Applications of combined DNA microarray and chromosome sorting technologies.

The sequencing of the human genome has led to the availability of an extensive mapped clone resource that is ideal for the construction of DNA microarrays. These genomic clone microarrays have largely been used for comparative genomic hybridisation studies of tumours to enable accurate measurement of copy number changes (array-CGH) at increased resolution. We have utilised these microarrays as the target for chromosome painting and reverse chromosome painting to provide a similar improvement in analysis resolution for these studies in a process we have termed array painting. In array painting, chromosomes are flow sorted, fluorescently labelled and hybridised to the microarray. The complete composition and the breakpoints of aberrant chromosomes can be analysed at high resolution in this way with a considerable reduction in time, effort and cytogenetic expertise required for conventional analysis using fluorescence in situ hybridisation. In a similar way, the resolution of cross-species chromosome painting can be improved and we present preliminary observations of the organisation of homologous DNA blocks between the white cheeked gibbon chromosome 14 and human chromosomes 2 and 17.

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).

In vitro and in vivo reconstitution and stability of vertebrate chromosome ends.

Telomeres are essential repetitive sequences at the ends of chromosomes that prevent chromosome fusion and degradation. We have successfully recapitulated these two protective functions in vivo and in vitro by incubating blunt-end DNA constructs having vertebrate telomeric ends in Xenopus eggs and egg extracts. Constructs with telomeric ends are stable as linear molecules; constructs with non-telomeric ends undergo intramolecular fusion. In extracts, 99.8% of the telomeric constructs from 78 to 700 bp in length are assembled into 'model telomeres' in <5 min and have an extra-polated half-life of >3.5 years. Non-telomeric constructs circularize with first order kinetics and a half-life of 4 h. In living eggs the telomeric constructs are protected from fusion and degradation. The stability of the telomeric constructs is not due to covalent processing. Extract can protect approximately 100 pM telomeric ends (equivalent to 1.7 x 10(7) ends/egg) even in the presence of a 20-fold excess of double-stranded telomeric DNA, suggesting that protection requires end-specific factors. Constructs with (TTGGGG) n repeats are unstable, suggesting that short tracts of this and other telomere-like sequences found within human telomeres could lead to genome instability if exposed by partial telomere erosion during aging.

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.

Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening.

The chromosomes of lower eukaryotes have short telomeric 3' extensions. Using a primer-extension/nick-translation technique and nondenaturing hybridization, we find long 3' G-rich tails at human chromosome ends in mortal primary fibroblasts, umbilical vein endothelial cells, and leukocytes, as well as in immortalized fibroblasts. For all cells tested, >80% of the telomeres have long G-rich overhangs, averaging 130-210 bases in length, in disagreement with the conventional model for incomplete lagging-strand replication, which predicts overhangs on 50% of the chromosome ends. The observed G tails must exist during most of the cell cycle and probably result from degradation of both chromosome ends. The average lengths of the G tails are quantitatively consistent with the observed rates of human chromosome shortening.

Condensation of rat telomere-specific nucleosomal arrays containing unusually short DNA repeats and histone H1.

Vertebrate telomeres contain arrays of nucleosomes with unusually short and regular repeat lengths (Makarov, V. L., Lejnine, S., Bedoyan, J., and Langmore, J. P.(1993) Cell 73, 775-787; Lejnine, S., Makarov, V., and Langmore, J. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2393-2397). In order to better define the specific structural features of telomere chromatin, we examined the condensation and H1 content of telomere nucleoproteins from rat liver. Velocity sedimentation analysis shows that telomeric nucleosome arrays condense with increasing ionic strength and molecular weight in a manner comparable with that of bulk chromatin despite the very short repeat length. However, these condensed structures do not exhibit the approximately 100-base pair deoxyribonuclease II repeat characteristic of condensed bulk chromatin. Frictional coefficient calculations suggest that telomere-specific higher order structure is more compact than bulk chromatin. Nucleoprotein gel electrophoresis shows that telomeric dinucleosomes from soluble chromatin contain H1. Finally, direct isolation and analysis of telomere nucleoproteins from formaldehyde-cross-linked nuclei indicate the presence of core histone proteins and H1. These results are consistent with the view that a major fraction of the long telomeres of rat are organized as specialized nucleosome arrays with features similar but not identical to those of bulk chromatin.

