Molecular cloning, gene localization, and structure of human cyclin B3

Here we describe the molecular cloning of human cyclin B3, its localization, and its structure. It is localized in the subcentromeric region of the X chromosome, still not completely sequenced by the Human Genome Project. This cyclin B3 is unusually large for a mitotic cyclin. Its mRNAs were found in all tissues and were particularly abundant in testis. At least three splice variants were found in the ORF and three variants in the 5'UTR.     

Electrostatic effects in highly charged proteins: salt sensitivity of pKa values of histidines in staphylococcal nuclease

The pK(a) values of most histidines in small peptides and in myoglobin increase on average by 0.30 unit between 0.02 and 1.5 M NaCl [Kao et al. (2000) Biophys. J. 79, 1637]. The DeltapK(a) values reflect primarily the ionic strength dependence of the solvation energy; screening of Coulombic interactions contributes only in a minor way. This implies that Coulombic interactions are weak, or that attractive and repulsive contributions to the pK(a) values are balanced. To distinguish experimentally between these two possibilities, and to further characterize the magnitude and salt sensitivity of surface electrostatic interactions in proteins, the salt dependence of pK(a) values of histidines in staphylococcal nuclease was measured by (1)H NMR spectroscopy. Three of the four histidines titrated with significantly depressed pK(a) values, and the salt sensitivity of all histidine pK(a) values was substantial. In three cases, the pK(a) values increased by a full unit between 0.01 and 1.5 M KCl. Anion-specific effects were found; the pK(a) values measured under equivalent ionic strengths in SCN(-) and SO(4)(2-) were higher than in Cl(-); the order of the sensitivity of pK(a) values to anions was SCN(-) > Cl(-) > SO(4)(2-). Structure-based pK(a) calculations with continuum methods were performed to interpret the measured effects structurally and to test their ability to capture the experimental behavior. Calculations in which the protein interior was treated empirically with a dielectric constant of 20 reproduced the pK(a) values and their dependence on the concentration of Cl(-). According to the calculations, the pK(a) values are depressed because of unfavorable self-energies and repulsive Coulombic interactions. Their striking salt sensitivity reflects screening of weak, repulsive, Coulombic interactions among charges separated by more than 10 A. Long-range Coulombic interactions on the surfaces of proteins are weak, but they can add up to produce substantial electrostatic effects when positive and negative charges are not balanced.     

Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria

Multiple regulatory mechanisms for coping with stress co-exist in low G+C Gram-positive bacteria. Among these, the HrcA and CtsR repressors control distinct regulons in the model organism, Bacillus subtilis. We recently identified an orthologue of the CtsR regulator of stress response in the major pathogen, Staphylococcus aureus. Sequence analysis of the S. aureus genome revealed the presence of potential CtsR operator sites not only upstream from genes encoding subunits of the Clp ATP-dependent protease, as in B. subtilis, but also, unexpectedly, within the promoter regions of the dnaK and groESL operons known to be specifically controlled by HrcA. The tandem arrangement of the CtsR and HrcA operators suggests a novel mode of dual heat shock regulation by these two repressors. The S. aureus ctsR and hrcA genes were cloned under the control of the PxylA xylose-inducible promoter and used to demonstrate dual regulation of the dnaK and groESL operons by both CtsR and HrcA, using B. subtilis as a heterologous host. Direct binding by both repressors was shown in vitro by gel mobility shift and DNase I footprinting experiments using purified S. aureus CtsR and HrcA proteins. DeltactsR, DeltahrcA and DeltactsRDeltahrcA mutants of S. aureus were constructed, indicating that the two repressors are not redundant but, instead, act together synergistically to maintain low basal levels of expression of the dnaK and groESL operons in the absence of stress. This novel regulatory mode appears to be specific to Staphylococci.     

The molecular chaperone Cdc37 is required for Ste11 function and pheromone-induced cell cycle arrest

The molecular chaperone Cdc37 is thought to act in part as a targeting subunit of the heat-shock protein 90 (Hsp90) chaperone complex. We demonstrate here that Cdc37 is required for activity of the kinase Ste11 in budding yeast. A cdc37 mutant strain is defective in Ste11-mediated pheromone signaling and in accumulation and functional maturation of the constitutively active Ste11 version Ste11DeltaN. Moreover, Cdc37, Ste11DeltaN and Hsp90 coprecipitate pairwise. Thus, Hsp90 and Cdc37 may transiently associate with Ste11 to promote proper folding and/or association with additional regulatory factors. Our results establish Ste11 as the first endogenous Cdc37 client protein in yeast.     

