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Eurofins complementary dna cdna libraries
Complementary Dna Cdna Libraries, supplied by Eurofins, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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complementary dna cdna libraries - by Bioz Stars, 2020-08
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Article Title: Unlocking the Transcriptomes of Two Carcinogenic Parasites, Clonorchis sinensis and Opisthorchis viverrini
Article Snippet: .. Sequencing and assembly of sequence data sets The transcriptomes of both C. sinensis and O. viverrini were characterized by 454 sequencing (Roche) from normalized, complementary DNA (cDNA) libraries (Eurofins MWG Operon, Ebersberg, Germany; www.eurofinsdna.com ) following the approach applied to F. hepatica . .. For the construction of the libraries, total RNA was isolated from ∼20 adult worms of each C. sinensis and O. viverrini , and polyadenylated (polyA+) RNA was then purified from 25 µg of pooled total RNA.

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    Eurofins single stranded dna library
    Assessment of the Selection Progression (A) <t>DNA</t> melt assay profiles assessing the complexity of the <t>RNA</t> pools plotted as first negative derivative (−dF/dT) against temperature. From left to right: rounds 2, 10, and 15. (B) DNA melt assay profiles assessing the progress of the selection plotted as raw fluorescence against the temperature. (C) Quantification of aptamer enrichment during selection in the denoted rounds. Relative enrichment is shown as the fold-difference of aptamer pools over untransfected cells (mean ± SD). (D) Representative confocal microscopy images showing the internalization of the fluorescein-labeled RNA pool (green) at the final round (scale bar, 45.92 μm). Nuclei were stained blue. Magnified view is of area indicated by asterisks (*). See also Figure S2 .
    Single Stranded Dna Library, supplied by Eurofins, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Eurofins mcr a gene library membranes
    Diversity of <t>mcr</t> A gene sequences from Colne Estuary sediments derived by PCR cloning (BR2, AR2, HY2 and HY30). Numbers of clones in each gene library are shown in parentheses.
    Mcr A Gene Library Membranes, supplied by Eurofins, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Eurofins sgd1 coding sequence
    The RNA binding domain of <t>Sgd1</t> and the interaction with Fal1 are required for the function of Sgd1 in SSU biogenesis . The Sgd1-HA-AID yeast strain was transformed with a pRS415 plasmid for expression of proteins from a MET25 promoter (EV) or derivative plasmids for the expression of full length Sgd1 (Sgd1 FL ) or different fragments of Sgd1. Exponentially growing cells were treated with auxin (IAA) to deplete endogenous Sgd1 and then total RNA was isolated. RNAs were separated by denaturing agarose gel electrophoresis then transferred for northern blotting using [ 32 P] labelled probes hybridizing within ITS1 and ITS2 of pre-rRNA transcripts. Pre-rRNAs were detected using a phosphorimager and mature rRNAs were visualized by methylene blue staining.
    Sgd1 Coding Sequence, supplied by Eurofins, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Assessment of the Selection Progression (A) DNA melt assay profiles assessing the complexity of the RNA pools plotted as first negative derivative (−dF/dT) against temperature. From left to right: rounds 2, 10, and 15. (B) DNA melt assay profiles assessing the progress of the selection plotted as raw fluorescence against the temperature. (C) Quantification of aptamer enrichment during selection in the denoted rounds. Relative enrichment is shown as the fold-difference of aptamer pools over untransfected cells (mean ± SD). (D) Representative confocal microscopy images showing the internalization of the fluorescein-labeled RNA pool (green) at the final round (scale bar, 45.92 μm). Nuclei were stained blue. Magnified view is of area indicated by asterisks (*). See also Figure S2 .

