Abstract. 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 sites in relationship to chromatin domains in the cell nucleus and the period of S phase were detected and termed type I (early to mid S), type II (mid to late S) and type III (late S). Similar patterns were seen with in vivo replicated DNA using antibodies to 5-bromodeoxyuridine. Extraction of the permeabilized cells with DNase I and 0.2 M ammonium sulfate revealed a striking maintenance of these replication granules and their distinct intranuclear arrangements with the remaining nuclear matrix structures despite the removal of >90 % of the total nuclear DNA. The in situ prepared nuclear matrix structures also incorporated biotinylated dUTP into replication granules that were indistinguishable from those detected within the intact nucleus. DSPITE considerable progress in defining specific molecular components involved in eucaryotic DNA replication such as DNA polymerases a, di, and DNA primase (Kornberg, 1988), our understanding of native replicational sites and their structural organization and associations in the cell nucleus has lagged behind. Most previous studies designed to localize the sites of DNA replication in eucaryotic cells have used autoradiographic microscopy. These techniques have been very useful in determining cells active in DNA replication and the general distribution of replication sites in the cell nucleus over peripheral versus internal sites and over condensed heterochromatin versus diffuse euchromatin (Hay and Revel, 1963;Milner, 1969; Huberman et al., 1973;Fakan and Hancock, 1974;Fakan, 1978;Smith et al., 1984). The level of resolution of these techniques, however, severely limits their potential usefulness for studying the structural organization of individual replicational sites in the cell nucleus.This has prompted us to explore more sensitive and higher-resolution approaches to this problem. Langer et al. (1981), who first synthesized biotin-labeled nucleotides for use as nucleic acid-affinity probes also demonstrated that 5-([N-biotinamidocaproyl]-3-aminoallyl)-2'-deoxyuridine-5'-triphosphate (biotin-ll-dUTP) ~ is effectively incorporated Dr. Nakayasu's present address is Department of Medical Biochemistry, Shiga, University of Medical Sciences, Seta, Otsu, 52021, Japan. Address correspondence to Dr. Ronald Berezney, Department of Biological Sciences, State University of New York, Buffalo, NY 14260. Abbreviations used in this paper:biotin-lI-dUTP, 5-([N-biotinamidocaproyl]-3-aminoallyl)-2'-deoxyuridine-5'-triphosphate; BrdU, 5-bromodeoxyuridine; glycerol buffer, 20 mM Tris-HCI, pH 7.4, 25% glycerol, 5 mM MgCt2, 0.5 mM EGTA, 0.5 mM PMSF; TBS buffer, 10 mM Tris-HC1, into DNA by a variety of DNA polymerases, including the...

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 hIo was localized to nuclear "speckle" domains enriched in splicing factors, indicating that it may also be associated with RNA processing in vivo. Moreover, pol IIo was retained in a similar pattern following in situ extraction of cells and was quantitatively recovered in the nuclear matrix fraction. The results implicate nuclear matrix-associated hyperphosphorylated pol IIo as a possible link in the coordination of transcription and splicing processes.with pre-mRNA processing that are related to the SR family.In the present study, a new anti-NM mAb, B3, is characterized that recognizes a 250-kDa NM protein concentrated in speckles. Similar to anti-NM mAbs which recognize SR proteins, B3 preferentially binds in vitro to a subset of splicing complexes containing exon sequences. Surprisingly, the B3 antigen corresponds to a hyperphosphorylated form of the large subunit of pol II (pol IIo). In addition to splicing complexes, pol IIo is associated with a subset of snRNP and SR protein splicing factors. The possible implications of these findings in relation to the regulation of RNA processing are discussed. MATERIALS AND METHODSIncreasing evidence suggests that transcription and processing of RNA polymerase II (pol II) transcripts are temporally and spatially linked. Visualization of chromatin spreads by electron microscopy has revealed that the majority of introns are removed cotranscriptionally from pre-mRNA (1, 2). These studies are supported by recent fluorescent in situ hybridization experiments, indicating that the synthesis and splicing of specific pol II transcripts are coincident at discrete foci (3-5).In several cases, transcript foci appear to be localized in association with specific nuclear domains that are highly enriched in splicing factors, referred to as "speckles" (3, 5-7).Although not mutually exclusive with evidence implicating speckle domains in splicing factor storage and/or assembly (8, 9), these transcript localization experiments indicate a possible direct role of speckle domains in the processing of pre-mRNAs (10, 11). Mammalian nuclei typically contain 20-50 speckle domains, which, in addition to the four spliceosomal small nuclear ribonucleoprotein particles (snRNPs; Ul, U2, U4/6, and U5), are also enriched for non-snRNP splicing factors and poly(A)+ RNA (8, 9, 11). Many of the non-snRNP splicing factors in speckles are related to the Ser-Arg (SR) family of proteins, all of which contain one or more domains rich in alternating serine and arginine residues (12). Besides splicing components, speckle structures also contain elevated concentrations of proteins involved in transcription and cellular transformation (13-15). Since these structure...

