EST Express: PHP/MySQL based automated annotation of ESTs from expression libraries

PubMed Central

Smith, Robin P; Buchser, William J; Lemmon, Marcus B; Pardinas, Jose R; Bixby, John L; Lemmon, Vance P

2008-01-01

Background Several biological techniques result in the acquisition of functional sets of cDNAs that must be sequenced and analyzed. The emergence of redundant databases such as UniGene and centralized annotation engines such as Entrez Gene has allowed the development of software that can analyze a great number of sequences in a matter of seconds. Results We have developed "EST Express", a suite of analytical tools that identify and annotate ESTs originating from specific mRNA populations. The software consists of a user-friendly GUI powered by PHP and MySQL that allows for online collaboration between researchers and continuity with UniGene, Entrez Gene and RefSeq. Two key features of the software include a novel, simplified Entrez Gene parser and tools to manage cDNA library sequencing projects. We have tested the software on a large data set (2,016 samples) produced by subtractive hybridization. Conclusion EST Express is an open-source, cross-platform web server application that imports sequences from cDNA libraries, such as those generated through subtractive hybridization or yeast two-hybrid screens. It then provides several layers of annotation based on Entrez Gene and RefSeq to allow the user to highlight useful genes and manage cDNA library projects. PMID:18402700

EST Express: PHP/MySQL based automated annotation of ESTs from expression libraries.

PubMed

Smith, Robin P; Buchser, William J; Lemmon, Marcus B; Pardinas, Jose R; Bixby, John L; Lemmon, Vance P

2008-04-10

Several biological techniques result in the acquisition of functional sets of cDNAs that must be sequenced and analyzed. The emergence of redundant databases such as UniGene and centralized annotation engines such as Entrez Gene has allowed the development of software that can analyze a great number of sequences in a matter of seconds. We have developed "EST Express", a suite of analytical tools that identify and annotate ESTs originating from specific mRNA populations. The software consists of a user-friendly GUI powered by PHP and MySQL that allows for online collaboration between researchers and continuity with UniGene, Entrez Gene and RefSeq. Two key features of the software include a novel, simplified Entrez Gene parser and tools to manage cDNA library sequencing projects. We have tested the software on a large data set (2,016 samples) produced by subtractive hybridization. EST Express is an open-source, cross-platform web server application that imports sequences from cDNA libraries, such as those generated through subtractive hybridization or yeast two-hybrid screens. It then provides several layers of annotation based on Entrez Gene and RefSeq to allow the user to highlight useful genes and manage cDNA library projects.

Preparing and Analyzing Expressed Sequence Tags (ESTs) Library for the Mammary Tissue of Local Turkish Kivircik Sheep

PubMed Central

Omeroglu Ulu, Zehra; Ulu, Salih; Un, Cemal; Ozdem Oztabak, Kemal; Altunatmaz, Kemal

2017-01-01

Kivircik sheep is an important local Turkish sheep according to its meat quality and milk productivity. The aim of this study was to analyze gene expression profiles of both prenatal and postnatal stages for the Kivircik sheep. Therefore, two different cDNA libraries, which were taken from the same Kivircik sheep mammary gland tissue at prenatal and postnatal stages, were constructed. Total 3072 colonies which were randomly selected from the two libraries were sequenced for developing a sheep ESTs collection. We used Phred/Phrap computer programs for analysis of the raw EST and readable EST sequences were assembled with the CAP3 software. Putative functions of all unique sequences and statistical analysis were determined by Geneious software. Total 422 ESTs have over 80% similarity to known sequences of other organisms in NCBI classified by Panther database for the Gene Ontology (GO) category. By comparing gene expression profiles, we observed some putative genes that may be relative to reproductive performance or play important roles in milk synthesis and secretion. A total of 2414 ESTs have been deposited to the NCBI GenBank database (GW996847–GW999260). EST data in this study have provided a new source of information to functional genome studies of sheep. PMID:28239610

Advanced colorectal adenoma related gene expression signature may predict prognostic for colorectal cancer patients with adenoma-carcinoma sequence.

PubMed

Li, Bing; Shi, Xiao-Yu; Liao, Dai-Xiang; Cao, Bang-Rong; Luo, Cheng-Hua; Cheng, Shu-Jun

2015-01-01

There are still no absolute parameters predicting progression of adenoma into cancer. The present study aimed to characterize functional differences on the multistep carcinogenetic process from the adenoma-carcinoma sequence. All samples were collected and mRNA expression profiling was performed by using Agilent Microarray high-throughput gene-chip technology. Then, the characteristics of mRNA expression profiles of adenoma-carcinoma sequence were described with bioinformatics software, and we analyzed the relationship between gene expression profiles of adenoma-adenocarcinoma sequence and clinical prognosis of colorectal cancer. The mRNA expressions of adenoma-carcinoma sequence were significantly different between high-grade intraepithelial neoplasia group and adenocarcinoma group. The biological process of gene ontology function enrichment analysis on differentially expressed genes between high-grade intraepithelial neoplasia group and adenocarcinoma group showed that genes enriched in the extracellular structure organization, skeletal system development, biological adhesion and itself regulated growth regulation, with the P value after FDR correction of less than 0.05. In addition, IPR-related protein mainly focused on the insulin-like growth factor binding proteins. The variable trends of gene expression profiles for adenoma-carcinoma sequence were mainly concentrated in high-grade intraepithelial neoplasia and adenocarcinoma. The differentially expressed genes are significantly correlated between high-grade intraepithelial neoplasia group and adenocarcinoma group. Bioinformatics analysis is an effective way to study the gene expression profiles in the adenoma-carcinoma sequence, and may provide an effective tool to involve colorectal cancer research strategy into colorectal adenoma or advanced adenoma.

Both positive and negative regulatory elements mediate expression of a photoregulated CAB gene from Nicotiana plumbaginifolia.

PubMed Central

Castresana, C; Garcia-Luque, I; Alonso, E; Malik, V S; Cashmore, A R

1988-01-01

We have analyzed promoter regulatory elements from a photoregulated CAB gene (Cab-E) isolated from Nicotiana plumbaginifolia. These studies have been performed by introducing chimeric gene constructs into tobacco cells via Agrobacterium tumefaciens-mediated transformation. Expression studies on the regenerated transgenic plants have allowed us to characterize three positive and one negative cis-acting elements that influence photoregulated expression of the Cab-E gene. Within the upstream sequences we have identified two positive regulatory elements (PRE1 and PRE2) which confer maximum levels of photoregulated expression. These sequences contain multiple repeated elements related to the sequence-ACCGGCCCACTT-. We have also identified within the upstream region a negative regulatory element (NRE) extremely rich in AT sequences, which reduces the level of gene expression in the light. We have defined a light regulatory element (LRE) within the promoter region extending from -396 to -186 bp which confers photoregulated expression when fused to a constitutive nopaline synthase ('nos') promoter. Within this region there is a 132-bp element, extending from -368 to -234 bp, which on deletion from the Cab-E promoter reduces gene expression from high levels to undetectable levels. Finally, we have demonstrated for a full length Cab-E promoter conferring high levels of photoregulated expression, that sequences proximal to the Cab-E TATA box are not replaceable by corresponding sequences from a 'nos' promoter. This contrasts with the apparent equivalence of these Cab-E and 'nos' TATA box-proximal sequences in truncated promoters conferring low levels of photoregulated expression. Images PMID:2901343

Cell-free translational screening of an expression sequence tag library of Clonorchis sinensis for novel antigen discovery.

PubMed

Kasi, Devi; Catherine, Christy; Lee, Seung-Won; Lee, Kyung-Ho; Kim, Yu Jung; Ro Lee, Myeong; Ju, Jung Won; Kim, Dong-Myung

2017-05-01

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite-related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell-free protein synthesis system for large-scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high-throughput expression and identification of individual genes comprising the library, a cell-free synthesis reaction was designed such that both the template DNA and the expressed proteins were co-immobilized on the same microbeads, leading to microbead-based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832-837, 2017. © 2017 American Institute of Chemical Engineers.

Comparative transcriptome analysis of microsclerotia development in Nomuraea rileyi

PubMed Central

2013-01-01

Background Nomuraea rileyi is used as an environmental-friendly biopesticide. However, mass production and commercialization of this organism are limited due to its fastidious growth and sporulation requirements. When cultured in amended medium, we found that N. rileyi could produce microsclerotia bodies, replacing conidiophores as the infectious agent. However, little is known about the genes involved in microsclerotia development. In the present study, the transcriptomes were analyzed using next-generation sequencing technology to find the genes involved in microsclerotia development. Results A total of 4.69 Gb of clean nucleotides comprising 32,061 sequences was obtained, and 20,919 sequences were annotated (about 65%). Among the annotated sequences, only 5928 were annotated with 34 gene ontology (GO) functional categories, and 12,778 sequences were mapped to 165 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) database. Furthermore, we assessed the transcriptomic differences between cultures grown in minimal and amended medium. In total, 4808 sequences were found to be differentially expressed; 719 differentially expressed unigenes were assigned to 25 GO classes and 1888 differentially expressed unigenes were assigned to 161 KEGG pathways, including 25 enrichment pathways. Subsequently, we examined the up-regulation or uniquely expressed genes following amended medium treatment, which were also expressed on the enrichment pathway, and found that most of them participated in mediating oxidative stress homeostasis. To elucidate the role of oxidative stress in microsclerotia development, we analyzed the diversification of unigenes using quantitative reverse transcription-PCR (RT-qPCR). Conclusion Our findings suggest that oxidative stress occurs during microsclerotia development, along with a broad metabolic activity change. Our data provide the most comprehensive sequence resource available for the study of N. rileyi. We believe that the transcriptome datasets will serve as an important public information platform to accelerate studies on N. rileyi microsclerotia. PMID:23777366

Molecular cloning and expression analysis of annexin A2 gene in sika deer antler tip.

PubMed

Xia, Yanling; Qu, Haomiao; Lu, Binshan; Zhang, Qiang; Li, Heping

2018-04-01

Molecular cloning and bioinformatics analysis of annexin A2 ( ANXA2 ) gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer ( Cervus Nippon hortulorum ) and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus . Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period). ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

Building a dictionary for genomes: Identification of presumptive regulatory sites by statistical analysis

PubMed Central

Bussemaker, Harmen J.; Li, Hao; Siggia, Eric D.

2000-01-01

The availability of complete genome sequences and mRNA expression data for all genes creates new opportunities and challenges for identifying DNA sequence motifs that control gene expression. An algorithm, “MobyDick,” is presented that decomposes a set of DNA sequences into the most probable dictionary of motifs or words. This method is applicable to any set of DNA sequences: for example, all upstream regions in a genome or all genes expressed under certain conditions. Identification of words is based on a probabilistic segmentation model in which the significance of longer words is deduced from the frequency of shorter ones of various lengths, eliminating the need for a separate set of reference data to define probabilities. We have built a dictionary with 1,200 words for the 6,000 upstream regulatory regions in the yeast genome; the 500 most significant words (some with as few as 10 copies in all of the upstream regions) match 114 of 443 experimentally determined sites (a significance level of 18 standard deviations). When analyzing all of the genes up-regulated during sporulation as a group, we find many motifs in addition to the few previously identified by analyzing the subclusters individually to the expression subclusters. Applying MobyDick to the genes derepressed when the general repressor Tup1 is deleted, we find known as well as putative binding sites for its regulatory partners. PMID:10944202

RNA-ID, a Powerful Tool for Identifying and Characterizing Regulatory Sequences.

PubMed

Brule, C E; Dean, K M; Grayhack, E J

2016-01-01

The identification and analysis of sequences that regulate gene expression is critical because regulated gene expression underlies biology. RNA-ID is an efficient and sensitive method to discover and investigate regulatory sequences in the yeast Saccharomyces cerevisiae, using fluorescence-based assays to detect green fluorescent protein (GFP) relative to a red fluorescent protein (RFP) control in individual cells. Putative regulatory sequences can be inserted either in-frame or upstream of a superfolder GFP fusion protein whose expression, like that of RFP, is driven by the bidirectional GAL1,10 promoter. In this chapter, we describe the methodology to identify and study cis-regulatory sequences in the RNA-ID system, explaining features and variations of the RNA-ID reporter, as well as some applications of this system. We describe in detail the methods to analyze a single regulatory sequence, from construction of a single GFP variant to assay of variants by flow cytometry, as well as modifications required to screen libraries of different strains simultaneously. We also describe subsequent analyses of regulatory sequences. © 2016 Elsevier Inc. All rights reserved.

Differential gene expression in dentate granule cells in mesial temporal lobe epilepsy with and without hippocampal sclerosis.

PubMed

Griffin, Nicole G; Wang, Yu; Hulette, Christine M; Halvorsen, Matt; Cronin, Kenneth D; Walley, Nicole M; Haglund, Michael M; Radtke, Rodney A; Skene, J H Pate; Sinha, Saurabh R; Heinzen, Erin L

2016-03-01

Hippocampal sclerosis is the most common neuropathologic finding in cases of medically intractable mesial temporal lobe epilepsy. In this study, we analyzed the gene expression profiles of dentate granule cells of patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis to show that next-generation sequencing methods can produce interpretable genomic data from RNA collected from small homogenous cell populations, and to shed light on the transcriptional changes associated with hippocampal sclerosis. RNA was extracted, and complementary DNA (cDNA) was prepared and amplified from dentate granule cells that had been harvested by laser capture microdissection from surgically resected hippocampi from patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis. Sequencing libraries were sequenced, and the resulting sequencing reads were aligned to the reference genome. Differential expression analysis was used to ascertain expression differences between patients with and without hippocampal sclerosis. Greater than 90% of the RNA-Seq reads aligned to the reference. There was high concordance between transcriptional profiles obtained for duplicate samples. Principal component analysis revealed that the presence or absence of hippocampal sclerosis was the main determinant of the variance within the data. Among the genes up-regulated in the hippocampal sclerosis samples, there was significant enrichment for genes involved in oxidative phosphorylation. By analyzing the gene expression profiles of dentate granule cells from surgically resected hippocampal specimens from patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis, we have demonstrated the utility of next-generation sequencing methods for producing biologically relevant results from small populations of homogeneous cells, and have provided insight on the transcriptional changes associated with this pathology. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene.

PubMed Central

de Vries, G E; Arfman, N; Terpstra, P; Dijkhuizen, L

1992-01-01

The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced. The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes. Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence. After purification of MDH from E. coli, the kinetic and biochemical properties of the enzyme were investigated. The physiological effect of MDH synthesis in E. coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed. Images PMID:1644761

Geoseq: a tool for dissecting deep-sequencing datasets.

PubMed

Gurtowski, James; Cancio, Anthony; Shah, Hardik; Levovitz, Chaya; George, Ajish; Homann, Robert; Sachidanandam, Ravi

2010-10-12

Datasets generated on deep-sequencing platforms have been deposited in various public repositories such as the Gene Expression Omnibus (GEO), Sequence Read Archive (SRA) hosted by the NCBI, or the DNA Data Bank of Japan (ddbj). Despite being rich data sources, they have not been used much due to the difficulty in locating and analyzing datasets of interest. Geoseq http://geoseq.mssm.edu provides a new method of analyzing short reads from deep sequencing experiments. Instead of mapping the reads to reference genomes or sequences, Geoseq maps a reference sequence against the sequencing data. It is web-based, and holds pre-computed data from public libraries. The analysis reduces the input sequence to tiles and measures the coverage of each tile in a sequence library through the use of suffix arrays. The user can upload custom target sequences or use gene/miRNA names for the search and get back results as plots and spreadsheet files. Geoseq organizes the public sequencing data using a controlled vocabulary, allowing identification of relevant libraries by organism, tissue and type of experiment. Analysis of small sets of sequences against deep-sequencing datasets, as well as identification of public datasets of interest, is simplified by Geoseq. We applied Geoseq to, a) identify differential isoform expression in mRNA-seq datasets, b) identify miRNAs (microRNAs) in libraries, and identify mature and star sequences in miRNAS and c) to identify potentially mis-annotated miRNAs. The ease of using Geoseq for these analyses suggests its utility and uniqueness as an analysis tool.

dictyExpress: a web-based platform for sequence data management and analytics in Dictyostelium and beyond.

PubMed

Stajdohar, Miha; Rosengarten, Rafael D; Kokosar, Janez; Jeran, Luka; Blenkus, Domen; Shaulsky, Gad; Zupan, Blaz

2017-06-02

Dictyostelium discoideum, a soil-dwelling social amoeba, is a model for the study of numerous biological processes. Research in the field has benefited mightily from the adoption of next-generation sequencing for genomics and transcriptomics. Dictyostelium biologists now face the widespread challenges of analyzing and exploring high dimensional data sets to generate hypotheses and discovering novel insights. We present dictyExpress (2.0), a web application designed for exploratory analysis of gene expression data, as well as data from related experiments such as Chromatin Immunoprecipitation sequencing (ChIP-Seq). The application features visualization modules that include time course expression profiles, clustering, gene ontology enrichment analysis, differential expression analysis and comparison of experiments. All visualizations are interactive and interconnected, such that the selection of genes in one module propagates instantly to visualizations in other modules. dictyExpress currently stores the data from over 800 Dictyostelium experiments and is embedded within a general-purpose software framework for management of next-generation sequencing data. dictyExpress allows users to explore their data in a broader context by reciprocal linking with dictyBase-a repository of Dictyostelium genomic data. In addition, we introduce a companion application called GenBoard, an intuitive graphic user interface for data management and bioinformatics analysis. dictyExpress and GenBoard enable broad adoption of next generation sequencing based inquiries by the Dictyostelium research community. Labs without the means to undertake deep sequencing projects can mine the data available to the public. The entire information flow, from raw sequence data to hypothesis testing, can be accomplished in an efficient workspace. The software framework is generalizable and represents a useful approach for any research community. To encourage more wide usage, the backend is open-source, available for extension and further development by bioinformaticians and data scientists.

Definition of Cis-Acting Elements Regulating Expression of the Drosophila Melanogaster Ninae Opsin Gene by Oligonucleotide-Directed Mutagenesis

PubMed Central

Mismer, D.; Rubin, G. M.

1989-01-01

We have analyzed the cis-acting regulatory sequences of the Rh1 (ninaE) gene in Drosophila melanogaster by P-element-mediated germline transformation of indicator genes transcribed from mutant ninaE promoter sequences. We have previously shown that a 200-bp region extending from -120 to +67 relative to the transcription start site is sufficient to obtain eye-specific expression from the ninaE promoter. In the present study, 22 different 4-13-bp sequences in the -120/+67 promoter region were altered by oligonucleotide-directed mutagenesis. Several of these sequences were found to be required for proper promoter function; two of these are conserved in the promoter of the homologous gene isolated from the related species Drosophila virilis. Alteration of a conserved 9-bp sequence results in aberrant, low level expression in the body. Alteration of a separate 11-bp sequence, found in the promoter regions of several photoreceptor-specific genes of Drosophila, results in an approximately 15-fold reduction in promoter efficiency but without apparent alteration of tissue-specificity. A protein factor capable of interacting with this 11-bp sequence has been detected by DNaseI footprinting in embryonic nuclear extracts. Finally, we have further characterized two separable enhancer sequences previously shown to be required for normal levels of expression from this promoter. PMID:2521839

Control of total GFP expression by alterations to the 3′ region nucleotide sequence

PubMed Central

2013-01-01

Background Previously, we distinguished the Escherichia coli type II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for unfolded and folded soluble target proteins. The translocation of folded protein to the periplasm for soluble expression via the Tat pathway was controlled by an N-terminal hydrophilic leader sequence. In this study, we investigated the effect of the hydrophilic C-terminal end and its nucleotide sequence on total and soluble protein expression. Results The native hydrophilic C-terminal end of GFP was obtained by deleting the C-terminal peptide LeuGlu-6×His, derived from pET22b(+). The corresponding clones induced total and soluble GFP expression that was either slightly increased or dramatically reduced, apparently through reconstruction of the nucleotide sequence around the stop codon in the 3′ region. In the expression-induced clones, the hydrophilic C-terminus showed increased Tat pathway specificity for soluble expression. However, in the expression-reduced clone, after analyzing the role of the 5′ poly(A) coding sequence with a substituted synonymous codon, we proved that the longer 5′ poly(A) coding sequence interacted with the reconstructed 3′ region nucleotide sequence to create a new mRNA tertiary structure between the 5′ and 3′ regions, which resulted in reduced total GFP expression. Further, to recover the reduced expression by changing the 3′ nucleotide sequence, after replacing selected C-terminal 5′ codons and the stop codon in the ORF with synonymous codons, total GFP expression in most of the clones was recovered to the undeleted control level. The insertion of trinucleotides after the stop codon in the 3′-UTR recovered or reduced total GFP expression. RT-PCR revealed that the level of total protein expression was controlled by changes in translational or transcriptional regulation, which were induced or reduced by the substitution or insertion of 3′ region nucleotides. Conclusions We found that the hydrophilic C-terminal end of GFP increased Tat pathway specificity and that the 3′ nucleotide sequence played an important role in total protein expression through translational and transcriptional regulation. These findings may be useful for efficiently producing recombinant proteins as well as for potentially controlling the expression level of specific genes in the body for therapeutic purposes. PMID:23834827

Expression of myostatin is not altered in lines of poultry exhibiting myofiber hyper- and hypoplasia.

PubMed

Mott, I; Ivarie, R

2002-06-01

Decades of selective breeding have yielded lines of poultry with substantial myofiber hyperplasia, vet little is known about what genes have been altered during the course of selection. Myostatin is a strong negative regulator of muscle mass in mice and cattle and could have been one of many genetic factors contributing to increased myofiber deposition in growth-selected lines of poultry. To test this hypothesis, the sequence and expression patterns of myostatin were analyzed in growth-selected lines of chickens and quail. The sequence of broiler myostatin cDNA, amplified via reverse transcription (RT)-PCR from embryonic muscle RNA, contained no missense mutations in the coding sequence when compared to that of White Leghorn layers, although two silent single nucleotide polymorphisms (SNP) were found. Northern analysis of myostatin transcripts from embryonic pectoralis and quadriceps showed no significant differences in expression levels between broiler and layer muscle RNA. However, levels of myostatin transcripts were greatly reduced in muscles of posthatch chicks compared to embryonic muscle. Myostatin protein was also present in broiler and layer embryonic muscle at similar levels. No significant polymorphisms or differences in RNA expression levels were found in embryonic muscles of divergently selected lines of Japanese quail. These results indicate that intense artificial selection in these growth-selected lines of poultry has neither silenced the expression of myostatin nor created null alleles via mutation in the lines analyzed.

ISRNA: an integrative online toolkit for short reads from high-throughput sequencing data.

PubMed

Luo, Guan-Zheng; Yang, Wei; Ma, Ying-Ke; Wang, Xiu-Jie

2014-02-01

Integrative Short Reads NAvigator (ISRNA) is an online toolkit for analyzing high-throughput small RNA sequencing data. Besides the high-speed genome mapping function, ISRNA provides statistics for genomic location, length distribution and nucleotide composition bias analysis of sequence reads. Number of reads mapped to known microRNAs and other classes of short non-coding RNAs, coverage of short reads on genes, expression abundance of sequence reads as well as some other analysis functions are also supported. The versatile search functions enable users to select sequence reads according to their sub-sequences, expression abundance, genomic location, relationship to genes, etc. A specialized genome browser is integrated to visualize the genomic distribution of short reads. ISRNA also supports management and comparison among multiple datasets. ISRNA is implemented in Java/C++/Perl/MySQL and can be freely accessed at http://omicslab.genetics.ac.cn/ISRNA/.

Molecular Cloning and Sequencing of Channel Catfish, Ictalurus punctatus, Cathepsin H and L cDNA

USDA-ARS?s Scientific Manuscript database

Cathepsin H and L, a lysosomal cysteine endopeptidase of the papain family, are ubiquitously expressed and involve in antigen processing. In this communication, the channel catfish cathepsin H and L transcripts were sequenced and analyzed. Total RNA from tissues was extracted and cDNA libraries we...

PROSPECT improves cis-acting regulatory element prediction by integrating expression profile data with consensus pattern searches

PubMed Central

Fujibuchi, Wataru; Anderson, John S. J.; Landsman, David

2001-01-01

Consensus pattern and matrix-based searches designed to predict cis-acting transcriptional regulatory sequences have historically been subject to large numbers of false positives. We sought to decrease false positives by incorporating expression profile data into a consensus pattern-based search method. We have systematically analyzed the expression phenotypes of over 6000 yeast genes, across 121 expression profile experiments, and correlated them with the distribution of 14 known regulatory elements over sequences upstream of the genes. Our method is based on a metric we term probabilistic element assessment (PEA), which is a ranking of potential sites based on sequence similarity in the upstream regions of genes with similar expression phenotypes. For eight of the 14 known elements that we examined, our method had a much higher selectivity than a naïve consensus pattern search. Based on our analysis, we have developed a web-based tool called PROSPECT, which allows consensus pattern-based searching of gene clusters obtained from microarray data. PMID:11574681

RNA sequencing to study gene expression and SNP variations associated with growth in zebrafish fed a plant protein-based diet.

PubMed

Ulloa, Pilar E; Rincón, Gonzalo; Islas-Trejo, Alma; Araneda, Cristian; Iturra, Patricia; Neira, Roberto; Medrano, Juan F

2015-06-01

The objectives of this study were to measure gene expression in zebrafish and then identify SNP to be used as potential markers in a growth association study. We developed an approach where muscle samples collected from low- and high-growth fish were analyzed using RNA-Sequencing (RNA-seq), and SNP were chosen from the genes that were differentially expressed between the low and high groups. A population of 24 families was fed a plant protein-based diet from the larval to adult stages. From a total of 440 males, 5 % of the fish from both tails of the weight gain distribution were selected. Total RNA was extracted from individual muscle of 8 low-growth and 8 high-growth fish. Two pooled RNA-Seq libraries were prepared for each phenotype using 4 fish per library. Libraries were sequenced using the Illumina GAII Sequencer and analyzed using the CLCBio genomic workbench software. One hundred and twenty-four genes were differentially expressed between phenotypes (p value < 0.05 and FDR < 0.2). From these genes, 164 SNP were selected and genotyped in 240 fish samples. Marker-trait analysis revealed 5 SNP associated with growth in key genes (Nars, Lmod2b, Cuzd1, Acta1b, and Plac8l1). These genes are good candidates for further growth studies in fish and to consider for identification of potential SNPs associated with different growth rates in response to a plant protein-based diet.

GENE-Counter: A Computational Pipeline for the Analysis of RNA-Seq Data for Gene Expression Differences

PubMed Central

Di, Yanming; Schafer, Daniel W.; Wilhelm, Larry J.; Fox, Samuel E.; Sullivan, Christopher M.; Curzon, Aron D.; Carrington, James C.; Mockler, Todd C.; Chang, Jeff H.

2011-01-01

GENE-counter is a complete Perl-based computational pipeline for analyzing RNA-Sequencing (RNA-Seq) data for differential gene expression. In addition to its use in studying transcriptomes of eukaryotic model organisms, GENE-counter is applicable for prokaryotes and non-model organisms without an available genome reference sequence. For alignments, GENE-counter is configured for CASHX, Bowtie, and BWA, but an end user can use any Sequence Alignment/Map (SAM)-compliant program of preference. To analyze data for differential gene expression, GENE-counter can be run with any one of three statistics packages that are based on variations of the negative binomial distribution. The default method is a new and simple statistical test we developed based on an over-parameterized version of the negative binomial distribution. GENE-counter also includes three different methods for assessing differentially expressed features for enriched gene ontology (GO) terms. Results are transparent and data are systematically stored in a MySQL relational database to facilitate additional analyses as well as quality assessment. We used next generation sequencing to generate a small-scale RNA-Seq dataset derived from the heavily studied defense response of Arabidopsis thaliana and used GENE-counter to process the data. Collectively, the support from analysis of microarrays as well as the observed and substantial overlap in results from each of the three statistics packages demonstrates that GENE-counter is well suited for handling the unique characteristics of small sample sizes and high variability in gene counts. PMID:21998647

Differential protein expression in alligator leukocytes in response to bacterial lipopolysaccharide injection.

PubMed

Merchant, Mark; Kinney, Clint; Sanders, Paige

2009-12-01

Blood was collected from three juvenile alligators (Alligator mississippiensis) before, and again 24h after, injection with bacterial lipopolysaccharide (LPS). The leukocytes were collected from both samples, and the proteins were extracted. Each group of proteins was labeled with a different fluorescent dye and the differences in protein expression were analyzed by two dimensional differential in-gel expressions (2D-DIGE). The proteins which appeared to be increased or decreased by treatment with LPS were selected and analyzed by MALDI-TOF to determine mass and LC-MS/MS to acquire the partial protein sequences. The peptide sequences were compared to the NCBI protein sequence database to determine homology with other sequences from other species. Several proteins of interest appeared to be increased upon LPS stimulation. Proteins with homology to human transgelin-2, fish glucose-6-phosphate dehydrogenase, amphibian α-enolase, alligator lactate dehydrogenase, fish ubiquitin-activating enzyme, and fungal β-tubulin were also increased after LPS injection. Proteins with homology to fish vimentin 4, murine heterogeneous nuclear ribonucleoprotein A3, and avian calreticulin were found to be decreased in response to LPS. In addition, five proteins, four of which were up-regulated (827, 560, 512, and 650%) and one that exhibited repressed expression (307%), did not show homology to any protein in the database, and thus may represent newly discovered proteins. We are using this biochemical approach to isolate and characterize alligator proteins with potential relevant immune function.

De novo Transcriptome Assembly of Chinese Kale and Global Expression Analysis of Genes Involved in Glucosinolate Metabolism in Multiple Tissues

PubMed Central

Wu, Shuanghua; Lei, Jianjun; Chen, Guoju; Chen, Hancai; Cao, Bihao; Chen, Changming

2017-01-01

Chinese kale, a vegetable of the cruciferous family, is a popular crop in southern China and Southeast Asia due to its high glucosinolate content and nutritional qualities. However, there is little research on the molecular genetics and genes involved in glucosinolate metabolism and its regulation in Chinese kale. In this study, we sequenced and characterized the transcriptomes and expression profiles of genes expressed in 11 tissues of Chinese kale. A total of 216 million 150-bp clean reads were generated using RNA-sequencing technology. From the sequences, 98,180 unigenes were assembled for the whole plant, and 49,582~98,423 unigenes were assembled for each tissue. Blast analysis indicated that a total of 80,688 (82.18%) unigenes exhibited similarity to known proteins. The functional annotation and classification tools used in this study suggested that genes principally expressed in Chinese kale, were mostly involved in fundamental processes, such as cellular and molecular functions, the signal transduction, and biosynthesis of secondary metabolites. The expression levels of all unigenes were analyzed in various tissues of Chinese kale. A large number of candidate genes involved in glucosinolate metabolism and its regulation were identified, and the expression patterns of these genes were analyzed. We found that most of the genes involved in glucosinolate biosynthesis were highly expressed in the root, petiole, and in senescent leaves. The expression patterns of ten glucosinolate biosynthetic genes from RNA-seq were validated by quantitative RT-PCR in different tissues. These results provided an initial and global overview of Chinese kale gene functions and expression activities in different tissues. PMID:28228764

APPLICATION OF DNA MICROARRAYS TO REPRODUCTIVE TOXICOLOGY AND THE DEVELOPMENT OF A TESTIS ARRAY

EPA Science Inventory

With the advent of sequence information for entire mammalian genomes, it is now possible to analyze gene expression and gene polymorphisms on a genomic scale. The primary tool for analysis of gene expression is the DNA microarray. We have used commercially available cDNA micro...

BIOMONITORING THE TOXICOGENOMIC RESPONSE TO ENDOCRINE DISRUPTING CHEMICALS IN HUMANS, LABORATORY SPECIES AND WILDLIFE

EPA Science Inventory

With the advent of sequence information for entire eukaryotic genomes, it is now possible to analyze gene expression on a genomic scale. The primary tool for genomic analysis of gene expression is the gene microarray. We have used commercially available and custom cDNA microarray...

The Silkworm (Bombyx mori) microRNAs and Their Expressions in Multiple Developmental Stages

PubMed Central

Luo, Qibin; Cai, Yimei; Lin, Wen-chang; Chen, Huan; Yang, Yue; Hu, Songnian; Yu, Jun

2008-01-01

Background MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species. Methodology/Principal Findings We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5′ and/or 3′ ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis. Conclusions/Significance Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms. PMID:18714353

Bioinformatic Analysis of the Human Recombinant Iduronate 2-Sulfate Sulfatase

PubMed Central

Morales-Álvarez, Edwin D.; Rivera-Hoyos, Claudia M.; Landázuri, Patricia; Poutou-Piñales, Raúl A.; Pedroza-Rodríguez, Aura M.

2016-01-01

Mucopolysaccharidosis type II is a human recessive disease linked to the X chromosome caused by deficiency of lysosomal enzyme Iduronate 2-Sulfate Sulfatase (IDS), which leads to accumulation of glycosaminoglycans in tissues and organs. The human enzyme has been expressed in Escherichia coli and Pichia pastoris in attempt to develop more successful expression systems that allow the production of recombinant IDS for Enzyme Replacement Therapy (ERT). However, the preservation of native signal peptide in the sequence has caused conflicts in processing and recognition in the past, which led to problems in expression and enzyme activity. With the main object being the improvement of the expression system, we eliminate the native signal peptide of human recombinant IDS. The resulting sequence showed two modified codons, thus, our study aimed to analyze computationally the nucleotide sequence of the IDSnh without signal peptide in order to determine the 3D structure and other biochemical properties to compare them with the native human IDS (IDSnh). Results showed that there are no significant differences between both molecules in spite of the two-codon modifications detected in the recombinant DNA sequence. PMID:27335624

Expression of young HERV-H loci in the course of colorectal carcinoma and correlation with molecular subtypes

PubMed Central

Naville, Magali; Bressan, Cédric; Hühns, Maja; Gock, Michael; Kühn, Florian; Volff, Jean-Nicolas; Trillet-Lenoir, Véronique

2015-01-01

Background Expression of the human endogenous retrovirus (HERV)-H family has been associated with colorectal carcinomas (CRC), yet no individual HERV-H locus expression has been thoroughly correlated with clinical data. Here, we characterized HERV-H reactivations in clinical CRC samples by integrating expression profiles, molecular patterns and clinical data. Expression of relevant HERV-H sequences was analyzed by qRT-PCR on two well-defined clinical cohorts (n = 139 pairs of tumor and adjacent normal colon tissue) including samples from adenomas (n = 21) and liver metastases (n = 16). Correlations with clinical and molecular data were assessed. Results CRC specific HERV-H sequences were validated and found expressed throughout CRC disease progression. Correlations between HERV-H expression and lymph node invasion of tumor cells (p = 0.0006) as well as microsatellite instable tumors (p < 0.0001) were established. No association with regard to age, tumor localization, grading or common mutations became apparent. Interestingly, CRC expressed elements belonged to specific young HERV-H subfamilies and their 5′ LTR often presented active histone marks. Conclusion These results suggest a functional role of HERV-H sequences in colorectal carcinogenesis. The pronounced connection with microsatellite instability warrants a more detailed investigation. Thus, HERV-H sequences in addition to tumor specific mutations may represent clinically relevant, truly CRC specific markers for diagnostic, prognostic and therapeutic purposes. PMID:26517682

Expression of young HERV-H loci in the course of colorectal carcinoma and correlation with molecular subtypes.

PubMed

Pérot, Philippe; Mullins, Christina Susanne; Naville, Magali; Bressan, Cédric; Hühns, Maja; Gock, Michael; Kühn, Florian; Volff, Jean-Nicolas; Trillet-Lenoir, Véronique; Linnebacher, Michael; Mallet, François

2015-11-24

Expression of the human endogenous retrovirus (HERV)-H family has been associated with colorectal carcinomas (CRC), yet no individual HERV-H locus expression has been thoroughly correlated with clinical data.Here, we characterized HERV-H reactivations in clinical CRC samples by integrating expression profiles, molecular patterns and clinical data. Expression of relevant HERV-H sequences was analyzed by qRT-PCR on two well-defined clinical cohorts (n = 139 pairs of tumor and adjacent normal colon tissue) including samples from adenomas (n = 21) and liver metastases (n = 16). Correlations with clinical and molecular data were assessed. CRC specific HERV-H sequences were validated and found expressed throughout CRC disease progression. Correlations between HERV-H expression and lymph node invasion of tumor cells (p = 0.0006) as well as microsatellite instable tumors (p < 0.0001) were established. No association with regard to age, tumor localization, grading or common mutations became apparent. Interestingly, CRC expressed elements belonged to specific young HERV-H subfamilies and their 5' LTR often presented active histone marks. These results suggest a functional role of HERV-H sequences in colorectal carcinogenesis. The pronounced connection with microsatellite instability warrants a more detailed investigation. Thus, HERV-H sequences in addition to tumor specific mutations may represent clinically relevant, truly CRC specific markers for diagnostic, prognostic and therapeutic purposes.

Genetic variation patterns of American chestnut populations at EST-SSRs

Treesearch

Oliver Gailing; C. Dana Nelson

2017-01-01

The objective of this study is to analyze patterns of genetic variation at genic expressed sequence tag - simple sequence repeats (EST-SSRs) and at chloroplast DNA markers in populations of American chestnut (Castanea dentata Borkh.) to assist in conservation and breeding efforts. Allelic diversity at EST-SSRs decreased significantly from southwest to northeast along...

Whole Transcriptome Sequencing Enables Discovery and Analysis of Viruses in Archived Primary Central Nervous System Lymphomas

PubMed Central

DeBoever, Christopher; Reid, Erin G.; Smith, Erin N.; Wang, Xiaoyun; Dumaop, Wilmar; Harismendy, Olivier; Carson, Dennis; Richman, Douglas; Masliah, Eliezer; Frazer, Kelly A.

2013-01-01

Primary central nervous system lymphomas (PCNSL) have a dramatically increased prevalence among persons living with AIDS and are known to be associated with human Epstein Barr virus (EBV) infection. Previous work suggests that in some cases, co-infection with other viruses may be important for PCNSL pathogenesis. Viral transcription in tumor samples can be measured using next generation transcriptome sequencing. We demonstrate the ability of transcriptome sequencing to identify viruses, characterize viral expression, and identify viral variants by sequencing four archived AIDS-related PCNSL tissue samples and analyzing raw sequencing reads. EBV was detected in all four PCNSL samples and cytomegalovirus (CMV), JC polyomavirus (JCV), and HIV were also discovered, consistent with clinical diagnoses. CMV was found to express three long non-coding RNAs recently reported as expressed during active infection. Single nucleotide variants were observed in each of the viruses observed and three indels were found in CMV. No viruses were found in several control tumor types including 32 diffuse large B-cell lymphoma samples. This study demonstrates the ability of next generation transcriptome sequencing to accurately identify viruses, including DNA viruses, in solid human cancer tissue samples. PMID:24023918

In silico Analysis of 2085 Clones from a Normalized Rat Vestibular Periphery 3′ cDNA Library

PubMed Central

Roche, Joseph P.; Cioffi, Joseph A.; Kwitek, Anne E.; Erbe, Christy B.; Popper, Paul

2005-01-01

The inserts from 2400 cDNA clones isolated from a normalized Rattus norvegicus vestibular periphery cDNA library were sequenced and characterized. The Wackym-Soares vestibular 3′ cDNA library was constructed from the saccular and utricular maculae, the ampullae of all three semicircular canals and Scarpa's ganglia containing the somata of the primary afferent neurons, microdissected from 104 male and female rats. The inserts from 2400 randomly selected clones were sequenced from the 5′ end. Each sequence was analyzed using the BLAST algorithm compared to the Genbank nonredundant, rat genome, mouse genome and human genome databases to search for high homology alignments. Of the initial 2400 clones, 315 (13%) were found to be of poor quality and did not yield useful information, and therefore were eliminated from the analysis. Of the remaining 2085 sequences, 918 (44%) were found to represent 758 unique genes having useful annotations that were identified in databases within the public domain or in the published literature; these sequences were designated as known characterized sequences. 1141 sequences (55%) aligned with 1011 unique sequences had no useful annotations and were designated as known but uncharacterized sequences. Of the remaining 26 sequences (1%), 24 aligned with rat genomic sequences, but none matched previously described rat expressed sequence tags or mRNAs. No significant alignment to the rat or human genomic sequences could be found for the remaining 2 sequences. Of the 2085 sequences analyzed, 86% were singletons. The known, characterized sequences were analyzed with the FatiGO online data-mining tool (http://fatigo.bioinfo.cnio.es/) to identify level 5 biological process gene ontology (GO) terms for each alignment and to group alignments with similar or identical GO terms. Numerous genes were identified that have not been previously shown to be expressed in the vestibular system. Further characterization of the novel cDNA sequences may lead to the identification of genes with vestibular-specific functions. Continued analysis of the rat vestibular periphery transcriptome should provide new insights into vestibular function and generate new hypotheses. Physiological studies are necessary to further elucidate the roles of the identified genes and novel sequences in vestibular function. PMID:16103642

Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

PubMed

He, Xiaoliang; Zhou, Xiaohui; Yang, Zi; Xu, Le; Yu, Yuxiu; Jia, Lingling; Li, Guoqing

2015-10-01

An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories. Copyright © 2015 Elsevier Inc. All rights reserved.

Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens.

PubMed

Liao, Wei; Jordaan, Gwen; Nham, Phillipp; Phan, Ryan T; Pelegrini, Matteo; Sharma, Sanjai

2015-10-16

To determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed. Ten CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system. An average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q < 0.05, fold change >2) were identified. Expression of selected DEGs (n = 32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90 % of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1). The RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis.

Identification of Fur, Aconitase, and Other Proteins Expressed by Mycobacterium tuberculosis under Conditions of Low and High Concentrations of Iron by Combined Two-Dimensional Gel Electrophoresis and Mass Spectrometry

PubMed Central

Wong, Diane K.; Lee, Bai-Yu; Horwitz, Marcus A.; Gibson, Bradford W.

1999-01-01

Iron plays a critical role in the pathophysiology of Mycobacterium tuberculosis. To gain a better understanding of iron regulation by this organism, we have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, and database searching to study protein expression in M. tuberculosis under conditions of high and low iron concentration. Proteins in cellular extracts from M. tuberculosis Erdman strain grown under low-iron (1 μM) and high-iron (70 μM) conditions were separated by 2-D polyacrylamide gel electrophoresis, which allowed high-resolution separation of several hundred proteins, as visualized by Coomassie staining. The expression of at least 15 proteins was induced, and the expression of at least 12 proteins was decreased under low-iron conditions. In-gel trypsin digestion was performed on these differentially expressed proteins, and the digestion mixtures were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Partial sequence data on some of the peptides were obtained by using after source decay and/or collision-induced dissociation. The fragmentation data were used to search computerized peptide mass and protein sequence databases for known proteins. Ten iron-regulated proteins were identified, including Fur and aconitase proteins, both of which are known to be regulated by iron in other bacterial systems. Our study shows that, where large protein sequence databases are available from genomic studies, the combined use of 2-D gel electrophoresis, mass spectrometry, and database searching to analyze proteins expressed under defined environmental conditions is a powerful tool for identifying expressed proteins and their physiologic relevance. PMID:9864233

Translating natural genetic variation to gene expression in a computational model of the Drosophila gap gene regulatory network

PubMed Central

Kozlov, Konstantin N.; Kulakovskiy, Ivan V.; Zubair, Asif; Marjoram, Paul; Lawrie, David S.; Nuzhdin, Sergey V.; Samsonova, Maria G.

2017-01-01

Annotating the genotype-phenotype relationship, and developing a proper quantitative description of the relationship, requires understanding the impact of natural genomic variation on gene expression. We apply a sequence-level model of gap gene expression in the early development of Drosophila to analyze single nucleotide polymorphisms (SNPs) in a panel of natural sequenced D. melanogaster lines. Using a thermodynamic modeling framework, we provide both analytical and computational descriptions of how single-nucleotide variants affect gene expression. The analysis reveals that the sequence variants increase (decrease) gene expression if located within binding sites of repressors (activators). We show that the sign of SNP influence (activation or repression) may change in time and space and elucidate the origin of this change in specific examples. The thermodynamic modeling approach predicts non-local and non-linear effects arising from SNPs, and combinations of SNPs, in individual fly genotypes. Simulation of individual fly genotypes using our model reveals that this non-linearity reduces to almost additive inputs from multiple SNPs. Further, we see signatures of the action of purifying selection in the gap gene regulatory regions. To infer the specific targets of purifying selection, we analyze the patterns of polymorphism in the data at two phenotypic levels: the strengths of binding and expression. We find that combinations of SNPs show evidence of being under selective pressure, while individual SNPs do not. The model predicts that SNPs appear to accumulate in the genotypes of the natural population in a way biased towards small increases in activating action on the expression pattern. Taken together, these results provide a systems-level view of how genetic variation translates to the level of gene regulatory networks via combinatorial SNP effects. PMID:28898266

Characteristics of the Lotus japonicus gene repertoire deduced from large-scale expressed sequence tag (EST) analysis.

PubMed

Asamizu, Erika; Nakamura, Yasukazu; Sato, Shusei; Tabata, Satoshi

2004-02-01

To perform a comprehensive analysis of genes expressed in a model legume, Lotus japonicus, a total of 74472 3'-end expressed sequence tags (EST) were generated from cDNA libraries produced from six different organs. Clustering of sequences was performed with an identity criterion of 95% for 50 bases, and a total of 20457 non-redundant sequences, 8503 contigs and 11954 singletons were generated. EST sequence coverage was analyzed by using the annotated L. japonicus genomic sequence and 1093 of the 1889 predicted protein-encoding genes (57.9%) were hit by the EST sequence(s). Gene content was compared to several plant species. Among the 8503 contigs, 471 were identified as sequences conserved only in leguminous species and these included several disease resistance-related genes. This suggested that in legumes, these genes may have evolved specifically to resist pathogen attack. The rate of gene sequence divergence was assessed by comparing similarity level and functional category based on the Gene Ontology (GO) annotation of Arabidopsis genes. This revealed that genes encoding ribosomal proteins, as well as those related to translation, photosynthesis, and cellular structure were more abundantly represented in the highly conserved class, and that genes encoding transcription factors and receptor protein kinases were abundantly represented in the less conserved class. To make the sequence information and the cDNA clones available to the research community, a Web database with useful services was created at http://www.kazusa.or.jp/en/plant/lotus/EST/.

Identification of genes differentially expressed during ripening of banana.

PubMed

Manrique-Trujillo, Sandra Mabel; Ramírez-López, Ana Cecilia; Ibarra-Laclette, Enrique; Gómez-Lim, Miguel Angel

2007-08-01

The banana (Musa acuminata, subgroup Cavendish 'Grand Nain') is a climacteric fruit of economic importance. A better understanding of the banana ripening process is needed to improve fruit quality and to extend shelf life. Eighty-four up-regulated unigenes were identified by differential screening of a banana fruit cDNA subtraction library at a late ripening stage. The ripening stages in this study were defined according to the peel color index (PCI). Unigene sequences were analyzed with different databases to assign a putative identification. The expression patterns of 36 transcripts confirmed as positive by differential screening were analyzed comparing the PCI 1, PCI 5 and PCI 7 ripening stages. Expression profiles were obtained for unigenes annotated as orcinol O-methyltransferase, putative alcohol dehydrogenase, ubiquitin-protein ligase, chorismate mutase and two unigenes with non-significant matches with any reported sequence. Similar expression profiles were observed in banana pulp and peel. Our results show differential expression of a group of genes involved in processes associated with fruit ripening, such as stress, detoxification, cytoskeleton and biosynthesis of volatile compounds. Some of the identified genes had not been characterized in banana fruit. Besides providing an overview of gene expression programs and metabolic pathways at late stages of banana fruit ripening, this study contributes to increasing the information available on banana fruit ESTs.

Bovine adipose triglyceride lipase is not altered and adipocyte fatty acid binding protein is increased by dietary flaxseed

USDA-ARS?s Scientific Manuscript database

In this paper, we report the full length coding sequence of bovine ATGL cDNA are reported and analyze its expression in bovine tissues. Similar to human, mouse, and pig ATGL sequences, bovine ATGL has a highly conserved patatin domain that is necessary for lipolytic function in mice and humans. Thi...

Analysis of Epstein-Barr Virus Genomes and Expression Profiles in Gastric Adenocarcinoma.

PubMed

Borozan, Ivan; Zapatka, Marc; Frappier, Lori; Ferretti, Vincent

2018-01-15

Epstein-Barr virus (EBV) is a causative agent of a variety of lymphomas, nasopharyngeal carcinoma (NPC), and ∼9% of gastric carcinomas (GCs). An important question is whether particular EBV variants are more oncogenic than others, but conclusions are currently hampered by the lack of sequenced EBV genomes. Here, we contribute to this question by mining whole-genome sequences of 201 GCs to identify 13 EBV-positive GCs and by assembling 13 new EBV genome sequences, almost doubling the number of available GC-derived EBV genome sequences and providing the first non-Asian EBV genome sequences from GC. Whole-genome sequence comparisons of all EBV isolates sequenced to date (85 from tumors and 57 from healthy individuals) showed that most GC and NPC EBV isolates were closely related although American Caucasian GC samples were more distant, suggesting a geographical component. However, EBV GC isolates were found to contain some consistent changes in protein sequences regardless of geographical origin. In addition, transcriptome data available for eight of the EBV-positive GCs were analyzed to determine which EBV genes are expressed in GC. In addition to the expected latency proteins (EBNA1, LMP1, and LMP2A), specific subsets of lytic genes were consistently expressed that did not reflect a typical lytic or abortive lytic infection, suggesting a novel mechanism of EBV gene regulation in the context of GC. These results are consistent with a model in which a combination of specific latent and lytic EBV proteins promotes tumorigenesis. IMPORTANCE Epstein-Barr virus (EBV) is a widespread virus that causes cancer, including gastric carcinoma (GC), in a small subset of individuals. An important question is whether particular EBV variants are more cancer associated than others, but more EBV sequences are required to address this question. Here, we have generated 13 new EBV genome sequences from GC, almost doubling the number of EBV sequences from GC isolates and providing the first EBV sequences from non-Asian GC. We further identify sequence changes in some EBV proteins common to GC isolates. In addition, gene expression analysis of eight of the EBV-positive GCs showed consistent expression of both the expected latency proteins and a subset of lytic proteins that was not consistent with typical lytic or abortive lytic expression. These results suggest that novel mechanisms activate expression of some EBV lytic proteins and that their expression may contribute to oncogenesis. Copyright © 2018 American Society for Microbiology.

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter.

PubMed

Li, Wanying; Yu, Dan; Yu, Jingjuan; Zhu, Dengyun; Zhao, Qian

2018-03-12

ZmbZIP25 ( Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5' RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from -2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from -2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5'-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5'-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5'-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5'-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from -1117 to -957 that were responsible for the specificity of the ZmbZIP25 5'-flanking sequence.

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter

PubMed Central

Li, Wanying; Yu, Dan; Yu, Jingjuan; Zhu, Dengyun; Zhao, Qian

2018-01-01

ZmbZIP25 (Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5′ RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from −2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from −2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5′-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5′-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5′-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5′-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from −1117 to −957 that were responsible for the specificity of the ZmbZIP25 5′-flanking sequence. PMID:29534529

End Joining-Mediated Gene Expression in Mammalian Cells Using PCR-Amplified DNA Constructs that Contain Terminator in Front of Promoter.

PubMed

Nakamura, Mikiko; Suzuki, Ayako; Akada, Junko; Tomiyoshi, Keisuke; Hoshida, Hisashi; Akada, Rinji

2015-12-01

Mammalian gene expression constructs are generally prepared in a plasmid vector, in which a promoter and terminator are located upstream and downstream of a protein-coding sequence, respectively. In this study, we found that front terminator constructs-DNA constructs containing a terminator upstream of a promoter rather than downstream of a coding region-could sufficiently express proteins as a result of end joining of the introduced DNA fragment. By taking advantage of front terminator constructs, FLAG substitutions, and deletions were generated using mutagenesis primers to identify amino acids specifically recognized by commercial FLAG antibodies. A minimal epitope sequence for polyclonal FLAG antibody recognition was also identified. In addition, we analyzed the sequence of a C-terminal Ser-Lys-Leu peroxisome localization signal, and identified the key residues necessary for peroxisome targeting. Moreover, front terminator constructs of hepatitis B surface antigen were used for deletion analysis, leading to the identification of regions required for the particle formation. Collectively, these results indicate that front terminator constructs allow for easy manipulations of C-terminal protein-coding sequences, and suggest that direct gene expression with PCR-amplified DNA is useful for high-throughput protein analysis in mammalian cells.

The organization and expression of the mdm2 gene.

PubMed

de Oca Luna, R M; Tabor, A D; Eberspaecher, H; Hulboy, D L; Worth, L L; Colman, M S; Finlay, C A; Lozano, G

1996-05-01

The mdm2 gene encodes a zinc finger protein that negatively regulates p53 function by binding and masking the p53 transcriptional activation domain. Two different promoters control expression of mdm2, one of which is also transactivated by p53. We cloned and characterized the mdm2 gene from a murine 129 library. It contained at least 12 exons and spanned approximately 25 kb of DNA. Sequencing of the mdm2 gene revealed three nucleotide differences that resulted in amino acid substitutions in the previously published mdm2 sequence. Sequencing of normal BalbC/J DNA and the original cosmid clone isolated from the 3T3DM cell line revealed that they are identical, suggesting that the published sequence is in error at these three positions. In addition, we analyzed the expression pattern of mdm2 and found ubiquitous low-level expression throughout embryo development and in adult tissues. Analysis of mRNA from numerous tissues for several mdm2 spliced variants that had been identified in the transformed 3T3DM cell line revealed that these variants could not be detected in the developing embryo or in adult tissues.

Gene discovery in the hamster: a comparative genomics approach for gene annotation by sequencing of hamster testis cDNAs

PubMed Central

Oduru, Sreedhar; Campbell, Janee L; Karri, SriTulasi; Hendry, William J; Khan, Shafiq A; Williams, Simon C

2003-01-01

Background Complete genome annotation will likely be achieved through a combination of computer-based analysis of available genome sequences combined with direct experimental characterization of expressed regions of individual genomes. We have utilized a comparative genomics approach involving the sequencing of randomly selected hamster testis cDNAs to begin to identify genes not previously annotated on the human, mouse, rat and Fugu (pufferfish) genomes. Results 735 distinct sequences were analyzed for their relatedness to known sequences in public databases. Eight of these sequences were derived from previously unidentified genes and expression of these genes in testis was confirmed by Northern blotting. The genomic locations of each sequence were mapped in human, mouse, rat and pufferfish, where applicable, and the structure of their cognate genes was derived using computer-based predictions, genomic comparisons and analysis of uncharacterized cDNA sequences from human and macaque. Conclusion The use of a comparative genomics approach resulted in the identification of eight cDNAs that correspond to previously uncharacterized genes in the human genome. The proteins encoded by these genes included a new member of the kinesin superfamily, a SET/MYND-domain protein, and six proteins for which no specific function could be predicted. Each gene was expressed primarily in testis, suggesting that they may play roles in the development and/or function of testicular cells. PMID:12783626

Simian virus 40 major late promoter: an upstream DNA sequence required for efficient in vitro transcription.

PubMed Central

Brady, J; Radonovich, M; Thoren, M; Das, G; Salzman, N P

1984-01-01

We have previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3' (simian virus 40 [SV40] map position 294 to 304), which is important in the control of SV40 late RNA expression in vitro and in vivo (Brady et al., Cell 31:625-633, 1982). We report here the identification of another domain of the SV40 late promoter. A series of mutants with deletions extending from SV40 map position 0 to 300 was prepared by nuclease BAL 31 treatment. The cloned templates were then analyzed for efficiency and accuracy of late SV40 RNA expression in the Manley in vitro transcription system. Our studies showed that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence were two of the six GGGCGG SV40 repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supported transcription with the same efficiency even when it was moved 72 nucleotides closer to the major late cap site. In vitro promoter competition analysis demonstrated that the upstream promoter sequence, independent of the 294 to 304 promoter element, is capable of binding polymerase-transcription factors required for SV40 late gene transcription. Finally, we show that DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie downstream of map position 294. Images PMID:6321950

The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPase alpha 3 subunit gene are required for brain expression in transgenic mice.

PubMed Central

Pathak, B G; Neumann, J C; Croyle, M L; Lingrel, J B

1994-01-01

The Na,K-ATPase is an integral plasma membrane protein consisting of alpha and beta subunits, each of which has discrete isoforms expressed in a tissue-specific manner. Of the three functional alpha isoform genes, the one encoding the alpha 3 isoform is the most tissue-restricted in its expression, being found primarily in the brain. To identify regions of the alpha 3 isoform gene that are involved in directing expression in the brain, a 1.6 kb 5'-flanking sequence was attached to a reporter gene, chloramphenicol acetyltransferase (CAT). The alpha 3-CAT chimeric gene construct was microinjected into fertilized mouse eggs, and transgenic mice were produced. Analysis of adult transgenic mice from different lines revealed that the transgene is expressed primarily in the brain. To further delineate regions that are needed for conferring expression in this tissue, systematic deletions of the 5'-flanking sequence of the alpha 3-CAT fusion constructs were made and analyzed, again using transgenic mice. The results from these analyses indicate that DNA sequences required for mediating brain-specific expression of the alpha 3 isoform gene are present within 210 bp upstream of the transcription initiation site. alpha 3-CAT promoter constructs containing scanning mutations in this region were also assayed in transgenic mice. These studies have identified both a functional neural-restrictive silencer element as well as a positively acting cis element. Images PMID:7984427

Transcriptional dynamics of the developing sweet cherry (Prunus avium L.) fruit: sequencing, annotation and expression profiling of exocarp-associated genes

PubMed Central

Alkio, Merianne; Jonas, Uwe; Declercq, Myriam; Van Nocker, Steven; Knoche, Moritz

2014-01-01

The exocarp, or skin, of fleshy fruit is a specialized tissue that protects the fruit, attracts seed dispersing fruit eaters, and has large economical relevance for fruit quality. Development of the exocarp involves regulated activities of many genes. This research analyzed global gene expression in the exocarp of developing sweet cherry (Prunus avium L., ‘Regina’), a fruit crop species with little public genomic resources. A catalog of transcript models (contigs) representing expressed genes was constructed from de novo assembled short complementary DNA (cDNA) sequences generated from developing fruit between flowering and maturity at 14 time points. Expression levels in each sample were estimated for 34 695 contigs from numbers of reads mapping to each contig. Contigs were annotated functionally based on BLAST, gene ontology and InterProScan analyses. Coregulated genes were detected using partitional clustering of expression patterns. The results are discussed with emphasis on genes putatively involved in cuticle deposition, cell wall metabolism and sugar transport. The high temporal resolution of the expression patterns presented here reveals finely tuned developmental specialization of individual members of gene families. Moreover, the de novo assembled sweet cherry fruit transcriptome with 7760 full-length protein coding sequences and over 20 000 other, annotated cDNA sequences together with their developmental expression patterns is expected to accelerate molecular research on this important tree fruit crop. PMID:26504533

Reproducibility of the dynamics of facial expressions in unilateral facial palsy.

PubMed

Alagha, M A; Ju, X; Morley, S; Ayoub, A

2018-02-01

The aim of this study was to assess the reproducibility of non-verbal facial expressions in unilateral facial paralysis using dynamic four-dimensional (4D) imaging. The Di4D system was used to record five facial expressions of 20 adult patients. The system captured 60 three-dimensional (3D) images per second; each facial expression took 3-4seconds which was recorded in real time. Thus a set of 180 3D facial images was generated for each expression. The procedure was repeated after 30min to assess the reproducibility of the expressions. A mathematical facial mesh consisting of thousands of quasi-point 'vertices' was conformed to the face in order to determine the morphological characteristics in a comprehensive manner. The vertices were tracked throughout the sequence of the 180 images. Five key 3D facial frames from each sequence of images were analyzed. Comparisons were made between the first and second capture of each facial expression to assess the reproducibility of facial movements. Corresponding images were aligned using partial Procrustes analysis, and the root mean square distance between them was calculated and analyzed statistically (paired Student t-test, P<0.05). Facial expressions of lip purse, cheek puff, and raising of eyebrows were reproducible. Facial expressions of maximum smile and forceful eye closure were not reproducible. The limited coordination of various groups of facial muscles contributed to the lack of reproducibility of these facial expressions. 4D imaging is a useful clinical tool for the assessment of facial expressions. Copyright © 2017 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Droplet barcoding for single cell transcriptomics applied to embryonic stem cells

PubMed Central

Klein, Allon M; Mazutis, Linas; Akartuna, Ilke; Tallapragada, Naren; Veres, Adrian; Li, Victor; Peshkin, Leonid; Weitz, David A; Kirschner, Marc W

2015-01-01

Summary It has long been the dream of biologists to map gene expression at the single cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after LIF withdrawal. The reproducibility of these high-throughput single cell data allowed us to deconstruct cell populations and infer gene expression relationships. PMID:26000487

Expressed sequence tags from larval gut of the european corn borer (Ostrinia nubilalis): exploring candidate genes potenially involved in Bacillus thuringiensis toxicity and resistance

USDA-ARS?s Scientific Manuscript database

Background: Knowledge of the genes that are expressed in the insect gut are crucial for understanding basic physiology of food digestion, their interactions with Bacillus thuringiensis (Bt) toxin and for discovering new targets for novel toxins for use in pest management. This study analyzed the ES...

In silico search, characterization and validation of new EST-SSR markers in the genus Prunus.

PubMed

Sorkheh, Karim; Prudencio, Angela S; Ghebinejad, Azim; Dehkordi, Mehrana Kohei; Erogul, Deniz; Rubio, Manuel; Martínez-Gómez, Pedro

2016-07-07

Simple sequence repeats (SSRs) are defined as sequence repeat units between 1 and 6 bp that occur in both coding and non-coding regions abundant in eukaryotic genomes, which may affect the expression of genes. In this study, expressed sequence tags (ESTs) of eight Prunus species were analyzed for in silico mining of EST-SSRs, protein annotation, and open reading frames (ORFs), and the identification of codon repetitions. A total of 316 SSRs were identified using MISA software. Dinucleotide SSR motifs (26.31 %) were found to be the most abundant type of repeats, followed by tri- (14.58 %), tetra- (0.53 %), and penta- (0.27 %) nucleotide motifs. An attempt was made to design primer pairs for 316 identified SSRs but these were successful for only 175 SSR sequences. The positions of SSRs with respect to ORFs were detected, and annotation of sequences containing SSRs was performed to assign function to each sequence. SSRs were also characterized (in terms of position in the reference genome and associated gene) using the two available Prunus reference genomes (mei and peach). Finally, 38 SSR markers were validated across peach, almond, plum, and apricot genotypes. This validation showed a higher transferability level of EST-SSR developed in P. mume (mei) in comparison with the rest of species analyzed. Findings will aid analysis of functionally important molecular markers and facilitate the analysis of genetic diversity.

Monoallelic Gene Expression in Mammals.

PubMed

Chess, Andrew

2016-11-23

Monoallelic expression not due to cis-regulatory sequence polymorphism poses an intriguing problem in epigenetics because it requires the unequal treatment of two segments of DNA that are present in the same nucleus and that can indeed have absolutely identical sequences. Here, I focus on a few recent developments in the field of monoallelic expression that are of particular interest and raise interesting questions for future work. One development is regarding analyses of imprinted genes, in which recent work suggests the possibility that intriguing networks of imprinted genes exist and are important for genetic and physiological studies. Another issue that has been raised in recent years by a number of publications is the question of how skewed allelic expression should be for it to be designated as monoallelic expression and, further, what methods are appropriate or inappropriate for analyzing genomic data to examine allele-specific expression. Perhaps the most exciting recent development in mammalian monoallelic expression is a clever and carefully executed analysis of genetic diversity of autosomal genes subject to random monoallelic expression (RMAE), which provides compelling evidence for distinct evolutionary forces acting on random monoallelically expressed genes.

Genetic Rearrangements Can Modify Chromatin Features at Epialleles

PubMed Central

Foerster, Andrea M.; Dinh, Huy Q.; Sedman, Laura; Wohlrab, Bonnie; Mittelsten Scheid, Ortrun

2011-01-01

Analogous to genetically distinct alleles, epialleles represent heritable states of different gene expression from sequence-identical genes. Alleles and epialleles both contribute to phenotypic heterogeneity. While alleles originate from mutation and recombination, the source of epialleles is less well understood. We analyze active and inactive epialleles that were found at a transgenic insert with a selectable marker gene in Arabidopsis. Both converse expression states are stably transmitted to progeny. The silent epiallele was previously shown to change its state upon loss-of-function of trans-acting regulators and drug treatments. We analyzed the composition of the epialleles, their chromatin features, their nuclear localization, transcripts, and homologous small RNA. After mutagenesis by T-DNA transformation of plants carrying the silent epiallele, we found new active alleles. These switches were associated with different, larger or smaller, and non-overlapping deletions or rearrangements in the 3′ regions of the epiallele. These cis-mutations caused different degrees of gene expression stability depending on the nature of the sequence alteration, the consequences for transcription and transcripts, and the resulting chromatin organization upstream. This illustrates a tight dependence of epigenetic regulation on local structures and indicates that sequence alterations can cause epigenetic changes at some distance in regions not directly affected by the mutation. Similar effects may also be involved in gene expression and chromatin changes in the vicinity of transposon insertions or excisions, recombination events, or DNA repair processes and could contribute to the origin of new epialleles. PMID:22028669

[Expression of human-mouse chimeric antibody directed against Chikungunya virus with site-specific integration system].

PubMed

Li, Jian-min; Chen, Wei; Jia, Xiu-jie; An, Xiao-ping; Li, Bing; Fan, Ying-ru; Tong, Yi-gang

2005-05-01

To obtain CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site and to express human-mouse chimeric antibody directed against Chikungunya Virus by using the cell line. The fusion gene of FRT and HBsAg was constructed by PCR and cloned into the MCS of pCI-neo to construct pCI-FRT-HBsAg. The pCI-FRT-HBsAg was transfected into CHO/dhfr(-) cells and cell clones with high expression of HBsAg were screened by detecting the amount of HBsAg with ELISA. A CHO cell clone with the highest expression was chosen and named as CHO/dhfr(-) FRT(+). pAFRT HFLF, a expression plasmid of chimeric antibody with RFT sequence was transfected into CHO/dhfr(-) FRT(+) cells and cell clones with high expression of the chimeric antibody were screened by increasing concentration of MTX. A CHO cell clone with high expression of the chimeric antibody was cultured in large scale and supernatant was collected from which the chimeric antibody was purified. The purified chimeric antibody was analyzed by SDS-PAGE, Western blot and IFA. A CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site was obtained successfully. A cell clone with yield of 5 mg/L of chimeric antibody was obtained, as compared with routine CHO cell expression system with a yield of 2 mg/L. A cell line with integrated FRT sequence in the chromosome transcription active site was obtained and with it human-mouse chimeric antibody directed against Chikungunya virus was expressed. This system lays a solid foundation which can be used for expressing antibodies and other proteins.

[Blue-light induced expression of S-adenosy-L-homocysteine hydrolase-like gene in Mucor amphibiorum RCS1].

PubMed

Gao, Ya; Wang, Shu; Fu, Mingjia; Zhong, Guolin

2013-09-04

To determine blue-light induced expression of S-adenosyl-L-homocysteine hydrolase-like (sahhl) gene in fungus Mucor amphibiorum RCS1. In the random process of PCR, a sequence of 555 bp was obtained from M. amphibiorum RCS1. The 555 bp sequence was labeled with digoxin to prepare the probe for northern hybridization. By northern hybridization, the transcription of sahhl gene was analyzed in M. amphibiorum RCS1 mycelia culture process from darkness to blue light to darkness. Simultaneously real-time PCR method was used to the sahhl gene expression analysis. Compared with the sequence of sahh gene from Homo sapiens, Mus musculus and some fungi species, a high homology of the 555 bp sequence was confirmed. Therefore, the preliminary confirmation has supported that the 555 bp sequence should be sahhl gene from M. amphibiorum RCS1. Under the dark pre-culture in 24 h, a large amounts of transcript of sahhl gene in the mycelia can be detected by northern hybridization and real-time PCR in the condition of 24 h blue light. But a large amounts of transcript of sahhl gene were not found in other detection for the dark pre-culture of 48 h, even though M. amphibiorum RCS1 mycelia were induced by blue light. Blue light can induce the expression of sahhl gene in the vigorous growth of M. amphibiorum RCS1 mycelia.

[The value of alpha-methylacyl-CoA racemase expression in the progression of colonic carcinoma].

PubMed

López-Valdivia, Cecilia M; González-Matea, Manuel; Mayordomo, Empar; Hervás, David; Ramos, David

Alpha-methylacyl-CoA racemase (AMACR) expression has been demonstrated in several normal tissues and in diverse types of carcinoma. Our aim was to analyze the immunohistochemical expression of AMACR in the sequence-progression of colonic cancer. We studied 237 cases, including samples of normal mucosa of the colon, adenomas with different degrees of dysplasia, colonic carcinomas, lymph nodes and liver metastases of colonic carcinomas. A scale of intensity and percentage of expression was used to analyze the AMACR immunohistochemical profile. The expression was nearly absent in samples of normal mucosa, increased in both adenomas and carcinomas, decreased in lymph node metastases but was significantly increased in liver metastases. Copyright © 2016 Sociedad Española de Anatomía Patológica. Publicado por Elsevier España, S.L.U. All rights reserved.

Linear Lepidopteran ambidensovirus 1 sequences drive random integration of a reporter gene in transfected Spodoptera frugiperda cells.

PubMed

Rizk, Francine; Laverdure, Sylvain; d'Alençon, Emmanuelle; Bossin, Hervé; Dupressoir, Thierry

2018-01-01

The Lepidopteran ambidensovirus 1 isolated from Junonia coenia (hereafter JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a potential tool for the biological control of insect pests. Previous works showed that JcDV-based circular plasmids experimentally integrate into insect cells genomic DNA. In order to approach the natural conditions of infection and possible integration, we generated linear JcDV- gfp based molecules which were transfected into non permissive Spodoptera frugiperda ( Sf9 ) cultured cells. Cells were monitored for the expression of green fluorescent protein (GFP) and DNA was analyzed for integration of transduced viral sequences. Non-structural protein modulation of the VP-gene cassette promoter activity was additionally assayed. We show that linear JcDV-derived molecules are capable of long term genomic integration and sustained transgene expression in Sf9 cells. As expected, only the deletion of both inverted terminal repeats (ITR) or the polyadenylation signals of NS and VP genes dramatically impairs the global transduction/expression efficiency. However, all the integrated viral sequences we characterized appear "scrambled" whatever the viral content of the transfected vector. Despite a strong GFP expression, we were unable to recover any full sequence of the original constructs and found rearranged viral and non-viral sequences as well. Cellular flanking sequences were identified as non-coding ones. On the other hand, the kinetics of GFP expression over time led us to investigate the apparent down-regulation by non-structural proteins of the VP-gene cassette promoter. Altogether, our results show that JcDV-derived sequences included in linear DNA molecules are able to drive efficiently the integration and expression of a foreign gene into the genome of insect cells, whatever their composition, provided that at least one ITR is present. However, the transfected sequences were extensively rearranged with cellular DNA during or after random integration in the host cell genome. Lastly, the non-structural proteins seem to participate in the regulation of p9 promoter activity rather than to the integration of viral sequences.

Metatranscriptomics of Soil Eukaryotic Communities.

PubMed

Yadav, Rajiv K; Bragalini, Claudia; Fraissinet-Tachet, Laurence; Marmeisse, Roland; Luis, Patricia

2016-01-01

Functions expressed by eukaryotic organisms in soil can be specifically studied by analyzing the pool of eukaryotic-specific polyadenylated mRNA directly extracted from environmental samples. In this chapter, we describe two alternative protocols for the extraction of high-quality RNA from soil samples. Total soil RNA or mRNA can be converted to cDNA for direct high-throughput sequencing. Polyadenylated mRNA-derived full-length cDNAs can also be cloned in expression plasmid vectors to constitute soil cDNA libraries, which can be subsequently screened for functional gene categories. Alternatively, the diversity of specific gene families can also be explored following cDNA sequence capture using exploratory oligonucleotide probes.

DSAP: deep-sequencing small RNA analysis pipeline.

PubMed

Huang, Po-Jung; Liu, Yi-Chung; Lee, Chi-Ching; Lin, Wei-Chen; Gan, Richie Ruei-Chi; Lyu, Ping-Chiang; Tang, Petrus

2010-07-01

DSAP is an automated multiple-task web service designed to provide a total solution to analyzing deep-sequencing small RNA datasets generated by next-generation sequencing technology. DSAP uses a tab-delimited file as an input format, which holds the unique sequence reads (tags) and their corresponding number of copies generated by the Solexa sequencing platform. The input data will go through four analysis steps in DSAP: (i) cleanup: removal of adaptors and poly-A/T/C/G/N nucleotides; (ii) clustering: grouping of cleaned sequence tags into unique sequence clusters; (iii) non-coding RNA (ncRNA) matching: sequence homology mapping against a transcribed sequence library from the ncRNA database Rfam (http://rfam.sanger.ac.uk/); and (iv) known miRNA matching: detection of known miRNAs in miRBase (http://www.mirbase.org/) based on sequence homology. The expression levels corresponding to matched ncRNAs and miRNAs are summarized in multi-color clickable bar charts linked to external databases. DSAP is also capable of displaying miRNA expression levels from different jobs using a log(2)-scaled color matrix. Furthermore, a cross-species comparative function is also provided to show the distribution of identified miRNAs in different species as deposited in miRBase. DSAP is available at http://dsap.cgu.edu.tw.

Efficient gusA transient expression in Porphyra yezoensis protoplasts mediated by endogenous beta-tubulin flanking sequences

NASA Astrophysics Data System (ADS)

Gong, Qianhong; Yu, Wengong; Dai, Jixun; Liu, Hongquan; Xu, Rifu; Guan, Huashi; Pan, Kehou

2007-01-01

Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were constructed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5'-and 3'-flanking regions ( Tub5' and Tub3') up-and down-stream of β-glucuronidase (GUS) gene ( gusA), respectively, into pA, a derivative of pCAT®3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3'. These constructs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5'-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when compared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

Characterization by Suppression Subtractive Hybridization of Transcripts That Are Differentially Expressed in Leaves of Anthracnose-Resistant Ramie Cultivar.

PubMed

Xuxia, Wang; Jie, Chen; Bo, Wang; Lijun, Liu; Hui, Jiang; Diluo, Tang; Dingxiang, Peng

2012-01-01

For the purpose of screening putative anthracnose resistance-related genes of ramie ( Boehmeria nivea L. Gaud), a cDNA library was constructed by suppression subtractive hybridization using anthracnose-resistant cultivar Huazhu no. 4. The cDNAs from Huazhu no. 4, which were infected with Colletotrichum gloeosporioides , were used as the tester and cDNAs from uninfected Huazhu no. 4 as the driver. Sequencing analysis and homology searching showed that these clones represented 132 single genes, which were assigned to functional categories, including 14 putative cellular functions, according to categories established for Arabidopsis . These 132 genes included 35 disease resistance and stress tolerance-related genes including putative heat-shock protein 90, metallothionein, PR-1.2 protein, catalase gene, WRKY family genes, and proteinase inhibitor-like protein. Partial disease-related genes were further analyzed by reverse transcription PCR and RNA gel blot. These expressed sequence tags are the first anthracnose resistance-related expressed sequence tags reported in ramie.

TEtools facilitates big data expression analysis of transposable elements and reveals an antagonism between their activity and that of piRNA genes

PubMed Central

Lerat, Emmanuelle; Fablet, Marie; Modolo, Laurent; Lopez-Maestre, Hélène

2017-01-01

Abstract Over recent decades, substantial efforts have been made to understand the interactions between host genomes and transposable elements (TEs). The impact of TEs on the regulation of host genes is well known, with TEs acting as platforms of regulatory sequences. Nevertheless, due to their repetitive nature it is considerably hard to integrate TE analysis into genome-wide studies. Here, we developed a specific tool for the analysis of TE expression: TEtools. This tool takes into account the TE sequence diversity of the genome, it can be applied to unannotated or unassembled genomes and is freely available under the GPL3 (https://github.com/l-modolo/TEtools). TEtools performs the mapping of RNA-seq data obtained from classical mRNAs or small RNAs onto a list of TE sequences and performs differential expression analyses with statistical relevance. Using this tool, we analyzed TE expression from five Drosophila wild-type strains. Our data show for the first time that the activity of TEs is strictly linked to the activity of the genes implicated in the piwi-interacting RNA biogenesis and therefore fits an arms race scenario between TE sequences and host control genes. PMID:28204592

Association of coral algal symbionts with a diverse viral community responsive to heat shock.

PubMed

Brüwer, Jan D; Agrawal, Shobhit; Liew, Yi Jin; Aranda, Manuel; Voolstra, Christian R

2017-08-17

Stony corals provide the structural foundation of coral reef ecosystems and are termed holobionts given they engage in symbioses, in particular with photosynthetic dinoflagellates of the genus Symbiodinium. Besides Symbiodinium, corals also engage with bacteria affecting metabolism, immunity, and resilience of the coral holobiont, but the role of associated viruses is largely unknown. In this regard, the increase of studies using RNA sequencing (RNA-Seq) to assess gene expression provides an opportunity to elucidate viral signatures encompassed within the data via careful delineation of sequence reads and their source of origin. Here, we re-analyzed an RNA-Seq dataset from a cultured coral symbiont (Symbiodinium microadriaticum, Clade A1) across four experimental treatments (control, cold shock, heat shock, dark shock) to characterize associated viral diversity, abundance, and gene expression. Our approach comprised the filtering and removal of host sequence reads, subsequent phylogenetic assignment of sequence reads of putative viral origin, and the assembly and analysis of differentially expressed viral genes. About 15.46% (123 million) of all sequence reads were non-host-related, of which <1% could be classified as archaea, bacteria, or virus. Of these, 18.78% were annotated as virus and comprised a diverse community consistent across experimental treatments. Further, non-host related sequence reads assembled into 56,064 contigs, including 4856 contigs of putative viral origin that featured 43 differentially expressed genes during heat shock. The differentially expressed genes included viral kinases, ubiquitin, and ankyrin repeat proteins (amongst others), which are suggested to help the virus proliferate and inhibit the algal host's antiviral response. Our results suggest that a diverse viral community is associated with coral algal endosymbionts of the genus Symbiodinium, which prompts further research on their ecological role in coral health and resilience.

Minimal doses of a sequence-optimized transgene mediate high-level and long-term EPO expression in vivo: challenging CpG-free gene design.

PubMed

Kosovac, D; Wild, J; Ludwig, C; Meissner, S; Bauer, A P; Wagner, R

2011-02-01

Advanced gene delivery techniques can be combined with rational gene design to further improve the efficiency of plasmid DNA (pDNA)-mediated transgene expression in vivo. Herein, we analyzed the influence of intragenic sequence modifications on transgene expression in vitro and in vivo using murine erythropoietin (mEPO) as a transgene model. A single electro-gene transfer of an RNA- and codon-optimized mEPOopt gene into skeletal muscle resulted in a 3- to 4-fold increase of mEPO production sustained for >1 year and triggered a significant increase in hematocrit and hemoglobin without causing adverse effects. mEPO expression and hematologic levels were significantly lower when using comparable amounts of the wild type (mEPOwt) gene and only marginal effects were induced by mEPOΔCpG lacking intragenic CpG dinucleotides, even at high pDNA amounts. Corresponding with these observations, in vitro analysis of transfected cells revealed a 2- to 3-fold increased (mEPOopt) and 50% decreased (mEPOΔCpG) erythropoietin expression compared with mEPOwt, respectively. RNA analyses demonstrated that the specific design of the transgene sequence influenced expression levels by modulating transcriptional activity and nuclear plus cytoplasmic RNA amounts rather than translation. In sum, whereas CpG depletion negatively interferes with efficient expression in postmitotic tissues, mEPOopt doses <0.5 μg were sufficient to trigger optimal long-term hematologic effects encouraging the use of sequence-optimized transgenes to further reduce effective pDNA amounts.

Two cis elements collaborate to spatially repress transcription from a sea urchin promoter

NASA Technical Reports Server (NTRS)

Frudakis, T. N.; Wilt, F.

