Transcriptional Analysis of Hydrogenase Genes in the Cyanobacteria Anacystis nidulans and Anabaena variabilis Monitored by RT-PCR

2000 ◽  
Vol 40 (5) ◽  
pp. 315-321 ◽  
Author(s):  
Gudrun Boison ◽  
Hermann Bothe ◽  
Oliver Schmitz
Keyword(s):  
Anacystis Nidulans ◽  
Anabaena Variabilis ◽  
Transcriptional Analysis ◽  
Rt Pcr

Identification of Molecular Risk Score in Advanced Hodgkin Lymphoma: Integrating Tumor and Microenvironment Signatures.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3660-3660
Author(s):  
Beatriz Sánchez-Espiridión ◽  
Carlos Montalbán ◽  
Mónica García-Cosio ◽  
Jose García-Laraña ◽  
Javier Menarguez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Treatment Response ◽  
Hodgkin Lymphoma ◽  
Risk Score ◽  
Molecular Techniques ◽  
Gene Set Enrichment Analysis ◽  
Transcriptional Analysis ◽  
Rank Statistic ◽  
Rt Pcr ◽  
Expression Levels

Abstract Abstract 3660 Poster Board III-596 Introduction Despite the major advances in the treatment of classical Hodgkin Lymphoma (cHL) patients, around 30% to 40% of cases in advanced stages may relapse or die as result of the disease. Current predictive systems, based on clinical and analytical parameters, fail to identify accurately this significant fraction of patients with short failure-free survival (FFS). Transcriptional analysis has identified genes and pathways associated with clinical failure, but the biological relevance and clinical applicability of these data await further development. Robust molecular techniques for the identification of biological processes associated with treatment response are necessary for developing new predictive tools. Patients and Methods We used a multistep approach to design a quantitative RT-PCR-based assay to be applied to routine formalin-fixed, paraffin-embedded samples (FFPEs), integrating genes known to be expressed either by the tumor cells and their reactive microenvironment, and related with clinical response to adriamycin-based chemotherapy. First, analysis of 29 patient samples allowed the identification of gene expression signatures related to treatment response and outcome and the design of an initial RT-PCR assay tested in 52 patient samples. This initial model included 60 genes from pathways related to cHL outcome that had been previously identified using Gene Set Enrichment Analysis (GSEA). Second, we selected the best candidate genes from the initial assay based on amplification efficiency, biological significance and treatment response correlation to set up a novel assay of 30 genes that was applied to a large series of 282 samples that were randomly split and assigned to either estimation (194) or validation series (88). The results of this assay were used to design an algorithm, based on the expression levels of the best predictive genes grouped in pathways, and a molecular risk score was calculated for each tumor sample. Results Adequate RT-PCR profiles were obtained in 264 of 282 (93,6%) cases. Normalized expression levels (DCt) of individual genes vary considerably among samples. The strongest predictor genes were selected and included in a multivariate 10-gene model integrating four gene expression pathway signatures, termed CellCycle, Apoptosis, NF-KB and Monocyte, which are able to predict treatment response with an overall accuracy of 68.5% and 73.4% in the estimation and validation sets, respectively. Patients were stratified by their molecular risk score and predicted probabilities identified two distinct risk groups associated with clinical outcome in the estimation (5-year FFS probabilities 75.6% vs. 45.9%, log rank statistic p≈0.000) and validation sets (5-year FFS probabilities 71.4% vs. 43.5%, log rank statistic p<0.004). Moreover, this biological model is independent of and complementary to the conventional International Prognostic Score using multivariate Cox proportional hazards analysis. Conclusions We have developed a molecular risk algorithm that includes genes expressed by tumoral cells and their reactive microenvironment. This makes it possible to classify advanced cHL patients with different risk of treatment failure using a method that could be applied to routinely prepared tumor blocks. These results could pave the way for more individualized and risk-adapted treatment strategies of cHL patients, enabling subsets of patients to be identified who might benefit from alternative approaches Disclosures: No relevant conflicts of interest to declare.

scholarly journals Assessing the role of the RND efflux pump in metronidazole resistance of Helicobacter pylori by RT-PCR assay

