Abstract/Details

LTR retrotransposons in plants: Mechanisms of targeted integration and applications in genome engineering


2008 2008

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Abstract (summary)

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.

Indexing (details)


Subject
Genetics
Classification
0369: Genetics
Identifier / keyword
Biological sciences; Centromere-specific retrotransposons; Chromodomains; Chromovirus; Integrase; LTR retrotransposon; Mini-Tnt1; Retrotransposons
Title
LTR retrotransposons in plants: Mechanisms of targeted integration and applications in genome engineering
Author
Hou, Yi
Number of pages
112
Publication year
2008
Degree date
2008
School code
0097
Source
DAI-B 71/07, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9780549997092
Advisor
Voytas, Daniel
Committee member
Howell, Stephen; Miller, W. Allen; Peterson, Thomas; Yin, Yanhai
University/institution
Iowa State University
Department
Genetics, Development and Cell Biology
University location
United States -- Iowa
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3342258
ProQuest document ID
734184126
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/734184126
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