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- Theodor-Boveri-Institut für Biowissenschaften (1834)
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Schriftenreihe
Sonstige beteiligte Institutionen
- Johns Hopkins School of Medicine (17)
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- Clinical Trial Center (CTC) / Zentrale für Klinische Studien Würzburg (ZKSW) (5)
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- Johns Hopkins University School of Medicine (5)
- Wilhelm-Conrad-Röntgen-Forschungszentrum für komplexe Materialsysteme (5)
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- Johns Hopkins School of Medicine, Baltimore, MD, U.S. (4)
- Technische Hochschule Nürnberg Georg Simon Ohm (3)
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Zentgraf, Hanswalter ; Trendelenburg, Michael F. ; Spring, Herbert ; Scheer, Ulrich ; Franke, Werner W. ; Müller, Ulrike ; Drury, Kenneth C. ; Rungger, Duri
Purified mitochondrial DNA (mitDNA) from ovaries ofXenopus lae vis was injected into the nuclei (germinal vesicles) of large viteUogenic oocytes of the same organism and examined by electron microscopy ofthe spread nuclear contents. Normally located nuclei of untreated oocytes as weil as peripherally translocated nuclei of centrifuged oocytes were used. In addition, oocyte nuclei isolated and incubated under liquid paraffin oil were injected with DNA. The integrity oftranscriptional structures of endogenous chromosomal (Iampbrush chromosomes) and extrachromosomal (nucleoli) genes of the injected nuclei was demonstrated. Microinjected mitDN A was identified as circles of chromatin exhibiting polynucleosome-like organization and a me an contour length of 2.6 J.Lm, corresponding to a compaction ratio of the mitDN A of about 2 : I. This DNA packing ratio is similar to that observed after preparation of various kinds of native chromatin in low salt buffers. The chromatin circles formed from injected mitDNA only very rarely exhibited lateral fibrils suggestive of transcriptional activity. These results suggest that purified mitDNA can be transformed to normally structured chromatin when exposed to oocyte nuclear contents but is rarely , if at all , transcribed in this form and in this environment.
Antibodies to calf thymus histone H2B were purified by chromatography on DEAE-cellulose and injected into oocyte nuclei of Pleurodeles waltlii. As shown by indirect immunofluorescence these antibodies cross-reacted strongly with corresponding histones associated with lampbrush chromosomes. Shortly after injection the lateral loops of the chromosomes retracted into the chromomeres and by 3 h postinjection the 'lampbrush' appearance was completely lost and the chromosomes appeared in light-microscopic preparations as rod-like structures consisting of 10ngitudina11y coalesced chromomeres. In control oocytes injected with non-immune immunoglobulins or antibodies against a ubiquitous transcript-associated protein no morphological alterations of the lampbrush chromosomes could be observed. Electron microscopic spreads of chromosomes prepared at various times after injection of anti-H2B revealed a progressive loss of transcriptional complexes from the loop axes. Finally, higher-order chromatin configurations, like supranuc1eosomal globules (' superbeads ') or cable-like chromatin strands 50- 60 nm thick predominated, indicating complete transcriptional inactivation of a11 chromosomal regions. The results indicate that H2B antibodies react specifically with his tones associated with the transcribed DNA of lateral loops in their native state. The resulting antigenantibody complexes seem to inhibit progression of the R A polymerases along the template, thus causing the premature release of transcripts, a process analogous to the stripping effect of actinomycin D. The demonstration of histones associated with heavily transcribed regions, which are not compacted into nucleosomes but largely extended, supports the current concept that unfolding of nucleosomes to a110w transcription of the DNA does not involve dissociation of histones. In contrast, amplified ribosomal RNA genes are unaffected by injected HzB antibodies. This does not necessarily indicate absence of his tones from nucleolar chromatin, since we do not know whether it is accessible in vivo to antibodies or whether the histone antigenie determinants are masked by the presence of other proteins. The technique of injecting specific antibodies should be widely applicable when analysing the in vivo distribution of chromosomal components at the electron-microscopic level and when studying complex metabolie processes, like the cleavage and modification of RNA, by selective inhibition of defined enzymic steps.
Xiphophorus andersi n. sp. from the Rio Atoyac, Vera Cruz, Mexico is described: lang head, moderately slender body, large dark black spar at the basis of the anal fin; adult male with short sword-like caudal appendage; rip of ray 5a of gonopodium without a developed claw. Xiphophorus andersi n. sp. differs by the combination of distinct characters from all the other species of the genus known so far. The new species shows features of both the so-called platyfish species group and the so-called swordtail species group.
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.
no abstract available
The A TPase eomplex has been isolated from mitoehondria of N eurospora crassa by immunologieal teehniques. The protein ean be obtained rapidly and qua ntitatively in high purity by miero- or large-seale immunopreeipitation. Immunopreeipitation has been applied to labeled and doubly labeled mitoehondrial proteins in order to investigate the number and moleeular weights of subunit polypeptides , the site of synthesis of subunit polypeptides, and the dieycIohexyIcarbodiimide-binding protein . The A TPase complex obtained by large-seale immunopreeipitation has been used as starting ma terial for the isolation of hydrophobie polypeptides.
The fungus Neurospora crassa represents a eukaryotic cell with high biosynthetic activities. Cell mass doubles in 2-4 hr during expone ntial growth , even in simple salt media with sucrose as the sole carbon source. The microorgani sm forms a mycelium of long hyphae durlng vegetative growth . The mitochondria can be isolated under relatively gentle condi tions since a few breaks in the threadlike hyphae are sufficient to cause the outflow of the organelles. This article describes two methods for the physical disruption of the hyphae : (I) The cell s are opened in a grind mill between two rotating corundum di sks. This is a continuous and fast procedure and allows large- and small-scale preparations of mitochondria. (2) Hyphae are ground with sand in a mortar and pestle. This procedure can be applied to microscale preparations of mitochondria starting with minute amounts of cells. Other procedures for the isolation of Neurospora mitochondria after the physical di sruption or the enzymatic degradation of the cell wall have been described elsewhere
Educational psychology
(1979)
This is a report on the more recent developments and the present state of research into educational psychology in German speaking countries. Particular emphasis is given to research on: parental upbringing and its effects on child development; the examination of socialization effects within and across different scbool systems; studies on teaching-leaming processes and on social interaction in the classroom; the systematic promotion of the development of cognitive abilities and motives in students; and, finally, the design of improved instruments in methods of describing, explaining and predicting school success. Subsequently, the report will look into problems in tbe practical application of research findings in educational psychology. Finally, there follows a sbort discussion of various metatheoretical positions in educational psycbology in German speaking countries and their possible effects on the future development of the field.
