Molecular Analysis of Chlamydomonas Mating-Type Locus

已经确定，位于mt-位点的单个基因mid是诱导负配子分化和抑制正特异配子基因表达的必要和充分条件。进一步推测，Mid蛋白通过与其他蛋白协作充当转录激活因子/抑制因子来实现这些结果。进一步推测，Mid可能与不同的蛋白质组协作，以在正配子和负配子融合后激活合子特异性基因的表达。本项目的第一个具体目标是检验这一“中间假设”。将针对重组Mid蛋白产生抗体，并用于免疫定位蛋白质，并在野生型和突变株中进行Mid加任何相关蛋白质的免疫沉淀。还将抗体添加到参与核连续转录的核中，以评估其是否阻断或增强配子和合子特异性基因的转录。将在体外对mid基因进行诱变，以产生温度敏感性等位基因，当将其转化到mid缺失菌株中时，应允许鉴定其表达随温度升高而上调或下调的中间调节转录物。人们也已经确定mt基因座充满了不寻常的遗传结构，这表明该区域一直是，也许继续是进化变化的核心。 该基因座的进化历史和潜力将使用几种方法进行研究。第二个具体的目标是继续进行一个正在进行的染色体步行通过一个密切相关的物种，C。incerta，评估其组织相对于C. reinhardtii轨迹估计100万年以来，这两个物种的分歧。第三个具体目标是继续评价最近（过去20年）与mt相关的异常，即中间基因和侧翼序列的串联重复以及该元件在CC-421株C.莱因哈德氏菌 第四个具体的目标是继续研究观察到的mid基因已经引起了异常高水平的非同义和同义密码子的变化，因为C。reinhardtii/C.不确定的分歧本研究的长期目标是了解单细胞绿色衣藻，莱茵衣藻的生命周期转换的遗传基础，特别是关于两个交配型基因座，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.