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Genome Remodeling and Developmental Programming
The ciliated protozoan, Tetrahymena, is a single celled organism that contains two nuclei: a diploid, germ line micronucleus and transcriptionally active macronucleus. During sexual reproduction, the macronucleus is degraded and a new one develops from a mitotic product of the micronucleus. The development of the new macronucleus involves extensive genome remodeling, including elimination of about 15% of the genome through 5000 or more site specific DNA deletions, de novo DNA methylation and multiple rounds of endoreduplication of the macronuclear genome. Since mating can be easily synchronized in Tetrahymena, the organism provides an unusually good system for analysis of the molecular machinery required for these events (7).
Our long term goal is to understand the molecular mechanisms of genome remodeling. We have characterized families of sequences that are eliminated from the macronuclear genome. One of these encodes homing endonucleases-like proteins with apetala2 DNA binding domains, the only example of this domain outside the plant kingdom (3). Another family, designated Tlr elements, were the first example of the Maverick elements, which are characterized by moderate copy number, long inverted terminal repeats, and a high degree of sequence identity among the family members (6). In vivo rearrangement experiments showed that any part of the Tlr element can serve to promote developmentally regulated deletion from a processing vector (4). This suggested that the machinery for genome remodeling operates via a genome surveillance mechanism. We proposed that rather than recognizing specific sequences, the cellular machinery targets some general feature of the germ line limited DNA, such as repetition of the sequence in the micronuclear genome. Recent experiments showed that foreign DNA sequences can be eliminated from the developing macronucleus and the efficiency of their deletion increases with the copy number of the element in the micronucleus (2).
The molecular machinery for genome remodeling is developmental stage specific. This characteristic was used to isolate genes that are up-regulated at the time of DNA rearrangement (1,5). One of them, ASI2, is nonessential for vegetative growth but is required in both the parental macronucleus and the germ line micronucleus to complete the program of sexual reproduction. Germ line knockouts of ASI2 with wild type macronuclei develop new macronuclei and undergo DNA rearrangement, but they fail to complete endoreduplication of the DNA and die before the first postzygotic division (1). The deduced protein encoded by ASI2 resembles a signal transduction receptor. We propose that it is required at a developmental checkpoint in the program of sexual reproduction. Current experiments are directed at testing the model that ASI2 encodes a signal transduction receptor that interacts with its downstream effector through an SH2 binding domain, and characterization of a protein ASI2 may interact with to transduce the signal.
Selected References
1. Li, S., L. Yin, E. S. Cole, R. A. Udani and K. M. Karrer (2006) Progeny of germ line knockouts of ASI2, a gene encoding a putative signal transduction receptor in Tetrahymena thermophila, fail to make the transition from sexual reproduction to vegetative growth. Developmental Biology 295:633-646.
2. Liu, Y., X. Song, M.A. Gorovsky and K.M. Karrer (2005) Elimination of foreign DNA during somatic differentiation in Tetrahymena is dosage dependent and involves double-stranded RNA. Euk Cell. 4:421-431
3. Wuitschick, J.D., P.R. Lindstrom, A.E. Meyer and K.M. Karrer (2004) Homing endonucleases encoded by germ line limited genes in the ciliate Tetrahymena have APETELA2 DNA binding domains. Euk. Cell 3:685-694.
4. Wuitschick, J.D. and K.M. Karrer (2003) Diverse sequences within Tlr elements target programmed DNA elimination in Tetrahymena thermophila. Euk. Cell 2:678-689.
5. Udani, R.A. and K.M. Karrer (2002) ASI1, a gene encoding a novel leucine zipper protein, is induced during development of the macronucleus in Tetrahymena. Mech. Dev. 118:215-218.
6. Wuitschick, J.D., J.A. Gershan, A.J. Lochowicz, S. Li and K.M. Karrer (2002) A novel family of mobile genetic elements are limited to the germ line genome in Tetrahymena thermophila Nucleic Acids Res. 30:2524-2537.
7. Karrer, K.M. (2000) Tetrahymena genetics: Two nuclei are better than one. In: Methods in Cell Biology series: Tetrahymena thermophila. Ed. D. Asai and J. Forney. Vol. 62 pp. 127-186.
Invited talks during the 2006/2007 Academic Year
Signal transduction and developmental programming in the ciliate Tetrahymena. Duquesne University, Pittsburgh, PA. November 10, 2006
ASI2, a gene encoding a putative signal transduction receptor, is required for sexual reproduction in Tetrahymena thermophila. Shuqiang Li, Lihui Yin, Eric S. Cole and Kathleen M. Karrer FASEB Summer Research Conference in Ciliate Molecular Biology. Tucson, AZ July 26, 2007.
Papers Presented at Meetings During the 2006/2007 Academic Year
Mutational anlaysis of ASI2, a gene encoding a predicted signal transduction receptor in Tetrahymena. Summer Research Conference in Ciliate Molecular Biology. Tucson, AZ July 24, 2007. (Presented by L. Yin).
