Hooper, Helen (1995) The emulation of fatty add synthetase genes in B.napus. Doctoral thesis, Durham University.
The main aim of this work was to analyse a cloned ACP gene promoter, which had been shown to contain regulatory information that directed both spatial and temporal expression. Gel retardation assays were used initially to define protein binding sites within the ACP05 promoter. Following such analysis, a DNA motif that interacted with a sequence specific binding factor was identified. This factor was detected in embryo extracts and was not present in leaf extracts. The stability and binding characteristics of the ACP promoter binding protein were studied after heparin agarose chromatography, using gel retardation assays to follow the protein. A binding site oligonucleotide was synthesised and used as a probe to screen an expression library, in a "South Western" cloning experiment. A single positive clone, λBS2xi, was isolated. The DNA binding specificity of the recombinant protein was determined by gel retardation. The clone was confirmed to encode a functional sequence specific DNA binding domain. Northern hybridisations demonstrated the mRNA was expressed and in a tissue specific manner: levels of message were high in seed, low in root and not detected in leaf tissue. Endonuclease restriction of the lambda clone with EcoRI excised a 3.0kbp fragment that corresponded to the entire cDNA. The fragment was subcloned in pSK(^+) and several strategies were used to characterise and sequence it. A set of nested deletions was generated, but reliable sequence data was not obtained from the first half of the cDNA. A section of readable sequence data was obtained approximately 1.5Kbp from the 5' end of the deleted cDNA. Exhaustive databank searches using the sequence data demonstrated that it corresponded to β- galactosidase. Southern analysis further demonstrated that this sequence was present in λBS2xi. Further work required to characterise XBS2xi is discussed. A second related subject of this thesis concerns a second member of the FAS complex, enoyl- ACP reductase (ER). Prior to this work it was shown that there were four ER isoforms, expressed in both leaf and seed. The levels of expression of individual isoforms were different, there being two major forms and two minor forms. One leaf expressed clone, pERL8 had been isolated and characterised. A DNA probe that encompassed the 3' untranslated region (3'UTR) was generated from pERL8 and used to screen a cDNA library generated from embryo. Eleven positives were isolated and ten were successfully subcloned by plasmid rescue. The clones were sequenced with internal primers. Exhaustive searches of databanks using the sequence data demonstrated that five clones corresponded to seed storage proteins and five were confirmed as ER. The ER clones were divided into two groups on the basis of sequence differences. One clone was identical to pERL8 and represented an isoform expressed in both leaf and seed. The sequences of the clones within a group was identical, apart from an "insert" present in the 3' UTR of one clone from both groups. Whether these inserts were real or artifacts could not be demonstrated conclusively as further sequencing showed both clones with inserts in the 3' UTR were 5' truncated. In order to determine whether all four ER isoforms had been isolated further work would-be required. This is also discussed.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||09 Oct 2012 11:51|