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Editor's Choice Collection

To celebrate the fine contributions of DNAR authors, the Executive Editor highlights the best article of each issue as the Editor's Choice.

Volume 23, Issue 6

Draft genome sequence of an inbred line of Chenopodium quinoa, an allotetraploid crop with great environmental adaptability and outstanding nutritional properties

By: Yasuo Yasui, Hideki Hirakawa, Tetsuo Oikawa, et al.

Historically, quite a few important plant species were ‘discovered’ in South America by Europeans during the Age of Exploration and were introduced into Europe. They were subsequently exported to other countries and became cultivated world wide because of their characteristic values. Of them, potato, tobacco, red pepper are said to be three major Solanacea crops that have influenced human life in a number of ways. Recently, another South American plant named quinoa (Chenopodium quinoa), which is regarded as a pseudocereal crop, has emerged to attract biologists’ as well as crop breeders’ attention for its excellent nutritional value. Quinoa is taxonomically quite different from ordinary cereal crops such as rice, corn, wheat etc., but has been similarly cultivated and consumed in the Andean region since ancient times. In addition to its nutritional value, quinoa is said to have an ability to tolerate stressful environments during cultivation. 
To understand its value more deeply, the authors of this article challenged the difficult task to decode the genomic information of allotetraploid quinoa. Like other crops, cultivated plants have been subjected to repeated outcrossings and selections and, consequently, their genomes are usually quite complex and difficult to analyze. 
Through their efforts, the authors have achieved de novo genome assembly of 25 k scaffolds totaling 1 Gbp, the results of which are now available by accessing the Quinoa Genome DataBase.

Volume 23, Issue 5

Discrepancy among the synonymous codons with respect to their selection as optimal codon in bacteria

By: Siddhartha Sankar Satapathy, Bhesh Raj Powdel, Alak Kumar Buragohain, and Suvendra Kumar Ray

Ever since 61 sense codons were successfully assigned to each of the 20 natural amino acids, biologists started to ask such a question as to which one of the several codons assigned to a particular amino acid (or, synonymous codons) is most frequently 'used' in individual organisms, and whether there are any differences amongst organisms with regard to that frequency. As genome analysis is rapidly progressing owing largely to the developments of more efficient DNA sequencers and software tools for sequence analysis, calculation of ‘codon adaptation index’ became widely adopted for determining the degree of codon distribution biases which was found quite different from one organism to another. Likewise, the most frequently used synonymous codon, termed 'optimal codon', was also different in different organisms. Factors such as G+C % of the genome and abundance of particular tRNA species have been known to affect codon usage biases. Based on their systematic analysis of codon usages in as many as 221 bacterial species, the authors of this article propose that some biases in codon usage for amino acids such as Glu, Gln, Lys and Leu seem to have been brought in under phylogenetic constraints. More interestingly, the authors further point out that their analyses confirm that some other biases may perhaps be related to the order of nucleotides within codons the possibility of which was originally proposed some 30 years ago.

Volume 23, Issue 4

Toward high-resolution population genomics using archaeological samples

By: Irina Morozova, Pavel Flegontov, Alexander S. Mikheyev, et al.

Biologists have long devoted themselves to extracting and combining pieces of information to deduce the evolutionary processes of living organisms that proliferated under diverse geological and environmental conditions at various times and places during the earth's history. Fossils have attracted paleontologists worldwide as they give remains with which the original structures and functions of living organisms and/or their parts could be reconstructed at least to some extent. Discovery of DNA along with its biological importance and development of methods including computer software to analyze the information encoded therein have revolutionized the way of performing research in almost every field of biology.

Based on the discussion in a meeting on 'ancient DNA' (or aDNA), the authors of this review article decided that they should survey research activities on aDNA further, as they were convinced that aDNA might become a newly opened window to visualize the past history of life. According to the authors, aDNA has so far been successfully extracted from ancient materials such as bones, teeth, eggshells as well as mummified, frozen, or artificially preserved tissues and analyzed. Recovery of DNA from such materials and its successful amplification by PCR followed by NGS sequencing will enable researchers to obtain as much information as possible from aDNA samples analyzed. Furthermore, the authors argue the possibility of performing epigenetic analysis of aDNA. To what extent we will be able to perform 'direct study of evolution for traits that are not associated with the fossil record' as stated by the authors remains to be seen.

Volume 23, Issue 3

All-in-one construct for genome engineering using Cre-lox technology

By: Ana M. Mariscal, Luis González-González, Enrique Querol, and Jaume Piñol

Recent remarkable developments in nucleotide sequencing technologies have accelerated the genome analysis of various organisms of interest in much less time and with much lower costs. Besides, the transcriptome of individual organisms and their derivatives can similarly be analyzed to identify genes actively expressed under a variety of biotic/abiotic conditions and to explore their functional diversity. These have resulted in accumulation of an enormous amount of data of various categories heretofore. One of the conceivable next steps would be ‘engineering’ of the genome structure to create progenies with more desirable phenotypes of various sorts. To this end, the authors of this article devised a simplified procedure by modifying the well known site-specific Cre-recombinase and its target sequence called loxP to introduce deletion, translocation, inversion, etc . The authors applied what they termed ‘all-in-one construct’ to engineer the genome of Mycoplasma genitalium , a small pathogenic bacterium with the genome of only 580 kb in size. Their construct contains a tetracycline-inducible promoter to control the Cre gene expression in the target organism. The authors emphasize that introduction of the Cre gene and the necessary genetic modification can be accomplished in a single transformation step. How efficient the method will be when applied to other organisms remains to be investigated further.

