Location of ribosome binding sites on the right end of lambda DNA.

The locations of ribosome binding sites on the right end of bacteriophage lambda DNA were determined by measurement of the positions of ribosomes bound to single-stranded DNA visualized by electron microscopy. A total of ten ribosome binding sites were found between map co-ordinates 0.90 and 1.0. Four of these ribosome binding sites probably correspond to the polypeptide initiation sites for genes Q (0.910), S (0.928), R (0.936) and Rz (0.945). Six other ribosome binding sites were found which presumably indicate the presence of new lambda genes. Four of these ribosome binding sites (0.958, 0.967, 0.975, 0.995) are located to the right of Rz, which is the most rightward known lambda gene. A ribosome binding site is located at 0.923, between genes Q and S, in or near the 6 S RNA sequence. Another is located left of gene Q at 0.900.     

Two-Electron Reactions S2QB → S0QB and S3QB → S1QB are Involved in Deactivation of Higher S States of the Oxygen-Evolving Complex of Photosystem II

The oxygen-evolving complex of Photosystem II cycles through five oxidation states (S₀-S₄), and dark incubation leads to 25% S₀ and 75% S₁. This distribution cannot be reached with charge recombination reactions between the higher S states and the electron acceptor Q_B⁻. We measured flash-induced oxygen evolution to understand how S₃ and S₂ are converted to lower S states when the electron required to reduce the manganese cluster does not come from Q_B⁻. Thylakoid samples preconditioned to make the concentration of the S₁ state 100% and to oxidize tyrosine Y_D were illuminated by one or two laser preflashes, and flash-induced oxygen evolution sequences were recorded at various time intervals after the preflashes. The distribution of the S states was calculated from the flash-induced oxygen evolution pattern using an extended Kok model. The results suggest that S₂ and S₃ are converted to lower S states via recombination from S₂Q_B⁻ and S₃Q_B⁻ and by a slow change of the state of oxygen-evolving complex from S₃ and S₂ to S₁ and S₀ in reactions with unspecified electron donors. The slow pathway appears to contain two-electron routes, S₂Q_B → S₀Q_B, and S₃Q_B → S₁Q_B. The two-electron reactions dominate in intact thylakoid preparations in the absence of chemical additives. The two-electron reaction was replaced by a one-electron-per-step pathway, S₃Q_B → S₂Q_B → S₁Q_B in PS II-enriched membrane fragments and in thylakoids measured in the presence of artificial electron acceptors. A catalase effect suggested that H₂O₂ acts as an electron donor for the reaction S₂Q_B → S₀Q_B but added H₂O₂ did not enhance this reaction.     

Nucleotide sequence analysis of <Emphasis Type="Italic">S</Emphasis>-locus genes to unify <Emphasis Type="Italic">S</Emphasis> haplotype nomenclature in radish

Radish, belonging to the family Brassicaceae, has a self-incompatibility which is controlled by multiple alleles on the S locus. To employ the self-incompatibility in an F₁ breeding system, identification of S haplotypes is necessary. Since collection of S haplotypes and determination of nucleotide sequences of SLG, SRK, and SCR alleles in cultivated radish have been conducted by different groups independently, the same or similar sequences with different S haplotype names and different sequences with the same S haplotype names have been registered in public databases, resulting in confusion of S haplotype names for researchers and breeders. In the present study, we developed S homozygous lines from radish F₁ hybrid cultivars in Japan and determined the nucleotide sequences of SCR, the S domain and the kinase domain of SRK, and the SLG of a large number of S haplotypes. Comparing these sequences with our previously published sequences, the haplotypes were ordered into 23 different S haplotypes. The sequences of the 23 S haplotypes were compared with S haplotype sequences registered by different groups, and we suggested a unification of these S haplotypes. Furthermore, dot-blot hybridization using SRK allele-specific probes was examined for developing a standard method for S haplotype identification.     

Determination of partial genomic sequences and development of a CAPS system of the S-RNase gene for the identification of 22 S haplotypes of apple (Malus × domestica Borkh.)

