DNA Sequence Composition Research Articles

TRNA genes protect a reporter gene from epigenetic silencing in mouse cells

It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.

CpG islands and the regulation of transcription

Vertebrate CpG islands (CGIs) are short interspersed DNA sequences that deviate significantly from the average genomic pattern by being GC-rich, CpG-rich, and predominantly nonmethylated. Most, perhaps all, CGIs are sites of transcription initiation, including thousands that are remote from currently annotated promoters. Shared DNA sequence features adapt CGIs for promoter function by destabilizing nucleosomes and attracting proteins that create a transcriptionally permissive chromatin state. Silencing of CGI promoters is achieved through dense CpG methylation or polycomb recruitment, again using their distinctive DNA sequence composition. CGIs are therefore generically equipped to influence local chromatin structure and simplify regulation of gene activity.

Bayesian Methods to Detect Dye-Labelled DNA Oligonucleotides in Multiplexed Raman Spectra

Summary Recent advances in the development of technology based on Raman scattering as a chemical analytical technique have made it possible to detect spectral mixtures of multiple DNA sequences quantitatively. However, to exploit these techniques fully, inferential methodologies are required which can deconvolute the observed mixture and infer the composition of distinct DNA sequences in the overall composite. Inferring the component spectra is posed as a model selection problem for a bilinear statistical model, and the Markov chain Monte Carlo inferential methodology required is developed. In particular a Gibbs sampler and reversible jump Markov chain Monte Carlo methods are presented along with techniques based on estimation of the marginal likelihood. The results reported are particularly encouraging, highlighting that, for multiplexed Raman spectra, inference of the composition of original sequences in the mixture is possible to acceptable levels of accuracy. This statistical methodology makes the exploitation of multiplexed surface-enhanced resonance Raman scattering spectra in disease identification a reality. A Web site containing supplementary material, the spectral data that are used in the paper as well as MATLAB scripts implementing the proposed statistical methods is available at http://www.ucl.ac.uk/stats/research/serrs.

Evidence of Activity-Specific, Radial Organization of Mitotic Chromosomes in Drosophila

Author SummaryMitotic chromosomes of eukaryotes are relatively large rod-like cellular organelles, about 1 µm in diameter and 10 µm long, of well-studied composition but unknown structure. The question of whether all DNA sequences equally contribute to the interactions leading to the formation of mitotic chromosomes has never been asked. To find an answer, we determined whether the radial positions of specific chromatin loci within mitotic chromosomes were reproduced at every cell cycle or were purely random. Based on fluorescence microscopy images of live or fixed chromosomes in cells from Drosophila embryos or Drosophila larval tissues expressing the MSL3-GFP fusion protein from a transgene, we report that the large-scale organization of mitotic chromosomes is reproduced not only longitudinally, as in the well-known chromosome banding phenomenon, but also radially. Actively transcribed, dosage-compensated genes of the Drosophila male X chromosome were always found at the periphery of mitotic chromosomes, starting from late prophase. Histone modifications specific to active chromatin were found to be more peripheral compared to silent chromatin that tended to be more central in the condensed chromosome. These findings are both exciting and significant for the field of cell and chromatin biology because they may help reconcile the old controversy between the existing models of chromosome structure that posit either radial loops of chromatin or consecutive coiling. In addition, we offer new insights into the mechanisms of mitotic condensation and suggest a link between structural and functional roles of different chromatin domains.

