Journal: Molecular Biology and Evolution

Article Title: Evolution of a Restriction Factor by Domestication of a Yeast Retrotransposon

doi: 10.1093/molbev/msae050

Figure Lengend Snippet: Ty1′ lacks self-encoded CNC. The 227.2 drt2Δ strains were populated with either Ty1′ (YBLWTy1-1) or Ty1c (Ty1-H3) elements. Ty1′ and Ty1c copy number was estimated by southern blotting ( online). A) Retromobility of pGTy1′ his3-AI and pGTy1c his3-AI was determined in the naïve strains in triplicate. B) Retromobility of pGTy1′ his3-AI and pGTy1c his3-AI was measured in triplicate in the populated strains. Fold change in retromobility of populated versus naïve strains is indicated. Retromobility measurements and statistics are reported in online. C) Western blot analysis of whole cell extracts from strains induced for expression was used to detect the level of Gag in populated strains. Ty1′ Gag was detected with α-Drt2 (black star indicates nonspecific band). Ty1c Gag was detected with α-Ty1c p18.

Article Snippet: Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blotted for Ty1′ Gag or Drt2 with α-Drt2 polyclonal primary antibody (Boster Bio cat. #DZ41275) and for Ty1c Gag with α-Ty1c p18 polyclonal primary antibody (Boster Bio cat. #DZ33975).

Techniques: Southern Blot, Western Blot, Expressing

Journal: RNA

Article Title: Retrotransposon Ty1 RNA contains a 5?-terminal long-range pseudoknot required for efficient reverse transcription

doi: 10.1261/rna.035535.112

Figure Lengend Snippet: In vitro RNA secondary structure model of (A) the first 388 nt of Ty1 genomic RNA and (B) the pseudoknot region. Each nucleotide is color-coded according to its normalized NMIA reactivity (color key, top left). Nonevaluated nucleotides are colored gray. The proposed pseudoknot region is boxed. The S1 stem is highlighted in green, S2 stem in blue. Sites of mutations in double/triple mutants GA6CU, UC264AG, CA258GC, UG322GC, CAU258GCC, and AUG321GGC are highlighted in gray. Sites of mutations in single mutants G6A, U269A, U260C, and A321G are highlighted by red circles. Neighboring stem–loop structure SL3 (nt 206–248) and SL4 (nt 275–316) are also designated. (C) Secondary structure model of the Ty2 pseudoknot region based on multiple sequence alignments of all known Saccharomyces cerevisiae Ty1 and Ty2 elements. Ty2 bases that differ from Ty1 are highlighted by light blue circles.

Article Snippet: “ Synthetic and biochemical studies of retrotransposon Ty1 .” PhD thesis, Johns Hopkins University School of Medicine, Baltimore, MD Yarrington RM, Richardson SM, Lisa Huang CR, Boeke JD 2012.

Techniques: In Vitro, Sequencing

Journal: RNA

Article Title: Retrotransposon Ty1 RNA contains a 5?-terminal long-range pseudoknot required for efficient reverse transcription

doi: 10.1261/rna.035535.112

Figure Lengend Snippet: Step plots of NMIA reactivity (left y-axis) of the Ty1 pseudoknot region. (A) S1a mutants, (B) S1b mutants, (C) S2 mutants, and (D) S1/S2 compensatory mutants. Each mutant (solid red line) is plotted against the WT (solid green line) for comparison. Nucleotide positions corresponding to reverse transcriptase stops in the DMSO-control reaction appear as gaps. Lowess smoother fit curves (right y-axis, in blue) over a 10-nt window size were plotted in dashed lines. Nucleotides within the pseudoknot are indicated by gray stripes. The gray horizontal line at 0.5 represents an arbitrarily defined threshold for reactive residues.

Article Snippet: “ Synthetic and biochemical studies of retrotransposon Ty1 .” PhD thesis, Johns Hopkins University School of Medicine, Baltimore, MD Yarrington RM, Richardson SM, Lisa Huang CR, Boeke JD 2012.

Techniques: Mutagenesis, Comparison, Reverse Transcription, Control

Journal: RNA

Article Title: Retrotransposon Ty1 RNA contains a 5?-terminal long-range pseudoknot required for efficient reverse transcription

doi: 10.1261/rna.035535.112

Figure Lengend Snippet: Clustering of Ty1 pseudoknot mutants. Each column consists of NMIA reactivity of the designated Ty1 RNA at single-nucleotide resolution (nt 1–388, color key at top). Green stripes on the left indicate the S1 stem. Blue stripes indicate the S2 stem. Nucleotide positions are marked on the right.

Article Snippet: “ Synthetic and biochemical studies of retrotransposon Ty1 .” PhD thesis, Johns Hopkins University School of Medicine, Baltimore, MD Yarrington RM, Richardson SM, Lisa Huang CR, Boeke JD 2012.

