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Download Viral replication factories/site(s) inside live host: Replication forks
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Viral replication factories/site(s) inside live host: Replication forks visualized in T4infected Escherichia coli Srivathsa Nallanchakravarthula Every living organism maintains its continuity by passing more or less accurate copies of its hereditary information to the next generation with the help of replication process. The replication process can be explained as “the process by which the genetic material of an organism copies itself in order to make a new genome to pass onto its daughter cell”. Recent scientific studies in bacteria have shown that the replicating chromosome (genetic material) moves through an anchored multiprotein structure, designated as a “replication factory”. As part of my degree project I attempted to locate and characterize the positions in the cell where the replication factory set up for replication of the T4 bacteriophage (a virus which infects bacteria) genomewere located. To visualize the phage development the DNA-specific stain DAPI (4’, 6-diamidino-2-phenylindole) was used to visualize the DNA and single strand DNA binding (SSB) protein (which binds to single stranded DNA regions at the site of replication fork) was fused to green fluorescent protein (GFP) and used to locate replication forks. By examining the periodic samples of the T4 infected culture, the development of the phage was followed using spectrophotometry (growth and lysis) and fluorescence microscope (total DNA and replication forks in individual cells). To measure the relative amounts of bacterial and phage DNA, flow cytometry (DNA content in cells), southern blotting (degrading host DNA) and TCA precipitation (relative amounts of bacterial or phage origin) were used. The results showed (a) T4-specific foci corresponding to the early replication mode appeared within 3-5 minutes following infection; (b) The early replication centers were located in cytoplasmic areas free from host DNA; (c) About 10-15 minutes after infection, the GFP spots changed from distinct to more fuzzy, indicating that late replication (with more replicative forks per chromosome) had take over from the early replication; (d) Phage DNA molecules formed clusters near the bacterial membrane for packaging and lysis. To my knowledge, this is the first time that phage infection and its development could be observed live in real time. As expected, this novel approach raised many interesting questions that need to be investigated further. A detailed knowledge of phage replication and development might provide insights that would be useful in designing approaches to phage therapy (the therapeutic use of lytic bacteriophages to treat pathogenic bacterial infections). Degree project in biology, Uppsala University, spring 2006 Examensarbete i biologi, 20p, Department of Biology Education and Department of Cell and Molecular Biology, Supervisior: Dr. Santanu Dasgupta