Downregulation of Barley Regulator of Telomere Elongation Helicase 1 Alters the Distribution of Meiotic Crossovers.

Programmed meiotic DNA double-strand breaks (DSBs), necessary for proper chromosomal segregation and viable gamete formation, are repaired by homologous recombination (HR) as crossovers (COs) or non-crossovers (NCOs). The mechanisms regulating the number and distribution of COs are still poorly understood. The regulator of telomere elongation helicase 1 (RTEL1) DNA helicase was previously shown to enforce the number of meiotic COs in Caenorhabditis elegans but its function in plants has been studied only in the vegetative phase. Here, we characterised barley RTEL1 gene structure and expression using RNA-seq data previously obtained from vegetative and reproductive organs and tissues. Using RNAi, we downregulated RTEL1 expression specifically in reproductive tissues and analysed its impact on recombination using a barley 50k iSelect SNP Array. Unlike in C. elegans, in a population segregating for RTEL1 downregulated by RNAi, high resolution genome-wide genetic analysis revealed a significant increase of COs at distal chromosomal regions of barley without a change in their total number. Our data reveal the important role of RTEL1 helicase in plant meiosis and control of recombination.

Potato miR828 Is Associated With Purple Tuber Skin and Flesh Color.

Anthocyanins are plant pigments responsible for the colors of many flowers, fruits and storage organs and have roles in abiotic and biotic stress resistance. Anthocyanins and polyphenols are bioactive compounds in plants including potato (Solanum tuberosum L.) which is the most important non-cereal crop in the world, cultivated for its tubers rich in starch and nutrients. The genetic regulation of the flavonoid biosynthetic pathway is relatively well known leading to the formation of anthocyanins. However, our knowledge of post-transcriptional regulation of anthocyanin biosynthesis is limited. There is increasing evidence that micro RNAs (miRNAs) and other small RNAs can regulate the expression level of key factors in anthocyanin production. In this study we have found strong associations between the high levels of miR828, TAS4 D4(-) and purple/red color of tuber skin and flesh. This was confirmed not only in different cultivars but in pigmented and non-pigmented sectors of the same tuber. Phytochemical analyses verified the levels of anthocyanins and polyphenols in different tissues. We showed that miR828 is able to direct cleavage of the RNA originating from Trans-acting siRNA gene 4 (TAS4) and initiate the production of phased small interfering RNAs (siRNAs) whose production depends on RNA-dependent RNA polymerase 6 (RDR6). MYB transcription factors were predicted as potential targets of miR828 and TAS4 D4(-) and their expression was characterized. MYB12 and R2R3-MYB genes showed decreased expression levels in purple skin and flesh in contrast with high levels of small RNAs in the same tissues. Moreover, we confirmed that R2R3-MYB and MYB-36284 are direct targets of the small RNAs. Overall, this study sheds light on the small RNA directed anthocyanin regulation in potato, which is an important member of the Solanaceae family.

Arabidopsis PTB1 and PTB2 proteins negatively regulate splicing of a mini-exon splicing reporter and affect alternative splicing of endogenous genes differentially.

This paper examines the function of Arabidopsis thaliana AtPTB1 and AtPTB2 as plant splicing factors. The effect on splicing of overexpression of AtPTB1 and AtPTB2 was analysed in an in vivo protoplast transient expression system with a novel mini-exon splicing reporter. A range of mutations in pyrimidine-rich sequences were compared with and without AtPTB and NpU2AF65 overexpression. Splicing analyses of constructs in protoplasts and RNA from overexpression lines used high-resolution reverse transcription polymerase chain reaction (RT-PCR). AtPTB1 and AtPTB2 reduced inclusion/splicing of the potato invertase mini-exon splicing reporter, indicating that these proteins can repress plant intron splicing. Mutation of the polypyrimidine tract and closely associated Cytosine and Uracil-rich (CU-rich) sequences, upstream of the mini-exon, altered repression by AtPTB1 and AtPTB2. Coexpression of a plant orthologue of U2AF65 alleviated the splicing repression of AtPTB1. Mutation of a second CU-rich upstream of the mini-exon 3' splice site led to a decline in mini-exon splicing, indicating the presence of a splicing enhancer sequence. Finally, RT-PCR of AtPTB overexpression lines with c. 90 known alternative splicing (AS) events showed that AtPTBs significantly altered AS of over half the events. AtPTB1 and AtPTB2 are splicing factors that influence alternative splicing. This occurs in the potato invertase mini-exon via the polypyrimidine tract and associated pyrimidine-rich sequence.

