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Expression
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
In parallel, Walter Fiers’ team performed the cloning of the apparently full-length DNA copy, lacking only 5′-terminal 14 nucleotides, of the MS2 RNA in a plasmid (Devos et al. 1979b). The history and methodology of this cloning were described in detail in the By-products and direct consequences of the MS2 sequencing section of Chapter 10. In a few words, the complementary DNA copy of the in vitro polyadenylated MS2 RNA was synthesized with avian myeloblastosis virus RNA-dependent DNA polymerase. After the MS2 RNA template was removed from the complementary DNA strand with T1 and pancreatic ribonuclease digestion, the complementary DNA became a good template for the synthesis of the double-stranded MS2 DNA with E. coli DNA polymerase I. The double-stranded MS2 DNA was inserted into the PstI restriction site of the classical pBR322 plasmid by means of the poly(dA)•poly(dT) tailing procedure. An E. coli transformant carrying a plasmid with the nearly full-length MS2 DNA insertion was named pMS2-7 and used further as the traditional source of the MS2 genes for the cloning and expression experiments. Remarkably, the restriction sites within the pMS2-7 plasmid correlated well with the predicted ones from the primary MS2 RNA structure. As mentioned in Chapter 10, the pMS2-7 plasmid contained an extra DNA insertion that was identified as the translocatable element IS1 (Devos et al. 1979a), a constant and imminent threat by the cloning procedures in future.
rDNA: Evolution Over a Billion Years
Published in S. K. Dutta, DNA Systematics, 2019
Fragment Β was originally cloned in λgtWES following an enrichment of rDNA EcoRI fragments using RPC5 chromatography.4 This fragment occurs in all strains of mice, and in recent years has provided the basis for the analysis of the internal transcribed spacer. A 3.7 kb fragment defined by EcoRI and Bam HI covers the ITS 1 and ITS 2 regions, the 5.8S rDNA, the 3′ end of the 18S gene, and the 5′ end of the 28S gene. This fragment has been cloned in pBR322 (pME B3200). The sequences surrounding the 4 rRNA processing sites in pre-RNA have been determined, and although the regions share little homology, sequences downstream from the 5.8S rDNA are conserved among vertebrates (85% for rodents and 73% comparing Mus and Xenopus). Bachellerie et al.200 suggest that such conserved regions may represent specific binding or recognition sites for U3 nucleolar RNA which may be involved in the maturation of precursor rRNA. The entire ITS 1 and ITS 2 regions have been sequenced201 and shown to be 999 and 1089 nucleotides long, respectively. The regions share little sequence homology but show some conservation of sequence when mouse and rat sequences are compared (ITS-1, 75% and ITS-2, 30%).
Transcriptionally Regulatory Sequences of Phylogenetic Significance
Published in S. K. Dutta, DNA Systematics, 2019
The existence of native Z-DNA has been further shown with immunochemical assays. Anti-Z-DNA antibodies were elicited by injecting rabbits with brominated Z polymer. The addition of bromine to the C-8 atom of guanosine blocks the ability of these bases to assume the “anti” conformation. Z-specific antibodies were purified by quantitative precipitation with unbrominated poly(dG-dC) poly (dG-dC), which ensured their being specific for Z-DNA.212,224 Such anti-Z-DNA antibodies react with poly(dG-dC)n sequences inserted into pBR322 as well as with other alternating purine-pyrimidine sequences normally present in this chimeric plasmid. In a more recent study using Z-DNA left-handed helical stretches in the DNA of native rabbit β-globin cDNA sequences were shown to be induced by torsional stress.225 This was demonstrated by inserting this cDNA into pBR322 and manipulating it with nick-closing enzymes. The Z-DNA antibody binds only to those DNA molecules that are stressed below linking number 5 = < −0.1, but not to form I or form II (relaxed circles). The bound antibody is released when the helix is unwound. This observation is further supported by circular dichroism and electron microscopy. The bindings were shown to be specific at 3 bp stretches enriched in alternating purine-pyrimidine and GC base pairs.
In vitro cytogenetic analysis of two different anti-phosphates (sevelamer hydrochloride and calcium carbonate) agents used by patients with hyperphosphatemia
Published in Drug and Chemical Toxicology, 2023
Goulzar Ulaya, Hasan Basri İla
Besides the controls, 5.0 µl of SH or CaCO3, 5.0 µl of pBR322 plasmid DNA (172 ng/µl), and 2.0 µl of 30% H2O2 were added to the tubes. The tubes containing the test substances were exposed to UV rays for 10 min. As the light source, a UV transilluminator (DNR-IS) was used which produces light at room temperature of 302 nm wavelength and density of 8000 μW/cm. After irradiation, 4.0 µl of loading buffer was added to the tubes and put on the agarose gel. Then after the application of the 1.0% agarose gel electrophoresis during 100 min of 100 Volts, the photographs were obtained by using the gel documentation system (Vilber Lourmat gel imaging system). In this test system, pBR322 plasmid DNA was used as the test material, and also H2O2 and UV agents were as the positive control. The results were interpreted by comparing the number of band patterns obtained in the control wells and the wells containing the test substances.
Butea monosperma (Lam.) Taub. Bark fractions protect against free radicals and induce apoptosis in MCF-7 breast cancer cells via cell-cycle arrest and ROS-mediated pathway
Published in Drug and Chemical Toxicology, 2020
Varinder Kaur, Manish Kumar, Ajay Kumar, Satwinderjeet Kaur
2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), gallic acid, rutin, 2′,7′-dichloroflourescein diacetate (DCFH-DA), and Rhodamine 123 were obtained from Sigma (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypsin, propidium iodide (PI), low-melting point agarose (LMPA), and normal melting point agarose (NMPA) were obtained from HiMedia Pvt. Limited (Mumbai, India). The pBR322 plasmid DNA was purchased from Genei Pvt. Ltd. (Bangalore, India). Escherichia coli PQ37 tester strain was obtained from Institut Pasteur (France). MCF-7 (human breast adenocarcinoma) cell line was procured from National Centre for Cell Sciences (NCCS) (Pune, India). Routine culturing of cells was performed in DMEM containing 10% FBS and antibiotic-antimycotic solution (sterile A-filtered), at 37 °C in a humidified incubator with 5% CO2. All the other reagents used in the experiments in the present study were also of analytical (AR) grade.
Analysis of bioactive components in Ghost chili (Capsicum chinense) for antioxidant, genotoxic, and apoptotic effects in mice
Published in Drug and Chemical Toxicology, 2020
Sarpras M, Sushil Satish Chhapekar, Ilyas Ahmad, Suresh K. Abraham, Nirala Ramchiary
Chemicals namely capsaicin (purity ≥95%), dihydrocapsaicin (purity ≥85%), ascorbic acid (purity ≥99%), quercetin (purity ≥95%), gallic acid (purity ≥99%), n-methyl-n-(trimethylsilyl) trifluoroacetamide (MSTFA), hydroxylamine hydrochloride, 1,1-diphenyl-2-picrylhydrazyl (DPPH), ethidium bromide (EtBr), acridine orange (AO), fetal calf serum (FCS), triton X-100, RNase A, propidium iodide (PI), 1-chloro 2,4-dinitrobenzene (CDNB), 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), 2,6-dichlorophenolindophenol (DCPIP), reduced glutathione (GSH), reduced nicotinamide adenine dinucleotide (NADH), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich Company (St. Louis, MO). The other chemicals were obtained from Merck (Kenilworth, NJ) except Folin–Ciocalteau reagent, which was purchased from Himedia (Mumbai, India). pBR322 plasmid DNA was obtained from Thermo Fisher Scientific (Waltham, MA).