Evaluation of gene prediction software using a genomic data set: application to Arabidopsis thaliana sequences

MOTIVATION: The annotation of the Arabidopsis thaliana genome remains a problem in terms of time and quality. To improve the annotation process, we want to choose the most appropriate tools to use inside a computer-assisted annotation platform. We therefore need evaluation of prediction programs with Arabidopsis sequences containing multiple genes. RESULTS: We have developed AraSet, a data set of contigs of validated genes, enabling the evaluation of multi-gene models for the Arabidopsis genome. Besides conventional metrics to evaluate gene prediction at the site and the exon levels, new measures were introduced for the prediction at the protein sequence level as well as for the evaluation of gene models. This evaluation method is of general interest and could apply to any new gene prediction software and to any eukaryotic genome. The GeneMark.hmm program appears to be the most accurate software at all three levels for the Arabidopsis genomic sequences. Gene modeling could be further improved by combination of prediction software. AVAILABILITY: The AraSet sequence set, the Perl programs and complementary results and notes are available at http://sphinx.rug.ac.be:8080/biocomp/napav/. CONTACT: Pierre.Rouze@gengenp.rug.ac.be.     

Multilocus analysis of variation using a large empirical data set: phenylpropanoid pathway genes in Arabidopsis thaliana

Detecting the signature of adaptation on nucleotide variation is often difficult in species that like Arabidopsis thaliana might have a complex demographic history. Recent re-sequencing surveys in this species provided genome-wide information that would mainly reflect its demographic history. We have used a large empirical data set (LED) as well as multilocus coalescent simulations to analyse sequence variation at loci involved in the phenylpropanoid pathway of this species. We surveyed and examined DNA sequence variation at nine of these loci (about 19.7 kb) in 23 accessions of A. thaliana and one accession of its closely related species Arabidopsis lyrata . Nucleotide variation was lower at nonsynonymous sites than at silent sites in all loci, indicating generalized functional constraint at the protein level. No association between variation and position in the metabolic pathway was detected. When the data were contrasted against the standard neutral model, significant deviations for silent variation were detected with Tajima's D , Fu's F_S and Fay and Wu's H multilocus test statistics. These deviations were in the same direction than in previous large-scale multilocus analyses, suggesting a genome-wide effect. When the nine-locus data set was contrasted against the large empirical data set, the level (Watterson's θ) and pattern of variation (Tajima's D ) detected in these loci did not deviate either at the single-locus or multilocus level from the corresponding empirical distributions. These results would support an important role of the demographic history of A. thaliana in shaping nucleotide variation at the nine studied phenylpropanoid loci. The potential and limitations of the empirical distribution approach are discussed.     

A set of Arabidopsis thaliana miRNAs involve shoot regeneration in vitro

Plant miRNAs, the critical regulator of gene expression, involve many development processes in vivo. However, the roles of miRNAs in plant cell proliferation and redifferntiation in vitro remain unknown. To determine better the molecular mechanism of these processes, we have recently reported that a set of miRNAs with different expression patterns between cells of totipotent and non-totipotent Arabidopsis calli. Some of these were specifically up- or downregulated during callus formation or shoot regeneration, and other development. Among them, miR160, and one of its target genes, ARF10, regulated Arabidopsis in vitro shoot regeneration via WUS, CLV3 and CUC1/2. The miR160-overexpressing, 35S transgenic lines, exhibited reduced shoot regeneration efficiency. The mARF10, a miR160-resistant form of ARF10, showed a high level of shoot regeneration ability. In the transgenic, expression of the above shoot meristem-specific genes was elevated, which is consistent with the improved shoot regeneration. In contrast, the ARF10 deficient knockout mutant produced fewer regenerated shoot. However, overexpressors of ARF10 were only marginally more efficient than the wild type with the respect to shoot regeneration. Our observation strongly supports that proper shoot regeneration from in vitro cultured cells requires the miR160-directed negative influence of ARF10. The enhanced expression of ARF10 is likely to have contributed to the improved regeneration ability.     

An Arabidopsis thaliana RFLP mapping set to localize mutations to chromosomal regions

Mapping of newly identified Arabidopsis thaliana mutants is an important step towards their molecular characterization and the attempt to saturate the genome by known mutations. The classical genetic analysis using phenotypic tester lines is well-established, but laborious, time-consuming and potentially ambiguous. An alternative molecular strategy was developed that is based on RFLPs. Subcloned DNA markers that detect only segregating RFLP bands distinguishing A. thaliana ecotype Landsberg from Columbia or Enkheim after EcoRI restriction digestion compose an Arabidopsis RFLP mapping set (ARMS). Up to 13 markers uniformly cover the five A. thaliana chromosomes and can be scored in only two successive Southern experiments on a single blot without mutual interference of the signals. Thus, this system allows a simple, reliable, rapid and especially inexpensive mapping of any monogenic mutant locus to the A. thaliana chromosomes. Several loci can be analysed in one experiment if the respective blots are hybridized together. This paper demonstrates the mapping of two recessive mutants affecting the development of A. thaliana leaves which had been generated in the Columbia and Enkheim ecotype by analysing less than 20 F₂ individuals. Further markers to refine or verify the result on the same blot can be chosen out of 14 additional probes detecting single segregating EcoRI polymorphic bands as well.     

