Fluorescence microscopic evaluation of newly replicated DNA has revealed discrete granular sites of replication (RS). calculations suggest that each RS contains an average of 1 mbp of DNA or ～6 average-sized replicons. Double pulse-double chase experiments demonstrate that this DNA sequences replicated at individual RS are precisely managed 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 this DNA synthesized at early S-phase is usually replicated at the same time and sites in the next round of replication. medium (Molecular Probes Inc. Eugene OR) and examined by laser scanning confocal microscopy. The confocal imaging was performed on a three channel laser scanning confocal imaging system (MRC-1024 ; Bio-Rad Laboratories Hercules CA) equipped with a Optiphot 2 microscope a 60× 1.4 NA objective and an argon/krypton laser used to excite FITC (488 nm) Texas red or PI (568) and Cy-5 (649 nm). In some cases we used an GB200 laser scanning confocal microscope system equipped with a plan apo 60× 1.4 NA objective and an argon laser (λ = 514 nm) to excite green and red fluorescence simultaneously. Optical sections of 512 pixels × 512 pixels × 8 bits/pixel (MRC-1024) or 1536 pixels × 1536 pixels × 24 bits/pixel (GB200) were collected through the samples at 0.5-μm intervals. 3-D images were reconstructed and pseudo-colored using Sterecon or VMLSM software. Results Measuring Replication Sites in the Mammalian Cell Nucleus We have combined double labeling techniques laser scanning confocal microscopy and a newly developed image segmentation method to investigate the three-dimensional spatial and temporal properties of in vivo DNA RS in SU-5402 the mammalian cell nucleus. In the first series of experiments we labeled mouse 3T3 fibroblasts with BrdU for 2-30 min. Then we analyzed the confocal images from cells engaged in early S-phase (type I; Nakayasu and Berezney 1989 DNA replication with an area based segmentation technique (Samarabandu et al. et al. 1995 This is distinct from your popular threshold based methods for segmentation and enabled us to section virtually all RS visible in the original images despite the wide range of intensities and the close proximity of many of the individual RS (Fig. ?(Fig.11 and > 12 0 Moreover neither the SU-5402 X-Y diameter nor the overall volume of a RS which represent the average SU-5402 of >12 0 Rabbit Polyclonal to CELSR3. sites for each pulse period significantly changed between 2-15-min pulses with only a slight increase after a 30-min pulse (Fig. ?(Fig.22 and SU-5402 and and and for details). Under these conditions we achieved a high degree of synchrony in early S-phase with >99% of the S-phase cells (507 counted) having type I early S-phase replication patterns immediately after launch and >90% of the cells entering S-phase within 2 h of liberating the aphidicolin block (data not demonstrated). The results demonstrated in Fig. ?Fig.88 demonstrate complete overlap of the RS labeled at two successive early S-phases. We conclude that DNA synthesized in early S-phase is definitely replicated at the same time and at the same site in the next round of replication. Number 8 Labeling replication sites SU-5402 at early S-phase for two consecutive cell decades. Replication sites in synchronized mouse 3T3 fibroblasts were 1st labeled for 30 min with CldU (FITC secondary antibody green sites) at the 1st G1/S border chased … Conversation Fluorescence microscopic analysis SU-5402 of newly replicated DNA offers exposed discrete granular sites of replication (Nakamura et al. 1986 Nakayasu and Berezney 1989 Mills et al. 1989 Berezney 1991 Destroy et al. 1991 Fox et al. 1991 O’Keefe et al. 1992 Moreover the pioneering studies of Nakamura et al. (1986) showed that increasing occasions of BrdU in vivo labeling of DNA RS prospects to progressively more elongated structures and to improved difficulty in resolving individual sites. We are using fluorescence laser scanning confocal microscopy in conjunction having a spot-based segmentation algorithm (Samarabandu et al. 1995 to address these issues and to gain more precise spatial information about individual RS and their potential business into higher order domains. We statement an average of ～1 100 replication sites after a 5-min pulse of 3T3 mouse fibroblasts synchronized in early S-phase. Related results were acquired in exponentially dividing cells where selection for.