Gene amplification in breast cancer – looking for the molecular mechanisms

Amplification of genes in breast cancer cells was shown to be coordinated in the majority of cases assuming the presence of common undisclosed mechanism underlying intrachromosomal gene amplification process. Overexpression and amplification of the HER-2/neu gene is observed in 20–30% of breast cancer cases and predicts more frequent relapse and shorter survival time. However, the initial events and molecular mechanisms of chromosomal aberrations in the region around HER2/neu as well as the precise boundaries of amplified regions remain to be obscure. This study was an attempt to define the boundaries of amplified chromosome 17q12-q21 region around HER2/neu gene. Structural peculiarities of amplification boundary regions were investigated by various techniques. Gene dosage analysis was performed for several genes located around HER2/neu gene in 154 breast cancer tissue samples. The copy number of the ppparbp, her2/neu, znfn1a3 Casc3 and top2a genes were analyzed by comparative TaqMan real time PCR. Fifty six samples (36 %) were found to be her2/neu-amplified (NHER2/neu ≥ 1,5), the boundaries for HER2-containing amplicons were mapped. Observed results suggest that similar positions of amplified region boundaries in different breast cancer samples are not accidental and may be related with the DNA structural and/or sequence peculiarities of boundary regions. Trying to find “special” sequences at the amplicon boundaries, we performed the analysis of sequence flexibility in the region TBC1D3– HER2/neu – TOP2A using the TwistFlex program. The analysis revealed several high flexible sequences in SOCS7, FBX047, HER2/neu and ZNFN1A3 genes. The locations of amplified region boundaries in 30% of cases were inside ZNFN1A3 gene. Organization of DNA sequence and nucleotide composition of FlexZNF-1 and FlexZNF-2 were similar to AT-rich islands of common fragile site (FS). Sequences of FlexZNF-1 and FlexZNF-2 have strong potential to form secondary structures according to the analysis by Mfold software (GCG Wisconsin PackageTM). Thus, the analysis of DNA sequence in TBC1D3– HER2/neu – TOP2A region revealed the existence of two flexible sequences in the ZNFN1A3 gene sequence with the strong potential to form stable secondary structures which may affect normal DNA replication at these sites and result in DNA double strand break appearance. The location of FlexZNF-1 and FlexZNF-2 strongly coincided with the location of amplified around HER2/neu gene fragment boundaries observed by real time PCR analysis. This observation indicates involvement of Flex sites in the process of intrachromosomal DNA amplification.

645 Array comparative genomic hybridization (aCGH) differentiates the major intrinsic subgroups of breast carcinomas

Circulating cell-free DNAs (cfDNAs) are DNA fragments which can be isolated from mammalian blood serum or plasma. In order to gain deeper insight into their origin(s), we have characterized the composition of human and cattle cfDNA via large-scale analyses of high-throughput sequencing data. We observed significant differences between the composition of cfDNA in serum/plasma and the corresponding DNA sequence composition of the human genome. Retrotransposable elements and non-telomeric satellite DNA were particularly overrepresented in the cfDNA population, while telomeric satellite DNA was underrepresented. This was consistently observed for human plasma, bovine serum and for the supernatant of human cancer cell cultures. Our results suggest that reverse transcription of retrotransposable elements and secondary-structure formation during the replication of satellite DNA are contributing to the composition of the cfDNA molecules in the mammalian blood stream. We believe that our work is an important step towards the understanding of the biogenesis of cfDNAs and thus may also facilitate the future exploitation of their diagnostic potential.

Variation Among Rainbow Trout (Oncorhynchus mykiss) Estrogen Receptor Isoform 3′ Untranslated Regions and the Effect of 17β-Estradiol on mRNA Stability in Hepatocyte Culture

Adenine and uridine (AU)-rich elements in the 3' untranslated region (3'UTR) have been implicated in the 17beta-estradiol (E2) stabilization of vertebrate estrogen receptor (ER) mRNAs. To date, fishes have the most complex arrangement of nuclear ERs with up to two isoforms of each of the two genes in some species (i.e., four different ERs). The objective of this study was to analyze the sequence variation of 3'UTRs among the four ER isoforms in the rainbow trout and determine to what degree it is responsible for the estrogen-induced increase of ER mRNAs in the liver of this fish. This was done by comparing the 3'UTR DNA sequence length and composition, and by measuring expression of ER isoform 3'UTR luciferase reporter constructs in primary cultures of trout hepatocytes treated with E2. There were large differences both in overall length and in sequence composition among the four ER isoform 3'UTRs. The ERalpha1 sequence was the longest and the only one of the four that contained multiple copies of the canonical AU-rich elements (AUUUA) as well as the stability sequence (GCUGAU). E2 treatment significantly increased the luciferase activity in cells transiently transfected with the ERalpha1 reporter construct, relative to cells transfected with reporter vectors containing the other three ER isoform 3'UTRs or the parental vector control. These results support the hypothesis that the E2-induced increase in hepatic ERalpha1 mRNA in rainbow trout is due in part to sequence variability among ER isoform 3'UTRs. We conclude that posttranscriptional stabilization of ER mRNA by E2 appears to be conserved among vertebrates.