The plasmid pBC16 (4.25 kbases), ongtnally isolated from Bacillus cereus, determines tetracycline resistance and can be transformed into competent cells of B. subtilis. A miniplasmid of pBCl6 (pBCI6-1), 2,7 kb) which has lost an EcoRI fragment of pBCI6 retains the replication functions and the tetracycline resistance. This plasmid which carries only one EcoRI site has been joined in vitro to pBS], a cryptic plasmid previously isolated from B. subtilis and shown to carry also a single EcoRI site (Bernhard et aI., 1978). The recombinant plasmid is unstable and dissociates into the plasmid pBSl61 (8.2 kb) and the smaller plasmid pBS162 (2. I kb). Plasmid pBS161 retains the tetracycline resistance. It possesses a single EcoRI site and 6 HindlII sites. The largest HindIII fragment of pBS161 carries the tetracycline resistance gene and the replication function. After circularization in vitro of this fragment a new plasmid, pBS161-l is generated, which can be used as a HindlII and EcoRI cloning vector in Bacillus suhtilis. Hybrid plasmids consisting of the E. coli plasmids pBR322, p WL 7 or pACl84 and different HindlII fragments of pBSI61 were constructed in vitro. Hybrids containing together with the E. coli plasmid the largest HindlII fragment of pBS161 can replicate in E. coli and B. sublilis. In E. coli only the replicon of the E. coli plasmid part is functioning whereas in B. suhtilis replication of the hybrid plasmid is under the control of the Bacillus replicon. The tetracycline resistance of the B. subtilis plasmid is expressed in E. coli, but several antibiotic resistances of the E. coli plasmids (ampicillin, kanamycin and chloramphenicol) are not expressed in B. suhtilis. The hybrid plasmids seem to be more unstable in B. subtilis than in E. coli.
The lnfluence of mlcrosomal and nuclear aryl hydrocarbon hydroxylase (AHH) actlvlty on the covalent blndlng of [G·3H]benzo(a )pyrene to rat llver DNA was evaluated in viWJ. lnductlon of mlcrosomal AHH was obtalned alter phenobarbltal treatment (160% of control), whlch also lncreased DNA blndlng to 190%, but left the nuclear actlvlty unchanged. Nuclear AHH was lnduced wlth dleldrln (150%), and the blndlng was decreased to 75%, whereaa the mlcrosomal AHH was at control Ievei. The lncreaslng effect of mlcrosomal AHH lnductlon as weil as the decreaslng effect of nuclear AHH lnductlon on the blndlng was shown clearly when the data of the Individual rata were uaed to solve the equatlon Binding = e•(mlcroeomal AHH) + b•(nuclear AHH) + c Multiple linear regresslon analysls wlth the data from 10 anlmala reaulted ln positive valuea for a and c, a negative value for b, and a good multiple correlatlon coefflclent of r = 0.974. Pretreatment wlth 3-methylcholanthrene ln· duced mlcrosomal AHH to 380% of control and nuclear AHH to 590% and lncreased the blndlng' to 175,.-o. The blndlng was hlgher than predlcted by the formula found, probably because the lncreaslng lnfluence of lnduced mlcrosomal AHH overahadowed the decreaslng effect of the nuclear AHH. The study ahows clearly that the blndlng of a forelgn compound to DNA in viWJ Ia dependent not only on mlcrosomal enzyme actlvltles but also on nuclear actlvltles even lf the latter are conslderably lower than thoae of mlcrosomes.
Thecovalent bindingof [6,7-\(^3\)H]ethinylestradiol (EE)and [6,7-\(^3\)H]estrone (E) to liver DNA of 200 g female ratswas measured 8 h after the administration of 80 \(\mu\)g (9.2 mCi) estrogen by gavage. The binding is 1.5 for EE and 1.1 for E, expressedas binding to DNA/dose, in units of \(\mu\)mol hormonefmol DNA phosphate/mmole honnone/kg body wt. It is in the same order of magnitude as for benzene and about 10 000 tim es below the binding of typical liver carcinogens, such as aflatoxin B\(_1\) or N,N-dimethylnitrosamine.
Wlth radioactive compound of high specific activity, the binding of carcinogene to DNA can be measured wlth doses that are ineffective ln long-term studies. The binding of tritiated benzo(a )pyrene to liver DNA of adult male rats has been determined 50 hr after a singie l.p. injection of doses between 40 1'9/kg and 4 mg/kg. The doseresponse relationship is linear up to 1 mg/kg, shows a step towards 2 mg/kg, and gives a shallow linear slope above that value. The observed binding ranges from 1.7 to 180 nmoles benzo(a)pyrene per mole DNA phosphate. The nonlinearity could be due to an induction of metabolizing enzymes. The microsomal aryl hydrocarbon hydroxylase activity increases significantly 24 hr after a single dose of 4 mg/kg and 48 hr after doses of 2 and 4 mg/kg, but no induction Ia found with 1 mg/kg. The binding from an equimolar dose is 35 times lower than the one found on mouse skin DNA and 300 times lower than that of N,Ndlmethylnitrosamine in rat liver. A good correlatlon exiats to the respective tumor formation in long-term studles.
Aryl hydrocarbon hydroxylase (AHH) has been measured in male rat Jiver nucJei and microsomes after treatment of adult animals with various inducers for up to 14 days. After daily i.p. injections of 3-methylcholanthrene (MC, 20 mg/kg) the nuclear activity increased to a maximum of 600 per cent of the control activity after 4 days whereas the microsomal activity was 400 per cent of control at the same date. After 12 days, both activities equilibrated at 400 per cent. A similar time course was found after a single i.p. injection of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD, 0.01 mg/kg) with an induction to .500 and 300 per cent for nuclei and microsomes, respectiveJy. after 2 days, and to 400 per cent for both after 12 days. PhenobarbitaJ (PB) was given continuously in the drinking water (I g/1) and induced the microsomal activity to 200 per cent after 8 days and 170 per cent after 14 days. The nuclear activity was only slightly induced to a constant Ievei of 130 per cent between day 8 and 14. Dieldrin did not significantly increase the microsomal activity after daiJy i.p. injections (20 mg/kg), but the nuclear activity raised to 200 per cent after 3 days and levelled down tocontrol valuesafter 12 days. Other inducers tested were benz[a)anthracene (BA), hexachlorobenzene (HCB} and 1,1.1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). The induction pattern with BA was similar tothat of MC, a modeJ compound for the group of cytochrome P448 inducers. The induction by HCB and DDT resembled that by PB. a typical cytochrome P450 inducer.
