochromatin formation at dispersed TCAST1 repeats and their proximal regions as a mechanism responsible for enhanced silencing of TCAST1-associated genes. Differences in the pattern of distribution of TCAST1 elements contribute to gene expression diversity among T. castaneum strains after long-term heat stress and might have an impact on adaptation to different environmental conditions. PMID:26275223

The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres.

PubMed

Zhang, Tao; Talbert, Paul B; Zhang, Wenli; Wu, Yufeng; Yang, Zujun; Henikoff, Jorja G; Henikoff, Steven; Jiang, Jiming

2013-12-10

Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO satellite repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90-100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have an ∼10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes.

Role for a region of helically unstable DNA within the Epstein-Barr virus latent cycle origin of DNA replication oriP in origin function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Polonskaya, Zhanna; Benham, Craig J.; Hearing, Janet

The minimal replicator of the Epstein-Barr virus (EBV) latent cycle origin of DNA replication oriP is composed of two binding sites for the Epstein-Barr virus nuclear antigen-1 (EBNA-1) and flanking inverted repeats that bind the telomere repeat binding factor TRF2. Although not required for minimal replicator activity, additional binding sites for EBNA-1 and TRF2 and one or more auxiliary elements located to the right of the EBNA-1/TRF2 sites are required for the efficient replication of oriP plasmids. Another region of oriP that is predicted to be destabilized by DNA supercoiling is shown here to be an important functional component ofmore » oriP. The ability of DNA fragments of unrelated sequence and possessing supercoiled-induced DNA duplex destabilized (SIDD) structures, but not fragments characterized by helically stable DNA, to substitute for this component of oriP demonstrates a role for the SIDD region in the initiation of oriP-plasmid DNA replication.« less

The organization and evolution of the Responder satellite in species of the Drosophila melanogaster group: dynamic evolution of a target of meiotic drive.

PubMed

Larracuente, Amanda M

2014-11-25

Satellite DNA can make up a substantial fraction of eukaryotic genomes and has roles in genome structure and chromosome segregation. The rapid evolution of satellite DNA can contribute to genomic instability and genetic incompatibilities between species. Despite its ubiquity and its contribution to genome evolution, we currently know little about the dynamics of satellite DNA evolution. The Responder (Rsp) satellite DNA family is found in the pericentric heterochromatin of chromosome 2 of Drosophila melanogaster. Rsp is well-known for being the target of Segregation Distorter (SD)- an autosomal meiotic drive system in D. melanogaster. I present an evolutionary genetic analysis of the Rsp family of repeats in D. melanogaster and its closely-related species in the melanogaster group (D. simulans, D. sechellia, D. mauritiana, D. erecta, and D. yakuba) using a combination of available BAC sequences, whole genome shotgun Sanger reads, Illumina short read deep sequencing, and fluorescence in situ hybridization. I show that Rsp repeats have euchromatic locations throughout the D. melanogaster genome, that Rsp arrays show evidence for concerted evolution, and that Rsp repeats exist outside of D. melanogaster, in the melanogaster group. The repeats in these species are considerably diverged at the sequence level compared to D. melanogaster, and have a strikingly different genomic distribution, even between closely-related sister taxa. The genomic organization of the Rsp repeat in the D. melanogaster genome is complex-it exists of large blocks of tandem repeats in the heterochromatin and small blocks of tandem repeats in the euchromatin. My discovery of heterochromatic Rsp-like sequences outside of D. melanogaster suggests that SD evolved after its target satellite and that the evolution of the Rsp satellite family is highly dynamic over a short evolutionary time scale (<240,000 years).

Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard.

PubMed

Kline, Margaret C; Duewer, David L; Travis, John C; Smith, Melody V; Redman, Janette W; Vallone, Peter M; Decker, Amy E; Butler, John M

2009-06-01

Modern highly multiplexed short tandem repeat (STR) assays used by the forensic human-identity community require tight control of the initial amount of sample DNA amplified in the polymerase chain reaction (PCR) process. This, in turn, requires the ability to reproducibly measure the concentration of human DNA, [DNA], in a sample extract. Quantitative PCR (qPCR) techniques can determine the number of intact stretches of DNA of specified nucleotide sequence in an extremely small sample; however, these assays must be calibrated with DNA extracts of well-characterized and stable composition. By 2004, studies coordinated by or reported to the National Institute of Standards and Technology (NIST) indicated that a well-characterized, stable human DNA quantitation certified reference material (CRM) could help the forensic community reduce within- and among-laboratory quantitation variability. To ensure that the stability of such a quantitation standard can be monitored and that, if and when required, equivalent replacement materials can be prepared, a measurement of some stable quantity directly related to [DNA] is required. Using a long-established conventional relationship linking optical density (properly designated as decadic attenuance) at 260 nm with [DNA] in aqueous solution, NIST Standard Reference Material (SRM) 2372 Human DNA Quantitation Standard was issued in October 2007. This SRM consists of three quite different DNA extracts: a single-source male, a multiple-source female, and a mixture of male and female sources. All three SRM components have very similar optical densities, and thus very similar conventional [DNA]. The materials perform very similarly in several widely used gender-neutral assays, demonstrating that the combination of appropriate preparation methods and metrologically sound spectrophotometric measurements enables the preparation and certification of quantitation [DNA] standards that are both maintainable and of practical utility.

Selective alkylation of T–T mismatched DNA using vinyldiaminotriazine–acridine conjugate

PubMed Central

Onizuka, Kazumitsu; Usami, Akira; Yamaoki, Yudai; Kobayashi, Tomohito; Hazemi, Madoka E; Chikuni, Tomoko; Sato, Norihiro; Sasaki, Kaname; Katahira, Masato

2018-01-01

Abstract The alkylation of the specific higher-order nucleic acid structures is of great significance in order to control its function and gene expression. In this report, we have described the T–T mismatch selective alkylation with a vinyldiaminotriazine (VDAT)–acridine conjugate. The alkylation selectively proceeded at the N3 position of thymidine on the T–T mismatch. Interestingly, the alkylated thymidine induced base flipping of the complementary base in the duplex. In a model experiment for the alkylation of the CTG repeats DNA which causes myotonic dystrophy type 1 (DM1), the observed reaction rate for one alkylation increased in proportion to the number of T–T mismatches. In addition, we showed that primer extension reactions with DNA polymerase and transcription with RNA polymerase were stopped by the alkylation. The alkylation of the repeat DNA will efficiently work for the inhibition of replication and transcription reactions. These functions of the VDAT–acridine conjugate would be useful as a new biochemical tool for the study of CTG repeats and may provide a new strategy for the molecular therapy of DM1. PMID:29309639

Recombinogenic Telomeres in Diploid Sorex granarius (Soricidae, Eulipotyphla) Fibroblast Cells

PubMed Central

Draskovic, I.; Minina, J. M.; Karamysheva, T. V.; Novo, C. L.; Liu, W.-Y.; Porreca, R. M.; Gibaud, A.; Zvereva, M. E.; Skvortsov, D. A.; Rubtsov, N. B.

2014-01-01

The telomere structure in the Iberian shrew Sorex granarius is characterized by unique, striking features, with short arms of acrocentric chromosomes carrying extremely long telomeres (up to 300 kb) with interspersed ribosomal DNA (rDNA) repeat blocks. In this work, we investigated the telomere physiology of S. granarius fibroblast cells and found that telomere repeats are transcribed on both strands and that there is no telomere-dependent senescence mechanism. Although telomerase activity is detectable throughout cell culture and appears to act on both short and long telomeres, we also discovered that signatures of a recombinogenic activity are omnipresent, including telomere-sister chromatid exchanges, formation of alternative lengthening of telomeres (ALT)-associated PML-like bodies, production of telomere circles, and a high frequency of telomeres carrying marks of a DNA damage response. Our results suggest that recombination participates in the maintenance of the very long telomeres in normal S. granarius fibroblasts. We discuss the possible interplay between the interspersed telomere and rDNA repeats in the stabilization of the very long telomeres in this organism. PMID:24842907

Target innervation is necessary for neuronal polyploidization in the terrestrial slug Limax.

PubMed

Matsuo, Ryota; Yamagishi, Miki; Wakiya, Kyoko; Tanaka, Yoko; Ito, Etsuro

2013-08-01

The brain of gastropod mollusks contains many giant neurons with polyploid genomic DNAs. Such DNAs are generated through repeated DNA endoreplication during body growth. However, it is not known what triggers DNA endoreplication in neurons. There are two possibilities: (1) DNAs are replicated in response to some unknown molecules in the hemolymph that reflect the nutritive status of the animal; or (2) DNAs are replicated in response to some unknown factors that are retrogradely transported through axons from the innervated target organs. We first tested whether hemolymph with rich nutrition could induce DNA endoreplication. We tested whether the transplanted brain exhibits enhanced DNA endoreplication like an endogenous brain does when transplanted into the homocoel of the body of a slug whose body growth is promoted by an increased food supply. However, no enhancement was observed in the frequency of DNA endoreplication when we compared the transplanted brains in the growth-promoted and growth-suppressed host slugs, suggesting that the humoral environment is irrelevant to triggering the body growth-dependent DNA endoreplication. Next, we tested the requirement of target innervation by surgically dissecting a unilateral posterior pedal nerve of an endogenous brain. Substantially lower number of neurons exhibited DNA endoreplication in the pedal ganglion ipsilateral to the dissected nerve. These results support the view that enhanced DNA endoreplication is mediated by target innervation and is not brought about through the direct effect of humoral factors in the hemolymph during body growth. Copyright © 2013 Wiley Periodicals, Inc.

Chromatin tethering effects of hNopp140 are involved in the spatial organization of nucleolus and the rRNA gene transcription

PubMed Central

Tsai, Yi-Tzang; Lin, Chen-I; Chen, Hung-Kai; Lee, Kuo-Ming; Hsu, Chia-Yi; Yang, Shun-Jen

2008-01-01

The short arms of five human acrocentric chromosomes contain ribosomal gene (rDNA) clusters where numerous mini-nucleoli arise at the exit of mitosis. These small nucleoli tend to coalesce into one or a few large nucleoli during interphase by unknown mechanisms. Here, we demonstrate that the N- and C-terminal domains of a nucleolar protein, hNopp140, bound respectively to α-satellite arrays and rDNA clusters of acrocentric chromosomes for nucleolar formation. The central acidic-and-basic repeated domain of hNopp140, possessing a weak self-self interacting ability, was indispensable for hNopp140 to build up a nucleolar round-shaped structure. The N- or the C-terminally truncated hNopp140 caused nucleolar segregation and was able to alter locations of the rDNA transcription, as mediated by detaching the rDNA repeats from the acrocentric α-satellite arrays. Interestingly, an hNopp140 mutant, made by joining the N- and C-terminal domains but excluding the entire central repeated region, induced nucleolar disruption and global chromatin condensation. Furthermore, RNAi knockdown of hNopp140 resulted in dispersion of the rDNA and acrocentric α-satellite sequences away from nucleolus that was accompanied by rDNA transcriptional silence. Our findings indicate that hNopp140, a scaffold protein, is involved in the nucleolar assembly, fusion, and maintenance. PMID:18253863

The role of DNA repair in herpesvirus pathogenesis.

PubMed

Brown, Jay C

2014-10-01

In cells latently infected with a herpesvirus, the viral DNA is present in the cell nucleus, but it is not extensively replicated or transcribed. In this suppressed state the virus DNA is vulnerable to mutagenic events that affect the host cell and have the potential to destroy the virus' genetic integrity. Despite the potential for genetic damage, however, herpesvirus sequences are well conserved after reactivation from latency. To account for this apparent paradox, I have tested the idea that host cell-encoded mechanisms of DNA repair are able to control genetic damage to latent herpesviruses. Studies were focused on homologous recombination-dependent DNA repair (HR). Methods of DNA sequence analysis were employed to scan herpesvirus genomes for DNA features able to activate HR. Analyses were carried out with a total of 39 herpesvirus DNA sequences, a group that included viruses from the alpha-, beta- and gamma-subfamilies. The results showed that all 39 genome sequences were enriched in two or more of the eight recombination-initiating features examined. The results were interpreted to indicate that HR can stabilize latent herpesvirus genomes. The results also showed, unexpectedly, that repair-initiating DNA features differed in alpha- compared to gamma-herpesviruses. Whereas inverted and tandem repeats predominated in alpha-herpesviruses, gamma-herpesviruses were enriched in short, GC-rich initiation sequences such as CCCAG and depleted in repeats. In alpha-herpesviruses, repair-initiating repeat sequences were found to be concentrated in a specific region (the S segment) of the genome while repair-initiating short sequences were distributed more uniformly in gamma-herpesviruses. The results suggest that repair pathways are activated differently in alpha- compared to gamma-herpesviruses. Copyright © 2014. Published by Elsevier Inc.

Variation in Ribosomal DNA among Isolates of the Mycorrhizal Fungus Cenococcum Geophilum FR.

NASA Astrophysics Data System (ADS)

Lobuglio, Katherine Frances

1990-01-01

Cenococcum geophilum Fr., a cosmopolitan mycorrhizal fungus, is well-known for its extremely wide host and habitat range. The ecological diversity of C. geophilum sharply contrasts its present taxonomic status as a monotypic form -genus. Restriction fragment length polymorphisms (RFLPs) in nuclear ribosomal DNA (rDNA) was used to assess the degree of genetic variation among 72 isolates of C. geophilum. The probe used in this study was the rDNA repeat cloned from C. geophilum isolate A145 (pCG15). Length of the rDNA repeat was approximately 9 kb. The rDNA clone was mapped for 5 restriction endonucleases. Hybridization with cloned Saccharomyces cerevisiae rDNA (pSR118, and pSR125 containing the 18S, and 5.8-25S rRNA genes respectively), and alignment of restriction endonuclease sites conserved in the rDNA genes of other fungi, were used to position the corresponding rDNAs of C. geophilum. Southern hybridizations with EcoRI, HindIII, XhoI, and PstI digested DNAs indicated extensive variation among the C. geophilum isolates, greater than has been previously reported to occur within a fungal species. Most of the rDNA polymorphisms occurred in the IGS region. Restriction endonuclease site and length polymorphisms were also observed in the 5.8S-26S genic regions. Sixteen size categories of length mutations, 6 restriction endonuclease site additions, and 4 restriction endonuclease site deletions were determined using isolate A145 as a reference. The rDNA repeat length among the isolates varied from approximately 8.5 to 10.2 kb. RFLPs were also observed in the mitochondrial (mt) 24S rRNA gene and flanking regions of HindIII digested DNAs of C. geophilum isolates representing both geographically distinct and similar origins. Among the C. geophilum isolates analyzed there were fewer RFLPs in mt-DNA than in nuclear rDNA. EcoRI rDNA phenotypes between C. geophilum and Elaphomyces anthracinus, its proposed teleomorph or sexual state, did not correspond. In addition, the four Elaphomyces species examined had smaller rDNA repeat sizes (approximately 7.3 to 8.0 kb) than that observed among C. geophilum isolates. UPGMA cluster analysis grouped C. geophilum isolates, on the basis of shared nuclear rDNA phenotypes, into a broad range of clusters ranging from 100% to 44% similarity. Limited correlation was observed among nuclear rDNA phenotypes and culture morphology, mycorrhizal characteristics, or geographic origins of the isolates. The amount of genetic variation demonstrated in this study indicates that C. geophilum is either an extremely heterogenous species or it represents more than one species and possibly more than one genus.

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution

USDA-ARS?s Scientific Manuscript database

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres comprise of megabase-scale arrays of tandem repeats. The true prevalence of centromere tandem repeats, and whether they exhibit conserved seque...

Plant genotyping using fluorescently tagged inter-simple sequence repeats (ISSRs): basic principles and methodology.

PubMed

Prince, Linda M

2015-01-01

Inter-simple sequence repeat PCR (ISSR-PCR) is a fast, inexpensive genotyping technique based on length variation in the regions between microsatellites. The method requires no species-specific prior knowledge of microsatellite location or composition. Very small amounts of DNA are required, making this method ideal for organisms of conservation concern, or where the quantity of DNA is extremely limited due to organism size. ISSR-PCR can be highly reproducible but requires careful attention to detail. Optimization of DNA extraction, fragment amplification, and normalization of fragment peak heights during fluorescent detection are critical steps to minimizing the downstream time spent verifying and scoring the data.

Chromosome ends: different sequences may provide conserved functions.