Techniques:

Journal: RNA

Article Title: Retrotransposon Ty1 RNA contains a 5?-terminal long-range pseudoknot required for efficient reverse transcription

doi: 10.1261/rna.035535.112

Figure Lengend Snippet: Ty1 pseudoknot formation is required for active transposition. (A) Transposition of full-length Ty1-mhis3AI elements containing designated mutations in the JB970 strain background was assayed at 22°C, 25°C, and 30°C. (B) Transposition of mini-Ty1-HIS3 elements containing designated mutations in the YQH055 strain background was assayed at 22°C, 25°C, and 30°C. Transposition under repressed conditions (glucose) is shown as a control. Quantitative retrotransposition frequency data are presented in Table 1.

Article Snippet: “ Synthetic and biochemical studies of retrotransposon Ty1 .” PhD thesis, Johns Hopkins University School of Medicine, Baltimore, MD Yarrington RM, Richardson SM, Lisa Huang CR, Boeke JD 2012.

Techniques: Control

Journal: RNA

Article Title: Retrotransposon Ty1 RNA contains a 5?-terminal long-range pseudoknot required for efficient reverse transcription

doi: 10.1261/rna.035535.112

Figure Lengend Snippet: Ty1 retrotransposition is affected by mutations in pseudoknot region

Article Snippet: “ Synthetic and biochemical studies of retrotransposon Ty1 .” PhD thesis, Johns Hopkins University School of Medicine, Baltimore, MD Yarrington RM, Richardson SM, Lisa Huang CR, Boeke JD 2012.

Techniques:

Journal: RNA

Article Title: Retrotransposon Ty1 RNA contains a 5?-terminal long-range pseudoknot required for efficient reverse transcription

doi: 10.1261/rna.035535.112

Figure Lengend Snippet: Mutations in the Ty1 pseudoknot junction do not affect pseudoknot formation or transposition efficiency. (A) Step plots of NMIA reactivity of the pseudoknot region in junction mutants C263A, C263U, and C263G. (B) Transposition of junction mutants compared with WT Ty1-mhis3AI in the JB740 strain background at 22°C. Transposition under repressed conditions (glucose) is shown as a control.

Article Snippet: “ Synthetic and biochemical studies of retrotransposon Ty1 .” PhD thesis, Johns Hopkins University School of Medicine, Baltimore, MD Yarrington RM, Richardson SM, Lisa Huang CR, Boeke JD 2012.

Techniques: Control

Journal: RNA

Article Title: Retrotransposon Ty1 RNA contains a 5?-terminal long-range pseudoknot required for efficient reverse transcription

doi: 10.1261/rna.035535.112

Figure Lengend Snippet: Ty1 RNA half-life and packaging into VLPs are not affected in pseudoknot mutants. (A) Steady state Ty1 RNA levels were measured by QRT-PCR in WT and S1 mutants. RNA levels were normalized to WT, and error bars represent the standard deviation of three biological replicates. (B) Ty1 RNA levels were measured by QRT-PCR at the indicated time points post-glucose exposure. RNA levels in each strain were normalized to the RNA level at the 0-h time point and plotted on a log scale. Solid lines are fitted using an exponential decay function. The slopes of the fitted lines reflect the decay rates of Ty1 RNA. Error bars represent the standard deviation of three replicates. (C) Ty1 RNA levels from samples with and without benzonase treatment were measured by QRT-PCR, and their ratios were plotted as a percentage of packaged Ty1 RNA. Error bars represent the standard deviation of two independently treated lysates. (D) Percentage of Ty1 RNA packaging is shown for WT and the UC264AG mutant at normal (22°C) and high (30°C) temperatures. (**) P < 0.01, (***) P < 0.001. P-values were determined by Student's t-test. (E) Immunoblot analysis of Ty1 Gag from JB970 strains expressing WT or designated mutant Ty1 element. Control lane is the JB970 strain with no plasmid transformed. Protein levels were normalized to α-tubulin levels.

Article Snippet: “ Synthetic and biochemical studies of retrotransposon Ty1 .” PhD thesis, Johns Hopkins University School of Medicine, Baltimore, MD Yarrington RM, Richardson SM, Lisa Huang CR, Boeke JD 2012.

Techniques: Quantitative RT-PCR, Standard Deviation, Mutagenesis, Western Blot, Expressing, Control, Plasmid Preparation, Transformation Assay

Journal: RNA

Article Title: Retrotransposon Ty1 RNA contains a 5?-terminal long-range pseudoknot required for efficient reverse transcription

doi: 10.1261/rna.035535.112

Figure Lengend Snippet: Decreased cDNA level in pseudoknot mutants. (A) Endogenous RT reactions for WT and pseudoknot mutants. (1) G6A, (2) U269A, (3) U260C, (4) GA6CU/UC264AG, (5) UC264AG, (6) U260C/A321G, (7) A321G, (M1, M2) DNA markers. Positions of −sss- and +sssDNA are indicated. (B) Ty1 cDNA levels were measured by Q-PCR and normalized to WT cDNA level; error bars represent standard deviation of three biological replicates. (**) P < 0.01, (****) P < 0.0001. P-values were determined by Student's t-test.

