Molecular Analysis of Chlamydomonas Mating-Type Locus

衣藻交配型基因座的分子分析

• 批准号: 9904667
• 负责人: Ursula Goodenough
• 金额: $ 42.7万
• 依托单位: Washington University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9904667&HistoricalAwards=false
• 关键词: Molecular; Analysis; Chlamydomonas; Mating; Type

AbstractIt has been established that a single gene, mid, located in the mt- locus, is necessary and sufficient to induce minus gametic differentiation and to suppress the expression of plus-specific gametic genes. It is further postulated that the Mid protein accomplishes these results by acting as a transcriptional activator/repressor in collaboration with other proteins. It is further postulated that Mid may collaborate with a different set of proteins to activate the expression of zygote specific genes following the fusion of plus and minus gametes. The first specific aim of this project is to test this "mid hypothesis". Antibodies will be raised against recombinant Mid protein and used to immunolocalize the protein and to perform immunoprecipitations of Mid plus any associated protein in wild type and mutant strains. The antibody will also be added to nuclei engaged in nuclear run-on transcription to assess whether it blocks or enhances transcription of gametic- and zygote-specific genes. The mid gene will be mutagenized in vitro to create temperature-sensitive alleles which, when transformed into a mid-deleted strain, should permit the identification of mid-regulated transcripts whose expression is up- or down-regulated with temperature increases. It has also been established that the mt locus is riddled with unusual genetic configurations suggesting that the region has been, and perhaps continues to be, a nucleus of evolutionary change. The evolutionary history and potential of this locus will be investigated using several approaches. The second specific aim is to continue an ongoing chromosome walk through the mt locus of a closely related species, C. incerta, evaluating the extent to which its organization has been conserved vs rearranged with respect to the C. reinhardtii locus during the estimated 1 million years since the two species diverged. The third specific aim is to continue the evaluation of a recent (past 20 years) mt-related anomaly, the tandem duplication of the mid gene and flanking sequences and the residency of this element in a non-mt location in the genome of the CC-421 strain of C. reinhardtii. The fourth specific aim is to continue to investigate the observation that the mid gene has incurred an unusually high level of nonsynonymous and synonymous codon changes since the C. reinhardtii/C. incerta divergence. The long-term goal of this research is to understand the genetic basis for life-cycle transitions in the unicellular green alga, Chlamydomonas reinhardtii, particularly with regard to the two mating-type loci and to analyze the evolutionary dynamics of these genetic systems.

摘要已经确定，位于mt位点的单个基因mid对于诱导负配子分化和抑制正特异配子基因的表达是必要且充分的。进一步推测，Mid 蛋白通过与其他蛋白合作充当转录激活剂/阻遏物来实现这些结果。进一步推测，Mid 可能与一组不同的蛋白质合作，在正负配子融合后激活受精卵特异性基因的表达。该项目的第一个具体目标是检验这个“中间假设”。将产生针对重组 Mid 蛋白的抗体，并用于免疫定位该蛋白，并对野生型和突变株中的 Mid 加上任何相关蛋白进行免疫沉淀。该抗体还将被添加到参与核连续转录的细胞核中，以评估它是否阻断或增强配子和合子特异性基因的转录。 mid基因将在体外诱变以产生温度敏感等位基因，当转化到mid缺失菌株中时，应允许鉴定其表达随着温度升高而上调或下调的中调节转录物。人们还确定，mt 基因座充满了不寻常的遗传配置，这表明该区域曾经是，也许仍然是进化变化的核心。 将使用多种方法研究该基因座的进化历史和潜力。第二个具体目标是继续对密切相关物种 C. incerta 的 mt 基因座进行染色体行走，评估自两个物种分化以来的估计 100 万年期间，其组织相对于 C. Reinhardtii 基因座的保守程度和重新排列程度。第三个具体目标是继续评估最近（过去 20 年）与 mt 相关的异常、中间基因和侧翼序列的串联重复以及该元件在莱茵衣藻 CC-421 菌株基因组中非 mt 位置的驻留情况。 第四个具体目标是继续研究自莱茵衣藻/C. 以来，mid 基因引起异常高水平的非同义和同义密码子变化的观察结果。不确定的分歧。这项研究的长期目标是了解单细胞绿藻莱茵衣藻生命周期转变的遗传基础，特别是两个交配型基因座，并分析这些遗传系统的进化动态。