Representational Difference Analysis Research Articles

Epigenetic Diversity Underlying Seasonal and Annual Variations in Brown Planthopper (BPH) Populations as Revealed by Methylation- sensitive Restriction Assay.

The brown planthopper (BPH) is a monophagous sap-sucking insect pest of rice that is responsible for massive yield loss. BPH populations, even when genetically homogenous, can display a vast range of phenotypes, and the development of effective pest-management strategies requires a good understanding of what generates this phenotypic variation. One potential source could be epigenetic differences. With this premise, we explored epigenetic diversity, structure and differentiation in field populations of BPH collected across the rice-growing seasons over a period of two consecutive years. Using a modified methylation-sensitive restriction assay (MSRA) and CpG island amplification-representational difference analysis, site-specific cytosine methylation of five stress-responsive genes (CYP6AY1, CYP6ER1, Carboxylesterase, Endoglucanase, Tf2-transposon) was estimated, for identifying methylation-based epiallelic markers and epigenetic variation across BPH populations. Using a cost-effective and rapid protocol, our study, for the first time, revealed the epigenetic component of phenotypic variations in the wild populations of BPH. Besides, results showed that morphologically indistinguishable populations of BPH can be epigenetically distinct. Screening field-collected BPH populations revealed the presence of previously unreported epigenetic polymorphisms and provided a platform for future studies aimed at investigating their significance for BPH. Furthermore, these findings can form the basis for understanding the contribution(s) of DNA methylation in providing phenotypic plasticity to BPH.

RDA coupled with deep sequencing detects somatic SVA-retrotranspositions and mosaicism in the human brain.

Cells of the developing human brain are affected by the progressive acquisition of genetic and epigenetic alterations that have been reported to contribute to somatic mosaicism in the adult brain and are increasingly considered a possible cause of neurogenetic disorders. A recent work uncovered that the copy-paste transposable element (TE) LINE-1 (L1) is mobilized during brain development, and thus mobile non-autonomous TEs like AluY and SINE-VNTR-Alu (SVA) families can use L1 activity in trans, leading to de novo insertions that may influence the variability of neural cells at genetic and epigenetic levels. In contrast to SNPs and when considering substitutional sequence evolution, the presence or absence of TEs at orthologous loci represents highly informative clade markers that provide insights into the lineage relationships between neural cells and how the nervous system evolves in health and disease. SVAs, as the 'youngest' class of hominoid-specific retrotransposons preferentially found in gene- and GC-rich regions, are thought to differentially co-regulate nearby genes and exhibit a high mobility in the human germline. Therefore, we determined whether this is reflected in the somatic brain and used a subtractive and kinetic enrichment technique called representational difference analysis (RDA) coupled with deep sequencing to compare different brain regions with respect to de novo SINE-VNTR-Alu insertion patterns. As a result, we detected somatic de novo SVA integrations in all human brain regions analyzed, and the majority of de novo insertions can be attributed to lineages of telencephalon and metencephalon, since most of the examined integrations are unique to different brain regions under scrutiny. The SVA positions were used as presence/absence markers, forming informative sites that allowed us to create a maximum parsimony phylogeny of brain regions. Our results largely recapitulated the generally accepted evo-devo patterns and revealed chromosome-wide rates of de novo SVA reintegration targets and preferences for specific genomic regions, e.g., GC- and TE-rich regions as well as close proximity to genes that tend to fall into neural-specific Gene Ontology pathways. We concluded that de novo SVA insertions occur in the germline and somatic brain cells at similar target regions, suggesting that similar retrotransposition modes are effective in the germline and soma.

