Chromatin fiber structure and plectonemic model of chromosome condensation in Drosophila cells

Reversible permeable cells have been used to isolate chromatin structures during the process of chromosome condensation. Analysis of individual structures slipping out from nuclei after reversal of permeabilization revealed that chromosomes of Drosophila cells consist of small units called rodlets. The fluorescent images of chromatin fibers were subjected to computer analysis allowing the computer-aided visualization of chromatin fibers. The zig-zag array of fibers consisting of 12-15 nucleosomes with a length of 270-330 nm (average 300 nm) showed decondensed extended strings, condensed loops, and coiled condensed loops. Theoretical considerations leading to the plectonemic model of chromatin condensation are based on experimental data, and give an explanation how the 30 chromatin fibers are formed and further condensed to the 300 nm chromatin loops in Drosophila cells.     

Higher levels of organization in chromosomes.

On the basis of recent results a unified view of different aspects of the higher levels in the organization of chromatin in chromosomes is presented. Basic to these forms of organization is the arrangement of DNA in the complex with nucleosomes and recent studies suggest that at least some species of satellite DNA may maintain a fixed DNA sequence relationship to the phasing of nucleosomes. Special proteins such as the high-mobility group (HMG) proteins or other non-histone proteins could serve specific functions in the recognition of satellite DNA sequences.In the presence of histone H1 the 110 Å nucleosome fiber formed from the basic string of nucleosomes can be further condensed into a thicker 250–300 Å fiber formed by a solenoidal coiling of the 110 Å fiber with about 6–8 nucleosomes per turn and the available evidence suggests that these structures are found in mitotic chromosomes as well as other forms of inactive chromatin. A further level of coiling of the 250–300 Å solenoid has been suggested by our recent studies of disintegrated mitotic chromosomes consisting of a thin-walled tube with an outer diameter of 4000 Å referred to as the unit fiber. This structure would account for a factor of 1400 × contraction of DNA in mitotic chromosomes which in their intact state are only 5-fold more contracted. The recently described “scaffold” proteins could be responsible for this final coiling of the unit fibers in intact chromosomes.Meiotic chromosomes are generally less contracted than mitotic chromosomes. An extreme example of this are lampbrush chromosomes that apart from the axial segments which might contain some structural proteins appear to consist of naked DNA arranged in lateral loops. In the later stages of meiosis more condensed structures arise as exemplified by the synaptonemal complex during the pachytene stage in many organisms. The characteristic features of this structure are interpreted to suggest that the structure consists of lateral components containing two parallel 110 Å nucleosome fibers each representing the axial segments of two sister chromatids. From these paired regions loops protrude laterally in a manner which closely resembles the less condensed lampbrush chromosomes. The implication of this structure in the process of crossingover is discussed.Less is known about the organization of chromatin in interphase nuclei, but structures analogous to the loop-like structures in meiotic chromosomes are suggested on the basis of the isolation of supercoiled DNA loops constrained by RNA-DNA and protein-DNA interactions. The position of these loops is suggested to be fixed by specific repeated DNA sequences that could be associated with specific tenacious non-histone or HMG proteins.     

Moving pictures of DNA released upon lysis from bacteria,chloroplasts, and mitochondria

Summary Cells and organelles suspended in gelled agarose agarose were lysed with detergent and protease, stained with ethidium bromide and their DNA was observed by fluorescence microscopy. The migration of individual DNA molecules during electrophoresis on a microscope slide was recorded on video tape so that moving pictures could be analyzed. The DNA from lysed bacteria (Escherichia coli andAgrobacterium tumefaciens) appeared as a rosette of at least twenty loops of varying size, whereas that from bacterial spheroplasts (E. coli andPseudomonas aeruginosa) appeared as circular forms or rods with many fine filaments of RNA extending toward the anode. The DNA from chloroplasts of watermelon (Citrullus vulgaris) and pea (Pisum sativum) did not appear as a rosette of loops. Many or most of the chloroplast DNA molecules per lysed chloroplast were immobile in the electric field, as if in circular form hooked on agarose fibers. The amount of DNA-fluorescence per watermelon mitochondrial particle was much less than that found for either chloroplasts or bacteria. The appearance of the mitochondrial DNA during electrophoresis was that of linear molecules, no obviously circular forms were evident and no rosette structures were observed.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole - kb kilobase pairs - mtDNA mitochondrial DNA - PFGE pulsed-field gel electrophoresis     

The structure of a mesokaryote chromosome

Condensed and dispersed forms of the chromosomes of the dinoflagellate, Prorocentrum micans, deposited on grids by the microcentrifugation technique were studied by electron microscopy. In the normally condensed form, the chromosomes appear as banded rods surrounded by a peripheral cloud of partially dispersed fibers. Single fibers in these and in extensively dispersed preparations appear as smooth threads of uniform diameter (55–65 Å). The chromosome fibers are contrasted by positive-group-specific stains, indicating the presence of cationic moieties associated with the DNA. Occasionally Y-shaped chromosomes are seen; these may be replicating structures. These observations are in general agreement with studies of dinoflagellate chromosomes by other techniques, and provide support for the suggestion that these organisms possess a genome organization whose structure is typical of neither prokaryotes nor eukaryotes, and hence may be intermediate forms.     

Fine Structural in Situ Analysis of Nascent DNA Movement Following DNA Replication

Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase α was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.     

Structure of viral φ29 DNA condensed by simple triamines: A light-scattering and electron-microscopy study

The structures of viral φ29 DNA condensed by triamines, principally spermidine, in 10^?3M NaCl were investigated by static and dynamic light scattering and electron microscopy. All of the results for DNA condensed in 30 μM spermidine at neutral pH are quantitatively consistent with a toroidal structure with a mean outer diameter of 1850 Å. At pH 10.2, however, condensed structures of a completely different size and shape are observed for the first time. These structures are also more irregular in shape and more polydisperse than those at neutral pH. This conformational change is believed to result from a change in the mode of spermidine binding that is coupled to, or associated with, the (premature) titration of protons on the base-ring nitrogens of guanine and thymine. Besides spermidine, certain homologs of spermidine, in which the butyl moiety of spermidine was replaced by longer pentyl through octyl moieties, were also studied. Though all of the triamines condensed the DNA at 30 μM, aggregation became a more prevalent occurrence as the length of the end chain increased. This suggests that crosslinking may play an important role in the condensation process. Finally, these aggregates are dissociated to a considerable extent at pH 10.2, and the resulting compact structures appear to be quite similar, independent of the triamine used to condense the DNA. The observed partial breakdown of aggregates is also consistent with the hypothesis of a change in mode of triamine binding at pH 10.2.     

DNA modeling reveals an extended lac repressor conformation in classic in vitro binding assays

Protein-mediated DNA looping, such as that induced by the lactose repressor (LacI) of Escherichia coli, is a well-known gene regulation mechanism. Although researchers have given considerable attention to DNA looping by LacI, many unanswered questions about this mechanism, including the role of protein flexibility, remain. Recent single-molecule observations suggest that the two DNA-binding domains of LacI are capable of splaying open about the tetramerization domain into an extended conformation. We hypothesized that if recent experiments were able to reveal the extended conformation, it is possible that such structures occurred in previous studies as well. In this study, we tested our hypothesis by reevaluating two classic in vitro binding assays using a computational rod model of DNA. The experiments and computations evaluate the looping of both linear DNA and supercoiled DNA minicircles over a broad range of DNA interoperator lengths. The computed energetic minima align well with the experimentally observed interoperator length for optimal loop stability. Of equal importance, the model reveals that the most stable loops for linear DNA occur when LacI adopts the extended conformation. In contrast, for DNA minicircles, optimal stability may arise from either the closed or the extended protein conformation depending on the degree of supercoiling and the interoperator length.     

Electron microscopy of surface spread polytene chromosomes of Drosophila and Stylonychia

The ultrastructure of polytene chromosomes of Drosophila and Stylonychia were compared in whole-mount spread preparations. In Drosophila the chromomeres appear as dense, unresolvable structures interconnected by 10-nm interband fibers. In contrast, chromomeres of Stylonychia polytene chromosomes are formed by aggregates of 30-nm loops laterally attached to 10-nm interband fibers. It is suggested that the polytene chromosomes in these two species are analogous rather than homologous structures.     

Localization of DNA in the condensed interphase chromosomes of Euglena

The localization of DNA in the condensed interphase chromosomes of Euglena was determined by immunoelectron microscopy. Deposits of gold particles that coincided with the localization of DNA followed threads that corresponded to the chromatin fibers. The threads were 55–80 nm in diameter and were assumed to be supersolenoids. The localization of gold deposits on chromosomes that had been sectioned in various directions suggested that the chromatin fibers coiled around the surface of chromosomes, with a wide central axial region of the chromosomes remaining free of DNA. These findings are discussed in relation to current models of chromosomal structure.     

