CLONING AND CHARACTERIZATION OF COMPLEMENTARY-DNA ENCODING THE EUKARYOTIC INITIATION FACTOR-2-ASSOCIATED 67-KDA PROTEIN (P(67))

The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa glycoprot ein (p67) protects eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, such as heme-regulated inhibitor and double-strande d RNA-activated inhibitor. This promotes protein synthesis in the pres ence 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, t he primary structure of rat p67 is determined by cDNA cloning. Based o n the partial amino acid sequences of overlapping tryptic and cyanogen bromide cleaved fragments, degenerate oligonucleotides were synthesiz ed and used as primers for the polymerase chain reaction to amplify th e corresponding p67 cDNA fragment from rat liver first strand cDNA. Th e amplified DNA was then used as a probe to screen a rat tumor hepatom a (KRC-7) cDNA library, and a positive clone covering the entire codin g region was obtained. From the cDNA sequence, an open reading frame t hat encodes p67 as a 480-amino acid protein with a molecular mass of 5 3 kilodaltons was predicted for the unglycosylated protein. The cloned cDNA was further characterized by in vitro transcription-coupled tran slation in micrococcal nuclease-treated reticulocyte lysate. The trans lated product migrated similarly to p67 in SDS-polyacrylamide gel elec trophoresis and was precipitated with antibodies against p67. Northern blot analysis of rat liver poly(A)+ RNA showed a single size class (a pproximately 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.