Alternative splicing in plants.

The impact of AS (alternative splicing) is well-recognized in animal systems as a key regulator of gene expression and proteome complexity. In plants, AS is of growing importance as more genes are found to undergo AS, but relatively little is known about the factors regulating AS or the consequences of AS on mRNA levels and protein function. We have established an accurate and reproducible RT (reverse transcription)-PCR system to analyse AS in multiple genes. Initial studies have identified new AS events confirming that current values for the frequency of AS in plants are likely to be underestimates.

E-2-ethylhexenal, E-2-ethyl-2-hexenol, mellein, and 4-hydroxymellein in Camponotus species from Brunei.

E-2-ethyl-2-hexen-1-ol (1), mellein (4), and 4-hydroxymellein (5) were identified as the major volatile compounds in the head and/or thorax of Camponotus quadrisectus. Neither 1 nor 5 have been previously detected in insects. Also identified were small amounts of m-cresol (2) and 6-methyl salicylic acid (3). E-2-ethylhexenal (6) and small amounts of 3 were identified in heads of Camponotus irritibilis from Kuala Belalong, Brunei. Compounds 2-4 occur in other Bornean camponotines with hypertrophied mandibular glands, and 4 is widespread in the tribe. The possibility of semiochemical parsimony (multiple functions) for these mandibular gland compounds is reviewed in the context of existing data on mandibular gland products of other camponotines, reported biological activities of the compounds, and secondary loss of metapleural glands in this ant group.

Monitoring changes in alternative precursor messenger RNA splicing in multiple gene transcripts.

Alternative splicing (AS) increases the proteomic and functional capacity of genomes through the generation of alternative mRNA transcripts from the same gene. AS is now estimated to occur in a third of Arabidopsis and rice genes, and includes genes involved in the control of growth and development, responses to stress and signalling. Regulation of AS reflects the interactions between positive and negative cis sequences in the precursor messenger RNA and a range of trans-acting factors. The levels and activities of these factors differ in different cells and growth conditions. To identify changes in AS in multiple genes simultaneously, we have established a reproducible RT-PCR panel that can analyse 96 alternative splicing events and accurately measure the ratio of alternatively spliced products. This procedure detected statistically significant changes in AS in different plant organs, in plants grown under different light and day-length conditions, and in plants overexpressing splicing factors. The system provides a convenient, medium-throughput means of monitoring changes in AS in multiple genes. It can readily be applied to much larger or targeted sets of gene transcripts to generate information on the significance and regulation of AS in plant growth and development, specific processes and responses to external stimuli.

Venom chemistry of the ant Myrmicaria melanogaster from Brunei.

Analysis of the extracts of the ant Myrmicaria melanogaster from Brunei in the Indonesian archipelago by GC-MS and GC-IR revealed the presence of five new alkaloids, identified as (9Z)-3-propylindolizidine (1), cis- and trans-2-butyl-5-propylpyrrolidine (2 and 3, respectively), (10E)-3-butyllehmizidine (7), and (5Z,8Z,9Z)-3-butyl-5-propyl-8-hydroxyindolizidine (10a), whose structures were established by comparison with synthetic samples. In addition the monoterpene hydrocarbons beta-pinene, myrcene, and limonene were detected along with all four isomers of 3-butyl-5-methylindolizidine (4a-d), cis- and trans-2-butyl-5-(4-pentenyl)pyrrolidine (5a and 5b), trans-2-butyl-5-pentylpyrrolidine (6), (5Z,9Z)-3-butyl-5-propylindolizidine (8), and (5Z,9E)-3-butyl-5-propylindolizidine (9), alkaloids well known from ants and frogs, whose structures were established on the basis of published spectra or comparison with authentic samples. This study utilized vapor-phase infrared analysis for the assignment of stereochemistry using Bohlmann bands for the bicyclic alkaloids and, in the case of 10a, the detection of an intramolecular hydrogen bond. A biogenetic relationship between the mono- and bicyclic ring systems is proposed.

