Somatic Chromosome Origins and Organization in the Genome of Tetrahymena thermophila

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Developmental genome rearrangements have been found at the gene level in a number of diverse organisms, including genes for vertebrate immunoglobin and yeast mating type. The ciliated protozoa provide an extreme case, in which an entire genome is subject to massive developmental alteration. All sexually competent ciliates contain two separate nuclei, a silent germinal micronucleus and an actively transcribed somatic macronucleus. Tetrahymena thermophila has a diploid micronucleus with five metacentric chromosomes that undergo meiosis and mitosis and are transcriptionally silent. It's macronucleus, derived from the micronucleus during conjugation following extensive genome alteration, contains about 45 copies of 200 amitotic chromosomes that have lost 10-15% of the genome, are maintained by a copy number correction system, are actively transcribed, and segregate haplotypes of the fragmented genome. In this two-part project, the sites for the rearrangement will be isolated and used to locate the macronuclear genome on the micronuclear genetic map, creating two relational genetic maps. In the first part, germinal deletions of various sizes will be created and mapped to specific micronuclear chromosomes. Because the micronucleus is not expressed, deletions will be maintained in heterokaryons, with homozygous mutant micronuclei and wild type macronuclei, and crossed to each other to identify overlaps. DNA from deletion strains will be used as template for PCR, with primers made from already mapped micronuclear specific RAPD sequences, to place the deletions on the existing micronuclear meiotic map. In the second part, the sequences that flank chromosome breakage sites (cbs) will be cloned and sequenced. These sites occur where macronuclear chromosomes are cut out of micronuclear chromosomes during development, and therefore mark the boundaries of adjacent macronuclear chromosomes in the micronuclear genome. The extent of micronuclear specific DNA will be determined in each clone and the clones will be used to identify adjoining macronuclear chromosomes, by probing pulse field electrophoresis gels. Length of macronuclear chromosomes and micronuclear specific sequences at the junctions will be combined to create a physical map of the macronuclear chromosomes projected on the micronuclear genome. The meiotic map location of the breakage sites will be identified by the mapped deletions, created in the first part, to relate the physical and genetic maps.The end result of this project will be a collection of extremely useful genetic reagents (the deletion set) and the addition of physical distances and specific macronuclear locations to the genetic map of Tetrahymena. This information will greatly facilitate positional cloning and cloning by complementation. In addition, the collection of sequences at all the break points will add to our knowledge this developmentally directed chromosome rearrangement.