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Title: Gene tagging in Arabidopsis
Author: Caryl, A. P.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1997
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The work described in this thesis takes two approaches towards the identification of tagged genes in Arabidopsis thaliana. A family screening approach was used to isolate mutants from the T-DNA transformed Feldmann lines of Arabidopsis. Co-segregation analysis was used to determine whether these mutants were tagged. None of the mutants isolated showed co-segregation with the T-DNA so this approach was abandoned. Enhancer trap constructs are designed to report the activity of enhancers near a transgene insert. Typically they contain a reporter gene with an weak promoter in a transformation vector. The second approach used in this study was to investigate a collection of 123 independently transformed lines of Arabidopsis containing such constructs which were available in the laboratory. The construct introduced into these lines contained a β-Glucuronidase reporter gene under the control of an attenuated Cauliflower Mosaic Virus promoter. The number of independent active inserts, T-DNA copy number and GUS staining patterns of the lines were investigated. One of the lines, Δ31, showed GUS staining associated with meristematic tissue (with the exception of the primary root meristem). The expression of the GUS reporter gene was presumably being controlled by a plant enhancer adjacent to the insert. Presumably the enhancer would normally act to regulate the expression of an endongenous plant gene. Spectrophotometric assays were used to measure the response to auxin in root tissues. IPCR was used to amplify plant DNA flanking the insert in line Δ31. A clone of this DNA was used to isolate four large overlapping clones from a lambda genomic library of wild-type plants which would be likely to contain the tagged enhancer, and any endogenous gene regulated by this enhancer.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available