Novel mutations of Escherichia coli that produce recombinogenic lesions in DNA. I. Identification and mapping of arl mutations

Hyper-rec mutants of Escherichia coli were originally identified as lac-diploid strains whose colonies exhibited unusually high numbers of Lac⁺ papillae during growth on indicator plates (Konrad, 1977). For this work, 38 hyper-rec strains with particularly high frequencies of papillation were selected and screened further, in order to identify those unusually proficient in recombination of bacteriophage λ. The screening procedure, plate-stock growth of λ duplication phages, yielded four strains that exhibited both enhanced recombination of λ and normal (or higher) yields of progeny phage. The mutants displayed the same novel phenotype: phage recombination was normal during the first lytic infection, but was stimulated four- to sixfold if the phages had previously been propagated for several cycles in the mutants. Phages thus appeared to accumulate an enhanced potential for recombination during growth in these four strains. The mutations responsible were designated arl. Enhanced recombination of the phages propagated on arl strains occurred in subsequent test infections of both arl and arl⁺ bacteria, but not in recA cells. Both the high frequency of Lac⁺ papillae and the effects on λ recombination appeared to result from the same mutations. The former phenotype was used for genetic analysis of two arl mutants; their location is near 2 minutes on the E. coli map. Known alleles of two nearby genes, polB and mutT, do not confer a hyper-rec phenotype (by the lac-diploid assay). High-level RecA-constitutive strains do not exhibit enhanced recombination of duplication phages.     

湖南道县后背山福岩洞2011年发掘报告

福岩洞位于湖南省道县乐福堂乡, 2011年9~10月间, 中国科学院古脊椎动物与古人类研究所和湖南省文物考古研究所在道县文物管理局的配合下, 对该洞穴进行了发掘, 揭露面积约20㎡。调查显示, 福岩洞属于大型管道型溶洞, 发育于上古生界碳酸盐岩系内, 遗址周边属于峰林和溶盆地貌, 处于岩溶发育的后期。洞内堆积发育, 以砂砾石层和红黏土为主。本次发掘出土5枚古人类牙齿化石和大量哺乳动物化石, 其中人类牙齿总体形态特征与现代人(Homo sapiens)接近; 哺乳动物化石中已鉴定出39种(含未定种), 其中灭绝种只有巴氏大熊猫、最后鬣狗、剑齿象、巨貘、南方猪等; 结合铀系法初步年代测定和动物群面貌判断, 古人类活动的年代大致为晚更新世。本次发掘表明, 该遗址仍有较大的工作潜力, 对其进一步发掘和研究将为探讨古人类在该区域的演化和生活方式提供有价值的科学信息。     

Docking of a single phage lambda to its membrane receptor maltoporin as a time-resolved event

We have been able to observe the first step in bacteriophage infection, the docking of phage lambda to its membrane receptor maltoporin, at the single-particle level. High-resolution conductance recording from a single trimeric maltoporin channel reconstituted into a planar lipid bilayer has allowed detection of the simultaneous and irreversible interaction of the phage tail with all three monomers of the receptor. The formation of a phage-maltoporin complex affects the channel transport properties. Our analysis demonstrates that phage attaches symmetrically to all three receptor monomers. The statistics of sugar binding to the phage-receptor complex on the side opposite to phage docking show that the monomers of maltoporin still bind sugar independently, with the kinetic constants expected from those of the phage-free receptor. This finding suggests that phage docking does not distort the structure of the receptor, and that the phage-binding regions are close to, but do not overlap with, the sugar-binding domains of the maltoporin monomers. However, ion fluxes through the pores of maltoporin in the phage-receptor complex share a new common pathway. We expect that the present study contributes to the current needs for structural information on the functional complexes involved in intercellular recognition.     

Transposition mutagenesis of bacteriophage lambda: a new gene affecting cell lysis.

