Full-length cDNA Collection Research Articles

Establishing a System for Functional Characterization of Full-Length cDNAs of Camellia sinensis.

Tea (Camellia sinensis) is enriched with bioactive secondary metabolites, and is one of the most popular nonalcoholic beverages globally. Two tea reference genomes have been reported; however, the functional analysis of tea genes has lagged, mainly due to tea’s recalcitrance to genetic transformation and the absence of alternative high throughput heterologous expression systems. A full-length cDNA collection with a streamlined cloning system is needed in this economically important woody crop species. RNAs were isolated from nine different vegetative tea tissues, pooled, then used to construct a normalized full-length cDNA library. The titer of unamplified and amplified cDNA library was 6.89 × 106 and 1.8 × 1010 cfu/mL, respectively; the library recombinant rate was 87.2%. Preliminary characterization demonstrated that this collection can complement existing tea reference genomes and facilitate rare gene discovery. In addition, to streamline tea cDNA cloning and functional analysis, a binary vector (pBIG2113SF) was reengineered, seven tea cDNAs isolated from this library were successfully cloned into this vector, then transformed into Arabidopsis. One FL-cDNA, which encodes a putative P1B-type ATPase 5 (CsHMA5), was characterized further as a proof of concept. We demonstrated that overexpression of CsHMA5 in Arabidopsis resulted in copper hyposensitivity. Thus, our data demonstrated that this represents an efficient system for rare gene discovery and functional characterization of tea genes. The integration of a tea FL-cDNA collection with efficient cloning and a heterologous expression system would facilitate functional annotation and characterization of tea genes.

Large Scale Full-Length cDNA Sequencing Reveals a Unique Genomic Landscape in a Lepidopteran Model Insect, Bombyx mori

The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.

Xenopus white papers and resources: Folding functional genomics and genetics into the frog

The frog Xenopus has been vital for biomedical science for over 80 years, contributing to diverse fields from cell signaling, cell and developmental biology, to ion channel physiology and toxicology. Its experimentally manipulable oocytes and embryos provide abundant material for molecular and biochemical approaches for a wide range of gene discovery and protein function studies. In recent years, the Xenopus community has invested in key resources for functional genomics, including genome-wide full-length cDNA collections and genome assemblies as well as genetic tools. These assets combine with Xenopus' extensive range of functional assays to create exciting new research avenues with medical as well as basic applications. This review describes how these resources were developed and what new tools are on the horizon.

Slow but not low: genomic comparisons reveal slower evolutionary rate and higher dN/dS in conifers compared to angiosperms

BackgroundComparative genomics can inform us about the processes of mutation and selection across diverse taxa. Among seed plants, gymnosperms have been lacking in genomic comparisons. Recent EST and full-length cDNA collections for two conifers, Sitka spruce (Picea sitchensis) and loblolly pine (Pinus taeda), together with full genome sequences for two angiosperms, Arabidopsis thaliana and poplar (Populus trichocarpa), offer an opportunity to infer the evolutionary processes underlying thousands of orthologous protein-coding genes in gymnosperms compared with an angiosperm orthologue set.ResultsBased upon pairwise comparisons of 3,723 spruce and pine orthologues, we found an average synonymous genetic distance (dS) of 0.191, and an average dN/dS ratio of 0.314. Using a fossil-established divergence time of 140 million years between spruce and pine, we extrapolated a nucleotide substitution rate of 0.68 × 10-9 synonymous substitutions per site per year. When compared to angiosperms, this indicates a dramatically slower rate of nucleotide substitution rates in conifers: on average 15-fold. Coincidentally, we found a three-fold higher dN/dS for the spruce-pine lineage compared to the poplar-Arabidopsis lineage. This joint occurrence of a slower evolutionary rate in conifers with higher dN/dS, and possibly positive selection, showcases the uniqueness of conifer genome evolution.ConclusionsOur results are in line with documented reduced nucleotide diversity, conservative genome evolution and low rates of diversification in conifers on the one hand and numerous examples of local adaptation in conifers on the other hand. We propose that reduced levels of nucleotide mutation in large and long-lived conifer trees, coupled with large effective population size, were the main factors leading to slow substitution rates but retention of beneficial mutations.