1995-01-01

The expression pattern of many territory-specific genes in metazoan embryos is maintained by an active process of negative spatial regulation. However, the mechanism of this strategy of gene regulation is not well understood in any system. Here we show that reporter constructs containing regulatory sequence for the SM30-alpha gene of Stronglyocentrotus purpuratus are expressed in a pattern congruent with that of the endogenous SM30 gene(s), largely as a result of active transcriptional repression in cell lineages in which the gene is not normally expressed. Chloramphenicol acetyl transferase assays of deletion constructs from the 2600-bp upstream region showed that repressive elements were present in the region from -1628 to -300. In situ hybridization analysis showed that the spatial fidelity of expression was severely compromised when the region from -1628 to -300 was deleted. Two highly repetitive sequence motifs, (G/A/C)CCCCT and (T/C)(T/A/C)CTTTT(T/A/C), are present in the -1628 to -300 region. Representatives of these elements were analyzed by gel mobility shift experiments and were found to interact specifically with protein in crude nuclear extracts. When oligonucleotides containing either sequence element were co-injected with a correctly regulated reporter as potential competitors, the reporter was expressed in inappropriate cells. When composite oligonucleotides, containing both sequence elements, were fused to a misregulated reporter, the expression of the reporter in inappropriate cells was suppressed. Comparison of composite oligonucleotides with oligonucleotides containing single constituent elements show that both sequence elements are required for effective spatial regulation. Thus, both individual elements are required, but only a composite element containing both elements is sufficient to function as a tissue-specific repressive element.

Deep sequencing-based transcriptome analysis of Plutella xylostella larvae parasitized by Diadegma semiclausum

PubMed Central

2011-01-01

Background Parasitoid insects manipulate their hosts' physiology by injecting various factors into their host upon parasitization. Transcriptomic approaches provide a powerful approach to study insect host-parasitoid interactions at the molecular level. In order to investigate the effects of parasitization by an ichneumonid wasp (Diadegma semiclausum) on the host (Plutella xylostella), the larval transcriptome profile was analyzed using a short-read deep sequencing method (Illumina). Symbiotic polydnaviruses (PDVs) associated with ichneumonid parasitoids, known as ichnoviruses, play significant roles in host immune suppression and developmental regulation. In the current study, D. semiclausum ichnovirus (DsIV) genes expressed in P. xylostella were identified and their sequences compared with other reported PDVs. Five of these genes encode proteins of unknown identity, that have not previously been reported. Results De novo assembly of cDNA sequence data generated 172,660 contigs between 100 and 10000 bp in length; with 35% of > 200 bp in length. Parasitization had significant impacts on expression levels of 928 identified insect host transcripts. Gene ontology data illustrated that the majority of the differentially expressed genes are involved in binding, catalytic activity, and metabolic and cellular processes. In addition, the results show that transcription levels of antimicrobial peptides, such as gloverin, cecropin E and lysozyme, were up-regulated after parasitism. Expression of ichnovirus genes were detected in parasitized larvae with 19 unique sequences identified from five PDV gene families including vankyrin, viral innexin, repeat elements, a cysteine-rich motif, and polar residue rich protein. Vankyrin 1 and repeat element 1 genes showed the highest transcription levels among the DsIV genes. Conclusion This study provides detailed information on differential expression of P. xylostella larval genes following parasitization, DsIV genes expressed in the host and also improves our current understanding of this host-parasitoid interaction. PMID:21906285

Comprehensive evaluation of AmpliSeq transcriptome, a novel targeted whole transcriptome RNA sequencing methodology for global gene expression analysis.

PubMed

Li, Wenli; Turner, Amy; Aggarwal, Praful; Matter, Andrea; Storvick, Erin; Arnett, Donna K; Broeckel, Ulrich

2015-12-16

Whole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis. However, RNA-seq carries a few limitations, e.g., the requirement of a significant amount of input RNA and complications led by non-specific mapping of short reads. The Ion AmpliSeq Transcriptome Human Gene Expression Kit (AmpliSeq) was recently introduced by Life Technologies as a whole-transcriptome, targeted gene quantification kit to overcome these limitations of RNA-seq. To assess the performance of this new methodology, we performed a comprehensive comparison of AmpliSeq with RNA-seq using two well-established next-generation sequencing platforms (Illumina HiSeq and Ion Torrent Proton). We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Using published data from two standard RNA reference samples, we observed a strong concordance of log2 fold change for all genes when comparing AmpliSeq to Illumina HiSeq (Pearson's r = 0.92) and Ion Torrent Proton (Pearson's r = 0.92). We used ROC, Matthew's correlation coefficient and RMSD to determine the overall performance characteristics. All three statistical methods demonstrate AmpliSeq as a highly accurate method for differential gene expression analysis. Additionally, for genes with high abundance, AmpliSeq outperforms the two RNA-seq methods. When analyzing four closely related hiPSC-CM lines, we show that both AmpliSeq and RNA-seq capture similar global gene expression patterns consistent with known sources of variations. Our study indicates that AmpliSeq excels in the limiting areas of RNA-seq for gene expression quantification analysis. Thus, AmpliSeq stands as a very sensitive and cost-effective approach for very large scale gene expression analysis and mRNA marker screening with high accuracy.

Scanning of Transposable Elements and Analyzing Expression of Transposase Genes of Sweet Potato [Ipomoea batatas

PubMed Central

Tao, Xiang; Lai, Xian-Jun; Zhang, Yi-Zheng; Tan, Xue-Mei; Wang, Haiyan

2014-01-01

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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. PMID:24608103

Spatial analysis of biomineralization associated gene expression from the mantle organ of the pearl oyster Pinctada maxima

PubMed Central

2011-01-01

Background Biomineralization is a process encompassing all mineral containing tissues produced within an organism. One of the most dynamic examples of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this architecture. However general understanding of how this process is achieved remains ambiguous. The mantle is a conserved organ involved in shell formation throughout molluscs. Specifically the mantle is thought to be responsible for secreting the protein component of the shell. This study employs molecular approaches to determine the spatial expression of genes within the mantle tissue to further the elucidation of the shell biomineralization. Results A microarray platform was custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from mantle tissue. This microarray was used to analyze the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and analyzed for differential gene expression with PmaxArray 1.0. Over 2000 ESTs were determined to be differentially expressed among the tissue sections, identifying five major expression regions. In situ hybridization validated and further localized the expression for a subset of these ESTs. Comparative sequence similarity analysis of these ESTs revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell related genes. Conclusions This investigation has mapped the spatial distribution for over 2000 ESTs present on PmaxArray 1.0 with reference to specific locations of the mantle. Expression profile clusters have indicated at least five unique functioning zones in the mantle. Three of these zones are likely involved in shell related activities including formation of nacre, periostracum and calcitic prismatic microstructure. A number of novel and known transcripts have been identified from these clusters. The development of PmaxArray 1.0, and the spatial map of its ESTs expression in the mantle has begun characterizing the molecular mechanisms linking the organics and inorganics of the molluscan shell. PMID:21936921

Novel green tissue-specific synthetic promoters and cis-regulatory elements in rice.

PubMed

Wang, Rui; Zhu, Menglin; Ye, Rongjian; Liu, Zuoxiong; Zhou, Fei; Chen, Hao; Lin, Yongjun

2015-12-11

As an important part of synthetic biology, synthetic promoter has gradually become a hotspot in current biology. The purposes of the present study were to synthesize green tissue-specific promoters and to discover green tissue-specific cis-elements. We first assembled several regulatory sequences related to tissue-specific expression in different combinations, aiming to obtain novel green tissue-specific synthetic promoters. GUS assays of the transgenic plants indicated 5 synthetic promoters showed green tissue-specific expression patterns and different expression efficiencies in various tissues. Subsequently, we scanned and counted the cis-elements in different tissue-specific promoters based on the plant cis-elements database PLACE and the rice cDNA microarray database CREP for green tissue-specific cis-element discovery, resulting in 10 potential cis-elements. The flanking sequence of one potential core element (GEAT) was predicted by bioinformatics. Then, the combination of GEAT and its flanking sequence was functionally identified with synthetic promoter. GUS assays of the transgenic plants proved its green tissue-specificity. Furthermore, the function of GEAT flanking sequence was analyzed in detail with site-directed mutagenesis. Our study provides an example for the synthesis of rice tissue-specific promoters and develops a feasible method for screening and functional identification of tissue-specific cis-elements with their flanking sequences at the genome-wide level in rice.

PARRoT- a homology-based strategy to quantify and compare RNA-sequencing from non-model organisms.

PubMed

Gan, Ruei-Chi; Chen, Ting-Wen; Wu, Timothy H; Huang, Po-Jung; Lee, Chi-Ching; Yeh, Yuan-Ming; Chiu, Cheng-Hsun; Huang, Hsien-Da; Tang, Petrus

2016-12-22

Next-generation sequencing promises the de novo genomic and transcriptomic analysis of samples of interests. However, there are only a few organisms having reference genomic sequences and even fewer having well-defined or curated annotations. For transcriptome studies focusing on organisms lacking proper reference genomes, the common strategy is de novo assembly followed by functional annotation. However, things become even more complicated when multiple transcriptomes are compared. Here, we propose a new analysis strategy and quantification methods for quantifying expression level which not only generate a virtual reference from sequencing data, but also provide comparisons between transcriptomes. First, all reads from the transcriptome datasets are pooled together for de novo assembly. The assembled contigs are searched against NCBI NR databases to find potential homolog sequences. Based on the searched result, a set of virtual transcripts are generated and served as a reference transcriptome. By using the same reference, normalized quantification values including RC (read counts), eRPKM (estimated RPKM) and eTPM (estimated TPM) can be obtained that are comparable across transcriptome datasets. In order to demonstrate the feasibility of our strategy, we implement it in the web service PARRoT. PARRoT stands for Pipeline for Analyzing RNA Reads of Transcriptomes. It analyzes gene expression profiles for two transcriptome sequencing datasets. For better understanding of the biological meaning from the comparison among transcriptomes, PARRoT further provides linkage between these virtual transcripts and their potential function through showing best hits in SwissProt, NR database, assigning GO terms. Our demo datasets showed that PARRoT can analyze two paired-end transcriptomic datasets of approximately 100 million reads within just three hours. In this study, we proposed and implemented a strategy to analyze transcriptomes from non-reference organisms which offers the opportunity to quantify and compare transcriptome profiles through a homolog based virtual transcriptome reference. By using the homolog based reference, our strategy effectively avoids the problems that may cause from inconsistencies among transcriptomes. This strategy will shed lights on the field of comparative genomics for non-model organism. We have implemented PARRoT as a web service which is freely available at http://parrot.cgu.edu.tw .

Expressed Sequence Tag Analysis of the Human Pathogen Paracoccidioides brasiliensis Yeast Phase: Identification of Putative Homologues of Candida albicans Virulence and Pathogenicity Genes

PubMed Central

Goldman, Gustavo H.; dos Reis Marques, Everaldo; Custódio Duarte Ribeiro, Diógenes; Ângelo de Souza Bernardes, Luciano; Quiapin, Andréa Carla; Vitorelli, Patrícia Marostica; Savoldi, Marcela; Semighini, Camile P.; de Oliveira, Regina C.; Nunes, Luiz R.; Travassos, Luiz R.; Puccia, Rosana; Batista, Wagner L.; Ferreira, Leslie Ecker; Moreira, Júlio C.; Bogossian, Ana Paula; Tekaia, Fredj; Nobrega, Marina Pasetto; Nobrega, Francisco G.; Goldman, Maria Helena S.

2003-01-01

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis. We present here a survey of expressed genes in the yeast pathogenic phase of P. brasiliensis. We obtained 13,490 expressed sequence tags from both 5′ and 3′ ends. Clustering analysis yielded the partial sequences of 4,692 expressed genes that were functionally classified by similarity to known genes. We have identified several Candida albicans virulence and pathogenicity homologues in P. brasiliensis. Furthermore, we have analyzed the expression of some of these genes during the dimorphic yeast-mycelium-yeast transition by real-time quantitative reverse transcription-PCR. Clustering analysis of the mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase, HSP70, HSP82, and HSP104. The first two groups displayed high mRNA expression in the mycelial phase, whereas the third group showed higher mRNA expression in the yeast phase. Our results suggest the possible conservation of pathogenicity and virulence mechanisms among fungi, expand considerably gene identification in P. brasiliensis, and provide a broader basis for further progress in understanding its biological peculiarities. PMID:12582121

Structural and functional analysis of mouse Msx1 gene promoter: sequence conservation with human MSX1 promoter points at potential regulatory elements.

PubMed

Gonzalez, S M; Ferland, L H; Robert, B; Abdelhay, E

1998-06-01

Vertebrate Msx genes are related to one of the most divergent homeobox genes of Drosophila, the muscle segment homeobox (msh) gene, and are expressed in a well-defined pattern at sites of tissue interactions. This pattern of expression is conserved in vertebrates as diverse as quail, zebrafish, and mouse in a range of sites including neural crest, appendages, and craniofacial structures. In the present work, we performed structural and functional analyses in order to identify potential cis-acting elements that may be regulating Msx1 gene expression. To this end, a 4.9-kb segment of the 5'-flanking region was sequenced and analyzed for transcription-factor binding sites. Four regions showing a high concentration of these sites were identified. Transfection assays with fragments of regulatory sequences driving the expression of the bacterial lacZ reporter gene showed that a region of 4 kb upstream of the transcription start site contains positive and negative elements responsible for controlling gene expression. Interestingly, a fragment of 130 bp seems to contain the minimal elements necessary for gene expression, as its removal completely abolishes gene expression in cultured cells. These results are reinforced by comparison of this region with the human Msx1 gene promoter, which shows extensive conservation, including many consensus binding sites, suggesting a regulatory role for them.

Identification of tissue-specific, abiotic stress-responsive gene expression patterns in wine grape (Vitis vinifera L.) based on curation and mining of large-scale EST data sets

PubMed Central

2011-01-01

Background Abiotic stresses, such as water deficit and soil salinity, result in changes in physiology, nutrient use, and vegetative growth in vines, and ultimately, yield and flavor in berries of wine grape, Vitis vinifera L. Large-scale expressed sequence tags (ESTs) were generated, curated, and analyzed to identify major genetic determinants responsible for stress-adaptive responses. Although roots serve as the first site of perception and/or injury for many types of abiotic stress, EST sequencing in root tissues of wine grape exposed to abiotic stresses has been extremely limited to date. To overcome this limitation, large-scale EST sequencing was conducted from root tissues exposed to multiple abiotic stresses. Results A total of 62,236 expressed sequence tags (ESTs) were generated from leaf, berry, and root tissues from vines subjected to abiotic stresses and compared with 32,286 ESTs sequenced from 20 public cDNA libraries. Curation to correct annotation errors, clustering and assembly of the berry and leaf ESTs with currently available V. vinifera full-length transcripts and ESTs yielded a total of 13,278 unique sequences, with 2302 singletons and 10,976 mapped to V. vinifera gene models. Of these, 739 transcripts were found to have significant differential expression in stressed leaves and berries including 250 genes not described previously as being abiotic stress responsive. In a second analysis of 16,452 ESTs from a normalized root cDNA library derived from roots exposed to multiple, short-term, abiotic stresses, 135 genes with root-enriched expression patterns were identified on the basis of their relative EST abundance in roots relative to other tissues. Conclusions The large-scale analysis of relative EST frequency counts among a diverse collection of 23 different cDNA libraries from leaf, berry, and root tissues of wine grape exposed to a variety of abiotic stress conditions revealed distinct, tissue-specific expression patterns, previously unrecognized stress-induced genes, and many novel genes with root-enriched mRNA expression for improving our understanding of root biology and manipulation of rootstock traits in wine grape. mRNA abundance estimates based on EST library-enriched expression patterns showed only modest correlations between microarray and quantitative, real-time reverse transcription-polymerase chain reaction (qRT-PCR) methods highlighting the need for deep-sequencing expression profiling methods. PMID:21592389

Discovery of cashmere goat (Capra hircus) microRNAs in skin and hair follicles by Solexa sequencing.

PubMed

Yuan, Chao; Wang, Xiaolong; Geng, Rongqing; He, Xiaolin; Qu, Lei; Chen, Yulin

2013-07-28

MicroRNAs (miRNAs) are a large family of endogenous, non-coding RNAs, about 22 nucleotides long, which regulate gene expression through sequence-specific base pairing with target mRNAs. Extensive studies have shown that miRNA expression in the skin changes remarkably during distinct stages of the hair cycle in humans, mice, goats and sheep. In this study, the skin tissues were harvested from the three stages of hair follicle cycling (anagen, catagen and telogen) in a fibre-producing goat breed. In total, 63,109,004 raw reads were obtained by Solexa sequencing and 61,125,752 clean reads remained for the small RNA digitalisation analysis. This resulted in the identification of 399 conserved miRNAs; among these, 326 miRNAs were expressed in all three follicular cycling stages, whereas 3, 12 and 11 miRNAs were specifically expressed in anagen, catagen, and telogen, respectively. We also identified 172 potential novel miRNAs by Mireap, 36 miRNAs were expressed in all three cycling stages, whereas 23, 29 and 44 miRNAs were specifically expressed in anagen, catagen, and telogen, respectively. The expression level of five arbitrarily selected miRNAs was analyzed by quantitative PCR, and the results indicated that the expression patterns were consistent with the Solexa sequencing results. Gene Ontology and KEGG pathway analyses indicated that five major biological pathways (Metabolic pathways, Pathways in cancer, MAPK signalling pathway, Endocytosis and Focal adhesion) accounted for 23.08% of target genes among 278 biological functions, indicating that these pathways are likely to play significant roles during hair cycling. During all hair cycle stages of cashmere goats, a large number of conserved and novel miRNAs were identified through a high-throughput sequencing approach. This study enriches the Capra hircus miRNA databases and provides a comprehensive miRNA transcriptome profile in the skin of goats during the hair follicle cycle.

Genome-Wide Identification and Expression Profiling of Cytokinin Oxidase/Dehydrogenase (CKX) Genes Reveal Likely Roles in Pod Development and Stress Responses in Oilseed Rape (Brassica napus L.).

PubMed

Liu, Pu; Zhang, Chao; Ma, Jin-Qi; Zhang, Li-Yuan; Yang, Bo; Tang, Xin-Yu; Huang, Ling; Zhou, Xin-Tong; Lu, Kun; Li, Jia-Na

2018-03-16

Cytokinin oxidase/dehydrogenases (CKXs) play a critical role in the irreversible degradation of cytokinins, thereby regulating plant growth and development. Brassica napus is one of the most widely cultivated oilseed crops worldwide. With the completion of whole-genome sequencing of B. napus , genome-wide identification and expression analysis of the BnCKX gene family has become technically feasible. In this study, we identified 23 BnCKX genes and analyzed their phylogenetic relationships, gene structures, conserved motifs, protein subcellular localizations, and other properties. We also analyzed the expression of the 23 BnCKX genes in the B. napus cultivar Zhong Shuang 11 ('ZS11') by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), revealing their diverse expression patterns. We selected four BnCKX genes based on the results of RNA-sequencing and qRT-PCR and compared their expression in cultivated varieties with extremely long versus short siliques. The expression levels of BnCKX5-1 , 5-2 , 6-1 , and 7-1 significantly differed between the two lines and changed during pod development, suggesting they might play roles in determining silique length and in pod development. Finally, we investigated the effects of treatment with the synthetic cytokinin 6-benzylaminopurine (6-BA) and the auxin indole-3-acetic acid (IAA) on the expression of the four selected BnCKX genes. Our results suggest that regulating BnCKX expression is a promising way to enhance the harvest index and stress resistance in plants.

A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries.

PubMed

Asamizu, E; Nakamura, Y; Sato, S; Tabata, S

2000-06-30

For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.

Towards the elements of successful insect Ribonucleic acid interference (RNAi)

USDA-ARS?s Scientific Manuscript database

Ribonucleic acid interference (RNAi), the sequence-specific suppression of gene expression, offers great opportunities for insect science, especially to analyze gene function, manage pest populations, and reduce disease pathogens. The accumulating body of literature on insect RNAi has revealed that ...

BNU-LSVED: a multimodal spontaneous expression database in educational environment

NASA Astrophysics Data System (ADS)

Sun, Bo; Wei, Qinglan; He, Jun; Yu, Lejun; Zhu, Xiaoming

2016-09-01

In the field of pedagogy or educational psychology, emotions are treated as very important factors, which are closely associated with cognitive processes. Hence, it is meaningful for teachers to analyze students' emotions in classrooms, thus adjusting their teaching activities and improving students ' individual development. To provide a benchmark for different expression recognition algorithms, a large collection of training and test data in classroom environment has become an acute problem that needs to be resolved. In this paper, we present a multimodal spontaneous database in real learning environment. To collect the data, students watched seven kinds of teaching videos and were simultaneously filmed by a camera. Trained coders made one of the five learning expression labels for each image sequence extracted from the captured videos. This subset consists of 554 multimodal spontaneous expression image sequences (22,160 frames) recorded in real classrooms. There are four main advantages in this database. 1) Due to recorded in the real classroom environment, viewer's distance from the camera and the lighting of the database varies considerably between image sequences. 2) All the data presented are natural spontaneous responses to teaching videos. 3) The multimodal database also contains nonverbal behavior including eye movement, head posture and gestures to infer a student ' s affective state during the courses. 4) In the video sequences, there are different kinds of temporal activation patterns. In addition, we have demonstrated the labels for the image sequences are in high reliability through Cronbach's alpha method.

Characterization of a novel ADAM protease expressed by Pneumocystis carinii.

PubMed

Kennedy, Cassie C; Kottom, Theodore J; Limper, Andrew H

2009-08-01

Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. Recent evidence has suggested that unidentified proteases are involved in Pneumocystis life cycle regulation. Proteolytically active ADAM (named for "a disintegrin and metalloprotease") family molecules have been identified in some fungal organisms, such as Aspergillus fumigatus and Schizosaccharomyces pombe, and some have been shown to participate in life cycle regulation. Accordingly, we sought to characterize ADAM-like molecules in the fungal opportunistic pathogen, Pneumocystis carinii (PcADAM). After an in silico search of the P. carinii genomic sequencing project identified a 329-bp partial sequence with homology to known ADAM proteins, the full-length PcADAM sequence was obtained by PCR extension cloning, yielding a final coding sequence of 1,650 bp. Sequence analysis detected the presence of a typical ADAM catalytic active site (HEXXHXXGXXHD). Expression of PcADAM over the Pneumocystis life cycle was analyzed by Northern blot. Southern and contour-clamped homogenous electronic field blot analysis demonstrated its presence in the P. carinii genome. Expression of PcADAM was observed to be increased in Pneumocystis cysts compared to trophic forms. The full-length gene was subsequently cloned and heterologously expressed in Saccharomyces cerevisiae. Purified PcADAMp protein was proteolytically active in casein zymography, requiring divalent zinc. Furthermore, native PcADAMp extracted directly from freshly isolated Pneumocystis organisms also exhibited protease activity. This is the first report of protease activity attributable to a specific, characterized protein in the clinically important opportunistic fungal pathogen Pneumocystis.

Identification and Characterization of MicroRNAs in Ovary and Testis of Nile Tilapia (Oreochromis niloticus) by Using Solexa Sequencing Technology

PubMed Central

Zhou, Yi; Yu, Fan; Gao, Yun; Luo, Yongju; Tang, Zhanyang; Guo, Zhongbao; Guo, Enyan; Gan, Xi; Zhang, Ming; Zhang, Yaping

2014-01-01

MicroRNAs (miRNAs) are endogenous non-coding small RNAs which play important roles in the regulation of gene expression by cleaving or inhibiting the translation of target gene transcripts. Thereinto, some specific miRNAs show regulatory activities in gonad development via translational control. In order to further understand the role of miRNA-mediated posttranscriptional regulation in Nile tilapia (Oreochromis niloticus) ovary and testis, two small RNA libraries of Nile tilapia were sequenced by Solexa small RNA deep sequencing methods. A total of 9,731,431 and 8,880,497 raw reads, representing 5,407,800 and 4,396,281 unique sequences were obtained from the sexually mature ovaries and testes, respectively. After comparing the small RNA sequences with the Rfam database, 1,432,210 reads in ovaries and 984,146 reads in testes were matched to the genome sequence of Nile tilapia. Bioinformatic analysis identified 764 mature miRNA, 209 miRNA-5p and 202 miRNA-3p were found in the two libraries, of which 525 known miRNAs are both expressed in the ovary and testis of Nile tilapia. Comparison of expression profiles of the testis, miR-727, miR-129 and miR-29 families were highly expressed in tilapia ovary. Additionally, miR-132, miR-212, miR-33a and miR-135b families, showed significant higher expression in testis compared with that in ovary. Furthermore, the expression patterns of the miRNAs were analyzed in different developmental stages of gonad. The result showed different expression patterns were observed during development of testis and ovary. In addition, the identification and characterization of differentially expressed miRNAs in the ovaries and testis of Nile tilapia provides important information on the role of miRNA in the regulation of the ovarian and testicular development and function. This data will be helpful to facilitate studies on the regulation of miRNAs during teleosts reproduction. PMID:24466258

Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.

PubMed

Klein, Allon M; Mazutis, Linas; Akartuna, Ilke; Tallapragada, Naren; Veres, Adrian; Li, Victor; Peshkin, Leonid; Weitz, David A; Kirschner, Marc W

2015-05-21

It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships. VIDEO ABSTRACT. Copyright © 2015 Elsevier Inc. All rights reserved.

Exploring single nucleotide polymorphism (SNP), microsatellite (SSR) and differentially expressed genes in the jellyfish (Rhopilema esculentum) by transcriptome sequencing.

PubMed

Li, Yunfeng; Zhou, Zunchun; Tian, Meilin; Tian, Yi; Dong, Ying; Li, Shilei; Liu, Weidong; He, Chongbo

2017-08-01

In this study, single nucleotide polymorphism (SNP), microsatellite (SSR) and differentially expressed genes (DEGs) in the oral parts, gonads, and umbrella parts of the jellyfish Rhopilema esculentum were analyzed by RNA-Seq technology. A total of 76.4 million raw reads and 72.1 million clean reads were generated from deep sequencing. Approximately 119,874 tentative unigenes and 149,239 transcripts were obtained. A total of 1,034,708 SNP markers were detected in the three tissues. For microsatellite mining, 5088 SSRs were identified from the unigene sequences. The most frequent repeat motifs were mononucleotide repeats, which accounted for 61.93%. Transcriptome comparison of the three tissues yielded a total of 8841 DEGs, of which 3560 were up-regulated and 5281 were down-regulated. This study represents the greatest sequencing effort carried out for a jellyfish and provides the first high-throughput transcriptomic resource for jellyfish. Copyright © 2017 Elsevier B.V. All rights reserved.

Amino acid sequence of a trypsin inhibitor from a Spirometra (Spirometra erinaceieuropaei).

PubMed

Sanda, A; Uchida, A; Itagaki, T; Kobayashi, H; Inokuchi, N; Koyama, T; Iwama, M; Ohgi, K; Irie, M

2001-12-01

A trypsin inhibitor that is highly homologous with bovine pancreatic trypsin inhibitor (BPTI) was co-purified along with RNase from Spirometra (Spirometra erinaceieuropaei). The amino acid sequence of this inhibitor (SETI) and the nucleotide sequence of the cDNA encoding this protein were determined by protein chemistry and gene technology. SETI contains 68 amino acid residues and has a molecular mass of 7,798 Da. SETI has 31 amino acid residues that are identical with BPTI's sequence, including 6 half-cystine and 5 aromatic amino acid residues. The active site Lys residue in BPTI is replaced by an Arg residue in SETI. SETI is an effective inhibitor of trypsin and moderately inhibits a-chymotrypsin, but less inhibits elastase or subtilisin. SETI was expressed by E. coli containing a PelB vector carrying the SETI encoding cDNA; an expression yield of 0.68 mg/l was obtained. The phylogenetic relationship of SETI and the other BPTI-like trypsin inhibitors was analyzed using most likelihood inference methods.

Identification and Expression Analysis of microRNAs at the Grain Filling Stage in Rice(Oryza sativa L.)via Deep Sequencing

PubMed Central

Yi, Rong; Zhu, Zhixuan; Hu, Jihong; Qian, Qian; Dai, Jincheng; Ding, Yi

2013-01-01

MicroRNAs (miRNAs) have been shown to play crucial roles in the regulation of plant development. In this study, high-throughput RNA-sequencing technology was used to identify novel miRNAs, and to reveal miRNAs expression patterns at different developmental stages during rice (Oryza sativa L.) grain filling. A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain. The expression profiles of these identified miRNAs were analyzed and the results showed that 161 known miRNAs were differentially expressed during grain development, a high proportion of which were up-regulated from 5 to 7 days after fertilization. In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally. Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation. In this study, differences were revealed in the composition and expression profiles of miRNAs among individual developmental stages during the rice grain filling process, and miRNA editing events were also observed, analyzed and validated during this process. The results provide novel insight into the dynamic profiles of miRNAs in developing rice grain and contribute to the understanding of the regulatory roles of miRNAs in grain filling. PMID:23469249

Identification and Characterization of 40 Isolated Rehmannia glutinosa MYB Family Genes and Their Expression Profiles in Response to Shading and Continuous Cropping

PubMed Central

Wang, Fengqing; Suo, Yanfei; Wei, He; Li, Mingjie; Xie, Caixia; Wang, Lina; Chen, Xinjian; Zhang, Zhongyi

2015-01-01

The v-myb avian myeloblastosis viral oncogene homolog (MYB) superfamily constitutes one of the most abundant groups of transcription factors (TFs) described in plants. To date, little is known about the MYB genes in Rehmannia glutinosa. Forty unique MYB genes with full-length cDNA sequences were isolated. These 40 genes were grouped into five categories, one R1R2R3-MYB, four TRFL MYBs, four SMH MYBs, 25 R2R3-MYBs, and six MYB-related members. The MYB DNA-binding domain (DBD) sequence composition was conserved among proteins of the same subgroup. As expected, most of the closely related members in the phylogenetic tree exhibited common motifs. Additionally, the gene structure and motifs of the R. glutinosa MYB genes were analyzed. MYB gene expression was analyzed in the leaf and the tuberous root under two abiotic stress conditions. Expression profiles showed that most R. glutinosa MYB genes were expressed in the leaf and the tuberous root, suggesting that MYB genes are involved in various physiological and developmental processes in R. glutinosa. Seven MYB genes were up-regulated in response to shading in at least one tissue. Two MYB genes showed increased expression and 13 MYB genes showed decreased expression in the tuberous root under continuous cropping. This investigation is the first comprehensive study of the MYB gene family in R. glutinosa. PMID:26147429

RAP: RNA-Seq Analysis Pipeline, a new cloud-based NGS web application

PubMed Central

2015-01-01

Background The study of RNA has been dramatically improved by the introduction of Next Generation Sequencing platforms allowing massive and cheap sequencing of selected RNA fractions, also providing information on strand orientation (RNA-Seq). The complexity of transcriptomes and of their regulative pathways make RNA-Seq one of most complex field of NGS applications, addressing several aspects of the expression process (e.g. identification and quantification of expressed genes and transcripts, alternative splicing and polyadenylation, fusion genes and trans-splicing, post-transcriptional events, etc.). Moreover, the huge volume of data generated by NGS platforms introduces unprecedented computational and technological challenges to efficiently analyze and store sequence data and results. Methods In order to provide researchers with an effective and friendly resource for analyzing RNA-Seq data, we present here RAP (RNA-Seq Analysis Pipeline), a cloud computing web application implementing a complete but modular analysis workflow. This pipeline integrates both state-of-the-art bioinformatics tools for RNA-Seq analysis and in-house developed scripts to offer to the user a comprehensive strategy for data analysis. RAP is able to perform quality checks (adopting FastQC and NGS QC Toolkit), identify and quantify expressed genes and transcripts (with Tophat, Cufflinks and HTSeq), detect alternative splicing events (using SpliceTrap) and chimeric transcripts (with ChimeraScan). This pipeline is also able to identify splicing junctions and constitutive or alternative polyadenylation sites (implementing custom analysis modules) and call for statistically significant differences in genes and transcripts expression, splicing pattern and polyadenylation site usage (using Cuffdiff2 and DESeq). Results Through a user friendly web interface, the RAP workflow can be suitably customized by the user and it is automatically executed on our cloud computing environment. This strategy allows to access to bioinformatics tools and computational resources without specific bioinformatics and IT skills. RAP provides a set of tabular and graphical results that can be helpful to browse, filter and export analyzed data, according to the user needs. PMID:26046471

Identification of immunity-related genes in the larvae of Protaetia brevitarsis seulensis (Coleoptera: Cetoniidae) by a next-generation sequencing-based transcriptome analysis.

PubMed

Bang, Kyeongrin; Hwang, Sejung; Lee, Jiae; Cho, Saeyoull

2015-01-01

To identify immune-related genes in the larvae of white-spotted flower chafers, next-generation sequencing was conducted with an Illumina HiSeq2000, resulting in 100 million cDNA reads with sequence information from over 10 billion base pairs (bp) and >50× transcriptome coverage. A subset of 77,336 contigs was created, and ∼35,532 sequences matched entries against the NCBI nonredundant database (cutoff, e < 10(-5)). Statistical analysis was performed on the 35,532 contigs. For profiling of the immune response, samples were analyzed by aligning 42 base sequence tags to the de novo reference assembly, comparing levels in immunized larvae to control levels of expression. Of the differentially expressed genes, 3,440 transcripts were upregulated and 3,590 transcripts were downregulated. Many of these genes were confirmed as immune-related genes such as pattern recognition proteins, immune-related signal transduction proteins, antimicrobial peptides, and cellular response proteins, by comparison to published data. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

Cloning of an ADP-ribosylation factor gene from banana (Musa acuminata) and its expression patterns in postharvest ripening fruit.

PubMed

Wang, Yuan; Wu, Jing; Xu, Bi-Yu; Liu, Ju-Hua; Zhang, Jian-Bin; Jia, Cai-Hong; Jin, Zhi-Qiang

2010-08-15

A full-length cDNA encoding an ADP-ribosylation factor (ARF) from banana (Musa acuminata) fruit was cloned and named MaArf. It contains an open reading frame encoding a 181-amino-acid polypeptide. Sequence analysis showed that MaArf shared high similarity with ARF of other plant species. The genomic sequence of MaArf was also obtained using polymerase chain reaction (PCR). Sequence analysis showed that MaArf was a split gene containing five exons and four introns in genomic DNA. Reverse-transcriptase PCR was used to analyze the spatial expression of MaArf. The results showed that MaArf was expressed in all the organs examined: root, rhizome, leaf, flower and fruit. Real-time quantitative PCR was used to explore expression patterns of MaArf in postharvest banana. There was differential expression of MaArf associated with ethylene biosynthesis. In naturally ripened banana, expression of MaArf was in accordance with ethylene biosynthesis. However, in 1-methylcyclopropene-treated banana, the expression of MaArf was inhibited and changed little. When treated with ethylene, MaArf expression in banana fruit significantly increased in accordance with ethylene biosynthesis; the peak of MaArf was 3 d after harvest, 11 d earlier than for naturally ripened banana fruits. These results suggest that MaArf is induced by ethylene in regulating postharvest banana ripening. Finally, subcellular localization assays showed the MaArf protein in the cytoplasm. Copyright 2010 Elsevier GmbH. All rights reserved.

Grouping of multicopper oxidases in Lentinula edodes by sequence similarities and expression patterns.

PubMed

Sakamoto, Yuichi; Nakade, Keiko; Yoshida, Kentaro; Natsume, Satoshi; Miyazaki, Kazuhiro; Sato, Shiho; van Peer, Arend F; Konno, Naotake

2015-12-01

The edible white rot fungus Lentinula edodes possesses a variety of lignin degrading enzymes such as manganese peroxidases and laccases. Laccases belong to the multicopper oxidases, which have a wide range of catalytic activities including polyphenol degradation and synthesis, lignin degradation, and melanin formation. The exact number of laccases in L. edodes is unknown, as are their complete properties and biological functions. We analyzed the draft genome sequence of L. edodes D703PP-9 and identified 13 multicopper oxidase-encoding genes; 11 laccases in sensu stricto, of which three are new, and two ferroxidases. lcc8, a laccase previously reported in L. edodes, was not identified in D703PP-9 genome. Phylogenetic analysis showed that the 13 multicopper oxidases can be classified into laccase sensu stricto subfamily 1, laccase sensu stricto subfamily 2 and ferroxidases. From sequence similarities and expression patterns, laccase sensu stricto subfamily 1 can be divided into two subgroups. Laccase sensu stricto subfamily 1 group A members are mainly secreted from mycelia, while laccase sensu stricto subfamily 1 group B members are expressed mainly in fruiting bodies during growth or after harvesting but are lowly expressed in mycelia. Laccase sensu stricto subfamily 2 members are mainly expressed in mycelia, and two ferroxidases are mainly expressed in the fruiting body during growth or after harvesting, and are expressed at very low levels in mycelium. Our data suggests that L. edodes laccases in same group share expression patterns and would have common biological functions.

Sugarcane transgenics expressing MYB transcription factors show improved glucose release

DOE PAGES

Poovaiah, Charleson R.; Bewg, William P.; Lan, Wu; ...

2016-07-15

In this study, sugarcane, a tropical C4 perennial crop, is capable of producing 30-100 tons or more of biomass per hectare annually. The lignocellulosic residue remaining after sugar extraction is currently underutilized and can provide a significant source of biomass for the production of second-generation bioethanol. As a result, MYB31 and MYB42 were cloned from maize and expressed in sugarcane with and without the UTR sequences. The cloned sequences were 98 and 99 % identical to the published nucleotide sequences. The inclusion of the UTR sequences did not affect any of the parameters tested. There was little difference in plantmore » height and the number of internodes of the MYB-overexpressing sugarcane plants when compared with controls. MYB transgene expression determined by qPCR exhibited continued expression in young and maturing internodes. MYB31 downregulated more genes within the lignin biosynthetic pathway than MYB42. MYB31 and MYB42 expression resulted in decreased lignin content in some lines. All MYB42 plants further analyzed showed significant increases in glucose release by enzymatic hydrolysis in 72 h, whereas only two MYB31 plants released more glucose than control plants. This correlated directly with a significant decrease in acid-insoluble lignin. Soluble sucrose content of the MYB42 transgenic plants did not vary compared to control plants. In conclusion, this study demonstrates the use of MYB transcription factors to improve the production of bioethanol from sugarcane bagasse remaining after sugar extraction.« less

Sugarcane transgenics expressing MYB transcription factors show improved glucose release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poovaiah, Charleson R.; Bewg, William P.; Lan, Wu

In this study, sugarcane, a tropical C4 perennial crop, is capable of producing 30-100 tons or more of biomass per hectare annually. The lignocellulosic residue remaining after sugar extraction is currently underutilized and can provide a significant source of biomass for the production of second-generation bioethanol. As a result, MYB31 and MYB42 were cloned from maize and expressed in sugarcane with and without the UTR sequences. The cloned sequences were 98 and 99 % identical to the published nucleotide sequences. The inclusion of the UTR sequences did not affect any of the parameters tested. There was little difference in plantmore » height and the number of internodes of the MYB-overexpressing sugarcane plants when compared with controls. MYB transgene expression determined by qPCR exhibited continued expression in young and maturing internodes. MYB31 downregulated more genes within the lignin biosynthetic pathway than MYB42. MYB31 and MYB42 expression resulted in decreased lignin content in some lines. All MYB42 plants further analyzed showed significant increases in glucose release by enzymatic hydrolysis in 72 h, whereas only two MYB31 plants released more glucose than control plants. This correlated directly with a significant decrease in acid-insoluble lignin. Soluble sucrose content of the MYB42 transgenic plants did not vary compared to control plants. In conclusion, this study demonstrates the use of MYB transcription factors to improve the production of bioethanol from sugarcane bagasse remaining after sugar extraction.« less

Hybrid Sequencing of Full-Length cDNA Transcripts of Stems and Leaves in Dendrobium officinale

PubMed Central

He, Liu; Fu, Shuhua; Xu, Zhichao; Yan, Jun; Xu, Jiang; Zhou, Hong; Zhou, Jianguo; Chen, Xinlian; Li, Ying; Au, Kin Fai; Yao, Hui

2017-01-01

Dendrobium officinale is an extremely valuable orchid used in traditional Chinese medicine, so sought after that it has a higher market value than gold. Although the expression profiles of some genes involved in the polysaccharide synthesis have previously been investigated, little research has been carried out on their alternatively spliced isoforms in D. officinale. In addition, information regarding the translocation of sugars from leaves to stems in D. officinale also remains limited. We analyzed the polysaccharide content of D. officinale leaves and stems, and completed in-depth transcriptome sequencing of these two diverse tissue types using second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing technology. The results of this study yielded a digital inventory of gene and mRNA isoform expressions. A comparative analysis of both transcriptomes uncovered a total of 1414 differentially expressed genes, including 844 that were up-regulated and 570 that were down-regulated in stems. Of these genes, one sugars will eventually be exported transporter (SWEET) and one sucrose transporter (SUT) are expressed to a greater extent in D. officinale stems than in leaves. Two glycosyltransferase (GT) and four cellulose synthase (Ces) genes undergo a distinct degree of alternative splicing. In the stems, the content of polysaccharides is twice as much as that in the leaves. The differentially expressed GT and transcription factor (TF) genes will be the focus of further study. The genes DoSWEET4 and DoSUT1 are significantly expressed in the stem, and are likely to be involved in sugar loading in the phloem. PMID:28981454

The Spatial and Temporal Transcriptomic Landscapes of Ginseng, Panax ginseng C. A. Meyer.

PubMed

Wang, Kangyu; Jiang, Shicui; Sun, Chunyu; Lin, Yanping; Yin, Rui; Wang, Yi; Zhang, Meiping

2015-12-11

Ginseng, including Asian ginseng (Panax ginseng C. A. Meyer) and American ginseng (P. quinquefolius L.), is one of the most important medicinal herbs in Asia and North America, but significantly understudied. This study sequenced and characterized the transcriptomes and expression profiles of genes expressed in 14 tissues and four different aged roots of Asian ginseng. A total of 265.2 million 100-bp clean reads were generated using the high-throughput sequencing platform HiSeq 2000, representing >8.3x of the 3.2-Gb ginseng genome. From the sequences, 248,993 unigenes were assembled for whole plant, 61,912-113,456 unigenes for each tissue and 54,444-65,412 unigenes for different year-old roots. We comprehensively analyzed the unigene sets and gene expression profiles. We found that the number of genes allocated to each functional category is stable across tissues or developmental stages, while the expression profiles of different genes of a gene family or involved in ginsenoside biosynthesis dramatically diversified spatially and temporally. These results provide an overall insight into the spatial and temporal transcriptome dynamics and landscapes of Asian ginseng, and comprehensive resources for advanced research and breeding of ginseng and related species.

Codon influence on protein expression in E. coli correlates with mRNA levels

PubMed Central

Boël, Grégory; Wong, Kam-Ho; Su, Min; Luff, Jon; Valecha, Mayank; Everett, John K.; Acton, Thomas B.; Xiao, Rong; Montelione, Gaetano T.; Aalberts, Daniel P.; Hunt, John F.

2016-01-01

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyze the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis

PubMed Central

2012-01-01

Background The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. Results Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. Conclusions By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand. PMID:22276739

Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis.

PubMed

Tu, Jing; Ge, Qinyu; Wang, Shengqin; Wang, Lei; Sun, Beili; Yang, Qi; Bai, Yunfei; Lu, Zuhong

2012-01-25

The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand.

Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

PubMed

Hussain, Hirra; Fisher, David I; Abbott, W Mark; Roth, Robert G; Dickson, Alan J

2017-10-01

Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant targets. Biotechnol. Bioeng. 2017;114: 2348-2359. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Sequence Evolution and Expression Regulation of Stress-Responsive Genes in Natural Populations of Wild Tomato

PubMed Central

Fischer, Iris; Steige, Kim A.; Stephan, Wolfgang; Mboup, Mamadou

2013-01-01

The wild tomato species Solanum chilense and S. peruvianum are a valuable non-model system for studying plant adaptation since they grow in diverse environments facing many abiotic constraints. Here we investigate the sequence evolution of regulatory regions of drought and cold responsive genes and their expression regulation. The coding regions of these genes were previously shown to exhibit signatures of positive selection. Expression profiles and sequence evolution of regulatory regions of members of the Asr (ABA/water stress/ripening induced) gene family and the dehydrin gene pLC30-15 were analyzed in wild tomato populations from contrasting environments. For S. chilense, we found that Asr4 and pLC30-15 appear to respond much faster to drought conditions in accessions from very dry environments than accessions from more mesic locations. Sequence analysis suggests that the promoter of Asr2 and the downstream region of pLC30-15 are under positive selection in some local populations of S. chilense. By investigating gene expression differences at the population level we provide further support of our previous conclusions that Asr2, Asr4, and pLC30-15 are promising candidates for functional studies of adaptation. Our analysis also demonstrates the power of the candidate gene approach in evolutionary biology research and highlights the importance of wild Solanum species as a genetic resource for their cultivated relatives. PMID:24205149

Genomic organization, expression, and chromosome localization of a third aurora-related kinase gene, Aie1.

PubMed

Hu, H M; Chuang, C K; Lee, M J; Tseng, T C; Tang, T K

2000-11-01

We previously reported two novel testis-specific serine/threonine kinases, Aie1 (mouse) and AIE2 (human), that share high amino acid identities with the kinase domains of fly aurora and yeast Ipl1. Here, we report the entire intron-exon organization of the Aie1 gene and analyze the expression patterns of Aie1 mRNA during testis development. The mouse Aie1 gene spans approximately 14 kb and contains seven exons. The sequences of the exon-intron boundaries of the Aie1 gene conform to the consensus sequences (GT/AG) of the splicing donor and acceptor sites of most eukaryotic genes. Comparative genomic sequencing revealed that the gene structure is highly conserved between mouse Aie1 and human AIE2. However, much less homology was found in the sequence outside the kinase-coding domains. The Aie1 locus was mapped to mouse chromosome 7A2-A3 by fluorescent in situ hybridization. Northern blot analysis indicates that Aie1 mRNA likely is expressed at a low level on day 14 and reaches its plateau on day 21 in the developing postnatal testis. RNA in situ hybridization indicated that the expression of the Aie1 transcript was restricted to meiotically active germ cells, with the highest levels detected in spermatocytes at the late pachytene stage. These findings suggest that Aie1 plays a role in spermatogenesis.

Method for Constructing Composite Response Surfaces by Combining Neural Networks with Polynominal Interpolation or Estimation Techniques

NASA Technical Reports Server (NTRS)

Rai, Man Mohan (Inventor); Madavan, Nateri K. (Inventor)

2007-01-01

A method and system for data modeling that incorporates the advantages of both traditional response surface methodology (RSM) and neural networks is disclosed. The invention partitions the parameters into a first set of s simple parameters, where observable data are expressible as low order polynomials, and c complex parameters that reflect more complicated variation of the observed data. Variation of the data with the simple parameters is modeled using polynomials; and variation of the data with the complex parameters at each vertex is analyzed using a neural network. Variations with the simple parameters and with the complex parameters are expressed using a first sequence of shape functions and a second sequence of neural network functions. The first and second sequences are multiplicatively combined to form a composite response surface, dependent upon the parameter values, that can be used to identify an accurate mode

Transcriptome de novo assembly from next-generation sequencing and comparative analyses in the hexaploid salt marsh species Spartina maritima and Spartina alterniflora (Poaceae)

PubMed Central

Ferreira de Carvalho, J; Poulain, J; Da Silva, C; Wincker, P; Michon-Coudouel, S; Dheilly, A; Naquin, D; Boutte, J; Salmon, A; Ainouche, M

2013-01-01

Spartina species have a critical ecological role in salt marshes and represent an excellent system to investigate recurrent polyploid speciation. Using the 454 GS-FLX pyrosequencer, we assembled and annotated the first reference transcriptome (from roots and leaves) for two related hexaploid Spartina species that hybridize in Western Europe, the East American invasive Spartina alterniflora and the Euro-African S. maritima. The de novo read assembly generated 38 478 consensus sequences and 99% found an annotation using Poaceae databases, representing a total of 16 753 non-redundant genes. Spartina expressed sequence tags were mapped onto the Sorghum bicolor genome, where they were distributed among the subtelomeric arms of the 10 S. bicolor chromosomes, with high gene density correlation. Normalization of the complementary DNA library improved the number of annotated genes. Ecologically relevant genes were identified among GO biological function categories in salt and heavy metal stress response, C4 photosynthesis and in lignin and cellulose metabolism. Expression of some of these genes had been found to be altered by hybridization and genome duplication in a previous microarray-based study in Spartina. As these species are hexaploid, up to three duplicated homoeologs may be expected per locus. When analyzing sequence polymorphism at four different loci in S. maritima and S. alterniflora, we found up to four haplotypes per locus, suggesting the presence of two expressed homoeologous sequences with one or two allelic variants each. This reference transcriptome will allow analysis of specific Spartina genes of ecological or evolutionary interest, estimation of homoeologous gene expression variation using RNA-seq and further gene expression evolution analyses in natural populations. PMID:23149455

Molecular characterization and functional analysis of chalcone synthase from Syringa oblata Lindl. in the flavonoid biosynthetic pathway.

PubMed

Wang, Yu; Dou, Ying; Wang, Rui; Guan, Xuelian; Hu, Zenghui; Zheng, Jian

2017-11-30

The flower color of Syringa oblata Lindl., which is often modulated by the flavonoid content, varies and is an important ornamental feature. Chalcone synthase (CHS) catalyzes the first key step in the flavonoid biosynthetic pathway. However, little is known about the role of S. oblata CHS (SoCHS) in flavonoid biosynthesis in this species. Here, we isolate and analyze the cDNA (SoCHS1) that encodes CHS in S. oblata. We also sought to analyzed the molecular characteristics and function of flavonoid metabolism by SoCHS1. We successfully isolated the CHS-encoding genomic DNA (gDNA) in S. oblata (SoCHS1), and the gene structural analysis indicated it had no intron. The opening reading frame (ORF) sequence of SoCHS1 was 1170bp long and encoded a 389-amino acid polypeptide. Multiple sequence alignment revealed that both the conserved CHS active site residues and CHS signature sequence were in the deduced amino acid sequence of SoCHS1. Crystallographic analysis revealed that the protein structure of SoCHS1 is highly similar to that of FnCHS1 in Freesia hybrida. The quantitative real-time polymerase chain reaction (PCR) performed to detect the SoCHS1 transcript expression levels in flowers, and other tissues revealed the expression was significantly correlated with anthocyanin accumulation during flower development. The ectopic expression results of Nicotiana tabacum showed that SoCHS1 overexpression in transgenic tobacco changed the flower color from pale pink to pink. In conclusion, these results suggest that SoCHS1 plays an essential role in flavonoid biosynthesis in S. oblata, and could be used to modify flavonoid components in other plant species. Copyright © 2017. Published by Elsevier B.V.

Epigenomics of Development in Populus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauss, Steve; Freitag, Michael; Mockler, Todd

2013-01-10

We conducted research to determine the role of epigenetic modifications during tree development using poplar (Populus trichocarpa), a model woody feedstock species. Using methylated DNA immunoprecipitation (MeDIP) or chromatin immunoprecipitation (ChIP), followed by high-throughput sequencing, we are analyzed DNA and histone methylation patterns in the P. trichocarpa genome in relation to four biological processes: bud dormancy and release, mature organ maintenance, in vitro organogenesis, and methylation suppression. Our project is now completed. We have 1) produced 22 transgenic events for a gene involved in DNA methylation suppression and studied its phenotypic consequences; 2) completed sequencing of methylated DNA from elevenmore » target tissues in wildtype P. trichocarpa; 3) updated our customized poplar genome browser using the open-source software tools (2.13) and (V2.2) of the P. trichocarpa genome; 4) produced summary data for genome methylation in P. trichocarpa, including distribution of methylation across chromosomes and in and around genes; 5) employed bioinformatic and statistical methods to analyze differences in methylation patterns among tissue types; and 6) used bisulfite sequencing of selected target genes to confirm bioinformatics and sequencing results, and gain a higher-resolution view of methylation at selected genes 7) compared methylation patterns to expression using available microarray data. Our main findings of biological significance are the identification of extensive regions of the genome that display developmental variation in DNA methylation; highly distinctive gene-associated methylation profiles in reproductive tissues, particularly male catkins; a strong whole genome/all tissue inverse association of methylation at gene bodies and promoters with gene expression; a lack of evidence that tissue specificity of gene expression is associated with gene methylation; and evidence that genome methylation is a significant impediment to tissue dedifferentiation and redifferentiation in vitro.« less

Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

PubMed Central

2010-01-01

Background Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. Results We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an isoflavone synthase gene) is co-expressed with several genes encoding isoflavonoid-related metabolic enzymes. We then focused on nodulation-induced P450s and found that CYP728H1 was co-expressed with the genes involved in phenylpropanoid metabolism. Similarly, CYP736A34 was highly co-expressed with lipoxygenase, lectin and CYP83D1, all of which are involved in root and nodule development. Conclusions The genome scale analysis of P450s in soybean reveals many unique features of these important enzymes in this crop although the functions of most of them are largely unknown. Gene co-expression analysis proves to be a useful tool to infer the function of uncharacterized genes. Our work presented here could provide important leads toward functional genomics studies of soybean P450s and their regulatory network through the integration of reverse genetics, biochemistry, and metabolic profiling tools. The identification of nodule-specific P450s and their further exploitation may help us to better understand the intriguing process of soybean and rhizobium interaction. PMID:21062474

The consensus sequence of FAMLF alternative splice variants is overexpressed in undifferentiated hematopoietic cells.

PubMed

Chen, W L; Luo, D F; Gao, C; Ding, Y; Wang, S Y

2015-07-01

The familial acute myeloid leukemia related factor gene (FAMLF) was previously identified from a familial AML subtractive cDNA library and shown to undergo alternative splicing. This study used real-time quantitative PCR to investigate the expression of the FAMLF alternative-splicing transcript consensus sequence (FAMLF-CS) in peripheral blood mononuclear cells (PBMCs) from 119 patients with de novo acute leukemia (AL) and 104 healthy controls, as well as in CD34+ cells from 12 AL patients and 10 healthy donors. A 429-bp fragment from a novel splicing variant of FAMLF was obtained, and a 363-bp consensus sequence was targeted to quantify total FAMLF expression. Kruskal-Wallis, Nemenyi, Spearman's correlation, and Mann-Whitney U-tests were used to analyze the data. FAMLF-CS expression in PBMCs from AL patients and CD34+ cells from AL patients and controls was significantly higher than in control PBMCs (P < 0.0001). Moreover, FAMLF-CS expression in PBMCs from the AML group was positively correlated with red blood cell count (rs =0.317, P=0.006), hemoglobin levels (rs = 0.210, P = 0.049), and percentage of peripheral blood blasts (rs = 0.256, P = 0.027), but inversely correlated with hemoglobin levels in the control group (rs = -0.391, P < 0.0001). AML patients with high CD34+ expression showed significantly higher FAMLF-CS expression than those with low CD34+ expression (P = 0.041). Our results showed that FAMLF is highly expressed in both normal and malignant immature hematopoietic cells, but that expression is lower in normal mature PBMCs.

Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

PubMed Central

Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jakob; Gilchrist, Michael J; Panitz, Frank; Jørgensen, Claus; Scheibye-Knudsen, Karsten; Arvin, Troels; Lumholdt, Steen; Sawera, Milena; Green, Trine; Nielsen, Bente J; Havgaard, Jakob H; Rosenkilde, Carina; Wang, Jun; Li, Heng; Li, Ruiqiang; Liu, Bin; Hu, Songnian; Dong, Wei; Li, Wei; Yu, Jun; Wang, Jian; Stærfeldt, Hans-Henrik; Wernersson, Rasmus; Madsen, Lone B; Thomsen, Bo; Hornshøj, Henrik; Bujie, Zhan; Wang, Xuegang; Wang, Xuefei; Bolund, Lars; Brunak, Søren; Yang, Huanming; Bendixen, Christian; Fredholm, Merete

2007-01-01

Background Knowledge of the structure of gene expression is essential for mammalian transcriptomics research. We analyzed a collection of more than one million porcine expressed sequence tags (ESTs), of which two-thirds were generated in the Sino-Danish Pig Genome Project and one-third are from public databases. The Sino-Danish ESTs were generated from one normalized and 97 non-normalized cDNA libraries representing 35 different tissues and three developmental stages. Results Using the Distiller package, the ESTs were assembled to roughly 48,000 contigs and 73,000 singletons, of which approximately 25% have a high confidence match to UniProt. Approximately 6,000 new porcine gene clusters were identified. Expression analysis based on the non-normalized libraries resulted in the following findings. The distribution of cluster sizes is scaling invariant. Brain and testes are among the tissues with the greatest number of different expressed genes, whereas tissues with more specialized function, such as developing liver, have fewer expressed genes. There are at least 65 high confidence housekeeping gene candidates and 876 cDNA library-specific gene candidates. We identified differential expression of genes between different tissues, in particular brain/spinal cord, and found patterns of correlation between genes that share expression in pairs of libraries. Finally, there was remarkable agreement in expression between specialized tissues according to Gene Ontology categories. Conclusion This EST collection, the largest to date in pig, represents an essential resource for annotation, comparative genomics, assembly of the pig genome sequence, and further porcine transcription studies. PMID:17407547

Small RNA Deep Sequencing and the Effects of microRNA408 on Root Gravitropic Bending in Arabidopsis

NASA Astrophysics Data System (ADS)

Li, Huasheng; Lu, Jinying; Sun, Qiao; Chen, Yu; He, Dacheng; Liu, Min

2015-11-01

MicroRNA (miRNA) is a non-coding small RNA composed of 20 to 24 nucleotides that influences plant root development. This study analyzed the miRNA expression in Arabidopsis root tip cells using Illumina sequencing and real-time PCR before (sample 0) and 15 min after (sample 15) a 3-D clinostat rotational treatment was administered. After stimulation was performed, the expression levels of seven miRNA genes, including Arabidopsis miR160, miR161, miR394, miR402, miR403, miR408, and miR823, were significantly upregulated. Illumina sequencing results also revealed two novel miRNAsthat have not been previously reported, The target genes of these miRNAs included pentatricopeptide repeat-containing protein and diadenosine tetraphosphate hydrolase. An overexpression vector of Arabidopsis miR408 was constructed and transferred to Arabidopsis plant. The roots of plants over expressing miR408 exhibited a slower reorientation upon gravistimulation in comparison with those of wild-type. This result indicate that miR408 could play a role in root gravitropic response.

Sequence analysis of PROTEOLYSIS 6 from Solanum lycopersicum

NASA Astrophysics Data System (ADS)

Roslan, Nur Farhana; Chew, Bee Lyn; Goh, Hoe-Han; Isa, Nurulhikma Md

2018-04-01

The N-end rule pathway is a protein degradation pathway that relates the protein half-life with the identity of its N-terminal residues. A destabilizing N-terminal residues is created by enzymatic reaction or chemical modifications. This destabilized substrate will be recognized by PROTEOLYSIS 6 (PRT6) protein, which encodes an E3 ligase enzyme and resulted in substrate degradation by proteasome. PRT6 has been studied in Arabidopsis thaliana and barley but not yet been studied in fleshy fruit plants. Hence, this study was carried out in tomato that is known as the model for fleshy fruit plants. BLASTX analysis identified that Solyc09g010830 which encodes for a PRT6 gene in tomato based on its sequence similarity with PRT6 in A. thaliana. In silico gene expression analysis shows that PRT6 gene was highly expressed in tomato fruits breaker +5. Co-expression analysis shows that PRT6 may not only involved in abiotic stresses but also in biotic stresses. The objective is to analyze the sequence and characterize PRT6 gene in tomato.

Xuhuai goat H-FABP gene clone, subcellular localization of expression products and the preparation of transgenic mice.

PubMed

Yin, Yan-hui; Li, Bi-chun; Wei, Guang-hui; Zhu, Cai-ye; Li, Wei; Zhang, Ya-ni; Du, Li-xin; Cao, Wen-guang

2012-05-01

The aim of this study was to clone the heart-type fatty acid binding protein (H-FABP) gene of Xuhuai goat, to explore it bioinformatically, and analyze the subcellular localization using enhanced green fluorescent protein (EGFP). The results showed that the coding sequence (CDS) length of Xuhuai goat H-FABP gene was 402 bp, encoding 133 amino acids (GenBank accession number AY466498.1). The H-FABP cDNA coding sequence was compared with the corresponding region of human, chicken, brown rat, cow, wild boar, donkey, and zebrafish. The similarity were 89%, 76%, 85%, 84%, 93%, 91%, 70%, respectively. For the corresponding amino acid sequences, the similarity were 90%, 79%, 88%, 97%, 95%, 94%, 72%, respectively. This study did not find the signal peptide region in the H-FABP protein; it revealed that H-FABP protein might be a nonsecreted protein. H-FABP expression was detected in vitro by reverse transcription-polymerase chain reaction (RT-PCR), and the EGFP-H-FABP fusion protein was localized to the cytoplasm. The gene could also be transiently and permanently expressed in mice.

MIPS: a calmodulin-binding protein of Gracilaria lemaneiformis under heat shock.

PubMed

Zhang, Xuan; Zhou, Huiyue; Zang, Xiaonan; Gong, Le; Sun, Hengyi; Zhang, Xuecheng

2014-08-01

To study the Ca(2+)/Calmodulin (CaM) signal transduction pathway of Gracilaria lemaneiformis under heat stress, myo-inositol-1-phosphate synthase (MIPS), a calmodulin-binding protein, was isolated using the yeast two-hybrid system. cDNA and DNA sequences of mips were cloned from G. lemaneiformis by using 5'RACE and genome walking procedures. The MIPS DNA sequence was 2,067 nucleotides long, containing an open reading frame (ORF) of 1,623 nucleotides with no intron. The mips ORF was predicted to encode 540 amino acids, which included the conserved MIPS domain and was 61-67 % similar to that of other species. After analyzing the amino acid sequence of MIPS, the CaM-Binding Domain (CaMBD) was inferred to be at a site spanning from amino acid 212 to amino acid 236. The yeast two-hybrid results proved that MIPS can interact with CaM and that MIPS is a type of calmodulin-binding protein. Next, the expression of CaM and MIPS in wild-type G. lemaneiformis and a heat-tolerant G. lemaneiformis cultivar, "981," were analyzed using real-time PCR under a heat shock of 32 °C. The expression level displayed a cyclical upward trend. Compared with wild type, the CaM expression levels of cultivar 981 were higher, which might directly relate to its resistance to high temperatures. This paper indicates that MIPS and CaM may play important roles in the high-temperature resistance of G. lemaneiformis.

Retrieval of Surface Lambert Albedos and Aerosols Optical Depths Using OMEGA Near-IR EPF Observations of Mars

NASA Astrophysics Data System (ADS)

Vincendon, M.; Langevin, Y.; Poulet, F.; Bibring, J.-P.; Gondet, B.

2007-03-01

We have analyzed five EPF sequences acquired by OMEGA/Mars Express in the near-IR over ice-free and ice-covered surfaces to retrieve simultaneously the Lambert albedo of the surface and the optical depth of aerosols.

Comparative analysis of the feline immunoglobulin repertoire.

PubMed

Steiniger, Sebastian C J; Glanville, Jacob; Harris, Douglas W; Wilson, Thomas L; Ippolito, Gregory C; Dunham, Steven A

2017-03-01

Next-Generation Sequencing combined with bioinformatics is a powerful tool for analyzing the large number of DNA sequences present in the expressed antibody repertoire and these data sets can be used to advance a number of research areas including antibody discovery and engineering. The accurate measurement of the immune repertoire sequence composition, diversity and abundance is important for understanding the repertoire response in infections, vaccinations and cancer immunology and could also be useful for elucidating novel molecular targets. In this study 4 individual domestic cats (Felis catus) were subjected to antibody repertoire sequencing with total number of sequences generated 1079863 for VH for IgG, 1050824 VH for IgM, 569518 for VK and 450195 for VL. Our analysis suggests that a similar VDJ expression patterns exists across all cats. Similar to the canine repertoire, the feline repertoire is dominated by a single subgroup, namely VH3. The antibody paratope of felines showed similar amino acid variation when compared to human, mouse and canine counterparts. All animals show a similarly skewed VH CDR-H3 profile and, when compared to canine, human and mouse, distinct differences are observed. Our study represents the first attempt to characterize sequence diversity in the expressed feline antibody repertoire and this demonstrates the utility of using NGS to elucidate entire antibody repertoires from individual animals. These data provide significant insight into understanding the feline immune system function. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Structure, inheritance, and expression of hybrid poplar (Populus trichocarpa x Populus deltoides) phenylalanine ammonia-lyase genes.

PubMed Central

Subramaniam, R; Reinold, S; Molitor, E K; Douglas, C J

1993-01-01

A heterologous probe encoding phenylalanine ammonia-lyase (PAL) was used to identify PAL clones in cDNA libraries made with RNA from young leaf tissue of two Populus deltoides x P. trichocarpa F1 hybrid clones. Sequence analysis of a 2.4-kb cDNA confirmed its identity as a full-length PAl clone. The predicted amino acid sequence is conserved in comparison with that of PAL genes from several other plants. Southern blot analysis of popular genomic DNA from parental and hybrid individuals, restriction site polymorphism in PAL cDNA clones, and sequence heterogeneity in the 3' ends of several cDNA clones suggested that PAL is encoded by at least two genes that can be distinguished by HindIII restriction site polymorphisms. Clones containing each type of PAL gene were isolated from a poplar genomic library. Analysis of the segregation of PAL-specific HindIII restriction fragment-length polymorphisms demonstrated the existence of two independently segregating PAL loci, one of which was mapped to a linkage group of the poplar genetic map. Developmentally regulated PAL expression in poplar was analyzed using RNA blots. Highest expression was observed in young stems, apical buds, and young leaves. Expression was lower in older stems and undetectable in mature leaves. Cellular localization of PAL expression by in situ hybridization showed very high levels of expression in subepidermal cells of leaves early during leaf development. In stems and petioles, expression was associated with subepidermal cells and vascular tissues. PMID:8108506

microRNA expression profiling in fetal single ventricle malformation identified by deep sequencing.

PubMed

Yu, Zhang-Bin; Han, Shu-Ping; Bai, Yun-Fei; Zhu, Chun; Pan, Ya; Guo, Xi-Rong

2012-01-01

microRNAs (miRNAs) have emerged as key regulators in many biological processes, particularly cardiac growth and development, although the specific miRNA expression profile associated with this process remains to be elucidated. This study aimed to characterize the cellular microRNA profile involved in the development of congenital heart malformation, through the investigation of single ventricle (SV) defects. Comprehensive miRNA profiling in human fetal SV cardiac tissue was performed by deep sequencing. Differential expression of 48 miRNAs was revealed by sequencing by oligonucleotide ligation and detection (SOLiD) analysis. Of these, 38 were down-regulated and 10 were up-regulated in differentiated SV cardiac tissue, compared to control cardiac tissue. This was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Predicted target genes of the 48 differentially expressed miRNAs were analyzed by gene ontology and categorized according to cellular process, regulation of biological process and metabolic process. Pathway-Express analysis identified the WNT and mTOR signaling pathways as the most significant processes putatively affected by the differential expression of these miRNAs. The candidate genes involved in cardiac development were identified as potential targets for these differentially expressed microRNAs and the collaborative network of microRNAs and cardiac development related-mRNAs was constructed. These data provide the basis for future investigation of the mechanism of the occurrence and development of fetal SV malformations.

Cinnamate-4-hydroxylase expression in arabidopsis. Regulation in response to development and the environment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bell-Lelong, D.A.; Cusumano, J.C.; Meyer, K.

1997-03-01

Cinnamate-r-hydroxylase (C4H) is the first Cyt P450-dependent monooxygenase of the phenylpropanoid pathway. To study the expression of this gene in Arabidopsis thaliana, a C4H cDNA clone from the Arabidopsis expressed sequence tag database was identified and used to isolate its corresponding genomic clone. The entire C4H coding sequence plus 2.9 kb of its promoter were isolated on a 5.4-kb HindIII fragment of this cosmid. Inspection of the promoter sequence revealed the presence of a number of putative regulatory motifs previously identified in the promoters of other phenylpropanoid pathway genes. The expression of C4H was analyzed by RNA blot hybridization analysismore » and in transgenic Arabidopsis carrying a C4H-{beta}-glucuronidase transcriptional fusion. C4H message accumulation was light-dependent, but was detectable even in dark-grown seedlings. Consistent with these data, C4H mRNA was accumulated to light-grown levels in etiolated det1-1 mutant seedlings. C4H is widely expressed in various Arabidopsis tissues, particularly in roots and cells undergoing lignification. The C4H-driven {beta}-glucuronidase expression accurately reflected the tissue-specificity and wound-inducibility of the C4H promoter indicated by RNA blot hybridization analysis. A modest increase in C4H expression was observed in the tt8 mutant of Arabidopsis. 77 refs., 5 figs.« less

Epigenetic Inactivation of GALR1 in Head and Neck Cancer

PubMed Central

Misawa, Kiyoshi; Ueda, Yo; Kanazawa, Takeharu; Misawa, Yuki; Jang, Ilwhan; Brenner, John Chadwick; Ogawa, Tetsuya; Takebayashi, Satoru; Grenman, Reidar A.; Herman, James G.; Mineta, Hiroyuki; Carey, Thomas E.

2011-01-01

Purpose One copy of the GALR1 locus on 18q is often deleted and expression is absent in some head and neck squamous cell carcinoma (HNSCC) cell lines. To determine if LOH and hypermethylation might silence the GALR1 gene, promoter methylation status and gene expression were assessed in a large panel of HNSCC cell lines and tumors. Experimental Design Promoter methylation of GALR1 in 72 cell lines and 100 primary tumor samples was analyzed using methylation-specific PCR (MSP). GALR1 expression and methylation status were analyzed further by real-time PCR and bisulfite sequencing analysis. Results The GALR1 promoter was fully or partially methylated in 38 of 72 HNSCC cell lines (52.7%) but not in the majority 18/20 (90.0%) of non-malignant lines. GALR1 methylation was also found in 38/100 (38%) primary tumor specimens. Methylation correlated with decreased GALR1 expression. In tumors methylation was significantly correlated with increased tumor size (P=0.0036), lymph-node status (P=0.0414), tumor stage (P=0.0037), cyclin D1 expression (P=0.0420), and p16 methylation (P=0.0494) and survival (P=0.045). Bisulfite sequencing of 36 CpG sites upstream of the transcription start site revealed that CpG methylation within transcription factor binding sites correlated with complete suppression of GALR1 mRNA. Treatment with TSA and 5-azacytidine restored GALR1 expression. In UM-SCC-23 cells that have total silencing of GALR1, exogenous GALR1 expression and stimulation with galanin suppressed cell proliferation. Conclusions Frequent promoter hypermethylation, gene silencing, association with prognosis, and growth suppression after re-expression support the hypothesis that GALR1 is a tumor suppressor gene in HNSCC. PMID:19047085

Development of unidentified dna-specific hif 1α gene of lizard (hemidactylus platyurus) which plays a role in tissue regeneration process

NASA Astrophysics Data System (ADS)

Novianti, T.; Sadikin, M.; Widia, S.; Juniantito, V.; Arida, E. A.

2018-03-01

Development of unidentified specific gene is essential to analyze the availability these genes in biological process. Identification unidentified specific DNA of HIF 1α genes is important to analyze their contribution in tissue regeneration process in lizard tail (Hemidactylus platyurus). Bioinformatics and PCR techniques are relatively an easier method to identify an unidentified gene. The most widely used method is BLAST (Basic Local Alignment Sequence Tools) method for alignment the sequences from the other organism. BLAST technique is online software from website https://blast.ncbi.nlm.nih.gov/Blast.cgi that capable to generate the similar sequences from closest kinship to distant kindship. Gecko japonicus is a species that it has closest kinship with H. platyurus. Comparing HIF 1 α gene sequence of G. japonicus with the other species used multiple alignment methods from Mega7 software. Conserved base areas were identified using Clustal IX method. Primary DNA of HIF 1 α gene was design by Primer3 software. HIF 1α gene of lizard (H. platyurus) was successfully amplified using a real-time PCR machine by primary DNA that we had designed from Gecko japonicus. Identification unidentified gene of HIF 1a lizard has been done successfully with multiple alignment method. The study was conducted by analyzing during the growth of tail on day 1, 3, 5, 7, 10, 13 and 17 of lizard tail after autotomy. Process amplification of HIF 1α gene was described by CT value in real time PCR machine. HIF 1α expression of gene is quantified by Livak formula. Chi-square statistic test is 0.000 which means that there is a different expression of HIF 1 α gene in every growth day treatment.

VH gene expression and regulation in the mutant Alicia rabbit. Rescue of VHa2 allotype expression.

PubMed

Chen, H T; Alexander, C B; Young-Cooper, G O; Mage, R G

1993-04-01

Rabbits of the Alicia strain, derived from rabbits expressing the VHa2 allotype, have a mutation in the H chain locus that has a cis effect upon the expression of VHa2 and VHa- genes. A small deletion at the most J-proximal (3') end of the VH locus leads to low expression of all the genes on the entire chromosome in heterozygous ali mutants and altered relative expression of VH genes in homozygotes. To study VH gene expression and regulation, we used the polymerase chain reaction to amplify the VH genes expressed in spleens of young and adult wild-type and mutant Alicia rabbits. The cDNA from reverse transcription of splenic mRNA was amplified and polymerase chain reaction libraries were constructed and screened with oligonucleotides from framework regions 1 and 3, as well as JH. Thirty-three VH-positive clones were sequenced and analyzed. We found that in mutant Alicia rabbits, products of the first functional VH gene (VH4a2), (or VH4a2-like genes) were expressed in 2- to 8-wk-olds. Expression of both the VHx and VHy types of VHa- genes was also elevated but the relative proportions of VHx and VHy, especially VHx, decreased whereas the relative levels of expression of VH4a2 or VH4a2-like genes increased with age. Our results suggest that the appearance of sequences resembling that of the VH1a2, which is deleted in the mutant ali rabbits, could be caused by alterations of the sequences of the rearranged VH4a2 genes by gene conversions and/or rearrangement of upstream VH1a2-like genes later in development.

Transcriptomic analysis of boron hyperaccumulation mechanisms in Puccinellia distans.

PubMed

Öztürk, Saniye Elvan; Göktay, Mehmet; Has, Canan; Babaoğlu, Mehmet; Allmer, Jens; Doğanlar, Sami; Frary, Anne

2018-05-01

Puccinellia distans, common alkali grass, is found throughout the world and can survive in soils with boron concentrations that are lethal for other plant species. Indeed, P. distans accumulates very high levels of this element. Despite these interesting features, very little research has been performed to elucidate the boron tolerance mechanism in this species. In this study, P. distans samples were treated for three weeks with normal (0.5 mg L -1 ) and elevated (500 mg L -1 ) boron levels in hydroponic solution. Expressed sequence tags (ESTs) derived from shoot tissue were analyzed by RNA sequencing to identify genes up and down-regulated under boron stress. In this way, 3312 differentially expressed transcripts were detected, 67.7% of which were up-regulated and 32.3% of which were down-regulated in boron-treated plants. To partially confirm the RNA sequencing results, 32 randomly selected transcripts were analyzed for their expression levels in boron-treated plants. The results agreed with the expected direction of change (up or down-regulation). A total of 1652 transcripts had homologs in A. thaliana and/or O. sativa and mapped to 1107 different proteins. Functional annotation of these proteins indicated that the boron tolerance and hyperaccumulation mechanisms of P. distans involve many transcriptomic changes including: alterations in the malate pathway, changes in cell wall components that may allow sequestration of excess boron without toxic effects, and increased expression of at least one putative boron transporter and two putative aquaporins. Elucidation of the boron accumulation mechanism is important in developing approaches for bioremediation of boron contaminated soils. Copyright © 2018 Elsevier Ltd. All rights reserved.

Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

PubMed Central

Yang, Christine; McLeod, Andrea J.; Cotton, Allison M.; de Leeuw, Charles N.; Laprise, Stéphanie; Banks, Kathleen G.; Simpson, Elizabeth M.; Brown, Carolyn J.

2012-01-01

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome. PMID:23023002

Comparison of global brain gene expression profiles between inbred long-sleep and inbred short-sleep mice by high-density gene array hybridization.

PubMed

Xu, Y; Ehringer, M; Yang, F; Sikela, J M

2001-06-01

Inbred long-sleep (ILS) and short-sleep (ISS) mice show significant central nervous system-mediated differences in sleep time for sedative dose of ethanol and are frequently used as a rodent model for ethanol sensitivity. In this study, we have used complementary DNA (cDNA) array hybridization methodology to identify genes that are differentially expressed between the brains of ILS and ISS mice. To carry out this analysis, we used both the gene discovery array (GDA) and the Mouse GEM 1 Microarray. GDA consists of 18,378 nonredundant mouse cDNA clones on a single nylon filter. Complex probes were prepared from total brain mRNA of ILS or ISS mice by using reverse transcription and 33P labeling. The labeled probes were hybridized in parallel to the gene array filters. Data from GDA experiments were analyzed with SQL-Plus and Oracle 8. The GEM microarray includes 8,730 sequence-verified clones on a glass chip. Two fluorescently labeled probes were used to hybridize a microarray simultaneously. Data from GEM experiments were analyzed by using the GEMTools software package (Incyte). Differentially expressed genes identified from each method were confirmed by relative quantitative reverse transcription-polymerase chain reaction (RT-PCR). A total of 41 genes or expressed sequence tags (ESTs) display significant expression level differences between brains of ILS and ISS mice after GDA, GEM1 hybridization, and quantitative RT-PCR confirmation. Among them, 18 clones were expressed higher in ILS mice, and 23 clones were expressed higher in ISS mice. The individual gene or EST's function and mapping information have been analyzed. This study identified 41 genes that are differentially expressed between brains of ILS and ISS mice. Some of them may have biological relevance in mediation of phenotypic variation between ILS and ISS mice for ethanol sensitivity. This study also demonstrates that parallel gene expression comparison with high-density cDNA arrays is a rapid and efficient way to discover potential genes and pathways involved in alcoholism and alcohol-related physiologic processes.

Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies.

PubMed

Villarreal, Michael A; Biediger, Nicole M; Bonner, Natalie A; Miller, Jennifer N; Zepeda, Samantha K; Ricard, Benjamin J; García, Dana M; Lewis, Karen A

2017-08-01

Antibodies raised against mammalian proteins may exhibit cross-reactivity with zebrafish proteins, making these antibodies useful for fish studies. However, zebrafish may express multiple paralogues of similar sequence and size, making them difficult to distinguish by traditional Western blot analysis. To identify the zebrafish proteins that are recognized by an antimammalian antibody, we developed a system to screen putative epitopes by cloning the sequences between the yeast SUMO protein and a C-terminal 6xHis tag. The recombinant fusion protein was expressed in Escherichia coli and analyzed by Western blot to conclusively identify epitopes that exhibit cross-reactivity with the antibodies of interest. This approach can be used to determine the species cross-reactivity and epitope specificity of a wide variety of peptide antigen-derived antibodies.

Simultaneous Characterization of Somatic Events and HPV-18 Integration in a Metastatic Cervical Carcinoma Patient Using DNA and RNA Sequencing

PubMed Central

Liang, Winnie S.; Aldrich, Jessica; Nasser, Sara; Kurdoglu, Ahmet; Phillips, Lori; Reiman, Rebecca; McDonald, Jacquelyn; Izatt, Tyler; Christoforides, Alexis; Baker, Angela; Craig, Christine; Egan, Jan B.; Chase, Dana M.; Farley, John H.; Bryce, Alan H.; Stewart, A. Keith; Borad, Mitesh J.; Carpten, John D.; Craig, David W.; Monk, Bradley J.

2014-01-01

Objective Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma. Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration. Given this need, the goal of this study was to use the next generation sequencing to simultaneously evaluate somatic alterations and expression changes in a patient’s cervical squamous carcinoma lesion metastatic to the lung and to detect and analyze HPV infection in the same sample. Materials and Methods We performed tumor and normal exome, tumor and normal shallow whole-genome sequencing, and RNA sequencing of the patient’s lung metastasis. Results We generated over 1.2 billion mapped reads and identified 130 somatic point mutations and indels, 21 genic translocations, 16 coding regions demonstrating copy number changes, and over 36 genes demonstrating altered expression in the tumor (corrected P < 0.05). Sequencing also revealed the HPV type 18 (HPV-18) integration in the metastasis. Using both DNA and RNA reads, we pinpointed 3 major events indicating HPV-18 integration into an intronic region of chromosome 6p25.1 in the patient’s tumor and validated these events with Sanger sequencing. This integration site has not been reported for HPV-18. Conclusions We demonstrate that DNA and RNA sequencing can be used to concurrently characterize somatic alterations and expression changes in a biopsy and delineate HPV integration at base resolution in cervical cancer. Further sequencing will allow us to better understand the molecular basis of cervical cancer pathogenesis. PMID:24418928

Utilization of next generation sequencing for analyzing transgenic insertions in plum

USDA-ARS?s Scientific Manuscript database

When utilizing transgenic plants, it is useful to know how many copies of the genes were inserted and the locations of these insertions in the genome. This information can provide important insights for the interpretation of transgene expression and the resulting phenotype. Traditionally, these qu...