Insertions of Tn903, a transposable kanamycin-resistance element, in bacteriophage lambda at 0.95 on the lambda physical map adversely affect growth of the phage. These insertion mutants are able to assemble particles, but are unable to lyse the infected cell properly. The mutants define a new genetic complementation group that we have designated as gene Rz. Cells infected with the λRz:: Tn903 isolates will, at the normal time of lysis, change their shape from a rod to a sphere. These spheres are stable in dilute buffers with Mg²⁺ but are lysed with EDTA. In addition, these results demonstrate the utility of transposition mutagenesis in refining the genetic map of even so intensely studied a genome as lambda.     

Nebulin: A Study of Protein Repeat Evolution

Protein domain repeats are common in proteins that are central to the organization of a cell, in particular in eukaryotes. They are known to evolve through internal tandem duplications. However, the understanding of the underlying mechanisms is incomplete. To shed light on repeat expansion mechanisms, we have studied the evolution of the muscle protein Nebulin, a protein that contains a large number of actin-binding nebulin domains.Nebulin proteins have evolved from an invertebrate precursor containing two nebulin domains. Repeat regions have expanded through duplications of single domains, as well as duplications of a super repeat (SR) consisting of seven nebulins. We show that the SR has evolved independently into large regions in at least three instances: twice in the invertebrate Branchiostoma floridae and once in vertebrates.In-depth analysis reveals several recent tandem duplications in the Nebulin gene. The events involve both single-domain and multidomain SR units or several SR units. There are single events, but frequently the same unit is duplicated multiple times. For instance, an ancestor of human and chimpanzee underwent two tandem duplications. The duplication junction coincides with an Alu transposon, thus suggesting duplication through Alu-mediated homologous recombination.Duplications in the SR region consistently involve multiples of seven domains. However, the exact unit that is duplicated varies both between species and within species. Thus, multiple tandem duplications of the same motif did not create the large Nebulin protein.Finally, analysis of segmental duplications in the human genome reveals that duplications are more common in genes containing domain repeats than in those coding for nonrepeated proteins. In fact, segmental duplications are found three to six times more often in long repeated genes than expected by chance.     

The major fibrinolytic proteases of human leukocytes

The major fibrinolytic enzymes present in leukocyte granules and active at physiological pH have been identified. The fibrinolytic activity in extracts of leukocyte granules was bound to fibrinogen-Sepharose and eluted with 8.0 M urea. Two distinct zones of fibrinolytic activity were detected upon electrophoresis of leukocyte extracts on fibrinogen polyacrylamide gels, and both were qualitatively recovered in the 8.0 M urea eluate. Quantitatively, greater than 95% of the fibrinolytic activity was recovered in the urea eluate. Two major leukocyte proteases, elastase (EC 3.4.21.11) and cathepsin G (EC 3.4.21.-), were quantitatively recovered in the urea eluate. Both enzymes, when purified separately by affinity chromatography, were shown to: (a) possess fibrinolytic activity; (b) coincide in mobility and generate the two zones of fibrinolytic activity on fibrinogen polyacrylamide gels; and (c) quantitatively reconstitute the fibrinolytic activity of the leukocyte granules when combined at activity levels present in granular extracts. A highly significant correlation (r = 0.98) was found between the fibrinolytic activity and the sum of elastase and cathepsin G activity in leukocytes from five donors. Thus, elastase and cathepsin G are the major enzymes of the leukocyte fibrinolytic pathway, and fibrinogen-Sepharose chromatography may be used to obtain these enzymes.     

Morphological and functional effects of extracellular matrix on pancreatic islet cell cultures

Extracellular matrix (ECM) has been reported to enhance epithelial cell attachment and proliferation as well as to induce differentiation in vitro. In an attempt to determine the benefits of culturing pancreatic islet cells on ECM, we studied the morphological and functional patterns of rat islet cells and an insulin-secreting tumor cell line. ECM enhanced islet cell attachment and proliferation when compared to plastic, as suggested by a higher specific activity of DNA synthesis and a higher mitotic index. Cells on ECM were heterogeneous in size and insulin content. They showed extended areas of confluence. Cultures on plastic demonstrated an organisation in clusters and low mitotic activity. However, ECM did not allow for reconstitution of an islet-like structure. When compared to plastic, an initial decrease in basal and stimulated insulin secretion per million cells was observed on ECM, but B-cell activity was restored after 6 days of culture. Glucagon and somatostatin secretion were similar on both substrates. These data suggest that ECM enhances markedly islet cells attachment and proliferation, as well as long-term culture maintenance.     

