Cloning of rainbow trout (Oncorhynchus mykiss) α-actin,myosin regulatory light chain genes and the 5′-flanking region ofα-tropomyosin. Functional assessment of promoters

Cloning and characterization of fish promoters is important for gene transfer research and studies of cell differentiation and regulation of gene expression. Identification of the genomic structure of fish genes provides valuable sequence information and adds to understanding of their molecular evolution. We aimed at cloning rainbow trout regulatory sequences that direct expression of genes in skeletal muscle. To address this task, we choseα-actin, myosin light regulatory chain and tropomyosin.

Actins are highly conserved structural proteins. Being components of contractile structures and cytoskeleton, actins are distributed ubiquitously. Of three major vertebrate actin groups (α, β and γ),α-actins are specific for striated muscle. Cardiac isoforms are predominant in heart, whereas skeletal α-actins are found in both skeletal and cardiac muscle. In pufferfish (Fugu rubripes), nine actin genes have been cloned, of which six were identified as α-isoforms(two muscle, three cardiac and one anomalous testis α-actin; Venkatesh et al., 1996).α-Actin genes have been cloned from three more fish species, zebrafish(Danio rerio; Higashijima et al.,1997), medaka (Oryzias latipes; Kusakabe et al., 1999) and channel catfish (Ictalurus punctatus; Kim et al., 2000), and their promoters have been functionally assessed. The myosin complex is a hexamer of two heavy and four light chains, of which regulatory chain is required for calcium binding. This gene has been cloned from one teleost species, zebrafish(Xu et al., 2000). Bothα-actin and MLC2 promoters have been an important model for identification of muscle-specific regulatory elements in vertebrates.

In skeletal muscle, tropomyosin is a dimer of α and β chains,mediating interaction between the troponin complex and actin that is required for regulation of contraction. Unlike actin and myosin, tissue-specific isoforms of mammalian tropomyosin are encoded by a single gene, and multiple proteins arise due to usage of different promoters and alternative splicing. Promoters of mammalian α-tropomyosins are characterized by a lack of canonical regulatory sequence elements, in this respect being similar to housekeeping genes (Wieczorek et al.,1988). In the α-tropomyosin gene from Xenopus laevis, two promoters flanking a pair of alternatively splicing exons were identified and a distal promoter generated muscle-specific isoforms(Gaillard et al., 1998). No fish tropomyosin gene has been cloned. In this study, we determined the genomic structure of rainbow trout α-actin (α-OnmyAct) and myosin regulatory light chain 2 (OnmyMLC2) genes. In addition, the 5′-flanking region of α-tropomyosin (α-OnmyTM) was cloned. Promoters of these genes were shown to direct expression of LacZ reporter and recombinant rainbow trout gene in rainbow trout embryos and cells.

The sequences of α-OnmyAct 5′- and 3′-untranslated regions (UTR) were determined by RACE (rapid amplification of cDNA ends) using PCR primers (Table 1) that were designed to the chum salmon (Oncorhynchus keta) α-actin cDNA(GenBank AB032464). RNA was extracted from skeletal muscle of adult rainbow trout, and RACE cloning was performed as described elsewhere(Teerijoki et al., 2001a). Genome walker kit (Clontech, Palo Alto, CA, USA) was used for PCR cloning of genomic sequences. High molecular mass genomic DNA was isolated from adult rainbow trout kidney (Sambrook et al.,1989). Four libraries were constructed by digestion of genomic DNA with EcoRV, DraI, PvuII and StuI followed by ligation to adaptor. To clone the 5′-flanking regions, three reverse gene-specific primers (GSP1-3) were designed to the 5′ termini ofα-OnmyAct, coho salmon (Oncorhynchus kisutch) MLC2 (GenBank AF251130) and Atlantic salmon (Salmo salar) fast myotomal muscleα-TM (GenBank L25609) cDNAs. A mixture of Taq (MBI Fermentas,Vilnius, Lithuania) and Pfu (Stratagene, La Jolla, CA, USA) DNA polymerases (50:1) was used for PCR. First, PCR was carried out with a combination of GSP1 (gene-specific primer) and AP1 (adaptor primer). PCR products were diluted 1:50 and reamplified with GSP2 or GSP3 and AP2. Touch-down PCR profile included five cycles at high temperature (2 s at 94°C and 3 min at 70°C) followed by 28-34 cycles at lower temperature(2 s at 94°C and 3 min at 67°C) and final extension (7 min at 67°C). α-OnmyAct gene upstream walk was continued and new primers were designed to the sequence of promoter that was determined in the first cloning step. The same approach was used for cloning of α-OnmyAct terminator and coding parts of α-OnmyAct and OnmyMLC2. Sequences were analysed with Blastn (Altschul et al.,1997). GenScan (Burge and Karlin, 1997) was used for prediction of transcription start and exon boundaries, and the TRANSFAC database(Wingender et al., 2000) was searched with MatInspector (Quandt et al.,1995) to find potential promoter regulatory elements.

