Satellitome Analysis of the Pacific Oyster Crassostrea gigas Reveals New Pattern of Satellite DNA Organization,Highly Scattered across the Genome

Several features already qualified the invasive bivalve species Crassostrea gigas as a valuable non-standard model organism in genome research. C. gigas is characterized by the low contribution of satellite DNAs (satDNAs) vs. mobile elements and has an extremely low amount of heterochromatin, predominantly built of DNA transposons. In this work, we have identified 52 satDNAs composing the satellitome of C. gigas and constituting about 6.33% of the genome. Satellitome analysis reveals unusual, highly scattered organization of relatively short satDNA arrays across the whole genome. However, peculiar chromosomal distribution and densities are specific for each satDNA. The inspection of the organizational forms of the 11 most abundant satDNAs shows association with constitutive parts of Helitron mobile elements. Nine of the inspected satDNAs are dominantly found in mobile element-associated form, two mostly appear standalone, and only one is present exclusively as Helitron-associated sequence. The Helitron-related satDNAs appear in more chromosomes than other satDNAs, indicating that these mobile elements could be leading satDNA propagation in C. gigas. No significant accumulation of satDNAs on certain chromosomal positions was detected in C. gigas, thus establishing a novel pattern of satDNA organization on the genome level.     

The Cytogenetic Map of the Nile Crocodile (Crocodylus niloticus,Crocodylidae, Reptilia) with Fluorescence In Situ Localization of Major Repetitive DNAs

Tandemly arranged and dispersed repetitive DNA sequences are important structural and functional elements that make up a significant portion of vertebrate genomes. Using high throughput, low coverage whole genome sequencing followed by bioinformatics analysis, we have identified seven major tandem repetitive DNAs and two fragments of LTR retrotransposons in the genome of the Nile crocodile (Crocodylus niloticus, 2n = 32). The repeats showed great variability in structure, genomic organization, and chromosomal distribution as revealed by fluorescence in situ hybridization (FISH). We found that centromeric and pericentromeric heterochromatin of C. niloticus is composed of previously described in Crocodylus siamensis CSI-HindIII and CSI-DraI repetitive sequence families, a satellite revealed in Crocodylus porosus, and additionally contains at least three previously unannotated tandem repeats. Both LTR sequences identified here belong to the ERV1 family of endogenous retroviruses. Each pericentromeric region was characterized by a diverse set of repeats, with the exception of chromosome pair 4, in which we found only one type of satellite. Only a few repeats showed non-centromeric signals in addition to their centromeric localization. Mapping of 18S–28S ribosomal RNA genes and telomeric sequences (TTAGGG)_n did not demonstrate any co-localization of these sequences with revealed centromeric and pericentromeric heterochromatic blocks.     

5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH)

We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.     

A Low-Activity Polymorphic Variant of Human NEIL2 DNA Glycosylase

Human NEIL2 DNA glycosylase (hNEIL2) is a base excision repair protein that removes oxidative lesions from DNA. A distinctive feature of hNEIL2 is its preference for the lesions in bubbles and other non-canonical DNA structures. Although a number of associations of polymorphisms in the hNEIL2 gene were reported, there is little data on the functionality of the encoded protein variants, as follows: only hNEIL2 R103Q was described as unaffected, and R257L, as less proficient in supporting the repair in a reconstituted system. Here, we report the biochemical characterization of two hNEIL2 variants found as polymorphisms in the general population, R103W and P304T. Arg103 is located in a long disordered segment within the N-terminal domain of hNEIL2, while Pro304 occupies a position in the β-turn of the DNA-binding zinc finger motif. Similar to the wild-type protein, both of the variants could catalyze base excision and nick DNA by β-elimination but demonstrated a lower affinity for DNA. Steady-state kinetics indicates that the P304T variant has its catalytic efficiency (in terms of k_cat/K_M) reduced ~5-fold compared with the wild-type hNEIL2, whereas the R103W enzyme is much less affected. The P304T variant was also less proficient than the wild-type, or R103W hNEIL2, in the removal of damaged bases from single-stranded and bubble-containing DNA. Overall, hNEIL2 P304T could be worthy of a detailed epidemiological analysis as a possible cancer risk modifier.     

