| Abstract |
Basement membranes are thin, sheet like arrangements of extracellular matrix proteins that serve a variety of functions, including the physical separation of cell layers and tissues, molecular ultrafiltration and tissue morphogenesis and display two distinct zones, the lamina lucida and the electron dense lamina densa which lies underneath the lamina lucida. Fras1 is expressed by various epithelia during mouse development, whereas the encoded protein is localized within the sub-lamina densa of the epithelial basement membranes. Mutations in Fras1/FRAS1 are responsible for the blebbed phenotype in mouse and the Fraser syndrome in man, respectively. Τhree of the 5 bleb mutations in mouse which exhibit similar phenotypes, have been ascribed to Fras1 or Fras1-related genes: Fras1 which has been linked to the blebbed phenotype, Frem1 responsible for the head-blebs, and Frem2 for the myelencephalic bleb mutation and only FRAS1 has been associated with Fraser syndrome. Also, the Grip1 gene, which encodes a cytoplasmic PDZ protein, has been linked to the eye-bleb mutant. The translation product of Frem3 exhibits significant homology to Fras1, and it could potentially be responsible for the fifth bleb mutation, however this has not yet been proved. All 5 bleb mutations are characterized by sub-epidermal hemorrhagic blisters, renal agenesis or dysplasia, fused eye lids and syndactyly. Aiming to understand the molecular basis of Fraser syndrome, as well as, the structural and functional defects underlying the phenotype and the relation, if any, of the Fras1/Frem family of proteins, in this project we performed phage library screenings in order to isolate part of the genomic DNA of Frem2 and Frem3, as well as the cDNA of Frem2. The cDNA of Frem2 gene will provide a useful tool in order to study protein-protein interactions, both between Fras1/Frem proteins and between proteins of this family with other proteins of the extracellular matrix. On the other hand, and since Frem3 has not yet been studied, the isolation of its genomic DNA will be used in order to inactivate it in mouse and study the outcome phenotype. Importantly, we isolated 6 putatively positive clones, which contain part of the cDNA of Frem2 and we now proceed further for their characterization. Ιn addition, we have also isolated a series of positive clones, for the isolation of Frem2 and Frem3 genomic DNA, which will be used for the second and third screening, respectively. Moreover, in order to characterize further the Frem3 gene we made antibodies against the protein, as well as RNA-probe, and studied its expression pattern in wild type mouse embryos, of various developmental stages, through immunohistochemical and RNA in situ hybridization (ISH) techniques. The results from ISH indicated the epithelial origin of Frem3, which is consistent with the pattern seen at the rest genes of the family. Also, immunohistochemistry showed expression of Frem3 protein at the basement membranes of various epithelia (e.g. epidermis, lungs, kidneys, gut), beginning at E11.5 and reaching the best expression pattern at E15.5. However, in the skin and choroids plexus, it was shown to surround the epithelial cell nucleus, resembling the expression pattern of Frem1 protein and indicating a possible cooperation between those proteins, as well as an intracellular localization, as opposed to Fras1 and Frem2.
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Frem3: ένα νέο μέλος της οικογένειας των γονιδίων Fras1/Frem. Εντοπισμός έκφρασης στον ποντικό και απομόνωση γενωμικών ενθεμάτων
