Cloning and sequencing of a ligninase gene from a lignindegrading basidiomycete,Phanerochaete chrysosporium

Summary A ligninase gene has been cloned from a Phanerochaete chrysosporium genomic DNA library. Nucleotide sequencing of the gene has revealed that the ligninase structural gene contains 1116 bp of the protein-encoding sequence, of which 84 bp encode the signal peptide. The protein-encoding sequence is interrupted by eight introns which conform to the universal G-T/A-G splicing rule observed for the 3 and 5 intron boundaries. The putative eukaryotic regulatory sequences, i.e. CAAT and TATA box-like sequences, are present in the 5 flanking region.     

Regulation of adenosine 5′-phosphosulfate sulfotransferase activity by H2S and cyst(e)ine in primary leaves of Phaseolus vulgaris L.

During chloroplast development in the primary leaves of Phaseolus vulgaris, the extractable activity of adenosine 5-phosphosulfate sulfotransferase increased ten-fold. When chloroplast development took place in air enriched with 3.5 l H₂S·l^-1 there was a decrease in adenosine 5-phosphosulfate sulfotransferase activity. Cyst(e)ine in concentrations up to 1 mM (in the external medium) did not affect the increase in adenosine 5-phosphosulfate sulfotransferase activity in intact plants. In plants with excised roots, 0.75 mM cyst(e)ine inhibited this increase. In green primary leaves, H₂S or cyst(e)ine treatment resulted in a decrease of extractable adenosine 5-phosphosulfate sulfotransferase activity. In intact plants, this effect of cyst(e)ine was observed at a concentration of 1 mM, and in plants with excised roots, 0.25 mM had a comparable effect.In developing plants, the extractable activities of O-acetyl-L-serine sulfhydrylase (EC 4.2.99.9) and ribulosebisphosphate carboxylase (EC 4.1.1.39.) were not affected by H₂S or cyst(e)ine.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5phosphosulfate sulfotransferase - BSA bovine serum albumin - DTE dithioerythritol - EDTA ethylenediaminetetra-acetic acid - OASSase O-acetyl-L-serine sulfhydrylase - PAPS adenosine 3-phosphate 5-phosphosulfate - POPOP 1,4 Di 2-(5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazol - RubP ribulose-bisphosphate - RubPCase ribulosebiphosphate carboxylase This is no. 8 in the series Regulation of Sulfate Assimilation in Plants. The term cysteine is used when it is clear that cystine is not involved; cyst(e)ine is used for an undefined mixture of cysteine and cystine. The concentrations are expressed in all cases relative to cysteine     

Microsatellite markers in the hexaploid Prunus domestica species and parentage lineage of three European plum cultivars using nuclear and chloroplast simple-sequence repeats

In a previous study, cDNA microsatellite markers were described in apricot (Prunus armeniaca L.). Specific PCR primers were designed to amplify the microsatellite-containing regions from genomic DNA in different Prunus species. In the present work, cDNA microsatellite markers were developed in the hexaploid Prunus domestica L. species and polymorphism was ascertained in a segregating plum population. Co-dominant mendelian segregation of alleles was demonstrated and microsatellite polymorphism displayed up to 6 alleles per SSR locus per individual. Parentage lineage of three full-sib European plum cultivars (cv. Cacanska najbolja, Cacanska rana and Cacanska lepotica) was reconstructed by the analysis of the above nuclear SSR markers, completed by four chloroplastic microsatellite loci. The six most informative nuclear loci enabled discrimination between the three Cacak cultivars and unrelated individuals as well as the previously proposed parents, Wangenheim and Pozegaca. Data obtained support previous evidence that these cultivars originated from the Stanley cultivar. However, SSR analysis finally excluded Wangenheim as the other possible parent. Based on the results obtained with nuclear and chloroplast SSR loci, we propose the origin of those three Cacak cultivars in a cross between Stanley as the mother plant and Ruth gerstetter as the pollinator. Furthermore, we demonstrate the utility of these apricot SSR markers for genotype fingerprinting of the hexaploid plum cultivars.     

