Physical properties of cytoplasmic membrane-associated DNA

Some of the physical properties of a cytoplasmic membrane-associated DNA isolated from a diploid human lymphocyte cell line have been examined. Cytoplasmic membrane-associated DNA extracted from lymphocytes labeled with either [³H]or [¹⁴C]thymidine had a specific activity lower than nuclear DNA extracted from the same cells. Analysis of cytoplasmic membrane-associated DNA in the electron microscope shows that the molecules are linear and have a mean length of 1·75 μm; the average sedimentation coefficient of this DNA is 16·6 S, which corresponds to a molecular weight of 4·2×10⁶. Cytoplasmic membrane-associated and nuclear DNA band at identical positions in both neutral and alkaline CsCl gradients with buoyant densities of 1·699 g/ml and 1·752 g/ml, respectively. Native cytoplasmic membrane-associated DNA is double-stranded and has a mole fraction of guanine plus cytosine of 40± l %. Sheared, denatured cytoplasmic membrane-associated DNA reassociates as two distinct fractions whose rates of reassociation differ by about four decades: the complexity of the reassociation of this DNA tends to rule out the possibility that it arises from either mycoplasmal or viral contamination of our cell cultures. The slowly reassociating fraction of cytoplasmic membrane-associated DNA reassociates about ten times faster than the unique sequences of nuclear DNA. This could represent potential genetic information for about 100,000 diverse genes of 1000 nucleotide pairs each. At present the function of cytoplasmic membrane-associated DNA in these cells is unknown.     

Appearance of virus-specific DNA in mammalian cells following transformation by Rous sarcoma virus

To detect Rous sarcoma virus-specific DNA in mammalian cells, we have measured the capacity of unlabeled cell DNA to accelerate the reassociation of labeled double-stranded DNA synthesized by the Rous sarcoma virus RNA directed DNA polymerase. Two populations of double-stranded polymerase products are identified by their reassociation kinetics and represent approximately 5% and 30% of the viral 70 S RNA genome. Using two strains of Rous sarcoma virus and four lines of transformed mammalian cells, we found two copies of DNA homologous to both DNA populations in Rous sarcoma virustransformed rat and mouse cells, but not in normal cells. The Rous sarcoma viruslike DNA can be demonstrated in the non-repeated fraction of transformed cell DNA and in nuclear DNA. The results are supported by evidence that the techniques employed detect the formation of extensive well-matched duplexes of cell DNA and viral polymerase products.     

Induction of Cellular Deoxyribonuleic Acid Synthesis by Simian Virus 40

The incorporation of (3)H-thymidine ((3)H-dT) into deoxyribonucleic acid (DNA) has been studied in uninfected confluent monolayer cultures of monkey kidney and mouse kidney cells, simian virus 40 (SV40)-infected cells, and in SV40-transformed mouse kidney cells. Radioautographic measurements revealed that during the period from 28 to 51 hr after productive SV40 infection of monkey kidney cultures about 80% of the cells synthesized DNA, compared to about 16% in uninfected cultures. At 28 to 43 hr after abortive SV40 infection of mouse kidney cultures, 24 to 37% of the cells synthesized DNA, compared to about 6 to 8% in uninfected cultures. The infected monkey kidney and mouse kidney cultures, respectively, incorporated about 5 to 10 times and 3 to 5 times as much (3)H-dT into DNA as did uninfected cultures. Moreover, the net DNA synthesized by SV40-infected monkey kidney cultures, estimated by colorimetric methods, substantially exceeded that of uninfected cultures.Nitrocellulose chromatography and band centrifugation experiments were performed to elucidate the kinds of DNA synthesized in the cultures. In uninfected monkey kidney cultures and at 2 to 12 hr after SV40 infection, almost all of the (3)H-dT labeled DNA sedimented more rapidly than SV40 DNA, and the radioactive DNA was denatured by heating for 12 min at 100 C (cellular DNA). Almost all of the labeled DNA obtained from abortively infected mouse kidney cultures and from SV40-transformed cells also had the properties of cellular DNA. However, approximately one-third to one-half of the labeled DNA obtained from monkey kidney cultures 28 to 51 hr after infection sedimented more slowly than cellular DNA and was not denatured by the heating (SV40 DNA). It is concluded that cellular DNA synthesis was induced during either the productive or abortive SV40 infections.     

