Cloning and characterization of complementary DNA encoding the eukaryotic initiation factor 2-associated 67-kDa protein (p67)
| Autor: | S, Wu, S, Gupta, N, Chatterjee, R E, Hileman, T G, Kinzy, N D, Denslow, W C, Merrick, D, Chakrabarti, J C, Osterman, N K, Gupta |
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| Rok vydání: | 1993 |
| Předmět: |
Transcription
Genetic Protein Conformation Molecular Sequence Data Saccharomyces cerevisiae Aminopeptidases Polymerase Chain Reaction Liver Neoplasms Experimental Sequence Homology Nucleic Acid Animals Humans Methionyl Aminopeptidases Amino Acid Sequence RNA Messenger Cloning Molecular Gene Library Glycoproteins Base Sequence Sequence Homology Amino Acid DNA Blotting Northern Chromatography Ion Exchange Peptide Fragments Rats Molecular Weight Liver Oligodeoxyribonucleotides Rabbits |
| Zdroj: | The Journal of biological chemistry. 268(15) |
| ISSN: | 0021-9258 |
| Popis: | The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa glycoprotein (p67) protects eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, such as heme-regulated inhibitor and double-stranded RNA-activated inhibitor. This promotes protein synthesis in the presence of eIF-2 kinases present in animal cells (Ray, M. K., Datta, B., Chakraborty, A., Chattopadhyay, A., Meza-Keuthen, S., and Gupta, N. K. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543). In this study, the primary structure of rat p67 is determined by cDNA cloning. Based on the partial amino acid sequences of overlapping tryptic and cyanogen bromide cleaved fragments, degenerate oligonucleotides were synthesized and used as primers for the polymerase chain reaction to amplify the corresponding p67 cDNA fragment from rat liver first strand cDNA. The amplified DNA was then used as a probe to screen a rat tumor hepatoma (KRC-7) cDNA library, and a positive clone covering the entire coding region was obtained. From the cDNA sequence, an open reading frame that encodes p67 as a 480-amino acid protein with a molecular mass of 53 kilodaltons was predicted for the unglycosylated protein. The cloned cDNA was further characterized by in vitro transcription-coupled translation in micrococcal nuclease-treated reticulocyte lysate. The translated product migrated similarly to p67 in SDS-polyacrylamide gel electrophoresis and was precipitated with antibodies against p67. Northern blot analysis of rat liver poly(A)+ RNA showed a single size class (approximately 2 kilobases) of mRNA. The deduced amino acid sequence of the protein showed a highly charged N-terminal region composed of two basic polylysine blocks and an acidic aspartic acid block. The protein also exhibits significant sequence identity in the N-terminal region with human eIF-2 beta-subunit. |
| Databáze: | OpenAIRE |
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