Volume 23, Issue 2

Spatially coordinated replication and minimization of expression noise constrain three-dimensional organization of yeast genome

By: Arashdeep Singh, Meenakshi Bagadia and Kuljeet Singh Sandhu et al.

Macroscopic and microscopic analysis of the ‘structure and function’ relationship of various cellular entities of living organisms has been one of the most extensively investigated issues in biological sciences. Of these, the dynamism of temporal regulation and structural and functional transitions of chromosomes have been amongst others at the center of attention and a number of physical, biochemical and genetic features associated with chromosomes during the cell cycle have been clarified to considerable extents. However, how they have evolved with one another into the genomes of eukaryotic organisms remains to be explored further. The authors of this article undertook their studies from a slightly different point of view and tried to unveil the function of three-dimensional chromatin structure in the unicellular model eukaryote Saccharomyces cerevisiae . They focused on the spatial interactions amongst various chromosomal regions in cis and trans configurations and the functional constraints underlying such interactions. As the chromosomes contain genes of varying degrees of importance in different regions, which are functionally interacting with one another, the authors paid their particular attention to the coordination of chromosomal replication and the minimization of expression noise which, they conclude, are evolutionary constraints in shaping the spatial organization of chromosomes in yeast as well as in other organisms.

Volume 23, Issue 1

The complete genome sequencing of Prevotella intermedia strain OMA14 and a subsequent fine-scale intra-species genomic comparison reveal an unusual amplification of conjugative and mobile transposons and identify a novel Prevotella -lineage specific repeat

By: Mariko Naito et al.

Comparative analysis of the genomes of two or more taxonomically related organisms are often quite effective in extracting and exploring as much information encoded in the nucleotide sequences as possible. The authors of this article isolated a strain of Prevotella intermedia from a human clinical sample and analyzed its genome, despite that the genome sequence of another strain of the same species had already been determined by another group and the data made publicly available for some time. By performing extensive comparative analysis of the genomes of the two strains, both of which are composed of two chromosomes of different sizes, it became clear that the larger chromosome was very similar between the two, while the smaller chromosome was distinctly different in various aspects, such as size, genetic architecture and abundance in strain-specific genes. Furthermore, the genomes of the two strains were remarkably rich in conjugative and mobilizable transposons which might have contributed to generate extensive differences in the arrangement of genes, especially in the smaller chromosome. In addition, the two strains were found to possess a lineage-specific repeat sequence. From these examples, it is thus apparent that analysis of only one strain could not have revealed the characteristic genomic features of P. intermedia which was only made possible through their comparative analysis.

Volume 22, Issue 6

AnABlast: a new in silico strategy for the genome-wide search of novel genes and fossil regions

By: Juan Jimenez et al.

Since the introduction of next generation sequencers (NGS) into genome analysis some ten years ago, the time and cost required for genome sequencing have been steadily improved and it is now a common practice to perform comparative analysis of genomic features amongst phylogenetically related species by sequencing their genomes with NGS. For such an analysis, it is essential to carry out reliable gene finding so that genes encoding proteins of different categories may be identified as precisely as possible which will subsequently be characterized and compared from various points of view. The authors of this article conceived an interesting idea to develop a computer program for gene finding by reducing the stringency in BLAST analysis so that sequences otherwise hidden may show up. One of their intentions was to thus identify what they term ‘protomotifs or ancient footprints’ buried in protein sequences. A foreseeable drawback in such a strategy is that a large number of false positives will be generated. Therefore, the authors employed various measures to lessen the effects and presented a software named AnABlast. The authors concluded that, by applying AnABlast, “the high number of sequence alignments accumulated at a specific strand and reading frame reveals a significant region in terms of biological function and/or evolution.”

Volume 22, Issue 5

Correction of Down syndrome and Edwards syndrome aneuploidies in human cell cultures

By: Tomokazu Amano et al.

In higher organisms including human, fusion of male and female gametes generates an embryo harboring cells of diploid karyotype. Occasionally, however, for some reasons the mechanism of which is not fully understood, an extra copy of chromosome is brought in as a result of unusual mitosis or meiosis and thus creates aneuploid cells. One of the best known cases of aneuploidy is the trisomy of chromosome 21 causing Down Syndrome. Until recently, aneuploidy has been regarded irremediable. In fact, elimination or inactivation of one copy of chromosome 21 has been attempted, but the process requires elaborate genetic manipulation and cannot easily be accomplished. During the course of analysis of the mammalian specific gene termed ZSCAN4 that encodes a zinc-finger protein, the authors of this article discovered that its forced expression in mouse embryonic stem cells showing aneuploidy and polyploidy dramatically increased the percentage of cells with normal karyotype within several days. Subsequently, the authors examined the effect of ZSCAN4 gene expression in human fibroblast cells derived from individuals with Down Syndrome. Within weeks after its application, the percentage of cells showing normal karyotype with two copies of chromosome 21 remarkably increased.

