On the nature of dominance in the ad2 locus of Saccharomyces cerevisiae

Summary A negative complementation experiment was used to study dominance in the ad₂ gene of Saccharomyces cerevisiae. The wild type allele showed near complete dominance over both the remedial and the inactive mutant alleles. The remedial allele was dominant to a lesser extent (in 77.4% of the combinations) over the inactive mutant allele. In 22.6% of the cases, the inactive allele was able, contrary to expectation, to dominate and impose its inactivity on its remedial partner (negative complementation).At the protein level, these results suggest that the wild type protein, which is the outcome of long evolutionary selection, has the most stabilized activity. The remedial protein, although superior to that of the completely inactive mutant, was not stable enough to always resist the inactivating influence of its defect partner.Part of a thesis of the Faculty of Mathematical and Natural Sciences of Freiburg University.     

Hydration of 3-cyanopyridine to nicotinamide by crude extract nitrile hydratase

Summary The kinetic and stability characteristics of crude extract nitrile hydratase fromBrevibacterium R-312 were studied for the hydration of 3-cyanopyridine to nicotinamide. The enzyme was substrate and product inhibited and had the following kinetic constants:K _m =28 mM;K _p =36 mM;K _s =155 mM;V _m =5.8 mol/min/mg protein (25°C). Itsmaximum temperature and pH (phosphate buffer) were 35°C and 8.0, respectively and it had half-lives of 50 days, 10 days and 1 day at 4°C, 10°C and 25°C, respectively. The crude extract also exhibited amidase activity on nicotinamide, but it became significant only at nicotinamide concentrations greater than 300 mM. Mathematical models for batch and fed-batch hydrations were developed to account for substrate and product inhibitions and for enzyme decay. They predicted to within 10% experimental results for initial substrate and final product concentrations up to 300 mM; the accuracies decreased at higher concentrations primarily because of the relatively rapid hydrolysis of nicotinamide.     

Improvement of<Emphasis Type="Italic"> Citrus clementina</Emphasis> Hort. ex Tan. microspore-derived embryoid induction and regeneration

An improvement of the protocol for haploid induction through anther culture of Citrus clementina Hort. ex Tan. cv. Nules was achieved following the evaluation of a number of the factors affecting androgenesis. The influence of thidiazuron (TDZ) and three temperature pre-treatments (4°C, 25°C, 32°C) on the floral buds with respect to anther culture of C. clementina Hort. ex Tan., cv. Nules was investigated. An increased embryoid production was induced in the medium supplemented with TDZ. Pre-treatment temperatures of 4°C and 25°C were more favorable for embryo production than 32°C. Regeneration of androgenic haploid plantlets from cv. SRA 63 of C. clementina is reported here for the first time.Abbreviations 6-BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - KI Kinetin - NAA -Naphthaleneacetic acid - TDZ Thidiazuron (N-phenyl-1,2,3,-thi-diazol-5-ylurea) - ZEA Zeatin Communicated by L. PeñaBoth authors have contributed equally to this article.     

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Recovery and its dependence on temperature

Recovery of photoinhibition in intact leaves of shade-grown kiwifruit was followed at temperatures between 10° and 35° C. Photoinhibition was initially induced by exposing the leaves for 240 min to a photon flux density (PFD) of 1 500 mol·m^-2·s^-1 at 20° C. In additional experiments to determine the effect of extent of photoinhibition on recovery, this period of exposure was varied between 90 and 400 min. The kinetics of recovery were followed by chlorophyll fluorescence at 77K. Recovery was rapid at temperatures of 25–35° and slow or negligible below 20° C. The results reinforce those from earlier studies that indicate chilling-sensitive species are particularly susceptible to photoinhibition at low temperatures because of the low rates of recovery. At all temperatures above 15° C, recovery followed pseudo first-order kinetics. The extent of photoinhibition affected the rate constant for recovery which declined in a linear fashion at all temperatures with increased photoinhibition. However, the extent of photoinhibition had little effect on the temperature-dependency of recovery. An analysis of the fluorescence characteristics indicated that a reduction in non-radiative energy dissipation and repair of damaged reaction centres contributed about equally to the apparent recovery though biochemical studies are needed to confirm this. From an interpretation of the kinetics of photoinhibition, we suggest that recovery occurring during photoinhibition is limited by factors different from those that affect post-photoinhibition recovery.Abbreviations and symbols F _o, F _m, F _v instantaneous, maximum, variable fluorescence - K _D, K _F, K _P, K _T rate constants for non-radiative energy dissipation, fluorescence, photochemistry, transfer to photosystem I - K(PI), k(R) rate constants for photoinhibition and recovery - PFD photon flux density - PSI, II photosystem I, II - _i photon yield of photosynthesis (incident light)     

