COORDINATE HORMONAL REGULATION OF THE FIBRINOGEN GENES

纤维蛋白原是血液凝块的主要结构蛋白，在 肝脏和分泌在血液中的三个多聚体 不同的亚基，分别命名为Apha、Bbeta和Gamma。这些亚单位是 由不同的基因编码，这些基因从共同的祖先序列中分离出来 在进化的早期。尽管它们的结构相距遥远 关系中，三个纤维蛋白原亚单位基因是高度调控的 以协调的方式对生理刺激作出反应。作为该计划的一部分 “急性时相反应”，一种对多种疾病的复杂生理反应。 应激和组织损伤，纤维蛋白原的产生通过 肾上腺类固醇激素，糖皮质激素。这项提案的重点是 确定糖皮质激素的分子机制 调节纤维蛋白原亚单位基因的转录。为了这些 调查中，我们使用的肝细胞培养系统源自 非洲爪蛙。高纯度非洲爪哇合成纤维蛋白原的研究 糖皮质激素对肝细胞的诱导作用约为20倍。 因此，这种可实验操作的系统非常适合于详细的 剖析与荷尔蒙反应有关的多种因素。 我们的基本实验策略是确定纤维蛋白原的区域 通过纯化纤维蛋白原基因片段对转录起重要作用的基因， 在该DNA中产生缺失突变，并分析对 将DNA导入非洲爪哇培养肝细胞后转录。 强大的基因转染法结合了精确的突变 体外实验，并分析生活中改变的功能后果 细胞。这项提案中概述的具体目标是：1)孤立和 纤维蛋白原基因上游调控DNA的特性及插入 2)将纤维蛋白原DNA导入 非洲爪哇原代肝细胞的转染法及应用 糖皮质激素诱导转录；3)缺失突变的产生 并分析对转基因肝细胞转录的影响；4) 与糖皮质激素受体结合的DNA序列的鉴定 体外；以及5)鉴定与其他基因结合的调控DNA序列 核蛋白对转录控制很重要。这些实验 将揭示DNA序列和蛋白质因子对基础和 糖皮质激素诱导纤维蛋白原基因的表达。我们的长期合作 我们的目标是了解这三种生物的高度协调激活 实现了独立的基因，即使不同的组合 使用转录因子。保持平衡是非常重要的 纤维蛋白原的合成，因为它在凝血中起重要作用 和伤口愈合。有几种病理状态 纤维蛋白原的产生是异常的。例如，纤维蛋白原水平是 血栓形成和心脏病的发生率升高。相反，纤维蛋白原和其他 新生儿的凝血因子往往不足， 尤其是那些早产的孩子。了解潜在的因素 调节纤维蛋白原基因转录有助于发育 治疗以纤维蛋白原产生异常为标志的疾病。 GRANT R01HL48268 一种新的M/r 28 kDa红细胞跨膜蛋白具有很强的 与晶状体的音量调节通道MIP同源。相关的28 kDa 在肾小管中也发现了蛋白质。这样做的长期目标是 建议对红鱼28 kDa蛋白的基本生物学特性进行研究 细胞，其他组织，并描绘28 kDa的分子病理学。 血液病和其他临床疾病。 I.遗传学研究.成人和胎儿红细胞和肾小管的cDNA 28 kDa的蛋白质将被分离和测序。28 kDa基因组组织 将确定和替代剪接异构体将被表征。 相关蛋白质将在非红系组织中寻找。 生化结构--28 kDa的高阶分子结构， 翻译后修饰和超微结构将被定义。 表达组装与功能--28 kDa的表达与组装 将在红系分化过程中研究肾脏中的亚基。 小管，以及在小鼠胚胎发育期间。功能研究将是 对插入28 kDa纯化红细胞的行为进行表征 进入平面膜，并在非洲爪哇卵母细胞中表达。 临床研究--采用I-III中发展的方法和知识， 将对患者样本进行先天性或获得性分子分析 28 kDa的缺陷。之后会特别注意红血球。 长期冷藏和先天性溶血性贫血患者， 红细胞生成障碍、营养性贫血和化疗后。肾和 在某些肾脏疾病中，尿液将被检测为28 kDa。

Fibrinogen, the major structural protein of a blood clot, is synthesized in the liver and secreted in the bloodstream as a multitimer of three different subunits, designated Aalpha, Bbeta, and gamma. The subunits are encoded by separate genes that diverged from a common ancestral sequence very early in evolution. In spite of their distant structural relationship, the three fibrinogen subunit genes are regulated in a highly coordinated manner in response to physiological stimuli. As part of the "acute-phase response", a complex physiological reaction to a variety of stresses and tissue injuries, production of fibrinogen is elevated by the adrenal steroid hormones, glucocorticoids. This proposal focuses on determining the molecular mechanisms by which glucocorticoid hormones regulate transcription of the fibrinogen subunit genes. For these investigations, we are using a liver cell culture system derived from the frog Xenopus Laevis. Fibrinogen synthesis in highly purified Xenopus hepatocytes is dramatically induced about 20-fold by glucocorticoids. Therefore, this experimentally-manipulable system is ideal for a detailed dissection of the multiple elements involved in hormone responsiveness. Our basic experimental strategy is to define regions of the fibrinogen genes important for transcription by purifying fibrinogen gene fragments, generating deletion mutations in this DNA, and assaying effects on transcription after introducing the DNA into cultured Xenopus hepatocytes. The powerful gene transfection approach combines precise mutagenesis in vitro with analysis of functional consequences of the alterations in living cells. The specific