Regulation And Function Of Retroelements

逆转录因子的调控和功能

基本信息

批准号：
7734715
负责人：
Henry L. LEVIN
金额：
$ 127.56万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7734715
关键词：
Acquired Immunodeficiency Syndrome Address Amino Acid Substitution Amino Acids Animal Model Binding Binding Sites Biological Assay Biological Models Cell Nucleus Cells Code Complex DNA DNA Polymerase II Data Disease Disruption Distant Elements Eukaryota Eukaryotic Cell Evolution Family Fission Yeast Gel Chromatography Genes Genetic Genetic Transcription Genome Goals Gypsies HIV-1 Human Indium Individual Insertional Mutagenesis Integrase Inverted Terminal Repeat Laboratories Lead Long Terminal Repeats Measures Mediating Methods Modeling Molecular Mus Musca domestica Mutagenesis Mutate Mutation Nuclear Pore Numbers Open Reading Frames Parasites Paste substance Pattern Peptides Phenotype Plasmids Play Positioning Attribute Process Property RNA-Directed DNA Polymerase Recombinants Regulation Research Resistance Retroelements Retrotransposon Retroviridae Reverse Transcription Role Saccharomyces cerevisiae Site Structure Study models Terminal Repeat Sequences Testing Transcription Coactivator Transposase Virus Work Yeasts antibiotic G 418 base crosslink dimer env Gene Products in vivo interest leukemia member novel novel strategies particle preference promoter research study tool transcription factor

项目摘要

Diseases such as AIDS and leukemia caused by retroviruses have intensified the need to understand the mechanisms of retrovirus replication. Our primary objective is to understand how retroviral cDNAs are integrated into the genome of infected cells. Because of their similarities to retroviruses, long terminal repeat (LTR)-retrotransposons are important models for retrovirus replication. The retrotransposon under study in our laboratory is the Tf1 element of the fission yeast Schizosaccharomyces pombe. We are particularly interested in Tf1 because of its strong preference for integrating into pol II promoters. This choice of target sites is similar to the strong preferences human immunodeficiency virus 1 (HIV-1) and murine leukemina virus (MLV) have for integration into pol II transcription units. Very little is known about how these viruses recognize their target sites. We therefore study the integration of Tf1 as a model system from which we hope uncover mechanisms general to the selection of integration sites. Such an understanding of the mechanisms responsible for targeted integration may lead to new approaches for blocking the replication of HIV-1. A key goal of our research this year was to identify the mechanism that directs integration to regions upstream of ORFs. To study insertion patterns in specific genes, a target plasmid assay was developed. Integration into plasmids containing various genes occurred upstream of the ORFs in insertion windows of approximately 150 nt. Deletion analyses of the plasmids indicated that the target positions were the only sequence features required for integration. Separate plasmids containing smaller sequences demonstrated that the windows of integration themselves were sufficient to direct integration. The prominent insertion sites in fbp1 were just 30 and 40 bp downstream of where the transcription activator Atf1p bound. These data suggested the model that transcription factors bound at their promoters, mediate integration. This model was supported by the finding that a functional binding site for the transcription factor Aft1p played a critical role in the targeting of Tf1 integration to the two major insertion sites in the fbp1 promoter. In addition, we found that Atf1p is required for integration in the fbp1 promoter and that Atf1p interacts with integrase. These data provide strong support for the role of Atf1p in directing integration to target sites. Another project conducted this year was to identify the function of the GPY domain present in the C-termini of some retrovirus and retrotransposon integrases. Based on sequence conservation, single amino acid substitutions were made in the GPY domain of Tf1 integrase. In vivo, these mutations greatly reduced transposition activity. Work with recombinant integrase revealed that mutating the conserved P365 greatly reduced catalytic activity. Gel filtration and cross-linking of a 71-amino acid fragment containing the GPY domain revealed this peptide formed dimers, trimers and tetramers. Mutations in the conserved amino acids of the GPY domain disrupted all multimerization. These results suggest that the GPY domain promotes the multimerization and catalytic activity of integrase. The integration of Tf1 occurs primarily into pol II promoters. Although we currently believe this preference is the result of a mechanism that actively targets Tf1, it is possible that the insertion bias is due to greater accessibility at the promoter sequences. We are currently testing this possibility by studying in S. pombe cells the integration pattern of hermes, a cut and paste transposon that was isolated from the house fly. Since hermes propagates in a host that is evolutionary distant from S. pombe, it is unlikely that a mechanism exists that would actively position insertion sites. Thus, any integration of hermes in S. pombe would likely occur at positions that were accessible to the transposase. In addition, unbiased activity of a transposon in S. pombe could be widely adapted as a tool for random mutagenesis. Since no methods currently exist for transposon mutagenesis of S. pombe, a method for insertional mutagenesis would be a significant contribution to the field. The transposase of hermes was expressed in S. pombe by fusing its gene to the promoter of nmt1. To measure transposition activity the cells that expressed the transposase also contained a plasmid encoded copy of neo flanked by the terminal inverted repeats (TIRs) of hermes. The ability of the transposase to cut out neo with the TIRs and insert this DNA into the pombe genome was tested. Twenty six independent strains that became G418 resistant were analyzed and each strain was found to have acquired a copy of hermes. Analysis of these inserted copies revealed that 54% of them disrupted ORFs. These results indicate that the insertion of hermes did not discriminate between coding and noncoding sequences. This is in strong contrast to the integration of Tf1 where virtually none of the inserts occur in ORFs. In pilot experiments cultures of mutagenized cells were screened for mutations in two specific genes, ade6, and ade7. Of 106,000 cells screened, seven produced red colonies, the phenotype expected for mutations in either ade7 or ade6. This number of ade- colonies was consistent with randomly distributed integration. Together, these data indicate that hermes can readily be used as a tool for the random disruption of pombe genes.

