Family Of DNA Sequences Research Articles

The Evolution of CRISPR Technology from Editing to Diagnostics

The Evolution of CRISPR Technology from Editing to Diagnostics

Large vs small genomes in Passiflora: the influence of the mobilome and the satellitome

While two lineages of retrotransposons were more abundant in larger Passiflora genomes, the satellitome was more diverse and abundant in the smallest genome analysed. Repetitive sequences are ubiquitous and fast-evolving elements responsible for size variation and large-scale organization of plant genomes. Within Passiflora genus, a tenfold variation in genome size, not attributed to polyploidy, is known. Here, we applied a combined in silico and cytological approach to study the organization and diversification of repetitive elements in three species of this genus representing its known range in genome size variation. Sequences were classified in terms of type and repetitiveness and the most abundant were mapped to chromosomes. We identified long terminal repeat (LTR) retrotransposons as the most abundant elements in the three genomes, showing a considerable variation among species. Satellite DNAs (satDNAs) were less representative, but highly diverse between subgenera. Our results clearly confirm that the largest genome species (Passiflora quadrangularis) presents a higher accumulation of repetitive DNA sequences, specially Angela and Tekay elements, making up most of its genome. Passiflora cincinnata, with intermediate genome and from the same subgenus, showed similarity with P. quadrangularis regarding the families of repetitive DNA sequences, but in different proportions. On the other hand, Passiflora organensis, the smallest genome, from a different subgenus, presented greater diversity and the highest proportion of satDNA. Altogether, our data indicates that while large genomes evolved by an accumulation of retrotransposons, the smallest genome known for the genus has evolved by diversification of different repeat types, particularly satDNAs.

CRISPR-Cas genome-editing tool in plant abiotic stress-tolerance

Adverse ambience caused by abiotic stresses affect the plant growth and increase the chances of low yield and resulting economic loss. To resolve this issue, several measures have been taken. Use of CRISPR/Cas gene-editing system to develop stress-resistant plant varieties has gained attention at present. Versatility of this user-friendly system has made it popular to the researchers. The DNA sequence family “CRISPR” and the endonuclease “Cas” work together in the gene-editing process following the direction by specific guide RNA. CRISPR/Cas system is applied in a number of plant species. Cas9 is frequently used in most of the cases. Several reports of the improvement of abiotic stress-tolerance in plants using CRISPR/Cas9 technology has been documented. Genome-editing efficiency of CRISPR/Cas9 system depends on some factors like Cas9 expression, RNA polymerase specificity, guide RNA expression, promoter sequence, types of transformation vectors etc. Sometimes, a few modifications of this system is needed to get expected outcomes. CRISPR/Cas9 system can be utilized in some prospective programmes like mutant library generation, reduction of agrochemical supplementation during cultivation, gene regulation, plant domestication etc. Basic and applied plant biology might be enriched through the further progress of CRISPR/Cas9 technology. The present review encompasses the applications of CRISPR-Cas genome-editing toolkit against abiotic stress-tolerance in many model as well as commercially important plants with notes on their genetic regulations. In addition, the article also outlines the existing shortcomings in past research with notes on future prospective.

Development of CRISPR technology for precise single-base genome editing: a brief review

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a family of DNA sequences originally discovered as a type of acquired immunity in prokaryotes such as bacteria and archaea. In many CRISPR systems, the functional ribonucleoproteins (RNPs) are composed of CRISPR protein and guide RNAs. They selectively bind and cleave specific target DNAs or RNAs, based on sequences complementary to the guide RNA. The specific targeted cleavage of the nucleic acids by CRISPR has been broadly utilized in genome editing methods. In the process of genome editing of eukaryotic cells, CRISPR-mediated DNA double-strand breaks (DSB) at specific genomic loci activate the endogenous DNA repair systems and induce mutations at the target sites with high efficiencies. Two of the major endogenous DNA repair machineries are non-homologous end joining (NHEJ) and homology-directed repair (HDR). In case of DSB, the two repair pathways operate in competition, resulting in several possible outcomes including deletions, insertions, and substitutions. Due to the inherent stochasticity of DSB-based genome editing methods, it was difficult to achieve defined single-base changes without unanticipated random mutation patterns. In order to overcome the heterogeneity in DSB-mediated genome editing, novel methods have been developed to incorporate precise single-base level changes without inducing DSB. The approaches utilized catalytically compromised CRISPR in conjunction with base-modifying enzymes and DNA polymerases, to accomplish highly efficient and precise genome editing of single and multiple bases. In this review, we introduce some of the advances in single-base level CRISPR genome editing methods and their applications.

