In-Yeast Meeting of Coronavirus Infectious cDNA Clones Utilizing a Artificial Genomics Pipeline
The Escherichia coli and vaccinia virus-based reverse genetics methods have been broadly utilized for the manipulation and engineering of coronavirus genomes. These methods, nonetheless, current a number of limitations and are generally tough to determine in a well timed method for (re-)rising viruses. On this chapter, we current a brand new common reverse genetics platform for the meeting and engineering of infectious full-length cDNAs utilizing yeast-based transformation-associated recombination cloning.
This novel meeting technique not solely ends in secure coronavirus infectious full-length cDNAs cloned within the yeast Saccharomyces cerevisiae but additionally fosters and accelerates the manipulation of their genomes.
Such a platform is broadly relevant for the scientific neighborhood, because it requires no particular gear and could be carried out in an ordinary laboratory setting. The protocol described could be simply tailored to just about all recognized or rising coronaviruses, reminiscent of Center East respiratory syndrome coronavirus (MERS-CoV).
RNA-cDNA hybrids mediate transposition via different mechanisms
Retrotransposons can represent half of eukaryotic genomes. Retrotransposon dysregulation destabilizes genomes and has been linked to various human diseases. Emerging regulators of retromobility include RNA-DNA hybrid-containing structures known as R-loops. Accumulation of these structures at the transposons of yeast 1 (Ty1) elements has been shown to increase Ty1 retromobility through an unknown mechanism.
Here, via a targeted genetic screen, we identified the rnh1Δ rad27Δ yeast mutant, which lacked both the Ty1 inhibitor Rad27 and the RNA-DNA hybrid suppressor Rnh1. The mutant exhibited elevated levels of Ty1 cDNA-associated RNA-DNA hybrids that promoted Ty1 mobility. Moreover, in this rnh1Δ rad27Δ mutant, but not in the double RNase H mutant rnh1Δ rnh201Δ, RNA-DNA hybrids preferentially existed as duplex nucleic acid structures and increased Ty1 mobility in a Rad52-dependent manner.
The data indicate that in cells lacking RNA-DNA hybrid and Ty1 repressors, elevated levels of RNA-cDNA hybrids, which are associated with duplex nucleic acid structures, boost Ty1 mobility via a Rad52-dependent mechanism.
In contrast, in cells lacking RNA-DNA hybrid repressors alone, elevated levels of RNA-cDNA hybrids, which are associated with triplex nucleic acid structures, boost Ty1 mobility via a Rad52-independent process. We propose that duplex and triplex RNA-DNA hybrids promote transposon mobility via Rad52-dependent or -independent mechanisms.
Identification of salinity responsive genes in lavender through cDNA-AFLP
Currently, a global demand exists forlavender as a significant medicinal plant and source of essential oils. Freshwater and arable lands are two major factors that inhibit extensive farming of medicinal plants in Iran. Saline water from seas and salty soil may be new resources for agricultural use, especially for medicinal plants.
We sought to extend our knowledge of the Lavandula angustifolia genome and molecular basis of its salinity tolerance by using cDNA amplified fragment length polymorphism (cDNA-AFLP) to investigate the changes in plant transcriptomes in response to NaCl.
All identified transcript derived fragments (TDF) were assigned as novel L. angustifolia genes related to signal transduction, regulation of gene expression, alternative splicing, autophagy, and secondary metabolite biosynthesis. qRT-PCR analysis of the TDFs in response to different concentrations of NaCl revealed various levels of mRNA of the identified genes in this plant. Our findings provided primary insights into the molecular response of L. angustifolia to salinity.
Description: Treponema Pallidum p47 (Syphilis) full length, recombinant protein from E. coli, fused with Beta-galactosidase (114 kDa) at the N-terminus, 1.00 mg/mL.
Recombinant Treponema Pallidum p17 Protein (Full Length)
Description: Treponema Pallidum p17 (Syphilis) full length, recombinant protein from E. coli, fused with Beta-galactosidase (114 kDa) at the N-terminus, 1.00 mg/mL.
Recombinant Treponema Pallidum p15 Protein (Full Length)
Description: Treponema Pallidum p15 (Syphilis) full length, recombinant protein from E. coli, fused with Beta-galactosidase (114 kDa) at the N-terminus, 1.00 mg/mL.
Description: Treponema Pallidum Treponemal Membrane Protein A (TmpA) Full Length, recombinant protein from E. coli, fused with Beta-galactosidase (114 kDa) at the N-terminus, MW 42 kDa, 1.00 mg/mL.
