GENETIC STUDIES OF PREGNANCY SPECIFIC B1 GLYCOPROTEIN

妊娠特异的β1糖蛋白(SP1)不仅是诊断妊娠的良好指标 监测异常妊娠，胎儿健康，并作为 滋养细胞肿瘤，也可能在植入过程中发挥非常重要的作用 胚胎和随后胎儿的生长。长期的 本研究的目的是了解SP1在脑内的功能作用。 以期提高我们对哪些因素的认识 对正在发育的胎儿的健康产生影响。要实现的主要障碍是 这在过去的目标是分子异质性的发生 SP1蛋白制备及其免疫化学检测方法的研究 量化不可靠。这一建议具体将(1)努力解决 通过克隆SP1的基因序列探讨SP1的分子异质性问题 蛋白。将通过筛选一个人胎盘获得SP1的cDNA克隆 利用标记抗体构建表达文库。这些人的身份 获得的克隆将通过包括蛋白质印迹在内的技术进行确认 杂交、基因组印迹杂交、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、大鼠和恒河猴作为研究SP1的模型。 这些系统中的类SP1分子将通过以下方式与人类SP1进行比较 首先进行Northern杂交。将通过以下方式进行进一步的比较 克隆了这些分子的基因，并与人SP1基因进行了比较 限制性内切酶图谱，并在需要时进行DNA测序； 探讨Meckel综合征患者SP1分泌增加的机制。 将使用来自患者的培养成纤维细胞。制作 这些成纤维细胞中SP1的表达将通过免疫沉淀法进行研究。如果 基因缺陷在成纤维细胞中的表达明显，其机制 SP1的过量生产将通过定量检测SP1的mRNA来进一步研究 SP1进行斑点杂交。然后将克隆并检测该cdna。 通过限制性内切酶图谱和DNA测序；(4)确定 SP1的基因结构。这将通过筛查一个人类来实现 用SP1基因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。我们将比较SP1的基因结构 与人胎盘催乳素和人绒毛膜促性腺激素β 链条。利用染色体行走、基因组印迹等技术 通过杂交等，这些基因在人类基因组上的排列将 也被确定；(5)最后，研究类SP1的性质 以葡萄胎为例，肿瘤组织中的分子。 将使用包括Northern杂交在内的技术来鉴定 SP1存在时，斑点杂交法定量检测 制作、克隆限制性内切酶图谱并进行DNA测序确定 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.