Evolution conservatively of SCAR DNA localization in genome isochores of warm-blooded vertebrates

In meiotic prophase I, chromatin fibrils attached to the lateral elements of the synaptonemal complexes form loops. Synaptonemal complex associated regions of DNA (SCARs DNA) are a family of genomic DNA sequences tightly associated with the synaptonemal complex; they are located at the chromatin loop basements. Isochore compositional fractions of the human and chicken genomes were used as 32P labeled probes for hybridization with SCAR DNA isolated previously from the spermatocyte nuclei of the golden hamster Mesocricetus auratus. Nucleotide sequences similar to the golden hamster’s SCAR DNA were found in human and chicken genome isochores. The localization of SCAR DNA in isochore compartments of the examined genomes was established to be evolutionary conservative.

Chromosome size-correlated and chromosome size-uncorrelated homogenization of centromeric repetitive sequences in New World quails

Many families of centromeric repetitive DNA sequences isolated from Struthioniformes, Galliformes, Falconiformes, and Passeriformes are localized primarily to microchromosomes. However, it is unclear whether chromosome size-correlated homogenization is a common characteristic of centromeric repetitive sequences in Aves. New World and Old World quails have the typical avian karyotype comprising chromosomes of two distinct sizes, and C-positive heterochromatin is distributed in centromeric regions of most autosomes and the whole W chromosome. We isolated six types of centromeric repetitive sequences from three New World quail species (Colinus virginianus, CVI; Callipepla californica, CCA; and Callipepla squamata, CSQ; Odontophoridae) and one Old World quail species (Alectoris chukar, ACH; Phasianidae), and characterized the sequences by nucleotide sequencing, chromosome in situ hybridization, and filter hybridization. The 385-bp CVI-MspI, 591-bp CCA-BamHI, 582-bp CSQ-BamHI, and 366-bp ACH-Sau3AI fragments exhibited tandem arrays of the monomer unit, and the 224-bp CVI-HaeIII and 135-bp CCA-HaeIII fragments were composed of minisatellite-like and microsatellite-like repeats, respectively. ACH-Sau3AI was a homolog of the chicken nuclear membrane repeat sequence, whose homologs are common in Phasianidae. CVI-MspI, CCA-BamHI, and CSQ-BamHI showed high homology and were specific to the Odontophoridae. CVI-MspI was localized to microchromosomes, whereas CVI-HaeIII, CCA-BamHI, and CSQ-BamHI were mapped to almost all chromosomes. CCA-HaeIII was localized to five pairs of macrochromosomes and most microchromosomes. ACH-Sau3AI was distributed in three pairs of macrochromosomes and all microchromosomes. Centromeric repetitive sequences may be homogenized in chromosome size-correlated and -uncorrelated manners in New World quails, although there may be a mechanism that causes homogenization of centromeric repetitive sequences primarily between microchromosomes, which is commonly observed in phasianid birds.

A comparative study of genome organization and inferences for the systematics of two large bushcricket genera of the tribe Barbitistini (Orthoptera: Tettigoniidae: Phaneropterinae)

BackgroundPoecilimon and Isophya are the largest genera of the tribe Barbitistini and among the most systematically complicated and evolutionarily intriguing groups of Palearctic tettigoniids. We examined the genomic organization of 79 taxa with a stable chromosome number using classical (C–banding, silver and fluorochrome staining) and molecular (fluorescence in situ hybridization with 18S rDNA and (TTAGG) n telomeric probes) cytogenetic techniques. These tools were employed to establish genetic organization and differences or similarities between genera or species within the same genus and determine if cytogenetic markers can be used for identifying some taxonomic groups of species.ResultsDifferences between the karyotypes of the studied genera include some general changes in the morphology of the X chromosome in Isophya (in contrast to Poecilimon). The number of major rDNA clusters per haploid genome divided Poecilimon into two main almost equal groups (with either one or two clusters), while two rDNA clusters predominated in Isophya. In both genera, rDNA loci were preferentially located in the paracentromeric region of the autosomes and rarely in the sex chromosomes. Our results demonstrate a coincidence between the location of rDNA loci and active NORs and GC-rich heterochromatin regions. The C/DAPI/CMA3 bands observed in most Poecilimon chromosomes suggest the presence of more families of repetitive DNA sequences as compared to the heterochromatin patterns in Isophya.ConclusionsThe results show both differences and similarities in genome organization among species of the same genus and between genera. Previous views on the systematics and phylogenetic grouping of certain lineages are discussed in light of the present cytogenetic results. In some cases, variation of chromosome markers was observed to correspond with variation in other evolutionary traits, which is related to the processes of ongoing speciation and hybridization in zones of secondary contact. It was concluded that the physical mapping of rDNA sequences and heterochromatin may be used as an additional marker for understanding interspecific relationships in these groups and their routes of speciation.