Synthesis of Full-Size cDNA Infectious Clones of Soybean Mosaic Virus and Purposeful Identification of a Key Amino Acid within the Silencing Suppressor Hc-Professional
Soybean mosaic virus (SMV), which belongs to the Potyviridae, causes vital reductions in soybean yield and seed high quality. On this examine, each tag-free and reporter gene inexperienced fluorescent protein (GFP)-containing infectious clones for the SMV N1 pressure have been constructed by Gibson meeting and with the yeast homologous recombination system, respectively.
Each infectious clones are appropriate for agroinfiltration on the mannequin host benthamiana and present robust infectivity for the pure host soybean and several other different legume species. Each infectious clones have been seed transmitted and prompted typical virus signs on seeds and progeny crops. We used the SMV-GFP infectious clone to additional examine the function of key amino acids within the silencing suppressor helper component-proteinase (Hc-Professional).
Amongst twelve amino acid substitution mutants, the co-expression of mutant 2-with an Asparagine→Leucine substitution at place 182 of the FRNK (Phe-Arg-Asn-Lys) motif-attenuated viral signs and alleviated the host progress retardation brought on by SMV.
Furthermore, the Hc-Prom2 mutant confirmed stronger oligomerization than wild-type Hc-Professional. Taken collectively, the SMV infectious clones can be helpful for research of host-SMV interactions and useful gene characterization in soybeans and associated legume species, particularly by way of seed transmission properties. Moreover, the SMV-GFP infectious clone will even facilitate useful research of each virus and host genes in an benthamiana transient expression system.
Profiling of rice Cd-tolerant genes by way of yeast-based cDNA library survival screening
The bioaccumulation of cadmium (Cd) in crop and the next meals chain has aroused intensive considerations. Nevertheless, the underlying molecular mechanisms of plant Cd tolerance stay to be clarified from the viewpoint of novel candidate genes.
Right here we described a extremely environment friendly strategy for preliminary figuring out rice Cd-tolerant genes by way of the yeast-based cDNA library survival screening mixed with high-throughput sequencing technique. About 690 gene isoforms have been recognized as being Cd-tolerant candidates utilizing this shotgun strategy.
Among the many Cd-tolerant genes recognized, a number of classes of genes reminiscent of BAX inhibitor (BI), NAC transcription elements and Fast ALkalinization Elements (RALFs) have been of specific curiosity, and their perform of Cd tolerance was additional validated through heterologous expression, which urged that SNAC1, RALF12, OsBI-1 can confer Cd tolerance in yeast and tobacco leaves.
Relating to the genes concerned in ion transport, the validated Cd-tolerant heavy metal-associated area (HMAD) isoprenylated protein HIPP42 was significantly noteworthy. Additional elucidation of those genes related to Cd tolerance in rice will profit agricultural actions.
Single-Cell Transcriptomics of Immune Cells: Cell Isolation and cDNA Library Era for scRNA-Seq
Single-cell RNA-sequencing (scRNA-seq) allows a complete evaluation of the transcriptome of particular person cells by next-generation sequencing. ScRNA-seq gives an unbiased strategy to analyze the mobile heterogeneity and dynamics of various organic methods, together with the immune system. Optimization of the technical procedures carried out previous to RNA-seq evaluation is crucial to the success of a scRNA-seq experiment.
Right here, three main experimental procedures are described: (1) the isolation of immune CD8a+ T cells from main murine tissue, (2) the technology of single-cell cDNA libraries utilizing the 10× Genomics Chromium Controller and the Chromium Single Cell 3′ Answer, and (3) cDNA library high quality management. On this protocol, CD8a+ T cells are remoted from murine spleen tissue, however any cell kind of curiosity could be enriched and used for single-cell cDNA library technology and subsequent RNA-seq experiments.
