Genome size in wild Pisum species

Genome size was measured in 75 samples of the wild pea species Pisum abyssinicum, P. elatius, P. fulvum and P. humile by ethidium-bromide (EB) flow cytometry (internal standard: Triticum monococcum) and Feulgen densitometry (internal standard: Pisum sativum Kleine Rheinländerin). Total variation of EB-DNA between samples covered 97.7% to 114.9% of the P. sativum value, and Feulgen DNA values were strongly correlated with EB-DNA values (r=0.9317, P < 0.001). Only P. fulvum was homogeneous in genome size (108.9% of P. sativum). Wide variation was observed between samples in P. abyssinicum (100.9–109.7%), P. elatius (97.7–114.9%) and P. humile (98.3–111.1% of P. sativum). In view of the world-wide genome size constancy in P. sativum, the present data are interpreted to show that the pea taxa with variable genome size are genetically inhomogeneous and that the current classification is not sufficient to describe the biological species groups adequately.     

Cryptopolyploidy inBunias (Brassicaceae) revisited — A flow-cytometric and densitometric study

The concern of the present analysis is the hypothetical cryptopolyploidy, a concept basically of historical interest only, but discussed again by Battaglia (1996) in a recent treatment of the term and its historical background. Melinossi (1935), while reanalyzing erratic observations on the crucifersBunias erucago andB. orientalis by Jaretzky (1928a), found 2n = 14 in both species but twice the chromosome volume inB. erucago compared withB. orientalis. Melinossi considered cryptopolyploidy inB. erucago, i.e., she discussed pairwise fused chromosomes on a tetraploid basis or endoreduplicated (and thus binemic) chromosomes in this species. Cryptopolyploidy has also been claimed by Pannocchia-Laj (1938) inVinca difformis (Vincaceae). Battaglia (1996) criticized the term cryptopolyploidy because, in his opinion, the genuinely polyploid status of these plants is not hidden (crypto) but phenotypically (from herbarium specimens) recognizable. He coins the term phenopolyploidy, i.e., phenotypic polyploidy disagreeing with the karyotype numerically evalated. We measured genome size ofB. orientalis andB. erucago (both 2n = 14) by Feulgen densitometry and propidium iodide flow cytometry. Surprisingly,B. erucago (the annual species with 2.13 pg, 1 C) turned out to have only 0.81-fold the DNA amount ofB. orientalis (the perennial species with 2.64 pg, 1 C). Therefore, any kind of genetically polyploid status inB. erucago is out of the question. Only speculative significance can be ascribed to the terms cryptopolyploidy and phenopolyploidy.     

Characterization ofGandalf, a new inverted-repeat transposable element ofDrosophila koepferae

The cloning and characterization ofGandalf, a new DNA-transposing mobile element obtained from theDrosophila koepferae (repleta group) genome is described. A fragment ofGandalf was found in a middle repetitive clone that shows variable chromosomal localization. Restriction, Southern blot, PCR and sequencing analyses have shown that mostGandalf copies are about 1 kb long, are flanked by 12 by inverted terminal repeats and contain subterminal repetitive regions on both sides of the element. As with other elements of the DNA-transposing type (known as the Ac family), theGandalf element generates 8 by direct duplications at the insertion point. Coding region analysis has shown that the longer open reading frame found inGandalf copies could encode part of a protein. However, whether or not the 1 kb copies of the element are actually the active transposons remains to be elucidated.Gandalf shows a very low copy number inD. buzzatii, a sibling species ofD. koepferae. An attempt to induce interspecific hybrid dysgenesis in hybrids of these two species has been unsuccessful.     

Flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum

A DAPI and ethidium bromide flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum was conducted. The material included 38 accessions of P. sativum of widely different geographic origin and altogether 14 samples of P. elatius, P. abyssinicum, P. humile and P. fulvum. The relative genome size values obtained with the three staining methods were strongly correlated. No evidence for genome size variation was found among P. sativum cultivars. In particular, certain Italian cultivars, for which strongly deviating C-values have been reported, proved to be invariant. The only occasion when ambiguous evidence for marginal genome size variation was found was when all 38 accessions taxonomically affiliated with P. sativum were considered. Pisum abyssinicum and P. fulvum differed from P. sativum by about 1.066-and 1.070-fold, respectively; 1 accession of P. humile differed by 1.089-fold, and 2 of P. elatius by 1.122- and 1.195-fold, respectively (ethidiumbromide comparison), while the other accessions of these taxa were not different from P. sativum. This variation may indicate taxonomic inhomogeneity and demands further investigation. Cultivated P. sativum has long been suspected of not being constant with respect to genome size. As shown here, these findings were not based on genuine differences, but rather were technical in origin.     

