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Molecular Biology and Bioinformatics in Industrial Microbiology and Biotechnology
Published in Nduka Okafor, Benedict C. Okeke, Modern Industrial Microbiology and Biotechnology, 2017
Nduka Okafor, Benedict C. Okeke
The mRNA is transcribed from one strand of the DNA of the gene; it is translated at the ribosome into a polypeptide sequence. Translation is the synthesis of protein from amino acids on a template of messenger RNA in association with a ribosome. The bases on mRNA code for amino acids in triplets or codons; that is three bases code for an amino acid. Sometimes, different triplet bases may code for the same amino acid. Thus, the amino acid glycine is coded for by four different codons: GGU, GGC, GGA, and GGG. There are 64 different codons; three of these UAA, UAG, and UGA are stop codons and end the process of translation. The remaining 61 codons code for the amino acids in proteins (Table 3.1). Translation of the message generally begins at AUG, which also codes for methionine. For AUG to act as a start codon, it must be preceded by a ribosome binding site. If that is not the case, it simply codes for methionine.
Genes and Genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
mRNA is involved in protein synthesis by carrying coded information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein. In mRNA, as in DNA, genetic information is encoded in the sequence of nucleotides arranged into codons consisting of three bases each. Each codon encodes for a specific amino acid, except the stop codons that terminate protein synthesis. This process requires two other types of RNA: transfer RNA (tRNA), which mediates recognition of the codon and provides the corresponding amino acid, and ribosomal RNA (rRNA), which is the central component of the ribosome’s protein manufacturing machinery.
Genes and genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
mRNA is involved in protein synthesis by carrying coded information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein. In mRNA, as in DNA, genetic information is encoded in the sequence of nucleotides arranged into codons consisting of three bases each. Each codon encodes for a specific amino acid, except the stop codons that terminate protein synthesis. This process requires two other types of RNA: tRNA, which mediates recognition of the codon and provides the corresponding amino acid, and rRNA, which is the central component of the ribosome’s protein manufacturing machinery.
Engineered Pseudomonas putida for biosynthesis of catechol from lignin-derived model compounds and biomass hydrolysate
Published in Preparative Biochemistry & Biotechnology, 2022
Synthetic codon-optimized aroY gene encoding protocatechuate decarboxylase from Enterobacter cloacae; and vanAB gene encoding vanillate-O-demethylase from Acinetobacter sp. ADP1 were procured from GenScript. GenScript provided the synthesized nucleotide sequences in multiple cloning site of pUC57 plasmid, containing codon-optimized gene along with the additional sequence such as restriction enzyme site; RBS (ribosome binding sequence) along with the spacer as GAATTCAGAGGAGGGAGA; and the stop codon as TGA. Homologous gene vanAB encoding vanillate-O-demethylase from Pseudomonas putida KT2440 as well as from Pseudomonas putida S12 (ATCC 700801) was PCR amplified from respective genomic DNA using the primer pairs mentioned in Table 3. The vanAB gene from Acinetobacter sp. ADP1; Pseudomonas putida KT2440 or Pseudomonas putida S12 was cloned along with the RBS in pSEVA 234 vector between the restriction site EcoRI and BamHI. Vanillic acid accumulation problem was addressed by the expression of vanAB gene from one of the organisms (Acinetobacter sp. ADP1; Pseudomonas putida KT2440 or Pseudomonas putida S12) in the clone containing ΔpcaHG knockout.
Characterization of the novel anti-TNF-α single-chain fragment antibodies using experimental and computational approaches
Published in Preparative Biochemistry and Biotechnology, 2019
Samira Pourtaghi-Anvarian, Samin Mohammadi, Maryam Hamzeh-Mivehroud, Ali Akbar Alizadeh, Siavoush Dastmalchi
Tomlinson I and J scFv libraries were used for identification of specific anti-TNF antibodies as described previously. In ELISA experiment, phage displaying scFv antibodies showed an adequate affinity towards TNF-α.[13] DNA sequencing of these antibodies revealed the existence of an extra amber stop codon in their coding sequence. In order to express and purify the selected scFv antibodies, the extra amber stop codon must be replaced by a normal amino acid codon. To do so, two sets of primers were used as indicated in Table 1. A pair of primers was used for full-length amplification of each scFv sequence (F1 and R1) and another pair of overlapping primers (F2 and R2) was designed using PrimerX web-based program to mutate the amber stop codon (TAG) into tyrosine (TAT) in the DNA sequence of the selected scFvs. The designed primers were used to perform two PCR reactions. First, in separate PCR reactions, F1 and R2, and F2 and R1 pairs of primers were used to perform two PCR reactions on pIT2 phage harboring the coding sequence for scFvs. The PCR products from the first step were used as the templates in the next PCR reaction using F1 & R1 primers. The final PCR product was digested using NcoI and NotI restriction enzymes and cloned into pIT2 vector cut with the same restriction enzymes. The constructed vectors were sent out for DNA sequencing. The amino acid sequences of scFvs after removing stop codons are available in Table 2. Finally, the validated constructs were transformed into Escherichia coli ER2738 strain for phage amplification according to the protocol described previously.[14]
Genetic variants affecting chemical mediated skin immunotoxicity
Published in Journal of Toxicology and Environmental Health, Part B, 2022
Isisdoris Rodrigues de Souza, Patrícia Savio de Araujo-Souza, Daniela Morais Leme
CASP14 is a product of the CASP14 gene located on chromosome 19p13 (Hoste et al. 2011). This gene is mainly expressed in the suprabasal layers of the epidermis, and its expression increases during the process of keratinocyte differentiation (Rendl et al. 2002). The small deletion c.462_463delCA in CASP14 leads to a frameshift on the second codon of a small non-catalytic subunit (p11) and a premature stop codon at amino acid position 180 (p.Asp154Glufs), resulting in a truncated protein, impaired skin barrier, and autosomal recessive inherited ichthyosis (Kirchmeier et al. 2017).