The <Emphasis Type="Italic">Tc1/mariner</Emphasis> DNA transposons in the genome of mollusk <Emphasis Type="Italic">Littorina saxatilis</Emphasis>

The Tc1/mariner superfamily is one of the most widely distributed among the DNA transposons in both terrestrial and aquatic organisms. We studied the abundance of the Tc1/mariner elements in the genome of the gastropod Littorina saxatilis Olivi, 1792 (Gastropoda: Littorinimorpha). For this purpose, nucleotide sequences with a total length of 358877 bp were analyzed. Six sequences were found to be similar to the Tc1/mariner DNA transposons. These sequences were studied for structure, the presence of functional transposase, and the systematic position within the superfamily. In addition, the loci with high homology to the DNA transposons of the hAT, Sola, Ginger, EnSpm/CACTA, ISL2EU, Kolobok, Novosib, Zisupton, and Helitron superfamilies were identified.     

Two active bamboo <Emphasis Type="Italic">mariner</Emphasis>-like transposable elements (<Emphasis Type="Italic">Ppmar1</Emphasis> and <Emphasis Type="Italic">Ppmar2</Emphasis>) identified as the transposon-based genetic tools for mutagenesis

Bamboo is one of the most important non-timber forest species in the world, but their molecular breeding lags far behind in contrast to other economic plants. Regarding the difficulties of hybridization and gene modification, the transposon-based insertional mutagenesis might be an alternative, feasible way for molecular breeding of bamboo. A systematic search for potential active transposons identified two full-length mariner-like elements (MLEs) (Ppmar1 and Ppmar2) from moso bamboo in the previous study. Both MLEs contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase. Two transposases contain intact DNA-binding motifs and a DD39D catalytic domain which indicates that Ppmar1 and Ppmar2 are likely active. Here, we deployed a heterologous transposition system of Arabidopsis thaliana to study the transposition activity of Ppmar1 and Ppmar2. The results show that both MLEs could transpose in A. thaliana. Excisions of Ppmar1 and Ppmar2 are usually unperfect as they leave 1–4 bp in excision sites. The reinsertions of both Ppmar1 and Ppmar2 occur at TA dinucleotides and prefer to insert into the TA-rich regions. The insertion sites are dispersed and non-linked. Two active bamboo transposons identified here not only could be applied to construction of the bamboo mutant libraries but also would provide another choice for other plant transposon-based gene tagging.     

Identification of active components from volatiles of Chinese bayberry, <Emphasis Type="Italic">Myrica rubra</Emphasis> attractive to <Emphasis Type="Italic">Drosophila suzukii</Emphasis>

Drosophila suzukii (Diptera: Drosophilidae) is native to Southeast Asia and now has become a severe pest of several soft fruits in Europe and the Americas. It causes considerable damage to Chinese bayberry, Myrica rubra, in China. In the present study, we employed gas chromatograph–electroantennographic detection (GC–EAD) together with behavioural bioassays and trapping experiments to identify volatile semiochemicals emitted by Chinese bayberry attracting D. suzukii. Electrophysiological experiments revealed the presence of six EAD-active compounds from ripe bayberry fruits, including methyl (E)-3-hexenoate, methyl (E)-2-hexenoate, ethyl (E)-2-hexenoate, α-ylangene, α-humulene and an unidentified compound that elicited consistent antennal response. In two-choice bioassays, bayberry fruits attracted all responding flies, and significantly more flies responded to the volatile extract of bayberry fruits. Four EAD-active compounds were attractive to mated female D. suzukii at lower doses (0.01 and 0.1 µg), but showed repellency at higher doses (10 and 100 µg). Mixtures of these four compounds at different ratios attracted D. suzukii flies at all test doses (0.1, 1 and 10 µg). Both male and female flies were trapped by a mixture of synthetic methyl (E)-3-hexenoate, methyl (E)-2-hexenoate, ethyl (E)-2-hexenoate and α-humulene in a ratio of 1:1.3:1:6.4 in the field trapping experiment. Significantly more males than females were captured in the trap baited with the synthetic blend, and the percentages of D. suzukii captured out of all flies by the traps baited with lure were higher than that baited with blank control. Our findings may provide insights into the olfactory responses of D. suzukii to specific host plant volatiles, and contribute to further development of an effective lure for monitoring D. suzukii in the field.     

