Ribozyme-mediated cleavage of bovine α-lactalbumin mRNA

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dc.contributor.advisor Bellamy, R. en
dc.contributor.author L'Huillier, Phil J. en
dc.date.accessioned 2007-07-30T08:03:20Z en
dc.date.available 2007-07-30T08:03:20Z en
dc.date.issued 1992 en
dc.identifier THESIS 93-180 en
dc.identifier.citation Thesis (PhD--Cellular and Molecular Biology)--University of Auckland, 1992 en
dc.identifier.uri http://hdl.handle.net/2292/1156 en
dc.description Full text is available to authenticated members of The University of Auckland only. en
dc.description.abstract 1. Alpha-lactalbumin and β 1, 4-galactosyltransferase form the enzyme complex lactose synthetase which is involved in synthesis of the milk sugar lactose. The ability of ribozymes to modify α-lactalbumin gene expression has been investigated using the T7-vaccinia delivery system to achieve high concentrations of ribozyme transcripts in the cytoplasm of Cl27I mouse mammary cells. 2. Five hammerhead ribozymes were designed that targeted selected sites along the bovine α-lactalbumin mRNA. At one site a hairpin ribozyme and a catalytically-inactive hammerhead molecule were also targeted. Three target sites were located in the α-lactalbumin coding region (nucleotides 15, 145, and 361; RZ1,RZ2 and RZ3 respectively) and two were located in the 3'non-coding region (nucleotides 442 and 694; RZ4 and RZ5 respectively). Target sequences at sites 361 and 442 were located in apparent stem-loop regions whilst target sequences at sites 15, 442 and 594 were near the initiation and termination codons and the polyadenylation signal, respectively. 3. Ribozyme and target RNA's were synthesised in vitro in 'run-off' transcription reactions. Cleavage reactions performed with this RNA at 37°C, in the presence of 20 mM MgCl2 and near-neutral pH demonstrated that each catalytically-active ribozyme specifically cleaved the target α-lactalbumin mRNA and produced (5'-) and (3'-) cleavage products of the anticipated size. The reactions produced cleavage fragments that possessed the anticipated sequences specific for the target molecule, and the rate of reaction was dependent on the concentration of ribozyme and substrate. The kinetics of the cleavage reaction indicated that only small differences existed between the ribozymes in respect of their Michaelis constants (values for the Km ranged from 0.17 to 0.62 μM). However, kcat values differed over a 5-fold range. RZ3 and RZ5 cleaved 1.1 and 1.5 molecules per minute respectively whilst RZ4a and RZ1 both cleaved only 0.3 molecules per minute. 4. When ribozyme sequences were delivered to the cytoplasm of. C127I mouse mammary cells using the T7-vaccinia virus transient expression system, the amount of α-lactalbumin mRNA present at 48 hrs post-transfection was reduced. At a target:ribozyme ratio of 1:1000, ribozymes targeting sites 361 and 694 reduced α-lactalbumin mRNA by greater than 80%; another two ribozymes (targeting nucleotides 442 and 145) reduced levels by 80% and 60% respectively; the fifth ribozyme (targeting nucleotide 15, near the AUG) was largely ineffective. 5. 'Down-regulation' of the intracellular levels of α-lactalbumin paralleled the ribozyme-dependent reduction achieved for mRNA. For site 442, the reduction in both mRNA and protein was attributed to the catalytic activity of the ribozyme rather than to the antisense effects of the flanking arms. Delivery of an engineered (catalytically-inactive) variant had only a small effect on mRNA levels and accounted for only 25% of the effect on the level of α-lactalbumin protein present in the cell. 6. The effect on the α-lactalbumin expression of ribozyme transcripts synthesized endogenously in the cell nucleus was investigated. The hammerhead ribozyme and the corresponding catalytically-inactive molecule that targeted site 442 (RZ4a and RZ4c) as well as RZ5 (targeting site 694) were sub-cloned into plasmids downstream of the cytomegalovirus immediate-early promoter (CMV). These constructs were used to transform MA104 monkey kidney cells to establish transformed cell lines that constitutively synthesised ribozyme molecules. In a single experiment, cell lines synthesising RZ4a and the catalytically-inactive variant RZ4c were found to contain the same level of α-lactalbumin protein as control cells that did not synthesise ribozyme transcripts. However, a cell line synthesising the RZ5 transcript (targeting site 694) exhibited levels of α-lactalbumin protein that were only ~10% of the control; synthesis of a control molecule (p53 protein) was unaffected. 7. These results are discussed in the context of the possible utility of ribozyme transcripts to modify expression of α-lactalbumin and the opportunity ribozymes targeting α-lactalbumin mRNA might present for the manipulation of the protein composition of milk. en
dc.language.iso en en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.relation.isreferencedby UoA9950264814002091 en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.title Ribozyme-mediated cleavage of bovine α-lactalbumin mRNA en
dc.type Thesis en
thesis.degree.discipline Cellular and Molecular Biology en
thesis.degree.grantor The University of Auckland en
thesis.degree.level Doctoral en
thesis.degree.name PhD en
dc.rights.holder Copyright: The author en

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