The Arabidopsis thaliana genome project

The genome of the model plant Arabidopsis thaliana is being analyzed in more and more detail. This paper reviews recent progress over the last 5 years. A first goal was to establish a catalogue of expressed genes using the EST (expressed sequence tag) strategy. Two consortia (French and American) have together released close to 30 000 EST representing approximately 10 000 genes. Such a catalogue has already facilitated a number of biological analyses. The next step, which is sequencing the whole genome, has already started with a European Union pilot project, which has demonstrated the feasability of the large scale sequencing of this genome. During the last 3 years 2.5 Mbp have been determined and data acquisition is accelerating tremendously. Two major questions remain for the future. What is the function of the genes with no known homology? How can this enormous information resource be used for the benefit of other plants? A few current ideas and perspectives are discussed.     

Characterization of the genome of Arabidopsis thaliana

The small crucifer Arabidopsis thaliana has many useful features as an experimental organism for the study of plant molecular biology. It has a four-week life-cycle, only five chromosomes and a genome size less than half that of Drosophila. To characterize the DNA sequence organization of this plant, we have randomly selected 50 recombinant lambda clones containing inserts with an average length of 12,800 base-pairs and analyzed their content of repetitive and unique DNA by various genome blot, restriction digestion and RNA blot procedures. The following conclusions can be drawn. The DNA represented in this random sample is composed predominantly of single-copy sequences. This presumably reflects the organization of the Arabidopsis genome as a whole and supports prior conclusions reached on the basis of kinetics of DNA reassociation. The DNA that encodes the ribosomal RNAs constitutes the only major class of cloned nuclear repetitive DNA. It consists of approximately 570 tandem copies of a heterogeneous 9900-base-pair repeat unit. There is an average of approximately 660 copies of the chloroplast genome per cell. Therefore, the chloroplast genome constitutes the major component of the repetitive sequences found in A. thaliana DNA made from whole plants. The inner cytosine residue in the sequence C-C-G-G is methylated more often than the outer in the tandem ribosomal DNA units, whereas very few differences in the methylation state of these two cytosine residues are detected in unique sequences.     

The shrunken genome of Arabidopsis thaliana

This paper examines macro and micro-level patterns of genome size evolution in the Brassicaceae. A phylogeny of 25 relatives of Arabidopsis thaliana was reconstructed using four molecular markers under both parsimony and Bayesian methods. Reconstruction of genome size (C value) evolution as a discrete character and as a continuous character was also performed. In addition, size dynamics in small chromosomal regions were assessed by comparing genomic clones generated for Arabidopsis lyrata and for Boechera stricta to the fully sequenced genome of A. thaliana. The results reveal a sevenfold variation in genome size among the taxa investigated and that the small genome size of A. thaliana is derived. Our results also indicate that the genome is free to increase or decrease in size across these evolutionary lineages without a directional bias. These changes are accomplished by insertions and deletions at both large and small-scales occurring mostly in intergenic regions, with repetitive sequences and transposable elements implicated in genome size increases. The focus upon taxa relatively closely related to the model organism A. thaliana, and the combination of complementary approaches, allows for unique insights into the processes driving genome size changes.     

The age of the Arabidopsis thaliana genome duplication

We estimate the timing of the Arabidopsis thaliana whole-genome duplication by means of phylogenetic and statistical analysis, and propose two possible scenarios for the duplication. The first one, based on the assumption that the duplicated segments diverged from an autotetraploid form, places the duplication at about 38 million years ago, after the Arabidopsislineage diverged from that of soybean (Glycine max) and before it diverged from its sister genus, Brassica. The second scenario assumes that the ancestor was allotetraploid, and suggests that the duplication is younger than 38 million years and may have contributed to the Arabidopsis-Brassica divergence. In each case, our estimate places the age of the genome duplication as significantly younger than previously reported.     

