Anomalies in sedimentation. II. Testing the entanglement hypothesis

Zones of T2 DNA were sedimented through uniform solutions of T7 DNA to determine if the smaller DNA molecules would become entangled in the larger. No entanglement could be demonstrated even at high DNA concentrations. It is suggested that molecular entanglement is not responsible for the sudden loss of DNA from solution which occurs in high centrifugal fields. This communication also includes observations on the effects of rotor speed on the sedimentation behavior of DNA in high centrifugal fields, distortion of zone shape at high concentrations, and hydrodynamic interactions between DNA and MS2 bacteriophage particles.     

Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. I. Effect of random double-strand breaks and centrifuge speed on sedimentation

A method is presented for preparing very large DNA from Bacillus subtilis protoplasts. When the DNA is characterized by sedimentation in neutral sucrose gradients, a fast-sedimenting component is found whose sedimentation coefficient varies with centrifuge speed. By use of [³H]thymine label for the DNA and a ¹⁴C-labeled amino acid, it is shown that less than 5% cellular material other than DNA is associated with this component. Irradiation of this DNA in solution with gamma rays forms a slower component, called the “main peak”, whose sedimentation coefficient also depends on centrifuge speed. More irradiation breaks down this main peak into even slower-sedimenting DNA; it is shown that for low doses, double-strand breaks are formed in both the B. subtilis DNA and in bacteriophage T2 DNA at the same rate linear in dose, 0.018 double-strand breaks per kilorad per mass equal to that of T2 DNA.The speed dependence of the DNA sedimenting at the main peak is compared with an approximate theory of the speed dependence of the sedimentation coefficient of linear DNA by B. H. Zimm (unpublished calculations). The comparison suggests that for sufficiently high centripedal acceleration, DNA molecules larger than a critical mass will sediment at much the same velocity. The theory, and data on the break-up of the DNA with gamma rays, are used to estimate that the DNA extracted is at least 13 times the mass of T2 DNA, and possibly larger.In the Appendix, data from the literature are put together with data taken during this work to make plausible the assumption that the usual theory for the sedimentation of DNA molecules, experimentally tested in salt solutions, may also be applied to sucrose solutions. If, in neutral sucrose gradients, the distance sedimented is proportional to a power α of the mass, the best value of α = 0.38.     

Rotor speed dependent sedimentation of circular and linear DNA.

The sedimentation rate of large, linear DNA molecules has been shown to be rotor speed dependent (Rubenstein and Leighton, Biophys. Chem. 1 (1974)). In this communication we report the first studies designed to measure the rotor speed effect with a homogenous, linear viral DNA larger than bacteriophage T2 DNA. We also report the first studies using a homogenous, circular episomal DNA of known molecular weight. For this circular DNA a small rotor speed effect, previously unsuspected, was discovered.     

Effect of centrifuge speed on the sedimentation of high-molecular-weight bacteriophage G DNA

Large molecular weight bacteriophage G DNA, about five times larger than T2 DNA, was used to test Zimm's theory [(1974) Biophys. Chem. 1 , 279–291] for the effect of rotor speed on the sedimentation of large linear monodisperse DNA. Sedimentation profiles from neutral sucrose gradinets at low and high rotor speeds show G DNA sedimenting from 1.8 to 0.7 times as fast as T2 DNA. Experimental measurements indicate that the sedimentation coefficient of G DNA decreases with increasing rotor speed about as fast as predicted by theory.     

Rotor speed dependent sedimentation of circular and linear DNA

The sedimentation rate of large, linear DNA molecules has been shown to be rotor speed dependent (Rubenstein and Leighton, Biophys. Chem. 1 (1974)). In this communication we report the first studies designed to measure the rotor speed effect with a homogenous, linear viral DNA larger than bacteriophage T2 DNA. We also report the first studies using a homogenous, circular episomal DNA of known molecular weight. For this circular DNA a small rotor speed effect, previously unsuspected, was discovered.     

Generation of supercoils in nicked and gapped DNA drives DNA unknotting and postreplicative decatenation

Due to the helical structure of DNA the process of DNA replication is topologically complex. Freshly replicated DNA molecules are catenated with each other and are frequently knotted. For proper functioning of DNA it is necessary to remove all of these entanglements. This is done by DNA topoisomerases that pass DNA segments through each other. However, it has been a riddle how DNA topoisomerases select the sites of their action. In highly crowded DNA in living cells random passages between contacting segments would only increase the extent of entanglement. Using molecular dynamics simulations we observed that in actively supercoiled DNA molecules the entanglements resulting from DNA knotting or catenation spontaneously approach sites of nicks and gaps in the DNA. Type I topoisomerases, that preferentially act at sites of nick and gaps, are thus naturally provided with DNA–DNA juxtapositions where a passage results in an error-free DNA unknotting or DNA decatenation.     

