To study this, corticosterone (CORT) levels were measured through the transition from light (inactive phase) to dark (active phase). One way to address this question is to delete a gene involved in regulating a central circadian gene such as Per2 in an animal model and to determine how this deletion may affect the HPA axis and behaviors that are altered when the HPA axis is dysregulated. Therefore, it is important to understand how disruption of the circadian rhythm alters the HPA axis. Dysregulation of the HPA axis is associated with neuropsychiatric disorders. Circadian control of the hypothalamus-pituitary-adrenal (HPA) axis is critical for regulation of hormones involved in the stress response. The gels were fixed and dried and the bands visualized using PhosphorImager screens scanned with Scanner Control SI software (Molecular Dynamics, Sunnyvale, CA).The Period Circadian Regulator 2 (Per2) gene is important for the modulation of circadian rhythms that influence biological processes. All products were analyzed by electrophoresis in 8% SDS–polyacrylamide gels (SDS-PAGE) with an acrylamide:bis-acrylamide ratio of 120:1 to enhance mobility shifts. At the end of the experiment, 20-μl aliquots were digested with 35 units of calf intestinal phosphatase in buffer (50 mM Tris HCl, pH 7.9, 10 mM MgCl 2, 0.1M NaCl, 1 mM DTT) for 30 min where indicated. Twenty-microliter aliquots were removed at selected time points and boiled with SDS gel-loading buffer (0.1% bromophenol blue, 50 mM Tris HCl, pH 6.8, 0.1 M DTT, 2% SDS, 10% glycerol) to stop the reaction. The labeled products were incubated with CKIε in buffer containing phosphatase inhibitors. Transcription and translation of h Per2 and m Per2 inserts were performed in vitro in the presence of 35S-methionine with the TnT SP6 Coupled Reticulocyte Lysate System (Promega) over a period of 90 min at 30☌. All constructs were confirmed by sequencing. A series of 3′ deletion mutations of m Per2 were constructed (encoding amino acids 1 to 554, 1 to 763, 1 to 810, and 1 to 904) for use in mapping the binding site for CKI ε, as previously described for m Per1 (25). Expression from an SP6 promoter generates 6-myc-tagged peptides. Eco RI–Xba I fragments encoding amino acids 474 to 815 of hPER2 (and the corresponding amino acids 472 to 804 of mPER2) were PCR-amplified with primers containing Eco RI and Xba I sites, gel-purified with the GENECLEAN kit (BIO 101, Vista, CA) and were directionally cloned into the Eco RI–Xba I sites of the pCS2 + MT vector. Mutagenesis was carried out with the QuikChange Site-directed Mutagenesis Kit (Stratagene) using the protocol outlined therein. Site-directed mutagenesis of the serine residue at position 662 of hPER2 and the homologous serine (659) of mPER2 were performed to substitute a glycine residue. Slides were allowed to dry in the dark at room temperature and were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (Vector labs, Burlingame, CA) for chromosome visualization.Ĭomplementary DNA clones encoding m Per2 and h Per2 were PCR amplified from the corresponding plasmids and cloned into the pCS2 + MT (myc-epitope tagged) vector as previously described (25). Hybridized slides were then washed in 0.4× SSC containing 0.1% Tween-20 at 74☌ for 2 min, and then 1 min at room temperature in 2× standard saline citrate (SSC). Slides were hybridized in a darkened, humidified chamber for 16 hours at 37☌. Twelve microliters of pre-annealed probe was applied per slide, a cover slip was added, and edges were sealed with rubber cement. The probe mixture was denatured at 74☌ for 5 min and then pre-annealed at 37☌ for 15 min. Two micrograms of labeled probe was blocked with 2 μg of human Cot-1 DNA in Hybrisol VI (ONCOR, Gaithersburg, MD). Chromosomes were denatured in 70% formamide in 2×SSC at 74☌ for 5 min, and slides were dehydrated again as above except in ice-cold EtOH. The slides were then washed in acetic acid for 35 min at room temperature and were dehydrated for 2 min each in 70, 85, and finally 100% EtOH. Slides were prepared by dropping fixed cells onto glass slides and washing with excess fixative. Human bacterial artificial chromosomes (BACs) were labeled with spectrum orange using a nick translation kit per the manufacturers protocol (Vysis, Downers Grove, IL). Cells were then fixed in 3:1 MeOH:acetic acid and stored at 4☌. Pellets were then resuspended with 0.075M KCl (3 ml per pellet) for 15 min at room temperature. Coloemid treated cultures were pelleted at 500 g at room temperature for 8 min. Human lymphoblast cultures were treated with cholcimid (0.025 mg/ml) at 37☌ for 1.5 hours.
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