OVINE PLACENTAL LACTOGEN: ITS REGULATION AND FUNCTION

羊胎盘泌乳素：其调节和功能

• 批准号: 3330480
• 负责人: RUSSELL V ANTHONY
• 金额: $ 8.05万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3330480
• 关键词: RNA splicing; complementary DNA; gene expression; genetic mapping; genetic regulatory element; hormone receptor; hormone regulation /control mechanism; ion exchange chromatography; messenger RNA; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; prolactin; protein sequence; protein structure function; reversed phase chromatography; secretion; sheep; somatomammotropin; vertebrate embryology

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.

长期目标：胎儿的生长速度不仅决定胎儿的大小 不仅影响后代的出生，而且还影响分娩的难易程度 后代出生后的生长速度。不幸的是，我们的理解 影响产前发育的因素及其机制 这些因素的作用至多是有限的。现有证据表明 绵羊胎盘催乳素 (oPL) 在 调节胎儿新陈代谢。长期目标旨在 (1) 了解生物功能、作用机制和 胎盘分泌的蛋白质的生化特性和 (2) 了解胎盘产品的监管机制 被制作。这些信息可能会导致以下方法：(1) 减少怀孕 失败和（2）调节胎儿生长速度。 具体目的和方法：（1）纯化至均质，得到氨基 OPL 受体的酸序列信息。 OPL 受体将是 纯化自妊娠 120 天时收集的胎儿肝脏 阴离子交换、亲和力、尺寸排阻和反向交换的组合 相色谱法。纯化的受体将受到有限的 NH2 末端气相氨基酸测序。 (2)生成、隔离 并将全长 cDNA 测序为 OPL 受体 mRNA。共识 绵羊生长激素 (OGH) 受体和牛之间的序列 催乳素 (bPRL) 受体 cDNA 将用作反向引物 胎儿转录酶聚合酶链式反应 (RT-PCR) 扩增 肝脏 mRNA 生成 OPL 受体特异性 cDNA 探针来筛选 胎儿肝脏 cDNA 文库。或者，GH 和 PRL 受体 cDNA'S 或从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。一次cDNA 编码 OPL 受体被分离出来，它将受到 通过双脱氧链终止程序进行核苷酸测序。这 将推断 OPL 受体的一级氨基酸序列，并且 然后与其他蛋白质激素比较序列相似性 受体。 (3) 分离基因并进行结构表征 OPL。 OPL 的基因组克隆将被映射，并且具有相邻的外显子 对侧翼区域进行测序以确定 mRNA 剪接位点。这 将确定转录起始位点和基因拷贝数，并且 5'-上游调控区（1 至 2 kb）将被测序 检查潜在的顺式作用调控元件。未来重点将 致力于检查 OPL 基因的组织特异性表达， 以及检查 OPL 的作用机制。