Degree Type

Dissertation

Date of Award

2008

Degree Name

Doctor of Philosophy

Department

Genetics, Development and Cell Biology

First Advisor

Daniel Voytas

Abstract

One important step in the replication of LTR retrotransposons is the selection of a favorable chromosomal site for integrating cDNA into the host genome. The data emerging from studies of retroviruses and the yeast LTR retrotransposons suggest that a tethering mechanism underlies target site choice: integration complexes are tethered to specific components of chromatin, and this determines where the retrotransposon integrates. The chromoviruses are Ty3/gypsy retrotransposons that have chromodomains located in their integrase C termini. Three groups of chromoviruses are described based on amino acid sequence relationships of their chromodomains and comparisons of their chromodomains to the heterochromain protein I (HP1) chromodomain, which typically recognizes histone H3-K9 methylation, an epigenetic mark characteristic of heterochromatin. When fused to fluorescent marker proteins, the chromoviral chromodomains target proteins to specific subnuclear foci coincident with heterochromatin. The chromodomain of Maggy, a fungal chromovirus, recognizes histone H3 dimethyl- and trimethyl-K9 in vitro. Furthermore, when fused to the integrase of the Schizosaccharomyces pombe Tf1 retrotransposon, the Maggy chromodomain directs integration to sites of H3-K9 methylation. A model was suggested in which mobile elements specifically target heterochromatin and then perpetuate heterochromatin by establishing epigenetic marks through the RNAi pathway.

The plant centromere-specific retrotransposons (CR elements) are chromoviruses that are highly associated with centromeres. An eleven amino acid sequence motif (the CR motif) was identified in the C-terminus of CR integrases from both monocots and dicots. When a larger domain containing the CR motif was fused to YFP and expressed in Arabidopsis protoplasts, specific nuclear loci were observed that were coincident with centromere-specific markers, namely the DAPI-staining chromocenters and CENP-C, a component of the kinetochore. Enrichment of the CR domain in the chromocenters was decreased in ddm1 and met1 mutant cells, which have reduced levels of heterochromatin. An arginine residue was identified in the CR motif that is required for its function. Mutations in this residue disrupt the ability of the CR motif to target proteins to centromeres. Collectively, these data suggest that plant CR elements adhere to the tethering model and that the integrase-encoded CR motif recognizes a specific component of plant centromeric heterochromatin to direct integration of CR elements to centromeres.

Understanding the mechanism by which plant retrotransposons target integration requires functional elements that can be genetically manipulated. Toward this end, a retrotransposon vector system was developed that is derived from the tobacco Tnt1 retrotransposon. Mini-Tnt1 vectors were constructed by replacing portions of the Tnt1 open reading frames with a selectable marker gene. Tnt1-encoded gene products required for transposition were provided in trans by endogenous Tnt1 elements, whose expression was induced during the generation of tobacco protoplasts. Two different mini-Tnt1 vectors were developed: transcription of one is driven by a complete 5' LTR; transcription of the other is driven by a chimeric 5' LTR in which the U3 region was replaced by the CaMV 35S promoter. It was shown that both vectors can be effectively complemented in trans by the endogenous helper Tnt1s after transformation into tobacco protoplasts. Also, like endogenous Tnt1, insertion sites of mini-Tnt1s were within or near coding sequences. Experimental evidence was obtained indicating that genetic recombination occurs during Tnt1 reverse transcription and that multiple copies of Tnt1 mRNA are packaged into virus-like particles. This supports an emerging picture indicating that multiple mRNAs are used during reverse transcription of diverse LTR retroelements, and that recombination and template switching are common occurrences during replication.

Copyright Owner

Yi Hou

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

112 pages

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