Dataset Information

Identifying and mapping novel retinal-expressed ESTs from humans.

ABSTRACT: The goal of this study was to develop efficient methods to identify tissue-specific expressed sequence tags (ESTs) and to map their locations in the human genome. Through a combination of database analysis and laboratory investigation, unique retina-specific ESTs were identified and mapped as candidate genes for inherited retinal diseases.DNA sequences from retina-specific EST clusters were obtained from the TIGR Human Gene Index Database. Further processing of the EST sequence data was necessary to ensure that each EST cluster represented a novel, non-redundant mapping candidate. Processing involved screening for homologies to known genes and proteins using BLAST, excluding known human gene sequences and repeat sequences, and developing primers for PCR amplification of the gene encoding each cDNA cluster from genomic DNA. The EST clusters were mapped using the GeneBridge 4.0 Radiation Hybrid Mapping Panel with standard PCR conditions.A total of 83 retinal-expressed EST clusters were examined as potential novel, non-redundant mapping candidates. Fifty-five clusters were mapped successfully and their locations compared to the locations of known retinal disease genes. Fourteen EST clusters localize to candidate regions for inherited retinal diseases.This pilot study developed methodology for mapping uniquely expressed retinal ESTs and for identifying potential candidate genes for inherited retinal disorders. Despite the overall success, several complicating factors contributed to the high failure rate (33%) for mapping EST-clustered sequences. These include redundancy in the sequence data, widely dispersed sequences, ambiguous nucleotides within the sequences, the possibility of amplifying through introns and the presence of repetitive elements within the sequence. However, the combination of database analysis and laboratory mapping is a powerful method for identification of candidate genes for inherited diseases.

SUBMITTER: Malone K

PROVIDER: S-EPMC2583080 | biostudies-literature | 1999 May

REPOSITORIES: biostudies-literature

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Identifying and mapping novel retinal-expressed ESTs from humans.

Malone K K Sohocki M M MM Sullivan L S LS Daiger S P SP

Molecular vision 19990504

<h4>Purpose</h4>The goal of this study was to develop efficient methods to identify tissue-specific expressed sequence tags (ESTs) and to map their locations in the human genome. Through a combination of database analysis and laboratory investigation, unique retina-specific ESTs were identified and mapped as candidate genes for inherited retinal diseases.<h4>Methods</h4>DNA sequences from retina-specific EST clusters were obtained from the TIGR Human Gene Index Database. Further processing of th ...[more]

PMID: 10228186

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Project description:BackgroundThe turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis.ResultsA total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value < or = 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot.ConclusionA collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot.

Project description:BACKGROUND: Oil palm (Elaeis guineensis Jacq.) is one of the most important oil bearing crops in the world. However, genetic improvement of oil palm through conventional breeding is extremely slow and costly, as the breeding cycle can take up to 10 years. This has brought about interest in vegetative propagation of oil palm. Since the introduction of oil palm tissue culture in the 1970s, clonal propagation has proven to be useful, not only in producing uniform planting materials, but also in the development of the genetic engineering programme. Despite considerable progress in improving the tissue culture techniques, the callusing and embryogenesis rates from proliferating callus cultures remain very low. Thus, understanding the gene diversity and expression profiles in oil palm tissue culture is critical in increasing the efficiency of these processes. RESULTS: A total of 12 standard cDNA libraries, representing three main developmental stages in oil palm tissue culture, were generated in this study. Random sequencing of clones from these cDNA libraries generated 17,599 expressed sequence tags (ESTs). The ESTs were analysed, annotated and assembled to generate 9,584 putative unigenes distributed in 3,268 consensi and 6,316 singletons. These unigenes were assigned putative functions based on similarity and gene ontology annotations. Cluster analysis, which surveyed the relatedness of each library based on the abundance of ESTs in each consensus, revealed that lipid transfer proteins were highly expressed in embryogenic tissues. A glutathione S-transferase was found to be highly expressed in non-embryogenic callus. Further analysis of the unigenes identified 648 non-redundant simple sequence repeats and 211 putative full-length open reading frames. CONCLUSION: This study has provided an overview of genes expressed during oil palm tissue culture. Candidate genes with expression that are modulated during tissue culture were identified. However, in order to confirm whether these genes are suitable as early markers for embryogenesis, the genes need to be tested on earlier stages of tissue culture and a wider range of genotypes. This collection of ESTs is an important resource for genetic and genome analyses of the oil palm, particularly during tissue culture development.

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Identifying and mapping novel retinal-expressed ESTs from humans.

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Identifying and mapping novel retinal-expressed ESTs from humans.

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