RAPID DNA SEQUENCING WITH LASER BASED MASS SPECTROMETRY

酵母基因组的功能分析是目前研究的重点 在大的开放阅读框架（ORF）上。 然而，大约 基因组的三分之一由“基因间区域”组成， 包括小的ORF和其他重要的非编码元件。 这项研究的总体目标是开发一种新的 在酵母中随机诱变的方法，可以应用于 无论是整个基因组，单个染色体，或 单个染色体的特定区域。 我们将测试 通过分析基因间区域， 第八号染色体 随机插入诱变（RIM）涉及构建 新整合载体（pHIT）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 酵母基因组的同源重组。 上 在这个项目的第一阶段，我们将构建这些新的集成 将允许：（I）直接在酵母中随机诱变的载体 并且还允许控制突变频率;（ii）选择 酵母中的突变;（iii）筛选框内融合;（iv） 突变的恢复和诱变DNA的扩增 序列在E.大肠杆菌或通过PCR;（v）去除整合的 序列;（vi）选择性酵母标记的再循环;（vii）重复的 用相同的文库进行诱变（例如寻找抑制子 （八）对新发现的开放式化学品进行独特标记; 用基因特异性寡核苷酸的阅读框（ORF）;和 最重要的是（ix）体内报告基因改组。 后一 该技术允许在一个细胞上快速和容易地交换报告基因， 大规模优化条件的测定细胞 定位或用于基因表达研究、分泌研究，两个- 混合分析等。此外，我们将确定 整合效率，特异性靶向诱变限定区域 在S288 C二倍体酵母的第八染色体上。 这将允许 对新颖的小开放式阅读框架和非开放式阅读框架的特定搜索 编码遗传元素 我们将描述选定的 RIM诱导突变的表型。 诱变的DNA 序列将被恢复和测序。 破坏菌株 质粒将被科学界所接受。

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.