Toxoplasma gondii Asexual Development: Identification of Developmentally Regulated Genes and Distinct Patterns of Gene Expression

ABSTRACT Asexual development in Toxoplasma gondii is a vital aspect of the parasite's life cycle, allowing transmission and avoidance of the host immune response. Differentiation of rapidly dividing tachyzoites into slowly growing, encysted bradyzoites involves significant changes in both physiology and morphology. We generated microarrays of ∼4,400 Toxoplasma cDNAs, representing a minimum of ∼600 genes (based on partial sequencing), and used these microarrays to study changes in transcript levels during tachyzoite-to-bradyzoite differentiation. This approach has allowed us to (i) determine expression profiles of previously described developmentally regulated genes, (ii) identify novel developmentally regulated genes, and (iii) identify distinct classes of genes based on the timing and magnitude of changes in transcript levels. Whereas microarray analysis typically involves comparisons of mRNA levels at different time points, we have developed a method to measure relative transcript abundance between genes at a given time point. This method was used to determine transcript levels in parasites prior to differentiation and to further classify bradyzoite-induced genes, thus allowing a more comprehensive view of changes in gene expression than is provided by standard expression profiles. Newly identified developmentally regulated genes include putative surface proteins (a SAG1-related protein, SRS9, and a mucin-domain containing protein), regulatory and metabolic enzymes (methionine aminopeptidase, oligopeptidase, aminotransferase, and glucose-6-phosphate dehydrogenase homologues), and a subset of genes encoding secretory organelle proteins (MIC1, ROP1, ROP2, ROP4, GRA1, GRA5, and GRA8). This analysis permits the first in-depth look at changes in gene expression during development of this complex protozoan parasite.

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Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00088.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. G875-G885 ◽

Cited By ~ 46

Author(s):

Carine Strup-Perrot ◽

Denis Mathé ◽

Christine Linard ◽

Dominique Violot ◽

Fabien Milliat ◽

...

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Expression Profiles ◽

Cdna Array ◽

Muscularis Propria ◽

Gelatin Zymography ◽

Tissue Inhibitors Of Metalloproteinases ◽

Radiation Enteritis ◽

Mrna Levels ◽

Fixed Tissue

Radiation enteritis, a common complication of radiation therapy for abdominal and pelvic cancers, is characterized by severe transmural fibrosis associated with mesenchymal cell activation, tissue disorganization, and deposition of fibrillar collagen. To investigate the mechanisms involved in this pathological accumulation of extracellular matrix, we studied gene expression of matrix components along with that of genes involved in matrix remodeling, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Hybrid selection on high-density cDNA array, real-time RT-PCR, gelatin zymography and imunohistochemistry were used to characterize the mRNA expression profile, activity, and tissue location of extracellular matrix-related genes in radiation enteritis compared with healthy ileum. cDNA array analysis revealed a strong induction of genes coding for collagens I, III, IV, VI, and VIII, SPARC, and tenascin-C, extracellular-matrix degrading enzymes (MMP-1, -2, -3, -14, -18+19), and metalloproteinase inhibitors (TIMP-1, -2, plasminogen activator inhibitor-1) in radiation enteritis. This increase was correlated with the degree of infiltration of the mucosa by inflammatory cells, and the presence of differentiated mesenchymal cells in the submucosa and muscularis propria. Despite the fact that expression of collagens, MMPs, and TIMPs simultaneously increase, quantification of net collagen deposition shows an overall accumulation of collagen. Our results indicate that late radiation enteritis tissues are subjected to active process of fibrogenesis as well as fibrolysis, with a balance toward fibrogenesis. This demonstrates that established fibrotic tissue is not scarred fixed tissue but is subjected to a dynamic remodeling process.

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Gene expression analysis of the pro-oestrous-stage rat uterus reveals neuroligin 2 as a novel steroid-regulated gene

Reproduction Fertility and Development ◽

10.1071/rd04040 ◽

2004 ◽

Vol 16 (8) ◽

pp. 763 ◽

Cited By ~ 8

Author(s):

Han-Seung Kang ◽

Chae-Kwan Lee ◽

Ju-Ran Kim ◽

Seong-Jin Yu ◽

Sung-Goo Kang ◽

...

