Pilot Study On Function Of The Novel Homo Gene MR-1

MR-1(myofibrillogenesis regulator 1)gene is a novel human gene cloned in the lab of Dept. of Pathway Engineering at the Institute of Medicinal Biotechnology. The complete cDNA of MR-1 is made up of 416 nucleotides, encoding 142 amino acid residues. Its EST sequence was cloned the first time from human ovarian tumor tissue and was found also presented in uterine endomembrane tissue and parathyroid tissue. Pilot studies indicate that MR-1 is related with the proteins involved in regulation of muscle contraction and cell signal transmission, and there is a transmembrane region in this protein.The first part of this research is to detect the mutation of hMR-1 gene by using non-isotopic polymerase reaction-based single-strand conformation polymorphism(PCR－SSCP). PCR- products of three exons were obtained by designing three pairs primers which were aimed to three exons of hMR-1 gene. PCR-SSCP analysis showed that the synonymous mutation was found only in exon3 of the hMR-1 gene. The study seems to indicate that the mutation of the hMR-1 was not necessarily related to cardiac muscle hypertrophy, heart failure, coronary heart disease and tumour. The second part is to find new proteins which interact with hMR-1 protein by using Yeast two-hybrid system and to clone their encoding sequences, and to research the interaction between these positive AD proteins and two segments which were from hMR-1. The yeast strain AH109 and bait vector (BD) pGBKT7 of MATCHMAKER GAL4 Two-Hybrid system 3, developed by CLONTECH Company of USA, were used in this research. Firstly, the full-length of hMR-1 gene was cloned into the GAL4 DNA-binding domain of the vector pGBKT7 to construct a bait plasmid. Then the bait plasmid was used to screen Human Skeleton Muscle cDNA Library which was constructed on the pACT2 vector, and the β-gal analysis was carried out to verify the interaction between proteins. As a result, 9 positive AD plasmids were obtained. The results of DNA sequencing and homologous comparison showed that the cDNA fragments in the positive AD plasmids encoding the proteins belong to eight types, among which three proteins were related to muscle contraction. Subsection Yeast two-hybrid indicate that the interactional locations between these AD proteins and hMR-1 were different. This study facilitated the further elucidation of MR-1 function in cell biology, especially its function involved in the regulation of muscle contraction.The third part is to further study function of the hMR-1 by constructing hMR-1-transgenic mice model . By microinjection techniques on germ nucleus of zygotes hMR-1 gene were injected into zygotes of CD-1?∕(ICR)BR mice and transplanted into the oviducts of pseudopregnancy female mice. The integration and expression of exogenous genes in offspring were detected by PCR and Western Blot, respectively. There were 6 founders of all injected mice, which had steadily inherited to the third generation, and three steady ancestry were obtained. Western Blot showed that there were obvious differences between transgenic mice and negative mice in the expression of hMR-1. The function of MR-1 need be further studied.