BIASES IN THE RATES AND PATTERNS OF MOLECULAR EVOLUTION

描述：霍华德·奥曼博士请求四年的支持， 研究分子进化的速率和模式的偏差 肠细菌。 拟议研究的具体目标是 确定DNA的前导/滞后或编码/非编码链是否 突变率不等，从而解释了置换的不对称性 rates. Wu和Maeda的早期工作发现了 某些相互替代，他们解释为差异 前导链和滞后链之间的突变率。 但 随后由Bulmer对相同数据进行分析，采用更严格的 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朗西诺女士分析了 E. coli和革兰氏阳性菌S.肠内，恕我直言 以检测替代率的不对称性。 即使他们当时并没有 检测超前和滞后碱基置换率的不对称性 他们确实检测到了两个编码序列之间的取代率不对称 和非编码链。 他们认为这种差别是最好的 通过非编码链上的转录偶联修复来解释。 博士 Ochman现在希望跟进这一观察，并检查 吴和前田的冲突索赔，和布尔默关于分歧 前导链和滞后链之间的突变率。 两条互补线 计划进行调查。 在第一个实验中，Ochman博士将采用实验方法。 他将 测量鼠伤寒沙门氏菌菌株的突变率 构建体，其含有整合到 染色体在相同的位置，但在相反的方向。 在这项工作中 他将利用Jeffrey构建的lacZ-菌株 Miller，其携带不同的置换或移码突变， 相同的密码子位置，在活性氨基酸序列的461位指定谷氨酸 β-半乳糖苷酶位点。 他将使用PCR分析来确定 整合方向，并使用mini-mu克隆和现在标准 将这些lacZ等位基因导入S. 鼠伤寒染色体上相同的位置。 突变率将 通过标准的Luria-Delbruck波动测试来测量。 既然lacZ 基因座在Ochman博士将使用的菌株中组成性表达，任何 突变率的差异可归因于整合的方向; 由转录引入的错误在所有菌株中是相同的。 博士Ochman的第二种方法是分析11个 E.杆菌 使用他已经开发的程序 他将寻找基因突变的不对称性， e.未转录的大肠杆菌染色体（包括两个隐性基因和 两个400-bp的非编码片段位于 位于两个所谓的"灰洞"中的复制）和 转录但未翻译。 每个区域或基因座将通过PCR扩增 然后测序。 所需的测序量相对适中， 以今天的标准来看。 奥奇曼医生会比较 已经获得的编码区域，结果，他将获得 对于已转录但未翻译的区域，以及对于未转录的区域。 这将使他能够区分 前导链/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性。

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.