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.     

Characterization of large mammalian DNA species sedimented in a reorienting zonal rotor

The characteristics of a Beckman-designed slow acceleration unit for the reorientation of alkaline sucrose gradients in a Ti-15 zonal rotor are described. The large DNA species (> 250S) obtained from cultured rat brain tumor cells with this system sediment linearly with time, have virtually no [³H]leucinelabeled or covalently bonded [³H]choline-labeled material sedimenting with them, sediment independently of smaller single-stranded DNA molecules (? 165S) and are 60–80% degraded by the single-strand-specific S1 nuclease. Therefore, it is postulated that these species are collapsed, partially denatured DNA molecules or a collapsed form of single-stranded DNA. When cells were labeled with [¹⁴C]TdR, then frozen and stored at ? 79°C, this system could detect radiation-induced DNA damage from decay of the incorporated label at accumulated doses as small as 18–126 rads.     

Fractionation and characterization of satellite DNAs of the kangaroo rat (Dipodomys ordii)

Nuclear DNA from liver cells of the kangaroo rat species Dipodomysordii was fractionated and characterized with the aid of buoyant density gradients in neutral and alkaline CsCl and in Ag⁺-Cs₂SO₄. More than one-half of the DNA was present in three density satellites, a greater proportion than in any other species yet reported; the purified satellite DNAs were denser than principal DNA. All satellite fractions revealed sharp isopycnic bands and narrow denaturation profiles. Two had identical buoyant densities but differed substantially in T_m, base composition, and reassociation kinetics. In alkaline CsCl all three satellites, as well as a shoulder of intermediate repetitive DNA on the heavy side of the principal band, revealed unique strand densities. The most highly repetitive satellite was unusually rich in (G + C) and contained 6.7% of 5-methylcytosine. A survey of internal organs and spermatozoa of an adult male revealed no significant differences in distribution of the satellites among tissues.     

Enzymatic determination of nonrandom incorporation of 5-bromodeoxyuridine in rat DNA.

Secondary cultures of normal rat embryo cells were synchronized by a double thymidine block and pulsed with 10(-7) M 5-[3H]bromodeoxyuridine (BrdUrd) OR 10(-7) M[3H]thymidine during an entire S phase (7.5 h). To examine the pattern of [3H]thymidine, DNA was immediately extracted and purified at the completion of the S phase, CsCl density gradient centrifugation revealed that substitution for thymine by bromouracil was less than 7%. Single-strand specific nucleases obtained from Aspergillus oryzae and Neurospora crassa were allowed to react with native and partially depurinated (24-29%) [3H]BrdUrd-labeled rat DNA samples, and the products were assayed by hydroxylapatite column chromatography. Approximately 4-6% of the native, nondepurinated rat DNA was hydrolyzed by both nucleases. However, 24-28% of the partially depurinated, [3H] thymidine-labeled rat DNA was hydrolyzed by both enzymes as determined by loss of mass as well as radioactivity. Whereas comparable levels of depurinated, [3H]BrdUrd-labeled DNA were physically hydrolyzed by both nucleases, nearly 65% of the radioactivity was not recovered. Native, as well as depurinated, enzyme-treated DNA samples were sequentially and preparatively reassociated into highly repetitive, middle repetitive, and nonrepetitive nucleotide sequence components. The absolute and relative specific activities of each subfraction of native [3H]thymidine-labeled DNA were comparable. [3H]BrdUrd was differentially concentrated in the middle repetitive sequences as compared to other reiteration frequency types. When depurinated, nuclease-treated DNA samples were similarly fractionated, [3H]thymine moieties were uniformly distributed thoughout all sequences. However, a differential loss of [3H]BrdUrd moieties was detected predominantly from the middle repetitive nucleotide fraction. Melting profiles of the renatured DNA samples were characteristic of each respective DNA subfraction regardless of isotopic precursor. These results suggest that [3H]BrdUrd may be differentially incorporated into A + T rich clusters of rat DNA, especially in the moderately repeated chromosomal elements.     

