In silico Analysis of 2085 Clones from a Normalized Rat Vestibular Periphery 3′ cDNA Library

Abstract

The inserts from 2400 cDNA clones isolated from a normalized Rattus norvegicus vestibular periphery cDNA library were sequenced and characterized. The Wackym-Soares vestibular 3′ cDNA library was constructed from the saccular and utricular maculae, the ampullae of all three semicircular canals and Scarpa’s ganglia containing the somata of the primary afferent neurons, microdissected from 104 male and female rats. The inserts from 2400 randomly selected clones were sequenced from the 5′ end. Each sequence was analyzed using the BLAST algorithm compared to the Genbank nonredundant, rat genome, mouse genome and human genome databases to search for high homology alignments. Of the initial 2400 clones, 315 (13%) were found to be of poor quality and did not yield useful information, and therefore were eliminated from the analysis. Of the remaining 2085 sequences, 918 (44%) were found to represent 758 unique genes having useful annotations that were identified in databases within the public domain or in the published literature; these sequences were designated as known characterized sequences. 1141 sequences (55%) aligned with 1011 unique sequences had no useful annotations and were designated as known but uncharacterized sequences. Of the remaining 26 sequences (1%), 24 aligned with rat genomic sequences, but none matched previously described rat expressed sequence tags or mRNAs. No significant alignment to the rat or human genomic sequences could be found for the remaining 2 sequences. Of the 2085 sequences analyzed, 86% were singletons. The known, characterized sequences were analyzed with the FatiGO online data-mining tool (http://fatigo.bioinfo.cnio.es/) to identify level 5 biological process gene ontology (GO) terms for each alignment and to group alignments with similar or identical GO terms. Numerous genes were identified that have not been previously shown to be expressed in the vestibular system. Further characterization of the novel cDNA sequences may lead to the identification of genes with vestibular-specific functions. Continued analysis of the rat vestibular periphery transcriptome should provide new insights into vestibular function and generate new hypotheses. Physiological studies are necessary to further elucidate the roles of the identified genes and novel sequences in vestibular function.

Glucose transporter 5 is undetectable in outer hair cells and does not contribute to cochlear amplification

“…In support, we found that Glut5 is absent in all accessible cDNA libraries constructed from various tissues (inner ear, cochlea, otocyst, organ of Corti, or hair cells) from several species including mouse and analyzed by various groups (dbEST ID: 18222; 9974; 11139; 10920; 4088; 14415; 16641) [6,23,27,[31][32][33][34]41]. Based on cochlear RT-PCR results, however, Glut5 mRNA is present in other cochlear cells of wildtype mice, although the cochlear cDNA used could have been contaminated with blood cells.…”

Drosophila Auditory Organ Genes and Genetic Hearing Defects

“…The same library also includes ZSCAN22, the homolog of gl, mutations in which impair JO function. DYX1C1, the homolog of the newly defined Drosophila deafness gene CG14921, in turn, is present in the Wackym-Soares normalized rat vestibular cDNA library (Roche et al, 2005; NCBI, library dbEST 16641), which also includes Ank2 and the Arr2 homolog Arrb1. DYX1C1 is also expressed in the zebrafish otic vesicle (Thisse and Thisse, 2004), as is zgc:63660, the zebrafish homolog of the Drosophila deafness gene CG11253.…”

Analysis and functional annotation of expressed sequence tags from in vitro cell lines of elasmobranchs: Spiny dogfish shark (Squalus acanthias) and little skate (Leucoraja erinacea)

“…The field of gene ontology attempts to make available descriptions of gene products from different databases in a manageable, reliable, classified and organized vocabulary, allowing a prediction of gene function (Ashburner et al, 2000; Roche et al, 2005). GO terms are represented in three ontologies: Biological Processes, Cellular Components and Molecular Functions.…”

“…Each classification can be organized as a directed acyclic graph. Use of GO terms allows uniformity in association of gene products identified in EST analysis independent of species, and is the most common approach to practical categorization of gene products (Ashburner et al, 2000; Conesa et al, 2005; Roche et al, 2005, Botton et al, 2008). …”