The origin of nascent single-stranded DNA extracted from mammalian cells.

In vitro cultured bovine liver cells were labelled with radioactive thymidine and dissolved in 0.5% sodium dodecyl sulphate. Centrifugation of the lysate through sucrose gradients in a zonal rotor revealed a slowly sedimenting fraction of preferentially pulse labelled DNA. The DNA of this zone was further analysed by chromatography on hydroxy-apatite, banding in CsCl density gradients, and sedimentation in neutral and alkaline sucrose gradients. It contained besides small amounts of fragmented bulk DNA, single-stranded nascent DNA and single-stranded pre-labelled DNA which could be separated from each other by using BrdU as a density label. The density labelling also revealed small amounts of nascent-nascent DNA duplexes. The slowly sedimenting fraction was practically absent from cell lysates which were prepared in 2 M NaCl - 50 microgram/ml pronase. The results suggest that nascent single-strands and nascent-nascent duplexes are released from the forks of replicating DNA by branch migration. Pre-labelled single strands may be released by the same branch migration. Pre-labelled single strands may be released by the same mechanism, but the in vivo structure from which they originate has yet to be elucidated.     

Bacteriophage T7 DNA packaging. III. A "hairpin" end formed on T7 concatemers may be an intermediate in the processing reaction

An unusual left end (M-end) has been identified on bacteriophage T7 DNA isolated from T7-infected cells. This end has a "hairpin" structure and is formed at a short inverted repeat sequence centered around nucleotide 39,587 of T7, 190 base-pairs to the left of the site where a mature left end is formed on the T7 concatemer. We do not detect the companion right end that would be formed if the M-end is produced by a double-stranded cut on the T7 concatemer. This suggests that the hairpin left end may be generated from a single-stranded cut in the DNA that is used to prime rightward DNA synthesis. The formation of M-end does not require the products of T7 genes 10, 18 or 19, proteins that are essential for the formation of mature T7 ends. During infection with a T7 gene 3 (endonuclease) mutant, phage DNA synthesis is reduced and the concatemers are not processed into unit length DNA molecules, but both M-end and the mature right end are formed on the concatemer DNA. These two ends are also found associated with the large, rapidly sedimenting concatemers formed during a normal T7 infection while the mature left end is present only on unit length T7 DNA molecules. We propose that DNA replication primed from the hairpin end produced by a nick in the inverted repeat sequence provides a mechanism to duplicate the terminal repeat before DNA packaging. Packaging is initiated with the formation of a mature right end on the branched concatemer and, as the phage head is filled, the T7 gene 3 endonuclease may be required to trim the replication forks from the DNA. Concatemer processing is completed by the removal of the 190 base-pair hairpin end to produce the mature left end.     

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.     

Studies on T4-head maturation. 2. Substrate specificity of gene-49-controlled endonuclease

The substrate specificity of 49+-enzyme was investigated in vitro. The enzyme showed a marked preference for rapidly sedimenting T4 DNA (greater than 1000 S) when helix-destabilizing proteins from Escherichia coli or phage T4 were added to the reaction. Regular replicative T4 DNA (200-S DNA) or denatured T4 DNA was not cleaved by the enzyme in the presence of these proteins but if they were omitted from the reaction both DNAs become good substrates for the enzyme. 200-S DNA was cleaved at its natural sites of single strandedness which occur at one-genome intervals. Gaps in T4 DNA which were constructed by treatment of a nicked DNA with exonuclease III were also cleaved by 49+-enzyme in the absence of helix-destabilizing proteins. Single-stranded T4 DNA was extensively degraded and up to 50% of the material was found to be acid-soluble in a limit digest. The degradation products were predominantly oligonucleotides of random size. No preference for a 5'-terminal nucleotide was observed in material from a limit digest with M13 DNA. Double-stranded DNA was nicked upon exposure to 49+-enzyme and double-strand breakage finally occurred by an accumulation of single-strand interruptions. No acid-soluble material was produced from native T4 DNA. The introduction of nicks in native DNA did not improve its properties as a substrate for the enzyme. Double-stranded DNA was about 100-fold less sensitive to the enzyme than single-stranded DNA.     

Subpopulations of chicken B lymphocytes.

