Construction and genetic characterization of lambda specialized transducing particles carrying therglB gene ofEscherichia coli K-12

TherglB gene ofEscherichia coli codes for a restriction activity that cleaves the hydroxymethylated DNA of T2 and T4 phages. Earlier mapping data placed the gene at 98.39 min counterclockwise to thehsd operon. Genetic analysis of the in vivo gene fusions with fusion-transducing phages established the location of therglB gene next to thehsdS gene of thehsdRMS cluster. The methodology used in this study could be extended to similar in vivo physical mapping of closely linked genes.     

Co-regulation of senescence-associated genes by oncogenic homeobox proteins and polycomb repressive complexes

Cellular senescence is a stable cell cycle arrest that can be induced by stresses such as telomere shortening, oncogene activation or DNA damage. Senescence is a potent anticancer barrier that needs to be circumvented during tumorigenesis. The cell cycle regulator p16^INK4a is a key effector upregulated during senescence. Polycomb repressive complexes (PRCs) play a crucial role in silencing the INK4/ARF locus, which encodes for p16^INK4a, but the mechanisms by which PRCs are recruited to this locus as well as to other targets remain poorly understood. Recently we discovered the ability of the homeobox proteins HLX1 (H2.0-like homeobox 1) and HOXA9 (Homeobox A9) to bypass senescence. We showed that HLX1 and HOXA9 recruit PRCs to repress INK4a, which constitutes a key mechanism explaining their effects on senescence. Here we provide evidence for the regulation of additional senescence-associated PRC target genes by HLX1 and HOXA9. As both HLX1 and HOXA9 are oncogenes implicated in leukemogenesis, we discuss the implications that the collaboration between Homeobox proteins and PRCs has for senescence and cancer.     

Intergeneric Conjugation in Streptomyces peucetius and Streptomyces sp. Strain C5: Chromosomal Integration and Expression of Recombinant Plasmids Carrying the chiC Gene

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the C31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of C31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.     

Development of MFC using sulphonated polyether ether ketone (SPEEK) membrane for electricity generation from waste water

Polyether ether ketone was sulphonated polyether ether ketone (SPEEK) and utilized as a proton exchange membrane (PEM) in a single chamber MFC (SCMFC). The SPEEK was compared with Nafion? 117 in the SCMFC using Escherichia coli. The MFC with the SPEEK membrane produced 55.2% higher power density than Nafion? 117. The oxygen mass transfer coefficient (K(O)) for SPEEK and Nafion? 117 was estimated to be 2.4 × 10(-6)cm/s and 1.6 × 10(-5)cm/s, respectively resulting in reduced substrate loss and increased columbic efficiency (CE) in the case of SPEEK. When the dairy and domestic waste water was treated in SPEEK-SCMFC, fitted with a membrane electrode assembly (MEA), a higher maximum power density was obtained for dairy waste water (5.7 W/m(3)). The results of this study indicate that SPEEK membrane has the potential to greatly enhance the efficiency of MFCs.     

Ameliorating Effect of Fish Oil on Acrylamide Induced Oxidative Stress and Neuronal Apoptosis in Cerebral Cortex

Acrylamide (ACR) is a known industrial toxic chemical that produce neurotoxicity characterized by progressive neuronal degeneration. This study was designed to investigate the protective effect of fish oil on ACR-induced neuronal damage in Wistar rats. ACR enhances the production of reactive oxygen species and potentially affects brain. ACR administered rats showed increased levels of lipid peroxidative product, protein carbonyl content, hydroxyl radical and hydroperoxide which were significantly modulated by the supplementation of fish oil. The activities of enzymic antioxidants and levels of reduced glutathione were markedly lowered in ACR-induced rats; fish oil treatment augmented these antioxidant levels in cortex. Free radicals generated during ACR administration reduced the activities of membrane adenosine triphosphatases and acetylcholine esterase. Fish oil enhanced the activities of these enzymes near normal level. Histological observation represented the protective role of fish oil in ACR-induced neuronal damage. Fish oil reduced the ACR-induced apoptosis through the modulation in expressions of B-cell lymphoma 2 (Bcl2)-associated X protein and Bcl2-associated death promoter. Further, fish oil increases the expression of heat shock protein 27 (Hsp27) in ACR-induced rats. This study provides evidence for the neuroprotective effect of fish oil on ACR-induced neurotoxicity by reducing oxidative stress and apoptosis with modulation in the expression of Hsp27.     

