Phylogeny of <Emphasis Type="Italic">Gunnera</Emphasis>

 The phylogeny of the genus Gunnera is investigated for the first time. Twelve species representing the six currently recognised subgenera are analysed. Two chloroplast DNA regions, the rbcL gene and the rps16 intron, together provide 46 informative characters out of 2335. A combined analysis of both genes gives four most parsimonious trees, firmly establishing the east South American G. herteri as sister group to the rest of the genus. The African G. perpensa is sister group to two well-supported clades, one including the South American subgenera Misandra and Panke, the other the Australian/New Zealand/Malayan species of subgenera Milligania and Pseudogunnera. Thus, South America is a composite area for Gunnera, showing up at two different levels in the cladogram. Our analysis supports a close biogeographic relationship between Australia and New Zealand. The evolution of some morphological characters is discussed. Lastly, the unusual structure of some of the rbcL sequences is reported. Received July 6, 2000 Accepted October 24, 2000     

Overexpression of <Emphasis Type="Italic">WUSCHEL</Emphasis> in <Emphasis Type="Italic">C. chinense</Emphasis> causes ectopic morphogenesis

Capsicum chinense is a recalcitrant species for in vitro morphogenesis, and up to date there is no efficient system for genetic transformation and regeneration of this species via somatic embryogenesis. Here, we carried out an in vitro transformation of C. chinense via Agrobacterium tumefaciens co-cultivation with a system that expresses the heterologous gene WUSCHEL from Arabidopsis thaliana. WUSCHEL has been shown to promote the transition from vegetative to embryogenic state when overexpressed. We tested if the expression of WUSCHEL in C. chinense would promote an embryogenic response in this species. After 15 days of induction, the segments of transformed stems begun to form globular structures, suggesting that heterologus WUSCHEL was active and involved in the process of morphogenesis.     

Interactions between endocarditis-derived <Emphasis Type="Italic">Streptococcus gallolyticus</Emphasis> subsp. <Emphasis Type="Italic">gallolyticus</Emphasis> isolates and human endothelial cells

Background  

Streptococcus gallolyticus subsp. gallolyticus is an important causative agent of infective endocarditis (IE) but the knowledge on virulence factors is limited and the pathogenesis of the infection is poorly understood. In the present study, we established an experimental in vitro IE cell culture model using EA.hy926 and HUVEC cells to investigate the adhesion and invasion characteristics of 23 Streptococcus gallolyticus subsp. gallolyticus strains from different origins (human IE-derived isolates, other human clinical isolates, animal isolates). Adhesion to eight components of the extracellular matrix (ECM) and the ability to form biofilms in vitro was examined in order to reveal features of S. gallolyticus subsp. gallolyticus endothelial infection. In addition, the strains were analyzed for the presence of the three virulence factors gtf, pilB, and fimB by PCR.     

Characterization and functional analysis of hsp21.8b: An orthologous small heat shock protein gene in Tribolium castaneum

Small heat shock proteins (sHSPs) act as molecular chaperones and are widely distributed in all kinds of organisms. Comparative analysis revealed that an orthologous shsp was present during insect evolution. Here, hsp21.8b, one insect orthologous shsp, had been identified in Tribolium castaneum. Quantitative real‐time PCR illustrated that Tchsp21.8b was expressed in all developmental stages, along with the lowest expression at early embryonic stage and relative high expression at other stages especially in late eggs and late pupae. In the adult period, Tchsp21.8b exhibited the highest expression level in central nervous system and followed in elytron, epidermis, ovary and fat body. Moreover, it was upregulated 3.39‐fold in response to enhanced heat stress (45°C) for 4 hr but not to cold stress (4°C) and was upregulated by 1.73‐ to 1.94‐fold under ultraviolet (UV) exposure during 4–6 hr. It was also downregulated by 20.8%–41.8% under starvation in 3 days and had a “down‐up‐down” trend under the pathogen stresses. Larval RNA interference of Tchsp21.8b caused 40.6% insects mortality and reduced the oviposition amount by 66.0% and only 21.0% of the ds‐Tchsp21.8b eggs could hatch into larvae. These results suggested that as an orthologous shsp, Tchsp21.8b not only plays important roles in the growth, development and fecundity of T. castaneum but with the competence to resist the environment stresses, although the response is relatively weak compared to other hsps. Results from this study also uncovered the functions of the orthologous shsp in the development and anti‐stresses ability of T. castanuem. It provided more scientific evidence for revealing the physiological mechanisms of shsps of the insects and enhanced the capabilities to control different pests.     

