CHARACTER AND ROLE OF HMG 1,2-DNA INTERACTIONS

HMG 1,2-DNA 相互作用的特征和作用

基本信息

批准号：
2094682
负责人：
WILLIAM M SCOVELL
金额：
$ 9.36万
依托单位：
BOWLING GREEN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-05-16 至 1995-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (adapted from the applicant's abstract and specific aims): The possible structural or functional role that the high mobility group (HMG) proteins 1,2 and E play in transcription will be examined. By using the anticancer drug and chemical cross-linking agent, cis-diamminedichloroplatinum (II) (cisplatin or DDP), which preferentially cross-links the HMG 1,2 and E proteins to DNA in chromatin, a unique route is available to determine if these proteins play a structural role in molding either transcriptionally active or inactive regions within the bulk of higher-order structure of chromatin. The Specific Aims are: (1) Using cis-diamminedichloroplatinum (II) as a selective cross-linking agent and specific polyclonal antibodies to the HMG proteins 1,2 and E, determine whether the HMG proteins are preferentially interacting with transcriptionally active or inactive sequences within the nucleus. (2) Use restriction endonuclease digestion to determine if transcriptionally active genes are preferentially accessible (limited digestion) within chromatin. Alternatively, carry out a limited digestion on intact nuclei to determine if the transcriptionally active sequences are associated with the nuclear matrix DNA. This restriction procedure will also be used following treatment of nuclei with various levels of cisplatin to determine if the HMG proteins are preferentially cross-linked to the excised or pelleted sequences. In objectives 1 and 2, erythrocyte nuclei from 11-day embryos obtained from blood, isolated with and without histone deacetylase inhibitor, will be compared to determine the influence of acetylation on HMG interaction in chromatin structure. The transcriptionally inactive erythrocytes from adult chickens will also be examined to further determine if the interactions (and therefore the possible role) of HMG proteins 1,2 and E change during development. (3) Using polyclonal antibodies to the HMG proteins, preliminary in vitro transcriptional assays will be carried out with nuclei from 11-day embryos to determine whether the HMG proteins are associated with the transcription complex. Different levels of alpha-amanitin will be incorporated in the assay to distinguish whether the HMG proteins exhibit any preferential effect on transcription by RNA polymerase I, II or III.

描述（改编自申请人的摘要和特定目的）：高迁移率组（HMG）可能的结构或功能作用将检查蛋白质1,2和E的转录中的E发挥。通过使用抗癌药物和化学交联剂，顺式 - 二氨基氯铂（II）（顺铂或DDP），优先将HMG 1,2和E蛋白交叉至染色质中的DNA，这是一种独特的途径可用于确定这些蛋白在成型散装内的转录活性区域或不活动区域染色质的高阶结构。具体目的是：（1）顺式二氨基氯铂（II）作为选择性交联剂，并且针对HMG蛋白1,2和E的特异性多克隆抗体，确定 HMG蛋白是否优先与细胞核内的转录活性或无效序列。（2）使用限制性核酸内切酶消化以确定转录活性是否有效基因在染色质中优先获得（有限的消化）。或者，对完整核进行有限的消化以确定如果转录活性序列与核序列相关基质DNA。此限制过程也将使用以下用各种水平的顺铂治疗核，以确定是否是否 HMG蛋白优先交联至切除的蛋白序列。在目标1和2中，来自11天胚胎的红细胞核从血液中获得，分离有和没有组蛋白脱乙酰基酶将比较抑制剂以确定乙酰化对染色质结构中的HMG相互作用。转录无活性还将检查成年鸡的红细胞以进一步确定如果HMG蛋白的相互作用（以及可能的作用）1,2 和在开发过程中的变化。（3）使用与多克隆抗体 HMG蛋白，将进行初步的体外转录测定从11天胚胎中脱离核，以确定HMG蛋白是否是否与转录复合物有关。不同的水平 α-氨氨酸蛋白将纳入分析中，以区分是否存在 HMG蛋白对RNA的转录表现出任何优先影响聚合酶I，II或III。