The 14-3-3 proteins in the teleost fish rainbow trout (Oncorhynchus mykiss)
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
* Author for correspondence (e-mail: krasnov{at}uku.fi)
Accepted 28 June 2004
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Summary |
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Key words: 14-3-3 proteins, rainbow trout, Oncorhynchus mykiss, duplicated genes, phylogeny, expression.
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Introduction |
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Taking into account remarkable structural conservation of the
14-3-3 genes (human and yeast proteins share 70% similarity), the
existence of a large number of isoforms may seem surprising. Thirteen
14-3-3 genes were found in the genome of Arabidopsis, and
expression of 12 isoforms has been confirmed
(Ferl et al., 2002). In
mammals, there are seven distinct 14-3-3 proteins. One gene belongs to the
epsilon type, which is common to plants and animals; the others are denoted as
sigma, gamma, eta, beta, zeta and tau. Are the functions of the
14-3-3 gene products redundant or does each gene have a unique
function? Microarray analyses and sequencing of cDNA libraries showed that
most human tissues harbor several or all of the 14-3-3 proteins, which do not
have any marked tissue specificity [expression profiles are deposited in the
GeneCards database
(http://bioinfo.weizmann.ac.il/cards/)].
In most in vitro studies, 14-3-3 proteins bound ligands with similar
affinities. Although several client proteins showed preferential interaction
with some of the 14-3-3 isoforms, the biological relevance of subtle
differences in affinities remains unknown (reviewed in
Fu et al., 2000
). At present,
there is not much evidence for the functional divergence of animal 14-3-3
isoforms. Studies of lower vertebrate 14-3-3 proteins are important for
understanding of evolution and structural and functional diversification of
this gene family. To date only three non-mammalian vertebrate 14-3-3
genes have been characterized. Two genes were identified in Xenopus
laevis tadpoles (Kousteni et al.,
1997
; Kumagai et al.,
1998
) and one 14-3-3 gene was characterized in the
teleost fish Fundulus heteroclitus
(Kultz et al., 2001
). In the
present study, we present 10 14-3-3 genes from rainbow trout, which
are likely to be duplicates of five ancestral genes. Their structure,
evolution and expression are reported.
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Materials and methods |
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Sequence analyses
EST sequences were analyzed with stand-alone blast
(Altschul et al., 1997).
Multiple sequence alignments were performed with ClustalW
(Thompson et al., 1994
).
Synonymous and nonsynonymous substitutions in genes were determined with Dnasp
(Rozas and Rozas, 1999
).
Phylogenetic analyses were performed with Phylip
(Felsenstein, 1989
). After
alignment of protein sequences, 1000 bootstrap datasets were generated and
Dayhoff's PAM matrix was computed. The consensus trees were constructed with
the neighbor-joining method. The rainbow trout, Oncorhynchus mykiss
(Omy), and zebrafish, Danio rerio (Dr), 14-3-3
proteins were denoted with respect to the result of phylogenetic study.
Expression in embryos and tissues of juvenile rainbow trout
Expression patterns of Omy14-3-3 genes in embryos and tissues of
rainbow trout were analyzed with RT-PCR. The PCR primers (Table S1 in
supplementary material) were designed from cDNA sequences to specifically
amplify diverged fragments.
In situ hybridization
Anti-sense probes were prepared for the 3'-UTRs of Omy14-3-3
genes. Plasmids were PCR amplified with gene-specific primers aligning to the
ends of protein coding sequences (Table S1 in supplementary material) and an
universal M13 primer. In vitro transcription was conducted using T7
RNA polymerase (MBI Fermentas, Vilinius, Lithuania) and DIG RNA labeling mix
(Roche Bioscience, Indianapolis, IN, USA). Samples were collected at the
40-somite stage and immediately after completion of somitogenesis. Embryos
were processed as described in Joly et al.
(1993). The probes were
detected with AP-conjugated antibodies using NBT/BCIP (nitroblue tetrazolium
chloride/5-bromo-4-chloro-3-indolyl-phosphate) substrate (Roche Bioscience).
Pictures were taken with an Olympus CK40 microscope.
