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Spatial and temporal dynamics of DNA replication sites in mammalian cells.

The Journal of cell biology | 1998

Fluorescence microscopic analysis of newly replicated DNA has revealed discrete granular sites of replication (RS). The average size and number of replication sites from early to mid S-phase suggest that each RS contains numerous replicons clustered together. We are using fluorescence laser scanning confocal microscopy in conjunction with multidimensional image analysis to gain more precise information about RS and their spatial-temporal dynamics. Using a newly improved imaging segmentation program, we report an average of approximately 1,100 RS after a 5-min pulse labeling of 3T3 mouse fibroblast cells in early S-phase. Pulse-chase-pulse double labeling experiments reveal that RS take approximately 45 min to complete replication. Appropriate calculations suggest that each RS contains an average of 1 mbp of DNA or approximately 6 average-sized replicons. Double pulse-double chase experiments demonstrate that the DNA sequences replicated at individual RS are precisely maintained temporally and spatially as the cell progresses through the cell cycle and into subsequent generations. By labeling replicated DNA at the G1/S borders for two consecutive cell generations, we show that the DNA synthesized at early S-phase is replicated at the same time and sites in the next round of replication.

Pubmed ID: 9852140 RIS Download

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Associated grants

  • Agency: NIGMS NIH HHS, United States
    Id: GM 23922

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ATCC (tool)

RRID:SCR_001672

Global nonprofit biological resource center (BRC) and research organization that provides biological products, technical services and educational programs to private industry, government and academic organizations. Its mission is to acquire, authenticate, preserve, develop and distribute biological materials, information, technology, intellectual property and standards for the advancement and application of scientific knowledge. The primary purpose of ATCC is to use its resources and experience as a BRC to become the world leader in standard biological reference materials management, intellectual property resource management and translational research as applied to biomaterial development, standardization and certification. ATCC characterizes cell lines, bacteria, viruses, fungi and protozoa, as well as develops and evaluates assays and techniques for validating research resources and preserving and distributing biological materials to the public and private sector research communities.

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Bio-Rad Laboratories (tool)

RRID:SCR_008426

Commercial instrument and chemical vendor. Developer and manufacturer of specialized technological products for life science research and clinical diagnostics markets.

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Molecular Probes (tool)

RRID:SCR_013318

An Antibody supplier and subset of ThermoFisher Scientific which provides fluorescence reagents for various experiments and methods.

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Zeiss Sigma VP Scanning Electron Microscope (tool)

RRID:SCR_020928

ZEISS SIGMA VP field emission scanning electron microscope (FE-SEM) for imaging of non-conducting samples. It images of bacteria, cells, plants and organisms. Uses ATLAS software and can be combined with 3View technology from Gatan Inc.

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