Dynamics of double stranded DNA reptation from bacteriophage.

The dynamics of dsDNA release process from a phage head has been analyzed theoretically. The process was considered as dsDNA reptation through the phage tail. The driving force is assumed to be the decrease of the DNA globule free energy on its releasing from the head in the surrounding medium. The results of the equilibrium theory on an intraphage DNA globule were applied. Three possible sources of friction were examined. The first one is in the inner channel of the tail. The second is the friction of DNA segments in the whole globule volume, which is essential when the globule decondensation is a collective process, at simultaneous moving of all the turns (mechanism 1). The third is the globule friction with the capsid inner surface, that is most important when decondensation proceeds via the globule rotation as a whole spool (mechanism 2). Mechanism 1 would require a lot of time for ejection. Mechanism 2 would lead to different ejection dynamics of short- and long-tailed phages. Comparison of the theoretical results with the published experimental data argues in favor of mechanism 2.     

A theoretical model of DNA packaging in the phage head]

Statistical model of dsDNA packaging to icosahedral bacteriophage capsid is presented. The model describes intraphage DNA as a globule, i.e. intramolecular liquid crystal. We analyse the free energy of DNA, which has a globulized part inside the phage capsid and coil-like tail outside it. Conditions when processes of DNA movement into capsid or back are thermodynamically favorable are investigated. These processes are not accompanied with any thermal effects. It is not "all or none" type process, i.e. intermediate stable states are possible. The role of DNA interaction with the capsid inner wall is considered. The essential model abilities for qualitative explanation of experimental data are exhibited.     

The Y chromosome of the mouse is decondensed in Sertoli cells

The condensation of the Y chromosome in mouse cells was studied with two repetitive DNA probes, pY353/B and M34. Both DNA probes are specific to the Y chromosome and hybridize in situ along the whole chromosome. Due to the high resolution of the in situ hybridization technique with non-radioactive labeled DNA probes it was possible to observe the degree of condensation of the Y chromosome in the interphase cell nuclei of various somatic tissues and on testes preparations. The Sertoli cells were the only cell type in which the Y chromosome was always observed to be in a highly decondensed state. The decondensation of the Y chromosome in the Sertoli cells supports the view that the genetic activity of the Y chromosome is cell autonomous and opens the way to its molecular analysis.     

Chromatin structure during the prereplicative phases in the life cycle of mammalian cells

The object of this study was to determine the kinetics of chromosome decondensation during the G1 period of the HeLa cell cycle. HeLa cells synchronized in the G1 period following the reversal of mitotic block were fused with Colcemid-arrested mitotic HeLa cells at 1.5, 3, 5, and 7 h after the reversal of N2O block. The resulting prematurely condensed chromosomes (PCC) were classified into six categories depending on the degree of their condensation. The frequency of occurrence of each category was plotted as a function of time after mitosis. The results of this study indicate that the process of chromosome decondensation, initiated during the telophase of mitosis continues throughout the G1 period without any interruption, thus the chromatin reaches an ultimate state of decondensation by the end of G1 period, when DNA synthesis is initiated.     

Trout sperm chromatin. II. Ultrastructural aspects after salt dissociation of proteins

The ultrastructural organization of the trout sperm nucleus was studied in ultrathin sections and spread preparations after partial decondensation of the nucleus with increasing NaCl concentrations. The obtained results suggest that the organization of the trout sperm chromatin is much more complex than a pure nucleoprotamine. Three types of complexes were observed. The first one results from the association of DNA with protamines. This complex appears as a fibrous network when partially decondensed nuclei are digested with DNase I indicating that at least a part of DNA remains protected by protamines and favours models accepting a colinear alignment of the latter on the DNA molecules. The second type of structures represent the DNA-protamine fibers compacted into dense clumps which appear as separate compaction units seen upon partial decondensation of the sperm nucleus. A third type are complexes of the ring-shaped granular bodies tightly associated with DNA and resisting high salt-urea and detergent treatment.     