GenBank.

The GenBank sequence database continues to expand its data coverage, quality control, annotation content and retrieval services. GenBank is comprised of DNA sequences submitted directly by authors as well as sequences from the other major public databases. An integrated retrieval system, known as Entrez, contains data from GenBank and from the major protein sequence and structural databases, as well as related MEDLINE abstracts. Users may access GenBank over the Internet through the World Wide Web and through special client-server programs for text and sequence similarity searching. FTP, CD-ROM and e-mail servers are alternate means of access.     

Flavonoid-specific staining of Arabidopsis thaliana.

Crop yields may be threatened by increases in UV-B radiation resulting from depletion of the ozone layer. In higher plants, the presence of flavonols provides a protective mechanism, and we report a novel staining procedure for the visualization of such protectants in plant tissue. It is shown that the proposed technique provides sensitive and specific fluorescence of flavonoids in chlorophyll-bleached tissue of Arabidopsis thaliana.     

Phosphatidylglycerophosphate synthases from Arabidopsis thaliana.

Two Arabidopsis thaliana genes were shown to encode phosphatidylglycerophosphate synthases (PGPS) of 25.4 and 32.2 kDa, respectively. Apart from their N-terminal regions, the two proteins exhibit high sequence similarity. Functional expression studies in yeast provided evidence that the 25.4 kDa protein is a microsomal PGPS while the 32.2 kDa protein represents a preprotein which can be imported into yeast mitochondria and processed to a mature PGPS. The two isozymes were solubilized and purified as fusion proteins carrying a His tag at their C-terminus. Enzyme assays with both membrane fractions and purified enzyme fractions revealed that the two A. thaliana isozymes have similar properties but differ in their CDP-diacylglycerol species specificity.     

Copper-sensitive mutant of Arabidopsis thaliana.

A Cu-sensitive mutant, cup1-1, of Arabidopsis thaliana has a pattern of heavy-metal sensitivity different from that of the cad1 and cad2 mutants, which are deficient in phytochelatin biosynthesis. The latter are significantly sensitive to Cd and Hg and only slightly sensitive to Cu, whereas the cup1-1 mutant is significantly sensitive to Cu, slightly sensitive to Cd, and not more sensitive to Hg, compared to the wild type. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus, which has been mapped to chromosome 1. Genetic and biochemical studies demonstrate that the cup1-1 mutant is not affected in phytochelatin biosynthesis or function. The sensitive phenotype of the cup1-1 mutant is associated with, and probably due to, increased accumulation of higher levels of Cd and Cu compared with the wild type. Consistent with this, a Cu-inducible, root-specific metallothionein gene, MT2a, is expressed in cup1-1 roots under conditions in which it is not expressed in the wild type. Undifferentiated cup1-1 callus tissue did not show the Cu-sensitive phenotype, suggesting that the mutant phenotype, in contrast to cad1 and cad2, is not expressed at the cellular level.     

Gene targeting in Arabidopsis thaliana.

Summary Gene targeting of a chromosomally integrated transgene in Arabidopsis thaliana is reported. A chimeric gene consisting of the promoter of the 35S RNA of CaMV, the polyadenylation signal of the octopine synthase gene and the coding region of the bacterial hygromycin phosphotransferase gene (hpt), which was rendered non-functional by deletion of 19 bp, was introduced into the genome of A. thaliana using Agrobacterium-mediated gene transfer. A total of 3.46 x 10⁸ protoplasts isolated from 17 independent transgenic Arabidopsis lines harbouring the defective chimeric hpt gene were transformed via direct gene transfer using various DNA forms containing only the intact coding region of the hpt gene. Out of 150 hygromycin-resistant colonies appearing in the course of these experiments, four were the result of targeted recombination of the incoming DNA with the defective chromosomal locus as revealed by PCR and Southern blot analysis. Comparison with the number of transformants obtained when an hpt gene controlled by a promoter and terminator from the nopaline synthase gene was employed results in a maximal ratio of homologous to non-homologous transformation in A. thaliana of 1 x 10^–4.     

GenBank.

