Highly Conserved Caspase and Bcl-2 Homologues from the Sea Anemone Aiptasia pallida: Lower Metazoans as Models for the Study of Apoptosis Evolution

Key insight into the complexities of apoptosis may be gained from the study of its evolution in lower metazoans. In this study we describe two genes from a cnidarian, Aiptasia pallida, that are homologous to key genes in the apoptotic pathway from vertebrates. The first is a novel ancient caspase, acasp, that displays attributes of both initiator and executioner caspases and includes a caspase recruitment domain (CARD). The second, a Bcl-2 family member, abhp, contains a BH1 and BH2 domain and shares structural characteristics and phylogenetic affinity with a group of antiapoptotic Bcl-2s including A1 and Bcl-2L10. The breadth of occurrence of other invertebrate homologues across the phylogenetic trees of both genes suggests that the complexity of apoptotic pathways is an ancient trait that predates the evolution of vertebrates and higher invertebrates such as nematodes and flies. This paves the way for establishing new lower metazoan model systems for the study of apoptosis. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Stuart Newfeld]     

Human-specific nonsense mutations identified by genome sequence comparisons

The comparative study of the human and chimpanzee genomes may shed light on the genetic ingredients for the evolution of the unique traits of humans. Here, we present a simple procedure to identify human-specific nonsense mutations that might have arisen since the human–chimpanzee divergence. The procedure involves collecting orthologous sequences in which a stop codon of the human sequence is aligned to a non-stop codon in the chimpanzee sequence and verifying that the latter is ancestral by finding homologs in other species without a stop codon. Using this procedure, we identify nine genes (CML2, FLJ14640, MT1L, NPPA, PDE3B, SERPINA13, TAP2, UIP1, and ZNF277) that would produce human-specific truncated proteins resulting in a loss or modification of the function. The premature terminations of CML2, MT1L, and SERPINA13 genes appear to abolish the original function of the encoded protein because the mutation removes a major part of the known active site in each case. The other six mutated genes are either known or presumed to produce functionally modified proteins. The mutations of five genes (CML2, FLJ14640, MT1L, NPPA, TAP2) are known or predicted to be polymorphic in humans. In these cases, the stop codon alleles are more prevalent than the ancestral allele, suggesting that the mutant alleles are approaching fixation since their emergence during the human evolution. The findings support the notion that functional modification or inactivation of genes by nonsense mutation is a part of the process of adaptive evolution and acquisition of species-specific features. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Biased distribution of single nucleotide polymorphisms (SNPs) in porcine Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR5, and TLR6 genes

Toll-like receptors (TLRs) recognize various microbial components and induce immune responses. Polymorphisms in TLRs may influence their recognition of pathogen-derived molecules; swine TLRs are predicted to be associated with responses to infectious diseases such as pneumonia. In this study, we searched for single nucleotide polymorphisms (SNPs) in the coding sequences of porcine TLR1, TLR2, TLR4, TLR5, and TLR6 genes in 96 pigs from 11 breeds and elucidated 21, 11, 7, 13, and 11 SNPs, respectively, which caused amino acid substitutions in the respective TLRs. Distribution of these nonsynonymous SNPs was biased; many were located in the leucine-rich repeats, particularly in TLR1. These data demonstrated that the heterogeneity of TLR genes was preserved in various porcine breeds despite intensive breeding that was carried out for livestock improvement. It suggests that the heterogeneity in TLR genes is advantageous in increasing the possibility of survival in porcine populations.Electronic SupplementaryMaterial Supplementary material is available for this article at     

Gene expression profile of Ralstonia Solanacearum for the rhizosphere ecological niche of Solanum tuberosum