Neuromuscular control of a single twitch muscle in wild type and mutant <Emphasis Type="Italic">Drosophila</Emphasis>, measured with an ergometer

How do deficits in neuronal growth, aging or synaptic function affect the final, mechanical output of a single muscle twitch? We address this in vivo (indeed in situ) with a novel ergometer that records the output of a large specialised muscle, the Drosophila jump muscle. Here, we describe in detail the ergometer, its construction and use. We evaluated the ergometer by showing that adult fly jump muscle output varies little between 3 h and 7 days; but newly eclosed flies produce only 65%. In a mutant with little octopamine (Tβh), jump muscle performance is reduced by 28%. The initial responses of synaptic growth mutants (highwire and spinster) do not differ from wild type, as expected on the homeostatic hypothesis. However, responses in highwire mutations gradually decline following repeated stimuli, suggesting physiological as well as anatomical abnormalities. We conclude that the assay is robust, sensitive and reliable with a good throughput.     

Thermotolerance and place memory in adult <Emphasis Type="Italic">Drosophila</Emphasis> are independent of natural variation at the <Emphasis Type="Italic">foraging</Emphasis> locus

Natural polymorphisms at the foraging (for) gene influence several behaviors. However, it is seldom clear how different for alleles could be selected. In one case, Drosophila with the rover allele (for ^r ) have higher locomotor activity in the presence of food than animals with the sitter allele (for ^s ), suggesting a complementary feeding strategy. There are, in addition, differences between for ^r and for ^s Drosophila in some tests of short-term memory (for ^r animals generally perform at higher levels) and thermotolerance (for ^s larvae are more resistant to the effects of high-temperature). We asked whether there could be a direct compensating advantages in adult for ^s flies that could maintain the natural for variants. First, are adult for ^s flies more thermotolerant? Second, do for ^r flies have a higher short-term place memory? Third, as an alternative, might for ^s flies have higher place memory? Our results do not confirm these possibilities. Thus, a thermotolerance advantage of for ^s flies does not compensate for a potential for ^r short-term memory advantage; for ^r flies do not have a universal advantage in short-term memory; and for ^s flies do not have an advantage in place memory that could compensate for for ^r advantages in other learning contexts.     

Subcutaneous administration CpG-ODNs acts as a potent adjuvant for an HIV-1-<Emphasis Type="Italic">tat</Emphasis>-based vaccine candidate to elicit cellular immunity in BALB/c mice

Objective

To evaluate the combined effects of CpG oligodeoxynucleotides (CpG-ODNs) adjuvant and subcutaneous injection route on efficacy of a HIV-1-tat DNA vaccine candidate using BALB/c mice as an animal model.

Results

Evaluation of cellular and humoral immunity of mice injected subcutaneously with HIV-1-tat gene cloned into a pcDNA3.1 vector indicated that significant levels of IFN-γ cytokine secretion (900 pg/ml), lymphocyte proliferation (2.5 stimulation index) and IgG_2a (1.45 absorbance 450 nm) production could be achieved. These indicators of stimulated cellular immunity were elicited 2 weeks after the last injection (P < 0.05).

Conclusions

Formulation of HIV-1-tat DNA vaccine candidate with CpG-ODNs as an adjuvant while administrated subcutaneously are a promising approach to induce effective cellular immunity responses against HIV-1 infection.

     

Molecular phylogenetics and anti-<Emphasis Type="Italic">Pythium</Emphasis> activity of endophytes from rhizomes of wild ginger congener, <Emphasis Type="Italic">Zingiber zerumbet</Emphasis> Smith