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers

    doi: 10.1016/j.omtn.2017.12.004

    Figure Lengend Snippet: Assessment of the Selection Progression (A) DNA melt assay profiles assessing the complexity of the RNA pools plotted as first negative derivative (−dF/dT) against temperature. From left to right: rounds 2, 10, and 15. (B) DNA melt assay profiles assessing the progress of the selection plotted as raw fluorescence against the temperature. (C) Quantification of aptamer enrichment during selection in the denoted rounds. Relative enrichment is shown as the fold-difference of aptamer pools over untransfected cells (mean ± SD). (D) Representative confocal microscopy images showing the internalization of the fluorescein-labeled RNA pool (green) at the final round (scale bar, 45.92 μm). Nuclei were stained blue. Magnified view is of area indicated by asterisks (*). See also Figure S2 .

    Article Snippet: Preparation of the RNA Library The initial single-stranded DNA library (sequence adapted from previous work; ) contained 40 nt of random sequence at an equimolar concentration and was synthesized by Eurofins Genomics (Wolverhampton, UK).

    Techniques: Selection, Fluorescence, Confocal Microscopy, Labeling, Staining

    Diversity of mcr A gene sequences from Colne Estuary sediments derived by PCR cloning (BR2, AR2, HY2 and HY30). Numbers of clones in each gene library are shown in parentheses.

    Journal: FEMS Microbiology Ecology

    Article Title: Archaeal community diversity and abundance changes along a natural salinity gradient in estuarine sediments

    doi: 10.1093/femsec/fiu025

    Figure Lengend Snippet: Diversity of mcr A gene sequences from Colne Estuary sediments derived by PCR cloning (BR2, AR2, HY2 and HY30). Numbers of clones in each gene library are shown in parentheses.

    Article Snippet: All archaeal 16S rRNA and mcr A gene library membranes were screened with 5′ and 3′ end-labeled digoxigenin (DIG) oligonucleotide probes (Eurofins MWG Operon) targeting Archaea and methanogen-specific 16S rRNA genes and mcr A genes (Table ), respectively, under optimized conditions.

    Techniques: Derivative Assay, Polymerase Chain Reaction, Clone Assay

    Depth profiles of geochemical data, total cell numbers and Archaea 16S rRNA gene copies for Colne Estuary sediment cores, (a) BR, (b) AR and (c) HY. Graph panels show data for (I) pore water chloride, sulphate and methane; shaded region denotes depths of samples used for archaeal 16S rRNA and mcr A gene libraries. (II) Log10 total cell numbers determined by AODC and prokaryotic 16S rRNA gene copy numbers determined by qPCR. The solid line shows Parkes, Cragg and Wellsbury (2000) general model for prokaryotic cell distributions in marine sediments, and dotted lines represent 95% prediction limits. (III) Log10 16S rRNA gene copy numbers for Bacteria , Archaea and Methanococcoides species. (IV) Percentage of Archaea and Methanococcoides species of the total prokaryotic and Archaea populations, respectively. All qPCR data points are means of three replicates.

    Journal: FEMS Microbiology Ecology

    Article Title: Archaeal community diversity and abundance changes along a natural salinity gradient in estuarine sediments

    doi: 10.1093/femsec/fiu025

    Figure Lengend Snippet: Depth profiles of geochemical data, total cell numbers and Archaea 16S rRNA gene copies for Colne Estuary sediment cores, (a) BR, (b) AR and (c) HY. Graph panels show data for (I) pore water chloride, sulphate and methane; shaded region denotes depths of samples used for archaeal 16S rRNA and mcr A gene libraries. (II) Log10 total cell numbers determined by AODC and prokaryotic 16S rRNA gene copy numbers determined by qPCR. The solid line shows Parkes, Cragg and Wellsbury (2000) general model for prokaryotic cell distributions in marine sediments, and dotted lines represent 95% prediction limits. (III) Log10 16S rRNA gene copy numbers for Bacteria , Archaea and Methanococcoides species. (IV) Percentage of Archaea and Methanococcoides species of the total prokaryotic and Archaea populations, respectively. All qPCR data points are means of three replicates.