DNA base composition heterogeneity in some agarophytes (Gracilariales,Rhodophyta) from Mexico and the Philippines

Estimates of nuclear DNA base composition by determination of thermal denaturation temperatures (T_m) indicate guanine + cytosine (G + C) levels of 35.4–46.8% for ten species of the Gracilariaceae, representing the generaGracilaria andHydropuntia. T_m values were found to be reproducible with variation among most samples and replicates of less than 1 °C and 2 mol%. Interspecific variation in G + C values was less than 11.4% amongGracilaria species. Calculation of intragenomic base pair composition distribution based on mid-resolution thermal denaturation (A 1 °C/min with 4s interval H and dT logging) indicated an inverse relationship between maximum similarity values and taxonomic rank. Intraspecific (population level) maximum similarity (homology) values were estimated to range from 79–90% inGracilaria tikvahiae (4 isolates). Interspecific values of 46–69% were found in 13 species ofGracilaria. Nucleotide distribution similarity values for the Gracilariaceae are compared with previous information for genome organization and complexity, genome size and karyotype patterns.Author for correspondence     

Analyses of<Emphasis Type="Italic"> cis</Emphasis> -acting elements that affect the transposition of<Emphasis Type="Italic"> Mos1 mariner</Emphasis> transposons in vivo

The left (5) inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3) ITR. The effects on the transposition frequency resulting from the use of two 3 ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3 ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3 ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3 ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.Communicated by G. P. Georgiev     

Differentiation betweenPhaeocystis pouchetii (Har.) Lagerheim andPhaeocystis globosa Scherffel

An Arctic clone ofPhaeocystis pouchetii LAGERHEIM was compared toPhaeocystis globosa SCHERFFEL isolated from the southern North Sea with regard to temperature tolerance and colony shapes. Already youngP.pouchetii colonies (<100 m) show the typical distribution of the cells in groups, separated from each other by wide zones of cell-free mucilage; the maximum colony size is ca 2 mm in diameter.P.pouchetii colonies form clouds with bubble-like vesicles, spherical colony-shapes are seldom found.P.globosa colonies are spherical up to a size of 2 mm; the cells are distributed homogeneously over the periphery of the colonies. A pouchetii-like distribution of cells never occurs either in the spherical young colonies or in the pear-shaped old colonies (size up to 8 mm). A development from the colony shape of the globosa-type to the pouchetii-type or vice versa was never found. Therefore the colony shape has to be considered a constant distinctive character. Single cells ofP.pouchetii andP.globosa cannot be separated from each other by using the light microscope; this also holds for the flagellates and the non-motile cells.P.pouchetii grows well between 0°C and 14°C,P.globosa between 4°C and 22°C, respectively. Because of the distinctive differences in the morphology of the colonies and the differences in temperature tolerances we propose thatPhaeocystis globosa should no longer be considered conspecific withPhaeocystis pouchetii.     

A comparative electrophoretic study of the seed albumins fromSophora microphylla andPisum sativum (Leguminosae)

The albumin proteins from seed ofSophora microphylla Ait. and from cotyledons ofPisum sativum L. (cv. Greenfeast) have been analysed electrophoretically using a range of gels of varied pore size. Plots of mobility [as 100 log₁₀ (R _f × 100)] vs.acrylamide content of gel indicate that very few of the albumins fromS. microphylla are homologous with albumins fromP. sativum. Despite the diverse compositions of the two fractions, their amino acid analyses were surprisingly similar.     

Effects of the potential allelochemical α-asarone on growth,physiology and ultrastructure of two unicellular green algae

The effects of the natural phenylpropanoid -asarone on growth pattern, photosynthesis, respiration and cell structure of two microalgae have been investigated. In cultures ofAnkistrodesmus braunii -asarone decreases in the medium and induces a lag in growth. Both phenomena were dependent on the number of cells inoculated. By contrast, in cultures ofSelenastrum capricornutum a constant decrease of the growth rate at all inocula was observed and only a slight decrease of -asarone in the medium occurred. In both algae -asarone caused an initial inhibition of photosynthesis, followed by a resumption of control values. The respiratory rate ofA. braunii was not significantly affected by -asarone, whereas inS. capricornutum respiration lowered to 60% of the control in the first 48 h and subsequently rose to values exceeding the controls by 20%. Ultrastructural observations carried out 24 and 72 h after the addition of -asarone showed modifications of cell wall inA. braunii, an increase in the number of mithocondrial profiles per cell section inS. capricornutum, and an accumulation of electron-dense deposits in the vacuoles of both algae.Author for correspondence     

