Transcribed Tc1-like transposons in salmonid fish
© Krasnov et al; licensee BioMed Central Ltd. 2005
Received: 31 January 2005
Accepted: 12 August 2005
Published: 12 August 2005
Mobile genetic elements comprise a substantial fraction of vertebrate genomes. These genes are considered to be deleterious, and in vertebrates they are usually inactive. High throughput sequencing of salmonid fish cDNA libraries has revealed a large number of transposons, which remain transcribed despite inactivation of translation. This article reports on the structure and potential role of these genes.
A search of EST showed the ratio of transcribed transposons in salmonid fish (i.e., 0.5% of all unique cDNA sequences) to be 2.4–32 times greater than in other vertebrate species, and 68% of these genes belonged to the Tc1-family of DNA transposons. A phylogenetic analysis of reading frames indicate repeated transposition of distantly related genes into the fish genome over protracted intervals of evolutionary time. Several copies of two new DNA transposons were cloned. These copies showed relatively little divergence (11.4% and 1.9%). The latter gene was transcribed at a high level in rainbow trout tissues, and was present in genomes of many phylogenetically remote fish species. A comparison of synonymous and non-synonymous divergence revealed remnants of divergent evolution in the younger gene, while the older gene evolved in a neutral mode. From a 1.2 MB fragment of genomic DNA, the salmonid genome contains approximately 105 Tc1-like sequences, the major fraction of which is not transcribed. Our microarray studies showed that transcription of rainbow trout transposons is activated by external stimuli, such as toxicity, stress and bacterial antigens. The expression profiles of Tc1-like transposons gave a strong correlation (r2 = 0.63–0.88) with a group of genes implicated in defense response, signal transduction and regulation of transcription.
Salmonid genomes contain a large quantity of transcribed mobile genetic elements. Divergent or neutral evolution within genomes and lateral transmission can account for the diversity and sustained persistence of Tc1-like transposons in lower vertebrates. A small part of transposons remain transcribed and their transcription is enhanced by responses to acute conditions.
A large fraction of repetitive sequences originate in eukaryotic genomes from mobile genetic elements (MGEs), which are grouped into 2 classes. Class I transposons require mRNA intermediates, whereas class II elements transpose directly as DNA. Tc1-like class II transposons, named after the founder gene in Caenorhabditis elegans, are probably the most widespread MGEs in nature, and are found in fungi, plant ciliates, nematodes, arthropods, fish, amphibians and mammals (reviewed in ). These genes contain a single reading frame that encodes for the enzyme transposase, which is flanked with terminal inverted repeating units. Transposition of class II MGEs is characterized by limited requirements for host cellular factors, which can account for their remarkable ability to undergo horizontal transfer across great taxonomic distances . MGEs are regarded as parasitic genes, and proliferation is deleterious for the host. Therefore, transposition is commonly followed by inactivation. MGEs could play an important role in the evolution of teleost fish, and comprise a substantial fraction of their genome. Multiple copies of Tc1-like transposons were found in several fish species from different orders [3–6], however transcription of teleost Tc1-like genes has not been documented. Recent high-throughput sequencing of salmonid cDNA libraries has revealed surprisingly large number of transposon transcripts. Most if not all these sequences contain incapacitating mutations in the reading frames, and can be regarded as transcribed pseudogenes or null-alleles. At present, rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) TIGR Gene Indices  contain 50773 and 31341 unique cDNA sequences, respectively among which we found several hundreds MGE, Tc1-like genes being most abundant. This wealth of sequence information provides insight into the structure and evolution of transposons. We also cloned several copies of two rainbow trout Tc1-like genes with complete reading frames, which adds to understanding the transposon life cycle. Multiple gene expression analyses with high-density cDNA microarray indicate stimulation of rainbow trout transposons transcription in response to stress, toxicity and pathogens.
Transcribed vertebrate transposons1.
