Low angle x-ray diffraction studies of HeLa metaphase chromosomes: effects of histone phosphorylation and chromosome isolation procedure.

To test whether gross changes in chromatin structure occur during the cell cycle, we compared HeLa mitotic metaphase chromosomes and interphase nuclei by low angle x-ray diffraction. Interphase nuclei and metaphase chromosomes differ only in the 30-40-nm packing reflection, but not in the higher angle part of the x-ray diffraction pattern. Our interpretation of these results is that the transition to metaphase affects only the packing of chromatin fibers and not, to the resolution of our method, the internal structure of nucleosomes or the pattern of nucleosome packing within chromatin fibers. In particular, phosphorylation of histones H1 and H3 at mitosis does not affect chromatin fiber structure, since the same x-ray results are obtained whether or not histone dephosphorylation is prevented by isolating metaphase chromosomes in the presence of 5,5'-dithiobis(2-nitrobenzoate) or low concentrations of p-chloromercuriphenylsulfonate (ClHgPhSO3). We also compared metaphase chromosomes isolated by several different published procedures, and found that the isolation procedure can significantly affect the x-ray diffraction pattern. High concentrations of ClHgPhSO3 can also profoundly affect the pattern.

Low angle x-ray diffraction studies of chromatin structure in vivo and in isolated nuclei and metaphase chromosomes.

Diffraction of x-rays from living cells, isolated nuclei, and metaphase chromosomes gives rise to several major low angle reflections characteristic of a highly conserved pattern of nucleosome packing within the chromatin fibers. We answer three questions about the x-ray data: Which reflections are characteristic of chromosomes in vivo? How can these reflections be preserved in vitro? What chromosome structures give rise to the reflections? Our consistent observation of diffraction peaks at 11.0, 6.0, 3.8, 2.7 and 2.1 nm from a variety of living cells, isolated nuclei, and metaphase chromosomes establishes these periodicities as characteristic of eukaryotic chromosomes in vivo. In addition, a 30-40- nm peak is observed from all somatic cells that have substantial amounts of condensed chromatin, and a weak 18-nm reflection is observed from nucleated erythrocytes. These observations provide a standard for judging the structural integrity of isolated nuclei, chromosomes, and chromatin, and thus resolve long standing controversy about the "tru" nature of chromosome diffraction. All of the reflection seen in vivo can be preserved in vitro provided that the proper ionic conditions are maintained. Our results show clearly that the 30-40-nm maximum is a packing reflection. The packing we observe in vivo is directly correlated to the side-by-side arrangement of 20- 30-nm fibers observed in thin sections of fixed and dehydrated cells and isolated chromosomes. This confirms that such packing is present in living cells and is not merely an artifact of electron microscopy. As expected, the packing reflection is shifted to longer spacings when the fibers are spread apart by reducing the concentration of divalent cations in vitro. Because the 18-, 11.0-, 6.0-, 3.8-, 2.7-, and 2.1-nm reflections are not affected by the decondensation caused by removal of divalent cations, these periodicities must reflect the internal structure of the chromaticn fibers.

Radial density distribution of chromatin: evidence that chromatin fibers have solid centers.

Fiber diameter, radial distribution of density, and radius of gyration were determined from scanning transmission electron microscopy (STEM) of unstained, frozen-dried chromatin fibers. Chromatin fibers isolated under physiological conditions (ionic strength, 124 mM) from Thyone briareus sperm (DNA linker length, n = 87 bp) and Necturus maculosus erythrocytes (n = 48 bp) were analyzed by objective image-processing techniques. The mean outer diameters were determined to be 38.0 nm (SD = 3.7 nm; SEM = 0.36 nm) and 31.2 nm (SD = 3.6 nm; SEM = 0.32 nm) for Thyone and Necturus, respectively. These data are inconsistent with the twisted-ribbon and solenoid models, which predict constant diameters of approximately 30 nm, independent of DNA linker length. Calculated radial density distributions of chromatin exhibited relatively uniform density with no central hole, although the 4-nm hole in tobacco mosaic virus (TMV) from the same micrographs was visualized clearly. The existence of density at the center of chromatin fibers is in strong disagreement with the hollow-solenoid and hollow-twisted-ribbon models, which predict central holes of 16 and 9 nm for chromatin of 38 and 31 nm diameter, respectively. The cross-sectional radii of gyration were calculated from the radial density distributions and found to be 13.6 nm for Thyone and 11.1 nm for Necturus, in good agreement with x-ray and neutron scattering. The STEM data do not support the solenoid or twisted-ribbon models for chromatin fiber structure. They do, however, support the double-helical crossed-linker models, which exhibit a strong dependence of fiber diameter upon DNA linker length and have linker DNA at the center.

