Intracellular localization of mouse DNA polymerase-alpha.

Immunoglobulin (IgG) and the F(ab')2 fragment of IgG were prepared from serum of a rabbit immunized with purified calf thymus DNA polymerase a (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7). An indirect immunofluorescent method based on these reagents was used to detect the intracellular localization of DNA polymerase a in primary fetal bovine fibroblasts. The results show that the bulk of DNA polymerase a is located in the perinuclear region of the cytoplasm. Immunofluorescent staining ofcytoplast and FicollPaque gradient-purified karyoplast fragments resulting from cytochalasin enucleation show the presence of DNA polymerase a in cytoplasts and the virtual absence of the enzyme in the nucleus of the karyoplast itself. The implication of this unusual intracellular location for DNA polymerase a is discussed.The original description of DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) in eukaryotic systems (1, 2) noted the presence of the major amount of its activity in the cytoplasmic fraction of buffered sucrose homogenates, with a minor amount of activity in the nucleus. This finding has been confirmed by other early investigators (3). A report suggesting transport of activity to the nucleus during S phase has been published also (4). Subsequent work with nonaqueous solvents (5, 6) has suggested a nuclear localization. Use of cytochalasin B to produce cytoplasts and karyoplasts also has been interpreted to prove a nuclear localization of DNA polymerase a (7). Biochemical studies routinely use a cytoplasmic fraction for isolation of DNA polymerase a'.DNA polymerase 18 has been found to be nuclear and cytoplasmic (8, 9) and DNA polymerase y has been localized in mitochondria (10,11).Because all of these studies have required breakage of cells, the arguments have centered on how the cells were disrupted and the quantitative analysis of the result. This matter could be settled by demonstrating localization of the enzyme in wholecell preparations. We Antibody to DNA Polymerase a. The antibody used for this study was a monospecific antibody against DNA polymerase a, prepared by immunizing a rabbit with calf thymus 'DNA polymerase a. Immunoglobulin (IgG) and F(ab')2 fragment of IgG were prepared from the immune serum. The preparation and characterization of this antibody have been described (12).Immunofluorescence. An indirect immunofluorescent technique was used to test for the presence of DNA polymerase a. Cells growing on cover slips, cytocentrifuge preparations of purified karyoplasts, and re-adhered cytoplasts were washed three times in medium F-10 without serum, fixed in absolute methanol for 10 min at 40C, and air dried. The IgG or F(ab')2 fragment of rabbit anti-calf thymus DNA polymerase a or preimmune IgG (diluted to 100-400 Ag/ml in phosphate-buffered saline) was used in the primary incubation for 30 min at room temperature. The secondary antibody was the fluorescein isothiocyanate-conjugated F(ab')2 fragment ofgoat anti-rabbit Ig...

Section: Discussioncontrasting

confidence: 97%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Intracellular localization of DNA polymerase alpha.

Brown¹,

Bollum²,

Chang³

1981

“…In the analysis of T7 lane c, the bands produced by RNase HI correspond to the T7 mRNA species previously observed (19) (20,31); and (iv) an RNase P, which, like that reported in KB cells (12) (33). Finally, the DNA polymerase a, initially recovered from the cytoplasm of various cell types (34), has more recently been found in association with the nucleus (35). Thus, although it appears that subcellular fractionation of eukaryotic cells may not preserve the in vivo distribution of enzymes involved in nucleic acid metabolism, this situation does not necessarily impede their study.…”

Section: Methodssupporting

confidence: 72%

Specific RNA-cleaving activities from HeLa cells.

