ABSTRACT
Avoiding biases in next generation sequencing (NGS) library preparation is crucial for obtaining reliable sequencing data. Recently, a new library preparation method has been introduced which has eliminated the need for the ligation step. This method, termed SMART (switching mechanism at the 5' end of the RNA transcript), is based on template switching reverse transcription. To date, there has been no systematic analysis of the additional biases introduced by this method. We analysed the genomic distribution of sequenced reads prepared from genomic DNA using the SMART methodology and found a strong bias toward long (≥12bp) poly dA/dT containing genomic loci. This bias is unique to the SMART-based library preparation and does not appear when libraries are prepared with conventional ligation based methods. Although this bias is obvious only when performing paired end sequencing, it affects single end sequenced samples as well. Our analysis demonstrates that sequenced reads originating from SMART-DNA libraries are heavily skewed toward genomic poly dA/dT tracts. This bias needs to be considered when deciding to use SMART based technology for library preparation.
Subject(s)
Gene Library , Genomics , High-Throughput Nucleotide Sequencing/methods , Poly dA-dT/genetics , Genome , RNA/genetics , Sequence Analysis, DNA/methodsABSTRACT
Poly(A) tails are critical for mRNA stability and translation. However, recent studies have challenged this view, showing that poly(A) tail length and translation efficiency are decoupled in non-embryonic cells. Using TAIL-seq and ribosome profiling, we investigate poly(A) tail dynamics and translational control in the somatic cell cycle. We find dramatic changes in poly(A) tail lengths of cell-cycle regulatory genes like CDK1, TOP2A, and FBXO5, explaining their translational repression in M phase. We also find that poly(A) tail length is coupled to translation when the poly(A) tail is <20 nucleotides. However, as most genes have >20 nucleotide poly(A) tails, their translation is regulated mainly via poly(A) tail length-independent mechanisms during the cell cycle. Specifically, we find that terminal oligopyrimidine (TOP) tract-containing transcripts escape global translational suppression in M phase and are actively translated. Our quantitative and comprehensive data provide a revised view of translational control in the somatic cell cycle.
Subject(s)
Mitosis , Poly A/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , Antigens, Neoplasm/biosynthesis , Antigens, Neoplasm/genetics , CDC2 Protein Kinase , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/genetics , Cyclin-Dependent Kinases/biosynthesis , Cyclin-Dependent Kinases/genetics , DNA Topoisomerases, Type II/biosynthesis , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , F-Box Proteins/biosynthesis , F-Box Proteins/genetics , Gene Expression Profiling/methods , Gene Expression Regulation , Gene Library , HeLa Cells , Humans , M Phase Cell Cycle Checkpoints , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/metabolism , Poly A/genetics , Poly dA-dT/genetics , Poly dA-dT/metabolism , Poly-ADP-Ribose Binding Proteins , RNA Stability , RNA, Messenger/genetics , Ribosomes/metabolism , S Phase Cell Cycle Checkpoints , Time FactorsABSTRACT
Nucleosomes regulate many DNA-dependent processes by controlling the accessibility of DNA, and DNA sequences such as the poly-dA:dT element are known to affect nucleosome binding. We demonstrate that poly-dA:dT tracts form an asymmetric barrier to nucleosome movement in vivo, mediated by ATP-dependent chromatin remodelers. We theorize that nucleosome transit over poly-A elements is more energetically favourable in one direction, leading to an asymmetric arrangement of nucleosomes around these sequences. We demonstrate that different arrangements of poly-A and poly-T tracts result in very different outcomes for nucleosome occupancy in yeast, mouse, and human, and show that yeast takes advantage of this phenomenon in its promoter architecture.