Research on plant abiotic stress responses in the post‐genome era: past, present and future

Understanding abiotic stress responses in plants is an important and challenging topic in plant research. Physiological and molecular biological analyses have allowed us to draw a picture of abiotic stress responses in various plants, and determination of the Arabidopsis genome sequence has had a great impact on this research field. The availability of the complete genome sequence has facilitated access to essential information for all genes, e.g. gene products and their function, transcript levels, putative cis-regulatory elements, and alternative splicing patterns. These data have been obtained from comprehensive transcriptome analyses and studies using full-length cDNA collections and T-DNA- or transposon-tagged mutant lines, which were also enhanced by genome sequence information. Moreover, studies on novel regulatory mechanisms involving use of small RNA molecules, chromatin modulation and genomic DNA modification have enabled us to recognize that plants have evolved complicated and sophisticated systems in response to complex abiotic stresses. Integrated data obtained with various 'omics' approaches have provided a more comprehensive picture of abiotic stress responses. In addition, research on stress responses in various plant species other than Arabidopsis has increased our knowledge regarding the mechanisms of plant stress tolerance in nature. Based on this progress, improvements in crop stress tolerance have been attempted by means of gene transfer and marker-assisted breeding. In this review, we summarize recent progress in abiotic stress studies, especially in the post-genomic era, and offer new perspectives on research directions for the next decade.

A full-length cDNA resource for the pea aphid, Acyrthosiphon pisum.

Large collections of full-length cDNAs are important resources for genome annotation and functional genomics. We report the creation of a collection of 50 599 full-length cDNA clones from the pea aphid, Acyrthosiphon pisum. Sequencing from 5' and 3' ends of the clones generated 97 828 high-quality expressed sequence tags, representing approximately 9000 genes. These sequences were imported to AphidBase and are shown to play crucial roles in both automatic gene prediction and manual annotation. Our detailed analyses demonstrated that the full-length cDNAs can further improve gene models and can even identify novel genes that are not included in the current version of the official gene set. This full-length cDNA collection can be utilized for a wide variety of functional studies, serving as a community resource for the study of the functional genomics of the pea aphid.