PubMed

Louis, Edward J; Vershinin, Alexander V

2005-07-01

The structures of specific chromosome regions, centromeres and telomeres, present a number of puzzles. As functions performed by these regions are ubiquitous and essential, their DNA, proteins and chromatin structure are expected to be conserved. Recent studies of centromeric DNA from human, Drosophila and plant species have demonstrated that a hidden universal centromere-specific sequence is highly unlikely. The DNA of telomeres is more conserved consisting of a tandemly repeated 6-8 bp Arabidopsis-like sequence in a majority of organisms as diverse as protozoan, fungi, mammals and plants. However, there are alternatives to short DNA repeats at the ends of chromosomes and for telomere elongation by telomerase. Here we focus on the similarities and diversity that exist among the structural elements, DNA sequences and proteins, that make up terminal domains (telomeres and subtelomeres), and how organisms use these in different ways to fulfil the functions of end-replication and end-protection. Copyright (c) 2005 Wiley Periodicals, Inc.

Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tuskan, Gerald A; Gunter, Lee E; DiFazio, Stephen P

The 18S-28S rDNA and 5S rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 18S-28S rDNA sites and one 5S rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis -type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones selected from 2 linkage groups based on genome sequence assembly (LG-I and LG-VI) were localized on 2 chromosomes, as expected. BACs from LG-I hybridized to the longest chromosome in the complement. All BAC positions were found to be concordant with sequencemore » assembly positions. BAC-FISH will be useful for delineating each of the Populus trichocarpa chromosomes and improving the sequence assembly of this model angiosperm tree species.« less

Repeatless and repeat-based centromeres in potato: implications for centromere evolution.

PubMed

Gong, Zhiyun; Wu, Yufeng; Koblízková, Andrea; Torres, Giovana A; Wang, Kai; Iovene, Marina; Neumann, Pavel; Zhang, Wenli; Novák, Petr; Buell, C Robin; Macas, Jirí; Jiang, Jiming

2012-09-01

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats. By contrast, most newly formed centromeres (neocentromeres) do not contain satellite repeats and instead include DNA sequences representative of the genome. An unknown question in centromere evolution is how satellite repeat-based centromeres evolve from neocentromeres. We conducted a genome-wide characterization of sequences associated with CENH3 nucleosomes in potato (Solanum tuberosum). Five potato centromeres (Cen4, Cen6, Cen10, Cen11, and Cen12) consisted primarily of single- or low-copy DNA sequences. No satellite repeats were identified in these five centromeres. At least one transcribed gene was associated with CENH3 nucleosomes. Thus, these five centromeres structurally resemble neocentromeres. By contrast, six potato centromeres (Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) contained megabase-sized satellite repeat arrays that are unique to individual centromeres. The satellite repeat arrays likely span the entire functional cores of these six centromeres. At least four of the centromeric repeats were amplified from retrotransposon-related sequences and were not detected in Solanum species closely related to potato. The presence of two distinct types of centromeres, coupled with the boom-and-bust cycles of centromeric satellite repeats in Solanum species, suggests that repeat-based centromeres can rapidly evolve from neocentromeres by de novo amplification and insertion of satellite repeats in the CENH3 domains.

Repeatless and Repeat-Based Centromeres in Potato: Implications for Centromere Evolution[C][W

PubMed Central

Gong, Zhiyun; Wu, Yufeng; Koblížková, Andrea; Torres, Giovana A.; Wang, Kai; Iovene, Marina; Neumann, Pavel; Zhang, Wenli; Novák, Petr; Buell, C. Robin; Macas, Jiří; Jiang, Jiming

2012-01-01

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats. By contrast, most newly formed centromeres (neocentromeres) do not contain satellite repeats and instead include DNA sequences representative of the genome. An unknown question in centromere evolution is how satellite repeat-based centromeres evolve from neocentromeres. We conducted a genome-wide characterization of sequences associated with CENH3 nucleosomes in potato (Solanum tuberosum). Five potato centromeres (Cen4, Cen6, Cen10, Cen11, and Cen12) consisted primarily of single- or low-copy DNA sequences. No satellite repeats were identified in these five centromeres. At least one transcribed gene was associated with CENH3 nucleosomes. Thus, these five centromeres structurally resemble neocentromeres. By contrast, six potato centromeres (Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) contained megabase-sized satellite repeat arrays that are unique to individual centromeres. The satellite repeat arrays likely span the entire functional cores of these six centromeres. At least four of the centromeric repeats were amplified from retrotransposon-related sequences and were not detected in Solanum species closely related to potato. The presence of two distinct types of centromeres, coupled with the boom-and-bust cycles of centromeric satellite repeats in Solanum species, suggests that repeat-based centromeres can rapidly evolve from neocentromeres by de novo amplification and insertion of satellite repeats in the CENH3 domains. PMID:22968715

Thermodynamic and spectroscopic investigations of TMPyP4 association with guanine- and cytosine-rich DNA and RNA repeats of C9orf72.

PubMed

Alniss, Hasan; Zamiri, Bita; Khalaj, Melisa; Pearson, Christopher E; Macgregor, Robert B

2018-01-22

An expansion of the hexanucleotide repeat (GGGGCC)n·(GGCCCC)n in the C9orf72 promoter has been shown to be the cause of Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). The C9orf72 repeat can form four-stranded structures; the cationic porphyrin (TMPyP4) binds and distorts these structures. Isothermal titration calorimetry (ITC), and circular dichroism (CD) were used to study the binding of TMPyP4 to the C-rich and G-rich DNA and RNA oligos containing the hexanucleotide repeat at pH 7.5 and 0.1 M K + . The CD spectra of G-rich DNA and RNA TMPyP4 complexes showed features of antiparallel and parallel G-quadruplexes, respectively. The shoulder at 260 nm in the CD spectrum becomes more intense upon formation of complexes between TMPyP4 and the C-rich DNA. The peak at 290 nm becomes more intense in the c-rich RNA molecules, suggesting induction of an i-motif structure. The ITC data showed that TMPyP4 binds at two independent sites for all DNA and RNA molecules. For DNA, the data are consistent with TMPyP4 stacking on the terminal tetrads and intercalation. For RNA, the thermodynamics of the two binding modes are consistent with groove binding and intercalation. In both cases, intercalation is the weaker binding mode. These findings are considered with respect to the structural differences of the folded DNA and RNA molecules and the energetics of the processes that drive site-specific recognition by TMPyP4; these data will be helpful in efforts to optimize the specificity and affinity of the binding of porphyrin-like molecules. Copyright © 2018 Elsevier Inc. All rights reserved.

Developmental validation of the PowerPlex(®) Fusion 6C System.

PubMed

Ensenberger, Martin G; Lenz, Kristy A; Matthies, Learden K; Hadinoto, Gregory M; Schienman, John E; Przech, Angela J; Morganti, Michael W; Renstrom, Daniel T; Baker, Victoria M; Gawrys, Kori M; Hoogendoorn, Marlijn; Steffen, Carolyn R; Martín, Pablo; Alonso, Antonio; Olson, Hope R; Sprecher, Cynthia J; Storts, Douglas R

2016-03-01

The PowerPlex(®) Fusion 6C System is a 27-locus, six-dye, multiplex that includes all markers in the expanded CODIS core loci and increases overlap with STR database standards throughout the world. Additionally, it contains two, rapidly mutating, Y-STRs and is capable of both casework and database workflows, including direct amplification. A multi-laboratory developmental validation study was performed on the PowerPlex(®) Fusion 6C System. Here, we report the results of that study which followed SWGDAM guidelines and includes data for: species specificity, sensitivity, stability, precision, reproducibility and repeatability, case-type samples, concordance, stutter, DNA mixtures, and PCR-based procedures. Where appropriate we report data from both extracted DNA samples and direct amplification samples from various substrates and collection devices. Samples from all studies were separated on both Applied Biosystems 3500 series and 6-dye capable 3130 series Genetic Analyzers and data is reported for each. Together, the data validate the design and demonstrate the performance of the PowerPlex(®) Fusion 6C System. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Short-Sequence DNA Repeats in Prokaryotic Genomes

PubMed Central

van Belkum, Alex; Scherer, Stewart; van Alphen, Loek; Verbrugh, Henri

1998-01-01

Short-sequence DNA repeat (SSR) loci can be identified in all eukaryotic and many prokaryotic genomes. These loci harbor short or long stretches of repeated nucleotide sequence motifs. DNA sequence motifs in a single locus can be identical and/or heterogeneous. SSRs are encountered in many different branches of the prokaryote kingdom. They are found in genes encoding products as diverse as microbial surface components recognizing adhesive matrix molecules and specific bacterial virulence factors such as lipopolysaccharide-modifying enzymes or adhesins. SSRs enable genetic and consequently phenotypic flexibility. SSRs function at various levels of gene expression regulation. Variations in the number of repeat units per locus or changes in the nature of the individual repeat sequences may result from recombination processes or polymerase inadequacy such as slipped-strand mispairing (SSM), either alone or in combination with DNA repair deficiencies. These rather complex phenomena can occur with relative ease, with SSM approaching a frequency of 10−4 per bacterial cell division and allowing high-frequency genetic switching. Bacteria use this random strategy to adapt their genetic repertoire in response to selective environmental pressure. SSR-mediated variation has important implications for bacterial pathogenesis and evolutionary fitness. Molecular analysis of changes in SSRs allows epidemiological studies on the spread of pathogenic bacteria. The occurrence, evolution and function of SSRs, and the molecular methods used to analyze them are discussed in the context of responsiveness to environmental factors, bacterial pathogenicity, epidemiology, and the availability of full-genome sequences for increasing numbers of microorganisms, especially those that are medically relevant. PMID:9618442

Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean.

PubMed

Demeke, Tigst; Ratnayaka, Indira; Phan, Anh

2009-01-01

The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

DNABIT Compress – Genome compression algorithm

PubMed Central

Rajarajeswari, Pothuraju; Apparao, Allam

2011-01-01

Data compression is concerned with how information is organized in data. Efficient storage means removal of redundancy from the data being stored in the DNA molecule. Data compression algorithms remove redundancy and are used to understand biologically important molecules. We present a compression algorithm, “DNABIT Compress” for DNA sequences based on a novel algorithm of assigning binary bits for smaller segments of DNA bases to compress both repetitive and non repetitive DNA sequence. Our proposed algorithm achieves the best compression ratio for DNA sequences for larger genome. Significantly better compression results show that “DNABIT Compress” algorithm is the best among the remaining compression algorithms. While achieving the best compression ratios for DNA sequences (Genomes),our new DNABIT Compress algorithm significantly improves the running time of all previous DNA compression programs. Assigning binary bits (Unique BIT CODE) for (Exact Repeats, Reverse Repeats) fragments of DNA sequence is also a unique concept introduced in this algorithm for the first time in DNA compression. This proposed new algorithm could achieve the best compression ratio as much as 1.58 bits/bases where the existing best methods could not achieve a ratio less than 1.72 bits/bases. PMID:21383923

Transcription of highly repetitive tandemly organized DNA in amphibians and birds: A historical overview and modern concepts.

PubMed

Trofimova, Irina; Krasikova, Alla

2016-12-01

Tandemly organized highly repetitive DNA sequences are crucial structural and functional elements of eukaryotic genomes. Despite extensive evidence, satellite DNA remains an enigmatic part of the eukaryotic genome, with biological role and significance of tandem repeat transcripts remaining rather obscure. Data on tandem repeats transcription in amphibian and avian model organisms is fragmentary despite their genomes being thoroughly characterized. Review systematically covers historical and modern data on transcription of amphibian and avian satellite DNA in somatic cells and during meiosis when chromosomes acquire special lampbrush form. We highlight how transcription of tandemly repetitive DNA sequences is organized in interphase nucleus and on lampbrush chromosomes. We offer LTR-activation hypotheses of widespread satellite DNA transcription initiation during oogenesis. Recent explanations are provided for the significance of high-yield production of non-coding RNA derived from tandemly organized highly repetitive DNA. In many cases the data on the transcription of satellite DNA can be extrapolated from lampbrush chromosomes to interphase chromosomes. Lampbrush chromosomes with applied novel technical approaches such as superresolution imaging, chromosome microdissection followed by high-throughput sequencing, dynamic observation in life-like conditions provide amazing opportunities for investigation mechanisms of the satellite DNA transcription.

Transcription of highly repetitive tandemly organized DNA in amphibians and birds: A historical overview and modern concepts

PubMed Central

Krasikova, Alla

2016-01-01

ABSTRACT Tandemly organized highly repetitive DNA sequences are crucial structural and functional elements of eukaryotic genomes. Despite extensive evidence, satellite DNA remains an enigmatic part of the eukaryotic genome, with biological role and significance of tandem repeat transcripts remaining rather obscure. Data on tandem repeats transcription in amphibian and avian model organisms is fragmentary despite their genomes being thoroughly characterized. Review systematically covers historical and modern data on transcription of amphibian and avian satellite DNA in somatic cells and during meiosis when chromosomes acquire special lampbrush form. We highlight how transcription of tandemly repetitive DNA sequences is organized in interphase nucleus and on lampbrush chromosomes. We offer LTR-activation hypotheses of widespread satellite DNA transcription initiation during oogenesis. Recent explanations are provided for the significance of high-yield production of non-coding RNA derived from tandemly organized highly repetitive DNA. In many cases the data on the transcription of satellite DNA can be extrapolated from lampbrush chromosomes to interphase chromosomes. Lampbrush chromosomes with applied novel technical approaches such as superresolution imaging, chromosome microdissection followed by high-throughput sequencing, dynamic observation in life-like conditions provide amazing opportunities for investigation mechanisms of the satellite DNA transcription. PMID:27763817

A Large Population Genetic Study of 15 Autosomal Short Tandem Repeat Loci for Establishment of Korean DNA Profile Database

PubMed Central

Yoo, Seong Yeon; Cho, Nam Soo; Park, Myung Jin; Seong, Ki Min; Hwang, Jung Ho; Song, Seok Bean; Han, Myun Soo; Lee, Won Tae; Chung, Ki Wha

2011-01-01

Genotyping of highly polymorphic short tandem repeat (STR) markers is widely used for the genetic identification of individuals in forensic DNA analyses and in paternity disputes. The National DNA Profile Databank recently established by the DNA Identification Act in Korea contains the computerized STR DNA profiles of individuals convicted of crimes. For the establishment of a large autosomal STR loci population database, 1805 samples were obtained at random from Korean individuals and 15 autosomal STR markers were analyzed using the AmpFlSTR Identifiler PCR Amplification kit. For the 15 autosomal STR markers, no deviations from the Hardy-Weinberg equilibrium were observed. The most informative locus in our data set was the D2S1338 with a discrimination power of 0.9699. The combined matching probability was 1.521 × 10-17. This large STR profile dataset including atypical alleles will be important for the establishment of the Korean DNA database and for forensic applications. PMID:21597912

A large population genetic study of 15 autosomal short tandem repeat loci for establishment of Korean DNA profile database.

PubMed

Yoo, Seong Yeon; Cho, Nam Soo; Park, Myung Jin; Seong, Ki Min; Hwang, Jung Ho; Song, Seok Bean; Han, Myun Soo; Lee, Won Tae; Chung, Ki Wha

2011-07-01

Genotyping of highly polymorphic short tandem repeat (STR) markers is widely used for the genetic identification of individuals in forensic DNA analyses and in paternity disputes. The National DNA Profile Databank recently established by the DNA Identification Act in Korea contains the computerized STR DNA profiles of individuals convicted of crimes. For the establishment of a large autosomal STR loci population database, 1805 samples were obtained at random from Korean individuals and 15 autosomal STR markers were analyzed using the AmpFlSTR Identifiler PCR Amplification kit. For the 15 autosomal STR markers, no deviations from the Hardy-Weinberg equilibrium were observed. The most informative locus in our data set was the D2S1338 with a discrimination power of 0.9699. The combined matching probability was 1.521 × 10(-17). This large STR profile dataset including atypical alleles will be important for the establishment of the Korean DNA database and for forensic applications.

Repeated administration of CGP 46381, a gamma-aminobutyric acidB antagonist, and ethosuximide suppresses seizure-associated cyclic adenosine 3'5' monophosphate response element- and activator protein-1 DNA-binding activities in lethargic (lh/lh) mice.

PubMed

Ishige, K; Endo, H; Saito, H; Ito, Y

2001-01-19

To characterize seizure-associated increases in cerebral cortical and thalamic cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities in lethargic (lh/lh) mice, a genetic model of absence seizures, we examined the effects of ethosuximide and CGP 46381 on these DNA-binding activities. Repeated administration (twice a day for 5 days) of ethosuximide (200 mg/kg) or CGP 46381 (60 mg/kg) attenuated both seizure behavior and the increased DNA-binding activities, and was more effective than a single administration of these drugs. These treatments did not affect either normal behavior or basal DNA-binding activities in non-epileptic control (+/+) mice. Gel supershift assays revealed that the increased CRE-binding activity was attributable to activation of the binding activity of CREB, and that the c-Fos-c-Jun complex was a component of the increased AP-1 DNA-binding activity.

