OVINE PLACENTAL LACTOGEN: ITS REGULATION AND FUNCTION

长期目标：胎儿生长速度不仅决定了胎儿的大小， 出生时的后代，但也影响分娩的容易程度， 后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和 这些因素的作用充其量是有限的。现有证据表明 绵羊胎盘催乳素（oPL）在 调节胎儿的新陈代谢长期目标是：（1） 了解生物学功能、作用机制和 胎盘分泌的蛋白质的生物化学性质和（2） 了解胎盘产物的调节机制 都是制造出来的这些信息可能会导致方法（1）减少怀孕 失败和（2）调节胎儿生长速率。 具体目的和方法：（1）纯化至均一，得到氨基 OPL受体的氨基酸序列信息。OPL受体将是 从妊娠120天收集的胎肝中纯化， 阴离子交换、亲和、尺寸排阻和反向离子交换的组合 相色谱法纯化的受体将受到有限的 NH 2-末端气相氨基酸测序。(2)生成、隔离 并对OPL受体mRNA的全长cDNA进行测序。共识 绵羊生长激素（OGH）受体和牛 催乳素（bPRL）受体cDNA将用作反向PCR的引物。 RT-PCR扩增胎儿 肝mRNA产生OPL受体特异性cDNA探针，以筛选 胎肝cDNA文库。或者，GH和PRL受体cDNA的 或从以下的氨基酸测序产生的寡核苷酸探针： OPL受体将用于筛选cDNA文库。曾经的cDNA 如果编码OPL受体的细胞被分离， 通过双脱氧链终止程序进行核苷酸测序。的 将推断OPL受体的一级氨基酸序列， 然后与其他蛋白质激素进行序列相似性比较， 受体。(3)分离并结构表征以下基因： OPL。将对OPL的基因组克隆进行定位，并将具有相邻外显子的外显子与OPL的外显子进行比较。 侧翼区将被测序以确定mRNA剪接位点。的 将确定转录起始位点和基因拷贝数，和 将对5 ′-上游调控区（1 - 2kb）进行测序， 检查潜在的顺式作用调节元件。今后的重点将是 放在检测OPL基因的组织特异性表达上， 以及研究OPL的作用机制。

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.