Zingiber zerumbet, a perennial rhizomatous herb exhibits remarkable disease resistance as well as a wide range of pharmacological activities. Towards characterizing the endophytic population of Z. zerumbet rhizomes, experiments were carried out during two different growing seasons viz., early-June of 2013 and late-July of 2014. A total of 34 endophytes were isolated and categorized into 11 morphologically distinct groups. Fungi were observed to predominate bacterial species with colonization frequency values ranging from 12.5 to 50 %. Among the 11 endophyte groups isolated, molecular analyses based on ITS/16S rRNA gene sequences identified seven isolate groups as Fusarium solani, two as F. oxysporum and one as the bacterium Rhizobium spp. Phylogenetic tree clustered the ITS sequences from Z. zerumbet endophytes into distinct clades consistent with morphological and sequence analysis. Dual culture assays were carried out to determine antagonistic activity of the isolated endophytes against Pythium myriotylum, an economically significant soil-borne phytopathogen of cultivated ginger. Experiments revealed significant P. myriotylum growth inhibition by F. solani and F. oxysporum isolates with percentage of inhibition (PoI) ranging from 45.17 ± 0.29 to 62.2 ± 2.58 with F. oxysporum exhibiting higher PoI values against P. myriotylum. Using ZzEF8 metabolite extract, concentration-dependent P. myriotylum hyphal growth inhibition was observed following radial diffusion assays. These observations were confirmed by scanning electron microscopy analysis wherein exposure to ZzEF8 metabolite extract induced hyphal deformities. Results indicate Z. zerumbet endophytes as promising resources for biologically active compounds and as biocontrol agents for soft rot disease management caused by Pythium spp.     

Identification of a novel <Emphasis Type="Italic">Drosophila</Emphasis> gene, <Emphasis Type="Italic">beltless</Emphasis>, using injectable embryonic and adult RNA interference (RNAi)

Background

RNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype.

Results

Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts.

Conclusions

We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system.

     

The <Emphasis Type="Italic">ecdysoneless</Emphasis><Superscript>1</Superscript> Gene Regulates Metabolism of the Juvenile Hormone and Dopamine in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The dopamine (DA) content and the level of juvenile hormone (JH) degradation were studied in females of the wild-type Canton S strain and the ecdysoneless ¹ (ecd ¹) mutant, which does not produce ecdysone at a restrictive temperature (29°C). Exposure at the restrictive temperature considerably increased the JH-hydrolyzing activity and the DA content in five-day ecd ¹ females compared with flies of both strains growing at 19°C and Canton S females exposed at 29°C. In one-day ecd ¹ females, the level of JH degradation also increased at the restrictive temperature, but the DA content was low. The effect of ecdysone deficiency on the stress reaction in Drosophila melanogaster females was studied using changes in DA content and JH degradation as the reaction indicators. The ecd ¹ mutation did not prevent the initiation of the stress reaction in females exposed at the restrictive temperature, but changed its intensity (stress reactivity). The interaction of 20-hydroxyecdysone with JH and DA in regulating Drosophila reproduction under normal conditions and in stress is discussed.     

Evidence for a role for the putative <Emphasis Type="Italic">Drosophila</Emphasis> hGRX1 orthologue in copper homeostasis

Glutaredoxins are a family of small molecular weight proteins that have a central role in cellular redox regulation. Human GRX1 (hGRX1) has also been shown to play an integral role in copper homeostasis by regulating the redox activity of the metalated sites of copper chaperones such as ATOX1 and SOD1, and the copper efflux proteins ATP7A and ATP7B. To further elucidate the role of hGRX1 in copper homeostasis, we examined the impact of RNA interference-mediated knockdown of CG6852, a putative Drosophila orthologue of hGRX1. CG6852 shares ~41 % amino acid identity with hGRX1 and key functional domains including the metal-binding CXXC motif are conserved between the two proteins. Knockdown of CG6852 in the adult midline caused a thoracic cleft and reduced scutellum, phenotypes that were exacerbated by additional knockdown of copper uptake transporters Ctr1A and Ctr1B. Knockdown of CG6852 in the adult eye enhanced a copper-deficiency phenotype caused by Ctr1A knockdown while ubiquitous knockdown of CG6852 resulted a mild systemic copper deficiency. Therefore we conclude that CG6852 is a putative orthologue of hGRX1 and may play an important role in Drosophila copper homeostasis.     