Keyword(s):

Gene Expression ◽

Northern Blot ◽

Blot Analysis ◽

Northern Blot Analysis ◽

Expression Profiles ◽

Expression Patterns ◽

Oestrous Cycle ◽

Mrna Levels ◽

Rat Uterus ◽

Ovx Rats

In the present study, differential gene expression in the uteri of ovariectomised (OVX) and pro-oestrous rats (OVX v. pro-oestrus pair) was investigated using cDNA expression array analysis. Differential uterine gene expression in OVX rats and progesterone (P4)-injected OVX rats (OVX v. OVX + P4 pair) was also examined. The uterine gene expression profiles of these two sets of animals were also compared for the effects of P4 treatment. RNA samples were extracted from uterine tissues and reverse transcribed in the presence of [α32P]-dATP. Membrane sets of rat arrays were hybridised with cDNA probe sets. Northern blot analysis was used to validate the relative gene expression patterns obtained from the cDNA array. Of the 1176 cDNAs examined, 23 genes showed significant (>two-fold) changes in expression in the OVX v. pro-oestrus pair. Twenty of these genes were upregulated during pro-oestrus compared with their expression in the OVX rat uterus. In the OVX v. OVX + P4 pair, 22 genes showed significant (>two-fold) changes in gene expression. Twenty of these genes were upregulated in the OVX + P4 animals. The genes for nuclear factor I–XI, afadin, neuroligin 2, semaphorin Z, calpain 4, cyclase-associated protein homologue, thymosin β-4X and p8 were significantly upregulated in the uteri of the pro-oestrus and OVX + P4 rats of both experimental pairs compared with the OVX rat uteri. These genes appear to be under the control of P4. One of the most interesting findings of the present study is the unexpected and marked expression of the neuroligin 2 gene in the rat uterus. This gene is expressed at high levels in the central nervous system and acts as a nerve cell adhesion factor. According to Northern blot analysis, neuroligin 2 gene expression was higher during the pro-oestrus and metoestrus stages than during the oestrus and dioestrus stages of the oestrous cycle. In addition, neuroligin 2 mRNA levels were increased by both 17β-oestradiol (E2) and P4, although P4 administration upregulated gene expression to a greater extent than injection of E2. These results indicate that neuroligin 2 gene expression in the rat uterus is under the control of both E2 and P4, which are secreted periodically during the oestrous cycle.

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Characterization of E93 in neometabolous thrips Frankliniella occidentalis and Haplothrips brevitubus

PLoS ONE ◽

10.1371/journal.pone.0254963 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0254963

Author(s):

Youhei Suzuki ◽

Takahiro Shiotsuki ◽

Akiya Jouraku ◽

Ken Miura ◽

Chieka Minakuchi

Keyword(s):

Larval Stage ◽

Hormonal Regulation ◽

Frankliniella Occidentalis ◽

Expression Profiles ◽

Mrna Levels ◽

Transcript Levels ◽

Adult Transition ◽

Thrips Species ◽

Insect Metamorphosis ◽

Krüppel Homolog 1

Insect metamorphosis into an adult occurs after the juvenile hormone (JH) titer decreases at the end of the juvenile stage. This generally coincides with decreased transcript levels of JH-response transcription factors Krüppel homolog 1 (Kr-h1) and broad (br), and increased transcript levels of the adult specifier E93. Thrips (Thysanoptera) develop through inactive and non-feeding stages referred to as “propupa” and “pupa”, and this type of distinctive metamorphosis is called neometaboly. To understand the mechanisms of hormonal regulation in thrips metamorphosis, we previously analyzed the transcript levels of Kr-h1 and br in two thrips species, Frankliniella occidentalis (Thripidae) and Haplothrips brevitubus (Phlaeothripidae). In both species, the transcript levels of Kr-h1 and br decreased in the “propupal” and “pupal” stages, and their transcription was upregulated by exogenous JH mimic treatment. Here we analyzed the developmental profiles of E93 in these two thrips species. Quantitative RT-PCR revealed that E93 expression started to increase at the end of the larval stage in F. occidentalis and in the “propupal” stage of H. brevitubus, as Kr-h1 and br mRNA levels decreased. Treatment with an exogenous JH mimic at the onset of metamorphosis prevented pupal-adult transition and caused repression of E93. These results indicated that E93 is involved in adult differentiation after JH titer decreases at the end of the larval stage of thrips. By comparing the expression profiles of Kr-h1, br, and E93 among insect species, we propose that the “propupal” and “pupal” stages of thrips have some similarities with the holometabolous prepupal and pupal stages, respectively.