The morphology of two forms of transcription ally active chromatin, the nucleoli and the loops of lampbrush chromosomes, has been examined after fixation in situ or after isolation and dispersion of the material in media of low ionic strengths, using a variety of electron microscopic preparation techniques (e.g. spread preparations with positive or negative staining or without any staining at all, with bright and dark field illumination, with autoradiography, after pretreatment of the chromatin with specific detergents such as Sarkosyl NL-30; transmission and scanning transmission electron microscopy of ultrathin sections). Nucleolar chromatin and chromosomes from oocytes of various amphibia and insects as well as from green algae of the family of the Dasycladaceae were studied in particular detail. The morphology of transcriptional units that are densely packed with lateral ribonucleoprotein fibrils, indicative of great transcriptional activity, was compared with that of chromatin of reduced lateral fibril density, including stages of drug-induced inhibition. The micrographs showed that under conditions which preserve the nucleosomal organization in condensed chromatin studied in parallel, nucleosomes are not recognized in transcriptionally active chromatin. This holds for the transcribed regions as well as for apparently untranscribed (i.e. fibril-free) regions interspersed between ('spacer') and/or adjacent to transcribed genes and for the fibril-free regions within transcriptional units of reduced fibril density. In addition, comparison oflengths of repeating units of isolated rDNA with those observed in spread nucleolar chromatin indicated that this DNA is not foreshortened and packed into nucleosomal structures. Granular particles which were observed, at irregular frequencies and in variable patterns, in some spacer regions, did not result in a proportional shortening of the spacer axis, and were found to be resistant to detergent treatment effective in removing most of the chromatin associated proteins including histones. Thus, these particles behave like RNA polymerases rather than nucleosomes. It is suggested that structural changes from nucleosomal packing to an extended form of DNA are involved in the transcriptional activation of chromatin.
The morphology of nucleolar and non-nucleolar (Iampbrush chromosome loops) chromatin was studied in the electron microscope during states of reduced transcriptional activity in amphibian oocytes (Xenopus laevis, Triturus alpestris, T. cristatus). Reduced transcriptional activity was observed in maturing stages of oocyte development and after treatment with an inhibitor, actinomycin D. Strands of nucleolar chromatin appear smooth and thin, and contain only few, if any, nucleosomal particles in the transcribed units. This is true whether they are densely or only sparsely covered with lateral ribonucleoprotein fibrils. This smooth and non-nucleosomal character is also predominant in the interspersed, apparently nontranscribed rDNA spacer regions. During inactivation, however, nucleolar chromatin frequently and progressively assumes a beaded appearance in extended fibril-free-that is, apparently nontranscribed - regions. I n either fUll-grown 00- cytes or late after drug treatment, most of the nucleolar chromatin is no longer smooth and thin, but rather shows a beaded configuration indistinguishable from inactive non - nucleolar chromatin. In many chromatin strands, transitions of fibril-associated regions of smooth character into beaded regions wihout lateral fibrils are seen. Similarly, in the non-nucleolar chromatin of the retracting lampbrush chromosome loops, reduced transcriptional activity is correlated with a change from smooth to beaded morphology. Here, however, beaded regions are also commonly found interspersed between the more or less distant bases of the lateral fibrils, the putative transcriptional complexes. I n both sorts of chromatin, detergents (in particular Sarkosyl) that remove most of the chromatin proteins including histones from the DNA axis but leave the RNA polymerases of the transcriptional complexes attached were used to discriminate between polymerases and nucleosomal particles. The results suggest that nucleosomes are absent in heavily transcribed chromatin regions but are reformed after inactivation. In contrast to the findings with inactivated nucleolar genes, in lampbrush chromosome loops the beaded nucleosomal configuration appears to be assumed also in regions within transcriptional units that, perhaps temporarily, are not involved in transcription.
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?
In the inhalation system described an animal can be kept in the same atmosphere of a 2-liter desiccator for up to 24 h. The expired carbon dioxide is adsorbed with soda lime and the resulting reduced pressure is balanced by a supply of oxygen also used for the inflow of the chemical to be investigated. Urine and faeces can be collected ~eparately and the system allows a periodical control of the concentration of the chemical by sampling the air with needle and syringe.
Under the intluence of 5-tluoro-uridine, the ultrastructure of the rDNA transcription units in Xenopus oocytes is altered. Whereas part of the matrix units maintains anormal aspect or shows various degrees of inhibition, in a strong proportion of the transcription units the alternating pattern of matrix units and fibril-free spacer regions is no longer recognized. Transcriptional complexes are found along the entire DNP axis, including the regions of the spacers. These observations support biochemical data on transcription in rDNA spacer region.
Nuclear envelopes of maturing oocytes of various amphibia contain an unusually high number of pore complexes in very close packing. Consequently, nuclear envelopes , which can be manually isolated in great purity, provide a remarkable enrichment of nuclear pore complex material, relative to membranous and other interporous structures. When the polypeptides of nuclear envelopes isolated from oocytes of Xenopl/s la evis and Triturus alpestris are examined by gel electrophoresis, visualized either by staining with Coomassie blue or by radiotluorography after in vitro reaction with [3H]dansyl chloride , a characteristic pattern is obtained (10 major and 15 minor bands). This polypeptide pattern is radically different from that of the nuclear contents isolated from the same cell. Extraction of the nuclear envelope with high salt concentrations and moderateIy ac tive detergents such as Triton X- 100 results in the removal of membrane material but leaves most of the non-membranous structure of the pore complexes. The dry weight of the pore complex (about 0.2 femtograms) remains essentially unchanged during such extractions as measured by quantitative electron microscopy . The extracted preparations which are highly enriched in nuclear pore complex material contain only two major polypeptide components with apparent molecular weights of 150000 and 73000. Components of such an electrophoretic mobility are not present as major bands , if at all , in nuclear contents extracted in the same way. lt is concluded that these two polypeptides are the major constituent protein(s) of the oocyte nuclear pore complex and are specific for this structure. When nuclear envelopes are isolated from rat liver and extracted with high salt buffers and Triton X- 100 similar bands are predominant, but two additional major components of molecular weights of 78000 and 66000 are also recognized. When the rat liver nuclear membranes are further subfractionated material enriched in the 66000 molecular weight component can be separated from the membrane material, indicating that this is relatively loosely associated material , probably a part of the nuclear matrix . The results suggest that the nuclear pore complex is not only a characteristic ubiquitous structure but also contains similar, if not identical , skeletal proteins that are remarkably re sistant to drastic changes of ionic strength as weil as to treatments with detergents and thiol reagents.
