GENETIC STUDIES OF PREGNANCY SPECIFIC B1 GLYCOPROTEIN

妊娠特异性β 1糖蛋白（SP1）除了是一个很好的指标， 监测异常妊娠、胎儿健康，并作为 滋养细胞肿瘤，也可能在植入中发挥非常重要的作用 以及胎儿的后续生长。 长期 本提案的目的是了解次级方案1在以下方面的职能作用： 人类怀孕，以提高我们的知识的因素， 影响胎儿的健康成长 实现的主要障碍 在过去，这一目标是发生分子异质性， SP1蛋白的免疫化学检测方法 定量不可靠。 该提案具体将（1）试图解决 通过克隆SP1的cDNA， 蛋白 将通过筛选人胎盘细胞获得SP1 cDNA克隆， 利用标记抗体构建cDNA表达文库。 的身份 获得的克隆将通过包括蛋白质印迹的技术进行确认 杂交、基因组印迹杂交以及DNA和蛋白质测序; (2)研究培养的人皮肤成纤维细胞的有效性， 胎盘细胞、小鼠、大鼠和狒狒作为研究SP1的模型。 将这些系统中的SP1样分子与人SP1进行比较， 先进行北方印迹杂交。 进一步的比较将由 克隆这些分子的cDNA，并与人SP1 cDNA进行比较， 限制性内切酶图谱，必要时通过DNA测序;（3） 探讨Meckel综合征中SP1产生增强的机制。 将使用来自患者的培养成纤维细胞。 生产 用免疫沉淀法研究SP1在这些成纤维细胞中的表达。 如果 基因缺陷在成纤维细胞中的表达是明显的， 将通过定量以下细胞的mRNA进一步研究SP1的过度产生： SP1的斑点杂交。 然后将克隆cDNA并进行检查 通过限制性酶切图谱和最终DNA测序;（4）确定 SP1的基因结构 这将通过筛选人类来实现 用SP1 cDNA探针构建基因组文库。 与探针杂交的克隆 将被挑选出来并进行排序 将比较SP1的基因结构 与人胎盘催乳素和人绒毛膜促性腺激素β 链 利用染色体步移、基因组印迹等技术 杂交等，这些基因在人类基因组中的排列 （5）最后，研究了类SP1的性质， 以葡萄胎为例， 使用的技术将包括北方印迹杂交以鉴定 存在SP1，斑点杂交以定量mRNA的量 生产，克隆限制性内切酶图谱和DNA测序，以确定 肿瘤中SP1蛋白的结构和性质。

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.