Cell cycle-specific changes in the ultrastructural organization of prematurely condensed chromosomes

Prematurely condensed chromosomes (PCC) of HeLa cells synchronized in different phases of the cell cycle were analyzed by high-resolution scanning electron microscopy. The purpose of this study was to examine changes in the arrangement of the basic 30-nm chromatin fiber within interphase chromosomes associated with progression through the cell cycle. These studies revealed that highly condensed metaphase chromosomes and early G1-PCC consisted of tightly packed looping fibers. Early to mid G1-PCC were more extended and exhibited gyres suggestive of a despiralized chromonema. Further attenuation of PCC during progression through G1 was associated with a gradual transition from packed looping fibers to single extended longitudinal fibers. This process occurs prior to the initiation of DNA synthesis which appears to be localized within single longitudinal fibers. Following replication of a chromosome segment, extended longitudinal fibers were rapidly reorganized into packed looping fiber clusters concomitant with the formation of a multifibered chromosome axis. This results in the characteristic “pulverized” appearance of S-PCC when viewed by light microscopy. Subsequently, adjacent looping fiber domains coalesce, resulting in the uniformly packed, looping fiber arrangement observed in G2-PCC. Spiralization of the chromonema during the G2-mitotic transition results in the formation of highly compact metaphase chromosomes.     

A fresh look at meiosis and centromeric heterochromatin in the red-backed salamander,Plethodon cinereus cinereus (Green)

Male meiosis, with special regard to the centromeric heterochromatin and to centromeric structure, has been studied in the salamander, Plethodon cinereus cinereus. In this salamander, n = 14. Early meiotic prophase proceeds as described by other authors. Pachytene is followed by a diffuse stage in which much of the chromosomal DNA becomes reorganized into fine lateral loops which spring from the bivalent axes. These loops can be seen along the bivalent axes as early as zygotene. Loops are maximally extended in the diffuse stage. The formation of diplotene bivalents involves a return of this extended DNA into the axes of the bivalents. — At leptotone, centromeric heterochromatin is in one or a few large masses. These masses break up during zygotene. At pachytene there is one mass of heterochromatin at the centromeric region of each bivalent. The heterochromatin remains condensed in the diffuse stage. During diplotene, centromeric heterochromatin becomes less conspicuous, and it is possible to see 4 centromere granules in each diplotene bivalent. These observations support the view that centromeres replicate at pre-meiotic S-phase when the associated hetero-chromatin is replicated. In the interphase before the 2nd division, the hetero-chromatin often forms a broken ring corresponding to the positions of the centromeres at the end of anaphase 1. There are 14 masses of heterochromatin in nuclei at prophase of the 2nd division. In spermatids, the heterochromatin appears as a single solid mass or a broken ring.     

Isolation of rosette-like structures from partially deproteinized chromatin in rat hepatocytes

The structure of partial deproteinized rat hepatocyte chromatin has been studied. Depending on the magnesium concentration the chromatin of isolated nuclei is present in the two conditions: diffuse (at 0-1.5 mM MgCl2) and condensed (at 2-5 mM MgCl2). The main components of nuclei with condensed chromatin are chromomers--globular structures about 100 nm in diameter. By treating such nuclei with heparin and dextransulfate one can observe a rosette-like structure with lateral loops having the following parameters: the length of the loops, 15-20 micron; the number of loops, 15-30. The rosette-like structures are sensitive to endogenous nuclease and DNase 1, but not to RNase. Pronase or higher concentration of polyanions give rise to unfolding of the rosette-like structures. The rosette structures cannot be isolated from the nuclei with diffuse chromatin. On the basis of these observations a hypothesis of chromatin structural organization in the interphase nucleus is proposed, and the connection of the rosette-like structures with some structural levels of chromatin organization is discussed.     

DNA-protein binding in interphase chromosomes

The metachromatic dye, azure B, was analyzed by microspectrophotometry when bound to DNA fibers and DNA in nuclei with condensed and dispersed chromatin. The interaction of DNA and protein was inferred from the amount of metachromasy (increased β/α-peak) of azure B that resulted after specific removal of various protein fractions. Dye bound to DNA-histone fibers and frog liver nuclei fixed by freeze-methanol substitution shows orthochromatic, blue-green staining under specific staining conditions, while metachromasy (blue or purple color) results from staining DNA fibers without histone or tissue nuclei after protein removal. The dispersed chromatin of hepatocytes was compared to the condensed chromatin of erythrocytes to see whether there were differences in DNA-protein binding in "active" and "inactive" nuclei. Extraction of histones with 0.02 N HCl, acidified alcohol, perchloric acid, and trypsin digestion all resulted in increased dye binding. The amount of metachromasy varied, however; removal of "lysine-rich" histone (extractable with 0.02 N HCl) caused a blue color, and a purplish-red color (µ-peak absorption) resulted from prolonged trypsin digestion. In all cases, the condensed and the dispersed chromatin behaved in the same way, indicating the similarity of protein bound to DNA in condensed and dispersed chromatin. The results appear to indicate that "lysine-rich" histone is bound to adjacent anionic sites of a DNA molecule and that nonhistone protein is located between adjacent DNA molecules in both condensed and dispersed chromatin.     

Concentration-dependent organization of DNA by the dinoflagellate histone-like protein HCc3

The liquid crystalline chromosomes of dinoflagellates are the alternative to the nucleosome-based organization of chromosomes in the eukaryotes. These nucleosome-less chromosomes have to devise novel ways to maintain active parts of the genome. The dinoflagellate histone-like protein HCc3 has significant sequence identity with the bacterial DNA-binding protein HU. HCc3 also has a secondary structure resembling HU in silico. We have examined HCc3 in its recombinant form. Experiments on DNA-cellulose revealed its DNA-binding activity is on the C-terminal domain. The N-terminal domain is responsible for intermolecular oligomerization as demonstrated by cross-linking studies. However, HCc3 could not complement Escherichia coli HU-deficient mutants, suggesting functional differences. In ligation assays, HCc3-induced DNA concatenation but not ring closure as the DNA-bending HU does. The basic HCc3 was an efficient DNA condensing agent, but it did not behave like an ordinary polycationic compound. HCc3 also induced specific structures with DNA in a concentration-dependent manner, as demonstrated by atomic force microscopy (AFM). At moderate concentration of HCc3, DNA bridging and bundling were observed; at high concentrations, the complexes were even more condensed. These results are consistent with a biophysical role for HCc3 in maintaining extended DNA loops at the periphery of liquid crystalline chromosomes.     

Rearrangements of large-insert T-DNAs in transgenic rice

Introduction of large-DNA fragments into cereals by Agrobacterium-mediated transformation is a useful technique for map-based cloning and molecular breeding. However, little is known about the organization and stability of large fragments of foreign DNA introduced into plant genomes. In this study, we produced transgenic rice plants by Agrobacterium-mediated transformation with a large-insert T-DNA containing a 92-kb region of the wheat genome. The structures of the T-DNA in four independent transgenic lines were visualized by fluorescence in situ hybridization on extended DNA fibers (fiber FISH). By using this cytogenetic technique, we showed that rearrangements of the large-insert T-DNA, involving duplication, deletion and insertion, had occurred in all four lines. Deletion of long stretches of the large-insert DNA was also observed in Agrobacterium.     

Formation of AraC-DNA sandwiches.

With the use of special DNA binding sites, but not the natural aral binding site, the dimeric AraC protein can be forced to make sandwich structures in which two DNA molecules are joined by two AraC protein dimers. Apparently one subunit from each dimer contacts each DNA molecule in an extended structure. These sandwich structures form only in the absence of arabinose. This behavior is consistent with the protein's ability to form DNA loops by binding to separated half sites in the absence of arabinose and its preference for binding to adjacent half-sites in the presence of arabinose.     

Differential accessibility of DNA in extended and condensed chromatin to pancreatic DNase I

Pancreatic DNase I has been used to study the interaction between DNA and chromosomal proteins in extended and condensed chromatin fractions isolated from mouse and Chinese hamster livers. It was found that DNase digests extended chromatin at a faster rate than condensed chromatin, and the evidence suggests that the chromosomal proteins are more tightly complexed to the DNA in condensed than in extended chromatin. This difference in DNA-protein interaction in extended and condensed chromatin may be related to the functional difference which characterizes these fractions, and might be one of the factors underlying the production of bands on metaphase chromosomes.