Oocytes of the water beetle, Dytiscus marginalis, contain large amounts of rDNA most of which is present in the form of rings containing one or several pre-rRNA genes. Electron microscopy of spread preparations of vitellogenic oocytes has shown that the rDNA is extended in chromatin rings with transcribed pre- rRNA genes and is not packed into nucleosomes (Trendelenburg eta!. , 1976). When similar preparations are made from previtellogenic ooytes in which a large proportion of the nuc1eolar chromatin is transcriptionally inactive, a different morphological form of this chromatin is recognized. In contrast to the transcribed chromatin rings the inactive nucleolar chromatin circles show the characteristic beaded configuration, indicative of nucleosomal packing. Nuc1eosomal packing is also indicated by the comparison of the lengths of these chromatin rings with both iso lated rDNA circ1es and transcribed chromatin rings. In addition, these inactive nuc1eofilaments often appear to be compacted into globular higher order structures of diameters from 21 to 34nm, each composed of an aggregate of 6-9 nuc1eosomes. While the estimated reduction of the overall length of rDNA, as seen in our preparations, is, on the average, only 2.2 - 2.4 fold in the nuc1eosomal state it is 10- 13 fold when supranuc1eosomal globules are present. These data show that the extrachromosomal rDNA of these oocytes goes through a cycle of condensation and extensio n, as a function of the specific transcriptional activity, and that the beaded state described here is exc1usively found in the non-transcribed state.
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.
A chromatographic procedure 1 is described by means of which cytochrome oxidase has been purified from a variety of organisms including the fungus N eurospora crassa,2,3 the unicellular alga Po/ytoma mirum, 4 the insect Locusta migratoria ,5 the frog Xenopus muel/eri,4 and the mammal Rattus norwegicus. 4 This procedure can be used to equal effect for large-scale preparations, starting from grams of mitochondrial protein, or for small-scale preparations starting from milligrams. The cytochrome oxidase preparations from the different organisms are enzymically active. They show similar subunit compositions.
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.
Quantitative anatomical studies on the postnatal development of the cerebellum of the albino rat
(1977)
The quantitative postnatal changes of the cerebella of 65 Wistar rats aged 2-120 days have been examined. The cerebellar volume increases in two phases: The first phase lasts from birth to the seventh postnatal week. The second phase begins ten weeks post parturn and lasts for a Ionger period than the first phase. The cerebellar surface increases continuously from birth to the end of the seventh week. The volume of the external granular layer is maximal when the organ grows rapidly. The external granular layer has nearly disappeared 24 days after birth; the volume of the interaal granular layer is maximal at this time. Later on, the volume and the width of the interaal granular layer decrease. Myelinization of the cerebellar fibers and growth of the molecular layer run parallel to this decrease. The second late, but protracted growth of the cerebellum, ten weeks after birth, is due to an increase of the molecular and medullary layer. These findings are in good accord with histological, histochemical, and ultrastructural observations of other authors.
The organization of the extrachromosomal nucleolar material in oocytes of two insect species with different ovary types, the house cricket Acheta domesticus (panoistic ovary) and the water beetle Dytiscus marginalis (meroistic ovary), was studied with light and electron microscopic techniques. Stages early in oogenesis were compared with fully vitellogenic stages (mid-to-Iate diplotene). The arrangement of the nucleolar material undergoes a marked change from a densely aggregated to a dispersed state. The latter was characterized by high transcriptional activity. In spread and positively stained preparations of isolated nucleolar material, a high frequency of small circular units of transcribed rDNA was observed and rings with small numbers (1-5) of pre-rRNA genes were predominant. The observations suggest that the "extra DNA body" observed in early oogenic stages of both species represents a dense aggregate of numerous short circular units of nucleolar chromatin, with morphological subcomponents identifiable in ultrathin sections. These apparently remain in close association with the chromosomal nucleolar organizer(s). The observations further indicate that the individual small nucleolar subunit circles dissociate and are dispersed as actively transcribed rDNA units later in diplotene. The results are discussed in relation to principles of the ultrastructural organization of nucleoli in other cell types as well as in relation to possible mechanisms of gene amplification.
Antibody against tubulin from porcine brain was used to evaluate the immunological cross reactivity of tubulin from a variety of animal and plant cells. Indirect immunofluorescence microscopy revealed microtubule-containing structures including cytoplasmic microtubules, spindle microtubules, cilia and fIagella. Thus tubulin from diverse species of both mammals and plants show immunological cross-reactivity with tubulin from porcine brain. Results obtained by immunofluorescence microscopy are whenever possible compared with previously known ultrastructural results obtained by electron microscopy.
The minor form of vallne tBNA from baker's yeaat - tRNA\(^{Val}_{2b}\) - purified by column chromatography was completely digesteft with guanylo-BNase and pancreatic ENase. The products of these digestions were separated by a combination of thin-layer chromatography on cellulose and high voltage electrophoresis on DEAE-paper and then identified. The halves of tRNA Val 2b were prepared by partial digestion with pancreatic Mass and their complete guanylo-BNase and pancreatic ENase, digests were analysed. Basing on the obtained data the primary structure of baker1s yeast tRNA\(^{Val}_{2b}\) was reconstructed.
Lengths and patterns of transcriptional units in the amplified nucleoli of oocytes of Xenopus laevis
(1977)
Transcriptionally active chromatin from peripheral amplified nuc1eoli of lampbrush-chromosome stage oocytes of Xenopus laevis was dispersed and spread in various solutions of low salt concentrations (incIuding some with additions of detergents) and examined by electron microscopy. Nucleolar material from oocytes of animals with normal (2-nu) and mutant (I-nu) genetical constitution of nucleolus organizers was compared. Histograms showing the distributions of the lengths of matrix units, apparent spacer intercepts, and the total repeating units of the rDNA containing chromatin axes revealed a significant degree of heterogeneity, with indications of subclasses and predominant repeat unit size c1asses of 3.3 and 3.8 11m length. The correspondence of matrix unit length to the molecular weight of the first stable product of rDNA transcription was studied using gel electrophoresis of labelIed pre-rRNA under non-denaturing and denaturing conditions. Evaluations of individual strands of nucleolar chromatin furt her demonstrated the existence of both (i) strands with obviously homogeneous repeating units and (ii) strands with intra-axial heterogeneity of rDNA subunits. " Preludecomplexes ", i.e. groups of transcriptional complexes in apparent spacer intercepts, were not infrequently noted. The data are compared with the measurements of lengths of repeating units in fragments of rDNA obtained by digestion with EcoRI endonuclease as described by Morrow et al. (1974) and Wellauer et al. (1974, 1976a, b). The results are discussed in relation to problems of variations in the modes of arrangement of the pre-rRNA genes, the state of packing of rDNA during transcription, and possible mechanisms of the amplification process.
