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Available data on the occurrence and expression of somatic histone HI during oogenesis and early embryogenesis of Xenopus laevis are contradictory. In particular the reported presence of a large storage pool of histone HIA in oocytes is difficult to reconcile with the high transcriptional activity of all gene classes in this specific cell type. In the present study we have used polyclonal antibodies raised against somatic Xenopus histone HI (HIA and HIA/B) for combined immunoblotting experiments to quantitate HI pools and immunolocalization studies to visualize chromosome- bound HI. Both approaches failed to detect soluble or chromosomal histone HI in vitellogenic oocytes, eggs, and cleavage-stage embryos up to early blastula. In addition, chromatin assembled in Xenopus egg extract was also negative for histone HI as revealed by immunofluorescence microscopy. Lampbrush chromosomes not only lacked histone HI but also the previously identified histone HI-like B4 protein (Smith et al., 1988, Genes Dev. 2,1284-1295). In contrast, chromosomes of eggs and early embryos fluoresced brightly with anti-B4 antibodies. Our results lend further support to the view that histone HI expression is developmentally regulated during Xenopus oogenesis and embryogenesis similar to what is known from other species.
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.
High sensitivity immunolocalization of double and single-stranded DNA by a monoclonal antibody
(1987)
A monoclonal antibody (AK 30-10) is described which specifically reacts with DNA both in double and single-stranded forms but not with other molecules and structures, including deoxyribonucleotides and RNAs. When used in immunocytochemical experiments on tissue sections and permeabilized cultured cells, this antibody detects DNA-containing structures, even when the DNA is present in very small amounts. Examples of high resolution detection include the DNA present in amplified extrachromosomal nucleoli, chromomeres of lampbrush chromosomes, mitochondria, chloroplasts and mycoplasmal particles. In immunoelectron microscopy using the immunogold technique, the DNA was localized in distinct substructures such as the "fibrillar centers" of nucleoli and certain stromal centers in chloroplasts. The antibody also reacts with DNA of chromatin of living cells, as shown by microinjection into cultured mitotic cells and into nuclei of amphibian oocytes. The potential value and the limitations of immunocytochemical DNA detection are discussed.
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.
Using a monoclonal antibody (No-194) we have identified, in Xenopus laevis and other amphibia, an acidic protein of M, 40,000 (ribocharin) which is specifically associated with the granular component of the nucleolus and nucleoplasmic 65S particles. These particles contain the nuclear 28S rRNA and apparently represent the precursor to the large ribosomal subunit in nucleocytoplasmic transit. By immunoelectron microscopy ribocharin has been localized in the granular component of the nucleolus and in interchromatin granules. During mitosis ribocharin-containing particles are associated with surfaces of chromosomes and are recollected in the reconstituting nucleoli in late telophase. We suggest that ribocharin is a specific component of precursor particles of the large ribosomal subunit, which dissociates from the 65S particle before passage through the nuclear envelope, and is reutilized in ribosome biogenesis.
A non-radioactive in situ hybridization method is described for the localization of transcription units of defined genes to lateral loops of Xenopus laevis lampbrush chromosomes. Two Xenopus cONA probes were used encoding the nucleolar protein N038/ B23 and cytokeratin 1(8). Both proteins are known to be synthesized in Xenopus oocytes, and Northern blot analysis revealed the presence of the corresponding mRNAs in different oogenic stages. The probes were enzymatically labeled with biotin-dCTP and hybridized to lampbrush chromosomes. The sites of hybridization were detected either by indirect immunofluorescence microscopy using rabbit antibodies against biotin and fluorescein-conjugated antirabbit IgG or enzymatically using peroxidase-conjugated streptavi din. The probe encoding the nucleolar protein hybridized to two sets of lateral loops on different bivalents, the cytokeratin probe to at least four. Our finding that each probe hybridized to more than one chromosomal locus may reflect the tetraploid nature of the Xenopus laevis genome or results from cross-hybridization to other transcriptionally active members of the N038/ B23-nucleoplasmin or the cytokeratin-Iamin gene families. The method described should facilitate further in situ hybridization studies with appropriate genomic clones in order to map specific DNA sequences to defined loop regions and to come to a better understanding of the relationship between loop organization and gene transcription unit.
Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtKz cells with WGA or antibody PIt and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1 . Although PtKz cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome- associated components.
In order to investigate the DNA localization within Ehrlich tumor cell nucleoli during mitosis, two recent immunocytochemical methods using either an anti-DNA or an anti-bromodeoxyuridine (BrdU) monoclonal antibody have been applied. In both cases, the immunogold labeling has been performed on ultrathin sections of cells embedded either in Lowicryl K4M or in Epon, respectively. Identical results are observed with both immunocytochemical approaches. In the interphase nucleolus, besides the labeling of the perinucleolar chromatin shell and of its intranucleolar invaginations which penetrate into the nucleolar body and often terminate at the fibrillar centers, a few gold particles are also preferentially found towards the peripheral region of the fibrillar centers. In contrast, the dense fibrillar component and the granular component are never labeled. During mitosis, the fibrillar centers persist at the chromosomal nucleolus organizing regions (NOR's) and can be selectively stained by the silver method. However, these metaphase fibrillar centers are no longer decorated by the DNA- or BrdU antibodies. These results indicate that until the end of prophase, rRNA genes are present inside the fibrillar center material, disappear during metaphase and reappear in reconstituting nucleoli during telophase. Thus, fibrillar centers appear to represent structures sui generis, which are populated by rRNA genes only when the nucleolus is functionally active. In segregated nucleoli after actinomycin D treatment, the DNA labeling is exclusively restricted to the perinucleolar chromatin blocks. These findings also suggest that the DNA content of the fibrillar center material varies according to the rRNA transcription level of the cells. The results are discussed in the light of the present knowledge of the functional organization of the nucleolus.
After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtKz) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed. These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent "nucleolar segregation". The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase.
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.
Thin section electron microscopy of Actinomycin D treated Tetrahymena cells and amphibian oocytes (Xenopus laevis, Triturus aZpestris) reveal no reduction in the central granules in the nuclear pore complexes. Possible reasons for the diversity between these results and earlier observations using negatively stained isolated nuclear envelopes from the same objects are discussed. The results clearly show that the presence of central granules within the nuclear pores does neither depend on nuclear RNA synthesis nor does indicate nucleocytoplasmic RNA transport. This conclusion leads to a reconsideration of the nature of the central granule. The functioning of the central granule of the nuclear pore complexes is further discussed in connection with recent studies on the ultrastructure of various types of cisternal pores.
A small fraction of HeLa cells within an exponentially growing culture showed cisternal differentiations, such as cytoplasmic as well as intranuclear annulate lamellae and special smooth surfaced endoplasmic reticulum aggregates with a typical "Cotte de maillet" appearance. Additionally, clusters of dense granules were observed in the cytoplasm which were often associated with polysomes and strongly resembled the so-called "heavy bodies" known in particular in diverse oocytes. The functional meaning of these structures is discussed. Moreover, it is deduced from the ultrastructural identity of the pore complexes in the nuclear envelope and the cytoplasmic and intranuclear annulate lamellae that the pore complex material with its highly ordered arrangement is not a structure characteristic for nucleocytoplasmically migrating material, but rather is a general structural expression of a tight binding of ribonucleoprotein (RNP) to cisternal membranes. The pore complexes are thought of as representing sites of a RNP-storage. A similar functioning is hypothesized for the "heavy body"like aggregates. To the current hypotheses on the formation of annulate lamellae and the nuclear envelope, which are based on the concept of membrane continuities and constancies, the alternative view of a self assembly mechanism of membrane constituents on nucleoprotein structures is added.
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.
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.