The diameters of frozen-hydrated chromatin fibers increase with DNA linker length: evidence in support of variable diameter models for chromatin.

The diameters of chromatin fibers from Thyone briareus (sea cucumber) sperm (DNA linker length, n = 87 bp) and Necturus maculosus (mudpuppy) erythrocytes (n = 48 bp) were investigated. Soluble fibers were frozen into vitrified aqueous solutions of physiological ionic strength (124 mM), imaged by cryo-EM, and measured interactively using quantitative computer image-processing techniques. Frozen-hydrated Thyone and Necturus fibers had significantly different mean diameters of 43.5 nm (SD = 4.2 nm; SEM = 0.61 nm) and 32.0 nm (SD = 3.0 nm; SEM = 0.36 nm), respectively. Evaluation of previously published EM data shows that the diameters of chromatin from a large number of sources are proportional to linker length. In addition, the inherent variability in fiber diameter suggests a relationship between fiber structure and the heterogeneity of linker length. The cryo-EM data were in quantitative agreement with space-filling double-helical crossed-linker models of Thyone and Necturus chromatin. The data, however, do not support solenoid or twisted-ribbon models for chromatin that specify a constant 30 nm diameter. To reconcile the concept of solenoidal packing with the data, we propose a variable-diameter solid-solenoid model with a fiber diameter that increases with linker length. In principle, each of the variable diameter models for chromatin can be reconciled with local variations in linker length.

Visibility of single atoms.

Theoretical and experimental studies indicate that, with a high-resolution scanning electron microscope, it is now possible to obtain pictures of a single heavy atom resting on a thin carbon substrate.

Crosslinking of proteins to DNA in human nuclei using a 60 femtosecond 266 nm laser.

We developed appropriate conditions to use a laser with 60 femtosecond pulses, a frequency of 1 KHz and a wavelength of 266 nm to efficiently crosslink proteins to DNA in human nuclei for the purpose of using immunoprecipitation to study the binding of specific proteins to specific sequences of DNA under native conditions. Irradiation of nuclei for 30 min with 1-3 GW/cm(2)pulses crosslinked 10-12% of total protein to DNA. The efficiency of crosslinking was dose and protein specific. Histones H1 and H3 were crosslinked by 15 min of irradiation with 20-25% efficiency, at least 10 times more strongly than the other histones, consistent with experiments using conventional UV light. Irradiation for 15 min did not damage proteins, as assayed by SDS-PAGE of Ku-70 and histones. Although the same level of irradiation did not cause double-strand breaks, it did make the DNA partially insensitive to Eco RI restriction enzyme, probably through formation of thymidine dimers. Immuno-analysis of crosslinked nucleoprotein showed that Ku crosslinking to nuclear DNA is detectable only in the presence of breaks in the DNA, and that nucleosomes are bound to a significant fraction of the telomeric repeat (TTAGGG) (n).

c-myb effects on kinetic events during MEL cell differentiation.

During dimethylsulfoxide (DMSO)-induced differentiation of Friend mouse erythroleukemia (MEL) cells there is a biphasic fall in c-myb mRNA levels. We have previously shown that constitutive expression of c-myb blocks differentiation. To delineate more accurately the point at which Myb blocks differentiation, MEL cells were transfected with a human c-myb construct under the control of the beta-globin promoter and enhancers. In concert with endogenous DMSO-induced globin transcription during MEL cell differentiation, the beta-globin c-myb transcription unit of the transfected plasmid is activated after 3-5 days of culture in media containing DMSO. Here we describe c-myb-transformed MEL clones which undergo delayed expression of the exogenous c-myb following 3-5 days of culture in DMSO. In contrast to wild-type MEL cells, both clones failed to display phenotypic markers of differentiation and continued to proliferate for up to 10 days of culture. These data suggest that the late fall in c-myb levels may be required in order for differentiation to occur. Additionally, we suggest that constitutive expression of c-myb does not block early commitment events such as activation of histone Hl', subsequent chromatin condensation, and alteration of proliferation-related gene expression. Taken together, these results show that c-myb acts very late in the process of differentiation.