Efficient Selection of Biomineralizing DNA Aptamers Using Deep Sequencing and Population Clustering

DNA-based information systems drive the combinatorial optimization processes of natural evolution, including the evolution of biominerals. Advances in high-throughput DNA sequencing expand the power of DNA as a potential information platform for combinatorial engineering, but many applications remain to be developed due in part to the challenge of handling large amounts of sequence data. Here we employ high-throughput sequencing and a recently developed clustering method (AutoSOME) to identify single-stranded DNA sequence families that bind specifically to ZnO semiconductor mineral surfaces. These sequences were enriched from a diverse DNA library after a single round of screening, whereas previous screening approaches typically require 5-15 rounds of enrichment for effective sequence identification. The consensus sequence of the largest cluster was poly d(T)30. This consensus sequence exhibited clear aptamer behavior and was shown to promote the synthesis of crystalline ZnO from aqueous solution at near-neutral pH. This activity is significant, as the crystalline form of this wide-bandgap semiconductor is not typically amenable to solution synthesis in this pH range. High-resolution TEM revealed that this DNA synthesis route yields ZnO nanoparticles with an amorphous-crystalline core-shell structure, suggesting that the mechanism of mineralization involves nanoscale coacervation around the DNA template. We thus demonstrate that our new method, termed Single round Enrichment of Ligands by deep Sequencing (SEL-Seq), can facilitate biomimetic synthesis of technological nanomaterials by accelerating combinatorial selection of biomolecular-mineral interactions. Moreover, by enabling direct characterization of sequence family demographics, we anticipate that SEL-Seq will enhance aptamer discovery in applications employing additional rounds of screening.

Highly Species-Specific Centromeric Repetitive DNA Sequences in Lizards: Molecular Cytogenetic Characterization of a Novel Family of Satellite DNA Sequences Isolated from the Water Monitor Lizard (Varanus salvator macromaculatus, Platynota)

Two novel repetitive DNA sequences, VSAREP1 and VSAREP2, were isolated from the water monitor lizard (Varanus salvator macromaculatus, Platynota) and characterized using molecular cytogenetics. The respective lengths and guanine-cytosine (GC) contents of the sequences were 190 bp and 57.5% for VSAREP1 and 185 bp and 59.7% for VSAREP2, and both elements were tandemly arrayed as satellite DNA in the genome. VSAREP1 and VSAREP2 were each located at the C-positive heterochromatin in the pericentromeric region of chromosome 2q, the centromeric region of chromosome 5, and 3 pairs of microchromosomes. This suggests that genomic compartmentalization between macro- and microchromosomes might not have occurred in the centromeric repetitive sequences of V. salvator macromaculatus. These 2 sequences did only hybridize to genomic DNA of V. salvator macromaculatus, but no signal was observed even for other squamate reptiles, including Varanus exanthematicus, which is a closely related species of V. salvator macromaculatus. These results suggest that these sequences were differentiated rapidly or were specifically amplified in the V. salvator macromaculatus genome.

Divergence of Repetitive DNA Sequences in the Heterochromatin of Medaka Fishes: Molecular Cytogenetic Characterization of Constitutive Heterochromatin in Two Medaka Species: Oryzias hubbsi and O. celebensis (Adrianichthyidae, Beloniformes)