Rainbow trout (Oncorhynchus mykiss)
Atlantic salmon (Salmo salar)
Medaka (Oryzias latipes)
Zebrafish (Danio rerio)
Killifish (Fundulus heteroclitus)
Pufferfish (Takifugu rubripes)
African clawed frog (Xenopus laevis)
Chicken (Gallus gallus)
Rat (Rattus norvegicus)
Presence of Glan in genomic DNA of fish from inland waters of Finland.
Arctic charr (Salvelinus alpinus)
Brown trout (Salmo trutta)
Smelt (Osmerus eperlanus)
Grayling (Thymallus thymallus)
Vendace (Coregonus albula)
Whitefish (Coregonus lavaretus)
Pikepearch (Sander lucioperca)
Perch (Perca fluviatilis)
Ruffe (Gymnocephalus cernuus)
Crucian carp (Carassius carassius)
Roach (Rutilus rutilus)
Bullhead (Cottus gobio)
Bream (Abramis brama)
Silver bream (Abramis bjoerkna)
Bleak (Alburnus alburnus)
Dace (Leuciscus leuciscus)
Rudd (Scardinius erythrophthalmus)
Ide (Leuciscus idus)
Burbot (Lota lota)
Northern pike (Esox lucius)
Eel (Anguilla anguilla)
Synonymous (Ks) and non-synonymous (Ka) divergences of the rainbow trout transposons Glan and Barb. Plaice transposase was used as a reference.
Rainbow trout (3 copies)
0.016 ± 0.007
0.022 ± 0.009
0.69 ± 0.05
0.69 ± 0.02
0.14 ± 0.01
4.85 ± 0.31
Rainbow trout (4 copies)
0.12 ± 0.02
0.11 ± 0.01
0.99 ± 0.13
0.68 ± 0.02
0.20 ± 0.00
3.35 ± 0.04
Over-presentation of Gene Ontology classes in a list of genes that showed co-ordinated expression with Tc1-like transposons. The composition of microarray was used as a reference. The gene names and expression profiles are shown in Figure 4.
Genes in list
Genes on chip
Antimicrobial humoral response
1 × 10-5
2 × 10-4
Cellular defense response
Response to oxidative stress
Transcription regulator activity
Having a large number of transposons and a preponderance of Tc1-like genes is a characteristic feature of salmonid genomes [3–5]. Sequence analysis of the transcribed genes (Figure 1) suggested repeated transpositions at protracted intervals. A wide distribution of Tc1 transposons is believed to account for the limited requirements in the host cellular factors. Sleeping Beauty, an artificially reconstructed salmon transposon  is capable of integration into genomes of a wide range of vertebrate species, however different efficiencies observed in various cell lines point to possible involvement of the recipient's proteins in transposition . This is in line with a wide, though limited, distribution of homologs for the transcribed salmonid DNA transposons, which have not been found among EST of warm-blood vertebrates. The variety of salmonid Tc1-like genes is truly remarkable. Phylogenetic analyses of 38 sequences, encoding homologous fragments of C-termini, found 14 distinct types of Tc1-like genes and the real number of different genes is probably much greater. Our search was based on the similarity between proteins that were available from Swissprot, and many transposons could remain unidentified due to the lack of known homologs. Furthermore, the rapid decay of transposons could impede the discovery of ancient transposed genes.
Despite the wide spread occurrence of Tc1-like transposons in vertebrates, not a single active gene has been identified to date . Inactivation of salmonid DNA transposons could take place within a relatively short period of time after transmission. Cloning of 2 transposons having a relatively low divergence rate indicates the rapid accumulation of incapacitating mutations, such as insertions or deletions, shifts of reading frames and premature stop codons (Figure 2). Analysis of synonymous and non-synonymous substitutions suggest that inactivation of younger transposon could be preceded by selective divergence within a limited period of time, whilst evolution of the older gene appeared entirely neutral. Results from a study on recent transpositions in insects from four different orders suggest that selective constraints operate exclusively by horizontal gene transfer . A comparison of rainbow trout genes with Tc1-like transposon from plaice confirm the conservation of functionally important domains in distantly related proteins, which is gradually obscured during the course of neutral evolution (Ks/Ka ratios in the younger Glan and older Barb genes are 4.85 ± 0.31 and 3.35 ± 0.04 respectively).