Identification of novel small molecule inhibitors of amyloid precursor protein synthesis as a route to lower Alzheimer's disease amyloid-beta peptide.

A wealth of independent research with transgenic mice, antibodies, and vaccines has pointed to a causative role of the amyloid-beta peptide (A beta) in Alzheimer's disease (AD). Based on these and earlier associative studies, A beta represents a promising target for development of therapeutics focused on AD disease progression. Interestingly, a cholinesterase inhibitor currently in clinical trials, phenserine, has been shown to inhibit production of both amyloid precursor protein (APP) and A beta. We have shown that this inhibition occurs at the post-transcriptional level with a specific blocking of the synthesis of APP relative to total protein synthesis (Shaw et al., 2001). However, the dose of phenserine necessary to block APP production is far higher than that needed to elicit its anticholinesterase activity, and it is these latter actions that are dose limiting in vivo. The focus of this study was to screen 144 analogs of phenserine to identify additional small molecules that inhibit APP protein synthesis, and thereby A beta production, without possessing potent acetylcholinesterase (AChE) inhibitory activity. An enzyme-linked immunosorbent assay was used to identify analogs capable of suppressing APP production following treatment of human neuroblastoma cells with 20 muM of compound. Eight analogs were capable of dose dependently reducing APP and A beta production without causing cell toxicity in further studies. Several of these analogs had little to no AChE activities. Translation of APP and A beta actions to mice was demonstrated with one agent. They thus represent interesting lead molecules for assessment in animal models, to define their tolerance and utility as potential AD therapeutics.

Human antibodies for immunotherapy development generated via a human B cell hybridoma technology.

Current strategies for the production of therapeutic mAbs include the use of mammalian cell systems to recombinantly produce Abs derived from mice bearing human Ig transgenes, humanization of rodent Abs, or phage libraries. Generation of hybridomas secreting human mAbs has been previously reported; however, this approach has not been fully exploited for immunotherapy development. We previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g., affinity and titers) of mAb-producing cell lines, including hybridomas. We reasoned that this process, named morphogenics, could be used to improve suboptimal hybridoma cells generated by means of ex vivo immunization and immortalization of antigen-specific human B cells for therapeutic Ab development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human mAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting mAbs with high binding specificity and biological activity. One mAb with strong neutralizing activity against human granulocyte-macrophage colony-stimulating factor was identified that is now considered for preclinical development for autoimmune disease indications. Moreover, these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large-scale manufacturing.

Ecological stoichiometry of ants in a New World rain forest.

C:N stoichiometry was investigated in relation to diet (delta(15)N), N-deprivation, and worker body size for a diverse assemblage of tropical Amazonian ants. Relative nitrogen (N) deprivation was assayed for 54 species as an exchange ratio (ER), defined as SUCmin/AAmin, or the minimum sucrose concentration, divided by the minimum amino acid concentration, accepted as food by >/=50% of tested workers. On average, N-deprivation (ER) was almost fivefold greater for N-omnivorous and N-herbivorous (N-OH) taxa than for N-carnivores. In two-way ANOVAs at three taxonomic levels (species and species groups, genera, and tribes), higher ER was associated with small body size and (marginally) with less carnivorous diets. ERs did not differ systematically between trophobiont-tending and "leaf-foraging" functional groups, but specialized wound-feeders and coccid-tenders were prominent among high ER taxa. Paradoxically, some high ER taxa were among the most predatory members of their genera or subfamilies. Biomass % N was lower in N-OH taxa than in carnivores and varied inversely with N-deprivation (log ER) in the former taxa only. In an expanded data set, N-content increased allometrically in N-OHs, N-carnivores, and all ants combined, and with carnivory in large-bodied ants only. Exceptional taxa included small-bodied and predaceous Wasmannia, with high % N despite high ER, and Linepithema, with the lowest % N despite high delta(15)N. Patterns in C:N stoichiometry are explained largely at the genus level and above by elemental composition of alarm/defensive/offensive chemical weaponry and, perhaps in some cases, by reduced N investment in cuticle in taxa with high surface:volume ratios. Several consequences of C:N stoichiometry identify Azteca, and possibly Crematogaster, as taxa preadapted for their roles as prominent associates of myrmecophytes. C:N stoichiometry of ants should be incorporated into models of strategic colony design and examined in a phylogenetic context as opportunities permit.

