Characterization and expression of <Emphasis Type="Italic">DjPreb</Emphasis> gene in the planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

In this study, we report the expression and identification of a PREB-related gene from the planarian Dugesia japonica, DjPreb. The planarian DjPreb cDNA is comprised of 1101 bp and contains a 972 bp open reading frame corresponding to a deduced protein of 323 amino acids with a 69 bp 5’-UTR and a 60 bp 3’-UTR. Phylogenetic analysis shows that DjPreb is PREB/PREB-like members. We examined its spatial and temporal expression and distribution in both intact and regenerating planarians by Relative quantitative real-time PCR and Whole-mount in situ hybridization. The analysis indicates that DjPreb shows a gradient express with peak levels present in the anterior and posterior regions and progressively lower levels in central regions in intact and regenerating planarians. During regeneration the expression of DjPreb is upregulated. Strong expression of DjPreb is observed in the anterior and posterior blastemas. These results suggest that DjPreb may participate in head and tail formation.     

Development of expressed sequence tag-derived microsatellite markers for <Emphasis Type="Italic">Saccharina</Emphasis> (<Emphasis Type="Italic">Laminaria</Emphasis>) <Emphasis Type="Italic">japonica</Emphasis>

Expressed sequence tag-derived microsatellite markers (EST-SSR) were generated and characterized in Laminaria japonica using data mining from updated public EST databases and polymorphism testing. Fifty-eight of 578 ESTs (10.0%) containing various repeat motifs were used to design polymerase chain reaction (PCR) amplification primers. A total of 12 pairs of primer were generated and used in the PCR amplification. Alleles per locus ranged from two to ten (average of 5.7). The observed heterozygosities and expected heterozygosities were from 0.045 to 0.543 and from 0.056 to 0.814, respectively. All loci were in Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. These robust, informative, and potentially transferable polymorphic markers appear suitable for population, genetic, parentage, and mapping studies of L. japonica.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

Characterization of the expression profile of <Emphasis Type="Italic">calpain</Emphasis>-<Emphasis Type="Italic">3</Emphasis> (<Emphasis Type="Italic">CAPN3</Emphasis>) gene in chicken

Calpain-3 is a skeletal muscle-specific protease and participates in the regulation of myogenesis. In this study, we quantified the expression of calpain-3 (CAPN3) mRNA in a Chinese local chicken breed (Sichuan Mountainous Black-boned chicken [MB]), to discern the tissue and ontogenic expression pattern. Meanwhile, we compared the CAPN3 mRNA expression pattern in MB chicken at 10 weeks with a commercial meat type chicken line (S01) of the same age to identify the unique expression pattern under different genetic background. A real time quantitative PCR (qRT-PCR) assay was developed for an accurate measurement of its expression in various tissues from chickens at different ages (0, 2, 4, 6, 8, 10, and 12 weeks). Expression of the CAPN3 mRNA was detected in the selected tissues, regardless of age. The breast muscle and leg muscle tissues had a significantly higher expression than the other tissues from the same individual (P < 0.01). Overall, the CAPN3 mRNA level exhibited a “rise-decline” developmental change in detected tissues except for brain. The S01 chicken had a higher expression of the CAPN3 mRNA in detected tissues than the MB chicken at 10 weeks. The present expression data of chicken CAPN3 gene may provide some information to shed light on the tissue and ontogenic expression pattern during chicken development.     

Expression of the<Emphasis Type="Italic"> lacZ</Emphasis> reporter gene in sporophytes of the seaweed<Emphasis Type="Italic"> Laminaria japonica</Emphasis> (Phaeophyceae) by gametophyte-targeted transformation

The seaweed Laminaria japonica (Phaeophyceae) has a two-generation life cycle consisting of haploid gametophytes and diploid sporophytes. Female and/or male gametophytes were transformed using particle bombardment and the histological LacZ assay was performed on sporophytes generated by either parthenogenesis or inbreeding. Female gametophyte-targeted transformation resulted in similar lower efficiencies in both parthenogenetic and zygotic sporophytes, and only a chimeric expression pattern was observed. Male gametophyte-targeted transformation led to a higher efficiency, with 3.5% of the zygotic sporophytes stained completely blue (all-blue), implying the integration of lacZ at the one-cell stage. Polymerase chain reaction analysis using primers specific for a lacZ-vector juncture fragment and subsequent blotting indicated the presence of the introduced gene in the sporophytes. The method reported here has a potential for seaweed transformation using spore-based bombardment followed by the developmental process.Abbreviations DPR Detected positive rate - ER Expression rateCommunicated by F. Sato     