Some properties of 2-5A binding/nucleolytic activities in gel filtered rabbit reticulocyte lysates

Rabbit reticulocyte lysates, gel filtered on Sephadex G-25 with or without ATP (or its analogs), were preincubated at 37°C and their subsequent binding to p₃A₄,3-[³²P]pCp was studied. Lysates filtered without ATP or in the presence of 0.1 mM 8-bromo-ATP, 1,N⁶-etheno-ATP, or ITP showed a time-dependent decrease in binding activity. This decrease was completely prevented when lysates were filtered with 0.1 mM ATP, 2-deoxy-ATP, --methylene-ATP, or ATP--S. The stability of binding provided by ATP or 2-deoxy-ATP analogs corresponds to a more active 2–5A dependent endonucleolytic (RNAase L) activity based on studies using [³H] viral mRNA. Chromatography on heparin-agarose showed that ATP-supplemented gel-filtered reticulocyte lysates had a different p₃A₄,3-[³²P]pCp binding activity elution-profile than lysates gel-filtered in the absence of ATP. Covalent cross-linking of periodate-oxidized p₃A₄,3-[³²P]pC to gelfiltered lysates, preincubated at 0°C or 37°C for 30 min, showed the following results: (1) all lysates gave a major cross-linking of the radioactive ligand to an 80 000 dalton polypeptide, regardless of the temperature of preincubation, (2) Iysates gel-filtered without ATP, with 0.1 mM ITP, or --methylene-ATP, showed a significant reduction in the cross-linking of the 80 000 dalton protein, after preincubation at 37°C for 30 min. This decrease was accompanied by an increase in the labeling of two smaller polypeptides.Abbreviations used 2 5-oligoadenylates oligonucleotides consisting of 5-adenylic acid residues joined by a 2 5-phosphodiester linkage     

Effects of data imbalance on estimation of heritability

Summary Effects of data imbalance on bias, sampling variance and mean square error of heritability estimated with variance components were examined using a random two-way nested classification. Four designs, ranging from zero imbalance (balanced data) to low, medium and high imbalance, were considered for each of four combinations of heritability (h²=0.2 and 0.4) and sample size (N=120 and 600). Observations were simulated for each design by drawing independent pseudo-random deviates from normal distributions with zero means, and variances determined by heritability. There were 100 replicates of each simulation; the same design matrix was used in all replications. Variance components were estimated by analysis of variance (Henderson's Method 1) and by maximum likelihood (ML). For the design and model used in this study, bias in heritability based on Method 1 and ML estimates of variance components was negligible. Effect of imbalance on variance of heritability was smaller for ML than for Method 1 estimation, and was smaller for heritability based on estimates of sire-plus-dam variance components than for heritability based on estimates of sire or dam variance components. Mean square error for heritability based on estimates of sire-plus-dam variance components appears to be less sensitive to data imbalance than heritability based on estimates of sire or dam variance components, especially when using Method 1 estimation. Estimation of heritability from sire-plus-dam components was insensitive to differences in data imbalance, especially for the larger sample size.Supported by grants from the Illinois Agricultural Experiment Station and the University of Illinois Research Board. Charles Smith, H. W. Norton and D. Gianola contributed valuable suggestions     

Studies on Tn951 (lac+) expression in Agrobacterium

Summary None of the Agrobacterium tumefaciens and A. rubi strains tested produces detectable amounts of -galactosidase although they are capable of utilizing lactose as sole source of carbon. This opportunity was taken to investigate the expression of lac transposon Tn951 (Cornelis et al. 1978) in Agrobacterium with the ultimate goal of using this system to investigate alien gene expression. When the transposon was introduced with the help of a broad-host range plasmid, RP1, the transconjugants produced significant quantities of -galactosidase which was inducible by isopropyl--D-thiogalactopyranoside. Tn951 was capable of restoring the Lac⁺ phenotype to an A. tumefaciens mutant not capable of using lactose. Cellobiose, a known inducer of aldohexopyranoside: cytochrome c oxidoreductase which regulates the characteristic 3-ketolactose production in Agrobacterium: van Beeumen and De Ley (1968), had no effect on -galactosidase activity.Abbreviations NCPPB National Collection of Plant Pathogenic Bacteria, Harpenden - km kanamycin resistance - str streptomycin resistance - rif ^r rifampicin resistance     

Confidence interval estimators for heritability for several mating and experiment designs

Summary Confidence interval estimators have not been described for several heritability (H) estimators relevant to recurrent family selection. Previously described H interval estimators do not apply to onefactor mating designs in split-plot in time experiment designs in one or more locations, one-factor mating designs for several experiment designs in two or more locations and years, and two-factor mating designs for several experiment designs in two or more locations or years. Our objective was to derive H interval estimators for these cases. H reduced to a function of constants and a single expected mean square ratio in every case; H=1–E(M)/E(M) where E(M) is a linear function of expected mean squares and E(M) is a single expected mean square. It was shown that F=[M/E(M)]/[M/E(M)] has an approximate F-distribution with df and df degrees of freedom, respectively, where M and M are mean squares corresponding to E(M) and E(M), respectively. H is a function of F, therefore, we used F to define an approximate (1–) interval estimator for H.Oregon Agricultural Experiment Station Technical Paper No. 7923     

