China
Africa
Explore chapters and articles related to this topic
Drug Discovery, Development, and Distribution
Published in Richard J. Sundberg, The Chemical Century, 2017
Molecular biology and biotechnology introduced other means of drug discovery. It became possible to identify, clone, and sequence the genes related to specific biological functions. These genes could then be used to prepare the corresponding proteins for bioassays. These efforts, including the completion of the sequence of the human genome in 2003, permitted identification of many more functional biological molecules. This genetic information provided the basis for identifying a wide variety of potential protein targets. For example, the effect of specific genes can be studied using “knock-out” or “knock-in” mice in which particular genes have been either inactivated or added. Beginning in the mid-1980s, it became possible to use genetic engineering to produce proteinbased drugs. Examples include insulin, human growth hormone, and granulocyte stimulating factor (see Section 21.3).
Engineered Pseudomonas putida for biosynthesis of catechol from lignin-derived model compounds and biomass hydrolysate
Published in Preparative Biochemistry & Biotechnology, 2022
Pseudomonas putida KT2440 mutant deficient in catechol and protocatechuate metabolism were generated by gene inactivation/knockout via homologous recombination protocol. In P. putida KT2440 the catA gene was first knocked out followed by catA2 gene and finally pcaHG gene. For e.g., ΔcatA knockout was carried out as depicted in Figure 2. It involved the construction of the gene replacement plasmid containing truncated catA gene corresponding to 726 bp, which upon introduction into the host organism results in site-specific integration of the entire cassette leading to merodiploid formation. Merodiploid clone contains two copies of a gene, one corresponding to wildtype catA gene with 936 bp while another one corresponding to the truncated catA gene with 726 bp as indicated in Figure 2a. Later upon second homologous recombination, mutant clone was obtained wherein the wildtype gene was replaced with the truncated variant making the gene nonfunctional. This was further confirmed by using internal set of primers where the wildtype PCR product corresponds to 500 bp while the mutant corresponds to 300 bp as indicated in Figure 2c.
Epigenotoxicity: a danger to the future life
Published in Journal of Environmental Science and Health, Part A, 2023
Farzaneh Kefayati, Atoosa Karimi Babaahmadi, Taraneh Mousavi, Mahshid Hodjat, Mohammad Abdollahi
Abnormal and excessive blood pressure in the pulmonary artery causes PHD, eventually leading to right ventricular dysfunction. In recent decades, the importance of epigenetic therapy mediated by DNA methylation alterations and histone modifications has been highlighted in treating lung disease. Using di-methyltransferase SUV4-20H1, knockout mouse caused a PHD phenotype that further confirm the role of histone methyltransferase in the etiology of this disease. The methyl-CpG-binding domain protein family, is a regulatory factor in DNA methylation that was shown to increase in expression in PHD patients’ pulmonary arteries, cigarette smoke (CS)-exposed rat models’ pulmonary arteries, and human pulmonary artery cells exposed to CS, indicating the role of epigenetic modulator in CS-induced PHD. Indeed, epigenetic factors play an essential role in the elevation of blood pressure in the pulmonary arteries (Table 2).[11] According to the previous research, there is a substantial decrease in histone modification of H4K20me2/3 in human patients with COPD, unlike patients with PHD that makes them responsive to epigenetic drug effect.[171] The methylation of H4K20me2/3 was attributed to the activity of the H4K20 di-methyltransferase SUV4-20H1. Smoking or exposure to environmental CS significantly alters gene methylation in COPD-related diseases, especially PHD. One of the regulatory factors of DNA methylation is the methyl-CpG-binding domain protein family (MBD). The MBD2 protein is a factor of the MBD protein family, which acts as a reader in DNA methylation. MBD2 can intervene in transcriptional repression or activation by merging methylated DNA or collecting proteins to form a suppressive combination.[172]
Impact of exoD gene knockout on the polyhydroxybutyrate overaccumulating mutant Mt_a24
Published in International Journal of Biobased Plastics, 2021
Sandra Mittermair, Juliane Richter, Philipp Doppler, Kevin Trenzinger, Cecilia Nicoletti, Christian Forsich, Oliver Spadiut, Christoph Herwig, Maximilian Lackner
In order to reduce the EPS production, the gene exoD, known to be part of its synthesis process, was knocked out. Therefore, a knockout plasmid carrying the flanking regions of the gene exoD referred to as upstream (US) and downstream (DS) and a chloramphenicol resistance cassette replacing exoD, was designed and cloned (shown in Figure 1(a)). Using electroporation, this knockout plasmid was taken up by the cells and the resistance cassette was integrated via double homologous recombination into the genome. The transformed cells were cultivated for 5 days without antibiotic selection pressure to recover from the electroporation. Then, they were transferred on agar supplemented with antibiotics until single colonies occurred. 233 clones of those single colonies were transferred on new plates, of which 13 grew after the transfer. Seven transformants, which constantly grow throughout the passages, were screened via Multiplex-PCR. The verification of the complete exoD-deletion in all genomes was done with the transformant genome-specific primers 6–48 F and 10–16 R as well as the wild type genome-specific primers 10–15 F and 10–16 R. Their binding sites are shown in Figure 1(a). The tested transformants are shown in Figure 1(b). In four out of the seven screened transformants only the transformant-specific fragment of 0.966 kb could be detected, verifying the complete deletion of the gene exoD out of each genome copy. Transformant #2 showed the transformant genome-specific (0.966 kb) as well as the wild type genome-specific fragment (0.676 kb), indicating some genome copies still have the gene exoD integrated. As the signal intensity of transformant KOexoD#4 and KOexoD#65 is the strongest, those were used for further analysis regarding growth behavior, storage compound, and EPS production.