MUTATOR TRANSPORTABLE ELEMENTS OF MAIZE

描述：本研究的长期目标是了解 突变体转座因子的活动在生命周期中受到调节 它的自然宿主玉米 Mutator系统包含自主MuDR 除了不同的非自主Mutator转座子共享之外， 只有~ 200 bp的末端反向重复序列（TIR）。 第一千一百零六章穆总动员 MuDR基因的正向突变频率最高， 真核转座子 最引人注目的是替代换位 Mutator系统表现出的机制：剪切或剪切并粘贴 分化的体细胞和生殖细胞中的复制转座。 与这种复杂的生活方式相一致的是， 潜在的和实际的MuDR RNA和蛋白质产物。 收敛泥浆A和 mudrB转录本起始于TIR内的启动子，终止于 重复基因间序列 MuRA与细菌同源 转座酶，MuRB是新颖的。 自然的MuDRB缺失导致更少或没有 体细胞切除 mudrA和B仅在含MuDR的品系中组成型表达， Walbot假设转录后事件负责 Mutator的发育晚期激活和cut的替代使用 以及粘贴和复制机制。 转录后机制可能 包括（i）通过反义核酸调节mudrA、B转录的比率， RNA，（ii）由长的含AUC前导序列介导的翻译控制 mudrA，（iii）选择性剪接产生的发育控制 不同的MURA、B蛋白和（iv）可选择的多聚腺苷酸化位点 产生不同的3 'UTR，从而影响RNA的稳定性。 这一建议有四个具体目标。 第一，明确哪些 MuDR转录物和蛋白质存在于Mutator之前和之后 体细胞和生殖细胞中的激活。 为此，具体 抗血清已经或将被提高到六种可能的MURA和B蛋白， 由选择性剪接产生。 选择性剪接 将被测试。 瞬时表达测定 建议确定是否两个mudrA转录本之一，与一个 上游AUG在翻译起始水平上受到调节。 最后， 表达（i）单独MuDR产物的14个转基因玉米品系 包括反义或（ii）myc标记的MuDR产物或（iii）TIR报告基因 构建体已经或将要构建并测定它们的能力， 单独或在杂交后促进突变体活性的各个方面， 合适的线。 第二个目的是表征反义 产品，看看它们是否受到发育调节，并评估其影响 转基因植物的诱导。 第三个目的是研究 使用含有质粒的转基因植物进行体内转座， 标记的Mu元件，其中供体位点和新的插入都可以是 回收并分析。 该测定系统将用于解决此类问题。 （i）在发展早期有效的差距修复是否掩盖了 切除和切除伴随着新的插入，（二）做所有穆 元件参与复制转座，（iii）哪些MuDr产物 防止TIR甲基化，以及（iv）MuDR产物的组合是什么 体细胞转座与胚细胞转座的启动子所必需的？ 第四和 最后一个目标集中于MURA/B与DNA模板结合的体外分析， 它们的核定位以及它们之间的相互作用， 甲基化底物。

DESCRIPTION: The long term goal of this study is to understand how the Mutator transposable element activities are regulated within the life cycle of its natural host, maize. The Mutator system contains the autonomous MuDR element in addition to diverse non-autonomous Mutator transposons sharing only the ~200bp terminal inverted repeats (TIRs). Mobilization of the Mu elements by MuDR produces the highest forward mutation frequency of any eukaryotic transposon. Most striking is the alternative transposition mechanisms exhibited by the Mutator system: cut or cut and paste in differentiated somatic cells and replicative transposition in germ cells. Consistent with this complex lifestyle is an extraordinary complexity of potential and actual MuDR RNA and protein products. Convergent mudrA and mudrB transcripts initiate at promoters within the TIRs and terminate at repetitive intergenic sequences. MuRA has homology with bacterial transposases, MuRB is novel. A natural MuDRB deletion leads to fewer or no somatic excision. Constitutive expression of mudrA and B only in MuDR-containing lines has led Walbot to hypothesize that post-transcriptional events are responsible for the developmental late activation of Mutator and the alternative use of cut and paste and replicative mechanisms. Post-transcriptional mechanisms may include (i) modulation of the ratio of mudrA,B transcription by antisense RNA, (ii) translational control mediated by the long, AUG-containing leader of mudrA, (iii) developmental control of alternative splicing producing different MURA,B proteins and (iv) alternative polyadenylation sites producing different 3'UTRs that could influence RNA stability. Four specific aims comprise this proposal. The first is to define which MuDR transcripts and proteins are present before and after Mutator activation in somatic and germinal cells. For this purpose, specific antisera has or will be raised to the six possible MURA and B proteins that result from alternative splicing. The possibility that alternative splicing is developmentally regulated will be tested. Transient expression assays are proposed to determine whether one of the two mudrA transcripts, with an upstream AUG, is regulated at the level of translation initiation. Finally, 14 lines of transgenic maize expressing (i) individual MuDR products including antisense or (ii) myc-tagged MuDR products or (iii) TIR reporter constructs have been or will be constructed and assayed for their ability to promote aspects of Mutator activity either alone or after crossing to appropriate lines. The second aim is to characterize the antisense products, see if they are developmentally regulated, and assess the effect of induction in transgenic plants. The third aim is study the mechanism of transposition in vivo using transgenic plants containing plasmids with marked Mu elements where both the donor site and new insertions can be recovered and analyzed. This assay system will be used to address such questions as (i) does efficient gap repair early in development mask excision and is excision accompanied by new insertions, (ii) do all Mu elements participate in replicative transposition, (iii) which MuDr products prevent TIR methylation, and (iv) what combination of MuDR products is necessary to promoter somatic vs. germinal transposition? The fourth and last aim focuses on an in vitro analysis of MURA/B binding to DNA templates, their nuclear localization and their interactions with each other and with methylated substrates.