Expression of<Emphasis Type="Italic"> FoxE</Emphasis> and<Emphasis Type="Italic"> FoxQ</Emphasis> genes in the endostyle of<Emphasis Type="Italic"> Ciona intestinalis</Emphasis>

We have analyzed the expression patterns of two Fox genes, FoxE and FoxQ, in the ascidian Ciona intestinalis. Expression of Ci-FoxE was specific to the endostyle of adults, being prominent in the thyroid-equivalent region of zone 7. Ci-FoxQ was expressed in several endodermal organs of adult ascidians, such as the endostyle, branchial sac and esophagus. In the endostyle, the pattern of Ci-FoxQ expression was similar to that of CiTTF-1, being prominent in the thyroid-equivalent regions of zones 7 and 8. Therefore, these Fox genes may perform thyroid-equivalent functions in the ascidian endostyle.Edited by N. Satoh     

Existence of<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> genomic fragment hybridizing with the<Emphasis Type="Italic">nifM</Emphasis> gene of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A novel finding that genomic restriction fragments of symbiotic nitrogen fixer S. meliloti hybridized with nifM gene probe of the free-living diazotroph Klebsiella pneumoniae is reported. When SmaI endonuclease was used to digest S. meliloti DNA, a unique hybridizing band was obtained.     

An early-flowering genotype of<Emphasis Type="Italic">Populus</Emphasis>

Unlike herbaceous, annual crops, trees are not highly domesticated and, therefore, have wild relatives with which they are interfertile. They are also long-lived perennials that produce copious amounts of pollen and seed, which are often disseminated over considerable distances by the wind. Federal regulators have made it clear that before transgenic trees can be grown commercially in the U.S., it will be necessary to develop a strategy to mitigate the risk of transgene spread into the environment. One way to satisfy this requirement is to genetically engineer reproductive sterility. Because of its many useful attributes, poplar has becomethe model tree species for research community. However, because of its relatively long juvenile period, the development of a reliable sterility system for poplar is taking longer than expected. By having an early-flowering genotype of poplar, it will be possible to make much faster progress in our efforts to develop a reliable transgene-confinement system. We have identified a genotype ofPopulus alba that can be induced to flower within nine months of being regenerated.     

Evaluation of<Emphasis Type="Italic">in Vitro</Emphasis> antifungal activity of<Emphasis Type="Italic">N</Emphasis>-Benzylsalicylamide derivatives

A series of 65 derivatives of N-benzylsalicylamide was tested against eight potentially human pathogenic fungi by microdilution broth method modified according to M27-A standard. The majority of these compounds showed only weak in vitro antifungal activity. The most significant effect was observed against filamentous fungi Trichophyton mentagrophytes, Absidia corymbifera, and Aspergillus fumigatus while yeasts, in general, were less susceptible. N-(4'-Chlorobenzyl) salicylamides, N-(3',4'-dichlorobenzyl)-salicylamides, and partially N-benzylsalicylamides exhibited relatively high in vitro antifungal activity. The most efficient derivatives had MIC < or = 7.8 mumol/L against T. mentagrophytes. Regression analysis suggested an indirect relationship between MIC values and lipophilicity (log P).     

Complementary expression of<Emphasis Type="Italic"> AP-2</Emphasis> and<Emphasis Type="Italic"> AP-2rep</Emphasis> in ectodermal derivatives of<Emphasis Type="Italic"> Xenopus</Emphasis> embryos

In an attempt to define the pattern of developmental expression of AP-2rep and AP-2 in Xenopus embryos, we cloned a Xenopus AP-2rep cDNA. The AP-2rep message was localized in the organizer region at the gastrula stage whereas AP-2 was expressed ventro-laterally in the animal hemisphere. Later, AP-2rep was expressed in the entire neural tissue at the neurula stage while AP-2 was predominantly expressed in the cranial neural crest areas. The endogenous expression of AP-2 in the neural crest area was diminished by ectopic injection of AP-2rep RNA, suggesting a role for AP-2rep in the differentiation of neural tissues by restricting the expression of AP-2 in the Xenopus embryo.     