In this work, a behavioural analysis of different mutants of the fruit fly Drosophila melanogaster has been carried out. Primarily, the gap climbing behaviour (Pick & Strauss, 2005) has been assayed as it lends itself for the investigation of decision making processes and the neuronal basis of adaptive behaviour. Furthermore it shows how basic motor actions can be combined into a complex motor behaviour. Thanks to the neurogenetic methods, Drosophila melanogaster has become an ideal study object for neurobiological questions. Two different modules of climbing control have been examined in detail. For the decision making, the mutant climbing sisyphus was analysed. While wild-type flies adapt the initiation of climbing behaviour to the width of the gap and the probability for a successful transition. climbing sisyphus flies initiate climbing behaviour even at clearly insurmountable gap widths. The climbing success itself is not improved in comparison to the wild-type siblings. The mutant climbing sisyphus is a rare example of a hyperactive mutant besides many mutants that show a reduced activity. Basic capabilities in vision have been tested in an optomotor and a distance-estimation paradigm. Since they are not affected, a defect in decision making is most probably the cause of this behavioural aberration. A second module of climbing control is keeping up orientation towards the opposite side of the gap during the execution of climbing behaviour. Mutants with a structural defect in the protocerebral bridge show abnormal climbing behaviour. During the climbing attempt, the longitudinal body axis does not necessarily point into the direction of the opposite side. Instead, many climbing events are initiated at the side edge of the walking block into the void and have no chance to ever succeed. The analysed mutants are not blind. In one of the mutants, tay bridge1 (tay1) a partial rescue attempt used to map the function in the brain succeeded such that the state of the bridge was restored. That way, a visual targeting mechanism has been activated, allowing the flies to target the opposite side. When the visibility of the opposing side was reduced, the rescued flies went back to a tay1 level of directional scatter. The results are in accord with the idea that the bridge is a central constituent of the visual targeting mechanism. The tay1 mutant was also analysed in other behavioural paradigms. A reduction in walking speed and walking activity in this mutant could be rescued by the expression of UAS-tay under the control of the 007Y-GAL4 driver line, which concomitantly restores the structure of the protocerebral bridge. The separation of bridge functions from functions of other parts of the brain of tay1 was accomplished by rescuing the reduced optomotor compensation in tay1 by the mb247-GAL4>UAS-tay driver. While still having a tay1-like protocerebral bridge, mb247-GAL4 rescue flies are able to compensate at wild-type levels. An intact compensation is not depended on the tay expression in the mushroom bodies, as mushroom body ablated flies with a tay1 background and expression of UAS-tay under the control of mb247-GAL4 show wild-type behaviour as well. The most likely substrate for the function are currently unidentified neurons in the fan-shaped body, that can be stained with 007Y-GAL4 and mb247-GAL4 as well.
So me species of the paleotropical tree genus Macaranga (Euphorbiaceae) live in elose association with ants. Thc genus comprises the full range of species from those not regularly inhabited by ants to obligate myrmecophytes. In Malaysia (peninsular and Borneo) 23 ofthe 52 species areknown to be ant-associated (44%). The simplest structural adaptation of plants to attract ants are extrafloral nectaries. We studied the distribution of extraflural nectaries in the genus Macaranga to assess the significance of this character as a possible predisposition for the evolution of obligate myrmecophytism. All species have marginal glands on the leaves. However, only the glands of nonmyrmecophytic species function as nectaries, whereas liquids secreted by these glands in myrmecophytic species did not contain sugar. Some non-myrmecophytic Macaranga and transitional Macaranga species in addition have extrafloral nectaries on the leaf blade near the petiole insertion. All obligatorily myrmecophytic Macaranga species, however, lack additional glands on the lamina. The non-myrmecophytic species are visited by a variety of different ant species, whereas myrmecophytic Macaranga are associated only with one specific ant-partner. Since these ants keep scale insects in the hollow sterns, reduction of nectary production in ant-inhabited Macaranga seems to be biologically significant. We interpret this as a means of (a) saving the assimilates and (b) stabilization of maintenance of the association's specificity. Competition with other ant species for food rewards is avoided and thereby danger ofweakening the protective function ofthe obligate antpartner for the plant is reduced. A comparison with other euphorb species living in the same habitats as Macaranga showed that in genera in which extrafloral nectaries are widespread, no myrmecophytes have evolved. Possession of extrafloral nectaries does not appear to be essential for the development of symbiotic ant-plant interactions. Other predispositions such as nesting space might have played a more important role.
Zanthoxylum myriacanthum, a small Rutaceous tree growing mainly in secondary hill forests in SE Asia, is a true myrmecophyte. It possesses stem domatia in the form of hollow branches with slitlike openings. Branch hollows and entrance slits are produced by the plant itself through pith degene~.tion ?u.d growth proceSses. If the entrance is not kept open by ants it closes again by growth ol the surrounding tissue after some time. The domatia are colonized opportunistic ally by different arboreous ants, e.g. Crematogaster and Campono tus. Additionally many small extrafloral nectaries are found on the leaflets of Zanthoxylum myriacanthum. Judging from herbarium studies and literature records at least four more true ant trees are found in the genus Zanthoxylum namely Z. rhetsa in SE Asia, Z. conspersipunctatum, Z. pluviatile and Z. vinkii in New Guinea. We could not confirm ant inhabitation in Drypetes pendula (Euphorbiaceae) on the Malay Peninsula, which has also been recorded to be an anttree.
