Novel mitochondrial alcohol metabolizing enzymes of <Emphasis Type="Italic">Euglena gracilis</Emphasis>

Ethanol is one of the most efficient carbon sources for Euglena gracilis. Thus, an in-depth investigation of the distribution of ethanol metabolizing enzymes in this organism was conducted. Cellular fractionation indicated localization of the ethanol metabolizing enzymes in both cytosol and mitochondria. Isolated mitochondria were able to generate a transmembrane electrical gradient (Δψ) after the addition of ethanol. However, upon the addition of acetaldehyde no Δψ was formed. Furthermore, acetaldehyde collapsed Δψ generated by ethanol or malate but not by D-lactate. Pyrazole, a specific inhibitor of alcohol dehydrogenase (ADH), abolished the effect of acetaldehyde on Δψ, suggesting that the mitochondrial ADH, by actively consuming NADH to reduce acetaldehyde to ethanol, was able to collapse Δψ. When mitochondria were fractionated, 27% and 60% of ADH and aldehyde dehydrogenase (ALDH) activities were found in the inner membrane fraction. ADH activity showed two kinetic components, suggesting the presence of two isozymes in the membrane fraction, while ALDH kinetics was monotonic. The ADH Km values were 0.64–6.5 mM for ethanol, and 0.16–0.88 mM for NAD⁺, while the ALDH Km values were 1.7–5.3 μM for acetaldehyde and 33–47 μM for NAD⁺. These novel enzymes were also able to use aliphatic substrates of different chain length and could be involved in the metabolism of fatty alcohol and aldehydes released from wax esters stored by this microorganism.     

Chromium uptake,retention and reduction in photosynthetic <Emphasis Type="Italic">Euglena gracilis</Emphasis>

Photosynthetic Euglena gracilis grown with different K₂CrO₄ concentrations was analyzed for its ability to take up, retain and reduce Cr(VI). For comparison, cells were also exposed to CrCl₃. Cellular Cr(VI) uptake at pH 7.2 showed a hyperbolic saturation pattern with K _m of 1.1 mM, V _m of 16 nmol (h × 10⁷ cells)⁻¹, and K _i sulfate of 0.4 mM. Kinetic parameters for sulfate uptake were similar, K _m = 0.83 mM, V _m = 15.9 nmol (h × 10⁷cells)⁻¹ and K _i chromate = 0.3 mM. The capacity to accumulate chromium depended on the ionic species, external concentration and pH of the incubation medium. Cr(VI) or Cr(III) accumulation was negligible in the acidic (pH 3.5) culture medium, in which Cr(VI) was abiotically reduced to Cr(III). At pH 7.2 Cr(VI) was fully stable and high accumulation (>170 nmol/1 × 10⁷ cells at 1 mM K₂CrO₄) was achieved; surprisingly, Cr(III) accumulation was also significant (>35 nmol/1 × 10⁷ cells at 1 mM CrCl₃). Cr(VI) was reduced by cells at pH 7.2, suggesting the presence of an external reductive activity. Cr(VI) induced an increased cysteine and glutathione content, but not in phytochelatins suggesting that chromium accumulation was mediated by monothiol compounds.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

A novel family of mitochondrial proteins is represented by the <Emphasis Type="Italic">Drosophila</Emphasis> genes <Emphasis Type="Italic">slmo</Emphasis>, <Emphasis Type="Italic">preli-like</Emphasis> and <Emphasis Type="Italic">real-time</Emphasis>

Mitochondria play essential roles in development and disease. The characterisation of mitochondrial proteins is therefore of particular importance. The slowmo (slmo) gene of Drosophila melanogaster has been shown to encode a novel type of mitochondrial protein, and is essential in the developing central nervous system. The Slmo protein contains a conserved PRELI/MSF1p domain, found in proteins from a wide variety of eukaryotic organisms. However, the function of the proteins of this family is currently unknown. In this study, the evolutionary relationships between members of the PRELI/MSF1p family are described, and we present the first analysis of two novel Drosophila genes predicted to encode proteins of this type. The first of these, preli-like (prel), is expressed ubiquitously during embryonic development, whilst the second, real-time (retm), is expressed dynamically in the developing gut and central nervous system. retm encodes a member of a novel conserved subclass of larger PRELI/MSF1p domain proteins, which also contain the CRAL-TRIO motif thought to mediate the transport of small hydrophobic ligands. Here we provide evidence that, like Slmo, both the Prel and Retm proteins are localised to the mitochondria, indicating that the function of the PRELI/MSF1p domain is specific to this organelle.Edited by P. Simpson     