Plant root secretion can be regarded as signal molecules, which exerts impact on microorganisms in the rhizosphere ecological niche. We obtained gene expression profile of Ralstonia solanacearumPO41 under the root secretions environment of Solanum tuberosum at the time points of 8 hrs, 16 hrs and 24 hrs, respectively, after infection with RNA microarray technology. Bioinformatics tools of differential genes expression analysis, GO functional analysis, cluster analysis and pathway analysis were conducted to find out the pathogenic genes and other related genes. We found that the virulence factors of R. solanacearum mainly focused on the output pathways of toxic protein (Sec pathway, Tat pathway and type III secretion system (T3SS)), the aggregation and transfer of exopolysaccharides and the chemotactic movement and adhesion of flagellum in the potato root secretion ecological niche, while the virulence factors in the atypical output pathway mainly distributed in Sec (secB, secDF, yidc) and Tat (tatA, tatC) pathways to promote the output of folded and unfolded toxic proteins. The fliIATPase was obviously upregulated 8 hrs postinoculation, suggesting that type III secretion system was only active at the early stage of PO41 infection. The upregulated expression of phosphoglucomutase and epimerase showed that the virulence factor of exopolysaccharides (EPS) was synthesized at the early stage of R. solanacearum infection. Chemotactic receptor and motor protein were obviously upregulated within 24 hrs postinoculation. Our study revealed that R. solanacearumPO41 had already colonized to the roots within 24 hrs with the stimulating of root secretion. Some pathogenic genes were upregulated during this period.     

Square-plate culture method allows detection of differential gene expression and screening of novel, region-specific genes in Aspergillus oryzae

When grown on solid agar medium, the mycelium of a filamentous fungus, Aspergillus oryzae, forms three morphologically distinct regions: the tip (T), white (W), and basal (B) regions. In this study, we developed the square-plate culture method, a novel culture method that enabled the extraction of mRNA samples from the three regions and analyzed the differential gene expression of the A. oryzae mycelium in concert with the microarray technique. Expression of genes involved in protein synthesis was predominant in the T region; relative expression was, at most, six times higher in the T region compared to the other regions. Genes encoding hypothetical proteins were expressed at high levels in the W and B regions. In addition, genes coding transporters/permeases were predominantly transcribed in the B region. By analyzing the expression patterns of genes in the three regions, we demonstrated the dynamic changes in the regulation of gene expression that occur along the mycelium of filamentous fungi. Consequently, our study established a method to analyze and screen for region-specific genes whose function may be essential for morphogenesis and differentiation in filamentous fungi and whose traits may be beneficial to the biotechnology industry.Electronic Supplementary Materials Supplementary material is available for this article at     

Gene expression profile analysis in astaxanthin-induced Haematococcus pluvialis using a cDNA microarray

The unicellular green alga Haematococcus pluvialis (Volvocales) is known for the ketocarotenoid astaxanthin (3, 3′-dihydroxy-β, β-carotene-4, 4′-dione) accumulation, which is induced under unfavorable culture conditions. In this work, we used cDNA microarray analysis to screen differentially expressed genes in H. pluvialis under astaxanthin-inductive culture conditions, such as combination of cell exposure to high irradiance and nutrient deprivation. Among the 965 genes in the cDNA array, there are 144 genes exhibiting differential expression (twofold changes) under these conditions. A significant decrease in the expression of photosynthesis-related genes was shown in astaxanthin-accumulating cells (red cells). Defense- or stress-related genes and signal transduction genes were also induced in the red cells. A comparison of microarray and real-time PCR analysis showed good correlation between the differentially expressed genes by the two methods. Our results indicate that the cDNA microarray approach, as employed in this work, can be relied upon and used to monitor gene expression profiles in H. pluvialis. In addition, the genes that were differentially expressed during astaxanthin induction are suitable candidates for further study and can be used as tools for dissecting the molecular mechanism of this unique pigment accumulation process in the green alga H. pluvialis. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Barley putative hypersensitive induced reaction genes: genetic mapping,sequence analyses and differential expression in disease lesion mimic mutants

The hypersensitive response (HR) is one of the most-efficient forms of plant defense against biotrophic pathogens, and results in localized cell death and the formation of necrotic lesions; however, the molecular components of pathways leading to HR remain largely unknown. Barley (Hordeum vulgare ssp. vulgare L.) cDNAs for putative hypersensitive-induced reaction (HIR) genes were isolated based on DNA and amino-acid homologies to maize HIR genes. Analyses of the cDNA and genomic sequences and genetic mapping found four distinct barley HIR genes, Hv-hir1, Hv-hir2, Hv-hir3 and Hv-hir4, on chromosomes 4(4H) bin10, 7(5H) bin04, 7(5H) bin07 and 1(7H) bin03, respectively. Hv-hir1, Hv-hir2 and Hv-hir3 genes were highly homologous at both DNA and the deduced amino-acid level, but the Hv-hir4 gene was similar to the other genes only at the amino-acid sequence level. Amino-acid sequence analyses of the barley HIR proteins indicated the presence of the SPFH protein-domain characteristic for the prohibitins and stomatins which are involved in control of the cell cycle and ion channels, as well as in other membrane-associated proteins from bacteria, plants and animals. HIR genes were expressed in all organs and developement stages analyzed, indicating a vital and non-redundant function. Barley fast-neutron mutants exhibiting spontaneous HR (disease lesion mimic mutants) showed up to a 35-fold increase in Hv-hir3 expression, implicating HIR genes in the induction of HR.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Wenzel     

