GC content of early metazoan genes and its impact on gene expression levels in mammalian cell lines

With the genomes available for many animal clades, including the early-branching metazoans, one can readily study the functional conservation of genes across a diversity of animal lineages. Ectopic expression of an animal protein in, for instance, a mammalian cell line is a generally used strategy in structure–function analysis. However, this might turn out to be problematic in case of distantly related species. Here we analyzed the GC content of the coding sequences of basal animals and show its impact on gene expression levels in human cell lines, and, importantly, how this expression efficiency can be improved. Optimization of the GC3 content in the coding sequences of cadherin, alpha-catenin, and paracaspase of Trichoplax adhaerens dramatically increased the expression of these basal animal genes in human cell lines

Robustness under Functional Constraint: The Genetic Network for Temporal Expression in Drosophila Neurogenesis

Precise temporal coordination of gene expression is crucial for many developmental processes. One central question in developmental biology is how such coordinated expression patterns are robustly controlled. During embryonic development of the Drosophila central nervous system, neural stem cells called neuroblasts express a group of genes in a definite order, which leads to the diversity of cell types. We produced all possible regulatory networks of these genes and examined their expression dynamics numerically. From the analysis, we identified requisite regulations and predicted an unknown factor to reproduce known expression profiles caused by loss-of-function or overexpression of the genes in vivo, as well as in the wild type. Following this, we evaluated the stability of the actual Drosophila network for sequential expression. This network shows the highest robustness against parameter variations and gene expression fluctuations among the possible networks that reproduce the expression profiles. We propose a regulatory module composed of three types of regulations that is responsible for precise sequential expression. This study suggests that the Drosophila network for sequential expression has evolved to generate the robust temporal expression for neuronal specification

Identification and Gene Expression Analysis of a Taxonomically Restricted Cysteine-Rich Protein Family in Reef-Building Corals

The amount of genomic sequence information continues to grow at an exponential rate, while the identification and characterization of genes without known homologs remains a major challenge. For non-model organisms with limited resources for manipulative studies, high-throughput transcriptomic data combined with bioinformatics methods provide a powerful approach to obtain initial insights into the function of unknown genes. In this study, we report the identification and characterization of a novel family of putatively secreted, small, cysteine-rich proteins herein named Small Cysteine-Rich Proteins (SCRiPs). Their discovery in expressed sequence tag (EST) libraries from the coral Montastraea faveolata required the performance of an iterative search strategy based on BLAST and Hidden-Markov-Model algorithms. While a discernible homolog could neither be identified in the genome of the sea anemone Nematostella vectensis, nor in a large EST dataset from the symbiotic sea anemone Aiptasia pallida, we identified SCRiP sequences in multiple scleractinian coral species. Therefore, we postulate that this gene family is an example of lineage-specific gene expansion in reef-building corals. Previously published gene expression microarray data suggest that a sub-group of SCRiPs is highly responsive to thermal stress. Furthermore, data from microarray experiments investigating developmental gene expression in the coral Acropora millepora suggest that different SCRiPs may play distinct roles in the development of corals. The function of these proteins remains to be elucidated, but our results from in silico, transcriptomic, and phylogenetic analyses provide initial insights into the evolution of SCRiPs, a novel, taxonomically restricted gene family that may be responsible for a lineage-specific trait in scleractinian corals

Incorporation of a Horizontally Transferred Gene into an Operon during Cnidarian Evolution

Genome sequencing has revealed examples of horizontally transferred genes, but we still know little about how such genes are incorporated into their host genomes. We have previously reported the identification of a gene (flp) that appears to have entered the Hydra genome through horizontal transfer. Here we provide additional evidence in support of our original hypothesis that the transfer was from a unicellular organism, and we show that the transfer occurred in an ancestor of two medusozoan cnidarian species. In addition we show that the gene is part of a bicistronic operon in the Hydra genome. These findings identify a new animal phylum in which trans-spliced leader addition has led to the formation of operons, and define the requirements for evolution of an operon in Hydra. The identification of operons in Hydra also provides a tool that can be exploited in the construction of transgenic Hydra strains

The p53 Tumor Suppressor-Like Protein nvp63 Mediates Selective Germ Cell Death in the Sea Anemone Nematostella vectensis

Here we report the identification and molecular function of the p53 tumor suppressor-like protein nvp63 in a non-bilaterian animal, the starlet sea anemone Nematostella vectensis. So far, p53-like proteins had been found in bilaterians only. The evolutionary origin of p53-like proteins is highly disputed and primordial p53-like proteins are variably thought to protect somatic cells from genotoxic stress. Here we show that ultraviolet (UV) irradiation at low levels selectively induces programmed cell death in early gametes but not somatic cells of adult N. vectensis polyps. We demonstrate with RNA interference that nvp63 mediates this cell death in vivo. Nvp63 is the most archaic member of three p53-like proteins found in N. vectensis and in congruence with all known p53-like proteins, nvp63 binds to the vertebrate p53 DNA recognition sequence and activates target gene transcription in vitro. A transactivation inhibitory domain at its C-terminus with high homology to the vertebrate p63 may regulate nvp63 on a molecular level. The genotoxic stress induced and nvp63 mediated apoptosis in N. vectensis gametes reveals an evolutionary ancient germ cell protective pathway which relies on p63-like proteins and is conserved from cnidarians to vertebrates

