Function and synthesis of small nucleolar RNAs. cautiously isolated to avoid cytoplasmic contamination. Intranuclear parts were isolated from nuclei by further dissection to separate nucleoli from your chromosomes and interchromatin. Protein Extraction and Western Blotting Nuclear and cytoplasmic components of tissue tradition cells were prepared essentially as explained by Wurtz (1996) . Nuclear draw out of HeLa cells was prepared as explained by Dignam (1983) . Cell draw out from was prepared as explained by Silve (1991) . Nuclear draw out from was prepared as explained by Petersen (1995) . Proteins were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride filters. HRP-labeled secondary antibodies were recognized by the enhanced chemiluminescence method (Amersham Biosciences Abdominal). Immunocytological Localization Cells.Cultured diploid cells were prepared and stained with antibodies essentially as explained previously (Baurn tissue culture cells. Isolated Polytene Chromosomes.Chromosomes were isolated from salivary glands and probed with antibodies essentially while described previously (Kiseleva cells. Cells expressing GFP-tagged proteins were fixed, mounted, and examined in the microscope. RNA-Protein Binding The coding part of the Ct-RBD-1 gene was cloned into the pET-15b manifestation vector (Novagen, Madison, WI) and indicated in (1996) . Then 2C3 fmol of RNA (in molecules) was heated at 60C in 20 mM Tris-HCl, pH 7.5, 200 mM KCl, 5 mM MgCl2 for 15 min, cooled to 20C, and incubated with different concentrations of purified protein in 60 l of binding buffer (25 mM Tris-HCl, pH 7.5, 200 mM KCl, 5 mM MgCl2, 20% glycerol, 50 g/ml tRNA, 10 g/ml bovine serum albumin) for 30 min at Treosulfan 20C. The reaction mixtures were filtered through damp nitrocellulose filters (0.45 m HA; Millipore, Bedford, MA), followed by three washes with 300 l of binding buffer. The RNA was essentially intact during the entire procedure as checked by electrophoresis in denaturing polyacrylamide gels. The percentage of certain RNA was determined by Cerenkov counting. The dissociation constant (fourth instar larvae and placed in a drop of hemolymph surrounded by paraffin oil. Anti-Ct-RBD-1 antibodies (12.5 g/l) or a control antibody (12.5 g/l) in PBS was injected into individual nuclei (AIS Micro Systems; Carl Zeiss). Approximately 10 Treosulfan cells/gland were injected with 0.01 nl of antibody solution per nucleus. Each injected gland was incubated in hemolymph comprising 3 M -[32P]ATP (400 Ci/mmol; Amersham Biosciences Abdominal) for 60 min at 18C. The gland was consequently incubated in hemolymph comprising 25 M unlabeled ATP for 60 min. The glands were then fixed in 70% ethanol for 30 min on snow and prepared for microdissection. The nucleoli from 10 injected cells as well as the nucleoli from 10 uninjected control cells were isolated from each gland. RNA was extracted by incubation in 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.5% SDS, 0.5 mg/ml proteinase K for 30 min at room temperature. After extraction with phenol:chloroform, the RNA was ethanol precipitated. The RNA was fractionated on 1% agarose gels, by using 20 mM Tris-HCl, pH 8, Treosulfan 20 mM NaCl, 2 mM EDTA, 0.2% SDS as operating buffer. The gel was treated with chilly 5% trichloroacetic acid, washed in water, dried, and exposed to x-ray film and to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) display for quantification analysis (Fujifilm FLA-3000, Image Gauge V3.45). In each experiment, RNA from injected cells was compared with RNA from noninjected cells from your same salivary gland. Injection of a control antibody did Rabbit polyclonal to AIBZIP not impact the proportions Treosulfan of the pre-rRNA species. Analysis of Polysomes, Ribosomes, and Ribosomal Subunits cells culture cells were washed in PBS.
