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Cloning and sequencing of myosin heavy chain isoform cDNAs in golden-mantled ground squirrels: effects of hibernation on mRNA expression

Departments of ¹Orthopaedics and ²Physiology and Biophysics, College of Medicine, University of California, Irvine, California 92697

Submitted 6 July 2004 ; accepted in final form 24 July 2004

ABSTRACT

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 whether pretranslational control mechanisms were responsible for differences in MHC_2x protein expression, as we previously observed between active and hibernating ground squirrels. We first cloned cDNA using the 3' rapid amplification of cDNA ends (3' RACE) technique and identified three sequences corresponding to MHC₁, MHC_2x, and MHC_2b. 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.

sequencing; Spermophilus lateralis; muscle atrophy; reverse transcriptase-polymerase chain reaction

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