KISEP Otology Korean J Otolaryngol 2001;44:239-45 유전성난청과동반된 GJB2 유전자변이의세포간극에대한기능적연구 정연훈 유상준 이준호 박홍준 Functional Study of Gap Junction in GJB2 Mutations Associated with Hereditary Hearing Loss Yun Hoon Choung, MD, DDS, Sang Jun Ryu, MD, Joon Ho Lee, MD and Hong-Joon Park, MD Department of Otolaryngology, Ajou University School of Medicine, Suwon, Korea ABSTRACT Background and ObjectivesGJB2 Connexin 26, the gene of the gap-junction proteins, was found to be the main causative gene of autosomal recessive nonsyndromic hearing loss DFNB1. Whereas 35delG was known as the major type mutation in the western countries, 235delC was reported as the specific form of mutation in Asian population. The objective of this study is to identify how two mutations 235delC, E114G found in the Korean population affect the function of GJB2 using the molecular biology techniques. Materials and Methods235delC and E114G types of mutations were cloned in the pcdna3 vector. HeLa cells were transfected with these cloned vectors by the liposome complex method. 1 The expression and subcellular localization of Cx26 were determined using antibodies against amino acid sequences in the intracellular loop IL and N-terminal NT portions of Cx26. 2 To analyze functions of the GJB2, we examined the lucifer yellow dye transfer between cells with scrape-loaded technique. We used the wild-type WT Cx26 of normal hearing as a positive control, and mock cells as a negative control. ResultsThe immunocytochemical analysis showed that cells transfected with E114G and WT gave characteristic punctuated patterns of reaction in the cell membrane with both antibodies. However, 235delC cells were not stained with the anti-il antibody but only with the anti-nt antibody slightly around the nucleus regions. In the functional study of GJB2, transfer of lucifer yellow dye into contiguous cells was detected in E114G but not in 235delC. ConclusionThe 235delC type of mutation showed loss of their targeting activity on the cell membrane. As a result, the function of gap junction channels were severely deteriorated. With the E114G type mutation, we didn t find any difference when compared with the WT transfected cells. Above data indicate that types of GJB2 mutation are closely related to the status of hearing loss due to altered function of gap junction protein. Korean J Otolaryngol 2001;44:239-55 KEY WORDSHereditary hearing loss Gap junction protein Connexin 26 Functional study. 239
240 효소중합반응 Transformation Fig. 1. Schema of this experiment. This study was carried out by 3 steps of transformation, transfection, subcellular localization of Cx26, and functional study of gap junction using 235delC, E114G, wild-type Cx26, and mock cells. The expression and subcellular localization of Cx26 was determined using antibodies against amino acid sequences in the intracellular loop and N-terminal of Cx26. To analyze functions of the gap junction, we examined Lucifer yellow dye transfer between cells with scrape-loaded technique. Korean J Otolaryngol 2001;44:239-45
Plasmid DNA preparation Cx26 발현및세포내위치확인 241
간극결합단백질의기능적연구 242 Fig. 2. The molecular structure of connexin 26. 235 delc and E114G are located in M2 and IL, respectively. Epitopes which are used in the immunocytochemistry A N- terminal, B int-racellular loop. M 1-4transmembrane domains, ILintra-cellular loop. Korean J Otolaryngol 2001;44:239-45
A B A B C D C D Fig. 3. Immunocytochemistry of HeLa cells using primary antibody against intracellular loop portion of Cx26. Awildtype WT BE114G C235delC Dmock transfected cells, respectively. Both WT and E114G transfected HeLa cells showed positive immunofl-uorescent reactions in the cell membranes. However, 235delC transfected cells showed no reaction like as mock transfected cells. Fig. 5. Functional study of GJB2 using scrape-loaded dye Lucifer yellow technique. Awild-type WT BE114G C 235delC trans-fected cells, respectively Fluorescent microscopic view. Transfer of Lucifer yellow dye into contiguous cells were detected in WT and E114G but not in 235delC transfected HeLa cells. DLight microscopic view of HeLa cells with scrape-loaded dye technique. A C Fig. 4. Immunocytochemistry of HeLa cells using primary antibody against N-terminal portion of Cx26. Awild-type WT BE114G C235delC Dmock transfected cells, respectively. Both WT and E114G transfected HeLa cells showed positive immunofluorescent reactions in the cell membranes. However, 235delC transfected cells showed weak reactions only around the nucleus regions. Mock transfected cells showed no reaction. B D 243
244 Korean J Otolaryngol 2001;44:239-45
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