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Sonstige beteiligte Institutionen
- Johns Hopkins School of Medicine, Baltimore, MD, U.S. (4)
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EU-Project number / Contract (GA) number
- 701983 (8)
The structural organization of transcriptionally active DNA that contains cistrons for precursor molecules of ribosomal RNA is described in positively stained spread preparations from nuclei and nucleoli isolated from the green alga, Acetabularia mediterranea Lmx. These nuclei contain large aggregates of nucleolar subunits in which fibril-covered regions, the putative active cistrons for precursors of ribosomal RNA, alternate with fibril-free intercepts, the "spacers". The length distribution of the different intercepts of this DNA is given, and the pattern is compared with those shown in animal cell systems. The data are discussed in relation to problems of transcription and of amplification of ribosomal RNA genes.
A significant contribution to the understanding of chromatin organization was the d iscovery of the nucleosome as a globular repeating unit of the package of DNA (Hewish and Burgoyne, 1973; Woodcock, 1973; Kornberg, 1974; Olins and Olins, 1974; for review see Oudet et al., 1978 a) . In accord with the original definition and in ag reement with most workers in this field of research we identify a nucleosome as a spheric alor slightly oblate gr anular particle 10-13 nm in diameter, containing about 200 base pairs of DNA and two of each of the four his tones H2a, H2b, H3 and H4. It is this structure in which the bulk of the nuclear chroma tin is organized in most eukaryotic cells, with the exception of the dinofl age llates (Rae and Steele, 1977; dinofl agellate DNA, however, c an be packed into nucleosoma l structures in vitro by addition of the appropriate amounts of histones;the same reference). Although it seems clear from the work reported that condensed and transcriptiona lly inactive chroma tin is contained in nucleosomes as the principle for first order p acking of DNA there are two important questions onto which we are focusing in the present study: ( i ) What is the higher order of p a cking present in - and perhaps typical-of - the condensed sta te of chromatin, and (ii) what is the specific form of arrangement of transcriptionally a ctive chromatin?
The binding of tritiated benzo(a)pyrene (BP) to liver DNA of 25 adult male rats (SIV 50) has been determined 50 h after a single intraperitoneal injection of doses between 40 ug/kg and 4; mg/kg. The dose-response relations~ ip is linear up to i mg/kg, shows a sigmoid step towards 2 mg/kg and a shallow linear. slope above that value. TlJe 0 bserved bin ding ranges from 1.7 to 180 nmoles BP per mole DNA phosphate. The non-linearity between 1 and 2 mg/kg could be explained 0):1 the basis of an induction of metabolizing enzymes. A pure1y mathematical extrapolation of therumour incidence from a carcinogenic dose (1 x 40mg/kg for a 20% hepatoma incidence in newborn mice) to human exposure levels (aboilt 0.1 ug/kg per day) would never have followed a step like the on~ found in our experiments. Our dose-effect study therefore shows how carcinogenitity data could be extrapolated in a biologically founded way to low doses.
Some decades ago it was noted by cytologists that within the interphase nucleus large portions of the transcriptionally ("genetically," in their terms) inactive chromosomal material are contained in aggregates of condensed chromatin, the "chromocenters," whereas transcriptionally active regions of chromosomes appear in a more dispersed form and are less intensely stained with DNA-directed staining procedures (Heitz 1929, 1932, 1956; Bauer 1933). The hypothesis that condensed chromatin is usually characterized by very low or no transcriptional activity, and that transcription occurs in loosely packed forms of chromatin (including, in most cells, the nucleolar chromatin) has received support from studies of ultrathin sections in the electron microscope and from the numerous attempts to separate transcriptionally active from inactive chromatin biochemically (for references, see Anderson et al. 1975; Berkowitz and Doty 1975; Krieg and Wells 1976; Rickwood and Birnie 1976; Gottesfeld 1977). Electron microscopic autoradiography has revealed that sites of RNA synthesis are enriched in dispersed chromatin regions located at the margins of condensed chromatin (Fakan and Bernhard 1971, 1973; Bouteille et al. 1974; Bachellerie et al. 1975) and are characterized by the occurrence of distinct granular and fibrillar ribonucleoprotein (RNP) structures, such as perichromatin granules and fibrils. The discovery that, in most eukaryotic nuclei, major parts of the chromatin are organized in the form of nucleosomes (Olins and Olins 1974; Kornberg 1974; Baldwin et al. 1975) has raised the question whether the same nucleosomal packing of DNA is also present in transcriptionally active chromatin strands. Recent detailed examination of the morphology of active and inactive chromatin involving a diversity of electron microscopic methods, particularly the spreading technique by Miller and coworkers (Miller and Beatty 1969; Miller and Bakken 1972), has indicated that the DNA of some actively transcribed regions is not packed into nucleosomal particles but is present in a rather extended form within a relatively thin (4-7 nm) chromatin fiber.
