CITRIC: cold-inducible translational readthrough in the chloroplast of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> using a novel temperature-sensitive transfer RNA

Background

The chloroplast of eukaryotic microalgae such as Chlamydomonas reinhardtii is a potential platform for metabolic engineering and the production of recombinant proteins. In industrial biotechnology, inducible expression is often used so that the translation or function of the heterologous protein does not interfere with biomass accumulation during the growth stage. However, the existing systems used in bacterial or fungal platforms do not transfer well to the microalgal chloroplast. We sought to develop a simple inducible expression system for the microalgal chloroplast, exploiting an unused stop codon (TGA) in the plastid genome. We have previously shown that this codon can be translated as tryptophan when we introduce into the chloroplast genome a trnW_UCA gene encoding a plastidial transfer RNA with a modified anticodon sequence, UCA.

Results

A mutated version of our trnW_UCA gene was developed that encodes a temperature-sensitive variant of the tRNA. This allows transgenes that have been modified to contain one or more internal TGA codons to be translated differentially according to the culture temperature, with a gradient of recombinant protein accumulation from 35 °C (low/off) to 15 °C (high). We have named this the CITRIC system, an acronym for cold-inducible translational readthrough in chloroplasts. The exact induction behaviour can be tailored by altering the number of TGA codons within the transgene.

Conclusions

CITRIC adds to the suite of genetic engineering tools available for the microalgal chloroplast, allowing a greater degree of control over the timing of heterologous protein expression. It could also be used as a heat-repressible system for studying the function of essential native genes in the chloroplast. The genetic components of CITRIC are entirely plastid-based, so no engineering of the nuclear genome is required.

     

Genetic structure and genetic diversity of the endangered grassland plant <Emphasis Type="Italic">Crepis mollis</Emphasis> (Jacq.) Asch. as a basis for conservation management in Germany

Plant diversity is decreasing mainly through anthropogenic factors like habitat fragmentation, which lead to spatial separation of remaining populations and thereby affect genetic diversity and structure within species. Twenty populations of the threatened grassland species Crepis mollis were studied across Germany (578 individual plants) based on microsatellite genotyping. Genetic diversity was significantly higher in populations from the Alpine region than from the Central Uplands. Furthermore, genetic diversity was significantly positively correlated with population size. Despite smaller populations in the Uplands there were no signs of inbreeding. Genetic differentiation between populations was moderate (F _ST?=?0.09) and no isolation by distance was found. In contrast, large-scale spatial genetic structure showed a significant decrease of individual pairwise relatedness, which was higher than in random pairs up to 50 km. Bayesian analyses detected three genetic clusters consistent with two regions in the Uplands and an admixture group in the Alpine region. Despite the obvious spatial isolation of the currently known populations, the absence of significant isolation by distance combined together with moderate population differentiation indicates that drift rather than inter-population gene flow drives differentiation. The absence of inbreeding suggests that pollination is still effective, while seed dispersal by wind is likely to be impaired by discontinuous habitats. Our results underline the need for maintaining or improving habitat quality as the most important short term measure for C. mollis. For maintaining long-term viability, establishing stepping stone habitats or, where this is not possible, assisted gene flow needs to be considered.     

Reproductive biology and management of two commercially important groupers in the SW Atlantic

The reproductive biology of Epinephelus morio (red grouper) and Mycteroperca bonaci (black grouper) were evaluated based on 533 specimens collected from artisanal fisheries landings in the Abrolhos Bank, Brazil, between May 2005 and September 2012. Sex ratio for the black grouper was 1:14 (n = 155 females and 11 males; 26.1–147 cm TL) and 1:10 for the red grouper (n = 334 females and 33 males; 15.0–96.0 cm TL). For both species, highest values of the gonadosomatic index (GSI) for females were recorded between July and October, indicating spawning during the austral winter. The length at first maturity (L₅₀) for females was estimated at 62.0 and 47.0 cm TL for the black and red grouper, respectively. Batch fecundity based on TL and TW ranged from 2 to 15.4^?10⁶ and 1.5 to 13.7^?10⁶ for the black and red grouper, respectively. Interviews with experienced fishers revealed that spawning seasons of both groupers are largely unrecognized. Results demonstrate a positive relationship between GSI peaks, lower temperatures and stronger winds. The information provided herein may help decision-making regarding fisheries management and conservation for E. morio and M. bonaci at various levels of governance in the Abrolhos Bank, the region with the largest and richest coralline reefs in the South Atlantic.     

