Isolation of Two cDNAs Encoding Functional Human Cytoplasmic Cysteinyl-tRNA Synthetase
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Edgar Davidson
Abstract
CysteinyltRNA synthetase catalyzes the addition cysteine to its cognate tRNA. The available eukaryotic sequences for this enzyme contain several insertions that are absent from bacterial sequences. gain insights into the differences between the bacterial and eukaryotic forms, we previously studied the E. coli cysteinyltRNA synthetase. In this study, we sought to clone and express the fulllength gene for the human cytoplasmic cysteinyltRNA synthetase. Although a gene encoding the human enzyme has been described, the predicted protein sequence, consisting of 638 amino acids, lacks homology with other eukaryotic enzymes in the carboxylterminus. This suggested that a further investigation was necessary to obtain the definitive sequence for the human enzyme. Here we report the isolation of a fulllength cDNA that encodes a protein of 748 amino acids. The predicted protein sequence shows considerable similarity to other eukaryotic cysteinyltRNA synthetases in the carboxylterminus. We also found that approximately 20% of the mRNA encoding the cytoplasmic cysteinyltRNA synthetase contained insertion of 8 bases in the 3 coding region of the mRNA. This insertion arises from an alternative splicing between the last two exons of the gene. The alternative splicing alters the reading frame and results the replacement of the carboxyterminal 44 amino acids with a novel sequence of 22 amino acids. Expression of the fulllength and alternative forms of the enzyme in E. coli generated functional proteins that were active in aminoacylation of human cytoplasmic tRNA with cysteine.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- Highlight: Fluorescence Correlation Spectroscopy
- Heuristic Statistical Analysis of Fluorescence Fluctuation Data with Bright Spikes: Application to Ligand Binding to the Human Serotonin Receptor Expressed in Escherichia coli Cells
- Fluorescence Correlation Spectroscopy as a Tool to Investigate Single Molecule Probe Dynamics in Thin Polymer Films
- Ligand-Receptor Interactions in the Membrane of Cultured Cells Monitored by Fluorescence Correlation Spectroscopy
- Fluorescence Fluctuation Analysis for the Study of Interactions between Oligonucleotides and Polycationic Polymers
- Determination of the Net Exchange Rate of Tubulin Dimer in Steady-State Microtubules by Fluorescence Correlation Spectroscopy
- UV-Fluorescence Correlation Spectroscopy of 2-Aminopurine
- Isolation of Two cDNAs Encoding Functional Human Cytoplasmic Cysteinyl-tRNA Synthetase
- Involvement of Intracellular Ca2+in the Regulation of Bovine Leukemia Virus Expression
- Nucleotide-Binding Sites in the Functional Unit of Sarcoplasmic Reticulum Ca2+-ATPase as Studied by Photoaffinity Spin-Labeled 2-N3-SL-ATP
- Interaction between Lipopolysaccharide (LPS), LPS-Binding Protein (LBP), and Planar Membranes
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- Effects of Cysteine to Serine Substitutions in the Two Inter-Chain Disulfide Bonds of Insulin
- Tricorn Protease in Bacteria: Characterization of the Enzyme from Streptomyces coelicolor
- Structures of Tryparedoxins Revealing Interaction with Trypanothione
- Quantitative Two-Color Fluorescence Cross-Correlation Spectroscopy in the Analysis of Polymerase Chain Reaction
- Fluorescence Correlation Spectroscopy as a New Method for the Investigation of Aptamer/Target Interactions
- Localization of Lysobisphosphatidic Acid-Rich Membrane Domains in Late Endosomes
- Fluorescence Correlation Spectroscopy for the Characterisation of Drug Delivery Systems
- Accessing Molecular Dynamics in Cells by Fluorescence Correlation Spectroscopy
- Tailor-Made Dyes for Fluorescence Correlation Spectroscopy (FCS)
- Synergistic Inhibition of the Glucocorticoid Receptor by Radicicol and Benzoquinone Ansamycins
