Biomedical subjects
Publications and source records attributed to Steve Bowra.
Several species of rhizobia were successfully transformed with broad-host-range plasmids of different replicons by using a modified freeze-thaw method. A generic binary vector (pPZP211) was maintained in Mesorhizobium loti without selection and stably inherited during nodulation. The method could extend the potential of rhizobia as a vehicle for plant transformation.
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The quantity and quality of first-strand cDNA directly influence the accuracy of transcriptional analysis and quantification. Using a plant-derived alpha-tubulin as a model system, the effect of oligo sequence and DTT on the quality and quantity of first-strand cDNA synthesis was assessed via a combination of semi-quantitative PCR and real-time PCR. The results indicated that anchored oligo dT significantly improved the quantity and quality of alpha-tubulin cDNA compared to the conventional oligo dT. Similarly, omitting DTT from the first-strand cDNA synthesis also enhanced the levels of transcript. This is the first time that a comparative analysis has been undertaken for a plant system and it shows conclusively that small changes to current protocols can have very significant impact on transcript analysis.
This paper presents the cloning and biochemical characterisation of the cysteine protease Tr-cp 14 from white clover (Trifolium repens). The predicted amino acid sequence of Tr-cp 14 is 71%, 74% and 74% identical to the cysteine proteases XCP1 and XCP2 from Arabidopsis thaliana, and p48h-17 from Zinnia elegans, respectively. These cysteine proteases have previously been shown to be involved in programmed cell death during tracheary element differentiation. The precursor polypeptide of Tr-cp 14 was expressed in Escherichia coli, purified from inclusion bodies and refolded. The precursor polypeptide could be processed to its active mature form autocatalytically at pH 5.0 and had a requirement for 20 mM l-cysteine for optimal activity. Mature Tr-cp 14 showed a preference for synthetic aminomethylcoumarin substrates with either Leu or Phe in the P2 position when tested with Arg in P1. A substrate with Arg in both the P1 and P2 position was not accepted as substrate.