We have studied the lowest magnetic excitation of Ni\(_{1-x}\)Co\(_x\)O mixed crystals for 0.94 \(\leq\) x \(\leq\) 1. Together with previous results for 0.02 \(\leq\) x \(\leq\) 0.07 and neutron data for x = 0.14 and x = 0.30, the results are discussed by means of a model, especially the variation of AFMR frequency and preferred spin direction with Co concentration x.
Silylation of cellulose
(1977)
Ethane-l:2-diol and propane-l:3-diol reaet with 1: 1:3:3-tetramethyl-l:3-dichlorodisiloxane forming the corresponding rings. However, no ring compounds could be traced tbrough the reaction between butane-l :4-diol, glycerol and the dichlorodisiloxane respectively, where only polymeric compounds are formed. The silylation products of the di- and trihydroxy alcohols, as model compounds, has confirmed that the ring formation during silylation of cellulose with dichlorodisiloxane is uncertain.
A video protocolling and retrieval system for the analysis of behaviour. Rec~nt developments in the data processing and video technologies have enabled the desIgn of automatic systems for the handling of video software. The system for the protocoIling and retrieval of audiovisual data (PRA VDA) described here has been developed for applications in behavioural observation and analysis. The facilities available, including time coding, search routines and automatic editing are explained and the advantages of computer aided operation are dealt with. Current applications of the system are briefly discussed.
The arrangement of genes of precursor molecules for ribosomal RNA (pre-rRNA) in primary nuclei from two green algae species, Acetabularia mediterranea and A. major, has been analyzed in an electron microscope study. The pattern of transcriptional units in individual strands of nucleolar chromatin was investigated using spread and positively stained preparations. The rDNA pattern is not uniform but differs in different strands. The predominant type of nucleolar chromatin exhibits a high degree of homogeneity in the sequence of matrix units (intercepts covered with fibrilst hat contain the pre-rRNA) and fibril-free spacer intercepts. Substantial differences, however, are observed between the patterns in different strands. In addition, there is evidence in some strands for intraaxial heterogeneity of both spacer and matrix units. The following major types can be distinguished: type la, ca. 2 micrometer long matrix units, extremely short spacer intercepts in A. mediterranea (ca. 1 micrometer long ones in A. major), completely homogeneous distribution; type Ib, as type la but with intercalated, isolated, significantly shorter and/or longer matrix units; type lIa, matrix unit sizes as in type la, but much longer spacer intercepts, high degree of homogeneity; type Ill, largely heterogeneous arrangements of matrix and spacer units of varying sizes. The matrix unit data are compared with the sizes of pre-rRNA as determined by polyacrylamide gelelectrophoresis under denaturing and non-denaturing conditions. The findings are discussed in relation to recent observations in amphibia and insects and with respect to current concepts of the species-specificity of rDNA arrangements.
The arrangement of transcriptional units in the loops of lampbrush chromosomes from oocyte nuclei of urodele amphibia and from primary nuclei of the green alga Acetabularia have been studied in the electron microscope using spread preparations. Loops with different patterns of arrangement of matrix units (i.e. to a first approximation, transcriptional units) can be distinguished: (i) loops consisting of one active transcriptional unit; (ii) loops containing one active transcriptional unit plus additional fibril-free, i.e. apparently untranscribed, intercepts that may include 'spacer' regions; (iii) loops containing two or more transcriptional units arranged in identical or changing polarities, with or without interspersed apparent spacer regions. Morphological details of the transcriptional complexes are described. The observations are not compatible with the concept that one loop reflects one and only one transcriptional unit but, rather, lead to a classification of loop types according to the arrangement of their transcriptional units. We propose that the lampbrush chromosome loop can represent a unit for the coordinate transcription of either one gene or a set of several (different) genes.
Natural changes in the transcription of rRNA genes were studied in nucleoli from three oogenic stages of the newt Triturus alpestris with electron microscope, autoradiographic, and biochemical techniques. From determinations of the uridine triphosphate pool sizes and [3H]uridine uptake, phosphorylation, and incorporation into 28S and 18S rRNAs in vivo it was estimated that the rate of rRNA synthesis was about 0.01% in previtellogenic oocytes and 13% in mature oocytes when compared to midvitellogenesis. Spread preparations of nucleoli showed significant morphological changes in the transcriptional complexes. The total number of lateral fibrils, i.e., ribonucleoproteins containing the nascent rRNA precursor, were drastically decreased in stages of reduced synthetic activity. This indicates that rRNA synthesis is regulated primarily at the level of transcription. The resulting patterns of fibril coverage of the nucleolar chromatin axes revealed a marked heterogeneity. On the same nucleolar axis occurred matrix units that were completely devoid of lateral fibrils, matrix units that were almost fully covered with lateral fibrils, and various forms of matrix units with a range of lateral fibril densities intermediate between the two extremes. Granular particles that were tentatively identified as RNA polymerase molecules were not restricted to the transcription l complexes. They were observed, although less regularly and separated by greater distances, in untranscribed spacer regions as well as in untranscribed gene intercepts. The results show that the pattern of transcriptional control of rRNA genes differs widely in different genes, even in the same genetic unit.
Scheer, Ulrich ; Kartenbeck, Jürgen ; Trendelenburg, Michael F. ; Stadler, Joachim ; Franke, Werner W.
The disintegration of the nuclear envelope has been examined in nuclei and nuclear envelopes isolated from amphibian oocytes and rat liver tissue, using different electron microscope techniques (ultrathin sections and negatively or positively stained spread preparations). Various treatments were studied, including disruption by surface tension forces, very low salt concentrations, and non ionic detergents such as Triton X-lOO and Nonidet P-40. The high local stability of the cylinders of nonmembranous pore complex material is emphasized. As progressive disintegration occurred in the membrane regions, a network of fibrils became apparent which interconnects the pore complexes and is distinguished from the pore complexassociated intranuclear fibrils. This network might correspond to an indistinct lamella, about 15 - 20 nm thick, located at the level of the inner nuclear membrane, which is recognized in thin sections to bridge the interpore distances. With all disintegration treatments a somewhat higher susceptibility of the outer nuclear membrane is notable, but a selective removal does not take place. Final stages of disintegration are generally characterized by the absence of identifiable, membrane- like structures. Analysis of detergent-treated nuclei and nuclear membrane fractions shows almost complete absence of lipid components but retention of significant amount of glycoproteins with a typical endomembrane-type carbohydrate pattern. Various alternative interpretations of these observations are discussed. From the present observations and those of Aaronson and Blobel (1,2), we favor the notion that threadlike intrinsic membrane components are stabilized by their attachment to the pore complexes, and perhaps also to peripheral nuclear structures, and constitute a detergent-resistant, interpore skeleton meshwork.