Microarrays
The Omy14-3-3 genes were selected for inclusion onto a glass
microarray containing 1300 rainbow trout and Baltic salmon (Salmo
salar) genes, and each clone was spotted in six replicates. Multiple gene
expression profiling was performed in experiments that addressed genomic
response to stress, toxicity, bacterial antigens and adaptation to cold. RNA
was extracted with Trizol reagent (Invitrogen), and four fish were pooled in
each sample. Labeling with Cy3- and Cy5-dCTP (Amersham Pharmacia, Little
Chalfont, UK) was made using SuperScript III reverse transcriptase
(Invitrogen) and oligo(dT) primer; cDNA was purified with Microcon YM30
(Millipore, Bedford, MA, USA). We used a dye-swap experimental design
(Kerr and Churchill, 2001).
Each sample was hybridized to two microarrays. For the first slide, test and
control cDNA were labeled with, respectively, Cy5 and Cy3. For the second
array, dye assignment was reversed. Slides were prehybridized with 1% bovine
serum albumin (BSA), fraction V, 5xSSC and 0.1% sodium dodecyl sulfate
(SDS) (30 min at 50°C) and washed with 2xSSC (3 min) and
0.2xSSC (3 min). Arrays were hybridized overnight; hybridization
cocktail contained 1.3xDenhardt's, 3xSSC, 0.3% SDS, 0.67 µg
µl-1 polyadenylate and 1.4 µg µl-1 yeast tRNA
(Sigma-Aldrich, St Louis, MO, USA). Slides were washed with 0.5xSSC,
0.1% SDS (15 min), 0.5xSSC, 0.01% SDS (15 min) and twice with
0.06xSSC (2 min). Scanning was conducted with ScanArray 5000 (Perkin
Elmer-Wallac, Turku, Finland), and images were processed with QuantArray (GSI
Luminomics, Munich, Germany). The spot measurements were filtered by criteria
I-B>3 and
(I-B)/(SI+SB)>0.6,
where I and B are the mean signal and background
intensities, respectively, and SI and
SB are their standard deviations. After subtraction of
mean background, lowess normalization was performed. Divergence of expression
profiles of 14-3-3 genes was analyzed using results of 31 microarray
experiments. For every pair, we estimated the number of genes that
significantly (P<0.05) correlated with both (Pearson r).
Then, similarity matrices were analyzed with multidimensional scaling. Genes
that significantly correlated with all 14-3-3 isoforms were
determined to characterize the stress response of the brain. Enrichment of the
functional classifications of these transcripts was observed by analysis with
Ease (Hosack et al., 2003
);
significance was determined with exact Fisher's test (P<0.05).
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Results |
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For phylogenetic analyses, we used 14-3-3 proteins of teleost fish (Fundulus and Danio) and mammals whose sequences were retrieved from Swiss-Prot (http://us.expasy.org/sprot/); the outroot was from ascidia (Ciona intestinalis). In addition to five Dr14-3-3 proteins from Swiss-Prot, five sequences were available from Ensembl (http://www.ensembl.org/) and four more were found by comparison of UniGene EST clusters (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene) with human 14-3-3 proteins using blastx (Table S2 in supplementary material). The total number of putative Dr14-3-3 genes was 14; however, two short sequences were not included in the analyses. Phylogenetic study provided additional evidence for duplication of ancestral rainbow trout genes, since 10 Omy14-3-3 proteins were divided into five separate clades (Fig. 2). Ten of 12 Dr14-3-3 genes were duplicates, and three Dr14-3-3G genes could arise as a result of two subsequent duplications. Some cyprinids are, like salmonids, tetraploid, and the possibility of ancient genome duplication in zebrafish is debated. All types of Omy14-3-3 proteins were found in zebrafish. In rainbow trout, there was no ortholog to the Fundulus (F) gene; however, this was assigned to the clade including Dr14-3-3F1 and F2. Three of six types of fish 14-3-3 proteins (E, G and B) were clustered with mammalian epsilon, gamma and beta proteins at high bootstrap values. The A, C and F types were specific for fish, whereas zeta, sigma, eta and tau 14-3-3 proteins were found exclusively in mammals. Interestingly, in four of five pairs of Omy14-3-3 proteins (B, C, G and E), one gene was assigned to a clade containing the second gene and either the mammalian or zebrafish ortholog. The 14-3-3 cladogram was markedly different from the organismic tree and this suggested rapid divergence of duplicated Omy14-3-3 genes. To verify this finding, we compared synonymous (Ks) and nonsynonymous (Ka) divergence in rainbow trout and mammalian (human and mouse) 14-3-3 genes (Table 1). The mean Ks in fish genes was 1.65 times greater than in mammalian 14-3-3 genes, whereas Ka was increased 10.5-fold.