Interstitial telomere-like repeats in the Arabidopsis thaliana genome

Eukaryotic chromosomal ends are protected by telomeres, which are thought to play an important role in ensuring the complete replication of chromosomes. On the other hand, non-functional telomere-like repeats in the interchromosomal regions (interstitial telomeric repeats; ITRs) have been reported in several eukaryotes. In this study, we identified eight ITRs in the Arabidopsis thaliana genome, each consisting of complete and degenerate 300- to 1200-bp sequences. The ITRs were grouped into three classes (class IA-B, class II, and class IIIA-E) based on the degeneracy of the telomeric repeats in ITRs. The telomeric repeats of the two ITRs in class I were conserved for the most part, whereas the single ITR in class II, and the five ITRs in class III were relatively degenerated. In addition, degenerate ITRs were surrounded by common sequences that shared 70-100% homology to each other; these are named ITR-adjacent sequences (IAS). Although the genomic regions around ITRs in class I lacked IAS, those around ITRs in class II contained IAS (IASa), and those around five ITRs in class III had nine types of IAS (IASb, c, d, e, f, g, h, i, and j). Ten IAS types in classes II and III showed no significant homology to each other. The chromosomal locations of ITRs and IAS were not category-related, but most of them were adjacent to, or part of, a centromere. These results show that the A. thaliana genome has undergone chromosomal rearrangements, such as end-fusions and segmental duplications.     

Whole genome transcriptome polymorphisms in Arabidopsis thaliana

ArrayPlex is a software package that centrally provides a large number of flexible toolsets useful for functional genomics, including microarray data storage, quality assessments, data visualization, gene annotation retrieval, statistical tests, genomic sequence retrieval and motif analysis. It uses a client-server architecture based on open source components, provides graphical, command-line, and programmatic access to all needed resources, and is extensible by virtue of a documented application programming interface. ArrayPlex is available at http://sourceforge.net/projects/arrayplex/.     

Gardening the genome: DNA methylation in Arabidopsis thaliana

DNA methylation has two essential roles in plants and animals - defending the genome against transposons and regulating gene expression. Recent experiments in Arabidopsis thaliana have begun to address crucial questions about how DNA methylation is established and maintained. One cardinal insight has been the discovery that DNA methylation can be guided by small RNAs produced through RNA-interference pathways. Plants and mammals use a similar suite of DNA methyltransferases to propagate DNA methylation, but plants have also developed a glycosylase-based mechanism for removing DNA methylation, and there are hints that similar processes function in other organisms.     

The Dictyostelium repertoire of seven transmembrane domain receptors

The availability of fully sequenced genomes allows the in silico analysis of whole gene families in a given genome. A particularly large and interesting gene family is the G-protein-coupled receptor family. These receptors detect a variety of extracellular signals and transduce them, generally via heterotrimeric G-proteins, to effector proteins inside the cell and thus elicit a physiological response. G-protein-coupled receptors are found in all eukaryotes and constitute in vertebrates 3-5% of all genes. They are also very important drug targets and approximately 25 of the top 100 selling drugs are directed against these receptors. The Dictyostelium discoideum genome contains a surprisingly high number of 55 such receptors, approximately 0.5% of the encoded genes. Besides the four well-studied cAMP receptors the genome encodes eight additional cAMP receptor-like proteins and one of these is distinguished by a novel domain structure, one secretin-like receptor, 17 GABA(B)-like and 25 Frizzled-like receptors. The existence of the latter three types of receptors in D. discoideum was surprising because they had not been observed outside the animal kingdom before. Their presence suggests unprecedentedly complex and so far unknown signaling activities in this lower eukaryote.     

Arabidopsis thaliana proteomics: from proteome to genome

Proteomics has become an important approach for investigating cellular processes and network functions. Significant improvements have been made during the last few years in technologies for high-throughput proteomics, both at the level of data analysis software and mass spectrometry hardware. As proteomics technologies advance and become more widely accessible, efforts of cataloguing and quantifying full proteomes are underway to complement other genomics approaches, such as RNA and metabolite profiling. Of particular interest is the application of proteome data to improve genome annotation and to include information on post-translational protein modifications with the annotation of the corresponding gene. This type of analysis requires a paradigm shift because amino acid sequences must be assigned to peptides without relying on existing protein databases. In this review, advances and current limitations of full proteome analysis are briefly highlighted using the model plant Arabidopsis thaliana as an example. Strategies to identify peptides are also discussed on the basis of MS/MS data in a protein database-independent approach.     

