mic genome sequences  (Broad Institute Inc)

Journal: International Journal of Oncology

Article Title: Association and regulation of protein factors of field effect in prostate tissues

doi: 10.3892/ijo.2016.3666

Figure Lengend Snippet: (A) Representative detection of EGR-1, PDGF-A, MIC-1, and FASN by immunofluorescence in tumor (panels i–iv), tumor-adjacent (panels v–viii), and disease-free (panels ix–xii) human prostate tissues. Unspecific IgG of mouse, rabbit, and goat origin were tested for absence of staining (panels xiii–xv). Images represent Alexa Fluor 633 immunostaining (yellow signals); the smaller insets represent corresponding nuclear staining by DAPI (blue); white bars, 10 μm. (B) Schematic representation of the whole-image (top) and ROI (bottom) quantitative acquisition modes for EGR-1 fluorescence intensity. Whole-image data acquisition includes three different settings as defined by DAPI staining, whole-cell/no selection (panel i), nuclear (panel ii), and cytoplasmic (panel iii), as indicated by the bright blue shading. ROI data acquisition includes nuclear (panel iv) and extranuclear/cytoplasmic (panel v), as indicated by the areas designated by the randomly placed yellow rectangle frames (~80 μm 2 ); white bars, 10 μm. EGR-1, early growth response-1; PDGF-A, platelet-derived growth factor-A; MIC-1, macrophage inhibitory cytokine-1; FASN, fatty acid synthase; ROI, region of interest.

Article Snippet: Genomic sequences for EGR-1, PDGF-A, MIC-1, and FASN were retrieved from the GRCh38 primary assembly of the gene database available at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/ ).

Techniques: Immunofluorescence, Staining, Immunostaining, Fluorescence, Selection, Derivative Assay

Journal: International Journal of Oncology

Article Title: Association and regulation of protein factors of field effect in prostate tissues

doi: 10.3892/ijo.2016.3666

Figure Lengend Snippet: (A and B) Ratios of PDGF-A, MIC-1, and FASN to EGR-1 expression (combined whole-cell, nuclear, cytoplasmic) in disease-free (DF), tumor-adjacent (ADJ), and tumor (TUM) tissues using images from all (left three bars) and matched only (right three bars) cases, acquired by the whole-image and the ROI mode, respectively. The bars represent average ratios + standard errors. The numbers by the bars represent the fold change in ADJ and TUM compared to DF tissues. * Statistical significance compared to DF tissues (p≤0.05). PDGF-A, platelet-derived growth factor-A; MIC-1, macrophage inhibitory cytokine-1; FASN, fatty acid synthase; EGR-1, early growth response-1; ROI, region of interest.

Article Snippet: Genomic sequences for EGR-1, PDGF-A, MIC-1, and FASN were retrieved from the GRCh38 primary assembly of the gene database available at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/ ).

Techniques: Expressing, Derivative Assay

Journal: International Journal of Oncology

Article Title: Association and regulation of protein factors of field effect in prostate tissues

doi: 10.3892/ijo.2016.3666

Figure Lengend Snippet: (A and C) Graphical representation of Pearson's correlation (r) between EGR-1 and PDGF-A, MIC-1, and FASN using data from digitized images acquired by the whole-image and the ROI mode, respectively. Within each type of tissue, disease-free (DF), tumor-adjacent (ADJ), and tumor (TUM), correlations were determined for all matched, and for EGR-1 above or below the median with the corresponding median-divided datasets of PDGF-A, MIC-1, and FASN. (A) Datasets consist of whole-cell, nuclear, and cytoplasmic EGR-1 measurements (a total of 15 correlations per factor). (B) Datasets consist of nuclear and cytoplasmic EGR-1 measurements (a total of 12 correlations per factor). Arrows depict the change of regulation by linking the mean Pearson's correlations (black dots) in the different types of tissues. (B and D) Average positive (pos; black bars) and negative (neg; grey bars) Pearson's correlations between EGR-1 and PDGF-A, MIC-1, and FASN in DF, ADJ, and TUM tissues acquired by the whole-image and the ROI mode, respectively. The bars represent average ratios + standard errors. The numbers represent the fold change in the ratio of positive/negative r in ADJ and TUM compared to DF tissues. * Statistical significance compared to DF tissues (p≤0.05). EGR-1, early growth response-1; PDGF-A, platelet-derived growth factor-A; MIC-1, macrophage inhibitory cytokine-1; FASN, fatty acid synthase; ROI, region of interest.

Article Snippet: Genomic sequences for EGR-1, PDGF-A, MIC-1, and FASN were retrieved from the GRCh38 primary assembly of the gene database available at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/ ).

