scholarly journals Expression of a symbiosis-specific gene in Symbiodinium type A1 associated with coral, nudibranch and giant clam larvae

2017 ◽  
Vol 4 (5) ◽  
pp. 170253 ◽  
Author(s):  
M. Mies ◽  
C. R. Voolstra ◽  
C. B. Castro ◽  
D. O. Pires ◽  
E. N. Calderon ◽  
...  
Keyword(s):  
Larval Development ◽  
Tissue Sample ◽  
Marker Gene ◽  
Rna Extraction ◽  
Specific Gene ◽  
Giant Clam ◽  
Symbiodinium Type ◽  
Symbiotic Associations ◽  
Mutualistic Symbiosis ◽  
Mutualistic Relationship

Symbiodinium are responsible for the majority of primary production in coral reefs and found in a mutualistic symbiosis with multiple animal phyla. However, little is known about the molecular signals involved in the establishment of this symbiosis and whether it initiates during host larval development. To address this question, we monitored the expression of a putative symbiosis-specific gene (H + -ATPase) in Symbiodinium A1 ex hospite and in association with larvae of a scleractinian coral ( Mussismilia hispida ), a nudibranch ( Berghia stephanieae ) and a giant clam ( Tridacna crocea ). We acquired broodstock for each host, induced spawning and cultured the larvae. Symbiodinium cells were offered and larval samples taken for each host during the first 72 h after symbiont addition. In addition, control samples including free-living Symbiodinium and broodstock tissue containing symbionts for each host were collected. RNA extraction and RT-PCR were performed and amplified products cloned and sequenced. Our results show that H + -ATPase was expressed in Symbiodinium associated with coral and giant clam larvae, but not with nudibranch larvae, which digested the symbionts. Broodstock tissue for coral and giant clam also expressed H + -ATPase, but not the nudibranch tissue sample. Our results of the expression of H + -ATPase as a marker gene suggest that symbiosis between Symbiodinium and M. hispida and T. crocea is established during host larval development. Conversely, in the case of B. stephanieae larvae, evidence does not support a mutualistic relationship. Our study supports the utilization of H + -ATPase expression as a marker for assessing Symbiodinium– invertebrate relationships with applications for the differentiation of symbiotic and non-symbiotic associations. At the same time, insights from a single marker gene approach are limited and future studies should direct the identification of additional symbiosis-specific genes, ideally from both symbiont and host.

scholarly journals Differential Regulation of Gene Expression of Alveolar Epithelial Cell Markers in Human Lung Adenocarcinoma-Derived A549 Clones

2015 ◽  
Vol 2015 ◽  
pp. 1-20 ◽  
Author(s):  
Hiroshi Kondo ◽  
Keiko Miyoshi ◽  
Shoji Sakiyama ◽  
Akira Tangoku ◽  
Takafumi Noma
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Mapk Pathway ◽  
Alveolar Epithelial Cell ◽  
Marker Gene ◽  
Regulation Of Gene Expression ◽  
Surfactant Protein ◽  
Specific Gene ◽  
Expression Levels ◽  
Alveolar Epithelial

Stem cell therapy appears to be promising for restoring damaged or irreparable lung tissue. However, establishing a simple and reproducible protocol for preparing lung progenitor populations is difficult because the molecular basis for alveolar epithelial cell differentiation is not fully understood. We investigated anin vitrosystem to analyze the regulatory mechanisms of alveolus-specific gene expression using a human alveolar epithelial type II (ATII) cell line, A549. After cloning A549 subpopulations, each clone was classified into five groups according to cell morphology and marker gene expression. Two clones (B7 and H12) were further analyzed. Under serum-free culture conditions,surfactant protein C(SPC), an ATII marker, was upregulated in both H12 and B7.Aquaporin 5(AQP5), an ATI marker, was upregulated in H12 and significantly induced in B7. When the RAS/MAPK pathway was inhibited,SPCandthyroid transcription factor-1(TTF-1) expression levels were enhanced. After treatment with dexamethasone (DEX), 8-bromoadenosine 3′5′-cyclic monophosphate (8-Br-cAMP), 3-isobutyl-1-methylxanthine (IBMX), and keratinocyte growth factor (KGF),surfactant protein BandTTF-1expression levels were enhanced. We found that A549-derived clones have plasticity in gene expression of alveolar epithelial differentiation markers and could be useful in studying ATII maintenance and differentiation.