Energy transfer dynamics in a red-shifted violaxanthin-chlorophyll a light-harvesting complex

Photosynthetic eukaryotes whose cells harbor plastids originating from secondary endosymbiosis of a red alga include species of major ecological and economic importance. Since utilization of solar energy relies on the efficient light-harvesting, one of the critical factors for the success of the red lineage in a range of environments is to be found in the adaptability of the light-harvesting machinery, formed by the proteins of the light-harvesting complex (LHC) family. A number of species are known to employ mainly a unique class of LHC containing red-shifted chlorophyll a (Chl a) forms absorbing above 690?nm. This appears to be an adaptation to shaded habitats. Here we present a detailed investigation of excitation energy flow in the red-shifted light-harvesting antenna of eustigmatophyte Trachydiscus minutus using time-resolved fluorescence and ultrafast transient absorption measurements. The main carotenoid in the complex is violaxanthin, hence this LHC is labeled the red-violaxanthin-Chl a protein, rVCP. Both the carotenoid-to-Chl a energy transfer and excitation dynamics within the Chl a manifold were studied and compared to the related antenna complex, VCP, that lacks the red-Chl a. Two spectrally defined carotenoid pools were identified in the red antenna, contributing to energy transfer to Chl a, mostly via S₂ and hot S₁ states. Also, Chl a triplet quenching by carotenoids is documented. Two separate pools of red-shifted Chl a were resolved, one is likely formed by excitonically coupled Chl a molecules. The structural implications of these observations are discussed.     

Viral fitness can influence the repertoire of virus variants selected by antibodies

Minority genomes in the mutant spectra of viral quasispecies may differ in relative fitness. Here, we report experiments designed to evaluate the contribution of relative fitness to selection by a neutralizing monoclonal antibody (mAb). We have reconstructed a foot-and-mouth disease virus (FMDV) quasispecies, with two matched pairs of distinguishable mAb-escape mutants as minority genomes of the mutant spectrum. Each mutant of a pair differs from the other by 11-fold or 33-fold in relative fitness. Analysis of the mutant spectra of virus populations selected with different concentrations of antibody in infections in liquid culture medium has documented a dominance of the high fitness counterpart in the selected population. Plaque development as a function of increasing concentration of the antibody has shown that each mutant of a matched pair yielded the same number of plaques, although the high fitness mutant required less time for plaque formation, and attained a larger plaque size at any given time-point. This result documents equal intrinsic resistance to the antibody of each mutant of a matched pair, confirming previous biochemical, structural, and genetic studies, which indicated that the epitopes of each mutant pair were indistinguishable regarding reactivity with the monoclonal antibody. Thus, relative viral fitness can influence in a significant way the repertoire of viral mutants selected from a viral quasispecies by a neutralizing antibody. We discuss the significance of these results in relation to antibody selection, and to other selective forces likely encountered by viral quasispecies in vivo.     

DARPins as Bispecific Receptor Antagonists Analyzed for Immunoglobulin E Receptor Blockage

The concept of multispecific antibodies is of high therapeutic interest but has failed to produce pharmaceutical products due to the poor biophysical properties of such molecules. Here, we propose an alternative and simple way to generate bispecific binding molecules using designed ankyrin repeat proteins (DARPins). For this purpose, monovalent DARPins with different epitope specificities were selected against the α chain of the high-affinity receptor for human immunoglobulin E (IgE) (Fc?RIα). Two of the isolated binders interfering with IgE binding to the receptor were joined to each other or to themselves via a flexible protein linker. The resulting bivalent and bispecific DARPins were tested for their ability to prevent allergen-induced cell degranulation using rat basophilic leukemia cells stably transfected with human Fc?RIα. The bispecific DARPin construct was the most potent one, efficiently blocking the IgE-Fc?RI interaction and preventing the release of proinflammatory mediators. Noteworthy, the multivalent and multispecific DARPin construct did not show any alteration of the beneficial biophysical properties of the monovalent parental DARPins. Hence, bispecific DARPins may be used to generate receptor antagonists simultaneously targeting different epitopes on the same molecule. Moreover, they easily overcome the limiting immunoglobulin binding paradigm (one binding molecule = one epitope) and thereby represent an alternative to monoclonal antibodies in cases where the immunoglobulin scaffold is unsuitable.     