Effect of temperature and photoperiod on efficiency of assimilated CO<Subscript>2</Subscript> conversion into the biomass of <Emphasis Type="Italic">Cucumis sativus</Emphasis>

The coefficient of effectiveness (K _e) of assimilated CO₂ conversion into dry matter of cucumber (Cucumis sativus L.) plants at the stage of four leaves as dependent on a photoperiod (8, 12, and 16 h) at an irradiance of 220 W/m² at the upper leaf level and the combinations of day and night temperatures: typical temperature of plant habitat (background temperature) of 25°C and heat- and cold-hardening temperatures (35 and 15°C, respectively) was determined in the multifactorial designed experiment. K _e reduced insignificantly at shortening of a photoperiod and greater at its lengthening. At background temperatures, K _e corresponded mainly to that of carbohydrate synthesis while the presence of cold-hardening temperatures in the thermoperiod increased K _e and heat-hardening temperature reduced it.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 203–208.Original Russian Text Copyright © 2005 by Talanov, Popov, Kurets, Drozdov.This revised version was published online in April 2005 with a corrected cover date.     

Temperature-conditional nuclear mutation of Chlamydomonas reinhardtii decreases the CO2/O2 specificity of chloroplast ribulosebisphosphate carboxylase/oxygenase

The Chlamydomonas reinhardtii (Dangeard) temperature-conditional mutant 68-11AR is phenotypically indistinguishable from the wild type at the permissive temperature (25°C), but has greatly reduced photosynthetic ability and requires acetate for growth at the restrictive temperature (35°C). The mutant strain is deficient in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) holoenzyme when grown at 35°C. This decrease in the level of enzyme appears to be due to degradation of assembled holoenzyme rather than to a reduction in the synthesis of enzyme subunits. When grown at 25°C, the mutant has a substantial amount of Rubisco. Enzyme purified from 25°C-grown mutant cells was found to have a 16% decrease in the CO₂/O₂ specificity factor when compared to the wild-type enzyme. This alteration was accompanied by changes in the kinetic constants for both carboxylation and oxygenation. Although the Rubisco active site is located on the chloroplast-encoded large subunit, genetic analysis showed that the 68-11AR strain arose from a nucleargene mutation. The two nuclear genes that encode the Rubisco small subunits (rbcS1 and rbcS2) were cloned from mutant 68-11AR and completely sequenced, but no mutation was found. Analysis of restriction-fragment length polymorphisms also failed to detect linkage between mutant and rbcS gene loci. These results indicate that nuclear genes can influence Rubisco catalysis without necessarily encoding polypeptides that reside within the holoenzyme.Abbreviations and Symbols K _c Michaelis constant for CO₂ - K _o Michaelis constant for O₂ - mt mating type - pf paralyzed flagella - RFLP restriction-fragment length polymorphism - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V _c V _max for carboxylation - V _o V _max for oxygenation - CO₂/O₂ specificity factor C. G. gratefully acknowledges fellowship support from the Consejo Superior de Investigaciones Cientificas (Spain). This work was supported by National Science Foundation grant MCB-9005547, and is published as Paper No. 10481, Journal Series, Nebraska Agricultural Research Division.     