A preparative two-dimensional polyacrylamide gel system was used to separate and purify the majorCoomassie blue-stained proteins from the isolated rat liver nuclear matrix. Approximately 12 major proteins were consistently found. Of these, 5 proteins represented identified proteins, including nuclear lamins A, B, and C, the nucleolar protein B-23, and residual components of core heterogeneous nuclear ribonucleoproteins. The remaining eight major proteins termed the nuclear matrins consisted of matrin 3 (125 kDa, slightly acidic), matrin 4 (105 kDa, basic), matrins D-G (60-75 kDa, basic), and matrins 12 and 13 (42-48 kDa, acidic). Peptide mapping and two-dimensional immunoblot studies indicate that matrins D-G compose two pairs of related proteins (matrins D/E and F/G) and that none of the matrins resemble the nuclear lamins or any of the other major proteins detected on our two-dimensional gels. Subfractionation immunoblot experiments demonstrated the nearly exclusive localization of matrins F/G and other matrins to the nuclear matrix fraction of the cell. These results were further supported by indirect immunofluorescence microscopy that showed a strictly interior nuclear localization of the matrins in intact cells in contrast to the peripherally located nuclear lamins. We conclude that the nuclear matrins are a major class of proteins of the nuclear matrix interior and are distinct from the nuclear lamins.There is a growing awareness that many of the important answers to questions concerning the expression and regulation of the eukaryotic genome will require an understanding of the higher-order arrangement and function of the genetic components as they interact within the complex threedimensional architecture of the cell nucleus (1-13). The nuclear matrix, a residual nuclear structure that has been isolated from a wide variety of eukaryotic cells throughout the phylogenetic scale (1-3, 6, 7, 13-16), offers a potentially valuable in vitro approach for studying nuclear processes in relation to nuclear structure. Indeed, numerous studies have implicated the matrix as a site of organization for virtually all known nuclear processes (1-11, 13, 16), such as, DNA loop attachment, DNA replication, transcription, RNA splicing and transport, hormone receptor function, carcinogen binding, oncogene proteins, viral proteins, and protein phosphorylation. Despite this progress, our knowledge of the proteins that compose this proteinaceous nucleoskeletal structure is still in its infancy. In this study we have used high-resolution preparative PAGE to identify and purify many of the major Coomassie blue-stained nuclear matrix proteins. A class of nuclear matrix proteins termed the nuclear matrins is identified and characterized by peptide maps, polyclonal antibodies generated against the individual purified matrins, and indirect immunofluorescence microscopy.

We prepared a monoclonal antibody (A-22) that recognizes a 60-kDa protein in the zebrafish brain. The antigen is distributed throughout the brain but is not found outside it. The antibody recognizes star-shaped cells with long processes in the spinal cord. All A-22-positive cells are also GFAP-immunopositive, but there are GFAP-positive cells that are A-22-negative. The cells are connected to small veins and to the surface of the spinal cord. Immunopositive cells are generally homogeneous in size and shape and are found not only in the spinal cord but also in several areas of the brain. These results indicate that the stained cell is an astrocyte. Most of these cells (88%) are distributed in the gray matter of the spinal cord; the remainder (12%) are found in the white matter. Most of the cells in the gray matter are found in the ventral and dorsal horns, but some are also present in the central area along the ventricle. Glial cell bodies form an array along the longitudinal axis and are connected to each other by thick projections. The cellular array is not visible in coronal sections. In contrast, thin processes from the cells extend to the surfaces of veins, to neurons, and to the periphery of the spinal cord. We estimate that there are about 13,500 A-22-positive astrocytes in the spinal cord; however, this represents only 26% of the total number of astrocytes in the spinal cord (approximately 52,000).

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