Subject(s)
DNA/genetics , Nucleosomes/genetics , Poly dA-dT/genetics , Adenosine Triphosphate/metabolism , Animals , Humans , Mice , Promoter Regions, Genetic , Yeasts/geneticsABSTRACT
DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41), a member of the DEXDc helicase family, was recently identified as an intracellular DNA sensor in mouse myeloid dendritic cells. In this study, porcine DDX41 (poDDX41) was cloned and its role in the type I interferon (IFN) signaling pathway was investigated in porcine kidney (PK-15) cells. Full-length poDDX41 cDNA encodes 622 amino acid residues and contains a DEADc domain and a HELICc domain. poDDX41 mRNA is widely expressed in different tissues, especially the stomach and liver. Overexpression of poDDX41 in PK-15 cells induced IFN-ß by activating transcription factors IRF3 and NF-κB. Knockdown of poDDX41 with siRNA significantly reduced IFN-ß expression induced by poly(dA:dT), a double-stranded DNA (dsDNA) analogue, or pseudorabies virus, a dsDNA swine virus. Therefore, poDDX41 is involved in the dsDNA- and dsDNA-virus-mediated type I IFN signaling pathway in porcine kidney cells.
Subject(s)
Cloning, Molecular , DEAD-box RNA Helicases/genetics , Epithelial Cells/immunology , Interferon-beta/immunology , Kidney/immunology , Amino Acid Sequence , Animals , DEAD-box RNA Helicases/antagonists & inhibitors , DEAD-box RNA Helicases/immunology , DNA/genetics , DNA/immunology , DNA, Complementary/genetics , DNA, Complementary/immunology , Epithelial Cells/cytology , Gene Expression Regulation , Herpesvirus 1, Suid/genetics , Herpesvirus 1, Suid/immunology , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/immunology , Interferon-beta/genetics , Kidney/cytology , Liver/immunology , Molecular Sequence Data , NF-kappa B/genetics , NF-kappa B/immunology , Open Reading Frames , Poly dA-dT/genetics , Poly dA-dT/immunology , Protein Structure, Tertiary , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Stomach/immunology , SwineABSTRACT
BACKGROUND: In eukaryotic organisms, packaging of DNA into nucleosomes controls gene expression by regulating access of the promoter to transcription factors. The human malaria parasite Plasmodium falciparum encodes relatively few transcription factors, while extensive nucleosome remodeling occurs during its replicative cycle in red blood cells. These observations point towards an important role of the nucleosome landscape in regulating gene expression. However, the relation between nucleosome positioning and transcriptional activity has thus far not been explored in detail in the parasite. RESULTS: Here, we analyzed nucleosome positioning in the asexual and sexual stages of the parasite's erythrocytic cycle using chromatin immunoprecipitation of MNase-digested chromatin, followed by next-generation sequencing. We observed a relatively open chromatin structure at the trophozoite and gametocyte stages, consistent with high levels of transcriptional activity in these stages. Nucleosome occupancy of genes and promoter regions were subsequently compared to steady-state mRNA expression levels. Transcript abundance showed a strong inverse correlation with nucleosome occupancy levels in promoter regions. In addition, AT-repeat sequences were strongly unfavorable for nucleosome binding in P. falciparum, and were overrepresented in promoters of highly expressed genes. CONCLUSIONS: The connection between chromatin structure and gene expression in P. falciparum shares similarities with other eukaryotes. However, the remarkable nucleosome dynamics during the erythrocytic stages and the absence of a large variety of transcription factors may indicate that nucleosome binding and remodeling are critical regulators of transcript levels. Moreover, the strong dependency between chromatin structure and DNA sequence suggests that the P. falciparum genome may have been shaped by nucleosome binding preferences. Nucleosome remodeling mechanisms in this deadly parasite could thus provide potent novel anti-malarial targets.
