Beta-ig. Molecular cloning and in situ hybridization in corneal tissues.

摘要: PURPOSE To identify a protein that copurifies with type VI collagen from rabbit cornea and to determine its cell source in corneal tissues by situ hybridization. METHODS Type was extracted urea purified ammonium sulfate precipitation gel chromatography. The purity of the assessed sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). On reduction mercaptoethanol or dithiothreitol, alpha chains ran into gel. In addition polypeptides, an extra 68-kDa band appeared, suggesting this is present as large molecular weight component before reduction. Amino acid sequencing indicated related beta ig-h3 humans. Western blot analysis used immunologic similarity human protein. A stromal cDNA library screened probe. Positive clones were sequenced analyzed for sequence homology. Oligonucleotide probes prepared sequences Northern hybridization tissues. RESULTS Electroblotting SDS-PAGE amino first 10 N-terminal acids gave 100% homology known produced adenocarcinoma cells, ig-h3. This identical immunologically analysis. Sequence clone contained whole coding region had high identity both mouse ig-m3. deduced 92% these species. An oligonucleotide probe detected single mRNA cultures cells consistent size mRNA. authors refer form ig because obtained normal comes primary not cloned line. tissue located primarily epithelium adult cornea, fetal endothelium- stroma-derived healing wounds. Normal endothelium stroma did show label. CONCLUSIONS highly conserved between human, mouse, proteins temporal expression message during development suggest plays important role morphogenesis