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Expression of rainbow trout 14-3-3 genes
Expression of 14-3-3 genes in tissues of rainbow trout was
analyzed by RT-PCR. The PCR primers were designed to distinguish between the
duplicated genes; however, we were unable to separate the Omy14-3-3A1
and A2 isoforms due to their high sequence similarity. Distribution
of 14-3-3 genes was ubiquitous, and transcripts of six genes
(Omy14-3-3B1, B2, C1, C2, G1 and G2) were found in 10 of 11
analyzed tissues (Fig. 3).
Brain, ovary and testis harbored a complete set of Omy14-3-3 genes.
The number of expressed isoforms in other tissues ranked from eight (gill and
kidney) to two (skin). Expression of Omy14-3-3 genes was also studied
in embryos. Omy14-3-3B1 and B2 were expressed at all
analyzed developmental stages (Fig.
4). Three more genes (Omy14-3-3C1, C2 and A)
were detectable in blastulas. This could be due to persistence of maternal
transcripts, because expression of these genes was interrupted at the
subsequent developmental stages. Six of nine analyzed Omy14-3-3 genes
were already expressed in the late gastrulas. Expression of
Omy14-3-3E2 and C2 began at early somitogenesis (15
somites), whereas G2 was the latest isoform (34 somites).
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|
We analysed differential expression of Omy14-3-3 genes in 31
microarray experiments that dealt with response of rainbow trout to stress,
environmental pollutants and bacterial antigens. Isoforms were compared by
correlation of expression profiles with other genes presented on the slide and
the data were analysed with multi-dimensional scaling. Distance metrics were
calculated for every pair of duplicated genes and differences of expression
profiles were clearly related to the non-synonymous divergence of duplicates
(Fig. 5;
Table 1). However, it is
possible that difference of expression between the A1 and A2
isoforms was underestimated. In contrast to other Omy14-3-3 genes,
the 3'-UTR sequences of these mRNAs are highly conserved, and
cross-hybridization could affect the results of microarray analyses. We
observed tight coordination of expression of 10 isoforms in studies of stress
response in the rainbow trout brain. All Omy14-3-3 genes were
downregulated after 1 day, which was followed by a subsequent increase of
expression levels (Fig. 6). We
selected genes with significantly similar profiles (Pearson r,
P<0.05) and analyzed over-representation of Gene Ontology functional
classes (Ashburner et al.,
2000) in this group (Table
2). This result suggested that Omy14-3-3 genes could
share regulatory mechanisms with nuclear proteins, chaperones and proteins
involved in signal transduction, nucleotide binding and metabolism.
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Expression of six Omy14-3-3 genes (A1, A2, E1, E2, G1 and G2) in somitic (40 somites) and postsomitic embryos was analyzed with in situ hybridization. To separate closely related isoforms, we used the PCR-amplified 3'-UTRs as templates for preparation of probes. We were unable to find any marked difference in the expression patterns of Omy14-3-3 genes; therefore Omy14-3-3B1 is shown as an example (Fig. 7). In somitic embryos (Fig. 7A,B), transcripts were found in the neural crest, eyes, yolk syncytium, tail bud and caudal somites. Interestingly, expression of 14-3-3 genes in the tail bud and caudal somites was seen in some of the analyzed embryos. In postsomitic embryos (Fig. 7C,D), transcripts were detected in the neural crest, gill covers and gill arches and in pectoral fins; however, there was no expression in the tail and eyes.
