Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.
All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.
Kolchanov, N A; Ponomarenko, M P; Frolov, A S; Ananko, E A; Kolpakov, F A; Ignatieva, E V; Podkolodnaya, O A; Goryachkovskaya, T N; Stepanenko, I L; Merkulova, T I; Babenko, V V; Ponomarenko, Y V; Kochetov, A V; Podkolodny, N L; Vorobiev, D V; Lavryushev, S V; Grigorovich, D A; Kondrakhin, Y V; Milanesi, L; Wingender, E; Solovyev, V; Overton, G C
The goal of the work was to develop a WWW-oriented computer system providing a maximal integration of informational and software resources on the regulation of gene expression and navigation through them. Rapid growth of the variety and volume of information accumulated in the databases on regulation of gene expression necessarily requires the development of computer systems for automated discovery of the knowledge that can be further used for analysis of regulatory genomic sequences. The GeneExpress system developed includes the following major informational and software modules: (1) Transcription Regulation (TRRD) module, which contains the databases on transcription regulatory regions of eukaryotic genes and TRRD Viewer for data visualization; (2) Site Activity Prediction (ACTIVITY), the module for analysis of functional site activity and its prediction; (3) Site Recognition module, which comprises (a) B-DNA-VIDEO system for detecting the conformational and physicochemical properties of DNA sites significant for their recognition, (b) Consensus and Weight Matrices (ConsFrec) and (c) Transcription Factor Binding Sites Recognition (TFBSR) systems for detecting conservative contextual regions of functional sites and their recognition; (4) Gene Networks (GeneNet), which contains an object-oriented database accumulating the data on gene networks and signal transduction pathways, and the Java-based Viewer for exploration and visualization of the GeneNet information; (5) mRNA Translation (Leader mRNA), designed to analyze structural and contextual properties of mRNA 5'-untranslated regions (5'-UTRs) and predict their translation efficiency; (6) other program modules designed to study the structure-function organization of regulatory genomic sequences and regulatory proteins. GeneExpress is available at http://wwwmgs.bionet.nsc. ru/systems/GeneExpress/ and the links to the mirror site(s) can be found at http://wwwmgs.bionet.nsc.ru/mgs/links/mirrors.html+ ++.
Cutroneo, Kenneth R; Ehrlich, H
The elucidation of molecular and signaling pathways in eukaryotic cells is often achieved by targeting regulatory element(s) found in the promoter or the enhancer region of eukaryotic gene(s) using a double-stranded (ds) oligodeoxynucleotide (ODN) containing a specific cis-element. Our laboratory is focusing on dsODN decoys containing the TGF-beta element as a novel nonsteroidal antifibrotic for achieving normal wound healing. In the model systems discussed, there is either a specific gene possessing a specific cis-element or a cluster of genes with one gene containing the consensus cis-element. The rest of the genes in the cluster contain the cis-elements homologous to this consensus element, which allows for dsODN decoy regulation of a gene cluster at one time.
Kuznetsov Vladimir A
Full Text Available The ability to simultaneously measure mRNA abundance for large number of genes has revolutionized biological research by allowing statistical analysis of global gene-expression data. Large-scale gene-expression data sets have been analyzed in order to identify the probability distributions of gene expression levels (or transcript copy numbers in eukaryotic cells. Determining such function(s may provide a theoretical basis for accurately counting all expressed genes in a given cell and for understanding gene expression control. Using the gene-expression libraries derived from yeast cells and from different human cell tissues we found that all observed gene expression levels data appear to follow a Pareto-like skewed frequency distribution. We produced a the skewed probability function, called the Binomial Differential distribution, that accounts for many rarely transcribed genes in a single cell. We also developed a novel method for estimating and removing major experimental errors and redundancies from the Serial Analysis Gene Expression (SAGE data sets. We successfully applied this method to the yeast transcriptome. A "basal" random transcription mechanism for all protein-coding genes in every eukaryotic cell type is predicted.
Full Text Available Abstract Background There is increasing evidence that gene order within the eukaryotic genome is not random. In yeast and worm, adjacent or neighboring genes tend to be co-expressed. Clustering of co-expressed genes has been found in humans, worm and fruit flies. However, in mice and rats, an effect of chromosomal distance (CD on co-expression has not been investigated yet. Also, no cross-species comparison has been made so far. We analyzed the effect of CD as well as normalized distance (ND using expression data in six eukaryotic species: yeast, fruit fly, worm, rat, mouse and human. Results We analyzed 24 sets of expression data from the six species. Highly co-expressed pairs were sorted into bins of equal sized intervals of CD, and a co-expression rate (CoER in each bin was calculated. In all datasets, a higher CoER was obtained in a short CD range than a long distance range. These results show that across all studied species, there was a consistent effect of CD on co-expression. However, the results using the ND show more diversity. Intra- and inter-species comparisons of CoER reveal that there are significant differences in the co-expression rates of neighboring genes among the species. A pair-wise BLAST analysis finds 8 – 30 % of the highly co-expressed pairs are duplic ated genes. Conclusion We confirmed that in the six eukaryotic species, there was a consistent tendency that neighboring genes are likely to be co-expressed. Results of pair-wised BLAST indicate a significant effect of non-duplicated pairs on co-expression. A comparison of CD and ND suggests the dominant effect of CD.
Pawan Kumar Jayaswal
Full Text Available Rapid advances in DNA sequencing technologies have resulted in the accumulation of large data sets in the public domain, facilitating comparative studies to provide novel insights into the evolution of life. Phylogenetic studies across the eukaryotic taxa have been reported but on the basis of a limited number of genes. Here we present a genome-wide analysis across different plant, fungal, protist, and animal species, with reference to the 36,002 expressed genes of the rice genome. Our analysis revealed 9831 genes unique to rice and 98 genes conserved across all 49 eukaryotic species analysed. The 98 genes conserved across diverse eukaryotes mostly exhibited binding and catalytic activities and shared common sequence motifs; and hence appeared to have a common origin. The 98 conserved genes belonged to 22 functional gene families including 26S protease, actin, ADP-ribosylation factor, ATP synthase, casein kinase, DEAD-box protein, DnaK, elongation factor 2, glyceraldehyde 3-phosphate, phosphatase 2A, ras-related protein, Ser/Thr protein phosphatase family protein, tubulin, ubiquitin and others. The consensus Bayesian eukaryotic tree of life developed in this study demonstrated widely separated clades of plants, fungi, and animals. Musa acuminata provided an evolutionary link between monocotyledons and dicotyledons, and Salpingoeca rosetta provided an evolutionary link between fungi and animals, which indicating that protozoan species are close relatives of fungi and animals. The divergence times for 1176 species pairs were estimated accurately by integrating fossil information with synonymous substitution rates in the comprehensive set of 98 genes. The present study provides valuable insight into the evolution of eukaryotes.
Qin, R F; Gu, X M; Chen, J W
To construct eukaryotic expression plasmid of mouse myogenic regulatory factor MyoD gene for further study on MyoD gene function in molecular regulatory mechanism in skeletal muscle repair. The plasmids PEMMBC2 beta 5 containing full cDNA length of MyoD inserted in EcoRI restriction site, were first propagated in Escherichia coli DH5a, then extracted and purified with the Wizard Plus Minipreps DNA Purification System (Promega, USA). The coding sequence of MyoD in PEMMBC2 beta 5 was confirmed by agarose gel electrophoresis and DNA sequence analysis. After plasmids PEMMBC2 beta 5 and plasmids pcDNA3-neo were prepared by digestion with EcoRI, the MyoD cDNA fragment was inserted into EcoRI site in pcDNA3-neo eukaryotic expression vector, and pcDNA3/MyoD was formed. The pcDNA3/MyoD, digested with restriction enzymes, was found to contain the MyoD cDNA sequence by agarose gel electrophoresis analysis. The extracted and purified PEMMBC2 beta 5 contained the correct nucleotide sequence for the full length of MyoD cDNA fragment. The MyoD cDNA fragment had been inserted into the eukaryotic expression plasmid pcDNA3-neo, which formed the pcDNA3/MyoD. The pcDNA3/MyoD, a eukaryotic expression plasmid, for MyoD is constructed successfully.
Raj, Dibyendu; Ghosh, Esha; Mukherjee, Avik K; Nozaki, Tomoyoshi; Ganguly, Sandipan
Giardia lamblia is a unicellular, early branching eukaryote causing giardiasis, one of the most common human enteric diseases. Giardia, a microaerophilic protozoan parasite has to build up mechanisms to protect themselves against oxidative stress within the human gut (oxygen concentration 60 μM) to establish its pathogenesis. G. lamblia is devoid of the conventional mechanisms of the oxidative stress management system, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. NADH oxidase is a major component of the electron transport chain of G. lamblia, which in concurrence with disulfide reductase, protects oxygen-labile proteins such as pyruvate: ferredoxin oxidoreductase against oxidative stress by sustaining a reduced intracellular environment. It also contains the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, includes substrate level phosphorylation and adequately active to make a major contribution to ATP production. To study differential gene expression under three types of oxidative stress, a Giardia genomic DNA array was constructed and hybridized with labeled cDNA of cells with or without stress. The transcriptomic data has been analyzed and further validated using real time PCR. We identified that out of 9216 genes represented on the array, more than 200 genes encoded proteins with functions in metabolism, oxidative stress management, signaling, reproduction and cell division, programmed cell death and cytoskeleton. We recognized genes modulated by at least ≥ 2 fold at a significant time point in response to oxidative stress. The study has highlighted the genes that are differentially expressed during the three experimental conditions which regulate the stress management pathway differently to achieve redox homeostasis. Identification of some unique genes in oxidative stress regulation may help in new drug designing for this common enteric parasite prone to
Full Text Available BACKGROUND: Herbivores rely on digestive tract lignocellulolytic microorganisms, including bacteria, fungi and protozoa, to derive energy and carbon from plant cell wall polysaccharides. Culture independent metagenomic studies have been used to reveal the genetic content of the bacterial species within gut microbiomes. However, the nature of the genes encoded by eukaryotic protozoa and fungi within these environments has not been explored using metagenomic or metatranscriptomic approaches. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a metatranscriptomic approach was used to investigate the functional diversity of the eukaryotic microorganisms within the rumen of muskoxen (Ovibos moschatus, with a focus on plant cell wall degrading enzymes. Polyadenylated RNA (mRNA was sequenced on the Illumina Genome Analyzer II system and 2.8 gigabases of sequences were obtained and 59129 contigs assembled. Plant cell wall degrading enzyme modules including glycoside hydrolases, carbohydrate esterases and polysaccharide lyases were identified from over 2500 contigs. These included a number of glycoside hydrolase family 6 (GH6, GH48 and swollenin modules, which have rarely been described in previous gut metagenomic studies. CONCLUSIONS/SIGNIFICANCE: The muskoxen rumen metatranscriptome demonstrates a much higher percentage of cellulase enzyme discovery and an 8.7x higher rate of total carbohydrate active enzyme discovery per gigabase of sequence than previous rumen metagenomes. This study provides a snapshot of eukaryotic gene expression in the muskoxen rumen, and identifies a number of candidate genes coding for potentially valuable lignocellulolytic enzymes.
Full Text Available Background and objective Raf is a key molecule in the Ras-Raf-MEK-ERK signal transduction pathway and is highly activated in different human carcinomas. However, its biological functions and regulation mechanisms are still unclear. The aims of this study were to construct eukaryotic expression vectors with Raf full encoding region, truncated amino-terminus and carboxyl-terminus, respectively. Methods Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-C-Raf were constructed by gene recombination technique and confirmed by restriction enzyme analysis and DNA sequencing. Furthermore, the expression of these fusion proteins was detected by western blot in transient transfected 293T cells. Results The sequences and open reading frames of these three vectors were completely consistent with experimental design. All target proteins can be detected in 293T cells. Conclusion Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-C-Raf were successfully constructed and can be expressed in 293T cells.
Full Text Available From the ENCODE project, it is realized that almost every base of the entire human genome is transcribed. One class of transcripts resulting from this arises from the conjoined gene, which is formed by combining the exons of two or more distinct (parent genes lying on the same strand of a chromosome. Only a very limited number of such genes are known, and the definition and terminologies used for them are highly variable in the public databases. In this work, we have computationally identified and manually curated 751 conjoined genes (CGs in the human genome that are supported by at least one mRNA or EST sequence available in the NCBI database. 353 representative CGs, of which 291 (82% could be confirmed, were subjected to experimental validation using RT-PCR and sequencing methods. We speculate that these genes are arising out of novel functional requirements and are not merely artifacts of transcription, since more than 70% of them are conserved in other vertebrate genomes. The unique splicing patterns exhibited by CGs reveal their possible roles in protein evolution or gene regulation. Novel CGs, for which no transcript is available, could be identified in 80% of randomly selected potential CG forming regions, indicating that their formation is a routine process. Formation of CGs is not only limited to human, as we have also identified 270 CGs in mouse and 227 in drosophila using our approach. Additionally, we propose a novel mechanism for the formation of CGs. Finally, we developed a database, ConjoinG, which contains detailed information about all the CGs (800 in total identified in the human genome. In summary, our findings reveal new insights about the functionality of CGs in terms of another possible mechanism for gene regulation and genomic evolution and the mechanism leading to their formation.
Full Text Available Eukaryotic organisms play essential roles in the biology and fertility of soils. For example the micro and mesofauna contribute to the fragmentation and homogenization of plant organic matter, while its hydrolysis is primarily performed by the fungi. To get a global picture of the activities carried out by soil eukaryotes we sequenced 2×10,000 cDNAs synthesized from polyadenylated mRNA directly extracted from soils sampled in beech (Fagus sylvatica and spruce (Picea abies forests. Taxonomic affiliation of both cDNAs and 18S rRNA sequences showed a dominance of sequences from fungi (up to 60% and metazoans while protists represented less than 12% of the 18S rRNA sequences. Sixty percent of cDNA sequences from beech forest soil and 52% from spruce forest soil had no homologs in the GenBank/EMBL/DDJB protein database. A Gene Ontology term was attributed to 39% and 31.5% of the spruce and beech soil sequences respectively. Altogether 2076 sequences were putative homologs to different enzyme classes participating to 129 KEGG pathways among which several were implicated in the utilisation of soil nutrients such as nitrogen (ammonium, amino acids, oligopeptides, sugars, phosphates and sulfate. Specific annotation of plant cell wall degrading enzymes identified enzymes active on major polymers (cellulose, hemicelluloses, pectin, lignin and glycoside hydrolases represented 0.5% (beech soil-0.8% (spruce soil of the cDNAs. Other sequences coding enzymes active on organic matter (extracellular proteases, lipases, a phytase, P450 monooxygenases were identified, thus underlining the biotechnological potential of eukaryotic metatranscriptomes. The phylogenetic affiliation of 12 full-length carbohydrate active enzymes showed that most of them were distantly related to sequences from known fungi. For example, a putative GH45 endocellulase was closely associated to molluscan sequences, while a GH7 cellobiohydrolase was closest to crustacean sequences, thus
Praz, Viviane; Périer, Rouaïda; Bonnard, Claude; Bucher, Philipp
The Eukaryotic Promoter Database (EPD) is an annotated, non-redundant collection of eukaryotic Pol II promoters, for which the transcription start site has been determined experimentally. Access to promoter sequences is provided by pointers to positions in nucleotide sequence entries. The annotation part of an entry includes a description of the initiation site mapping data, exhaustive cross-references to the EMBL nucleotide sequence database, SWISS-PROT, TRANSFAC and other databases, as well as bibliographic references. EPD is structured in a way that facilitates dynamic extraction of biologically meaningful promoter subsets for comparative sequence analysis. World Wide Web-based interfaces have been developed which enable the user to view EPD entries in different formats, to select and extract promoter sequences according to a variety of criteria, and to navigate to related databases exploiting different cross-references. The EPD web site also features yearly updated base frequency matrices for major eukaryotic promoter elements. EPD can be accessed at http://www.epd.isb-sib.ch.
Wang, Jianping; Liu, Dejie; Liang, Xiaohong; Gao, Lifen; Yue, Xuetian; Yang, Yang; Ma, Chunhong; Liu, Jun
The zinc-fingers and homeoboxes protein 1 (ZHX1) consists of 873 amino acid residues, is localized in the cell nucleus and appears to act as a transcriptional repressor. Previous studies have shown that ZHX1 interacts with nuclear factor Y subunit α (NF-YA), DNA methyltransferases (DNMT) 3B and ZHX2, all of which are involved in tumorigenesis. However, the exact role of ZHX1 in tumorigenesis remains unknown. The aim of the current study was to construct a recombinant eukaryotic expression plasmid containing the human ZHX1 (hZHX1) gene and to investigate the biological activities of ZHX1 in hepatocellular carcinoma (HCC). Reverse transcription-polymerase chain reaction (RT‑PCR) was used to amplify the N- and C-terminal fragments (ZHX1‑N and ZHX1‑C, respectively) of the hZHX1 gene. The two PCR fragments were cloned into the pEASY-T1 vector and subcloned into the pcDNA3 plasmid to generate a recombinant pcDNA3‑ZHX1 plasmid. Following identification by enzyme digestion and DNA sequencing, the recombinant pcDNA3‑ZHX1 plasmid was transfected into SMMC-7721 cells. The level of ZHX1 expression was detected by RT-PCR and western blot analysis. Cell growth curve assays were used to evaluate the effect of ZHX1 on cell proliferation. Moreover, the differential expression of ZHX1 between cancer and adjacent cirrhotic liver tissue was investigated by quantitative PCR (qPCR). Enzyme digestion and DNA sequencing confirmed the successful construction of the recombinant plasmid, pcDNA3‑ZHX1. qPCR and western blot analysis demonstrated that ZHX1 was efficiently expressed in SMMC-7721 cells and overexpression of ZHX1 may inhibit the proliferation of SMMC-7721 cells. In addition, reduced ZHX1 expression is widespread among cancer tissues from HCC patients. In conclusion, a recombinant eukaryotic expression plasmid, pcDNA3‑ZHX1, was successfully constructed. In addition, the current results indicate that a low expression of ZHX1 may be responsible for hepatocarcinogenesis.
Mayfield, Stephen P.
The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.
Zhang, Hanbang; Ehrenkaufer, Gretchen M; Pompey, Justine M; Hackney, Jason A; Singh, Upinder
Small interfering RNAs regulate gene expression in diverse biological processes, including heterochromatin formation and DNA elimination, developmental regulation, and cell differentiation. In the single-celled eukaryote Entamoeba histolytica, we have identified a population of small RNAs of 27 nt size that (i) have 5'-polyphosphate termini, (ii) map antisense to genes, and (iii) associate with an E. histolytica Piwi-related protein. Whole genome microarray expression analysis revealed that essentially all genes to which antisense small RNAs map were not expressed under trophozoite conditions, the parasite stage from which the small RNAs were cloned. However, a number of these genes were expressed in other E. histolytica strains with an inverse correlation between small RNA and gene expression level, suggesting that these small RNAs mediate silencing of the cognate gene. Overall, our results demonstrate that E. histolytica has an abundant 27 nt small RNA population, with features similar to secondary siRNAs from C. elegans, and which appear to regulate gene expression. These data indicate that a silencing pathway mediated by 5'-polyphosphate siRNAs extends to single-celled eukaryotic organisms.
Zhang, Hanbang; Ehrenkaufer, Gretchen M.; Pompey, Justine M.; Hackney, Jason A.; Singh, Upinder
Small interfering RNAs regulate gene expression in diverse biological processes, including heterochromatin formation and DNA elimination, developmental regulation, and cell differentiation. In the single-celled eukaryote Entamoeba histolytica, we have identified a population of small RNAs of 27 nt size that (i) have 5′-polyphosphate termini, (ii) map antisense to genes, and (iii) associate with an E. histolytica Piwi-related protein. Whole genome microarray expression analysis revealed that essentially all genes to which antisense small RNAs map were not expressed under trophozoite conditions, the parasite stage from which the small RNAs were cloned. However, a number of these genes were expressed in other E. histolytica strains with an inverse correlation between small RNA and gene expression level, suggesting that these small RNAs mediate silencing of the cognate gene. Overall, our results demonstrate that E. histolytica has an abundant 27 nt small RNA population, with features similar to secondary siRNAs from C. elegans, and which appear to regulate gene expression. These data indicate that a silencing pathway mediated by 5′-polyphosphate siRNAs extends to single-celled eukaryotic organisms. PMID:19043551
Full Text Available Small interfering RNAs regulate gene expression in diverse biological processes, including heterochromatin formation and DNA elimination, developmental regulation, and cell differentiation. In the single-celled eukaryote Entamoeba histolytica, we have identified a population of small RNAs of 27 nt size that (i have 5'-polyphosphate termini, (ii map antisense to genes, and (iii associate with an E. histolytica Piwi-related protein. Whole genome microarray expression analysis revealed that essentially all genes to which antisense small RNAs map were not expressed under trophozoite conditions, the parasite stage from which the small RNAs were cloned. However, a number of these genes were expressed in other E. histolytica strains with an inverse correlation between small RNA and gene expression level, suggesting that these small RNAs mediate silencing of the cognate gene. Overall, our results demonstrate that E. histolytica has an abundant 27 nt small RNA population, with features similar to secondary siRNAs from C. elegans, and which appear to regulate gene expression. These data indicate that a silencing pathway mediated by 5'-polyphosphate siRNAs extends to single-celled eukaryotic organisms.
Full Text Available Abstract Background The costs and benefits of spliceosomal introns in eukaryotes have not been established. One recognized effect of intron splicing is its known enhancement of gene expression. However, the mechanism regulating such splicing-mediated expression enhancement has not been defined. Previous studies have shown that intron splicing is a time-consuming process, indicating that splicing may not reduce the time required for transcription and processing of spliced pre-mRNA molecules; rather, it might facilitate the later rounds of transcription. Because the densities of active RNA polymerase II on most genes are less than one molecule per gene, direct interactions between the splicing apparatus and transcriptional complexes (from the later rounds of transcription are infrequent, and thus unlikely to account for splicing-mediated gene expression enhancement. Presentation of the hypothesis The serine/arginine-rich protein SF2/ASF can inhibit the DNA topoisomerase I activity that removes negative supercoiling of DNA generated by transcription. Consequently, splicing could make genes more receptive to RNA polymerase II during the later rounds of transcription, and thus affect the frequency of gene transcription. Compared with the transcriptional enhancement mediated by strong promoters, intron-containing genes experience a lower frequency of cut-and-paste processes. The cleavage and religation activity of DNA strands by DNA topoisomerase I was recently shown to account for transcription-associated mutagenesis. Therefore, intron-mediated enhancement of gene expression could reduce transcription-associated genome instability. Testing the hypothesis Experimentally test whether transcription-associated mutagenesis is lower in intron-containing genes than in intronless genes. Use bioinformatic analysis to check whether exons flanking lost introns have higher frequencies of short deletions. Implications of the hypothesis The mechanism of intron
Torabi, Azam; Tahmoorespour, Mojtaba; Vahedi, Fatemeh; Mosavari, Nader; Nassiri, Mohammadreza
Mycobacterium (M.) bovis is the agent of bovine tuberculosis (TB) in a range of animal species, including humans. Recent advances in immunology and the molecular biology of Mycobacterium have allowed identification of a large number of antigens with the potential for the development of a new TB vaccine. The ESAT-6 and CFP-10 proteins of M. bovis are important structural and functional proteins known to be important immunogens. In the current study, the DNAs encoding these genes were utilized in the construction of pcDNA 3.1+/ESAT-6 and pcDNA3.1+/CFP-10 plasmids. After intramuscular injection of BALB/c mice with these plasmids, ESAT-6 and CFP-10 mRNA expression was assessed by RT-PCR. Mice were inoculated and boosted with the plasmids to evaluate their effects on lymphocyte proliferation. Our results indicate the plasmids are expressed at the RNA level and can induce lymphocyte proliferation. Further study is needed to characterize the effect of these antigens on the immune system and determine whether they are effective vaccine candidates against M. bovis.
Soanes, Darren; Richards, Thomas A
Gene transfer has been identified as a prevalent and pervasive phenomenon and an important source of genomic innovation in bacteria. The role of gene transfer in microbial eukaryotes seems to be of a reduced magnitude but in some cases can drive important evolutionary innovations, such as new functions that underpin the colonization of different niches. The aim of this review is to summarize published cases that support the hypothesis that horizontal gene transfer (HGT) has played a role in the evolution of phytopathogenic traits in fungi and oomycetes. Our survey of the literature identifies 46 proposed cases of transfer of genes that have a putative or experimentally demonstrable phytopathogenic function. When considering the life-cycle steps through which a pathogen must progress, the majority of the HGTs identified are associated with invading, degrading, and manipulating the host. Taken together, these data suggest HGT has played a role in shaping how fungi and oomycetes colonize plant hosts.
Alsmark, Cecilia; Foster, Peter G; Sicheritz-Pontén, Thomas
BACKGROUND: The influence of lateral gene transfer on gene origins and biology in eukaryotes is poorly understood compared with those of prokaryotes. A number of independent investigations focusing on specific genes, individual genomes, or specific functional categories from various eukaryotes have...... indicated that lateral gene transfer does indeed affect eukaryotic genomes. However, the lack of common methodology and criteria in these studies makes it difficult to assess the general importance and influence of lateral gene transfer on eukaryotic genome evolution. RESULTS: We used a phylogenomic...... approach to systematically investigate lateral gene transfer affecting the proteomes of thirteen, mainly parasitic, microbial eukaryotes, representing four of the six eukaryotic super-groups. All of the genomes investigated have been significantly affected by prokaryote-to-eukaryote lateral gene transfers...
The significance of horizontal gene transfer (HGT) in eukaryotic evolution remains controversial. Although many eukaryotic genes are of bacterial origin, they are often interpreted as being derived from mitochondria or plastids. Because of their fixed gene pool and gene loss, however, mitochondria and plastids alone cannot adequately explain the presence of all, or even the majority, of bacterial genes in eukaryotes. Available data indicate that no insurmountable barrier to HGT exists, even in complex multicellular eukaryotes. In addition, the discovery of both recent and ancient HGT events in all major eukaryotic groups suggests that HGT has been a regular occurrence throughout the history of eukaryotic evolution. A model of HGT is proposed that suggests both unicellular and early developmental stages as likely entry points for foreign genes into multicellular eukaryotes. PMID:24037739
Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F
Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation.
Full Text Available Abstract Background Calpains are Ca2+-dependent cysteine proteases that participate in a range of crucial cellular processes. Dysfunction of these enzymes may cause, for instance, life-threatening diseases in humans, the loss of sex determination in nematodes and embryo lethality in plants. Although the calpain family is well characterized in animal and plant model organisms, there is a great lack of knowledge about these genes in unicellular eukaryote species (i.e. protists. Here, we study the distribution and evolution of calpain genes in a wide range of eukaryote genomes from major branches in the tree of life. Results Our investigations reveal 24 types of protein domains that are combined with the calpain-specific catalytic domain CysPc. In total we identify 41 different calpain domain architectures, 28 of these domain combinations have not been previously described. Based on our phylogenetic inferences, we propose that at least four calpain variants were established in the early evolution of eukaryotes, most likely before the radiation of all the major supergroups of eukaryotes. Many domains associated with eukaryotic calpain genes can be found among eubacteria or archaebacteria but never in combination with the CysPc domain. Conclusions The analyses presented here show that ancient modules present in prokaryotes, and a few de novo eukaryote domains, have been assembled into many novel domain combinations along the evolutionary history of eukaryotes. Some of the new calpain genes show a narrow distribution in a few branches in the tree of life, likely representing lineage-specific innovations. Hence, the functionally important classical calpain genes found among humans and vertebrates make up only a tiny fraction of the calpain family. In fact, a massive expansion of the calpain family occurred by domain shuffling among unicellular eukaryotes and contributed to a wealth of functionally different genes.
Steven W Paugh
Full Text Available MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10(-16 for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.
Thiergart, Thorsten; Landan, Giddy; Schenk, Marc; Dagan, Tal; Martin, William F.
To test the predictions of competing and mutually exclusive hypotheses for the origin of eukaryotes, we identified from a sample of 27 sequenced eukaryotic and 994 sequenced prokaryotic genomes 571 genes that were present in the eukaryote common ancestor and that have homologues among eubacterial and archaebacterial genomes. Maximum-likelihood trees identified the prokaryotic genomes that most frequently contained genes branching as the sister to the eukaryotic nuclear homologues. Among the archaebacteria, euryarchaeote genomes most frequently harbored the sister to the eukaryotic nuclear gene, whereas among eubacteria, the α-proteobacteria were most frequently represented within the sister group. Only 3 genes out of 571 gave a 3-domain tree. Homologues from α-proteobacterial genomes that branched as the sister to nuclear genes were found more frequently in genomes of facultatively anaerobic members of the rhiozobiales and rhodospirilliales than in obligate intracellular ricketttsial parasites. Following α-proteobacteria, the most frequent eubacterial sister lineages were γ-proteobacteria, δ-proteobacteria, and firmicutes, which were also the prokaryote genomes least frequently found as monophyletic groups in our trees. Although all 22 higher prokaryotic taxa sampled (crenarchaeotes, γ-proteobacteria, spirochaetes, chlamydias, etc.) harbor genes that branch as the sister to homologues present in the eukaryotic common ancestor, that is not evidence of 22 different prokaryotic cells participating at eukaryote origins because prokaryotic “lineages” have laterally acquired genes for more than 1.5 billion years since eukaryote origins. The data underscore the archaebacterial (host) nature of the eukaryotic informational genes and the eubacterial (mitochondrial) nature of eukaryotic energy metabolism. The network linking genes of the eukaryote ancestor to contemporary homologues distributed across prokaryotic genomes elucidates eukaryote gene origins in a
Full Text Available The ability to predict gene content is highly desirable for characterization of not-yet sequenced genomes like those of dinoflagellates. Using data from completely sequenced and annotated genomes from phylogenetically diverse lineages, we investigated the relationship between gene content and genome size using regression analyses. Distinct relationships between log(10-transformed protein-coding gene number (Y' versus log(10-transformed genome size (X', genome size in kbp were found for eukaryotes and non-eukaryotes. Eukaryotes best fit a logarithmic model, Y' = ln(-46.200+22.678X', whereas non-eukaryotes a linear model, Y' = 0.045+0.977X', both with high significance (p0.91. Total gene number shows similar trends in both groups to their respective protein coding regressions. The distinct correlations reflect lower and decreasing gene-coding percentages as genome size increases in eukaryotes (82%-1% compared to higher and relatively stable percentages in prokaryotes and viruses (97%-47%. The eukaryotic regression models project that the smallest dinoflagellate genome (3x10(6 kbp contains 38,188 protein-coding (40,086 total genes and the largest (245x10(6 kbp 87,688 protein-coding (92,013 total genes, corresponding to 1.8% and 0.05% gene-coding percentages. These estimates do not likely represent extraordinarily high functional diversity of the encoded proteome but rather highly redundant genomes as evidenced by high gene copy numbers documented for various dinoflagellate species.
Pallisgaard, N; Pedersen, FS; Birkelund, Svend
Here, we describe the construction of plasmid vectors facilitating expression of cloned genes in bacteria and in cells of mammalian and insect origin. Two types of multiple cloning site (MCS) were designed based on the MCS in the expression vector lambda gt11Sfi-Not. In the first set of vectors...... a start Met codon was included in the same reading frame as in lambda gt11Sfi-Not to support expression of partial cDNA clones. Thus a cDNA insert of lambda gt11Sfi-Not could be shuttled among the new vectors for expression. The other set of vectors without a start codon were suitable for expression of cDNA...... carrying their own start Met codon. By Western blot analysis and by transactivation of a reporter plasmid in co-transfections we show that cDNA is very efficiently expressed in NIH 3T3 cells under control of the elongation factor 1 alpha promoter....
Chen, Lifeng; Liu, Hongfang; Friedman, Carol
With more and more scientific literature published online, the effective management and reuse of this knowledge has become problematic. Natural language processing (NLP) may be a potential solution by extracting, structuring and organizing biomedical information in online literature in a timely manner. One essential task is to recognize and identify genomic entities in text. 'Recognition' can be accomplished using pattern matching and machine learning. But for 'identification' these techniques are not adequate. In order to identify genomic entities, NLP needs a comprehensive resource that specifies and classifies genomic entities as they occur in text and that associates them with normalized terms and also unique identifiers so that the extracted entities are well defined. Online organism databases are an excellent resource to create such a lexical resource. However, gene name ambiguity is a serious problem because it affects the appropriate identification of gene entities. In this paper, we explore the extent of the problem and suggest ways to address it. We obtained gene information from 21 organisms and quantified naming ambiguities within species, across species, with English words and with medical terms. When the case (of letters) was retained, official symbols displayed negligible intra-species ambiguity (0.02%) and modest ambiguities with general English words (0.57%) and medical terms (1.01%). In contrast, the across-species ambiguity was high (14.20%). The inclusion of gene synonyms increased intra-species ambiguity substantially and full names contributed greatly to gene-medical-term ambiguity. A comprehensive lexical resource that covers gene information for the 21 organisms was then created and used to identify gene names by using a straightforward string matching program to process 45,000 abstracts associated with the mouse model organism while ignoring case and gene names that were also English words. We found that 85.1% of correctly retrieved mouse
Terkelsen, Kasper Munch; Krogh, A.
Background The number of sequenced eukaryotic genomes is rapidly increasing. This means that over time it will be hard to keep supplying customised gene finders for each genome. This calls for procedures to automatically generate species-specific gene finders and to re-train them as the quantity...... length distributions. The performance of each individual gene predictor on each individual genome is comparable to the best of the manually optimised species-specific gene finders. It is shown that species-specific gene finders are superior to gene finders trained on other species....
Wen, Ling; Yue, Ling; Shi, Yi; Ren, Lili; Chen, Tingting; Li, Na; Zhang, Shuyu; Yang, Wei; Yang, Zhanshan
PprI accelerates radiation-induced DNA damage repair via regulating the expression of DNA repair genes and enhances antioxidative enzyme activity in Deinococcus radiodurans after radiation. The main aim of our study was to determine whether the expression of pprI gene could fulfil its DNA repair function in eukaryotes and enhance the radioresistance of eukaryotic organism or not. In this study, we constructed pEGFP-c1-pprI eukaryotic expression vector and established a human lung epithelial cell line BEAS-2B with stable integration of pprI gene. We found that pprIexpression enhanced radioresistance of BEAS-2B cells, decreased γ-H2AX foci formation and apoptosis in irradiated BEAS-2B cells and alleviated radiation induced G2/M arrest of BEAS-2B cells. Moreover, we transferred pEGFP-c1-pprI vector into muscle of BALB/c mice by in vivo electroporation and studied the protective effect of prokaryotic pprI gene in irradiated mice. We found that pprI expression alleviated acute radiation induced hematopoietic system, lung, small intestine and testis damage and increased survival rate of irradiated mice via regulating Rad51 expression in different organs. These findings suggest that prokaryotic pprI gene expression in mammalian cells could enhance radioresistance in vitro and in vivo.
Weber Andreas PM
Full Text Available Abstract Background Euglenophytes are a group of photosynthetic flagellates possessing a plastid derived from a green algal endosymbiont, which was incorporated into an ancestral host cell via secondary endosymbiosis. However, the impact of endosymbiosis on the euglenophyte nuclear genome is not fully understood due to its complex nature as a 'hybrid' of a non-photosynthetic host cell and a secondary endosymbiont. Results We analyzed an EST dataset of the model euglenophyte Euglena gracilis using a gene mining program designed to detect laterally transferred genes. We found E. gracilis genes showing affinity not only with green algae, from which the secondary plastid in euglenophytes evolved, but also red algae and/or secondary algae containing red algal-derived plastids. Phylogenetic analyses of these 'red lineage' genes suggest that E. gracilis acquired at least 14 genes via eukaryote-to-eukaryote lateral gene transfer from algal sources other than the green algal endosymbiont that gave rise to its current plastid. We constructed an EST library of the aplastidic euglenid Peranema trichophorum, which is a eukaryovorous relative of euglenophytes, and also identified 'red lineage' genes in its genome. Conclusions Our data show genome mosaicism in E. gracilis and P. trichophorum. One possible explanation for the presence of these genes in these organisms is that some or all of them were independently acquired by lateral gene transfer and contributed to the successful integration and functioning of the green algal endosymbiont as a secondary plastid. Alternative hypotheses include the presence of a phagocytosed alga as the single source of those genes, or a cryptic tertiary endosymbiont harboring secondary plastid of red algal origin, which the eukaryovorous ancestor of euglenophytes had acquired prior to the secondary endosymbiosis of a green alga.
Schönknecht, Gerald; Weber, Andreas P M; Lercher, Martin J
In contrast to vertical gene transfer from parent to offspring, horizontal (or lateral) gene transfer moves genetic information between different species. Bacteria and archaea often adapt through horizontal gene transfer. Recent analyses indicate that eukaryotic genomes, too, have acquired numerous genes via horizontal transfer from prokaryotes and other lineages. Based on this we raise the hypothesis that horizontally acquired genes may have contributed more to adaptive evolution of eukaryotes than previously assumed. Current candidate sets of horizontally acquired eukaryotic genes may just be the tip of an iceberg. We have recently shown that adaptation of the thermoacidophilic red alga Galdieria sulphuraria to its hot, acid, toxic-metal laden, volcanic environment was facilitated by the acquisition of numerous genes from extremophile bacteria and archaea. Other recently published examples of horizontal acquisitions involved in adaptation include ice-binding proteins in marine algae, enzymes for carotenoid biosynthesis in aphids, and genes involved in fungal metabolism. Editor's suggested further reading in BioEssays Jumping the fine LINE between species: Horizontal transfer of transposable elements in animals catalyses genome evolution Abstract. © 2014 WILEY Periodicals, Inc.
Ma, L J; Wang, G J; Li, L; Hong, X; Pei, X T
Bone marrow stromal cell line-HFCL were transfected with the recombinant eukaryotic expressing vector-pIRESlneo/hFL by using liposome-mediated gene transfer method and get a stable expression. HFCL cells were transfected with the recombinant eukaryotic expressing vector-pIRESlneo/hFL by using liposome lipofectamine. Integration of hFL in the genome, transcription of hFL mRNA and expression of hFL protein in the transfected HFCL cells were assayed by Southern blot, Northern blot, Western blot and ELISA, the experiment of the human umbilical blood CD34+ cell multiplication. hFL cDNA was integrated into HFCL genome successfully, hFL mRNA was transcripted, hFL protein was expressed with (60.3 +/- 0.1) ng. 10(6) cell(-1) x d(-1) and the experiment of the human umbilical blood CD34+ cell multiplication shows that hFL has obvious biological activity in the supernatant. The recombinant plasmid is proved to be stably expressed in HFCL cells and obvious biological activity of hFL was detectable in the supernatant of the transfected cells.
Yuri M. Moshkin
Full Text Available Most of eukaryotic DNA is embedded into nucleosome arrays formed by DNA wrapped around a core histone octamer. Nucleosome is a fundamental repeating unit of chromatin guarding access to the genetic information. Here, I will discuss two facets of nucleosome in eukaryotic gene control. On the one hand, nucleosome acts as a regulatory unit, which controls gene switches through a set of post-translational modifications occurring on histone tails. On the other hand, global configuration of nucleosome arrays with respect to nucleosome positioning, spacing and turnover acts as a tuning parameter for all genomic functions. A “histone code” hypothesis extents the Jacob-Monod model for eukaryotic gene control; however, when considering factors capable of reconfiguring entire nucleosome array, such as ATP-dependent chromatin remodelers, this model becomes limited. Global changes in nucleosome arrays will be sensed by every gene, yet the transcriptional responses might be specific and appear as gene targeted events. What determines such specificity is unclear, but it’s likely to depend on initial gene settings, such as availability of transcription factors, and on configuration of new nucleosome array state.
Richards, Thomas A; Dacks, Joel B; Jenkinson, Joanna M; Thornton, Christopher R; Talbot, Nicholas J
Filamentous fungi and oomycetes are eukaryotic microorganisms that grow by producing networks of thread-like hyphae, which secrete enzymes to break down complex nutrients, such as wood and plant material, and recover the resulting simple sugars and amino acids by osmotrophy. These organisms are extremely similar in both appearance and lifestyle and include some of the most economically important plant pathogens . However, the morphological similarity of fungi and oomycetes is misleading because they represent some of the most distantly related eukaryote evolutionary groupings, and their shared osmotrophic growth habit is interpreted as being the result of convergent evolution . The fungi branch with the animals, whereas the oomycetes branch with photosynthetic algae as part of the Chromalveolata . In this report, we provide strong phylogenetic evidence that multiple horizontal gene transfers (HGT) have occurred from filamentous ascomycete fungi to the distantly related oomycetes. We also present evidence that a subset of the associated gene families was initially the product of prokaryote-to-fungi HGT. The predicted functions of the gene products associated with fungi-to-oomycete HGT suggest that this process has played a significant role in the evolution of the osmotrophic, filamentous lifestyle on two separate branches of the eukaryote tree.
Full Text Available Abstract Background In addition to vertical transmission, organisms can also acquire genes from other distantly related species or from their extra-chromosomal elements (plasmids and viruses via horizontal gene transfer (HGT. It has been suggested that phages represent substantial forces in prokaryotic evolution. In eukaryotes, retroviruses, which can integrate into host genome as an obligate step in their replication strategy, comprise approximately 8% of the human genome. Unlike retroviruses, few members of other virus families are known to transfer genes to host genomes. Results Here we performed a systematic search for sequences related to circular single-stranded DNA (ssDNA viruses in publicly available eukaryotic genome databases followed by comprehensive phylogenetic analysis. We conclude that the replication initiation protein (Rep-related sequences of geminiviruses, nanoviruses and circoviruses have been frequently transferred to a broad range of eukaryotic species, including plants, fungi, animals and protists. Some of the transferred viral genes were conserved and expressed, suggesting that these genes have been coopted to assume cellular functions in the host genomes. We also identified geminivirus-like and parvovirus-like transposable elements in genomes of fungi and lower animals, respectively, and thereby provide direct evidence that eukaryotic transposons could derive from ssDNA viruses. Conclusions Our discovery extends the host range of circular ssDNA viruses and sheds light on the origin and evolution of these viruses. It also suggests that ssDNA viruses act as an unforeseen source of genetic innovation in their hosts.
Full Text Available In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i the nasF transcriptional attenuator, which reduces basal expression levels, (ii a strong ribosome binding site, and (iii the Type III Secretion System (TTSS signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.
Roger Andrew J
Full Text Available Abstract Background Lateral gene transfer can introduce genes with novel functions into genomes or replace genes with functionally similar orthologs or paralogs. Here we present a study of the occurrence of the latter gene replacement phenomenon in the four gene families encoding different classes of glutamate dehydrogenase (GDH, to evaluate and compare the patterns and rates of lateral gene transfer (LGT in prokaryotes and eukaryotes. Results We extend the taxon sampling of gdh genes with nine new eukaryotic sequences and examine the phylogenetic distribution pattern of the various GDH classes in combination with maximum likelihood phylogenetic analyses. The distribution pattern analyses indicate that LGT has played a significant role in the evolution of the four gdh gene families. Indeed, a number of gene transfer events are identified by phylogenetic analyses, including numerous prokaryotic intra-domain transfers, some prokaryotic inter-domain transfers and several inter-domain transfers between prokaryotes and microbial eukaryotes (protists. Conclusion LGT has apparently affected eukaryotes and prokaryotes to a similar extent within the gdh gene families. In the absence of indications that the evolution of the gdh gene families is radically different from other families, these results suggest that gene transfer might be an important evolutionary mechanism in microbial eukaryote genome evolution.
Full Text Available Abstract Background The ferlin gene family possesses a rare and identifying feature consisting of multiple tandem C2 domains and a C-terminal transmembrane domain. Much currently remains unknown about the fundamental function of this gene family, however, mutations in its two most well-characterised members, dysferlin and otoferlin, have been implicated in human disease. The availability of genome sequences from a wide range of species makes it possible to explore the evolution of the ferlin family, providing contextual insight into characteristic features that define the ferlin gene family in its present form in humans. Results Ferlin genes were detected from all species of representative phyla, with two ferlin subgroups partitioned within the ferlin phylogenetic tree based on the presence or absence of a DysF domain. Invertebrates generally possessed two ferlin genes (one with DysF and one without, with six ferlin genes in most vertebrates (three DysF, three non-DysF. Expansion of the ferlin gene family is evident between the divergence of lamprey (jawless vertebrates and shark (cartilaginous fish. Common to almost all ferlins is an N-terminal C2-FerI-C2 sandwich, a FerB motif, and two C-terminal C2 domains (C2E and C2F adjacent to the transmembrane domain. Preservation of these structural elements throughout eukaryotic evolution suggests a fundamental role of these motifs for ferlin function. In contrast, DysF, C2DE, and FerA are optional, giving rise to subtle differences in domain topologies of ferlin genes. Despite conservation of multiple C2 domains in all ferlins, the C-terminal C2 domains (C2E and C2F displayed higher sequence conservation and greater conservation of putative calcium binding residues across paralogs and orthologs. Interestingly, the two most studied non-mammalian ferlins (Fer-1 and Misfire in model organisms C. elegans and D. melanogaster, present as outgroups in the phylogenetic analysis, with results suggesting
Chew Chieng Yeo
Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.
Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann
Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.
Marcela Dávila López
Full Text Available The order of genes in eukaryotes is not entirely random. Studies of gene order conservation are important to understand genome evolution and to reveal mechanisms why certain neighboring genes are more difficult to separate during evolution. Here, genome-wide gene order information was compiled for 64 species, representing a wide variety of eukaryotic phyla. This information is presented in a browser where gene order may be displayed and compared between species. Factors related to non-random gene order in eukaryotes were examined by considering pairs of neighboring genes. The evolutionary conservation of gene pairs was studied with respect to relative transcriptional direction, intergenic distance and functional relationship as inferred by gene ontology. The results show that among gene pairs that are conserved the divergently and co-directionally transcribed genes are much more common than those that are convergently transcribed. Furthermore, highly conserved pairs, in particular those of fungi, are characterized by a short intergenic distance. Finally, gene pairs of metazoa and fungi that are evolutionary conserved and that are divergently transcribed are much more likely to be related by function as compared to poorly conserved gene pairs. One example is the ribosomal protein gene pair L13/S16, which is unusual as it occurs both in fungi and alveolates. A specific functional relationship between these two proteins is also suggested by the fact that they are part of the same operon in both eubacteria and archaea. In conclusion, factors associated with non-random gene order in eukaryotes include relative gene orientation, intergenic distance and functional relationships. It seems likely that certain pairs of genes are conserved because the genes involved have a transcriptional and/or functional relationship. The results also indicate that studies of gene order conservation aid in identifying genes that are related in terms of transcriptional
Hoff, Katharina J.; Stanke, Mario
The prediction of protein coding genes is an important step in the annotation of newly sequenced and assembled genomes. AUGUSTUS is one of the most accurate tools for eukaryotic gene prediction. Here, we present WebAUGUSTUS, a web interface for training AUGUSTUS and predicting genes with AUGUSTUS. Depending on the needs of the user, WebAUGUSTUS generates training gene structures automatically. Besides a genome file, either a file with expressed sequence tags or a file with protein sequences is required for this step. Alternatively, it is possible to submit an externally generated training gene structure file and a genome file. The web service optimizes AUGUSTUS parameters and predicts genes with those parameters. WebAUGUSTUS is available at http://bioinf.uni-greifswald.de/webaugustus. PMID:23700307
Hoff, Katharina J; Stanke, Mario
The prediction of protein coding genes is an important step in the annotation of newly sequenced and assembled genomes. AUGUSTUS is one of the most accurate tools for eukaryotic gene prediction. Here, we present WebAUGUSTUS, a web interface for training AUGUSTUS and predicting genes with AUGUSTUS. Depending on the needs of the user, WebAUGUSTUS generates training gene structures automatically. Besides a genome file, either a file with expressed sequence tags or a file with protein sequences is required for this step. Alternatively, it is possible to submit an externally generated training gene structure file and a genome file. The web service optimizes AUGUSTUS parameters and predicts genes with those parameters. WebAUGUSTUS is available at http://bioinf.uni-greifswald.de/webaugustus.
Brian L Zielinski
Full Text Available The Amazon River has the largest discharge of all rivers on Earth, and its complex plume system fuels a wide array of biogeochemical processes, across a large area of the western tropical North Atlantic. The plume thus stimulates microbial processes affecting carbon sequestration and nutrient cycles at a global scale. Chromosomal gene expression patterns of the 2.0 to 156 μm size-fraction eukaryotic microbial community were investigated in the Amazon River Plume, generating a robust dataset (more than 100 million mRNA sequences that depicts the metabolic capabilities and interactions among the eukaryotic microbes. Combining classical oceanographic field measurements with metatranscriptomics yielded characterization of the hydrographic conditions simultaneous with a quantification of transcriptional activity and identity of the community. We highlight the patterns of eukaryotic gene expression for 31 biogeochemically significant gene targets hypothesized to be valuable within forecasting models. An advantage to this targeted approach is that the database of reference sequences used to identify the target genes was selectively constructed and highly curated optimizing taxonomic coverage, throughput, and the accuracy of annotations. A coastal diatom bloom highly expressed nitrate transporters and carbonic anhydrase presumably to support high growth rates and enhance uptake of low levels of dissolved nitrate and CO2. Diatom-diazotroph association (DDA: diatoms with nitrogen fixing symbionts blooms were common when surface salinity was mesohaline and dissolved nitrate concentrations were below detection, and hence did not show evidence of nitrate utilization, suggesting they relied on ammonium transporters to aquire recently fixed nitrogen. These DDA blooms in the outer plume had rapid turnover of the photosystem D1 protein presumably caused by photodegradation under increased light penetration in clearer waters, and increased expression of silicon
Chu, Jennifer; Cajal, Santiago Ramon Y; Sonenberg, Nahum; Pelletier, Jerry
There is enormous diversity in the genetic makeup and gene expression profiles between and within tumors. This heterogeneity leads to phenotypic variation and is a major mechanism of resistance to molecular targeted therapies. Here we describe a conceptual framework for targeting eukaryotic initiation factor (eIF) 4F in cancer-an essential complex that drives and promotes multiple Cancer Hallmarks. The unique nature of eIF4F and its druggability bypasses several of the heterogeneity issues that plague molecular targeted drugs developed for cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.
Manna, Sam; Harman, Ashley
tRNA-guanine transglycosylases are found in all domains of life and mediate the base exchange of guanine with queuine in the anticodon loop of tRNAs. They can also regulate virulence in bacteria such as Shigella flexneri, which has prompted the development of drugs that inhibit the function of these enzymes. Here we report a group of tRNA-guanine transglycosylases in eukaryotic microbes (algae and protozoa) which are more similar to their bacterial counterparts than previously characterized eukaryotic tRNA-guanine transglycosylases. We provide evidence demonstrating that the genes encoding these enzymes were acquired by these eukaryotic lineages via horizontal gene transfer from the Chlamydiae group of bacteria. Given that the S. flexneri tRNA-guanine transglycosylase can be targeted by drugs, we propose that the bacterial-like tRNA-guanine transglycosylases could potentially be targeted in a similar fashion in pathogenic amoebae that possess these enzymes such as Acanthamoeba castellanii. This work also presents ancient prokaryote-to-eukaryote horizontal gene transfer events as an untapped resource of potential drug target identification in pathogenic eukaryotes. Copyright © 2015 Elsevier Inc. All rights reserved.
Haas, B J; Salzberg, S L; Zhu, W; Pertea, M; Allen, J E; Orvis, J; White, O; Buell, C R; Wortman, J R
EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.