Abstract 163: DTMETH: A novel method for increased length quantitative CpG methylation analysis

Abstract DNA methylation analysis has been blooming in the last decade as there is increasing evidence for epigenetic control of new pathways involved in normal cell function as well as the pathogenesis of human disease. A large number of methods have been devised to address the needs of DNA methylation detection in clinical samples. A DNA methylation detection method of preference should be able to interrogate multiple CpGs over long stretches of DNA, quantify DNA methylation, include multiple bisulphite conversion controls, provide increased sensitivity and low background. Here we present DTMETH, a novel method for DNA methylation analysis that bears the above characteristics. In principle, the desired locus is amplified from bisulphite treated-DNA using primers at CpG-free areas. Primers bear short 5’ tails which contain two cytosines (forward) or guanines (reverse) that will serve as normalisation tools. After a spin-column cleanup process, the PCR product is subjected to DTMETH. This is a thermostable reaction utilising FS Taq and labelled ddC (Applied Biosystems). The latter will be only incorporated at methylated cytosines, as post-bisulphite unmethylated cytosines are converted to uracils. The DTMETH products are cleaned by ethanol precipitation and subjected to analysis on a capillary sequencer. ddC peak areas corresponding to methylated Cs are normalised by the peak areas of the control Cs on the primer tail. As proof of principle we present DNA methylation analysis for the p16 gene. We amplified a 269 bp region of the gene's 5’UTR containing 28 CpGs. The assays provide excellent linearity and reproducibility. We used it to screen 90 non-small cell lung carcinomas for p16 methylation in comparison to an established pyrosequencing assay. p16 hypermethylation was detected in 23 samples with both methods, 3 samples with DTMETH only, while 64 samples were negative. DTMETH combines the read lengths of dideoxy terminator sequencing, and enables quantitation without the need of cloning. It is highly versatile, offering easy assay design and due to its thermostable nature can easily overcome secondary structure issues arising from the low complexity bisulphite DNA sequence composition. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 163.

Analysis of Carica papaya Telomeres and Telomere-Associated Proteins: Insights into the Evolution of Telomere Maintenance in Brassicales

Telomeres are terminal regions of linear eukaryotic chromosomes that are critical for genome stability and continued cell proliferation. The draft assembly of the papaya genome provides an opportunity to analyze and compare the evolution of telomeric DNA sequence composition and telomere maintenance machinery in this and other organisms of the Brassicales Order, which includes Arabidopsis. Here we investigate telomere size and sequence variation at papaya chromosome ends. As with most other plant species, papaya telomeres consist of TTTAGGG repeats. However, in contrast to members of the closely related Brassicaceae family, telomeres in papaya are ~10-fold longer. Sequence analysis reveals that many centromereproximal telomere repeats in papaya harbor nucleotide substitutions and insertions of Gs and Ts. In contrast, we found very few N-to-C substitutions, and even fewer instances of nucleotide deletion, suggesting that a six-nucleotide telomere repeat is not well tolerated. The papaya genome encodes single-copy sequence homologues of several genes involved in telomere maintenance and chromosome end protection, including the Telomerase Reverse Transcriptase (TERT) and Protection Of Telomeres (POT1). Notably, unlike Arabidopsis, which encodes six Telomere Repeat binding Factor-like (TRFL) proteins that bind double-stranded telomere DNA, papaya appears to encode only two such proteins. Thus, the more streamlined genome of papaya will provide an excellent resource for comparative and functional analysis of telomeres in plants.

Vibrio choleraeO1 Hybrid El Tor Strains, Asia and Africa

<i>Vibrio cholerae</i>O1 Hybrid El Tor Strains, Asia and Africa

Response of microbial communities to phytoremediation of nickel contaminated soils

Through pot experiment, effects of phytoremediation on microbial communities in soils at different nickel treatment levels were studied. Two Ni hyperaccumulating and one Ni tolerant species were planted in paddy soils different in Ni concentration, ranging from 100 to 1 600 mg/kg. After 110 days of incubation, soil microbial activities were analyzed. Results showed that populations of bacteria, fungus, and actinomycetes and biomass of the microorganisms were stimulated when nickel was added at a rate of 100 mg/kg in non-rhizospheric soil. When the rate was over 100 mg/kg in the soil, adverse effects on the soil microbial communities were observed. The plantation of Ni hyperaccumulating species could increase both the population and biomass of soil microorganisms, because, by absorbing nickel from the soil and excreting root exudates, the plants reduced nickel toxicity and improved the living environment of the microbes. However, different plant species had different effects on microorganisms in soil. Randomly Amplified Polymorphic DNA (RAPD) with five primers was used in this study in 25 soil samples of four types of soils. A total of 947 amplified bands were obtained, including 888 polymorphic bands and 59 non-polymorphic bands. The results indicated that the composition of microbial DNA sequences had changed because of the addition of nickel to the treated soils. Shannon-Weaver index of soil microbial DNA sequences reduced in the nickel contaminated soils with increasing nickel concentration. The changes in Shannon-Weaver index in the four types of soils ranged from 1.65 to 2.32 for Alyssum corsicum, 1.37 to 2.27 for Alyssum murale, 1.37 to 1.96 for Brassica juncea, and 1.19 to 1.85 for nonrhizospheric soil. With the same amount of nickel added to soils, the Shannon-Weaver index in rhizospheric soil with plants was higher than that in non-rhizospheric soil.

