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A functional study of genes essential for autogamy and nuclear reorganization in Paramecium
Get PDFLike all ciliates, Paramecium tetraurelia is a unicellular eukaryote that harbors two kinds of nuclei within its cytoplasm. At each sexual cycle, a new somatic macronucleus (MAC) develops from the germline micronucleus (MIC) through a sequence of complex events, which includes meiosis, karyogamy and assembly of the MAC genome from MIC sequences. The latter process involves developmentally programmed genome rearrangements controlled by non-coding RNAs and a specialized RNA interference machinery. We describe our first attempts to identify genes and biological processes that contribute to the progression of the sexual cycle. Given the high percentage of unknown genes annotated in the P. tetraurelia genome, we applied a global strategy to monitor gene expression profiles during autogamy, a self-fertilization process. We focused this pilot study on the genes carried by the largest somatic chromosome and designed dedicated DNA arrays covering 484 genes from this chromosome (1.2% of all genes annotated in the genome). Transcriptome analysis revealed four major patterns of gene expression, including two successive waves of gene induction. Functional analysis of 15 up-regulated genes revealed four that are essential for vegetative growth, one of which is involved in the maintenance of MAC integrity and another in cell division or membrane trafficking. Two additional genes, encoding a MIC-specific protein and a putative RNA helicase localizing to the old, then to the new MAC, are specifically required during sexual processes. Our work provides a proof of principle that genes essential for meiosis and nuclear reorganization can be uncovered following genome-wide transcriptome analysis
SNF1-RELATED PROTEIN KINASES 2 ARE NEGATIVELY REGULATED BY A PLANT-SPECIFIC CALCIUM SENSOR
Get PDFSNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb3+ as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration was 2.5±0.9x105 M-1. The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor which we named SCS (SnRK2-interacting Calcium Sensor). Our results suggest that SCS is involved in response to ABA during seed germination most probably by a negative regulation of SnRK2s activity
Dynamical insight into Caenorhabditis elegans eIF4E recognition specificity for mono-and trimethylated structures of mRNA 5′ cap
Get PDFAbstract Specific recognition and binding of the ribonucleic
acid 5′ termini (mRNA 5′ cap) by the eukaryotic translation initiation factor 4E (eIF4E) is a key, rate limiting
step in translation initiation. Contrary to mammalian and
yeast eIF4Es that discriminate in favor of 7-methylguanosine
cap, three out of five eIF4E isoforms from the nematode
Caenorhabditis elegans as well as eIF4Es from the parasites
Schistosome mansoni and Ascaris suum, exhibit dual binding
specificity for both 7-methylguanosine-and N2,N2,7-trimethylguanosine cap. To address the problem of the differences in the mechanism of the cap recognition by those highly homologic proteins, we carried out molecular dynamics simulations in water of three factors, IFE-3 and IFE-5 isoforms from C. elegans and murine eIF4E, in the apo form as well as in the complexes with 7-methyl-GDP and N2,
N2,7-trimethyl-GDP. The results clearly pointed to a dynamical mechanism of discrimination between each type of the cap, viz. differences in mobility of the loops located at the entrance into the protein binding pockets during the cap association and dissociation. Additionally, our data showed that the hydrogen bond involving the N2-amino group of 7-methylguanosine and the carboxylate of glutamic acid was not stable. The dynamic mechanism proposed here differs
from a typical, static one in that the differences in the
protein-ligand binding specificity cannot be ascribed to
formation and/or disruption of well defined stabilizing
contacts
Cyclic enkephalin-deltorphin hybrids containing a carbonyl bridge: structure and opioid activity
Get PDFSix hybrid N-ureidoethylamides of octapeptides in which
an N-terminal cyclic structure related to enkephalin was
elongated by a C-terminal fragment of deltorphin were
synthesized on MBHA resin. The synthetic procedure involved
deprotection of Boc groups with HCl/dioxane and
cleavage of the peptide resin with 45 % TFA in DCM. d-
Lys and d-Orn were incorporated in position 2, and Lys,
Orn, Dab, or Dap in position 5. The side chains of the
dibasic amino function were protected with the Fmoc
group. This protection was removed by treatment with
55 % piperidine in DMF, and cyclization was achieved
by treatment with bis-(4-nitrophenyl)carbonate. Using
various combinations of dibasic amino acids, peptides
containing a 17-, 18-, 19- or 20-membered ring structure
were obtained. The peptides were tested in the
guinea-pig ileum (GPI) and mouse vas deferens (MVD)
assays. Diverse opioid activities were observed, depending
on the size of the ring. Extension of the enkephalin
sequence at the C-terminus by a deltorphin fragment resulted
in a change of receptor selectivity in favor of the
δ receptor. The conformational propensities of selected
peptides were determined using the EDMC method in
conjunction with data derived from NMR experiments
carried out in water. This approach allowed proper examination of the dynamical behavior of these small peptides. The results were compared with those obtained
earlier with corresponding N-(ureidoethyl)pentapeptide
amides
Extending the aerolysin family: from bacteria to vertebrates.
