| The Glutamine/Glutamic acid-rich proteins (GRP) are highly abundant salivary proteins exclusively expressed in the acinar cells of the rat submandibular gland (SMG). Two variants of the gene encoding GRPs, GRP-Ca and GRP-Cb, were identified at the cDNA level. In this study, we have cloned the GRP-Cb gene from a rat genomic library. The gene structure is similar to other members of the GRP gene family consisting of four exons and three introns. Sequence comparison between GRP-Ca and Cb shows a high degree of homology between the exons (99-100%) and the introns (88-90%) except in exon 3 where there is a 90-96 bp region of no homology. The high degree of homology in the coding sequence of these two genes strongly suggests that they arose by gene duplication. In addition, primer extension analysis identified two putative transcription initiation sites that correspond to 38 and 35 bases upstream from the translation start site of each gene.; To determine if nuclear proteins from rat SMG specifically bind the upstream region of the GRP genes, six subclones of the GRP-Ca 5{dollar}spprime{dollar} flanking region were tested using the electrophoretic mobility shift assay (EMSA). Using rat SMG nuclear extract, specific binding activity was found to a fragment containing the region between {dollar}-{dollar}1544 bp to {dollar}-{dollar}1251 bp. This binding activity was absent in HeLa cell nuclear extract and could not be competed by oligonucleotides containing general transcription factor consensus binding sites. We hypothesize that this represents a SMG-specific regulatory element.; To evaluate the feasibility of using adenovirus-mediated gene transfer to study salivary gene expression in vivo, we tested a series of GRP-Cb/CAT and GRP-Cb/luciferase reporter constructs in rat SMGs. However, the variability of the results, even where using positive control plasmids, called into question the reliability of this technique for studying gene expression. At this time, the use of this technique is not considered an adequate alternative to other functional assays such as transgenic mice and transient transfection of cell lines. |
