Proteins Required for Zygotic Nuclear Fusion

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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.