Use of a cDNA library for the study of mRNA changes during muscle differentiation.

ABSTRACT

A cDNA library was used to measure changes in many individual mRNAs during muscle differentiation in culture. A library of 1000 clones was constructed from total myofiber poly(A) RNA. About 23% of these clones gave a detectable colony hybridization signal using end-labeled myofiber mRNA, the remainder containing muscle sequences too rare to be detected with this assay. The 230 positive clones were grouped into four classes based on relative visual intensity. Reconstruction experiments using pure globin mRNA enable us to determine the approximate percentage of total RNA made up by each mRNA hybridizing to a cDNA clone. Those clones containing sequences complementary to developmentally regulated mRNAs were identified by a differential hybridization procedure. The cDNA library was screened with end-labeled mRNA from both undifferentiated myoblasts and differentiated myofibers. Although the bulk of the clones hybridized essentially the same with both RNA populations, several dozen were found which hybridized differentially. Some clones contained sequences which were not present at all in myoblasts and present in very high quantities in myofibers. Others contained sequences found in both myoblasts and myofibers but in increased quantities in the differentiated cells. Still others contained sequences which decreased in quantity during muscle differentiation. The clones in the first group were chosen for immediate analysis since they likely contain contractile protein mRNA sequences. However, all the characterized cDNA clones can now be used as probes to study the chromosomal organization and developmental expression of genes active during muscle differentiation.