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Etude sur des lamelles obtenues par cristallisation a partir de solutions diluees. Traitement de monocristaux entre 100 et 270 o C. Transition hexagonale-tetragonale

Recently the relationship between the B and C forms of DNA has been questioned. We have found some amino acids and related amines which form complexes with DNA and favour the C form. We have studied such complexes by X-ray diffraction. With some of these counterions the transition between the B and C forms occurs smoothly, whereas in other cases there is a discontinuous transition. We conclude that the C form is a well-defined variant of the B form favoured by some counterions under low humidity conditions.

It is shown that acid treated histones H1 and H3 are susceptible to specific degradation by an associated acid resistant protease. Dialysis against distilled water (pH 5.5–6) of the acid treated histones enhances proteolysis. On the other hand, no degradation is observed in nucleohistone either in the presence of Ca++ or Na++ ions. The conditions required to avoid degradation during nucleohistone and histone manipulation are described.

Nuclei of spermatozoa of the sea cucumber Holothuria tubulosa contain the five somatic-type histones plus a sperm-specific histone H1 and a unique basic protein ϑ0, which is related to H1 in amino acid composition. No proteins of the High Mobility Group (HMG) type have been detected. The structure of this chromatin has been probed by nuclease digestion. Its behaviour is anomalous, since two distinct fractions of chromatin are recovered from these spermatozoa, which differ either in the presence or absence of the sperm-specific proteins H1 and ϑ0. This heterogeneous distribution is not found in conventional materials, such as calf thymus or chicken erythrocytes. Proteins H1 and ϑ0 are not uniformly distributed and may be localized in special regions of chromatin. Fragments containing long stretches of nucleosomes lacking both proteins can be recovered. At the same time, the chromatin fractions which contain these two proteins are shown to be less soluble. When an extensive digestion of chromatin is carried out yielding only nucleosomes and small oligomers, the H1 and ϑ0 proteins redistribute themselves on chromatin, the two proteins acting in a cooperative fashion in this process. Cross-linking experiments carried out in whole cells indicate a proximity of ϑ0 and H1, whereas no crosslinks have been detected between ϑ0 and any of the four nucleosomal histones. The ϑ0 protein may thus play a role similar to histone H1 and be only loosely associated with nucleosomal histones, but contribute to the structuration of chromatin during spermiogenesis.

It is shown that spermatozoa from the marine snails Gibbula divaricata and Haliotis tuberculata contain a protamine plus a histone component. The composition of the latter is similar to H2 b histone from calf thymus.

We have studied by X-ray diffraction fibers of complexes of poly(dG).poly(dC) with N-alpha-acetyl-L-arginine ethylamide. Although these polynucleotides favour the A form of DNA, in this complex it is never found, thus confirming that arginine prevents the appearance of this form of DNA. At high relative humidity the B form is present. Upon dehydration two new structures appear. One of them is a triple helix, most likely formed by poly(dC+).poly(dG).poly(dC). The other structure found also has features which indicate a multistranded conformation.

In this paper we show that the extent of DNA denaturation achieved by dialysis against N,N-dimethyl formamide depends on both the source of DNA and the buffer used. The stability against denaturation increases with the guanine plus cytosine content. The base sequence also plays a minor role. This behavior is due to differences in the solubility of DNA in the denaturing solvent. The DNAs with a high guanine-cytosine content have a lower solubility in dimethyl formamide and this fact results in a higher stability against denaturation. As a practical consequence, in order to achieve complete denaturation of DNA by dialysis against dimethyl formamide, it is necessary to start with DNA dissolved in a buffer of low ionic strength, preferably below 10(-3) M.

Protein phi 0 is a unique protein which is present in the sperm of the sea cucumber, Holothuria tubulosa. It associates with histones, but its physiological role is unknown. From its amino acid composition and sequence, protein phi 0 can be considered as an H1-related protein. In this paper, we have studied its interaction with chicken erythrocyte chromatin particles of different complexity, from core particles to polynucleosomes. Addition of protein phi 0 results in marked chromatin insolubilization. The higher the molecular weight of the chromatin fragment, the lower is the phi 0/nucleosome molar ratio at which precipitation occurs, so that complete insolubilization of polynucleosomes is achieved at a phi 0/nucleosome molar ratio which is identical to that found in mature H. tubulosa spermatozoa. We have also found that the interaction of protein phi 0 with chromatin is cooperative. These findings contribute to clarification of the peculiar physico-chemical properties shown by H. tubulosa sperm chromatin and the role played by the phi 0 protein.