Silencing of transposons takes place at the transcriptional or post-transcriptional levels , and both of these mechanisms could act in salmonid fish. Based on frequency in a 1.2 MB gene fragment, we can assume that Tc1-like genes comprise nearly 5% of the Atlantic salmon genome and only a minor fraction preserved transcription after inactivation of translation. A survey of salmonid EST found untranslated transposons in both sense and anti-sense polarities, which is the main prerequisite for the formation of double-stranded RNA. RNA interference (RNAi) is implicated in the control of transposition in germ cell lines of the nematode C. elegans , and existence of an RNAi pathway in rainbow trout was recently demonstrated . Suppression of intact transposases with mutant genes was also reported in insects, and this control mechanism is referred to as dominant-negative complementation .
Given efficient protection against transposition in animals, the tenacity and variety of transposons may seem surprising. Sustained persistence of transposons can, in theory, account for their residence in unknown reservoir species; e.g, the role of parasites as potential vectors of horizontal transfer across phylogenetically remote organisms has been hypothesized . However this can hardly explain the remarkable diversity of these genes. The ML tree (Figure 1) suggests that at each transposition event, the rainbow trout genome was invaded with a new transposon, although several genes could have a common ancestor. If expression of translated genes is under control of RNAi, successful recurring transposition of identical or highly similar genes appears unlikely. Hence, the combination of neutral or divergent evolution within a genome with transfer across phylogenetic boundaries can be the most efficient strategy for the survival and diversification of transposons. PCR screen detected Glan in genomes of many fish species from phylogenetically remote taxonomic groups (Table 2). Clades I-VII of the ML tree (Figure 1) can correspond to genes that evolved independently. However it is also possible that descendants of a founder gene has returned several times into the rainbow trout genome, after passage through a chain of co-evolutionary hosts.
Results of our microarray studies suggested that a large fraction of transcribed Tc1-genes can be stimulated under acute conditions, but it remains unclear whether or not the transposon transcripts have any functional importance. In theory, they can be transcribed from cryptic promoters, which are activated by the remodelling of chromatin. However, input from stress-responsive promoters is also plausible. Transcripts can be required for the control of transposition through RNAi, however such explanation appears unlikely for highly mutated genes that were probably silenced long ago in evolutionary time. Currently, there is a growing body of evidence to support the involvement of non-coding RNA into the regulation of gene expression at different levels. The role of small and large RNA in modification of the chromatin structure was reviewed recently [21–23]. Stress-induced transcription of short interspersed repeated sequences (SINE) was reported in human, mouse and silkworm [24–27]; and SINE transcripts were shown to enhance translation of reporter genes [28, 29]. Stress also activates the transcription of satellite III repeat . Because this large non-coding RNA is consistently associated with chromatin, it can be required for the protection of sensitive regions from stress-induced damage. Synthetic double-stranded RNA enhances the expression of anti-viral proteins in salmonid fish [31, 32] and, in theory, endogenous dsRNA can mimic a viral infection by launching protective reactions.