Induction of mutations in bacteriophage T7 by gamma-rays: no influence of the SOS repair pathway

Under conditions where the reversion of an amber mutant of bacteriophage lambda by gamma-rays is enhanced by subjecting the irradiated phage to SOS repair, gamma-ray-induced reversion of two T7 ambers is not influenced by this error-prone bacterial repair system. The survival of T7 gamma-irradiated under anoxic conditions is somewhat enhanced by SOS repair, whereas the survival of phage irradiated under oxygen is not affected.     

Regulatory light-chains and scallop myosin. Full dissociation, reversibility and co-operative effects

Lambda duplication phages grown for several rounds on Escherichia coli strains containing arl mutations were recombined at elevated frequencies (3 to 6-fold higher) in subsequent test infections. Enhanced recombination of Arl^? phages (grown on arl bacteria) was demonstrable by assays for altered genetic linkages as well as by the standard assay, which measures the conversion of duplication phages (EDTA-sensitive) to single-copy phages (EDTA-resistant). The accumulated potential for enhanced recombination was lost during subsequent growth of the phages on arl⁺ bacteria. Arl^? phages had the same mutation frequencies, at a variety of loci, as control phages; arl bacteria themselves exhibited normal mutation rates. Arl^? phages had normal plating efficiencies and buoyant densities. DNA extracted from Arl^? phages exhibited the same frequency of strand interruption, the same superhelical density (when circularized in vivo), and the same thermal denaturation profile as DNA from phages grown on arl⁺ bacteria. Recombination of Arl^? phages in the presence of λ repressor was very low, as is the case for normal phages. The recombination frequency of ultraviolet light irradiated (80 J/m²) Arl^? phages was more than twice the sum of the frequencies for unirradiated Arl^? phages and irradiated control phages. Substantially increased recombination of Arl^? phages was observed when either the E. coli RecBC, or RecE (but not RecF) pathway was active.     

Stylized facts in microalgal growth: interpretation in a dynamic energy budget context

A dynamic energy budget (DEB) model for microalgae is proposed. This model deviates from the standard DEB model as it needs more reserves to cope with the variation of assimilation pathways, requiring a different approach to growth based on the synthesizing unit (SU) theory for multiple substrates. It is shown that the model is able to accurately predict experimental data in constant and light-varying conditions with most of the parameter values taken directly from the literature. Also, model simulations are shown to be consistent with stylized facts (SFs) concerning NC ratio. These SFs are reinterpreted and the general conclusion is that all forcing variables (dilution rate, temperature and irradiance) impose changes in the nitrogen or carbon limitation status of the population, and consequently on reserve densities. Model predictions are also evaluated in comparison with SFs on chlorophyll concentration. It is proposed that an extra structure, more dependent on the nitrogen reserve, is required to accurately model chlorophyll dynamics. Finally, SFs concerning extracellular polymeric substances (EPSs) production by benthic diatoms are collected and interpreted and a formulation based on product synthesis and rejection flux is proposed for the EPSs production rate.     

A high-throughput assay for the detection of Tyr-phosphorylated proteins in urine of bladder cancer patients

Background

Bladder cancer has the peculiarity of shedding neoplastic cells and their components in urine representing a valuable opportunity to detect diagnostic markers. Using a semi-quantitative method we previously demonstrated that the levels of Tyr-phosphorylated proteins (TPPs) are highly increased in bladder cancer tissues and that soluble TPPs can also be detected in patient's urine samples. Although the preliminary evaluation showed very promising specificity and sensitivity, insufficient accuracy and very low throughput of the method halted the diagnostic evaluation of the new marker. To overcome this problem we developed a quantitative methodology with high sensitivity and accuracy to measure TPPs in urine.