The nuclei of sperm of the mussel Mytilus edulis contain three specific proteins: (1) a major protamine-like component (φ1); (2) a lysine-rich protein (φ3); and (3) a histone-like component (φ2b). The latter is characterized here for the first time. It is also shown that the φ3 protein does not contain modified lysines, but has some phosphorylated serines. Nuclease digestion of whole nuclei gives a continuous distribution of sizes for DNA, without any evidence of a nucleosome structure. X-ray diffraction indicates that DNA is organized as bundles of parallel molecules without any signs of either crystallinity or higher order structure.

We have developed a method for embedding biological materials for electron microscopy in the water soluble polymer polyacrylamide (PAM). The fixed samples are infiltrated with an aqueous solution of acrylamide, which is then polymerized to form a hydrated gel. The gel is ready for sectioning after insolubilization by cross-linking with glutaraldehyde. Water is retained throughout the whole embedding process and contact of the samples with organic solvents is completely avoided. Sections are best stained with silicotungstic acid. The potential of this technique has been evaluated by detailed examination of several representative tissues. Observation of the native periodicity of 17 nm in PAM embedded myelin indicates a very good structural preservation. The method gives optimal results with samples which are easily infiltrated, such as pellets of cells.

The acid-soluble proteins from the sperm of several species of echinoderms are described. All of them are analogous to calf thymus histones, as judged by their amino acid composition, although some minor differences appear. The electrophoretic pattern is very similar for organisms belonging to the same class, but it differs among the four classes of echinoderms studied. Three to five bands appear on polyacrylamide gel electrophoresis. Two of them (γ and δ) are present and have similar mobilities for all the species studied and for calf thymus, but their relative intensity varies. On the other hand, the additional bands differ in mobility among the four classes studied.

The kinetics of renaturation of heat- or formamide-denatured DNA have been studied by following the change of optical density at a constant temperature. Solvents of different ionic strength and various DNA samples have been used. At the lower ionic strengths studied, the reaction follows second-order kinetics, substantiating the hypothesis that strands of native DNA separate upon denaturation and recombine during renaturation. As the ionic strength is increased at a constant temperature, the reaction deviates from simple second-order behavior. This appears to be the result of the inhibition to rewinding caused by short helical segments in the denatured DNA which are more stable at the higher ionic strenth.

Histone fractions have been obtained from the haemal system and male gonads of the echinoderm Holothuria tubulosa . Fractionation was achieved by the methods of Johns [5], although some anomalies were found. The fractions were purified by precipitation in adequate solvent systems. The histones obtained are similar to those found in calf thymus, although f 1, and to a lesser extent f 2 b , show significant differences. In the gonads an additional minor component with high electrophoretic mobility is found. The other histone fractions appear to be identical with those obtained from somatic tissues. The relevance of this result towards an understanding of the role played by nuclear proteins in the sperm is discussed. Some of the acidic proteins present in the nuclei of these tissues are also preliminarily characterized.

No data are available on the protamines of birds, with the exception of galline. We have characterized the protamines from four species of birds belonging to four different orders. All of them have very similar properties. They have been purified by carboxymethylcellulose chromatography and analyzed with respect to amino acid composition and electrophoretic behaviour. They are very arginine-rich proteins (63.4–67.3%) but do not contain lysine. Serine (12.0–18.2%), tyrosine (5.8–9.0%) and glycine (4.5–7.1%), along with arginine, make up the bulk of the amino acid residues in these molecules. The electrophoretic mobility of bird protamines in acetic acid-urea-polyacrylamide gels is intermediate between that of somatic histones and salmine. The molecular size, estimated from amino acid analysis and electrophoretic migration, is 65 ± 5 amino acid residues.

