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Tay-Sachs disease/hexosaminidase A deficiency
Published in William L. Nyhan, Georg F. Hoffmann, Aida I. Al-Aqeel, Bruce A. Barshop, Atlas of Inherited Metabolic Diseases, 2020
Hexosaminidase A is a heterodimer containing the α and β subunits. Hexosaminidase B is composed of two β subunits. Cultured cells of patients with Tay-Sachs disease lack the α chain. The genes for the α and β chains have been cloned and the locus for the α chain and for Tay-Sachs disease is on chromosome 15q23 [8]. The gene is common in Ashkenazi Jews. A considerable number and variety of mutations have been described, most in patients with the classic infantile phenotype [9, 10]. The most frequent mutation in the Ashkenazi Jewish population is a four-nucleotide insertion in exon 11, which introduces a frameshift and a downstream premature termination signal that results in a deficiency of mRNA.
Nijmegen Breakage Syndrome
Published in Dongyou Liu, Handbook of Tumor Syndromes, 2020
The most notable NBS pathogenic variants include c.330T>G (p.Tyr110Ter), c.643C>T (p.Arg215Trp), c.657_661del5 (657del5) (p.Lys219AsnfsTer15), c.681delT (p.Phe227LeufsTer4), c.698_701del4 (p.Lys233SerfsTer4), c.741_742dup (742insGG) (p.Glu248GlyfsTer5), c.835_838del4 (p.Gln279ProfsTer1), c.842insT (p.Leu281PhefsTer3), c.900del25 (p.Gly301LysfsTer5), c.976C>T (p.Gln326Ter), c.1089C>A (p.Tyr363Ter), c.1125G>A (p.Trp375Ter), and c.1142delC (p.Pro381GlnfsTer22). Among these, the founder mutation c.657_661del5, of Slavic origin, creates a five base pair deletion in exon 6 and inserts a premature termination signal at codon 219, resulting in a 26 kDa amino-terminal fragment (p26-nibrin, containing the FHA domain and one BRCT domain) and a 70 kDa carboxy-terminal fragment (p70-nibrin, containing the second BRCT domain), which is responsible for a majority of clinical cases reported. Homozygous c.1089C>A pathogenic variant may show features of Fanconi anemia. Compound heterozygous mutations involving c.657_661del5 and c.643C>T are observed in monozygotic twins, who presented with severe neurologic features (microcephaly, mildly asymmetric lateral ventricles, enlarged subarachnoid areas and poor gyrification of the brain, and retarded psychomotor development) without chromosomal instability and radiation sensitivity [1].
RNA
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
The other intercistronic region was located between the A protein gene and the coat gene. As the amino acid sequence at the carboxyl end of the A protein was not known at that time, it was not possible to identify directly the nucleotide sequence containing the termination signal of the A protein gene. This termination codon, however, must be a single UAG codon because it was suppressible in various Su+ strains with a normal efficiency (Remaut and Fiers 1972a,b). Such readthrough resulted in a prolonged A protein containing some 30 additional amino acids, which did not incorporate into phage particles. The sequence of 160 nucleotides preceding the initiating AUG of the coat gene was identified (Contreras et al. 1973). As Fiers (1975) noted, inspection of this sequence for a UAG codon, which would not be preceded by another nonsense codon in the same reading frame and which would only be followed by another nonsense codon in the same phase some 90 nucleotides farther on, made it possible to identify the termination signal of the A protein unambiguously (see Figure 10.3). This assignment was confirmed by Vandekerckhove et al. (1973a,b), who isolated and sequenced some peptides derived from the carboxyl end of the A protein. It was concluded that the untranslated intercistronic region was 26 nucleotides long.
Correlation of Staphylococcus Epidermidis Phenotype and Its Corneal Virulence
Published in Current Eye Research, 2021
Armando R. Caballero, Aihua Tang, Michael Bierdeman, Richard O’Callaghan, Mary Marquart
The gene sequence for the S. epidermidis Esp protease was obtained from the mass spectrometry data using the GenBank database. Based on these data, primers for the complete gene were designed. The restriction enzyme site NcoI was incorporated into the 5ʹ primer (AACC ATG GCTAAAAAGAGATTTTTATCT) and Bgl II site into the 3ʹ primer (TT AGA TCT CTGAATATTTATATCAGG) for subsequent cloning. The TAA termination signal was eliminated from the 3ʹ primer to allow read-through of the poly- histidine tail. S. epidermidis strain 30111 genomic DNA was used as the template DNA. PCR reactions were conducted with Taq polymerase according to the manufacturer’s instructions (Promega, Madison, WI). The resulting PCR product was purified from agarose gels and cloned into the E. coli expression vector pQE-60 (QIAGEN), which was then transformed into E coli M15 [pREP4].
Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli
Published in mAbs, 2018
Prasad T. Reddy, Robert G. Brinson, J. Todd Hoopes, Colleen McClung, Na Ke, Lila Kashi, Mehmet Berkmen, Zvi Kelman
The genes encoding for the heavy and light chains of the eNISTmAb were synthesized at GeneArt Gene Synthesis and obtained as a gene cloned into the Bgl II and Bpu1102 I/Blp I restriction endonuclease sites of pET-21a vector (Merck Millipore, 69740-3) (Fig. S7). The construct contained the DNA sequence encoding the heavy and light chains of the eNISTmAb, each with a promoter (cyan in Fig. S7), its own regulatory sequences and transcription termination signal (gray in Fig. S7). The plasmid was transformed into E. coli SHuffle T7 express cells (New England Biolabs, C3029J) and selected on Luria-Bertani (LB) agar media containing 100 µg/mL ampicillin (Sigma-Aldrich, A9518). Incubation of cells post transformation and subsequent growth was at 30°C as specified by the supplier.
Identification and characterization of an IgG sequence variant with an 11 kDa heavy chain C-terminal extension using a combination of mass spectrometry and high-throughput sequencing analysis
Published in mAbs, 2019
Claire Harris, Weichen Xu, Luigi Grassi, Chunlei Wang, Abigail Markle, Colin Hardman, Richard Stevens, Guillermo Miro-Quesada, Diane Hatton, Jihong Wang
Heavy chain extensions produced by splicing require transcript read through the downstream termination signal sequence, producing an extended transcript that is then spliced. It is possible that the strong promoter used in the heavy chain expression cassette may increase the frequency of transcripts reading through beyond the termination signal sequences. Extension products could be prevented by either increasing the level of transcript termination37 or by reducing mis-splicing. Engineering of the DNA to remove the cryptic donor site at the end of the CH3 domain whilst maintaining the amino acid sequence was reported by Spahr et al. to prevent the formation C-terminal heavy chain extension products.19