At present, the precise mechanism of the apparent ‘karyotype correction’ by ZSCAN4 is not known. However, the results reported in this article will enhance our understanding as to how genes such as ZSCAN4 interact with karyotype maintenance. Perhaps, the phenomenon the same group of authors observed (Akiyama et al. “Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells”) might play a role there.

Volume 22, Issue 4

A comprehensive, genome-wide analysis of autophagy-related genes identified in tobacco suggests a central role of autophagy in plant response to various environmental cues

By: Xue-Mei Zhou et al.

Next generation sequencing generates a huge amount of partial or complete genomic data of target organisms, thereby enabling discovery and analysis of differences amongst individuals, such as, for example, domesticated animals and cultivated plants. The data thus generated would expand possible identification of mutations in the protein-coding as well as non-coding regions many of which are not known if they manifest any phenotypically detectable traits. Such data, when provided in millions, will be of extensive usefulness not only in evolutionary sciences, but particularly in broad fields of agriculture and other applied sciences, since combination of those with subtle genetic differences will often create progenies with unexpected phenotypes. The authors of this article characterized 15 million SNPs from the chicken genome of both commercial and research chicken lines, aiming to obtain ‘variants with potential functional implications’ in addition to genetic and linkage studies. They classified the resultant SNPs into various categories such as those predicted to encode proteins with altered amino acids, likely to alter RNA secondary structures and splicing variants, etc. For these purposes, the authors attempted to reduce false positive SNPs by applying different filtration methods and evaluated. The results presented in this article are largely descriptive, but are expected to be useful for exploration of functional variants of various categories in chicken lines.

Volume 22, Issue 3

Functional classification of 15 million SNPs detected from diverse chicken populations

By: A. Gheyas et al.

Next generation sequencing generates a huge amount of partial or complete genomic data of target organisms, thereby enabling discovery and analysis of differences amongst individuals, such as, for example, domesticated animals and cultivated plants. The data thus generated would expand possible identification of mutations in the protein-coding as well as non-coding regions many of which are not known if they manifest any phenotypically detectable traits. Such data, when provided in millions, will be of extensive usefulness not only in evolutionary sciences, but particularly in broad fields of agriculture and other applied sciences, since combination of those with subtle genetic differences will often create progenies with unexpected phenotypes. The authors of this article characterized 15 million SNPs from the chicken genome of both commercial and research chicken lines, aiming to obtain ‘variants with potential functional implications’ in addition to genetic and linkage studies. They classified the resultant SNPs into various categories such as those predicted to encode proteins with altered amino acids, likely to alter RNA secondary structures and splicing variants, etc. For these purposes, the authors attempted to reduce false positive SNPs by applying different filtration methods and evaluated. The results presented in this article are largely descriptive, but are expected to be useful for exploration of functional variants of various categories in chicken lines.

Volume 22, Issue 1

Highly sensitive targeted methylome sequencing by post-bisulfite adaptor tagging

By: Fumihito Miura and Takashi Ito

One of the most challenging issues in studies of the molecular mechanism underlying cellular differentiation in multicellular organisms is to uncover how the genetic information is modified and inherited in differentiating cells, which results in terminal expression of altered phenotypes. Of various factors involved in differential modification of genetic information, 5-methylcytosine (5-mC) has been regarded as one of the key players and, therefore, methods to locate 5-mC within the genome, or methylome analysis, have been enthusiastically explored. One of the most standard and commonly used methods to analyze DNA methylation is ‘whole-genome bisulfite sequencing’ or WGBS, in which 5-mC in the genomic DNA is analyzed by virtue of next generation sequencing after treatment with bisulfite. The authors of this article have developed a new modification to the WGBS method termed ‘post-bisulfite adaptor tagging’ in which WGBS can be performed with as little as 125 pg of DNA as opposed to the conventional method in which some 100-fold more DNA is needed. They achieved such a drastic reduction in the amount of DNA required for WGBS by reversing the steps of adaptor tagging and bisulfite treatment, thereby eliminating DNA fragments damaged by bisulfite prior to enrichment of the target DNA.

Volume 21, Issue 6

Optimized whole-genome amplification strategy for extremely AT-biased template

By: Samuel O. Oyola et al.

Recent remarkable advancement in next generation sequencing technologies has made it possible to acquire information encoded in the genomes of various organisms through a variety of analytical approaches. It has enabled nucleotide sequencing of the genome of an organism with much less costs and in a much shorter time than before. In addition, by performing sequence analysis of RNAs expressed in cells of individual and/or closely related organisms, it is now possible to analyze the transcriptome much easier and to uncover sequence differences amongst members of an organism that may be used for the construction of a genetic map of high resolution. Therefore, if a reliable method is available to amplify the entire genome of an organism starting with a very small amount of DNA, such as 1 ng or less, then genome analysis can easily be expanded into hitherto unexplored fields. The authors of this article have established a powerful method for whole genome amplification using the φ29 system optimized for the AT-rich genome of Plasmodium falciparum by including tetramethylammonium chloride in the amplification reaction. In this way, they were also able to reduce chimera formation and to improve the quality of sequence reads. The method will thus be suited for sequence analysis of clinical samples or for single cell genome analysis etc .

