Sequences and genomic organization of the human myosin heavy chain family
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The aim of this thesis research is to characterize the human sarcomeric myosin heavy chain (MyHC) gene family. MyHCs are actin-based molecular motors that convert chemical energy released from the hydrolysis of ATP into mechanical force in eukaryotic cells. The conventional or class III myosins that function to drive contraction in sarcomeres of mammalian striated (cardiac and skeletal) muscle include 8 sarcomeric MyHC isoforms. Six skeletal MyHCs are encoded by genes found in tightly linked clusters on human and mouse chromosomes 17 and 11, respectively.;This thesis presents full coding sequences for the entire human sarcomeric gene family. This involved isolating two previously unidentified human genes, MyHC-IIb and MyHC-extraocular, and completing the sequencing of MyHC-IIa and MyHC-IIx/d. It provides the first opportunity to compare the complete sequences of all sarcomeric MyHC isoforms within a vertebrate organism. In addition to calculations of overall sequence identity, sequence diversity in specific functional domains including 3 flexible loop regions, the 25/50-kDa, 50/20-kDa (Loops 1 and 2), and secondary actin binding loop, the regulatory or light chain binding domain, and the entire myosin rod was compared. The observed differences have been analyzed in the context of available structure/function data with an emphasis on potential functional diversity within the family. Results indicate that it is likely that functional diversity among MyHCs is accomplished by having small pockets of sequence diversity in an otherwise highly conserved molecule.;Also presented is the high-resolution physical mapping of the 6 human skeletal MyHC genes on chromosome 17 via PCR-based marker content mapping of P1-artifical chromosome (PAC) clones. These results indicate that unlike many clustered gene families, the MyHC genes are not arranged according to their temporal patterns of expression, but their organization does reflect the extent of sequence similarity among family members. Furthermore, comparison of the human map with the corresponding mouse MyHC physical map, reveals that the organization of the MyHC locus is highly conserved between these species. The finding that this organization has been maintained for ∼60--80 million years, suggests that it is important for the function, regulation, and/or molecular evolution of this gene family.
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