DNA-directed RNA polymerase from HeLa cells. Isolation, characterization and cell-cycle distribution of three enzymes.

DNA-directed RNA polymerase was solubilized from total HeLa cells. Three distinct classes of the enzyme could be clearly differentiated by their sensitivity toward alpha-amanitin. While form A is completely resistant to high concentrations (133 mug/ml) of this toxin, enzyme B is highly sensitive and is completely inhibited by concentrations of 0.1 mug/ml. In contrast, RNA polymerase C shows an intermediate behaviour (50% inhibition at 30% mug/ml). Separation of the three individual enzymes was achieved by chromatography on DEAE-cellulose (to separate enzyme B from A and C) and DEAE-Sephadex (to separate polymerase A from C). All three RNA polymerases were subsequently purified by phosphocellulose chromatography followed by sedimentation through glycerol gradients. Analysis of the purified enzymes by gel electrophoresis under denaturating conditions showed that the A enzyme consists of five subunits with molecular weights of 185, 128, 65, 41 and 32 X 10(3). In contrast, polymerase B is composed of seven subunits in variable stoichiometry with molecular weights of 215, 175, 145, 123, 68, 43 and 31 X 10(3) respectively. The subunit structure of enzyme C is not entirely clear at present and remains to be established. In addition, RNA polymerase activities were solubilized from mitotic and middle-S phase cells in comparison to controls. With respect to amounts and/or activities of all three RNA polymerases A,B and C no significant differences were detectable between logarithmically growing, mitotic and middle-S phase cells.     

A web services choreography scenario for interoperating bioinformatics applications

Background  

Very often genome-wide data analysis requires the interoperation of multiple databases and analytic tools. A large number of genome databases and bioinformatics applications are available through the web, but it is difficult to automate interoperation because: 1) the platforms on which the applications run are heterogeneous, 2) their web interface is not machine-friendly, 3) they use a non-standard format for data input and output, 4) they do not exploit standards to define application interface and message exchange, and 5) existing protocols for remote messaging are often not firewall-friendly. To overcome these issues, web services have emerged as a standard XML-based model for message exchange between heterogeneous applications. Web services engines have been developed to manage the configuration and execution of a web services workflow.     

Immobilized pH gradient-driven paper-based IEF: a new method for fractionating complex peptide mixtures before MS analysis

Introduction  

The vast difference in the abundance of different proteins in biological samples limits the determination of the complete proteome of a cell type, requiring fractionation of proteins and peptides before MS analysis.     

Transcription of specific genes in isolated nuclei from HeLa cells in vitro.

Isolated HeLa cell nuclei were employed to catalyze the synthesis of RNA in vitro. In the presence of low concentrations of alpha-amanitin (1 mug/ml), used to suppress the formation heterogeneous nRNA, these nuclei synthesize RNA very efficiently for extended periods of time (at least 60 min) at an elongation rate of about seven nucleotides per second. The product, analyzed on sucrose density gradients and polyacrylamide gels was found to exist of two predominant size classes. Synthesis of the 45-S ribosomal precursor was completely resistant even to high concentrations of alpha-amanitin (150 mug/ml) and hence was catalyzed by enzyme A (or I). A limited degree of processing of the 45-S precursor occurred in vitro. In addition, a second RNA class of low molecular weight (4-8 S) was synthesized by HeLa cell nuclei in the presence of 1 mug/ml alpha-amanitin in vitro. Analysis on 8% polyacrylamide gels resolved the RNA into four distinct components. Their synthesis was resistant to low (1 mug/ml) but clearly sensitive to high (150 mug/ml) concentrations of alpha-amanitin. Consequently the synthesis of all these small-molecular-weight RNA species is catalyzed by RNA polymerase C (or III). For the assessment of the initiation frequency of the individual classes of RNA, a new technique was developed independent of labelling the 5' end of the RNA molecule with the gamma-phosphate of the initiating nucleotide. It employs the double labelling of an RNA molecule with two different isotopes added sequentially at different stages of completion of the chain. From the incorporation ratio of the two isotopes into a particular class of RNA, conclusions can be drawn concerning their initiation frequency. The results obtained have shown a high reinitiation frequency for the small-molecular-weight RNA species at all stages of the incubation reaction. In contrast, reinitiation of the 45-S precursor RNA occurs only to a limited extent in isolated HeLa cell nuclei in vitro.