The tendency of DNA to form fibers upon condensation with counterions is reviewed. It is shown that chromatin fibers may acquire a relatively constant diameter of about 30 nm simply as an optimal size achieved upon neutralization of DNA, without requiring a repetitive internal structure. Thus the size of chromatin fibers would not be determined by any specific spatial interaction between DNA and histones. The driving force for the formation of fibers in chromatin would be similar to that found in proteins when they acquire a compact globular shape.

The complexes formed by DNA and different types of protamines are studied by X-ray diffraction. The protamines used have considerable differences in their molecular weight and in the content of various amino acids. However, in all cases it is found that protamines stabilize DNA in the B form. The molecules pack in the hexagonal system with a single DNA molecule per unit cell. It is concluded that bridges of protamine between DNA molecules are an essential feature in the formation of nucleoprotamine complexes. The salts of DNA with arginine and L-arginyl-L-arginine are also studied. Their X-ray diffraction patterns show features intermediate between those of nucleoprotamine and the alkaline metal salts of DNA.

Helices are expected to scatter circularly polarized light preferentially. According to theory, the scattering depends on the pitch, radius and sense of helix1–4; however, experimental data have been lacking. We now report quantitative measurements of the differential scattering of left and right circularly polarized light from the sperm head of the octopus Eledone cirrhosa. This sperm head is a left-handed helix with pitch and diameter of about 1 µm. It preferentially scatters left circularly polarized light versus right circularly polarized light in the forward direction, with a maximum differential scattering of 2% at 35° to the incident beam (wavelength = 0.442 µm). The preferential forward scattering of left circularly polarized light is consistent with theory1–4. As a differential scattering of 0.1% is easily measured, this can be a useful new method to characterize helical biological objects.

The molecular structure of nucleoprotamine from Gibbula divaricata and its packing in oriented fibers has been modelled both to fit the X-ray diffraction pattern and to avoid steric compression. The representative model consists of 51 poly(dinucleotide) B-DNA helices with 51 poly(hexapeptide) chains associated with the major grooves. The prevailing peptide conformation is β, The four arginine residues present are hydrogen-bonded to DNA phosphates while neutral peptides protrude into the minor grooves of neighboring nucleoprotamine molecules which are packed 2.61 nm apart in a screw-disordered, quasi-hexagonal lattice. This model reconciles a number of earlier, apparently conflicting experimental results and explains the remarkable stability of nucleoprotamines.

Crystals of self complementary DNA hexamers d(CACGTG), d(CCGCGG) and d(GGCGCC) were grown by vapour diffusion technique and studied by microRaman and microIR spectroscopies. The oligonucleotides were studied in parallel in solution by vibrational spectroscopy. A B- greater than Z transition was detected by Raman spectroscopy during the crystallization process for d(CACGTG). Vibrational spectroscopy shows that the d(GGCGCC) crystals adopt a B geometry. On the contrary the d(CCGCGG) sequence which is shown to be able to undergo in solution or in films quite easily the B- greater than Z transition, remains trapped in crystals in a geometry which may correspond to an intermediate conformation often proposed in models of the B- greater than Z transition. The crystals used in this study were characterized by X-ray diffraction. The unit cell and space group have been determined.

It is shown that when tension is applied to DNA it can change its conformation. In the relaxed state, DNA adopts the A form in the absence of excess salt. When tension is applied, either the B, C or stretched conformations of DNA are observed, depending on the humidity of the sample.

Model calculations are presented in order to interpret the X-ray diffraction diagrams given by chromatin gels. It is shown that by taking into account the hydration of chromatin subunits, the problem of calculating the interference function in concentrated gels is greatly simplified. In this way it is spossible to fully interpret the influence of concentration on the position and intensity of the various rings present in the X-ray diffraction patterns. The possibilities and limitations of models based on spherical symmetry are also discussed. It is concluded that each chromatin subunit most likely contains three turns of DNA in each 200 base pairs segment surrounding a central protein core. With the method presented here it is possible to test if other models of chromatin based on different kinds of evidence are compatible with the X-ray diffraction data.

We have studied the diameter of chromatin fibres embedded in epoxy resins for three different materials: mouse thymus, chicken erythrocytes and sea cucumber spermatozoa. We confirm that the diameter of chromatin fibres increases with linker length, both values being influenced by the protein composition of chromatin.