The human genome has been sequenced since 2001. Most proteins have been characterized now and with everyday more bioinformatical predictions are experimentally verified. A project is underway to sequence thousand humans. But still, little is known about the evolution of the human proteome itself. Domains and their combinations are analysed in detail but not all of the human domain architectures at once. Like no one before, we have large datasets of high quality human protein-protein-protein interactions and complexes available which allow us to characterize the human proteome with unmatched accuracy. Advanced clustering algorithms and computing power enable us to gain new information about protein interactions without touching a pipette. In this work, the human proteome is analysed at three different levels. First, the origin of the different types of proteins was analysed based on their domain architectures. The second part focuses on the protein-protein interactions. Finally, in the third part, proteins are clustered based on their interactions and non-interactions. Most proteins are built of domains and their function is the sum of their domain functions. Proteins that share the same domain architecture, the linear order of domains are homologues and should have originated from one common ancestral protein. This ancestor was calculated for roughly 750 000 proteins from 1313 species. The relations between the species are based on the NCBI Taxonomy and additional molecular data. The resulting data set of 5817 domains and 32868 domain architectures was used to estimate the origin of these proteins based on their architectures. It could be observed, that new domain architectures are only in a small fraction composed of domains arisen at the same taxon. It was also found that domain architectures increase in length and complexity in the course of evolution and that different organisms like worm, and human share nearly the same amount of proteins but differ in their number of distinct domain architectures. The second part of this thesis focuses on protein-protein interactions. This chapter addresses the question how new evolved proteins form connections within the existing network. The network built of protein-protein interactions was shown to be scale free. Scale free networks, like the internet, consist of few hubs with many connections and many nodes with few connections. They are thought to arise by two mechanisms. First, newly emerged proteins interact with proteins of the network. Second, according to the theory of preferential attachment, new proteins have a higher chance to interact with already interaction rich proteins. The Human Protein Reference Database provides an on in-vivo interaction data based network for human. With the data obtained from chapter one, proteins were marked with their taxon of origin based on their domain architectures. The interaction ratio of proteins of the same taxa compared to all interactions was calculated and higher values than the random model showed for nearly every taxa. On the other hand, there was no enrichment of proteins originated at the taxon of cellular organisms for the node degree found. The node degree is the number of links for this node. According to the theorie of preferential attachment the oldest nodes should have the most interactions and newly arisen proteins should be preferably attached to them not together. Both could not be shown in this analysis, preferential attachment could therefore not be the only explanation for the forming of the human protein interaction network. Finally in part three, proteins and all their interactions in the network are analysed. Protein networks can be divided into smaller highly interacting parts carrying out specific functions. This can be done with high statistical significance but still, it does not reflect the biological significance. Proteins were clustered based on their interactions and non-interactions with other proteins. A version with eleven clusters showed high gene ontology based ratings and clusters related to specific cell parts. One cluster consists of proteins having very few interactions together but many to proteins of two other clusters. This first cluster is significantly enriched with transport proteins and the two others are enriched with extracellular and cytoplasm/membrane located proteins. The algorithm seems therefore well suited to reflect the biological importance behind functional modules. Although we are still far from understanding the origin of species, this work has significantly contributed to a better understanding of evolution at the protein level and has, in particular, shown the relation of protein domains and protein architectures and their preferences for binding partners within interaction networks.
