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Genes and Genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
Chromatin is the complex combination of DNA, RNA, and protein that makes up chromosomes. It is found inside the nuclei in eukaryotic cells, and within the nucleoid in prokaryotic cells. It is divided between heterochromatin (condensed) and euchromatin (extended) forms. The major components of chromatin are DNA and histone proteins, although many other chromosomal proteins have prominent roles too. The functions of chromatin are to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis, and to serve as a mechanism to control expression and DNA replication. Chromatin contains genetic material instructions to direct cell functions. Changes in chromatin structure are affected by chemical modifications of histone proteins such as methylation (DNA and proteins) and acetylation (proteins), and by non-histone DNA-binding proteins.
Genes and genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Chromatin is the complex combination of DNA, RNA, and protein that makes up chromosomes. It is found inside the nuclei of eukaryotic cells and within the nucleoid in prokaryotic cells. It is divided between heterochromatin (condensed) and euchromatin (extended) forms. The major components of chromatin are DNA and his-tone proteins, although many other chromosomal proteins have prominent roles, too. The functions of chromatin are to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis, and to serve as a mechanism to control expression and DNA replication. Chromatin contains genetic material instructions to direct cell functions. Changes in chromatin structure are affected by chemical modifications of histone proteins such as methylation (DNA and proteins) and acetylation (proteins) and by nonhistone DNA-binding proteins.
General Introductory Topics
Published in Vadim Backman, Adam Wax, Hao F. Zhang, A Laboratory Manual in Biophotonics, 2018
Vadim Backman, Adam Wax, Hao F. Zhang
Transcription factors are DNA-binding proteins. Some DNA-binding proteins are activators, while others are repressors. Their names tell it all: Activators increase the rate of gene transcription, while repressors do the opposite. These factors are critically important in transcription, as without their help, only a very low level of transcriptional activity would be possible. Other modulator molecules include coactivators—proteins that assist transcription factors to increase the rate of gene transcription—and corepressors—proteins that work with transcription factors to decrease the rate of transcription.
Direct and cost-effective method for histone isolation from cultured mammalian cells
Published in Preparative Biochemistry & Biotechnology, 2023
Anja Batel, Mirjana Polović, Mateo Glumac, Andrea Gelemanović, Matilda Šprung, Ivana Marinović Terzić
Next, we applied immunoblotting to detect if the amount of individual histones and histone post-translational modifications differ between histone fractions in the G1, S, and G2 phases. Histone fractions from cells arrested in the G1, S, or G2 phases were tested for γH2AX, phospho-H3, H2A, H2B, H3, and H4. In addition to histones, one non-histone DNA binding protein, DHX9 was tested. Different amounts of isolated proteins and protein modifications through the cell cycle suggest that our protocol is specific and sensitive to detect differences in histone variations (Figure 4B). Ponceau S staining of the membrane is shown in Figure S4G. To verify the effectiveness of synchronization, total cellular fractions isolated from the cells synchronized in the G1, S, and G2 phases of the cell cycle, as well as unsynchronized cells, were analyzed by flow cytometry with propidium iodide staining. Results presented in Figure 4C show a successful synchronization of cells in the mentioned phases of the cell cycle.
Developing mitochondrial DNA field-compatible tests
Published in Critical Reviews in Environmental Science and Technology, 2022
Bidhan C. Dhar, Christina E. Roche, Jay F. Levine
Another innovative isothermal nucleic acid amplification approach, the RPA could be used to detect mtDNA and enhance opportunities for on-site field use (Yin et al., 2017). The assay uses a recombinase complex to embed primers into specific DNA regions and start the amplification response by using a strand displacement of DNA polymerase. By adding a RT enzyme to an RPA reaction, it can detect RNA as well as DNA, without the need to generate cDNA. The RPA assay utilizes three core enzymes – a recombinase, a single-stranded DNA-binding protein (SSB) and strand-displacing polymerase (e.g. a large fragment of Bacillus subtilis Pol 1, Bsu). A recombinase complex is used to insert primers into specific DNA regions and start the amplification response with a DNA polymerase with strand displacement activity. The enzyme Bsu binds to the 3′ end of the primer to elongate it in the presence of dNTPs. The key advantage of the RPA assay over LAMP is that the RPA method requires a single set of primers to produce specific amplicons that can easily be observed by gel electrophoresis. Additional advantages supporting potential development of assays for on-site field assay development include: (1) Simple primer design; (2) Assays temperatures of 37–42 °C without a heat-denaturing step; (3) Rapid amplification of DNA in less than 30 min; and (4) No expensive laboratory instrumentation is needed. These features make RPA an excellent platform for developing low-cost, rapid POC mtDNA detection molecular tests for environmental monitoring and conservation biology.
Binuclear ruthenium(II) complexes of 4,4′-azopyridine bridging ligand as anticancer agents: synthesis, characterization, and in vitro cytotoxicity studies
Published in Journal of Coordination Chemistry, 2019
Priyanka Khanvilkar, Ramadevi Pulipaka, Kavita Shirsath, Ranjitsinh Devkar, Debjani Chakraborty
Four new homobinuclear Ru(II)–arene complexes with 4,4′-azopyridine as the bridging ligand have been synthesized and characterized. Single-crystal X-ray diffraction study of one of the precursor mononuclear complexes revealed that the complex attains a pseudo-octahedral “piano-stool” geometry, where the p-cymene ligand forms the seat of the piano stool and the chloride as well as the chelating ferrocenyl amino acid ligand resemble the legs. All the synthesized complexes were evaluated for DNA binding, protein binding and cytotoxicity studies. The DNA-binding of the complexes studied by absorption and fluorescence spectral techniques revealed an intercalative interaction between them and CT-DNA with binding constants ranging from 103 to 105 M−1. Among the investigated complexes, C4, C3 and C1 showed better binding efficacy due to the presence of additional hydrophobic planar aromatic moieties which facilitates cell permeation and intercalation. Binding of the metal complexes with BSA monitored by fluorescence spectroscopy revealed a single quenching mechanism, probably static quenching is operative in the systems. The results of cytotoxicity study reveal a linear relationship between the concentration of the metal complexes and the percentage inhibition of HeLa cell growth without apparent damage to the cells in the absence of the complexes.