CLONING AND EXPRESSION OF CDNAS FOR THE BETA-SUBUNIT OF EUKARYOTIC INITIATION FACTOR-2B, THE GUANINE-NUCLEOTIDE EXCHANGE FACTOR FOR EUKARYOTIC INITIATION FACTOR-II

A key control point in the initiation of protein synthesis in mammalia n cells is the recycling of eukaryotic initiation factor (eIF)-2 by th e guanine nucleotide exchange factor eIF-2B. In mammalian cells, eIF-2 B is a complex of five different subunits termed epsilon, delta,gamma, beta and alpha. To clone cDNAs for the beta subunit of rabbit eIF-2B, amino acid sequence data was first obtained and used to design redund ant oligonucleotide primers for use in PCR. PCR products were used to screen a rabbit liver cDNA library in lambda gt11 to obtain full-lengt h cDNAs for eIF-2B beta. The cDNAs were sequenced completely on both s trands and revealed an open reading frame encoding a predicted 351-ami no acid polypeptide of 39.0 kDa. The molecular mass and pI (5.99) of t he predicted protein agree well with the properties of eIF-2B beta pur ified from rabbit reticulocytes. In vitro transcription/translation of the cDNAs gave rise to a product that migrated at a position indistin guishable from that of this subunit of the purified protein. The amino acid sequence shows a high degree of similarity to that of GCD7, a Sa ccharomyces cerevisiae protein thought to be equivalent to mammalian e IF-2B beta. Northern-blot analysis revealed a single major mRNA specie s for eIF-2B beta in each of the four rabbit tissues tested.