Cold-sensitive mutations in the Z gene of prophage P2 that result in increased sensitivity of the lysogens to a low molecular weight product of the host bacteria

Summary Z mutants of bacteriophage P2 form clear plaques and are unable to give rise to stable lysogens in Escherichia coli C. To study the function of the Z gene in lysogenization by P2, temperature-sensitive mutants were isolated. Those that were classified as Z mutants by complementation were all cold-sensitive (cs); they were unable to form lysogens at 30° C, but had wild type phenotype at 42° C. When lysogens carrying such mutants, prepared at 42° C, were shifted to the lower temperature, the bacteria continued to multiply at the normal rate until they reached concentrations of about 5 × 10⁷ per ml, at which point the viable titer began to decrease. Inactivation of the bacteria at even lower concentrations occurred if they were transferred to medium taken from overnight cultures of the same strain, suggesting that they were sensitive to some material that had accumulated in the culture medium.The lethal material was produced not only by csZ lysogens, but by all derivatives of Escherichia coli C tested, including non-lysogens, and at both 30° C and 42° C. Only csZ lysogens were sensitive to it, however, and only at the lower temperature. A preliminary characterization of the material indicates that it is heat-stable, of low molecular weight and does not adsorb to activated charcoal.This work was supported by Research Grant 72 from the Swedish Medical Research Council     

Stimulation of embryogenesis and haploid production in Brassica campestris anther cultures by elevated temperature treatments

Summary Culture of Brassica campestris anthers at 35°C for one or three days prior to culture at 25°C significantly stimulated the yield of microspore-derived embryos. More than 100 plants were regenerated from cultured embryos and haploids were identified amongst them. The haploid frequency was greater than 70% if all small-flowered sterile plants were considered to be haploid. The yield of microspore-derived plants in B. campestris is approaching the level where anther culture may be utilized as a practical breeding tool.     

Effect of temperature and oxygen on cell growth and recombinant protein production in insect cell cultures

The effect of temperature and O₂ saturation on the production of recombinant proteins -galactosidase and human glucocerebrosidase by Spodoptera frugiperda cells (Sf9) infected with recombinant Autographa californica nuclear polyhedrosis virus was investigated. The rates of cell growth, glucose consumption, O₂ consumption and product expression were measured at temperatures between 22° C and 35° C. The results indicated that possible O₂ limitation may be alleviated without compromising the maximum cell yield by lowering the incubation temperature from 27° C to 25° C. The expression level of the recombinant proteins at 27° C was similar to that obtained at 22° C and 25° C; lower protein yields were obtained at 30° C. An increase in temperature from 22° C to 27° C led to earlier production of the proteins and to an increase in the proportion of the product released outside the cells. Correspondence to: J. Shiloach     

Mutagen specificity in the induction of karyotic versus cytoplasmic respiratory deficient mutants in yeast by nitrous acid and alkylating nitrosamides

Summary In the haploid eukaryotic organism Saccharomyces cerevisiae the induction of cytoplasmic and genic (karyotic) RD mutants was studied, using nitrous acid, nitrosomethylurethane (NMU) and nitrosoimidazolidone (NIL).The cytoplasmic or genic origin of the induced RD mutants was determined by prescreening in complementation tests with ^– and wild type tester strains. Among the mutants of all three agents we could thus score the incidence of three RD mutant types: genic, suppressive and cytoplasmic (both primary and secondary). The final identification of the cytoplasmic type was only possible through tetrad analysis, performed in the cases of HNO₂ and NMU.A distinct difference in cytoplasmic versus genic mutagen specificity was observed between HNO₂ and NMU. HNO₂ was unable to induce cytoplasmic RD mutants but it proved to be highly efficient in the induction of genic RD mutants. In contrast, NMU induced more cytoplasmic effects was it possible to detect mutagenic specificities which, solely on the basis of karyotic action, were not detectable.     

