Abundance and DNA sequence of two-base repeat regions in tropical tree genomes

Tandem DNA repeats of two-base pairs are potentially important tools for population genetic studies because of their abundance and length variation. As part of our research into the ecology of tropical forest plants, we began a study of dinucleotide repeat regions in several genera of tropical trees. Genomic libraries in bacteriophage lambda were screened with the oligonucleotide probes poly(GT) and poly(AG). Both types of repeat regions were abundant in the genomes of all six plant species examined. Using the size of inserts in the phage libraries and number of phage screened, we estimated that there were 5 x 10(3) to 3 x 10(5) poly(AC) sites per genome, with slightly more AG than AC sites. When libraries were made from smaller fragments of genomic DNA, abundance estimates were higher, suggesting that two-base repeat sites were clustered in the genome. Poly(AC) sites were 16-22 bp in length, and four of the five sequenced were adjacent to either poly(AG)or poly(AT) sites. Other repeat region s appeared in DNA flanking the AC sites. This further demonstrated that two-base repeats and other repetitive DNA were clustered in the genome. Two-base repeats are abundant in plant genomes and could provide a large number of polymorphic markers for studies of plant population genetics.     

Selective constraint in intergenic regions of human and mouse genomes.

We aligned and analyzed 100 pairs of complete, orthologous intergenic regions from the human and mouse genomes (average length approximately 12 000 nucleotides). The alignments alternate between highly similar segments and dissimilar segments, indicating a wide variation of selective constraint. The average number of selectively constrained nucleotides within a mammalian intergenic region is at least 2000. This is threefold higher than within a nematode intergenic region and at least twofold higher than the number of selectively constrained nucleotides coding for an average protein. Because mammals possess only two- to threefold more proteins than Caenorhabditis elegans, the higher complexity of mammals might be primarily because of the functioning of intergenic DNA.     

Identification of compositionally distinct regions in genomes using the centroid method

MOTIVATION: It is known that most genomic regions of special interest, e.g. horizontally acquired sequences, genomic islands, etc. have distinct word (m-mer) compositions. Most of the earlier work along this direction, addressed di- and tri-nucleotide compositions. We present an approach that can be applied to analyze compositions of any given word size. The method, called the centroid approach, can reveal compositionally distinct regions in genomic sequences for any given word size. RESULTS: We applied our method to 50 bacterial genomes and demonstrated its ability to identify embedded sequences of varying lengths from distantly related organisms. We also investigated the genetic makeup of the regions identified as compositionally distinct by our method, for four organisms from our dataset. Pathogenicity island (PAI) components and genes encoding strain-specific proteins are all frequently seen to be constituents of these regions. AVAILABILITY: Program is available on request from the authors. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Analysis of collinear regions of Oryza AA and CC genomes

Comparative analyses of genome structure and sequence of closely related species have yielded insights into the evolution and function of plant genomes. A total of 103,844 BAC end sequences delegated -73.8 Mb of O. officinalis that belongs to the CC genome type of the rice genus Oryza were obtained and compared with the genome sequences office cultivar, O. sativa ssp.japonica cv. Nipponbare. We found that more than 45% of O. officinalis genome consists of repeat sequences, which is higher than that of Nipponbare cultivar. To further investigate the evolutionary divergence of AA and CC genomes, two BAC-contigs of O. officinalis were compared with the collinear genomic regions of Nipponbare. Of 57 genes predicted in the AA genome orthologous regions, 39 had orthologs in the regions of the CC genome. Alignment of the orthologous regions indicated that the CC genome has undergone expansion in both genic and intergenic regions through primarily retroelement insertion. Particularly, the density of RNA transposable elements was 17.95% and 1.78% in O. officinalis and O. sativa, respectively. This explains why the orthologous region is about 100 kb longer in the CC genome in comparison to the AA genome.     

Multiplexed DNA sequence capture of mitochondrial genomes using PCR products

Background

To utilize the power of high-throughput sequencers, target enrichment methods have been developed. The majority of these require reagents and equipment that are only available from commercial vendors and are not suitable for the targets that are a few kilobases in length.

Methodology/Principal Findings

We describe a novel and economical method in which custom made long-range PCR products are used to capture complete human mitochondrial genomes from complex DNA mixtures. We use the method to capture 46 complete mitochondrial genomes in parallel and we sequence them on a single lane of an Illumina GA_II instrument.

Conclusions/Significance

This method is economical and simple and particularly suitable for targets that can be amplified by PCR and do not contain highly repetitive sequences such as mtDNA. It has applications in population genetics and forensics, as well as studies of ancient DNA.     