Information about self-incompatibility (S) genotypes of apple cultivars is important for the selection of pollen donors for fruit production and breeding. Although S genotyping systems using S haplotype-specific PCR of S-RNase, the pistil S gene, are useful, they are sometimes associated with false-positive/negative problems and are unable to identify new S haplotypes. The CAPS (cleaved amplified polymorphic sequences) system is expected to overcome these problems, however, the genomic sequences needed to establish this system are not available for many S-RNases. Here, we determined partial genomic sequences of eight S-RNases, and used the information to design new primer and to select 17 restriction enzymes for the discrimination of 22 S-RNases by CAPS. Using the system, the S genotypes of three cultivars were determined. The genomic sequence-based CAPS system would be useful for S genotyping and analyzing new S haplotypes of apple.     

Molecular Diagnosis of Pathogenic Sporothrix Species

Background

Sporotrichosis is a chronic (sub)cutaneous infection caused by thermodimorphic fungi in the order, Ophiostomatales. These fungi are characterized by major differences in routes of transmission, host predilections, species virulence, and susceptibilities to antifungals. Sporothrix species emerge in the form of outbreaks. Large zoonoses and sapronoses are ongoing in Brazil and China, respectively. Current diagnostic methods based on morphology and physiology are inaccurate due to closely related phenotypes with overlapping components between pathogenic and non-pathogenic Sporothrix. There is a critical need for new diagnostic tools that are specific, sensitive, and cost-effective.

Methodology

We developed a panel of novel markers, based on calmodulin (CAL) gene sequences, for the large-scale diagnosis and epidemiology of clinically relevant members of the Sporothrix genus, and its relative, Ophiostoma. We identified specific PCR-based markers for S. brasiliensis, S. schenckii, S. globosa, S. mexicana, S. pallida, and O. stenoceras. We employed a murine model of disseminated sporotrichosis to optimize a PCR assay for detecting Sporothrix in clinical specimens.

Results

Primer-BLAST searches revealed candidate sequences that were conserved within a single species. Species-specific primers showed no significant homology with human, mouse, or microorganisms outside the Sporothrix genus. The detection limit was 10–100 fg of DNA in a single round of PCR for identifying S. brasiliensis, S. schenckii, S. globosa, S. mexicana, and S. pallida. A simple, direct PCR assay, with conidia as a source of DNA, was effective for rapid, low-cost genotyping. Samples from a murine model of disseminated sporotrichosis confirmed the feasibility of detecting S. brasiliensis and S. schenckii DNA in spleen, liver, lungs, heart, brain, kidney, tail, and feces of infected animals.

Conclusions

This PCR-based method could successfully detect and identify a single species in samples from cultures and from clinical specimens. The method proved to be simple, high throughput, sensitive, and accurate for diagnosing sporotrichosis.     

What can we still learn from the electrochromic band-shifts in Photosystem II?

Electrochromic band-shifts have been investigated in Photosystem II (PSII) from Thermosynechoccocus elongatus. Firstly, by using Mn-depleted PsbA1-PSII and PsbA3-PSII in which the Q_X absorption of Phe_D1 differs, a band-shift in the Q_X region of Phe_D2 centered at ~ 544 nm has been identified upon the oxidation, at pH 8.6, of Tyr_D. In contrast, a band-shift due to the formation of either Q_A^•- or Tyr_Z^• is observed in PsbA3-PSII at ~ 546 nm, as expected with E130 H-bonded to Phe_D1 and at ~ 544 nm as expected with Q130 H-bonded to Phe_D1. Secondly, electrochromic band-shifts in the Chla Soret region have been measured in O₂-evolving PSII in PsbA3-PSII, in the PsbA3/H198Q mutant in which the Soret band of P_D1 is blue shifted and in the PsbA3/T179H mutant. Upon Tyr_Z^•Q_A^•- formation the Soret band of P_D1 is red shifted and the Soret band of Chl_D1 is blue shifted. In contrast, only P_D1 undergoes a detectable S-state dependent electrochromism. Thirdly, the time resolved S-state dependent electrochromism attributed to P_D1 is biphasic for all the S-state transitions except for S₁ to S₂, and shows that: i) the proton release in S₀ to S₁ occurs after the electron transfer and ii) the proton release and the electron transfer kinetics in S₂ to S₃, in T. elongatus, are significantly faster than often considered. The nature of S₂Tyr_Z^• is discussed in view of the models in the literature involving intermediate states in the S₂ to S₃ transition.     