Methods

The Immobilized Metal Affinity Chromatography (IMAC) was miniaturized in a 96 well format. Luminescence, visible and infrared fluorescence antibody-based detection methods were comparatively evaluated.

Results

Due to their low abundance we evidenced that both phosphoprotein enrichment step and very sensitive detection methods are required to detect TPPs in urine samples. To pursue high throughput, reproducibility and cost containment, which are required for bladder cancer screening programs, we coupled the pre-analytical IMAC procedure with high sensitive detection phases (infrared fluorescence or chemiluminescence) in an automated platform.

Conclusions

A high throughput method for measuring with high sensitivity TPP levels in urine samples is now available for large clinical trial for the establishment of the diagnostic and predictive power of TPPs as bladder cancer marker.

General significance

The new assay represents the first quantitative and high throughput method for the measurement of TPPs in urine.     

Consistency of mate choice in eye and hair colour: Testing possible mechanisms

Partner preferences are formed by several mechanisms, including an imprinting-like effect (parent-similarity) and homogamy (self-similarity). It is still unknown, however, whether these preferences remain stable throughout an individual’s lifetime. We have therefore tested the consistency of mate choice in eye and hair colour both in a shortand long-term context. In other words, we tested whether people systematically choose partners with a particular eye and hair colour. We asked 1,048 respondents to indicate the eye and hair colour of themselves, their opposite-sex and same-sex parent, and all the romantic partners they had in their lives. Our results show that people consistently choose partners of a particular eye and hair colour in both short- and long-term contexts, which suggests that people do have their ‘types’. Nevertheless, the consistency was significantly higher in a long-term context than in a short-term context. Furthermore, the eye colour of one's partner was predicted by the eye colour of one's opposite-sex as well as same-sex parent, but the strongest parental effect was found when both parents had same eye colour. There were no significant results for hair colour. Our results thus suggest that preferences for eye colour are determined by the imprinting-like effect rather than by homogamy, and that they remain stable over time. These findings also indirectly support an assumption of stability of this imprinting-like effect in humans, since people consistently choose partners with their opposite-sex parent's eye-colour.     

Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop alpha-8 of domain II

Bacillus thuringiensis subs israelensis produces Cry toxins active against mosquitoes. Receptor binding is a key determinant for specificity of Cry toxins composed of three domains. We found that exposed loop alpha-8 of Cry11Aa toxin, located in domain II, is an important epitope involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop alpha-8, beta-4 and loop 3) competed binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. The role of loop alpha-8 of Cry11A in receptor interaction was demonstrated by phage display and site-directed mutagenesis. We isolated a peptide-displaying phage (P5.tox), that recognizes loop alpha-8 in Cry11Aa, interferes interaction with the midgut receptor and attenuates toxicity in bioassay. Loop alpha-8 mutants affected in toxicity and receptor binding were characterized.     

Modelling mitochondrial site polymorphisms to infer the number of segregating units and mutation rate

We present a mathematical model of mitochondrial inheritance evolving under neutral evolution to interpret the heteroplasmies observed at some sites. A comparison of the levels of heteroplasmies transmitted from mother to her offspring allows us to estimate the number N_x of inherited mitochondrial genomes (segregating units). The model demonstrates the necessity of accounting for both the multiplicity of an unknown number N_x, and the threshold θ, below which heteroplasmy cannot be detected reliably, in order to estimate the mitochondrial mutation rate μ_m in the maternal line of descent. Our model is applicable to pedigree studies of any eukaryotic species where site heteroplasmies are observed in regions of the mitochondria, provided neutrality can be assumed. The model is illustrated with an analysis of site heteroplasmies in the first hypervariable region of mitochondrial sequence data sampled from Adélie penguin families, providing an estimate N_x and μ_m. This estimate of μ_m was found to be consistent with earlier estimates from ancient DNA analysis.