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     

Reverse genetic analysis of the glutathione metabolic pathway suggests a novel role of<Emphasis Type="Italic"> PHGPX</Emphasis> and<Emphasis Type="Italic"> URE2</Emphasis> genes in aluminum resistance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

We have taken a systematic genetic approach to study the potential role of glutathione metabolism in aluminum (Al) toxicity and resistance, using disruption mutants available in Saccharomyces cerevisiae. Yeast disruption mutants defective in phospholipid hydroperoxide glutathione peroxidases (PHGPX; phgpx1 , phgpx2 , and phgpx3), were tested for their sensitivity to Al. The triple mutant, phgpx1 /2/3, was more sensitive to Al (55% reduction in growth at 300 M Al) than any single phgpx mutant, indicating that the PHGPX genes may collectively contribute to Al resistance. The hypersensitivity of phgpx3 to Al was overcome by complementation with PHGPX3, and all PHGPX genes showed increased expression in response to Al in the wild-type strain (YPH250), with maximum induction of approximately 2.5-fold for PHGPX3. Both phgpx3 and phgpx1/2/3 mutants were sensitive to oxidative stress (exposure to H₂O₂ or diamide). Lipid peroxidation was also increased in the phgpx1/2/3 mutant compared to the parental strain. Disruption mutants defective in genes for glutathione S-transferases (GSTs) (gtt1 and gtt2), glutathione biosynthesis (gsh1 and gsh2), glutathione reductase (glr1) and a glutathione transporter (opt1) did not show hypersensitivity to Al relative to the parental strain BY4741. Interestingly, a strain deleted for URE2, a gene which encodes a prion precursor with homology to GSTs, also showed hypersensitivity to Al. The hypersensitivity of the ure2 mutant could be overcome by complementation with URE2. Expression of URE2 in the parental strain increased approximately 2-fold in response to exposure to 100 M Al. Intracellular oxidation levels in the ure2 mutant showed a 2-fold (non-stressed) and 3-fold (when exposed-to 2 mM H₂O₂) increase compared to BY4741; however, the ure2 mutant showed no change in lipid peroxidation compared to the control. The phgpx1/2/3 and ure2 mutants both showed increased accumulation of Al. These findings suggest the involvement of PHGPX genes and a novel role of URE2 in Al toxicity/resistance in S. cerevisiae.Communicated by D.Y. Thomas     

Effect of<Emphasis Type="Italic"> p</Emphasis>CO<Subscript>2</Subscript> and temperature on the boron isotopic composition of the zooxanthellate coral<Emphasis Type="Italic"> Acropora</Emphasis> sp.

Culture experiments were carried out with Acropora sp. (a branching scleractinian coral) in seawater at two pCO₂ conditions (438 and 725 µatm) and two temperatures (25 and 28 °C) in order to establish the pH and temperature dependence of the boron isotopic composition of the skeleton. A clear pCO₂ effect, but no temperature effect, on the coral boron isotope composition is seen. For corals cultured at normal pCO₂ (438 µatm), the ¹¹B of the skeleton was 24.0±0.2 at 25 °C, and 23.9±0.3 at 28 °C. The values of ¹¹B measured for corals cultured at higher pCO₂ (725 µatm) were lower: 22.5±0.1, and 22.8±0.1 at 25 and 28 °C, respectively. The ¹¹B of corals cultivated at both high and normal pCO₂ conditions are consistent with a dominant pH control, and are very close to that calculated from theoretical considerations. Thus, the corals do not seem to significantly alter ambient seawater for calcification with respect to pH. Co-variation between boron and carbon isotope values is explored.Communicated by: Guest Editor A. Grottoli     

Characterization of an unusual <Emphasis Type="Italic">Ds</Emphasis> transposable element in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: Insertion of an abortive circular transposition intermediate

The maize Ac/Dstransposable elements, which belong to the hAT transposon superfamily, are widely used as insertional mutagens in numerous plant species. Molecular studies suggest that Ac/Ds elements transpose in a conservative non-replicative fashion; however the molecular mechanism of transposition remains unclear. We describe here the identification of an unusual Ds element, Ds-mmd1, in a transgenic Arabidopsis line. Ds-mmd1 is rearranged relative to the original Ds element, such that the original 5 and 3 ends are internal and previously internal sequences are the new 5 and 3 termini of Ds-mmd1. Short duplications of plant genomic DNA and Ds sequences are present at the Ds-mmd1 junctions, suggesting that a circular Dsmolecule was part of the events that created the Ds-mmd1 element. In addition, a revertant analysis on mmd1 plants demonstrated that Ds-mmd1 can be eliminated from the genome in an Ac-dependent process.     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

