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

Abstract

SUMMARYWe report PCR cloning of rainbow trout α-actin (α-OnmyAct),myosin regulatory light chain (OnmyMLC2) genes and the 5′-flanking region of α-tropomyosin (α-OnmyTM). Being expressed in skeletal and cardiac muscle, α-OnmyAct was a predominant isoform in trunk muscle of adult rainbow trout. Exon structure of this gene was identical to all known vertebrate skeletal and to some of the cardiac α-Act genes. Two distinct OnmyMLC2 promoters were cloned and both included transposon-like sequences. The coding part of OnmyMLC2 consisted of seven exons whose length was typical for vertebrate MLC2 genes. The upstream regions of α-OnmyAct and OnmyMLC2 included a TATA box and a number of putative regulatory motifs(E-boxes in all three sequences and CArG-boxes in α-OnmyAct), whereas there were no canonical motifs in the α-OnmyTM promoter. LacZ reporter gene was fused with the 5′-flanking regions of α-OnmyAct, two OnmyMLC2 genes and α-OnmyTM promoters. These constructs were transferred into rainbow trout eggs. At the stage of 39 somite pairs, LacZ reporter was detected in the myotomes, neural plate and neural crest, brain and yolk syncytial layer of all analysed embryos. α-OnmyTMLacZ was also expressed in the heart. Functionality of promoters and the α-OnmyAct terminator was confirmed in rainbow trout primary embryonic cell cultures. We cloned rainbow trout glucose transporter type I (OnmyGLUT1) into vectors including the α-OnmyAct and OnmyMLC2 promoters and the α-SkAct terminator. Recombinant OnmyGLUT1 transcripts were detected in rainbow trout embryos during somitogenesis.