Intracellular trehalose improves osmotolerance but not desiccation tolerance in mammalian cells

AbstractTrehalose has been shown to play a role in osmotolerance or desiccation tolerance in some microorganisms, anhydrobiotic invertebrates and resurrection plants. To test whether trehalose could improve stress responses of higher eukaryotes, a mouse cell line was genetically engineered to express bacterial trehalose synthase genes. We report that the resulting levels of intracellular trehalose (∼80 mM) are able to confer increased resistance to the partial dehydration resulting from hypertonic stress, but do not enable survival of complete desiccation due to air drying

Plant desiccation gene found in a nematode

Included in tex

The majority of human CD3 epitopes are conferred by the epsilon chain

Transgenic mouse T cells expressing the human CD3ε chain bind the majority (29/36) of monoclonal antibodies (mAbs) specific for human CD3. A proportion of these mAbs are also able to recognize isolated CD3ε in a soluble, recombinant form. Thus, CD3ε can confer most CD3 epitopes on the TCR-CD3 complex, but many determinants may require assembly of the complex for their formation. A number ot mAbs did not recognize ε-transgenic T cells and probably need other CD3 subunits for binding. CD3-specific mAbs from each of the three groups defined here, as well as mAbs directed against the TCRαβ heterodimer, are all able to activate T cells. Therefore mAb attachment at several different sites on the TCR-CD3 complex can give rise to activation signals. This suggests that the cross-linking function of mitogenic antibodies may be their most significant property, rather than the perturbation of a particular ‘functional epitope

Gene expression dynamics underlying diversification and expansion of an actin gene family

During the evolution of gene families, functional diversification of proteins often follows gene duplication. However, some gene families expand while preserving protein sequence. Why would a cell need to maintain multiple copies of the same gene? In this thesis I have addressed this question for an actin gene family containing 17 genes encoding an identical protein in the social amoeba Dictyostelium discoideum. Using bioinformatics I identified several highly conserved sequence elements as potential regulatory motifs, yet found that gene expression patterns during development are broadly similar across the gene family. Turning to live cell imaging I showed that family members display different transcription dynamics, with strong 'bursty' behaviours contrasted by more steady, continuous transcriptional activity. By switching promoters I showed that different dynamics are directly determined by endogenous promoter sequences, rather than genomic context. I have explored how cell-to-cell variability in gene expression introduced by bursty transcription propagates to resultant cytoplasmic mRNA and protein and showed that population variance of these molecules is reduced compared to nascent transcription. Finally, I generated cell lines with up to 6 genes knocked out and showed that these cells potentially display a minor defect in growth. Overall these data suggest that expanded gene families are utilised not only to generate sufficient protein for normal cell physiology, but also to enable both robustness and responsiveness to a range of stimuli regulating the expression of essential genes

Expression of multiple horizontally acquired genes is a hallmark of both vertebrate and invertebrate genomes.

BACKGROUND: A fundamental concept in biology is that heritable material, DNA, is passed from parent to offspring, a process called vertical gene transfer. An alternative mechanism of gene acquisition is through horizontal gene transfer (HGT), which involves movement of genetic material between different species. HGT is well-known in single-celled organisms such as bacteria, but its existence in higher organisms, including animals, is less well established, and is controversial in humans. RESULTS: We have taken advantage of the recent availability of a sufficient number of high-quality genomes and associated transcriptomes to carry out a detailed examination of HGT in 26 animal species (10 primates, 12 flies and four nematodes) and a simplified analysis in a further 14 vertebrates. Genome-wide comparative and phylogenetic analyses show that HGT in animals typically gives rise to tens or hundreds of active 'foreign' genes, largely concerned with metabolism. Our analyses suggest that while fruit flies and nematodes have continued to acquire foreign genes throughout their evolution, humans and other primates have gained relatively few since their common ancestor. We also resolve the controversy surrounding previous evidence of HGT in humans and provide at least 33 new examples of horizontally acquired genes. CONCLUSIONS: We argue that HGT has occurred, and continues to occur, on a previously unsuspected scale in metazoans and is likely to have contributed to biochemical diversification during animal evolution.This work was supported by the European Research Council (AdG233232).This is the final published version. It first appeared at http://genomebiology.com/2015/16/1/50