The importance of<Emphasis Type="Italic"> porE</Emphasis> and<Emphasis Type="Italic"> porF</Emphasis> in the anabolic pyruvate oxidoreductase of<Emphasis Type="Italic"> Methanococcus maripaludi</Emphasis>s

The operon of the anabolic pyruvate oxidoreductase (POR) of Methanococcus maripaludis encodes two genes (porEF) whose functions are unknown. Because these genes possess sequence similarity to polyferredoxins, they may be electron carriers to the POR. To elucidate whether the methanococcal POR requires PorEF for activity, a deletion mutant, strain JJ150, lacking porEF was constructed. Compared to the wild-type strain JJ1, the mutant grew more slowly in minimal medium and minimal plus acetate medium, and pyruvate-dependent methanogenesis was inhibited. In contrast, the methyl-viologen-dependent pyruvate-oxidation activity of POR, carbon monoxide dehydrogenase, and hydrogenase activities of the mutant were similar to those of the wild-type. Upon genetic complementation of the mutant with porEF in the methanococcal shuttle vector pMEV2+porEF, growth in minimal medium and pyruvate-dependent methanogenesis were restored to wild-type levels. Complementation with porE alone restored methanogenesis from pyruvate but not growth in minimal medium. Complementation with porF alone partially restored growth but not methanogenesis from pyruvate. Although the specific roles of porE and porF have not been determined, these results suggest that PorEF play important roles in the anabolic POR in vivo even though they are not required for the dye-dependent activity.Abbreviations CODH/ACS Carbon monoxide dehydrogenase/acetyl-CoA synthase - POR Pyruvate oxidoreductase     

Stable genetic transformation of<Emphasis Type="Italic"> Vigna mungo</Emphasis> L. Hepper via<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>

Vigna mungo is one of the large-seeded grain legumes that has not yet been transformed. We report here for the first time the production of morphologically normal and fertile transgenic plants from cotyledonary-node explants inoculated with Agrobacterium tumefaciens carrying binary vector pCAMBIA2301, the latter of which contains a neomycin phosphotransferase ( nptII) gene and a beta-glucuronidase (GUS) gene ( uidA) interrupted with an intron. The transformed green shoots, selected and rooted on medium containing kanamycin, tested positive for nptII and uidA genes by polymerase chain reaction (PCR) analysis. These shoots were established in soil and grown to maturity to collect the seeds. Mechanical wounding of the explants prior to inoculation with Agrobacterium, time lag in regeneration due to removal of the cotyledons from explants and a second round of selection at the rooting stage were found to be critical for transformation. Analysis of T(0) plants showed the expression and integration of uidA into the plant genome. GUS activity in leaves, roots, flowers, anthers and pollen grains was detected by histochemical assay. PCR analysis of T(1) progeny revealed a Mendelian transgene inheritance pattern. The transformation frequency was 1%, and 6-8 weeks were required for the generation of transgenics.     

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     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-<Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Helminthosporium turcicum</Emphasis>, the maize leaf-blight fungus

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.     

Genetic analysis of<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> susceptibility in<Emphasis Type="Italic"> Brassica oleracea</Emphasis>

The genetic control and heritability of Agrobacterium tumefaciens susceptibility was investigated using a doubled haploid (DH) mapping population of Brassica oleracea and the associated RFLP map. Preliminary studies were carried out by analysis of an 8×8 diallel, for which the parental lines were selected to include a range of susceptibilities to A. tumefaciens. The variation observed within the diallel was attributed to both additive and dominant gene effects, with additive gene effects being more important. A broad sense heritability value of 0.95 suggested that 95% of the observed variation was due to genetic effects, with just 5% attributed to non-genetic or environmental effects. A high narrow-sense heritibility value of 0.79 suggested that 79% of this trait was controlled by additive gene effects and, therefore, the potential to introduce this trait into breeding material is high. Fifty-nine DH lines from the mapping population were screened for susceptibility towards A. tumefaciens. Variation in susceptibility was observed across the population. The results of the DH screen were entered into the mapping programme MAPQTL and a highly significant quantitative trait loci (QTL) associated with susceptibility to A. tumefaciens was identified on linkage group 09. The use of substitution lines covering this region confirmed the location of this QTL. This work shows that susceptibility to A. tumefaciens is a heritable trait, and the transfer of susceptibility into resistant lines is demonstrated. These findings may help to overcome genotype restrictions to genetic transformation.Communicated by G. Wenzel     