Complete chloroplast genome sequence of <Emphasis Type="Italic">Capsicum</Emphasis><Emphasis Type="Italic">baccatum</Emphasis> var. <Emphasis Type="Italic">baccatum</Emphasis>

Molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution. Complete chloroplast (cp) genome sequences of Capsicum species have been reported. We herein report the complete chloroplast genome sequence of Capsicum baccatum var. baccatum, a wild Capsicum species. The total length of the chloroplast genome is 157,145 bp with 37.7 % overall GC content. One pair of inverted repeats, 25,910 bp in length, was separated by a small single-copy region (17,974 bp) and large single-copy region (87,351 bp). This region contains 86 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 11 genes contain one or two introns. Pair-wise alignments of chloroplast genome were performed for genome-wide comparison. Analysis revealed a total of 134 simple sequence repeat (SSR) motifs and 282 insertions or deletions variants in the C. baccatum var. baccatum cp genome. The types and abundances of repeat units in Capsicum species were relatively conserved, and these loci could be used in future studies to investigate and conserve the genetic diversity of the Capsicum species.     

P-glycoprotein-like protein contributes to cadmium resistance in<Emphasis Type="Italic"> Euglena gracilis</Emphasis>

Selective pressures from polluted environments have led to the development of resistance systems in aquatic organisms. Using different techniques, this study examined a cadmium defense mechanism of the freshwater unicellular protozoa Euglena gracilis, and found it to be an efflux pump similar to the multidrug resistance P-glycoprotein. Cd²⁺-treated E. gracilis were able to extrude Rhodamine 123 at 21 °C, but not at 4 °C. Furthermore, verapamil, a P-glycoprotein modulator, partially blocked the efflux process (at 21 °C), and enhanced the Cd²⁺ toxic effects on these cells. Western immunoblots of cell lysates, using the anti-P-glycoprotein antibody JSB-1, revealed a 120-KDa protein, which was expressed, in high amounts on Cd²⁺-exposed cells (74% above the control values). Moreover, cells treated with JSB-1 became more sensitive to the harmful effects of cadmium, showing a decreased survival rate. Taken together, these results suggest that a MDR phenotype has evolved in Euglena as one of the mechanisms for cadmium detoxification.Abbreviations DTT dithiothreitol - mAb JSB-1 anti-human P-gp monoclonal antibody JSB-1 - MDR multidrug resistance - MRP MDR-associated protein - PBS phosphate buffered saline - P-gp P-glycoproteinCommunicated by G. HeldmaierA.J.F.O. and F.L.S.S. are undergraduate students under a CNPq special program for research training     

The involvement of a protein kinase in phototaxis and gravitaxis of <Emphasis Type="Italic">Euglena gracilis</Emphasis>

The unicellular flagellate Euglena gracilis shows positive phototaxis at low-light intensities (<10 W/m²) and a negative one at higher irradiances (>10 W/m²). Phototaxis is based on blue light-activated adenylyl cyclases, which produce cAMP upon irradiation. In the absence of light the cells swim upward in the water column (negative gravitaxis). The results of sounding rocket campaigns and of a large number of ground experiments led to the following model of signal perception and transduction in gravitaxis of E. gracilis: The body of the cell is heavier than the surrounding medium, sediments and thereby exerts a force onto the lower membrane. Upon deviation from a vertical swimming path mechano-sensitive ion channels are activated. Calcium is gated inwards which leads to an increase in the intracellular calcium concentration and causes a change of the membrane potential. After influx, calcium activates one of several calmodulins found in Euglena, which in turn activates an adenylyl cyclase (different from the one involved in phototaxis) to produce cAMP from ATP. One further element in the sensory transduction chain of both phototaxis and gravitaxis is a specific protein kinase A. We found five different protein kinases A in E. gracilis. The blockage of only one of these (PK.4, accession No. EU935859) by means of RNAi inhibited both phototaxis and gravitaxis, while inhibition of the other four affected neither phototaxis nor gravitaxis. It is assumed that cAMP directly activates this protein kinase A which may in turn phosphorylate a protein involved in the flagellar beating mechanism.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">IGHV1</Emphasis>,<Emphasis Type="Italic"> IGHV5</Emphasis> and<Emphasis Type="Italic"> IGHV7</Emphasis> subgroup genes in the Rhesus macaque