Helicobacter Pylori's Plasticity Zones Are Novel Transposable Elements

Background

Genes present in only certain strains of a bacterial species can strongly affect cellular phenotypes and evolutionary potentials. One segment that seemed particularly rich in strain-specific genes was found by comparing the first two sequenced Helicobacter pylori genomes (strains 26695 and J99) and was named a “plasticity zone”.

Principal Findings

We studied the nature and evolution of plasticity zones by sequencing them in five more Helicobacter strains, determining their locations in additional strains, and identifying them in recently released genome sequences. They occurred as discrete units, inserted at numerous chromosomal sites, and were usually flanked by direct repeats of 5′AAGAATG, a sequence generally also present in one copy at unoccupied sites in other strains. This showed that plasticity zones are transposable elements, to be called TnPZs. Each full length TnPZ contained a cluster of type IV protein secretion genes (tfs3), a tyrosine recombinase family gene (“xerT”), and a large (≥2800 codon) orf encoding a protein with helicase and DNA methylase domains, plus additional orfs with no homology to genes of known function. Several TnPZ types were found that differed in gene arrangement or DNA sequence. Our analysis also indicated that the first-identified plasticity zones (in strains 26695 and J99) are complex mosaics of TnPZ remnants, formed by multiple TnPZ insertions, and spontaneous and transposable element mediated deletions. Tests using laboratory-generated deletions showed that TnPZs are not essential for viability, but identified one TnPZ that contributed quantitatively to bacterial growth during mouse infection and another that affected synthesis of proinflammatory cytokines in cell culture.

Conclusions

We propose that plasticity zone genes are contained in conjugative transposons (TnPZs) or remnants of them, that TnPZ insertion is mediated by XerT recombinase, and that some TnPZ genes affect bacterial phenotypes and fitness.     

HOCTAR database: a unique resource for microRNA target prediction

An Antarctic strain (NJ-7) of Chlorella vulgaris possesses the same 18S rRNA sequence as that of a temperate strain (UTEX259), but shows significantly higher freezing tolerance than the latter. Suppression subtractive hybridization (SSH) was performed to identify genes of intensified expression in NJ-7 relative to UTEX259. Among the genes identified, Ccor1 and Ccor2, co-organized in the same gene cluster Ccor1-Ccor2-Ccor1-Ccor2, showed much higher expression levels in NJ-7 than in UTEX259 at both 20°C and 4°C. As detected by Northern blot and Western blot analyses, the two genes were cold-inducible in NJ-7 but almost not expressed in UTEX259. Their encoded products are predicted to share 55.7% identity to each other and possess physicochemical characteristics similar to that of late embryogenesis abundant (LEA) proteins in plants. The purified recombinant Ccor1 and Ccor2 showed high heat-stability and could act as cryoprotectants to lactate dehydrogenase in vitro. Based on their expression patterns and protein characteristics, we propose that Ccor1 and Ccor2 are two novel LEA proteins and are related to the greatly enhanced freezing tolerance in the Antarctic strain.     

Induction of the inhibitor of RecBCD enzyme inEscherichia coli is alexA-independent SOS response

Exonuclease V (ExoV), an enzyme involved in the RecBCD pathway of recombination, was inhibited in cells induced for SOS functions. In vitro experiments showed that an ExoV inhibitor (Exi) induced after SOS induction was responsible for the inhibition of ExoV. Unlike other SOS functions, Exi protein was induced even inlexA(Ind^–) mutants. Phage Mud(amp^r,lac) was fused to the promoter of theexi gene in alexA(Ind^–) strain, and in these fusion strains-galactosidase was inducible five- to six-fold after DNA damage. The Exi protein, in addition to the inhibition of ATP-dependent DNase activity of ExoV, appeared to repress the synthesis of polypeptide subunits of ExoV as well. Further, Exi protein appeared to be an inducible repressor of a number of other genes in SOS-induced cells.