The influence of microsomal (mAHH) and nuclear (nAHH) aryl hydrocarbon hydroxylase activity on the covalent binding of t:titiated benzo(a)pyrene to rat liver DNA was evaluated in vivo. Induction ofmAHH was obtained after phenobarbitone treatment (180% of control), which increased DNA binding to 210%, but left the nAHH unchanged. mAHH and nAHH were slightly indilced with dieldrin (130% and 120%), but the binding remairred unchanged. The increasing effect of mAHlt as weil as the possibly decreasing effect of nAHH induction on the binding became obvious when the data of 11 individual rats were used to solve the equation Binding = aX(mAHH) + bX(nAHH) + c. Multiple linear regression analysis resulted in positive values for a and c, a negative value for b, and a multiple correlation coefficient R = 0.82. An influence of other enzymes involved in the metabolism of benzo(a)pyrene cannot be excluded. The Study shows clearly that the binding of a foreign compound to DNA in vivo is not only dependent on microsomal enzyme activities but also on nuclear activities even if the latter are considerably lower than those of mic'rosomes.
Neoplasia in Xiphophorus can be classified into: a) a Jarge group triggered by carcinogens; b) a large group triggered by promoters; and c) a small group that develops "spontaneously" according to Mendelian Jaw. The process leading to susceptibility for neoplasia is represented by the disintegration of gene systems that normally protect the fish from neoplasia. Interpopulational arid interracial hybridization is the most effective process that Ieads to disintegration of the protective gene systems. Environmental factors may complete disintegration in somatic cells and thus may trigger neoplasia. The applications of the findings on Xiphophorus to humans are discussed.
Neoplasia in Xiphophorus can be classified into a) a large group that is triggered by carcinogens; b) a large group triggered by promoters; c) a small group that develops "spontaneously" following interpopulational and interracial hybridizations; and d) a small group that develops "spontaneously" following germ line mutation. The process leading to susceptibility for neoplasia is represented by the disintegration of gene systems that normally protect the fish from neoplasia. Hybridization is the most effective process that leads to disintegration of the protection gene systems. Environmental factors may complete disintegration and thus may trigger neoplasia. It is discussed whether the findings on Xiphophorus may also apply to humans.
Rabbit antibodies to RNA polymerase I from a rat hepatoma have been used to localize the enzyme in a variety of cells at the light and electron microscopic level. In interphase cells the immunofluorescence pattern indicated that polymerase I is contained exclusively within the nucleolus. That this fluorescence, which appeared punctated rather than uniform, represented transcriptional complexes of RNA polymerase I and rRNA genes was suggested by the observation that it was enhanced in regenerating liver and in a hepatoma and was markedly diminished in cells treated with actinomycin D. Electron microscopic immunolocalization using gold-coupled second antibodies showed that transcribed rRNA genes are located in, and probably confined to, the fibrillar centers of the nucleolus. In contrast, the surrounding dense fibrillar component, previously thought to be the site of nascent prerRNA, did not contain detectable amounts of polymerase I. During mitosis, polymerase I molecules were detected by immunofluorescence microscopy at the chromosomal nucleolus organizer region, indicating that a considerable quantity of the enzyme remains bound to the rRNA genes. From this we conclude that rRNA genes loaded with polymerase I molecules are transmitted from one cell generation to the next one and that factors other than the polymerase itself are involved in the modulation of transcription of DNA containing rRNA genes during the cell cycle.