逆转录病毒引起的艾滋病和白血病等疾病加剧了了解逆转录病毒复制机制的需求。我们的主要目标是了解逆转录病毒CDNA如何整合到感染细胞的基因组中。由于它们与逆转录病毒的相似性，长期重复（LTR） - 返回转座子是逆转录病毒复制的重要模型。在我们的实验室研究的逆转录座子是裂变酵母菌酵母酸酯POMBE的TF1元素。我们对TF1特别感兴趣，因为它强烈偏爱整合到Pol II启动子中。目标位点的这种选择类似于强烈的偏好人类免疫缺陷病毒1（HIV-1）和鼠白血病病毒（MLV）将其整合到POL II转录单元中。这些病毒如何识别其目标部位知之甚少。因此，我们研究了TF1作为模型系统的整合，我们希望从中发现整合位点选择的机制。对负责有针对性整合的机制的这种理解可能会导致阻止HIV-1复制的新方法。我们今年研究的一个关键目标是确定将整合到ORF上游地区的机制。为了研究特定基因中的插入模式，开发了靶质粒测定。在大约150 nt的插入窗口中，ORF上游发生了包含各种基因的质粒。质粒的缺失分析表明，目标位置是整合所需的唯一序列特征。包含较小序列的单独质粒表明集成窗口本身足以直接集成。 FBP1中突出的插入位点仅在转录激活剂ATF1P结合的下游30和40 bp。这些数据表明，转录因子绑定在其启动子上的模型，介导整合。该模型得到了以下发现，即转录因子AFT1P的功能结合位点在FBP1启动子中两个主要插入位点的TF1整合靶向中起关键作用。此外，我们发现在FBP1启动子中积分需要ATF1P，并且ATF1P与整合酶相互作用。这些数据为ATF1P在将集成到目标站点中的作用提供了强有力的支持。今年进行的另一个项目是确定某些逆转录病毒和逆转录座集成酶中存在的GPY结构域的功能。基于序列保守，在TF1积分酶的GPY结构域中进行了单个氨基酸取代。在体内，这些突变大大降低了换位活性。与重组集成酶一起工作表明，对保守的p365突变大大降低了催化活性。含有GPY结构域的71个氨基酸片段的凝胶过滤和交联，显示了这种肽形成的二聚体，三聚体和四聚体。 GPY结构域的保守氨基酸中的突变破坏了所有多聚化。这些结果表明，GPY结构域促进了积分酶的多聚化和催化活性。 TF1的整合主要发生在Pol II启动子中。尽管我们目前认为这种偏好是积极针对TF1的机制的结果，但插入偏差可能是由于启动子序列上更大的可访问性所致。我们目前正在通过在S. pombe细胞中研究爱马仕（Hermes）的整合模式来测试这种可能性。由于爱马仕（Hermes）在距离庞贝（S. 因此，在pombe中，爱马仕的任何整合都可能发生在转座酶可以使用的位置。此外，可以广泛适应转座子的无偏活性作为随机诱变的工具。由于目前尚无用于pombe链球子诱变的方法，因此插入诱变的方法将对该领域做出重要贡献。爱马仕的转座酶通过将其基因与NMT1的启动子融合来表达。为了测量换位活性，表达转座酶的细胞还包含一个编码的质粒编码的新侧面，侧面是爱马仕的末端反复重复（TIR）。测试了转座酶用TIRS切除NEO并将该DNA插入POMBE基因组的能力。分析了二十六个具有G418耐药性的独立菌株，发现每种菌株都获得了爱马仕的副本。对这些插入副本的分析表明，其中有54％破坏了ORF。这些结果表明，爱马仕的插入没有区分编码和非编码序列。这与TF1的整合形成鲜明对比，在ORF中几乎没有插入物。在试点实验中，筛选了两个特定基因ADE6和ADE7中的突变。在筛选的106,000个细胞中，有7个产生了红色菌落，这是ADE7或ADE6突变的表型。这种数量的菌落与随机分布的集成一致。这些数据一起表明，爱马仕可以很容易地用作随机破坏POMBE基因的工具。