Get PDFA number of bacterial virulence factors have been observed to adopt structures similar to that of aerolysin, the principal toxin of Aeromonas species. However, a comprehensive description of architecture and structure of the aerolysin-like superfamily has not been determined. In this study, we define a more compact aerolysin-like domain - or aerolysin fold - and show that this domain is far more widely spread than anticipated since it can be found throughout kingdoms. The aerolysin-fold could be found in very diverse domain and functional contexts, although a toxic function could often be assigned. Due to this diversity, the borders of the superfamily could not be set on a sequence level. As a border-defining member, we therefore chose pXO2-60 - a protein from the pathogenic pXO2 plasmid of Bacillus anthracis. This fascinating protein, which harbors a unique ubiquitin-like fold domain at the C-terminus of the aerolysin-domain, nicely illustrates the diversity of the superfamily. Its putative role in the virulence of B. anthracis and its three dimensional model are discussed
Rhythmic diel pattern of gene expression in juvenile maize leaf.
Get PDFCo-expressed genes belonging to individual clusters are likely to be regulated by common mechanisms. The nocturnal phase of the diurnal cycle involves gross induction of fundamental biochemical processes and should be studied more thoroughly than was appreciated in most earlier physiological studies. Although some general mechanisms responsible for the diel regulation of gene expression might be shared among plants, details of the diurnal regulation of gene expression seem to differ between taxa
Novel mutations reveal two important regions in Aspergillus nidulans transcriptional activator MetR
Get PDFExpression of the sulfur assimilation pathway in Aspergillus nidulans is under control of sulfur metabolite repression, which is composed of scon genes encoding subunits of ubiquitin ligase and the metR gene coding for a transcriptional activator. In this paper we report three dominant suppressors of methionine requirement isolated from a metB3 diploid strain. All three mutations lead to the substitution of phenylalanine 48 by serine or leucine in the conserved N-terminal region of the MetR protein. Strains carrying the dominant suppressor mutations exhibit increased activities of homocysteine synthase and sulfur assimilation enzymes as well as elevated levels of the corresponding transcripts. These changes are observed even under conditions of methionine repression, which suggests that the mutated MetR protein may be resistant to inactivation or degradation mediated by sulfur metabolite repression. We also found that a mutant impaired in sulfite reductase activity, known until now as sG8, has a frameshift which changes 41 C-terminal amino acids. Therefore, it is now designated metR18. This mutant has elevated levels of MetR-regulated transcripts and of activities of sulfur assimilation enzymes (except sulfite reductase), which can be repressed to the wild type level by exogenous methionine. Thus, metR18 and the three dominant suppressors represent new types of mutations affecting different parts of the A. nidulans MetR protein
Effect of Medicago sativa Mhb1gene expression on defense response of Arabidopsis thaliana plants