Determination of Water Content in Dehydrated Mammalian Cells Using Terahertz Pulsed Imaging: A Feasibility Study

This is the accepted version of the following article: Chau, D., Dennis, A. R., Lin, H., Zeitler, J. A., Tunnacliffe, A., ‘Determination of Water Content in Dehydrated Mammalian Cells Using Terahertz Pulsed Imaging: A Feasibility Study’, Current Pharmaceutical Biotechnology 17(2): 200-207, February 2016. Subject to 12 months' embargo. Embargo end date: 1 February 2017. The published manuscript is available at http://www.eurekaselect.com/136239/articleMammalian cells are involved in a range of biotechnological applications and more recently have been increasingly exploited in regenerative medicine. Critical to successful applications involving mammalian cells are their long-term storage and transport, for which cryopreservation in liquid nitrogen is the most frequently used strategy. However, cryopreservation suffers from high costs, difficulties in transport logistics and the use of undesirable additives (e.g. animal sera or DMSO). An alternative approach, proposed as low cost, low maintenance and process-compatible, is viable desiccation of mammalian cells. Several groups claim to have achieved this, but the extent of desiccation in the cell samples concerned is not always clear, in part because of difficulties in determining very low water content. Although several techniques exist that are frequently used to quantify the amount of water in samples (e.g. FTIR spectroscopy, thermogravimetric analysis (TGA), NMR spectroscopy), the complexity of sample preparation, as well as the costs and time constraints involved are disadvantageous. Here, we assess a novel, rapid and low cost technique, i.e. terahertz (THz) spectroscopy, for the quantification of water content within dehydrated mammalian cell samples.Peer reviewe

Transition from Natively Unfolded to Folded State Induced by Desiccation in an Anhydrobiotic Nematode Protein

Late embryogenesis abundant (LEA) proteins are associated with desiccation tolerance in resurrection plants and in plant seeds, and the recent discovery of a dehydration- induced Group 3 LEA-like gene in the nematode Aphelenchus avenae suggests a similar association in anhydrobiotic animals. Despite their importance, little is known about the structure of Group 3 LEA proteins, although computer modeling and secondary structure algorithms predict a largely -helical monomer that forms coiled coil oligomers. We have therefore investigated the structure of the nematode protein, AavLEA1, in the first such analysis of a well characterized Group 3 LEA-like protein. Immunoblotting and subunit cross-linking experiments demonstrate limited oligomerization of AavLEA1, but analytical ultracentrifugation and gel filtration show that the vast majority of the protein is monomeric. Moreover, CD, fluorescence emission, and Fourier transform-infrared spectroscopy indicate an unstructured conformation for the nematode protein. Therefore, in solution, no evidence was found to support structure predictions; instead, AavLEA1 seems to be natively unfolded with a high degree of hydration and low compactness. Such proteins can, however, be induced to fold into more rigid structures by partner molecules or by altered physiological conditions. Because AavLEA1 is associated with desiccation stress, its Fourier transform-infrared spectrum in the dehydrated state was examined. A dramatic but reversible increase in -helix and, possibly, coiled coil formation was observed on drying, indicating that computer predictions of secondary structure may be correct for the solid state. This unusual finding offers the possibility that structural shifts in Group 3 LEA proteins occur on dehydration, perhaps consistent with their role in anhydrobiosis

Preferential adsorption to air-water interfaces: a novel cryoprotective mechanism for LEA proteins