DNA rearrangement in orthologous orp regions of the maize, rice and sorghum genomes

The homeologous Orp1 and Orp2 regions of maize and the orthologous regions in sorghum and rice were compared by generating sequence data for >486 kb of genomic DNA. At least three genic rearrangements differentiate the maize Orp1 and Orp2 segments, including an insertion of a single gene and two deletions that removed one gene each, while no genic rearrangements were detected in the maize Orp2 region relative to sorghum. Extended comparison of the orthologous Orp regions of sorghum and japonica rice uncovered numerous genic rearrangements and the presence of a transposon-rich region in rice. Only 11 of 27 genes (40%) are arranged in the same order and orientation between sorghum and rice. Of the 8 genes that are uniquely present in the sorghum region, 4 were found to have single-copy homologs in both rice and Arabidopsis, but none of these genes are located near each other, indicating frequent gene movement. Further comparison of the Orp segments from two rice subspecies, japonica and indica, revealed that the transposon-rich region is both an ancient and current hotspot for retrotransposon accumulation and genic rearrangement. We also identify unequal gene conversion as a mechanism for maize retrotransposon rearrangement.     

Multiple transforming regions of human cytomegalovirus DNA.

The transforming (focus forming) activity of defined cloned DNA fragments from human cytomegalovirus Towne and AD169 was carried out in immortalized rodent cells. The frequency of focus formation in NIH 3T3 cells by Towne XbaI fragment E was 80- to 100-fold higher than that observed with Towne XbaI fragments AO, O, C, or carrier DNA alone but was similar to that observed with pCM4127, a transforming fragment from HCMV AD169 (J. A. Nelson, B. Fleckenstein, D. A. Galloway, and J. K. McDougall, J. Virol. 43:83-91, 1982; J. A. Nelson, B. Fleckenstein, G. Jahn, D. A. Galloway, and J. K. McDougall, J. Virol. 49:109-115, 1984). Foci were first detected in Towne XbaI fragment E-transfected NIH 3T3 cells at 5 to 6 weeks posttransfection, whereas foci were detected at 2 to 3 weeks after transfection with AD169 pCM4127. Digestion of Towne XbaI fragment E with BamHI did not significantly reduce its focus-forming activity. When BamHI subclones of Towne XbaI fragment E were assayed individually for focus formation in NIH 3T3 and Rat-2 cells, transforming activity was localized within each terminal fragment (EJ and EM). Foci induced by EJ or EM DNA alone were smaller compared with those induced by Towne XbaI fragment E. Isolated focal lines exhibited growth in soft agar and were tumorigenic in immunocompetent syngeneic animals. High-molecular-weight DNAs from transformed and tumor-derived lines were analyzed by Southern blot hybridization with intact EM and a 1.5-kilobase subfragment lacking cell-related sequences. Virus-specific EM sequences were detected at less than one copy per cell in Towne XbaI fragment E-transformed NIH 3T3 cells and at multiple copies in rat tumor-derived cell lines. In contrast, virus-specific EJ sequences were barely detected in EJ-transformed and tumor-derived lines with intact EJ as probe.     

Selective DNA amplification from complex genomes using universal double-sided adapters

There is a rapidly developing need for new technologies to amplify millions of different targets from genomic DNA for high throughput genotyping and population gene-sequencing from diverse species. Here we describe a novel approach for the specific selection and amplification of genomic DNA fragments of interest that eliminates the need for costly and time consuming synthesis and testing of potentially millions of amplicon-specific primers. This technique relies upon Type IIs restriction enzyme digestion of genomic DNA and ligation of the fragments to double-sided adapters to form closed-circular DNA molecules. The novel use of double-sided adapters, assembled through the combinatorial use of two small universal sets of oligonucleotide building blocks, provides greater selection capacity by utilizing both sides of the adapter in a sequence-specific ligation event. As demonstrated, formation of circular structures results in protection of the desired molecules from nuclease treatment and enables a level of selectivity high enough to isolate single, or multiple, pre-defined fragments from the human genome when digested at over five million sites. Priming sites incorporated into the adapter allows the utilization of a common pair of primers for the amplification of any adapter-captured DNA fragment of interest.     

Comparative analyses of DNA methylation and sequence evolution using Nasonia genomes

The functional and evolutionary significance of DNA methylation in insect genomes remains to be resolved. Nasonia is well situated for comparative analyses of DNA methylation and genome evolution, since the genomes of a moderately distant outgroup species as well as closely related sibling species are available. Using direct sequencing of bisulfite-converted DNA, we uncovered a substantial level of DNA methylation in 17 of 18 Nasonia vitripennis genes and a strong correlation between methylation level and CpG depletion. Notably, in the sex-determining locus transformer, the exon that is alternatively spliced between the sexes is heavily methylated in both males and females, whereas other exons are only sparsely methylated. Orthologous genes of the honeybee and Nasonia show highly similar relative levels of CpG depletion, despite ~190 My divergence. Densely and sparsely methylated genes in these species also exhibit similar functional enrichments. We found that the degree of CpG depletion is negatively correlated with substitution rates between closely related Nasonia species for synonymous, nonsynonymous, and intron sites. This suggests that mutation rates increase with decreasing levels of germ line methylation. Thus, DNA methylation is prevalent in the Nasonia genome, may participate in regulatory processes such as sex determination and alternative splicing, and is correlated with several aspects of genome and sequence evolution.     

