RAPID DNA SEQUENCING WITH LASER BASED MASS SPECTROMETRY

酵母基因组的功能分析是目前关注的焦点。 在大的开放阅读框(ORF)上。然而，大约 三分之一的基因组由“基因间隔区”组成， 包括小的ORF和其他重要的非编码元件。 这项拟议研究的总体目标是开发一种新的 可在酵母中应用的随机诱变方法 或者是整个基因组，在单个染色体上，或者在 单个染色体的特定区域。我们将测试 通过分析基因间隔区来分析这项新技术的实用性 酵母菌染色体。 随机插入突变(RIM)涉及构建 新整合载体(PHITs)中的DNA文库 在插入序列内通过用 独特的III型限制性内切酶和随后整合到 通过同源重组获得酵母基因组。在第一个 在这个项目的阶段，我们将构建这些新的集成 允许：(I)在酵母中直接进行随机诱变的载体 并且还允许控制突变频率；(Ii)选择 酵母中的突变；(Iii)框内融合的筛选；(Iv) 突变的恢复和突变DNA的扩增 大肠杆菌中的序列或通过聚合酶链式反应；(V)去除整合的 序列；(Vi)选择性酵母标记的回收；(Vii)重复 使用相同文库的诱变(例如，搜索抑制子 或合成致死物)；(Viii)新确定的开放的独特标签 带有基因特异性寡核苷酸的阅读框架(ORF)； 最重要的是，(九)活体内的报告基因改组。后者 这项技术允许快速、轻松地交换报告基因 规模化优化细胞测定条件 定位或用于基因表达研究、分泌物研究，两个- 混合分析等。此外，我们将确定 整合效率，目标明确地突变定义的区域 在S288C二倍体酵母的第VIII染色体上。这将允许 对新型小开放阅读框和非开放阅读框的具体搜索 对遗传元素进行编码。我们将对选定的 RIM诱导突变的表型。诱变DNA 序列将被恢复和测序。破坏性菌株 并将向科学界提供质粒。

The functional analysis of the yeast genome is currently focused on large open reading frames (ORFs). However, approximately one third of the genome is comprised of "intergenic regions," including small ORFs and other important non-coding elements. The overall goal of this proposed research is to develop a new means of random mutagenesis in yeast that can be applied on either the whole genome, on individual chromosomes, or on defined regions of individual chromosomes. We will test the usefulness of this new technology by analyzing intergenic regions of yeast chromosome VIII. Random insertion mutagenesis (RIM) involves the construction of DNA libraries in new integration vectors (pHITs) that allows linearization within the insert sequences via digestion with a unique type III restriction enzyme and subsequent integration into the yeast genome via homologous recombination. In the first phase of this project, we will construct these new integration vectors that will permit: (I) random mutagenesis directly in yeast and also allow control of the mutation frequency; (ii) selection of mutations in yeast; (iii) screening for in-frame fusions; (iv) recovery of mutations and amplification of mutagenized DNA sequences in E. Coli or via PCR; (v) removal of integrated sequences; (vi) recycling of selective yeast markers; (vii) repeated mutagenesis with the same library (e.g. to search for suppressors or synthetic lethals); (viii) unique tagging of newly identified open reading frames (ORFs) with a gene-specific oligonucleotide; and most importantly, (ix) reporter gene shuffling in vivo. This latter technique allows a rapid and easy exchange of reporter genes on a large scale to optimize conditions for determination of the cellular localization or for gene expression studies, secretion studies, two- hybrid analysis, etc. Furthermore, we will determine the integration efficiency, target specifically mutagenize defined areas in chromosome VIII of S288C diploid yeast. This will allow a specific search for novel small open reading frames and non- coding genetic elements. We will characterize selected phenotypes of RIM-induced mutations. Mutagenized DNA sequences will be recovered and sequenced. Disruption strains and plasmids will be made available to the scientific community.