BIASES IN THE RATES AND PATTERNS OF MOLECULAR EVOLUTION

描述：霍华德·奥克曼博士请求四年支持 研究分子进化的速度和模式中的偏差 肠道细菌。拟议研究的具体目标是 确定领先/滞后或编码/非编码的DNA链是否具有 不相等的突变率，从而解释了替代中的不对称 费率。吴和前田的早期工作发现了 某些互换，他们将其解释为 领先和落后链之间的突变率。然而，a 布尔默随后对相同数据进行了分析，采用了更具限制性的 对祖先序列的推断，未能检测到这样的差异。 最近，奥奇曼博士和他的学生弗兰西诺女士分析了DNA序列 大肠埃希氏菌和肠杆菌自然分离株的变异 来检测替代率的不对称性。尽管他们无法 检测领先和滞后之间的碱基替换率不对称 链，他们确实检测到不同编码之间的替换率不对称 和非编码链。他们认为这种差异是最好的 可通过非编码链上的转录偶联修复来解释。Dr。 奥克曼现在希望跟进这一观察结果，并研究 吴和前田以及布尔默关于 领先和落后链之间的突变率。两条互补线路 正在计划中的调查。 在第一个实验中，奥克曼博士将采取一种实验方法。他会的 他将测量鼠伤寒沙门氏菌菌株的突变率 构建，包含整合到LacZ基因座的特定等位基因 染色体位于相同的位置，但方向相反。在这部作品中 他将利用杰弗里构建的LacZ菌株集 Miller在基因中存在不同的替换或移码突变 指定活性基因中第461位谷氨酸的相同密码子位置 -半乳糖苷酶部位。他将使用一种聚合酶链式反应分析来确定 定位融合，并采用迷你亩克隆和现在标准 细菌遗传程序将这些LacZ等位基因引入S. 小鼠染色体位于相同的位置。突变率将是 通过标准的Luria-Delbruck波动检验来衡量。自从Lacz 基因座在奥克曼博士将使用的菌株中有结构性表达，任何 突变率的差异可归因于整合性取向； 由转录引起的错误在所有菌株中都是相同的。 奥赫曼博士的第二种方法是分析11个基因的序列变异 大肠埃希菌的天然分离物。使用他已经开发的程序 和报道，他将寻找突变不对称的区域 未转录的大肠杆菌染色体(包括两个隐含基因和 位于原点两侧的两个400bp的非编码片段 位于两个所谓的“灰洞”中的复制)和位于 已转录但未翻译。每个区域或基因座都将通过聚合酶链式反应进行扩增 然后进行测序。所需的测序量相对较小， 以今天的标准来看。奥克曼博士将比较他的研究结果 已经获得了编码区的结果，以及他将获得的结果 对于转录但未翻译的区域，以及对于未转录的区域。 这应该使他能够区分相互竞争的假设 领先/滞后链突变率差异，以及转录偶联 修复，在产生观察到的不对称。

DESCRIPTION: Dr. Howard Ochman requests four years of support to investigate biases in the rates and patterns of molecular evolution in the enterobacteria. The specific goals of the proposed research are to determine if leading/lagging or coding/non-coding strands of DNA have unequal mutation rates, thereby explaining an asymmetry in substitution rates. Early work by Wu and Maeda detected inequalities in the rate of certain reciprocal substitutions, which they interpreted as differences in mutation rates between the leading and lagging strands. However, a subsequent analysis of the same data by Bulmer, employing a more restrictive inference of the ancestral sequences, failed to detect such a difference. Recently, Dr. Ochman and his student Ms. Francino have analyzed DNA sequence variation in natural isolates of E. coli and S. enterica, and with respect to detect asymmetries in substitution rates. Although they were unable to detect an asymmetry in base substitution rates between leading and lagging strands, they did detect an asymmetry in substitution rates between coding and non-coding strands. They consider that this difference is best explained by transcription-coupled repair on the non-coding strand. Dr. Ochman now wishes to follow up on this observation, and to examine the conflicting claims of Wu and Maeda, and Bulmer regarding differences in mutation rates between leading and lagging strands. Two complementary lines of investigation are planned. In the first, Dr. Ochman will take an experimental approach. He will measure mutation rates in strains of Salmonella typhimurium that he will construct, containing specific alleles at the lacZ locus integrated into the chromosome at the same position, but in opposite orientations. In this work he will take advantage of sets of lacZ- strains constructed by Jeffrey Miller that harbor a different substitution or frameshift mutations in the same codon position specifying glutamic acid at position 461 in the active site of -galactosidase. He will use a PCR assay to determine the orientation of integration, and use mini-mu cloning and now-standard bacterial genetic procedures to introduce these lacZ alleles into the S. typhimurium chromosome at the same locations. Mutation rate will be measured by a standard Luria-Delbruck fluctuation test. Since the lacZ locus is expressed constitutively in the strains Dr. Ochman will use, any differences in mutation rate can be ascribed to orientation of integration; errors introduced by transcription will be the same in all strains. Dr. Ochman's second approach will be to analyze sequence variation in 11 natural isolates of E. coli. Using procedures that he has already developed and reported on, he will look for mutational asymmetries in regions of the E. coli chromosome that are untranscribed (including two cryptic genes and two 400-bp non-coding segments on opposite sides of the origin of replication located in two so-called "gray holes") and in regions that are transcribed but untranslated. Each region or loci will be amplified by PCR and then sequenced. The amount of sequencing required is relatively modest, by today's standards. Dr. Ochman will compare the results that he has already obtained for coding regions, with the results that he will obtain for transcribed but untranslated regions, and for untranscribed regions. This should allow him to distinguish between the competing hypotheses of leading/lagging strand mutation rate differences, and transcription-coupled repair, in generating the observed asymmetries.