scholarly journals Transcriptional milestones in Dictyostelium development

2021 ◽  
Author(s):  
Mariko Katoh-Kurasawa ◽  
Karin Hrovatin ◽  
Shigenori Hirose ◽  
Amanda Webb ◽  
Hsing-I Ho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genetic Background ◽  
Web Application ◽  
Morphological Changes ◽  
Single Cells ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Gene Annotations ◽  
Developmental Conditions

Development of the social amoeba Dictyostelium discoideum begins by starvation of single cells and ends in multicellular fruiting bodies 24 hours later. These major morphological changes are accompanied by sweeping gene expression changes, encompassing nearly half of the 13,000 genes in the genome. To explore the relationships between the transcriptome and developmental morphogenesis, we performed time-series RNA-sequencing analysis of the wild type and 20 mutant strains with altered morphogenesis. These strains exhibit arrest at different developmental stages, accelerated development, or terminal morphologies that are not typically seen in the wild type. Considering eight major morphological transitions, we identified 1,371 milestone genes whose expression changes sharply between two consecutive transitions. We also identified 1,099 genes as members of 21 regulons, which are groups of genes that remain coordinately regulated despite the genetic, temporal, and developmental perturbations in the dataset. The gene annotations in these milestones and regulons validate known transitions and reveal several new physiological and functional transitions during development. For example, we found that DNA replication genes are co-regulated with cell division genes, so they are co-expressed in mid-development even though chromosomal DNA is not replicated at that time. Altogether, the dataset includes 486 transcriptional profiles, across developmental and genetic conditions, that can be used to identify new relationships between gene expression and developmental processes and to improve gene annotations. We demonstrate the utility of this resource by showing that the cycles of aggregation and disaggregation observed in allorecognition-defective mutants involve a dedifferentiation process. We also show unexpected variability and sensitivity to genetic background and developmental conditions in two commonly used genes, act6 and act15, and robustness of the coaA gene. Finally, we propose that gpdA should be used as a standard for mRNA quantitation because it is less sensitive to genetic background and developmental conditions than commonly used standards. The dataset is available for democratized exploration without the need for programming skills through the web application dictyExpress and the data mining environment Orange.

scholarly journals Inactivation of the Stress- and Starvation-Inducible gls24 Operon Has a Pleiotrophic Effect on Cell Morphology, Stress Sensitivity, and Gene Expression inEnterococcus faecalis

2000 ◽  
Vol 182 (16) ◽  
pp. 4512-4520 ◽  
Author(s):  
Jean-Christophe Giard ◽  
Alain Rince ◽  
Herve Capiaux ◽  
Yanick Auffray ◽  
Axel Hartke
Keyword(s):  
Gene Expression ◽  
Bile Salts ◽  
Morphological Changes ◽  
Tap Water ◽  
Stress Protein ◽  
Biological Significance ◽  
Stress Sensitivity ◽  
Northern Analysis ◽  
Alkaline Stress ◽  
Wild Type

ABSTRACT Enterococcus faecalis induces the synthesis of at least 42 proteins during 24 h of glucose starvation. Because of its induction during carbohydrate and complete starvation (incubation in tap water) and CdCl2 and bile salts stresses, one of these proteins (Gls24) was qualified as a “general stress protein” and was analyzed at the molecular level. Its corresponding gene,gls24, seems to be the penultimate gene of an operon composed, altogether, of six open reading frames (ORFs). The ORF preceding gls24 (orf4) showed very strong identity with gls24. The deduced polypeptides of these two genes showed similarity with a 20-kDa hypothetical protein fromLactococcus lactis and an alkaline stress protein fromStaphylococcus aureus with no previously known biological significance. Data from the operon sequence and Northern analysis led to the conclusions that (i) gls24 possesses its own promoter which is especially induced at the onset of starvation and (ii) the operon promoter is stress inducible in exponential-phase cells. A mutation in the gls24 gene led to a severe reduction of growth rate and reduction of survival against 0.3% bile salts in the 24-h-starved cells compared to the wild-type strain. Moreover, the chain length of the mutant is significantly reduced during growth. These results argue strongly for a role of the protein Gls24 and/or GlsB in morphological changes and in stress tolerance inE. faecalis. Comparison of two-dimensional protein gels from wild-type cells with those from gls24 mutant cells revealed a pleiotropic effect of the mutation on gene expression. At least nine proteins were present in larger amounts in the mutant. For six of them, the corresponding N-terminal microsequence has been obtained. Three of these sequences map in genes coding forl-lactate dehydrogenase, lipoamide dehydrogenase, and pyruvate decarboxylase, all involved in pyruvate metabolism.