Evaluation of four automated protocols for extraction of DNA from FTA cards.

PubMed

Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura; Frank-Hansen, Rune; Poulsen, Lena; Hansen, Anders J; Morling, Niels

2013-10-01

Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already extracted from the FTA cards.

DNA synthesis arrest sites at the right terminus of rat long interspersed repeated (LINE or L1Rn) DNA family members.

PubMed Central

d'Ambrosio, E; Furano, A V

1987-01-01

An approximately equal to 150-bp GC-rich (approximately equal to 60%) region is at the right end of rat long interspersed repeated DNA (LINE or L1Rn) family members. We report here that one of the DNA strands from this region contains several non-palindromic sites that strongly arrest DNA synthesis in vitro by the prokaryotic Klenow and T4 DNA polymerases, the eukaryotic alpha polymerase, and AMV reverse transcriptase. The strongest arrest sites are G-rich (approximately equal to 70%) homopurine stretches of 18 or more residues. Shorter homopurine stretches (12 residues or fewer) did not arrest DNA synthesis even if the stretch contains 11/12 G residues. Arrest of the prokaryotic polymerases was not affected by their respective single strand binding proteins or polymerase accessory proteins. The region of duplex DNA which contains DNA synthesis arrest sites reacts with bromoacetaldehyde when present in negatively supercoiled molecules. By contrast, homopurine stretches that do not arrest DNA synthesis do not react with bromoacetaldehyde. The presence of bromoacetaldehyde-reactive bases in a G-rich homopurine-containing duplex under torsional stress is thought to be caused by base stacking in the homopurine strand. Therefore, we suggest that base-stacked regions of the template arrest DNA synthesis. Images PMID:2436148

Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes

PubMed Central

Lee, Michael; Hills, Mark; Conomos, Dimitri; Stutz, Michael D.; Dagg, Rebecca A.; Lau, Loretta M.S.; Reddel, Roger R.; Pickett, Hilda A.

2014-01-01

Telomeres are terminal repetitive DNA sequences on chromosomes, and are considered to comprise almost exclusively hexameric TTAGGG repeats. We have evaluated telomere sequence content in human cells using whole-genome sequencing followed by telomere read extraction in a panel of mortal cell strains and immortal cell lines. We identified a wide range of telomere variant repeats in human cells, and found evidence that variant repeats are generated by mechanistically distinct processes during telomerase- and ALT-mediated telomere lengthening. Telomerase-mediated telomere extension resulted in biased repeat synthesis of variant repeats that differed from the canonical sequence at positions 1 and 3, but not at positions 2, 4, 5 or 6. This indicates that telomerase is most likely an error-prone reverse transcriptase that misincorporates nucleotides at specific positions on the telomerase RNA template. In contrast, cell lines that use the ALT pathway contained a large range of variant repeats that varied greatly between lines. This is consistent with variant repeats spreading from proximal telomeric regions throughout telomeres in a stochastic manner by recombination-mediated templating of DNA synthesis. The presence of unexpectedly large numbers of variant repeats in cells utilizing either telomere maintenance mechanism suggests a conserved role for variant sequences at human telomeres. PMID:24225324

Analysis on the DNA Fingerprinting of Aspergillus Oryzae Mutant Induced by High Hydrostatic Pressure

NASA Astrophysics Data System (ADS)

Wang, Hua; Zhang, Jian; Yang, Fan; Wang, Kai; Shen, Si-Le; Liu, Bing-Bing; Zou, Bo; Zou, Guang-Tian

2011-01-01

The mutant strains of aspergillus oryzae (HP300a) are screened under 300 MPa for 20 min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively.

The Self Primer of the Long Terminal Repeat Retrotransposon Tf1 Is Not Removed during Reverse Transcription

PubMed Central

Atwood-Moore, Angela; Yan, Kenneth; Judson, Robert L.; Levin, Henry L.

2006-01-01

The long terminal repeat retrotransposon Tf1 of Schizosaccharomyces pombe uses a unique mechanism of self priming to initiate reverse transcription. Instead of using a tRNA, Tf1 primes minus-strand synthesis with an 11-nucleotide RNA removed from the 5′ end of its own transcript. We tested whether the self primer of Tf1 was similar to tRNA primers in being removed from the cDNA by RNase H. Our analysis of Tf1 cDNA extracted from virus-like particles revealed the surprising observation that the dominant species of cDNA retained the self primer. This suggests that integration of the cDNA relies on mechanisms other than reverse transcription to remove the primer. PMID:16873283

A ranking index for quality assessment of forensic DNA profiles forensic DNA profiles

PubMed Central

2010-01-01

Background Assessment of DNA profile quality is vital in forensic DNA analysis, both in order to determine the evidentiary value of DNA results and to compare the performance of different DNA analysis protocols. Generally the quality assessment is performed through manual examination of the DNA profiles based on empirical knowledge, or by comparing the intensities (allelic peak heights) of the capillary electrophoresis electropherograms. Results We recently developed a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles, the forensic DNA profile index (FI) (Hedman et al. Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles, Biotechniques 47 (2009) 951-958). FI uses electropherogram data to combine the intensities of the allelic peaks with the balances within and between loci, using Principal Components Analysis. Here we present the construction of FI. We explain the mathematical and statistical methodologies used and present details about the applied data reduction method. Thereby we show how to adapt the ranking index for any Short Tandem Repeat-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles. Conclusions The developed tool provides unbiased quality assessment of forensic DNA profiles. It can be applied for any DNA profiling system based on Short Tandem Repeat markers. Apart from crime related DNA analysis, FI can therefore be used as a quality tool in paternal or familial testing as well as in disaster victim identification. PMID:21062433

Evolutionary dynamics and sites of illegitimate recombination revealed in the interspersion and sequence junctions of two nonhomologous satellite DNAs in cactophilic Drosophila species.

PubMed

Kuhn, G C S; Teo, C H; Schwarzacher, T; Heslop-Harrison, J S

2009-05-01

Satellite DNA (satDNA) is a major component of genomes but relatively little is known about the fine-scale organization of unrelated satDNAs residing at the same chromosome location, and the sequence structure and dynamics of satDNA junctions. We studied the organization and sequence junctions of two nonhomologous satDNAs, pBuM and DBC-150, in three species from the neotropical Drosophila buzzatii cluster (repleta group). In situ hybridization to microchromosomes, interphase nuclei and extended DNA fibers showed frequent interspersion of the two satellites in D. gouveai, D. antonietae and, to a lesser extent, D. seriema. We isolated by PCR six pBuM x DBC-150 junctions: four are exclusive to D. gouveai and two are exclusive to D. antonietae. The six junction breakpoints occur at different positions within monomers, suggesting independent origin. Four junctions showed abrupt transitions between the two satellites, whereas two junctions showed a distinct 10 bp tandem duplication before the junction. Unlike pBuM, DBC-150 junction repeats are more variable than randomly cloned monomers and showed diagnostic features in common to a 3-monomer higher-order repeat seen in the sister species D. serido. The high levels of interspersion between pBuM and DBC-150 repeats suggest extensive rearrangements between the two satellites, maybe favored by specific features of the microchromosomes. Our interpretation is that the junctions evolved by multiples events of illegitimate recombination between nonhomologous satDNA repeats, with subsequent rounds of unequal crossing-over expanding the copy number of some of the junctions.

Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes.

PubMed

Al-Attar, Sinan; Westra, Edze R; van der Oost, John; Brouns, Stan J J

2011-04-01

Many prokaryotes contain the recently discovered defense system against mobile genetic elements. This defense system contains a unique type of repetitive DNA stretches, termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs). CRISPRs consist of identical repeated DNA sequences (repeats), interspaced by highly variable sequences referred to as spacers. The spacers originate from either phages or plasmids and comprise the prokaryotes' 'immunological memory'. CRISPR-associated (cas) genes encode conserved proteins that together with CRISPRs make-up the CRISPR/Cas system, responsible for defending the prokaryotic cell against invaders. CRISPR-mediated resistance has been proposed to involve three stages: (i) CRISPR-Adaptation, the invader DNA is encountered by the CRISPR/Cas machinery and an invader-derived short DNA fragment is incorporated in the CRISPR array. (ii) CRISPR-Expression, the CRISPR array is transcribed and the transcript is processed by Cas proteins. (iii) CRISPR-Interference, the invaders' nucleic acid is recognized by complementarity to the crRNA and neutralized. An application of the CRISPR/Cas system is the immunization of industry-relevant prokaryotes (or eukaryotes) against mobile-genetic invasion. In addition, the high variability of the CRISPR spacer content can be exploited for phylogenetic and evolutionary studies. Despite impressive progress during the last couple of years, the elucidation of several fundamental details will be a major challenge in future research.

Transposon-like properties of the major, long repetitive sequence family in the genome of Physarum polycephalum

PubMed Central

Pearston, Douglas H.; Gordon, Mairi; Hardman, Norman

1985-01-01

A family of long, highly-repetitive sequences, referred to previously as `HpaII-repeats', dominates the genome of the eukaryotic slime mould Physarum polycephalum. These sequences are found exclusively in scrambled clusters. They account for about one-half of the total complement of repetitive DNA in Physarum, and represent the major sequence component found in hypermethylated, 20-50 kb segments of Physarum genomic DNA that fail to be cleaved using the restriction endonuclease HpaII. The structure of this abundant repetitive element was investigated by analysing cloned segments derived from the hypermethylated genomic DNA compartment. We show that the `HpaII-repeat' forms part of a larger repetitive DNA structure, ∼8.6 kb in length, with several structural features in common with recognised eukaryotic transposable genetic elements. Scrambled clusters of the sequence probably arise as a result of transposition-like events, during which the element preferentially recombines in either orientation with target sites located in other copies of the same repeated sequence. The target sites for transposition/recombination are not related in sequence but in all cases studied they are potentially capable of promoting the formation of small `cruciforms' or `Z-DNA' structures which might be recognised during the recombination process. ImagesFig. 3.Fig. 4. PMID:16453652

A model for genesis of transcription systems.

PubMed

Burton, Zachary F; Opron, Kristopher; Wei, Guowei; Geiger, James H

2016-01-01

Repeating sequences generated from RNA gene fusions/ligations dominate ancient life, indicating central importance of building structural complexity in evolving biological systems. A simple and coherent story of life on earth is told from tracking repeating motifs that generate α/β proteins, 2-double-Ψ-β-barrel (DPBB) type RNA polymerases (RNAPs), general transcription factors (GTFs), and promoters. A general rule that emerges is that biological complexity that arises through generation of repeats is often bounded by solubility and closure (i.e., to form a pseudo-dimer or a barrel). Because the first DNA genomes were replicated by DNA template-dependent RNA synthesis followed by RNA template-dependent DNA synthesis via reverse transcriptase, the first DNA replication origins were initially 2-DPBB type RNAP promoters. A simplifying model for evolution of promoters/replication origins via repetition of core promoter elements is proposed. The model can explain why Pribnow boxes in bacterial transcription (i.e., (-12)TATAATG(-6)) so closely resemble TATA boxes (i.e., (-31)TATAAAAG(-24)) in archaeal/eukaryotic transcription. The evolution of anchor DNA sequences in bacterial (i.e., (-35)TTGACA(-30)) and archaeal (BRE(up); BRE for TFB recognition element) promoters is potentially explained. The evolution of BRE(down) elements of archaeal promoters is potentially explained.

Coactivator-associated arginine methyltransferase 1 enhances transcriptional activity of the human T-cell lymphotropic virus type 1 long terminal repeat through direct interaction with Tax.

PubMed

Jeong, Soo-Jin; Lu, Hanxin; Cho, Won-Kyung; Park, Hyeon Ung; Pise-Masison, Cynthia; Brady, John N

2006-10-01

In this study, we demonstrate that the coactivator-associated arginine methyltransferase 1 (CARM1), which methylates histone H3 and other proteins such as p300/CBP, is positively involved in the regulation of Tax transactivation. First, transfection studies demonstrated that overexpression of CARM1 wild-type protein resulted in increased Tax transactivation of the human T-cell lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR). In contrast, transfection of a catalytically inactive CARM1 methyltransferase mutant did not enhance Tax transactivation. CARM1 facilitated Tax transactivation of the CREB-dependent cellular GEM promoter. A direct physical interaction between HTLV-1 Tax and CARM1 was demonstrated using in vitro glutathione S-transferase-Tax binding assays, in vivo coimmunoprecipitation, and confocal microscopy experiments. Finally, chromatin immunoprecipitation analysis of the activated HTLV-1 LTR promoter showed the association of CARM1 and methylated histone H3 with the template DNA. In vitro, Tax facilitates the binding of CARM1 to the transcription complex. Together, our data provide evidence that CARM1 enhances Tax transactivation of the HTLV-1 LTR through a direct interaction between CARM1 and Tax and this binding promotes methylation of histone H3 (R2, R17, and R26).

Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples

PubMed Central

Pérez Santángelo, Agustín; Corti Bielsa, Rodrigo M.; Sala, Andrea; Ginart, Santiago; Corach, Daniel

2017-01-01

Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5’ region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA). PMID:29099837

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity

PubMed Central

Traverse, Charles C.

2017-01-01

ABSTRACT Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola, which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. PMID:28851848

Competition between B-Z and B-L transitions in a single DNA molecule: Computational studies

NASA Astrophysics Data System (ADS)

Kwon, Ah-Young; Nam, Gi-Moon; Johner, Albert; Kim, Seyong; Hong, Seok-Cheol; Lee, Nam-Kyung

2016-02-01

Under negative torsion, DNA adopts left-handed helical forms, such as Z-DNA and L-DNA. Using the random copolymer model developed for a wormlike chain, we represent a single DNA molecule with structural heterogeneity as a helical chain consisting of monomers which can be characterized by different helical senses and pitches. By Monte Carlo simulation, where we take into account bending and twist fluctuations explicitly, we study sequence dependence of B-Z transitions under torsional stress and tension focusing on the interaction with B-L transitions. We consider core sequences, (GC) n repeats or (TG) n repeats, which can interconvert between the right-handed B form and the left-handed Z form, imbedded in a random sequence, which can convert to left-handed L form with different (tension dependent) helical pitch. We show that Z-DNA formation from the (GC) n sequence is always supported by unwinding torsional stress but Z-DNA formation from the (TG) n sequence, which are more costly to convert but numerous, can be strongly influenced by the quenched disorder in the surrounding random sequence.

Independent mechanisms recruit the cohesin loader protein NIPBL to sites of DNA damage.

PubMed

Bot, Christopher; Pfeiffer, Annika; Giordano, Fosco; Manjeera, Dharani E; Dantuma, Nico P; Ström, Lena

2017-03-15

NIPBL is required to load the cohesin complex on to DNA. While the canonical role of cohesin is to couple replicated sister chromatids together until the onset of mitosis, it also promotes tolerance to DNA damage. Here, we show that NIPBL is recruited to DNA damage throughout the cell cycle via independent mechanisms, influenced by type of damage. First, the heterochromatin protein HP1γ (also known as CBX3) recruits NIPBL to DNA double-strand breaks (DSBs) through the corresponding HP1-binding motif within the N-terminus. By contrast, the C-terminal HEAT repeat domain is unable to recruit NIPBL to DSBs but independently targets NIPBL to laser microirradiation-induced DNA damage. Each mechanism is dependent on the RNF8 and RNF168 ubiquitylation pathway, while the recruitment of the HEAT repeat domain requires further ATM or ATR activity. Thus, NIPBL has evolved a sophisticated response to damaged DNA that is influenced by the form of damage, suggesting a highly dynamic role for NIPBL in maintaining genomic stability. © 2017. Published by The Company of Biologists Ltd.

A polymerase chain reaction strategy for the diagnosis of camelpox.

