The use of antinucleoside antibodies to probe the organization of chromosomes denatured by ultraviolet irradiation

Ultraviolet irradiation of methanol: acetic acid-fixed human and mouse metaphase chromosomes rendered them capable of binding antibodies specific for purine or pyrimidine bases. Since these antibodies react with single-stranded but not with native DNA, our results indicate that UV irradiation generated single-stranded regions in chromosomal DNA. Using an indirect immuno-fluorescence technique to detect antibody binding, highly characteristic, nonrandom patterns of antibody binding were observed. Antibodies to adenosine (anti-A) and thymidine (anti-T) produced identical patterns of binding which in most respects matched the chromosome banding patterns produced by quinacrine. However, additional foci of intense fluorescence were seen in the paracentromeric regions of constitutive heterochromatin on chromosomes 1, 9 and 16, regions which had been shown by in situ DNA-RNA hybridization to be the locations of AT-rich human satellite DNA. Antibodies to cytidine also bound to the same region of chromosome 9. In mouse chromosome preparations, both anti-A and anti-T produced bright fluorescence of the region containing centromeric heterochromatin, which had been shown to be the location of the AT-rich satellite DNA of this species.     

Relationship between sister-chromatid exchanges and heterochromatin or DNA replication in chromosomes of Crepis capillaris

The C-band patterns, DNA late replication patterns and distribution patterns of spontaneous and γ-ray-induced SCEs in Crepis capillaris chromosomes were studied. The fluorescence plus Giemsa (FPG) technique was used for detection of SCEs and late-replicating chromosome regions after unifilar incorporation of BrdU into DNA. An asynchronous replication of both euchromatic and heterochromatic chromosome regions was established. The frequency of SCEs is increased about 2-fold by 1.5 Gy γ-rays. The localization of the sites of SCEs was analyzed with special reference to eu- and heterochromatin and early- and late-replicating regions. The data obtained showed that SCEs were distributed nonrandomly along the chromosomes. Preferential occurrence of SCEs was observed in the following chromosome regions: at the junction between eu- and heterochromatic regions, the latter being rich in late-replicating DNA; at the junction between early- and late-replicating regions, the latter not being C-band positive. Certain heterochromatic regions were more rarely involved in SCEs than expected on the basis of their length. The lowest incidence of SCEs was found in the centromeric regions. Very similar distribution patterns of spontaneous and γ-ray-induced SCEs were observed. The possible role of the differences in the time of replication of the different chromosome regions in the formation of SCEs is discussed.     

Inter-individual variation of DNA methylation and its implications for large-scale epigenome mapping

Genomic DNA methylation profiles exhibit substantial variation within the human population, with important functional implications for gene regulation. So far little is known about the characteristics and determinants of DNA methylation variation among healthy individuals. We performed bioinformatic analysis of high-resolution methylation profiles from multiple individuals, uncovering complex patterns of inter-individual variation that are strongly correlated with the local DNA sequence. CpG-rich regions exhibit low and relatively similar levels of DNA methylation in all individuals, but the sequential order of the (few) methylated among the (many) unmethylated CpGs differs randomly across individuals. In contrast, CpG-poor regions exhibit substantially elevated levels of inter-individual variation, but also significant conservation of specific DNA methylation patterns between unrelated individuals. This observation has important implications for experimental analysis of DNA methylation, e.g. in the context of epigenome projects. First, DNA methylation mapping at single-CpG resolution is expected to uncover informative DNA methylation patterns for the CpG-poor bulk of the human genome. Second, for CpG-rich regions it will be sufficient to measure average methylation levels rather than assaying every single CpG. We substantiate these conclusions by an in silico benchmarking study of six widely used methods for DNA methylation mapping. Based on our findings, we propose a cost-optimized two-track strategy for mammalian methylome projects.     

Repetitive plasmid sequences generate DNA fingerprinting patterns in mammals

Bacterial plasmids with stringently regulated copy numbers have directly repeated DNA sequences, termed iterons, in the vicinity of their replication origins. These sequences bind a specific protein exerting a key role in the initiation of plasmid replication. Plasmids P1, pSC101 and RFS1010 have different iteron sequences and belong to three different incompatibility groups. Used as DNA probes each of these plasmids generates specific patterns in mammals similar to those obtained by the DNA fingerprinting technique. The iteron-containing regions were identified as the part of the plasmids responsible for those patterns by using polymerase chain reaction (PCR) amplified DNA segments that contained the iteron regions as probes.     

Arrangement of Dam methylation sites (GATC) in the Escherichia coli chromosome.

