Hydroxyapatite Chromatography and Formamide Denaturation of Adenovirus DNA

Denatured adenovirus DNA was retained by hydroxyapatite columns under conditions generally used for selective retention of double-stranded DNA, probably due to several partially complementary sequences within single-stranded DNA. It was found that addition of formamide reduced the fraction of sonically treated, denatured adenovirus DNA bound to hydroxyapatite from about 30% to less than 1%. This led to a study of the effect of formamide on the melting temperature (T(m)) of double-stranded DNA in solution or bound to hydroxyapatite. The T(m) of DNA decreases 0.56 C/1% formamide, a value determined in buffered solutions with purified formamide.     

Conformational transition of DNA induced by cationic lipid vesicle in acidic solution: spectroscopy investigation

The conformational transition of DNA induced by the interaction between DNA and a cationic lipid vesicle, didodecyldimethylammonium bromide (DDAB), had been investigated by circular dichroism (CD) and UV spectroscopy methods. We used singular value decomposition least squares method (SVDLS) to analyze the experimental CD spectra. Although pH value influenced the conformation of DNA in solution, the results showed that upon binding to double helical DNA, positively charged liposomes induced a conformational transition of DNA molecules from the native B-form to more compact conformations. At the same time, no obvious conformational changes occurred at single-strand DNA (ssDNA). While the cationic lipid vesicles and double-strand DNA (dsDNA) were mixed at a high molar ratio of DDAB vesicles to dsDNA, the conformation of dsDNA transformed from the B-form to the C-form resulting in an increase in duplex stability (DeltaT(m)=8+/-0.4 degrees C). An increasing in T(m) was also observed while the cationic lipid vesicles interacted with ssDNA.     

INCREASE OF DNA POLYMERASE ACTIVITY FIRMLY BOUND TO NUCLEI DURING THE DNA SYNTHETIC PHASE OF THE CELL CYCLE

DNA polymerase activities were measured on nuclear and supernatant fractions obtained from hamster fibroblast cells (the Don-C clone) grown in tissue culture and mitotically synchronized by selective removal of cells arrested in metaphase following a brief exposure to colcemid. A reproducible fraction (5–10%) of the polymerase activity was found to remain bound in the nuclear pellet after repeated cycles of freezing and thawing. The specific activity of this firmly-bound nuclear DNA polymerase was found to increase during S-phase in proportion to DNA synthesis. The bulk of this activity, after extraction in 1 m salt, exhibited an S value of 8·7 on neutral high salt sucrose gradients and was 24 times more active with poly dA. dT₁₀ as template than with heat denatured DNA. The rest of the cellular DNA polymerase activity showed no significant variation correlated with the cell cycle. This activity also had an S value from 8 to 9 but it was only 2·8 times more active with the homopolymer template than with heat denatured DNA. DNA polymerase activity similar to the firmly-bound activity was found in extracts prepared from metaphase chromosomes.     

Detection and typing of Helicobacter pylori cagA/vacA genes by radioactive,one-step polymerase chain reaction in stool samples from children

The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian children using the QIAamp Kit following the manufacturer's instructions but with modifications. DNA templates were amplified for the vacA s and m regions and for the cagA gene by PCR using radioactively labeled (32P) primers. The modifications in the standard Qiagen protocol of stool DNA extraction increased the final concentration of eluted total stool DNA 4.7 times (117 +/- 17 versus 22 +/- 3 ng/microl; P < 0.0001). Nevertheless, its amplification by regular PCR programs (30-40 cycles) did not generate visible signals because of the very low ratio of H. pylori DNA to other DNA. PCR for 80 cycles successfully amplified vacA in 36/39 samples (sensitivity, 92.3%) and cagA fragments in 21/39 (53.8%) fecal DNA samples. Both s and m vacA regions were amplified in 33/36 (91.7%) DNA samples. The s1m1 genotype was the most commonly isolated variant, accounting for 17/36 or 47.2% of positive samples. The s2m2 genotype was ascertained to be frequent also (14/36 or 38.9%). Almost all (94.1%) s1m1 genotypes were cagA positive. The majority of s2m2 genotypes (78.6%) were not associated with the cagA gene. Neither cagA nor vacA fragments were amplified from DNA isolates of H. pylori-uninfected children nor from DNA isolated from six gastrointestinal bacterial strains (specificity, 100%). The data suggest that the proposed modified technique of DNA extraction and PCR assay of stool samples may be an effective and reliable noninvasive tool for the detection and typing of H. pylori cagA/vacA virulence genes in infected individuals.     

