| Summary: | 碩士 === 淡江大學 === 生命科學研究所碩士班 === 95 === MRF4 belongs to the family of myogenic regulatory factors（MRFs）and participates in the differentiation and proliferation of myoblasts. Previous studies in Zebrafish indicated the existence of two different types of MRF4 gene, namely MRF4a and MRF4b. This situation is the same as the three other members of MRF family: Myf5, MyoD, Myogenin of which they share a conserved bHLH domain. Other studies indicated another functional protein sequence is present at the carboxyl end following the bHLH domain. It is predicted to be an amiphipathic α-helix, and is referred to as「Helix 3 domain」. It was also verified that this α-helix is capable of promoting expression of endogenous muscle gene during skeletal myogenesis. Because「Helix 3 domain」of carboxy-terminal region of MRFs family exhibits highly conserved sequence﹐I substituted amino acid 219 for proline in the carboxyl terminal sequence of MRF4b by site-directed mutagenesis PCR to determine any change in the secondary structure. I also utilized yeast one-hybrid analysis to investigate the role of「Helix 3 domain」in the transcription activity of MRFs. I used E.coli BL21-CodonPlus (DE3) RIL strain to produce MRF4b C-terminal protein, MRF4b C-terminal protein and MRF4b C-terminal mutant protein. I then compared the difference of secondary structures of these three different recombinant proteins by circular dichroism. The results indicated「Helix 3 domain」contains a「β-sheet」. In addition, yeast one-hybrid system utilizes GAL1-HIS3 reporter gene to observe the transcription activity of MRFs in the yeast host cell. I found MRF4b is the best of all three MRFs. I infer that MRF4b with「Helix 3 domain」has a higher activity than the other two forms of MRF.
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