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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.

SUBMITTER: Roche JP

PROVIDER: S-EPMC1421512 | biostudies-literature | 2005 Nov-Dec

REPOSITORIES: biostudies-literature

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In silico analysis of 2085 clones from a normalized rat vestibular periphery 3' cDNA library.

Roche Joseph P JP Wackym P Ashley PA Cioffi Joseph A JA Kwitek Anne E AE Erbe Christy B CB Popper Paul P

Audiology & neuro-otology 20050805 6

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 ...[more]

PMID: 16103642

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Project description:Constitutively active background or "leak" two-pore-domain potassium (K(+)) channels (Kcnk family), as defined by lack of voltage and time dependency are central to electrical excitability of cells by controlling resting membrane potential and membrane resistance. Inhibition of these channels by several neurotransmitters, e.g. glutamate, or acetylcholine, induces membrane depolarization and subsequent action potential firing as well as increases membrane resistance amplifying responses to synaptic inputs. In contrast, their opening contributes to hyperpolarization. Because of their central role in determining cellular excitability and response to synaptic stimulation, these channels likely play a role in the differential effects of vestibular efferent neurons on afferent discharge. Microarray data from previous experiments showed Kcnk 1, 2, 3, 6, 12 and 1 5 mRNA in Scarpa's ganglia. Real-time RT-PCR showed Kcnk 1, 2, 3, 6, 12 and 15 mRNA expression in Scarpa's ganglia and Kcnk 1, 2, 3, 6, 12 but not 15 mRNA expression in the crista ampullaris. We studied the distribution of two-pore-domain potassium channels K(2P)1.1, 2.1, 3.1 and 6.1 like immunoreactivity (corresponding to Kcnk genes 1, 2, 3 and 6) in the vestibular periphery. K(2P)1.1 (TWIK 1) immunoreactivity was detected along nerve terminals, supporting cells and blood vessels of the crista ampullaris and in the cytoplasm of neurons of the Scarpa's ganglia. K(2P)2.1 (TREK 1) immunoreactivity was detected in nerve terminals and transitional cells of the crista ampullaris, in the vestibular dark cells and in neuronal fibers and somata of neurons of Scarpa's ganglia. K(2P)3.1 (TASK 1) immunoreactivity was detected in supporting cells and transitional cells of the crista ampullaris, in vestibular dark cells and in neuron cytoplasm within Scarpa's ganglia. K(2P)6.1 (TWIK 2) immunoreactivity was detected in nerve terminals, blood vessels hair cells and transitional cells of the crista ampullaris and in the somata and neuron fibers of Scarpa's ganglia.

Project description:BackgroundPlants often face multiple stresses including drought, extreme temperature, salinity, nutrition deficiency and biotic stresses during growth and development. All the stresses result in a series of physiological and metabolic reactions and then generate reversible inhibition of metabolism and growth and can cause seriously irreversible damage, even death. At each stage of cotton growth, environmental stress conditions pose devastating threats to plant growth and development, especially yield and quality. Due to the complex stress conditions and unclear molecular mechanisms of stress response, there is an urgent need to explore the mechanisms of cotton response against abiotic stresses.Methodology and principal findingsA normalized cDNA library was constructed using Gossypium barbadense Hai-7124 treated with different stress conditions (heat, cold, salt, drought, potassium and phosphorus deficit and Verticillium dahliae infection). Random sequencing of this library generated 6,047 high-quality expressed sequence tags (ESTs). The ESTs were clustered and assembled into 3,135 uniESTs, composed of 2,497 contigs and 638 singletons. The blastx results demonstrated 2,746 unigenes showing significant similarity to known genes, 74 uniESTs displaying significant similarity to genes of predicted proteins, and 315 uniESTs remain uncharacterized. Functional classification unveiled the abundance of uniESTs in binding, catalytic activity, and structural molecule activity. Annotations of the uniESTs by the plant transcription factor database (PlantTFDB) and Plant Stress Protein Database (PSPDB) disclosed that transcription factors and stress-related genes were enriched in the current library. The expression of some transcription factors and specific stress-related genes were verified by RT-PCR under various stress conditions.Conclusions/significanceAnnotation results showed that a huge number of genes respond to stress in our study, such as MYB-related, C2H2, FAR1, bHLH, bZIP, MADS, and mTERF. These results will improve our knowledge of stress tolerance in cotton. In addition, they are also helpful in discovering candidate genes related to stress tolerance. The publicly available ESTs from G. barbadense are a valuable genomic resource that will facilitate further molecular study and breeding of stress-tolerant cotton.

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In silico analysis of 2085 clones from a normalized rat vestibular periphery 3' cDNA library.

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In silico analysis of 2085 clones from a normalized rat vestibular periphery 3' cDNA library.

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