Identification of candidate genes responsible for chasmogamy in wheat

BackgroundThe flowering biology of wheat plants favours self-pollination which causes obstacles in wheat hybrid breeding. Wheat flowers can be divided into two groups, the first one is characterized by flowering and pollination within closed flowers (cleistogamy), while the second one possesses the ability to open flowers during processes mentioned above (chasmogamy). The swelling of lodicules is involved in the flowering of cereals and among others their morphology, calcium and potassium content differentiate between cleistogamic and non-cleistogamous flowers. A better understanding of the chasmogamy mechanism can lead to the development of tools for selection of plants with the desired outcrossing rate. To learn more, the sequencing of transcriptomes (RNA-Seq) and Representational Difference Analysis products (RDA-Seq) were performed to investigate the global transcriptomes of wheat lodicules in two highly chasmogamous (HCH, Piko and Poezja) and two low chasmogamous (LCH, Euforia and KWS Dacanto) varieties at two developmental stages—pre-flowering and early flowering.ResultsThe differentially expressed genes were enriched in five, main pathways: “metabolism”, “organismal systems”, “genetic information processing”, “cellular processes” and “environmental information processing”, respectively. Important genes with opposite patterns of regulation between the HCH and LCH lines have been associated with the lodicule development i.e. expression levels of MADS16 and MADS58 genes may be responsible for quantitative differences in chasmogamy level in wheat.ConclusionsWe conclude that the results provide a new insight into lodicules involvement in the wheat flowering process. This study generated important genomic information to support the exploitation of the chasmogamy in wheat hybrid breeding programs.

Insights into the Seasonal Olfactory Mechanism of Geosmin in Raw Water of Huangpu River.

Since the 1990s, the raw water of Huangpu River in Shanghai, China, has intermittently encountered off-flavor contamination. In this work, the concentrations of typical odor, geosmin, in raw water of Huangpu River are found to shift along with the seasons. However, microbes recognized as the producer of geosmin such as Cyanobacteria and Actinobacteria are not consistent with the shift of geosmin. Cyanobacteria blooms in summer rather than winter, whereas Actinobacteria thrives in winter. Representational difference analysis (RDA) reveals that microbes associated with blooming algae have positive co-occurrence correlations with the concentrations of geosmin and nutrients in winter, whereas those within Cyanobacteria and Planctomycete are in a positive correlation with temperature and thrive in summer. This causes the concentration of geosmin in raw water to appear to depend on the abundance of Actinobacteria rather than that of Cyanobacteria. However, combining with the synthesis and storage properties of geosmin in algae, as well as the decomposition properties of algae with Actinobacteria, geosmin might be synthesized by Cyanobacteria in summer, which is stored in cells of Cyanobacteria and released only via the decomposition of Actinobacteria in winter. This potential olfactory mechanism of geosmin is quite different from that derived from pure culture of odor producers or correlation analysis of bacteria and odors; thus, providing insights into the mechanism of practical off-flavor events.

Identification of gene transcripts contributing to trunking and non-trunking sago palm (Metroxylon sagu Rottb.)

Sago palm (Metroxylon sagu Rottb.) is an economically valuable crop and cultivated widely in Sarawak, Malaysia, for its storage starch. Under certain conditions, such as deep peat areas, some sago palms do not develop well and become stunted, described as non-trunking sago palms. Morphologically, this type of sago palm is abnormal, leading to wastage of various resources and affecting starch production. The research objective was to understand the molecular process that affects non-trunking and starch synthesis in sago palm. Representational Difference Analysis was performed to identify the differences between trunking and non-trunking sago palm transcriptome. This subtractive hybridisation technique detects the differences in gene expression. After three rounds of hybridisation, the differentiated RDA products were cloned and 24 DNA sequences obtained were compared with the sequences in the NCBI database. Results showed that the differentiated genes in response to the non-trunking sago palm have similarities to genes implicated in plant growth, cell metabolism, and salt or osmotic stress. Some of these genes are related to growth, development and signaling involving neutralising the imbalance in plant stress. The information obtained through this work could be used to develop a database on related genes and enzymes contributing to trunking and non-trunking sago palm.

Early Changes in Skeletal Muscle of Young C22 Mice, A Model of Charcot-Marie-Tooth 1A.

Background:The C22 mouse is a Charcot-Marie-Tooth 1A transgenic model with minimal axonal loss.Objective:To analyse early skeletal muscle changes resulting from this dysmyelinating neuropathy.Methods:Histology of tibialis anterior muscles of C22 mice and wild type litter mate controls for morphometric analysis and (immuno-)histochemistry for known denervation markers and candidate proteins identified by representational difference analysis (RDA) based on mRNA from the same muscles; quantitative PCR and Western blotting for confirmation of RDA findings.Results:At age 10 days, morphometry was not different between groups, while at 21 days, C22 showed significantly more small diameter fibres, indicating the onset of atrophy at an age when weakness becomes detectable. Neither (immuno-)histochemistry nor RDA detected extrajunctional expression of acetylcholine receptors by age 10 and 21 days, respectively. RDA identified some mRNA up-regulated in C22 muscles, among them at 10 days, prior to detectable weakness or atrophy, integral membrane protein 2a (Itm2a), eukaryotic initiation factor 2, subunit 2 (Eif2s2) and cytoplasmic phosphatidylinositol transfer protein 1 (Pitpnc1). However, qPCR failed to measure significant differences. In contrast, Itm2a and Eif2s2 mRNA were significantly down-regulated comparing 21 versus 10 days of age in both groups, C22 and controls. Western blotting confirmed significant down-regulation of ITM2A protein in C22 only.Conclusion:Denervation-like changes in this model develop slowly with onset of atrophy and weakness at about three weeks of age, before detection of extrajunctional acetylcholine receptors. Altered Itm2a expression seems to begin early as an increase, but becomes distinct as a decrease later.

