Stm1p alters the ribosome association of eukaryotic elongation factor 3 and affects translation elongation

Stm1p is a Saccharomyces cerevisiae protein that is primarily associated with cytosolic 80S ribosomes and polysomes. Several lines of evidence suggest that Stm1p plays a role in translation under nutrient stress conditions, although its mechanism of action is not yet known. In this study, we show that yeast lacking Stm1p (stm1Δ) are hypersensitive to the translation inhibitor anisomycin, which affects the peptidyl transferase reaction in translation elongation, but show little hypersensitivity to other translation inhibitors such as paromomycin and hygromycin B, which affect translation fidelity. Ribosomes isolated from stm1Δ yeast have intrinsically elevated levels of eukaryotic elongation factor 3 (eEF3) associated with them. Overexpression of eEF3 in cells lacking Stm1p results in a growth defect phenotype and increased anisomycin sensitivity. In addition, ribosomes with increased levels of Stm1p exhibit decreased association with eEF3. Taken together, our data indicate that Stm1p plays a complementary role to eEF3 in translation.     

Positional effects of monofluorinated phenylalanines on histone acetyltransferase stability and activity

To explore the impact of global incorporation of fluorinated aromatic amino acids on protein function, we investigated the effects of three monofluorinated phenylalanine analogs para-fluorophenylalanine (pFF), meta-fluorophenylalanine (mFF), and ortho-fluorophenylalanine (oFF) on the stability and enzymatic activity of the histone acetyltransferase (HAT), tGCN5. We selected this set of fluorinated amino acids because they bear the same size and overall polarity but alter in side chain shape and dipole direction. Our experiments showed that among three fluorinated amino acids, the global incorporation of pFF affords the smallest perturbation to the structure and function of tGCN5.     

BchY-Based Degenerate Primers Target All Types of Anoxygenic Photosynthetic Bacteria in a Single PCR

