Most ciliates are predators in the microbial world, eating bacteria and other protists. Many ciliates can form cysts when starved or otherwise stressed. The cysts are very strong, and can be dried and survive for years-when put back into water with food, the ciliate hatches out again. Ciliates are characterized by the presence of two types of nuclei in each cell: a somatic nucleus - macronucleus (MAC) - which is very large, and provides templates for the transcription of all genes required for vegetative growth, and a germline nucleus - micronucleus (MIC) - used for the exchange of meiotic products during sexual reproduction. The macronucleus is what allows ciliates to grow so large, since it can carry thousands of copies of each gene. Ciliates, like many protists, undergo sexual reproduction, even though they are single celled. Two cells of different mating types meet, and partially fuse, so that there is a cytoplasmic bridge connecting the two cells. Through this bridge the cells exchange gametic nuclei, equivalent to metazoan sperm and eggs. The cells then separate, and the gametic nuclei fuse and develop into the new macronucleus and micronucleus. DNA in the micronucleus remains organized in the form typical of most eukaryotes, with large chromosomes, each containing thousands of different genes. In contrast, chromosomes of the macronucleus genome undergo extensive DNA fragmentation, elimination and amplification. The extent of MAC genome reorganization varies greatly among ciliate species. In ciliates such as Oxytricha, the level of DNA processing in the formation of a new macronucleus is extraordinary: the original micronucleus chromosomes are fragmented at tens of thousands of positions, 95% of the DNA complexity is lost, and the resulting chromosomes - sometimes referred to as "nanochromosomes" - are amplified to thousands of copies each. Each macronucleus chromosome typically contains a single gene flanked by very short telomeres. The size of these molecules ranges from 0.25 to 35 kb, and each is present at an average of 1000 copies.
Oxytricha is a ciliated protozoan. The protozoa are single celled eukaryotes, and so unlike bacteria, and like animals and plants, have a nucleus. Most ciliates inhabit the free-living microbial world (anywhere where there is water, at least occasionally) and are some of the most obvious, since they are both large and generally very motile, propelled though the water or scuttling along surfaces with the aid of numerous cilia. Most ciliates are predators in the microbial world, eating bacteria and other protists. Many ciliates can from cysts when starved or otherwise stressed. The cysts are very tough, and can be dried and survive for years-when put back into water with food, the ciliate hatches out again. This not only allows ciliates to survive hard times, but probably is also a way to disperse, stuck to duck feathers, for example. Paramecium is a ciliate, and perhaps the best known protist, since so many have seen them as part of a microscope course in school. But ciliates are related to two other major groups of protists - the dinoflagellates and apicomplexans - because all three groups possess membrane-bound sacs or alveoli beneath their plasma membrane. The dinoflagellates are important single-celled algae in the oceans, while apicomplexans are very deadly pathogens - Plasmodium causes malaria. We hope that these comparisons will teach us more about all three groups.
The Oxytricha sequencing plan aims to provide information for both the MAC and MIC genomes. For the MAC genome, we plan to clone and sequence to finished quality all MAC chromosomes, utilizing a variety of subcloning and finishing strategies, according to the chromosomal size range. In addition to the MAC genomic sequence, we will prepare a fosmid library from the micronuclear genome that will provide 2X coverage of the MIC genome, to be printed onto high-density nitrocellulose filters for distribution to the end-user community by request. Each fosmid subclone will be end-sequenced to generate paired end reads that characterize the subclone and this information will be linked to the corresponding position on the fosmid filter grid. Based on screening the gridded library with MAC DNA or with specific probes, a selection of ~1400 fosmid clones will be sequenced and finished. A bioinformatics collaboration with members of the user community will result in a blending of the fosmid sequence information and the finished sequences of MAC chromosomes. The National Human Genome Research Institute (NHGRI), National Institutes of Health (NIH) is providing funding for the sequence characterization of the Oxytricha trifallax genome.