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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.
No abstract available
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.
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 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.
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
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.
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.
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 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.
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.
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.
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 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.
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.
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.
Incubation of mitochondria from Neuraspara crassa and Saccharomyces cerevisiae with the radioactive ATPase inhibitor [14C]dicyclohexylcarbodiimide results in the irreversible and rather specific labelling of a low-molecular-weight polypeptide. This dicyclohexylcarbodiimide-binding protein is identical with the smallest subunit (Mr 8000) of the mitochondrial ATPase complex, and it occurs as oligomer, probably as hexamer, in the enzyme protein. The dicyclohexylcarbodiimide-binding protein is extracted from whole mitochondria with neutral chloroformjmethanol both in the free and in the inhibitor-modified form. In Neuraspara and yeast, this extraction is highly selective and the protein is obtained in homogeneaus form when the mitochondria have been prewashed with certain organic solvents. The bound dicyclohexylcarbodiimide Iabel is enriched in the purified protein up to 50-fold compared to whole mitochondria. Based on the amino acid analysis, the dicyclohexylcarbodiimide-binding protein from Neurospora and yeast consists of at least 81 and 76 residues, respectively. The content of hydrophobic residues is extremely high. Histidine and tryptophan are absent. The N-terminal ~mino acid is tyrosine in Neuraspara and formylmethionine in yeast.
The desert isopod, Hemilepistus reaumuri, extremely common in the arid regions of North Africa and Asia Minor, depends upon the burrows it itself digs for survival during the hotter parts of the year. The dig-ging of new burrows is limited by chmatic conditions to a short period during the spring. Burrows must be constantly defendet - especially against roving eonspecifics. The decisive problem of a connnuous burrow defense is solved through cooperative behavior: the adult woodlice form monogamous pairs whose partners recognize one another individually. Here, questions on the binding of partners, especially the problem of the binding of male to female will be treated upon, along with questions on the evolution of monogamy, wherein the purely maternal families of Porcellio species will be taken as models for intermediäre stages. At first, males olHemilepistus are not permitted to copulate at all; later, for a relatively long period, they are only permitted incomplete copulations, the females alone have control over the partunal ecdysis; they alone determine the moment of final copulations. Under the thermal conditions prevalent during the season of pair formation, a female irreversibly induces a parturial ecdysis only when it has spent a minimum of sev-eral days in her own burrow with a specific male. At higher average temperatures, the number of females which undergo parturial ecdyses without these preconditions increases sharply. Males cannot greatly lnrlu-ence the willingness of females to reproduce with the investment they make in the digging of burrows; the factors deciding this are the male's presence and its role as guard. The first condition necessary for the genesis of monogamy might have been the evolution of a stncüy lo-cation-dependent copulatory behavior, which guaranteed the male exclusive mating pnveliges with the female whose location - the burrow - he acheived control of. A male must, under these conditions, serve guard duty in his own interest, and defend the burrow against competitors (Cf or 2) seeking an already-dug burrow. The decisive advantage for the female in the beginning of the development was probably that she could leave the burrow for extended feeding excursions, whereas alone it would have to either completely forego nourishment or, as is the case with the Porcellio species mentioned, must greatly restrict the spectrum of food that it can use (to that which is to be found only a short distance from the burrow and which can eas-ily be carried inside the burrow). This could be a disadvantage, especially during egg production. Necessary to the male's successful defense of the burrow is that he recognises his female. Studies of the Canary Island Porcellio species have shown over which pathways and under what selection pressures the recopinon of individuals, as is realized mHemilepistus, could have evolved. Females can bind males longer, the longer the period of their attraction is extended: Females olHemilepistus reaumuri have been proven to be al·ready att-ractive before they are ready to copulate and still remain attractive after they have copulated. The conse-quences of the last fact will be discussed. The question of why the males remain with the females after the parturial ecdysis will also be discussed: The great danger to the male's investment resulting from a tooi early abandoning, and the low probability of successfully finding another partner after a later abandomng should prevent a positive balance in the males' cost-effecriveness calculations.
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.
