Behaviors of Naïve vs. Pheromone-Exposed Leafroller Moths in Plumes from High-Dosage Pheromone Dispensers in a Sustained-Flight Wind Tunnel: Implications for Mating Disruption of These Species

Brief exposures of male Choristoneura rosaceana and Argyrotaenia velutinana to the plumes generated by lures releasing 3-component pheromone blends specifically tuned for each species or by commercially distributed Isomate OBLR/PLR Plus pheromone rope dispensers induced markedly different subsequent behavioral responses to pheromone. A greater proportion of C. rosaceana males took flight and successfully oriented toward lures 24 h after preexposure to a lure, a rope, or the lure–rope combination in a sustained-flight wind tunnel compared to naïve moths. Flights were also longer for preexposed than naïve moths. Preexposed male C. rosaceana were not more likely to fly toward ropes 24 h after preexposure. By contrast, fewer male A. velutinana oriented to lures 24 h after preexposure than did naïve moths. Those preexposed A. velutinana successfully locking onto plumes from lures flew for significantly shorter intervals than did unexposed moths. Electroantennograms revealed no changes at the periphery 15 min and 24 h after preexposure. For A. velutinana, the long-lasting effect was decreased attraction to a lure and increased attraction to a rope. For C. rosaceana, pheromone preexposure increased responsiveness to its authentic blend. This behavioral evidence is sufficient to explain why sexual communication of C. rosaceana is more difficult to disrupt than that of A. velutinana. Furthermore, it suggests a more complete blend of pheromone may be necessary to disrupt the former species but not the latter when using rope dispensers.     

The interplay of fold recognition and experimental structure determination in structural genomics

Achieving the goals of structural genomics initiatives depends on the outcomes of two groups of factors: the number and distribution of experimentally determined protein structures, and our ability to assign novel proteins to known structures (fold recognition) and use them to build models (modeling). The quality of the tools used for fold recognition defines the scope of experimental effort - the more distant the templates that can be recognized, the smaller the number of proteins that have to be solved. Recent improvements in fold recognition may have suggested that the goals of structural genomics initiatives are getting closer. However, problems that surfaced during the first few years of active work have put many of the early estimates in doubt and new ones are still slow in coming.     

Sequence clustering strategies improve remote homology recognitions while reducing search times

Sequence databases are rapidly growing, thereby increasing the coverage of protein sequence space, but this coverage is uneven because most sequencing efforts have concentrated on a small number of organisms. The resulting granularity of sequence space creates many problems for profile-based sequence comparison programs. In this paper, we suggest several strategies that address these problems, and at the same time speed up the searches for homologous proteins and improve the ability of profile methods to recognize distant homologies. One of our strategies combines database clustering, which removes highly redundant sequence, and a two-step PSI-BLAST (PDB-BLAST), which separates sequence spaces of profile composition and space of homology searching. The combination of these strategies improves distant homology recognitions by more than 100%, while using only 10% of the CPU time of the standard PSI-BLAST search. Another method, intermediate profile searches, allows for the exploration of additional search directions that are normally dominated by large protein sub-families within very diverse families. All methods are evaluated with a large fold-recognition benchmark.     

Mapping QTLs for resistance against <Emphasis Type="Italic">Globodera pallida</Emphasis> (Stone) Pa2/3 in a diploid potato progeny originating from <Emphasis Type="Italic">Solanum spegazzinii</Emphasis>

A "F1" diploid population between Solanum tuberosum 2 x and the wild Solanum spegazzinii was studied. It segregated for resistance against the potato cyst nematode Globodera pallida derived from the wild species. The inheritance had a quantitative nature. Linkage maps of AFLP and RFLP markers were constructed for both parents. Three QTLs were identified on the map of the resistant parent on chromosomes V, VI and XII, respectively. The first one had a major effect and explained more than 50% of the total variance of resistance. It is located in a cluster of resistance genes and may be the same locus as Gpa which has been described formerly. The two others explained about 20% of the total variance each. The QTL on chromosome XII is also in a cluster of resistance genes, and in an orthologous position with resistance genes against nematodes in tomato and pepper.     

