Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes

Summary When a non-selected DNA sequence was added during the transformation of amdS320 deletion strains of Aspergillus nidulans with a vector containing the wild-type amdS gene the AmdS⁺ transformants were cotransformed at a high frequency. Cotransformation of an amdS320, trpC801 double mutant strain showed that both the molar ratio of the two vectors and the concentration of the cotransforming vector affected the cotransformation frequency. The maximum frequency obtained was defined by the gene chosen as selection marker for transformation. Cotransformation was used to induce a gene replacement in A. nidulans. An amdS320 strain was transformed to AmdS⁺ and cotransformed with a DNA fragment containing a fusion between a non-functional A. nidulans trpC gene and the Escherichia coli lacZ gene. Ten AmdS⁺, LacZ⁺ transformants with a Trp^– mutant phenotype were selected. All of these strains could be transformed with a functional copy of the A. nidulans trpC gene, but only two strains yielded TrpC⁺ transformants which, with a low frequency, had a LacZ^– phenotype. These latter transformants had also lost the AmdS⁺ phenotype. Southern blotting analysis of DNA from these transformants confirmed the inactivation of the wild-type trpC gene, but revealed that amdS vector sequences were also involved in the gene replacement events.     

An improved method for light- and electron microscopial studies of nematode/fungal interactions

A method is presented that enables studies to be made of single nematode-fungal interactions under conditions where fungal growth at the expense of external nutrients is prevented. The nematophagous fungus Arthrobotrys ologospora was used as a model organism in these studies. The method is based on removal of the traps from the vegetative mycelium, immediately after a nematode was captured and transfer of the trap with the captured nematode into a droplet of sterile distilled water placed in a moisture chamber. In the absence of external nutrients, such isolated traps of A. oligospora were fully effective in penetrating and subsequently digesting the captured nematode. Solely vegetative mycelium was formed at the expense of the digested nematode; this developed from the trap that originally had captured the nematode. One advantage of the present method is that studies on various stages of the nematode-fungal interaction can now be performed under conditions that exclude major influences of external nutrients which otherwise could be communicated to the trap cells by way of the vegetative mycelium.     

A Kdp-like,high-affinity,K+-translocating ATPase is expressed during growth of Rhodobacter sphaeroides in low potassium media

Cells of the purple non-sulphur bacterium Rhodobacter sphaeroides express a high-affinity K⁺ uptake system when grown in media with low K⁺ concentrations. Antibodies againts the catalytic KdpB protein or the whole KdpABC complex of Escherichia coli crossreact with a 70.0 kDa R. sphaeroides protein that was expressed only in cells grown in media with low K⁺ concentrations. In membranes derived from R. sphaeroides cells grown with low K⁺ concentrations (induced cells), a high ATPase activity could be detected when assayed in Tris-HCl pH 8.0 containing 1 mM MgSO₄. This ATPase activity increased upon addition of 1 mM KCl from 166 to 289 mol ATP hydrolysed x min^-1 x g protein^-1 (1.7-fold stimulation). The K⁺-stimulated ATPase activity was inhibited approximately 93% by 0.5 mM vanadate but hardly by N,N-dicyclohexylcarbo-diimide (DCCD). These results indicate that the inducible K⁺-ATPase in R. sphaeroides resembles the Kdp K⁺-translocating ATPase of Escherichia coli. This Kdp-like transport system is also expressed in R. capsulatus and Rhodospirillum rubrum during growth in media with low K⁺ concentrations suggesting a wide distribution of this transport system among phototrophic bacteria.Abbreviations electrical potential difference across the cytoplasmic membrane - pH pH difference across the cytoplasmic membrane - BSA bovine serum albumine - PAGE polyacrylamide gel electrophoresis - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - PMSF phenyl-methyl-sulfonyl fluoride - DCCD N,N-dicyclohexylcarbodiimide - AIB 2--aminoisobutyric acid - TMG methyl--d-thiogalactopyranoside     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

The synthesis of some polyether bridged diphosphines and their reaction with Rh(COD)acac

The polyether bridged diphosphines,

(n = 1,2) have been prepared in 60–70% yield by reduction of the corresponding diphosphinedioxides with Si₂Cl₆ or (i-Bu)₂AlH. These diphosphinedioxides have been prepared in 75–90% yield by reaction of two equivalents of the appropriate

with one equivalent of di- and triethylene glycol ditosylate.In general, reaction between the diphosphines, Rh(COD)acac and HClO₄ gives a mixture of species, cis-[Rh(COD)($\text{PP}$)] [ClO₄] being the main complex. This complex reacts with CO to η³-trans- [$\text{Rh(CO)(}\text{PO}$$\text{P}$)] [ClO₄].     