The large biarmed chromosomes of Oryzias celebensis [2n = 36, fundamental arm number (FN) = 48] are considered to have resulted from fusions of acrocentric chromosomes in the ancestral karyotype of Oryzias, which is retained in O. hubbsi (2n = 48, FN = 48). To understand the molecular evolution of heterochromatin associated with karyotype reorganization in medaka fishes, we cloned 3 and 6 novel families of heterochromatin-related repetitive DNA sequences from O. hubbsi and O. celebensis, respectively, and characterized them using molecular cytogenetics. Two AT-rich repetitive sequences isolated from the genomic DNA of O. hubbsi, a 164-bp satellite DNA (OHU-RsaI-Scen) and a 177-bp telomere-specific repeat (OHU-RsaI-Stelo), were shown to be major components of the constitutive heterochromatin of centromeres and telomeres, respectively. A GC-rich 326-bp sequence, named OHU-AluI-M1, was colocalized with the 18S-28S ribosomal RNA gene cluster to a single autosomal pair of chromosomes and the W chromosome. In O. celebensis, 2 major satellite DNA sequences (the AT-rich 157-bp OCE-AluI-Scen sequence and the 186-bp OCE-HinfI-Scen sequence) were identified in the centromeric regions of almost all chromosomes. The 197-bp OCE-HinfI-S6 sequence was located in the centromeric and distal and/or interstitial heterochromatin of almost all chromosomes, and the 191-bp OCE-HinfI-S8 sequence was located in 6 pairs of chromosomes. Constitutive heterochromatin on the short arm of large submetacentric chromosome 5 was composed of at least 3 different repetitive sequences: the 171-bp OCE-AluI-S18 sequence, the 197-bp OCE-HinfI-S6 sequence and the 172-bp OCE-HinfI-S11 sequence. All families of repeated sequences showed no nucleotide sequence similarity with each other and high species-specificity among 7 different species. These results suggest that the heterochromatin of O. hubbsi and O. celebensis consists of various types of repetitive sequence and that the sequences evolved independently and were then amplified site-specifically in each lineage after karyotype reorganization occurred in the ancestral karyotype.

Karyotype Reorganization with Conserved Genomic Compartmentalization in Dot-Shaped Microchromosomes in the Japanese Mountain Hawk-Eagle (Nisaetus nipalensis orientalis, Accipitridae)

The karyotype of the Japanese mountain hawk-eagle (Nisaetus nipalensis orientalis) (2n = 66) consists of a large number of medium-sized and small chromosomes but only 4 pairs of dot-shaped microchromosomes, in contrast to the typical avian karyotype with a small number of macrochromosomes and many indistinguishable microchromosomes. To investigate the drastic karyotype reorganization in this species, we performed a molecular cytogenetic characterization employing chromosome in situ hybridization and molecular cloning of centromeric heterochromatin. Cross-species chromosome painting with chicken chromosome-specific probes 1-9 and Z and a paint pool of 20 microchromosome pairs revealed that the N. n. orientalis karyotype differs from chicken by at least 13 fissions of macrochromosomes and 15 fusions between microchromosomes and between micro- and macrochromosomes. A novel family of satellite DNA sequences (NNO-ApaI) was isolated, consisting of a GC-rich 173-bp repeated sequence element. The NNO-ApaI sequence was localized to the C-positive centromeric heterochromatin of 4 pairs of microchromosomes, which evolved concertedly by homogenization between the microchromosomes. These results suggest that the 4 pairs of dot-shaped microchromosomes have retained their genomic compartmentalization from other middle-sized and small chromosomes.

A clustering approach for estimating parameters of a profile hidden Markov model

A Profile Hidden Markov Model (PHMM) is a standard form of a Hidden Markov Models used for modeling protein and DNA sequence families based on multiple alignment. In this paper, we implement Baum-Welch algorithm and the Bayesian Monte Carlo Markov Chain (BMCMC) method for estimating parameters of small artificial PHMM. In order to improve the prediction accuracy of the estimation of the parameters of the PHMM, we classify the training data using the weighted values of sequences in the PHMM then apply an algorithm for estimating parameters of the PHMM. The results show that the BMCMC method performs better than the Maximum Likelihood estimation.

Enlightenment of yeast mitochondrial homoplasmy: diversified roles of gene conversion.