Compositional evolution of noncoding DNA in the human and chimpanzee genomes

We have examined the compositional evolution of noncoding DNA in the primate genome by comparison of lineage-specific substitutions observed in 1.8 Mb of genomic alignments of human, chimpanzee, and baboon with 6542 human single-nucleotide polymorphisms (SNPs) rooted using chimpanzee sequence. The pattern of compositional evolution, measured in terms of the numbers of GC-->AT and AT-->GC changes, differs significantly between fixed and polymorphic sites, and indicates that there is a bias toward fixation of AT-->GC mutations, which could result from weak directional selection or biased gene conversion in favor of high GC content. Comparison of the frequency distributions of a subset of the SNPs revealed no significant difference between GC-->AT and AT-->GC polymorphisms, although AT-->GC polymorphisms in regions of high GC segregate at slightly higher frequencies on average than GC-->AT polymorphisms, which is consistent with a fixation bias favoring high GC in these regions. However, the substitution data suggest that this fixation bias is relatively weak, because the compositional structure of the human and chimpanzee genomes is becoming homogenized, with regions of high GC decreasing in GC content and regions of low GC increasing in GC content. The rate and pattern of nucleotide substitution in 333 Alu repeats within the human-chimpanzee-baboon alignments are not significantly affected by the GC content of the region in which they are inserted, providing further evidence that, since the time of the human-chimpanzee ancestor, there has been little or no regional variation in mutation bias.     

Intra- and interpopulation differentiation of human genomes by molecular DNA hybridization

The DNA-DNA hybridization method was used to compare the repetitive sequences with a low degree of intragenomic divergence in various etno-territorial groups (Russians, Bouriats and Paleoasiats). Values of intergenomic divergence within groups and between them were estimated by a decrease in melting temperature of hybrid duplexes in relation to homologous 3H-labeled thermostable fraction reassociates of DNA of a Russian. Statistically valid differences in melting temperature were revealed when Russian, Bouriat and Paleoasiatic groups were compared. No such differences were found within each of the groups. Though the thermostability profiles had much in common in each case, some quantitative differences in melting temperature allowed to differentiate local groups in humans.     

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays

Whole-genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG-Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction-digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1-RD10) relative to M. tuberculosis. Seven of these regions, RD4-RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG-specific deletions being identical to the RD1-RD3 loci described previously. The distribution of RD4-RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT-6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.     

OWEN: aligning long collinear regions of genomes

OWEN is an interactive tool for aligning two long DNA sequences that represents similarity between them by a chain of collinear local similarities. OWEN employs several methods for constructing and editing local similarities and for resolving conflicts between them. Alignments of sequences of lengths over 10(6) can often be produced in minutes. OWEN requires memory below 20 L, where L is the sum of lengths of the compared sequences.     

Local DNA dynamics shape mutational patterns of mononucleotide repeats in human genomes

Single base substitutions (SBSs) and insertions/deletions are critical for generating population diversity and can lead both to inherited disease and cancer. Whereas on a genome-wide scale SBSs are influenced by cellular factors, on a fine scale SBSs are influenced by the local DNA sequence-context, although the role of flanking sequence is often unclear. Herein, we used bioinformatics, molecular dynamics and hybrid quantum mechanics/molecular mechanics to analyze sequence context-dependent mutagenesis at mononucleotide repeats (A-tracts and G-tracts) in human population variation and in cancer genomes. SBSs and insertions/deletions occur predominantly at the first and last base-pairs of A-tracts, whereas they are concentrated at the second and third base-pairs in G-tracts. These positions correspond to the most flexible sites along A-tracts, and to sites where a ‘hole’, generated by the loss of an electron through oxidation, is most likely to be localized in G-tracts. For A-tracts, most SBSs occur in the direction of the base-pair flanking the tracts. We conclude that intrinsic features of local DNA structure, i.e. base-pair flexibility and charge transfer, render specific nucleotides along mononucleotide runs susceptible to base modification, which then yields mutations. Thus, local DNA dynamics contributes to phenotypic variation and disease in the human population.     

