Soluble fibrin consists of fibrin oligomers of heterogeneous distribution

Soluble fibrin is observed in patients with intravascular coagulation and represents an intermediary product of conversion of fibrin monomers into a fibrin clot whereby the presence of fibrinogen may suppress fibrin clot formation. The interactions between fibrin and fibrinogen and the occurrence of fibrin oligomers in soluble fibrin were studied by sucrose density ultracentrifugation. Different concentrations of soluble fibrin, prepared by mixing 125I-fibrin (24 nM - 1.5 microM) with a constant concentration of 131I-fibrinogen (6 microM) were analyzed at 37 degrees C in stable linear sucrose density gradients containing a uniform concentration of unlabelled fibrinogen (6 microM) and calcium ions in order to mimic the physiological situation. At any fibrin concentration, 125I-fibrin sedimented faster than 131I-fibrinogen through 5-30% (w/v) sucrose gradients. Sedimentation rates of fibrin increased from 9 S to 23 S depending on the initial fibrin concentration. The relative amount of residual fibrin monomer not incorporated into oligomers was calculated from the sedimentation profiles. At any fibrin concentration, the portion of free monomer was always more than twofold higher for batroxobin-generated (desAA-) fibrin than for thrombin-generated (desAABB-) fibrin. Apparent association constants for desAABB-fibrin were 3-10 times higher than those for desAA-fibrin indicating a stronger interaction between monomers of the former type of fibrin. In the presence of excess fibrinogen the predominant species in soluble desAA-fibrin were monomers and dimers, whereas dimers, trimers and higher-molecular-mass oligomers were present in soluble desAABB-fibrin. Strong interactions between both types of fibrin were demonstrated from their cosedimentation, whereby the size of these copolymers were shown to be governed by the oligomer size of the desAABB-fibrin type. These results provide evidence for the occurrence of differently sized oligomers of fibrin in soluble fibrin and for the concept of a cooperative polymerization process between both types of fibrin devoid of any stable complexes between fibrin and fibrinogen.     

Defined oligomeric SV40 DNA: A sensitive probe of general recombination in somatic cells

We have constructed well defined oligomeric molecules of simian virus 40 (SV40) DNA as probes for investigating mechanisms by which cultured somatic cells recombine DNA. Restriction enzyme fragments from different temperature-sensitive mutants were joined in a head-to-tail orientation to create partial dimers 1.84 genome lengths in size. These molecules are too large to fit into a viral capsid. Therefore an assay that depends on production of progeny virus after infection with oligomeric DNA is a selective measure of precise conversion of oligomers to monomers. By constructing oligomers from appropriate combinations of temperature-sensitive DNAs, we have been able to study the conversion process in several defined regions of the SV40 genome. Our results indicate that conversion of oligomers to monomers occurs uniformly throughout the genome and is not dependent on normal viral DNA replication. These data indicate that conversion occurs primarily by general, homology-dependent recombination. At least one secondary mechanism that generates a low level of wild-type progeny was also detected. Studies with heteroduplex molecules indicate that repair of mismatched bases may be the secondary mechanism.     

Biological activity of different forms of bacteriophage lambda DNA]

Hershey circles and linear tandem aggregated forms of DNA have been obtained in vitro and treated with polynucleotide ligase to form phosphodiester bond. Using zone centrifugation in glycerol gradient covalently closed circles and linear dimers have been purified and their biological activity investigated. It was found that closed circular molecules lost most, if not all, of their activity in CaCl2-dependent system. In order to investigate the biological activity of tandem dimer molecules, hybrid dimers consisting of DNA's from lambda C1857 and lambda 1434 have been obtained. In plaque assay with the appropriate non-permissive strains of E. coli the efficiency of infectivity of hybrid dimers was measured. Biological activity of dimer molecules sealed with ligase was about 5% of the activity of linear monomers. Ig has been suggested that tandem dimers of lambda DNA joined by phosphodiester bond are able to penetrate into the CaCl2-treated host cells and both components of dimers are active during subsequent multiplication.     

Preparation of trimers and tetramers of mixed sequence oligodeoxynucleoside methylphosphonates and assignment of configurations at the chiral phosphorus.