Several types of "irregular" structures in the arrangement of lateral fibrils were noted in electron microscopic preparations of transcriptionally active nucleolar chromatin from various plant and animal cells. Such forms include: I. Disproportionately long lateral fibrils which occur either as individual fibrils or in groups; 2. "Prelude complexes" and other arrangements of lateral fibrils in apparent spacer intercepts; 3. Thickening of the rDNA chromatin axis at the starting end of pre-rRNA matrix units; 4. Extremely long matrix units , the length of which exceeds that of the rDNA (double-strand) sequence complementary to the specific pre-rRN A (for abbreviations see text). In addition, the stability of high molecular weight RNAs contained in the nucleolar ribonucleoproteins during the preparation for electron microscopy was demonstrated by gel electrophoresis. The observations indicate that the morphological starting point of a pre-rRNA matrix unit is not necessarily identical with the initiation site for synthesis of pre-rRNA, but they rather suggest that the start of the transcriptional unit is located at least O.2-D.8 JLm before the matrix unit and that parts of the "apparent spacer" are transcribed. It is proposed that the pre-rRN A molecules do not represent the primary product of rDNA transcription but rather relatively stable intermediate products that have already been processed during transcription.
The photoconductivity of both undeformed and deformed Te samples has been investigated at liquid He temperature by means of a Fourier spectrometer. Three peaks were usually found in the spectra of undeformed samples at 11, 24 and 46 cm\(^{-1}\). These are shown to be due to three different chemical impurities. The deformed samples are characterized by additional structure at higher frequencies.
Franke, Werner W. ; Scheer, Ulrich ; Trendelenburg, Michael F. ; Spring, Herbert ; Zentgraf, Hanswalter
The ultrastructure of twO kinds of transcription ally active chromatin, the lampbrush chromosome loops and the nucleoli from amphibian oocytes and primary nuclei of the green alga Acetabularia, has been examined after manual isolation and dispersion in low salt media of slightly alkaline pH using various electron microscopic staining techniques (positive staining, metal shadowing, negative staining, preparation on positively charged films, etc.) and compared with the appearance of chromatin from various somatic cells (hen erythrocytes, rat hepatocytes, ClIltured murine sarcoma cells) prepared in parallel. While typical nucleosomes were revealed with all the techniques for chromatin from the latter three cell system, no nucleosomes were identified in either the lampbrush chromosome structures or the nucleolar chromatin. Nucleosomal arrays were absent not only in maximally fibril-covered matrix units but also in fibril-free regions between transcriptional complexes, including the apparent spacer intercepts between different transcriptional units. Moreover, comparisons of the length of the repeating units of rDNA in the transcribed state with those determined in the isolated rDNA and with the lengths of the first stable product of rDNA transcription, the pre-rRNA, demonstrated that the transcribed rDNA was not significantly shortened and/or condensed but rather extended in the transcriptional units. Distinct granules of about nucleosomal size which were sometimes found in apparent spacer regions as well as within matrix units of reduced fibril density were shown not to represent nucleosomes since their number per spacer unit was not inversely correlated with the length of the specific unit and also on the basis of their resistance to treatment with the detergent Sarkosyl NL-30. It is possible to structurally distinguish between transcriptionally active chromatin in which the DNA is extended in a non-nucleosomal form of chromatin and condensed, inactive chromatin within the typical nucleosomal package. The characteristic extended structure of transcriptionally active chromatin is found not only in the transcribed genes but also in non-transcribed regions within or between ("spacer") transcriptional units as well as in transcriptional units that are untranscribed amidst transcribed ones and/or have been inactivated for relatively short time. It is hypothesized that activation of transcription involves a transition from a nucleosomal to an extended chromatin organisation and that this structural transition is not specific for single "activated" genes but may involve larger chromatin regions, including adjacent untranscribed intercepts.
Soluble mitochondrial ATPase (F1) isolated from Neurospora crassa is resolved by dodecylsulfate- gel electrophoresis into five polypeptide bands with apparent molecular weights of 59000, 55000, 36000, 15000 and 12000. At least nine further polypeptides remain associated with ATPase after disintegration of mitochondria with Triton X-100 as shown by the analysis of an immunoprecipitate obtained with antiserum to F 1 A TPase. Two of the associated polypeptides with apparent molecular weights of 19000 and 11000 are translated on mitochondrial ribosomes, as demonstrated by incorporation in vivo of radioactive leueine in the presence of specific inhibitors of mitochondrial (chloramphenicol) and extramitochondrial ( cycloheximide) protein synthesis. The appearance of mitochondrial translation products in the immunoprecipitated A TPase complex is inhibited by' cycloheximide. The same applies for some of the extramitochondrial translation products in the presence of chloramphenicol. This suggests that both types of polypeptides are necessary for the assembly of the A TPase complex.
Via reduction of benzvalene (1) with diirnine tricyclo[3.1.0.02•6]hexane is obtained in good yield. The procedure renders 3, which has already been synthesized by Lemal and Shim, accessible much easier and in larger quantities. IH and 13C n.m.r. spectroscopic data are discussed. Both the thermal and the AgBF4-catalyzed rearrangernent of 3 yield 1,3-cyclohexadiene (8). - The ozonolysis of 1 with subsequent LiAIH4-reduction results in cis-I,3- bis(hydroxyrnethyl)cyclobutane (13a).
Incubation of the colicinogenic Escherichia coli strain JC 411 (ColE1) at elevated temperatures (47-49°) leads to the accumulation of catenated molecules and replicative intermediates of this plasmid. Mature supercoiled OolE1 DNA molecules synthesized under these conditions have an increased number of tertiary turns as shown by electron microscopy. The monomeric tightly supercoiled molecules possess a slightly slower sedimentation rate and a higher binding capacity for ethidium bromide than supercoiJed monomers synthesized at lower temperatures. Recombination deficient mutants of E. coli recA, recB and recC, which carry the ColE1 plasmid, form about the same amount of catenated molecules at the elevated temperature as a rec+ strain. In addition, we have observed by electron microscopy a small percentage (.--.5% of the circular DNA molecules) of minicircular DNA molecules in all preparations of JC 411 (CoIE1). They are homogenous in size, with a molecular weight of 1.4 X 106 daltons. Addition of chloramphenicol to a culture of Proteus mirabilis (ColE1) leads to an increased amount of higher multiple circular oligomers and to a stimulated accumulation of catenated OolE1 DNA molecules of varying sizes. ColE1 DNA synthesis is more thermosensitive than chromosomal DNA replication in P. mirabili8. Plasmid replication stops completely at temperatures above 43°C.