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
The amino acid sequence of the proteolipid subunit of the A TP synthase was analyzed in six mutant strains from Escherichia coli K 12, selected for their increased resistance towards the inhibitor N,N'-dicyclohexylcarbodiimide. All six inhibitor-resistant mutants were found to be altered at the same position of the proteolipid, namely at the isoleucine at residue 28. Two substitutions could be identified. In type I this residue was substituted by a valine resulting in a moderate decrease in sensitivity to dicyclohexylcarbodiimide. Type II contained a threonine residue at this position. Here a strong resistance was observed. These two amino acid substitutions did not influence functional properties of the ATPase complex. ATPase as well as A TP-dependent proton-translocating activities of mutant membranes were indistinguishable from the wild type. At elevated concentrations, dicyclohexylcarbodiimide still bound specifically to the aspartic acid at residue 61 of the mutant proteolipid as in the wild type, and thereby inhibited the activity of the ATPase complex. It is suggested that the residue 28 substituted in the resistant mutants interacts with dicyclohexylcarbodiimide during the reactions leading to the covalent attachment of the inhibitor to the aspartic acid at residue 61. This could indicate that these two residues are in close vicinity and would thus provide a first hint on the functional conformation of the proteolipid. Its polypeptide chain would have to fold back to bring together these two residues separated by a segment of 32 residues.
When reovirus-infected Hela cells are incubated at 43°C virus-specific messenger RNA is released ~rom the polysomes. It accumulates free in the cytoplasm as messenger ribonucleoprotem partIcles (mRNPs). The:e part~cles have a sedimentati~n rate of about 50S and a buoyant densIty m CsCI of 1.42 g/cm . ReovIrus mRNPs contam, beSIdes all three size classes of reovirus messenger RNA, the same spectrum of proteins found in the polysomal mRNPs from uninfected cells, plus t~o addi~ional pr?teins with molecular masses of 7000~ d and 110000 d, respectively. Electron mIcroscoPIc exammatlOn of the reovIrus mRNP fractIOn reveals specific Y-shaped structures wIth a total mean length ofO.5Ilm.
The assembly of DNA into nucleosomal and supranucleosomal chromatin structures has been studied (i) by injection of circular DNA molecules (plasmids) into nuclei of Pleurodeles waltlii oocytes; and (ii) by in vitro incubation of plasmid molecules with the supernatant fraction from oocyte nuclei of Pleurodeles and Xenopus laevis, followed by purification of nucleoprotein structures formed with sucrose gradient centrifugation. [n both types of experiments , spread preparations of the newly assembled and transcriptionally inactive chromatin , examined by electron microscopy , show dense globular higher order (supranucleosomal) packing forms. Under partially relaxing (low salt) preparation conditions granular chromatin subunits of about 30 nm diameter can be seen either as widely spaced particles or in closely packed aggregates. The transcriptionally inactive endogenous chromatin of chromomeres of lampbrush chromosomes is arranged in similar higher order chromatin units. A correlation is found between the sizes of the DN A molecule probes used and the numbers of nucleosomes and higher order globules in the assembled chromatin structures. After prolonged dispersion in low salt buffers , these globular chromatin units unfold into chains of7-12 nucleosomes. The results support the concept that chromatin is arranged , under physiological ion concentrations as they are present in the nucleus , in supranucleosomal units of globular morphology.
Transcribed nucleolar chomatin, including the spacer regions interspersed between the rRNA genes, is different from the bulk of nontranscribed chromatin in that the DNA of these regions appears to be in an extended (B) conformation when examined by electron microscopy. The possibility that this may reflect artificial unfolding of nucleosomes during incubation in very low salt buffers as routinely used in such spread preparations has been examined by studying the influence of various ion concentrations on nucleolar chromatin structure. Amplified nucleolar chromatin of amphibian oocytes (Xenopus laevis, Pleurodeles waltlii, Triturus cristatus) was spread in various concentrations of NaCl (range 0 to 20 mM). Below 1 mM salt spacer chromatin frequently revealed a variable number of irregularly shaped beads, whereas above this concentration the chromatin axis appeared uniformly smooth. At all salt concentrations studied, however, the length distribution of spacer and gene regions was identical. Preparations fixed with glutaraldehyde instead of formaldehyde, or unftxed preparations, were indistinguishable in this respect. The observations indicate that (i) rDNA spacer regions are not compacted into nucleosomal particles and into supranucleosomal structures when visualized at chromatin stabilizing salt concentrations (e.g., 20 mM NaCl), and (ii) spacer DNA is covered by a uniform layer of proteins of unknown nature which, at very low salt concentrations (below 1 mM NaCl), can artificially give rise to the appearance of small granular particles of approximately nucleosome-like sizes. These particles, however, are different from nucleosomes in that they do not foreshorten the associated spacer DNA. The data support the concept of an altered nucleohistone conformation not only in transcribed chromatin but also in the vicinity of transcriptional events.