Subject(s)
DNA/metabolism , Malaria/parasitology , Nucleosomes/metabolism , Plasmodium falciparum/genetics , Chromatin/metabolism , Chromatin Immunoprecipitation , Erythrocytes/parasitology , High-Throughput Nucleotide Sequencing , Histones/genetics , Histones/metabolism , Humans , Malaria/pathology , Micrococcal Nuclease/metabolism , Poly dA-dT/genetics , Promoter Regions, Genetic , Sequence Analysis, DNA , Transcription, Genetic , Trophozoites/metabolismABSTRACT
Although the genomic pattern of nucleosome positioning is broadly conserved, quantitative aspects vary over evolutionary timescales. We identify the cis and trans determinants of nucleosome positioning using a functional evolutionary approach involving S. cerevisiae strains containing large genomic regions from other yeast species. In a foreign species, nucleosome depletion at promoters is maintained over poly(dA:dT) tracts, whereas internucleosome spacing and all other aspects of nucleosome positioning tested are not. Interestingly, the locations of the +1 nucleosome and RNA start sites shift in concert. Strikingly, in a foreign species, nucleosome-depleted regions occur fortuitously in coding regions, and they often act as promoters that are associated with a positioned nucleosome array linked to the length of the transcription unit. We suggest a three-step model in which nucleosome remodelers, general transcription factors, and the transcriptional elongation machinery are primarily involved in generating the nucleosome positioning pattern in vivo.
Subject(s)
Evolution, Molecular , Models, Biological , Models, Genetic , Nucleosomes/genetics , Nucleosomes/metabolism , Base Sequence , Chromatin Assembly and Disassembly , Chromosomes, Artificial, Yeast/genetics , DNA, Fungal/genetics , Genome, Fungal , Molecular Sequence Data , Poly dA-dT/genetics , Promoter Regions, Genetic , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription Initiation, GeneticABSTRACT
Evolutionary theory assumes that mutations occur randomly in the genome; however, studies performed in a variety of organisms indicate the existence of context-dependent mutation biases. Sources of mutagenesis variation across large genomic contexts (e.g., hundreds of bases) have not been identified. Here, we use high-coverage whole-genome sequencing of a conditional mismatch repair mutant line of diploid yeast to identify mutations that accumulated after 160 generations of growth. The vast majority of the mutations accumulated as insertion/deletions (in/dels) in homopolymeric [poly(dA:dT)] and repetitive DNA tracts. Surprisingly, the likelihood of an in/del mutation in a given poly(dA:dT) tract is increased by the presence of nearby poly(dA:dT) tracts in up to a 1,000 bp region centered on the given tract. Our work suggests that specific mutation hot spots can contribute disproportionately to the genetic variation that is introduced into populations and provides long-range genomic sequence context that contributes to mutagenesis.
Subject(s)
Mutation/genetics , Poly dA-dT/genetics , Saccharomyces cerevisiae/genetics , Base Sequence , Chromosome Segregation/genetics , Cluster Analysis , Endopeptidases/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Genome, Fungal/genetics , Genotype , Heterozygote , INDEL Mutation/genetics , Molecular Sequence Data , Mutagenesis/genetics , Saccharomyces cerevisiae Proteins/genetics , Spores, Fungal/genetics , Ubiquitin Thiolesterase/geneticsABSTRACT
The chemokine RANTES plays an essential role in inflammation and immune response. In this study, we cloned the nucleotide sequence of the 5'-flanking region of the porcine RANTES (poRANTES) gene and characterized the regulatory elements that activate transcription. Analyses of a series of 5' deletion constructs demonstrated that a 266 bp region (-220/+46) that spanned the potential transcription start site of the poRANTES gene was sufficient to activate transcription in PK-15 cells. Furthermore, our results indicated that dsDNA/dsRNA significantly induced poRANTES promoter activity and expression of mRNA levels in a time- and dose-dependent manner. Promoter deletions and mutagenesis experiments indicated that an interferon-stimulated responsive element (ISRE) was critical for dsDNA/dsRNA-induced poRANTES transcription. In addition, porcine interferon regulatory factor 3 (IRF-3) and IRF-7 play important roles in dsDNA/dsRNA-induced poRANTES expression.