The occurrence of GATC (Dam-recognition) sites in available E. coli DNA sequences (representing about 2% of the chromosome) has been determined by a simple numerical analysis. Our approach was to analyze the nucleotide composition of nine large sequenced DNA stretches ("cantles") in order to identify patterns of GATC distribution and to rationalize such patterns in biological/structural terms. The following observations were made: (i) In addition to oriC, GATC-rich regions are present in numerous locations. (ii) There is a wide variation in GATC frequency both between and within DNA cantles which led to the identification of a void-cluster pattern of GATC arrangement. The distance between two GATCs was never greater than 2 kb. (iii) GATC sites are found more frequently in translated regions than (in decreasing order) non-coding or non-translated regions. In particular, rRNA and tRNA encoding genes exhibit the lowest GATC content.     

Visualization of the protein-coding regions with a self adaptive spectral rotation approach

Identifying protein-coding regions in DNA sequences is an active issue in computational biology. In this study, we present a self adaptive spectral rotation (SASR) approach, which visualizes coding regions in DNA sequences, based on investigation of the Triplet Periodicity property, without any preceding training process. It is proposed to help with the rough coding regions prediction when there is no extra information for the training required by other outstanding methods. In this approach, at each position in the DNA sequence, a Fourier spectrum is calculated from the posterior subsequence. Following the spectrums, a random walk in complex plane is generated as the SASR's graphic output. Applications of the SASR on real DNA data show that patterns in the graphic output reveal locations of the coding regions and the frame shifts between them: arcs indicate coding regions, stable points indicate non-coding regions and corners' shapes reveal frame shifts. Tests on genomic data set from Saccharomyces Cerevisiae reveal that the graphic patterns for coding and non-coding regions differ to a great extent, so that the coding regions can be visually distinguished. Meanwhile, a time cost test shows that the SASR can be easily implemented with the computational complexity of O(N).     

A role for poly(ADP-ribosyl)ation in DNA methylation.

The aberrant DNA methylation of promoter regions of housekeeping genes leads to gene silencing. Additional epigenetic events, such as histone methylation and acetylation, also play a very important role in the definitive repression of gene expression by DNA methylation. If the aberrant DNA methylation of promoter regions is the starting or the secondary event leading to the gene silencing is still debated. Mechanisms controlling DNA methylation patterns do exist although they have not been ultimately proven. Our data suggest that poly(ADP-ribosyl)ation might be part of this control mechanism. Thus an additional epigenetic modification seems to be involved in maintaining tissue and cell-type methylation patterns that when formed during embryo development, have to be rigorously conserved in adult organisms.     

Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences

Five distinct patterns of DNA replication have been identified during S-phase in asynchronous and synchronous cultures of mammalian cells by conventional fluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. During early S-phase, replicating DNA (as identified by 5-bromodeoxyuridine incorporation) appears to be distributed at sites throughout the nucleoplasm, excluding the nucleolus. In CHO cells, this pattern of replication peaks at 30 min into S-phase and is consistent with the localization of euchromatin. As S-phase continues, replication of euchromatin decreases and the peripheral regions of heterochromatin begin to replicate. This pattern of replication peaks at 2 h into S-phase. At 5 h, perinucleolar chromatin as well as peripheral areas of heterochromatin peak in replication. 7 h into S-phase interconnecting patches of electron-dense chromatin replicate. At the end of S-phase (9 h), replication occurs at a few large regions of electron-dense chromatin. Similar or identical patterns have been identified in a variety of mammalian cell types. The replication of specific chromosomal regions within the context of the BrdU-labeling patterns has been examined on an hourly basis in synchronized HeLa cells. Double labeling of DNA replication sites and chromosome-specific alpha-satellite DNA sequences indicates that the alpha-satellite DNA replicates during mid S-phase (characterized by the third pattern of replication) in a variety of human cell types. Our data demonstrates that specific DNA sequences replicate at spatially and temporally defined points during the cell cycle and supports a spatially dynamic model of DNA replication.     

