A long-term aim is to delineate the mechanisms by which chronic changes in contractile activity alter the phenotypic expression of skeletal muscle. In the regrowth of of atrophied skeletal muscle after ending limb immobilization, all proteins increase in response to normal muscle usage. Further increases in all proteins in normal adult skeletal muscles are only possible if it undergoes maximal loading exercise, such as weight-lifting. In contrast, repetitive, low-intensity exercise like running results in no enlargement of normal muscle, but causes an preferential expression of mitochondrial proteins, such as cytochrome c. A strategy of the present application is to compare the responses of mrna levels, protein synthetic machinery and potential molecular signals controlling the upregulation of specific protein quantities in different types of exercises known to cause various phenotypic expressions. The following methods will be employed in the comparison among exercises. In situ hybridization will permit the localization of transcripts to nuclei by using intron probes, so a determination can be made as the time when changes in the levels of specific nuclear transcripts occur after the exercise and for which transcripts. The four cytochrome c mRNAs will be isolated from a cDNA library, cloned, sequenced and translated so as to provide information on the functional significance of shifts in their relative profiles which occur during regrowth from atrophy and during a program of repeated daily bouts of running. A rat genomic library will be screened for fragments containing 5' upstream and coding sequences for somatic cytochrome c, a skeletal actin, fast myosin-heavy-chain and rRNA for the purposes of obtaining intron probes for in situ hybridization. Also, the 5' stream sequences will be used for a further proposal to study regulatory sequences for gene expression. In accordance with this long-range plan, studies are initiated in the present application to identify potential candidates for signals which may interact,directly r indirectly through a cascade, with the 5' untranscribed regulatory region of the genome. The potential roles of the oncogenes, ATP, creatine phosphate, H+, and Ca++as the linkers from increased muscle usage to differential gene expression will be considered.
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