CLONING AND CHARACTERIZATION OF CDNAS ENCODING THE EPSILON-SUBUNIT OFEUKARYOTIC INITIATION FACTOR-2B FROM RABBIT AND HUMAN

A rabbit reticulocyte lysate cDNA library was screened with a polyclon al antiserum directed against eukaryotic initiation factor eIF-2B (eIF -2B), A 2508 base pair cDNA (pA1) was isolated and determined to encod e the epsilon-subunit of eIF-2B based on the immunoreactivity of the f usion protein expressed from the cDNA in Escherichia coli and the pres ence of two peptide sequences obtained from two V8 fragments of purifi ed nonrecombinant eIF-2B epsilon in the deduced amino acid sequence of the cDNA. The open reading frame of the cDNA began with the third nuc leotide of the cDNA with the first AUG codon at nucleotide 522. Mutati onal analysis of pA1 indicated that the cDNA did not code for full-len gth eIF-2B epsilon. Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2B epsilon mRNA were obt ained by 5' RACE. A human eIF-2B epsilon cDNA fragment, which correspo nded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma ( U-937) cell cDNA library constructed in lambda gt10. The nucleotide an d amino acid sequences were highly conserved between the rabbit and hu man cDNAs, showing approx. 90% sequence identity within the open readi ng frame. Northern and Western blot analyses of reticulocyte lysate an d other rabbit tissue extracts indicated that the eIF-2B epsilon polyp eptide has a similar apparent molecular weight in all tissues examined , and is coded for by a single similar to 2.8 kilobase mRNA species wh ich is ubiquitously expressed.