Characterization of a sterile dwarf mutant and the cloning of zeaxanthin epoxidase in Asian cotton (<Emphasis Type="Italic">Gossypium arboreum</Emphasis> L.)

Amino acids are constituents of proteins, precursors of many secondary metabolites and nitrogen carriers in plants. Transport across intracellular membranes and translocation of amino acids within the plant is mediated by membrane amino acid transporters. However, the amino acid transport in tea plant is rarely reported. In this study, six cationic amino acid transporter (CAT) family genes were cloned. Phylogenetic analysis categorized these CsCATs into four subgroups. These CsCATs all contain the 12–14 transmembrane domains and the conserved CAT motifs. Their expression was tissue-specific, with higher expression levels in root and stem and correlated to the abundances of key free amino acids such as Theanine. Some CsCATs expression responded to some abiotic stress conditions and to the exogenous application of theanine (Thea), glutamine or ethylamine hydrochloride, an ethylamine precursor for Thea biosynthesis. Our results indicated that the CsCATs expression is regulated by amino acid contents and is sensitive to abiotic stresses. These findings shed light on the mechanism of amino acid transport in tea plants.     

Male-Driven Differences in Chimpanzee (<Emphasis Type=Italic>Pan troglodytes</Emphasis>) Population Genetic Structure Across Three Habitats in Cameroon and Nigeria

Complex ecological pressures affect the social dynamics of many primate species, but it is unclear how they affect primate speciation. Molecular tools are often used to answer questions about the evolutionary histories and social systems of primates. Mitochondrial DNA (mtDNA), in particular, is frequently used to answer many of these questions, but because it is passed from mothers to offspring it reveals only the histories of females. In many species, including chimpanzees, females generally disperse from their natal groups while males are philopatric, and thus differences in dispersal patterns likely leave different signatures in the genome. We previously analyzed samples from 187 unrelated male and female chimpanzees in Nigeria and Cameroon using 21 autosomal microsatellites and mtDNA sequences. Here, we examine the contributions of males and females in shaping the genetic history of these chimpanzees by genotyping a subset of 56 males at 12 Y-chromosome microsatellites. We found that Y-chromosome population structure differed from the results of analysis of mtDNA haplotypes. The results also revealed that males in rainforest habitats (Guinean and Congolian rainforests) are more closely related to one another than those inhabiting the savanna-woodland mosaic ecotone in central Cameroon. In contrast, the pattern of female relatedness did not differ across habitats. We hypothesize that these differences in population structure and patterns of relatedness among males in different habitat types may be due to differences in the community dynamics of chimpanzees in the ecotone vs. rainforests, and that these factors contribute to making Cameroon an engine of diversification for chimpanzees. Broadly, these results demonstrate the importance of habitat variation in shaping social systems, population genetics, and primate speciation.     

An Experimental Test of Trade-Offs Associated with the Adaptation to Alternate Host Plants in the Introduced Herbivorous Beetle, <Emphasis Type="Italic">Ophraella communa</Emphasis>

The adaptation to alternate host plants of introduced herbivorous insects can be vital to agriculture due to the emergence of crop pests. Historically, it is assumed that there are trade-offs associated with the adaptation to new host plants; a generalist genotype that adapts to an alternate host is expected to have a relatively lower fitness on the ancestral host than a specialist genotype (physiological cost) or a relatively lower host-searching ability for the ancestral host plant (behavioral cost). In this study, we tested the costs of adaptation to a new host plant in the introduced herbivorous insect, Ophraella communa LeSage (Coleoptera: Chrysomelidae). In its native range (United States), O. communa feeds mostly on Ambrosia artemisiifolia L. (Asterales: Asteraceae) and cannot utilize the related species, Ambrosia trifida L. (Asterales: Asteraceae), as a host plant. On the other hand, the introduced O. communa population in Japan utilizes A. trifida extensively, and is adapting to it, both physiologically and behaviorally. We compared larval performance on the ancestral and alternate plants and adult host-searching ability between the native and introduced beetle populations. The introduced O. communa showed higher larval survival and adult feeding preference for the alternate host plant A. trifida than did the native O. communa, indicating that the introduced O. communa has rapidly adapted to the alternate host plant. However, there are no differences in either larval performance on the ancestral host A. artemisiifolia or host-searching accuracy between the native and introduced O. communa.     