WebWatch

BioTechniquesVol. 38, No. 5 WebWatchOpen AccessWebWatchKevin AhernKevin AhernSearch for more papers by this authorPublished Online:30 May 2018https://doi.org/10.2144/05385WW01AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Well-Managed CareLooking for a better healthcare provider? Consider self-analysis. The best system in the world, hands down, has many layers, thousands of workers, and is an incredible microman-agerto boot—your immune system. From its innate to its adaptive defense strategies, the immune system is your body's main protection against a cold, cruel world. Complexity is no barrier to providing excellent healthcare, as a perusal of topics at Interhealth Molecular Immunology quickly reveals. The site's hierachical, hyperlinked headings span the entirety of immunology, thus tackling a complicated topic head on. In doing do, Molecular Immunology educates newbies as much as it aids researchers. Besides a primary focus on human immunology, there's also a considerable body of information on the immune systems of insects, mice, and even plants.www.mi.interhealth.infoEpilepsigeneticsEpilepsy can be a debilitating medical condition. Affecting about 3% of the population, the affliction is characterized by several different forms of seizures that usually begin in adolescence. Epileptic seizures can arise from many causes, including injury, infection, and other brain trauma. What many people may not know, however, is that symptoms of the disease have been demonstrated in organisms as simple as nematodes. This evolutionary aspect of epilepsy provides researchers with important opportunities for understanding its genesis and developing treatments. Enter CarpeDB, the epilepsy database hosted at the University of Alabama, whose aim is exposing the disease's underlying genetics. At the web site, visitors can retrieve information by gene name, chromosome, description, keyword, or species. Returned records contain the massively hyperlinked gene information (NCBI, ENTREZ, LocusLink, Pfam, Swiss-Prot, UniProt, and others) now so commonly available for many genes.www.carpedb.ua.eduTuning ImmunityLooking for a better understanding of how the immune system works its magic? Check out the Cytokine Family Database and learn about this fascinating class of polypeptides/glycopolypeptides and their associated cellular receptors. Often described as “hormones of the immune system,” cytokines help to regulate cellular replication and differentiation by activation of organismal defenses. Cytokines help to control immune cells and regulate secretion of antibodies or even other cytokines. Sharing many properties of hormones and growth factors, cytokines are implicated in everything from primary and secondary immune responses. The site's offerings link to important cytokine information contained in other databases, such as NCBI/GenBank®, UniGene, and others.cytokine.medic.kumamoto-u.ac.jpcDNAs In TotoOnce the bane of RNA workers, cDNAs are essential substrates for expressing eukaryotic messages in prokaryotic cells. The technology for making full-length versions of these important templates has improved to the point that today they are almost commodities available for trading. Through the Mammalian Gene Collection (MGC) site at the NIH, for example, full-length cDNAs for over 25,000 nonredun-dant human, mouse, and rat genes can now be purchased. All of the sequences have been deposited in GenBank® and are available for researchers to use. In addition, full-length cDNA collections from at least two other model organisms (Xenopus and Zebrafish) are, in the familiar words of the web, “under construction.”mgc.nci.nih.govVirtual Life for RealIt's hard to imagine what molecular biology would be like today if not for information from Escherichia coli. The framework for much groundbreaking molecular research, this tiny cell is being positioned to become the first living system to leap from its native environment to the computer. Virtual life has long been a fascination for science fiction writers and computer programmers, but new modelers, systems biologists, are entering the picture in a big way via Project Cybercell. This mammoth effort will combine molecular structure/function data from genomics and proteomics with the mathematics/kinetics of biochemistry. Using computational/ biological methods, scientists hope to more accurately mimic life and obtain new perspectives on life and develop alternative strategies for combating infection. Can biocomputing emulate life? Stay tuned.www.projectcybercell.caFiguresReferencesRelatedDetails Vol. 38, No. 5 Follow us on social media for the latest updates Metrics Downloaded 199 times History Published online 30 May 2018 Published in print May 2005 Information© 2005 Author(s)PDF download

Large-Scale Identification of Expressed Sequence Tags Involved in Rice and Rice Blast Fungus Interaction

To better understand the molecular basis of the defense response against the rice blast fungus (Magnaporthe grisea), a large-scale expressed sequence tag (EST) sequencing approach was used to identify genes involved in the early infection stages in rice (Oryza sativa). Six cDNA libraries were constructed using infected leaf tissues harvested from 6 conditions: resistant, partially resistant, and susceptible reactions at both 6 and 24 h after inoculation. Two additional libraries were constructed using uninoculated leaves and leaves from the lesion mimic mutant spl11. A total of 68,920 ESTs were generated from 8 libraries. Clustering and assembly analyses resulted in 13,570 unique sequences from 10,934 contigs and 2,636 singletons. Gene function classification showed that 42% of the ESTs were predicted to have putative gene function. Comparison of the pathogen-challenged libraries with the uninoculated control library revealed an increase in the percentage of genes in the functional categories of defense and signal transduction mechanisms and cell cycle control, cell division, and chromosome partitioning. In addition, hierarchical clustering analysis grouped the eight libraries based on their disease reactions. A total of 7,748 new and unique ESTs were identified from our collection compared with the KOME full-length cDNA collection. Interestingly, we found that rice ESTs are more closely related to sorghum (Sorghum bicolor) ESTs than to barley (Hordeum vulgare), wheat (Triticum aestivum), and maize (Zea mays) ESTs. The large cataloged collection of rice ESTs in this study provides a solid foundation for further characterization of the rice defense response and is a useful public genomic resource for rice functional genomics studies.

Plant genomics: the third wave.