Description: A polyclonal antibody against CCR6. Recognizes CCR6 from Human. This antibody is Unconjugated. Tested in the following application: ELISA, WB, IHC;ELISA:1:2000-1:10000, WB:1:1000-1:5000, IHC:1:50-1:200
Description: A polyclonal antibody against CCR6. Recognizes CCR6 from Human. This antibody is HRP conjugated. Tested in the following application: ELISA
Description: A polyclonal antibody against CCR6. Recognizes CCR6 from Human. This antibody is FITC conjugated. Tested in the following application: ELISA
Description: A polyclonal antibody against CCR6. Recognizes CCR6 from Human. This antibody is Biotin conjugated. Tested in the following application: ELISA
Description: A polyclonal antibody raised in Rabbit that recognizes and binds to Human CCR6 (C-Terminus). This antibody is tested and proven to work in the following applications:
Description: A polyclonal antibody raised in Rabbit that recognizes and binds to Human CCR6 (Cytoplasmic Domain). This antibody is tested and proven to work in the following applications:
Description: A polyclonal antibody raised in Rabbit that recognizes and binds to Human CCR6 (Extracellular Domain). This antibody is tested and proven to work in the following applications:
Description: A polyclonal antibody raised in Goat that recognizes and binds to Human CCR6 (aa18-46). This antibody is tested and proven to work in the following applications:
Description: Description of target: This gene encodes a member of the beta chemokine receptor family, which is predicted to be a seven transmembrane protein similar to G protein-coupled receptors. The gene is preferentially expressed by immature dendritic cells and memory T cells. The ligand of this receptor is macrophage inflammatory protein 3 alpha (MIP-3 alpha). This receptor has been shown to be important for B-lineage maturation and antigen-driven B-cell differentiation, and it may regulate the migration and recruitment of dentritic and T cells during inflammatory and immunological responses. Alternatively spliced transcript variants that encode the same protein have been described for this gene.;Species reactivity: Human;Application: ELISA;Assay info: ;Sensitivity: < 0.058ng/mL
Description: Description of target: Receptor for the C-C type chemokine CCL20. Binds to CCL20 and subsequently transduces a signal by increasing the intracellular calcium ion levels (PubMed:20068036). Although CCL20 is its major ligand it can also act as a receptor for non-chemokine ligands such as beta-defensins (PubMed:25122636). Binds to defensin DEFB1 leading to increase in intracellular calcium ions and cAMP levels. Its binding to DEFB1 is essential for the function of DEFB1 in regulating sperm motility and bactericidal activity. Binds to defensins DEFB4 and DEFB4A/B and mediates their chemotactic effects (PubMed:20068036). The ligand-receptor pair CCL20-CCR6 is responsible for the chemotaxis of dendritic cells (DC), effector/memory T-cells and B-cells and plays an important role at skin and mucosal surfaces under homeostatic and inflammatory conditions, as well as in pathology, including cancer and various autoimmune diseases. CCR6-mediated signals are essential for immune responses to microbes in the intestinal mucosa and in the modulation of inflammatory responses initiated by tissue insult and trauma (PubMed:21376174). CCR6 is essential for the recruitment of both the proinflammatory IL17 producing helper T-cells (Th17) and the regulatory T-cells (Treg) to sites of inflammation (PubMed:19050256). Required for the normal migration of Th17 cells in Peyers patches and other related tissue sites of the intestine and plays a role in regulating effector T-cell balance and distribution in inflamed intestine (PubMed:19129757). Plays an important role in the coordination of early thymocyte precursor migration events important for normal subsequent thymocyte precursor development, but is not required for the formation of normal thymic natural regulatory T-cells (nTregs). Required for optimal differentiation of DN2 and DN3 thymocyte precursors (PubMed:24638065). Essential for B-cell localization in the subepithelial dome of Peyers-patches and for efficient B-cell isotype switching to IgA in the Peyers-patches (PubMed:27174992). Essential for appropriate anatomical distribution of memory B-cells in the spleen and for the secondary recall response of memory B-cells (PubMed:25505290). Positively regulates sperm motility and chemotaxis via its binding to CCL20 (PubMed:23765988).;Species reactivity: Mouse;Application: ELISA;Assay info: ;Sensitivity: < 0.053ng/mL
Description: Description of target: Receptor for the C-C type chemokine CCL20. Binds to CCL20 and subsequently transduces a signal by increasing the intracellular calcium ion levels (PubMed:20068036). Although CCL20 is its major ligand it can also act as a receptor for non-chemokine ligands such as beta-defensins (PubMed:25122636). Binds to defensin DEFB1 leading to increase in intracellular calcium ions and cAMP levels. Its binding to DEFB1 is essential for the function of DEFB1 in regulating sperm motility and bactericidal activity. Binds to defensins DEFB4 and DEFB4A/B and mediates their chemotactic effects (PubMed:20068036). The ligand-receptor pair CCL20-CCR6 is responsible for the chemotaxis of dendritic cells (DC), effector/memory T-cells and B-cells and plays an important role at skin and mucosal surfaces under homeostatic and inflammatory conditions, as well as in pathology, including cancer and various autoimmune diseases. CCR6-mediated signals are essential for immune responses to microbes in the intestinal mucosa and in the