Immunoglobulin-synthesizing cells from the spleen and bursa were fractionated by the 1 X G sedimentation velocity technique and characterized by their ability to synthesize immunoglobulin and by staining with fluorescent anti-light chain chain. Four subpopulations of immunoglobulin-synthesizing cells were identified. In the bursa, slowly sedimenting (S 2.3 mm/hr) and rapidly sedimenting (S greater than 3.5 mm/hr) subpopulations with surface immunoglobulin were present; in the spleen, a slowly sedimenting (S 2.3 mm/hr) subpopulation with surface immunoglobulin and plasma cells (S greater than 3.5 mm/hr) with large concentrations of intracellular immunoglobulin existed. The subpopulations differed most markedly in their ability to synthesize immunoglobulin (cpm Ig synthesized/10(6) Ig-positive cells); the rates of immunoglobulin synthesis were in the ratio 1:2:1:900. The slowly sedimenting B cells from the spleen and both subpopulations of B cells from the bursa released small amounts of immunoglobulin into the culture media, whereas, the plasma cells released immunoglobulin at a rate as much as 3700 times greater. Bursal B cells could be further distinguished from splenic B cells by a greater amount of DNA synthesis.     

Physical and topological properties of circular DNA

Several types of circular DNA molecules are now known. These are classified as single-stranded rings, covalently closed duplex rings, and weakly bonded duplex rings containing an interruption in one or both strands. Single rings are exemplified by the viral DNA from φX174 bacteriophage. Duplex rings appear to exist in a twisted configuration in neutral salt solutions at room temperature. Examples of such molecules are the DNA''s from the papova group of tumor viruses and certain intracellular forms of φX and λ-DNA. These DNA''s have several common properties which derive from the topological requirement that the winding number in such molecules is invariant. They sediment abnormally rapidly in alkaline (denaturing) solvents because of the topological barrier to unwinding. For the same basic reason these DNA''s are thermodynamically more stable than the strand separable DNA''s in thermal and alkaline melting experiments. The introduction of one single strand scission has a profound effect on the properties of closed circular duplex DNA''s. In neutral solutions a scission appears to generate a swivel in the complementary strand at a site in the helix opposite to the scission. The twists are then released and a slower sedimenting, weakly closed circular duplex is formed. Such circular duplexes exhibit normal melting behavior, and in alkali dissociate to form circular and linear single strands which sediment at different velocities. Weakly closed circular duplexes containing an interruption in each strand are formed by intramolecular cyclization of viral λ-DNA. A third kind of weakly closed circular duplex is formed by reannealing single strands derived from circularly permuted T2 DNA. These reconstituted duplexes again contain an interruption in each strand though not necessarily regularly spaced with respect to each other.     

Flow birefringence and intrinsic viscosity of a T2 bacteriophage DNA–methylated bovine serum albumin complex

The flow birefringence, extinction angles, and intrinsic viscosity have been determined at low velocity gradients for a complex of T2 bacteriophage DNA and methylated serum albumin prepared in dilute solution to a stoichiometry of approximately 90 proteins per DNA molecule. Comparative data upon equivalent solutions of pure uncomplexed T2 DNA are also presented, and these data are completely in accord with the results of previous study. The experimental data are interpreted in terms of current dynamical theory and indicate that the complex has an essentially linear chain structure, consisting of approximately two DNA molecules, which is hydrodynamically indistinguishable from the pure DNA and that extensive internal or intramolecular binding in the complex does not occur. Although interpretation of the results is hampered by an apparent moderate degree of polydispersity in the complex preparations and by relatively large shear extrapolations, the data for both DNA and the complex are substantially in accord with dynamical theory for a nondraining bead subchain model having high kinetic segmental rigidity.     

Early Intracellular Events in the Replication of Bacteriophage T4 Deoxyribonucleic Acid: V. Further Studies on the T4 Protein-Deoxyribonucleic Acid Complex 1

Soon after infection parental deoxyribonucleic acid (DNA) enters a structure sedimenting fast to the bottom of a sucrose gradient. The addition of chloramphenicol (CM) prevents formation of this structure, whereas treatment with Pronase releases DNA which sediments thereafter with the speed characteristic of phenol-extracted replicative DNA. It is assumed therefore that the structure responsible for fast sedimentation of replicative DNA is a newly synthesized protein. Those fast-sedimenting complexes contain preferentially the replicative form of parental DNA; this was proven by density labeling experiments. Progeny DNA labeled with (3)H-thymidine added after infection can also be detected preferentially in this fast-sedimenting moiety. The association of the DNA with the complexing protein is of a colinear or quasi-colinear type. This was proven by introducing double-strand scissions into DNA embedded in the replicative complex; double-strand scissions do not liberate DNA from the fast-sedimenting complex. Despite the apparent intimate relation between protein and DNA, DNA residing in complexes is fully sensitive to the action of nucleases. Shortly prior to the appearance of the fast-sedimenting complex, parental DNA displays still another characteristic: at about 3 min after infection, it sediments faster than reference, but sizeably slower than the complex which appears at roughly 4 to 5 min after infection. The transition between these two fast-sedimenting types of moieties is not continuous. This fast-sedimenting intermediate, which appears at 3 min after infection, cannot be inhibited by the addition of CM either at the moment or prior to infection. Fast-sedimenting intermediate can be destroyed by sodium dodecyl sulfate, Pronase, or phenol extraction. The progeny DNA labeled with (3)H-thymidine between 3 and 3.5 min after infection can be recovered in fast-sedimenting intermediate. The contribution of newly synthesized progeny DNA is so small that it cannot be detected as a shift of the parental density in a density labeling experiment. Small fragments of progeny DNA recovered in fast-sedimenting intermediate are not covalentlv attached to parental molecules and represent both strands of T4 DNA.     

