Pregnancy specific Beta 1 glycoprotein (SP1) besides being a good index for monitoring abnormal pregnancies, fetal well being and as a marker for trophoblastic tumors, may also play a very important role in implantation of the embryo and the subsequent growth of the fetus. The long-term objective of this proposal is to understand the functional roles of SP1 in human pregnancy with the view to enhance our knowledge of factors which effect the well-being of the growing fetus. The major obstacle to achieve this goal in the past is the occurrence of molecular heterogeneity of the SP1 protein making all the immunochemical assaying methods for its quantitation unreliable. This proposal specifically will (1) Try to solve the question of molecular heterogeneity of SP1 by cloning the cDNA of the protein. SP1 cDNA clones will be obtained by screening a human placental cDNA expression library using labelled antibody. The identity of the clones obtained will be confirmed by techniques including Western blot hybridization, genomic blot hybridization and DNA and protein sequencing; (2) Investigate the validity of cultured human skin fibroblasts, cultured placental cells, mouse, rat and baboon serving as models for studying SP1. The SP1 like molecules in these systsems will be compared with human SP1 by Northern blot hybridization first. Further comparison will be made by cloning the cDNA of these molecules and compared with human SP1 cDNA by restriction enzyme mapping and, when needed, by DNA sequencing; (3) Investigate the mechanism of enhanced SP1 production in Meckel's syndrome. Cultured fibroblasts derived from a patient will be used. The production of SP1 in these fibroblasts will be studied by immunoprecipitation. If expression of the genetic defect in the fibroblast is obvious, mechanism of SP1 overproduction will be further investigated by quantitating the mRNA of SP1 with dot blot hybridization. The cDNA will then be cloned and examined by restriction enzyme mapping and eventually DNA sequencing; (4) Determine the gene structure of SP1. This will be achieved by screening a human genomic library with SP1 cDNA probe. The clones hybridizing to the probe will be picked and sequenced. The gene structure of SP1 will be compared with that of human placental lactogen and human chorionic gonadotropin Beta chain. With techniques such as chromosomal walking, genomic blot hybridizing etc., the arrangement of these genes on the human genome will also be determined; (5) Lastly, investigate the properties of SP1 like molecules in tumor tissues using hydatidiform mole as an example. Techniques used will include Northern blot hybridization to identify the presence of SP1, dot blot hybridization to quantitate the quantity of mRNA produced, cloning restriction enzyme map and DNA sequencing to determine the structure and nature of the SP1 protein in tumor.
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