Mitochondria have their own genomic DNA. Unlike the nuclear genome, each cell contains hundreds to thousands of copies of mitochondrial DNA (mtDNA). The copies of mtDNA tend to have heterogeneous sequences, due to the high frequency of mutagenesis, but are quickly homogenized within a cell (“homoplasmy”) during vegetative cell growth or through a few sexual generations. Heteroplasmy is strongly associated with mitochondrial diseases, diabetes and aging. Recent studies revealed that the yeast cell has the machinery to homogenize mtDNA, using a common DNA processing pathway with gene conversion; i.e., both genetic events are initiated by a double-stranded break, which is processed into 3′ single-stranded tails. One of the tails is base-paired with the complementary sequence of the recipient double-stranded DNA to form a D-loop (homologous pairing), in which repair DNA synthesis is initiated to restore the sequence lost by the breakage. Gene conversion generates sequence diversity, depending on the divergence between the donor and recipient sequences, especially when it occurs among a number of copies of a DNA sequence family with some sequence variations, such as in immunoglobulin diversification in chicken. MtDNA can be regarded as a sequence family, in which the members tend to be diversified by a high frequency of spontaneous mutagenesis. Thus, it would be interesting to determine why and how double-stranded breakage and D-loop formation induce sequence homogenization in mitochondria and sequence diversification in nuclear DNA. We will review the mechanisms and roles of mtDNA homoplasmy, in contrast to nuclear gene conversion, which diversifies gene and genome sequences, to provide clues toward understanding how the common DNA processing pathway results in such divergent outcomes.

Characterization, Evolution and Chromosomal Distribution of Two Satellite DNA Sequence Families in Lathyrus species

DNA clones containing highly repetitive DNA sequences were selected from partial libraries of Lathyrus sativus and L. sylvestris. Two satellite DNA sequence families were isolated from the genome of the former species. A first family was made up of repeats that varied in length from 54–56 bp, and shared 51.7–94.8% nucleotide sequence similarity. The repeats of the second sequence family were 52–62 bp in length, and shared a 58.5–78.5% nucleotide sequence similarity. All the repeat units contained in a clone from L. sylvestris were 41 bp in length and showed an almost perfect structural conservation (95.1–100% nucleotide sequence similarity). The evolution of the first sequence family from L. sativus and of that isolated from L. sylvestris was studied by dot-blot hybridization to the genomic DNA of these species and 3 other Lathyrus species, L. clymenum, L. latifolius and L. odoratus. The former repeats were found to be species-specific and their redundancy was calculated to be 2.9 × 10⁷. The satellite DNA sequence isolated from L. sylvestris was present also in L.latifolius, with a redundancy of 1.4 × 10⁷ and 1.1 × 10⁷, respectively. Fluorescent in situ hybridization (FISH) was used to investigate the chromosomal distribution of the two sequence families and of 45S and 5S ribosomal genes. The species-specific sequences of L. sativus were located around the centromere of chromosome pair IV, where they occupied a very broad region, and, in a much smaller amount, close to the centromere in the short arm of pair II. Sequences related to the repeat units isolated from L. sylvestris were found, both in this species and L. latifolius, in all of the chromosome pairs at terminal and interstitial regions, where they co-localize with the vast majority of DAPI bands. The pattern of distribution of the satellite DNA sequences investigated, together with that of DAPI bands and ribosomal DNA, allowed each chromosome pair of the 3 complements studied to be identified unambiguously.

Transposable elements in natural populations ofDrosophila melanogaster

Transposable elements (TEs) are families of small DNA sequences found in the genomes of virtually all organisms. The sequences typically encode essential components for the replicative transposition sequences of that TE family. Thus, TEs are simply genomic parasites that inflict detrimental mutations on the fitness of their hosts. Several models have been proposed for the containment of TE copy number in outbreeding host populations such as Drosophila. Surveys of the TEs in genomes from natural populations of Drosophila have played a central role in the investigation of TE dynamics. The early surveys indicated that a typical TE insertion is rare in a population, which has been interpreted as evidence that each TE is selected against. The proposed mechanisms of this natural selection are reviewed here. Subsequent and more targeted surveys identify heterogeneity among types of TEs and also highlight the large role of homologous and possibly ectopic crossing over in the dynamics of the Drosophila TEs. The recent discovery of germline-specific RNA interference via the piwi-interacting RNA pathway opens yet another interesting mechanism that may be critical in containing the copy number of TEs in natural populations of Drosophila. The expected flood of Drosophila population genomics is expected to rapidly advance understanding of the dynamics of TEs.

Transposon identification using profile HMMs.

