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14C-labeled uracil (2 μCi/ml), and labeling was continued for indicated times (Fig. 2). Agarose plugs for CHEF analyses (6.5 V/cm, a linear pulse ramp of 3 to 17 s for 20 hours, 14°C) were prepared from 5-ml cultures by using a modification of standard protocols. Gels and filters (1 to 2 weeks of exposure to Storage Phosphor screens) were analyzed with a Storm imaging system (Molecular Dynamics, Sunnyvale, CA). Radioactivity of each Not I fragment was determined by using ImageQuant software and following the manufacturer's recommendations. To ensure that the results obtained were not an artifact of the puromycin treatment, the relative steady-state levels of Not I fragments were also investigated in untreated early exponential stage cultures. In this case, CHEF gels were stained with VistraGreen (Molecular Dynamics), a DNA-specific fluorescence dye, and were directly scanned with a blue fluorescence mode of a Storm system. Relative frequencies for fragments were obtained by dividing the amount of radioactivity/fluorescence of a given band by its size calculated on the basis of the determined genome sequence. All analyzed signals were within a linear range of a Storm system. The given values were normalized to the minimal value of one arbitrary unit and are averages of two independent measurements.
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This region is interrupted by several stop codons in the three Pyrococcus genomes, indicating that it does not contain genes that could have been missed during the genome annotation.
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-33. The use of the TBLASTX program indicates that the high scores of the remaining six intergenic regions might be due to some hard-to-identify open reading frames.
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This work was supported by research grants from the European Union (BIO-CT 96-0488) and the Association de Recherche contre le Cancer. H.M. acknowledges financial support from the European Union and thanks U. Liebl for helpful discussions. We thank J. Weissenbach and his staff for sequencing the complete genome of P. abyssi.
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