b-Type cytochromes
(1980)
T~e N,N'-dicrclohexylcarbodiimide-binding proteolipid subumt of the mitochondrial adenosinetriphosphatases (ATP phosphohydrolase, EC 3.6.1.3) of Neurosporacrassa and Saccharomyces cerevisiae were purified from mitochondria incubated with the radioactively labeled inhibitor. The specifically labeled subunit was cleaved with cyanogen bromide and N-bromosuccinimide, and the resultant fragments were separated by gel chromatography in the presence of 80% (vol/vol) formic acid. The N,N'-dicyclohexylcarbodiimide label was recovered in each organism exclusively in a 17-residue fragment. Further analysis by automated solid-phase Edman degrada.ti.on revealed tha~ the bound label was present at only one positIOn, correspondmg to a glutamyl residue. The NN'~ icyc~ohexyl~a~bodiiJ?1~de-'!l0dified glutamyl residue is the ~nly Id~ntIcal aCidic posItIon m both proteins and occurs in the middle of a hydrophobic sequence of about 25 residues.
Nonnucleolar chromatin from interphase nuclei of Physarum polycephalum plasmodia occurs in two different structural configurations as seen in electron microscopic spread preparations. While the majority of the chromatin is devoid of nascent ribonucleoprotein (RNP) fibrils and compacted into nucleosomal particles, a minor proportion (10- 20%) is organized differently and reveals a smooth contour. It is this form of smooth chromatin which is rich in transcription units (mean length: 1.36±0.21 11m). Only occasionally are solitary nascent RNP fibrils observed which are associated with beaded strands of chromatin. In transcribed smooth chromatin nucleosomal particles are not only absent from the transcription units but also from their nontranscribed flan king regions, indicating that this special structural aspect is not merely a direct consequence of the transcriptional process. The existence of ca. 10- 20% of Physarum chromatin in the smoothly contoured form is discussed in relation to reports of a preferential digestibility of a similar proportion of Physarum chromatin by DNAse I (Jalouzot et al. , 1980) and to the altered configuration of "peak A" chromatin subunits after micrococcal nuclease digestion (Johnson et al., 1978a, b).
Comparisons ofrelative lengths oflampbrush loops, nascent RNP transcripts and hnRNA molecules from oocytes of amphibia with different C-values show that there is an increasing trend in loop, and transcriptional unit, length with increase in genome size but no increasing trend with respect to RN A contour length.The formation of duplex regions and circles in RNP fibrils indicates that RNA processing may occur within the nascent fibrils. The hnRNA molecules from oocytes of the various amphibia readily form intermolecular duplex structures. These complementary sequences have a low kinetic complexity and are transcribed from highly repetitive sequences distributed throughout the genome. Their possible function is considered.
Antibodies directed against RNA polymerase II (B) from Drosophila melanogaster were obtained from rabbit sera and, as monoclonal immunoglobulins, from mouse hybridomas and shown to cross-react with the amphibian enzyme protein. Localization by indirect immunofluorescence microscopy revealed the association of this enzyme with chromatin of interphase nuclei of amphibian cells and its absence in nucleoli. Purified immunoglobulins were microinjected in to nuclei ofliving vitellogenic oocytes of Ple1lrodeles waltlii and X enopus laevis and their effects on transcriptional processes were monitored by biochemical and light and electron microscopic stud ies. RNA polymerase II antibodies from rabbit sera caused a rapid and almost complete release of nascent transcripts from the chromatin axis of the loops of lampbrush chromosomes, followed by collapse of the loops and their retraction on the main chromosome axis. Monoclonal murine antibodies to the Iarge RNA polymerase II subunits also inhibited transcription in chromosome Ioops but appeared to inhibit initiation rather than elongation events. Activities of class land III RNA polymerases were not significantly affected by injection of antibodies to polymerase II, indicating immunological differences between the three RNA polymerases. The potential value of the in vitro test system described , as a very sensitive assay for detecting proteins involved in transcription in living cells, is discussed. 1
A nucleolar skeleton of protein filaments demonstrated in amplified nucleoli of Xenopus laevis
(1981)
