Dataset Information

Expression profile of the first 24 hrs of Drosophila embryonic development using tiling arrays

ABSTRACT: Many animal and plant genomes are transcribed much more extensively than current annotations predict. However, the biological function of these unannotated transcribed regions is largely unknown. To begin to address this question, we have generated a series of transcription maps at an interrogation resolution of 35 base pairs (bp) across the Drosophila genome for the first 24 hrs of development in 2 hour increments, similar to maps we have created for regions of the human genome. Using a single tiling array, 3,075,693 probe pairs of twenty-five nucleotides (nts) in length were used to interrogate 105,897,358 non-repeat bp of the fly genome. Maps of the sites of transcription were made using total RNA greater than 200 nts. Approximately 7% and 23% of the detected transcribed nucleotides during D. melanogaster embryogenesis map to unannotated intergenic and intronic regions, respectively. Based on computational analysis of coordinated transcription, we conservatively estimate that 29% of all unannotated transcribed sequences function as missed or alternative exons of well-characterized protein coding genes. 15.6 % of intergenic transcribed regions function as missed or alternative transcription start sites utilized by 11.4% of the expressed protein-coding genes. Identification of P element mutations within or near newly identified 5' exons provides a strategy for mapping previously uncharacterized mutations to their respective genes. Collectively, these data indicate that at least 85% of the fly genome is transcribed and processed into mature transcripts representing at least 30% of the fly genome. Keywords: time course, Drosphila embryonic development, tiling array, expression profile Overall design: Total RNA was extracted from dechorionated whole embryos collected in 2hr intervals (0-2 hr, 2-4hr..etc)for 12 time points (first 24hr of development). RNA was then converted to cDNA using standard protocols. 3 replicate chips were used for each sample (time point).

INSTRUMENT(S): Affymetrix Drosophila Melanogaster Antisense Tiling Array

ORGANISM(S): Drosophila Melanogaster

SUBMITTER: Thomas R Gingeras

PROVIDER: GSE5514 | GEO | 2006-08-14

SECONDARY ACCESSION(S): PRJNA96067

REPOSITORIES: GEO

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Publications

Biological function of unannotated transcription during the early development of Drosophila melanogaster.

Manak J Robert JR Dike Sujit S Sementchenko Victor V Kapranov Philipp P Biemar Frederic F Long Jeff J Cheng Jill J Bell Ian I Ghosh Srinka S Piccolboni Antonio A Gingeras Thomas R TR

Nature genetics 20060903 10

Many animal and plant genomes are transcribed much more extensively than current annotations predict. However, the biological function of these unannotated transcribed regions is largely unknown. Approximately 7% and 23% of the detected transcribed nucleotides during D. melanogaster embryogenesis map to unannotated intergenic and intronic regions, respectively. Based on computational analysis of coordinated transcription, we conservatively estimate that 29% of all unannotated transcribed sequenc ...[more]

PMID: 16951679

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Project description:BACKGROUND: The 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. RESULTS: Bioinformatic 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. CONCLUSION: Using 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.