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.
Franke, Werner W. ; Berger, S. ; Falk, Heinz ; Spring, H. ; Scheer, Ulrich ; Trendelenburg, Michael F. ; Schweiger, H. G. ; Herth, W.
The ultrastructure of th e growin g and ma turing primary nucleus of Acetabularia medite rranea and Acetabularia major has been studied with the use of various fi xation procedures. Particular interest has been focused on the deta ils of the nuclear periphery and the perinuclear region. It is demonstrated that early in nuclear grow th a characteristic perinucl ear structura l complex is formed which is, among the eukaryotic cells, unique to Acetabularia and re lated genera. This perinuclear system consists essentially of a) the nuclear envelope with a very hi gh pore frequency and various pore complex assoc iat ion s w ith granular and/or threadlike structures some of which are continuous with the nucleolus; b) an approx imate ly 100 nm thick intermediate zone densely filled with a filam entOus material and occasional sma ll membraneous structures from which the typical cytOplasmic and nuclear organe lles and particles are excl ud ed ; c) an adjacent Iacunar labyrinthum which is interrupted by many plasmatic junction channels between the intermed iate zone and the free cytOplasm; d) numerous dense perinuclear bodies in the juxtanuclear cytOplasm which a re especia lly frequent at the junction channels and reveal a composition of aggregated fibrillar and granul ar structures; e) very dense exclusively fibrill ar agg regates which occur either in assoc iation with t he perinuclear region of the lacunar labyrinthum or, somewhat further out, in the cytOplasmic strands between the bra nches of the lacun ar labyrinthum in the form of slender, characteristic rods or "sausages".
The present article had originally been conceived as a review on endomembranes, the plasma membrane, and the major product of membrane-bound activities, the cell wall material. However, limitations of space and the cascading number of pertinent literature articles made it necessary to confine this to one group of membranes and one type of cell wall components. Therefore, we shall begin our survey on the biochemical and cytological aspects of membranes by a review of the class of the pore complex bearing endomembranes, i.e. the nuclear envelope and the annulate lamellae (AL). Next year the membranes of the endoplasmic reticulum and the dictyosomes will be dealt with in conjunction with a discussion of the various intracellular vesicles, the tonoplast and the plasmalemma.
The preparation of Bi2Sea single crystals with low free carrier densities allowed an investigation of the lattice vibrations to be carried out from the reflectivity of cleavage planes at nearly normal incidence of the radiation (E ~ c). The experimental results ean be explained with two classical oscillators, whose eigenfrequencies oceur at 92 and 69.5 cm-I. The static dielectric constant was determined to be 100 ± 10 for E ~ c.
Primary (giant) nuclei of the green algae Acetabularia mediterranea and A. major were studied by light and electron microscopy using in situ fixed material as well as manually isolated nuclear components. In addition, cytochemical reactions of nuclear structures and biochemical determinations of nuclear and cytoplasmic RNA and of genome DNA content were performed. The data obtained and the structures observed are interpreted as demonstralions of transcriptional activities of different gene classes. The most prominent class is the nucleolar cistrons of precursors of ribosomal RNA which occur highly repeated in clusters in the form of regularly alternating intercepts on deoxyribonucleoprotein axes of transcribed rDNA, the fibril-covered matrix units, and the fibril-free "spacer" segments. A description and a classification of the various structural complexes which seem to represent transcriptional activities is given. Quantitative evaluations of these arrangements are presented. The morphology and the dimensions of such structures are compared with the RNA molecular weight determinations and with the corresponding data reported from various animal cell systems. It is suggested that the formation of the giant nucleus is correlated with, and probably due to, an enormous amplification of transcriptionally active rDNA and packing of the extrachromosomal copies into the large nucleolar aggregate bodies.
no abstract available
Cytochrome oxidase isolated from N eurospora crassa was resolved into seven protein eomponents by eleetrophoresis in polyaerylamide gels eontaining sodium dodeeylsulfate. The apparent molecular weights were determined tobe 41000, 28500, 21000, 16000, 14000, 11500 and 10000 for the eomponents 1, 2, 3, 4, 5, 6, and 7, respectively. The components 1, 2 and 3 are synthesized on mitochondrial ribosomes as shown by the incorporation of radioactive amino aeids in the presenee of cyeloheximide. Amino-acidanalysis of the isolated components 1, 2 and 3 revealed a high content of apolar amino acids and a low eontent of basic amino aeids compared to an average amino-aeid eomposition of components 4-7. Components 1, 2 and 3 eontribute 27.9°/0, 18°/0 and 14.2°/0 to the whole eytoehrome oxidase protein. This was calculated from the contributions of the single eomponents to the totalleueine eontent of the enzyme and the leueine eontents (nmol leueine per mg protein) of the single eomponents as determined by amino-aeid analysis. Equimolar relations of the components 1, 2 and 3 are found by dividing the amounts of protein by their apparent molecular weights. A stoichiometry of 1:1:1 results assuming a minimal molecular weight of 150000 for the whole cytochrome oxidase protein. On the basis of the heme a content a molecular weight of about 70000 per heme group was determined, using an absorption coeffieient L1e605 (redueed minus oxidized) of 12 mM-1 cm-1• It is concluded that the smallest structural unit of eytochrome oxidase contains two heme groups.
A cytochrome b preparation from Neurospora crassa mitochondria is found to consist of three polypeptides (apparent molecular weight 10 000, 11 000 and 32 000), a cytochrome aa3 preparation of six to seven polypeptides (apparent molecular weight 8 000, 11 000, 13 000, 18 000, 28 000 and 36 000). Selective incorporation of radioactive amino acids by eilher mitochondrial protein synthesis when the cytoplasmic one is blocked or by the cytoplasmic protein synthesis, when the mitochondrial one is blocked, indicates that one cytochrome b polypeptide (mw 32 000) and one to three cytochrome aa3 polypeptides (mw 36 000, 28 000 and 18 000) are mitochondrial translation products, the other cytochrome b and cytochrome aa3 polypeptides cytoplasmic translation products. The delayed appearance of labeling in the cytochrome b and cytochrome aa3 polypeptides compared to the average cell protein after a pulse of <~H leueine revealed that these polypeptides are derived from separate pools of precursor polypeptides. The pool sizes range from 2 p. cent to 25 p. cent of the amount of the corresponding polypeptide present in the cytochromes. The 32 000 molecular weight polypeptide of cytochrome band at least the 18 000 molecular weight polypeptide of cytochrome aa\(_3\) are mitochondrial translation products as well in the fungus Neurospora crassa as in the insect Locusta migratoria. So, despite the fact that the size of mitochondrial DNA and mitochondrial ribosomes is reduced in insects, the products have maintained their characteristics.