Full Text Available AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2, transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 in vitro and in vivo and to assess the probability of defensins as a new application for infectious corneal diseases in the future. METHODS: The synthetic rBD-2 DNA fragment was inserted between the XhoI and BamHI restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into E.coli DH5α, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in
Keeling Patrick J
Full Text Available Abstract Background Horizontal or lateral transfer of genetic material between distantly related prokaryotes has been shown to play a major role in the evolution of bacterial and archaeal genomes, but exchange of genes between prokaryotes and eukaryotes is not as well understood. In particular, gene flow from eukaryotes to prokaryotes is rarely documented with strong support, which is unusual since prokaryotic genomes appear to readily accept foreign genes. Results Here, we show that abundant marine cyanobacteria in the related genera Synechococcus and Prochlorococcus acquired a key Calvin cycle/glycolytic enzyme from a eukaryote. Two non-homologous forms of fructose bisphosphate aldolase (FBA are characteristic of eukaryotes and prokaryotes respectively. However, a eukaryotic gene has been inserted immediately upstream of the ancestral prokaryotic gene in several strains (ecotypes of Synechococcus and Prochlorococcus. In one lineage this new gene has replaced the ancestral gene altogether. The eukaryotic gene is most closely related to the plastid-targeted FBA from red algae. This eukaryotic-type FBA once replaced the plastid/cyanobacterial type in photosynthetic eukaryotes, hinting at a possible functional advantage in Calvin cycle reactions. The strains that now possess this eukaryotic FBA are scattered across the tree of Synechococcus and Prochlorococcus, perhaps because the gene has been transferred multiple times among cyanobacteria, or more likely because it has been selectively retained only in certain lineages. Conclusion A gene for plastid-targeted FBA has been transferred from red algae to cyanobacteria, where it has inserted itself beside its non-homologous, functional analogue. Its current distribution in Prochlorococcus and Synechococcus is punctate, suggesting a complex history since its introduction to this group.
Dadashipour, Mohammad; Fukuta, Yasuhisa; Asano, Yasuhisa
Low protein solubility and inclusion body formation represent big challenges in production of recombinant proteins in Escherichia coli. We have recently reported functional expression of hydroxynitrile lyase from Manihot esculenta, MeHNL, in E. coli with high in vivo solubility and activity using directed evolution. As a part of attempts to clarify the mechanism of this phenomenon, we have described the possibility of expression of the highly active and soluble mutant MeHNL-His103Leu as well as wild-type enzyme in several expression systems. Methylotrophic yeast Pichia pastoris, protozoan host Leishmania tarentolae and two cell-free translations, including an E. coli lysate (WakoPURE system) and wheat germ translation system were used to compare expression profiles of the genes. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryotic-based systems. The wild-type and mutant enzyme showed high activity for both genes (up to 10 U/ml) in eukaryotic hosts P. pastoris and L. tarentolae, while those of E. coli exhibited about 1 and 15 U/ml, respectively. The different activity level in prokaryotic systems but the same level among the eukaryotic hosts indicate the phenomenon is specific to the E. coli system. Both the wild-type and mutant enzymes were functionally expressed in eukaryotic systems, probably using the folding assistants such as chaperones. Properties of expression systems used in this study were precisely compared, too. Copyright © 2011 Elsevier Inc. All rights reserved.
Ghanbarian, Avazeh T.; Hurst, Laurence D.
When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543
Cui, Hongli; Yu, Xiaona; Wang, Yan; Cui, Yulin; Li, Xueqin; Liu, Zhaopu; Qin, Song
. Protein domain structures and expression analyses in green alga H. pluvialis indicate that various chy genes are in different manners response to light. The knowledge of evolution of chy genes in photosynthetic eukaryotes provided information of gene cloning and functional investigation of chy genes in algae in the future.
green algae and higher plants. Protein domain structures and expression analyses in green alga H. pluvialis indicate that various chy genes are in different manners response to light. The knowledge of evolution of chy genes in photosynthetic eukaryotes provided information of gene cloning and functional investigation of chy genes in algae in the future. PMID:23834441
Watkins, Russell F; Gray, Michael W
Single-celled bacterivorous eukaryotes offer excellent test cases for evaluation of the frequency of prey-to-predator lateral gene transfer (LGT). Here we use analysis of expressed sequence tag (EST) data sets to quantify the extent of LGT from eubacteria to two amoebae, Acanthamoeba castellanii and Hartmannella vermiformis. Stringent screening for LGT proceeded in several steps intended to enrich for authentic events while at the same time minimizing the incidence of false positives due to factors such as limitations in database coverage and ancient paralogy. The results were compared with data obtained when the same methodology was applied to EST libraries from a number of other eukaryotic taxa. Significant differences in the extent of apparent eubacterium-to-eukaryote LGT were found between taxa. Our results indicate that there may be substantial inter-taxon variation in the number of LGT events that become fixed even between amoebozoan species that have similar feeding modalities.
da Silva Junior, Haroldo Cid; da Silva E Mouta Junior, Sérgio; de Mendonça, Marcos César Lima; de Souza Pereira, Mirian Claudia; da Rocha Nogueira, Alanderson; de Azevedo, Maria Luiza Borges; Leite, José Paulo Gagliardi; de Moraes, Márcia Terezinha Baroni
The VP6 protein of rotavirus A (RVA) is a target antigen used for diagnostic assays and also for the development of new RVA vaccines. We have compared the expression of VP6 protein in human embryonic kidney (HEK293-T) cells with results obtained using a well-established insect cell-baculovirus system. The recombinant VP6 (rVP6) expressed in HEK293-T cells did not present degradation and also retained the ability to form trimers. In the insect cell-baculovirus system, rVP6 was expressed at higher levels and with protein degradation as well as partial loss of ability to form trimers was observed. Therefore, HEK293-T cells represent a less laborious alternative system than insect cells for expression of rVP6 from human RVA.
Sun, Da-Kang; An, Xin-Ye; Hu, Feng-Ai; Li, Cai-Yu; Zheng, Jing
To construct the recombinant eukaryotic expression vector pcDNA3.1 (+)-Trim6, and observe its expression in HEK293T cells in vitro. The total RNA was isolated from HeLa cells. After amplification with reverse transcription polymerase chain reaction (RT-PCR), the target sequences were cloned into the pcDNA3.1(+). The recombinant vector was confirmed by restriction enzyme digestion, PCR and sequencing. Then it was transfected into HEK293T cells.After 24 hours, the Trim6 expression was detected by Western blot. The results of the restriction enzyme digestion, PCR and sequencing confirmed the vector was constructed successfully, and it can express Trim6 protein in HEK293T cells. The vector is constructed successfully, which establishes the foundation for future research on the effect of Trim6.
Transposable elements (TEs) are the major components of eukaryotic genomes. Their propensity to densely populate and in some cases invade the genomes of plants and animals is in contradiction with the fact that transposition is strictly controlled by several molecular pathways acting at either transcriptional or post-transcriptional levels. Horizontal transfers, defined as the transmission of genetic material between sexually isolated species, have long been considered as rare phenomena. Here, we show that the horizontal transfers of transposable elements (HTTs) are very frequent in ecosystems. The exact mechanisms of such transfers are not well understood, but species involved in close biotic interactions, like parasitism, show a propensity to exchange genetic material horizontally. We propose that HTTs allow TEs to escape the silencing machinery of their host genome and may therefore be an important mechanism for their survival and their dissemination in eukaryotes. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.
Xu, Ming-Yan; Liu, Ju-Li; Zhang, Ren-Li; Fu, Yu-cai
The Ras subfamily proteins are small, monomeric GTP-binding proteins with vital roles in regulating eukaryotic signal transduction pathways. Gene duplication and divergence have been postulated as the mechanism by which such family members have evolved their specific functions. A cDNA clone of TvRsp was isolated and sequenced from a cDNA expression library of the primitive eukaryote Trichomonas vaginalis. The genomic DNA corresponding to the cDNA sequence was amplified by PCR and sequenced. Sequence analysis suggested that TvRsp was an intronless gene. This gene encoded a protein of 181 amino acids and contained the 5 conserved G domains that designated it as a Ras or Rap subfamily member. However, the deduced amino acid sequence shared only 34%-37% overall identity with other Ras subfamily members of different species, and the presence of motifs characteristic of both the Ras and Rap families of GTPase confused the familial classification of this gene. Phylogenetic analysis showed its origins at the divergence point of the Ras/Rap families and suggested that TvRsp was a possible evolutionary ancestral gene of the ras/rap genes of higher eukaryotes. This information was of importance not only from the perspective of understanding the evolution and diversity of eukaryotic signal transduction pathways but also in providing a framework by which to understand protein processing in the growth and differentiation of single-celled microorganisms.
Whitaker, John W; McConkey, Glenn A; Westhead, David R
Metabolic networks are responsible for many essential cellular processes, and exhibit a high level of evolutionary conservation from bacteria to eukaryotes. If genes encoding metabolic enzymes are horizontally transferred and are advantageous, they are likely to become fixed. Horizontal gene transfer (HGT) has played a key role in prokaryotic evolution and its importance in eukaryotes is increasingly evident. High levels of endosymbiotic gene transfer (EGT) accompanied the establishment of plastids and mitochondria, and more recent events have allowed further acquisition of bacterial genes. Here, we present the first comprehensive multi-species analysis of E/HGT of genes encoding metabolic enzymes from bacteria to unicellular eukaryotes. The phylogenetic trees of 2,257 metabolic enzymes were used to make E/HGT assertions in ten groups of unicellular eukaryotes, revealing the sources and metabolic processes of the transferred genes. Analyses revealed a preference for enzymes encoded by genes gained through horizontal and endosymbiotic transfers to be connected in the metabolic network. Enrichment in particular functional classes was particularly revealing: alongside plastid related processes and carbohydrate metabolism, this highlighted a number of pathways in eukaryotic parasites that are rich in enzymes encoded by transferred genes, and potentially key to pathogenicity. The plant parasites Phytophthora were discovered to have a potential pathway for lipopolysaccharide biosynthesis of E/HGT origin not seen before in eukaryotes outside the Plantae. The number of enzymes encoded by genes gained through E/HGT has been established, providing insight into functional gain during the evolution of unicellular eukaryotes. In eukaryotic parasites, genes encoding enzymes that have been gained through horizontal transfer may be attractive drug targets if they are part of processes not present in the host, or are significantly diverged from equivalent host enzymes.
Primer 5.0 software. To adjust for RNA quality and diffe- rences in cDNA concentration, we amplified actin as an internal control with the following primers: PtActin-F (5′-TG. AAGGAGAAACTTGCGTAT-3′) and PtActin-R (5′-GCA. CAATGTTACCGTACAGAT-3′). These genes were ampli- fied from first-strand cDNA using ...
Irimia, Manuel; Rukov, Jakob Lewin; Penny, David
, and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional...... classes, cellular locations, intron/exon structures and evolutionary origins. RESULTS: For each species, we find that genes from most functional categories are alternatively spliced. Ancient genes (shared between animals, fungi and plants) show high levels of alternative splicing. Genes with products...... expressed in the nucleus or plasma membrane are generally more alternatively spliced while those expressed in extracellular location show less alternative splicing. We find a clear correspondence between incidence of alternative splicing and intron number per gene both within and between genomes. In general...
Ros, V.I.D.; Hurst, G.D.D.
The expansion of genome sequencing projects has produced accumulating evidence for lateral transfer of genes between prokaryotic and eukaryotic genomes. However, it remains controversial whether these genes are of functional importance in their recipient host. Nikoh and Nakabachi, in a recent paper
Li, Jin-Fu; Chen, Jian-Ping; Yang, Zhi-Wei; Tian, Yu; Ma, Ying; Hu, Xiao-Su
To construct recombinant eukaryotic expression plasmid of amastin gene of Leishmania Donovani and detect expression of the gene in NIH3T3 cells. Amastin gene was amplified from nuclear DNA of Leishmania Donovani isolates and cloned into an eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid was named pcDNA3.1-amastin. NIH3T3 cell was transfected by pcDNA3.1-amastin. Transient and stable expression of amastin gene were detected by immunofluoresence and RT-PCR. It was found that there was high green fluorescence on the cell membrane and inside the cell. It showed that NIH3T3 cell was transfected by pcDNA3.1-amastin successfully. A recombinant eukaryotic expression plasmid of amastin gene of Leishmania Donovani was successfully constructed, and can be expressed stably in the NIH3T3 cells.
Majewska, Małgorzata; Wysokińska, Halina; Kuźma, Łukasz; Szymczyk, Piotr
The complete exploration of the regulation of gene expression remains one of the top-priority goals for researchers. As the regulation is mainly controlled at the level of transcription by promoters, study on promoters and findings are of great importance. This review summarizes forty selected databases that centralize experimental and theoretical knowledge regarding the organization of promoters, interacting transcription factors (TFs) and microRNAs (miRNAs) in many eukaryotic and prokaryotic species. The presented databases offer researchers valuable support in elucidating the regulation of gene transcription. Copyright © 2017 Elsevier B.V. All rights reserved.
Full Text Available Photosynthesis in eukaryotes occurs in the plastid, an organelle that is derived from a single cyanobacterial primary endosymbiosis in the common ancestor of the supergroup Plantae (or Archaeplastida that includes green, red, and glaucophyte algae and plants. However a variety of other phytoplankton such as the chlorophyll c-containing diatoms, dinoflagellates, and haptophytes contain a red alga-derived plastid that traces its origin to secondary or tertiary (eukaryote engulfs eukaryote endosymbiosis. The hypothesis of Plantae monophyly has only recently been substantiated, however the extent and role of endosymbiotic and horizontal gene transfer (EGT and HGT in algal genome evolution still remain to be fully understood. What is becoming clear from analysis of complete genome data is that algal gene complements can no longer be considered essentially eukaryotic in provenance; i.e., with the expected addition of several hundred cyanobacterial genes derived from EGT and a similar number derived from the mitochondrial ancestor. For example, we now know that foreign cells such as Chlamydiae and other prokaryotes have made significant contributions to plastid functions in Plantae. Perhaps more surprising is the recent finding of extensive bacterium-derived HGT in the nuclear genome of the unicellular red alga Porphyridium purpureum that does not relate to plastid functions. These non-endosymbiont gene transfers not only shaped the evolutionary history of Plantae but also were propagated via secondary endosymbiosis to a multitude of other phytoplankton. Here we discuss the idea that Plantae (in particular red algae are one of the major players in eukaryote genome evolution by virtue of their ability to act as sinks and sources of foreign genes through HGT and endosymbiosis, respectively. This hypothesis recognizes the often under-appreciated Rhodophyta as major sources of genetic novelty among photosynthetic eukaryotes.
Artamonova, Irena I; Lappi, Tanya; Zudina, Liudmila; Mushegian, Arcady R
Assessment of phylogenetic positions of predicted gene and protein sequences is a routine step in any genome project, useful for validating the species' taxonomic position and for evaluating hypotheses about genome evolution and function. Several recent eukaryotic genome projects have reported multiple gene sequences that were much more similar to homologues in bacteria than to any eukaryotic sequence. In the spirit of the times, horizontal gene transfer from bacteria to eukaryotes has been invoked in some of these cases. Here, we show, using comparative sequence analysis, that some of those bacteria-like genes indeed appear likely to have been horizontally transferred from bacteria to eukaryotes. In other cases, however, the evidence strongly indicates that the eukaryotic DNA sequenced in the genome project contains a sample of non-integrated DNA from the actual bacteria, possibly providing a window into the host microbiome. Recent literature suggests also that common reagents, kits and laboratory equipment may be systematically contaminated with bacterial DNA, which appears to be sampled by metagenome projects non-specifically. We review several bioinformatic criteria that help to distinguish putative horizontal gene transfers from the admixture of genes from autonomously replicating bacteria in their hosts' genome databases or from the reagent contamination. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.
Vaněk, O.; Celadová, P.; Kolenko, Petr; Dohnálek, Jan; Bezouška, Karel
Roč. 276, Suppl. 1 (2009), s. 170 ISSN 1742-464X. [FEBS Congress "Life´s Molecular Interactions /34./. 04.07.2009-09.07.2009, Praha] Institutional research plan: CEZ:AV0Z40500505 Keywords : NK cell lectin receptors * HEK293T * eukaryotic expression system Subject RIV: CD - Macromolecular Chemistry
Shiba, Hiroshi; Takayama, Seiji
Monoallelic expression from biallelic genes is frequently observed in diploid eukaryotic organisms. Classic examples of this phenomenon include the well-characterized cases of genomic imprinting and X-chromosome inactivation. However, recent studies have shown that monoallelic expression is widespread in autosomal genes. This discovery was met with great interest because it represents another mechanism to generate diversity in gene expression that can affect cell fate and physiology. To date, the molecular mechanisms underlying this phenomenon are largely unknown. In our original study describing the dominant ⁄ recessive relationships of pollen- determinant alleles in Brassica self-incompatibility, we found that the recessive allele was specifically methylated and silenced through the action of small RNA derived from the dominant allele. In this review, we focus on recent studies of monoallelic expression in autosomal genes, and discuss the possible mechanisms driving this form of monoallelic gene suppression.
Moran, Yehu; Fredman, David; Szczesny, Pawel; Grynberg, Marcin; Technau, Ulrich
In this work, we report likely recurrent horizontal (lateral) gene transfer events of genes encoding pore-forming toxins of the aerolysin family between species belonging to different kingdoms of life. Clustering based on pairwise similarity and phylogenetic analysis revealed several distinct aerolysin sequence groups, each containing proteins from multiple kingdoms of life. These results strongly support at least six independent transfer events between distantly related phyla in the evolutionary history of one protein family and discount selective retention of ancestral genes as a plausible explanation for this patchy phylogenetic distribution. We discuss the possible roles of these proteins and show evidence for a convergent new function in two extant species. We hypothesize that certain gene families are more likely to be maintained following horizontal gene transfer from commensal or pathogenic organism to its host if they 1) can function alone; and 2) are immediately beneficial for the ecology of the organism, as in the case of pore-forming toxins which can be utilized in multicellular organisms for defense and predation.
Schönknecht, Gerald; Chen, Wei-Hua; Ternes, Chad M; Barbier, Guillaume G; Shrestha, Roshan P; Stanke, Mario; Bräutigam, Andrea; Baker, Brett J; Banfield, Jillian F; Garavito, R Michael; Carr, Kevin; Wilkerson, Curtis; Rensing, Stefan A; Gagneul, David; Dickenson, Nicholas E; Oesterhelt, Christine; Lercher, Martin J; Weber, Andreas P M
Some microbial eukaryotes, such as the extremophilic red alga Galdieria sulphuraria, live in hot, toxic metal-rich, acidic environments. To elucidate the underlying molecular mechanisms of adaptation, we sequenced the 13.7-megabase genome of G. sulphuraria. This alga shows an enormous metabolic flexibility, growing either photoautotrophically or heterotrophically on more than 50 carbon sources. Environmental adaptation seems to have been facilitated by horizontal gene transfer from various bacteria and archaea, often followed by gene family expansion. At least 5% of protein-coding genes of G. sulphuraria were probably acquired horizontally. These proteins are involved in ecologically important processes ranging from heavy-metal detoxification to glycerol uptake and metabolism. Thus, our findings show that a pan-domain gene pool has facilitated environmental adaptation in this unicellular eukaryote.
He, Ding; Fu, Cheng-Jie; Baldauf, Sandra L
The most gene-rich and bacterial-like mitochondrial genomes known are those of Jakobida (Excavata). Of these, the most extreme example to date is the Andalucia godoyi mitochondrial DNA (mtDNA), including a cox15 gene encoding the respiratory enzyme heme A synthase (HAS), which is nuclear-encoded in nearly all other mitochondriate eukaryotes. Thus cox15 in eukaryotes appears to be a classic example of mitochondrion-to-nucleus (endosymbiotic) gene transfer, with A. godoyi uniquely retaining the ancestral state. However, our analyses reveal two highly distinct HAS types (encoded by cox15-1 and cox15-2 genes) and identify A. godoyi mitochondrial cox15-encoded HAS as type-1 and all other eukaryotic cox15-encoded HAS as type-2. Molecular phylogeny places the two HAS types in widely separated clades with eukaryotic type-2 HAS clustering with the bulk of α-proteobacteria (>670 sequences), whereas A. godoyi type-1 HAS clusters with an eclectic set of bacteria and archaea including two α-proteobacteria missing from the type-2 clade. This wide phylogenetic separation of the two HAS types is reinforced by unique features of their predicted protein structures. Meanwhile, RNA-sequencing and genomic analyses fail to detect either cox15 type in the nuclear genome of any jakobid including A. godoyi. This suggests that not only is cox15-1 a relatively recent acquisition unique to the Andalucia lineage but also the jakobid last common ancestor probably lacked both cox15 types. These results indicate that uptake of foreign genes by mtDNA is more taxonomically widespread than previously thought. They also caution against the assumption that all α-proteobacterial-like features of eukaryotes are ancient remnants of endosymbiosis. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: email@example.com.
U.S. Department of Health & Human Services — Gene Expression Omnibus is a public functional genomics data repository supporting MIAME-compliant submissions of array- and sequence-based data. Tools are provided...
Xiong, Qiang; Xu, Zheng; Xu, Lu; Yao, Zhong; Li, Sha; Xu, Hong
γ-Aminobutyric acid (γ-GABA) is a non-proteinogenic amino acid, which acts as a major regulator in the central nervous system. Glutamate decarboxylase (namely GAD, EC 184.108.40.206) is known to be an ideal enzyme for γ-GABA production using L-glutamic acid as substrate. In this study, we cloned and expressed GAD gene from eukaryote Saccharomyces cerevisiae (ScGAD) in E. coli BL21(DE3). This enzyme was further purified and its optimal reaction temperature and pH were 37 °C and pH 4.2, respectively. The cofactor of ScGAD was verified to be either pyridoxal 5'-phosphate (PLP) or pyridoxal hydrochloride. The optimal concentration of either cofactor was 50 mg/L. The optimal medium for E. coli-ScGAD cultivation and expression were 10 g/L lactose, 5 g/L glycerol, 20 g/L yeast extract, and 10 g/L sodium chloride, resulting in an activity of 55 U/mL medium, three times higher than that of using Luria-Bertani (LB) medium. The maximal concentration of γ-GABA was 245 g/L whereas L-glutamic acid was near completely converted. These findings provided us a good example for bio-production of γ-GABA using recombinant E. coli expressing a GAD enzyme derived from eukaryote.
Marchisio, Mario Andrea; Colaiacovo, Moreno; Whitehead, Ellis; Stelling, Jörg
The modular design of synthetic gene circuits via composable parts (DNA segments) and pools of signal carriers (molecules such as RNA polymerases and ribosomes) has been successfully applied to bacterial systems. However, eukaryotic cells are becoming a preferential host for new synthetic biology applications. Therefore, an accurate description of the intricate network of reactions that take place inside eukaryotic parts and pools is necessary. Rule-based modeling approaches are increasingly used to obtain compact representations of reaction networks in biological systems. However, this approach is intrinsically non-modular and not suitable per se for the description of composable genetic modules. In contrast, the Model Description Language (MDL) adopted by the modeling tool ProMoT is highly modular and it enables a faithful representation of biological parts and pools. We developed a computational framework for the design of complex (eukaryotic) gene circuits by generating dynamic models of parts and pools via the joint usage of the BioNetGen rule-based modeling approach and MDL. The framework converts the specification of a part (or pool) structure into rules that serve as inputs for BioNetGen to calculate the part's species and reactions. The BioNetGen output is translated into an MDL file that gives a complete description of all the reactions that take place inside the part (or pool) together with a proper interface to connect it to other modules in the circuit. In proof-of-principle applications to eukaryotic Boolean circuits with more than ten genes and more than one thousand reactions, our framework yielded proper representations of the circuits' truth tables. For the model-based design of increasingly complex gene circuits, it is critical to achieve exact and systematic representations of the biological processes with minimal effort. Our computational framework provides such a detailed and intuitive way to design new and complex synthetic gene circuits.
Butler Margaret I
Full Text Available Abstract Background Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi. Results We identified seven intein coding sequences within nuclear genes coding for the second largest subunits of RNA polymerase. These sequences were found in diverse eukaryotes: one is in the second largest subunit of RNA polymerase I (RPA2 from the ascomycete fungus Phaeosphaeria nodorum, one is in the RNA polymerase III (RPC2 of the slime mould Dictyostelium discoideum and four intein coding sequences are in RNA polymerase II genes (RPB2, one each from the green alga Chlamydomonas reinhardtii, the zygomycete fungus Spiromyces aspiralis and the chytrid fungi Batrachochytrium dendrobatidis and Coelomomyces stegomyiae. The remaining intein coding sequence is in a viral relic embedded within the genome of the oomycete Phytophthora ramorum. The Chlamydomonas and Dictyostelium inteins are the first nuclear-encoded inteins found outside of the fungi. These new inteins represent a unique dataset: they are found in homologous proteins that form a paralogous group. Although these paralogues diverged early in eukaryotic evolution, their sequences can be aligned over most of their length. The inteins are inserted at multiple distinct sites, each of which corresponds to a highly conserved region of RNA polymerase. This dataset supports earlier work suggesting that inteins preferentially occur in highly conserved regions of their host proteins. Conclusion The identification of these new inteins
Applications of Recombinant Dna Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part B: Eukaryotic Gene Transcription and Post-Transcripional Rna Processing
Gary E Wild
Full Text Available The transcription of DNA into RNA is the primary level at which gene expression is controlled in eukaryotic cells. Eukaryotic gene transcription involves several different RNA polymerases that interact with a host of transcription factors to initiate transcription. Genes that encode proteins are transcribed into messenger RNA (mRNA by RNA polymerase II. Ribosomal RNAs (rRNAs and transfer RNAs (tRNAs are transcribed by RNA polymerase I and III, respectively. The production of each mRNA in human cells involves complex interactions of proteins (ie, trans-acting factors with specific sequences on the DNA (ie, cis-acting elements. Cis-acting elements are short base sequences adjacent to or within a particular gene. While the regulation of transcription is a pivotal step in the control of gene expression, a variety of molecular events, collectively known as ’RNA processing’ add an additional level of control of gene expression in eukaryotic cells.
Müller, Carolin A; Nieduszynski, Conrad A
Eukaryotic genomes are replicated in a reproducible temporal order; however, the physiological significance is poorly understood. We compared replication timing in divergent yeast species and identified genomic features with conserved replication times. Histone genes were among the earliest replicating loci in all species. We specifically delayed the replication of HTA1 - HTB1 and discovered that this halved the expression of these histone genes. Finally, we showed that histone and cell cycle genes in general are exempt from Rtt109-dependent dosage compensation, suggesting the existence of pathways excluding specific loci from dosage compensation mechanisms. Thus, we have uncovered one of the first physiological requirements for regulated replication time and demonstrated a direct link between replication timing and gene expression. © 2017 Müller and Nieduszynski.
Iwabe, N; Kuma, K; Hasegawa, M; Osawa, S; Miyata, T
All extant organisms are though to be classified into three primary kingdoms, eubacteria, eukaryotes, and archaebacteria. The molecular evolutionary studies on the origin and evolution of archaebacteria to date have been carried out by inferring a molecular phylogenetic tree of the primary kingdoms based on comparison of a single molecule from a variety of extant species. From such comparison, it was not possible to derive the exact evolutionary relationship among the primary kingdoms, because the root of the tree could not be determined uniquely. To overcome this difficulty, we compared a pair of duplicated genes, elongation factors Tu and G, and the alpha and beta subunits of ATPase, which are thought to have diverged by gene duplication before divergence of the primary kingdoms. Using each protein pair, we inferred a composite phylogenetic tree with two clusters corresponding to different proteins, from which the evolutionary relationship of the primary kingdoms is determined uniquely. The inferred composite trees reveal that archaebacteria are more closely related to eukaryotes than to eubacteria for all the cases. By bootstrap resamplings, this relationship is reproduced with probabilities of 0.96, 0.79, 1.0, and 1.0 for elongation factors Tu and G and for ATPase subunits alpha and beta, respectively. There are also several lines of evidence for the close sequence similarity between archaebacteria and eukaryotes. Thus we propose that this tree topology represents the general evolutionary relationship among the three primary kingdoms. PMID:2531898
Erives, Albert J
While the genomes of eukaryotes and Archaea both encode the histone-fold domain, only eukaryotes encode the core histone paralogs H2A, H2B, H3, and H4. With DNA, these core histones assemble into the nucleosomal octamer underlying eukaryotic chromatin. Importantly, core histones for H2A and H3 are maintained as neofunctionalized paralogs adapted for general bulk chromatin (canonical H2 and H3) or specialized chromatin (H2A.Z enriched at gene promoters and cenH3s enriched at centromeres). In this context, the identification of core histone-like "doublets" in the cytoplasmic replication factories of the Marseilleviridae (MV) is a novel finding with possible relevance to understanding the origin of eukaryotic chromatin. Here, we analyze and compare the core histone doublet genes from all known MV genomes as well as other MV genes relevant to the origin of the eukaryotic replisome. Using different phylogenetic approaches, we show that MV histone domains encode obligate H2B-H2A and H4-H3 dimers of possible proto-eukaryotic origin. MV core histone moieties form sister clades to each of the four eukaryotic clades of canonical and variant core histones. This suggests that MV core histone moieties diverged prior to eukaryotic neofunctionalizations associated with paired linear chromosomes and variant histone octamer assembly. We also show that MV genomes encode a proto-eukaryotic DNA topoisomerase II enzyme that forms a sister clade to eukaryotes. This is a relevant finding given that DNA topo II influences histone deposition and chromatin compaction and is the second most abundant nuclear protein after histones. The combined domain architecture and phylogenomic analyses presented here suggest that a primitive origin for MV histone genes is a more parsimonious explanation than horizontal gene transfers + gene fusions + sufficient divergence to eliminate relatedness to eukaryotic neofunctionalizations within the H2A and H3 clades without loss of relatedness to each of
Figge, R M; Schubert, M; Brinkmann, H; Cerff, R
Cyanobacteria contain up to three highly divergent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes: gap1, gap2, and gap3. Genes gap1 and gap2 are closely related at the sequence level to the nuclear genes encoding cytosolic and chloroplast GAPDH of higher plants and have recently been shown to play distinct key roles in catabolic and anabolic carbon flow, respectively, of the unicellular cyanobacterium Synechocystis sp. PCC6803. In the present study, sequences of 10 GAPDH genes distributed across the cyanobacteria Prochloron didemni, Gloeobacter violaceus PCC7421, and Synechococcus PCC7942 and the alpha-proteobacterium Paracoccus denitrificans and the beta-proteobacterium Ralstonia solanacearum were determined. Prochloron didemni possesses homologs to the gap2 and gap3 genes from Anabaena, Gloeobacter harbors gap1 and gap2 homologs, and Synechococcus possesses gap1, gap2, and gap3. Paracoccus harbors two highly divergent gap genes that are related to gap3, and Ralstonia possesses a homolog of the gap1 gene. Phylogenetic analyses of these sequences in the context of other eubacterial and eukaryotic GAPDH genes reveal that divergence across eubacterial gap1, and gap2, and gap3 genes is greater than that between eubacterial gap1 and eukaroytic glycolytic GapC or between eubacterial gap2 and eukaryotic Calvin cycle GapAB. These data strongly support previous analyses which suggested that eukaryotes acquired their nuclear genes for GapC and GapAB via endosymbiotic gene transfer from the antecedents of mitochondria and chloroplasts, and extend the known range of sequence diversity of the antecedent eubacterial genes. Analyses of available GAPDH sequences from other eubacterial sources indicate that the glycosomal gap gene from trypanosomes (cytosolic in Euglena) and the gap gene from the spirochete Treponema pallidum are each other's closest relatives. This specific relationship can therefore not reflect organismal evolution but must be the result of an
Madiha Bou Ali
Full Text Available Recombinant DNA methods are being widely used to express proteins in both prokaryotic and eukaryotic cells for both fundamental and applied research purposes. Expressed protein must be well characterized to be sure that it retains the same properties as the native one, especially when expressed protein will be used in the pharmaceutical field. In this aim, interfacial and kinetic properties of native, untagged recombinant and tagged recombinant forms of a pancreatic lipase were compared using the monomolecular film technique. Turkey pancreatic lipase (TPL was chosen as model. A kinetic study on the dependence of the stereoselectivity of these three forms on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The heterologous expression and the N-His-tag extension were found to modify the pressure preference and decrease the catalytic hydrolysis rate of three dicaprin isomers. Besides, the heterologous expression was found to change the TPL regioselectivity without affecting its stereospecificity contrary to the N-tag extension which retained that regioselectivity and changed the stereospecificity at high surface pressures. The study of parameters, termed Recombinant expression Effects on Catalysis (REC, N-Tag Effects on Catalysis (TEC, and N-Tag and Recombinant expression Effects on Catalysis (TREC showed that the heterologous expression effects on the catalytic properties of the TPL were more deleterious than the presence of an N-terminal tag extension.
Full Text Available Abstract Sterols are key components of eukaryotic cellular membranes that are synthesized by multi-enzyme pathways that require molecular oxygen. Because prokaryotes fundamentally lack sterols, it is unclear how the vast diversity of bacterivorous eukaryotes that inhabit hypoxic environments obtain, or synthesize, sterols. Here we show that tetrahymanol, a triterpenoid that does not require molecular oxygen for its biosynthesis, likely functions as a surrogate of sterol in eukaryotes inhabiting oxygen-poor environments. Genes encoding the tetrahymanol synthesizing enzyme squalene-tetrahymanol cyclase were found from several phylogenetically diverged eukaryotes that live in oxygen-poor environments and appear to have been laterally transferred among such eukaryotes. Reviewers This article was reviewed by Eric Bapteste and Eugene Koonin.
Balestrini, Raffaella; Lanfranco, Luisa
Arbuscular mycorrhizas (AMs) are a unique example of symbiosis between two eukaryotes, soil fungi and plants. This association induces important physiological changes in each partner that lead to reciprocal benefits, mainly in nutrient supply. The symbiosis results from modifications in plant and fungal cell organization caused by specific changes in gene expression. Recently, much effort has gone into studying these gene expression patterns to identify a wider spectrum of genes involved. We aim in this review to describe AM symbiosis in terms of current knowledge on plant and fungal gene expression profiles.
Ricardo M Ferreira
Full Text Available Whole genome protein-protein association networks are not random and their topological properties stem from genome evolution mechanisms. In fact, more connected, but less clustered proteins are related to genes that, in general, present more paralogs as compared to other genes, indicating frequent previous gene duplication episodes. On the other hand, genes related to conserved biological functions present few or no paralogs and yield proteins that are highly connected and clustered. These general network characteristics must have an evolutionary explanation. Considering data from STRING database, we present here experimental evidence that, more than not being scale free, protein degree distributions of organisms present an increased probability for high degree nodes. Furthermore, based on this experimental evidence, we propose a simulation model for genome evolution, where genes in a network are either acquired de novo using a preferential attachment rule, or duplicated with a probability that linearly grows with gene degree and decreases with its clustering coefficient. For the first time a model yields results that simultaneously describe different topological distributions. Also, this model correctly predicts that, to produce protein-protein association networks with number of links and number of nodes in the observed range for Eukaryotes, it is necessary 90% of gene duplication and 10% of de novo gene acquisition. This scenario implies a universal mechanism for genome evolution.
Swarts, D.C.; Koehorst, J.J.; Westra, E.R.; Schaap, P.J.; Oost, van der J.
BACKGROUND: Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a
Full Text Available Abstract Background The origin and importance of exon-intron architecture comprises one of the remaining mysteries of gene evolution. Several studies have investigated the variations of intron length, GC content, ordinal position in a gene and divergence. However, there is little study about the structural variation of exons and introns. Results We investigated the length, GC content, ordinal position and divergence in both exons and introns of 13 eukaryotic genomes, representing plant and animal. Our analyses revealed that three basic patterns of exon-intron variation were present in nearly all analyzed genomes (P Conclusion Although the factors contributing to these patterns have not been identified, our results provide three important clues: common factor(s exist and may shape both exons and introns; the ordinal reduction patterns may reflect a time-orderly evolution; and the larger first and last exons may be splicing-required. These clues provide a framework for elucidating mechanisms involved in the organization of eukaryotic genomes and particularly in building exon-intron structures.
Willenbrock, Hanni; Ussery, David
BACKGROUND: It is well known that gene expression is dependent on chromatin structure in eukaryotes and it is likely that chromatin can play a role in bacterial gene expression as well. Here, we use a nucleosomal position preference measure of anisotropic DNA flexibility to predict highly expressed...
Billon, Pierre; Bryant, Eric E; Joseph, Sarah A; Nambiar, Tarun S; Hayward, Samuel B; Rothstein, Rodney; Ciccia, Alberto
Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP. Copyright © 2017 Elsevier Inc. All rights reserved.
Chiba, Yoko; Kamikawa, Ryoma; Nakada-Tsukui, Kumiko; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi
Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO2 to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PPi)-type PEPCK (PPi-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PPi-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PPi-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PPi-PEPCK activity. The primary structure of PPi-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PPi-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PPi-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply. PMID:26269598
Chiba, Yoko; Kamikawa, Ryoma; Nakada-Tsukui, Kumiko; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi
Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO2 to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PPi)-type PEPCK (PPi-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PPi-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PPi-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PPi-PEPCK activity. The primary structure of PPi-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PPi-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PPi-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Yang, Qing-Qing; Yang, Si-Si; Tan, Jiang-Lin; Luo, Gao-Xing; He, Wei-Feng; Wu, Jun
Background: Hypertrophic scar is one of the most common complications and often causes the disfigurement or deformity in burn or trauma patients. Therapeutic methods on hypertrophic scar treatment have limitations due to the poor understanding of mechanisms of hypertrophic scar formation. To throw light on the molecular mechanism of hypertrophic scar formation will definitely improve the outcome of the treatment. This study aimed to illustrate the negative role of eukaryotic initiation factor 6 (eIF6) in the process of human hypertrophic scar formation, and provide a possible indicator of hypertrophic scar treatment and a potential target molecule for hypertrophic scar. Methods: In the present study, we investigated the protein expression of eIF6 in the human hypertrophic scar of different periods by immunohistochemistry and Western blot analysis. Results: In the hypertrophic scar tissue, eIF6 expression was significantly decreased and absent in the basal layer of epidermis in the early period, and increased slowly and began to appear in the basal layer of epidermis by the scar formation time. Conclusions: This study confirmed that eIF6 expression was significantly related to the development of hypertrophic scar, and the eIF6 may be a target molecule for hypertrophic scar control or could be an indicator of the outcomes for other treatment modalities. PMID:26481747
Uhlig, Christiane; Kilpert, Fabian; Frickenhaus, Stephan; Kegel, Jessica U; Krell, Andreas; Mock, Thomas; Valentin, Klaus; Beszteri, Bánk
Ice-binding proteins (IBPs) have been isolated from various sea-ice organisms. Their characterisation points to a crucial role in protecting the organisms in sub-zero environments. However, their in situ abundance and diversity in natural sea-ice microbial communities is largely unknown. In this study, we analysed the expression and phylogenetic diversity of eukaryotic IBP transcripts from microbial communities of Arctic and Antarctic sea ice. IBP transcripts were found in abundances similar to those of proteins involved in core cellular processes such as photosynthesis. Eighty-nine percent of the IBP transcripts grouped with known IBP sequences from diatoms, haptophytes and crustaceans, but the majority represented novel sequences not previously characterized in cultured organisms. The observed high eukaryotic IBP expression in natural eukaryotic sea ice communities underlines the essential role of IBPs for survival of many microorganisms in communities living under the extreme conditions of polar sea ice. PMID:25885562
Uhlig, Christiane; Kilpert, Fabian; Frickenhaus, Stephan; Kegel, Jessica U; Krell, Andreas; Mock, Thomas; Valentin, Klaus; Beszteri, Bánk
Ice-binding proteins (IBPs) have been isolated from various sea-ice organisms. Their characterisation points to a crucial role in protecting the organisms in sub-zero environments. However, their in situ abundance and diversity in natural sea-ice microbial communities is largely unknown. In this study, we analysed the expression and phylogenetic diversity of eukaryotic IBP transcripts from microbial communities of Arctic and Antarctic sea ice. IBP transcripts were found in abundances similar to those of proteins involved in core cellular processes such as photosynthesis. Eighty-nine percent of the IBP transcripts grouped with known IBP sequences from diatoms, haptophytes and crustaceans, but the majority represented novel sequences not previously characterized in cultured organisms. The observed high eukaryotic IBP expression in natural eukaryotic sea ice communities underlines the essential role of IBPs for survival of many microorganisms in communities living under the extreme conditions of polar sea ice.
Zhang, Zhang; Liu, Jingxing; Wu, Jiayan; Yu, Jun
The regulation of gene expression is essential for eukaryotes, as it drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types in multicellular organisms. RNA-Sequencing (RNA-Seq) provides researchers with a powerful toolbox for characterization and quantification of transcriptome. Many different human tissue/cell transcriptome datasets coming from RNA-Seq technology are available on public data resource. The fundamental issue here is how to develop an effective analysis method to estimate expression pattern similarities between different tumor tissues and their corresponding normal tissues. We define the gene expression pattern from three directions: 1) expression breadth, which reflects gene expression on/off status, and mainly concerns ubiquitously expressed genes; 2) low/high or constant/variable expression genes, based on gene expression level and variation; and 3) the regulation of gene expression at the gene structure level. The cluster analysis indicates that gene expression pattern is higher related to physiological condition rather than tissue spatial distance. Two sets of human housekeeping (HK) genes are defined according to cell/tissue types, respectively. To characterize the gene expression pattern in gene expression level and variation, we firstly apply improved K-means algorithm and a gene expression variance model. We find that cancer-associated HK genes (a HK gene is specific in cancer group, while not in normal group) are expressed higher and more variable in cancer condition than in normal condition. Cancer-associated HK genes prefer to AT-rich genes, and they are enriched in cell cycle regulation related functions and constitute some cancer signatures. The expression of large genes is also avoided in cancer group. These studies will help us understand which cell type-specific patterns of gene expression differ among different cell types, and particularly for cancer. PMID:23382867
Chen, Meili; Xiao, Jingfa; Zhang, Zhang; Liu, Jingxing; Wu, Jiayan; Yu, Jun
The regulation of gene expression is essential for eukaryotes, as it drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types in multicellular organisms. RNA-Sequencing (RNA-Seq) provides researchers with a powerful toolbox for characterization and quantification of transcriptome. Many different human tissue/cell transcriptome datasets coming from RNA-Seq technology are available on public data resource. The fundamental issue here is how to develop an effective analysis method to estimate expression pattern similarities between different tumor tissues and their corresponding normal tissues. We define the gene expression pattern from three directions: 1) expression breadth, which reflects gene expression on/off status, and mainly concerns ubiquitously expressed genes; 2) low/high or constant/variable expression genes, based on gene expression level and variation; and 3) the regulation of gene expression at the gene structure level. The cluster analysis indicates that gene expression pattern is higher related to physiological condition rather than tissue spatial distance. Two sets of human housekeeping (HK) genes are defined according to cell/tissue types, respectively. To characterize the gene expression pattern in gene expression level and variation, we firstly apply improved K-means algorithm and a gene expression variance model. We find that cancer-associated HK genes (a HK gene is specific in cancer group, while not in normal group) are expressed higher and more variable in cancer condition than in normal condition. Cancer-associated HK genes prefer to AT-rich genes, and they are enriched in cell cycle regulation related functions and constitute some cancer signatures. The expression of large genes is also avoided in cancer group. These studies will help us understand which cell type-specific patterns of gene expression differ among different cell types, and particularly for cancer.
Novo, Maite; Bigey, Frédéric; Beyne, Emmanuelle; Galeote, Virginie; Gavory, Frédérick; Mallet, Sandrine; Cambon, Brigitte; Legras, Jean-Luc; Wincker, Patrick; Casaregola, Serge; Dequin, Sylvie
Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1118 chromosome. These regions encompass 34 genes involved in key wine fermentation functions. Phylogeny and synteny analyses showed that 1 of these regions originated from a species closely related to the Saccharomyces genus, whereas the 2 other regions were of non-Saccharomyces origin. We identified Zygosaccharomyces bailii, a major contaminant of wine fermentations, as the donor species for 1 of these 2 regions. Although natural hybridization between Saccharomyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and non-Saccharomyces species. We show that the regions identified are frequent and differentially distributed among S. cerevisiae clades, being found almost exclusively in wine strains, suggesting acquisition through recent transfer events. Overall, these data show that the wine yeast genome is subject to constant remodeling through the contribution of exogenous genes. Our results suggest that these processes are favored by ecologic proximity and are involved in the molecular adaptation of wine yeasts to conditions of high sugar, low nitrogen, and high ethanol concentrations.
Lundblad, Eirik Wasmuth; Altman, Sidney
The ability to interfere with gene expression is of crucial importance to unravel the function of genes and is also a promising therapeutic strategy. Here we discuss methodologies for inhibition of target RNAs based on the cleavage activity of the essential enzyme, Ribonuclease P (RNase P). RNase P-mediated cleavage of target RNAs can be directed by external guide sequences (EGSs) or by the use of the catalytic M1 RNA from E. coli linked to a guide sequence (M1GSs). These are not only basic tools for functional genetic studies in prokaryotic and eukaryotic cells but also promising antibacterial, anticancer and antiviral agents. Copyright (c) 2010 Elsevier B.V. All rights reserved.
Full Text Available Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA. CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9 Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.
Full Text Available Geographic partitioning is postulated to foster divergence of Helicobacter pylori populations as an adaptive response to local differences in predominant host physiology. H. pylori's ability to establish persistent infection despite host inflammatory responses likely involves active management of host defenses using bacterial proteins that may themselves be targets for adaptive evolution. Sequenced H. pylori genomes encode a family of eight or nine secreted proteins containing repeat motifs that are characteristic of the eukaryotic Sel1 regulatory protein, whereas the related Campylobacter and Wolinella genomes each contain only one or two such "Sel1-like repeat" (SLR genes ("slr genes". Signatures of positive selection (ratio of nonsynonymous to synonymous mutations, dN/dS = omega > 1 were evident in the evolutionary history of H. pylori slr gene family expansion. Sequence analysis of six of these slr genes (hp0160, hp0211, hp0235, hp0519, hp0628, and hp1117 from representative East Asian, European, and African H. pylori strains revealed that all but hp0628 had undergone positive selection, with different amino acids often selected in different regions. Most striking was a divergence of Japanese and Korean alleles of hp0519, with Japanese alleles having undergone particularly strong positive selection (omegaJ > 25, whereas alleles of other genes from these populations were intermingled. Homology-based structural modeling localized most residues under positive selection to SLR protein surfaces. Rapid evolution of certain slr genes in specific H. pylori lineages suggests a model of adaptive change driven by selection for fine-tuning of host responses, and facilitated by geographic isolation. Characterization of such local adaptations should help elucidate how H. pylori manages persistent infection, and potentially lead to interventions tailored to diverse human populations.
Full Text Available Abstract Background Free-living amoebae serve as a natural reservoir for some bacteria that have evolved into «amoeba-resistant» bacteria. Among these, some are strictly intra-amoebal, such as Candidatus "Protochlamydia amoebophila" (Candidatus "P. amoebophila", whose genomic sequence is available. We sequenced the genome of Legionella drancourtii (L. drancourtii, another recently described intra-amoebal bacterium. By comparing these two genomes with those of their closely related species, we were able to study the genetic characteristics specific to their amoebal lifestyle. Findings We identified a sterol delta-7 reductase-encoding gene common to these two bacteria and absent in their relatives. This gene encodes an enzyme which catalyses the last step of cholesterol biosynthesis in eukaryotes, and is probably functional within L. drancourtii since it is transcribed. The phylogenetic analysis of this protein suggests that it was acquired horizontally by a few bacteria from viridiplantae. This gene was also found in the Acanthamoeba polyphaga Mimivirus genome, a virus that grows in amoebae and possesses the largest viral genome known to date. Conclusion L. drancourtii acquired a sterol delta-7 reductase-encoding gene of viridiplantae origin. The most parsimonious hypothesis is that this gene was initially acquired by a Chlamydiales ancestor parasite of plants. Subsequently, its descendents transmitted this gene in amoebae to other intra-amoebal microorganisms, including L. drancourtii and Coxiella burnetii. The role of the sterol delta-7 reductase in prokaryotes is as yet unknown but we speculate that it is involved in host cholesterol parasitism.
Ámon, Judit; Fernández-Martín, Rafael; Bokor, Eszter; Cultrone, Antonietta; Kelly, Joan M.; Flipphi, Michel; Scazzocchio, Claudio
Nicotinate degradation has hitherto been elucidated only in bacteria. In the ascomycete Aspergillus nidulans, six loci, hxnS/AN9178 encoding the molybdenum cofactor-containing nicotinate hydroxylase, AN11197 encoding a Cys2/His2 zinc finger regulator HxnR, together with AN11196/hxnZ, AN11188/hxnY, AN11189/hxnP and AN9177/hxnT, are clustered and stringently co-induced by a nicotinate derivative and subject to nitrogen metabolite repression mediated by the GATA factor AreA. These genes are strictly co-regulated by HxnR. Within the hxnR gene, constitutive mutations map in two discrete regions. Aspergillus nidulans is capable of using nicotinate and its oxidation products 6-hydroxynicotinic acid and 2,5-dihydroxypyridine as sole nitrogen sources in an HxnR-dependent way. HxnS is highly similar to HxA, the canonical xanthine dehydrogenase (XDH), and has originated by gene duplication, preceding the origin of the Pezizomycotina. This cluster is conserved with some variations throughout the Aspergillaceae. Our results imply that a fungal pathway has arisen independently from bacterial ones. Significantly, the neo-functionalization of XDH into nicotinate hydroxylase has occurred independently from analogous events in bacteria. This work describes for the first time a gene cluster involved in nicotinate catabolism in a eukaryote and has relevance for the formation and evolution of co-regulated primary metabolic gene clusters and the microbial degradation of N-heterocyclic compounds. PMID:29212709
Gary E Wild
Full Text Available Progress in the basic sciences of cell and molecular biology has provided an exciting dimension that has translated into clinically relevant information in every medical subspecialty. Importantly, the application of recombinant DNA technology has played a major role in unravelling the intricacies related to the molecular pathophysiology of disease. This series of review articles constitutes a framework for the integration of the database of new information into the core knowledge base of concepts related to the pathogenesis of gastrointestinal disorders and liver disease. The goal of this series of three articles is to review the basic principles of eukaryotic gene expression. The first article examines the role of DNA in directing the flow of genetic information in eukaryotic cells.
Ortega, Alvaro D; Gonzalo-Asensio, Jesús; García-del Portillo, Francisco
Small non-coding regulatory RNAs (sRNAs) have been studied in many bacterial pathogens during infection. However, few studies have focused on how intracellular pathogens modulate sRNA expression inside eukaryotic cells. Here, we monitored expression of all known sRNAs of Salmonella enterica serovar Typhimurium (S. Typhimurium) in bacteria located inside fibroblasts, a host cell type in which this pathogen restrains growth. sRNA sequences known in S. Typhimurium and Escherichia coli were searched in the genome of S. Typhimurium virulent strain SL1344, the subject of this study. Expression of 84 distinct sRNAs was compared in extra- and intracellular bacteria. Non-proliferating intracellular bacteria upregulated six sRNAs, including IsrA, IsrG, IstR-2, RyhB-1, RyhB-2 and RseX while repressed the expression of the sRNAs DsrA, GlmZ, IsrH-1, IsrI, SraL, SroC, SsrS(6S) and RydC. Interestingly, IsrH-1 was previously reported as an sRNA induced by S. Typhimurium inside macrophages. Kinetic analyses unraveled changing expression patterns for some sRNAs along the infection. InvR and T44 expression dropped after an initial induction phase while IstR-2 was induced exclusively at late infection times (> 6 h). Studies focused on the Salmonella-specific sRNA RyhB-2 revealed that intracellular bacteria use this sRNA to regulate negatively YeaQ, a cis-encoded protein of unknown function. RyhB-2, together with RyhB-1, contributes to attenuate intracellular bacterial growth. To our knowledge, these data represent the first comprehensive study of S. Typhimurium sRNA expression in intracellular bacteria and provide the first insights into sRNAs that may direct pathogen adaptation to a non-proliferative state inside the host cell.
