Cloning of rainbow trout (Oncorhynchus mykiss) -actin, myosin regulatory light chain genes and the 5'-flanking region of
-tropomyosin. Functional assessment of promoters
1 Institute of Applied Biotechnology, University of Kuopio, P.O.B. 1627,
FIN-Kuopio 70211, Finland
2 Biological Institute, University of Sanct Petersburg, Oranienbaum Chaussee
2, Stary Peterhof, Sanct Petersburg 198504, Russia
* Author for correspondence (e-mail: krasnov{at}uku.fi)
Accepted 30 October 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: rainbow trout, Oncorhynchus mykiss, -actin,
-OnmyAct, myosin regulatory light chain,
-tropomyosin, promoter, skeletal muscle
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Actins are highly conserved structural proteins. Being components of
contractile structures and cytoskeleton, actins are distributed ubiquitously.
Of three major vertebrate actin groups (, ß and
),
-actins are specific for striated muscle. Cardiac isoforms are
predominant in heart, whereas skeletal
-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
-isoforms
(two muscle, three cardiac and one anomalous testis
-actin;
Venkatesh et al., 1996
).
-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
). Both
-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 and ß 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
-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
-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
-actin (
-OnmyAct) and myosin
regulatory light chain 2 (OnmyMLC2) genes. In addition, the 5'-flanking
region of
-tropomyosin (
-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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Expression vectors
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 -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,
-OnmyAct terminator was inserted into the XbaI/SmaI
sites, and then
-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.
|
Functional assessment of regulatory sequences
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-1
potassium 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
-OnmyAct (ActR1-3). These were designed to the
sequences upstream (ActR1-2) and downstream (ActR3) from the polyadenylation
signal (Fig. 1).
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Myosin regulatory light chain (OnmyMLC2)
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.
|
-Tropomyosin (
-OnmyTM)
We cloned a 700-bp 5'-flanking region of -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,
-OnmyTM was similar to the known vertebrate
-TM genes.
Transgene expression in rainbow trout embryos and cells
The ability of -OnmyAct,
-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
-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
-OnmyTM; similar patterns were observed with
-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
-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
-OnmyAct
and
-OnmyTM promoters was greater than that of MLC2-1 and MLC2-2.
Difference in expression levels of vectors with
-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 -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 containing
-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).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
-OnmyAct. No canonical
CArG-boxes were found in OnmyMLC2 promoters and there were only four E-boxes,
whereas there were 24 in
-OnmyAct. These elements, which play a key
part in regulation of expression of mammalian and avian skeletal muscle genes,
were found in other fish
-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
-OnmyTM region
flanking the first exon (3'-UTR). Expression of
-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
-OnmyAct and OnmyMLC2, we did not reveal any common structural
features in promoters of mammalian and rainbow trout
-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. -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
-Act,
-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 -OnmyAct,
OnmyMLC2 and
-OnmyTM promoters. Mosaic expression impeded quantitative
analyses, which made accurate evaluation of promoter strength impossible.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J.,
Zhang, Z., Miller, W. and Lipman, D. J. (1997). Gapped BLAST
and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res. 25,3389
-3402.
Baker, C. V. H. and Bronner-Fraser, M. (1997). The origins of the neural crest. II. An evolutionary perspective. Mech. Dev. 69,13 -29.[CrossRef][Medline]
Betchaku, T. and Trinkaus, J. P. (1978). Contact relations, surface activity, and cortical microfilaments of marginal cells of the enveloping layer and of the yolk syncytial and yolk cytoplasmic layers of fundulus before and during epiboly. J. Exp. Zool. 206,381 -426.[Medline]
Braun, T., Tannich, E., Buschhausen-Denker, G. and Arnold, H. H. (1989). Promoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription. Mol. Cell. Biol. 9,2513 -2525.[Medline]
Burge, C. and Karlin, S. (1997). Prediction of complete gene structures in human genomic DNA. J. Mol. Biol. 268,78 -94.[CrossRef][Medline]
Gaillard, C., Thézé, N., Hardy, S., Allo, M. R., Ferrasson, E. and Thiébaud, P. (1998). Alpha-tropomyosin gene expression in Xenopus laevis: differential promoter usage during development and controlled expression by myogenic factors. Dev. Genes. Evol. 207,435 -445.[CrossRef][Medline]
Gorodilov, Y. N. (1996). Description of the early ontogeny of the Atlantic salmon, Salmo salar, with a novel system of interval (state) identification. Envir. Biol. Fish. 47,109 -127.