The crystal and molecular structure of the sodium salt of deoxycytidylyl-(3H-5H)-deoxyguanosine has been determined from X-ray diffraction data. The crystal, obtained from an aqueous gamma-butyrolactone solution at pH = 5.3 are orthorhombic, P212121, a = 10.640(2), b = 11.184(2) and c = 44.618(4)A. The structure was refined to an R = 0.041. The d(CpG) structure is similar to the ammonium salt solved by Cruse et al.(1). Both structures form a parallel self base paired mini-double helix. In d(CpG).Na+ one of the two paired cytosines is protonated on N(3). The cytosines form 3 hydrogen bonds while the guanines form only 2. The Na+ ion is coordinated with five groups: two water molecules, O(6) of guanine A, N(7) of guanine B and 0(5') of cytosine B, forming a square pyramid. The hydration shell around the mini-helix is analysed and compared with that of the ammonium salt, d(CpG).Na+ is the second d(CpG) oligonucleotide found with a self base pairing arrangement despite of the fact that the crystallization conditions and counterion were different in both cases. The hypothesis that self base pairing is not only a crystallization artifact but may play a role under physiological conditions as a source of transversion mutations is discussed.

Histones in chromatin from germ cells of the echinoderm Holothuria tubulosa are retained throughout spermatogenesis. However, some alterations occur in the histone complement of the mature sperm, including the presence of a germ-line-specific H1 subcomponent unusually rich in arginine, and the appearance of a basic component termed φ 0 . Histones from ripe sperm have been extracted in a preparative scale to allow for isolation and purification of protein φ 0 . Polyclonal antibodies against φ 0 have been produced and purified by affinity chromatography. The specificity of the antibodies to φ 0 has been assessed by enzyme-linked immunosorbent assays, competition experiments, and Western immunoblotting analysis. No cross-reactivity of the antibodies with the remainder histone fractions has been observed. Immunocytolocalization of protein φ 0 by immunogold labeling has revealed that this protein is essentially confined to chromatin from ripe sperm, whereas it is wholly absent from less advanced germ cell types. From these observations, together with biochemical studies previously reported, it is inferred that protein φ 0 may well be instrumental in the known chromatin transitions occurrring in this organism during germ cell development.

It is shown that histones are the nuclear proteins present in spermatozoa of the horseshoe crab Limmulus polyphemus , an arthropod which is considered a living fossil. They have been characterized and found to be closely related to calf thymus histones. The only difference is the presence of an additional histone in small amounts (2−3% of the whole histones) which has intermediate properties between H1 and H2b.

Ram spermatid nuclei and caput epididymal sperm nuclei were prepared and treated with DTT under conditions avoiding proteolysis. Whole-mount preparations for the electron microscope were made in the presence or absence of the detergent Joy. The chromatin of the less mature, non-round spermatid nuclei displayed a nucleosomal organization that gradually disappeared at the time the histones leave the nuclei (elongating spermatids). Digestion with micrococcal nuclease suggests that polynucleosome arrays are scarcer and more accessible to nuclease in the elongating than in the round nuclei, with increasing amounts of DNA becoming devoid of nucleosomes. In the protamine-containing nuclei (elongated spermatids), only smooth filaments were observed, which formed thick fibers by parallel aggregation. The change from a nucleosomal organization to bundles of smooth filaments appeared to result from a complex process involving the transitory presence of conspicuous "knobby fibers" that suggest a periodicity in the organization of the spermatidal proteins along the DNA molecules. X-ray diffraction patterns obtained with protamine-containing spermatid nuclei and with sperm nuclei confirm that the DNA is arranged in smoothly bent bundles of parallel molecules. No higher-order reflections that might correspond to nucleosome structures were detected in the 30-200 A region.

The study of the interactions between proteins and DNA is one of the crucial problems in molecular biology given the central role they play in the biological activity of the genetic material. However the nature of the forces and mechanisms involved has remained elusive. Only very recently the first two structures of proteins which interact specifically with DNA has been reported1,2, but the nature of the complex they form with DNA is based only on speculation. We can hope that this situation will change in the near future, since there are now methods to obtain oligonucleotides suitable to form specific complexes with proteins.

