High-resolution mapping of satellite DNA using biotin-labeled DNA probes.

We have developed a novel method for high resolution mapping of specific DNA sequences after in situ hybridization. DNA probes, labeled with biotin-nucleotides in conventional nick-translation reactions, are hybridized to cytological preparations and detected with affinity-purified rabbit antibiotin antibodies followed by antibodies to rabbit IgG that are conjugated to fluorescent or enzymatic reagents. Using peroxidase labeled anti-rabbit IgG, we are able to detect and localize specific sequences at both the light and electron microscopic levels. Initial studies were done with repeated DNA sequences previously mapped by light microscope autoradiography to assess the fidelity and resolution of this method. An analysis using biotin-labeled mouse satellite DNA is presented here.

A view of interphase chromosomes.

Metaphase chromosomes are dynamically modified in interphase. This review focuses on how these structures can be modified, and explores the functional mechanisms and significance of these changes. Current analyses of genes often focus on relatively short stretches of DNA and consider chromatin conformations that incorporate only a few kilobases of DNA. In interphase nuclei, however, orderly transcription and replication can involve highly folded chromosomal domains containing hundreds of kilobases of DNA. Specific "junk" DNA sequences within selected chromosome domains may participate in more complex levels of chromosome folding, and may index different genetic compartments for orderly transcription and replication. Three-dimensional chromosome positions within the nucleus may also contribute to phenotypic expression. Entire chromosomes are maintained as discrete, reasonably compact entities in the nucleus, and heterochromatic coiled domains of several thousand kilobases can acquire unique three-dimensional positions in differentiated cell types. Some aspects of neoplasia may relate to alterations in chromosome structure at several higher levels of organization.

Movement of the X chromosome in epilepsy.

The position of selected chromosomes was assessed in samples of normal and epileptic human cortex with biotinylated probes specific for individual chromosome domains. Optical sectioning provided a rapid method for three-dimensional resolution of in situ hybridization signals in interphase cells, and solid models were reconstructed from digitized images for detailed rotational studies. There was a dramatic repositioning of the X chromosome in neurons of both males and females in electrophysiologically defined seizure foci. Other chromosomes (1, 9, and Y) showed more subtle positional changes. Specifically altered nuclear patterns involving the X chromosome may become established and create the genetic memory for intractable seizure activity.

Cholera toxin-peroxidase: changes in surface labeling of glioblastoma cells with increased time in tissue culture.

Cholera toxin coupled to peroxidase yielded a highly specific ultrastructural marker of plasma membrane monosialogangliosides. Studies with cultures of brain and brain tumors suggested that long-term culture of tissue in monolayers results in eventual loss of surface monosialogangliosides.

Evolution of a strain of CJD that induces BSE-like plaques.

Bovine spongiform encephalopathy (BSE) has become a public health issue because a recently evolved BSE agent has infected people, yielding an unusual form of Creutzfeld-Jakob disease (CJD). A new CJD agent that provokes similar amyloid plaques and cerebellar pathology was serially propagated. First-passage rats showed obvious clinical signs and activated microglia but had negligible PrP-res (the more protease-resistant form of host PrP) or cerebellar lesions. Microglia and astrocytes may participate in strain selection because the agent evolved, stabilized, and reproducibly provoked BSE-like disease in subsequent passages. Early vacuolar change involving activated microglia and astrocytes preceded significant PrP-res accumulation by more than 50 days. These studies reveal several inflammatory host reactions to an exogenous agent.

Viremia in experimental Creutzfeldt-Jakob disease.

Inoculation of the buffy coat of blood from guinea pigs infected with Creutzfeldt-Jakob disease resulted in passage of this disease to recipient animals. This demonstrates that there is a viremia in experimental Creutzfeldt-Jakob disease. These findings suggest that the hematogenous route may be implicated in the human infection and that the disease may possibly be transmitted by blood transfusions.

Amount of satellite DNA in four experimentally induced tumors of the central nervous system. Quantitative changes in a glioblastoma producing C-type particles.