Genome-wide identification and evolution of <Emphasis Type="Italic">TC1</Emphasis>/<Emphasis Type="Italic">Mariner</Emphasis> in the silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>) genome

TC1/Mariner transposons belong to class II transposable elements (TEs) that use DNA-mediated “cut and paste” mechanism to transpose, and they have been identified in almost all organisms. Although silkworm (Bombyx mori) has a large amount of TC1/Mariner elements, the genome wide information of this superfamily in the silkworm is unknown. In this study, we have identified 2670 TC1/Mariner (Bmmar) elements in the silkworm genome. All the TEs were classified into 22 families by means of fgclust, a tool of repetitive sequence classification, seven of which was first reported in this study. Phylogenetic and structure analyses based on the catalytic domain (DDxD/E) of transposase sequences indicated that all members of TC1/Mariner were grouped into five subgroups: Mariner, Tc1, maT, DD40D and DD41D/E. Of these five subgroups, maT rather than Mariner possessed most members of TC1/Mariner (51.23%) in the silkworm genome. In particular, phylogenetic analysis and structure analysis revealed that Bmmar15 (DD40D) formed a new basal subgroup of TC1/Mariner element in insects, which was referred to as bmori. Furthermore, we concluded that DD40D appeared to intermediate between mariner and Tc1. Finally, we estimated the insertion time for each copy of TC1/Mariner in the silkworm and found that most of members were dramatically amplified during a period from 0 to 1 mya. Moreover, the detailed functional data analysis showed that Bmmar1, Bmmar6 and Bmmar9 had EST evidence and intact transposases. These implied that TC1/Mariner might have potential transpositional activity. In conclusion, this study provides some new insights into the landscape, origin and evolution of TC1/Mariner in the insect genomes.     

Activity of phosphino palladium(II) and platinum(II) complexes against HIV-1 and <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Treatment of human immunodeficiency virus (HIV) is currently complicated by increased prevalence of co-infection with Mycobacterium tuberculosis. The development of drug candidates that offer the simultaneous management of HIV and tuberculosis (TB) would be of great benefit in the holistic treatment of HIV/AIDS, especially in sub-Saharan Africa which has the highest global prevalence of HIV-TB coinfection. Bis(diphenylphosphino)-2-pyridylpalladium(II) chloride (1), bis(diphenylphosphino)-2-pyridylplatinum(II) chloride (2), bis(diphenylphosphino)-2-ethylpyridylpalladium(II) chloride (3) and bis(diphenylphosphino)-2-ethylpyridylplatinum(II) (4) were investigated for the inhibition of HIV-1 through interactions with the viral protease. The complexes were subsequently assessed for biological potency against Mycobacterium tuberculosis H37Rv by determining the minimal inhibitory concentration (MIC) using broth microdilution. Complex (3) showed the most significant and competitive inhibition of HIV-1 protease (p = 0.014 at 100 µM). Further studies on its in vitro effects on whole virus showed reduced viral infectivity by over 80 % at 63 µM (p < 0.05). In addition, the complex inhibited the growth of Mycobacterium tuberculosis at an MIC of 5 µM and was non-toxic to host cells at all active concentrations (assessed by tetrazolium dye and real time cell electronic sensing). In vitro evidence is provided here for the possibility of utilizing a single metal-based compound for the treatment of HIV/AIDS and TB.     

Chemical and molecular identification of the invasive termite <Emphasis Type="Italic">Zootermopsis nevadensis</Emphasis> (Isoptera: Archotermopsidae) in Japan

Numerous termite species have been introduced outside their native ranges by human transport, and some have become invasive. The dampwood termite Zootermopsis nevadensis (Hagen), which is native to western North America, has been introduced to and become established in Kawanishi City, Hyogo Prefecture, Japan. Zootermopsis nevadensis is subdivided into two subspecies based on cuticular hydrocarbon (CHC) phenotypes: Z. nevadensis nevadensis and Z. nevadensis nuttingi (Haverty and Thorne). Here, we identified Z. nevadensis in Japan as hybrids between the two subspecies. Chemical analysis showed the presence of 7,15-dimethylhenicosane and 5,17-dimethylhenicosane in the CHCs of Z. nevadensis in Japan, corresponding to the CHC phenotype of Z. n. nevadensis. Conversely, all mitochondrial cytochrome c oxidase subunit I sequences of Z. nevadensis in Japan were identical to sequences from Z. n. nuttingi and hybrids between the two subspecies from a native hybrid zone in California, USA. In addition, phylogenetic analysis showed that Z. nevadensis in Japan formed a clade with Z. n. nuttingi and hybrids between the two subspecies. Our results show discordance between the chemical and genetic features of Z. nevadensis in Japan, indicating that individuals of Z. nevadensis in Japan are hybrids between the two subspecies.     

