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Translation
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
The binding site for the Qβ coat protein on the Qβ RNA was mapped by Hans Weber (1976). Again, the complexes of the coat protein and 32P-RNA were subjected to the ribonuclease T1 degradation. The three main fragments obtained, 88, 71, and 27 nucleotides in length, all consisted of sequences extending from the intergenic region to the beginning of the replicase gene. This finding confirmed the universal character of the translational regulation in the RNA phages: in the Qβ replication, as in the case of the R17 and other group I phages, the coat protein acted as a translational repressor by binding to the ribosomal initiation site of the replicase gene. Figure 16.6 presents the original picture from the paper (Weber 1976). By analogy with the R17 coat, the Qβ coat protein was bound to the replicase initiation site of the Qβ RNA (Weber 1975).
Robert W. Holley 1968
Published in Daniel M. Fox, Marcia Meldrum, Ira Rezak, Nobel Laureates in Medicine or Physiology, 2019
Daniel M. Fox, Marcia Meldrum, Ira Rezak
In 1965 Holley announced that he and his colleagues had determined the complete nucleotide sequence of a tRNA specific for the amino acid alanine. He had obtained pure alanine tRNA by the countercurrent-distribution technique. To solve the primary structure of the purified tRNA Holley devised procedures similar in concept to those introduced by Nobel laureate Fred Sanger for the determination of the sequence of amino acids in proteins. Purified tRNA was broken into pieces by different enzymes that were known to catalyze chain scission only at bonds occupied by a particular type of nucleotide residue. For example, ribonuclease T1 splits the chain at guanylic acid residues. Pancreatic ribonuclease splits the chain at both uridylic and cytidylic acid residues. Holley and his coworkers had to work out methods for separating and sequencing the small polynucleotide fragments released from tRNA by enzymatic digestion. When the fragments had been analyzed, they had to be put together in the unique linear array that constituted the original alanine tRNA. Holley noted that the nucleotide sequence for alanine tRNA was not just the first nucleotide for a nucleic acid; with appropriate modifications for DNA, the sequence also provided the first nucleotide sequence for a gene.
Affinity Modification — Experimental Methods
Published in Dmitri G. Knorre, Valentin V. Vlassov, Affinity Modification of Biopolymers, 1989
Dmitri G. Knorre, Valentin V. Vlassov
The identification of the modified sites in nucleic acids can also be carried out using fragmentation of the biopolymers with specific enzymes. Thus, the first step of localization of modified residues in double-stranded DNAs consists in the digestion of the nucleic acid with restriction enzymes which cleave DNA in sequence-specific way, separation of the restriction fragments by gel electrophoresis, and identification of the fragments which carry the modified residues. Similarly, modified RNAs are hydrolyzed by specific ribonucleases and identification of the modified oligonucleotides is very often the final level of resolution achieved. For example, in experiments on direct UV cross-linking of tRNAs to aminoacyl- tRNA-synthetases the cross-linked proteins and tRNAs were treated with ribonuclease T1 which cleaves RNA at positions of guanosine residues and the released oligonucleotides were separated and quantitized. Comparison of the data with results of similar oligonucleotide analysis of the free tRNA allowed the oligonucleotides which were lacking from the digests to be revealed. These oligonucleotides were considered as the protein-cross-linked ones.335
Non-invasive targeted iontophoretic delivery of cetuximab to skin
Published in Expert Opinion on Drug Delivery, 2020
Maria Lapteva, Marwa A. Sallam, Alexandre Goyon, Davy Guillarme, Jean-Luc Veuthey, Yogeshvar N. Kalia
The EM contribution is dependent on the electrophoretic mobility of the molecule, which, in turn, depends on the mass-to-charge ratio. Therefore, it was considered that as molecular weight increased, EM would become less important and would be superseded by EO as the dominant electrotransport mechanism. It was originally thought that EO would already govern the iontophoretic transport of peptides in the 3–4 kDa range [15,17]. However, this hypothesis was put into question by the successful iontophoresis of cytochrome C, a small protein with an MW of 12.4 kDa that is positively charged at physiological pH (pI 9.6), across intact skin and the confirmation that EM was the dominant electrotransport mechanism [18]. Subsequent experiments with ribonuclease A (13.6 kDa, pI 8.64) [19], ribonuclease T1 (11.1 kDa, pI 4.27 – and thus negatively charged at physiological pH) [20] and human basic fibroblast growth factor (17.4 kDa, pI 9.6) [17]), showed that EM was still the principal electrotransport mechanism and confirmed that biological activity was retained post-iontophoresis. Most therapeutic mAbs have isoelectric points in the range of 7.0–9.5, meaning that they are positively charged at physiological pH. Although there are no reports on the iontophoretic delivery of a mAb across intact skin, the feasibility of iontophoresing bevacizumab (149.2 kDa) across human-isolated sclera, a more permeable biological barrier, has been demonstrated [21].