<Emphasis Type="Italic">Septobasidium parviflorae</Emphasis> sp. nov. on <Emphasis Type="Italic">Pinus parviflora</Emphasis> from Japan

Septobasidium parviflorae sp. nov. on Pinus parviflora is described and illustrated. This species is characterized by its whitish-gray, gray to dark gray-colored fungus body with an indeterminate margin, hyphal strands, and cylindrical basidia with long sterigmata. This is the first report of Septobasidium occurring on a member of the genus Pinus in Japan.     

Ndst1, a heparan sulfate modification enzyme, regulates neuroectodermal patterning by enhancing Wnt signaling in Xenopus

Neural tissue is derived from three precursor regions: neural plate, neural crest, and preplacodal ectoderm. These regions are determined by morphogen-mediated signaling. Morphogen distribution is generally regulated by binding to an extracellular matrix component, heparan sulfate (HS) proteoglycan. HS is modified by many enzymes, such as N-deacetyl sulfotransferase 1 (Ndst1), which is highly expressed in early development. However, functions of HS modifications in ectodermal patterning are largely unknown. In this study, we analyzed the role of Ndst1 using Xenopus embryos. We found that ndst1 was expressed in anterior neural plate and the trigeminal region at the neurula stage. ndst1 overexpression expanded the neural crest (NC) region, whereas translational inhibition reduced not only the trigeminal region, but also the adjacent NC region, especially the anterior part. At a later stage, ndst1 knocked-down embryos showed defects in cranial ganglion formation. We also found that Ndst1 activates Wnt signaling pathway at the neurula stage. Taken together, our results suggest that N-sulfonated HS accumulates Wnt ligand and activates Wnt signaling in ndst1-expressing cells, but that it inhibits signaling in non-ndst1-expressing cells, leading to proper neuroectodermal patterning.     

The isolation and analysis of a soybean <Emphasis Type="Italic">CO</Emphasis> Homologue <Emphasis Type="Italic">GmCOL10</Emphasis>

The CONSTANS (CO) gene is a key regulator of the response to photoperiod in the model plant Arabidopsis thaliana, and its homologues are present in many plant species. We describe here the isolation of the CO homologue for zinc finger protein gene GmCOL10 (Glycine max CONSTANS-Like 10) from the soybean cultivar Kennong18. Sequence comparisons showed that the closest A. thaliana gene to GmCOL10 is COL5. The expression of GmCOL10 was regulated in a circadian manner, especially under short-day conditions. The expression of GmCOL10 was concentrated in vegetative organs, and in particular in the unifoliolates and cotyledons. An analysis of subcellular localization found GmCOL10 in the nucleus. Our data suggested that GmCOL10 was not related to the photoperiodic pathway of floral transition as Arabidopsis CO does.     

