HORMONAL REGULATION OF GALANIN GENE EXPRESSION

甘丙肽是一种分布广泛的调节脑下垂体的多肽， 胰腺和胃部激素的分泌。在大鼠脑下垂体内， 甘丙素的生产仅限于营养乳汁和生长的细胞 其中合成和分泌都受到循环雌激素的影响。 雌激素对脑下垂体甘丙素基因的表达有显著影响。 在正常的发情周期中，稳定的mRNA水平变化了30倍 成年女性的比例，并在怀孕期间再增加10倍。 甘丙素特异性信使核糖核酸占雌激素总信使核糖核酸的0.5% 对这些细胞中甘丙素基因的表达施加转录控制。 这些观察结果表明，甘丙肽基因提供了一种极好的 激素与细胞特异性相互作用的研究模型 转录的特定调节因子。 拟议中的研究将调查大鼠 甘丙肽基因在脑垂体细胞中受到调控。雌激素对大鼠血管紧张素转换酶的影响 甘丙素基因转录将进一步鉴定。DNA 具有赋予雌激素反应性的序列将被鉴定为 利用缺失突变体进行瞬时表达分析。然后我们将评估 这些序列与雌激素结合并被雌激素激活的能力 受体蛋白。 表达的细胞谱系特异性的基础将通过 甘丙肽产生细胞系和ON产生细胞系的转基因。我们会 试图辨别甘丙肽基因中赋予细胞的DNA序列 类型特异性。一旦确定，这些序列将被用于分离 蛋白质(S)负责观察到的特异性并进行鉴定 编码这种蛋白的cDNA克隆(S)。我们将特别注意 用于评价PIT-1(GHF-1)在控制赤潮中的作用 甘丙肽的表达。PIT-1/GHF-1是一种转录激活剂，它 似乎决定了催乳素的细胞类型特异性和生长 激素基因在大鼠脑垂体中的表达。PIT-1/GHF-1基因的表达 仅限于脑下垂体促乳素细胞和促生长激素细胞 受雌激素的负面调节。我们将确定这种蛋白质是否 调节甘丙素的表达以及雌激素对其活性的影响。 我们还将评估甘丙素是否会对雌激素做出反应。 调节PIT-1/GHF-1基因表达。 细胞和激素特异性调节蛋白之间的相互作用将是 顺式基因突变分析在瞬时表达分析中的应用 元素。如果我们成功地确定了一种细胞特异性的调控因子 甘丙素基因的表达，我们将考察该基因的表达 调节蛋白本身受雌激素和产量的调节 影响甘丙肽基因的雌激素反应性 表情。

Galanin is a widely-distributed peptide which modulates pituitary, pancreatic, and gastric hormone secretion. Within the rat pituitary, galanin production is limited to the lactotrophic and somatotrophic cells where both synthesis and secretion are influenced by circulating estrogens. Estrogens have a dramatic effect on pituitary galanin gene expression. Steady-state mRNA levels vary 30-fold during the normal estrous cycle of adult female rates, and increase another 10-fold during gestation. Galanin-specific mRNA accounts for up to 0.5% of total mRNA in estrogen exerts transcriptional control over galanin gene expression in these cells. These observations suggest that the galanin gene provides an excellent model for the study of the specific interaction of hormonal and cell- specific regulators of transcription. The proposed studies will investigate the mechanisms by which the rat galanin gene is regulated in pituitary cells. The effects of estrogen on galanin gene transcription will be further characterized. The DNA sequences with confer estrogen-responsiveness will be identified by transient expression assays using deletion mutants. We will then evaluate the ability of these sequences to bind and be activated by estrogen receptor protein. The basis for cell lineage specificity of expression will be examined by transfection of galanin-producing and on-producing cell lines. We will attempt to discern DNA sequences within the galanin gene which confer cell type specificity. Once identified, those sequences will be used to isolate the protein(s) responsible for the observed specificity and to identify cDNA clones which encode such protein(s). Particular attention will be given to the evaluation of the role of pit-1(GHF-1) in the control of galanin expression. Pit-1/GHF-1 is a transcriptional activator which appears to determine the cell-type specificity of prolactin and growth hormone gene expression in the rat pituitary. Pit-1/GHF-1 gene expression is limited to pituitary lactotrophs and somatotrophs and appears to be negatively regulated by estrogen. We will determine whether this protein regulates galanin expression and how its activity is affected by estrogen. We will also evaluate whether galanin secreted in response to estrogen regulates pit-1/GHF-1 gene expression. The interaction of cell- and hormone-specific regulatory proteins will be evaluated in transient expression assays by mutational analysis of cis- elements. If we are successful in identifying a cell-specific regulator of galanin gene expression, we will examine whether the expression of the regulatory protein is itself regulated by estrogen and whether production of the protein affects the estrogen-responsiveness of galanin gene expression.