OVINE PLACENTAL LACTOGEN: ITS REGULATION AND FUNCTION

长期目标：胎儿生长速度不仅决定着胎儿的大小 出生时的后代，但也影响分娩的便利性和 子代的出生后生长速度。不幸的是，我们的理解 影响产前发育的因素及其作用机制 这些因素的作用充其量也是有限的。现有证据表明 绵羊胎盘催乳素(OPL)在 调节胎儿新陈代谢。远期目标的目标是(1) 了解生物功能、作用机制和 胎盘分泌蛋白质的生化性质及(2) 了解胎盘产品的调节机制 都是制造出来的。这些信息可能导致以下方法：(1)减少怀孕 失败和(2)调节胎儿生长速度。 具体目标和方法：(1)纯化同质，获得氨基 OPL受体上的酸序列信息。OPL受体将是 从妊娠120天的胎儿肝脏中提纯 阴离子交换法、亲和力、排除法和反萃取法的结合。 相色谱。纯化的受体将受到有限的 氨基末端气相氨基酸测序。(2)生成、隔离 并对OPL受体的全长CDNA进行了测序。共识 绵羊生长激素(OGH)受体与牛之间的序列 催乳素(BPRL)受体CDNA将作为逆转录酶的引物 逆转录聚合酶链式反应(RT-PCR)扩增胎儿 肝脏mRNA将产生OPL受体特异性CDNA探针以筛选A 胎肝CDNA文库。或者，生长激素和催乳素受体CDNA 或从氨基酸序列中产生的寡核苷酸探针 OPL受体将用于筛选CDNA文库。曾经的CDNA 编码OPL受体是分离的，它将受到 用双脱氧链终止法进行核苷酸测序。这个 将推断OPL受体的初级氨基酸序列，并 然后与其他蛋白质激素进行序列相似性比较 感受器。(3)分离和结构鉴定S基因。 OPL。OPL的基因组克隆将被定位，并将外显子与相邻的 侧翼区域将被测序，以确定信使核糖核酸剪接位点。这个 将确定转录起始点和基因拷贝数，并 5‘-上游调控区域(1至2 kb)将被测序至 检查潜在的顺式作用的监管要素。未来的重点将是 被放在检测OPL基因的组织特异性表达上， 以及对OPL的作用机制进行研究。

Long-Term Objectives: Fetal growth rate not only determine the size of the offspring at birth, but also influences the ease of delivery and postnatal growth rate of the offspring. Unfortunately, our understanding of what factors influence prenatal development and the mechanism of action of these factors is limited at best. Existing evidence suggests that ovine placental lactogen (oPL) plays a significant role in modulating fetal metabolism. The long-term objectives are aimed at (1) understanding the biological functions, mechanisms of action and biochemical properties of proteins secreted by the placenta and (2) understanding the regulatory mechanisms under which placental products are made. This information could lead to methods to (1) reduce pregnancy failure and (2) modulate fetal growth rate. Specific Aims and Methods: (1) Purify to homogeneity and obtain amino acid sequence information on the OPL receptor. The OPL receptor will be purified from fetal liver collected at 120 days of gestation by a combination of anion-exchange, affinity, size-exclusion and reverse- phase chromatography. The purified receptor will be subjected to limited NH2-terminal vapor-phase amino acid sequencing. (2) Generate, isolate and sequence full-length CDNA'S to OPL receptor MRNA. Consensus sequences between the ovine growth hormone (OGH) receptor and bovine prolactin (bPRL) receptor CDNA'S will be used as primers for reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of fetal liver MRNA to generate OPL receptor specific CDNA probes to screen a fetal liver CDNA library. Alternatively, the GH and PRL receptor CDNA'S or oligonucleotide probes generated from the amino acid sequencing of the OPL receptor will be used to screen the CDNA library. Once a CDNA encoding the OPL receptor is isolated, it will be subjected to nucleotide sequencing by the dideoxy-chain terminating procedure. The primary amino acid sequence of the OPL receptor will be inferred, and then compared for sequence similarity with other protein hormone receptors. (3) Isolate and structurally characterize the gene(s) for OPL. Genomic clones for OPL will be mapped and the exons with adjacent flanking regions will be sequenced to define the MRNA splice sites. The transcriptional start site and gene copy number will be determined, and the 5'-upstream regulatory region (1 to 2 kb) will be sequenced to examine potential cis-acting regulatory elements. Future emphasis will be placed on examining the tissue-specific expression of the OPL gene, as well as examining the mechanism of action of OPL.