scholarly journals An integrated platform for genome engineering and gene expression perturbation in Plasmodium falciparum

2019 ◽  
Author(s):  
Armiyaw S. Nasamu ◽  
Alejandra Falla ◽  
Charisse Flerida A. Pasaje ◽  
Bridget A. Wall ◽  
Jeffrey C. Wagner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Genome Engineering ◽  
Malarial Parasite ◽  
Dna Assembly ◽  
Biological Processes ◽  
Linear Plasmids ◽  
Functional Genetics ◽  
Integrated Platform ◽  
Significant Barrier

ABSTRACTEstablishing robust genome engineering methods in the malarial parasite, Plasmodium falciparum, has the potential to substantially improve the efficiency with which we gain understanding of this pathogen’s biology to propel treatment and elimination efforts. Methods for manipulating gene expression and engineering the P. falciparum genome have been validated. However, a significant barrier to fully leveraging these advances is the difficulty associated with assembling the extremely high AT content DNA constructs required for modifying the P. falciparum genome. These are frequently unstable in commonly-used circular plasmids. We address this bottleneck by devising a DNA assembly framework leveraging the improved reliability with which large AT-rich regions can be efficiently manipulated in linear plasmids. This framework integrates several key functional genetics outcomes via CRISPR/Cas9 and other methods from a common, validated framework. Overall, this molecular toolkit enables P. falciparum genetics broadly and facilitates deeper interrogation of parasite genes involved in diverse biological processes.

scholarly journals Serial Analysis of Gene Expression in Plasmodium falciparum Reveals the Global Expression Profile of Erythrocytic Stages and the Presence of Anti-Sense Transcripts in the Malarial Parasite

2001 ◽  
Vol 12 (10) ◽  
pp. 3114-3125 ◽  
Author(s):  
Swati Patankar ◽  
Anusha Munasinghe ◽  
Azadeh Shoaibi ◽  
Leda M. Cummings ◽  
Dyann F. Wirth
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Low Complexity ◽  
Culture Conditions ◽  
Open Reading Frames ◽  
High Abundance ◽  
Malarial Parasite ◽  
Northern Analysis ◽  
Local Alignment ◽  
Open Platform

Serial analysis of gene expression (SAGE) was applied to the malarial parasite Plasmodium falciparum to characterize the comprehensive transcriptional profile of erythrocytic stages. A SAGE library of ∼8335 tags representing 4866 different genes was generated from 3D7 strain parasites. Basic local alignment search tool analysis of high abundance SAGE tags revealed that a majority (88%) corresponded to 3D7 sequence, and despite the low complexity of the genome, 70% of these highly abundant tags matched unique loci. Characterization of these suggested the major metabolic pathways that are used by the organism under normal culture conditions. Furthermore several tags expressed at high abundance (30% of tags matching to unique loci of the 3D7 genome) were derived from previously uncharacterized open reading frames, demonstrating the use of SAGE in genome annotation. The open platform “profiling” nature of SAGE also lead to the important discovery of a novel transcriptional phenomenon in the malarial pathogen: a significant number of highly abundant tags that were derived from annotated genes (17%) corresponded to antisense transcripts. These SAGE data were validated by two independent means, strand specific reverse transcription-polymerase chain reaction and Northern analysis, where antisense messages were detected in both asexual and sexual stages. This finding has implications for transcriptional regulation ofPlasmodium gene expression.

Review of Progress in Predicting Protein Methylation Sites

2019 ◽  
Vol 23 (15) ◽  
pp. 1663-1670 ◽  
Author(s):  
Chunyan Ao ◽  
Shunshan Jin ◽  
Yuan Lin ◽  
Quan Zou
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Transcriptional Activity ◽  
Regulation Of Gene Expression ◽  
Protein Methylation ◽  
Biological Processes ◽  
Post Translational Modification ◽  
Regulatory Enzymes ◽  
Lysine Residues ◽  
Arginine Residues

Protein methylation is an important and reversible post-translational modification that regulates many biological processes in cells. It occurs mainly on lysine and arginine residues and involves many important biological processes, including transcriptional activity, signal transduction, and the regulation of gene expression. Protein methylation and its regulatory enzymes are related to a variety of human diseases, so improved identification of methylation sites is useful for designing drugs for a variety of related diseases. In this review, we systematically summarize and analyze the tools used for the prediction of protein methylation sites on arginine and lysine residues over the last decade.

scholarly journals Cocaine induces paradigm-specific changes to the transcriptome within the ventral tegmental area

2021 ◽  
Author(s):  
Rianne R. Campbell ◽  
Siwei Chen ◽  
Joy H. Beardwood ◽  
Alberto J. López ◽  
Lilyana V. Pham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ventral Tegmental Area ◽  
Home Cage ◽  
Expression Patterns ◽  
Energy Regulation ◽  
Biological Processes ◽  
Cocaine Exposure ◽  
Self Administration ◽  
Drug Reinforcement ◽  
Ventral Tegmental