Written Language Expression: Assessment Instruments and Teacher Tools

ERIC Educational Resources Information Center

Penner-Williams, Janet; Smith, Tom E. C.; Gartin, Barbara C.

2009-01-01

Written language is a complex set of skills that facilitate communication and that are developed in a predictable sequence. It is therefore possible to analyze current skills, identify deficits, plan interventions, and determine the effectiveness of the intervention. To effectively accomplish these tasks, educators need to choose appropriate…

The opportunities and challenges of large-scale molecular approaches to songbird neurobiology

PubMed Central

Mello, C.V.; Clayton, D.F.

2014-01-01

High-through put methods for analyzing genome structure and function are having a large impact in song-bird neurobiology. Methods include genome sequencing and annotation, comparative genomics, DNA microarrays and transcriptomics, and the development of a brain atlas of gene expression. Key emerging findings include the identification of complex transcriptional programs active during singing, the robust brain expression of non-coding RNAs, evidence of profound variations in gene expression across brain regions, and the identification of molecular specializations within song production and learning circuits. Current challenges include the statistical analysis of large datasets, effective genome curations, the efficient localization of gene expression changes to specific neuronal circuits and cells, and the dissection of behavioral and environmental factors that influence brain gene expression. The field requires efficient methods for comparisons with organisms like chicken, which offer important anatomical, functional and behavioral contrasts. As sequencing costs plummet, opportunities emerge for comparative approaches that may help reveal evolutionary transitions contributing to vocal learning, social behavior and other properties that make songbirds such compelling research subjects. PMID:25280907

Characterization of microRNAs from goat (Capra hircus) by Solexa deep-sequencing technology.

PubMed

Ling, Y H; Ding, J P; Zhang, X D; Wang, L J; Zhang, Y H; Li, Y S; Zhang, Z J; Zhang, X R

2013-06-13

MicroRNAs (miRNAs) are an important class of small noncoding RNAs that are highly conserved in plants and animals. Many miRNAs are known to mediate a myriad of cell processes, including proliferation and differentiation, via the regulation of some transcription and signaling factors, which are closely related to muscle development and disease. In this study, small RNA cDNA libraries of Boer goats were constructed. In addition, we obtained the goat muscle miRNAs by using Solexa deep-sequencing technology and analyzed these miRNA characteristics by combining it with the bioinformatics technology. Based on Solexa sequencing and bioinformatics analysis, 562 species-conserved and 5 goat genome-specific miRNAs were identified, 322 of which exceeded 100 in the expression levels. The results of real-time quantitative polymerase chain reaction from 8 randomly selected miRNAs showed that the 8 miRNAs were expressed in goat muscle, and the expression patterns were consistent with the Solexa sequencing results. The identification and characterization of miRNAs in goat muscle provide important information on the role of miRNA regulation in muscle growth and development. These data will help to facilitate studies on the regulatory roles played by miRNAs during goat growth and development.

Sequence analysis and gene expression of putative exo- and endo-glucanases from oil palm (Elaeis guineensis) during fungal infection.

PubMed

Yeoh, Keat-Ai; Othman, Abrizah; Meon, Sariah; Abdullah, Faridah; Ho, Chai-Ling

2012-10-15

Glucanases are enzymes that hydrolyze a variety β-d-glucosidic linkages. Plant β-1,3-glucanases are able to degrade fungal cell walls; and promote the release of cell-wall derived fungal elicitors. In this study, three full-length cDNA sequences encoding oil palm (Elaeis guineensis) glucanases were analyzed. Sequence analyses of the cDNA sequences suggested that EgGlc1-1 is a putative β-d-glucan exohydolase belonging to glycosyl hydrolase (GH) family 3 while EgGlc5-1 and EgGlc5-2 are putative glucan endo-1,3-β-glucosidases belonging to GH family 17. The transcript abundance of these genes in the roots and leaves of oil palm seedlings treated with Ganoderma boninense and Trichoderma harzianum was profiled to investigate the involvement of these glucanases in oil palm during fungal infection. The gene expression of EgGlc1-1 in the root of oil palm seedlings was increased by T. harzianum but suppressed by G. boninense; while the gene expression of both EgGlc5-1 and EgGlc5-2 in the roots of oil palm seedlings was suppressed by G. boninense or/and T. harzianum. Copyright © 2012 Elsevier GmbH. All rights reserved.

RNA Deep Sequencing Reveals Differential MicroRNA Expression during Development of Sea Urchin and Sea Star

PubMed Central

Kadri, Sabah; Hinman, Veronica F.; Benos, Panayiotis V.

2011-01-01

microRNAs (miRNAs) are small (20–23 nt), non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc.) to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads). Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common). We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html. PMID:22216218

The sugar transporter inventory of tomato: genome-wide identification and expression analysis.

PubMed

Reuscher, Stefan; Akiyama, Masahito; Yasuda, Tomohide; Makino, Haruko; Aoki, Koh; Shibata, Daisuke; Shiratake, Katsuhiro

2014-06-01

The mobility of sugars between source and sink tissues in plants depends on sugar transport proteins. Studying the corresponding genes allows the manipulation of the sink strength of developing fruits, thereby improving fruit quality for human consumption. Tomato (Solanum lycopersicum) is both a major horticultural crop and a model for the development of fleshy fruits. In this article we provide a comprehensive inventory of tomato sugar transporters, including the SUCROSE TRANSPORTER family, the SUGAR TRANSPORTER PROTEIN family, the SUGAR FACILITATOR PROTEIN family, the POLYOL/MONOSACCHARIDE TRANSPORTER family, the INOSITOL TRANSPORTER family, the PLASTIDIC GLUCOSE TRANSLOCATOR family, the TONOPLAST MONOSACCHARIDE TRANSPORTER family and the VACUOLAR GLUCOSE TRANSPORTER family. Expressed sequence tag (EST) sequencing and phylogenetic analyses established a nomenclature for all analyzed tomato sugar transporters. In total we identified 52 genes in tomato putatively encoding sugar transporters. The expression of 29 sugar transporter genes in vegetative tissues and during fruit development was analyzed. Several sugar transporter genes were expressed in a tissue- or developmental stage-specific manner. This information will be helpful to better understand source to sink movement of photoassimilates in tomato. Identification of fruit-specific sugar transporters might be a first step to find novel genes contributing to tomato fruit sugar accumulation. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Transcriptomic analysis of Ruditapes philippinarum hemocytes reveals cytoskeleton disruption after in vitro Vibrio tapetis challenge.

PubMed

Brulle, Franck; Jeffroy, Fanny; Madec, Stéphanie; Nicolas, Jean-Louis; Paillard, Christine

2012-10-01

The Manila clam, Ruditapes philippinarum, is an economically-important, commercial shellfish; harvests are diminished in some European waters by a pathogenic bacterium, Vibrio tapetis, that causes Brown Ring disease. To identify molecular characteristics associated with susceptibility or resistance to Brown Ring disease, Suppression Subtractive Hybridization (SSH) analyzes were performed to construct cDNA libraries enriched in up- or down-regulated transcripts from clam immune cells, hemocytes, after a 3-h in vitro challenge with cultured V. tapetis. Nine hundred and ninety eight sequences from the two libraries were sequenced, and an in silico analysis identified 235 unique genes. BLAST and "Gene ontology" classification analyzes revealed that 60.4% of the Expressed Sequence Tags (ESTs) have high similarities with genes involved in various physiological functions, such as immunity, apoptosis and cytoskeleton organization; whereas, 39.6% remain unidentified. From the 235 unique genes, we selected 22 candidates based upon physiological function and redundancy in the libraries. Then, Real-Time PCR analysis identified 3 genes related to cytoskeleton organization showing significant variation in expression attributable to V. tapetis exposure. Disruption in regulation of these genes is consistent with the etiologic agent of Brown Ring disease in Manila clams. Copyright © 2012 Elsevier Ltd. All rights reserved.

Insertion sequence transposition determines imipenem resistance in Acinetobacter baumannii.

PubMed

Kuo, Han-Yueh; Chang, Kai-Chih; Liu, Chih-Chin; Tang, Chuan Yi; Peng, Jhih-Hua; Lu, Chia-Wei; Tu, Chi-Chao; Liou, Ming-Li

2014-10-01

This study employed genomewide analysis to investigate potential resistance mechanisms in Acinetobacter baumannii following imipenem exposure. Imipenem-selected mutants were generated from the imipenem-susceptible strain ATCC 17978 by multistep selection resistance. Antibiotic susceptibilities were examined, and the selected mutants originated from the ATCC 17978 strain were confirmed by pulsed-field gel electrophoresis. The genomic sequence of a resistant mutant was analyzed using a next-generation sequencing platform, and genetic recombination was further confirmed by PCR. The result showed that phenotypic resistance was observed with carbapenem upon exposure to various concentrations of imipenem. Genomewide analysis showed that ISAba1 transposition was initiated by imipenem exposure at concentrations up to 0.5 mg/L. Transposition of ISAba1 upstream of blaOXA-95 was detected in all the selected mutants. The expression of blaOXA-95 was further analyzed by quantitative PCR, and the results demonstrated that a 200-fold increase in gene expression was required for resistance to imipenem. This study concluded that imipenem exposure at a concentration of 0.5 mg/L mediated the transposition of ISAba1 upstream of the blaOXA-95 gene and resulted in the overexpression of blaOXA-95 gene, which may play a major role in the resistance to imipenem in A. baumannii.

[Research progress in neuropsychopharmacology updated for the post-genomic era].

PubMed

Nakanishi, Toru

2009-11-01

Neuropsychopharmacological research in the post genomic (genomic sequence) era has been developing rapidly through the use of novel techniques including DNA chips. We have applied these techniques to investigate the anti-tumor effect of NSAIDs, isolate novel genes specifically expressed in rheumatoid arthritis, and analyze gene expression profiles in mesenchymal stem cells. Recently, we have developed a novel system of quantitative PCR for detection of BDNF mRNA isoforms. By using this system, we identified the exon-specific mode of expression in acute and chronic pain. In addition, we have made gene expression profiles of KO mice of beta2 subunits in acetylcholine receptors.

Transcriptome Profiling of Human FoxP3+ Regulatory T Cells

PubMed Central

Bhairavabhotla, Ravikiran; Kim, Yong C.; Glass, Deborah D.; Escobar, Thelma M.; Patel, Mira C.; Zahr, Rami; Nguyen, Cuong K.; Kilaru, Gokhul K.; Muljo, Stefan A.; Shevach, Ethan M.

2015-01-01

The major goal of this study was to perform an in depth characterization of the “gene signature” of human FoxP3+ T regulatory cells (Tregs). Highly purified Tregs and T conventional cells (Tconvs) from multiple healthy donors (HD), either freshly explanted or activated in vitro, were analyzed via RNA sequencing (RNA-seq) and gene expression changes validated using the nCounter system. Additionally, we analyzed microRNA (miRNA) expression using TaqMan low-density arrays. Our results confirm previous studies demonstrating selective gene expression of FoxP3, IKZF2, and CTLA4 in Tregs. Notably, a number of yet uncharacterized genes (RTKN2, LAYN, UTS2, CSF2RB, TRIB1, F5, CECAM4, CD70, ENC1 and NKG7) were identified and validated as being differentially expressed in human Tregs. We further characterize the functional roles of RTKN2 and LAYN by analyzing their roles in vitro human Treg suppression assays by knocking them down in Tregs and overexpressing them in Tconvs. In order to facilitate a better understanding of the human Treg gene expression signature, we have generated from our results a hypothetical interactome of genes and miRNAs in Tregs and Tconvs, PMID:26686412

TranslatomeDB: a comprehensive database and cloud-based analysis platform for translatome sequencing data

PubMed Central

Liu, Wanting; Xiang, Lunping; Zheng, Tingkai; Jin, Jingjie

2018-01-01

Abstract Translation is a key regulatory step, linking transcriptome and proteome. Two major methods of translatome investigations are RNC-seq (sequencing of translating mRNA) and Ribo-seq (ribosome profiling). To facilitate the investigation of translation, we built a comprehensive database TranslatomeDB (http://www.translatomedb.net/) which provides collection and integrated analysis of published and user-generated translatome sequencing data. The current version includes 2453 Ribo-seq, 10 RNC-seq and their 1394 corresponding mRNA-seq datasets in 13 species. The database emphasizes the analysis functions in addition to the dataset collections. Differential gene expression (DGE) analysis can be performed between any two datasets of same species and type, both on transcriptome and translatome levels. The translation indices translation ratios, elongation velocity index and translational efficiency can be calculated to quantitatively evaluate translational initiation efficiency and elongation velocity, respectively. All datasets were analyzed using a unified, robust, accurate and experimentally-verifiable pipeline based on the FANSe3 mapping algorithm and edgeR for DGE analyzes. TranslatomeDB also allows users to upload their own datasets and utilize the identical unified pipeline to analyze their data. We believe that our TranslatomeDB is a comprehensive platform and knowledgebase on translatome and proteome research, releasing the biologists from complex searching, analyzing and comparing huge sequencing data without needing local computational power. PMID:29106630

Inferring the expression variability of human transposable element-derived exons by linear model analysis of deep RNA sequencing data.

PubMed

Zhang, Wensheng; Edwards, Andrea; Fan, Wei; Fang, Zhide; Deininger, Prescott; Zhang, Kun

2013-08-28

The exonization of transposable elements (TEs) has proven to be a significant mechanism for the creation of novel exons. Existing knowledge of the retention patterns of TE exons in mRNAs were mainly established by the analysis of Expressed Sequence Tag (EST) data and microarray data. This study seeks to validate and extend previous studies on the expression of TE exons by an integrative statistical analysis of high throughput RNA sequencing data. We collected 26 RNA-seq datasets spanning multiple tissues and cancer types. The exon-level digital expressions (indicating retention rates in mRNAs) were quantified by a double normalized measure, called the rescaled RPKM (Reads Per Kilobase of exon model per Million mapped reads). We analyzed the distribution profiles and the variability (across samples and between tissue/disease groups) of TE exon expressions, and compared them with those of other constitutive or cassette exons. We inferred the effects of four genomic factors, including the location, length, cognate TE family and TE nucleotide proportion (RTE, see Methods section) of a TE exon, on the exons' expression level and expression variability. We also investigated the biological implications of an assembly of highly-expressed TE exons. Our analysis confirmed prior studies from the following four aspects. First, with relatively high expression variability, most TE exons in mRNAs, especially those without exact counterparts in the UCSC RefSeq (Reference Sequence) gene tables, demonstrate low but still detectable expression levels in most tissue samples. Second, the TE exons in coding DNA sequences (CDSs) are less highly expressed than those in 3' (5') untranslated regions (UTRs). Third, the exons derived from chronologically ancient repeat elements, such as MIRs, tend to be highly expressed in comparison with those derived from younger TEs. Fourth, the previously observed negative relationship between the lengths of exons and the inclusion levels in transcripts is also true for exonized TEs. Furthermore, our study resulted in several novel findings. They include: (1) for the TE exons with non-zero expression and as shown in most of the studied biological samples, a high TE nucleotide proportion leads to their lower retention rates in mRNAs; (2) the considered genomic features (i.e. a continuous variable such as the exon length or a category indicator such as 3'UTR) influence the expression level and the expression variability (CV) of TE exons in an inverse manner; (3) not only the exons derived from Alu elements but also the exons from the TEs of other families were preferentially established in zinc finger (ZNF) genes.

Serial analysis of gene expression (SAGE) in normal human trabecular meshwork.

PubMed

Liu, Yutao; Munro, Drew; Layfield, David; Dellinger, Andrew; Walter, Jeffrey; Peterson, Katherine; Rickman, Catherine Bowes; Allingham, R Rand; Hauser, Michael A

2011-04-08

To identify the genes expressed in normal human trabecular meshwork tissue, a tissue critical to the pathogenesis of glaucoma. Total RNA was extracted from human trabecular meshwork (HTM) harvested from 3 different donors. Extracted RNA was used to synthesize individual SAGE (serial analysis of gene expression) libraries using the I-SAGE Long kit from Invitrogen. Libraries were analyzed using SAGE 2000 software to extract the 17 base pair sequence tags. The extracted sequence tags were mapped to the genome using SAGE Genie map. A total of 298,834 SAGE tags were identified from all HTM libraries (96,842, 88,126, and 113,866 tags, respectively). Collectively, there were 107,325 unique tags. There were 10,329 unique tags with a minimum of 2 counts from a single library. These tags were mapped to known unique Unigene clusters. Approximately 29% of the tags (orphan tags) did not map to a known Unigene cluster. Thirteen percent of the tags mapped to at least 2 Unigene clusters. Sequence tags from many glaucoma-related genes, including myocilin, optineurin, and WD repeat domain 36, were identified. This is the first time SAGE analysis has been used to characterize the gene expression profile in normal HTM. SAGE analysis provides an unbiased sampling of gene expression of the target tissue. These data will provide new and valuable information to improve understanding of the biology of human aqueous outflow.

Identification of Differentially Expressed miRNAs between White and Black Hair Follicles by RNA-Sequencing in the Goat (Capra hircus)

PubMed Central

Wu, Zhenyang; Fu, Yuhua; Cao, Jianhua; Yu, Mei; Tang, Xiaohui; Zhao, Shuhong

2014-01-01

MicroRNAs (miRNAs) play a key role in many biological processes by regulating gene expression at the post-transcriptional level. A number of miRNAs have been identified from livestock species. However, compared with other animals, such as pigs and cows, the number of miRNAs identified in goats is quite low, particularly in hair follicles. In this study, to investigate the functional roles of miRNAs in goat hair follicles of goats with different coat colors, we sequenced miRNAs from two hair follicles samples (white and black) using Solexa sequencing. A total of 35,604,016 reads were obtained, which included 30,878,637 clean reads (86.73%). MiRDeep2 software identified 214 miRNAs. Among them, 205 were conserved among species and nine were novel miRNAs. Furthermore, DESeq software identified six differentially expressed miRNAs. Quantitative PCR confirmed differential expression of two miRNAs, miR-10b and miR-211. KEGG pathways were analyzed using the DAVID website for the predicted target genes of the differentially expressed miRNAs. Several signaling pathways including Notch and MAPK pathways may affect the process of coat color formation. Our study showed that the identified miRNAs might play an essential role in black and white follicle formation in goats. PMID:24879525

An exonic missense mutation c.28G>A is associated with weak B blood group by affecting RNA splicing of the ABO gene.

PubMed

Cai, Xiaohong; Qian, Chengrui; Wu, Wenman; Lei, Hang; Ding, Qiulan; Zou, Wei; Xiang, Dong; Wang, Xuefeng

2017-09-01

The amino acid substitutions caused by ABO gene mutations are usually predicted to impact glycosyltransferase's function or its biosynthesis. Here we report an ABO exonic missense mutation that affects B-antigen expression by decreasing the mRNA level of the ABO gene rather than the amino acid change. Serologic studies including plasma total GTB transfer capacity were performed. The exon sequences of the ABO gene were analyzed by Sanger sequencing. B 310 cDNA with c.28G>A (p.G10R) mutation was expressed in HeLa cells and total GTB transfer capacity in cell supernatant was measured. Flow cytometry was performed on these HeLa cells after transfection, and agglutination of Hela-B weak cells was also examined. The mRNA of the ABO gene was analyzed by direct sequencing and real-time reverse transcriptase-polymerase chain reaction. A minigene construct was prepared to evaluate the potential of splicing. While plasma total GTB transfer capacity was undetectable in this B 3 -like individual, the relative percentage of antigen-expressing cells and mean fluorescence index of the B weak red blood cells (RBCs) were 19 and 14% of normal B RBCs, respectively. There was no significant difference of total GTB transfer capacity in cell supernatant and B-antigen expression on cell surfaces between HeLa cells transfected with B 310 cDNA and B cDNA. The mRNA expression level of B 310 in peripheral whole blood was significantly reduced. The amount of splicing is significantly lower in c.28G>A construct compared to that in wild-type construct after transfection in K562 cells. ABO c.28G>A mutation may cause B 3 -like subgroup by affecting RNA splicing of the ABO gene. © 2017 AABB.

SPARTA: Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis.

PubMed

Johnson, Benjamin K; Scholz, Matthew B; Teal, Tracy K; Abramovitch, Robert B

2016-02-04

Many tools exist in the analysis of bacterial RNA sequencing (RNA-seq) transcriptional profiling experiments to identify differentially expressed genes between experimental conditions. Generally, the workflow includes quality control of reads, mapping to a reference, counting transcript abundance, and statistical tests for differentially expressed genes. In spite of the numerous tools developed for each component of an RNA-seq analysis workflow, easy-to-use bacterially oriented workflow applications to combine multiple tools and automate the process are lacking. With many tools to choose from for each step, the task of identifying a specific tool, adapting the input/output options to the specific use-case, and integrating the tools into a coherent analysis pipeline is not a trivial endeavor, particularly for microbiologists with limited bioinformatics experience. To make bacterial RNA-seq data analysis more accessible, we developed a Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis (SPARTA). SPARTA is a reference-based bacterial RNA-seq analysis workflow application for single-end Illumina reads. SPARTA is turnkey software that simplifies the process of analyzing RNA-seq data sets, making bacterial RNA-seq analysis a routine process that can be undertaken on a personal computer or in the classroom. The easy-to-install, complete workflow processes whole transcriptome shotgun sequencing data files by trimming reads and removing adapters, mapping reads to a reference, counting gene features, calculating differential gene expression, and, importantly, checking for potential batch effects within the data set. SPARTA outputs quality analysis reports, gene feature counts and differential gene expression tables and scatterplots. SPARTA provides an easy-to-use bacterial RNA-seq transcriptional profiling workflow to identify differentially expressed genes between experimental conditions. This software will enable microbiologists with limited bioinformatics experience to analyze their data and integrate next generation sequencing (NGS) technologies into the classroom. The SPARTA software and tutorial are available at sparta.readthedocs.org.

The Physcomitrella patens gene atlas project: large-scale RNA-seq based expression data.

PubMed

Perroud, Pierre-François; Haas, Fabian B; Hiss, Manuel; Ullrich, Kristian K; Alboresi, Alessandro; Amirebrahimi, Mojgan; Barry, Kerrie; Bassi, Roberto; Bonhomme, Sandrine; Chen, Haodong; Coates, Juliet C; Fujita, Tomomichi; Guyon-Debast, Anouchka; Lang, Daniel; Lin, Junyan; Lipzen, Anna; Nogué, Fabien; Oliver, Melvin J; Ponce de León, Inés; Quatrano, Ralph S; Rameau, Catherine; Reiss, Bernd; Reski, Ralf; Ricca, Mariana; Saidi, Younousse; Sun, Ning; Szövényi, Péter; Sreedasyam, Avinash; Grimwood, Jane; Stacey, Gary; Schmutz, Jeremy; Rensing, Stefan A

2018-07-01

High-throughput RNA sequencing (RNA-seq) has recently become the method of choice to define and analyze transcriptomes. For the model moss Physcomitrella patens, although this method has been used to help analyze specific perturbations, no overall reference dataset has yet been established. In the framework of the Gene Atlas project, the Joint Genome Institute selected P. patens as a flagship genome, opening the way to generate the first comprehensive transcriptome dataset for this moss. The first round of sequencing described here is composed of 99 independent libraries spanning 34 different developmental stages and conditions. Upon dataset quality control and processing through read mapping, 28 509 of the 34 361 v3.3 gene models (83%) were detected to be expressed across the samples. Differentially expressed genes (DEGs) were calculated across the dataset to permit perturbation comparisons between conditions. The analysis of the three most distinct and abundant P. patens growth stages - protonema, gametophore and sporophyte - allowed us to define both general transcriptional patterns and stage-specific transcripts. As an example of variation of physico-chemical growth conditions, we detail here the impact of ammonium supplementation under standard growth conditions on the protonemal transcriptome. Finally, the cooperative nature of this project allowed us to analyze inter-laboratory variation, as 13 different laboratories around the world provided samples. We compare differences in the replication of experiments in a single laboratory and between different laboratories. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Global analysis of gene expression profiles in developing physic nut (Jatropha curcas L.) seeds.

PubMed

Jiang, Huawu; Wu, Pingzhi; Zhang, Sheng; Song, Chi; Chen, Yaping; Li, Meiru; Jia, Yongxia; Fang, Xiaohua; Chen, Fan; Wu, Guojiang

2012-01-01

Physic nut (Jatropha curcas L.) is an oilseed plant species with high potential utility as a biofuel. Furthermore, following recent sequencing of its genome and the availability of expressed sequence tag (EST) libraries, it is a valuable model plant for studying carbon assimilation in endosperms of oilseed plants. There have been several transcriptomic analyses of developing physic nut seeds using ESTs, but they have provided limited information on the accumulation of stored resources in the seeds. We applied next-generation Illumina sequencing technology to analyze global gene expression profiles of developing physic nut seeds 14, 19, 25, 29, 35, 41, and 45 days after pollination (DAP). The acquired profiles reveal the key genes, and their expression timeframes, involved in major metabolic processes including: carbon flow, starch metabolism, and synthesis of storage lipids and proteins in the developing seeds. The main period of storage reserves synthesis in the seeds appears to be 29-41 DAP, and the fatty acid composition of the developing seeds is consistent with relative expression levels of different isoforms of acyl-ACP thioesterase and fatty acid desaturase genes. Several transcription factor genes whose expression coincides with storage reserve deposition correspond to those known to regulate the process in Arabidopsis. The results will facilitate searches for genes that influence de novo lipid synthesis, accumulation and their regulatory networks in developing physic nut seeds, and other oil seeds. Thus, they will be helpful in attempts to modify these plants for efficient biofuel production.

Genome-Scale Transcriptome Analysis in Response to Nitric Oxide in Birch Cells: Implications of the Triterpene Biosynthetic Pathway

PubMed Central

Zeng, Fansuo; Sun, Fengkun; Li, Leilei; Liu, Kun; Zhan, Yaguang

2014-01-01

Evidence supporting nitric oxide (NO) as a mediator of plant biochemistry continues to grow, but its functions at the molecular level remains poorly understood and, in some cases, controversial. To study the role of NO at the transcriptional level in Betula platyphylla cells, we conducted a genome-scale transcriptome analysis of these cells. The transcriptome of untreated birch cells and those treated by sodium nitroprusside (SNP) were analyzed using the Solexa sequencing. Data were collected by sequencing cDNA libraries of birch cells, which had a long period to adapt to the suspension culture conditions before SNP-treated cells and untreated cells were sampled. Among the 34,100 UniGenes detected, BLASTX search revealed that 20,631 genes showed significant (E-values≤10−5) sequence similarity with proteins from the NR-database. Numerous expressed sequence tags (i.e., 1374) were identified as differentially expressed between the 12 h SNP-treated cells and control cells samples: 403 up-regulated and 971 down-regulated. From this, we specifically examined a core set of NO-related transcripts. The altered expression levels of several transcripts, as determined by transcriptome analysis, was confirmed by qRT-PCR. The results of transcriptome analysis, gene expression quantification, the content of triterpenoid and activities of defensive enzymes elucidated NO has a significant effect on many processes including triterpenoid production, carbohydrate metabolism and cell wall biosynthesis. PMID:25551661

BAC and RNA sequencing reveal the brown planthopper resistance gene BPH15 in a recombination cold spot that mediates a unique defense mechanism.

PubMed

Lv, Wentang; Du, Ba; Shangguan, Xinxin; Zhao, Yan; Pan, Yufang; Zhu, Lili; He, Yuqing; He, Guangcun

2014-08-11

Brown planthopper (BPH, Nilaparvata lugens Stål), is the most destructive phloem-feeding insect pest of rice (Oryza sativa). The BPH-resistance gene BPH15 has been proved to be effective in controlling the pest and widely applied in rice breeding programs. Nevertheless, molecular mechanism of the resistance remain unclear. In this study, we narrowed down the position of BPH15 on chromosome 4 and investigated the transcriptome of BPH15 rice after BPH attacked. We analyzed 13,000 BC2F2 plants of cross between susceptible rice TN1 and the recombinant inbred line RI93 that carrying the BPH15 gene from original resistant donor B5. BPH15 was mapped to a 0.0269 cM region on chromosome 4, which is 210-kb in the reference genome of Nipponbare. Sequencing bacterial artificial chromosome (BAC) clones that span the BPH15 region revealed that the physical size of BPH15 region in resistant rice B5 is 580-kb, much bigger than the corresponding region in the reference genome of Nipponbare. There were 87 predicted genes in the BPH15 region in resistant rice. The expression profiles of predicted genes were analyzed. Four jacalin-related lectin proteins genes and one LRR protein gene were found constitutively expressed in resistant parent and considered the candidate genes of BPH15. The transcriptomes of resistant BPH15 introgression line and the susceptible recipient line were analyzed using high-throughput RNA sequencing. In total, 2,914 differentially expressed genes (DEGs) were identified. BPH-responsive transcript profiles were distinct between resistant and susceptible plants and between the early stage (6 h after infestation, HAI) and late stage (48 HAI). The key defense mechanism was related to jasmonate signaling, ethylene signaling, receptor kinase, MAPK cascades, Ca(2+) signaling, PR genes, transcription factors, and protein posttranslational modifications. Our work combined BAC and RNA sequencing to identify candidate genes of BPH15 and revealed the resistance mechanism that it mediated. These results increase our understanding of plant-insect interactions and can be used to protect against this destructive agricultural pest.

STAT5A and STAT5B have opposite correlations with drug response gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lamba, V., E-mail: vlamba@ufl.edu; Jia, B.; Liang, F.

Introduction: STAT5A and STAT5B are important transcription factors that play a key role in regulation of several important physiological processes including proliferation, survival, mediation of responses to cytokines and in regulating gender differences in drug response genes such as the hepatic cytochrome P450s (CYPs) that are responsible for a large majority of drug metabolism reactions in the human body. STAT5A and STAT5b have a high degree of sequence homology and have been reported to have largely similar functions. Recent studies have, however, indicated that they can also often have distinct and unique roles in regulating gene expression. Objective: In thismore » study, we evaluated the association of STAT5A and STAT5B mRNA expression levels with those of several key hepatic cytochrome P450s (CYPs) and hepatic transcription factors (TFs) and evaluated the potential roles of STAT5A and 5b in mediating gender differences in these CYPs and TFs. Methods: Expression profiling for major hepatic CYP isoforms and transcription factors was performed using RNA sequencing (RNA-seq) in 102 human liver samples (57 female, 45 male). Real time PCR gene expression data for selected CYPs and TFs was available on a subset of 50 human liver samples (25 female, 25 male) and was used to validate the RNA-seq findings. Results: While STAT5A demonstrated significant negative correlation with expression levels of multiple hepatic transcription factors (including NR1I2 and HNF4A) and DMEs such as CYP3A4 and CYP2C19, STAT5B expression was observed to demonstrate positive associations with several CYPs and TFs analyzed. As STAT5A and STAT5B have been shown to be important in regulation of gender differences in CYPs, we also analyzed STAT5A and 5b associations with CYPs and TFs separately in males and females and observed gender dependent differential associations of STATs with several CYPs and TFs. Results from the real time PCR validation largely supported our RNA-seq findings. Conclusions: Using both RNA sequencing and real time PCR, we examined the association of STAT5A and STAT5B mRNA expression with CYP and TF gene expression. While STAT5A demonstrated significant negative correlations with expression levels of multiple hepatic TFs (including NR1I2 and HNF4α) and CYPs (eg. CYP3A4, CYP2C19), STAT5B expression was observed to demonstrate positive association with most of the CYPs/TFs analyzed suggesting that STAT5A and STAT5b have potentially different and distinct roles in regulating expression of hepatic drug response genes. Further studies are needed to elucidate the potential roles of STAT5A and 5b in regulation of CYPs/TFs and the potential implications of these findings.« less

GESearch: An Interactive GUI Tool for Identifying Gene Expression Signature.

PubMed

Ye, Ning; Yin, Hengfu; Liu, Jingjing; Dai, Xiaogang; Yin, Tongming

2015-01-01

The huge amount of gene expression data generated by microarray and next-generation sequencing technologies present challenges to exploit their biological meanings. When searching for the coexpression genes, the data mining process is largely affected by selection of algorithms. Thus, it is highly desirable to provide multiple options of algorithms in the user-friendly analytical toolkit to explore the gene expression signatures. For this purpose, we developed GESearch, an interactive graphical user interface (GUI) toolkit, which is written in MATLAB and supports a variety of gene expression data files. This analytical toolkit provides four models, including the mean, the regression, the delegate, and the ensemble models, to identify the coexpression genes, and enables the users to filter data and to select gene expression patterns by browsing the display window or by importing knowledge-based genes. Subsequently, the utility of this analytical toolkit is demonstrated by analyzing two sets of real-life microarray datasets from cell-cycle experiments. Overall, we have developed an interactive GUI toolkit that allows for choosing multiple algorithms for analyzing the gene expression signatures.

Transcriptome Analysis of Orbital Adipose Tissue in Active Thyroid Eye Disease Using Next Generation RNA Sequencing Technology

PubMed Central

Lee, Bradford W.; Kumar, Virender B.; Biswas, Pooja; Ko, Audrey C.; Alameddine, Ramzi M.; Granet, David B.; Ayyagari, Radha; Kikkawa, Don O.; Korn, Bobby S.

2018-01-01

Objective: This study utilized Next Generation Sequencing (NGS) to identify differentially expressed transcripts in orbital adipose tissue from patients with active Thyroid Eye Disease (TED) versus healthy controls. Method: This prospective, case-control study enrolled three patients with severe, active thyroid eye disease undergoing orbital decompression, and three healthy controls undergoing routine eyelid surgery with removal of orbital fat. RNA Sequencing (RNA-Seq) was performed on freshly obtained orbital adipose tissue from study patients to analyze the transcriptome. Bioinformatics analysis was performed to determine pathways and processes enriched for the differential expression profile. Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) was performed to validate the differential expression of selected genes identified by RNA-Seq. Results: RNA-Seq identified 328 differentially expressed genes associated with active thyroid eye disease, many of which were responsible for mediating inflammation, cytokine signaling, adipogenesis, IGF-1 signaling, and glycosaminoglycan binding. The IL-5 and chemokine signaling pathways were highly enriched, and very-low-density-lipoprotein receptor activity and statin medications were implicated as having a potential role in TED. Conclusion: This study is the first to use RNA-Seq technology to elucidate differential gene expression associated with active, severe TED. This study suggests a transcriptional basis for the role of statins in modulating differentially expressed genes that mediate the pathogenesis of thyroid eye disease. Furthermore, the identification of genes with altered levels of expression in active, severe TED may inform the molecular pathways central to this clinical phenotype and guide the development of novel therapeutic agents. PMID:29760827

Delayed diagnosis in X-linked agammaglobulinemia and its relationship to the occurrence of mutations in BTK non-kinase domains.

PubMed

Carrillo-Tapia, Eduardo; García-García, Elizabeth; Herrera-González, Norma Estela; Yamazaki-Nakashimada, Marco Antonio; Staines-Boone, Aidee Tamara; Segura-Mendez, Nora Hilda; Scheffler-Mendoza, Selma Cecilia; O Farrill-Romanillos, Patricia; Gonzalez-Serrano, Maria E; Rodriguez-Alba, Juan Carloa; Santos-Argumedo, Leopoldo; Berron-Ruiz, Laura; Sanchez-Flores, Alejandro; López-Herrera, Gabriela

2018-01-01

X-linked agammaglobulinemia (XLA) is characterized by the absence of immunoglobulin and B cells. Patients suffer from recurrent bacterial infections from early childhood, and require lifelong immunoglobulin replacement therapy. Mutations in BTK (Bruton's Tyrosine Kinase) are associated with this phenotype. Some patients that present XLA do not show typical clinical symptoms, resulting in delayed diagnosis due to the lack of a severe phenotype. This study presents a report of five XLA patients from four different families and attempts to determine a relationship between delayed diagnosis and the occurrence of BTK mutations. Samples from patients with antibody deficiency were analyzed to determine BTK expression, immunophenotyping and mutation analysis. Clinical and laboratory data was analyzed and presented for each patient. Most patients presented here showed atypical clinical and laboratory data for XLA, including normal IgM, IgG, or IgA levels. Most patients expressed detectable BTK protein. Sequencing of BTK showed that these patients harbored missense mutations in the pleckstrin homology and Src-homology-2 domains. When it was compared to public databases, BTK sequencing exhibited a new change, along with three other previously reported changes. Delayed diagnosis and atypical manifestations in XLA might be related to mutation type and BTK expression.

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalra, Rajkumar S., E-mail: renu-wadhwa@aist.go.jp; Wadhwa, Renu, E-mail: renu-wadhwa@aist.go.jp

2015-02-27

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylationmore » target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.« less

Gene Expression Profiling of Development and Anthocyanin Accumulation in Kiwifruit (Actinidia chinensis) Based on Transcriptome Sequencing

PubMed Central

Zeng, Shaohua; Xiao, Gong; Wang, Gan; Wang, Ying; Peng, Ming; Huang, Hongwen

2015-01-01

Red-fleshed kiwifruit (Actinidia chinensis Planch. ‘Hongyang’) is a promising commercial cultivar due to its nutritious value and unique flesh color, derived from vitamin C and anthocyanins. In this study, we obtained transcriptome data of ‘Hongyang’ from seven developmental stages using Illumina sequencing. We mapped 39–54 million reads to the recently sequenced kiwifruit genome and other databases to define gene structure, to analyze alternative splicing, and to quantify gene transcript abundance at different developmental stages. The transcript profiles throughout red kiwifruit development were constructed and analyzed, with a focus on the biosynthesis and metabolism of compounds such as phytohormones, sugars, starch and L-ascorbic acid, which are indispensable for the development and formation of quality fruit. Candidate genes for these pathways were identified through MapMan and phylogenetic analysis. The transcript levels of genes involved in sucrose and starch metabolism were consistent with the change in soluble sugar and starch content throughout kiwifruit development. The metabolism of L-ascorbic acid was very active, primarily through the L-galactose pathway. The genes responsible for the accumulation of anthocyanin in red kiwifruit were identified, and their expression levels were investigated during kiwifruit development. This survey of gene expression during kiwifruit development paves the way for further investigation of the development of this uniquely colored and nutritious fruit and reveals which factors are needed for high quality fruit formation. This transcriptome data and its analysis will be useful for improving kiwifruit genome annotation, for basic fruit molecular biology research, and for kiwifruit breeding and improvement. PMID:26301713

Deep sequencing of small RNA repertoires in mice reveals metabolic disorders-associated hepatic miRNAs.

PubMed

Liang, Tingming; Liu, Chang; Ye, Zhenchao

2013-01-01

Obesity and associated metabolic disorders contribute importantly to the metabolic syndrome. On the other hand, microRNAs (miRNAs) are a class of small non-coding RNAs that repress target gene expression by inducing mRNA degradation and/or translation repression. Dysregulation of specific miRNAs in obesity may influence energy metabolism and cause insulin resistance, which leads to dyslipidemia, steatosis hepatis and type 2 diabetes. In the present study, we comprehensively analyzed and validated dysregulated miRNAs in ob/ob mouse liver, as well as miRNA groups based on miRNA gene cluster and gene family by using deep sequencing miRNA datasets. We found that over 13.8% of the total analyzed miRNAs were dysregulated, of which 37 miRNA species showed significantly differential expression. Further RT-qPCR analysis in some selected miRNAs validated the similar expression patterns observed in deep sequencing. Interestingly, we found that miRNA gene cluster and family always showed consistent dysregulation patterns in ob/ob mouse liver, although they had various enrichment levels. Functional enrichment analysis revealed the versatile physiological roles (over six signal pathways and five human diseases) of these miRNAs. Biological studies indicated that overexpression of miR-126 or inhibition of miR-24 in AML-12 cells attenuated free fatty acids-induced fat accumulation. Taken together, our data strongly suggest that obesity and metabolic disturbance are tightly associated with functional miRNAs. We also identified hepatic miRNA candidates serving as potential biomarkers for the diagnose of the metabolic syndrome.

Spanish Subject Personal Pronoun Use by Monolinguals, Bilinguals and Second Language Learners

ERIC Educational Resources Information Center

Abreu, Laurel

2009-01-01

Various studies analyzing pronominal subject expression in Spanish have found that switch-reference, the relationship between two consecutive subjects, is the factor that most commonly constrains speakers' choice of a null or explicit subject personal pronoun (SPP) (Cameron, 1995; Flores-Ferran, 2002). When the second subject in a sequence differs…

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts.

PubMed

Imin, Nijat; De Jong, Femke; Mathesius, Ulrike; van Noorden, Giel; Saeed, Nasir A; Wang, Xin-Ding; Rose, Ray J; Rolfe, Barry G

2004-07-01

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.

Insilico profiling of microRNAs in Korean ginseng (Panax ginseng Meyer)

PubMed Central

Mathiyalagan, Ramya; Subramaniyam, Sathiyamoorthy; Natarajan, Sathishkumar; Kim, Yeon Ju; Sun, Myung Suk; Kim, Se Young; Kim, Yu-Jin; Yang, Deok Chun

2013-01-01

MicroRNAs (miRNAs) are a class of recently discovered non-coding small RNA molecules, on average approximately 21 nucleotides in length, which underlie numerous important biological roles in gene regulation in various organisms. The miRNA database (release 18) has 18,226 miRNAs, which have been deposited from different species. Although miRNAs have been identified and validated in many plant species, no studies have been reported on discovering miRNAs in Panax ginseng Meyer, which is a traditionally known medicinal plant in oriental medicine, also known as Korean ginseng. It has triterpene ginseng saponins called ginsenosides, which are responsible for its various pharmacological activities. Predicting conserved miRNAs by homology-based analysis with available expressed sequence tag (EST) sequences can be powerful, if the species lacks whole genome sequence information. In this study by using the EST based computational approach, 69 conserved miRNAs belonging to 44 miRNA families were identified in Korean ginseng. The digital gene expression patterns of predicted conserved miRNAs were analyzed by deep sequencing using small RNA sequences of flower buds, leaves, and lateral roots. We have found that many of the identified miRNAs showed tissue specific expressions. Using the insilico method, 346 potential targets were identified for the predicted 69 conserved miRNAs by searching the ginseng EST database, and the predicted targets were mainly involved in secondary metabolic processes, responses to biotic and abiotic stress, and transcription regulator activities, as well as a variety of other metabolic processes. PMID:23717176

Predicting protein crystallization propensity from protein sequence

PubMed Central

2011-01-01

The high-throughput structure determination pipelines developed by structural genomics programs offer a unique opportunity for data mining. One important question is how protein properties derived from a primary sequence correlate with the protein’s propensity to yield X-ray quality crystals (crystallizability) and 3D X-ray structures. A set of protein properties were computed for over 1,300 proteins that expressed well but were insoluble, and for ~720 unique proteins that resulted in X-ray structures. The correlation of the protein’s iso-electric point and grand average hydropathy (GRAVY) with crystallizability was analyzed for full length and domain constructs of protein targets. In a second step, several additional properties that can be calculated from the protein sequence were added and evaluated. Using statistical analyses we have identified a set of the attributes correlating with a protein’s propensity to crystallize and implemented a Support Vector Machine (SVM) classifier based on these. We have created applications to analyze and provide optimal boundary information for query sequences and to visualize the data. These tools are available via the web site http://bioinformatics.anl.gov/cgi-bin/tools/pdpredictor. PMID:20177794

THAP1/DYT6 sequence variants in non-DYT1 early-onset primary dystonia in China and their effects on RNA expression.

PubMed

Cheng, Fu Bo; Ozelius, Laurie J; Wan, Xin Hua; Feng, Jia Chun; Ma, Ling Yan; Yang, Ying Mai; Wang, Lin

2012-02-01

Mutations in the THAP1 gene were recently identified as the cause of DYT6 primary dystonia. More than 40 mutations in this gene have been described in different populations. However, no previous report has identified sequence variations that affect the transcript process of the THAP1 gene. In addition, the mutation frequency in Chinese early-onset primary dystonia has not been well characterized. One hundred and two unrelated patients with non-DYT1 early-onset primary dystonia (age at onset <26 years), family members of participants with mutations, and 200 neurologically normal controls were screened for THAP1 gene mutations. The effects of the identified mutations on RNA expression were analyzed using semi-quantitative real-time PCR. Seven sequence variants (c.63_66del TTTC, c.161G>T, c.224A>T, c.267G>A, c.339T>C, c.449A>C, and c.539T>C) were identified in this group of patients (6.9%). In this cohort, 15 subjects (seven unrelated patients and eight family members) were detected to have THAP1 sequence variants. Among these 15 subjects, 11 were manifested (penetrance of DYT6 was 73.3%) and seven presented with craniocervical involvement (63.6%). However, one patient manifested paroxysmal headshake, and one presented with essential hand tremor. Semi-quantitative real-time PCR indicated that a novel silent mutation (c.267G>A) decreased the expression of THAP1 in human lymphocytes. Our findings indicated that THAP1 sequence variants are not common in non-DYT1 early-onset primary dystonia in China and that the clinical manifestation may vary. One silent mutation (c.267G>A) was shown to affect THAP1 expression.

Characterization and expression analysis of Toll-like receptor 3 cDNA from Atlantic salmon (Salmo salar).

PubMed

Vidal, R; González, R; Gil, F

2015-06-10

Innate pathway activation is fundamental for early anti-viral defense in fish, but currently there is insufficient understanding of how salmonid fish identify viral molecules and activate these pathways. The Toll-like receptor (TLR) is believed to play a crucial role in host defense of pathogenic microbes in the innate immune system. In the present study, the full-length cDNA of Salmo salar TLR3 (ssTLR3) was cloned. The ssTLR3 cDNA sequence was 6071 bp long, containing an open reading frame of 2754 bp and encoding 971 amino acids. The TLR group motifs, such as leucine-rich repeat (LRR) domains and Toll-interleukin-1 receptor (TIR) domains, were maintained in ssTLR3, with sixteen LRR domains and one TIR domain. In contrast to descriptions of the TLR3 in rainbow trout and the murine (TATA-less), we found a putative TATA box in the proximal promoter region 29 bp upstream of the transcription start point of ssTLR3. Multiple-sequence alignment analysis of the ssTLR3 protein-coding sequence with other known TLR3 sequences showed the sequence to be conserved among all species analyzed, implying that the function of the TLR3 had been sustained throughout evolution. The ssTLR3 mRNA expression patterns were measured using real-time PCR. The results revealed that TLR3 is widely expressed in various healthy tissues. Individuals challenged with infectious pancreatic necrosis virus and immunostimulated with polyinosinic:polycytidylic acid exhibited increased expression of TLR3 at the mRNA level, indicating that ssTLR3 may be involved in pathogen recognition in the early innate immune system.

Predicting earthquakes by analyzing accelerating precursory seismic activity

USGS Publications Warehouse

Varnes, D.J.

1989-01-01

During 11 sequences of earthquakes that in retrospect can be classed as foreshocks, the accelerating rate at which seismic moment is released follows, at least in part, a simple equation. This equation (1) is {Mathematical expression},where {Mathematical expression} is the cumulative sum until time, t, of the square roots of seismic moments of individual foreshocks computed from reported magnitudes;C and n are constants; and tfis a limiting time at which the rate of seismic moment accumulation becomes infinite. The possible time of a major foreshock or main shock, tf,is found by the best fit of equation (1), or its integral, to step-like plots of {Mathematical expression} versus time using successive estimates of tfin linearized regressions until the maximum coefficient of determination, r2,is obtained. Analyzed examples include sequences preceding earthquakes at Cremasta, Greece, 2/5/66; Haicheng, China 2/4/75; Oaxaca, Mexico, 11/29/78; Petatlan, Mexico, 3/14/79; and Central Chile, 3/3/85. In 29 estimates of main-shock time, made as the sequences developed, the errors in 20 were less than one-half and in 9 less than one tenth the time remaining between the time of the last data used and the main shock. Some precursory sequences, or parts of them, yield no solution. Two sequences appear to include in their first parts the aftershocks of a previous event; plots using the integral of equation (1) show that the sequences are easily separable into aftershock and foreshock segments. Synthetic seismic sequences of shocks at equal time intervals were constructed to follow equation (1), using four values of n. In each series the resulting distributions of magnitudes closely follow the linear Gutenberg-Richter relation log N=a-bM, and the product n times b for each series is the same constant. In various forms and for decades, equation (1) has been used successfully to predict failure times of stressed metals and ceramics, landslides in soil and rock slopes, and volcanic eruptions. Results of more recent experiments and theoretical studies on crack propagation, fault mechanics, and acoustic emission can be closely reproduced by equation (1). Rate-process theory and continuum damage mechanics offer leads toward understanding the physical processes. ?? 1989 Birkha??user Verlag.

Long- and short-time analysis of heartbeat sequences: correlation with mortality risk in congestive heart failure patients.

PubMed

Allegrini, P; Balocchi, R; Chillemi, S; Grigolini, P; Hamilton, P; Maestri, R; Palatella, L; Raffaelli, G

2003-06-01

We analyze RR heartbeat sequences with a dynamic model that satisfactorily reproduces both the long- and the short-time statistical properties of heart beating. These properties are expressed quantitatively by means of two significant parameters, the scaling delta concerning the asymptotic effects of long-range correlation, and the quantity 1-pi establishing the amount of uncorrelated fluctuations. We find a correlation between the position in the phase space (delta, pi) of patients with congestive heart failure and their mortality risk.

Lupin nad9 and nad6 genes and their expression: 5' termini of the nad9 gene transcripts differentiate lupin species.

PubMed

Rurek, Michał; Nuc, Katarzyna; Raczyńska, Katarzyna Dorota; Augustyniak, Halina

2003-10-02

The mitochondrial nad9 and nad6 genes were analyzed in four lupin species: Lupinus luteus, Lupinus angustifolius, Lupinus albus and Lupinus mutabilis. The nucleotide sequence of these genes confirmed their high conservation, however, higher number of nucleotide substitution was observed in the L. albus genes. Southern hybridizations confirmed the presence of single copy number of these genes in L. luteus, L. albus and L. angustifolius. The expression of nad9 and nad6 genes was analyzed by Northern in different tissue types of analyzed lupin species. Transcription analyses of the two nad genes displayed single predominant mRNA species of about 0.6 kb in L. luteus and L. angustifolius. The L. albus transcripts were larger in size. The nad9 and nad6 transcripts were modified by RNA editing at 8 and 11 positions, in L. luteus and L. angustifolius, respectively. The gene order, rps3-rpl16-nad9, found in Arabidopsis thaliana is also conserved in L. luteus and L. angustifolius mitochondria. L. luteus and L. angustifolius showed some variability in the sequence of the nad9 promoter region. The last feature along with the differences observed in nad9 mRNA 5' termini of two lupins differentiate L. luteus and L. angustifolius species.

CAPA-peptides of praying mantids (Mantodea).

PubMed

Koehler, Rene; Predel, Reinhard

2010-03-01

Dictyoptera which consist of cockroaches, termites, and praying mantids are among the oldest pterygote insects known. Whereas the localization and sequences of neuropeptides from a number of cockroaches are very well known, nearly nothing is known about the neuropeptides typical of praying mantids. In this study, the neuroanatomy of the median neuroendocrine system in the abdominal ventral nerve cord and the sequences of the CAPA-peptides which are expressed in the respective neuroendocrine cells were analyzed. Altogether, 40 species belonging to different families of Mantodea were included. In contrast to cockroaches, the mantids mostly express two CAPA-periviscerokinins (PVKs), only in Mantis religiosa a third PVK was identified. These PVKs are sequence-related to the PVKs of basal cockroaches (Polyphagidae). In a group of closely related Mantodea (Paramantinae), extended forms of PVK-2 were observed. As shown, these forms are possibly the result of substitutions in the N-terminal cleavage sites of the respective PVKs. No trace of a CAPA-pyrokinin was found in any of the praying mantids. (c) 2009 Elsevier Inc. All rights reserved.

Shotgun Bisulfite Sequencing of the Betula platyphylla Genome Reveals the Tree’s DNA Methylation Patterning

PubMed Central

Su, Chang; Wang, Chao; He, Lin; Yang, Chuanping; Wang, Yucheng

2014-01-01

DNA methylation plays a critical role in the regulation of gene expression. Most studies of DNA methylation have been performed in herbaceous plants, and little is known about the methylation patterns in tree genomes. In the present study, we generated a map of methylated cytosines at single base pair resolution for Betula platyphylla (white birch) by bisulfite sequencing combined with transcriptomics to analyze DNA methylation and its effects on gene expression. We obtained a detailed view of the function of DNA methylation sequence composition and distribution in the genome of B. platyphylla. There are 34,460 genes in the whole genome of birch, and 31,297 genes are methylated. Conservatively, we estimated that 14.29% of genomic cytosines are methylcytosines in birch. Among the methylation sites, the CHH context accounts for 48.86%, and is the largest proportion. Combined transcriptome and methylation analysis showed that the genes with moderate methylation levels had higher expression levels than genes with high and low methylation. In addition, methylated genes are highly enriched for the GO subcategories of binding activities, catalytic activities, cellular processes, response to stimulus and cell death, suggesting that methylation mediates these pathways in birch trees. PMID:25514241

Molecular Cloning, Expression Profile and 5′ Regulatory Region Analysis of Two Chemosensory Protein Genes from the Diamondback Moth, Plutella xylostella

PubMed Central

Gong, Liang; Zhong, Guo-Hua; Hu, Mei-Ying; Luo, Qian; Ren, Zhen-Zhen

2010-01-01

Chemosensory proteins play an important role in transporting chemical compounds to their receptors on dendrite membranes. In this study, two full-length cDNA codings for chemosensory proteins of Plutella xylostella (Lepidoptera: Plutellidae) were obtained by RACE-PCR. PxylCSP3 and Pxyl-CSP4, with GenBank accession numbers ABM92663 and ABM92664, respectively, were cloned and sequenced. The gene sequences both consisted of three exons and two introns. RT-PCR analysis showed that Pxyl-CSP3 and Pxyl-CSP4 had different expression patterns in the examined developmental stages, but were expressed in all larval stages. Phylogenetic analysis indicated that lepidopteran insects consist of three branches, and Pxyl-CSP3 and Pxyl-CSP4 belong to different branches. The 5′regulatory regions of Pxyl-CSP3 and Pxyl-CSP4 were isolated and analyzed, and the results consist of not only the core promoter sequences (TATA-box), but also several transcriptional elements (BR-C Z4, Hb, Dfd, CF2-II, etc.). This study provides clues to better understanding the various physiological functions of CSPs in P. xylostella and other insects. PMID:21073345

Genome-wide analyses of long noncoding RNA expression profiles correlated with radioresistance in nasopharyngeal carcinoma via next-generation deep sequencing.

PubMed

Li, Guo; Liu, Yong; Liu, Chao; Su, Zhongwu; Ren, Shuling; Wang, Yunyun; Deng, Tengbo; Huang, Donghai; Tian, Yongquan; Qiu, Yuanzheng

2016-09-06

Radioresistance is one of the major factors limiting the therapeutic efficacy and prognosis of patients with nasopharyngeal carcinoma (NPC). Accumulating evidence has suggested that aberrant expression of long noncoding RNAs (lncRNAs) contributes to cancer progression. Therefore, here we identified lncRNAs associated with radioresistance in NPC. The differential expression profiles of lncRNAs associated with NPC radioresistance were constructed by next-generation deep sequencing by comparing radioresistant NPC cells with their parental cells. LncRNA-related mRNAs were predicted and analyzed using bioinformatics algorithms compared with the mRNA profiles related to radioresistance obtained in our previous study. Several lncRNAs and associated mRNAs were validated in established NPC radioresistant cell models and NPC tissues. By comparison between radioresistant CNE-2-Rs and parental CNE-2 cells by next-generation deep sequencing, a total of 781 known lncRNAs and 2054 novel lncRNAs were annotated. The top five upregulated and downregulated known/novel lncRNAs were detected using quantitative real-time reverse transcription-polymerase chain reaction, and 7/10 known lncRNAs and 3/10 novel lncRNAs were demonstrated to have significant differential expression trends that were the same as those predicted by deep sequencing. From the prediction process, 13 pairs of lncRNAs and their associated genes were acquired, and the prediction trends of three pairs were validated in both radioresistant CNE-2-Rs and 6-10B-Rs cell lines, including lncRNA n373932 and SLITRK5, n409627 and PRSS12, and n386034 and RIMKLB. LncRNA n373932 and its related SLITRK5 showed dramatic expression changes in post-irradiation radioresistant cells and a negative expression correlation in NPC tissues (R = -0.595, p < 0.05). Our study provides an overview of the expression profiles of radioresistant lncRNAs and potentially related mRNAs, which will facilitate future investigations into the function of lncRNAs in NPC radioresistance.

Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera

PubMed Central

2010-01-01

Background The AP2/ERF protein family contains transcription factors that play a crucial role in plant growth and development and in response to biotic and abiotic stress conditions in plants. Grapevine (Vitis vinifera) is the only woody crop whose genome has been fully sequenced. So far, no detailed expression profile of AP2/ERF-like genes is available for grapevine. Results An exhaustive search for AP2/ERF genes was carried out on the Vitis vinifera genome and their expression profile was analyzed by Real-Time quantitative PCR (qRT-PCR) in different vegetative and reproductive tissues and under two different ripening stages. One hundred and forty nine sequences, containing at least one ERF domain, were identified. Specific clusters within the AP2 and ERF families showed conserved expression patterns reminiscent of other species and grapevine specific trends related to berry ripening. Moreover, putative targets of group IX ERFs were identified by co-expression and protein similarity comparisons. Conclusions The grapevine genome contains an amount of AP2/ERF genes comparable to that of other dicot species analyzed so far. We observed an increase in the size of specific groups within the ERF family, probably due to recent duplication events. Expression analyses in different aerial tissues display common features previously described in other plant systems and introduce possible new roles for members of some ERF groups during fruit ripening. The presented analysis of AP2/ERF genes in grapevine provides the bases for studying the molecular regulation of berry development and the ripening process. PMID:21171999

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

PubMed

Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

2014-08-01

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. © 2014 Remy et al.; Published by Cold Spring Harbor Laboratory Press.

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases

PubMed Central

Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J.; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

2014-01-01

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%–5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. PMID:24989021

RNA Sequencing and Bioinformatics Analysis Implicate the Regulatory Role of a Long Noncoding RNA-mRNA Network in Hepatic Stellate Cell Activation.

PubMed

Guo, Can-Jie; Xiao, Xiao; Sheng, Li; Chen, Lili; Zhong, Wei; Li, Hai; Hua, Jing; Ma, Xiong

2017-01-01

To analyze the long noncoding (lncRNA)-mRNA expression network and potential roles in rat hepatic stellate cells (HSCs) during activation. LncRNA expression was analyzed in quiescent and culture-activated HSCs by RNA sequencing, and differentially expressed lncRNAs verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) were subjected to bioinformatics analysis. In vivo analyses of differential lncRNA-mRNA expression were performed on a rat model of liver fibrosis. We identified upregulation of 12 lncRNAs and 155 mRNAs and downregulation of 12 lncRNAs and 374 mRNAs in activated HSCs. Additionally, we identified the differential expression of upregulated lncRNAs (NONRATT012636.2, NONRATT016788.2, and NONRATT021402.2) and downregulated lncRNAs (NONRATT007863.2, NONRATT019720.2, and NONRATT024061.2) in activated HSCs relative to levels observed in quiescent HSCs, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that changes in lncRNAs associated with HSC activation revealed 11 significantly enriched pathways according to their predicted targets. Moreover, based on the predicted co-expression network, the relative dynamic levels of NONRATT013819.2 and lysyl oxidase (Lox) were compared during HSC activation both in vitro and in vivo. Our results confirmed the upregulation of lncRNA NONRATT013819.2 and Lox mRNA associated with the extracellular matrix (ECM)-related signaling pathway in HSCs and fibrotic livers. Our results detailing a dysregulated lncRNA-mRNA network might provide new treatment strategies for hepatic fibrosis based on findings indicating potentially critical roles for NONRATT013819.2 and Lox in ECM remodeling during HSC activation. © 2017 The Author(s). Published by S. Karger AG, Basel.

Let-7 miRNA Precursors Co-express with LIN28B in Cervical Cells.

PubMed

Zamora-Contreras, Aida Margarita; Alvarez-Salas, Luis Marat

2018-01-01

The let-7 microRNAs (miRNAs) are frequently dysregulated in carcinogenic processes, including cervical cancer. LIN28 proteins regulate let-7 biogenesis by binding to conserved sequences within the pre-miRNA structure. Nevertheless, recent research has shown that some let-7 miRNAs may escape LIN28 regulation. Correlate pre-let-7 miRNAs and LIN28B levels in cervical cell lines with different malignancy and HPV content. Pre-let-7 levels were determined by RTqPCR. LIN28B and other let-7 targets were analyzed by immunoblot. In silico tools were used to correlate let-7 and LIN28B expression and to analyze prelet- 7 sequences and structures. Lin28B protein was detected in all tested cell lines although it was more expressed in tumor cell lines. High levels of pre-let-7c/f-1 and pre-miR-98 were present in almost all cell lines regardless malignancy and LIN28B expression. Pre-let-7g/i were mainly expressed in tumor cell lines, pre-let-7e and pre-let-7-a3 were absent in all cell lines and pre-let-7a-2 showed indistinct expression. LIN28B showed positive correlation with pre-let-7i/g/f-1 and pre-miR-98 in tumor cell lines, suggesting escape from regulation. Sequence alignment and analysis of pre-let-7 miRNAs showed distinctive structural features within the preE region that may influence the ideal pre-let-7 structuring for LIN28B interaction. Short preE-stems were present in pre-let-7 that may escape LIN28B regulation, but long preEstems were mostly associated with high-level pre-let-7 miRNAs. The observed differences of pre-let-7 levels in cervical cell lines may be the result of alternative preE structuring affecting interaction with LIN28B thus resulting in differential let-7 regulation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads.

PubMed

Sasagawa, Yohei; Danno, Hiroki; Takada, Hitomi; Ebisawa, Masashi; Tanaka, Kaori; Hayashi, Tetsutaro; Kurisaki, Akira; Nikaido, Itoshi

2018-03-09

High-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. We thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts, at a rate of 30-50%, and detect more genes. To demonstrate the power of Quartz-Seq2, we analyzed approximately 10,000 transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads.

(abstract) Synthesis of Speaker Facial Movements to Match Selected Speech Sequences

NASA Technical Reports Server (NTRS)

Scott, Kenneth C.

1994-01-01

We are developing a system for synthesizing image sequences the simulate the facial motion of a speaker. To perform this synthesis, we are pursuing two major areas of effort. We are developing the necessary computer graphics technology to synthesize a realistic image sequence of a person speaking selected speech sequences. Next, we are developing a model that expresses the relation between spoken phonemes and face/mouth shape. A subject is video taped speaking an arbitrary text that contains expression of the full list of desired database phonemes. The subject is video taped from the front speaking normally, recording both audio and video detail simultaneously. Using the audio track, we identify the specific video frames on the tape relating to each spoken phoneme. From this range we digitize the video frame which represents the extreme of mouth motion/shape. Thus, we construct a database of images of face/mouth shape related to spoken phonemes. A selected audio speech sequence is recorded which is the basis for synthesizing a matching video sequence; the speaker need not be the same as used for constructing the database. The audio sequence is analyzed to determine the spoken phoneme sequence and the relative timing of the enunciation of those phonemes. Synthesizing an image sequence corresponding to the spoken phoneme sequence is accomplished using a graphics technique known as morphing. Image sequence keyframes necessary for this processing are based on the spoken phoneme sequence and timing. We have been successful in synthesizing the facial motion of a native English speaker for a small set of arbitrary speech segments. Our future work will focus on advancement of the face shape/phoneme model and independent control of facial features.

[Effects of bushen yinao tablet on physiology and cerebral gene expression in senescence-accelerated mice].

PubMed

Zhang, Chong; Wang, Jin-gang; Yang, Ting

2006-06-01

To study the effects of Bushen Yin' ao Tablet (BSYNT) on physiology and cerebral gene expression in senescence-accelerated mice (SAM). The change of cerebral tissues mRNA expression in SAM was analyzed and compared by messenger ribonucleic acids reverse transcription differential display polymerase chain reaction (mRNA DDRT-PCR) between the medicated group and the control group. BSYNT could increase the level of hemoglobin (Hb) and amount of erythrocyte (RBC) of blood deficiency mice, improve the spatial learning and memory function and the escape response by conditional stimulus. In this study, 14 differential display bands had been discerned, and three of them had been sequenced. The sequence of the three fragments was similar to fatty acid binding protein 7, ubiquinol-cytochrome C reductase complex (7. 2 kD) and 60S ribosomal protein L21 respectively. And the homogeneity was 97% , 100% , and 99% , respectively. BSYNT has effect on the physiological changing of mice, and its effect on cerebral tissues mRNA expression maybe play an important role in anti-aging on the molecular level.

Detection of Sirtuin-1 protein expression in peripheral blood leukocytes in dogs.

PubMed

Yoshimura, Kuniko; Matsuu, Aya; Sasaki, Kai; Momoi, Yasuyuki

2018-05-11

Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase with a large number of protein substrates. It has attracted a lot of attention in association with extending lifespan. The objective of this study was to enable the evaluation of SIRT1 expression in peripheral blood mononuclear cells (PBMCs) from dogs by flow cytometry. Three transcript variants were amplified from PBMCs by reverse transcription PCR and the nucleotide sequences were analyzed. On the basis deduced amino acid sequence, a monoclonal antibody against human SIRT1, 1F3, was selected to detect canine SIRT1. Canine SIRT1 in peripheral blood mononuclear cells was successfully detected by western blotting using this antibody. Intracellular canine SIRT1 was also detected in permeabilized 293T cells transfected with a canine SIRT1 expression plasmid by flow cytometry using this antibody. SIRT1 was detected in all leukocyte subsets including lymphocytes, granulocytes and monocytes. The expression level was markedly different among individual dogs. These results indicated that the method applied in this study is useful for evaluating canine SIRT1 levels in PBMCs from dogs.

Structure, Expression, Chromosomal Location and Product of the Gene Encoding Adh2 in Petunia

PubMed Central

Gregerson, R. G.; Cameron, L.; McLean, M.; Dennis, P.; Strommer, J.

1993-01-01

In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes. PMID:8096485

Class B CpG-ODN2006 is highly associated with IgM and antimicrobial peptide gene expression through TLR9 pathway in yellowtail Seriola lalandi.

PubMed

Angulo, Carlos; Alamillo, Erika; Hirono, Ikuo; Kondo, Hidehiro; Jirapongpairoj, Walissara; Perez-Urbiola, Juan Carlos; Reyes-Becerril, Martha

2018-06-01

The purpose of this study was to characterize the TLR9 gene from yellowtail (Seriola lalandi) and evaluate its functional activity using the class B Cytosine-phosphate-guanine-oligodeoxynucleotide2006 (CpG-ODN2006) in an in vivo experiment after one-week immunostimulation. The gene expressions of TLR9, Immunoglobulin M (IgM), antimicrobial peptides and cytokines were evaluated by real time PCR, and humoral immune parameters were analyzed in serum. The TLR9 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. The yellowtail full-length cDNA sequence of SlTLR9 was 3789 bp in length, including a 66-bp 5'-untranslated region (UTR), a 3'-UTR of 528 bp, and an open reading frame (ORF) of 3192 bp translatable to 1064 amino acid showing a high degree of similarity with the counterparts of other fish species and sharing common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR9 mRNA in all analyzed tissues; the highest levels were observed in intestine, liver and spleen where they play an important role in the fish immune system. The expression levels of TLR9 after B class CpG-ODN2006 (the main TLR9-agonist) was significantly up-regulated in all analyzed tissues, with the high expression observed in spleen followed by intestine and skin. The CpG-B has been shown as a potent B cell mitogen, and interestingly, IgM mRNA transcript was up-regulated in spleen and intestine, which was highly correlated with TLR9 after CpG-ODN2006 stimulation. The antimicrobial peptides, piscidin and NK-lysine, were up-regulated in spleen and gill after CpG-ODN2006 injection with a high correlation (r ≥ 0.82) with TLR9 gene expression. Cytokine genes were up-regulated in spleen, intestine and skin after CpG-ODN was compared with the control group. No significant correlation was observed between TLR9 and IL-1β, TNF-α and Mx gene expressions. The results showed that CpG-ODN2006 intraperitoneal injection enhanced lysozyme, peroxidase and superoxide dismutase activities in serum and demonstrated that CpG-ODN2006 can induce a specific immune response via TLR9 in which IgM and antimicrobial peptides must have an important role in the defense mechanisms against infections in yellowtail. Copyright © 2018 Elsevier Ltd. All rights reserved.

Strong positive selection and recombination drive the antigenic variation of the PilE protein of the human pathogen Neisseria meningitidis.

PubMed

Andrews, T Daniel; Gojobori, Takashi

2004-01-01

The PilE protein is the major component of the Neisseria meningitidis pilus, which is encoded by the pilE/pilS locus that includes an expressed gene and eight homologous silent fragments. The silent gene fragments have been shown to recombine through gene conversion with the expressed gene and thereby provide a means by which novel antigenic variants of the PilE protein can be generated. We have analyzed the evolutionary rate of the pilE gene using the nucleotide sequence of two complete pilE/pilS loci. The very high rate of evolution displayed by the PilE protein appears driven by both recombination and positive selection. Within the semivariable region of the pilE and pilS genes, recombination appears to occur within multiple small sequence blocks that lie between conserved sequence elements. Within the hypervariable region, positive selection was identified from comparison of the silent and expressed genes. The unusual gene conversion mechanism that operates at the pilE/pilS locus is a strategy employed by N. meningitidis to enhance mutation of certain regions of the PilE protein. The silent copies of the gene effectively allow "parallelized" evolution of pilE, thus enabling the encoded protein to rapidly explore a large area of sequence space in an effort to find novel antigenic variants.

TranslatomeDB: a comprehensive database and cloud-based analysis platform for translatome sequencing data.

PubMed

Liu, Wanting; Xiang, Lunping; Zheng, Tingkai; Jin, Jingjie; Zhang, Gong

2018-01-04

Translation is a key regulatory step, linking transcriptome and proteome. Two major methods of translatome investigations are RNC-seq (sequencing of translating mRNA) and Ribo-seq (ribosome profiling). To facilitate the investigation of translation, we built a comprehensive database TranslatomeDB (http://www.translatomedb.net/) which provides collection and integrated analysis of published and user-generated translatome sequencing data. The current version includes 2453 Ribo-seq, 10 RNC-seq and their 1394 corresponding mRNA-seq datasets in 13 species. The database emphasizes the analysis functions in addition to the dataset collections. Differential gene expression (DGE) analysis can be performed between any two datasets of same species and type, both on transcriptome and translatome levels. The translation indices translation ratios, elongation velocity index and translational efficiency can be calculated to quantitatively evaluate translational initiation efficiency and elongation velocity, respectively. All datasets were analyzed using a unified, robust, accurate and experimentally-verifiable pipeline based on the FANSe3 mapping algorithm and edgeR for DGE analyzes. TranslatomeDB also allows users to upload their own datasets and utilize the identical unified pipeline to analyze their data. We believe that our TranslatomeDB is a comprehensive platform and knowledgebase on translatome and proteome research, releasing the biologists from complex searching, analyzing and comparing huge sequencing data without needing local computational power. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

PubMed

Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

2014-10-01

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing

PubMed Central

Yum, Soo-Young; Lee, Song-Jeon; Kim, Hyun-Min; Choi, Woo-Jae; Park, Ji-Hyun; Lee, Won-Wu; Kim, Hee-Soo; Kim, Hyeong-Jong; Bae, Seong-Hun; Lee, Je-Hyeong; Moon, Joo-Yeong; Lee, Ji-Hyun; Lee, Choong-Il; Son, Bong-Jun; Song, Sang-Hoon; Ji, Su-Min; Kim, Seong-Jin; Jang, Goo

2016-01-01

Here, we efficiently generated transgenic cattle using two transposon systems (Sleeping Beauty and Piggybac) and their genomes were analyzed by next-generation sequencing (NGS). Blastocysts derived from microinjection of DNA transposons were selected and transferred into recipient cows. Nine transgenic cattle have been generated and grown-up to date without any health issues except two. Some of them expressed strong fluorescence and the transgene in the oocytes from a superovulating one were detected by PCR and sequencing. To investigate genomic variants by the transgene transposition, whole genomic DNA were analyzed by NGS. We found that preferred transposable integration (TA or TTAA) was identified in their genome. Even though multi-copies (i.e. fifteen) were confirmed, there was no significant difference in genome instabilities. In conclusion, we demonstrated that transgenic cattle using the DNA transposon system could be efficiently generated, and all those animals could be a valuable resource for agriculture and veterinary science. PMID:27324781

Conservation and divergence of plant LHP1 protein sequences and expression patterns in angiosperms and gymnosperms.

PubMed

Guan, Hexin; Zheng, Zhengui; Grey, Paris H; Li, Yuhua; Oppenheimer, David G

2011-05-01

Floral transition is a critical and strictly regulated developmental process in plants. Mutations in Arabidopsis LIKE HETEROCHROMATIN PROTEIN 1 (AtLHP1)/TERMINAL FLOWER 2 (TFL2) result in early and terminal flowers. Little is known about the gene expression, function and evolution of plant LHP1 homologs, except for Arabidopsis LHP1. In this study, the conservation and divergence of plant LHP1 protein sequences was analyzed by sequence alignments and phylogeny. LHP1 expression patterns were compared among taxa that occupy pivotal phylogenetic positions. Several relatively conserved new motifs/regions were identified among LHP1 homologs. Phylogeny of plant LHP1 proteins agreed with established angiosperm relationships. In situ hybridization unveiled conserved expression of plant LHP1 in the axillary bud/tiller, vascular bundles, developing stamens, and carpels. Unlike AtLHP1, cucumber CsLHP1-2, sugarcane SoLHP1 and maize ZmLHP1, rice OsLHP1 is not expressed in the shoot apical meristem (SAM) and the OsLHP1 transcript level is consistently low in shoots. "Unequal crossover" might have contributed to the divergence in the N-terminal and hinge region lengths of LHP1 homologs. We propose an "insertion-deletion" model for soybean (Glycine max L.) GmLHP1s evolution. Plant LHP1 homologs are more conserved than previously expected, and may favor vegetative meristem identity and primordia formation. OsLHP1 may not function in rice SAM during floral induction.

Suppressive subtractive hybridization approach revealed differential expression of hypersensitive response and reactive oxygen species production genes in tea (Camellia sinensis (L.) O. Kuntze) leaves during Pestalotiopsis thea infection.

PubMed

Senthilkumar, Palanisamy; Thirugnanasambantham, Krishnaraj; Mandal, Abul Kalam Azad

2012-12-01

Tea (Camellia sinensis (L.) O. Kuntze) is an economically important plant cultivated for its leaves. Infection of Pestalotiopsis theae in leaves causes gray blight disease and enormous loss to the tea industry. We used suppressive subtractive hybridization (SSH) technique to unravel the differential gene expression pattern during gray blight disease development in tea. Complementary DNA from P. theae-infected and uninfected leaves of disease tolerant cultivar UPASI-10 was used as tester and driver populations respectively. Subtraction efficiency was confirmed by comparing abundance of β-actin gene. A total of 377 and 720 clones with insert size >250 bp from forward and reverse library respectively were sequenced and analyzed. Basic Local Alignment Search Tool analysis revealed 17 sequences in forward SSH library have high degree of similarity with disease and hypersensitive response related genes and 20 sequences with hypothetical proteins while in reverse SSH library, 23 sequences have high degree of similarity with disease and stress response-related genes and 15 sequences with hypothetical proteins. Functional analysis indicated unknown (61 and 59 %) or hypothetical functions (23 and 18 %) for most of the differentially regulated genes in forward and reverse SSH library, respectively, while others have important role in different cellular activities. Majority of the upregulated genes are related to hypersensitive response and reactive oxygen species production. Based on these expressed sequence tag data, putative role of differentially expressed genes were discussed in relation to disease. We also demonstrated the efficiency of SSH as a tool in enriching gray blight disease related up- and downregulated genes in tea. The present study revealed that many genes related to disease resistance were suppressed during P. theae infection and enhancing these genes by the application of inducers may impart better disease tolerance to the plants.

Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Fariss, Robert N; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics. A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins. Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping. The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.

Clinical Implications of Promoter Hypermethylation in RASSF1A and MGMT in Retinoblastoma1

PubMed Central

Choy, Kwong Wai; Lee, Tom C; Cheung, Kin Fai; Fan, Dorothy S P; Lo, Kwok Wai; Beaverson, Katherine L; Abramson, David H; Lam, Dennis S C; Yu, Christopher B O; Pang, Chi Pui

2005-01-01

Abstract We investigated the epigenetic silencing and genetic changes of the RAS-associated domain family 1A (RASSF1A) gene and the O6-methylguanine-DNA methyltransferase (MGMT) gene in retinoblastoma. We extracted DNA from microdissected tumor and normal retina tissues of the same patient in 68 retinoblastoma cases. Promoter methylation in RASSF1A and MGMT was analyzed by methylation-specific PCR, RASSF1A sequence alterations in all coding exons by direct DNA sequencing, and RASSF1A expression by RT-PCR. Cell cycle staging was analyzed by flow cytometry. We detected RASSF1A promoter hypermethylation in 82% of retinoblastoma, in tumor tissues only but not in adjacent normal retinal tissue cells. There was no expression of RASSF1A transcripts in all hypermethylated samples, but RASSF1A transcripts were restored after 5-aza-2′-deoxycytidine treatment with no changes in cell cycle or apoptosis. No mutation in the RASSF1A sequence was found. MGMT hypermethylation was present in 15% of theretinoblastoma samples, and the absence of MGMT hypermethylation was associated (P = .002) with retinoblastoma at advanced Reese-Ellsworth tumor stage. Our results revealed a high RASSF1A hypermethylation frequency in retinoblastoma. The correlation of MGMT inactivation by promoter hypermethylation with lower-stage diseases indicated that MGMT hypermethylation provides useful prognostic information. Epigenetic mechanism plays an important role in the progression of retinoblastoma. PMID:15799820

Global Analysis of Gene Expression Profiles in Developing Physic Nut (Jatropha curcas L.) Seeds

PubMed Central

Jiang, Huawu; Wu, Pingzhi; Zhang, Sheng; Song, Chi; Chen, Yaping; Li, Meiru; Jia, Yongxia; Fang, Xiaohua; Chen, Fan; Wu, Guojiang

2012-01-01

Background Physic nut (Jatropha curcas L.) is an oilseed plant species with high potential utility as a biofuel. Furthermore, following recent sequencing of its genome and the availability of expressed sequence tag (EST) libraries, it is a valuable model plant for studying carbon assimilation in endosperms of oilseed plants. There have been several transcriptomic analyses of developing physic nut seeds using ESTs, but they have provided limited information on the accumulation of stored resources in the seeds. Methodology/Principal Findings We applied next-generation Illumina sequencing technology to analyze global gene expression profiles of developing physic nut seeds 14, 19, 25, 29, 35, 41, and 45 days after pollination (DAP). The acquired profiles reveal the key genes, and their expression timeframes, involved in major metabolic processes including: carbon flow, starch metabolism, and synthesis of storage lipids and proteins in the developing seeds. The main period of storage reserves synthesis in the seeds appears to be 29–41 DAP, and the fatty acid composition of the developing seeds is consistent with relative expression levels of different isoforms of acyl-ACP thioesterase and fatty acid desaturase genes. Several transcription factor genes whose expression coincides with storage reserve deposition correspond to those known to regulate the process in Arabidopsis. Conclusions/Significance The results will facilitate searches for genes that influence de novo lipid synthesis, accumulation and their regulatory networks in developing physic nut seeds, and other oil seeds. Thus, they will be helpful in attempts to modify these plants for efficient biofuel production. PMID:22574177

Transcriptome sequencing of rhizome tissue of Sinopodophyllum hexandrum at two temperatures.