Two phases of chromatin decondensation during dedifferentiation of plant cells: distinction between competence for cell fate switch and a commitment for S phase

Cellular dedifferentiation is the major process underlying totipotency, regeneration, and formation of new stem cell lineages in multicellular organisms. In animals it is often associated with carcinogenesis. Here, we used tobacco protoplasts (plant cells devoid of cell wall) to study changes in chromatin structure in the course of dedifferentiation of mesophyll cells. Using flow cytometry and micrococcal nuclease analyses, we identified two phases of chromatin decondensation prior to entry of cells into S phase. The first phase takes place in the course of protoplast isolation, following treatment with cell wall degrading enzymes, whereas the second occurs only after protoplasts are induced with phytohormones to re-enter the cell cycle. In the absence of hormonal application, protoplasts undergo cycles of chromatin condensation/decondensation and die. The ubiquitin proteolytic system was found indispensable for protoplast progression into S phase, being required for the second but not the first phase of chromatin decondensation. The emerging model suggests that cellular dedifferentiation proceeds by two functionally distinct phases of chromatin decondensation: the first is a transitory phase that confers competence for cell fate switch, which is followed, under appropriate conditions, by a second proteasome-dependent phase representing a commitment for the mitotic cycle. These findings might have implications for a wide range of dedifferentiation-driven cellular processes in higher eukaryotes.     

Theory of cooperative transitions in protein molecules. II. Phase diagram for a protein molecule in solution

The thermodynamically stable states of denatured protein in solution are investigated. These states are distinguished from the native state by the absence of tight packing of side chains while the compactness of denatured protein may vary within a wide region. The following regimes are outlined: 1. the "wet" molten globule, i.e., the compact state with pores occupied by solvent; 2. the swollen globule ("wet," of course); and 3. the coil. The "dry" molten globule, when solvent does not penetrate inside the protein, is excluded for all experimental conditions. All the transitions within the denatured globule state are gradual while the denatured globule-coil phase transition is a second order one. The conditions of protein denaturation as well as conditions of transitions and crossovers within the denatured state are outlined.     

The phage phi29 membrane protein p16.7, involved in DNA replication, is required for efficient ejection of the viral genome

It is becoming clear that in vivo phage DNA ejection is not a mere passive process. In most cases, both phage and host proteins seem to be involved in pulling at least part of the viral DNA inside the cell. The DNA ejection mechanism of Bacillus subtilis bacteriophage phi29 is a two-step process where the linear DNA penetrates the cell with a right-left polarity. In the first step approximately 65% of the DNA is pushed into the cell. In the second step, the remaining DNA is actively pulled into the cytoplasm. This step requires protein p17, which is encoded by the right-side early operon that is ejected during the first push step. The membrane protein p16.7, also encoded by the right-side early operon, is known to play an important role in membrane-associated phage DNA replication. In this work we show that, in addition, p16.7 is required for efficient execution of the second pull step of DNA ejection.     

Chromatin structure during the prereplicative phases in the life cycle of mammalian cells

The object of this study was to determine the kinetics of chromosome decondensation during the G₁ period of the HeLa cell cycle. HeLa cells synchronized in the G₁ period following the reversal of mitotic block were fused with Colcemid-arrested mitotic HeLa cells at 1.5, 3, 5, and 7 h after the reversal of N₂O block. The resulting prematurely condensed chromosomes (PCC) were classified into six categories depending on the degree of their condensation. The frequency of occurrence of each category was plotted as a function of time after mitosis. The results of this study indicate that the process of chromosome decondensation, initiated during the telophase of mitosis continues throughout the G₁ period without any interruption, thus the chromatin reaches an ultimate state of decondensation by the end of G₁ period, when DNA synthesis is initiated.     