Phenotypic and Genotypic Characteristics of Members of the Genus Streptobacillus

The genus Streptobacillus (S.) remained monotypic for almost 90 years until two new species were recently described. The type species, S. moniliformis, is one of the two etiological agents of rat bite fever, an under-diagnosed, worldwide occurring zoonosis. In a polyphasic approach field isolates and reference strains of S. moniliformis, S. hongkongensis, S. felis as well as divergent isolates were characterized by comparison of molecular data (n = 29) and from the majority also by their physiological as well as proteomic properties (n = 22). Based on growth-independent physiological profiling using VITEK2-compact, API ZYM and the Micronaut system fastidious growth-related difficulties could be overcome and streptobacilli could definitively be typed despite generally few differences. While differing in their isolation sites and dates, S. moniliformis isolates were found to possess almost identical spectra in matrix-assisted laser desorption ionization—time of flight mass spectrometry and Fourier transform infrared spectroscopy. Spectroscopic methods facilitated differentiation of S. moniliformis, S. hongkongensis and S. felis as well as one divergent isolate. Sequencing of 16S rRNA gene as well as functional genes groEL, recA and gyrB revealed only little intraspecific variability, but generally proved suitable for interspecies discrimination between all three taxa and two groups of divergent isolates.     

S genotyping in Japanese plum and sweet cherry by allele-specific hybridization using streptavidin-coated magnetic beads

Key message

We report a rapid and reliable method for S genotyping of Rosaceae fruit trees, which would to be useful for successful planting of cross-compatible cultivars in orchards.

Abstract

Japanese plum (Prunus salicina) and sweet cherry (Prunus avium), belonging to the family Rosaceae, possess gametophytic self-incompatibility controlled by a single polymorphic locus containing at least two linked genes, S-RNase and SFB (S-haplotype-specific F-box gene). For successful planting of cross-compatible cultivars of Rosaceae fruit trees in commercial orchards, it is necessary to obtain information on S genotypes of cultivars. Recently, a method of dot-blot analysis utilizing allele-specific oligonucleotides having sequences of SFB-HVa region has been developed for identification of S haplotypes in Japanese plum and sweet cherry. However, dot-blot hybridization requires considerable time and skill for analysis even of a small number of plant samples. Thus, a quick and efficient method for S genotyping was developed in this study. In this method, instead of a nylon membrane used for dot-blot hybridization, streptavidin-coated magnetic beads are used to immobilize PCR products, which are hybridized with allele-specific oligonucleotide probes. Our improved method allowed us to identify 10 S haplotypes (S-a, S-b, S-c, S-d, S-e, S-f, S–h, S-k, S-7 and S-10) of 13 Japanese plum cultivars and 10 S haplotypes (S-1, S-2, S-3, S-4, S-4′, S-5, S-6, S-7, S-9 and S-16) of 13 sweet cherry cultivars utilizing SFB or S-RNase gene polymorphism. This method would be suitable for identification of S genotypes of a small number of plant samples.     

Dissimilarity of Species and Forms of Planktonic Neocalanus Copepods Using Mitochondrial COI, 12S,Nuclear ITS,and 28S Gene Sequences

Background

A total of six Neocalanus species inhabit the oceans of the world. Of these, three species plus form variants (N. cristatus, N. plumchrus, N. flemingeri large form, and N. flemingeri small form), which constitute a monophyletic group among Neocalanus copepods, occur in the Northwestern Pacific off Japan. In the present study, we have tried to discriminate the three species plus form variants of Neocalanus copepods based on sequences of four DNA marker regions.

Methodology/Principal Findings

Discrimination was performed based on the DNA sequence information from four genetic markers, including the mitochondrial COI, 12S, nuclear ITS, and 28S gene regions. Sequence dissimilarity was compared using both distance- and character-based approaches. As a result, all three species were confirmed to be distinct based on the four genetic marker regions. On the contrary, distinction of the form variants was only confirmed based on DNA sequence of the mitochondrial COI gene region.

