1?mM EDTA, 5% glycerol and 1?mM
dithiothreitol), at 4��C for 30?min. For competition assays, recombinant RpaB in binding buffer was preincubated with a 25- or 250-fold excess amount of unlabelled competitor DNA fragment mTOR inhibitor
at 4��C for 30?min. DNA�Cprotein complexes were resolved on a native 6% polyacrylamide gel, run at 100?V and 4��C for approximately 1?h and visualized on the Fuji Film FLA3000 gel imaging system (Ex 473?nm; Y520 filter). Strategies used for construction of nblS, rpaB and srrA derivative strains, and description of plasmids are provided in the Experimental Procedures, Table?S2 and Figs?S2�CS5 in Supporting information. Synechococcus elongatus cells were routinely grown until they reached mid-exponential phase (OD750???0.5) in LL conditions. For HL treatments the cultures were exposed to 1000??mol photons m?2?s?1 white light and at the indicated times, 50?ml of aliquots were removed for RNA extractions. The samples were rapidly chilled on ice, centrifuged and pellets stored at ?20��C. Total RNA was isolated using a hot phenol method and treated with DNase I (Turbo DNase, Ambion). After PCR verification that no contaminant DNA was present, 0.5??g of total RNA was retrotranscribed in a total volume of 30??l with RevertAid H Minus M-MuL V Reverse Transcriptase (Fermentas) using primers RnpB-R, RpaB7942-2R,
SrrA-OV-1R, NewhliA-R, NblA-R and Sip1-BTH-R. Five microlitres of sample of retrotranscription reactions were subjected to different amplification cycles to optimize reactions, using Biotools DNA Polymerase (Biotools B&M Laboratories), with the following primer pairs: RpaB-BYTH-1F/RpaB7942-2R,
SrrAD64A-F/SrrA-OV-1R, NewhliA-F/NewhliA-R, NblA-F/NblA-R, Sip1-BTH-F/Sip1-BTH-R and RnpB-F/RnpB-R. This work was supported by Ministerio de Ciencia e Innovaci��n (Grants BFU2009-07371, BIO2009-10872 and Prometeo/2009/051) and Ministerio de Educaci��n (PR2009-0378). Thanks to Sociedad de Relaciones Internacionales, Universidad de Alicante, for Felix Moronta's grant. ""Thirty-two Aedes aegypti populations collected throughout Thailand and five populations of Aedes albopictus from southern Thailand were subjected to standard WHO contact bioassays to assess susceptibility to three commonly used synthetic pyrethroids: permethrin, deltamethrin, and lambda-cyhalothrin. A wide degree of physiological response to permethrin was detected in Ae. aegypti, ranging from 56.5% survival (Lampang, northern Thailand) to only 4% (Kalasin in northeastern and Phuket in southern Thailand). All 32 populations of Ae. aegypti were found to have evidence of incipient resistance (62.5%) or levels of survival deemed resistant (37.5%) to permethrin. Four populations of Ae. albopictus were found with incipient resistance (97 �C 80% mortality) and one with resistance (< 80%) to permethrin. The majority of Ae. aegypti populations (68.