Molecular Analysis of Chlamydomonas Mating-Type Locus

摘要已经证实，一个位于mt-位点的基因mid是诱导负配子分化和抑制正配子基因表达的必要和充分条件。进一步的假设是，Mid蛋白通过与其他蛋白合作作为转录激活因子/抑制因子来实现这些结果。进一步假设，在正配子和负配子融合后，Mid可能与一组不同的蛋白质合作，激活受精卵特异性基因的表达。这个项目的第一个具体目标是检验这个“中间假设”。将产生针对重组Mid蛋白的抗体，用于对该蛋白进行免疫定位，并对野生型和突变株的Mid及任何相关蛋白进行免疫沉淀。该抗体还将被添加到参与核运行转录的细胞核中，以评估它是阻断还是增强配子和合子特异性基因的转录。中间基因将在体外诱变以产生温度敏感的等位基因，当将其转化为中间缺失的菌株时，应该允许鉴定表达随温度升高而上调或下调的中间调控转录本。mt基因座充满了不寻常的遗传结构，这也表明该区域曾经是，也许将继续是进化变化的核心。该基因座的进化历史和潜力将通过几种方法进行研究。第二个具体目标是继续通过密切相关的物种，c.a incerta的mt位点进行染色体行走，评估其组织在大约100万年的时间里相对于c.a reinhardtii位点的保存和重排程度。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素在莱茵c -421菌株基因组中非mt位置的驻留。第四个具体目标是继续研究中基因发生异常高水平的非同义和同义密码子变化的观察，因为C. reinhardtii/C。incerta散度。本研究的长期目标是了解单细胞绿藻莱茵衣藻（Chlamydomonas reinhardtii）生命周期转变的遗传基础，特别是关于两个交配型位点，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.