Psoralen crosslinking of active and inactive sea urchin histone and rRNA genes.

Chromatin structure is highly correlated with the transcriptional activity of specific genes. For example, it has been found that the regularity of nucleosome spacing is compromised when genes are transcribed. The rRNA genes from fungi, plants, and animals give distinctly bimodal distributions of psoralen crosslinking, which has led to the suggestion that these genes might be largely devoid of nucleosomes when transcriptionally active. We investigated the chromatin structure of the multicopy rRNA and histone genes during sea urchin early embryogenesis. The rRNA genes, which are weakly expressed, give a unimodal distribution of weak psoralen crosslinking, in contrast to the situation in all other organisms studied. The early histone genes were more accessible to psoralen crosslinking when active than inactive. The pattern of crosslinking suggests that these polII genes have a homogeneous structure and are still highly protected by nucleosomes when in the active conformation, unlike the situation in polI genes.

The major component of a large, intracellular proteinase accumulated by inhibitors is a complex of alpha 2-macroglobulin and thrombin.

A large, intracellular proteinase accumulated by inhibitors (PABI) was found in cultured mammalian cells as a large, multicatalytic proteinase with a greatly elevated concentration in the presence of small peptide proteinase inhibitors (Tsuji and Kurachi (1989) J. Biol. Chem. 264, 16093). Electron microscopic analysis showed that the tertiary structure of PABI highly resembled that of alpha 2-macroglobulin complexed with a proteinase(s). Isolation of the anti-PABI cross-reacting material from calf serum added to the culture media of baby hamster kidney cells further supported that the primary component of PABI was alpha 2-macroglobulin. Immunoblot analyses and the substrate specificity of PABI indicated that the major proteinase component contained in PABI was thrombin. When alpha 2-macroglobulin was added to the PABI-depleted serum, a significant accumulation or a degradation of the intracellular alpha 2-macroglobulin was observed in the presence or absence of leupeptin, respectively. Similarly, when thrombin was added to the PABI-depleted fetal calf serum supplemented with fresh alpha 2-macroglobulin, a significant amount of intracellular thrombin was found only in the presence of leupeptin. These results indicate that the major component of the intracellular PABI molecules is a complex of alpha 2-macroglobulin with thrombin which is internalized from the culture media. Intracellular accumulation of PABI, therefore, is a phenomenon primarily relevant to the culture cells. Whether or not PABI is also generated in certain physiological or pathological conditions requires further study.

Turnover of histone acetyl groups during sea urchin early development is not required for histone, heat shock and actin gene transcription.

Histone acetylation is an extremely complex, reversible and specific process. In order to evaluate the importance of this modification for gene expression during sea urchin development, acetyl group turnover of histone lysine residues was blocked by sodium butyrate. The continuous presence of 15 Mm sodium butyrate in the incubation medium from the onset of development blocked gastrulation and resulted in chromatin containing hyperacetylated histone molecules in amounts usually not found in nature. At the mesenchyme blastula stage, the expression of the early histone genes was shut off and the expression of the late genes was switched on both in control and sodium butyrate-treated embryos. Investigation of the early histone gene chromatin structure in butyrate-treated embryos revealed a random distribution of nucleosomes when the genes were transcriptionally active as compared to regular nucleosomal packaging when genes were inactive. These changes in chromatin structure during development mimicked the chromatin structural transition of the early histone genes in control embryos. In addition, the ability of heat shock genes to be induced at elevated temperature and repressed at normal temperature was unaffected in butyrate treatment of embryos. Finally, the developmental profiles of the cytoskeletal CyIIIa actin gene expression in control and butyrate-treated embryos were very similar. The data presented suggest that turnover of histone acetyl groups and the overall level of histone acetylation are not determining factors in the up and down regulation of a number of genes during early development of sea urchin.

Quantitation of molecular densities by cryo-electron microscopy. Determination of the radial density distribution of tobacco mosaic virus.