For functional assessment, the PCR-amplified 5′-flanking regions of rainbow trout genes were cloned into TOPO pBlue vector (Invitrogen, Groningen,The Netherlands), which contains the LacZ reporter gene and bovine growth hormone (bGH) terminator. Five vectors with rainbow trout promoters were prepared (Table 2). Strong constitutive cytomegalovirus (CMV) promoter was PCR amplified and cloned into the same vector. This construct was used as a control for transfection and detection of reporter. To verify functionality of α-OnmyAct terminator,it was inserted into the CMVLacZ plasmid to substitute the bGH sequence. Next,we constructed all-trout expression vectors using a pUC18 backbone(Table 2). First,α-OnmyAct terminator was inserted into the XbaI/SmaI sites, and then α-OnmyAct and OnmyMLC2 promoters were cloned as HindIII fragments. To verify the capability of these vectors for transgene expression in rainbow trout embryos, we used fish glucose transporter type 1 (OnmyGLUT1). The protein-coding region of this gene was amplified with PCR from plasmid that had been shown to express a functional glucose transporter (Teerijoki et al.,2001b). PCR primers included convenient restriction sites for cloning into the expression vectors.

Vectors containing LacZ reporter were tested in rainbow trout embryos and primary embryonic cell cultures. Plasmids were transferred into fertilized eggs by microinjections as described previously(Krasnov et al., 1999). Primary cell cultures were prepared from embryos at the stage of somitogenesis. Embryos were excised from chorions and washed with phosphate-buffered saline (PBS) to remove yolk. Then they were triturated with a Pasteur pipette and incubated in PBS for 30 min. Complete dissociation of cells was achieved by passing embryos through 1 ml and 0.2 ml plastic tips using an automatic pipette. Cells were cultivated in 6-well plates in minimum essential medium (MEM) with Hanks' salts supplemented with 10% foetal bovine serum and antibiotics (all reagents were from Life Technologies, Paisley, UK). Transfection was performed with FuGENE reagent (Boehringer-Mannheim, Mannheim,Germany) according to the manufacturer's instructions. Embryos and cells were fixed with 1% glutaraldehyde in PBS for 30 min, washed three times with an excess of PBS and incubated at 37°C with substrate (4 mmol l^-1potassium ferricyanide, 4 mol l^-1 potassium ferrocyanide, 2 mmol l^-1 magnesium chloride and 1 mg ml^-1 X-gal in PBS). Plasmids, including OnmyGLUT1, were microinjected into fertilized eggs and transgene expression was analysed by RT-PCR at the stage of 35 somite pairs. RNA extracted from microinjected embryos was treated with RNase-free DNase(Promega, Madison, WI, USA), and synthesis of cDNA was primed with oligo(dT)₁₈ (Promega). To distinguish recombinant and endogenous transcripts, we used GSP to OnmyGLUT1 in conjunction with primers to the 3′-UTR of α-OnmyAct (ActR1-3). These were designed to the sequences upstream (ActR1-2) and downstream (ActR3) from the polyadenylation signal (Fig. 1).

