An Oxytricha trifallax micronuclear BAC library

We propose construction of a BAC library for the germline micronuclear genome (MIC) of the ciliate protist Oxytricha trifallax (recently renamed Sterkiella histriomuscorum; alveolate; class Spirotrichia). This library will primarily be used as a tool to characterize the structure and function of the ciliate micronucleus and its relationship to that of the somatic macronucleus, rather than for complete genome sequencing. Micronuclear differentiation into macronuclei requires specialized DNA replication, cleavage and splicing, telomere addition, specific DNA destruction, and highly dramatic DNA rearrangement events, including the unscrambling of protein-coding gene fragments dispersed in the MIC. As many as 25-30% of the 20,000-30,000 genes in the MIC appear to be scrambled in complex patterns ranging from few to over 50 fragments, present at one or more micronuclear loci. These fragments become properly reordered and joined into linear gene-sized " chromosomes " in the MAC. Studying these DNA processing events requires the detailed comparison of micronuclear with macronuclear sequences, which will be greatly aided by the availability of a micronuclear BAC library. Ciliates have been important experimental organisms for over 100 years, contributing to the discovery and understanding of many essential cellular processes—including self-splicing RNA, telomere biochemistry, and transcriptional regulation by histone modification—with Oxytricha representing the lineage with the surprising discoveries of gene-sized macronuclear mini-chromosomes and scrambled genes. Among Spirotrichs, O. trifallax also appears to have the highest density of scrambled genes in its micronucleus. • INTRODUCTION TO CILATE BIOLOGY Ciliates diverged from other microbial eukaryotes quite late in the history of life, as part of a monophyletic lineage (alveolates) with apicomplexans (e.g. Plasmodium) and dinoflagellates (Wright & Lynn 1997; Baldauf e t al. 2000). Therefore, as phylogenetic outgroups, ciliates provide a foil to studies of the crown eukaryotes plants, animals, and fungi. Molecular biologists have focused on several ciliates, of one of two classes, the Oligohymenophorans Tetrahymena and Paramecium, and the Spirotrichs, with Euplotes and several Stichotrichs (Stylonychia and Oxytricha); each has its experimental virtues. Ciliates have a typical diploid, meiotic germ-line genome in the micronucleus (MIC), except that its genes are not directly expressed, and those that are scrambled in the MIC have an exceptional organization (see figure). A highly specialized expression organelle, the macronucleus (MAC), develops from a mitotic copy of the MIC immediately after cells mate and exchange gametic nuclei. Stichotrich MAC differentiation is the premier showcase of somatic genome alterations (Jahn & Klobutcher 2002). As the MAC differentiates: MIC …