Subject(s)
Chemokine CCL5/biosynthesis , Gene Expression Regulation/genetics , Promoter Regions, Genetic/genetics , Response Elements/genetics , Swine/genetics , Animals , Base Sequence , Cell Line , Chemokine CCL5/genetics , Cloning, Molecular , DNA/genetics , Humans , Molecular Sequence Data , Poly I-C/genetics , Poly dA-dT/genetics , RNA, Double-Stranded/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Transcription, Genetic , TransfectionABSTRACT
Previous studies have shown that both hepatitis A virus and hepatitis C virus inhibit innate immunity by cleaving the mitochondrial antiviral signaling (MAVS) protein, an essential component of the virus-activated signaling pathway that activates NF-kappaB and IFN regulatory factor-3 to induce the production of type I IFN. For human hepatitis B virus (HBV), hepatitis B s-Ag, hepatitis B e-Ag, or HBV virions have been shown to suppress TLR-induced antiviral activity with reduced IFN-beta production and subsequent induction of IFN-stimulated genes. However, HBV-mediated suppression of the RIG-I-MDA5 pathway is unknown. In this study, we found that HBV suppressed poly(deoxyadenylate-thymidylate)-activated IFN-beta production in hepatocytes. Specifically, hepatitis B virus X (HBX) interacted with MAVS and promoted the degradation of MAVS through Lys(136) ubiquitin in MAVS protein, thus preventing the induction of IFN-beta. Further analysis of clinical samples revealed that MAVS protein was downregulated in hepatocellular carcinomas of HBV origin, which correlated with increased sensitivities of primary murine hepatocytes isolated from HBX knock-in transgenic mice upon vesicular stomatitis virus infections. By establishing a link between MAVS and HBX, this study suggests that HBV can target the RIG-I signaling by HBX-mediated MAVS downregulation, thereby attenuating the antiviral response of the innate immune system.
Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Immunity, Innate/immunology , Mitochondrial Proteins/immunology , Trans-Activators/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line , Chlorocebus aethiops , DEAD Box Protein 58 , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/immunology , DEAD-box RNA Helicases/metabolism , Down-Regulation , Hep G2 Cells , Humans , Immunoblotting , Interferon-gamma/genetics , Interferon-gamma/immunology , Interferon-gamma/metabolism , Kinetics , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Poly dA-dT/genetics , Poly dA-dT/immunology , Poly dA-dT/metabolism , Protein Binding , Receptors, Immunologic , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/immunology , Trans-Activators/genetics , Trans-Activators/metabolism , Transfection , Vero Cells , Viral Regulatory and Accessory ProteinsABSTRACT
The vaccinia virus E3 protein is an important intracellular modulator of innate immunity that can be split into distinct halves. The C terminus contains a well defined dsRNA-binding domain, whereas the N terminus contains a Z-DNA-binding domain, and both domains are required for virulence. In this study, we investigated whether the E3 Z-DNA-binding domain functions by sequestering cytoplasmic dsDNA thereby preventing the induction of type I interferon (IFN). In line with this hypothesis, expression of E3 ablated both IFN-beta expression and NF-kappaB activity in response to the dsDNA, poly(dA-dT). However, surprisingly, the ability of E3 to block poly(dA-dT) signalling was independent of the N terminus, whereas the dsRNA-binding domain was essential, suggesting that the Z-DNA-binding domain does not bind immunostimulatory dsDNA. This was confirmed by the failure of E3 to co-precipitate with biotinylated dsDNA, whereas the recruitment of several cytoplasmic DNA-binding proteins could be detected. Recently, AT-rich dsDNA was reported to be transcribed into 5'-triphosphate poly(A-U) RNA by RNA polymerase III, which then activates retinoic acid-inducible gene I (RIG-I). Consistent with this, RNA from poly(dA-dT) transfected cells induced IFN-beta and expression of the E3 dsRNA-binding domain was sufficient to ablate this response. Given the well documented function of the E3 dsRNA-binding domain we propose that E3 blocks signalling in response to poly(dA-dT) by binding to transcribed poly(A-U) RNA preventing RIG-I activation. This report describes a DNA virus-encoded inhibitor of the RNA polymerase III-dsDNA-sensing pathway and extends our knowledge of E3 as a modulator of innate immunity.
