Enhancement of in vitro transcription by addition of cloned, overexpressed major sigma factor of Chlamydia psittaci 6BC

Douglas, Annemarie L.; Saxena, Neerja; Hatch, Thomas P.

doi:10.1128/jb.176.13.4196.1994

J Bacteriol

1994

DOI: 10.1128/jb.176.13.4196.1994

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Enhancement of in vitro transcription by addition of cloned, overexpressed major sigma factor of Chlamydia psittaci 6BC

Annemarie L. Douglas

Neerja Saxena

Thomas P. Hatch

Abstract: Volume 176, no. 10, p. 3036: Figure 1 should appear as shown below. Sequence from amino acids 601 to 644 was omitted in the original figure.

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Cited by 13 publications

(28 citation statements)

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“…, a homolog of the Escherichia coli major sigma factor, 70 (3). The major 5Ј ends that we identified for transcripts generated in vivo were identical to those identified by Stephens et al (8) (Fig.…”

supporting

confidence: 69%

“…3). This pattern of multiple bands is characteristic of the imprecise termination at the spf terminator and is not the result of initiation of transcription at multiple sites (3). The length of the transcripts generated from pALR210C was within the range predicted (145 nucleotides) for transcripts initiated from the P2 promoter.…”

mentioning

confidence: 81%

“…The P1 and P2 promoter regions were cloned into the transcription assay vector pUC19-spfЈ, and radiolabelled RNA was generated by using chlamydial RNA polymerase with and without recombinant 66 , [␣-32 P]UTP, and unlabelled nucleoside triphosphates as described previously (3). Radiolabelled transcripts initiated from the cloned promoters and terminated at the E. coli spf gene terminator were detected directly on DNA sequencing gels.…”

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confidence: 99%

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Functional analysis of the major outer membrane protein gene promoters of Chlamydia trachomatis

Douglas

Hatch

1995

J Bacteriol

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We determined that the putative upstream promoter of the long transcript (P1) of ompA, the gene encoding the major outer membrane protein of Chlamydia trachomatis L2, is recognized in vitro by the major sigma factor of chlamydiae, 66 . We found no evidence that the putative downstream promoter of the short ompA transcript (P1) is functional in vitro. RNase protection of guanylyltransferase-capped transcripts made in vivo confirmed that P2 is a primary transcript while P1 is a processed product of a longer transcript, possibly P2.The major outer membrane protein (MOMP) of Chlamydia trachomatis constitutes about 50% of the total protein content of the outer membrane in both developmental forms of this obligately intracellular parasite (2, 6). Although the MOMP is present during all stages of the chlamydial life cycle, transcription of the gene encoding the MOMP, ompA, appears to be developmentally regulated. A short transcript (designated P1), with a 5Ј end 25 bp upstream from the AUG translation initiation codon, predominates early in the cycle, and an additional long transcript (designated P2), with a 5Ј end 247 bp upstream from the AUG codon, can be detected during the middle and late stages (8). By 15 to 18 h postinfection (p.i.), the steadystate ratio of short to long transcripts, as measured by RNA Northern blot analysis, is about 1 to 1 (8). Stephens et al. (8) suggested that the two transcripts are initiated from different promoters, P1 for the P1 transcript and P2 for the P2 transcript.Analysis of ompA transcripts generated in vitro. A map of the ompA upstream region, with the locations of the 5Ј ends of transcripts P2 and P1, is shown at the top of Fig. 1. The putative Ϫ10 and Ϫ35 promoter sequences of the P2 and P1 transcripts (8) are shown at the bottom of the figure. We used the oligonucleotides LMPE1 and LMPE2 ( Fig. 1) to map by primer extension (PE) the 5Ј ends of ompA transcripts generated in vivo (by using RNA isolated from infected HeLa cells harvested at 20 h p.i.) and in vitro (by using RNA polymerase prepared from reticulate bodies harvested and purified at 20 h p.i.). The in vitro transcription and PE assays were carried out as described previously (3, 7). The reaction mixtures included pFEN50 (1), a DNA template that encodes the entire MOMP gene and over 1 kb of upstream sequence, and recombinant Chlamydia psittaci 66 , a homolog of the Escherichia coli major sigma factor, 70 (3). The major 5Ј ends that we identified for transcripts generated in vivo were identical to those identified by Stephens et al. (8) (Fig. 2). A strong PE signal was observed when in vitro-generated RNA served as the template and LMPE2 was used as the primer (Fig. 2B); this signal was identical to the 5Ј end of the long in vivo transcript, suggesting that the P2 promoter region functions in our in vitro system. The same 5Ј end was also identified when LMPE1 was used as the primer (Fig. 2A). In addition, several bands which roughly correspond to the 5Ј end of the short P1 transcript were noted when RNA generated i...