Male Horn Dimorphism and its Function in the Neotropical Dung Beetle <Emphasis Type="Italic">Sulcophanaeus velutinus</Emphasis>

Horned beetles are emerging models in the study of coevolution between novel morphologies and behavior. In Onthophagus beetles, large males use horns to fight other males in brood tunnels while small males with higher mobility sneak around the large males to gain access to females. Mating tactics have rarely been described in other dung beetle genera. We studied the horned dung beetle Sulcophanaeus velutinus that exhibits two parallel horns on the prothorax and one on the head. We put two males of different horn lengths, but similar mass, in observation chambers and found that the large male with longer horns won access to the female in physical competition. Speed tests in artificial tunnels show that locomotion is impeded in large males, suggesting an advantage in mobility for males with small horns. This work contributes to the limited existing evidence on the function of alternative morphologies in horned dung beetles taxa.     

A Peptide Based Pro-drug Disrupts Alzheimer’s Amyloid into Non-toxic Species and Reduces Aβ Induced Toxicity In Vitro

Aggregation of Amyloid β (Aβ) in the interneuronal spaces is a major etiopathological factor for onset and progression of Alzheimer’s disease (AD). Since the mechanism of aggregation is not fully understood, control and modulation of the aggregation process is a challenging task. Although, several strategies were developed for the past few decades, yet there is no proper therapeutics available. Herein, we report a peptide based pro-drug, termed as a conformational Pro-Drug peptide (PDp), which disrupts existing Aβ fibrils, but does not produce toxic soluble oligomers, through a series of spontaneous chemical reactions resulting in in situ generation of β-sheet destabilizing factors. Furthermore, PDp reduces Aβ mediated toxicity examined on an in vitro model consisting of the human neuroblastoma SH-SY5Y cells. PDp also disrupts fibrils originated from AD affected human cerebrospinal fluid. These findings will help to understand the process of amyloidogenesis better and also indicate a novel approach for therapeutically important peptide design.     

Hepatocyte nuclear factors as possible C-reactive protein transcriptional inducer in the liver and white adipose tissue of rats with experimental chronic renal failure

Little is known about the effects of coffee that are not related to the presence of caffeine. The aim of the study was to analyse changes in kidney function and nucleotide metabolism related to high intake of decaffeinated coffee. Mice consumed decaffeinated coffee extract for two weeks. Activities of AMP deaminase, ecto5′-nucleotidase, adenosine deaminase, purine nucleoside phosphorylase were measured in kidney cortex and medulla by analysis of conversion of substrates into products using HPLC. Concentration of nucleotides in kidney cortex, kidney medulla and serum were estimated by HPLC. Activity of ecto5′-nucleotidase increased from 0.032 ± 0.006 to 0.049 ± 0.014 nmol/mg tissue/min in kidney cortex of mice administered high-dose decaffeinated coffee (HDC) together with increase in cortex adenosine concentration and decrease in plasma creatinine concentration. HDC leads to increased activity of ecto5′-nucleotidase in kidney cortex that translates to increase in concentration of adenosine. Surprisingly this caused improved kidney excretion function.     

The Expression Alteration of BC1 RNA and its Interaction with Eukaryotic Translation Initiation Factor eIF4A Post-Status Epilepticus

Abnormal dendritic sprouting and synaptic remodelling are important pathological features of temporal lobe epilepsy. BC1 RNA is a translation repressor involved in the regulation of the dendritic protein synthesis and mRNA transport, which is essential for dendritic development and plasticity. The expression alteration of BC1 RNA in the pilocarpine induced epilepsy model remains unknown. It is unclear if the interactions between BC1 RNA and eukaryotic initiation factor 4A (eIF4A) exists in this model. The purpose of this study was to investigate the expression changes of BC1 RNA and its interactions with eIF4A post-status epilepticus (SE). Chloride lithium and pilocarpine were used to induce the SE rat model. Either a whole brain or hippocampus tissues were collected at different time points after SE. The expression patterns of BC1 was detected by qPCR and in situ hybridization. The levels of eIF4AI/II protein expression were analyzed via western blotting and immunohistochemistry. The BC1 RNA-eIF4AI/II interaction was determined by electrophoretic mobility shift assay (EMSA). We found that the BC1 RNA levels decreased in hippocampus 3d, 1w and 2w post-SE before the levels recovered. The eIF4AI/II began to rise 3d post-SE and reached the maximum level 1w post-SE. After 1w post-SE the levels decreased in the hippocampal CA1, CA3 and DG subregions. EMSA analysis showed that BC1 RNA specifically interacted with the eIF4AI/II. The BC1 RNA-eIF4AI/II complex reduced to the lowest level 1w post-SE. Our results suggested that BC1 has a negative regulatory correlation with eIF4AI/II, where BC1 RNA could be involved in epileptogenesis by regulating dendritic protein synthesis.