A DNA sequence of Drosophila melanogaster with a differential telomeric distribution

A DNA sequence (8–19T) of 2.3 kilobase pairs (kb) of Drosophila melanogaster was localized by in situ hybridization to the extreme ends of polytene chromosomes and to the chromocenter. The relative abundance of this sequence at the ends of polytene chromosomes X2L2R3L3R is 13.41.902.7. This differential distribution is probably due to different copy numbers at the individual telomeric regions. Restriction enzyme analysis of genomic DNA shows that 8–19T sequences are interspersed with other sequences. The clone 8–19T, which contains most of this interspersed repetitive sequence, is itself not internally repetitive but has a complex sequence composition. Some of these sequences are transcribed into poly(A)⁺RNA. We suggest that the ends of Drosophila chromosomes are of a complex arrangement with some sequences common to all ends.     

Identification and DNA sequence analysis of a γ-type gliadin cDNA plasmid from winter wheat

Summary Recombinant cDNA plasmids possessing the coding sequences for the -type gliadins were isolated from a cDNA library prepared from wheat seed poly (A⁺) RNA. One of these plasmids, pGliB48, specifically hybridizes to poly (A⁺) RNA molecules 1 400–1 500 bases in length that direct the synthesis of polypeptides at 38 Kd and 46 Kd, the latter size characteristic of the -type gliadins. The cDNA sequence of pGliB48 was determined and encompasses the 3 untranslated region as well as 245 amino acids from the C-terminus of the -type gliadin polypeptide. The 5-end of the DNA coding sequence consists of a tandem repeat unit composed of eight amino acids. Localized regions of homology are observed for the /-type and -type gliadin cDNA sequences.     

Genetic Distance in Housekeeping Genes Between <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> and <Emphasis Type="Italic">Plasmodium reichenowi</Emphasis> and Within <Emphasis Type="Italic">P. falciparum</Emphasis>

The time to the most recent common ancestor of the extant populations of Plasmodium falciparum is controversial. The controversy primarily stems from the limited availability of sequences from Plasmodium reichenowi, a chimpanzee malaria parasite closely related to P. falciparum. Since the rate of nucleotide substitution differs in different loci and DNA regions, the estimation of genetic distance between P. falciparum and P. reichenowi should be performed using orthologous sequences that are evolving neutrally. Here, we obtained full-length sequences of two housekeeping genes, sarcoplasmic and endoplasmic reticulum Ca²⁺-ATPase (serca) and lactate dehydrogenase (ldh), from 11 isolates of P. falciparum and 1 isolate of P. reichenowi and estimate the interspecific genetic distance (divergence) between the two species and intraspecific genetic distance (polymorphism) within P. falciparum. Interspecific distance and intraspecific distance at synonymous sites of interspecies-conserved regions of serca and ldh were 0.0672±0.0088 and 0.0011±0.0007, respectively, using the Nei and Gojobori method. Based on the ratio of interspecific distance to intraspecific distance, the time to the most recent common ancestor of P. falciparum was estimated to be (8.30±5.40) × 10⁴ and (11.62±7.56) × 10⁴ years ago, assuming the divergence time of the two parasite species to be 5 and 7 million years ago, respectively.This article contains an online supplementary table.Reviewing Editor: Dr. Martin Kreitman     

<Emphasis Type="Italic">In vitro</Emphasis> screening of sugarcane to evaluate smut susceptibility

Tissue-cultured plantlets of three sugarcane (Saccharum spp.) cultivars having a known field smut reaction were screened for susceptibility to Ustilago scitaminea H&P Sydow. Plantlets were inoculated with 0.5 l of a suspension of equally mixed quantities of plus and minus mating type sporidia of U. scitaminea at concentrations ranging from 1×10¹ to 1×10⁶ cells. Fungal sori (whips) were produced in cultivar N12 (intermediate) 6weeks following inoculation with 1×10⁵ mixed sporidia and thereafter in cultivar NCo310 (susceptible) but not in cultivar N19 (resistant). Sori bearing teliospores were produced up to 3months following inoculation and incubation at 26°C. No sori were produced at mixed sporidial concentrations lower than 1×10⁵cells. The in vitro soral production in cultivars N19, N12 and NCo310 was 0, 27.5 and 47.5% respectively. Plantlets inoculated with 1×105sporidia of only one mating-type did not produce sori in any of the three cultivars tested. Blind scoring of an unknown sugarcane cultivar by this method corresponded exactly with its field smut rating.     