    Article Snippet: All archaeal 16S rRNA and mcr A gene library membranes were screened with 5′ and 3′ end-labeled digoxigenin (DIG) oligonucleotide probes (Eurofins MWG Operon) targeting Archaea and methanogen-specific 16S rRNA genes and mcr A genes (Table ), respectively, under optimized conditions.

    Techniques: Real-time Polymerase Chain Reaction

    The RNA binding domain of Sgd1 and the interaction with Fal1 are required for the function of Sgd1 in SSU biogenesis . The Sgd1-HA-AID yeast strain was transformed with a pRS415 plasmid for expression of proteins from a MET25 promoter (EV) or derivative plasmids for the expression of full length Sgd1 (Sgd1 FL ) or different fragments of Sgd1. Exponentially growing cells were treated with auxin (IAA) to deplete endogenous Sgd1 and then total RNA was isolated. RNAs were separated by denaturing agarose gel electrophoresis then transferred for northern blotting using [ 32 P] labelled probes hybridizing within ITS1 and ITS2 of pre-rRNA transcripts. Pre-rRNAs were detected using a phosphorimager and mature rRNAs were visualized by methylene blue staining.

    Journal: RNA Biology

    Article Title: Sgd1 is an MIF4G domain-containing cofactor of the RNA helicase Fal1 and associates with the 5’ domain of the 18S rRNA sequence

    doi: 10.1080/15476286.2020.1716540

    Figure Lengend Snippet: The RNA binding domain of Sgd1 and the interaction with Fal1 are required for the function of Sgd1 in SSU biogenesis . The Sgd1-HA-AID yeast strain was transformed with a pRS415 plasmid for expression of proteins from a MET25 promoter (EV) or derivative plasmids for the expression of full length Sgd1 (Sgd1 FL ) or different fragments of Sgd1. Exponentially growing cells were treated with auxin (IAA) to deplete endogenous Sgd1 and then total RNA was isolated. RNAs were separated by denaturing agarose gel electrophoresis then transferred for northern blotting using [ 32 P] labelled probes hybridizing within ITS1 and ITS2 of pre-rRNA transcripts. Pre-rRNAs were detected using a phosphorimager and mature rRNAs were visualized by methylene blue staining.

    Article Snippet: For recombinant expression of Sgd1 in E. coli , a codon-optimized version of the Sgd1 coding sequence was synthesized by MWG Eurofins and sub-cloned into a pQE80-derived plasmid for the expression of an N-terminally MBP-TEV protease cleavage site, C-terminally His tagged protein.

    Techniques: RNA Binding Assay, Transformation Assay, Plasmid Preparation, Expressing, Isolation, Agarose Gel Electrophoresis, Northern Blot, Staining

    Sgd1 crosslinks to helix H12 of the 18S rRNA sequence . (A) Wild type yeast (WT) or a yeast strain expressing C-terminally His-TEV protease cleavage site-Protein A (HTP) tagged Sgd1 from its genomic locus were crosslinked in culturo using irradiation at 254 nm. The tagged protein and crosslinked RNAs were retrieved under native conditions on IgG sepharose and subjected to a partial RNase digest. Complexes were then enriched under denaturing conditions on NiNTA, and co-purified RNA fragments were [ 32 P] labelled and ligated to sequencing adaptors. Protein-RNA complexes were separated by denaturing PAGE, transferred to a nitrocellulose membrane and radiolabelled RNAs were detected by autoradiography. A non-specific signal is indicated by an asterisk. (B) The region of the membrane containing crosslinked Sgd1-HTP complexes, and a corresponding region of the lane containing the WT sample, were excised and RNAs were release by protease treatment. Purified RNAs were converted to a cDNA library that was subjected to deep sequencing. The obtained sequencing reads were mapped to the S. cerevisiae genome and the relative proportions of reads mapped to gene features encoding different classes of RNA was determined. Abbreviations – ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), non-coding RNA (ncRNA), small nuclear RNA (snRNA). (C) After normalization, the number of reads mapping to each nucleotide of RDN37 , which encodes the 35S pre-rRNA transcript, was determined for the WT and Sgd1-HTP samples and is shown above a schematic view of the pre-rRNA transcript. The number of mutations, induced by the presence of crosslinked nucleotides, mapping to each nucleotide is also shown. (D) The number of sequencing reads in the Sgd1-HTP CRAC dataset mapping to each nucleotide of the 18S rRNA sequence is shown on the secondary structure of the mature rRNA using a colour scale in which the maximum number of reads (100%) is highlighted in red and lesser numbers of reads ( > 20%) are shown in yellow. A magnified view of the region of the 18S rRNA to which Sgd1 binds is presented. (E) The number of sequencing reads mapping to each nucleotide of the 18S rRNA sequence was mapped onto the tertiary structure of the SSU processome (PBD: 5WLC) using a colour scale as in (D). Pre-rRNA sequences are shown in cartoon mode, and ribosomal proteins and ribosome assembly factors are shown in surface view in pale cyan. The densities corresponding to specific ribosomal proteins and ribosome assembly factors that are present in close proximity to the crosslinking site of Sgd1 are highlighted.