Identification of genes differentially expressed between a somatotrope and a lactotrope pituitary cell lines by representational difference analysis

The molecular and cellular processes leading to the development and differentiation of the anterior pituitary gland is well characterized both in rodents and in human and rely on the highly regulated expression of transcription and growth factors. Yet regulatory mechanisms and signature molecules implicated in postnatal pituitary homeostasis, cell activation in physiological conditions and uncontrolled cell proliferation leading to pituitary tumorigenesis are still evasive and mediators of these events are still missing. To address this issue, representational difference analysis was applied to two established tumoral rodent cell lines, the GC cells secreting growth hormone and the 235-1 cells producing prolactin. A limited number of genes were expressed in only one cell line and not in the other. Several genes implicated in cell fate determination (Mb21d2, Gpc4, Creg1 and Wls) were identified. Their function in somatotrope or lactotrope cell specification remains to be determined. Half of the genes detected by RDA are potential candidate genes for pituitary tumorigenesis as they are implicated in signaling pathways associated with cell proliferation, oncogenic or tumor-suppressor function, epithelial to mesemchymal transition or invasiveness. These poorly characterized genes may encode new biomarkers relevant to the understanding of the molecular mechanisms associated with the development, proliferation or invasiveness of pituitary adenomas. The specific role of these new genes in the context of pituitary cell homeostasis or tumorigenesis will required further investigation as some of the identified genes were not reported to be associated with pituitary function in normal or pathological condition. The present work identified new genes encoding potential biomarkers that could turned out to be key intrinsic factors in pituitary tumorigenesis and molecular therapeutic targets in the treatment of human pituitary adenomas.

Transcribed sex-specific markers on the Y chromosome of the oriental fruit fly, Bactrocera dorsalis

BackgroundThe Oriental fruit fly, Bactrocera dorsalis, is a highly polyphagous invasive species with a high reproductive potential. In many tropical and subtropical parts of the world it ranks as one of the major pests of fruits and vegetables. Due to its economic importance, genetic, cytogenetic, genomic and biotechnological approaches have been applied to understand its biology and to implement the Sterile Insect Technique, currently a part of area-wide control programmes against this fly. Its chromosome complement includes five pairs of autosomes and the sex chromosomes. The X and Y sex chromosomes are heteromorphic and the highly heterochromatic and degenerate Y harbours the male factor BdMoY. The characterization of the Y chromosome in this fly apart from elucidating its role as primary sex determination system, it is also of crucial importance to understand its role in male biology. The repetitive nature of the Y chromosome makes it challenging to sequence and characterise.ResultsUsing Representational Difference Analysis, fluorescent in situ hybridisation on mitotic chromosomes and in silico genome resources, we show that the B. dorsalis Y chromosome harbours transcribed sequences of gyf, (typo-gyf) a homologue of the Drosophila melanogaster Gigyf gene, and of a non-LTR retrotransposon R1. Similar sequences are also transcribed on the X chromosome. Paralogues of the Gigyf gene are also present on the Y and X chromosomes of the related species B. tryoni. Another identified Y-specific repetitive sequence linked to BdMoY appears to be specific to B. dorsalis.ConclusionsOur random scan of the Y chromosome provides a broad picture of its general composition and represents a starting point for further applicative and evolutionary studies. The identified repetitive sequences can provide a useful Y-marking system for molecular karyotyping of single embryos. Having a robust diagnostic marker associated with BdMoY will facilitate studies on how BdMoY regulates the male sex determination cascade during the embryonic sex-determination window. The Y chromosome, despite its high degeneracy and heterochromatic nature, harbours transcribed sequences of typo-gyf that may maintain their important function in post-transcriptional mRNA regulation. That transcribed paralogous copies of Gigyf are present also on the X and that this genomic distribution is maintained also in B. tryoni raises questions on the evolution of sex chromosomes in Bactrocera and other tephritids.

