Molecular cloning and sequence of Sparus aurata skeletal myosin light chains expressed in white muscle: developmental expression and thyroid regulation
Katerina A. Moutou1,*,
Adelino V. M. Canario2,
Zissis Mamuris1 and
Deborah M. Power2
1 Department of Biochemistry and Biotechnology, University of Thessaly, 26 Ploutonos Street, 41221 Larissa, Greece and
2 Centre of Marine Sciences (CCMAR), University of Algarve, Campus de Gambelas, 8000 Faro, Portugal
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Fig. 1. Nucleotide and deduced amino acid sequence of the Sparus aurata myosin light chain 2. The polyadenylation signals are underlined and the start codon is bold.
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Fig. 2. Alignment of the deduced amino acid sequences of the myosin light chain 2 (MLC2) proteins. The Ca2+-binding domain is indicated in bold type. Conserved residues are marked by an asterisk; conservative differences are marked by a full point. Sources, references and accession numbers are described in Materials and methods.
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Fig. 3. Nucleotide and deduced amino acid sequence of the Sparus aurata myosin light chain 3. The polyadenylation signal is underlined and the start codon is bold.
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Fig. 4. Alignment of the deduced amino acid sequences of the myosin light chain 3 (MLC3) proteins. The MLC3-specific domain of avian and mammalian sequences and the ancestral Ca2+-binding domain are indicated in bold type. Conserved residues are marked by an asterisk; conservative differences are marked by a full point. Sources, references and accession numbers are described in Materials and methods.
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Fig. 5. (A) Tissue-specific transcription of MLC2 and MLC3 genes. Northern blot analysis of total RNA (5µg) from the brain, gills, liver, intestine (smooth muscle), white muscle, red muscle, heart (cardiac muscle) and kidney. (B) RNA expression analysis of MLC2 and MLC3 genes during the development of Sparus aurata; northern blot analysis of total RNA (5µg) from larvae at different stage of development. kbp, kilobase-pairs; hpf, hours post-fertilization.
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Fig. 6. Effects of experimental hypothyroidism (thiourea treatment) or hyperthyroidism (T3 and T4 treatment) on MLC2 (A) and MLC3 (B) RNA expression of Sparus aurata juvenile or adult individuals. The MLC transcript levels were determined by northern blot analysis, and densitometry was carried out with the resulting autoradiograph for each probe and normalised against the expression of cytoplasmic ß-actin. Values are means + S.E.M. (N=4).
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Fig. 7. A phylogenetic tree of myosin light chain 2 (MLC2) sequences constructed using the neighbour-joining method. The lengths of branches are proportional to the phylogenetic distances estimated using Kimura’s empirical method for protein distances (Kimura, 1983). Danio rerio cardiac MLC2 is included as an outgroup. Numbers indicate percentages of 100 bootstrap replicates in which the same internal branch was recovered.
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Fig. 8. A phylogenetic tree of myosin light chain 3 (MLC3) sequences constructed using the neighbour-joining method. The lengths of branches are proportional to the phylogenetic distances estimated using Kimura’s empirical method for protein distances (Kimura, 1983). Mus musculus non-muscle MLC3 is included as an outgroup. Numbers indicate percentages of 100 bootstrap replicates in which the same internal branch was recovered.
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© The Company of Biologists Ltd 2001