Plant DNA Repair and Agrobacterium T−DNA Integration

Agrobacterium species transfer DNA (T−DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T−DNA often contains, at its junctions with plant DNA, deletions of T−DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T−DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T−DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T−DNA integration into the plant genome.     

The interaction of water-soluble iron porphyrins with DNA films and the electrocatalytic properties for inorganic and organic nitro compounds

The electrochemistry of water-soluble iron porphyrins (Fe(n-TMPyP)) (where n=2 and 4) was studied as an electrochemically active film on DNA modified glassy carbon, gold, platinum, and transparent semiconductor tin oxide electrodes in solutions of various pH values. The two layers of the modified electrode containing the iron porphyrin and the DNA film were prepared by depositing the iron porphyrin on a DNA film modified electrode. The Fe(4-TMPyP)/DNA film was electrocatalytic reductive for p-nitrobenzoic acid in a weak acidic, or neutral aqueous solution through an Fe^II species, and the electrocatalytic reduction peak potential became more negative than the cathodic peak of the Fe^III/II redox couple. The electrocatalytic reduction properties by the Fe(2-TMPyP)/DNA film as catalysts for nitrite reduction have also been determined, and shown to be active through an Fe^I species and to be pH-dependent. The electrocatalytic oxidation properties of nitrite by Fe(n-TMPyP)/DNA (for n=2 and 4) film have also been determined and shown to be active through an Fe^IV species with the electrocatalytic oxidation efficiency of NO₂⁻ with Fe^IV(O)(n-TMPyP) being higher than with (HO)Fe^IV(O)(n-TMPyP). The electrocatalytic oxidation efficiency of NO₂⁻ by iron porphyrin is pH-dependent. The electrocatalytic reduction of p-nitrophenol by Fe(2-TMPyP)/DNA film are also discussed.     

Sequence,Chromatin and Evolution of Satellite DNA

Satellite DNA consists of abundant tandem repeats that play important roles in cellular processes, including chromosome segregation, genome organization and chromosome end protection. Most satellite DNA repeat units are either of nucleosomal length or 5–10 bp long and occupy centromeric, pericentromeric or telomeric regions. Due to high repetitiveness, satellite DNA sequences have largely been absent from genome assemblies. Although few conserved satellite-specific sequence motifs have been identified, DNA curvature, dyad symmetries and inverted repeats are features of various satellite DNAs in several organisms. Satellite DNA sequences are either embedded in highly compact gene-poor heterochromatin or specialized chromatin that is distinct from euchromatin. Nevertheless, some satellite DNAs are transcribed into non-coding RNAs that may play important roles in satellite DNA function. Intriguingly, satellite DNAs are among the most rapidly evolving genomic elements, such that a large fraction is species-specific in most organisms. Here we describe the different classes of satellite DNA sequences, their satellite-specific chromatin features, and how these features may contribute to satellite DNA biology and evolution. We also discuss how the evolution of functional satellite DNA classes may contribute to speciation in plants and animals.     

Structure‐Specific Recognition of Friedreich's Ataxia (GAA)n Repeats by Benzoquinoquinoxaline Derivatives

Expansion of GAA triplet repeats in intron 1 of the FXN gene reduces frataxin expression and causes Friedreich's ataxia. (GAA)_n repeats form non‐B‐DNA structures, including triple helix H‐DNA and higher‐order structures (sticky DNA). In the proposed mechanisms of frataxin gene silencing, central unanswered questions involve the characterization of non‐B‐DNA structure(s) that are strongly suggested to play a role in frataxin expression. Here we examined (GAA)_n binding by triplex‐stabilizing benzoquinoquinoxaline (BQQ) and the corresponding triplex‐DNA‐cleaving BQQ‐1,10‐phenanthroline (BQQ‐OP) compounds. We also examined the ability of these compounds to act as structural probes for H‐DNA formation within higher‐order structures at pathological frataxin sequences in plasmids. DNA‐complex‐formation analyses with a gel‐mobility‐shift assay and sequence‐specific probing of H‐DNA‐forming (GAA)_n sequences by single‐strand oligonucleotides and triplex‐directed cleavage demonstrated that a parallel pyrimidine (rather than purine) triplex is the more stable motif formed at (GAA)_n repeats under physiologically relevant conditions.     