Conclusions/Significance

Although discrimination was not successful for the form variants based on the mitochondrial 12S, nuclear ITS, and 28S genes, diagnostic nucleotide sequence characters were observed in their mitochondrial COI gene sequences. Therefore, these form variants are considered to be an important unit of evolution below the species level, and constitute a part of the Neocalanus biodiversity.     

Construction of various bacteriophage λ mutants for stable and efficient production of recombinant protein in Escherichia coli

Three λ mutants were constructed based on the Q⁻ mutant in order to enhance their productivity and stability in an Escherichia coli/bacteriophage λ system. The newly constructed bacteriophage mutants named λSNU1, λSNU2, and λSNU3 were Q⁻S⁻, Q⁻W⁻E⁻, and Q⁻S⁻W⁻E⁻ mutants, respectively. Compared to all of the mutants, λSNU1 turned out to be the best with regards to higher protein expression and better genetic stability. Mechanisms by which these attributes are achieved have been discussed. The high productivity of P90c/λSNU1 for the recombinant protein was due to the high copy number of λ DNA and high translational efficiency. This mutant phage λSNU1 can be used to provide a high level of stability and productivity of the cloned gene particularly for long-term continuous operation.     

Silver(I) derivatives with new functionalised acylpyrazolonates

Silver(I) acylpyrazolonate derivatives of formula [Ag(Q)(R₃P)]₂ and [Ag(Q)(R₃P)₂], (QH=1-phenyl-3-methyl-4-R′(CO)-pyrazol-5-one; Q^OH, R′=furane; Q^SH, R′=thiophene; R=Ph, Cy, o-tol), have been synthesised and characterised, both in the solid state and in solution. The derivatives [Ag(Q)(R₃P)]₂ contain dinuclear AgO₂NP units with the acylpyrazolonate coordinating in a bridging O,O′-Q-N fashion. The [Ag(Q)(R₃P)₂] are tetrahedral species, with the distortion from ideal geometry increasing with the bulk of the phosphine. The [Ag(Q)(R₃P)₂] derivatives are fluxional in chloroform solution when R₃P is sterically hindered (R=Cy or o-tol), dissociating partially to the [Ag(Q)(R₃P)] fragment and free R₃P. [Ag(Q^S)(Ph₃P)]₂ reacts with 1-methyl-2-mercaptoimidazole (Hmimt) affording the compound [Ag(Hmimt)(Ph₃P)(Q^S)] and [Ag(Q^O)(Ph₃P)]₂ reacts with 1-methyl-imidazole (Meim) affording the compound [Ag(Meim)(Ph₃P)(Q^O)], whereas [Ag(Q^S)(Ph₃P)]₂ reacts with 1,10-phenanthroline (phen), affording the compound [Ag(phen)(Ph₃P)](Q^S). Finally [Ag(Q^S)(Ph₃P)₂] reacts with phen producing the ionic species [Ag(phen)(Ph₃P)₂](Q^S).     

Sequence Analysis of New S-RNase and SFB alleles in Japanese Apricot (Prunus mume)

As with other self-incompatible Prunus species, cultivars of Japanese apricot (Prunus mume Sieb. et Zucc.) display the S-RNase-based gametophytic self-incompatibility system. In this study, S-genotypes of ten Japanese apricot cultivars native to China were subjected to polyacrylamide gel electrophoresis (PAGE) analysis using an efficient Prunus S-RNase primer pair, Pru-C2 and PCE-R. In addition, three new S-RNase genes (S ₃₄ , S ₃₅ and S ₃₆ -RNase) and six new SFB genes (PmSFB14, PmSFB18, PmSFB22, PmSFB24, PmSFB31 and PmSFB34) were identified and their sequences were characterized and deposited in the GenBank database.     

