Our previous experiments indicate that the transcription of ribosomal DNA (rDNA) and ribosomal protein (r-protein) genes are reduced 3-6 fold and the translational efficiencies or r-protein mRNAs are reduced 1.3-1.6 fold after proliferating mouse myoblasts differentiate into muscle fibers. This grant proposes to identify the extracellular signals triggering these decreases and to identify the sequences in r-protein genes and r-protein mRNAs that are involved in regulating these decreases. To determine if these decreases actually occur coordinately, the transcription of S16, L18 and L32 r-protein genes and the translational efficiencies of S16, L18, and L32 r-protein mRNAs will be measured in non-proliferating cultures of myoblasts undergoing highly synchronous differentiation. These measurements will also be made in non-dividing, but non-differentiating myoblasts cultures to determine if these decreases are dependent on myoblast differentiation or upon the cessation of myoblast cell division. In these experiments transcriptional rates will be measured in nuclear transcription reactions, and the translational efficiencies of r-protein mRNAs will be inferred from the polysomal distribution of r-protein mRNAs. The sequences in the L32 r-protein gene that are involved in regulating its transcription during myoblast differentiation will be identified using deletion/replacement mapping and cell transformation techniques. A series of L32 r-protein-neomycin chimeric plasmids will be constructed that differ into the sequence content of the L32 r-protein gene. These will be stably introduced in myoblasts. The transcription of the r-protein-neomycin chimeric gene will be measured in nuclear transcription reactions from these transformed myoblasts and fibers. The same strategy will be used to identify the sequences in L32 r-protein mRNA that control the decreased translational efficiency of this mRNA during myoblast differentiation. In this case the translational efficiency of the chimeric l32 r-protein-neomycin mRNAs will be measured. Further, to determine if the transcription of rRNA or r-protein genes and the translation of r-protein mRNAs are regulated by the intracellular concentrations of rRNA, r-protein mRNAs or r-proteins, myoblast cell lines will be constructed that overproduce either rRNA or r-proteins. The transcription of rRNA and S16, L18, and L32 r-protein genes and the translational efficiencies of S16, L18, and L32 r-protein mRNAs will be measured in myoblasts and fibers of these overproducing lines.
| Hammond, M L; Merrick, W; Bowman, L H (1991) Sequences mediating the translation of mouse S16 ribosomal protein mRNA during myoblast differentiation and in vitro and possible control points for the in vitro translation. Genes Dev 5:1723-36 |
| Hammond, M L; Bowman, L H (1988) Insulin stimulates the translation of ribosomal proteins and the transcription of rDNA in mouse myoblasts. J Biol Chem 263:17785-91 |
| Bowman, L H (1987) The synthesis of ribosomal proteins S16 and L32 is not autogenously regulated during mouse myoblast differentiation. Mol Cell Biol 7:4464-71 |
