一株东方醋酸杆菌分离与其促进果蝇生长发育

【目的】分离与鉴定黑腹果蝇体内醋酸杆菌,并研究其对宿主生长发育的促进作用。【方法】利用醋酸杆菌选择性培养基分离果蝇肠道醋酸杆菌;通过革兰氏染色和16S rRNA基因比对鉴定菌种;肠道定植实验验证共生关系;发育历期和生长速率实验检测其促进果蝇生长作用;免疫荧光染色技术检测肠道细胞增殖;RT-PCR法检测促生长的分子标志物和相关的信号通路。【结果】菌株为东方醋酸杆菌(Acetobacter orientalis),可以持续地定植在果蝇肠道及其培养基中,并且明显促进果蝇的生长。东方醋酸杆菌通过胰岛素信号通路增加肠分裂细胞的数量和促进蜕皮激素的分泌。【结论】东方醋酸杆菌是果蝇的一种共生菌,对果蝇肠道结构和机体发育具有重要的作用。     

Use of the Cre-loxP recombination system as an estimate for Agrobacterium-mediated co-transformation of tobacco leaves

Agrobacterium tumefaciens-mediated transformation of tobacco leaves (Nicotiana tabacum) is used to study gene expression in a heterologous genetic background. Here, the Cre-loxP recombination system was used to detect T-DNA transfer by two A. tumefaciens cells harboring different binary vectors. Cre, under the control of the CaMV 35S promoter, was cloned into one vector, and a loxP cassette into another vector. A mixture of A. tumefaciens, in which each cell contained either a Cre- or loxP-vector, was co-infiltrated into tobacco leaves. After two days, excision of loxP-flanked DNA was detected by PCR and used as an estimate for co-transformation events. Strongest excision (> 50%) was observed when the loxP cassette was cloned into vector pPZP112 and Cre into pISV2678. This fast and easy technique can be used to assess the co-transformation efficiency of tobacco cells in future studies.     

Production of saikosaponins by tissue culture of Bupleurum falcatum L.

The studies for production of saikosaponins by tissue culture of Bupleurum falcatum L. were carried out to produce saikosaponins with several kinds of media and plant hormones. Among the media and plant hormones studied, Gamborg's B-5 [23] medium containing 0.5 ppm kinetin (k) and 1.0 ppm 3-indolebutyric acid (IBA) was the most effective medium and hormone for production of saikosaponins. The highest content of saikosaponin-d in the dried cells was 0.26%, which was similar to a concentration of Bupleuri Radix.Abbreviations MS medium used by Murashige and Skoog [22] - G medium used by Gamborg (B 5) [23] - W medium used by White [24] - NN medium used by Nitsch and Nitsch [25] - k Kinetin - BAP 6-Benzylaminopurine - 2,4 D 2,4-Dichlorophenoxyacetic acid - NAA -Naphtylacetic acid - IBA 3-Indolebutyric acid - IAA 3-Indoleacetic acid - ssd saikosaponin-d - PM Production medium - dw Dry weight     

A note on the structure of system state spaces and its implications on the existence of non-repeatable experiments

The implication of state space structure on the existence of a repeatable experimentE designed to determine if a states∈L has propertyP or notP is investigated. It is shown that if a state spaceL is connected, then no experimentE is repeatable. This formalism is used to demonstrate that if a propertyP has an associated set of points inL which is dense with dense complement inL, then there exists no repeatable experimentE which can be used to test whethers has propertyP or notP. Other consequences of this formalization are discussed.     