Synthesis of stereoregular DNA methylphosphonates has been accomplished for homo-oligomers, but remains a formidable problem for oligomers of a defined antisense target sequence. In this work, four trimer and tetramer deoxynucleoside methylphosphonates of mixed sequence (dACA, dCCAA, dAGGG, and dGCAT) were prepared by block coupling of diastereomerically pure dimers with either monomers or other diastereomerically pure dimers. These oligomers were separated chromatographically into individual diastereomers, and the configurations of the chiral methylphosphonate linkages were assigned. Three types of methods were used to assign configuration of a new methylphosphonate linkage: preparation of the same diastereomer through multiple synthetic pathways, base hydrolysis, and acid hydrolysis. Hydrolysis of the diastereomerically pure oligomers into component dimers and monomers was followed by chromatographic comparison with control dimers of known configuration. In all cases studied, oligomers with R configurations displayed faster elution from silica gel than did oligomers with the respective S configuration. NMR spectra of individual diastereomers of dACA were studied, revealing characteristic differences in chemical shifts which may prove useful in configurational assignments of longer oligomers. Thus, these data provide a methodological basis for synthesis and configurational assignment of longer methylphosphonate oligomers to use as antisense probes.     

Association and dissociation properties of natural human interferon γ

The properties of natural human interferon γ (IFN-γ) molecules dissolved in protein-denaturing and non-denaturing solvents were examined by high-performance size-exclusion chromatography on a gel permeation column. IFN-γ and tritium-labeled IFN-γ molecules formed either dimers (>90.5%) with the molecular mass of 60 kDa or probably tetramers (<9.5%) with the molecular mass of approximately 100 kDa in non-denaturing solvents, and no monomer was detected. These oligomers were dissociated in protein-denaturing solvents such as 6 M guanidine hydrochloride, and IFN-γ existed as monomers. There is no effect on formation of the monomer based on the dissociation of oligomers by acid treatment at pH 4.0. The monomers in protein-denaturing solvents formed dimers by association when applied to a column equilibrated with a non-denaturing solvent of phosphate buffer, pH 7.0. In conclusion, natural human IFN-γ forms oligomers, particularly dimers, in non-denaturing solution, and this oligomer formation is a reversible reaction.     

Structure and formation of circular dimers of simian virus 40 DNA.

Most of the viral DNA extracted from simian virus 40 (SV40)-infected African green monkey kidney cells consists of circular molecules about 5.3 kilobases in contour length. However, about 1% of the viral DNA was found to occur as closed circular dimers that appeared to be formed, preferentially, late in infection. The monomeric units of dimers were organized in a head-to-tail, tandem arrangement; moreover, the monomeric units were not defective; i.e., they lacked deletions or other rearrangements. After infections with dimer DNA, nondefective monomers were formed. These findings suggest that dimers are not intermediates in the production of defective SV40 genomes. The majority of the dimers formed in mixed infections with two mutants were homodimers, but about 5% of the circular dimers were heterodimers and must have arisen by intermolecular recombination.     

Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity

RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPγS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 Å × 130 Å with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding.     

Circular replicating baculovirus genome

Free Malacosoma neustria nuclear polyhedrosis virus preparations contain nucleocapsids typical of Baculoviruses' morphology and size as well as long virus-like particles. Viral DNA is circular and covalently closed. Its preparations contain rings with single-stranded breaks, catenanes and circular dimers as well. Replicating pulse-labelled DNA preparations have been obtained from cells with the highest virus DNA synthesis. Theta-forms of replicating DNA are found in heavy fractions of sucrose gradients. Theta-forms, catenanes and circular dimers are discussed as intermediate molecules. Catenanes or (sometimes) circular dimers appear to form protein-containing complexes and long virus-like particles if the monomerization process is inhibited.     

Self-association of the yeast nucleosome assembly protein 1

The self-association properties of the yeast nucleosome assembly protein 1 (yNAP1) have been investigated using biochemical and biophysical methods. Protein cross-linking and calibrated gel filtration chromatography of yNAP1 indicate the protein exists as a complex mixture of species at physiologic ionic strength (75-150 mM). Sedimentation velocity reveals a distribution of species of 4.5-12 Svedbergs (S) over a 50-fold range of concentrations. The solution-state complexity is reduced at higher ionic strength, allowing for examination of the fundamental oligomer. Sedimentation equilibrium of a homogeneous 4.5 S population at 500 mM sodium chloride reveals these species to be yNAP1 dimers. These dimers self-associate to form higher order oligomers at more moderate ionic strength. Titration of guanidine hydrochloride converts the higher order oligomers to the homogeneous 4.5 S dimer and then converts the 4.5 S dimers to 2.5 S monomers. Circular dichroism shows that guanidine-mediated dissociation of higher order oligomers into yNAP1 dimers is accompanied by only slight changes in secondary structure. Dissociation of the dimer requires a nearly complete denaturation event.     