Eye movements in Daphnia magna

Summary Three types of behavior of the compound eye of Daphnia magna are characterized: flick, a transient rotation elicited by a brief flash of light; fixation, a maintained eye orientation in response to a stationary light stimulus of long-duration; tracking, the smooth pursuit of a moving stimulus. The magnitudes of the flick and fixation responses vary with stimulus position and are generally proportional to stimulus intensity, although at high intensities there is an attenuation of both behaviors. When the stimulus is placed at a position 80° dorsal to the eye axis, there is no response; this area is called the null region. For stationary stimuli in other positions, the direction of the response is such as to bring the stimulus closer to the null region. During tracking, the relative positions of the eye and stimulus change; the eye velocity is approximately half that of the moving stimulus. The regions of the eye in which these behaviors may be induced are different, being largest for flick and smallest for tracking. It is proposed that flick and fixation responses are a means for rotating the eye so that the stimulus is within the area surrounding the null region which is used for tracking.     

Bias in genetic variance estimates due to spatial autocorrelation

Summary A central problem in the analysis of genetic field trials is the dichotomy of genetic and environmental effects because one cannot be defined without the other. Results from 768,000 simulated family trials in complete randomized block designs demonstrated a serious upward bias in estimates of family variance components from multi-unit plot designs when the phenotypic observations were compatible with a first-order autoregressive (AR1) process. The inflation of family variances and, thus, additive genetic variance and narrow sense individual heritabilities progressed exponentially with an increase in the nearest neighbor correlation () in the AR1 process. Significant differences in inflation rates persisted among various plot configurations. At = 0.2 the inflation of family variances reached 48–73%. Inflation rates were independent of the level of heritability. Modified Papadakis nearest neighbor (NN) adjustment procedures were tested for their ability to remove the bias in family variances. A NN-adjustment based on Mead's coefficient of interplant interaction and one derived from Bartlett's simultaneous autoregressive scheme removed up to 97% of the bias introduced by the phenotypic correlations. NN-adjusted estimates had slightly (5–8%) higher relative errors than did unadjusted estimates.     

The demonstration of chloride ions in the “chloride cells” of the gills of eels (Anguilla anguilla L.) adapted to sea water

Summary The ultrastructure of chloride cells in the gills of eels kept in artificial sea water and of a control animal kept in fresh water was studied. In addition to glutaraldehydeosmium tetroxide and simple osmium tetroxide fixation, a special method for the demonstration of chloride ions was used (Komnick, 1962, 1963). Based on the principle of silver chloride precipitation in the presence of chloride ions, the procedure showed positive results in the chloride cells of eels adapted to sea water. Smooth-surfaced tubules of the endoplasmic reticulum contained a material of medium to strong electron density, that was often in communication with the plasma membrane. The same material, always of very high density, was present in the intercellular spaces, thus forming conspicuous lines around cells. The silver precipitate was found very often in large quantities in the pits of chloride cells, having thereby the aspect of a secretory product. However, a direct communication between the system of endoplasmic reticulum tubules containing the silver reaction product with the above-mentioned masses of silver chloride was not demonstrated. Schultz (1958) first published an electron micrograph of these cells. Two features, numerous mitochondria and highly developed agranular endoplasmic reticulum, affirmed by all later investigators, have remained the most important criteria for the identification of chloride cells (Kessel and Beams, 1962; Philpott, 1962; Philpott and Copeland, 1963; Rhodin, 1964; Henrikson and Matoltsy, 1968). Kessel and Beams (1962) and Philpott and Copeland (1963) demonstrated pits, or apical cavities (Oberg, 1967) filled with an amorphous granular substance of medium electron density. (Threadgold and Houston, 1964). These cavities are found in animals adapted to sea water only, and correspond to the excretory vesicles described earlier by Copeland (1948). Philpott and Copeland demonstrated numerous vesicles and tubules in the cytoplasm surrounding the apical cavities and opening into them, possibly contributing to their granular material. Several authors attempted to demonstrate a chloride excretory function of chloride cells with the aid of some histochemical reactions. Copeland (1948), Datta Munshi (1964) and Philpott (1966) used silver techniques     

A frequency-time domain 3D COSY-J technique for measurement of1H−31P coupling constants in oligonucleotides

Summary A frequency-time domain 3D NMR technique has been developed for measurement of heteronuclear coupling constants in oligonucleotides employing a combination of COSY andJ-resolved techniques. The method employs frequency-selective excitation to generate the ₁ axis and 2D FT to generate the ₂ and ₃ axes. The procedure yields high resolution, especially along the ₁ axis. The technique is demonstrated on a dinucleotide.     