Genetic fine structure and complementation at the albino locus in spider mites (Tetranychus species: Acarina)

Two mutations inTetranychus urticae and nine inTetranychus pacificus, all originating spontaneously, block the production of red and yellow carotenoid pigments in these spider mite species. Inter-mutant crosses were carried out to study complementation and recombination relationships between the mutations. InT. urticae, the two albino mutants complement one another completely, i.e., crosses between them produce wild-type hybrid females; while they recombine with a frequency of 0.05%. Of the nine mutants inT. pacificus, fivep mutants in general are complementary to a high degree with foura mutants.p mutants fail to complement one another, while somea mutants are mutually complementary to a slight degree. Scoring the degree of complementation produced by all possible combinations of mutants permits the construction of a linear complementation map. Certain combinations, however, are exceptional to such a representation. Moreover, marked reciprocal differences in complementation indicate that maternal effects are involved, implying that the albino locus may control more than one enzymatic step. Attempts to derive a genetic map were impeded by the absence of suitable linked markers, by a pronounced maternal effect (high pigmentation) in the haploid F₂ males, and by the appearance of pseudowild type F₂ males. The given genetic sequence, although comparable in a limited fashion to the complementation map, is considered tentative. Pink types appeared in crosses with certainp mutants. These were due to mutation at a separate locus, called rose, and seem to involve the production of pink pigments in an alternative or substitute pathway. A scheme attempting to orientate the present state of understanding of pigmentation in spider mites is presented.This work forms part of a thesis submitted in fulfillment of the requirements for the Ph. D. degree at the University of Amsterdam (1969).     

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35°C for a few days and by then incubating them in the dark at 25°C. Pre-culturing anthers at 35°C for 4days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15°C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.     

The effect of temperature on glycollate decarboxylation in leaf peroxisomes

[1-¹⁴C]glycollate was oxidised to¹⁴CO₂ by peroxisomes isolated from leaves of spinach beet about 3 times as rapidly at 35°C as at 25°C; the rate was further increased with rise in temperature to a maximum at 55°C. These increases are shown to be mainly due to the increased H₂O₂ available to oxidise glyoxylate non-enzymically as a result of the higher temperature coefficient of glycollate oxidase activity relative to that of catalase. These results are compared with similar increases in the rate of¹⁴CO₂ release between 25°C and 35°C when [1-¹⁴C]glycollate was supplied to leaf discs in light or darkness. The role of these reactions in accounting for the temperature effect on the release of photorespiratory CO₂ is discussed.Abbreviations PHMS Pyrid-2-yl--hydroxymethane sulphonate - FMN flavin mononucleotide     

Chlorophyll a Fluorescence Analysis in Response to Excitation Irradiance in Bean Plants (Phaseolus vulgaris L. and Vigna unguiculata L. Walp) Submitted to High Temperature Stress

Bean plants Phaseolus vulgaris L. (cv. Carioca and Negro Huasteco) and Vigna unguiculata L. Walp (cv. Epace-10) were grown in a growth chamber with a photosynthetic photon flux density of 200 mol m^–2 s^–1 at leaf level and air temperature of 25+1 °C. Fully expanded, first pair leaves of 12-d-old plants were submitted for 90 min to high temperature (25, 30, 35, 40, 45, and 48 °C). Chlorophyll a fluorescence parameters (ETR, q_P, q_N, and F₀) were investigated using a modulated fluorimeter at 25 °C during recovery considered here as 48 h after stress induction period. An accentuated decrease in q_P and an increase in q_N at 48 °C in Carioca and Negro Huasteco was not observed in Epace-10. In response to excitation irradiance a great potential for ETR was found in Negro Huasteco at 25 °C, also demonstrated by net photosynthetic rate. At 48 °C ETR was high for Epace-10 while it was equal to zero for Carioca and Negro Huasteco. Tolerance to high temperature observed in Epace-10 provided important information about the adaptative characteristics of Vigna cultivars to warm climates.     