The amplified, extrachromosomal nucleoli of Xenopus oocytes contain a meshwork of -4-nm-thick filaments, which are densely coiled into higher-order fibrils of diameter 30-40 nm and are resistant to treatment with high- and low-salt concentrations, nucleases (DNase I, pancreatic RNase, micrococcal nuclease), sulfhydryl agents, and various nonionic detergents. This filamentous "skeleton" has been prepared from manually isolated nuclear contents and nucleoli as weil as from nucleoli isolated by fluorescence-activated particle sorting. The nucleolar skeletons are observed in light and electron microscopy and are characterized by ravels of filaments that are especially densely packed in the nucleolar cortex. DNA as weil as RNA are not constituents of this structure, and precursors to ribosomal RNAs are completely removed from the extraction-resistant filaments by treatment with high-salt buffer or RN ase. Fractions of isolated nucleolar skeletons show specific enrichment of an acidic major protein of 145,000 mol wt and an apparent pi value of -6.15, accompanied in some preparations by various amounts of minor proteins. The demonstration of this skeletal structure in "free" extrachromosomal nucleoli excludes the problem of contaminations by nonnucleolar material such as perinucleolar heterochromatin normally encountered in studies of nucleoli from somatic cells. It is suggested that this insoluble protein filament complex forms a skeleton specific to the nucleolus proper that is different from other extraction-resistant components of the nucleus such as matrix and lamina and is involved in the spatial organization of the nucleolar chromatin and its transcriptional products. In studies of the organization of the interphase nucleus, considerable progress has been made in the elucidation of the arrangement of chromatin components and transcriptional products. However, relatively little is known about the composition and function of another category of nuclear structures, the nonnucleoproteinaceous architectural components that are insoluble in solutions of low and high ionic strength, despite numerous studies dedicated to this problem. Such structures include (a) the nuclear envelope and its pore complexes (I, 15, 18, 23, 37, 41), (b) a peripheral layer of insoluble protein ("lamina"; I, 15, 22, 23, 59), (e) certain skeletal proteins related to the chromosome "scaffold" described by Laemmli and coworkers (see references 2 and 3), and (d) ill-defined tangles of fibrillar structures of the nuclear interior that are collectively described as residual "matrix" (6, 21 ; for reviews, see references THE JOURNAL OF CEll BrOlOGY . VOlUME 90 AUGUST 1981 289-299 © The RockefeIler University Press · 0021 -9525/ 81 / 08/ 0289/ 11 $1 .00 4 and 12). The latter, preparatively
Electron microscope preparations of lampbrush chromosomes from oocytes of Pleurodeles waltl;; have revealed a new class of tandemly repeated genes. These genes are highly active, as judged by the close spacing of nascent transcripts. They occur in clusters of >100 copies and are transcribed in units containing roughly 940 base pairs of DNA that are separated by nontranscribed spacers of an estimated DNA content of 2,410 base pairs. The size and the pattern of arrangement of these transcription units can not be correlated with any of the repetitious genes so far described.
no abstract available
The isolated H\(^+\) conductor, F\(_0\) , of the Escherichia co1i ATP-synthase consists of three subunits, a, b, and c. H\(^+\) -permeable liposomes can be reconstit~ted with F\(_0\) and lipids; addition of F\(_1\)-ATPase reconstitutes a functional ATP-synthase. Mutants with altered or misslng F\(_0\) subunits are defective in H\(^+\) conduction. Thus, all three subunits are necessary for the expression of H\(^+\) conduction. The subunits a and b contain binding sites for F\(_1\)• Computer calculations, cross-links, membrane-permeating photo-reactive labels, and proteases were used to develop tentative structural models for the individual F\(_0\) subunits.
The ATP synthase occurs in remarkably conserved form in procaryotic and eucaryotic cells. Thus, our present knowledge of ATP synthase is derived from sturlies of the enzyme from different organisms, each affering specific experimental possibilities. In recent tim es, research on the H\(^+\) -conducting F0 part of the ATP synthase has been greatly stimulated by two developments in the Escherichio coli system. Firstly, the purification and reconstitution of the whole ATP synthase as weil as the proton conductor Fa from E. coli have been achieved. These functionally active preparations are well defined in terms of subunit composition, similar to the thermophilic enzyme from PS-3 studied by Kagawa's group.u Secondly, the genetics and the molecular cloning of the genes of all the F\(_0\) subunits from E. coli yielded information on the function of subunit polypeptides and essential amino acid residues. Furthermore, the amino acid sequence of hydrophobic F\(_0\) subunits, which are difficult to analyze by protein-chemical techniques, could be derived from the nucleotide sequence of the genes. These achievements, which shall be briefly summarized in the next part of this communication, provide the framework to study specific aspects of the structure and function of the F\(_0\) subunits.