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Cloning and evaluation of reference genes for quantitative real-time PCR analysis inAmorphophallus

PeerJ ◽

10.7717/peerj.3260 ◽

2017 ◽

Vol 5 ◽

pp. e3260 ◽

Cited By ~ 4

Author(s):

Kai Wang ◽

Yi Niu ◽

Qijun Wang ◽

Haili Liu ◽

Yi Jin ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Real Time ◽

Reference Genes ◽

Dynamic Range ◽

Expression Profiles ◽

Mrna Levels ◽

Expression Stability ◽

Degenerate Primers ◽

Different Tissues

Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes.Amorphophallusis a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes inAmorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genesAmorphophalluswere cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) inA. albusandA. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated thatEF1-a,EIF4A,H3andUBQwere the best reference genes under heat stress inAmorphophallus. Furthermore,EF1-a,EIF4A,TUB, andRPwere the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined thatEF1-α,EIF4A,andCYPwere stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock proteinSHSP, which is related to heat stress inAmorphophallus. In sum,EF1-αandEIF4Awere the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to normalize gene expression. Our results will provide researchers with appropriate reference genes for further gene expression quantification using RT-qPCR inAmorphophallus.

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Rewiring of gene expression in circulating white blood cells is associated with pregnancy outcome in heifers (Bos taurus)

Scientific Reports ◽

10.1038/s41598-020-73694-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Sarah E. Moorey ◽

Bailey N. Walker ◽

Michelle F. Elmore ◽

Joshua B. Elmore ◽

Soren P. Rodning ◽

...

Keyword(s):

Gene Expression ◽

Pregnancy Outcome ◽

Artificial Insemination ◽

Blood Cells ◽

Expression Profiles ◽

White Blood Cells ◽

Transcript Abundance ◽

Prediction Algorithm ◽

Differential Coexpression ◽

Micro Rnas

Abstract Infertility is a challenging phenomenon in cattle that reduces the sustainability of beef production worldwide. Here, we tested the hypothesis that gene expression profiles of protein-coding genes expressed in peripheral white blood cells (PWBCs), and circulating micro RNAs in plasma, are associated with female fertility, measured by pregnancy outcome. We drew blood samples from 17 heifers on the day of artificial insemination and analyzed transcript abundance for 10,496 genes in PWBCs and 290 circulating micro RNAs. The females were later classified as pregnant to artificial insemination, pregnant to natural breeding or not pregnant. We identified 1860 genes producing significant differential coexpression (eFDR < 0.002) based on pregnancy outcome. Additionally, 237 micro RNAs and 2274 genes in PWBCs presented differential coexpression based on pregnancy outcome. Furthermore, using a machine learning prediction algorithm we detected a subset of genes whose abundance could be used for blind categorization of pregnancy outcome. Our results provide strong evidence that transcript abundance in circulating white blood cells is associated with fertility in heifers.

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Effects of Adeno-Associated Virus on Adenovirus Replication and Gene Expression during Coinfection

Journal of Virology ◽

10.1128/jvi.00198-06 ◽

2006 ◽

Vol 80 (16) ◽

pp. 7807-7815 ◽

Cited By ~ 30

Author(s):

Jennifer M. Timpe ◽

Kristin C. Verrill ◽

James P. Trempe

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Dna Synthesis ◽

Virus Production ◽

Dna Amplification ◽

Early Gene ◽

Mrna Levels ◽

Dependent Manner ◽

Adeno Associated Virus ◽

Transcript Levels

ABSTRACT Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.

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Gene Expression Profiles Involved in γ to β Globin Gene Switching during Erythroid Maturation.