    Journal: RNA Biology

    Article Title: Sgd1 is an MIF4G domain-containing cofactor of the RNA helicase Fal1 and associates with the 5’ domain of the 18S rRNA sequence

    doi: 10.1080/15476286.2020.1716540

    Figure Lengend Snippet: Sgd1 crosslinks to helix H12 of the 18S rRNA sequence . (A) Wild type yeast (WT) or a yeast strain expressing C-terminally His-TEV protease cleavage site-Protein A (HTP) tagged Sgd1 from its genomic locus were crosslinked in culturo using irradiation at 254 nm. The tagged protein and crosslinked RNAs were retrieved under native conditions on IgG sepharose and subjected to a partial RNase digest. Complexes were then enriched under denaturing conditions on NiNTA, and co-purified RNA fragments were [ 32 P] labelled and ligated to sequencing adaptors. Protein-RNA complexes were separated by denaturing PAGE, transferred to a nitrocellulose membrane and radiolabelled RNAs were detected by autoradiography. A non-specific signal is indicated by an asterisk. (B) The region of the membrane containing crosslinked Sgd1-HTP complexes, and a corresponding region of the lane containing the WT sample, were excised and RNAs were release by protease treatment. Purified RNAs were converted to a cDNA library that was subjected to deep sequencing. The obtained sequencing reads were mapped to the S. cerevisiae genome and the relative proportions of reads mapped to gene features encoding different classes of RNA was determined. Abbreviations – ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), non-coding RNA (ncRNA), small nuclear RNA (snRNA). (C) After normalization, the number of reads mapping to each nucleotide of RDN37 , which encodes the 35S pre-rRNA transcript, was determined for the WT and Sgd1-HTP samples and is shown above a schematic view of the pre-rRNA transcript. The number of mutations, induced by the presence of crosslinked nucleotides, mapping to each nucleotide is also shown. (D) The number of sequencing reads in the Sgd1-HTP CRAC dataset mapping to each nucleotide of the 18S rRNA sequence is shown on the secondary structure of the mature rRNA using a colour scale in which the maximum number of reads (100%) is highlighted in red and lesser numbers of reads ( > 20%) are shown in yellow. A magnified view of the region of the 18S rRNA to which Sgd1 binds is presented. (E) The number of sequencing reads mapping to each nucleotide of the 18S rRNA sequence was mapped onto the tertiary structure of the SSU processome (PBD: 5WLC) using a colour scale as in (D). Pre-rRNA sequences are shown in cartoon mode, and ribosomal proteins and ribosome assembly factors are shown in surface view in pale cyan. The densities corresponding to specific ribosomal proteins and ribosome assembly factors that are present in close proximity to the crosslinking site of Sgd1 are highlighted.

