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Flies were reared in 12-hour cycles of light and darkness (LD 12:12) at 25°C with lights on at 9 a.m. and off at 9 p.m. Males 2 to 7 days old were placed in the locomotor activity tubes at either 18°C or 29°C and exposed to two additional LD cycles, after which data collection in constant darkness (DD) was initiated. Locomotor activity data was collected in 30-min "time windows" for 7 days. Autocorrelation and spectral analysis were carried out on each male's activity record (14, 15).
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2642658036
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note
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+ to further produce two transformant lines. The (Thr-Gly)1 construct was made by amplifying a 716-base pair (bp) fragment from the (Thr-Gly)20 clone and incorporating a deletion encoding 19 Thr-Gly pairs. This was done with 5′ primer (A), 5′-AACTATAACGAGAACCTGCT-3′ (4874 to 4893); with 3′ primer (B), 5′-ATTGCCGGTACCACCAGTGCCGGCAATGCT-3′ (5094 to 5113); with 5′ primer (C), 5′-GCACTGGTGGTACCGGCAATGGAACAAATTCCGG-3′ (5231 to 5250); and with 3′ primer (D), 5′-GCTACGCCTGTTCCGGATCC-3′ (5627 to 5646), using hybrid polymerase chain reaction (PCR) methods (25). Numbers in brackets denote the nucleotide positions corresponding to the per sequence described (26). Primer B and C create a Kpn I site (GGTACC) using the two underlined mismatch bases. Primer A ends 37 bp upstream of an Sst I site, and primer D incorporates a Bam HI site (underlined); these sites were used to substitute a 607-bp Thr-Gly deleted fragment into a 7-kb Xba fragment, which encodes the 3′ half of the per gene. This Xba fragment was then ligated to a 5′ Bam HI-Xba fragment in several cloning steps, thereby reconstituting the 13.2-kb per transcription unit within the pW8 transformation vector. The (Thr-Gly)17 construct was generated by amplifying a 364-bp Thr-Gly fragment using a natural (Thr-Gly) 17a variant as template and 5′ primer (A), in conjunction with 3′ primer 5′-CATTGCCGGTACCAGTGCCT-3′ (5199 to 5215 and 5233 to 5236), which carried two mismatch bases (underlined) forming a Kpn I site. The amplified product was digested with Sst I (cuts at position 4930) and Kpn I, and was used to replace the Sst I-Kpn I fragment in the (Tnr-Gly)1 construct.
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2642685253
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The mean free-running spectrally derived periods of the two Δ(Thr-Gly) transformant lines were 25.02 and 24.58 hours at 18°C, and 26.16 and 27.38 hours at 29°C, respectively; those for the three (Thr-Gly)1 lines were 24.76, 24.34, and 24.93 hours at 18°C, and 26.05, 26.25, and 26.58 hours at 29°C, respectively; for the two (Thr-Gly)17 lines, periods were 23.4 and 24.3 hours at 18°C, and 24.27 and 25.48 hours at 29°C, respectively; periods for the four (Thr-Gly)20 lines were 24.61, 24.07, 24.21, and 23.51 hours at 18°C, and 24.80, 24.18, 24.92, and 25.43 hours at 29°C, respectively. Periods derived from autocorrelation were almost identical.
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0001757026
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2642635510
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note
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Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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unpublished results
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L. A. Sawyer et al., unpublished results.
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Sawyer, L.A.1
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We thank the U.K. Biotechnology and Biological Sciences Research Council (BBSRC), the U.K. National Environment Research Council (NERC), and the Human Frontier Science Program for grants to C.P.K. and the European Commission for a grant to C.P.K and R.C. We acknowledge a NERC studentship to L.S., a BBSRC studentship to H.P., a Brazilian CNPq scholarship to A.A.P., and a Ministero Universitá Ricerca Scientifica Tecnologica-British Council award for Anglo-Italian cooperation to C.P.K. and R.C.
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