Blood ◽

10.1182/blood.v106.11.820.820 ◽

2005 ◽

Vol 106 (11) ◽

pp. 820-820

Author(s):

Wei Li ◽

Betty S. Pace

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Globin Gene ◽

Gene Expression Profiles ◽

Phase System ◽

Mrna Levels ◽

Erythroid Progenitors ◽

Globin Mrna ◽

Gene Switching ◽

Globin Gene Expression

Abstract The design and evaluation of therapies for sickle cell disease (SCD) rely on our understanding of hemoglobin accumulation during erythropoiesis and sequential globin gene expression (ε → Gγ → Aγ → δ → β) during development. To gain insights into globin gene switching, we completed time course micorarray analyses of erythroid progenitors to identify trans-factors involved in γ gene activation. Studies were completed to map the pattern of γ and β globin gene expression in progenitors grown from normal peripheral blood mononuclear cells. We compared cells grown in a 2-phase (phase 1, d0-6: SCF, IL-3, IL-6, and GM-CSF and phase 2, d7-25: SCF and EPO) vs. 1-phase (d0-34: SCF, IL-3, and EPO) liquid culture system. From day 0 to 34 in either system cell viability remained >99%. Total RNA was isolated using Trizol and column cleanup (Qiagen). Globin mRNA levels were measured at 2–3 day intervals by quantitative PCR (qPCR). In the 2-phase system γ-globin mRNA>β-globin mRNA up to d14, 4 days of approximately equal expression then β mRNA > γ mRNA by d20. By contrast, in 1-phase studies there was a rapid switch around d20(see graph). We speculate that this difference may be due to the early addition of EPO on d0 therefore we continued our detailed analysis in this system. To confirm that our in vitro system recapitulates in vivo gene expression patterns, we completed studies to ascertain Gγ - vs. Aγ globin mRNA levels. The normalized Gγ:Aγ ratio decreased from ~3:1 on d7 to ~1:1 by d34; These findings were confirmed using two sets of Gγ and Aγ globin primers. We concluded that the 1-phase system recapitulated normal γ/β globin switching and that gene profiling studies to identify the trans-factor involved in switching mechanisms were feasible. We used Discover oligo chips (ArrayIt, Sunnyvale, CA) containing 380 human genes selected from 30 major functional groups including hematopoiesis. To aide interpretation of chip data, cell populations were rated morphologically using Giemsa stained cytospin preps. From d16 on we observed an increase in late erythroid progenitors (normoblasts) from 1% to 71% by d31. After verifying RNA quality by gel inspection of ribosomal molecules, we prepared Cy3 and Cy5 probes for early and late time-point RNA samples respectively. Chip analysis was performed at several time points but d0/21, d7/21, and d21/28 were most informative. Based on Axon GenePixPro 6.0 and Acuity 4.0 software analysis we found the following genes with >1.5-fold change in expression profile (shown as down-regulated/up-regulated genes): d0/21: 33/73, d7/21: 13/25, and d21/28:35/26. Principal component analysis (PCA), hierarchical clusters and self organizing maps were constructed. Gene profiles were correlated with the γ/β switching curve using d7 (γ >β), d21 (γ ~ β), and d28 (γ <β) data. Hematopoietic dataset analysis at d21 revealed 4 candidate γ-globin gene activators including v-myb, upsteam binding transfactor -RNApol1 and 2 zinc finger proteins. Analysis of a d28 dataset revealed 12 proteins involved in γ-globin gene silencing including IL-3, SCF, MAPKKK3, v-raf-1, ATF-2, and glucocorticoid receptor DNA binding factor 1 among others. Gene expression profiles will be validated using qPCR and promising candidates will be tested by forced expression in transient and stable reporter systems. Figure Figure

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TRAP-based allelic translation efficiency imbalance analysis to identify genetic regulation of ribosome occupancy in specific cell types in vivo

10.1101/2020.08.24.265389 ◽

2020 ◽

Author(s):

Yating Liu ◽

Anthony D. Fischer ◽

Celine L. St. Pierre ◽

Juan F. Macias-Velasco ◽

Heather A. Lawson ◽

...

