Delauney, Ashton Joseph (1984) Cloning and characterisation of cDNAs encoding the major, pea storage proteins, and expression of vicilin in E.coli. Doctoral thesis, Durham University.
A cDNA library was constructed using mRNA from developing seeds of pea (Pisim s'ativum L.). Clones encoding legumin and vicilin, the major storage proteins, were isolated and characterised, in several cases to the extent of complete DNA sequencing. The composite DNA sequence of the two longest legumin cDNAs extended over almost 90% of a complete legumin gene coding sequence. Both these clones contained three ~54bp tandem repeats in the region1encoding the acidic subunit. Evidence is presented that these repeats may be present in all chromosomal legumin genes and consequently, that the absence of the repeats from a previously isolated legumin cDNA probably represented a cloning artefact. Two, near full-length, vicilin cDNAs encoding 50000-Mr vicilin subunits, and another encoding a 47000-Mr subunit were sequenced. The 50000-Mr vicilin cDNAs were almost identical over most of their lengths, but one contained an artefactual, inverse repeat at its 3' terminus, while sequence differences at the 3' termini indicated the use of alternative polyadenylation sites. Comparisons of protein and cDNA-encoded amino acid sequences indicated that vicilins are synthesised as polypeptide precursors which subsequently undergo the removal of an N-terminal signal pep tide and possibly a C-terminal extension, as well as being susceptible to endo-proteolytic processing. Extending these comparisons to legumin and lectin sequences suggests that endo-proteolysis of these seed proteins occurs on the C-terminal side of asparagine residues located within B-turn conformations in hydrophilic regions of the proteins. Two vicilin cDNAs were expressed as both fused and unfused products in Escherichia coli under the influence of the phage lambda, leftward promoter (λP(_L)). Levels of expression obtained with different expression plasmid constructions supported previous hypotheses that translational efficiencies were lowered when the Shine-Dalgarno sequence was sequestered into double-stranded regions of the mRNA. There was also some indication that synthesis of a vicilin polypeptide bearing a signal peptide had a deleterious effect on the viability of the host strain.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||18 Sep 2013 09:19|