Ferrari¹,

Yehle²,

Robertson³

et al. 1980

It is now evident that in primary transcripts of most prokaryotic and eukaryotic RNAs the regions destined to occur in the mature molecules are interspersed among regions that are removed during the maturation process (1-5). The role of RNA processing enzymes is the correct removal of these "extra" regions, leading to the formation of functional mature RNAs. Because of the availability of numerous well-defined prokaryotic RNA precursors, the enzymatic RNA processing reactions that occur in prokaryotes have been the most extensively studied (6-10). From these studies the conclusion has emerged that such enzymes retain the ability to cleave their natural substrates properly in vitro, even after extensive purification of both the enzyme and the RNA precursors. Considerably less information concerning the enzymes involved in specific cleavage of eukaryotic RNA precursors (11, 12) is available.We have carried out gentle fractionation of HeLa cells to minimize release of random nucleases, and we have assayed nuclear and cytoplasmic fractions for their ability to cleave a wide spectrum of well-defined prokaryotic and eukaryotic RNA molecules. Four specific RNA-cleaving activities were detected: cleavage of heterogeneous nuclear RNA (hnRNA) in a characteristic fashion, specific cleavage of 45S rRNA precursor, cleavage of RNA-DNA hybrids (RNase H), and cleavage of a tRNA precursor (RNase P). Only the latter two activities have been described previously in mammalian cells. MATERIALS AND METHODSCell Growth and Subcellular Fractionation. HeLa (human) cells (strain S3) from the collection of Igor Tamm, The Rockefeller University, were maintained in suspension culture [Joklik's modified Eagle's medium (GIBCO) containing 5% fetal calf serum (GIBCO)]. Cells from 1-3 liters of culture at 5-6 X 105 cells per ml were harvested and fractionated according to Bothwell and Altman's modifications (13) of methods published by Penman (14) and Amaldi and Attardi (15). Worthington DNase DPFF was used without further treatment in the preparation of nuclear lysates. Ammonium sulfate precipitation was performed by adding neutralized saturated ammonium sulfate (40C) to obtain the desired final concentration, with constant stirring, incubating at 40C for 20 min, and centrifuging at 20,000 X g for 20 min. Precipitates were resuspended in 10 mM Tris-HCl, pH 7.5/20 mM NaCI/2 mM MgCl2/1 mM dithiothreitol, and dialyzed for 2 hr at 40C against this buffer.Labeled Enzyme Assays. Protein concentrations were determined by measuring the spectral shift caused by Coomassie blueprotein interaction as described by Bradford (21). Comparable proportions of subcellular fractions were assayed for their ability to cleave RNA substrates by incubation in 10 ,l of a standard buffer containing 30 mM Tris-HCI at pH 8.0, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, and 0.1 mM dithiothreitol for 30 min at 37°C. Reactions were terminated by adding 3 ,ul of 20% sucrose/1% sodium dodecyl sulfate/0.2% bromphenol blue/0.2 M EDTA. The extent of substrate cleavage was determined by ...

“…For example, by a detergent method, DNA polymerase is found in the cytoplasm. However, by the cytochalasin B technique, this enzyme activity is found to be associated with the nucleus (3). The cytochalasin B technique has the additional feature in that the cytoplasm can be recovered in intact particles, the cytoplasts.…”

mentioning

confidence: 99%

Chondrocytes contain a growth factor that is localized in the nucleus and is associated with chromatin.

Azizkhan¹,

Khagsbrun²

1980

Bovine scapular and articular chondrocytes were isolated from fresh cartilage and disrupted by sonication. The disrupted cells had the ability to stimulate DNA synthesis and cell division in vitro in chondrocytes and in 3T3 cells. Subcellular fractions were prepared by two methods, enucleation with cytochalasin B and lysis of cells with Nonidet P-40. After enucleation of chondrocytes, karyoplasts and cytoplasts were collected, disrupted by sonication, and tested for their ability to stimulate DNA synthesis. Over 95% of the cellular growth factor activity was localized in the karyoplast. In addition, after lysis of chondrocytes in Nonidet P-40, over 95% of the growth factor activity was recovered in the nuclear fraction. Chondrocyte chromatin was prepared by low ionic strength detergent treatment of karyoplasts. All of the growth factor activity of the karyoplast was found to be associated with chromatin. The growth factor activity of chondrocytes, cytoplasts, karyoplasts, and chromatin was analyzed by gel filtration on Bio-Gel A-0.5 m equilibrated with 4 M guanidine'HCI and 5 mM dithiothreitol. Chondrocytes, chondrocyte karyoplasts, and chondrocyte chromatin had similar column elution profiles, with molecular weights in the range of 12,000-22,000.A growth factor that stimulates DNA synthesis and cell division in BALB/c 3T3 cells and in chrondocytes can be extracted from cartilage with guanidine-HCl (1). Purification of the growth factor indicates that it has a molecular weight between 16,000 and 17,000 and an isoelectric point between 9 and 10 (unpublished data). Growth factor activity can also be obtained from chondrocytes. A homogeneous cell population of chondrocytes can be prepared by digesting cartilage with collagenase and separating the cells from the extracellular matrix by centrifugation. Growth-promoting activity is released by sonication of chondrocytes or by collection of medium conditioned by chondrocytes that have been cultured in the absence of serum. Thus, the cartilage-derived growth factor is probably an endogenous polypeptide that is synthesized by the chondrocyte.The subcellular origin of most growth factors is unknown. One exception is the platelet-derived growth factor, which is found within the a granules of platelets (2). However, platelets are atypical cells because they contain no nuclei. The presence of the cartilage-derived growth factor in chondrocytes affords a unique opportunity to study the localization of a growth factor within a nucleated cell. Cells were fractionated by two methods, enucleation of cells with cytochalasin B and lysis of cells with Nonidet P-40. The cytochalasin B method of fractionating cells was chosen because of reports that detergent lysis of cells sometimes leads to leakage of proteins from one cellular compartment to another. For example, by a detergent method, DNA polymerase is found in the cytoplasm. However, by the cytochalasin B technique, this enzyme activity is found to be associated with the nucleus (3). The cytochalasin B technique has the a...