Subject(s)
Immunity, Innate , RNA Polymerase III/antagonists & inhibitors , RNA-Binding Proteins/immunology , Vaccinia virus/immunology , Viral Proteins/immunology , Cell Line , DNA, Z-Form/metabolism , HeLa Cells , Host-Pathogen Interactions/immunology , Humans , Interferon-beta/biosynthesis , NF-kappa B/metabolism , Poly dA-dT/genetics , Poly dA-dT/metabolism , Protein Structure, Tertiary , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Signal Transduction , Toll-Like Receptor 9/metabolism , Transfection , Vaccinia virus/genetics , Vaccinia virus/pathogenicity , Vaccinia virus/physiology , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Eukaryotic transcriptional regulation is mediated by the organization of nucleosomes in promoter regions. Most Saccharomyces cerevisiae promoters have a highly stereotyped chromatin organization, where nucleosome-free regions (NFR) are flanked by well-ordered nucleosomes. We have found that yeast promoters fall into two classes differing in NFR sharpness, and that this distinction follows a known transcriptional dichotomy in yeast genes. A class of yeast promoters having well-defined NFRs are characterized by positioned patterns of poly(dA:dT) tracts with several novel features. First, poly(dA:dT) tracts are localized in a strand-dependent manner, with poly(dA) tracts lying proximal to transcriptional start sites and poly(dT) tracts lying distal, and collectively define a symmetry axis that is coincident with NFR centers. Second, poly(dA:dT) tracts are preferentially "capped" by G:C residues on the terminus proximal to the symmetry axis. Both signature features co-vary with fine positional variations between NFRs, establishing a closely knit relationship between poly(dA:dT) tracts, their capping patterns, and the central coordinates of NFRs. We found that these features are unique to promoters with well-defined NFRs, and that these promoters display significant difference between in vitro and in vivo nucleosome occupancy patterns. These observations are consistent with a model in which localized and G:C-capped poly(dA:dT) tracts initiate or facilitate the formation of NFRs at their center, possibly with chromatin remodeling and transcriptional machines involved.
Subject(s)
Gene Order/physiology , Poly dA-dT/genetics , Promoter Regions, Genetic/genetics , RNA Caps , Yeasts/genetics , Base Composition , Base Sequence , Chromatin , Chromosomes, Fungal , DNA, Fungal/chemistry , DNA, Fungal/genetics , Gene Expression Regulation, Fungal , Models, Biological , Molecular Sequence Data , Nucleic Acid Conformation , Nucleosomes/genetics , RNA Caps/chemistry , RNA Caps/genetics , Saccharomyces cerevisiae/geneticsABSTRACT
The interaction of the Cu(II) drugs CuL(NO(3)) and CuL'(NO(3)) (HL is pyridine-2-carbaldehyde thiosemicarbazone and HL' is pyridine-2-carbaldehyde 4N-methylthiosemicarbazone, in water named [CuL](+) and [CuL'](+)) with [poly(dA-dT)](2), [poly(dG-dC)](2), and calf thymus (CT) DNA has been probed in aqueous solution at pH 6.0, I = 0.1 M, and T = 25 degrees C by absorbance, fluorescence, circular dichroism, and viscosity measurements. The results reveal that these drugs act as groove binders with [poly(dA-dT)](2), with a site size n = 6-7, whereas they act as external binders with [poly(dG-dC)](2) and/or CT-DNA, thus establishing overall electrostatic interaction with n = 1. The binding constants with [CuL'](+) were slightly larger than with [CuL](+). The title compounds display some cleavage activity in the presence of thiols, bringing about the rupture of the DNA strands by the reactive oxygen species formed by reoxidation of Cu(I) to Cu(II); this feature was not observed in the absence of thiols. Mutagenic assays performed both in the presence and in the absence of S9 mix, probed by the Ames test on TA 98, TA 100, and TA 102, were negative. Weak genotoxic activity was detected for [CuL](+) and [CuL'](+), with a significative dose-response effect for [CuL'](+), which was shown to be more cytotoxic in the Ames test and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assays. Methylation of the terminal NH(2) group enhances the antiproliferative activity of the pyridine-2-carbaldehyde thiosemicarbazones.
