Studies on the aroA gene of Escherichia coli

Duncan, Kenneth (1984) Studies on the aroA gene of Escherichia coli. PhD thesis, University of Glasgow.

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1. The E.coli aroB gene, encoding 3-dehydroquinate synthase has been sub-cloned from pLC29-47, a member of the Clarke-Carbon E.coli gene bank. Cells harbouring an aroB recombinant plasmid pKD101, overexpress the enzyme 20-25 fold. Further sub-cloning of a 3.5 kb EcoRI-Bg1ll fragment, and comparison with an independently isolated aroB clone, pJB14, has located the coding sequence of the gene. 2. The E.coli structural gene encoding 3-dehydroquinase (aroD) has been subcloned on a 1.8 kb Clal fragment from the plasmid pJKK12. The resulting recombinant, pKD201, overexpresses the enzyme approximately 100-fold. The entire DNA sequence of the Clal fragment has been determined and the coding sequence of the aroD gene identified. The aroD gene encodes a 240 amino acid polypeptide of calculated Mr 28117. 3. The aroA gene of E. coli, encoding 5-enolpyruvylshikimate phosphate synthase was sub-cloned from the tranducing bacteriophage ApserC. Further sub-cloning located the gene on a 1.9 kb Clal-PvuII fragment. Cells harbouring a number of aroA recombinant plasmids overexpress EPSP synthase approximately 100-fold, including pKD501 which is now used as a source of the purified enzyme; these cells are also tolerant to high levels of the herbicide glyphosate, the target of which is EPSP synthase. It was found, however, that cloning from a C1al site in the phage reduced the level of expression to that found in wild type cells. Polyacrylamide gel electrophoresis in the presence of SDS of crude extracts of plasmid carrying cells showed that in those clones which overexpress EPSP synthase at a high level, a heavily stained 40 000 Mr band is present. 4. The DNA sequence of the aroA gene has been determined. The gene encodes a 427 amino acid polypeptide with a calculated of 46112. The location of the coding sequence of aroA in the DNA sequence revealed that it is positioned 700 bp away from the C1al site and examination of the sequence revealed that there was no obvious promoter for the gene. Further cloning experiments showed that the aroA promoter was located at least 1 200 bp upstream of the initiation codon. DNA sequencing and analysis of the sequences upstream of aroA revealed the presence of another gene. This gene encodes a polypeptide of 362 amino acids and calculated 39834, corresponding to the 40 000 band seen on SDS-PAGE of the crude extracts of cells carrying EPSP synthase overexpressing plasmids. The gene was identified as serC, which encodes the enzyme phosphoserine aminotransferase, the second enzyme on the three-step serine biosynthetic pathway. 6. Northern blot analysis and S1 nuclease mapping have been used to show that the two genes are linked to form the serC-aroA operon. The operon is expressed from a single promoter, located 55 bp upstream of the initiation codon for serC. Two messages are produced: the first is monocistronic, encoding only the serC gene; the second is polycistronic, and encodes both genes.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics
Date of Award: 1984
Depositing User: Enlighten Team
Unique ID: glathesis:1984-76529
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 14:12
Last Modified: 19 Nov 2019 14:12

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