Conserved nucleoprotein structure at the ends of vertebrate and invertebrate chromosomes.

Eukaryotic chromosomes terminate with telomeres, nucleoprotein structures that are essential for chromosome stability. Vertebrate telomeres consist of terminal DNA tracts of sequence (TTAGGG)n, which in rat are predominantly organized into nucleosomes regularly spaced by 157 bp. To test the hypothesis that telomeres of other animals have nucleosomes, we compared telomeres from eight vertebrate tissues and cell cultures, as well as two tissues from an invertebrate. All telomeres have substantial tracts of (TTAGGG)n comprising 0.01-0.2% of the genome. All telomeres are long (20-100 kb), except for those of sea urchin, human, and some chicken chromosomes, which are 3-10 kb in length. All of the animal telomeres contained nucleosome arrays, consistent with the original hypothesis. The telomere repeat lengths vary from 151 to 205 bp, seemingly uncorrelated with telomere size, regularity of nucleosome spacing, species, or state of differentiation but surprisingly correlated with the repeat of bulk chromatin within the same cells. The telomere nucleosomes were consistently approximately 40 bp smaller than bulk nucleosomes. Thus, animal telomeres have highly conserved sequences and unusually short nucleosomes with cell-specific structure.

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.

Embryonic regulation of histone ubiquitination in the sea urchin.

We have used quantitative 2-D protein electrophoresis and immunoprecipitation to study the patterns of histone ubiquitination at 10 h and 36 h of embryonic development in Strongylocentrotus purpuratus. Variants csH2A, alpha H2A, beta H2A, gamma H2A, delta HA, H2AF./Z, alpha H2B, beta H2B, and gamma H2B showed up to sevenfold differences in level of monoubiquitination between variants, and individual variants showed up to sixfold changes during development. At 36 h of embryogenesis, the late variants were less ubiquitinated than the early variants, although the overall level of ubiquitination was appreciably greater than at 10 h. Antiubiquitin antibodies were used to precipitate formaldehyde-fixed chromatin fragments in order to estimate the degree of ubiquitination of the early histone genes. The 5' regulatory region of the active H3 gene appeared to be at least twice as ubiquitinated as the adjacent upstream spacer. However, the absolute level of ubiquitination of the early histone gene repeat seemed to be independent of transcriptional activity. These results show that variant-specific ubiquitination of histones is a part of the developmental program in sea urchin embryos, but is not clearly correlated with transcriptional activity of the early histone genes, except perhaps in the regulatory regions.

Nucleosomal organization of telomere-specific chromatin in rat.

Rat liver interphase chromosomes have telomeres 20-100 kb in length. Micrococcal nuclease digestion of nuclei cleaves telomeres with a uniform 157 bp periodicity, producing soluble particles that sediment in sucrose gradients exactly like oligonucleosomes. The monomeric telomere particles comigrate with nucleosome core particles on nucleoprotein and DNA gels but do not bind H1. DNAase I cleaves telomere nucleoprotein into a series of bands spaced by about 10.4 bp and with the same intensity distribution as bands from bulk nucleosomes. Removal of H1 from chromatin alters the sedimentation properties of telomeres in parallel with bulk chromatin. Thus, telomeres of mammals are constructed of closely spaced nucleosomes, in contrast with the telomeres of lower eukaryotes, which show no evidence of nucleosomal structure.

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.

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.

DNA methylation pattern changes during development of a sea urchin.

Cytosine methylation of developmentally regulated genes of the sea urchin Strongylocentrotus purpuratus was studied by using restriction-endonuclease digestion and Southern blotting. The single-copy bindin gene, the family of five cytoplasmic actin genes and the 400-fold-repeated set of five early histone genes were mostly unmethylated, but some sites exhibited partial methylation that varied throughout development. This shows that in echinoderms the methylation of DNA is not confined to the non-transcribed portion of the genome, as previously believed [Bird, Tagart & Smith (1979) Cell 17, 889-901], and may play a role in transcriptional regulation.

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.

