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Quantitative Real-time PCR Analysis (quantitative + real-time_pcr_analysis)
Selected AbstractsModulation of Mycobacterium tuberculosis proliferation by MtrA, an essential two-component response regulatorMOLECULAR MICROBIOLOGY, Issue 3 2006Marek Fol Summary Paired two-component regulatory systems consisting of a sensor kinase and a response regulator are the major means by which bacteria sense and respond to different stimuli. The role of essential response regulator, MtrA, in Mycobacterium tuberculosis proliferation is unknown. We showed that elevating the intracellular levels of MtrA prevented M. tuberculosis from multiplying in macrophages, mice lungs and spleens, but did not affect its growth in broth. Intracellular trafficking analysis revealed that a vast majority of MtrA overproducing merodiploids were associated with lysosomal associated membrane protein (LAMP-1) positive vacuoles, indicating that intracellular growth attenuation is, in part, due to an impaired ability to block phagosome,lysosome fusion. A merodiploid strain producing elevated levels of phosphorylation-defective MtrA (MtrAD53N) was partially replicative in macrophages, but was attenuated in mice. Quantitative real-time PCR analyses revealed that expression of dnaA, an essential replication gene, was sharply upregulated during intramacrophage growth in the MtrA overproducer in a phosphorylation-dependent manner. Chromatin immunoprecipitation using anti-MtrA antibodies provided direct evidence that MtrA regulator binds to dnaA promoter in vivo indicating that dnaA promoter is a MtrA target. Simultaneous overexpression of mtrA regulator and its cognate mtrB kinase neither inhibited growth nor sharply increased the expression levels of dnaA in macrophages. We propose that proliferation of M. tuberculosis in vivo depends, in part, on the optimal ratio of phosphorylated to non-phosphorylated MtrA response regulator. [source] The Legionella pneumophila Dps homolog is regulated by iron and involved in multiple stress toleranceJOURNAL OF BASIC MICROBIOLOGY, Issue S1 2009Ming-jia Yu Abstract Iron homeostasis is essential to almost all organisms. In this study, we identified the putative homolog of the iron-storage protein-encoding gene, dpsL, in the intracellular pathogen Legionella pneumophila and demonstrated its expression under iron-limited conditions and its responses to multiple stresses. Quantitative real-time PCR analysis indicated that the expression of dpsL was enhanced under iron limitation regardless of the growth phase. Compared with the wild-type cells, the cells devoid of dpsL were heat and H2O2 -sensitive. In contrast to the dps mutants of other bacteria, the growth of the dpsL mutant in an iron-deprived medium was delayed but finally reached the same cell density as wild-type cells during the stationary phase of growth. The finding that the dpsL mutant is salt resistant suggested the involvement of DpsL in virulence. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Molecular characterization of two nicotinic acetylcholine receptor subunits from Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2009Pei-An Tang Abstract Two nicotinic acetylcholine receptor (nAChR) subunit genes, Lb,1 and Lb,8, were isolated and characterized from psocid, Liposcelis bostrychophila Badonnel, using the rapid amplification of cDNA ends (RACE) technique. They are the first two nAChR family members isolated from the insect order of Psocoptera. The full-length cDNAs of Lb,1 (GenBank accession number: EU871527) and Lb,8 (EU871526) consist of 2,025 and 1,763 nucleotides, respectively, and an open reading frame of 1,644 and 1,608,bp encoding 547 and 535 amino acid proteins, respectively. Both genes have typical features of nAChR family members, though they share only 56% identity in amino acid sequence. The dendrogram generated by the MEGA 3.1 program shows that the protein deduced by Lb,1 had the closest phylogenetic relationship to Agam,1 from Anopheles gambiae and Amel,1 from Apis mellifera, and Lb,8 shares the highest identity with Agam,8 from An. gambiae and Amel,8 from A. mellifera. Quantitative real-time PCR analysis showed that Lb,1 was expressed 2.03,6.54-fold higher than Lb,8 at the different developmental stages of L. bostrychophila. The highest expression levels of Lb,1 and Lb,8 were both detected at adult stage and the lowest were at the third and fourth nymphal stages, respectively. There was a stable and relatively low expression level for Lb,1, whereas there was a descending expression pattern for Lb,8 in the 1st through the 4th nymphal stadia. © 2009 Wiley Periodicals Inc. [source] Gossypium arboreum GHSP26 enhances drought tolerance in Gossypium hirsutumBIOTECHNOLOGY PROGRESS, Issue 1 2010Asma Maqbool Abstract Heat-shock proteins (HSP) are molecular chaperones for protein molecules. These proteins play an important role in protein,protein interactions such as, folding and assisting in the establishment of proper protein conformation and prevention of unwanted protein aggregation. A small HSP gene GHSP26 present in Gossypium arboreum responds to dehydration. In the present study, an attempt was made to overcome the problem of drought stress in cotton. A cDNA of GHSP26 was isolated from G. arboreum, cloned in plant expression vector, pCAMBIA-1301 driven by the cauliflower mosaic virus 35S promoter and introduced into Gossypium hirsutum. The integration and expression studies of putative transgenic plants were performed through GUS assay; PCR from genomic DNA, and quantitative real-time PCR analysis. Transgenic cotton plants showed an enhanced drought tolerance, suggesting that GHSP26 may play a role in plant responsiveness to drought. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] | ||||||||||