Structural and functional analysis of the killer element pPin1-3 from Pichia inositovora

Strains of the yeast Pichia inositovora that carry the linear plasmids pPin1-1 (18 kb) and pPin1-3 (10 kb) display a killer activity towards Saccharomyces cerevisiae. Cloning and sequencing of the smaller plasmid, pPin1-3, revealed that it is 9683 bp long and has 154-bp terminal inverted repeats. Comparison of pPin1-3 with the only other completely sequenced killer plasmid, pGKL1 of Kluyveromyces lactis, revealed differences in genome organization. The Pichia element has four ORFs that account for 95% of the sequence. ORF1 is homologous to the putative immunity gene of the K. lactis system. A viral B-type DNA polymerase is encoded by ORF2. The predicted product of ORF3 displays similarities to the - and -subunits of the heterotrimeric K. lactis killer toxin, also known as zymocin. A cysteine-rich chitin-binding site and a chitinase signature, characteristic for the -subunit of zymocin were identified in Orf3p. Chitin affinity chromatography and Western analysis confirmed the plasmid specific expression and secretion of a protein that cross-reacts with an antibody raised against the -subunit of K. lactis zymocin. Disruption of the major chitin synthase-gene ( CHS3) renders S. cerevisiae resistant to the toxin, providing further evidence that chitin is the cellular receptor for the P. inositovora toxin. Orf4p of pPin1-3 displays only weak similarities to the -subunit of zymocin, which causes a G1 cell-cycle arrest in S. cerevisiae. However, disruption of the S. cerevisiae gene ELP3/TOT3, which encodes a histone-acetyltransferase that is essential for zymocin action, resulted in reduced sensitivity to the P. inositovora toxin also. Thus, despite obvious differences in genome organization and protein architecture, both killer systems very probably have similar modes of action.Communicated by C. P. Hollenberg     

Genome size variation and C-band polymorphism inAlstroemeria aurea, A. ligtu andA. magnifica (Alstroemeriaceae)

Among a total of 43 accessions ofAlstroemeria aurea, A. ligtu andA. magnifica nuclear DNA amounts (2C-values) showed significant intraspecific variation, 1.09, 1.21 and 1.15 fold, respectively, when determined through flow cytometric measurements of fluorescence of propidium iodide (PI) stained nuclei. After staining with another fluorochrome, 4,6-diamidino-2-phenylindole (DAPI), an intraspecific variation of 1.10, 1.11 and 1.12 fold, respectively, was found. C-band polymorphisms were present among and within the accessions of all three species. In some cases only very small differences in C-banding pattern were observed. In other cases, however, differences were more prominent. Besides C-band polymorphism, there were also instances of chromosome length polymorphism, which concerned the total chromosome complement or single chromosomes. The variation in nuclear DNA amount inA. aurea andA. ligtu was more or less continuous, except for one accession ofA. ligtu subsp.simsii. Artificial selection and possibly introgression of chromosomes from other species may have moulded the karyotypes of some of the accessions ofA. aurea, a species that has been under cultivation for more than 160 years. The variation as observed inA. magnifica subsp.magnifica was discontinuous and could be due to a broad species concept.     

Polytene chromosomes in cultured pea roots (Pisum,Fabaceae)

The experiments on cultured pea roots (Pisum sativum cv. Alaska and Dwarf Telephone) are summarized in Table 1. Nuclear growth and mitosis occurred mainly in cultures in which the medium of Torrey and Shigemura was supplemented with 6 p.p.m. 2,4-D and 1 p.p.m. kinetin. The greatest reaction was observed in 16-day cultures of cv. Alaska. Nuclei had increased in size, and prophases diploid and polyploid, with normal chromosomes, diplochromosomes or larger bundles of chromatids were visible. Metaphases which ranged from 2 n to an estimated 32 n had normal chromosomes with two chromatids. Polytene chromosomes, in diploid, rarely in tetraploid number, occurred in numerous cortex cells. They did not show banding, and their telomeres, spread into individual chromatids, were attached to the nuclear membrane. In some cells the polytene chromosomes were condensed into spherical structures, obviously a stage in their falling apart; the last stage of this process is a polyploid metaphase.     