Chemical crosslinking of different forms of the simian virus 40 large T antigen using bifunctional reagents

Chemical crosslinking was used for a direct analysis of the different forms of large tumor (T) antigen, the simian virus 40 A gene product. The first subclass, sedimenting at 14-16S, is composed of monomeric to tetrameric units, whereas the second, sedimenting at 5-6S, only contains dimers and monomers of T. The occurrence of oligomeric structures of T in solution which are higher than dimers suggests the possibility of direct binding of such trimers or tetramers to the origin of replication of the viral DNA as an alternative to the formation of these structures by aggregation of bound dimers or monomers after their sliding along the DNA.     

Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface

Single green fluorescent protein (GFP) molecules were successfully imaged for the first time in living cells. GFP linked to the cytoplasmic carboxyl terminus of E-cadherin (E-cad-GFP) was expressed in mouse fibroblast L cells, and observed using an objective-type total internal reflection fluorescence microscope. Based on the fluorescence intensity of individual fluorescent spots, the majority of E-cad-GFP molecules on the free cell surface were found to be oligomers of various sizes, many of them greater than dimers, suggesting that oligomerization of E-cadherin takes place before its assembly at cell-cell adhesion sites. The translational diffusion coefficient of E-cad-GFP is reduced by a factor of 10 to 40 upon oligomerization. Because such large decreases in translational mobility cannot be explained solely by increases in radius upon oligomerization, an oligomerization-induced trapping model is proposed in which, when oligomers are formed, they are trapped in place due to greatly enhanced tethering and corralling effects of the membrane skeleton on oligomers (compared with monomers). The presence of many oligomers greater than dimers on the free surface suggests that these greater oligomers are the basic building blocks for the two-dimensional cell adhesion structures (adherens junctions).     

Replicative intermediates in UV-irradiated simian virus 40

We have used Simian virus 40 (SV40) as a probe to study the replication of UV-damaged DNA in mammalian cells. Viral DNA replication in infected monkey kidney cells was synchronized by incubating a mutant of SV40 (tsA58) temperature-sensitive for the initiation of DNA synthesis at the restrictive temperature and then adding aphidicolin to temporarily inhibit DNA synthesis at the permissive temperature while permitting pre-replicative events to occur. After removal of the drug, the infected cells were irradiated at 100 J/m2 (254 nm) to produce 6-7 pyrimidine dimers per SV40 genome, and returned to the restrictive temperature to prevent reinitiation of replication from the SV40 origin. Replicative intermediates (RI) were labeled with [3H]thymidine, and isolated by centrifugation in CsCl/ethidium bromide gradients followed by BND-cellulose chromatography. The size distribution of daughter DNA strands in RI isolated shortly after irradiation was skewed towards lengths less than the interdimer spacing in parental DNA; this bias persisted for at least 1 h after irradiation, but disappeared within 3 h, by which time the size of the newly-synthesized DNA exceeded the interdimer distance. No significant excision of dimers from parental strands in either replicative intermediates or Form I (closed circular) DNA molecules was detected. These data are consistent with the hypothesis that replication forks are temporarily blocked by dimers encountered on the leading strand side of the fork, but that daughter strand continuity opposite dimers is eventually established. Evidence was obtained for the generation at late times after irradiation, of Form I molecules in which the daughter DNA strands contain dimers. Thus DNA strand exchange as well as trans-dimer synthesis may be involved in the generation of supercoiled Form I DNA from UV-damaged SV40 replicative intermediates.     

Arrangement of nucleotide sequences in adeno-associated virus DNA

There are two types of adeno-associated virus virions which contain complementary single-stranded DNA genomes of about 1.4 × 10⁶ daltons. The purified complementary single polynucleotide chains anneal to form duplex linear monomers, circular monomers, and linear dimers, in addition to other less well-defined structures, as identified by sedimentation and electron microscopy. All duplex species are formed by linear single polynucleotide chains of unit length, thus duplex circles and linear dimers are assumed to be held together by relatively short overlapping hydrogen-bonded regions. The initial linear monomers present after annealing the complementary single strands do not form duplex circles or oligomers when re-exposed to annealing conditions, but DNA which sediments as linear monomers after heating linear dimers at a temperature from 7 to 25 deg. C below the T_m of duplex AAV² DNA does re-form oligomers and circles when exposed to annealing conditions. Denaturation and reannealing of any duplex species leads to the formation of all forms, indicating that the over-all single strand composition of all species is equivalent. Disruption of duplex circles by limited exonucleolytic digestion using either 3′ or 5′ exonucleases leads to the conclusion that the overlap region may have either 3′ or 5′ termini and that the overlap region represents less than 6% of the length of the genome. Exonucleolytic digestion of linear monomers to the extent of 50% leaves polynucleotide chains which cannot reanneal after denaturation, thus AAV DNA is not randomly circularly permuted. Duplex linear monomers which do not form circles when exposed to annealing conditions do form duplex circles after 1% exonuclease III digestion. A model consistent with these data is one in which the linear single polynucleotide chains present in AAV virions consist of two or more permutations. All of these chains contain terminal repetitions and their start points occur within a limited region, representing < 6% of the length of the genome. According to this interpretation AAV DNA would exist within the virion as a linear single polynucleotide chain or is cleaved at a few specific sites during extraction.     

