Journal: mSystems

Article Title: Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

doi: 10.1128/msystems.01039-25

Figure Lengend Snippet: Functional metagenomics and METa assembly. (A) The general steps in the preparation and application of a functional metagenomic library consist of the following. (1) Extraction of metagenomic DNA from a source microbiome. (2) Fragmentation of metagenomic DNA to the preferred size range. (3) Packaging of inserts into expression vectors. (4) Transformation of host cells with vector library. (5) Screen or selection of a functional metagenomic library for a phenotype of interest. (6) Collection of screened or selected metagenomic fragments. (7) Sequencing of selected inserts. (8) Open reading frame calling and annotation to identify potential genes underlying phenotypes of interest. (B) Steps 2 and 3 above are modified in Mosaic ends tagmentation (METa) assembly. Fragmentation is achieved using Tn5 transposase tagmentation with mosaic end sequence oligos. Tagmented DNA is gap-filled by polymerase and directly cloned (without amplification) into an expression vector with matching mosaic end sequences defining the cloning site using assembly cloning.

Article Snippet: The ZymoBIOMICS fecal standard, the source of our stool functional metagenomic library input DNA, has been sequenced and annotated for known antibiotic resistance genes by the Zymo corporation, but the data associated with this product do not list any known streptothricin resistance genes.

Techniques: Functional Assay, Extraction, Expressing, Transformation Assay, Plasmid Preparation, Selection, Sequencing, Modification, Clone Assay, Amplification, Cloning

Journal: mSystems

Article Title: Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

doi: 10.1128/msystems.01039-25

Figure Lengend Snippet: Tetracycline selected metagenomic DNA fragments from an aquarium microbiome. (A) Gene schematics of the tetracycline-selected TET1, TET3, and TET13 metagenomic DNA fragments with predicted efflux pumps highlighted (TET1 teal, TET3 red, and TET13 blue). Other predicted open reading frames are shown as empty arrows, and plasmid backbone markers are highlighted as follows: promoter (green), mosaic end sequences (orange), and terminator (red). (B) Maximum likelihood consensus phylogenetic tree of MFS efflux pumps with predicted efflux pump amino acid sequences from TET1 (teal), TET3 (blue), and TET13 (red) fragments highlighted.

Article Snippet: The ZymoBIOMICS fecal standard, the source of our stool functional metagenomic library input DNA, has been sequenced and annotated for known antibiotic resistance genes by the Zymo corporation, but the data associated with this product do not list any known streptothricin resistance genes.

Techniques: Plasmid Preparation

Journal: mSystems

Article Title: Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

doi: 10.1128/msystems.01039-25

Figure Lengend Snippet: Efflux pump containing metagenomic DNA fragments from an aquarium confers tetracycline resistance. Microbroth dilution assay curves and calculated 50% inhibitory concentration (IC50) values for E. coli clones carrying the indicated metagenomic DNA fragments (TET1, TET3, or TET13). (A and B) tetracycline, (C and D) chloramphenicol, or (E and F) azithromycin. **** P < 0.0001, ** P < 0.005, * P < 0.05, n = 4 for all.

Article Snippet: The ZymoBIOMICS fecal standard, the source of our stool functional metagenomic library input DNA, has been sequenced and annotated for known antibiotic resistance genes by the Zymo corporation, but the data associated with this product do not list any known streptothricin resistance genes.

Techniques: Dilution Assay, Concentration Assay, Clone Assay

Journal: mSystems

Article Title: Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

doi: 10.1128/msystems.01039-25

Figure Lengend Snippet: Predicted novel streptothricin acetyltransferase from the human gut microbiome. (A) Gene schematics of the NTC1 metagenomic DNA fragment with the predicted acetyltransferase gene highlighted (SatB, blue). Predicted genes captured on the same DNA fragment are shown as empty arrows. Plasmid backbone elements are: Promoter (green), mosaic end sequence (orange), and terminator (red). (B) Maximum likelihood consensus phylogenetic tree of the predicted SatB protein (blue) in the context of known streptothricin acetyltransferases (SATs, NAT, and STAT) and aminoglycoside, chloramphenicol, virginiamycin, apramycin, and capreomycin acetyltransferases.

Article Snippet: The ZymoBIOMICS fecal standard, the source of our stool functional metagenomic library input DNA, has been sequenced and annotated for known antibiotic resistance genes by the Zymo corporation, but the data associated with this product do not list any known streptothricin resistance genes.

Techniques: Plasmid Preparation, Sequencing

Journal: mSystems

Article Title: Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

doi: 10.1128/msystems.01039-25

Figure Lengend Snippet: Streptothricin resistance is conferred by the NTC1 metagenomic DNA fragment and satB . (A) The results of a microbroth dilution assay of E. coli clones grown in the presence of variable nourseothricin concentrations (NTC1, E. coli carrying the NTC1 metagenomic DNA fragment; stat , E. coli expressing the stat streptothricin acetyltransferase; satB , E. coli expressing the predicted satB open reading frame from the NTC1 fragment). (B) IC50 values calculated from dose-response curves. **** P < 0.0001, *** P < 0.0005, ** P < 0.005, n = 4 for all.

