Anaerobically grown <Emphasis Type="Italic">Thauera aromatica</Emphasis>, <Emphasis Type="Italic">Desulfococcus multivorans</Emphasis>, <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis> are more sensitive towards organic solvents than aerobic bacteria

The effect of seven important pollutants and three representative organic solvents on growth of Thauera aromatica K172, as reference strain for nitrate-reducing anaerobic bacteria, was investigated. Toxicity in form of the effective concentrations (EC50) that led to 50% growth inhibition of potential organic pollutants such as BTEX (benzene, toluene, ethylbenzene, and xylene), chlorinated phenols and aliphatic alcohols on cells was tested under various anaerobic conditions. Similar results were obtained for Geobacter sulfurreducens and Desulfococcus multivorans as representative for Fe³⁺-reducing and sulphate-reducing bacteria, respectively, leading to a conclusion that anaerobic bacteria are far more sensitive to organic pollutants than aerobic ones. Like for previous studies for aerobic bacteria, yeast and animal cell cultures, a correlation between toxicity and hydrophobicity (log P values) of organic compounds for different anaerobic bacteria was ascertained. However, compared to aerobic bacteria, all three tested anaerobic bacteria were shown to be about three times more sensitive to the tested substances.     

Mutation in<Emphasis Type="Italic"> slowmo</Emphasis> causes defects in<Emphasis Type="Italic"> Drosophila</Emphasis> larval locomotor behaviour

We have identified a mutant slowmotion phenotype in first instar larval peristaltic behaviour of Drosophila. By the end of embryogenesis and during early first instar phases, slowmo mutant animals show a marked decrease in locomotory behaviour, resulting from both a reduction in number and rate of peristaltic contractions. Inhibition of neurotransmitter release, using targeted expression of tetanus toxin light chain (TeTxLC), in the slowmo neurons marked by an enhancer-trap results in a similar phenotype of largely absent or uncoordinated contractions. Cloning of the slowmo gene identifies a product related to a family of proteins of unknown function. We show that Slowmo is associated with mitochondria, indicative of it being a mitochondrial protein, and that during embryogenesis and early larval development is restricted to the nervous system in a subset of cells. The enhancer-trap marks a cellular component of the CNS that is seemingly required to regulate peristaltic movement.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Structural and transcriptional analysis of<Emphasis Type="Italic"> S</Emphasis>-locus F-box genes in<Emphasis Type="Italic"> Antirrhinum</Emphasis>

A class of ribonucleases termed S-RNases, which control the pistil expression of self-incompatibility, represents the only known functional products encoded by the S locus in species from the Solanaceae, Scrophulariaceae and Rosaceae. Previously, we identified a pollen-specific F-box gene, AhSLF (S locus F-box)-S₂, very similar to S₂-RNase in Antirrhinum, a member of the Scrophulariaceae. In addition, AhSLF-S₂ also detected the presence of its homologous DNA fragments. To identify these fragments, we constructed two genomic DNA libraries from Antirrhinum self-incompatible lines carrying alleles S₁S₅ and S₂S₄, respectively, using a transformation-competent artificial chromosome (TAC) vector. With AhSLF-S₂-specific primers, TAC clones containing both AhSLF-S₂ and its homologs were subsequently identified (S₂TAC, S₅TACa, S₄TAC, and S₁TACa). DNA blot hybridization, sequencing and segregation analyses revealed that they are organized as single allelic copies (AhSLF-S₂, -S₁, -S₄ and -S₅) tightly linked to the S-RNases. Furthermore, clusters of F-box genes similar to AhSLF-S₂ were identified. In total, three F-box genes (AhSLF-S₂, -S₂A and -S₂C) in S₂TAC (51 kb), three (AhSLF-S₄, -S₄A and -S₄D) in S₄TAC (75 kb), two (AhSLF-S₅ and -S₅A) in S₅TACa (55 kb), and two (AhSLF-S₁ and -S₁E) in S₁TACa (71 kb), respectively, were identified. Paralogous copies of these genes show 38–54% identity, with allelic copies sharing 90% amino acid identity. Among these genes, three (AhSLF-S₂C, -S₄D and -S₁E) were specifically expressed in pollen, similar to AhSLF-S₂, implying that they likely play important roles in pollen, whereas three AhSLF-SA alleles showed no detectable expression. In addition, several types of retroelements and transposons were identified in the sequenced regions, revealing some detailed information on the structural diversity of the S locus region. Taken together, these results indicate that both single allelic and tandemly duplicated genes are associated with the S locus in Antirrhinum. The implications of these findings in evolution and possible roles of allelic AhSLF-S genes in the self-incompatible reaction are discussed in species like Antirrhinum.Sequence data from this article have been deposited with the EMBL/GenBank databases under accession numbers AJ300474, AJ515534, AJ515536 and AJ515535     