To detect anoxygenic bacteria containing either type 1 or type 2 photosynthetic reaction centers in a single PCR, we designed a degenerate primer set based on the bchY gene. The new primers were validated in silico using the GenBank nucleotide database as well as by PCR on pure strains and environmental DNA.Anoxygenic photosynthetic bacteria are diverse and important members of microbial communities (11, 13, 17, 20). There are five bacterial phyla containing anoxygenic phototrophs: Proteobacteria (purple bacteria), Chlorobi (green sulfur bacteria), Chloroflexi (green nonsulfur bacteria), Acidobacteria (“Candidatus Chloracidobacterium thermophilum” [7]), and Firmicutes (heliobacteria). While Heliobacterium modesticaldum, Chlorobi, and “Ca. Chloracidobacterium thermophilum” have a type 1 reaction center (RC1) similar to photosystem I in Cyanobacteria and higher plants, Chloroflexi and Proteobacteria possess a type 2 reaction center (RC2) similar to photosystem II of oxygenic phototrophs (7, 16).Primers based on pufM, the gene encoding the M subunit of RC2, have been widely used to detect phototrophic purple bacteria (1, 4, 12, 19). However, phototrophic bacteria that do not possess RC2 are not retrieved when pufM is used as the target. Achenbach and coworkers (1) developed primers targeting rRNA genes of Chlorobi, Chloroflexi, and heliobacteria, while Alexander and coworkers (2) have developed primers to specifically detect green sulfur bacteria (Chlorobi) by using 16S rRNA and fmoA as gene targets and applied these primers in environmental studies (3). No currently available primer set can simultaneously target phototrophs containing either RC1 or RC2.Since it is well established that both RC1- and RC2-containing anoxygenic phototrophs synthesize bacteriochlorophylls (BChls), we searched for a universal anoxygenic photosynthesis gene marker among all enzymes involved in BChl biosynthetic pathways. All known pathways for chlorophyll and BChl biosynthesis branch from the heme biosynthesis pathway at protoporphyrin IX and continue to chlorophyllide a (Chlide a) through the same intermediates (9). Chlide a is the branching point that separates chlorophyll and BChl biosynthetic pathways. Moreover, pathways for the synthesis of different BChls are also split at this stage: chlorophyllide oxidoreductase converts Chlide a to 3-vinyl-bacteriophyllide a, which is the precursor for BChls a, b, and g, while a yet unknown enzyme reduces Chlide a to 3-vinyl-bacteriophyllide d, a precursor for antenna BChls c, d, and e in Chlorobium spp. (9). Since 3-vinyl-bacteriophyllide a is the last common intermediate in the synthesis of BChl a and BChl g, and the latter is the only BChl in heliobacteria (14, 15), chlorophyllide oxidoreductase is the only enzyme that is (i) present in anoxygenic phototrophic bacteria and not in oxygenic phototrophs and (ii) common to all known anoxygenic phototrophic bacterial species (with the exception of “Ca. Chloracidobacterium thermophilum,” where the pathway for BChl synthesis is not yet known). Analyzing multiple alignments of the subunits of chlorophyllide oxidoreductase, we found that only the Y subunit (encoded by the BchY gene) had two conserved regions distinguishing this protein from its closest homologs; therefore, the bchY gene was chosen as a universal marker for anoxygenic photosynthesis.Due to likely codon variations coding identical amino acid sequences in different genomes (19), degenerate BchY primers were designed by reverse translation of two conserved regions of the BchY alignment (Fig. ​(Fig.1):1): bchY_fwd (5′-CCNCARACNATGTGYCCNGCNTTYGG-3′ [26 bases; 2,048 variants; corresponding amino acid sequence, PQTMCPAFG]) and bchY_rev (5′-GGRTCNRCNGGRAANATYTCNCC-3′ [23 bases; 4,096 variants; corresponding amino acid sequence, GE{I/M}FP{A/ V}DP]). Each primer had no more than two bases deviating from known bchY sequences in the GenBank nr database (except for H. modesticaldum) as well as to environmental BchY variants in the GenBank env_nr database. None of these deviations were located in the 3′ ends of the primers (see Tables S2 and S3 in the supplemental material). These primers, therefore, were predicted to amplify a wide diversity of bchY genes under nonstringent PCR conditions (50 to 52°C annealing temperature). The lengths of the expected PCR products were either 480 bp (for green sulfur, green nonsulfur bacteria, and heliobacteria) or 510 bp (for purple bacteria).Open in a separate windowFIG. 1.Multiple-amino-acid alignment of BchY proteins. Sequence abbreviations: R.den, Roseobacter denitrificans (gi|110677524); R.gel, Rubrivivax gelatinosus (gi|29893484); R.cap, Rhodobacter capsulatus (gi|114868); C.lit, Congregibacter litoralis KT 71 (gi|88706663); H.hal, Halorhodospira halophila (gi|121998388); C.aur, Chloroflexus aurantiacus (gi|163849328); C.tep, Chlorobium tepidum (gi|66576270); and H.mod, Heliobacterium modesticaldum (gi|167629410).In order to check primer specificity in silico, a screening procedure was developed. Putative primer sites (tags) for both the bchY_fwd and the bchY_rev primers were gathered from the GenBank nucleotide collection (nt) by BLAST with relaxed search conditions; the tags having mismatches at the 3′ end or more than five overall mismatches from their primer were filtered out, and the remaining tags were mapped to their sequences mimicking PCR primer annealing. Fragments ranging from 300 to 700 bp (virtual “PCR products”) were retrieved from GenBank and annotated (see Table S4 in the supplemental material). All bchY genes present in the GenBank nt database were virtually “amplified,” pointing to the robustness of the primers and our in silico PCR analysis. On the other hand, all nonspecific “amplicons” have major deviations from the primer sequences and would likely not be amplified by a real PCR. The same screening procedure was performed against the GenBank environmental nucleotide collection (env_nt) (see Table S5 in the supplemental material), and as in the case with the nt database, only bchY fragments were virtually “amplified.”The BchY primer set was validated using five key control organisms, including the RC2-containing the purple sulfur bacterium Allochromatium vinosum and the purple nonsulfur bacterium Rhodobacter capsulatus as well as the RC1-containing green sulfur bacterium Chlorobium limicola, green nonsulfur bacterium Chloroflexus aurantiacus, and the heliobacterium H. modesticaldum. Amplifications yielded the predicted products of 510 bp from the purple bacteria and 480 bp from the green sulfur and nonsulfur bacteria and H. modesticaldum. Negative-control Escherichia coli and Synechocystis sp. strain PCC 6803 did not yield amplification products when the bchY primers were used.The designed BchY primer set successfully amplified bchY genes from DNA obtained from both marine (East Mediterranean Sea) and freshwater (Lake Kinneret) environments (see Table S6 in the supplemental material for best BLASTX hits for selected sequenced fragments). These habitats were chosen for testing due to the previously reported wide diversity of their anoxygenic phototrophs (8, 10, 18, 19). A phylogenetic tree of bchY gene fragments amplified from both freshwater and marine DNA samples is shown in Fig. ​Fig.22.Open in a separate windowFIG. 2.BchY phylogenetic tree based on a maximum likelihood tree to which short sequences were added by ARB parsimony. The branches that appeared on the original maximum likelihood tree are shown with thicker lines. Bootstrap values greater than 50% are indicated next to the branches. Sequences obtained in this study are shown in bold. For reasons of clarity, not all BchY sequences retrieved are shown in the tree. For cases in which a BchY fragment was found in more than three clones, the numbers of clones are given in parentheses. Clones m21_2 and m21_3 are identical to the bchY gene of Hoeflea phototrophica strain DFL-43 (6); the m20_2 clone was identical to the bchY gene of Dinoroseobacter shibae (5).Our study underlines the utility of the bchY gene as a molecular marker for revealing genetic heterogeneity in phototrophic microbial populations. Using both wide-scale bioinformatic analysis and PCR on control strains and naturally occurring microbial community DNA, we have confirmed the specificity and coverage of the proposed degenerate BchY primers.     

