Molecular Analysis of Chlamydomonas Mating-Type Locus

已有研究表明，位于mt基因座上的单个基因MID是诱导负配子分化和抑制正配子基因表达的必要条件和充分条件。进一步推测，Mid蛋白是通过与其他蛋白质合作作为转录激活/抑制因子来实现这些结果的。进一步推测，在正负配子融合后，Mid可能与一组不同的蛋白质合作激活受精卵特异基因的表达。这个项目的第一个具体目标是检验这个“中间假说”。将产生针对重组Mid蛋白的抗体，并用于免疫定位该蛋白，并在野生型和突变株中对Mid和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，以评估它是阻止还是增强配子和受精卵特异基因的转录。MID基因将在体外被诱变，以产生温度敏感的等位基因，当这些等位基因转化为MID缺失菌株时，应该可以识别其表达随着温度的升高而上调或下调的MID调节转录本。也已经确定，mt基因座充满了不寻常的遗传配置，这表明该地区一直是，或许还将继续是进化变化的核心。将使用几种方法来研究该基因座的进化历史和潜力。第二个具体目标是继续正在进行的染色体遍历密切相关物种C.Incerta的mt基因座，评估其组织在自两个物种分化以来的大约100万年中相对于C.reinhardtii基因座的保守和重新排列的程度。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、MID基因和侧翼序列的串联复制以及该元件在CC-421菌株基因组中的非mt位置的驻留。第四个具体目标是继续调查观察到的MID基因自C.reinhardtii/C.Incerta分歧以来引起了异常高水平的非同义和同义密码子变化。本研究的长期目标是了解单细胞绿藻-莱茵衣藻生活史转变的遗传基础，特别是关于这两个交配型基因座的遗传基础，并分析这些遗传系统的进化动力学。

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.