Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injuryReportar como inadecuado




Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Genomics

, 8:249

First Online: 24 July 2007Received: 13 November 2006Accepted: 24 July 2007

Abstract

BackgroundThe sequencing of the D.melanogaster genome revealed an unexpected small number of genes ~ 14,000 indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1-3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags ORESTES methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome.

ResultsBioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version 4.3. Of these, a set of 38 was analysed by polyA northern blot hybridization, validating 17 50% new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury.

ConclusionUsing the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-8-249 contains supplementary material, which is available to authorized users.

Rafaela M Maia, Valeria Valente contributed equally to this work.

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Autor: Rafaela M Maia - Valeria Valente - Marco AV Cunha - Josane F Sousa - Daniela D Araujo - Wilson A SilvaJr - Marco A Za

Fuente: https://link.springer.com/article/10.1186/1471-2164-8-249







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