2010 ◽  
Vol 5 (02) ◽  
pp. 88-93 ◽  
Author(s):  
Jalil Fallah Mehrabadi ◽  
Mehrandokht Sirous ◽  
Naser Ebrahimi Daryani ◽  
Saeed Eshraghi ◽  
Bahram Akbari ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Efflux Pumps ◽  
Clinical Isolates ◽  
Transcriptional Analysis ◽  
Rt Pcr ◽  
Helicobactor Pylori ◽  
Homologous Genes ◽  
Metronidazole Resistance ◽  
Rnd Efflux Pump ◽  
H Pylori

Introduction: Metronidazole is a significant antibiotic used for eradication of Helicobactor pylori infections and it is of notice that metronidazole-resistant clinical isolates have been found in high rates worldwide. While the RND family of efflux pumps plays a central role in drug resistance among Gram-negative bacteria, this is questionable for H. pylori. Methodology: To understand whether TolC homologues of RND pumps contribute to metronidazole resistance in H. pylori isolates, expression of four TolC homologous genes of five resistant clinical isolates exposed to varying concentrations of metronidazole were evaluated by RT-PCR and transcriptional analysis. Results: The results indicate that excess amounts of metronidazole are able to increase the expression level of these genes at the transcriptional stage. Conclusions: Therefore, it may be hypothesized that use of metronidazole in H. pyori infection can induce metronidazole resistance. Furthermore, the RND family of efflux pumps may contribute to metronidazole resistance in clinical isolates of H. pylori.

scholarly journals Genomic Sequence and Transcriptional Analysis of a 23-Kilobase Mycobacterial Linear Plasmid: Evidence for Horizontal Transfer and Identification of Plasmid Maintenance Systems

2001 ◽  
Vol 183 (7) ◽  
pp. 2157-2164 ◽  
Author(s):  
Corinne Le Dantec ◽  
Nathalie Winter ◽  
Brigitte Gicquel ◽  
Véronique Vincent ◽  
Mathieu Picardeau
Keyword(s):  
Horizontal Transfer ◽  
Genomic Sequence ◽  
Opportunistic Pathogen ◽  
Open Reading Frames ◽  
Transcriptional Analysis ◽  
Linear Plasmid ◽  
Rt Pcr ◽  
Maintenance Systems ◽  
Function Sequence ◽  
Reading Frames

ABSTRACT Linear plasmids were unknown in mycobacteria until recently. Here, we report the complete nucleotide sequence of 23-kb linear plasmid pCLP from Mycobacterium celatum, an opportunistic pathogen. The sequence of pCLP revealed at least 19 putative open reading frames (ORFs). Expression of pCLP genes in exponential-phase cultures was determined by reverse transcriptase PCR (RT-PCR). Twelve ORFs were expressed, whereas no transcription of the 7 other ORFs of pCLP was detected. Five of the 12 transcribed ORFs detected by RT-PCR are of unknown function. Sequence analysis revealed similar loci in bothM. celatum pCLP and the Mycobacterium tuberculosis chromosome, including transposase-related sequences. This result suggests horizontal transfer between these two organisms. pCLP also contains ORFs that are similar to genes of bacterial circular plasmids involved in partition (par operon) and postsegregational (pem operon) mechanisms. Functional analysis of these ORFs suggests that they probably carry out similar maintenance roles in pCLP.

scholarly journals Transcriptional Regulation of the Equol Biosynthesis Gene Cluster in Adlercreutzia equolifaciens DSM19450T

Nutrients ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 993 ◽  
Author(s):  
Flórez ◽  
Vázquez ◽  
Rodríguez ◽  
Redruello ◽  
Mayo
Keyword(s):  
Gene Cluster ◽  
Expression Patterns ◽  
Transcriptional Analysis ◽  
Whole Genome Sequence ◽  
Intestinal Bacterium ◽  
Rt Pcr ◽  
Biotechnological Production ◽  
Biosynthesis Gene Cluster ◽  
Intergenic Regions ◽  
Metabolic Transformation

