Molecular mechanisms that lead to the progressive loss of muscle size and strength with age in humans have not been elucidated. Age-related changes in the ability of adult myoblasts to proliferate and differentiate in response to muscle damage could reduce muscle size. Beta-enolase, a muscle-specific glycolytic enzyme, is expressed in proliferating myoblasts of adult, but not fetal muscle. This is in contrast to most muscle- specific genes which are expressed upon differentiation, under the control of the MyoD family of regulators. The goal of this research is to identify the molecular mechanisms that control beta-enolase gene expression in adult myoblasts and determine if changes in gene regulation occur with age. This will be accomplished by defining the cis-regulatory DNA sequences responsible for controlling the beta-enolase gene. Expression of reporter gene constructs containing different amounts of DNA flanking the beta- enolase gene will be assayed following transfection into muscle and nonmuscle cells. Proteins that bind to the regulatory DNA sequences will be isolated from an adult human myoblast cDNA expression library. Beta- enolase is also expressed in differentiated myofibers and accumulation of beta-enolase mRNA changes with age in muscle tissue. This may be indicative of more global changes in the metabolic activity of the muscle since several lines of evidence suggest that genes encoding glycolytic enzymes may be coordinately regulated. The molecular mechanisms that control beta-enolase gene expression in myofibers will also be explored. The role of the MyoD family of proteins in regulating beta-enolase expression following differentiation will be ascertained. Finally, the human homology to the yeast GCR1 gene product, which has been shown to be necessary for the coordinate regulation of yeast glycolytic enzyme genes, will be isolated. A GCR1 DNA probe, generated using oligonucleotide primers to published sequence and PCR, will be used to screen a human myoblast cDNA library under low stringency. Therefore, in addition to identifying factors involved in muscle-specific expression, factors involved in the coordinate regulation of metabolic enzyme gene expression will be identified. Changes in the activity of these factors may contribute to the loss of muscle function with age.
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