Volume 21, Issue 5

A high-density genetic map with array-based markers facilitates structural and quantitative trait locus analyses of the common wheat genome

By: Julio Iehisa et al.

Common wheat ( Triticum aestivum L.) is one of the most important grain crops in the world. According to the statistical data released from USDA, wheat is currently cultivated in some 220 million hectares of fields world-wide, yielding more than 700 million tons of grain. During domestication of the ancestral plants into today’s wheat, genetic manipulations of various sorts were performed to obtain plants possessing better characters and yields. The resultant common wheat cultivated today is allohexaploid possessing the genomic architecture of AABBDD, which was brought in from the tetraploid wheat Triticum turgidum L. (AABB) and a wild diploid relative Aegilops tauschii Coss. (DD). However, due to the large genome size of 17 Gb, its molecular analysis is quite difficult and its complete genomic structure remains to be determined. To facilitate further analyses of wheat, the authors of this article constructed a high-resolution genetic map based on the data of two common wheat cultivars obtained by using the next generation sequencing technologies along with development of a microarray-based system for detection of polymorphisms. They were thus successful in adding 13,056 new markers, about 40% of which were derived from within genes and 11% from repetitive regions. Consequently, quantitative trait loci affecting such important agronomical characters as flowering etc. have been mapped more accurately than before.

Volume 21, Issue 4

Evolutionary origin of higher-order repeat structure in alpha-satellite DNA of primate centromeres

By: Akihiko Koga et al .

Eukaryotic chromosomes, in particular those of primates including human, are characterized by the presence of tandem repeat sequences called alpha satellite in the centromeric regions. In human alpha satellites, a block of basic repeat units forms a larger unit which constitutes a higher order repeat (HOR) structure. It has been postulated that alpha satellites with HOR play a more important role in chromosome dynamics than simple monomeric alpha satellites that are often present in peripheral centromeric regions. Despite their apparent importance, the precise role of HOR in alpha satellites has not been fully elucidated yet. To approach this problem, the authors of this article paid attention to a small ape called siamang ( Symphalangus syndactylus ) in the alpha satellites of which they had previously revealed the presence of HOR. By performing various comparative analyses, these authors conclude that HOR is present not only in the alpha satellites of human and great apes but also in those of other primates including siamang and other gibbons. They argue in the end that it will be of interest to investigate whether within-species variation in HOR of gibbons is large or not, as it is known that variation in HOR is quite large in human.

Volume 21, Issue 3

Comparative genome analysis of the closely related Synechocystis strains PCC 6714 and PCC 6803

By: Mattias Kopf et al.

As the name ‘blue green algae’ given to cyanobacteria indicates, cyanobacteria used to be classified into ‘algae’. Indeed, some cyanobacteria appear to be algae-like with their cells concatenated with one another along with differentiated cyst cells in between. Phylogenetically, cyanobacteria are quite ancient and diverse, possessing the ability to perform photosynthesis. Whereas some of them cause problems, many others are useful for biofuels, foods, nitrogen fixation, etc. Of a variety of cyanobacteria, Synechocyctis sp. PCC 6803, which grows autotrophically or heterotrophically with its photosynthetic apparatus resembling that of higher plants, was chosen as a model for genome analysis in 1996. To further characterize the model cyanobacterium, the authors of this article performed draft genome analysis of another Synechocyctis isolate named PCC 6714 and performed genomic and transcriptomic comparisons with 6803. They noticed quite a few structural and functional differences between the two closely related cyanobacteria such as lack of a transport system in 6714 for the compatible solute glucosylglycerol, causing its growth retardation above 2% salinity unlike 6803 which is able to grow at 4%. These and other results presented will be useful in understanding the diversity of cyanobacteria and their future exploitation in various fields.

Volume 21, Issue 2

Dissection of the octoploid strawberry genome by deep-sequencing of the genomes of Fragaria species

By: Hideki Hirakawa, et al.

Strawberry is undoubtedly one of the most popular fruits cultivated and consumed all over the world. Wild plants belonging to the genus Fragaria bearing edible fruits can be found in Eurasia and in North and South Americas with di-, tetra-, hexa-, octo- and decaploid genomes. The widely cultivated strawberry, Fragaria x ananassa , is octoploid which was obtained in 1700s from a natural hybrid between two octoploid species. Based on various cytological analyses, its karyotype has been inferred to be of either AABBBBCC, AAA’A’BBBB, or AAA’A’BBB’B’, though its molecular details remain to be established. The authors of this article challenged molecular dissection of the cultivated strawberry genome by using next generation sequencers, hoping to elucidate its molecular architecture and phylogenetic relationship amongst wild Fragaria species. They established that the genome size of F. x ananassa was 692 Mb and, based on the four homeologous subgenomes, constructed a virtual reference genome of 173.2 Mb termed FANhybrid_r1.2. The work presented is the first example of draft genome analysis of a polyploid species and is, therefore, expected to contribute to analysis of other polyploid plants.

