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We have used fluorescent microscopy to map DNA replication sites in the interphase cell nucleus after incorporation of biotinylated dUTP into permeabilized PtK-1 kangaroo kidney or 3T3 mouse fibroblast cells. Discrete replication granules were found distributed throughout the nuclear interior and along the periphery. Three distinct patterns of replication(More)
To study the possible role of the nuclear matrix in chromosome territory organization, normal human fibroblast cells are treated in situ via classic isolation procedures for nuclear matrix in the absence of nuclease (e.g., DNase I) digestion, followed by chromosome painting. We report for the first time that chromosome territories are maintained intact on(More)
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(More)
The largest subunit of RNA polymerase II contains a unique C-terminal domain (CTD) consisting of tandem repeats of the consensus heptapeptide sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Two forms of the largest subunit can be separated by SDS-polyacrylamide gel electrophoresis. The faster migrating form termed IIA contains little or no phosphate on the(More)
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(More)
Different agents have been employed to extract the histones and other soluble components from isolated HeLa S3 nuclei during nuclear matrix isolation. We report that 0.2M (NH4)2SO4 is a milder extracting agent than NaCl and LIS (lithium 3,5-diiodosalicylate), on the basis of the apparent preservation of the elaborate fibrogranular network and the residual(More)
A new view of the cell nucleus is emerging based on the functional dynamics of nuclear architecture. The striking structural preservation of a variety of genomic processes on the nuclear matrix provides an important approach for correlating nuclear form and function. In situ labeling coupled with three-dimensional microscopy and computer imaging techniques(More)
While significant progress has been made in elucidating molecular properties of specific genes and their regulation, our understanding of how the whole genome is coordinated has lagged behind. To understand how the genome functions as a coordinated whole, we must understand how the nucleus is put together and functions as a whole. An important step in that(More)
A hyperphosphorylated form of the largest subunit of RNA polymerase II (pol IIo) is associated with the pre-mRNA splicing process. Pol IIo was detected in association with a subset of small nuclear ribonucleoprotein particle and Ser-Arg protein splicing factors and also with pre-mRNA splicing complexes assembled in vitro. A subpopulation of pol IIo was(More)