Cloning of α-OnmyAct began with identification of the 5′-UTR sequence using RACE. We analysed five clones, and their sequences were identical except for minor variation in the splicing site of the first non-coding exon, the length of the 5′-UTR being 63-69 bp. Primer ACTF1 was designed to this region, and RT-PCR analyses detected α-Act transcripts in skeletal muscle and heart. Furthermore, the whole coding part of this gene (2900 bp) was amplified with PCR using ACTGF1-2 and ACTGR1-2 primers. Alignment of genomic sequence with mRNA and computer analysis(GenScan) predicted one non-coding and six coding exons, whose size was identical to those reported for all vertebrate α-SkAct and some of theα-CardAct genes (Fig. 2A). To amplify the α-OnmyAct promoter, we designed primers to the 5′-UTR, and a 2018-bp 5′-flanking region was cloned in two steps. A TATA box was located 28 bp upstream from the transcription start, and a search across the TRANSFAC database revealed many putative transcription factor binding sites in the promoter and the first intron(Fig. 2B). Of 24 E-boxes(CANNTG), 12 were predicted to be binding sites of MyoD, a myogenic regulatory factor that plays a key part in muscle differentiation. Two perfect CarG-boxes(CC(A/T)₆GG) were located 117 bp and 244 bp upstream from the transcription start, and two more CarG-box-like motifs were found(Fig. 2B). Cloning ofα-OnmyAct terminator was preceded by 3′-RACE. The sequence of 305-bp 3′-UTR was 96% identical to that of O. keta α-Act. Interestingly, a 53-bp sequence was well conserved in α-Act 3′-UTRs of both salmonid species and tilapia (Tilapia mossambica). Furthermore, we cloned a 588-bp genomic sequence, which included 305 bp downstream from the 3′-end of SkAct cDNA and contained a polyadenylation signal (AATAAA).

Using primers to the 5′ terminus of MLC2 cDNA, we cloned two different genomic 5′-flanking sequences designated as OnmyMLC2-1 (1635 bp) and OnmyMLC2-2 (1082 bp). Blastn analysis revealed Tc1 transposon-like fragments at the distal ends of these sequences(Fig. 3A). MLC2-1 included a sequence resembling the 3′-UTR of plaice (Pleuronectes platessa) Tc1 transposon tn5 gene (GenBank AJ303068) in direct orientation, whereas a reverse sequence similar to the coding part of Salmo salar transposase pseudogene (GenBank L22865) was found in MLC2-2. Approximately 800 bp at the 3′ ends of OnmyMLC2 flanking regions were nearly identical with the exception of a 55-bp deletion in MLC2-2(Fig. 3A). A TATA box was found 31 bp upstream from the putative transcription start, and there were four E-boxes. The whole protein-coding part of OnmyMLC2 was obtained by PCR, and only a fragment of the second exon remained uncloned. As is true of all known vertebrate MLC2 genes, this gene consisted of seven exons, whose length was conserved (Fig. 3B). Simple(TG)₄₄ and (AG)₁₆ repeats arranged in tandem were found in the third intron of OnmyMLC2.

We cloned a 700-bp 5′-flanking region of α-OnmyTM that included a 102-bp 3′-UTR. Neither a TATA box nor muscle-specific regulatory elements were found in the promoter sequence and, in this respect,α-OnmyTM was similar to the known vertebrate α-TM genes.

The ability of α-OnmyAct, α-OnmyTM and OnmyMLC2 promoters to direct LacZ expression was assessed in rainbow trout embryos. Five constructs with rainbow trout sequences (Table 2) and control plasmid (CMVLacZ) were delivered into one-cell embryos using microinjection. Vector containing viral promoter was expressed at high level in blastulas, whereas none of the rainbow trout promoters were active at this stage. At early somitogenesis (5 somite pairs), two constructs with rainbow trout promoters (OnmyMLC2-2 and α-OnmyTM) were expressed at low level, and in both groups LacZ-positive cells were detected in four out of 10 analysed embryos. Next, 25-30 embryos from each experimental group were analysed at the stage of 39 somite pairs, and all five rainbow trout promoters were active (Table 3). Fig. 4 presents the expression of α-OnmyTM; similar patterns were observed with α-OnmyAct and OnmyMLC2. Spatial distribution of LacZ-positive cells was mosaic, which is common for transient transgene expression in fish embryos. Nevertheless, all embryos microinjected with constructs containing rainbow trout promoters showed reporter activity in the differentiating somites, presumably myotomes,neural structures (brain, neural plate and neural crest) and the yolk syncytial layer. Cardiac expression of reporter was found in one embryo microinjected with α-OnmyTMLacZ (Fig. 4D). The stage of 39 somite pairs precedes the beginning of heart contraction (Gorodilov, 1996). LacZ-positive cells were not detected in notochord and visceral organs. The reporter constructs were also tested in primary rainbow trout embryonic cell cultures, and four rainbow trout promoters were active(Table 3). By numbers of LacZ-positive cells in embryos and cell cultures, activity of α-OnmyAct and α-OnmyTM promoters was greater than that of MLC2-1 and MLC2-2. Difference in expression levels of vectors with α-OnmyAct promoter sequences of different length suggested that the -800/2019 bp region could include important regulatory elements.