PubMed

Balamurugan, Vinayagamurthy; Bhanuprakash, Veerakyathappa; Hosamani, Madhusudhan; Jayappa, Kallesh Danappa; Venkatesan, Gnanavel; Chauhan, Bina; Singh, Raj Kumar

2009-03-01

Camelpox is a contagious viral skin disease that is mostly seen in young camels. The disease is caused by the Camelpox virus (CMLV). In the present study, a polymerase chain reaction (PCR) assay based on the C18L gene (encoding ankyrin repeat protein) and a duplex PCR based on the C18L and DNA polymerase (DNA pol) genes were developed. The former assay yields a specific amplicon of 243 bp of the C18L gene, whereas the duplex PCR yields 243- and 96-bp products of the C18L and DNA pol genes, respectively, in CMLV, and only a 96-bp product of the DNA pol gene in other orthopoxviruses. The limit of detection was as low as 0.4 ng of viral DNA. Both PCR assays were employed successfully for the direct detection and differentiation of CMLV from other orthopoxviruses, capripoxviruses, and parapoxviruses in both cell culture samples and clinical material. Furthermore, a highly sensitive SYBR Green dye-based, real-time PCR was optimized for quantitation of CMLV DNA. In the standard curve of the quantitative assay, the melting temperature of the specific amplicon at 77.6 degrees C with peak measured fluorescence in dissociation plot was observed with an efficiency of 102%. To the authors' knowledge, this is the first report to describe a C18L gene-based PCR for specific diagnosis of camelpox infection.

A new yeast gene with a myosin-like heptad repeat structure.

PubMed

Kölling, R; Nguyen, T; Chen, E Y; Botstein, D

1993-03-01

We isolated a gene encoding a 218 kDa myosin-like protein from Saccharomyces cerevisiae using a monoclonal antibody directed against human platelet myosin as a probe. The protein sequence encoded by the MLP1 gene (for myosin-like protein) contains extensive stretches of a heptad-repeat pattern suggesting that the protein can form coiled coils typical of myosins. Immunolocalization experiments using affinity-purified antibodies raised against a TrpE-MLP1 fusion protein showed a dot-like structure adjacent to the nucleus in yeast cells bearing the MLP1 gene on a multicopy plasmid. In mouse epithelial cells the yeast anti-MLP1 antibodies stained the nucleus. Mutants bearing disruptions of the MLP1 gene were viable, but more sensitive to ultraviolet light than wild-type strains, suggesting an involvement of MLP1 in DNA repair. The MLP1 gene was mapped to chromosome 11, 25 cM from met1.

Minimal and Contributing Sequence Determinants of the cis-Acting Locus of Transfer (clt) of Streptomycete Plasmid pIJ101 Occur within an Intrinsically Curved Plasmid Region

PubMed Central

Ducote, Matthew J.; Prakash, Shubha; Pettis, Gregg S.

2000-01-01

Efficient interbacterial transfer of streptomycete plasmid pIJ101 requires the pIJ101 tra gene, as well as a cis-acting plasmid function known as clt. Here we show that the minimal pIJ101 clt locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3′ end of the korB regulatory gene. Evidence that sequences extending beyond the minimal locus and into the korB open reading frame influence clt transfer function and demonstration that clt-korB sequences are intrinsically curved raise the possibility that higher-order structuring of DNA and protein within this plasmid region may be an inherent feature of efficient pIJ101 transfer. PMID:11073933

Minimal and contributing sequence determinants of the cis-acting locus of transfer (clt) of streptomycete plasmid pIJ101 occur within an intrinsically curved plasmid region.

PubMed

Ducote, M J; Prakash, S; Pettis, G S

2000-12-01

Efficient interbacterial transfer of streptomycete plasmid pIJ101 requires the pIJ101 tra gene, as well as a cis-acting plasmid function known as clt. Here we show that the minimal pIJ101 clt locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3' end of the korB regulatory gene. Evidence that sequences extending beyond the minimal locus and into the korB open reading frame influence clt transfer function and demonstration that clt-korB sequences are intrinsically curved raise the possibility that higher-order structuring of DNA and protein within this plasmid region may be an inherent feature of efficient pIJ101 transfer.

The coactivator CBP stimulates human T-cell lymphotrophic virus type I Tax transactivation in vitro.

PubMed

Kashanchi, F; Duvall, J F; Kwok, R P; Lundblad, J R; Goodman, R H; Brady, J N

1998-12-18

Tax interacts with the cellular cyclic AMP-responsive element binding protein (CREB) and facilitates the binding of the coactivator CREB binding protein (CBP), forming a multimeric complex on the cyclic AMP-responsive element (CRE)-like sites in the human T-cell lymphotrophic virus type I (HTLV-I) promoter. The trimeric complex is believed to recruit additional regulatory proteins to the HTLV-I long terminal repeat, but there has been no direct evidence that CBP is required for Tax-mediated transactivation. We present evidence that Tax and CBP activate transcription from the HTLV-I 21 base pair repeats on naked DNA templates. Transcriptional activation of the HTLV-I sequences required both Tax and CBP and could be mediated by either the N-terminal activation domain of CBP or the full-length protein. Fluorescence polarization binding assays indicated that CBP does not markedly enhance the affinity of Tax for the trimeric complex. Transcription analyses suggest that CBP activates Tax-dependent transcription by promoting transcriptional initiation and reinitiation. The ability of CBP to activate the HTLV-I promoter does not involve the stabilization of Tax binding, but rather depends upon gene activation properties of the co-activator that function in the context of a naked DNA template.

TAD-free analysis of architectural proteins and insulators.

PubMed

Mourad, Raphaël; Cuvier, Olivier

2018-03-16

The three-dimensional (3D) organization of the genome is intimately related to numerous key biological functions including gene expression and DNA replication regulations. The mechanisms by which molecular drivers functionally organize the 3D genome, such as topologically associating domains (TADs), remain to be explored. Current approaches consist in assessing the enrichments or influences of proteins at TAD borders. Here, we propose a TAD-free model to directly estimate the blocking effects of architectural proteins, insulators and DNA motifs on long-range contacts, making the model intuitive and biologically meaningful. In addition, the model allows analyzing the whole Hi-C information content (2D information) instead of only focusing on TAD borders (1D information). The model outperforms multiple logistic regression at TAD borders in terms of parameter estimation accuracy and is validated by enhancer-blocking assays. In Drosophila, the results support the insulating role of simple sequence repeats and suggest that the blocking effects depend on the number of repeats. Motif analysis uncovered the roles of the transcriptional factors pannier and tramtrack in blocking long-range contacts. In human, the results suggest that the blocking effects of the well-known architectural proteins CTCF, cohesin and ZNF143 depend on the distance between loci, where each protein may participate at different scales of the 3D chromatin organization.

A theory that may explain the Hayflick limit--a means to delete one copy of a repeating sequence during each cell cycle in certain human cells such as fibroblasts.

PubMed

Naveilhan, P; Baudet, C; Jabbour, W; Wion, D

1994-09-01

A model that may explain the limited division potential of certain cells such as human fibroblasts in culture is presented. The central postulate of this theory is that there exists, prior to certain key exons that code for materials needed for cell division, a unique sequence of specific repeating segments of DNA. One copy of such repeating segments is deleted during each cell cycle in cells that are not protected from such deletion through methylation of their cytosine residues. According to this theory, the means through which such repeated sequences are removed, one per cycle, is through the sequential action of enzymes that act much as bacterial restriction enzymes do--namely to produce scissions in both strands of DNA in areas that correspond to the DNA base sequence recognition specificities of such enzymes. After the first scission early in a replicative cycle, that enzyme becomes inhibited, but the cleavage of the first site exposes the closest site in the repetitive element to the action of a second restriction enzyme after which that enzyme also becomes inhibited. Then repair occurs, regenerating the original first site. Through this sequential activation and inhibition of two different restriction enzymes, only one copy of the repeating sequence is deleted during each cell cycle. In effect, the repeating sequence operates as a precise counter of the numbers of cell doubling that have occurred since the cells involved differentiated during development.

Repeated oral dosing of TAS-102 confers high trifluridine incorporation into DNA and sustained antitumor activity in mouse models

PubMed Central

TANAKA, NOZOMU; SAKAMOTO, KAZUKI; OKABE, HIROYUKI; FUJIOKA, AKIO; YAMAMURA, KEISUKE; NAKAGAWA, FUMIO; NAGASE, HIDEKI; YOKOGAWA, TATSUSHI; OGUCHI, KEI; ISHIDA, KEIJI; OSADA, AKIKO; KAZUNO, HIROMI; YAMADA, YUKARI; MATSUO, KENICHI

2014-01-01

TAS-102 is a novel oral nucleoside antitumor agent containing trifluridine (FTD) and tipiracil hydrochloride (TPI). The compound improves overall survival of colorectal cancer (CRC) patients who are insensitive to standard chemotherapies. FTD possesses direct antitumor activity since it inhibits thymidylate synthase (TS) and is itself incorporated into DNA. However, the precise mechanisms underlying the incorporation into DNA and the inhibition of TS remain unclear. We found that FTD-dependent inhibition of TS was similar to that elicited by fluorodeoxyuridine (FdUrd), another clinically used nucleoside analog. However, washout experiments revealed that FTD-dependent inhibition of TS declined rapidly, whereas FdUrd activity persisted. The incorporation of FTD into DNA was significantly higher than that of other antitumor nucleosides. Additionally, orally administered FTD had increased antitumor activity and was incorporated into DNA more effectively than continuously infused FTD. When TAS-102 was administered, FTD gradually accumulated in tumor cell DNA, in a TPI-independent manner, and significantly delayed tumor growth and prolonged survival, compared to treatment with 5-FU derivatives. TAS-102 reduced the Ki-67-positive cell fraction, and swollen nuclei were observed in treated tumor tissue. The amount of FTD incorporation in DNA and the antitumor activity of TAS-102 in xenograft models were positively and significantly correlated. These results suggest that TAS-102 exerts its antitumor activity predominantly due to its DNA incorporation, rather than as a result of TS inhibition. The persistence of FTD in the DNA of tumor cells treated with TAS-102 may underlie its ability to prolong survival in cancer patients. PMID:25230742

Mitochondrial Mutations in Subjects with Psychiatric Disorders

PubMed Central

Magnan, Christophe; van Oven, Mannis; Baldi, Pierre; Myers, Richard M.; Barchas, Jack D.; Schatzberg, Alan F.; Watson, Stanley J.; Akil, Huda; Bunney, William E.; Vawter, Marquis P.

2015-01-01

A considerable body of evidence supports the role of mitochondrial dysfunction in psychiatric disorders and mitochondrial DNA (mtDNA) mutations are known to alter brain energy metabolism, neurotransmission, and cause neurodegenerative disorders. Genetic studies focusing on common nuclear genome variants associated with these disorders have produced genome wide significant results but those studies have not directly studied mtDNA variants. The purpose of this study is to investigate, using next generation sequencing, the involvement of mtDNA variation in bipolar disorder, schizophrenia, major depressive disorder, and methamphetamine use. MtDNA extracted from multiple brain regions and blood were sequenced (121 mtDNA samples with an average of 8,800x coverage) and compared to an electronic database containing 26,850 mtDNA genomes. We confirmed novel and rare variants, and confirmed next generation sequencing error hotspots by traditional sequencing and genotyping methods. We observed a significant increase of non-synonymous mutations found in individuals with schizophrenia. Novel and rare non-synonymous mutations were found in psychiatric cases in mtDNA genes: ND6, ATP6, CYTB, and ND2. We also observed mtDNA heteroplasmy in brain at a locus previously associated with schizophrenia (T16519C). Large differences in heteroplasmy levels across brain regions within subjects suggest that somatic mutations accumulate differentially in brain regions. Finally, multiplasmy, a heteroplasmic measure of repeat length, was observed in brain from selective cases at a higher frequency than controls. These results offer support for increased rates of mtDNA substitutions in schizophrenia shown in our prior results. The variable levels of heteroplasmic/multiplasmic somatic mutations that occur in brain may be indicators of genetic instability in mtDNA. PMID:26011537

Capturing the Biofuel Wellhead and Powerhouse: The Chloroplast and Mitochondrial Genomes of the Leguminous Feedstock Tree Pongamia pinnata

PubMed Central

Kazakoff, Stephen H.; Imelfort, Michael; Edwards, David; Koehorst, Jasper; Biswas, Bandana; Batley, Jacqueline; Scott, Paul T.; Gresshoff, Peter M.

2012-01-01

Pongamia pinnata (syn. Millettia pinnata) is a novel, fast-growing arboreal legume that bears prolific quantities of oil-rich seeds suitable for the production of biodiesel and aviation biofuel. Here, we have used Illumina® ‘Second Generation DNA Sequencing (2GS)’ and a new short-read de novo assembler, SaSSY, to assemble and annotate the Pongamia chloroplast (152,968 bp; cpDNA) and mitochondrial (425,718 bp; mtDNA) genomes. We also show that SaSSY can be used to accurately assemble 2GS data, by re-assembling the Lotus japonicus cpDNA and in the process assemble its mtDNA (380,861 bp). The Pongamia cpDNA contains 77 unique protein-coding genes and is almost 60% gene-dense. It contains a 50 kb inversion common to other legumes, as well as a novel 6.5 kb inversion that is responsible for the non-disruptive, re-orientation of five protein-coding genes. Additionally, two copies of an inverted repeat firmly place the species outside the subclade of the Fabaceae lacking the inverted repeat. The Pongamia and L. japonicus mtDNA contain just 33 and 31 unique protein-coding genes, respectively, and like other angiosperm mtDNA, have expanded intergenic and multiple repeat regions. Through comparative analysis with Vigna radiata we measured the average synonymous and non-synonymous divergence of all three legume mitochondrial (1.59% and 2.40%, respectively) and chloroplast (8.37% and 8.99%, respectively) protein-coding genes. Finally, we explored the relatedness of Pongamia within the Fabaceae and showed the utility of the organellar genome sequences by mapping transcriptomic data to identify up- and down-regulated stress-responsive gene candidates and confirm in silico predicted RNA editing sites. PMID:23272141

Capturing the biofuel wellhead and powerhouse: the chloroplast and mitochondrial genomes of the leguminous feedstock tree Pongamia pinnata.

PubMed

Kazakoff, Stephen H; Imelfort, Michael; Edwards, David; Koehorst, Jasper; Biswas, Bandana; Batley, Jacqueline; Scott, Paul T; Gresshoff, Peter M

2012-01-01

Pongamia pinnata (syn. Millettia pinnata) is a novel, fast-growing arboreal legume that bears prolific quantities of oil-rich seeds suitable for the production of biodiesel and aviation biofuel. Here, we have used Illumina® 'Second Generation DNA Sequencing (2GS)' and a new short-read de novo assembler, SaSSY, to assemble and annotate the Pongamia chloroplast (152,968 bp; cpDNA) and mitochondrial (425,718 bp; mtDNA) genomes. We also show that SaSSY can be used to accurately assemble 2GS data, by re-assembling the Lotus japonicus cpDNA and in the process assemble its mtDNA (380,861 bp). The Pongamia cpDNA contains 77 unique protein-coding genes and is almost 60% gene-dense. It contains a 50 kb inversion common to other legumes, as well as a novel 6.5 kb inversion that is responsible for the non-disruptive, re-orientation of five protein-coding genes. Additionally, two copies of an inverted repeat firmly place the species outside the subclade of the Fabaceae lacking the inverted repeat. The Pongamia and L. japonicus mtDNA contain just 33 and 31 unique protein-coding genes, respectively, and like other angiosperm mtDNA, have expanded intergenic and multiple repeat regions. Through comparative analysis with Vigna radiata we measured the average synonymous and non-synonymous divergence of all three legume mitochondrial (1.59% and 2.40%, respectively) and chloroplast (8.37% and 8.99%, respectively) protein-coding genes. Finally, we explored the relatedness of Pongamia within the Fabaceae and showed the utility of the organellar genome sequences by mapping transcriptomic data to identify up- and down-regulated stress-responsive gene candidates and confirm in silico predicted RNA editing sites.

From America to Eurasia: a multigenomes history of the genus Abies.

PubMed

Semerikova, Svetlana A; Khrunyk, Yuliya Y; Lascoux, Martin; Semerikov, Vladimir L

2018-03-15

The origin of conifer genera, the main components of mountain temperate and boreal forests, was deemed to arise in the Mesozoic, although paleontological records and molecular data point to a recent diversification, presumably related to Neogene cooling. The geographical area(s) where the modern lines of conifers emerged remains uncertain, as is the sequence of events leading to their present distribution. To gain further insights into the biogeography of firs (Abies), we conducted phylogenetic analyses of chloroplast, mitochondrial and nuclear markers. The species tree, generated from ten single-copy nuclear genes, yielded probably the best phylogenetic hypothesis available for Abies. The tree obtained from five regions of chloroplast DNA largely corresponded to the nuclear species tree. Ancestral area reconstructions based on fossil calibrated chloroplast DNA and nuclear DNA trees pointed to repeated intercontinental migrations. The mitochondrial DNA haplotype tree, however, disagreed with nuclear and chloroplast DNA trees. It consisted of two clusters: one included mainly American haplotypes, while the other was composed of only Eurasian haplotypes. Presumably, this conflict is due to inter-continental migrations and introgressive hybridization, accompanied by the capture of the mitotypes from aboriginal species by the invading firs. Given that several species inhabiting Northeastern Asia carry American mitotypes and mutations typical for the American cluster, whereas no Asian mitotypes were detected within the American species, we hypothesize that Abies migrated from America to Eurasia, but not in the opposite direction. The direction and age of intercontinental migrations in firs are congruent with other conifers, such as spruces and pines of subsection Strobus, suggesting that these events had the same cause. Copyright © 2018 Elsevier Inc. All rights reserved.