Garcia, Nelson; Zhang, Wei; Wu, Yongrui; Messing, Joachim
We took a rather unique approach to investigate the conservation of gene expression of prolamin storage protein genes across two different subfamilies of the Poaceae. We took advantage of oat plants carrying single maize chromosomes in different cultivars, called oat-maize addition (OMA) lines, which permitted us to determine whether regulation of gene expression was conserved between the two species. We found that γ-zeins are expressed in OMA7.06, which carries maize chromosome 7 even in the absence of the trans-acting maize prolamin-box-binding factor (PBF), which regulates their expression. This is likely because oat PBF can substitute for the function of maize PBF as shown in our transient expression data, using a γ-zein promoter fused to green fluorescent protein (GFP). Despite this conservation, the younger, recently amplified prolamin genes in maize, absent in oat, are not expressed in the corresponding OMAs. However, maize can express the oldest prolamin gene, the wheat high-molecular weight glutenin Dx5 gene, even when maize Pbf is knocked down (through PbfRNAi), and/or another maize transcription factor, Opaque-2 (O2) is knocked out (in maize o2 mutant). Therefore, older genes are conserved in their regulation, whereas younger ones diverged during evolution and eventually acquired a new repertoire of suitable transcriptional activators. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Palmgren, Michael Broberg; Engström, Karin; Hallström, Björn M
the evolutionary origin of AS3MT and assessed the ability of different genotypes to produce methylated arsenic metabolites. Phylogenetic analysis suggests that multiple, independent horizontal gene transfers between different bacteria, and from bacteria to eukaryotes, increased tolerance to environmental arsenic...
DNA methylation and histone acetylation are important for controlling gene expression in all eukaryotes. Microarray analysis revealed an altered gene expression profile after treatment with the DNA methylation inhibitor 5-aza-2’ deoxyctidine (5-AC), which included the upregulation of many transposab...
Full Text Available Abstract Background The main function of gene promoters appears to be the integration of different gene products in their biological pathways in order to maintain homeostasis. Generally, promoters have been classified in two major classes, namely TATA and CpG. Nevertheless, many genes using the same combinatorial formation of transcription factors have different gene expression patterns. Accordingly, we tried to ask ourselves some fundamental questions: Why certain genes have an overall predisposition for higher gene expression levels than others? What causes such a predisposition? Is there a structural relationship of these sequences in different tissues? Is there a strong phylogenetic relationship between promoters of closely related species? Results In order to gain valuable insights into different promoter regions, we obtained a series of image-based patterns which allowed us to identify 10 generic classes of promoters. A comprehensive analysis was undertaken for promoter sequences from Arabidopsis thaliana, Drosophila melanogaster, Homo sapiens and Oryza sativa, and a more extensive analysis of tissue-specific promoters in humans. We observed a clear preference for these species to use certain classes of promoters for specific biological processes. Moreover, in humans, we found that different tissues use distinct classes of promoters, reflecting an emerging promoter network. Depending on the tissue type, comparisons made between these classes of promoters reveal a complementarity between their patterns whereas some other classes of promoters have been observed to occur in competition. Furthermore, we also noticed the existence of some transitional states between these classes of promoters that may explain certain evolutionary mechanisms, which suggest a possible predisposition for specific levels of gene expression and perhaps for a different number of factors responsible for triggering gene expression. Our conclusions are based on
Full Text Available Tandemly arrayed genes (TAGs account for about one third of the duplicated genes in eukaryotic genomes, yet there has not been any systematic study of their gene expression patterns. Taking advantage of recently published large-scale microarray data sets, we studied the expression divergence of 361 two-member TAGs in human and 212 two-member TAGs in mouse and examined the effect of sequence divergence, gene orientation, and chromosomal proximity on the divergence of TAG expression patterns. Our results show that there is a weak negative correlation between sequence divergence of TAG members and their expression similarity. There is also a weak negative correlation between chromosomal proximity of TAG members and their expression similarity. We did not detect any significant relationship between gene orientation and expression similarity. We also found that downstream TAG members do not show significantly narrower expression breadth than upstream members, contrary to what we predict based on TAG expression divergence hypothesis that we propose. Finally, we show that both chromosomal proximity and expression correlation in TAGs do not differ significantly from their neighboring non-TAG gene pairs, suggesting that tandem duplication is unlikely to be the cause for the higher-than-random expression association between neighboring genes on a chromosome in human and mouse.
Mei, Qiming; Dvornyk, Volodymyr
The PAS domains are signal modules, which are widely distributed in proteins across all kingdoms of life. They are common in photoreceptors and transcriptional regulators of eukaryotic circadian clocks q(bHLH-PAS proteins and PER in animals; PHY and ZTL in plants; and WC-1, 2, and VVD in fungi) and possess mainly protein-protein interaction and light-sensing functions. We conducted several evolutionary analyses of the PAS superfamily. Although the whole superfamily evolved primarily under strong purifying selection (average ω ranges from 0.0030 to 0.1164), some lineages apparently experienced strong episodic positive selection at some periods of the evolution. Although the PAS domains from different proteins vary in sequence and length, but they maintain a fairly conserved 3D structure, which is determined by only eight residues. The WC-1 and WC- 2, bHLH-PAS, and P er genes probably originated in the Neoproterozoic Era (1000-542 Mya), plant P hy and ZTL evolved in the Paleozoic (541-252 Mya), which might be a result of adaptation to the major climate and global light regime changes having occurred in those eras.
Corrado, Giandomenico; Karali, Marianthi
Plant biotechnology relies heavily on the genetic manipulation of crops. Almost invariantly, the gene of interest is expressed in a constitutive fashion, although this may not be strictly necessary for several applications. Currently, there are several regulatable expression systems for the temporal, spatial and quantitative control of transgene activity. These molecular switches are based on components derived from different organisms, which range from viruses to higher eukaryotes. Many inducible systems have been designed for fundamental and applied research and since their initial development, they have become increasingly popular in plant molecular biology. This review covers a broad number of inducible expression systems examining their properties and relevance for plant biotechnology in its various guises, from molecular breeding to pharmaceutical and industrial applications. For each system, we examine some advantages and limitations, also in relation to the strategy on which they rely. Besides being necessary to control useful genes that may negatively affect crop yield and quality, we discuss that inducible systems can be also used to increase public acceptance of GMOs, reducing some of the most common concerns. Finally, we suggest some directions and future developments for their further diffusion in agriculture and biotechnology.
Altenburger, Andreas; Martinez, Pedro; Wanninger, Andreas
The molecular control that underlies brachiopod ontogeny is largely unknown. In order to contribute to this issue we analyzed the expression pattern of two homeobox containing genes, Not and Cdx, during development of the rhynchonelliform (i.e., articulate) brachiopod Terebratalia transversa....... Not is a homeobox containing gene that regulates the formation of the notochord in chordates, while Cdx (caudal) is a ParaHox gene involved in the formation of posterior tissues of various animal phyla. The T. transversa homolog, TtrNot, is expressed in the ectoderm from the beginning of gastrulation until...... formation. TtrNot expression is absent in unfertilized eggs, in embryos prior to gastrulation, and in settled individuals during and after metamorphosis. Comparison with the expression patterns of Not genes in other metazoan phyla suggests an ancestral role for this gene in gastrulation and germ layer...
Vinay Kumar Aakalu
Full Text Available The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development.We performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium.The analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described.Differential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas.
Dunn, Casey W; Luo, Xi; Wu, Zhijin
Phylogenetic analyses of gene expression have great potential for addressing a wide range of questions. These analyses will, for example, identify genes that have evolutionary shifts in expression that are correlated with evolutionary changes in morphological, physiological, and developmental characters of interest. This will provide entirely new opportunities to identify genes related to particular phenotypes. There are, however, 3 key challenges that must be addressed for such studies to realize their potential. First, data on gene expression must be measured from multiple species, some of which may be field-collected, and parameterized in such a way that they can be compared across species. Second, it will be necessary to develop comparative phylogenetic methods suitable for large multidimensional datasets. In most phylogenetic comparative studies to date, the number n of independent observations (independent contrasts) has been greater than the number p of variables (characters). The behavior of comparative methods for these classic problems is now well understood under a wide variety of conditions. In studies of gene expression, and in studies based on other high-throughput tools, the number n of samples is dwarfed by the number p of variables. The estimated covariance matrices will be singular, complicating their analysis and interpretation, and prone to spurious results. Third, new approaches are needed to investigate the expression of the many genes whose phylogenies are not congruent with species phylogenies due to gene loss, gene duplication, and incomplete lineage sorting. Here we outline general considerations of project design for phylogenetic analyses of gene expression and suggest solutions to these three categories of challenges. These topics are relevant to high-throughput phenotypic data well beyond gene expression.
Full Text Available Reverse transcription-qPCR (RT-qPCR has become a popular method for gene expression studies. Its results require data normalization by housekeeping genes. No single gene is proved to be stably expressed under all experimental conditions. Therefore, systematic evaluation of reference genes is necessary. With the aim to identify optimum reference genes for RT-qPCR analysis of gene expression in different tissues of Panax ginseng and the seedlings grown under heat stress, we investigated the expression stability of eight candidate reference genes, including elongation factor 1-beta (EF1-β, elongation factor 1-gamma (EF1-γ, eukaryotic translation initiation factor 3G (IF3G, eukaryotic translation initiation factor 3B (IF3B, actin (ACT, actin11 (ACT11, glyceraldehyde-3-phosphate dehydrogenase (GAPDH and cyclophilin ABH-like protein (CYC, using four widely used computational programs: geNorm, Normfinder, BestKeeper, and the comparative ΔCt method. The results were then integrated using the web-based tool RefFinder. As a result, EF1-γ, IF3G and EF1-β were the three most stable genes in different tissues of P. ginseng, while IF3G, ACT11 and GAPDH were the top three-ranked genes in seedlings treated with heat. Using three better reference genes alone or in combination as internal control, we examined the expression profiles of MAR, a multiple function-associated mRNA-like non-coding RNA (mlncRNA in P. ginseng. Taken together, we recommended EF1-γ/IF3G and IF3G/ACT11 as the suitable pair of reference genes for RT-qPCR analysis of gene expression in different tissues of P. ginseng and the seedlings grown under heat stress, respectively. The results serve as a foundation for future studies on P. ginseng functional genomics.
Su, Xiaoyun; Schmitz, George; Zhang, Meiling; Mackie, Roderick I; Cann, Isaac K O
Filamentous fungi are critical to production of many commercial enzymes and organic compounds. Fungal-based systems have several advantages over bacterial-based systems for protein production because high-level secretion of enzymes is a common trait of their decomposer lifestyle. Furthermore, in the large-scale production of recombinant proteins of eukaryotic origin, the filamentous fungi become the vehicle of choice due to critical processes shared in gene expression with other eukaryotic organisms. The complexity and relative dearth of understanding of the physiology of filamentous fungi, compared to bacteria, have hindered rapid development of these organisms as highly efficient factories for the production of heterologous proteins. In this review, we highlight several of the known benefits and challenges in using filamentous fungi (particularly Aspergillus spp., Trichoderma reesei, and Neurospora crassa) for the production of proteins, especially heterologous, nonfungal enzymes. We review various techniques commonly employed in recombinant protein production in the filamentous fungi, including transformation methods, selection of gene regulatory elements such as promoters, protein secretion factors such as the signal peptide, and optimization of coding sequence. We provide insights into current models of host genomic defenses such as repeat-induced point mutation and quelling. Furthermore, we examine the regulatory effects of transcript sequences, including introns and untranslated regions, pre-mRNA (messenger RNA) processing, transcript transport, and mRNA stability. We anticipate that this review will become a resource for researchers who aim at advancing the use of these fascinating organisms as protein production factories, for both academic and industrial purposes, and also for scientists with general interest in the biology of the filamentous fungi. Copyright © 2012 Elsevier Inc. All rights reserved.
Rockman, Matthew V
Hidden among the myriad nucleotide variants that constitute each species' gene pool are a few variants that contribute to phenotypic variation. Many of these differences that make a difference are non-coding cis-regulatory variants, which, unlike coding variants, can only be identified through laborious experimental analysis. Recently, Cowles et al.1 described a screening method that does an end-run around this problem by searching for genes whose cis regulation varies without having to find the polymorphic nucleotides that influence transcription. While we will continue to require a diverse arsenal of experimental methods, this versatile method will speed the identification of functional genetic variation. Copyright 2003 Wiley Periodicals, Inc.
John N. Reeve
, designated TFE, that had sequences in common with the eukaryotic general transcription factor TFIIE, stimulated archaeal transcription initiation and that the archaeal TATA-box binding protein (TBP) remained attached to the promoter region whereas the transcription factor TFB dissociated from the template DNA following initiation. DNA sequences that directed the localized assembly of archaeal histones into archaeal nucleosomes were identified, and we established that transcription by an archaeal RNA polymerase was slowed but not blocked by archaeal nucleosomes. We developed a new protocol to purify archaeal RNA polymerases and with this enzyme and additional improvements to the in vitro transcription system, we established the template requirements for archaeal transcription termination, investigated the activities of proteins predicted to be methane gene regulators, and established how TrpY, a novel archaeal regulator of expression of the tryptophan biosynthetic operon functions in M. thermautotrophicus. This also resulted in the discovery that almost all M. thermautotrophicus mutants isolated as spontaneously resistant to 5-methyl tryptophan (5MTR) had mutations in trpY and were therefore 5MTR through de-repressed trp operon expression. This established a very simple, practical procedure to determine and quantify the DNA sequence changes that result from exposure of this Archaeon to any experimental mutagenesis protocol. Following the discovery that the Thermococcus kodakaraensis was amenable to genetic manipulation, we established this technology at OSU and subsequently added plasmid expression, a reporter system and additional genetic selections to the T. kodakaraensis genetic toolbox. We established that transcription and translation are coupled in this Archaeon, and by combining in vitro transcription and in vivo genetics, we documented that both TFB1 and TFB2 support transcription initiation in T. kodakaraensis. We quantified the roles of ribosome binding sequences
Darbani Shirvanehdeh, Behrooz; Stewart, C. Neal, Jr.; Noeparvar, Shahin
This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies....... For maximal reliability of analysis, therefore, comparisons should be performed at the cellular level. This could be accomplished using an appropriate correction method that can detect and remove the inter-treatment bias for cell-number. Based on inter-treatment variations of reference genes, we introduce...
da Costa Souza, Annie; Ribeiro, Sidarta
Sleep occurs in a wide range of animal species as a vital process for the maintenance of homeostasis, metabolic restoration, physiological regulation, and adaptive cognitive functions in the central nervous system. Long-term perturbations induced by the lack of sleep are mostly mediated by changes at the level of transcription and translation. This chapter reviews studies in humans, rodents, and flies to address the various ways by which sleep deprivation affects gene expression in the nervous system, with a focus on genes related to neuronal plasticity, brain function, and cognition. However, the effects of sleep deprivation on gene expression and the functional consequences of sleep loss are clearly not restricted to the cognitive domain but may include increased inflammation, expression of stress-related genes, general impairment of protein translation, metabolic imbalance, and thermal deregulation.
Qing-Qing Yang; Si-Si Yang; Jiang-Lin Tan; Gao-Xing Luo; Wei-Feng He; Jun Wu
Background: Hypertrophic scar is one of the most common complications and often causes the disfigurement or deformity in burn or trauma patients. Therapeutic methods on hypertrophic scar treatment have limitations due to the poor understanding of mechanisms of hypertrophic scar formation. To throw light on the molecular mechanism of hypertrophic scar formation will definitely improve the outcome of the treatment. This study aimed to illustrate the negative role of eukaryotic initiation factor...
Vulcani-Freitas, Tânia M; Saba-Silva, Najsla; Cappellano, Andréa; Cavalheiro, Sérgio; Marie, Sueli K N; Oba-Shinjo, Sueli M; Malheiros, Suzana M F; de Toledo, Sílvia Regina Caminada
Medulloblastomas are the most common malignant tumors of the central nervous system in childhood. The incidence is about 19-20% between children younger than 16 years old with peak incidence between 4 and 7 years. Despite its sensibility to no specific therapeutic means like chemotherapy and radiotherapy, the treatment is very aggressive and frequently results in regression, growth deficit, and endocrine dysfunction. From this point of view, new treatment approaches are needed such as molecular targeted therapies. Studies in glioblastoma demonstrated that ASPM gene was overexpressed when compared to normal brain and ASPM inhibition by siRNA-mediated inhibits tumor cell proliferation and neural stem cell proliferation, supporting ASPM gene as a potential molecular target in glioblastoma. The aim of this work was to evaluate ASPM expression in medulloblastoma fragment samples, and to compare the results with the patient clinical features. Analysis of gene expression was performed by quantitative PCR real time using SYBR Green system in tumor samples from 37 children. The t test was used to analyze the gene expression, and Mann-Whitney test was performed to analyze the relationship between gene expressions and clinical characteristics. Kaplan-Meier test evaluated curve survival. All samples overexpressed ASPM gene more than 40-fold. However, we did not find any association between the overexpressed samples and the clinical parameters. ASPM overexpression may modify the ability of stem cells to differentiate during the development of the central nervous system, contributing to the development of medulloblastoma, a tumor of embryonic origin from cerebellar progenitor cells.
Andersen, Kasper Røjkjær; Jonstrup, Anette Thyssen; Van, Lan Bich
When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems to be the ......When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems...
Gene expression, the process by which the genetic code comes alive in the form of proteins, is one of the most important biological processes in living cells, and begins when transcription factors bind to specific DNA sequences in the promoter region upstream of a gene. The relationship between gene expression output and transcription factor input which is termed the gene regulation function is specific to each promoter, and predicting this gene regulation function from the locations of transcription factor binding sites is one of the challenges in biology. In eukaryotic organisms (for example, animals, plants, fungi etc), DNA is highly compacted into nucleosomes, 147-bp segments of DNA tightly wrapped around histone protein core, and therefore, the accessibility of transcription factor binding sites depends on their locations with respect to nucleosomes - sites inside nucleosomes are less accessible than those outside nucleosomes. To understand how transcription factor binding sites contribute to gene expression in a quantitative manner, we obtain gene regulation functions of promoters with various configurations of transcription factor binding sites by using fluorescent protein reporters to measure transcription factor input and gene expression output in single yeast cells. In this talk, I will show that the affinity of a transcription factor binding site inside and outside the nucleosome controls different aspects of the gene regulation function, and explain this finding based on a mass-action kinetic model that includes competition between nucleosomes and transcription factors.
Full Text Available Stra8 (Stimulated by Retinoic Acid 8 is considered a meiotic gatekeeper gene. Using reverse transcriptase PCR and rapid amplification of cDNA ends (RACE, the complete sequence of the pig Stra8 gene was cloned. Bioinformatics analyses of this sequence were performed. Using semi-quantitative methods, the expression characteristics of Stra8 in Testis, cauda epididymis, body epididymis, caput epididymis, seminal vesicles, prostate gland, Cowper’s gland, heart, liver, spleen, lung, kidney, stomach, hypothalamus, pituitary gland, cerebrum, cerebellum, and hippocampus of adult Meishan boar and sow tissues were examined. The expression pattern in the testis of 2-, 30-, 60-, 90-, and 150-day old Meishan boars were analyzed using real-time PCR. We constructed a eukaryotic expression vector for the Stra8 gene and used it to transfect NIH-3T3 cells and third generation pig spermatogonial stem cells (SSCs cultured in vitro. Testes weight and sperm count in the cauda epididymis were evaluated at various time points. The results showed that the length of the pig Stra8 gene cDNA was 1444 bp encoding 366 amino acids with one typical helix-loop-helix (HLH domain. It is testes-specific expression. Expression was first detected in boar testis starting at day 2, and its expression significantly (p < 0.05 increased with age and body weight. When NIH-3T3 cells and pig SSCs were transfected with the eukaryotic expression vector EGFP (enhanced green fluorescent protein-N1-pStra8, it was expressed in the cytoplasm of NIH-3T3 cells. However, in SSCs, Stra8 was expressed predominantly in cytoplasm and few in nucleus. Our data suggest that perhaps Stra8 acts as a transcription factor to initiate meiosis in young boar.
Sara Tarek; Reda Abd Elwahab; Mahmoud Shoman
Cancer classification based on molecular level investigation has gained the interest of researches as it provides a systematic, accurate and objective diagnosis for different cancer types. Several recent researches have been studying the problem of cancer classification using data mining methods, machine learning algorithms and statistical methods to reach an efficient analysis for gene expression profiles. Studying the characteristics of thousands of genes simultaneously offered a deep in...
Ferl, Robert; Paul, Anna-Lisa
The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.
Birkenkamp-Demtroder, Karin; Christensen, Lise Lotte; Olesen, Sanne Harder
Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each......%). Fifteen nuclear encoded mitochondrial proteins were all down-regulated in CRC. We identified several chromosomal locations with clusters of either potential oncogenes or potential tumor suppressors. Some of these, such as aminopeptidase N/CD13 and sigma B3 protein on chromosome 15q25, coincided...
Jebanathirajah, J.A.; Andersen, S.; Blagoev, B.
Rapid methods using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to monitor recombinant protein expression from various prokaryotic and eukaryotic cell culture systems were devised. Intracellular as well as secreted proteins from both induced and constitutive...... expression systems were measured and monitored from whole cells and growth media, thus providing an alternative to time-consuming traditional methods for screening and monitoring of protein expression. The methods described here involve minimal processing of samples and are therefore relevant to high...
Inupakutika, Madhuri A.; Sengupta, Soham; Nechushtai, Rachel; Jennings, Patricia A.; Onuchic, Jose' N.; Azad, Rajeev K.; Padilla, Pamela; Mittler, Ron
NEET proteins belong to a unique family of iron-sulfur proteins in which the 2Fe-2S cluster is coordinated by a CDGSH domain that is followed by the “NEET” motif. They are involved in the regulation of iron and reactive oxygen metabolism, and have been associated with the progression of diabetes, cancer, aging and neurodegenerative diseases. Despite their important biological functions, the evolution and diversification of eukaryotic NEET proteins are largely unknown. Here we used the three members of the human NEET protein family (CISD1, mitoNEET; CISD2, NAF-1 or Miner 1; and CISD3, Miner2) as our guides to conduct a phylogenetic analysis of eukaryotic NEET proteins and their evolution. Our findings identified the slime mold Dictyostelium discoideum’s CISD proteins as the closest to the ancient archetype of eukaryotic NEET proteins. We further identified CISD3 homologs in fungi that were previously reported not to contain any NEET proteins, and revealed that plants lack homolog(s) of CISD3. Furthermore, our study suggests that the mammalian NEET proteins, mitoNEET (CISD1) and NAF-1 (CISD2), emerged via gene duplication around the origin of vertebrates. Our findings provide new insights into the classification and expansion of the NEET protein family, as well as offer clues to the diverged functions of the human mitoNEET and NAF-1 proteins.
Daniel J Kliebenstein
Full Text Available BACKGROUND: Most eukaryotic genomes have undergone whole genome duplications during their evolutionary history. Recent studies have shown that the function of these duplicated genes can diverge from the ancestral gene via neo- or sub-functionalization within single genotypes. An additional possibility is that gene duplicates may also undergo partitioning of function among different genotypes of a species leading to genetic differentiation. Finally, the ability of gene duplicates to diverge may be limited by their biological function. METHODOLOGY/PRINCIPAL FINDINGS: To test these hypotheses, I estimated the impact of gene duplication and metabolic function upon intraspecific gene expression variation of segmental and tandem duplicated genes within Arabidopsis thaliana. In all instances, the younger tandem duplicated genes showed higher intraspecific gene expression variation than the average Arabidopsis gene. Surprisingly, the older segmental duplicates also showed evidence of elevated intraspecific gene expression variation albeit typically lower than for the tandem duplicates. The specific biological function of the gene as defined by metabolic pathway also modulated the level of intraspecific gene expression variation. The major energy metabolism and biosynthetic pathways showed decreased variation, suggesting that they are constrained in their ability to accumulate gene expression variation. In contrast, a major herbivory defense pathway showed significantly elevated intraspecific variation suggesting that it may be under pressure to maintain and/or generate diversity in response to fluctuating insect herbivory pressures. CONCLUSION: These data show that intraspecific variation in gene expression is facilitated by an interaction of gene duplication and biological activity. Further, this plays a role in controlling diversity of plant metabolism.
Gerald, Wiliam L
... to identify genes, gene expression profiles and molecular pathways associated with metastatic BC we have performed genome-wide gene expression analysis of a large number of breast cancer samples...
Guo, Ji-hua; Fan, Ming-wen; Jia, Rong; Bian, Zhuan; Chen, Zhi; Yu, Fei
To investigate and compare the expression pattern and level of targeted anti-caries plasmids encoding different-size antigens in eukaryotic cells. The A-P fragment of PAc (surface protein antigen) was removed from pGJA-P encoding the signal peptide, extracellular domains of human CTLA-4, human Ig hinge, CH2 and CH3 domains, A-P fragment of PAc and GLU (glucan binding domain) region of GTF-I of Streptococcus mutans, to obtain the plasmid pGJGLU. pCI vector skeleton of pGJA-P or pGJGLU was replaced by pVAX1 to construct plasmids pGJA-P/VAX and pGJGLU/VAX. CTLA4-Ig-GLU fragment was removed from pGJGLU and inserted into the vector pEGFP-N1 to obtain the recombinant plasmid pGJGLU/GFP. The CHO cells were transfected with those plasmids by using liposome and the expression of fusion protein was observed with fluorescence microscope. ELISA was used to detect the expression level of fusion proteins in cultured supernatants. Specific vesicles with green fluorescence could be observed in the CHO cells transfected with pGJGLU/GFP. The recombinant fusion protein could be detected in the cultured supernatants of CHO cells transfected with pGJA-P/VAX, pGJGLU/VAX and pGJGLU/GFP, of which the concentration was different. The highest concentration of recombinant fusion protein was observed in the supernatants of CHO cells transfected with pGJGLU/VAX. CTLA-4 targeted fusion protein could be expressed and secreted by eukaryotic cells. The size of antigen may affect the expression level of CTLA-4 targeted anti-caries DNA vaccine.
Angélica Concepción eMartínez-Navarro
Full Text Available The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular tissues. This tissue-specific expression in Arabidopsis is predicted by the overrepresentation of GA/CT-rich motifs in gene promoters. In this work we have searched for common motifs in upstream regions of the homologous genes from plants considered to possess a primitive vascular tissue (a lycophyte, as well as from others that lack a true vascular tissue (a bryophyte, and finally from chlorophytes. Both lycophyte and bryophyte display motifs similar to those found in Arabidopsis with a significantly low E-value, while the chlorophytes showed either a different conserved motif or no conserved motif at all. These results suggest that these same genes are expressed coordinately in non- vascular plants; this coordinate expression may have been one of the prerequisites for the development of conducting tissues in plants. We have also analyzed the phylogeny of conserved proteins that may be involved in phloem function and development. The presence of CmPP16, APL, FT and YDA in chlorophytes suggests the recruitment of ancient regulatory networks for the development of the vascular tissue during evolution while OPS is a novel protein specific to vascular plants.
Ju Yun Bae; Jose Laplaza; Thomas W. Jeffries
Orientation of adjacent genes has been reported to affect their expression in eukaryotic systems, and metabolic engineering also often makes repeated use of a few promoters to obtain high expression. To improve transcriptional control in heterologous expression, we examined how these factors affect gene expression and enzymatic activity in Saccharomyces cerevisiae. We...
Full Text Available Abstract Background Perennial ryegrass (Lolium perenne L. is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. Results Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2 were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L. samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h, a moderately, but stably expressed eEF1A (s, and combined expression of multigene eEF1A (m. NormFinder identified eEF1A (s and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. Conclusions This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples
Galicia, Johnah C.; Henson, Brett R.; Parker, Joel S.; Khan, Asma A.
The cost, prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis. This study was aimed to identify the genetic contributors to pulpal pain and inflammation. Inflamed pulps were collected from patients diagnosed with irreversible pulpitis (n=20). Normal pulps from teeth extracted for various reasons served as controls (n=20). Pain level was assessed using a visual analog scale (VAS). Genome-wide microarray analysis was performed using Affymetrix GeneTitan Multichannel Instrument. The difference in gene expression levels were determined by the Significance Analysis of Microarray program using a false discovery rate (q-value) of 5%. Genes involved in immune response, cytokine-cytokine receptor interaction and signaling, integrin cell surface interactions, and others were expressed at relatively higher levels in the in the pulpitis group. Moreover, several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients (≥30mm on VAS) compared to those with moderate to severe pain. This exploratory study provides a molecular basis for the clinical diagnosis of pulpitis. With an enhanced understanding of pulpal inflammation, future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology. PMID:27052691
Full Text Available The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa.
Galicia, J C; Henson, B R; Parker, J S; Khan, A A
The cost, prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis. This study was aimed to identify the genetic contributors to pulpal pain and inflammation. Inflamed pulps were collected from patients diagnosed with irreversible pulpitis (n=20). Normal pulps from teeth extracted for various reasons served as controls (n=20). Pain level was assessed using a visual analog scale (VAS). Genome-wide microarray analysis was performed using Affymetrix GeneTitan Multichannel Instrument. The difference in gene expression levels were determined by the significance analysis of microarray program using a false discovery rate (q-value) of 5%. Genes involved in immune response, cytokine-cytokine receptor interaction and signaling, integrin cell surface interactions, and others were expressed at relatively higher levels in the pulpitis group. Moreover, several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients (⩾30 mm on VAS) compared with those with moderate to severe pain. This exploratory study provides a molecular basis for the clinical diagnosis of pulpitis. With an enhanced understanding of pulpal inflammation, future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology.
Vilar, Jose M.G.; Saiz, Leonor
Gene expression is a process central to any form of life. It involves multiple temporal and functional scales that extend from specific protein-DNA interactions to the coordinated regulation of multiple genes in response to intracellular and extracellular changes. This diversity in scales poses fundamental challenges to the use of traditional approaches to fully understand even the simplest gene expression systems. Recent advances in computational systems biophysics have provided promising avenues to reliably integrate the molecular detail of biophysical process into the system behavior. Here, we review recent advances in the description of gene regulation as a system of biophysical processes that extend from specific protein-DNA interactions to the combinatorial assembly of nucleoprotein complexes. There is now basic mechanistic understanding on how promoters controlled by multiple, local and distal, DNA binding sites for transcription factors can actively control transcriptional noise, cell-to-cell variability, and other properties of gene regulation, including precision and flexibility of the transcriptional responses. PMID:23790365
Glenn, Sean T.; Jones, Craig A.; Gross, Kenneth W.; Pan, Li
Renin, as part of the renin-angiotensin system, plays a critical role in the regulation of blood pressure, electrolyte homeostasis, mammalian renal development and progression of fibrotic/hypertrophic diseases. Renin gene transcription is subject to complex developmental and tissue-specific regulation. Initial studies using the mouse As4.1 cell line, which has many characteristics of the renin-expressing juxtaglomerular cells of the kidney, have identified a proximal promoter region (−197 to −50 bp) and an enhancer (−2866 to −2625 bp) upstream of the Ren-1c gene, which are critical for renin gene expression. The proximal promoter region contains several transcription factor-binding sites including a binding site for the products of the developmental control genes Hox. The enhancer consists of at least 11 transcription factor-binding sites and is responsive to various signal transduction pathways including cAMP, retinoic acid, endothelin-1, and cytokines, all of which are known to alter renin mRNA levels. Furthermore, in vivo models have validated several of these key components found within the proximal promoter region and the enhancer as well as other key sites necessary for renin gene transcription. PMID:22576577
Full Text Available Promoters process signals through recruitment of transcription factors and RNA polymerase, and dynamic changes in promoter activity constitute a major noise source in gene expression. However, it is barely understood how complex promoter architectures determine key features of promoter dynamics. Here, we employ prototypical promoters of yeast ribosomal protein genes as well as simplified versions thereof to analyze the relations among promoter design, complexity, and function. These promoters combine the action of a general regulatory factor with that of specific transcription factors, a common motif of many eukaryotic promoters. By comprehensively analyzing stationary and dynamic promoter properties, this model-based approach enables us to pinpoint the structural characteristics underlying the observed behavior. Functional tradeoffs impose constraints on the promoter architecture of ribosomal protein genes. We find that a stable scaffold in the natural design results in low transcriptional noise and strong co-regulation of target genes in the presence of gene silencing. This configuration also exhibits superior shut-off properties, and it can serve as a tunable switch in living cells. Model validation with independent experimental data suggests that the models are sufficiently realistic. When combined, our results offer a mechanistic explanation for why specific factors are associated with low protein noise in vivo. Many of these findings hold for a broad range of model parameters and likely apply to other eukaryotic promoters of similar structure.
Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal
The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene ...
Full Text Available Abstract Background Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes. Results Here we show that the introduction of Erv1p, a sulfhydryl oxidase and a disulfide isomerase allows the efficient formation of natively folded eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli. The production of disulfide bonded proteins was also aided by the use of an appropriate fusion protein to keep the folding intermediates soluble and by choice of media. By combining the pre-expression of a sulfhydryl oxidase and a disulfide isomerase with these other factors, high level expression of even complex disulfide bonded eukaryotic proteins is possible Conclusions Our results show that the production of eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli is possible. The required exogenous components can be put onto a single plasmid vector allowing facile transfer between different prokaryotic strains. These results open up new avenues for the use of E. coli as a microbial cell factory.
Full Text Available Abstract Background Native as well as recombinant bacterial cell surface layer (S-layer protein of Geobacillus (G. stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099 of the S-layer protein and EGFP (enhanced green fluorescent protein, in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. Results Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S. cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE promoter. Conclusion The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of
Full Text Available Alternative splicing generates an enormous amount of functional and proteomic diversity in metazoan organisms. This process is probably central to the macromolecular and cellular complexity of higher eukaryotes. While most studies have focused on the molecular mechanism triggering and controlling alternative splicing, as well as on its incidence in different species, its maintenance and evolution within populations has been little investigated. Here, we propose to address these questions by comparing the structural characteristics as well as the functional and transcriptional profiles of genes with monomorphic or polymorphic splicing, referred to as MS and PS genes, respectively. We find that MS and PS genes differ particularly in the number of tissues and cell types where they are expressed.We find a striking deficit of PS genes on the sex chromosomes, particularly on the Y chromosome where it is shown not to be due to the observed lower breadth of expression of genes on that chromosome. The development of a simple model of evolution of cis-regulated alternative splicing leads to predictions in agreement with these observations. It further predicts the conditions for the emergence and the maintenance of cis-regulated alternative splicing, which are both favored by the tissue specific expression of splicing variants. We finally propose that the width of the gene expression profile is an essential factor for the acquisition of new transcript isoforms that could later be maintained by a new form of balancing selection.
Landgrebe, Jobst; Dierks, Thomas; Schmidt, Bernhard; von Figura, Kurt
Recently, the human C(alpha)-formylglycine (FGly)-generating enzyme (FGE), whose deficiency causes the autosomal-recessively transmitted lysosomal storage disease multiple sulfatase deficiency (MSD), has been identified. In sulfatases, FGE posttranslationally converts a cysteine residue to FGly, which is part of the catalytic site and is essential for sulfatase activity. FGE is encoded by the sulfatase modifying factor 1 (SUMF1) gene, which defines a new gene family comprising orthologs from prokaryotes to higher eukaryotes. The genomes of E. coli, S. cerevisiae and C. elegans lack SUMF1, indicating a phylogenetic gap and the existence of an alternative FGly-generating system. The genomes of vertebrates including mouse, man and pufferfish contain a sulfatase modifying factor 2 (SUMF2) gene encoding an FGE paralog of unknown function. SUMF2 evolved from a single exon SUMF1 gene as found in diptera prior to divergent intron acquisition. In several prokaryotic genomes, the SUMF1 gene is cotranscribed with genes encoding sulfatases which require FGly modification. The FGE protein contains a single domain that is made up of three highly conserved subdomains spaced by nonconserved sequences of variable lengths. The similarity among the eukaryotic FGE orthologs varies between 72% and 100% for the three subdomains and is highest for the C-terminal subdomain, which is a hotspot for mutations in MSD patients.
Li, Tao; Li, Zhan; Chen, Fanghong; Liu, Xiong; Ning, Nianzhi; Huang, Jie; Wang, Hui
Enterohemorrhagic Escherichia coli (EHEC) or other attaching/effacing pathogen infections often cause host intestinal inflammation and pathology, which is thought to result in part from a host aggressive innate immune response. However, few effectors that play an important role in this pathology change have been reported. In this study, we discovered a previously unknown EHEC effector, Stk (putative serine/threonine kinase), which induces host aggressive inflammatory response during EHEC infection. Interestingly, homologous proteins of Stk are widely distributed in many pathogens. After translocating into the infected host cells, Stk efficiently phosphorylates IκBα and activates the NF-κB pathway. In EHEC-infected mice, Stk increases serum keratinocyte-derived cytokine (KC) levels and hyperactivates the inflammatory response of the colon, intensifying pathological injury of the colon. The virulence of Stk is based on its eukaryotic-like kinase activity. In conclusion, our data suggest that Stk is a new effector that induces the host aggressive inflammatory response during EHEC infection. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: firstname.lastname@example.org.
Full Text Available Objective To construct a eukaryotic expression vector for HuR and analyze its expression and biological function in NIH3T3 cells.Methods The total RNA was extracted from NIH3T3 cells and reverse transcribed to cDNAs.The coding region sequence of mouse HuR was then amplified by PCR and subcloned into the pcDNA3.1-FLAG plasmid.The recombinant plasmid pcDNA3.1-HuR-FLAG was verified by PCR and restriction endonuclease analysis,confirmed by DNA sequence analysis,and then transiently transfected into NIH3T3 cells with Lipofectamine LTX.The expression of HuR protein was determined by Western blotting,and the mRNA level of HuR and DUSP1 were analyzed by using real-time PCR.Result The recombinant plasmid pcDNA3.1-HuR-FLAG was correctly constructed.Twenty-four hours after transfection of the recombinant plasmid into NIH3T3 cells,the fusion protein was found to have highly expressed in the cells as revealed by Western blotting.Real-time PCR results detected that the over-expression of HuR could up-regulate the expression of DUSP1.Conclusion The eukaryotic expression vector for HuR-FLAG fusion protein has been successfully constructed and transiently expressed in NIH3T3 cells.It can be used in further analysis of the posttranscriptional regulation of DUSP1 by HuR in cancer cells.
Pan, Qiuling; Wang, Ling; Dang, Xiaoqun; Ma, Zhengang; Zhang, Xiaoyan; Chen, Shiliang; Zhou, Zeyang; Xu, Jinshan
The microsporidia Nosema bombycis is the insect pathogen of pebrine disease severely destructive to sericulture production. Here, we describe the use of Escherichia coli HT115 strain (DE3) to express double-strand RNAs targeting the gene encoding ADP/ATP protein in N. bombycis. The results showed that dsRNAs deferentially suppressed the gene expression during N. bombycis infection in the silkworm, and the effect waned gradually. Our results, for the first time, provide a tool to utilize the dsRNA expressed by recombinant E. coli to control the pebrine disease of the domestic silkworm. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.
Full Text Available The complexity and diversity of eukaryotic organisms are a feat of nature’s engineering. Pulling the strings of such an intricate machinery requires an even more masterful and crafty approach. Only the number and type of responses that they generate exceed the staggering proportions of environmental signals perceived and processed by eukaryotes. Hence, at first glance, the cell’s sparse stockpile of controlling factors does not seem remotely adequate to carry out this response. The question as to how eukaryotes sense and respond to environmental cues has no single answer. It is an amalgamation, an interplay between several processes, pathways, and factors—a combinatorial control. A short description of some of the most important elements that operate this entire conglomerate is given in this paper.
Full Text Available Rice (Oryza sativa L. is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE genes, 33K annotated non-expressed (ANE genes, and 5.5K non-annotated expressed (NAE genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria.
Munsky, B.; Neuert, G.; van Oudenaarden, A.
Phenotypic variation is ubiquitous in biology and is often traceable to underlying genetic and environmental variation. However, even genetically identical cells in identical environments display variable phenotypes. Stochastic gene expression, or gene expression "noise," has been suggested as a
Haqq, Christopher; Nosrati, Mehdi; Sudilovsky, Daniel; Crothers, Julia; Khodabakhsh, Daniel; Pulliam, Brian L.; Federman, Scot; Miller, James R.; Allen, Robert E.; Singer, Mark I.; Leong, Stanley P L; Ljung, Britt-Marie; Sagebiel, Richard W.; Kashani-Sabet, Mohammed
Because of the paucity of available tissue, little information has previously been available regarding the gene expression profiles of primary melanomas. To understand the molecular basis of melanoma progression, we compared the gene expression profiles of a series of nevi, primary melanomas, and melanoma metastases. We found that metastatic melanomas exhibit two dichotomous patterns of gene expression, which unexpectedly reflect gene expression differences already apparent in comparing laser...
Iglesias-Platas, Isabel; Monk, David
The purpose of this review is to highlight the recent advances in epigenetic regulation and chromatin biology for a better understanding of gene regulation related to human disease. Alterations to chromatin influence genomic function, including gene transcription. At its most simple level, this involves DNA methylation and posttranscriptional histone modifications. However, recent developments in biochemical and molecular techniques have revealed that transcriptional regulation is far more complex, involving combinations of histone modifications and discriminating transcription factor binding, and long-range chromatin loops with enhancers, to generate a multifaceted code. Here, we describe the most recent advances, culminating in the example of genomic imprinting, the parent-of-origin monoallelic expression that utilizes the majority of these mechanisms to attain one active and one repressed allele. It is becoming increasingly evident that epigenetic mechanisms work in unison to maintain tight control of gene expression and genome function. With the wealth of knowledge gained from recent molecular studies, future goals should focus on the application of this information in deciphering their role in developmental diseases.
Full Text Available Plants synthesize and accumulate a series of stress-resistance proteins to protect normal physiological activities under adverse conditions. Chimonanthus praecox which blooms in freezing weather accumulates late embryogenesis abundant proteins (LEAs in flowers, but C. praecox LEAs are little reported. Here, we report a group of five LEA genes of C. praecox (CpLEA5, KT727031. Prokaryotic-expressed CpLEA5 was employed in Escherichia coli to investigate bioactivities and membrane permeability at low-temperature. In comparison with the vacant strains, CpLEA5-containing strains survived in a 20% higher rate; and the degree of cell membrane damage in CpLEA5-containing strains was 55% of that of the vacant strains according to a conductivity test, revealing the low-temperature resistance of CpLEA5 in bacteria. CpLEA5 was also expressed in Pichia pastoris. Interestingly, besides low-temperature resistance, CpLEA5 conferred high resistance to salt and alkali in CpLEA5 overexpressing yeast. The CpLEA5 gene was transferred into Arabidopsis thaliana to also demonstrate CpLEA5 actions in plants. As expected, the transgenic lines were more resistant against low-temperature and drought while compared with the wild type. Taken together, CpLEA5-conferred resistances to several conditions in prokaryote and eukaryotes could have great value as a genetic technology to enhance osmotic stress and low-temperature tolerance.
Noort, Vera van
This thesis focuses on developing comparative genomics methods in eukaryotes, with an emphasis on applications for gene function prediction and regulatory element detection. In the past, methods have been developed to predict functional associations between gene pairs in prokaryotes. The challenge
Bedbrook, Claire N; Yang, Kevin K; Rice, Austin J; Gradinaru, Viviana; Arnold, Frances H
There is growing interest in studying and engineering integral membrane proteins (MPs) that play key roles in sensing and regulating cellular response to diverse external signals. A MP must be expressed, correctly inserted and folded in a lipid bilayer, and trafficked to the proper cellular location in order to function. The sequence and structural determinants of these processes are complex and highly constrained. Here we describe a predictive, machine-learning approach that captures this complexity to facilitate successful MP engineering and design. Machine learning on carefully-chosen training sequences made by structure-guided SCHEMA recombination has enabled us to accurately predict the rare sequences in a diverse library of channelrhodopsins (ChRs) that express and localize to the plasma membrane of mammalian cells. These light-gated channel proteins of microbial origin are of interest for neuroscience applications, where expression and localization to the plasma membrane is a prerequisite for function. We trained Gaussian process (GP) classification and regression models with expression and localization data from 218 ChR chimeras chosen from a 118,098-variant library designed by SCHEMA recombination of three parent ChRs. We use these GP models to identify ChRs that express and localize well and show that our models can elucidate sequence and structure elements important for these processes. We also used the predictive models to convert a naturally occurring ChR incapable of mammalian localization into one that localizes well.
Snel, B.; Noort, V. van; Huynen, M.A.
Differences between species have been suggested to largely reside in the network of connections among the genes. Nevertheless, the rate at which these connections evolve has not been properly quantified. Here, we measure the extent to which co-regulation between pairs of genes is conserved over
Hansson, Bengt; Naurin, Sara; Hasselquist, Dennis
Inbreeding increases homozygosity, exposes genome-wide recessive deleterious alleles and often reduces fitness. The physiological and reproductive consequences of inbreeding may be manifested already during gene regulation, but the degree to which inbreeding influences gene expression is unknown in most organisms, including in birds. To evaluate the pattern of inbreeding-affected gene expression over the genome and in relation to sex, we performed a transcriptome-wide gene expression (10 695 genes) study of brain tissue of 10-day-old inbred and outbred, male and female zebra finches. We found significantly lower gene expression in females compared with males at Z-linked genes, confirming that dosage compensation is incomplete in female birds. However, inbreeding did not affect gene expression at autosomal or sex-linked genes, neither in males nor in females. Analyses of single genes again found a clear sex-biased expression at Z-linked genes, whereas only a single gene was significantly affected by inbreeding. The weak effect of inbreeding on gene expression in zebra finches contrasts to the situation, for example, in Drosophila where inbreeding has been found to influence gene expression more generally and at stress-related genes in particular. © 2014 The Author(s) Published by the Royal Society. All rights reserved.
Huberman, Eliezer [Chicago, IL; Baccam, Mekhine J [Woodridge, IL
The present invention relates to a nucleic acid sequence and its corresponding protein sequence useful as a dominant selectable marker in eukaryotes. More specifically the invention relates to a nucleic acid encoding a bacterial IMPDH gene that has been engineered into a eukaryotic expression vectors, thereby permitting bacterial IMPDH expression in mammalian cells. Bacterial IMPDH expression confers resistance to MPA which can be used as dominant selectable marker in eukaryotes including mammals. The invention also relates to expression vectors and cells that express the bacterial IMPDH gene as well as gene therapies and protein synthesis.
Solem, Christian; Jensen, Peter Ruhdal
A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example...... that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the las operon, containing the genes pfk, pyk, and ldh, was modulated by integrating a truncated copy of the pfk gene. Importantly, the modulation affected...
Wang, Yuanyuan; Hu, Jieli; Cui, Jing; Huang, Ailong; Ruan, Xiongzhong; Chen, Yaxi
The experiment was designed to investigate the function of SREBP cleavage-activating protein (SCAP) mutant (D443N) by constructing an eukaryotic expressive vector using a smooth muscle specific promoter SM22 (pGL3-SM22-SCAP(D443N)). SM22 promoter (pSM22) was amplified from genome DNA of mice by nested PCR, and then cloned into pMD-T vector. The SM22 promoter fragment released from the vector by Kpn I and Hind III digestion was sub-cloned into pGL3-control-Luc vector, to form pGL3-SM22-Luc. The activity of pSM22 in human vascular smooth muscle cells (VSMCs) was tested using Dual-Luciferase Reporter System. SCAP(D443) mutant amplified from plasmid pTK-HSV-SCAP(D443N) and pSM22 from mice liver were cloned into pGL3-control vector to construct pGL3-SM22-SCAP(D443N) which was transfected into Chinese hamster ovary cells (CHO) to test SCAP(D443) expression by real-time PCR and Western blot. The sequence and construction of pGL3-SM22-SCAP(D443N) were correct. SM22 promoter activity initiated the expression of luciferase in VSMCs and also drove SCAP(D443) expression in transfected CHO cells. The pGL3-SM22-SCAP(D443N) eukaryotic expression vector was successfully constructed and the recombinant vector provides a powerful approach in investigating the function and regulation of SCAP and also in producing vascular smooth muscle specific SCAP transgenic mice.
Bohuski Elizabeth; Colbourne John K; Eads Brian D; Andrews Justen
Abstract Background Sexual reproduction is a core biological function that is conserved throughout eukaryotic evolution, yet breeding systems are extremely variable. Genome-wide comparative studies can be effectively used to identify genes and regulatory patterns that are constrained to preserve core functions from those that may help to account for the diversity of animal reproductive strategies. We use a custom microarray to investigate gene expression in males and two reproductive stages o...
Full Text Available Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. Here, we present evidence that adaptation dominates the evolution of gene expression levels in flies. We show that 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. Our results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, we identify a large group of genes with sex-specific adaptation of expression, which predominantly occurs in males. Our analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis.
Fehrmann, Rudolf S. N.; Karjalainen, Juha M.; Krajewska, Malgorzata
Many cancer-associated somatic copy number alterations (SCNAs) are known. Currently, one of the challenges is to identify the molecular downstream effects of these variants. Although several SCNAs are known to change gene expression levels, it is not clear whether each individual SCNA affects gene...... expression. We reanalyzed 77,840 expression profiles and observed a limited set of 'transcriptional components' that describe well-known biology, explain the vast majority of variation in gene expression and enable us to predict the biological function of genes. On correcting expression profiles...... for these components, we observed that the residual expression levels (in 'functional genomic mRNA' profiling) correlated strongly with copy number. DNA copy number correlated positively with expression levels for 99% of all abundantly expressed human genes, indicating global gene dosage sensitivity. By applying...
A. P. Kozlov
The evolutionarily novel genes originated through different molecular mechanisms are expressed in tumors. Sometimes the expression of evolutionarily novel genes in tumors is highly specific. Moreover positive selection of many human tumor-related genes in primate lineage suggests their involvement in the origin of new functions beneficial to organisms. It is suggested to consider the expression of evolutionarily young or novel genes in tumors as a new biological phenomenon, a phenomenon of TS...
Full Text Available Abstract Background In order to improve gene prediction, extrinsic evidence on the gene structure can be collected from various sources of information such as genome-genome comparisons and EST and protein alignments. However, such evidence is often incomplete and usually uncertain. The extrinsic evidence is usually not sufficient to recover the complete gene structure of all genes completely and the available evidence is often unreliable. Therefore extrinsic evidence is most valuable when it is balanced with sequence-intrinsic evidence. Results We present a fairly general method for integration of external information. Our method is based on the evaluation of hints to potentially protein-coding regions by means of a Generalized Hidden Markov Model (GHMM that takes both intrinsic and extrinsic information into account. We used this method to extend the ab initio gene prediction program AUGUSTUS to a versatile tool that we call AUGUSTUS+. In this study, we focus on hints derived from matches to an EST or protein database, but our approach can be used to include arbitrary user-defined hints. Our method is only moderately effected by the length of a database match. Further, it exploits the information that can be derived from the absence of such matches. As a special case, AUGUSTUS+ can predict genes under user-defined constraints, e.g. if the positions of certain exons are known. With hints from EST and protein databases, our new approach was able to predict 89% of the exons in human chromosome 22 correctly. Conclusion Sensitive probabilistic modeling of extrinsic evidence such as sequence database matches can increase gene prediction accuracy. When a match of a sequence interval to an EST or protein sequence is used it should be treated as compound information rather than as information about individual positions.
Full Text Available ABSTRACT Safflower (Carthamus tinctorius L., Asteraceae is an important oil crop and medicinal plant. Gene expression analysis is gaining importance in the research of safflower. Quantitative PCR has become a powerful method for gene study. Reference genes are one of the major qualification requirements of qPCR because they can reduce the variability. To identify the reference genes in safflower, nine candidate genes of the housekeeping genes were selected from the EST library of safflower constructed by our lab: CtACT (actin, CtGAPDH (glyceraldehyde 3-phosphate dehydrogenase, CtE1F4A (elongation factor 1 alpha, CtTUA (alpha-tubulin, CtTUB (beta-tubulin, CtPP2A (serine/threonine-protein phosphatase, CtE1F4A (eukaryotic initiation factor 4A, CtUBI (Ubiquitin, and Ct60S (60S acidic ribosomal protein. Expression stability was examined by qPCR across 54 samples, representing tissues at different flowering stages and two chemotype of safflower lines. We assessed the expression stability of these candidate genes by employing four different algorithms (geNorm, NormFinder, ΔCt approach, and BestKeeper and found that CtUBI and Ct60S were the highly ranked candidate genes. CtUBI and Ct60S were used as reference genes to evaluate the expression of CtFAD2-10 and CtKASII. Our data suggest CtUBI and Ct60S could be used as internal controls to normalize gene expression in safflower.
