Physical and linkage mapping of mammary-derived expressed sequence tags in cattle

This study describes the physical and linkage mapping of 42 gene-associated markers developed from mammary gland-derived expressed sequence tags to the cattle genome. Of the markers, 25 were placed on the USDA reference linkage map and 37 were positioned on the Roslin 3000-rad radiation hybrid (RH) map, with 20 assignments shared between the maps. Although no novel regions of conserved synteny between the cattle and the human genomes were identified, the coverage was extended for bovine chromosomes 3, 7, 15, and 29 compared with previously published comparative maps between human and bovine genomes. Overall, these data improve the resolution of the human-bovine comparative maps and will assist future efforts to integrate bovine RH and linkage map data.     

Deletion mapping of homoeologous group 6-specific wheat expressed sequence tags

To localize wheat (Triticum aestivum L.) ESTs on chromosomes, 882 homoeologous group 6-specific ESTs were identified by physically mapping 7965 singletons from 37 cDNA libraries on 146 chromosome, arm, and sub-arm aneuploid and deletion stocks. The 882 ESTs were physically mapped to 25 regions (bins) flanked by 23 deletion breakpoints. Of the 5154 restriction fragments detected by 882 ESTs, 2043 (loci) were localized to group 6 chromosomes and 806 were mapped on other chromosome groups. The number of loci mapped was greatest on chromosome 6B and least on 6D. The 264 ESTs that detected orthologous loci on all three homoeologs using one restriction enzyme were used to construct a consensus physical map. The physical distribution of ESTs was uneven on chromosomes with a tendency toward higher densities in the distal halves of chromosome arms. About 43% of the wheat group 6 ESTs identified rice homologs upon comparisons of genome sequences. Fifty-eight percent of these ESTs were present on rice chromosome 2 and the remaining were on other rice chromosomes. Even within the group 6 bins, rice chromosomal blocks identified by 1-6 wheat ESTs were homologous to up to 11 rice chromosomes. These rice-block contigs were used to resolve the order of wheat ESTs within each bin.     

Characterization and radiation hybrid mapping of expressed sequence tags from the canine brain

Abstract Maps of the canine genome are now developing rapidly. Most of the markers on the current integrated canine radiation hybrid/genetic linkage/cytogenetic map are highly polymorphic microsatellite (type II) markers that are very useful for mapping disease loci. However, there is still an urgent need for the mapping of gene-based (type I) markers that are required for comparative mapping, as well as identifying candidate genes for disease loci that have been genetically mapped. We constructed an adult brain cDNA library as a resource to increase the number of gene-based markers on the canine genome map. Eighty-one percent of the 2700 sequenced expressed sequence tags (ESTs) represented unique sequences. The canine brain ESTs were compared with sequences in public databases to identify putative canine orthologs of human genes. One hundred nine of the canine ESTs were mapped on the latest canine radiation hybrid (RH) panel to determine the location of the respective canine gene. The addition of these new gene-based markers revealed three conserved segments (CS) between human and canine genomes previously detected by fluorescence in situ hybridization (FISH), but not by RH mapping. In addition, five new CS between dog and human were identified that had not been detected previously by RH mapping or FISH. This work has increased the number of gene-based markers on the canine RH map by approximately 30% and indicates the benefit to be gained by increasing the gene content of the current canine comparative map.     

Alternative splicing and expression profile analysis of expressed sequence tags in domestic pig

Domestic pig （Sus scrofa domestica） is one of the most important mammals to humans. Alternative splicing is a cellular mechanism in eukaryotes that greatly increases the diversity of gene products. Expression sequence tags （ESTs） have been widely used for gene discovery, expression profile analysis, and alternative splicing detection. In this study, a total of 712,905 ESTs extracted from 101 different nonnormalized EST libraries of the domestic pig were analyzed. These EST libraries cover the nervous system, digestive system, immune system, and meat production related tissues from embryo, newborn, and adult pigs, making contributions to the analysis of alternative splicing variants as well as expression profiles in various stages of tissues. A modified approach was designed to cluster and assemble large EST datasets, aiming to detect alternative splicing together with EST abundance of each splicing variant. Much efforts were made to classify alternative splicing into different types and apply different filters to each type to get more reliable results. Finally, a total of 1,223 genes with average 2.8 splicing variants were detected among 16,540 unique genes. The overview of expression profiles would change when we take alternative splicing into account.     

