Cloning of rainbow trout (Oncorhynchus mykiss) alpha-actin, myosin regulatory light chain genes and the 5'-flanking region of alpha-tropomyosin. Functional assessment of promoters.
We report PCR cloning of rainbow trout alpha-actin (alpha-OnmyAct), myosin regulatory light chain (OnmyMLC2) genes and the 5'-flanking region of alpha-tropomyosin (alpha-OnmyTM). Being expressed in skeletal and cardiac muscle, alpha-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 alpha-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 alpha-OnmyAct and OnmyMLC2 included a TATA box and a number of putative regulatory motifs (E-boxes in all three sequences and CArG-boxes in alpha-OnmyAct), whereas there were no canonical motifs in the alpha-OnmyTM promoter. LacZ reporter gene was fused with the 5'-flanking regions of alpha-OnmyAct, two OnmyMLC2 genes and alpha-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. alpha-OnmyTMLacZ was also expressed in the heart. Functionality of promoters and the alpha-OnmyAct terminator was confirmed in rainbow trout primary embryonic cell cultures. We cloned rainbow trout glucose transporter type I (OnmyGLUT1) into vectors including the alpha-OnmyAct and OnmyMLC2 promoters and the alpha-SkAct terminator. Recombinant OnmyGLUT1 transcripts were detected in rainbow trout embryos during somitogenesis.
Krasnov
Aleksei
A
Institute of Applied Biotechnology, University of Kuopio, POB 1627, FIN-Kuopio 70211, Finland. krasnov@uku.fi
Teerijoki
Heli
H
Gorodilov
Yuri
Y
Mölsä
Hannu
H
D024506:5' Flanking Region / Q000235:genetics*; D000199:Actins / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D007763:Lac Operon / Q000235:genetics; D009124:Muscle Proteins / Q000235:genetics* / Q000378:metabolism; D018994:Myosin Light Chains / Q000235:genetics* / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019076:Transgenes / Q000235:genetics; D014335:Tropomyosin / Q000235:genetics* / Q000378:metabolism
D000199:Actins; D009124:Muscle Proteins; D018994:Myosin Light Chains; D011993:Recombinant Fusion Proteins; D014335:Tropomyosin