An analysis of the bovine genome by Cs2SO4-Ag density gradient centrifugation

Calf DNA preparations having molecular weights of 5 to 7 × 10⁶ have been fractionated by preparative Cs₂SO₄—Ag⁺ density gradient centrifugation into a number of components. These may be divided into three groups: (1) the main DNA component (1.697 g/cm³; all densities quoted are those determined in CsCl density gradients), the 1.704 and 1.709 g/cm³ components form about 50, 25 and 10% of the genome, respectively; they are characterized by having symmetrical CsCl bands and melting curves, both of which have standard deviations close to those of bacterial DNAs of comparable molecular weight, and by their G + C contents being equal to 39, 48 and 54%, respectively; after heat-denaturation and reannealing, their buoyant densities in CsCl are greater than native DNA by 12, 10 and 3 mg/cm³, respectively. (2) The 1.705, 1.710, 1.714 and 1.723 g/cm³ components represent 4, 1.5, 7 and 1.5% of the DNA, respectively, and exhibit the properties of “satellite” DNAs; their CsCl bands and melting curves have standard deviations lower than those of bacterial DNAs; after heat-denaturation and reannealing, their buoyant densities are identical to native DNA, except for the 1.705 g/cm³ component, which remains heavier by 5 mg/cm³; in alkaline CsCl, only the 1.714 g/cm³ component shows a strand separation. (3) A number of minor components, forming 1% of the DNA, have been recognized, but they have not been investigated in detail; two of them (1.719 and 1.699 g/cm³) might correspond to ribosomal cistrons and mitochondrial DNA, respectively.     

Characterization of Astasia longa ribonsomal DNA.

Ehrlich tumour cell nucleoli contain fibrillar and granular components and low electron density areas called “fibrillar centres”. Analysis by high-resolution autoradiography using [³H]actinomycin D or [³H]TdR reveals that a small amount of DNA is present inside the fibrillar centres. Newly synthesized RNA is also present within the fibrillar centres or at their periphery, already 3 min after the precursor is given. According to these results, RNA is synthesized on DNA in the fibrillar centres. It is possible that the latter contain dispersed genetically active chromatin. These observations add further support to the hypothesis that fibrillar centres have a chromosomal origin and are related to the nucleolar organizers.     

DIFFERENT LABELLING PATTERNS IN MOUSE LYMPHOID TISSUES WITH [3H]DEOXYCYTIDINE AND [3H]THYMIDINE

The percentages of labelled lymphocytes in smear preparations of mouse thymus were higher than those in similar preparations of mesenteric lymph nodes with either generally labelled tritiated deoxycytidine, [³H]CdR, or tritiated thymidine, [³H]TdR. Lymphocytes in the thymus cortex and in germinal centres of mesenteric lymph nodes were intensely labelled with [³H]CdR, whereas with [³H]TdR lymphocytes in the peripheral region of thymus and medullary cords of mesenteric lymph nodes were heavily labelled. The majority of lymphocytes in thymic cortex and germinal centres of mesenteric lymph nodes were labelled weakly with [³H]TdR. Thus, labelling patterns with [³H]CdR differed from those with [³H]TdR in lymphoid tissues of the mouse. Mouse lymphocytes can utilize [³H]CdR as a precursor molecule for cytosine and thymine in DNA. The ratio of radioactivity of thymine to that of cytosine was measured biochemically in DNA extracted from lymphocytes labelled with [³H]CdR. This radioactivity ratio in thymus was higher than that in mesenteric lymph nodes. These results suggest that the metabolic activities of utilizing CdR for DNA synthesis differ within lymphocyte populations in various lymphoid tissues in the mouse.     

Inorganic Phosphorus Stimulation of Bacterioplankton Production in a Meso-Eutrophic Lake

Experiments were conducted to determine whether production of heterotrophic bacterioplankton in a small meso-eutrophic lake was influenced by the dissolved inorganic phosphorus (DIP) supply. DIP may indirectly limit bacterial production by limiting phytoplankton, which in turn may limit the carbon available to bacteria. Direct DIP limitation of bacteria occurs where the availability of DIP for bacteria is insufficient to maintain growth. This work examined direct DIP limitation of bacteria by removing phytoplankton and incubating flasks with or without added P in the dark. Bacterial production was measured via the rate of incorporation of [³H]thymidine ([³H]TdR) into DNA. Bacterial abundance was followed with epifluorescent direct counts. Rates of [³H]TdR incorporation were significantly greater in flasks with added DIP, and changes in cell abundances generally paralleled increases in [³H]TdR incorporation. Even very small additions of P (0.05 μM) were sufficient to stimulate production. DIP addition to whole lakewater also stimulated [³H]TdR incorporation relative to that in zero-addition controls, but there was not a concurrent increase in bacterial cell numbers. The stimulation of [³H]TdR incorporation after DIP addition to whole lakewater was significantly less than the stimulation due to DIP addition to 1-μm-pore-size-filtered lakewater. In this study, addition of DIP caused as much as an eightfold stimulation of [³H]TdR incorporation.     

The isolation of a low molecular weight inhibitor of [H]TdR incorporation into hepatic DNA

An inhibitor of [³H]TdR incorporation into rat liver DNA has been partially purified from bovine and rat livers and from bovine serum. The material isolated does not contain TdR: it is species non-specific, tissue-specific, and non-cytotoxic, and may contain a hepatic chalone.     

The isolation of a low molecular weight inhibitor of [3H]TdR incorporation into hepatic DNA.