Subject(s)
Copper/chemistry , DNA/metabolism , Organometallic Compounds/chemistry , Organometallic Compounds/metabolism , Poly dA-dT/metabolism , Polydeoxyribonucleotides/metabolism , Thiosemicarbazones/chemistry , 3-Mercaptopropionic Acid/metabolism , Animals , Base Sequence , Cattle , Cell Line , DNA/genetics , DNA Breaks/drug effects , Dithiothreitol/metabolism , Glutathione/metabolism , Hydrogen-Ion Concentration , Mutagenicity Tests , Organometallic Compounds/pharmacology , Oxidation-Reduction , Poly dA-dT/genetics , Polydeoxyribonucleotides/genetics , Spectrum Analysis , Temperature , ViscosityABSTRACT
Recognition of foreign DNA by cytosolic innate immune receptors triggers the production of IFN-beta. However, it is unclear whether different types of DNA ligands are recognized by similar receptors and whether the resulting response is distinct from the endosomal TLR response. To address these questions, we compared the two most commonly used types of DNA ligands (IFN-stimulatory DNA (ISD) and poly(dAdT)) and assessed the minimal structural requirements for stimulatory capacity in RAW264.7 cells. Gene expression signatures and competition experiments suggest that ISD and poly(dAdT) are qualitatively indistinguishable and differ from the CpG-containing oligonucleotides triggering the TLR9 pathway. Structure - activity relationship analyses revealed that a minimal length of two helical turns is sufficient for ISD-mediated IFN-beta induction, while phosphorylation at the 5'-end is dispensable. Altogether, our data suggest that, in murine macrophages, only one major cytosolic DNA recognition pathway is operational.
Subject(s)
DNA/genetics , Interferon-beta/genetics , Macrophages/metabolism , Signal Transduction , Toll-Like Receptor 9/metabolism , Animals , Cell Line , Chemokine CXCL11/genetics , CpG Islands/genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression/drug effects , Immunoblotting , Interferon-beta/metabolism , Intracellular Signaling Peptides and Proteins , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Mice , Monomeric GTP-Binding Proteins/genetics , Oligonucleotide Array Sequence Analysis , Oligonucleotides/genetics , Phosphorylation , Poly dA-dT/genetics , Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Serpins/genetics , TransfectionABSTRACT
Homopolymeric stretches of deoxyadenosine nucleotides (A's) on one strand of double-stranded DNA, referred to as poly(dA:dT) tracts or A-tracts, are overabundant in eukaryotic genomes. They have unusual structural, dynamic, and mechanical properties, and may resist sharp bending. Such unusual material properties, together with their overabundance in eukaryotes, raised the possibility that poly(dA:dT) tracts might function in eukaryotes to influence the organization of nucleosomes at many genomic regions. Recent genome-wide studies strongly confirm these ideas and suggest that these tracts play major roles in chromatin organization and genome function. Here we review what is known about poly(dA:dT) tracts and how they work.
Subject(s)
Nucleosomes/chemistry , Poly dA-dT/chemistry , Animals , Base Sequence , DNA/chemistry , DNA/genetics , Humans , Models, Biological , Molecular Sequence Data , Nucleic Acid Conformation , Nucleosomes/metabolism , Poly dA-dT/geneticsABSTRACT
The eukaryote, Dictyostelium discoideum, has one of the most (A+T) rich genomes studied to date. Isolated nuclear D. discoideum DNA (AX3 strain) was used to qualitatively determine the frequency and length distribution of long (dA).(dT) homopolymer tracts in this genome, in comparison to the less (A+T) rich calf thymus and Schistosoma mansoni DNAs that had few observable long tracts. These experimental data accurately reflect the significantly elevated frequencies of long tracts found computationally within the D. discoideum intron and flanking sequences, but not exons. PCR amplification of long (dA).(dT) homopolymer tract containing sequences was carried out. Then experimental biotinylated (dT)18 probe hybridization to the PCR amplified DNA showed that the long (dA).(dT) homopolymer tracts were enriched in D. discoideum sequences only hundreds of base pair in length, under conditions where no equivalent hybridization was observed to S. mansoni DNA or calf DNA sequences. Similar probe hybridization to DNA isolated following micrococcal nuclease digestion of D. discoideum chromatin demonstrated that long (dA).(dT) homopolymer tracts were more highly enriched in nucleosomal DNA lengths that included the internucleosomal linker as compared to shorter linker free mononucleosomal lengths. This observation is in agreement with the frequency of tract spacing results calculated from GenBank sequence data. These frequency data indicate that adjacent long tracts plus the intervening spacer DNA are found at peak lengths (average 42 bp), exactly characteristic of the internucleosomal spacer region of D. discoideum chromatin and are in sufficient number to be found in nearly half of all nucleosomes. Compared to shuffled tract sequence controls, these lengths of adjacent long tracts plus the intervening spacer DNA were found to be significantly enriched. Lesser enrichments are observed at lengths corresponding to adjacent tracts being separated by nucleosomal core length DNA sequences (145-185 bp). These data strongly suggest that adjacent long tracts occur spaced at selected lengths so as to avoid the central core regions of nucleosomes and instead are found localized within internucleosomal DNA linker and core edge regions in D. discoideum chromatin.