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supporting

confidence: 69%

mentioning

confidence: 81%

mentioning

confidence: 99%

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Functional analysis of the major outer membrane protein gene promoters of Chlamydia trachomatis

Douglas

Hatch

1995

J Bacteriol

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“…Using a high-salt extract from C. psittaci and C. trachomatis reticulate bodies (RBs), they were able to show A -dependent transcription of several chlamydial genes, including PCT, hctB, ltuA, and ltuB (6,14,23). Single mutations in the Ϫ10 and Ϫ35 regions of the putative PCT promoter region had no effect on promoter activity (24).…”

mentioning

confidence: 99%

Identification of sequences necessary for transcription in vitro from the Chlamydia trachomatis rRNA P1 promoter

Tan

Engel

1996

J Bacteriol

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Chlamydia trachomatis RNA polymerase was partially purified by heparin-agarose chromatography and used in conjunction with a plasmid-borne G-less cassette template to characterize the C. trachomatis rRNA P1 promoter in vitro. Stepwise mutational analysis revealed that sequences in the ؊10, ؊25, and ؊35 regions are necessary for promoter activity, but no sequence upstream of position ؊40 is required. Partially purified C. trachomatis RNA polymerase and purified Escherichia coli holoenzyme exhibited some differences in promoter specificity.Chlamydia trachomatis is a gram-negative obligate intracellular pathogen with a complex life cycle characterized by two serial morphologic forms (reviewed in references 26 and 34). Stage-specific expression has been demonstrated in early (38) and late (1-3, 8, 14, 19, 28) periods of the developmental cycle, but the molecular basis of this gene regulation is not understood. Direct genetic studies of promoter structures have not been feasible; transient transformation of chlamydiae has been described (35), but a practical DNA-mediated transformation system does not exist.In the absence of a genetic system, several approaches have been taken to identify and characterize chlamydial promoters. The transcription initiation sites of several abundant transcripts have been identified, and their upstream regions have been examined for putative promoter sequences. By inspection, appropriately spaced canonical Escherichia coli 70 -type promoter sequences are present in a few of these upstream regions. These include the C. trachomatis plasmid countertranscript (PCT) (15), envA P1 (13, 22), and the dnaK operon of the C. trachomatis mouse pneumonitis biovar (11). Others, including the hctB, ltuA, and ltuB genes, have 70 promoterlike sequences at the putative Ϫ10 region only. Interestingly, these three genes are all transcribed late in the developmental cycle (14). However, many chlamydial genes are not preceded by 70 -like promoter sequences (i.e., there is no more than a 50% match), and no consensus chlamydial promoter sequence is apparent (8). Explanations for this sequence diversity include the possibilities that (i) some of the presumptive chlamydial transcription initiation sites are instead processing sites, (ii) the putative promoters belong to more than one class of promoters, and (iii) there is latitude in the promoter specificity of C. trachomatis A RNA polymerase (RNAP). The subunits of C. trachomatis RNAP have been cloned and sequenced (9,10,18,21).A , a C. trachomatis 70 homolog (9, 21), is the only sigma factor identified in chlamydiae to date.A and 70 share striking amino acid sequence conservation in subregions 2.4 and 4.2 (9, 21) of 70 , including the specific amino acid residues which have been shown in E. coli to be involved in promoter recognition at the Ϫ10 and Ϫ35 positions (reviewed in reference 5). This conservation of amino acid residues involved in promoter recognition contrasts with the observed variability in the DNA sequences of the putative C. trachomatis promote...

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“…The nucleotide sequence was 875 nucleotides long including the 5 '-region of the sigA and the putative 3' -region of the ORF D616 homolog sequenced in C. trachomatis D strain (2,5,16,23). We aligned 10 chlamydial sequences, 7 of which were determined in this study and 3 cited from DDBJ.…”

mentioning

confidence: 99%

Conservation of Putative Promoter Sequences Located Upstream of Chlamydial Major Sigma Factor Gene, sigA among Chlamydia Spp.

Ochiai

Fukushi

Cai

et al. 1999

Microbiology and Immunology

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Enhancement of in vitro transcription by addition of cloned, overexpressed major sigma factor of Chlamydia psittaci 6BC

Abstract: Volume 176, no. 10, p. 3036: Figure 1 should appear as shown below. Sequence from amino acids 601 to 644 was omitted in the original figure.

Cited by 13 publications

References 0 publications

Functional analysis of the major outer membrane protein gene promoters of Chlamydia trachomatis

Functional analysis of the major outer membrane protein gene promoters of Chlamydia trachomatis

Identification of sequences necessary for transcription in vitro from the Chlamydia trachomatis rRNA P1 promoter

Conservation of Putative Promoter Sequences Located Upstream of Chlamydial Major Sigma Factor Gene, sigA among Chlamydia Spp.

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