Many mutagens and carcinogens act via covalent interaction of metabolic intermediates with DNA in the target cell. This report groups those structural elements which are often found to form the basis for a metabolism to such chemically reactive metabolites. ~mpounds which are chemically reactive per se and which do not require metabolic activation form group 1. Group 2 compri~es of olefins and aromatic hydrocarbons where the oxidation via an epoxide can be responsible for the generation of reactive species. Aromatic amines, hydrazines, and nitrosamirres form group 3 requiring an oxidation of a nitrogen atom or of a carbon atom in alpha position to a nitrosated amine. Group 4 compounds are halogenated hydrocarbons which can either give rise to radicals or can form an ·olefin (group 2) upon dehydrohalogenation. Group 5 compounds depend upon some preceding enzymatic activity either not available in the target cell or acting on positions in the molecule which are not directly involved in the subsequent formation of electrophilic atoms. Examples for each group are taken from the "List of Chemieals and Irrdustrial Processes Associated with Cancer in Humans" as compiled by the International Agency for the Research on Cancer, and it is shown that 91% of the organic carcinogens would have been detected on the basis of structural elements characteristic for group 1-5. As opposed to this very high sensitivity, the specificity ( the true negative fraction) of using this approach as a short-term test for carcinogenicity is shown to be bad because detoxification pathways have so far not been taken into account. These competing processes are so complex, however, that either only very extensive knowledge about pharmacokinetics, stability, and reactivity will be required or that in vivo systems have to be used to predict, on a quantitative basis, the darnage expected on the DNA. DNA-binding experiments in vivo are presented with benzene and toluene to demonstrate one possible way for an experimental assessment and it is shown that the detoxification reaction at the methyl group available only in toluene gives rise to a reduction by at least a factor of forty for the binding to rat liver DNA. This quantitative approach available with DNA-binding tests in vivo, also allows evaluation as to whether reactive metabolites and their DNA binding are always the most important single activities contributing to the overall carcinogenicity of a chemical. With the example of the livertumor inducing hexachlorocyclohexane isomers it is shown that situations will be found where reactive metabolites are formed and DNA binding in vivo is measurable but where this activity cannot be the decisive mode of carcinogenic action. It is concluded that the lack of structural elements known to become potentially reactive does not guarantee the lack of a carcinogenic potential.
The capacity of Xiphophorus to develop neoplasia can be formally assigned to a "tumor gene" (Tu), which appears to be a normal part of the genome of all individuals. The wild fish have evolved population-specific and cell type-specific systems of regulatory genes (R) for Tu that protect the fish from neoplasia. Hybridization of members of different wild populations in the laborstory followed by treatment of the hybrids with carcinogens led to disintegration of the R systems permitting excessive expression of Tu and thus resulting in neoplasia. Certain hybrids developed neoplasia even spontaneously. Observations on the genuine phenotypic effect of the derepressed Tu in the early embryo indicated an essential normal function of this oncogene in cell differentiation, proliferation and cell-cell communication. Tu appeared to be indispensable in the genome but may also be present in accessory copics. Recently, c-src, the cellular homolog of the Rous sarcoma virus oncogene v-src, was detected in Xiphophorus. The protein product of c-src, pp60c-src, was identified and then examined by its associated kinase activity. This pp60c-src was found in all individuals tested, but, depending on the genotype, its kinase activity was different. The genetic characters of c-src, such as linkage relations, dosage relations, expression, etc., correspond to those of Tu. From a systematic study which showed that pp60c-src was present in all metazoa tested ranging from mammals down to sponges, we concluded that c-src has evolved with the multicellular organization of animals. Neoplasia of animals and humans is a characteristic closely related to this evolution. Our data showed that small aquariurn fish, besides being used successfully because they are time-, space-, and money-saving systems for carcinogenicity testing, are also highly suitable for basic studies on neoplasia at the populational, morphological, developmental, cell biological, and molecular levels.
Melanotic melanoma (MM) of Xiphophorus (Teleostei: Poeciliidae) was studied by conventional preparations and freeze-etch preparations for electron microscopy. MM of Xiphophorus exhibits tightly packed pigment cells with prominent dendritic processes and interdigitations of their plasma membranes. The most impressive feature of MM cells is the occurrence of Iarge lobulated nuclei with numerous nuclear pores and some nuclear pockets. Abundant spheroidal or ellipsoidal melanosomes (diameter 200-650 nm) and vesicular structures are distributed throughout the cellular dendrites, whereas the perinucJear cytoplasm is free of melanosomes.
