- Email: [email protected]
2.P2. Molecular cloning and expression pattern of elav-like genes from silkmoth, Bombyx mori
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Abstracts / Comparative Biochemistry and Physiology, Part A 148 (2007) S7–S12
sprouting in the hippocampus and hippocampal slice cultures of animals (Scorza et al., 2003; Thomas et al., 2005). Pirocarpine-treated slice cultures have been widely used as an in vitro model of morphological changes observed in epileptic brains. Pilocarpine increases the firing rate of hippocampal neurons. Recently Koyama et al. (2004) have reported enhanced mossy fiber sprouting in picrotoxin-treated hippocampus. In these studies, mossy fibers in the hippocampal dentate gyrus have been observed with Neo-Timm staining after chemical fixation of the tissue. Therefore, the time course of the morphological changes of mossy fibers induced by pilocarpine has not been examined in detail. In this study we found that the neurites of GFP transfected neurons in the rat hippocampal dentate gyrus were transiently retracted and followed by extension when pilocarpine was applied; the retraction peaked at approximately 12 h and the elongation started at approximately 24 h after the drug application. A detailed analysis of 29 cases showed that neurites retracted toward the soma along the path of the original neuritis, the neurites started to regrow after the retraction, they almost restored their original shape during regrowing processes, and often extended more than the original length. doi:10.1016/j.cbpa.2007.06.022
2.P2. Molecular cloning and expression pattern of elav-like genes from silkmoth, Bombyx mori Sakurai, T., Yamagata, T., Uchino, K., Sezutsu, H., Tamura, T., and Kanzaki, R. Research Center for Advanced Science and The University of Tokyo, Japan [email protected] Drosophila elav gene encodes neuron-specific RNA binding protein, and is expressed in all neurons. Using this specific gene expression pattern, elav and its orthologs are used as a pan-neuronal marker in Drosophila and other species. In this study, to identify a neuronal marker gene in the silkmoth, Bombyx mori, we cloned the elav-like genes in B. mori. The silkworm genome database searches led to the discovery of several sequencings that showed similarity with Drosophila elav. Of these, two genes with the highest similarity were isolated from adult brain by RT-PCR, and named Bmelav1 and Bmelav2. Bmelav1 and Bmelav2 encode 353 and 358 amino acids protein, respectively, and both contain three RNA binding motifs characteristic to the member of elav family. Molecular phylogenetic analysis showed that Bmelav1 and Bmelav2 form an isolated cluster with Drosophila elav genes among the known elav gene family. RT-PCR analysis revealed that the Bmelav1 mRNA expression was restricted to brain and neural ganglions, while Bmelav2 was
expressed in testis in addition to brain and neural ganglions. These results suggest that Bmelav1 encodes a neuron-specific elav homolog in B. mori. We are currently examining the Bmelav1 expression pattern in the brain by in situ hybridization. In addition, construction of transgenic moths that express GFP under the control of putative Bmelav1 promoter is ongoing. This work was supported by JSPS (Scientific Research (B) 18370028). doi:10.1016/j.cbpa.2007.06.023
2.P3. Cloning and gene expression of two novel stanniocalcin genes in the puffer fish Tetraodon nigroviridis Schein, V., Pinto, P., and Canário, A.V.M. Molecular and Comparative Endocrinology, Centre of Marine Science, CCMAR, Portugal [email protected] Stanniocalcin 1 (STC1) is a hypocalcemic hormone first described in fishes. Recently a second gene (STC2) was isolated from mammals and fishes. However, in silico analysis of Tetraodon nigroviridis and Takifugu rubripes genomes revealed the presence of a third and a fourth gene. In this study STC1 was cloned from de Corpuscles of Stannius (CS), STC2 from the muscle, and the two novel genes identified, STC1B and STC2B from the brain of Tetraodon. The STC-1B gene encodes an ORF of 287aa and contains 11 conserved cyteine residues and the Nlinked glycosylation site, present in all identified STC1. The predicted sequence protein share 65% sequence homology with Tetraodon STC1 and 67% with human STC1. In contrast with Tetraodon STC2, which contain 297 aa, 14 cysteine residues and the conserved N-linked glycosylation site, STC2-B contain an ORF of 426 aa, 20 cysteine residues and four glycosylation sites. The analysis of STC2-B reveals Tetraodon STC2B share 63% of homology with Fugu STC2B and only 32% with hSTC2. STC1-B gene expression in Tetraodon was found in the kidney, gills and notochord, in contrast with STC1, only expressed in the CS. STC2 was expressed in liver, kidney, gills, midgut, brain, pituitary, gonad, notochord, heart and bone. However, more then one transcript for STC2B was detected in the Tetraodon tissues, namely, in neural and bone tissues. In conclusion, the phylogenetic analyses suggest that STC1 and STC2 were duplicated in fishes and our findings raise novel questions about the understanding of STC functions in fishes. Acknowledgements: The study was made possible with grants support from the Fundação para Ciência e Tecnologia of Portugal (VS*: SFRH/BPD/ 25353/2005; PP*: SFRH/BPD/25247/2005). doi:10.1016/j.cbpa.2007.06.024