Small angle x-ray scattering of chromatin. Radius and mass per unit length depend on linker length.

Analyses of low angle x-ray scattering from chromatin, isolated by identical procedures but from different species, indicate that fiber diameter and number of nucleosomes per unit length increase with the amount of nucleosome linker DNA. Experiments were conducted at physiological ionic strength to obtain parameters reflecting the structure most likely present in living cells. Guinier analyses were performed on scattering from solutions of soluble chromatin from Necturus maculosus erythrocytes (linker length 48 bp), chicken erythrocytes (linker length 64 bp), and Thyone briareus sperm (linker length 87 bp). The results were extrapolated to infinite dilution to eliminate interparticle contributions to the scattering. Cross-sectional radii of gyration were found to be 10.9 +/- 0.5, 12.1 +/- 0.4, and 15.9 +/- 0.5 nm for Necturus, chicken, and Thyone chromatin, respectively, which are consistent with fiber diameters of 30.8, 34.2, and 45.0 nm. Mass per unit lengths were found to be 6.9 +/- 0.5, 8.3 +/- 0.6, and 11.8 +/- 1.4 nucleosomes per 10 nm for Necturus, chicken, and Thyone chromatin, respectively. The geometrical consequences of the experimental mass per unit lengths and radii of gyration are consistent with a conserved interaction among nucleosomes. Cross-linking agents were found to have little effect on fiber external geometry, but significant effect on internal structure. The absolute values of fiber diameter and mass per unit length, and their dependencies upon linker length agree with the predictions of the double-helical crossed-linker model. A compilation of all published x-ray scattering data from the last decade indicates that the relationship between chromatin structure and linker length is consistent with data obtained by other investigators.

Nucleoprotein hybridization: a method for isolating active and inactive genes as chromatin.

The developmentally regulated sea urchin early histone gene repeat (SUEHGR) from Strongylocentrotus purpuratus was isolated as chromatin by nucleoprotein hybridization. This technique is a novel method to isolate specific sequences as chromatin. Because the purification scheme is based only on the gene sequence and is independent of other physical properties such as protein composition and transcriptional activity, we were able to isolate the same gene in different functional states. Gene size chromatin fragments were solubilized by restriction endonuclease digestion of cell nuclei. Using T7 gene 6 exonuclease, the 3'termini of the fragments were exposed and then hybridized in solution to a biotinylated oligonucleotide complementary to one end of the SUEHGR fragment. The hybrids were bound to an Avidin D matrix. DTT cleavage of the biotin linker yielded a chromatin fraction greater than 700 fold enriched in SUEHGR. Overall yields were between 2% and 15%. The purity of the isolated material was independently measured to be greater than 80%. The homogeneous native structure of the inactive genes was preserved as shown by electron microscopy and micrococcal nuclease digestion of the purified SUEHGR. Minor heterogeneity was observed for the purified active genes by micrococcal nuclease digestion but the main features of the active chromatin were preserved during isolation. This isolation offers the first opportunity to study the structure of an RNA polymerase II gene at different stages of the cell cycle and development.

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.

Chromatin structure of the developmentally regulated early histone genes of the sea urchin Strongylocentrotus purpuratus.

Chromatin organization of the early histone gene repeat was studied at the early embryonic stages of the sea urchin S. purpuratus. Micrococcal nuclease digestion showed a highly irregular packaging of the whole repeat at the period of transcriptional activity, which was progressively replaced by more regular nucleosomal arrays upon developmentally programmed inactivation. No evidence for unique positioning of the nucleosomes was found. Regions upstream of each of the genes were hypersensitive to DNAase I digestion in the active state. These regions contained one (H2A and H2B), or two (H3 and H4) well-defined DNAase I cutting sites, or two poorly-defined sites (H1). They mapped within DNA sequences shown previously to be required for proper expression of the genes. Hypersensitivity continued in the hatching blastula, which have a conventional nucleosomal structure and a much reduced transcriptional activity. Hypersensitivity of these regions during morula and early blastula was not dependent on the torsional strain in chromatin, as it was not influenced by extensive gamma ray-induced nicking of the DNA in nuclei. By late blastula no hypersensitive regions were present.

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.