A novel chromatin configuration is described in lampbrush chromosomes of Pleurodeles waltlii oocytes which is different from transcriptionally inactive chromatin as weil as from the various forms of transcribed chromatin hitherto described. This novel type of chromatin is not arranged in Christmas tree-Iike configurations of densely packed lateral ribonucleoprotein (RNP) fibriIs but is characterized by a periodic alternating pattern of thick and thin regions which occur in clusters 01 some 10,000 repeats. Each thickened unit with an average length of 45 nm contains two c10sely spaced particles, the putative RNA polymerases, and each thickened unit is separated from the next one by a beaded chromatin spacer with a length of about 80 nm. This chromatin spacer contains on average two particles of approximately 14 nm in diameter, assumed to be nucleosomes. The thickened regions are interpreted to represent short transcriptional units containing approximately 130 base pairs of DNA which are separated from each other by nontranscribed spacers of 240-400 base pairs of DNA. The possibility is discussed that these transcriptional units represent 5S rRNA or tRNA genes.
Repeat sequences are transcribed in the germinal vesicles of amphibian oocytes. In the hnRNA population both complements of the repeats are found and can be readily detected because they form intermolecular duplex structures. The structure and formation of duplex regions have been studied in the hnRNA of Xenopus laevis, Triturus cristatus, Amphiuma means and Necturus maculosus, a series of amphibians of increasing genome size (C-value). In T. cristatus, the duplex structures are mostly 600- 1200 bp in length, whereas in X. laevis they are shorter and in N. maculosus they tend to be longer. Although the proportion of RNA sequence capable of rapidly forming duplex structures is different in different organisms, this property bears no relationship to C-value. However the sequence complexity of complementary repeats, as estimated from the rate of duplex formation, does show an increasing trend with C-value. The complementary repeats found in oocyte hnRNA are transcribed from families of DNA sequence that are each represented in the genome by thousands of copies. The extent of cross-species hybridization is low, indicating that the repeat sequences transcribed in different amphibian genera are not the same. In situ hybridization experiments indicate that the repeat sequences are spread throughout the genome. The evolution and possible function of complementary repeats are considered.
Visualizing nucleic acids (DNA, RNA), nucleoprotein complexes and chromatin requires the use of special electron microscopicspreading techniques. In part 4 (27 refs.), methods are outlined for spreading DNA and RNA molecules for electron microscopic observation, these methods using modifications of the basic protein film method developed by A. Kleinschmidt and R. K. Zahn (1959). Hybridization techniques that allow the observation of heteroduplexes formed between two DNA molecules or between DNA and RNA molecules are reviewed, with special emphasis being placed on the DNA-RNA hybrids as a tool for elucidating RNA splicing. Techniques for studying DNA-protein interactions without the use of a protein monolayer film are mentioned. Finally, the "Miller spreading technique" for visualizing the nucleosomal organization of eukaryotic chromatin as well as the transcription of genes is discribed and illustrated.
Sizes of chromosome loops and hnRNA molecules in oocytes of amphibia of different genome sizes
(1982)
The lengths of lampbrush chromosome loops and their tran scription units show a positive correlation with genome size in oocytes of amphibia with different C values. However, there is no such correlation with contour lengths of hnRNA molecu les isolated from these oocytes. These results indi cate th at more ON A sequences are transcribed in amphibia of higher C value , but that processing of RNA transc ripts occurs while they are still attached to the chromosomes as nascent ribonucleoprotein fibrils.
The expression in eukaryotes of a tyrosine kinase which is reactive with pp60v-src antibodies
(1982)
All specimens of Eumetazoa and Parazoa, ranging from mammals, birds, teleosts, sharks, lampreys, amphioxus, insects, down to sponges showed the pp60c-src associated kinase activity, indicating that c-src, which is the cellular homologue of the oncogene v-src of Rous sarcoma virus (RSV) is probably present in all multicellular animals. Protozoa and plants did not show pp60c-src: kinase activity.
The degree of c-src expression depends on the taxonomic rank of the Eumetazoa tested, and is organ-specific with nervaus tissues displaying the highest kinase activities. In the central nervous system of mammals and birds we found a high c-src expression, and in that of the lampreys, amphioxus, and insects the lowest. Unexpectedly, total extracts of sponges showed an amount of pp60c-src kinase activity similar to that of brain cell extracts of mammals and birds. These findings suggest that pp60c-src is a phylogenetic old protein that might have evolved together with the multicellular organisation of Metazoa, and that might be of importance in proliferation and differentiation of nontransformed cells.