Chlorosomes of green sulfur bacteria: Pigment composition and energy transfer

The pigment composition and energy transfer pathways in isolated chlorosomes ofChlorobium phaeovibrioides andChlorobium vibrioforme were studied by means of high performance liquid chromatography (HPLC) and picosecond absorbance difference spectroscopy. Analysis of pigment extracts of the chlorosomes revealed that they contain small amounts of bacteriochlorophyll (BChl)a esterified with phytol, whereas the BChlsc, d ande are predominantly esterified with farnesol. The chlorosomal BChla content inC. phaeovibrioides andC. vibrioforme was found to be 1.5% and 0.9%, respectively. The time resolved absorbance difference spectra showed a bleaching shifted to longer wavelengths as compared to the Q_y absorption maxima and in chlorosomes ofC. vibrioforme also an absorbance increase at shorter wavelengths was observed. These spectral features were ascribed to excitation of oligomers of BChle and BChlc/d, respectively. One-color and two-color pump-probe kinetics ofC. phaeovibrioides showed rapid energy transfer to long-wavelength absorbing BChle oligomers, followed by trapping of excitations by BChla with a time constant of about 60 ps. Time resolved anisotropy measurements inC. vibrioforme showed randomization of excitations among BChla molecules with a time constant of about 20 ps, indicating that BChla in the baseplate is organized in clusters. One-color and two-color pump-probe measurements inC. vibrioforme showed rapid energy transfer from short-wavelength to long-wavelength absorbing oligomers with a time constant of about 11 ps. Trapping of excitations by BChla in this species could not be resolved unambiguously due to annihilation processes in the BChla clusters, but may occur with time constants of 15, 70 and 200 ps.     

Ecophysiological aspects of growth and nitrogen fixation inAzospirillum spp.

The nitrogenase activity ofAzospirillum spp. is efficiently regulated by environmental factors. InA. brasilense andA. lipoferum a rapid switch off of nitrogenase activity occurs after the addition of ammonium chloride. As in photosynthetic bacteria, a covalent modification of nitrogenase reductase (Fe-protein) is involved. InA. amazonense, a non-covalent mechanism causes only a partial inhibition of nitrogenase activity after ammonium chloride is added. In anaerobic conditions, nitrogenase reductase is also switched off by a covalent modification inA. brasilense andA. lipoferum. Short-time exposure ofAzospirillum to increased oxygen levels causes a partially reversible inhibition of nitrogenase activity, but no covalent modification is involved.Azospirillum spp. show variations in their oxygen tolerance. High levels of carotenoids confer a slightly improved oxygen tolerance. Certain amino acids (e. g. glutamate, aspartate, histidine and serine) affect growth and nitrogen fixation differently inAzospirillum spp. Amino acids may influence growth and nitrogen fixation ofAzospirillum in the association with plants.Azospirillum brasilense andA. halopraeferens are the more osmotolerant species. They utilize most amino acids poorly and accumulate glycine betaine, which also occurs in osmotically stressed grasses as a compatible solute to counteract osmotic stress. Nitrogen fixation is stimulated by glycine betaine and choline. Efficient iron acquisition is a prerequisite for competitive and aerotoleran growth and for high nitrogenase activity.Azospirillum halopraeferens andA. amazonense assimilate iron reasonably well, whereas growth of someA. brasilense andA. lipoferum strains is severely inhibited by iron limitation and by competition with foreign microbial iron chelators. However, growth of certain iron-limitedA. brasilense strains is stimulated by the phytosiderophore mugineic acid. Thus, various plant-derived substances may stimulate growth and nitrogen fixation ofAzospirillum.     

A novel eye morphology induced by a P element in somatic tissue of Drosophila melanogaster.

Summary We found a specific eye morphology designated as Square, which is induced when some Drosophila melanogaster strains harboring P elements are crossed with the 2–3 strain carrying a modified P element, P[ry ⁺, 2–3], which produces transposase in somatic tissue. This phenotype was dominant and also induced in the reciprocal crosses. Square was induced when the 2–3 strain was crossed with Q and M strains such as the sn^w (M) strain carrying three small P elements but not with P strains. Inheritance of Square was also tested and its phenotype was not transmitted to the next generation. These results suggest that Square is caused by the transposition of P elements in somatic cells.     

