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Transposon-Induced Homologous Recombination at the Maize P Locus and in Transgenic Arabidopsis Yongli Xiao Iowa State University

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Transposon-Induced Homologous Recombination at the Maize P Locus and in Transgenic Arabidopsis Yongli Xiao Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1999 Transposon-induced homologous recombination at the maize P locus and in transgenic Arabidopsis Yongli Xiao Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Genetics Commons, and the Molecular Biology Commons Recommended Citation Xiao, Yongli, "Transposon-induced homologous recombination at the maize P locus and in transgenic Arabidopsis " (1999). Retrospective Theses and Dissertations. 12626. https://lib.dr.iastate.edu/rtd/12626 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfihn master. UMI films the text directly fix)m the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter &ce, while others may be fi^m any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photogr^hs, print bleedthrough, substandard margins, and improper alignment can adverse^ affect reproduction. In the unlikely event that the author did not said UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, b^inning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photogr^hed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. EBgher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contsict UMI directly to order. UMI A Bell & Howell lofijrmation Company 300 North Zeeb Road, Ann Aiiwr MI 48106-1346 USA 313/761-4700 800/521-0600 Transposon-induced homologous recombination at the maize P locus and in transgenic Arabidopsis by Yongli Xiao A dissertation submitted to the graduate faculty in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major Genetics Major Professon Thomas A. Peterson Iowa State University Ames, Iowa 1999 DMJ Ntunber: 9924780 UMI Microform 9924780 Copyriglit 1999, by UMI Company. AU rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ami Arbor, MI 48103 Graduate College Iowa State University This is to certify the Doctoral dissertation of YongliXiao Has met the dissertation requirement of Iowa State University Signature was redacted for privacy. Major Professor Signature was redacted for privacy. For the Major Program Signature was redacted for privacy. iii TABLE OF CONTENTS CHAPTER 1. INTRODUCTION 1 Dissertation Organization I Literature Review 2 References 32 CHAPTER 2. Ac INSERTION SITE AFFECTS THE FREQUENCY OF TRANSPOSON- INDUCED HOMOLOGOUS RECOMBINATION AT THE MAIZE P LOCUS 51 Abstract 51 Introduction 52 Materials and Methods 55 Results 57 Discussion 61 References 66 CHAPTER 3. INTRACHROMOSOMAL HOMOLOGOUS RECOMBINATION IN ARABIDOPSIS INDUCED BY A MAIZE TRANSPOSON 81 Abstract 81 Results and Discussion 82 Methods 87 References 89 Acknowledgements 91 CHAPTER 4. GENERAL SUMMARY 99 References 103 ACKNOWLEDGEMENTS 107 1 CHAPTER 1. GENERAL INTRODUCTION Dissertation Organization This dissertation is organized in an alternative formaL It contains four chapters including general introduction and general sununary. References are placed at the end of each chapter. Two chapters include papers: Chapter 3 is paper reviewed by Nature Genetics, and Chapter 2 is in preparation for submission to Genetics for formal publication. Chapter 2 presents an analysis of the effects of a maize transposable element (transposon). Activator (Ac), on homologous recombination at maize P locus. The results show that the insertion position of the Ac transposon relative to two homologous direct repeats at the P locus can affect the frequency of the homologous recombination, whereas the orientation of the transposon insertion may not have a significant effect on recombination. The possible mechanism is discussed. This is the first report to demonstrate that a transposon insertion position can affect the frequency of homologous recombination in plants. Chapter 3 demonstrates that transposon-induced homologous recombination can occur on an artificial construct in transgenic Arabidopsis, a dicotyledonous model plant. Here, the maize transposon (Ac/Ds system) increases homologous recombination frequency over 1000 fold in plants with an active transposon than in control plants with the same reporter construct but lacking an active transposon. In Arabidopsis, transposon-induced recombination was observed in different organs during different developmental stages, and several germinally transmitted events were recovered. Therefore, transposon-induced 2 homologous recombination appears to be a general phenomenon in plants and may play an important role in genome evolution. Literature Review Transposable elements are segments of DNA that can promote their own insertion into new locations via element-encoded transposase proteins. Transposable elements are widespread among different organisms and play an important role in genome evolution. (Ginzburg era/. 1984; Mackay 1986; Finnegan 1989; Kidwell and Lisch 1997). Transposable elements in bacteria In bacteria, there are three type of transposable elements: Insertion seguenceJlS) elements, transposons (Tn) and Mu bacteriophage. IS elements were discovered during the study of mutations in the galactose and lactose operons (Jordan et aL 1968; Malamy 1966, 1970; Shapiro 1969) and in the early genes of bacteriophage k (Brachet etal. 1970). Electron microscopic analysis of DNA/DNA heteroduplexes indicated that many of these mutations carried distinct DNA segment insertions; hence they are called insertion sequences (Fiandt et al, 1972; Hirsch et aL 1972; Malamy et al. 1972). After the initial discovery of IS elements (ISl, IS2, IS3, IS4), many others were found in the genomes, plasmids and bacteriophages of a wide range of bacterial genera and species. The length of IS elements are from 800 to 2500 bp, and their copy number is from 0 to a few hundred per genome. All IS elements have perfect or nearly perfect terminal inverted repeats (IRs) of about 9-41 bp. These are recognition sites for transposase, which is the enzyme for transposition produced by the transposable element Upon insertion into a new position, an IS element generates small (2 to 3 13bp) directly repeated duplications of the target DNA at the insertion point This sequence is called target site duplication (TSD). The original IS element remains in place (Ohtsubo and Sekine 1996). Prokaryotic transposons (Tn), like IS elements, encode transposase, have inverted terminal repeats and generate target site duplications. Unlike IS element, Tns usually contain other genes, such as drug resistance genes. In addition, Tns can be classified into two groups: 1) Composite transposons are complex transposons with a central region containing genes (including drug resistance genes) flanked by IS elements on both sides. For example, TnlO has two IS 10 elements located in opposite orientation at both ends. Between the IS 10 elements, there is -6.7 kb of unique sequence including a tetracycline resistance gene. TnlO generates a 9 bp target site duplication after transposition (Kleckner 1979, 1989). 2) Noncomposite transposons also carry drug resistance genes like composite transposons, but they do not terminate with IS elements. They have their own terminal inverted repeats required for transposition. Tn3 is one example of a noncomposite transposon; it contains an ampicillin resistance gene and it is the first described transposon carrying an antibiotic resistance gene (Hedges and Jacob, 1974). Tn3 is 4957 bp and has 38 bp terminal inverted repeats. In addition to the ampicillin resistance gene, Tn3 contains the tnpA and tnpB genes encoding the transposase and resolvase for Tn3 transposition in the central region. Tn3 produces a 3 bp target site duplication (Heffron et al. 1979; Grindley 1983). There are two types of transposition mechanisms used by bacterial transposons. 1) Replicative transposition involves the fusion of the donor DNA carrying the transposon and the recipient DNA into a single molecule known as a co-integrate. The transposon becomes duplicated with one copy located at each junction between donor and recipient DNA 4 Subsequently, the co-integrate resolves into two products, each containing one copy of the transposon. 2) In nonreplicative transposition (or simple insertion), the transposon moves from one location to another without replication. The element is lost fr-om the original position. DiSerent transposons can use different mechanisms; for example, Tn3 uses replicative transposition, while TnlO transposes by a nonreplicative mechanism. Most
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