For preparation of all-trout vectors we used α-OnmyAct terminator. Functionality of this sequence was assessed preliminarily by substitution of the bGH terminator in the CMVLacZ plasmid. Vector with rainbow trout terminator was expressed in embryonic cell culture. Plasmids containingα-OnmyAct and OnmyMLC2-1 promoters and OnmyGLUT1 were microinjected into rainbow trout embryos. Transgene expression was analysed at the stage of 35 somite pairs by RT-PCR. Plasmid DNA was degraded by DNase, and synthesis of cDNA was initiated using oligo(dT)₁₈. As expected, cDNA was amplified with ActR1 or ActR2 but not with ActR3; the ActR3 primer was designed to the sequence downstream from the polyadenylation signal(Fig. 1).

The goal of this study was to clone rainbow trout genes that are expressed at a high level in skeletal muscle and to assess the functionality of their regulatory sequences. We chose to clone α-skeletal actin because, in contrast to many other muscle-specific genes, the structure of vertebrateα-SkAct promoters is relatively simple and short sequences can be capable of high-level expression of transgenes. Cloning of skeletalα-Act promoter can be complicated by the fact that vertebrate genomes contain several α-actin genes characterized by different expression patterns. Because the coding regions of α-actin cDNAs are highly conserved, we preferred not to use these sequences to design PCR primers for cloning of skeletal α-Act promoter. Therefore, it was necessary to identify the 5′-UTR sequence of α-Act cDNA expressed in skeletal muscle of rainbow trout; usually, these regions show greater divergence than the coding parts. Because a TATA box was found 28-bp upstream from the end of the 5′-UTR, it was likely that we obtained a complete sequence of the 5′ terminus of α-OnmyAct cDNA. Sequences of five clones were nearly identical, suggesting that this isoform was predominant in skeletal muscle of rainbow trout. RT-PCR analyses detected expression ofα-OnmyAct in trunk muscle and heart, which was common for all skeletal and cardiac actin isoforms. Exon structure of α-OnmyAct was identical to all known vertebrate α-SkAct genes, being slightly different from that of pufferfish α-cardiac actin genes(Fig. 2A). α-OnmyAct contained six introns, whereas there are seven introns in pufferfishα-cardiac2 and 3 (Venkatesh et al.,1996). One more pufferfish α-Act gene, designated asα-cardiac1, lacked the first non-coding exon, and its tissue specificity remained unknown because no expression was detected in fish. Medakaα-CardAct gene (GenBank AB016259) is also characterized by the absence of a 5′-UTR. Mammalian smooth muscle (aortic) α-Act genes contain eight introns, and their exon pattern is clearly different from that ofα-OnmyAct. By its abundance in trunk muscle, its tissue distribution and its exon structure, α-OnmyAct could probably be designated as a skeletal actin.