The quantitative preservation of satellite NA was studied in several central nervous system (CNS) neoplasms; four tumor lines deriveo from 3-methylcholanthrene implantation into the CNS of mice were compared with brain and tissue cultures of normal mouse cells by analytical centrifugation in cesium chlorie. Three tumors showed no detectable difference from normal cells; nuclear and whole cell preparations were comparable. Only a glioblastoma line proucing C-type particles (TC509) revealed a significant difference from normal cells and exhibited a decrease of approximately 20% in satellite DNA or 2% of the total DNA on repeated examination for 1 year. C-type RNA virus may be related to relative decreases in satellite DNA observed in TC509.

Active nucleolus organizers are precisely positioned in adult central nervous system cells but not in neuroectodermal tumor cells.

Active nucleolus organizing regions (NOR) were stained with silver in isolated nuclei and chromosomes, monolayer cultures, and tissues for electron microscopy. In nuclei isolated in 0.8-1.3 M urea, only the NOR stained with silver, and round or fiber-like regions with a diameter of 200-250 nm with a substructure were delineated at appropriate nucleolar or acrocentric chromosomal sites. In whole cells, additional (non NOR) silver binding regions were noted. Electrophoresis studies on nuclear isolates indicated at least two different nuclear protein subsets were responsible for the observed silver binding; the NOR protein(s) was most tightly bound with respect to urea. Interphase cells displayed a more extensive NOR network than mitotic cells, suggesting an increase in rDNA transcription during interphase. Mature sperm cells showed no active NOR regions. Interphase neuroectodermal tumor cells generally contained large multiple NOR indicating extensive activity; the position of NOR in the nucleus was variable from cell to cell, even in cloned lines. In contrast to tumor lines, central nervous system (CNS) cells from various species all showed highly reproducible or non-random NOR locations within the nucleus of each cell type. The NOR of large neurons were always central and single, whereas small granule cell neurons displayed a few small NOR that were positioned more peripherally. These findings suggest that in highly differentiated cells, the NOR region is precisely positioned in the nucleus, regardless of non-identical chromosome locations of the NOR in different species. The variability of NOR in tumor cells indicates profound changes in nuclear structure that may be part of the neoplastic transformation.

In vitro transformation elicited by Creutzfeldt-Jakob-infected brain material.

Previous studies indicated that tissue culture cells derived from Creutzfeldt-Jakob disease (CJD) brains possess neoplastic properties. In order to see if CJD brain material could itself transform cells in vitro, BALB/C-3T3 and normal hamster brain monolayer cultures were exposed to whole brain homogenate or synaptosomal-mitochondrial fractions derived from CJD or control brains. Cells were exposed for seven days to CJD or control brain material, washed, and then passaged at weekly intervals. Transformation was evaluated by loss of contact inhibition, replication of cells with absent or low serum content of the medium and significant colony formation in soft agar. Seven parallel experiments were done, and six were viable. All cultures exposed to CJD material containing greater than or equal to 10(4) LD50 infectious units were positive for transformation (five of six studies); the single negative experiment contained only 2 X 10(3) infectious units. Definitive transformation could be observed at 12-16 weeks after application of both mouse and hamster CJD material. BALB/C-3T3 cells exposed to control hamster or mouse brain material were studied for greater than 120 passages in vitro and showed no comparable changes or evidence of spontaneous transformation, i.e. they required serum for replication and produced essentially no colonies in soft agar. Similarly, a normal adult hamster brain culture exposed to control brain material showed no evidence of transformation, whereas the identically passaged normal brain culture exposed to CJD material not only became a permanent line, able to grow in soft agar, but also was capable of producing tumors in nude mice. Thus CJD transformation was not confined to special potentials of "permanent" BALB/C-3T3 cells. These experiments suggest that a genomic alteration can be effected by CJD material.

A novel cDNA/PCR strategy for efficient cloning of small amounts of undefined RNA.

In this report we present a strategy for generating a representative cDNA library from prohibitively low amounts of mRNA template. A defined DNA adapter, which carries an EcoRI site, is ligated to both ends of the products of a cDNA synthesis reaction. This allows low levels of cDNA to be amplified by a polymerase chain reaction. In studies with pg amounts of rabbit globin mRNA, the amplified cDNA product is shown to be full-length. Globin cDNA recombinants are positively identified in lambda gt10. The protocol should be widely applicable to mRNAs of low abundance, whose sequences have not been determined, and to limited samples from patients or animals. It may also be useful for generating representative libraries of low titer or variant viral sequences.