Discontinuous DNA replication in mouse P-815 cells.

The length of newly synthesized DNA strands from mouse P-815 cells was analyzed after denaturation both by electrophoresis and by sedimentation in alkaline sucrose gradients. [3-H]-Thymidine pulses of 2-8 min at 37 degrees C predominantly label molecules of 20-60 S. With 30-s pulses at 25 degrees C, all the [3-H]thymidine appears in short DNA strands of 50-200 nucleotides. Thus, DNA strand elongation occurs discontinuously via Okazaki fragments at both the 5' end and the 3' end. In dodecylsulfate lysates, only 10% of the Okazaki fragments are found as single-stranded molecules. About 90% are resistant to hydrolysis by the single-strand-specific nuclease S-1 and band in isopycnic gradients at the buoyant density of double-stranded DNA. No evidence for ribonucleotides at the 5' end of Okazaki fragments was obtained either in isopycnic CsCl or Cs2SO4 gradients or after incubation with polynucleotide kinase and [gamma-32P]ATP.     

Study of the interaction of DNA and acridine orange by various optical methods

The degree of binding of acridine orange to DNA, native or denatured, has been determined by equilibrium dialysis in 0.1M and 0.001M NaCl at 20°. The nature of the binding process has been investigated by studying various optical properties of the dye–DNA complexes and by relating them to the binding ratio. All these properties were found to vary quantitatively and qualitatively according to the successive stages of the process. These stages were assumed to be a strong binding of intercalated monomers followed by formation of bound dimers and finally by external binding of aggregates of native DNA. Absorption spectra of the complexes could be interpreted on that basis. Circular dichroism spectra were resolved into components: one band for intercalated monomers without interactions, two excition splittings for interacting monomers and bound dimers, respectively, weak bands and exciton splitting for external aggregates. The fluorescence intensity was greatly enhanced in intercallated monomers; its quenching at higher binding ratio was quantitatively related to dimer fixation. The value of the anisotropy of fluorescence at low binding ratio suggested a limited mobility of intercalated monomers; the decrease of polarization at higher binding was attributed to energy transfer between monomers. Electric dichroism displayed by the complexes in the dye absorption bands indicated an orientation of the bound molecules quite parallel to the base rings at low binding. In the range of fixation of dimers and external molecules, the dichroism was lower but still indicated an important degree of ordering.     

Interactions and aggregation of apoferritin molecules in solution: effects of added electrolytes

We have studied the structure of the protein species and the protein-protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of Na(+) and Cd(2+) ions. We used chromatographic, and static and dynamic light scattering techniques, and atomic force microscopy (AFM). Size-exclusion chromatography was used to isolate these two protein fractions. The sizes and shapes of the monomers and dimers were determined by dynamic light scattering and AFM. Although the monomer is an apparent sphere with a diameter corresponding to previous x-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was applied to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The results for the monomers indicate that Na(+) ions cause strong intermolecular repulsion even at concentrations higher than 0.15 M, contrary to the predictions of the commonly applied Derjaguin-Landau-Verwey-Overbeek theory. We argue that the reason for such behavior is hydration force due to the formation of a water shell around the protein molecules with the help of the sodium ions. The addition of even small amounts of Cd(2+) changes the repulsive interactions to attractive but does not lead to oligomer formation, at least at the protein concentrations used. Thus, the two ions provide examples of strong specificity of their interactions with the protein molecules. In solutions of the apoferritin dimer, the molecules attract even in the presence of Na(+) only, indicating a change in the surface of the apoferritin molecule. In view of the strong repulsion between the monomers, this indicates that the dimers and higher oligomers form only after partial denaturation of some of the apoferritin monomers. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization and other phase transitions in the protein solution.     

Segregation of relaxed replicated dimers when DNA ligase and DNA polymerase I are limited during oriC-specific DNA replication.