Liquid-feeding performances of ants (Formicidae): ecological and evolutionary implications.

Disparities in liquid-feeding performances of major ant taxa have likely been important to resource partitioning among ants, to the nature and composition of ant partnerships with plants and sap-feeding trophobionts, and to ecological and evolutionary diversification of ant taxa. We measured performance volumetrically for individual workers of 77 ant species from lowland rain forests of Amazonia and Borneo and three key North American taxa. In trials with 9% sucrose solution, performances were strongly related to body size (and alitrunk length) and to proventricular structure at generic to subfamilial levels. Highly modified proventriculi were associated with disproportionately large load sizes in Formicinae and certain small-bodied Dolichoderinae. These same taxa also ingested liquids more rapidly during foraging than did similar-sized species with plesiomorphic proventriculi. Secondarily reduced foraging performances of several formicines likely reflect ecological or evolutionary trade-offs related to dietary specialization or anti-predator defenses. Across formicines and dolichoderines, performances differed by functional group. Relatively small loads and slow uptake characterized species tending trophobionts (mainly Hemiptera) day and night in large worker aggregations. Large loads and rapid uptake typified solitary, diurnal "leaf-foragers." Intermediate feeding performances characterized a few species that both tended trophobionts in small aggregates and frequently foraged alone.

Explaining the abundance of ants in lowland tropical rainforest canopies.

The extraordinary abundance of ants in tropical rainforest canopies has led to speculation that numerous arboreal ant taxa feed principally as "herbivores" of plant and insect exudates. Based on nitrogen (N) isotope ratios of plants, known herbivores, arthropod predators, and ants from Amazonia and Borneo, we find that many arboreal ant species obtain little N through predation and scavenging. Microsymbionts of ants and their hemipteran trophobionts might play key roles in the nutrition of taxa specializing on N-poor exudates. For plants, the combined costs of biotic defenses and herbivory by ants and tended Hemiptera are substantial, and forest losses to insect herbivores vastly exceed current estimates.

Myrmecophytic Cecropia: antiherbivore defenses under different nutrient treatments.

In greenhouse experiments, we compared putative biotic, chemical, physical and phenological defenses of six myrmecophytic Cecropia species cultivated under high and low nutrient regimes. We tested the intraspecific predictions of the C:N balance hypothesis for a broader range of defenses than included in other studies to date. Treatment effects on defenses appear to depend on the nutrient constituents of those defenses. Only strictly carbon-based defenses such as tannins and phenolics reached higher concentrations at the lower nutrient level. The production of glycogen-rich and membrane-bound Müllerian body ant rewards (MBs) increased with greater levels of both nutrients (this study) and light (Folgarait and Davidson 1994). In contrast, lipid- and amino acid-rich pearl body food rewards (PBs) were produced in greater numbers under conditions of high nutrient levels (this study) and low light (Folgarait and Davidson 1994), both of which should have contributed to a relative excess of nitrogen. Nutrient effects on toughness and leaf expansion rates (perhaps serving as phenological defenses) were inconsistent with the predictions of the C:N balance hypothesis. Mature leaves are protected principally by chemical and physical defenses, and new leaves, by biotic defenses. As in a previous study, interspecific comparisons agreed with the resource availability theory of plant defense. Plant investment in immobile defenses (tannins and phenolics, and leaf toughness), and in a defense with high initial construction costs (trichilia differentiated to produce MBs) were greater in each of three comparatively slow-growing "gap" Cecropia typical of small openings in primary forest, than in closely related and fast-growing "pioneer" species of large-scale disturbances (riparian edge and land slips). In contrast, both production of PBs (with negligible initial construction costs) and leaf expansion rates were greater in pioneers than in gap species. Rapid onset of biotic defenses during new leaf development (earlier in pioneers) may reduce new leaf herbivory in all species.