A comparison of the autecology of two seed-taking ant genera, <Emphasis Type="Italic">Rhytidoponera</Emphasis> and <Emphasis Type="Italic">Melophorus</Emphasis>

Members of the genus Rhytidoponera and, to a lesser extent, certain Melophorus spp. are keystone mutualists for the dispersal of seeds in the southwest of Western Australia, with important ramifications for the ecology and speciation of plants in this biodiversity hotspot. For this reason, it is important to understand the autecology of the relevant ant species and the way in which they interact with plant seeds. This paper addresses key aspects of the ecology of three such ant species, Rhytidoponera violacea (Forel), R. inornata Crawley and Melophorus turneri perthensis Wheeler. Data are presented on their geographic distribution, seasonality of foraging, diurnal activity, response to fire, nest site preference, nest structure, colony size, feeding habits, foraging response to seed availability, and seedling emergence from nests. The role of all three species as seed dispersers is confirmed, and all three species have ecologies that are well-suited for dispersal and survival of native plant seeds. Preservation of this interaction is important for the conservation of plants, and it is fortuitous that all three species are able to survive disturbance and return to rehabilitated areas. However, the smaller R. inornata, and to a lesser extent, the larger R. violacea, are vulnerable to invasive ant [Pheidole megacephala (Fabricius)] incursions. M. turneri perthensis is able to coexist with the invasive ant unless this is at high densities, probably as a result of its ability to forage during high temperatures when the invasive species is inactive.     

A role of <Emphasis Type="Italic">ygfZ</Emphasis> in the <Emphasis Type="Italic">Escherichia coli</Emphasis> response to plumbagin challenge

Plumbagin is found in many herbal plants and inhibits the growth of various bacteria. Escherichia coli strains are relatively resistant to this drug. The mechanism of resistance is not clear. Previous findings showed that plumbagin treatment triggered up-regulation of many genes in E. coli including ahpC, mdaB, nfnB, nfo, sodA, yggX and ygfZ. By analyzing minimal inhibition concentration and inhibition zones of plumbagin in various gene-disruption mutants, ygfZ and sodA were found critical for the bacteria to resist plumbagin toxicity. We also found that the roles of YgfZ and SodA in detoxifying plumbagin are independent of each other. This is because of the fact that ectopically expressed SodA reduced the superoxide stress but not restore the resistance of bacteria when encountering plumbagin at the absence of ygfZ. On the other hand, an ectopically expressed YgfZ was unable to complement and failed to rescue the plumbagin resistance when sodA was perturbed. Furthermore, mutagenesis analysis showed that residue Cys228 within YgfZ fingerprint region was critical for the resistance of E. coli to plumbagin. By solvent extraction and HPLC analysis to follow the fate of the chemical, it was found that plumbagin vanished apparently from the culture of YgfZ-expressing E. coli. A less toxic form, methylated plumbagin, which may represent one of the YgfZ-dependent metabolites, was found in the culture supernatant of the wild type E. coli but not in the ΔygfZ mutant. Our results showed that the presence of ygfZ is not only critical for the E coli resistance to plumbagin but also facilitates the plumbagin degradation.     

Duplicate Gene Evolution Toward Multiple Fates at the <Emphasis Type="Italic">Drosophila melanogaster HIP</Emphasis>/<Emphasis Type="Italic">HIP-Replacement</Emphasis> Locus

Hsc/Hsp70-interacting protein (HIP) is a rapidly evolving Hsp70 cofactor. Analyses of multiple Drosophila species indicate that the HIP gene is duplicated only in D. melanogaster. The HIP region, in fact, contains seven distinctly evolving duplicated genes. The regional duplication occurred in two steps, fixed rapidly, and illustrates multiple modes of duplicate gene evolution. HIP and its duplicate HIP-R are adaptively evolving in a manner unique to the region: they exhibit elevated divergence from other drosophilids and low polymorphism within D. melanogaster. HIP and HIP-R are virtually identical, share polymorphisms, and are subject to gene conversion. In contrast, two other duplicate genes in the region, CG33221 and GP-CG32779, are pseudogenes, and the chimeric gene Crg1 is subject to balancing selection. HIP and HIP-R are evolving rapidly and adaptively; however, positive selection is not sufficient to explain the molecular evolution of the region as a whole. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reduction of drosopterin content caused by a 45-nt insertion in <Emphasis Type="Italic">Henna</Emphasis> pre-mRNA of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Phenylalanine hydroxylase is assumed to be a key enzyme in drosopterin metabolism, but direct in vivo evidence to support this hypothesis is still absent. In the present study, we found a new natural recessive purple eye mutant of Drosophila melanogaster, Hn ^bp , which was a 45-nt insertion mutant in the second exon of Henna. The insertion resulted in a predicted protein with 15 additional amino acids as compared to the wild-type protein. Further analysis of protein structure showed that the predicted mutant protein probably had two more β-sheets, which may cause instability of two α-helices near the catalytic centre of the enzyme in the Biopterin-Hydroxyl binding domain. Hn ^bp mutant showed eye color defect with decrease of mRNA level, as well as drosopterin content reduction. The drosopterin defect could be fully rescued by expression of wild type Henna in the Hn ^bp background by GMR-GAL4 UAS-Henna/UAS-Henna:Hn ^bp /Hn ^bp transgenic line. All taken together, it can be concluded that the mutation in Henna is responsible for drosopterin reduction in mutant Hn ^bp , which provides key in vivo evidence to support the hypothesis that Henna is involved in drosopterin synthesis.     

