Journal: bioRxiv

Article Title: Bioinformatic and fine-scale chromosomal mapping uncover the essence and evolution of eliminated chromosomes in the Japanese hagfish, Eptatretus burgeri , through repetitive DNA family analysis

doi: 10.1101/2023.05.28.542657

Figure Lengend Snippet: Identification of four novel eliminated repetitive DNA families. (a) Amplification of four novel repetitive DNA families. PCR products using germline DNA (lane G) and somatic DNA (lane S) of E. burgeri as templates were separated on 3.0% agarose gel (family 0) or 2.0% agarose gel (families 38, 10, and 5). The left lane in the image contains the DNA molecular size marker. The corresponding sizes of the monomers and multimers of each family are indicated by arrows. ( b ) The consensus sequences of the four repetitive DNA families. Each consensus nucleotide sequence was derived from the inserts of germline DNA clones and somatic DNA clones of family 0 (F0G and F0S), family 38 (F38G and F38S), family 10 (F10G and F10S), and family 5 (F5G and F5S). Germline sequences (top) and somatic sequences (bottom) are aligned. Nucleotides identical to those in the consensus sequence at the top are represented by dots (.), while base substitutions are indicated by the respective bases. Primer regions are marked with italic letters and direct repeats by arrows. The obtained sequence data were deposited in GenBank ( LC731298 – LC731305 ). ( c ) Chromosomal mapping of the four DNA families in E. burgeri . The digoxigenin-labeled EEEb1 probes (green) and biotin-labeled families 0, 38, 10, and 5 (red) were hybridized on the metaphase chromosomes in the spermatocytes. The chromosomal localization of family 5 in somatic cells is also shown at the bottom panel. Chromosomes were counterstained with Hoechst 33342 (blue). The magnified images of E-chromosomes with each signal are shown in insets. The scale bar was set to 5 μm.

Article Snippet: After pretreatment with 1/2- diluted Blocking One Histo (Nacalai Tesque) in double-distilled water for 10 min at room temperature, the slides were incubated with 4 µg/mL of anti-digoxigenin Fab fragments conjugated with FITC (Merck) and 1/2,000-diluted streptavidin conjugated with alexa405 (Vector Laboratories) or 1/2,000- diluted streptavidin conjugated with cyanine-3 (Vector Laboratories) in TNT for 1 hour at room temperature in a dark humid chamber.

Techniques: Amplification, Agarose Gel Electrophoresis, Marker, Sequencing, Derivative Assay, Clone Assay, Labeling

Journal: bioRxiv

Article Title: Bioinformatic and fine-scale chromosomal mapping uncover the essence and evolution of eliminated chromosomes in the Japanese hagfish, Eptatretus burgeri , through repetitive DNA family analysis

doi: 10.1101/2023.05.28.542657

Figure Lengend Snippet: Chromosomal mapping of 10 eliminated DNA families in E. burgeri . Metaphase chromosomes in spermatocyte were hybridized using cyanine-5-labeled EEEb1 probes (gray) along with biotin-labeled EEEb2 ( a and e ), EEEb4 ( b ), EEEb7 ( c ), and EEEb8 ( d ) (blue) as well as digoxigenin-labeled EEEb3 ( a ), EEEb5 ( b ), EEEb6 ( c ), EEEb9 ( d ), and EEEb10 ( e ) (green) (top). Fluorescence intensity histograms, derived from XY-sections, were displayed below representative individual E-chromosomes (bottom). Chromosomes were counterstained with propidium iodide (red). The scale bar was set to 5 μm.

Article Snippet: After pretreatment with 1/2- diluted Blocking One Histo (Nacalai Tesque) in double-distilled water for 10 min at room temperature, the slides were incubated with 4 µg/mL of anti-digoxigenin Fab fragments conjugated with FITC (Merck) and 1/2,000-diluted streptavidin conjugated with alexa405 (Vector Laboratories) or 1/2,000- diluted streptavidin conjugated with cyanine-3 (Vector Laboratories) in TNT for 1 hour at room temperature in a dark humid chamber.

