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Quantitative Shift Model and Generation of Single-Cell Heterogeneity
Published in Ovide Arino, David E. Axelrod, Marek Kimmel, Mathematical population dynamics, 2020
From a biological point of view, it is important to consider possible mechanisms by which quantitative shifts of a factor of 2 can occur with equal probability both up and down. If structural gene amplification were involved, it would be easy to envision how a doubling of gene copy number can occur by amplification, but how halving the gene copy number could occur is unclear. From studies with variation in albumin syntheses in hepatoma cells it is doubtful that structural gene amplification is involved since there appears to be a unique copy of the albumin gene and phenotypic variation occurs without changes in structural gene copy number (Peterson, 1984). Previously, we proposed an alternative mechanism involving reamplification of some regulatory sequence from a single copy at each cell division, where the number of consecutive duplications would be controlled but where there would be a high probability that it would miss by one more or one less duplication, thus resulting in the factor-of-2 shift proposed in the model (Peterson, 1984). Alternatively, the folding of some repeated regulatory sequence could determine the level of transcription, and at each cell division it is refolded and alternates among several different folding configurations. Although these proposed mechanisms may seem speculative, it is yet all but clear how transcription is quantitatively regulated and why it has a stochastic component.
Role of Microbes in Environmental Sustainability and Food Preservation
Published in Ram Chandra, R.C. Sobti, Microbes for Sustainable Development and Bioremediation, 2019
Huang En, Ravi Kr. Gupta, Fangfei Lou, Sun Hee Moon
Lantibiotic variants can be produced by manipulation of the structural gene lanA. In the whole-cell system, lantibiotics variants can be generated in vivo in the natural host organism or in a genetically well-characterized host (Cortés, Appleyard, & Dawson, 2009). To avoid production of the mixture of wild-type and mutated lantibiotics, a lanA− host is usually created by inactivation of the lanA gene. Inactivation can be achieved by deletion of lanA, gene replacement through recombination, or generating a frameshift mutation (Cortés et al., 2009). The lanA− gene can be replaced by a mutated lanA gene in the same locus (in cis) or complemented in trans with a separate mutated lanA on a plasmid. The in cis complementation system has been used to produce mutants such as subtilin, mutacin II, pep5, mersacidin, cinnamycin, and lacticin 3147 (Cortés et al., 2009). Example of lantibiotic variants generated by in trans complementation system included nisin, mersacidin, gallidermin/epidermin, actagardine, lacticin 3417, and nukacin ISK-1 (Cortés et al., 2009).
Improvement of Useful Enzymes by Protein Engineering
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
In a separate study, a strain B. stearothermophilus MK232 was isolated that produced a highly thermostable neutral proteinase. Thermostability and specific activity of the new proteinase (NprM) were about 10 and 40%, respectively, higher than those of thermolysin, the neutral proteinase from B. thermoproteolyticus [8]. The structural gene of NprM was cloned and sequenced [9]. The amino acid sequence of NprM was identical with that of thermolysin, except for two amino acid substitutions (Asp-37 to Asn-37 and Glu-119 to Gln-119, both corresponding to the addition of one amino group). Both NprM and thermolysin were crystallized, and the tertiary structures were determined by x-ray diffraction (Fig. 9).
High-level production and purification of bioactive recombinant human activin A in Chinese hamster ovary cells
Published in Preparative Biochemistry & Biotechnology, 2023
Changin Kim, Hyunjoo Kim, Jeong Soo Park, Jiwon Park, Jeongmin Oh, Jaeseung Yoon, Kwanghee Baek
For the construction of the rhActivin A expression plasmid, the nucleotide sequence encoding the human activin A preproprotein (INHBA, GeneBank Accession No. BC007858) synthesized by Bioneer Corporation (Korea) was cloned into the mammalian expression vector, pC(F)mEGM(R)-TA, which was reported to induce high expression of recombinant proteins in CHO cells.[23] The expression vector, pC(F)mEGM(R)-TA, contains cytotoxic serine protease-B(CSP-B) scaffold attachment region (SAR) and β-globin matrix attachment region (MAR) to promote the position-independent expression of a recombinant gene and strong promoter of mouse EF1 α modified with the TATA box of CHO EF1 α promoter. In addition, it has an efficient polyadenylation signal and transcription terminator. Modification of the translation initiation site of rhActivin A with the Kozak sequence was performed during the DNA cloning process.[24] Following the adaptation in EX-CELL® CD CHO medium (Merck) supplemented with 8 mM L-glutamine and HT, DHFR-deficient CHO cells (CHO DG44) were used as host cells for the transfections. Transfection of the host cells with rhActivin A expression plasmid DNA (5 µg), along with the pDCH1P plasmid (28 ng) containing the dhfr gene as a selection marker, was performed by electroporation as previously described.[25] The pDCH1P plasmid is 5614 bp DNA and able to express dhfr with the structural gene of CHO dhfr and its original promoter.
Nitrite removal by Acinetobacter sp.TX: a candidate of curbing N2O emission
Published in Environmental Technology, 2022
Shuqian Sun, Xiaohui Bi, Bin Yang, Weihong Zhang, Xinyu Zhang, Shujing Sun, Jibo Xiao, Yunlong Yang, Zhida Huang
Recent research showed that N2OR, the terminal enzyme of denitrification, is a multi-copper protein consisting of seven sequentially encoded components, nosRZDFYL [32, 35], out of which nosZ is the structural gene of N2OR and encodes the catalytic subunit [36]. Figure 2 showed the amplification results for the gene encoding nitrous oxide reductase, and clearly a fragment of 415 bp was obtained. Through cloning and sequencing, a high similarity could be observed between this nosZ gene and other nosZ genes by the BLAST analysis in NCBI, indicating that TX5 had nos genes. In general, higher density of nosZ gene copies was in correspondence with more bacteria capable of reducing N2O to N2 [37], which was also evidenced by a report where the reduction of N2O emission from biological nitrogen removal processes was achieved by Alcaligenes faecalis augmentation [38]. As such, the augmentation of TX5 into nitrogen removal processes could undoubtedly decrease N2O emission, indicative of a potential candidate for the wastewater treatment with a little and even no accumulation of N2O.