Comparative residual toxicities of pesticides to the predator Agistemus industani (Acari: Stigmaeidae) on citrus in Florida

Residual toxicities of registered and selected experimental pesticides used on citrus against Agistemus industani Gonzalez (Acari: Stigmaeidae) were compared. Pesticides considered highly toxic to A. industani were: abamectin 0.15 EC at 731ml/ha+FC 435-66 petroleum oil at 46.8l/ha, pyridaben 75WP at 469g/ha, ethion 4EC at 7.01l/ha+FC 435-66 petroleum oil at 46.8l/ha, propargite 6.55 EC at 3.51l/ha, chlorfenapyr 2SC at 1.46l/ha applied alone or in combination with FC 435-66 petroleum oil at 46.8l/ha, sulphur 80DF at 16.81kg/ha, dicofol 4EC at 7.01l/ha, fenbutatin oxide 50WP at 2.24kg/ha, benomyl 50WP at 2.24kg/ha, benomyl 50WP at 1.68kg/ha+ferbam 76 GF at 5.60kg/ha, ferbam 76GF at 11.21kg/ha, neem oil 90EC at 46.8l/ha, and copper hydroxide DF (40% metallic copper) at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha. Pesticides that were moderately to slightly toxic included: copper sulphate 98% at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha, fenbuconazole 2F at 280ml/ha+FC 435-66 petroleum oil at 46.8l/ha, FC 435-66 petroleum oil applied alone at 46.8l/ha or 23.4l/ha, and diflubenzuron 25WP at 1.40kg/ha. Pesticides that were non-toxic included: fenbuconazole 2F at 585ml/ha, malathion 57EC at 5.85l/ha, FC 435-66 petroleum oil at 46.8l/ha, carbaryl 80S at 3.36kg/ha, chlorpyrifos 4EC at 4.68l/ha, and formetanate 92SP at 1.12kg/ha. Understanding the toxic effects of field weathered pesticides against key predacious mite species is important for effective IPM. The results of this study provide a comparison of direct and indirect toxic effects of various pesticides to A. industani under field conditions.     

Dihydropteridine reductase variation in man and the characid fish “Cheirodon axelrodi”: Evidence for a dimeric enzyme structure

Summary Genetic evidence for a dimeric structure of dihydropteridine reductase in man and in the fish species Cheirodon axelrodi and Salmo irideus is presented. A single locus in man and two loci in the fishes examined encode this enzyme. Zymograms revealed two alleles for the locus in man and two alleles for each locus in the fish Cheirodon axelrodi. The liver homogenate of a patient with dihydropteridine reductase deficiency showed no detectable activity in the gel, while his parents showed the normal electrophoretic phenotype.     

Breeding systems and population structure in Limnanthes

Summary The breeding systems of seven Limanthes (Limanthaceae) populations, including one inbreeding and three outbreeding taxa, were quantified using a multilocus outcrossing rate estimator (t_m) and autofertility estimates. Along with the assays of heterozygosity levels, these data were used to separate components of effective outcrossing in terms of Wright's equilibrium inbreeding coefficient (F_e) and adult (F_A) and zygotic (F_Z) fixation indices. The patchy distribution of alleles as a potential source of substructure inbreeding was tested from the allelic frequencies mapped along a linear transect. Evidence for consanguineous matings in restricted neighborhoods and for selection at two different life cycle stages, and the efficiency of the protandrous breeding system were noted and discussed. Multilocus estimates of outcrossing are useful for their greater precision and unbiased nature while single locus estimates can help in detecting the effects of selection and population substructure. The data generally support the heterozygosity paradox noted by Brown (1979) but further suggest that the paradox may often result from a lack of precision of outcrossing estimates and from overlooking the stages of the life cycle being sampled.     