Techniques: Labeling, Fluorescence, Derivative Assay

Journal: bioRxiv

Article Title: Bioinformatic and fine-scale chromosomal mapping uncover the essence and evolution of eliminated chromosomes in the Japanese hagfish, Eptatretus burgeri , through repetitive DNA family analysis

doi: 10.1101/2023.05.28.542657

Figure Lengend Snippet: Chromosomal mapping of similarly distributed DNA families in E. burgeri . Metaphase chromosomes in spermatocytes were hybridized using cyanine-5-labeled EEEb1 probes (gray) in combination with biotin-labeled EEEb2 (blue) and digoxigenin-labeled EEEb4 (green) ( a ), digoxigenin-labeled EEEb4 (green), and biotin-labeled EEEb7 (blue) ( b ), biotin-labeled EEEb7 (blue) and digoxigenin-labeled EEEb2 (green) ( c ), biotin-labeled EEEb6 (red) and digoxigenin- labeled EEEb9 (green) ( d ), biotin-labeled EEEb3 (red) and digoxigenin-labeled EEEb5 (green) ( e ), biotin-labeled EEEb5 (red) and digoxigenin-labeled EEEb10 (green) ( f ), or digoxigenin-labeled EEEb10 (green) and biotin-labeled EEEb3 (red) ( g ). Chromosomes were counterstained with propidium iodide ( a – c ) (red) or Hoechst33342 ( d – g ) (blue). The other notations correspond to those in .

Article Snippet: After pretreatment with 1/2- diluted Blocking One Histo (Nacalai Tesque) in double-distilled water for 10 min at room temperature, the slides were incubated with 4 µg/mL of anti-digoxigenin Fab fragments conjugated with FITC (Merck) and 1/2,000-diluted streptavidin conjugated with alexa405 (Vector Laboratories) or 1/2,000- diluted streptavidin conjugated with cyanine-3 (Vector Laboratories) in TNT for 1 hour at room temperature in a dark humid chamber.

Techniques: Labeling

Journal: bioRxiv

Article Title: Bioinformatic and fine-scale chromosomal mapping uncover the essence and evolution of eliminated chromosomes in the Japanese hagfish, Eptatretus burgeri , through repetitive DNA family analysis

doi: 10.1101/2023.05.28.542657

Figure Lengend Snippet: Fiber-FISH analysis of 10 eliminated DNA families in E. burgeri . Extended chromatin fibers derived from testicular cells were subjected to hybridization using cyanine-5-labeled EEEb1 probes (gray) with biotin-labeled EEEb2 ( a and e ), EEEb4 ( b ), EEEb7 ( c ), and EEEb8 ( d ) (blue). Additionally, digoxigenin-labeled EEEb3 ( a ), EEEb5 ( b ), EEEb6 ( c ), EEEb9 ( d ), and EEEb10 ( e ) were also utilized for the hybridization and were detected (green). Schematic diagrams corresponding to each signal image appear below. Scale bar=20 µm.

Article Snippet: After pretreatment with 1/2- diluted Blocking One Histo (Nacalai Tesque) in double-distilled water for 10 min at room temperature, the slides were incubated with 4 µg/mL of anti-digoxigenin Fab fragments conjugated with FITC (Merck) and 1/2,000-diluted streptavidin conjugated with alexa405 (Vector Laboratories) or 1/2,000- diluted streptavidin conjugated with cyanine-3 (Vector Laboratories) in TNT for 1 hour at room temperature in a dark humid chamber.

Techniques: Derivative Assay, Hybridization, Labeling

Journal: bioRxiv

Article Title: Bioinformatic and fine-scale chromosomal mapping uncover the essence and evolution of eliminated chromosomes in the Japanese hagfish, Eptatretus burgeri , through repetitive DNA family analysis

doi: 10.1101/2023.05.28.542657

Figure Lengend Snippet: Fiber-FISH analysis of similarly distributed DNA families in E. burgeri . Extended chromatin fibers from testicular cells were hybridized using cyanine-5-labeled EEEb1 probes (gray) with biotin-labeled EEEb2 (red) and digoxigenin-labeled EEEb4 (green) ( a ), digoxigenin-labeled EEEb4 (green) and biotin-labeled EEEb7 (red) ( b ), digoxigenin-labeled EEEb2 (green) and biotin-labeled EEEb7 (red) ( c ), biotin-labeled EEEb6 (red) and digoxigenin-labeled EEEb9 (green) ( d ), biotin-labeled EEEb3 (red) and digoxigenin-labeled EEEb5 (green) ( e ), biotin- labeled EEEb5 (red) and digoxigenin-labeled EEEb10 (green) ( e ), or biotin-labeled EEEb3 (red) and digoxigenin-labeled EEEb10 (green) ( g ). The other notations correspond to those in .

Article Snippet: After pretreatment with 1/2- diluted Blocking One Histo (Nacalai Tesque) in double-distilled water for 10 min at room temperature, the slides were incubated with 4 µg/mL of anti-digoxigenin Fab fragments conjugated with FITC (Merck) and 1/2,000-diluted streptavidin conjugated with alexa405 (Vector Laboratories) or 1/2,000- diluted streptavidin conjugated with cyanine-3 (Vector Laboratories) in TNT for 1 hour at room temperature in a dark humid chamber.

Techniques: Labeling