The GenBank sequence database incorporates DNA sequences from all available public sources, primarily through the direct submission of sequence data from authors and from large-scale sequencing projects. Data exchange with the EMBL Data Library and the DNA Data Bank of Japan helps ensure comprehensive coverage. GenBank continues to focus on quality control and annotation while expanding data coverage and retrieval services. An integrated retrieval system, known asEntrez, incorporates data from the major DNA and protein sequence databases, along with genome maps and protein structure information. MEDLINE abstracts from published articles describing the sequences are also included as an additional source of biological annotation. Sequence similarity searching is offered through the BLAST family of programs. All of NCBI's services are offered through the World Wide Web. In addition, there are specialized server/client versions as well as FTP and e-mail server access.     

GenBank.

The GenBank(R) sequence database (http://www.ncbi.nlm.nih.gov/) incorporates DNA sequences from all available public sources, primarily through the direct submission of sequence data from individual laboratories and from large-scale sequencing projects. Most submitters use the BankIt (WWW) or Sequin programs to send their sequence data. Data exchange with the EMBL Data Library and the DNA Data Bank of Japan helps ensure comprehensive worldwide coverage. GenBank data is accessible through NCBI's integrated retrieval system, Entrez , which integrates data from the major DNA and protein sequence databases along with taxonomy, genome and protein structure information. MEDLINE(R) abstracts from published articles describing the sequences are also included as an additional source of biological annotation. Sequence similarity searching is offered through the BLAST series of database search programs. In addition to FTP, e-mail and server/client versions of Entrez and BLAST, NCBI offers a wide range of World Wide Web retrieval and analysis services of interest to biologists.     

Systematic prediction of autophagy-related proteins using Arabidopsis thaliana interactome data

Autophagy is a self-degradative process that is crucial for maintaining cellular homeostasis by removing damaged cytoplasmic components and recycling nutrients. Such an evolutionary conserved proteolysis process is regulated by the autophagy-related (Atg) proteins. The incomplete understanding of plant autophagy proteome and the importance of a proteome-wide understanding of the autophagy pathway prompted us to predict Atg proteins and regulators in Arabidopsis. Here, we developed a systems-level algorithm to identify autophagy-related modules (ARMs) based on protein subcellular localization, protein–protein interactions, and known Atg proteins. This generates a detailed landscape of the autophagic modules in Arabidopsis. We found that the newly identified genes in each ARM tend to be upregulated and coexpressed during the senescence stage of Arabidopsis. We also demonstrated that the Golgi apparatus ARM, ARM13, functions in the autophagy process by module clustering and functional analysis. To verify the in silico analysis, the Atg candidates in ARM13 that are functionally similar to the core Atg proteins were selected for experimental validation. Interestingly, two of the previously uncharacterized proteins identified from the ARM analysis, AGD1 and Sec14, exhibited bona fide association with the autophagy protein complex in plant cells, which provides evidence for a cross-talk between intracellular pathways and autophagy. Thus, the computational framework has facilitated the identification and characterization of plant-specific autophagy-related proteins and novel autophagy proteins/regulators in higher eukaryotes.     

GenBank.

The GenBank sequence database has undergone an expansion in data coverage, annotation content and the development of new services for the scientific community. In addition to nucleotide sequences, data from the major protein sequence and structural databases, and from U.S. and European patents is now included in an integrated system. MEDLINE abstracts from published articles describing the sequences provide an important new source of biological annotation for sequence entries. In addition to the continued support of existing services, new CD-ROM and network-based systems have been implemented for literature retrieval and sequence similarity searching. Major releases of GenBank are now more frequent and the data are distributed in several new forms for both end users and software developers.     

Submission of sequence data to GenBank

Editorial Announcement

Submission of sequence data to GenBank     

GenBank.

The GenBank sequence database continues to expand its data coverage, quality control, annotation content and retrieval services for the scientific community. Besides handling direct submissions of sequence data from authors, GenBank also incorporates DNA sequences from all available public sources; an integrated retrieval system, known as Entrez, also makes available data from the major protein sequence and structural databases, and from U.S. and European patents. MIDLINE abstracts from published articles describing the sequences are also included as an additional source of biological annotation for sequence entries. GenBank supports distribution of the data via FTP, CD-ROM, and E-mail servers. Network server-client programs provide access to an integrated database for literature retrieval and sequence similarity searching.     

GenBank.

The GenBank (Registered Trademark symbol) sequence database incorporates DNA sequences from all available public sources, primarily through the direct submission of sequence data from individual laboratories and from large-scale sequencing projects. Most submitters use the BankIt (Web) or Sequin programs to format and send sequence data. Data exchange with the EMBL Data Library and the DNA Data Bank of Japan helps ensure comprehensive worldwide coverage. GenBank data is accessible through NCBI's integrated retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome and protein structure information. MEDLINE (Registered Trademark symbol) s from published articles describing the sequences are included as an additional source of biological annotation through the PubMed search system. Sequence similarity searching is offered through the BLAST series of database search programs. In addition to FTP, Email, and server/client versions of Entrez and BLAST, NCBI offers a wide range of World Wide Web retrieval and analysis services based on GenBank data. The GenBank database and related resources are freely accessible via the URL: http://www.ncbi.nlm.nih.gov     

A metal-accumulator mutant of Arabidopsis thaliana.