AbstractDuring the initial stages of drug use, cocaine-induced neuroadaptations within the ventral tegmental area (VTA) are critical for drug-associated cue learning and drug reinforcement processes. These neuroadaptations occur, in part, from alterations to the transcriptome. Although cocaine-induced transcriptional mechanisms within the VTA have been examined, various regimens and paradigms have been employed to examine candidate target genes. In order to identify key genes and biological processes regulating cocaine-induced processes, we employed genome-wide RNA-sequencing to analyze transcriptional profiles within the VTA from male mice that underwent one of four commonly used paradigms: acute home cage injections of cocaine, chronic home cage injections of cocaine, cocaine-conditioning, or intravenous-self administration of cocaine. We found that cocaine alters distinct sets of VTA genes within each exposure paradigm. Using behavioral measures from cocaine self-administering mice, we also found several genes whose expression patterns corelate with cocaine intake. In addition to overall gene expression levels, we identified several predicted upstream regulators of cocaine-induced transcription shared across all paradigms. Although distinct gene sets were altered across cocaine exposure paradigms, we found, from Gene Ontology (GO) term analysis, that biological processes important for energy regulation and synaptic plasticity were affected across all cocaine paradigms. Coexpression analysis also identified gene networks that are altered by cocaine. These data indicate that cocaine alters networks enriched with glial cell markers of the VTA that are involved in gene regulation and synaptic processes. Our analyses demonstrate that transcriptional changes within the VTA depend on the route, dose and context of cocaine exposure, and highlight several biological processes affected by cocaine. Overall, these findings provide a unique resource of gene expression data for future studies examining novel cocaine gene targets that regulate drug-associated behaviors.

scholarly journals Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nathan J. VanDusen ◽  
Julianna Y. Lee ◽  
Weiliang Gu ◽  
Catalina E. Butler ◽  
Isha Sethi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genetic Screen ◽  
Forward Genetics ◽  
Epigenetic Mark ◽  
Biological Processes ◽  
Dynamic Changes ◽  
Forward Genetic Screen ◽  
High Resource ◽  
Organ Systems

AbstractThe forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.

scholarly journals Analysis of the transcriptome of bovine endometrial cells isolated by laser micro-dissection (1): specific signatures of stromal, glandular and luminal epithelial cells

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Wiruntita Chankeaw ◽  
Sandra Lignier ◽  
Christophe Richard ◽  
Theodoros Ntallaris ◽  
Mariam Raliou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Localization ◽  
Expression Profiles ◽  
Cell Types ◽  
Molecular Signature ◽  
Receptor Protein ◽  
Specific Gene ◽  
Specific Cell ◽  
Biological Processes ◽  
Endometrial Cells

Abstract Background A number of studies have examined mRNA expression profiles of bovine endometrium at estrus and around the peri-implantation period of pregnancy. However, to date, these studies have been performed on the whole endometrium which is a complex tissue. Consequently, the knowledge of cell-specific gene expression, when analysis performed with whole endometrium, is still weak and obviously limits the relevance of the results of gene expression studies. Thus, the aim of this study was to characterize specific transcriptome of the three main cell-types of the bovine endometrium at day-15 of the estrus cycle. Results In the RNA-Seq analysis, the number of expressed genes detected over 10 transcripts per million was 6622, 7814 and 8242 for LE, GE and ST respectively. ST expressed exclusively 1236 genes while only 551 transcripts were specific to the GE and 330 specific to LE. For ST, over-represented biological processes included many regulation processes and response to stimulus, cell communication and cell adhesion, extracellular matrix organization as well as developmental process. For GE, cilium organization, cilium movement, protein localization to cilium and microtubule-based process were the only four main biological processes enriched. For LE, over-represented biological processes were enzyme linked receptor protein signaling pathway, cell-substrate adhesion and circulatory system process. Conclusion The data show that each endometrial cell-type has a distinct molecular signature and provide a significantly improved overview on the biological process supported by specific cell-types. The most interesting result is that stromal cells express more genes than the two epithelial types and are associated with a greater number of pathways and ontology terms.

scholarly journals Transcription Factors as the “Blitzkrieg” of Plant Defense: A Pragmatic View of Nitric Oxide’s Role in Gene Regulation

2021 ◽  
Vol 22 (2) ◽  
pp. 522
Author(s):  
Noreen Falak ◽  
Qari Muhammad Imran ◽  
Adil Hussain ◽  
Byung-Wook Yun
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Plant Defense ◽  
Systemic Acquired Resistance ◽  
Defense Mechanism ◽  
Regulation Of Gene Expression ◽  
Acquired Resistance ◽  
Biological Processes ◽  
Genetic Components ◽  
Transcriptional Changes

