Degree Type

Dissertation

Date of Award

2009

Degree Name

Doctor of Philosophy

Department

Genetics, Development and Cell Biology

First Advisor

Thomas Peterson

Abstract

Traditional Ac/Ds transposable elements have been used in gene isolation and functional analysis in a variety of plants. Alternative transposition refers to transposition events involving the ends of different transposons; alternative transposition can induce a variety of genome rearrangement, including deletion, duplication, and inversion. The underlying aim of this thesis study is to gain a greater understanding of the alternative transposition process and the genome rearrangements thereby generated.

Composite or closely linked maize Ac/Ds transposable elements can induce chromosome breakage, but the precise configurations of Ac/Ds elements that can lead to chromosome breakage are not completely defined. Here, we determined the structures and chromosome breakage properties of 14 maize p1 alleles: each allele contains a fixed fractured Ac (fAc) element and a closely linked full-length Ac at various flanking sites. Our results show that pairs of directly or reversely oriented Ac/Ds termini can induce chromosome breakage, whereas elements arranged in a macrotransposon configuration do not. Among the structures that can lead to chromosome breaks, breakage frequency is inversely correlated with the distance between the interacting Ac/Ds termini. These results provide new insight into the mechanism of transposition-induced chromosome breakage, which is one outcome of the chromosome-restructuring ability of alternative transposition events.

The alternative transposition model predicts that a variety of chromosome rearrangements could be generated. Indeed, Barbara McClintock reported that the Ac/Ds transposable element system can generate major chromosomal rearrangements (MCRs). However, evidence that alternative transposition can directly generate chromosome rearrangements was not previuously reported. In this study, we identified a series of chromosome rearrangements derived from maize lines containing pairs of closely-linked Ac transposable element termini. Molecular and cytogenetic analyses showed that the MCRs in these lines comprised 17 reciprocal translocations and two large inversions. The breakpoints of all 19 MCRs are delineated by Ac termini and characteristic 8 bp target site duplications, indicating that the MCRs were generated by precise transposition reactions involving the Ac termini of two closely-linked elements. This alternative transposition mechanism may have contributed to chromosome evolution, and may also occur during V(D)J recombination resulting in oncogenic translocations.

To investigate the types and frequencies of the genome rearrangements caused by a pair of reversed Ac/Ds termini, we screened 100 mutant alleles induced by an intact Ac and a fractured Ac (fAc) structure at the maize p1 locus from two alleles P1-rr11 and P1-rr910. The rearrangement types were characterized by PCR pattern analysis and/or by direct sequencing. The results show that 20/100 mutant alleles display deletion patterns and 52/100 exhibit inversions, translocations or local rearrangements. Sequence analysis results show the majority of another set of 17/100 is flanking Ac or fAc small deletion. Interestingly one deletion allele, with only one nucleotide distance between Ac and fAc, is not competent for alternative transposition, evaluated by chromosome breakage frequency. 11/100 alleles are possible simple Ac excision or unknown mechanism. The reversed ends inserting to sister chromatid model was proposed to explain how the flanking Ac/fAc deletion alleles were generated from their parental lines.

Finally, to assess the extent to which alternative transposition can be applied in non-maize plants, we tested the ability of Ac/Ds alternative transposition to induce genome rearrangements in rice plant. In this study, we transformed a reverse-orientated Ac/Ds ends construct together with an Ac transposase gene in cis into rice (Oryza sativa ssp. japonica cv. Nipponbare). A green fluorescence protein (GFP) marker between the reversed Ds termini was used for efficient screening of rearrangement events. Molecular analyses indicate that a total of 25 independent rearrangements were obtained from three different chromosome loci. The rearrangements include chromosome deletions and inversions, and one translocation. Most of the deletions occurred within the T-DNA region, but two cases removed 72.5 kilobase pairs (kb) and 79 kb of rice genomic DNA flanking the transgene. The 79 kb deletion can be maintained as a heterozygote, but appears to be homozygous lethal as no homozygous deletion plants could be recovered. In addition to deletions, a variety of inversions were obtained from one transgene locus. The inversions range from very small (within the transgene DNA) to over 1 million base pairs (Mb) in size. For a subset of inversions both breakpoints were sequenced, and all of these contained the typical 8 base pairs (bp) Ac/Ds targeted site duplication (TSD), confirming their origin as transposition products. Together, our results indicate that alternative Ac/Ds transposition can be an effective tool for functional genomics and chromosome manipulation in rice.

DOI

https://doi.org/10.31274/etd-180810-1020

Copyright Owner

Chuanhe Yu

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

162 pages

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