In this paper we will review the different types of proteins found in animal sperm nuclei, as well as the structure of the corresponding DNA-protein complexes. We will neither discuss the biological role of these proteins (Subirana, 1975), nor consider the specific protein components which appear at intermediate stages of spermiogenesis.

Optimal conditions for studying the ultrastructure of chromatin fibers of histone-containing spermatozoa in thin sections have been determined. Better results for preservation in sperm of the sea cucumber Holothuria tubulosa, have been found than in different frog species studied. The fine structure of chromatin fibers after different treatments was studied by computer materials. A clear superbead structure was found under all conditions which preserve the chromatin fibres. These have a diameter of 30 nm, with superbeads about 33 nm long. In the best preserved cases an additional periodicity of 11 nm along the fibres was found. There is no clear relationship of this periodicity with an eventual solenoidal structure of the chromatin fibers.

The possibility that nucleohistone is constituted by a helical arrangement of DNA molecules is analyzed in this paper. A polyhelical model of nucleohistone in plectanemic double coils of variable dimensions is found to be compatible with the x-ray diffraction results obtained in this and other laboratories. The radius of the coils varies between 35 and 45 Å, whereas the pitch varies between 200 and 120 Å. Another part of nucleohistone is much less coiled and contributes to the “background” scattering of the sample. This model is compatible with a lampbrush organization of the chromosome.

We have obtained tilt series of thin sections of chromatin fibers embedded in araldite-epon and stained with uranyl acetate. The tilt series contain between 12 and 18 micrographs, spanning tilt ranges between 84 and 117 degrees. Reconstructions in three dimensions have been obtained by filtered back-projection from each tilt series. The reconstructions have been low-pass filtered in order to reduce the amount of noise. In the reconstructions it is possible to approximately localize the nucleosomes. In several regions they show a clear zigzag arrangement, but in other regions it is difficult to determine the sequence of the nucleosomes. In any case there is no clear indication of a solenoidal arrangement. We discuss the rules which may give rise to a 3-D arrangement of the nucleosomal zigzag.

The conformation of DNA is influenced both by its sequence and by the counterions and other small molecules (in particular water) which interact with it. In this article we will review the effect on DNA of counterions, including basic proteins. We will not deal with sequence effects and thus we will not consider specific protein-DNA interactions. We will also not consider the influence of ions on the denaturation of DNA in solution.

The structural organization of the histone-containing spermatozoa of the goldfish C. auratus is studied. Chemical fractionation shows that the five standard histones are present. Histone H1 subfractions are also similar to those found in other organisms. This is the first organism studied in which no peculiar basic protein is found in spermatozoa, since in all the organisms described up to now, unique protein components are always present, either alone or accompanied by somatic-like histones. Ultrastructural studies show that this chromatin is organized as a bundle of fibers of about 25 nm diameter, which upon spreading give the typical ‘beads-on-a-string’ appearance. Nuclease digestion demonstrates a repeat length of 205 base pairs (bp), slightly longer than in somatic tissues, but shorter than in echinoderm spermatozoa.

A molecular model is presented to substantiate the interpretation of mitosis given by Ostergren, Mole-Bajer & Bajer (1960). It is based on active shearing forces produced at the surface of the microtubules. The model explains the formation of the metaphase plate as a result of the same forces which produce the movement of the chromosomes. The shearing forces may be produced either by an actin-myosin like interaction or by a similar mechanism involving a single protein. Some of the points which can be tested by experiment are discussed. The consequences of the model for other microtubular systems are briefly presented.

Frequently during the characterization of histones by gel electrophoresis, sharply defined slow moving bands are observed. These bands may correspond either to contaminating non-histone proteins or to specific aggregation products cf histone fractions. In this paper some of these specific aggregation products are studied in order to obtain information on histone-histone interactions. It is found that cysteine bridges are involved in the formation of some of these aggregat&;,whereas in other cases specific hydrophobic interactions occur. At the same time some details of the chemical structure of the histones are elucidated. It is shown that fraction F2al in echinoderms contains cysteine as a constituent amino acid.

