Asymmetrical progression of replication forks just after initiation on Mycoplasma capricolum chromosome revealed by two-dimensional gel electrophoresis |
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| Affiliation: | 1. Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany;2. Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom;1. Department of Chemistry, Chair of Organic Chemistry II, Center for Integrated Protein Science (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany;2. Department of Biociences, Center for Integrated Protein Science (CIPSM), Technische Universität München, Emil-Erlenmeyer-Forum 8, 85354 Freising, Germany;1. Global Health Institute, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland;2. Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland;1. Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune 411008, India;2. INRAE, University of Bordeaux, UMR 1332 BFP, Villenave d’Ornon, Bordeaux, France;1. CIRAD, UMR CMAEE, F-34398 Montpellier, France;2. INRA, UMR1309 CMAEE, F-34398 Montpellier, France;3. INRA, Joint Research Unit 385 UMR BGPI, Campus International de Baillarguet, Montpellier, France;4. INSERM U1051—Hôpital Saint Eloi INM. 80, rue Augustin Fliche, 34091 Montpellier cedex 5, France |
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| Abstract: | Previously, we mapped the replication initiation site of the Mycoplasma capricolum chromosome into a region containing the dnaA gene [M. Miyata et al., 1993a. Nucleic Acids Res. 21, 4816–4823]. In this study, various regions including this functional domain were analyzed by two complementary two-dimensional (2D) gel electrophoretic methods. Sizes of nascent strands in a 10.7-kb and a 5.6-kb region were examined by a neutral/alkaline (N/A) method. The shortest nascent strand was detected in an 875-bp region composed of the 3′ end of the dnaA gene and its downstream non-coding sequence. The shortest nascent strand detected became longer in an asymmetrical manner as position of the probe became further from the putative initiation site in both directions. The intermediate forms of eight regions restricted at different sites were examined by a neutral/neutral (N/N) method. Bubble arcs were observed in four regions including the 875-bp region. The region containing the 875-bp region at about its center showed an asymmetrical arc, although that containing the 875-bp region at its end showed a symmetrical arc. These results show that the replication forks develop in the 875-bp region and proceed bidirectionally in an asymmetrical manner around the initiation site. The results of N/A analysis of the 5.6-kb region showed a shift of intensity in the nascent strand signal, which suggests an upshift of fork progression velocity. |
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