Combining sap flow and trunk diameter measurements to assess water needs in mature olive orchards

Sap flux (Q) and trunk diameter variation (TDV) are among the most useful plant-based measurements to detect water stress and to evaluate plant water consumption. The usefulness of both methods decreases, however, when applied to species that, like olive, have an outstanding tolerance to drought and a remarkable capacity to take up water from drying soils. Evidence shows that this problem is greater in old, big trees with heavy fruit load. Our hypothesis is that the analysis of simultaneous measurements of Q and TDV made in the same trees is more useful for assessing irrigation needs in old olive orchards than the use of any of these two methods alone. To test our hypothesis, we analysed relations between Q, TDV, midday stem water potential (Ψ_stem), relative extractable water and atmospheric demand in an olive orchard of 38-year-old ‘Manzanilla’ trees with heavy fruit load. Measurements were made during one irrigation season (May-October), in fully irrigated trees (FI, 107% of the crop evapotranspiration, ET_c, supplied by irrigation), and in trees under two levels of deficit irrigation (DI60, 61% ET_c; DI30, 29% ET_c). Time courses of Q and TDV measured on days of contrasting weather and soil water conditions were analysed to evaluate the usefulness of both methods to assess the crop water status. We calculated the daily tree water consumption (E_p) from Q measurements. For both DI treatments we calculated a signal intensity by dividing daily E_p values of each DI tree by those of the FI tree (SI−Ep). We did the same with the maximum daily shrinkage (MDS) values (S_I−MDS). Neither SI−Ep nor S_I−MDS rendered useful information for assessing the crop water needs. On the contrary, the daily difference for maximum trunk diameter (MXTD) between each of the DI trees and the FI tree (D_MXTD) clearly indicated the onset and severity of water stress. A similar analysis with the E_p values, from which DEp values were derived, showed the effect of water stress on the water consumption of the trees. We concluded that the simultaneous use of D_MXTD and DEp values provides more detailed information to assess water needs in mature olive orchards than the use of Q or TDV records alone.     

Evolutionary genetics of an S-like polymorphism in Papaveraceae with putative function in self-incompatibility

Background

Papaver rhoeas possesses a gametophytic self-incompatibility (SI) system not homologous to any other SI mechanism characterized at the molecular level. Four previously published full length stigmatic S-alleles from the genus Papaver exhibited remarkable sequence divergence, but these studies failed to amplify additional S-alleles despite crossing evidence for more than 60 S-alleles in Papaver rhoeas alone.

Methodology/Principal Findings

Using RT-PCR we identified 87 unique putative stigmatic S-allele sequences from the Papaveraceae Argemone munita, Papaver mcconnellii, P. nudicuale, Platystemon californicus and Romneya coulteri. Hand pollinations among two full-sib families of both A. munita and P. californicus indicate a strong correlation between the putative S-genotype and observed incompatibility phenotype. However, we also found more than two S-like sequences in some individuals of A. munita and P. californicus, with two products co-segregating in both full-sib families of P. californicus. Pairwise sequence divergence estimates within and among taxa show Papaver stigmatic S-alleles to be the most variable with lower divergence among putative S-alleles from other Papaveraceae. Genealogical analysis indicates little shared ancestral polymorphism among S-like sequences from different genera. Lack of shared ancestral polymorphism could be due to long divergence times among genera studied, reduced levels of balancing selection if some or all S-like sequences do not function in incompatibility, population bottlenecks, or different levels of recombination among taxa. Preliminary estimates of positive selection find many sites under selective constraint with a few undergoing positive selection, suggesting that self-recognition may depend on amino acid substitutions at only a few sites.

Conclusions/Significance

Because of the strong correlation between genotype and SI phenotype, sequences reported here represent either functional stylar S-alleles, tightly linked paralogs of the S-locus or a combination of both. The considerable complexity revealed in this study shows we have much to learn about the evolutionary dynamics of self-incompatibility systems.     

Co-operative response of chemically excitable membrane III. Three-state model

The general three-state model is formulated first, which is the direct extension of the unified two-state model previously formulated (Kijima & Kijima, 1978). In this model, each protomer in a symmetrically interacting system (oligomers or lattices) can take three states, S, R and Q, where S and R states are the same as in the two-state model and Q state is another state either corresponding to a different open-state of ionophore from R open-state or corresponding to another closed state of ionophore. The model has no restriction on the value of Hill coefficient at the midpoint of the dose-response curves in contrast to two-state models. It is applied on GABA sensitive inhibitory synapse of crayfish muscle to account for anomalous behaviour of the membrane in I^? solution.The simplified versions of the above general three-state model are also formulated (simplified three-state model), in which it is assumed that R and Q state are equivalent in regard to the nearest neighbor interaction. By this assumption, R and Q state are collectively treated as state A and mathematical formula obtained on Ising model are applicable on this model. This model is applied on the insect sugar receptor which was shown to be incompatible with the two-state models (Kijima & Kijima, 1980). Further simplification of the above simplified model results in two convenient models: three-state KNF model and three-state MWC model, which have minimum parameters but sufficient to account for most experiments. They give plausible physico-chemical base on the “classical model” in which the existence of both inactive and active ligand-receptor complex is assumed.     