An inhibitor of [³H]TdR incorporation into rat liver DNA has been partially purified from bovine and rat livers and from bovine serum. The material isolated does not contain TdR: it is species non-specific, tissue-specific, and non-cytotoxic, and may contain a hepatic chalone.     

Reciprocal induction of cell division by cells of complementary mating types in Tetrahymena

Chick embryo fibroblasts in monolayer culture were synchronized by contact inhibition and serum starvation. Nuclear DNA isolated from the [³H] thymidine pulse-labelled cells throughout the period of DNA synthesis (S phase) was analysed by hydroxylapatite chromatography after renaturation at different C₀t values. It is shown that repeated sequences having different frequencies of reassociation, replicate differently throughout the S period. In order to study the distribution of the repeated sequences, DNA isolated during the S period was fractionated according to its buoyant density. It is shown that only some of the highly reiterated sequences which are included in the high buoyant density DNA fractions, replicate equally well during the early and the late S periods. By contrast, reiterated sequences of the low buoyant density DNA fractions replicate mainly during the late S period.     

Analysis of plant genomes. V. Comparative study of molecular properties of DNAs of seven Allium species

The genomes of seven plant species belonging to the genus Allium and exhibiting a threefold variation in their nuclear DNA content were analyzed by studying their reassociation kinetics, equilibrium centrifugation behavior in neutral CsCl gradients, and melting properties. The reassociation kinetics experiments revealed the presence of 44–65% repeated DNA sequences. A comparison between DNA contents and the proportion of repeated DNA sequences indicated that, in Allium, increase in the genome size is not exclusively due to variations in the proportions of repetitive DNA. The total DNA as well as the various repetitive DNA fractions in all the Allium species examined exhibited, in spite of a few differences, a gross similarity in their behavior in neutral CsCl gradients and in their melting properties.     

The effect of natural selection on enzymic catalysis.

The karyotype of Drosophila nasutoides reveals a very large autosome pair at the metaphase plate. The application of the C-banding technique shows that this chromosome is almost entirely heterochromatic and an isochromosome (Cordeiro et al., 1975). Examination of the DNA isolated from purified nuclei of D. nasutoides in neutral CsCl gradients reveals four major satellites. As much as 60% of the total DNA appears as satellites in the DNA from larval brains. The buoyant densities of the four satellites, designated as I through IV in the order of descending density, are 1.687, 1.682, 1.669 and 1.665 g/cm³, respectively. All four satellites show strand separations in alkaline CsCl gradients with the least separation in satellite III. Thermal denaturation studies with purified native satellites show that satellites I and IV consist of repeats of identical sequences, whereas satellites II and III show a large sequence variation between repeating units. As much as 10 to 24% base-pair mis-matching is observed in the reassociated satellite II. The sequence complexities obtained from DNA reassociation kinetics data are 5, 103, 2.3 × 10⁶ and 46 nucleotide pairs for the satellites I, II, III and IV, respectively. The complexity of satellite III is almost as large as that of Escherichia coli, when the reassociation rate is corrected according to the amount of mis-matching in this satellite. All four satellite sequences are localized in one chromosome (dot chromosome) according to in situ hybridizations to polytene chromosomes. The large heterochromatic chromosome seen at the metaphase plate appears as the dot chromosome after polytenization. Therefore, the large heterochromatic chromosome contains all four satellite DNA components.     

Synthesis of herpes simplex virus DNA in a soluble nuclear extract.

A soluble extract prepared from nuclei of HeLa cells infected with herpes simplex virus type 1 has been found to synthesize herpes DNA in a process comparable to that observed in intact nuclei. The incorporation of [³H]dTTP has an absolute requirement for Mg⁺⁺ and for the other three deoxyribonucleoside triphosphates, and is relatively independent of added ATP. The reaction product, although of relatively short chain length, bands in CsCl density gradients at the density of herpes DNA and is essentially free of labeled cell DNA. Incorporation of BrdUTP results in a density shift suggesting extensive replication of endogenous DNA sequences.     

EFFECT OF TRITIATED THYMIDINE ON THE KINETICS AND VIABILITY OF 9L CELLS IN VITRO

The colony-forming efficiency of 9L rat gliosarcoma cells was unaffected by treatment with 0.1 μCi/ml of [³H]TdR. However, when cells were treated with 1 or 10 μCi/ml of [³H]Tdr, cell growth was reduced and cell survival decreased. When monolayer 9L cells were treated with 1 μCi/ml of [³H]TdR for up to 72 hr, approximately 5% survived, which is closely related to the percentage of non-cycling cells in this system. When cells were treated with 10 μCi/ml of [³H]TdR for 72 hr, less survival was observed. the additional cell kill observed may be induced by [³H]TdR released from doomed cells into petri dishes during the incubation period of the colony-formation assay.     