PubMed

Kumari, Anita; Singh, Heikham Russiachand; Jha, Ashwani; Swarnkar, Mohit Kumar; Shankar, Ravi; Kumar, Sanjay

2014-10-07

Sinopodophyllum hexandrum is an endangered medicinal herb, which is commonly present in elevations ranging between 2,400-4,500 m and is sensitive to temperature. Medicinal property of the species is attributed to the presence of podophyllotoxin in the rhizome tissue. The present work analyzed transcriptome of rhizome tissue of S. hexandrum exposed to 15°C and 25°C to understand the temperature mediated molecular responses including those associated with podophyllotoxin biosynthesis. Deep sequencing of transcriptome with an average coverage of 88.34X yielded 60,089 assembled transcript sequences representing 20,387 unique genes having homology to known genes. Fragments per kilobase of exon per million fragments mapped (FPKM) based expression analysis revealed genes related to growth and development were over-expressed at 15°C, whereas genes involved in stress response were over-expressed at 25°C. There was a decreasing trend of podophyllotoxin accumulation at 25°C; data was well supported by the expression of corresponding genes of the pathway. FPKM data was validated by quantitative real-time polymerase chain reaction data using a total of thirty four genes and a positive correlation between the two platforms of gene expression was obtained. Also, detailed analyses yielded cytochrome P450s, methyltransferases and glycosyltransferases which could be the potential candidate hitherto unidentified genes of podophyllotoxin biosynthesis pathway. The present work revealed temperature responsive transcriptome of S. hexandrum on Illumina platform. Data suggested expression of genes for growth and development and podophyllotoxin biosynthesis at 15°C, and prevalence of those associated with stress response at 25°C.

Development of a Stable Cell Line, Overexpressing Human T-cell Immunoglobulin Mucin 1

PubMed Central

Ebrahimi, Mina; Kazemi, Tohid; Ganjalikhani-hakemi, Mazdak; Majidi, Jafar; khanahmad, Hossein; Rahimmanesh, Ilnaz; Homayouni, Vida; Kohpayeh, Shirin

2015-01-01

Background Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 (TIM-1) glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore, targeting TIM-1 could be a potential therapeutic approach against such diseases. Objectives In this study, we aimed to express TIM-1 protein on Human Embryonic kidney (HEK) 293T cell line in order to have an available source of the TIM-1 antigen. Materials and Methods The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells (PBMC) and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA™3.1/Hygro (+) and transformed in Escherichia coli TOP 10 F’. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR. Results The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells. Conclusions Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific monoclonal antibodies, nanobodies and aptamers against human TIM-1. PMID:28959306

MicroRNA-21 promotes proliferation of rat hepatocyte BRL-3A by targeting FASLG.

PubMed

Li, J J; Chan, W H; Leung, W Y; Wang, Y; Xu, C S

2015-04-27

Rat liver regeneration (RLR) induced by partial hepatectomy involves cell proliferation regulated by numerous factors, including microRNAs (miRNAs). miRNA high-throughput sequencing has been established and used to analyze miRNA expression profiles. This study showed that 39 miRNAs were related to RLR through the analysis of miRNA high-throughput sequencing. Their role toward rat normal hepatocyte line BRL-3A was studied by gain- and loss-of-function analyses, and one of them, microRNA-21 (miR-21), obviously upregulated and promoted BRL-3A cell proliferation. Using bioinformatics to search for miR-21 targets revealed that Fas ligand (FASLG) is one of miR-21's target genes. A dual-luciferase report assay and Western blot assay showed that miR-21 directly targeted the 3'-untranslated region of FASLG and inhibited the expression of FASLG, which suggests that miR-21 promoted BRL-3A cell proliferation by reducing FASLG expression.

Cloning and Characterization of 5′ Flanking Regulatory Sequences of AhLEC1B Gene from Arachis Hypogaea L.

PubMed Central

Tang, Guiying; Xu, Pingli; Liu, Wei; Liu, Zhanji; Shan, Lei

2015-01-01

LEAFY COTYLEDON1 (LEC1) is a B subunit of Nuclear Factor Y (NF-YB) transcription factor that mainly accumulates during embryo development. We cloned the 5′ flanking regulatory sequence of AhLEC1B gene, a homolog of Arabidopsis LEC1, and analyzed its regulatory elements using online software. To identify the crucial regulatory region, we generated a series of GUS expression frameworks driven by different length promoters with 5′ terminal and/or 3′ terminal deletion. We further characterized the GUS expression patterns in the transgenic Arabidopsis lines. Our results show that both the 65bp proximal promoter region and the 52bp 5′ UTR of AhLEC1B contain the key motifs required for the essential promoting activity. Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements. PMID:26426444

Cross species analysis of microarray expression data

PubMed Central

Lu, Yong; Huggins, Peter; Bar-Joseph, Ziv

2009-01-01

Motivation: Many biological systems operate in a similar manner across a large number of species or conditions. Cross-species analysis of sequence and interaction data is often applied to determine the function of new genes. In contrast to these static measurements, microarrays measure the dynamic, condition-specific response of complex biological systems. The recent exponential growth in microarray expression datasets allows researchers to combine expression experiments from multiple species to identify genes that are not only conserved in sequence but also operated in a similar way in the different species studied. Results: In this review we discuss the computational and technical challenges associated with these studies, the approaches that have been developed to address these challenges and the advantages of cross-species analysis of microarray data. We show how successful application of these methods lead to insights that cannot be obtained when analyzing data from a single species. We also highlight current open problems and discuss possible ways to address them. Contact: zivbj@cs.cmu.edu PMID:19357096

Negative effect of the 5'-untranslated leader sequence on Ac transposon promoter expression.

PubMed

Scortecci, K C; Raina, R; Fedoroff, N V; Van Sluys, M A

1999-08-01

Transposable elements are used in heterologous plant hosts to clone genes by insertional mutagenesis. The Activator (Ac) transposable element has been cloned from maize, and introduced into a variety of plants. However, differences in regulation and transposition frequency have been observed between different host plants. The cause of this variability is still unknown. To better understand the activity of the Ac element, we analyzed the Ac promoter region and its 5'-untranslated leader sequence (5' UTL). Transient assays in tobacco NT1 suspension cells showed that the Ac promoter is a weak promoter and its activity was localized by deletion analyses. The data presented here indicate that the core of the Ac promoter is contained within 153 bp fragment upstream to transcription start sites. An important inhibitory effect (80%) due to the presence of the 5' UTL was found on the expression of LUC reporter gene. Here we demonstrate that the presence of the 5' UTL in the constructs reduces the expression driven by either strong or weak promoters.

αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

PubMed Central

Buitrago, Lorena; Rendon, Augusto; Liang, Yupu; Simeoni, Ilenia; Negri, Ana; Filizola, Marta; Ouwehand, Willem H.; Coller, Barry S.; Alessi, Marie-Christine; Ballmaier, Matthias; Bariana, Tadbir; Bellissimo, Daniel; Bertoli, Marta; Bray, Paul; Bury, Loredana; Carrell, Robin; Cattaneo, Marco; Collins, Peter; French, Deborah; Favier, Remi; Freson, Kathleen; Furie, Bruce; Germeshausen, Manuela; Ghevaert, Cedric; Gomez, Keith; Goodeve, Anne; Gresele, Paolo; Guerrero, Jose; Hampshire, Dan J.; Hadinnapola, Charaka; Heemskerk, Johan; Henskens, Yvonne; Hill, Marian; Hogg, Nancy; Johnsen, Jill; Kahr, Walter; Kerr, Ron; Kunishima, Shinji; Laffan, Michael; Natwani, Amit; Neerman-Arbez, Marguerite; Nurden, Paquita; Nurden, Alan; Ormiston, Mark; Othman, Maha; Ouwehand, Willem; Perry, David; Vilk, Shoshana Ravel; Reitsma, Pieter; Rondina, Matthew; Simeoni, Ilenia; Smethurst, Peter; Stephens, Jonathan; Stevenson, William; Szkotak, Artur; Turro, Ernest; Van Geet, Christel; Vries, Minka; Ward, June; Waye, John; Westbury, Sarah; Whiteheart, Sidney; Wilcox, David; Zhang, Bi

2015-01-01

Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbβ3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbβ3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and β3 C547G severely reduced αIIbβ3 expression, whereas αIIb P943A partially reduced αIIbβ3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69–98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or β3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbβ3 and highlight the challenges in predicting the clinical significance of novel missense variants. PMID:25827233

Quantitative analysis of lentiviral transgene expression in mice over seven generations.

PubMed

Wang, Yong; Song, Yong-tao; Liu, Qin; Liu, Cang'e; Wang, Lu-lu; Liu, Yu; Zhou, Xiao-yang; Wu, Jun; Wei, Hong

2010-10-01

Lentiviral transgenesis is now recognized as an extremely efficient and cost-effective method to produce transgenic animals. Transgenes delivered by lentiviral vectors exhibited inheritable expression in many species including those which are refractory to genetic modification such as non-human primates. However, epigenetic modification was frequently observed in lentiviral integrants, and transgene expression found to be inversely correlated with methylation density. Recent data showed that about one-third lentiviral integrants exhibited hypermethylation and low expression, but did not demonstrate whether those integrants with high expression could remain constant expression and hypomethylated during long term germline transmission. In this study, using lentiviral eGFP transgenic mice as the experimental animals, lentiviral eGFP expression levels and its integrant numbers in genome were quantitatively analyzed by fluorescent quantitative polymerase-chain reaction (FQ-PCR), using the house-keeping gene ribosomal protein S18 (Rps18) and the single copy gene fatty acid binding protein of the intestine (Fabpi) as the internal controls respectively. The methylation densities of the integrants were quantitatively analyzed by bisulfite sequencing. We found that the lentiviral integrants with high expression exhibited a relative constant expression level per integrant over at least seven generations. Besides, the individuals containing these integrants exhibited eGFP expression levels which were positively and almost linearly correlated with the integrant numbers in their genomes, suggesting that no remarkable position effect on transgene expression of the integrants analyzed was observed. In addition, over seven generations the methylation density of these integrants did not increase, but rather decreased remarkably, indicating that these high expressing integrants were not subjected to de novo methylation during at least seven generations of germline transmission. Taken together, these data suggested that transgenic lines with long term stable expression and no position effect can be established by lentiviral transgenesis.

Development of a transgenic zebrafish model expressing GFP in the notochord, somite and liver directed by the hfe2 gene promoter.

PubMed

Bian, Yue-Hong; Xu, Cheng; Li, Junling; Xu, Jin; Zhang, Hongwei; Du, Shao Jun

2011-08-01

Hemojuvelin, also known as RGMc, is encoded by hfe2 gene that plays an important role in iron homeostasis. hfe2 is specifically expressed in the notochord, developing somite and skeletal muscles during development. The molecular regulation of hfe2 expression is, however, not clear. We reported here the characterization of hfe2 gene expression and the regulation of its tissue-specific expression in zebrafish embryos. We demonstrated that the 6 kb 5'-flanking sequence upstream of the ATG start codon in the zebrafish hfe2 gene could direct GFP specific expression in the notochord, somites, and skeletal muscle of zebrafish embryos, recapitulating the expression pattern of the endogenous gene. However, the Tg(hfe2:gfp) transgene is also expressed in the liver of fish embryos, which did not mimic the expression of the endogenous hfe2 at the early stage. Nevertheless, the Tg(hfe2:gfp) transgenic zebrafish provides a useful model to study liver development. Treating Tg(hfe2:gfp) transgenic zebrafish embryos with valproic acid, a liver development inhibitor, significantly inhibited GFP expression in zebrafish. Together, these data indicate that the tissue specific expression of hfe2 in the notochord, somites and muscles is regulated by regulatory elements within the 6 kb 5'-flanking sequence of the hfe2 gene. Moreover, the Tg(hfe2:gfp) transgenic zebrafish line provides a useful model system for analyzing liver development in zebrafish.

Gene expression in grass ovaries infected with seed born fungal endophyte Neotyphodium occultans analyzed by a next-generation sequencing system

USDA-ARS?s Scientific Manuscript database

Fungal endophytes of the genus Neotyphodium form symbiotic associations with many grass species of the subfamily Pooideae, including some important forage and turf species such as Lolium grasses. The endophytes are maintained in host plant communities by seed transmission from maternal plants to off...

Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library

PubMed Central

Bhore, Subhash J; Kassim, Amelia; Loh, Chye Ying; Shah, Farida H

2010-01-01

It is well known that the nutritional quality of the American oil-palm (Elaeis oleifera) mesocarp oil is superior to that of African oil-palm (Elaeis guineensis Jacq. Tenera) mesocarp oil. Therefore, it is of important to identify the genetic features for its superior value. This could be achieved through the genome sequencing of the oil-palm. However, the genome sequence is not available in the public domain due to commercial secrecy. Hence, we constructed a cDNA library and generated expressed sequence tags (3,205) from the mesocarp tissue of the American oil-palm. We continued to annotate each of these cDNAs after submitting to GenBank/DDBJ/EMBL. A rough analysis turned our attention to the beta-carotene hydroxylase (Chyb) enzyme encoding cDNA. Then, we completed the full sequencing of cDNA clone for its both strands using M13 forward and reverse primers. The full nucleotide and protein sequence was further analyzed and annotated using various Bioinformatics tools. The analysis results showed the presence of fatty acid hydroxylase superfamily domain in the protein sequence. The multiple sequence alignment of selected Chyb amino acid sequences from other plant species and algal members with E. oleifera Chyb using ClustalW and its phylogenetic analysis suggest that Chyb from monocotyledonous plant species, Lilium hubrid, Crocus sativus and Zea mays are the most evolutionary related with E. oleifera Chyb. This study reports the annotation of E. oleifera Chyb. Abbreviations ESTs - expressed sequence tags, EoChyb - Elaeis oleifera beta-carotene hydroxylase, MC - main cluster PMID:21364789

Organization and annotation of the Xcat critical region: elimination of seven positional candidate genes.

PubMed

Huang, Kristen M; Geunes-Boyer, Scarlett; Wu, Sufen; Dutra, Amalia; Favor, Jack; Stambolian, Dwight

2004-05-01

Xcat mice display X-linked congenital cataracts and are a mouse model for the human X-linked cataract disease Nance Horan syndrome (NHS). The genetic defect in Xcat mice and NHS patients is not known. We isolated and sequenced a BAC contig representing a portion of the Xcat critical region. We combined our sequencing data with the most recent mouse sequence assemblies from both Celera and public databases. The sequence of the 2.2-Mb Xcat critical region was then analyzed for potential Xcat candidate genes. The coding regions of the seven known genes within this area (Rai2, Rbbp7, Ctps2, Calb3, Grpr, Reps2, and Syap1) were sequenced in Xcat mice and no mutations were detected. The expression of Rai2 was quantitatively identical in wild-type and Xcat mutant eyes. These results indicate that the Xcat mutation is within a novel, undiscovered gene.

Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

PubMed

Ogasawara, Shun; Shimada, Nao; Kawata, Takefumi

2009-02-01

Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins. Promoter fragments of the expL7 were fused to a lacZ reporter and the expression patterns determined. As expected from the behavior of the endogenous expL7 gene, the expL7/lacZ fusion gene was downregulated in Dd-STATa null slugs. In the parental strain, the expL7 promoter was activated in the anterior tip region. Mutational analysis of the promoter identified a sequence that was necessary for expression in tip cells. In addition, an activator sequence for pstAB cells was identified. These sequences act in combination with the repressor region to prevent ectopic expL7 expression in the prespore and prestalk regions of the slug and culminant. Although the expL7 null mutant showed no phenotypic change, the expL7 overexpressor showed aberrant stalk formation. These results indicate that the expansin-like molecule is important for morphogenesis in Dictyostelium.

Defining the Transcriptional Landscape during Cytomegalovirus Latency with Single-Cell RNA Sequencing

PubMed Central

2018-01-01

ABSTRACT Primary infection with human cytomegalovirus (HCMV) results in a lifelong infection due to its ability to establish latent infection, with one characterized viral reservoir being hematopoietic cells. Although reactivation from latency causes serious disease in immunocompromised individuals, our molecular understanding of latency is limited. Here, we delineate viral gene expression during natural HCMV persistent infection by analyzing the massive transcriptome RNA sequencing (RNA-seq) atlas generated by the Genotype-Tissue Expression (GTEx) project. This systematic analysis reveals that HCMV persistence in vivo is prevalent in diverse tissues. Notably, we find only viral transcripts that resemble gene expression during various stages of lytic infection with no evidence of any highly restricted latency-associated viral gene expression program. To further define the transcriptional landscape during HCMV latent infection, we also used single-cell RNA-seq and a tractable experimental latency model. In contrast to some current views on latency, we also find no evidence for any highly restricted latency-associated viral gene expression program. Instead, we reveal that latency-associated gene expression largely mirrors a late lytic viral program, albeit at much lower levels of expression. Overall, our work has the potential to revolutionize our understanding of HCMV persistence and suggests that latency is governed mainly by quantitative changes, with a limited number of qualitative changes, in viral gene expression. PMID:29535194

High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallaher, Sean D.; Fitz-Gibbon, Sorel T.; Strenkert, Daniela

Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Gen-ome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genomemore » and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar tran-scripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3’-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellargenomes, which is 1/10th as much genetic diversity as is found in the nucleus.« less

Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated.

PubMed

Reddy, M K; Nair, S; Singh, B N; Mudgil, Y; Tewari, K K; Sopory, S K

2001-01-24

We report the cloning and sequencing of both cDNA and genomic DNA of a 33 kDa chloroplast ribonucleoprotein (33RNP) from pea. The analysis of the predicted amino acid sequence of the cDNA clone revealed that the encoded protein contains two RNA binding domains, including the conserved consensus ribonucleoprotein sequences CS-RNP1 and CS-RNP2, on the C-terminus half and the presence of a putative transit peptide sequence in the N-terminus region. The phylogenetic and multiple sequence alignment analysis of pea chloroplast RNP along with RNPs reported from the other plant sources revealed that the pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RNP and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively. The pea 33RNP was expressed in Escherichia coli and purified to homogeneity. The in vitro import of precursor protein into chloroplasts confirmed that the N-terminus putative transit peptide is a bona fide transit peptide and 33RNP is localized in the chloroplast. The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed that 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDNA and dsDNA. The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated. Sequence analysis of the genomic clone revealed that the gene contains four exons and three introns. We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene.

Transcriptional Analysis of Flowering Time in Switchgrass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tornqvist, Carl-Erik; Vaillancourt, Brieanne; Kim, Jeongwoon

Over the past two decades, switchgrass (Panicum virgatum) has emerged as a priority biofuel feedstock. The bulk of switchgrass biomass is in the vegetative portion of the plant; therefore, increasing the length of vegetative growth will lead to an increase in overall biomass yield. The goal of this study was to gain insight into the control of flowering time in switchgrass that would assist in development of cultivars with longer vegetative phases through delayed flowering. RNA sequencing was used to assess genome-wide expression profiles across a developmental series between switchgrass genotypes belonging to the two main ecotypes: upland, typically earlymore » flowering, and lowland, typically late flowering. Leaf blades and tissues enriched for the shoot apical meristem (SAM) were collected in a developmental series from emergence through anthesis for RNA extraction. RNA from samples that flanked the SAM transition stage was sequenced for expression analyses. The analyses revealed differential expression patterns between early- and late-flowering genotypes for known flowering time orthologs. Namely, genes shown to play roles in photoperiod response and the circadian clock in other species were identified as potential candidates for regulating flowering time in the switchgrass genotypes analyzed. Based on their expression patterns, many of the differentially expressed genes could also be classified as putative promoters or repressors of flowering. The candidate genes presented here may be used to guide switchgrass improvement through marker-assisted breeding and/or transgenic or gene editing approaches.Over the past two decades, switchgrass (Panicum virgatum) has emerged as a priority biofuel feedstock. The bulk of switchgrass biomass is in the vegetative portion of the plant; therefore, increasing the length of vegetative growth will lead to an increase in overall biomass yield. The goal of this study was to gain insight into the control of flowering time in switchgrass that would assist in development of cultivars with longer vegetative phases through delayed flowering. RNA sequencing was used to assess genome-wide expression profiles across a developmental series between switchgrass genotypes belonging to the two main ecotypes: upland, typically early flowering, and lowland, typically late flowering. Leaf blades and tissues enriched for the shoot apical meristem (SAM) were collected in a developmental series from emergence through anthesis for RNA extraction. RNA from samples that flanked the SAM transition stage was sequenced for expression analyses. The analyses revealed differential expression patterns between early- and late-flowering genotypes for known flowering time orthologs. Namely, genes shown to play roles in photoperiod response and the circadian clock in other species were identified as potential candidates for regulating flowering time in the switchgrass genotypes analyzed. Based on their expression patterns, many of the differentially expressed genes could also be classified as putative promoters or repressors of flowering. The candidate genes presented here may then be used to guide switchgrass improvement through marker-assisted breeding and/or transgenic or gene editing approaches.« less

Transcriptional Analysis of Flowering Time in Switchgrass

DOE PAGES

Tornqvist, Carl-Erik; Vaillancourt, Brieanne; Kim, Jeongwoon; ...

2017-04-27

Over the past two decades, switchgrass (Panicum virgatum) has emerged as a priority biofuel feedstock. The bulk of switchgrass biomass is in the vegetative portion of the plant; therefore, increasing the length of vegetative growth will lead to an increase in overall biomass yield. The goal of this study was to gain insight into the control of flowering time in switchgrass that would assist in development of cultivars with longer vegetative phases through delayed flowering. RNA sequencing was used to assess genome-wide expression profiles across a developmental series between switchgrass genotypes belonging to the two main ecotypes: upland, typically earlymore » flowering, and lowland, typically late flowering. Leaf blades and tissues enriched for the shoot apical meristem (SAM) were collected in a developmental series from emergence through anthesis for RNA extraction. RNA from samples that flanked the SAM transition stage was sequenced for expression analyses. The analyses revealed differential expression patterns between early- and late-flowering genotypes for known flowering time orthologs. Namely, genes shown to play roles in photoperiod response and the circadian clock in other species were identified as potential candidates for regulating flowering time in the switchgrass genotypes analyzed. Based on their expression patterns, many of the differentially expressed genes could also be classified as putative promoters or repressors of flowering. The candidate genes presented here may be used to guide switchgrass improvement through marker-assisted breeding and/or transgenic or gene editing approaches.Over the past two decades, switchgrass (Panicum virgatum) has emerged as a priority biofuel feedstock. The bulk of switchgrass biomass is in the vegetative portion of the plant; therefore, increasing the length of vegetative growth will lead to an increase in overall biomass yield. The goal of this study was to gain insight into the control of flowering time in switchgrass that would assist in development of cultivars with longer vegetative phases through delayed flowering. RNA sequencing was used to assess genome-wide expression profiles across a developmental series between switchgrass genotypes belonging to the two main ecotypes: upland, typically early flowering, and lowland, typically late flowering. Leaf blades and tissues enriched for the shoot apical meristem (SAM) were collected in a developmental series from emergence through anthesis for RNA extraction. RNA from samples that flanked the SAM transition stage was sequenced for expression analyses. The analyses revealed differential expression patterns between early- and late-flowering genotypes for known flowering time orthologs. Namely, genes shown to play roles in photoperiod response and the circadian clock in other species were identified as potential candidates for regulating flowering time in the switchgrass genotypes analyzed. Based on their expression patterns, many of the differentially expressed genes could also be classified as putative promoters or repressors of flowering. The candidate genes presented here may then be used to guide switchgrass improvement through marker-assisted breeding and/or transgenic or gene editing approaches.« less

Deep sequencing of the Mexican avocado transcriptome, an ancient angiosperm with a high content of fatty acids.

PubMed

Ibarra-Laclette, Enrique; Méndez-Bravo, Alfonso; Pérez-Torres, Claudia Anahí; Albert, Victor A; Mockaitis, Keithanne; Kilaru, Aruna; López-Gómez, Rodolfo; Cervantes-Luevano, Jacob Israel; Herrera-Estrella, Luis

2015-08-13

Avocado (Persea americana) is an economically important tropical fruit considered to be a good source of fatty acids. Despite its importance, the molecular and cellular characterization of biochemical and developmental processes in avocado is limited due to the lack of transcriptome and genomic information. The transcriptomes of seeds, roots, stems, leaves, aerial buds and flowers were determined using different sequencing platforms. Additionally, the transcriptomes of three different stages of fruit ripening (pre-climacteric, climacteric and post-climacteric) were also analyzed. The analysis of the RNAseqatlas presented here reveals strong differences in gene expression patterns between different organs, especially between root and flower, but also reveals similarities among the gene expression patterns in other organs, such as stem, leaves and aerial buds (vegetative organs) or seed and fruit (storage organs). Important regulators, functional categories, and differentially expressed genes involved in avocado fruit ripening were identified. Additionally, to demonstrate the utility of the avocado gene expression atlas, we investigated the expression patterns of genes implicated in fatty acid metabolism and fruit ripening. A description of transcriptomic changes occurring during fruit ripening was obtained in Mexican avocado, contributing to a dynamic view of the expression patterns of genes involved in fatty acid biosynthesis and the fruit ripening process.

Characterization and Expression Analysis of Receptor for Activated C Kinase from Silk-producing Insect Antheraea pernyi.

PubMed

Zhu, Bao-Jian; Yu, Hao; Tian, Sen; Dai, Li-Shang; Sun, Yu; Liu, Chao-Liang

2016-01-01

The receptor for activated C kinase (RACK) is an important scaffold protein with regulatory functions in cells. However, its role in the immune response of Antheraea pernyi to pathogen challenge remains unclear. To investigate the biological functions of RACK in the wild silkworm A. pernyi, cloning was performed and the expression patterns of the RACK gene were analyzed. Sequence analysis revealed that the RACK gene was 1120 bp containing a 960-bp open reading frame. The deduced RACK protein sequence reveals the higher identity with its homologs from other insects. SDS-PAGE and western blot analysis demonstrated successful expression of a 36-kDa recombinant RACK protein in Escherichia coli. The titer of a rabbit-raised antibody against recombinant RACK protein was about 1: 20000, determined by ELISA. Real-time PCR analysis showed that RACK expression was higher in fat bodies than in other examined A. pernyi tissues. The expression of RACK mRNA in fat bodies of fifth larvae of A. pernyi was obviously induced after nucleopolyhedrovirus, E. coli or Beauveria bassiana challenge. However, the expression patterns of RACK were different in response to these pathogens. Our data suggest that RACK may play a role in the innate immune responses of A. pernyi.

Krüppel Like Factors Family Expression in Cervical Cancer Cells.

PubMed

Marrero-Rodríguez, Daniel; la Cruz, Hugo Arreola-De; Taniguchi-Ponciano, Keiko; Gomez-Virgilio, Laura; Huerta-Padilla, Victor; Ponce-Navarrete, Gustavo; Andonegui-Elguera, Sergio; Jimenez-Vega, Florinda; Romero-Morelos, Pablo; Rodriguez-Esquivel, Miriam; Meraz-Rios, Marco; Figueroa-Corona, Ma Del Pilar; Monroy, Alberto; Pérez-González, Oscar; Salcedo, Mauricio

2017-05-01

Krüppel Like Factors (KLF) refers to a family of seventeen members of transcription factors. Involved in several cellular processes. As other cancer types, Cervical Cancer (CC) presents molecular deregulations in transcription factors, but especially Human Papilloma Virus (HPV) sequences. Here in this work we analyzed the mRNA expression of all KLF family members in CC-derived cell lines and CC tissues. The cell lines used were HeLa, INBL, RoVa, C4-I, Ms751, ViPa, CaLo, SiHa, CaSki, C33a and ViBo and the non-tumorigenic HaCaT. mRNA expression was analyzed by means of expression microarray and RT-PCR, and KLF5 protein by immunofluorescence. The cell lines were grouped according to HPV genotype as HPV16, HPV18 positive or HPV negative cells. Heterogeneous expression was observed among the cell lines. Despite the heterogeneous expression profile, KLF3, -5, -12, -15 and -16 transcripts were present in all cell lines, KLF4 and -10 which were not expressed in CaSki; KLF11 and 13 were not expressed by Vipa and C4-I, and KLF7 was not expressed by C4-I and Rova. The CC tissue analysis shows expression of most of the KLF members, such as KLF5. KLF5 immunosignal was positive in the three cell lines analyzed. We suggest that KLF expression could not be related to HPV presence/genotype, at least at transcriptional level, and the expression of KLF family members may be necessary in the biology of the CC cells. Copyright © 2017 IMSS. Published by Elsevier Inc. All rights reserved.

Transcriptome dynamics through alternative polyadenylation in developmental and environmental responses in plants revealed by deep sequencing

PubMed Central

Shen, Yingjia; Venu, R.C.; Nobuta, Kan; Wu, Xiaohui; Notibala, Varun; Demirci, Caghan; Meyers, Blake C.; Wang, Guo-Liang; Ji, Guoli; Li, Qingshun Q.

2011-01-01

Polyadenylation sites mark the ends of mRNA transcripts. Alternative polyadenylation (APA) may alter sequence elements and/or the coding capacity of transcripts, a mechanism that has been demonstrated to regulate gene expression and transcriptome diversity. To study the role of APA in transcriptome dynamics, we analyzed a large-scale data set of RNA “tags” that signify poly(A) sites and expression levels of mRNA. These tags were derived from a wide range of tissues and developmental stages that were mutated or exposed to environmental treatments, and generated using digital gene expression (DGE)–based protocols of the massively parallel signature sequencing (MPSS-DGE) and the Illumina sequencing-by-synthesis (SBS-DGE) sequencing platforms. The data offer a global view of APA and how it contributes to transcriptome dynamics. Upon analysis of these data, we found that ∼60% of Arabidopsis genes have multiple poly(A) sites. Likewise, ∼47% and 82% of rice genes use APA, supported by MPSS-DGE and SBS-DGE tags, respectively. In both species, ∼49%–66% of APA events were mapped upstream of annotated stop codons. Interestingly, 10% of the transcriptomes are made up of APA transcripts that are differentially distributed among developmental stages and in tissues responding to environmental stresses, providing an additional level of transcriptome dynamics. Examples of pollen-specific APA switching and salicylic acid treatment-specific APA clearly demonstrated such dynamics. The significance of these APAs is more evident in the 3034 genes that have conserved APA events between rice and Arabidopsis. PMID:21813626

Changes in rat spinal cord gene expression after inflammatory hyperalgesia of the joint and manual therapy.

PubMed

Ruhlen, Rachel L; Singh, Vineet K; Pazdernik, Vanessa K; Towns, Lex C; Snider, Eric J; Sargentini, Neil J; Degenhardt, Brian F

2014-10-01

Mobilization of a joint affects local tissue directly but may also have other effects that are mediated through the central nervous system. To identify differential gene expression in the spinal cords of rats with or without inflammatory joint injury after manual therapy or no treatment. Rats were randomly assigned to 1 of 4 treatment groups: no injury and no touch (NI/NT), injury and no touch (I/NT), no injury and manual therapy (NI/MT), and injury and manual therapy (I/MT). We induced acute inflammatory joint injury in the rats by injecting carrageenan into an ankle. Rats in the no-injury groups did not receive carrageenan injection. One day after injury, rats received manual therapy to the knee of the injured limb. Rats in the no-touch groups were anesthetized without receiving manual therapy. Spinal cords were harvested 30 minutes after therapy or no touch, and spinal cord gene expression was analyzed by microarray for 3 comparisons: NI/NT vs I/NT, I/MT vs I/NT, and NI/NT vs NI/MT. Three rats were assigned to each group. Of 38,875 expressed sequence tags, 755 were differentially expressed in the NI/NT vs I/NT comparison. For the other comparisons, no expressed sequence tags were differentially expressed. Cluster analysis revealed that the differentially expressed sequence tags were over-represented in several categories, including ion homeostasis (enrichment score, 2.29), transmembrane (enrichment score, 1.55), and disulfide bond (enrichment score, 2.04). An inflammatory injury to the ankle of rats caused differential expression of genes in the spinal cord. Consistent with other studies, genes involved in ion transport were among those affected. However, manual therapy to the knees of injured limbs or to rats without injury did not alter gene expression in the spinal cord. Thus, evidence for central nervous system mediation of manual therapy was not observed. © 2014 The American Osteopathic Association.

Biallelic expression of the L-arginine:glycine amidinotransferase gene with different methylation status between male and female primordial germ cells in chickens.

PubMed

Jang, H J; Lee, M O; Kim, S; Kim, T H; Kim, S K; Song, G; Womack, J E; Han, J Y

2013-03-01

The basic functions of DNA methylation include in gene silencing by methylation of specific gene promoters, defense of the host genome from retrovirus, and transcriptional suppression of transgenes. In addition, genomic imprinting, by which certain genes are expressed in a parent-of-origin-specific manner, has been observed in a wide range of plants and animals and has been associated with differential methylation. However, imprinting phenomena of DNA methylation effects have not been revealed in chickens. To analyze whether genomic imprinting occurs in chickens, methyl-DNA immunoprecipitation array analysis was applied across the entire genome of germ cells in early chick embryos. A differentially methylated region (DMR) was detected in the eighth intron of the l-arginine:glycine amidinotransferase (GATM) gene. When the DMR in GATM was analyzed by bisulfite sequencing, the methylation in male primordial germ cells (PGC) of 6-d-old embryos was higher than that in female PGC (57.5 vs. 35.0%). At 8 d, the DMR methylation of GATM in male PGC was 3.7-fold higher than that in female PGC (65.0 vs. 17.5%). Subsequently, to investigate mono- or biallelic expression of the GATM gene during embryo development, we found 2 indel sequences (GTTTAATGC and CAAAAA) within the GATM 3'-untranslated region in Korean Oge (KO) and White Leghorn (WL) chickens. When individual WL and KO chickens were genotyped for indel sequences, 3 allele combinations (homozygous insertion, homozygous deletion, and heterozygotes) were detected in both breeds using a gel shift assay and high-resolution melt assay. The deletion allele was predominant in KO, whereas the insertion allele was predominant in WL. Heterozygous animals were evenly distributed in both breeds (P < 0.01). Despite the different methylation status between male and female PGC, the GATM gene conclusively displayed biallelic expression in PGC as well as somatic embryonic, extraembryonic, and adult chicken tissues.

In Search for Pheromone Receptors: Certain Members of the Odorant Receptor Family in the Desert Locust Schistocerca gregaria (Orthoptera: Acrididae) Are Co-expressed with SNMP1.

PubMed

Pregitzer, Pablo; Jiang, Xingcong; Grosse-Wilde, Ewald; Breer, Heinz; Krieger, Jürgen; Fleischer, Joerg

2017-01-01

Under given environmental conditions, the desert locust ( Schistocera gregaria ) forms destructive migratory swarms of billions of animals, leading to enormous crop losses in invaded regions. Swarm formation requires massive reproduction as well as aggregation of the animals. Pheromones that are detected via the olfactory system have been reported to control both reproductive and aggregation behavior. However, the molecular basis of pheromone detection in the antennae of Schistocerca gregaria is unknown. As an initial step to disclose pheromone receptors, we sequenced the antennal transcriptome of the desert locust. By subsequent bioinformatical approaches, 119 distinct nucleotide sequences encoding candidate odorant receptors (ORs) were identified. Phylogenetic analyses employing the identified ORs from Schistocerca gregaria (SgreORs) and OR sequences from the related species Locusta migratoria revealed a group of locust ORs positioned close to the root, i.e. at a basal site in a phylogenetic tree. Within this particular OR group (termed basal or b-OR group), the locust OR sequences were strictly orthologous, a trait reminiscent of pheromone receptors from lepidopteran species. In situ hybridization experiments with antennal tissue demonstrated expression of b-OR types from Schistocerca gregaria in olfactory sensory neurons (OSNs) of either sensilla trichodea or sensilla basiconica, both of which have been reported to respond to pheromonal substances. More importantly, two-color fluorescent in situ hybridization experiments showed that most b-OR types were expressed in cells co-expressing the "sensory neuron membrane protein 1" (SNMP1), a marker indicative of pheromone-sensitive OSNs in insects. Analyzing the expression of a larger number of SgreOR types outside the b-OR group revealed that only a few of them were co-expressed with SNMP1. In summary, we have identified several candidate pheromone receptors from Schistocerca gregaria that could mediate responses to pheromones implicated in controlling reproduction and aggregation behavior.

PirAB protein from Xenorhabdus nematophila HB310 exhibits a binary toxin with insecticidal activity and cytotoxicity in Galleria mellonella.

PubMed

Yang, Qing; Zhang, Jie; Li, Tianhui; Liu, Shen; Song, Ping; Nangong, Ziyan; Wang, Qinying

2017-09-01

PirAB (Photorhabdus insect-related proteins, PirAB) toxin was initially found in the Photorhabdus luminescens TT01 strain and has been shown to be a binary toxin with high insecticidal activity. Based on GenBank data, this gene was also found in the Xenorhabdus nematophila genome sequence. The predicted amino acid sequence of pirA and pirB in the genome of X. nematophila showed 51% and 50% identity with those gene sequences from P. luminescens. The purpose of this experiment is to identify the relevant information for this toxin gene in X. nematophila. The pirA, pirB and pirAB genes of X. nematophila HB310 were cloned and expressed in Escherichia coli BL21 (DE3) using the pET-28a vector. A PirAB-fusion protein (PirAB-F) was constructed by linking the pirA and pirB genes with the flexible linker (Gly) 4 DNA encoding sequence and then efficiently expressed in E. coli. The hemocoel and oral insecticidal activities of the recombinant proteins were analyzed against the larvae of Galleria mellonella. The results show that PirA/B alone, PirA/B mixture, co-expressed PirAB protein, and PirAB-F all had no oral insecticidal activity against the second-instar larvae of G. mellonella. Only PirA/B mixture and co-expressed PirAB protein had hemocoel insecticidal activity against G. mellonella fifth-instar larvae, with an LD 50 of 2.718μg/larva or 1.566μg/larva, respectively. Therefore, we confirmed that PirAB protein of X. nematophila HB310 is a binary insecticidal toxin. The successful expression and purification of PirAB laid a foundation for further studies on the function, insecticidal mechanism and expression regulation of the binary toxin. Copyright © 2017 Elsevier Inc. All rights reserved.

Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, CY; Yang, H; Wei, CL

Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes. Using high-throughput Illumina RNA-seq, the transcriptome from poly (A){sup +} RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled intomore » 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real time PCR (qRT-PCR). An extensive transcriptome dataset has been obtained from the deep sequencing of tea plant. The coverage of the transcriptome is comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomic studies in C. sinensis.« less

Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds

PubMed Central

2011-01-01

Background Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes. Results Using high-throughput Illumina RNA-seq, the transcriptome from poly (A)+ RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real time PCR (qRT-PCR). Conclusions An extensive transcriptome dataset has been obtained from the deep sequencing of tea plant. The coverage of the transcriptome is comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomic studies in C. sinensis. PMID:21356090

expVIP: a Customizable RNA-seq Data Analysis and Visualization Platform1[OPEN

PubMed Central

2016-01-01

The majority of transcriptome sequencing (RNA-seq) expression studies in plants remain underutilized and inaccessible due to the use of disparate transcriptome references and the lack of skills and resources to analyze and visualize these data. We have developed expVIP, an expression visualization and integration platform, which allows easy analysis of RNA-seq data combined with an intuitive and interactive interface. Users can analyze public and user-specified data sets with minimal bioinformatics knowledge using the expVIP virtual machine. This generates a custom Web browser to visualize, sort, and filter the RNA-seq data and provides outputs for differential gene expression analysis. We demonstrate expVIP’s suitability for polyploid crops and evaluate its performance across a range of biologically relevant scenarios. To exemplify its use in crop research, we developed a flexible wheat (Triticum aestivum) expression browser (www.wheat-expression.com) that can be expanded with user-generated data in a local virtual machine environment. The open-access expVIP platform will facilitate the analysis of gene expression data from a wide variety of species by enabling the easy integration, visualization, and comparison of RNA-seq data across experiments. PMID:26869702

Digital detection of multiple minority mutants and expression levels of multiple colorectal cancer-related genes using digital-PCR coupled with bead-array.

PubMed

Huang, Huan; Li, Shuo; Sun, Lizhou; Zhou, Guohua

2015-01-01

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed "multiplex ligation-dependent probe amplification-digital amplification coupled with hydrogel bead-array" (MLPA-DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA-DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA-DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.

De novo assembly and characterization of the garlic (Allium sativum) bud transcriptome by Illumina sequencing.

PubMed

Sun, Xiudong; Zhou, Shumei; Meng, Fanlu; Liu, Shiqi

2012-10-01

Garlic is widely used as a spice throughout the world for the culinary value of its flavor and aroma, which are created by the chemical transformation of a series of organic sulfur compounds. To analyze the transcriptome of Allium sativum and discover the genes involved in sulfur metabolism, cDNAs derived from the total RNA of Allium sativum buds were analyzed by Illumina sequencing. Approximately 26.67 million 90 bp paired-end clean reads were achieved in two libraries. A total of 127,933 unigenes were generated by de novo assembly and were compared with the sequences in public databases. Of these, 45,286 unigenes had significant hits to the sequences in the Nr database, 29,514 showed significant similarity to known proteins in the Swiss-Prot database and, 20,706 and 21,952 unigenes had significant similarity to existing sequences in the KEGG and COG databases, respectively. Moreover, genes involved in organic sulfur biosynthesis were identified. These unigenes data will provide the foundation for research on gene expression, genomics and functional genomics in Allium sativum. Key message The obtained unigenes will provide the foundation for research on functional genomics in Allium sativum and its closely related species, and fill the gap of the existing plant EST database.

Genome-wide survey of Aux/IAA gene family members in potato (Solanum tuberosum): Identification, expression analysis, and evaluation of their roles in tuber development.

PubMed

Gao, Junpeng; Cao, Xiaoli; Shi, Shandang; Ma, Yuling; Wang, Kai; Liu, Shengjie; Chen, Dan; Chen, Qin; Ma, Haoli

2016-03-04

The Auxin/indole-3-acetic acid (Aux/IAA) genes encode short-lived nuclear proteins that are known to be involved in the primary cellular responses to auxin. To date, systematic analysis of the Aux/IAA genes in potato (Solanum tuberosum) has not been conducted. In this study, a total of 26 potato Aux/IAA genes were identified (designated from StIAA1 to StIAA26), and the distribution of four conserved domains shared by the StIAAs were analyzed based on multiple sequence alignment and a motif-based sequence analysis. A phylogenetic analysis of the Aux/IAA gene families of potato and Arabidopsis was also conducted. In order to assess the roles of StIAA genes in tuber development, the results of RNA-seq studies were reformatted to analyze the expression patterns of StIAA genes, and then verified by quantitative real-time PCR. A large number of StIAA genes (12 genes) were highly expressed in stolon organs and in during the tuber initiation and expansion developmental stages, and most of these genes were responsive to indoleacetic acid treatment. Our results suggested that StIAA genes were involved in the process of tuber development and provided insights into functional roles of potato Aux/IAA genes. Copyright © 2016 Elsevier Inc. All rights reserved.

A Guide for Designing and Analyzing RNA-Seq Data.

PubMed

Chatterjee, Aniruddha; Ahn, Antonio; Rodger, Euan J; Stockwell, Peter A; Eccles, Michael R

2018-01-01

The identity of a cell or an organism is at least in part defined by its gene expression and therefore analyzing gene expression remains one of the most frequently performed experimental techniques in molecular biology. The development of the RNA-Sequencing (RNA-Seq) method allows an unprecedented opportunity to analyze expression of protein-coding, noncoding RNA and also de novo transcript assembly of a new species or organism. However, the planning and design of RNA-Seq experiments has important implications for addressing the desired biological question and maximizing the value of the data obtained. In addition, RNA-Seq generates a huge volume of data and accurate analysis of this data involves several different steps and choices of tools. This can be challenging and overwhelming, especially for bench scientists. In this chapter, we describe an entire workflow for performing RNA-Seq experiments. We describe critical aspects of wet lab experiments such as RNA isolation, library preparation and the initial design of an experiment. Further, we provide a step-by-step description of the bioinformatics workflow for different steps involved in RNA-Seq data analysis. This includes power calculations, setting up a computational environment, acquisition and processing of publicly available data if desired, quality control measures, preprocessing steps for the raw data, differential expression analysis, and data visualization. We particularly mention important considerations for each step to provide a guide for designing and analyzing RNA-Seq data.

Discovering Single Nucleotide Polymorphisms Regulating Human Gene Expression Using Allele Specific Expression from RNA-seq Data

PubMed Central

Kang, Eun Yong; Martin, Lisa J.; Mangul, Serghei; Isvilanonda, Warin; Zou, Jennifer; Ben-David, Eyal; Han, Buhm; Lusis, Aldons J.; Shifman, Sagiv; Eskin, Eleazar

2016-01-01

The study of the genetics of gene expression is of considerable importance to understanding the nature of common, complex diseases. The most widely applied approach to identifying relationships between genetic variation and gene expression is the expression quantitative trait loci (eQTL) approach. Here, we increased the computational power of eQTL with an alternative and complementary approach based on analyzing allele specific expression (ASE). We designed a novel analytical method to identify cis-acting regulatory variants based on genome sequencing and measurements of ASE from RNA-sequencing (RNA-seq) data. We evaluated the power and resolution of our method using simulated data. We then applied the method to map regulatory variants affecting gene expression in lymphoblastoid cell lines (LCLs) from 77 unrelated northern and western European individuals (CEU), which were part of the HapMap project. A total of 2309 SNPs were identified as being associated with ASE patterns. The SNPs associated with ASE were enriched within promoter regions and were significantly more likely to signal strong evidence for a regulatory role. Finally, among the candidate regulatory SNPs, we identified 108 SNPs that were previously associated with human immune diseases. With further improvements in quantifying ASE from RNA-seq, the application of our method to other datasets is expected to accelerate our understanding of the biological basis of common diseases. PMID:27765809

Novel kinase fusion transcripts found in endometrial cancer

PubMed Central

Tamura, Ryo; Yoshihara, Kosuke; Yamawaki, Kaoru; Suda, Kazuaki; Ishiguro, Tatsuya; Adachi, Sosuke; Okuda, Shujiro; Inoue, Ituro; Verhaak, Roel G. W.; Enomoto, Takayuki

2015-01-01

Recent advances in RNA-sequencing technology have enabled the discovery of gene fusion transcripts in the transcriptome of cancer cells. However, it remains difficult to differentiate the therapeutically targetable fusions from passenger events. We have analyzed RNA-sequencing data and DNA copy number data from 25 endometrial cancer cell lines to identify potential therapeutically targetable fusion transcripts, and have identified 124 high-confidence fusion transcripts, of which 69% are associated with gene amplifications. As targetable fusion candidates, we focused on three in-frame kinase fusion transcripts that retain a kinase domain (CPQ-PRKDC, CAPZA2-MET, and VGLL4-PRKG1). We detected only CPQ-PRKDC fusion transcript in three of 122 primary endometrial cancer tissues. Cell proliferation of the fusion-positive cell line was inhibited by knocking down the expression of wild-type PRKDC but not by blocking the CPQ-PRKDC fusion transcript expression. Quantitative real-time RT-PCR demonstrated that the expression of the CPQ-PRKDC fusion transcript was significantly lower than that of wild-type PRKDC, corresponding to a low transcript allele fraction of this fusion, based on RNA-sequencing read counts. In endometrial cancers, the CPQ-PRKDC fusion transcript may be a passenger aberration related to gene amplification. Our findings suggest that transcript allele fraction is a useful predictor to find bona-fide therapeutic-targetable fusion transcripts. PMID:26689674

Novel kinase fusion transcripts found in endometrial cancer.

PubMed

Tamura, Ryo; Yoshihara, Kosuke; Yamawaki, Kaoru; Suda, Kazuaki; Ishiguro, Tatsuya; Adachi, Sosuke; Okuda, Shujiro; Inoue, Ituro; Verhaak, Roel G W; Enomoto, Takayuki

2015-12-22

Recent advances in RNA-sequencing technology have enabled the discovery of gene fusion transcripts in the transcriptome of cancer cells. However, it remains difficult to differentiate the therapeutically targetable fusions from passenger events. We have analyzed RNA-sequencing data and DNA copy number data from 25 endometrial cancer cell lines to identify potential therapeutically targetable fusion transcripts, and have identified 124 high-confidence fusion transcripts, of which 69% are associated with gene amplifications. As targetable fusion candidates, we focused on three in-frame kinase fusion transcripts that retain a kinase domain (CPQ-PRKDC, CAPZA2-MET, and VGLL4-PRKG1). We detected only CPQ-PRKDC fusion transcript in three of 122 primary endometrial cancer tissues. Cell proliferation of the fusion-positive cell line was inhibited by knocking down the expression of wild-type PRKDC but not by blocking the CPQ-PRKDC fusion transcript expression. Quantitative real-time RT-PCR demonstrated that the expression of the CPQ-PRKDC fusion transcript was significantly lower than that of wild-type PRKDC, corresponding to a low transcript allele fraction of this fusion, based on RNA-sequencing read counts. In endometrial cancers, the CPQ-PRKDC fusion transcript may be a passenger aberration related to gene amplification. Our findings suggest that transcript allele fraction is a useful predictor to find bona-fide therapeutic-targetable fusion transcripts.

Global survey of genomic imprinting by transcriptome sequencing.

PubMed

Babak, Tomas; Deveale, Brian; Armour, Christopher; Raymond, Christopher; Cleary, Michele A; van der Kooy, Derek; Johnson, Jason M; Lim, Lee P

2008-11-25

Genomic imprinting restricts gene expression to a paternal or maternal allele. To date, approximately 90 imprinted transcripts have been identified in mouse, of which the majority were detected after intense interrogation of clusters of imprinted genes identified by phenotype-driven assays in mice with uniparental disomies [1]. Here we use selective priming and parallel sequencing to measure allelic bias in whole transcriptomes. By distinguishing parent-of-origin bias from strain-specific bias in embryos derived from a reciprocal cross of mice, we constructed a genome-wide map of imprinted transcription. This map was able to objectively locate over 80% of known imprinted loci and allowed the detection and confirmation of six novel imprinted genes. Even in the intensely studied embryonic day 9.5 developmental stage that we analyzed, more than half of all imprinted single-nucleotide polymorphisms did not overlap previously discovered imprinted transcripts; a large fraction of these represent novel noncoding RNAs within known imprinted loci. For example, a previously unnoticed, maternally expressed antisense transcript was mapped within the Grb10 locus. This study demonstrates the feasibility of using transcriptome sequencing for mapping of imprinted gene expression in physiologically normal animals. Such an approach will allow researchers to study imprinting without restricting themselves to individual loci or specific transcripts.

Phylogeny and expression profiling of CAD and CAD-like genes in hybrid Populus (P. deltoides × P. nigra): evidence from herbivore damage for subfunctionalization and functional divergence

PubMed Central

2010-01-01

Background Cinnamyl Alcohol Dehydrogenase (CAD) proteins function in lignin biosynthesis and play a critical role in wood development and plant defense against stresses. Previous phylogenetic studies did not include genes from seedless plants and did not reflect the deep evolutionary history of this gene family. We reanalyzed the phylogeny of CAD and CAD-like genes using a representative dataset including lycophyte and bryophyte sequences. Many CAD/CAD-like genes do not seem to be associated with wood development under normal growth conditions. To gain insight into the functional evolution of CAD/CAD-like genes, we analyzed their expression in Populus plant tissues in response to feeding damage by gypsy moth larvae (Lymantria dispar L.). Expression of CAD/CAD-like genes in Populus tissues (xylem, leaves, and barks) was analyzed in herbivore-treated and non-treated plants by real time quantitative RT-PCR. Results CAD family genes were distributed in three classes based on sequence conservation. All the three classes are represented by seedless as well as seed plants, including the class of bona fide lignin pathway genes. The expression of some CAD/CAD-like genes that are not associated with xylem development were induced following herbivore damage in leaves, while other genes were induced in only bark or xylem tissues. Five of the CAD/CAD-like genes, however, showed a shift in expression from one tissue to another between non-treated and herbivore-treated plants. Systemic expression of the CAD/CAD-like genes was generally suppressed. Conclusions Our results indicated a correlation between the evolution of the CAD gene family and lignin and that the three classes of genes may have evolved in the ancestor of land plants. Our results also suggest that the CAD/CAD-like genes have evolved a diversity of expression profiles and potentially different functions, but that they are nonetheless co-regulated under stress conditions. PMID:20509918

Transcriptome Profiling of Bovine Milk Oligosaccharide Metabolism Genes Using RNA-Sequencing

PubMed Central

Wickramasinghe, Saumya; Hua, Serenus; Rincon, Gonzalo; Islas-Trejo, Alma; German, J. Bruce; Lebrilla, Carlito B.; Medrano, Juan F.

2011-01-01

This study examines the genes coding for enzymes involved in bovine milk oligosaccharide metabolism by comparing the oligosaccharide profiles with the expressions of glycosylation-related genes. Fresh milk samples (n = 32) were collected from four Holstein and Jersey cows at days 1, 15, 90 and 250 of lactation and free milk oligosaccharide profiles were analyzed. RNA was extracted from milk somatic cells at days 15 and 250 of lactation (n = 12) and gene expression analysis was conducted by RNA-Sequencing. A list was created of 121 glycosylation-related genes involved in oligosaccharide metabolism pathways in bovine by analyzing the oligosaccharide profiles and performing an extensive literature search. No significant differences were observed in either oligosaccharide profiles or expressions of glycosylation-related genes between Holstein and Jersey cows. The highest concentrations of free oligosaccharides were observed in the colostrum samples and a sharp decrease was observed in the concentration of free oligosaccharides on day 15, followed by progressive decrease on days 90 and 250. Ninety-two glycosylation-related genes were expressed in milk somatic cells. Most of these genes exhibited higher expression in day 250 samples indicating increases in net glycosylation-related metabolism in spite of decreases in free milk oligosaccharides in late lactation milk. Even though fucosylated free oligosaccharides were not identified, gene expression indicated the likely presence of fucosylated oligosaccharides in bovine milk. Fucosidase genes were expressed in milk and a possible explanation for not detecting fucosylated free oligosaccharides is the degradation of large fucosylated free oligosaccharides by the fucosidases. Detailed characterization of enzymes encoded by the 92 glycosylation-related genes identified in this study will provide the basic knowledge for metabolic network analysis of oligosaccharides in mammalian milk. These candidate genes will guide the design of a targeted breeding strategy to optimize the content of beneficial oligosaccharides in bovine milk. PMID:21541029

Intestinal flora of FAP patients containing APC-like sequences.

PubMed

Hainova, K; Adamcikova, Z; Ciernikova, S; Stevurkova, V; Tyciakova, S; Zajac, V

2014-01-01

Colorectal cancer mortality is one of the most common cause of cancer-related mortality. A multiple risk factors are associated with colorectal cancer, including hereditary, enviromental and inflammatory syndromes affecting the gastrointestinal tract. Familial adenomatous polyposis (FAP) is characterized by the emergence of hundreds to thousands of colorectal adenomatous polyps and FAP syndrome is caused by mutations within the adenomatous polyposis coli (APC) tumor suppressor gene. We analyzed 21 rectal bacterial subclones isolated from FAP patient 41-1 with confirmed 5bp ACAAA deletion within codons 1060-1063 for the presence of APC-like sequences in longest exon 15. The studied section was defined by primers 15Efor-15Erev, what correlates with mutation cluster region (MCR) in which the 75% of all APC germline mutations were detected. More than 90% homology was showed by sequencing and subsequent software comparison. The expression of APC-like sequences was demostrated by Western blot analysis using monoclonal and polyclonal antibodies against APC protein. To study missing link between the DNA analysis (PCR, DNA sequencing) and protein expresion experiments (Western blotting) we analyzed bacterial transcripts containing the 15Efor-15Erev sequence of APC gene by reverse transcription-PCR, what indicated that an APC gene derived fragment may be produced. We observed 97-100 % homology after computer comparison of cDNA PCR products. Our results suggest that presence of APC-like sequences in intestinal/rectal bacteria is enrichment of bacterial genetic information in which horizontal gene transfer between humans and microflora play an important role.

Differential expression of myogenic regulatory factor MyoD in pacu skeletal muscle (Piaractus mesopotamicus Holmberg 1887: Serrasalminae, Characidae, Teleostei) during juvenile and adult growth phases.

PubMed

de Almeida, Fernanda Losi Alves; Carvalho, Robson Francisco; Pinhal, Danillo; Padovani, Carlos Roberto; Martins, Cesar; Dal Pai-Silva, Maeli

2008-12-01

Skeletal muscle is the edible part of the fish. It grows by hypertrophy and hyperplasia, events regulated by differential expression of myogenic regulatory factors (MRFs). The study of muscle growth mechanisms in fish is very important in fish farming development. Pacu (Piaractus mesopotamicus) is one of the most important food species farmed in Brazil and has been extensively used in Brazilian aquaculture programs. The aim of this study was to analyze hyperplasia and hypertrophy and the MRF MyoD expression pattern in skeletal muscle of pacu (P. mesopotamicus) during juvenile and adult growth stages. Juvenile (n=5) and adult (n=5) fish were anaesthetized, sacrificed, and weight (g) and total length (cm) determined. White dorsal region muscle samples were collected and immersed in liquid nitrogen. Transverse sections (10 microm thick) were stained with Haematoxilin-Eosin (HE) for morphological and morphometric analysis. Smallest fiber diameter from 100 muscle fibers per animal was calculated in each growth phase. These fibers were grouped into three classes (<20, 20-50, and >50 microm) to evaluate hypertrophy and hyperplasia in white skeletal muscle. MyoD gene expression was determined by semi-quantitative RT-PCR. PCR products were cloned and sequenced. Juvenile and adult pacu skeletal muscle had similar morphology. The large number of <20 microm diameter muscle fibers observed in juvenile fish confirms active hyperplasia. In adult fish, most fibers were over 50 microm diameter and denote more intense muscle fiber hypertrophy. The MyoD mRNA level in juveniles was higher than in adults. A consensus partial sequence for MyoD gene (338 base pairs) was obtained. The Pacu MyoD nucleotide sequence displayed high similarity among several vertebrates, including teleosts. The differential MyoD gene expression observed in pacu white muscle is possibly related to differences in growth patterns during the phases analyzed, with hyperplasia predominant in juveniles and hypertrophy in adult fish. These results should provide a foundation for understanding the molecular control of skeletal muscle growth in economically important Brazilian species, with a view to improving production quality.

Spatio-temporal analysis of aftershock sequences in terms of Non Extensive Statistical Physics.

NASA Astrophysics Data System (ADS)

Chochlaki, Kalliopi; Vallianatos, Filippos

2017-04-01

Earth's seismicity is considered as an extremely complicated process where long-range interactions and fracturing exist (Vallianatos et al., 2016). For this reason, in order to analyze it, we use an innovative methodological approach, introduced by Tsallis (Tsallis, 1988; 2009), named Non Extensive Statistical Physics. This approach introduce a generalization of the Boltzmann-Gibbs statistical mechanics and it is based on the definition of Tsallis entropy Sq, which maximized leads the the so-called q-exponential function that expresses the probability distribution function that maximizes the Sq. In the present work, we utilize the concept of Non Extensive Statistical Physics in order to analyze the spatiotemporal properties of several aftershock series. Marekova (Marekova, 2014) suggested that the probability densities of the inter-event distances between successive aftershocks follow a beta distribution. Using the same data set we analyze the inter-event distance distribution of several aftershocks sequences in different geographic regions by calculating non extensive parameters that determine the behavior of the system and by fitting the q-exponential function, which expresses the degree of non-extentivity of the investigated system. Furthermore, the inter-event times distribution of the aftershocks as well as the frequency-magnitude distribution has been analyzed. The results supports the applicability of Non Extensive Statistical Physics ideas in aftershock sequences where a strong correlation exists along with memory effects. References C. Tsallis, Possible generalization of Boltzmann-Gibbs statistics, J. Stat. Phys. 52 (1988) 479-487. doi:10.1007/BF01016429 C. Tsallis, Introduction to nonextensive statistical mechanics: Approaching a complex world, 2009. doi:10.1007/978-0-387-85359-8. E. Marekova, Analysis of the spatial distribution between successive earthquakes in aftershocks series, Annals of Geophysics, 57, 5, doi:10.4401/ag-6556, 2014 F. Vallianatos, G. Papadakis, G. Michas, Generalized statistical mechanics approaches to earthquakes and tectonics. Proc. R. Soc. A, 472, 20160497, 2016.

Cloning and analysis of DnaJ family members in the silkworm, Bombyx mori.

PubMed

Li, Yinü; Bu, Cuiyu; Li, Tiantian; Wang, Shibao; Jiang, Feng; Yi, Yongzhu; Yang, Huipeng; Zhang, Zhifang

2016-01-15

Heat shock proteins (Hsps) are involved in a variety of critical biological functions, including protein folding, degradation, and translocation and macromolecule assembly, act as molecular chaperones during periods of stress by binding to other proteins. Using expressed sequence tag (EST) and silkworm (Bombyx mori) transcriptome databases, we identified 27 cDNA sequences encoding the conserved J domain, which is found in DnaJ-type Hsps. Of the 27 J domain-containing sequences, 25 were complete cDNA sequences. We divided them into three types according to the number and presence of conserved domains. By analyzing the gene structures, intron numbers, and conserved domains and constructing a phylogenetic tree, we found that the DnaJ family had undergone convergent evolution, obtaining new domains to expand the diversity of its family members. The acquisition of the new DnaJ domains most likely occurred prior to the evolutionary divergence of prokaryotes and eukaryotes. The expression of DnaJ genes in the silkworm was generally higher in the fat body. The tissue distribution of DnaJ1 proteins was detected by western blotting, demonstrating that in the fifth-instar larvae, the DnaJ1 proteins were expressed at their highest levels in hemocytes, followed by the fat body and head. We also found that the DnaJ1 transcripts were likely differentially translated in different tissues. Using immunofluorescence cytochemistry, we revealed that in the blood cells, DnaJ1 was mainly localized in the cytoplasm. Copyright © 2015 Elsevier B.V. All rights reserved.

Differential gene expression in the siphonophore Nanomia bijuga (Cnidaria) assessed with multiple next-generation sequencing workflows.

PubMed

Siebert, Stefan; Robinson, Mark D; Tintori, Sophia C; Goetz, Freya; Helm, Rebecca R; Smith, Stephen A; Shaner, Nathan; Haddock, Steven H D; Dunn, Casey W

2011-01-01

We investigated differential gene expression between functionally specialized feeding polyps and swimming medusae in the siphonophore Nanomia bijuga (Cnidaria) with a hybrid long-read/short-read sequencing strategy. We assembled a set of partial gene reference sequences from long-read data (Roche 454), and generated short-read sequences from replicated tissue samples that were mapped to the references to quantify expression. We collected and compared expression data with three short-read expression workflows that differ in sample preparation, sequencing technology, and mapping tools. These workflows were Illumina mRNA-Seq, which generates sequence reads from random locations along each transcript, and two tag-based approaches, SOLiD SAGE and Helicos DGE, which generate reads from particular tag sites. Differences in expression results across workflows were mostly due to the differential impact of missing data in the partial reference sequences. When all 454-derived gene reference sequences were considered, Illumina mRNA-Seq detected more than twice as many differentially expressed (DE) reference sequences as the tag-based workflows. This discrepancy was largely due to missing tag sites in the partial reference that led to false negatives in the tag-based workflows. When only the subset of reference sequences that unambiguously have tag sites was considered, we found broad congruence across workflows, and they all identified a similar set of DE sequences. Our results are promising in several regards for gene expression studies in non-model organisms. First, we demonstrate that a hybrid long-read/short-read sequencing strategy is an effective way to collect gene expression data when an annotated genome sequence is not available. Second, our replicated sampling indicates that expression profiles are highly consistent across field-collected animals in this case. Third, the impacts of partial reference sequences on the ability to detect DE can be mitigated through workflow choice and deeper reference sequencing.

Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows

PubMed Central

Siebert, Stefan; Robinson, Mark D.; Tintori, Sophia C.; Goetz, Freya; Helm, Rebecca R.; Smith, Stephen A.; Shaner, Nathan; Haddock, Steven H. D.; Dunn, Casey W.

2011-01-01

We investigated differential gene expression between functionally specialized feeding polyps and swimming medusae in the siphonophore Nanomia bijuga (Cnidaria) with a hybrid long-read/short-read sequencing strategy. We assembled a set of partial gene reference sequences from long-read data (Roche 454), and generated short-read sequences from replicated tissue samples that were mapped to the references to quantify expression. We collected and compared expression data with three short-read expression workflows that differ in sample preparation, sequencing technology, and mapping tools. These workflows were Illumina mRNA-Seq, which generates sequence reads from random locations along each transcript, and two tag-based approaches, SOLiD SAGE and Helicos DGE, which generate reads from particular tag sites. Differences in expression results across workflows were mostly due to the differential impact of missing data in the partial reference sequences. When all 454-derived gene reference sequences were considered, Illumina mRNA-Seq detected more than twice as many differentially expressed (DE) reference sequences as the tag-based workflows. This discrepancy was largely due to missing tag sites in the partial reference that led to false negatives in the tag-based workflows. When only the subset of reference sequences that unambiguously have tag sites was considered, we found broad congruence across workflows, and they all identified a similar set of DE sequences. Our results are promising in several regards for gene expression studies in non-model organisms. First, we demonstrate that a hybrid long-read/short-read sequencing strategy is an effective way to collect gene expression data when an annotated genome sequence is not available. Second, our replicated sampling indicates that expression profiles are highly consistent across field-collected animals in this case. Third, the impacts of partial reference sequences on the ability to detect DE can be mitigated through workflow choice and deeper reference sequencing. PMID:21829563

Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.

PubMed

Vukmirovic, Milica; Herazo-Maya, Jose D; Blackmon, John; Skodric-Trifunovic, Vesna; Jovanovic, Dragana; Pavlovic, Sonja; Stojsic, Jelena; Zeljkovic, Vesna; Yan, Xiting; Homer, Robert; Stefanovic, Branko; Kaminski, Naftali

2017-01-12

Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues. We isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four. Our results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.

Dissecting the Root Nodule Transcriptome of Chickpea (Cicer arietinum L.)

PubMed Central

Kant, Chandra; Pradhan, Seema; Bhatia, Sabhyata

2016-01-01

A hallmark trait of chickpea (Cicer arietinum L.), like other legumes, is the capability to convert atmospheric nitrogen (N2) into ammonia (NH3) in symbiotic association with Mesorhizobium ciceri. However, the complexity of molecular networks associated with the dynamics of nodule development in chickpea need to be analyzed in depth. Hence, in order to gain insights into the chickpea nodule development, the transcriptomes of nodules at early, middle and late stages of development were sequenced using the Roche 454 platform. This generated 490.84 Mb sequence data comprising 1,360,251 reads which were assembled into 83,405 unigenes. Transcripts were annotated using Gene Ontology (GO), Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways analysis. Differential expression analysis revealed that a total of 3760 transcripts were differentially expressed in at least one of three stages, whereas 935, 117 and 2707 transcripts were found to be differentially expressed in the early, middle and late stages of nodule development respectively. MapMan analysis revealed enrichment of metabolic pathways such as transport, protein synthesis, signaling and carbohydrate metabolism during root nodulation. Transcription factors were predicted and analyzed for their differential expression during nodule development. Putative nodule specific transcripts were identified and enriched for GO categories using BiNGO which revealed many categories to be enriched during nodule development, including transcription regulators and transporters. Further, the assembled transcriptome was also used to mine for genic SSR markers. In conclusion, this study will help in enriching the transcriptomic resources implicated in understanding of root nodulation events in chickpea. PMID:27348121

De novo transcriptome sequencing in Frankliniella occidentalis to identify genes involved in plant virus transmission and insecticide resistance.

PubMed

Zhang, Zhijun; Zhang, Pengjun; Li, Weidi; Zhang, Jinming; Huang, Fang; Yang, Jian; Bei, Yawei; Lu, Yaobin

2013-05-01

The western flower thrips (WFT), Frankliniella occidentalis, a world-wide invasive insect, causes agricultural damage by directly feeding and by indirectly vectoring Tospoviruses, such as Tomato spotted wilt virus (TSWV). We characterized the transcriptome of WFT and analyzed global gene expression of WFT response to TSWV infection using Illumina sequencing platform. We compiled 59,932 unigenes, and identified 36,339 unigenes by similarity analysis against public databases, most of which were annotated using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Within these annotated transcripts, we collected 278 sequences related to insecticide resistance. GO and KEGG analysis of different expression genes between TSWV-infected and non-infected WFT population revealed that TSWV can regulate cellular process and immune response, which might lead to low virus titers in thrips cells and no detrimental effects on F. occidentalis. This data-set not only enriches genomic resource for WFT, but also benefits research into its molecular genetics and functional genomics. Copyright © 2013 Elsevier Inc. All rights reserved.

Differential expression of a WRKY gene between wild and cultivated soybeans correlates to seed size.

PubMed

Gu, Yongzhe; Li, Wei; Jiang, Hongwei; Wang, Yan; Gao, Huihui; Liu, Miao; Chen, Qingshan; Lai, Yongcai; He, Chaoying

2017-05-17

Soybean (Glycine max) probably originated from the wild soybean (Glycine soja). Glycine max has a significantly larger seed size, but the underlying genomic changes are largely unknown. Candidate regulatory genes were preliminarily proposed by data co-localizing RNA sequencing with the quantitative loci (QTLs) for seed size. The soybean gene locus SoyWRKY15a and its orthologous genes from G. max (GmWRKY15a) and G. soja (GsWRKY15a) were analyzed in detail. The coding sequences were nearly identical between the two orthologs, but GmWRKY15a was significantly more highly expressed than GsWRKY15a. Four haplotypes (H1-H4) were found and they varied in the size of a CT-core microsatellite locus in the 5'-untranslated region of this gene. H1 (with six CT-repeats) was the only allelic version found in G. max, while H3 (with five CT-repeats) was the dominant G. soja allele. Differential expression of this gene in soybean pods was correlated with CT-repeat variation, and manipulation of the CT copy number altered the reporter gene expression, suggesting a regulatory role for the simple sequence repeats. Seed weight of wild soybeans harboring H1 was significantly greater than that of soybeans having haplotypes H2, H3, or H4, and seed weight was correlated with gene expression, suggesting the influence of GsWRKY15a in controlling seed size. However, the seed size might be refractory to increased SoyWRKY15a expression in cultivated soybeans. The evolutionary significance of SoyWRKY15a variation in soybean seed domestication is discussed. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Molecular Characterization and Expression Analysis of Chloroplast Protein Import Components in Tomato (Solanum lycopersicum)

PubMed Central

Yan, Jianmin; Campbell, James H.; Glick, Bernard R.; Smith, Matthew D.; Liang, Yan

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and initial import into the organelle of thousands of nucleus-encoded proteins. These proteins are translated in the cytosol as precursor proteins with cleavable amino-terminal targeting sequences called transit peptides. The majority of the known Toc components that mediate chloroplast protein import were originally identified in pea, and more recently have been studied most extensively in Arabidopsis. With the completion of the tomato genome sequencing project, it is now possible to identify putative homologues of the chloroplast import components in tomato. In the work reported here, the Toc GTPase cDNAs from tomato were identified, cloned and analyzed. The analysis revealed that there are four Toc159 homologues (slToc159-1, -2, -3 and -4) and two Toc34 homologues (slToc34-1 and -2) in tomato, and it was shown that tomato Toc159 and Toc34 homologues share high sequence similarity with the comparable import apparatus components from Arabidopsis and pea. Thus, tomato is a valid model for further study of this system. The expression level of Toc complex components was also investigated in different tissues during tomato development. The two tomato Toc34 homologues are expressed at higher levels in non-photosynthetic tissues, whereas, the expression of two tomato Toc159 homologues, slToc159-1 and slToc159-4, were higher in photosynthetic tissues, and the expression patterns of slToc159-2 was not significantly different in photosynthetic and non-photosynthetic tissues, and slToc159-3 expression was limited to a few select tissues. PMID:24751891

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

PubMed Central

Hirsh, J; Morgan, B A; Scholnick, S B

1986-01-01

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity. Images PMID:3099170

High-Throughput Sequencing of microRNAs in Glucocorticoid Sensitive Paediatric Inflammatory Bowel Disease Patients.

PubMed

De Iudicibus, Sara; Lucafò, Marianna; Vitulo, Nicola; Martelossi, Stefano; Zimbello, Rosanna; De Pascale, Fabio; Forcato, Claudio; Naviglio, Samuele; Di Silvestre, Alessia; Gerdol, Marco; Stocco, Gabriele; Valle, Giorgio; Ventura, Alessandro; Bramuzzo, Matteo; Decorti, Giuliana

2018-05-08

The aim of this research was the identification of novel pharmacogenomic biomarkers for better understanding the complex gene regulation mechanisms underpinning glucocorticoid (GC) action in paediatric inflammatory bowel disease (IBD). This goal was achieved by evaluating high-throughput microRNA (miRNA) profiles during GC treatment, integrated with the assessment of expression changes in GC receptor (GR) heterocomplex genes. Furthermore, we tested the hypothesis that differentially expressed miRNAs could be directly regulated by GCs through investigating the presence of GC responsive elements (GREs) in their gene promoters. Ten IBD paediatric patients responding to GCs were enrolled. Peripheral blood was obtained at diagnosis (T0) and after four weeks of steroid treatment (T4). MicroRNA profiles were analyzed using next generation sequencing, and selected significantly differentially expressed miRNAs were validated by quantitative reverse transcription-polymerase chain reaction. In detail, 18 miRNAs were differentially expressed from T0 to T4, 16 of which were upregulated and 2 of which were downregulated. Out of these, three miRNAs (miR-144, miR-142, and miR-96) could putatively recognize the 3’UTR of the GR gene and three miRNAs (miR-363, miR-96, miR-142) contained GREs sequences, thereby potentially enabling direct regulation by the GR. In conclusion, we identified miRNAs differently expressed during GC treatment and miRNAs which could be directly regulated by GCs in blood cells of young IBD patients. These results could represent a first step towards their translation as pharmacogenomic biomarkers.

Molecular cloning, mRNA expression and tissue distribution analysis of Slc7a11 gene in alpaca (Lama paco) skins associated with different coat colors.

PubMed

Tian, Xue; Meng, Xiaolin; Wang, Liangyan; Song, Yunfei; Zhang, Danli; Ji, Yuankai; Li, Xuejun; Dong, Changsheng

2015-01-25

Slc7a11 encoding solute carrier family 7 member 11 (amionic amino acid transporter light chain, xCT), has been identified to be a critical genetic regulator of pheomelanin synthesis in hair and melanocytes. To better understand the molecular characterization of Slc7a11 and the expression patterns in skin of white versus brown alpaca (lama paco), we cloned the full length coding sequence (CDS) of alpaca Slc7a11 gene and analyzed the expression patterns using Real Time PCR, Western blotting and immunohistochemistry. The full length CDS of 1512bp encodes a 503 amino acid polypeptide. Sequence analysis showed that alpaca xCT contains 12 transmembrane regions consistent with the highly conserved amino acid permease (AA_permease_2) domain similar to other vertebrates. Sequence alignment and phylogenetic analysis revealed that alpaca xCT had the highest identity and shared the same branch with Camelus ferus. Real Time PCR and Western blotting suggested that xCT was expressed at significantly high levels in brown alpaca skin, and transcripts and protein possessed the same expression pattern in white and brown alpaca skins. Additionally, immunohistochemical analysis further demonstrated that xCT staining was robustly increased in the matrix and root sheath of brown alpaca skin compared with that of white. These results suggest that Slc7a11 functions in alpaca coat color regulation and offer essential information for further exploration on the role of Slc7a11 in melanogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel chromosomal rearrangements and break points at the t(6;9) in salivary adenoid cystic carcinoma: association with MYB-NFIB chimeric fusion, MYB expression, and clinical outcome.

PubMed

Mitani, Yoshitsugu; Rao, Pulivarthi H; Futreal, P Andrew; Roberts, Dianna B; Stephens, Philip J; Zhao, Yi-Jue; Zhang, Li; Mitani, Mutsumi; Weber, Randal S; Lippman, Scott M; Caulin, Carlos; El-Naggar, Adel K

2011-11-15

To investigate the molecular genetic heterogeneity associated with the t(6:9) in adenoid cystic carcinoma (ACC) and correlate the findings with patient clinical outcome. Multimolecular and genetic techniques complemented with massive pair-ended sequencing and single-nucleotide polymorphism array analyses were used on tumor specimens from 30 new and 52 previously analyzed fusion transcript-negative ACCs by reverse transcriptase PCR (RT-PCR). MYB mRNA expression level was determined by quantitative RT-PCR. The results of 102 tumors (30 new and 72 previously reported cases) were correlated with the clinicopathologic factors and patients' survival. The FISH analysis showed 34 of 82 (41.5%) fusion-positive tumors and molecular techniques identified fusion transcripts in 21 of the 82 (25.6%) tumors. Detailed FISH analysis of 11 out the 15 tumors with gene fusion without transcript formation showed translocation of NFIB sequences to proximal or distal sites of the MYB gene. Massive pair-end sequencing of a subset of tumors confirmed the proximal translocation to an NFIB sequence and led to the identification of a new fusion gene (NFIB-AIG1) in one of the tumors. Overall, MYB-NFIB gene fusion rate by FISH was in 52.9% whereas fusion transcript forming incidence was 38.2%. Significant statistical association between the 5' MYB transcript expression and patient survival was found. We conclude that: (i) t(6;9) results in complex genetic and molecular alterations in ACC, (ii) MYB-NFIB gene fusion may not always be associated with chimeric transcript formation, (iii) noncanonical MYB-NFIB gene fusions occur in a subset of tumors, (iv) high MYB expression correlates with worse patient survival.

Novel Chromosomal Rearrangements and breakpoints at the t(6;9) in Salivary Adenoid Cystic Carcinoma: association with MYB-NFIB chimeric fusion, MYB expression, and clinical outcome

PubMed Central

Mitani, Yoshitsugu; Rao, Pulivarthi H.; Futreal, P. Andrew; Roberts, Dianna B.; Stephens, Philip J.; Zhao, Yi-Jue; Zhang, Li; Mitani, Mutsumi; Weber, Randal S.; Lippman, Scott M.; Caulin, Carlos; El-Naggar, Adel K.

2011-01-01

Objective To investigate the molecular-genetic heterogeneity associated with the t(6:9) in adenoid cystic carcinoma (ACC) and correlate the findings with patient clinical outcome. Experimental Design Multi-molecular and genetic techniques complemented with massive pair-ended sequencing and SNP array analyses were used on tumor specimens from 30 new and 52 previously RT-PCR analyzed fusion transcript negative ACCs. MYB mRNA expression level was determined by quantitative RT-PCR. The results of 102 tumors (30 new and 72 previously reported cases) were correlated with the clinicopathologic factors and patients’ survival. Results The FISH analysis showed 34/82 (41.5%) fusion positive tumors and molecular techniques identified fusion transcripts in 21 of the 82 (25.6%) tumors. Detailed FISH analysis of 11 out the 15 tumors with gene fusion without transcript formation showed translocation of NFIB sequences to proximal or distal sites of the MYB gene. Massive pair-end sequencing of a subset of tumors confirmed the proximal translocation to an NFIB sequence and led to the identification of a new fusion gene (NFIB-AIG1) in one of the tumors. Overall, MYB-NFIB gene fusion rate by FISH was in 52.9% while fusion transcript forming incidence was 38.2%. Significant statistical association between the 5′ MYB transcript expression and patient survival was found. Conclusions We conclude that: 1) t(6;9) results in a complex genetic and molecular alterations in ACC, 2) MYB-NFIB gene fusion may not always be associated with chimeric transcript formation, 3) non-canonical MYB, NFIB gene fusions occur in a subset of tumors, 4) high MYB expression correlates with worse patient survival. PMID:21976542

RNA-Seq Analysis of Human Trigeminal and Dorsal Root Ganglia with a Focus on Chemoreceptors

PubMed Central

Flegel, Caroline; Schöbel, Nicole; Altmüller, Janine; Becker, Christian; Tannapfel, Andrea; Hatt, Hanns; Gisselmann, Günter

2015-01-01

The chemosensory capacity of the somatosensory system relies on the appropriate expression of chemoreceptors, which detect chemical stimuli and transduce sensory information into cellular signals. Knowledge of the complete repertoire of the chemoreceptors expressed in human sensory ganglia is lacking. This study employed the next-generation sequencing technique (RNA-Seq) to conduct the first expression analysis of human trigeminal ganglia (TG) and dorsal root ganglia (DRG). We analyzed the data with a focus on G-protein coupled receptors (GPCRs) and ion channels, which are (potentially) involved in chemosensation by somatosensory neurons in the human TG and DRG. For years, transient receptor potential (TRP) channels have been considered the main group of receptors for chemosensation in the trigeminal system. Interestingly, we could show that sensory ganglia also express a panel of different olfactory receptors (ORs) with putative chemosensory function. To characterize OR expression in more detail, we performed microarray, semi-quantitative RT-PCR experiments, and immunohistochemical staining. Additionally, we analyzed the expression data to identify further known or putative classes of chemoreceptors in the human TG and DRG. Our results give an overview of the major classes of chemoreceptors expressed in the human TG and DRG and provide the basis for a broader understanding of the reception of chemical cues. PMID:26070209

Polycythemia in transgenic mice expressing the human erythropoietin gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Semenza, G.L.; Traystman, M.D.; Gearhart, J.D.