Key to the eggs of the equid stomach bot flies Gasterophilus Leach 1817 (Diptera: Gasterophilidae) utilizing scanning electron microscopy

Abstract. A key to the eggs of the equid stomach bot flies is presented. Scanning electron photomicrographs of eggs are used to illustrate differences among the eight Gasterophilus species. The eggs include those of G.haemorrhoidalis (Linnaeus, 1758), G.inermis (Brauer, 1858), G.intestinalis (De Geer, 1776), G.meridionalis (Piller and Evans, 1926), G.nasalis (Linnaeus, 1758), G.nigricornis (Loew, 1863), G.pecorum (Fabricius, 1794), and G.ternicinctus Gedoelst, 1912. The eggs of G.meridionalis and G.ternicinctus are shown for the first time. Egg profile is the same for a particular species and is used as a key character for egg identification. Colour of eggs is used in some couplets but only as a supplemental character. Absence or presence of striae on the eggs is used as a primary contrasting character to separate G.pecorum from the other seven species. Shape of the striae varies on eggs of the same species, even those dissected from the same specimen, and is therefore deemed an unreliable taxonomic character for further separation of the Gasterophilus species. Eggs of the same species taken from specimens throughout the world appear the same in profile. Two sets of eggs require close inspection for adequate identification: G.inermis and G.nigricornis separated primarily by the shape of the microphylar region; and G.intestinalis and G.ternicinctus separated by the shape of the egg ventrum. All other eggs have very unique and distinctive profiles. Only G.pecorum was found to possess the Type-II egg attachment organ (AO) used for adherence of the egg to plants or flat surfaces. The eggs of the remaining seven species possess a Type-I AO used to attach the eggs to hair shafts. The type of AO and the degree that the Type-I AO is extended posteriorly were used as key characters in the first and second couplet respectively.     

Development of gfp Vectors for Expression in Listeria monocytogenes and Other Low G+C Gram Positive Bacteria

The gfp (green fluorescent protein) gene has previously been used to construct a variety of reporter plasmids for Gram-positive bacteria for bacterial localization and gene expression studies. When a native red-shifted gfp variant (gfp3) was cloned into an expression vector using the P _xyn promoter and used to transform the soil-borne pathogen Listeria monocytogenes, only a small proportion of the population was seen to fluoresce when examined by epifluorescence microscopy. When the P _xyn promoter was replaced with the P _xylA promoter, with accompanying modification of the translation initiation region of the gfp3 gene, a homogeneously fluorescent population of cells was obtained. When expressed in other Gram-positive organisms, such as Staphylococcus aureus and Bacillus subtilis, the translationally enhanced gene also resulted in high-level and homogeneous GFP production within the bacterial population. High-level expression of these reporter constructs in L. monocytogenes was evaluated to determine if it had any detrimental biological effect during intracellular infection of eukaryotic cell lines. The gfp3 ⁺ Listeria were found to invade equally as well as the wild-type cells; showing that these expression systems can be used to monitor the bacterium in natural environments. Based on these results, similar translationally enhanced vectors were also developed using unstable GFP3 variants, which retain their short-half life characteristics in L. monocytogenes and therefore can be used as a sensitive monitor of gene expression.     

Use of random amplified polymorphic DNA to identify several novel markers for sex identification in the crested serpent eagle and crested goshawk

The crested serpent eagle (Spilornis cheela hoya) has no distinct sexual dimorphic traits. In the current study, we report the results of an EE0.6 (EcoRI 0.6-kb fragment) sequence applied to S. cheela hoya and a novel random amplified polymorphic DNA (RAPD) marker that can be used to sex individuals within the species S. cheela hoya and Accipiter trivigatus formosae (crested goshawk). We used sex-specific primers for the avian CHD1 (chromo-helicase-DNA-binding 1) gene and the EE0.6 sequence in PCR assays to determine sex. In addition, 120 random primers were used for RAPD fingerprinting to search for novel sex-specific fragments of S. cheela hoya. The OPBB08 random primer generated a 1241-bp sex-specific fragment in all female S. cheela hoya. From the nucleotide sequence, PCR primers were designed to amplify 553-, 895-, and 194-bp sex-specific fragments present in all female S. cheela hoya. One of these primer pairs (ScBB08-7F/R) also amplified a male/female common fragment that can be used as an internal control (543 bp). Moreover, one of the primer pairs (ScBB08-5aF/5bR) could be used to identify genders of A. trivigatus formosae. In conclusion, we identified novel sex-specific DNA markers of S. cheela hoya and A. trivigatus formosae that can be used for rapid and accurate sex identification.     