Stimulation of Chloride In→Out Permeation Across the Deiters' Neuron Membrane by Pentobarbital on the Cytoplasmic Side: Additional Evidence of GABAA Receptors Acting as Chloride Extrusion Pumps

Pentobarbital stimulates ³⁶Cl^– permeation across single Deiters' membranes in a microchamber system, acting on classical, extracellularly facing, GABA_A receptors. However, when applied on the membrane cytoplasmic side it activates per se labeled chloride inout permeation. No effect was found on chloride outin permeation. Similarly, at lower concentrations it facilitates the increase of ³⁶Cl^– inout permeation by application of GABA on the membrane inside, again via asymmetric chloride channels allowing inout but not outin passage. These data confirm that on the Deiters' membrane cytoplasmic side there are structures behaving pharmacologically as GABA_A receptors whose function is that of a Cl^– extrusion pump. This mechanism involves a cycle of activation-phosphorylation/desensitization-reactivation of the receptor complexes     

Evaluation and first-year field testing of efficient vesicular arbuscular mycorrhizal fungi for inoculation of wetland rice seedlings

Grain yields of the rice cultivar Prakash were improved upon inoculation with Glomus intraradices and G. fasciculatum, by 11% and 8%, respectively, compared with an uninoculated control. The results indicate that the amount of phosphate fertilizer usually applied to rice may be decreased by 50%, without affecting yield, if G. intraradices is inoculated.The authors are with the Department of Agricultural Microbiology, University of Agricultural Sciences, GKVK Campus, Bangalore 560 065, India. ing author.     

Fruiting studies in species ofCapronia (Herpotrichiellaceae)

A recently described protocol for thein vitro production of ascomata was employed to determine the sexual incompatibility systems of five species ofCapronia. The formation of mature ascomata in isolates derived from single ascospores demonstrated thatC. epimyces, C. mansonii, andC. munkii n. sp. are homothallic. In contrast, fertile ascomata were observed only in mass-ascospore isolates and pairwise crosses between specific single-ascospore isolates inC. dactylotricha n. sp. andC. moravica. TheExophiala anamorphs ofC. dactylotricha andC. munkii are described and aPhialophora-like synanamorph is reported for the former species. Germinating ascospores ofC. munkii formed conidiogenous cells directly, while the ascospores of the remaining species germinated to produce germ tubes and hyphae. The application of the terms microcyclic conidiation to secondary conidium production and sclerotial body and stroma to the multicellular structures produced by species ofCapronia andExophiala are discussed.     

Quantitative analyses of C-banded karyotypes,and systematics in the cultivated species of theScilla siberica group (Liliaceae)

Quantified C-band karyograms are presented for the related speciesScilla siberica, S. mordakiae, S. ingridae, S. amoena, andS. mischtschenkoana. Chromosome structure, banding style, and heterochromatin characters suggest a systematic grouping of two more closely related species pairs:S. siberica andS. mordakiae, S. ingridae andS. amoena; they are part of a larger aggregate, well separated fromS. mischtschenkoana. Four different heterochromatin fractions can be recognized inS. siberica and its relatives, but only two inS. mischtschenkoana. C-bands do not replace, but they are added to euchromatin. The particular origin and history of the cultivatedS. amoena and the triploidS. siberica cv. Spring Beauty appear to be responsible for their karyotype constancy, but chromosome conservatism obviously is genuine inS. mischtschenkoana. The banding data support the systematic grouping proposed on a morphological basis, and provide additional evolutionary evidence.Evolution ofScilla and Related Genera, IV.     

Mutations that are synthetically lethal with a<Emphasis Type="Italic"> gas1Δ</Emphasis> allele cause defects in the cell wall of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The GAS1 -related genes of fungi encode GPI-anchored proteins with -1,3-glucanosyltransferase activity. Loss of this activity results in defects in the assembly of the cell wall. We isolated mutants that show a synthetic defect when combined with a gas1 allele in Saccharomyces cerevisiae, and identified nine wild-type genes that rescue this defect. The indispensability of BIG1 and KRE6 for the viability of gas1 cells confirmed the important role of -1,6-glucan in cells that are defective in the processing of -1,3-glucan. The identification of the Wsc1p hypo-osmotic stress sensor and components of the PKC signal transduction pathway in our screen also confirmed that the cell wall integrity response attenuates the otherwise lethal gas1 defect. Unexpectedly, we found that the KEX2 gene is also required for the viability of the gas1 mutant. Kex2p is a Golgi/endosome-membrane-anchored protease that processes secretory preproteins. A cell wall defect was also found in the kex2 mutant, which was suppressible by multiple copies of the MKC7 or YAP3 gene, both of which encode other GPI-anchored proteases. Therefore, normal cell wall assembly requires proteolytic processing of secretory preproteins. Furthermore, the genes CSG2 and IPT1 were found to be required for normal growth of gas1 cells in the presence of 1 M sorbitol. This finding suggests that complex sphingolipids play a role in the hyper-osmotic response.Communicated by C. P. Hollenberg