Construction of shuttle vectors for cloning in Vibrio cholerae and Escherichia coli

Starting from a naturally occurring cryptic plasmid pVC540 of Vibrio cholerae non-OI. strain 1095, a number of plasmid vectors have been constructed for cloning genes inVibrio cholerae by introducing antibiotic resistance markers containing a set of unique cloning sites. The constructs pVC810 and pVE920 have the origins of both Vibrio cholerae and Escherichia coli replicons and are stable in both organisms in the absence of selective pressure. These plasmids can serve as shuttle vectors between Escherichia coli and Vibrio cholerae. The plasmid vectors reported here along with the demonstration of transformation in Vibrio cholerae by plasmid DNA will facilitate genetic analysis of this important human pathogen

Inhibition of nucleoporin member Nup214 expression by miR-133b perturbs mitotic timing and leads to cell death

Background: Nucleoporins mediate nucleocytoplasmic exchange of macromolecules and several have been assigned active mitotic functions. Nucleoporins can participate in various mitotic functions like spindle assembly, kinetochore organisation and chromosome segregation- important for genome integrity. Pathways to genome integrity are frequently deregulated in cancer and many are regulated in part by microRNAs. Indeed, altered levels of numerous microRNAs have frequently been associated with tumorigenesis. Here, we unveil a microRNA-mediated regulation of the nucleoporin Nup214 and its downstream effect on genome integrity. Methods: Databases/bioinformatic tools such as miRBase, Oncomine and RNAhybrid predicted Nup214 as a miR-133b target. To validate this, we used luciferase reporter assays, Real-Time PCR and immuno-blotting. Flow cytometry and immuno-blots of mitotic markers were used to analyse cell cycle pattern upon thymidine synchronization and miR-133b treatment. Mitotic indices and chromosomal abnormalities were assessed by immuno-fluorescence for FITC-tagged phospho-H3 as well as video-microscopy for GFP-tagged histone H4. Annexin V/propidium iodide staining, caspase3/ PARP cleavage and colony formation assays were done to investigate cell death upon either miR-133b transfection or NUP214 knockdown by siRNA. UPCI:SCC084, HCT116, HeLa-H4-pEGFP and HEK293 (human oral squamous cell carcinoma, colorectal, cervical carcinomas and embryonic kidney cell lines, respectively) were used. miR-133b and NUP214 expressions were validated in cancer cell lines and tissues by Real-Time PCR. Results: Examination of head and neck tumour tissues and cancer cell lines revealed that Nup214 and miR-133b expressions are negatively correlated. In vitro, Nup214 was significantly downregulated by ectopic miR-133b. This downregulation elevated mitotic indices and delayed degradation of mitotic marker proteins cyclinB1 and cyclinA and dephosphorylation of H3. Moreover, this mitotic delay enhanced chromosomal abnormalities and apoptosis. Conclusions: We have identified NUP214, a member of the massive nuclear pore complex, as a novel miR-133b target. Thus, we have shown a hitherto unknown microRNA regulation of mitosis mediated by a member of the nucleoporin family. Based on observations, we also raise some hypotheses regarding transport-dependent/independent functions of Nup214 in this study. Our results hence attempt to explain why miR-133b is generally downregulated in tumours and lay out the potential for Nup214 as a therapeutic target in the treatment of cancer

Lack of umuDC Gene Functions in Vibrio Cholerae Cells

Attempts to identify an umuDC analog, using interspecific complementation of Escherichia coli mutants with plasmids containing a gene bank of Vibrio cholerae, were not successful. The DNA from none of the vibrio species examined including marine vibrios hybridized to E. coli umuC and umuD gene sequences. These cells arenot mutable by ultraviolet (UV) light and cannot Weigle-reactivate UV-irradiated choleraphages, suggesting that vibrios are deficient in the umuDC operon. This possibility is supported by the fact that when the plasmid pKM101 carrying the mucAB genes is introduced into V. cholerae cells, they acquire the UV-mutable phenotype and UV-irradiated choleraphages can be Weigle-reactivate

Transformation of Vibrio cholerae by plasmid DNA

The lack of an eficient transformation system in Vibrio c~oler~e was a handicap in the genetic manipulation of this important human pathogen. Since V. cholerae cells secrete DNases, this may interfere with the uptake of DNA. The present report describes the approaches taken for transforming V’. cholerae cells with plasmid DNA, by overcoming this DNase barrier. The partial success of transforming DNase-negative mutants confirmed the role of DNase in the nontransformability of the wild-type cells. Successful transformation was carried out following removal of DNases from the periplasmic space. This was achieved by treating the cells with Mg2 + and Ca* + ions to allow the DNase to be released, and then holding them under conditions where the remaining DNase activity was minimized before adding DNA to the competent cells. Transformation effxciencies of the order of 10 5 per recipient cell were observed

Recombinant Derivative of a Naturally Occurring Non-Toxinogenic Vibrio cholerae Ol Expressing the B Subunit of Cholera Toxin: a Potential Oral Vaccine Strain

A clinical isolate of Vibrio cholerae O1 was identified which did not possess the heat-labile ( CT), the heat-stable (ST) or the zonula occludens ( Zot) toxin genes. Rabbit ileal loop assays showed that no other CT-like toxin was produced by this strain. The partly deleted cholera toxin gene which carries the intact gene for the B subunit was cloned and the recombinant plasmid, pURD110, was introduced into this non-toxinogenic natural human isolate. The transformed cells (strain URD2) secreted the B subunit gene product which competed with the holotoxin secreted by the hypertoxinogenic strain 569B of V. cholerae for the GM1 ganglioside binding sites in vivo. This strain can colonize the rabbit intestine as detected by the removable intestinal tie adult rabbit diarrhoea ( RITARD ) model. This construct has an advantage over other live oral attenuated V. cholerae strains used as vaccines in that the latter strains were made non-toxinogenic by only deleting part of the gene coding for the A subunit of cholera toxin while the strain described here is naturally non-toxinogenic

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Not AvailablePurpose Potassium (K)-fractions, thresholds of K release and fixation, quantity-intensity (Q/I) parameters of K, K-release kinetics, and K-fixation capacity were compared for their effectiveness in differentiating the effect of various nutrient management practices on K supplying capacity of an Aeric Endoaquept soil after 45 years of puddled rice cultivation. Materials and methods Soil samples (0–15 cm) were collected after the completion of 45 rice-rice cycles from an on-going longterm fertilizer experiment located in ICAR-National Rice Research Institute, Cuttack, India. The treatments involved control (unfertilized), N (nitrogen fertilizer), NP (N+ phosphorus fertilizer), NK (N+ potassium fertilizer), NPK (N + P + K fertilizer), FYM (farmyard manure), N + FYM, NP + FYM, NK + FYM, and NPK + FYM. Results and discussion Rice cultivation without K fertilizer application resulted in lower values of soil K parameters than the Kfertilized treatments. Treatment effects were most prominent on release threshold concentration (RTC), followed by cumulativeK release, K-release rate constants, and K-fixation capacity. Parameters of K-release kinetics and Q/I relationships showed better correlation with rice grain yields than soil-K fractions. Soil K thresholds were closely related with exchangeable (Kex) and nonexchangeable K (Knx), but not clay minerals. Conclusions Among the soil K parameters, RTC, cumulative K release (Kf) with 0.01MCaCl2, release rate constants (bR and bS) of parabolic diffusion equation, and K-fixation capacity were most effective in revealing the nutrient management induced variations in soil K fertility. In the studied soil, K-thresholds were significantly related to Kex and Knx.Not Availabl