Expressed Sequence Tag Libraries Research Articles

Analysis of gene expression in arbuscular mycorrhizas: new approaches and challenges

SummaryThis review summarizes the most recent results obtained using molecular biological techniques in the understanding of the arbuscular mycorrhizal symbiosis. On the plant side, cDNA libraries have been established from mycorrhizal RNA using several techniques, such as suppressive subtractive hybridization, and a large number of clones are being sequenced to obtain expressed sequence tags (ESTs). These ESTs can be transferred to solid supports (arrays) and hybridized with cDNA from different sources to obtain RNA accumulation profiles of genes expressed during the mycorrhizal symbiosis. On the fungal side, EST libraries are also being established, both using direct cloning and suppressive subtractive hybridization techniques. For both partners, the next steps are the detailed analysis of some relevant genes concerned with regulation and function, as well as the study of their role in the symbiosis. At the end of this review, we discuss how results about gene expression and functioning from molecular analyses can help in understanding the ecology of the symbiosis and the way in which it can be further exploited for application in plant production systems.

Characterization of the cDNA encoding the 90 kDa heat-shock protein in the Lepidoptera Bombyx mori and Spodoptera frugiperda

This report presents the first hsp90 complete cDNA sequences from two Lepidoptera. The Bombyx mori full sequence was reconstituted from 15 partial cDNA clones belonging to expressed sequence tag libraries obtained from different tissues or cultured cells, thus showing the ubiquitous expression of the hsp90 gene. The Spodoptera frugiperda cDNA was isolated as a full-length clone from a cDNA library established from the Sf9 cell line. Both cDNAs are highly homologous and display the classical amino acid (aa) stretches representing the HSP90 signature. They potentially encode a 716 aa ( B. mori) and a 717 aa ( S. frugiperda) protein, with a calculated molecular mass of 83 kDa similar to the Drosophila homologous protein. We show that, unlike the vertebrates, hsp90 is a unique gene in both S. frupiperda and B. mori genomes. Sequencing of the corresponding genomic region shows that, contrary to the dipteran homologous gene, the lepidopteran hsp90 gene does not display any intron. Phylogenetic analysis based on the two lepidopteran and 23 other HSP90 aa sequences displays a high consistency with known phylogeny at both high and low taxonomic levels. Transcriptional analysis performed in S. frugiperda shows that the induction of the hsp90 gene only occurs 14°C above physiological growth conditions (42°C).

Microsatellite markers from sugarcane ( Saccharum spp.) ESTs cross transferable to erianthus and sorghum

Analysis of a sugarcane ( Saccharum spp.) EST (expressed sequence tag) library of 8678 sequences revealed approximately 250 microsatellite or simple sequence repeats (SSRs) sequences. A diversity of dinucleotide and trinucleotide SSR repeat motifs were present although most were of the (CGG) n trinucleotide motif. Primer sets were designed for 35 sequences and tested on five sugarcane genotypes. Twenty-one primer pairs produced a PCR product and 17 pairs were polymorphic. Primer pairs that produced polymorphisms were mainly located in the coding sequence with only a single pair located within the 5′ untranslated region. No primer pairs producing a polymorphic product were found in the 3′ untranslated region. The level of polymorphism (PIC value) in cultivars detected by these SSRs was low in sugarcane (0.23). However, a subset of these markers showed a significantly higher level of polymorphism when applied to progenitor and related genera ( Erianthus sp. and Sorghum sp.). By contrast, SSRs isolated from sugarcane genomic libraries amplify more readily, show high levels of polymorphism within sugarcane with a higher PIC value (0.72) but do not transfer to related species or genera well.

EST-library construction using spore RNA of the arbuscular mycorrhizal fungus Gigaspora rosea

RNA was extracted from activated spores of the arbuscular mycorrhizal fungus Gigaspora rosea. Double-stranded cDNA was synthesised, digested and cloned into the vector lambda-ZAP express. Of the 1,500 clones obtained, 1.5% carried inserts of the rRNA gene cluster. After excision, inserts from 50 randomly selected clones were sequenced. Database searches revealed that 62% of the clones had similarities to already known sequences. These mainly code for proteins involved in translation and protein processing, replication and the cell cycle and cell signal transduction. One fragment probably belonged to a metallothionein-encoding gene which may be involved in heavy-metal binding. The method presented is an easy and rapid way to obtain short fragments of coding regions for expressed sequence tag libraries.

Mass spectrometry and proteomics.

the 15th asilomar conference on Mass Spectrometry this October was devoted to the role of mass spectrometry (MS) in proteomics. The Asilomar Conference site is in a picturesque national park in Pacific Grove, CA, overlooking the Pacific Ocean. The conference aims to bring together scientists from a

Assembly and functional expression of murine glutathione reductase cDNA: a sequence missing in expressed sequence tag libraries

Glutathione reductase (GR) is a chemotherapeutic target. Murine GRcDNA, which contains 85% GC in the 38 codons following the start codon, was assembled from the PCR-amplified exon 1 and a downstream cDNA prior to expression in Escherichia coli as a His 6-tagged protein. Recombinant GR, an FAD-containing homodimer, corresponds in its enzymic and spectral properties to GR isolated from murine Ehrlich ascites tumor cells. Another cDNA, representing GR with a mitochondrial targeting sequence, yielded two distinct enzymically active expression products.