Compartmentation of dCTP pools. Evidence from deoxyliponucleotide synthesis

The nucleotide fraction of cultured 3T6 and 3T3 mouse fibroblasts contains deoxy-CDP choline and deoxy-CDP ethanolamine as well as the corresponding riboliponucleotides. In permeabilized cells both deoxyliponucleotides were formed from dCTP. In intact cells they could be labeled from [5-3H] deoxycytidine or cytidine via transformation of the nucleosides to dCTP. Their turnover was slow compared to that of dCTP. When rapidly growing 3T3 cells were labeled during 90 min from deoxycytidine the specific activity of dCDP choline was 2.4 times higher than that of dCTP while after labeling from cytidine both nucleotides (and CTP) reached the same specific activity under steady state conditions. Also dCDP ethanolamine was labeled more rapidly from deoxycytidine than from cytidine. Our results suggest that the deoxyliponucleotides were synthesized from a dCTP pool that was labeled preferentially from deoxycytidine. Earlier work (Nicander, B., and Reichard, P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1347-1351) had demonstrated synthesis of DNA from a dCTP pool labeled preferentially from cytidine. Taken together our results suggest that deoxyliponucleotides and DNA are synthesized from separate dCTP pools.     

Diversity of sequences in total and polyadenylated nuclear RNA from Drosophila cells.

Complementary DNA was synthesized using polyadenylated nuclear RNA of cultured Drosophila cells as template. The kinetics of hybridization of this cDNA with nuclear RNA indicated that the complexity of this RNA population is five to ten times greater than that of cytoplasmic mRNA. The same difference in the fraction of DNA represented was obtained when nuclear and cytoplasmic RNA were hybridized with labeled unique sequence DNA. The fraction of the DNA sequences represented in total number of polyadenylated nuclear RNA is much higher than that represented in cytoplasmic RNA.     

Reassociation of human lymphoblastoid cell DNA repair replicated following methyl methanesulfonate treatment

The reassociation rates of repair replicated DNA of two human lymphoblastoid cell lines, the WIL₂-A3 ‘normal’ line and the RAJI line of Burkitt's lymphoma, were examined using the DNA/DNA ‘C₀t’ hybridization technique. The cells were treated with methyl methanesulfonate (MMS), an alkylating agent and mutagen, to induce the repair.The incorporated repair replication radioactivity in highly repetitive sequences of WIL₂-A3 cell DNA reassociates as expected for a randomly distributed incorporation. The reassociation of repair radioactivity in sequences of fewer numbers of copies, however, is less than expected for a random distribution. It is less than that occurring for semiconservatively synthesized DNA of WIL₂-A3 cells co-incubated with the repair labeled DNA as an internal control.The observed difference could be due to an over-representation of repair replication radioactivity in DNA sequences with fewer copies. It is unlikely to be due to residual alkali labile damage resulting from MMS treatment, since a similar difference was not observed when semiconservatively labeled DNA from cells which had been treated with MMS for the same time and at the same concentration as in the repair experiments was substituted for repair replicated DNA in the reassociation reactions. Other possible causes of the apparent difference in the reassociation rates observed are discussed.     

The anchoring of newly synthesized adenovirus DNA to the nuclear matrix

After adenovirus infected HeLa cells were pulse labeled and pulse-chase labeled with 3H-thymidine, the nuclear matrix and DNA remaining tightly bound to the matrix were obtained by sequential cell fractionation. Measuring the radioactivity of labeled DNA indicated that newly synthesized viral DNA specifically attach to the nuclear matrix and the amount of binding DNA is in direct proportion to the viral DNA replication activity: then the DNA gradually detach from the matrix and are involved in viral assembly. Electron microscopic autoradiography of the extracted cells showed the virion and viral DNA associated with the nuclear matrix, and thus further confirmed the anchoring of newly synthesized viral DNA to the nuclear matrix.     