BackgroundTransposons are "jumping genes" that account for large quantities of repetitive content in genomes. They are known to affect transcriptional regulation in several different ways, and are implicated in many human diseases. Transposons are related to microRNAs and viruses, and many genes, pseudogenes, and gene promoters are derived from transposons or have origins in transposon-induced duplication. Modeling transposon-derived genomic content is difficult because they are poorly conserved. Profile hidden Markov models (profile HMMs), widely used for protein sequence family modeling, are rarely used for modeling DNA sequence families. The algorithm commonly used to estimate the parameters of profile HMMs, Baum-Welch, is prone to prematurely converge to local optima. The DNA domain is especially problematic for the Baum-Welch algorithm, since it has only four letters as opposed to the twenty residues of the amino acid alphabet.ResultsWe demonstrate with a simulation study and with an application to modeling the MIR family of transposons that two recently introduced methods, Conditional Baum-Welch and Dynamic Model Surgery, achieve better estimates of the parameters of profile HMMs across a range of conditions.ConclusionsWe argue that these new algorithms expand the range of potential applications of profile HMMs to many important DNA sequence family modeling problems, including that of searching for and modeling the virus-like transposons that are found in all known genomes.

Genomic Subtraction Recovers Rye-Specific DNA Elements Enriched in the Rye Genome

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3-1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.

Characterization of two families of tandem repeated DNA sequences inPotamogeton pectinatusL.

DNA sequences belonging to two families of tandem repeats, PpeRsa1 (362-364 bp in length, 62% A+T residues) and PpeRsa2 (355-359 bp in length, 59% A+T residues), have been isolated from the Potamogeton pectinatus L. genome. The two sequence families do not share significant nucleotide sequence similarity, even if an evolutionary relationship between them could be assumed. The comparison of the cleaving activity of isoschizomeres that are either sensitive or insensitive to methylation of cytosine residues in the target sequence revealed high methylation in both sequence families. The copy number per 1C DNA of PpeRsa1- and PpeRsa2-related sequences is estimated to be 4.92 x 10(4) and 7.96 x 10(4), respectively. Taken together, these sequences account for about 7.5% of the entire genome of P. pectinatus. The chromosomal organization of these sequences was investigated by fluorescent in situ hybridization. PpeRsa1 and PpeRsa2 repeats found related sequences in 52 chromosomes of the P. pectinatus complement (2n = 78). The related sequences were localized around the centromeres and at the chromosome ends in three pairs of chromosomes, while they were found only at the chromosome ends in the remaining pairs. Twenty-six chromosomes did not show any hybridization signal. The hypothesis that the species is a hybrid between a diploid parent and an allotetraploid parent is put forward.

Modeling the sequence-dependent diffusion coefficients of short DNA molecules

A boundary element model for the computation of sequence-dependent hydrodynamic properties of short DNA molecules is introduced. The hydrated surface is modeled as a curved tube of uniform radius with ends capped by hemispheres, and the axis of the tube is a general space curve whose length and curvature are determined locally by the sequence using a rigid basepair model of double-helical DNA with parameters based on x-ray crystallography. Diffusion coefficients for families of random and periodic DNA sequences are computed and compared with theories for straight tubes and experimental data. Our results indicate that sequence-dependent curvature can have a measurable impact on both the translational and rotational diffusion coefficients, even for relatively short fragments of lengths less than about 150 basepairs, and that previous estimates of the hydrated radius of DNA are likely to be underestimates. Moreover, our results suggest a possible method for refining the rigid basepair model parameters for DNA in solution as well as the hydrated radius.

Molecular cloning and characterization of the repetitive DNA sequences that comprise the constitutive heterochromatin of the A and B chromosomes of the Korean field mouse (Apodemus peninsulae, Muridae, Rodentia)

Three novel families of repetitive DNA sequences were molecularly cloned from the Korean field mouse (Apodemus peninsulae) and characterized by chromosome in-situ hybridization and filter hybridization. They were all localized to the centromeric regions of all autosomes and categorized into major satellite DNA, type I minor, and type II minor repetitive sequences. The type II minor repetitive sequence also hybridized interspersedly in the non-centromeric regions. The major satellite DNA sequence, which consisted of 30 bp elements, was organized in tandem arrays and constituted the majority of centromeric heterochromatin. Three families of repetitive sequences hybridized with B chromosomes in different patterns, suggesting that the B chromosomes of A. peninsulae were derived from A chromosomes and that the three repetitive sequences were amplified independently on each B chromosome. The minor repetitive sequences are present in the genomes of the other seven Apodemus species. In contrast, the major satellite DNA sequences that had a low sequence homology are present only in a few species. These results suggest that the major satellite DNA was amplified with base substitution in A. peninsulae after the divergence of the genus Apodemus from the common ancestor and that the B chromosomes of A. peninsulae might have a species-specific origin.