1989-04-01

Erythropoietin is a glycoprotein hormone that regulates mammalian erythropoiesis. To study the expression of the human erythropoietin gene, EPO, 4 kilobases of DNA encompassing the gene with 0.4 kilobase of 5{prime} flanking sequence and 0.7 kilobase of 3{prime} flanking sequence was microinjected into fertilized mouse eggs. Transgenic mice were generated that are polycythemic, with increased erythrocytic indices in peripheral blood, increased numbers of erythroid precursors in hematopoietic tissue, and increased serum erythropoietin levels. Transgenic homozygotes show a greater degree of polycythemia than do heterozygotes as well as striking extramedullary erythropoiesis. Human erythropoietin RNA was found not only in fetal liver,more » adult liver, and kidney but also in all other transgenic tissues analyzed. Anemia induced increased human erythropoietin RNA levels in liver but not kidney. These transgenic mice represent a unique model of polycythemia due to increased erythropoietin levels.« less

A pipeline for the de novo assembly of the Themira biloba (Sepsidae: Diptera) transcriptome using a multiple k-mer length approach.

PubMed

Melicher, Dacotah; Torson, Alex S; Dworkin, Ian; Bowsher, Julia H

2014-03-12

The Sepsidae family of flies is a model for investigating how sexual selection shapes courtship and sexual dimorphism in a comparative framework. However, like many non-model systems, there are few molecular resources available. Large-scale sequencing and assembly have not been performed in any sepsid, and the lack of a closely related genome makes investigation of gene expression challenging. Our goal was to develop an automated pipeline for de novo transcriptome assembly, and to use that pipeline to assemble and analyze the transcriptome of the sepsid Themira biloba. Our bioinformatics pipeline uses cloud computing services to assemble and analyze the transcriptome with off-site data management, processing, and backup. It uses a multiple k-mer length approach combined with a second meta-assembly to extend transcripts and recover more bases of transcript sequences than standard single k-mer assembly. We used 454 sequencing to generate 1.48 million reads from cDNA generated from embryo, larva, and pupae of T. biloba and assembled a transcriptome consisting of 24,495 contigs. Annotation identified 16,705 transcripts, including those involved in embryogenesis and limb patterning. We assembled transcriptomes from an additional three non-model organisms to demonstrate that our pipeline assembled a higher-quality transcriptome than single k-mer approaches across multiple species. The pipeline we have developed for assembly and analysis increases contig length, recovers unique transcripts, and assembles more base pairs than other methods through the use of a meta-assembly. The T. biloba transcriptome is a critical resource for performing large-scale RNA-Seq investigations of gene expression patterns, and is the first transcriptome sequenced in this Dipteran family.

Transcriptomic Profiling of Extracellular RNAs Present in Cerebrospinal Fluid Identifies Differentially Expressed Transcripts in Parkinson’s Disease

PubMed Central

Hossein-nezhad, Arash; Fatemi, Roya Pedram; Ahmad, Rili; Peskind, Elaine R.; Zabetian, Cyrus P.; Hu, Shu-Ching; Shi, Min; Wahlestedt, Claes; Zhang, Jing; Faghihi, Mohammad Ali

2016-01-01

Background: Parkinson’s disease (PD) is a debilitating neurological disorder for which prognostic and diagnostic biomarkers are lacking. Cerebrospinal fluid (CSF) is an accessible body fluid that comes into direct contact with the central nervous system (CNS) and acts as a nuclease-free repository where RNA transcripts shed by brain tissues can reside for extended periods of time. Objective: We studied the RNA species present in the CSF of PD patients to identify novel diagnostic biomarkers. Methods: Small volumes of CSF from 27 PD patients and 30 healthy age- and sex-matched controls were used for RNA extraction followed by next-generation sequencing (RNA-seq) using the Illumina platform. CSF contains a number of fragmented RNA species that were individually sequenced and analyzed. Comparing PD to control subjects, we observed a pool of dysregulated sequencing tags that were further analyzed and validated by quantitative real-time PCR (qRT-PCR). Results: A total of 201 differentially expressed sequencing tags (DETs), including 92 up-regulated and 109 down-regulated DETs were identified. We validated the following DETs by real time PCR in the patient samples: Dnmt1, Ezh2, CCR3, SSTR5,PTPRC, UBC, NDUFV2, BMP7, SCN9, SCN9 antisense (AC010127.3), and long noncoding RNAs AC079630 and UC001lva.4 (close to the LRRK2 gene locus), as potential PD biomarkers. Conclusions: The CSF is a unique environment that contains many species of RNA. Our work demonstrates that CSF can potentially be used to identify biomarkers for the detection and tracking of disease progression and evaluation of therapeutic outcomes. PMID:26889637

Variant ribosomal RNA alleles are conserved and exhibit tissue-specific expression

PubMed Central

Parks, Matthew M.; Kurylo, Chad M.; Dass, Randall A.; Bojmar, Linda; Lyden, David; Vincent, C. Theresa; Blanchard, Scott C.

2018-01-01

The ribosome, the integration point for protein synthesis in the cell, is conventionally considered a homogeneous molecular assembly that only passively contributes to gene expression. Yet, epigenetic features of the ribosomal DNA (rDNA) operon and changes in the ribosome’s molecular composition have been associated with disease phenotypes, suggesting that the ribosome itself may possess inherent regulatory capacity. Analyzing whole-genome sequencing data from the 1000 Genomes Project and the Mouse Genomes Project, we find that rDNA copy number varies widely across individuals, and we identify pervasive intra- and interindividual nucleotide variation in the 5S, 5.8S, 18S, and 28S ribosomal RNA (rRNA) genes of both human and mouse. Conserved rRNA sequence heterogeneities map to functional centers of the assembled ribosome, variant rRNA alleles exhibit tissue-specific expression, and ribosomes bearing variant rRNA alleles are present in the actively translating ribosome pool. These findings provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human disease. PMID:29503865

Molecular Characterization, Tissue Distribution and Expression, and Potential Antiviral Effects of TRIM32 in the Common Carp (Cyprinus carpio).

PubMed

Wang, Yeda; Li, Zeming; Lu, Yuanan; Hu, Guangfu; Lin, Li; Zeng, Lingbing; Zhou, Yong; Liu, Xueqin

2016-10-09

Tripartite motif-containing protein 32 (TRIM32) belongs to the tripartite motif (TRIM) family, which consists of a large number of proteins containing a RING (Really Interesting New Gene) domain, one or two B-box domains, and coiled coil motif followed by different C-terminal domains. The TRIM family is known to be implicated in multiple cellular functions, including antiviral activity. However, it is presently unknown whether TRIM32 of common carp ( Cyprinus carpio ) has the antiviral effect. In this study, the sequence, expression, and antiviral function of TRIM32 homolog from common carp were analyzed. The full-length coding sequence region of trim32 was cloned from common carp. The results showed that the expression of TRIM32 (mRNA) was highest in the brain, remained stably expressed during embryonic development, and significantly increased following spring viraemia of carp virus (SVCV) infection. Transient overexpression of TRIM32 in affected Epithelioma papulosum cyprinid cells led to significant decrease of SVCV production as compared to the control group. These results suggested a potentially important role of common carp TRIM32 in enhancing host immune response during SVCV infection both in vivo and in vitro.

Molecular Characterization, Tissue Distribution and Expression, and Potential Antiviral Effects of TRIM32 in the Common Carp (Cyprinus carpio)

PubMed Central

Wang, Yeda; Li, Zeming; Lu, Yuanan; Hu, Guangfu; Lin, Li; Zeng, Lingbing; Zhou, Yong; Liu, Xueqin

2016-01-01

Tripartite motif-containing protein 32 (TRIM32) belongs to the tripartite motif (TRIM) family, which consists of a large number of proteins containing a RING (Really Interesting New Gene) domain, one or two B-box domains, and coiled coil motif followed by different C-terminal domains. The TRIM family is known to be implicated in multiple cellular functions, including antiviral activity. However, it is presently unknown whether TRIM32 of common carp (Cyprinus carpio) has the antiviral effect. In this study, the sequence, expression, and antiviral function of TRIM32 homolog from common carp were analyzed. The full-length coding sequence region of trim32 was cloned from common carp. The results showed that the expression of TRIM32 (mRNA) was highest in the brain, remained stably expressed during embryonic development, and significantly increased following spring viraemia of carp virus (SVCV) infection. Transient overexpression of TRIM32 in affected Epithelioma papulosum cyprinid cells led to significant decrease of SVCV production as compared to the control group. These results suggested a potentially important role of common carp TRIM32 in enhancing host immune response during SVCV infection both in vivo and in vitro. PMID:27735853

Capsule null locus meningococci: typing of antigens used in an investigational multicomponent meningococcus serogroup B vaccine.

PubMed

Claus, Heike; Jördens, Markus S; Kriz, Pavla; Musilek, Martin; Jarva, Hanna; Pawlik, Marie-Christin; Meri, Seppo; Vogel, Ulrich

2012-01-05

The investigational multicomponent meningococcus serogroup B vaccine (4CMenB) targets the antigenetically variable population of serogroup B meningococci. Forty-one strains of capsule null locus (cnl) meningococci, which are frequent among healthy carriers, were selected from nine sequence types (ST), which belong to four clonal complexes (cc), and three countries. They were antigen sequence typed and analyzed for antigen expression to predict whether these strains harbor the genes and express the four vaccine antigens of 4CMenB as measured by the meningococcal antigen typing system (MATS). The PorA variant used in the vaccine was not found. The nadA gene was absent in all but one strain, which did not express the antigen in vitro. Only strains of clonal complex ST-198 harbored a factor H binding protein (FHBP) allele of the cross-reactive variant 1 family which is included in the vaccine. All these strains expressed the antigen. Five variants of the Neisserial heparin binding antigen (NHBA) gene were identified. Expression of NHBA was observed in all strains with highest levels in ST-198 cc and ST-845. The data suggest a potential impact of 4CMenB immunization at least on cnl meningococci of the ST-198 cc and ST-845. Copyright © 2011 Elsevier Ltd. All rights reserved.

Preferential susceptibility of Th9 and Th2 CD4+ T cells to X4-tropic HIV-1 infection.

PubMed

Orlova-Fink, Nina; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

2017-10-23

The functional polarization of CD4 T cells determines their antimicrobial effector profile, but may also impact the susceptibility to infection with HIV-1. Here, we analyzed the susceptibility of CD4 T cells with different functional polarization to infection with X4 and R5-tropic HIV-1. CD4 T cells with a Th1, Th2, Th17, and Th9 polarization were subjected to in-vitro infection assays with X4, R5, or vesicular stomatitis virus-G protein-pseudotyped HIV-1. In addition, we sorted differentially polarized CD4 T-cell subsets from individuals treated with antiretroviral therapy and analyzed the tropism of viral env sequences. Th9-polarized CD4 T cells and, to a lesser extent, Th2-polarized CD4 T cells expressed higher surface levels of CXCR4, and are more permissive to X4-tropic infection in vitro. In contrast, Th1 and Th17 CD4 T cells exhibited stronger surface expression of CCR5, and were more susceptible to infection with R5-tropic viruses. Correspondingly, the distribution of X4-tropic viral sequences in antiretroviral therapy-treated HIV-1-infected patients was biased toward Th9/Th2 cells, whereas R5-tropic sequences were more frequently observed in Th17 cells. CD4 T-cell polarization is associated with a distinct susceptibility to X4 and R5-tropic HIV-1 infection.

Faster-X Evolution of Gene Expression in Drosophila

PubMed Central

Meisel, Richard P.; Malone, John H.; Clark, Andrew G.

2012-01-01

DNA sequences on X chromosomes often have a faster rate of evolution when compared to similar loci on the autosomes, and well articulated models provide reasons why the X-linked mode of inheritance may be responsible for the faster evolution of X-linked genes. We analyzed microarray and RNA–seq data collected from females and males of six Drosophila species and found that the expression levels of X-linked genes also diverge faster than autosomal gene expression, similar to the “faster-X” effect often observed in DNA sequence evolution. Faster-X evolution of gene expression was recently described in mammals, but it was limited to the evolutionary lineages shortly following the creation of the therian X chromosome. In contrast, we detect a faster-X effect along both deep lineages and those on the tips of the Drosophila phylogeny. In Drosophila males, the dosage compensation complex (DCC) binds the X chromosome, creating a unique chromatin environment that promotes the hyper-expression of X-linked genes. We find that DCC binding, chromatin environment, and breadth of expression are all predictive of the rate of gene expression evolution. In addition, estimates of the intraspecific genetic polymorphism underlying gene expression variation suggest that X-linked expression levels are not under relaxed selective constraints. We therefore hypothesize that the faster-X evolution of gene expression is the result of the adaptive fixation of beneficial mutations at X-linked loci that change expression level in cis. This adaptive faster-X evolution of gene expression is limited to genes that are narrowly expressed in a single tissue, suggesting that relaxed pleiotropic constraints permit a faster response to selection. Finally, we present a conceptional framework to explain faster-X expression evolution, and we use this framework to examine differences in the faster-X effect between Drosophila and mammals. PMID:23071459

eRNA: a graphic user interface-based tool optimized for large data analysis from high-throughput RNA sequencing

PubMed Central

2014-01-01

Background RNA sequencing (RNA-seq) is emerging as a critical approach in biological research. However, its high-throughput advantage is significantly limited by the capacity of bioinformatics tools. The research community urgently needs user-friendly tools to efficiently analyze the complicated data generated by high throughput sequencers. Results We developed a standalone tool with graphic user interface (GUI)-based analytic modules, known as eRNA. The capacity of performing parallel processing and sample management facilitates large data analyses by maximizing hardware usage and freeing users from tediously handling sequencing data. The module miRNA identification” includes GUIs for raw data reading, adapter removal, sequence alignment, and read counting. The module “mRNA identification” includes GUIs for reference sequences, genome mapping, transcript assembling, and differential expression. The module “Target screening” provides expression profiling analyses and graphic visualization. The module “Self-testing” offers the directory setups, sample management, and a check for third-party package dependency. Integration of other GUIs including Bowtie, miRDeep2, and miRspring extend the program’s functionality. Conclusions eRNA focuses on the common tools required for the mapping and quantification analysis of miRNA-seq and mRNA-seq data. The software package provides an additional choice for scientists who require a user-friendly computing environment and high-throughput capacity for large data analysis. eRNA is available for free download at https://sourceforge.net/projects/erna/?source=directory. PMID:24593312

eRNA: a graphic user interface-based tool optimized for large data analysis from high-throughput RNA sequencing.

PubMed

Yuan, Tiezheng; Huang, Xiaoyi; Dittmar, Rachel L; Du, Meijun; Kohli, Manish; Boardman, Lisa; Thibodeau, Stephen N; Wang, Liang

2014-03-05

RNA sequencing (RNA-seq) is emerging as a critical approach in biological research. However, its high-throughput advantage is significantly limited by the capacity of bioinformatics tools. The research community urgently needs user-friendly tools to efficiently analyze the complicated data generated by high throughput sequencers. We developed a standalone tool with graphic user interface (GUI)-based analytic modules, known as eRNA. The capacity of performing parallel processing and sample management facilitates large data analyses by maximizing hardware usage and freeing users from tediously handling sequencing data. The module miRNA identification" includes GUIs for raw data reading, adapter removal, sequence alignment, and read counting. The module "mRNA identification" includes GUIs for reference sequences, genome mapping, transcript assembling, and differential expression. The module "Target screening" provides expression profiling analyses and graphic visualization. The module "Self-testing" offers the directory setups, sample management, and a check for third-party package dependency. Integration of other GUIs including Bowtie, miRDeep2, and miRspring extend the program's functionality. eRNA focuses on the common tools required for the mapping and quantification analysis of miRNA-seq and mRNA-seq data. The software package provides an additional choice for scientists who require a user-friendly computing environment and high-throughput capacity for large data analysis. eRNA is available for free download at https://sourceforge.net/projects/erna/?source=directory.

[Studies on antigencity of human immunodeficiency virus type 1 (HIV-1) external glycoprotein as well as its expression in Pichia pastoris].

PubMed

Zhao, Li-Hui; Yu, Xiang-Hui; Jiang, Chun-Lai; Wu, Yong-Ge; Shen, Jia-Cong; Kong, Wei

2007-05-01

Based on the computer simulation, we analyzed hydrophobicity, potential epitope of recombined subtypes HIV-1 Env protein (851 amino acids) from Guangxi in China. Compared with conservative peptides of other subtypes in env protein, three sequences (469-511aa, 538-674aa, 700-734aa) were selected to recombine into a chimeric gene that codes three conservative epitope peptides with stronger antigencity, and was constructed in the yeast expression plasmid pPICZB. Chimeric proteins were expressed in Pichia pastoris under the induction of methanol, and were analyzed by SDS-PAGE and Westernblot. The results showed that fusion proteins of three-segment antigen were expressed in Pichia pastoris and that specific protein band at the site of 40kD was target protein, which is interacted with HIV-1 serum. The target proteins were purified by metal Ni-sepharose 4B, and were demonstrated to possess good antigenic specificity from the data of ELISA. This chimeric antigen may be used as research and developed into HIV diagnostic reagents.

Effects of Gene Duplication, Positive Selection, and Shifts in Gene Expression on the Evolution of the Venom Gland Transcriptome in Widow Spiders

PubMed Central

Haney, Robert A.; Clarke, Thomas H.; Gadgil, Rujuta; Fitzpatrick, Ryan; Hayashi, Cheryl Y.; Ayoub, Nadia A.; Garb, Jessica E.

2016-01-01

Gene duplication and positive selection can be important determinants of the evolution of venom, a protein-rich secretion used in prey capture and defense. In a typical model of venom evolution, gene duplicates switch to venom gland expression and change function under the action of positive selection, which together with further duplication produces large gene families encoding diverse toxins. Although these processes have been demonstrated for individual toxin families, high-throughput multitissue sequencing of closely related venomous species can provide insights into evolutionary dynamics at the scale of the entire venom gland transcriptome. By assembling and analyzing multitissue transcriptomes from the Western black widow spider and two closely related species with distinct venom toxicity phenotypes, we do not find that gene duplication and duplicate retention is greater in gene families with venom gland biased expression in comparison with broadly expressed families. Positive selection has acted on some venom toxin families, but does not appear to be in excess for families with venom gland biased expression. Moreover, we find 309 distinct gene families that have single transcripts with venom gland biased expression, suggesting that the switching of genes to venom gland expression in numerous unrelated gene families has been a dominant mode of evolution. We also find ample variation in protein sequences of venom gland–specific transcripts, lineage-specific family sizes, and ortholog expression among species. This variation might contribute to the variable venom toxicity of these species. PMID:26733576

Comparative antennal transcriptome of Apis cerana cerana from four developmental stages.

PubMed

Zhao, Huiting; Peng, Zhu; Du, Yali; Xu, Kai; Guo, Lina; Yang, Shuang; Ma, Weihua; Jiang, Yusuo

2018-06-20

Apis cerana cerana, an important endemic honey bee species in China, possesses valuable characteristics such as a sensitive olfactory system, good foraging ability, and strong resistance to parasitic mites. Here, we performed transcriptome sequencing of the antenna, the major chemosensory organ of the bee, using an Illumina sequencer, to identify typical differentially expressed genes (DEGs) in adult worker bees of different ages, namely, T1 (1 day); T2 (10 days); T3 (15 days); and T4 (25 days). Surprisingly, the expression levels of DEGs changed significantly between the T1 period and the other three periods. All the DEGs were classified into 26 expression profiles by trend analysis. Selected trend clusters were analyzed, and valuable information on gene expression patterns was obtained. We found that the expression levels of genes encoding cuticle proteins declined after eclosion, while those of immunity-related genes increased. In addition, genes encoding venom proteins and major royal jelly proteins were enriched at the T2 stage; small heat shock proteins showed significantly higher expression at the T3 stage; and some metabolism-related genes were more highly expressed at the T4 stage. The DEGs identified in this study may serve as a valuable resource for the characterization of expression patterns of antennal genes in A. cerana cerana. Furthermore, this study provides insights into the relationship between labor division in social bees and gene function. Copyright © 2018. Published by Elsevier B.V.

Bayesian Inference of Allele-Specific Gene Expression Indicates Abundant Cis-Regulatory Variation in Natural Flycatcher Populations

PubMed Central

Wang, Mi

2017-01-01

Abstract Polymorphism in cis-regulatory sequences can lead to different levels of expression for the two alleles of a gene, providing a starting point for the evolution of gene expression. Little is known about the genome-wide abundance of genetic variation in gene regulation in natural populations but analysis of allele-specific expression (ASE) provides a means for investigating such variation. We performed RNA-seq of multiple tissues from population samples of two closely related flycatcher species and developed a Bayesian algorithm that maximizes data usage by borrowing information from the whole data set and combines several SNPs per transcript to detect ASE. Of 2,576 transcripts analyzed in collared flycatcher, ASE was detected in 185 (7.2%) and a similar frequency was seen in the pied flycatcher. Transcripts with statistically significant ASE commonly showed the major allele in >90% of the reads, reflecting that power was highest when expression was heavily biased toward one of the alleles. This would suggest that the observed frequencies of ASE likely are underestimates. The proportion of ASE transcripts varied among tissues, being lowest in testis and highest in muscle. Individuals often showed ASE of particular transcripts in more than one tissue (73.4%), consistent with a genetic basis for regulation of gene expression. The results suggest that genetic variation in regulatory sequences commonly affects gene expression in natural populations and that it provides a seedbed for phenotypic evolution via divergence in gene expression. PMID:28453623

Flp and Cre expressed from Flp–2A–Cre and Flp–IRES–Cre transcription units mediate the highest level of dual recombinase-mediated cassette exchange

PubMed Central

Anderson, Rachelle P.; Voziyanova, Eugenia; Voziyanov, Yuri

2012-01-01

Recombinase-mediated cassette exchange (RMCE) is a powerful tool for unidirectional integration of DNA fragments of interest into a pre-determined genome locale. In this report, we examined how the efficiency of dual RMCE catalyzed by Flp and Cre depends on the nature of transcription units that express the recombinases. The following recombinase transcription units were analyzed: (i) Flp and Cre genes expressed as individual transcription units located on different vectors, (ii) Flp and Cre genes expressed as individual transcription units located on the same vector, (iii) Flp and Cre genes expressed from a single promoter and separated by internal ribosome entry sequence and (iv) Flp and Cre coding sequences separated by the 2A peptide and expressed as a single gene. We found that the highest level of dual RMCE (35–45% of the transfected cells) can be achieved when Flp and Cre recombinases are expressed as Flp–2A–Cre and Flp–IRES–Cre transcription units. In contrast, the lowest level of dual RMCE (∼1% of the transfected cells) is achieved when Flp and Cre are expressed as individual transcription units. The analysis shows that it is the relative Flp–to–Cre ratio that critically affects the efficiency of dual RMCE. Our results will be helpful for maximizing the efficiency of dual RMCE aimed to engineer and re-engineer genomes. PMID:22270085

Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

To explore the expression profile of the human lens and to provide a resource for microarray studies, expressed sequence tag (EST) analysis has been performed on cDNA libraries from adult lenses. A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering. The lens library (by) contains a low percentage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described. The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases.

High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2.

PubMed

Abécassis, V; Pompon, D; Truan, G

2000-10-15

The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 +/- 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure-function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.

Mating-type genes from the homothallic fungus Sordaria macrospora are functionally expressed in a heterothallic ascomycete.

PubMed

Pöggeler, S; Risch, S; Kück, U; Osiewacz, H D

1997-10-01

Homokaryons from the homothallic ascomycte Sordaria macrospora are able to enter the sexual pathway and to form fertile fruiting bodies. To analyze the molecular basis of homothallism and to elucidate the role of mating-products during fruiting body development, we cloned and sequenced the entire S. macrospora mating-type locus. Comparison of the Sordaria mating-type locus with mating-type idiomorphs from the heterothallic ascomycetes Neurospora crassa and Podospora anserina revealed that sequences from both idiomorphs (A/a and mat-/mat+, respectively) are contiguous in S. macrospora. DNA sequencing of the S. macrospora mating-type region allowed the identification of four open reading frames (ORFs), which were termed Smt-a1, SmtA-1, SmtA-2 and SmtA-3. While Smt-a1, SmtA-1, and SmtA-2 show strong sequence similarities with the corresponding N. crassa mating-type ORFs, SmtA-3 has a chimeric character. It comprises sequences that are similar to the A and a mating-type idiomorph from N. crassa. To determine functionality of the S. macrospora mating-type genes, we show that all ORFs are transcriptionally expressed. Furthermore, we transformed the S. macrospora mating-type genes into mat- and mat+ strains of the closely related heterothallic fungus P. anserina. The transformation experiments show that mating-type genes from S. macrospora induce fruiting body formation in P. anserina.

Enhanced expression of EGFP gene in CHSE-214 cells by an ARS element from mud loach (Misgurnus mizolepis).

PubMed

Kim, Moo-Sang; Lim, Hak-Seob; Ahn, Sang Jung; Jeong, Yong-Kee; Kim, Chul Geun; Lee, Hyung Ho

2007-11-01

The origins of replication are associated with nuclear matrices or are found in close proximity to matrix attachment regions (MARs). In this report, fish MARs were cloned into an autonomously replicating sequence (ARS) cloning vector and were screened for ARS elements in Saccharomyces cerevisiae. Sixteen clones were isolated that were able to grow on the selective plates. In particular, an ARS905 that shows high efficiency among them was selected for this study. Southern hybridization indicated the autonomous replication of the transformation vector containing the ARS905 element. DNA sequences analysis showed that the ARS905 contained two ARS consensus sequences as well as MAR motifs, such as AT tracts, ORI patterns, and ATC tracts. In vitro matrix binding analysis, major matrix binding activity and ARS function coincided in a subfragment of the ARS905. To analyze the effects of ARS905 on expression of a reporter gene, an ARS905(E1158) with ARS activity was inserted into pBaEGFP(+) containing mud loach beta-actin promoter, EGFP as a reporter gene, and SV40 poly(A) signal. The pBaEGFP(+)-ARS905(E1158) was transfected into a fish cell line, CHSE-214. The intensity of EGFP transfected cells was a 7-fold of the control at 11days post-transfection. These results indicate that ARS905 enhances the expression of the EGFP gene and that it should be as a component of expression vectors in further fish biotechnological studies.

Conservation of regulatory sequences and gene expression patterns in the disintegrating Drosophila Hox gene complex

PubMed Central

Negre, Bárbara; Casillas, Sònia; Suzanne, Magali; Sánchez-Herrero, Ernesto; Akam, Michael; Nefedov, Michael; Barbadilla, Antonio; de Jong, Pieter; Ruiz, Alfredo

2005-01-01

Homeotic (Hox) genes are usually clustered and arranged in the same order as they are expressed along the anteroposterior body axis of metazoans. The mechanistic explanation for this colinearity has been elusive, and it may well be that a single and universal cause does not exist. The Hox-gene complex (HOM-C) has been rearranged differently in several Drosophila species, producing a striking diversity of Hox gene organizations. We investigated the genomic and functional consequences of the two HOM-C splits present in Drosophila buzzatii. Firstly, we sequenced two regions of the D. buzzatii genome, one containing the genes labial and abdominal A, and another one including proboscipedia, and compared their organization with that of D. melanogaster and D. pseudoobscura in order to map precisely the two splits. Then, a plethora of conserved noncoding sequences, which are putative enhancers, were identified around the three Hox genes closer to the splits. The position and order of these enhancers are conserved, with minor exceptions, between the three Drosophila species. Finally, we analyzed the expression patterns of the same three genes in embryos and imaginal discs of four Drosophila species with different Hox-gene organizations. The results show that their expression patterns are conserved despite the HOM-C splits. We conclude that, in Drosophila, Hox-gene clustering is not an absolute requirement for proper function. Rather, the organization of Hox genes is modular, and their clustering seems the result of phylogenetic inertia more than functional necessity. PMID:15867430

Identification of mediator complex 26 (Crsp7) gametologs on platypus X1 and Y5 sex chromosomes: a candidate testis-determining gene in monotremes?

PubMed

Tsend-Ayush, Enkhjargal; Kortschak, R Daniel; Bernard, Pascal; Lim, Shu Ly; Ryan, Janelle; Rosenkranz, Ruben; Borodina, Tatiana; Dohm, Juliane C; Himmelbauer, Heinz; Harley, Vincent R; Grützner, Frank

2012-01-01

The basal lineage of monotremes features an extraordinarily complex sex chromosome system which has provided novel insights into the evolution of mammalian sex chromosomes. Recently, sequence information from autosomes, X chromosomes, and XY-shared pseudoautosomal regions has become available. However, no gene has so far been described on any of the Y chromosome-specific regions. We analyzed sequences derived from Y-specific BAC clones to identify genes with potentially male-specific function. Here, we report the identification and characterization of the mediator complex protein gametologs on platypus Y5 (Crspy). We also identified the X-chromosomal copy which unexpectedly maps to X1 (Crspx). Sequence comparison shows extensive divergence between the X and Y copy, but we found no significant positive selection on either gametolog. Expression analysis shows widespread expression of Crspx. Crspy is expressed exclusively in males with particularly strong expression in testis and kidney. Reporter gene assays to investigate whether Crspx/y can act on the recently discovered mouse Sox9 testis-specific enhancer element did reveal a modest effect together with mouse Sox9 + Sf1, but showed overall no significant upregulation of the reporter gene. This is the first report of a differentiated functional male-specific gene on platypus Y chromosomes, providing new insights into sex chromosome evolution and a candidate gene for male-specific function in monotremes.

Gene duplication, silencing and expression alteration govern the molecular evolution of PRC2 genes in plants.

PubMed

Furihata, Hazuka Y; Suenaga, Kazuya; Kawanabe, Takahiro; Yoshida, Takanori; Kawabe, Akira

2016-10-13

PRC2 genes were analyzed for their number of gene duplications, d N /d S ratios and expression patterns among Brassicaceae and Gramineae species. Although both amino acid sequences and copy number of the PRC2 genes were generally well conserved in both Brassicaceae and Gramineae species, we observed that some rapidly evolving genes experienced duplications and expression pattern changes. After multiple duplication events, all but one or two of the duplicated copies tend to be silenced. Silenced copies were reactivated in the endosperm and showed ectopic expression in developing seeds. The results indicated that rapid evolution of some PRC2 genes is initially caused by a relaxation of selective constraint following the gene duplication events. Several loci could become maternally expressed imprinted genes and acquired functional roles in the endosperm.

Differential expression of catalase genes in Nicotiana plumbaginifolia (L.).

PubMed Central

Willekens, H; Langebartels, C; Tiré, C; Van Montagu, M; Inzé, D; Van Camp, W

1994-01-01

We have analyzed the expression of three catalase (Cat; EC 1.11.1.6) genes from Nicotiana plumbaginifolia by means of RNA blot and in situ hybridizations. Our data demonstrate that the expression of each catalase is associated with a particular H2O2-producing process. Cat1 appears to be specifically involved in the scavenging of photorespiratory H2O2 and is under control of a circadian rhythm, Cat2 is uniformly expressed in different organs with a cellular preference for vascular tissues, and the expression profile of Cat3 points to a role in glyoxysomal processes. Differential expression of these catalases is also manifested in response to temperature changes. DNA sequence comparison with other dicotyledonous catalases led to the identification of at least three distinct classes, which indicates that the functional organization of catalases is generally conserved in dicotyledonous plants. Images PMID:7937973

Differential expression of catalase genes in Nicotiana plumbaginifolia (L.).

PubMed

Willekens, H; Langebartels, C; Tiré, C; Van Montagu, M; Inzé, D; Van Camp, W

1994-10-25

We have analyzed the expression of three catalase (Cat; EC 1.11.1.6) genes from Nicotiana plumbaginifolia by means of RNA blot and in situ hybridizations. Our data demonstrate that the expression of each catalase is associated with a particular H2O2-producing process. Cat1 appears to be specifically involved in the scavenging of photorespiratory H2O2 and is under control of a circadian rhythm, Cat2 is uniformly expressed in different organs with a cellular preference for vascular tissues, and the expression profile of Cat3 points to a role in glyoxysomal processes. Differential expression of these catalases is also manifested in response to temperature changes. DNA sequence comparison with other dicotyledonous catalases led to the identification of at least three distinct classes, which indicates that the functional organization of catalases is generally conserved in dicotyledonous plants.

Young infants' generalization of emotional expressions: effects of familiarity.

PubMed

Walker-Andrews, Arlene S; Krogh-Jespersen, Sheila; Mayhew, Estelle M Y; Coffield, Caroline N

2011-08-01

From birth, infants are exposed to a wealth of emotional information in their interactions. Much research has been done to investigate the development of emotion perception, and factors influencing that development. The current study investigates the role of familiarity on 3.5-month-old infants' generalization of emotional expressions. Infants were assigned to one of two habituation sequences: in one sequence, infants were visually habituated to parental expressions of happy or sad. At test, infants viewed either a continuation of the habituation sequence, their mother depicting a novel expression, an unfamiliar female depicting the habituated expression, or an unfamiliar female depicting a novel expression. In the second sequence, a new sample of infants was matched to the infants in the first sequence. These infants viewed the same habituation and test sequences, but the actors were unfamiliar to them. Only those infants who viewed their own mothers and fathers during the habituation sequence increased looking. They dishabituated looking to maternal novel expressions, the unfamiliar female's novel expression, and the unfamiliar female depicting the habituated expression, especially when sad parental expressions were followed by an expression change to happy or to a change in person. Infants are guided in their recognition of emotional expressions by the familiarity of their parents, before generalizing to others. 2011 APA, all rights reserved

Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors*

PubMed Central

WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei

2013-01-01

Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237

De novo sequencing and analysis of the transcriptome during the browning of fresh-cut Luffa cylindrica 'Fusi-3' fruits.

PubMed

Zhu, Haisheng; Liu, Jianting; Wen, Qingfang; Chen, Mindong; Wang, Bin; Zhang, Qianrong; Xue, Zhuzheng

2017-01-01

Fresh-cut luffa (Luffa cylindrica) fruits commonly undergo browning. However, little is known about the molecular mechanisms regulating this process. We used the RNA-seq technique to analyze the transcriptomic changes occurring during the browning of fresh-cut fruits from luffa cultivar 'Fusi-3'. Over 90 million high-quality reads were assembled into 58,073 Unigenes, and 60.86% of these were annotated based on sequences in four public databases. We detected 35,282 Unigenes with significant hits to sequences in the NCBInr database, and 24,427 Unigenes encoded proteins with sequences that were similar to those of known proteins in the Swiss-Prot database. Additionally, 20,546 and 13,021 Unigenes were similar to existing sequences in the Eukaryotic Orthologous Groups of proteins and Kyoto Encyclopedia of Genes and Genomes databases, respectively. Furthermore, 27,301 Unigenes were differentially expressed during the browning of fresh-cut luffa fruits (i.e., after 1-6 h). Moreover, 11 genes from five gene families (i.e., PPO, PAL, POD, CAT, and SOD) identified as potentially associated with enzymatic browning as well as four WRKY transcription factors were observed to be differentially regulated in fresh-cut luffa fruits. With the assistance of rapid amplification of cDNA ends technology, we obtained the full-length sequences of the 15 Unigenes. We also confirmed these Unigenes were expressed by quantitative real-time polymerase chain reaction analysis. This study provides a comprehensive transcriptome sequence resource, and may facilitate further studies aimed at identifying genes affecting luffa fruit browning for the exploitation of the underlying mechanism.

In vitro resistance to 5-nitroimidazoles and benzimidazoles in Giardia duodenalis: variability and variation in gene expression.

PubMed

Argüello-García, Raúl; Cruz-Soto, Maricela; Romero-Montoya, Lydia; Ortega-Pierres, Guadalupe

2009-12-01

The susceptibility of Giardia duodenalis trophozoites exposed in vitro to sublethal concentrations of metronidazole (MTZ) and albendazole (ABZ) may exhibit inter-culture (variability) and intra-culture (variation) differences in drug susceptibility. It was previously reported that MTZ-resistant trophozoites may display changes in pyruvate:ferredoxin oxidoreductase (PFOR) expression while changes at the beta-tubulin molecule are apparently absent in ABZ-resistant cultures. To assess the levels of gene expression of these molecules, we obtained cloned cultures growing at concentrations up to 23 microM MTZ (WBRM23) and up to 8muM ABZ (WBRA8) and gene sequence and expression of pfor and beta-tubulin loci were compared with these of drug-susceptible clone WB1. Neither the pfor nor the beta-tubulin genes showed changes at sequence level but the MTZ-resistant clones WBRM21 and WBRM23 showed up-regulation of the pfor RNA using the gdh gene as reference. By using WB1 and WBRA8 clones in representational difference analyses of gene expression (RDA) an insert referred to as ARR-VSP was selected and sequenced. It showed the highest homology to one VSP molecule in the Giardia Genome Database (orf GL50803_101765). This isogene was up-regulated in five ABZ-resistant clones and the clone WBRA8 exhibited the highest RNA expression level. When successive progenies of clones WB1, WBRM23 and WBRA8 were analyzed in Northern blot assays to detect pfor and ARR-VSP RNAs respectively, the expression patterns showed variation for both genes but it was much lower in the clone WBRA8. These results suggest that G. duodenalis cultures either susceptible or resistant to MTZ and ABZ may display variability and variation at RNA expression levels albeit these were more marked in the MTZ-resistant parasites. These data might have further implications defining major mechanisms involved in drug resistance of Giardia.

Systematic elucidation and in vivo validation of sequences enriched in hindbrain transcriptional control

PubMed Central

Burzynski, Grzegorz M.; Reed, Xylena; Taher, Leila; Stine, Zachary E.; Matsui, Takeshi; Ovcharenko, Ivan; McCallion, Andrew S.

2012-01-01

Illuminating the primary sequence encryption of enhancers is central to understanding the regulatory architecture of genomes. We have developed a machine learning approach to decipher motif patterns of hindbrain enhancers and identify 40,000 sequences in the human genome that we predict display regulatory control that includes the hindbrain. Consistent with their roles in hindbrain patterning, MEIS1, NKX6-1, as well as HOX and POU family binding motifs contributed strongly to this enhancer model. Predicted hindbrain enhancers are overrepresented at genes expressed in hindbrain and associated with nervous system development, and primarily reside in the areas of open chromatin. In addition, 77 (0.2%) of these predictions are identified as hindbrain enhancers on the VISTA Enhancer Browser, and 26,000 (60%) overlap enhancer marks (H3K4me1 or H3K27ac). To validate these putative hindbrain enhancers, we selected 55 elements distributed throughout our predictions and six low scoring controls for evaluation in a zebrafish transgenic assay. When assayed in mosaic transgenic embryos, 51/55 elements directed expression in the central nervous system. Furthermore, 30/34 (88%) predicted enhancers analyzed in stable zebrafish transgenic lines directed expression in the larval zebrafish hindbrain. Subsequent analysis of sequence fragments selected based upon motif clustering further confirmed the critical role of the motifs contributing to the classifier. Our results demonstrate the existence of a primary sequence code characteristic to hindbrain enhancers. This code can be accurately extracted using machine-learning approaches and applied successfully for de novo identification of hindbrain enhancers. This study represents a critical step toward the dissection of regulatory control in specific neuronal subtypes. PMID:22759862

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library.

PubMed

Yu, Bing; Ni, Ming; Li, Wen-Han; Lei, Ping; Xing, Wei; Xiao, Dai-Wen; Huang, Yu; Tang, Zhen-Jie; Zhu, Hui-Fen; Shen, Guan-Xin

2005-07-14

To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M(r) value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.

Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas

PubMed Central

Hou, Yu; Guo, Huahu; Cao, Chen; Li, Xianlong; Hu, Boqiang; Zhu, Ping; Wu, Xinglong; Wen, Lu; Tang, Fuchou; Huang, Yanyi; Peng, Jirun

2016-01-01

Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells. PMID:26902283

Combined hairpin-antisense compositions and methods for modulating expression

DOEpatents

Shanklin, John; Nguyen, Tam

2014-08-05

A nucleotide construct comprising a nucleotide sequence that forms a stem and a loop, wherein the loop comprises a nucleotide sequence that modulates expression of a target, wherein the stem comprises a nucleotide sequence that modulates expression of a target, and wherein the target modulated by the nucleotide sequence in the loop and the target modulated by the nucleotide sequence in the stem may be the same or different. Vectors, methods of regulating target expression, methods of providing a cell, and methods of treating conditions comprising the nucleotide sequence are also disclosed.

Combined hairpin-antisense compositions and methods for modulating expression

DOEpatents

Shanklin, John; Nguyen, Tam Huu

2015-11-24

A nucleotide construct comprising a nucleotide sequence that forms a stem and a loop, wherein the loop comprises a nucleotide sequence that modulates expression of a target, wherein the stem comprises a nucleotide sequence that modulates expression of a target, and wherein the target modulated by the nucleotide sequence in the loop and the target modulated by the nucleotide sequence in the stem may be the same or different. Vectors, methods of regulating target expression, methods of providing a cell, and methods of treating conditions comprising the nucleotide sequence are also disclosed.

Isolation, structural analysis, and expression characteristics of the maize (Zea mays L.) hexokinase gene family.

PubMed

Zhang, Zhongbao; Zhang, Jiewei; Chen, Yajuan; Li, Ruifen; Wang, Hongzhi; Ding, Liping; Wei, Jianhua

2014-09-01

Hexokinases (HXKs, EC 2.7.1.1) play important roles in metabolism, glucose (Glc) signaling, and phosphorylation of Glc and fructose and are ubiquitous in all organisms. Despite their physiological importance, the maize HXK (ZmHXK) genes have not been analyzed systematically. We isolated and characterized nine members of the ZmHXK gene family which were distributed on 3 of the 10 maize chromosomes. A multiple sequence alignment and motif analysis revealed that the maize ZmHXK proteins share three conserved domains. Phylogenetic analysis revealed that the ZmHXK family can be divided into four subfamilies. We identified putative cis-elements in the ZmHXK promoter sequences potentially involved in phytohormone and abiotic stress responses, sugar repression, light and circadian rhythm regulation, Ca(2+) responses, seed development and germination, and CO2-responsive transcriptional activation. To study the functions of maize HXK isoforms, we characterized the expression of the ZmHXK5 and ZmHXK6 genes, which are evolutionarily related to the OsHXK5 and OsHXK6 genes from rice. Analysis of tissue-specific expression patterns using quantitative real time-PCR showed that ZmHXK5 was highly expressed in tassels, while ZmHXK6 was expressed in both tassels and leaves. ZmHXK5 and ZmHXK6 expression levels were upregulated by phytohormones and by abiotic stress.

A Brassica oleracea gene expressed in a variety-specific manner may encode a novel plant transmembrane receptor.

PubMed

Palmer, J E; Dikeman, D A; Fujinuma, T; Kim, B; Jones, J I; Denda, M; Martínez-Zapater, J M; Cruz-Alvarez, M

2001-04-01

The species Brassica oleracea includes several agricultural varieties characterized by the proliferation of different types of meristems. Using a combination of subtractive hybridization and PCR (polymerase chain reaction) techniques we have identified several genes which are expressed in the reproductive meristems of the cauliflower curd (B. oleracea var. botrytis) but not in the vegetative meristems of Brussels sprouts (B. oleracea var. gemmifera) axillary buds. One of the cloned genes, termed CCE1 (CAULIFLOWER CURD EXPRESSION 1) shows specific expression in the botrytis variety. Preferential expression takes place in this variety in the meristems of the curd and in the stem throughout the vegetative and reproductive stages of plant growth. CCE1 transcripts are not detected in any of the organs of other B. oleracea varieties analyzed. Based on the nucleotide sequence of a cDNA encompassing the complete coding region, we predict that this gene encodes a transmembrane protein, with three transmembrane domains. The deduced amino acid sequence includes motifs conserved in G-protein-coupled receptors (GPCRs) from yeast and animal species. Our results suggest that the cloned gene encodes a protein belonging to a new, so far unidentified, family of transmembrane receptors in plants. The expression pattern of the gene suggests that the receptor may be involved in the control of meristem development/arrest that takes place in cauliflower.

Molecular cloning and expression profile of an ATP-binding cassette (ABC) transporter gene from the hemipteran insect Nilaparvata lugens.

PubMed

Zha, W J; Li, S H; Zhou, L; Chen, Z J; Liu, K; Yang, G C; Hu, G; He, G C; You, A Q

2015-03-30

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. In insects, ABC transporters have important functions in the transport of molecules, and are also involved in insecticide resistance, metabolism, and development. In this study, the Nilaparvata lugens Stal (Hemiptera: Delphacidae) ABCG (NlABCG) gene was identified and characterized. The complete mRNA sequence of NlABCG was 2608-bp long, with an open reading frame of 2064 bp encoding a protein comprised of 687 amino acids. The conserved regions include three N-glycosylation and 34 phosphorylation sites, as well as seven transmembrane domains. The amino acid identity with the closely related species Acyrthosiphon pisum was 42.8%. Developmental expression analysis using quantitative real-time reverse transcriptase PCR suggested that the NlABCG transcript was expressed at all developmental stages of N. lugens. The lowest expression of NlABCG was in the 1st instar, and levels increased with larval growth. The transcript profiles of NlABCG were analyzed in various tissues from a 5th instar nymph, and the highest expression was observed in the midgut. These results suggest that the sequence, characteristics, and expression of NlABCG are highly conserved, and basic information is provided for its functional analysis.

Molecular mechanisms of pathogenesis in hepatocellular carcinoma revealed by RNA‑sequencing.

PubMed

Liu, Yao; Yang, Zhe; Du, Feng; Yang, Qiao; Hou, Jie; Yan, Xiaohong; Geng, Yi; Zhao, Yaning; Wang, Hua

2017-11-01

The present study aimed to explore the underlying molecular mechanisms of hepatocellular carcinoma (HCC). RNA‑sequencing profiles GSM629264 and GSM629265, from the GSE25599 data set, were downloaded from the Gene Expression Omnibus database and processed by quality evaluation. GSM629264 and GSM629265 were from HCC and adjacent non‑cancerous tissues, respectively. TopHat software was used for alignment analysis, followed by the detection of novel splicing sites. In addition, the Cufflinks software package was used to analyze gene expressions, and the Cuffdiff program was used to screen for differently expressed genes (DEGs) and differentially expressed splicing variants. Gene ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of DEGs were also performed. Transcription factors (TFs) and microRNAs (miRNAs) that regulate DEGs were identified, and a protein‑protein interaction (PPI) network was constructed. The hub node in the PPI network was obtained, and the TFs and miRNAs that regulated the hub node were further predicted. The quality of the sequencing data met the standards for analysis, and the clean reads were ~65%. Most sequencing reads mapped into coding sequence exons (CDS_exons), whereas other reads mapped into exon 3' untranslated regions (UTR_Exons), 5'UTR_Exons and Introns. Upregulated and downregulated DEGs between HCC and adjacent non‑cancerous tissues were screened. Genes of differentially expressed splicing variants were identified, including vesicle‑associated membrane protein 4, phosphatidylinositol glycan anchor biosynthesis class C, protein disulfide isomerase family A member 4 and growth arrest specific 5. Screened DEGs were enriched in the complement pathway. In the PPI network, ubiquitin C (UBC) was the hub node. UBC was predicted to be regulated by several TFs, including specificity protein 1 (SP1), FBJ murine osteosarcoma viral oncogene homolog (FOS), proto‑oncogene c‑JUN (JUN), FOS‑like antigen 2 (FOSL2) and SWI/SNF‑related, matrix‑associated, actin‑dependent regulator of chromatin, subfamily A, member 4 (SMARCA4), and several miRNAs, including miR‑30 and miR‑181. Results from the present study demonstrated that UBC, SP1, FOS, JUN, FOSL2, SMARCA4, miR‑30 and miR‑181 may participate in the development of HCC.

Large-scale gene function analysis with the PANTHER classification system.

PubMed

Mi, Huaiyu; Muruganujan, Anushya; Casagrande, John T; Thomas, Paul D

2013-08-01

The PANTHER (protein annotation through evolutionary relationship) classification system (http://www.pantherdb.org/) is a comprehensive system that combines gene function, ontology, pathways and statistical analysis tools that enable biologists to analyze large-scale, genome-wide data from sequencing, proteomics or gene expression experiments. The system is built with 82 complete genomes organized into gene families and subfamilies, and their evolutionary relationships are captured in phylogenetic trees, multiple sequence alignments and statistical models (hidden Markov models or HMMs). Genes are classified according to their function in several different ways: families and subfamilies are annotated with ontology terms (Gene Ontology (GO) and PANTHER protein class), and sequences are assigned to PANTHER pathways. The PANTHER website includes a suite of tools that enable users to browse and query gene functions, and to analyze large-scale experimental data with a number of statistical tests. It is widely used by bench scientists, bioinformaticians, computer scientists and systems biologists. In the 2013 release of PANTHER (v.8.0), in addition to an update of the data content, we redesigned the website interface to improve both user experience and the system's analytical capability. This protocol provides a detailed description of how to analyze genome-wide experimental data with the PANTHER classification system.

Comprehensive analysis of Arabidopsis expression level polymorphisms with simple inheritance

PubMed Central

Plantegenet, Stephanie; Weber, Johann; Goldstein, Darlene R; Zeller, Georg; Nussbaumer, Cindy; Thomas, Jérôme; Weigel, Detlef; Harshman, Keith; Hardtke, Christian S

2009-01-01

In Arabidopsis thaliana, gene expression level polymorphisms (ELPs) between natural accessions that exhibit simple, single locus inheritance are promising quantitative trait locus (QTL) candidates to explain phenotypic variability. It is assumed that such ELPs overwhelmingly represent regulatory element polymorphisms. However, comprehensive genome-wide analyses linking expression level, regulatory sequence and gene structure variation are missing, preventing definite verification of this assumption. Here, we analyzed ELPs observed between the Eil-0 and Lc-0 accessions. Compared with non-variable controls, 5′ regulatory sequence variation in the corresponding genes is indeed increased. However, ∼42% of all the ELP genes also carry major transcription unit deletions in one parent as revealed by genome tiling arrays, representing a >4-fold enrichment over controls. Within the subset of ELPs with simple inheritance, this proportion is even higher and deletions are generally more severe. Similar results were obtained from analyses of the Bay-0 and Sha accessions, using alternative technical approaches. Collectively, our results suggest that drastic structural changes are a major cause for ELPs with simple inheritance, corroborating experimentally observed indel preponderance in cloned Arabidopsis QTL. PMID:19225455

LuFLA1PRO and LuBGAL1PRO promote gene expression in the phloem fibres of flax (Linum usitatissimum).

PubMed

Hobson, Neil; Deyholos, Michael K

2013-04-01

Cell type-specific promoters were identified that drive gene expression in an industrially important product. To identify flax (Linum usitatissimum) gene promoters, we analyzed the genomic regions upstream of a fasciclin-like arabinogalactan protein (LuFLA1) and a beta-galactosidase (LuBGAL1). Both of these genes encode transcripts that have been found to be highly enriched in tissues bearing phloem fibres. Using a beta-glucuronidase (GUS) reporter construct, we found that a 908-bp genomic sequence upstream of LuFLA1 (LuFLA1PRO) directed GUS expression with high specificity to phloem fibres undergoing secondary cell wall development. The DNA sequence upstream of LuBGAL1 (LuBGAL1PRO) likewise produced GUS staining in phloem fibres with developing secondary walls, as well as in tissues of developing flowers and seed bolls. These data provide further evidence of a specific role for LuFLA1 in phloem fibre development, and demonstrate the utility of LuFLA1PRO and LuBGAL1PRO as tools for biotechnology and further investigations of phloem fibre development.

Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array

PubMed Central

Huang, Huan; Li, Shuo; Sun, Lizhou; Zhou, Guohua

2015-01-01

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC. PMID:25880764

Ebbie: automated analysis and storage of small RNA cloning data using a dynamic web server

PubMed Central

Ebhardt, H Alexander; Wiese, Kay C; Unrau, Peter J

2006-01-01

Background DNA sequencing is used ubiquitously: from deciphering genomes[1] to determining the primary sequence of small RNAs (smRNAs) [2-5]. The cloning of smRNAs is currently the most conventional method to determine the actual sequence of these important regulators of gene expression. Typical smRNA cloning projects involve the sequencing of hundreds to thousands of smRNA clones that are delimited at their 5' and 3' ends by fixed sequence regions. These primers result from the biochemical protocol used to isolate and convert the smRNA into clonable PCR products. Recently we completed a smRNA cloning project involving tobacco plants, where analysis was required for ~700 smRNA sequences[6]. Finding no easily accessible research tool to enter and analyze smRNA sequences we developed Ebbie to assist us with our study. Results Ebbie is a semi-automated smRNA cloning data processing algorithm, which initially searches for any substring within a DNA sequencing text file, which is flanked by two constant strings. The substring, also termed smRNA or insert, is stored in a MySQL and BlastN database. These inserts are then compared using BlastN to locally installed databases allowing the rapid comparison of the insert to both the growing smRNA database and to other static sequence databases. Our laboratory used Ebbie to analyze scores of DNA sequencing data originating from an smRNA cloning project[6]. Through its built-in instant analysis of all inserts using BlastN, we were able to quickly identify 33 groups of smRNAs from ~700 database entries. This clustering allowed the easy identification of novel and highly expressed clusters of smRNAs. Ebbie is available under GNU GPL and currently implemented on Conclusion Ebbie was designed for medium sized smRNA cloning projects with about 1,000 database entries [6-8].Ebbie can be used for any type of sequence analysis where two constant primer regions flank a sequence of interest. The reliable storage of inserts, and their annotation in a MySQL database, BlastN[9] comparison of new inserts to dynamic and static databases make it a powerful new tool in any laboratory using DNA sequencing. Ebbie also prevents manual mistakes during the excision process and speeds up annotation and data-entry. Once the server is installed locally, its access can be restricted to protect sensitive new DNA sequencing data. Ebbie was primarily designed for smRNA cloning projects, but can be applied to a variety of RNA and DNA cloning projects[2,3,10,11]. PMID:16584563

Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy

PubMed Central

Levin, Mattias; King, Jasmine J.; Glanville, Jacob; Jackson, Katherine J. L.; Looney, Timothy J.; Hoh, Ramona A.; Mari, Adriano; Andersson, Morgan; Greiff, Lennart; Fire, Andrew Z.; Boyd, Scott D.; Ohlin, Mats

2016-01-01

Background Specific immunotherapy (SIT) is the only treatment with proven long-term curative potential in allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B cell repertoires are not well understood. Objective To characterize the IgE sequences expressed by allergen-specific B cells, and track the fate of these B cell clones during SIT. Methods We have used high-throughput antibody gene sequencing and identification of allergen-specific IgE using combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and nasal mucosa of aeroallergen-sensitized individuals before and during the first year of subcutaneous SIT. Results Of 52 distinct allergen-specific IgE heavy chains from eight allergic donors, 37 were also detected by high-throughput antibody gene sequencing of blood, nasal mucosa, or both sample types. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships. Conclusion Combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies may in the future aid assessment of SIT mechanisms and efficacy. PMID:26559321

Next Generation Sequencing of Prostate Cancer from a Patient Identifies a Deficiency of Methylthioadenosine Phosphorylase (MTAP), an Exploitable Tumor Target

PubMed Central

Collins, Colin C; Volik, Stanislav V; Lapuk, Anna V; Wang, Yuwei; Gout, Peter W; Wu, Chunxiao; Xue, Hui; Cheng, Hongwei; Haegert, Anne; Bell, Robert H; Brahmbhatt, Sonal; Anderson, Shawn; Fazli, Ladan; Hurtado-Coll, Antonio; Rubin, Mark A.; Demichelis, Francesca; Beltran, Himisha; Hirst, Martin; Marra, Marco; Maher, Christopher A.; Chinnaiyan, Arul M.; Gleave, Martin; Bertino, Joseph R.; Lubin, Martin; Wang, Yuzhuo

2013-01-01

Castrate resistant prostate cancer (CRPC) and neuroendocrine carcinoma of the prostate are invariably fatal diseases for which only palliative therapies exist. As part of a prostate tumour sequencing program, a patient tumour was analyzed using Illumina genome sequencing and a matched renal capsule tumour xenograft was generated. Both tumour and xenograft had a homozygous 9p21 deletion spanning the MTAP, CDKN2 and ARF genes. It is rare for this deletion to occur in primary prostate tumours yet approximately 10% express decreased levels of MTAP mRNA. Decreased MTAP expression is a prognosticator for poor outcome. Moreover, it appears that this deletion is more common in CRPC than in primary prostate cancer. We show for the first time that treatment with methylthioadenosine and high dose 6-thioguanine causes marked inhibition of a patient derived neuroendocrine xenograft growth while protecting the host from 6-thioguanine toxicity. This therapeutic approach can be applied to other MTAP-deficient human cancers since deletion or hypermethylation of the MTAP gene occurs in a broad spectrum of tumours at high frequency. The combination of genome sequencing and patient-derived xenografts can identify candidate therapeutic agents and evaluate them for personalized oncology. PMID:22252602

STINGRAY: system for integrated genomic resources and analysis.

PubMed

Wagner, Glauber; Jardim, Rodrigo; Tschoeke, Diogo A; Loureiro, Daniel R; Ocaña, Kary A C S; Ribeiro, Antonio C B; Emmel, Vanessa E; Probst, Christian M; Pitaluga, André N; Grisard, Edmundo C; Cavalcanti, Maria C; Campos, Maria L M; Mattoso, Marta; Dávila, Alberto M R

2014-03-07

The STINGRAY system has been conceived to ease the tasks of integrating, analyzing, annotating and presenting genomic and expression data from Sanger and Next Generation Sequencing (NGS) platforms. STINGRAY includes: (a) a complete and integrated workflow (more than 20 bioinformatics tools) ranging from functional annotation to phylogeny; (b) a MySQL database schema, suitable for data integration and user access control; and (c) a user-friendly graphical web-based interface that makes the system intuitive, facilitating the tasks of data analysis and annotation. STINGRAY showed to be an easy to use and complete system for analyzing sequencing data. While both Sanger and NGS platforms are supported, the system could be faster using Sanger data, since the large NGS datasets could potentially slow down the MySQL database usage. STINGRAY is available at http://stingray.biowebdb.org and the open source code at http://sourceforge.net/projects/stingray-biowebdb/.

STINGRAY: system for integrated genomic resources and analysis

PubMed Central

2014-01-01

Background The STINGRAY system has been conceived to ease the tasks of integrating, analyzing, annotating and presenting genomic and expression data from Sanger and Next Generation Sequencing (NGS) platforms. Findings STINGRAY includes: (a) a complete and integrated workflow (more than 20 bioinformatics tools) ranging from functional annotation to phylogeny; (b) a MySQL database schema, suitable for data integration and user access control; and (c) a user-friendly graphical web-based interface that makes the system intuitive, facilitating the tasks of data analysis and annotation. Conclusion STINGRAY showed to be an easy to use and complete system for analyzing sequencing data. While both Sanger and NGS platforms are supported, the system could be faster using Sanger data, since the large NGS datasets could potentially slow down the MySQL database usage. STINGRAY is available at http://stingray.biowebdb.org and the open source code at http://sourceforge.net/projects/stingray-biowebdb/. PMID:24606808

DeepBase: annotation and discovery of microRNAs and other noncoding RNAs from deep-sequencing data.

PubMed

Yang, Jian-Hua; Qu, Liang-Hu

2012-01-01

Recent advances in high-throughput deep-sequencing technology have produced large numbers of short and long RNA sequences and enabled the detection and profiling of known and novel microRNAs (miRNAs) and other noncoding RNAs (ncRNAs) at unprecedented sensitivity and depth. In this chapter, we describe the use of deepBase, a database that we have developed to integrate all public deep-sequencing data and to facilitate the comprehensive annotation and discovery of miRNAs and other ncRNAs from these data. deepBase provides an integrative, interactive, and versatile web graphical interface to evaluate miRBase-annotated miRNA genes and other known ncRNAs, explores the expression patterns of miRNAs and other ncRNAs, and discovers novel miRNAs and other ncRNAs from deep-sequencing data. deepBase also provides a deepView genome browser to comparatively analyze these data at multiple levels. deepBase is available at http://deepbase.sysu.edu.cn/.

BigFoot: Bayesian alignment and phylogenetic footprinting with MCMC

PubMed Central

Satija, Rahul; Novák, Ádám; Miklós, István; Lyngsø, Rune; Hein, Jotun

2009-01-01

Background We have previously combined statistical alignment and phylogenetic footprinting to detect conserved functional elements without assuming a fixed alignment. Considering a probability-weighted distribution of alignments removes sensitivity to alignment errors, properly accommodates regions of alignment uncertainty, and increases the accuracy of functional element prediction. Our method utilized standard dynamic programming hidden markov model algorithms to analyze up to four sequences. Results We present a novel approach, implemented in the software package BigFoot, for performing phylogenetic footprinting on greater numbers of sequences. We have developed a Markov chain Monte Carlo (MCMC) approach which samples both sequence alignments and locations of slowly evolving regions. We implement our method as an extension of the existing StatAlign software package and test it on well-annotated regions controlling the expression of the even-skipped gene in Drosophila and the α-globin gene in vertebrates. The results exhibit how adding additional sequences to the analysis has the potential to improve the accuracy of functional predictions, and demonstrate how BigFoot outperforms existing alignment-based phylogenetic footprinting techniques. Conclusion BigFoot extends a combined alignment and phylogenetic footprinting approach to analyze larger amounts of sequence data using MCMC. Our approach is robust to alignment error and uncertainty and can be applied to a variety of biological datasets. The source code and documentation are publicly available for download from PMID:19715598

BigFoot: Bayesian alignment and phylogenetic footprinting with MCMC.

PubMed

Satija, Rahul; Novák, Adám; Miklós, István; Lyngsø, Rune; Hein, Jotun

2009-08-28

We have previously combined statistical alignment and phylogenetic footprinting to detect conserved functional elements without assuming a fixed alignment. Considering a probability-weighted distribution of alignments removes sensitivity to alignment errors, properly accommodates regions of alignment uncertainty, and increases the accuracy of functional element prediction. Our method utilized standard dynamic programming hidden markov model algorithms to analyze up to four sequences. We present a novel approach, implemented in the software package BigFoot, for performing phylogenetic footprinting on greater numbers of sequences. We have developed a Markov chain Monte Carlo (MCMC) approach which samples both sequence alignments and locations of slowly evolving regions. We implement our method as an extension of the existing StatAlign software package and test it on well-annotated regions controlling the expression of the even-skipped gene in Drosophila and the alpha-globin gene in vertebrates. The results exhibit how adding additional sequences to the analysis has the potential to improve the accuracy of functional predictions, and demonstrate how BigFoot outperforms existing alignment-based phylogenetic footprinting techniques. BigFoot extends a combined alignment and phylogenetic footprinting approach to analyze larger amounts of sequence data using MCMC. Our approach is robust to alignment error and uncertainty and can be applied to a variety of biological datasets. The source code and documentation are publicly available for download from http://www.stats.ox.ac.uk/~satija/BigFoot/

In silico characterization and expression analysis of the multigene family encoding the Bowman-Birk protease inhibitor in soybean.

PubMed

de Almeida Barros, Beatriz; da Silva, Wiliane Garcia; Moreira, Maurilio Alves; de Barros, Everaldo Gonçalves

2012-01-01

The Bowman-Birk (BBI) protease inhibitors can be used as source of sulfur amino acids, can regulate endogenous protease activity during seed germination and during the defense response of plants to pathogens. In soybean this family has not been fully described. The goal of this work was to characterize in silico and analyze the expression of the members of this family in soybean. We identified 11 potential BBI genes in the soybean genome. In each one of them at least a characteristic BBI conserved domain was detected in addition to a potential signal peptide. The sequences have been positioned in the soybean physical map and the promoter regions were analyzed with respect to known regulatory elements. Elements related to seed-specific expression and also to response to biotic and abiotic stresses have been identified. Based on the in silico analysis and also on quantitative RT-PCR data it was concluded that BBI-A, BBI-CII and BBI-DII are expressed specifically in the seed. The expression profiles of these three genes are similar along seed development. Their expressions reach a maximum in the intermediate stages and decrease as the seed matures. The BBI-DII transcripts are the most abundant ones followed by those of BBI-A and BBI-CII.

Identification and profiling of growth-related microRNAs of the swimming crab Portunus trituberculatus by using Solexa deep sequencing.

PubMed

Ren, Xianyun; Cui, Yanting; Gao, Baoquan; Liu, Ping; Li, Jian

2016-08-01

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that regulate gene expression by post-transcriptional repression of mRNAs. The swimming crab Portunus trituberculatus is one of the most important crustacean species for aquaculture in China. However, to date no miRNAs have been reported to for modulating growth in P. trituberculatus. To investigate miRNAs involved in the growth of this species, we constructed six small RNA libraries for big individuals (BIs) and small individuals (SIs) from a highly inbred family. Six mixed RNA pools of five tissues (eyestalk, gill, heart, hepatopancreas, and muscle) were obtained. By aligning sequencing data with those for known miRNAs, a total of 404 miRNAs, including 339 known and 65 novel miRNAs, were identified from the six libraries. MiR-100 and miR-276a-3p were among the most prominent miRNA species. We identified seven differentially expressed miRNAs between the BIs and SIs, which were validated using real-time PCR. Preliminary analyzes of their putative target genes and GO and KEGG pathway analyzes showed that these differentially expressed miRNAs could play important roles in global transcriptional depression and cell differentiation of P. trituberculatus. This study reveals the first miRNA profile related to the body growth of P. trituberculatus, which would be particularly useful for crab breeding programs. Copyright © 2016 Elsevier B.V. All rights reserved.

High-Throughput Sequencing Identifies MicroRNAs from Posterior Intestine of Loach (Misgurnus anguillicaudatus) and Their Response to Intestinal Air-Breathing Inhibition.

PubMed

Huang, Songqian; Cao, Xiaojuan; Tian, Xianchang; Wang, Weimin

2016-01-01

MicroRNAs (miRNAs) exert important roles in animal growth, immunity, and development, and regulate gene expression at the post-transcriptional level. Knowledges about the diversities of miRNAs and their roles in accessory air-breathing organs (ABOs) of fish remain unknown. In this work, we used high-throughput sequencing to identify known and novel miRNAs from the posterior intestine, an important ABO, in loach (Misgurnus anguillicaudatus) under normal and intestinal air-breathing inhibited conditions. A total of 204 known and 84 novel miRNAs were identified, while 47 miRNAs were differentially expressed between the two small RNA libraries (i.e. between the normal and intestinal air-breathing inhibited group). Potential miRNA target genes were predicted by combining our transcriptome data of the posterior intestine of the loach under the same conditions, and then annotated using COG, GO, KEGG, Swissprot and Nr databases. The regulatory networks of miRNAs and their target genes were analyzed. The abundances of nine known miRNAs were validated by qRT-PCR. The relative expression profiles of six known miRNAs and their eight corresponding target genes, and two novel potential miRNAs were also detected. Histological characteristics of the posterior intestines in both normal and air-breathing inhibited group were further analyzed. This study contributes to our understanding on the functions and molecular regulatory mechanisms of miRNAs in accessory air-breathing organs of fish.

Tissue-specific expression of human CD4 in transgenic mice.

PubMed Central

Gillespie, F P; Doros, L; Vitale, J; Blackwell, C; Gosselin, J; Snyder, B W; Wadsworth, S C

1993-01-01

The gene for the human CD4 glycoprotein, which serves as the receptor for human immunodeficiency virus type 1, along with approximately 23 kb of sequence upstream of the translational start site, was cloned. The ability of 5' flanking sequences to direct tissue-specific expression was tested in cell culture and in transgenic mice. A 5' flanking region of 6 kb was able to direct transcription of the CD4 gene in NIH 3T3 cells but did not result in detectable expression in the murine T-cell line EL4 or in four lines of transgenic mice. A larger 5' flanking region of approximately 23 kb directed high-level CD4 transcription in the murine T-cell line EL4 and in three independent lines of transgenic mice. Human CD4 expression in all tissues analyzed was tightly correlated with murine CD4 expression; the highest levels of human CD4 RNA expression were found in the thymus and spleen, with relatively low levels detected in other tissues. Expression of human CD4 protein in peripheral blood mononuclear cells was examined by flow cytometry in these transgenic animals and found to be restricted to the murine CD4+ subset of lymphocytes. Human CD4 protein, detected with an anti-human CD4 monoclonal antibody, was present on the surface of 45 to 50% of the peripheral blood mononuclear cells from all transgenic lines. Images PMID:8474453

[Prokaryotic expression of Leptospira interrogans groEL gene and immunoprotection of its products in hamsters].

PubMed

Li, Xiaoyu; Wang, Yinhuan; Yan, Jie; Cheng, Dongqing

2013-03-01

To construct a prokaryotic expression system of groEL gene of Leptospira interrogans serogroup Icterohaemorrhagia serovar Lai strain Lai, and to determine the immunoprotective effect of recombinant GroEL protein (rGroEL) in LVG hamsters. The groEL gene was amplified by high fidelity PCR and the amplification products were then sequenced. A prokaryotic expression system of groEL gene was constructed using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Gel Image Analyzer was applied to examine the expression and dissolubility of rGroEL protein while Ni-NTA affinity chromatography was used to extract the expressed rGroEL. The immunoprotective rate in rGroEL-immunized LVG hamsters was determined after challenge with L.interrogans strain Lai. The cross agglutination titers of sera from immunized hamsters with different L.interrogans serogroups were detected using MAT. The nucleotide and amino acid sequences of the cloned groEL gene were the same as those reported in GenBank. The constructed prokaryotic expression system of groEL gene expressed soluble rGroEL. The immunoprotective rates of 100 and 200 μg rGroEL in LVG hamsters were 50.0 % and 75.0%, respectively. The sera from the rGroEL-immunized LVG hamsters agglutinated all the L.interrogans serogroups tested with different levels. The GroEL protein is a genus-specific immunoprotective antigen of L.interrogans and can be used to develop an universal genetically engineering vaccine of Leptospira.

Expression and copy number gains of the RET gene in 631 early and mid stage non‐small cell lung cancer cases

PubMed Central

Tan, Ling; Hu, Yerong; Tao, Yongguang; Wang, Bin; Xiao, Jun; Tang, Zhenjie; Lu, Ting

2018-01-01

Background To identify whether RET is a potential target for NSCLC treatment, we examined the status of the RET gene in 631 early and mid stage NSCLC cases from south central China. Methods RET expression was identified by Western blot. RET‐positive expression samples were verified by immunohistochemistry. RET gene mutation, copy number variation, and rearrangement were analyzed by DNA Sanger sequencing, TaqMan copy number assays, and reverse transcription‐PCR. ALK and ROS1 expression levels were tested by Western blot and EGFR mutation using Sanger sequencing. Results The RET‐positive rate was 2.5% (16/631). RET‐positive expression was related to poorer tumor differentiation (P < 0.05). In the 16 RET‐positive samples, only two samples of moderately and poorly differentiated lung adenocarcinomas displayed RET rearrangement, both in RET‐KIF5B fusion partners. Neither ALK nor ROS1 translocation was found. The EGFR mutation rate in RET‐positive samples was significantly lower than in RET‐negative samples (P < 0.05). Conclusion RET‐positive expression in early and mid stage NSCLC cases from south central China is relatively low and is related to poorer tumor differentiation. RET gene alterations (copy number gain and rearrangement) exist in all RET‐positive samples. RET‐positive expression is a relatively independent factor in NSCLC patients, which indicates that the RET gene may be a novel target site for personalized treatment of NSCLC. PMID:29473341

Identification and functional analysis of flowering related microRNAs in common wild rice (Oryza rufipogon Griff.).

PubMed

Chen, Zongxiang; Li, Fuli; Yang, Songnan; Dong, Yibo; Yuan, Qianhua; Wang, Feng; Li, Weimin; Jiang, Ying; Jia, Shirong; Pei, Xinwu

2013-01-01

MicroRNAs (miRNAs) is a class of non-coding RNAs involved in post- transcriptional control of gene expression, via degradation and/or translational inhibition. Six-hundred sixty-one rice miRNAs are known that are important in plant development. However, flowering-related miRNAs have not been characterized in Oryza rufipogon Griff. It was approved by supervision department of Guangdong wild rice protection. We analyzed flowering-related miRNAs in O. rufipogon using high-throughput sequencing (deep sequencing) to understand the changes that occurred during rice domestication, and to elucidate their functions in flowering. Three O. rufipogon sRNA libraries, two vegetative stage (CWR-V1 and CWR-V2) and one flowering stage (CWR-F2) were sequenced using Illumina deep sequencing. A total of 20,156,098, 21,531,511 and 20,995,942 high quality sRNA reads were obtained from CWR-V1, CWR-V2 and CWR-F2, respectively, of which 3,448,185, 4,265,048 and 2,833,527 reads matched known miRNAs. We identified 512 known rice miRNAs in 214 miRNA families and predicted 290 new miRNAs. Targeted functional annotation, GO and KEGG pathway analyses predicted that 187 miRNAs regulate expression of flowering-related genes. Differential expression analysis of flowering-related miRNAs showed that: expression of 95 miRNAs varied significantly between the libraries, 66 are flowering-related miRNAs, such as oru-miR97, oru-miR117, oru-miR135, oru-miR137, et al. 17 are early-flowering -related miRNAs, including osa-miR160f, osa-miR164d, osa-miR167d, osa-miR169a, osa-miR172b, oru-miR4, et al., induced during the floral transition. Real-time PCR revealed the same expression patterns as deep sequencing. miRNAs targets were confirmed for cleavage by 5'-RACE in vivo, and were negatively regulated by miRNAs. This is the first investigation of flowering miRNAs in wild rice. The result indicates that variation in miRNAs occurred during rice domestication and lays a foundation for further study of phase change and flowering in O. rufipogon. Complicated regulatory networks mediated by multiple miRNAs regulate the expression of flowering genes that control the induction of flowering.

Identification and expression analysis of two pro-inflammatory cytokines, TNF-α and IL-8, in cobia (Rachycentron canadum L.) in response to Streptococcus dysgalactiae infection.

PubMed

Nguyen, Thuy Thi Thu; Nguyen, Hai Trong; Wang, Pei-Chyi; Chen, Shih-Chu

2017-08-01

Tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8/CXCL8) play pivotal roles in mediating inflammatory responses to invading pathogens. In this study, we identified and analyzed expressions of cobia TNF-α and IL-8 during Streptococcus dysgalactiae infection. The cloned cDNA transcript of cobia TNF-α comprised of 1281 base pairs (bp), with a 774 bp open reading frame (ORF) encoding 257 amino acids. The deduced amino acid sequence of cobia TNF-α showed a close relationship (84% similarity) with TNF-α of yellowtail amberjack. The cloned IL-8 cDNA sequence was 828 bp long, including a 300-bp ORF encoding 99 amino acids. The deduced amino acid sequence of cobia IL-8 shared 90% identity with IL-8 of striped trumpeter. Cobia challenged with a virulent S. dysgalactiae strain displayed an early significant up-regulation of TNF-α and IL-8 in head kidney, liver, and spleen. Notably, IL-8 expression level increased dramatically in the liver at the severe stage of infection (72 h). In conclusion, a better understanding of TNF-α and IL-8 allows more detailed investigation of immune responses in cobia and furthers study on controlling the infectious disease caused by S. dysgalactiae. Copyright © 2017 Elsevier Ltd. All rights reserved.

Application of the verona coding definitions of emotional sequences (VR-CoDES) on a pediatric data set.

PubMed

Vatne, Torun M; Finset, Arnstein; Ørnes, Knut; Ruland, Cornelia M

2010-09-01

Adult patients present concerns as defined in the Verona Coding Definitions of Emotional Sequences (VR-CoDES), but we do not know how children express their concerns during medical consultations. This study aimed to evaluate the applicability of VR-CoDES to pediatric oncology consultations. Twenty-eight pediatric consultations were coded with the Verona Coding Definitions of Emotional Sequences (VR-CoDES), and the material was also qualitatively analyzed for descriptive purposes. Five consultations were randomly selected for reliability testing and descriptive statistics were computed. Perfect inter-rater reliability for concerns and moderate reliability for cues were obtained. Cues and/or concerns were present in over half of the consultations. Cues were more frequent than concerns, with the majority of cues being verbal hints to hidden concerns or non-verbal cues. Intensity of expressions, limitations in vocabulary, commonality of statements, and complexity of the setting complicated the use of VR-CoDES. Child-specific cues; use of the imperative, cues about past experiences, and use of onomatopoeia were observed. Children with cancer express concerns during medical consultations. VR-CoDES is a reliable tool for coding concerns in pediatric data sets. For future applications in pediatric settings an appendix should be developed to incorporate the child-specific traits. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Sequencing, annotation, and characterization of the influenza ferret infectome.

PubMed

León, Alberto J; Banner, David; Xu, Luoling; Ran, Longsi; Peng, Zhiyu; Yi, Kang; Chen, Chao; Xu, Fengping; Huang, Jinrong; Zhao, Zhen; Lin, Zhen; Huang, Stephen H S; Fang, Yuan; Kelvin, Alyson A; Ross, Ted M; Farooqui, Amber; Kelvin, David J

2013-02-01

Ferrets have become an indispensable tool in the understanding of influenza virus virulence and pathogenesis. Furthermore, ferrets are the preferred preclinical model for influenza vaccine and therapeutic testing. Here we characterized the influenza infectome during the different stages of the infectious process in ferrets with and without prior specific immunity to influenza. RNA from lung tissue and lymph nodes from infected and naïve animals was subjected to next-generation sequencing, followed by de novo data assembly and annotation of the resulting sequences; this process generated a library comprising 13,202 ferret mRNAs. Gene expression profiles during pandemic H1N1 (pdmH1N1) influenza virus infection were analyzed by digital gene expression and solid support microarrays. As expected during primary infection, innate immune responses were triggered in the lung tissue; meanwhile, in the lymphoid tissue, genes encoding antigen presentation and maturation of effector cells of adaptive immunity increased dramatically. After 5 days postinfection, the innate immune gene expression was replaced by the adaptive immune response, which correlates with viral clearance. Reinfection with homologous pandemic influenza virus resulted in a diminished innate immune response, early adaptive immune gene regulation, and a reduction in clinical severity. The fully annotated ferret infectome will be a critical aid to the understanding of the molecular events that regulate disease severity and host-influenza virus interactions among seasonal, pandemic, and highly pathogenic avian influenzas.

miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades.

PubMed

Friedländer, Marc R; Mackowiak, Sebastian D; Li, Na; Chen, Wei; Rajewsky, Nikolaus

2012-01-01

microRNAs (miRNAs) are a large class of small non-coding RNAs which post-transcriptionally regulate the expression of a large fraction of all animal genes and are important in a wide range of biological processes. Recent advances in high-throughput sequencing allow miRNA detection at unprecedented sensitivity, but the computational task of accurately identifying the miRNAs in the background of sequenced RNAs remains challenging. For this purpose, we have designed miRDeep2, a substantially improved algorithm which identifies canonical and non-canonical miRNAs such as those derived from transposable elements and informs on high-confidence candidates that are detected in multiple independent samples. Analyzing data from seven animal species representing the major animal clades, miRDeep2 identified miRNAs with an accuracy of 98.6-99.9% and reported hundreds of novel miRNAs. To test the accuracy of miRDeep2, we knocked down the miRNA biogenesis pathway in a human cell line and sequenced small RNAs before and after. The vast majority of the >100 novel miRNAs expressed in this cell line were indeed specifically downregulated, validating most miRDeep2 predictions. Last, a new miRNA expression profiling routine, low time and memory usage and user-friendly interactive graphic output can make miRDeep2 useful to a wide range of researchers.

The low expression of Dmrt7 is associated with spermatogenic arrest in cattle-yak.

PubMed

Yan, Ping; Xiang, Lin; Guo, Xian; Bao, Peng-Jia; Jin, Shuai; Wu, Xiao-Yun

2014-11-01

Dmrt7 is a member of the DM domain family of genes. Dmrt7 deficiency is also a strong candidate as a cause for male cattle-yak infertility, as it is regarded as essential for male spermatogenesis, between the pachynema and diplonema stages. In our study, the coding region sequence of yak and cattle-yak Dmrt7 was cloned by molecular cloning techniques, and the sequence, conserved domains, functional sites, and secondary and tertiary structures of the Dmrt7-encoded protein were predicted and analyzed using bioinformatics methods. The coding region sequences of the Dmrt7 gene, encoding 370 amino acids, were consistent in yak and cattle-yak. The protein encoded by yak and cattle-yak Dmrt7 contains a DM domain. We detected Dmrt7 mRNA expression in testis, but not in any other tissue. Dmrt7 mRNA and protein expression was significantly higher in testis of cattle and yak than that in cattle-yak (p < 0.01). Histological analysis indicated that seminiferous tubules in male cattle-yak were highly vacuolated and contained primarily Sertoli cells and spermatogonia, while those of cattle and yak contained abundant primary spermatocytes. Male cattle-yak testis contained a significantly larger number of apoptotic cells than those in cattle and yak assessed by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) analysis. The accumulation of SCP3-positive spermatocytes indicated the arrest of spermatogenesis at the pachynema stage in the cattle-yak. These results suggest low levels of Dmrt7 expression lead to male sterility in cattle-yak. The molecular function of Dmrt7 and the regulation of its expression warrant need to be examined in future studies.