Cytological effects of cellulases in the parasitism of Phytophthora parasitica by Pythium oligandrum

The ubiquitous oomycete Pythium oligandrum is a potential biocontrol agent for use against a wide range of pathogenic fungi and an inducer of plant disease resistance. The ability of P. oligandrum to compete with root pathogens for saprophytic colonization of substrates may be critical for pathogen increase in soil, but other mechanisms, including antibiosis and enzyme production, also may play a role in the antagonistic process. We used transmission electron microscopy and gold cytochemistry to analyze the intercellular interaction between P. oligandrum and Phytophthora parasitica. Growth of P. oligandrum towards Phytophthora cells correlated with changes in the host, including retraction of the plasma membrane and cytoplasmic disorganization. These changes were associated with the deposition onto the inner host cell surface of a cellulose-enriched material. P. oligandrum hyphae could penetrate the thickened host cell wall and the cellulose-enriched material, suggesting that large amounts of cellulolytic enzymes were produced. Labeling of cellulose with gold-complexed exoglucanase showed that the integrity of the cellulose was greatly affected both along the channel of fungal penetration and also at a distance from it. We measured cellulolytic activity of P. oligandrum in substrate-free liquid medium. The enzymes present were almost as effective as those from Trichoderma viride in degrading both carboxymethyl cellulose and Phytophthora wall-bound cellulose. P. oligandrum and its cellulolytic enzymes may be useful for biological control of oomycete pathogens, including Phytophthora and Pythium spp., which are frequently encountered in field and greenhouse production.     

Early locomotor behaviour in genetic stocks of chickens with different growth rates

Reduction in exercise increases the occurrence of lameness in meat-type chickens. Locomotor activity is dramatically reduced during the finishing period in chickens from fast-growing genetic types compared to slow-growing genetic types, but it is not known whether this difference is already present during the starting period and may be influenced by genetic factors. In order to define the effect of genetic origin on early locomotor behaviour, exercise was compared from 1 to 22 days of age in two meat-type chicken stocks differing in growth rate: male broilers (B) which grow fast and are often lame, and male "label rouge" chickens (L) which grow slowly and are rarely lame.Time budget (lying, standing, drinking, eating, walking) was measured by scanning in six repetitions of five birds (density=2.5 birds/m(2)) at 1, 8, 15 and 17 days of age. Standing bouts were analysed by focal sampling at 2-3, 6-7, 13-14 and 20-21 days of age.B chicks spent less time standing than L chicks at 15 days of age (B=13+/-2%, L=24+/-1%, P<0.01) and 17 days of age, and spent more time lying at 17 days of age (B=73+/-3%, L=60+/-4%, P<0.05).The major part (74%) of the total active time observed by focal sampling was linked to feeding activity. At 2 and 3 days, the activity of B chicks was half that of L chicks during standing bouts (duration of walking per bout: 19+/-4 s for B; 45+/-4 s for L, P<0.05). The activity observed by focal sampling during non-feeding bouts at 20-21 days was significantly correlated with the corresponding data recorded at 2-3 days in the same chicks in the B stock but not in the L stock.We concluded that (1) both B and L genetic stocks have the same overall activity during the first 3 days of age (scanning) but they exhibit different organisation and composition of standing bouts (focal sampling). (2) Genetic factors are probably involved in the expression of locomotor behaviour in very young chicks. (3) The correlations between the levels of activity at early and later ages suggest that selection of young mobile broiler chicks might increase activity at a later age and might therefore reduce the occurrence of leg abnormalities.     

Behavioral syndrome in a native and an invasive hymenoptera species

Recent studies have focused on the role of behavior in biological invasions. Individuals may differ consistently in time for several behavioral traits (personality) which covary (behavioral syndrome) resulting in different behavioral types, some of them favoring invasion. Social hymenopterans have a strong potential to be invaders and their success depends primarily on the foundresses’ ability to found viable colonies. They are expected to be active, explorative and bold for optimally establishing their nest. In Europe, 2 hornet species coexist: the native Vespa crabro and the invasive Vespa velutina. These 2 species may compete for nesting sites and we suggest that the initial success of V. velutina has been favored by its behavior in outperforming V. crabro for the traits involved in nest initiation. Here, we (i) defined the personality of V. crabro and V. velutina, (ii) tested for the existence of behavioral syndrome in these species, and (iii) compared their performances using an open‐field test. Our results show that V. crabro foundresses behave consistently but not V. velutina; this lack of consistency being mainly due to reduced variance among individuals. This result questions the possibility of detecting consistent behavioral differences in species having recently undergone a strong bottleneck. Both species exhibit the same correlations between activity, boldness and exploration and V. velutina clearly outperforms V. crabro for all traits. Our results suggest that activity, boldness, and exploration are implicated in both hornet nest initiation and invasion process which contributed to explain why social hymenopterans are so successful at colonization.     

Topographic prominence as a method for cluster identification in single‐molecule localisation data

Single‐molecule localisation based super‐resolution fluorescence imaging produces maps of the coordinates of fluorescent molecules in a region of interest. Cluster analysis algorithms provide information concerning the clustering characteristics of these molecules, often through the generation of cluster heat maps based on local molecular density. The goal of this study was to generate a new cluster analysis method based on a topographic approach. In particular, a topographic map of the level of clustering across a region is generated based on Getis' variant of Ripley's K‐function. By using the relative heights (topographic prominence, TP) of the peaks in the map, cluster characteristics can be identified more accurately than by using previously demonstrated height thresholds. Analogous to geological TP, the concepts of wet and dry TP and topographic isolation are introduced to generate binary maps. The algorithm is validated using simulated and experimental data and found to significantly outperform previous cluster identification methods.

Illustration of the topographic prominence based cluster analysis algorithm.