Determination of Subcellular Localization of Brambleberry Homolog in Tetrahymena thermophila.

Abstract

During early embryonic development in the Danio rerio, maternal-effect factor Brambleberry(bmb) was shown to assist in pronuclear fusion, is localized on the nuclear membrane. The process of the fusion of chromatin-containing structures known as karyomeres has also been shown to be facilitated by Brambleberry, and is essential in the formation of a single mononucleus following fertilization. Questions concerning the conservation of the function of Brambleberry arose, and an in-silico Basic Local Alignment Sequence Tool search unveiled a homolog of bmb in the ciliate Tetrahymena thermophila. Depending on nutrient levels in the environment, T. thermophila is capable of performing either sexual or asexual reproduction. The sexual reproductive process resembles that of higher vertebrates, and involves the exchange of haploid nuclei, of which there are a migratory and residential pair. The migratory nuclei is exchanged with the mating pair, and the migratory and residential pairs then fuse, forming a diploid nucleus with genetic material from both mating pairs. It is this fusion event, and the formation of the diploid nucleus that will be studied through molecular techniques. Through the use of Polymerase Chain Reaction (PCR), a construct will be created containing upstream and downstream homology arms, along with a fluorescent marker, and a selectivity marker. This approach involves observation of the localization of Brambleberry in real time. The function of Brambleberry in T. thermophila will be compared to the function of Brambleberry in Danio rerio.