Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerase chain reaction

J N Engel,D Ganem,J Pollack,F Malik

doi:10.1128/jb.172.10.5732-5741.1990

J N Engel, D Ganem + Show 2 more

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https://doi.org/10.1128/jb.172.10.5732-5741.1990

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Abstract

Taking advantage of sequence conservation of portions of the alpha, beta, and beta' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome. The polymerase chain reaction products were used as a probe to recover the genomic fragments encoding the beta subunit and the 5' portion of the beta' subunit from a library of cloned murine Chlamydia trachomatis DNA. Similar attempts to recover the alpha subunit were unsuccessful. Sequence analysis demonstrated that the beta subunit of RNA polymerase was located between genes encoding the L7/L12 ribosomal protein and the beta' subunit of RNA polymerase; this organization is reminiscent of the rpoBC operon of Escherichia coli. The C. trachomatis beta subunit overproduced in E. coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit. Although this polyclonal antibody specifically immunoprecipitated the beta subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme. Immunoblots with this antibody demonstrated that the beta subunit appeared early in infection.

Highlights

Taking advantage of sequence conservation of portions of the a, I1, and O' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome
Significant homologies to the E. coli RNA polymerase a chain were found in the RNA polymerases of vaccinia virus [7], B. subtilis [5], liverwort [18], and common tobacco plant chloroplast [28]; significant homologies to the E. coli RNA polymerase f chain were found in the chloroplast RNA polymerase of the liverwort [18] and the common tobacco plant [17]; and significant homologies to the E. coli RNA polymerase 3' chain were found in the chloroplast RNA polymerase of liverwort [18], RNA polymerase II of Drosophila melanogaster [4] and Saccharomyces cerevisiae [1], and the vaccinia virus RNA polymerase [7] (Fig. 1)
Reasoning that regions of the protein conserved between enterobacteria, plant chloroplast, and eucaryotic RNA polymerase would probably be conserved in the RNA polymerase subunit homologs of other gram-negative organisms, we synthesized degenerate oligonucleotides from these regions to use as primers (Fig. 1 and Materials and Methods) in a polymerase chain reaction (PCR) reaction to amplify the corresponding region from chlamydiae

Summary

Introduction

Taking advantage of sequence conservation of portions of the a, I1, and O' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome. The C. trachomatis i subunit overproduced in E. coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit This polyclonal antibody immunoprecipitated the subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme. Immunoblots with this antibody demonstrated that the ji subunit appeared early in infection. Chlamydia trachomatis is an obligate intracellular parasite of eucaryotic cells (for reviews, see references 3, 26, and 27). This medically important gram-negative bacterium causes an array of ocular and genital disorders which rank among the most prevalent diseases of humans.

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