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Discussion |
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Continuous emergence of novel 14-3-3 isoforms in the vertebrate evolution
suggested a strong tendency towards functional diversification of this
multi-gene family. This was supported by rapid divergence of rainbow trout
genes. All Omy14-3-3 genes seem to be duplicates, and at least four
of five pairs most likely appeared due to the duplication of the salmonid
genome, which took place about 50 million years ago
(Bailey et al., 1978). It is
possible that duplication of Omy14-3-3A occurred independently and
later, since sequence divergence in this pair was markedly lower than in other
genes (Fig. 1;
Table 1). In organisms of
tetraploid origin, functionally redundant duplicated genes are gradually
silenced, becoming pseudogenes in a process referred to as diploidisation.
Active genes may evolve in a neutral mode or, alternatively, they are selected
for conservation or divergence. In rainbow trout, all duplicated
14-3-3 genes are expressed at relatively high level. Phylogenetic
analysis suggested that in four of five pairs of Omy14-3-3 genes one
was closer to the mammalian or zebrafish ortholog than to the homologous gene.
The only exclusion was the least diverged Omy14-3-3A pair. A similar
result was reported by Taylor et al.
(2001
) for nine of 27 analyzed
duplicate zebrafish genes. Furthermore, nonsynonymous divergence in the
duplicated rainbow trout 14-3-3 genes was one order of magnitude
greater than in human and mouse 14-3-3 genes. Structural conservation
of orthologous 14-3-3 genes in the phylogenetically remote mammalian
species is likely to be due to their indispensable cellular functions.
Duplication of genes can relax these constraints, since one isoform is
sufficient for preservation of function whereas the second one can acquire
novel features. Activity and rapid divergence of the duplicated rainbow trout
14-3-3 genes strongly suggests that appearance of new distinct
isoforms was favored.
Rapid divergence of rainbow trout 14-3-3 proteins suggests functional
diversification of the duplicated genes. Microarray analyses in yeast showed
differential expression in most pairs with nonsynonymous divergence
(Gu et al., 2002). Recent
study of the zebrafish annexin gene family found that difference in
expression patterns of duplicated genes was closely related to the degree of
their sequence divergence (Farber et al.,
2003
). Analyses of tissue distribution and embryonic expression of
Omy14-3-3 revealed only minor differences between isoforms. Thus,
expression of Omy14-3-3C1 but not of C2 was found in the
intestine whereas the opposite was observed in skin
(Fig. 3B). One of two
Omy14-3-3B isoforms was active in skin (B1) and in muscle
(B2). In embryonic development, Omy14-3-3E1 and G1
were activated earlier than Omy14-3-3E2 and G2, respectively
(Fig. 4). Furthermore, we
compared expression profiles of Omy14-3-3 genes in microarray
experiments. Distance between isoforms was determined by numbers of genes that
showed similar expression profiles with both genes of each pair and it
decreased in the range
C>E=B>G>A. Noteworthy,
the duplicated genes were ranked in nearly the same order by nonsynonymous
divergence (Table 1). This
result indicated that structural diversification of the duplicated genes could
be due to adaptation to different client proteins.
In most microarray experiments, differential expression was shown by one or
several Omy14-3-3 isoforms. We demonstrated close concordance of
expression profiles of all 10 Omy14-3-3 genes in the brain of
stressed rainbow trout (Fig.
6), and similar responses were shown by a large group of genes,
which were significantly over-represented by nuclear proteins and proteins
involved in cell communication and signal transduction. In line with this
finding, we observed coordinated expression of all Omy14-3-3 isoforms
in parts of embryos that are known for rapid growth and differentiation.
Stable expression was seen in the neural crest, which is characterized by
exclusively high morphogenetic potential. This structure gives rise to
connective tissue, some muscle, dermal and pigmented tissues and many
structures including the cranium, branchial skeleton and sensory ganglia
(reviewed in Baker and Bronner-Fraser,
1997; Gorodilov,
2000
). We did not find tail bud expression after completion of
somitogenesis. Noteworthy, only some of the analyzed somitic embryos expressed
Omy14-3-3 genes in the tail buds, which could indicate cyclic
activity of these genes. At the subsequent stages, expression began to
decrease in most tissues and the marked differences were observed between
early and differentiated mesenchyme.
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Acknowledgments |
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Footnotes |
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