Variant‐specific and reciprocal Hsp40 functions in Hsp104‐mediated prion elimination

The amyloid‐based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded proteins, passed to daughter cells following fragmentation by molecular chaperones including the J‐protein Sis1, Hsp70 and Hsp104. Overexpression of Hsp104 efficiently cures cell populations of the prion [PSI⁺] by an alternative Sis1‐dependent mechanism that is currently the subject of significant debate. Here, we broadly investigate the role of J‐proteins in this process by determining the impact of amyloid polymorphisms (prion variants) on the ability of well‐studied Sis1 constructs to compensate for Sis1 and ask whether any other S. cerevisiae cytosolic J‐proteins are also required for this process. Our comprehensive screen, examining all 13 members of the yeast cytosolic/nuclear J‐protein complement, uncovered significant variant‐dependent genetic evidence for a role of Apj1 (antiprion DnaJ) in this process. For strong, but not weak [PSI⁺] variants, depletion of Apj1 inhibits Hsp104‐mediated curing. Overexpression of either Apj1 or Sis1 enhances curing, while overexpression of Ydj1 completely blocks it. We also demonstrated that Sis1 was the only J‐protein necessary for the propagation of at least two weak [PSI⁺] variants and no J‐protein alteration, or even combination of alterations, affected the curing of weak [PSI⁺] variants, suggesting the possibility of biochemically distinct, variant‐specific Hsp104‐mediated curing mechanisms.     

Microbial Formation of Dimethyl Sulfide in Anoxic Sphagnum Peat

Peat bogs dominated by Sphagnum spp. have relatively high areal rates of dimethyl sulfide (DMS) emission to the atmosphere. DMS was produced in anoxic slurries of Sphagnum peat with a linear time course and with an average rate of 40.4 (range, 22.0 to 68.6) nmol per liter of slurry (middot) day(sup-1) observed in nine batches of slurry. Methanethiol (MeSH) was produced at roughly similar rates over the typical 4- to 8-day incubations. DMS and MeSH production in these acidic (pH 4.2 to 4.6) peats were biological, as they were stopped completely by autoclaving and inhibited strongly by addition of antibiotics and 500 (mu)M chloroform. Endogenous DMS production may be due to the degradation of S-methyl-methionine, dimethyl sulfoxide, or methoxyaromatic compounds (e.g., syringic acid), each of which stimulated DMS formation when added at 5 to 10 (mu)M concentrations. However, on the basis of the high rates of thiol (MeSH and ethanethiol) methylation activity that we observed and the availability of endogenous MeSH, we suggest that methylation of MeSH is the major pathway leading to DMS formation in anaerobic peat. Solid-phase adsorption of MeSH plays a key role in its availability for biomethylation reactions. Additions of acetate (1.5 mM) or compounds which could cause acetate to accumulate (e.g., glucose, alanine, and 2-bromoethanesulfonate) suppressed DMS formation. It is likely that acetogenic bacteria are involved in DMS formation, but our data are insufficient to allow firm conclusions about the metabolic pathways or organisms involved. Our observations are the first which point to the methylation of MeSH as the major mechanism for endogenous DMS production in any environment. The rates of net DMS production observed are sufficient to explain the relatively high fluxes of DMS emitted to the atmosphere from Sphagnum sp.-dominated wetlands.     

Disruption of the Crithidia fasciculata RNH1 gene results in the loss of two active forms of ribonuclease H.

Both prokaryotic and eukaryotic cells contain multiple forms of ribonuclease H, a ribonuclease that specifically degrades the RNA strand of RNA-DNA hybrids and which has been implicated in the processing of initiator RNAs and in the removal of RNA primers from Okazaki fragments. The Crithidia fasciculata RNH1 gene encodes an RNase H and was shown to be a single-copy gene in this diploid trypanosomatid. The RNH1 gene has been disrupted by targeted gene disruption using hygromycin or G418 drug-resistance cassettes. Major active forms of RNase H (38 and 45 kDa) were observed on activity gels of extracts of wild-type cells or cells in which one allele of RNH1 was disrupted. Both the 38 and 45 kDa activities were absent in extracts of cells in which both alleles of RNH1 were disrupted indicating that both forms of the C.fasciculata RNase H are encoded by the RNH1 gene.     