Gene expression in<Emphasis Type="Italic"> Escherichia coli</Emphasis> biofilms

DNA microarrays were used to study the gene expression profile of Escherichia coli JM109 and K12 biofilms. Both glass wool in shake flasks and mild steel 1010 plates in continuous reactors were used to create the biofilms. For the biofilms grown on glass wool, 22 genes were induced significantly (p0.05) compared to suspension cells, including several genes for the stress response (hslS, hslT, hha, and soxS), type I fimbriae (fimG), metabolism (metK), and 11 genes of unknown function (ybaJ, ychM, yefM, ygfA, b1060, b1112, b2377, b3022, b1373, b1601, and b0836). The DNA microarray results were corroborated with RNA dot blotting. For the biofilm grown on mild steel plates, the DNA microarray data showed that, at a specific growth rate of 0.05/h, the mature biofilm after 5 days in the continuous reactors did not exhibit differential gene expression compared to suspension cells although genes were induced at 0.03/h. The present study suggests that biofilm gene expression is strongly associated with environmental conditions and that stress genes are involved in E. coli JM109 biofilm formation.     

Heterologous expression of the<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferase genes in<Emphasis Type="Italic"> Clostridium acetobutylicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis> for the production of isoamyl acetate

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production.     

Callus induction and regeneration in<Emphasis Type="Italic"> Spirodela</Emphasis> and<Emphasis Type="Italic"> Lemna</Emphasis>

The development of tissue culture systems in duckweeds has, to date, been limited to species of the genus Lemna. We report here the establishment of an efficient tissue culture cycle (callus induction, callus growth and plant regeneration) for Spirodela oligorrhiza Hegelm SP, Spirodela punctata 8717 and Lemna gibba var. Hurfeish. Significant differences were found among the three duckweed species pertaining to carbohydrate and phytohormone requirements for callus induction, callus growth and frond regeneration. In vitro incubation with poorly assimilated carbohydrates such as galactose (S. oligorrhiza SP and L. gibba var. Hurfeish) and sorbitol (S. punctata 8717) as sole carbon source yielded high levels of callus induction on phytohormone-supplemented medium. Sorbitol is required for optimal callus growth of S. oligorrhiza SP and S. punctata 8717, while sucrose is required for callus growth of L. gibba var. Hurfeish. Sucrose either alone (S. oligorrhiza SP, L. gibba var. Hurfeish) or in addition to sorbitol (S. punctata 8717) is required for frond regeneration.Abbreviations ABA: (±)-Abscisic acid - BA: N⁶-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - Dicamba: 3,6-Dichloro-2-methoxybenzoic acid - 2iP: N⁶-(2-Isopentenyl)adenine - NAA: -Naphthaleneacetic acid - PCA: p-Chlorophenoxy acetic acid - Picloram: 4-Amino-3,5,6-trichloropicolinic acid - TDZ: Thidiazuron Communicated by A. AltmanJ. Li and M. Jain contributed equally to the research reported in this article.     

Identification and genomic distribution of<Emphasis Type="Italic"> gypsy</Emphasis> like retrotransposons in<Emphasis Type="Italic"> Citrus</Emphasis> and<Emphasis Type="Italic"> Poncirus</Emphasis>

Transposable elements might be importantly involved in citrus genetic instability and genome evolution. The presence of gypsy like retrotransposons, their heterogeneity and genomic distribution in Citrus and Poncirus, have been investigated. Eight clones containing part of the POL coding region of gypsy like retrotransposons have been isolated from a commercial variety of Citrus clementina, one of the few sexual species in Citrus. Four of the eight clones might correspond to active elements given that they present all the conserved motifs described in the literature as essential for activity, no in-frame stop codon and no frame-shift mutation. High homology has been found between some of these citrus elements and retroelements within a resistance-gene cluster from potato, another from Poncirus trifoliata and two putative resistance polyproteins from rice. Nested copies of gypsy like elements are scattered along the Citrus and Poncirus genomes. The results on genomic distribution show that these elements were introduced before the divergence of both genera and evolved separately thereafter. IRAPs based on gypsy and copia types of retrotransposons seem to distribute differently, therefore gypsy based IRAPs prove a new, complementary set of molecular markers in Citrus to study and map genetic variability, especially for disease resistance. Similarly to copia-derived IRAPs, the number of copies and heterozygosity values found for gypsy derived IRAPs are lower in Poncirus than in Citrus aurantium, which is less apomictic and the most usual rootstock for clementines until 1970.Communicated by C. Möllers     