Germ line insertion of mtDNA at the breakpoint junction of a reciprocal constitutional translocation.

PubMed

Willett-Brozick, J E; Savul, S A; Richey, L E; Baysal, B E

2001-08-01

Constitutional chromosomal translocations are relatively common causes of human morbidity, yet the DNA double-strand break (DSB) repair mechanisms that generate them are incompletely understood. We cloned, sequenced and analyzed the breakpoint junctions of a familial constitutional reciprocal translocation t(9;11)(p24;q23). Within the 10-kb region flanking the breakpoints, chromosome 11 had 25% repeat elements, whereas chromosome 9 had 98% repeats, 95% of which were L1-type LINE elements. The breakpoints occurred within an L1-type repeat element at 9p24 and at the 3'-end of an Alu sequence at 11q23. At the breakpoint junction of derivative chromosome 9, we discovered an unusually large 41-bp insertion, which showed 100% identity to 12S mitochondrial DNA (mtDNA) between nucleotides 896 and 936 of the mtDNA sequence. Analysis of the human genome failed to show the preexistence of the inserted sequence at normal chromosomes 9 and 11 breakpoint junctions or elsewhere in the genome, strongly suggesting that the insertion was derived from human mtDNA and captured into the junction during the DSB repair process. To our knowledge, these findings represent the first observation of spontaneous germ line insertion of modern human mtDNA sequences and suggest that DSB repair may play a role in inter-organellar gene transfer in vivo. Our findings also provide evidence for a previously unrecognized insertional mechanism in human, by which non-mobile extra-chromosomal fragments can be inserted into the genome at DSB repair junctions.

The rolling-circle melting-pot model for porcine circovirus DNA replication

USDA-ARS?s Scientific Manuscript database

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

Checking of individuality by DNA profiling.

PubMed

Brdicka, R; Nürnberg, P

1993-08-25

A review of methods of DNA analysis used in forensic medicine for identification, paternity testing, etc. is provided. Among other techniques, DNA fingerprinting using different probes and polymerase chain reaction-based techniques such as amplified sequence polymorphisms and minisatellite variant repeat mapping are thoroughly described and both theoretical and practical aspects are discussed.

Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System

PubMed Central

Cooper, Lauren A.; Stringer, Anne M.

2018-01-01

ABSTRACT In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5′ end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing. PMID:29666291

The minisatellite of the GPI/AMF/NLK/MF gene: interspecies conservation and transcriptional activity.

PubMed

Williams, R R; Hassan-Walker, A F; Lavender, F L; Morgan, M; Faik, P; Ragoussis, J

2001-05-16

Minisatellites are tandemly repeated DNA sequences found throughout the genomes of all eukaryotes. They are regions often prone to instability and hence hypervariability; thus repeat unit sequence is generally not conserved beyond closely related species. We have studied the minisatellite located in intron 9 of the human glucose phosphate isomerase (GPI) gene (also known as neuroleukin, autocrine motility factor, maturation and differentiation factor) and have found, by Zoo blotting coupled with PCR amplification and DNA sequencing, that similar repeat units are present in seven other species of mammal. There is also evidence for the presence of the minisatellite in chicken. The repeat unit does not appear to be present at any other locus in these genomes. Minisatellite DNA has been reported to be involved in recombination activity, control of gene expression of nearby gene(s) (both transcriptional and translational), whilst others form protein coding regions. The high level of conservation exhibited by the GPI minisatellite, coupled with the unique location, strongly suggests a functional role. Our results from transient and stable transfections using luciferase reporter constructs have shown that the GPI minisatellite region can act to increase transcription from the SV40 promoter, CMV promoter and the human GPI promoter.

Mutational Dynamics of Aroid Chloroplast Genomes

PubMed Central

Ahmed, Ibrar; Biggs, Patrick J.; Matthews, Peter J.; Collins, Lesley J.; Hendy, Michael D.; Lockhart, Peter J.

2012-01-01

A characteristic feature of eukaryote and prokaryote genomes is the co-occurrence of nucleotide substitution and insertion/deletion (indel) mutations. Although similar observations have also been made for chloroplast DNA, genome-wide associations have not been reported. We determined the chloroplast genome sequences for two morphotypes of taro (Colocasia esculenta; family Araceae) and compared these with four publicly available aroid chloroplast genomes. Here, we report the extent of genome-wide association between direct and inverted repeats, indels, and substitutions in these aroid chloroplast genomes. We suggest that alternative but not mutually exclusive hypotheses explain the mutational dynamics of chloroplast genome evolution. PMID:23204304

Inhibition of RNA polymerase II allows controlled mobilisation of retrotransposons for plant breeding.

PubMed

Thieme, Michael; Lanciano, Sophie; Balzergue, Sandrine; Daccord, Nicolas; Mirouze, Marie; Bucher, Etienne

2017-07-07

Retrotransposons play a central role in plant evolution and could be a powerful endogenous source of genetic and epigenetic variability for crop breeding. To ensure genome integrity several silencing mechanisms have evolved to repress retrotransposon mobility. Even though retrotransposons fully depend on transcriptional activity of the host RNA polymerase II (Pol II) for their mobility, it was so far unclear whether Pol II is directly involved in repressing their activity. Here we show that plants defective in Pol II activity lose DNA methylation at repeat sequences and produce more extrachromosomal retrotransposon DNA upon stress in Arabidopsis and rice. We demonstrate that combined inhibition of both DNA methylation and Pol II activity leads to a strong stress-dependent mobilization of the heat responsive ONSEN retrotransposon in Arabidopsis seedlings. The progenies of these treated plants contain up to 75 new ONSEN insertions in their genome which are stably inherited over three generations of selfing. Repeated application of heat stress in progeny plants containing increased numbers of ONSEN copies does not result in increased activation of this transposon compared to control lines. Progenies with additional ONSEN copies show a broad panel of environment-dependent phenotypic diversity. We demonstrate that Pol II acts at the root of transposon silencing. This is important because it suggests that Pol II can regulate the speed of plant evolution by fine-tuning the amplitude of transposon mobility. Our findings show that it is now possible to study induced transposon bursts in plants and unlock their use to induce epigenetic and genetic diversity for crop breeding.

RECG Maintains Plastid and Mitochondrial Genome Stability by Suppressing Extensive Recombination between Short Dispersed Repeats

PubMed Central

Odahara, Masaki; Masuda, Yuichi; Sato, Mayuko; Wakazaki, Mayumi; Harada, Chizuru; Toyooka, Kiminori; Sekine, Yasuhiko

2015-01-01

Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO) mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8–79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA) instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12–63 bp) in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions. PMID:25769081

In Depth Characterization of Repetitive DNA in 23 Plant Genomes Reveals Sources of Genome Size Variation in the Legume Tribe Fabeae.

PubMed

Macas, Jiří; Novák, Petr; Pellicer, Jaume; Čížková, Jana; Koblížková, Andrea; Neumann, Pavel; Fuková, Iva; Doležel, Jaroslav; Kelly, Laura J; Leitch, Ilia J

2015-01-01

The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55-83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57%) of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%). Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes.

Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development.

PubMed

Sun, Cheng; Wyngaard, Grace; Walton, D Brian; Wichman, Holly A; Mueller, Rachel Lockridge

2014-03-11

Chromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution--some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 - 75 Gb, 12-74 Gb of which are lost from pre-somatic cell lineages at germline--soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown. Here, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes of Mesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome. Our results suggest that germline genome expansion in M. edax reflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms.

Visual ModuleOrganizer: a graphical interface for the detection and comparative analysis of repeat DNA modules

PubMed Central

2014-01-01

Background DNA repeats, such as transposable elements, minisatellites and palindromic sequences, are abundant in sequences and have been shown to have significant and functional roles in the evolution of the host genomes. In a previous study, we introduced the concept of a repeat DNA module, a flexible motif present in at least two occurences in the sequences. This concept was embedded into ModuleOrganizer, a tool allowing the detection of repeat modules in a set of sequences. However, its implementation remains difficult for larger sequences. Results Here we present Visual ModuleOrganizer, a Java graphical interface that enables a new and optimized version of the ModuleOrganizer tool. To implement this version, it was recoded in C++ with compressed suffix tree data structures. This leads to less memory usage (at least 120-fold decrease in average) and decreases by at least four the computation time during the module detection process in large sequences. Visual ModuleOrganizer interface allows users to easily choose ModuleOrganizer parameters and to graphically display the results. Moreover, Visual ModuleOrganizer dynamically handles graphical results through four main parameters: gene annotations, overlapping modules with known annotations, location of the module in a minimal number of sequences, and the minimal length of the modules. As a case study, the analysis of FoldBack4 sequences clearly demonstrated that our tools can be extended to comparative and evolutionary analyses of any repeat sequence elements in a set of genomic sequences. With the increasing number of sequences available in public databases, it is now possible to perform comparative analyses of repeated DNA modules in a graphic and friendly manner within a reasonable time period. Availability Visual ModuleOrganizer interface and the new version of the ModuleOrganizer tool are freely available at: http://lcb.cnrs-mrs.fr/spip.php?rubrique313. PMID:24678954

Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development

PubMed Central

2014-01-01

Background Chromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution — some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 – 75 Gb, 12–74 Gb of which are lost from pre-somatic cell lineages at germline – soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown. Results Here, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes of Mesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome. Conclusions Our results suggest that germline genome expansion in M. edax reflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms. PMID:24618421

Recent advances in therapeutic recruitment of mammalian RNAi and bacterial CRISPR-Cas DNA interference pathways as emerging antiviral strategies.

PubMed

Chin, Wei-Xin; Ang, Swee Kim; Chu, Justin Jang Hann

2017-01-01

In invertebrate eukaryotes and prokaryotes, respectively, the RNAi and clustered regularly interspaced short palindromic repeats-CRISPR-associated (CRISPR-Cas) pathways are highly specific and efficient RNA and DNA interference systems, and are well characterised as potent antiviral systems. It has become possible to recruit or reconstitute these pathways in mammalian cells, where they can be directed against desired host or viral targets. The RNAi and CRISPR-Cas systems can therefore yield ideal antiviral therapeutics, capable of specific and efficient viral inhibition with minimal off-target effects, but development of such therapeutics can be slow. This review covers recent advances made towards developing RNAi or CRISPR-Cas strategies for clinical use. These studies address the delivery, toxicity or target design issues that typically plague the in vivo or clinical use of these technologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Direct CRISPR spacer acquisition from RNA by a natural reverse-transcriptase-Cas1 fusion protein

PubMed Central

Sidote, David J.; Markham, Laura M.; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M.; Fire, Andrew Z.

2016-01-01

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in Type I and II CRISPR systems by the acquisition of short segments of DNA (“spacers”) from invasive elements. In several Type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we show that an RT-Cas1 fusion enables the acquisition of RNA spacers in vivo in an RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze ligation of RNA segments into the CRISPR array, followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data.

PubMed

Chin, Chen-Shan; Alexander, David H; Marks, Patrick; Klammer, Aaron A; Drake, James; Heiner, Cheryl; Clum, Alicia; Copeland, Alex; Huddleston, John; Eichler, Evan E; Turner, Stephen W; Korlach, Jonas

2013-06-01

We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.

Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

PubMed Central

van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D.

2015-01-01

The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.

PubMed

Shewale, Jaiprakash G; Schneida, Elaine; Wilson, Jonathan; Walker, Jerilyn A; Batzer, Mark A; Sinha, Sudhir K

2007-03-01

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.

Reductionist Approach in Peptide-Based Nanotechnology.

PubMed

Gazit, Ehud

2018-06-20

The formation of ordered nanostructures by molecular self-assembly of proteins and peptides represents one of the principal directions in nanotechnology. Indeed, polyamides provide superior features as materials with diverse physical properties. A reductionist approach allowed the identification of extremely short peptide sequences, as short as dipeptides, which could form well-ordered amyloid-like β-sheet-rich assemblies comparable to supramolecular structures made of much larger proteins. Some of the peptide assemblies show remarkable mechanical, optical, and electrical characteristics. Another direction of reductionism utilized a natural noncoded amino acid, α-aminoisobutryic acid, to form short superhelical assemblies. The use of this exceptional helix inducer motif allowed the fabrication of single heptad repeats used in various biointerfaces, including their use as surfactants and DNA-binding agents. Two additional directions of the reductionist approach include the use of peptide nucleic acids (PNAs) and coassembly techniques. The diversified accomplishments of the reductionist approach, as well as the exciting future advances it bears, are discussed.

Recurrent herpetic keratitis: failure to detect herpes simplex virus infection using the Syva MicroTrak HSV1/HSV2 direct specimen identification/typing test.

PubMed

Kumano, Y; Yamamoto, M; Inomata, H; Sakuma, S; Hidaka, Y; Minagawa, H; Mori, R

1990-01-01

A 35-year-old man had developed recurrent herpetic keratitis characterized by dendritic keratitis at intervals of a year. We were able to culture cytopathic agents repeatedly from his lesions by inoculating Vero cells. The cultures yielded definitive evidence of a virus that caused a cytopathic effect within 3 days. However, these virus strains could not be identified as herpes simplex virus (HSV) in immunofluorescence assays using the Syva MicroTrak HSV1/HSV2 direct specimen identification/typing test. Rather they were identified as strains of HSV type 1 (HSV-1) on the basis of plaque morphology, neutralization tests, electron-microscopic examination and DNA restriction endonuclease analysis. Our results allow us to assume the existence of HSV-1 strains isolated clinically that are negative to analysis using the Syva Micro-Trak HSV1/HSV2 direct specimen identification/typing test.

[Analysis on genetic polymorphism of 5 STR loci selected from X chromosome].

PubMed

Liu, Qi-ji; Gong, Yao-qin; Zhang, Xi-yu; Gao, Gui-min; Li, Jiang-xia; Guo, Yi-shou

2005-02-01

To select short tandem repeats(STR) from X chromosome. STR is a universal genetic marker that has changeable polymorphism and stable heredity in human genome. It is a specific DNA segment composed of 2-6 base pairs as its core sequence. It is an ideal DNA marker used in linkage analysis and gene mapping. In this study, 8 short tandem repeats were selected from two genomic clones on X chromosome by using BCM Search Launcher. Primers amplifying the STR loci were designed by using Primer 3.0 according to the unique sequence flanking the STRs. Polymorphisms of the short tandem repeats in Chinese population were evaluated by PCR amplification and PAGE. Five of these STRs were polymorphic. Chi-square test indicated that the distribution of genotypes agreed with Hardy-Weinberg equilibrium (P>0.05). Five polymorphic short tandem repeats have been identified on chromosome X and will be useful for linkage analysis and gene mapping.

Chromosome evolution in the Thermotogales: large-scale inversions and strain diversification of CRISPR sequences.

PubMed

DeBoy, Robert T; Mongodin, Emmanuel F; Emerson, Joanne B; Nelson, Karen E

2006-04-01

In the present study, the chromosomes of two members of the Thermotogales were compared. A whole-genome alignment of Thermotoga maritima MSB8 and Thermotoga neapolitana NS-E has revealed numerous large-scale DNA rearrangements, most of which are associated with CRISPR DNA repeats and/or tRNA genes. These DNA rearrangements do not include the putative origin of DNA replication but move within the same replichore, i.e., the same replicating half of the chromosome (delimited by the replication origin and terminus). Based on cumulative GC skew analysis, both the T. maritima and T. neapolitana lineages contain one or two major inverted DNA segments. Also, based on PCR amplification and sequence analysis of the DNA joints that are associated with the major rearrangements, the overall chromosome architecture was found to be conserved at most DNA joints for other strains of T. neapolitana. Taken together, the results from this analysis suggest that the observed chromosomal rearrangements in the Thermotogales likely occurred by successive inversions after their divergence from a common ancestor and before strain diversification. Finally, sequence analysis shows that size polymorphisms in the DNA joints associated with CRISPRs can be explained by expansion and possibly contraction of the DNA repeat and spacer unit, providing a tool for discerning the relatedness of strains from different geographic locations.