Use of RNA Sequencing to Perform Comprehensive Analysis of Long Noncoding RNA Expression Profiles in Macrophages Infected with Trichosporon asahii

Trichosporon asahii (T. asahii) is a clinically important opportunistic pathogenic fungus capable of causing systemic lethal infection in immunosuppressive and immunodeficient hosts. However, the mechanism of the host immune response upon T. asahii infection has not been elucidated. Recent evidence has shown that long noncoding RNAs (lncRNAs) play key roles in regulating the immune response to resist microbial infections. In this study, we analyzed the expression profiles of lncRNAs at 12 and 24 h post-infection (hpi) in THP-1 cells infected with T. asahii using RNA sequencing (RNA-Seq). A total of 64 and 160 lncRNAs displayed significant differentially expressed (DE) at 12 h and 24 hpi, respectively. Among these lncRNAs, 18 lncRNAs were continuous DE at two time points. The DE of eight candidate lncRNAs were verified by real time quantitative polymerase chain reaction (RT-qPCR). Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to analyze the cis-target genes of 18 DE lncRNAs. The results showed that they were enriched in signaling pathways related to the host immune response, indicating that these lncRNAs might play important roles in fungi–host interactions. Finally, we explored the function of lncRNA NEAT1 and found that the expression of TNF-α and IL-1β declined after NEAT1 knockdown in T. asahii-infected THP-1 cells. To our knowledge, this is the first report of a expression analysis of lncRNAs in macrophages infected with T. asahii. Our study helps to elucidate the role of lncRNAs in the host immune response to early infection by T. asahii.

     

Constitutive knox1 gene expression in dandelion (Taraxacum officinale, Web.) changes leaf morphology from simple to compound

Seed plants with compound leaves constitute a polyphyletic group, but studies of diverse taxa show that genes of the class 1 KNOTTED-LIKE HOMEOBOX (KNOX1) family are often involved in compound leaf development. This suggests that knox1 genes have been recruited on multiple occasions during angiosperm evolution (Bharathan et al. in Science 296:1858–1860, 2002). In agreement with this, we demonstrate that the simple leaf of dandelion (Taraxacum officinale Web.) can be converted into a compound leaf by the constitutive expression of heterologous knox1 genes. Dandelion is a rosette plant of the family Asteraceae, characterised by simple leaves with deeply lobed margins and endogenous knox1 gene expression. Transgenic dandelion plants constitutively expressing the barley (Hordeum vulgare L.) hooded gene (bkn3, barley knox3) or the related bkn1 gene, developed compound leaves featuring epiphyllous rosettes. We discuss these results in the context of two current models of compound leaf formation.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The “Window <Emphasis Type="Italic">t</Emphasis> test”: a simple and powerful approach to detect differentially expressed genes in microarray datasets

This work focuses on differential expression analysis of microarray datasets. One way to improve such statistical analyses is to integrate biological information in the design of these analyses. In this paper, we will use the relationship between the level of gene expression and variability. Using this biological information, we propose to integrate the information from multiple genes to get a better estimate of individual gene variance, when a small number of replicates are available, to increase the power of the statistical analysis. We describe a strategy named the “Window t test” that uses multiple genes which share a similar expression level to compute the variance which is then incorporated a classic t test. The performances of this new method are evaluated by comparison with classic and widely-used methods for differential expression analysis (the classic Student t test, the Regularized t test (reg t test), SAM, Limma, LPE and Shrinkage t). In each case tested, the results obtained were at least equivalent to the best performing method and, in most cases, outperformed it. Moreover, the Window t test relies on a very simple procedure requiring small computing power compared with other methods designed for microarray differential expression analysis. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.