Initiation of translation in eukaryotes is mediated by a set of initiation factors. Mammalian initiation factor 3 is composed of at least 8 subunits, with the largest being about 180 kDa in size. Here we report the cloning of the p180 subunit of human eukaryotic translation initiation factor (eIF) 3. The amino acid sequence deduced from the cDNA agrees with the sequences of CNBr fragments of eIF-3, confirming the identity of the clone. The 1382 amino acid open reading frame contains a high percentage of charged residues (48%) and an unusual repetitive domain near the carboxyl terminus composed of 25 repeats of 10 amino acids each. Data base searches identified related sequences found in members of the plant and fungal kingdoms as well as in other mammals and the nematode Caenorhabditis elegans. These sequences share significant identity with the human clone and probably represent the homologues of the p180 subunit in these organisms. This is the first report identifying the sequence of the large subunit of eIF-3. Eukaryotic translation initiation factor (eIF)1 3 is the largest of the protein synthesis initiation factors, with a size of about 650 kDa. eIF-3 purified from rabbit reticulocyte lysate consists of at least eight individual polypeptide chains (1, 2). Originally, eIF-3 was identified as a factor that binds to the 40 S ribosomal subunit and thereby prevents the association of the 40 and 60 S subunits with one another. This results in a pool of 40 S subunits, which are then able to participate in the initiation process.However, eIF-3 has also been implicated in a number of additional roles. One example is the association of eIF-3 with eIF-4F, where the interaction is sufficiently stable such that 0.5 M KCl is required to separate the two initiation factors (3). eIF-3 interacts with a number of initiation factors in addition to eIF-4F. This suggests that eIF-3 may be the major protein which aligns the factors so that the mRNA is correctly positioned for initial binding to the 40 S subunit and the subsequent identification of the initiating AUG.Of the many associations of eIF-3 with other translational components, most appear to be via the p180 subunit. The ability of eIF-3 to bind to and stabilize the ternary complex (eIF-2⅐GTP⅐Met-tRNA i ) is dependent on the p180 subunit, since preparations that have been depleted of p180 fail to promote the formation of the ternary complex (4). The p180 subunit of eIF-3 has also been shown to interact with eIF-4B using "Far Western" blotting. 2 As noted above, eIF-3 interacts with eIF-4F. The site of interaction between eIF-3/eIF-4F has been mapped to the middle region (amino acids 480 -886) of the ␥ subunit of eIF-4F (5), but the binding site in eIF-3 has not yet been identified. The limited biochemical evidence indicates that the p180 subunit is important for all of these interactions and processes.Many mammalian factors can replace their yeast counterparts in vivo (1), suggesting extensive homology between the factors. For eIF-3, the only detailed comparison that can...

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BruceUa abortus proteins from virulent S2308 expressed from a pBluescript II SK-genomic library stimulated peripheral blood mononuclear (PBM) cell proliferation from cattle vaccinated with B. abortus S19. The method described here permits a rapid and directed approach to isolate genes encoding antigens of B. abortus that interact with lymphocytes primed to the living bacterium. The supernatants from the bacterial host JM109 (DE3) were cultured with freshly isolated bovine PBM cells. A total of 300 clones were evaluated. Ten clones were identified that stimulated T-lymphocyte proliferation. Among them, one clone with a 2.5-kb insert stimulated T-lymphocyte proliferation in all three animals, suggesting that the proteins encoded by genes within this fragment may represent immunodominant antigens. DNA sequencing of this clone reveals two large open reading frames (ORFs). ORF II has a high degree of similarity to the Escherichia coli ssb gene, which codes for the single-stranded DNA binding protein. ORF I, in the opposite direction to ORF II, shows similarity to the N terminus of the E. coli uvrA gene, which codes for one of the three subunits of the E. coli ABC excision nuclease. The observation that the PBM cells recognized and proliferated in response to proteins expressed from single clones provides a novel strategy to select bacterial antigens that may prove useful in designing alternative vaccines against brucellosis.B. abortus genomic library. The library of B. abortus S2308, a virulent strain of B. abortus, was constructed in the HindIII restriction site of the pBluescript II SK-phagemid. 5339 on July 16, 2020 by guest http://iai.asm.org/ Downloaded from