Representational Difference Analysis of Transcripts Involved in Jervine Biosynthesis.

Veratrum-type steroidal alkaloids (VSA) are the major bioactive ingredients that strongly determine the pharmacological activities of Veratrum nigrum. Biosynthesis of VSA at the molecular and genetic levels is not well understood. Next-generation sequencing of representational difference analysis (RDA) products after elicitation and precursor feeding was applied to identify candidate genes involved in VSA biosynthesis. A total of 12,048 contigs with a median length of 280 bases were received in three RDA libraries obtained after application of methyl jasmonate, squalene and cholesterol. The comparative analysis of annotated sequences was effective in identifying candidate genes. GABAT2 transaminase and hydroxylases active at C-22, C-26, C-11, and C-16 positions in late stages of jervine biosynthesis were selected. Moreover, genes coding pyrroline-5-carboxylate reductase and enzymes from the short-chain dehydrogenases/reductases family (SDR) associated with the reduction reactions of the VSA biosynthesis process were proposed. The data collected contribute to better understanding of jervine biosynthesis and may accelerate implementation of biotechnological methods of VSA biosynthesis.

Factors promoting the natural regeneration of Larix principis-rupprechtii plantation in the Lvliang Mountains of central China.

Given their complexity, targeted care and management of different areas and tree species are necessary for enhancing the natural regeneration of forests. Thus, an understanding of changes in the overstory and understory is essential for ensuring successful regeneration. Promoting the natural regeneration of Larix principis-rupprechtii plantations is considered challenging; indeed, regional sustainable development through natural regeneration of many stands has often been considered unattainable. Here, we studied several plots with varying extents of regeneration to identify the most important factors that affect regeneration. The plots were divided into three forest types based on the number of regenerating plants. For each type of plot, we measured various factors that might potentially affect regeneration. Representational difference analysis was used to identify the most important factors >9% contribution). Based on these factors, multiple corrections were made to construct a structural equation model of topography, stand structure, soil properties and litter to identify the most important factors driving variation in regeneration. Positive correlations were detected between regeneration with diameter at breast height (0.21) and litter thickness (0.57). Regeneration was negatively correlated with soil (−0.54) and slope (−0.48). Additionally, the number of regenerating plants and the height of regenerating plants were strongly positively correlated. However, there was no significant relationship between regeneration and litter accumulation, stand density, altitude, average tree height, total P and total N. Overall, our study showed that key factors for promoting natural regeneration include appropriate litter thickness, strong parent trees, a gentle slope and sufficient quantities of soil nutrients. Moreover, our findings provide a reference for the design of effective management and restoration plans.

Nitrogen fertilization alters the effects of earthworms on soil physicochemical properties and bacterial community structure

Although it is known that earthworms can improve the granular structure of the soil and enrich the nutrient content, the effects of earthworms in combination with inorganic fertilization are not well understood. In the current work, 16S rRNA high-throughput sequencing and quantitative PCR (qPCR) approaches were used to reveal the effects of earthworms (Pheretima guillelm) and N fertilization on soil nutrient availability and to clarify their interactions with soil bacterial community composition. Pot experiments involving nine treatments were conducted using a split-plot design. Earthworm throw-in density was used as the whole-plot factor and N fertilization rate as the split-plot factor. The results showed that earthworms increased the proportion of macroaggregates in the soil, and the mean weight diameter and geometric mean diameter of soil aggregates, whereas N fertilization decreased these parameters. The qPCR results revealed a significantly higher bacterial abundance in soil treated with a high earthworm density without nitrogen addition (E2N0) than in the other treatments. The microbial biomass C content (CMic) of the soil in the low earthworm density with high N fertilization treatment (E1N2) was significantly higher than in the other treatments (P < 0.05). However, no significant differences were found in the microbial biomass N (NMic) of the soil among N fertilization treatments. The hierarchically clustered heatmap analysis based on the bacterial beta diversity revealed a higher proportion of some beneficial bacteria in E1N2, including Flavobacterium (11%) and Pseudomonas (5%). Furthermore, CMic (P = 0.006), available K (P = 0.016), NMic (P = 0.018) and available N (P = 0.027) were major factors affecting the variance in the bacterial community structure according to the representational difference analysis. This study suggests that earthworms can enhance the availability of soil CMic and NMic by increasing the number of soil macroaggregates and 16S rRNA at appropriate N fertilization rates.