Completing the primary genomic sequence of Arabidopsis thaliana was a major milestone, being the first plant genome and only the third high-quality finished eukaryotic genome sequence. Understanding how the genome sequence comprehensively encodes developmental programs and environmental responses is the next major challenge for all plant genome projects. This requires fully characterizing the genes, the regulatory sequences, and their functions. We discuss several functional genomics approaches to decode the linear sequence of the reference plant Arabidopsis thaliana, including full-length cDNA collections, microarrays, natural variation, knockout collections, and comparative sequence analysis. Genomics provides the essential tools to speed the work of the traditional molecular geneticist and is now a scientific discipline in its own right.

From masking repeats to identifying functional repeats in the mouse transcriptome.

The back-to-back release of the mouse genome and the functionally annotated RIKEN mouse full-length cDNA collection was an important milestone in mammalian genomics. Yet much of the data remain to be explored in terms of biological effects and mechanisms. For example, interspersed repeats account for 39 per cent of the mouse genome sequence and 11 per cent of representative transcripts. A considerable number of transposable repeat elements are still active and propagating in mouse compared with human. While existing repeat databases and tools assist the classification of repeats or identification of new repeats, there is little bioinformatic support towards exploring the extent and role of repeats in transcriptional variation, modulation of protein function, or gene regulatory events. Since the mouse is used as a model organism to study human genes and their disease associations, this review focuses on information extraction and collation that captures the functional context of repeats in mouse transcripts to facilitate the biological interpretation and extrapolation of findings to the human.

The Riken mouse genome encyclopedia project

The Riken mouse genome encyclopedia a comprehensive full-length cDNA collection and sequence database. High-level functional annotation is based on sequence homology search, expression profiling, mapping and protein–protein interactions. More than 1 000 000 clones prepared from 163 tissues were end-sequenced and classified into 128 000 clusters, and 60 000 representative clones were fully sequenced representing 24 000 clear protein-encoding genes. The application of the mouse genome database for positional cloning and gene network regulation analysis is reported. To cite this article: Y. Hayashizaki, C. R. Biologies 326 (2003).

Identification of putative noncoding RNAs among the RIKEN mouse full-length cDNA collection.

With the sequencing and annotation of genomes and transcriptomes of several eukaryotes, the importance of noncoding RNA (ncRNA)-RNA molecules that are not translated to protein products-has become more evident. A subclass of ncRNA transcripts are encoded by highly regulated, multi-exon, transcriptional units, are processed like typical protein-coding mRNAs and are increasingly implicated in regulation of many cellular functions in eukaryotes. This study describes the identification of candidate functional ncRNAs from among the RIKEN mouse full-length cDNA collection, which contains 60,770 sequences, by using a systematic computational filtering approach. We initially searched for previously reported ncRNAs and found nine murine ncRNAs and homologs of several previously described nonmouse ncRNAs. Through our computational approach to filter artifact-free clones that lack protein coding potential, we extracted 4280 transcripts as the largest-candidate set. Many clones in the set had EST hits, potential CpG islands surrounding the transcription start sites, and homologies with the human genome. This implies that many candidates are indeed transcribed in a regulated manner. Our results demonstrate that ncRNAs are a major functional subclass of processed transcripts in mammals.

