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First published online August 23, 2004
Journal of Experimental Biology 207, 3361-3368 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01165

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The 14-3-3 proteins in the teleost fish rainbow trout (Oncorhynchus mykiss)

Heikki Koskinen¹, Aleksei Krasnov^1,*, Caird Rexroad², Yuri Gorodilov³, Sergey Afanasyev⁴ and Hannu Mölsä¹

¹ Institute of Applied Biotechnology, University of Kuopio, POB 1627, Kuopio 70211, Finland,
² National Center for Cool and Cold Water Aquaculture, USDA-ARS, 11861 Leetown Road, Kearneysville, WV 25430, USA,
³ Biological Institute, University of Sanct Petersburg, Oranienbaum Chaussee 2, Stary Peterhof, Sanct Petersburg 198504, Russia
⁴ Sechenov Institute of Evolutionary Physiology and Biochemistry, M. Toreza av. 44, Petersburg 194223, Russia

View larger version (126K): [in a new window]   Fig. 1. Structure of rainbow trout 14-3-3 proteins. The sequences were aligned with ClustalW and the conserved amino acids were highlighted with Boxshade.

View larger version (16K): [in a new window]   Fig. 2. Cladogram of vertebrate 14-3-3 proteins. Protein distant matrix was computed and the tree was constructed using the neighbor-joining method. The bootstrap values for 1000 replicates are indicated at the nodes of clades that include rainbow trout proteins. Accession numbers of proteins used in phylogenetic analyses are given in Tables S2, S3 (supplementary material).

View larger version (19K): [in a new window]   Fig. 3. (A) Expression of Omy14-3-3 genes was analyzed with RTPCR in rainbow trout spleen (sp), kidney (k), intestine (i), liver (l), gill (g), skin (sk), heart (h), skeletal muscle (sm), ovary (o), testis (t) and brain (b). In each sample, RNA was pooled from three individuals and analyses were repeated twice. (B) The gel picture presents differential expression of duplicated Omy14-3-3 genes.

View larger version (19K): [in a new window]   Fig. 4. (A) Expression of Omy14-3-3 genes in rainbow trout embryos was determined with RT-PCR at different developmental stages: blastula (b), early gastrula (eg), late gastrula (eg), 15 somites (15 sp) and 34 somites (34 sp). In each sample, RNA was pooled from 10 embryos and analyses were repeated twice. (B) The gel picture presents differential expression of duplicated Omy14-3-3 genes.

View larger version (36K): [in a new window]   Fig. 5. Duplicated Omy14-3-3 genes were compared by similarity of expression profiles in 31 microarray experiments. The distances were determined with multidimensional scaling.

View larger version (37K): [in a new window]   Fig. 6. Expression of Omy14-3-3 genes in the brain of stressed rainbow trout. One-year-old fish were stressed by netting (2 min) and confinement (20 min) and this treatment was repeated over 5 days. The brain samples were collected 1, 3 and 5 days after the first stress exposure. In each sample, RNA was pooled from four individuals. A dye-swap design was used for hybridization, and expression of genes was measured in 12 replicates.

View larger version (75K): [in a new window]   Fig. 7. Expression of Omy14-3-3B1 was analyzed in somitic (A,B) and postsomitic (C,D) rainbow trout embryos with in situ hybridization. 15 embryos were analyzed at each developmental stage, and representative examples are shown. Transcripts were detected in the neural crest (nc), eyes (e), yolk syncytium layer (ysl), caudal somites (s), tail bud (tb), gill covers (gc), gill arches (ga) and pectoral fins (pf). The areas of active expression are shown at higher magnification in B and D.