    Article Snippet: For recombinant expression of Sgd1 in E. coli , a codon-optimized version of the Sgd1 coding sequence was synthesized by MWG Eurofins and sub-cloned into a pQE80-derived plasmid for the expression of an N-terminally MBP-TEV protease cleavage site, C-terminally His tagged protein.

    Techniques: Sequencing, Expressing, Irradiation, Purification, Polyacrylamide Gel Electrophoresis, Autoradiography, cDNA Library Assay

    Fal1 and Sgd1 are required for pre-rRNA cleavages at sites A 0 , A 1 and A 2 . (A-C) Exponentially growing yeast cells expressing Fal1-HA (A), Fal1-HA-AID (B) or Sgd1-HA-AID (C) were treated with IAA for the indicated times (t) or left untreated (-IAA) before harvesting. Total proteins were separated by SDS-PAGE followed by western blotting using antibodies against the HA tag or Pgk1. (D) Simplified schematic overview of pre-rRNA processing in S. cerevisiae . Mature rRNA sequences are indicated by black rectangles, and internal transcribed spacers (ITS1 and ITS2) and external transcribed spacers (5ʹ ETS and 3ʹ ETS) are represented by black lines. Selected pre-rRNA cleavage sites and the hybridization positions of probes used for northern blotting are marked on the 35S pre-rRNA transcript. (E) Exponentially growing yeast cells expressing Fal1-HA, Fal1-HA-AID or Sgd1-HA-AID were treated with IAA (+) for 60 min or left untreated (-) before harvesting. Total RNA was extracted, separated by denaturing agarose gel electrophoresis and transferred to a nylon membrane. The mature 25S and 18S rRNAs were visualized by methylene blue staining (MB) and pre-rRNAs were detected by northern blotting using probes hybridizing to different regions of the pre-rRNA transcript.

    Journal: RNA Biology

    Article Title: Sgd1 is an MIF4G domain-containing cofactor of the RNA helicase Fal1 and associates with the 5’ domain of the 18S rRNA sequence

    doi: 10.1080/15476286.2020.1716540

    Figure Lengend Snippet: Fal1 and Sgd1 are required for pre-rRNA cleavages at sites A 0 , A 1 and A 2 . (A-C) Exponentially growing yeast cells expressing Fal1-HA (A), Fal1-HA-AID (B) or Sgd1-HA-AID (C) were treated with IAA for the indicated times (t) or left untreated (-IAA) before harvesting. Total proteins were separated by SDS-PAGE followed by western blotting using antibodies against the HA tag or Pgk1. (D) Simplified schematic overview of pre-rRNA processing in S. cerevisiae . Mature rRNA sequences are indicated by black rectangles, and internal transcribed spacers (ITS1 and ITS2) and external transcribed spacers (5ʹ ETS and 3ʹ ETS) are represented by black lines. Selected pre-rRNA cleavage sites and the hybridization positions of probes used for northern blotting are marked on the 35S pre-rRNA transcript. (E) Exponentially growing yeast cells expressing Fal1-HA, Fal1-HA-AID or Sgd1-HA-AID were treated with IAA (+) for 60 min or left untreated (-) before harvesting. Total RNA was extracted, separated by denaturing agarose gel electrophoresis and transferred to a nylon membrane. The mature 25S and 18S rRNAs were visualized by methylene blue staining (MB) and pre-rRNAs were detected by northern blotting using probes hybridizing to different regions of the pre-rRNA transcript.

    Article Snippet: For recombinant expression of Sgd1 in E. coli , a codon-optimized version of the Sgd1 coding sequence was synthesized by MWG Eurofins and sub-cloned into a pQE80-derived plasmid for the expression of an N-terminally MBP-TEV protease cleavage site, C-terminally His tagged protein.

