ABSTRACT
Formation of the initiation translation complex containing the three initiation factors, IF1, IF2, and IF3, tRNA(fMet), and GTP constitutes the earliest event in the protein synthesis. IF2, a GTP-binding protein, is the principal factor involved in selecting and binding fMet-tRNA(fMet) to the 30 S ribosomal subunit. Although some chloroplast initiation translational factors have been identified and purified from algae, none of these factors have been characterized from plants. In this work, we report the molecular characterization of a nuclear-encoded chloroplastic IF2 gene from common bean (PvIF2cp). We show that the PvIF2cp gene encodes a protein containing a chloroplast translocation signal peptide, able to target a green fluorescent protein fusion protein to chloroplasts. A high accumulation of PvIF2cp transcript was found in photosynthetic tissues, whereas low mRNA levels were detected in etiolated plants and in nonphotosynthetic organs. Additional data indicate that the PvIF2cp transcript accumulation is modulated by light. The PvIF2cp gene encodes a functional factor, since the PvIF2cp conserved region, containing the G-domain and the C-terminal end, complements an Escherichia coli infB null mutation. Phylogenetic analysis using the PvIF2cp conserved region suggests that the PvIF2cp gene originated via endosymbiotic gene transfer to the nucleus and that it may be a useful marker for phylogeny reconstruction.
Subject(s)
Cell Nucleus/metabolism , Chloroplasts/metabolism , Escherichia coli/metabolism , Mutation , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/genetics , Protein Biosynthesis , Active Transport, Cell Nucleus , Amino Acid Sequence , Biological Transport , Blotting, Northern , Cloning, Molecular , DNA, Complementary/metabolism , Gene Library , Genes, Plant , Genetic Complementation Test , Genetic Markers , Green Fluorescent Proteins , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Models, Genetic , Molecular Sequence Data , Photosynthesis/genetics , Phylogeny , Plants, Toxic , Prokaryotic Initiation Factor-2 , Protein Structure, Tertiary , RNA, Messenger/metabolism , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNA , Time Factors , Tissue Distribution , Nicotiana/geneticsABSTRACT
Conducting intervention research with culturally diverse, underserved, and often hard to reach populations in naturalistic or field settings presents investigators with a number of practical challenges. This article describes four special challenges and strategies for dealing with them that clients, service providers, and researchers experienced in conducting a prevention intervention to reduce substance use and sexual risky behaviors with low-income Latina young women. The challenges are (a) building community partnerships; (b) developing interventions that are acceptable and relevant; (c) promoting successful recruitment, participation, and retention of participants; and (d) developing a diverse, cohesive, and committed research team and effective managerial information support systems.
Subject(s)
Cultural Diversity , Nursing Research , Female , Humans , MaleSubject(s)
Adolescent Health Services/organization & administration , Health Promotion , Hispanic or Latino , Women's Health , Adolescent , Female , Humans , Income , Male , Pregnancy , Pregnancy in Adolescence , Risk-Taking , Sexual Behavior , Substance-Related Disorders/prevention & control , United StatesABSTRACT
The mature 19-amino acid STa heat-stable enterotoxin of E. coli has a preceding peptide of 53 amino acids which contains two domains called Pre (aa 1-19) and Pro (aa 20-53) sequences, proposed to be essential for extracellular toxin release by this host. The Pro sequence, however, has been proven not be indispensable for this process since Pro deletion mutants secrete STa. To find out if Pre and/or other unremoved natural STa flanking sequences are responsible for toxin secretion in those mutants we genetically fused mature STa directly to the leader peptide of the periplasmic E. coli heat-labile enterotoxin B-subunit (LTB). Expression of this gene fusion resulted in extracellular secretion of biologically active STa by E. coli independently of natural STa neighboring genetic sequences. Moreover, these results suggest that STa might be able to gain access to the extracellular milieu simply upon its entry into the E. coli periplasm once guided into this compartment by the LTB leader peptide. To test if extracellular secretion in this fashion might be extended to other disulfide bond-rich small peptides, the 13 amino acid conotoxin GI and a non-enterotoxic STa-related decapeptide were cloned. None of the two peptides was found in culture supernatants, in spite of high structural homology to the toxin. Failure to be secreted most likely leads to degradation as peptides were also not detected in bacterial sonicates. We hypothesize that cysteine-rich peptides must have an amino acid length and/or number of disulfide bridges closer to those in STa for them to follow this toxin secretory pathway in E. coli.
Subject(s)
Bacterial Toxins/metabolism , Conotoxins , Enterotoxins/metabolism , Escherichia coli/metabolism , Protein Sorting Signals/metabolism , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial , Escherichia coli Proteins , Molecular Sequence Data , Mollusk Venoms/metabolism , Peptides, Cyclic/genetics , Plasmids , Protein Sorting Signals/geneticsABSTRACT
In this paper a new approach to create antigens through genetic engineering is discussed. In this particular case the subunits of V. cholerae toxin are used as heterologous epitope carries. In this paper the manipulation of A and B subunits is described. This manipulation allows both the insertion of epitopes to the B subunit and the use of subunit A in the construction of recombinant antigens similar to the ones derived from subunit B.