A further characteristic feature of melanoma cells in fish is the occurrence of melanosome complexes (i.e., "compound melanosomes"). These melanosome complexes consist of a few to numerous melanosomes, which are enveloped by a separate rnembrane. Pinocytotic vesicles couJd be demonstrated with distinct differences in frequency and distribution patterns, indicating differences in the metabolic activities of the cells in the same melanoma. Intercellular junctions are lacking in the MM cells.
The conventional TEM technique showed clear advantages in the demonstration of intemal architecture of organelles, whereas FE bad considerable potential in respect to the visualization of membrane surface specializations.
The \(\alpha\)-Sialyl-\(\beta\) 2-3-Galactosyl-specific adhesin (S adhesin) was isolated from cells of a recombinant Escherichia coli K-12 strain expressing the S-flmbrial adhesin complex. A crude cell extract was partiaUy dissociated into fimbriae and an adhesin-enriched fraction by heating to 7O°C. From the latter, adhesin was purified to apparent homogeneity (by fast protein liquid chromatography, immunoblot, and NaDodSO\(_4\)/PAGE) by differential ammonium sulfate precipitation, dissociation in 8 M guanidine hydrochloride, and high-resolution anion-exchange chromatography in 8 M urea. The purified adhesin formed an aggregate of M\(_r\)\(\approx\)10\(^6\) that was made up of one type of 12-kDa polypeptide (fimbrillin is 16.5 kDa). It had pI value of 4.7 (fimbriae has a pI value of 6). Adhesin and fimbrillin had different amino add compositions. The purified adhesins agglutinated human and bovine erythrocytes with the same speclfkity as the whole bacteria; purified fimbriae were not adhesive. Monoclonal anti-adhesin and anti-fimbriae antibodies were obtained. Monoclonal antiadhesin, but none of the anti-fimbriae, antibodies inhibited the agglutination of erythrocytes. The anti-adhesive antibodies were used in immuno-gold electron microscopy to localize adhesin exclusively on the fimbriae, with a possible preference to their tips.
no abstract available
no abstract available
K-Ar dating on hornblendes and micas from the Tepla Domazlice zone revealed a pattern of dates which significantly deviates from the mid-Carboniferous to early Permian one that is found in the adjacent low-pressure metamorphic Moldanubian and Saxothuringian. Especially for the Marianske Lazne metabasic complex, confirming early Czech determinations, the dates resemble the early Devonian pattern determined for the Munchberg Gneiss Massif and the Erbendorf-Vohenstrau zone of northeastern Bavaria. This supports the idea that all three units are remnants of a huge' complex which suffered a metamorphic overprint under medium-pressure conditions, probably in the early Devonian. Strong rejuvenation is found in the southern part of the Tepla-Domazlice zone by which micas and even two hornblendes were reset to mid-Carboniferous ages. According to the geological setting, part of the apparently preDevonian dates may be explained by inherited argon from earlier metamorphic and magmatic events, e.g. the high-pressure metamorphism documented in eciogitic relics. However, excess argon, caused by the mid-Carboniferous overprint cannot be excluded.
Characterization of the env gene and of two novel coding regions of the human spumaretrovirus
(1988)
Recombinant clones harboring retroviral DNA were established. The nucleotide sequence of the central and 3' region of the genome of the human spumaretrovirus was determined. The 5' end of the deduced protein sequence was homologaus to the endonuclease domain of retroviral reverse transcriptases. A small intergenic region is followed by a lang open reading frame of 985 aminoacid residues that according to its genomic location and structural features is a typical retroviral env gene. Surprisingly, the postenv region contains two open reading frames that encodes two novel retroviral genes, termed bel-l and bel-2. The 3' LTR is 963 nucleotides lang and contains the signal sequences characteristic for transcriptional regulation of retrovirus genomes.