A new method for gene expression analysis of pure lymphocytes recovered from heterogeneous fixed mouse tissue.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e23089-e23089
Author(s):  
Jennifer Chow ◽  
Ana Paula Galvão Da Silva ◽  
Gianni Medoro ◽  
Nicolò Manaresi ◽  
Paul David Lira ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Tissue Sample ◽  
Critical Role ◽  
Rna Extraction ◽  
Response To Treatment ◽  
Mouse Tissue ◽  
Fixed Tissue ◽  
Mouse Splenocytes

e23089 Background: Tumor infiltrating lymphocytes (TILs) are biomarkers that play a critical role in cancer diseases, including differential diagnosis, determination of prognosis, prediction of response to treatment, and evaluation of disease progression. Gene expression analysis in TILs derived from fresh tissue may not accurately depict the gene profile of the tissue microenvironment as it can change aggressively during lymphocyte isolation and RNA extraction. In addition, tissue sample size can limit the isolation of TILs with current technologies. In this study, we demonstrate the use of the DEPArray™platform to isolate pure populations of lymphocytes from a fixed mouse tissue for RNA analysi. Methods: Mouse splenocytes were activated in vitro with anti-CD3 and -CD28 for 72hs. Cells were harvested, fixed with 2% paraformaldehyde (PFA) for 20 min at RT, and stained for either CD4 or CD8 expression. Gene expression analysis of CD45, ADORA2A, GLS and GAPDH was performed in CD4+ and CD8+ DEPArray™sorted cells using the TaqMan PreAmp Cells-to-Ct kit. Results: The table below summarizes the Ct values for CD45, ADORA2A, GLS and GAPDH expression in 300 fixed unsorted control and DEPArray™sorted lymphocytes. Conclusions: We have demonstrated the feasibility of gene expression analysis on pure populations of CD4+ and CD8+ cells isolated from a fixed tissue using the DEPArray™ platform. The advantage of this approach is the DEPArray’s ability to identify and isolate subpopulations of cells from complex heterogeneous samples and/or specimens that are limited by size or content. This methodology will be applied for isolation of TILs in syngeneic and xenograft models of cancers for downstream RNA applications. [Table: see text]

scholarly journals The Medicago truncatula MtAnn1 Gene Encoding an Annexin Is Induced by Nod Factors and During the Symbiotic Interaction with Rhizobium meliloti

1998 ◽  
Vol 11 (6) ◽  
pp. 504-513 ◽  
Author(s):  
Fernanda de Carvalho Niebel ◽  
Nicole Lescure ◽  
Julie V. Cullimore ◽  
Pascal Gamas
Keyword(s):  
Medicago Truncatula ◽  
Rhizobium Meliloti ◽  
Marker Gene ◽  
Distal Region ◽  
Infection Process ◽  
Biologically Active ◽  
Nod Factors ◽  
Symbiotic Interaction ◽  
Symbiotic Associations ◽  
Infection Threads

Here we report the characterization of a new Nod factor-induced gene from Medicago truncatula identified by mRNA differential display. This gene, designated MtAnn1, encodes a protein homologous to the annexin family of calcium- and phospholipid-binding proteins. We further show that the MtAnn1 gene is also induced during symbiotic associations with Rhizobium meliloti, both at early stages in bacterial-inoculated roots and in nodule structures. By in situ hybridization, we demonstrate that MtAnn1 expression in nodules is mainly associated with the distal region of invasion zone II not containing infection threads, revealing MtAnn1 as a new marker gene of the pre-infection zone. Moreover, analyses of MtAnn1 expression in response to bacterial symbiotic mutants suggest that the expression of MtAnn1 during nodulation requires biologically active Nod factors and is independent of the infection process.

scholarly journals The Cytoskeletal Network Controls c-Jun Expression and Glucocorticoid Receptor Transcriptional Activity in an Antagonistic and Cell-Type-Specific Manner

1999 ◽  
Vol 19 (3) ◽  
pp. 1742-1750 ◽  
Author(s):  
Anat Oren ◽  
Avia Herschkovitz ◽  
Iris Ben-Dror ◽  
Vered Holdengreber ◽  
Yehuda Ben-Shaul ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Glucocorticoid Receptor ◽  
Cell Separation ◽  
Transcriptional Activity ◽  
Marker Gene ◽  
Mitogen Activated Protein Kinase ◽  
Cell Contact ◽  
Specific Gene ◽  
Functional Link ◽  
Cytoskeletal Network