Polyhydroxybutyrate in <Emphasis Type="Italic">Rhizobium</Emphasis> and <Emphasis Type="Italic">Bradyrhizobium</Emphasis>: quantification and <Emphasis Type="Italic">phbC</Emphasis> gene expression

Polyhydroxyalkanoates (PHAs) are hydroxyalkanoate polymers that are produced and accumulate by many kinds of bacteria. These polymers act as an energy store for bacteria. Polyhydroxybutyrate (PHB) is the most studied polymer in the PHA family. These polymers have awakened interest in the environmental and industrial research areas because they are biodegradable and have thermoplastic qualities, like polypropylene. In this work, we analyzed the PHB production in Bradyrhizobium sp., Rhizobium leguminosarum bv. phaseoli, and Rhizobium huautlense cultured with two different carbon sources. We did biochemical quantification of PHB production during the three phases of growth. Moreover, these samples were used for RNA extraction and phbC gene expression analysis via real-time PCR. The bacteria showed different manner of growth, PHB accumulation and phbC gene expression when different quantity and quality of carbon sources were used. These results showed that under different growth media conditions, the growth and metabolism of different species of bacteria were influenced. These differences reflect the increase or decrease in PHB accumulation.     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.     

Identification of SCAR marker linking to longer frond length of <Emphasis Type="Italic">Saccharina japonica</Emphasis> (Laminariales,Phaeophyta) using bulked-segregant analysis

To construct a molecular-marker-assisted selection (MAS) system, research was done on identifying molecular markers linking to longer frond length, a crucial selection index in the breeding of the commercially important seaweed Saccharina japonica. An F₂-segregant population of 92 individuals was obtained by crossing two prominent S. japonica strains. Genomic DNA from ten individuals with the longest frond and ten individuals with the shortest frond in the F₂-segregant population were mixed to create two DNA pools for screening polymorphic markers. In bulked-segregant analysis (BSA), out of 100 random amplified polymorphic DNA (RAPD) primers only two produced three polymorphic RAPD markers between the two DNA pools. In conversion of the three RAPD markers into sequence-characterized amplified region (SCAR) markers, only one was successfully converted into a SCAR marker FL-569 linking to the trait of longer frond. Test of the marker FL-569 showed that 80% of the individuals with longest fronds in a wild population and 87.5% of individuals with the longest fronds in an inbred line “Zhongke No. 2” could be detected by FL-569. Additionally, genetic linkage analysis showed that the SCAR marker could be integrated into the reported genetic map and QTL mapping showed that FL-569 linking to qL1-1. The obtained marker FL-569 will be beneficial to MAS in S. japonica breeding.     

Production of transgenic <Emphasis Type="Italic">Pinus armandii</Emphasis> plants harbouring <Emphasis Type="Italic">btCry</Emphasis>III(<Emphasis Type="Italic">A</Emphasis>) gene

A synthetic chimeric gene SbtCryIII(A) encoding the insecticidal protein btCryIII(A), was transformed into Pinus armandii embryos and embryogenic calli using Agrobacterium tumefaciens. Polymerase chain reaction and genomic DNA Southern blot analysis showed that the SbtCryIII(A) gene was integrated into the genome of transgenic Pinus armandii plants, and Northern blot analysis indicated that the SbtCryIII(A) gene was transcribed.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

<Emphasis Type="Italic">Lacuna vincta</Emphasis> (Mollusca,Neotaenioglossa) herbivory on juvenile and adult <Emphasis Type="Italic">Nereocystis luetkeana</Emphasis> (Heterokontophyta,Laminariales)

Herbivory can be an important factor structuring coastal algal communities. Herbivores may preferentially graze particular algal species or tissue types. Mesograzers, despite their small size, can critically weaken kelp thalli and impact entire kelp beds. We propose that when kelp beds are composed of several kelp cohorts, mesograzers will selectively choose to inhabit younger plants and grazing activities will have a greater impact on younger plants. This study investigated the effects of grazing by the littorinid gastropod, Lacuna vincta, on different age classes of the bull kelp, Nereocystis luetkeana by (1) testing food preference of L. vincta on juvenile, first-year adult, and second-year adult Nereocystis blades in the laboratory, (2) determining substrate (blades of different ages) preference of L. vincta in the laboratory, and by (3) estimating in-situ herbivore abundances and densities on juvenile and adult Nereocystis. Results demonstrated that grazing by L. vincta produced greater damage on juvenile than older Nereocystis tissues. Although L. vincta did not select juvenile versus older kelps as substrate in the laboratory, in situ surveys showed that differences existed between age classes with higher L. vincta densities on juvenile than adult kelp. We conclude that at a local scale, L. vincta can be an important structuring factor in Nereocystis populations due to its high density and grazing ability. Handling editor: K. Martens     