The accessibility of the three F\(_0\) subunits a, b and c from the Escherichia coli Kll A TP synthase to various proteases was studied in F\(_1\)-depleted inverted membrane vesicles. Subunit b was very sensitive to all applied proteases. Chymotrypsin produced a defined fragment of mol. wt. 1S 000 which remained tightly bound to the membrane. The cleavage site was located at the C-terminal region of subunit b. Larger amounts of proteases were necessary to attack subunit a (mol. wt. 30 000). There was no detectable deavage of subunit c. It is suggested that the major hydrophilic part of subunit b extends from the membrane into the cytoplasm and is in contact with the F\(_1\) sector. The F\(_1\) sector was found to afford some protection against proteolysis oftheb subunit in vitro andin vivo. Protease digestion bad no influence on the electro-impelled H\(^+\) conduction via F\(_0\) bot ATP-dependent H\(^+\) translocation could not be reconstituted upon binding of F\(_1\)• A possible role for subunit b as a linker between catalytic events on the F\(_1\) component and the proton pathway across the membrane is discussed.
Bifunctional recombinant plasmids were constructed, comprised of the E. coli vectors pBR322, pBR325 and pACYC184 and different plasmids from Gram-positive bacteria, e.g. pBSU161-1 of B. subtilis and pUB110 and pC221 of S. aureus. The beta-lactamase (bla) gene and the chloramphenicol acetyltransferase (cat) gene from the E. coli plasmids were not transcribed and therefore not expressed in B. subtilis. However, tetracycline resistance from the E. coli plasmids was expressed in B. subtilis. Transcription of the tetracycline resistance gene(s) started in B. subtilis at or near the original E. coli promoter, the sequence of which is almost identical with the sequence recognized by σ<sup>55</sup> of B. subtilis RNA polymerase.
From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertionwas recloned as aPstl fragment into pJKK3-1, a shuttle vector which repücates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no extemal or intemal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at Ievels comparable to those ofthe B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin 0-producing strain of Streptococcus pyogenes or from üsteriolysin-producing strains of Usteria monoeytogenes, no positive hybridization signals were obtained. These data soggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.
Transcriptionally inactive chick erythrocyte nudei were reactivated by Sendai virusinduced fusion of erythrocytes with rat L6j1 myoblasts. We used antibodies to trace the appearance of a specific protein engaged in transcription of a defined dass of genes, those coding for rRNA, during reactivation. Using immunofluorescence microscopy, we found increasing amounts of rat RNA polymerase I to appear, during a certain period of time after fusion, in the reforming nudeoli of the chick nudei. Amounts of rat RNA polymerase I sufficient to be detected by immunofluorescence microscopy had accumulated in the newly developed chick nudeoli 72- 190 h after fusion was initiated. This time interval coincides with the time when chick rRNA synthesis can first be detected. The results raise the possibility that during these stages of the reactivation process chick rRNA genes are transcribed by heterologous RNA polymerase I moleeules of rat origin.
no abstract available
The cellular onc-genes c-src and c-yes are expressed very differently during chicken embryonic development. The c-src mRNA and its translational product are detectable at high levels in brain extracts of chicken embryos and adult chickens, whereas muscle extracts show an age-dependent decrease in the amounts of c-src-specific mRNA and pp60<sup>c-src</sup> kinase activity. In contrast, the Ievels of c-yes mRNA in brain, heart, and muscle are relatively low in early embryonic stages and increase later on to values comparable to those found for liver, while in adult animals the pattern of c-yes expression is similar to that of the c-src gene. From the close correlation between the Ievels of pp60<sup>c-src</sup>, its enzymatic activity, and its corresponding mRNA at a given stage of development and in given tissues, it appears that the expression of pp60<sup>c-src</sup> is primarily controlled at the level of transcription. It is suggested that because of the different patterns of expression, the two cellular oncogenes, c-src and c-yes, play different roles in cell proliferation during early embryonic stages as weil as in ensuing differentiation processes.
Nucleotide sequence of the cloned mRNA and gene of the ADP/ATP carrier from Neurospora crassa
(1984)
A cDNA complementary to the mRNA of the ADPIATP carrier from Neurospora crassa was identified among ordered cDNA clones by hybridizing total polyadenylated RNA to pools of 96 cDNA recombinant plasmids and subsequent cellfree translation of hybridization-selected mRNA. Further carrier cDNAs were found by colony fdter hybridization at a frequency of 0.2-0.3%. The gene of the carrier was cloned and isolated on a 4.6-kbp EcoRl fragment of total Neurospora DNA, and the start of the mRNA was determined by Sl nuclease mapping. From the nucleotide sequence of the cDNA and the genomic DNA, the primary structure of the gene, of the mRNA and of the ADP I ATP carrier protein could be deduced. The gene occurs in a single copy in the genome and related genes are absent. It contains two short introns, and a pyrimidine-rieb promoter region. The mRNA has a 46-bp 5 1 end and a 219-bp 3 1 end. There is an open reading frame coding for the 313 amino acid residues of the Neurospora carrier protein. The amino acid sequence is homologous in 148 positions with the established primary structure of the beef heart carrier.