Xiao Li Shi
Full Text Available The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX, which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.
Lee, Andrew Loyd [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry
Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the δ-Al-ε activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a βαβ-βαβ pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel β-sheet. In addition 15N T1, T2, and 15N/1H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone 1H, 13C, and 15N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and 15N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.
Michael F. Duffy
Full Text Available Histone modifications are important in regulating gene expression in eukaryotes. Of the numerous histone modifications which have been identified, acetylation is one of the best characterised and is generally associated with active genes. Histone acetylation can directly affect chromatin structure by neutralising charges on the histone tail, and can also function as a binding site for proteins which can directly or indirectly regulate transcription. Bromodomains specifically bind to acetylated lysine residues on histone tails, and bromodomain proteins play an important role in anchoring the complexes of which they are a part to acetylated chromatin. Bromodomain proteins are involved in a diverse range of functions, such as acetylating histones, remodeling chromatin, and recruiting other factors necessary for transcription. These proteins thus play a critical role in the regulation of transcription.
Full Text Available Background & Objectives: hcpD gene in Helicobacter pylori is a member of cysteine-rich proteins family which triggers the host's immune system and antibody production. H. pylori is found in human's stomach and causes digestive diseases such as duodenal ulcer, chronic gastritis and stomach cancer. The objectives of this study were to isolate, amplify and clone H. pylori's hcpD gene in pcDNA3.1 (- vector and to study its expression in eukaryotic system. Methods: H. pylori genomic DNA was isolated by extraction kit. The hcpD gene was amplified using PCR reaction and then purified from gel, followed by pTZ cloning. Subcloning of hcpD was performed in pcDNA3.1 (- eukaryotic expression vector. The accuracy of cloning steps was investigated through PCR, enzymatic digestion by BamHI and EcoRV enzymes, and sequencing, respectively. Transfer of expression construct into CHO cells was done by electroporation. The gene expression in these cells was analyzed using RT-PCR and SDS-PAGE. Results: PCR results showed amplification of a 933bp segment related to hcpD gene. Successful cloning of the gene in pTZ vector and construction of pTZ-hcpD recombinant vector were achieved. Enzymatic digestion and sequencing confirmed the correctness of subcloning and creation of pcDNA3.1 (--hcpD construct. hcpD was expressed in eukaryotic system, and its protein product was observed on SDS-PAGE gel. Conclusion: pTZ-hcpD construct can be used as a source of H. pylori's hcpD gene for future research, like production of recombinant protein and vaccine in different systems. Furthermore, successful expression of the gene using pcDNA3.1 (--hcpD in CHO animal cells shows the potential of vector as a gene vaccine against H. pylori.
Kourmpetis, Y.A.I.; Dijk, van A.D.J.; Braak, ter C.J.F.
Gene Ontology (GO) is a hierarchical vocabulary for the description of biological functions and locations, often employed by computational methods for protein function prediction. Due to the structure of GO, function predictions can be self- contradictory. For example, a protein may be predicted to
Stranger, Barbara E; Raj, Towfique
A steadily growing number of studies have identified and characterized expression quantitative trait loci (eQTLs) in human cell-lines, primary cells, and tissues. This class of variation has been shown to play a role in complex traits, including disease. Here, we discuss how eQTLs have the potential to accelerate discovery of disease genes and functional mechanisms underlying complex traits. We discuss how context-specificity of eQTLs is being characterized at an unprecedented scale and breadth, and how this both informs on the intricacy of human genome function, and has important ramifications for elucidating function of genetic variants of interest, particularly for those contributing to disease. Copyright © 2013 Elsevier Ltd. All rights reserved.
Bai, Shuangyi; Wallis, James G; Denolf, Peter; Browse, John
Directed evolution of a cyanobacterial Δ9 fatty acid desaturase (DSG) from Synechococcus elongatus, PCC6301 created new, more productive desaturases and revealed the importance of certain amino acid residues to increased desaturation. A codon-optimized DSG open reading frame with an endoplasmic-reticulum retention/retrieval signal appended was used as template for random mutagenesis. Increased desaturation was detected using a novel screen based on complementation of the unsaturated fatty acid auxotrophy of Saccharomyces cerevisiae mutant ole1Δ. Amino acid residues whose importance was discovered by the random processes were further examined by saturation mutation to determine the best amino acid at each identified location in the peptide chain and by combinatorial analysis. One frequently-detected single amino acid change, Q240R, yielded a nearly 25-fold increase in total desaturation in S. cerevisiae. Several other variants of the protein sequence with multiple amino acid changes increased total desaturation more than 60-fold. Many changes leading to increased desaturation were in the vicinity of the canonical histidine-rich regions known to be critical for electron transfer mediated by these di-iron proteins. Expression of these evolved proteins in the seed of Arabidopsis thaliana altered the fatty acid composition, increasing monounsaturated fatty acids and decreasing the level of saturated fatty acid, suggesting a potential application of these desaturases in oilseed crops. Biotechnol. Bioeng. 2016;113: 1522-1530. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
De Marco, N; Tussellino, M; Carotenuto, R; Ronca, R; Rizzolio, S; Biffo, S; Campanella, C
The eukaryotic initiation translation factor eIF6 is a highly conserved, essential protein implicated in translation. eIF6 is regulated in vivo by extracellular signals, such as IGF signaling (for a review see Miluzio et al., 2009). In Xenopus, eif6 over-expression causes a delay in eye development (De Marco et al., 2011). In this study we showed that eif6 co-immunoprecipitates with the insulin-like growth factor receptor (igfr) and may function downstream of igf in eye formation. The relationship between eif6 and gipc2, a protein partner of a variety of molecules including membrane proteins, was investigated. gipc2 is required for maintaining igf-induced akt activation on eye development (Wu et al., 2006). Significantly eif6 and gipc2 have opposite effects in eye development. While eif6 is required for eye formation below threshold levels, gipc2 knockdown impairs eye development (De Marco et al., 2011; Wu et al., 2006). In this study, it was shown that in eif6 over-expressors, the delay in eye morphogenesis is reversed by gipc2 injection, while the injection of eif6 down-regulates gipc2 expression. Real-time-PCR indicates that eif6 regulates gipc2 expression in a dose-dependent manner. In contrast, gipc2 knockdown has no significant effect on eif6 mRNA levels. These results suggest that eif6 regulation of gipc2 enables correct morphogenesis of Xenopus eye and stimulate questions on the molecular network implicated in this process. Copyright © 2017 Elsevier Inc. All rights reserved.
Xiaoyu, Yang; Shiyi, Li; Di, Zhang; Ying, Wu; Tao, Yang; Changhong, Liu
To clone and construct a eukaryotic expression vector of human bone morphogenetic protein (BMP) 2 and histidine in vitro and synthesize chitosan (CS)/pIRES2-EGFP-hBMP2-His nanoparticles. pMD18T-hBMP2-His was digested by EcoR I and BamH I to obtain the hBMP2-His gene, which was inserted into pIRES2-EGFP to form pIRES2-EGFP-hBMP2-His. Afterward, CS, which exhibited five different molecular weights and deacetylation degrees, was complexed with pIRES2-EGFP-hBMP2-His to form CS/pIRES2-EGFP-hBMP2-His nanoparticles; in this procedure, a desolvent method was used at different N/P ratios (amino in CS to phospho in plasmid DNA). The gene-encapsulating ability of CS was evaluated by agarose gel electrophoresis and fluorescence spectrophotometry; size, distribution, and potential were analyzed using a ZetaPALS analyzer. The shape of the nanoparticles was observed under an atomic force microscope. 1) pIRES2-EGFP-hBMP2-His was constructed after the cloned hBMP2-His gene was confirmed by sequencing. 2) CS/pIRES2-EGFP-hBMP2-His nanoparticles were synthesized and pIRES2-EGFP-hBMP2-His was packaged by CS. 3) CS/pIRES2-EGFP-hBMP2-His nanoparticles were globular with an average size of 111.7 nm to 3,214.2 nm and an average zeta-potential of 4.93 mV to 16.79 mV. CS/pIRES2-EGFP-hBMP2-His nanospheres are successfully synthesized.
Berka, Randy [Davis, CA; Bachkirova, Elena [Davis, CA; Rey, Michael [Davis, CA
The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.
Berka, Randy; Bachkirova, Elena; Rey, Michael
The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.
Daan C Swarts
Full Text Available Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a role in host defense. Argonaute of the bacterium Thermus thermophilus (TtAgo targets invading plasmid DNA during and after transformation. Using small interfering DNA guides, TtAgo can cleave single and double stranded DNAs. Although TtAgo additionally has been demonstrated to cleave RNA targets complementary to its DNA guide in vitro, RNA targeting by TtAgo has not been demonstrated in vivo.To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (Δago, and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels.In the absence of exogenous DNA (plasmid, TtAgo presence or absence had no effect on gene expression levels. When plasmid DNA is present, TtAgo reduces plasmid DNA levels 4-fold, and a corresponding reduction of plasmid gene transcript levels was observed. We therefore conclude that TtAgo interferes with plasmid DNA, but not with plasmid-encoded RNA. Interestingly, TtAgo presence stimulates expression of specific endogenous genes, but only when exogenous plasmid DNA was present. Specifically, the presence of TtAgo directly or indirectly stimulates expression of CRISPR loci and associated genes, some of which are involved in CRISPR adaptation. This suggests that TtAgo-mediated interference with plasmid DNA stimulates CRISPR adaptation.
Full Text Available The activity state of a gene is determined by a complex regulatory network of co-acting factors affecting the structure of the chromatin into which the gene is embedded. While significant changes of the transcriptome occur during cell differentiation in apicomplexan parasites, basic mechanisms controlling gene expression are still unknown. Recent studies support and expand the concept of the chromatin environment being key factor for the control of transcriptional activity in these lower eukaryotes organisms. Here, we review recent advances in the field of epigenetic gene regulation in Toxoplasma gondii, the model apicomplexan.
Chen, Yifei; Li, Yi; Narayan, Rajiv; Subramanian, Aravind; Xie, Xiaohui
Large-scale gene expression profiling has been widely used to characterize cellular states in response to various disease conditions, genetic perturbations, etc. Although the cost of whole-genome expression profiles has been dropping steadily, generating a compendium of expression profiling over thousands of samples is still very expensive. Recognizing that gene expressions are often highly correlated, researchers from the NIH LINCS program have developed a cost-effective strategy of profiling only ∼1000 carefully selected landmark genes and relying on computational methods to infer the expression of remaining target genes. However, the computational approach adopted by the LINCS program is currently based on linear regression (LR), limiting its accuracy since it does not capture complex nonlinear relationship between expressions of genes. We present a deep learning method (abbreviated as D-GEX) to infer the expression of target genes from the expression of landmark genes. We used the microarray-based Gene Expression Omnibus dataset, consisting of 111K expression profiles, to train our model and compare its performance to those from other methods. In terms of mean absolute error averaged across all genes, deep learning significantly outperforms LR with 15.33% relative improvement. A gene-wise comparative analysis shows that deep learning achieves lower error than LR in 99.97% of the target genes. We also tested the performance of our learned model on an independent RNA-Seq-based GTEx dataset, which consists of 2921 expression profiles. Deep learning still outperforms LR with 6.57% relative improvement, and achieves lower error in 81.31% of the target genes. D-GEX is available at https://github.com/uci-cbcl/D-GEX CONTACT: email@example.com Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: firstname.lastname@example.org.
Full Text Available Objective To investigate the expression of green fluorescent protein plasmid of human papilloma virus 6b L1 gene(HPV6bL1 in eukaryotic cells.Methods The L1 gene of PQE40-HPV6bL1 was amplified by PCR,purified by restriction enzyme digestion,and then connected to eukaryotic expression plasmid PEGFP-C1.The recombinant expression vector was then transformed into E.coli DH5a,which was identified by BamH Ⅰ and Hand Ⅲ digestion and the positive vector was selected.The recombinant plasmid PEGFP-HPV6bL1 was transfected into COS-7 cells by liposomal transfection technique and the expression of fusion protein was observed under fluorescence microscope.The generation of HPV6bL1 mRNA was detected by RT-PCR.Results Identification of PEGFP-HPV6bL1 by enzyme digestion and sequencing showed that the length,direction and inserted location of target,which was inserted into the recombinant,was correct and the expression of EGFP in transfected cell was observed.Conclusions A new type of green fluorescent HPV6bL1 eukaryotic expression system has been established.It may provide a research foundation for the study of the protein.
Brahmachari Samir K
Full Text Available Abstract Background Poly purine.pyrimidine sequences have the potential to adopt intramolecular triplex structures and are overrepresented upstream of genes in eukaryotes. These sequences may regulate gene expression by modulating the interaction of transcription factors with DNA sequences upstream of genes. Results A poly purine.pyrimidine sequence with the potential to adopt an intramolecular triplex DNA structure was designed. The sequence was inserted within a nucleosome positioned upstream of the β-galactosidase gene in yeast, Saccharomyces cerevisiae, between the cycl promoter and gal 10Upstream Activating Sequences (UASg. Upon derepression with galactose, β-galactosidase gene expression is reduced 12-fold in cells carrying single copy poly purine.pyrimidine sequences. This reduction in expression is correlated with reduced transcription. Furthermore, we show that plasmids carrying a poly purine.pyrimidine sequence are not specifically lost from yeast cells. Conclusion We propose that a poly purine.pyrimidine sequence upstream of a gene affects transcription. Plasmids carrying this sequence are not specifically lost from cells and thus no additional effort is needed for the replication of these sequences in eukaryotic cells.
Sturrock, Marc; Li, Shiyu; Shahrezaei, Vahid
Gene expression is an inherently noisy process. This noise is generally thought to be deleterious as precise internal regulation of biochemical reactions is essential for cell growth and survival. Self-repression of gene expression, which is the simplest form of a negative feedback loop, is commonly believed to be employed by cellular systems to decrease the stochastic fluctuations in gene expression. When there is some delay in autoregulation, it is also believed that this system can generate oscillations. In eukaryotic cells, mRNAs that are synthesised in the nucleus must be exported to the cytoplasm to function in protein synthesis, whereas proteins must be transported into the nucleus from the cytoplasm to regulate the expression levels of genes. Nuclear transport thus plays a critical role in eukaryotic gene expression and regulation. Some recent studies have suggested that nuclear retention of mRNAs can control noise in mRNA expression. However, the effect of nuclear transport on protein noise and its interplay with negative feedback regulation is not completely understood. In this paper, we systematically compare four different simple models of gene expression. By using simulations and applying the linear noise approximation to the corresponding chemical master equations, we investigate the influence of nuclear import and export on noise in gene expression in a negative autoregulatory feedback loop. We first present results consistent with the literature, i.e., that negative feedback can effectively buffer the variability in protein levels, and nuclear retention can decrease mRNA noise levels. Interestingly we find that when negative feedback is combined with nuclear retention, an amplification in gene expression noise can be observed and is dependant on nuclear translocation rates. Finally, we investigate the effect of nuclear compartmentalisation on the ability of self-repressing genes to exhibit stochastic oscillatory dynamics. Copyright © 2017 Elsevier
Anaya-Bergman, C; Rosato, A; Lewis, J P
grown with eukaryotic cells. Taken together, the results of this study demonstrated the utility of whole-genome microarray analysis for the identification of genes with altered expression profiles during varying growth conditions and provided a framework for the detailed analysis of the molecular mechanisms of iron and hemin acquisition, metabolism and virulence of P. gingivalis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Francisco J. Romero-Campero
Full Text Available Phototrophic eukaryotes are among the most successful organisms on Earth due to their unparalleled efficiency at capturing light energy and fixing carbon dioxide to produce organic molecules. A conserved and efficient network of light-dependent regulatory modules could be at the bases of this success. This regulatory system conferred early advantages to phototrophic eukaryotes that allowed for specialization, complex developmental processes and modern plant characteristics. We have studied light-dependent gene regulatory modules from algae to plants employing integrative-omics approaches based on gene co-expression networks. Our study reveals some remarkably conserved ways in which eukaryotic phototrophs deal with day length and light signaling. Here we describe how a family of Arabidopsis transcription factors involved in photoperiod response has evolved from a single algal gene according to the innovation, amplification and divergence theory of gene evolution by duplication. These modifications of the gene co-expression networks from the ancient unicellular green algae Chlamydomonas reinhardtii to the modern brassica Arabidopsis thaliana may hint on the evolution and specialization of plants and other organisms.
Maria A dos Santos silva Landin
Full Text Available The aim of this study was to describe the expression of genes, including ameloblastin (Ambn, amelogenin X chromosome (Amelx and enamelin (Enam during early (pre-secretory tooth development. The expression of these genes has predominantly been studied at post-secretory stages. Deoxyoligonucleotide microarrays were used to study gene expression during development of the murine first molar tooth germ at 24h intervals, starting at the eleventh embryonic day (E11.5 and up to the seventh day after birth (P7. The profile search function of Spotfire software was used to select genes with similar expression profile as the enamel genes (Ambn, Amelx and Enam. Microarray results where validated using real-time Reverse Transcription-Polymerase Chain Reaction (real-time RT-PCR, and translated proteins identified by Western blotting. In situ localisation of the Ambn, Amelx and Enam mRNAs were monitored from E12.5 to E17.5 using deoxyoligonucleotide probes. Bioinformatics analysis was used to associate biological functions with differentially (p ≤0.05 expressed (DE genes.Microarray results showed a total of 4362 genes including Ambn, Amelx and Enam to be significant differentially expressed throughout the time-course. The expression of the three enamel genes was low at pre-natal stages (E11.5-P0 increasing after birth (P1-P7. Profile search lead to isolation of 87 genes with significantly similar expression to the three enamel proteins. The mRNAs expressed in dental epithelium and epithelium derived cells. Although expression of Ambn, Amelx and Enam were lower during early tooth development compared to secretory stages enamel proteins were detectable by Western blotting. Bioinformatic analysis associated the 87 genes with multiple biological functions. Around thirty-five genes were associated with fifteen transcription factors.
Holwerda, S.; de Laat, W.
Technological developments and intense research over the last years have led to a better understanding of the 3D structure of the genome and its influence on genome function inside the cell nucleus. We will summarize topological studies performed on four model gene loci: the alpha- and beta-globin
Conclusion: Progressive therapy with Dendrobium mixture, which has glucose- and lipid-lowering effects, is associated with multi-gene expression pathways. By treating diabetic r and wild-type rats with the mixture, the disorder is further understood at the transcriptomic level. Keywords: Diabetes, Gene expression, ...
van Ruissen, Fred; Baas, Frank
In 1995, serial analysis of gene expression (SAGE) was developed as a versatile tool for gene expression studies. SAGE technology does not require pre-existing knowledge of the genome that is being examined and therefore SAGE can be applied to many different model systems. In this chapter, the SAGE
Ravarani, Charles N J; Chalancon, Guilhem; Breker, Michal; de Groot, Natalia Sanchez; Babu, M Madan
Cell-to-cell variation in gene expression levels (noise) generates phenotypic diversity and is an important phenomenon in evolution, development and disease. TATA-box binding protein (TBP) is an essential factor that is required at virtually every eukaryotic promoter to initiate transcription. While the presence of a TATA-box motif in the promoter has been strongly linked with noise, the molecular mechanism driving this relationship is less well understood. Through an integrated analysis of multiple large-scale data sets, computer simulation and experimental validation in yeast, we provide molecular insights into how noise arises as an emergent property of variable binding affinity of TBP for different promoter sequences, competition between interaction partners to bind the same surface on TBP (to either promote or disrupt transcription initiation) and variable residence times of TBP complexes at a promoter. These determinants may be fine-tuned under different conditions and during evolution to modulate eukaryotic gene expression noise.
Vera, Maria; Biswas, Jeetayu; Senecal, Adrien; Singer, Robert H; Park, Hye Yoon
Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells. Definition of heterogeneity has revealed stochastic processes, determined characteristics of under-represented cell types or transitional states, and integrated cellular behaviors in the context of multicellular organisms. In this review, we discuss novel aspects of gene expression of eukaryotic cells and multicellular organisms revealed by the latest advances in single-cell and single-molecule imaging technology.
KAWASAKI, KATSUSHIGE; KAWASAKI, MAIKO; WATANABE, MOMOKO; IDRUS, ERIK; NAGAI, TAKAHIRO; OOMMEN, SHELLY; MAEDA, TAKEYASU; HAGIWARA, NOBUKO; QUE, JIANWEN; SHARPE, PAUL T.; OHAZAMA, ATSUSHI
Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development. PMID:26864488
Kawasaki, Katsushige; Kawasaki, Maiko; Watanabe, Momoko; Idrus, Erik; Nagai, Takahiro; Oommen, Shelly; Maeda, Takeyasu; Hagiwara, Nobuko; Que, Jianwen; Sharpe, Paul T; Ohazama, Atsushi
Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development.
Aldea, M; Garrido, T; Tormo, A
Regulation of gene expression in prokaryotic cells usually takes place at the level of transcription initiation. Different forms of RNA polymerase recognizing specific promoters are engaged in the control of many prokaryotic regulons. This also seems to be the case for some Escherichia coli genes that are induced at low growth rates and by nutrient starvation. Their gene products are synthesized at levels inversely proportional to growth rate, and this mode of regulation has been termed gearbox gene expression. This kind of growth-rate modulation is exerted by specific transcriptional initiation signals, the gearbox promoters, and some of them depend on a putative new σ factor (RpoS). Gearbox promoters drive expression of morphogenetic and cell division genes at constant levels per cell and cycle to meet the demands of cell division and septum formation. A mechanism is proposed that could sense the growth rate of the cell to alter gene expression by the action of specific σ factors.
Gagoski, Dejan; Mureev, Sergey; Giles, Nichole; Johnston, Wayne; Dahmer-Heath, Mareike; Škalamera, Dubravka; Gonda, Thomas J; Alexandrov, Kirill
Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximize throughput and minimize the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1 ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in Leishmania tarentolae and Escherichia coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era. Copyright © 2014 Elsevier B.V. All rights reserved.
Vonk Freek J
Full Text Available Abstract Background Actin is a cytoskeletal protein which exerts a broad range of functions in almost all eukaryotic cells. In higher vertebrates, six primary actin isoforms can be distinguished: alpha-skeletal, alpha-cardiac, alpha-smooth muscle, gamma-smooth muscle, beta-cytoplasmic and gamma-cytoplasmic isoactin. Expression of these actin isoforms during vertebrate development is highly regulated in a temporal and tissue-specific manner, but the mechanisms and the specific differences are currently not well understood. All members of the actin multigene family are highly conserved, suggesting that there is a high selective pressure on these proteins. Results We present here a model for the evolution of the genomic organization of alpha-skeletal actin and by molecular modeling, illustrate the structural differences of actin proteins of different phyla. We further describe and compare alpha-skeletal actin expression in two developmental stages of five vertebrate species (mouse, chicken, snake, salamander and fish. Our findings confirm that alpha-skeletal actin is expressed in skeletal muscle and in the heart of all five species. In addition, we identify many novel non-muscular expression domains including several in the central nervous system. Conclusion Our results show that the high sequence homology of alpha-skeletal actins is reflected by similarities of their 3 dimensional protein structures, as well as by conserved gene expression patterns during vertebrate development. Nonetheless, we find here important differences in 3D structures, in gene architectures and identify novel expression domains for this structural and functional important gene.
Full Text Available Abstract Background Gene set analysis (GSA has become a successful tool to interpret gene expression profiles in terms of biological functions, molecular pathways, or genomic locations. GSA performs statistical tests for independent microarray samples at the level of gene sets rather than individual genes. Nowadays, an increasing number of microarray studies are conducted to explore the dynamic changes of gene expression in a variety of species and biological scenarios. In these longitudinal studies, gene expression is repeatedly measured over time such that a GSA needs to take into account the within-gene correlations in addition to possible between-gene correlations. Results We provide a robust nonparametric approach to compare the expressions of longitudinally measured sets of genes under multiple treatments or experimental conditions. The limiting distributions of our statistics are derived when the number of genes goes to infinity while the number of replications can be small. When the number of genes in a gene set is small, we recommend permutation tests based on our nonparametric test statistics to achieve reliable type I error and better power while incorporating unknown correlations between and within-genes. Simulation results demonstrate that the proposed method has a greater power than other methods for various data distributions and heteroscedastic correlation structures. This method was used for an IL-2 stimulation study and significantly altered gene sets were identified. Conclusions The simulation study and the real data application showed that the proposed gene set analysis provides a promising tool for longitudinal microarray analysis. R scripts for simulating longitudinal data and calculating the nonparametric statistics are posted on the North Dakota INBRE website http://ndinbre.org/programs/bioinformatics.php. Raw microarray data is available in Gene Expression Omnibus (National Center for Biotechnology Information with
Ramirez-Prado, Juan Sebastian; Rodriguez-Granados, Natalia Yaneth; Ariel, Federico; Raynaud, Cécile; Benhamed, Moussa
As the most recent evidence of eukaryotic cell complexity, genome architecture has astounded the scientific community and prompted a variety of technical and cognitive challenges. Several technologies have emerged and evidenced the integration of chromatin packaging and topology, epigenetic processes, and transcription for the pertinent regulation of gene expression. In the present addendum we present and discuss some of our recent research, directed toward the holistic comprehension of the p...
Full Text Available Abstract Background The regulatory processes that govern cell proliferation and differentiation are central to developmental biology. Particularly well studied in this respect is the lymphoid system due to its importance for basic biology and for clinical applications. Gene expression measured in lymphoid cells in several distinguishable developmental stages helps in the elucidation of underlying molecular processes, which change gradually over time and lock cells in either the B cell, T cell or Natural Killer cell lineages. Large-scale analysis of these gene expression trees requires computational support for tasks ranging from visualization, querying, and finding clusters of similar genes, to answering detailed questions about the functional roles of individual genes. Results We present the first statistical framework designed to analyze gene expression data as it is collected in the course of lymphoid development through clusters of co-expressed genes and additional heterogeneous data. We introduce dependence trees for continuous variates, which model the inherent dependencies during the differentiation process naturally as gene expression trees. Several trees are combined in a mixture model to allow inference of potentially overlapping clusters of co-expressed genes. Additionally, we predict microRNA targets. Conclusion Computational results for several data sets from the lymphoid system demonstrate the relevance of our framework. We recover well-known biological facts and identify promising novel regulatory elements of genes and their functional assignments. The implementation of our method (licensed under the GPL is available at http://algorithmics.molgen.mpg.de/Supplements/ExpLym/.
Peng, Liang; Guo, Chuang; Wang, Tao; Li, Baoman; Gu, Li; Wang, Zhanyou
Traditionally, astrocytic mRNA and protein expression are studied by in situ hybridization (ISH) and immunohistochemically. This led to the concept that astrocytes lack aralar, a component of the malate-aspartate-shuttle. At least similar aralar mRNA and protein expression in astrocytes and neurons isolated by fluorescence-assisted cell sorting (FACS) reversed this opinion. Demonstration of expression of other astrocytic genes may also be erroneous. Literature data based on morphological methods were therefore compared with mRNA expression in cells obtained by recently developed methods for determination of cell-specific gene expression. All Na,K-ATPase-α subunits were demonstrated by immunohistochemistry (IHC), but there are problems with the cotransporter NKCC1. Glutamate and GABA transporter gene expression was well determined immunohistochemically. The same applies to expression of many genes of glucose metabolism, whereas a single study based on findings in bacterial artificial chromosome (BAC) transgenic animals showed very low astrocytic expression of hexokinase. Gene expression of the equilibrative nucleoside transporters ENT1 and ENT2 was recognized by ISH, but ENT3 was not. The same applies to the concentrative transporters CNT2 and CNT3. All were clearly expressed in FACS-isolated cells, followed by biochemical analysis. ENT3 was enriched in astrocytes. Expression of many nucleoside transporter genes were shown by microarray analysis, whereas other important genes were not. Results in cultured astrocytes resembled those obtained by FACS. These findings call for reappraisal of cellular nucleoside transporter expression. FACS cell yield is small. Further development of cell separation methods to render methods more easily available and less animal and cost consuming and parallel studies of astrocytic mRNA and protein expression by ISH/IHC and other methods are necessary, but new methods also need to be thoroughly checked.
Zusman, Tal; Feldman, Michal; Halperin, Einat; Segal, Gil
Legionella pneumophila, the causative agent of Legionnaires' disease, replicates intracellularly within a specialized phagosome of mammalian and protozoan host cells, and the Icm/Dot type IV secretion system has been shown to be essential for this process. Unlike all the other known Icm/Dot proteins, the IcmF protein, which was described before, and the IcmH protein, which is characterized here, have homologous proteins in many bacteria (such as Yersinia pestis, Salmonella enterica, Rhizobium leguminosarum, and Vibrio cholerae), all of which associate with eukaryotic cells. Here, we have characterized the L. pneumophila icmH and icmF genes and found that both genes are present in 16 different Legionella species examined. The icmH and icmF genes were found to be absolutely required for intracellular multiplication in Acanthamoeba castellanii and partially required for intracellular growth in HL-60-derived human macrophages, for immediate cytotoxicity, and for salt sensitivity. Mutagenesis of the predicted ATP/GTP binding site of IcmF revealed that the site is partially required for intracellular growth in A. castellanii. Analysis of the regulatory region of the icmH and icmF genes, which were found to be cotranscribed, revealed that it contains at least two regulatory elements. In addition, an icmH::lacZ fusion was shown to be activated during stationary phase in a LetA- and RelA-dependent manner. Our results indicate that although the icmH and icmF genes probably have a different evolutionary origin than the rest of the icm/dot genes, they are part of the icm/dot system and are required for L. pneumophila pathogenesis.
Pablo S Gutierrez
Full Text Available In the present work we extend and analyze the scope of our recently proposed stochastic model for transcriptional regulation, which considers an arbitrarily complex cis-regulatory system using only elementary reactions. Previously, we determined the role of cooperativity on the intrinsic fluctuations of gene expression for activating transcriptional switches, by means of master equation formalism and computer simulation. This model allowed us to distinguish between two cooperative binding mechanisms and, even though the mean expression levels were not affected differently by the acting mechanism, we showed that the associated fluctuations were different. In the present generalized model we include other regulatory functions in addition to those associated to an activator switch. Namely, we introduce repressive regulatory functions and two theoretical mechanisms that account for the biphasic response that some cis-regulatory systems show to the transcription factor concentration. We have also extended our previous master equation formalism in order to include protein production by stochastic translation of mRNA. Furthermore, we examine the graded/binary scenarios in the context of the interaction energy between transcription factors. In this sense, this is the first report to show that the cooperative binding of transcription factors to DNA promotes the "all-or-none" phenomenon observed in eukaryotic systems. In addition, we confirm that gene expression fluctuation levels associated with one of two cooperative binding mechanism never exceed the fluctuation levels of the other.
Zhou, Yuan; Jia, Run-Qing; Teng, Zhi-Ping; Zhang, Xiao-Mei; Zeng, Yi
To Construction of P and NP genes eukaryotic expression vectors of Newcastle Disease Virus LaSota strain,study its reverse genetics and functional genome of NDV. P, NP genes were amplified and cloned into pGEM-T easy vector and then subcloned into pcDNA3.1 (+) expression vector respectively, the recombinant plasmids were named pcDNA3.1 (+)-P and pcDNA3.1 (+)-NP, Recombinant plasmids were transfected into 293 and BHK-21 cells respectively and were detected using IE and Western blot analysis. Expression of P, NP genes were detected and confirmed by the IE and WB analysis. The recombinant eukaryotic plasmids pcDNA3. 1(+)-P, pcDNA3.1 (+)-NP were expressed in 293 and BHK-21 cells successfully. This research may be helpful for further study of reverse genetics and functional genome of NDV.
Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.
Full Text Available To understand fully cell behaviour, biologists are making progress towards cataloguing the functional elements in the human genome and characterising their roles across a variety of tissues and conditions. Yet, functional information - either experimentally validated or computationally inferred by similarity - remains completely missing for approximately 30% of human proteins. FFPred was initially developed to bridge this gap by targeting sequences with distant or no homologues of known function and by exploiting clear patterns of intrinsic disorder associated with particular molecular activities and biological processes. Here, we present an updated and improved version, which builds on larger datasets of protein sequences and annotations, and uses updated component feature predictors as well as revised training procedures. FFPred 2.0 includes support vector regression models for the prediction of 442 Gene Ontology (GO terms, which largely expand the coverage of the ontology and of the biological process category in particular. The GO term list mainly revolves around macromolecular interactions and their role in regulatory, signalling, developmental and metabolic processes. Benchmarking experiments on newly annotated proteins show that FFPred 2.0 provides more accurate functional assignments than its predecessor and the ProtFun server do; also, its assignments can complement information obtained using BLAST-based transfer of annotations, improving especially prediction in the biological process category. Furthermore, FFPred 2.0 can be used to annotate proteins belonging to several eukaryotic organisms with a limited decrease in prediction quality. We illustrate all these points through the use of both precision-recall plots and of the COGIC scores, which we recently proposed as an alternative numerical evaluation measure of function prediction accuracy.
Full Text Available Abstract Background As whole genome and transcriptome sequencing gets cheaper and faster, a great number of 'exotic' animal models are emerging, rapidly adding valuable data to the ever-expanding Evo-Devo field. All these new organisms serve as a fantastic resource for the research community, but the sheer amount of data, some published, some not, makes detailed comparison of gene expression patterns very difficult to summarize - a problem sometimes even noticeable within a single lab. The need to merge existing data with new information in an organized manner that is publicly available to the research community is now more necessary than ever. Description In order to offer a homogenous way of storing and handling gene expression patterns from a variety of organisms, we have developed the first web-based comparative gene expression database for invertebrates that allows species-specific as well as cross-species gene expression comparisons. The database can be queried by gene name, developmental stage and/or expression domains. Conclusions This database provides a unique tool for the Evo-Devo research community that allows the retrieval, analysis and comparison of gene expression patterns within or among species. In addition, this database enables a quick identification of putative syn-expression groups that can be used to initiate, among other things, gene regulatory network (GRN projects.
Marco Aurelio Krieger
Full Text Available The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE. The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells, while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.
Full Text Available Strains of the Pseudovibrio genus have been detected worldwide, mainly as part of bacterial communities associated with marine invertebrates, particularly sponges. This recurrent association has been considered as an indication of a symbiotic relationship between these microbes and their host. Until recently, the availability of only two genomes, belonging to closely related strains, has limited the knowledge on the genomic and physiological features of the genus to a single phylogenetic lineage.Here we present 10 newly sequenced genomes of Pseudovibrio strains isolated from marine sponges from the west coast of Ireland, and including the other two publicly available genomes we performed an extensive comparative genomic analysis. Homogeneity was apparent in terms of both the orthologous genes and the metabolic features shared amongst the 12 strains. At the genomic level, a key physiological difference observed amongst the isolates was the presence only in strain P. axinellae AD2 of genes encoding proteins involved in assimilatory nitrate reduction, which was then proved experimentally. We then focused on studying those systems known to be involved in the interactions with eukaryotic and prokaryotic cells. This analysis revealed that the genus harbors a large diversity of toxin-like proteins, secretion systems and their potential effectors. Their distribution in the genus was not always consistent with the phylogenetic relationship of the strains. Finally, our analyses identified new genomic islands encoding potential toxin-immunity systems, previously unknown in the genus.Our analyses shed new light on the Pseudovibrio genus, indicating a large diversity of both metabolic features and systems for interacting with the host. The diversity in both distribution and abundance of these systems amongst the strains underlines how metabolically and phylogenetically similar bacteria may use different strategies to interact with the host and find a niche
Eveland, Andrea L; Satoh-Nagasawa, Namiko; Goldshmidt, Alexander; Meyer, Sandra; Beatty, Mary; Sakai, Hajime; Ware, Doreen; Jackson, David
Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks. In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect the determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait. In this work, we developed and tested a framework for analysis of tag-based, digital gene expression profiles using Illumina's high-throughput sequencing technology and the newly assembled B73 maize reference genome. We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in axillary meristem determinacy. The RA3 gene encodes a trehalose-6-phosphate phosphatase and may act at the interface between developmental and metabolic processes. Deep sequencing of digital gene expression libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20- to 21-nucleotide reads with frequencies spanning 4 orders of magnitude. Unique sequence tags were anchored to 3'-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing. We mapped 86% of nonredundant signature tags to the maize genome, which associated with 37,117 gene models and unannotated regions of expression. In total, 66% of genes were detected by at least nine reads in immature maize ears. We used comparative genomics to leverage existing information from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) in functional analyses of differentially expressed maize genes. Results from this study provide a basis for the analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene.
Stranger, Barbara E.; Nica, Alexandra C.; Forrest, Matthew S.; Dimas, Antigone; Bird, Christine P.; Beazley, Claude; Ingle, Catherine E.; Dunning, Mark; Flicek, Paul; Koller, Daphne; Montgomery, Stephen; Tavaré, Simon; Deloukas, Panagiotis; Dermitzakis, Emmanouil T.
Genetic variation influences gene expression, and this can be efficiently mapped to specific genomic regions and variants. We used gene expression profiling of EBV-transformed lymphoblastoid cell lines of all 270 individuals of the HapMap consortium to elucidate the detailed features of genetic variation underlying gene expression variation. We find gene expression levels to be heritable and differentiation between populations in agreement with earlier small-scale studies. A detailed association analysis of over 2.2 million common SNPs per population (5% frequency HapMap) with gene expression identified at least 1348 genes with association signals in cis and at least 180 in trans. Replication in at least one independent population was achieved for 37% of cis- signals and 15% of trans- signals, respectively. Our results strongly support an abundance of cis- regulatory variation in the human genome. Detection of trans- effects is limited but suggests that regulatory variation may be the key primary effect contributing to phenotypic variation in humans. Finally, we explore a variety of methodologies that improve the current state of analysis of gene expression variation. PMID:17873874
Full Text Available Abstract Background In periodontitis, treatment aimed at controlling the periodontal biofilm infection results in a resolution of the clinical and histological signs of inflammation. Although the cell types found in periodontal tissues following treatment have been well described, information on gene expression is limited to few candidate genes. Therefore, the aim of the study was to determine the expression profiles of immune and inflammatory genes in periodontal tissues from sites with severe chronic periodontitis following periodontal therapy in order to identify genes involved in tissue homeostasis. Gingival biopsies from 12 patients with severe chronic periodontitis were taken six to eight weeks following non-surgical periodontal therapy, and from 11 healthy controls. As internal standard, RNA of an immortalized human keratinocyte line (HaCaT was used. Total RNA was subjected to gene expression profiling using a commercially available microarray system focusing on inflammation-related genes. Post-hoc confirmation of selected genes was done by Realtime-PCR. Results Out of the 136 genes analyzed, the 5% most strongly expressed genes compared to healthy controls were Interleukin-12A (IL-12A, Versican (CSPG-2, Matrixmetalloproteinase-1 (MMP-1, Down syndrome critical region protein-1 (DSCR-1, Macrophage inflammatory protein-2β (Cxcl-3, Inhibitor of apoptosis protein-1 (BIRC-1, Cluster of differentiation antigen 38 (CD38, Regulator of G-protein signalling-1 (RGS-1, and Finkel-Biskis-Jinkins murine osteosarcoma virus oncogene (C-FOS; the 5% least strongly expressed genes were Receptor-interacting Serine/Threonine Kinase-2 (RIP-2, Complement component 3 (C3, Prostaglandin-endoperoxide synthase-2 (COX-2, Interleukin-8 (IL-8, Endothelin-1 (EDN-1, Plasminogen activator inhibitor type-2 (PAI-2, Matrix-metalloproteinase-14 (MMP-14, and Interferon regulating factor-7 (IRF-7. Conclusion Gene expression profiles found in periodontal tissues following
Holter, N. S.; Maritan, A.; Cieplak, M.; Fedoroff, N. V.; Banavar, J. R.
We describe the time evolution of gene expression levels by using a time translational matrix to predict future expression levels of genes based on their expression levels at some initial time. We deduce the time translational matrix for previously published DNA microarray gene expression data sets by modeling them within a linear framework by using the characteristic modes obtained by singular value decomposition. The resulting time translation matrix provides a measure of the relationships among the modes and governs their time evolution. We show that a truncated matrix linking just a few modes is a good approximation of the full time translation matrix. This finding suggests that the number of essential connections among the genes is small.
Brown, Christopher R; Boeger, Hinrich
Gene product molecule numbers fluctuate over time and between cells, confounding deterministic expectations. The molecular origins of this noise of gene expression remain unknown. Recent EM analysis of single PHO5 gene molecules of yeast indicated that promoter molecules stochastically assume alternative nucleosome configurations at steady state, including the fully nucleosomal and nucleosome-free configuration. Given that distinct configurations are unequally conducive to transcription, the nucleosomal variation of promoter molecules may constitute a source of gene expression noise. This notion, however, implies an untested conjecture, namely that the nucleosomal variation arises de novo or intrinsically (i.e., that it cannot be explained as the result of the promoter's deterministic response to variation in its molecular surroundings). Here, we show--by microscopically analyzing the nucleosome configurations of two juxtaposed physically linked PHO5 promoter copies--that the configurational variation, indeed, is intrinsically stochastic and thus, a cause of gene expression noise rather than its effect.
Bovin, Lone Frier; Brynskov, Jørn; Hegedüs, Laszlo
A central issue in autoimmune disease is whether the underlying inflammation is a repeated stereotypical process or whether disease specific gene expression is involved. To shed light on this, we analysed whether genes previously found to be differentially regulated in rheumatoid arthritis (RA......) patients and healthy individuals were specific for the arthritic process or likewise altered in other chronic inflammatory diseases such as chronic autoimmune thyroiditis (Hashimoto's thyroiditis, HT) and inflammatory bowel disease (IBD). Using qPCR for 18 RA-discriminative genes, there were no significant...... differences in peripheral blood mononuclear cell (MNC) gene expression patterns between 15 newly diagnosed HT patients and 15 matched healthy controls. However, the MNC expression levels of five genes were significantly upregulated in 25 IBD patients, compared to 18 matched healthy controls (CD14, FACL2, FCN1...
Needham, Chris J; Manfield, Iain W; Bulpitt, Andrew J; Gilmartin, Philip M; Westhead, David R
The elucidation of networks from a compendium of gene expression data is one of the goals of systems biology and can be a valuable source of new hypotheses for experimental researchers. For Arabidopsis, there exist several thousand microarrays which form a valuable resource from which to learn. A novel Bayesian network-based algorithm to infer gene regulatory networks from gene expression data is introduced and applied to learn parts of the transcriptomic network in Arabidopsis thaliana from a large number (thousands) of separate microarray experiments. Starting from an initial set of genes of interest, a network is grown by iterative addition to the model of the gene, from another defined set of genes, which gives the 'best' learned network structure. The gene set for iterative growth can be as large as the entire genome. A number of networks are inferred and analysed; these show (i) an agreement with the current literature on the circadian clock network, (ii) the ability to model other networks, and (iii) that the learned network hypotheses can suggest new roles for poorly characterized genes, through addition of relevant genes from an unconstrained list of over 15,000 possible genes. To demonstrate the latter point, the method is used to suggest that particular GATA transcription factors are regulators of photosynthetic genes. Additionally, the performance in recovering a known network from different amounts of synthetically generated data is evaluated. Our results show that plausible regulatory networks can be learned from such gene expression data alone. This work demonstrates that network hypotheses can be generated from existing gene expression data for use by experimental biologists.
Full Text Available Microarray produces a large amount of gene expression data, containing various biological implications. The challenge is to detect a panel of discriminative genes associated with disease. This study proposed a robust classification model for gene selection using gene expression data, and performed an analysis to identify disease-related genes using multiple sclerosis as an example.Gene expression profiles based on the transcriptome of peripheral blood mononuclear cells from a total of 44 samples from 26 multiple sclerosis patients and 18 individuals with other neurological diseases (control were analyzed. Feature selection algorithms including Support Vector Machine based on Recursive Feature Elimination, Receiver Operating Characteristic Curve, and Boruta algorithms were jointly performed to select candidate genes associating with multiple sclerosis. Multiple classification models categorized samples into two different groups based on the identified genes. Models' performance was evaluated using cross-validation methods, and an optimal classifier for gene selection was determined.An overlapping feature set was identified consisting of 8 genes that were differentially expressed between the two phenotype groups. The genes were significantly associated with the pathways of apoptosis and cytokine-cytokine receptor interaction. TNFSF10 was significantly associated with multiple sclerosis. A Support Vector Machine model was established based on the featured genes and gave a practical accuracy of ∼86%. This binary classification model also outperformed the other models in terms of Sensitivity, Specificity and F1 score.The combined analytical framework integrating feature ranking algorithms and Support Vector Machine model could be used for selecting genes for other diseases.
Cicek, Mustafa; Mutlu, Ozal; Erdemir, Aysegul; Ozkan, Ebru; Saricay, Yunus; Turgut-Balik, Dilek
One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5'-GGAGGC-3' sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5'-GGAGGA-3', by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.
Full Text Available Abstract Background Comparative genomics brings insight into sequence evolution, but even more may be learned by coupling sequence analyses with experimental tests of gene function and regulation. However, the reliability of such comparisons is often limited by biased sampling of expression conditions and incomplete knowledge of gene functions across species. To address these challenges, we previously systematically generated expression profiles in Saccharomyces bayanus to maximize functional coverage as compared to an existing Saccharomyces cerevisiae data repository. Results In this paper, we take advantage of these two data repositories to compare patterns of ortholog expression in a wide variety of conditions. First, we developed a scalable metric for expression divergence that enabled us to detect a significant correlation between sequence and expression conservation on the global level, which previous smaller-scale expression studies failed to detect. Despite this global conservation trend, between-species gene expression neighborhoods were less well-conserved than within-species comparisons across different environmental perturbations, and approximately 4% of orthologs exhibited a significant change in co-expression partners. Furthermore, our analysis of matched perturbations collected in both species (such as diauxic shift and cell cycle synchrony demonstrated that approximately a quarter of orthologs exhibit condition-specific expression pattern differences. Conclusions Taken together, these analyses provide a global view of gene expression patterns between two species, both in terms of the conditions and timing of a gene's expression as well as co-expression partners. Our results provide testable hypotheses that will direct future experiments to determine how these changes may be specified in the genome.
Tânia Maria Vulcani-Freitas
Full Text Available Medulloblastoma is the most common malignant tumors of central nervous system in the childhood. The treatment is severe, harmful and, thus, has a dismal prognosis. As PRAME is present in various cancers, including meduloblastoma, and has limited expression in normal tissues, this antigen can be an ideal vaccine target for tumor immunotherapy. In order to find a potential molecular target, we investigated PRAME expression in medulloblastoma fragments and we compare the results with the clinical features of each patient. Analysis of gene expression was performed by real-time quantitative PCR from 37 tumor samples. The Mann-Whitney test was used to analysis the relationship between gene expression and clinical characteristics. Kaplan-Meier curves were used to evaluate survival. PRAME was overexpressed in 84% samples. But no statistical association was found between clinical features and PRAME overexpression. Despite that PRAME gene could be a strong candidate for immunotherapy since it is highly expressed in medulloblastomas.
The nuclear envelope (NE) separates the key mechanisms of transcription and translation, and as such is a critical control point in all eukaryotic cells. In plants, the proteins of the NE influence a number of processes including the control of nucleo-cytoplasmic transport of RNA and protein, chromatin localization to the nuclear periphery, and direct chromatin binding by members of the nuclear pore complex (NPC). In this review I attempt to bring these roles under the umbrella of their effect on gene expression, even though the complex nature of this cellular environment means there is considerable overlap of effects. Although the volume of research in plant cells has greatly improved over recent years, it is clear that our understanding of how the components of the NE either directly or indirectly influence gene expression is still in its infancy. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: email@example.com.
Szweykowska-Kulińska, Zofia; Jarmołowski, Artur; Figlerowicz, Marek
Several years ago it was discovered that plant transformation with a transcribed sense transgene could shut down the expression of a homologous endogenous gene. Moreover, it was shown that the introduction into the cell of dsRNA (double-stranded RNA) containing nucleotide sequence complementary to an mRNA sequence causes selective degradation of the latter and thus silencing of a specific gene. This phenomenon, called RNA interference (RNAi) was demonstrated to be present in almost all eukaryotic organisms. RNAi is also capable of silencing transposons in germ line cells and fighting RNA virus infection. Enzymes involved in this process exhibit high homology across species. Some of these enzymes are involved in other cellular processes, for instance developmental timing, suggesting strong interconnections between RNAi and other metabolic pathways. RNAi is probably an ancient mechanism that evolved to protect eukaryotic cells against invasive forms of nucleic acids.
The modeling of gene networks from transcriptional expression data is an important tool in biomedical research to reveal signaling pathways and to identify treatment targets. Current gene network modeling is primarily based on the use of Gaussian graphical models applied to continuous data, which give a closedformmarginal likelihood. In this paper,we extend network modeling to discrete data, specifically data from serial analysis of gene expression, and RNA-sequencing experiments, both of which generate counts of mRNAtranscripts in cell samples.We propose a generalized linear model to fit the discrete gene expression data and assume that the log ratios of the mean expression levels follow a Gaussian distribution.We restrict the gene network structures to decomposable graphs and derive the graphs by selecting the covariance matrix of the Gaussian distribution with the hyper-inverse Wishart priors. Furthermore, we incorporate prior network models based on gene ontology information, which avails existing biological information on the genes of interest. We conduct simulation studies to examine the performance of our discrete graphical model and apply the method to two real datasets for gene network inference. © The Author 2013. Published by Oxford University Press. All rights reserved.
Nielsen, Jennifer L.; Pavey, Scott A.
Functional genes and gene expression have been connected to physiological traits linked to effective production and broodstock selection in aquaculture, selective implications of commercial fish harvest, and adaptive changes reflected in non-commercial fish populations subject to human disturbance and climate change. Gene mapping using single nucleotide polymorphisms (SNPs) to identify functional genes, gene expression (analogue microarrays and real-time PCR), and digital sequencing technologies looking at RNA transcripts present new concepts and opportunities in support of effective and sustainable fisheries. Genomic tools have been rapidly growing in aquaculture research addressing aspects of fish health, toxicology, and early development. Genomic technologies linking effects in functional genes involved in growth, maturation and life history development have been tied to selection resulting from harvest practices. Incorporating new and ever-increasing knowledge of fish genomes is opening a different perspective on local adaptation that will prove invaluable in wild fish conservation and management. Conservation of fish stocks is rapidly incorporating research on critical adaptive responses directed at the effects of human disturbance and climate change through gene expression studies. Genomic studies of fish populations can be generally grouped into three broad categories: 1) evolutionary genomics and biodiversity; 2) adaptive physiological responses to a changing environment; and 3) adaptive behavioral genomics and life history diversity. We review current genomic research in fisheries focusing on those that use microarrays to explore differences in gene expression among phenotypes and within or across populations, information that is critically important to the conservation of fish and their relationship to humans.