Expression of a rice <Emphasis Type="Italic">CYP81A6</Emphasis> gene confers tolerance to bentazon and sulfonylurea herbicides in both <Emphasis Type="Italic">Arabidopsis</Emphasis> and tobacco

Bentazon and sulfonylurea are two different classes of herbicides that have been widely used to kill broad-leaf weeds in rice fields. A cytochrome P450 gene, CYP81A6, encoding a monooxygenase has been previously identified to confer resistance to these two classes of herbicides in wild-type rice. In this study, we introduced the rice CYP81A6 gene into Arabidopsis and tobacco plants to test the possibility of engineering tolerance to these two types of herbicides in other susceptible plants. Arabidopsis and tobacco plants expressing CYP81A6 showed tolerance to both bentazon and bensulfuron-methyl (BSM), a widely applied sulfonylurea herbicide. The optimal concentrations of bentazon and BSM for the selection of CYP81A6 transgenic plants were also determined. In addition, we also demonstrated that CYP81A6 can be used as a selection marker to effectively screen for positive transgenic Arabidopsis plants. The selection efficiency of CYP81A6 was comparable to that of the bar gene in Arabidopsis. These results suggest that CYP81A6 can not only be used to produce transgenic plants tolerant to both bentazon and sulfonylureas, but that it can also be used as a novel plant-derived selection marker in plant transformation.     

Autonomy of the wingless mutation in Drosophila melanogaster

Summary T(Y;2) translocations were used to cytologically localise the wingless locus of Drosophila melanogaster. We found that an existing T(Y;2), which is an insertion of a segment of 2L into the Y chromosome, has wg ⁺ within this insert. This Y chromosome was used to generate an attached XY chromosome containing wg ⁺. The mutation claret-nondisjunctional (ca ^nd) was used to induce the loss of this XY chromosome and thus generate gynandromorphs with wg ¹/wg ¹ male tissue and wg ⁺/wg ¹/wg ¹ female tissue. Analysis of these gynanders demonstrated that a genotypically wingless mutant hemithorax is usually also phenotypically mutant in these half body mosaics; thus wg ¹ is discautonomous. This observation is of interest as it is known that wg is not cell autonomous.     

From macro to micro: a combined bioluminescence-fluorescence approach to monitor bacterial localization

Bacterial bioluminescence is widely used to study the spatiotemporal dynamics of bacterial populations and gene expression in vivo at a population level but cannot easily be used to study bacterial activity at the level of individual cells. In this study, we describe the development of a new library of mini-Tn7-lux and lux::eyfp reporter constructs that provide a wide range of lux expression levels, and which combine the advantages of both bacterial bioluminescence and fluorescent proteins to bridge the gap between macro- and micro-scale imaging techniques. We demonstrate that a dual bioluminescence-fluorescence approach using the lux operon and eYFP can be used to monitor bacterial movement in plants both macro- and microscopically and demonstrate that Pseudomonas syringae pv phaseolicola can colonize the leaf vascular system and systemically infect leaves of common bean (Phaseolus vulgaris). We also show that bacterial bioluminescence can be used to study the impact of plant immune responses on bacterial multiplication, viability and spread within plant tissues. The constructs and approach described in this study can be used to study the spatiotemporal dynamics of bacterial colonization and to link population dynamics and cellular interactions in a wide range of biological contexts.     

Roosting Ecology of the Tent‐Roosting Bat Artibeus watsoni (Chiroptera: Phyllostomidae) in Southwestern Costa Rica1

We described the plants used as roost resources by Artibeus watsoni in southwestern Costa Rica, assessed roost fidelity, and compared roosting ecology between two sites, Golfito and Corcovado, which vary in the degree of human influence. A total of 349 tents from 25 different plant species were used by A. watsoni as roosts; some plant species (e.g., Carludovica palmata, Asplundia alata, Heliconia imbricata and Calathea lutea) were modified into tents with significantly higher frequency than others. The highest tents above the ground were observed in Philodendron popenoei and Rhodospatha wendlandii, whereas tents in Philodendron grandipes and A. alata were significantly lower than any other species. Asplundia alata and R. wendlandii also had the highest frequency of leaves modified per plant. Fidelity of bats to tents was low, although bats used several tents intermittently within a restricted area. Males generally were more faithful to tents than females, although not significantly so. This observation, along with indirect evidence of leaf modification, suggests that males are primarily responsible for tent construction. The two study sites differed in the plants used for roosting and in tent fidelity. Bats in Corcovado used a greater variety of plant species for tent roosting, whereas bats in Golfito were more faithful, suggesting that roosting resources were scarcer at the latter site.     

