Conserved sequences in the beta subunit of archaeal and eukaryal translation initiation factor 2 (eIF2), absent from eIF5, mediate interaction with eIF2gamma.

The eukaryotic translation initiation factor 2 (eIF2) binds the methionyl-initiator tRNA in a GTP-dependent mode. This complex associates with the 40 S ribosomal particle, which then, with the aid of other factors, binds to the 5' end of the mRNA and migrates to the first AUG codon, where eIF5 promotes GTP hydrolysis, followed by the formation of the 80 S ribosome. Here we provide a comparative sequence analysis of the beta subunit of eIF2 and its archaeal counterpart (aIF2beta). aIF2beta differs from eIF2beta in not possessing an N-terminal extension implicated in binding RNA, eIF5 and eIF2B. The remaining sequences are highly conserved, and are shared with eIF5. Previously isolated mutations in the yeast eIF2beta, which allow initiation of translation at UUG codons due to the uncovering of an intrinsic GTPase activity in eIF2, involve residues that are conserved in aIF2beta, but not in eIF5. We show that the sequence of eIF2beta homologous to aIF2beta is sufficient for binding eIF2gamma, the only subunit with which it interacts, and comprises, at the most, 78 residues. eIF5 does not interact with eIF2gamma, despite its similarity with eIF2beta, probably because of a gap in homology in this region. These observations have implications for the evolution of the mechanism of translation initiation.

A Plasmodium vivax vaccine candidate displays limited allele polymorphism, which does not restrict recognition by antibodies.

BACKGROUND: The 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)) has been suggested as candidate for part of a subunit vaccine against malaria. A major concern in vaccine development is the polymorphism observed in different plasmodial strains. The present study examined the extension and immunological relevance of the allelic polymorphism of the MSP1(19) from Plasmodium vivax, a major human malaria parasite. MATERIALS AND METHODS: We cloned and sequenced 88 gene fragments representing the MSP1(19) from 28 Brazilian isolates of P. vivax. Subsequently, we evaluated the reactivity of rabbit polyclonal antibodies, a monoclonal antibody, and a panel of 80 human sera to bacterial and yeast recombinant proteins representing the two allelic forms of P. vivax MSP1(19) described thus far. RESULTS: We observed that DNA sequences encoding MSP1(19) were not as variable as the equivalent region of other species of Plasmodium, being conserved among Brazilian isolates of P. vivax. Also, we found that antibodies are directed mainly to conserved epitopes present in both allelic forms of the protein. CONCLUSIONS: Our findings suggest that the use of MSP1(19) as part of a subunit vaccine against P. vivax might be greatly facilitated by the limited genetic polymorphism and predominant recognition of conserved epitopes by antibodies.

Adhesion of Escherichia coli to HeLa cells mediated by Trypanosoma cruzi surface glycoprotein-derived peptides inserted in the outer membrane protein LamB.

Peptides derived from the surface glycoprotein gp82 of Trypanosoma cruzi, previously implicated in the parasite's invasion of host cells, were expressed as fusions to the protein LamB of Escherichia coli in a region known to be exposed on the cell surface. Bacteria expressing these proteins adhered to HeLa cells in a manner that mimics the pattern of parasite invasion of mammalian cells. Purified LamB fusion proteins were shown to bind to HeLa cells and to inhibit infection by T. cruzi, supporting the notion that these gp82-derived peptides can mediate interaction of the parasite with its host.

The beta subunit of eukaryotic translation initiation factor 2 binds mRNA through the lysine repeats and a region comprising the C2-C2 motif.

Eukaryotic translation initiation factor 2 (eIF2) has been implicated in the selection of the AUG codon as the start site for eukaryotic translation initiation, since mutations in its three subunits in yeast that allow the recognition of a UUG codon by the anticodon of the initiator Met-tRNAMet have been identified. All such mutations in the beta subunit of eIF2 (eIF2beta) mapped to a region containing a putative zinc finger structure of the C2-C2 type, indicating that these sequences could be involved in RNA recognition. Another feature of eIF2beta that could mediate an interaction with RNA is located in the amino-terminal sequences and is composed of three repeats of seven lysine residues which are highly conserved in other species. We show here the ability of eIF2beta, purified from Escherichia coli as a fusion to glutathione S-transferase, to bind mRNA in vitro. Through a deletion analysis, mRNA binding was found to be dependent on the lysine repeats and a region encompassing the C2-C2 motif. Strong mRNA binding in vitro could be maintained by the presence of only one lysine or one arginine run but not one alanine run. We further show that only one run of lysine residues is sufficient for the in vivo function of eIF2beta, probably through charge interaction, since its replacement by arginines did not impair cell viability, whereas substitution for alanines resulted in inviable cells. mRNA binding, but not GTP-dependent initiator Met-tRNAMet binding, by the eIF2 complex was determined to be dependent on the presence of the lysine runs of the beta subunit.