A novel trehalose synthase gene from Deinococcus geothermalis (DSMZ 11300) containing 1692 bp reading-frame encoding 564 amino acids was amplified using polymerase chain reaction (PCR). The gene was ligated into pET30Ek/LIC vector and expressed after isopropyl β-D-thiogalactopyranoside induction in ...
firmed difference in expression profiles of the identified genes in musculus longissimus muscle tissues between the two Lan- ..... Discussion. The EEF1A2, TSG101 and TTN identified as upregulated genes in high-growth group have been reported to be involved in myotube survival and .... cDNA probes and libraries. Proc.
dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana
Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis. PMID:26393928
Wang, Xuefel (University of New Mexico, Albuquerque, NM); Kang, Huining (University of New Mexico, Albuquerque, NM); Fields, Chris (New Mexico State University, Las Cruces, NM); Cowie, Jim R. (New Mexico State University, Las Cruces, NM); Davidson, George S.; Haaland, David Michael; Sibirtsev, Valeriy (New Mexico State University, Las Cruces, NM); Mosquera-Caro, Monica P. (University of New Mexico, Albuquerque, NM); Xu, Yuexian (University of New Mexico, Albuquerque, NM); Martin, Shawn Bryan; Helman, Paul (University of New Mexico, Albuquerque, NM); Andries, Erik (University of New Mexico, Albuquerque, NM); Ar, Kerem (University of New Mexico, Albuquerque, NM); Potter, Jeffrey (University of New Mexico, Albuquerque, NM); Willman, Cheryl L. (University of New Mexico, Albuquerque, NM); Murphy, Maurice H. (University of New Mexico, Albuquerque, NM)
Molecular analysis of cancer, at the genomic level, could lead to individualized patient diagnostics and treatments. The developments to follow will signal a significant paradigm shift in the clinical management of human cancer. Despite our initial hopes, however, it seems that simple analysis of microarray data cannot elucidate clinically significant gene functions and mechanisms. Extracting biological information from microarray data requires a complicated path involving multidisciplinary teams of biomedical researchers, computer scientists, mathematicians, statisticians, and computational linguists. The integration of the diverse outputs of each team is the limiting factor in the progress to discover candidate genes and pathways associated with the molecular biology of cancer. Specifically, one must deal with sets of significant genes identified by each method and extract whatever useful information may be found by comparing these different gene lists. Here we present our experience with such comparisons, and share methods developed in the analysis of an infant leukemia cohort studied on Affymetrix HG-U95A arrays. In particular, spatial gene clustering, hyper-dimensional projections, and computational linguistics were used to compare different gene lists. In spatial gene clustering, different gene lists are grouped together and visualized on a three-dimensional expression map, where genes with similar expressions are co-located. In another approach, projections from gene expression space onto a sphere clarify how groups of genes can jointly have more predictive power than groups of individually selected genes. Finally, online literature is automatically rearranged to present information about genes common to multiple groups, or to contrast the differences between the lists. The combination of these methods has improved our understanding of infant leukemia. While the complicated reality of the biology dashed our initial, optimistic hopes for simple answers from
Bagger, Frederik Otzen
Acute Myeloid Leukaemia (AML) is an aggressive cancer of the bone marrow, affecting formation of blood cells during haematopoiesis. This thesis presents investigation of AML using mRNA gene expression profiles (GEP) of samples extracted from the bone marrow of healthy and diseased subjects. Here...... signatures and for reducing dimensionally of gene expression data. Next, we have used machine-learning methods to predict survival and to assess important predictors based on these results. General application of a number of these methods has been implemented into two public query-based gene...
Bagger, Frederik Otzen
Summary Acute Myeloid Leukaemia (AML) is an aggressive cancer of the bone marrow, affecting formation of blood cells during haematopoiesis. This thesis presents investigation of AML using mRNA gene expression profiles (GEP) of samples extracted from the bone marrow of healthy and diseased subjects...... genes and genetic signatures and for reducing dimensionally of gene expression data. Next, we have used machine-learning methods to predict survival and to assess important predictors based on these results. General application of a number of these methods has been implemented into two public query...
Shimizu Kentaro; Nakai Yuji; Kadota Koji
Abstract Background To identify differentially expressed genes (DEGs) from microarray data, users of the Affymetrix GeneChip system need to select both a preprocessing algorithm to obtain expression-level measurements and a way of ranking genes to obtain the most plausible candidates. We recently recommended suitable combinations of a preprocessing algorithm and gene ranking method that can be used to identify DEGs with a higher level of sensitivity and specificity. However, in addition to th...
Hojman, Pernille; Zibert, John R; Gissel, Hanne
BACKGROUND: Gene transfer by electroporation (DNA electrotransfer) to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of DNA electrotransfer on muscle fibres. We have...... therefore investigated transcriptional changes through gene expression profile analyses, morphological changes by histological analysis, and physiological changes by force generation measurements. DNA electrotransfer was obtained using a combination of a short high voltage pulse (HV, 1000 V/cm, 100 mus......) followed by a long low voltage pulse (LV, 100 V/cm, 400 ms); a pulse combination optimised for efficient and safe gene transfer. Muscles were transfected with green fluorescent protein (GFP) and excised at 4 hours, 48 hours or 3 weeks after treatment. RESULTS: Differentially expressed genes were...
Zhang, Yu; Zhang, Xiao-Dong; Liu, Xing; Li, Yun-Sheng; Ding, Jian-Ping; Zhang, Xiao-Rong; Zhang, Yun-Hai
Real-time quantitative PCR (qRT-PCR) is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2) in ten tissues types sourced from Boer goats. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in genes expression stability. When all tissues were considered, 18S, TBP and HMBS is the optimal reference combination for calibrating quantitative PCR analysis of gene expression from goat tissues. Dividing data set by tissues, ACTB was the most stable in stomach, small intestine and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney and GAPDH in muscle. Overall, this study provided valuable information about the goat reference genes that can be used in order to perform a proper normalisation when relative quantification by qRT-PCR studies is undertaken.
Full Text Available Real-time quantitative PCR (qRT-PCR is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2 in ten tissues types sourced from Boer goats. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in genes expression stability. When all tissues were considered, 18S, TBP and HMBS is the optimal reference combination for calibrating quantitative PCR analysis of gene expression from goat tissues. Dividing data set by tissues, ACTB was the most stable in stomach, small intestine and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney and GAPDH in muscle. Overall, this study provided valuable information about the goat reference genes that can be used in order to perform a proper normalisation when relative quantification by qRT-PCR studies is undertaken.
Bagger, Frederik Otzen
Summary Acute Myeloid Leukaemia (AML) is an aggressive cancer of the bone marrow, affecting formation of blood cells during haematopoiesis. This thesis presents investigation of AML using mRNA gene expression profiles (GEP) of samples extracted from the bone marrow of healthy and diseased subjects...... model to investigate the role of telomerase in AML, we were able to translate the observed effect into human AML patients and identify specific genes involved, which also predict survival patterns in AML patients. During these studies we have applied methods for investigating differentially expressed...... genes and genetic signatures and for reducing dimensionally of gene expression data. Next, we have used machine-learning methods to predict survival and to assess important predictors based on these results. General application of a number of these methods has been implemented into two public query...
Zhang, Chengwei; Guo, Linlin; Wang, Xiulan; Zhang, Hui; Shi, Haiyan; Xu, Wenliang; Li, Xuebao
Actin depolymerizing factor (ADF), highly conserved in all eukaryotic cells, is a low molecular mass of actin-binding protein, which plays a key role in modulating the polymerizing and depolymerizing of the actin filaments. Four cDNAs (designated GhADF2, GhADF3, GhADF4, and GhADF5, respectively) encoding ADF proteins were isolated from cotton (Gossypium hirsutum) fiber cDNA library. GhADF2 cDNA is 705 bp in length and deduces a protein with 139 amino acids. GhADF3 cDNA is 819 bp in length and encodes a protein of 139 amino acids. GhADF4 cDNA is 804 bp in length and deduces a protein with 143 amino acids. GhADF5 cDNA is 644 bp in length and encodes a protein of 141 amino acids. The molecular evolutionary relationship of these genes was analyzed by means of bioinformatics. GhADF2 is closely related to GhADF3 (99% identity) and PetADF2 (89% identity). GhADF4 is closely related to AtADF6 (78% identity), and GhADF5 is closely related to AtADF5 (83% identity). These results demonstrated that the plant ADF genes are highly conserved in structure. RT-PCR analysis showed that GhADF2 is predominantly expressed in fiber, whereas, GhADF5 is mainly expressed in cotyledons. On the other hand, it seems that GhADF3 and GhADF4 have no tissue specificity. Expression levels of different ADF genes may vary considerably in the same cell type, suggesting that they might be involved in regulating tissue development of cotton and the each ADF isoform may diverge to form the functional difference from the other ADFs during evolution.
Yin, Jun; Bai, Jie; Wang, Wei; Song, Wei; Wang, Zhongze
To express human soluble CD14 (sCD14) in eukaryotic cells. Human sCD14 cDNA was amplified from U937 cells with RT-PCR method. The recombinant expression plasmid pEF1/HisC/sCD14(348aa) was constructed and the expression in COS-7 cells was carried out using liposome transfection method. The yield was examined with scanning map identification. The expressed product was purified by immuno-affinity chromatography. Sequence analysis demonstrated that the amplified gene sequence and those reported by documents were completely identical. sCD14 was expressed with high-yield. The expressed product was purified to above 90%. Recombinant sCD14, specifically combinable with endotoxins, had a natural biological activity. Human sCD14 was expressed in COS-7 cells, which laid a foundation for further study.
Archambault Joanne M
Full Text Available Abstract Background Chronic tendon injuries, also known as tendinopathies, are common among professional and recreational athletes. These injuries result in a significant amount of morbidity and health care expenditure, yet little is known about the molecular mechanisms leading to tendinopathy. Methods We have used histological evaluation and molecular profiling to determine gene expression changes in 23 human patients undergoing surgical procedures for the treatment of chronic tendinopathy. Results Diseased tendons exhibit altered extracellular matrix, fiber disorientation, increased cellular content and vasculature, and the absence of inflammatory cells. Global gene expression profiling identified 983 transcripts with significantly different expression patterns in the diseased tendons. Global pathway analysis further suggested altered expression of extracellular matrix proteins and the lack of an appreciable inflammatory response. Conclusions Identification of the pathways and genes that are differentially regulated in tendinopathy samples will contribute to our understanding of the disease and the development of novel therapeutics.
Schembri, Mark; Kjærgaard, K.; Klemm, Per
It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance...... to antimicrobial treatments and host immune defence responses. Escherichia coli has been used as a model organism to study the mechanisms of growth within adhered communities. In this study, we use DNA microarray technology to examine the global gene expression profile of E. coli during sessile growth compared...... the transition to biofilm growth, and these included genes expressed under oxygen-limiting conditions, genes encoding (putative) transport proteins, putative oxidoreductases and genes associated with enhanced heavy metal resistance. Of particular interest was the observation that many of the genes altered...
Stolf-Moreira, R; Medri, M E; Neumaier, N; Lemos, N G; Pimenta, J A; Tobita, S; Brogin, R L; Marcelino-Guimarães, F C; Oliveira, M C N; Farias, J R B; Abdelnoor, R V; Nepomuceno, A L
Soybean genotypes MG/BR46 (Conquista) and BR16, drought-tolerant and -sensitive, respectively, were compared in terms of morphophysiological and gene-expression responses to water stress during two stages of development. Gene-expression analysis showed differential responses in Gmdreb1a and Gmpip1b mRNA expression within 30 days of water-deficit initiation in MG/BR46 (Conquista) plants. Within 45 days of initiating stress, Gmp5cs and Gmpip1b had relatively higher expression. Initially, BR16 showed increased expression only for Gmdreb1a, and later (45 days) for Gmp5cs, Gmdefensin and Gmpip1b. Only BR16 presented down-regulated expression of genes, such as Gmp5cs and Gmpip1b, 30 days after the onset of moisture stress, and Gmgols after 45 days of stress. The faster perception of water stress in MG/BR46 (Conquista) and the better maintenance of up-regulated gene expression than in the sensitive BR16 genotype imply mechanisms by which the former is better adapted to tolerate moisture deficiency.
Liu, Yongmei; Ding, Jingzhong; Reynolds, Lindsay M.; Lohman, Kurt; Register, Thomas C.; De La Fuente, Alberto; Howard, Timothy D.; Hawkins, Greg A.; Cui, Wei; Morris, Jessica; Smith, Shelly G.; Barr, R. Graham; Kaufman, Joel D.; Burke, Gregory L.; Post, Wendy; Shea, Steven; Mccall, Charles E.; Siscovick, David; Jacobs, David R.; Tracy, Russell P.; Herrington, David M.; Hoeschele, Ina
DNA methylation is one of several epigenetic mechanisms that contribute to the regulation of gene expression; however, the extent to which methylation of CpG dinucleotides correlates with gene expression at the genome-wide level is still largely unknown. Using purified primary monocytes from subjects in a large community-based cohort (n = 1264), we characterized methylation (>485 000 CpG sites) and mRNA expression (>48K transcripts) and carried out genome-wide association analyses of 8370 expression phenotypes. We identified 11 203 potential cis-acting CpG loci whose degree of methylation was associated with gene expression (eMS) at a false discovery rate threshold of 0.001. Most of the associations were consistent in effect size and direction of effect across sex and three ethnicities. Contrary to expectation, these eMS were not predominately enriched in promoter regions, or CpG islands, but rather in the 3′ UTR, gene bodies, CpG shores or ‘offshore’ sites, and both positive and negative correlations between methylation and expression were observed across all locations. eMS were enriched for regions predicted to be regulatory by ENCODE (Encyclopedia of DNA Elements) data in multiple cell types, particularly enhancers. One of the strongest association signals detected (P < 2.2 × 10−308) was a methylation probe (cg17005068) in the promoter/enhancer region of the glutathione S-transferase theta 1 gene (GSTT1, encoding the detoxification enzyme) with GSTT1 mRNA expression. Our study provides a detailed description of the epigenetic architecture in human monocytes and its relationship to gene expression. These data may help prioritize interrogation of biologically relevant methylation loci and provide new insights into the epigenetic basis of human health and diseases. PMID:23900078
Fatemeh, Ghaffarifar; Fatemeh, Tabatabaie; Zohreh, Sharifi; Abdolhosein, Dalimiasl; Mohammad Zahir, Hassan; Mehdi, Mahdavi
TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting. The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.
Arnaiz, Olivier; Van Dijk, Erwin; Bétermier, Mireille; Lhuillier-Akakpo, Maoussi; de Vanssay, Augustin; Duharcourt, Sandra; Sallet, Erika; Gouzy, Jérôme; Sperling, Linda
The 15 sibling species of the Paramecium aurelia cryptic species complex emerged after a whole genome duplication that occurred tens of millions of years ago. Given extensive knowledge of the genetics and epigenetics of Paramecium acquired over the last century, this species complex offers a uniquely powerful system to investigate the consequences of whole genome duplication in a unicellular eukaryote as well as the genetic and epigenetic mechanisms that drive speciation. High quality Paramecium gene models are important for research using this system. The major aim of the work reported here was to build an improved gene annotation pipeline for the Paramecium lineage. We generated oriented RNA-Seq transcriptome data across the sexual process of autogamy for the model species Paramecium tetraurelia. We determined, for the first time in a ciliate, candidate P. tetraurelia transcription start sites using an adapted Cap-Seq protocol. We developed TrUC, multi-threaded Perl software that in conjunction with TopHat mapping of RNA-Seq data to a reference genome, predicts transcription units for the annotation pipeline. We used EuGene software to combine annotation evidence. The high quality gene structural annotations obtained for P. tetraurelia were used as evidence to improve published annotations for 3 other Paramecium species. The RNA-Seq data were also used for differential gene expression analysis, providing a gene expression atlas that is more sensitive than the previously established microarray resource. We have developed a gene annotation pipeline tailored for the compact genomes and tiny introns of Paramecium species. A novel component of this pipeline, TrUC, predicts transcription units using Cap-Seq and oriented RNA-Seq data. TrUC could prove useful beyond Paramecium, especially in the case of high gene density. Accurate predictions of 3' and 5' UTR will be particularly valuable for studies of gene expression (e.g. nucleosome positioning, identification of cis
Mori, Hiroshi; Maruyama, Fumito; Kato, Hiromi; Toyoda, Atsushi; Dozono, Ayumi; Ohtsubo, Yoshiyuki; Nagata, Yuji; Fujiyama, Asao; Tsuda, Masataka; Kurokawa, Ken
The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.
Full Text Available Abstract Background Segmentation is a hallmark of the arthropods; most knowledge about the molecular basis of arthropod segmentation comes from work on the fly Drosophila melanogaster. In this species a hierarchic cascade of segmentation genes subdivides the blastoderm stepwise into single segment wide regions. However, segmentation in the fly is a derived feature since all segments form virtually simultaneously. Conversely, in the vast majority of arthropods the posterior segments form one at a time from a posterior pre-segmental zone. The pair rule genes (PRGs comprise an important level of the Drosophila segmentation gene cascade and are indeed the first genes that are expressed in typical transverse stripes in the early embryo. Information on expression and function of PRGs outside the insects, however, is scarce. Results Here we present the expression of the pair rule gene orthologs in the pill millipede Glomeris marginata (Myriapoda: Diplopoda. We find evidence that these genes are involved in segmentation and that components of the hierarchic interaction of the gene network as found in insects may be conserved. We further provide evidence that segments are formed in a single-segment periodicity rather than in pairs of two like in another myriapod, the centipede Strigamia maritima. Finally we show that decoupling of dorsal and ventral segmentation in Glomeris appears already at the level of the PRGs. Conclusions Although the pair rule gene network is partially conserved among insects and myriapods, some aspects of PRG interaction are, as suggested by expression pattern analysis, convergent, even within the Myriapoda. Conserved expression patterns of PRGs in insects and myriapods, however, may represent ancestral features involved in segmenting the arthropod ancestor.
Patel, Minesh; Berry, James O
In leaves of most C(4) plants, ribulose 1,5 bisphosphate carboxylase (Rubisco) accumulates only in bundle sheath (bs) cells that surround the vascular centres, and not in mesophyll (mp) cells. It has been shown previously that in the C(4) dicots amaranth and Flaveria bidentis, post-transcriptional control of mRNA translation and stability mediate the C(4) expression patterns of genes encoding the large and small Rubisco subunits (chloroplast rbcL and nuclear RbcS, respectively). Translational control appears to regulate bs cell-specific Rubisco gene expression during early dicot leaf development, while control of mRNA stability appears to mediate bs-specific accumulation of RbcS and rbcL transcripts in mature leaves. Post-transcriptional control is also involved in the regulation of Rubisco gene expression by light, and in response to photosynthetic activity. Transgenic and transient expression studies in F. bidentis provide direct evidence for post-transcriptional control of bs cell-specific RbcS expression, which is mediated by the 5' and 3' untranslated regions (UTRs) of the mRNA. Comparisons of Rubisco gene expression in these dicots and in the monocot maize indicates possible commonalities in the regulation of RbcS and rbcL genes in these divergent C(4) species. Now that the role of post-transcriptional regulation in C(4) gene expression has been established, it is likely that future studies of mRNA-protein interactions will address long-standing questions about the establishment and maintenance of cell type-specificity in these plants. Some of these regulatory mechanisms may have ancestral origins in C(3) species, through modification of pre-existing factors, or by the acquisition of novel C(4) processes.
There is certain degree of frustration and discontent in the area of microarray gene expression data analysis of cancer datasets. It arises from the mathematical problem called 'curse of dimensionality,' which is due to the small number of samples available in training sets, used for calculating transcriptional signatures from the large number of differentially expressed (DE) genes, measured by microarrays. The new generation of causal reasoning algorithms can provide solutions to the curse of dimensionality by transforming microarray data into activity of a small number of cancer hallmark pathways. This new approach can make feature space dimensionality optimal for mathematical signature calculations. The author reviews the reasons behind the current frustration with transcriptional signatures derived from DE genes in cancer. He also provides an overview of the novel methods for signature calculations based on differentially variable genes and expression regulators. Furthermore, the authors provide perspectives on causal reasoning algorithms that use prior knowledge about regulatory events described in scientific literature to identify expression regulators responsible for the differential expression observed in cancer samples. The author advocates causal reasoning methods to calculate cancer pathway activity signatures. The current challenge for these algorithms is in ensuring quality of the knowledgebase. Indeed, the development of cancer hallmark pathway collections, together with statistical algorithms to transform activity of expression regulators into pathway activity, are necessary for causal reasoning to be used in cancer research.
Full Text Available Superoxide dismutases (SODs are critical antioxidant enzymes that protect organisms from reactive oxygen species (ROS caused by adverse conditions, and have been widely found in the cytoplasm, chloroplasts, and mitochondria of eukaryotic and prokaryotic cells. Tomato (Solanum lycopersicum L. is an important economic crop and is cultivated worldwide. However, abiotic and biotic stresses severely hinder growth and development of the plant, which affects the production and quality of the crop. To reveal the potential roles of SOD genes under various stresses, we performed a systematic analysis of the tomato SOD gene family and analyzed the expression patterns of SlSOD genes in response to abiotic stresses at the whole-genome level. The characteristics of the SlSOD gene family were determined by analyzing gene structure, conserved motifs, chromosomal distribution, phylogenetic relationships, and expression patterns. We determined that there are at least nine SOD genes in tomato, including four Cu/ZnSODs, three FeSODs, and one MnSOD, and they are unevenly distributed on 12 chromosomes. Phylogenetic analyses of SOD genes from tomato and other plant species were separated into two groups with a high bootstrap value, indicating that these SOD genes were present before the monocot-dicot split. Additionally, many cis-elements that respond to different stresses were found in the promoters of nine SlSOD genes. Gene expression analysis based on RNA-seq data showed that most genes were expressed in all tested tissues, with the exception of SlSOD6 and SlSOD8, which were only expressed in young fruits. Microarray data analysis showed that most members of the SlSOD gene family were altered under salt- and drought-stress conditions. This genome-wide analysis of SlSOD genes helps to clarify the function of SlSOD genes under different stress conditions and provides information to aid in further understanding the evolutionary relationships of SOD genes in plants.
A large number of proteins are specifically synthesized in the hepatocyte. Only the adult liver expresses the complete repertoire of functions which are required at various stages during development. There is therefore a complex series of regulatory mechanisms responsible for the maintenance of the differentiated state and for the developmental and physiological variations in the pattern of gene expression. Human hepatoma cell lines HepG2 and Hep3B display a pattern of gene expression similar to adult and fetal liver, respectively; in contrast, cultured fibroblasts or HeLa cells do not express most of the liver specific genes. They have used these cell lines for transfection experiments with cloned human liver specific genes. DNA segments coding for alpha1-antitrypsin and retinol binding protein (two proteins synthesized both in fetal and adult liver) are expressed in the hepatoma cell lines HepG2 and Hep3B, but not in HeLa cells or fibroblasts. A DNA segment coding for haptoglobin (a protein synthesized only after birth) is only expressed in the hepatoma cell line HepG2 but not in Hep3B nor in non hepatic cell lines. The information for tissue specific expression is located in the 5' flanking region of all three genes. In vivo competition experiments show that these DNA segments bind to a common, apparently limiting, transacting factor. Conventional techniques (Bal deletions, site directed mutagenesis, etc.) have been used to precisely identify the DNA sequences responsible for these effects. The emerging picture is complex: they have identified multiple, separate transcriptional signals, essential for maximal promoter activation and tissue specific expression. Some of these signals show a negative effect on transcription in fibroblast cell lines.
Nevozhay, Dmitry; Adams, Rhys M.; Murphy, Kevin F.; Josić, Krešimir; Balázsi, Gábor
Although several recent studies have focused on gene autoregulation, the effects of negative feedback (NF) on gene expression are not fully understood. Our purpose here was to determine how the strength of NF regulation affects the characteristics of gene expression in yeast cells harboring chromosomally integrated transcriptional cascades that consist of the yEGFP reporter controlled by (i) the constitutively expressed tetracycline repressor TetR or (ii) TetR repressing its own expression. Reporter gene expression in the cascade without feedback showed a steep (sigmoidal) dose–response and a wide, nearly bimodal yEGFP distribution, giving rise to a noise peak at intermediate levels of induction. We developed computational models that reproduced the steep dose–response and the noise peak and predicted that negative autoregulation changes reporter expression from bimodal to unimodal and transforms the dose–response from sigmoidal to linear. Prompted by these predictions, we constructed a “linearizer” circuit by adding TetR autoregulation to our original cascade and observed a massive (7-fold) reduction of noise at intermediate induction and linearization of dose–response before saturation. A simple mathematical argument explained these findings and indicated that linearization is highly robust to parameter variations. These findings have important implications for gene expression control in eukaryotic cells, including the design of synthetic expression systems. PMID:19279212
Full Text Available Neighboring genes in the eukaryotic genome have a tendency to express concurrently, and the proximity of two adjacent genes is often considered a possible explanation for their co-expression behavior. However, the actual contribution of the physical distance between two genes to their co-expression behavior has yet to be defined. To further investigate this issue, we studied the co-expression of neighboring genes in zebrafish, which has a compact genome and has experienced a whole genome duplication event. Our analysis shows that the proportion of highly co-expressed neighboring pairs (Pearson’s correlation coefficient R>0.7 is low (0.24% ~ 0.67%; however, it is still significantly higher than that of random pairs. In particular, the statistical result implies that the co-expression tendency of neighboring pairs is negatively correlated with their physical distance. Our findings therefore suggest that physical distance may play an important role in the co-expression of neighboring genes. Possible mechanisms related to the neighboring genes’ co-expression are also discussed.
Full Text Available Abstract Background Euglenophyta and Chlorarachniophyta are groups of photosynthetic eukaryotes harboring secondary plastids of distinct green algal origins. Although previous phylogenetic analyses of genes encoding Calvin cycle enzymes demonstrated the presence of genes apparently not derived from green algal endosymbionts in the nuclear genomes of Euglena gracilis (Euglenophyta and Bigelowiella natans (Chlorarachniophyta, the origins of these "non-green" genes in "green" secondary phototrophs were unclear due to the limited taxon sampling. Results Here, we sequenced five new phosphoribulokinase (PRK genes (from one euglenophyte, two chlorarachniophytes, and two glaucophytes and performed an extended phylogenetic analysis of the genes based on a phylum-wide taxon sampling from various photosynthetic eukaryotes. Our phylogenetic analyses demonstrated that the PRK sequences form two genera of Euglenophyta formed a robust monophyletic group within a large clade including stramenopiles, haptophytes and a cryptophyte, and three genera of Chlorarachniophyta were placed within the red algal clade. These "non-green" affiliations were supported by the taxon-specific insertion/deletion sequences in the PRK alignment, especially between euglenophytes and stramenopiles. In addition, phylogenetic analysis of another Calvin cycle enzyme, plastid-targeted sedoheptulose-bisphosphatase (SBP, showed that the SBP sequences from two genera of Chlorarachniophyta were positioned within a red algal clade. Conclusions Our results suggest that PRK genes may have been transferred from a "stramenopile" ancestor to Euglenophyta and from a "red algal" ancestor to Chlorarachniophyta before radiation of extant taxa of these two "green" secondary phototrophs. The presence of two of key Calvin cycle enzymes, PRK and SBP, of red algal origins in Chlorarachniophyta indicate that the contribution of "non-green" algae to the plastid proteome in the "green" secondary phototrophs is
Full Text Available Abstract Background Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. Results We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages seed coats (globular and torpedo stages and endosperm (pooled globular to torpedo stages and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST (GenBank accessions LIBEST_026995 to LIBEST_027011 were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152 had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. Conclusions We have developed a foundational database of expressed sequences and collection of plasmid
Venglat, Prakash; Xiang, Daoquan; Qiu, Shuqing; Stone, Sandra L; Tibiche, Chabane; Cram, Dustin; Alting-Mees, Michelle; Nowak, Jacek; Cloutier, Sylvie; Deyholos, Michael; Bekkaoui, Faouzi; Sharpe, Andrew; Wang, Edwin; Rowland, Gordon; Selvaraj, Gopalan; Datla, Raju
Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as
Visualizing bacterial cells and describing their responses to the environment are difficult tasks. Their small size is the chief reason for the difficulty, which means that we must often use many millions of cells in a sample in order to determine what the average response of the bacteria is. However, an average response can sometimes mask important events in bacterial physiology, which means that our understanding of these organisms will suffer. We have used a variety of instruments to visualize bacterial cells, all of which tell us something different about the sample. We use a fluorescence activated cell sorter to sort cells based on the fluorescence provided by bioreporter genes, and these can be used to select for particular genetic mutations. Cells can be visualized by epifluorescent microscopy, and sensitive photodetectors can be added that allow us to find a single bacterial cell that is fluorescent or bioluminescent. We have also used standard photomultipliers to examine cell aggregates as field bioreporter microorganisms. Examples of each of these instruments show how our understanding of bacterial physiology has changed with the technology.
Heather E. Driscoll
Full Text Available Here we describe microarray expression data (raw and normalized, experimental metadata, and gene-level data with expression statistics from Saccharomyces cerevisiae exposed to simulated asbestos mine drainage from the Vermont Asbestos Group (VAG Mine on Belvidere Mountain in northern Vermont, USA. For nearly 100 years (between the late 1890s and 1993, chrysotile asbestos fibers were extracted from serpentinized ultramafic rock at the VAG Mine for use in construction and manufacturing industries. Studies have shown that water courses and streambeds nearby have become contaminated with asbestos mine tailings runoff, including elevated levels of magnesium, nickel, chromium, and arsenic, elevated pH, and chrysotile asbestos-laden mine tailings, due to leaching and gradual erosion of massive piles of mine waste covering approximately 9 km2. We exposed yeast to simulated VAG Mine tailings leachate to help gain insight on how eukaryotic cells exposed to VAG Mine drainage may respond in the mine environment. Affymetrix GeneChip® Yeast Genome 2.0 Arrays were utilized to assess gene expression after 24-h exposure to simulated VAG Mine tailings runoff. The chemistry of mine-tailings leachate, mine-tailings leachate plus yeast extract peptone dextrose media, and control yeast extract peptone dextrose media is also reported. To our knowledge this is the first dataset to assess global gene expression patterns in a eukaryotic model system simulating asbestos mine tailings runoff exposure. Raw and normalized gene expression data are accessible through the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO Database Series GSE89875 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE89875.
Peters, Dorien J.M.; Lookeren Campagne, Michiel M. van; Haastert, Peter J.M. van; Spek, Wouter; Schaap, Pauline
We investigated the effect of Li+ on two types of cyclic AMP-regulated gene expression and on basal and cyclic AMP-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Li+ effectively inhibits cyclic AMP-induced prespore gene expression, half-maximal inhibition occurring at about 2mM-LiCl.
van Ruissen, Fred; Schaaf, Gerben J.; Kool, Marcel; Baas, Frank; Ruijter, Jan M.
Serial analysis of gene expression (SAGE) and microarrays have found a widespread application, but much ambiguity exists regarding the amalgamation of the data resulting from these technologies. Cross-platform utilization of gene expression data from the SAGE and microarray technology could reduce
Van Dijck Patrick
Full Text Available Abstract Background Compendia of gene expression profiles under chemical and genetic perturbations constitute an invaluable resource from a systems biology perspective. However, the perturbational nature of such data imposes specific challenges on the computational methods used to analyze them. In particular, traditional clustering algorithms have difficulties in handling one of the prominent features of perturbational compendia, namely partial coexpression relationships between genes. Biclustering methods on the other hand are specifically designed to capture such partial coexpression patterns, but they show a variety of other drawbacks. For instance, some biclustering methods are less suited to identify overlapping biclusters, while others generate highly redundant biclusters. Also, none of the existing biclustering tools takes advantage of the staple of perturbational expression data analysis: the identification of differentially expressed genes. Results We introduce a novel method, called ENIGMA, that addresses some of these issues. ENIGMA leverages differential expression analysis results to extract expression modules from perturbational gene expression data. The core parameters of the ENIGMA clustering procedure are automatically optimized to reduce the redundancy between modules. In contrast to the biclusters produced by most other methods, ENIGMA modules may show internal substructure, i.e. subsets of genes with distinct but significantly related expression patterns. The grouping of these (often functionally related patterns in one module greatly aids in the biological interpretation of the data. We show that ENIGMA outperforms other methods on artificial datasets, using a quality criterion that, unlike other criteria, can be used for algorithms that generate overlapping clusters and that can be modified to take redundancy between clusters into account. Finally, we apply ENIGMA to the Rosetta compendium of expression profiles for
Baniushin, B F
Like in bacteria, DNA in these organisms is subjected to enzymatic modification (methylation) both at adenine and cytosine residues. There is an indirect evidence that adenine DNA methylation takes place also in animals. In plants m6A was detected in total, mitochondrial and nuclear DNAs; in plants one and the same gene (DRM2) can be methylated both at adenine and cytosine residues. ORF homologous to bacterial adenine DNA-methyltransferases are present in nuclear DNA of protozoa, yeasts, insects, nematodes, higher plants, vertebrates and other eukaryotes. Thus, adenine DNA-methyltransferases can be found in the various evolutionary distant eukaryotes. First N6-adenine DNA-methyltransferase (wadmtase) of higher eukaryotes was isolated from vacuolar fraction of vesicles obtained from aging wheat coleoptiles; in the presence of S-adenosyl-L-methionine this Mg2+ -, Ca2+ -dependent enzyme de novo methylates first adenine residue in TGATCA sequence in single- and double-stranded DNA but it prefers single-stranded DNA structures. Adenine DNA methylation in eukaryotes seems to be involved in regulation of both gene expression and DNA replication including replication of mitochondrial DNA. It can control persistence of foreign DNA in a cell and seems to be an element of R-M system in plants. Thus, in eukaryotic cell there are, at least, two different systems of the enzymatic DNA methylations (adenine and cytosine ones) and a special type of regulation of gene functioning based on the combinatory hierarchy of these interdependent genome modifications.
Zhang, Qingzhou; Yang, Bo; Chen, Xujiao; Xu, Jing; Mei, Changlin; Mao, Zhiguo
We present a bioinformatics database named Renal Gene Expression Database (RGED), which contains comprehensive gene expression data sets from renal disease research. The web-based interface of RGED allows users to query the gene expression profiles in various kidney-related samples, including renal cell lines, human kidney tissues and murine model kidneys. Researchers can explore certain gene profiles, the relationships between genes of interests and identify biomarkers or even drug targets in kidney diseases. The aim of this work is to provide a user-friendly utility for the renal disease research community to query expression profiles of genes of their own interest without the requirement of advanced computational skills. Availability and implementation: Website is implemented in PHP, R, MySQL and Nginx and freely available from http://rged.wall-eva.net. Database URL: http://rged.wall-eva.net PMID:25252782
Amann, P M; Eichmüller, S B; Schmidt, J; Bazhin, A V
Vitamin A serves as substrate for the biosynthesis of several derivates (retinoids) which are important for cell growth and cell differentiation as well as for vision. Retinoic acid is the major physiologically active form of vitamin A regulating the expression of different genes. At present, hundreds of genes are known to be regulated by retinoic acid. This regulation is very complex and is, in turn, regulated on many levels. To date, two families of retinoid nuclear receptors have been identified: retinoic acid receptors and retinoid X receptors, which are members of the steroid hormone receptor superfamily of ligand-activated transcription factors. In order to regulate gene expression, all-trans retinal needs to be oxidized to retinoic acid. All-trans retinal, in turn, can be produced during oxidation of all-trans retinol or in a retinol-independent metabolic pathway through cleavage of β-carotene with all-trans retinal as an intermediate metabolite. Recently it has been shown that not only retinoic acid is an active form of vitamin A, but also that all-trans retinal can play an important role in gene regulation. In this review we comprehensively summarize recent literature on regulation of gene expression by retinoids, biochemistry of retinoid receptors, and molecular mechanisms of retinoid-mediated effects on gene regulation.
Zhang, Hong-Yu; Xu, Pei-Zhou; Yang, Hua; Wu, Xian-Jun
Genomic imprinting refers to the phenomenon that the expression of a gene copy depends on its parent of origin. The Arabidopsis imprinted FIS (Fertilisation-independent seed) genes, mea, fis2, and fie, play essential roles in the repression of central cell and the regulation of early endosperm development. fis mutants display two phenotypes: autonomous diploid endosperm development when fertilization is absent and un-cellularised endosperm formation when fertilization occurs. The FIS Polycomb protein complex including the above three FIS proteins catalyzes histone H3 K27 tri-methylation on target loci. DME (DEMETER), a DNA glycosylase, and AtMET1 (Methyltransferase1), a DNA methyltransferase, are involved in the regulation of imprinted expression of both mea and fis2. This review summarizes the studies on the Arabidopsis imprinted FIS genes and other related genes. Recent works have shown that the insertion of transposons may affect nearby gene expression, which may be the main driving force behind the evolution of genomic imprinting. This summary covers the achievements on Arabidopsis imprinted genes will provide important information for studies on genomic imprinting in the important crops such as rice and maize.
Full Text Available Metal nanomaterials are shown to enhance gene expression for rice -galactosidase gene (-Gal in yeast cells. Au and Ag nanoparticles and their nanocomposites, silica-Au and silica-Ag, were prepared and characterized by UV-vis spectroscopy and TEM technique. The rice -galactosidase gene was cloned into the yeast chromosome, where the cloned cells were precultured and induced into a medium containing each of the testing nanomaterials. The nanomaterials were observed to incorporate inside the cells, and no cell death has been detected during the course of gene expression. The enzyme activity was determined by a synthetic substrate, p-nitrophenyl--D-galctopyranoside, and the yellow product yield was recorded in a spectrophotometer at 400 nm. When Au and Ag nanoparticles were incorporated with the culture, a 3–5 fold enhancement in -galactosidase was observed for intracellular activity as well as the secreted activity into the medium. The secreted protein was analyzed to have a pure form and displayed as a single protein band in the SDS-gel electrophoresis. The effects of size and chemical nature of nanomaterials on gene expression for the rice -galactosidase gene in yeast cells are discussed.
Ren, X.Y.; Vorst, O.F.J.; Fiers, M.W.E.J.; Stiekema, W.J.; Nap, J.P.H.
In both the monocot rice and the dicot Arabidopsis, highly expressed genes have more and longer introns and a larger primary transcript than genes expressed at a low level: higher expressed genes tend to be less compact than lower expressed genes. In animal genomes, it is the other way round.
Shimizu, Akifumi; Yano, Kentaro
Microarrays provide genome-wide gene expression changes. In current analyses, the majority of genes on the array are frequently eliminated for further analysis just in order for computational effort to be affordable. This strategy risks failure to discover whole sets of genes related to a quantitative trait of interest, which is generally controlled by several loci that might be eliminated in current approaches. Here, we describe a high-throughput gene discovery method based on correspondence analysis with a new index for expression ratios [arctan (1/ratio)] and three artificial marker genes. This method allows us to quickly analyze the whole microarray dataset without elimination and discover up/down-regulated genes related to a trait of interest. We employed an example dataset to show the theoretical advantage of this method. We then used the method to identify 88 cancer-related genes from a published microarray data from patients with breast cancer. This method can be easily performed and the result is also visible in three-dimensional viewing software that we have developed. Our method is useful for revaluating the wealth of microarray data available from web-sites.
Full Text Available Abstract Background The determination of the right model structure describing a gene regulation network and the identification of its parameters are major goals in systems biology. The task is often hampered by the lack of relevant experimental data with sufficiently low noise level, but the subset of genes whose concentration levels exhibit an oscillatory behavior in time can readily be analyzed on the basis of their Fourier spectrum, known to turn complex signals into few relatively noise-free parameters. Such genes therefore offer opportunities of understanding gene regulation quantitatively. Results Fourier analysis is applied to data on gene expression levels in mouse liver cells that oscillate according to the circadian rhythm. Several model structures in the form of linear and nonlinear differential equations are matched to the data and it is shown that although the considered models can reproduce many features of the oscillatory patterns, some can be excluded on the basis of Fourier analysis without appeal to prior knowledge of regulatory pathways. A systematic method for testing models is also proposed based on measuring the effects of variations in gene copy-number on the expression levels of coupled genes. Conclusions Fourier analysis is a technique that is well-adapted to the study of biological oscillators and can be used instead or in addition to conventional modeling techniques. Its usefulness will increase as more high-resolution data become available.
Castanera, Raúl; Pérez, Gúmer; López, Leticia; Sancho, Rubén; Santoyo, Francisco; Alfaro, Manuel; Gabaldón, Toni; Pisabarro, Antonio G; Oguiza, José A; Ramírez, Lucía
Helitrons are class-II eukaryotic transposons that transpose via a rolling circle mechanism. Due to their ability to capture and mobilize gene fragments, they play an important role in the evolution of their host genomes. We have used a bioinformatics approach for the identification of helitrons in two Pleurotus ostreatus genomes using de novo detection and homology-based searching. We have analyzed the presence of helitron-captured genes as well as the expansion of helitron-specific helicases in fungi and performed a phylogenetic analysis of their conserved domains with other representative eukaryotic species. Our results show the presence of two helitron families in P. ostreatus that disrupt gene colinearity and cause a lack of synteny between their genomes. Both putative autonomous and non-autonomous helitrons were transcriptionally active, and some of them carried highly expressed captured genes of unknown origin and function. In addition, both families contained eukaryotic, bacterial and viral domains within the helitron's boundaries. A phylogenetic reconstruction of RepHel helicases using the Helitron-like and PIF1-like helicase conserved domains revealed a polyphyletic origin for eukaryotic helitrons. P. ostreatus helitrons display features similar to other eukaryotic helitrons and do not tend to capture host genes or gene fragments. The occurrence of genes probably captured from other hosts inside the helitrons boundaries pose the hypothesis that an ancient horizontal transfer mechanism could have taken place. The viral domains found in some of these genes and the polyphyletic origin of RepHel helicases in the eukaryotic kingdom suggests that virus could have played a role in a putative lateral transfer of helitrons within the eukaryotic kingdom. The high similarity of some helitrons, along with the transcriptional activity of its RepHel helicases indicates that these elements are still active in the genome of P. ostreatus.
Lundin, A-C; Aspenberg, P; Eliasson, P
The pathogenesis of trigger finger has generally been ascribed to primary changes in the first annular ligament. In contrast, we recently found histological changes in the tendons, similar to the findings in Achilles tendinosis or tendinopathy. We therefore hypothesized that trigger finger tendons would show differences in gene expression in comparison to normal tendons in a pattern similar to what is published for Achilles tendinosis. We performed quantitative real-time polymerase chain reaction on biopsies from finger flexor tendons, 13 trigger fingers and 13 apparently healthy control tendons, to assess the expression of 10 genes which have been described to be differently expressed in tendinosis (collagen type 1a1, collagen 3a1, MMP-2, MMP-3, ADAMTS-5, TIMP-3, aggrecan, biglycan, decorin, and versican). In trigger finger tendons, collagen types 1a1 and 3a1, aggrecan and biglycan were all up-regulated, and MMP-3and TIMP-3 were down-regulated. These changes were statistically significant and have been previously described for Achilles tendinosis. The remaining four genes were not significantly altered. The changes in gene expression support the hypothesis that trigger finger is a form of tendinosis. Because trigger finger is a common condition, often treated surgically, it could provide opportunities for clinical research on tendinosis. © 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Samadani, Uzma; Judkins, Alexander R.; Akpalu, Albert; Aronica, Eleonora; Crino, Peter B.
PURPOSE: Gangliogliomas (GGs) are neuronal-glial tumors highly associated with epilepsy. We hypothesized that the expression of select gene families including neurotransmitter receptor subunits and growth factors would be distinct in neurons and astrocytes within GG compared with adjacent cortex and
Full Text Available Mammalian hearts cannot regenerate. In contrast, zebrafish hearts regenerate even when up to 20% of the ventricle is amputated. The mechanism of zebrafish heart regeneration is not understood. To systematically characterize this process at the molecular level, we generated transcriptional profiles of zebrafish cardiac regeneration by microarray analyses. Distinct gene clusters were identified based on temporal expression patterns. Genes coding for wound response/inflammatory factors, secreted molecules, and matrix metalloproteinases are expressed in regenerating heart in sequential patterns. Comparisons of gene expression profiles between heart and fin regeneration revealed a set of regeneration core molecules as well as tissue-specific factors. The expression patterns of several secreted molecules around the wound suggest that they play important roles in heart regeneration. We found that both platelet-derived growth factor-a and -b (pdgf-a and pdgf-b are upregulated in regenerating zebrafish hearts. PDGF-B homodimers induce DNA synthesis in adult zebrafish cardiomyocytes. In addition, we demonstrate that a chemical inhibitor of PDGF receptor decreases DNA synthesis of cardiomyocytes both in vitro and in vivo during regeneration. Our data indicate that zebrafish heart regeneration is associated with sequentially upregulated wound healing genes and growth factors and suggest that PDGF signaling is required.
Gene Expression and Microarray Investigation of Dendrobium Mixture as Progressive Therapy for the Treatment of Type 2 Diabetes Mellitus. ... Those with random blood glucose > 16.7 mmol/L were used as the model group and treated with Dendrobium mixture (DEN, containing Dendrobium, Astragalus, Schisandra, etc) in ...
Horowitz, Jordan M.; Kulkarni, Rahul V.
The intrinsic stochasticity of gene expression can give rise to large fluctuations and rare events that drive phenotypic variation in a population of genetically identical cells. Characterizing the fluctuations that give rise to such rare events motivates the analysis of large deviations in stochastic models of gene expression. Recent developments in non-equilibrium statistical mechanics have led to a framework for analyzing Markovian processes conditioned on rare events and for representing such processes by conditioning-free driven Markovian processes. We use this framework, in combination with approaches based on queueing theory, to analyze a general class of stochastic models of gene expression. Modeling gene expression as a Batch Markovian Arrival Process (BMAP), we derive exact analytical results quantifying large deviations of time-integrated random variables such as promoter activity fluctuations. We find that the conditioning-free driven process can also be represented by a BMAP that has the same form as the original process, but with renormalized parameters. The results obtained can be used to quantify the likelihood of large deviations, to characterize system fluctuations conditional on rare events and to identify combinations of model parameters that can give rise to dynamical phase transitions in system dynamics.
Whole blood from five Mycobacterium bovis-sensitised hyenas was incubated in Nil and TB antigen tubes of the QuantiFERON®-TB Gold (QFT) system. Using qPCR, the relative expression stability of the reference genes ACTB, GAPDH, YWHAZ and TBP in these samples was determined as well as the mean fold change in ...
Suppression subtractive hybridization was used to identify genes showing differential expression profile associated withgrowth rate in skeletal muscle tissue of Landrace weanling pig. Two subtracted cDNA populations were generated from mus-culus longissimus muscle tissues of selected pigs with extreme expected ...
Wong, Darren C J; Sweetman, Crystal; Ford, Christopher M
The genus Citrus encompasses major cultivated plants such as sweet orange, mandarin, lemon and grapefruit, among the world's most economically important fruit crops. With increasing volumes of transcriptomics data available for these species, Gene Co-expression Network (GCN) analysis is a viable option for predicting gene function at a genome-wide scale. GCN analysis is based on a "guilt-by-association" principle whereby genes encoding proteins involved in similar and/or related biological processes may exhibit similar expression patterns across diverse sets of experimental conditions. While bioinformatics resources such as GCN analysis are widely available for efficient gene function prediction in model plant species including Arabidopsis, soybean and rice, in citrus these tools are not yet developed. We have constructed a comprehensive GCN for citrus inferred from 297 publicly available Affymetrix Genechip Citrus Genome microarray datasets, providing gene co-expression relationships at a genome-wide scale (33,000 transcripts). The comprehensive citrus GCN consists of a global GCN (condition-independent) and four condition-dependent GCNs that survey the sweet orange species only, all citrus fruit tissues, all citrus leaf tissues, or stress-exposed plants. All of these GCNs are clustered using genome-wide, gene-centric (guide) and graph clustering algorithms for flexibility of gene function prediction. For each putative cluster, gene ontology (GO) enrichment and gene expression specificity analyses were performed to enhance gene function, expression and regulation pattern prediction. The guide-gene approach was used to infer novel roles of genes involved in disease susceptibility and vitamin C metabolism, and graph-clustering approaches were used to investigate isoprenoid/phenylpropanoid metabolism in citrus peel, and citric acid catabolism via the GABA shunt in citrus fruit. Integration of citrus gene co-expression networks, functional enrichment analysis and gene
Background The genus Citrus encompasses major cultivated plants such as sweet orange, mandarin, lemon and grapefruit, among the world’s most economically important fruit crops. With increasing volumes of transcriptomics data available for these species, Gene Co-expression Network (GCN) analysis is a viable option for predicting gene function at a genome-wide scale. GCN analysis is based on a “guilt-by-association” principle whereby genes encoding proteins involved in similar and/or related biological processes may exhibit similar expression patterns across diverse sets of experimental conditions. While bioinformatics resources such as GCN analysis are widely available for efficient gene function prediction in model plant species including Arabidopsis, soybean and rice, in citrus these tools are not yet developed. Results We have constructed a comprehensive GCN for citrus inferred from 297 publicly available Affymetrix Genechip Citrus Genome microarray datasets, providing gene co-expression relationships at a genome-wide scale (33,000 transcripts). The comprehensive citrus GCN consists of a global GCN (condition-independent) and four condition-dependent GCNs that survey the sweet orange species only, all citrus fruit tissues, all citrus leaf tissues, or stress-exposed plants. All of these GCNs are clustered using genome-wide, gene-centric (guide) and graph clustering algorithms for flexibility of gene function prediction. For each putative cluster, gene ontology (GO) enrichment and gene expression specificity analyses were performed to enhance gene function, expression and regulation pattern prediction. The guide-gene approach was used to infer novel roles of genes involved in disease susceptibility and vitamin C metabolism, and graph-clustering approaches were used to investigate isoprenoid/phenylpropanoid metabolism in citrus peel, and citric acid catabolism via the GABA shunt in citrus fruit. Conclusions Integration of citrus gene co-expression networks
Tarbell, John M; Shi, Zhong-Dong; Dunn, Jessilyn; Jo, Hanjoong
This review places modern research developments in vascular mechanobiology in the context of hemodynamic phenomena in the cardiovascular system and the discrete localization of vascular disease. The modern origins of this field are traced, beginning in the 1960s when associations between flow characteristics, particularly blood flow-induced wall shear stress, and the localization of atherosclerotic plaques were uncovered, and continuing to fluid shear stress effects on the vascular lining endothelial) cells (ECs), including their effects on EC morphology, biochemical production, and gene expression. The earliest single-gene studies and genome-wide analyses are considered. The final section moves from the ECs lining the vessel wall to the smooth muscle cells and fibroblasts within the wall that are fluid me chanically activated by interstitial flow that imposes shear stresses on their surfaces comparable with those of flowing blood on EC surfaces. Interstitial flow stimulates biochemical production and gene expression, much like blood flow on ECs.
Matthew S Hestand
Full Text Available Cellular mechanisms that achieve protein diversity in eukaryotes are multifaceted, including transcriptional components such as RNA splicing. Through alternative splicing, a single protein-coding gene can generate multiple mRNA transcripts and protein isoforms, some of which are tissue-specific. We have conducted qualitative and quantitative analyses of the Bodymap 2.0 messenger RNA-sequencing data from 16 human tissue samples and identified 209,363 splice junctions. Of these, 22,231 (10.6% were not previously annotated and 21,650 (10.3% were expressed in a tissue-restricted pattern. Tissue-restricted alternative splicing was found to be widespread, with approximately 65% of expressed multi-exon genes containing at least one tissue-specific splice junction. Interestingly, we observed many tissue-specific splice junctions not only in genes expressed in one or a few tissues, but also from gene loci with a broad pattern of expression.
O. M. Krivtsova
Full Text Available Recent large-scale genomic studies established the occurrence of multiple DNA sequence variants in genomes of healthy individuals that differ from the reference sequence. Among these variants mostly represented by germline single nucleotide polymorphisms disease-related alleles are detected including alleles which are associated with monogenic disorders, and putative deleterious genetic variants. Apart from functional significance of a particular variant and of a gene harboring it, the penetrance of these allelic variants depends on their expression level and can be determined by preferential expression of a particular allele, or allele-specific expression. It is estimated that 20–30 % of genes present in the human genome display allelic bias in a tissue-specific manner. Allele-specific expression is defined by a range of genetic and epigenetic mechanisms including cis-regulatory polymorphisms, allele-specific binding of transcription factors, allele-specific DNA methylation and regulation through non-coding RNA.Although the data on the issue are scarce, allele-specific expression has been reported to be implicated in several hereditary disorders including benign and malignant tumors of the large intestine. Recent studies that estimate allele-specific expression incidence in tumors and identify wide range of genes displaying allelic imbalance indicate that allele-specific expression might play a significant role in carcinogenesis. Eventually, estimation of transcriptional rate of allelic variants which cause dysfunction of oncogenes and tumor suppressors may prove to be essential for rational choice of antitumor therapeutic strategy. In this review, we outline the main concepts and mechanisms of allele-specific expression and the data on allelic imbalance in tumors.