Effect of ectopic expression of the eutypine detoxifying gene <Emphasis Type="Italic">Vr</Emphasis>-<Emphasis Type="Italic">ERE</Emphasis> in transgenic apple plants

The development of alternative selection systems without antibiotic resistance genes is a key issue to produce safer and more acceptable transgenic plants. Eutypine is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevine, which is detoxified in mung bean (Vigna radiata) by the gene Vr-ERE. Many phytotoxic compounds containing an aldehyde group can act as substrates for the Vr-ERE enzyme. The aim of the present work was to evaluate the effects of the overexpression of Vr-ERE in transgenic apple plants, as a first step towards the development of an alternative selection system. Viable transgenic apple clones expressing Vr-ERE were produced from the cultivar Greensleeves under kanamycin selection. Although the Vr-ERE transgene was normally expressed at the RNA and protein levels, the increase in aldehyde reductase activity tested on a range of potential substrates was very low in these clones. None of them revealed a significant increase in tolerance to toxic aldehydes compared to their non-transgenic control. This work with transgenic apple plants overexpressing the detoxifying gene Vr-ERE illustrates some of the difficulties in developing an alternative selection pressure.     

Origin of the Genus <Emphasis Type="Italic">Homo</Emphasis>

The origin of the genus Homo in Africa signals the beginning of the shift from increasingly bipedal apes to primitive, large-brained, stone tool-making, meat-eaters that traveled far and wide. This early part of the human genus is represented by three species: Homo habilis, Homo rudolfensis, and Homo erectus. H. habilis is known for retaining primitive features that link it to australopiths and for being the first stone tool makers. Little is known about H. rudolfensis except that it had a relatively large brain and large teeth compared to H. habilis and that it overlapped in time and space with other early Homo. Our understanding of the paleobiology and evolution of the larger-brained H. erectus is enhanced due to its rich fossil record. H. erectus was the first obligate, fully committed biped, and with a body adapted for modern striding locomotion, it was also the first in the human lineage to disperse outside of Africa. The early members of the genus Homo are the first to tip the scale from the more apish side of our evolutionary history toward the more human one.     

High-frequency<Emphasis Type="Italic">in vitro</Emphasis> flowering in six species of<Emphasis Type="Italic">ceropegia</Emphasis>

High-frequencyin vitro flowering is reported here fromin vitro regenerated shoots ofin vitro-raised seedlings of rare and endemicCeropegia lawii, Ceropegia maccannii, Ceropegia oculata, andCeropegia sahyadrica, as well as the widely distributedCeropegia bulbosa var.bulbosa andCeropegia hirsuta. In our first set of experiments, the MS medium contained 87 mM sucrose and was supplemented with varying concentrations of BAP (4.4 to 26.6 μM). For the second set of trials, varying concentrations of sucrose (87 to 233 mM) were tested in MS media containing a constant 4.4 p.M BAP. Sub-cultured apical as well as axillary buds flowered with similar frequencies after 30 d of incubation. For all six species, the highest percentage of flowering shoots was obtained with either 26.6 μM BAP or 175 mM sucrose. Although smaller in size, theirin vitro flowers were morphologically comparable within wVo-derived flowers. Variations among species were noted for the number of flower buds per shoot and the percentage of flower formation. Because all six species showed similar responses in both experiments, we can suggest that this protocol is applicable across the wide range ofCeropegia species.     