Late embryogenesis abundant (LEA) proteins comprise a diverse family whose members play a key role in abiotic stress tolerance. As intrinsically disordered proteins, LEA proteins are highly hydrophilic and inherently stress tolerant. They have been shown to stabilize multiple client proteins under a variety of stresses, but current hypotheses do not fully explain how such broad range stabilization is achieved. Here, using neutron reflection and surface tension experiments, we examine in detail the mechanism by which model LEA proteins, AavLEA1 and ERD10, protect the enzyme citrate synthase from aggregation during freeze-thaw. We find that a major contributing factor to citrate synthase aggregation is the formation of air bubbles during the freeze-thaw process. This greatly increases the air-water interfacial area, which is known to be detrimental to folded protein stability. Both model LEA proteins preferentially adsorb to this interface and compete with citrate synthase, thereby reducing surface induced aggregation. This novel surface activity provides a general mechanism by which diverse members of the LEA protein family might function to provide aggregation protection that is not specific to the client protein.Canadian Research Council for PhD studentship + ERC gran

Multiple functionally divergent and conserved copies of alpha tubulin in bdelloid rotifers.

BACKGROUND: Bdelloid rotifers are microscopic animals that have apparently survived without sex for millions of years and are able to survive desiccation at all life stages through a process called anhydrobiosis. Both of these characteristics are believed to have played a role in shaping several unusual features of bdelloid genomes discovered in recent years. Studies into the impact of asexuality and anhydrobiosis on bdelloid genomes have focused on understanding gene copy number. Here we investigate copy number and sequence divergence in alpha tubulin. Alpha tubulin is conserved and normally present in low copy numbers in animals, but multiplication of alpha tubulin copies has occurred in animals adapted to extreme environments, such as cold-adapted Antarctic fish. Using cloning and sequencing we compared alpha tubulin copy variation in four species of bdelloid rotifers and four species of monogonont rotifers, which are facultatively sexual and cannot survive desiccation as adults. Results were verified using transcriptome data from one bdelloid species, Adineta ricciae. RESULTS: In common with the typical pattern for animals, monogonont rotifers contain either one or two copies of alpha tubulin, but bdelloid species contain between 11 and 13 different copies, distributed across five classes. Approximately half of the copies form a highly conserved group that vary by only 1.1% amino acid pairwise divergence with each other and with the monogonont copies. The other copies have divergent amino acid sequences that evolved significantly faster between classes than within them, relative to synonymous changes, and vary in predicted biochemical properties. Copies of each class were expressed under the laboratory conditions used to construct the transcriptome. CONCLUSIONS: Our findings are consistent with recent evidence that bdelloids are degenerate tetraploids and that functional divergence of ancestral copies of genes has occurred, but show how further duplication events in the ancestor of bdelloids led to proliferation in both conserved and functionally divergent copies of this gene.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Comparative genomics of bdelloid rotifers: Insights from desiccating and nondesiccating species.

Bdelloid rotifers are a class of microscopic invertebrates that have existed for millions of years apparently without sex or meiosis. They inhabit a variety of temporary and permanent freshwater habitats globally, and many species are remarkably tolerant of desiccation. Bdelloids offer an opportunity to better understand the evolution of sex and recombination, but previous work has emphasised desiccation as the cause of several unusual genomic features in this group. Here, we present high-quality whole-genome sequences of 3 bdelloid species: Rotaria macrura and R. magnacalcarata, which are both desiccation intolerant, and Adineta ricciae, which is desiccation tolerant. In combination with the published assembly of A. vaga, which is also desiccation tolerant, we apply a comparative genomics approach to evaluate the potential effects of desiccation tolerance and asexuality on genome evolution in bdelloids. We find that ancestral tetraploidy is conserved among all 4 bdelloid species, but homologous divergence in obligately aquatic Rotaria genomes is unexpectedly low. This finding is contrary to current models regarding the role of desiccation in shaping bdelloid genomes. In addition, we find that homologous regions in A. ricciae are largely collinear and do not form palindromic repeats as observed in the published A. vaga assembly. Consequently, several features interpreted as genomic evidence for long-term ameiotic evolution are not general to all bdelloid species, even within the same genus. Finally, we substantiate previous findings of high levels of horizontally transferred nonmetazoan genes in both desiccating and nondesiccating bdelloid species and show that this unusual feature is not shared by other animal phyla, even those with desiccation-tolerant representatives. These comparisons call into question the proposed role of desiccation in mediating horizontal genetic transfer