Cloning and analysis of the nucleotide sequence of the cryptic plasmid of Chlamydia trachomatis serovar L1

Hatt, Christopher (1990) Cloning and analysis of the nucleotide sequence of the cryptic plasmid of Chlamydia trachomatis serovar L1. University of Southampton, Doctoral Thesis.

Abstract

Chlamydia tachomatis serovar L1/440/LN possesses a 7498bp plasmid which was designated pLGV440. Plasmid cut at its unique BamHI site was cloned into bacteriophage lambda and then into the BamHI site of plasmid pAT153 in Eschericia coli. The recombinant plasmid was designated pCTL1. A detailed restriction endonuclease map of pCTL1 was constructed. The plasmid was also cloned at its unique BamHI, PstI and PvuII sites into the vector pUC18. A fragment of the chlamydial plasmid was shown to function as a promoter in E. coli when placed upstream of the lacZ gene in this vector. In vitro coupled transription/translation experiments demonstrated the production of polypeptides in E. coli cell-free extracts. The entire plasmid was sequenced by the chain termination method. Open reading frames were identified from the resulting consensus sequence together with a candidate for the plasmid origin of replication consisting of four perfect tandem repeats of a 22bp sequence, an A:T rich sequence and an open reading frame which could generate a 34.8kdal product. The predicted polypeptide products of the open reading frames were compared by computer with all reported protein sequences. Homology of the predicted polypeptide product of open reading frame P-3 to the E. coli dnaB protein and the analogous product of gene 12 of bacteriophage P22 is described. Homology to several enzymes which bind adenosine tri-phosphate was shown. The sequence of the plasmid was compared to the published sequences of plasmids from two isolates from other serovars of C. trachomatis.

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