Search results for "Polymerase"
showing 10 items of 2127 documents
Estimation of the density of the protocatechuate-degrading bacterial community in soil by real-time PCR
2008
Summary The β-ketoadipate pathway is the major route for degradation of aromatic compounds by various soil microorganisms. Protocatechuate 3,4-dioxygenase, a key enzyme of this pathway and which is encoded by pcaGH genes, catalyses the ring cleavage of protocatechuate. Microorganisms harbouring pcaGH genes are widely distributed in the environment but little is known about their relative abundance within the total microflora. Hence, this paper reports the development of a real-time PCR assay to quantify the bacterial pcaH sequence that encodes the β sub-unit of the protocatechuate 3,4-dioxygenase. This real-time PCR assay was linear over seven orders of magnitude with a calculated efficienc…
Biochemical and Kinetic Analyses of NS5B RNA-Dependent RNA Polymerase of the Hepatitis C Virus
1998
The biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagged NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of the purified protein was inhibited by KCl or high concentrations of NaCl and was absolutely dependent on Mg2+, which could be replaced by Mn2+. NS5B was found to be processive and able to copy long heteropolymeric templates with an elongation rate of 150-200 nucleotides/min at 22 degreesC. Kinetic constants were determined for all four nucleoside triphosphates and different templates. In case of a heteropolymeric RNA…
Two novel oxetane containing lignans and a new megastigmane from Paronychia arabica and in silico analysis of them as prospective SARS-CoV-2 inhibito…
2021
The chemical characterization of the extract of the aerial parts of Paronychia arabica afforded two oxetane containing lignans, paronychiarabicine A (1) and B (2), and one new megastigmane, paronychiarabicastigmane A (3), alongside a known lignan (4), eight known phenolic compounds (5–12), one known elemene sesquiterpene (13) and one steroid glycoside (14). The chemical structures of the isolated compounds were constructed based upon the HRMS, 1D, and 2D-NMR results. The absolute configurations were established via NOESY experiments as well as experimental and TDDFT-calculated electronic circular dichroism (ECD). Utilizing molecular docking, the binding scores and modes of compounds 1–3 tow…
Inhibition of herpesvirus DNA synthesis by 9-beta-D-arabinofuranosyladenine in cellular and cell-free systems.
1977
9-beta-D-Arabinofuranosyladenine 5'-triphosphate (ara-ATP) is an inhibitor both of DNA polymerase-alpha and -beta from noninfected rabbit kidney cells and of the DNA-dependent DNA polymerase induced by herpes simplex virus Type 1 (strain IES). The studies were performed with partially purified enzymes, and each of the different polymerase preparations contained only one DNA-dependent DNA polymerase species. These enzymes were inhibited in a competitive manner. The HSV-induced DNA-dependent DNA polymerase was 39-fold more sensitive to ara-ATP than was cellular DNA polymerase-beta and 116-fold more sensitive than cellular DNA polymerase-alpha. The affinity of the HSV-induced enzyme for ara-AT…
Self-instructed condensation of amino acids and the origin of biological information
1984
In contemporary cells biological information is largely stored in nucleic acids. Therefore, a prerequisite in many theories on the origin of cellular life is the pre-existence of self-replicating polynucleotides that had to be formed by abiotic processes on the prebiotic Earth. It is usually assumed that the spontaneous synthesis of a self-replicating polynucleotide could take place readily. However, serious stereochemical obstacles exist which make such a synthesis extremely improbable. Amino acids, on the other hand, which are abundantly formed in prebiotic simulation experiments, are relatively easily polymerized to macromolecules (protoproteins) that share with modern proteins many prop…
Bleomycin, an Antibiotic That Removes Thymine from Double-Stranded DNA
1977
Publisher Summary This chapter reviews that bleomycins are members of a new class of DNA-modifying agents, the quasi-enzymes. In in vitro systems, bleomycin first removes thymines from native DNA by hydrolysis of the N-glycosidic bonds without modifying the deoxyribose moiety. In a second step, single-strand scissions occur at the sites of the nonglycosidic deoxyribose moieties, resulting in the formation of 3'-OH and 5'-P termini. It is suggested that bleomycin is bound to DNA by interaction of the positively charged terminal amine moiety with the negatively charged phosphate group in DNA; intercalation seems to be involved in binding. Bleomycin is inactivated by copper and zinc ions, prob…
RNA In Vitro Synthesis by Phage T7 DNA-Dependent RNA Polymerase
1998
The purpose of this chapter is to introduce beginners in molecular biology to RNA transcription by phage T7 DNA-dependent RNA polymerase. The work outlined here includes the transcription procedure of plasmid vectors or PCR-amplified DNA templates, the purification and identification of RNA products by sequencing with reverse transcriptase.
Absence of mutation at the GAP-related domain of the neurofibromatosis type 1 gene in sporadic neurofibrosarcomas and other bone and soft tissue sarc…
1995
The NF1 gene encodes neurofibromin, a GTPase-activating protein containing a GAP-related domain (NF1-GRD) that is capable of downregulating ras by stimulating ras intrinsic GTPase activity. We tested 44 sarcomas, nine of which corresponded to sporadic neurofibrosarcomas, for mutations at the NF1-GRD by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique, finding no mutation in every sample tested. We suggest that inactivation of the NF1-GRD by gene mutation seems not to be an important event in the tumorigenesis of sarcomas.
Bleomycin, a selective inhibitor of DNA-dependent DNA polymerase from oncogenic RNA viruses.
1972
Abstract Bleomycin, an antibiotic, inhibits the DNA-dependent DNA polymerase from Rauscher murine leukemia virus. Higher concentrations of BLM ∗ are required to inhibit it's RNA-dependent DNA polymerase. These inhibition effects of the non-competitive type are not altered by preincubation of the DNA with BLM. Under comparable conditions neither the DNA-dependent DNA polymerase activity from E. coli and mouse liver nor the DNA-dependent RNA polymerase activity from mouse lymphoma cells are affected by BLM.
Activity and kinetics of DNA dependent DNA and RNA polymerases n xeroderma pigmentosum and in normal human skin.
1971
1. DNA dependent DNA polymerase (E.C.2.7.7.7) was prepared from human normal and from Xeroderma pigmentosum skin. 2. DNA polymerase from normal skin has the same Michaelis constant with native and denatured DNA as templateKm= 120 ± 11 µg DNA/ml, with differing maximum reaction velocities. 3. The enzyme from Xeroderma pigmentosum has the same Michaelis constant for denatured DNA as the enzyme from normal skin, but with native DNA as template, theKmvalue is lower (97.2 ± 9.8). The maximum reaction velocities of the Xeroderma pigmentosum enzyme with native resp. denatured DNA as template are the same. 4. DNA dependent RNA polymerases (E.C.2.7.7.6) from normal and Xeroderma pigmentosum skin wer…