Genome rearrangements occur on an evolutionary scale, as well as on a developmental scale, in a wide range of eukaryotes. Even in cancer cells, dramatic DNA rearrangements are frequently observed in somatic cell lineages. This project uses certain species of ciliates as a model system to study gene rearrangements. These organisms undergo massive recombination during differentiation of an archival germline micronucleus into a somatic macronucleus capable of gene expression. The objective of this project is to increase the understanding of this process of assembly, starting from the basic principles of molecular DNA - RNA - enzyme interactions, through general knowledge of the possible pathways and intermediate steps that lead to the final product. Through mutual reinforcement, the project interlaces biological experiments with mathematical findings. The theoretical model utilizes spatial graphs with 4-valent vertices as a physical representation of the DNA at the time of recombination, and smoothing of the vertices models the actual recombination. Specific aims include: (a) examine the interactions of the template RNA and the involvement of possible cutting and splicing enzymes at the moment of recombination, (b) identify steps in the process by pinpointing putative intermediate molecules during unscrambling a variety of genes, and theoretically analyze sets of pointers leading to these intermediates (c) characterize theoretically and confirm experimentally all possible gene unscrambling pathways for a subset of genes, and (d) develop mathematical techniques based on methods from knot theory and formal languages to study the theoretical model.
Our understanding of genome-wide DNA rearrangements is still very limited. These processes appear on an evolutionary scale, where they can lead to species-specific differences and even the creation of species boundaries. On a developmental scale, genome rearrangements appear in a wide range of differentiating eukaryotic cells. The findings of this project will offer insight into the process of gene descrambling in ciliates and may also extend more generally to other model systems. This project seeks to learn from both theory and experiments how a mechanism for RNA-templated DNA recombination sculpts the assembly of genetic information in ciliates. This mechanism of template-mediated DNA rearrangement can be visualized as a scaffold for controlling and programming certain types of genetic information within a cell. Such control of information processing could facilitate biomolecular computing in vivo, as well as methods for epigenetic control of development.
In a wide range of species, from unicellular eukaryotes to mammals, genome rearrangements have been observed on an evolutionary scale, as well as on a developmental scale. Even in cancer cells, dramatic DNA rearrangements are frequently observed in somatic cell lineages. Some of the most excessive gene rearrangements and DNA recombination events appear in certain species of ciliates, a group of eukaryotic unicellular organisms. This project combined theory and experiments to improve our understanding of the mechanism for RNA-template guided DNA recombination in the assembly of genetic information. An interdisciplinary team of biologists at Princeton and mathematicians at the University of South Florida developed and experimentally tested a theoretical model for RNA-templates guided DNA rearrangement in the single-celled ciliate, Oxytricha trifallax. We developed a mathematical model based on spatial graphs and ideas from knot theory to study these rearrangements. These recombination events involve excision of certain sequences, rearrangement and inversion of other type of sequences. The recombination processes happen in certain succession, possibly with some recombination events performed at the same time, but others in a prescribed order. Our model provides a method for deriving all possible pathways of gene rearrangements. We applied our model to experimental data available from Princeton's lab that gave all possible intermediate molecules in the gene assembly. We have observed some previously unreported rearrangement events. Broader impacts include a book "Discrete and Topological Models in Molecular Biology" edited by the PIs and published by Springer in 2014, journal publications, public lectures and conference presentations given by the PIs. The USF-Princeton team also developed a variety of computational resources for the community, most of them available at the project website: http://knot.math.usf.edu. The project provided a broad cross-disciplinary training and mentorship of several graduate and undergraduate students, including hands-on training of USF math graduate students in molecular biology, with semester-long visits at the Princeton laboratory. The math graduate and undergraduate students have been exposed to an extensive literature in biology. They are trained in many research related skills, including writing papers and making presentations. Total of 18 students have been involved in this project, from summer undergraduate research projects to PhD dissertations. Many undergraduates have continued their studies in graduate or medical schools after their graduation at USF.