Subject(s)
DNA, Protozoan/chemistry , DNA, Protozoan/genetics , Dictyostelium/chemistry , Dictyostelium/genetics , Poly dA-dT/chemistry , Poly dA-dT/genetics , Animals , Base Composition , DNA, Intergenic/chemistry , DNA, Intergenic/genetics , Nucleosomes/chemistry , Nucleosomes/genetics , Polymerase Chain ReactionABSTRACT
The dynamics of the B-A transition of DNA double helices with different GC contents and various chain lengths has been characterized by an electric field pulse technique. The field-induced B-A reaction is separated from orientation effects using the magic angle technique. Amplitudes reflecting the B-A reaction are observed selectively in the limited range of ethanol contents, where CD spectra demonstrate the B-A transition. The maximum amplitude appears at 1-2% higher ethanol content than the center of the B-A transition observed by CD because electric field pulses induce a relatively large perturbation from the A- toward the B-form. The relaxation curves measured after pulse termination reflect a spectrum of up to three relaxation processes. For DNA's with approximately 50% GC, the main part of the amplitude ( approximately 75%) is associated with time constants of approximately 2 micros, and another major component appears with time constants of 50-100 micros. These relaxation effects have been observed for DNA samples with 859, 2629, 7160, and 48501 bp. The time constant associated with the main amplitude increases with decreasing GC content from approximately 2 micros at 50% GC to approximately 3 mus at 41% GC and approximately 10 micros at 0% GC at the center of the B-A transition. Model calculations on the kinetics of cooperative linear Ising lattices predict the appearance of a distinct maximum of the mean relaxation time at the center of the transition. The absence of such maximum in our experimental data indicates a low cooperativity of the B-A transition with a nucleation parameter of approximately 0.1. The rate of the B-A transition is lower by approximately 3 orders of magnitude than that predicted by molecular dynamics simulations.
Subject(s)
DNA, A-Form/chemistry , DNA/chemistry , Nucleic Acid Conformation , Animals , Bacteriophage lambda/genetics , Base Composition , DNA/genetics , DNA, A-Form/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , Electrons , Male , Plasmids/chemistry , Plasmids/genetics , Poly dA-dT/chemistry , Poly dA-dT/genetics , Salmon/genetics , Spectrum Analysis , TestisABSTRACT
The interaction of newly synthesised water-soluble planar complexes of general structure [Pt(diimine)(N,N-dihydroxyethyl-N'-benzoylthioureato)]+Cl- with DNA was investigated by means of DNA melting studies, CD spectroscopy, and DNA gel mobility studies. Addition of stoichometric amounts of [Pt(diimine)H2L-S,O]Cl complexes to polynucleotides caused a significant increase in the melting temperature of poly(dA-dT) and calf-thymus DNA, respectively, indicating that these complexes interacted with DNA and stabilised the double helical structure. The CD spectra confirmed the relatively strong binding of three related Pt(II) complexes ([Pt(2,2'-bipyridine)H2L-S,O]Cl, [Pt(4,4'-dimethyl-2,2'-bipyridine)H2L-S,O]Cl, and [Pt(1,10-phenanthroline)H2L-S,O]Cl), to DNA. Comparison with the published CD spectra of ethidium bromide/DNA complex suggests a similar intercalation mode of binding. cis-[(4,4'-di-tert-butyl-2,2'-bipyridyl)N,N-di(2-hydroxyethyl)-N'-benzoylthioureatoplatinum(II)] chloride, with its very bulky tert-butyl groups, did not intercalate into the polynucleotide double helix. In DNA mobility studies in the presence of the four [Pt(diimine)H2L-S,O]Cl complexes, only [Pt(2,2'-bipyridine)H2L-S,O]Cl affected the DNA mobility to any detectable extent. Finally, in vivo studies on the biological activity of the complexes, using an Escherichia coli DNA excision repair deficient uvrA mutant strain, indicated that only the [Pt(2,2'-bipyridine)H2L-S,O]Cl complex showed significant cellular toxicity and that this was, in part, linked to DNA damage.
