Development of a quantitative gene expression assay for <i>Chlamydia trachomatis</i> identified temporal expression of σ factors

Mathews, Sarah A.; Volp, Kym M.; Timms, Peter

doi:10.1016/s0014-5793(99)01182-5

Cited by 78 publications

(73 citation statements)

References 27 publications

(40 reference statements)

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“…The range of applications for the Lightcycler are similarly wide, and include detection of minimal residual disease (Eckert et al 2000), cancer cells in the peritoneal cavity of patients with gastrointestinal and gynaecological malignancies (Nakanishi et al 1999) and pathogens (Mathews et al 1999), and monitoring delivery, expression and maintenance of transgenes (Thorpe & Porteous 1999).…”

Section: Applications Quantificationmentioning

confidence: 99%

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

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The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

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Section: Applications Quantificationmentioning

confidence: 99%

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

3,397

2,448

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“…The unique developmental cycle of Chlamydia involves the interconversion between the infectious elementary body and the metabolically active reticulate body (23). Although the key morphological stages of chlamydial development are understood (19,23) and the developmental expression of over 20 genes has been determined (12,18), the elements which regulate this developmental gene expression are yet to be elucidated. Our recent investigations (18) identified temporal expression of the three C. trachomatis RNA polymerase sigma factors, 66 (major 70 homolog), 28 , and 54 .…”

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confidence: 99%

“…Although the key morphological stages of chlamydial development are understood (19,23) and the developmental expression of over 20 genes has been determined (12,18), the elements which regulate this developmental gene expression are yet to be elucidated. Our recent investigations (18) identified temporal expression of the three C. trachomatis RNA polymerase sigma factors, 66 (major 70 homolog), 28 , and 54 . Several reports have characterized 66 promoters (8,17,29), but to date, no promoters have been identified for the developmental-stage-specific sigma factors, 28 and 54 .…”

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confidence: 99%

Identification and Mapping of Sigma-54 Promoters in Chlamydia trachomatis

Mathews

2000

J Bacteriol

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The first 54 promoters in Chlamydia trachomatis L2 were mapped upstream of hypothetical proteins CT652.1 and CT683. Comparative genomics indicated that these 54 promoters and potential upstream activation binding sites are conserved in orthologous C. trachomatis D, C. trachomatis mouse pneumonitis strain, and Chlamydia pneumoniae (CWL029 and AR39) genes.Chlamydia is an organism of major medical and veterinary significance; however, its obligate intracellular existence makes genetic investigations a challenge. The unique developmental cycle of Chlamydia involves the interconversion between the infectious elementary body and the metabolically active reticulate body (23). Although the key morphological stages of chlamydial development are understood (19, 23) and the developmental expression of over 20 genes has been determined (12, 18), the elements which regulate this developmental gene expression are yet to be elucidated. Our recent investigations (18) 28 and 54 . The present study utilized the complete Chlamydia trachomatis genome sequence (27) and a modified fluorescence-based primer extension (PE) assay to identify and map the first 54 promoters for Chlamydia.Transcription initiation from 54 promoters is a multistep process involving the recognition of the promoter by 54 , binding of the core RNA polymerase to the 54 to form a closed complex, and subsequent activation to an open complex following binding by an enhancer binding protein (EBP) (20,21). In most cases the EBP binds an upstream activator sequence (UAS) located within 200 bp of the promoter (15, 21) and is brought into contact with the 54 -RNA polymerase complex by DNA looping, an event mediated by the integration host factor (IHF) or intrinsic DNA bends (9). In addition to the 54 gene (rpoN), recently identified in the C. trachomatis genome, genes for the NtrC family EBP (ntrC) and IHF (ihfA) were also found to be present (27). More detailed analysis of the translated amino acid sequences identified that RpoN ( 54 ) contains a perfect RpoN box (ARRTVAKYR), which is responsible for recognition of the cognate promoter (30), and the chlamydial NtrC homolog has an exact match to the 54 -binding domain, GAFTGA (7). Furthermore, the chlamydial NtrC has six of seven conserved amino acids of the UAS binding domain, GESGCGK (7) (the underlined amino acid is nonconserved). We previously reported the late-stage-specific expression of rpoN (18) and subsequently confirmed that ntrC was transcribed by reverse transcription-PCR analysis (data not shown). These observations led us to hypothesize that some chlamydial genes would be regulated by NtrC-activated 54 -mediated transcription initiation. Of the cognate promoters for all eubacterial sigma factors, 54 promoters are the most highly conserved (2) and hence lend themselves to a computational search of the full chlamydial genome. We therefore used the Findpatterns database searching program of the Australian National Genome Information Service to search the C. trachomatis D genome for sequences corresponding to ...

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“…TPR-containing proteins are often associated with multiprotein complexes and may be involved in the functioning of chaperones, cell-cycle, transcription and proteintransport complexes (2). Matthews et al (10) propose that σ N may be involved in the conversion from reticulate bodies (RB) to elementary bodies (EB) in chlamydias and thus it may be that these genes have some role in that developmental process.…”

Section: N -Dependent Promoters In Bacterial Genomesmentioning

confidence: 99%