[Expression of mutation type GJA8 gene and wild type GJA8 gene of a congenital inherited nuclear cataract family in eukaryotic cells].

OBJECTIVE: To clone the sequence of mutation type GJA8 gene (mGJA8) and wild type GJA8 gene (wGJA8) of a congenital inherited nuclear cataract family and study their expression in eukaryotic cell lines in vitro. METHODS: The mGJA8 and wGJA8 were amplified from this family's DNA and healthy people's DNA by PCR respectively. The mGJA8 and wGJA8 were recombined with plasmid pEGFP-N1 respectively. The accuracy of pEGFP-N1-GJA8 was confirmed by restriction enzyme digestion and DNA sequencing. Finally pEGFP-N1- mGJA8 and pEGFP-N1- wGJA8 and GFP protein were transfected into COS7 cells by lipofectin. The expression of pEGFP-N1-GJA8 and GFP fusion protein were to observe under fluorescence microscope, and to detect by Western-blotting and immunohistochemical staining. RESULTS: The mGJA8 and wGJA8 were cloned successfully. With restricting enzyme digestion analysis and DNA sequencing, recombinant plasmid pEGFP-N1-mGJA8 and pEGFP-N1-wGJA8 were constructed correctly and their GFP fusions were expressed in transfected COS7 cells. The expression of pEGFP-N1-mGJA8 and pEGFP-N1-wGJA8 fusion protein were observed under fluorescence microscope, and detected by Western-blotting and immunohistochemical staining successfully. CONCLUSIONS: The mGJA8 gene and wGJA8 gene are cloned successfully, and pEGFP-N1-mGJA8 and pEGFP-N1-mGJA8 fusion protein can be expressed in COS7 cells, which establish the foundation for further studying the mechanism of this congenital inherited nuclear cataract family.

[Comparative study of eukaryotic cell expression of wild-type and Pro370Leu mutation type myocilin gene].

OBJECTIVE: To compare the eukaryotic cell expression of wild-type myocilin (MYOC) (wMYOC) gene and Pro370Leu mutation type MYOC (mMYOC) gene so as to understand the mechanism of primary open angle glaucoma (POAG). METHODS: HeLa cells were cultured and then transfected with the vector pEGFP-N3-wMYOC, recombinant plasmid with wild type MYOC gene and enhanced green fluorescein gene, or the vector pDsRed2-N1-mMYOC, recombinant plasmid with mutation MYOC gene and red fluorescein gene, respectively, or co-transfected with these 2 plasmids. Corresponding blank vectors pEGFP-N3 and pDsRed2-N1 were used as markers. Fluorescence microscopy was used to observe the localization of red fluorescence and green fluorescence. Laser co-focusing microscopy was used to observe the effect of co-transfection. The green and red fluorescein antibodies were examined by Western blotting. RESULTS: Green fluorescence was observed in the cytoplasm of the HeLa cells transfected with the blank vector pEGFP-N3, in an even distribution; and red fluorescence was observed in the cytoplasm of the HeLa cells transfected with the blank vector pDsRed2-N1, in an even distribution too. The cells transfected with pEGFP-N3-wMYOC showed evenly-distributed green fluorescence in the cytoplasm, and the cells transfected with pDsRed2-N1-mMYOC showed red fluorescence in the cytoplasm in a state of aggregation. Both red and green fluorescence could be seen in the cells co-transfected with pEGFP-N3-wMYOC and pDsRed2-N1-mMYOC, both in a state of aggregation and co-localization. Laser co-focusing microscopy showed the same results. Protein with the relative molecular weight of 83,000, identical to that of the recombinant protein of wMYOC protein and green fluorescein, could be found in the culture fluid and cell lysate of the pEGFP-N3-wMYOC-transfected cells; however, could be found in the lysate only but not in the culture fluid of the co-transfected cells. Protein with the relative molecular weight of 81,000, identical to that of the recombinant protein of mMYOC protein and red fluorescein, could be found in the cell lysates of the pDsRed2-N1-mMYOC-transfected cells and the pEGFP-N3-wMYOC and pDsRed2-N1-mMYOC co-transfected cells, but not in the culture fluid of both cells. CONCLUSION: Both the WMYOC and mMYOC genes can be expressed and localized in the cytoplasm. mMYOC protein shoes a tendency of aggregation and disorder in secretion, and affects the expression and secretion of wMYOC, thus influencing the factions of the trabecular meshwork and causing POAG.

[A heterozygous transversion of connexin 50 in a family with congenital nuclear cataract in the northeast of China].

OBJECTIVE: To identify the genetic defect causing autosomal dominant congenital cataract (ADCC) in a five-generation family in the northeast of China. METHODS: Linkage analysis was carried out with polymorphic microsatellites on the Human MapPairs marker set, special known loci. Mutation analysis of the candidate gene in the critical region was performed to detect the potential mutation. RESULTS: The maximum Lod score (2.44 at recombination fraction theta=0) was obtained for markers D1S498,D1S305, and D1S2844. The cataract locus in this family constellation was mapped to 1q21.1 and 21.44 cM interval between D1S2344 and D1S2844, which were known to flank the gene coding Connexin 50 (Cx50) or gap junction protein alpha-8 (GJA8). Sequencing of the coding region of GJA8 gene showed a heterozygous transversion T>G in exon 2, which resulted in the substitution of glycine for valine at amino acid 64, and this position was in the first connexin signature region that characterized this protein. CONCLUSION: This is the first report on a mutation in the first connexin signature region of the GJA8 and a different mutation within Cx50 revealed in this family, which might account for the phenotypic differences observed. Furthermore, this study confirmed that GJA8 plays a vital role in the maintenance of human lens transparency.