Early changes in the synthesis of acidic nuclear proteins in human diploid fibroblasts stimulated to synthesize DNA by changing the medium

When resting WI-38 cells in a confluent monolayer were stimulated to proliferate by changing the medium, the incorporation of leucine-³H into nuclear acidic proteins was promptly stimulated, although its incorporation into total cellular proteins was unchanged or even decreased. Three fractions, all acidic by aminoacid analysis, were extracted from the nuclei: (1) ribonucleoproteins (RNP); (2) a fraction extractable with 0.15 M NaC1; and (3) a fraction tenaciously bound to the insoluble residue (residual fraction). A first increase occurred between one and three hours after stimulation in all three fractions. The synthesis of NaCl-soluble proteins then returned to control levels, while the synthesis of residual and RNP proteins remained high between 6 and 12 hours and increased even further at 18 hours, the peak of DNA synthesis. Pulse chase experiments indicated that the proteins synthesized in the first hour after stimulation have a turnover time of less than four hours, while the same fractions in non-proliferating cells were stable for at least 12 hours. 2-mercapto-1-(β-4-pyridethyl) benzimidazole, when added at the same time as the fresh medium, produced an inhibition of the increase in nuclear protein synthesis at one hour, but, if added at five hours after stimulation, it did not inhibit the increase in nuclear protein synthesis occurring at six hours. Actinomycin D (0.01 μg/ml) inhibited both the stimulation of DNA synthesis and the increases in nuclear acidic protein synthesis occurring at one and six hours after stimulation. These results seem to indicate that the serum factors responsible for the stimulation of WI-38 cells, after binding to cells, induce an early synthesis of acidic nuclear proteins which is sensitive to very low doses of actinomycin D. In turn, the newly synthesized proteins could in some way activate in the nuclei the genes that control DNA synthesis and cell division.     

DNA replication and the nuclear envelope

Upon isolation of the nuclear membrane from cultured mouse leukemia L5178Y cells, approximately 1% of the total nuclear DNA was found to be attached to this structure. After pulse labeling of DNA and isolation of the nuclear membrane, the ratio of labeled DNA in the membrane fraction and in the rest of chromatin was compared. Results indicate that with a 3 min pulse, DNA in the membrane fraction showed slightly higher specific activity, but when the pulse was longer than 5 min there was no difference in the specific activities. Since the DNA fragment associated with the membrane fraction was found to be long enough to contain most of the DNA labeled during a 5 min pulse, the results obtained indicate that there is no preferential association of DNA to the nuclear envelope during initiation or elongation of DNA.     

Dynamics of the activity of the normal mouse spleen killers after partial splenectomy

The activity of natural killers from the mouse spleen was determined by 51Cr clearance from labeled target cells (YAC-1) at different times after resection of the two-thirds of the spleen. In the early periods after operation, there was a decrease in the natural cytotoxic activity of splenocytes followed by a relatively rapid rise for 8 days until the maximum exceeding the cytotoxicity of intact spleen cells was reached. However, the total lytic potential of the regenerating spleen did not return to normal during 28 days.     

DNA synthesized in the hepatitis B Dane particle DNA polymerase reaction.

Radioactive DNA was prepared in extensive (4 h) Dane particle DNA polymerase reactions. In different experiments the amount of new DNA, determined by the amount of nucleotide incorporation into an acid-insoluble form, was between 29 and 45% of the total circular DNA isolated from Dane particle preparations after the reaction. DNA reassociation kinetics were used to determine the complexity of the newly synthesized DNA. In different experiments COt1/2 values, corresponding to between 625 and 1,250 nucleotide pairs, were obtained for the radioactive Dane particle DNA. These results suggest that a unique region (or regions), corresponsing to approximately one-fourth to one-half of the circular Dane particle DNA template, was copied one time during the reaction. DNA and RNA extracted from hepatitis B virus-infected liver but not from uninfected liver accelerated the rate of reassociation of radioactive DNA from Dane particles. These Dane particle DNA base sequences were found in alkali-stable, rapidly sedimenting DNA from infected liver as well as in DNA sedimenting at a rate similar to the DNA extracted from Dane particles. These findings are consistent with Dane particle DNA being hepatitis B virus DNA that is integrated into high-molecular-weight cellular DNA and transcribed into RNA in infected liver.     

Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization Assay for Replication Origin Deoxyribonucleic Acid of Escherichia coli

Deoxyribonucleic acid (DNA)-DNA hybridization on nitrocellulose filters can be used to assay for replication origin DNA from Escherichia coli if the DNA attached to the filters is enriched for the replication origin sequences. Such DNA can be readily isolated from very rapidly growing cells. When low amounts of this DNA were attached to filters, radioactively labeled DNA from the replication origin hybridized 1.7 times as well as radioactive replication terminus DNA. Under identical conditions, radioactively labeled DNA from exponentially growing cells hybridized only 1.3 times as well as radioactive replication terminus DNA. The replication origin, replication terminus, and randomly labeled DNA hybridized with similar efficiencies to filters containing DNA isolated from cells incubated in the absence of required amino acids. This DNA appeared to have all sequences present at equal frequencies. The hybridization assay was used to demonstrate that the DNA synthesized shortly after the addition of amino acids to cells previously deprived of required amino acids was primarily from the replication origin and then rapidly became similar to DNA synthesized by exponentially growing cells.     

Activity of deoxyribonucleoside-activated nucleotidase in cells from various lymphoid mouse tissues and in concanavalin A-stimulated lymphocytes

The specific activity in cells from lymph nodes, spleen and thymus was 32, 28 and 25 nmol/min per mg of cytosol protein, respectively, whereas that in bone marrow cells was significantly lower (10 units/mg of protein). No difference in specific DAN activity between isolated B- and T-lymphocytes was observed. Two types of lymphoid mouse cell lines (MOPC-31C plasmacytoma cells, S49 Cyc- lymphoma cells) showed specific activities similar to the normal lymphoid cells. In concanavalin A- stimulated spleen lymphocytes in culture there was a rapid increase in DAN activity shortly after maximum DNA synthesis, reaching a plateau 2-3 times the original level. The enzyme (DAN) of mouse tissues possessed the characteristic properties previously detected for the rat enzyme.     

Effects of Mitogenic and Antigenic Stimulation on the Thymidine Kinases of Mouse Spleen Cells in vivo and in vitro

The DNA-synthesizing ability of mouse spleen cells in vitro and in vivo is paralleled by their levels of cytoplasmic thymidine kinase. However, a very high level of nuclear-associated enzyme activity developed in cultures of both non-stimulated and mitogen-transformed lymphocytes. This activity did not appear in the spleens of mice during the primary immune response to sheep erythrocytes (SRC) or following administration of Convanavalin A (Con A).
Feedback inhibition studies with TTP demonstrated that the nuclear enzymes were more sensitive than the cytoplasmic activities. The thermal stabilities of the nuclear enzymes were also found to be greater than the corresponding cytoplasmic ones. Furthermore, analysis of the rate of thermal inacti-vation indicated that both the cytoplasmic and nuclear enzymes present in transformed lymphocytes in vitro were far more heat-stable than those activities normally found in the mouse spleen, even after antigenic challenge with SRC in vivo or incubation in vitro in the absence of mitogenic stimulation.     

Generation of cytotoxic T lymphocytes in vitro. VI. Effect of cell density on response in mixed leukocyte cultures.

Reexposure of day 14 murine mixed leukocyte culture (MLC) populations to the original irradiated allogeneic stimulating spleen cells has previously been found to result in the ratpid generation of cytolytic T lymphocytes (CTL) associated with a net increase in cultured cell number. Under the experimental conditions used, day 5 MLC cells appeared unable to respond to the allogeneic stimulus. In order to characterize further the development of the potential for anamnestic reactivity during the course of MLC, C57BL/6 spleen cells were incubated with irradiated (1000 rads) DBA/2 spleen cells (primary MLC) for up to 3 weeks. At various time intervals after the onset of the primary MLC, the surviving cells were collected and reexposed, at varying cell concentrations, to irradiated DBA/2 spleen cells (secondary MLC). At daily intervals thereafter, CTL activity was assessed using a quantitative 51Cr-release assay system. A paradoxic effect of responding cell concentration on generation of CTL activity was observed; relatively greater increase in CTL activity was observed as the concentration of responding cells was decreased over a 100-fold range. This effect was more pronounced with responding cells reexposed to antigen after primary MLC for 20 days, but was observed even with normal cells. The apparent unresponsiveness of day 5 MLC cells to alloantigen restimulation could be overcome by simple dilution of responding cells. Cytotoxic activity at the time of restimulation with antigen seems to be a major factor determining the magnitude of the secondary response. Since intact cells bearing alloantigens are required for the generation of CTL in MLC, residual cytotoxic cells reduce the effective antigenic stimulus by destroying stimulating cells. This effect of concentration of responding cells on generation of CTL in MLC complicates interpretation of experiments investigating the role of "inhibitor" and "helper" cell in cell-mediated immune responses occurring in vitro. Under optimal conditions, the highest CTL activity and the largest increase in total cell number was observed 4 days after restimulation of day 10 MLC cells. On a per cell basis, the lytic activity was up to 4 times greater than that observed at the peak of a primary response, and the number of viable cells recovered was nearly 20 times higher than that at the onset. Such secondary MLC are thus a convenient source of lymphoid cells selected primarily on the basis of proliferation induced by alloantigens.     

