Cloning and characterization of complementary DNA encoding the eukaryotic initiation factor-2 associated 67 kDa polypeptide (p[sup 67])
An eukaryotic initiation factor-2 (eIF-2) associated 67 kDa polypeptide (p[sup 67]) plays a critical role in the regulation of protein synthesis initiation in mammalian cells. The eIF-2 [alpha]-subunit can be phosphorylated by several protein kinases, leading to eIF-2 inactivation and hence protein synthesis inhibition. As p[sup 67] can protect the eIF-2 [alpha]-subunit from phosphorylation, it promotes protein synthesis in the presence of active eIF-2 kinases. The authors cloned the p[sup 67] gene: (1) A cDNA library in [lambda]gt 11 was constructed from a cultured rat hepatoma (KRC-7) cell line highly enriched in p[sup 67]. (2) P[sup 67] was cleaved using chemical and enzymatic methods. Stretches of amino acid sequence were obtained from p[sup 67] using Edman degradation. (3) Oligonucleotides that corresponded to the peptides sequence of p[sup 67] were synthesized and used to amplify the gene encoding p[sup 67] with the polymerase chain reaction (PCR). Southern blot analysis and DNA sequencing proved that one of the PCR products was part of the p[sup 67] gene. (4) The PCR product was [sup 32]P labeled and used as a probe to screen the [lambda]gt 11 library. One p[sup 67] gene, which contained approximately 2 kilobases (kb), was cloned and characterized. (5) The gene was subcloned into pGEM5zf(+) vector and sequenced by the Sanger method. The gene contains a total of 1953 base-pairs and had a 1440 bp open reading frame that encodes a protein with a calculated molecular weight of 53190.78 kDa. The amino acid sequence deduced from the DNA sequence matches the sequence of the p[sup 67] peptide fragments. It contains a strong initiation sequence (AACATGG). It has a poly(A) additional signal (AATAAA) 14 bases upstream of a poly(A)[sub 9] tail. (6) The subcloned p[sup 67] gene was translated in vitro. The product had a molecular weight of 65 kDa and was immunoprecipitated by anti-p[sup 67] polyclonal antibodies. This 65 kDa polypeptide interacted in vitro with eIF-2.
- Research Organization:
- Nebraska Univ., Lincoln, NE (United States)
- OSTI ID:
- 7233842
- Resource Relation:
- Other Information: Thesis (Ph.D.)
- Country of Publication:
- United States
- Language:
- English
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