modulation of inflammatory responses initiated by tissue insult and trauma (PubMed:21376174). CCR6 is essential for the recruitment of both the proinflammatory IL17 producing helper T-cells (Th17) and the regulatory T-cells (Treg) to sites of inflammation (PubMed:19050256). Required for the normal migration of Th17 cells in Peyers patches and other related tissue sites of the intestine and plays a role in regulating effector T-cell balance and distribution in inflamed intestine (PubMed:19129757). Plays an important role in the coordination of early thymocyte precursor migration events important for normal subsequent thymocyte precursor development, but is not required for the formation of normal thymic natural regulatory T-cells (nTregs). Required for optimal differentiation of DN2 and DN3 thymocyte precursors (PubMed:24638065). Essential for B-cell localization in the subepithelial dome of Peyers-patches and for efficient B-cell isotype switching to IgA in the Peyers-patches (PubMed:27174992). Essential for appropriate anatomical distribution of memory B-cells in the spleen and for the secondary recall response of memory B-cells (PubMed:25505290). Positively regulates sperm motility and chemotaxis via its binding to CCL20 (PubMed:23765988).;Species reactivity: Mouse;Application: ;Assay info: Assay Methodology: Quantitative Sandwich ELISA;Sensitivity: 0.092 ng/mL
Description: Description of target: This gene encodes a member of the beta chemokine receptor family, which is predicted to be a seven transmembrane protein similar to G protein-coupled receptors. The gene is preferentially expressed by immature dendritic cells and memory T cells. The ligand of this receptor is macrophage inflammatory protein 3 alpha (MIP-3 alpha). This receptor has been shown to be important for B-lineage maturation and antigen-driven B-cell differentiation, and it may regulate the migration and recruitment of dentritic and T cells during inflammatory and immunological responses. Alternatively spliced transcript variants that encode the same protein have been described for this gene.;Species reactivity: Human;Application: ELISA;Assay info: Assay Methodology: Quantitative Sandwich ELISA;Sensitivity: 0.058 ng/mL
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Human Chemokine C-C-Motif Receptor 6 (CCR6) in tissue homogenates, cell lysates and other biological fluids.
Human Chemokine C-C-Motif Receptor 6 (CCR6) ELISA Kit
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Human Chemokine C-C-Motif Receptor 6 (CCR6) in tissue homogenates, cell lysates and other biological fluids.
Human Chemokine C-C-Motif Receptor 6 (CCR6) ELISA Kit
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Human Chemokine C-C-Motif Receptor 6 (CCR6) in tissue homogenates, cell lysates and other biological fluids.
Human Chemokine C-C-Motif Receptor 6 (CCR6) ELISA Kit
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Human Chemokine C-C-Motif Receptor 6 (CCR6) in tissue homogenates, cell lysates and other biological fluids.
Human Chemokine C-C-Motif Receptor 6 (CCR6) ELISA Kit
Description: Enzyme-linked immunosorbent assay based on the Double-antibody Sandwich method for detection of Human Chemokine C-C-Motif Receptor 6 (CCR6) in samples from tissue homogenates, cell lysates and other biological fluids with no significant corss-reactivity with analogues from other species.
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Mouse Chemokine C-C-Motif Receptor 6 (CCR6) in Tissue homogenates, cell lysates and other biological fluids.
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Mouse Chemokine C-C-Motif Receptor 6 (CCR6) in Tissue homogenates, cell lysates and other biological fluids.
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Mouse Chemokine C-C-Motif Receptor 6 (CCR6) in Tissue homogenates, cell lysates and other biological fluids.
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Mouse Chemokine C-C-Motif Receptor 6 (CCR6) in Tissue homogenates, cell lysates and other biological fluids.
Description: Enzyme-linked immunosorbent assay based on the Double-antibody Sandwich method for detection of Mouse Chemokine C-C-Motif Receptor 6 (CCR6) in samples from Tissue homogenates, cell lysates and other biological fluids. with no significant corss-reactivity with analogues from other species.
Description: Can be used for various studies in the realm of gene expression, both normal and pathological. It is an excellent control and suitable for educational purposes.
cDNA from Plant Normal Tissue: cDNA from Plant: Corn
Description: Can be used for various studies in the realm of gene expression, both normal and pathological. It is an excellent control and suitable for educational purposes.
cDNA from Plant Normal Tissue: cDNA from Plant: Orange
Description: Can be used for various studies in the realm of gene expression, both normal and pathological. It is an excellent control and suitable for educational purposes.
cDNA from Plant Normal Tissue: cDNA from Plant: Potato
Description: Can be used for various studies in the realm of gene expression, both normal and pathological. It is an excellent control and suitable for educational purposes.
cDNA from Plant Normal Tissue: cDNA from Plant: Rice
Description: Can be used for various studies in the realm of gene expression, both normal and pathological. It is an excellent control and suitable for educational purposes.
cDNA from Plant Normal Tissue: cDNA from Plant: Wheat
Description: Can be used for various studies in the realm of gene expression, both normal and pathological. It is an excellent control and suitable for educational purposes.