Application of random amplified polymorphic DNA (RAPD) assays in identifying conserved regions of actinomycete genomes

Abstract Various arbitrary primers as well as pUC18/19 'reverse' sequencing primers were used for random amplified polymorphic DNA assays. Use of a modified reverse primer led to amplification of one major approx. 1100-bp band from the chromosomal DNA of all actinomycetes tested; however, the band was not found when DNAs from other bacteria were used in comparable experiments. Hybridization experiments showed that these bands all contained similar genomic regions. Subsequent sequencing of four of these fragments showed they each contained the sequence of the 3' end of the 23S rRNA gene, the intergenic region and the start of the 5S rRNA gene.     

Testing for archaic hominin admixture on the X chromosome: model likelihoods for the modern human RRM2P4 region from summaries of genealogical topology under the structured coalescent

A 2.4-kb stretch within the RRM2P4 region of the X chromosome, previously sequenced in a sample of 41 globally distributed humans, displayed both an ancient time to the most recent common ancestor (e.g., a TMRCA of approximately 2 million years) and a basal clade composed entirely of Asian sequences. This pattern was interpreted to reflect a history of introgressive hybridization from archaic hominins (most likely Asian Homo erectus) into the anatomically modern human genome. Here, we address this hypothesis by resequencing the 2.4-kb RRM2P4 region in 131 African and 122 non-African individuals and by extending the length of sequence in a window of 16.5 kb encompassing the RRM2P4 pseudogene in a subset of 90 individuals. We find that both the ancient TMRCA and the skew in non-African representation in one of the basal clades are essentially limited to the central 2.4-kb region. We define a new summary statistic called the minimum clade proportion (pmc), which quantifies the proportion of individuals from a specified geographic region in each of the two basal clades of a binary gene tree, and then employ coalescent simulations to assess the likelihood of the observed central RRM2P4 genealogy under two alternative views of human evolutionary history: recent African replacement (RAR) and archaic admixture (AA). A molecular-clock-based TMRCA estimate of 2.33 million years is a statistical outlier under the RAR model; however, the large variance associated with this estimate makes it difficult to distinguish the predictions of the human origins models tested here. The pmc summary statistic, which has improved power with larger samples of chromosomes, yields values that are significantly unlikely under the RAR model and fit expectations better under a range of archaic admixture scenarios.     

Gene-containing regions of wheat and the other grass genomes

Deletion line-based high-density physical maps revealed that the wheat (Triticum aestivum) genome is partitioned into gene-rich and -poor compartments. Available deletion lines have bracketed the gene-containing regions to about 10% of the genome. Emerging sequence data suggest that these may further be partitioned into "mini" gene-rich and gene-poor regions. An average of about 10% of each gene-rich region seem to contain genes. Sequence analyses in various species suggest that uneven distribution of genes may be a characteristic of all grasses and perhaps all higher organisms. Comparison of the physical maps with genetic linkage maps showed that recombination in wheat and barley (Hordeum vulgare) is confined to the gene-containing regions. Number of genes, gene density, and the extent of recombination vary greatly among the gene-rich regions. The gene order, relative region size, and recombination are highly conserved within the tribe Triticeae and moderately conserved within the family. Gene-poor regions are composed of retrotransposon-like non-transcribing repeats and pseudogenes. Direct comparisons of orthologous regions indicated that gene density in wheat is about one-half compared with rice (Oryza sativa). Genome size difference between wheat and rice is, therefore, mainly because of amplification of the gene-poor regions. Presence of species-, genera-, and family-specific repeats reveal a repeated invasion of the genomes by different retrotransposons over time. Preferential transposition to adjacent locations and presence of vital genes flanking a gene-rich region may have restricted retrotransposon amplification to gene-poor regions, resulting into tandem blocks of non-transcribing repeats. Insertional inactivation by adjoining retro-elements and selection seem to have played a major role in stabilizing genomes.     

Identification of genes with fast-evolving regions in microbial genomes

Complete sequences of multiple strains of the same microbial species provide an invaluable source for studying the evolutionary dynamics between orthologous genes over a relatively short time scale. Usually the intensity of the selection pressure is inferred from a comparison between the nonsynonymous substitution rate and the synonymous substitution rate. In this paper, we propose an alternative method for detecting genes with one or more fast-evolving regions from pairwise comparisons of orthologous genes. Our method looks for regions with overrepresented nonsynonymous mutations along the alignment, and requires a higher nonsynonymous evolution rate in those regions than the neutral evolution rate. It identifies gene targets under intensive selection pressure that are not detected from the conventional rate comparison analysis. For those identified genes with known annotations, most of them have a clear role in processes such as bacterial defense and host–pathogen interactions. Gene sets reported from our method provide a measure of the phenotypic divergence between two closely related genomes.