The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the A TP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 8011/o fonnie acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 5011/o homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 genein Aspergillus nidulans.
Processing peptidase of Neurospora mitochondria. Two-step cleavage of imported ATPase subunit 9
(1984)
Subunit 9 (dicyclohexylcarbodümide binding protein, 'proteolipid') of the mitochondrial F 1F0-ATPase is a nuclearly coded protein in Neurospora crassa. lt is synthesized on free cytoplasmic ribosomes as a larger precursor with an NH2-terminal peptide extension. The peptide extension is cleaved ofT after transport of the protein into the mitochondria. A processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and characterized using a cell-free system. Precursor synthesized in vitro was incubated with extracts of mitochondria. Processing peptidase required Mn2 + for its activity. Localization studies suggested that it is a soluble component of the mitochondrial matrix. The precursor was cleaved in two sequential steps via an intermediate-sized polypeptide. The intermediate form in the processing of subunit 9 was also seen in vivo and upon import of the precursor into isolated mitochondria in vitro. The two dcavage sites in the precursor molecule were determined. The data indicate that: {a) the correct NH2-terminus of the mature protein was generated, (b) the NH2-terminal amino acid of the intermediate-sized polypeptide is isoleueine in position -31. The cleavage sites show similarity ofprimary structure. It is concluded that processing peptidase removes the peptide extension from the precursor to subunit 9 (and probably other precursors) after translocation of these polypeptides (or the NHrterminal part of these polypeptides) into the matrix space of mitochondria.
Malignant melanomas (MM) in the fish Xiphophorus and in humans were studied both by transmission electron microscopy (TEM) and freeze-etching (FE). In both fish and human melanomas the cells show interdigitations of the,plasma membranes. The nuclei are large and lobulated and have many nuclear pores. Melanosomes are abundant and melanosome complexes ("compound melanosomes") occur regularly. Pinocytotic vesicles could be demonstrated in fish and human melanomas showing iocal differences in frequency and distribution patterns in the tumor. lntercellular junctions are lacking in MM cells from fish and humans. The FE technique showed considerable advantages in demonstrating membrane-surface peculiarities such as nuclear pores or pinocytotic vesicles. The FE replicas of fish melanomas are like those of humans. These findings may support the hypothesis that melanoma in fish and humans reflect the same biological phenomenon.
A monoclonal murine antibody (No-I 14) is described which reacts specifically with a polypeptide of molecular weight (M,) 180000 present in low-speed nuclear pellets from oocytes and somatic cells of Xenopus laevis and X. borealis and in isolated amplified nucleoli. Two-dimensional gel electrophoresis has revealed the acidic nature of this polypeptide (isoelectric at pH of ca 4.2 in the presence of 9.5 M urea). A relatively large proportion of the protein is extracted at elevated ionic strength( i.e., at 0.4-0.5 M alkali salt) in a form sedimenting at approx. 7-8S , compatible with a monomeric state. It is also extracted by digestion with RNase but not with DNase. In immunofluorescence microscopy, antibody No-114 stains intensely nucleoli of oocytes and all somatic cells examined , including the residual nucleolar structure of Xenopus erythrocytes which are transcriptionally inactive. During mitosis the antigen does not remain associated with the nucleolar organizer regions (NOR) of chromosomes but is released and dispersed over the cytoplasm until telophase when it re-associates with the reforming interphase nucleoli. At higher resolution the immunofluorescent region is often resolved into a number of distinct subnucleolar components of varied size and shape. Immunoelectron microscopy using colloidal gold-coupled secondary antibodies reveals that the M, 180000 protein is confined to the dense fibrillar component of the nucleolus. This conclusion is also supported by its localization in the fibrillar part of segregated nucleoli of cells treated with actinomycin D. We conclude that nucleoli contain a prominent protein of M, 180000 which contributes to the general structure of the dense fibrillar component of the interphase nucleolus , independent of its specific transcriptional activity.