Segregation of the nucleolar components is described in the differentiated nucleus of the generative cell in the growing Clivia and Lilium pollen tubes. This finding of a natural nucleolar segregation is discussed against the background of current views of the correlations of nucleolar morphology and transcriptional activity.
Electron microscopic spread preparations of oocyte nucleoli (lampbrush stage) of various amphibians are quantitatively evaluated and the length distributions of repeat-, matrix-, and spacer-units along the rRNA cistron containing axes are given. The correlation of the matrix unit data with the gel electrophoretic pattern of labelled nuclear RNA from the same oocytes is examined. The mean value of the matrix unit corresponds fairly well to a 2.6 million D peak of pre-rRNA but the distribution of both matrix units and labelled pre-rRNAs shows an asymmetrical heterogeneity indicating the existence of some larger primary transcription products of rDNA. Novel structural aspects are described in the spacer regions which suggest that transcription does also take place in DNP regions between the matrix units. A special "prelude piece" coding for approx. 0.5 million D of RNA is frequently visualized in the spacer segments at the beginning of a matrix unit. Possible artifacts resulting from the preparation, the relative congruence between the data obtained using both methods, and the functional meaning of the findings are discussed against the background of current concepts of structural organization and transcription products of nucleolar DNA.
The number of ribosomal RNA molecules which are transferred through an average nuclear pore complex per minute into the cytoplasm (nuclear pore flow rate, NPFR) during oocyte growth of Xenopus laevis is estimated. The NPFR calculations are based on determinations of the increase of cytoplasmic rRNA content during defined time intervals and of the total number of pore complexes in the respective oogenesis stages. In the mid-la mpbrush stage (500:"700 I'm oocyte diameter) the NPFR is maximal with 2.62 rRNA molecules/ pore/ minute. Then it decreases to zero at the end of oogenesis. The nucleocytoplasmic RNA f10w rates determined are compared with corresponding values of other cell types. The molecular weight of the rRNA precursor transcribed in the extrachromosomal nucleoli of Xenopus lampbrush stage oocytes is determined by acrylamide gel electrophoresis to be 2.5 x 10· daltons. From the temporal increase of cytoplasmic rRNA (3.8 I'g per oocyte in 38 days) and the known number of simultaneously growing precursor molecules in the nucleus the chain growth rate of the 40 S precursor RNA is estimated to be 34 nucleotides per second.
Electron-opaque material is shown in the perinuclear cisternae of various cell types to connect the inner and outer nuclear membrane faces. Similar bridges were observed between the outer nuclear membrane and the outer mitochondrial membrane. The intracisternal bridges of the nuclear envelope appear to be important for the structural stability of the perinuclear cisterna. Stable structural linkage of mitochondria to the outer nuclear membrane might be relevant to the understanding of the characteristic juxtanuclear accumulation of mitochondria and also provide arguments for the discussions of certain biochemical activities found in nuclear and nuclear membrane fractions.
Different pool sizes of the precursor polypeptides of cytochrome oxidase from Neurospora crassa.
(1972)
Pulse-labelling experiments with growing Neurospora crassa revealed that the polypeptides composing the protein moiety of a cytochrome oxidase preparation are derived from at least four independent pools of precursor polypeptides. The pool sizes range from 2 ° f 0 to 25 °/0 of the amount of the corresponding polypeptide present in cytochrome oxidase. The smallest pool is assigned to a polypeptide of mitochondrial origm. Serial pools were found for one of the polypeptides.
Cytochrome oxidasewas prepared from Neurospora crassa by chromatography on oleyl polymethacrylic acid resin and separated into seven polypeptides by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Incorporation oflabelled amino acids into the single polypeptideswas investigated after a pulse labelling in the absence and presence of chloramphenicol, and afterwashing out the inhibitor. Chloramphenicol (4 mg/ml) inhibited amino acid incorporation into all polypeptides 90-95%• while labeHing of the whole membrane protein was inhibited only 30%• Mter washing out the inhibitor and further growth of the cells. the four smaller polypeptides were highly labelled, whereas the other polypeptides showed only a. small increase in radioactivity. It is concluded that the four small-sized polypeptides of cytochrome oxidase are synthesized but not integrated into the functional enzyme under the action of chloramphenicol.
The 130 chemical shifts were determined of the carbons in 12 cycloheptanes, 21 cycloheptanols, and 8 cycloheptanones. In some cyc1oheptanols and cyc1oheptanones, the assignments have been obtained unambiguously by the synthesis of deuterated derivatives and the use of paramagnetic-shift reagents. Substituent effects for the different types of groups have been calculated. The most informative data about the cyc10heptane conformations were provided by the relatively well understood I' effects. The results are generally in,good agreement with predictions based on the twist-chair form, which has been predicted by Hendrickson to be the most stable conformation. Pairs of cis-trans isomers are found to have rather characteristic differences in their 130 spectra. This fact was used to assign the resonances found for cis-trans mixtures of methyl-substituted cyc1oheptanols to specific isomers.
The occurrence of stacked annulate tamellae is documented for a plant cell system, namely for pollen mother cells and developing pollen grains of Canna generalis. Their structural subarchiteeture and relationship to endoplasmie reticulum (ER) and nuclear envelope cisternae is described in detail. The results demonstrate structural homology between plant and animal annulate lamellae and are compatible with, though do not prove, the view that annulate lamcllar cisternae may originate as a degenerative form of endoplasmic retieulum.
Structural details of the dictyosomal pores in several plant cell types are described from tangential and cross sections of Golgi cisternae. Frequency distributions of the sizes of such Golgi pores are given and compared with the corresponding values of nuclear pores in the same cells. Golgi pore inner diameters are less homogeneously distributed and can be as small as 100 A or less. They are not simply cisterna I holes, but are often associated with centrally located electron dense granules or rods and with inner pore filaments. This organization, which is very common in dictyosomal pores in plant and animal cells, has some similarities with the structural architecture of nuclear envelope and annulate lamellar pore complexes. The particulate material associated with the dictyosomal pores shows spatial and structural relationship to cytoplasmic ribosomes. Possible modes of Golgi pore formation and some consequences of these observations for interpretation of nuclear pore structures are discussed.