Search

Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA polymerase with RT activity, K4pol L329A from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis. By optimizing assay conditions for three enzymes using Taguchi's method, 100 to 1000-fold higher sensitivity was achieved for cDNA synthesis than conventional assay condition using only RT. Our results suggest that DNA polymerase with RT activity and DNA/RNA helicase are useful to increase the sensitivity of cDNA synthesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: Dialister pneumosintes is a nonmotile, nonfermentative, non-spore-forming, obligately anaerobic, gram-negative bacillus that has been associated with some infections in the human body. A species-specific nested polymerase chain reaction assay was used to investigate the occurrence of D pneumosintes in primary root canal infections., Study Design: Samples were collected from 32 teeth with carious lesions, necrotic pulps, and radiographic evidence of periradicular bone destruction. Twenty-two teeth were asymptomatic, and 10 cases were diagnosed as acute apical periodontitis. DNA extracted from the samples was initially amplified with universal 16S ribosomal DNA primers. A second round of amplification used the first polymerase chain reaction products to specifically detect D pneumosintes., Results: This bacterial species was detected in 17 of 22 asymptomatic cases (77.3%) and in 4 of 10 root canals associated with acute apical periodontitis (40%). No relationship was found between the presence of this bacterial species and the occurrence of symptoms. In general, D pneumosintes was detected in 21 of 32 root canal samples (65.6%)., Conclusion: This study is the first to report a high prevalence of D pneumosintes in root canal infections of humans. Because of this high prevalence and the apparent pathogenicity of the microorganism, we suggest the inclusion of D pneumosintes in the selected group of putative endodontic pathogens.

Arginine-mediated regulation is remarkably well conserved in very divergent bacteria, and shows a number of unusual features that distinguish arginine regulation from other transcriptional control mechanisms. The arginine repressor subunit consists of a basic N-terminal DNA-binding domain, which belongs to the winged helix-turn-helix family, connected through a flexible linker to an acidic C-terminal domain responsible for binding of arginine and assembly of the high-affinity holohexamer, which binds an approximately 40 bp target. To gain further insight into the molecular details of arginine repressor-operator interactions we have established a high resolution contact map of the argC operator from Bacillus stearothermophilus, a moderate thermophilic Gram-positive bacterium, and the argR operator from Thermotoga neapolitana, a Gram-negative hyperthermophile, with the corresponding ArgR proteins. Enzymatic and chemical footprinting have been combined with missing contact, pre-modification, base substitution, and small ligand binding interference techniques to gather information on backbone and base-specific contacts with major and minor groove determinants of the operators. Wild-type and mutant argC operators have been compared for their interaction with the repressor, using both in vivo and in vitro approaches. Our results indicate that the operators of B. stearothermophilus and T. neapolitana consist of two ARG box-like sequences, 18 bp imperfect palindromes, separated by two and three base-pairs, respectively, and that the repressors from thermophilic origin establish base-specific contacts with two major groove segments and the intervening minor groove of each ARG box, all aligned on one face of the helix. In contrast, no specific contacts are established in the minor groove facing the repressor in the centre of the operator, nevertheless this region plays a crucial structural role in complex formation, as indicated by mutant studies. This picture is reminiscent of arginine repressor binding in Escherichia coli, and therefore reinforces the uniform view of arginine regulation, but also reveals a number of striking differences at particular positions of the boxes and in the length and base-pair composition of the spacer connecting two ARG boxes in the operator. These might be responsible, in part, for subtle but important functional and mechanistic differences in the way species-specific repressors interact with their cognate target sites. These variations are underlined by the different behaviour of the repressors from E. coli, B. stearothermophilus and T. neapolitana in their potential to bind heterologous operators, their requirement for arginine, and the resistance of complex formation to non-specific competitor DNA. Our findings are discussed in view of the crystal structure of the arginine repressor from B. stearothermophilus., (Copyright 2002 Academic Press.)