Relationship of R6K replicating forms to the folded chromosome of Escherichia coli.

An examination of the relationship of both nonreplicating and replicating forms of R6K plasmid DNA to the Escherichia coli folded chromosome showed that both forms cosediment with the chromosome in neutral sucrose gradients. Approximately 20% of the nonreplicatin molecules was found as freely sedimenting forms when the folded-configuration of the chromosomes was preserved. However, under the same conditions negligible amounts of the replicating forms were found as freely sedimenting molecules. Thus, it is concluded that the replicating forms, when compared with nonreplicating molecules, are preferentially associated with the folded chromosomal structure. Exposure of the folded chromosomal structure to RNase resulted in an unfolding of the chromosome and a concomitant increase in the amount of freely sedimenting replicating and nonreplicating forms of R6K DNA. Analyses of the single-stranded length of RNase-released nascent molecules suggest that they replicate in continuous association with the folded chromsome complex. Nonenzymatic unfolding of the chromosomes by progressively lowering the sodium ion concentration during lysis resulted in a progressive increase in the release of nonreplicating molecules. Replicating molecules wer not released by unfolding the chromosome in this fashion.     

Functional interactions of gastric mucus glycoprotein

The concentration-dependence of viscosity in solutions of purified glycoprotein from pig gastric mucus is of the form expected for simple polymer entanglement. At higher concentrations, however, a weak viscoelastic gel is formed, whose mechanical spectrum (over the frequency range 10⁻²---10² rad s⁻¹) indicates a more stable mechanism of interchain association, and is closely similar to that of native mucus. On prolonged exposure to solvent, reconstituted gels redissolve, while native mucus retains its structural integrity (as characterized by the storage modulus, G′) but releases a significant, variable amount of glycoprotein. On proteolytic digestion or disulphide reduction of the glycoprotein to its component subunits, network structure is lost, but the mechanical spectra of the resulting solutions show interactions beyond simple entanglement. From this evidence we suggest that in the sub-micrometre-sized ‘domains’ in which native mucus is secreted, the carbohydrate side chains of component glycoprotein molecules are interdigitated in a comparatively stable arrangement, with the polymeric subunit structure of the glycoprotein conferring the branching required for development of a three-dimensional network, and with a substantial, variable sol-fraction of free glycoprotein within the interstices of the gel. On solubilization of native mucus, the ‘domain’ structure is destroyed irreversibly. Interaction between domains, and between individual molecules in gels reconstituted from the component glycoprotein after extraction and purification, is by more transient, non-specific interdigitation and entanglement, to confer the overall flow and spreading characteristics of the gel.     

Isolation and characterization of poly(A)-containing polyoma "early" and "late" messenger RNAs.

During late lytic infection of mouse kidney cell cultures polyoma 16S and 19S (late 19S RNA) were isolated by oligo(dT)-cellulose chromatography. Approximately 60-80% of total cytoplasmic polyoma RNA contained tracts of poly(A) which were retained by oligo(dT)-cellulose. Early in lytic infection when viral DNA synthesis and the production of capsid protein are blocked by the addition of 5-fluorodeoxyuridine, approximately 100% of polyoma "early" 19S RNA was quantitatively retained by oligo(dT)-cellulose indicating the presence of poly(A) tracts on most 19S mRNA molecules. In addition, 2 classes polyoma RNA, synthesized after the onset of cellular RNA synthesis under conditions where DNA synthesis is inhibited with 5-fluorodeoxyuridine, were found to contain tracts of poly(A). These species sedimenting at 16S and 19S in aqueous sucrose density gradients were also quantitatively retained by oligo (dT)-cellulose.     

Subcellular fractionation of wheat leaf protoplasts by centrifugal elutriation

A new method using centrifugal elutriation for subcellular fractionation of plant cells has been developed. This method takes advantage of the fact that particles sedimenting in a gravitational field can be eluted by flow against the field. A wheat protoplast homogenate was fed into an elutriation rotor spinning at high speed and the flow rate into the rotor was gradually increased. The smaller and less dense materials such as mitochondria, microbodies, endoplasmic reticulum, and cytoplasm were elutriated earlier than the larger and denser nuclei and chloroplasts. The intact chloroplasts, free of mitochondria, microbodies, endoplasmic reticulum, and cytoplasm, could be obtained within 40 min following the rupture of protoplasts. The chlorophyll-free mitochondria could be obtained within 80 min.