BackgroundExpressed Sequence Tags (ESTs) are a source of simple sequence repeats (SSRs) that can be used to develop molecular markers for genetic studies. The availability of ESTs for Quercus robur and Quercus petraea provided a unique opportunity to develop microsatellite markers to accelerate research aimed at studying adaptation of these long-lived species to their environment. As a first step toward the construction of a SSR-based linkage map of oak for quantitative trait locus (QTL) mapping, we describe the mining and survey of EST-SSRs as well as a fast and cost-effective approach (bin mapping) to assign these markers to an approximate map position. We also compared the level of polymorphism between genomic and EST-derived SSRs and address the transferability of EST-SSRs in Castanea sativa (chestnut).ResultsA catalogue of 103,000 Sanger ESTs was assembled into 28,024 unigenes from which 18.6% presented one or more SSR motifs. More than 42% of these SSRs corresponded to trinucleotides. Primer pairs were designed for 748 putative unigenes. Overall 37.7% (283) were found to amplify a single polymorphic locus in a reference full-sib pedigree of Quercus robur. The usefulness of these loci for establishing a genetic map was assessed using a bin mapping approach. Bin maps were constructed for the male and female parental tree for which framework linkage maps based on AFLP markers were available. The bin set consisting of 14 highly informative offspring selected based on the number and position of crossover sites. The female and male maps comprised 44 and 37 bins, with an average bin length of 16.5 cM and 20.99 cM, respectively. A total of 256 EST-SSRs were assigned to bins and their map position was further validated by linkage mapping. EST-SSRs were found to be less polymorphic than genomic SSRs, but their transferability rate to chestnut, a phylogenetically related species to oak, was higher.ConclusionWe have generated a bin map for oak comprising 256 EST-SSRs. This resource constitutes a first step toward the establishment of a gene-based map for this genus that will facilitate the dissection of QTLs affecting complex traits of ecological importance.
Defining the molecular mechanisms involved in cancer formation and progression is still a major challenge in colorectal-cancer research. Our strategy was to characterize genes whose expression is altered during colorectal carcinogenesis. To this end, the phenotype of a colorectal tumour was previously established by partial sequencing of a large number of its transcripts and the genes of interest were selected by differential screening on high-density filters with mRNA of colorectal cancer and normal adjacent mucosa. Fifty-one clones were found over-expressed and 23 were underexpressed in the colorectal-cancer tissues of the 5 analyzed patients. Among the latter, clones 6G2 and 32D6 were found of particular interest, since they had significant homology with several homeodomain-containing genes. The highest degree of similarity was with the murine Cdx1 for 6G2, and with the murine Cdx2 and hamster Cdx3 for 32D6. Using a RT-PCR approach, complete sequence of both types of homeobox-containing cDNA was obtained. The amino-acid sequence of the human Cdx1 is 85% identical to the mouse protein, and human Cdx2 has 94% identity with the mouse Cdx2 and hamster Cdx3. Tissue-distribution analysis of Cdx1 and Cdx2 mRNA showed that both transcripts were specifically expressed in small intestine, in colon and rectum. Colorectal cancer is the second most common tumour in men and the third in women in the Western countries. Although much is known about the epidemiology, morphology and genetics of colorectal tumorigenesis, our knowledge of the perturbations of gene expression that occur in colorectal tumours remains limited. Such tumours may arise from benign adenomatous polyps, which later progress to adenocarcinomas through several molecular events. They thus provide a very useful paradigm for studying the molecular genetic bases of cancer. The multistep process leading to colorectal tumorigenesis probably involves the loss of function of tumour-suppressor genes, as well as the activation of oncogenes. Several important genes have already been identified, but they do not account for the whole process, and other genes are probably involved.Efforts have been made to characterize these genes. Differential hybridization techniques and screening of substracted libraries allowed the elucidation of some of them (Bartsch et al., 1986;Denis et al., 1993;Yow et al., 1988;Schweinfest et al., 1993;Kondoh et al., 1992; Barnard et al., 1992a, b). We developed an alternative strategy, in which the phenotype of a colorectal tumour was established by partial sequencing of a large number of randomly selected transcripts (Frigerio et al., 1995). This repertory of ESTs should therefore contain most of the differentially expressed genes. Recently, Nguyen et al. (1995) have developed an efficient method of differential screening in which cDNA clones are gridded on high-density colony filters and hybridized with complex probes derived from poly (A) 1 RNA from different cells or tissues. The signals observed are measured, providing a ''hybridization s...
Wood is one of our most important natural resources. Surprisingly, we know hardly anything about the details of the process of wood formation. The aim of this work was to describe the main proteins expressed in wood forming tissue of a conifer species (Pinus pinaster Ait.). Using high resolution 2-DE with linear pH gradient ranging from 4 to 7, a total of 1039 spots were detected. Out of the 240 spots analyzed by MS/MS, 67.9% were identified, 16.7% presented no homology in the databases, and 15.4% corresponded to protein mixtures. Out of the 57 spots analyzed by MALDI-MS, only 15.8% were identified. Most of the 175 identified proteins play a role in either defense (19.4%), carbohydrates (16.6%) and amino acid (14.9%) metabolisms, genes and proteins expression (13.1%), cytoskeleton (8%), cell wall biosynthesis (5.7%), secondary (5.1%) and primary (4%) metabolisms. A summary of the identified proteins, their putative functions, and behavior in different types of wood are presented. This information was introduced into the PROTICdb database and is accessible at http://cbib1.cbib.u-bordeaux2.fr/Protic/Protic/home/index.php. Finally, the average protein amount was compared with their respective transcript abundance as quantified through EST counting in a cDNA-library constructed with mRNA extracted from wood forming tissue.
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