Given the emerging evidence of equol’s benefit to human health, understanding its synthesis and regulation in equol-producing bacteria is of paramount importance. Adlercreutzia equolifaciens DSM19450T is a human intestinal bacterium —for which the whole genome sequence is publicly available— that produces equol from the daidzein isoflavone. In the present work, daidzein (between 50 to 200 μM) was completely metabolized by cultures of A. equolifaciens DSM19450T after 10 h of incubation. However, only about one third of the added isoflavone was transformed into dihydrodaidzein and then into equol. Transcriptional analysis of the ORFs and intergenic regions of the bacterium’s equol gene cluster was therefore undertaken using RT-PCR and RT-qPCR techniques with the aim of identifying the genetic elements of equol biosynthesis and its regulation mechanisms. Compared to controls cultured without daidzein, the expression of all 13 contiguous genes in the equol cluster was enhanced in the presence of the isoflavone. Depending on the gene and the amount of daidzein in the medium, overexpression varied from 0.5- to about 4-log10 units. Four expression patterns of transcription were identified involving genes within the cluster. The genes dzr, ddr and tdr, which code for daidzein reductase, dihydrodaidzein reductase and tetrahydrodaidzein reductase respectively, and which have been shown involved in equol biosynthesis, were among the most strongly expressed genes in the cluster. These expression patterns correlated with the location of four putative ρ-independent terminator sequences in the cluster. All the intergenic regions were amplified by RT-PCR, indicating the operon to be transcribed as a single RNA molecule. These findings provide new knowledge on the metabolic transformation of daidzein into equol by A. equolifaciens DSM19450T, which might help in efforts to increase the endogenous formation of this compound and/or its biotechnological production.

scholarly journals RT-PCR as a tool for systematic transcriptional analysis of large regions of the Bacillus subtilis genome

Microbiology ◽  
2000 ◽  
Vol 146 (4) ◽  
pp. 823-828 ◽  
Author(s):  
Alicia Hernández ◽  
Angélica Figueroa ◽  
Luis A. Rivas ◽  
Vı́ctor Parro ◽  
Rafael P. Mellado
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Analysis ◽  
Rt Pcr ◽  
Subtilis Genome

scholarly journals The hanR/hanI quorum-sensing system of Halomonas anticariensis, a moderately halophilic bacterium

Microbiology ◽  
2011 ◽  
Vol 157 (12) ◽  
pp. 3378-3387 ◽  
Author(s):  
Ali Tahrioui ◽  
Emilia Quesada ◽  
Inmaculada Llamas
Keyword(s):  
Quorum Sensing ◽  
Halophilic Bacterium ◽  
Transcriptional Analysis ◽  
Signal Molecules ◽  
Rt Pcr ◽  
Significant Group ◽  
Signalling Molecules ◽  
Sensing System ◽  
Moderately Halophilic Bacterium ◽  
Quorum Sensing System

Quorum sensing is a cell density-dependent gene expression mechanism found in many Gram-negative bacteria which involves the production of signal molecules such as N-acylhomoserine lactones (AHLs). One significant group of micro-organisms in which quorum sensing has not been previously studied, however, are the moderate halophiles. We describe here the results of our studies of the quorum-sensing system in Halomonas anticariensis FP35T, which is composed of luxR/luxI homologues: hanR (the putative transcriptional regulator gene) and hanI (the autoinducer synthase gene). To understand how the hanR/hanI system is organized and regulated we conducted RT-PCR and quantitative real-time PCR assays. Transcriptional analysis indicated that the hanR and hanI genes are on the same transcript and that their transcription is growth phase-dependent. HanI seems to be the only autoinducer synthase responsible for the synthesis of AHLs by the bacterium, since the inactivation of hanI resulted in the complete loss of its AHLs. We also found that the hanI gene appears to be transcribed from its own promoter and that its expression does not depend upon HanR. This finding was supported by the fact that the FP35hanR mutant showed AHL-producing activity and hanI expression similar to that of the wild-type strain, the latter being measured by RT-PCR. Moreover, hanR is expressed from its own promoter and appears to be independent of the AHL signalling molecules produced by HanI.

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