Optimized Probe Masking for Comparative Transcriptomics of Closely Related Species

PubMed Central

Poeschl, Yvonne; Delker, Carolin; Trenner, Jana; Ullrich, Kristian Karsten; Quint, Marcel; Grosse, Ivo

2013-01-01

Microarrays are commonly applied to study the transcriptome of specific species. However, many available microarrays are restricted to model organisms, and the design of custom microarrays for other species is often not feasible. Hence, transcriptomics approaches of non-model organisms as well as comparative transcriptomics studies among two or more species often make use of cost-intensive RNAseq studies or, alternatively, by hybridizing transcripts of a query species to a microarray of a closely related species. When analyzing these cross-species microarray expression data, differences in the transcriptome of the query species can cause problems, such as the following: (i) lower hybridization accuracy of probes due to mismatches or deletions, (ii) probes binding multiple transcripts of different genes, and (iii) probes binding transcripts of non-orthologous genes. So far, methods for (i) exist, but these neglect (ii) and (iii). Here, we propose an approach for comparative transcriptomics addressing problems (i) to (iii), which retains only transcript-specific probes binding transcripts of orthologous genes. We apply this approach to an Arabidopsis lyrata expression data set measured on a microarray designed for Arabidopsis thaliana, and compare it to two alternative approaches, a sequence-based approach and a genomic DNA hybridization-based approach. We investigate the number of retained probe sets, and we validate the resulting expression responses by qRT-PCR. We find that the proposed approach combines the benefit of sequence-based stringency and accuracy while allowing the expression analysis of much more genes than the alternative sequence-based approach. As an added benefit, the proposed approach requires probes to detect transcripts of orthologous genes only, which provides a superior base for biological interpretation of the measured expression responses. PMID:24260119

High-Throughput Sequencing Reveals Differential Expression of miRNAs in Intestine from Sea Cucumber during Aestivation

PubMed Central

Chen, Muyan; Zhang, Xiumei; Liu, Jianning; Storey, Kenneth B.

2013-01-01

The regulatory role of miRNA in gene expression is an emerging hot new topic in the control of hypometabolism. Sea cucumber aestivation is a complicated physiological process that includes obvious hypometabolism as evidenced by a decrease in the rates of oxygen consumption and ammonia nitrogen excretion, as well as a serious degeneration of the intestine into a very tiny filament. To determine whether miRNAs play regulatory roles in this process, the present study analyzed profiles of miRNA expression in the intestine of the sea cucumber (Apostichopus japonicus), using Solexa deep sequencing technology. We identified 308 sea cucumber miRNAs, including 18 novel miRNAs specific to sea cucumber. Animals sampled during deep aestivation (DA) after at least 15 days of continuous torpor, were compared with animals from a non-aestivation (NA) state (animals that had passed through aestivation and returned to the active state). We identified 42 differentially expressed miRNAs [RPM (reads per million) >10, |FC| (|fold change|) ≥1, FDR (false discovery rate) <0.01] during aestivation, which were validated by two other miRNA profiling methods: miRNA microarray and real-time PCR. Among the most prominent miRNA species, miR-200-3p, miR-2004, miR-2010, miR-22, miR-252a, miR-252a-3p and miR-92 were significantly over-expressed during deep aestivation compared with non-aestivation animals. Preliminary analyses of their putative target genes and GO analysis suggest that these miRNAs could play important roles in global transcriptional depression and cell differentiation during aestivation. High-throughput sequencing data and microarray data have been submitted to GEO database. PMID:24143179

Investigation of Endogenous Retrovirus Sequences in the Neighborhood of Genes Up-regulated in a Neuroblastoma Model after Treatment with Hypoxia-Mimetic Cobalt Chloride

PubMed Central

Brütting, Christine; Narasimhan, Harini; Hoffmann, Frank; Kornhuber, Malte E.; Staege, Martin S.; Emmer, Alexander

2018-01-01

Human endogenous retroviruses (ERVs) have been found to be associated with different diseases, e.g., multiple sclerosis (MS). Most human ERVs integrated in our genome are not competent to replicate and these sequences are presumably silent. However, transcription of human ERVs can be reactivated, e.g., by hypoxia. Interestingly, MS has been linked to hypoxia since decades. As some patterns of demyelination are similar to white matter ischemia, hypoxic damage is discussed. Therefore, we are interested in the association between hypoxia and ERVs. As a model, we used human SH-SY5Y neuroblastoma cells after treatment with the hypoxia-mimetic cobalt chloride and analyzed differences in the gene expression profiles in comparison to untreated cells. The vicinity of up-regulated genes was scanned for endogenous retrovirus-derived sequences. Five genes were found to be strongly up-regulated in SH-SY5Y cells after treatment with cobalt chloride: clusterin, glutathione peroxidase 3, insulin-like growth factor 2, solute carrier family 7 member 11, and neural precursor cell expressed developmentally down-regulated protein 9. In the vicinity of these genes we identified large (>1,000 bp) open reading frames (ORFs). Most of these ORFs showed only low similarities to proteins from retro-transcribing viruses. However, we found very high similarity between retrovirus envelope sequences and a sequence in the vicinity of neural precursor cell expressed developmentally down-regulated protein 9. This sequence encodes the human endogenous retrovirus group FRD member 1, the encoded protein product is called syncytin 2. Transfection of syncytin 2 into the well-characterized Ewing sarcoma cell line A673 was not able to modulate the low immunostimulatory activity of this cell line. Future research is needed to determine whether the identified genes and the human endogenous retrovirus group FRD member 1 might play a role in the etiology of MS. PMID:29515560

Investigation of Endogenous Retrovirus Sequences in the Neighborhood of Genes Up-regulated in a Neuroblastoma Model after Treatment with Hypoxia-Mimetic Cobalt Chloride.

PubMed

Brütting, Christine; Narasimhan, Harini; Hoffmann, Frank; Kornhuber, Malte E; Staege, Martin S; Emmer, Alexander

2018-01-01

Human endogenous retroviruses (ERVs) have been found to be associated with different diseases, e.g., multiple sclerosis (MS). Most human ERVs integrated in our genome are not competent to replicate and these sequences are presumably silent. However, transcription of human ERVs can be reactivated, e.g., by hypoxia. Interestingly, MS has been linked to hypoxia since decades. As some patterns of demyelination are similar to white matter ischemia, hypoxic damage is discussed. Therefore, we are interested in the association between hypoxia and ERVs. As a model, we used human SH-SY5Y neuroblastoma cells after treatment with the hypoxia-mimetic cobalt chloride and analyzed differences in the gene expression profiles in comparison to untreated cells. The vicinity of up-regulated genes was scanned for endogenous retrovirus-derived sequences. Five genes were found to be strongly up-regulated in SH-SY5Y cells after treatment with cobalt chloride: clusterin, glutathione peroxidase 3, insulin-like growth factor 2, solute carrier family 7 member 11, and neural precursor cell expressed developmentally down-regulated protein 9. In the vicinity of these genes we identified large (>1,000 bp) open reading frames (ORFs). Most of these ORFs showed only low similarities to proteins from retro-transcribing viruses. However, we found very high similarity between retrovirus envelope sequences and a sequence in the vicinity of neural precursor cell expressed developmentally down-regulated protein 9. This sequence encodes the human endogenous retrovirus group FRD member 1, the encoded protein product is called syncytin 2. Transfection of syncytin 2 into the well-characterized Ewing sarcoma cell line A673 was not able to modulate the low immunostimulatory activity of this cell line. Future research is needed to determine whether the identified genes and the human endogenous retrovirus group FRD member 1 might play a role in the etiology of MS.

Phylogenetic and expression analysis of the NPR1-like gene family from Persea americana (Mill.).

PubMed

Backer, Robert; Mahomed, Waheed; Reeksting, Bianca J; Engelbrecht, Juanita; Ibarra-Laclette, Enrique; van den Berg, Noëlani

2015-01-01

The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) forms an integral part of the salicylic acid (SA) pathway in plants and is involved in cross-talk between the SA and jasmonic acid/ethylene (JA/ET) pathways. Therefore, NPR1 is essential to the effective response of plants to pathogens. Avocado (Persea americana) is a commercially important crop worldwide. Significant losses in production result from Phytophthora root rot, caused by the hemibiotroph, Phytophthora cinnamomi. This oomycete infects the feeder roots of avocado trees leading to an overall decline in health and eventual death. The interaction between avocado and P. cinnamomi is poorly understood and as such limited control strategies exist. Thus uncovering the role of NPR1 in avocado could provide novel insights into the avocado - P. cinnamomi interaction. A total of five NPR1-like sequences were identified. These sequences were annotated using FGENESH and a maximum-likelihood tree was constructed using 34 NPR1-like protein sequences from other plant species. The conserved protein domains and functional motifs of these sequences were predicted. Reverse transcription quantitative PCR was used to analyze the expression of the five NPR1-like sequences in the roots of avocado after treatment with salicylic and jasmonic acid, P. cinnamomi infection, across different tissues and in P. cinnamomi infected tolerant and susceptible rootstocks. Of the five NPR1-like sequences three have strong support for a defensive role while two are most likely involved in development. Significant differences in the expression profiles of these five NPR1-like genes were observed, assisting in functional classification. Understanding the interaction of avocado and P. cinnamomi is essential to developing new control strategies. This work enables further classification of these genes by means of functional annotation and is a crucial step in understanding the role of NPR1 during P. cinnamomi infection.

Phylogenetic and expression analysis of the NPR1-like gene family from Persea americana (Mill.)

PubMed Central

Backer, Robert; Mahomed, Waheed; Reeksting, Bianca J.; Engelbrecht, Juanita; Ibarra-Laclette, Enrique; van den Berg, Noëlani

2015-01-01

The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) forms an integral part of the salicylic acid (SA) pathway in plants and is involved in cross-talk between the SA and jasmonic acid/ethylene (JA/ET) pathways. Therefore, NPR1 is essential to the effective response of plants to pathogens. Avocado (Persea americana) is a commercially important crop worldwide. Significant losses in production result from Phytophthora root rot, caused by the hemibiotroph, Phytophthora cinnamomi. This oomycete infects the feeder roots of avocado trees leading to an overall decline in health and eventual death. The interaction between avocado and P. cinnamomi is poorly understood and as such limited control strategies exist. Thus uncovering the role of NPR1 in avocado could provide novel insights into the avocado – P. cinnamomi interaction. A total of five NPR1-like sequences were identified. These sequences were annotated using FGENESH and a maximum-likelihood tree was constructed using 34 NPR1-like protein sequences from other plant species. The conserved protein domains and functional motifs of these sequences were predicted. Reverse transcription quantitative PCR was used to analyze the expression of the five NPR1-like sequences in the roots of avocado after treatment with salicylic and jasmonic acid, P. cinnamomi infection, across different tissues and in P. cinnamomi infected tolerant and susceptible rootstocks. Of the five NPR1-like sequences three have strong support for a defensive role while two are most likely involved in development. Significant differences in the expression profiles of these five NPR1-like genes were observed, assisting in functional classification. Understanding the interaction of avocado and P. cinnamomi is essential to developing new control strategies. This work enables further classification of these genes by means of functional annotation and is a crucial step in understanding the role of NPR1 during P. cinnamomi infection. PMID:25972890

Towards decrypting cryptobiosis--analyzing anhydrobiosis in the tardigrade Milnesium tardigradum using transcriptome sequencing.

PubMed

Wang, Chong; Grohme, Markus A; Mali, Brahim; Schill, Ralph O; Frohme, Marcus

2014-01-01

Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair.

Towards Decrypting Cryptobiosis—Analyzing Anhydrobiosis in the Tardigrade Milnesium tardigradum Using Transcriptome Sequencing

PubMed Central

Wang, Chong; Grohme, Markus A.; Mali, Brahim; Schill, Ralph O.; Frohme, Marcus

2014-01-01

Background Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. Results A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. Conclusions Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair. PMID:24651535

[Molecular cloning and characterization of a novel Clonorchis sinensis antigenic protein containing tandem repeat sequences].

PubMed

Liu, Qian; Xu, Xue-Nian; Zhou, Yan; Cheng, Na; Dong, Yu-Ting; Zheng, Hua-Jun; Zhu, Yong-Qiang; Zhu, Yong-Qiang

2013-08-01

To find and clone new antigen genes from the lambda-ZAP cDNA expression library of adult Clonorchis sinensis, and determine the immunological characteristics of the recombinant proteins. The cDNA expression library of adult C. sinensis was screened by pooled sera of clonorchiasis patients. The sequences of the positive phage clones were compared with the sequences in EST database, and the full-length sequence of the gene (Cs22 gene) was obtained by RT-PCR. cDNA fragments containing 2 and 3 times tandem repeat sequences were generated by jumping PCR. The sequence encoding the mature peptide or the tandem repeat sequence was respectively cloned into the prokaryotic expression vector pET28a (+), and then transformed into E. coli Rosetta DE3 cells for expression. The recombinant proteins (rCs22-2r, rCs22-3r, rCs22M-2r, and rCs22M-3r) were purified by His-bind-resin (Ni-NTA) affinity chromatography. The immunogenicity of rCs22-2r and rCs22-3r was identified by ELISA. To evaluate the immunological diagnostic value of rCs22-2r and rCs22-3r, serum samples from 35 clonorchiasis patients, 31 healthy individuals, 15 schistosomiasis patients, 15 paragonimiasis westermani patients and 13 cysticercosis patients were examined by ELISA. To locate antigenic determinants, the pooled sera of clonorchiasis patients and healthy persons were analyzed for specific antibodies by ELISA with recombinant protein rCs22M-2r and rCs22M-3r containing the tandem repeat sequences. The full-length sequence of Cs22 antigen gene of C. sinensis was obtained. It contained 13 times tandem repeat sequences of EQQDGDEEGMGGDGGRGKEKGKVEGEDGAGEQKEQA. Bioinformatics analysis indicated that the protein (Cs22) belonged to GPI-anchored proteins family. The recombinant proteins rCs22-2r and rCs22-3r showed a certain level of immunogenicity. The positive rate by ELISA coated with the purified PrCs22-2r and PrCs22-3r for sera of clonorchiasis patients both were 45.7% (16/35), and 3.2% (1/31) for those of healthy persons. There was no cross reaction with sera of schistosomiasis and cysticercosis patients. The cross reaction with sera of paragonimiasis westermani patients was 1/15. The recombinant proteins rCs22M-2r and rCs22M-3r which only contained tandem repeats were specifically recognized by pooled sera of clonorchiasis patients. The Cs22 antigen gene of Clonorchis sinensis is obtained, and the recombinant proteins have certain diagnostic value. The antigenic determinant is located in tandem repeat sequences.

Overexpression and Purification of C-terminal Fragment of the Passenger Domain of Hap Protein from Nontypeable Haemophilus influenzae in a Highly Optimized Escherichia coli Expression System

PubMed Central

Tabatabaee, Akram; Siadat, Seyed Davar; Moosavi, Seyed Fazllolah; Aghasadeghi, Mohammad Reza; Memarnejadian, Arash; Pouriayevali, Mohammad Hassan; Yavari, Neda

2013-01-01

Background Nontypeable Haemophilus influenzae (NTHi) is a common cause of respiratory tract disease and initiates infection by colonization in nasopharynx. The Haemophilus influenzae (H. influenzae) Hap adhesin is an auto transporter protein that promotes initial interaction with human epithelial cells. Hap protein contains a 110 kDa internal passenger domain called “HapS” and a 45 kDa C-terminal translocator domain called “Hapβ”. Hap adhesive activity has been recently reported to be connected to its Cell Binding Domain (CBD) which resides within the 311 C-terminal residues of the internal passenger domain of the protein. Furthermore, immunization with this CBD protein has been shown to prevent bacterial nasopharynx colonization in animal models. Methods To provide enough amounts of pure HapS protein for vaccine studies, we sought to develop a highly optimized system to overexpress and purify the protein in large quantities. To this end, pET24a-cbd plasmid harboring cbd sequence from NTHi ATCC49766 was constructed and its expression was optimized by testing various expression parameters such as growth media, induction temperature, IPTG inducer concentration, induction stage and duration. SDS-PAGE and Western-blotting were used for protein analysis and confirmation and eventually the expressed protein was easily purified via immobilized metal affinity chromatography (IMAC) using Ni-NTA columns. Results The highest expression level of target protein was achieved when CBD expressing E. coli BL21 (DE3) cells were grown at 37°C in 2xTY medium with 1.0 mM IPTG at mid-log phase (OD600 nm equal to 0.6) for 5 hrs. Amino acid sequence alignment of expressed CBD protein with 3 previously published CBD amino acid sequences were more than %97 identical and antigenicity plot analysis further revealed 9 antigenic domains which appeared to be well conserved among different analyzed CBD sequences. Conclusion Due to the presence of high similarity among CBD from NTHi ATCC49766 and other NTHi strains, CBD protein expressed here sounds to be theoretically ideal as a universal candidate for being used in vaccine studies against NTHi strains of various geographical areas. Further investigations to corroborate the potency of this protein as a vaccine candidate are under process. PMID:23919121

Denitrification gene expression in clay-soil bacterial community

NASA Astrophysics Data System (ADS)

Pastorelli, R.; Landi, S.

2009-04-01

Our contribution in the Italian research project SOILSINK was focused on microbial denitrification gene expression in Mediterranean agricultural soils. In ecosystems with high inputs of nitrogen, such as agricultural soils, denitrification causes a net loss of nitrogen since nitrate is reduced to gaseous forms, which are released into the atmosphere. Moreover, incomplete denitrification can lead to emission of nitrous oxide, a potent greenhouse gas which contributes to global warming and destruction of ozone layer. A critical role in denitrification is played by microorganisms and the ability to denitrify is widespread among a variety of phylogenetically unrelated organisms. Data reported here are referred to wheat cultivation in a clay-rich soil under different environmental impact management (Agugliano, AN, Italy). We analysed the RNA directly extracted from soil to provide information on in situ activities of specific populations. The expression of genes coding for two nitrate reductases (narG and napA), two nitrite reductases (nirS and nirK), two nitric oxide reductases (cnorB and qnorB) and nitrous oxide reductase (nosZ) was analyzed by reverse transcription (RT)-nested PCR. Only napA, nirS, nirK, qnorB and nosZ were detected and fragments sequenced showed high similarity with the corresponding gene sequences deposited in GenBank database. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples and they offered us the possibility to perform the denaturing gradient gel electrophoresis (DGGE) analyzes for denitrification genes.. Earlier conclusions showed nirK gene is more widely distributed in soil environment than nirS gene. The results concerning the nosZ expression indicated that microbial activity was clearly present only in no-tilled and no-fertilized soils.

RAP: RNA-Seq Analysis Pipeline, a new cloud-based NGS web application.

PubMed

D'Antonio, Mattia; D'Onorio De Meo, Paolo; Pallocca, Matteo; Picardi, Ernesto; D'Erchia, Anna Maria; Calogero, Raffaele A; Castrignanò, Tiziana; Pesole, Graziano

2015-01-01

The study of RNA has been dramatically improved by the introduction of Next Generation Sequencing platforms allowing massive and cheap sequencing of selected RNA fractions, also providing information on strand orientation (RNA-Seq). The complexity of transcriptomes and of their regulative pathways make RNA-Seq one of most complex field of NGS applications, addressing several aspects of the expression process (e.g. identification and quantification of expressed genes and transcripts, alternative splicing and polyadenylation, fusion genes and trans-splicing, post-transcriptional events, etc.). In order to provide researchers with an effective and friendly resource for analyzing RNA-Seq data, we present here RAP (RNA-Seq Analysis Pipeline), a cloud computing web application implementing a complete but modular analysis workflow. This pipeline integrates both state-of-the-art bioinformatics tools for RNA-Seq analysis and in-house developed scripts to offer to the user a comprehensive strategy for data analysis. RAP is able to perform quality checks (adopting FastQC and NGS QC Toolkit), identify and quantify expressed genes and transcripts (with Tophat, Cufflinks and HTSeq), detect alternative splicing events (using SpliceTrap) and chimeric transcripts (with ChimeraScan). This pipeline is also able to identify splicing junctions and constitutive or alternative polyadenylation sites (implementing custom analysis modules) and call for statistically significant differences in genes and transcripts expression, splicing pattern and polyadenylation site usage (using Cuffdiff2 and DESeq). Through a user friendly web interface, the RAP workflow can be suitably customized by the user and it is automatically executed on our cloud computing environment. This strategy allows to access to bioinformatics tools and computational resources without specific bioinformatics and IT skills. RAP provides a set of tabular and graphical results that can be helpful to browse, filter and export analyzed data, according to the user needs.

Sequence homology and expression profile of genes associated with DNA repair pathways in Mycobacterium leprae.

PubMed

Sharma, Mukul; Vedithi, Sundeep Chaitanya; Das, Madhusmita; Roy, Anindya; Ebenezer, Mannam

2017-01-01

Survival of Mycobacterium leprae, the causative bacteria for leprosy, in the human host is dependent to an extent on the ways in which its genome integrity is retained. DNA repair mechanisms protect bacterial DNA from damage induced by various stress factors. The current study is aimed at understanding the sequence and functional annotation of DNA repair genes in M. leprae. T he genome of M. leprae was annotated using sequence alignment tools to identify DNA repair genes that have homologs in Mycobacterium tuberculosis and Escherichia coli. A set of 96 genes known to be involved in DNA repair mechanisms in E. coli and Mycobacteriaceae were chosen as a reference. Among these, 61 were identified in M. leprae based on sequence similarity and domain architecture. The 61 were classified into 36 characterized gene products (59%), 11 hypothetical proteins (18%), and 14 pseudogenes (23%). All these genes have homologs in M. tuberculosis and 49 (80.32%) in E. coli. A set of 12 genes which are absent in E. coli were present in M. leprae and in Mycobacteriaceae. These 61 genes were further investigated for their expression profiles in the whole transcriptome microarray data of M. leprae which was obtained from the signal intensities of 60bp probes, tiling the entire genome with 10bp overlaps. It was noted that transcripts corresponding to all the 61 genes were identified in the transcriptome data with varying expression levels ranging from 0.18 to 2.47 fold (normalized with 16SrRNA). The mRNA expression levels of a representative set of seven genes ( four annotated and three hypothetical protein coding genes) were analyzed using quantitative Polymerase Chain Reaction (qPCR) assays with RNA extracted from skin biopsies of 10 newly diagnosed, untreated leprosy cases. It was noted that RNA expression levels were higher for genes involved in homologous recombination whereas the genes with a low level of expression are involved in the direct repair pathway. This study provided preliminary information on the potential DNA repair pathways that are extant in M. leprae and the associated genes.

Peritransplant Treg-Based Immunomodulation to Improve VCA Outcomes

DTIC Science & Technology

2017-10-01

function as assessed in vitro assays (mean ± SD, n=4/group) using cells analyzed at day 5. (D) Western blots of Foxp3 protein expression in Tregs from...mice and undertaking bisulphite conversion, cloning and sequencing . WT Tregs were largely demethylated at the TSDR site (open circles, Fig. 2...term murine limb vascularized composite allotransplantation (VCA) survival. • Aim 2 - Determine if histone/ protein deacetylase (HDAC) inhibitor

A theoretical and methodological proposal for the descriptive assessment of therapeutic interactions.

PubMed

Froján-Parga, María Xesús; Ruiz-Sancho, Elena M; Calero-Elvira, Ana

2016-01-01

The goal of this study is to show the development of a strategy for a descriptive assessment of the therapeutic interaction. In this study, we develop an observational methodology to analyze the dialogues that took place during 92 sessions conducted in a psychological center in Madrid, Spain, in which 19 adults were treated for various psychological problems by 9 behavioral therapists. A system was developed to codify vocal behavior of both the therapists and the clients; the software The Observer XT was used for recording. Therapeutic interactions were analyzed using sequential analysis. There are three main sequences that synthesize the therapist-client interaction: first, an utterance by the client preceded by a therapist's verbalization, specifically a question (discriminative morphology) and followed by an expression of approval (reinforcement morphology); second, verbalizations of failure or discomfort uttered by the client, followed most often by verbalizations of disapproval (punishing morphology) uttered by the therapist; and third, verbalizations uttered by the client that are discriminated by the therapist after an in-depth explanation and followed by different therapist's utterances (expressions of approval, technical information, etc.). Depending on how the client responds the results in this study present a starting point for the study of the functional sequences that form the basis of therapeutic change.

Plasmids for variable expression of proteins targeted to the mitochondrial matrix or intermembrane space.

PubMed

Newman, Laura E; Schiavon, Cara; Kahn, Richard A

2016-01-01

We describe the construction and uses of a series of plasmids for directing expression to varied levels of exogenous proteins targeted to the mitochondrial matrix or intermembrane space. We found that the level of protein expression achieved, the kinetics of expression and mitochondrial import, and half-life after import can each vary with the protein examined. These factors should be considered when directing localization of an exogenous protein to mitochondria for rescue, proteomics, or other approaches. We describe the construction of a collection of plasmids for varied expression of proteins targeted to the mitochondrial matrix or intermembrane space, using previously defined targeting sequences and strength CMV promoters. The limited size of these compartments makes them particularly vulnerable to artifacts from over-expression. We found that different proteins display different kinetics of expression and import that should be considered when analyzing results from this approach. Finally, this collection of plasmids has been deposited in the Addgene plasmid repository to facilitate the ready access and use of these tools.

Transcriptomic investigation of meat tenderness in two Italian cattle breeds.

PubMed

Bongiorni, S; Gruber, C E M; Bueno, S; Chillemi, G; Ferrè, F; Failla, S; Moioli, B; Valentini, A

2016-06-01

Our objectives for this study were to understand the biological basis of meat tenderness and to provide an overview of the gene expression profiles related to meat quality as a tool for selection. Through deep mRNA sequencing, we analyzed gene expression in muscle tissues of two Italian cattle breeds: Maremmana and Chianina. We uncovered several differentially expressed genes that encode for proteins belonging to a family of tripartite motif proteins, which are involved in growth, cell differentiation and apoptosis, such as TRIM45, or play an essential role in regulating skeletal muscle differentiation and the regeneration of adult skeletal muscle, such as TRIM32. Other differentially expressed genes (SCN2B, SLC9A7 and KCNK3) emphasize the involvement of potassium-sodium pumps in tender meat. By mapping splice junctions in RNA-Seq reads, we found significant differences in gene isoform expression levels. The PRKAG3 gene, which is involved in the regulation of energy metabolism, showed four isoforms that were differentially expressed. This distinct pattern of PRKAG3 gene expression could indicate impaired glycogen storage in skeletal muscle, and consequently, this gene very likely has a role in the tenderization process. Furthermore, with this deep RNA-sequencing, we captured a high number of expressed SNPs, for example, we found 1462 homozygous SNPs showing the alternative allele with a 100% frequency when comparing tender and tough meat. SNPs were then classified into categories by their position and also by their effect on gene coding (174 non-synonymous polymorphisms) based on the available UMD_3.1 annotations. © 2016 Stichting International Foundation for Animal Genetics.

Digital gene expression analysis of gene expression differences within Brassica diploids and allopolyploids.

PubMed

Jiang, Jinjin; Wang, Yue; Zhu, Bao; Fang, Tingting; Fang, Yujie; Wang, Youping

2015-01-27

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.

Optimal Scaling of Digital Transcriptomes

PubMed Central

Glusman, Gustavo; Caballero, Juan; Robinson, Max; Kutlu, Burak; Hood, Leroy

2013-01-01

Deep sequencing of transcriptomes has become an indispensable tool for biology, enabling expression levels for thousands of genes to be compared across multiple samples. Since transcript counts scale with sequencing depth, counts from different samples must be normalized to a common scale prior to comparison. We analyzed fifteen existing and novel algorithms for normalizing transcript counts, and evaluated the effectiveness of the resulting normalizations. For this purpose we defined two novel and mutually independent metrics: (1) the number of “uniform” genes (genes whose normalized expression levels have a sufficiently low coefficient of variation), and (2) low Spearman correlation between normalized expression profiles of gene pairs. We also define four novel algorithms, one of which explicitly maximizes the number of uniform genes, and compared the performance of all fifteen algorithms. The two most commonly used methods (scaling to a fixed total value, or equalizing the expression of certain ‘housekeeping’ genes) yielded particularly poor results, surpassed even by normalization based on randomly selected gene sets. Conversely, seven of the algorithms approached what appears to be optimal normalization. Three of these algorithms rely on the identification of “ubiquitous” genes: genes expressed in all the samples studied, but never at very high or very low levels. We demonstrate that these include a “core” of genes expressed in many tissues in a mutually consistent pattern, which is suitable for use as an internal normalization guide. The new methods yield robustly normalized expression values, which is a prerequisite for the identification of differentially expressed and tissue-specific genes as potential biomarkers. PMID:24223126

Identification, expression and phylogenetic analysis of EgG1Y162 from Echinococcus granulosus.

PubMed

Zhang, Fengbo; Ma, Xiumin; Zhu, Yuejie; Wang, Hongying; Liu, Xianfei; Zhu, Min; Ma, Haimei; Wen, Hao; Fan, Haining; Ding, Jianbing

2014-01-01

This study was to clone, identify and analyze the characteristics of egG1Y162 gene from Echinococcus granulosus. Genomic DNA and total RNAs were extracted from four different developmental stages of protoscolex, germinal layer, adult and egg of Echinococcus granulosus, respectively. Fluorescent quantitative PCR was used for analyzing the expression of egG1Y162 gene. Prokaryotic expression plasmid of pET41a-EgG1Y162 was constructed to express recombinant His-EgG1Y162 antigen. Western blot analysis was performed to detect antigenicity of EgG1Y162 antigen. Gene sequence, amino acid alignment and phylogenetic tree of EgG1Y162 were analyzed by BLAST, online Spidey and MEGA4 software, respectively. EgG1Y162 gene was expressed in four developmental stages of Echinococcus granulosus. And, egG1Y162 gene expression was the highest in the adult stage, with the relative value of 19.526, significantly higher than other three stages. Additionally, Western blot analysis revealed that EgG1Y162 recombinant protein had good reaction with serum samples from Echinococcus granulosus infected human and dog. Moreover, EgG1Y162 antigen was phylogenetically closest to EmY162 antigen, with the similarity over 90%. Our study identified EgG1Y162 antigen in Echinococcus granulosus for the first time. EgG1Y162 antigen had a high similarity with EmY162 antigen, with the genetic differences mainly existing in the intron region. And, EgG1Y162 recombinant protein showed good antigenicity.

Asymmetric purine-pyrimidine distribution in cellular small RNA population of papaya

PubMed Central

2012-01-01

Background The small RNAs (sRNA) are a regulatory class of RNA mainly represented by the 21 and 24-nucleotide size classes. The cellular sRNAs are processed by RNase III family enzyme dicer (Dicer like in plant) from a self-complementary hairpin loop or other type of RNA duplexes. The papaya genome has been sequenced, but its microRNAs and other regulatory RNAs are yet to be analyzed. Results We analyzed the genomic features of the papaya sRNA population from three sRNA deep sequencing libraries made from leaves, flowers, and leaves infected with Papaya Ringspot Virus (PRSV). We also used the deep sequencing data to annotate the micro RNA (miRNA) in papaya. We identified 60 miRNAs, 24 of which were conserved in other species, and 36 of which were novel miRNAs specific to papaya. In contrast to the Chargaff’s purine-pyrimidine equilibrium, cellular sRNA was significantly biased towards a purine rich population. Of the two purine bases, higher frequency of adenine was present in 23nt or longer sRNAs, while 22nt or shorter sRNAs were over represented by guanine bases. However, this bias was not observed in the annotated miRNAs in plants. The 21nt species were expressed from fewer loci but expressed at higher levels relative to the 24nt species. The highly expressed 21nt species were clustered in a few isolated locations of the genome. The PRSV infected leaves showed higher accumulation of 21 and 22nt sRNA compared to uninfected leaves. We observed higher accumulation of miRNA* of seven annotated miRNAs in virus-infected tissue, indicating the potential function of miRNA* under stressed conditions. Conclusions We have identified 60 miRNAs in papaya. Our study revealed the asymmetric purine-pyrimidine distribution in cellular sRNA population. The 21nt species of sRNAs have higher expression levels than 24nt sRNA. The miRNA* of some miRNAs shows higher accumulation in PRSV infected tissues, suggesting that these strands are not totally functionally redundant. The findings open a new avenue for further investigation of the sRNA silencing pathway in plants. PMID:23216749

Asymmetric purine-pyrimidine distribution in cellular small RNA population of papaya.

PubMed

Aryal, Rishi; Yang, Xiaozeng; Yu, Qingyi; Sunkar, Ramanjulu; Li, Lei; Ming, Ray

2012-12-05

The small RNAs (sRNA) are a regulatory class of RNA mainly represented by the 21 and 24-nucleotide size classes. The cellular sRNAs are processed by RNase III family enzyme dicer (Dicer like in plant) from a self-complementary hairpin loop or other type of RNA duplexes. The papaya genome has been sequenced, but its microRNAs and other regulatory RNAs are yet to be analyzed. We analyzed the genomic features of the papaya sRNA population from three sRNA deep sequencing libraries made from leaves, flowers, and leaves infected with Papaya Ringspot Virus (PRSV). We also used the deep sequencing data to annotate the micro RNA (miRNA) in papaya. We identified 60 miRNAs, 24 of which were conserved in other species, and 36 of which were novel miRNAs specific to papaya. In contrast to the Chargaff's purine-pyrimidine equilibrium, cellular sRNA was significantly biased towards a purine rich population. Of the two purine bases, higher frequency of adenine was present in 23nt or longer sRNAs, while 22nt or shorter sRNAs were over represented by guanine bases. However, this bias was not observed in the annotated miRNAs in plants. The 21nt species were expressed from fewer loci but expressed at higher levels relative to the 24nt species. The highly expressed 21nt species were clustered in a few isolated locations of the genome. The PRSV infected leaves showed higher accumulation of 21 and 22nt sRNA compared to uninfected leaves. We observed higher accumulation of miRNA* of seven annotated miRNAs in virus-infected tissue, indicating the potential function of miRNA* under stressed conditions. We have identified 60 miRNAs in papaya. Our study revealed the asymmetric purine-pyrimidine distribution in cellular sRNA population. The 21nt species of sRNAs have higher expression levels than 24nt sRNA. The miRNA* of some miRNAs shows higher accumulation in PRSV infected tissues, suggesting that these strands are not totally functionally redundant. The findings open a new avenue for further investigation of the sRNA silencing pathway in plants.

De novo sequencing and analysis of the transcriptome during the browning of fresh-cut Luffa cylindrica 'Fusi-3' fruits

PubMed Central

Chen, Mindong; Wang, Bin; Zhang, Qianrong; Xue, Zhuzheng

2017-01-01

Fresh-cut luffa (Luffa cylindrica) fruits commonly undergo browning. However, little is known about the molecular mechanisms regulating this process. We used the RNA-seq technique to analyze the transcriptomic changes occurring during the browning of fresh-cut fruits from luffa cultivar ‘Fusi-3’. Over 90 million high-quality reads were assembled into 58,073 Unigenes, and 60.86% of these were annotated based on sequences in four public databases. We detected 35,282 Unigenes with significant hits to sequences in the NCBInr database, and 24,427 Unigenes encoded proteins with sequences that were similar to those of known proteins in the Swiss-Prot database. Additionally, 20,546 and 13,021 Unigenes were similar to existing sequences in the Eukaryotic Orthologous Groups of proteins and Kyoto Encyclopedia of Genes and Genomes databases, respectively. Furthermore, 27,301 Unigenes were differentially expressed during the browning of fresh-cut luffa fruits (i.e., after 1–6 h). Moreover, 11 genes from five gene families (i.e., PPO, PAL, POD, CAT, and SOD) identified as potentially associated with enzymatic browning as well as four WRKY transcription factors were observed to be differentially regulated in fresh-cut luffa fruits. With the assistance of rapid amplification of cDNA ends technology, we obtained the full-length sequences of the 15 Unigenes. We also confirmed these Unigenes were expressed by quantitative real-time polymerase chain reaction analysis. This study provides a comprehensive transcriptome sequence resource, and may facilitate further studies aimed at identifying genes affecting luffa fruit browning for the exploitation of the underlying mechanism. PMID:29145430

Identification of human skeletal muscle miRNA related to strength by high-throughput sequencing.

PubMed

Mitchell, Cameron J; D'Souza, Randall F; Schierding, William; Zeng, Nina; Ramzan, Farha; O'Sullivan, Justin M; Poppitt, Sally D; Cameron-Smith, David

2018-06-01

The loss of muscle size, strength, and quality with aging is a major determinant of morbidity and mortality in the elderly. The regulatory pathways that impact the muscle phenotype include the translational regulation maintained by microRNAs (miRNA). Yet the miRNAs that are expressed in human skeletal muscle and relationship to muscle size, strength, and quality are unknown. Using next-generation sequencing, we selected the 50 most abundantly expressed miRNAs and then analyzed them in vastus lateralis muscle, obtained by biopsy from middle-aged males ( n = 48; 50.0 ± 4.3 yr). Isokinetic strength testing and midthigh computed tomography was undertaken for muscle phenotype analysis. Muscle attenuation was measured by computerized tomography and is inversely proportional to myofiber lipid content. miR-486-5p accounted for 21% of total miR sequence reads, with miR-10b-5p, miR-133a-3p, and miR-22-3p accounting for a further 15, 12, and 10%, respectively. Isokinetic knee extension strength and muscle cross-sectional area were positively correlated with miR-100-5p, miR-99b-5p, and miR-191-5p expression. Muscle attenuation was negatively correlated to let-7f-5p, miR-30d-5p, and miR-125b-5p expression. In silico analysis implicates miRNAs related to strength and muscle size in the regulation of mammalian target of rapamycin, while miRNAs related to muscle attenuation may have potential roles regulating the transforming growth factor-β/SMAD3 pathway.

Molecular cloning and expression of Cro s 1: an occupational allergen from saffron pollen (Crocus sativus)

PubMed Central

Varasteh, Abdol-Reza; Sankian, Mojtaba; Midoro-Horiuti, Terumi; Moghadam, Malihe; Shakeri, Mohamad Taghi; Brooks, Edward G.; Goldblum, Randall M.; Chapman, Martin D.; Pomés, Anna

2012-01-01

Background: The cultivation of saffron is expanding through the southeast of Iran, and allergy to saffron pollen occurs in workers involved in processing this plant. We aimed to clone, sequence and express a major allergen involved in saffron pollen allergy, and to compare the recombinant with the natural allergen. Methods: The N-terminal amino acid sequence of Cro s 1, an allergen from saffron pollen, was determined after immunoblotting. The cDNA encoding for this allergen was cloned by PCR utilizing a primer based on the N-terminal amino acid sequence. Recombinant Cro s 1 (rCro s 1) was expressed as a soluble protein in Pichia pastoris and purified to homogeneity by gel filtration. Inhibition of IgE binding to rCro s 1 by pollen extract was analyzed by ELISA. Section Title The allergen Cro s 1 was identified from saffron pollen extracts and cloned by PCR. Cro s 1 cDNA defined an acidic polypeptide with homology to pollen proteins from Chenopodium album and Ligastrum vulgaris. The rCro s 1 was expressed in P. pastoris at 28 mg/l. Saffron pollen extract inhibited the binding of patient serum IgE to rCro s 1. Conclusion: We identified and cloned the first Crocus sativus pollen allergen. rCro s 1 cDNA shows a very high homology with Che a 1, the major allergen of lamb's-quarter, Chenopodium album, Caryophyllales, pollen (97%). Cro s 1 is a useful tool for specific diagnosis and structural studies of occupational allergy to saffron. PMID:26989701

TP53 Mutation Status of Tubo-ovarian and Peritoneal High-grade Serous Carcinoma with a Wild-type p53 Immunostaining Pattern.

PubMed

Na, Kiyong; Sung, Ji-Youn; Kim, Hyun-Soo

2017-12-01

Diffuse and strong nuclear p53 immunoreactivity and a complete lack of p53 expression are regarded as indicative of missense and nonsense mutations, respectively, of the TP53 gene. Tubo-ovarian and peritoneal high-grade serous carcinoma (HGSC) is characterized by aberrant p53 expression induced by a TP53 mutation. However, our experience with some HGSC cases with a wild-type p53 immunostaining pattern led us to comprehensively review previous cases and investigate the TP53 mutational status of the exceptional cases. We analyzed the immunophenotype of 153 cases of HGSC and performed TP53 gene sequencing analysis in those with a wild-type p53 immunostaining pattern. Immunostaining revealed that 109 (71.3%) cases displayed diffuse and strong p53 expression (missense mutation pattern), while 39 (25.5%) had no p53 expression (nonsense mutation pattern). The remaining five cases of HGSC showed a wild-type p53 immunostaining pattern. Direct sequencing analysis revealed that three of these cases harbored nonsense TP53 mutations and two had novel splice site deletions. TP53 mutation is almost invariably present in HGSC, and p53 immunostaining can be used as a surrogate marker of TP53 mutation. In cases with a wild-type p53 immunostaining pattern, direct sequencing for TP53 mutational status can be helpful to confirm the presence of a TP53 mutation. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Molecular cloning and expression of Cro s 1: an occupational allergen from saffron pollen (Crocus sativus).

PubMed

Varasteh, Abdol-Reza; Sankian, Mojtaba; Midoro-Horiuti, Terumi; Moghadam, Malihe; Shakeri, Mohamad Taghi; Brooks, Edward G; Goldblum, Randall M; Chapman, Martin D; Pomés, Anna

2012-10-01

The cultivation of saffron is expanding through the southeast of Iran, and allergy to saffron pollen occurs in workers involved in processing this plant. We aimed to clone, sequence and express a major allergen involved in saffron pollen allergy, and to compare the recombinant with the natural allergen. The N-terminal amino acid sequence of Cro s 1, an allergen from saffron pollen, was determined after immunoblotting. The cDNA encoding for this allergen was cloned by PCR utilizing a primer based on the N-terminal amino acid sequence. Recombinant Cro s 1 (rCro s 1) was expressed as a soluble protein in Pichia pastoris and purified to homogeneity by gel filtration. Inhibition of IgE binding to rCro s 1 by pollen extract was analyzed by ELISA. The allergen Cro s 1 was identified from saffron pollen extracts and cloned by PCR. Cro s 1 cDNA defined an acidic polypeptide with homology to pollen proteins from Chenopodium album and Ligastrum vulgaris. The rCro s 1 was expressed in P. pastoris at 28 mg/l. Saffron pollen extract inhibited the binding of patient serum IgE to rCro s 1. We identified and cloned the first Crocus sativus pollen allergen. rCro s 1 cDNA shows a very high homology with Che a 1, the major allergen of lamb's-quarter, Chenopodium album, Caryophyllales, pollen (97%). Cro s 1 is a useful tool for specific diagnosis and structural studies of occupational allergy to saffron.

Cloning of the anhidrotic ectodermal dysplasia gene: Identification of cDNAs associated with CpG islands mapped near translocation breakpoint in two female patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srivastava, A.K.; Schlessinger, D.; Kere, J.

1994-09-01

The gene for the X chromosomal developmental disorder anhidrotic ectodermal dysplasia (EDA) has been mapped to Xq12-q13 by linkage analysis and is expressed in a few females with chromosomal translocations involving band Xq12-q13. A yeast artificial chromosome (YAC) contig (2.0 Mb) spanning two translocation breakpoints has been assembled by sequence-tagged site (STS)-based chromosomal walking. The two translocation breakpoints (X:autosome translocations from the affected female patients) have been mapped less than 60 kb apart within a YAC contig. Unique probes and intragenic STSs (mapped between the two translocations) have been developed and a somatic cell hybrid carrying the translocated X chromosomemore » from the AK patient has been analyzed by isolating unique probes that span the breakpoint. Several STSs made from intragenic sequences have been found to be conserved in mouse, hamster and monkey, but we have detected no mRNAs in a number of tissues tested. However, a probe and STS developed from the DNA spanning the AK breakpoint is conserved in mouse, hamster and monkey, and we have detected expressed sequences in skin cells and cDNA libraries. In addition, unique sequences have been obtained from two CpG islands in the region that maps proximal to the breakpoints. cDNAs containing these sequences are being studied as candidates for the gene affected in the etiology of EDA.« less

GobyWeb: Simplified Management and Analysis of Gene Expression and DNA Methylation Sequencing Data

PubMed Central

Dorff, Kevin C.; Chambwe, Nyasha; Zeno, Zachary; Simi, Manuele; Shaknovich, Rita; Campagne, Fabien

2013-01-01

We present GobyWeb, a web-based system that facilitates the management and analysis of high-throughput sequencing (HTS) projects. The software provides integrated support for a broad set of HTS analyses and offers a simple plugin extension mechanism. Analyses currently supported include quantification of gene expression for messenger and small RNA sequencing, estimation of DNA methylation (i.e., reduced bisulfite sequencing and whole genome methyl-seq), or the detection of pathogens in sequenced data. In contrast to previous analysis pipelines developed for analysis of HTS data, GobyWeb requires significantly less storage space, runs analyses efficiently on a parallel grid, scales gracefully to process tens or hundreds of multi-gigabyte samples, yet can be used effectively by researchers who are comfortable using a web browser. We conducted performance evaluations of the software and found it to either outperform or have similar performance to analysis programs developed for specialized analyses of HTS data. We found that most biologists who took a one-hour GobyWeb training session were readily able to analyze RNA-Seq data with state of the art analysis tools. GobyWeb can be obtained at http://gobyweb.campagnelab.org and is freely available for non-commercial use. GobyWeb plugins are distributed in source code and licensed under the open source LGPL3 license to facilitate code inspection, reuse and independent extensions http://github.com/CampagneLaboratory/gobyweb2-plugins. PMID:23936070

Molecular cloning and characterization of beluga whale (Delphinapterus leucas) interleukin-1beta and tumor necrosis factor-alpha.

PubMed Central

Denis, F; Archambault, D

2001-01-01

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are cytokines produced primarily by monocytes and macrophages with regulatory effects in inflammation and multiple aspects of the immune response. As yet, no molecular data have been reported for IL-1beta and TNF-alpha of the beluga whale. In this study, we cloned and determined the entire cDNA sequence encoding beluga whale IL-1beta and TNF-alpha. The genetic relationship of the cytokine sequences was then analyzed with those from several mammalian species, including the human and the pig. The homology of beluga whale IL-1beta nucleic acid and deduced amino acid sequences with those from these mammalian species ranged from 74.6 to 86.0% and 62.7 to 77.1%, respectively, whereas that of TNF-alpha varied from 79.3 to 90.8% and 75.3 to 87.7%, respectively. Phylogenetic analyses based on deduced amino acid sequences showed that the beluga whale IL-1beta and TNF-alpha were most closely related to those of the ruminant species (cattle, sheep, and deer). The beluga whale IL-1beta- and TNF-alpha-encoding sequences were thereafter successfully expressed in Escherichia coli as fusion proteins by using procaryotic expression vectors. The fusion proteins were used to produce beluga whale IL-1beta- and TNF-alpha-specific rabbit antisera. Images Figure 3. Figure 4. Figure 5. PMID:11768130

Transcriptome-wide investigation of genomic imprinting in chicken.

PubMed

Frésard, Laure; Leroux, Sophie; Servin, Bertrand; Gourichon, David; Dehais, Patrice; Cristobal, Magali San; Marsaud, Nathalie; Vignoles, Florence; Bed'hom, Bertrand; Coville, Jean-Luc; Hormozdiari, Farhad; Beaumont, Catherine; Zerjal, Tatiana; Vignal, Alain; Morisson, Mireille; Lagarrigue, Sandrine; Pitel, Frédérique

2014-04-01

Genomic imprinting is an epigenetic mechanism by which alleles of some specific genes are expressed in a parent-of-origin manner. It has been observed in mammals and marsupials, but not in birds. Until now, only a few genes orthologous to mammalian imprinted ones have been analyzed in chicken and did not demonstrate any evidence of imprinting in this species. However, several published observations such as imprinted-like QTL in poultry or reciprocal effects keep the question open. Our main objective was thus to screen the entire chicken genome for parental-allele-specific differential expression on whole embryonic transcriptomes, using high-throughput sequencing. To identify the parental origin of each observed haplotype, two chicken experimental populations were used, as inbred and as genetically distant as possible. Two families were produced from two reciprocal crosses. Transcripts from 20 embryos were sequenced using NGS technology, producing ∼200 Gb of sequences. This allowed the detection of 79 potentially imprinted SNPs, through an analysis method that we validated by detecting imprinting from mouse data already published. However, out of 23 candidates tested by pyrosequencing, none could be confirmed. These results come together, without a priori, with previous statements and phylogenetic considerations assessing the absence of genomic imprinting in chicken.

Cloning and expression of a small heat and salt tolerant protein (Hsp22) from Chaetomium globosum.

PubMed

Aggarwal, Rashmi; Gupta, Sangeeta; Sharma, Sapna; Banerjee, Sagar; Singh, Priyanka

2012-11-01

The present study reports molecular characterization of small heat shock protein gene in Indian isolates of Chaetomium globosum, C. perlucidum, C. reflexum, C. cochlioides and C. cupreum. Six isolates of C. globosum and other species showed a band of 630bp using specific primers. Amplified cDNA product of C. globosum (Cg 1) cloned and sequenced showed 603bp open reading frame encoding 200 amino-acids. The protein sequence had a molecular mass of 22 kDa and was therefore, named Hsp22. BlastX analysis revealed that the gene codes for a protein homologous to previously characterized Hsp22.4 gene from C. globosum (AAR36902.1, XP 001229241.1) and shared 95% identity in amino acid sequence. It also showed varying degree of similarities with small Hsp protein from Neurospora spp. (60%), Myceliophthora sp. (59%), Glomerella sp. (50%), Hypocrea sp. (52%), and Fusarium spp. (51%). This gene was further cloned into pET28a (+) and transformed E. coli BL21 cells were induced by IPTG, and the expressed protein of 30 kDa was analyzed by SDS-PAGE. The IPTG induced transformants displayed significantly greater resistance to NaCl and Na2CO3 stresses.

Transcriptional Analysis of Drought-Induced Genes in the Roots of a Tolerant Genotype of the Common Bean (Phaseolus vulgaris L.)

PubMed Central

Recchia, Gustavo Henrique; Caldas, Danielle Gregorio Gomes; Beraldo, Ana Luiza Ahern; da Silva, Márcio José; Tsai, Siu Mui

2013-01-01

In Brazil, common bean (Phaseolus vulgaris L.) productivity is severely affected by drought stress due to low technology cultivation systems. Our purpose was to identify differentially expressed genes in roots of a genotype tolerant to water deficit (BAT 477) when submitted to an interruption of irrigation during its development. A SSH library was constructed taking as “driver” the genotype Carioca 80SH (susceptible to drought). After clustering and data mining, 1572 valid reads were obtained, resulting in 1120 ESTs (expressed sequence tags). We found sequences for transcription factors, carbohydrates metabolism, proline-rich proteins, aquaporins, chaperones and ubiquitins, all of them organized according to their biological processes. Our suppressive subtractive hybridization (SSH) library was validated through RT-qPCR experiment by assessing the expression patterns of 10 selected genes in both genotypes under stressed and control conditions. Finally, the expression patterns of 31 ESTs, putatively related to drought responses, were analyzed in a time-course experiment. Our results confirmed that such genes are more expressed in the tolerant genotype during stress; however, they are not exclusive, since different levels of these transcripts were also detected in the susceptible genotype. In addition, we observed a fluctuation in gene regulation over time for both the genotypes, which seem to adopt and adapt different strategies in order to develop tolerance against this stress. PMID:23538843

Novel splice mutation in microthalmia-associated transcription factor in Waardenburg Syndrome.

PubMed

Brenner, Laura; Burke, Kelly; Leduc, Charles A; Guha, Saurav; Guo, Jiancheng; Chung, Wendy K

2011-01-01

Waardenburg Syndrome (WS) is a syndromic form of hearing loss associated with mutations in six different genes. We identified a large family with WS that had previously undergone clinical testing, with no reported pathogenic mutation. Using linkage analysis, a region on 3p14.1 with an LOD score of 6.6 was identified. Microthalmia-Associated Transcription Factor, a gene known to cause WS, is located within this region of linkage. Sequencing of Microthalmia-Associated Transcription Factor demonstrated a c.1212 G>A synonymous variant that segregated with the WS in the family and was predicted to cause a novel splicing site that was confirmed with expression analysis of the mRNA. This case illustrates the need to computationally analyze novel synonymous sequence variants for possible effects on splicing to maximize the clinical sensitivity of sequence-based genetic testing.

The structure of the coding and 5'-flanking region of the type 1 iodothyronine deiodinase (dio1) gene is normal in a patient with suspected congenital dio1 deficiency.

PubMed

Toyoda, N; Kleinhaus, N; Larsen, P R

1996-06-01

We analyzed the exon-intron structure of the human type 1 deiodinase gene (dio1) and compared it with that of a patient with suspected congenital type 1 deiodinase (D1) deficiency. The hdio1 gene is identical in exon-intron arrangement to the mouse gene, with coding sequences and a selenocysteine insertion sequence (SECIS) element contained in four exons. There were no mutations in the sequences of exons 1-4 of the patient's genomic DNA. Functional studies by transient expression techniques showed no difference in basal promoter activity or T3 responsiveness between the patient's and the normal dio1 gene. A structural abnormality in the dio1 gene is not a likely explanation for this patient's D1-deficient phenotype.

Contingency Table Browser - prediction of early stage protein structure.

PubMed

Kalinowska, Barbara; Krzykalski, Artur; Roterman, Irena

2015-01-01

The Early Stage (ES) intermediate represents the starting structure in protein folding simulations based on the Fuzzy Oil Drop (FOD) model. The accuracy of FOD predictions is greatly dependent on the accuracy of the chosen intermediate. A suitable intermediate can be constructed using the sequence-structure relationship information contained in the so-called contingency table - this table expresses the likelihood of encountering various structural motifs for each tetrapeptide fragment in the amino acid sequence. The limited accuracy with which such structures could previously be predicted provided the motivation for a more indepth study of the contingency table itself. The Contingency Table Browser is a tool which can visualize, search and analyze the table. Our work presents possible applications of Contingency Table Browser, among them - analysis of specific protein sequences from the point of view of their structural ambiguity.

Molecular Characterization of Voltage-Gated Sodium Channels and Their Relations with Paralytic Shellfish Toxin Bioaccumulation in the Pacific Oyster Crassostrea gigas

PubMed Central

Boullot, Floriane; Castrec, Justine; Bidault, Adeline; Dantas, Natanael; Payton, Laura; Perrigault, Mickael; Tran, Damien; Amzil, Zouher; Boudry, Pierre; Soudant, Philippe; Hégaret, Hélène; Fabioux, Caroline

2017-01-01

Paralytic shellfish toxins (PST) bind to voltage-gated sodium channels (Nav) and block conduction of action potential in excitable cells. This study aimed to (i) characterize Nav sequences in Crassostrea gigas and (ii) investigate a putative relation between Nav and PST-bioaccumulation in oysters. The phylogenetic analysis highlighted two types of Nav in C. gigas: a Nav1 (CgNav1) and a Nav2 (CgNav2) with sequence properties of sodium-selective and sodium/calcium-selective channels, respectively. Three alternative splice transcripts of CgNav1 named A, B and C, were characterized. The expression of CgNav1, analyzed by in situ hybridization, is specific to nervous cells and to structures corresponding to neuromuscular junctions. Real-time PCR analyses showed a strong expression of CgNav1A in the striated muscle while CgNav1B is mainly expressed in visceral ganglia. CgNav1C expression is ubiquitous. The PST binding site (domain II) of CgNav1 variants possess an amino acid Q that could potentially confer a partial saxitoxin (STX)-resistance to the channel. The CgNav1 genotype or alternative splicing would not be the key point determining PST bioaccumulation level in oysters. PMID:28106838

Hysteretic energy prediction method for mainshock-aftershock sequences

NASA Astrophysics Data System (ADS)

Zhai, Changhai; Ji, Duofa; Wen, Weiping; Li, Cuihua; Lei, Weidong; Xie, Lili

2018-04-01

Structures located in seismically active regions may be subjected to mainshock-aftershock (MSAS) sequences. Strong aftershocks significantly affect the hysteretic energy demand of structures. The hysteretic energy, E H,seq, is normalized by mass m and expressed in terms of the equivalent velocity, V D,seq, to quantitatively investigate aftershock effects on the hysteretic energy of structures. The equivalent velocity, V D,seq, is computed by analyzing the response time-history of an inelastic single-degree-of-freedom (SDOF) system with a varying vibration period subjected to 309 MSAS sequences. The present study selected two kinds of MSAS sequences, with one aftershock and two aftershocks, respectively. The aftershocks are scaled to maintain different relative intensities. The variation of the equivalent velocity, V D,seq, is studied for consideration of the ductility values, site conditions, relative intensities, number of aftershocks, hysteretic models, and damping ratios. The MSAS sequence with one aftershock exhibited a 10% to 30% hysteretic energy increase, whereas the MSAS sequence with two aftershocks presented a 20% to 40% hysteretic energy increase. Finally, a hysteretic energy prediction equation is proposed as a function of the vibration period, ductility value, and damping ratio to estimate hysteretic energy for mainshock-aftershock sequences.

Clinical significance of miR-146a in gastric cancer cases.

PubMed

Kogo, Ryunosuke; Mimori, Koshi; Tanaka, Fumiaki; Komune, Shizuo; Mori, Masaki

2011-07-01

The profiles of microRNAs change significantly in gastric cancer. MiR-146a is reported to be a tumor suppressor in pancreatic cancer, breast cancer, and prostate cancer. We investigated the clinical significance of miR-146a in gastric cancer, in particular focusing on hypothetical miR-146a target genes, such as epidermal growth factor receptor (EGFR) and interleukin-1 receptor-associated kinase (IRAK1). We examined miR-146a levels in 90 gastric cancer samples by q-real-time (qRT)-PCR and analyzed the association between miR-146a levels and clinicopathologic factors and prognosis. The regulation of EGFR and IRAK1 by miR-146a was examined with miR-146a-transfected gastric cancer cells. Moreover, we analyzed the association between miR-146a levels and the G/C single nucleotide polymorphism (SNP) within pre-miR-146a seed sequences in 76 gastric cancer samples, using direct sequencing of genomic DNA. In 90 clinical samples of gastric cancer, miR-146a levels in cancer tissues were significantly lower than those in the corresponding noncancerous tissue (P < 0.001). Lower levels of miR-146a were associated with lymph node metastasis and venous invasion (P < 0.05). Moreover, a lower level of miR-146a was an independent prognostic factor for overall survival (P = 0.003). Ectopic expression of miR-146a inhibited migration and invasion and downregulated EGFR and IRAK1 expression in gastric cancer cells. In addition, G/C SNP within the pre-miR-146a seed sequence significantly reduced miR-146a levels in the GG genotype compared with the CC genotype. MiR-146a contains an SNP, which is associated with mature miR-146a expression. MiR-146a targeting of EGFR and IRAK1 is an independent prognostic factor in gastric cancer cases.

Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets

PubMed Central

Macosko, Evan Z.; Basu, Anindita; Satija, Rahul; Nemesh, James; Shekhar, Karthik; Goldman, Melissa; Tirosh, Itay; Bialas, Allison R.; Kamitaki, Nolan; Martersteck, Emily M.; Trombetta, John J.; Weitz, David A.; Sanes, Joshua R.; Shalek, Alex K.; Regev, Aviv; McCarroll, Steven A.

2015-01-01

Summary Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-Seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. Drop-Seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts’ cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-Seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. PMID:26000488

[Expression profiles of the exosomal miRNAs in the chronic hepatitis B patients with persistently normal ALT].

PubMed

Li, Ronghua; Fu, Xiaoyu; Tang, Yujing; Fu, Lei; Tan, Deming; Ouyang, Yi; Peng, Shifang

2018-05-28

To investigate expression profiles of the plasma exosomal miRNAs of the chronic hepatitis B (CHB) patients with persistently normal alamine aminotransferase (PNALT) for the first time and try to find exosomal miRNAs which could reflect liver inflammation better. 
 Methods: Five CHB patients with liver tissue inflammation grade ≥A2 of PNALT and 5 CHB patients with liver tissue inflammation grade

Down-regulation of long non-coding RNA RP11-708H21.4 is associated with poor prognosis for colorectal cancer and promotes tumorigenesis through regulating AKT/mTOR pathway.

PubMed

Sun, Longci; Jiang, Chunhui; Xu, Chunjie; Xue, Hanbing; Zhou, Hong; Gu, Lei; Liu, Ye; Xu, Qing

2017-04-25

Long non-coding RNAs (lncRNAs) serve critical roles in cancer development and progression. Herein, through next generation RNA sequencing and experimental validations, we determined the expression status of RP11-708H21.4 in colorectal cancer (CRC) and explored its clinical significance and biological functions in CRC. Differentially expressed lncRNAs from CRC samples and corresponding normal mucosa tissues was screened through RNA sequencing, and RP11-708H21.4 was selected for further experimental validation. The expression levels of RP11-708H21.4 in CRC tissues and cell lines were determined using qRT-PCR. Also, the relationship between the clinicopathological features and RP11-708H21.4 expression was analyzed. Cell viability was examined by CCK-8 and colony assays; cell migration and invasion were detected by transwell assays; cell cycle and cell apoptosis were analyzed by flow cytometry. The chemosensitivity of CRC cells to 5-Fluorouracil (5-FU) was also determined using CCK-8 assay. CRC xenograft tumor models were established to determine the biological functions of RP11-708H21.4 in vivo. Levels of cell cycle-related proteins and AKT/mTOR pathway-related proteins were detected by western blot assay. RP11-708H21.4 expression was aberrantly decreased in CRC, and its expression was closely associated with aggressive clinicopathologic features and unfavorable prognosis of CRC patients. Overexpressed RP11-708H21.4 suppresses CRC cell proliferation through inducing G1 arrest. Moreover, up-regulation of RP11-708H21.4 inhibits cell migration and invasion, causes cell apoptosis, and enhances 5-FU sensitivity of CRC cells. Finally, increased RP11-708H21.4 expression blocked AKT/mTOR pathway, and repressed in vivo CRC xenograft tumor growth. The results indicated that RP11-708H21.4 might have potential roles as a biomarker and a therapeutic target for CRC.

Microgravity elicits reproducible alterations in cytoskeletal and metabolic gene and protein expression in space-flown Caenorhabditis elegans

PubMed Central

Higashibata, Akira; Hashizume, Toko; Nemoto, Kanako; Higashitani, Nahoko; Etheridge, Timothy; Mori, Chihiro; Harada, Shunsuke; Sugimoto, Tomoko; Szewczyk, Nathaniel J; Baba, Shoji A; Mogami, Yoshihiro; Fukui, Keiji; Higashitani, Atsushi

2016-01-01

Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P⩽0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts. PMID:28725720

De novo characterization of the Chinese fir (Cunninghamia lanceolata) transcriptome and analysis of candidate genes involved in cellulose and lignin biosynthesis

PubMed Central

2012-01-01

Background Chinese fir (Cunninghamia lanceolata) is an important timber species that accounts for 20–30% of the total commercial timber production in China. However, the available genomic information of Chinese fir is limited, and this severely encumbers functional genomic analysis and molecular breeding in Chinese fir. Recently, major advances in transcriptome sequencing have provided fast and cost-effective approaches to generate large expression datasets that have proven to be powerful tools to profile the transcriptomes of non-model organisms with undetermined genomes. Results In this study, the transcriptomes of nine tissues from Chinese fir were analyzed using the Illumina HiSeq™ 2000 sequencing platform. Approximately 40 million paired-end reads were obtained, generating 3.62 gigabase pairs of sequencing data. These reads were assembled into 83,248 unique sequences (i.e. Unigenes) with an average length of 449 bp, amounting to 37.40 Mb. A total of 73,779 Unigenes were supported by more than 5 reads, 42,663 (57.83%) had homologs in the NCBI non-redundant and Swiss-Prot protein databases, corresponding to 27,224 unique protein entries. Of these Unigenes, 16,750 were assigned to Gene Ontology classes, and 14,877 were clustered into orthologous groups. A total of 21,689 (29.40%) were mapped to 119 pathways by BLAST comparison against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The majority of the genes encoding the enzymes in the biosynthetic pathways of cellulose and lignin were identified in the Unigene dataset by targeted searches of their annotations. And a number of candidate Chinese fir genes in the two metabolic pathways were discovered firstly. Eighteen genes related to cellulose and lignin biosynthesis were cloned for experimental validating of transcriptome data. Overall 49 Unigenes, covering different regions of these selected genes, were found by alignment. Their expression patterns in different tissues were analyzed by qRT-PCR to explore their putative functions. Conclusions A substantial fraction of transcript sequences was obtained from the deep sequencing of Chinese fir. The assembled Unigene dataset was used to discover candidate genes of cellulose and lignin biosynthesis. This transcriptome dataset will provide a comprehensive sequence resource for molecular genetics research of C. lanceolata. PMID:23171398

Divergent and nonuniform gene expression patterns in mouse brain

PubMed Central

Morris, John A.; Royall, Joshua J.; Bertagnolli, Darren; Boe, Andrew F.; Burnell, Josh J.; Byrnes, Emi J.; Copeland, Cathy; Desta, Tsega; Fischer, Shanna R.; Goldy, Jeff; Glattfelder, Katie J.; Kidney, Jolene M.; Lemon, Tracy; Orta, Geralyn J.; Parry, Sheana E.; Pathak, Sayan D.; Pearson, Owen C.; Reding, Melissa; Shapouri, Sheila; Smith, Kimberly A.; Soden, Chad; Solan, Beth M.; Weller, John; Takahashi, Joseph S.; Overly, Caroline C.; Lein, Ed S.; Hawrylycz, Michael J.; Hohmann, John G.; Jones, Allan R.

2010-01-01

Considerable progress has been made in understanding variations in gene sequence and expression level associated with phenotype, yet how genetic diversity translates into complex phenotypic differences remains poorly understood. Here, we examine the relationship between genetic background and spatial patterns of gene expression across seven strains of mice, providing the most extensive cellular-resolution comparative analysis of gene expression in the mammalian brain to date. Using comprehensive brainwide anatomic coverage (more than 200 brain regions), we applied in situ hybridization to analyze the spatial expression patterns of 49 genes encoding well-known pharmaceutical drug targets. Remarkably, over 50% of the genes examined showed interstrain expression variation. In addition, the variability was nonuniformly distributed across strain and neuroanatomic region, suggesting certain organizing principles. First, the degree of expression variance among strains mirrors genealogic relationships. Second, expression pattern differences were concentrated in higher-order brain regions such as the cortex and hippocampus. Divergence in gene expression patterns across the brain could contribute significantly to variations in behavior and responses to neuroactive drugs in laboratory mouse strains and may help to explain individual differences in human responsiveness to neuroactive drugs. PMID:20956311

A Children's Oncology Group and TARGET initiative exploring the genetic landscape of Wilms tumor. | Office of Cancer Genomics

Cancer.gov

We performed genome-wide sequencing and analyzed mRNA and miRNA expression, DNA copy number, and DNA methylation in 117 Wilms tumors, followed by targeted sequencing of 651 Wilms tumors. In addition to genes previously implicated in Wilms tumors (WT1, CTNNB1, AMER1, DROSHA, DGCR8, XPO5, DICER1, SIX1, SIX2, MLLT1, MYCN, and TP53), we identified mutations in genes not previously recognized as recurrently involved in Wilms tumors, the most frequent being BCOR, BCORL1, NONO, MAX, COL6A3, ASXL1, MAP3K4, and ARID1A.

SEXCMD: Development and validation of sex marker sequences for whole-exome/genome and RNA sequencing.

PubMed

Jeong, Seongmun; Kim, Jiwoong; Park, Won; Jeon, Hongmin; Kim, Namshin

2017-01-01

Over the last decade, a large number of nucleotide sequences have been generated by next-generation sequencing technologies and deposited to public databases. However, most of these datasets do not specify the sex of individuals sampled because researchers typically ignore or hide this information. Male and female genomes in many species have distinctive sex chromosomes, XX/XY and ZW/ZZ, and expression levels of many sex-related genes differ between the sexes. Herein, we describe how to develop sex marker sequences from syntenic regions of sex chromosomes and use them to quickly identify the sex of individuals being analyzed. Array-based technologies routinely use either known sex markers or the B-allele frequency of X or Z chromosomes to deduce the sex of an individual. The same strategy has been used with whole-exome/genome sequence data; however, all reads must be aligned onto a reference genome to determine the B-allele frequency of the X or Z chromosomes. SEXCMD is a pipeline that can extract sex marker sequences from reference sex chromosomes and rapidly identify the sex of individuals from whole-exome/genome and RNA sequencing after training with a known dataset through a simple machine learning approach. The pipeline counts total numbers of hits from sex-specific marker sequences and identifies the sex of the individuals sampled based on the fact that XX/ZZ samples do not have Y or W chromosome hits. We have successfully validated our pipeline with mammalian (Homo sapiens; XY) and avian (Gallus gallus; ZW) genomes. Typical calculation time when applying SEXCMD to human whole-exome or RNA sequencing datasets is a few minutes, and analyzing human whole-genome datasets takes about 10 minutes. Another important application of SEXCMD is as a quality control measure to avoid mixing samples before bioinformatics analysis. SEXCMD comprises simple Python and R scripts and is freely available at https://github.com/lovemun/SEXCMD.

[Recombinant expression of Schistosoma japonicum fructose-1, 6-bisphos- phate aldolase and its expression in different developmental stages of S. japonicum].

PubMed

Yan, Ke; Zhong, Zheng-rong; Xu, Yun-xia; Ding, Shu-qin; Hu, Jian-guo; Xu, Yuan-hong; Luo, Qing-lie; Shen, Ji-long

2015-06-01

To clone, express and purify Schistosoma japonicum fructose-1, 6-bisphosphate aldolase (SjFBPA) in E. coli and observe its expression in different developmental stages of S. japonicum. FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid, and inducibly expressed with IPTG in E. coli BL21. SDS-PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA (rSjFBPA). Then, rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS- PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore, SjFBPA mRNA was ana- lyzed in different developmental stages of S. japonicum by RT-PCR. SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro- tein could specifically reactive to the anti-His-tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT-PCR showed that SjFBPA mRNA was expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum. SjFBPA is successfully recombined and expressed in a prokaryotic system, and SjFBPA mRNA is expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum.

Correlation between mutations and mRNA expression of APC and MUTYH genes: new insight into hereditary colorectal polyposis predisposition.

PubMed

Aceto, Gitana Maria; Fantini, Fabiana; De Iure, Sabrina; Di Nicola, Marta; Palka, Giandomenico; Valanzano, Rosa; Di Gregorio, Patrizia; Stigliano, Vittoria; Genuardi, Maurizio; Battista, Pasquale; Cama, Alessandro; Curia, Maria Cristina

2015-10-28

Transcript dosage imbalance may influence the transcriptome. To gain insight into the role of altered gene expression in hereditary colorectal polyposis predisposition, in the present study we analyzed absolute and allele-specific expression (ASE) of adenomatous polyposis coli (APC) and mutY Homolog (MUTYH) genes. We analyzed DNA and RNA extracted from peripheral blood mononuclear cells (PBMC) of 49 familial polyposis patients and 42 healthy blood donors selected according similar gender and age. Patients were studied for germline alterations in both genes using dHPLC, MLPA and automated sequencing. APC and MUTYH mRNA expression levels were investigated by quantitative Real-Time PCR (qRT-PCR) analysis using TaqMan assay and by ASE assays using dHPLC-based primer extension. Twenty out of 49 patients showed germline mutations: 14 in APC gene and six in MUTYH gene. Twenty-nine patients did not show mutations in both genes. Results from qRT-PCR indicated that gene expression of both APC and MUTYH was reduced in patients analyzed. In particular, a significant reduction in APC expression was observed in patients without APC germline mutation vs control group (P < 0.05) while APC expression in the mutation carrier patients, although lower compared to control individuals, did not show statistical significance. On the other hand a significant reduced MUTYH expression was detected in patients with MUTYH mutations vs control group (P < 0.05). Altered ASE of APC was detected in four out of eight APC mutation carriers. In particular one case showed a complete loss of one allele. Among APC mutation negative cases, 4 out of 13 showed a moderate ASE. ASE of MUTYH did not show any altered expression in the cases analyzed. Spearman's Rho Test analysis showed a positive and significant correlation between APC and MUTYH genes both in cases and in controls (P = 0.020 and P < 0.001). APC and MUTYH showed a reduced germline expression, not always corresponding to gene mutation. Expression of APC is decreased in mutation negative cases and this appears to be a promising indicator of FAP predisposition, while for MUTYH gene, mutation is associated to reduced mRNA expression. This study could improve the predictive genetic diagnosis of at-risk individuals belonging to families with reduced mRNA expression regardless of presence of mutation.

Cooperative DNA binding and sequence discrimination by the Opaque2 bZIP factor.

PubMed Central

Yunes, J A; Vettore, A L; da Silva, M J; Leite, A; Arruda, P

1998-01-01

The maize Opaque2 (O2) protein is a basic leucine zipper transcription factor that controls the expression of distinct classes of endosperm genes through the recognition of different cis-acting elements in their promoters. The O2 target region in the promoter of the alpha-coixin gene was analyzed in detail and shown to comprise two closely adjacent binding sites, named O2u and O2d, which are related in sequence to the GCN4 binding site. Quantitative DNase footprint analysis indicated that O2 binding to alpha-coixin target sites is best described by a cooperative model. Transient expression assays showed that the two adjacent sites act synergistically. This synergy is mediated in part by cooperative DNA binding. In tobacco protoplasts, O2 binding at the O2u site is more important for enhancer activity than is binding at the O2d site, suggesting that the architecture of the O2-DNA complex is important for interaction with the transcriptional machinery. PMID:9811800

Cooperative DNA binding and sequence discrimination by the Opaque2 bZIP factor.

PubMed

Yunes, J A; Vettore, A L; da Silva, M J; Leite, A; Arruda, P

1998-11-01

The maize Opaque2 (O2) protein is a basic leucine zipper transcription factor that controls the expression of distinct classes of endosperm genes through the recognition of different cis-acting elements in their promoters. The O2 target region in the promoter of the alpha-coixin gene was analyzed in detail and shown to comprise two closely adjacent binding sites, named O2u and O2d, which are related in sequence to the GCN4 binding site. Quantitative DNase footprint analysis indicated that O2 binding to alpha-coixin target sites is best described by a cooperative model. Transient expression assays showed that the two adjacent sites act synergistically. This synergy is mediated in part by cooperative DNA binding. In tobacco protoplasts, O2 binding at the O2u site is more important for enhancer activity than is binding at the O2d site, suggesting that the architecture of the O2-DNA complex is important for interaction with the transcriptional machinery.

Comprehensive analysis of the dynamic structure of nuclear localization signals.

PubMed

Yamagishi, Ryosuke; Okuyama, Takahide; Oba, Shuntaro; Shimada, Jiro; Chaen, Shigeru; Kaneko, Hiroki

2015-12-01

Most transcription and epigenetic factors in eukaryotic cells have nuclear localization signals (NLSs) and are transported to the nucleus by nuclear transport proteins. Understanding the features of NLSs and the mechanisms of nuclear transport might help understand gene expression regulation, somatic cell reprogramming, thus leading to the treatment of diseases associated with abnormal gene expression. Although many studies analyzed the amino acid sequence of NLSs, few studies investigated their three-dimensional structure. Therefore, we conducted a statistical investigation of the dynamic structure of NLSs by extracting the conformation of these sequences from proteins examined by X-ray crystallography and using a quantity defined as conformational determination rate (a ratio between the number of amino acids determining the conformation and the number of all amino acids included in a certain region). We found that determining the conformation of NLSs is more difficult than determining the conformation of other regions and that NLSs may tend to form more heteropolymers than monomers. Therefore, these findings strongly suggest that NLSs are intrinsically disordered regions.

A worldwide survey of genome sequence variation provides insight into the evolutionary history of the honeybee Apis mellifera.

PubMed

Wallberg, Andreas; Han, Fan; Wellhagen, Gustaf; Dahle, Bjørn; Kawata, Masakado; Haddad, Nizar; Simões, Zilá Luz Paulino; Allsopp, Mike H; Kandemir, Irfan; De la Rúa, Pilar; Pirk, Christian W; Webster, Matthew T

2014-10-01

The honeybee Apis mellifera has major ecological and economic importance. We analyze patterns of genetic variation at 8.3 million SNPs, identified by sequencing 140 honeybee genomes from a worldwide sample of 14 populations at a combined total depth of 634×. These data provide insight into the evolutionary history and genetic basis of local adaptation in this species. We find evidence that population sizes have fluctuated greatly, mirroring historical fluctuations in climate, although contemporary populations have high genetic diversity, indicating the absence of domestication bottlenecks. Levels of genetic variation are strongly shaped by natural selection and are highly correlated with patterns of gene expression and DNA methylation. We identify genomic signatures of local adaptation, which are enriched in genes expressed in workers and in immune system- and sperm motility-related genes that might underlie geographic variation in reproduction, dispersal and disease resistance. This study provides a framework for future investigations into responses to pathogens and climate change in honeybees.

Discovery of genes related to insecticide resistance in Bactrocera dorsalis by functional genomic analysis of a de novo assembled transcriptome.

PubMed

Hsu, Ju-Chun; Chien, Ting-Ying; Hu, Chia-Cheng; Chen, Mei-Ju May; Wu, Wen-Jer; Feng, Hai-Tung; Haymer, David S; Chen, Chien-Yu

2012-01-01

Insecticide resistance has recently become a critical concern for control of many insect pest species. Genome sequencing and global quantization of gene expression through analysis of the transcriptome can provide useful information relevant to this challenging problem. The oriental fruit fly, Bactrocera dorsalis, is one of the world's most destructive agricultural pests, and recently it has been used as a target for studies of genetic mechanisms related to insecticide resistance. However, prior to this study, the molecular data available for this species was largely limited to genes identified through homology. To provide a broader pool of gene sequences of potential interest with regard to insecticide resistance, this study uses whole transcriptome analysis developed through de novo assembly of short reads generated by next-generation sequencing (NGS). The transcriptome of B. dorsalis was initially constructed using Illumina's Solexa sequencing technology. Qualified reads were assembled into contigs and potential splicing variants (isotigs). A total of 29,067 isotigs have putative homologues in the non-redundant (nr) protein database from NCBI, and 11,073 of these correspond to distinct D. melanogaster proteins in the RefSeq database. Approximately 5,546 isotigs contain coding sequences that are at least 80% complete and appear to represent B. dorsalis genes. We observed a strong correlation between the completeness of the assembled sequences and the expression intensity of the transcripts. The assembled sequences were also used to identify large numbers of genes potentially belonging to families related to insecticide resistance. A total of 90 P450-, 42 GST-and 37 COE-related genes, representing three major enzyme families involved in insecticide metabolism and resistance, were identified. In addition, 36 isotigs were discovered to contain target site sequences related to four classes of resistance genes. Identified sequence motifs were also analyzed to characterize putative polypeptide translational products and associate them with specific genes and protein functions.

Single Cell Transcriptomics of Hypothalamic Warm Sensitive Neurons that Control Core Body Temperature and Fever Response

PubMed Central

Eberwine, James; Bartfai, Tamas

2011-01-01

We report on an ‘unbiased’ molecular characterization of individual, adult neurons, active in a central, anterior hypothalamic neuronal circuit, by establishing cDNA libraries from each individual, electrophysiologically identified warm sensitive neuron (WSN). The cDNA libraries were analyzed by Affymetrix microarray. The presence and frequency of cDNAs was confirmed and enhanced with Illumina sequencing of each single cell cDNA library. cDNAs encoding the GABA biosynthetic enzyme. GAD1 and of adrenomedullin, galanin, prodynorphin, somatostatin, and tachykinin were found in the WSNs. The functional cellular and in vivo studies on dozens of the more than 500 neurotransmitter -, hormone- receptors and ion channels, whose cDNA was identified and sequence confirmed, suggest little or no discrepancy between the transcriptional and functional data in WSNs; whenever agonists were available for a receptor whose cDNA was identified, a functional response was found.. Sequencing single neuron libraries permitted identification of rarely expressed receptors like the insulin receptor, adiponectin receptor2 and of receptor heterodimers; information that is lost when pooling cells leads to dilution of signals and mixing signals. Despite the common electrophysiological phenotype and uniform GAD1 expression, WSN- transcriptomes show heterogenity, suggesting strong epigenetic influence on the transcriptome. Our study suggests that it is well-worth interrogating the cDNA libraries of single neurons by sequencing and chipping. PMID:20970451

Coordinate cytokine regulatory sequences

DOEpatents

Frazer, Kelly A.; Rubin, Edward M.; Loots, Gabriela G.

2005-05-10

The present invention provides CNS sequences that regulate the cytokine gene expression, expression cassettes and vectors comprising or lacking the CNS sequences, host cells and non-human transgenic animals comprising the CNS sequences or lacking the CNS sequences. The present invention also provides methods for identifying compounds that modulate the functions of CNS sequences as well as methods for diagnosing defects in the CNS sequences of patients.

Metabolism and Genetics of Helicobacter pylori: the Genome Era

PubMed Central

Marais, Armelle; Mendz, George L.; Hazell, Stuart L.; Mégraud, Francis

1999-01-01

The publication of the complete sequence of Helicobacter pylori 26695 in 1997 and more recently that of strain J99 has provided new insight into the biology of this organism. In this review, we attempt to analyze and interpret the information provided by sequence annotations and to compare these data with those provided by experimental analyses. After a brief description of the general features of the genomes of the two sequenced strains, the principal metabolic pathways are analyzed. In particular, the enzymes encoded by H. pylori involved in fermentative and oxidative metabolism, lipopolysaccharide biosynthesis, nucleotide biosynthesis, aerobic and anaerobic respiration, and iron and nitrogen assimilation are described, and the areas of controversy between the experimental data and those provided by the sequence annotation are discussed. The role of urease, particularly in pH homeostasis, and other specialized mechanisms developed by the bacterium to maintain its internal pH are also considered. The replicational, transcriptional, and translational apparatuses are reviewed, as is the regulatory network. The numerous findings on the metabolism of the bacteria and the paucity of gene expression regulation systems are indicative of the high level of adaptation to the human gastric environment. Arguments in favor of the diversity of H. pylori and molecular data reflecting possible mechanisms involved in this diversity are presented. Finally, we compare the numerous experimental data on the colonization factors and those provided from the genome sequence annotation, in particular for genes involved in motility and adherence of the bacterium to the gastric tissue. PMID:10477311

[Cloning and bioinformatics analysis of abscisic acid 8'-hydroxylase from Pseudostellariae Radix].

PubMed

Li, Jun; Long, Deng-Kai; Zhou, Tao; Ding, Ling; Zheng, Wei; Jiang, Wei-Ke

2016-07-01

Abscisic acid 8'-hydroxylase was one of key enzymes genes in the metabolism of abscisic acid (ABA). Seven menbers of abscisic acid 8'-hydroxylase were identified from Pseudostellaria heterophylla transcriptome sequencing results by using sequence homology. The expression profiles of these genes were analyzed by transcriptome data. The coding sequence of ABA8ox1 was cloned and analyzed by informational technology. The full-length cDNA of ABA8ox1 was 1 401 bp,with 480 encoded amino acids. The predicated isoelectric point (pI) and relative molecular mass (MW) were 8.55 and 53 kDa,respectively. Transmembrane structure analysis showed that there were 21 amino acids in-side and 445 amino acids out-side. High level of transcripts can detect in bark of root and fibrous root. Multi-alignment and phylogenetic analysis both show that ABA8ox1 had a high similarity with the CYP707As from other plants,especially with AtCYP707A1 and AtCYP707A3 in Arabidopsis thaliana. These results lay a foundation for molecular mechanism of tuberous root expanding and response to adversity stress. Copyright© by the Chinese Pharmaceutical Association.

RHOMBOID DOMAIN CONTAINING 2 (RHBDD2)

PubMed Central

Abba, MC; Lacunza, E; Nunez, MI; Colussi, A; Isla-Larrain, M; Segal-Eiras, A; Croce, MV; Aldaz, CM

2009-01-01

In the course of breast cancer global gene expression studies, we identified an uncharacterized gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly over-expressed in primary tumors from patients with recurrent disease. In this study, we identified RHBDD2 mRNA and protein expression significantly elevated in breast carcinomas compared with normal breast samples as analyzed by SAGE (n=46) and immunohistochemistry (n=213). Interestingly, specimens displaying RHBDD2 over-expression were predominantly advanced stage III breast carcinomas (p=0.001). Western-blot, RT-PCR and cDNA sequencing analyses allowed us to identify two RHBDD2 alternatively spliced mRNA isoforms expressed in breast cancer cell lines. We further investigated the occurrence and frequency of gene amplification and over-expression affecting RHBDD2 in 131 breast samples. RHBDD2 gene amplification was detected in 21% of 98 invasive breast carcinomas analyzed. However, no RHBDD2 amplification was detected in normal breast tissues (n=17) or breast benign lesions (n=16) (p=0.014). Interestingly, siRNA mediated silencing of RHBDD2 expression results in a decrease of MCF7 breast cancer cells proliferation compared with the corresponding controls (p=0.001). In addition, analysis of publicly available gene expression data showed a strong association between high RHBDD2 expression and decreased overall survival (p=0.0023), relapse-free survival (p= 0.0013), and metastasis-free interval (p=0.006) in patients with primary ER-negative breast carcinomas. In conclusion, our findings suggest that RHBDD2 over-expression behaves as an indicator of poor prognosis and may play a role facilitating breast cancer progression. PMID:19616622

Deep sequencing of small RNA libraries from human prostate epithelial and stromal cells reveal distinct pattern of microRNAs primarily predicted to target growth factors.

PubMed

Singh, Savita; Zheng, Yun; Jagadeeswaran, Guru; Ebron, Jey Sabith; Sikand, Kavleen; Gupta, Sanjay; Sunker, Ramanjulu; Shukla, Girish C

2016-02-28

Complex epithelial and stromal cell interactions are required during the development and progression of prostate cancer. Regulatory small non-coding microRNAs (miRNAs) participate in the spatiotemporal regulation of messenger RNA (mRNA) and regulation of translation affecting a large number of genes involved in prostate carcinogenesis. In this study, through deep-sequencing of size fractionated small RNA libraries we profiled the miRNAs of prostate epithelial (PrEC) and stromal (PrSC) cells. Over 50 million reads were obtained for PrEC in which 860,468 were unique sequences. Similarly, nearly 76 million reads for PrSC were obtained in which over 1 million were unique reads. Expression of many miRNAs of broadly conserved and poorly conserved miRNA families were identified. Sixteen highly expressed miRNAs with significant change in expression in PrSC than PrEC were further analyzed in silico. ConsensusPathDB showed the target genes of these miRNAs were significantly involved in adherence junction, cell adhesion, EGRF, TGF-β and androgen signaling. Let-7 family of tumor-suppressor miRNAs expression was highly pervasive in both, PrEC and PrSC cells. In addition, we have also identified several miRNAs that are unique to PrEC or PrSC cells and their predicted putative targets are a group of transcription factors. This study provides perspective on the miRNA expression in PrEC and PrSC, and reveals a global trend in miRNA interactome. We conclude that the most abundant miRNAs are potential regulators of development and differentiation of the prostate gland by targeting a set of growth factors. Additionally, high level expression of the most members of let-7 family miRNAs suggests their role in the fine tuning of the growth and proliferation of prostate epithelial and stromal cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

[Cloning, Expression and Immunodiagnostic Evaluation of the Fasciola gigantica Thioredoxin Peroxidase].

PubMed

Wang, Yue-qi; Zhou, Yan; Cheng, Na; Chen, Mu-xin; Ai, Lin; Liu, Yu-hua; Zhang, Jian-guo; Luo, Jia-jun; Xu, Xue-nian

2015-04-01

To immunoscreen the gene encoding thioredoxin peroxidase (TPx) from a cDNA library made from adult Fasciola gigantica worms, clone and express the gene, and evaluate the immunodiagnostic value of TPx recombinant protein. The A ZAP cDNA library was immunoscreened with pooled serum of fascioliasis gigantica patients. The obtained positive clones were sequenced and analyzed by multiple sequence alignment. The full-length (rFgTPx) and N-termianal truncated (rFgTPx_nt) sequence of FgTPx was subcloned into prokaryotic plasmid pET28a(+) with a non-fusion expression technique, respectively. The recombinant proteins of rFgTPx and rFgTPx_nt were purified by His-bind affinity column (Ni-NTA). rFgTPx and rFgTPx_nt were used in indirect ELISA to test the antibody response of the serum samples. Sera of 27 fascioliasis gigantica patients, 15 patients with schistosomaisis japonica, 15 clonorchiasis sinensis patients, and 32 healthy donors were tested by using the recombinant protein based ELISA. The TPx recombinant proteins were obtained through expression, purification and renaturation, the relative molecular mass of rFgTPx and rFgTPx_nt were Mr 30,000 and Mr 26,000, respectively. The total diagnostic coincidence rate, sensitivity and specificity of rFgTPx_nt-based ELISA was 87.6% (78/89), 66.7% (18/27), and 96.8% (60/62), respectively. The cross reaction with Schistosoma japonicum and Clonorchis sinensis was 0 and 1/15 for rFgTPx_nt, respectively. Before and after treatment, A450 value of the serum samples from fascioliasis patients was 0.233 ± 0.088 and 0.129 ± 0.072, respectively (t = 4.27, P < 0.01). The gene encoding TPx is expressed in the prokaryotic expression system. The recombinant protein shows proper sensitivity and high specificity for the serodiagnosis of Fasciola gigantica infection.

Study on the zona pellucida 4 (ZP4) gene sequence and its expression in the ovaries of patients with polycystic ovary syndrome.

PubMed

Meczekalski, B; Nawrot, R; Nowak, W; Czyzyk, A; Kedzia, H; Gozdzicka-Jozefiak, A

2015-07-01

Polycystic ovary syndrome (PCOS) is a common endocrine disorder of unknown pathology, involving reproductive and metabolic abnormalities. Oocyte-specific genes are a group of genes expressed exclusively in ovarian tissue; therefore, they can play an important role in ovarian pathologies such as PCOS. The zona pellucida 4 (ZP4) gene encodes glycoprotein which is a part of the extracellular matrix of oocyte. We analyzed 87 patients with PCOS, which were divided into four groups depending on their phenotype. In each patient, we performed profound clinical and biochemical analysis, including the measurement of serum androgens. The ovarian tissue samples were used to perform a real-time polymerase chain reaction and immunohistochemical staining using anti-ZP4 monoclonal antibodies. The ZP4 gene was sequenced from peripheral lymphocytes. The expression of ZP4 was present in early antral follicles and was stronger in mature follicles. The subgroup of patients with eumenorrhea and without hyperandrogenism presented the highest expression of ZP4 in ovarian tissue. In one case, we found a mutation of the ZP4 gene. No correlations were found between the ZP4 expression level and biochemical or clinical indices. Data from this and animal studies suggest a possible relationship between androgens and ZP4 expression. ZP4 expression is highest among patients with PCOS and a regular cycle, and this is a consequence of the presence of mature follicles in this group. In some patients with PCOS and infertility, ZP4 mutation can be found.

Gene expression analysis of E. coli strains provides insights into the role of gene regulation in diversification

PubMed Central

Vital, Marius; Chai, Benli; Østman, Bjørn; Cole, James; Konstantinidis, Konstantinos T; Tiedje, James M

2015-01-01

Escherichia coli spans a genetic continuum from enteric strains to several phylogenetically distinct, atypical lineages that are rare in humans, but more common in extra-intestinal environments. To investigate the link between gene regulation, phylogeny and diversification in this species, we analyzed global gene expression profiles of four strains representing distinct evolutionary lineages, including a well-studied laboratory strain, a typical commensal (enteric) strain and two environmental strains. RNA-Seq was employed to compare the whole transcriptomes of strains grown under batch, chemostat and starvation conditions. Highly differentially expressed genes showed a significantly lower nucleotide sequence identity compared with other genes, indicating that gene regulation and coding sequence conservation are directly connected. Overall, distances between the strains based on gene expression profiles were largely dependent on the culture condition and did not reflect phylogenetic relatedness. Expression differences of commonly shared genes (all four strains) and E. coli core genes were consistently smaller between strains characterized by more similar primary habitats. For instance, environmental strains exhibited increased expression of stress defense genes under carbon-limited growth and entered a more pronounced survival-like phenotype during starvation compared with other strains, which stayed more alert for substrate scavenging and catabolism during no-growth conditions. Since those environmental strains show similar genetic distance to each other and to the other two strains, these findings cannot be simply attributed to genetic relatedness but suggest physiological adaptations. Our study provides new insights into ecologically relevant gene-expression and underscores the role of (differential) gene regulation for the diversification of the model bacterial species. PMID:25343512

Molecular cloning, sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system.

PubMed

Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Kumar, Surender; Kumar, Sudarshan; Mohanty, Ashok K; Kaushik, Jai K; Malakar, Dhruba

2014-01-01

Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.

Interpretable dimensionality reduction of single cell transcriptome data with deep generative models.

PubMed

Ding, Jiarui; Condon, Anne; Shah, Sohrab P

2018-05-21

Single-cell RNA-sequencing has great potential to discover cell types, identify cell states, trace development lineages, and reconstruct the spatial organization of cells. However, dimension reduction to interpret structure in single-cell sequencing data remains a challenge. Existing algorithms are either not able to uncover the clustering structures in the data or lose global information such as groups of clusters that are close to each other. We present a robust statistical model, scvis, to capture and visualize the low-dimensional structures in single-cell gene expression data. Simulation results demonstrate that low-dimensional representations learned by scvis preserve both the local and global neighbor structures in the data. In addition, scvis is robust to the number of data points and learns a probabilistic parametric mapping function to add new data points to an existing embedding. We then use scvis to analyze four single-cell RNA-sequencing datasets, exemplifying interpretable two-dimensional representations of the high-dimensional single-cell RNA-sequencing data.

Analysis of differentially expressed genes between fluoride-sensitive and fluoride-endurable individuals in midgut of silkworm, Bombyx mori.

PubMed

Qian, Heying; Li, Gang; He, Qingling; Zhang, Huaguang; Xu, Anying

2016-08-15

Fluoride tolerance is an economically important trait of silkworm. Near-isogenic lines (NILs) of the dominant endurance to fluoride (Def) gene in Bombyx mori has been constructed before. Here, we analyzed the gene expression profiles of midgut of fluoride-sensitive and fluoride-endurable individuals of Def NILs by using high-throughput Illumina sequencing technology and bioinformatics tools, and identified differentially expressed genes between these individuals. A total of 3,612,399 and 3,567,631 clean tags for the libraries of fluoride-endurable and fluoride-sensitive individuals were obtained, which corresponded to 32,933 and 43,976 distinct clean tags, respectively. Analysis of differentially expressed genes indicates that 241 genes are differentially expressed between the two libraries. Among the 241 genes, 30 are up-regulated and 211 are down-regulated in fluoride-endurable individuals. Pathway enrichment analysis demonstrates that genes related to ribosomes, pancreatic secretion, steroid biosynthesis, glutathione metabolism, steroid biosynthesis, and glycerolipid metabolism are down-regulated in fluoride-endurable individuals. qRT-PCR was conducted to confirm the results of the DGE. The present study analyzed differential expression of related genes and tried to find out whether the crucial genes were related to fluoride detoxification which might elucidate fluoride effect and provide a new way in the fluorosis research. Copyright © 2016 Elsevier B.V. All rights reserved.

Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

PubMed Central

Ngô, V; Gourdji, D; Laverrière, J N

1996-01-01

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo. PMID:8668139

Cloud-scale genomic signals processing classification analysis for gene expression microarray data.

PubMed

Harvey, Benjamin; Soo-Yeon Ji

2014-01-01

As microarray data available to scientists continues to increase in size and complexity, it has become overwhelmingly important to find multiple ways to bring inference though analysis of DNA/mRNA sequence data that is useful to scientists. Though there have been many attempts to elucidate the issue of bringing forth biological inference by means of wavelet preprocessing and classification, there has not been a research effort that focuses on a cloud-scale classification analysis of microarray data using Wavelet thresholding in a Cloud environment to identify significantly expressed features. This paper proposes a novel methodology that uses Wavelet based Denoising to initialize a threshold for determination of significantly expressed genes for classification. Additionally, this research was implemented and encompassed within cloud-based distributed processing environment. The utilization of Cloud computing and Wavelet thresholding was used for the classification 14 tumor classes from the Global Cancer Map (GCM). The results proved to be more accurate than using a predefined p-value for differential expression classification. This novel methodology analyzed Wavelet based threshold features of gene expression in a Cloud environment, furthermore classifying the expression of samples by analyzing gene patterns, which inform us of biological processes. Moreover, enabling researchers to face the present and forthcoming challenges that may arise in the analysis of data in functional genomics of large microarray datasets.

Cloning and functional analysis of the promoter region of the human Disc large gene.

PubMed

Cavatorta, Ana Laura; Giri, Adriana A; Banks, Lawrence; Gardiol, Daniela

2008-11-15

A number of studies have demonstrated the involvement of human Disc large (DLG1) in the control of both cell polarity and maintenance of tissue architecture. However, the mechanisms controlling DLG1 transcription are not fully understood. This is relevant since DLG1 is lost in many tumours during the later stages of malignant progression. Therefore, we performed the cloning and functional analysis of a genomic 5' flanking region of the DLG1 open reading frame with promoter activity. We analyzed the activity of a series of 5' deletion constructs of the DLG1 promoter and determined the minimal essential sequences that are required for promoter activity as well as cis-elements that regulate transcription. We found, within the DLG1 promoter sequences, consensus-binding sites for the Snail family of transcription factors that repress the expression of epithelial markers and are up-regulated in a variety of tumours. Snail transcription factors repress the transcriptional activity of the DLG1 promoter and, ectopically expressed Snail proteins bind to the native DLG1 promoter. These data suggest a role for Snail transcription factors in the control of DLG1 expression and provide a basis for understanding the transcriptional regulation of DLG1.

Transcriptome responses to heat- and cold-stress in ladybirds (Cryptolaemus montrouzieri Mulasnt) analyzed by deep-sequencing.

PubMed

Zhang, Yuhong; Wu, Hongsheng; Xie, Jiaqin; Jiang, Ruixin; Deng, Congshuang; Pang, Hong

2015-11-19

Changed temperature not only threaten agricultural production, but they also affect individual biological behavior, population and community of many insects, and consequently reduce the stability of our ecosystem. Insect's ability to respond to temperature stress evolved through a complex adaptive process, thus resulting in varied temperature tolerance among different insects. Both high and low extreme temperatures are detrimental to insect development since they constitute an important abiotic stress capable of inducing abnormal biological responses. Many studies on heat or cold tolerance of ladybirds have focused on measurements of physiological and biochemical indexes such as supercooling point, higher/lower lethal temperatures, survival rate, dry body weight, water content, and developmental duration. And studies of the molecular mechanisms of ladybird responses to heat or cold stress have focused on single genes, such as those encoding heat shock proteins, but has not been analyzed by transcriptome profiling. In this study, we report the use of Digital Gene Expression (DGE) tag profiling to gain insight into transcriptional events associated with heat- and cold-stress in C. montrouzieri. About 6 million tags (49 bp in length) were sequenced in a heat stress group, a cold stress group and a negative control group. We obtained 687 and 573 genes that showed significantly altered expression levels following heat and cold shock treatments, respectively. Analysis of the global gene expression pattern suggested that 42 enzyme-encoding genes mapped to many Gene Ontology terms are associated with insect's response to heat- and cold-stress. These results provide a global assessment of genes and molecular mechanisms involved in heat and cold tolerance.

Identification of a novel prophage-like gene cluster actively expressed in both virulent and avirulent strains of Leptospira interrogans serovar Lai.

PubMed

Qin, Jin-Hong; Zhang, Qing; Zhang, Zhi-Ming; Zhong, Yi; Yang, Yang; Hu, Bao-Yu; Zhao, Guo-Ping; Guo, Xiao-Kui

2008-06-01

DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains but concomitantly upregulated in strain 56601 in contrast to that of IPAV. Reverse transcription-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 of 34 sequences encoding prophage-like proteins, of which the LA0195 protein is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II, and III, all proximal to the LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT) activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to the LA0195 protein in vitro. These results suggested that the LA0195 protein is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems residing outside of this region within the genome may be responsible for the differential expression profiling in these two strains.

Development and confirmation of potential gene classifiers of human clear cell renal cell carcinoma using next-generation RNA sequencing.

PubMed

Eikrem, Oystein S; Strauss, Philipp; Beisland, Christian; Scherer, Andreas; Landolt, Lea; Flatberg, Arnar; Leh, Sabine; Beisvag, Vidar; Skogstrand, Trude; Hjelle, Karin; Shresta, Anjana; Marti, Hans-Peter

2016-12-01

A previous study by this group demonstrated the feasibility of RNA sequencing (RNAseq) technology for capturing disease biology of clear cell renal cell carcinoma (ccRCC), and presented initial results for carbonic anhydrase-9 (CA9) and tumor necrosis factor-α-induced protein-6 (TNFAIP6) as possible biomarkers of ccRCC (discovery set) [Eikrem et al. PLoS One 2016;11:e0149743]. To confirm these results, the previous study is expanded, and RNAseq data from additional matched ccRCC and normal renal biopsies are analyzed (confirmation set). Two core biopsies from patients (n = 12) undergoing partial or full nephrectomy were obtained with a 16 g needle. RNA sequencing libraries were generated with the Illumina TruSeq ® Access library preparation protocol. Comparative analysis was done using linear modeling (voom/Limma; R Bioconductor). The formalin-fixed and paraffin-embedded discovery and confirmation data yielded 8957 and 11,047 detected transcripts, respectively. The two data sets shared 1193 of differentially expressed genes with each other. The average expression and the log 2 -fold changes of differentially expressed transcripts in both data sets correlated, with R²   =   .95 and R²   =   .94, respectively. Among transcripts with the highest fold changes were CA9, neuronal pentraxin-2 and uromodulin. Epithelial-mesenchymal transition was highlighted by differential expression of, for example, transforming growth factor-β 1 and delta-like ligand-4. The diagnostic accuracy of CA9 was 100% and 93.9% when using the discovery set as the training set and the confirmation data as the test set, and vice versa, respectively. These data further support TNFAIP6 as a novel biomarker of ccRCC. TNFAIP6 had combined accuracy of 98.5% in the two data sets. This study provides confirmatory data on the potential use of CA9 and TNFAIP6 as biomarkers of ccRCC. Thus, next-generation sequencing expands the clinical application of tissue analyses.

Identification and characterization of calcium transporter gene family in finger millet in relation to grain calcium content.

PubMed

Singh, Uma M; Metwal, Mamta; Singh, Manoj; Taj, Gohar; Kumar, Anil

2015-07-15

Calcium (Ca) is an essential mineral for proper growth and development of plants as well as animals. In plants including cereals, calcium is deposited in seed during its development which is mediated by specialized Ca transporters. Common cereal seeds contain very low amounts of Ca while the finger millet (Eleusine coracana) contains exceptionally high amounts of Ca in seed. In order to understand the role of Ca transporters in grain Ca accumulation, developing seed transcriptome of two finger millet genotypes (GP-1, low Ca and GP-45 high Ca) differing in seed Ca content was sequenced using Illumina paired-end sequencing technology and members of Ca transporter gene family were identified. Out of 109,218 and 120,130 contigs, 86 and 81 contigs encoding Ca transporters were identified in GP-1 and GP-45, respectively. After removal of redundant sequences, a total of 19 sequences were confirmed as Ca transporter genes, which includes 11 Ca(2+) ATPases, 07 Ca(2+)/cation exchangers and 01 Ca(2+) channel. The differential expressions of all genes were analyzed from transcriptome data and it was observed that 9 and 3 genes were highly expressed in GP-45 and GP-1 genotypes respectively. Validation of transcriptome expression data of selected Ca transporter genes was performed on different stages of developing spikes of both genotypes grown under different concentrations of exogenous Ca. In both genotypes, significant correlation was observed between the expression of these genes, especially EcCaX3, and on the amount of Ca accumulated in seed. The positive correlation of seed mass with the amount of Ca concentration was also observed. The efficient Ca transport property and responsiveness of EcCAX3 towards exogenous Ca could be utilized in future biofortification program. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteomic Analysis of Parkin Isoforms Expression in Different Rat Brain Areas.

PubMed

D'Amico, Agata Grazia; Maugeri, Grazia; Reitano, Rita; Cavallaro, Sebastiano; D'Agata, Velia

2016-10-01

PARK2 gene's mutations are related to the familial form of juvenile Parkinsonism, also known as the autosomic recessive juvenile Parkinsonism. This gene encodes for parkin, a 465-amino acid protein. To date, a large number of parkin isoforms, generated by an alternative splicing mechanism, have been described. Currently, Gene Bank lists 27 rat PARK2 transcripts, which matches to 20 exclusive parkin alternative splice variants. Despite the existence of these isoforms, most of the studies carried out so far, have been focused only on the originally cloned parkin. In this work we have analyzed the expression profile of parkin isoforms in some rat brain areas including prefrontal cortex, hippocampus, substantia nigra and cerebellum. To discriminate among these isoforms, we detected their localization through the use of two antibodies that are able to identify different domains of the parkin canonical sequence. Our analysis has revealed that at least fourteen parkin isoforms are expressed in rat brain with a various distribution in the regions analyzed. Our study might help to elucidate the pathophysiological role of these proteins in the central nervous system.

SAMSA2: a standalone metatranscriptome analysis pipeline.

PubMed

Westreich, Samuel T; Treiber, Michelle L; Mills, David A; Korf, Ian; Lemay, Danielle G

2018-05-21

Complex microbial communities are an area of growing interest in biology. Metatranscriptomics allows researchers to quantify microbial gene expression in an environmental sample via high-throughput sequencing. Metatranscriptomic experiments are computationally intensive because the experiments generate a large volume of sequence data and each sequence must be compared with reference sequences from thousands of organisms. SAMSA2 is an upgrade to the original Simple Annotation of Metatranscriptomes by Sequence Analysis (SAMSA) pipeline that has been redesigned for standalone use on a supercomputing cluster. SAMSA2 is faster due to the use of the DIAMOND aligner, and more flexible and reproducible because it uses local databases. SAMSA2 is available with detailed documentation, and example input and output files along with examples of master scripts for full pipeline execution. SAMSA2 is a rapid and efficient metatranscriptome pipeline for analyzing large RNA-seq datasets in a supercomputing cluster environment. SAMSA2 provides simplified output that can be examined directly or used for further analyses, and its reference databases may be upgraded, altered or customized to fit the needs of any experiment.

Methods and compositions for regulating gene expression in plant cells

NASA Technical Reports Server (NTRS)

Dai, Shunhong (Inventor); Beachy, Roger N. (Inventor); Luis, Maria Isabel Ordiz (Inventor)

2010-01-01

Novel chimeric plant promoter sequences are provided, together with plant gene expression cassettes comprising such sequences. In certain preferred embodiments, the chimeric plant promoters comprise the BoxII cis element and/or derivatives thereof. In addition, novel transcription factors are provided, together with nucleic acid sequences encoding such transcription factors and plant gene expression cassettes comprising such nucleic acid sequences. In certain preferred embodiments, the novel transcription factors comprise the acidic domain, or fragments thereof, of the RF2a transcription factor. Methods for using the chimeric plant promoter sequences and novel transcription factors in regulating the expression of at least one gene of interest are provided, together with transgenic plants comprising such chimeric plant promoter sequences and novel transcription factors.

Translational bioinformatics: linking the molecular world to the clinical world.

PubMed

Altman, R B

2012-06-01

Translational bioinformatics represents the union of translational medicine and bioinformatics. Translational medicine moves basic biological discoveries from the research bench into the patient-care setting and uses clinical observations to inform basic biology. It focuses on patient care, including the creation of new diagnostics, prognostics, prevention strategies, and therapies based on biological discoveries. Bioinformatics involves algorithms to represent, store, and analyze basic biological data, including DNA sequence, RNA expression, and protein and small-molecule abundance within cells. Translational bioinformatics spans these two fields; it involves the development of algorithms to analyze basic molecular and cellular data with an explicit goal of affecting clinical care.

Next-generation sequencing facilitates quantitative analysis of wild-type and Nrl−/− retinal transcriptomes

PubMed Central

Brooks, Matthew J.; Rajasimha, Harsha K.; Roger, Jerome E.

2011-01-01

Purpose Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis. Methods Retinal mRNA profiles of 21-day-old wild-type (WT) and neural retina leucine zipper knockout (Nrl−/−) mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. PMID:22162623

Endo-beta-N-acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol.

PubMed

Suzuki, Tadashi; Yano, Keiichi; Sugimoto, Seiji; Kitajima, Ken; Lennarz, William J; Inoue, Sadako; Inoue, Yasuo; Emori, Yasufumi

2002-07-23

Formation of oligosaccharides occurs both in the cytosol and in the lumen of the endoplasmic reticulum (ER). Luminal oligosaccharides are transported into the cytosol to ensure that they do not interfere with proper functioning of the glycan-dependent quality control machinery in the lumen of the ER for newly synthesized glycoproteins. Once in the cytosol, free oligosaccharides are catabolized, possibly to maximize the reutilization of the component sugars. An endo-beta-N-acetylglucosaminidase (ENGase) is a key enzyme involved in the processing of free oligosaccharides in the cytosol. This enzyme activity has been widely described in animal cells, but the gene encoding this enzyme activity has not been reported. Here, we report the identification of the gene encoding human cytosolic ENGase. After 11 steps, the enzyme was purified 150,000-fold to homogeneity from hen oviduct, and several internal amino acid sequences were analyzed. Based on the internal sequence and examination of expressed sequence tag (EST) databases, we identified the human orthologue of the purified protein. The human protein consists of 743 aa and has no apparent signal sequence, supporting the idea that this enzyme is localized in the cytosol. By expressing the cDNA of the putative human ENGase in COS-7 cells, the enzyme activity in the soluble fraction was enhanced 100-fold over the basal level, confirming that the human gene identified indeed encodes for ENGase. Careful gene database surveys revealed the occurrence of ENGase homologues in Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana, indicating the broad occurrence of ENGase in higher eukaryotes. This gene was expressed in a variety of human tissues, suggesting that this enzyme is involved in basic biological processes in eukaryotic cells.

Whole genome sequencing of an African American family highlights toll like receptor 6 variants in Kawasaki disease susceptibility.

PubMed

Kim, Jihoon; Shimizu, Chisato; Kingsmore, Stephen F; Veeraraghavan, Narayanan; Levy, Eric; Ribeiro Dos Santos, Andre M; Yang, Hai; Flatley, Jay; Hoang, Long Truong; Hibberd, Martin L; Tremoulet, Adriana H; Harismendy, Olivier; Ohno-Machado, Lucila; Burns, Jane C

2017-01-01

Kawasaki disease (KD) is the most common acquired pediatric heart disease. We analyzed Whole Genome Sequences (WGS) from a 6-member African American family in which KD affected two of four children. We sought rare, potentially causative genotypes by sequentially applying the following WGS filters: sequence quality scores, inheritance model (recessive homozygous and compound heterozygous), predicted deleteriousness, allele frequency, genes in KD-associated pathways or with significant associations in published KD genome-wide association studies (GWAS), and with differential expression in KD blood transcriptomes. Biologically plausible genotypes were identified in twelve variants in six genes in the two affected children. The affected siblings were compound heterozygous for the rare variants p.Leu194Pro and p.Arg247Lys in Toll-like receptor 6 (TLR6), which affect TLR6 signaling. The affected children were also homozygous for three common, linked (r2 = 1) intronic single nucleotide variants (SNVs) in TLR6 (rs56245262, rs56083757 and rs7669329), that have previously shown association with KD in cohorts of European descent. Using transcriptome data from pre-treatment whole blood of KD subjects (n = 146), expression quantitative trait loci (eQTL) analyses were performed. Subjects homozygous for the intronic risk allele (A allele of TLR6 rs56245262) had differential expression of Interleukin-6 (IL-6) as a function of genotype (p = 0.0007) and a higher erythrocyte sedimentation rate at diagnosis. TLR6 plays an important role in pathogen-associated molecular pattern recognition, and sequence variations may affect binding affinities that in turn influence KD susceptibility. This integrative genomic approach illustrates how the analysis of WGS in multiplex families with a complex genetic disease allows examination of both the common disease-common variant and common disease-rare variant hypotheses.

Characterization of Stearoyl-CoA Desaturases from a Psychrophilic Antarctic Copepod, Tigriopus kingsejongensis.

PubMed

Jung, Woongsic; Kim, Eun Jae; Han, Se Jong; Choi, Han-Gu; Kim, Sanghee

2016-10-01

Stearoyl-CoA desaturase is a key regulator in fatty acid metabolism that catalyzes the desaturation of stearic acid to oleic acid and controls the intracellular levels of monounsaturated fatty acids (MUFAs). Two stearoyl-CoA desaturases (SCD, Δ9 desaturases) genes were identified in an Antarctic copepod, Tigriopus kingsejongensis, that was collected in a tidal pool near the King Sejong Station, King George Island, Antarctica. Full-length complementary DNA (cDNA) sequences of two T. kingsejongensis SCDs (TkSCDs) were obtained from next-generation sequencing and isolated by reverse transcription PCR. DNA sequence lengths of the open reading frames of TkSCD-1 and TkSCD-2 were determined to be 1110 and 681 bp, respectively. The molecular weights deduced from the corresponding genes were estimated to be 43.1 kDa (TkSCD-1) and 26.1 kDa (TkSCD-2). The amino acid sequences were compared with those of fatty acid desaturases and sterol desaturases from various organisms and used to analyze the relationships among TkSCDs. As assessed by heterologous expression of recombinant proteins in Escherichia coli, the enzymatic functions of both stearoyl-CoA desaturases revealed that the amount of C16:1 and C18:1 fatty acids increased by greater than 3-fold after induction with isopropyl β-D-thiogalactopyranoside. In particular, C18:1 fatty acid production increased greater than 10-fold in E. coli expressing TkSCD-1 and TkSCD-2. The results of this study suggest that both SCD genes from an Antarctic marine copepod encode a functional desaturase that is capable of increasing the amounts of palmitoleic acid and oleic acid in a prokaryotic expression system.

Comparative transgenic analysis of enhancers from the human SHOX and mouse Shox2 genomic regions.

PubMed

Rosin, Jessica M; Abassah-Oppong, Samuel; Cobb, John

2013-08-01

Disruption of presumptive enhancers downstream of the human SHOX gene (hSHOX) is a frequent cause of the zeugopodal limb defects characteristic of Léri-Weill dyschondrosteosis (LWD). The closely related mouse Shox2 gene (mShox2) is also required for limb development, but in the more proximal stylopodium. In this study, we used transgenic mice in a comparative approach to characterize enhancer sequences in the hSHOX and mShox2 genomic regions. Among conserved noncoding elements (CNEs) that function as enhancers in vertebrate genomes, those that are maintained near paralogous genes are of particular interest given their ancient origins. Therefore, we first analyzed the regulatory potential of a genomic region containing one such duplicated CNE (dCNE) downstream of mShox2 and hSHOX. We identified a strong limb enhancer directly adjacent to the mShox2 dCNE that recapitulates the expression pattern of the endogenous gene. Interestingly, this enhancer requires sequences only conserved in the mammalian lineage in order to drive strong limb expression, whereas the more deeply conserved sequences of the dCNE function as a neural enhancer. Similarly, we found that a conserved element downstream of hSHOX (CNE9) also functions as a neural enhancer in transgenic mice. However, when the CNE9 transgenic construct was enlarged to include adjacent, non-conserved sequences frequently deleted in LWD patients, the transgene drove expression in the zeugopodium of the limbs. Therefore, both hSHOX and mShox2 limb enhancers are coupled to distinct neural enhancers. This is the first report demonstrating the activity of cis-regulatory elements from the hSHOX and mShox2 genomic regions in mammalian embryos.

Insights in KIR2.1 channel structure and function by an evolutionary approach; cloning and functional characterization of the first reptilian inward rectifier channel KIR2.1, derived from the California kingsnake (Lampropeltis getula californiae).

PubMed

Houtman, Marien J C; Korte, Sanne M; Ji, Yuan; Kok, Bart; Vos, Marc A; Stary-Weinzinger, Anna; van der Heyden, Marcel A G

2014-10-03

Potassium inward rectifier KIR2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the KIR2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of KIR2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. KIR2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian KIR2.1 channels. Using a degenerated primer set we cloned the KCNJ2 coding regions from muscle tissue of turtle, snake, bear, quail and bream, and compared their deduced amino acid sequences with those of KIR2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed KIR2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle KIR2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 KIR2.1 sequences showed that all disease causing KIR2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake KIR2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC50 ∼6μM) inward rectifier currents. Copyright © 2014 Elsevier Inc. All rights reserved.

Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

PubMed

Booman, Marije; Borza, Tudor; Feng, Charles Y; Hori, Tiago S; Higgins, Brent; Culf, Adrian; Léger, Daniel; Chute, Ian C; Belkaid, Anissa; Rise, Marlies; Gamperl, A Kurt; Hubert, Sophie; Kimball, Jennifer; Ouellette, Rodney J; Johnson, Stewart C; Bowman, Sharen; Rise, Matthew L

2011-08-01

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.

Sequences 5' to translation start regulate expression of petunia rbcS genes.

PubMed Central

Dean, C; Favreau, M; Bedbrook, J; Dunsmuir, P

1989-01-01

The promoter sequences that contribute to quantitative differences in expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase have been characterized. The promoter regions of the two most abundantly expressed petunia rbcS genes, SSU301 and SSU611, show sequence similarity not present in other rbcS genes. We investigated the significance of these and other sequences by adding specific regions from the SSU301 promoter (the most strongly expressed gene) to equivalent regions in the SSU911 promoter (the least strongly expressed gene) and assaying the expression of the fusions in transgenic tobacco plants. In this way, we characterized an SSU301 promoter region (either from -285 to -178 or -291 to -204) which, when added to SSU911, in either orientation, increased SSU911 expression 25-fold. This increase was equivalent to that caused by addition of the entire SSU301 5'-flanking region. Replacement of SSU911 promoter sequences between -198 and the start codon with sequences from the equivalent region of SSU301 did not increase SSU911 expression significantly. The -291 to -204 SSU301 promoter fragment contributes significantly to quantitative differences in expression between the petunia rbcS genes. PMID:2535543

Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene.

PubMed

Dale, Rodney M; Topczewski, Jacek

2011-09-15

Zebrafish (Danio rerio) is an excellent model organism for the study of vertebrate development including skeletogenesis. Studies of mammalian cartilage formation were greatly advanced through the use of a cartilage specific regulatory element of the Collagen type II alpha 1 (Col2a1) gene. In an effort to isolate such an element in zebrafish, we compared the expression of two col2a1 homologues and found that expression of col2a1b, a previously uncharacterized zebrafish homologue, only partially overlaps with col2a1a. We focused our analysis on col2a1a, as it is expressed in both the stacked chondrocytes and the perichondrium. By comparing the genomic sequence surrounding the predicted transcriptional start site of col2a1a among several species of teleosts we identified a small highly conserved sequence (R2) located 1.7 kb upstream of the presumptive transcriptional initiation site. Interestingly, neither the sequence nor location of this element is conserved between teleost and mammalian Col2a1. We generated transient and stable transgenic lines with just the R2 element or the entire 1.7 kb fragment 5' of the transcriptional initiation site. The identified regulatory elements enable the tracking of cellular development in various tissues by driving robust reporter expression in craniofacial cartilage, ear, notochord, floor plate, hypochord and fins in a pattern similar to the expression of endogenous col2a1a. Using a reporter gene driven by the R2 regulatory element, we analyzed the morphogenesis of the notochord sheath cells as they withdraw from the stack of initially uniform cells and encase the inflating vacuolated notochord cells. Finally, we show that like endogenous col2a1a, craniofacial expression of these reporter constructs depends on Sox9a transcription factor activity. At the same time, notochord expression is maintained after Sox9a knockdown, suggesting that other factors can activate expression through the identified regulatory element in this tissue. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene

PubMed Central

Dale, Rodney M.; Topczewski, Jacek

2011-01-01

Zebrafish (Danio rerio) is an excellent model organism for the study of vertebrate development including skeletogenesis. Studies of mammalian cartilage formation were greatly advanced through the use of a cartilage specific regulatory element of the Collagen type II alpha 1 (Col2a1) gene. In an effort to isolate such an element in zebrafish, we compared the expression of two col2a1 homologues and found that expression of col2a1b, a previously uncharacterized zebrafish homologue, only partially overlaps with col2a1a. We focused our analysis on col2a1a, as it is expressed in both the stacked chondrocytes and the perichondrium. By comparing the genomic sequence surrounding the predicted transcriptional start site of col2a1a among several species of teleosts we identified a small highly conserved sequence (R2) located 1.7 kb upstream of the presumptive transcriptional initiation site. Interestingly, neither the sequence nor location of this element is conserved between teleost and mammalian Col2a1. We generated transient and stable transgenic lines with just the R2 element or the entire 1.7 kb fragment 5’ of the transcriptional initiation site. The identified regulatory elements enable the tracking of cellular development in various tissues by driving robust reporter expression in craniofacial cartilage, ear, notochord, floor plate, hypochord and fins in a pattern similar to the expression of endogenous col2a1a. Using a reporter gene driven by the R2 regulatory element, we analyzed the morphogenesis of the notochord sheath cells as they withdraw from the stack of initially uniform cells and encase the inflating vacuolated notochord cells. Finally, we show that like endogenous col2a1a, craniofacial expression of these reporter constructs depends on Sox9a transcription factor activity. At the same time, notochord expression is maintained after Sox9a knockdown, suggesting that other factors can activate expression through the identified regulatory element in this tissue. PMID:21723274

Evolutionary conservation of vertebrate notochord genes in the ascidian Ciona intestinalis.

PubMed

Kugler, Jamie E; Passamaneck, Yale J; Feldman, Taya G; Beh, Jeni; Regnier, Todd W; Di Gregorio, Anna

2008-11-01

To reconstruct a minimum complement of notochord genes evolutionarily conserved across chordates, we scanned the Ciona intestinalis genome using the sequences of 182 genes reported to be expressed in the notochord of different vertebrates and identified 139 candidate notochord genes. For 66 of these Ciona genes expression data were already available, hence we analyzed the expression of the remaining 73 genes and found notochord expression for 20. The predicted products of the newly identified notochord genes range from the transcription factors Ci-XBPa and Ci-miER1 to extracellular matrix proteins. We examined the expression of the newly identified notochord genes in embryos ectopically expressing Ciona Brachyury (Ci-Bra) and in embryos expressing a repressor form of this transcription factor in the notochord, and we found that while a subset of the genes examined are clearly responsive to Ci-Bra, other genes are not affected by alterations in its levels. We provide a first description of notochord genes that are not evidently influenced by the ectopic expression of Ci-Bra and we propose alternative regulatory mechanisms that might control their transcription. Copyright 2008 Wiley-Liss, Inc.

Liver Transcriptome and miRNA Analysis of Silver Carp (Hypophthalmichthys molitrix) Intraperitoneally Injected With Microcystin-LR

PubMed Central

Qu, Xiancheng; Hu, Menghong; Shang, Yueyong; Pan, Lisha; Jia, Peixuan; Fu, Chunxue; Liu, Qigen; Wang, Youji

2018-01-01

Next-generation sequencing was used to analyze the effects of toxic microcystin-LR (MC-LR) on silver carp (Hypophthalmichthys molitrix). Silver carps were intraperitoneally injected with MC-LR, and RNA-seq and miRNA-seq in the liver were analyzed at 0.25, 0.5, and 1 h. The expression of glutathione S-transferase (GST), which acts as a marker gene for MC-LR, was tested to determine the earliest time point at which GST transcription was initiated in the liver tissues of the MC-LR-treated silver carps. Hepatic RNA-seq/miRNA-seq analysis and data integration analysis were conducted with reference to the identified time point. Quantitative PCR (qPCR) was performed to detect the expression of the following genes at the three time points: heme oxygenase 1 (HO-1), interleukin-10 receptor 1 (IL-10R1), apolipoprotein A-I (apoA-I), and heme binding protein 2 (HBP2). Results showed that the liver GST expression was remarkably decreased at 0.25 h (P < 0.05). RNA-seq at this time point revealed that the liver tissue contained 97,505 unigenes, including 184 significantly different unigenes and 75 unknown genes. Gene Ontology (GO) term enrichment analysis suggested that 35 of the 145 enriched GO terms were significantly enriched and mainly related to the immune system regulation network. KEGG pathway enrichment analysis showed that 18 of the 189 pathways were significantly enriched, and the most significant was a ribosome pathway containing 77 differentially expressed genes. miRNA-seq analysis indicated that the longest miRNA had 22 nucleotides (nt), followed by 21 and 23 nt. A total of 286 known miRNAs, 332 known miRNA precursor sequences, and 438 new miRNAs were predicted. A total of 1,048,575 mRNA–miRNA interaction sites were obtained, and 21,252 and 21,241 target genes were respectively predicted in known and new miRNAs. qPCR revealed that HO-1, IL-10R1, apoA-I, and HBP2 were significantly differentially expressed and might play important roles in the toxicity and liver detoxification of MC-LR in fish. These results were consistent with those of high-throughput sequencing, thereby verifying the accuracy of our sequencing data. RNA-seq and miRNA-seq analyses of silver carp liver injected with MC-LR provided valuable and new insights into the toxic effects of MC-LR and the antitoxic mechanisms of MC-LR in fish. The RNA/miRNA data are available from the NCBI database Registration No. : SRP075165. PMID:29692738

Comprehensive RNA-Seq Expression Analysis of Sensory Ganglia with a Focus on Ion Channels and GPCRs in Trigeminal Ganglia

PubMed Central

Manteniotis, Stavros; Lehmann, Ramona; Flegel, Caroline; Vogel, Felix; Hofreuter, Adrian; Schreiner, Benjamin S. P.; Altmüller, Janine; Becker, Christian; Schöbel, Nicole; Hatt, Hanns; Gisselmann, Günter

2013-01-01

The specific functions of sensory systems depend on the tissue-specific expression of genes that code for molecular sensor proteins that are necessary for stimulus detection and membrane signaling. Using the Next Generation Sequencing technique (RNA-Seq), we analyzed the complete transcriptome of the trigeminal ganglia (TG) and dorsal root ganglia (DRG) of adult mice. Focusing on genes with an expression level higher than 1 FPKM (fragments per kilobase of transcript per million mapped reads), we detected the expression of 12984 genes in the TG and 13195 in the DRG. To analyze the specific gene expression patterns of the peripheral neuronal tissues, we compared their gene expression profiles with that of the liver, brain, olfactory epithelium, and skeletal muscle. The transcriptome data of the TG and DRG were scanned for virtually all known G-protein-coupled receptors (GPCRs) as well as for ion channels. The expression profile was ranked with regard to the level and specificity for the TG. In total, we detected 106 non-olfactory GPCRs and 33 ion channels that had not been previously described as expressed in the TG. To validate the RNA-Seq data, in situ hybridization experiments were performed for several of the newly detected transcripts. To identify differences in expression profiles between the sensory ganglia, the RNA-Seq data of the TG and DRG were compared. Among the differentially expressed genes (> 1 FPKM), 65 and 117 were expressed at least 10-fold higher in the TG and DRG, respectively. Our transcriptome analysis allows a comprehensive overview of all ion channels and G protein-coupled receptors that are expressed in trigeminal ganglia and provides additional approaches for the investigation of trigeminal sensing as well as for the physiological and pathophysiological mechanisms of pain. PMID:24260241

Proteogenomic characterization of human colon and rectal cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Bing; Wang, Jing; Wang, Xiaojing

2014-09-18

We analyzed proteomes of colon and rectal tumors previously characterized by the Cancer Genome Atlas (TCGA) and performed integrated proteogenomic analyses. Protein sequence variants encoded by somatic genomic variations displayed reduced expression compared to protein variants encoded by germline variations. mRNA transcript abundance did not reliably predict protein expression differences between tumors. Proteomics identified five protein expression subtypes, two of which were associated with the TCGA "MSI/CIMP" transcriptional subtype, but had distinct mutation and methylation patterns and associated with different clinical outcomes. Although CNAs showed strong cis- and trans-effects on mRNA expression, relatively few of these extend to the proteinmore » level. Thus, proteomics data enabled prioritization of candidate driver genes. Our analyses identified HNF4A, a novel candidate driver gene in tumors with chromosome 20q amplifications. Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords novel insights into cancer biology.« less

Molecular analysis of the ependymin gene and functional test of its promoter region by transient expression in Brachydanio rerio.

PubMed

Rinder, H; Bayer, T A; Gertzen, E M; Hoffmann, W

1992-01-01

Ependymins are secretory products of meningeal cells and represent the predominant glycoproteins in the cerebrospinal fluid from various orders of teleost fish. In the zebrafish, their expression starts between 48 and 72 h post-fertilization. Generally, they share characteristics with proteins involved in cell-contact phenomena. Here, we characterize the ependymin gene from Brachydanio rerio and its flanking regions. The sequence was obtained from clones generated using the polymerase chain reaction (PCR), including a variation of an "anchored" PCR. Also, clones from a conventional phage library were analyzed. We found that the transcribed portion is arranged in six exons. Transient expression of an ependymin-promoter-lacZ gene fusion in zebrafish embryos revealed that the 2.0-kb upstream regulatory region used is sufficient to direct the ependymin-specific correct temporal and spatial expression pattern of the lacZ reporter gene.

STARD13 promotes hepatocellular carcinoma apoptosis by acting as a ceRNA for Fas.

PubMed

Zhang, Hai; Wang, Fang; Hu, Yahua

2017-02-01

To study the roles of STARD13 in cellular apoptosis of hepatocellular carcinoma (HCC). Quantitative real-time PCR and immunohistochemistry analyses showed that the expression levels of STARD13 and Fas were lower in clinical HCC tissues than in normal tissues and were positively correlated, which is consistent with the results analyzed by The Cancer Genome Atlas (TCGA) data. Patients with higher STARD13 or Fas expression levels had longer overall survival. Additionally, STARD13 3'-UTR enhanced cellular apoptosis and the 3'-UTRs of STARD13 and Fas were predicted to harbor nine similar miRNA binding sites. And STARD13 3'-UTR promoted Fas expression in a 3'-UTR- and miRNA-dependent way and increased the sensitivity of HCC cells to chemotherapy. Importantly, the coding sequence of STARD13 did not increase Fas expression. STARD13 3'-UTR promotes HCC apoptosis through acting as a ceRNA for Fas.

Gene expression changes in subcutaneous adipose tissue due to Cushing's disease

PubMed Central

Hochberg, Irit; Harvey, Innocence; Tran, Quynh T; Stephenson, Erin J; Barkan, Ariel L; Saltiel, Alan R; Chandler, William F; Bridges, Dave

2015-01-01

Glucocorticoids have major effects on adipose tissue metabolism. To study tissue mRNA expression changes induced by chronic elevated endogenous glucocorticoids, we performed RNA sequencing on the subcutaneous adipose tissue from patients with Cushing's disease (n=5) compared to patients with nonfunctioning pituitary adenomas (n=11). We found a higher expression of transcripts involved in several metabolic pathways, including lipogenesis, proteolysis and glucose oxidation as well as a decreased expression of transcripts involved in inflammation and protein synthesis. To further study this in a model system, we subjected mice to dexamethasone treatment for 12 weeks and analyzed their inguinal (subcutaneous) fat pads, which led to similar findings. Additionally, mice treated with dexamethasone showed drastic decreases in lean body mass as well as increased fat mass, further supporting the human transcriptomic data. These data provide insight to transcriptional changes that may be responsible for the comorbidities associated with chronic elevations of glucocorticoids. PMID:26150553

Transcriptional Response to Lactic Acid Stress in the Hybrid Yeast Zygosaccharomyces parabailii

PubMed Central

2017-01-01

ABSTRACT Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH and a fermentative metabolism with a high growth rate. Here we used mRNA sequencing (RNA-seq) to analyze Z. parabailii's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than under control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/Aft2, previously described as lactic acid responsive in Saccharomyces cerevisiae. Furthermore, formate dehydrogenase (FDH) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii in comparison to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production. IMPORTANCE Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression under conditions of interest and relating gene expression patterns to protein functions. However, hybrid organisms present a challenge to the standard use of mRNA sequencing (RNA-seq) to study transcriptional responses to stress, because their genomes contain two similar copies of almost every gene. Here we used stringent mapping methods and a high-quality genome sequence to study the transcriptional response to lactic acid stress in Zygosaccharomyces parabailii ATCC 60483, a natural interspecies hybrid yeast that contains two complete subgenomes that are approximately 7% divergent in sequence. Beyond the insights we gained into lactic acid tolerance in this study, the methods we developed will be broadly applicable to other yeast hybrid strains. PMID:29269498

Transcriptional Response to Lactic Acid Stress in the Hybrid Yeast Zygosaccharomyces parabailii.

PubMed

Ortiz-Merino, Raúl A; Kuanyshev, Nurzhan; Byrne, Kevin P; Varela, Javier A; Morrissey, John P; Porro, Danilo; Wolfe, Kenneth H; Branduardi, Paola

2018-03-01

Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH and a fermentative metabolism with a high growth rate. Here we used mRNA sequencing (RNA-seq) to analyze Z. parabailii 's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than under control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/Aft2, previously described as lactic acid responsive in Saccharomyces cerevisiae Furthermore, formate dehydrogenase ( FDH ) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii in comparison to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production. IMPORTANCE Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression under conditions of interest and relating gene expression patterns to protein functions. However, hybrid organisms present a challenge to the standard use of mRNA sequencing (RNA-seq) to study transcriptional responses to stress, because their genomes contain two similar copies of almost every gene. Here we used stringent mapping methods and a high-quality genome sequence to study the transcriptional response to lactic acid stress in Zygosaccharomyces parabailii ATCC 60483, a natural interspecies hybrid yeast that contains two complete subgenomes that are approximately 7% divergent in sequence. Beyond the insights we gained into lactic acid tolerance in this study, the methods we developed will be broadly applicable to other yeast hybrid strains. Copyright © 2018 Ortiz-Merino et al.

High-resolution identification and abundance profiling of cassava (Manihot esculenta Crantz) microRNAs.

PubMed

Khatabi, Behnam; Arikit, Siwaret; Xia, Rui; Winter, Stephan; Oumar, Doungous; Mongomake, Kone; Meyers, Blake C; Fondong, Vincent N

2016-01-28

Small RNAs (sRNAs) are endogenous sRNAs that play regulatory roles in plant growth, development, and biotic and abiotic stress responses. In plants, one subset of sRNAs, microRNAs (miRNAs) exhibit tissue-differential expression and regulate gene expression mainly through direct cleavage of mRNA or indirectly via production of secondary phased siRNAs (phasiRNAs) that silence cognate target transcripts in trans. Here, we have identified cassava (Manihot esculenta Crantz) miRNAs using high resolution sequencing of sRNA libraries from leaf, stem, callus, male and female flower tissues. To analyze the data, we built a cassava genome database and, via sequence analysis and secondary structure prediction, 38 miRNAs not previously reported in cassava were identified. These new cassava miRNAs included two miRNAs not previously been reported in any plant species. The miRNAs exhibited tissue-differential accumulation as confirmed by quantitative RT-PCR and Northern blot analysis, largely reflecting levels observed in sequencing data. Some of the miRNAs identified were predicted to trigger production of secondary phased siRNAs (phasiRNAs) from 80 PHAS loci. Cassava is a woody perennial shrub, grown principally for its starch-rich storage roots, which are rich in calories. In this study, new miRNAs were identified and their expression was validated using qRT-PCR of RNA from five different tissues. The data obtained expand the list of annotated miRNAs and provide additional new resources for cassava improvement research.

Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in Flavonoid Biosynthetic Pathway

PubMed Central

Sun, Wei; Meng, Xiangyu; Liang, Lingjie; Jiang, Wangshu; Huang, Yafei; He, Jing; Hu, Haiyan; Almqvist, Jonas; Gao, Xiang; Wang, Li

2015-01-01

Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants. PMID:25742495

Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway.

PubMed

Sun, Wei; Meng, Xiangyu; Liang, Lingjie; Jiang, Wangshu; Huang, Yafei; He, Jing; Hu, Haiyan; Almqvist, Jonas; Gao, Xiang; Wang, Li

2015-01-01

Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

Whole-Exome Sequencing in a South American Cohort Links ALDH1A3, FOXN1 and Retinoic Acid Regulation Pathways to Autism Spectrum Disorders.

PubMed

Moreno-Ramos, Oscar A; Olivares, Ana María; Haider, Neena B; de Autismo, Liga Colombiana; Lattig, María Claudia

2015-01-01

Autism spectrum disorders (ASDs) are a range of complex neurodevelopmental conditions principally characterized by dysfunctions linked to mental development. Previous studies have shown that there are more than 1000 genes likely involved in ASD, expressed mainly in brain and highly interconnected among them. We applied whole exome sequencing in Colombian-South American trios. Two missense novel SNVs were found in the same child: ALDH1A3 (RefSeq NM_000693: c.1514T>C (p.I505T)) and FOXN1 (RefSeq NM_003593: c.146C>T (p.S49L)). Gene expression studies reveal that Aldh1a3 and Foxn1 are expressed in ~E13.5 mouse embryonic brain, as well as in adult piriform cortex (PC; ~P30). Conserved Retinoic Acid Response Elements (RAREs) upstream of human ALDH1A3 and FOXN1 and in mouse Aldh1a3 and Foxn1 genes were revealed using bioinformatic approximation. Chromatin immunoprecipitation (ChIP) assay using Retinoid Acid Receptor B (Rarb) as the immunoprecipitation target suggests RA regulation of Aldh1a3 and Foxn1 in mice. Our results frame a possible link of RA regulation in brain to ASD etiology, and a feasible non-additive effect of two apparently unrelated variants in ALDH1A3 and FOXN1 recognizing that every result given by next generation sequencing should be cautiously analyzed, as it might be an incidental finding.

[Analysis of FABP4 expression pattern in rump fat deposition and metabolism of Altay sheep].

PubMed

Ruixia, Xu; Lei, Gao; Weili, Zhao; Wei, Zhang; Guangchao, Song; Shangquan, Gan; Guoqing, Shi

2015-02-01

FABP4 (Fatty acid binding protein 4) is a hot candidate gene in fat deposition and lipid metabolism and participates in the transport and metabolism of intracellular free fatty acids. We aim to study the role of FABP4 in fat deposition and metabolism of the rump fat in Altay sheep. In this study, bioinformatics method was used to analyze the protein sequence homology among 10 species, and RT-PCR was employed to detect FABP4 tissue profiling of Altay sheep. An animal model simulating the rump fat deposition and metabolism of Altay sheep was established by continuous starvation, and qPCR and iTRAQ (isobaric tags for relative and absolute quantitation) were used to detecte FABP4 mRNA and protein expression changes in the control and continuous starvation groups, respectively. Sequence analysis showed that FABP4 protein sequence is highly conserved among species, suggesting an important biological function during evolution for FABP4. The RT-PCR result confirmed that FABP4 mRNA was highly expressed in intestinal and rump fat, suggesting that FABP4 plays an important physiological role in fat tissues. We did not find significant differences in FABP4 mRNA and protein between control and continuous starvation groups (P>0.05), which indicates that FABP4 may not be the key gene in fat deposition and metabolism in Altay sheep.The results above lay a foundation for further studies of FABP4 in rump or tail fat.

IVAG: An Integrative Visualization Application for Various Types of Genomic Data Based on R-Shiny and the Docker Platform.

PubMed

Lee, Tae-Rim; Ahn, Jin Mo; Kim, Gyuhee; Kim, Sangsoo

2017-12-01

Next-generation sequencing (NGS) technology has become a trend in the genomics research area. There are many software programs and automated pipelines to analyze NGS data, which can ease the pain for traditional scientists who are not familiar with computer programming. However, downstream analyses, such as finding differentially expressed genes or visualizing linkage disequilibrium maps and genome-wide association study (GWAS) data, still remain a challenge. Here, we introduce a dockerized web application written in R using the Shiny platform to visualize pre-analyzed RNA sequencing and GWAS data. In addition, we have integrated a genome browser based on the JBrowse platform and an automated intermediate parsing process required for custom track construction, so that users can easily build and navigate their personal genome tracks with in-house datasets. This application will help scientists perform series of downstream analyses and obtain a more integrative understanding about various types of genomic data by interactively visualizing them with customizable options.

Genome-wide methylation analysis identified sexually dimorphic methylated regions in hybrid tilapia

PubMed Central

Wan, Zi Yi; Xia, Jun Hong; Lin, Grace; Wang, Le; Lin, Valerie C. L.; Yue, Gen Hua

2016-01-01

Sexual dimorphism is an interesting biological phenomenon. Previous studies showed that DNA methylation might play a role in sexual dimorphism. However, the overall picture of the genome-wide methylation landscape in sexually dimorphic species remains unclear. We analyzed the DNA methylation landscape and transcriptome in hybrid tilapia (Oreochromis spp.) using whole genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq). We found 4,757 sexually dimorphic differentially methylated regions (DMRs), with significant clusters of DMRs located on chromosomal regions associated with sex determination. CpG methylation in promoter regions was negatively correlated with the gene expression level. MAPK/ERK pathway was upregulated in male tilapia. We also inferred active cis-regulatory regions (ACRs) in skeletal muscle tissues from WGBS datasets, revealing sexually dimorphic cis-regulatory regions. These results suggest that DNA methylation contribute to sex-specific phenotypes and serve as resources for further investigation to analyze the functions of these regions and their contributions towards sexual dimorphisms. PMID:27782217

De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.

PubMed

Haas, Brian J; Papanicolaou, Alexie; Yassour, Moran; Grabherr, Manfred; Blood, Philip D; Bowden, Joshua; Couger, Matthew Brian; Eccles, David; Li, Bo; Lieber, Matthias; MacManes, Matthew D; Ott, Michael; Orvis, Joshua; Pochet, Nathalie; Strozzi, Francesco; Weeks, Nathan; Westerman, Rick; William, Thomas; Dewey, Colin N; Henschel, Robert; LeDuc, Richard D; Friedman, Nir; Regev, Aviv

2013-08-01

De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms. We also present Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes. In the procedure, we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sourceforge.net. The run time of this protocol is highly dependent on the size and complexity of data to be analyzed. The example data set analyzed in the procedure detailed herein can be processed in less than 5 h.

Pru du 2S albumin or Pru du vicilin?

PubMed

Garino, Cristiano; De Paolis, Angelo; Coïsson, Jean Daniel; Arlorio, Marco

2015-06-01

A short partial sequence of 28 amino acids is all the information we have so far about the putative allergen 2S albumin from almond. The aim of this work was to analyze this information using mainly bioinformatics tools, in order to verify its rightness. Based on the results reported in the paper describing this allergen from almond, we analyzed the original data of amino acids sequencing through available software. The degree of homology of the almond 12kDa protein with any other known 2S albumin appears to be much lower than the one reported in the paper that firstly described it. In a publicly available cDNA library we discovered an expressed sequence tag which translation generates a protein that perfectly matches both of the sequencing outputs described in the same paper. A further analysis indicated that the latter protein seems to belong to the vicilin superfamily rather than to the prolamin one. The fact that also vicilins are seed storage proteins known to be highly allergenic would explain the IgE reactivity originally observed. Based on our observations we suggest that the IgE reactive 12kDa protein from almond currently known as Pru du 2S albumin is in reality the cleaved N-terminal region of a 7S vicilin like protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340.

PubMed

Prakobphol, A; Xu, F; Hoang, V M; Larsson, T; Bergstrom, J; Johansson, I; Frängsmyr, L; Holmskov, U; Leffler, H; Nilsson, C; Borén, T; Wright, J R; Strömberg, N; Fisher, S J

2000-12-22

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.

Identification of molecular tumor markers in renal cell carcinomas with TFE3 protein expression by RNA sequencing.

PubMed

Pflueger, Dorothee; Sboner, Andrea; Storz, Martina; Roth, Jasmine; Compérat, Eva; Bruder, Elisabeth; Rubin, Mark A; Schraml, Peter; Moch, Holger

2013-11-01

TFE3 translocation renal cell carcinoma (tRCC) is defined by chromosomal translocations involving the TFE3 transcription factor at chromosome Xp11.2. Genetically proven TFE3 tRCCs have a broad histologic spectrum with overlapping features to other renal tumor subtypes. In this study, we aimed for characterizing RCC with TFE3 protein expression. Using next-generation whole transcriptome sequencing (RNA-Seq) as a discovery tool, we analyzed fusion transcripts, gene expression profile, and somatic mutations in frozen tissue of one TFE3 tRCC. By applying a computational analysis developed to call chimeric RNA molecules from paired-end RNA-Seq data, we confirmed the known TFE3 translocation. Its fusion partner SFPQ has already been described as fusion partner in tRCCs. In addition, an RNA read-through chimera between TMED6 and COG8 as well as MET and KDR (VEGFR2) point mutations were identified. An EGFR mutation, but no chromosomal rearrangements, was identified in a control group of five clear cell RCCs (ccRCCs). The TFE3 tRCC could be clearly distinguished from the ccRCCs by RNA-Seq gene expression measurements using a previously reported tRCC gene signature. In validation experiments using reverse transcription-PCR, TMED6-COG8 chimera expression was significantly higher in nine TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in 24 ccRCCs (P < .001) and 22 papillary RCCs (P < .05-.07). Immunohistochemical analysis of selected genes from the tRCC gene signature showed significantly higher eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) and Contactin 3 (CNTN3) expression in 16 TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in over 200 ccRCCs (P < .0001, both).

DNA polymerase β variant Ile260Met generates global gene expression changes related to cellular transformation

PubMed Central

Sweasy, Joann B.

2012-01-01

Maintenance of genomic stability is essential for cellular survival. The base excision repair (BER) pathway is critical for resolution of abasic sites and damaged bases, estimated to occur 20,000 times in cells daily. DNA polymerase β (Pol β) participates in BER by filling DNA gaps that result from excision of damaged bases. Approximately 30% of human tumours express Pol β variants, many of which have altered fidelity and activity in vitro and when expressed, induce cellular transformation. The prostate tumour variant Ile260Met transforms cells and is a sequence-context-dependent mutator. To test the hypothesis that mutations induced in vivo by Ile260Met lead to cellular transformation, we characterized the genome-wide expression profile of a clone expressing Ile260Met as compared with its non-induced counterpart. Using a 1.5-fold minimum cut-off with a false discovery rate (FDR) of <0.05, 912 genes exhibit altered expression. Microarray results were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and revealed unique expression profiles in other clones. Gene Ontology (GO) clusters were analyzed using Ingenuity Pathways Analysis to identify altered gene networks and associated nodes. We determined three nodes of interest that exhibited dysfunctional regulation of downstream gene products without themselves having altered expression. One node, peroxisome proliferator-activated protein γ (PPARG), was sequenced and found to contain a coding region mutation in PPARG2 only in transformed cells. Further analysis suggests that this mutation leads to dominant negative activity of PPARG2. PPARG is a transcription factor implicated to have tumour suppressor function. This suggests that the PPARG2 mutant may have played a role in driving cellular transformation. We conclude that PPARG induces cellular transformation by a mutational mechanism. PMID:22914675

Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol

PubMed Central

2017-01-01

Tight and tunable control of gene expression is a highly desirable goal in synthetic biology for constructing predictable gene circuits and achieving preferred phenotypes. Elucidating the sequence–function relationship of promoters is crucial for manipulating gene expression at the transcriptional level, particularly for inducible systems dependent on transcriptional regulators. Sort-seq methods employing fluorescence-activated cell sorting (FACS) and high-throughput sequencing allow for the quantitative analysis of sequence–function relationships in a robust and rapid way. Here we utilized a massively parallel sort-seq approach to analyze the formaldehyde-inducible Escherichia coli promoter (Pfrm) with single-nucleotide resolution. A library of mutated formaldehyde-inducible promoters was cloned upstream of gfp on a plasmid. The library was partitioned into bins via FACS on the basis of green fluorescent protein (GFP) expression level, and mutated promoters falling into each expression bin were identified with high-throughput sequencing. The resulting analysis identified two 19 base pair repressor binding sites, one upstream of the −35 RNA polymerase (RNAP) binding site and one overlapping with the −10 site, and assessed the relative importance of each position and base therein. Key mutations were identified for tuning expression levels and were used to engineer formaldehyde-inducible promoters with predictable activities. Engineered variants demonstrated up to 14-fold lower basal expression, 13-fold higher induced expression, and a 3.6-fold stronger response as indicated by relative dynamic range. Finally, an engineered formaldehyde-inducible promoter was employed to drive the expression of heterologous methanol assimilation genes and achieved increased biomass levels on methanol, a non-native substrate of E. coli. PMID:28463494

Heterogeneous conservation of Dlx paralog co-expression in jawed vertebrates.

PubMed

Debiais-Thibaud, Mélanie; Metcalfe, Cushla J; Pollack, Jacob; Germon, Isabelle; Ekker, Marc; Depew, Michael; Laurenti, Patrick; Borday-Birraux, Véronique; Casane, Didier

2013-01-01

The Dlx gene family encodes transcription factors involved in the development of a wide variety of morphological innovations that first evolved at the origins of vertebrates or of the jawed vertebrates. This gene family expanded with the two rounds of genome duplications that occurred before jawed vertebrates diversified. It includes at least three bigene pairs sharing conserved regulatory sequences in tetrapods and teleost fish, but has been only partially characterized in chondrichthyans, the third major group of jawed vertebrates. Here we take advantage of developmental and molecular tools applied to the shark Scyliorhinus canicula to fill in the gap and provide an overview of the evolution of the Dlx family in the jawed vertebrates. These results are analyzed in the theoretical framework of the DDC (Duplication-Degeneration-Complementation) model. The genomic organisation of the catshark Dlx genes is similar to that previously described for tetrapods. Conserved non-coding elements identified in bony fish were also identified in catshark Dlx clusters and showed regulatory activity in transgenic zebrafish. Gene expression patterns in the catshark showed that there are some expression sites with high conservation of the expressed paralog(s) and other expression sites with events of paralog sub-functionalization during jawed vertebrate diversification, resulting in a wide variety of evolutionary scenarios within this gene family. Dlx gene expression patterns in the catshark show that there has been little neo-functionalization in Dlx genes over gnathostome evolution. In most cases, one tandem duplication and two rounds of vertebrate genome duplication have led to at least six Dlx coding sequences with redundant expression patterns followed by some instances of paralog sub-functionalization. Regulatory constraints such as shared enhancers, and functional constraints including gene pleiotropy, may have contributed to the evolutionary inertia leading to high redundancy between gene expression patterns.

Modeling bias and variation in the stochastic processes of small RNA sequencing

PubMed Central

Etheridge, Alton; Sakhanenko, Nikita; Galas, David

2017-01-01

Abstract The use of RNA-seq as the preferred method for the discovery and validation of small RNA biomarkers has been hindered by high quantitative variability and biased sequence counts. In this paper we develop a statistical model for sequence counts that accounts for ligase bias and stochastic variation in sequence counts. This model implies a linear quadratic relation between the mean and variance of sequence counts. Using a large number of sequencing datasets, we demonstrate how one can use the generalized additive models for location, scale and shape (GAMLSS) distributional regression framework to calculate and apply empirical correction factors for ligase bias. Bias correction could remove more than 40% of the bias for miRNAs. Empirical bias correction factors appear to be nearly constant over at least one and up to four orders of magnitude of total RNA input and independent of sample composition. Using synthetic mixes of known composition, we show that the GAMLSS approach can analyze differential expression with greater accuracy, higher sensitivity and specificity than six existing algorithms (DESeq2, edgeR, EBSeq, limma, DSS, voom) for the analysis of small RNA-seq data. PMID:28369495

The Comprehensive Phytopathogen Genomics Resource: a web-based resource for data-mining plant pathogen genomes.

PubMed

Hamilton, John P; Neeno-Eckwall, Eric C; Adhikari, Bishwo N; Perna, Nicole T; Tisserat, Ned; Leach, Jan E; Lévesque, C André; Buell, C Robin

2011-01-01

The Comprehensive Phytopathogen Genomics Resource (CPGR) provides a web-based portal for plant pathologists and diagnosticians to view the genome and trancriptome sequence status of 806 bacterial, fungal, oomycete, nematode, viral and viroid plant pathogens. Tools are available to search and analyze annotated genome sequences of 74 bacterial, fungal and oomycete pathogens. Oomycete and fungal genomes are obtained directly from GenBank, whereas bacterial genome sequences are downloaded from the A Systematic Annotation Package (ASAP) database that provides curation of genomes using comparative approaches. Curated lists of bacterial genes relevant to pathogenicity and avirulence are also provided. The Plant Pathogen Transcript Assemblies Database provides annotated assemblies of the transcribed regions of 82 eukaryotic genomes from publicly available single pass Expressed Sequence Tags. Data-mining tools are provided along with tools to create candidate diagnostic markers, an emerging use for genomic sequence data in plant pathology. The Plant Pathogen Ribosomal DNA (rDNA) database is a resource for pathogens that lack genome or transcriptome data sets and contains 131 755 rDNA sequences from GenBank for 17 613 species identified as plant pathogens and related genera. Database URL: http://cpgr.plantbiology.msu.edu.

Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects.

PubMed

Zhang, Xiaoyan; Marjani, Sadie L; Hu, Zhaoyang; Weissman, Sherman M; Pan, Xinghua; Wu, Shixiu

2016-03-15

Advances in genomic technology have enabled the faithful detection and measurement of mutations and the gene expression profile of cancer cells at the single-cell level. Recently, several single-cell sequencing methods have been developed that permit the comprehensive and precise analysis of the cancer-cell genome, transcriptome, and epigenome. The use of these methods to analyze cancer cells has led to a series of unanticipated discoveries, such as the high heterogeneity and stochastic changes in cancer-cell populations, the new driver mutations and the complicated clonal evolution mechanisms, and the novel identification of biomarkers of variant tumors. These methods and the knowledge gained from their utilization could potentially improve the early detection and monitoring of rare cancer cells, such as circulating tumor cells and disseminated tumor cells, and promote the development of personalized and highly precise cancer therapy. Here, we discuss the current methods for single cancer-cell sequencing, with a strong focus on those practically used or potentially valuable in cancer research, including single-cell isolation, whole genome and transcriptome amplification, epigenome profiling, multi-dimensional sequencing, and next-generation sequencing and analysis. We also examine the current applications, challenges, and prospects of single cancer-cell sequencing. ©2016 American Association for Cancer Research.

miRDis: a Web tool for endogenous and exogenous microRNA discovery based on deep-sequencing data analysis.

PubMed

Zhang, Hanyuan; Vieira Resende E Silva, Bruno; Cui, Juan

2018-05-01

Small RNA sequencing is the most widely used tool for microRNA (miRNA) discovery, and shows great potential for the efficient study of miRNA cross-species transport, i.e., by detecting the presence of exogenous miRNA sequences in the host species. Because of the increased appreciation of dietary miRNAs and their far-reaching implication in human health, research interests are currently growing with regard to exogenous miRNAs bioavailability, mechanisms of cross-species transport and miRNA function in cellular biological processes. In this article, we present microRNA Discovery (miRDis), a new small RNA sequencing data analysis pipeline for both endogenous and exogenous miRNA detection. Specifically, we developed and deployed a Web service that supports the annotation and expression profiling data of known host miRNAs and the detection of novel miRNAs, other noncoding RNAs, and the exogenous miRNAs from dietary species. As a proof-of-concept, we analyzed a set of human plasma sequencing data from a milk-feeding study where 225 human miRNAs were detected in the plasma samples and 44 show elevated expression after milk intake. By examining the bovine-specific sequences, data indicate that three bovine miRNAs (bta-miR-378, -181* and -150) are present in human plasma possibly because of the dietary uptake. Further evaluation based on different sets of public data demonstrates that miRDis outperforms other state-of-the-art tools in both detection and quantification of miRNA from either animal or plant sources. The miRDis Web server is available at: http://sbbi.unl.edu/miRDis/index.php.

Effect of read-mapping biases on detecting allele-specific expression from RNA-sequencing data

PubMed Central

Degner, Jacob F.; Marioni, John C.; Pai, Athma A.; Pickrell, Joseph K.; Nkadori, Everlyne; Gilad, Yoav; Pritchard, Jonathan K.

2009-01-01

Motivation: Next-generation sequencing has become an important tool for genome-wide quantification of DNA and RNA. However, a major technical hurdle lies in the need to map short sequence reads back to their correct locations in a reference genome. Here, we investigate the impact of SNP variation on the reliability of read-mapping in the context of detecting allele-specific expression (ASE). Results: We generated 16 million 35 bp reads from mRNA of each of two HapMap Yoruba individuals. When we mapped these reads to the human genome we found that, at heterozygous SNPs, there was a significant bias toward higher mapping rates of the allele in the reference sequence, compared with the alternative allele. Masking known SNP positions in the genome sequence eliminated the reference bias but, surprisingly, did not lead to more reliable results overall. We find that even after masking, ∼5–10% of SNPs still have an inherent bias toward more effective mapping of one allele. Filtering out inherently biased SNPs removes 40% of the top signals of ASE. The remaining SNPs showing ASE are enriched in genes previously known to harbor cis-regulatory variation or known to show uniparental imprinting. Our results have implications for a variety of applications involving detection of alternate alleles from short-read sequence data. Availability: Scripts, written in Perl and R, for simulating short reads, masking SNP variation in a reference genome and analyzing the simulation output are available upon request from JFD. Raw short read data were deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE18156. Contact: jdegner@uchicago.edu; marioni@uchicago.edu; gilad@uchicago.edu; pritch@uchicago.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19808877

High-resolution mapping of the 11q13 amplicon and identification of a gene, TAOS1, that is amplified and overexpressed in oral cancer cells

PubMed Central

Huang, Xin; Gollin, Susanne M.; Raja, Siva; Godfrey, Tony E.

2002-01-01

Amplification of chromosomal band 11q13 is a common event in human cancer. It has been reported in about 45% of head and neck carcinomas and in other cancers including esophageal, breast, liver, lung, and bladder cancer. To understand the mechanism of 11q13 amplification and to identify the potential oncogene(s) driving it, we have fine-mapped the structure of the amplicon in oral squamous cell carcinoma cell lines and localized the proximal and distal breakpoints. A 5-Mb physical map of the region has been prepared from which sequence is available. We quantified copy number of sequence-tagged site markers at 42–550 kb intervals along the length of the amplicon and defined the amplicon core and breakpoints by using TaqMan-based quantitative microsatellite analysis. The core of the amplicon maps to a 1.5-Mb region. The proximal breakpoint localizes to two intervals between sequence-tagged site markers, 550 kb and 160 kb in size, and the distal breakpoint maps to a 250 kb interval. The cyclin D1 gene maps to the amplicon core, as do two new expressed sequence tag clusters. We have analyzed one of these expressed sequence tag clusters and now report that it contains a previously uncharacterized gene, TAOS1 (tumor amplified and overexpressed sequence 1), which is both amplified and overexpressed in oral cancer cells. The data suggest that TAOS1 may be an amplification-dependent candidate oncogene with a role in the development and/or progression of human tumors, including oral squamous cell carcinomas. The approach described here should be useful for characterizing amplified genomic regions in a wide variety of tumors. PMID:12172009

Two alternative ways of start site selection in human norovirus reinitiation of translation.

PubMed

Luttermann, Christine; Meyers, Gregor

2014-04-25

The calicivirus minor capsid protein VP2 is expressed via termination/reinitiation. This process depends on an upstream sequence element denoted termination upstream ribosomal binding site (TURBS). We have shown for feline calicivirus and rabbit hemorrhagic disease virus that the TURBS contains three sequence motifs essential for reinitiation. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18 S rRNA leading to the model that hybridization between motif 1 and 18 S rRNA tethers the post-termination ribosome to the mRNA. Motif 2 and motif 2* are proposed to establish a secondary structure positioning the ribosome relative to the start site of the terminal ORF. Here, we analyzed human norovirus (huNV) sequences for the presence and importance of these motifs. The three motifs were identified by sequence analyses in the region upstream of the VP2 start site, and we showed that these motifs are essential for reinitiation of huNV VP2 translation. More detailed analyses revealed that the site of reinitiation is not fixed to a single codon and does not need to be an AUG, even though this codon is clearly preferred. Interestingly, we were able to show that reinitiation can occur at AUG codons downstream of the canonical start/stop site in huNV and feline calicivirus but not in rabbit hemorrhagic disease virus. Although reinitiation at the original start site is independent of the Kozak context, downstream initiation exhibits requirements for start site sequence context known for linear scanning. These analyses on start codon recognition give a more detailed insight into this fascinating mechanism of gene expression.

