Beth Berta

Defense Date

4-16-2004

Graduation Date

Spring 2004

Availability

Immediate Access

Submission Type

thesis

Degree Name

MS

Department

Biological Sciences

Committee Chair

David J. Lampe

Committee Member

Mary Alleman

Committee Member

Nancy Trun

Keywords

Himar1, mariner, transposon

Abstract

Himar1, found in the horn fly Haematobia irritans, belongs to the mariner family of transposable elements. Mariners are a diverse and widespread family of eukaryotic transposons that transpose via a cut-and-paste mechanism. Mariner transposition not only requires the transposase, but also specific cis-acting DNA sequences known as inverted terminal repeats (ITRs). Very little is known about the fine structure of mariner ITRs, particularly which bases are important for transposition. A mariner family ITR sequence logo representing 20 different mariners from six different subfamilies suggests that more conserved regions of the logo are required for transposase binding while the more variable regions of the logo are likely to be involved in secondary transposition functions such as DNA cleavage and element insertion. To test this hypothesis, point mutations were introduced at 27 positions of the Himar1 ITR plus the two flanking nucleotides, "T" and "A". An Escherichia coli based genetic screen yielded results that lacked a clear pattern similar to that of the sequence logo. Therefore, gel mobility shift DNA-binding, strand-specific cleavage, and in vitro transposition assays were performed to test certain positions in the ITR that exhibited null phenotypes in the genetic screen. Positions 2, 12, 15, 22, and 24 of the ITR are required for DNA-binding. Positions 5, 8, 11, 20, 26, 27, and the second flanking nucleotide (A) exhibited inefficient DNA cleavage in comparison to the wild type ITR. Position 26 of the ITR is required for element insertion into the target DNA. Overall, my results indicate that the transposase is making base-specific contacts with the DNA on one face of the Himar1 ITR. However, these contacts appear to be occurring along the variable regions of the mariner ITR sequence logo.

Format

PDF

Language

English

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