Metabolic turnover of proliferation-related nuclear proteins in serum-stimulated Swiss mouse 3T3 cells

Stimulation of quiescent Swiss mouse 3T3 cells either by serum or by pure growth factors induces DNA synthesis after a lag period of about 15 h. Following restimulation by serum or by growth factors there is an overall increase of 2-4-fold in the rate of biosynthesis of nuclear proteins. Two nuclear polypeptides show specific temporal correlations with the transition from quiescence to proliferation. The synthesis of p30 (30 kDa, pI 5.2) is at a maximum within 5 h of restimulation, while the synthesis of p36 (36 kDa, pI 4.25) is first seen at 10-12 h after restimulation. The synthesis of p36 correlates well with the initiation of DNA biosynthesis. The metabolic turnover of both of these proteins has been estimated by pulse-chase and by cycloheximide inhibition experiments. They both have a half-life of 10-15 h and appear to be cell-cycle related.     

Characterization of cytoplasmic DNA of mouse-myeloma cells.

Cytoplasmic DNA from mouse myeloma cells comprised between 1% and 2% of the total cellular DNA. Detergent-prepared cytoplasmic lysate consisted mainly of 8-S and 22-S species. While these DNA species were present in the 13000 times g pellet of the detergent-prepared cytoplasmic lysate, only the light DNA species was present in the 13000 times g supernatant fraction. In neutral CsCl gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm3, which is identical to that of nuclear DNA. The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed-circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent-prepared cytoplasmic lysate. It was found that poly (A)-containing mRNA and 28-S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA.     

Changes in mitochondrial DNA levels during development of pea (Pisum sativum L.)

The percentage of mitochondrial DNA (mtDNA) present in total DNA isolated from pea tissues was determined using labeled mtDNA in reassociation kinetics reactions. Embryos contained the highest level of mtDNA, equal to 1.5% of total DNA. This value decreased in light- and dark-grown shoots and leaves, and roots. The lowest value found was in dark-grown shoots; their total DNA contained only 0.3% mtDNA. This may be a reflection of increased nuclear ploidy levels without concomitant mtDNA synthesis. It was possible to compare the mtDNA values directly with previous estimates of the amount of chloroplast DNA (ctDNA) per cell because the same preparations of total DNA were used for both analyses. The embryo contained 1.5% of both mtDNA and ctDNA; this equals 410 copies of mtDNA and 1200 copies of ctDNA per diploid cell. Whereas mtDNA levels decreased to 260 copies in leaf cells of pea, the number of copies of ctDNA increased to 10300. In addition, the levels of ctDNA in first leaves of dark-grown and light-transferred pea were determined, and it was found that leaves of plants maintained in the dark had the same percentage of ctDNA as those transferred to the light.Abbreviations ctDNA chloroplast DNA - mtDNA mitochondrial DNA     

Evidence for attachment of interphase chromatin to the nuclear matrix via matrix-bound nucleosomes

Chromatin structure has been studied in the sites of attachment to the nuclear matrix in interphase mouse liver and spleen nuclei. The patterns of fragmentation of the DNA belonging to these sites (0.3-2% of total DNA in spleen and liver, respectively) with staphylococcal nuclease and DNAase I were very close to those of usual nucleosomal chains. Moreover, the nuclear matrix preparations contained all five major histones, including H1, in almost stoichiometric amounts. The histone/DNA ratios for the matrix were also similar to those found in nuclei. These findings and the size of the matrix-protected DNA indicated that interphase chromatin was attached to the nuclear matrix via matrix-bound nucleosomes and, to a much lesser extent, oligonucleosomes up to 5-6 units long. Two-dimensional electrophoretic separation of the matrix-bound histones revealed that modifications of histone H1 and, probably, of other histones were distinguished from those in bulk chromatin. Study of binding of exogenously added labeled histone octamers or mononucleosomal size DNA to nuclear matrix excluded the possibility of their artifactual trapping during the isolation procedure.