A Portrait of Ribosomal DNA Contacts with Hi-C Reveals 5S and 45S rDNA Anchoring Points in the Folded Human Genome

PubMed Central

Yu, Shoukai; Lemos, Bernardo

2016-01-01

Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. PMID:27797956

Microsatellite DNA capture from enriched libraries.

PubMed

Gonzalez, Elena G; Zardoya, Rafael

2013-01-01

Microsatellites are DNA sequences of tandem repeats of one to six nucleotides, which are highly polymorphic, and thus the molecular markers of choice in many kinship, population genetic, and conservation studies. There have been significant technical improvements since the early methods for microsatellite isolation were developed, and today the most common procedures take advantage of the hybrid capture methods of enriched-targeted microsatellite DNA. Furthermore, recent advents in sequencing technologies (i.e., next-generation sequencing, NGS) have fostered the mining of microsatellite markers in non-model organisms, affording a cost-effective way of obtaining a large amount of sequence data potentially useful for loci characterization. The rapid improvements of NGS platforms together with the increase in available microsatellite information open new avenues to the understanding of the evolutionary forces that shape genetic structuring in wild populations. Here, we provide detailed methodological procedures for microsatellite isolation based on the screening of GT microsatellite-enriched libraries, either by cloning and Sanger sequencing of positive clones or by direct NGS. Guides for designing new species-specific primers and basic genotyping are also given.

Design and performance testing of a real-time PCR assay for sensitive and reliable direct quantification of Brettanomyces in wine.

PubMed

Tessonnière, H; Vidal, S; Barnavon, L; Alexandre, H; Remize, F

2009-02-28

Because the yeast Brettanomyces produces volatile phenols and acetic acid, it is responsible for wine spoilage. The uncontrolled accumulation of these molecules in wine leads to sensorial defects that compromise wine quality. The need for a rapid, specific, sensitive and reliable method to detect this spoilage yeast has increased over the last decade. All these requirements are met by real-time PCR. We here propose improvements of existing methods to enhance the robustness of the assay. Six different protocols to isolate DNA from a wine and three PCR mix compositions were tested, and the best method was selected. Insoluble PVPP addition during DNA extraction by a classical phenol:chloroform protocol succeeded in the relief of PCR inhibitors from wine. We developed an internal control which was efficient to avoid false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The method was evaluated by an intra-laboratory study for its specificity, linearity, repeatability and reproducibility. A standard curve was established from 14 different wines artificially inoculated. The quantification limit was 31 cfu/mL.

Detection of Variation in Long-Term Micropropagated Mature Pistachio via DNA-Based Molecular Markers.

PubMed

Akdemir, Hülya; Suzerer, Veysel; Tilkat, Engin; Onay, Ahmet; Çiftçi, Yelda Ozden

2016-12-01

Determination of genetic stability of in vitro-grown plantlets is needed for safe and large-scale production of mature trees. In this study, genetic variation of long-term micropropagated mature pistachio developed through direct shoot bud regeneration using apical buds (protocol A) and in vitro-derived leaves (protocol B) was assessed via DNA-based molecular markers. Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) were employed, and the obtained PIC values from RAPD (0.226), ISSR (0.220), and AFLP (0.241) showed that micropropagation of pistachio for different periods of time resulted in "reasonable polymorphism" among donor plant and its 18 clones. Mantel's test showed a consistence polymorphism level between marker systems based on similarity matrices. In conclusion, this is the first study on occurrence of genetic variability in long-term micropropagated mature pistachio plantlets. The obtained results clearly indicated that different marker approaches used in this study are reliable for assessing tissue culture-induced variations in long-term cultured pistachio plantlets.

Target innervation is necessary for neuronal polyploidization in the terrestrial slug Limax.

PubMed

Matsuo, Ryota; Yamagishi, Miki; Wakiya, Kyoko; Tanaka, Yoko; Ito, Etsuro

2013-05-30

The brain of gastropod mollusks contains many giant neurons with polyploid genomic DNAs. Such DNAs are generated through repeated DNA endoreplication during body growth. However, it is not known what triggers DNA endoreplication in neurons. There are two possibilities: (1) DNAs are replicated in response to some unknown molecules in the hemolymph that reflect the nutritive status of the animal; or (2) DNAs are replicated in response to some unknown factors that are retrogradely transported through axons from the innervated target organs. We first tested whether hemolymph with rich nutrition could induce DNA endoreplication. We tested whether the transplanted brain exhibits enhanced DNA endoreplication like an endogenous brain does when transplanted into the homocoel of the body of a slug whose body growth is promoted by an increased food supply. However, no enhancement was observed in the frequency of DNA endoreplication when we compared the transplanted brains in the growth-promoted and growth-suppressed host slugs, suggesting that the humoral environment is irrelevant to triggering the body growth-dependent DNA endoreplication. Next, we tested the requirement of target innervation by surgically dissecting a unilateral posterior pedal nerve of an endogenous brain. Substantially lower number of neurons exhibited DNA endoreplication in the pedal ganglion ipsilateral to the dissected nerve. These results support the view that enhanced DNA endoreplication is mediated by target innervation and is not brought about through the direct effect of humoral factors in the hemolymph during body growth. © 2013 Wiley Periodicals, Inc. Develop Neurobiol, 2013. Copyright © 2013 Wiley Periodicals, Inc.

Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction.

PubMed

Hicks, Matt N; Gunasekara, Sanjiva; Serate, Jose; Park, Jin; Mosharaf, Pegah; Zhou, Yue; Lee, Jin-Won; Youn, Hwan

2017-10-01

The Escherichia coli cAMP receptor protein (CRP) utilizes the helix-turn-helix motif for DNA binding. The CRP's recognition helix, termed F-helix, includes a stretch of six amino acids (Arg180, Glu181, Thr182, Val183, Gly184, and Arg185) for direct DNA contacts. Arg180, Glu181 and Arg185 are known as important residues for DNA binding and specificity, but little has been studied for the other residues. Here we show that Gly184 is another F-helix residue critical for the transcriptional activation function of CRP. First, glycine was repeatedly selected at CRP position 184 for its unique ability to provide wild type-level transcriptional activation activity. To dissect the glycine requirement, wild type CRP and mutants G184A, G184F, G184S, and G184Y were purified and their in vitro DNA-binding activity was measured. G184A and G184F displayed reduced DNA binding, which may explain their low transcriptional activation activity. However, G184S and G184Y displayed apparently normal DNA affinity. Therefore, an additional factor is needed to account for the diminished transcriptional activation function in G184S and G184Y, and the best explanation is perturbations in their interaction with RNA polymerase. The fact that glycine is the smallest amino acid could not fully warrant its suitability, as shown in this study. We hypothesize that Gly184 fulfills the dual functions of DNA binding and RNA polymerase interaction by conferring conformational flexibility to the F-helix.

Repetitive DNA in the pea (Pisum sativum L.) genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula

PubMed Central

Macas, Jiří; Neumann, Pavel; Navrátilová, Alice

2007-01-01

Background Extraordinary size variation of higher plant nuclear genomes is in large part caused by differences in accumulation of repetitive DNA. This makes repetitive DNA of great interest for studying the molecular mechanisms shaping architecture and function of complex plant genomes. However, due to methodological constraints of conventional cloning and sequencing, a global description of repeat composition is available for only a very limited number of higher plants. In order to provide further data required for investigating evolutionary patterns of repeated DNA within and between species, we used a novel approach based on massive parallel sequencing which allowed a comprehensive repeat characterization in our model species, garden pea (Pisum sativum). Results Analysis of 33.3 Mb sequence data resulted in quantification and partial sequence reconstruction of major repeat families occurring in the pea genome with at least thousands of copies. Our results showed that the pea genome is dominated by LTR-retrotransposons, estimated at 140,000 copies/1C. Ty3/gypsy elements are less diverse and accumulated to higher copy numbers than Ty1/copia. This is in part due to a large population of Ogre-like retrotransposons which alone make up over 20% of the genome. In addition to numerous types of mobile elements, we have discovered a set of novel satellite repeats and two additional variants of telomeric sequences. Comparative genome analysis revealed that there are only a few repeat sequences conserved between pea and soybean genomes. On the other hand, all major families of pea mobile elements are well represented in M. truncatula. Conclusion We have demonstrated that even in a species with a relatively large genome like pea, where a single 454-sequencing run provided only 0.77% coverage, the generated sequences were sufficient to reconstruct and analyze major repeat families corresponding to a total of 35–48% of the genome. These data provide a starting point for further investigations of legume plant genomes based on their global comparative analysis and for the development of more sophisticated approaches for data mining. PMID:18031571

Characterization and assessment of an avian repetitive DNA sequence as an icterid phylogenetic marker.

PubMed

Quinn, J S; Guglich, E; Seutin, G; Lau, R; Marsolais, J; Parna, L; Boag, P T; White, B N

1992-02-01

The first tandemly repeated sequence examined in a passerine bird, a 431-bp PstI fragment named pMAT1, has been cloned from the genome of the brown-headed cowbird (Molothrus ater). The sequence represents about 5-10% of the genome (about 4 x 10(5) copies) and yields prominent ethidium bromide stained bands when genomic DNA cut with a variety of restriction enzymes is electrophoresed in agarose gels. A particularly striking ladder of fragments is apparent when the DNA is cut with HinfI, indicative of a tandem arrangement of the monomer. The cloned PstI monomer has been sequenced, revealing no internal repeated structure. There are sequences that hybridize with pMAT1 found in related nine-primaried oscines but not in more distantly related oscines, suboscines, or nonpasserine species. Little sequence similarity to tandemly repeated PstI cut sequences from the merlin (Falco columbarius), saurus crane (Grus antigone), or Puerto Rican parrot (Amazona vittata) or to HinfI digested sequence from the Toulouse goose (Anser anser) was detected. The isolated sequence was used as a probe to examine DNA samples of eight members of the tribe Icterini. This examination revealed phylogenetically informative characters. The repeat contains cutting sites from a number of restriction enzymes, which, if sufficiently polymorphic, would provide new phylogenetic characters. Sequences like these, conserved within a species, but variable between closely related species, may be very useful for phylogenetic studies of closely related taxa.

Conserved DNA motifs in the type II-A CRISPR leader region.

PubMed

Van Orden, Mason J; Klein, Peter; Babu, Kesavan; Najar, Fares Z; Rajan, Rakhi

2017-01-01

The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas) systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer) site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3' end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3' leader end: ATTTGAG (noted previously in the CRISPR1 locus of Streptococcus thermophilus DGCC7710) and a newly defined CTRCGAG, associated with the CRISPR3 locus of S. thermophilus DGCC7710. A third group with a very short CG DNA conservation at the 3' leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci.

Conserved DNA motifs in the type II-A CRISPR leader region

PubMed Central

Babu, Kesavan; Najar, Fares Z.

2017-01-01

The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas) systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer) site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3′ end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3′ leader end: ATTTGAG (noted previously in the CRISPR1 locus of Streptococcus thermophilus DGCC7710) and a newly defined CTRCGAG, associated with the CRISPR3 locus of S. thermophilus DGCC7710. A third group with a very short CG DNA conservation at the 3′ leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci. PMID:28392985

CRISPR-mediated direct mutation of cancer genes in the mouse liver.

PubMed

Xue, Wen; Chen, Sidi; Yin, Hao; Tammela, Tuomas; Papagiannakopoulos, Thales; Joshi, Nikhil S; Cai, Wenxin; Yang, Gillian; Bronson, Roderick; Crowley, Denise G; Zhang, Feng; Anderson, Daniel G; Sharp, Phillip A; Jacks, Tyler

2014-10-16

The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.

Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences*

PubMed Central

Cilli, Piera; Minoprio, Anna; Bossa, Cecilia; Bignami, Margherita; Mazzei, Filomena

2015-01-01

The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase β (POL β) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL β incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL β was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences. PMID:26338705

MiniX-STR multiplex system population study in Japan and application to degraded DNA analysis.

PubMed

Asamura, H; Sakai, H; Kobayashi, K; Ota, M; Fukushima, H

2006-05-01

We sought to evaluate a more effective system for analyzing X-chromosomal short tandem repeats (X-STRs) in highly degraded DNA. To generate smaller amplicon lengths, we designed new polymerase chain reaction (PCR) primers for DXS7423, DXS6789, DXS101, GATA31E08, DXS8378, DXS7133, DXS7424, and GATA165B12 at X-linked short tandem repeat (STR) loci, devising two miniX-multiplex PCR systems. Among 333 Japanese individuals, these X-linked loci were detected in amplification products ranging in length from 76 to 169 bp, and statistical analyses of the eight loci indicated a high usefulness for the Japanese forensic practice. Results of tests on highly degraded DNA indicated the miniX-STR multiplex strategies to be an effective system for analyzing degraded DNA. We conclude that analysis by the current miniX-STR multiplex systems offers high effectiveness for personal identification from degraded DNA samples.

Uncoupling of Satellite DNA and Centromeric Function in the Genus Equus

PubMed Central

Magnani, Elisa; Bertoni, Livia; Attolini, Carmen; Khoriauli, Lela; Raimondi, Elena; Giulotto, Elena

2010-01-01

In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs. PMID:20169180

Peculiarities of RFLP of highly repetitive DNA in crow genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chelomina, G.N.; Kryukov, A.P.; Ivanov, S.V.

1995-02-01

We present a study of the structural organization of highly repetitive DNA in genomes of hooded crow Corvus cornix L., carrion crow C. corone L., and jungle crow C. macrorhynchos Wagl. RFLP and blot-hybridization with {sup 32}P-labeled Msp I fragment from hooded crow nDNA suggest the interspecific structural conservatism of the most repetitive DNA. The family of repeats we studied had tandem organization and the same (210 bp) period of reiteration for a set of restriction enzymes. However, in parallel to the general similarity of restriction patterns, there are species-specific peculiarities. The repetitive family revealed (Alu I, BsuR I, andmore » Msp I fragments) has quantitative RFLP of nDNA and interspecific differences in the extent of the multimer {open_quotes}ladder{close_quotes} pattern of Msp I fragments. The latter is more pronounced in nDNA of carrion crow than in that of phylogenetically distant jungle crow and closely related hooded crow. This suggests a recent amplification event for highly organized homological repeats in crow genomes. 10 refs., 2 figs.« less

DNA Methylation Analysis of the Angiotensin Converting Enzyme (ACE) Gene in Major Depression

PubMed Central

Zill, Peter; Baghai, Thomas C.; Schüle, Cornelius; Born, Christoph; Früstück, Clemens; Büttner, Andreas; Eisenmenger, Wolfgang; Varallo-Bedarida, Gabriella; Rupprecht, Rainer; Möller, Hans-Jürgen; Bondy, Brigitta

2012-01-01

Background The angiotensin converting enzyme (ACE) has been repeatedly discussed as susceptibility factor for major depression (MD) and the bi-directional relation between MD and cardiovascular disorders (CVD). In this context, functional polymorphisms of the ACE gene have been linked to depression, to antidepressant treatment response, to ACE serum concentrations, as well as to hypertension, myocardial infarction and CVD risk markers. The mostly investigated ACE Ins/Del polymorphism accounts for ∼40%–50% of the ACE serum concentration variance, the remaining half is probably determined by other genetic, environmental or epigenetic factors, but these are poorly understood. Materials and Methods The main aim of the present study was the analysis of the DNA methylation pattern in the regulatory region of the ACE gene in peripheral leukocytes of 81 MD patients and 81 healthy controls. Results We detected intensive DNA methylation within a recently described, functional important region of the ACE gene promoter including hypermethylation in depressed patients (p = 0.008) and a significant inverse correlation between the ACE serum concentration and ACE promoter methylation frequency in the total sample (p = 0.02). Furthermore, a significant inverse correlation between the concentrations of the inflammatory CVD risk markers ICAM-1, E-selectin and P-selectin and the degree of ACE promoter methylation in MD patients could be demonstrated (p = 0.01 - 0.04). Conclusion The results of the present study suggest that aberrations in ACE promoter DNA methylation may be an underlying cause of MD and probably a common pathogenic factor for the bi-directional relationship between MD and cardiovascular disorders. PMID:22808171

Protein domain analysis of genomic sequence data reveals regulation of LRR related domains in plant transpiration in Ficus.