Expression pattern of <Emphasis Type="Italic">DjPreb</Emphasis> gene during the planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis> embryonic development

Prolactin regulatory element binding (PREB) protein belongs to the family of WD-repeat proteins which are regulatory and versatile proteins for diverse functions. In this study we have shown the expression pattern of the planarian Dugesia japonica PREB-related gene (DjPreb) during embryonic development by whole-mount in situ hybridization. Genomic analysis reveals that the DjPreb gene consists of two exons and one intron. Expression of the DjPreb mRNA was not observed as early as in stage 3 embryos. DjPreb positive signal was first found in stage 4. It is expressed in some embryonic cells in the periphery of the embryo. The number of DjPreb positive embryonic cells grows in stage 5. DjPreb is expressed in the dorsolateral regions and part of the anterior regions in stage 6. In stage 7, DjPreb positive signals are detected in the dorsolateral regions along the A-P axis and from stage 8 to the juvenile stage DjPreb mRNA is strongly expressed not only in the differentiating tissues of the anterior and posterior regions, but also in the parenchyma of the dorsolateral regions, and generates the gradient in the head of the juvenile. These results on the DjPreb expression pattern suggest its potential role in the specification of many cell types; in particular, DjPreb may play an essential role in spatial and temporal regulation during the head and tail formation and the anterior/posterior patterning formation.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Regulation of gamete release in the economic brown seaweed <Emphasis Type="Italic">Hizikia fusiforme</Emphasis> (Phaeophyta)

Gamete release is an essential event in artificial seeding of the economic brown seaweed, Hizikia fusiforme. Mass egg release occurred in the dark, with few eggs being discharged in the light. Release of eggs was elicited with eight practical salinity units (one PSU ≡ 1 g sea salts l⁻¹) and was inhibited by salinity levels >32 PSU. Egg release was optimal at 23 °C, and was decreased by 72% in agitated seawater compared to unstirred seawater. Inhibitors of photosynthesis and ions channels suppressed egg release, indicating that this process was physiologically associated with photosynthetic activity and ion transport.     

<Emphasis Type="Italic">In planta</Emphasis> gene expression analysis of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pathovar <Emphasis Type="Italic">oryzae</Emphasis>, African strain MAI1

Background  

Bacterial leaf blight causes significant yield losses in rice crops throughout Asia and Africa. Although both the Asian and African strains of the pathogen, Xanthomonas oryzae pv. oryzae (Xoo), induce similar symptoms, they are nevertheless genetically different, with the African strains being more closely related to the Asian X. oryzae pv. oryzicola (Xoc).     

Polysaccharide and lipid composition of the brown seaweed <Emphasis Type="Italic">Laminaria gurjanovae</Emphasis>

Polysaccharide and lipid composition of the Pacific brown seaweed Laminaria gurjanovae is determined. Alginic acid is shown to be the main polysaccharide of its biomass (about 28%); it consists of mannuronic and guluronic acid residues at a ratio of 3: 1. The yield of water-soluble polymannuronic acid is low and does not exceed 1.1% of dry biomass. High laminaran content (about 22%) is found, whereas the yield of fucoidan is no more than 3.6%. Laminaran consists of two fractions, soluble and insoluble in cold water, their ratio is 2.5: 1. Insoluble laminaran is a practically linear 1,3-β-D-glucan, and the soluble fraction was shown to be 1,3;1,6-β-D-glucan. The oligosaccharide products of desulfation or partial acidic hydrolysis of fucoidan were studied by MALDI TOF MS; they were found to be fuco- and galactooligosaccharides. The fucoidan is suggested to be a highly sulfated partially acetylated galactofucan (Fuc/Gal is ～1: 1). The main lipid components of the dried L. gurjanovae are neutral lipids and glyceroglycolipids, whereas phospholipids are found in minor amounts. The main fatty acid components of lipids are 14:0, 16:0, 16:1 ω-7, 18:1 ω-7 and 18:2 ω-6 acids.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.