Physicochemical studies of human O6-methylguanine-DNA methyltransferase

O6-Methylguanine-DNA methyltransferase, present in most organisms, removes mutagenic and carcinogenic O6-alkylguanine from DNA by accepting the alkyl group in a stoichiometric reaction. The protein has been partially purified from human placenta. It reacts with second-order rate constants of 2.20 x 10(8) and 0.067 x 10(8) lmol-1 min-1 at 37 degrees C for duplex and single-stranded DNA substrates, respectively. The corresponding value for the alkylated base in synthetic poly(dC, dG, m6dG) is 0.02 x 10(8) l mol-1 min-1. The native protein is monomeric with a molecular mass of 22-24 kDa. Methylation of the protein does not lead to a gross change in its conformation but causes a slight reduction in its isoelectric point of 6.2. Although DNA protects the protein from heat inactivation, both duplex and single-stranded DNAs inhibit its activity in a concentration-dependent manner. The transferase reaction rate is also strongly inhibited by salt with about 20% of the maximum rate observed in physiological ionic strength. This inhibition is nonspecific with respect to the ions of univalent salts.     

Concentration of viruses and dissolved DNA from aquatic environments by vortex flow filtration.

Vortex flow filtration (VFF) was used to concentrate viruses and dissolved DNA from freshwater and seawater samples taken in Florida, the Gulf of Mexico, and the Bahamas Bank. Recoveries of T2 phage and calf thymus DNA added to artificial seawater and concentrated by VFF were 72.8 and 80%, respectively. Virus concentrations determined by transmission electron microscopy of VFF-concentrated samples ranged from 3.4 x 10(7)/ml for a eutrophic Tampa Bay sample to 2.4 x 10(5) for an oligotrophic oceanic surface sample from the southeastern Gulf of Mexico. Viruslike particles were also observed in a sample taken from a depth of 1,500 m in the subtropical North Atlantic Ocean. Filtration of samples through Nuclepore or Durapore filters (pore size, 0.2 micron) prior to VFF reduced phage counts by an average of two-thirds. Measurement of dissolved-DNA content by Hoechst 33258 fluorescence in environmental samples concentrated by VFF yielded values only ca. 35% of those obtained for samples concentrated by ethanol precipitation (the standard dissolved-DNA method). However, ethanol precipitation of VFF-concentrated extracts resulted in an increase in measurable DNA, reaching 80% of the value obtained by the standard method. These results indicate that a portion of the naturally occurring dissolved DNA is in a form inaccessible to nucleases and Hoechst stain, perhaps bound to protein or other polymeric material, and is released upon ethanol precipitation. Viral DNA contents estimated from viral counts averaged only 3.7% (range, 0.9 to 12.3%) of the total dissolved DNA for samples from freshwater, estuarine, and offshore oligotrophic environments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentration of viruses and dissolved DNA from aquatic environments by vortex flow filtration.

Vortex flow filtration (VFF) was used to concentrate viruses and dissolved DNA from freshwater and seawater samples taken in Florida, the Gulf of Mexico, and the Bahamas Bank. Recoveries of T2 phage and calf thymus DNA added to artificial seawater and concentrated by VFF were 72.8 and 80%, respectively. Virus concentrations determined by transmission electron microscopy of VFF-concentrated samples ranged from 3.4 x 10(7)/ml for a eutrophic Tampa Bay sample to 2.4 x 10(5) for an oligotrophic oceanic surface sample from the southeastern Gulf of Mexico. Viruslike particles were also observed in a sample taken from a depth of 1,500 m in the subtropical North Atlantic Ocean. Filtration of samples through Nuclepore or Durapore filters (pore size, 0.2 micron) prior to VFF reduced phage counts by an average of two-thirds. Measurement of dissolved-DNA content by Hoechst 33258 fluorescence in environmental samples concentrated by VFF yielded values only ca. 35% of those obtained for samples concentrated by ethanol precipitation (the standard dissolved-DNA method). However, ethanol precipitation of VFF-concentrated extracts resulted in an increase in measurable DNA, reaching 80% of the value obtained by the standard method. These results indicate that a portion of the naturally occurring dissolved DNA is in a form inaccessible to nucleases and Hoechst stain, perhaps bound to protein or other polymeric material, and is released upon ethanol precipitation. Viral DNA contents estimated from viral counts averaged only 3.7% (range, 0.9 to 12.3%) of the total dissolved DNA for samples from freshwater, estuarine, and offshore oligotrophic environments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the DNA of a Nonoccluded Baculovirus, Hz-1V