Nitrate reductase from yeast: cultivation,partial purification and characterization

Summary Several yeast strains were assayed for occurence of nitrate reductase after growth in a defined medium with nitrate as the sole nitrogen source, Candida boidinii DSM 70026, showing the highest specific activity, was further investigated. The procedures for yeast fermentation and nitrate reductase purfication are described in detail. Nitrate reductase from this yeast was characterized as NAD(P)H: nitrate oxidoreductase (E.C.1.6.6.2). The enzyme activity with NADH (NADPH) was highest at pH 7.0 (7.1) and 30° C (25° C). The values of K _m determinations with NADH/NADPH were both 4 × 10^–4 mol/l; values for the substrate inhibition constant (K _i) were 6 × 10^–4 mol/l. The molecular mass of the native enzyme was estimated by gel permeation chromatography to be approximately 350 kDa. Offprint requests to: R. Gromes     

Fast determination of operational stability of the soluble acetylacetone-cleaving enzyme Dke1 in an enzyme membrane reactor

The main aim of this study was the determination of the operational stability of soluble Dke1 (EC 1.13.11.50) in an enzyme membrane reactor. In order to calculate the half-life of soluble Dke1, the K _M of oxygen must be known. The determination of this constant was done using progress curve analysis (K _M=260 μmol l⁻¹). In a next step, the reactor system was studied by building a mathematical model for calculation of the reactor system, using Berkeley Madonna ver. 8.0.1 software. After that, the determination of the half-life of Dke1 under operational conditions at different temperatures (5, 10, 15, 25, 30, 35°C) was performed. The quantitative criterion for stability was the value of the first-order rate constant of monomolecular inactivation. The experiments showed that soluble Dke1 is poorly stable. The half-life ranged from 308 min at 5°C to 9 min at 35°C. This method for determining the half-life is quite applicable for enzymes which are poorly stable. In addition, both the storage stability and the operational stability can be determined.     

Effects of temperature and CO2 enrichment on kinetic properties of phospho-enol-pyruvate carboxylase in two ecotypes of Echinochloa crus-galli (L.) Beauv., a C4 weed grass species

Summary Two populations of Echinochloa crus-galli (Québec, Mississippi) were grown at the Duke University Phytotron under 2 thermoperiods (28°/22°C, 21°/15°C day/night) and 2 CO₂ regimes (350 and 675 l l^-1). Thermostability, energy of activation (E _a ),K _m (PEP), K _m (Mg⁺⁺), and specific activity of phospho-enol-pyruvate carboxylase (PEP_c) were analyzed in partially purified enzyme preparations of plants grown for 5 weeks. Thermostability of PEP_c from extracts (in vitro) and leaves (in situ) was significantly higher in Mississippi plants. In vitro denaturation was not appreciably modified by thermal acclimation but CO₂ enrichment elicited higher thermostability of PEP_c. In situ thermostability was significantly higher than that of in vitro assays and was higher in Mississippi plants acclimated at 28°/22°C and in plants of the two ecotypes grown at 675 l l^-1 CO₂. E _a (Q ₁₀ 30°/20°C) for PEP_c was significantly lower in Québec plants as compared to Mississippi and no acclimatory shifts were observed. Significantly higher K _m's (PEP) in 20°C assays were obtained for Mississippi as compared to Québec plants but values were similar at 30°C and 40°C assays. K _m (Mg⁺⁺) decreased at higher assay temperatures and were significantly lower for PEP_c of the Québec ecotype. No significant changes in K _m (Mg⁺⁺) values were associated with modifications in temperature on CO₂ regimes. PEP_c activity measured at 30°C was significantly higher for Québec plants when measured on a leaf fresh weight, leaf area or protein basis but not on a chlorophyll basis. Significantly higher PEP_c activity for both genotypes was observed for plants acclimated at 21°/15°C or grown at 675 l l^-1 CO₂. Net photosynthesis (Ps) and net assimilation rates (NAR) were higher in Québec plants and were enhanced by CO₂ enrichment. NAR was higher in plants acclimated at low temperature, while an opposite trend was observed for Ps. PEP_c activities were always in excess of the amounts required to support observed rates of CO₂ assimilation.     

A highly thermostable ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus

A ferritin from the obligate anaerobe and hyperthermophilic archaeon Pyrococcus furiosus (optimal growth at 100°C) has been cloned and overproduced in Escherichia coli to one-fourth of total cell-free extract protein, and has been purified in one step to homogeneity. The ferritin (PfFtn) is structurally similar to known bacterial and eukaryal ferritins; it is a 24-mer of 20 kDa subunits, which add up to a total Mr 480 kDa. The protein belongs to the non-heme type of ferritins. The 24-mer contains approximately 17 Fe (as isolated), 2,700 Fe (fully loaded), or <1 Fe (apoprotein). Fe-loaded protein exhibits an EPR spectrum characteristic for superparamagnetic core formation. At 25°C V_max=25 mole core Fe³⁺ formed per min per mg protein when measured at 315 nm, and the K_0.5=5 mM Fe(II). At 0.3 mM Fe(II) activity increases 100-fold from 25 to 85°C. The wild-type ferritin is detected in P. furiosus grown on starch. PfFtn is extremely thermostable; its activity has a half-life of 48 h at 100°C and 85 min at 120°C. No apparent melting temperature was found up to 120°C. The extreme thermostability of PfFtn has potential value for biotechnological applications.     

Conditional lysis ofEscherichia coli by the fusion of extracellular (STA) to periplasmic (LTB) enterotoxins: Apparent phenotypic suppression of lactose permease

The expression of a methanol-soluble, heat-stable enterotoxin (ST_A) fused to the B subunit of the heat-labile enterotoxin (LT_B) at 35°C or higher temperatures caused strains ofEscherichia coli deficient in lactose permease to behave on indicator media as Lac⁺; however, at 33°C or lower temperatures the original Lac^– phenotype of the host strains was maintained. The apparent phenotypic suppression oflacY was shown to be due to lysis of a fraction of the bacteria and the consequent release of active-galactosidase to the culture supernatant. After incubation at 37°C for 1 h, the cultures were committed to lyse. Plasmid and chromosomal mutants that do not show this phenotype were isolated by selecting Lac^– colonies at the unpermissive temperature. The mutations on the plasmids were localized in both the heat-stable and the heat-labile enterotoxin genes. Chromosomal mutants that show normal levels of-galactosidase and fused toxins have also been isolated.     

Effects of paraquat on selected microbial activities in soil

Paraquat, applied as Gramoxone, to a nonamended sandy loam soil at five times the suggested field application rate (10 lb/A 115g/cm²) increased the numbers of bacteria, actinomycetes, and fungi during a 14-day incubation at 25°C. This increase was attributed to the use of compounds in the Gramoxone formulation rather than the use of paraquat. Treatment at one and five times the normal rate reduced CO₂ evolution by 44% and 67%, respectively, in soil amended with 2% glucose during a 12-day incubation. Similar treatments reduced CO₂ evolution in 1% straw-amended soil by 39% and 58%, respectively, during a 28-day incubation. Cellulose decomposition of cotton duck containing 13 and 176g of paraquat per milligram of material was inhibited for 15 and 28 days, respectively, in soil containing a large population of cellulolytic microorganisms. A concentration of 5000g/gm of paraquat was necessary to inhibit nitrification in soil by 44% druing a 28-day incubation at 20°C. Paraquat inhibited C₂H₂ reduction in artificial aggregates of soil amended with 2% glucose and incubated anaerobically at 25°C. Nitrogenase activity in aggregates was inhibited by 43% and 52% at concentrations of 580 and 720g/gm of paraquat respectively. The inhibitory effects of the herbicide were reduced when soil was amended with organic matter in the form of peat or straw. The availability of paraquat controlled the toxicity of the herbicide to soil microorganisms.