PWP2, a member of the WD-repeat family of proteins, is an essentialSaccharomyces cerevisiae gene involved in cell separation

WD-repeat proteins contain four to eight copies of a conserved motif that usually ends with a tryptophan-aspartate (WD) dipeptide. TheSaccharomyces cerevisiae PWP2 gene, identified by sequencing of chromosome III, is predicted to contain eight so-called WD-repeats, flanked by nonhomologous extensions. This gene is expressed as a 3.2-kb mRNA in all cell types and encodes a protein of 104 kDa. ThePWP2 gene is essential for growth because spores carrying thepwp21::HIS3 disruption germinate before arresting growth with one or two large buds. The growth defect ofpwp21::HIS3 cells was rescued by expression ofPWP2 or epitope-taggedHA-PWP2 using the galactose-inducibleGAL1 promoter. In the absence of galactose, depletion of Pwp2p resulted in multibudded cells with defects in bud site selection, cytokinesis, and hydrolysis of the septal junction between mother and daughter cells. In cell fractionation studies, HA-Pwp2p was localized in the particulate component of cell lysates, from which it would be solubulized by high salt and alkaline buffer but not by nonionic detergents or urea. Indirect immunofluorescence microscopy indicated that HA-Pwp2p was clustered at multiple points in the cytoplasm. These results suggest that Pwp2p exists in a proteinaceous complex, possibly associated with the cytoskeleton, where it functions in control of cell growth and separation.     

Identification of a surface on the beta-propeller protein RACK1 that interacts with the cAMP-specific phosphodiesterase PDE4D5

A strategy of mutagenesis followed by yeast two-hybrid assay was used to determine the sites on the WD-repeat protein Receptor for Activated C Kinase 1 (RACK1) necessary for it to interact with the cAMP-specific phosphodiesterase isoform PDE4D5. Analysis of deletion mutations demonstrated that WD-repeats 5-7, inclusively, of RACK1 contained the major site for interaction with PDE4D5. A reverse two-hybrid screen focusing on WD-repeats 5-7 of RACK1 isolated 11 single amino acid mutations from within this region that blocked the interaction. The ability of these mutations to block the interaction was confirmed by "pull-down" assays using bacterially expressed glutathione-S-transferase (GST)-RACK1 and mammalian cell-expressed PDE4D5. A model of RACK1 structure, based on the structural similarity of RACK1 to other beta-propeller WD-repeat proteins, indicated that the majority of the amino acids identified by mutagenesis are clustered in a discrete surface of RACK1. We propose that this surface of RACK1 is the major site for its interaction with the unique amino-terminal region of PDE4D5.     

The WD-repeats of Net2p interact with Dnm1p and Fis1p to regulate division of mitochondria

The Net2, Fis1, and Dnm1 proteins are required for the division of mitochondria in the yeast Saccharomyces cerevisiae. Net2p has an amino-terminal region that contains predicted coiled-coil motifs and a carboxyl-terminal domain composed of WD-40 repeats. We found that the amino-terminal part of Net2p interacts with Fis1p, whereas the carboxyl-terminal region interacts with both Dnm1p and Fis1p. Overproduction of either domain of Net2p in yeast cells poisons mitochondrial fission, and the dominant-negative effect caused by the WD-repeats of Net2p is suppressed by increased levels of Dnm1p. Point mutations in the WD-region of Net2p or in the GTPase region of Dnm1p disrupt the normal Net2p-Dnm1p interaction, causing Net2p to lose its normal punctate distribution. Our results suggest that Dnm1p interacts with the WD-repeats of Net2p and in a GTP-dependent manner recruits Net2p to sites of mitochondrial division. Furthermore, our results indicate that Net2p is required for proper assembly of the mitochondrial fission components to regulate organelle division.     

Identification of the versatile scaffold protein RACK1 on the eukaryotic ribosome by cryo-EM

RACK1 serves as a scaffold protein for a wide range of kinases and membrane-bound receptors. It is a WD-repeat family protein and is predicted to have a beta-propeller architecture with seven blades like a Gbeta protein. Mass spectrometry studies have identified its association with the small subunit of eukaryotic ribosomes and, most recently, it has been shown to regulate initiation by recruiting protein kinase C to the 40S subunit. Here we present the results of a cryo-EM study of the 80S ribosome that positively locate RACK1 on the head region of the 40S subunit, in the immediate vicinity of the mRNA exit channel. One face of RACK1 exposes the WD-repeats as a platform for interactions with kinases and receptors. Using this platform, RACK1 can recruit other proteins to the ribosome.     

PWP2, a member of the WD-repeat family of proteins,is an essentialSaccharomyces cerevisiae gene involved in cell separation

WD-repeat proteins contain four to eight copies of a conserved motif that usually ends with a tryptophan-aspartate (WD) dipeptide. TheSaccharomyces cerevisiae PWP2 gene, identified by sequencing of chromosome III, is predicted to contain eight so-called WD-repeats, flanked by nonhomologous extensions. This gene is expressed as a 3.2-kb mRNA in all cell types and encodes a protein of 104 kDa. ThePWP2 gene is essential for growth because spores carrying thepwp2Δ1::HIS3 disruption germinate before arresting growth with one or two large buds. The growth defect ofpwp2Δ1::HIS3 cells was rescued by expression ofPWP2 or epitope-taggedHA-PWP2 using the galactose-inducibleGAL1 promoter. In the absence of galactose, depletion of Pwp2p resulted in multibudded cells with defects in bud site selection, cytokinesis, and hydrolysis of the septal junction between mother and daughter cells. In cell fractionation studies, HA-Pwp2p was localized in the particulate component of cell lysates, from which it would be solubulized by high salt and alkaline buffer but not by nonionic detergents or urea. Indirect immunofluorescence microscopy indicated that HA-Pwp2p was clustered at multiple points in the cytoplasm. These results suggest that Pwp2p exists in a proteinaceous complex, possibly associated with the cytoskeleton, where it functions in control of cell growth and separation.     

A WD-FYVE protein binds to the kinases Akt and PKCzeta/lambda

WD (tryptophan-aspartic acid dipeptide)-repeat proteins play a central role in signal transduction cascades by co-ordinating the interaction of key signalling molecules. We identified a novel propeller-FYVE [domain identified in Fab1p, YOTB, Vac1p and EEA1 (early endosome antigen 1)] protein, ProF, which is expressed in various cell lines and tissues and consists of seven WD-repeats and a FYVE domain. WD-repeat proteins offer a platform for protein-protein interactions by folding into a seven-bladed propeller-like structure, while the FYVE domain binds to phosphatidylinositol 3-phosphate present mainly on intracellular membranes. The ProF protein partially co-localizes with EEA1 on vesicular structures and binds to the protein kinases Akt and PKCzeta/lambda (protein kinase Czeta/lambda) via its WD-repeat propeller. ProF interacts more strongly with the kinases after hormonal stimulation. Endogenously expressed ProF and the two kinases interact in brain and in the preadipocyte cell line 3T3-L1, suggesting a role in secretory vesicular processes. In summary, we describe a new binding partner for kinases, located on vesicular structures in specialized cells, which may play a role for the spatial organization of signalling cascades.     

IC138 is a WD-repeat dynein intermediate chain required for light chain assembly and regulation of flagellar bending

Increased phosphorylation of dynein IC IC138 correlates with decreases in flagellar microtubule sliding and phototaxis defects. To test the hypothesis that regulation of IC138 phosphorylation controls flagellar bending, we cloned the IC138 gene. IC138 encodes a novel protein with a calculated mass of 111 kDa and is predicted to form seven WD-repeats at the C terminus. IC138 maps near the BOP5 locus, and bop5-1 contains a point mutation resulting in a truncated IC138 lacking the C terminus, including the seventh WD-repeat. bop5-1 cells display wild-type flagellar beat frequency but swim slower than wild-type cells, suggesting that bop5-1 is altered in its ability to control flagellar waveform. Swimming speed is rescued in bop5-1 transformants containing the wild-type IC138, confirming that BOP5 encodes IC138. With the exception of the roadblock-related light chain, LC7b, all the other known components of the I1 complex, including the truncated IC138, are assembled in bop5-1 axonemes. Thus, the bop5-1 motility phenotype reveals a role for IC138 and LC7b in the control of flagellar bending. IC138 is hyperphosphorylated in paralyzed flagellar mutants lacking radial spoke and central pair components, further indicating a role for the radial spokes and central pair apparatus in control of IC138 phosphorylation and regulation of flagellar waveform.     

Characterization, cloning and immunolocalization of a coronin homologue in Trichomonas vaginalis

On adhesion to host cells the flagellate Trichomonas vaginalis switches to an amoeboid form rich in actin microfilaments. We have undertaken the identification of actin-associated proteins that regulate actin dynamics. A monoclonal antibody 4C12 raised against a cytoskeletal fraction of T. vaginalis labeled a protein doublet at circa 50 kDa. These two bands were recognized by the antibody against Dictyostelium discoideum coronin. During cell extraction and actin polymerization, T. vaginalis coronin cosedimented with F-actin. By two-dimensional gel electrophoresis, the protein doublet was separated into two sets of isoforms covering two Ip zones around 6 and 7. By screening a T. vaginalis library with 4C12, two clones Cor 1 and Cor 2 were isolated. This gene duplicity is a particularity among unicellular organisms examined. The complete sequence of the gene Cor 1 encodes a 435-residue protein with a calculated molecular mass of 48 kDa and Ip of 5.58. The incomplete sequence Cor 2 was very similar but with a more basic calculated Ip than Cor 1 on the same region. T. vaginalis coronin had 50% similarity with the coronin family, possessing the five WD-repeats and a leucine zipper in its C-terminal part. Double immunofluorescence labeling showed that coronin mainly colocalized with actin at the periphery of the adherent amoeboid cells. However, coronin labeling displayed patches within a reticular array. Immunogold electron microscopy confirmed the coronin labeling in the actin-rich microfilamentous fringe beneath the plasma membrane, with accumulation in phagocytic zones and pseudopodial extensions. In T. vaginalis, one of the first emerging lineage of eukaryotes, coronin seems to play an important role in actin dynamics and may be a downstream target of a signaling mechanism for the cytoskeleton reorganization.     

Interspecific interference and the functional response of four species of carnivorous stoneflies

1. A previous study compared the functional responses to their prey and intraspecific interference in mature larvae of Perlodes microcephalus, Isoperla grammatica, Dinocras cephalotes and Perla bipunctata. The present study extends this work by assessing interspecific interference between pairs of these species in equal numbers (one, two or three larvae per species) to provide total predator densities of two, four or six larvae. Baetis larvae as prey were replaced as they were eaten, and their density per predator was varied between 20 and 200 larvae. 2. The number of prey eaten by each competing species increased curvilinearly with prey density, the relationship being well described by a Type II model. Of the two constants in the model, handling time varied considerably between species, mean values being shortest for Perlodes, slightly higher for Isoperla, and much higher for Dinocras and Perla. It was not affected significantly either by predator density or the identity of the competing species. 3. Attack rate also varied between species and decreased with predator density. This decrease was slight for Perlodes, and also for Dinocras and Perla in competition with Isoperla. The decrease in Dinocras and Perla was similar to that for intraspecific interference. 4. The decrease in attack rate was described by a convex curve for Perlodes with the other three species and for Dinocras/Perla with Isoperla, but by a concave curve (negative power function) for Isoperla competing with the other three species, and for both Dinocras and Perla in competition with Perlodes. Prey consumption also decreased with predator density, the severity of competition with different species reflecting that for attack rate. 5. A comparison with previous results for intraspecific interference showed that the latter was dominant for Perlodes in all contests and for Dinocras or Perla competing with Isoperla, whilst interspecific interference dominated for Isoperla in all contests and for Dinocras and Perla competing with Perlodes. Both types of interference were applicable to competition between Dinocras and Perla. Isoperla was the least, and Perlodes the most, aggressive of the four species with Dinocras and Perla intermediate.     

An Examination of the Differential Sensitivity to Ketolide Antibiotics in <Emphasis Type="Italic">ermB</Emphasis> Strains of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Several reports in the literature have described a differential sensitivity to ketolide antibiotics in ermB strains of Streptococcus pyogenes and Streptococcus pneumoniae resistant to erythromycin. Strains of S. pyogenes and S. pneumoniae carrying different erm gene alleles were examined for their susceptibility to the ketolide antibiotics cethromycin (ABT-773) and telithromycin. The effect of the antibiotics on cell growth and viability was assessed as were effects on protein synthesis and 50S ribosomal subunit formation. The susceptibility of wild-type strains of both organisms was compared with effects in strains containing the ermA and ermB methyltransferase genes. A wild-type antibiotic-susceptible strain of S. pyogenes was comparable to an ermA strain of the organism in its ketolide sensitivity, with IC₅₀ values for 50% inhibition of protein synthesis and 50S ribosomal subunit formation of 10 ng/mL for cethromycin and 16 ng/mL for telithromycin. An S. pneumoniae strain with the ermB gene and an S. pyogenes strain with the ermA gene were also similar in their sensitivity to ketolide inhibition. IC₅₀ values for inhibition of translation and subunit formation in S. pneumoniae (ermB) were 30 ng/mL and 55 ng/mL and for the ermA strain of S. pyogenes they were 15 ng/mL and 35 ng/mL respectively. By contrast, an S. pyogenes ermB strain was significantly more resistant to both ketolides, with IC₅₀ values for inhibition of 50S synthesis of 215 and 380 ng/mL for the two ketolides. Experiments were conducted to examine ribosome synthesis and translational activity in the two ermB strains at intervals during growth in the presence of each antibiotic. Cell viability and 50S subunit formation were dramatically reduced in the S. pneumoniae strain during continued growth with either drug. By contrast, the ketolides had little effect on the S. pyogenes strain growing with the antibiotics. The results indicate that ketolides have a reduced inhibitory effect on translation and 50S subunit synthesis in S. pyogenes with the ermB gene compared with the other strains examined.     

RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains

Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae. Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS. The molecular mechanism by which Gle2p and the Gle2p-Nup116p interaction function in mRNA export is unknown. Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non-WD-repeat extension. This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking. Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner. Docking of RAE1 to the NE is highly dependent on new mRNA synthesis. Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA. Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98-RAE1 interaction in mRNA export. Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.     

Competition of cyclooctenes and cyclooctadienes for ethylene binding and activity in plants

trans-Cyclooctene, cis,trans-1,5-cyclooctadiene, and cis,trans-1,3-cyclooctadiene have been compared with the cis and cis,cis isomers and with 2,5-norbornadiene for competition with ethylene for binding in mung bean sprouts and tobacco and for action (induction of chlorophyll degradation) in banana. The compounds containing a trans double bond were much more effective in competition for binding and action than the cis and cis,cis compounds. trans-Cyclooctene and cis,trans-1,3-cyclooctadiene were in the general range of 50–90 times more effective than 2,5-norbornadiene.R.J. Reynolds Research Apprentice     

Dynamic light-scattering studies of internal motions in DNA. I. Applicability of the rouse-zimm model

The intermediate scattering function G(K,t) for any polymer model obeying a linear separable Langevin equation can be expressed in terms of the eigenvalues and eigenvectors of its normal coordinate transformation. An algorithm for the extract numerical evaluation of G(K,t) for linear Rouse-Zimm chains in the presence of hydrodynamic interaction has been developed. The computed G(K,t)² were fit to C(t) = A exp(?t/τA) + B, and apparent diffusion coefficients calculated according to D_app ≡ 1/(2τ_AK²). G(K,t)² was surprisingly well-fit by single-exponential decays, especially at both small and large values of Kb, where K is the scattering vector and b the root-mean-squared subunit extension. Plots of D_app vs K² in-variably showed a sigmoidal rise from D₀ at K² = O up to a constant plateau value at large K²b². Analytical expression for G(K,t), exact in the limit of short times, were obtained for circular Rouse-Zimm chains with and without hydrodynamic interaction, and also for free-draining linear chains, and in addition for the independent-segment-mean-force (ISMF) model. The predicted behaviors for G(K,t) at large Kb (or KR_G) was found in all cases to be single-exponential with 1/τ ∝ K² at large Kb, in agreement with the computational results. A simple procedure for estamating all parameter of the Rouse-Zimm model from a plot of D_app vs K² is proposed. Experimental data for both native and pH-denatured calf-thymus DNA in 1.0M Nacl with and without EDTA clearly plateau behavior of D_app at large values of K, in harmony with the present Rouse-Zimm and ISMF theories, and in sharp contrast to previous predictions based on the Rouse-Zimm model.     

Nuclear magnetic resonance study on the model compounds for poly(N-alkylglycine)s

Cis-trans isomerism was investigated with N-acetyl and N-propionyl, N-alkylglycine dimethylamides as model compounds for poly(N-alkylglycine dimethlamides as model compounds for (N-alkylglycine)s using n.m.r. spectroscopy. The population of the cis isomer measured in benzene and methylene chloride solutions did not show any marked dependence on the bulkiness of N-alkyl substituents. This contrasts with polyN-alkylglycine)s, whose cis isomer population increased with the introduction of bulky N-alkyl groups. Kinetics of the Cis-trans isomerization was also investigated with N-acetyldimethylamides of sacrosine, N-n-propylglycine, and N-isopropylglycine in tetrachloroethane solution. The δG? values for Cis-trans isomerization in these amides were 18 ～ 19 kcal/mole, which were virtually the same as that of polysacrosine.     

Bioprospecting of Saprobe Fungi from the Semi‐Arid North‐East of Brazil for the Control of Anthracnose on Sorghum

Anthracnose, caused by the hemiotrophic fungus Colletotrichum sublineolum, is one of the most important diseases affecting sorghum production worldwide. The main goal of this study was to select saprobe fungi from the semi‐arid north‐east of Brazil that could increase sorghum resistance to anthracnose and investigate this increased resistance at both physiological and biochemical levels. Plants were sprayed with Curvularia inaequalis, Gonytrichum macroladum, Memnoniella levispora, Pithomyces chartarum, Periconia hispidula, Phaeoisaria clematidia, Dictyochaeta heteroderae, Sarcopodium circinatum, Periconia byssoides, Moorella speciosa, Stachybotrys chartarum, Pseudobotrytis terrestres, Memnoniella echinata, Stachybotrys globosa and Gonytrichum clamydosporium 24 h before inoculation with C. sublineolum. Plants sprayed with water served as the control treatment. The area under the anthracnose progress curve was significantly reduced in comparison with the control treatment only for plants sprayed with C. inaequalis. Therefore, C. inaequalis was selected for physiological and biochemical evaluations. The peroxidases, chitinases and β‐1,3‐glucanases activities were significantly higher for plants sprayed with C. inaequalis and inoculated with C. sublineolum than for plants not sprayed with C. inaequalis and inoculated with C. sublineolum. There was no apparent decrease in the values of net carbon assimilation rate, stomatal conductance to water vapour or transpiration rate for plants sprayed with C. inaequalis and infected by C. sublineolum in comparison with plants not sprayed with C. inaequalis and infected by C. sublineolum. In conclusion, sorghum resistance against C. sublineolum infection was greatly potentiated by C. inaequalis without any apparent change in the photosynthetic capacity of the infected plants.     

Association of <Emphasis Type="Italic">iceA</Emphasis> and <Emphasis Type="Italic">babA</Emphasis> genotypes in <Emphasis Type="Italic">Helicobacter pylori</Emphasis> strains with patient and strain characteristics

Data on the geographic prevalence of Helicobacter pylori iceA and babA alleles in Eastern Europe are still relatively scant. The aim of this study was to evaluate the prevalence of iceA and babA genotypes in Bulgarian symptomatic patients. The iceA and babA genotypes were evaluated by PCR with pure cultures in strains from 196 and 181 patients, respectively. Mixed infections were found in 10.2% of all 196 patients. Prevalence of H. pylori genotypes in patients with single-strain infections was 69.3% for iceA1, 30.7% for iceA2, 82.4% for cagA ⁺, 89.2% for vacA s1, 10.8% for vacA s2, 39.8% for vacA m1, 60.2% for vacA m2 and 48.8% for babA2. Within the iceA1 positive strains, 94.3% and 88.5% were also vacA s1a and cagA positive, respectively. Of the babA2 positive strains, 100.0%, 92.4% and 72.2% were also vacA s1a, cagA and iceA1 positive, respectively. Ulcer patients had more often strains with cagA positive status and vacA s1a allele. Although neither iceA1 nor babA2 were more common in ulcer patients, the combination of both alleles was more frequent (48.1%) in the ulcer patients than in the rest (28.7%). Clarithromycin susceptible strains had more often iceA1 allele (74.4%) than the resistant strains (55.3%). In conclusion, the results demonstrated a high prevalence of virulent H. pylori in Bulgaria. Both iceA1 and babA2 genotypes were associated with other virulence factors of H. pylori and, in addition, the iceA1 allele was associated with the strain susceptibility.     

Control of Amino Acid Permease Sorting in the Late Secretory Pathway of Saccharomyces Cerevisiae by Sec13, Lst4, Lst7 and Lst8

The SEC13 gene was originally identified by temperature-sensitive mutations that block all protein transport from the ER to the Golgi. We have found that at a permissive temperature for growth, the sec13-1 mutation selectively blocks transport of the nitrogen-regulated amino acid permease, Gap1p, from the Golgi to the plasma membrane, but does not affect the activity of constitutive permeases such as Hip1p, Can1p, or Lyp1p. Different alleles of SEC13 exhibit different relative effects on protein transport from the ER to the Golgi, or on Gap1p activity, indicating distinct requirements for SEC13 function at two different steps in the secretory pathway. Three new genes, LST4, LST7, and LST8, were identified that are also required for amino acid permease transport from the Golgi to the cell surface. Mutations in LST4 and LST7 reduce the activity of the nitrogen-regulated permeases Gap1p and Put4p, whereas mutations in LST8 impair the activities of a broader set of amino acid permeases. The LST8 gene encodes a protein composed of WD-repeats and has a close human homologue. The LST7 gene encodes a novel protein. Together, these data indicate that SEC13, LST4, LST7, and LST8 function in the regulated delivery of Gap1p to the cell surface, perhaps as components of a post-Golgi secretory-vesicle coat.