Real-time observation of nucleoplasmin-mediated DNA decondensation and condensation reveals its specific functions as a chaperone

Fertilization requires decondensation of promatine-condensed sperm chromatin, a dynamic process serving as an attractive system for the study of chromatin reprogramming. Nucleoplasmin is a key factor in regulating nucleosome assembly as a chaperone during fertilization process. However, knowledge on nucleoplasmin in chromatin formation remains elusive. Herein, magnetic tweezers (MT) and a chromatin assembly system were used to study the nucleoplasmin-mediated DNA decondensation/condensation at the single-molecular level in vitro. We found that protamine induces DNA condensation in a stepwise manner. Once DNA was condensed, nucleoplasmin, polyglutamic acid, and RNA could remove protamine from the DNA at different rates. The affinity binding of the different polyanions with protamine suggests chaperone-mediated chromatin decondensation activity occurs through protein–protein interactions. After decondensation, both RNA and polyglutamic acid prevented the transfer of histones onto the naked DNA. In contrast, nucleoplasmin is able to assist the histone transfer process, even though it carries the same negative charge as RNA and polyglutamic acid. These observations imply that the chaperone effects of nucleoplasmin during the decondensation/condensation process may be driven by specific spatial configuration of its acidic pentamer structure, rather than by electrostatic interaction. Our findings offer a novel molecular understanding of nucleoplasmin in sperm chromatin decondensation and subsequent developmental chromatin reprogramming at individual molecular level.     

Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.     

Secretory protein decondensation as a distinct, Ca2+-mediated event during the final steps of exocytosis in Paramecium cells

The contents of secretory vesicles ("trichocysts") were isolated in the condensed state from Paramecium cells. It is well known that the majority portion of trichocysts perform a rapid decondensation process during exocytosis, which is visible in the light microscope. We have analyzed this condensed leads to decondensed transition in vitro and determined some relevant parameters. In the condensed state, free phosphate (and possibly magnesium) ions screen local surplus charges. This is supported by x-ray spectra recorded from individual trichocysts (prepared by physical methods) in a scanning transmission electron microscope. Calcium, as well as other ions that eliminate phosphate by precipitation, produces decondensation in vitro. Under in vivo conditions, Ca2+ enters the vesicle lumen from the outside medium, once an exocytic opening has been formed. Consequently, within the intact cell, membrane fusion and protein decondensation take place with optimal timing. Ca2+ might then trigger decondensation in the same way by precipitating phosphate ions (as it does in vitro) and, indeed, such precipitates (again yielding Ca and P signals in x-ray spectra) can be recognized in situ under trigger conditions. As decondensation is a unidirectional, rapid process in Paramecium cells, it would contribute to drive the discharge of the secretory contents to the outside. Further implications on the energetics of exocytosis are discussed.     

Amyloid fibril formation by human stefin B: influence of the initial pH-induced intermediate state

The amyloid fibril field is briefly described, with some stress put on differences between various proteins and possible role for domain swapping. In the main body of the text, first, a short review is given of the folding properties of both human stefins, alpha/beta-type globular proteins of 53% identity with a known three-dimensional fold. Second, in vitro study of amyloid fibril formation by human stefin B (type I cystatin) is described. Solvents of pH 4.8 and pH 3.3 with and without 2,2,2-trifluoroethanol (TFE) were probed, as it has been shown previously that stefin B forms acid intermediates, a native-like and molten globule intermediate, respectively. The kinetics of fibrillation were measured by thioflavin T fluorescence and CD. At pH 3.3, the protein is initially in the molten globule state. The fibrillation is faster than at pH 4.8; however, there is more aggregation observed. On adding TFE at each pH, the fibril formation is further accelerated.     

Initiation of DNA replication cycle in fertilized eggs of the starfish, Asterina pectinifera

Starfish oocytes or eggs were inseminated at various times between first prometaphase and pronuclear stage, and were subsequently labeled with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) in order to detect the onset of DNA synthesis phase (S phase) during the first cell cycle using a monoclonal antibody against BrdU. The interval between fertilization and the first S phase was found to be constant (30-45 min, depending on batches) in eggs fertilized after formation of the first polar body. Eggs fertilized before first polar body formation, however, always entered the S phase 10-20 min after the second polar body formation. On the basis of these observations we conclude that (i) the chain of events triggered by fertilization, collectively called "postactivation process" for the first S phase, goes on in parallel with the process of maturation and (ii) only the final step of the postactivation process is arrested until the termination of meiosis. In eggs that had been fertilized before the first polar body formation, the female and male pronuclei exhibited uniformly distributed chromatin soon after the second polar body formation. In eggs that had been fertilized after the second polar body formation, however, the chromatin of the pronuclei remained fibrillar even during the S phase. Thus full decondensation of chromatin appears to depend on a certain advance in the postactivation process.     