Special FANTOM2 Issue

It is my great pleasure to be able to publish a special issue of Genome Research focusing on the functional annotation of mouse cDNA (FANTOM). Publication of this FANTOM special issue is quite timely,as not only the mouse full-length cDNA collection but also the first draft of mouse genome sequences were just published at the end of last year. Moreover,completion of the human genome sequence was announced this spring and entered the next phase. This is a very exciting series of events. In the history of life sciences,the mouse is the first organism for which large-scale analyses of the genome and transcriptome were undertaken at the same time. This data will form the basic platform of life sciences in the 21st century. In this issue,we present papers on FANTOM activity, describing analyses based on the first data for the genome and transcriptome. The international FANTOM consortium was established to annotate functions for sequences in the full-length cDNA sequence database,which was developed by the Riken Mouse Gene Encyclopedia Project. The background of this project should first be explained. The project was started in 1995 and originally aimed at the overall collection of full-length cDNAs for all genes supported by the Science Technology Agency (currently the Ministry of Education,Culture,Sports,and Technology [MEXT]). At that time,the international human genome consortium started large-scale sequencing analysis of the entire human genome,and the EST project was almost saturated. Under circumstances in which genome science was rapidly gaining prominence in the world,we faced the following question: “What significant contribution would Japan make to this scientific field?” Our answer was mouse full-length cDNA. We started this mouse gene encyclopedia project by developing original technologies for full-length cDNA library preparation and a high-throughput system for sequence determination. Our system,the RIKEN integrated sequence analysis (RISA) system,achieved throughput of 40,000 samples per day,facilitating the establishment of the largest collection of full-length cDNAs for the mouse in the world. The full-length cDNA library was prepared by Spring 2000 from 263 tissues,finishing sequence analysis of 60,770 clones in total with full sequences selected from about 2 million end-sequenced clones in Summer 2001. After determining full-length cDNAs,functional annotation of these clones required the kind of enormous effort that can only be achieved with worldwide cooperation. We contacted researchers from various fields of biology and advertised in journals and on the Internet,opening our meetings to anyone who wished to participate,contribute,and help. Many researchers agreed with the goal of founding an international de facto standard of well-annotated full-length cDNA resources,and they voluntarily joined the FANTOM consortium. We greatly appreciate the dedication of the more than 80 researchers in FANTOM1 and 100 researchers in FANTOM2. Integrated analysis of the genome and transcriptome created a synergistic effect,providing extremely valuable insights into the nature of life. The transcriptome provides the exact location of genes,whereas genome sequences provide information such as promoters and exon–intron junctions. Analyses of the mouse genome sequence and 60,770 mouse transcripts were fortunately completed at the same time,with results published late last year. After publication,further studies by the FANTOM consortium based on the enormous amounts of data aimed for systematic understanding of the mouse transcriptome as a model for the human system,and they form the contents of this special issue. One notable feature of this special issue is a new proposal for clone transportation. With the development of genome science,handling of numerous clones as a platform for research has become indispensable. A new type of transportation system to facilitate such handling has long been warranted. The proposal described herein,in a paper entitled “DNA Book,” may well represent a timely solution, and is expected to work very effectively. The DNA book forms an easily transposable bank of full-length cDNA clones printed on paper and bound like a book,and DNA is actually incorporated onto paper that is included in this report. This issue marks the first large-scale trial of the technique,and the cooperation of subscribers worldwide with this project would be deeply appreciated. Subscribers can get involved by filling out the feedback form at the end of this issue for submission to the URL provided. We would like to report the results of subscriber feedback and assist in further improvements to this new technology. RIKEN and others who are concerned with the large-scale distribution of clones hope that the DNA book will offer a new standard of clone distribution that will soon see practical use.

RIKEN mouse genome encyclopedia

We have been working to establish the comprehensive mouse full-length cDNA collection and sequence database to cover as many genes as we can, named Riken mouse genome encyclopedia. Recently we are constructing higher-level annotation (Functional ANnoTation Of Mouse cDNA; FANTOM) not only with homology search based annotation but also with expression data profile, mapping information and protein–protein database. More than 1,000,000 clones prepared from 163 tissues were end-sequenced to classify into 159,789 clusters and 60,770 representative clones were fully sequenced. As a conclusion, the 60,770 sequences contained 33,409 unique. The next generation of life science is clearly based on all of the genome information and resources. Based on our cDNA clones we developed the additional system to explore gene function. We developed cDNA microarray system to print all of these cDNA clones, protein–protein interaction screening system, protein–DNA interaction screening system and so on. The integrated database of all the information is very useful not only for analysis of gene transcriptional network and for the connection of gene to phenotype to facilitate positional candidate approach. In this talk, the prospect of the application of these genome resourced should be discussed. More information is available at the web page: http://genome.gsc.riken.go.jp/.