Volume 21, Issue 1

High-Resolution Linkage and Quantitative Trait Locus Mapping Aided by Genome Survey Sequencing: Building Up An Integrative Genomic Framework for a Bivalve Mollusc

By Wenqian Jiao, Xiaoteng Fu, Jinzhuang Dou, Hengde Li, Hailin Su, Junxia Mao, Qian Yu, Lingling Zhang, Xiaoli Hu, Xiaoting Huang, Yangfan Wang, Shi Wang, and Zhenmin Bao

Genetic linkage map is a basic resource of high utility to be exploited in genetic and genomic studies as well as in breeding etc. A linkage map used to be based on isolation and analysis of mutations and, therefore, its construction required a lot of time and efforts. However, recent developments in genomic sequencing technologies with very much reduced running costs have enabled such a method as 'genotyping-by-sequencing' feasible as the authors of this article as well as many others have adopted. The method is quite powerful in characterizing the genotype of an organism the genome sequencing of which has not been completed yet due to its structural complexity: the results thus obtained are then subjected to identification of traits of interest encoded in the genome. In this article, the authors performed draft sequencing of the genome of a bivalve mollusk named Chlamys farreri and used the data to construct a linkage map of 3,806 loci including quantitative trait loci (QTL) for various phenotypes. Through its analysis, the authors were able to identify, for example, a QTL named PROP1 the orthologues of which are known to function in growth hormone production in vertebrates. The linkage map is expected to serve as a milestone for further analysis of the mollusk.

Volume 20, Issue 6

Large, Male Germ Cell-Specific Hypomethylated DNA Domains With Unique Genomic and Epigenomic Features on the Mouse X Chromosome

By Rieko Ikeda, Hirosuke Shiura, Koji Numata, Michiko Sugimoto, Masayo Kondo, Nathan Mise, Masako Suzuki, John M. Greally and Kuniya Abe

Of a large number of differentiated cells constituting an organism, only germ cells can transmit their genetic information to the progeny. Germ cells are known to possess a specialized transcription system effected through marked changes in their global epigenetic state during development, although details of the changes have only been poorly understood due largely to technical difficulties, as the availability of primodial germ cells for analysis is very much limited. Moreover, importance of DNA methylation in regions other than CpG islands has recently become increasingly apparent in regards to gene expression and nuclear organization of chromatin. Bearing these in mind, the authors of this article devised an elegant system termed ‘HELP assay’ ( H pa II tiny fragment e nrichment by l igation-mediated P CR) to analyze the epigenetic changes in the duplicated regions of the X-chromosome in developing mouse germ cells. The authors conclude that the specialized gene expression in the domains of mouse X-chromosome may be attributed to their unique epigenetic state.

Volume 20, Issue 5

Heterochromatin blocks constituting the entire short arms of acrocentric chromosomes of Azara's owl monkey: formation processes inferred from chromosomal locations

By Ornjira Prakhongcheep, et al.

Eukaryotic chromosomes, especially those of higher eukaryotes, are remarkable entities and the number, shape, size and behavior of individual chromosomes have been analyzed in detail in a large number of organisms to establish their overall pictures as well as how each of them differs from one another. For this purpose, a variety of staining methods have been developed so as to differentiate the resultant patterns such as G-, R-, C-bands, etc . of each chromosome. These patterns are caused by the presence of heterochromatic regions rich in repeat sequences of different sorts. The authors of this article paid particular attention to the location of heterochromatic regions in acrocentric chromosomes of a primate species called owl monkey. They have cloned and sequenced the heterochromatic DNA and revealed the presence of a repeat sequence unit termed OwlRep as well as some other units in the owl monkey chromosomes. They analyzed distribution of OwlRep and other repeat units and compared the results amongst primate species. Based on the information thus obtained, they propose that the heterochromatic regions have possibly been transferred from chromosome to chromosome and amplified there to generate the short arms of acrocentric chromosomes in owl monkey as well as in other primates.

Volume 20, Issue 4

Next Generation Annotation of prokaryotic genomes with EuGene-P: application to Sinorhizobium meliloti 2011

By Schiex, Thomas, et al.

Genomic sequences available from the public databases are most fundamental data that are essential resources in genetic, genomic, evolutionary as well as various other fields of current biological sciences. Since the establishment of public databases (EMBL in 1981, GenBank in 1982 and DDBJ in 1986), the sequence data are accompanied by brief annotation on various features of the sequence to facilitate easier handling of the data by users. A number of bioinformatic tools have since been developed to accomplish automated annotation of genomic sequences, but none of them has so far been completely satisfactory to all organisms. The authors of this article created a tool termed EuGene-P for gene finding in prokaryotic genomes by adapting the one they developed earlier for eukaryotic genomes. They made use of proteome data that have recently become easier to produce and the tool thus developed seems quite satisfactory in annotating the genome of the nitrogen-fixing rhizobium, Sinorhizobium meliloti 2011: 4,077 transcription start sites upstream of protein-coding or non-coding genes were precisely mapped, demonstrating that the tool will produce a reliable automatic annotation of prokaryotic genomes.

Volume 20, Issue 3

Genome-wide SNP genotyping to infer the effects on gene functions in tomato

By Hirakawa et al.

