Feature Extraction Using Genetic Algorithms

Abstract

Nature © Macmillan Publishers Ltd 1998 8 letters to nature NATURE | VOL 391 | 5 FEBRUARY 1998 601 with 10% fetal calf serum. Transfections, CAT and luciferase assays were done using standard techniques 15. TSA (Wako BioProducts) was added at 330 nM 24 h before collecting cells. Recombinant GST fusion and histidine-tagged proteins. Recombinant proteins were expressed in and purified from E. coli XA90 as described 20. For competition experiments, GST-E7 proteins were cleaved from GST using thrombin. GST microcolumns, pull-down assays and in vitro translation. Micro-columns were prepared by placing a glass bead in a yellow Gilson tip and applying 30-50 l GST fusion protein prebound to glutathione beads. GST-E2F380-437 columns were loaded with recombinant Rb protein and washed 4 times with IPH buffer. Beads were then transferred to a fresh reaction tube for affinity purification of histone deacetylase activity. In vitro translation reactions and GST pull-down experiments have been described 15. HDAC1 was translated in vitro from pING14A-HDAC1. The sequences of the competitor peptides were LQPGTTDLYCYEQLNDSS (LXCXE) and YPYDVPDYA (HA), respectively. Plasmids. pING14A-HDAC1 was subcloned from pBJ5-HD1-F by PCR (ref. 4). pGEX-Rb, pGEX-E7 and pGEX-E2F expression vectors, pCMV-Rb379-928, pHKG-E2F380-437 and pCycE-luc have all been described 15-17 .