Renaturation of ovotransferrin under two-step conditions allowing primary folding of the fully reduced form and the subsequent regeneration of the intramolecular disulfides

A two-step procedure was found to be useful for the efficient refolding of a complex protein, ovotransferrin. In the first step, the reduced and denatured form of the protein was incubated at a low temperature in a nondenaturing buffer containing reduced glutathione; in the second step, the reduced form was reoxidized at a higher temperature in the presence of oxidized glutathione. Under these conditions, the fully reduced forms of ovotransferrin and its half-molecules were almost quantitatively reoxidized to regain iron-binding abilities and conformations, very similar to the native form. The circular dichroism spectra revealed that at low temperatures the fully reduced forms have partially folded conformations, which are fluctuating like "molten globule" states. The reoxidization kinetics compared between whole ovotransferrin and the two half-molecules supported independent refolding of the N- and C-terminal domains.     

Developmental changes in chromatin organization in rat cerebral hemisphere neurons and analysis of DNA reassociation kinetics

Previous reports have demonstrated that neuronal nuclei of rabbit, mouse and rat cerebral hemispheres exhibit a short DNA repeat length of 160 bp compared to the more typical repeat size of 200 bp found in glial nuclei and other cell types of higher eukaryotes. In this study we report that the conversion of chromatin to a short DNA repeat length in rat cerebral hemisphere neurons is a gradual process which begins between the first and second day after birth and is complete by 8 days. In these neurons, histone H1 appears to be less accessible to degradation by trypsin in the newborn rat brain compared to the 8 day old rat. This suggests that the developmental shift to a short DNA repeat length may be accompanied by a dispersal or decondensation of neuronal chromatin which results in an increased accessibility of neuronal histone H1 to degradation by trypsin. The increase in nuclear DNA content to 3.5C which has been reported in rat cortical neurons during early postnatal development does not appear to be associated with a selective amplification of a subset of DNA sequences as determined by DNA reassociation kinetics.     

Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258

A benzimidazole derivative, Hoechst 33258 can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when the compound is given in sufficiently high concentration (40 micrograms/ml) to the L cell culture. Hoechst 33258 at low concentration (1 micrograms/ml, 16 h) cannot produce this effect on L cell chromosomes. Bromodeoxyuridine (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258 treatment at low concentration could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incorporation for one cell cycle, the association of A-T rich satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.     

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme.

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.     

A model of chromatin-dependent DNA replication sequences based on the decondensation units hypothesis

A model of chromatin-dependent DNA replication sequences was developed on the previously reported "decondensation units" hypothesis and its kinetic properties were examined by way of calculating various numerical indices using a Monte Carlo procedure. The model has much in common with the previous one but a fundamental difference is that the unit is assumed to consist of linearly arranged H-, D-, A- and S-zones each containing genes of different functional categories which are called H-, D-, A- and S-genes, respectively. The units are decondensed by the action of D-factors, i.e. decondensation factors, from H-zone to the end of S-zone and the genes in decondensed regions release signals to produce housekeeping enzymes, D-factors, A-factors and S-factors. These products are stored and at the same time degraded. A-factors activate replication origins in the decondensed regions and S-factors induce DNA synthesis at the activated origins. Replicated DNA is recondensed and gene activities are shut down in the recondensed chromatin. The factors are produced under the control of chromosome cycle and in turn affect chromosomes. Thus, dual control mechanism operates as Mazia and Prescott have argued. Biochemical and cytogenetic basis of this model was reviewed briefly and some results of simulation presented which include DNA synthesis rate vs. DNA content relationships. An outstanding characteristic of the model is the constancy of cellular state in A-subphase located in the late G1.