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An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries.

ABSTRACT: BACKGROUND: The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. RESULTS: Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. CONCLUSIONS: Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online.

SUBMITTER: Habermann B

PROVIDER: S-EPMC522874 | biostudies-literature | 2004

REPOSITORIES: biostudies-literature

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An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries.

Habermann Bianca B Bebin Anne-Gaelle AG Herklotz Stephan S Volkmer Michael M Eckelt Kay K Pehlke Kerstin K Epperlein Hans Henning HH Schackert Hans Konrad HK Wiebe Glenis G Tanaka Elly M EM

Genome biology 20040813 9

<h4>Background</h4>The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum.<h4>Results</h4>Two cDNA libraries, one made from stage ...[more]

PMID: 15345051

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Project description:BACKGROUND:Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. RESULT:We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot plants. Codon usages of melon full-length transcripts were largely similar to those of Arabidopsis coding sequences. CONCLUSION:The collection of melon ESTs generated from full-length enriched and standard cDNA libraries is expected to play significant roles in annotating the melon genome. The ESTs and associated analysis results will be useful resources for gene discovery, functional analysis, marker-assisted breeding of melon and closely related species, comparative genomic studies and for gaining insights into gene expression patterns.

Project description:Some organisms, such as the Mexican axolotl, have the capacity to regenerate complicated biological structures throughout their lives. Which molecular pathways are sufficient to induce a complete endogenous regenerative response in injured tissue is an important question that remains unanswered. Using a gain-of-function regeneration assay, known as the Accessory Limb Model (ALM), we and others have begun to identify the molecular underpinnings of the three essential requirements for limb regeneration; wounding, neurotrophic signaling, and the induction of pattern from cells that retain positional memory. We have previously shown that treatment of Mexican axolotls with exogenous retinoic acid (RA) is sufficient to induce the formation of complete limb structures from blastemas that were generated by deviating a nerve bundle into an anterior-located wound site on the limb. Here we show that these ectopic structures are capable of regenerating and inducing new pattern to form when grafted into new anterior-located wounds. We additionally found that the expression of Alx4 decreases, and Shh expression increases in these anterior located blastemas, but not in the mature anterior tissues, supporting the hypothesis that RA treatment posteriorizes blastema tissue. Based on these and previous observations, we used the ALM assay to test the hypothesis that a complete regenerative response can be generated by treating anterior-located superficial limb wounds with a specific combination of growth factors at defined developmental stages. Our data shows that limb wounds that are first treated with a combination of FGF-2, FGF-8, and BMP-2, followed by RA treatment of the resultant mid-bud stage blastema, will result in the generation of limbs with complete proximal/distal and anterior/posterior limb axes. Thus, the minimal signaling requirements from the nerve and a positional disparity are achieved with the application of this specific combination of signaling molecules.

Dataset Information

An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries.

Publications

An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries.

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