EUKARYOTIC GENE ORGANIZATION

本项目的长期目标是为分析做出贡献 真核生物基因组组织的DNA核苷酸 序列级。 拟议的工作旨在深入了解 哺乳动物的基因调控，并应增加了解 遗传疾病的基础。 两个遗传因素 将研究实验室小鼠： 1)小鼠中的Hbb复合物基因座。 这个基因座编码 血红蛋白的β和β样链基因。 的DNA 一个65 Kb的区域，包括七个已知的β- 来自BALB/c小鼠的珠蛋白基因和假基因将被 完成 将对序列进行广泛分析。 的 将小鼠序列与人Hbb序列进行比较， 识别功能和进化意义的特征。 将研究保守序列元件以确定 它们的基因组拷贝数，转录活性， 特定的蛋白质结合位点。 2)L1（LINES-1）逆转录子。 这是主要的长期 在哺乳动物基因组中散布的重复元件。 充分 长度L1元件的长度约为7 Kb，具有两个开口 阅读框（ORF）长度分别为1137-bp和3900-bp。 这些 ORF在E.大肠杆菌和酵母中。 的 将检测ORF产物的可疑功能， 实例逆转录酶活性、核酸结合和 蛋白酶活性 将对包装在以下物质中的L1 RNA进行检索： 病毒样颗粒 针对L1 ORF产物的抗体 将用于探测小鼠组织中的L1编码蛋白， 培养细胞 200-bp的可能的启动子和增强子活性 在小鼠中L1的5'末端发现的重复序列将被 研究了 将开发一个实验系统来检测 L1转位，长期目标是利用L1作为 哺乳动物系统插入诱变剂。 提出 方法包括L1过表达和L1的检测， 插入疱疹病毒TK基因。 搜索可能的 例如，将制备KL 1繁殖中的中间体 DNA/RNA杂合体和染色体外L1 DNA。

The long term goal of this project is to contribute to the analysis of eukaryotic genome organization at the DNA nucleotide sequence level. The proposed work aims to provide insights into mammalian gene regulation, and should add to understanding of the basis of genetic diseases. Two genetic elements in the laboratory mouse will be studied: 1) The Hbb complex locus in the mouse. This locus encodes the genes for the beta and beta-like chains of hemoglobin. The DNA sequences for a 65 Kb region including the seven known beta- globin genes and pseudogenes from the BALB/c mouse will be completed. The sequence will be extensively analyzed. The mouse sequence will be compared to the human Hbb sequence to identify features of functional and evolutionary significance. Conserved sequence elements will be investigated to determine their genomic copy number, transcriptional activity, and to look for specific protein binding sites. 2) The L1 (LINES-1) retroposon. This is the major long interspersed repetitive element in the mammalian genome. Full length L1 elements are about 7 Kb in length with two open reading frames (ORFs) 1137-bp and 3900-bp in length. These ORF's will be translated at high levels in E. coli and in yeast. The ORF products will be assayed for suspected functions, for example reverse transcriptase activity, nucleic acid binding, and protease activity. A search will be made for L1 RNA packaged in virus-like particles. Antibodies directed against L1 ORF products will be used to probe for L1 coded proteins in mouse tissues and cultured cells. Possible promoter and enhancer activity of 200-bp repeated sequences found at the 5' end of L1 in mouse will be investigated. An experimental system will be developed to detect L1 transposition, with a long term goal of harnessing L1 as an insertional mutagen for mammalian systems. Proposed approaches include L1 overexpression, and detection of L1 insertion into the herpes virus TK gene. A search for likely intermediates in KL1 propagation will be made, for example DNA/RNA hybrids and extrachromosomal L1 DNA.