Alkanes of four related moth species,Helicoverpa and Heliothis

Comparison of the presence and quantities of cuticular hydrocarbons has been used successfully for identifying sibling species and races of several groups of insects. This approach has been extended to four species of moths previously regarded as belonging to the same genus, Heliothis. Gas chromatography was used to quantify the numerous high-molecular weight alkanes found on the cuticle of two pairs of closely related species: Helicoverpa zea and Helicoverpa armigera, and Heliothis virescens and Heliothis subflexa. Both sexes of H. zea and H. armigera contained different quantities of several alkanes that could be used for unambiguous identification. Similar comparisons of H. subflexa and H. virescens showed four peak ratios that were different for each species. Sexual dimorphism was minor in H. subflexa and H. virescens.     

Biodegradation of aliphatic hydrocarbon by indigenous fungi isolated from used motor oil contaminated sites

Eighteen indigenous fungal isolates has been successfully isolated from samples of used motor oil, top five centimetres of soil and drainage water contaminated with used motor oil. All of the pure fungal isolates obtained were identified, characterized and subjected to preliminary screening by evaluating the average growth rate of each fungal isolates on minimal media containing 1% (v/v) used motor oil. Trichoderma asperellum strain TUB F-1067 (SA4), Trichoderma asperellum strain Tr48 (SA5), Trichoderma asperellum strain TUB F-756 (SA6), Penicillium species (P1), and Aspergillus species (P9) were further selected for their hydrocarbon biodegradation potential. Among these five fungal isolates selected, P1 strain presented a significant degree of degradation by degrading almost all of the n-alkanes (n-C-₁₅ to n-C-₂₃ range) present in the used motor oil, thus of greater potential in degrading the aliphatic hydrocarbon compounds of used motor oil. The authors would like to certify that the work have not been sent/considered to be published in other journals.     

Goodness-of-Fit Testing for the Logistic Regression Model when the Estimated Probabilities are Small

The distribution of the Hosmer-Lemeshow chi-square type goodness-of-fit tests (?_g, ?_g) for the logistic regression model are examined via simulations designed to examine their behavior when most of the estimated probabilities are small or are expected to fall in a few deciles. The results of the simulations show statistic ?_g should be used when the two outcome groups (y = 0, 1) are not well separated, Δ≤2, where Δ² is the Mahalanobis distance. Statistic ?_g should be used when Δ ≥ 8. Either statistic may be used when 2 ≦ Δ ≦ 8. All tests should be used with caution when the proportion in the sample with y = 1 is less than 0.1.     

Phylogenetic measurement in procaryotes by primary structural characterization

Summary Oligonucleotide cataloguing has been used to characterize a number of 5S RNA species from various Procaryotes. Such catalogs can be used to establish certain and detailed phylogenetic relationships among organisms. Confining attention at present to four Families of Procaryotes, theEnterobacteriaceae, theBacillaceae, theAchromobacteraceae, and thePseudomonadaceae, we have shown that the conventionally accepted classification of these organisms which places the first three in the orderEubacteriales, and the last in the orderPseudomonadales, is not phylogenetically valid.     

Non-specificity of a colorimetric method for the estimation of N-acetyl-d-glucosamine

The colorimetric method of Reissig et al. for the estimation of N-acetylamino sugars, is often used as a specific method for the quantification of the N-acetyl-d-glucosamine. Although this assay is more sensitive to the monomer, it recognizes all soluble N-acetyl-d-glucosamine oligomers. This result is very important because this method is extensively used in biology for the estimation of chitinolytic activity.     

