The long term objective of this proposal is to understand the functional roles of the different species of """"""""pregnancy-specific beta 1-glycoprotein (SP1)"""""""" in human pregnancy with a view to enhance the knowledge of factors which affect the establishment of pregnancy and the well-being of the growing fetus. The immediate goals of this proposal include elucidating the structure of the different SP1 cDNAs and genes, demonstrating the factors regulating gene expression, and defining the potential physiological functions of these proteins. Initially, the nucleotide sequence of the different SP1 CDNAs will be determined. Gene-specific oligonucleotide probes will be synthesized basing on the structural differences among these cDNAs. The different species of human SP1 proteins will be prepares by in vitro expression. Specific monoclonal antibodies (MAb) to each species of SP1 will also be prepared. Factors affecting the expression of RNA dot blotting techniques. Gestational effects will be studied using placental tissues and maternal serum. Tissue specificity of SP1 gene products will be examined in tissue cultures as well as using cDNA libraries derived from different tissues. Effects of hormones and other molecules will be examined using trophoblastic cultures. SP1 genes will be characterized with respect to the intron/exon location as well as the transcription initiation and termination sites using techniques such as restriction enzyme mapping, Northern and Southern blot analysis, S1 nuclease protection assay, primer extension and nuclear run-on assay. Organization of SP1 gene family will be studied by chromosomal localization using gene-specific probes and chromosomal walking. Functional studies of SP1 include identification of its receptor, examination of its immunological functions and growth enhancing properties. Existence of specific receptors for SP1 will be examined in cultured cells. The potential immunosuppressive effect of SP1 will be investigated using stimulated lymphocyte systems. The ability of SP1 in blocking cytotoxic T cell action and in binding immunoglobulins will also be investigated. THe growth effect of Sp1 will be studied using cultured cells.
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