Article Snippet: “ Synthetic and biochemical studies of retrotransposon Ty1 .” PhD thesis, Johns Hopkins University School of Medicine, Baltimore, MD Yarrington RM, Richardson SM, Lisa Huang CR, Boeke JD 2012.

Techniques: Standard Deviation

Journal: RNA

Article Title: Retrotransposon Ty1 RNA contains a 5?-terminal long-range pseudoknot required for efficient reverse transcription

doi: 10.1261/rna.035535.112

Figure Lengend Snippet: 3D structure model of Ty1 RNA (nt 1–362). (A) Relative orientation of the PBS and the pseudoknot. S1 is indicated in green, S2 in blue, C263 in the junction is shown in yellow, Ty1 PBS is shown in red. (B) View rotated 90° along y-axis and pseudoknot with the central segment (nt 7, 262–264, 319) shown as sticks.

Article Snippet: “ Synthetic and biochemical studies of retrotransposon Ty1 .” PhD thesis, Johns Hopkins University School of Medicine, Baltimore, MD Yarrington RM, Richardson SM, Lisa Huang CR, Boeke JD 2012.

Techniques:

Journal: bioRxiv

Article Title: Depolymerized lamins link nuclear envelope breakdown to mitotic transcriptional quiescence

doi: 10.1101/334110

Figure Lengend Snippet: a. pS22-LMNA and UnP-LMNA ChIP-seq signals at LADs. LADs are defined by UnP-LMNA ChIP-seq ( Methods ). A subset of LADs (885 of total 2,178) that do not have adjacent LADs within 250 kb downstream of LADs are shown. Inset shows location of strong ChIP-seq signals (marked in red). FE, fold enrichment. b. pS22-LMNA and UnP-LMNA ChIP-seq signals at 34,489 dLASs. c. Feature size of dLASs and LADs. d. Location of dLASs relative to LADs. e. Ty1 ChIP-seq signals at dLASs. Ty1 ChIP-seq was performed in BJ-5ta cells overexpressing Ty1-LMNA, Ty1-LMNC (Lamin C), phospho-deficient Ty1-LMNA-S22A/S392, or phospho-mimetic Ty1-LMNA-S22D/S392D. f. (Left) Fraction of dLASs overlapping anti-Ty1 ChIP-seq peaks in the overexpression cells shown in e . (Right) Fraction of anti-Ty1 ChIP-seq peaks overlapping dLASs. g. ATAC-seq, K27ac ChIP-seq, K4me3 ChIP-seq, and pS22-LMNA ChIP-seq signals at 73,933 ATAC-defined accessible sites in wild-type BJ-5ta cells.

Article Snippet: Antibodies used in ChIP are: rabbit monoclonal anti-phospho-Ser22-LMNA antibody D2B2E (Cell Signaling 13448S, Lot # 1; 5 µL per IP); mouse monoclonal anti-unphospho-Ser22-LMNA antibody E1 (Santa Cruz Biotechnology sc-376248, Lot # H2812; 10 µL per IP); mouse monoclonal anti-acetyl-Lys27 histone H3 antibody (Wako MABI0309, Lot # 14007; 2 µL per IP); mouse monoclonal anti-trimethyl-Lys4 histone H3 antibody (Wako MABI14004, Lot # 14004; 2 µL per IP); mouse monoclonal anti-Ty1 antibody (Diagenode C15200054, Lot # 005; 1 µL per IP).

Techniques: ChIP-sequencing, Over Expression

Journal: bioRxiv

Article Title: Finding novel vulnerabilities of hypomorphic BRCA1 alleles

doi: 10.1101/2024.05.24.595688

Figure Lengend Snippet: (A) The expression of BRCA1 and Cas9 was assessed by Western blotting of the indicated complemented RPE1 hTERT TP53 -/- BRCA1-mAID + doxycycline inducible Cas9 cells lines. Data shown represent three independent experiments. (B) Ty1 immunoprecipitation on RPE1 hTERT TP53 -/- BRCA1 -/- cells complemented with BRCA1-Ty1 and BRCA1 R1699Q -Ty1. Dashed line indicates removal of non-relevant lanes post-imaging. (C) The eficiency of virally integrated inducible Cas9 cassette was assessed by survival of the cells after transduction with a sgRNA against PSMD1 , an essential gene. Data shown represent two independent replicates. (D) The growth speed of each indicated RPE1 hTERT TP53 -/- cell line was assessed by monitoring cell growth and calculating the doubling time. Data shown is the average of two independent replicates.

Article Snippet: Dynabeads protein-G (ThermoFisher Scientific) were washed 3 times with NETT buffer and mixed with α-Ty1 (Diagenode, Liege, Belgium), followed by incubated for 2.5 hours in this buffer.

Techniques: Expressing, Western Blot, Immunoprecipitation, Imaging, Transduction