Patterns of evolution of mitochondrial cytochrome c oxidase I and II DNA and implications for DNA barcoding

DNA barcoding has focused increasing attention on the use of specific regions of mitochondrial cytochrome c oxidase I and II genes (COI-COII) to diagnose and delimit species. However, our understanding of patterns of molecular evolution within these genes is limited. Here we examine patterns of nucleotide divergence in COI-COII within species and between species pairs of Lepidoptera and Diptera using a sliding window analysis. We found that: (1) locations of maximum divergence within COI-COII were highly variable among taxa surveyed in this study; (2) there was major overlap in divergence within versus between species, including within individual COI-COII profiles; (3) graphical DNA saturation analysis showed variation in percent nucleotide transitions throughout COI-COII and only limited association with levels of DNA divergence. Ultimately, no single optimally informative 600 bp location was found within the 2.3 kb of COI-COII, and the DNA barcoding region was no better than other regions downstream in COI. Consequently, we recommend that researchers should maximize sequence length to increase the probability of sampling regions of high phylogenetic informativeness, and to minimize stochastic variation in estimating total divergence.     

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova & Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.     

Strain identification in Rhizobium trifolii using DNA restriction analysis, plasmid DNA profiles and intrinsic antibiotic resistances

Abstract Indigenous strains of R. trifolii , originating from different geographical regions, were compared using total DNA restriction analysis, plasmid DNA profiles and intrinsic antibiotic resistance patterns. The results of strain relatedness using 2 of these techniques (DNA restriction and antibiotic resistance patterns) were similar, and indicated that these strains formed a heterogeneous group. A large variation in plasmid DNA profiles was observed among the R. trifolii isolates investigated, indicating heterogeneity of plasmid DNA pools. This variation occurred even in some strains that were shown to be related on the basis of their antibiotic resistance patterns. The application of these techniques to differentiate indigenous R. trifolii strains is discussed in this report.     

Comprehensive DNA methylation analysis of human peripheral blood leukocytes and lymphoblastoid cell lines

DNA methylation is involved in development and in human diseases. Genomic DNA derived from lymphoblastoid cell lines (LCLs) is commonly used to study DNA methylation. There are potential confounding factors regarding the use of LCL-derived DNA, however, such as Epstein-Barr (EB) viral infection and artifacts induced during cell culture. Recently, several groups compared the DNA methylation status of peripheral blood leukocytes (PBLs) and LCLs and concluded that the DNA methylation profiles between them might be consistent. To confirm and extend theses results, we performed a comprehensive DNA methylation analysis using both PBLs and LCLs derived from the same individuals. Using the luminometric methylation assay, we revealed that the global DNA methylation level was different between PBLs and LCLs. Furthermore, the direction of change was not consistent. Comparisons of genome-wide DNA methylation patterns of promoter regions revealed that methylation profiles were largely conserved between PBLs and LCLs. A preliminary analysis in a small number of samples suggested that the methylation status of an LCL may be better correlated with PBLs from the same individual than with LCLs from other individuals. Expectedly, DNA methylation in promoter regions overlapping with CpG islands was associated with gene silencing in both PBLs and LCLs. With regard to methylation differences, we found that hypermethylation was more predominant than hypomethylation in LCLs compared with PBLs. These findings suggest that LCLs should be used for DNA methylation studies with caution as the methylation patterns of promoter regions in LCLs are not always the same as those in PBLs.     

Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with ³H-thymidine and chromosome samples were prepared for the next 32 h at 1–2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.     

Inheritance Patterns and Stability of DNA Methylation Variation in Maize Near-Isogenic Lines

DNA methylation is a chromatin modification that contributes to epigenetic regulation of gene expression. The inheritance patterns and trans-generational stability of 962 differentially methylated regions (DMRs) were assessed in a panel of 71 near-isogenic lines (NILs) derived from maize (Zea mays) inbred lines B73 and Mo17. The majority of DMRs exhibit inheritance patterns that would be expected for local (cis) inheritance of DNA methylation variation such that DNA methylation level was coupled to local genotype. There are few examples of DNA methylation that exhibit trans-acting control or paramutation-like patterns. The cis-inherited DMRs provide an opportunity to study the stability of inheritance for DNA methylation variation. There was very little evidence for alterations of DNA methylation levels at these DMRs during the generations of the NIL population development. DNA methylation level was associated with local genotypes in nearly all of the >30,000 potential cases of inheritance. The majority of the DMRs were not associated with small RNAs. Together, our results suggest that a significant portion of DNA methylation variation in maize exhibits locally (cis) inherited patterns, is highly stable, and does not require active programming by small RNAs for maintenance.DNA methylation may contribute to heritable epigenetic information in many eukaryotic genomes. In this study, we have documented the inheritance patterns and trans-generational stability for nearly 1000 DNA methylation variants in a segregating maize population. At most loci studied, the DNA methylation differences are locally inherited and are not influenced by the other allele or other genomic regions. The inheritance of DNA methylation levels across generations is quite robust with almost no examples of unstable inheritance, suggesting that DNA methylation differences can be quite stably inherited, even in segregating populations.     

异源DNA导入水稻诱发活跃反转子Tos17发生可遗传DNA甲基化变异

用水稻(Oryza sativa L.)内源反转座子Tos17为探针,经Southern杂交在5种含有野生稻(Zizania latifolia Griseb)(菰)DNA片段的水稻渐渗杂交系中检测到了可遗传DNA甲基化变异.在分析的4种甲基化敏感限制性内切酶中,每种酶切都发生了亲本杂交片段的消失和新片段的出现.发生甲基化变异的位点包括对称和不对称的胞嘧啶碱基,也包括腺嘌呤碱基.序列分析表明,与水稻亲本比较,所研究的5种渐渗杂交系在Tos17的2个重要区域(5'-LTR和RT)均未发生序列变异.但甲基化敏感-序列特异性PCR分析证实,每种渐渗杂交系在这2个区域内均发生了广泛的DNA甲基化变异.而且,在2种渐渗杂交系中发现5'-LTR和RT区域的甲基化变异存在协同性.甲基化变异可稳定遗传给后代.因为已有的研究表明,在这5种渐渗杂交系中异源DNA导入均导致了Tos17的激活和转座,因此可以推测DNA甲基化在调控Tos17活性中可能具有一定作用.但反转座子激活和甲基化变异之间的确切关系尚有待进一步研究.     

Complex high-resolution linkage disequilibrium and haplotype patterns of single-nucleotide polymorphisms in 2.5 Mb of sequence on human chromosome 21.

One approach to identify potentially important segments of the human genome is to search for DNA regions with nonrandom patterns of human sequence variation. Previous studies have investigated these patterns primarily in and around candidate gene regions. Here, we determined patterns of DNA sequence variation in 2.5 Mb of finished sequence from five regions on human chromosome 21. By sequencing 13 individual chromosomes, we identified 1460 single-nucleotide polymorphisms (SNPs) and obtained unambiguous haplotypes for all chromosomes. For all five chromosomal regions, we observed segments with high linkage disequilibrium (LD), extending from 1.7 to>81 kb (average 21.7 kb), disrupted by segments of similar or larger size with no significant LD between SNPs. At least 25% of the contig sequences consisted of segments with high LD between SNPs. Each of these segments was characterized by a restricted number of observed haplotypes,with the major haplotype found in over 60% of all chromosomes. In contrast, the interspersed segments with low LD showed significantly more haplotype patterns. The position and extent of the segments of high LD with restricted haplotype variability did not coincide with the location of coding sequences. Our results indicate that LD and haplotype patterns need to be investigated with closely spaced SNPs throughout the human genome, independent of the location of coding sequences, to reliably identify regions with significant LD useful for disease association studies.     

Variability in allelic DNA methylation in spermatozoa

In certain segments of human DNA, the methylation of deoxycytidine residues has been found to be highly specific and interindividually conserved. Imprinted DNA sequences in diploid primary cells show allele-specific differences in DNA methylation, usually with the active chromosomal regions being unmethylated and the inactive regions being methylated. We show here that DNA from spermatozoa exhibits variations in allelic methylation patterns. Since germ cells are haploid, individual spermatozoa can differ in DNA methylation patterns not only in the maternally or paternally derived allele, but also within each allele.     

A method for fast database search for all k-nucleotide repeats.

A significant portion of DNA consists of repeating patterns of various sizes, from very small (one, two and three nucleotides) to very large (over 300 nucleotides). Although the functions of these repeating regions are not well understood, they appear important for understanding the expression, regulation and evolution of DNA. For example, increases in the number of trinucleotide repeats have been associated with human genetic disease, including Fragile-X mental retardation and Huntington's disease. Repeats are also useful as a tool in mapping and identifying DNA; the number of copies of a particular pattern at a site is often variable among individuals (polymorphic) and is therefore helpful in locating genes via linkage studies and also in providing DNA fingerprints of individuals. The number of repeating regions is unknown as is the distribution of pattern sizes. It would be useful to search for such regions in the DNA database in order that they may be studied more fully. The DNA database currently consists of approximately 150 million basepairs and is growing exponentially. Therefore, any program to look for repeats must be efficient and fast. In this paper, we present some new techniques that are useful in recognizing repeating patterns and describe a new program for rapidly detecting repeat regions in the DNA database where the basic unit of the repeat has size up to 32 nucleotides. It is our hope that the examples in this paper will illustrate the unrealized diversity of repeats in DNA and that the program we have developed will be a useful tool for locating new and interesting repeats.