A Molecular Basis for Natural Selection at the timeless Locus in Drosophila melanogaster

doi:10.1126/science.1138426

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Biology

Science 29 June 2007:
Vol. 316. no. 5833, pp. 1898 - 1900
DOI: 10.1126/science.1138426

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A Molecular Basis for Natural Selection at the timeless Locus in Drosophila melanogaster

Federica Sandrelli¹^*, Eran Tauber²^*, Mirko Pegoraro¹^*, Gabriella Mazzotta¹, Paola Cisotto¹, Johannes Landskron³, Ralf Stanewsky³^,4, Alberto Piccin¹, Ezio Rosato², Mauro Zordan¹, Rodolfo Costa¹ and Charalambos P. Kyriacou²

¹ Department of Biology, University of Padova, 35131 Padova, Italy.
² Department of Genetics, University of Leicester, Leicester LE1 7RH, UK.
³ Institut für Zoologie, Lehrstuhl für Entwicklungsbiologie, University of Regensburg, Regensburg 93040, Germany.
⁴ School of Biological and Chemical Sciences, Queen Mary College, University of London, London E1 4NS, UK.

Fig. 1. Phase response to light pulses of tim genotypes. Mean phase responses [hours (h) ±SEM] to 20-min light pulses delivered at ZT15, giving phase delays (–, below), and ZT21, giving phase advances (+, above), are shown. (A) Natural lines: Bitetto (Bit), southern Italy; Houten (Hu), Netherlands; and Moscow (Mos), Russia at 18° and 24°C. Black bars, s-tim; white bars, ls-tim; h, hours. (B) Transformants at 18°, 24°, and 28°C. Black bars, P[S-tim]; gray bars, P[L-tim]; white bars, P[LS-tim]. [View Larger Version of this Image (18K GIF file)]

Fig. 2. TIM Western blots reveal different TIM isoforms. Fly heads harvested at ZT1 or ZT 13 are shown. The upper row shows natural lines: columns 1 and 5, GAB (ls-tim); 2 and 6, ATDD (s-tim); 3 and 7, Nov75 (ls-tim); and 4 and 8, B16 (s-tim). The broader "doublet" TIM band in ls-tim genotypes is shown with arrows. The upper middle row shows CS, Canton-S (ls-tim); y w(s), yw (s-tim); T27L and T29L (P[L-TIM]); T28S and T30S (P[S-TIM]); and (P[LS-TIM]) from fly heads harvested at ZT1. There are doublets in CS and P[LS-tim] transformants, with single bands in (P[S-TIM]) and (P[L-TIM]) transformants. The lower middle row shows larger-scale figures of yw (s-tim), Canton-S (ls-tim) and their heterozygote s/ls [(s/ls-tim)] at ZT1. The bottom row shows the results of a phosphatase (P) treatment applied to ls-tim and s-tim samples at ZT1 and ZT13. Blots were performed with anti-TIM and anti-PER. ls-tim genotypes maintain the higher–molecular weight isoform after phosphatase treatment. [View Larger Version of this Image (54K GIF file)]

Fig. 3. Circadian TIM profiles in natural lines and transformants. (A) Natural variants. Left panels show mean ± SEM TIM/TUB ratios from Western blots of the Moscow line (23°C in LD12:12, n = 6 blots for each variant); right panels show examples of corresponding Western blots. (B) Transformants. Left panels show mean ± SEM TIM/TUB ratios for Western blots (right) of each of the P[S-tim] (n = 11) and P[L-tim] (n = 10) transformants. [View Larger Version of this Image (45K GIF file)]

Fig. 4. TIM interactions with CRY and PER in the yeast two-hybrid system. In light, L-TIM shows a diminished interaction with CRY in (A) plate assays (pJG4-5, empty vector control) and (B) liquid assays (mean ± SD), as compared with S-TIM [F_(1,16) = 141.4, P < 0.001] for at least nine cultures derived from at least eight independent clones are shown. (C and D) L-TIM and S-TIM show equally robust interactions with the PER_(233-685) fragment in light or darkness in both plate and liquid assays [F_{(1, 20)} = 0.04, NS]. [View Larger Version of this Image (25K GIF file)]