PubMed

Lang, Tiange; Yin, Kangquan; Liu, Jinyu; Cao, Kunfang; Cannon, Charles H; Du, Fang K

2014-01-01

Predicting protein domains is essential for understanding a protein's function at the molecular level. However, up till now, there has been no direct and straightforward method for predicting protein domains in species without a reference genome sequence. In this study, we developed a functionality with a set of programs that can predict protein domains directly from genomic sequence data without a reference genome. Using whole genome sequence data, the programming functionality mainly comprised DNA assembly in combination with next-generation sequencing (NGS) assembly methods and traditional methods, peptide prediction and protein domain prediction. The proposed new functionality avoids problems associated with de novo assembly due to micro reads and small single repeats. Furthermore, we applied our functionality for the prediction of leucine rich repeat (LRR) domains in four species of Ficus with no reference genome, based on NGS genomic data. We found that the LRRNT_2 and LRR_8 domains are related to plant transpiration efficiency, as indicated by the stomata index, in the four species of Ficus. The programming functionality established in this study provides new insights for protein domain prediction, which is particularly timely in the current age of NGS data expansion.

The SIDER2 elements, interspersed repeated sequences that populate the Leishmania genomes, constitute subfamilies showing chromosomal proximity relationship.

PubMed

Requena, Jose M; Folgueira, Cristina; López, Manuel C; Thomas, M Carmen

2008-06-02

Protozoan parasites of the genus Leishmania are causative agents of a diverse spectrum of human diseases collectively known as leishmaniasis. These eukaryotic pathogens that diverged early from the main eukaryotic lineage possess a number of unusual genomic, molecular and biochemical features. The completion of the genome projects for three Leishmania species has generated invaluable information enabling a direct analysis of genome structure and organization. By using DNA macroarrays, made with Leishmania infantum genomic clones and hybridized with total DNA from the parasite, we identified a clone containing a repeated sequence. An analysis of the recently completed genome sequence of L. infantum, using this repeated sequence as bait, led to the identification of a new class of repeated elements that are interspersed along the different L. infantum chromosomes. These elements turned out to be homologues of SIDER2 sequences, which were recently identified in the Leishmania major genome; thus, we adopted this nomenclature for the Leishmania elements described herein. Since SIDER2 elements are very heterogeneous in sequence, their precise identification is rather laborious. We have characterized 54 LiSIDER2 elements in chromosome 32 and 27 ones in chromosome 20. The mean size for these elements is 550 bp and their sequence is G+C rich (mean value of 66.5%). On the basis of sequence similarity, these elements can be grouped in subfamilies that show a remarkable relationship of proximity, i.e. SIDER2s of a given subfamily locate close in a chromosomal region without intercalating elements. For comparative purposes, we have identified the SIDER2 elements existing in L. major and Leishmania braziliensis chromosomes 32. While SIDER2 elements are highly conserved both in number and location between L. infantum and L. major, no such conservation exists when comparing with SIDER2s in L. braziliensis chromosome 32. SIDER2 elements constitute a relevant piece in the Leishmania genome organization. Sequence characteristics, genomic distribution and evolutionarily conservation of SIDER2s are suggestive of relevant functions for these elements in Leishmania. Apart from a proved involvement in post-transcriptional mechanisms of gene regulation, SIDER2 elements could be involved in DNA amplification processes and, perhaps, in chromosome segregation as centromeric sequences.

Repeated bouts of exhaustive exercise increase circulating cell free nuclear and mitochondrial DNA without development of tolerance in healthy men

PubMed Central

Stawski, Robert; Walczak, Konrad; Kosielski, Piotr; Meissner, Pawel; Budlewski, Tomasz; Padula, Gianluca; Nowak, Dariusz

2017-01-01

Objective Acute single strenuous exercise increases circulating cell free DNA (cf DNA). We tested whether three repeated bouts of exhaustive exercise induced the cf DNA response without development of tolerance in healthy men. Methods Eleven average-trained men (age 34.0±5.2 years, body mass index 26.2±3.1 kg/m2, maximal oxygen consumption—VO2max 49.6±4.5 ml/kg*min) performed three treadmill exercise tests to exhaustion at speed corresponding to 70% VO2max separated by 72 hours of resting. Blood was collected before and after each bout of exercise for determination of cell free nuclear and mitochondrial DNA (cf n-DNA, cf mt-DNA) by real-time PCR, selected markers of muscle damage, and blood cell count. Results Each bout induced the increase (p<0.05) in plasma cf n-DNA: from 3.4±1.4 to 38.5±27.5, from 4.1±3.3 to 48.5±26.2, and 3.1±1.6 to 53.8±39.9 ng/mL after the first, second, and third exercise, respectively. In a congruent way, cf mt-DNA rose significantly after the second (from 229±216 to 450±228*103 GE/mL) and third bout of exercise (from 173±120 to 462±314*103 GE/mL). Pre-exercise cf mt-DNA decreased (p<0.05) by 2-times (from 355±219 before the first bout to 173±120*103 GE/mL before the third bout) over the study period and were accompanied by significant increase in white blood cells, platelets, creatine kinase, creatinine and lactate after each bout. However, the exercise induced percentage increment of cf n-DNA was always many times higher than corresponding increments of the afore-mentioned markers at any occasion. Conclusions Repeated bouts of exhaustive exercise induced remarkable increase in circulating cf n-DNA without signs of tolerance development. Baseline cf mt-DNA decreased in response to series of strenuous exercise. Since percentage increments of cf n-DNA in response to exercise were many times higher than those observed for other markers, measurement of circulating cf n-DNA could be a sensitive tool for monitoring acute exercise effects in human body. PMID:28542490

Risk score predicts high-grade prostate cancer in DNA-methylation positive, histopathologically negative biopsies.

PubMed

Van Neste, Leander; Partin, Alan W; Stewart, Grant D; Epstein, Jonathan I; Harrison, David J; Van Criekinge, Wim

2016-09-01

Prostate cancer (PCa) diagnosis is challenging because efforts for effective, timely treatment of men with significant cancer typically result in over-diagnosis and repeat biopsies. The presence or absence of epigenetic aberrations, more specifically DNA-methylation of GSTP1, RASSF1, and APC in histopathologically negative prostate core biopsies has resulted in an increased negative predictive value (NPV) of ∼90% and thus could lead to a reduction of unnecessary repeat biopsies. Here, it is investigated whether, in methylation-positive men, DNA-methylation intensities could help to identify those men harboring high-grade (Gleason score ≥7) PCa, resulting in an improved positive predictive value. Two cohorts, consisting of men with histopathologically negative index biopsies, followed by a positive or negative repeat biopsy, were combined. EpiScore, a methylation intensity algorithm was developed in methylation-positive men, using area under the curve of the receiver operating characteristic as metric for performance. Next, a risk score was developed combining EpiScore with traditional clinical risk factors to further improve the identification of high-grade (Gleason Score ≥7) cancer. Compared to other risk factors, detection of DNA-methylation in histopathologically negative biopsies was the most significant and important predictor of high-grade cancer, resulting in a NPV of 96%. In methylation-positive men, EpiScore was significantly higher for those with high-grade cancer detected upon repeat biopsy, compared to those with either no or low-grade cancer. The risk score resulted in further improvement of patient risk stratification and was a significantly better predictor compared to currently used metrics as PSA and the prostate cancer prevention trial (PCPT) risk calculator (RC). A decision curve analysis indicated strong clinical utility for the risk score as decision-making tool for repeat biopsy. Low DNA-methylation levels in PCa-negative biopsies led to a NPV of 96% for high-grade cancer. The risk score, comprising DNA-methylation intensity and traditional clinical risk factors, improved the identification of men with high-grade cancer, with a maximum avoidance of unnecessary repeat biopsies. This risk score resulted in better patient risk stratification and significantly outperformed current risk prediction models such as PCPTRC and PSA. The risk score could help to identify patients with histopathologically negative biopsies harboring high-grade PCa. Prostate 76:1078-1087, 2016. © 2016 The Authors. The Prostate Published by Wiley Periodicals, Inc. © 2016 The Authors. The Prostate Published by Wiley Periodicals, Inc.

All five host-range variants of Xanthomonas citri carry one pthA homolog with 17.5 repeats that determines pathogenicity on citrus, but none determine host-range variation.

PubMed

Al-Saadi, Abdulwahid; Reddy, Joseph D; Duan, Yong P; Brunings, Asha M; Yuan, Qiaoping; Gabriel, Dean W

2007-08-01

Citrus canker disease is caused by five groups of Xanthomonas citri strains that are distinguished primarily by host range: three from Asia (A, A*, and A(w)) and two that form a phylogenetically distinct clade and originated in South America (B and C). Every X. citri strain carries multiple DNA fragments that hybridize with pthA, which is essential for the pathogenicity of wide-host-range X. citri group A strain 3213. DNA fragments that hybridized with pthA were cloned from a representative strain from all five groups. Each strain carried one and only one pthA homolog that functionally complemented a knockout mutation of pthA in 3213. Every complementing homolog was of identical size to pthA and carried 17.5 nearly identical, direct tandem repeats, including three new genes from narrow-host-range groups C (pthC), A(w) (pthAW), and A* (pthA*). Every noncomplementing paralog was of a different size; one of these was sequenced from group A* (pthA*-2) and was found to have an intact promoter and full-length reading frame but with 15.5 repeats. None of the complementing homologs nor any of the noncomplementing paralogs conferred avirulence to 3213 on grapefruit or suppressed avirulence of a group A* strain on grapefruit. A knockout mutation of pthC in a group C strain resulted in loss of pathogenicity on lime, but the strain was unaffected in ability to elicit an HR on grapefruit. This pthC- mutant was fully complemented by pthA, pthB, or pthC. Analysis of the predicted amino-acid sequences of all functional pthA homologs and nonfunctional paralogs indicated that the specific sequence of the 17th repeat may be essential for pathogenicity of X. citri on citrus.

Forensic DNA testing.

PubMed

Butler, John M

2011-12-01

Forensic DNA testing has a number of applications, including parentage testing, identifying human remains from natural or man-made disasters or terrorist attacks, and solving crimes. This article provides background information followed by an overview of the process of forensic DNA testing, including sample collection, DNA extraction, PCR amplification, short tandem repeat (STR) allele separation and sizing, typing and profile interpretation, statistical analysis, and quality assurance. The article concludes with discussions of possible problems with the data and other forensic DNA testing techniques.

Self-organisation of an oligodeoxynucleotide containing the G- and C-rich stretches of the direct repeats of the human mitochondrial DNA.

PubMed

Nonin-Lecomte, Sylvie; Dardel, Frédéric; Lestienne, Patrick

2005-08-01

Stretches of cytosines and guanosines have been shown in vitro to adopt non-canonical structures known as i-motifs and G-quartets, respectively. When combined, such sequences are expected to either retain their structure or form duplexes or triple helices. All these structures may occur in vivo whenever the sequence criteria are met. Such stretches are present in the circular genome of human mitochondria, as two 10 nucleotide-long perfect tandem direct repeats (DR1 and DR2). The DR1 and DR2 repeats are G-rich on the heavy strand and C-rich on the light strand. Previous results suggested that during replication, transient formation of a parallel GGC triple helix between the neo-synthesised G-rich DR1 and the double-stranded homologous DR2 could be involved in a rearrangement process leading to genome instability. In order to get structural insights into the interaction between the two repeats, we have studied by nuclear magnetic resonance (NMR) the assembly properties of a 24-mer oligodeoxyribonucleotide in which the C- and G-rich segments of the DRs are covalently tethered by a TTTT linker. We show here that this 24-mer self-associates into a triplex-containing symmetrical tetramer. The core of the structure is composed of anti-parallel Watson-Crick (WC) base pairs. Two additional strands are hydrogen-bonded to the Hoogsteen side of the Gs, thus forming CGC(+) triple helices, with G-rich ends folding into G-quartets. These results suggest that such structures could occur when the two DRs are put to close proximity in a biological context.

Molecular Identification of Date Palm Cultivars Using Random Amplified Polymorphic DNA (RAPD) Markers.

PubMed

Al-Khalifah, Nasser S; Shanavaskhan, A E

2017-01-01

Ambiguity in the total number of date palm cultivars across the world is pointing toward the necessity for an enumerative study using standard morphological and molecular markers. Among molecular markers, DNA markers are more suitable and ubiquitous to most applications. They are highly polymorphic in nature, frequently occurring in genomes, easy to access, and highly reproducible. Various molecular markers such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) markers have been successfully used as efficient tools for analysis of genetic variation in date palm. This chapter explains a stepwise protocol for extracting total genomic DNA from date palm leaves. A user-friendly protocol for RAPD analysis and a table showing the primers used in different molecular techniques that produce polymorphisms in date palm are also provided.

Missing genes, multiple ORFs, and C-to-U type RNA editing in Acrasis kona (Heterolobosea, Excavata) mitochondrial DNA.

PubMed

Fu, Cheng-Jie; Sheikh, Sanea; Miao, Wei; Andersson, Siv G E; Baldauf, Sandra L

2014-08-21

Discoba (Excavata) is an ancient group of eukaryotes with great morphological and ecological diversity. Unlike the other major divisions of Discoba (Jakobida and Euglenozoa), little is known about the mitochondrial DNAs (mtDNAs) of Heterolobosea. We have assembled a complete mtDNA genome from the aggregating heterolobosean amoeba, Acrasis kona, which consists of a single circular highly AT-rich (83.3%) molecule of 51.5 kb. Unexpectedly, A. kona mtDNA is missing roughly 40% of the protein-coding genes and nearly half of the transfer RNAs found in the only other sequenced heterolobosean mtDNAs, those of Naegleria spp. Instead, over a quarter of A. kona mtDNA consists of novel open reading frames. Eleven of the 16 protein-coding genes missing from A. kona mtDNA were identified in its nuclear DNA and polyA RNA, and phylogenetic analyses indicate that at least 10 of these 11 putative nuclear-encoded mitochondrial (NcMt) proteins arose by direct transfer from the mitochondrion. Acrasis kona mtDNA also employs C-to-U type RNA editing, and 12 homologs of DYW-type pentatricopeptide repeat (PPR) proteins implicated in plant organellar RNA editing are found in A. kona nuclear DNA. A mapping of mitochondrial gene content onto a consensus phylogeny reveals a sporadic pattern of relative stasis and rampant gene loss in Discoba. Rampant loss occurred independently in the unique common lineage leading to Heterolobosea + Tsukubamonadida and later in the unique lineage leading to Acrasis. Meanwhile, mtDNA gene content appears to be remarkably stable in the Acrasis sister lineage leading to Naegleria and in their distant relatives Jakobida. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genetic variation among the Mapuche Indians from the Patagonian region of Argentina: mitochondrial DNA sequence variation and allele frequencies of several nuclear genes.

PubMed

Ginther, C; Corach, D; Penacino, G A; Rey, J A; Carnese, F R; Hutz, M H; Anderson, A; Just, J; Salzano, F M; King, M C

1993-01-01

DNA samples from 60 Mapuche Indians, representing 39 maternal lineages, were genetically characterized for (1) nucleotide sequences of the mtDNA control region; (2) presence or absence of a nine base duplication in mtDNA region V; (3) HLA loci DRB1 and DQA1; (4) variation at three nuclear genes with short tandem repeats; and (5) variation at the polymorphic marker D2S44. The genetic profile of the Mapuche population was compared to other Amerinds and to worldwide populations. Two highly polymorphic portions of the mtDNA control region, comprising 650 nucleotides, were amplified by the polymerase chain reaction (PCR) and directly sequenced. The 39 maternal lineages were defined by two or three generation families identified by the Mapuches. These 39 lineages included 19 different mtDNA sequences that could be grouped into four classes. The same classes of sequences appear in other Amerinds from North, Central, and South American populations separated by thousands of miles, suggesting that the origin of the mtDNA patterns predates the migration to the Americas. The mtDNA sequence similarity between Amerind populations suggests that the migration throughout the Americas occurred rapidly relative to the mtDNA mutation rate. HLA DRB1 alleles 1602 and 1402 were frequent among the Mapuches. These alleles also occur at high frequency among other Amerinds in North and South America, but not among Spanish, Chinese or African-American populations. The high frequency of these alleles throughout the Americas, and their specificity to the Americas, supports the hypothesis that Mapuches and other Amerind groups are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)

Rosa26-GFP Direct Repeat (RaDR-GFP) Mice Reveal Tissue- and Age-Dependence of Homologous Recombination in Mammals In Vivo

PubMed Central

Kay, Jennifer E.; Na, Li; Rowland, Elizabeth A.; Winther, Kelly E.; Chow, Danielle N.; Kimoto, Takafumi; Matsuguchi, Tetsuya; Jonnalagadda, Vidya S.; Maklakova, Vilena I.; Singh, Vijay R.; Wadduwage, Dushan N.; Rajapakse, Jagath; So, Peter T. C.; Collier, Lara S.; Engelward, Bevin P.