The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment

Single-cell and DNA fiber autoradiography, cytophotometry and velocity sedimentation in alkaline sucrose gradients were used to analyse DNA replication and nascent replicon maturation in 5-fluorodeoxyuridine (FUdR)-synchronized cells of Pisum sativum. The replicon size was not significantly changed by the protracted FUdR treatment. When the synchronized cells were released from the inhibitor, labeled with [³H]TdR for 30 min, and chased in medium containing 1 × 10⁻⁶ M or lower concentrations of cold thymidine, DNA replication stopped after approx. 25% of the genome had replicated, and the nascent strands failed to grow above 9–12 × 10⁶ D single-stranded (ss) DNA. When the cells were chased in medium with 1 × 10⁻⁵ M cold thymidine, the DNA content of the labeled cells steadily increased with time and the size of the nascent molecules grew continuously until replicon size was achieved; then they were accumulated at replicon size until the cells arrived in late S or G2. When the FUdR-synchronized cells were chased in medium containing 1 × 10⁻⁴ M cold thymidine, the size of the nascent strands increased continuously with time, indicating that some neighbouring nascent replicons were joined as soon as they completed their replication. These observations led us to postulate that in FUdR-synchronized cells the rates of chain elongation, cell progression through the S phase and nascent replicon maturation are controlled by thymidine availability.     

The replication of mouse skin epidermal DNA to which is bound 7,12-dimethylbenz(a)anthracene

Experiments were carried out to determine in the intact mouse whether or not mouse skin epidermal DNA to which the polycyclic hydrocarbon DMBA was bound could serve as a template for further DNA replication. Mice which were treated topically with [³H]7,12-dimethylbenz(a)anthracene ([³H]DMBA) received 5-bromodeoxyuridine (BUdR) and 5-fluorouracil (5-FU) in order to incorporate BUdR into replicating DNA which was stimulated to undergo synthesis one or two days later. Epidermal DNA was put on a neutral CsCl gradient and binding of [³H]DMBA was found to both replicated and non-replicated DNA. Separation of the BUdR substituted and non-substituted parental strands of newly replicated DNA an on alkaline CsCl, Cs₂SO₄ gradient showed that the great majority of DMBA was bound to parental strand DNA. The possibility that [³H]DMBA binding was taking place at the same time that labeling with BUdR occurred was eliminated. Thus, these experiments showed that in the intact mouse, skin epidermal DNA to which DMBA is bound can serve as a template for further DNA synthesis.     

Measuring bacterial production via rate of incorporation of [3H]thymidine into DNA

Thymidine (TdR) incorporation into DNA as a measure of bacterial production in environmental samples relies on assumptions about what organisms incorporate exogenous thymidine, extent of dilution of labelled thymidine by internal and external pools, and analytical methods for recovery and purification of bacterial DNA. We have examined these assumptions with regard to the feasibility of using [³H]TdR incorporation in the water column and sediments of a blackwater river. The extent of dilution of added [³H]TdR may be determined with isotope dilution plots (Moriarty and Pollard, 1981 and 1982) and these indicate a wide range of degree of participation of added [³H]TdR. Previously described methods for extracting DNA from sediment bacteria may lead to underestimates and we described a more efficient recovery scheme.     

Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies

The present experiments with [14C]-thymidine (TdR) and [3H]-bromodeoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 microgram g body weight-1 and that of BrdU is about 5.0 micrograms g body weight-1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [3H]-TdR doses commonly used in cell kinetic studies this proportion is only 0.1-1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [3H]-TdR.     

Repair replication and unscheduled DNA synthesis in mammalian cell lines showing differential sensitivity to alkylating agents

Repair replication has been measured by CsCl density gradient centrifugation in cell lines showing differential sensitivity to mono- and bifunctional alkylating agents. A correlation between cellular sensitivity as measured by the D₀ value and amount of repair replication was demonstrated after exposure of Yoshida cells to nitrogen mustard (HN2) and methylene dimethanesulphonate (MDMS). No differences in the amount of repair replication after methyl methanesulphonate (MMS) were observed in two L5178Y cell lines which differed in sensitivity by virtue of the shoulder size only. The Yoshida cell lines showed no difference in sensitivity to MMS and no difference in amount of repair replication. Incorporation of tritiated thymidine 9[³H]TdR) after drug treament was also measured by autoradiography. The qualitative differences observed between the two cell lines were similar to those obtained in density gradient experiments. The temporal pattern of [³H]TdR uptake indicated that the reduced repair replication observed in the sensitive line after HN2 and MDMS is not due to slower synthesis. The kinetics of [^3H]TdR incorporation differed for all three mutagens suggesting that different enzymes may be involved in each case.