Complete structure of the chloroplast genome of Arabidopsis thaliana.

The complete nucleotide sequence of the chloroplast genome of Arabidopsis thaliana has been determined. The genome as a circular DNA composed of 154,478 bp containing a pair of inverted repeats of 26,264 bp, which are separated by small and large single copy regions of 17,780 bp and 84,170 bp, respectively. A total of 87 potential protein-coding genes including 8 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acid species were assigned to the genome on the basis of similarity to the chloroplast genes previously reported for other species. The translated amino acid sequences from respective potential protein-coding genes showed 63.9% to 100% sequence similarity to those of the corresponding genes in the chloroplast genome of Nicotiana tabacum, indicating the occurrence of significant diversity in the chloroplast genes between two dicot plants. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

Comparative genome analysis reveals extensive conservation of genome organisation for Arabidopsis thaliana and Capsella rubella

Genome colinearity has been studied for two closely related diploid species of the Brassicaceae family, Arabidopsis thaliana and Capsella rubella. Markers mapping to chromosome 4 of A. thaliana were found on two linkage groups in Capsella and colinear segments spanning more than 10 cM were revealed. Detailed analysis of a 60 kbp region in A. thaliana and its counterpart in C. rubella showed virtually complete conservation of gene repertoire, order and orientation. The comparison of orthologous genes revealed very similar exon-intron structures and sequence identities of 90% or more were found for exon sequences. This extensive genome colinearity at the genetic and molecular level allows the efficient transfer of data from the well-studied A. thaliana genome to other species in the Brassicaceae family, substantially facilitating genome analysis studies for species of this family.     

A census of carbohydrate-active enzymes in the genome of Arabidopsis thaliana

The synthesis, modification, and breakdown of carbohydrates is one of the most fundamentally important reactions in nature. The structural and functional diversity of glycosides is mirrored by a vast array of enzymes involved in their synthesis (glycosyltransferases), modification (carbohydrate esterases) and breakdown (glycoside hydrolases and polysaccharide lyases). The importance of these processes is reflected in the dedication of 1-2% of an organism's genes to glycoside hydrolases and glycosyltransferases alone. In plants, these processes are of particular importance for cell-wall synthesis and expansion. starch metabolism, defence against pathogens, symbiosis and signalling. Here we present an analysis of over 730 open reading frames representing the two main classes of carbohydrate-active enzymes, glycoside hydrolases and glycosyltransferases, in the genome of Arabidopsis thaliana. The vast importance of these enzymes in cell-wall formation and degradation is revealed along with the unexpected dominance of pectin degradation in Arabidopsis, with at least 170 open-reading frames dedicated solely to this task.     

Chromosome landmarks as tools to study the genome of Arabidopsis thaliana

The model plant Arabidopsis thaliana has long been used for genetic, cellular and molecular studies. Whereas this plant was used as a model of genetics in the 1940's, the first cytogenetic observation of A. thaliana chromosomes was published in the beginning of the 20th century. Although Arabidopsis was not originally considered to be a good plant model for cytogenetics due to smallness of its genome, the number of published chromosome studies has expanded enormously in recent years. The advent of fluorescence in situ hybridization techniques on meiotic chromosomes together with indirect immuno-fluorescence localization of key chromosomal and nuclear proteins and wide accessibility of Arabidopsis mutants have resulted in a synergistic boost in Arabidopsis cytogenetics. In comparison to other plant species, the small genome with under-represented DNA repeats together with a small number of chromosomes makes this model plant easy to comprehend for a cytologist.     

