Molecular and genetic analysis of purine genes in Escherichia coli K-12
Molecular and genetic analysis of purine genes in Escherichia coli K-12
The expression and regulation of two open reading frames (ORF23 and ORF15) upstream and adjacent to the purB gene in E.coli has been investigated. The expression of ORF23 and ORF15 was selectively directed from a T7 bacteriophage promoter by T7 RNA polymerase. Expression from E. coli promoters was inhibited by rifampicin, while that from the T7 promoter remained unaffected. Expression of ORF23 was demonstrated and the analysis of cellular fractions showed P23 to be localized to the membrane. ORF15 was found not be be expressed, possibly beacuse of an incomplete identification of this open reading frame.
ORF23 and purB form a two gene operon. A promoter 64 nucleotides upstream of ORF23, within the coding sequence of ORF15, was identified by primer extension analysis. Co-transcription of ORD23 and purB was demonstrated by fusing the purB gene in-frame to lacZ carried on a plasmid. β-galactosidase expression was measured in these constructs. Disruption of ORF23, by insertional mutageneis in vitro, totally abolished β-galactosidase activity while disruption of ORF15, upstream of the promoter identified by primer extension, reduced β-galactosidase activity to half that of the undisrupted construct. Thus two promoters located within ORF15 regulate expression of ORF23 and purB.
The chromosomal copy of ORF 23 was disrupted by inserting the chloramphenicol acetyl transferase (Cm^R) gene to investigate a possible role for P23 in purine biosynthesis. Insertion of the Cm^R gene had no polar effect on purB. These ORF23 insertion mutants were not purine auxotrophs, but P23 may represent one of a number of purine transporter proteins. Chromosomal mutations of ORF15 resulted in loss of viability, and hence P15 serves an essential function in the cell. ORF15 may correspond to part of the asuE gene coding for a tRNA modifying enzyme.
Repressor-operator interactions at the guaBA regulatory region were investigated. Footprinting analysis in vivo was used to locate bases protected by a repressor protein, but no footprint at the operator-like sequence was observed.
University of Southampton
1994
Malik, Tahir Habib
(1994)
Molecular and genetic analysis of purine genes in Escherichia coli K-12.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The expression and regulation of two open reading frames (ORF23 and ORF15) upstream and adjacent to the purB gene in E.coli has been investigated. The expression of ORF23 and ORF15 was selectively directed from a T7 bacteriophage promoter by T7 RNA polymerase. Expression from E. coli promoters was inhibited by rifampicin, while that from the T7 promoter remained unaffected. Expression of ORF23 was demonstrated and the analysis of cellular fractions showed P23 to be localized to the membrane. ORF15 was found not be be expressed, possibly beacuse of an incomplete identification of this open reading frame.
ORF23 and purB form a two gene operon. A promoter 64 nucleotides upstream of ORF23, within the coding sequence of ORF15, was identified by primer extension analysis. Co-transcription of ORD23 and purB was demonstrated by fusing the purB gene in-frame to lacZ carried on a plasmid. β-galactosidase expression was measured in these constructs. Disruption of ORF23, by insertional mutageneis in vitro, totally abolished β-galactosidase activity while disruption of ORF15, upstream of the promoter identified by primer extension, reduced β-galactosidase activity to half that of the undisrupted construct. Thus two promoters located within ORF15 regulate expression of ORF23 and purB.
The chromosomal copy of ORF 23 was disrupted by inserting the chloramphenicol acetyl transferase (Cm^R) gene to investigate a possible role for P23 in purine biosynthesis. Insertion of the Cm^R gene had no polar effect on purB. These ORF23 insertion mutants were not purine auxotrophs, but P23 may represent one of a number of purine transporter proteins. Chromosomal mutations of ORF15 resulted in loss of viability, and hence P15 serves an essential function in the cell. ORF15 may correspond to part of the asuE gene coding for a tRNA modifying enzyme.
Repressor-operator interactions at the guaBA regulatory region were investigated. Footprinting analysis in vivo was used to locate bases protected by a repressor protein, but no footprint at the operator-like sequence was observed.
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Published date: 1994
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Local EPrints ID: 462626
URI: http://eprints.soton.ac.uk/id/eprint/462626
PURE UUID: 6315f7b7-0999-485c-902b-a440f30409b9
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Date deposited: 04 Jul 2022 19:33
Last modified: 04 Jul 2022 19:33
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Author:
Tahir Habib Malik
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