The tetramine spermine forms a complex with DNA, and thereby serves to protect it against denaturation and breakage. X-ray fibre photographs of the DNA-spermine complex were taken at various relative humidities. The most detailed pattern was obtained at 92% relative humidity. It shows the complex to have the same helical parameters as the naturally occurring B form of DNA, and presumably a closely similar conformation. However, at 76% and lower relative humidities, the complex conforms to the DNA C configuration. The intermolecular distance was found to vary with the degree of hydration of the specimen, but to have a definite upper limit. This and the range of intermolecular distance observed are consistent with spermine cross-links between DNA molecules. Studies of molecular models have shown that it is also possible for all four basic groups of a spermine molecule to make hydrogen bonds with phosphate groups on one molecule of DNA. The spermine molecules would lie across the small groove of the DNA and serve to bind its two strands together. Calculation of intensities for various models for the DNA-spermine complex indicates that no one model can account for the observed X-ray pattern. However, the assumption of spermine in the small groove of DNA as well as in intermolecular cross-links, or of an even greater variety of modes of attachment between DNA and spermine, does lead to satisfactory agreement between calculated and observed intensities.

Gonads of the male Holothuria tubulosa contain two families of lysine-rich histones. One of these families resembles the lysine-rich histones found in somatic tissues of higher organisms (e.g. calf and rabbit). The other family, which may be restricted to the male gonad, is recognizably related to the first family and yet is quite distinct. About 35% of the tryptic peptides differ between these families. These findings support the notion that a broad spectrum of structural variation may exist in lysine-rich histones, perhaps even merging into structures of the slightly lysine-rich class.

Some aspects of the irreversible denaturation of bacteriophage DNA, equivalent to strand separation, have been studied by the density-gradient technique using CsCl. The anomalous loss of denaturated DNA observed when this technique is used has been attributed to adsorption on to solid particles and on to the walls of the container. Two main conclusions have been obtained: (1) the irreversible denaturation of DNA becomes more cooperative as the ionic strength of the solvent is increased; and (2) when denaturation is achieved by the action of formamide, a certain amount of the DNA stays resistant to denaturation. This result has been explained by assuming that the two strands are held together by small amounts of residual protein. This residual protein could be relevant to the understanding of the unique regions recently found in some bacteriophage DNA molecules.

The structure of renatured T4 DNA has been studied by CsCl density-gradient centrifugation. It has been found that the products of the reaction differ, depending on the method used for denaturation of the DNA. If denaturation is carried out without taking precautions to prevent chain degradation, for example, by heat, the DNA formed by renaturation shows approximately 70% recovery of the native structure as judged by its density. With long times of annealing, the DNA can recover the native density. This behavior is also observed with bacterial DNA samples. On the other hand, if precautions arc taken to prevent chain degradation during denaturation, two products appear as a result of renaturation. One of them is undistinguishable from native T4 DNA, whereas the second one consists of highly aggregated DNA which shows only a partial recovery of the native structure. With long times of annealing, this second species recovers the native density but retains its highly aggregated nature. At higher ionic-strengths, renaturation follows a different pattern and a single product is formed. The relevance of all these observations to the kinetic anomalies reported in the previous communication is discussed.

The thermal denaturation profiles of nucleohistone from calf thymus, sea urchin sperm and sea cucumber male gonad, are studied and compared under a variety of conditions. These include melting in the presence of either one of the following agents: urea, methanol, divalent cations or excess histones. The influence of ionic strength, pH, formaldehyde treatment and partial denaturation is also studied. Particular attention is given to the factors which influence the bimodal appearance of the profiles. The melting curves of the three materials used are qualitatively similar under all conditions, although they show quantitative differences. The histone:DNA ratio appears to be the most important parameter to define the denaturation properties of a given nucleohistone preparation. It is shown that redistribution of histones may determine the melting profile, since during denaturation histones can migrate from locally denatured regions towards those regions which contain native DNA. It is also shown that there are regions of phosphate negative charges of DNA not protected by histone. These regions can be protected against denaturation either by additional histones or by certain divalent cations. The results are interpreted in terms of the various models possible for the distribution of histones on DNA in native nucleohistone. Their biological significance is also discussed.