scholarly journals ID: 1027 High-Throughput, Single Cell Whole Transcriptome Sequencing Analysis of Cancer Cells with the New BD FACSMelody™ Cell Sorter and BD™ Precise assay

2017 ◽  
Vol 4 (S) ◽  
pp. 102
Author(s):  
Xiaoyang (Alice) Wang ◽  
Chip Lomas ◽  
Craig Betts ◽  
Aaron Walker ◽  
Christina Fan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cancer Cells ◽  
Cell Line ◽  
Selection Criteria ◽  
Single Cells ◽  
Principal Component ◽  
Jurkat Cells ◽  
Sequencing Analysis ◽  
Whole Transcriptome

Gene expression studies performed on bulk samples might obscure the understanding of complex samples. Gene expression analyses performed on single cells, however, can offer a powerful method to resolve sample heterogeneity and reveal hidden biology. Optimal sample preparation is critical to obtain high quality gene expression data from single cells.Historically, single cells or small numbers of cells were isolated and prepared by limiting dilutions, laser capture microdissection, or microfluidics technologies, or fluorescence-activated cell sorting (FACS). FACS sorting enables highthroughput processing of a heterogeneous mixture of cells and ensures the delivery of single cells or a small number ofcells into a chosen receptacle to meet the selection criteria at a purity level that is unmatched by other approaches.Furthermore, by FACS, the single cell selection criteria can be based on surface marker expression, cell size, and granularity(represented by scatter). Sorted cells can be used for any downstream application including next generation sequencing(NGS).In this study, the new, easy-to-use BD FACSMelody™ sorter was applied to sort individual cancer cells. Jurkat cells (a Tleukemia cell line), and T47D cells (a breast cancer cell line) were mixed, stained, analyzed, and sorted on a BD FACSMelody system. The individual cell’s whole transcriptome was interrogated using BD™ Precise Single Cell WTA (whole transcriptome amplification) Assay. Principal component analysis was applied to cluster the sorted Jurkat and T47D-cell populations.

scholarly journals A Nonessential HP1-Like Protein Affects Starvation-Induced Assembly of Condensed Chromatin and Gene Expression in Macronuclei of Tetrahymena thermophila

1999 ◽  
Vol 19 (5) ◽  
pp. 3624-3634 ◽  
Author(s):  
Hui Huang ◽  
James F. Smothers ◽  
Emily A. Wiley ◽  
C. David Allis
Keyword(s):  
Gene Expression ◽  
Tetrahymena Thermophila ◽  
Morphological Changes ◽  
Condensed Chromatin ◽  
Induced Response ◽  
Wild Type ◽  
Eukaryotic Genes ◽  
Associated Proteins ◽  
Induced Genes ◽  
Dense Chromatin

ABSTRACT Heterochromatin represents a specialized chromatin environment vital to both the repression and expression of certain eukaryotic genes. One of the best-studied heterochromatin-associated proteins isDrosophila HP1. In this report, we have disrupted all somatic copies of the Tetrahymena HHP1 gene, which encodes an HP1-like protein, Hhp1p, in macronuclei (H. Huang, E. A. Wiley, R. C. Lending, and C. D. Allis, Proc. Natl. Acad. Sci. USA 95:13624–13629, 1998). Unlike the Drosophila HP1 gene,HHP1 is not essential in Tetrahymena spp., and during vegetative growth no clear phenotype is observed in cells lacking Hhp1p (ΔHHP1). However, during a shift to nongrowth conditions, the survival rate of ΔHHP1 cells is reduced compared to that of wild-type cells. Upon starvation, Hhp1p becomes hyperphosphorylated concomitant with a reduction in macronuclear volume and an increase in the size of electron-dense chromatin bodies; neither of these morphological changes occurs in the absence of Hhp1p. Activation of two starvation-induced genes (ngoA and CyP) is significantly reduced in ΔHHP1 cells while, in contrast, the expression of several growth-related or constitutively expressed genes is comparable to that in wild-type cells. These results suggest that Hhp1p functions in the establishment and/or maintenance of a specialized condensed chromatin environment that facilitates the expression of certain genes linked to a starvation-induced response.

scholarly journals CREB-binding protein gene, HAC701, negatively regulates WRKY45-dependent immunity in rice

2020 ◽  
Author(s):  
Nino A. Espinas ◽  
Tu Ngoc Le ◽  
Miura Saori ◽  
Yasuka Shimajiri ◽  
Ken Shirasu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Responses ◽  
Binding Protein ◽  
Large Set ◽  
Sequencing Analysis ◽  
Creb Binding Protein ◽  
Transcriptional Coactivator ◽  
Wild Type ◽  
Genome Wide ◽  
H3k9 Acetylation