We have determined the absolute mass and radial scattering density distribution of tobacco mosaic virus in the frozen-hydrated state by energy-filtered low-dose bright-field transmission electron microscopy. The absolute magnitude of electron scattering from tobacco mosaic virus in 150 nm of ice was within 3.0% of that predicted, with inelastic scattering accounting for approximately 80% of the scattering contrast. In order to test the accuracy of the radial reconstruction, a computer model of tobacco mosaic virus was built from the atomic co-ordinates assuming uniform solvent density. The validity of the model was confirmed by comparison of X-ray scattering and predictions of the model (R factor = 0.05). First-order corrections for the microscope contrast transfer function were necessary and sufficient for conversion of the cryo-electron microscopy images into accurate representations of the mass density. At 1.9 nm resolution the compensated reconstruction and model had density peaks of similar magnitude at 2.4, 4.2, 6.0 and 7.8 nm radius and a central hole of 2 nm radius. Equatorial Fourier transforms of the corrected electron images were in excellent agreement with predictions of the model (R factor = 0.12). Thus, the uniform solvent approximation was adequate at 1.9 nm resolution to describe quantitatively X-ray scattering in liquid water and electron imaging in vitreous ice. This is the first demonstration that cryo-electron microscopy images can be used to quantitate the absolute mass, mass per unit length and internal density distributions of proteins and nucleic acids.

A polarized photobleaching study of chromatin reorientation in intact nuclei.

Polarized fluorescence recovery after photobleaching (pFRAP) was used to monitor the effects that condensation, i.e. compaction and aggregation, have on the (microseconds and ms) internal dynamics of chromatin in intact nuclei. When divalent cations were present with physiological (approximately 90 mM) monovalent salt the chromatin was found to exist in a compact and aggregated state which was characterized by rotational immobilization over timescales that range from 10 microseconds to 40 milliseconds. This immobilization is attributed to suppression of internal dynamics by intermolecular interactions. When the divalent cations were removed, the compact fibers no longer aggregated and were free to reorient with a characteristic decay time of about 1.2 milliseconds. It is shown that this millisecond relaxation could represent rigid rotation of topologically independent structural domains. Dilution of the monovalent salt induced a gradual change in the structural state of the chromatin that was manifest as a dramatic increase in internal flexibility. At the lowest salt concentration studied (11 mM-monovalent salt) the chromatin reorients in fewer than ten microseconds. These changes in flexibility are continuous with salt concentration, indicating that there are no well-defined endpoints to structural transitions and that the microsecond-millisecond internal dynamics of chromatin are a sensitive measure of structure. Measurements made on nuclei from cells that are either transcriptionally quiescent or active indicate that the dynamics mirrors biological activity.

Quantitative energy-filtered electron microscopy of biological molecules in ice.

The theoretical and experimental bases for quantitative electron microscopy of frozen-hydrated specimens are described, with special considerations of energy filtration to improve the images. The elastic and inelastic scattering from molecules in vacuum and in ice are calculated, and simple methods to approximate scattering are introduced. Multiple scattering calculations are used to describe the scattering from vitreous ice and to predict the characteristics of images of frozen-hydrated molecules as a function of ice thickness and accelerating voltage. Energy filtration is predicted to improve image contrast and signal-to-noise ratio. Experimental values for the inelastic scattering of ice, the energy spectrum of thick ice, and the contrast of biological specimens are determined. The principles of compensation for the contrast transfer function are presented. Tobacco mosaic virus is used to quantify the accuracy of interpreting image intensities to derive the absolute mass, mass per unit length, and internal mass densities of biological molecules. It is shown that compensation for the contrast transfer function is necessary and sufficient to convert the images into accurate representations of molecular density. At a resolution of 2 nm, the radial density reconstructions of tobacco mosaic virus are in quantitative agreement with the atomic model derived from X-ray results.

Implementation of an NSOM system for fluorescence microscopy.

We describe our progress toward an NSOM system intended for fluorescence imaging of biological samples. This process included integration of shear-force feedback into an existing NSOM system. Topographic images acquired using uncoated tips are presented. We also present our initial effort at simultaneous acquisition of topographic and fluorescence data using an aluminum coated tip.

Global population growth.

