Submitted on July 6, 2004
Accepted on July 24, 2004
Cloning and sequencing of myosin heavy chain isoform cDNAs in golden-mantled ground squirrels (Spermophilus lateralis): Effects of hibernation on mRNA expression
Bryan C Rourke1, Anqi Qin2, Fadia Haddad2, Kenneth M Baldwin2, and Vince Caiozzo3*
1 Department of Orthopaedics, University of California, Irvine, CA, USA
2 Department of Physiology and Biophysic, University of California, Irvine, CA, USA
3 Department of Orthopaedics, University of California, Irvine, CA, USA; Department of Physiology and Biophysic, University of California, Irvine, CA, USA
* To whom correspondence should be addressed. E-mail: vjcaiozz{at}uci.edu.
The golden-mantled ground squirrel is a small rodent hibernator that demonstrates unusual myosin heavy chain (MHC) isoform plasticity during several months of torpor, punctuated by bouts of rewarming and shivering thermogenesis. We measured MHC mRNA levels to determine if pretranslational control mechanisms were responsible for differences in type IIx MHC protein expression, as we previously observed between active and hibernating ground squirrels. We first cloned cDNA using the 3' prime Rapid Amplification of cDNA Ends (3 prime RACE) technique, and identified three sequences corresponding to type I, IIx and IIb MHC. A DNA control fragment was developed to be used in conjunction with a coupled RT-PCR reaction to simultaneously measure MHC mRNA levels for each isoform in the skeletal muscle of ground squirrels. MHC mRNA and protein expression were strongly correlated, and type IIx and IIb mRNA levels were significantly different between active and hibernating ground squirrels. Pretranslational control of MHC protein is apparently an important process during hibernation, although the exact stimulus is not known. The techniques presented can be used to obtain MHC cDNA sequences and to measure mRNA expression in many vertebrate groups.