2014-01-01

Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals. PMID:24901438

The profile of repeat-associated histone lysine methylation states in the mouse epigenome

PubMed Central

Martens, Joost H A; O'Sullivan, Roderick J; Braunschweig, Ulrich; Opravil, Susanne; Radolf, Martin; Steinlein, Peter; Jenuwein, Thomas

2005-01-01

Histone lysine methylation has been shown to index silenced chromatin regions at, for example, pericentric heterochromatin or of the inactive X chromosome. Here, we examined the distribution of repressive histone lysine methylation states over the entire family of DNA repeats in the mouse genome. Using chromatin immunoprecipitation in a cluster analysis representing repetitive elements, our data demonstrate the selective enrichment of distinct H3-K9, H3-K27 and H4-K20 methylation marks across tandem repeats (e.g. major and minor satellites), DNA transposons, retrotransposons, long interspersed nucleotide elements and short interspersed nucleotide elements. Tandem repeats, but not the other repetitive elements, give rise to double-stranded (ds) RNAs that are further elevated in embryonic stem (ES) cells lacking the H3-K9-specific Suv39h histone methyltransferases. Importantly, although H3-K9 tri- and H4-K20 trimethylation appear stable at the satellite repeats, many of the other repeat-associated repressive marks vary in chromatin of differentiated ES cells or of embryonic trophoblasts and fibroblasts. Our data define a profile of repressive histone lysine methylation states for the repetitive complement of four distinct mouse epigenomes and suggest tandem repeats and dsRNA as primary triggers for more stable chromatin imprints. PMID:15678104

Recent advances in assays for the fragile X-related disorders.

PubMed

Hayward, Bruce E; Kumari, Daman; Usdin, Karen

2017-10-01

The fragile X-related disorders are a group of three clinical conditions resulting from the instability of a CGG-repeat tract at the 5' end of the FMR1 transcript. Fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI) are disorders seen in carriers of FMR1 alleles with 55-200 repeats. Female carriers of these premutation (PM) alleles are also at risk of having a child who has an FMR1 allele with >200 repeats. Most of these full mutation (FM) alleles are epigenetically silenced resulting in a deficit of the FMR1 gene product, FMRP. This results in fragile X Syndrome (FXS), the most common heritable cause of intellectual disability and autism. The diagnosis and study of these disorders is challenging, in part because the detection of alleles with large repeat numbers has, until recently, been either time-consuming or unreliable. This problem is compounded by the mosaicism for repeat length and/or DNA methylation that is frequently seen in PM and FM carriers. Furthermore, since AGG interruptions in the repeat tract affect the risk that a FM allele will be maternally transmitted, the ability to accurately detect these interruptions in female PM carriers is an additional challenge that must be met. This review will discuss some of the pros and cons of some recently described assays for these disorders, including those that detect FMRP levels directly, as well as emerging technologies that promise to improve the diagnosis of these conditions and to be useful in both basic and translational research settings.

CRISPR/Cas9-Induced (CTG⋅CAG)n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing.

PubMed

van Agtmaal, Ellen L; André, Laurène M; Willemse, Marieke; Cumming, Sarah A; van Kessel, Ingeborg D G; van den Broek, Walther J A A; Gourdon, Geneviève; Furling, Denis; Mouly, Vincent; Monckton, Darren G; Wansink, Derick G; Wieringa, Bé

2017-01-04

Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG) n -repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5' or 3' unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Insights into mutagenesis using Escherichia coli chromosomal lacZ strains that enable detection of a wide spectrum of mutational events.

PubMed

Seier, Tracey; Padgett, Dana R; Zilberberg, Gal; Sutera, Vincent A; Toha, Noor; Lovett, Susan T

2011-06-01

Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3' exonucleases targeted to the lagging strand. The loss of 3' exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F' element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes.

Somatic mosaicism of androgen receptor CAG repeats in colorectal carcinoma epithelial cells from men.

PubMed

Di Fabio, Francesco; Alvarado, Carlos; Gologan, Adrian; Youssef, Emad; Voda, Linda; Mitmaker, Elliot; Beitel, Lenore K; Gordon, Philip H; Trifiro, Mark

2009-06-01

The X-linked human androgen receptor gene (AR) contains an exonic polymorphic trinucleotide CAG. The length of this encoded CAG tract inversely affects AR transcriptional activity. Colorectal carcinoma is known to express the androgen receptor, but data on somatic CAG repeat lengths variations in malignant and normal epithelial cells are still sporadic. Using laser capture microdissection (LCM), epithelial cells from colorectal carcinoma and normal-appearing mucosa were collected from the fresh tissue of eight consecutive male patients undergoing surgery (mean age, 70 y; range, 54-82). DNA isolated from each LCM sample underwent subsequent PCR and DNA sequencing to precisely determine AR CAG repeat lengths and the presence of microsatellite instability (MSI). Different AR CAG repeat lengths were observed in colorectal carcinoma (ranging from 0 to 36 CAG repeats), mainly in the form of multiple shorter repeat lengths. This genetic heterogeneity (somatic mosaicism) was also found in normal-appearing colorectal mucosa. Half of the carcinoma cases examined tended to have a higher number of AR CAG repeat lengths with a wider range of repeat size variation compared to normal mucosa. MSI carcinomas tended to have longer median AR CAG repeat lengths (n = 17) compared to microsatellite stable carcinomas (n = 14), although the difference was not significant (P = 0.31, Mann-Whitney test). Multiple unique somatic mutations of the AR CAG repeats occur in colorectal mucosa and in carcinoma, predominantly resulting in shorter alleles. Colorectal epithelial cells carrying AR alleles with shorter CAG repeat lengths may be more androgen-sensitive and therefore have a growth advantage.

Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome.

PubMed

Waye, J S; Willard, H F

1986-09-01

The centromeric regions of all human chromosomes are characterized by distinct subsets of a diverse tandemly repeated DNA family, alpha satellite. On human chromosome 17, the predominant form of alpha satellite is a 2.7-kilobase-pair higher-order repeat unit consisting of 16 alphoid monomers. We present the complete nucleotide sequence of the 16-monomer repeat, which is present in 500 to 1,000 copies per chromosome 17, as well as that of a less abundant 15-monomer repeat, also from chromosome 17. These repeat units were approximately 98% identical in sequence, differing by the exclusion of precisely 1 monomer from the 15-monomer repeat. Homologous unequal crossing-over is suggested as a probable mechanism by which the different repeat lengths on chromosome 17 were generated, and the putative site of such a recombination event is identified. The monomer organization of the chromosome 17 higher-order repeat unit is based, in part, on tandemly repeated pentamers. A similar pentameric suborganization has been previously demonstrated for alpha satellite of the human X chromosome. Despite the organizational similarities, substantial sequence divergence distinguishes these subsets. Hybridization experiments indicate that the chromosome 17 and X subsets are more similar to each other than to the subsets found on several other human chromosomes. We suggest that the chromosome 17 and X alpha satellite subsets may be related components of a larger alphoid subfamily which have evolved from a common ancestral repeat into the contemporary chromosome-specific subsets.

DNA residence time is a regulatory factor of transcription repression

PubMed Central

Clauß, Karen; Popp, Achim P.; Schulze, Lena; Hettich, Johannes; Reisser, Matthias; Escoter Torres, Laura; Uhlenhaut, N. Henriette

2017-01-01

Abstract Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation. PMID:28977492

Cloning and restriction enzyme mapping of ribosomal DNA of Giardia duodenalis, Giardia ardeae and Giardia muris.

PubMed

van Keulen, H; Campbell, S R; Erlandsen, S L; Jarroll, E L

1991-06-01

In an attempt to study Giardia at the DNA sequence level, the rRNA genes of three species, Giardia duodenalis, Giardia ardeae and Giardia muris were cloned and restriction enzyme maps were constructed. The rDNA repeats of these Giardia show completely different restriction enzyme recognition patterns. The size of the rDNA repeat ranges from approximately 5.6 kb in G. duodenalis to 7.6 kb in both G. muris and G. ardeae. These size differences are mainly attributable to the variation in length of the spacer. Minor differences exist among these Giardia in the sizes of their small subunit rRNA and the internal transcribed spacer between small and large subunit rRNA. The genetic maps were constructed by sequence analysis of the DNA around the 5' and 3' ends of the mature rRNA genes and between the rRNA covering the 5.8S rRNA gene and internal transcribed spacer. Comparison of the 5.8S rDNA and 3' end of large subunit rDNA from these three Giardia species showed considerable sequence variation, but the rDNA sequences of G. duodenalis and G. ardeae appear more closely related to each other than to G. muris.

Comparing the Properties of Electrochemical-Based DNA Sensors Employing Different Redox Tags

PubMed Central

Kang, Di; Zuo, Xiaolei; Yang, Renqiang; Xia, Fan; Plaxco, Kevin W.; White, Ryan J.

2009-01-01

Many electrochemical biosensor approaches developed in recent years utilize redox labeled (most commonly methylene blue or ferrocene) oligonucleotide probes site-specifically attached to an interrogating electrode. Sensors in this class have been reported employing a range of probe architectures, including single- and double-stranded DNA, more complex DNA structures, DNA and RNA aptamers and, most recently, DNA-small molecule chimeras. Signaling in this class of sensors is generally predicated on binding-induced changes in the efficiency with which the covalently attached redox label transfers electrons with the interrogating electrode. Here we have investigated how the properties of the redox tag affect the performance of such sensors. Specifically, we compare the differences in signaling and stability of electrochemical DNA sensors (E-DNA sensors) fabricated using either ferrocene or methylene blue as the signaling redox moiety. We find that while both tags support efficient E-DNA signaling, ferrocene produces slightly improved signal gain and target affinity. These small advantages, however, come at a potentially significant price: the ferrocene-based sensors are far less stable than their methylene blue counterparts, particularly with regards to stability to long-term storage, repeated electrochemical interrogations, repeated sensing/regeneration iterations, and employment in complex sample matrices such as blood serum. PMID:19810694

[Variation of mini- and microsatellite DNA repeats in parthenogenetic lizard Darevskia armeniaca as revealed by DNA fingerprinting analysis].

PubMed

Martirosian, I A; Kan, N G; Petrosian, V G; Malysheva, D N; Trofimova, A A; Danielian, F D; Darevskiĭ, I S; Korochkin, L I; Ryskov, A P; Tokarskaia, O N

2003-02-01

Population and family samples of two morphological forms (mutant and normal with respect to dorsal color) of pathogenetic lizard Darevskia armeniaca were examined by means of DNA fingerprinting using M13 mini- and (GATA)n and (TCC)n microsatellite DNA markers. The morphological forms examined were characterized by clonally inherited, species-specific patterns of the DNA markers, which were different from the species-specific DNA fingerprints of the other parthenogenetic species of the genus Darevskia (D. dahli. D. unisexualis, and D. rostombekovi). The mean index of similarity (S) obtained for a sample of 36 individuals from three isolated populations using three types of DNA markers was 0.966. This was similar to the variability level observed in D. dahli (0.962) (P > 0.05), but higher than that in D. unisexualis (0.950) (P < 0.05) and D. rostombekovi (0.875) (P < 0.01). Inheritance of M13 minisatellite and (TCC)n microsatellite DNA markers in the F1 offspring of parthenogenetic lizards was examined. It was shown that variability and clonal diversity of the fingerprint phenotypes observed in the populations and families of D. armeniaca could be at least partly explained by RFLP mutations in microsatellite repeats.

Identification and chromosome mapping of repetitive elements in the Astyanax scabripinnis (Teleostei: Characidae) species complex.

PubMed

Barbosa, Patrícia; de Oliveira, Luiz Antonio; Pucci, Marcela Baer; Santos, Mateus Henrique; Moreira-Filho, Orlando; Vicari, Marcelo Ricardo; Nogaroto, Viviane; de Almeida, Mara Cristina; Artoni, Roberto Ferreira

2015-02-01

Most part of the eukaryotic genome is composed of repeated sequences or multiple copies of DNA, which were considered as "junk DNA", and may be associated to the heterochromatin. In this study, three populations of Astyanax aff. scabripinnis from Brazilian rivers of Guaratinguetá and Pindamonhangaba (São Paulo) and a population from Maringá (Paraná) were analyzed concerning the localization of the nucleolar organizer regions (Ag-NORs), the As51 satellite DNA, the 18S ribosomal DNA (rDNA), and the 5S rDNA. Repeated sequences were also isolated and identified by the Cot - 1 method, which indicated similarity (90%) with the LINE UnaL2 retrotransposon. The fluorescence in situ hybridization (FISH) showed the retrotransposon dispersed and more concentrated markers in centromeric and telomeric chromosomal regions. These sequences were co-localized and interspaced with 18S and 5S rDNA and As51, confirmed by fiber-FISH essay. The B chromosome found in these populations pointed to a conspicuous hybridization with LINE probe, which is also co-located in As51 sequences. The NORs were active at unique sites of a homologous pair in the three populations. There were no evidences that transposable elements and repetitive DNA had influence in the transcriptional regulation of ribosomal genes in our analyses.

Optimization of HIV-1 Envelope DNA Vaccine Candidates within Three Different Animal Models, Guinea Pigs, Rabbits and Cynomolgus Macaques.

PubMed

Borggren, Marie; Vinner, Lasse; Andresen, Betina Skovgaard; Grevstad, Berit; Repits, Johanna; Melchers, Mark; Elvang, Tara Laura; Sanders, Rogier W; Martinon, Frédéric; Dereuddre-Bosquet, Nathalie; Bowles, Emma Joanne; Stewart-Jones, Guillaume; Biswas, Priscilla; Scarlatti, Gabriella; Jansson, Marianne; Heyndrickx, Leo; Grand, Roger Le; Fomsgaard, Anders

2013-07-19

HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques.

Optimization of HIV-1 Envelope DNA Vaccine Candidates within Three Different Animal Models, Guinea Pigs, Rabbits and Cynomolgus Macaques

PubMed Central

Borggren, Marie; Vinner, Lasse; Andresen, Betina Skovgaard; Grevstad, Berit; Repits, Johanna; Melchers, Mark; Elvang, Tara Laura; Sanders, Rogier W; Martinon, Frédéric; Dereuddre-Bosquet, Nathalie; Bowles, Emma Joanne; Stewart-Jones, Guillaume; Biswas, Priscilla; Scarlatti, Gabriella; Jansson, Marianne; Heyndrickx, Leo; Le Grand, Roger; Fomsgaard, Anders

2013-01-01

HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques. PMID:26344115

Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame

PubMed Central

Rajendran, Arivazhagan; Endo, Masayuki; Hidaka, Kumi; Lan Thao Tran, Phong; Mergny, Jean-Louis; Sugiyama, Hiroshi

2013-01-01

Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G–G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex. PMID:23863846

Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana

PubMed Central

Simon, Lauriane; Rabanal, Fernando A; Dubos, Tristan; Oliver, Cecilia; Lauber, Damien; Poulet, Axel; Vogt, Alexander; Mandlbauer, Ariane; Le Goff, Samuel; Sommer, Andreas; Duborjal, Hervé; Tatout, Christophe

2018-01-01

Abstract Organized in tandem repeat arrays in most eukaryotes and transcribed by RNA polymerase III, expression of 5S rRNA genes is under epigenetic control. To unveil mechanisms of transcriptional regulation, we obtained here in depth sequence information on 5S rRNA genes from the Arabidopsis thaliana genome and identified differential enrichment in epigenetic marks between the three 5S rDNA loci situated on chromosomes 3, 4 and 5. We reveal the chromosome 5 locus as the major source of an atypical, long 5S rRNA transcript characteristic of an open chromatin structure. 5S rRNA genes from this locus translocated in the Landsberg erecta ecotype as shown by linkage mapping and chromosome-specific FISH analysis. These variations in 5S rDNA locus organization cause changes in the spatial arrangement of chromosomes in the nucleus. Furthermore, 5S rRNA gene arrangements are highly dynamic with alterations in chromosomal positions through translocations in certain mutants of the RNA-directed DNA methylation pathway and important copy number variations among ecotypes. Finally, variations in 5S rRNA gene sequence, chromatin organization and transcripts indicate differential usage of 5S rDNA loci in distinct ecotypes. We suggest that both the usage of existing and new 5S rDNA loci resulting from translocations may impact neighboring chromatin organization. PMID:29518237