Abstracts / Comparative Biochemistry and Physiology, Part A 148 (2007) S7–S12
sprouting in the hippocampus and hippocampal slice cultures of animals (Scorza et al., 2003; Thomas et al., 2005). Pirocarpine-treated slice cultures have been widely used as an in vitro model of morphological changes observed in epileptic brains. Pilocarpine increases the firing rate of hippocampal neurons. Recently Koyama et al. (2004) have reported enhanced mossy fiber sprouting in picrotoxin-treated hippocampus. In these studies, mossy fibers in the hippocampal dentate gyrus have been observed with Neo-Timm staining after chemical fixation of the tissue. Therefore, the time course of the morphological changes of mossy fibers induced by pilocarpine has not been examined in detail. In this study we found that the neurites of GFP transfected neurons in the rat hippocampal dentate gyrus were transiently retracted and followed by extension when pilocarpine was applied; the retraction peaked at approximately 12 h and the elongation started at approximately 24 h after the drug application. A detailed analysis of 29 cases showed that neurites retracted toward the soma along the path of the original neuritis, the neurites started to regrow after the retraction, they almost restored their original shape during regrowing processes, and often extended more than the original length. doi:10.1016/j.cbpa.2007.06.022
2.P2. Molecular cloning and expression pattern of elav-like genes from silkmoth, Bombyx mori Sakurai, T., Yamagata, T., Uchino, K., Sezutsu, H., Tamura, T., and Kanzaki, R. Research Center for Advanced Science and The University of Tokyo, Japan [email protected] Drosophila elav gene encodes neuron-specific RNA binding protein, and is expressed in all neurons. Using this specific gene expression pattern, elav and its orthologs are used as a pan-neuronal marker in Drosophila and other species. In this study, to identify a neuronal marker gene in the silkmoth, Bombyx mori, we cloned the elav-like genes in B. mori. The silkworm genome database searches led to the discovery of several sequencings that showed similarity with Drosophila elav. Of these, two genes with the highest similarity were isolated from adult brain by RT-PCR, and named Bmelav1 and Bmelav2. Bmelav1 and Bmelav2 encode 353 and 358 amino acids protein, respectively, and both contain three RNA binding motifs characteristic to the member of elav family. Molecular phylogenetic analysis showed that Bmelav1 and Bmelav2 form an isolated cluster with Drosophila elav genes among the known elav gene family. RT-PCR analysis revealed that the Bmelav1 mRNA expression was restricted to brain and neural ganglions, while Bmelav2 was
expressed in testis in addition to brain and neural ganglions. These results suggest that Bmelav1 encodes a neuron-specific elav homolog in B. mori. We are currently examining the Bmelav1 expression pattern in the brain by in situ hybridization. In addition, construction of transgenic moths that express GFP under the control of putative Bmelav1 promoter is ongoing. This work was supported by JSPS (Scientific Research (B) 18370028). doi:10.1016/j.cbpa.2007.06.023
2.P3. Cloning and gene expression of two novel stanniocalcin genes in the puffer fish Tetraodon nigroviridis Schein, V., Pinto, P., and Canário, A.V.M. Molecular and Comparative Endocrinology, Centre of Marine Science, CCMAR, Portugal [email protected] Stanniocalcin 1 (STC1) is a hypocalcemic hormone first described in fishes. Recently a second gene (STC2) was isolated from mammals and fishes. However, in silico analysis of Tetraodon nigroviridis and Takifugu rubripes genomes revealed the presence of a third and a fourth gene. In this study STC1 was cloned from de Corpuscles of Stannius (CS), STC2 from the muscle, and the two novel genes identified, STC1B and STC2B from the brain of Tetraodon. The STC-1B gene encodes an ORF of 287aa and contains 11 conserved cyteine residues and the Nlinked glycosylation site, present in all identified STC1. The predicted sequence protein share 65% sequence homology with Tetraodon STC1 and 67% with human STC1. In contrast with Tetraodon STC2, which contain 297 aa, 14 cysteine residues and the conserved N-linked glycosylation site, STC2-B contain an ORF of 426 aa, 20 cysteine residues and four glycosylation sites. The analysis of STC2-B reveals Tetraodon STC2B share 63% of homology with Fugu STC2B and only 32% with hSTC2. STC1-B gene expression in Tetraodon was found in the kidney, gills and notochord, in contrast with STC1, only expressed in the CS. STC2 was expressed in liver, kidney, gills, midgut, brain, pituitary, gonad, notochord, heart and bone. However, more then one transcript for STC2B was detected in the Tetraodon tissues, namely, in neural and bone tissues. In conclusion, the phylogenetic analyses suggest that STC1 and STC2 were duplicated in fishes and our findings raise novel questions about the understanding of STC functions in fishes. Acknowledgements: The study was made possible with grants support from the Fundação para Ciência e Tecnologia of Portugal (VS*: SFRH/BPD/ 25353/2005; PP*: SFRH/BPD/25247/2005). doi:10.1016/j.cbpa.2007.06.024