Plants are in continuous conflict with the environmental constraints and their sessile nature demands a fine-tuned, well-designed defense mechanism that can cope with a multitude of biotic and abiotic assaults. Therefore, plants have developed innate immunity, R-gene-mediated resistance, and systemic acquired resistance to ensure their survival. Transcription factors (TFs) are among the most important genetic components for the regulation of gene expression and several other biological processes. They bind to specific sequences in the DNA called transcription factor binding sites (TFBSs) that are present in the regulatory regions of genes. Depending on the environmental conditions, TFs can either enhance or suppress transcriptional processes. In the last couple of decades, nitric oxide (NO) emerged as a crucial molecule for signaling and regulating biological processes. Here, we have overviewed the plant defense system, the role of TFs in mediating the defense response, and that how NO can manipulate transcriptional changes including direct post-translational modifications of TFs. We also propose that NO might regulate gene expression by regulating the recruitment of RNA polymerase during transcription.

scholarly journals The crucial roles of N6-methyladenosine (m6A) modification in the carcinogenesis and progression of colorectal cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zhihao Fang ◽  
Yiqiu Hu ◽  
Jinhui Hu ◽  
Yanqin Huang ◽  
Shu Zheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colorectal Cancer ◽  
Nuclear Export ◽  
Messenger Rna ◽  
Regulatory Proteins ◽  
Biological Processes ◽  
The Past ◽  
Common Cancer ◽  
Potential Applications ◽  
Regulated Gene Expression

AbstractAs the predominant modification in RNA, N6-methyladenosine (m6A) has attracted increasing attention in the past few years since it plays vital roles in many biological processes. This chemical modification is dynamic, reversible and regulated by several methyltransferases, demethylases and proteins that recognize m6A modification. M6A modification exists in messenger RNA and affects their splicing, nuclear export, stability, decay, and translation, thereby modulating gene expression. Besides, the existence of m6A in noncoding RNAs (ncRNAs) could also directly or indirectly regulated gene expression. Colorectal cancer (CRC) is a common cancer around the world and of high mortality. Increasing evidence have shown that the changes of m6A level and the dysregulation of m6A regulatory proteins have been implicated in CRC carcinogenesis and progression. However, the underlying regulation laws of m6A modification to CRC remain elusive and better understanding of these mechanisms will benefit the diagnosis and therapy. In the present review, the latest studies about the dysregulation of m6A and its regulators in CRC have been summarized. We will focus on the crucial roles of m6A modification in the carcinogenesis and development of CRC. Moreover, we will also discuss the potential applications of m6A modification in CRC diagnosis and therapeutics.

scholarly journals High-performance chemical- and light-inducible recombinases in mammalian cells and mice

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Benjamin H. Weinberg ◽  
Jang Hwan Cho ◽  
Yash Agarwal ◽  
N. T. Hang Pham ◽  
Leidy D. Caraballo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
High Performance ◽  
Genome Engineering ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Expression Control ◽  
Gene Expression Control ◽  
High Performance Systems ◽  
Spatiotemporal Control

Abstract Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

scholarly journals Localization of ferrochelatase in Plasmodium falciparum

2004 ◽  
Vol 384 (2) ◽  
pp. 429-436 ◽  
Author(s):  
Sundaramurthy VARADHARAJAN ◽  
B. K. Chandrashekar SAGAR ◽  
Pundi N. RANGARAJAN ◽  
Govindarajan PADMANABAN
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
Enzymic Activity ◽  
Malarial Parasite ◽  
Parasite Genome ◽  
Marker Proteins ◽  
Parasite Plasmodium ◽  
Theoretical Predictions ◽  
Parasite Plasmodium Falciparum

Our previous studies have demonstrated de novo haem biosynthesis in the malarial parasite (Plasmodium falciparum and P. berghei). It has also been shown that the first enzyme of the pathway is the parasite genome-coded ALA (δ-aminolaevulinate) synthase localized in the parasite mitochondrion, whereas the second enzyme, ALAD (ALA dehydratase), is accounted for by two species: one species imported from the host red blood cell into the parasite cytosol and another parasite genome-coded species in the apicoplast. In the present study, specific antibodies have been raised to PfFC (parasite genome-coded ferrochelatase), the terminal enzyme of the haem-biosynthetic pathway, using recombinant truncated protein. With the use of these antibodies as well as those against the hFC (host red cell ferrochelatase) and other marker proteins, immunofluorescence studies were performed. The results reveal that P. falciparum in culture manifests a broad distribution of hFC and a localized distribution of PfFC in the parasite. However, PfFC is not localized to the parasite mitochondrion. Immunoelectron-microscopy studies reveal that PfFC is indeed localized to the apicoplast, whereas hFC is distributed in the parasite cytoplasm. These results on the localization of PfFC are unexpected and are at variance with theoretical predictions based on leader sequence analysis. Biochemical studies using the parasite cytosolic and organellar fractions reveal that the cytosol containing hFC accounts for 80% of FC enzymic activity, whereas the organellar fraction containing PfFC accounts for the remaining 20%. Interestingly, both the isolated cytosolic and organellar fractions are capable of independent haem synthesis in vitro from [4-14C]ALA, with the cytosol being three times more efficient compared with the organellar fraction. With [2-14C]glycine, most of the haem is synthesized in the organellar fraction. Thus haem is synthesized in two independent compartments: in the cytosol, using the imported host enzymes, and in the organellar fractions, using the parasite genome-coded enzymes.

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