The electrochemical H+ gradient in the yeastRhodotorula glutinis

The electrochemical gradient of protons, , was estimated in the obligatory aerobic yeastRhodotorula glutinis in the pH₀ range from 3 to 8.5. The membrane potential, , was measured by steady-state distribution of the hydrophobic ions, tetraphenylphosphonium (TPP⁺) for negative above pH₀ 4.5, and thiocyanate (SCN^–) for positive below pH₀ 4.5. The chemical gradient of H⁺ was determined by measuring the chemical shift of intracellular P_i by³¹P-NMR at given pH₀ values. The values of pH_i increased almost linearly from 7.3 at pH₀ 3 to 7.8 at pH₀ 8.5. In the physiological pH₀ range from 3.5 to 6, was fairly constant at values between 17–18 KJ mol^–1, gradually decreasing at pH₀ above 6. In deenergized cells, the intracellular pH_i decreased to values as low as 6, regardless of whether the cell suspension was buffered at pH₀ 4.5 or 7.5. There was no membrane potential detectable in deenergized cells.     

The semiquinone cycle. A hypothesis of electron transfer and proton translocation in cytochromebc-type complexes

The Q cycle and theb cycle are the main current models of action of the cytochromebc-type complexes of mitochondria, bacteria, and chloroplasts. Both are based on the concept, proposed in 1972, of two sequential one-electron oxidations of (ubi)quinol along two discrete pathways which operate at different redox potentials, and with bound semiubiquinone as an intermediate. The models differ in two respects, viz. in the pathway of electron transfer and the principle of linkage of electron transfer to proton translocation. In this article we outline a new model, called the semiquinone or, simply, SQ cycle, which is based on the electron transfer principles of theb cycle but which incorporates the Q cycle concept of direct coupling between electron transfer and proton translocation through action of ubiquinone.This paper is dedicated to the memory of Bob Casey, who died in Helsinki on the 2nd of August 1985.     

The quantum efficiency of photosystem II and its relation to non-photochemical quenching of chlorophyll fluorescence; the effect of measuring-and growth temperature

The relation between the quantum yield of oxygen evolution of open photosystem II reactions centers (_p), calculated according to Weis and Berry (1987), and non-photochemical quenching of chlorophyll fluorescence of plants grown at 19°C and 7°C was measured at 19°C and 7°C. The relation was linear when measured at 19°C, but when measured at 7°C a deviation from linearity was observed at high values of non-photochemical quenching. In plants grown at 7°C this deviation occurred at higher values of non-photochemical quenching than in plants grown at 19°C. The deviations at high light intensity and low temperature are ascribed to an increase in an inhibition-related, non-photochemical quenching component (q_I).The relation between the quantum yield of excitation capture of open photosystem II reaction centers (_exe), calculated according to Genty et al. (1989), and non-photochemical quenching of chlorophyll fluorescence was found to be non-linear and was neither influenced by growth temperature nor by measuring temperature.At high PFD the efficiency of overall steady state electron transport measured by oxygen-evolution, correlated well with the product of q _N and the efficiency of excitation capture (_exe) but it deviated at low PFD. The deviations at low light intensity are attributed to the different populations of chloroplasts measured by gas exchange and chlorophyll fluorescence and to the light gradient within the leaf.Abbreviations F₀ basic fluorescence - F₀ basic fluorescence, thylakoid in energized state - F_m maximal fluorescence - F_m maximum fluorescence in energized state - F_s steady state fluorescence - F_v maximal variable fluorescence - PFD photon flux density - PS II_rc Photosystem II reaction center - q_F0 quenching of basic fluorescence - q_E energy related quenching - q_N non-photochemical quenching:-q_f-total quenching - q_I inhibition-related quenching - q_p photochemical quenching - q_r quenching due to state transition - R_d dark respiration - _p PS II efficiency of excitation capture of open PS II_rc - _pe extrapolated minimal value of _p - _p0 extrapolated maximal value of _p - _si quantum efficiency of linear electron transport, calculated from gas exchange measurements based on incident light - _sf quantum efficiency of linear electron transport, calculated from fluorescence measurements, based on incident measuring light     

Chloroplast DNA restriction analysis and the infrageneric grouping ofAllium (Alliaceae)

The utility of chloroplast DNA variation for checking a recently proposed infrageneric classification of the genusAllium was tested. cpDNA restriction patterns of 49 species representing the main subgenera, sections, and subsections of the existing classification were compared. 363 different fragments generated by 4 restriction enzymes were identified and analysed by UPGMA clustering. The resulting phenogram largely confirms the subgeneric classification based on an integration of morphological and other methods.     

Prediction of the Reaction Equilibrium of Biocatalytic Reactions in Aqueous-Organic Two-Phase Systems

Biocatalytic reactions can be carried out in aqueous-organic two-phase systems. Several models to describe the thermodynamically-determined equilibrium position in such systems have appeared in the literature. Some of these models are only valid for dilute systems, whereas others can also be used for nondilute systems. In this paper, these models are described and compared. It is explained in what way the equilibrium constants of each model can be used to predict the product concentration in different organic solvents.