The diversity of the antibody response is achieved, in part, by rearrangement of different immunoglobulin (Ig) genes. The Ig heavy chain is made up of a variable region (IGHV), a diversity region (IGHD) and a joining region (IGHJ). Human germline IGHV genes have been grouped into seven multigene subgroups. Size and usage of these subgroups is not equal, the IGHV3 subgroup is the most commonly used (36%), followed by IGHV1/7 (26%), then IGHV4, IGHV5, IGHV2, IGHV6 (15%, 12%, 4%, 3% respectively). The rhesus macaque (Macaca mulatta) is a useful non-human primate model for studies of infection and the database of germline Ig genes for the macaque is gradually growing to become a useful tool in the study of B-cell responses. The proportions of IGHV subgroup usage in the macaque are similar to those in man. Representatives from IGHV3 and IGHV4 subgroups for the macaque have been published, as have germline sequences of the IGHD and IGHJ genes. However, to date there have been no sequences published from the second largest IGHV subgroup, IGHV1. We report the isolation and sequencing of a genomic fragment containing an IGHV1 gene from the macaque. Polymerase chain reaction (PCR) primers designed from this sequence enabled us to amplify and sequence 25 new IGHV1 germline genes. We also isolated two IGHV7 genes, using the same primers, and two IGHV5 genes, using human IGHV5 primers.     

Mercury pretreatment selects an enhanced cadmium-accumulating phenotype in<Emphasis Type="Italic"> Euglena gracilis</Emphasis>

Pre-treatment of heterotrophic cultures of Euglena gracilis with 1.5 microM HgCl(2) for at least 60 generations resulted in a cell population that showed both increased resistance to Cd(2+) and ability to accumulate it, when compared to non-Hg(2+)-pretreated Euglena. These Hg(2+)-enhanced capacities were evident in cells cultured in the dark in a medium with lactate, but not in cells cultured with glutamate plus malate. After culturing with 0.1 mM CdCl(2) through three consecutive transfers, the mercury-pretreated cells still grew and maintained high levels of glutathione-related metabolites, while the non-Hg(2+)-pretreated cells died. Cultures of Hg(2+)-pretreated cells, after transfer to media with or without cadmium, did not alter either their enhanced Cd(2+) accumulation or their increased production of glutathione-related metabolites. These observations suggested that the Hg(2+)-pretreated population underwent a permanent change that improved its Cd(2+) resistance. Several factors that contributed to the improved capacities included: (a) higher cellular malate, cysteine and glutathione levels induced by Hg(2+) before and after Cd(2+) exposure; and (b) increased storage of Cd(2+) in mitochondria along with increased intramitochondrial citrate, cysteine, and glutathione levels. These characteristics suggested that this Cd(2+) hyper-accumulating strain of E. gracilis might be a suitable candidate for Cd(2+)-bioremediation of polluted water systems.     

Genomewide analysis of <Emphasis Type="Italic">ABCB</Emphasis>s with a focus on <Emphasis Type="Italic">ABCB1</Emphasis> and <Emphasis Type="Italic">ABCB19</Emphasis> in <Emphasis Type="Italic">Malus domestica</Emphasis>

The B subfamily of ATP-binding cassette (ABC) proteins (ABCB) plays a vital role in auxin efflux. However, no systematic study has been done in apple. In this study, we performed genomewide identification and expression analyses of the ABCB family in Malus domestica for the first time. We identified a total of 25 apple ABCBs that were divided into three clusters based on the phylogenetic analysis. Most ABCBs within the same cluster demonstrated a similar exon–intron organization. Additionally, the digital expression profiles of ABCB genes shed light on their functional divergence. ABCB1 and ABCB19 are two well-studied auxin efflux carrier genes, and we found that their expression levels are higher in young shoots of M106 than in young shoots of M9. Since young shoots are the main source of auxin synthesis and auxin efflux involves in tree height control. This suggests that ABCB1 and ABCB19 may also take a part in the auxin efflux and tree height control in apple.