Techniques: Derivative Assay

Journal: International Journal of Oncology

Article Title: Association and regulation of protein factors of field effect in prostate tissues

doi: 10.3892/ijo.2016.3666

Figure Lengend Snippet: (A) Computational analysis of the EGR-1 recognition sequence [GCG(G/T)GGCG] in the genomic sequence 1,500 bp upstream and 500 bp downstream of the transcription initiation site of PDGF-A, MIC-1, and FASN. Black vertical lines and black rectangular boxes denote genomic sequences and exons, respectively; vertical arrow heads indicate EGR-1 recognition sequences. (B) EGR-1, PDGF-A, MIC-1, and FASN protein expression in RWPE-1 cells transiently transfected with pcDNA3.1/EGR-1 (EGR-1 overexpression) or pLKO.1/EGR-1 shRNA (EGR-1 suppression), and their empty plasmid controls. Double bands in EGR-1 represent post-translational modifications . The fold change difference compared to empty plasmid control and determined by densitometry as a ratio with β-actin signal is indicated in the small bar graphs (left bar, EGR-1 overexpression; right bar, EGR-1 suppression). (C) Relative mRNA expression of PDGF-A, MIC-1, and FASN in RWPE-1 cells transiently transfected with pcDNA3.1/EGR-1 (EGR-1 overexpression) or pLKO.1/EGR-1 shRNA (EGR-1 suppression), and their empty plasmid controls. Bars represent averages of triplicates ± standard deviation; * Statistical significance (p≤0.05) from pcDNA3.1 and pLKO.1 plasmid vector control, respectively. EGR-1, early growth response-1; PDGF-A, platelet-derived growth factor-A; MIC-1, macrophage inhibitory cytokine-1; FASN, fatty acid synthase.

Article Snippet: Genomic sequences for EGR-1, PDGF-A, MIC-1, and FASN were retrieved from the GRCh38 primary assembly of the gene database available at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/ ).

Techniques: Sequencing, Genomic Sequencing, Expressing, Transfection, Over Expression, shRNA, Plasmid Preparation, Control, Standard Deviation, Derivative Assay

Journal: Developmental Cell

Article Title: Phosphorylation of an HP1-like Protein Regulates Heterochromatin Body Assembly for DNA Elimination

doi: 10.1016/j.devcel.2015.11.017

Figure Lengend Snippet: Identification of Heterochromatin Body Components (A) A single Tetrahymena thermophila cell possesses a MAC (a) and a MIC (i). During vegetative growth, these nuclei divide and are segregated independently into daughter cells. Nutritional starvation induces the conjugation of two cells carrying different mating types. In the early conjugation stage (∼1–4 hpm), the MICs undergo meiosis. In the mid-stage, one of the meiotic products is exchanged between the cells (∼5 hpm) and fuses with the stationary meiotic product to form a zygotic nucleus (∼6 hpm), which then divides twice to form two new MACs and two MICs (∼7 hpm). At the late-stage, the new MACs (na) are enlarged (∼8 hpm). The pair is dissolved and the parental MAC (pa) and one of the MICs are degraded in the exconjugants (∼12–16 hpm). The exconjugants resume vegetative growth when nutrients are available. (B) Summary of the screen for heterochromatin body components. (C) WT cells at 10, 12, 14, and 16 hpm were hybridized with a probe complementary to the Tlr1 element (green) and stained with an anti-Pdd1p antibody (red). DNA was stained with DAPI (blue). Arrowheads indicate the MIC (i), the new MAC (na), and the parental MAC (pa). The scale bars represent 10 μm. (D) Exconjugants expressing the indicated proteins tagged with EGFP (green) and Pdd1p-mCherry (red) were counterstained with DAPI (blue). Arrowheads indicate the new MACs (na). All pictures share the scale bar, representing 10 μm. See also Figure S1 .

Article Snippet: The MIC genome sequence (version 2) was obtained from the Tetrahymena Comparative Sequencing Project (Broad Institute of MIT and Harvard).

Techniques: Conjugation Assay, Staining, Expressing

Journal: Molecular Cell

Article Title: Small-RNA-Mediated Genome-wide trans -Recognition Network in Tetrahymena DNA Elimination