    Techniques: Expressing, SDS Page, Western Blot, Hybridization, Northern Blot, Agarose Gel Electrophoresis, Staining

    Sgd1 interacts directly with Fal1 and stimulates its ATPase activity . (A) Soluble whole cell extracts were prepared from exponentially growing yeast cells expressing HA tagged Fal1 or Rok1 from their endogenous promoters and either treated with RNase (+) or left untreated (-). Recombinant MBP-Sgd1-His or the MBP-His tag alone purified from E. coli were immobilized on amylose resin and unbound proteins were washed away. The cell extracts were incubated with the Sgd1-bound beads or empty amylose resin (A) and co-purified complexes were retrieved. Input samples (25%) and recovered proteins (Eluate) were separated by SDS-PAGE and transferred for western blotting using antibodies against the MBP and HA tags. (B) Schematic views of N-terminally MBP, C-terminally His tagged fragments of Sgd1 showing the positions of the predicted MIF4G and MA3 domains. Amino acid numbers corresponding to the boundaries of domains and fragments are given above each scheme. (C) MBP-His tagged full length Sgd1 (Sgd1 FL ) or its fragments were recombinantly expressed in E. coli and purified. Proteins were separated by SDS-PAGE and detected by Coomassie staining. (D) Recombinant MBP-His tagged Sgd1 FL or its fragments, or the MBP-His tag, were immobilized on amylose resin. Excess, unbound proteins were removed before addition of His-Fal1. After thorough washing steps, co-purified proteins were eluted. Eluates were separated by SDS-PAGE and proteins were detected by Coomassie staining. The band corresponding to His-Fal1 is indicated by an arrow head. (E) The ATPase activity of Fal1 alone or together with the His-ZZ-tagged MIF4G domain of Sgd1 (Sgd1 MIF4G ) in the presence or absence of RNA was monitored in vitro using NADH-coupled assays. The ATPase activity of Sgd1 MIF4G in the presence or absence of RNA and background (no protein) ATP hydrolysis were also monitored. Data from three independent experiments are presented as mean ± standard deviation.

    Journal: RNA Biology

    Article Title: Sgd1 is an MIF4G domain-containing cofactor of the RNA helicase Fal1 and associates with the 5’ domain of the 18S rRNA sequence

    doi: 10.1080/15476286.2020.1716540

    Figure Lengend Snippet: Sgd1 interacts directly with Fal1 and stimulates its ATPase activity . (A) Soluble whole cell extracts were prepared from exponentially growing yeast cells expressing HA tagged Fal1 or Rok1 from their endogenous promoters and either treated with RNase (+) or left untreated (-). Recombinant MBP-Sgd1-His or the MBP-His tag alone purified from E. coli were immobilized on amylose resin and unbound proteins were washed away. The cell extracts were incubated with the Sgd1-bound beads or empty amylose resin (A) and co-purified complexes were retrieved. Input samples (25%) and recovered proteins (Eluate) were separated by SDS-PAGE and transferred for western blotting using antibodies against the MBP and HA tags. (B) Schematic views of N-terminally MBP, C-terminally His tagged fragments of Sgd1 showing the positions of the predicted MIF4G and MA3 domains. Amino acid numbers corresponding to the boundaries of domains and fragments are given above each scheme. (C) MBP-His tagged full length Sgd1 (Sgd1 FL ) or its fragments were recombinantly expressed in E. coli and purified. Proteins were separated by SDS-PAGE and detected by Coomassie staining. (D) Recombinant MBP-His tagged Sgd1 FL or its fragments, or the MBP-His tag, were immobilized on amylose resin. Excess, unbound proteins were removed before addition of His-Fal1. After thorough washing steps, co-purified proteins were eluted. Eluates were separated by SDS-PAGE and proteins were detected by Coomassie staining. The band corresponding to His-Fal1 is indicated by an arrow head. (E) The ATPase activity of Fal1 alone or together with the His-ZZ-tagged MIF4G domain of Sgd1 (Sgd1 MIF4G ) in the presence or absence of RNA was monitored in vitro using NADH-coupled assays. The ATPase activity of Sgd1 MIF4G in the presence or absence of RNA and background (no protein) ATP hydrolysis were also monitored. Data from three independent experiments are presented as mean ± standard deviation.