ABSTRACT The physical and functional link between adhesion molecules and the cytoskeletal network suggests that the cytoskeleton might mediate the transduction of cell-to-cell contact signals, which often regulate growth and differentiation in an antagonistic manner. Depolymerization of the cytoskeleton in confluent cell cultures is reportedly sufficient to initiate DNA synthesis. Here we show that depolymerization of the cytoskeleton is also sufficient to repress differentiation-specific gene expression. Glutamine synthetase is a glia-specific differentiation marker gene whose expression in the retinal tissue is regulated by glucocorticoids and is ultimately dependent on glia-neuron cell contacts. Depolymerization of the actin or microtubule network in cells of the intact retina mimics the effects of cell separation, repressing glutamine synthetase induction by a mechanism that involves induction of c-Jun and inhibition of glucocorticoid receptor transcriptional activity. Depolymerization of the cytoskeleton activates JNK and p38 mitogen-activated protein kinase and induces c-Jun expression by a signaling pathway that depends on tyrosine kinase activity. Induction of c-Jun expression is restricted to Müller glial cells, the only cells in the tissue that express glutamine synthetase and maintain the ability to proliferate upon cell separation. Our results suggest that the cytoskeletal network might play a part in the transduction of cell contact signals to the nucleus.

scholarly journals Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs

2018 ◽  
Author(s):  
Georg Oberhofer ◽  
Tobin Ivy ◽  
Bruce A. Hay
Keyword(s):  
Gene Function ◽  
Homing Endonuclease ◽  
Marker Gene ◽  
Specific Gene ◽  
Gene Drive ◽  
Loss Of Function ◽  
Complex Activity ◽  
Guide Rnas ◽  
Carry Over ◽  
Population Suppression

AbstractA gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous progeny. Earlier work inDrosophilaand mosquitoes utilized HEGs consisting of Cas9 and a single gRNA that together target a specific gene for cleavage. Homing was observed, but resistant alleles, immune to cleavage, while retaining wildtype gene function, were also created through non-homologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs, targeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and guide RNAs (gRNAs) designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments the HEGs failed to spread, due to the high fitness load induced in offspring as a result of maternal carry over of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.Significance statementHEG-based gene drive can bring about population suppression when genes required for viability or fertility are targeted. However, these strategies are vulnerable to failure through mechanisms that create alleles resistant to cleavage, but that retain wildtype gene function. We show that resistance allele creation can be prevented through the use of gRNAs designed to cleave a gene at four target sites. However, homing rates were modest, and the HEGs were unstable during homing. In addition, use of a promoter active in the female germline resulted in levels of HEG carryover that compromised the viability or fertility of HEG-bearing heterozygotes, thereby preventing drive. We propose strategies that can help to overcome these problems in next generation HEG systems.

scholarly journals A shift away from mutualism under food-deprived conditions in an anemone-dinoflagellate association

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9745
Author(s):  
Shao-En Peng ◽  
Alessandro Moret ◽  
Cherilyn Chang ◽  
Anderson B. Mayfield ◽  
Yu-Ting Ren ◽  
...  
Keyword(s):  
Coral Reef ◽  
Symbiotic Association ◽  
Physiological Conditions ◽  
Functional Basis ◽  
Mutualistic Symbiosis ◽  
Metabolic Burden ◽  
The Family ◽  
Mutualistic Relationship ◽  
Coral Reef Ecosystems ◽  
Exaiptasia Pallida

The mutualistic symbiosis between anthozoans and intra-gastrodermal dinoflagellates of the family Symbiodiniaceae is the functional basis of all coral reef ecosystems, with the latter providing up to 95% of their fixed photosynthate to their hosts in exchange for nutrients. However, recent studies of sponges, jellyfish, and anemones have revealed the potential for this mutualistic relationship to shift to parasitism under stressful conditions. Over a period of eight weeks, we compared the physiological conditions of both inoculated and aposymbiotic anemones (Exaiptasia pallida) that were either fed or starved. By the sixth week, both fed groups of anemones were significantly larger than their starved counterparts. Moreover, inoculated and starved anemones tended to disintegrate into “tissue balls” within eight weeks, and 25% of the samples died; in contrast, starved aposymbiotic anemones required six months to form tissue balls, and no anemones from this group died. Our results show that the dinoflagellates within inoculated anemones may have posed a fatal metabolic burden on their hosts during starvation; this may be because of the need to prioritize their own metabolism and nourishment at the expense of their hosts. Collectively, our study reveals the potential of this dynamic symbiotic association to shift away from mutualism during food-deprived conditions.

scholarly journals Light-Dependent Phenomena and Related Molecular Mechanisms in Giant Clam-Dinoflagellate Associations: A Review

2021 ◽  
Vol 8 ◽  
Author(s):  
Yuen K. Ip ◽  
Shit F. Chew
Keyword(s):  
Molecular Mechanisms ◽  
Anthropogenic Activities ◽  
Giant Clam ◽  
Ambient Seawater ◽  
Growth And Survival ◽  
Giant Clams ◽  
Gene And Protein Expression ◽  
Mutualistic Relationship ◽  
New Ideas ◽  
Symbiotic Dinoflagellates

Giant clams can grow to large sizes despite living in oligotrophic waters of the tropical Indo-Pacific as they maintain a mutualistic relationship with symbiotic dinoflagellates (zooxanthellae) and receive photosynthate from them. The phototrophic dinoflagellates live extracellularly inside a tubular system located mainly in the colorful outer mantle and have no access to the ambient seawater. Hence, the clam host needs to absorb exogenous inorganic carbon (Ci), nitrogen (N) and phosphorus (P), and supply them to the symbionts. As photosynthesizing symbionts need more nutrients in light than in the dark, the uptake rates of these exogenous nutrients by the host must increase during illumination, implying that the host’s transporters involved need to be regulated by some kind of light-responsive mechanisms. Furthermore, the growth and development of the host can also be augmented by light, because of the photosynthate donated by the photosynthesizing symbionts. Consequently, giant clams display many light-dependent phenomena related to phototrophy, antioxidative defense, biomineralization, as well as absorption of exogenous Ci, N, and P. These phenomena may involve collaborations among enzymes and transporters in several organs of the host, whereby the gene and protein expression levels of these biocatalysts are up- or down-regulated during illumination. This review aims to examine the molecular mechanisms of light-dependent physiological phenomena that occur in intact giant clam-dinoflagellate associations, and to highlight the differences between giant clams and scleractinian corals in those regards. As the population of giant clams in nature are dwindling due to climate change and anthropogenic activities, a good understanding of their light-dependent processes may generate new ideas to improve their growth and survival under rapidly changing environmental conditions.

scholarly journals Transcriptome Profiling Reveals Differential Gene Expression of Secreted Proteases and Highly Specific Gene Repertoires Involved in Lactarius–Pinus Symbioses

2021 ◽  
Vol 12 ◽  
Author(s):  
Nianwu Tang ◽  
Annie Lebreton ◽  
Wenjun Xu ◽  
Yucheng Dai ◽  
Fuqiang Yu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Time Course ◽  
Developmental Stages ◽  
Transcriptome Profiling ◽  
Specific Gene ◽  
Gene Repertoire ◽  
Symbiotic Gene ◽  
Mutualistic Symbiosis ◽  
Secreted Proteases ◽  
Fungal Genes

Ectomycorrhizal fungi establish a mutualistic symbiosis in roots of most woody plants. The molecular underpinning of ectomycorrhizal development was only explored in a few lineages. Here, we characterized the symbiotic transcriptomes of several milkcap species (Lactarius, Russulales) in association with different pine hosts. A time-course study of changes in gene expression during the development of L. deliciosus–Pinus taeda symbiosis identified 6 to 594 differentially expressed fungal genes at various developmental stages. Up- or down-regulated genes are involved in signaling pathways, nutrient transport, cell wall modifications, and plant defenses. A high number of genes coding for secreted proteases, especially sedolisins, were induced during root colonization. In contrast, only a few genes encoding mycorrhiza-induced small secreted proteins were identified. This feature was confirmed in several other Lactarius species in association with various pines. Further comparison among all these species revealed that each Lactarius species encodes a highly specific symbiotic gene repertoire, a feature possibly related to their host-specificity. This study provides insights on the genetic basis of symbiosis in an ectomycorrhizal order, the Russulales, which was not investigated so far.

scholarly journals Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs

2018 ◽  
Vol 115 (40) ◽  
pp. E9343-E9352 ◽  
Author(s):  
Georg Oberhofer ◽  
Tobin Ivy ◽  
Bruce A. Hay
Keyword(s):  
Homing Endonuclease ◽  
Marker Gene ◽  
Specific Gene ◽  
Gene Drive ◽  
Loss Of Function ◽  
End Joining ◽  
Complex Activity ◽  
Guide Rna ◽  
Type Gene ◽  
Population Suppression

A gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific, nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous, progeny. Earlier work inDrosophilaand mosquitoes utilized HEGs consisting of Cas9 and a single guide RNA (gRNA) that together target a specific gene for cleavage. Homing was observed, but resistant alleles immune to cleavage, while retaining wild-type gene function, were also created through nonhomologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs inDrosophila melanogastertargeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and gRNAs designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments, the HEGs failed to spread due to the high fitness load induced in offspring as a result of maternal carryover of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.

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