Two functional S100A4 monomers are necessary for regulating nonmuscle myosin-IIA and HCT116 cell invasion

S100A4, a member of the Ca(2+)-activated S100 protein family, regulates the motility and invasiveness of cancer cells. Moreover, high S100A4 expression levels correlate with poor patient survival in several cancers. Although biochemical, biophysical, and structural data indicate that S100A4 is a noncovalent dimer, it is unknown if two functional S100A4 monomers are required for the productive recognition of protein targets and the promotion of cell invasion. To address this question, we created covalently linked S100A4 dimers using a glycine rich flexible linker. The single-chain S100A4 (sc-S100A4) proteins exhibited wild-type affinities for calcium and nonmuscle myosin-IIA, retained the ability to regulate nonmuscle myosin-IIA assembly, and promoted tumor cell invasion when expressed in S100A4-deficient colon carcinoma cells. Mutation of the two calcium-binding EF-hands in one monomer, while leaving the other monomer intact, caused a 30-60-fold reduction in binding affinity for nonmuscle myosin-IIA concomitant with a weakened ability to regulate the monomer-polymer equilibrium of nonmuscle myosin-IIA. Moreover, sc-S100A4 proteins with one monomer deficient in calcium responsiveness did not support S100A4-mediated colon carcinoma cell invasion. Cross-linking and titration data indicate that the S100A4 dimer binds a single myosin-IIA target peptide. These data are consistent with a model in which a single peptide forms interactions in the vicinity of the canonical target binding cleft of each monomer in such a manner that both target binding sites are required for the efficient interaction with myosin-IIA.     

Silencing activity of 2'-O-methyl modified anti-MDR1 siRNAs with mismatches in the central part of the duplexes

The thermodynamic properties of siRNA duplexes are important for their silencing activity. siRNAs with high thermodynamic stability of both the central part of the duplex and in the whole, usually display low silencing activity. Destabilization of the central part of the siRNA duplex could increase its silencing activity. However, mismatches located in the central part of the duplex could substantially decrease the amount of RNAi efficacy, hindering active RISC formation and function. In this study, we examined the impact of duplex destabilization by nucleotide substitutions in the central part (7-10 nt counting from the 5'-end of the antisense strand) of the nuclease-resistant siRNA on its silencing activity.     

Dynamic lipid-protein stoichiometry on E1 and E2 conformations of the Na+/K+ -ATPase

Annular lipid-protein stoichiometry in native pig kidney Na+/K+ -ATPase preparation was studied by [125I]TID-PC/16 labeling. Our data indicate that the transmembrane domain of the Na+/K+ -ATPase in the E1 state is less exposed to the lipids than in E2, i.e., the conformational transitions are accompanied by changes in the number of annular lipids but not in the affinity of these lipids for the protein. The lipid-protein stoichiometry was 23 ± 2 (α subunit) and 5.0 ± 0.4 (β subunit) in the E1 conformation and 32 ± 2 (α subunit) and 7 ± 1 (β subunit) in the E2 conformation.     