Adam Rodney D
Full Text Available Abstract Background Giardia intestinalis is a protist found in freshwaters worldwide, and is the most common cause of parasitic diarrhea in humans. The phylogenetic position of this parasite is still much debated. Histones are small, highly conserved proteins that associate tightly with DNA to form chromatin within the nucleus. There are two classes of core histone genes in higher eukaryotes: DNA replication-independent histones and DNA replication-dependent ones. Results We identified two copies each of the core histone H2a, H2b and H3 genes, and three copies of the H4 gene, at separate locations on chromosomes 3, 4 and 5 within the genome of Giardia intestinalis, but no gene encoding a H1 linker histone could be recognized. The copies of each gene share extensive DNA sequence identities throughout their coding and 5' noncoding regions, which suggests these copies have arisen from relatively recent gene duplications or gene conversions. The transcription start sites are at triplet A sequences 1–27 nucleotides upstream of the translation start codon for each gene. We determined that a 50 bp region upstream from the start of the histone H4 coding region is the minimal promoter, and a highly conserved 15 bp sequence called the histone motif (him is essential for its activity. The Giardia core histone genes are constitutively expressed at approximately equivalent levels and their mRNAs are polyadenylated. Competition gel-shift experiments suggest that a factor within the protein complex that binds him may also be a part of the protein complexes that bind other promoter elements described previously in Giardia. Conclusion In contrast to other eukaryotes, the Giardia genome has only a single class of core histone genes that encode replication-independent histones. Our inability to locate a gene encoding the linker histone H1 leads us to speculate that the H1 protein may not be required for the compaction of Giardia's small and gene-rich genome.
Yao, Jianxiu; Saenkham, Panatda; Levy, Julien; Ibanez, Freddy; Noroy, Christophe; Mendoza, Azucena; Huot, Ordom; Meyer, Damien F; Tamborindeguy, Cecilia
"Candidatus Liberibacter solanacearum" (Lso) has emerged as a serious threat world-wide. Five Lso haplotypes have been identified so far. Haplotypes A and B are present in the Americas and/or New Zealand, where they are vectored to solanaceous plants by the potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae). The fastidious nature of these pathogens has hindered the study of the interactions with their eukaryotic hosts (vector and plant). To understand the strategies used by these pathogens to infect their vector, the effects of each Lso haplotype (A or B) on psyllid fitness was investigated, and genome-wide transcriptomic and RT-qPCR analyses were performed to evaluate Lso gene expression in association with its vector. Results showed that psyllids infected with haplotype B had significantly lower percentage of nymphal survival compared to psyllids infected with haplotype A. Although overall gene expression across Lso genome was similar between the two Lso haplotypes, differences in the expression of key candidate genes were found. Among the 16 putative type IV effector genes tested, four of them were differentially expressed between Lso haplotypes, while no differences in gene expression were measured by qPCR or transcriptomic analysis for the rest of the genes. This study provides new information regarding the pathogenesis of Lso haplotypes in their insect vector.
Meiosis and gamete formation are processes that are essential for sexual reproduction in all eukaryotic organisms. Multiple intracellular and extracellular signals feed into pathways that converge on transcription factors that induce the expression of meiosis-specific genes. Once triggered the meiosis-specific gene expression program proceeds in a cascade that drives progress through the events of meiosis and gamete formation. Meiosis-specific gene expression is tightly controlled by a balance of positive and negative regulatory factors that respond to a plethora of signaling pathways. The budding yeast Saccharomyces cerevisiae has proven to be an outstanding model for the dissection of gametogenesis owing to the sophisticated genetic manipulations that can be performed with the cells. It is possible to use a variety selection and screening methods to identify genes and their functions. High-throughput screening technology has been developed to allow an array of all viable yeast gene deletion mutants to be screened for phenotypes and for regulators of gene expression. This chapter describes a protocol that has been used to screen a library of homozygous diploid yeast deletion strains to identify regulators of the meiosis-specific IME1 gene.
Zhao, Mei-Xia; Zhang, Biao; Liu, Sheng-Yi; Ma, Jian-Xin
Transposons or transposable elements (TEs) are ubiquitous and most abundant DNA components in higher eukaryotes. Recent sequencing of the Brassica rapa and B. oleracea genomes revealed that the amplification of TEs is one of the main factors inducing the difference in genome size. However, the expressions of TEs and the TE effects on gene regulation and functions of these two Brassica diploid species were unclear. Here, we analyzed the RNA sequencing data of leaves, roots, and stems from B. rapa and B. oleracea. Our data showed that overall TEs in either genome expressed at very low levels, and the expression levels of different TE categories and families varied among different organs. Moreover, even for the same TE category or family, the expression activities were distinct between the two Brassica diploids. Forty-one and nine LTR retrotransposons with the transcripts that read into their adjacent sequences have the distances shorter than 2 kb and 100 bp compared to the downstream genes. These LTR retrotransposon readout transcriptions may produce sense or antisense transcripts of nearby genes, with the effects on activating or silencing corresponding genes. Meanwhile, intact LTRs were detected at stronger readout activities than solo LTRs. Of the TEs inserted into genes, the frequencies were ob-served at a higher level in B. rapa than in B. oleracea. In addition, DNA transposons were prone to insert or retain in the intronic regions of genes in either Brassica genomes. These results revealed that the TEs may have potential effects on regulating protein coding genes.
Doevendans, P A; Jukema, W; Spiering, W; Defesche, J C; Kastelein, J J
is available. Recent studies suggest that such treatment should be genotype specific, as the genetic makeup can determine the outcome of a pharmacological intervention (pharmacogenetics). Once the trigger for atherosclerosis has initiated disease development, various genes are activated or silenced and contribute to lesion progression. Every stage of lesion development depends on a different gene expression programme (genomics). In this review paper an introduction is provided into genetics, pharmacogenetics and gene expression with respect to atherosclerotic disease.
Minemura, Shoko; Tanaka, Takeshi; Arai, Makoto; Okimoto, Kenichiro; Oyamada, Arata; Saito, Keiko; Maruoka, Daisuke; Matsumura, Tomoaki; Nakagawa, Tomoo; Katsuno, Tatsuro; Kishimoto, Takashi; Yokosuka, Osamu
Laterally spreading tumors (LSTs) are generally defined as lesions >10 mm in diameter, are characterized by lateral expansion along the luminal wall with a low vertical axis. In contrast to other forms of tumor, LSTs are generally considered to have a superficial growth pattern and the potential for malignancy. We focused on this morphological character of LSTs, and analyzed the gene expression profile of LSTs. The expression of 168 genes in 41 colorectal tumor samples (17 LST-adenoma, 12 LST-carcinoma, 12 Ip [pedunculated type of the Paris classification)-adenoma, all of which were 10 mm or more in diameter] was analyzed by PCR array. Based on the results, we investigated the expression levels of genes up-regulated in LST-adenoma, compared to Ip-adenoma, by hierarchical and K-means clustering. To confirm the results of the array analysis, using an additional 60 samples (38 LST-adenoma, 22 Ip-adenoma), we determined the localization of the gene product by immunohistochemical staining. The expression of 129 genes differed in colorectal tumors from normal mucosa by PCR array analysis. As a result of K-means clustering, the expression levels of five genes, AKT1, BCL2L1, ERBB2, MTA2 and TNFRSF25, were found to be significantly up-regulated (p < 0.05) in LST-adenoma, compared to Ip-adenoma. Immunohistochemical analysis showed that the BCL2L1 protein was significantly and meaningfully up-regulated in LST-adenoma compared to Ip-adenoma (p = 0.010). With respect to apoptosis status in LST-Adenoma, it assumes that BCL2L1 is anti-apoptotic protein, the samples such as BCL2L1 positive and TUNEL negative, or BCL2L1 negative and TUNEL positive are consistent with the assumption. 63.2 % LST-adenoma samples were consistent with the assumption. LSTs have an unusual profile of gene expression compared to other tumors and BCL2L1 might be concerned in the organization of LSTs.
Gojkovic, Zoran; Sandrini, Michael; Piskur, Jure
activity was used to clone analogous genes from different eukaryotes. Putative PYD3 sequences from the yeast S. kluyveri, the slime mold Dictyostelium discoideum, and the fruit fly Drosophila melanogaster complemented the pyd3 defect. When the S. kluyveri PYD3 gene was expressed in S. cerevisiae, which has...
Jiang, Jinjin; Wang, Yue; Zhu, Bao; Fang, Tingting; Fang, Yujie; Wang, Youping
Brassica includes many successfully cultivated crop species of polyploid origin, either by ancestral genome triplication or by hybridization between two diploid progenitors, displaying complex repetitive sequences and transposons. The U's triangle, which consists of three diploids and three amphidiploids, is optimal for the analysis of complicated genomes after polyploidization. Next-generation sequencing enables the transcriptome profiling of polyploids on a global scale. We examined the gene expression patterns of three diploids (Brassica rapa, B. nigra, and B. oleracea) and three amphidiploids (B. napus, B. juncea, and B. carinata) via digital gene expression analysis. In total, the libraries generated between 5.7 and 6.1 million raw reads, and the clean tags of each library were mapped to 18547-21995 genes of B. rapa genome. The unambiguous tag-mapped genes in the libraries were compared. Moreover, the majority of differentially expressed genes (DEGs) were explored among diploids as well as between diploids and amphidiploids. Gene ontological analysis was performed to functionally categorize these DEGs into different classes. The Kyoto Encyclopedia of Genes and Genomes analysis was performed to assign these DEGs into approximately 120 pathways, among which the metabolic pathway, biosynthesis of secondary metabolites, and peroxisomal pathway were enriched. The non-additive genes in Brassica amphidiploids were analyzed, and the results indicated that orthologous genes in polyploids are frequently expressed in a non-additive pattern. Methyltransferase genes showed differential expression pattern in Brassica species. Our results provided an understanding of the transcriptome complexity of natural Brassica species. The gene expression changes in diploids and allopolyploids may help elucidate the morphological and physiological differences among Brassica species.
Romier, Christophe; Ben Jelloul, Marouane; Albeck, Shira; Buchwald, Gretel; Busso, Didier; Celie, Patrick H N; Christodoulou, Evangelos; De Marco, Valeria; van Gerwen, Suzan; Knipscheer, Puck; Lebbink, Joyce H; Notenboom, Valerie; Poterszman, Arnaud; Rochel, Natacha; Cohen, Serge X; Unger, Tamar; Sussman, Joel L; Moras, Dino; Sixma, Titia K; Perrakis, Anastassis
Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells.
Johnston, Rachel A; Paxton, Kristina L; Moore, Frank R; Wayne, Robert K; Smith, Thomas B
The annual migration of a bird can involve thousands of kilometres of nonstop flight, requiring accurately timed seasonal changes in physiology and behaviour. Understanding the molecular mechanisms controlling this endogenous programme can provide functional and evolutionary insights into the circannual biological clock and the potential of migratory species to adapt to changing environments. Under naturally timed photoperiod conditions, we maintained captive Swainson's thrushes (Catharus ustulatus) and performed RNA sequencing (RNA-Seq) of the ventral hypothalamus and optic chiasma to evaluate transcriptome-wide gene expression changes of individuals in migratory condition. We found that 188 genes were differentially expressed in relation to migratory state, 86% of which have not been previously linked to avian migration. Focal hub genes were identified that are candidate variables responsible for the occurrence of migration (e.g. CRABP1). Numerous genes involved in cell adhesion, proliferation and motility were differentially expressed (including RHOJ, PAK1 and TLN1), suggesting that migration-related changes are regulated by seasonal neural plasticity. © 2016 John Wiley & Sons Ltd.
Janiak, Agnieszka; Kwaśniewski, Mirosław; Szarejko, Iwona
Stress signalling and regulatory networks controlling expression of target genes are the basis of plant response to drought. Roots are the first organs exposed to water deficiency in the soil and are the place of drought sensing. Signalling cascades transfer chemical signals toward the shoot and initiate molecular responses that lead to the biochemical and morphological changes that allow plants to be protected against water loss and to tolerate stress conditions. Here, we present an overview of signalling network and gene expression regulation pathways that are actively induced in roots under drought stress. In particular, the role of several transcription factor (TF) families, including DREB, AP2/ERF, NAC, bZIP, MYC, CAMTA, Alfin-like and Q-type ZFP, in the regulation of root response to drought are highlighted. The information provided includes available data on mutual interactions between these TFs together with their regulation by plant hormones and other signalling molecules. The most significant downstream target genes and molecular processes that are controlled by the regulatory factors are given. These data are also coupled with information about the influence of the described regulatory networks on root traits and root development which may translate to enhanced drought tolerance. This is the first literature survey demonstrating the gene expression regulatory machinery that is induced by drought stress, presented from the perspective of roots. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: firstname.lastname@example.org.
Full Text Available Abstract Background Sinonasal adenocarcinomas are uncommon tumors which develop in the ethmoid sinus after exposure to wood dust. Although the etiology of these tumors is well defined, very little is known about their molecular basis and no diagnostic tool exists for their early detection in high-risk workers. Methods To identify genes involved in this disease, we performed gene expression profiling using cancer-dedicated microarrays, on nine matched samples of sinonasal adenocarcinomas and non-tumor sinusal tissue. Microarray results were validated by quantitative RT-PCR and immunohistochemistry on two additional sets of tumors. Results Among the genes with significant differential expression we selected LGALS4, ACS5, CLU, SRI and CCT5 for further exploration. The overexpression of LGALS4, ACS5, SRI, CCT5 and the downregulation of CLU were confirmed by quantitative RT-PCR. Immunohistochemistry was performed for LGALS4 (Galectin 4, ACS5 (Acyl-CoA synthetase and CLU (Clusterin proteins: LGALS4 was highly up-regulated, particularly in the most differentiated tumors, while CLU was lost in all tumors. The expression of ACS5, was more heterogeneous and no correlation was observed with the tumor type. Conclusion Within our microarray study in sinonasal adenocarcinoma we identified two proteins, LGALS4 and CLU, that were significantly differentially expressed in tumors compared to normal tissue. A further evaluation on a new set of tissues, including precancerous stages and low grade tumors, is necessary to evaluate the possibility of using them as diagnostic markers.
Tabaka, Marcin; Kalwarczyk, Tomasz; Szymanski, Jedrzej; Hou, Sen; Hołyst, Robert
We discuss a quantitative influence of macromolecular crowding on biological processes: motion, bimolecular reactions, and gene expression in prokaryotic and eukaryotic cells. We present scaling laws relating diffusion coefficient of an object moving in a cytoplasm of cells to a size of this object and degree of crowding. Such description leads to the notion of the length scale dependent viscosity characteristic for all living cells. We present an application of the length-scale dependent viscosity model to the description of motion in the cytoplasm of both eukaryotic and prokaryotic living cells. We compare the model with all recent data on diffusion of nanoscopic objects in HeLa, and E. coli cells. Additionally a description of the mobility of molecules in cell nucleus is presented. Finally we discuss the influence of crowding on the bimolecular association rates and gene expression in living cells.
Full Text Available Triacylglycerols (TAG are the major molecules of energy storage in eukaryotes. TAG are packed in subcellular structures called oil bodies or lipid droplets. Oleosins (OLE are the major proteins in plant oil bodies. Multiple isoforms of OLE are present in plants such as tung tree (Vernicia fordii, whose seeds are rich in novel TAG with a wide range of industrial applications. The objectives of this study were to identify OLE genes, classify OLE proteins and analyze OLE gene expression in tung trees. We identified five tung tree OLE genes coding for small hydrophobic proteins. Genome-wide phylogenetic analysis and multiple sequence alignment demonstrated that the five tung OLE genes represented the five OLE subfamilies and all contained the "proline knot" motif (PX5SPX3P shared among 65 OLE from 19 tree species, including the sequenced genomes of Prunus persica (peach, Populus trichocarpa (poplar, Ricinus communis (castor bean, Theobroma cacao (cacao and Vitis vinifera (grapevine. Tung OLE1, OLE2 and OLE3 belong to the S type and OLE4 and OLE5 belong to the SM type of Arabidopsis OLE. TaqMan and SYBR Green qPCR methods were used to study the differential expression of OLE genes in tung tree tissues. Expression results demonstrated that 1 All five OLE genes were expressed in developing tung seeds, leaves and flowers; 2 OLE mRNA levels were much higher in seeds than leaves or flowers; 3 OLE1, OLE2 and OLE3 genes were expressed in tung seeds at much higher levels than OLE4 and OLE5 genes; 4 OLE mRNA levels rapidly increased during seed development; and 5 OLE gene expression was well-coordinated with tung oil accumulation in the seeds. These results suggest that tung OLE genes 1-3 probably play major roles in tung oil accumulation and/or oil body development. Therefore, they might be preferred targets for tung oil engineering in transgenic plants.
Adomas, Aleksandra B; Lopez-Giraldez, Francesc; Clark, Travis A; Wang, Zheng; Townsend, Jeffrey P
Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays. We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA. Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression. Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and precise assay of the transcribed sequences within the genome.
Full Text Available Glioblastomas are highly aggressive brain tumors of adults with poor clinical outcome. Despite a broad range of new and more specific treatment strategies, therapy of glioblastomas remains challenging and tumors relapse in all cases. Recent work demonstrated that the posttranslational hypusine modification of the eukaryotic initiation factor 5A (eIF-5A is a crucial regulator of cell proliferation, differentiation and an important factor in tumor formation, progression and maintenance. Here we report that eIF-5A as well as the hypusine-forming enzymes deoxyhypusine synthase (DHS and deoxyhypusine hydroxylase (DOHH are highly overexpressed in glioblastoma patient samples. Importantly, targeting eIF-5A and its hypusine modification with GC7, a specific DHS-inhibitor, showed a strong antiproliferative effect in glioblastoma cell lines in vitro, while normal human astrocytes were not affected. Furthermore, we identified p53 dependent premature senescence, a permanent cell cycle arrest, as the primary outcome in U87-MG cells after treatment with GC7. Strikingly, combined treatment with clinically relevant alkylating agents and GC7 had an additive antiproliferative effect in glioblastoma cell lines. In addition, stable knockdown of eIF-5A and DHS by short hairpin RNA (shRNA could mimic the antiproliferative effects of GC7. These findings suggest that pharmacological inhibition of eIF-5A may represent a novel concept to treat glioblastomas and may help to substantially improve the clinical course of this tumor entity.
Hustinx, Steven R; Cao, Dengfeng; Maitra, Anirban
Serial analysis of gene expression (SAGE) is a powerful tool for the discovery of novel tumor markers. The publicly available online SAGE libraries of normal and neoplastic tissues (http://www.ncbi.nlm.nih.gov/SAGE/) have recently been expanded; in addition, a more complete annotation of the human...... 16 additional differentially expressed genes. The differential expression of seven genes, involved in multiple cellular processes such as signal transduction (MIC-1), differentiation (DMBT1 and Neugrin), immune response (CD74), inflammation (CXCL2), cell cycle (CEB1) and enzymatic activity...... of this program. Novel differentially expressed genes in a cancer type can be identified by revisiting updated and expanded SAGE databases. TAGmapper should prove to be a powerful tool for the discovery of novel tumor markers through assignment of uncharacterized SAGE tags....
de Vries, Ronald P.; van den Broeck, Hetty C.; Dekkers, Ester; Manzanares, Paloma; de Graaff, Leo H.; Visser, Jaap
A gene encoding a third α-galactosidase (AglB) from Aspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other α-galactosidases revealed that it belongs to a subfamily of α-galactosidases that also includes A. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic α-galactosidases. The expression of aglA, aglB, aglC, and lacA, the latter of which encodes an A. niger β-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins. PMID:10347026
Bailly, Julie; Fraissinet-Tachet, Laurence; Verner, Marie-Christine; Debaud, Jean-Claude; Lemaire, Marc; Wésolowski-Louvel, Micheline; Marmeisse, Roland
To appreciate the functional diversity of communities of soil eukaryotic micro-organisms we evaluated an experimental approach based on the construction and screening of a cDNA library using polyadenylated mRNA extracted from a forest soil. Such a library contains genes that are expressed by each of the different organisms forming the community and represents its metatranscriptome. The diversity of the organisms that contributed to this library was evaluated by sequencing a portion of the 18S rDNA gene amplified from either soil DNA or reverse-transcribed RNA. More than 70% of the sequences were from fungi and unicellular eukaryotes (protists) while the other most represented group was the metazoa. Calculation of richness estimators suggested that more than 180 species could be present in the soil samples studied. Sequencing of 119 cDNA identified genes with no homologues in databases (32%) and genes coding proteins involved in different biochemical and cellular processes. Surprisingly, the taxonomic distribution of the cDNA and of the 18S rDNA genes did not coincide, with a marked under-representation of the protists among the cDNA. Specific genes from such an environmental cDNA library could be isolated by expression in a heterologous microbial host, Saccharomyces cerevisiae. This is illustrated by the functional complementation of a histidine auxotrophic yeast mutant by two cDNA originating possibly from an ascomycete and a basidiomycete fungal species. Study of the metatranscriptome has the potential to uncover adaptations of whole microbial communities to local environmental conditions. It also gives access to an abundant source of genes of biotechnological interest.
Micklem David R
Full Text Available Abstract Background Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and post-transcriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter. Results By exploiting a new method of producing the retroviral genome in vitro it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods. We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both a conventional MMLV retroviral vector and a vector designed to allow in vitro transcription of the retroviral genome by T7 RNA polymerase. When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of the in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when used to infect target primary human cells led to very high GFP expression – up to 3.5 times greater than conventional retroviral LTR-driven expression. Conclusion Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA
Zhang, Lin; Weng, Wenya; Guo, Jinhu
The circadian clock is essential in almost all living organisms to synchronise biochemical, metabolic, physiological and behavioural cycles to daily changing environmental factors. In a highly conserved fashion, the circadian clock is primarily controlled by multiple positive and negative molecular circuitries that control gene expression. More recently, research in Neurospora and other eukaryotes has uncovered the involvement of additional regulatory components that operate at the posttranslational level to fine tune the circadian system. Though it remains poorly understood, a growing body of evidence has shown that posttranscriptional regulation controls the expression of both circadian oscillator and output gene transcripts at a number of different steps. This regulation is crucial for driving and maintaining robust circadian rhythms. Here we review recent advances in circadian rhythm research at the RNA level. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Full Text Available Abstract Background The bacterium Pseudomonas aeruginosa is capable of three types of motilities: swimming, twitching and swarming. The latter is characterized by a fast and coordinated group movement over a semi-solid surface resulting from intercellular interactions and morphological differentiation. A striking feature of swarming motility is the complex fractal-like patterns displayed by migrating bacteria while they move away from their inoculation point. This type of group behaviour is still poorly understood and its characterization provides important information on bacterial structured communities such as biofilms. Using GeneChip® Affymetrix microarrays, we obtained the transcriptomic profiles of both bacterial populations located at the tip of migrating tendrils and swarm center of swarming colonies and compared these profiles to that of a bacterial control population grown on the same media but solidified to not allow swarming motility. Results Microarray raw data were corrected for background noise with the RMA algorithm and quantile normalized. Differentially expressed genes between the three conditions were selected using a threshold of 1.5 log2-fold, which gave a total of 378 selected genes (6.3% of the predicted open reading frames of strain PA14. Major shifts in gene expression patterns are observed in each growth conditions, highlighting the presence of distinct bacterial subpopulations within a swarming colony (tendril tips vs. swarm center. Unexpectedly, microarrays expression data reveal that a minority of genes are up-regulated in tendril tip populations. Among them, we found energy metabolism, ribosomal protein and transport of small molecules related genes. On the other hand, many well-known virulence factors genes were globally repressed in tendril tip cells. Swarm center cells are distinct and appear to be under oxidative and copper stress responses. Conclusions Results reported in this study show that, as opposed to
Hovav, Ran; Udall, Joshua A; Hovav, Einat; Rapp, Ryan; Flagel, Lex; Wendel, Jonathan F
Multicellular eukaryotes contain a diversity of cell types, presumably differing from one another in the suite of genes expressed during development. At present, little is known about the proportion of the genome transcribed in most cell types, nor the degree to which global patterns of expression change during cellular differentiation. To address these questions in a model plant system, we studied the unique and highly exaggerated single-celled, epidermal seed trichomes ("cotton") of cultivated cotton (Gossypium hirsutum). By taking advantage of advances in expression profiling and microarray technology, we evaluated the transcriptome of cotton fibers across a developmental time-course, from a few days post-anthesis through primary and secondary wall synthesis stages. Comparisons of gene expression in populations of developing cotton fiber cells to genetically complex reference samples derived from 6 different cotton organs demonstrated that a remarkably high proportion of the cotton genome is transcribed, with 75-94% of the total genome transcribed at each stage. Compared to the reference samples, more than half of all genes were up-regulated during at least one stage of fiber development. These genes were clustered into seven groups of expression profiles that provided new insight into biological processes governing fiber development. Genes implicated in vesicle coating and trafficking were found to be overexpressed throughout all stages of fiber development studied, indicating their important role in maintaining rapid growth of this unique plant cell.
Wu, Gene PK; Chan, Keith CC; Wong, Andrew KC
Background Discovering patterns from gene expression levels is regarded as a classification problem when tissue classes of the samples are given and solved as a discrete-data problem by discretizing the expression levels of each gene into intervals maximizing the interdependence between that gene and the class labels. However, when class information is unavailable, discovering gene expression patterns becomes difficult. Methods For a gene pool with large number of genes, we first cluster the ...
Manijak, Mieszko P.; Nielsen, Henrik Bjørn
BACKGROUND: Although, systematic analysis of gene annotation is a powerful tool for interpreting gene expression data, it sometimes is blurred by incomplete gene annotation, missing expression response of key genes and secondary gene expression responses. These shortcomings may be partially...... circumvented by instead matching gene expression signatures to signatures of other experiments. FINDINGS: To facilitate this we present the Functional Association Response by Overlap (FARO) server, that match input signatures to a compendium of 242 gene expression signatures, extracted from more than 1700...... Arabidopsis microarray experiments. CONCLUSIONS: Hereby we present a publicly available tool for robust characterization of Arabidopsis gene expression experiments which can point to similar experimental factors in other experiments. The server is available at http://www.cbs.dtu.dk/services/faro/....
Manijak, Mieszko P; Nielsen, Henrik B
Although, systematic analysis of gene annotation is a powerful tool for interpreting gene expression data, it sometimes is blurred by incomplete gene annotation, missing expression response of key genes and secondary gene expression responses. These shortcomings may be partially circumvented by instead matching gene expression signatures to signatures of other experiments. To facilitate this we present the Functional Association Response by Overlap (FARO) server, that match input signatures to a compendium of 242 gene expression signatures, extracted from more than 1700 Arabidopsis microarray experiments. Hereby we present a publicly available tool for robust characterization of Arabidopsis gene expression experiments which can point to similar experimental factors in other experiments. The server is available at http://www.cbs.dtu.dk/services/faro/.
Hervé, Cécile; Siméon, Amandine; Jam, Murielle; Cassin, Andrew; Johnson, Kim L; Salmeán, Armando A; Willats, William G T; Doblin, Monika S; Bacic, Antony; Kloareg, Bernard
Arabinogalactan proteins (AGPs) are highly glycosylated, hydroxyproline-rich proteins found at the cell surface of plants, where they play key roles in developmental processes. Brown algae are marine, multicellular, photosynthetic eukaryotes. They belong to the phylum Stramenopiles, which is unrelated to land plants and green algae (Chloroplastida). Brown algae share common evolutionary features with other multicellular organisms, including a carbohydrate-rich cell wall. They differ markedly from plants in their cell wall composition, and AGPs have not been reported in brown algae. Here we investigated the presence of chimeric AGP-like core proteins in this lineage. We report that the genome sequence of the brown algal model Ectocarpus siliculosus encodes AGP protein backbone motifs, in a gene context that differs considerably from what is known in land plants. We showed the occurrence of AGP glycan epitopes in a range of brown algal cell wall extracts. We demonstrated that these chimeric AGP-like core proteins are developmentally regulated in embryos of the order Fucales and showed that AGP loss of function seriously impairs the course of early embryogenesis. Our findings shine a new light on the role of AGPs in cell wall sensing and raise questions about the origin and evolution of AGPs in eukaryotes. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.
Full Text Available Bronchiolitis obliterans syndrome (BOS, the main manifestation of chronic lung allograft dysfunction, leads to poor long-term survival after lung transplantation. Identifying predictors of BOS is essential to prevent the progression of dysfunction before irreversible damage occurs. By using a large set of 107 samples from lung recipients, we performed microarray gene expression profiling of whole blood to identify early biomarkers of BOS, including samples from 49 patients with stable function for at least 3 years, 32 samples collected at least 6 months before BOS diagnosis (prediction group, and 26 samples at or after BOS diagnosis (diagnosis group. An independent set from 25 lung recipients was used for validation by quantitative PCR (13 stables, 11 in the prediction group, and 8 in the diagnosis group. We identified 50 transcripts differentially expressed between stable and BOS recipients. Three genes, namely POU class 2 associating factor 1 (POU2AF1, T-cell leukemia/lymphoma protein 1A (TCL1A, and B cell lymphocyte kinase, were validated as predictive biomarkers of BOS more than 6 months before diagnosis, with areas under the curve of 0.83, 0.77, and 0.78 respectively. These genes allow stratification based on BOS risk (log-rank test p < 0.01 and are not associated with time posttransplantation. This is the first published large-scale gene expression analysis of blood after lung transplantation. The three-gene blood signature could provide clinicians with new tools to improve follow-up and adapt treatment of patients likely to develop BOS.
Martinez, Salette; Yang, Xinhang; Bennett, Brian; Holz, Richard C
Nitrile hydratase (NHase), an industrially important enzyme that catalyzes the hydration of nitriles to their corresponding amides, has only been characterized from prokaryotic microbes. The putative NHase from the eukaryotic unicellular choanoflagellate organism Monosiga brevicollis (MbNHase) was heterologously expressed in Escherichia coli. The resulting enzyme expressed as a single polypeptide with fused α- and β-subunits linked by a seventeen-histidine region. Size-exclusion chromatography indicated that MbNHase exists primarily as an (αβ)2 homodimer in solution, analogous to the α2β2 homotetramer architecture observed for prokaryotic NHases. The NHase enzyme contained its full complement of Co(III) and was fully functional without the co-expression of an activator protein or E. coli GroES/EL molecular chaperones. The homology model of MbNHase was developed identifying Cys400, Cys403, and Cys405 as active site ligands. The results presented here provide the first experimental data for a mature and active eukaryotic NHase with fused subunits. Since this new member of the NHase family is expressed from a single gene without the requirement of an activator protein, it represents an alternative biocatalyst for industrial syntheses of important amide compounds. Copyright Â© 2016 Elsevier B.V. All rights reserved.
Full Text Available Keratoconus is a progressive corneal thinning disease associated with significant tissue remodeling activities and activation of a variety of signaling networks. However, it is not understood how differential gene and protein expression direct function in keratoconus corneas to drive the underlying pathology, ectasia. Research in the field has focused on discovering differentially expressed genes and proteins and quantifying their levels and activities in keratoconus patient samples. In this study, both microarray analysis of total ribonucleic acid (RNA and whole proteome analyses are carried out using corneal epithelium and tears from keratoconus patients and compared to healthy controls. A number of structural proteins, signaling molecules, cytokines, proteases, and enzymes have been found to be deregulated in keratoconus corneas. Together, the data provide clues to the complex process of corneal degradation which suggest novel ways to clinically diagnose and manage the disease. This review will focus on discussing these recent advances in the knowledge of keratoconus biology from a gene expression and function point-of-view.
Stein, Wilfred D; Litman, Thomas; Fojo, Tito
It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies that comp......It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies...... that compare cells grown in suspension to similar cells grown attached to one another as aggregates have suggested that it is adhesion to the extracellular matrix of the basal membrane that confers resistance to apoptosis and, hence, resistance to cytotoxins. The genes whose expression correlates with poor...... survival might, therefore, act through such a matrix-to-cell suppression of apoptosis. Indeed, correlative mining of gene expression and patient survival databases suggests that poor survival in patients with metastatic cancer correlates highly with tumor expression of a common theme: the genes involved...
Ondrey, Jakob M; Visick, Karen L
The marine bacterium Vibrio fischeri serves as a model organism for a variety of natural phenomena, including symbiotic host colonization. The ease with which the V. fischeri genome can be manipulated contributes greatly to our ability to identify the factors involved in these phenomena. Here, we have adapted genetic tools for use in V. fischeri to promote our ability to conditionally control the expression of genes of interest. Specifically, we modified the commonly used mini-Tn5 transposon to contain an outward-facing, LacI-repressible/IPTG-inducible promoter, and inserted the lacI gene into the V. fischeri chromosome. Used together, these tools permit the identification and induction of genes that control specific phenotypes. To validate this approach, we identified IPTG-controllable motility mutants. We anticipate that the ability to randomly insert an inducible promoter into the genome of V. fischeri will advance our understanding of various aspects of the physiology of this microbe.
Full Text Available Increasing consumer knowledge of the link between diet and health has raised the demand for high quality food. Meat and meat products may be considered as irreplaceable in human nutrition. Breeding livestock to higher content of lean meat and the use of modern hybrids entails problems with the quality of meat. Analysing of livestock genomes could get us a great deal of important information, which may significantly affect the improvement process. Domestic animals are invaluable resources for study of the molecular architecture of complex traits. Although the mapping of quantitative trait loci (QTL responsible for economically important traits in domestic animals has achieved remarkable results in recent decades, not all of the genetic variation in the complex traits has been captured because of the low density of markers used in QTL mapping studies. The genome wide association study (GWAS, which utilizes high-density single-nucleotide polymorphism (SNP, provides a new way to tackle this issue. New technologies now allow producing microarrays containing thousands of hybridization probes on a single membrane or other solid support. We used microarray analysis to study gene expression in rabbit muscle during different developmental age stages. The outputs from GeneSpring GX sotware are presented in this work. After the evaluation of gene expression in rabbits, will be selected genes of interest in relation to meat quality parameters and will be further analyzed by the available methods of molecular biology and genetics.
Microarray profiling of chemical-induced effects is being increasingly used in medium and high-throughput formats. In this study, we describe computational methods to identify molecular targets from whole-genome microarray data using as an example the estrogen receptor α (ERα), often modulated by potential endocrine disrupting chemicals. ERα biomarker genes were identified by their consistent expression after exposure to 7 structurally-diverse ERα agonists and 3 ERα antagonists in ERα-positive MCF-7 cells. Most of the biomarker genes were shown to be directly regulated by ERα as determined by ESR1 gene knockdown using siRNA as well as through ChIP-Seq analysis of ERα-DNA interactions. The biomarker was evaluated as a predictive tool using the fold-change rank-based Running Fisher algorithm by comparison to annotated gene expression data sets from experiments using MCF-7 cells, including those evaluating the transcriptional effects of hormones and chemicals. Using 141 comparisons from chemical- and hormone-treated cells, the biomarker gave a balanced accuracy for prediction of ERα activation or suppression of 94% and 93%, respectively. The biomarker was able to correctly classify 18 out of 21 (86%) ER reference chemicals including “very weak” agonists. Importantly, the biomarker predictions accurately replicated predictions based on 18 in vitro high-throughput screening assays that queried different steps in ERα signaling. For 114 chemicals,
Full Text Available Abstract Background The aim of the present study was to analyze the expression of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1 in nasopharyngeal carcinoma (NPC and its correlation with clinicopathologic features, including patients' survival time. Methods Using real-time PCR, we detected the expression of EIF4G1 in normal nasopharyngeal tissues, immortalized nasopharyngeal epithelial cell lines NP69, NPC tissues and cell lines. EIF4G1 protein expression in NPC tissues was examined using immunohistochemistry. Survival analysis was performed using Kaplan-Meier method. The effect of EIF4G1 on cell invasion and tumorigenesis were investigated. Results The expression levels of EIF4G1 mRNA were significantly greater in NPC tissues and cell lines than those in the normal nasopharyngeal tissues and NP69 cells (P EIF4G1 protein was higher in NPC tissues than that in the nasopharyngeal tissues (P EIF4G1 protein in tumors were positively correlated with tumor T classification (P = 0.039, lymph node involvement (N classification, P = 0.008, and the clinical stages (P = 0.003 of NPC patients. Patients with higher EIF4G1 expression had shorter overall survival time (P = 0.019. Multivariate analysis showed that EIF4G1 expression was an independent prognostic indicator for the overall survival of NPC patients. Using shRNA to knock down the expression of EIF4G1 not only markedly inhibited cell cycle progression, proliferation, migration, invasion, and colony formation, but also dramatically suppressed in vivo xenograft tumor growth. Conclusion Our data suggest that EIF4G1 can serve as a biomarker for the prognosis of NPC patients.
Salem, Tamer Z. [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza 12619 (Egypt); Division of Biomedical Sciences, Zewail University, Zewail City of Science and Technology, Giza 12588 (Egypt); Zhang, Fengrui [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Thiem, Suzanne M., E-mail: email@example.com [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States)
Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.
Sederoff, Heike; Heber, Steffen; Howard, Brian; Myburg-Nichols, Henrietta; Hammond, Rebecca; Salinas-Mondragon, Raul; Brown, Christopher S.
Plants sense changes in their orientation towards the vector of gravity and respond with directional growth. Several metabolites in the signal transduction cascade have been identified. However, very little is known about the interaction between these sensing and signal transduction events and even less is known about their role in the differential growth response. Gravity induced changes in transcript abundance have been identified in Arabidopsis whole seedlings and root apices (Moseyko et al. 2002; Kimbrough et al. 2004). Gravity induced transcript abundance changes can be observed within less than 1 min after stimulation (Salinas-Mondragon et al. 2005). Gene expression however requires not only transcription but also translation of the mRNA. Translation can only occur when mRNA is associated with ribosomes, even though not all mRNA associated with ribosomes is actively translated. To approximate translational capacity we quantified whole genome transcript abundances in corn stem pulvini during the first hour after gravity stimulation in total and poly-ribosomal fractions. As in Arabidopsis root apices, transcript abundances of several clusters of genes responded to gravity stimulation. The vast majority of these transcripts were also found to associate with polyribosomes in the same temporal and quantitative pattern. These genes are transcriptionally regulated by gravity stimulation, but do not exhibit translational regulation. However, a small group of genes showed increased transcriptional regulation after gravity stimulation, but no association with polysomes. These transcripts likely are translationally repressed. The mechanism of translational repression for these transcripts is unknown. Based on the hypothesis that the genes essential for gravitropic responses should be expressed in most or all species, we compared the temporal gravity induced expression pattern of all orthologs identified between maize and Arabidopsis. A small group of genes showed high
Bowden Nikola A
Full Text Available Abstract Xeroderma pigmentosum (XP is a rare recessive disorder that is characterized by extreme sensitivity to UV light. UV light exposure results in the formation of DNA damage such as cyclobutane dimers and (6-4 photoproducts. Nucleotide excision repair (NER orchestrates the removal of cyclobutane dimers and (6-4 photoproducts as well as some forms of bulky chemical DNA adducts. The disease XP is comprised of 7 complementation groups (XP-A to XP-G, which represent functional deficiencies in seven different genes, all of which are believed to be involved in NER. The main clinical feature of XP is various forms of skin cancers; however, neurological degeneration is present in XPA, XPB, XPD and XPG complementation groups. The relationship between NER and other types of DNA repair processes is now becoming evident but the exact relationships between the different complementation groups remains to be precisely determined. Using gene expression analysis we have identified similarities and differences after UV light exposure between the complementation groups XP-A, XP-C, XP-D, XP-E, XP-F, XP-G and an unaffected control. The results reveal that there is a graded change in gene expression patterns between the mildest, most similar to the control response (XP-E and the severest form (XP-A of the disease, with the exception of XP-D. Distinct differences between the complementation groups with neurological symptoms (XP-A, XP-D and XP-G and without (XP-C, XP-E and XP-F were also identified. Therefore, this analysis has revealed distinct gene expression profiles for the XP complementation groups and the first step towards understanding the neurological symptoms of XP.
Modern taxonomy is largely relying on DNA barcoding, a nucleotide sequence-based approach that provides automated species identification using short orthologous DNA regions, mainly of organellar origin when applied to multicellular Eukaryotic species. Target DNA loci have been selected that contain a minimal amount of nucleotide sequence variation within species while diverging among species. This strategy is quite effective for the identification of vertebrates and other animal lineages but poses a problem in plants where different combinations of two or three loci are constantly used. Even so, species discrimination in such plant categories as ornamentals and herbals remain problematic as well as the confident identification of subspecies, ecotypes, and closely related or recently evolved species. All these limitations may be successfully solved by the application of a different strategy, based on the use of a multi-locus, ubiquitous, nuclear marker, that is tubulin. In fact, the tubulin-based polymorphism method can release specific genomic profiles to any plant species independently from its taxonomic group. This offers the rare possibility of an effective yet generic genomic fingerprint. In a more general context, the issue is raised about the possibility that approaches alternative to systematic DNA sequencing may still provide useful and simple solutions.
Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan
Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...
Lee, Jae Myung; Lee, Junhyeong; Kim, Taesung; Lee, Sung Kuk
We present a light-switchable gene expression system for both inducible and switchable control of gene expression at a single cell level in Escherichia coli using a previously constructed light-sensing system. The λ...
Kaushik, N; Fear, D; Richards, S C M; McDermott, C R; Nuwaysir, E F; Kellam, P; Harrison, T J; Wilkinson, R J; Tyrrell, D A J; Holgate, S T; Kerr, J R
Chronic fatigue syndrome (CFS) is a multisystem disease, the pathogenesis of which remains undetermined. To test the hypothesis that there are reproducible abnormalities of gene expression in patients with CFS compared with normal healthy persons. To gain further insight into the pathogenesis of this disease, gene expression was analysed in peripheral blood mononuclear cells from 25 patients with CFS diagnosed according to the Centers for Disease Control criteria and 25 normal blood donors matched for age, sex, and geographical location, using a single colour microarray representing 9522 human genes. After normalisation, average difference values for each gene were compared between test and control groups using a cutoff fold difference of expression > or = 1.5 and a p value of 0.001. Genes showing differential expression were further analysed using Taqman real time polymerase chain reaction (PCR) in fresh samples. Analysis of microarray data revealed differential expression of 35 genes. Real time PCR confirmed differential expression in the same direction as array results for 16 of these genes, 15 of which were upregulated (ABCD4, PRKCL1, MRPL23, CD2BP2, GSN, NTE, POLR2G, PEX16, EIF2B4, EIF4G1, ANAPC11, PDCD2, KHSRP, BRMS1, and GABARAPL1) and one of which was downregulated (IL-10RA). This profile suggests T cell activation and perturbation of neuronal and mitochondrial function. Upregulation of neuropathy target esterase and eukaryotic translation initiation factor 4G1 may suggest links with organophosphate exposure and virus infection, respectively. These results suggest that patients with CFS have reproducible alterations in gene regulation.
Volpert, Marianna; Mangum, Jonathan E.; Jamsai, Duangporn; D'Sylva, Rebecca; O'Bryan, Moira K.; McIntyre, Peter
While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.
Preiss, Thomas; W Hentze, Matthias
The final assembly of the protein synthesis machinery occurs during translation initiation. This delicate process involves both ends of eukaryotic messenger RNAs as well as multiple sequential protein-RNA and protein-protein interactions. As is expected from its critical position in the gene expression pathway between the transcriptome and the proteome, translation initiation is a selective and highly regulated process. This synopsis summarises the current status of the field and identifies intriguing open questions. Copyright 2003 Wiley Periodicals, Inc.
Tan, Fen-Lai; Moravec, Christine S.; Li, Jianbo; Apperson-Hansen, Carolyn; McCarthy, Patrick M.; Young, James B.; Bond, Meredith
Multiple pathways are responsible for transducing mechanical and hormonal stimuli into changes in gene expression during heart failure. In this study our goals were (i) to develop a sound statistical method to establish a comprehensive cutoff point for identification of differentially expressed genes, (ii) to identify a gene expression fingerprint for heart failure, (iii) to attempt to distinguish different etiologies of heart failure by their gene expression fingerprint, and (iv) to identify...
Wechter, W Patrick; Levi, Amnon; Harris, Karen R; Davis, Angela R; Fei, Zhangjun; Katzir, Nurit; Giovannoni, James J; Salman-Minkov, Ayelet; Hernandez, Alvaro; Thimmapuram, Jyothi; Tadmor, Yaakov; Portnoy, Vitaly; Trebitsh, Tova
Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR. High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon genotype with a similar phenotype, i.e. seeded
Full Text Available Abstract Background Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb. Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR. Results High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon
Founds, Sandra A; Terhorst, Lauren A; Conrad, Kirk P; Hogge, W Allen; Jeyabalan, Arun; Conley, Yvette P
The goal of this study was to further validate eight candidate genes identified in a microarray analysis of first trimester placentas in preeclampsia. Surplus chorionic villus sampling (CVS) specimens of 4 women subsequently diagnosed with preeclampsia (PE) and 8 control women (C) without preeclampsia analyzed previously by microarray and 24 independent additional control samples (AS) were submitted for confirmatory studies by quantitative real-time polymerase chain reaction (qRT-PCR). Downregulation was significant in FSTL3 in PE as compared to C and AS (p = .04). PAEP was downregulated, but the difference was only significant between C and AS (p = .002) rather than between PE and either of the control groups. Expression levels for CFH, EPAS1, IGFBP1, MMP12, and SEMA3C were not statistically different among groups, but trends were consistent with microarray results; there was no anti-correlation. S100A8 was not measurable in all samples, probably because different probes and primers were needed. This study corroborates reduced FSTL3 expression in the first trimester of preeclampsia. Nonsignificant trends in the other genes may require follow-up in studies powered for medium or medium/large effect sizes. qRT-PCR verification of the prior microarray of CVS may support the placental origins of preeclampsia hypothesis. Replication is needed for the candidate genes as potential biomarkers of susceptibility, early detection, and/or individualized care of maternal-infant preeclampsia.
Su, Shian; Law, Charity W; Ah-Cann, Casey; Asselin-Labat, Marie-Liesse; Blewitt, Marnie E; Ritchie, Matthew E
Leon, J.; Guerrero, I; Pellicer, A
We compared the expression of the ras gene family (H-ras, K-ras, and N-ras) in adult mouse tissues and during development. We found substantial variations in expression among different organs and in the amounts of the different transcripts originating from each gene, especially for the N-ras gene. The expression patterns were consistent with the reported preferential tissue activation of ras genes and suggested different cellular functions for each of the ras genes.
Full Text Available Organisms simplify the orchestration of gene expression by coregulating genes whose products function together in the cell. The use of clustering methods to obtain sets of coexpressed genes from expression arrays is very common; nevertheless there are no appropriate tools to study the expression networks among these sets of coexpressed genes. The aim of the developed tools is to allow studying the complex expression dependences that exist between sets of coexpressed genes. For this purpose, we start detecting the nonlinear expression relationships between pairs of genes, plus the coexpressed genes. Next, we form networks among sets of coexpressed genes that maintain nonlinear expression dependences between all of them. The expression relationship between the sets of coexpressed genes is defined by the expression relationship between the skeletons of these sets, where this skeleton represents the coexpressed genes with a well-defined nonlinear expression relationship with the skeleton of the other sets. As a result, we can study the nonlinear expression relationships between a target gene and other sets of coexpressed genes, or start the study from the skeleton of the sets, to study the complex relationships of activation and deactivation between the sets of coexpressed genes that carry out the different cellular processes present in the expression experiments.
Xie, Jun; Bentler, Peter M.
Covariance structure models are applied to gene expression data using a factor model, a path model, and their combination. The factor model is based on a few factors that capture most of the expression information. A common factor of a group of genes may represent a common protein factor for the transcript of the co-expressed genes, and hence, it…
Quinn, John P; Bubb, Vivien J
Endogenous mobile genetic elements can give rise to de novo germline or somatic mutations that can have dramatic consequences for genome regulation both local and possibly more globally based on the site of integration. However if we consider them as "normal genetic" components of the reference genome then they are likely to modify local chromatin structure which would have an effect on gene regulation irrelevant of their ability to further transpose. As such they can be treated as any other domain involved in a gene × environment interaction. Similarly their evolutionary appearance in the reference genome would supply a driver for species specific responses/traits. Our recent data would suggest the hominid specific subset of retrotransposons, SINE-VNTR-Alu (SVA), can function as transcriptional regulatory domains both in vivo and in vitro when analyzed in reporter gene constructs. Of particular interest in the SVA element, were the variable number tandem repeat (VNTR) domains which as their name suggests can be polymorphic. We and others have previously shown that VNTRs can be both differential regulators and biomarkers of disease based on the genotype of the repeat. Here, we provide an overview of why polymorphism in the SVA elements, in particular the VNTRs, could alter gene expression patterns that could be mechanistically associated with different traits in evolution or disease progression in humans.
Carinci, F; Piattelli, A; Guida, L; Perrotti, V; Laino, G; Oliva, A; Annunziata, M; Palmieri, A; Pezzetti, F
Emdogain (EMD) is a protein extract purified from porcine enamel and has been introduced in clinical practice to obtain periodontal regeneration. EMD is composed mainly of amelogenins (90%), while the remaining 10% is composed of non-amelogenin enamel matrix proteins such as enamelins, tuftelin, amelin and ameloblastin. Enamel matrix proteins seem to be involved in root formation. EMD has been reported to promote proliferation, migration, adhesion and differentiation of cells associated with healing periodontal tissues in vivo. How this protein acts on osteoblasts is poorly understood. We therefore attempted to address this question by using a microarray technique to identify genes that are differently regulated in osteoblasts exposed to enamel matrix proteins. By using DNA microarrays containing 20,000 genes, we identified several upregulated and downregulated genes in the osteoblast-like cell line (MG-63) cultured with enamel matrix proteins (Emd). The differentially expressed genes cover a broad range of functional activities: (i) signaling transduction, (ii) transcription, (iii) translation, (iv) cell cycle regulation, proliferation and apoptosis, (v) immune system, (vi) vesicular transport and lysosome activity, and (vii) cytoskeleton, cell adhesion and extracellular matrix production. The data reported are the first genome-wide scan of the effect of enamel matrix proteins on osteoblast-like cells. These results can contribute to our understanding of the molecular mechanisms of bone regeneration and as a model for comparing other materials with similar clinical effects.
Bagger, Frederik Otzen
Summary Acute Myeloid Leukaemia (AML) is an aggressive cancer of the bone marrow, affecting formation of blood cells during haematopoiesis. This thesis presents investigation of AML using mRNA gene expression profiles (GEP) of samples extracted from the bone marrow of healthy and diseased subjects......-based gene-lookup webservices, called HemaExplorer and BloodSpot. These web-services support the aim of making data and analysis of haematopoietic cells from mouse and human accessible for researchers without bioinformatics expertise. Finally, in order to aid the analysis of the very limited number...... of haematopoietic progenitor cells obtainable from bone marrow aspirations, this thesis presents a method developed to investigate transcription factor binding and histone modifications by ChIP-Seq using pico-scale amounts of DNA....
Mattick, J S
Around 98% of all transcriptional output in humans is non-coding RNA. RNA-mediated gene regulation is widespread in higher eukaryotes and complex genetic phenomena like RNA interference, co-suppression, transgene silencing, imprinting, methylation, and possibly position-effect variegation and transvection, all involve intersecting pathways based on or connected to RNA signaling. I suggest that the central dogma is incomplete, and that intronic and other non-coding RNAs have evolved to comprise a second tier of gene expression in eukaryotes, which enables the integration and networking of complex suites of gene activity. Although proteins are the fundamental effectors of cellular function, the basis of eukaryotic complexity and phenotypic variation may lie primarily in a control architecture composed of a highly parallel system of trans-acting RNAs that relay state information required for the coordination and modulation of gene expression, via chromatin remodeling, RNA-DNA, RNA-RNA and RNA-protein interactions. This system has interesting and perhaps informative analogies with small world networks and dataflow computing.