Regulation of cytokine transcription in the context of chromatin

Understanding the transcriptional regulation of an important class of innate and adaptive immune system effector molecules, the cytokines, is increasingly important given the promise cytokine regulation holds for treating various autoimmune and inflammatory diseases. Studies defining the mechanisms regulating cytokine transcription initially focused on identifying the cis-acting elements and trans-acting factors that activate cytokine promoters and enhancers. In the past, these studies were largely completed in the absence of constraints instituted by cellular chromatin. Over the past decade it has become obvious that changes in chromatin accessibility critically control, rather than simply correlate with, the transcriptional activation of most genes, including cytokines. Hence candidate transcriptional activators are being re-evaluated for potency in the context of cellular chromatin. Several distinct mechanisms for manipulating the generally repressive context of chromatin have been identified for cytokine genes. Most recently, single nucleotide polymorphisms in cytokine transcriptional regulatory elements have been shown to play measurable roles in regulating cytokine levels in the context of naturally selected haplotypes. Overall, subtle differences in DNA sequence and nucleoprotein complex composition, including protein post-translational modification, come together in cell type-specific combinations to explain the normal variation in cytokine transcription throughout the human populace.

Agrobacterium T-DNA integration in Arabidopsis is correlated with DNA sequence compositions that occur frequently in gene promoter regions

Mobile insertion elements such as transposons and T-DNA generate useful genetic variation and are important tools for functional genomics studies in plants and animals. The spectrum of mutations obtained in different systems can be highly influenced by target site preferences inherent in the mechanism of DNA integration. We investigated the target site preferences of Agrobacterium T-DNA insertions in the chromosomes of the model plant Arabidopsis thaliana. The relative frequencies of insertions in genic and intergenic regions of the genome were calculated and DNA composition features associated with the insertion site flanking sequences were identified. Insertion frequencies across the genome indicate that T-strand integration is suppressed near centromeres and rDNA loci, progressively increases towards telomeres, and is highly correlated with gene density. At the gene level, T-DNA integration events show a statistically significant preference for insertion in the 5' and 3' flanking regions of protein coding sequences as well as the promoter region of RNA polymerase I transcribed rRNA gene repeats. The increased insertion frequencies in 5' upstream regions compared to coding sequences are positively correlated with gene expression activity and DNA sequence composition. Analysis of the relationship between DNA sequence composition and gene activity further demonstrates that DNA sequences with high CG-skew ratios are consistently correlated with T-DNA insertion site preference and high gene expression. The results demonstrate genomic and gene-specific preferences for T-strand integration and suggest that DNA sequences with a pronounced transition in CG- and AT-skew ratios are preferred targets for T-DNA integration.

SeeDNA: A Visualization Tool for K-string Content of Long DNA Sequences and Their Randomized Counterparts

An interactive tool to visualize the K-string composition of long DNA sequences including bacterial complete genomes is described. It is especially useful for exploring short palindromic structures in the sequences. The SeeDNA program runs on Red Hat Linux with GTK+ support. It displays two-dimensional (2D) or one-dimensional (1D) histograms of the K-string distribution of a given sequence and/or its randomized counterpart. It is also capable of showing the difference of K-string distributions between two sequences. The C source code using the GTK+ package is freely available.

Sequence analysis of a functional Drosophila centromere.

Centromeres are the site for kinetochore formation and spindle attachment and are embedded in heterochromatin in most eukaryotes. The repeat-rich nature of heterochromatin has hindered obtaining a detailed understanding of the composition and organization of heterochromatic and centromeric DNA sequences. Here, we report the results of extensive sequence analysis of a fully functional centromere present in the Drosophila Dp1187 minichromosome. Approximately 8.4% (31 kb) of the highly repeated satellite DNA (AATAT and TTCTC) was sequenced, representing the largest data set of Drosophila satellite DNA sequence to date. Sequence analysis revealed that the orientation of the arrays is uniform and that individual repeats within the arrays mostly differ by rare, single-base polymorphisms. The entire complex DNA component of this centromere (69.7 kb) was sequenced and assembled. The 39-kb "complex island" Maupiti contains long stretches of a complex A+T rich repeat interspersed with transposon fragments, and most of these elements are organized as direct repeats. Surprisingly, five single, intact transposons are directly inserted at different locations in the AATAT satellite arrays. We find no evidence for centromere-specific sequences within this centromere, providing further evidence for sequence-independent, epigenetic determination of centromere identity and function in higher eukaryotes. Our results also demonstrate that the sequence composition and organization of large regions of centric heterochromatin can be determined, despite the presence of repeated DNA.

"SPATIAL-TEMPORAL" PATTERNS IN PROKARYOTE GENOMES

We have developed a simple scheme to visualize the string composition of long DNA sequences and applied it to all available prokaryote complete genomes. Each species has a specific "portrait" and most clearly seen patterns in these portraits are determined by short avoided and under-represented strings. The "spatial" patterns in the space of short strings might reflect some "temporal" events in the evolutionary history.

Label-free probing of genes by time-domain terahertz sensing

A label-free sensing approach for the label-free characterization of genetic material with terahertz (THz) electromagnetic waves is presented. Time-resolved THz analysis of polynucleotides demonstrates a strong dependence of the complex refractive index of DNA molecules in the THz frequency range on their hybridization state. By monitoring THz signals one can thus infer the binding state (hybridized or denatured) of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a free-space analytic configuration, as well as a higher-sensitivity approach using integrated THz sensors reaching femtomol detection levels and demonstrating the capability to detect single-base mutations, are presented. The potential application for next generation high-throughput label-free genetic analytic systems is discussed.

Label-Free Probing of the Binding State of DNA by Time-Domain Terahertz Sensing

We present a new approach for the label-free characterization of genetic material. Time-resolved THz transmission analysis of polynucleotides demonstrate a strong dependence of the complex refractive index of DNA molecules on their hybridization state (hybridized / denatured). By monitoring THz signals one can thus infer the binding state of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a free-space configuration, as well as a first demonstration of a higher-sensitivity integrated approach using planar THz resonator sensors, are elucidated. The potential application for next generation high-throughput label-free genetic analytic systems is discussed.

Stretching Single-Stranded DNA: Interplay of Electrostatic, Base-Pairing, and Base-Pair Stacking Interactions

Recent single-macromolecule observations revealed that the force/extension characteristics of single-stranded DNA (ssDNA) are closely related to solution ionic concentration and DNA sequence composition. To understand this, we studied the elastic property of ssDNA through the Monte Carlo implementation of a modified freely jointed chain (FJC), with electrostatic, base-pairing, and base-pair stacking interactions all incorporated. The simulated force-extension profiles for both random and designed sequences have attained quantitative agreements with the experimental data. In low-salt solution, electrostatic interaction dominates, and at low forces, the molecule can be more easily aligned than an unmodified FJC. In high-salt solution, secondary hairpin structure appears in ssDNA by the formation of base pairs between complementary bases, and external stretching causes a hairpin-coil structural transition, which is continuous for ssDNA made of random sequences. In designed sequences such as poly(dA-dT) and poly(dG-dC), the stacking potential between base pairs encourages the aggregation of base pairs into bulk hairpins and makes the hairpin-coil transition a discontinuous (first-order) process. The sensitivity of elongation to the base-pairing rule is also investigated. The comparison of modeling calculations and the experimental data suggests that the base pairing of single-stranded polynucleotide molecules tends to form a nested and independent planar hairpin structure rather than a random intersecting pattern.

Integration of telomere sequences with the draft human genome sequence

Telomeres are the ends of linear eukaryotic chromosomes. To ensure that no large stretches of uncharacterized DNA remain between the ends of the human working draft sequence and the ends of each chromosome, we would need to connect the sequences of the telomeres to the working draft sequence. But telomeres have an unusual DNA sequence composition and organization that makes them particularly difficult to isolate and analyse. Here we use specialized linear yeast artificial chromosome clones, each carrying a large telomere-terminal fragment of human DNA, to integrate most human telomeres with the working draft sequence. Subtelomeric sequence structure appears to vary widely, mainly as a result of large differences in subtelomeric repeat sequence abundance and organization at individual telomeres. Many subtelomeric regions appear to be gene-rich, matching both known and unknown expressed genes. This indicates that human subtelomeric regions are not simply buffers of nonfunctional 'junk DNA' next to the molecular telomere, but are instead functional parts of the expressed genome.