Horticultural characterization of<Emphasis Type="Italic"> Angelonia salicariifolia</Emphasis> plants transformed with wild-type strains of<Emphasis Type="Italic"> Agrobacterium rhizogenes</Emphasis>

Genetic transformation was carried out with wild-type strains of Agrobacterium rhizogenes for introducing a dwarf trait into the Scrophulariaceous ornamental plant, angelonia (Angelonia salicariifolia). Leaf segments of two angelonia genotypes (Ang.1 and Ang.2) were co-cultivated with mikimopine-type strains of A. rhizogenes. Adventitious roots that showed vigorous growth and increased lateral branching when cultured on half-strength Murashige and Skoog's (MS) basal salts medium lacking plant growth regulators (PGRs) after co-cultivation were selected as putatively transformed lines. All of these selected lines produced mikimopine. Adventitious shoots were efficiently induced from putatively transformed root segments on half-strength MS basal salts medium containing 1 mg l(-1) benzyladenine (BA) under continuous illumination (24-h photoperiod), and the shoots easily rooted following their transfer to half-strength MS basal salts medium lacking PGRs. The transgenic nature of regenerated plants was confirmed by Southern hybridization. Transformed plants frequently died during their acclimatization, and acclimatized plants of eight transformed lines grew very slowly for 1-5 months after transplantation to the greenhouse. Plants of two transformed lines of Ang.2 flowered 4-6 months after transplantation. These transformed plants exhibited phenotypic alterations such as dwarfness and smaller leaves. There were no apparent alterations observed in the number, shape, and size of the flowers. Pollen fertility of the transformed plants was 60-80% based on aceto-carmine staining. These results indicate the possibility of applying A. rhizogenes-mediated transformation for introducing a dwarf trait into angelonia.     

An improved protocol for<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Antirrhinum majus</Emphasis> L.

Efficient Agrobacterium -mediated transformation of Antirrhinum majus L. was achieved via indirect shoot organogenesis from hypocotyl explants of seedlings. Stable transformants were obtained by inoculating explants with A. tumefaciens strain GV2260 harboring the binary vector pBIGFP121, which contains the neomycin phosphotransferase gene (NPT II) as a selectable marker and the gene for the Green Fluorescent Protein (GFP) as a visual marker. Putative transformants were identified by selection for kanamycin resistance and by examining the shoots using fluorescence microscopy. PCR and Southern analyses confirmed integration of the GFP gene into the genomes of the transformants. The transformants had a morphologically normal phenotype. The transgene was shown to be inherited in a Mendelian manner. This improved method requires only a small number of seeds for explant preparation, and three changes of medium; the overall transformation efficiency achieved, based on the recovery of transformed plants after 4–5 months of culture, reached 8–9%. This success rate makes the protocol very useful for producing transgenic A. majus plants.Communicated by G. Jürgens     

Mutation of<Emphasis Type="Italic">gyrA</Emphasis> and<Emphasis Type="Italic">parC</Emphasis> in clinical isolates of<Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> and its relationship with antimicrobial drugs resistance in Taiwan

Acinetobacter baumannii is a species of non fermentative Gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Herein we have determined the mutation frequency of two hot spot residue 83 in gyrA of gyrase and residue 80 in parC of topoisomerase IV and performed a comparative screen the drug resistance ability in 128 clinical isolates ofAcinetobacter baumannii in Taiwan region. Low frequency of mutation was found (11.7%, 11.7%, and 10.2% ingyrA, parC, or both, respectively). Mutation of these sites was not correlated with drug resistance. Our study suggested that mutation ofgyrA andparC may play a minor role in quinolone resistance and other mechanisms may contribute to the drug resistance ofA. Baumannii.     

