Cloning and characterisation of S-adenosyl-L-homocysteine hydrolase.

摘要

This thesis concerns S-adenosyl-L-homocysteine hydrolase (SHH), the enzyme responsible for the reversible hydrolysis of S-adenosyl-L-homocysteine (SAH) to adenosine and homocysteine. SAH is formed as a direct product of transmethylation reactions involving S-adenosyl-L-methionine (SAM) and is known to be a potent inhibitor of most SAM mediated methyltransfer reactions. An Asparagus officinalis cDNA showing strong homology to previously cloned SHH cDNAs was identified by the random sequencing of cDNAs from a library enriched for wound induced clones. PCR primers were designed which allowed the amplification of a region of the SHH gene from any plant species tested to date. The alignment of sequences from both cloned PCR products, A. officinalis cDNAs, and previously isolated SHH clones, highlights the high level of sequence homology retained between divergent species as well as the presence of an extra polypeptide motif in the amino acid sequence of SHH genes from photosynthetic species. Northern analysis using RNA isolated from asparagus suggested SHH is transcriptionally upregulated from its constitutive low level by a wound stimulus. An Arabidopsis thaliana genomic SHH clone was isolated and used in promoter/reporter gene fusion studies to facilitate a detailed study of the temporal and spatial expression pattern of the SHH gene. Data obtained from this study corroborated evidence obtained from northern, western and enzyme activity analysis, suggesting SHH was expressed in all plant organs. Perturbation of SHH gene expression was also attempted within transgenic plants to try and further elucidate biochemical pathways requiring SHH enzyme activity.