Exploiting plant somatic radiation hybrids for physical mapping of expressed sequence tags

Methods are described for the optimisation of the generation of radiation hybrids suitable for physical mapping of a plant (barley) genome. A combination of PCR-based technologies, involving the use of whole genome, mixed primer and hemi-nested primer amplifications, can greatly extend their utility for the physical mapping of expressed sequence tags (ESTs). Using panels of hybrids and ESTs, donor DNA retention and individual marker retention frequencies for the expressed portion of the barley genome in the hybrids were estimated.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by P. Langridge     

Analysis and chromosomal mapping of Leishmania (Leishmania) amazonensis amastigote expressed sequence tags

The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32% are not related to any other sequences in database and 68% presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis.     

Analysis of expressed sequence tags of Porphyra yezoensis

Single direct partial sequencing of anonymous cDNA clones was performed to obtain genetic information on red algae Porphyra yezoensis of which genetic information is not available. This expressed sequence tags (EST) analysis revealed 81 clones (42%) had significant homologies to known genes in GenBank. Of these clones, eight are related to known algal genes, whereas above 90% of the EST clones were newly identified in algae. Putative functional categories of these clones showed that the most abundant genes were involved in stress and defense mechanisms and that the next abundant genes were associated with protein synthetic pathways.     

Analysis of expressed genes in ethylene-treated potato leaves using expressed sequence tags

To isolate useful and interesting plant genes in large quantities, random sequencing of cDNA clones from potato leaf library treated with ethylene was performed. Partial sequences of randomly selected 210 clones with the insert of longer than 500 base pair (bp) as well as poly (A) tail have been compared with sequences in GeneBank, EMBL and DDBJ nucleic acid databases and fostered 193 expressed sequence tags (ESTs). The 210 cDNA clones identified are related to various aspect of metabolic pathways such as glycolysis, amino acid synthesis, translation mechanism, ribosome synthesis, hormone response, stress response, regulation of gene expression, and signal transduction. Among the 193 ESTs, 12 ESTs (29 cDNA clones) appeared more than once and 181 ESTs appeared once regarded as a solitary group. Out of 210 clones, 29 clones (13.8%) have no similarity to the known nucleotide sequences and could serve as a potentially useful resource for plant molecular biology referring to particular genes. Nucleotide sequencing to generate more ESTs from ethylene-induced as well as non-induced potato leaf is in progress as well.     

Comparative analysis of expressed sequence tags from Sesamum indicum and Arabidopsis thaliana developing seeds

Sesame (Sesamum indicum) is an important oilseed crop which produces seeds with 50% oil that have a distinct flavor and contains antioxidant lignans. Because sesame lignans are known to have antioxidant and health-protecting properties, metabolic pathways for lignans have been of interest in developing sesame seeds. As an initial approach to identify genes involved in accumulation of storage products and in the biosynthesis of antioxidant lignans, 3328 expressed sequence tags (ESTs) were obtained from a cDNA library of immature seeds 5-25 days old. ESTs were clustered and analyzed by the BLASTX or FASTAX program against the GenBank NR and Arabidopsis proteome databases. To compare gene expression profiles during development of green and non-green seeds, a comparative analysis was carried out between developing sesame and Arabidopsis seed ESTs. Analyses of these two seed EST sets have helped to identify similar and different gene expression profiles during seed development, and to identify a large number of sesame seed-specific genes. In particular, we have identified EST candidates for genes possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin, and also suggest a possible metabolic pathway for the generation of cofactors required for synthesis of storage lipid in non-green oilseeds. Seed-specific expression of several candidate genes has been confirmed by northern blot analysis.