Gorodilov, Y. N. (2000). The fate of Spemann's organizer. Zool. Sci. 17,1197 -1220.
Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y. and Eguchi, G. (1997). High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev. Biol. 192,289 -299.[CrossRef][Medline]
Hirayama, Y., Kobiyama, A., Ochiai, Y. and Watabe, S. (1998). Two types of mRNA encoding myosin regulatory light chain in carp fast skeletal muscle differ in their 3' non-coding regions and expression patterns following temperature acclimation. J. Exp. Biol. 201,2815 -2820.
Kim, S., Karsi, A., Dunham, R. A. and Liu, Z. (2000). The skeletal muscle alpha-actin gene of channel catfish (Ictalurus punctatus) and its association with piscine specific SINE elements. Gene 252,173 -181.[CrossRef][Medline]
Krasnov, A., Pitkänen, T. I., Reinisalo, M. and Mölsä, H. (1999). Expression of human glucose transporter type 1 and rat hexokinase type II cDNAs in rainbow trout embryos; effects on glucose metabolism. Mar. Biotechnol. 1, 25-32.[Medline]
Kusakabe, R., Kusakabe, T. and Suzuki, N. (1999). In vivo analysis of two striated muscle actin promoters reveals combinations of multiple regulatory modules required for skeletal and cardiac muscle-specific gene expression. Int. J. Dev. Biol. 43,541 -554.[Medline]
Moutou, K. A., Canario, A. V., Mamuris, Z. and Power, D. M.
(2001). Molecular cloning and sequence of Sparus aurata
skeletal myosin light chains expressed in white muscle: developmental
expression and thyroid regulation. J. Exp. Biol.
204,3009
-3018.
Quandt, K., Frech, K., Karas, H., Wingender, E. and Werner, T. (1995). MatInd and MatInspector new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res. 23,4878 -4884.[Abstract]
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning. A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press.
Teerijoki, H., Krasnov, A., Pitkänen, T. I. and Mölsä, H. (2001a). Monosaccharide uptake in common carp (Cyprinus carpio) EPC cells is mediated by facilitative glucose transporter. Comp. Biochem. Physiol. B 128,483 -491.[CrossRef][Medline]
Teerijoki, H., Krasnov, A., Gorodilov, Y., Krishna, S.,
Pitkänen, T. I. and Mölsä, H. (2001b). Rainbow
trout glucose transporter (OnmyGLUT1): translation in Xenopus oocytes
and expression patterns in fish embryos. J. Exp. Biol.
204,2667
-2673.
Thiebaud, P., Rescan, P. Y., Barillot, W., Ralliere, C. and Theze N. (2001). Developmental program expression of myosin alkali light chain and skeletal actin genes in the rainbow trout Oncorhynchus mykiss. Biochim. Biophys. Acta 1519,139 -142.[Medline]
Trinkaus, J. P. (1993). The yolk syncytial layer of Fundulus: its origin and history and its significance for early embryogenesis. J Exp. Zool. 265,258 -284.[Medline]
Venkatesh, B., Tay, B. H., Elgar, G. and Brenner, S. (1996). Isolation, characterization and evolution of nine pufferfish (Fugu rubripes) actin genes. J. Mol. Biol. 259,655 -665.[CrossRef][Medline]
Wieczorek, D. F., Smith, C. W. and Nadal-Ginard, B. (1988). The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing. Mol. Cell. Biol. 8, 679-694.[Medline]
Williams, D. W., Muller, F., Lavender, F. L., Orban, L. and Maclean N. (1996). High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish (Danio rerio) embryos. Transgenic Res. 6, 433-442.
Wingender, E., Chen, X., Hehl, R., Karas, H., Liebich, I.,
Matys, V., Meinhardt, T., Prüß, M., Reuter, I. and Schacherer,
F. (2000). TRANSFAC: an integrated system for gene expression
regulation. Nucleic Acids Res.
28,316
-319.
Xu, Y., He, J., Tian, H. L., Chan, C. H., Liao, J., Yan, T., Lam, T. J. and Gong Z. (1999). Fast skeletal muscle-specific expression of a zebrafish myosin light chain 2 gene and characterization of its promoter by direct injection into skeletal muscle. DNA Cell. Biol. 18,85 -95.[CrossRef][Medline]
Xu, Y., He, J., Wang, X., Lim, T. M. and Gong, Z. (2000) Asynchronous activation of 10 muscle-specific protein (MSP) genes during zebrafish somitogenesis. Dev. Dyn. 219,201 -215.[CrossRef][Medline]