The DNA of the nonoccluded baculovirus (Hz-1V) obtained from the IMC-Hz-1 cell line was characterized by physicochemical and restriction endonuclease techniques. Hz-1V DNA isolated from purified virus had buoyant densities of 1.58 and 1.54 g/ml in CsCl-ethidium bromide density gradients, which corresponded to supercoiled and to relaxed circular and linear DNA, respectively. Neutral CsCl equilibrium centrifugation indicated that the Hz-1V DNA had a buoyant density of 1.7024 g/ml, which corresponded to a guanine-plus-cytosine (G+C) content of 43%. Thermal denaturation indicated a high G+C domain(s) in the Hz-1V genomic DNA. The domain(s), which included about 11% of the total genomic DNA, exhibited a T(m) of 97 degrees C. The remaining portion (89%) of the DNA had a T(m) of 86.5 degrees C. The T(m)s corresponded to G+C contents of 42 and 67%, respectively. The mean genetic complexity of Hz-1V DNA determined by DNA reassociation kinetic analysis was found to be 152 x 10(6). A possible rapidly reassociating component comprising approximately 13% of the genome was observed. The mean molecular weights from restriction endonuclease digests were 159 x 10(6) for both HindIII and EcoRI. Genomic heterogeneity was found in both the wild-type Hz-1V stock and in two plaque isolates. Of 12 single-plaque isolates, 3 basic restriction endonuclease DNA fragment patterns were observed. The molecular size estimates from electron microscopic contour lengths of uncloned viral DNA ranged from 70 to 158 megadaltons, and the mode was the 130- to 140-megadalton class.     

Semi-conservative synthesis of DNA in UV-sensitive mutant cells of Chinese hamster after UV-irradiation

A study was made of the rate of semi-conservative DNA synthesis in asynchronous UV-resistant (clone V79) and UV-sensitive clones (VII and XII) of Chinese hamster cells after UV-irradiation. In all 3 clones studied, UV-irradiation (5-30 J/m2) induced a decrease in the rate of DNA synthesis during the subsequent 1-2 h. In the resistant clone (V79) recovery of DNA synthesis rate started after the first 2 h post-irradiation (5 J/m2) and by the 3rd hour reached its maximum value, which constituted 70% of that observed in control, non-irradiated cells. The UV-sensitive mutant clones VII and XII showed no recovery in the rate of DNA synthesis during 6-7 h post-irradiation. The results obtained show that the survival of cells is correlated with the ability of DNA synthesis to recover after UV-irradiation in 3 clones studied. The observed recovery of UV-inhibited DNA synthesis in mutant clones may be due to certain defects in DNA repair.     

Levels and stability of DNA methylation in random surviving cell clones derived from a Chinese hamster cell line after prolonged treatment with 5-aza-2'-deoxycytidine

SCC30 cells (derived from a single cell from the Chinese hamster ovary CHO-K1 cell line, selected on the basis of a stable chromosome complement) were used to select cell variants with hypomethylated DNA. Cells were treated with 5-aza-2'-deoxycytidine (5azadCyd) at 0.1, 1, or 5 microM for two weeks with the medium and drug renewed twice weekly. From the few surviving cells, 25 random single cell-derived clones were grown for freezing cell stocks, and for DNA isolation for 5-methyldeoxycytidine (5medCyd) estimations. After a minimum of one month's recovery from the drug, these cells showed a continuum of 5medCyd levels ranging from ones with the same as the parental clone (2.93%) to ones having lost almost 50% of their DNA methylation. The modal value corresponded to a loss of one third to one quarter of methylated sites. Five subclones with hypomethylated DNA were grown from the frozen stocks. These cells were shown not to be 5azaCyd-resistant cell variants. By the time sufficient cells had been grown to determine DNA methylation levels, the average percentage of 5medCyd had increased to 76% of the SCC30 value compared to 67% at the time of freezing cell stocks. However, this level of DNA hypomethylation remained constant over two months of continuous culture. Cells of one of these hypomethylated subclones were subjected to a second cycle of 5azaCyd treatment. Six random clones from the survivors showed a further decrease averaging 11% in the level of DNA methylation but, by two months in continuous culture, 5medCyd levels had returned to that present before the second cycle of selection. Hence, cell variants can be readily obtained which have lost some 8-10 million methylated sites (pairs of methylated deoxycytidines), and this loss does not compromise cell viability in in vitro culture. This is consistent with mammalian genomes containing a high level of background methylation in non-essential sites. The usefulness of such single cell-derived clones with stably hypomethylated genomes is discussed in relation to understanding the functions of deoxycytidine methylation in mammalian DNA.     

