SUPPLEMENTARY FIGURE AND TABLE LEGENDS SUPPLEMENTARY FIGURE S1 GENERATION

PRACTICE NOTE SOURCING SUPPLEMENTARY EMERGENCY RESPONSE RESOURCES
SUPPLEMENTARY MATERIAL (ESI) FOR CHEMICAL COMMUNICATIONS THIS
(SUPPLEMENTARY ORDER PAPER) 19 DÁIL ÉIREANN DÉ MÁIRT 2

1 SUPPLEMENTARY MATERIAL FOR PERKIN TRANSACTIONS 1 THIS JOURNAL
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Supplementary Figure and Table Legends


Supplementary Figure and Table Legends

Supplementary Figure S1. Generation of recombinant yeast nucleosomes. Purified 601 DNA was mixed with reconstituted yeast octamers in a 1:1.5 ratio and dialyzed in a stepwise fashion. Retarded migration rate of DNA in nucleosome compared to free DNA demonstrates the presence of reconstituted nucleosomes. Coomassie staining of nucleosomes show equimolar amounts of H2A, H2B, H3 and H4.

Supplementary Figure S2. Enhanced RNAi targeting dTRRAP (Nipped-A) leads the reduced expression of wingless target gene senseless. Using the UAS-GAL4 system, en-GAL4 was used to drive the co-expression of Dicer-2 and dTRRAP(Nipped-A) RNAi constructs in the posterior half of wing imaginal discs (A-A’’). Dicer-2 co-expression with UAS-GAL4 driving the RNAi hairpin constructs has been found to enhance the effectiveness of RNAi significantly in flies ADDIN EN.CITE ADDIN EN.CITE.DATA ﾧﾵ(Dietzl et al. 2007)ﾧ. This enhanced effectiveness of dTRRAP (Nipped-A) RNAi results in significant reduction of senseless expression. The domain of knockdown is marked by GFP. (A’) Arrows point to the location where senseless is reduced. (A’’) Merged image of GFP-expressing domain and Senseless antibody labeling.

Supplementary Figure S3. Effect of knockdown of members of dDotCom on Wingless expression in wing discs. Using the UAS-GAL4 system, en-GAL4 was used to drive the RNAi constructs in the posterior half of wing imaginal discs as in figure 5, and stained for Wingless protein. Knockdown of dDot1 leads to small reduction in Wingless expression in the DV boundary, but reduction in other members of the dDotCom have no effect on Wingless expression. Arrow points to the DV boundary where wingless is expressed.

Supplementary Figure S4. dDot1 (gpp), dAF10 (Alhambra), ENL/AF9-related (ear), dSkp1 (skpB) and dTRRAP (Nipped-A) regulate the expression of a number of Wg target genes in flies. (A) dDot1 (gpp1A) is a non-lethal allele (generated in an ethyl methanesulfonate screen) ADDIN EN.CITE ADDIN EN.CITE.DATA ﾧﾵ(Shanower et al. 2005)ﾧ. qRT PCR was performed on RNA extracted from third instar homozygous larvae. There is no alteration in the expression level of dDot1 (gpp) mRNA, and the expression of all Wingless target genes tested were affected. (B) dDot1 (gpp-v16001) RNAi affects expression of all Wingless target genes tested. qRT PCR was performed on total RNA extracted from dissected 3rd instar larval wing discs from a cross of A9-Gal4 and UAS-ddot1 (gpp-v16001) RNAi. A9-Gal4 is a wing imaginal disc specific driver. (C) dAF10 (Alhambra-1070R-3) RNAi, performed as in (B), affects the expression of CG5895-, CG6234-, Notum-, and Homothorax-, but not of dFz3-mRNA.(D) ENL/AF9-related (4913R-1) RNAi leads to a reduction of CG5895- and CG6234-mRNA. Total RNA from whole 3rd instar ubiquitously expressing dsRNA with the Actin 5C driver. (E) dSkp1 (skpB- 8881R-4)) RNAi, performed as in (D), leads to reduction of CG5895-, CG6234-, and Notum-, but not of Homothorax-mRNA. (F) dTRRAP (Nipped-A-40789) RNAi, performed as in (D), leads to a reduced expression of CG6234 and Homothorax.

Supplementary Table S1. Ubiquitous knockdown of components of the dDot1/GPP complex using Act5C-Gal4. Ubiquitous expression of RNAi constructs targeting dDot1 (gpp), dAF10 (Alh), EAR and dTRRAP (Nipped-A) results in lethality in early pupal stages. Ubiquitous knockdown of ddot1 (gpp) using ddot1(gpp-v16001) line lead to lethality at stage earlier than third instar larvae, suggesting that it has stronger effect on dDot1 (Gpp) function, though we can not rule out off targets effects of the RNAi. dSkp1 (skpB) knockdown only showed a weak phenotype, with about 10% of the flies showing wing notching phenotype (data not shown). Notched-wing phenotype in the adult has been seen when dominant-negative form of Lgs (Lgs17E) is expressed in wing discs, and has been attributed to compromised transcriptional output of the Armadillo activation complex ADDIN EN.CITE ADDIN EN.CITE.DATA ﾧﾵ(Mosimann et al. 2006)ﾧ. It is possible that down regulation of dSkp1 (skpB) also affects the Armadillo activation complex. Also, SkpA, which has extensive homologuey to SkpB at the protein level, might rescue functions of SkpB.






10 SUPPLEMENTARY DATA FOR “THE VACUUM UV PHOTOABSORPTION SPECTROSCOPY
11 Supplementary Record Form Advanced Livestock Record Rabbits 20to
12 SUPPLEMENTARY MATERIALS THIS FILE CONTAINS THREE SUPPLEMENTARY TABLES


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