Using one set of PCR primers designed to the 5′ terminus of MLC2 cDNA, we cloned two different genomic 5′-flanking regions. The finding of two rainbow trout MLC2 genes could be expected, as two distinct MLC2 transcripts were reported in carp (Cyprinus carpio; Hirayama et al., 1998) and in gilthead seabream (Sparus aurata; Moutou et al., 2001). Both promoters included a TATA box and sequences similar to fish transposon-like elements. Although transposon-like sequences were located in the vicinity of the transcription start, these genes are likely to be functional. Conservation of the 800-bp 5′-flanking region sequences implied their importance. Beside this, it was known that promoters of vertebrate MLC2 genes could be relatively short. For example, the length of promoter required for efficient muscle-specific expression of MLC2 was only 64 bp in chicken(Braun et al., 1989) and 79 bp in zebrafish (Xu et al.,1999). The length of coding region of OnmyMLC2 was greater than 4500 bp and we were unable to derive the whole sequence from a single clone. Nonetheless, PCR cloning allowed us to determine the exon structure of this gene. The sequence of cDNA was split into seven exons, which is typical for known vertebrate MLC2 genes. The number of putative transcription factor binding sites in OnmyMLC2 was less than in α-OnmyAct. No canonical CArG-boxes were found in OnmyMLC2 promoters and there were only four E-boxes,whereas there were 24 in α-OnmyAct. These elements, which play a key part in regulation of expression of mammalian and avian skeletal muscle genes,were found in other fish α-Act promoters(Kusakabe et al., 1999; Kim et al., 2000). Sequence analyses found putative transcription factor binding sites in rainbow trout promoters, and special studies will be required to determine the elements involved in regulation of expression. We cloned the α-OnmyTM region flanking the first exon (3′-UTR). Expression of α-TM in Xenopus muscle is driven by the distal promoter(Gaillard et al., 1998) and it is possible that the rainbow trout gene has a similar structure. In contrast to α-OnmyAct and OnmyMLC2, we did not reveal any common structural features in promoters of mammalian and rainbow trout α-TM.

The ability of rainbow trout promoters to control reporter expression in embryos was detected earliest at the stage of 5 somite pairs, and then only two out of five constructs were expressed. α-OnmyAct and OnmyMLC2 promoters were activated in the course of somitogenesis, which was in concordance with the temporal patterns of expression of these genes in fish embryos (Xu et al., 2000; Thiebaud et al., 2001). At the stage of 39 somite pairs, the transformation rate was within the range of 45-90%, being equal to 89% in the control group (CMVLacZ). As expected,reporter activity was found in the dorsal parts of segments, the sites of myogenic differentiation. High-level expression was also seen in the yolk syncytial layer. This structure, which is formed during blastulation, plays an important part in epiboly (Trinkaus,1993). The yolk syncytial layer is divided into the internal(under the blastoderm) and external parts. Cortical cytoplasm of the external yolk syncytial layer (E-YSL) is proposed to be generously endowed with actin-containing microfilaments (Betchaku and Trinkaus, 1978), which cause contraction of the E-YSL and narrowing of the underlying yolk to give expansion of the blastodermal germ ring. E-YSL is apparently a major motor for epiboly that advances across the yolk. Rainbow trout embryos complete epiboly by the stage of 17-20 somite pairs. Then, the germ ring becomes narrow and is eventually compressed,whereas the yolk syncytial layer retains. It is likely that LacZ-positive cells seen beneath the embryo (Fig. 4E) were located in this structure. Interestingly, YSL is characterized by an exclusively high capacity for transgene expression(Williams et al., 1996). In all analysed embryos, LacZ-positive cells were detected in neural tissues. Fig. 4B shows reporter expression in the neural crest, which lies at the boundary between the neural plate and the lateral ectoderm. This structure gives rise to connective,skeletal and some muscle tissues of the head, skeleton, pigmented cells and nervous roots with sensory and motor ganglia in the trunk-tail part (reviewed in Baker and Bronner-Fraser,1997; Gorodilov,2000). LacZ was also detected in different parts of the brain. The finding of reporter expression in neural tissues was not surprising. High-throughput sequencing of cDNA libraries documented in the dbEST division of Genbank(http://www.ncbi.nlm.nih.gov/dbEST/index.html)provides compelling evidence that muscle isoforms of α-Act, α-TM and MLC are expressed in various fish tissues. For instance, transcripts of these genes were found in the brain library of zebrafish. High levels of neural expression observed in this study could be characteristic of the analysed developmental stage.

Our study was designed to assess functionality of rainbow trout promoters and their ability to control transgene expression in embryonic muscle. Transient reporter expression in fish embryos made it possible to locate the major sites of promoters' activity. However, due to mosaicism, this model system did not allow more detailed analyses. We were unable to find any major difference in the expression patterns of constructs with α-OnmyAct,OnmyMLC2 and α-OnmyTM promoters. Mosaic expression impeded quantitative analyses, which made accurate evaluation of promoter strength impossible.

This study was funded by Tekes, the National Technology Agency,Finland.