Effect of the number of nucleic acid oligomer charges on the salt dependence of stability (DeltaG 37degrees) and melting temperature (Tm): NLPB analysis of experimental data

For nucleic acid oligomers with variable chain lengths, the salt concentration ([salt]) dependences of the denaturation temperature (T(m)) and of the free energy of helix formation at 37 degrees C (Delta) are predicted using nonlinear Poisson-Boltzmann (NLPB) calculations. Analysis of experimental data reveals that the ratio of the [salt] derivative of melting temperature (ST(m) = dT(m)/d log[salt]) to the value for a polymer with the same base composition (ST(m)/ST(m, infinity)) is independent of base composition but strongly dependent on the number of DNA charges (/Z/) below approximately 8 bp for two-strand helices (formed from association of two complementary strands) and below approximately 18 bp for hairpin helices (formed from folding of one self-complementary strand). We interpret these ST(m)/ST(m, infinity) ratios in terms of the ratio of thermodynamic ion release from the oligomer (Deltan(u), per charge) to that from the same oligomer embedded in polymeric DNA (Deltan(u, infinity), per charge). Experimental values of ST(m)/ST(m, infinity) and its dependence on /Z/ are in good agreement with NLPB predictions for a preaveraged (essential structural) model of DNA. In particular, the NLPB calculations describe the stronger /Z/ dependence of ST(m) observed for melting of oligomeric hairpin helices than for melting of two-strand helices. These calculations predict an experimentally detectable (>or=10%) difference between ST(m) and ST(m, infinity) which increases strongly with decreasing length for two-strand helix lengths of <15 bp and for hairpin helix lengths of <30 bp. From NLPB values of Deltan(u)/Deltan(u, infinity), we predict Delta as a function of [salt] and /Z/. Predictions of thermodynamic and thermal stabilities of oligomeric helices as functions of length and [salt] are consistent with and represent a significant refinement of the average oligomer salt effect currently in use in nearest neighbor stability predictions.     

Thermal denaturation of calf thymus DNA: existence of a GC-richer fraction

In 2.5 x 10(-4)M EDTA buffer, the derivative melting curve of calf thymus DNA shows a major band at 47 degrees with a shoulder at about 54 degrees . The fraction of melting area of this shoulder is about 13%. For reconstituted polylysine-calf thymus DNA complexes, in addition to the melting of free DNA regions at about 50 degrees (T(m)) there is another melting at about 106 degrees (T(m)) of polylysine-bound regions. The melting band of the complex at T(m) is not symmetrical. As more polylysine is bound to DNA the melting amplitude is diminished greatly on the major band at 47 degrees but only slightly on the shoulder at 54 degrees . The insensitivity of this shoulder appears to result from the existence of a 13% fraction of calf thymus DNA containing 55% GC. It is not favorably bound by polylysine. It remains in the supernatant after centrifugation and melts at about 54-56 degrees . This conclusion is further supported by two facts: the reconstitution method provides a condition for selective binding of polylysine to AT-rich DNA, and it yields a fully symmetric melting band at T(m) for complexes of polylysine with homogeneous bacterial DNA such as the one from M. luteus.     

