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 ∼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 ∼45 min to complete replication. Appropriate 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 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.

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Microscopy shows that individual sites of DNA replication and transcription of mammalian nuclei segregate into sets of roughly 22 and 16 higher order domains, respectively. Each domain set displayed a distinct network-like appearance, including regions of individual domains and interdigitation of domains between the two networks. These data support a dynamic mosaic model for the higher order arrangement of genomic function inside the cell nuclei.

Transcription sites are detected by labeling nascent transcripts with BrUTP in permeabilized 3T3 mouse fibroblasts followed by laser scanning confocal microscopy. Inhibition and enzyme digestion studies confirm that the labeled sites are from RNA transcripts and that RNA polymerase I (RP I) and II (RP II) are responsible for nucleolar and extranucleolar transcription, respectively. An average of 2,000 sites are detected per nucleus with over 90% in the extranucleolar compartment where they are arranged in clusters and three-dimensional networklike arrays. The number of transcription sites, their three-dimensional organization and arrangement into functional zones (Wei et al. 1998) is strikingly maintained after extraction for nuclear matrix. Significant levels of total RP II mediated transcription sites (45%) were associated with splicing factor–rich nuclear speckles even though the speckles occupied <10% of the total extranucleolar space. Moreover, the vast majority of nuclear speckles (>90%) had moderate to high levels of associated transcription activity. Transcription sites were found along the periphery as well as inside the speckles themselves. These spatial relations were confirmed in optical sections through individual speckles and after in vivo labeling of nascent transcripts. Our results demonstrate that nuclear speckles and their surrounding regions are major sites of RP II-mediated transcription in the cell nucleus, and support the view that both speckle- and nonspeckle-associated regions of the nucleus contain sites for the coordination of transcription and splicing processes.

The authors generated and tested a semiautomated carotid artery VWV measurement tool to provide measurements with reduced operator time and interaction, with high Dice coefficients, and with necessary required precision.

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Text that appears in images contains important and useful information. Detection and extraction of text in images have been used in many applications. In this paper, we propose a multiscale edge-based text extraction algorithm, which can automatically detect and extract text in complex images. The proposed method is a general-purpose text detection and extraction algorithm, which can deal not only with printed document images but also with scene text. It is robust with respect to the font size, style, color, orientation, and alignment of text and can be used in a large variety of application fields, such as mobile robot navigation, vehicle license detection and recognition, object identification, document retrieving, page segmentation, etc.