PMC

(a) Cu0, annealed ribbon, (b) Cu0.3, annealed ribbon, (c) Cu0.7, annealed ribbon, (d) Cu0, annealed rod, (e) Cu0.3, annealed rod, (f) Cu0.7, annealed rod.

Back-scattering SEM images of the as-cast Mo_0.3, Mo_0.4, and Mo_0.5 high entropy alloys and the alloys annealed at different temperatures for 10 h. DR is for dendrite FCC, WP1 is for white (Cr,Mo)-rich precipitate with relative low Mo content, WP2 is the whit (Cr,Mo)-rich precipitate with relative high Mo content, and BP is for black (Al,Ni)-rich precipitate. (a) as-cast Mo_0.3 alloy; (b) as-cast Mo_0.4 alloy; (c) as-cast Mo_0.5 alloy; (d) Mo_0.3 alloy annealed at 600 °C; (e) Mo_0.4 alloy annealed at 600 °C; (f) Mo_0.5 alloy annealed at 600 °C; (g) Mo_0.3 alloy annealed at 800 °C; (h) Mo_0.4 alloy annealed at 800 °C; (i) Mo_0.5 alloy annealed at 800 °C; (j) Mo_0.3 alloy annealed at 1000 °C; (k) Mo_0.4 alloy annealed at 1000 °C; (l) Mo_0.5 alloy annealed at 1000 °C; (m) Mo_0.3 alloy annealed at 1100 °C; (n) Mo_0.4 alloy annealed at 1100 °C; (o) Mo_0.5 alloy annealed at 1100 °C; (p) Mo_0.3 alloy annealed at 1200 °C; (q) Mo_0.4 alloy annealed at 1200 °C; (r) Mo_0.5 alloy annealed at 1200 °C.

Back-scattering scanning electron microscope (SEM) images of the as-cast Mo₀, Mo_0.1 and Mo_0.2 high entropy alloys and the alloys annealed at different temperatures for 10 h. DR is for FCC dendrite phase, ID is for (Al,Ni)-rich interdendrite phase, NLP is for needle-like (Al,Ni)-rich recipitate, WP is for white (Cr,Mo)-rich precipitate, and BP is for black (Al,Ni)-rich precipitate. (a) as-cast Mo₀ alloy; (b) as-cast Mo_0.1 alloy; (c) as-cast Mo_0.2 alloy; (d) Mo₀ alloy annealed at 600 °C; (e) Mo_0.1 alloy annealed at 600 °C; (f) Mo_0.2 alloy annealed at 600 °C; (g) Mo₀ alloy annealed at 800 °C; (h) Mo_0.1 alloy annealed at 800 °C; (i) Mo_0.2 alloy annealed at 800 °C; (j) Mo₀ alloy annealed at 1000 °C; (k) Mo_0.1 alloy annealed at 1000 °C; (l) Mo_0.2 alloy annealed at 1000 °C; (m) Mo₀ alloy annealed at 1100 °C; (n) Mo_0.1 alloy annealed at 1100 °C; (o) Mo_0.2 alloy annealed at 1100 °C; (p) Mo₀ alloy annealed at 1200 °C; (q) Mo_0.1 alloy annealed at 1200 °C; (r) Mo_0.2 alloy annealed at 1200 °C.

Unwinding of the ssDNA/ssRNA duplex by p33 in an in vitro strand separation assay. (A) Schematic representation of the known domains in the tombusvirus replication proteins p33 and p92^pol. Note that the N-terminal segment in p92^pol contains the same sequence as in p33 due to the overlapping expression strategy of TBSV genome, while the C-terminal region of p92^pol carries the RdRp domain. TMD, trans-membrane domains; P, phosphorylation sites; RPR, arginine-proline-rich RNA-binding domain; S1 and S2 are subdomains of the p33:p33/p92 interaction domain. The closely related TCV p88 is similar to p92^pol and carries the p33 similarity domain as well as the RdRp domain. P88C is an N-terminally truncated p88 derivative, which has strong RdRp activity in vitro. (B) Scheme of the strand separation assay showing the hybridized ssDNA/ssRNA duplex. The 5′ end labeled, 21 base ssDNA oligo was annealed to the unlabeled DI-72(−)RNA prior to the addition of purified recombinant p33. (C) In vitro strand separation assay with p33 using an ssDNA oligo/ssRNA template duplex. Lane 1: 5′ end labeled-ssDNA oligo; Lanes 2 and 8: annealed ssDNA/ssRNA, no p33; Lane 3: annealed ssDNA/ssRNA template plus 4 pmol purified recombinant TBSV MBP-p33; Lane 4: annealed ssDNA/ssRNA plus 4 pmol purified recombinant TCV MBP-p88C; Lanes 5–7: annealed ssDNA/ssRNA plus 0.4, 1 and 4 pmol purified recombinant MBP-Ded1p, respectively. The assay was performed in the absence of ATP. The ³²P-labeled free ssDNA and ssDNA/ssRNA duplex were separated on non-denaturing 5% acrylamide gels. Quantification of the ssDNA/ssRNA duplex was done with ImageQuant. Bottom image shows an SDS-PAGE analysis of purified recombinant proteins used in these assays. Each experiment was repeated at least three times. (D) In vitro strand separation assay with recombinant proteins using an ssDNA oligo/ssRNA template duplex in the absence of ATP. Lane 1: 5′ end labeled-ssDNA oligo; Lanes 2–3: annealed ssDNA/ssRNA, plus 1 and 4 pmol of p88; Lanes 4–6: annealed ssDNA/ssRNA template plus 0.4, 1 and 4 pmol purified recombinant TBSV MBP-p33; Lanes 7–8: annealed ssDNA/ssRNA plus 1 and 4 pmol purified recombinant TCV MBP-p88C; Lanes 9–11: annealed ssDNA/ssRNA plus 0.4, 1 and 4 pmol purified recombinant MBP-Ded1p, respectively; Lane 12: annealed ssDNA/ssRNA, no p33. The percentage of unwound ssDNA/ssRNA duplex is shown. The lower image shows a shorter exposure for the ssDNA/ssRNA duplex. (E) In vitro strand separation assay with recombinant proteins using an ssDNA oligo/ssRNA template duplex in the presence of ATP. Lane 1: 5′ end labeled-ssDNA oligo; Lane 2: annealed ssDNA/ssRNA, no p33; Lanes 3–4: annealed ssDNA/ssRNA, plus 1 and 4 pmol of p88; Lanes 5–6: annealed ssDNA/ssRNA template plus 1 and 4 pmol purified recombinant TBSV MBP-p33; Lane 7: annealed ssDNA/ssRNA, no p33; Lanes 8–9: annealed ssDNA/ssRNA plus 1 and 4 pmol purified recombinant TCV MBP-p88C; lanes 10–11: annealed ssDNA/ssRNA plus 1 and 4 pmol purified recombinant MBP-Ded1p, respectively; Lane 12: annealed ssDNA/ssRNA, no p33. The percentage of unwound ssDNA/ssRNA duplex is shown. See additional details in panel C.