Creutzfeldt-Jakob infection increases adenylate cyclase activity in specific regions of guinea pig brain.

Creutzfeldt-Jakob disease is a slow, infectious, progressive neurological disorder which results in human dementia. Synaptic membranes from various brain regions of guinea pigs infected with Creutzfeldt-Jakob disease show increased guanyl nucleotide- or 5-hydroxytryptamine-mediated activation of adenylate cyclase. This increased enzyme activity appears due, primarily, to facilitated 'coupling' between the GTP-binding protein which stimulates adenylate cyclase (GNs) and the catalytic moiety of that enzyme rather than increased sensitivity to 5-hydroxytryptamine. It is possible that this phenomenon is due to direct effects of the Creutzfeldt-Jakob infectious agent, or a pathological product resulting from that agent, upon synaptic membrane adenylate cyclase.

Analysis of Creutzfeldt-Jakob disease infectious fractions by gel permeation chromatography and sedimentation field flow fractionation.

Gel permeation chromatography and sedimentation field flow fractionation (SF3) were used to further analyze highly infectious fractions from Creutzfeldt-Jakob disease (CJD) infected hamster brain. These analyses defined the relative molecular mass and physical size of the Creutzfeldt-Jakob disease (CJD) agent with greater precision than previously possible. Highly purified disaggregated fractions yielded single, homogeneous Gaussian peaks with both methods. The relevant analytical peaks contained protein-nucleic acid complexes with an M(r) of approximately 1.5 x 10(7) daltons and a mean radius of approximately 30 nm. The experimental evidence further solidifies the concept of an infectious agent that resembles a viral core rather than a simple protein.

Non myelin-forming perineuronal Schwann cells in rat trigeminal ganglia express P0 myelin glycoprotein mRNA during postnatal development.

To determine whether P0 myelin glycoprotein mRNA is expressed in Schwann cells that ensheath neurons and do not form myelin, we probed aldehyde-fixed vibratome sections of developing and adult trigeminal ganglia with a biotinylated P0 cDNA. For probe detection, vibratome sections were treated with nickel-enhanced horseradish peroxidase (HRP). At each age, some vibratome sections were used to count numbers of HRP-positive and -negative satellite cells. The percentages of HRP-positive satellite cells at 2, 7, and 15 days were 22%, 30% and 14%. None was positive at 30 days or in the adult. Other vibratome sections were embedded for light and electron microscopic study. In semithin sections from ganglia removed from 2-day-old rats, small dot-like densities of HRP were located in perinuclear regions of a few perineuronal Schwann cells. In 7-day-old ganglia, more of these Schwann cells contained HRP. In thin sections studied with the electron microscope, peroxidase was found in cytoplasmic regions enriched in granular endoplasmic reticulum and ribosomes. At 2 and 7 days, HRP densities in perinuclear regions were larger and more numerous than at 15 days. No signal was detected in 30 day or adult perineuronal Schwann cells. The results show that early in postnatal development, P0 mRNA is expressed in some Schwann cells that ensheath neurons, that do not contain immunocytochemically detectable levels of P0 and that do not ever form myelin.

Dementias, neurodegeneration, and viral mechanisms of disease from the perspective of human transmissible encephalopathies.

Our transmission experiments with human CJD emphasize the centrality of an exogenous infectious pathogen that can exist in symbiosis with its host for extended periods. Many latent or persistent viruses can cause neurodegenerative disease and may have a role in late onset dementias. There are reasons to believe that CJD infections may share properties with some of these latent viruses in causing dementia, and several retroviral mechanisms may be operative in CJD. In order to clarify viral-like attributes of the CJD agent we have closely followed infectivity and find the following: 1) the CJD agent has a virus-like size and density, and is biochemically separable from most host-encoded prion protein (PrP); 2) Endogenous retroviral IAP RNA sequences of 5,000 bases, as well as several gag-like nucleic acid binding proteins, co-purify with infectivity in preparations treated with high concentrations of anionic detergents and exhaustive nuclease digestion. They signify the purification of true viral cores rather than aggregation artifacts, and diminish claims that there are no protected nucleic acids of > 50 bases in highly purified infectious preparations; 3) In established hamster CJD, temporal studies show the agent has an effective doubling time of approximately 7.5 days in brain, consistent with complex host-viral interactions common to slow viral infections; 4) PrP-res does not correspond to titered levels of infectivity either in a biochemical or an in vivo setting but may function as a viral receptor that can modulate disease expression. Interestingly, functional changes in glial cells occur earlier than PrP-res changes, and indicate an important role for glial cells in evolving infections; 5) Human-rodent transmission studies suggest that CJD, or a CJD-like variant can be a common but latent infection of humans, with relatively infrequent expression of neurological disease. Susceptibility to disease can rest on host attributes and possibly age-related co-factors. Nonetheless, fundamental viral principles are also operative. Agent strain variants, viral burden, and the routes of infection are critical parameters for latency and disease expression. The properties described above have led me to return to the inclusion of CJD (and scrapie) in the panorama of conventional slow viral infections of the brain, as originally proposed by Sigurdsson. Identification of virus-specific molecules are essential for elucidating the role of these agents in the spectrum of human dementias.

Indications of centromere movement during interphase and differentiation.

Mouse and human DNA sequences from centromeric and ribosomal domains were labeled with biotinylated deoxynucleotides and hybridized in situ to paraformaldehyde-fixed tissue culture cells. Centromeres were widely dispersed in most of these interphase nuclei. At late G2 phases of the cell cycle, centromeres appeared to coalesce and then to align in an orderly pattern, with discrete positional assignments for individuals chromosomes in metaphase and anaphase. Ribosomal cistrons were also organized in an orderly and defined fashion during mitosis. As soon as the nuclear membrane forms in early G1, centromeres rapidly disperse throughout the nucleus. Centromere patterns during G1 and S were indistinguishable in cultured cells, as determined by double-labeling experiments. Antibodies that bind to centric chromosomal proteins revealed the same patterns in cultured cells as those obtained with DNA sequence-specific probes. Large differentiated neurons display reproducible collections of centromeres in interphase that are very different from those seen in cultured cells. Neurons in widely divergent mammalian species, despite large differences in centromeric DNA sequences, maintain similar nuclear positions for these chromosomal segments. Similarly, ribosomal cistrons are positioned in comparable nuclear locales in neurons of divergent species. It is suggested that such arrangements reflect, or are necessary for, the function of a given cell type. Studies of large cerebellar neurons at critical times in development indicated a relative "movement" of centromeric domains, away from the nuclear membrane and toward the central nucleolar region. It is possible that the orderly and temporal positioning of centromeric, as well as of other chromosomal regions, is based on protein-nucleic acid interactions. Implications for trisomy 21 and other disorders involving chromosomal rearrangements, such as transposition, are considered from this perspective.

Soft x-ray lithographic studies of interphase chromosomes.

Soft x-ray absorption lithograph patterns of purified interphase human nuclei and chromosome arrays, imaged on PMMA resists, were examined by scanning EM. The patterns obtained were compared to those utilizing more conventional sources, including transmission EM, scanning EM, high voltage EM, and various light microscopic techniques. The x-ray resist images revealed orderly arrays of absorption profiles in the 3-dimensional specimen with both mild and more extensive developments of the resist. Dense chromatin at the edge of interphase nuclei revealed aligned periodic peaks on the order of 2200 A diameter, with substructure. The periodicity and alignment of interphase chromosomes were entirely consistent with birefringent data on nuclei indicating a high degree of 3-dimensional order. This degree of 3-dimensional order was observed in nuclei containing essentially DNA and histones with only very few other minor (probably structural) proteins. Sonication and nuclease treatment to disperse interphase chromosomes revealed similar absorption periodicities in individual chromosome fibers. Analysis of x-ray absorption profiles thus appears to offer significant new insights into the ordered structure of these defined biological specimens.