S1 Supplemental Data A Novel C-Terminal Domain of Drosophila PERIOD Inhibits dCLOCK:CYCLE-Mediated Transcription Dennis C. Chang and Steven M. Reppert Supplemental Experimental Procedures together, and the ligation product used to transform DH5 cells. The mutations were verified by diagnostic digest and sequencing. Plasmids per, tim, and dClk cDNAs under control of a Drosophila actin pro- S2 Cell Culture and Transfections S2 cells were maintained at 25°C in Schneider’s Drosophila medium moter (pAct) and a hsp70-luciferase reporter construct regulated by four per E boxes (perE-luc) were generously provided by Steve Kay (Gibco) supplemented with 9% heat-inactivated fetal bovine serum (FBS; Gibco). Transient transfection of plasmids into S2 cells was [8]. per was subcloned into the S2 cell expression vector, pAc5.1V5/ HisA (Invitrogen), by PCR from the pAct-per template with primers carried out by mixing the DNA with 5–10 l CellFECTIN reagent (Gibco), incubating for 15 min at room temperature, and applying containing appropriate restriction sites, followed by digestion and ligation. Deletion mutants of per were similarly generated by PCR the mixture to the cells in Drosophila serum-free medium (Gibco) supplemented with 2 mM L-glutamine. Four hours later, the cells and subcloned into pAc5.1V5/HisA. were fed with an equal volume of 9% FBS in Schneider’s medium and incubated for 48 hr at 25°C before use. Site-Directed Mutagenesis NLS mutants per 513–1224 M822-3 and per 513–1224 M835-7 were generated by designing complementary primers (both sense and Transcription Assays To test the ability of TIM and/or various PER constructs to inhibit antisense) containing the desired mutation (along with a diagnostic restriction site) and PCR-amplifying appropriate template (per 513– dCLK:CYC transcription, we used a modified version of the tran- scription assay of Darlington and coworkers [S1]. 10 ng, 100 ng, 1224 in pAc5.1V5/HisA). The PCR reactions were treated with DpnI to remove the template DNA and then used to transform chemically 200 ng, or 600 ng of per/tim was cotransfected with: 10 ng perE- luc, 30 ng gal pAc5.1V5/HisA (gal-V5), and 1 ng pAct-dClk. Cotran- competent E. coli cells (DH5). Plasmid minipreps (QIAGEN) pre- pared from these cells were digested using the diagnostic site and sfection of cyc was unnecessary because S2 cells endogenously express cyc [S1]. In each assay, two controls were included: (1) the sequenced to verify the mutagenesis. NLS mutants per 1–450 M73-7 and per 513–1224 M788-91 were reporters, perE-luc and gal-V5, in the absence of other constructs to establish baseline reporter activity, (2) the reporters and pAct- generated by designing primers (both sense and antisense) con- taining the mutation and a restriction site for subcloning. Fragments dClk to measure the relative activation of perE-luc. All V5-tagged proteins were expressed using the pAc5.1 promoter (Invitrogen). N-terminal of the mutation and C-terminal of the mutation were generated by PCR, subcloned, and sequenced. Then the two frag- The total amount (ng) of DNA in each transfection was normalized by adding empty vector (pAc5.1V5) to the control mixes. In each ments were digested at the introduced subcloning site (and with an appropriate enzyme for the other end of the fragment), ligated assay, the same transfection mix was applied to three replicate sets Table S1. Immunocytochemical Analysis of PER Constructs Both Nuclear and Nuclear Staining, N Cytoplasmic Staining, C Cytoplasmic Staining, B V5-Tagged PER Constructs (% Cells  SEM) (% Cells  SEM) (% Cells  SEM) PER 1–1224 5  2 69  6 26  5 PER 1–516 3  3 59  28 38  24 PER 1–450 5  4 12  2 83  6 PER 1–450 M73-7 2  2 27  7 71  6 PER 447–1224 6  4 38  4 57  5 PER 513–1224 91  2 0  0 9  2 PER 513–1224 M788-91 72  2 5  5 23  7 PER 586–984 90  2 0  0 10  2 PER 764–1224 88  5 2  2 10  3 PER 807–1224 80  4 1  1 19  3 PER 843–1224 0  0 48  13 52  13 PER 513–813 1  1 24  3 75  2 PER 513–840 71  10 0  0 29  10 PER 513–892 80  3 0  0 20  3 PER 513–1224 M822-3 0  0 95  2 5  2 PER 513–1224 M835-7 0  0 80  4 20  4 PER 1–516 + NLS 4  3 36  11 60  8 PER 1–450 + NLS 83  4 1  1 16  3 PER 513–1224 M822-3 + NLS 89  4 1  1 10  4 PER 513–1224 M835-7 + NLS 90  5 2  2 8  3 S2 cells seeded onto glass coverslips were transiently transfected with ca. 300 ng plasmid for the expression of the indicated C-terminal V5- tagged PER constructs. Numerical intervals in the leftmost column indicate which amino acids in the 1224 aa form of PER are present in the expressed protein. Two days after transfection, the cells were processed for immunocytochemistry using a monoclonal anti-V5 primary antibody and a Cy3-conjugated secondary antibody. The cells were also DAPI stained to visualize the nuclei. Slides were viewed under a fluorescence microscope (Olympus IX70) and 30 cells per slide were categorized as having primarily nuclear staining (N), cytoplasmic staining (C), or staining in both nucleus and cytoplasm (B). Cells were scored blind to which construct was transfected into them. The scores were converted into percentages and the mean and standard error of two to seven replicates are shown in the table.