Enhanced locomotion caused by loss of the Drosophila DEG/ENaC protein Pickpocket1

Coordination of rhythmic locomotion depends upon a precisely balanced interplay between central and peripheral control mechanisms. Although poorly understood, peripheral proprioceptive mechanosensory input is thought to provide information about body position for moment-to-moment modifications of central mechanisms mediating rhythmic motor output. Pickpocket1 (PPK1) is a Drosophila subunit of the epithelial sodium channel (ENaC) family displaying limited expression in multiple dendritic (md) sensory neurons tiling the larval body wall and a small number of bipolar neurons in the upper brain. ppk1 null mutant larvae had normal external touch sensation and md neuron morphology but displayed striking alterations in crawling behavior. Loss of PPK1 function caused an increase in crawling speed and an unusual straight path with decreased stops and turns relative to wild-type. This enhanced locomotion resulted from sustained peristaltic contraction wave cycling at higher frequency with a significant decrease in pause period between contraction cycles. The mutant phenotype was rescued by a wild-type PPK1 transgene and duplicated by expressing a ppk1RNAi transgene or a dominant-negative PPK1 isoform. These results demonstrate that the PPK1 channel plays an essential role in controlling rhythmic locomotion and provide a powerful genetic model system for further analysis of central and peripheral control mechanisms and their role in movement disorders.     

Amperometric biosensors for detection of phenol using chemically modified electrodes containing immobilized bacteria

Eight strains of Pseudomonas were studied for development of phenol sensor. The immobilization of cells was performed by absorbing them on the working part of mediator-modified screen-printed electrodes (SPEs). Only three Pseudomonas strains were able to transfer electrons resulting from specific oxidation of phenol to the electrode by means of mediators; ferrocene, duroquinone and dimethyferrocene were successfully used with the strains 394 (p20), 74-III and 83-IV (working names), respectively. The lower limits for detection of phenol were 1 micro M for the strain 74-III and 10 micro M for the strain 83-IV and 394 (p20). Calibrations were obtained as the dependencies of logarithm of current changes (log deltaI) on logarithm of concentration (logC), log delta I vs. logC. Among all substrates tested (phenol, catechol, hydroquinone, ethanol, methanol, propanol, isopropanol, isobutanol, isoamylalcohol, acetate, glucose, xylose, vanillin, 2,4,6-trichlorphenol, 2,3,6-trichlorphenol, 4-hydroxy-3-methoxybenzoic acid, coumarin, pentafluorophenol), bacterial sensor demonstrated a good selectivity with respect to phenol and lower responses to catechol and hydroquinone (10-times lower). The dependence of signals on operating conditions was studied. The biosensor should be used during the day of preparation. The operational stability was satisfactory to perform up to 10 consecutive measurements. Low cost and very simple manufacturing procedure allow for bacterial sensor to be applied as disposable devices.     

Extraordinary Thermal Stability of an Oligodeoxynucleotide Octamer Constructed from Alternating 7‐Deaza‐7‐iodo guanine and 5‐Iodocytosine Base Pairs – DNA Duplex Stabilization by Halogen Bonds?

A reinvestigation of the published X‐ray crystal‐structure analyses of 7‐halogenated (Br, I) 8‐aza‐7‐deaza‐2′‐deoxyguanosines Br⁷c⁷z⁸G_d; 1a and I⁷c⁷z⁸G_d, 1b , as well as of the structurally related 7‐deaza‐7‐iodo‐2′‐deoxy‐β‐D ‐ribofuranosyladenine (β‐I⁷c⁷A_d; 2 = 6e in Table 1) and its α‐D ‐anomer (α‐I⁷c⁷A_d; 3 ) clearly revealed the existence of halogen bonds between corresponding halogen substituents and the adjacent N(3)‐atoms of neighboring nucleoside molecules within the single crystals. These halogen bonds can be rationalized by the presence of a region of positive electrostatic potential, the σ‐hole, on the outermost portion the halogen's surface, while the three unshared pairs of electrons produce a belt of negative electrostatic potential around the central part of the halogen substituent. The N(3) atoms of the halogenated nucleosides carry a partial negative charge. This novel type of bonding between nucleosides was tentatively used to explain the extraordinary high stability of oligodeoxynucleotides constructed from halogenated nucleotide building blocks.     

BtrI, a novel restriction endonuclease, recognises the non-palindromic sequence 5′-CACGTC(-3/-3)-3′

The recognition sequence and cleavage positions of a new restriction endonuclease BtrI isolated from Bacillus stearothermophilus SE-U62 have been determined. BtrI belongs to a rare type IIQ of restriction endonucleases, which recognise non-palindromic nucleotide sequences and cleave DNA symmetrically within them.