The major outer membrane protein gene of chlamydia as a phylogenetic chronometer

Carter, Michael William (1993) The major outer membrane protein gene of chlamydia as a phylogenetic chronometer. University of Southampton, Doctoral Thesis.

Abstract

The major outer membrane protein (MOMP) of the genus Chlamydia accounts for 60% of the outer envelope and via disulphide cross linkage with other cysteine-rich proteins appears to be responsible for the structural integrity of the outer envelope in the absence of peptidoglycan. In addition this protein also appears to function as a porin. Successful molecular characterization of 13 MOMP genes and their proteins prior to this study revealed; five constant regions interspersed by four variable segments (VS1 to VS4); a signal peptide and conserved cysteine residues.

A molecular approach was adopted to isolate and characterize the C.pneumoniae MOMP gene for use as a phylogenetic marker. Previous studies had revealed the potential lethal nature of the complete gene in Escherichia coli. Therefore, an 8.5Kb Bg/II MOMP gene fragment was cloned into λ EMBL3 and subcloned as two recombinant λ EMBL3 EcoRI fragments into the λ ZAP^◯II cloning vector. The nucleotide sequence was determined from the resulting plasmids and used to generate a novel phylogenetic tree for the Chlamydia which supported the designation of C.pneumoniae as a new species and proved the MOMP gene acceptable as a phylogenetic chronometer.

A successful mouse model of salpingitis used the human C.trachomatis serovar F strain NI-1. The nucleotide sequence of the NI-1 MOMP gene was determined as described for the C.pneumoniae IOL-207 and was found to be identical to that of another C.trachomatis serovar F strain.

The prerequisites of isolation and cloning the MOMP gene were overcome using solid phase DNA sequencing of DNA amplified by PCR in the presence of biotinylated primers.(DX178141)

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