A new tetracycline resistance determinant cloned from Proteus mirabilis.

The chromosomal inducible Tcr determinant from Proteus mirabilis was cloned and the nucleotide sequence of both the structural and repressor genes determined. The deduced amino acid sequence of the structural protein shows the highest similarity to TetA(H) from Pasteurella multocida (78.4%), followed by TetA(B) from Tn10 (50.9%). Based on this analysis, we suggest that this new determinant can be assigned to a new class, TetJ.

Mapping of B cell epitopes in an immunodominant antigen of Trypanosoma cruzi using fusions to the Escherichia coli LamB protein.

The JL8 protein antigen from Trypanosoma cruzi, a dominant immunogen in man, has been characterized as containing tandem amino acid repeats. Here, we describe the use of the LamB protein of Escherichia coli as a carrier of JL8 derived sequences in order to map the immunodominant B cell epitopes in this antigen. Five different sequences of JL8 were inserted in the LamB protein and the JL8-LamB fusion proteins were tested by ELISA with human chronic chagasic sera. The fusion carrying the sequence AEKQKAAEATKVAE was recognized by most sera. This protein was also capable of inhibiting the binding of human chagasic antibodies to GST-JL8 in competitive ELISA suggesting that it contains an immunodominant B cell epitope of JL8.

Mini-Mu insertions in the tetracycline resistance determinant from Proteus mirabilis.

The inducible tetracycline resistance determinant isolated from Proteus mirabilis cloned into the plasmid pACYC177 was mutagenized by insertion of a mini-Mu-lac phage in order to define the regions in the cloned sequences encoding the structural and regulatory proteins. Three different types of mutants were obtained: one lost the resistance phenotype and became Lac+; another expressed the resistance at lower levels and constitutively; the third was still dependent on induction but showed a lower minimal inhibitory concentration. The mutant phenotypes and the locations of the insertions indicate that the determinant is composed of a repressor gene and a structural gene which are not transcribed divergently as are other known tetracycline determinants isolated from Gram-negative bacteria.

Mini-Mu insertions in the tetracycline resistence determinant from proteus mirabilis

The inducible tetracycline resistance determinant isolated from Proteus mirabilis cloned into the plasmid pACYC177 was mutagenized by insertion of a mini-Mu-lac phage in order to define the regions in the cloned sequences encoding the structural and regulatory proteins. Three different types of mutants were obtained: one lost the resistance phenotype and became Lac+; another expressed the resistance at lowerlevels and constitutively; the third was still dependent on induction but showed a lower minimal inhibitory concentration. The mutant phenotypes and the locations of the insertions indicate that the determinant is composed of a repressor gene and a structural gene which are not transcribed divergently as are other known tetracycline determinants isolated from Gram-negative bacteria.

Specificity of mini-Mu bacteriophage insertions in a small plasmid.

Target site selection for bacteriophage Mu transposition was studied in pools of over 10(7) independent mini-Mu insertions in pUC9, selected by transduction of the plasmid. Insertions in both orientations were clustered in three regions and, within these, at preferred sites.

Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons.

Small bacteriophage Mu transposable elements containing the lac operon structural genes were constructed to facilitate the isolation and use of Mu insertions and lac gene fusions. These mini-Mu elements have selectable genes for either ampicillin or kanamycin resistance and can be used to form both transcriptional and translational lac gene fusions. Some of the mini-Mu-lac elements constructed are deleted for the Mu A and B transposition genes and form stable insertions that cannot undergo transposition unless complemented for these functions. A procedure was developed for selecting mini-Mu insertions specifically into plasmids, including commonly used high-copy-number cloning vectors such as pBR322. Mu insertions in pBR322 were found to be distributed around the plasmid, but insertions in certain regions occurred more frequently than in others.

In vivo DNA cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon.

A mini-Mu bacteriophage containing a high copy number plasmid replicon was constructed to clone genes in vivo. A chloramphenicol resistance gene for independent selection and the lacZYA operon to form gene fusions were also incorporated into this phage. This mini-Mu element can transpose at a high frequency when derepressed, and it can be complemented by a helper Mu prophage for lytic growth. DNA sequences that are flanked by two copies of this mini-Mu can be packaged along with them. After infection, homologous recombination can occur between the mini-Mu sequences, resulting in the formation of plasmids carrying the transduced sequences. lac operon fusions can be formed with promoters and translation initiation sites on the cloned sequences in the resulting plasmids. The utility of this system was demonstrated by cloning genes from eight different Escherichia coli operons and by identifying lac fusions to the regulated araBAD operon among clones selected for the nearby leu operon.