Keyword(s):

Gene Expression ◽

Genetic Regulation ◽

Transcript Abundance ◽

Cell Types ◽

Regulatory Elements ◽

Model Organisms ◽

Specific Cell ◽

Translation Efficiency ◽

Mrna Levels

AbstractThe alteration of gene expression due to variations in the sequences of transcriptional regulatory elements has been a focus of substantial inquiry in humans and model organisms. However, less is known about the extent to which natural variation contributes to post-transcriptional regulation. Allelic Expression Imbalance (AEI) is a classical approach for studying the association of specific haplotypes with relative changes in transcript abundance. Here, we piloted a new TRAP based approach to associate genetic variation with transcript occupancy on ribosomes in specific cell types, to determine if it will allow examination of Allelic Translation Imbalance (ATI), and Allelic Translation Efficiency Imbalance, using as a test case mouse astrocytes in vivo. We show that most changes of the mRNA levels on ribosomes were reflected in transcript abundance, though ∼1.5% of transcripts have variants that clearly alter loading onto ribosomes orthogonally to transcript levels. These variants were often in conserved residues and altered sequences known to regulate translation such as upstream ORFs, PolyA sites, and predicted miRNA binding sites. Such variants were also common in transcripts showing altered abundance, suggesting some genetic regulation of gene expression may function through post-transcriptional mechanisms. Overall, our work shows that naturally occurring genetic variants can impact ribosome occupancy in astrocytes in vivo and suggests that mechanisms may also play a role in genetic contributions to disease.

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Validation of Reference Genes for Quantitative Real-time PCR in Diaphanosoma Celebensis: For Chemical Exposure Conditions and Different Ages

10.21203/rs.3.rs-847536/v1 ◽

2021 ◽

Author(s):

Young-Mi Lee ◽

Soyeon In ◽

Se-Joo Kim ◽

Eun-Ji Won ◽

Hayoung Cho ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Expression Profiles ◽

Chemical Exposure ◽

Mrna Levels ◽

Qrt Pcr ◽

Different Ages ◽

Diaphanosoma Celebensis

Abstract Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a primary approach for evaluating gene expression, requires an appropriate normalization strategy to rule out variations in gene expression among samples. The best option is to use a reference gene whose expression level is stable across various experimental conditions to compare the mRNA levels of a target gene. However, there is limited information on how the reference gene is differentially expressed at different ages (growth) in small invertebrates with notable changes such as molting. In this study, expression profiles of nine candidate reference genes from the brackish water flea, Diaphanosoma celebensis, were evaluated under diverse exposure to toxicants and according to growth. As a result, four different algorithms showed similar stabilities of genes for chemical exposures in the case of limited conditions using the same developmental stage (e.g., adult), while the results according to age showed a significantly different pattern in suite of candidate reference genes. This affected the results of genes EcRA and GST, which are involved in development and detoxification mechanisms, respectively. Our finding is the first step towards establishing a standardized real-time qRT-PCR analysis of this environmentally important invertebrate that has potential for aquatic ecotoxicology, particularly in estuarine environments.

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Using LongSAGE to Detect Biomarkers of Cervical Cancer Potentially Amenable to Optical Contrast Agent Labelling

Biomarker Insights ◽

10.1177/117727190700200020 ◽

2007 ◽

Vol 2 ◽

pp. 117727190700200 ◽

Cited By ~ 2

Author(s):

Julie M. Kneller ◽

Thomas Ehlen ◽

Jasenka P. Matisic ◽

Dianne Miller ◽

Dirk Van Niekerk ◽

...

Keyword(s):

Gene Expression ◽

Cell Surface ◽

Contrast Agent ◽

Expression Profiles ◽

Physiological State ◽

Disease Process ◽

Intraepithelial Neoplasia ◽

Surface Proteins ◽

Optical Contrast ◽

Optical Contrast Agent

Sixteen longSAGE libraries from four different clinical stages of cervical intraepithelial neoplasia have enabled us to identify novel cell-surface biomarkers indicative of CIN stage. By comparing gene expression profiles of cervical tissue at early and advanced stages of CIN, several genes are identified to be novel genetic markers. We present fifty-six cell-surface gene products differentially expressed during progression of CIN. These cell surface proteins are being examined to establish their capacity for optical contrast agent binding. Contrast agent visualization will allow real-time assessment of the physiological state of the disease process bringing vast benefit to cancer care. The data discussed in this publication have been submitted to NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/ ) and are accessible through GEO Series accession number GSE6252.

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