Get PDFBesides the previously described nitric oxide-detoxification activity we identified new features of class-1 non-symbiotic hemoglobin from Medicago sativa (Mhb1). Under in vitro conditions, using peroxidase in-gel activity assay, the Mhb1 protein was shown to possess also peroxidase-like activity. Due to this activity, in the presence of nitrite and hydrogen peroxide, the protein can mediate autonitration and nitration of other proteins at tyrosine residues, as revealed by tandem mass spectrometry and immune assay approaches. Mhb1 through its multifunctional activities can affect different components of signal transduction cascades operating during plant response to infections. This influence is manifested by Mhb1-mediated selective up-regulation of expression of certain pathogen inducible genes in Pseudomonas syringae infected Arabidopsis thaliana plants which overproduce Mhb1, as revealed by reverse transcription–quantitative real-time PCR analysis. Changes in expression level of these genes can influence such processes as synthesis of secondary metabolites, protein degradation and biosynthesis of ethylene. They can also result in alteration of pathogen-induced defense response of Mhb1 transgenic plants
Proofreading deficiency of Pol I increases the levels of spontaneous rpoB mutations in E. coli
Get PDFThe fidelity role of DNA polymerase I in chromosomal DNA replication in E. coli was investigated using the rpoB forward target. These experiments indicated that in a strain carrying a proofreading-exonuclease-defective form of Pol I (polAexo mutant) the frequency of rpoB mutations increased by about 2-fold, consistent with a model that the fidelity of DNA polymerase I is important in controlling the overall fidelity of chromosomal DNA replication. DNA sequencing of rpoB mutants revealed that the Pol I exonuclease deficiency lead to an increase in a variety of base-substitution mutations. A polAexo mutator effect was also observed in strains defective in DNA mismatch repair and carrying the dnaE915 antimutator allele. Overall, the data are consistent with a proposed role of Pol I in the faithful completion of Okazaki fragment gaps at the replication fork
NHE3 modulates the severity of colitis in IL-10-deficient mice.
No full textNHE3, the major intestinal Na(+)/H(+) exchanger, was shown to be downregulated and/or inhibited in patients with inflammatory bowel disease (IBD), a phenomenon believed to contribute to inflammation-associated diarrhea. NHE3(-/-) mice spontaneously develop colitis and demonstrate high susceptibility to dextran sulfate-induced mucosal injury. We investigated the effects of NHE3 deficiency on the development of chronic colitis in an IL-10 knockout (KO) mouse model of Crohn's disease. NHE3(-/-) mice were first backcrossed to 129/SvEv mice for >10 generations, with no apparent changes in their survival or phenotype. These mice were crossed with IL-10(-/-) mice on the same genetic background, and the phenotypes of 10-wk-old wild-type (WT), IL-10(-/-), NHE3(-/-), and IL-10(-/-)/NHE3(-/-) (double-KO) mice were studied. Histological and immunohistochemical examination of the colon established important architectural alterations, including increased neutrophilic and mononuclear cell infiltration in double- compared with single-KO mice. Double-KO mice demonstrated increased colonic expression of neutrophil collagenase matrix metalloproteinase-8 and the chemokines macrophage inflammatory protein-2, CXCL1, CXCL10, and CXCL11. Colonic IFNγ, IL-17, and IL-12/23 p40 protein secretion was significantly increased in double- compared with single-KO mice. IL-10(-/-)/NHE3(-/-) mouse colonic epithelium exhibited increased hallmarks of apoptosis, including a significantly increased number of cleaved caspase-3-positive surface epithelial cells. These results highlight the importance of NHE3 in the maintenance of intestinal barrier integrity and in modulating the inflammatory process in IL-10-deficient mice. Chronic NHE3 inhibition or underexpression observed in IBD may therefore contribute to the pathogenesis of IBD by influencing the extent of the epithelial barrier defect and affect the ultimate degree of inflammation