Fang Feng
2011
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Journal
Journal of Applied Clinical Pediatrics
Abstract
Objective To find the domains of M122 involved in the interaction with Myst4. Methods The DNA fragments encoding various truncated M122 were amplified by polymerase chain reaction using pGBKT7-M122 as a template,and then inserted into pMD-T smiple vector,respectively.After verified with restriction endonuclease digestion of EcoRI and SalI,the right fragments of various truncated M122 gene determined by sequencing were subcloned into pGBKT7-BD vector,respectively.All the recombinant plasmids were verified by restriction enzyme digestion and DNA sequencing.Then recombinant plasmids were transformed respectively into yeast strain AH109 together with plasmid pGADT7-Myst4.The transformed yeast cells were plated on nutrient deficiency medium SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade/X-α-Gal.The M122 domains involved in the interaction were identified by auxotrophic selection. Results DNA fragments encoding deletion mutants of the M122 protein were amplified and cloned into bait vector.The reconstructed bait plasmids containing various truncated M122 were successfully constructed.The binding site of M122 and Myst4 was mapped to the region(residues 1-148) of M122. Conclusions The region(residues 1-148) of M122 is responsible for the interactions of M122 with Myst4.It provides an experimental basis for further studying the actions of M122 in molecular neuropathogenesis of mouse cytomegalovirus.