Expression, Characterization of 3-AS Novel Gene in Human Tooth Tissues

Cementum is a unique mineralized tissue that covers the root surface of teeth, however very little is known about its growth and differentiation because the lack of specific markers. Objectives: the aim of this research was to characterize a novel gene which we have named 3-AS (GenBank accession Num. EU220031). Methods: in silico analysis of NCBI; PROSITE and ExPASSY data bases were used to determine the homologies of 3-AS gene sequence. RT-PCR and in situ hybridization were used to study the temporal and spatial expression of 3-AS gene in human and periodontal tissues. We cloned the 3-AS gene sequence onto an expression vector for Drosophila melanogaster system. Results: analysis of 3-AS sequence gave us a theoretical protein of 195 aminoacids with a Mr of 17,760 with possible phosphorylations in amino acids Ser, Thr; O-glycosilation and C-manosilations. Purification of the rh3-AS protein were analyzed by Western blot using specified antibodies against the 6xHis-tag and V5 epitope showed a band with a 35 kDa MW. Antibodies against phosphorilations crossreact with this prominent band. RT-PCR and in situ hybridization determined that the expression 3-AS mRNA in human adult periodontal tissues showed strong signal of the hybrids RNA-RNA in cellular cementum, fibroblasts of periodontal ligament and some cells around blood vessels. Conclusions: we have isolated a new gene that is highly expressed in cementum and periodontal ligament. This could contribute to determine its possible role during cementum and periodontal ligament formation. This project was partially supported by Grants from DGAPA-UNAM: IN204705, IN200808, IN200908, CONACYT 48638.