Interaction with Pantoea agglomerans Modulates Growth and Melanization of Sporothrix brasiliensis and Sporothrix schenckii.

Sporothrix brasiliensis and Sporothrix schenckii stand as the most virulent agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. The origin of Sporothrix virulence seems to be associated with fungal interactions with organisms living in the same environment. To assess this hypothesis, the growth of these two species in association with Pantoea agglomerans, a bacterium with a habitat similar to Sporothrix spp., was evaluated. Growth, melanization, and gene expression of the fungus were compared in the presence or absence of the bacterium in the same culture medium. Both S. brasiliensis and S. schenckii grew in contact with P. agglomerans yielding heavily melanized conidia after 5days of incubation at 30°C in Sabouraud agar. This increased melanin production occurred around bacterial colonies, suggesting that fungal melanization is triggered by a diffusible bacterial product, which is also supported by a similar pattern of melanin production during Sporothrix spp. growth in contact with heat-killed P. agglomerans. Growth of P. agglomerans was similar in the presence or absence of the fungus. However, the growth of S. brasiliensis and S. schenckii was initially inhibited, but further enhanced when these species were co-cultured with P. agglomerans. Moreover, fungi were able to use killed bacteria as both carbon and nitrogen sources for growth. Representational difference analysis identified overexpressed genes related to membrane transport when S. brasiliensis was co-cultured with the bacteria. The down-regulation of metabolism-related genes appears to be related to nutrient availability during bacterial exploitation. These findings can lead to a better knowledge on Sporothrix ecology and virulence.

Accessory gland proteins of males in the male‐diphenic ant Cardiocondyla obscurior

AbstractSeminal fluid proteins are important agents in sperm competition and sexual conflict. In solitary insects, males and females may have different interests concerning the outcome of a mating episode, and males may use seminal fluid to manipulate the physiology and behaviour of the female against the latter's fitness interests. Less is known about the role of seminal fluids in social insects, where the interests of mating partners are more aligned because of life‐long pair‐bonding. In the male‐diphenic ant Cardiocondyla obscurior, mating even with sterilized males appears to prolong the lifespan of queens, suggesting a positive role of seminal fluids. In the present study, using two approaches (two‐dimensional gel electrophoresis/mass spectrometry and representational difference analysis), we analyze the seminal fluid and the male accessory glands of both winged and wingless males of C. obscurior. The results obtained reveal the presence of hundreds of proteins, of which proteases and protease inhibitors are also widespread in the seminal fluids of other insects.

Occurrence of subdioecy and scarcity of gender-specific markers reveal an ongoing transition to dioecy in Himalayan seabuckthorn (Hippophae rhamnoides ssp. turkestanica).

Dioecy and the dynamics of its evolution are intensely investigated aspects of plant reproduction. Seabuckthorn (Hippophae rhamnoides ssp. turkestanica) is an alpine shrub growing wild in certain parts of western Himalaya. The previous studies have reported heteromorphic sex chromosomes in the species and yet marker-based studies indicate high similarity between the male and female genomes. Lack of information on sexual system in the species has further complicated the situation. A systematic study was thus undertaken to understand the sexual system in seabuckthorn and to discern the extent of similarity/dissimilarity between the male and female genomes by generating a large number of markers using amplified fragment length polymorphism and representational difference analysis. Floral biology and regular monitoring of species revealed the presence of polygamomonoecious (PGM) plants in most populations at a low percentage (~2-4%). PGM plants showed low pollen production and overall low fertility, suggesting a monoecy-paradioecy pathway at function. The results of the marker study demonstrated that there are limited differences between male and female genomes and these differences were not uniform across the populations in the Leh-Ladakh region, especially when the geographical distance increases. Results also suggest that a dynamic partitioning of genomes is operational between the two genders of seabuckthorn and differences are not homogenized across the populations. Both reproductive biology-based and DNA marker-based studies indicate that genders have separated recently. The present study proposes seabuckthorn as a promising model system to study evolution of dioecy and sex determination.

