Proteins Required for Zygotic Nuclear Fusion

该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。如果上述任何一个步骤出错，受精卵就不能发育。在受精卵内产生一个二倍体核，消除了来自两个不同生物体的染色体混合的最后障碍，从而使遗传多样性的产生成为可能。然而，尽管它具有核心重要性，但对核聚变或核肌力的详细了解才刚刚开始。本提案的具体目的是：1)免疫定位两种受精卵特异性cdna的基因产物，这两种cdna是参与单细胞藻类莱茵衣藻（Chlamydomonas reinhardtii）核融合的候选基因；2)筛选莱茵衣藻文库中与该过程中已经涉及的酵母KAR基因同源的序列。第一个目标将通过生成针对融合蛋白的抗体来实现，该融合蛋白使用一种表达载体，将胶原连接物置于b -半乳糖苷酶和感兴趣的cDNA之间。然后，这些抗体将被用来免疫定位发育中的受精卵中各自的蛋白质。据推测，至少有一种多肽由于参与单倍体核的融合而只定位于内质网和核膜。第二个目标将通过基于酵母KAR基因的预测氨基酸序列生成寡核苷酸，然后筛选C. reinhardtii文库中是否存在类似序列来实现。这样就有可能确定这些基因是在哪个发育阶段表达的，以及它们在莱因哈德梭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.