Chewing side preference is associated with hemispheric laterality in healthy adults

Purpose To investigate if chewing side preference (CSP) can be used as an indicator of hemispheric laterality in healthy adults. Materials and methods Seventy-five individuals were included. The visual analogue scale (VAS) was used to determine CSP and laterality test for preferred peripheral organs. Results Significant correlation between CSP and hand, foot, ear, and eye side preference was found (r?=?.41, p?r?=?.34, p?=?.003; r?=?.35, p?=?.03; r?=?.36, p?=?.002). Conclusion Besides peripheral organs, the CSP can also be used in determination of hemispheric lateralization.     

High efficiency electrotransformation of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>cells pretreated with lithium acetate and dithiothreitol

Background  

A goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis). Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria.     

Genetic diversity and phylogeny analysis of Anabaena azollae based on RFLPs detected in Azolla-Anabaena azollae DNA complexes using nif gene probes

The cyanobacterium Anabaena has both symbiotic and free-living forms. The genetic diversity of Anabaena strains symbiotically associated with the aquatic fern Azolla and the evolutionary relationships among these symbionts were evaluated by means of RFLP (restriction fragment length polymorphism) experiments. Three DNA fragments corresponding to nif genes were cloned from the free-living cyanobacterium Anabaena PCC 7120 and used as probes. A mixture of Azolla, Anabaena and bacterial DNA was extracted from Azolla fronds and digested with two restriction enzymes. Single-copy RFLP signals were detected with two of the probes in all Azolla Anabaena examined. Multiple-copy RFLP signals were obtained from the third probe which corresponded to a part of the nif N gene. A total of 46 probe/enzyme combinations were scored as present or absent and used to calculate pairwise Nei's genetic distances among symbiotic Anaebaena strains. Phylogenetic trees summarizing phenetic and cladistic relationships among strains were generated according to three different evolutionary scenarios: parsimony, UPGMA and neighbour joining. All trees revealed identical phylogenetic relationships. Principal component analysis was also used to evaluate genetic similarities and revealed three groups: group one contains the cyanobacteria associated with plants from the Azolla section, group two contains those associated with plants from the pinnata species and group three contains those associated with plants from the nilotica species. The same groups had already been identified earlier in a random amplified polymorphic DNA (RAPD) analysis of Azolla-Anbaena DNA complexes, suggesting that the present Azolla taxonomy should be revised. We now suggest a taxonomy of Anabaena azollae that is parallel to such a revised Azolla taxonomy. An Azolla chloroplast DNA sequence derived from Oryza sativa was also used as an RFLP probe on Azolla DNA to confirm the presence of plant DNA in the total genomic DNA extracted from ferns with or without the symbiont. Our results also suggest that total DNA extracted from the Azolla-Anabaena complexes includes both plant and symbiont DNA and can be used equally well for RFLP analysis of host plant or symbiotic cyanobacteria.     

Challenges Associated with Heterologous Expression of <Emphasis Type="Italic">Flavobacterium psychrophilum</Emphasis> Proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A two-parameter statistical model was used to predict the solubility of 96 putative virulence-associated proteins of Flavobacterium psychrophilum (CSF259-93) upon over expression in Escherichia coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates (inclusion bodies). These solubility predictions were verified experimentally by colony filtration blot for six different F. psychrophilum proteins. A comprehensive analysis of codon usage identified over a dozen codons that are used frequently in F. psychrophilum, but that are rarely used in E. coli. Expression of F. psychrophilum proteins in E. coli was often associated with production of minor molecular weight products, presumably because of the codon usage bias between these two organisms. Expression of recombinant protein in the presence of rare tRNA genes resulted in marginal improvements in the expressed products. Consequently, Vibrio parahaemolyticus was developed as an alternative expression host because its codon usage is similar to F. psychrophilum. A full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified from V. parahaemolyticus in soluble form, whereas this protein was insoluble upon expression in E. coli. We show that V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy challenges associated with expression and production of F. psychrophilum recombinant proteins.