Relative affinities of divalent polyamines and of their N-methylated analogues for helical DNA determined by 23Na NMR

Interactions of divalent polyamines with double-helical DNA in aqueous solution are investigated by monitoring the decrease in 23Na NMR relaxation rates as NaDNA is titrated with H3N(+)-(CH2)m-+NH3, where m = 3, 4, 5, or 6. Analogous measurements are made for the same homologous series of methylated polyamines (methonium ions). The dependence of the 23Na relaxation rates on the amount of added divalent cation (M2+) is analyzed quantitatively in terms of a two-state model. The sodium ions are assumed to be in rapid exchange between a "bound" state, where they are close enough to DNA so that it affects their relaxation rate, and a "free" state in bulk solution, where their relaxation rate is the same as in solutions containing no DNA. The distribution of Na+ and M2+ between these states is described quantitatively in terms of an ion-exchange parameter: DM = (pMB)(1-pNaB)n/(pNaB)n(1-pMB), where pNaB and pMB are the fractions of Na+ and M2+ that are close enough to DNA to be considered bound (by the NMR criterion), and n is the number of sodium ions displaced from DNA by the binding of one M2+ ion. For each of the polyamines and methonium ions investigated here, equations derived from this two-state model yield acceptable fittings of the titration curves if roNa, the number of sodium ions bound per DNA phosphate when no competing cations are present, is assigned a value between 0.6 and 1.00. Within this range, changing the value assigned to roNa does change the best-fitted values of DM determined for these polyamines (DH) and for the methonium ions (DMe) but does not alter the following conclusions about the trends in these parameters. (1) For polyamines and methonium ions of the same m, DH exceeds DMe by factors that are significantly larger for m = 3 and 4 than for m = 5 and 6. (2) DH for m = 3 and 4 is larger than DH for m = 5 and 6. (3) DMe for m = 3 and 4 is smaller than DMe for m = 5 and 6.     

A simple epigenetic method for the diagnosis and classification of brain tumors

The new, simple, and reliable method for the diagnosis of brain tumors is described. It is based on a TLC quantitative determination of 5-methylcytosine (m(5)C) in relation to its damage products of DNA from tumor tissue. Currently, there is evidence that oxidative stress through reactive oxygen species (ROS) plays an important role in the etiology and progression of several human diseases. Oxidative damage of DNA, lipids, and proteins is deleterious for the cell. m(5)C, along with other basic components of DNA, is the target for ROS, which results in the appearance of new modified nucleic acid bases. If so, m(5)C residue constitutes a mutational hotspot position, whether it occurs within a nucleotide sequence of a structural gene or a regulatory region. Here, we show the results of the analysis of 82 DNA samples taken from brain tumor tissues. DNA was isolated and hydrolyzed into nucleotides, which, after labeling with [gamma-(32)P]ATP, were separated on TLC. Chromatograms were evaluated using PhosphorImager and the amounts of 5-methyldeoxycytosine (m(5)dC) were calculated as a ratio (R) of m(5)dC to m(5)dC + deoxycytosine + deoxythymidine spot intensities. The R value could not only be a good diagnostic marker for brain tumors but also a factor differentiating low-grade and high-grade gliomas. Therefore, DNA methylation pattern might be a useful tool to give a primary diagnosis of a brain tumor or as a marker for the early detection of the relapse of the disease. This method has several advantages over those existing nowadays.     

Peptide nucleic acid-DNA duplexes containing the universal base 3-nitropyrrole

A peptide nucleic acid (PNA) monomer containing the universal base 3-nitropyrrole was synthesized by coupling 1-carboxymethyl-3-nitropyrrole to ethyl N-[2-(tert-butoxycarbonylamino)ethyl]glycinate. The PNA sequence H-TGTACGTXACAACTA-NH2 (X = 3-nitropyrrole and C) and DNA sequence 5'-TGTACGTXACAACTA-3' were synthesized and thermal melting studies with the complementary DNA sequence 5'-TAGTTGTYACGTACA-3' (Y = A,C, G, T) compared. The T(m) data show that 3-nitropyrrole pairs indiscriminately with all four natural nucleobases as a constituent of either DNA or PNA. However, 3-nitropyrrole-containing PNA-DNA (average T(m) value = 51.1 degrees C) is significantly more thermally stable than 3-nitropyrrole-containing DNA-DNA (average T(m) value = 39.6 degrees C). From circular dichroism measurements, it is apparent that 3-nitropyrrole in the PNA strand causes a significant change in duplex structure.     

