1. Origin of the Reflectin Gene and Hierarchical Assembly of Its Protein
Zhe Guan, Tiantian Cai, Zhongmin Liu, Yunfeng Dou, Xuesong Hu, Peng Zhang, Xin Sun, Hongwei Li, Yao Kuang, Qiran Zhai, Hao Ruan, Xuanxuan Li, Zeyang Li, Qihui Zhu, Jingeng Mai, Qining Wang, Luhua Lai, Jianguo Ji, Haiguang Liu, Bin Xia, Taijiao Jiang, Shu- Jin Luo, Hong-Wei Wang, Can Xie 
Current Biology 
Volume 27, Issue 18, Pages e6 (September 2017)
DOI: /j.cub
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2. Current Biology 2017 27, 2833-2842.e6DOI: (10.1016/j.cub.2017.07.061)
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3. Figure 1
Reflectin Originated from a Transposon in the Symbiotic Bacterium V. fischeri
(A) Schematic diagram of the architecture of reflectin (SoRef2). Protopeptide (orange boxes), core sequences (Core 1–Core 4), and domains (D1–D4) are shown.
(B) The amino acid percentage composition of SoRef2. Four aromatic residues (Y, F, W, and H) are shown with R group.
(C) Schematic diagram showing the genetic traces of transposon TnlD in the octopus (O. bimaculoides) genome. Top: five transposons (gray boxes for TnlA, TnlB, TnlC, and TnlE; orange box for TnlD) on plasmid pKB1A97-67 from V. fischeri. The colored boxes in TnlD diagram indicate gene fragments identified in the O. bimaculoides genome, representing the genetic traces of transposition. The gene fragment coding the protopeptide is shown as an orange box and labeled as a star in TnlD, and amino acid sequence is shown below. Bottom: all gene fragments identified in the O. bimaculoides genome (shown as scaffolds) with sequence similarity to TnlD from symbiotic bacteria. The color schemes remain the same as in the TnlD diagram in the upper panel.
(D–I) Experimental validation of transposase activity of TnlD. HeLa cells were transfected EGFP fused with N-terminal TnlD with (D and E) or without inverted repeats (IRs) (F and G) and mCherry with IRs (H and I). EGFP fluorescence indicates the excision of N-terminal sequences via transposition. (D), (F), and (H) are bright field images; (E), (G), and (I) are fluorescent images. The scale bars represent 100 μm.
(J and K) Schematic diagram (J) and RT-PCR (K) showing the excision of TnlD from EGFP expression cassette in transfected HeLa cells (D and E). Lanes in (K): 1, marker; 2, negative control, untransfected cells; 3, positive control, TnlD-EGFP-pEF1-puro plasmid as PCR template; 4, cells transfected with TnlD-EGFP-pEF1-puro.
(L) Schematic diagram of proposed reflectin gene origin. The transposon on plasmid pKB1A97-67 (orange circle) may have translocated from V. fischeri to the cephalopod genome through horizontal gene transfer. A 24-bp fragment (orange box) coding YMDMSGYQ (here named protopeptide) was retained and underwent duplications and divergence leading to the new gene reflectin gene.
See also Figure S1 and Tables S1 and S2.
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4. Figure 2 Building Bricks Self-Assembled by Reflectin
(A) Size-exclusion chromatography purification of SoRef2. Panel insert shows the SDS-PAGE of protein fractions in peak 1 and 2.
(B) Representative raw image of SoRef2 from peak 1 in (A). Cyan boxes represent globular single particles (bricks), and yellow boxes represent elongated particles. The scale bar represents 100 nm.
(C–E) Negative-staining EM structure of “reflectin bricks” from 2D class average. Hexapetalous (group 1; C) and pentapetalous (group 2; D) flower-shaped and double-layer structures (group 3; E) of reflectin bricks are shown, respectively.
(F) Structural features of reflectin bricks summarized from groups 1–3 and proposed structural architectures illustrated as cartoons.
(G–J) EM structures of elongated forms (G–I) and branched forms (J) of reflectin particles from 2D class average (upper panels of G and H) and raw images (lower panels of G and H and images of I and J).
The scale bars represent 10 nm. See also Figure S2.
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5. Figure 3 Higher-Ordered Assembly Triggered by Small Molecules
(A–C) The reversible regulation of regular hexagonal structural formation by imidazole. Hexagonal structures assembled from reflectin bricks in the presence of imidazole (A), and removal of imidazole by dialysis led to the dissociation of hexagonal structure and reappearance of reflectin bricks (B). This process can be revsed by re-adding imidazole to the same protein preparation (C). The top right corner is the building brick at the same scale.
(D–F) Different morphologies of SoRef2 assembly when incubated with histamine (D), octopamine (E), and 5-hydroxytryptamine (F).
Isolated reflectin bricks are highlighted in cyan boxes in (A)–(F). The scale bars represent 100 nm. See also Figure S3 and Table S3.
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6. Figure 4 The Origin of Reflectin Assembly
(A) Schematic diagram of reflectin, MBP-D1, D1, and protopeptide.
(B) Size-exclusion chromatography purification of MBP (blue curve) and MBP-D1 (orange curve) in Superose 6 Increase 10/300 GL. Panel inset shows the SDS-PAGE of protein fractions in peaks 1–3.
(C and D) Representative raw image of MBP-D1 (C) and MBP (D).
(E and F) Representative raw image of D1 before (E) and after (F) imidazole is added.
(G) Protopeptide in Superdex 75 10/300 GL. Orange curve indicates 215 nm absorption, and blue curve indicates 280 nm absorption.
(H and I) Representative raw image of protopeptide before (H) and after (I) imidazole added.
The scale bars represent 100 nm. See also Figure S4.
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