Recent technological developments in nucleotide sequencing have resulted in generation of huge amounts of genomic and transcriptomic data for various organisms with speed and ease. By performing systematic comparison of the resultant data with those of related organisms, it is possible to identify and genotype SNPs and other structural differences, which will serve as useful markers in basic as well as applied fields of genetics. Tomato is one of the Solanaceae crops commonly cultivated in almost every country and at the same time is regarded as an important model plant. The authors of this article analyzed and classified a large number of SNPs in various tomato lines including wild relatives and annotated genes harboring SNPs to characterize the effects of SNPs on gene functions. For that purpose, they performed homology and domain searches against public databases and in addition performed homology modeling of three-dimensional structures of the proteins encoded by individual genes to infer their functions. Quite a few proteins thus analyzed were found to possess SNPs in the segments predicted to be involved in their functions.

Volume 20, Issue 2

Gene capture coupled to high-throughput sequencing as a strategy for targeted metagenome exploration

By Peyret Pierre et al .

Analysis of microbial diversity in various environments has recently been remarkably advanced due largely to the development of next generation sequencing (NGS) technologies. Environmental DNA samples are subjected to NGS and the resultant metagenomic data are analyzed with bioinformatic tools to extract useful information. To perform ecological analysis in this way, however, enormous diversity of microorganisms often makes it difficult to explore the data to a full extent. Pre-filtration of the environmental DNA samples to reduce the diversity before sequencing is apparently needed. To cope with the situation, the authors of this article developed a method termed ‘Solution Hybrid Selection’ to capture sequence data homologous to the mcrA gene of a methanogen, Methanosarcina acetivorans , that encodes the alpha subunit of methyl coenzyme M reductase. The method adopts two sets of ‘capture probes’ specifically designed to facilitate efficient enrichment of the target. Consequently, the authors succeeded in capturing DNA fragments containing the target gene with high efficiencies and thus were able to identify a novel genetic organization surrounding the mcrA gene.

Volume 20, Issue 1

Polygenic Molecular Architecture Underlying Non-Sexual Cell Aggregation in
Budding Yeast

By Jiarui Li et al .

Cells in eukaryotic organisms are intricately interacting with each other to constitute and sustain the structure and function of individually differentiated tissues, organs, nervous systems, etc. Although the yeast, Saccharomyces cerevisiae , is generally known as a unicellular organism, it also possesses the capacity to form a ‘multicellular structure’ to enhance its response to various environmental stresses. Analysis of the cell aggregation in yeast is expected to deepen our understanding of the mechanism involved in cellular ‘communication’ leading to the establishment of such a ‘multicellular structure’. The authors of this article dissected molecular basis underlying the asexual cell aggregation in yeast on the basis of genome-wide mapping of the responsible quantitative trait loci (QTL). They approached to the problem in that way as cell aggregation is likely to be controlled by several genes in a co-operative manner. By performing high-resolution mapping of the QTL through knockout and allele replacement of the candidate genes, the authors identified two new genes, in addition to some of the known genes, that are responsible for the observed QTL.

Volume 19, Issue 6

Discovery of high-confidence SNPs from large-scale de novo analysis of leaf transcripts of Aegilops tauschii , a wild wheat progenitor

by Julio Cesar Masaru Iehisa, Akifumi Shimizu, Kazuhiro Sato, Shuhei Nasuda and Shigeo Takumi

Wheat is often referred to as one of the three most important crops together with rice and maize. It is said to be originated from central Asia and has been cultivated for thousands of years worldwide during which the original plant was successively crossed with several related plant species for various improvements. The common wheat cultivated today is a hexaploid consisting of A, B and D genomes of which the D genome was derived from Aegilops taushii . In this article the authors analyzed transcriptomes of two accessions of Ae. taushii , representing each of its two major genealogical lineages, by using the next generation sequencing technique. They have identified 9,435 SNPs and 735 indels of which 31 SNPs and 6 indels were commonly found in 20 diverse accessions of Ae. taushii analyzed. In addition, based on the conserved synteny between wheat and barley chromosomes, the authors propose that the high-density expression sequence tag maps established for barley may be exploited for further genetic/genomic analyses and improvements of wheat.

Volume 19, Issue 5

Mate Pair Sequencing of Whole Genome Amplified DNA following Laser Capture Microdissection of Prostate Cancer

by Stephen J. Murphy, John C. Cheville, Shabnam Zarei, Sarah H. Johnson, Robert A. Sikkink, Farhad Kosari, Andrew L. Feldman, Bruce W. Eckloff, R. Jeffery Karnes and George Vasmatzis

Recent advancement in next generation sequencing has made it possible to perform genome-wide comparative analysis of nucleotide changes that occur in an individual during the course of development. The technology has been proven to be quite powerful in dissecting aberrations of various sorts and can further be applied in finding sequence heterogeneity often concealed in groups and subgroups of cells within particular tissues. Thus, the technology is anticipated to uncover early changes that will eventually result in development of serious diseases. In practice, however, it is often difficult to apply the technology as the available number of cells harboring changes is limited. To overcome the problem and accomplish the goal, the authors of this article adopted use of the laser capture microdissection method to select particular types of cells and performed in situ whole genome amplification of DNA to detect the causative changes by nucleotide sequencing. The successful combination of methods enabled the authors to reveal genomic aberrations responsible for initiating prostate cancer and those driving its progression. The methods may be further modified and applied for analysis of various features in other organisms.

Volume 19, Issue 4

A set of Lotus japonicus Gifu x Lotus burttii recombinant inbred lines facilitate map-based cloning and QTL mapping

by Niels Sandal, Haojie Jin, Dulce Nombre Rodriguez-Navarro, Francisco Temprano, Cristina Cvitanich, Andreas Brachmann, Shusei Sato, Masayoshi Kawaguchi, Satoshi Tabata, Martin Parniske, Jose E. Ruiz-Sainz, Stig U. Andersen and Jens Stougaard

Identification of QTL, or quantitative trait loci, has become greatly facilitated by the development of new generation sequencing technologies. Amongst various plant species in which QTL analysis is extensively performed, legume plants are of particular interest as some of them are of high agronomical importance. Moreover, they are known to interact with rhizobacteria for symbiotic nitrogen fixation, a process that includes the functions of a variety of genes of both the host and symbiotic bacteria for their cooperative interactions. To perform extensive QTL analysis, it is vital to have a set of suitable cultivars/species to obtain recombinants possessing differences at a variety of genetic loci in comparison to the target plant species. In this article, the authors chose a model legume, Lotus japonicus cultivar Gifu, the genome sequence of which is largely known, to seek candidates to generate recombinant inbred lines that facilitate more efficient QTL analysis for a wide range of traits. They found that, amongst the candidates examined, L. burttii would serve as a valid alternative to those that have been used till now. By using L. burttii , the authors were able to identify a rhizobacterial nodulation-related QTL.

Volume 19, Issue 3

Characterization of species-specific repeats in 613 prokaryotic species

by Triinu Koressaar and Maido Remm

In general, it seems natural to assume that bacteria have evolved to possess compact genomes by discarding sequences unnecessary for their survival. In fact, some of the symbiotic bacteria are known to harbor very small genomes, lacking even those genes indispensable for their independent life. In this article, the authors performed a systematic survey of bacterial genomes to clarify to what extent the species-specific repeat sequences they had identified previously might be present in bacterial species the genomic sequences of which are currently available. For that purpose, the authors analyzed the genomes of as many as 613 bacterial species and consequently found that 97% of them contained at least one species-specific repeat. They further confirmed that the repeat sequences were not of a common origin: some were associated with duplicated protein-coding genes, while some others with rRNA and tRNA genes. Based on their analysis, the authors emphasize that the species-specific repeat sequences are functionally variable and only one-fourth of them are associated with mobile genetic elements. Why a high percentage of bacterial genomes possess species-specific repeats remains to be elucidated further.

Volume 19, Issue 2

Draft Genome of the Pearl Oyster Pinctada fucata : A Platform for Understanding Bivalve Biology

by Takeshi Takeuchi et al.

The pearl oyster, Pinctada fucata , is a bivalve mollusc growing in the tropical to subtropical coastal regions including central to south-western Japan. The shell of P. fucata has been well known to yield pearls since ancient times, and in particular since Koukichi Mikimoto developed the method to harvest cultured pearls. Amongst various molluscan organisms subjected to genome analysis, P. fucata was the first to accomplish the goal and its draft genome sequence is now made available. With the sequence data presented in this article, the authors aimed to elucidate the mechanisms by which pearls are formed through bio-mineralization processes within the layers of oyster shells, and further to understand the biology of bivalve molluscs, especially features that characterize molluscs and their evolution amongst metazoans. The presented genome data of some 1,000 Mb of P. fucata contain approximately 23,000 genes including those involved in bio-mineralization. The data will become a platform for basic as well as pearl-related industrial studies.

Volume 19, Issue 1

Discovery and Mapping of a New EST-SNP and SSR Panel for Large-scale Genetic Studies and Breeding of Theobroma cacao L.

by Mathilde Allegre et al.

Recent remarkable progresses in the DNA sequencing technology have made it possible to obtain massive nucleotide sequence data of individual organisms easily and within a short time. This has made it possible to perform comprehensive genomic/genetic analyses of various sorts to establish which genes or groups of genes are important for the hereditary traits that characterize an organism to be analysed. The DNA sequence data accumulated will serve as an indispensable reservoir for various genetic markers, which in turn will contribute to efficient analysis of the organism. Bearing these in mind, the authors of this article have taken advantage of DNA sequencing of ESTs obtained from each of the cultivars of Theobroma cacao L., a tree that has been grown in as many as 50 tropical countries, and performed extensive in silico analysis of the data obtained to identify SNPs and SSRs (simple sequence repeats). The resultant map is densely populated with markers and thus can be exploited for genetic analysis at the genomic level as well as markerassisted breeding.

Volume 18, Issue 6

Mutation bias is the driving force of codon usage in the Gallus gallus genome

by Yousheng Rao, Guozuo Wu, Zhangfeng Wang, Xuewen Chai, Qinghua Nie and Xiquan Zhang

The occurrence of synonymous codons is considerably deviated from randomness and the degree of deviation varies from one gene to another within an organism and similarly from the genes in one organism to those in another. The phenomenon is often called ‘codon usage bias’ and is partly attributed to factors such as differences in the abundance level of corresponding tRNA species. Although the phenomenon and its biological implications have been quite extensively investigated in prokaryotes, much less has been clarified in eukaryotes, in particular in multicellular organisms. In this article, the authors have studied the evolutionary background of codon usage biases in the chicken genome by analyzing the relative synonymous codon usage and identified eleven ‘optimal codons’, all ending with uracil, an observation not seen in other eukaryotes. Upon further characterization, the authors found that characteristics such as the regional G+C content are negatively correlated with the optimal codon frequency, while the gene length and gene expression level are positively correlated. The authors propose that at least one of the driving forces is likely to be mutational biases.

Volume 18, Issue 5

In silico analysis of transcription factor repertoires and prediction of stressresponsive transcription factors from six major Gramineae plants.

by Keiichi Mochida, Takuhiro Yoshida, Tetsuya Sakurai, Kazuko Yamaguchi-Shinozaki, Kazuo Shinozaki and Lam-Son Phan Tran

Genomic sequences of closely related organisms enable detailed comparative analysis of homologous genes. As genes acquire divergent functions through duplications followed by alterations in their structure as well as in their regulatory elements, close comparison of the genomic sequences would lead to understanding of the events that have possibly occurred during the course of evolution. Thus, several important grass plants including rice, maize and wheat can now be targets of studies of this kind. In this article, Mochida et al . focused on the analysis of transcription factors and cis -regulatory elements in six Gramineae plants and created a specialized database termed GramineaeTFDB. The database provides sequence features, promoter regions, domain alignments, GO assignment, and cDNA information of related transcription factors along with cross- references to public databases and related genetic resources. It also provides hyperlinks to expression profiles of the transcription factor genes as well as information for available mutant lines, which would facilitate survey of the regulatory characteristics of genes in grass plants under different environmental conditions.

Volume 18, Issue 4

Identification of tammar wallaby SIRH12 , derived from a marsupial-specific retrotransposition event

By: Ryuichi Ono, Yoko Kuroki, Mie Naruse, Masayuki Ishii, Sawa Iwasaki, Atsushi Toyoda, Asao Fujiyama, Geoff Shaw, Marilyn B. Renfree, Tomoko Kaneko-Ishino and Fumitoshi Ishino

Recent advances in nucleotide sequence technology have facilitated detailed comparison between the genomes of related organisms. This enables identification and analysis of genes that play crucial roles in the development of structures specific to organisms of a particular lineage. Some of these originate from transposable elements. For example, genes derived from the sushi-ichi family of retrotransposons are known to be essential in placental development. The authors of this article previously discovered that at least one such gene was conserved in marsupials and eutherians - even though - marsupials do not develop placenta. In this article, Ono et al . describe a novel gene of related origin, SIRH12 , that is present in the Australian tammar wallaby and, as a mutated pseudogene, in the South American opossum. Whether the SIRH12 gene carries a placenta-related function in the wallaby remains to be determined.

Volume 18, Issue 3

Eukaryotic-like Ser/Thr Protein Kinases SpkC/F/K Are Involved in Phosphorylation of GroES in the Cyanobacterium Synechocystis

By Anna Zorina, Natalia Stepanchenko, Galina V. Novikova, Maria Sinetova, Vladimir B. Panichkin, Igor E. Moshkov, Vladislav V. Zinchenko, Sergey V. Shestakov, Iwane Suzuki, Norio Murata and Dmitry A. Los

Transfer of signals to induce particular reactions in response to internal/external changes has been extensively studied in a variety of organisms, the most common process of which is the one mediated by phosphorylation/dephosphorylation of some of the amino acid residues of the proteins involved. In this article, the authors investigated protein phosphorylation in Synechocystis and found that Ser/Thr protein kinases similar to those present in eukaryotes play their roles in protein phophorylation in cyanobacteria. The results reported are of particular interest, because the targets include several proteins that appear to be key to the functions characteristic to cyanobacteria such as the large subunit of RuBisCo, translation elongation factor Tu, etc . The authors reached the conclusion by analysing mutants in which each one of the 12 genes coding for Ser/Thr protein kinases has been knocked out. Furthermore, three of the genes appear to function toward the phosphorylation of the same target, i.e . chaperonin GroES, suggesting that perhaps they function in a cascade-like manner as in eukaryotes.

Volume 18, Issue 2

Prediction of Directional Changes of Influenza A Virus Genome Sequences with Emphasis on Pandemic H1N1/09 as a Model Case

by Yuki Iwasaki, Takashi Abe, Kennosuke Wada, Masae Itoh and Toshimichi Ikemura

Influenza virus is known to infect various host organisms such as migrating birds, poultry, domestic animals and human. The viral coat encapsulates eight segments of RNA molecules, each possessing a gene of different function necessary for the viral life cycle. It has been reported that viruses of avian as well as of human origin can both infect swine, thereby resulting in ‘hybrid virus’ formation upon re-assortment of the RNA segments and accumulation of mutations. Apart from medical importance, it is quite interesting to investigate as to whether there are any characteristic features associated with the individual RNA segments (genes) of the viruses from different hosts. The report by Abe et al. in this issue of DNA Research focuses on this point from a bioinformatics point of view by analysing a large number of sequences of influenza viruses isolated from different hosts and at different times and locations. The authors have successfully developed a method termed BLSOM for clustering a large number of viral genomes. This method makes it possible to visualize how viral genomes of different origins are clustered, thereby enabling one to follow the general tendency of genomic/segmental changes within respective virus populations and how they are correlated with one another. Analysis of this sort has not previously been performed and, therefore, the report by Abe et al. is expected to deepen the discussion in the related areas.

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