A mutation designated man1 (for manganese accumulator) was found to cause Arabidopsis thaliana seedlings to accumulate a range of metals. The man1 mutation segregated as a single recessive locus located on chromosome 3. When grown on soil, mutant seedlings accumulated Mn (7.5 times greater than wild type), Cu (4.6 times greater than wild type), Zn (2.8 times greater than wild type), and Mg (1.8 times greater than wild type) in leaves. In addition to these metals, the man1 mutant accumulated 2.7-fold more S in leaves, primarily in the oxidized form, than wild-type seedlings. Analysis of seedlings grown by hydroponic culture showed a similar accumulation of metals in leaves of man1 mutants. Roots of man1 mutants also accumulated metals, but unlike leaves they accumulated 10-fold more total Fe (symplasmic and apoplasmic combined) than wild-type roots. Roots of man1 mutants possessed greater (from 1.8- to 20-fold) ferric-chelate reductase activity than wild-type seedings, and this activity was not responsive to changes of Mn nutrition in either genotype. Taken together, these results suggest that the man1 mutation disrupts the regulation of metal-ion uptake or homeostasis in Arabidopsis.     

Global protein interactome exploration through mining genome-scale data in Arabidopsis thaliana

Background

Many essential cellular processes, such as cellular metabolism, transport, cellular metabolism and most regulatory mechanisms, rely on physical interactions between proteins. Genome-wide protein interactome networks of yeast, human and several other animal organisms have already been established, but this kind of network reminds to be established in the field of plant.

Results

We first predicted the protein protein interaction in Arabidopsis thaliana with methods, including ortholog, SSBP, gene fusion, gene neighbor, phylogenetic profile, coexpression, protein domain, and used Naïve Bayesian approach next to integrate the results of these methods and text mining data to build a genome-wide protein interactome network. Furthermore, we adopted the data of GO enrichment analysis, pathway, published literature to validate our network, the confirmation of our network shows the feasibility of using our network to predict protein function and other usage.

Conclusions

Our interactome is a comprehensive genome-wide network in the organism plant Arabidopsis thaliana, and provides a rich resource for researchers in related field to study the protein function, molecular interaction and potential mechanism under different conditions.

     

One-step analysis of seed storage data and the longevity of Arabidopsis thaliana seeds

Seeds of two ecotypes of Arabidopsis thaliana, NW20 and N1601, were aged over a range of saturated salt solutions at temperatures between 6 degrees C and 55 degrees C. For each ecotype, the results from 37 storage experiments were summarized using the Ellis and Roberts viability equations and a modified version of these equations which allows for a proportion of 'non-respondents'. For both models, two approaches were taken in order to model the effect of moisture content (MC) and temperature on seed longevity. The first, a two-step approach, involved fitting individual survival curves and then multiple regression analysis of the fitted parameters on moisture content and temperature. For the second approach, the full viability models were fitted in one step, including the multiple regression for the effects of MC and temperature within the generalized linear model used to describe each survival curve. This one-step approach takes into account the full variability of the data and provides the best predictions of seed longevity based on the original assumptions of the Ellis and Roberts viability equations. As a consequence of taking into account all the variation, this one-step approach is more sensitive and thus more likely to detect changes due to reducing the number of parameters in the model as being significant. Whilst both approaches indicated that seeds from the two Arabidopsis ecotypes have the same response to MC and temperature, parameter values did differ between the approaches, with the one-step approach providing the better fit. The best model for these two ecotypes, from the one-step approach, confirmed a quadratic relationship between temperature and longevity, but the magnitude of the non-linearity is not as large as indicated by the universal value for the quadratic term.     

Unified display of Arabidopsis thaliana physical maps from AtDB, the A.thaliana database.

In the past several years, there has been a tremendous effort to construct physical maps and to sequence the genome of Arabidopsis thaliana. As a result, four of the five chromosomes are completely covered by overlapping clones except at the centromeric and nucleolus organizer regions (NOR). In addition, over 30% of the genome has been sequenced and completion is anticipated by the end of the year 2000. Despite these accomplishments, the physical maps are provided in many formats on laboratories' Web sites. These data are thus difficult to obtain in a coherent manner for researchers. To alleviate this problem, AtDB (Arabidopsis thaliana DataBase, URL: http://genome-www.stanford.edu/Arabidopsis/) has constructed a unified display of the physical maps where all publicly available physical-map data for all chromosomes are presented through the Web in a clickable, 'on-the-fly' graphic, created by CGI programs that directly consult our relational database.