Pressure effects on the photochemistry of bacterial photosynthetic reaction centers from Rhodobacter sphaeroides R-26 and Rhodopseudomonas viridis

Electron-transfer reactions and triplet decay rates have been studied at pressures up to 300 MPa. In reaction centers from Rhodobacter sphaeroides R-26, high pressure hastened the electron transfers from both the primary and secondary quinones (Q_A and Q_B) to the primary electron donor bacteriochlorophyll, P. Motion of Q_A between two sites, one nearer to P and the other nearer to Q_B, could account for these pressure effects. In reaction centers from Rhodopseudomonas viridis, charge recombination was slowed by high pressure. Decay rates were also studied for the triplet state, P^R. In Rb. sphaeroides R-26 with Q_A reduced with Na₂S₂O₄, the decay was hastened by pressure. This could be explained if P^R decays through a charge-transfer triplet state, or if the decay kinetics of P^R are sensitive to the distance between P and Q_A⁻. In Rps. viridis reaction centers, and in Rb. sphaeroides reaction centers that were depleted of Q_A, the lifetime of P^R was not altered by pressure.     

Evidence from thermoluminescence for bicarbonate action on the recombination reactions involving the secondary quinone electron acceptor of Photosystem II

In this paper, we present the first measurements on thermoluminescence from isolated thylakoids to probe the recombination reactions of S₂ (or possibly S₃) with Q^?_B or Q^?_A, after bicarbonate depletion and its readdition. The effects of bicarbonate depletion on the S₂Q^?_B (or S₃O^?_B) thermoluminescence band was (1) a 6–10°C shift to a higher temperature; (2) a reduction in its intensity upon prolonged depletion; and (3) elimination after the first few flashes of the characteristic period four oscillations in its intensity as a function of the flash number. On the other hand, addition of diuron (3-(3′,4′-dichlorophenyl)-1,1-dimethylurea), which blocks electron flow from Q^?_A to Q_B, produced the same thermoluminescence band, at about + 20°C, assigned to S₂Q^?_A recombination, in both depleted and reconstituted samples. These results suggest (1) the initial effect of bicarbonate depletion is to increase the activation energy for S₂(S₃)Q^?_B recombination; (2) with further depletion, the incidence of this recombination decreases and the cycling of the S₂Q^?_B and S₃Q^?_B recombination is inhibited through effects at the Q_B apoprotein; and (3) the depletion effects are fully reversible. It is suggested that a conformational change of the PS II complex in the region of the Q_B apoprotein is responsible for these effects.     

Diversity and Systematics of Schizomavella Species (Bryozoa: Bitectiporidae) from the Bathyal NE Atlantic

Eight NE Atlantic and Mediterranean species, which were originally assigned to the genus Schizoporella (Family Schizoporellidae) when introduced, are redescribed and stabilized by typification. Seven of these species are transferred to the bitectiporid genus Schizomavella: S. fischeri, S. glebula, S. neptuni, S. obsoleta, S. richardi, S. triaviculata, and S. triaviculata var. paucimandibulata, which is here raised to species rank. The eighth species, Schizoporella fayalensis, is transferred to the lanceoporid genus Stephanotheca. Schizomavella obsoleta and S. glebula are considered junior subjective synonyms of S. fischeri and S. richardi, respectively. Two new species are described: Schizomavella rectangularis n. sp. from the Strait of Gibraltar, and Schizomavella phterocopa n. sp. from the Great Meteor Bank. A new subgenus, Calvetomavella n. subgen. is established as a result of a phylogenetic analysis based on morphological characters; it includes S. neptuni, S. triaviculata, S. paucimandibulata and S. phterocopa n. sp., together with Schizomavella discoidea and Schizomavella noronhai. The rest of the species remain in the nominotypical subgenus Schizomavella.