Recently, we have isolated cDNA clones encoding the low molecular weight human salivary mucin, MG2 (MUC7) and deduced the amino acid sequence of its protein core from the nucleotide sequence. Further, we have determined that the expression of MUC7 gene appears to be extremely tissue- (submandibular and sublingual gland) and cell-(mucous acini) specific. Little is known about the regulation of mucin gene expression in general and nothing regarding the human salivary mucin genes. We hypothesize that expression of MUC7 is regulated at the transcriptional level by a combination of specific cis-acting elements in the DNA and trans-acting molecules (transcription factors) found in the salivary glands. In this project, studies will be carried out towards understanding the molecular mechanism underlying the tissue- and cell- specific expression of the MUC7. Before these studies can be initiated, however, the MUC7 gene(s) and the flanking sequences will be isolated and characterized. Also, with MUC7 cDNA probe, we will examine the MUC7 cell-specific expression by in situ hybridization, determine if MUC7 gene exhibits genetic polymorphism and examine MUC7 homologue(s) in phylogenetically close and distant species; in particular we will study a mouse homologue with intention of using it as a marker for our future studies. Since appropriate cell lines for studies of regulation of MUC7 gene expression in vitro are not available, we will examine the MUC7 expression in vivo by transgenic animal technology. Transgenic animals can, however, also be used for establishing new cell lines. Thus, the goals of the transgenic approach will be to define the cis-acting regulatory elements controlling the tissue- and cell-specificity of MUC7 expression and use the transgene format to develop novel mouse cell lines capable of MUC7 expression. These cell lines should provide an excellent tool for studying mucin gene regulation. We would like to perform similar studies with the high molecular weight mucin, MG1.However, the primary structure of this mucin still remains to be determined. Thus, in this part of the project, we propose to use molecular cloning techniques in order to obtain the structural information about the protein core of the MG1 Several strategies will be developed and pursued simultaneously that will allow us to isolate apo-MG1 cDNA sequences from a cDNA library or generate apo-MG1 cDNA by the PCR-based approach. Amino acid sequence of the apo-MG1 will then be deduced from the nucleotide sequences.
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