The objective of this study is to understand the structure, function and control of expression of a group of structurally related macromolecules, the pregnancy-associated glycoproteins (or PAG), that are secreted by the trophoblast of sheep, cattle and possibly other mammals, beginning in the pre-implantation period and continuing to term. The PAG, which are detectable in maternal serum and whose presence there can be used to diagnose pregnancy, have marked sequence similarity to pepsin and other aspartyl proteinases but appear to be catalytically inactive because of critical changes in amino acid sequence in the active site region. However, because they still retain the large substrate-binding cleft and are capable of binding peptide, it is hypothesized that these molecules may be a novel kind of hormone or binding protein.
The specific aims are: 1) to determine the peptide-binding specificity of PAG by screening a bacteriophage epitope library that expresses a very large number of hexapeptides; 2) to describe the organization of the PAG genes and to study their number and distribution among mammals; 3) to define the sequences upstream of the transcription start site of the PAG genes that may be responsible for their expression in binucleate cells. The latter aim will entail completion of the sequencing the upstream regions of the genes, defining conserved regions, transfection of promoter constructs linked to reported genes into trophoblast cells, and gel shift and footprinting assays. 4) To develop lines of mice transgenic for reporter genes under the control of the PAG promoter and to determine whether trophoblast-specific expression is retained and which cells of the trophoblast are implicated. It is anticipated that the study may lead to characterization of a novel group of proteins likely to have an important but presently undefined role in pregnancy.
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