doi: 10.1016/j.molcel.2015.05.024

Figure Lengend Snippet: Two Types of scnRNAs (A) A Tetrahymena cell contains a macronucleus (MAC) and a micronucleus (MIC). During vegetative growth, both the MAC and the MIC divide and segregate to daughter cells. Mixing starved cells of different mating types induces conjugation (i). The MICs undergo meiosis (ii), and one of the selected products divides mitotically to form two pronuclei (iii). One of the pronuclei crosses the conjugation bridge (iv) and fuses with the stationary pronucleus to produce the zygotic nucleus (v), which then divides twice (vi) to form two new MACs and two MICs (vii). The parental MAC is degraded, and the pair is dissolved (viii). The exconjugants resume vegetative growth when the nutrient supply is restored (ix). The approximate time when each event occurs is indicated (hpm, hours post-mixing). (B) 1,464 MIC genome supercontigs (SCs, blue bars) were ordered by their lengths (longest to shortest) and concatenated. Normalized numbers (reads per kilobase per million reads [RPKM]) of sequenced 26- to 32-nt RNAs from WT cells at the indicated time points that map uniquely to the MIC genome are shown as histograms with 50-kb bins. The densities of IESs and mappable (unique) sequences are also shown. The drop in IES density in the region containing very short SCs is probably because these SCs are shorter than most of the IESs, and the prediction of IESs from them failed. The regions enlarged in (C and D) are marked with green lines. Longer (1–50) and shorter (51–1,464) MIC SCs represent B- and A-regions of the MIC genome, respectively. (C and D) Small RNA expression from the indicated 300-kb windows (shown and analyzed as in B, except with 100-nt bins). Colored boxes indicate the positions of IESs (magenta, type A; sky blue, type B; see Figure 3 for the IES classification). In (D), the arrows mark MAC-destined regions that are the origins of Early-scnRNAs that accumulated prominently at early stages (3 hpm) but were degraded later.

Article Snippet: The draft MIC genome sequence (version 2) was obtained from the Tetrahymena Comparative Sequencing Project (Broad Institute of Harvard and MIT).

Techniques: Conjugation Assay, RNA Expression

Journal: Molecular Cell

Article Title: Small-RNA-Mediated Genome-wide trans -Recognition Network in Tetrahymena DNA Elimination

doi: 10.1016/j.molcel.2015.05.024

Figure Lengend Snippet: Three Types of IESs (A) Classification of IESs according to the expression of Early- and Late-scnRNAs. (B) Localization of different types of IESs, transposons (TEs), and coding sequences (CDSs) in the MIC genome are shown in a histogram with 50-kb bins. (C and D) Mean lengths (C) and GC contents (D) of IESs in different IES classes. (E) Distributions of TE-related sequences among MIC genome components (left) and IES types (right). All possible 25-mers from the MIC genome sequences were classified as sequences that were complementary to only IESs (red), only MAC-destined sequences (MDSs, blue), or both (yellow). All possible 25-mers from the total IESs were classified as sequences that were complementary to only Type-A IESs (magenta), only Type-B IESs (sky blue), or both (purple). The fraction of TE-derived 25-nt sequences complementary to these DNA classes was calculated. (F–I) Analysis of DNA elimination efficiency. The retention indexes (RIs) of individual IESs in the purified new MACs of the indicated strains at 36 hpm were plotted. IESs are ordered according to their LEIs (on the x axis). The red lines indicate RI = 1 (no DNA elimination).

Article Snippet: The draft MIC genome sequence (version 2) was obtained from the Tetrahymena Comparative Sequencing Project (Broad Institute of Harvard and MIT).

Techniques: Expressing, Derivative Assay, Purification

Journal: Molecular Cell

Article Title: Small-RNA-Mediated Genome-wide trans -Recognition Network in Tetrahymena DNA Elimination

doi: 10.1016/j.molcel.2015.05.024

Figure Lengend Snippet: trans Recognition of IESs (A–C) Top two panels: normalized numbers (RPM) of sequenced 26- to 32-nt RNAs from wild-type cells at 3 hpm mapping to the three representative Type-B IES loci (10 kb) are shown as histograms with 50-nt bins. For the top histograms, only the numbers of sequences uniquely mapping to the MIC genome are shown (unique mappers). The middle histograms show the numbers of sequence reads mapping to each position within the loci divided by the total numbers of sites in the entire MIC genome to which the sequence reads map (weighted). Arrows indicate regions to which Early-scnRNAs map. Sky-blue boxes represent the IESs. Bottom panels (repeats): all possible 25-mers from the entire MIC genome sequence (gray), from Type-A IESs (magenta), or from Type-B IESs (sky blue) were mapped to the three Type-B IES regions, and their frequencies of occurrence are shown as histograms with 50-nt bins. (D) All possible 25-mers were extracted from the indicated IESs, and their frequencies of occurrence (hits per kilobase [HPK]) on the MIC genome are shown as histograms with 50-kb bins. The locations of the IESs are marked with red dots. The density of the Type-A IESs is shown at the top. (E–G) Three representative Type-A IES (magenta boxes) loci were analyzed as in (A)–(C). See also Figure S2 .

Article Snippet: The draft MIC genome sequence (version 2) was obtained from the Tetrahymena Comparative Sequencing Project (Broad Institute of Harvard and MIT).

Techniques: Sequencing