Identification and analysis of pig chimeric mRNAs using RNA sequencing data

PubMed Central

2012-01-01

Background Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. Results The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. Conclusions The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs. PMID:22925561

RNA deep sequencing as a tool for selection of cell lines for systematic subcellular localization of all human proteins.

PubMed

Danielsson, Frida; Wiking, Mikaela; Mahdessian, Diana; Skogs, Marie; Ait Blal, Hammou; Hjelmare, Martin; Stadler, Charlotte; Uhlén, Mathias; Lundberg, Emma

2013-01-04

One of the major challenges of a chromosome-centric proteome project is to explore in a systematic manner the potential proteins identified from the chromosomal genome sequence, but not yet characterized on a protein level. Here, we describe the use of RNA deep sequencing to screen human cell lines for RNA profiles and to use this information to select cell lines suitable for characterization of the corresponding gene product. In this manner, the subcellular localization of proteins can be analyzed systematically using antibody-based confocal microscopy. We demonstrate the usefulness of selecting cell lines with high expression levels of RNA transcripts to increase the likelihood of high quality immunofluorescence staining and subsequent successful subcellular localization of the corresponding protein. The results show a path to combine transcriptomics with affinity proteomics to characterize the proteins in a gene- or chromosome-centric manner.

Performance Analysis of Direct-Sequence Code-Division Multiple-Access Communications with Asymmetric Quadrature Phase-Shift-Keying Modulation

NASA Technical Reports Server (NTRS)

Wang, C.-W.; Stark, W.

2005-01-01

This article considers a quaternary direct-sequence code-division multiple-access (DS-CDMA) communication system with asymmetric quadrature phase-shift-keying (AQPSK) modulation for unequal error protection (UEP) capability. Both time synchronous and asynchronous cases are investigated. An expression for the probability distribution of the multiple-access interference is derived. The exact bit-error performance and the approximate performance using a Gaussian approximation and random signature sequences are evaluated by extending the techniques used for uniform quadrature phase-shift-keying (QPSK) and binary phase-shift-keying (BPSK) DS-CDMA systems. Finally, a general system model with unequal user power and the near-far problem is considered and analyzed. The results show that, for a system with UEP capability, the less protected data bits are more sensitive to the near-far effect that occurs in a multiple-access environment than are the more protected bits.

Exome sequencing identifies SUCO mutations in mesial temporal lobe epilepsy.

PubMed

Sha, Zhiqiang; Sha, Longze; Li, Wenting; Dou, Wanchen; Shen, Yan; Wu, Liwen; Xu, Qi

2015-03-30

Mesial temporal lobe epilepsy (mTLE) is the main type and most common medically intractable form of epilepsy. Severity of disease-based stratified samples may help identify new disease-associated mutant genes. We analyzed mRNA expression profiles from patient hippocampal tissue. Three of the seven patients had severe mTLE with generalized-onset convulsions and consciousness loss that occurred over many years. We found that compared with other groups, patients with severe mTLE were classified into a distinct group. Whole-exome sequencing and Sanger sequencing validation in all seven patients identified three novel SUN domain-containing ossification factor (SUCO) mutations in severely affected patients. Furthermore, SUCO knock down significantly reduced dendritic length in vitro. Our results indicate that mTLE defects may affect neuronal development, and suggest that neurons have abnormal development due to lack of SUCO, which may be a generalized-onset epilepsy-related gene. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The Aquaporin Channel Repertoire of the Tardigrade Milnesium tardigradum

PubMed Central

Grohme, Markus A.; Mali, Brahim; Wełnicz, Weronika; Michel, Stephanie; Schill, Ralph O.; Frohme, Marcus

2013-01-01

Limno-terrestrial tardigrades are small invertebrates that are subjected to periodic drought of their micro-environment. They have evolved to cope with these unfavorable conditions by anhydrobiosis, an ametabolic state of low cellular water. During drying and rehydration, tardigrades go through drastic changes in cellular water content. By our transcriptome sequencing effort of the limno-terrestrial tardigrade Milnesium tardigradum and by a combination of cloning and targeted sequence assembly, we identified transcripts encoding eleven putative aquaporins. Analysis of these sequences proposed 2 classical aquaporins, 8 aquaglyceroporins and a single potentially intracellular unorthodox aquaporin. Using quantitative real-time PCR we analyzed aquaporin transcript expression in the anhydrobiotic context. We have identified additional unorthodox aquaporins in various insect genomes and have identified a novel common conserved structural feature in these proteins. Analysis of the genomic organization of insect aquaporin genes revealed several conserved gene clusters. PMID:23761966

O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation

PubMed Central

Kreth, Simone; Thon, Niklas; Eigenbrod, Sabina; Lutz, Juergen; Ledderose, Carola; Egensperger, Rupert; Tonn, Joerg C.; Kretzschmar, Hans A.; Hinske, Ludwig C.; Kreth, Friedrich W.

2011-01-01

Background We analyzed prospectively whether MGMT (O6-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. Methodology/Principal Findings Adult patients with a histologically proven malignant astrocytoma (glioblastoma: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression. Conclusions/Significance MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined. PMID:21365007

Identification, expression and phylogenetic analysis of EgG1Y162 from Echinococcus granulosus

PubMed Central

Zhang, Fengbo; Ma, Xiumin; Zhu, Yuejie; Wang, Hongying; Liu, Xianfei; Zhu, Min; Ma, Haimei; Wen, Hao; Fan, Haining; Ding, Jianbing

2014-01-01

Objective: This study was to clone, identify and analyze the characteristics of egG1Y162 gene from Echinococcus granulosus. Methods: Genomic DNA and total RNAs were extracted from four different developmental stages of protoscolex, germinal layer, adult and egg of Echinococcus granulosus, respectively. Fluorescent quantitative PCR was used for analyzing the expression of egG1Y162 gene. Prokaryotic expression plasmid of pET41a-EgG1Y162 was constructed to express recombinant His-EgG1Y162 antigen. Western blot analysis was performed to detect antigenicity of EgG1Y162 antigen. Gene sequence, amino acid alignment and phylogenetic tree of EgG1Y162 were analyzed by BLAST, online Spidey and MEGA4 software, respectively. Results: EgG1Y162 gene was expressed in four developmental stages of Echinococcus granulosus. And, egG1Y162 gene expression was the highest in the adult stage, with the relative value of 19.526, significantly higher than other three stages. Additionally, Western blot analysis revealed that EgG1Y162 recombinant protein had good reaction with serum samples from Echinococcus granulosus infected human and dog. Moreover, EgG1Y162 antigen was phylogenetically closest to EmY162 antigen, with the similarity over 90%. Conclusion: Our study identified EgG1Y162 antigen in Echinococcus granulosus for the first time. EgG1Y162 antigen had a high similarity with EmY162 antigen, with the genetic differences mainly existing in the intron region. And, EgG1Y162 recombinant protein showed good antigenicity. PMID:25337206

Intrinsic and extrinsic approaches for detecting genes in a bacterial genome.

PubMed Central

Borodovsky, M; Rudd, K E; Koonin, E V

1994-01-01

The unannotated regions of the Escherichia coli genome DNA sequence from the EcoSeq6 database, totaling 1,278 'intergenic' sequences of the combined length of 359,279 basepairs, were analyzed using computer-assisted methods with the aim of identifying putative unknown genes. The proposed strategy for finding new genes includes two key elements: i) prediction of expressed open reading frames (ORFs) using the GeneMark method based on Markov chain models for coding and non-coding regions of Escherichia coli DNA, and ii) search for protein sequence similarities using programs based on the BLAST algorithm and programs for motif identification. A total of 354 putative expressed ORFs were predicted by GeneMark. Using the BLASTX and TBLASTN programs, it was shown that 208 ORFs located in the unannotated regions of the E. coli chromosome are significantly similar to other protein sequences. Identification of 182 ORFs as probable genes was supported by GeneMark and BLAST, comprising 51.4% of the GeneMark 'hits' and 87.5% of the BLAST 'hits'. 73 putative new genes, comprising 20.6% of the GeneMark predictions, belong to ancient conserved protein families that include both eubacterial and eukaryotic members. This value is close to the overall proportion of highly conserved sequences among eubacterial proteins, indicating that the majority of the putative expressed ORFs that are predicted by GeneMark, but have no significant BLAST hits, nevertheless are likely to be real genes. The majority of the putative genes identified by BLAST search have been described since the release of the EcoSeq6 database, but about 70 genes have not been detected so far. Among these new identifications are genes encoding proteins with a variety of predicted functions including dehydrogenases, kinases, several other metabolic enzymes, ATPases, rRNA methyltransferases, membrane proteins, and different types of regulatory proteins. Images PMID:7984428

Identification of a novel splice variant of human PD-L1 mRNA encoding an isoform-lacking Igv-like domain.

PubMed

He, Xian-hui; Xu, Li-hui; Liu, Yi

2005-04-01

To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). The cDNA encoding human PD-L1 precursor was cloned from the total RNA extracted from the resting and phorbol dibutyrate plus ionomycin- or phytohemagglutinin-activated PBMC, by reverse transcription polymerase chain reaction (RT-PCR), and independent clones were sequenced and analyzed. The expression and subcellular localization were examined in transiently transfected cells. The PD-L1 gene expression in different PBMC was also analyzed by RT-PCR. A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon? encoding an immunoglobulin variable domain (Igv)-like domain but retaining all other exons without a frame-shift. Consequently, the putative translated protein contained all other domains including the transmembrane region except for the Igv-like domain. Furthermore, the conventional isoform was expressed on the plasma surface whereas the novel isoform showed a pattern of intracellular membrane distribution in transiently transfected K562 cells. In addition, the expression pattern of the PD-L1 splice variant was variable in different individuals and in different cellular status. PD-L1 expression may be regulated at the posttranscriptional level through alternative splicing, and modulation of the PD-L1 isoform expression may influence the outcome of specific immune responses in the peripheral tissues.

Changes in the Peripheral Blood Gene Expression Profile Induced by 3 Months of Valproate Treatment in Patients with Newly Diagnosed Epilepsy

PubMed Central

Rakitin, Aleksei; Kõks, Sulev; Reimann, Ene; Prans, Ele; Haldre, Sulev

2015-01-01

Valproic acid (VPA) is a widely used antiepileptic drug with a broad range of effects and broad clinical efficacy. As a well-known histone deacetylase (HDAC) inhibitor, VPA regulates epigenetic programming by altering the expression of many genes. The aim of study was to analyze differences in gene expression profiles before and after the start of VPA treatment in patients with newly diagnosed epilepsy. RNA sequencing was used to compare whole-genome gene expression patterns of peripheral blood from nine patients with epilepsy before and 3 months after the start of treatment with VPA. Of the 23,099 analyzed genes, only 11 showed statistically significant differential expression with false discovery rate-adjusted p-values below 0.1. Functional annotation and network analyses showed activation of only one genetic network (enrichment score = 30), which included genes for cardiovascular system development and function, cell morphology, and hematological system development and function. The finding of such a small number of differently expressed genes between before and after the start of treatment suggests a lack of HDAC inhibition in these patients, which could be explained by the relatively low doses of VPA that were used. In conclusion, VPA at standard therapeutic dosages modulates the expression of a small number of genes. Therefore, to minimize the potential side effects of HDAC inhibition, it is recommended that the lowest effective dose of VPA be used for treating epilepsy. PMID:26379622

Identification and Functional Analysis of Flowering Related microRNAs in Common Wild Rice (Oryza rufipogon Griff.)

PubMed Central

Dong, Yibo; Yuan, Qianhua; Wang, Feng; Li, Weimin; Jiang, Ying; Jia, Shirong; Pei, XinWu

2013-01-01

Background MicroRNAs (miRNAs) is a class of non-coding RNAs involved in post- transcriptional control of gene expression, via degradation and/or translational inhibition. Six-hundred sixty-one rice miRNAs are known that are important in plant development. However, flowering-related miRNAs have not been characterized in Oryza rufipogon Griff. It was approved by supervision department of Guangdong wild rice protection. We analyzed flowering-related miRNAs in O. rufipogon using high-throughput sequencing (deep sequencing) to understand the changes that occurred during rice domestication, and to elucidate their functions in flowering. Results Three O. rufipogon sRNA libraries, two vegetative stage (CWR-V1 and CWR-V2) and one flowering stage (CWR-F2) were sequenced using Illumina deep sequencing. A total of 20,156,098, 21,531,511 and 20,995,942 high quality sRNA reads were obtained from CWR-V1, CWR-V2 and CWR-F2, respectively, of which 3,448,185, 4,265,048 and 2,833,527 reads matched known miRNAs. We identified 512 known rice miRNAs in 214 miRNA families and predicted 290 new miRNAs. Targeted functional annotation, GO and KEGG pathway analyses predicted that 187 miRNAs regulate expression of flowering-related genes. Differential expression analysis of flowering-related miRNAs showed that: expression of 95 miRNAs varied significantly between the libraries, 66 are flowering-related miRNAs, such as oru-miR97, oru-miR117, oru-miR135, oru-miR137, et al. 17 are early-flowering -related miRNAs, including osa-miR160f, osa-miR164d, osa-miR167d, osa-miR169a, osa-miR172b, oru-miR4, et al., induced during the floral transition. Real-time PCR revealed the same expression patterns as deep sequencing. miRNAs targets were confirmed for cleavage by 5′-RACE in vivo, and were negatively regulated by miRNAs. Conclusions This is the first investigation of flowering miRNAs in wild rice. The result indicates that variation in miRNAs occurred during rice domestication and lays a foundation for further study of phase change and flowering in O. rufipogon. Complicated regulatory networks mediated by multiple miRNAs regulate the expression of flowering genes that control the induction of flowering. PMID:24386120

Evaluation of anonymous and expressed sequence tag derived polymorphic microsatellite markers in the tobacco budworm Heliothis virescens (Lepidoptera: noctuidae)

USDA-ARS?s Scientific Manuscript database

Polymorphic genetic markers were identified and characterized using a partial genomic library of Heliothis virescens enriched for simple sequence repeats (SSR) and nucleotide sequences of expressed sequence tags (EST). Nucleotide sequences of 192 clones from the partial genomic library yielded 147 u...

Widespread and evolutionary analysis of a MITE family Monkey King in Brassicaceae.

PubMed

Dai, Shutao; Hou, Jinna; Long, Yan; Wang, Jing; Li, Cong; Xiao, Qinqin; Jiang, Xiaoxue; Zou, Xiaoxiao; Zou, Jun; Meng, Jinling

2015-06-19

Miniature inverted repeat transposable elements (MITEs) are important components of eukaryotic genomes, with hundreds of families and many copies, which may play important roles in gene regulation and genome evolution. However, few studies have investigated the molecular mechanisms involved. In our previous study, a Tourist-like MITE, Monkey King, was identified from the promoter region of a flowering time gene, BnFLC.A10, in Brassica napus. Based on this MITE, the characteristics and potential roles on gene regulation of the MITE family were analyzed in Brassicaceae. The characteristics of the Tourist-like MITE family Monkey King in Brassicaceae, including its distribution, copies and insertion sites in the genomes of major Brassicaceae species were analyzed in this study. Monkey King was actively amplified in Brassica after divergence from Arabidopsis, which was indicated by the prompt increase in copy number and by phylogenetic analysis. The genomic variations caused by Monkey King insertions, both intra- and inter-species in Brassica, were traced by PCR amplification. Genomic sequence analysis showed that most complete Monkey King elements are located in gene-rich regions, less than 3kb from genes, in both the B. rapa and A. thaliana genomes. Sixty-seven Brassica expressed sequence tags carrying Monkey King fragments were also identified from the NCBI database. Bisulfite sequencing identified specific DNA methylation of cytosine residues in the Monkey King sequence. A fragment containing putative TATA-box motifs in the MITE sequence could bind with nuclear protein(s) extracted from leaves of B. napus plants. A Monkey King-related microRNA, bna-miR6031, was identified in the microRNA database. In transgenic A. thaliana, when the Monkey King element was inserted upstream of 35S promoter, the promoter activity was weakened. Monkey King, a Brassicaceae Tourist-like MITE family, has amplified relatively recently and has induced intra- and inter-species genomic variations in Brassica. Monkey King elements are most abundant in the vicinity of genes and may have a substantial effect on genome-wide gene regulation in Brassicaceae. Monkey King insertions potentially regulate gene expression and genome evolution through epigenetic modification and new regulatory motif production.

An Automated Pipeline for Engineering Many-Enzyme Pathways: Computational Sequence Design, Pathway Expression-Flux Mapping, and Scalable Pathway Optimization.

PubMed

Halper, Sean M; Cetnar, Daniel P; Salis, Howard M

2018-01-01

Engineering many-enzyme metabolic pathways suffers from the design curse of dimensionality. There are an astronomical number of synonymous DNA sequence choices, though relatively few will express an evolutionary robust, maximally productive pathway without metabolic bottlenecks. To solve this challenge, we have developed an integrated, automated computational-experimental pipeline that identifies a pathway's optimal DNA sequence without high-throughput screening or many cycles of design-build-test. The first step applies our Operon Calculator algorithm to design a host-specific evolutionary robust bacterial operon sequence with maximally tunable enzyme expression levels. The second step applies our RBS Library Calculator algorithm to systematically vary enzyme expression levels with the smallest-sized library. After characterizing a small number of constructed pathway variants, measurements are supplied to our Pathway Map Calculator algorithm, which then parameterizes a kinetic metabolic model that ultimately predicts the pathway's optimal enzyme expression levels and DNA sequences. Altogether, our algorithms provide the ability to efficiently map the pathway's sequence-expression-activity space and predict DNA sequences with desired metabolic fluxes. Here, we provide a step-by-step guide to applying the Pathway Optimization Pipeline on a desired multi-enzyme pathway in a bacterial host.

Transcriptome Analysis of Beta macrocarpa and Identification of Differentially Expressed Transcripts in Response to Beet Necrotic Yellow Vein Virus Infection.

PubMed

Fan, Huiyan; Zhang, Yongliang; Sun, Haiwen; Liu, Junying; Wang, Ying; Wang, Xianbing; Li, Dawei; Yu, Jialin; Han, Chenggui

2015-01-01

Rhizomania is one of the most devastating diseases of sugar beet. It is caused by Beet necrotic yellow vein virus (BNYVV) transmitted by the obligate root-infecting parasite Polymyxa betae. Beta macrocarpa, a wild beet species widely used as a systemic host in the laboratory, can be rub-inoculated with BNYVV to avoid variation associated with the presence of the vector P. betae. To better understand disease and resistance between beets and BNYVV, we characterized the transcriptome of B. macrocarpa and analyzed global gene expression of B. macrocarpa in response to BNYVV infection using the Illumina sequencing platform. The overall de novo assembly of cDNA sequence data generated 75,917 unigenes, with an average length of 1054 bp. Based on a BLASTX search (E-value ≤ 10-5) against the non-redundant (NR, NCBI) protein, Swiss-Prot, the Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, there were 39,372 unigenes annotated. In addition, 4,834 simple sequence repeats (SSRs) were also predicted, which could serve as a foundation for various applications in beet breeding. Furthermore, comparative analysis of the two transcriptomes revealed that 261 genes were differentially expressed in infected compared to control plants, including 128 up- and 133 down-regulated genes. GO analysis showed that the changes in the differently expressed genes were mainly enrichment in response to biotic stimulus and primary metabolic process. Our results not only provide a rich genomic resource for beets, but also benefit research into the molecular mechanisms of beet- BNYV Vinteraction.

An ordered EST catalogue and gene expression profiles of cassava (Manihot esculenta) at key growth stages.

PubMed

Li, You-Zhi; Pan, Ying-Hua; Sun, Chang-Bin; Dong, Hai-Tao; Luo, Xing-Lu; Wang, Zhi-Qiang; Tang, Ji-Liang; Chen, Baoshan

2010-12-01

A cDNA library was constructed from the root tissues of cassava variety Huanan 124 at the root bulking stage. A total of 9,600 cDNA clones from the library were sequenced with single-pass from the 5'-terminus to establish a catalogue of expressed sequence tags (ESTs). Assembly of the resulting EST sequences resulted in 2,878 putative unigenes. Blastn analysis showed that 62.6% of the unigenes matched with known cassava ESTs and the rest had no 'hits' against the cassava database in the integrative PlantGDB database. Blastx analysis showed that 1,715 (59.59%) of the unigenes matched with one or more GenBank protein entries and 1,163 (40.41%) had no 'hits'. A cDNA microarray with 2,878 unigenes was developed and used to analyze gene expression profiling of Huanan 124 at key growth stages including seedling, formation of root system, root bulking, and starch maturity. Array data analysis revealed that (1) the higher ratio of up-regulated ribosome-related genes was accompanied by a high ratio of up-regulated ubiquitin, proteasome-related and protease genes in cassava roots; (2) starch formation and degradation simultaneously occur at the early stages of root development but starch degradation is declined partially due to decrease in UDP-glucose dehydrogenase activity with root maturity; (3) starch may also be synthesized in situ in roots; (4) starch synthesis, translocation, and accumulation are also associated probably with signaling pathways that parallel Wnt, LAM, TCS and ErbB signaling pathways in animals; (5) constitutive expression of stress-responsive genes may be due to the adaptation of cassava to harsh environments during long-term evolution.

Identification of Genes Associated with Lemon Floral Transition and Flower Development during Floral Inductive Water Deficits: A Hypothetical Model

PubMed Central

Li, Jin-Xue; Hou, Xiao-Jin; Zhu, Jiao; Zhou, Jing-Jing; Huang, Hua-Bin; Yue, Jian-Qiang; Gao, Jun-Yan; Du, Yu-Xia; Hu, Cheng-Xiao; Hu, Chun-Gen; Zhang, Jin-Zhi

2017-01-01

Water deficit is a key factor to induce flowering in many woody plants, but reports on the molecular mechanisms of floral induction and flowering by water deficit are scarce. Here, we analyzed the morphology, cytology, and different hormone levels of lemon buds during floral inductive water deficits. Higher levels of ABA were observed, and the initiation of floral bud differentiation was examined by paraffin sections analysis. A total of 1638 differentially expressed genes (DEGs) were identified by RNA sequencing. DEGs were related to flowering, hormone biosynthesis, or metabolism. The expression of some DEGs was associated with floral induction by real-time PCR analysis. However, some DEGs may not have anything to do with flowering induction/flower development; they may be involved in general stress/drought response. Four genes from the phosphatidylethanolamine-binding protein family were further investigated. Ectopic expression of these genes in Arabidopsis changed the flowering time of transgenic plants. Furthermore, the 5′ flanking region of these genes was also isolated and sequence analysis revealed the presence of several putative cis-regulatory elements, including basic elements and hormone regulation elements. The spatial and temporal expression patterns of these promoters were investigated under water deficit treatment. Based on these findings, we propose a model for citrus flowering under water deficit conditions, which will enable us to further understand the molecular mechanism of water deficit-regulated flowering in citrus. Highlight: Based on gene activity during floral inductive water deficits identified by RNA sequencing and genes associated with lemon floral transition, a model for citrus flowering under water deficit conditions is proposed. PMID:28659956

Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank

PubMed Central

Simpanya, Mukoma F.; Wistow, Graeme; Gao, James; David, Larry L.; Giblin, Frank J.

2008-01-01

Purpose To characterize gene expression patterns in guinea pig ocular tissues and identify orthologs of human genes from NEIBank expressed sequence tags. Methods RNA was extracted from dissected eye tissues of 2.5-month-old guinea pigs to make three unamplified and unnormalized cDNA libraries in the pCMVSport-6 vector for the lens, retina, and eye minus lens and retina. Over 4,000 clones were sequenced from each library and were analyzed using GRIST for clustering and gene identification. Lens crystallin EST data were validated using two-dimensional electrophoresis (2-DE), matrix assisted laser desorption (MALDI), and electrospray ionization mass spectrometry (ESIMS). Results Combined data from the three libraries generated a total of 6,694 distinctive gene clusters, with each library having between 1,000 and 3,000 clusters. Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank. Complete cDNA sequences were obtained for many guinea pig lens proteins, including αA/αAinsert-, γN-, and γS-crystallins, lengsin and GRIFIN. The ratio of αA- to αB-crystallin on 2-DE gels was 8: 1 in the lens nucleus and 6.5: 1 in the cortex. Analysis of ESTs, genome sequence, and proteins (by MALDI), did not reveal any evidence for the presence of γD-, γE-, and γF-crystallin in the guinea pig. Predicted masses of many guinea pig lens crystallins were confirmed by ESIMS analysis. For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin. The guinea-pig ortholog of NRL, a key rod photoreceptor-specific transcription factor, was also represented in EST data. In the ‘rest-of-eye’ library, the most abundant transcripts included decorin and keratin 12, representative of the cornea. Conclusions Genomic analysis of guinea pig eye tissues provides sequence-verified clones for future studies. Guinea pig orthologs of many human eye specific genes were identified. Guinea pig gene structures were similar to their human and rodent gene counterparts. Surprisingly, no orthologs of γD-, γE-, and γF-crystallin were found in EST, proteomic, or the current guinea pig genome data. PMID:19104676

Sequencing, bioinformatic characterization and expression pattern of a putative amino acid transporter from the parasitic cestode Echinococcus granulosus.

PubMed

Camicia, Federico; Paredes, Rodolfo; Chalar, Cora; Galanti, Norbel; Kamenetzky, Laura; Gutierrez, Ariana; Rosenzvit, Mara C

2008-03-31

We have sequenced and partially characterized an Echinococcus granulosus cDNA, termed egat1, from a protoscolex signal sequence trap (SST) cDNA library. The isolated 1627 bp long cDNA contains an ORF of 489 amino acids and shows an amino acid identity of 30% with neutral and excitatory amino acid transporters members of the Dicarboxylate/Amino Acid Na+ and/or H+ Cation Symporter family (DAACS) (TC 2.A.23). Additional bioinformatics analysis of EgAT1, confirmed the results obtained by similarity searches and showed the presence of 9 to 10 transmembrane domains, consensus sequences for N-glycosylation between the third and fourth transmembrane domain, a highly similar hydropathy profile with ASCT1 (a known member of DAACS family), high score with SDF (Sodium Dicarboxilate Family) and similar motifs with EDTRANSPORT, a fingerprint of excitatory amino acid transporters. The localization of the putative amino acid transporter was analyzed by in situ hybridization and immunofluorescence in protoscoleces and associated germinal layer. The in situ hybridization labelling indicates the distribution of egat1 mRNA throughout the tegument. EgAT1 protein, which showed in Western blots a molecular mass of approximately 60 kD, is localized in the subtegumental region of the metacestode, particularly around suckers and rostellum of protoscoleces and layers from brood capsules. The sequence and expression analyses of EgAT1 pave the way for functional analysis of amino acids transporters of E. granulosus and its evaluation as new drug targets against cystic echinococcosis.

Poly A- transcripts expressed in HeLa cells.

PubMed

Wu, Qingfa; Kim, Yeong C; Lu, Jian; Xuan, Zhenyu; Chen, Jun; Zheng, Yonglan; Zhou, Tom; Zhang, Michael Q; Wu, Chung-I; Wang, San Ming

2008-07-30

Transcripts expressed in eukaryotes are classified as poly A+ transcripts or poly A- transcripts based on the presence or absence of the 3' poly A tail. Most transcripts identified so far are poly A+ transcripts, whereas the poly A- transcripts remain largely unknown. We developed the TRD (Total RNA Detection) system for transcript identification. The system detects the transcripts through the following steps: 1) depleting the abundant ribosomal and small-size transcripts; 2) synthesizing cDNA without regard to the status of the 3' poly A tail; 3) applying the 454 sequencing technology for massive 3' EST collection from the cDNA; and 4) determining the genome origins of the detected transcripts by mapping the sequences to the human genome reference sequences. Using this system, we characterized the cytoplasmic transcripts from HeLa cells. Of the 13,467 distinct 3' ESTs analyzed, 24% are poly A-, 36% are poly A+, and 40% are bimorphic with poly A+ features but without the 3' poly A tail. Most of the poly A- 3' ESTs do not match known transcript sequences; they have a similar distribution pattern in the genome as the poly A+ and bimorphic 3' ESTs, and their mapped intergenic regions are evolutionarily conserved. Experiments confirmed the authenticity of the detected poly A- transcripts. Our study provides the first large-scale sequence evidence for the presence of poly A- transcripts in eukaryotes. The abundance of the poly A- transcripts highlights the need for comprehensive identification of these transcripts for decoding the transcriptome, annotating the genome and studying biological relevance of the poly A- transcripts.

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies.

PubMed

Harris, Katherine E; Aldred, Shelley Force; Davison, Laura M; Ogana, Heather Anne N; Boudreau, Andrew; Brüggemann, Marianne; Osborn, Michael; Ma, Biao; Buelow, Benjamin; Clarke, Starlynn C; Dang, Kevin H; Iyer, Suhasini; Jorgensen, Brett; Pham, Duy T; Pratap, Payal P; Rangaswamy, Udaya S; Schellenberger, Ute; van Schooten, Wim C; Ugamraj, Harshad S; Vafa, Omid; Buelow, Roland; Trinklein, Nathan D

2018-01-01

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Genome-Wide Identification and Evolution Analysis of Trehalose-6-Phosphate Synthase Gene Family in Nelumbo nucifera

PubMed Central

Jin, Qijiang; Hu, Xin; Li, Xin; Wang, Bei; Wang, Yanjie; Jiang, Hongwei; Mattson, Neil; Xu, Yingchun

2016-01-01

Trehalose-6-phosphate synthase (TPS) plays a key role in plant carbohydrate metabolism and the perception of carbohydrate availability. In the present work, the publicly available Nelumbo nucifera (lotus) genome sequence database was analyzed which led to identification of nine lotus TPS genes (NnTPS). It was found that at least two introns are included in the coding sequences of NnTPS genes. When the motif compositions were analyzed we found that NnTPS generally shared the similar motifs, implying that they have similar functions. The dN/dS ratios were always less than 1 for different domains and regions outside domains, suggesting purifying selection on the lotus TPS gene family. The regions outside TPS domain evolved relatively faster than NnTPS domains. A phylogenetic tree was constructed using all predicted coding sequences of lotus TPS genes, together with those from Arabidopsis, poplar, soybean, and rice. The result indicated that those TPS genes could be clearly divided into two main subfamilies (I-II), where each subfamily could be further divided into 2 (I) and 5 (II) subgroups. Analyses of divergence and adaptive evolution show that purifying selection may have been the main force driving evolution of plant TPS genes. Some of the critical sites that contributed to divergence may have been under positive selection. Transcriptome data analysis revealed that most NnTPS genes were predominantly expressed in sink tissues. Expression pattern of NnTPS genes under copper and submergence stress indicated that NNU_014679 and NNU_022788 might play important roles in lotus energy metabolism and participate in stress response. Our results can facilitate further functional studies of TPS genes in lotus. PMID:27746792

hSAGEing: an improved SAGE-based software for identification of human tissue-specific or common tumor markers and suppressors.

PubMed

Yang, Cheng-Hong; Chuang, Li-Yeh; Shih, Tsung-Mu; Chang, Hsueh-Wei

2010-12-17

SAGE (serial analysis of gene expression) is a powerful method of analyzing gene expression for the entire transcriptome. There are currently many well-developed SAGE tools. However, the cross-comparison of different tissues is seldom addressed, thus limiting the identification of common- and tissue-specific tumor markers. To improve the SAGE mining methods, we propose a novel function for cross-tissue comparison of SAGE data by combining the mathematical set theory and logic with a unique "multi-pool method" that analyzes multiple pools of pair-wise case controls individually. When all the settings are in "inclusion", the common SAGE tag sequences are mined. When one tissue type is in "inclusion" and the other types of tissues are not in "inclusion", the selected tissue-specific SAGE tag sequences are generated. They are displayed in tags-per-million (TPM) and fold values, as well as visually displayed in four kinds of scales in a color gradient pattern. In the fold visualization display, the top scores of the SAGE tag sequences are provided, along with cluster plots. A user-defined matrix file is designed for cross-tissue comparison by selecting libraries from publically available databases or user-defined libraries. The hSAGEing tool provides a combination of friendly cross-tissue analysis and an interface for comparing SAGE libraries for the first time. Some up- or down-regulated genes with tissue-specific or common tumor markers and suppressors are identified computationally. The tool is useful and convenient for in silico cancer transcriptomic studies and is freely available at http://bio.kuas.edu.tw/hSAGEing.

Two novel mutations in the PPIB gene cause a rare pedigree of osteogenesis imperfecta type IX.

PubMed

Jiang, Yu; Pan, Jingxin; Guo, Dongwei; Zhang, Wei; Xie, Jie; Fang, Zishui; Guo, Chunmiao; Fang, Qun; Jiang, Weiying; Guo, Yibin

2017-06-01

Osteogenesis imperfecta (OI) is a rare genetic skeletal disorder characterized by increased bone fragility and vulnerability to fractures. PPIB is identified as a candidate gene for OI-IX, here we detect two pathogenic mutations in PPIB and analyze the genotype-phenotype correlation in a Chinese family with OI. Next-generation sequencing (NGS) was used to screen the whole exome of the parents of proband. Screening of variation frequency, evolutionary conservation comparisons, pathogenicity evaluation, and protein structure prediction were conducted to assess the pathogenicity of the novel mutations. Sanger sequencing was used to confirm the candidate variants. RTQ-PCR was used to analyze the PPIB gene expression. All mutant genes screened out by NGS were excluded except PPIB. Two novel heterozygous PPIB mutations (father, c.25A>G; mother, c.509G>A) were identified in relation to osteogenesis imperfecta type IX. Both mutations were predicted to be pathogenic by bioinformatics analysis and RTQ-PCR analysis revealed downregulated PPIB expression in the two carriers. We report a rare pedigree with an autosomal recessive osteogenesis imperfecta type IX (OI-IX) caused by two novel PPIB mutations identified for the first time in China. The current study expands our knowledge of PPIB mutations and their associated phenotypes, and provides new information on the genetic defects associated with this disease for clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Transcriptomic variation of eyestalk reveals the genes and biological processes associated with molting in Portunus trituberculatus

PubMed Central

Zhang, Longtao; Liu, Ping; Li, Jian

2017-01-01

Background Molting is an essential biological process throughout the life history of crustaceans, which is regulated by many neuropeptide hormones expressed in the eyestalk. To better understand the molting mechanism in Portunus trituberculatus, we used digital gene expression (DGE) to analyze single eyestalk samples during the molting cycle by high-throughput sequencing. Results We obtained 14,387,942, 12,631,508 and 13,060,062 clean sequence reads from inter-molt (InM), pre-molt (PrM) and post-molt (PoM) cDNA libraries, respectively. A total of 1,394 molt-related differentially expressed genes (DEGs) were identified. GO and KEGG enrichment analysis identified some important processes and pathways with key roles in molting regulation, such as chitin metabolism, peptidase inhibitor activity, and the ribosome. We first observed a pattern associated with the neuromodulator-related pathways during the molting cycle, which were up-regulated in PrM and down-regulated in PoM. Four categories of important molting-related transcripts were clustered and most of them had similar expression patterns, which suggests that there is a connection between these genes throughout the molt cycle. Conclusion Our work is the first molt-related investigation of P. trituberculatus focusing on the eyestalk at the whole transcriptome level. Together, our results, including DEGs, identification of molting-related biological processes and pathways, and observed expression patterns of important genes, provide a novel insight into the function of the eyestalk in molting regulation. PMID:28394948

Analysis of the grapevine moth Lobesia botrana antennal transcriptome and expression of odorant-binding and chemosensory proteins.

PubMed

Rojas, Valentina; Jiménez, Héctor; Palma-Millanao, Rubén; González-González, Angélica; Machuca, Juan; Godoy, Ricardo; Ceballos, Ricardo; Mutis, Ana; Venthur, Herbert

2018-04-30

The grapevine moth, Lobesia botrana, is considered a harmful pest for vineyards in Chile as well as in North America and Europe. Currently, monitoring and control methods of L. botrana are based on its main sex pheromone component, being effective for low population densities. In order to improve control methods, antennal olfactory proteins in moths, such as odorant-binding proteins (OBPs) and odorant receptors (ORs) have been studied as promising targets for the discovery of new potent semiochemicals, which have not been reported for L. botrana. Therefore, the objective of this study was to identify the repertoire of proteins related to chemoreception in L. botrana by antennal transcriptome and analyze the relative expression of OBPs and CSPs in male and female antennae. Through next-generation sequencing of the antennal transcriptome by Ilumina HiSeq2500 we identified a total of 118 chemoreceptors, from which 61, 42 and 15 transcripts are related to ORs, ionotropic receptors (IRs) and gustatory receptors (GRs), respectively. Furthermore, RNA-Seq data revealed 35 transcripts for OBPs and 18 for chemosensory proteins (CSPs). Analysis by qRT-PCR showed 20 OBPs significantly expressed in female antennae, while 5 were more expressed in males. Similarly, most of the CSPs were significantly expressed in female than male antennae. All the olfactory-related sequences were compared with homologs and their phylogenetic relationships elucidated. Finally, our findings in relation to the improvement of L. botrana management are discussed. Copyright © 2018 Elsevier Inc. All rights reserved.

Characterization and expression analysis of genes encoding ubiquitin conjugating domain-containing enzymes in Carica papaya.

PubMed

Jue, Dengwei; Sang, Xuelian; Shu, Bo; Liu, Liqin; Wang, Yicheng; Jia, Zhiwei; Zou, Yu; Shi, Shengyou

2017-01-01

Ripening affects the quality and nutritional contents of fleshy fruits and is a crucial process of fruit development. Although several studies have suggested that ubiquitin-conjugating enzyme (E2s or UBC enzymes) are involved in the regulation of fruit ripening, little is known about the function of E2s in papaya (Carica papaya). In the present study, we searched the papaya genome and identified 34 putative UBC genes, which were clustered into 17 phylogenetic subgroups. We also analyzed the nucleotide sequences of the papaya UBC (CpUBC) genes and found that both exon-intron junctions and sequence motifs were highly conserved among the phylogenetic subgroups. Using real-time PCR analysis, we also found that all the CpUBC genes were expressed in roots, stems, leaves, male and female flowers, and mature fruit, although the expression of some of the genes was increased or decreased in one or several specific organs. We also found that the expression of 13 and two CpUBC genes were incresesd or decreased during one and two ripening stages, respectively. Expression analyses indicates possible E2s playing a more significant role in fruit ripening for further studies. To the best of our knowledge, this is the first reported genome-wide analysis of the papaya UBC gene family, and the results will facilitate further investigation of the roles of UBC genes in fruit ripening and will aide in the functional validation of UBC genes in papaya.

Olfactomedin-like 2 A and B (OLFML2A and OLFML2B) expression profile in primates (human and baboon).

PubMed

Pérez-Ibave, Diana Cristina; González-Alvarez, Rafael; de La Luz Martinez-Fierro, Margarita; Ruiz-Ayma, Gabriel; Luna-Muñoz, Maricela; Martínez-De-Villarreal, Laura Elia; De Lourdes Garza-Rodríguez, María; Reséndez-Pérez, Diana; Mohamed-Noriega, Jibran; Garza-Guajardo, Raquel; Bautista-De-Lucío, Víctor Manuel; Mohamed-Noriega, Karim; Barboza-Quintana, Oralia; Arámburo-De-La-Hoz, Carlos; Barrera-Saldaña, Hugo Alberto; Rodríguez-Sánchez, Irám Pablo

2016-11-08

The olfactomedin-like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon. The aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology. OLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT-PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays. OLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human's orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear. Expression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high similarity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye.

Co-Occurrence of Two Allelic Variants of CYP51 in Erysiphe necator and Their Correlation with Over-Expression for DMI Resistance

PubMed Central

Rallos, Lynn Esther E.; Baudoin, Anton B.

2016-01-01

Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136th codon of the predicted EnCYP51 sequence: the wild-type (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other. PMID:26839970

Genome-wide evidence for local DNA methylation spreading from small RNA-targeted sequences in Arabidopsis.

PubMed

Ahmed, Ikhlak; Sarazin, Alexis; Bowler, Chris; Colot, Vincent; Quesneville, Hadi

2011-09-01

Transposable elements (TEs) and their relics play major roles in genome evolution. However, mobilization of TEs is usually deleterious and strongly repressed. In plants and mammals, this repression is typically associated with DNA methylation, but the relationship between this epigenetic mark and TE sequences has not been investigated systematically. Here, we present an improved annotation of TE sequences and use it to analyze genome-wide DNA methylation maps obtained at single-nucleotide resolution in Arabidopsis. We show that although the majority of TE sequences are methylated, ∼26% are not. Moreover, a significant fraction of TE sequences densely methylated at CG, CHG and CHH sites (where H = A, T or C) have no or few matching small interfering RNA (siRNAs) and are therefore unlikely to be targeted by the RNA-directed DNA methylation (RdDM) machinery. We provide evidence that these TE sequences acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, we show that although both methylated and unmethylated TE sequences located in euchromatin tend to be more abundant closer to genes, this trend is least pronounced for methylated, siRNA-targeted TE sequences located 5' to genes. Based on these and other findings, we propose that spreading of DNA methylation through promoter regions explains at least in part the negative impact of siRNA-targeted TE sequences on neighboring gene expression.

A recombinant isoform of the Ole e 7 olive pollen allergen assembled by de novo mass spectrometry retains the allergenic ability of the natural allergen.

PubMed

Oeo-Santos, Carmen; Mas, Salvador; Benedé, Sara; López-Lucendo, María; Quiralte, Joaquín; Blanca, Miguel; Mayorga, Cristobalina; Villalba, Mayte; Barderas, Rodrigo

2018-06-05

The allergenic non-specific lipid transfer protein Ole e 7 from olive pollen is a major allergen associated with severe symptoms in areas with high olive pollen levels. Despite its clinical importance, its cloning and recombinant production has been unable by classical approaches. This study aimed at determining by mass-spectrometry based proteomics its complete amino acid sequence for its subsequent expression and characterization. To this end, the natural protein was in-2D-gel tryptic digested, and CID and HCD fragmentation spectra obtained by nLC-MS/MS analyzed using PEAKS software. Thirteen out of the 457 de novo sequenced peptides obtained allowed assembling its full-length amino acid sequence. Then, Ole e 7-encoding cDNA was synthesized and cloned in pPICZαA vector for its expression in Pichia pastoris yeast. The analyses by Circular Dichroism, and WB, ELISA and cell-based tests using sera and blood from olive pollen-sensitized patients showed that rOle e 7 mostly retained the structural, allergenic and antigenic properties of the natural allergen. In summary, rOle e 7 allergen assembled by de novo peptide sequencing by MS behaved immunologically similar to the natural allergen scarcely isolated from pollen. Olive pollen is an important cause of allergy. The non-specific lipid binding protein Ole e 7 is a major allergen with a high incidence and a phenotype associated to severe clinical symptoms. Despite its relevance, its cloning and recombinant expression has been unable by classical techniques. Here, we have inferred the primary amino acid sequence of Ole e 7 by mass-spectrometry. We separated Ole e 7 isolated from pollen by 2DE. After in-gel digestion with trypsin and a direct analysis by nLC-MS/MS in an LTQ-Orbitrap Velos, we got the complete de novo sequenced peptides repertoire that allowed the assembling of the primary sequence of Ole e 7. After its protein expression, purification to homogeneity, and structural and immunological characterization using sera from olive pollen allergic patients and cell-based assays, we observed that the recombinant allergen retained the antigenic and allergenic properties of the natural allergen. Collectively, we show that the recombinant protein assembled by proteomics would be suitable for a better in vitro diagnosis of olive pollen allergic patients. Copyright © 2018. Published by Elsevier B.V.

Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between Nicotiana attenuata and Rhizophagus irregularis.

PubMed

Groten, Karin; Pahari, Nabin T; Xu, Shuqing; Miloradovic van Doorn, Maja; Baldwin, Ian T

2015-01-01

Most land plants live in a symbiotic association with arbuscular mycorrhizal fungi (AMF) that belong to the phylum Glomeromycota. Although a number of plant genes involved in the plant-AMF interactions have been identified by analyzing mutants, the ability to rapidly manipulate gene expression to study the potential functions of new candidate genes remains unrealized. We analyzed changes in gene expression of wild tobacco roots (Nicotiana attenuata) after infection with mycorrhizal fungi (Rhizophagus irregularis) by serial analysis of gene expression (SuperSAGE) combined with next generation sequencing, and established a virus-induced gene-silencing protocol to study the function of candidate genes in the interaction. From 92,434 SuperSAGE Tag sequences, 32,808 (35%) matched with our in-house Nicotiana attenuata transcriptome database and 3,698 (4%) matched to Rhizophagus genes. In total, 11,194 Tags showed a significant change in expression (p<0.05, >2-fold change) after infection. When comparing the functions of highly up-regulated annotated Tags in this study with those of two previous large-scale gene expression studies, 18 gene functions were found to be up-regulated in all three studies mainly playing roles related to phytohormone metabolism, catabolism and defense. To validate the function of identified candidate genes, we used the technique of virus-induced gene silencing (VIGS) to silence the expression of three putative N. attenuata genes: germin-like protein, indole-3-acetic acid-amido synthetase GH3.9 and, as a proof-of-principle, calcium and calmodulin-dependent protein kinase (CCaMK). The silencing of the three plant genes in roots was successful, but only CCaMK silencing had a significant effect on the interaction with R. irregularis. Interestingly, when a highly activated inoculum was used for plant inoculation, the effect of CCaMK silencing on fungal colonization was masked, probably due to trans-complementation. This study demonstrates that large-scale gene expression studies across different species induce of a core set of genes of similar functions. However, additional factors seem to influence the overall pattern of gene expression, resulting in high variability among independent studies with different hosts. We conclude that VIGS is a powerful tool with which to investigate the function of genes involved in plant-AMF interactions but that inoculum strength can strongly influence the outcome of the interaction.

Analysis of differential gene expression by bead-based fiber-optic array in nonfunctioning pituitary adenomas.

PubMed

Jiang, Z; Gui, S; Zhang, Y

2011-05-01

Nonfunctioning pituitary adenomas (NFPAs) are relatively common, accounting for 30% of all pituitary adenomas; however, their pathogenesis remains enigmatic. To explore the possible pathogenesis of NFPAs, we used fiber-optic BeadArray to examine gene expression in 5 NFPAs compared with 3 normal pituitaries. 4 differentially expressed genes were chosen randomly for validation by reverse transcriptase-real time quantitative polymerase chain reaction (RT-qPCR). We then analyzed the differentially expressed gene profile with Kyoto Encyclopedia of Genes and Genomes (KEGG). The array analysis indentified significant increases in the expression of 1,402 genes and 383 expressed sequence tags (ESTs), and decreases in 1,697 genes and 113 ESTs in the NFPAs. Bioinformatic and pathway analysis showed that the genes HIGD1B, FAM5C, PMAIP1 and the pathway cell-cycle regulation may play an important role in tumorigenesis and progression of NFPAs. Our data suggest fiber-optic BeadArray combined with pathway analysis of differential gene expression profile appears to be a valid approach for investigating the pathogenesis of tumors. © Georg Thieme Verlag KG Stuttgart · New York.

Identification of genes differentially expressed during adventitious shoot induction in Pinus pinea cotyledons by subtractive hybridization and quantitative PCR.

PubMed

Alonso, Pablo; Cortizo, Millán; Cantón, Francisco R; Fernández, Belén; Rodríguez, Ana; Centeno, Maria L; Cánovas, Francisco M; Ordás, Ricardo J

2007-12-01

As part of a study aimed at understanding the physiological and molecular mechanisms involved in adventitious shoot bud formation in pine cotyledons, we conducted a transcriptome analysis to identify early-induced genes during the first phases of adventitious caulogenesis in Pinus pinea L. cotyledons cultured in the presence of benzyladenine. A subtractive cDNA library with more than 700 clones was constructed. Of these clones, 393 were sequenced, analyzed and grouped according to their putative function. Quantitative real-time PCR analysis was performed to confirm the differential expression of 30 candidate genes. Results are contrasted with available data for other species.

Identification of small secreted peptides (SSPs) in maize and expression analysis of partial SSP genes in reproductive tissues.

PubMed

Li, Ye Long; Dai, Xin Ren; Yue, Xun; Gao, Xin-Qi; Zhang, Xian Sheng

2014-10-01

Maize 1,491 small secreted peptides were identified, which were classified according to the character of peptide sequences. Partial SSP gene expressions in reproductive tissues were determined by qRT-PCR. Small secreted peptides (SSPs) are important cell-cell communication messengers in plants. Most information on plant SSPs come from Arabidopsis thaliana and Oryza sativa, while little is known about the SSPs of other grass species such as maize (Zea mays). In this study, we identified 1,491 SSP genes from maize genomic sequences. These putative SSP genes were distributed throughout the ten maize chromosomes. Among them, 611 SSPs were classified into 198 superfamilies according to their conserved domains, and 725 SSPs with four or more cysteines at their C-termini shared similar cysteine arrangements with their counterparts in other plant species. Moreover, the SSPs requiring post-translational modification, as well as defensin-like (DEFL) proteins, were identified. Further, the expression levels of 110 SSP genes were analyzed in reproductive tissues, including male flower, pollen, silk, and ovary. Most of the genes encoding basal-layer antifungal peptide-like, small coat proteins-like, thioredoxin-like proteins, γ-thionins-like, and DEFL proteins showed high expression levels in the ovary and male flower compared with their levels in silk and mature pollen. The rapid alkalinization factor-like genes were highly expressed only in the mature ovary and mature pollen, and pollen Ole e 1-like genes showed low expression in silk. The results of this study provide basic information for further analysis of SSP functions in the reproductive process of maize.

Tissue distribution and early developmental expression patterns of aldolase A, B, and C in grass carp Ctenopharyngodon idellus.

PubMed

Fan, J J; Bai, J J; Ma, D M; Yu, L Y; Jiang, P

2017-09-27

Aldolase is a key enzyme involved in glycolysis, gluconeogenesis, and the pentose phosphate pathway. To establish the expression patterns of all three aldolase isozyme genes in different tissues and during early embryogenesis in lower vertebrates, as well as to explore the functional differences between these three isozymes, the grass carp was selected as a model owing to its relatively high glucose-metabolizing capability. Based on the cDNA sequences of the aldolase A, B, and C genes, the expression patterns of these three isozymes were analyzed in different tissues and during early embryogenesis using quantitative real-time polymerase chain reaction (qRT-PCR). Sequence analysis of cDNAs indicated that aldolase A, B, and C (GenBank accession numbers: KM192250, KM192251, and KM192252) consist of 364, 364, and 363 amino acids, respectively. The qRT-PCR results showed that the expression levels of aldolase A, B, and C were highest in the muscle, liver, and brain, respectively. Aldolase A and C exhibited similar expression patterns during embryogenesis, with high levels observed in unfertilized and fertilized eggs and at the blastocyst stage, followed by a decline and then increase after organogenesis. In contrast, aldolase B transcript was not detected during the unfertilized egg stage, and appeared only from gastrulation; the expression increased markedly during the feeding period (72 h after hatching), at which point the level was higher than those of aldolase A and C. These data suggest that the glucose content of grass carp starter feed should be adjusted according to the metabolic activity of aldolase B.

Characterization and differential expression of microRNAs elicited by sulfur deprivation in Chlamydomonas reinhardtii

PubMed Central

2012-01-01

Background microRNAs (miRNAs) have been found to play an essential role in the modulation of numerous biological processes in eukaryotes. Chlamydomonas reinhardtii is an ideal model organism for the study of many metabolic processes including responses to sulfur-deprivation. We used a deep sequencing platform to extensively profile and identify changes in the miRNAs expression that occurred under sulfur-replete and sulfur-deprived conditions. The aim of our research was to characterize the differential expression of Chlamydomonas miRNAs under sulfur-deprived conditions, and subsequently, the target genes of miRNA involved in sulfur-deprivation were further predicted and analyzed. Results By using high-throughput sequencing, we characterized the microRNA transcriptomes under sulphur-replete and sulfur-deprived conditions in Chlamydomonas reinhardtii. We predicted a total of 310 miRNAs which included 85 known miRNAs and 225 novel miRNAs. 13 miRNAs were the specific to the sulfur-deprived conditions. 47 miRNAs showed significantly differential expressions responding to sulfur-deprivation, and most were up-regulated in the small RNA libraries with sulfur-deprivation. Using a web-based integrated system (Web MicroRNAs Designer 3) and combing the former information from a transcriptome of Chlamydomonas reinhardtii, 22 miRNAs and their targets involved in metabolism regulation with sulfur-deprivation were verified. Conclusions Our results indicate that sulfur-deprivation may have a significant influence on small RNA expression patterns, and the differential expressions of miRNAs and interactions between miRNA and its targets might further reveal the molecular mechanism responding to sulfur-deprivation in Chlamydomonas reinhardtii. PMID:22439676

Large-scale identification and comparative analysis of miRNA expression profile in the respiratory tree of the sea cucumber Apostichopus japonicus during aestivation.

PubMed

Chen, Muyan; Storey, Kenneth B

2014-02-01

The sea cucumber Apostichopus japonicus withstands high water temperatures in the summer by suppressing its metabolic rate and entering a state of aestivation. We hypothesized that changes in the expression of miRNAs could provide important post-transcriptional regulation of gene expression during hypometabolism via control over mRNA translation. The present study analyzed profiles of miRNA expression in the sea cucumber respiratory tree using Solexa deep sequencing technology. We identified 279 sea cucumber miRNAs, including 15 novel miRNAs specific to sea cucumber. Animals sampled during deep aestivation (DA; after at least 15 days of continuous torpor) were compared with animals from a non-aestivation (NA) state (animals that had passed through aestivation and returned to an active state). We identified 30 differentially expressed miRNAs ([RPM (reads per million) >10, |FC| (|fold change|)≥1, FDR (false discovery rate)<0.01]) during aestivation, which were validated by two other miRNA profiling methods: miRNA microarray and real-time PCR. Among the most prominent miRNA species, miR-124, miR-124-3p, miR-79, miR-9 and miR-2010 were significantly over-expressed during deep aestivation compared with non-aestivation animals, suggesting that these miRNAs may play important roles in metabolic rate suppression during aestivation. High-throughput sequencing data and microarray data have been submitted to the GEO database with accession number: 16902695. Copyright © 2014 Elsevier B.V. All rights reserved.

Screening and analyzing genes associated with Amur tiger placental development.

PubMed

Li, Q; Lu, T F; Liu, D; Hu, P F; Sun, B; Ma, J Z; Wang, W J; Wang, K F; Zhang, W X; Chen, J; Guan, W J; Ma, Y H; Zhang, M H

2014-09-26

The Amur tiger is a unique endangered species in the world, and thus, protection of its genetic resources is extremely important. In this study, an Amur tiger placenta cDNA library was constructed using the SMART cDNA Library Construction kit. A total of 508 colonies were sequenced, in which 205 (76%) genes were annotated and mapped to 74 KEGG pathways, including 29 metabolism, 29 genetic information processing, 4 environmental information processing, 7 cell motility, and 5 organismal system pathways. Additionally, PLAC8, PEG10 and IGF-II were identified after screening genes from the expressed sequence tags, and they were associated with placental development. These findings could lay the foundation for future functional genomic studies of the Amur tiger.

Identification of Differentially Expressed Genes Associated with Apple Fruit Ripening and Softening by Suppression Subtractive Hybridization

PubMed Central

Zhang, Zongying; Jiang, Shenghui; Wang, Nan; Li, Min; Ji, Xiaohao; Sun, Shasha; Liu, Jingxuan; Wang, Deyun; Xu, Haifeng; Qi, Sumin; Wu, Shujing; Fei, Zhangjun; Feng, Shouqian; Chen, Xuesen

2015-01-01

Apple is one of the most economically important horticultural fruit crops worldwide. It is critical to gain insights into fruit ripening and softening to improve apple fruit quality and extend shelf life. In this study, forward and reverse suppression subtractive hybridization libraries were generated from ‘Taishanzaoxia’ apple fruits sampled around the ethylene climacteric to isolate ripening- and softening-related genes. A set of 648 unigenes were derived from sequence alignment and cluster assembly of 918 expressed sequence tags. According to gene ontology functional classification, 390 out of 443 unigenes (88%) were assigned to the biological process category, 356 unigenes (80%) were classified in the molecular function category, and 381 unigenes (86%) were allocated to the cellular component category. A total of 26 unigenes differentially expressed during fruit development period were analyzed by quantitative RT-PCR. These genes were involved in cell wall modification, anthocyanin biosynthesis, aroma production, stress response, metabolism, transcription, or were non-annotated. Some genes associated with cell wall modification, anthocyanin biosynthesis and aroma production were up-regulated and significantly correlated with ethylene production, suggesting that fruit texture, coloration and aroma may be regulated by ethylene in ‘Taishanzaoxia’. Some of the identified unigenes associated with fruit ripening and softening have not been characterized in public databases. The results contribute to an improved characterization of changes in gene expression during apple fruit ripening and softening. PMID:26719904

The molecular characterization of porcine egg precursor cells

PubMed Central

Tsai, Te-Sha; Johnson, Jacqueline; White, Yvonne; John, Justin C.

2017-01-01

Female-factor infertility can be caused by poor oocyte quality and depleted ovarian reserves. Egg precursor cells (EPCs), isolated from the ovarian cortex, have the potential to be used to overcome female infertility. We aimed to define the origins of EPCs by analyzing their gene expression profiles and mtDNA content using a mini-pig model. We characterized FAC-sorted DDX4+-derived porcine EPCs by performing RNA-sequencing and determined that they utilize pathways important for cell cycle and proliferation, which supports the existence of adult mitotically active oogonial cells. Expression of the pluripotent markers Sox2 and Oct4, and the primitive germ cell markers Blimp1 and Stella were not detected. However, Nanog and Ddx4 were expressed, as were the primitive germ cell markers Fragilis, c-Kit and Tert. Moreover, porcine EPCs expressed self-renewal and proliferation markers including Myc, Esrrb, Id2, Klf4, Klf5, Stat3, Fgfr1, Fgfr2 and Il6st. The presence of Zp1, Zp2, Zp3 and Nobox were not detected, indicating that porcine EPCs are not indicative of mature primordial oocytes. We performed mitochondrial DNA Next Generation Sequencing and determined that one mtDNA variant harbored by EPCs was present in oocytes, preimplantation embryos and somatic tissues over three generations in our mini-pig model indicating the potential germline origin of EPCs. PMID:28969006

The WRKY Transcription Factor Family in Citrus: Valuable and Useful Candidate Genes for Citrus Breeding.

PubMed

Ayadi, M; Hanana, M; Kharrat, N; Merchaoui, H; Marzoug, R Ben; Lauvergeat, V; Rebaï, A; Mzid, R

2016-10-01

WRKY transcription factors belong to a large family of plant transcriptional regulators whose members have been reported to be involved in a wide range of biological roles including plant development, adaptation to environmental constraints and response to several diseases. However, little or poor information is available about WRKY's in Citrus. The recent release of completely assembled genomes sequences of Citrus sinensis and Citrus clementina and the availability of ESTs sequences from other citrus species allowed us to perform a genome survey for Citrus WRKY proteins. In the present study, we identified 100 WRKY members from C. sinensis (51), C. clementina (48) and Citrus unshiu (1), and analyzed their chromosomal distribution, gene structure, gene duplication, syntenic relation and phylogenetic analysis. A phylogenetic tree of 100 Citrus WRKY sequences with their orthologs from Arabidopsis has distinguished seven groups. The CsWRKY genes were distributed across all ten sweet orange chromosomes. A comprehensive approach and an integrative analysis of Citrus WRKY gene expression revealed variable profiles of expression within tissues and stress conditions indicating functional diversification. Thus, candidate Citrus WRKY genes have been proposed as potentially involved in fruit acidification, essential oil biosynthesis and abiotic/biotic stress tolerance. Our results provided essential prerequisites for further WRKY genes cloning and functional analysis with an aim of citrus crop improvement.

Quadriphase DS-CDMA wireless communication systems employing the generalized detector

NASA Astrophysics Data System (ADS)

Tuzlukov, Vyacheslav

2012-05-01

Probability of bit-error Per performance of asynchronous direct-sequence code-division multiple-access (DS-CDMA) wireless communication systems employing the generalized detector (GD) constructed based on the generalized approach to signal processing in noise is analyzed. The effects of pulse shaping, quadriphase or direct sequence quadriphase shift keying (DS-QPSK) spreading, aperiodic spreading sequences are considered in DS-CDMA based on GD and compared with the coherent Neyman-Pearson receiver. An exact Per expression and several approximations: one using the characterristic function method, a simplified expression for the improved Gaussian approximation (IGA) and the simplified improved Gaussian approximation are derived. Under conditions typically satisfied in practice and even with a small number of interferers, the standard Gaussian approximation (SGA) for the multiple-access interference component of the GD statistic and Per performance is shown to be accurate. Moreover, the IGA is shown to reduce to the SGA for pulses with zero excess bandwidth. Second, the GD Per performance of quadriphase DS-CDMA is shown to be superior to that of bi-phase DS-CDMA. Numerical examples by Monte Carlo simulation are presented to illustrate the GD Per performance for square-root raised-cosine pulses and spreading factors of moderate to large values. Also, a superiority of GD employment in CDMA systems over the Neyman-Pearson receiver is demonstrated

Comparison of the theoretical and real-world evolutionary potential of a genetic circuit

NASA Astrophysics Data System (ADS)

Razo-Mejia, M.; Boedicker, J. Q.; Jones, D.; DeLuna, A.; Kinney, J. B.; Phillips, R.

2014-04-01

With the development of next-generation sequencing technologies, many large scale experimental efforts aim to map genotypic variability among individuals. This natural variability in populations fuels many fundamental biological processes, ranging from evolutionary adaptation and speciation to the spread of genetic diseases and drug resistance. An interesting and important component of this variability is present within the regulatory regions of genes. As these regions evolve, accumulated mutations lead to modulation of gene expression, which may have consequences for the phenotype. A simple model system where the link between genetic variability, gene regulation and function can be studied in detail is missing. In this article we develop a model to explore how the sequence of the wild-type lac promoter dictates the fold-change in gene expression. The model combines single-base pair resolution maps of transcription factor and RNA polymerase binding energies with a comprehensive thermodynamic model of gene regulation. The model was validated by predicting and then measuring the variability of lac operon regulation in a collection of natural isolates. We then implement the model to analyze the sensitivity of the promoter sequence to the regulatory output, and predict the potential for regulation to evolve due to point mutations in the promoter region.

Oasis 2: improved online analysis of small RNA-seq data.

PubMed

Rahman, Raza-Ur; Gautam, Abhivyakti; Bethune, Jörn; Sattar, Abdul; Fiosins, Maksims; Magruder, Daniel Sumner; Capece, Vincenzo; Shomroni, Orr; Bonn, Stefan

2018-02-14

Small RNA molecules play important roles in many biological processes and their dysregulation or dysfunction can cause disease. The current method of choice for genome-wide sRNA expression profiling is deep sequencing. Here we present Oasis 2, which is a new main release of the Oasis web application for the detection, differential expression, and classification of small RNAs in deep sequencing data. Compared to its predecessor Oasis, Oasis 2 features a novel and speed-optimized sRNA detection module that supports the identification of small RNAs in any organism with higher accuracy. Next to the improved detection of small RNAs in a target organism, the software now also recognizes potential cross-species miRNAs and viral and bacterial sRNAs in infected samples. In addition, novel miRNAs can now be queried and visualized interactively, providing essential information for over 700 high-quality miRNA predictions across 14 organisms. Robust biomarker signatures can now be obtained using the novel enhanced classification module. Oasis 2 enables biologists and medical researchers to rapidly analyze and query small RNA deep sequencing data with improved precision, recall, and speed, in an interactive and user-friendly environment. Oasis 2 is implemented in Java, J2EE, mysql, Python, R, PHP and JavaScript. It is freely available at https://oasis.dzne.de.

Transcriptome-wide investigation of genomic imprinting in chicken

PubMed Central

Frésard, Laure; Leroux, Sophie; Servin, Bertrand; Gourichon, David; Dehais, Patrice; Cristobal, Magali San; Marsaud, Nathalie; Vignoles, Florence; Bed'hom, Bertrand; Coville, Jean-Luc; Hormozdiari, Farhad; Beaumont, Catherine; Zerjal, Tatiana; Vignal, Alain; Morisson, Mireille; Lagarrigue, Sandrine; Pitel, Frédérique

2014-01-01

Genomic imprinting is an epigenetic mechanism by which alleles of some specific genes are expressed in a parent-of-origin manner. It has been observed in mammals and marsupials, but not in birds. Until now, only a few genes orthologous to mammalian imprinted ones have been analyzed in chicken and did not demonstrate any evidence of imprinting in this species. However, several published observations such as imprinted-like QTL in poultry or reciprocal effects keep the question open. Our main objective was thus to screen the entire chicken genome for parental-allele-specific differential expression on whole embryonic transcriptomes, using high-throughput sequencing. To identify the parental origin of each observed haplotype, two chicken experimental populations were used, as inbred and as genetically distant as possible. Two families were produced from two reciprocal crosses. Transcripts from 20 embryos were sequenced using NGS technology, producing ∼200 Gb of sequences. This allowed the detection of 79 potentially imprinted SNPs, through an analysis method that we validated by detecting imprinting from mouse data already published. However, out of 23 candidates tested by pyrosequencing, none could be confirmed. These results come together, without a priori, with previous statements and phylogenetic considerations assessing the absence of genomic imprinting in chicken. PMID:24452801

Expressed Sequence Reference Standards for Evaluating Stage-specific Gene Expression in Southern Green Lacewings, Chrysoperla rufilabris

USDA-ARS?s Scientific Manuscript database

Five developmental stages of Chrysoperla rufilabris were tested using nine primer pairs. Three sequences were highly expressed at all life stages and six were differentially expressed. These primer pairs may be used as standards to quantitate functional gene expression associated with physiological ...

Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer).

PubMed

Chelomina, Galina N; Rozhkovan, Konstantin V; Voronova, Anastasia N; Burundukova, Olga L; Muzarok, Tamara I; Zhuravlev, Yuri N

2016-04-01

Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440-640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine.

Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer)

PubMed Central

Chelomina, Galina N.; Rozhkovan, Konstantin V.; Voronova, Anastasia N.; Burundukova, Olga L.; Muzarok, Tamara I.; Zhuravlev, Yuri N.

2015-01-01

Background Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. Methods The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. Results In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440–640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. Conclusion This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine. PMID:27158239

Positive Selection Underlies Faster-Z Evolution of Gene Expression in Birds

PubMed Central

Dean, Rebecca; Harrison, Peter W.; Wright, Alison E.; Zimmer, Fabian; Mank, Judith E.

2015-01-01

The elevated rate of evolution for genes on sex chromosomes compared with autosomes (Fast-X or Fast-Z evolution) can result either from positive selection in the heterogametic sex or from nonadaptive consequences of reduced relative effective population size. Recent work in birds suggests that Fast-Z of coding sequence is primarily due to relaxed purifying selection resulting from reduced relative effective population size. However, gene sequence and gene expression are often subject to distinct evolutionary pressures; therefore, we tested for Fast-Z in gene expression using next-generation RNA-sequencing data from multiple avian species. Similar to studies of Fast-Z in coding sequence, we recover clear signatures of Fast-Z in gene expression; however, in contrast to coding sequence, our data indicate that Fast-Z in expression is due to positive selection acting primarily in females. In the soma, where gene expression is highly correlated between the sexes, we detected Fast-Z in both sexes, although at a higher rate in females, suggesting that many positively selected expression changes in females are also expressed in males. In the gonad, where intersexual correlations in expression are much lower, we detected Fast-Z for female gene expression, but crucially, not males. This suggests that a large amount of expression variation is sex-specific in its effects within the gonad. Taken together, our results indicate that Fast-Z evolution of gene expression is the product of positive selection acting on recessive beneficial alleles in the heterogametic sex. More broadly, our analysis suggests that the adaptive potential of Z chromosome gene expression may be much greater than that of gene sequence, results which have important implications for the role of sex chromosomes in speciation and sexual selection. PMID:26067773

Cis-regulatory somatic mutations and gene-expression alteration in B-cell lymphomas.

PubMed

Mathelier, Anthony; Lefebvre, Calvin; Zhang, Allen W; Arenillas, David J; Ding, Jiarui; Wasserman, Wyeth W; Shah, Sohrab P

2015-04-23

With the rapid increase of whole-genome sequencing of human cancers, an important opportunity to analyze and characterize somatic mutations lying within cis-regulatory regions has emerged. A focus on protein-coding regions to identify nonsense or missense mutations disruptive to protein structure and/or function has led to important insights; however, the impact on gene expression of mutations lying within cis-regulatory regions remains under-explored. We analyzed somatic mutations from 84 matched tumor-normal whole genomes from B-cell lymphomas with accompanying gene expression measurements to elucidate the extent to which these cancers are disrupted by cis-regulatory mutations. We characterize mutations overlapping a high quality set of well-annotated transcription factor binding sites (TFBSs), covering a similar portion of the genome as protein-coding exons. Our results indicate that cis-regulatory mutations overlapping predicted TFBSs are enriched in promoter regions of genes involved in apoptosis or growth/proliferation. By integrating gene expression data with mutation data, our computational approach culminates with identification of cis-regulatory mutations most likely to participate in dysregulation of the gene expression program. The impact can be measured along with protein-coding mutations to highlight key mutations disrupting gene expression and pathways in cancer. Our study yields specific genes with disrupted expression triggered by genomic mutations in either the coding or the regulatory space. It implies that mutated regulatory components of the genome contribute substantially to cancer pathways. Our analyses demonstrate that identifying genomically altered cis-regulatory elements coupled with analysis of gene expression data will augment biological interpretation of mutational landscapes of cancers.

[Knockdown of STAT3 inhibits proliferation and migration of HepG2 hepatoma cells induced by IFN1].

PubMed

Li, Xiaofang; Wang, Yuqi; Yan, Ben; Fang, Peipei; Ma, Chao; Xu, Ning; Fu, Xiaoyan; Liang, Shujuan

2018-02-01

Objective To prepare lentiviruses expressing shRNA sequences targeting human signal transducer and activator of transcription 3 (STAT3) and detect the effect of STAT3 knockdown on type I interferon (IFN1)-induced proliferation and migration in HepG2 cells. Methods Four STAT3-targeting shRNA sequences (shRNA1-shRNA4) and one control sequence (Ctrl shRNA) were selected and cloned respectively into pLKO.1-sp6-pgk-GFP to construct shRNA-expressing vectors. Along with backbone psPAX2 and pMD2.G vectors, they were separately transfected into HEK293T cells to prepare lentiviruses. HepG2 cells were infected with the lentiviruses. Cytoplastic STAT3 level was detected by Western blotting to screen effective shRNA sequence(s) targeting STAT3. Proliferation and migration of HepG2 cells were analyzed by CCK-8 assay and Transwell TM migration and scratching assay, respectively. To detect the effect of IFN1 on cell proliferation and migration of HepG2 cells, the cells were treated with 2000 U/mL IFNα2b for indicated time and the activation of IFN-triggered STAT1 signal transduction was assayed by Western blotting. Results Two most effective STAT3-targeting shRNA sequences shRNA1 and shRNA2 were selected, and the expression of both STAT3 shRNA significantly decreased proliferation and migration of HepG2 cells. When treated with IFNα2b, 2000 U/mL of IFN1 showed more competent in attenuating growth and migration of HepG2 cells. Our data further proved that knockdown of STAT3 increased the phosphorylation of STAT1, and IFNα2b further enhanced the activation of STAT1 signaling in HepG2 cells. Conclusion Knockdown of STAT3 inhibits cell migration and growth, and rescues IFN response through up-regulating STAT1 signal transduction in HepG2 hepatoma cells.

Transcriptomic resources for the medicinal legume Mucuna pruriens: de novo transcriptome assembly, annotation, identification and validation of EST-SSR markers.

PubMed

Sathyanarayana, N; Pittala, Ranjith Kumar; Tripathi, Pankaj Kumar; Chopra, Ratan; Singh, Heikham Russiachand; Belamkar, Vikas; Bhardwaj, Pardeep Kumar; Doyle, Jeff J; Egan, Ashley N

2017-05-25

The medicinal legume Mucuna pruriens (L.) DC. has attracted attention worldwide as a source of the anti-Parkinson's drug L-Dopa. It is also a popular green manure cover crop that offers many agronomic benefits including high protein content, nitrogen fixation and soil nutrients. The plant currently lacks genomic resources and there is limited knowledge on gene expression, metabolic pathways, and genetics of secondary metabolite production. Here, we present transcriptomic resources for M. pruriens, including a de novo transcriptome assembly and annotation, as well as differential transcript expression analyses between root, leaf, and pod tissues. We also develop microsatellite markers and analyze genetic diversity and population structure within a set of Indian germplasm accessions. One-hundred ninety-one million two hundred thirty-three thousand two hundred forty-two bp cleaned reads were assembled into 67,561 transcripts with mean length of 626 bp and N50 of 987 bp. Assembled sequences were annotated using BLASTX against public databases with over 80% of transcripts annotated. We identified 7,493 simple sequence repeat (SSR) motifs, including 787 polymorphic repeats between the parents of a mapping population. 134 SSRs from expressed sequenced tags (ESTs) were screened against 23 M. pruriens accessions from India, with 52 EST-SSRs retained after quality control. Population structure analysis using a Bayesian framework implemented in fastSTRUCTURE showed nearly similar groupings as with distance-based (neighbor-joining) and principal component analyses, with most of the accessions clustering per geographical origins. Pair-wise comparison of transcript expression in leaves, roots and pods identified 4,387 differentially expressed transcripts with the highest number occurring between roots and leaves. Differentially expressed transcripts were enriched with transcription factors and transcripts annotated as belonging to secondary metabolite pathways. The M. pruriens transcriptomic resources generated in this study provide foundational resources for gene discovery and development of molecular markers. Polymorphic SSRs identified can be used for genetic diversity, marker-trait analyses, and development of functional markers for crop improvement. The results of differential expression studies can be used to investigate genes involved in L-Dopa synthesis and other key metabolic pathways in M. pruriens.

Molecular characterization and expression of ADAMTS8 (a disintegrin and metalloproteinase with thrombospondin motifs 8) in chicken.

PubMed

Lee, Ra Ham; Lee, Seokhyun; Kim, Yu Ra; Lee, Hak-Kyo; Song, Ki-Duk

2018-05-31

A disintegrin and metallopeptidase with thrombospondin motifs type 8 (ADAMTS8) is crucial for diverse physiological processes, such as inflammation, tissue morphogenesis, and tumorigenesis. The chicken ADAMTS8 (chADAMTS8) gene was differentially expressed in the kidney following exposure to different calcium concentrations, suggesting a pathological role of this protein in metabolic diseases. We aimed to examine the molecular characteristics of chADAMTS8 and analyze the gene-expression differences in response to Toll-like receptor 3 (TLR3) stimulation. The ADAMTS8 mRNA and amino acid sequences of various species (chicken, duck, cow, mouse, rat, human, chimpanzee, pig, and horse) were retrieved from the Ensembl database and subjected to bioinformatics analyses. Reverse-transcription polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) experiments were performed with various chicken tissues and the chicken fibroblast DF-1 cell line, which was stimulated with polyinosinic-polycytidylic acid (poly [I:C]; a TLR3 ligand). The ChADAMTS8 gene was predicted to contain three thrombospondin type 1 (TSP1) domains, whose amino acid sequences shared homology among the different species, whereas sequences outside the TSP1 domains (especially the amino-terminal region) were very different. Phylogenetic analysis revealed that chADAMTS8 is evolutionarily clustered in the same clade with that of the duck. chADAMTS8 mRNA was broadly expressed in chicken tissues, and the expression was significantly up-regulated in the DF-1 cells in response to poly (I:C) stimulation (p < 0.05). These results showed that chADAMTS8 may be a target gene for TLR3 signaling. In this report, the genetic information of chicken ADAMTS8 gene, its expression in chicken tissues, and chicken DF-1 cells under the stimulation of TLR3 were shown. The result suggests that chADAMTS8 expression may be induced by viral infection and correlated with TLR3-mediated signaling pathway. Further study of the function of chADAMTS8 during TLR3-dependent inflammation (which represents RNA viral infection) is needed and it will also be important.

Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues.

PubMed

Viana, Antonio A B; Fragoso, Rodrigo R; Guimarães, Luciane M; Pontes, Naiara; Oliveira-Neto, Osmundo B; Artico, Sinara; Nardeli, Sarah M; Alves-Ferreira, Marcio; Batista, João A N; Silva, Maria C M; Grossi-de-Sa, Maria F

2011-11-24

Cotton (Gossypium spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization. Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.

Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues

PubMed Central

2011-01-01

Background Cotton (Gossypium spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization. Results Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. Conclusions uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues. PMID:22115195

Expression Profiles and DNA-Binding Affinity of Five ERF Genes in Bunches of Vitis vinifera cv. Cardinal Treated with High Levels of CO2 at Low Temperature

PubMed Central

Romero, Irene; Vazquez-Hernandez, Maria; Escribano, M. I.; Merodio, Carmen; Sanchez-Ballesta, M. T.

2016-01-01

Ethylene response factors (ERFs) play an important role in plants by regulating defense response through interaction with various stress pathways. After harvest, table grapes (Vitis vinifera L.) are subject to a range of problems associated with postharvest storage at 0°C, such as fungal attack, water loss and rachis browning. The application of a 3-day high CO2 treatment maintained fruit quality and activated the induction of transcription factors belonging to different families such as ERF. In this paper, we have isolated five VviERFs from table grapes cv. Cardinal, whose deduced amino acid sequence contained the conserved apetalous (AP2)/ERF domain. The phylogeny and putative conserved motifs in VviERFs were analyzed and compared with those previously reported in Vitis. VviERFs-c gene expression was studied by quantitative real-time RT-PCR in the different tissues of bunches stored at low temperature and treated with high levels of CO2. The results showed that in most of the tissues analyzed, VviERFs-c gene expression was induced by the storage under normal atmosphere although the application of high levels of CO2 caused a greater increase in the VviERFs-c transcript accumulation. The promoter regions of two PRs (pathogenesis related proteins), Vcchit1b and Vcgns1, were obtained and the in silico analysis revealed the presence of a cis-acting ethylene response element (GCC box). In addition, expression of these two PR genes was analyzed in the pulp and rachis of CO2-treated and non-treated table grapes stored at 0°C and results showed significant correlations with VviERF2-c and VviERF6L7-c gene expression in rachis, and between VviERF11-c and Vcchit1b in pulp. Finally by using electro mobility shift assays, we denoted differences in binding of VviERFs to the GCC sequences present in the promoters of both PRs, with VviERF6L7-c being the only member which did not bind to any tested probe. Overall, our results suggest that the beneficial effect of high CO2 treatment maintaining table grape quality seems to be mediated by the regulation of ERFs and in particular VviERF2-c might play an important role by modulating the expression of PR genes. PMID:27965678

Comparative study of four interleukin 17 cytokines of tongue sole Cynoglossus semilaevis: Genomic structure, expression pattern, and promoter activity.

PubMed

Chi, Heng; Sun, Li

2015-11-01

The interleukin (IL)-17 cytokine family participates in the regulation of many cellular functions. In the present study, we analyzed the genomic structure, expression, and promoter activity of four IL-17 members from the teleost fish tongue sole (Cynoglossus semilaevis), i.e. CsIL-17C CsIL-17D, CsIL-17F, and IL-17F like (IL-17Fl). We found that CsIL-17C, CsIL-17D, CsIL-17F, and CsIL-17Fl share 21.2%-28.6% overall sequence identities among themselves and 31.5%-71.2% overall sequence identities with their counterparts in other teleost. All four CsIL-17 members possess an IL-17 domain and four conserved cysteine residues. Phylogenetic analysis classified the four CsIL-17 members into three clusters. Under normal physiological conditions, the four CsIL-17 expressed in multiple tissues, especially non-immune tissues. Bacterial infection upregulated the expression of all four CsIL-17, while viral infection upregulated the expression of CsIL-17D and CsIL-17Fl but downregulated the expression of CsIL-17C and CsIL-17F. The 1.2 kb 5'-flanking regions of the four CsIL-17 exhibited apparent promoter activity and contain a number of putative transcription factor-binding sites. Furthermore, the promoter activities of CsIL-17C, CsIL-17D, and CsIL-17F, but not CsIL-17Fl, were modulated to significant extents by lipopolysaccharide, PolyI:C, and PMA. This study provides the first evidence that in teleost, different IL-17 members differ in expression pattern and promoter activity. Copyright © 2015 Elsevier Ltd. All rights reserved.