Functional analysis on the 5'-flanking region of carp fast skeletal myosin heavy chain genes for their expression at different temperatures.
Two types of the fast skeletal myosin heavy chain (MYH) genes were cloned from a genomic DNA library of carp (Cyprinus carpio L.) and named MYH10 and MYH30, which showed the sequence similarity to the MYH cDNAs predominantly expressed in carp acclimated to 10 and 30 degrees C, respectively. The 5'-flanking region of about 3 kbp in size each from MYH10 and MYH30 contained various cis-elements to bind to transcriptional regulatory factors such as MyoD family and myocyte enhancer factor 2 (MEF2) family members. To localize functional regions responsible for the MYH gene expression in a temperature-dependent manner, a series of deletion constructs were prepared from the 5'-flanking region, inserted upstream the luciferase gene in a commercially available plasmid, and injected into the dorsal fast muscle of carp acclimated to 10 and 30 degrees C. The sequence of -1004 to -995 bp with the transcriptional activity in MYH30 was identified as an MEF2 binding site. While the activity given by a sequence of -921 to -824 bp in MYH10 contained only a GATA box, that of the activity of the -1 kbp construct from MYH10 was markedly higher in carp reared at 10 degrees C than fish reared at 30 degrees C. On the other hand, no temperature-dependent expressional regulation was observed for MYH30 even with the full-length construct of -3 kbp. The DNA fragment of -921 to -824 bp in MYH10 and MEF2 binding site in MYH30 interacted with nuclear proteins extracted from carp fast skeletal muscle as revealed by electrophoretic mobility shift assay. The signal intensity of a complex formed between the DNA fragment of MYH10 and nuclear extracts from the 10 degrees C-acclimated carp were higher than those with extracts from the 30 degrees C-acclimated fish. Although MEF2-binding site in MYH30 could form complex with nuclear extracts from the 30 degrees C-acclimated carp, the same or stronger signals were detected in complex formed with extracts from the 10 degrees C-acclimated fish.
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan.
Atsushi
A
Kobiyama
Makoto
M
Hirayama
Maiko
M
Muramatsu-Uno
Shugo
S
Watabe
D024506:5' Flanking Region / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D005399:Fishes / Q000235:genetics*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000235:genetics*; D024501:Nonmuscle Myosin Type IIB / Q000737:chemistry / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D024501:Nonmuscle Myosin Type IIB; C103815:nonmuscle myosin type IIB heavy chain; D018995:Myosin Heavy Chains