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Title: Studies of the glyoxylate bypass of Streptomyces coelicolor A3(2) : purification of isocitrate lyase and cloning of the ICL gene
Author: Chapman, Paul G.
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1994
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Abstract:
This thesis is concerned with the glyoxylate bypass of Streptomyces coelicolor A3(2) and in particular the enzyme isocitrate lyase (ICL) and its corresponding gene (icl). At the start of this project oligonucleotides were designed against regions of other ICL proteins which were found to be highly conserved across a variety of species. These oligonucleotides were used for Southern analysis of genomic DNA from S.coelicolor and also as primers for use in PCR amplification of genomic DNA from S.coelicolor. Sequencing of DNA which was cloned using these techniques revealed that the S.coelicolor icl gene had not been cloned. In order to facilitate in cloning of the S.coelicolor icl gene, it was decided to purify the S.coelicolor ICL protein. Acetate was found to be an extremely poor carbon source, resulting in poor growth and low ICL activity. Growth on Tween (polyoxyethylensorbitan) containing compounds was found to result in high activities of the ICL enzyme and purification of the enzyme was facilitated using a five step protocol employing hydrophobic chromatography and anion exchange chromatography. Amino acid sequence from the purified protein and also from chymotrypsin digested peptides was obtained. This amino acid sequence was then used to design oligonucleotides, which were used as probes to facilitate in cloning the S.coelicolor icl gene. An amplified fragment of DNA from S.coelicolor was obtained using the oligonucleotides and sequencing of this DNA revealed that it encoded part of the icl gene. The amplified DNA was then used in Southern analysis in order to clone the entire S.coelicolor icl gene. Sequencing of the gene was carried out and the sequence of the regions surrounding the gene were also determined. The icl gene sequence shows that it is similar to the previously sequenced icl's and in particular to the E.coli icl., notably lacking the similar internal region as the E.coli ICL protein. Sequencing downstream from the icl has revealed the ms gene from S.coelicolor This is the opposite to E.coli, where the gene order is ms/icl. It remains to be determined if these genes form part of an operon in S.coelicolor. Some work on the S.coelicolor idh gene was carried out and 30-fold overexpression of the idh gene in S.coelicolor was achieved. Preliminary experiments to overexpress idh in Escherichia coli and an attempt to disrupt the S.coelicolor idh were also carried out, but had still to be completed by the end of this project.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796938  DOI: Not available
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