Evolutionary stability in strategic models of single-locus frequency-dependent viability selection

The dynamic stability of an evolutionarily stable strategy (ESS) is analyzed for a diploid species under individual viability selection. An individual's viability depends on the genotypic frequencies at a single autosomal locus through a payoff matrix determined by phenotypic behaviours (i.e. strategies). It is shown that an ESS of this payoff matrix is dynamically stable if there are at most three alleles — an intuitive result that strengthens the importance of static game-theoretic methods in genetic models.Author for correspondence     

Estimating indices of range shifts in birds using dynamic models when detection is imperfect

There is intense interest in basic and applied ecology about the effect of global change on current and future species distributions. Projections based on widely used static modeling methods implicitly assume that species are in equilibrium with the environment and that detection during surveys is perfect. We used multiseason correlated detection occupancy models, which avoid these assumptions, to relate climate data to distributional shifts of Louisiana Waterthrush in the North American Breeding Bird Survey (BBS) data. We summarized these shifts with indices of range size and position and compared them to the same indices obtained using more basic modeling approaches. Detection rates during point counts in BBS surveys were low, and models that ignored imperfect detection severely underestimated the proportion of area occupied and slightly overestimated mean latitude. Static models indicated Louisiana Waterthrush distribution was most closely associated with moderate temperatures, while dynamic occupancy models indicated that initial occupancy was associated with diurnal temperature ranges and colonization of sites was associated with moderate precipitation. Overall, the proportion of area occupied and mean latitude changed little during the 1997–2013 study period. Near‐term forecasts of species distribution generated by dynamic models were more similar to subsequently observed distributions than forecasts from static models. Occupancy models incorporating a finite mixture model on detection – a new extension to correlated detection occupancy models – were better supported and may reduce bias associated with detection heterogeneity. We argue that replacing phenomenological static models with more mechanistic dynamic models can improve projections of future species distributions. In turn, better projections can improve biodiversity forecasts, management decisions, and understanding of global change biology.     

Ichthyophthiriasis in the mirror carp Cyprinus carpio (L.) IV. Physiological dysfunction*

Changes in blood ion concentration, oxygen absorption, blood haemoglobin concentration and blood urea-ammonia nitrogen level were measured in a group of mirror carp, Cyprinus carpio (L.) infected with measured numbers of Ichthyophthirius multifiliis under standardized conditions. The disease was characterized by a drop in serum sodium and magnesium and increase in serum potassium. The ability of infected fish to absorb oxygen was reduced as was their ability to tolerate dissolved oxygen concentrations under 5 ppm. Blood ureaammonia level was elevated during the disease but blood haemoglobin level remained unchanged.     

Characterization of proline endopeptidase from rat brain

A homogeneous proline endopeptidase from rat brain is characterized with respect to its substrate specificity and the residues essential for catalysis. The two fluorogenic substrate analogues tested, pyroglutamylhistidylprolyl-beta-naphthylamide and pyroglutamy(N-benzylimidazolyl)-histidylprolyl-beta-naphthylamide, have higher Vmax values (19.5 and 26.9 mumol . min-1 . mg-1, respectively) and considerably lower Km values (0.034 and 0.020 mM, respectively) than pyroglutamylhistidylprolylamide (Vmax = 2.9 mumol . min-1 . mg-1 and Km = 4.1 mM). Both fluorogenic substrates give rise to pH optima and pH-rate profiles similar to those of the amide. Values of Km and kcat are determined as a function of pH. Km is pH independent, with the titration curve for kcatKm-1 implicating an active-site residue(s) with a pKa of 6.2. Proline endopeptidase can be completely inactivated by low concentrations of diisopropyl fluorophosphate with an observed second-order rate constant of 2.5 x 10(4) min-1 . M-1. The stoichiometry of the alkylphosphorylation is 0.83 mol/mol of enzyme. The pH dependence of the inactivation by diisopropylfluorophosphate implicates a residue(s) involved in covalent bond formation having a pKa of 6.0. These data suggest that proline endopeptidase is a serine proteinase.     

Construction and characterization of new coliphage M13 cloning vectors

New single-stranded DNA cloning vectors have been constructed by the insertion of additional DNA fragments into a HaeII restriction site in the bacteriophage M13 duplex replicative form (RF). These inserts into the M13 genome bring a single restriction sites useful for cloning, including PstI, XorII, EcoRI, SstI, XhoI, KpnI, and PvuII. Drug-resistance genes cloned into M13 include the beta-lactamase (bla) gene and the chloramphenicol acetyl transferase (cat) gene. These vectors provide a convenient means of easily obtaining the separated strands of a cloned duplex DNA fragment by cloning the fragment in each of the two possible orientations. Standard cloning techniques commonly applied to double-stranded DNAs can be utilized to insert foreign DNAs into the duplex RF DNAs of these vectors. Cells transformed by chimeric DNAs extrude filamentous phage particles carrying a circular single-stranded copy of the chimeric viral strand. Because M13-infected cells continue to grow and divide, cells can be transformed to yield either plaques or drug-resistant colonies. Specific inserts are readily detected by plaque hybridization techniques using an appropriate probe. Chimeric viral single strands from virus particles in the supernatant of small volumes of infected cultures can be rapidly and sensitively analyzed by agarose gel electrophoresis to determine the size of an insert.