The moss<Emphasis Type="Italic"> Physcomitrella patens</Emphasis> releases a tetracyclic diterpene

The presence of the tetracyclic diterpene 16-hydroxykaurane (16-hydroxy-ent-kaurane, C₂₀H₃₄O, CAS 5524–17–4) was detected in sterile cell cultures of the moss Physcomitrella patens (Hedw.) B.S.G. using gas chromatography and mass spectrometry. 16-hydroxykaurane was found to be a major lipid compound in P. patens, with an estimated intracellular concentration of up to 0.84 mmol/l and an extracellular concentration of up to 9.3 µmol/l. The overall content of 16-hydroxykaurane (in milligrams) produced per culture reached 0.37-fold that of chlorophyll a+b. In agar cultures with low air exchange, 16-hydroxykaurane forms needle-like crystals on tissue and on the inner surface of the culture vessels, indicating that it is being released into the atmosphere. Solid phase microextraction confirmed the air-bound release of 16-hydroxykaurane. To our knowledge this is the first report on the release of a plant-derived tetracyclic diterpene into the air.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. ReskiThis work is dedicated to the 65th birthday of Prof. Heinz Hahn.     

Karyosystematics of thePhleum alpinum polyploid complex (Poaceae)

The karyotypes of three taxa from thePhleum alpinum group of sect.Phleum (P. alpinum subsp.rhaeticum, 2n = 14,P. commutatum, 2n = 28, and informally namedP. commutatum, 2n = 14) were investigated by Giemsa C-banding. The overall similarity of diploid genomes suggests thatP. alpinum subsp.rhaeticum andP. commutatum are closely related — their karyotypes vary only with respect to their average amounts of telomeric heterochromatin. TheP. commutatum genome contains less telomeric heterochromatin than the genome ofP. alpinum subsp.rhaeticum, but in theP. alpinum group as a whole almost fluent transition between low (1.5%) and high (25.5%) amounts of telomeric heterochromatin was observed among populations. In the karyotype of tetraploidP. commutatum, seven distinguishable chromosome types were observed. Each of them is represented at somatic metaphase by four chromosomes. C-band structure of karyotype and average amount of telomeric heterochromatin suggest that this taxon has originated from hybridization between two related diploid forms of theP. commutatum — P. alpinum complex.     

A Motility Revertant of the <Emphasis Type="Italic">ndvB</Emphasis> Mutant of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

A motility revertant of a Bradyrhizobium japonicum ndvB mutant was isolated and characterized. The ndvB mutants of B. japonicum have been reported to be osmotically sensitive, as well as defective in motility, periplasmic cyclic -(13), (16)-D-glucan synthesis, and symbiosis with soybean. The motility revertant was restored for osmotic tolerance but not for cyclic -glucan production or effective symbiosis. These results support our hypothesis that cyclic -glucans have an important role in symbiosis—the suppression of a plant defense response—in addition to their role in periplasmic osmoprotection.     

(T2AG3)n Telomeric Sequence Hybridization Indicating Centric Fusion Rearrangements in the Karyotype of the Rodent Oryzomys Subflavus

Chromosome preparations of 30 specimens of Oryzomys subflavus trapped in eight Brazilian localities were C-, and G-banded and analyzed by fluorescence in situ hybridization (FISH). Two karyotypes were found, 2n=50/FN=64, at three coastal localities of the Atlantic Forest domain, and 2n=58/FN=70 at two sites located in the Cerrado biome, Brazil Central. Two fluorescence in situ hybridization (FISH) patterns of the telomeric sequence (T₂AG₃)_n were observed: in both karyotypes the probes hybridized to the telomeres of all chromosomes and also a hybridization signal in the centromeric regions of two autosome pairs was seen in the 2n=50 karyotype. These results, together with the occurrence of other diploid numbers described in the literature, suggest that O. subflavus is a complex species, bearing fusion/fission rearrangements proper to the different biomes which it inhabits.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the genus <Emphasis Type="Italic">Clivia</Emphasis>