An integrated genetic/RFLP map of the Arabidopsis thaliana genome

We have assembled an integrated genetic/restriction fragment length polymorphism (RFLP) linkage map of the nuclear genome of the flowering plant Arabidopsis thaliana . The map is based on two independent sets of RFLP data, RFLP data for 123 new markers, and pair-wise segregation data of 125 classical genetic markers. Mathematical integration of the independent data sets was performed using the joinmap computer package. Sixty-two markers common to two or more data sets were exploited to facilitate integration of the individual maps. The current map, which encompasses a total genetic distance of 520 cM, contains 125 classical genetic markers and 306 RFLP markers. Comparison of the integrated consensus map with the individual maps demonstrates that the overall linear order of the integrated map is in good agreement with the component maps. It must be emphasized, however, that the integrated map represents the 'best fit' which is clearly subject to the statistical limitations of the available data. We present several examples where local differences in map order are observed between the integrated and component maps. It is likely, given the problems associated with statistical integration of mapping data from different populations, that the integrated map will contain additional local inconsistencies and problematic regions. None the less, the unified map provides a framework for building an increasingly accurate and useful map. Subsequent refinements of the map will be available electronically end researchers are invited to submit revised map data to the corresponding author for inclusion in future updates (see Appendix 1).     

Clathrin facilitates the internalization of seven transmembrane segment receptors for mating pheromones in yeast

The role of clathrin in endocytosis of the yeast phermone receptors was examined using strains expressing a temperature-sensitive clathrin heavy chain. The yeast phermone receptors belong to the family of seven transmembrane segment, G-protein-coupled receptors. A rapid and reversible defect in uptake of radiolabeled alpha-factor pheromone occurred when the cells were transferred to the nonpermissive temperature. Constitutive, pheromone-independent internalization of newly synthesized a-factor phermone receptor was also rapidly inhibited in mutant strains at the nonpermissive temperature. In both cases residual endocytosis, 30-50% of wild-type levels, was detected in the absence of functional clathrin heavy chain. Once internalized, the a- factor receptor was delivered to the vacuole at comparable rates in chc1-ts and wild-type cells at the nonpermissive temperature. Clathrin heavy chain was also required for maximal uptake of a mutant a-factor receptor which is dependent on pheromone for internalization. In the presence of a-factor, the internalization rate of the mutant receptor in chc1-ts cells at the nonpermissive temperature was 2.5 times slower than the rate observed for endocytosis of the mutant receptor in wild- type cells. These experiments provide in vivo evidence that clathrin plays an important role in the endocytosis of the seven trans-membrane segment pheromone receptors in yeast.     

The word landscape of the non-coding segments of the Arabidopsis thaliana genome

Background

Genome sequences can be conceptualized as arrangements of motifs or words. The frequencies and positional distributions of these words within particular non-coding genomic segments provide important insights into how the words function in processes such as mRNA stability and regulation of gene expression.

Results

Using an enumerative word discovery approach, we investigated the frequencies and positional distributions of all 65,536 different 8-letter words in the genome of Arabidopsis thaliana. Focusing on promoter regions, introns, and 3' and 5' untranslated regions (3'UTRs and 5'UTRs), we compared word frequencies in these segments to genome-wide frequencies. The statistically interesting words in each segment were clustered with similar words to generate motif logos. We investigated whether words were clustered at particular locations or were distributed randomly within each genomic segment, and we classified the words using gene expression information from public repositories. Finally, we investigated whether particular sets of words appeared together more frequently than others.

Conclusion

Our studies provide a detailed view of the word composition of several segments of the non-coding portion of the Arabidopsis genome. Each segment contains a unique word-based signature. The respective signatures consist of the sets of enriched words, 'unwords', and word pairs within a segment, as well as the preferential locations and functional classifications for the signature words. Additionally, the positional distributions of enriched words within the segments highlight possible functional elements, and the co-associations of words in promoter regions likely represent the formation of higher order regulatory modules. This work is an important step toward fully cataloguing the functional elements of the Arabidopsis genome.