K-Ar dating on hornblendes and micas from the TepläDomazlice zone revealed a pattern of dates which significantly deviates from the mid-Carboniferous to early Permian one that is found in the adjacent low-pressure metamorphic Moldanubian and Saxothuringian. Especially for the Mariänske Läzne metabasic complex, confirming early Czech determinations, the dates resemble the early Devonian pattern determined for the Münchberg Gneiss Massif and the Erbendorf-Vohenstrauß zone of northeastern Bavaria. This supports the idea that all three units are remnants of a huge complex which suffered a metamorphic overprint under medium-pressure conditions, probably in the early Devonian. Streng rejuvenation is found in the southern part of the Teplä-Domailice zone by which micas and even two hornblendes were reset to mid-Carboniferous ages. According to the geological setting, part of the apparently preDevonian dates may be explained by inherited argon from earlier metamorphic and magmatic events, e.g. the high-pressure metamorphism documented in eclogitic relics. However, excess argon, caused by the mid-Carboniferous overprint cannot be excluded.
Various amphibolites, metagabbros and eclogitic relics of the Mariänske Läzne complex, and amphibolites from the Cernä Hora Massif exhibit an uniform geochemical character which compares weil with modern mid-ocean ridge basalts. Geochemically these metabasites are similar to the amphibolites of the Myto area and to schistose, partly striped amphibolites of the neighbouring Tirschenreuth-Mähring Zone and the Erbendorf-Vohenstrauss Zone (Bavaria). Greenschists and amphibolites from the Domazlice metamorphic complex show an alkaline-basaltic tendency conforming to modern within-plate basalts or basalts from anomalaus midocean ridge segments. In their chemical character, these metabasites compare weil with the flaseramphibolites of the Erbendorf-Vohenstrauss Zone. Fine-grained amphibolites in the Warzenrieth area and (gabbro-) amphibolites in the Blätterberg-Hoher Bogen area show normal MORB character. The metamorphosed gabbroic rocks in the southern part of the Neukirchen-Kdyne (meta-) igneous complex are subalkaline - tholeiitic and exhibit a magmatic differentiation trend. They differ from the neighbouring amphibolites by generally lower contents of incompatible elements.
This project had two goals: (1) to examine the impact of strategy training on memory performance in German and American children, and (2) to search for environmental correlates of individual differences in cognitive processes. Following pretesting, 437 children were divided into training and control groups, with the former receiving training in clustering strategies. Trained children showed sizable strategy maintenance and transfer effects two weeks and six months later. Parents and teachers completed questionnaires about the teaching of strategies and their attributional beliefs about children's academic successes and failures. The differences in strategie behavior and attributions of German and American children were due, in part, to differences in strategy-enriched environments.
Non-Formal Education
(1989)
To study the activation of HIV by human spumaretrovirus (HSRV) the long terminal repeats (LTRs) of HSRV, HIVl and HIV2 were examined with respect to their ability to function as transcriptional promoters in virus infected and uninfected cells. Transient transfections using plasmids in which the L TRs of the three viruses were coupled to the bacterial chloramphenicol acetyltransferase (CA T) gene revealed (i) the level of cat gene expression directed by the HSRV LTR was markedly increased in HSRV infected cells compared to uninfected cells, (ii) cat gene expression driven by the HIV1 LTR, but not by the HIV2 LTR could be enhanced upon HSRV infection, whereas (iii) neither in HIV1 nor in HIV2 infected cells an effect on HSRV LTR driven cat geneexpression was detected.
Pandinus imperator is a forest dweller of tropical West Africa. In the field, lobserved aggregations of up to 15 individuals. In the laboratory, mixed age groups of related and also unrelated animals lived jointly in terraria rarely showing within-group aggression or cannibalism. Brood-caring behavior of the mother influenced growth rate and survival probability of the young. With birth, mothers became very aggressive. To study family cohesion in Pandinus, experiments with family groups were conducted. Siblings aggregated around their mother. In choice experiments with two family groups, mothers were placed in enclosures that only the young were able to enter or to leave. Second instars significantly preferred the enclosure containing their own mother. Aggression among unrelated young of the same age was not observed. Feeding experiments studied the possible advantages of long-Iasting group living with regard to enhanced success in prey capture and its effect on growth of the young. Even groups of second instars were unable to subdue large prey on their own. Sibling groups with their mother removed suffered high mortality due to starvation and cannibalism compared to groups with mothers present. Here, young grew significantly faster: they shared the prey that only the mother was able to kill and dismember. Pandinus imperator has to be considered an intermediate subsocial scorpion.