Biotic factors in induced defence revisited: cell aggregate formation in the toxic cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC 7806 is triggered by spent <Emphasis Type="Italic">Daphnia</Emphasis> medium and disrupted cells

Bioassays with the toxic cyanobacterium Microcystis aeruginosa PCC 7806, its non-toxic mutant ΔmcyB, and Daphnia magna as grazer were used to evaluate biotic factors in induced defence, in particular cyanobacterial and grazer-released info-chemicals. Three main questions were addressed in this study: Does Daphnia grazing lead to a loss of cyanobaterial biomass? Is the survival time of Daphnia shorter in a culture of the toxic cyanobacterium? Does direct grazing or the presence of spent Daphnia medium or a high number of disrupted toxic Microcystis cells in the assays lead to an increase in the cellular microcystin content in the remaining intact cells? The biovolume (growth) as well as size and abundance of Microcystis aggregates were determined by particle analysis, while the survival time of Daphnia individuals was recorded by daily observation and counting, with the relative concentration of cell-bound microcystin-LR, was measured by HPLC analysis. Compared to some recent studies in the field of induced defence, in this study, evidence was found for a direct grazing effect, i.e. the loss of biovolume in the toxic culture. In addition, Daphnia magna ingested more non-toxic than toxic cells, and survived longer with non-toxic cells. In terms of increased cell-bound toxin concentration as a means of defence reported in some studies, a higher cell-bound microcystin-LR content was not measured in this study in any of the treatments (P > 0.05). Under low light conditions with impaired growth of Microcystis, and the presence of a high number of particles with less than 1-μm diameter (possibly heterotrophic bacteria), Daphnia medium was associated with a strong reduction in cell-bound toxin concentration (P < 0.05). This study showed no increased cell aggregation under direct grazing (P > 0.05), but increased aggregation with spent Daphnia medium under high light conditions (P < 0.05). Further, the addition of cell-free extract from disrupted toxic Microcystis cells strongly increased the aggregation of the intact cells under low light (P < 0.05). These findings are discussed with the possible role of microcystin and other infochemicals in the expression of proteins and morphology changes in Microcystis.     

Development and validation of multiplex-PCR assay for simultaneous detection of rare alleles of <Emphasis Type="Italic">crtRB1</Emphasis> and <Emphasis Type="Italic">lcyE</Emphasis> governing higher accumulation of provitamin A in maize

Vitamin A deficiency is a widely prevalent health disorder among millions of people worldwide. Introgression of crtRB1 and lcyE favourable alleles that enhance concentration of provitamin A in maize endosperm have been employed in maize biofortification programmes. To make marker-assisted selection (MAS) more effective, we have developed rapid and convenient multiplex-polymerase chain reaction (PCR) assay to simultaneously discover the allelic combinations among the segregants. Validation of the multiplex assay was done in two backcross-derived populations developed using elite inbreds viz., HKI193-1 and HKI193-2 carrying unfavourable alleles of crtRB1 (296 bp) and lcyE (300 bp) and HarvestPlus inbreds viz., HP704-22 and HP704-23 possessing favourable alleles of crtRB1 (543 bp) and lcyE (650 bp). We also standardized the uniplex-PCR assays for both the genes that gave robust and reproducible results in sub-tropical populations. Gel profiles of BC₁F₁, BC₂F₁ and BC₂F₂ revealed that these assays identified the backcross progenies homo-or hetero-zygous for the favourable- or unfavourable-alleles. Multiplex-PCR assay also precisely confirmed the results of individual uniplex assays in different backcross generations. Cost and time analyses showed that multiplex-PCR assay has potential to save 41% of cost, and 50% of time compared to two uniplex assays in a MAS programme. It has also saved 50% of the manpower. The multiplex assay possesses significant advantage over uniplex assays and enhances the efficiency of selection. This is the first report of development and validation of multiplex-PCR assay of crtRB1 and lcyE for utilization in maize biofortification programme.     