Microbial ecological associations in the surface sediments of Bohai Strait

Microbial communities play key roles in the marine ecosystem. Despite a few studies on marine microbial communities in deep straits, ecological associations among microbial communities in the sediments of shallow straits have not been fully investigated. The Bohai Strait in northern China (average depth less than 20 m) separates the Bohai Sea from the Yellow Sea and has organic-rich sediments. In this study, in the summer of 2014, six stations across the strait were selected to explore the taxonomic composition of microbial communities and their ecological associations. The four most abundant classes were Gammaproteobacteria, Deltaproteobacteria, Bacilli and Flavobacteriia. Temperature, total carbon, depth, nitrate, fishery breeding and cold water masses influenced the microbial communities, as suggested by representational difference and composition analyses. Network analysis of microbial associations revealed that key families included Flavobacteriaceae, Pirellulaceae and Piscirickettsiaceae. Our findings suggest that the families with high phylogenetic diversity are key populations in the microbial association network that ensure the stability of microbial ecosystems. Our study contributes to a better understanding of microbial ecology in complex hydrological environments.

Bacillus Calmette-Guérin induces rapid gene expression changes in human bladder cancer cell lines that may modulate its survival.

Bacillus Calmette-Guérin (BCG) immunotherapy is the standard therapy for non-muscle invasive bladder cancer. The aim of the present study was to identify genes that are induced in response to BCG immunotherapy, as these may be potential biomarkers for the response to clinical therapy. To model clinical therapy, human bladder cancer cell lines were incubated with BCG (live or lyophilized BCG Connaught) for 2 h. RNA was extracted and evaluated by Representational Differential Analysis (RDA) and oligo arrays. Gene expression was confirmed by reverse transcription polymerase chain reaction on fresh cell lines with differential abilities to internalize BCG. The effect of 2 major BCG soluble proteins, antigen 85B (Ag85B) and Mycobacterium protein tyrosine phosphatase A (MptpA) and BCG Tice® on gene expression was also determined. GAPDH and β-actin, which are normally used as control genes, were upregulated by BCG. Therefore, the ribosomal RNA gene ribosomal protein S27a was used to normalize gene expression. The genes likely to be induced by BCG internalization and soluble factors were: GSTT2, MGST2, CCL20, TNFα, CCNE1 and IL10RB. Those induced by BCG membrane interactions and/or soluble factors were: MGST1, CXCL6, IL12A, CSF2, IL1β and TOLLIP. MptpA decreased GSTT2 expression, and Ag85B increased TNFα expression. The two BCG strains significantly increased GSTT2, TNFα and TOLLIP levels in MGH cells. However, in J82 cells there was a BCG strain-dependent difference in TNFα expression. An important outcome of the present study was the determination that neither GAPDH nor β-actin were suitable control genes for the analysis of BCG-induced gene expression. BCG Connaught and Tice® induced similar expression levels of genes in bladder cancer cell lines. BCG soluble proteins modulated gene expression and therefore may affect therapeutic outcomes. The genes identified may be novel biomarkers of the response to BCG therapy.

Discovery of leucokinin-like neuropeptides that modulate a specific parameter of feeding motor programs in the molluscan model, Aplysia

A better understanding of neuromodulation in a behavioral system requires identification of active modulatory transmitters. Here, we used identifiable neurons in a neurobiological model system, the mollusc Aplysia, to study neuropeptides, a diverse class of neuromodulators. We took advantage of two types of feeding neurons, B48 and B1/B2, in the Aplysia buccal ganglion that might contain different neuropeptides. We performed a representational difference analysis (RDA) by subtraction of mRNAs in B48 versus mRNAs in B1/B2. The RDA identified an unusually long (2025 amino acids) peptide precursor encoding Aplysia leucokinin-like peptides (ALKs; e.g. ALK-1 and ALK-2). Northern blot analysis revealed that, compared with other ganglia (e.g. the pedal-pleural ganglion), ALK mRNA is predominantly present in the buccal ganglion, which controls feeding behavior. We then used in situ hybridization and immunohistochemistry to localize ALKs to specific neurons, including B48. MALDI-TOF MS on single buccal neurons revealed expression of 40 ALK precursor-derived peptides. Among these, ALK-1 and ALK-2 are active in the feeding network; they shortened the radula protraction phase of feeding motor programs triggered by a command-like neuron. We also found that this effect may be mediated by the ALK-stimulated enhancement of activity of an interneuron, which has previously been shown to terminate protraction. We conclude that our multipronged approach is effective for determining the structure and defining the diverse functions of leucokinin-like peptides. Notably, the ALK precursor is the first verified nonarthropod precursor for leucokinin-like peptides with a novel, marked modulatory effect on a specific parameter (protraction duration) of feeding motor programs.