An in vitro Escherichia coli oriC-specific DNA replication system was used to investigate the DNA replication pathways of oriC plasmids. When this system was perturbed by the DNA ligase inhibitor nicotinamide mononucleotide (NMN), alterations occurred in the initiation of DNA synthesis and processing of intermediates and DNA products. Addition of high concentrations of NMN soon after initiation resulted in the accumulation of open circular dimers (OC-OC). These dimers were decatenated to open circular monomers (form II or OC), which were then processed to closed circular supercoiled monomers (form I or CC) products. After a delay, limited ligation of the interlinked dimers (OC-OC to CC-OC and CC-CC) also occurred. Similar results were obtained with replication protein extracts from polA mutants. The presence of NMN before any initiation events took place prolonged the existence of nicked template DNA and promoted, without a lag period, limited incorporation into form II molecules. This DNA synthesis was nonspecific with respect to oriC, as judged by DnaA protein dependence, and presumably occurred at nicks in the template DNA. These results are consistent with oriC-specific initiation requiring closed supercoiled molecules dependent on DNA ligase activity. The results also show that decatenation of dimers occurs readily on nicked dimer and represents an efficient pathway for processing replication intermediates in vitro.     

Characterization of a new class of circular DNA molecules in yeast

This paper describes the isolation of a pure population of covalently closed circular twisted DNA molecules from yeast. These molecules are homogeneous in size, that is consist of monomers of 2.2μ and of multiple length oligomers of n x 2.2μ. While no data rule out the mitochondrial origin of this DNA, its actual intracellular localization remains unknown; it displays the same buoyant density as the main nuclear DNA and therefore is not the heavy nuclear satellite DNA (γ-DNA described by Moustacchi and Williamson (1966)); although circular molecules represent only 1 to 5 % of the total DNA, they can be prepared in sizable and reproducible amounts by a method based on the use of mechanical disruption of yeast cells rather than lysis by snail gut juice.     

Behavior of bacteriophage Mu DNA upon infecton of Escherichia coli cells.

The question whether the ends of bacteriophage Mu DNA are fused to form a ring in host cells is critical to the understanding of the mechanism of integrative recombination between Mu DNA and host DNA. We have examined the fate of ³²P-labeled Mu DNA, after infection of sensitive and immune (lysogenic) cells, by sedimentation in sucrose gradients, ethidium bromide/CsCl density centrifugation and by electrophoresis of parental Mu DNA and its fragments in agarose gels. We find that the parental Mu DNA cannot be detected as covalently closed circles at any stage during the Mu life cycle. An interesting form of Mu DNA can be seen after superinfection of immune cells. This form sediments about twice as fast as the mature phage DNA marker in neutral sucrose gradients but yields linear molecules upon phenol extraction. Upon infection of sensitive cells, most of the parental DNA associates with a large complex, presumably containing the host chromosome. When Mu-sensitive cells are infected with unlabeled Mu particles and Mu DNA examined at different times after infection by fractionation in 0.3% agarose gels and hybridization with ³²P-labeled Mu DNA, Mu sequences are found to appear with the bulk host DNA as the phage lytic cycle progresses. However, no distinct replicative or integrative intermediate of Mu, that behaves differently from linear Mu DNA and is separate from the host DNA, can be detected.     

DNA sequence of the Bryan high-titer strain of Rous sarcoma virus: extent of env deletion and possible genealogical relationship with other viral strains

Raji cells, collected at various times from a synchronized culture, were gently lysed, and the high-molecular-weight DNA was enriched ca. 10-fold for latent Epstein-Barr virus (EBV) genomes by equilibrium density gradient centrifugation in neutral CsCl. The heavy-density DNA pool, which included more than 90% of the total intracellular EBV DNA sequences, was further fractionated by velocity sedimentation on neutral glycerol gradients, and material from fractions containing potential EBV DNA replicative forms was examined in the electron microscope. Early in the cellular S phase, when the EBV DNA content was found to be doubling in parallel with host chromosome replication, half of the 50- to 55-micron circular EBV genomes were observed to have two or more DNA branch points or forks. Most molecules were in a relaxed theta configuration, indicative of the Cairns mode of DNA replication. In the supercoiled state, the two daughter strands of the partially replicated molecules were seen to be wrapped around each other. Two theta structures had more than two DNA forks, indicating that DNA replication can initiate more than once on the same DNA molecule. Late in the S phase, the EBV DNA sedimenting at positions where theta structures were found with early S phase samples was composed of catenated dimers rather than partially replicated genomes. It is concluded that the circular EBV genomes, which are the major intracellular form in latently infected cells, are maintained as independent replicons and are not synthesized from an integrated template.