Tetrahymena is a ciliated protozoan which contains two nuclei, a diploid, germinal micronucleus (mic) and a polyploid macronucleus (mac) which is transcriptionally active in the vegetative cell. At conjugation (sexual reproduction), the macronucleus is degraded and a new macronucleus is made from one of the mitotic products of the zygotic micronucleus. The development of the new macronucleus involves 1.) polyploidization of the bulk of the Tetrahymena genome to the level of 45C 2.) elimination of specific DNA sequences and rearrangement of some others 3.) methylation of the macronuclear DNA and 4.) the initiation of transcription from a genome which was previously silent. We plan to study several aspects of the development of the somatic macronucleus from the germ line micronucleus. We have cloned a fragment of Tetrahymena micronuclear DNA which is rearranged during development of the macronucleus. Molecular cloning techniques will be used to analyze the nature of the rearrangement, to determine whether any of the various DNA sequences which have been implicated in genome rearrangements are in close proximity to this particular rearranged sequence and to probe for transcription of this sequence in vegetative and conjugating cells. Macronuclear DNA in Tetrahymena contains 0.8% methyladenine. Micronuclear DNA is unmethylated. In vivo labelling techniques will be used to determine the developmental stage at which methylation occurs. We have isolated several mic-specific DNA sequences. They will be used to probe RNA preparations to determine whether they are transcribed during conjugation. Several predominant in vivo labelled proteins are specifically synthesized in conjugating cells. Phage wil be selected which contain Tetrahymena DNA sequences homologous to RNAs translated during conjugation. Filter hybridization will be used to determine whether the DNA sequences are mic-specific and/or linked on the Tetrahymena chromosomes and to determine the stage(s) at which the conjugation specific RNAs are transcribed.
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