Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336746
Title: Analysis of variant cytosolic serine hydroxymethyltransferases
Author: Chave, Karen Judy
ISNI:       0000 0001 3530 6905
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 1997
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
Serine hydroxymethyltransferase (SHMT) is a major enzyme for serine, glycine and one carbon group metabolism and aberrant SHMT activity has been implicated in some diseases such as cancer and schizophrenia. cDNAs encoding the cytosolic isozyme of human and rabbit SHMT (cSHMT) were expressed in E. coli using the pET vector system which produces SHMT proteins fused to an amino terminal motif of six histidine residues. This allows rapid purification of cSHMT protein by metal ion chromatography. Enzymatically active SHMT was purified. Mutant SHMT proteins were produced by site directed mutagenesis of the cDNA coding for human cSHMT The amino acids mutated had been implicated in SHMT activity by chemical modification. Mutant proteins were expressed in E. coli, purified as above and were assayed for enzyme activity. Whereas chemical modification had in all cases completely inactivated enzyme activity, none of the mutants created completely inactivated the protein suggesting that chemical modification of proteins only has a limited role in determining residues important in SHMT activity. Two bacteriophage lambda clones containing human genomic DNA hybridising to cSHMT cDNA were characterised. Sequence data was obtained showing although the sequence had 90% homology to cSHMT cDNA there were many point mutations, deletions and insertions into the genomic DNA including a mutated translation initiation codon. No introns were present in the genomic sequence and a 16 base pair direct repeat was found either side of the cSHMT homologous sequence. It was concluded that the clones contained DNA encoding a processed pseudogene of cSHMT. PCR amplification of human genomic DNA was performed and sequences from the cSHMT gene were identified. Five introns were identified when compared to the known cDNA sequence and a further intron may be present.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.336746  DOI: Not available
Keywords: Serine; Glycine; Metabolism
Share: