Correction: Initial sequencing and analysis of the human genome

Abstract

565 transfection, cells were collected and processed for CAT or luciferase activity using standard techniques 14 . GST pull downs and immunoprecipitationsGST±Rb (wild type and mutants 15) and other GST fusion proteins were expressed and puri®ed from Escherichia coli XA90 (ref. 16). GST fusion proteins that were immobilized on glutathione-sepharose (Pharmacia), or H3-derived peptides 3 bound to Sulfolink beads (Pierce), were incubated with extract in IPH buffer 16 . Complexes were washed four times in IPH buffer before processing for methylase assays or western blotting. Antibodies against HA (12CA5, Roche), Gal4±DBD (DNA-binding domain; sc-510, Santa Cruz), SUV39H1 (M. Cleary), Rb (G3-245; XZ55, PharMingen) or HP1 (ref. 3) were used. For immunoprecipitations antibodies were incubated with HeLa nuclear extract (Cell Culture Center) or U2OS nuclear extract in IPH buffer at 4 8C (ref. 17). After 2 h a 50:50 mixture of protein A/G-sepharose beads (Pharmacia) was added. To avoid the possibility that DNA mediates the interaction between SUV39H1 and Rb, the immunoprecipitations were probed for the presence of histones with negative results. Histone methylase assays and protein sequencingPrecipitations from pull downs or immunoprecipitations were incubated with 20 mg histones (Sigma) and 1 ml [3H-Me]-S-adenosyl methionine (NEN, 80 Ci mmol -1 ) in PBS at 30 8C for 1 h. Assays were analysed by SDS±PAGE followed by western blotting and autoradiography or were spotted onto P-81 cationic exchange paper (Whatman), washed in carbonate buffer and quanti®ed by scintillation counting 3 . For amino-terminal sequencing, radiolabelled H3 was blotted to polyvinylidene¯uoride and sequenced by Edman degradation (Protein Sequencing Facility, University of Cambridge). We counted fractions for the presence of tritium. RNA puri®cation and RT-PCR analysisTotal RNA (0.5 mg) was isolated from W12 (wild type) and D3 (SUV39H1 and SUV39H2 double knockout; D.O. and T.J., unpublished observations) female mouse cells, and was used for quantitative RT-PCR, following the Qiagen One Step protocol, for 20, 25 and 30 PCR cycles. Antibody generationRabbits were immunized with a H3 N-terminal lysine-methylated peptide corresponding to amino acids 1±16. Immunoreactive serum was applied to a H3 Lys-9-methylated peptide column to af®nity purify speci®c antibodies, as the antiserum crossreacted with H3 methylated at Lys 4. Chromatin immunoprecipitationChromatin immunoprecipitations were performed using HeLa cells and MEF cells essentially as described 18,19 . Immunoprecipitates were analysed for the presence of cyclin E or Cdc25C promoter fragments by PCR using primers speci®c for single nucleosomes. PCR reactions were repeated exhaustively using varying cycle numbers and different amounts of templates to ensure that results were within the linear range of the PCR. Cell 92, 463±473 (1998). 8. Firestein, R., Cui, X., Huie, P. & Cleary, M. L. Set domain-dependent regulation of transcriptional silencing and growth control by SUV39H1, a mammalian ortholog of Dro...

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