Recombination within mouse <Emphasis Type="Italic">t</Emphasis> haplotypes has replaced significant segments of <Emphasis Type="Italic">t</Emphasis>-specific DNA

Previous studies on the fourth inversion of the t complex, In17(4), suggest that loci near the center of this inversion have been subjected to segmental recombination during the past 1–2 million years. We have used a combination of PCR-based restriction site (PBR) analysis and DNA sequencing to perform a high-resolution analysis of a 2-million base pair (Mbp) segment in the middle of In17(4). We examined 21 restriction sites that are polymorphic between t haplotypes and their wild-type homologs, over nine distinct loci. In addition, we examined several other polymorphic sites through DNA sequence analysis of two of these nine loci. We analyzed several haplotypes in this way, including the “complete” t haplotypes t ^w2 , t ⁰ , t ^w32 , t ^w71 , and t ^w75 . We show that only t ^w32 is a true “complete” t haplotype; the remaining four t haplotypes have segments of wild-type DNA ranging from less than 100 bp to 2 Mbp. The sizes of these wild-type DNA segments are consistent with their being generated by gene-conversion events. The 2-Mbp segment is located in a region that may contain the t-complex distorter gene Tcd2. One of the nine loci examined in this study is Fgd2, a gene that has been proposed to encode Tcd2. Sequencing and PBR data show that at least a portion of the Fgd2 gene has been converted to the wild-type within t ^w71 and t ^w75 mice.     

<Emphasis Type="Italic">Byssoloma rubromarginatum</Emphasis> (Pilocarpaceae: Ascomycota), a new corticolous species from <Emphasis Type="Italic">Nothofagus</Emphasis> forests in Argentina

A new corticolous species of Byssoloma, B. rubromarginatum Messuti and de la Rosa from Nothofagus dombeyi forests is described and illustrated. The new species is characterized by the morphology of the thallus and apothecia, the 3–5 (7)-septate ascospores, the presence of an anthraquinone in the exciple, and its ecology. It is the first species within the genus known to have gyrophoric acid in the apothecial margin. A key to Byssoloma species known from Nothofagus temperate forests in South America is provided.     

Target site selection by the <Emphasis Type="Italic">mariner</Emphasis>-like element, <Emphasis Type="Italic">Mos1</Emphasis>

The eukaryotic transposon Mos1 is a class-II transposable element that moves using a “cut-and-paste” mechanism in which the transposase is the only protein factor required. The formation of the excision complex is well documented, but the integration step has so far received less investigation. Like all mariner-like elements, Mos1 was thought to integrate into a TA dinucleotide without displaying any other target selection preferences. We set out to synthesize what is currently known about Mos1 insertion sites, and to define the characteristics of Mos1 insertion sequences in vitro and in vivo. Statistical analysis can be used to identify the TA dinucleotides that are non-randomly targeted for transposon integration. In vitro, no specific feature determining target choice other than the requirement for a TA dinucleotide has been identified. In vivo, data were obtained from two previously reported integration hotspots: the bacterial cat gene and the Caenorhabditis elegans rDNA locus. Analysis of these insertion sites revealed a preference for TA dinucleotides that are included in TATA or TA × TA motifs, or located within AT-rich regions. Analysis of the physical properties of sequences obtained in vitro and in vivo do not help to explain Mos1 integration preferences, suggesting that other characteristics must be involved in Mos1 target choice.