    Article Snippet: For recombinant expression of Sgd1 in E. coli , a codon-optimized version of the Sgd1 coding sequence was synthesized by MWG Eurofins and sub-cloned into a pQE80-derived plasmid for the expression of an N-terminally MBP-TEV protease cleavage site, C-terminally His tagged protein.

    Techniques: Activity Assay, Expressing, Recombinant, Purification, Incubation, SDS Page, Western Blot, Staining, In Vitro, Standard Deviation

    The C-terminal region of Sgd1 binds RNA . (A) Schematic view of HTP tagged fragments of Sgd1 showing the positions of the predicted MIF4G and MA3 domains. Amino acid numbers corresponding to the boundaries of domains and fragments are given above each scheme. (B) The Sgd1-HA-AID yeast strain was transformed with plasmids for expression of the fragments depicted in (A). Exponentially growing cells were depleted of endogenous Sgd1 by auxin treatment before crosslinking in culturo using irradiation of 254 nm. Protein-RNA complexes were enriched by tandem affinity purification under native and denaturing conditions, and RNAs were partially digested and 5ʹ labelled with [ 32 P]. Complexes were separated by denaturing PAGE and transferred to nitrocellulose membranes. Proteins were detected by western blotting using an anti-PAP antibody (upper panel) and radiolabelled RNAs were detected by autoradiography (lower panel). Signals corresponding to specifically crosslinked RNAs are indicated by boxes and non-specific signals are indicated with asterisks. (C) Anisotropy measurements of an 11 nt, Atto647 labelled RNA in the presence of different amounts of purified, MBP/His tagged Sgd1 fragments were performed. The data from three independent experiments is presented as mean ± standard deviation.

    Journal: RNA Biology

    Article Title: Sgd1 is an MIF4G domain-containing cofactor of the RNA helicase Fal1 and associates with the 5’ domain of the 18S rRNA sequence

    doi: 10.1080/15476286.2020.1716540

    Figure Lengend Snippet: The C-terminal region of Sgd1 binds RNA . (A) Schematic view of HTP tagged fragments of Sgd1 showing the positions of the predicted MIF4G and MA3 domains. Amino acid numbers corresponding to the boundaries of domains and fragments are given above each scheme. (B) The Sgd1-HA-AID yeast strain was transformed with plasmids for expression of the fragments depicted in (A). Exponentially growing cells were depleted of endogenous Sgd1 by auxin treatment before crosslinking in culturo using irradiation of 254 nm. Protein-RNA complexes were enriched by tandem affinity purification under native and denaturing conditions, and RNAs were partially digested and 5ʹ labelled with [ 32 P]. Complexes were separated by denaturing PAGE and transferred to nitrocellulose membranes. Proteins were detected by western blotting using an anti-PAP antibody (upper panel) and radiolabelled RNAs were detected by autoradiography (lower panel). Signals corresponding to specifically crosslinked RNAs are indicated by boxes and non-specific signals are indicated with asterisks. (C) Anisotropy measurements of an 11 nt, Atto647 labelled RNA in the presence of different amounts of purified, MBP/His tagged Sgd1 fragments were performed. The data from three independent experiments is presented as mean ± standard deviation.

    Article Snippet: For recombinant expression of Sgd1 in E. coli , a codon-optimized version of the Sgd1 coding sequence was synthesized by MWG Eurofins and sub-cloned into a pQE80-derived plasmid for the expression of an N-terminally MBP-TEV protease cleavage site, C-terminally His tagged protein.

    Techniques: Transformation Assay, Expressing, Irradiation, Affinity Purification, Polyacrylamide Gel Electrophoresis, Western Blot, Autoradiography, Purification, Standard Deviation