Article Snippet: The ZymoBIOMICS fecal standard, the source of our stool functional metagenomic library input DNA, has been sequenced and annotated for known antibiotic resistance genes by the Zymo corporation, but the data associated with this product do not list any known streptothricin resistance genes.

Techniques: Dilution Assay, Clone Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: Reduced DDX1 protein level leads to increased R-loop amount. ( A ) Slot blot of 0.5 and 1 μg of genomic DNA from MRC5 cells transfected with scrambled control (CTR) or DDX1 siRNA in the absence (−) or presence (+) of RNase H1 and hybridized with antibodies recognizing the RNA/DNA hybrids (S9.6 antibody) or dsDNA (left). The intensity of the bands was measured with ImageJ. The amount of S9.6 signal was normalized to the amount of the loading control dsDNA (right). The values are the mean of at least three independent experiments (* P < .05; Student’s t -test). ( B ) DRIP analysis with the S9.6 antibody at the β-actin locus ( ACTB ) of MRC5 cells treated with scrambled (CTR) or DDX1 siRNA for 120 h. The amount of RNA/DNA hybrids at a, b, c, d, and e positions, indicated as horizontal bars within the β-actin locus (schematic representation on the top), was evaluated by real-time PCR. When applied, the RNase H1 treatment is indicated. Data are expressed as fold enrichment over the input. The values are the mean of at least three independent experiments (* P < .05, ** P < .01; Student’s t -test). ( C ) Density plot of DDX1 ChIP-seq peak distribution relative to the gene TSS in Mus musculus . The plot shows the distribution of DDX1 occupancy across 8504 genes, each showing at least one DDX1 ChIP-seq peak within −1000 to +1000 bp of TSS. Peaks were mapped separately to both the plus strand and the minus strand. The x -axis indicates the upstream and downstream distance in bp from the TSS corresponding to position 0 bp. The y -axis indicates the density of DDX1 peaks.

Article Snippet: A fraction of the genomic DNA was stored as ‘genomic DNA input.’ As a negative control, 10 μg of DNA were digested with 20 U RNase H1 (Invitrogen) O/N at 37°C and then processed in parallel to the samples.

Techniques: Dot Blot, Transfection, Control, Real-time Polymerase Chain Reaction, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: TFIIH plays a role in R-loop processing. ( A ) Immunoblot analysis with antibodies raised against the RPB1 subunit of Pol II and various TFIIH subunits (XPB and p62 of the core-TFIIH, XPD, CDK7, and CycH of the CAK) in MRC5 whole cell extracts transfected with scrambled control (CTR) or XPD siRNA. Protein levels were normalized to the amount of γ-tubulin and reported as arbitrary units (au). The diagram reports the mean values of three independent experiments (right). Bars indicate the standard errors (* P < .05, ** P < .01, *** P < .001; Student’s t -test). ( B ) Slot blot of 0.5 and 1 μg of genomic DNA from MRC5 cells transfected with scrambled control (CTR) or XPD siRNAs in the absence (−) or presence (+) of RNase H1 and hybridized with antibodies recognizing the RNA/DNA hybrids (S9.6 antibody) or dsDNA (left panel). The intensity of the bands was measured with ImageJ. The amount of S9.6 signal was normalized to the amount of the loading control, the dsDNA signal (right panel). The values are the mean of at least three independent experiments. Bars indicate the standard error (* P < .05; Student’s t -test). ( C ) DRIP analysis with the S9.6 antibody at the β-actin locus ( ACTB ) of MRC5 cells treated with scrambled control (CTR) or XPD siRNA for 72 h. The amount of RNA/DNA hybrids at the a, b, c, d, and e positions of the β-actin locus was evaluated by real-time PCR. When applied, the RNase H1 treatment is indicated. Data are expressed as fold enrichment over the input. The values are the mean of at least three independent experiments. Bars indicate the standard errors (* P < .05, ** P < .01; Student’s t -test). ( D ) Immunoblot analysis of XPD and the CDK7 subunits of TFIIH, NONO, SFPQ, and DDX1 proteins in the chromatin-enriched fractions of MRC5 cells transfected with scrambled control (CTR) or XPD siRNAs before (Input) and after immunoprecipitation (IP) with anti-CDK7 or IgG antibodies. Two independent IPs are shown (upper and lower left panels). In the cells treated with XPD -siRNA, the amount of co-immunoprecipitated proteins has been normalized to the amount of the corresponding immunoprecipitated CDK7 and expressed as fold increased relative to the sample CTR siRNA (right panel). The values are the mean of at least three independent experiments. When depicted, bars indicate standard errors (* P < .05; Student’s t -test).

Article Snippet: A fraction of the genomic DNA was stored as ‘genomic DNA input.’ As a negative control, 10 μg of DNA were digested with 20 U RNase H1 (Invitrogen) O/N at 37°C and then processed in parallel to the samples.

Techniques: Western Blot, Transfection, Control, Dot Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation