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[en] RNA silencing is used as a common method for investigating loss-of-function effects of genes of interest. In mammalian cells, RNA interference (RNAi) or RNA silencing can be achieved by transient siRNA (small or short interfering RNA) transfection or by stable shRNA (short hairpin RNA) systems. Various vectors are used for efficient delivery of shRNA. Lentiviral vectors offer an efficient delivery system for stable and long-term expression of the shRNA in mammalian cells. The widely used lentiviral pLKO.1 plasmid vector is very popular in RNAi studies. A large RNAi database, a TRC (the RNAi Consortium) library, was established based on the pLKO.1-TRC plasmid vector. This plasmid (also called pLKO.1-puro) has a puromycin-resistant gene for selection in mammalian cells along with designs for generating lentiviral particles as well for RNA silencing. While using the pLKO.1-puro TRC control shRNA plasmid for transfection in murine P19 embryonic stem (ES) cells, it was unexpectedly discovered that this plasmid vector induced robust endodermal differentiation. Since P19 ES cells are pluripotent and respond to external stimuli that have the potential to alter the phenotype and thus its stemness, other cell types used in RNA silencing studies do not display the obvious effect and therefore, may affect experiments in subtle ways that would go undetected. This study for the first time provides evidence that raises concern and warrants extreme caution while using the pLKO.1-puro control shRNA vector because of its unexpected non-specific effects on cellular integrity. - Highlights: • In P19 ES cells the pLKO.1-puro lentiviral control shRNA vector induced endodermal differentiation. • P19 ES cells harboring the pCDNA3 plasmid vector retained their stem-ness as opposed to those harboring the pLKO.1-puro vector. • P19 ES cells can serve as a sensor to determine vector safety. • Extreme caution is warranted while using the widely used pLKO.1-puro lentiviral vector for experimental and therapeutic designs.
[en] The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG–HIF–1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. - Highlights: • HIF-1α protein is constitutively degraded in hypoxic conditions. • Requirement of ubiquitination for HIF-1α degradation in hypoxia. • Hypoxic HIF-1α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization.
[en] Transformation of human cells requires both SV40 large T and small t antigens. Plasmids that contained mutations in the amino-terminal dnaJ domain of the early region fail to transform human diploid fibroblasts. However, large T dnaJ mutants can be rescued by plasmids that express early region products other than large T antigen. The protein found to be responsible for such complementation was the third early region product, 17KT. Similar to large T, this protein reduces levels of the retinoblastoma-related protein, p130, and stimulates cell-cycle progression of quiescent fibroblasts, two activities of large T that are disrupted by dnaJ mutations
[en] Restriction fragments of pUC19 DNA were irradiated by various doses of UV light and analyzed by denaturing (alkaline) agarose gel electrophoresis. The irradiation generated retarded species whose mobility indicated two crosslinked DNA strands. Quantitative analysis of the experimental data provided an empirical equation relating the fraction of crosslinked DNA molecules to their length and to the dose of their irradiation by UV light. This equation can be used to predict the crosslinking behavior of pUC19-like DNA molecules whose primary structures do not much differ from a random nucleotide sequence. The amount of interstrand crosslinks increased with the (A + T) content of the pUC19 DNA fragments but the dependence was not clear-cut to indicate that oligonucleotide composition of DNA played a significant role as well. (author)
[en] Nanodiamonds synthesized by detonation have been found not to immobilize the ring form of pUC19 plasmid DNA. Linear pUC19 molecules with blunt ends, prepared by restriction of the initial ring form of pUC19 DNA, and linear 0.25-10 kb DNA fragments are adsorbed on nanodiamonds. The amount of adsorbed linear DNA molecules depends on the size of the molecules and the size of the nanodiamond clusters
[en] We describe a general and simple modification to the standard M9 minimal medium recipe that leads to an approximate twofold increase in the yield of heterologously expressed proteins in Escherichia coli BL21(DE3) bacteria. We monitored the growth of bacteria transformed with plasmids for three different test proteins in five minimal media with different concentrations of buffering salts and/or initial media pH. After purification of the over-expressed proteins, we found a clear correlation between the protein yield and change in media pH over time, where the minimal media that were the most buffered and therefore most resistant to change in pH produced the most protein. And in all three test protein cases, the difference in yield was nearly twofold between the best and worst buffering media. Thus, we propose that increasing the buffering capacity of M9 minimal media will generally lead to a similar increase for most of the proteins currently produced by this standard protein expression protocol. Moreover, we have qualitatively found that this effect also extends to deuterated M9 minimal media growths, which could lead to significant cost savings in these preparations.
[en] We describe a novel virus, AFV1, of the hyperthermophilic archaeal genus Acidianus. Filamentous virions are covered with a lipid envelope and contain at least five different proteins with molecular masses in the range of 23-130 kDa and a 20.8-kb-long linear double-stranded DNA. The virus has been assigned to the family Lipothrixviridae on the basis of morphotypic characteristics. Host range is confined to several strains of Acidianus and the virus persists in its hosts in a stable carrier state. The latent period of virus infection is about 4 h. Viral DNA was sequenced and sequence similarities were found to the lipothrixvirus SIFV, the rudiviruses SIRV1 and SIRV2, as well as to conjugative plasmids and chromosomes of the genus Sulfolobus. Exceptionally for the linear genomes of archaeal viruses, many short direct repeats, with the sequence TTGTT or close variants thereof, are closely clustered over 300 bp at each end of the genome. They are reminiscent of the telomeric ends of linear eukaryal chromosomes
[en] STAT3 is constitutively activated in several cancers, including prostate cancer, and is therefore, a potential target for cancer therapy. DU-145 prostate cancer cells were stably co-transfected with STAT3 reporter and puromycin resistant plasmids to create a stable STAT3 reporter cell line that can be used for high throughput screening of STAT3 modulators. The applicability of this cell line was tested with two known activators and inhibitors of STAT3. As expected, EGF and IL-6 increased STAT3 reporter activity and enhanced the nuclear localization of phosphorylated STAT3 (pSTAT3); whereas Cucurbitacin I and AG490 decreased STAT3 reporter activity dose and time-dependently and reduced the localization of pSTAT3 in the nuclei of prostate cancer cells. Given the importance of STAT3 in cancer initiation and progression, the development of a stable STAT3 reporter cell line in prostate cancer cells provides a rapid, sensitive, and cost effective method for the screening of potential STAT3 modulators.
[en] Five strains belong to Pseudomonas aeruginosa were chosen for this study. Quantitative resistance to antibiotics showed wide range of variation among the tested strains. The preliminary ultraviolet radiation responses data, pointed to the major envolvement of chromosomal genes in the overall radiation response. The cytoplasmic factors, however, were partially envolved and may demonstrate an active or passive participation. (32 tabs., 22 figs., 125 refs.)