EUKARYOTIC GENE ORGANIZATION

这个项目的长期目标是为分析做出贡献 在DNA核苷酸上的真核基因组组织 序列级别。拟议的工作旨在为以下方面提供见解 哺乳动物的基因调控，并应增加对 遗传性疾病的基础。基因中的两个遗传因素 将对实验室小鼠进行研究： 1)小鼠的Hbb复合体基因座。这个位置编码了 贝塔和类贝塔血红蛋白链的基因。DNA 一个65kb的区域的序列，包括7个已知的β- 来自BALB/c小鼠的珠蛋白基因和假基因将是 完成。将对序列进行广泛的分析。这个 将鼠标序列与人类Hbb序列进行比较，以 确定具有功能和进化意义的特征。 保守的序列元件将被研究以确定 它们的基因组拷贝数、转录活性和外观 用于特定的蛋白质结合部位。 2)L1(LINES-1)反转录。这就是龙少校 哺乳动物基因组中散布的重复元件。饱满 长度L1元素的长度约为7KB，其中有两个开放 阅读框(ORF)长度为1137-bp和3900-bp。这些 ORF将在大肠杆菌和酵母中得到高水平的翻译。这个 将对ORF产品进行可疑功能检测，以确定 反转录酶活性、核酸结合和 水解酶活性。将搜索包装在 像病毒一样的颗粒。针对L1 ORF产物的抗体 将用于探测小鼠组织中的L1编码蛋白和 培养的细胞。200-bp可能的启动子和增强子活性 在小鼠L1的5‘端发现的重复序列将是 调查过了。将开发一个实验系统来检测 L1转位，长期目标是将L1作为一种 用于哺乳动物系统的插入诱变剂。建议 方法包括L1过表达和L1的检测 插入疱疹病毒TK基因。对可能的搜索 例如，将在KL1繁殖中制作中间体 DNA/RNA杂交体和染色体外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.