PIP: The global population passed 5 billion in 1987. In the year 2000 the world's population will be more than 6 billion, increasing by 90-100 million each year. About 95% of future demographic growth will take place in developing countries. The number of school age children is projected to increase from 940 million in 1980 to 1280 million by the year 2000. Under current labor force growth projections in developing countries, around 1.6 billion new jobs will have to be created between 1980 and 2025, with nearly 1 billion of them in Asia. Population often increases at a more rapid rate than agricultural growth. Food production per capita has declined in 70 developing countries. Much of the projected population increase will take place in environmentally fragile regions of the developing world. Population pressures contribute to deforestation, desertification, and scarcity of clean water. The United Nations Population Fund has estimated that in Asia over 43% of women not using family planning would like to postpone, space, or limit their childbearing. Over half of the world's couples of reproductive age are now using contraception. Family planning to postpone the first birth and to eliminate late child bearing would reduce both child loss and maternal illness and death. Both infant and maternal mortality are greater with higher order births. Reducing average family size is an effective way of reducing infant and maternal mortality. The World Bank has given high priority to population assistance, with large programs in Bangladesh, Egypt, India, Indonesia, the Philippines, and Thailand. Population assistance provided by the Australian International Development Assistance Bureau totaled about $4.5 million during 1989-90 and is expected to be about $8 million during 1991-92. Australia should increase the proportion of its development assistance budget devoted to population, and family planning programs should increase to around $26 million in line with other major donors.^ieng

Globalisation and social policy.

This paper discusses six major themes: that economic and social issues are closely interdependent and that the appropriate stance is to work on both together, simultaneously; that though the threats from globalisation have been exaggerated, there can be substantial costs as well as considerable benefits; that constraints on national policy are significant but are less severe than is commonly considered; that the vitality-the vigour-of national and international political processes must be increased to cope effectively with the changes which are underway; that the private sector, unions and civil society have crucial roles in the provision of services and in advocating socially responsible values, standards and policies; and that one of the most effective means of addressing the erosion of national autonomy from globalisation is for countries to cooperate in setting and implementing shared objectives and international standards and establishing more global public goods.

Nucleoprotein hybridization: a method for isolating specific genes as high molecular weight chromatin.

We describe a new technique designed to isolate specific eukaryotic genes as native oligonucleosome fragments. The isolation method consists of hybridization of single-stranded termini of chromatin restriction fragments to complementary mercurated DNA probes, followed by isolation of the hybrids by sulfhydryl-Sepharose chromatography. SV40 minichromosomes were used to test the effectiveness of the technique. About 80% of KpnI- or BamHI-restricted and lambda exonuclease treated SV40 minichromosomes hybridized to an appropriate DNA probe after a 12-h hybridization reaction under mild conditions (0.1 M aqueous salt, 37 degrees C, pH 8). When the restricted minichromosomes were mixed with a 15-fold excess of "background" chromatin from sea urchin embryos, nucleoprotein hybridization was able to reisolate the SV40 chromatin to 88% purity with a 63% yield. This represented a 115-fold enrichment of specific genes as chromatin. Results of electron microscopy and polyacrylamide gel electrophoresis indicate that the hybridized SV40 chromatin has not lost the major chromosomal proteins characteristic of SV40 nor acquired significant amounts of protein due to exchange with background chromatin. Our experimental results show that it is currently possible to isolate repeated genes from higher eukaryotes for structural and biochemical study of the proteins involved with gene regulation.

Efficient solubilization and partial purification of sea urchin histone genes as chromatin.

Soluble chromatin fragments are rapidly and efficiently produced when nuclei are digested with restriction endonucleases in buffers containing very low concentrations of magnesium. Under these conditions, the sequence specificity of the restriction endonucleases is maintained, resulting in release of specific genes as fragments with discrete molecular weights that can be fractionated by size on glycerol gradients. Gradient fractions can be chosen to be significantly enriched in specific genes and their associated proteins. For instance, we can achieve a 16-fold enrichment of the chromatin containing the early histone genes of sea urchin. The enrichments produced by these methods are useful as a first step in techniques to purify specific genes as chromatin. Glycerol gradient analyses can also be used to test whether putative gene-specific proteins are actually bound to the same sequences in vivo.