Panning for Genes—A Visual Strategy for Identifying Novel Gene Orthologs and Paralogs

We have developed a rapid visual method for identifying novel members of gene families. Starting with an evolutionary tree, 20-50 protein query sequences for a gene family are selected from different branches of the tree. These query sequences are used to search the GenBank and expressed sequence tag (EST) DNA databases and their nightly updates using the tfastx3 or tfasty3 programs. The results of all 20-50 searches are collated and resorted to highlight EST or genomic sequences that share significant similarity with the query sequences. The statistical significance of each DNA/protein alignment is plotted, highlighting the portion of the query sequence that is present in the database sequence and the percent identity in the aligned region. The collated results for database sequences are linked using the WWW to the underlying scores and alignments; these links can also be used to perform additional searches to characterize the novel sequence further. With traditional "deep" scoring matrices (BLOSUM50) one can search for previously unrecognized families of large protein superfamilies. Alternatively, by using query sequences and EST libraries from the same species (e. g., human or mouse) together with "shallow" scoring matrices and filters that remove high-identity sequences, one can highlight new paralogs of previously described subfamilies. Using query sequences from the glutathione transferase superfamily, we identified two novel mammalian glutathione transferase families that were recognized previously only in plants. Using query sequences from known mammalian glutathione transferase subfamilies, we identified new candidate paralogs from the mouse class-mu, class-pi, and class-theta families.

THE USE OF CHLAMYDOMONAS (CHLOROPHYTA: VOLVOCALES) AS A MODEL ALGAL SYSTEM FOR GENOME STUDIES AND THE ELUCIDATION OF PHOTOSYNTHETIC PROCESSES

Chlamydomonas reinhardtii Dangeard is a unicellular, haploid green alga that has been used as a model genetic system to investigate basic biological processes in photosynthetic eukaryotes. This organism has been particularly useful for dissecting photosynthesis because it can grow heterotrophically as well as photoautotrophically; this has allowed for the isolation and characterization of a large number of mutants that are completely devoid of photosynthetic CO2 fixation. Over the past 10 years there has been a dramatic increase in the number of molecular technologies that can be applied to studies of Chlamydomonas, enhancing its value as a model organism. Nuclear transformation has enabled researchers to generate and complement a range of different mutants and to investigate transcriptional control of gene expression in vivo. The development of methods for chloroplast transformation has helped elucidate the functions of chloroplast genes and the factors that modulate their expression. Genome‐wide studies will further enhance the value of Chlamydomonas as a resource for the biological community and will have a profound effect on the types of experimentation for which this alga is used. For example, the generation and characterization of a library of expressed sequence tags (ESTs) would identify cDNAs for most of the genes present on the Chlamydomonas genome. DNA microarray technology could be used to place these cDNAs on “chips,” configured from microscope slides, that would enable investigators to monitor changes in gene expression of essentially the entire genome simultaneously. The development of methods to identify strains harboring insertions in specific genes would help elucidate the functions of the genes that are represented in the EST library and that have been characterized for expression via the microarray technology. The production of linked physical and genetic maps would facilitate the map‐based cloning of any gene identified by a mutant phenotype. Finally, completing the sequence of the chloroplast genome and analyzing the function and expression of individual chloroplast genes would enhance our knowledge of the activities and biogenesis of this organelle. In thisreview we discuss the application of genomic technologies to studies of Chlamydomonas and how they would help elucidate the metabolism of plants, especially with respect to photosynthetic processes.

Construction of a Lotus japonicus late nodulin expressed sequence tag library and identification of novel nodule-specific genes.

A range of novel expressed sequence tags (ESTs) associated with late developmental events during nodule organogenesis in the legume Lotus japonicus were identified using mRNA differential display; 110 differentially displayed polymerase chain reaction products were cloned and analyzed. Of 88 unique cDNAs obtained, 22 shared significant homology to DNA/protein sequences in the respective databases. This group comprises, among others, a nodule-specific homolog of protein phosphatase 2C, a peptide transporter protein, and a nodule-specific form of cytochrome P450. RNA gel-blot analysis of 16 differentially displayed ESTs confirmed their nodule-specific expression pattern. The kinetics of mRNA accumulation of the majority of the ESTs analyzed were found to resemble the expression pattern observed for the L. japonicus leghemoglobin gene. These results indicate that the newly isolated molecular markers correspond to genes induced during late developmental stages of L. japonicus nodule organogenesis and provide important, novel tools for the study of nodulation.

Pruritus after administration of hetastarch

We isolated the full-length chicken IL-10 (chIL-10) cDNA from an expressed sequence tag library derived from RNA from cecal tonsils of <i>Eimeria tenella</i>-infected chickens. It encodes a 178-aa polypeptide, with a predicted 162-aa mature peptide. Chicken IL-10 has 45 and 42% aa identity with human and murine IL-10, respectively. The structures of the chIL-10 gene and its promoter were determined by direct sequencing of a bacterial artificial chromosome containing chIL-10. The chIL-10 gene structure is similar to (five exons, four introns), but more compact than, that of its mammalian orthologues. The promoter is more similar to that of <i>Fugu</i> IL-10 than human IL-10. Chicken IL-10 mRNA expression was identified mainly in the bursa of Fabricius and cecal tonsils, with low levels of expression also seen in thymus, liver, and lung. Expression was also detected in PHA-activated thymocytes and LPS-stimulated monocyte-derived macrophages, with high expression in an LPS-stimulated macrophage cell line. Recombinant chIL-10 was produced and bioactivity demonstrated through IL-10-induced inhibition of IFN-γ synthesis by mitogen-activated lymphocytes. We measured the expression of mRNA for chIL-10 and other signature cytokines in gut and spleen of resistant (line C.B12) and susceptible (line 15I) chickens during the course of an <i>E. maxima</i> infection. Susceptible chickens showed higher levels of chIL-10 mRNA expression in the spleen, both constitutively and after infection, and in the small intestine after infection than did resistant chickens. These data indicate a potential role for chIL-10 in changing the Th bias during infection with an intracellular protozoan, thereby contributing to susceptibility of line 15I chickens.