ABSTRACTCREB-binding protein (CBP) is a known transcriptional coactivator and an acetyltransferase that functions in several cellular processes by regulating gene expression. However, how it functions in plant immunity remains unexplored. By characterizing hac701, we demonstrate that HAC701 negatively regulates the immune responses in rice. hac701 shows enhanced disease resistance against a bacterial pathogen, Pseudomonas syringae pv. oryzae (Pso), which causes bacterial halo blight of rice. Our transcriptomic analysis revealed that rice WRKY45, one of the main regulators of rice immunity, is upregulated in hac701 and possibly conferring the resistance phenotype against Pso. The morphological phenotypes of hac701 single mutants were highly similar to WRKY45 overexpression transgenic lines reported in previous studies. In addition, we also compared the list of genes in these studies when WRKY45 is overexpressed and chemically induced transiently with the differentially expressed genes (DEGs) in hac701, and found that they largely overlap. When we investigated for cis-elements found 1kb upstream of WRKY45 gene and WRKY45-dependent DEGs, we found that WRKY45 promoter contains the CRE motif, a possible target of HAC701-mediated regulation. Genome-wide H3K9 acetylation profiling showed depletion of acetylation at large set of genes in hac701. However, consistent with the upregulation of WRKY45 gene expression, our ChIP-sequencing analysis demonstrated that regions of WRKY45 promoter are enriched in H3K9 acetylation in hac701 compared to the segregated wild type control in the mock condition. WRKY45 promoter might be on the receiving end for possible genome-wide compensatory effects when a global regulator like HAC701 is mutated. Finally, we show that HAC701 may have roles in systemic immune signaling. We therefore propose that wild type HAC701 negatively regulates WRKY45 gene expression, thereby suppressing immune responses.SIGNIFICANCEHAC701 is a member of CREB-binding protein (CBP) family that acts as transcriptional coactivator and acetyltransferase. However, little is known how it regulates innate immunity in plants. Herein we reported that rice HAC701 suppresses WRKY45-dependent defense pathway. Our study showed that HAC701 seemingly interacts genetically with WRKY45 in rice to modulate immune responses against pathogens.

scholarly journals Phosphorylation Events in the Multiple Gene Regulator of Group A Streptococcus Significantly Influence Global Gene Expression and Virulence

2015 ◽  
Vol 83 (6) ◽  
pp. 2382-2395 ◽  
Author(s):  
Misu Sanson ◽  
Nishanth Makthal ◽  
Maire Gavagan ◽  
Concepcion Cantu ◽  
Randall J. Olsen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Mutant Strain ◽  
Group A Streptococcus ◽  
Global Gene Expression ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Content Type ◽  
Group A

Whole-genome sequencing analysis of ∼800 strains of group AStreptococcus(GAS) found that the gene encoding themultiple virulencegene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenicmgamutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidinesin vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations inmga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis.

scholarly journals Gene expression profiles of Spo11−/− mouse testes with spermatocytes arrested in meiotic prophase I

Reproduction ◽  
2006 ◽  
Vol 132 (1) ◽  
pp. 67-77 ◽  
Author(s):  
Natalya A Smirnova ◽  
Peter J Romanienko ◽  
Pavel P Khil ◽  
R Daniel Camerini-Otero
Keyword(s):  
Gene Expression ◽  
Meiotic Recombination ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Specific Protein ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Before And After ◽  
Genes Encoding ◽  
Protein Components

Spo11, a meiosis-specific protein, introduces double-strand breaks on chromosomal DNA and initiates meiotic recombination in a wide variety of organisms. Mouse null Spo11 spermatocytes fail to synapse chromosomes and progress beyond the zygotene stage of meiosis. We analyzed gene expression profiles in Spo11−/ −adult and juvenile wild-type testis to describe genes expressed before and after the meiotic arrest resulting from the knocking out of Spo11. These genes were characterized using the Gene Ontology data base. To focus on genes involved in meiosis, we performed comparative gene expression analysis of Spo11−/ −and wild-type testes from 15-day mice, when spermatocytes have just entered pachytene. We found that the knockout of Spo11 causes dramatic changes in the level of expression of genes that participate in meiotic recombination (Hop2, Brca2, Mnd1, FancG) and in the meiotic checkpoint (cyclin B2, Cks2), but does not affect genes encoding protein components of the synaptonemal complex. Finally, we discovered unknown genes that are affected by the disruption of the Spo11 gene and therefore may be specifically involved in meiosis and spermatogenesis.

scholarly journals Population Heterogeneity in Corynebacterium glutamicum ATCC 13032 Caused by Prophage CGP3