New sections in<Emphasis Type="Italic">Taraxacum</Emphasis>

Sectional taxonomy ofTaraxacum in steppe or subsaline habitats in Central Asia is revised based on material collected during expeditions, cultivated or studied in herbarium. Two new sections are described from that area:T. sect.Stenoloba similar toT. sect.Leucantha (syn.:T. sect.Sinensia), andT. sect.Suavia allied toT. sect.Dissecta. The type species of the sectionSuavia is described asTaraxacum formosissimum Kirschner etŠtěpánek. Widespread mountain dandelions of the Caucasus, intermediate between the sect.Piesis andT. stevenii, are described asT. sect.Confusa. Taraxacum species dominating dry habitats in S Ukraine and Crimea are described asT. sect.Borysthenica. Species belonging to the new sections were found to be polyploid and agamospermous.     

Subcellular localization of the homocitrate synthase in<Emphasis Type="Italic"> Penicillium chrysogenum</Emphasis>

There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.     

<Emphasis Type="Italic">ZWILLE</Emphasis> buffers meristem stability in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The shoot apical meristem of higher plants consists of a population of stem cells at the tip of the plant body that continuously gives rise to organs such as leaves and flowers. Cells that leave the meristem differentiate and must be replaced to maintain the integrity of the meristem. The balance between differentiation and maintenance is governed both by the environment and the developmental status of the plant. In order to respond to these different stimuli, the meristem has to be plastic thus ensuring the stereotypic shape of the plant body. Meristem plasticity requires the ZWILLE (ZLL) gene. In zll mutant embryos, the apical cells are misspecified causing a variability of the meristems size and function. Using specific antibodies against ZLL, we show that the zll phenotype is due to the complete absence of the ZLL protein. In immunohistochemical experiments we confirm the observation that ZLL is solely localized in vascular tissue. For a better understanding of the role of ZLL in meristem stability, we analysed the genetic interactions of ZLL with WUSCHEL (WUS) and the CLAVATA1, 2 and 3 (CLV) genes that are involved in size regulation of the meristem. In a zll loss-of-function background wus has a negative effect whereas clv mutations have a positive effect on meristem size. We propose that ZLL buffers meristem stability non-cell-autonomously by ensuring the critical number of apical cells required for proper meristem function.Edited by G. JürgensAn erratum to this article can be found at     

Identification and characterization of the<Emphasis Type="Italic"> carAB</Emphasis> genes responsible for encoding carbamoylphosphate synthetase in<Emphasis Type="Italic"> Halomonas eurihalina</Emphasis>

Halomonas eurihalina is a moderately halophilic bacterium which produces exopolysaccharides potentially of great use in many fields of industry and ecology. Strain F2-7 of H. eurihalina synthesizes an anionic exopolysaccharide known as polymer V2-7, which not only has emulsifying activity but also becomes viscous under acidic conditions, and therefore we consider it worthwhile making a detailed study of the genetics of this strain. By insertional mutagenesis using the mini-Tn 5 Km2 transposon we isolated and characterized a mutant strain, S36 K, which requires both arginine and uracil for growth and does not excrete EPS. S36 K carries a mutation within the carB gene that encodes the synthesis of the large subunit of the carbamoylphosphate synthetase enzyme, which in turn catalyzes the synthesis of carbamoylphosphate, an important precursor of arginine and pyrimidines. We describe here the cloning and characterization of the carAB genes, which encode carbamoylphosphate synthetase in Halomonas eurihalina, and discuss this enzyme's possible role in the pathways for the synthesis of exopolysaccharides in strain F2-7.     

The<Emphasis Type="Italic"> ThioredoxinT</Emphasis> and<Emphasis Type="Italic"> deadhead</Emphasis> gene pair encode testis- and ovary-specific thioredoxins in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis>

So far, two thioredoxin proteins, DHD and Trx-2, have been biochemically characterized in Drosophila melanogaster. Here, with the cloning and characterization of TrxT we describe an additional thioredoxin with testis-specific expression. TrxT and dhd are arranged as a gene pair, transcribed in opposite directions and sharing a 471 bp regulatory region. We show that this regulatory region is sufficient for correct expression of the two genes. This gene pair makes a good model for unraveling how closely spaced promoters are differentially regulated by a short common control region. Both TrxT and DHD proteins are localized within the nuclei in testes and ovaries, respectively. Use of a transgenic construct expressing TrxT fused to Enhanced Yellow Fluorescent Protein reveals a clear association of TrxT with the Y chromosome lampbrush loops ks-1 and kl-5 in primary spermatocytes. The association is lost in the absence of the Y chromosome. Our results suggest that nuclear thioredoxins may have regulatory functions in the germline.Sequence data from this paper have been deposited with the EMBL/GenBank Data Libraries under Accession number AJ507731