RESUMEN
An entirely automated 96-well microplate-based system to perform procedures for the measurement of protein is described. This single instrument system utilizes a series of computer-controlled mechanical subsystems to move the plate, control incubations and dispense samples or reagents in order to perform the assay. This system allows the investigator to reproducibly perform these protein assays on large numbers of biological samples with minimal effort.
Asunto(s)
Autoanálisis/métodos , Proteínas/análisis , Autoanálisis/instrumentación , Autoanálisis/estadística & datos numéricos , Unión Competitiva , Biotina , Biuret , Quinolinas , Albúmina Sérica BovinaRESUMEN
We show assembly of low and high multimers of HeLa cell nuclear protein, RIP60, at the origin of bidirectional replication (OBR) identified by Burhans, Vassilev, Caddle, Heintz and DePamphilis in Chinese hamster ovary cells. RIP60 binds a 5'-ATT-3' reiterated sequence downstream of the OBR and a second, homologous ATT sequence of opposite orientation situated within the OBR zone. Specifically bound structures were studied by conventional electron microscopy (EM) and quantitative scanning transmission electron microscopy (STEM). Dimers and multiples of dimers link the downstream binding site that overlaps a bent DNA sequence and the homologous upstream OBR sequence, looping out 700 bp of intervening DNA. Superposed dimers are found at individual unlinked sites, stabilized presumably through protein-protein interaction, and such superposition appears to occur also in the basic link structure. Along the loop, single crossovers and extended twists are observed by conventional EM. By STEM, loop DNA is laterally compacted, with diameter and mass equivalent to double-duplex DNA strands. Supercoiled 736 bp and 5243 bp circular DNAs assume similar laterally compacted geometries that are mostly absent from relaxed forms. These observations parallel the compacted, interwound superhelices viewed by cryo-electron microscopy in vitrified solutions containing magnesium ions, and provide structural evidence in agreement with that from conventional EM for superhelical tension in RIP60 loop DNA. Loop superhelicity could arise as a topological response to linking and suggests a functional role for link formation.
Asunto(s)
Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas Nucleares/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Animales , Sitios de Unión , Biopolímeros , Células CHO , Cricetinae , ADN/ultraestructura , Proteínas de Unión al ADN/ultraestructura , Células HeLa , Humanos , Microscopía Electrónica , Microscopía Electrónica de Transmisión de Rastreo , Proteínas Nucleares/ultraestructura , Conformación de Ácido Nucleico , Conformación ProteicaRESUMEN
A novel method of synchronizing monolayer tissue culture cells is described. By limiting the period of attachment of trypsinized cells and the subsequent removal of unattached cells a G1 population of cells is isolated. Evaluation of the degree of synchrony has been carried out by measuring the labeling index and incorporation of [3H]thymidine into DNA. Further conformation of synchrony, as well as a comparison with synchrony by isoleucine deprivation, was obtained by flow cytometry. The expected peak in DNA synthesis rate following limited attachment was observed. This peak becomes more prominent and shifts to earlier times with shorter attachment intervals. The synchronization method described is simple, rapid, yields a substantial number of cells, and is applicable to many cell lines.