The production of extrafloral nectar and food bodies plays an important role in many tropical ant-plant mutualisms. In Malaysia, a close association exists between ants and some species of the pioneer tree genus Macaranga (Euphorbiaccac). Macaranga is a very diverse genus which exhibits all stages ofintcraction with ants, from facultative to obligatory associations. The ants nest inside the hollow inlcrnodes and reed mainly on food budies provided by the plants. Food body production had previously been reported only in myrrnecophytic Macaranga species, where it is usually coneentrated on protected parts or the plants such as recurved stipules. We found that non-myrmecophytic Macaranga species also produce food bodies on leaves and stems, where they are collected by a variety or ants. Levels of food body production differ between facultatively and obligatorily ant-associated species but also among the various non-myrmecophytes. This may he rdated to the degree of interaction with ants. Food body production starts at a younger age in the myrmccophytic species than in the transitional or non-myrmcccophytic Macaranga. Although food bodies of the non-inhabited Macaranga species are collected by a variety of ants, there is nu evidence of association with specific ant species. Our observations suggest that food bodies enhance the evolution of ant-plant interactions. Production of food bodies alone, however, does not appear to be the most important factor for the development of obligate myrmccopllytism in Macaranga.
Nucleoli provide the fascinating possibility of linking morphologically distinct structures such as those seen in the electron microscope with biochemical f eatures of the formation and step wise maturation of ribosomes. Localization of proteins by immunocytochemistry and of rRNA genes and their transcripts by in situ hybridization has greatly improved our understanding of the structural-functional relationships of the nucleolus. The present review describes some recent results obtained by electron microscopic in situ hybridization and argues that this approach has the potential to correlate each step of the complex pre-rRNA maturation pathway with nucleolar structures. Evidence is accumulating that the nucleolus-specific U3 snRNPs (small nuclear ribonucleoprotein particles) participate in rRNA processing events, similar to the role played by the nucleoplasmic snRNPs in mRNA maturation. The intranucleolar distribution of U3 snRNA is consistent with the view that it is involved in both early and late stages of pre-rRNA processing.
Lampbrush chromosomes of amphibian oocytes were isolated in the presence of near-physiological salt concentrations, to preserve their native state, and studied by electron microscopy of ultrathin s~dions. The transcriptional state of the lampbrush chromosomes was experimentally modulated by incubating the oocytes for various time periods in medium containing actinomycin D. The observations show that the structure of the lateral loops changes rapidly in response to alterations in transcriptional activity. During decreasing transcriptional activity and reduced packing density of transcripts, the chromatin axis first condensed into nucleosomes and then into an approximately 30 nm thick higher order chromatin fiber. Packaging of the loop axis into supranucleosomal structures may contribute to the foreshortening and retraction of the loops observed during inhibition of transcription and in later stages of meiotic prophase. The increasing packing density of the DNA during the retraction process of the loops could also be visualized by immunofluorescence microscopy using antibodies to DNA. The dependence of the loop chromatin structure on transcriptional activity is discussed in relation to current views of mechanisms involved in gene activation.
The distribution of DNA in Ehrlich tumour cell nucleoli was investigated by means of an immunocytochemical approach , involving a monoclonal antibody directed against double- and single-stranded DNA. Immunolabelling was performed . either before or after the embedding process. The postembedding labelling method allows better ultrastructural preservation than the preembedding labelling method. In particular, the various nucleolar components are well preserved and identifiable. In the nucleolus, labelling is particularly concentrated over the perinucleolar chromatin and over its intranucleolar invaginations, which penetrate the nucleolar body and often terminate at the fibrillar centres. In addition, aggregates of gold particles are found in the fibrillar centres, preferentially towards the peripheral regions. By contrast, the dense fibrillar component is completely devoid of labelling. The results seem to indicate that DNA containing the rDNA genes is located in the fibrillar centres, with a preference for the peripheral regions. This finding suggests that transcription of the rDNA genes should occur within the confines of the fibrillar centre, probably close to the boundary region of the surrounding dense fibrillar component. The results are discussed in the light of present knowledge of the functional organization of the nucleolus.
Small nucleolus-related bodies which occur in the nUcleoplasm of " micronuclei" lacking nucleolar organizers have been studied by immunofluorescence microscopy. These bodies stained specifically with three different antibodies directed against proteins that are normally associated with the dense fibrillar component of functional nucleoli, but not with antibodies specific for certain proteins of the granular component or the fibrillar centers. Our data show that, in the absence of rRNA genes, the various constituent proteins characteristic of the dense fibrillar component spontaneously assemble into spherical entities but that the subsequent fusion of these bodies into larger structures is prevented in these micronuclei. The similarity between these nucleolus-related bodies of micronuclei and the prenucleolar bodies characteristic of early stages of nucleologenesis during mitotic telophase is discussed.