2008 ◽  
Vol 190 (14) ◽  
pp. 5111-5119 ◽  
Author(s):  
Julia Frunzke ◽  
Marc Bramkamp ◽  
Jens-Eric Schweitzer ◽  
Michael Bott
Keyword(s):  
Corynebacterium Glutamicum ◽  
Copy Number ◽  
Iron Homeostasis ◽  
Single Cells ◽  
Population Heterogeneity ◽  
Mrna Levels ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Intracellular Iron ◽  
Prophage Region

ABSTRACT The genome of Corynebacterium glutamicum type strain ATCC 13032 (accession number BX927147) contains three prophages, CGP1, CGP2, and CGP3. We recently observed that many genes within the CGP3 prophage region have increased mRNA levels in a dtxR deletion mutant that lacks the master regulator of iron homeostasis (J. Wennerhold and M. Bott, J. Bacteriol. 188:2907-2918, 2006). Here, we provide evidence that this effect is due to the increased induction of the prophage CGP3 in the dtxR mutant, possibly triggered by DNA damage caused by elevated intracellular iron concentrations. Upon induction, the CGP3 prophage region is excised from the genome and forms a circular double-stranded DNA molecule. Using quantitative real-time PCR, an average copy number of about 0.1 per chromosome was determined for circular CGP3 DNA in wild-type C. glutamicum. This copy number increased about 15-fold in the dtxR mutant. In order to visualize the CGP3 DNA within single cells, a derivative of the wild type was constructed that contained an array of tet operators integrated within the CGP3 region and a plasmid-encoded YFP-TetR fusion protein. As expected, one to two fluorescent foci that represented the chromosomally integrated CGP3 prophage were detected in the majority of cells. However, in a small fraction (2 to 4%) of the population, 4 to 10 CGP3 DNA molecules could be observed in a single cell. Interestingly, the presence of many CGP3 copies in a cell often was accompanied by an efflux of chromosomal DNA, indicating the lysis of the corresponding cell. However, evidence for the formation of functional infective CGP3 phage particles could not be obtained.

scholarly journals Argonaute binding within 3′-untranslated regions poorly predicts gene repression

2020 ◽  
Author(s):  
Yongjun Chu ◽  
Audrius Kilikevicius ◽  
Jing Liu ◽  
Krystal C Johnson ◽  
Shinnichi Yokota ◽  
...  
Keyword(s):  
Gene Expression ◽  
Steady State ◽  
Mammalian Cells ◽  
Gene Repression ◽  
Type Versus ◽  
Untranslated Regions ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Knock Out ◽  
Steady State Levels

Abstract Despite two decades of study, the full scope of RNAi in mammalian cells has remained obscure. Here we combine: (i) Knockout of argonaute (AGO) variants; (ii) RNA sequencing analysis of gene expression changes and (iii) Enhanced Crosslinking Immunoprecipitation Sequencing (eCLIP-seq) using anti-AGO2 antibody to identify potential microRNA (miRNA) binding sites. We find that knocking out AGO1, AGO2 and AGO3 together are necessary to achieve full impact on steady state levels of mRNA. eCLIP-seq located AGO2 protein associations within 3′-untranslated regions. The standard mechanism of miRNA action would suggest that these associations should repress gene expression. Contrary to this expectation, associations between AGO and RNA are poorly correlated with gene repression in wild-type versus knockout cells. Many clusters are associated with increased steady state levels of mRNA in wild-type versus knock out cells, including the strongest cluster within the MYC 3′-UTR. Our results suggest that assumptions about miRNA action should be re-examined.

scholarly journals Argonaute Binding within 3’-Untranslated Regions Does Not Predict Gene Repression

2020 ◽  
Author(s):  
Yongjun Chu ◽  
Audrius Kilikevicius ◽  
Jing Liu ◽  
Krystal C. Johnson ◽  
Shinnichi Yakota ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Gene Repression ◽  
Type Versus ◽  
Untranslated Regions ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Knock Out

ABSTRACTDespite two decades of study, the full scope of RNAi in mammalian cells has remained obscure. Here we combine: 1) Knockout of argonaute (AGO) variants; 2) RNA sequencing analysis of gene expression changes; and 3) Crosslinking Immunoprecipitation Sequencing (CLIP-seq) using anti-AGO2 antibody to identify potential microRNA (miRNA) binding sites. We find that knocking out AGO1, AGO2, and AGO3 are necessary to achieve full impact on gene expression. CLIP-seq reveals several hundred significant AGO2 associations within the 3’-untranslated regions of cytoplasmic transcripts. The standard mechanism of miRNA action would suggest that these associations repress gene expression. Contrary to this expectation, clusters are poorly correlated with gene repression in wild-type versus knockout cells. Many clusters are associated with increased gene expression in wild-type versus knock out cells, including the strongest cluster within the MYC 3’-UTR. Our results suggest that assumptions about miRNA action should be re-examined.

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