Die Verteilung der Chromosomen auf die Tochterzellkerne multipolarer Mitosen in euploiden Gewebekulturen von Microtus agrestis

In kidney epithelial cultures from female Microtus agrestis, 3,55% of all mitoses are multipolar, 94% of them tripolar. Feulgen photometric measurements of 21 tripolar mitoses reveal a total DNA amount corresponding to the mitotic diploid value (4c) in 5 cases, and to the tetraploid value (8c) in 16 cases, Diploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei each with a haploid DNA value (1c). Most tetraploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei with a triploid DNA value (3c). Also the sex chromosomes are distributed to the daughter nuclei in the relation of 2∶3∶3. This can be seen in anaphase figures as well as in interphase nuclei presumably derived from tripolar mitoses, showing chromocenters according to the number of X-chromosomes. In two cases of tripolar tetraploid mitoses the resulting nuclei have a haploid, a triploid and a tetraploid DNA value. The DNA replication pattern is always identical in the daughter nuclei of diploid and tetraploid tripolar mitoses. — Our observations suggest segregation and distribution of haploid chromosome sets or multiples of haploid sets to the daughter nuclei of multipolar mitoses. They also show a possible way of formation of haploid and triploid cells in a basically diploid tissue. Presumably triploid nuclei (with 3 chromocenters) are capable of DNA synthesis.     

Pelagic-benthic coupling of nucleic acids in an abyssal location of the northeastern Atlantic Ocean.

Spatial and temporal changes in sedimentary nucleic acid concentrations in an abyssal locality of the northeastern Atlantic Ocean were investigated in relation to fluxes of nucleic acids produced in the photic layer. Sediment trap material, collected between 1996 and 1998 at depths of 1,000, 3,000, and 4,700 m, and sediment samples were analyzed for DNA and RNA content. Nucleic acid concentrations in the sediments were very high and displayed significant temporal changes, whereas mesoscale variability was low. DNA and RNA concentrations generally displayed opposite temporal patterns, which are likely to be dependent on the nature and characteristics of DNA and RNA molecules. Nucleic acid fluxes were high and displayed clear seasonal changes apparently coupled with seasonal pulses of primary production. However, while median values of DNA fluxes were relatively similar in all sediment traps, median values of RNA fluxes almost doubled from the 1,000- to the 4,700-m depth, suggesting differences in the metabolic activity of microbes associated with sinking particles. Significant relationships between DNA concentrations in the sediments and DNA fluxes and between RNA concentrations and RNA fluxes, indicating the presence of a clear pelagic-benthic coupling of particulate nucleic acids, were observed. The benthic system investigated was not steady state since we estimated that, from September 1996 to October 1998, nucleic acid concentration in the sediments decreased by about 165 mg of DNA m(-2). Vertical profiles revealed a significant decrease in DNA concentration with depth in the sediments, reaching an asymptotic value of about 5 microg g(-1). This DNA fraction constitutes a pool of potentially refractory DNA (accounting for 16 to 40% of the total DNA pool) that might be buried in the sediments.     

The cyclization of Xenopus laevis 5 S DNA

DNA from Xenopus laevis containing the sequences complementary to 5 S RNA has been studied by the formation of folded rings. Maximal cyclization for fragments 1 to 2 μm in length is 45 to 55%. Thus the efficiency of folded ring formation from this tandemly-repeating DNA is about 50%, assuming that all fragments are 5 S DNA. From the ring frequency as a function of the number of nucleotides removed from the 3′ terminals of the shear-broken fragments, one may calculate that the repeating sequence is approximately 750 nucleotides long, a number that agrees with earlier partial denaturation mapping. The circumference of the folded rings confirms this repeating length since most rings correspond to modular size classes of 0.25-μm increments. Fragments 12 μm long cyclize almost as readily as 1 to 2-μm fragments do. Therefore, the length of the regions (g-regions) containing the tandemly-repeating 5 S DNA is more than 12 μm. The folded rings are about as stable to linearization by increasing concentrations of formamide as the duplex DNA is to denaturation. This indicates that the local, non-transcribed, spacer portions which represent the majority (83%) of the nucleotides in the tandemly-repeating unit, are probably homogeneous in sequence. The exonuclease-treated 5 S DNA fragments cyclize more rapidly than phage T7 DNA, and the kinetics are in accord with theoretical expectation.