Characterization of the complete mitochondrial genome of flower-breeding <Emphasis Type="Italic">Drosophila incompta</Emphasis> (Diptera,Drosophilidae)

Drosophila incompta belongs to the flavopilosa group of Drosophila, and has a restricted ecology, being adapted to flowers of Cestrum as feeding and oviposition sites. We sequenced, assembled, and characterized the complete mitochondrial genome (mtDNA) of D. incompta. In addition, we performed phylogenomic and polymorphism analyses to assess evolutionary diversification of this species. Our results suggest that this genome is syntenic with the other published mtDNA of Drosophila. This molecule contains 15,641 bp and encompasses two rRNA, 22 tRNA and 13 protein-coding genes. Regarding nucleotide composition, we found a high A?T bias (76.6 %). The recovered phylogenies indicate D. incompta in the virilis–repleta radiation, as sister to the virilis or repleta groups. The most interesting result is the high degree of polymorphism found throughout the D. incompta mitogenome, revealing pronounced intrapopulational variation. Furthermore, intraspecific nucleotide diversity levels varied between different regions of the genome, thus allowing the use of different mitochondrial molecular markers for analysis of population structure of this species.     

Identification of <Emphasis Type="Italic">r</Emphasis> mutations conferring white flowers in the Japanese morning glory (<Emphasis Type="Italic">Ipomoea nil</Emphasis>)

The wild-type Japanese morning glory [Ipomoea nil (L.) Roth.] exhibits blue flowers with red stems, and spontaneous r mutants display white flowers with green stems. We have identified two r mutations, r1-1 and r1-2, that are caused by insertions of Tpn1-related DNA transposable elements, Tpn3 (5.6 kb) and Tpn6 (4.7 kb), respectively, into a unique intron of the CHS-D gene, which is responsible for flower and stem pigmentation. Both Tpn3 and Tpn6, which belong to the En/Spm or CACTA superfamily, are nonautonomous elements lacking transposase genes but containing unrelated cellular DNA segments including exons and introns. Interestingly, r1-2 contains an additional 4-bp insertion at the Tpn3 integration site in r1-1, presumably a footprint caused by the excision of Tpn3. The results strengthen the previous notion that Tpn1 and its relatives are major spontaneous mutagens for generating various floriculturally important traits in I. nil. Since I. nil has an extensive history of genetic studies, molecular identification of classical spontaneous mutations would also facilitate reinterpretation of the abundant classical genetic data available.     

Delivery of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> HpaA to gastrointestinal mucosal immune sites using <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and its immune efficacy in mice

Objective

To develop a safe and effective oral vaccine against Helicobacter pylori using its HpaA protein expressed in Lactococcus lactis.

Results

The gene encoding HpaA was obtained by PCR and ligated to pNZ8110-lysM following digestion with NaeI + SphI. The recombinant plasmid was transferred into E. coli for multiplication, and then into L. lactis. The recombinant L. lactis was induced to express HpaA, resulting in two products of 29 and 25 kDa, both of which yielded positive immunoreaction with mouse antisera against H. pylori, as confirmed by immunoblot assays. The 29 kDa product constituted 12% of the cell lysates. Oral inoculation with the engineered L. lactis evoked significantly elevated serum IgG level in mice (P < 0.05).

Conclusions

A novel engineered L. lactis strain was developed that efficiently produces whole HpaA protein with desired antigenicity and potent immunogenicity. It provides a basis for approaches to L. lactis-delivered anti-H. pylori vaccination.

     

Fingerprinting by amplified fragment length polymorphism (AFLP) and barcoding by three plastidic markers in the genus <Emphasis Type="Italic">Wolffiella</Emphasis> Hegelm

Amplified fragment length polymorphism (AFLP) fingerprinting and three different plastidic DNA regions (rpl16, rps16, atpF-atpH) were used to investigate species identity in the genus Wolffiella. For this purpose, clones (67 in total) belonging to all ten species were selected. Almost all the species were represented by more than one clone. The fingerprinting technique, AFLP, clearly distinguished the species, W. caudata, W. gladiata, W. neotropica, W. rotunda, and W. welwitschii. Apart from confirming the molecular identity of these five species, the plastidic markers could delineate two additional species, W. hyalina and W. denticulata, although the conclusion concerning the latter is restricted by the availability of only one clone. The efficiency of the plastid-derived markers in identifying the number of species-specific clusters followed the sequence rps16 > rpl16 > atpF-atpH. The species W. lingulata, W. oblonga, and W. repanda could not be identified by any of the molecular methods presented here, but could be strictly defined on a morphological basis. In several clones, high amounts of genetic admixtures between different species were detected. Further, simulation studies demonstrated that these clones are genetic hybrids. This might be one of the obstacles in molecular identification of species in the genus Wolffiella.