A novel class of heat-responsive small RNAs derived from the chloroplast genome of Chinese cabbage (Brassica rapa)

<p>Abstract</p> <p>Background</p> <p>Non-coding small RNAs play critical roles in various cellular processes in a wide spectrum of eukaryotic organisms. Their responses to abiotic stress have become a popular topic of economic and scientific importance in biological research. Several studies in recent years have reported a small number of non-coding small RNAs that map to chloroplast genomes. However, it remains uncertain whether small RNAs are generated from chloroplast genome and how they respond to environmental stress, such as high temperature. Chinese cabbage is an important vegetable crop, and heat stress usually causes great losses in yields and quality. Under heat stress, the leaves become etiolated due to the disruption and disassembly of chloroplasts. In an attempt to determine the heat-responsive small RNAs in chloroplast genome of Chinese cabbage, we carried out deep sequencing, using heat-treated samples, and analysed the proportion of small RNAs that were matched to chloroplast genome.</p> <p>Results</p> <p>Deep sequencing provided evidence that a novel subset of small RNAs were derived from the chloroplast genome of Chinese cabbage. The chloroplast small RNAs (csRNAs) include those derived from mRNA, rRNA, tRNA and intergenic RNA. The rRNA-derived csRNAs were preferentially located at the 3'-ends of the rRNAs, while the tRNA-derived csRNAs were mainly located at 5'-termini of the tRNAs. After heat treatment, the abundance of csRNAs decreased in seedlings, except those of 24 nt in length. The novel heat-responsive csRNAs and their locations in the chloroplast were verified by Northern blotting. The regulation of some csRNAs to the putative target genes were identified by real-time PCR. Our results reveal that high temperature suppresses the production of some csRNAs, which have potential roles in transcriptional or post-transcriptional regulation.</p> <p>Conclusions</p> <p>In addition to nucleus, the chloroplast is another important organelle that generates a number of small RNAs. Many members of csRNA families are highly sensitive to heat stress. Some csRNAs respond to heat stress by silencing target genes. We suggest that proper temperature is important for production of chloroplast small RNAs, which are associated with plant resistance to abiotic stress.</p

To Be or Not to Be a Flatworm: The Acoel Controversy

Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa - the Catenulida, the Acoelomorpha and the Rhabditophora - have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives

Ancient Origin of the New Developmental Superfamily DANGER

Developmental proteins play a pivotal role in the origin of animal complexity and diversity. We report here the identification of a highly divergent developmental protein superfamily (DANGER), which originated before the emergence of animals (∼850 million years ago) and experienced major expansion-contraction events during metazoan evolution. Sequence analysis demonstrates that DANGER proteins diverged via multiple mechanisms, including amino acid substitution, intron gain and/or loss, and recombination. Divergence for DANGER proteins is substantially greater than for the prototypic member of the superfamily (Mab-21 family) and other developmental protein families (e.g., WNT proteins). DANGER proteins are widely expressed and display species-dependent tissue expression patterns, with many members having roles in development. DANGER1A, which regulates the inositol trisphosphate receptor, promotes the differentiation and outgrowth of neuronal processes. Regulation of development may be a universal function of DANGER family members. This family provides a model system to investigate how rapid protein divergence contributes to morphological complexity

Activation of Neural and Pluripotent Stem Cell Signatures Correlates with Increased Malignancy in Human Glioma

The presence of stem cell characteristics in glioma cells raises the possibility that mechanisms promoting the maintenance and self-renewal of tissue specific stem cells have a similar function in tumor cells. Here we characterized human gliomas of various malignancy grades for the expression of stem cell regulatory proteins. We show that cells in high grade glioma co-express an array of markers defining neural stem cells (NSCs) and that these proteins can fulfill similar functions in tumor cells as in NSCs. However, in contrast to NSCs glioma cells co-express neural proteins together with pluripotent stem cell markers, including the transcription factors Oct4, Sox2, Nanog and Klf4. In line with this finding, in high grade gliomas mesodermal- and endodermal-specific transcription factors were detected together with neural proteins, a combination of lineage markers not normally present in the central nervous system. Persistent presence of pluripotent stem cell traits could only be detected in solid tumors, and observations based on in vitro studies and xenograft transplantations in mice imply that this presence is dependent on the combined activity of intrinsic and extrinsic regulatory cues. Together these results demonstrate a general deregulated expression of neural and pluripotent stem cell traits in malignant human gliomas, and indicate that stem cell regulatory factors may provide significant targets for therapeutic strategies