Nucleic acid biosensor for DNA hybridization detection using rutin-Cu as an electrochemical indicator

The complex of rutin-Cu (C₈₁H₈₆Cu₂O₄₈, abbreviated by Cu₂R₃, R = rutin) was synthesized and characterized by elemental analysis and IR spectra. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction of Cu₂R₃ with salmon sperm DNA. It was revealed that Cu₂R₃ could interact with double-stranded DNA (dsDNA) by a major intercalation role. Using Cu₂R₃ as a novel electroactive indicator, an electrochemical DNA biosensor for the detection of specific DNA fragment was developed and its selectivity for the recognition with different target DNA was assessed by differential pulse voltammetry (DPV). The target DNA related to coliform virus gene could be quantified ranged from 1.62 × 10⁻⁸ mol L⁻¹ to 8.10 × 10⁻⁷ mol L⁻¹ with a good linearity (r = 0.9989) and a detection limit of 2.3 × 10⁻⁹ mol L⁻¹ (3σ, n = 7) was achieved by the constructed electrochemical DNA biosensor.     

A Cross between Bread Wheat and a 2D(2R) Disomic Substitution Triticale Line Leads to the Formation of a Novel Disomic Addition Line and Provides Information of the Role of Rye Secalins on Breadmaking Characteristics

A bread wheat line (N11) and a disomic 2D(2R) substitution triticale line were crossed and backrossed four times. At each step electrophoretic selection for the seeds that possessed, simultaneously, the complete set of high molecular weight glutenin subunits of N11 and the two high molecular weight secalins of rye, present in the 2D(2R) line, was carried out. Molecular cytogenetic analyses of the BC₄F₈ generation revealed that the selection carried out produced a disomic addition line (2n = 44). The pair of additional chromosomes consisted of the long arm of chromosome 1R (1RL) from rye fused with the satellite body of the wheat chromosome 6B. Rheological analyses revealed that the dough obtained by the new addition line had higher quality characteristics when compared with the two parents. The role of the two additional high molecular weight secalins, present in the disomic addition line, in influencing improved dough characteristics is discussed.     

Identification of Novel Chromosomal Aberrations Induced by 60Co-γ-Irradiation in Wheat-Dasypyrum villosum Lines

Mutations induced by radiation are widely used for developing new varieties of plants. To better understand the frequency and pattern of irradiation-induced chromosomal rearrangements, we irradiated the dry seeds of Chinese Spring (CS)-Dasypyrum villosum nullisomic-tetrasomic (6A/6D) addition (6V) line (2n = 44), WD14, with ⁶⁰Co-γ-rays at dosages of 100, 200, and 300 Gy. The M₀ and M₁ generations were analyzed using Feulgen staining and non-denaturing fluorescence in situ hybridization (ND-FISH) by using oligonucleotide probes. Abnormal mitotic behavior and chromosomes with structural changes were observed in the M₀ plants. In all, 39 M₁ plants had structurally changed chromosomes, with the B genome showing the highest frequency of aberrations and tendency to recombine with chromosomes of the D genome. In addition, 19 M₁ plants showed a variation in chromosome number. The frequency of chromosome loss was considerably higher for 6D than for the alien chromosome 6V, indicating that 6D is less stable after irradiation. Our findings suggested that the newly obtained γ-induced genetic materials might be beneficial for future wheat breeding programs and functional gene analyses.     

Integrating Genetic and Chromosome Maps of Allium cepa: From Markers Visualization to Genome Assembly Verification

The ability to directly look into genome sequences has opened great opportunities in plant breeding. Yet, the assembly of full-length chromosomes remains one of the most difficult problems in modern genomics. Genetic maps are commonly used in de novo genome assembly and are constructed on the basis of a statistical analysis of the number of recombinations. This may affect the accuracy of the ordering and orientation of scaffolds within the chromosome, especially in the region of recombination suppression. Moreover, it is impossible to assign contigs lacking DNA markers. Here, we report the use of Tyr-FISH to determine the position of the short DNA sequence of markers and non-mapped unique copy sequence on the physical chromosomes of a large-genome onion (Allium cepa L.). In order to minimize potential background masking of the target signal, we improved our earlier developed pipeline for probe design. A total of 23 markers were located on physical chromosomes 2 and 6. The order of markers was corrected by the integration of genetic, pseudochromosome maps and cytogenetic maps. Additionally, the position of the mlh1 gene, which was not on the genetic map, was defined on physical chromosome 2. Tyr-FISH mapping showed that the order of 23.1% (chromosome 2) and 27.3% (chromosome 6) of the tested genes differed between physical chromosomes and pseudochromosomes. The results can be used for the improvement of pseudochromosome 2 and 6 assembly. The present study aims to demonstrate the value of the in situ visualization of DNA sequences in chromosome-scaffold genome assembly.     

