Cloning and characterization of complementary DNA encoding the eukaryotic initiation factor-2 associated 67 kDa polypeptide (p(67))

Abstract

An eukaryotic initiation factor-2 (eIF-2) associated 67 kDa polypeptide (p$\sp{67}$) has been known to play a critical role in the regulation of protein synthesis initiation in mammalian cells. Under certain physiological conditions, the eIF-2 $\alpha$-subunit can be phosphorylated by several protein kinases, leading to eIF-2 inactivation and hence protein synthesis inhibition. As p$\sp{67}$ can protect the eIF-2 $\alpha$-subunit from phosphorylation, it promotes protein synthesis in the presence of active eIF-2 kinases.

In order to further study the activities of p$\sp{67},$ we cloned the p$\sp{67}$ gene using the following procedures: (1) A cDNA library in $\lambda$gt 11 was constructed from a cultured rat hepatoma (KRC-7) cell line which is highly enriched in p$\sp{67}.$ (2) P$\sp{67}$ was cleaved using chemical and enzymatic methods. Stretches of amino acid sequence were obtained from p$\sp{67}$ using Edman degradation. (3) Oligonucleotides that corresponded to the peptides sequence of p$\sp{67}$ were synthesized and used to amplify the gene encoding p$\sp{67}$ with the polymerase chain reaction (PCR). Southern blot analysis and DNA sequencing proved that one of the PCR products was part of the p$\sp{67}$ gene. (4) The PCR product was $\sp{32}$P labeled and used as a probe to screen the $\lambda$gt 11 library. One p$\sp{67}$ 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. It was found that: (a) The gene contains a total of 1953 base-pairs (bp). (b) It has a 1440 bp open reading frame that encodes a protein with a calculated molecular weight of 53190.78 kDa. (c) The amino acid sequence deduced from the DNA sequence matches the sequence of the p$\sp{67}$ peptide fragments. (d) It contains a strong initiation sequence (AACATGG). (e) It has a poly(A) additional signal (AATAAA) 14 bases upstream of a poly(A)$\sb9$ tail. (6) The subcloned p$\sp{67}$ gene was translated in vitro. The product had a molecular weight of 65 kDa on SDS-PAGE and was immunoprecipitated by anti-p$\sp{67}$ polyclonal antibodies. This 65 kDa polypeptide interacted in vitro with eIF-2.