We have developed a protocol for the in vitro propagation of the genus Clivia. Shoots were regenerated when fragments of the peduncle-pedicel junction (PP junction) from young inflorescences were used as explants. The optimal media for PP junction were Murashige and Skoog (MS)-based medium containing 10 M of 6-benzyladenine (BA) and 10 M of 2,4-dichlorophenoxyacetic acid (2,4-D) or MS supplemented with 5 M BA, 10 M -naphthaleneacetic acid (NAA), 250 mg l^-1 glutamine and 500 mg l^–1 casein hydrolysate and their usage depended on the breeding lines. Multiplication from initiations and in vitro seedlings was the best when the explants were cut longitudinally through the meristem and placed on MS plus 44 M BA. Plantlets were transferred on to hormone -free MS medium with charcoal for rooting.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Consequences of harvesting for genetic diversity in American ginseng (<Emphasis Type="Italic">Panax quinquefolius</Emphasis> L.): a simulation study

American ginseng, Panax quinquefolius L., is one of the most heavily traded medicinal plants in North America. The effect of harvest on genetic diversity in ginseng was measured with a single generation culling simulation program. Culling scenarios included random harvest at varying levels, legal limit random harvest and legal limit mature plant harvest. The legal limit was determined by the proportion of legally harvestable plants per population (% mature plants per population). Random harvest at varying levels resulted in significant loss of genetic diversity, especially allelic richness. Relative to initial levels, average within-population genetic diversity (H _e) was significantly lower when plants were culled randomly at the legal limit (Mann–Whitney U=430, p<0.001) or when only mature plants were culled (Mann–Whitney U=394, p<0.01). Within-population genetic diversity was significantly higher with legal limit mature plant harvest (H _e=0.068) than when plants were culled randomly at the legal limit (H _e=0.064; U=202, p<0.01). Based on these simulations of harvest over one generation, we recommend that harvesting fewer than the proportion of mature plants could reduce the negative genetic effects of harvest on ginseng populations.     

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.     

Photon- and carbon-use efficiency in<Emphasis Type="Italic"> Ulva rigida</Emphasis> at different CO<Subscript>2</Subscript> and N levels

The seaweed Ulva rigida C. Agardh (Chlorophyta) was cultured under two CO₂ conditions supplied through the air bubbling system: non-manipulated air and 1% CO₂-enriched aeration. These were also combined with N sufficiency and N limitation, using nitrate as the only N source. High CO₂ in U. rigida led to higher growth rates without increasing the C fixed through photosynthesis under N sufficiency. Quantum yields for charge separation at photosystem II (PSII) reaction centres (_PSII) and for oxygen evolution (_O2) decreased at high CO₂ even in N-sufficient thalli. Cyclic electron flow around PSII as part of a photoprotection strategy accompanied by decreased antennae size was suspected. The new re-arrangement of the photosynthetic energy at high CO₂ included reduced investment in processes other than C fixation, as well as in carbon diverted to respiration. As a result, quantum yield for new biomass-C production (^growth) increased. The calculation of the individual quantum yields for the different processes involved allowed the completion of the energy flow scheme through the cell from incident light to biomass production for each of the CO₂ and N-supply conditions studied.Abbreviations A total thallus absorptance - A_pig absorptance due to pigments - A_str Absorptance due to non-pigmented structures - a* spectrally averaged in vivo absorption cross-section of chlorophyll a - CCM carbon-concentrating mechanism - Chl chlorophyll - DOC dissolved organic carbon - ETR electron transport rate - F_v/F_m optimum quantum yield for PSII charge separation - GP gross O₂ evolution rate - k_pig specific light absorption coefficient for pigments - k_str specific light absorption coefficient for non-pigmented structures - OP optimum O₂ evolution rate - PFR photon fluence rate - POC particulate organic carbon - PS photosystem - q_N non-photochemical quenching - q_P photochemical quenching - ^growth quantum yield for new biomass-C production - _O2 quantum yield for gross O₂ evolution - _PSII quantum yield for PSII charge separation