Tc1-like transposons are co-regulated with a group of genes that are implicated in the defense response, signal transduction and regulation of transcription. In this respect, it is noteworthy to mention that Tc1-like fragments reside in a number of immune and stress-related salmonid genes, such as the non-classical MHC class I antigen [Genbank:AF091779, Genbank:AF091780], immunoglobulin heavy chain, IgD [Genbank:AF141605, Genbank:AF278717], inducible nitric oxide synthase iNOS/NOS2 [Genbank:AJ295231] and aryl hydrocarbon receptor 2b, AhR2 [Genbank:AY463929]. Multiple copies of Glan in sense and anti-sense polarity are found in rainbow trout MHC class Ia [Genbank: AB162342.1] and b [AB162343.1] regions, in the vicinity of genes encoding the complement proteasome subunit and several MHCI loci. Modulation of gene expression that was due to the insertion of transposons has been documented in many studies (reviewed in ), and involvement of dispersed repeated sequences into the co-ordination of gene expression with similar functions was hypothesized more than three decades ago . The role of transposon transcripts in the regulation of gene expression was recently discovered in yeast , where the induction of an RNAi-dependent silent chromatin configuration resulted in reduced transcription of several meiotic genes. A possible involvement of transposon transcripts in the regulation of gene expression in salmonid fish remains to be studied.
Information produced by the sequencing of salmonid fish cDNA libraries and identification of recently transmitted transposons provide new insights into the structure, diversity and molecular evolution and life cycle of mobile genetic elements. High expression levels in rainbow trout tissues and marked responses to external stimuli indicate potential functional roles of transposon pseudogenes, which requires further investigation. These genes can be used as sensitive molecular biomarkers of acute conditions in salmonid fish.
The expressed transposons were analysed in rainbow trout and Atlantic salmon TIGR Gene indices, and sequence comparison was conduced with stand-alone blast . Multiple sequence alignments were performed with ClustalW  and the conserved protein domains were searched in Interpro . Synonymous and non-synonymous substitutions in newly cloned genes were determined by Dnasp . Maximum Likelihood (ML) phylogenetic analyses were performed with Phylip .
The conserved sequence in the untranslated regions of rainbow trout Tc1-like transposases were inferred from EST sequences. RNA was extracted from rainbow trout brain and treated with Rnase-free Dnase (Promega). Reverse transcription with SuperScriptIII (Invitrogen) was primed with oligo(dT). PCR was performed with primer 5'-ATACAGTGCCTTGCGAGAGTATTC-3' using a TripleMaster kit (Eppendorf), and the product was cloned into pcDNA3.1/V5-His-TOPO (Invitrogen). Seven of nine sequenced clones contained complete reading frames.
PCR analyses of genomic DNA
The fish samples were collected from inland reservoirs in Finland, and DNA from fin clips was prepared with salt extraction . In brief, fin samples were digested at 60°C in 440 μl of buffer (1.8 mM EDTA, 9 mM Tris-HCl, pH 8; 1.8% SDS) containing 160 μg of proteinase K. After addition of 300 μl of 6 M NaCl, lysates were centrifuged at 12,000 g for 30 min. DNA in the supernatants was precipitated with isopropanol, washed with 70% aqueous ethanol and dissolved in water. The 654-base fragments of Glan PCR were amplified using the Hot Master Taq kit (Eppendorf). Primers (5'-TGAAGAATCGACAACAAGTGGGACA-3' and 5'-GCTTTCTTCTTGCCACTCTTCCATA-3') were annealed to templates at 68°C.
Fish experiments, design of the rainbow trout cDNA microarray, hybridization protocol and data analyses are described in detail elsewhere [10, 11]. In brief, the platform included 1,300 genes printed in 6 replicates each. The dye swap design was used; each sample containing RNA from 4 individuals was hybridized to slides with reverse assignment of fluorescent dyes (Cy3- and Cy5-dCTP from Amersham Pharmacia). Labels were incorporated at the stage of cDNA synthesis. The measurements in spots were filtered by criteria I/B ≥ 3 and (I-B)/(S I +S B ) ≥ 0.6, where I and B were the mean signal and background intensities, respectively, and SI,S B were the standard deviations. Lowess normalization was performed and differential expression was analysed with the Student's t-test (p < 0.01). The genes were ranked by the log(p-level).
- MGE –:
mobile genetic element
- ML –:
This study was supported by the National Agency of Technology, Finland. We wish to thank Rolf Sara for preparation of the microarrays and Miia Antikainen for assistance in cloning the rainbow trout transposons.
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