Full Text Available Abstract Background Although the sequence of events leading to wound repair has been described at the cellular and, to a limited extent, at the protein level this process has yet to be fully elucidated. Genome wide transcriptional analysis tools promise to further define the global picture of this complex progression of events. Study Design This study was part of a placebo-controlled double-blind clinical trial in which basal cell carcinomas were treated topically with an immunomodifier – toll-like receptor 7 agonist: imiquimod. The fourteen patients with basal cell carcinoma in the placebo arm of the trial received placebo treatment consisting solely of vehicle cream. A skin punch biopsy was obtained immediately before treatment and at the end of the placebo treatment (after 2, 4 or 8 days. 17.5K cDNA microarrays were utilized to profile the biopsy material. Results Four gene signatures whose expression changed relative to baseline (before wound induction by the pre-treatment biopsy were identified. The largest group was comprised predominantly of inflammatory genes whose expression was increased throughout the study. Two additional signatures were observed which included preferentially pro-inflammatory genes in the early post-treatment biopsies (2 days after pre-treatment biopsies and repair and angiogenesis genes in the later (4 to 8 days biopsies. The fourth and smallest set of genes was down-regulated throughout the study. Early in wound healing the expression of markers of both M1 and M2 macrophages were increased, but later M2 markers predominated. Conclusion The initial response to a cutaneous wound induces powerful transcriptional activation of pro-inflammatory stimuli which may alert the host defense. Subsequently and in the absence of infection, inflammation subsides and it is replaced by angiogenesis and remodeling. Understanding this transition which may be driven by a change from a mixed macrophage population to predominately M2
Full Text Available We tackle the problem of completing and inferring genetic networks under stationary conditions from static data, where network completion is to make the minimum amount of modifications to an initial network so that the completed network is most consistent with the expression data in which addition of edges and deletion of edges are basic modification operations. For this problem, we present a new method for network completion using dynamic programming and least-squares fitting. This method can find an optimal solution in polynomial time if the maximum indegree of the network is bounded by a constant. We evaluate the effectiveness of our method through computational experiments using synthetic data. Furthermore, we demonstrate that our proposed method can distinguish the differences between two types of genetic networks under stationary conditions from lung cancer and normal gene expression data.
Møller, Martin Nue; Kirkeby, Svend; Vikeså, Jonas
OBJECTIVES/HYPOTHESIS: The purpose of the present study is to explore, demonstrate, and describe the expression of genes related to the solute carrier (SLC) molecules of ion transporters in the human endolymphatic sac. STUDY DESIGN: cDNA microarrays and immunohistochemistry were used for analyses......a1 sodium-bicarbonate transporter, SLC9a2 sodium-hydrogen transporter, SLC12a3 thiazide-sensitive Na-Cl transporter, and SLC34a2 sodium-phosphate transporter. CONCLUSIONS: Several important ion transporters of the SLC family are expressed in the human endolymphatic sac, including Pendrin......, the thiazide-sensitive Na-Cl transporter, and the Na-phosphate transporter SLC34a2. The data provide a new knowledge base considering the ion-dependent metabolic mechanisms maintaining inner ear homeostasis. More specifically, the results indicate a strong similarity with the ion transportation occurring...
Full Text Available The vertical segments of Populus stems are an ideal experimental system for analyzing the gene expression patterns involved in primary and secondary growth during wood formation. Suitable internal control genes are indispensable to quantitative real time PCR (qRT-PCR assays of gene expression. In this study, the expression stability of eight candidate reference genes was evaluated in a series of vertical stem segments of Populus tomentosa. Analysis through software packages geNorm, NormFinder and BestKeeper showed that genes ribosomal protein (RP and tubulin beta (TUBB were the most unstable across the developmental stages of P. tomentosa stems, and the combination of the three reference genes, eukaryotic translation initiation factor 5A (eIF5A, Actin (ACT6 and elongation factor 1-beta (EF1-beta can provide accurate and reliable normalization of qRT-PCR analysis for target gene expression in stem segments undergoing primary and secondary growth in P. tomentosa. These results provide crucial information for transcriptional analysis in the P. tomentosa stem, which may help to improve the quality of gene expression data in these vertical stem segments, which constitute an excellent plant system for the study of wood formation.
Wang, Ying; Chen, Yajuan; Ding, Liping; Zhang, Jiewei; Wei, Jianhua; Wang, Hongzhi
The vertical segments of Populus stems are an ideal experimental system for analyzing the gene expression patterns involved in primary and secondary growth during wood formation. Suitable internal control genes are indispensable to quantitative real time PCR (qRT-PCR) assays of gene expression. In this study, the expression stability of eight candidate reference genes was evaluated in a series of vertical stem segments of Populus tomentosa. Analysis through software packages geNorm, NormFinder and BestKeeper showed that genes ribosomal protein (RP) and tubulin beta (TUBB) were the most unstable across the developmental stages of P. tomentosa stems, and the combination of the three reference genes, eukaryotic translation initiation factor 5A (eIF5A), Actin (ACT6) and elongation factor 1-beta (EF1-beta) can provide accurate and reliable normalization of qRT-PCR analysis for target gene expression in stem segments undergoing primary and secondary growth in P. tomentosa. These results provide crucial information for transcriptional analysis in the P. tomentosa stem, which may help to improve the quality of gene expression data in these vertical stem segments, which constitute an excellent plant system for the study of wood formation.
Wada, Masayoshi; Takahashi, Hiroki; Altaf-Ul-Amin, Md; Nakamura, Kensuke; Hirai, Masami Y; Ohta, Daisaku; Kanaya, Shigehiko
Operon-like arrangements of genes occur in eukaryotes ranging from yeasts and filamentous fungi to nematodes, plants, and mammals. In plants, several examples of operon-like gene clusters involved in metabolic pathways have recently been characterized, e.g. the cyclic hydroxamic acid pathways in maize, the avenacin biosynthesis gene clusters in oat, the thalianol pathway in Arabidopsis thaliana, and the diterpenoid momilactone cluster in rice. Such operon-like gene clusters are defined by their co-regulation or neighboring positions within immediate vicinity of chromosomal regions. A comprehensive analysis of the expression of neighboring genes therefore accounts a crucial step to reveal the complete set of operon-like gene clusters within a genome. Genome-wide prediction of operon-like gene clusters should contribute to functional annotation efforts and provide novel insight into evolutionary aspects acquiring certain biological functions as well. We predicted co-expressed gene clusters by comparing the Pearson correlation coefficient of neighboring genes and randomly selected gene pairs, based on a statistical method that takes false discovery rate (FDR) into consideration for 1469 microarray gene expression datasets of A. thaliana. We estimated that A. thaliana contains 100 operon-like gene clusters in total. We predicted 34 statistically significant gene clusters consisting of 3 to 22 genes each, based on a stringent FDR threshold of 0.1. Functional relationships among genes in individual clusters were estimated by sequence similarity and functional annotation of genes. Duplicated gene pairs (determined based on BLAST with a cutoff of Emetabolism, containing P450 genes restricted to the Brassica family and predicted to be involved in secondary metabolism. Operon-like clusters tend to include genes encoding bio-machinery associated with ribosomes, the ubiquitin/proteasome system, secondary metabolic pathways, lipid and fatty-acid metabolism, and the lipid
Background Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue specific expression differences. However, different types of gene expression alteration should have different effects on an organism, the evolutionary forces that act on them might be different, and different types of genes might show different types of differential expression between species. To confirm this, we studied differentially expressed (DE) genes among closely related groups that have extensive gene expression atlases, and clarified characteristics of different types of DE genes including the identification of regulating loci for differential expression using expression quantitative loci (eQTL) analysis data. Results We detected differentially expressed (DE) genes between rice subspecies in five homologous tissues that were verified using japonica and indica transcriptome atlases in public databases. Using the transcriptome atlases, we classified DE genes into two types, global DE genes and changed-tissues DE genes. Global type DE genes were not expressed in any tissues in the atlas of one subspecies, however changed-tissues type DE genes were expressed in both subspecies with different tissue specificity. For the five tissues in the two japonica-indica combinations, 4.6 ± 0.8 and 5.9 ± 1.5 % of highly expressed genes were global and changed-tissues DE genes, respectively. Changed-tissues DE genes varied in number between tissues, increasing linearly with the abundance of tissue specifically expressed genes in the tissue. Molecular evolution of global DE genes was rapid, unlike that of changed-tissues DE genes. Based on gene ontology, global and changed-tissues DE genes were different, having no common GO terms. Expression differences of most global DE genes were regulated by cis-eQTLs. Expression
Jiang, Fan; Chen, Xiu-Ping; Hu, Wen-Shun; Zheng, Shao-Quan
Nowadays, there are few reports about regulatory genes implicated in peel color of longan. The basic genetic research of longan has been in stagnation for a long time as a lack of transcriptomic and genetic information. To predict candidate genes associated with peel color, Gene Functional Annotation and Coding Sequence prediction were used to perform functional annotation for our assembled unigenes and investigate differentially expressed genes (DEGs) of fruitlet peels from Longli (Dimocarpus confinis). Finally, a total of 24,044 (44.19 %) unigenes were annotated at least in one database after BLAST search to NCBI non-redundant protein sequence, NCBI non-redundant nucleotide sequences, Kyoto Encyclopedia of Genes and Genomes (KEGG) Ortholog, manually annotated and reviewed protein sequence database (Swiss-Prot), Protein family, Gene Ontology, euKaryotic Ortholog Groups databases. After searching against the KEGG-GENE protein database, a result of 6228 (11.45 %) unigenes were assigned to 245 KEGG pathways. Via comparing the distributions of expression value of all corresponding unigenes from red peel and green peel fruit, it could be intuitively concluded that high similarity was existed in the two distributions; however, on the whole, between two distributions of log RPKM expression value, some differences indicated that expression level in green-peel fruit group is slightly higher than values in red-peel fruit group. Finally, a total of 1349 unigenes were identified as DEGs after blasting the DEGs to public sequence databases, and 32 peel-color-related genes were identified in longan. Our results suggest that a number of unigenes involved in longan metabolic process, including anthocyanin biosynthesis. In addition, DRF, F3H, ANS, CYP75A1 and C1 may be the key ones. The study on key genes related to peel color will be contributed to revealing the molecular mechanisms of regulating peel color in woody plants.
Eads, Brian D; Colbourne, John K; Bohuski, Elizabeth; Andrews, Justen
Sexual reproduction is a core biological function that is conserved throughout eukaryotic evolution, yet breeding systems are extremely variable. Genome-wide comparative studies can be effectively used to identify genes and regulatory patterns that are constrained to preserve core functions from those that may help to account for the diversity of animal reproductive strategies. We use a custom microarray to investigate gene expression in males and two reproductive stages of females in the crustacean Daphnia pulex. Most Daphnia species reproduce by cyclical parthenogenesis, alternating between sexual and clonal reproduction. Both sex determination and the switch in their mode of reproduction is environmentally induced, making Daphnia an interesting comparative system for the study of sex-biased and reproductive genes. Patterns of gene expression in females and males reveal that 50% of assayed transcripts show some degree of sex-bias. Female-biased transcription is enriched for translation, metabolic and regulatory genes associated with development. Male-biased expression is enriched for cuticle and protease function. Comparison with well studied arthropods such as Drosophila melanogaster and Anopheles gambiae suggests that female-biased patterns tend to be conserved, whereas male-biased genes are evolving faster in D. pulex. These findings are based on the proportion of female-biased, male-biased, and unbiased genes that share sequence similarity with proteins in other animal genomes. Some transcriptional differences between males and females appear to be conserved across Arthropoda, including the rapid evolution of male-biased genes which is observed in insects and now in a crustacean. Yet, novel patterns of male-biased gene expression are also uncovered. This study is an important first step towards a detailed understanding of the genetic basis and evolution of parthenogenesis, environmental sex determination, and adaptation to aquatic environments.
Full Text Available Abstract Background Sexual reproduction is a core biological function that is conserved throughout eukaryotic evolution, yet breeding systems are extremely variable. Genome-wide comparative studies can be effectively used to identify genes and regulatory patterns that are constrained to preserve core functions from those that may help to account for the diversity of animal reproductive strategies. We use a custom microarray to investigate gene expression in males and two reproductive stages of females in the crustacean Daphnia pulex. Most Daphnia species reproduce by cyclical parthenogenesis, alternating between sexual and clonal reproduction. Both sex determination and the switch in their mode of reproduction is environmentally induced, making Daphnia an interesting comparative system for the study of sex-biased and reproductive genes. Results Patterns of gene expression in females and males reveal that 50% of assayed transcripts show some degree of sex-bias. Female-biased transcription is enriched for translation, metabolic and regulatory genes associated with development. Male-biased expression is enriched for cuticle and protease function. Comparison with well studied arthropods such as Drosophila melanogaster and Anopheles gambiae suggests that female-biased patterns tend to be conserved, whereas male-biased genes are evolving faster in D. pulex. These findings are based on the proportion of female-biased, male-biased, and unbiased genes that share sequence similarity with proteins in other animal genomes. Conclusion Some transcriptional differences between males and females appear to be conserved across Arthropoda, including the rapid evolution of male-biased genes which is observed in insects and now in a crustacean. Yet, novel patterns of male-biased gene expression are also uncovered. This study is an important first step towards a detailed understanding of the genetic basis and evolution of parthenogenesis, environmental sex
Full Text Available Abstract Background Fluctuations in external salinity force eukaryotic cells to respond by changes in the gene expression of proteins acting in protective biochemical processes, thus counteracting the changing osmotic pressure. The high-osmolarity glycerol (HOG signaling pathway is essential for the efficient up-regulation of the osmoresponsive genes. In this study, the differential gene expression of the extremely halotolerant black yeast Hortaea werneckii was explored. Furthermore, the interaction of mitogen-activated protein kinase HwHog1 and RNA polymerase II with the chromatin in cells adapted to an extremely hypersaline environment was analyzed. Results A cDNA subtraction library was constructed for H. werneckii, adapted to moderate salinity or an extremely hypersaline environment of 4.5 M NaCl. An uncommon osmoresponsive set of 95 differentially expressed genes was identified. The majority of these had not previously been connected with the adaptation of salt-sensitive S. cerevisiae to hypersaline conditions. The transcriptional response in hypersaline-adapted and hypersaline-stressed cells showed that only a subset of the identified genes responded to acute salt-stress, whereas all were differentially expressed in adapted cells. Interaction with HwHog1 was shown for 36 of the 95 differentially expressed genes. The majority of the identified osmoresponsive and HwHog1-dependent genes in H. werneckii have not been previously reported as Hog1-dependent genes in the salt-sensitive S. cerevisiae. The study further demonstrated the co-occupancy of HwHog1 and RNA polymerase II on the chromatin of 17 up-regulated and 2 down-regulated genes in 4.5 M NaCl-adapted H. werneckii cells. Conclusion Extremely halotolerant H. werneckii represents a suitable and highly relevant organism to study cellular responses to environmental salinity. In comparison with the salt-sensitive S. cerevisiae, this yeast shows a different set of genes being expressed at
Patrignani, Paola; Tacconelli, Stefania; Bruno, Annalisa
Novel concepts of disease susceptibility and development suggest an important role of gastrointestinal microbiota and microbial pathogens. They can contribute to physiological systems and disease processes, even outside of the gastrointestinal tract. There is increasing evidence that genetics of the host influence and interact with gut microbiota. Moreover, aging-associated oxidative stress may cause morphologic alterations of bacterial cells, thus influencing the aggressive potential and virulence markers of an anaerobic bacterium and finally the type of interaction with the host. At the same time, microbiota may influence host gene expression and it is becoming apparent that it may occur through the regulation of microRNAs. They are short single-stranded noncoding RNAs that regulate posttranscriptional gene expression by affecting mRNA stability and/or translational repression of their target mRNAs. The introduction of -omics approaches (such as metagenomics, metaproteomics, and metatranscriptomics) in microbiota research will certainly advance our knowledge of this area. This will lead to greatly deepen our understanding of the molecular targets in the homeostatic interaction between the gut microbiota and the host and, thereby, promises to reveal new ways to treat diseases and maintain health.
Westenberg, Michel A.; Hijum, Sacha A.F.T. van; Lulko, Andrzej T.; Kuipers, Oscar P.; Roerdink, Jos B.T.M.; Linsen, L; Hagen, H; Hamann, B
We present GENeVis, an application to visualize gene expression time series data in a gene regulatory network context. This is a network of regulator proteins that regulate the expression of their respective target genes. The networks are represented as graphs, in which the nodes represent genes,
Khaitovich, Philipp; Tang, Kun; Franz, Henriette
Recent work has shown that the expression levels of genes transcribed in the brains of humans and chimpanzees have changed less than those of genes transcribed in other tissues  . However, when gene expression changes are mapped onto the evolutionary lineage in which they occurred, the brain...... shows more changes than other tissues in the human lineage compared to the chimpanzee lineage  ,  and  . There are two possible explanations for this: either positive selection drove more gene expression changes to fixation in the human brain than in the chimpanzee brain, or genes expressed...... in the brain experienced less purifying selection in humans than in chimpanzees, i.e. gene expression in the human brain is functionally less constrained. The first scenario would be supported if genes that changed their expression in the brain in the human lineage showed more selective sweeps than other genes...
Edoardo M Airoldi
Full Text Available Maintaining balanced growth in a changing environment is a fundamental systems-level challenge for cellular physiology, particularly in microorganisms. While the complete set of regulatory and functional pathways supporting growth and cellular proliferation are not yet known, portions of them are well understood. In particular, cellular proliferation is governed by mechanisms that are highly conserved from unicellular to multicellular organisms, and the disruption of these processes in metazoans is a major factor in the development of cancer. In this paper, we develop statistical methodology to identify quantitative aspects of the regulatory mechanisms underlying cellular proliferation in Saccharomyces cerevisiae. We find that the expression levels of a small set of genes can be exploited to predict the instantaneous growth rate of any cellular culture with high accuracy. The predictions obtained in this fashion are robust to changing biological conditions, experimental methods, and technological platforms. The proposed model is also effective in predicting growth rates for the related yeast Saccharomyces bayanus and the highly diverged yeast Schizosaccharomyces pombe, suggesting that the underlying regulatory signature is conserved across a wide range of unicellular evolution. We investigate the biological significance of the gene expression signature that the predictions are based upon from multiple perspectives: by perturbing the regulatory network through the Ras/PKA pathway, observing strong upregulation of growth rate even in the absence of appropriate nutrients, and discovering putative transcription factor binding sites, observing enrichment in growth-correlated genes. More broadly, the proposed methodology enables biological insights about growth at an instantaneous time scale, inaccessible by direct experimental methods. Data and tools enabling others to apply our methods are available at http://function.princeton.edu/growthrate.
Smith Desmond J
Full Text Available Abstract Background Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we present an approach for identifying the relation between gene expression maps obtained by voxelation and gene functions. Results To analyze the dataset, we chose typical genes as queries and aimed at discovering similar gene groups. Gene similarity was determined by using the wavelet features extracted from the left and right hemispheres averaged gene expression maps, and by the Euclidean distance between each pair of feature vectors. We also performed a multiple clustering approach on the gene expression maps, combined with hierarchical clustering. Among each group of similar genes and clusters, the gene function similarity was measured by calculating the average gene function distances in the gene ontology structure. By applying our methodology to find similar genes to certain target genes we were able to improve our understanding of gene expression patterns and gene functions. By applying the clustering analysis method, we obtained significant clusters, which have both very similar gene expression maps and very similar gene functions respectively to their corresponding gene ontologies. The cellular component ontology resulted in prominent clusters expressed in cortex and corpus callosum. The molecular function ontology gave prominent clusters in cortex, corpus callosum and hypothalamus. The biological process ontology resulted in clusters in cortex, hypothalamus and choroid plexus. Clusters from all three ontologies combined were most prominently expressed in
Germanguz, I; Listgarten, J; Cinkornpumin, J.; Solomon, A; Gaeta, X.; Lowry, W. E.
Using a compendium of cell-state-specific gene expression data, we identified genes that uniquely define cell states, including those thought to represent various developmental stages. Our analysis sheds light on human cell fate through the identification of core genes that are altered over several developmental milestones, and across regional specification. Here we present cell-type specific gene expression data for 17 distinct cell states and demonstrate that these modules of genes can in f...
Wu, Gene P K; Chan, Keith C C; Wong, Andrew K C
Discovering patterns from gene expression levels is regarded as a classification problem when tissue classes of the samples are given and solved as a discrete-data problem by discretizing the expression levels of each gene into intervals maximizing the interdependence between that gene and the class labels. However, when class information is unavailable, discovering gene expression patterns becomes difficult. For a gene pool with large number of genes, we first cluster the genes into smaller groups. In each group, we use the representative gene, one with highest interdependence with others in the group, to drive the discretization of the gene expression levels of other genes. Treating intervals as discrete events, association patterns of events can be discovered. If the gene groups obtained are crisp gene clusters, significant patterns overlapping different gene clusters cannot be found. This paper presents a new method of "fuzzifying" the crisp gene clusters to overcome such problem. To evaluate the effectiveness of our approach, we first apply the above described procedure on a synthetic data set and then a gene expression data set with known class labels. The class labels are not being used in both analyses but used later as the ground truth in a classificatory problem for assessing the algorithm's effectiveness in fuzzy gene clustering and discretization. The results show the efficacy of the proposed method. The existence of correlation among continuous valued gene expression levels suggests that certain genes in the gene groups have high interdependence with other genes in the group. Fuzzification of a crisp gene cluster allows the cluster to take in genes from other clusters so that overlapping relationship among gene clusters could be uncovered. Hence, previously unknown hidden patterns resided in overlapping gene clusters are discovered. From the experimental results, the high order patterns discovered reveal multiple gene interaction patterns in cancerous
Welburn Susan C
Full Text Available Trypanosoma brucei rhodesiense can be induced to undergo apoptosis after stimulation with Con A. As cell death in these parasites is associated with de novo gene expression we have applied a differential display technique, Randomly Amplified Differential Expressed Sequence-Polymerase Chain Reaction (RADES-PCR to the study of gene expression during Con A induced cell death in these organisms. Twenty-two differentially displayed products have been cloned and sequenced. These represent the first endogenous genes to be identified as implicated in cellular death in trypanosomatids (the most primitive eukaryote in which apoptosis has been described. Evidence for an ancestral death machinery, `proto-apoptosis' in single celled organisms is discussed.
Full Text Available Temporal gene expression data are of particular interest to researchers as they contain rich information in characterization of gene function and have been widely used in biomedical studies. However, extracting information and identifying efficient treatment effects without loss of temporal information are still in problem. In this paper, we propose a method of classifying temporal gene expression curves in which individual expression trajectory is modeled as longitudinal data with changeable variance and covariance structure. The method, mainly based on generalized mixed model, is illustrated by a dense temporal gene expression data in bacteria. We aimed at evaluating gene effects and treatments. The power and time points of measurements are also characterized via the longitudinal mixed model. The results indicated that the proposed methodology is promising for the analysis of temporal gene expression data, and that it could be generally applicable to other high-throughput temporal gene expression analyses.
Paul, Prosenjit; Malakar, Arup Kumar; Chakraborty, Supriyo
Highly expressed genes in any species differ in the usage frequency of synonymous codons. The relative recurrence of an event of the favored codon pair (amino acid pairs) varies between gene and genomes due to varying gene expression and different base composition. Here we propose a new measure for predicting the gene expression level, i.e., codon plus amino bias index (CABI). Our approach is based on the relative bias of the favored codon pair inclination among the genes, illustrated by analyzing the CABI score of the Medicago truncatula genes. CABI showed strong correlation with all other widely used measures (CAI, RCBS, SCUO) for gene expression analysis. Surprisingly, CABI outperforms all other measures by showing better correlation with the wet-lab data. This emphasizes the importance of the neighboring codons of the favored codon in a synonymous group while estimating the expression level of a gene.
Kristensen, Louise; Kielsgaard Kristensen, Thomas; Abildgaard, Niels
Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world. Autophagy is a highly conserved process in eukaryotic cells. In CLL autophagy is involved in mediating the effect of chemotherapy but the role of autophagy in CLL pathogenesis remains unknown....... In the present study, we used real-time RT-PCR to analyze expression of the PIK3C3, PIK3R4, and BECN1 genes. These genes encode the components of the PI3K core complex, which is central to initiation of autophagy. A consecutive series of 149 well-characterized CLL cases from Region of Southern Denmark were...... included in the study. All three genes were observed to be independent markers of prognosis in CLL with high expression being associated with more aggressive disease. With this clear association with outcome in CLL, these genes thereby represent promising candidates for future functional studies...
Abou-Zied, Akram M.; Lüppert, Martin; Dethlefsen, Johan; Mukherjee, Krishanu; Tong, Yong Guang; Tang, Lois; Gangishetti, Umesh; Baillie, David L.; Bürglin, Thomas R.
Homeobox genes play crucial roles for the development of multicellular eukaryotes. We have generated a revised list of all homeobox genes for Caenorhabditis elegans and provide a nomenclature for the previously unnamed ones. We show that, out of 103 homeobox genes, 70 are co-orthologous to human homeobox genes. 14 are highly divergent, lacking an obvious ortholog even in other Caenorhabditis species. One of these homeobox genes encodes 12 homeodomains, while three other highly divergent homeobox genes encode a novel type of double homeodomain, termed HOCHOB. To understand how transcription factors regulate cell fate during development, precise spatio-temporal expression data need to be obtained. Using a new imaging framework that we developed, Endrov, we have generated spatio-temporal expression profiles during embryogenesis of over 60 homeobox genes, as well as a number of other developmental control genes using GFP reporters. We used dynamic feedback during recording to automatically adjust the camera exposure time in order to increase the dynamic range beyond the limitations of the camera. We have applied the new framework to examine homeobox gene expression patterns and provide an analysis of these patterns. The methods we developed to analyze and quantify expression data are not only suitable for C. elegans, but can be applied to other model systems or even to tissue culture systems. PMID:26024448
Full Text Available The availability of highly active homologous promoters is critical in the development of a transformation system and improvement of the transformation efficiency. To facilitate transformation of green microalga Ankistrodesmus convolutus which is considered as a potential candidate for many biotechnological applications, a highly-expressed native promoter sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (AcRbcS has been used to drive the expression of β-glucuronidase (gusA gene in this microalga. Besides the determination of the transcription start site by 5¢-RACE, sequence analysis revealed that AcRbcS promoter contained consensus TATA-box and several putative cis-acting elements, including some representative light-regulatory elements (e.g., G-box, Sp1 motif and SORLIP2, which confer light responsiveness in plants, and several potential conserved motifs (e.g., CAGAC-motif, YCCYTGG-motifs and CACCACA-motif, which may be involved in light responsiveness of RbcS gene in green microalgae. Using AcRbcS promoter::gusA translational fusion, it was demonstrated that this promoter could function as a light-regulated promoter in transgenic A. convolutus, which suggested that the isolated AcRbcS promoter was a full and active promoter sequence that contained all cis-elements required for developmental and light-mediated control of gene expression, and this promoter can be used to drive the expression of heterologous genes in A. convolutus. This achievement therefore advances the development of A. convolutus as an alternative expression system for the production of recombinant proteins. This is the first report on development of gene manipulation system for unicellular green alga A. convolutus.
Thanh, Tran; Chi, Vu Thi Quynh; Omar, Hishamuddin; Abdullah, Mohd Puad; Napis, Suhaimi
The availability of highly active homologous promoters is critical in the development of a transformation system and improvement of the transformation efficiency. To facilitate transformation of green microalga Ankistrodesmus convolutus which is considered as a potential candidate for many biotechnological applications, a highly-expressed native promoter sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (AcRbcS) has been used to drive the expression of β-glucuronidase (gusA) gene in this microalga. Besides the determination of the transcription start site by 5′-RACE, sequence analysis revealed that AcRbcS promoter contained consensus TATA-box and several putative cis-acting elements, including some representative light-regulatory elements (e.g., G-box, Sp1 motif and SORLIP2), which confer light responsiveness in plants, and several potential conserved motifs (e.g., CAGAC-motif, YCCYTGG-motifs and CACCACA-motif), which may be involved in light responsiveness of RbcS gene in green microalgae. Using AcRbcS promoter::gusA translational fusion, it was demonstrated that this promoter could function as a light-regulated promoter in transgenic A. convolutus, which suggested that the isolated AcRbcS promoter was a full and active promoter sequence that contained all cis-elements required for developmental and light-mediated control of gene expression, and this promoter can be used to drive the expression of heterologous genes in A. convolutus. This achievement therefore advances the development of A. convolutus as an alternative expression system for the production of recombinant proteins. This is the first report on development of gene manipulation system for unicellular green alga A. convolutus. PMID:22489117
Bagger, Frederik Otzen
Acute Myeloid Leukaemia (AML) is an aggressive cancer of the bone marrow, affecting formation of blood cells during haematopoiesis. This thesis presents investigation of AML using mRNA gene expression profiles (GEP) of samples extracted from the bone marrow of healthy and diseased subjects. Here......-lookup webservices, called HemaExplorer and BloodSpot. These web-services support the aim of making data and analysis of haematopoietic cells from mouse and human accessible for researchers without bioinformatics expertise. Finally, in order to aid the analysis of the very limited number of haematopoietic progenitor...... cells obtainable from bone marrow aspirations, this thesis presents a method developed to investigate transcription factor binding and histone modifications by ChIP-Seq using pico-scale amounts of DNA....
Full Text Available Sutanuka Mitra,1 Suvendra Kumar Ray,2 Rajat Banerjee1 1Department of Biotechnology, University of Calcutta, Kolkata, West Bengal, 2Department of Molecular Biology and Biotechnology, Tezpur University, Napaam, Tezpur, Assam, India Abstract: Nowadays, it is beyond doubt that synonymous codons are not the same with respect to expression of a gene. In favor of this, ribosome profiling experiments in vivo and in vitro have suggested that ribosome occupancy time is not the same for different synonymous codons. Therefore, synonymous codons influence differently the speed of translation elongation, which guides further cotranslational folding kinetics of a protein. It is now realized that the position of each codon in a coding sequence is important. The effect of synonymous codons on protein structure is an exciting field of research nowadays. This review discusses the recent developments in this field. Keywords: codon usage bias, synonymous codons, ribosome profiling, cotranslational protein folding, protein structure
In order to isolate peanut genes, an expressed sequence tag (EST) sequencing project was carried out using a peanut seed cDNA library. From 6258 ESTs, 19 LEA-encoding genes were identified and could be classified into eight distinct groups. Expression of these genes in seeds at different developmental stages and in ...
transcription and translation at individual molecule and single base-pair resolution. By directly representing the position and activity of individual molecules on DNA, Tabasco can directly test the effects of detailed molecular processes on system-wide gene expression. Tabasco would also be useful for studying the complex regulatory mechanisms controlling eukaryotic gene expression. The computational engine underlying Tabasco could also be adapted to represent other types of processive systems in which individual reaction events are organized across a single spatial dimension (e.g., polysaccharide synthesis.
Tomancak, Pavel; Berman, Benjamin P; Beaton, Amy; Weiszmann, Richard; Kwan, Elaine; Hartenstein, Volker; Celniker, Susan E; Rubin, Gerald M
Cell and tissue specific gene expression is a defining feature of embryonic development in multi-cellular organisms. However, the range of gene expression patterns, the extent of the correlation of expression with function, and the classes of genes whose spatial expression are tightly regulated have been unclear due to the lack of an unbiased, genome-wide survey of gene expression patterns. We determined and documented embryonic expression patterns for 6,003 (44%) of the 13,659 protein-coding genes identified in the Drosophila melanogaster genome with over 70,000 images and controlled vocabulary annotations. Individual expression patterns are extraordinarily diverse, but by supplementing qualitative in situ hybridization data with quantitative microarray time-course data using a hybrid clustering strategy, we identify groups of genes with similar expression. Of 4,496 genes with detectable expression in the embryo, 2,549 (57%) fall into 10 clusters representing broad expression patterns. The remaining 1,947 (43%) genes fall into 29 clusters representing restricted expression, 20% patterned as early as blastoderm, with the majority restricted to differentiated cell types, such as epithelia, nervous system, or muscle. We investigate the relationship between expression clusters and known molecular and cellular-physiological functions. Nearly 60% of the genes with detectable expression exhibit broad patterns reflecting quantitative rather than qualitative differences between tissues. The other 40% show tissue-restricted expression; the expression patterns of over 1,500 of these genes are documented here for the first time. Within each of these categories, we identified clusters of genes associated with particular cellular and developmental functions.
Gerosa, Luca; Kochanowski, Karl; Heinemann, Matthias; Sauer, Uwe
Gene expression is regulated by specific transcriptional circuits but also by the global expression machinery as a function of growth. Simultaneous specific and global regulation thus constitutes an additional-but often neglected-layer of complexity in gene expression. Here, we develop an
Darieva, Zoulfia; Han, Namshik; Warwood, Stacey; Doris, Kathryn S.; Morgan, Brian A.
The control of the cell cycle in eukaryotes is exerted in part by the coordinated action of a series of transcription factor complexes. This is exemplified by the Mcm1p-Fkh2p-Ndd1p complex in Saccharomyces cerevisiae, which controls the cyclical expression of the CLB2 cluster of genes at the G2/M phase transition. The activity of this complex is positively controlled by cyclin-dependent kinase (CDK) and polo kinases. Here, we demonstrate that the protein kinase Pkc1p works in the opposite manner to inhibit the activity of the Mcm1p-Fkh2p-Ndd1p complex and the expression of its target genes. In particular, Pkc1p causes phosphorylation of the coactivator protein Ndd1p. Reductions in Pkc1p activity and the presence of Pkc1p-insensitive Ndd1p mutant proteins lead to changes in the timing of CLB2 cluster expression and result in associated late cell cycle defects. This study therefore identifies an important role for Pkc1p in controlling the correct temporal expression of genes in the cell cycle. PMID:22966207
Frey, Steffen; Görlich, Dirk
During autophagy, members of the ubiquitin-like Atg8 protein family get conjugated to phosphatidylethanolamine and act as protein-recruiting scaffolds on the autophagosomal membrane. The Atg4 protease produces mature Atg8 from C-terminally extended precursors and deconjugates lipid-bound Atg8. We now found that Xenopus laevis Atg4B (xAtg4B) is ideally suited for proteolytic removal of N-terminal tags from recombinant proteins. To implement this strategy, an Atg8 cleavage module is inserted in between tag and target protein. An optimized xAtg4B protease fragment includes the so far uncharacterized C-terminus, which crucially contributes to recognition of the Xenopus Atg8 homologs xLC3B and xGATE16. xAtg4B-mediated tag cleavage is very robust in solution or on-column, efficient at 4°C and orthogonal to TEV protease and the recently introduced proteases bdSENP1, bdNEDP1 and xUsp2. Importantly, xLC3B fusions are stable in wheat germ extract or when expressed in Saccharomyces cerevisiae, but cleavable by xAtg4B during or following purification. We also found that fusions to the bdNEDP1 substrate bdNEDD8 are stable in S. cerevisiae. In combination, or findings now provide a system, where proteins and complexes fused to xLC3B or bdNEDD8 can be expressed in a eukaryotic host and purified by successive affinity capture and proteolytic release steps.
Li, Bo; Fang, Lusheng; Li, Bo
is proposed and analyzed, as well as its three categories i.e., the operator gene, the structural gene and the regulator gene. Second, the trigger mechanism that design objectives and constraints trigger the operator gene is constructed. Third, the expression principle of structural gene is analyzed...... with the example of design management gene. Last, the regulation mode that the regulator gene regulates the expression of the structural gene is established and it is illustrated by taking the design process management gene as an example. © (2011) Trans Tech Publications.......To improve the design process efficiency, this paper proposes the principle and methodology that design process gene controls the characteristics of design process under the framework of design process reuse and optimization based on design process gene. First, the concept of design process gene...
Full Text Available Pre-mRNA in eukaryotes is subjected to mRNA processing, which includes capping, polyadenylation, and splicing. Transcription and mRNA processing are coupled, and this coupling stimulates mRNA processing; however, the effects of mRNA processing on transcription are not fully understood. In this study, we found that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA, or by an antisense oligonucleotide to U2 snRNA, caused gene-specific 3'-end down-regulation. Removal of SSA from the culture media restored expression of the 3' ends of genes, suggesting that U2 snRNP is required for expression of the 3' end of genes. Finally, we found that SSA treatment caused accumulation of Pol II near the 5' end of 3'-end down regulated genes, such as CDK6, SMEK2 and EGFR, indicating that SSA treatment led to transcription elongation arrest on these genes. These findings suggest that U2 snRNP is important for production of full length mRNA probably through regulation of transcription elongation, and that a novel checkpoint mechanism prevents pre-mRNA from accumulating as a result of splicing deficiencies, and thereby prevents production of aberrant proteins that might be translated from pre-mRNAs through the arrest of transcription elongation.
Andrew D Strand
Full Text Available Many neurodegenerative diseases have a hallmark regional and cellular pathology. Gene expression analysis of healthy tissues may provide clues to the differences that distinguish resistant and sensitive tissues and cell types. Comparative analysis of gene expression in healthy mouse and human brain provides a framework to explore the ability of mice to model diseases of the human brain. It may also aid in understanding brain evolution and the basis for higher order cognitive abilities. Here we compare gene expression profiles of human motor cortex, caudate nucleus, and cerebellum to one another and identify genes that are more highly expressed in one region relative to another. We separately perform identical analysis on corresponding brain regions from mice. Within each species, we find that the different brain regions have distinctly different expression profiles. Contrasting between the two species shows that regionally enriched genes in one species are generally regionally enriched genes in the other species. Thus, even when considering thousands of genes, the expression ratios in two regions from one species are significantly correlated with expression ratios in the other species. Finally, genes whose expression is higher in one area of the brain relative to the other areas, in other words genes with patterned expression, tend to have greater conservation of nucleotide sequence than more widely expressed genes. Together these observations suggest that region-specific genes have been conserved in the mammalian brain at both the sequence and gene expression levels. Given the general similarity between patterns of gene expression in healthy human and mouse brains, we believe it is reasonable to expect a high degree of concordance between microarray phenotypes of human neurodegenerative diseases and their mouse models. Finally, these data on very divergent species provide context for studies in more closely related species that address
Brunner Amy M
Full Text Available Abstract Background Real-time reverse transcription PCR (RT-PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Results Using real-time RT-PCR to study the expression of 10 poplar (genus Populus housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Conclusion Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.
Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H
Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.
Geeven, G.; van Kesteren, R.E.; Smit, A.B.; de Gunst, M.C.M.
Motivation: Gene regulatory networks, in which edges between nodes describe interactions between transcriptional regulators and their target genes, determine the coordinated spatiotemporal expression of genes. Especially in higher organisms, context-specific combinatorial regulation by transcription
Zhao, Jingjing; Shi, Hongbo; Ahituv, Nadav
Tissue-specific gene expression is thought to be one of the major forces shaping mammalian gene order. A recent study that used whole-genome chromosome conformation assays has shown that the mammalian genome is divided into specific topological domains that are shared between different tissues and organisms. Here, we wanted to assess whether gene expression and regulation are involved in shaping these domains and can be used to classify them. We analyzed gene expression and regulation levels in these domains by using RNA-seq and enhancer-associated ChIP-seq datasets for 17 different mouse tissues. We found 162 domains that are active (high gene expression and regulation) in all 17 tissues. These domains are significantly shorter, contain less repeats, and have more housekeeping genes. In contrast, we found 29 domains that are inactive (low gene expression and regulation) in all analyzed tissues and are significantly longer, have more repeats, and gene deserts. Tissue-specific active domains showed some correlation with tissue-type and gene ontology. Domain temporal gene regulation and expression differences also displayed some gene ontology terms fitting their temporal function. Combined, our results provide a catalog of shared and tissue-specific topological domains and suggest that gene expression and regulation could have a role in shaping them.
Jørgensen, Anne; Morthorst, Jane Ebsen; Andersen, Ole
BACKGROUND: The mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine...... the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation. RESULTS...... "female" genes (fig alpha and cyp19a1a). When comparing all five genes with expected sex related expression 56% show expression expected for either male or female. Furthermore, the expression of all genes was investigated in different tissue of adult male and female zebrafish. CONCLUSION: In zebrafish...
Bowman Rayleen V
Full Text Available Abstract Chronic obstructive pulmonary disease (COPD is a major public health problem. The aim of this study was to identify genes involved in emphysema severity in COPD patients. Gene expression profiling was performed on total RNA extracted from non-tumor lung tissue from 30 smokers with emphysema. Class comparison analysis based on gas transfer measurement was performed to identify differentially expressed genes. Genes were then selected for technical validation by quantitative reverse transcriptase-PCR (qRT-PCR if also represented on microarray platforms used in previously published emphysema studies. Genes technically validated advanced to tests of biological replication by qRT-PCR using an independent test set of 62 lung samples. Class comparison identified 98 differentially expressed genes (p p Gene expression profiling of lung from emphysema patients identified seven candidate genes associated with emphysema severity including COL6A3, SERPINF1, ZNHIT6, NEDD4, CDKN2A, NRN1 and GSTM3.
Georg K Gerber
Full Text Available An important research problem in computational biology is the identification of expression programs, sets of co-expressed genes orchestrating normal or pathological processes, and the characterization of the functional breadth of these programs. The use of human expression data compendia for discovery of such programs presents several challenges including cellular inhomogeneity within samples, genetic and environmental variation across samples, uncertainty in the numbers of programs and sample populations, and temporal behavior. We developed GeneProgram, a new unsupervised computational framework based on Hierarchical Dirichlet Processes that addresses each of the above challenges. GeneProgram uses expression data to simultaneously organize tissues into groups and genes into overlapping programs with consistent temporal behavior, to produce maps of expression programs, which are sorted by generality scores that exploit the automatically learned groupings. Using synthetic and real gene expression data, we showed that GeneProgram outperformed several popular expression analysis methods. We applied GeneProgram to a compendium of 62 short time-series gene expression datasets exploring the responses of human cells to infectious agents and immune-modulating molecules. GeneProgram produced a map of 104 expression programs, a substantial number of which were significantly enriched for genes involved in key signaling pathways and/or bound by NF-kappaB transcription factors in genome-wide experiments. Further, GeneProgram discovered expression programs that appear to implicate surprising signaling pathways or receptor types in the response to infection, including Wnt signaling and neurotransmitter receptors. We believe the discovered map of expression programs involved in the response to infection will be useful for guiding future biological experiments; genes from programs with low generality scores might serve as new drug targets that exhibit minimal
Hackam, Abigail S; Qian, Jiang; Liu, Dongmei; Gunatilaka, Tushara; Farkas, Ronald H; Chowers, Itay; Kageyama, Masaaki; Parmigiani, Giovanni; Zack, Donald J
Several high-throughput studies have described gene expression in the central nervous system (CNS), and recently there has been increasing interest in analyzing how gene expression compares in different regions of the CNS. As the retina is often used as a model system to study CNS development and function, we compared retina and brain gene expression using microarray analyses. Mouse retina, brain and liver RNA was hybridized to a custom cDNA microarray containing 5,376 genes and ESTs, and the data from the quantified scanned images were analyzed using Bioconductor and SAM. Preferential retina expression was confirmed by real-time PCR. The cellular distribution of genes newly identified as retina enriched genes was determined by immunohistochemistry. Using stringent statistical analyses we identified 733 genes that were preferentially expressed in retina and 389 in brain. The retina-liver hybridizations identified an additional 837 retina enriched genes. The cellular distribution in the retina was determined for two genes that had not previously been reported to be expressed in the retina, the transcription regulatory proteins EWS and PCPB1. Both proteins were found primarily in the inner nuclear layer. Finally, a comparison of the microarray data to publicly available SAGE and EST library databases demonstrated only limited overlap of the sets of retina enriched genes identified by the different methodologies. The preferential retinal expression of a subset of genes from the microarray, which were not identified as differentially expressed by other methods, was confirmed by quantitative PCR. The finding of differences in the groups of identified retina enriched genes from the various profiling techniques supports the use of multiple approaches to obtain a more complete description of retinal gene expression. Characterization of gene expression profiles of retina and brain may facilitate the understanding of the processes that underlie differences between the retina
Hou, Shu-xun; Sun, Da-ming; Du, Gui-xin; Tong, Yi-gang; Fu, Xiao-bing
To construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs. The BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1. At the same time, mesenchymal stem cells (MSCs) were isolated and cultured in vitro. The plasmid carrying the BMP7 gene was transfected into MSCs. PCR and digesting demonstrated that the eucaryotic expression plasmid -pcDNA-BMP7 was obtained. RT-PCR and immunohistochemical methods showed that the BMP7 gene was expressed in MSCs. Construction of an eucaryotic expression plasmid carrying BMP7 gene and expression in MSCs provide a sound basis for gene therapy using the BMP7 gene and the ideal seeds for tissue engineering.
Agarwal Rachana; Hewitt Kyle J; Morin Patrice J
Abstract Background The claudin (CLDN) genes encode a family of proteins important in tight junction formation and function. Recently, it has become apparent that CLDN gene expression is frequently altered in several human cancers. However, the exact patterns of CLDN expression in various cancers is unknown, as only a limited number of CLDN genes have been investigated in a few tumors. Methods We identified all the human CLDN genes from Genbank and we used the large public SAGE database to as...
Yu, Yao; Xu, Tao; Yu, Yongtao; Hao, Pei; Li, Xuan
Embryogenesis is the process by which the embryo is formed, develops, and establishes developmental hierarchies of tissues. The recent advance in microarray technology made it possible to investigate the tissue specific patterns of gene expression and their relationship with tissue lineages. This study is focused on how tissue specific functions, tissue lineage, and cell differentiation are correlated, which is essential to understand embryonic development and organism complexity. We performed individual gene and gene set based analysis on multiple tissue expression data, in association with the classic topology of mammalian fate maps of embryogenesis. For each sub-group of tissues on the fate map, conservatively, differentially and correlatively expressed genes or gene sets were identified. Tissue distance was found to correlate with gene expression divergence. Tissues of the ectoderm or mesoderm origins from the same segments on the fate map shared more similar expression pattern than those from different origins. Conservatively expressed genes or gene sets define common functions in a tissue group and are related to tissue specific diseases, which is supported by results from Gene Ontology and KEGG pathway analysis. Gene expression divergence is larger in certain human tissues than in the mouse homologous tissues. The results from tissue lineage and gene expression analysis indicate that common function features of neighbor tissue groups were defined by the conservatively expressed genes and were related to tissue specific diseases, and differentially expressed genes contribute to the functional divergence of tissues. The difference of gene expression divergence in human and mouse homologous tissues reflected the organism complexity, i.e. distinct neural development levels and different body sizes.
Muthukaruppan, Anita; Lasham, Annette; Blenkiron, Cherie; Woad, Kathryn J; Black, Michael A; Knowlton, Nicholas; McCarthy, Nicole; Findlay, Michael P; Print, Cristin G; Shelling, Andrew N
New Zealand has one of the highest rates of breast cancer incidence in the world. We investigated the gene expression profiles of breast tumours from New Zealand patients, compared them to gene expression profiles of international breast cancer cohorts and identified any associations between altered gene expression and the clinicopathological features of the tumours. Affymetrix microarrays were used to measure the gene expression profiles of 106 breast tumours from New Zealand patients. Gene expression data from six international breast cancer cohorts were collated, and all the gene expression data were analysed using standard bioinformatic and statistical tools. Gene expression profiles associated with tumour ER and ERBB2 status, molecular subtype and selected gene expression signatures within the New Zealand cohort were consistent with those found in international cohorts. Significant differences in clinicopathological features such as tumour grade, tumour size and lymph node status were also observed between the New Zealand and international cohorts. Gene expression profiles, which are a sensitive indicator of tumour biology, showed no clear difference between breast tumours from New Zealand patients and those from non-New Zealand patients. This suggests that other factors may contribute to the high and increasing breast cancer incidence in New Zealand compared to international populations.
Wang, An-ping; Sun, Huai-chang; Wang, Jian-ye; Wang, Yong-juan; Yuan, Wei-feng
To generate recombinant avian adeno-associated virus (rAAAV) for gene transfer studies in avian cells, the recombinant plasmid containing the whole genome of AAAV was digested with restriction enzymes to remove the Rep and Cap genes, resulting in AAAV transfer vector pAITR. GFP-expressing cassette was amplified by PCR and inserted into the AAAV transfer vector. The Rep-Cap gene of AAAV amplified by high fidelity PCR was subcloned into eukaryotic expression vector pcDNA3, resulting in an AAAV helper vector pcDNA-ARC. The Rep and Cap genes amplified by high fidelity PCR were subcloned separately into the co-expression vector pVITRO2-mcs, resulting in another AAAV helper vector pVITRO2-ARC. Using calcium phosphate precipitation method, rAAAV-GFP was generated by co-transfecting AAV-293 cells with a cocktail of pAITR-GFP, pcDNA-ARC or pVITRO2-ARC, and adenovirus helper vector pHelper. The three structural proteins VP1, VP2 and VP3 of correct molecular masses were detected by SDS-PAGE and the GFP reporter gene was detected by PCR in purified rAAAV-GFP virions. Chicken embryonic fibroblast (CEF) cells and CEL cell line were transduced with the recombinant virus, the GFP-positive cells were easily observed under fluorescent microscope, expression of which lasted for at least two weeks. These data demonstrate that an efficient helper virus-free packaging system has been established for generating recombinant AAAV particles for gene transfer studies in avian cells and for development of recombinant vaccines against avian diseases.
Lee, Jae Myung; Lee, Junhyeong; Kim, Taesung; Lee, Sung Kuk
.... This system also ensures homogenous expression across the entire cell population. We also report the design of a miniaturized photobioreactor to be used in combination with the light-switchable gene expression system...
Jun 12, 2012 ... Key words: Mammalian cells, plasmid vector, stable gene expression, protein therapeutics, woodchuck hepatitis ... embryonic kidney (HEK293) cell expression system, have ..... Animal cell cultures: recent achievements and.
Pagani, S; Meazza, C; Gertosio, C; Bozzola, E; Bozzola, M
The mechanisms regulating the synergic effect of growth hormone and other hormones during pubertal spurt are not completely clarified. We enrolled 64 females of Caucasian origin and normal height including 22 prepubertal girls, 26 pubertal girls, and 16 adults to evaluate the role of Growth Hormone/Insulin-like growth factor-I axis (GH/IGF-I) during the pubertal period. In these subjects both serum IGF-I and growth hormone binding protein levels, as well as quantitative growth hormone receptor (GHR) gene expression were evaluated in peripheral lymphocytes of all individuals by real-time PCR. Our results showed significantly lower IGF-I levels in women (148±10 ng/ml) and prepubertal girls (166.34±18.85 ng/ml) compared to pubertal girls (441.95±29.42 ng/ml; p<0.0001). Serum GHBP levels were significantly higher in prepubertal (127.02±20.76 ng/ml) compared to pubertal girls (16.63±2.97 ng/ml; p=0.0001) and adult women (19.95±6.65 ng/ml; p=0.0003). We also found higher GHR gene expression levels in pubertal girls [174.73±80.22 ag (growth hormone receptor)/5×10(5) ag (glyceraldehyde 3-phosphate dehydrogenase)] compared with other groups of subjects [women: 42.52±7.66 ag (growth hormone receptor)/5×10(5) ag (glyceraldehyde 3-phosphate dehydrogenase); prepubertal girls: 58.45±0.18.12 ag (growth hormone receptor)/5×10(5) ag (glyceraldehyde 3-phosphate dehydrogenase)], but the difference did not reach statistical significance. These results suggest that sexual hormones could positively influence GHR action, during the pubertal period, in a dual mode, that is, increasing GHR mRNA production and reducing GHR cleavage leading to GHBP variations. © Georg Thieme Verlag KG Stuttgart · New York.