Evaluation of microbial testing methods for the mutagenicity of quinoline and its derivatives

Using the mutational enhancement method and the Ames test, the mutagenicity and potential carcinogenicity of quinoline and its derivatives were determined and compared. Quinoline, 8-hydroxyquinoline, 5-hydroxyquinoline, 8-hydroxyquinoline sulfate, 6-nitroquinoline, 8-nitroquinoline, and 3-methylquinoline were mutagenic in the Ames direct plating test on TA 98 and TA 100 with activating system (S-9) from the rat liver. These compounds were not mutagenic in the mutational enchancement test onEscherichia coli HCR+ strain. 5,7-Dichloro-8-hydroxyquinoline, isoquinoline, and 2-chloroquinoline were nonmutagenic without or with S-9 in both the Ames and mutational enhancement test system. The compounds chloroquine, primaquine diphosphate, quinine hydrobromide, quinine hydrochloride, quinine lactate, quinine urea hydrochloride, quinine ethylcarbonate, quinine dihydrochloride, beta quinine quinine valerate, and quinine glycerophosphate were nonmutagenic with and without S-9 in the Ames test but mutagenic (20–60 g/ml) in the mutational enhancement test method onEscherichia coli HCR+ strains. The observations reported here point out that the Ames test responds negatively to several quinoline derivatives that are positive in the mutational enchancement test method.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

A posteriori time-varying filtering of averaged evoked potentials

The problem of estimating an unknown transient signal, given an ensemble of waveforms, in which this signal appears as a nonrandom component in the presence of additive noise is considered. This problem is solved by generalizing the method of a posteriori Wiener filtering. In the new method, the ensemble average is filtered by a time-varying system which is based on estimated time-varying power spectra of signal and noise. The nature of this system, and the computational procedures involved, are discussed in detail. A software package for time-varying filtering is briefly described. Application of the method is illustrated by a simulation example, which also provides a comparison to time-invariant a posteriori Wiener filtering.     

Populational Heritability: Extending Punnett Square Concepts to Evolution at the Metapopulation Level

In a previous study, using experimental metapopulations of the flour beetle, Tribolium castaneum, we investigated phase III of Wright's shifting balance process (Wade and Griesemer 1998). We experimentally modeled migration of varying amounts from demes of high mean fitness into demes of lower mean fitness (as in Wright's characterization of phase III) as well as the reciprocal (the opposite of phase III). We estimated the meta-populational heritability for this level of selection by regression of offspring deme means on the weighted parental deme means.Here we develop a Punnett Square representation of the inheritance of the group mean to place our empirical findings in a conceptual context similar to Mendelian inheritance of individual traits. The comparison of Punnett Squares for individual and group inheritance shows how the latter concept can be rigorously defined and extended despite the lack of explicitly formulated, simple Mendelian laws of inheritance at the group level. Whereas Wright's phase III combines both interdemic selection and meta-populational inheritance, our formulation separates the issue of meta-populational heritability from that of interdemic selection. We use this conceptual context to discuss the controversies over the levels of selection and the units of inheritance.     

Image processing technique for measuring specific growth rates of Gibberella fujikuroi

Summary Two methods were compared for estimating of Gibberella fujikuroi grown with different proportions of glucose and starch. They were, =Ln2 (V_r/Le) on Petri dish (V_r= rate of tip extension and L_e= mean hyphal length) and, ₂=d (LnX)/dt in stirred fermenter. Values of ₁ and ₂ were in close agreement with each other.     

A method for the estimation of gene flow parameters from a population structure caused by restricted gene flow and genetic drift

Summary A method has been developed which enables the estimation of the plant gene flow parameters _p (pollen dispersal), _s (seed dispersal) and t (outcrossing rate) from a selection-free continuously structured population in equilibrium. The method uses Wright's F-coefficients and introduces a new F-function which describes the genetic similarity as a function of the spatial distance. The method has been elaborated for wind pollinated plant species but can be modified for insect pollination and for animal species. In practice allozymes will provide for the necessary neutral genetic variation. The more loci used and the more intermediate the gene frequencies, the more reliable the results. For the estimation of _p and t together (when the outcrossing rate is not known) at least two chromosomally unlinked loci are required. The method for estimating _s depends on whether the plant species is annual or perennial. The mechanism of selfing has been analysed by the explanation of the value of t by three components: population density (d), pollen flow (_p) and relative fertilization potential of own pollen (Z). The concepts of neighbourhood size and isolation by distance, developed by Wright, who used a single gene flow parameter , have been extended to the situation which is realistic for seed plants, using all three parameters _p, _s and t. When _p is large with respect to _s, _s largely determines the value of the neighbourhood size, whereas _p is the most dominating factor in isolation by distance. The use of local effective population size and mean gene transport per generation instead of neighbourhood size and neighbourhood area, respectively, is proposed to avoid confusion. Computer simulations have been carried out to check the validity and the reliability of the method. Populations of 200 plants, using two or three loci with intermediate allele frequencies, gave good results in the calculation of _p with known value of t and of _s and N_e. With unknown t, especially with lower values of t, larger populations of at least 1,000 plants are necessary to obtain reasonably accurate results for _p and mean gene transport per generation M.Grassland Species Research Group Publication No. 81