Sexual identification of<Emphasis Type="Italic">Galemys pyrenaicus</Emphasis>

The absence of sexual dimorphism in Pyrenean desmanGalemys pyrenaicus (E. Geoffroy, 1811) makes it difficult to sex live animals. This paper presents a method of sexing desmans observing their external genitals. Based in a sampling of 49 animals from the north of Spain, it was possible to identify the penis of males, independently of the age or season.     

Biallelic expression of<Emphasis Type="Italic"> HRAS</Emphasis> and<Emphasis Type="Italic"> MUCDHL</Emphasis> in human and mouse

At least eight genes clustered in 1 Mb of DNA on human chromosome (Chr) 11p15.5 are subject to parental imprinting, with monoallelic expression in one or more tissues. Orthologues of these genes show conserved linkage and imprinting on distal Chr 7 of mice. The extended imprinted region has a bipartite structure, with at least two differentially methylated DNA elements (DMRs) controlling the imprinting of two sub-domains. We previously described three biallelically expressed genes ( MRPL23, 2G7 and TNNT3) in 100 kb of DNA immediately downstream of the imprinted H19 gene, suggesting that H19 marks one border of the imprinted region. Here we extend this analysis to two additional downstream genes, HRAS and MUCDHL (mu-protocadherin). We find that these genes are biallelically expressed in multiple fetal and adult tissues, both in humans and in mice. The mouse orthologue of a third gene, DUSP8, located between H19 and MUCDHL, is also expressed biallelically. The DMR immediately upstream of H19 frequently shows a net gain of methylation in Wilms tumors, either via Chr 11p15.5 loss of heterozygosity (LOH) or loss of imprinting (LOI), but changes in methylation in CpG-rich sequences upstream and within the MUCDHL gene are rare in these tumors and do not correlate with LOH or LOI. These findings are further evidence for a border of the imprinted region immediately downstream of H19, and the data allow the construction of an imprinting map that includes more than 20 genes, distributed over 3 Mb of DNA on Chr 11p15.5.     

Cerebrosides of<Emphasis Type="Italic"> Candida lipolytica</Emphasis> yeast

Candida lipolytica yeast was grown batchwise on glucose medium. Cerebrosides were isolated from the sphingolipid fraction of total lipids using column chromatography and separated into two compounds by high-performance thin-layer chromatography. Glucose was detected as the sole sugar constituent in cerebrosides. The fatty acid composition of cerebrosides was characterised by a predominance of saturated fatty acids and by a high proportion of fatty acids with 16 carbon atoms. The dominant fatty acid was h16:0. The principal long-chain base components of both cerebroside species were trihydroxy bases, 18- and 20-phytosphinosine. The unique characteristic of cerebrosides was the presence of a high proportion of sphingosine (one-fourth of the total long-chain bases), which is a common characteristic of mammalian sphingolipids and rarely occurs in yeast cerebrosides. The ceramide moiety profile of cerebrosides is similar to that of epidermal ceramides, which implies a possibility for their application in care cosmetics.     

Physiology of<Emphasis Type="Italic">Anabaena khannae</Emphasis> and<Emphasis Type="Italic">Chlorococcum humicola</Emphasis> under fluoride stress

Sodium fluoride showed pH-dependent physiological responses in the two test microalgae Anabaena khannae and Chlorococcum humicola. A. khannae showed severe membrane damage with fluoride at low pH with leakage of pigments and electrolytes. Annihilation of photosynthesis along with inhibition in 14C uptake was observed at pH 6 with 50 mg/L fluoride. While respiration was less affected in the cyanobacterium, C. humicola showed 30 % inhibition in respiratory activity. Resistance of C. humicola to fluoride toxicity has been attributed to the hindrance provided by the thick cell envelope, intracellular compartmentation and increase in extracellular pH as a consequence of its metabolism.     

A comparative map viewer integrating genetic maps for <Emphasis Type="Italic">Brassica</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

Molecular genetic maps provide a means to link heritable traits with underlying genome sequence variation. Several genetic maps have been constructed for Brassica species, yet to date, there has been no simple means to compare this information or to associate mapped traits with the genome sequence of the related model plant, Arabidopsis.