Proteins Required for Zygotic Nuclear Fusion

这个项目的长期目标是了解新形成的受精卵中的单倍体核如何识别彼此作为最终融合的合适伙伴，它们如何相互移动，以及融合事件本身是如何启动的。如果前面的任何一个步骤出错，受精卵就不能发育。在受精卵中创建一个二倍体细胞核，消除了来自两个不同生物的染色体混合的最后障碍，从而能够产生遗传多样性。然而，尽管它具有核心重要性，但对核聚变或核配子的详细了解才刚刚开始。这一建议的具体目的是：1)免疫定位参与单细胞藻核融合的两个受精卵特异cDNA的基因产物，莱茵衣藻；2)筛选莱茵衣藻文库，寻找与这一过程中已涉及的酵母KAR基因同源的序列。第一个目标将通过产生针对融合蛋白的抗体来实现，融合蛋白是利用表达载体在B-半乳糖苷酶和目的基因之间定位胶原连接物而产生的。然后，这些抗体将被用来在发育中的受精卵中免疫定位它们各自的蛋白质。推测至少有一种多肽参与单倍体核的融合，只定位于内质网和核膜。第二个目标将通过根据预测的酵母KAR基因的氨基酸序列产生寡核苷酸，然后筛选存在相似序列的莱茵哈迪尔酵母文库来实现。然后将有可能确定这些基因在哪些发育阶段表达，以及它们在莱茵葡萄球菌中的功能是否与酵母中的功能同源。

The long-term objectives of this project are to understand how haploid nuclei within a newly formed zygote recognize each other as appropriate partners for eventual fusion, how they move towards one another, and how the fusion event itself is initiated. If any one of the preceding steps goes awry, the zygote can not develop. The creation of a single diploid nucleus within the zygote eliminates the last barriers to an intermingling of the chromosomes from two different organisms and thus enables the generation of genetic diversity. Despite its central importance, however, a detailed understanding of nuclear fusion or karyogamy has only just begun. The specific aims of this proposal are: to 1) immunolocalize the gene products of two zygote-specific cDNAs which are candidates for involvement in nuclear fusion in the unicellular alga, Chlamydomonas reinhardtii and 2) to screen C. reinhardtii libraries for sequences that are homologous to the yeast KAR genes already implicated in this process. The first goal will be accomplished by generating antibodies against fusion proteins created using an expression vector that positions a collagen linker between B-galactosidase and the cDNA of interest. These antibodies will then be used to immunolocalize their respective proteins within the developing zygote. It is hypothesized that at least one of the polypeptides will localize solely to the endoplasmic reticulum and the nuclear envelope due to its involvement in the fusion of the haploid nuclei. The second goal will be achieved by generating oligonucleotides based on the predicted amino acid sequences of the yeast KAR genes and then screening C. reinhardtii libraries for the presence of similar sequences. It will then be possible to determine during which developmental stages these genes are expressed and whether their function in C. reinhardtii is homologous to that in yeast.