A Bni4-Glc7 phosphatase complex that recruits chitin synthase to the site of bud emergence

Bni4 is a scaffold protein in the yeast Saccharomyces cerevisiae that tethers chitin synthase III to the bud neck by interacting with septin neck filaments and with Chs4, a regulatory subunit of chitin synthase III. We show herein that Bni4 is also a limiting determinant for the targeting of the type 1 serine/threonine phosphatase (Glc7) to the bud neck. Yeast cells containing a Bni4 variant that fails to associate with Glc7 fail to tether Chs4 to the neck, due in part to the failure of Bni4(V831A/F833A) to localize properly. Conversely, the Glc7-129 mutant protein fails to bind Bni4 properly and glc7-129 mutants exhibit reduced levels of Bni4 at the bud neck. Bni4 is phosphorylated in a cell cycle-dependent manner and Bni4(V831A/F833A) is both hyperphosphorylated and mislocalized in vivo. Yeast cells lacking the protein kinase Hsl1 exhibit increased levels of Bni4-GFP at the bud neck. GFP-Chs4 does not accumulate at the incipient bud site in either a bni4::TRP1 or a bni4(V831A/F833A) mutant but does mobilize to the neck at cytokinesis. Together, these results indicate that the formation of the Bni4-Glc7 complex is required for localization to the site of bud emergence and for subsequent targeting of chitin synthase.     

Enzymatic function of loop movement in enolase: preparation and some properties of H159N,H159A,H159F,and N207A enolases

The hypothesis that His159 in yeast enolase moves on a polypeptide loop to protonate the phosphoryl of 2-phosphoglycerate to initiate its conversion to phosphoenolpyruvate was tested by preparing H159N, H159A, and H159F enolases. These have 0.07%–0.25% of the native activity under standard assay conditions and the pH dependence of maximum velocities of H159A and H159N mutants is markedly altered. Activation by Mg²⁺ is biphasic, with the smaller Mg²⁺ activation constant closer to that of the catalytic Mg²⁺ binding site of native enolase and the larger in the mM range in which native enolase is inhibited. A third Mg²⁺ may bind to the phosphoryl, functionally replacing proton donation by His159. N207A enolase lacks an intersubunit interaction that stabilizes the closed loop(s) conformation when 2-phosphoglycerate binds. It has 21% of the native activity, also exhibits biphasic Mg²⁺ activation, and its reaction with the aldehyde analogue of the substrate is more strongly inhibited than is its normal enzymatic reaction. Polypeptide loop(s) closure may keep a proton from His159 interacting with the substrate phosphoryl oxygen long enough to stabilize a carbanion intermediate.     

Evaluation and comparison of random amplification of polymorphic DNA, pulsed-field gel electrophoresis and ADSRRS-fingerprinting for typing Serratia marcescens outbreaks

Amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) is a novel assay based on suppression of polymerase chain reaction (PCR). This phenomenon allows the amplification of only a limited subset of DNA fragments, since only those with two different oligonucleotides ligated at the ends of complementary DNA strands are amplified in the PCR. The DNA fragments can be easily analyzed on polyacrylamide gels, stained with ethidium bromide. We have implemented this method using a set of clinical Serratia marcescens isolates from three outbreaks ongoing in the Public Hospital in Gdańsk (Poland). Clustering of ADSRRS-fingerprinting data matched epidemiological, microbiological, random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) data. Based on this study, we found that there is at least a similar power of discrimination between the present 'gold-standard' PFGE and the novel method, ADSRRS-fingerprinting. Although the ADSRRS-fingerprinting method may appear to be more complex than the RAPD technique, we found it fast and reproducible.     

Computational methods of analysis of protein-protein interactions

Computational methods play an important role at all stages of the process of determining protein-protein interactions. They are used to predict potential interactions, to validate the results of high-throughput interaction screens and to analyze the protein networks inferred from interaction databases.