Nuclei of amphibian oocytes contain large amounts of actin, mostly in unpolymerized or short-polymer form. When antibodies to actin or actin-binding proteins (fragmin and the actin modulator from mammalian smooth muscle) are injected into nuclei of living oocytes of Pleurodeles waltlii, transcription of the lampbrush chromosomes, but not of the rRNA genes, is inhibited. When transcription is repressed by drugs or RNA is digested by microinjection of RNAase into oocyte nuclei, an extensive meshwork of actin filament bundles is seen in association with the isolated lampbrush chromosomes. These observations indicate a close relationship between the state of nuclear actin and transcriptional activity and suggest that nuclear actin may be involved in transcriptional events concerning protein-coding genes.
Upon incubation of cultured rat cells with the adenosine analogue 5,6-dichloro-l-β- D-ribofuranosylbenzimidazole (DRB), nucleoli reversibly dissociate into their substructures, disperse throughout the nuclear interior, and form nucleolar "necklaces". We have used this experimental system, which does not inhibit transcription of the rRNA genes, to study by immunocytochemistry the distribution of active rRNA genes and their transcriptional products during nucleolar dispersal and recovery to normal morphology. Antibodies to RNA polymerase I allow detection of template-engaged polymerase, and monoclonal antibodies to a ribosomal protein (S 1) of the small ribosomal subunit permit localization of nucleolar preribosomal particles. The results show that, under the action of DRB transcribed rRNA, genes spread throughout the nucleoplasm and finally appear in the form of several rows, each containing several (up to 30) granules positive for RNA polymerase land argyrophilic proteins. Nucleolar material containing preribosomal particles also appears in granular structures spread over the nucleoplasm but its distribution is distinct from that of rRNA gene-containing granules. We conclude that, although transcriptional units and preribosomal particles are both redistributed in response to DRB, these entities retain their individuality as functionally defined subunits. We further propose that each RNA polymerase-positive granular unit represents a single transcription unit and that each continuous array of granules ("string of nucleolar beads") reflects the linear distribution of rRNA genes along a nucleolar organizer region. Based on the total number of polymerase I-positive granules we estimate that a minimum of 60 rRNA genes are active during interphase of DRB-treated rat cells.
Behavioural adaptations have made the desert isopod Hemilepistus reaumuri the most successful herbivore and detritivore of the macrofauna of many arid areas in North Africa and Asia Minor. For survival and reproduction Hemilepistus is dependent on burrows. New burrows can only be dug during spring. With the time-consuming digging of a burrow, Hemilepistus has only made the first step towards solving its ecological problems. The burrows are vital and have to be continuously defended against competitors. This requirement is met by co-operation of individuals within the framework of a highly developed social behaviour. In spring adults form monogamous pairs in which partners recognize each other individually and later form, with their progeny, strictly closed family communities. Hemilepistus is compared with a Porcellio' sp. which has developed, convergently, a social behaviour which resembles that of Hemilepistus in many respects, but differs essentially in some aspects, partly reflecting differences in ecological requirements. This and a few other Porcellio species demonstrate some possible steps in the evolution of the social behaviour of Hemilepistus. The female Hemilepistus is-in contrast to Porcellio sp. - semelparous and the selective advantages of monogamy in its environment are not difficult to recognize. This chapter discusses how this mating system could have evolved and especially why monogamous behaviour is also the best method for the Hemilepistus male to maximize its reproductive success. The cohesion of pairs and of family communities in Hemilepistus is based on a highly developed chemical communication system. Individual- and family-specific badges owe their specificity to genetically determined discriminating substances. The nature of the badges raises a series of questions: e.g. since alien badges release aggression, how do parents avoid cannibalizing their young? Similar problems arise from the fact that family badges are mixtures of chemical compounds of very low volatility with the consequence that they can only be transferred by direct contact and that during moulting all substances are lost which an individual does not produce itself. It is shown that in solving these problems inhibiting properties (presumably substances) and learning play a dominant role.
The Xiphophorus tumor system has provided the opportunity to reduce the enormous complexity of cancer etiology to a few biological elements basically involved in neoplasia. The development of a tumor requires an oncogene which, after impairment, deletion, or elimination of its regulatory genes is permitted to mediate neoplastic transformation. Emphasis is being placed today in cancer research on the actual oncogenes themselves, but, in our opinion, the most important genes involved in neoplasia are these regulatory genes. However, although detected by c1assical genetics in the Xiphophorus system, th ese genes are not at present open to a more fin ely detailed molecular biological analysis. Their actual mode of action is therefore still far from being understood.