Full Text Available BACKGROUND: Numerous biochemical and physiological parameters of living organisms follow a circadian rhythm. Although such rhythmic behavior is particularly pronounced in plants, which are strictly dependent on the daily photoperiod, data on the molecular aspects of the diurnal cycle in plants is scarce and mostly concerns the model species Arabidopsis thaliana. Here we studied the leaf transcriptome in seedlings of maize, an important C4 crop only distantly related to A. thaliana, throughout a cycle of 10 h darkness and 14 h light to look for rhythmic patterns of gene expression. RESULTS: Using DNA microarrays comprising ca. 43,000 maize-specific probes we found that ca. 12% of all genes showed clear-cut diel rhythms of expression. Cluster analysis identified 35 groups containing from four to ca. 1,000 genes, each comprising genes of similar expression patterns. Perhaps unexpectedly, the most pronounced and most common (concerning the highest number of genes expression maxima were observed towards and during the dark phase. Using Gene Ontology classification several meaningful functional associations were found among genes showing similar diel expression patterns, including massive induction of expression of genes related to gene expression, translation, protein modification and folding at dusk and night. Additionally, we found a clear-cut tendency among genes belonging to individual clusters to share defined transcription factor-binding sequences. CONCLUSIONS: Co-expressed genes belonging to individual clusters are likely to be regulated by common mechanisms. The nocturnal phase of the diurnal cycle involves gross induction of fundamental biochemical processes and should be studied more thoroughly than was appreciated in most earlier physiological studies. Although some general mechanisms responsible for the diel regulation of gene expression might be shared among plants, details of the diurnal regulation of gene expression seem to differ
Samee, Md Abul Hassan; Sinha, Saurabh
Modeling a gene's expression from its intergenic locus and trans-regulatory context is a fundamental goal in computational biology. Owing to the distributed nature of cis-regulatory information and the poorly understood mechanisms that integrate such information, gene locus modeling is a more challenging task than modeling individual enhancers. Here we report the first quantitative model of a gene's expression pattern as a function of its locus. We model the expression readout of a locus in two tiers: 1) combinatorial regulation by transcription factors bound to each enhancer is predicted by a thermodynamics-based model and 2) independent contributions from multiple enhancers are linearly combined to fit the gene expression pattern. The model does not require any prior knowledge about enhancers contributing toward a gene's expression. We demonstrate that the model captures the complex multi-domain expression patterns of anterior-posterior patterning genes in the early Drosophila embryo. Altogether, we model the expression patterns of 27 genes; these include several gap genes, pair-rule genes, and anterior, posterior, trunk, and terminal genes. We find that the model-selected enhancers for each gene overlap strongly with its experimentally characterized enhancers. Our findings also suggest the presence of sequence-segments in the locus that would contribute ectopic expression patterns and hence were "shut down" by the model. We applied our model to identify the transcription factors responsible for forming the stripe boundaries of the studied genes. The resulting network of regulatory interactions exhibits a high level of agreement with known regulatory influences on the target genes. Finally, we analyzed whether and why our assumption of enhancer independence was necessary for the genes we studied. We found a deterioration of expression when binding sites in one enhancer were allowed to influence the readout of another enhancer. Thus, interference between enhancer
Xu, Ke; Doak, Thomas G; Lipps, Hans J; Wang, Jingmei; Swart, Estienne C; Chang, Wei-Jen
Ciliated protozoa are peculiar for their nuclear dimorphism, wherein two types of nuclei divide nuclear functions: a germline micronucleus (MIC) is transcriptionally inert during vegetative growth, but serves as the genetic blueprint for the somatic macronucleus (MAC), which is responsible for all transcripts supporting cell growth and reproduction. While all the advantages/disadvantages associated with nuclear dimorphism are not clear, an essential advantage seems to be the ability to produce a highly polyploid MAC, which then allows for the maintenance of extremely large single cells - many ciliate cells are larger than small metazoa. In some ciliate classes, chromosomes in the MAC are extensively fragmented to create extremely short chromosomes that often carry single genes, and these chromosomes may be present in different copy numbers, resulting in different ploidies. While using gene copy number to regulate gene expression is limited in most eukaryotic systems, the extensive fragmentation in some ciliate classes provides this opportunity to every MAC gene. However, it is still unclear if this mechanism is in fact used extensively in these ciliates. To address this, we have quantified copy numbers of 11 MAC chromosomes and their gene expression in Oxytricha trifallax (CI: Spirotrichea). We compared copy numbers between two subpopulations of O. trifallax, and copy numbers of 7 orthologous genes between O. trifallax and the closely related Stylonychia lemnae. We show that copy numbers of MAC chromosomes are variable, dynamic, and positively correlated to gene expression. These features might be conserved in all spirotrichs, and might exist in other classes of ciliates with heavily fragmented MAC chromosomes. Copyright © 2012 Elsevier B.V. All rights reserved.
Dionicia Gloria León-Martínez
Full Text Available To explore the molecular mechanisms that prevail during the establishment of the arbuscular mycorrhiza symbiosis involving the genus Glomus, we transcriptionally analysed spores of Glomus intraradices BE3 during early hyphal growth. Among 458 transcripts initially identified as being expressed at presymbiotic stages, 20% of sequences had homology to previously characterized eukaryotic genes, 30% were homologous to fungal coding sequences, and 9% showed homology to previously characterized bacterial genes. Among them, GintPbr1a encodes a homolog to Phenazine Biosynthesis Regulator (Pbr of Burkholderia cenocepacia, an pleiotropic regulatory protein that activates phenazine production through transcriptional activation of the protein D isochorismatase biosynthetic enzyme phzD (Ramos et al., 2010. Whereas GintPbr1a is expressed during the presymbiotic phase, the G. intraradices BE3 homolog of phzD (BGintphzD is transcriptionally active at the time of the establishment of the arbuscular mycorrhizal symbiosis. DNA from isolated bacterial cultures found in spores of G. intraradices BE3 confirmed that both BGintPbr1a and BGintphzD are present in the genome of its potential endosymbionts. Taken together, our results indicate that spores of G. intraradices BE3 express bacterial phenazine biosynthetic genes at the onset of the fungal-plant symbiotic interaction.
Nobutaka Hanagata, Taro Takemura and Takashi Minowa
Full Text Available Comprehensive gene expression analysis using DNA microarrays has become a widespread technique in molecular biological research. In the biomaterials field, it is used to evaluate the biocompatibility or cellular toxicity of metals, polymers and ceramics. Studies in this field have extracted differentially expressed genes in the context of differences in cellular responses among multiple materials. Based on these genes, the effects of materials on cells at the molecular level have been examined. Expression data ranging from several to tens of thousands of genes can be obtained from DNA microarrays. For this reason, several tens or hundreds of differentially expressed genes are often present in different materials. In this review, we outline the principles of DNA microarrays, and provide an introduction to methods of extracting information which is useful for evaluating and designing biomaterials from comprehensive gene expression data.
Hanagata, Nobutaka; Takemura, Taro; Minowa, Takashi, E-mail: HANAGATA.Nobutaka@nims.go.j [Nanotechnology Innovation Center and Biomaterials Center, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047 (Japan)
Comprehensive gene expression analysis using DNA microarrays has become a widespread technique in molecular biological research. In the biomaterials field, it is used to evaluate the biocompatibility or cellular toxicity of metals, polymers and ceramics. Studies in this field have extracted differentially expressed genes in the context of differences in cellular responses among multiple materials. Based on these genes, the effects of materials on cells at the molecular level have been examined. Expression data ranging from several to tens of thousands of genes can be obtained from DNA microarrays. For this reason, several tens or hundreds of differentially expressed genes are often present in different materials. In this review, we outline the principles of DNA microarrays, and provide an introduction to methods of extracting information which is useful for evaluating and designing biomaterials from comprehensive gene expression data. (topical review)
Full Text Available Troxerutin, a semi-synthetic derivative of the natural bioflavanoid rutin, has been reported to possess many beneficial effects in human bodies, such as vasoprotection, immune support, anti-inflammation and anti-aging. However, the effects of troxerutin on genome-wide transcription in blood cells are still unknown. In order to find out effects of troxerutin on gene transcription, a high-throughput RNA sequencing was employed to analysis differential gene expression in blood cells consisting of leucocytes, erythrocytes and platelets isolated from the mice received subcutaneous injection of troxerutin. Transcriptome analysis demonstrated that the expression of only fifteen genes was significantly changed by the treatment with troxerutin, among which 5 genes were up-regulated and 10 genes were down-regulated. Bioinformatic analysis of the fifteen differentially expressed genes was made by utilizing the Gene Ontology (GO, and the differential expression induced by troxerutin was further evaluated by real-time quantitative PCR (Q-PCR.
Zhou, Shenghu; Du, Guocheng; Kang, Zhen; Li, Jianghua; Chen, Jian; Li, Huazhong; Zhou, Jingwen
Production of useful chemicals by industrial microorganisms has been attracting more and more attention. Microorganisms screened from their natural environment usually suffer from low productivity, low stress resistance, and accumulation of by-products. In order to overcome these disadvantages, rational engineering of microorganisms to achieve specific industrial goals has become routine. Rapid development of metabolic engineering and synthetic biology strategies provide novel methods to improve the performance of industrial microorganisms. Rational regulation of gene expression by specific promoters is essential to engineer industrial microorganisms for high-efficiency production of target chemicals. Identification, modification, and application of suitable promoters could provide powerful switches at the transcriptional level for fine-tuning of a single gene or a group of genes, which are essential for the reconstruction of pathways. In this review, the characteristics of promoters from eukaryotic, prokaryotic, and archaea microorganisms are briefly introduced. Identification of promoters based on both traditional biochemical and systems biology routes are summarized. Besides rational modification, de novo design of promoters to achieve gradient, dynamic, and logic gate regulation are also introduced. Furthermore, flexible application of static and dynamic promoters for the rational engineering of industrial microorganisms is highlighted. From the perspective of powerful promoters in industrial microorganisms, this review will provide an extensive description of how to regulate gene expression in industrial microorganisms to achieve more useful goals.
Tashireva L. A.
Full Text Available Aim. To investigate the expression of the genes TGFB1, TNF, CSF1, CSF2, VEGFA and HIF1A in the patients with invasive breast carcinoma of no special type considering the intratumoral morphological heterogeneity. Methods. The technology of laser capture microdissection PALM was used to isolate five types of morphological tumor structures from three patients with invasive carcinoma of no special type (IC NST, luminal A subtype, T1-2NxMx. The level of expression of the cytokine (TNF, growth factor genes (TGFB1, CSF1, CSF2, VEGFA and the HIF1A gene was assessed in the samples obtained using real-time PCR, TaqMan-probes and specific oligonucleotides. Results. The study demonstrated the absence of the expression of the growth factor gene CSF2 in tumor cells of IC NST, and the expression of the gene CSF1, independent from the metastasis status and tumor structure type. The prevalence of the expression of the genes VEGFA and TGFB1 was revealed in the alveolar and solid structures along with the rare expression of the gene TNF. Conclusions. The expression of pre-metastatic niche genes in the tumors of patients with IC NST is heterogeneous. The hypoxia-mediated change in the cytokine gene expression may be expected in the alveolar and solid structures, which ultimately results in the formation of microenvironment, facilitating tumor growth and the formation of tumor metastatic potential.
Tomfohr, John; Lu, Jun; Kepler, Thomas B
A promising direction in the analysis of gene expression focuses on the changes in expression of specific predefined sets of genes that are known in advance to be related (e.g., genes coding for proteins involved in cellular pathways or complexes). Such an analysis can reveal features that are not easily visible from the variations in the individual genes and can lead to a picture of expression that is more biologically transparent and accessible to interpretation. In this article, we present a new method of this kind that operates by quantifying the level of 'activity' of each pathway in different samples. The activity levels, which are derived from singular value decompositions, form the basis for statistical comparisons and other applications. We demonstrate our approach using expression data from a study of type 2 diabetes and another of the influence of cigarette smoke on gene expression in airway epithelia. A number of interesting pathways are identified in comparisons between smokers and non-smokers including ones related to nicotine metabolism, mucus production, and glutathione metabolism. A comparison with results from the related approach, 'gene-set enrichment analysis', is also provided. Our method offers a flexible basis for identifying differentially expressed pathways from gene expression data. The results of a pathway-based analysis can be complementary to those obtained from one more focused on individual genes. A web program PLAGE (Pathway Level Analysis of Gene Expression) for performing the kinds of analyses described here is accessible at http://dulci.biostat.edu/pathways.
Hussein, Gihan M; Abu El-Heba, Ghada A; Abdou, Sara M; Abdallah, Naglaa A
Low transformation efficiency and long generation time for production of transgenic Gerbera jemosonii plants leads to vulnerable gene function studies. Thus, transient expression of genes would be an efficient alternative. In this investigation, a transient expression system for gerbera petals based on the Agrobacterium infiltration protocol was developed using the reporter genes β-glucuronidase (gus) and green florescence protein (gfp). Results revealed the incapability of using the gfp gene as a reporter gene for transient expression study in gerbera flowers due to the detection of green fluorescent color in the non-infiltrated gerbera flower petals. However, the gus reporter gene was successfully utilized for optimizing and obtaining the suitable agroinfiltration system in gerbera flowers. The expression of GUS was detectable after three days of agroinfiltration in gerbera cultivars "Express" and "White Grizzly" with dark pink and white flower colors, respectively. The vacuum agroinfiltration protocol has been applied on the cultivar "Express" for evaluating the transient expression of the two genes involved in the anthocyanin pathway (iris-dfr and petunia-f3' 5'h), which is responsible for the color in flowers. In comparison to the control, transient expression results showed change in the anthocyanin pigment in all infiltrated flowers with color genes. Additionally, blue color was detected in the stigma and pollen grains in the infiltrated flowers. Moreover, blue colors with variant intensities were observed in produced calli during the routine work of stable transformation with f3' 5'h gene.
Anna K Greenwood
Full Text Available Identifying genes that are differentially expressed in response to social interactions is informative for understanding the molecular basis of social behavior. To address this question, we described changes in gene expression as a result of differences in the extent of social interactions. We housed threespine stickleback (Gasterosteus aculeatus females in either group conditions or individually for one week, then measured levels of gene expression in three brain regions using RNA-sequencing. We found that numerous genes in the hindbrain/cerebellum had altered expression in response to group or individual housing. However, relatively few genes were differentially expressed in either the diencephalon or telencephalon. The list of genes upregulated in fish from social groups included many genes related to neural development and cell adhesion as well as genes with functions in sensory signaling, stress, and social and reproductive behavior. The list of genes expressed at higher levels in individually-housed fish included several genes previously identified as regulated by social interactions in other animals. The identified genes are interesting targets for future research on the molecular mechanisms of normal social interactions.
Zambon, Alexander C.; Zhang, Lingzhi; Minovitsky, Simon; Kanter, Joan R.; Prabhakar, Shyam; Salomonis, Nathan; Vranizan, Karen; Dubchak Inna,; Conklin, Bruce R.; Insel, Paul A.
Although a substantial number of hormones and drugs increase cellular cAMP levels, the global impact of cAMP and its major effector mechanism, protein kinase A (PKA), on gene expression is not known. Here we show that treatment of murine wild-type S49 lymphoma cells for 24 h with 8-(4-chlorophenylthio)-cAMP (8-CPTcAMP), a PKA-selective cAMP analog, alters the expression of approx equal to 4,500 of approx. equal to 13,600 unique genes. By contrast, gene expression was unaltered in Kin- S49 cells (that lack PKA) incubated with 8-CPTcAMP. Changes in mRNA and protein expression of several cell cycle regulators accompanied cAMP-induced G1-phase cell-cycle arrest of wild-type S49 cells. Within 2h, 8-CPT-cAMP altered expression of 152 genes that contain evolutionarily conserved cAMP-response elements within 5 kb of transcriptional start sites, including the circadian clock gene Per1. Thus, cAMP through its activation of PKA produces extensive transcriptional regulation in eukaryotic cells. These transcriptional networks include a primary group of cAMP-response element-containing genes and secondary networks that include the circadian clock.
Andreuzza, Sébastien; Nishal, Bindu; Singh, Aparna; Siddiqi, Imran
Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast.
Full Text Available Abstract Background Renal cell carcinoma (RCC is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays. Methods Total RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC. Results Selected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR. Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved. Conclusions This is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most
An, Li; Obradovic, Zoran; Smith, Desmond; Bodenreider, Olivier; Megalooikonomou, Vasileios
Association rules mining methods have been recently applied to gene expression data analysis to reveal relationships between genes and different conditions and features. However, not much effort has focused on detecting the relation between gene expression maps and related gene functions. Here we describe such an approach to mine association rules among gene functions in clusters of similar gene expression maps on mouse brain. The experimental results show that the detected association rules make sense biologically. By inspecting the obtained clusters and the genes having the gene functions of frequent itemsets, interesting clues were discovered that provide valuable insight to biological scientists. Moreover, discovered association rules can be potentially used to predict gene functions based on similarity of gene expression maps.
Hori, Manabu; Fujishima, Masahiro
The bacterium Holospora obtusa is a macronuclear-specific symbiont of the ciliate Paramecium caudatum. H. obtusa-bearing paramecia could survive even after the cells were quickly heated from 25 degrees C to 35 degrees C. To determine whether infection with H. obtusa confers heat shock resistance on its host, we isolated genes homologous to the heat shock protein genes hsp60 and hsp70 from P. caudatum. The deduced amino acid sequences of both cDNAs were highly homologous to hsp family sequences from other eukaryotes. Competitive PCR showed that H. obtusa-free paramecia expressed only trace amounts of hsp60 and hsp70 mRNA at 25 degrees C, but that expression of hsp70 was enhanced immediately after the cells were transferred to 35 degrees C. H. obtusa-bearing paramecia expressed high levels of hsp7O mRNA even at 25 degrees C and the level was further enhanced when the cells were incubated at 35 degrees C. In contrast, the expression pattern of hsp60 mRNA was the same in H. obtusa-bearing as in H. obtusa-free paramecia. These results indicate that infection with its endosymbiont can confer a heat-shock resistant nature on its host cells.
Full Text Available The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3 in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information
Rasmussen Lene J
Full Text Available Abstract Background The mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation. Results In the present study, we have used quantitative real-time PCR to investigate the expression of ar, sox9a, dmrt1, fig alpha, cyp19a1a and cyp19a1b during the expected sex determination and gonadal sex differentiation period. The expression of the genes expected to be high in males (ar, sox9a and dmrt1a and high in females (fig alpha and cyp19a1a was segregated in two groups with more than 10 times difference in expression levels. All of the investigated genes showed peaks in expression levels during the time of sex determination and gonadal sex differentiation. Expression of all genes was investigated on cDNA from the same fish allowing comparison of the high and low expressers of genes that are expected to be highest expressed in either males or females. There were 78% high or low expressers of all three "male" genes (ar, sox9a and dmrt1 in the investigated period and 81% were high or low expressers of both "female" genes (fig alpha and cyp19a1a. When comparing all five genes with expected sex related expression 56% show expression expected for either male or female. Furthermore, the expression of all genes was investigated in different tissue of adult male and female zebrafish. Conclusion In zebrafish, the first significant peak in gene expression during the investigated period (2–40 dph was dmrt1 at 10 dph which indicates involvement of this gene
Karouia, Fathi; Ricco, Antonio; Pohorille, Andrew; Peyvan, Kianoosh
The capability to measure gene expression on board spacecrafts opens the doors to a large number of experiments on the influence of space environment on biological systems that will profoundly impact our ability to conduct safe and effective space travel, and might also shed light on terrestrial physiology or biological function and human disease and aging processes. Measurements of gene expression will help us to understand adaptation of terrestrial life to conditions beyond the planet of origin, identify deleterious effects of the space environment on a wide range of organisms from microbes to humans, develop effective countermeasures against these effects, determine metabolic basis of microbial pathogenicity and drug resistance, test our ability to sustain and grow in space organisms that can be used for life support and in situ resource utilization during long-duration space exploration, and monitor both the spacecraft environment and crew health. These and other applications hold significant potential for discoveries in space biology, biotechnology and medicine. Accordingly, supported by funding from the NASA Astrobiology Science and Technology Instrument Development Program, we are developing a fully automated, miniaturized, integrated fluidic system for small spacecraft capable of in-situ measuring microbial expression of thousands of genes from multiple samples. The instrument will be capable of (1) lysing bacterial cell walls, (2) extracting and purifying RNA released from cells, (3) hybridizing it on a microarray and (4) providing electrochemical readout, all in a microfluidics cartridge. The prototype under development is suitable for deployment on nanosatellite platforms developed by the NASA Small Spacecraft Office. The first target application is to cultivate and measure gene expression of the photosynthetic bacterium Synechococcus elongatus, i.e. a cyanobacterium known to exhibit remarkable metabolic diversity and resilience to adverse conditions
Lidie, Kristy B; Ryan, James C; Barbier, Michele; Van Dolah, Frances M
Karenia brevis (Davis) is the dinoflagellate responsible for nearly annual red tides in the Gulf of Mexico. Although the mechanisms regulating the growth and toxicity of this problematic organism are of considerable interest, little information is available on its molecular biology. We therefore constructed a complementary DNA library from which to gain insight into its expressed genome and to develop tools for studying its gene expression. Large-scale sequencing yielded 7001 high-quality expressed sequence tags (ESTs), which clustered into 5280 unique gene groups. The vast majority of genes expressed fell into a low-abundance class, with the highest expressed gene accounting for only 1% of the total ESTs. Approximately 29% of genes were found to have similarity to known sequences in other organisms after BLAST similarity comparisons to the GenBank public protein database using a cutoff of P < 10e(-4). We identified for the first time in a dinoflagellate a suite of conserved eukaryotic genes involved in cell cycle control, intracellular signaling, and the transcription and translation machinery. At least 40% of gene clusters displayed single nucleotide polymorphisms, suggesting the presence of multiple gene copies. The average GC content of ESTs was 51%, with a slight preference for G or C in the third codon position (53.5%). The ESTs were used to develop an oligonucleotide microarray containing 4629 unique features and 3462 replicate probes. Microarray labeling has been optimized, and the microarray has been validated for probe specificity and reproducibility. This is the first information to be developed on the expressed genome of K. brevis and provides the basis from which to begin functional genomic studies on this harmful algal bloom species.
Full Text Available The 14-3-3 gene family, which is conserved in eukaryotes, is involved in protein-protein interactions and mediates signal transduction. However, detailed investigations of the 14-3-3 gene family in Medicago truncatula are largely unknown. In this study, the identification and study of M. truncatula 14-3-3-family genes were performed based on the latest M. truncatula genome. In the M. truncatula genome, 10 14-3-3 family genes were identified, and they can be grouped into ε and non-ε groups. An exon-intron analysis showed that the gene structures are conserved in the same group. The protein structure analysis showed that 14-3-3 proteins in M. truncatula are composed of nine typical antiparallel α-helices. The expression patterns of Mt14-3-3 genes indicated that they are expressed in all tissues. Furthermore, the gene expression levels of Mt14-3-3 under hormone treatment and Sinorhizobium meliloti infection showed that the Mt14-3-3 genes were involve in nodule formation. Our findings lay a solid foundation for further functional studies of 14-3-3 in M. truncatula.
Turner Renee J
Full Text Available Abstract Background Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT, 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder. Conclusion The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.
Lim, Han N.; Lee, Yeong; Hussein, Razika
Half a century has passed since the discovery of operons (groups of genes that are transcribed together as a single mRNA). Despite the importance of operons in bacterial gene networks, the relationship between their organization and gene expression remains poorly understood. Here we show using synthetic operons in Escherichia coli that the expression of a given gene increases with the length of the operon and as its position moves farther from the end of the operon. These findings can be explained by a common mechanism; increasing the distance from the start of a gene to the end of the operon (termed the “transcription distance”) provides more time for translation to occur during transcription, resulting in increased expression. We confirmed experimentally that the increased expression is indeed due to increased translation. Furthermore our analysis indicates the translation initiation rate for an mRNA is sixfold greater during transcription than after its release, which amplifies the impact of the transcription distance on gene expression. As a result of these mechanisms, gene expression increases by ∼40% for each 1,000 nucleotides of transcription distance. In summary, we demonstrate that a fundamental relationship exists between gene expression and the number, length, and order of the genes in an operon. This relationship has important implications for understanding the functional basis of genome organization and practical applications for synthetic biology. PMID:21670266
Leach, Sonia M; Feng, Weiguo; Williams, Trevor
This article contains data related to the research articles "Spatial and Temporal Analysis of Gene Expression during Growth and Fusion of the Mouse Facial Prominences" (Feng et al., 2009)  and "Systems Biology of facial development: contributions of ectoderm and mesenchyme" (Hooper et al., 2017 In press) . Embryonic mammalian craniofacial development is a complex process involving the growth, morphogenesis, and fusion of distinct facial prominences into a functional whole. Aberrant gene regulation during this process can lead to severe craniofacial birth defects, including orofacial clefting. As a means to understand the genes involved in facial development, we had previously dissected the embryonic mouse face into distinct prominences: the mandibular, maxillary or nasal between E10.5 and E12.5. The prominences were then processed intact, or separated into ectoderm and mesenchyme layers, prior analysis of RNA expression using microarrays (Feng et al., 2009, Hooper et al., 2017 in press) , . Here, individual gene expression profiles have been built from these datasets that illustrate the timing of gene expression in whole prominences or in the separated tissue layers. The data profiles are presented as an indexed and clickable list of the genes each linked to a graphical image of that gene׳s expression profile in the ectoderm, mesenchyme, or intact prominence. These data files will enable investigators to obtain a rapid assessment of the relative expression level of any gene on the array with respect to time, tissue, prominence, and expression trajectory.
Sonia M. Leach
Full Text Available This article contains data related to the research articles "Spatial and Temporal Analysis of Gene Expression during Growth and Fusion of the Mouse Facial Prominences" (Feng et al., 2009  and “Systems Biology of facial development: contributions of ectoderm and mesenchyme” (Hooper et al., 2017 In press . Embryonic mammalian craniofacial development is a complex process involving the growth, morphogenesis, and fusion of distinct facial prominences into a functional whole. Aberrant gene regulation during this process can lead to severe craniofacial birth defects, including orofacial clefting. As a means to understand the genes involved in facial development, we had previously dissected the embryonic mouse face into distinct prominences: the mandibular, maxillary or nasal between E10.5 and E12.5. The prominences were then processed intact, or separated into ectoderm and mesenchyme layers, prior analysis of RNA expression using microarrays (Feng et al., 2009, Hooper et al., 2017 in press [1,2]. Here, individual gene expression profiles have been built from these datasets that illustrate the timing of gene expression in whole prominences or in the separated tissue layers. The data profiles are presented as an indexed and clickable list of the genes each linked to a graphical image of that gene׳s expression profile in the ectoderm, mesenchyme, or intact prominence. These data files will enable investigators to obtain a rapid assessment of the relative expression level of any gene on the array with respect to time, tissue, prominence, and expression trajectory.
In order to identify differentially expressed genes involved in heat shock response, cDNA amplified fragment length polymorphism (cDNA-AFLP) and quantitative real-time polymerase chain reaction (QPCR) were used to study gene expression of eggplant seedlings subjected to 0, 6 and 12 h at 43°C. A total of 53 of over ...
The role of mast cells in allergic diseases and innate immunity has been widely researched and much is known about the expression profiles of immune-related genes in mast cells after bacterial challenges. However, little is known about the gene expression profiles of mast cells in response to adenosine. Herein, we ...
The prognostic value of autotaxin activity and gene expression, matrix metalloproteinase-9 and p53 antibodies in breast cancer patients. ... included in this study and subjected to determination of ATX (both activity by colorimetric method and gene expression by RT-PCR) and both p53 Abs and MMP-9 by ELISA technique.
The relationship between the structure of genes and their expression is a relatively new aspect of genome organization and regulation. With more genome sequences and expression data becoming available, bioinformatics approaches can help the further elucidation of the relationships between gene
Abstract. Background: Human cytomegalovirus (HCMV) is a virus which has the potential to alter cellular gene expression through multiple mechanisms. Objective: With the application of DNA microarrays, we could monitor the effects of pathogens on host-cell gene expression programmes in great depth and on a broad ...
Although there is a rich literature on gene ordering in hierarchical clustering framework for gene expression analysis, there is no .... Step 1: Create the string representation (chromosome of. GA) for a .... The expression profiles are represented as lines of coloured boxes using Expander (Sharan et al 2003), each of which.
1 Department of Gene and Cell Engineering, Institute of Animal Science, Chinese Academy of Agricultural Sciences,. Beijing 100094 ... quantitative RT-PCR methods, we compared the mRNA expression patterns in porcine oocytes from two ... Keywords: Differential gene expression, DD-RT-PCR, porcine oocytes, cumulus.
Beurden, van S.J.; Peeters, B.P.H.; Rottier, P.J.M.; Davison, A.A.; Engelsma, M.Y.
Background Whereas temporal gene expression in mammalian herpesviruses has been studied extensively, little is known about gene expression in fish herpesviruses. Here we report a genome-wide transcription analysis of a fish herpesvirus, anguillid herpesvirus 1, in cell culture, studied during the
Background: Human cytomegalovirus (HCMV) is a virus which has the potential to alter cellular gene expression through multiple mechanisms. Objective: With the application of DNA microarrays, we could monitor the effects of pathogens on host-cell gene expression programmes in great depth and on a broad scale.
Background: Human cytomegalovirus (HCMV) is a virus which has the potential to alter cellular gene expression through multiple mechanisms. Objective: With the application of DNA microarrays, we could monitor the effects of pathogens on host-cell gene expression programmes in great depth and on a broad scale.
Mposhi, Archibold; van der Wijst, Monique G. P.; Faber, Klaas Nico; Rots, Marianne G.
Epigenetics provides an important layer of information on top of the DNA sequence and is essential for establishing gene expression profiles. Extensive studies have shown that nuclear DNA methylation and histone modifications influence nuclear gene expression. However, it remains unclear whether
There are great differences in silk production efficiency and quality between the male and female domestic silkworm (Bombyx mori). Many genes act together but are differentially expressed between the sexes during silk biosynthesis. Two long serial analyses of gene expression (SAGE) libraries were constructed from the ...
A decrease in mRNA levels for cytochrome c oxidase (COX) subunits was observed in skeletal muscle of hypothyroid rats. However, the precise expression mechanisms of the related genes in hypothyroid state still remain unclear. This study investigated gene expressions of DNA methyltransferases (Dnmts), DNA ...
This is also applied to a retrotransposon protein encoding gene whose expression was strongly induced following extract treatment. The induced expression of all these defence-related genes suggests that the crude A. africanus extract has the ability to prime the resistance response of wheat prior to leaf rust infection.
Conclusion: The expression of KLK2 gene in people with prostate cancer is the higher than the healthy person; finally, according to the results, it could be mentioned that the KLK2 gene considered as a useful factor in prostate cancer, whose expression is associated with progression and development of the prostate cancer.
Full Text Available AIM: To discuss the expression and clinical significance of Pax6 gene in retinoblastoma(Rb. METHODS: Totally 15 cases of fresh Rb organizations were selected as observation group and 15 normal retinal organizations as control group. Western-Blot and reverse transcriptase polymerase chain reaction(RT-PCRmethods were used to detect Pax6 protein and Pax6 mRNA expressions of the normal retina organizations and Rb organizations. At the same time, Western Blot method was used to detect the Pax6 gene downstream MATH5 and BRN3b differentiation gene protein level expression. After the comparison between two groups, the expression and clinical significance of Pax6 gene in Rb were discussed. RESULTS: In the observation group, average value of mRNA expression of Pax6 gene was 0.99±0.03; average value of Pax6 gene protein expression was 2.07±0.15; average value of BRN3b protein expression was 0.195±0.016; average value of MATH5 protein expression was 0.190±0.031. They were significantly higher than the control group, and the differences were statistically significant(PCONCLUSION: Abnormal expression of Pax6 gene is likely to accelerate the occurrence of Rb.
Nannapaneni, Kishore; Ben-Shahar, Yehuda; Keen, Henry L; Welsh, Michael J; Casavant, Thomas L; Scheetz, Todd E
Operons are primarily a bacterial phenomenon, not commonly observed in eukaryotes. However, new research indicates that operons are found in higher organisms as well. There are instances of operons found in C. elegans, Drosophila melanogaster and other eukaryotic species. We developed a prototype using positional, structural and gene expression information to identify candidate operons. We focused our efforts on "trans-spliced" operons in which the pre-mRNA is trans-spliced into individual transcripts and subsequently translated, as widely observed in C. elegans and some instances in Drosophila. We identify several candidate operons in Drosophila melanogaster of which two have been subsequently molecularly validated. Copyright © 2013 Elsevier Ltd. All rights reserved.
Jessica C Seeliger
Full Text Available Research on the human pathogen Mycobacterium tuberculosis (Mtb would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb.
Hoegh, Anne Mette; Borup, Rehannah; Nielsen, Finn Cilius
Several studies point to the placenta as the primary cause of pre-eclampsia. Our objective was to identify placental genes that may contribute to the development of pre-eclampsia. RNA was purified from tissue biopsies from eleven pre-eclamptic placentas and eighteen normal controls. Messenger RNA...... expression from pooled samples was analysed by microarrays. Verification of the expression of selected genes was performed using real-time PCR. A surprisingly low number of genes (21 out of 15,000) were identified as differentially expressed. Among these were genes not previously associated with pre-eclampsia...... as bradykinin B1 receptor and a 14-3-3 protein, but also genes that have already been connected with pre-eclampsia, for example, inhibin beta A subunit and leptin. A low number of genes were repeatedly identified as differentially expressed, because they may represent the endpoint of a cascade of events...
The FANTOM consortium has generated a large gene expression dataset of different cell lines and tissue cultures using the single-molecule sequencing technology of HeliscopeCAGE. This provides a unique opportunity to investigate novel associations between gene expression over time and different cell types. Here, we create a MatLab wrapper for a powerful and computationally intensive set of statistics known as Maximal Information Coefficient, and then calculate this statistic for a large, comprehensive dataset containing gene expression of a variety of differentiating tissues. We then distinguish between linear and nonlinear associations, and then create gene association networks. Following this analysis, we are then able to identify clusters of linear gene associations that then associate nonlinearly with other clusters of linearity, providing insight to much more complex connections between gene expression patterns than previously anticipated.
Gauda, Estelle B
The peripheral arterial chemoreceptors of the carotid body participate in the ventilatory responses to hypoxia and hypercapnia, the arousal responses to asphyxial apnea, and the acclimatization to high altitude. In response to an excitatory stimuli, glomus cells in the carotid body depolarize, their intracellular calcium levels rise, and neurotransmitters are released from them. Neurotransmitters then bind to autoreceptors on glomus cells and postsynaptic receptors on chemoafferents of the carotid sinus nerve. Binding to inhibitory or excitatory receptors on chemoafferents control the electrical activity of the carotid sinus nerve, which provides the input to respiratory-related brainstem nuclei. We and others have used gene expression in the carotid body as a tool to determine what neurotransmitters mediate the response of peripheral arterial chemoreceptors to excitatory stimuli, specifically hypoxia. Data from physiological studies support the involvement of numerous putative neurotransmitters in hypoxic chemosensitivity. This article reviews how in situ hybridization histochemistry and other cellular localization techniques confirm, refute, or expand what is known about the role of dopamine, norepinephrine, substance P, acetylcholine, adenosine, and ATP in chemotransmission. In spite of some species differences, review of the available data support that 1). dopamine and norepinephrine are synthesized and released from glomus cells in all species and play an inhibitory role in hypoxic chemosensitivity; 2). substance P and acetylcholine are not synthesized in glomus cells of most species but may be made and released from nerve fibers innervating the carotid body in essentially all species; 3). adenosine and ATP are ubiquitous molecules that most likely play an excitatory role in hypoxic chemosensitivity. Copyright 2002 Wiley-Liss, Inc.
Yan, Lang; Gu, Ying-Hong; Tao, Xiang; Lai, Xian-Jun; Zhang, Yi-Zheng; Tan, Xue-Mei; Wang, Haiyan
Transposable elements (TEs) are the most abundant genomic components in eukaryotes and affect the genome by their replications and movements to generate genetic plasticity. Sweet potato performs asexual reproduction generally and the TEs may be an important genetic factor for genome reorganization. Complete identification of TEs is essential for the study of genome evolution. However, the TEs of sweet potato are still poorly understood because of its complex hexaploid genome and difficulty in genome sequencing. The recent availability of the sweet potato transcriptome databases provides an opportunity for discovering and characterizing the expressed TEs. We first established the integrated-transcriptome database by de novo assembling four published sweet potato transcriptome databases from three cultivars in China. Using sequence-similarity search and analysis, a total of 1,405 TEs including 883 retrotransposons and 522 DNA transposons were predicted and categorized. Depending on mapping sets of RNA-Seq raw short reads to the predicted TEs, we compared the quantities, classifications and expression activities of TEs inter- and intra-cultivars. Moreover, the differential expressions of TEs in seven tissues of Xushu 18 cultivar were analyzed by using Illumina digital gene expression (DGE) tag profiling. It was found that 417 TEs were expressed in one or more tissues and 107 in all seven tissues. Furthermore, the copy number of 11 transposase genes was determined to be 1-3 copies in the genome of sweet potato by Real-time PCR-based absolute quantification. Our result provides a new method for TE searching on species with transcriptome sequences while lacking genome information. The searching, identification and expression analysis of TEs will provide useful TE information in sweet potato, which are valuable for the further studies of TE-mediated gene mutation and optimization in asexual reproduction. It contributes to elucidating the roles of TEs in genome evolution.
Ramirez-Prado, Juan Sebastian; Rodriguez-Granados, Natalia Yaneth; Ariel, Federico; Raynaud, Cécile; Benhamed, Moussa
ABSTRACT As the most recent evidence of eukaryotic cell complexity, genome architecture has astounded the scientific community and prompted a variety of technical and cognitive challenges. Several technologies have emerged and evidenced the integration of chromatin packaging and topology, epigenetic processes, and transcription for the pertinent regulation of gene expression. In the present addendum we present and discuss some of our recent research, directed toward the holistic comprehension of the processes by which plants respond to environmental and developmental stimuli. We propose that the study of genome topology and genomic interactions is essential for the understanding of the molecular mechanisms behind a phenotype. Even though our knowledge and understanding of genome architecture and hierarchy has improved substantially in the last few years -in Arabidopsis and other eukaryotes -, there is still a long way ahead in this relatively new field of study. For this, it is necessary to take advantage of the high resolution of the emerging available techniques, and perform integrative approaches with which it will be possible to depict the role of chromatin architecture in the regulation of transcription and ultimately, physiological processes. PMID:27611230
Ramirez Prado, Juan Sebastian
As the most recent evidence of eukaryotic cell complexity, genome architecture has astounded the scientific community and prompted a variety of technical and cognitive challenges. Several technologies have emerged and evidenced the integration of chromatin packaging and topology, epigenetic processes, and transcription for the pertinent regulation of gene expression. In the present addendum we present and discuss some of our recent research, directed toward the holistic comprehension of the processes by which plants respond to environmental and developmental stimuli. We propose that the study of genome topology and genomic interactions is essential for the understanding of the molecular mechanisms behind a phenotype. Even though our knowledge and understanding of genome architecture and hierarchy has improved substantially in the last few years -in Arabidopsis and other eukaryotes -, there is still a long way ahead in this relatively new field of study. For this, it is necessary to take advantage of the high resolution of the emerging available techniques, and perform integrative approaches with which it will be possible to depict the role of chromatin architecture in the regulation of transcription and ultimately, physiological processes.
Hausman, Gary J; Barb, C Rick; Fairchild, Brian D; Gamble, John; Lee-Rutherford, Laura
In this study, total RNA was collected from abdominal adipose tissue samples obtained from ten broiler chickens at 3, 4, 5, and 6 weeks of age and prepared for gene microarray analysis with Affymetrix GeneChip Chicken Genome Arrays (Affymetrix) and quantitative real-time PCR analysis. Studies of global gene expression in chicken adipose tissue were initiated since such studies in many animal species show that adipose tissue expresses and secretes many factors that can influence growth and physiology. Microarray results indicated 333 differentially expressed adipose tissue genes between 3 and 6 wk, 265 differentially expressed genes between 4 and 6 wk and 42 differentially expressed genes between 3 and 4 wk. Enrichment scores of Gene Ontology Biological Process categories indicated strong age upregulation of genes involved in the immune system response. In addition to microarray analysis, quantitative real-time PCR analysis was used to confirm the influence of age on the expression of adipose tissue CC chemokine ligands (CCL), toll-like receptor (TLR)-2, lipopolysaccharide-induced TNF factor (LITAF), chemokine (C-C motif) receptor 8 (CCR8), and several other genes. Between 3 and 6 wk of age CCL5, CCL1, and CCR8 expression increased (P = 0.0001) with age. Furthermore, TLR2, CCL19, and LITAF expression increased between 4 and 6 wk of age (P = 0.001). This is the first demonstration of age related changes in CCL, LITAF, and TLR2 gene expression in chicken adipose tissue. Future studies are needed to elucidate the role of these adipose tissue genes in growth and the immune system. PMID:26317054
Lü, Xiaoying; Wang, Jiandan; Li, Bin; Zhang, Zhiwei; Zhao, Lifeng
The aim of this study was to investigate the osteoinductive effect of natural hydroxyapatite (NHA). NHA was extracted from pig bones and prepared into disk-like samples. Then, proliferation of mouse bone mesenchymal stem cells (MSCs) cultured on NHA was assessed by the methylthiazoltetrazolium (MTT) assay. Furthermore, microarray technology was applied to obtain the gene expression profiles of MSCs cultured on NHA at 24, 48, and 72 h. The gene expression profile was then comprehensively analyzed by clustering, Gene Ontology (GO), Gene Microarray Pathway Profiler (GenMAPP) and Ingenuity Pathway Analysis (IPA). According to the results of microarray experiment, 8992 differentially expressed genes were obtained. 90 differential expressed genes related to HA osteogenic differentiation were determined by GO analysis. These genes included not only 6 genes related to HA osteogenic differentiation as mentioned in the literatures but also newly discovered 84 genes. Some important signaling pathways (TGF-β, MAPK, Wnt, etc.) were influenced by these genes. Gene interaction networks were obtained by IPA software, in which the scoring values of two networks were highest, and their main functions were related to cell development. The comprehensive analysis of these results indicate that NHA regulate some crucial genes (e.g., Bmp2, Spp1) and then activate some pathways such as TGF-β signaling pathway, and ultimately osteogenic differentiation was induced. © 2013 Wiley Periodicals, Inc.
Aug 4, 2009 ... contrary to most studies, this research showed no significant correlation was found between copy number and expression level of ... insecticidal toxin gene from the spider (Atrax robustus). *Corresponding author: ... of spider insecticidal peptide gene and the C peptide sequence of. Bt gene were used as ...
Hosfield, T; Lu, Q
We have constructed an epitope-tagging vector, pCMV-Tag1, for gene expression in mammalian cells. This vector, which allows for N-terminal, C-terminal and internal tagging of the gene product of interest with the FLAG and/or c-myc epitopes, enables researchers to rapidly and efficiently characterize gene products in vivo.
These genes may play a major role in promoting excessive proliferation and accumulation of lipid droplets, which contribute to the development of obesity. By using microarray-based technology, we examined differential gene expression in early differentiated adipocytes and late differentiated adipocytes. Validated genes ...
The authors reported the identification of genes associated with embryonic development and microsatellite sequences. The future direction will entail characterization of these genes using gene over-expression and mutant assays. Key words: Namibia, simple sequence repeats (SSR), data mining, homology searches, ...
Jan 17, 2011 ... rapidly after switching from gluconeogenesis to glycolysis. (Regelmann et al., 2003). The identification of gene transcripts for RDM5 in G. arboreum and G. barbadense indicates that this gene might be involved in some specialized function in the two cotton species. M2 gene expression was detected in G.
Expression profiles of genes involved in tanshinone biosynthesis of two. Salvia miltiorrhiza genotypes with different tanshinone contents. Zhenqiao Song, Jianhua Wang and Xingfeng Li. J. Genet. 95, 433–439. Table 1. S. miltiorrhiza genes and primer pairs used for qRT-PCR. Gene. GenBank accession. Primer name.
Germanguz, I; Listgarten, J; Cinkornpumin, J; Solomon, A; Gaeta, X; Lowry, W E
Using a compendium of cell-state-specific gene expression data, we identified genes that uniquely define cell states, including those thought to represent various developmental stages. Our analysis sheds light on human cell fate through the identification of core genes that are altered over several developmental milestones, and across regional specification. Here we present cell-type specific gene expression data for 17 distinct cell states and demonstrate that these modules of genes can in fact define cell fate. Lastly, we introduce a web-based database to disseminate the results. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
Full Text Available The analysis of genes in evolutionarily distant but morphologically similar species is of major importance to unravel the changes in genomes over millions of years, which led to gene silencing and functional diversification. We report the analysis of Wnt8a gene expression in the medakafish and provide a detailed comparison to other vertebrates. In all teleosts analyzed there are two paralogous Wnt8a copies. These show largely overlapping expression in the early developing zebrafish embryo, an evolutionarily distant relative of medaka. In contrast to zebrafish, we find that both maternal and zygotic expression of particularly one Wnt8a paralog has diverged in medaka. While Wnt8a1 expression is mostly conserved at early embryonic stages, the expression of Wnt8a2 differs markedly. In addition, both genes are distinctly expressed during organogenesis unlike the zebrafish homologs, which may hint at the emergence of functional diversification of Wnt8a ligands during evolution.
Three studies of gene expression during the division cycle of Schizosaccharomyces pombe led to the proposal that a large number of genes are expressed at particular times during the S. pombe cell cycle. Yet only a small fraction of genes proposed to be expressed in a cell-cycle-dependent manner are reproducible in all three published studies. In addition to reproducibility problems, questions about expression amplitudes, cell-cycle timing of expression, synchronization artifacts, and the problem with methods for synchronizing cells must be considered. These problems and complications prompt the idea that caution should be used before accepting the conclusion that there are a large number of genes expressed in a cell-cycle-dependent manner in S. pombe.
Johansen, Steinar D; Haugen, Peik; Nielsen, Henrik
Ribosomal DNA (rDNA) is a specialised chromosomal location that is dedicated to high-level transcription of ribosomal RNA genes. Interestingly, rDNAs are frequently interrupted by parasitic elements, some of which carry protein genes. These are non-LTR retrotransposons and group II introns...... that encode reverse transcriptase-like genes, and group I introns and archaeal introns that encode homing endonuclease genes (HEGs). Although rDNA-embedded protein genes are widespread in nuclei, organelles and bacteria, there is surprisingly little information available on how these genes are expressed....... Exceptions include a handful of HEGs from group I introns. Recent studies have revealed unusual and essential roles of group I and group I-like ribozymes in the endogenous expression of HEGs. Here we discuss general aspects of rDNA-embedded protein genes and focus on HEG expression from group I introns...
Full Text Available During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that can be divided into three general stages: immediate-early (IE, early (E and late (L. This expression program is the result of a complex interplay between viral and cellular factors at both the transcriptional and post-transcriptional levels, as well as structural differences within the promoter architecture for each of the three gene classes. Since the cellular enzyme RNA polymerase II (RNAP-II is responsible for the transcription of herpesvirus genes, most viral promoters contain DNA motifs that are common with those of cellular genes, although promoter complexity decreases from immediate-early to late genes. Immediate-early and early promoters contain numerous cellular and viral cis-regulating sequences upstream of a TATA box, whereas late promoters differ significantly in that they lack cis-acting sequences upstream of the Transcription Start Site (TSS. Moreover, in the case of the β- and γ-herpesviruses, a TATT box motif is frequently found in the position where the consensus TATA box of eukaryotic promoters usually localizes. The mechanisms of transcriptional regulation of the late viral gene promoters appear to be different between α-herpesviruses and the two other herpesvirus subfamilies ( and . In this review, we will compare the mechanisms of late gene transcriptional regulation between HSV-1, for which the viral IE transcription factors - especially ICP4 - play an essential role, and the two other subfamilies of herpesviruses, with a particular emphasis on EBV, which has recently been found to code for its own specific TATT-binding protein.
Gui, Shupeng; Rice, Andrew P; Chen, Rui; Wu, Liang; Liu, Ji; Miao, Hongyu
Gene regulatory interactions are of fundamental importance to various biological functions and processes. However, only a few previous computational studies have claimed success in revealing genome-wide regulatory landscapes from temporal gene expression data, especially for complex eukaryotes like human. Moreover, recent work suggests that these methods still suffer from the curse of dimensionality if a network size increases to 100 or higher. Here we present a novel scalable algorithm for identifying genome-wide gene regulatory network (GRN) structures, and we have verified the algorithm performances by extensive simulation studies based on the DREAM challenge benchmark data. The highlight of our method is that its superior performance does not degenerate even for a network size on the order of 10(4), and is thus readily applicable to large-scale complex networks. Such a breakthrough is achieved by considering both prior biological knowledge and multiple topological properties (i.e., sparsity and hub gene structure) of complex networks in the regularized formulation. We also validate and illustrate the application of our algorithm in practice using the time-course gene expression data from a study on human respiratory epithelial cells in response to influenza A virus (IAV) infection, as well as the CHIP-seq data from ENCODE on transcription factor (TF) and target gene interactions. An interesting finding, owing to the proposed algorithm, is that the biggest hub structures (e.g., top ten) in the GRN all center at some transcription factors in the context of epithelial cell infection by IAV. The proposed algorithm is the first scalable method for large complex network structure identification. The GRN structure identified by our algorithm could reveal possible biological links and help researchers to choose which gene functions to investigate in a biological event. The algorithm described in this article is implemented in MATLAB (Ⓡ) , and the source code is
Johnson, Steven B.; Lissauer, Matthew; Bochicchio, Grant V.; Moore, Richard; Cross, Alan S.; Scalea, Thomas M.
Introduction: The systemic inflammatory response syndrome (SIRS) occurs frequently in critically ill patients and presents similar clinical appearances despite diverse infectious and noninfectious etiologies. Despite similar phenotypic expression, these diverse SIRS etiologies may induce divergent genotypic expressions. We hypothesized that gene expression differences are present between sepsis and uninfected SIRS prior to the clinical appearance of sepsis. Methods: Critically ill uninfected SIRS patients were followed longitudinally for the development of sepsis. All patients had whole blood collected daily for gene expression analysis by Affymetrix Hg_U133 2.0 Plus microarrays. SIRS patients developing sepsis were compared with those remaining uninfected for differences in gene expression at study entry and daily for 3 days prior to conversion to sepsis. Acceptance criteria for differentially expressed genes required: >1.2 median fold change between groups and significance on univariate and multivariate analysis. Differentially expressed genes were annotated to pathways using DAVID 2.0/EASE analysis. Results: A total of 12,782 (23.4%) gene probes were differentially expressed on univariate analysis 0 to 48 hours before clinical sepsis. 626 (1.1%) probes met acceptance criteria, corresponding to 459 unique genes, 65 (14.2%) down and 395 (85.8%) up expressed. These genes annotated to 10 pathways that functionally categorized to 4 themes involving innate immunity, cytokine receptors, T cell differentiation, and protein synthesis regulation. Conclusions: Sepsis has a unique gene expression profile that is different from uninfected inflammation and becomes apparent prior to expression of the clinical sepsis phenotype. PMID:17414611
Johnson Dianna A
Full Text Available Abstract Background Retinal pigment epithelium cells play an important role in the pathogenesis of age related macular degeneration. Their morphological, molecular and functional phenotype changes in response to various stresses. Functional profiling of genes can provide useful information about the physiological state of cells and how this state changes in response to disease or treatment. In this study, we have constructed a functional profile of the genes expressed by the ARPE-19 cell line of retinal pigment epithelium. Methods Using Affymetrix MAS 5.0 microarray analysis, genes expressed by ARPE-19 cells were identified. Using GeneChip® annotations, these genes were classified according to their known functions to generate a functional gene expression profile. Results We have determined that of approximately 19,044 unique gene sequences represented on the HG-U133A GeneChip® , 6,438 were expressed in ARPE-19 cells irrespective of the substrate on which they were grown (plastic, fibronectin, collagen, or Matrigel. Rather than focus our subsequent analysis on the identity or level of expression of each individual gene in this large data set, we examined the number of genes expressed within 130 functional categories. These categories were selected from a library of HG-U133A GeneChip® annotations linked to the Affymetrix MAS 5.0 data sets. Using this functional classification scheme, we were able to categorize about 70% of the expressed genes and condense the original data set of over 6,000 data points into a format with 130 data points. The resulting ARPE-19 Functional Gene Expression Profile is displayed as a percentage of ARPE-19-expressed genes. Conclusion The Profile can readily be compared with equivalent microarray data from other appropriate samples in order to highlight cell-specific attributes or treatment-induced changes in gene expression. The usefulness of these analyses is based on the assumption that the numbers of genes