aims outlined in this proposal are: 1) Isolation and characterization of fibrinogen gene upstream regulatory DNA and insertion into a transfection vector; 2) Introduction of the fibrinogen DNA into primary Xenopus liver cells by transfection and demonstration of glucocorticoid-inducible transcription; 3) Generation of deletion mutations and analysis of effects on transcription in transfected liver cells; 4) Identification of DNA sequences that bind the glucocorticoid receptor in vitro; and 5) Identification of regulatory DNA sequences that bind other nuclear proteins important for control of transcription. These experiments will reveal DNA sequences and protein factors critical for basal and glucocorticoid-induced expression of the fibrinogen genes. Our long-term goal is to understand how highly coordinated activation of these three independent genes is achieved, even though different combinations of transcription factors are used. It is very important to maintain balanced synthesis of fibrinogen since it plays a vital role in blood coagulation and wound healing. There are several pathological states in which fibrinogen production is abnormal. For example, fibrinogen levels are elevated in thrombosis and heart disease. Conversely, fibrinogen and other blood-clotting factors are often insufficient in newborn infants, particularly those born prematurely. Understanding the factors underlying regulation of fibrinogen gene transcription will aid in developing treatments for conditions marked by aberrant fibrinogen production. GRANT R01HL48268 A novel M/r 28kDa red cell transmembrane protein was found to have a strong homology with MIP, the volume regulatory channel of lens. A related 28kDa protein was also found in renal tubules. The long term objectives of this proposal are to characterize the basic biology of the 28kDa protein in red cells, other tissues, and to delineate molecular pathology of 28kDa in blood diseases and other clinical disorders. I. GENETIC STUDIES-cDNAs for adult and fetal red cell and renal tubule 28kDa proteins will be isolated and sequenced. 28kDa genomic organization will determined and alternate splice isoforms will be characterized. Related proteins will be sought in nonerythroid tissues. II. BIOCHEMICAL STRUCTURAL--The higher order molecular structure of 28kDa, posttranslational modifications, and ultrastructure will be defined. III. EXPRESSION ASSEMBLY, AND FUNCTION--Expression and assembly of 28kDa subunits will be studied during erythroid differentiation, in renal tubules, and during mouse fetal development. Functional studies will be undertaken to characterize the behavior of purified red cell 28kDa inserted into planar membranes and after expression in Xenopus oocytes. IV. CLINICAL STUDIES--Employing methods and knowledge developed in I-III, patient samples will be analyzed for congenital or acquired molecular defects in 28kDa. Particular attention will be paid to red cells after prolonged cold storage and from patients with congenital hemolytic anemias, dyserythropoiesis, nutritional anemias and after chemotherapy. Kidney and urine will be examined for 28kDa in certain renal diseases.