Subject(s)
DNA/drug effects , Intercalating Agents/chemistry , Platinum/chemistry , Water/chemistry , Animals , Binding Sites , Circular Dichroism , DNA/metabolism , Electrophoretic Mobility Shift Assay , Escherichia coli/genetics , Escherichia coli/metabolism , Intercalating Agents/pharmacology , Mutation , Nucleic Acid Conformation , Nucleic Acid Denaturation , Organometallic Compounds/chemistry , Organometallic Compounds/pharmacology , Poly dA-dT/genetics , Poly dA-dT/metabolism , Solubility , Stereoisomerism , Temperature , Time FactorsABSTRACT
We extracted phosphorus atom coordinates from the database of DNA crystal structures and calculated geometrical parameters needed to reproduce the crystal structures in the phosphorus atom representation. Using the geometrical parameters we wrote a piece of software assigning the phosphorus atom coordinates to the DNA of any nucleotide sequence. The software demonstrates non-negligible influence of the primary structure on DNA helicity, which may stand behind the heteromonous double helices of poly(dA).poly(dT) and poly(dG).poly(dC). In addition, the software is so simple that it makes possible to simulate the "crystal" structures of not only viral DNAs, but also the whole genome of Saccharomyces cerevisiae as well as the DNA human chromosome 22 having dozens of megabases in length.
Subject(s)
Chromosomes, Human, Pair 22/genetics , DNA/chemistry , Phosphorus/chemistry , Saccharomyces cerevisiae/genetics , Computational Biology/methods , DNA, Fungal/chemistry , Genome , Humans , Models, Molecular , Nucleic Acid Conformation , Poly C/genetics , Poly G/genetics , Poly dA-dT/genetics , Protein Structure, TertiaryABSTRACT
Gilbert's syndrome is characterized by mild unconjugated hyperbilirubinemia. The molecular basis of this syndrome usually concerns an additional dinucleotide insertion (TA) in the A(TA)(n)TAA configuration residing in the promoter region of the UGT1 A1 gene. This configuration may vary in length; the "n" represents the different number of TA repeats. The homozygosity A(TA)(7)TAA/A(TA)(7)TAA is involved in Gilbert's syndrome. In many cases of patients with thalassemia intermedia and sickle cell disease considerable variation in bilirubin levels is observed. In this study we investigated the contribution of the A(TA)(7)TAA/A(TA)(7)TAA genotype in the variable unconjugated serum bilirubin levels in 31 Greek patients with thalassemia intermedia and 27 Greek compound heterozygotes for beta thalassemia and sickle cell anemia. Analysis of the A(TA)(n)TAA configuration in the promoter region of the latter patients showed that those who were carrying the homozygosity A(TA)(7)TAA/A(TA)(7)TAA had higher levels of unconjugated bilirubin. These findings suggest that the coexistence of Gilbert's syndrome in patients with thalassemia intermedia and sickle cell disease may be the cause of the elevated values of unconjugated bilirubin, reducing the possibility of excessive hemolysis in these patients.