DnaJC18, a Novel Type III DnaJ Family Protein, is Expressed Specifically in Rat Male Germ Cells.

ABSTRACTMammalian spermatogenesis occurs in a precise and coordinated manner in the seminiferous tubules. One of the attempts to understand the detailed biological process during mammalian spermatogenesis at the molecular level has been to identify the testis specific genes followed by study of the testicular expression pattern of the genes. From the subtracted cDNA library of rat testis prepared using representational difference analysis (RDA) method, a complimentary DNA clone encoding type III member of a DnaJ family protein, DnaJC18, was cloned (GenBank Accession No. DQ158861). The full-length DnaJC18 cDNA has the longest open reading frame of 357 amino acids. Tissue and developmental Northern blot analysis revealed that the DnaJC18 gene was expressed specifically in testis and began to express from postnatal week 4 testis, respectively. In situ hybridization studies showed that DnaJC18 mRNA was expressed only during the maturation stages of late pachy- tene, round and elongated spermatids of adult rat testis. Western blot analysis with DnaJC18 antibody revealed that 41.2 kDa DnaJC18 protein was detected only in adult testis. Immunohistochemistry study further confirmed that DnaJC18 protein, was expressed in developing germ cells and the result was in concert with the in situ hybridization result. Confocal microscopy with GFP tagged DnaJC18 protein revealed that it was localized in the cytoplasm of cells. Taken together, these results suggested that testis specific DnaJC18, a member of the type III DnaJ protein family, might play a role during germ cell maturation in adult rat testis.

Next-generation sequencing of representational difference analysis products for identification of genes involved in diosgenin biosynthesis in fenugreek (Trigonella foenum-graecum)

Main conclusionRepresentational difference analysis of cDNA was performed and differential products were sequenced and annotated. Candidate genes involved in biosynthesis of diosgenin in fenugreek were identified. Detailed mechanism of diosgenin synthesis was proposed.Fenugreek (Trigonella foenum-graecum L.) is a valuable medicinal and crop plant. It belongs to Fabaceae family and has a unique potential to synthesize valuable steroidal saponins, e.g., diosgenin. Elicitation (methyl jasmonate) and precursor feeding (cholesterol and squalene) were used to enhance the content of sterols and steroidal sapogenins in in vitro grown plants for representational difference analysis of cDNA (cDNA-RDA). To identify candidate genes involved in diosgenin biosynthesis, differential, factor-specific libraries were subject to the next-generation sequencing. Approximately 9.9 million reads were obtained, trimmed, and assembled into 31,491 unigenes with an average length of 291 bp. Then, functional annotation and gene ontogeny enrichment analysis was performed by aligning all-unigenes with public databases. Within the transcripts related to sterol and steroidal saponin biosynthesis, we discovered novel candidate genes of diosgenin biosynthesis and validated their expression using quantitative RT-PCR analysis. Based on these findings, we supported the idea that diosgenin is biosynthesized from cycloartenol via cholesterol. This is the first report on the next-generation sequencing of cDNA-RDA products. Analysis of the transcriptomes enriched in low copy sequences contributed substantially to our understanding of the biochemical pathways of steroid synthesis in fenugreek.

Determining Phylogenetic Relationships Among Date Palm Cultivars Using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) Markers.

Investigation of genetic variation and phylogenetic relationships among date palm (Phoenix dactylifera L.) cultivars is useful for their conservation and genetic improvement. Various molecular markers such as restriction fragment length polymorphisms (RFLPs), simple sequence repeat (SSR), representational difference analysis (RDA), and amplified fragment length polymorphism (AFLP) have been developed to molecularly characterize date palm cultivars. PCR-based markers random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) are powerful tools to determine the relatedness of date palm cultivars that are difficult to distinguish morphologically. In this chapter, the principles, materials, and methods of RAPD and ISSR techniques are presented. Analysis of data generated from these two techniques and the use of these data to reveal phylogenetic relationships among date palm cultivars are also discussed.