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Analysis of Variant Cytosolic Serine Hydroxymethyltransferases.

Chave, Karen Judy. (1997) Analysis of Variant Cytosolic Serine Hydroxymethyltransferases. Doctoral thesis, University of Surrey (United Kingdom)..

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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.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Chave, Karen Judy.
Date : 1997
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1997.
Depositing User : EPrints Services
Date Deposited : 30 Apr 2019 08:08
Last Modified : 20 Aug 2019 15:33
URI: http://epubs.surrey.ac.uk/id/eprint/851504

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