Identification and DNA Marker Development for a Wheat-Leymus mollis 2Ns (2D) Disomic Chromosome Substitution

Leymus mollis (2n = 4x = 28, NsNsXmXm), a wild relative of common wheat (Triticum aestivum L.), carries numerous loci which could potentially be used in wheat improvement. In this study, line 17DM48 was isolated from the progeny of a wheat and L. mollis hybrid. This line has 42 chromosomes forming 21 bivalents at meiotic metaphase I. Genomic in situ hybridization (GISH) demonstrated the presence of a pair chromosomes from the Ns genome of L. mollis. This pair substituted for wheat chromosome 2D, as shown by fluorescence in situ hybridization (FISH), DNA marker analysis, and hybridization to wheat 55K SNP array. Therefore, 17DM48 is a wheat-L. mollis 2Ns (2D) disomic substitution line. It shows longer spike and a high level of stripe rust resistance. Using specific-locus amplified fragment sequencing (SLAF-seq), 13 DNA markers were developed to identify and trace chromosome 2Ns of L. mollis in wheat background. This line provides a potential bridge germplasm for genetic improvement of wheat stripe rust resistance.     

Kinetic model of single boron particle ignition based upon both oxygen and (BO) n diffusion mechanism

A comprehensive ignition model for single boron particles in an oxygenated environment containing O₂ and H₂O is developed. Microcharacteristics of the boron oxide layer on the surface of boron particles at elevated temperatures are studied. Two typical distributions of species inside the surface oxide layer are detected. One is composed of three layers [B₂O₃, (BO) _n , and B₂O₃], while the other is composed of two layers [(BO) _n and B₂O₃], both according to the order from the internal to external side of the layer. In the model development, two submodels, submodel I and submodel II, are developed with regard to two different observed species distributions in the surface oxide layer. For submodel I, it is assumed that both (BO) _n and O₂ are the governing species diffusing into the liquid oxide layer. For submodel II, only (BO) _n is the governing species. These two submodels are combined into a new bi-directional model consisting of four key kinetic processes: evaporation of the liquid oxide layer, global surface reaction between oxygen from the environment and boron, reaction between the inner boron core and oxygen, and global reaction of boron with water vapor. The ignition time predicted by the model is in good agreement with previous experimental data.     

Responses of DNA Mismatch Repair Proteins to a Stable G-Quadruplex Embedded into a DNA Duplex Structure

DNA mismatch repair (MMR) plays a crucial role in the maintenance of genomic stability. The main MMR protein, MutS, was recently shown to recognize the G-quadruplex (G4) DNA structures, which, along with regulatory functions, have a negative impact on genome integrity. Here, we studied the effect of G4 on the DNA-binding activity of MutS from Rhodobacter sphaeroides (methyl-independent MMR) in comparison with MutS from Escherichia coli (methyl-directed MMR) and evaluated the influence of a G4 on the functioning of other proteins involved in the initial steps of MMR. For this purpose, a new DNA construct was designed containing a biologically relevant intramolecular stable G4 structure flanked by double-stranded regions with the set of DNA sites required for MMR initiation. The secondary structure of this model was examined using NMR spectroscopy, chemical probing, fluorescent indicators, circular dichroism, and UV spectroscopy. The results unambiguously showed that the d(GGGT)₄ motif, when embedded in a double-stranded context, adopts a G4 structure of a parallel topology. Despite strong binding affinities of MutS and MutL for a G4, the latter is not recognized by E. coli MMR as a signal for repair, but does not prevent MMR processing when a G4 and G/T mismatch are in close proximity.