Nucleoli are the sites of ribosome biogenesis. Transcription of the ribosomal RNA genes as well as processing and initial packaging of their transcripts with ribosomal and non-ribosomal proteins all occur within the nucleolus in an ordered manner and under defined topological conditions. Components of the nucleolus have been localized by immunocytochemistry and their functional aspects investigated by microinjection of antibodies directed against the enzyme responsible for rDNA transcription, RNA polymerase I. The role of nascent transcripts in postmitotic formation of nucleoli will be discussed.
A significant contribution to the understanding of chromatin organization was the d iscovery of the nucleosome as a globular repeating unit of the package of DNA (Hewish and Burgoyne, 1973; Woodcock, 1973; Kornberg, 1974; Olins and Olins, 1974; for review see Oudet et al., 1978 a) . In accord with the original definition and in ag reement with most workers in this field of research we identify a nucleosome as a spheric alor slightly oblate gr anular particle 10-13 nm in diameter, containing about 200 base pairs of DNA and two of each of the four his tones H2a, H2b, H3 and H4. It is this structure in which the bulk of the nuclear chroma tin is organized in most eukaryotic cells, with the exception of the dinofl age llates (Rae and Steele, 1977; dinofl agellate DNA, however, c an be packed into nucleosoma l structures in vitro by addition of the appropriate amounts of histones;the same reference). Although it seems clear from the work reported that condensed and transcriptiona lly inactive chroma tin is contained in nucleosomes as the principle for first order p acking of DNA there are two important questions onto which we are focusing in the present study: ( i ) What is the higher order of p a cking present in - and perhaps typical-of - the condensed sta te of chromatin, and (ii) what is the specific form of arrangement of transcriptionally a ctive chromatin?
The 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.
In order to investigate the chemical composition of the nuclear pore complexes isolated nuclei from mature Xenopus laevis oocytes were manually fractioned into nucleo· plasmic aggregates and the nuclear envelopes. The whole isolation procedure takes no more than 60- 90 sec, and the pore complexes of the isolated envelopes are well preserved as demonstrated by electron microscopy. Minor nucleoplasmic and cytoplasmic contaminations associated with the isolated nuclear envelopes were determined with electron microscopic morphometry and were found to be quantitatively negligible as far as their mass and nucleic acid content is concerned. The RNA content of the fractions was determined by direct phosphorus analysis after differential alkaline hydrolysis. Approximately 9% of the total nuclear RNA of the mature Xenopus egg was found to be attached to the nuclear envelope. The nonmembranous elements of one pore complex contain 0.41 X 10- 16 g RNA. This value agrees well with the content estimated from morphometric data. The RNA package density in the pore complexes (270 X 10- 15 g/fJ-3) is compared with the nucleolar, nucleoplasmic and cytoplasmic RNA concentration and is discussed in context with the importance of the pore complexes for the nucleo-cytoplasmic transport of RNA-containing macromolecules. Additionally, the results of the chemical analyses as well as of the 3H-actinomycin D autoradiography and of the nucleoprotein staining method of Bernhard (1969) speak against the occurence of considerable amounts of DNA in the nuclear pore complex structures.
Electron opaque cross-bridge structures span the inter- and intracisternal spaces and provide membrane-to-membrane connections between adjacent cisternae of dictyosomes of pollen tubes of Clivia and Lilium. Additionally, the classic intercisternal rods, characteristic of intercisternal regions near the maturing face of dictyosomes, are connected with the adjacent membranes through similar cross-bridge elements. We suggest that these structural links are responsible for maintaining the flattened appearance of the central parts of Golgi apparatus cisternae as well as for the coherence of cisternae within the stack. Observations on other plant (e.g. microsporocytes of Canna) and animal cells (e.g. rodent liver and hepatoma cells, newt spermatocytes) show that such an array of membrane cross-links is a universal feature of Golgi apparatus architecture. The cross-bridges appear as part of the complex "zone of exclusion" which surrounds dictyosomes, entire Golgi apparatus and Golgi apparatus equivalents in a variety of cell types.
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.
In somatic cells DNA topoisomerase II (topo II) is thought to be involved in the domain Organization of the genome by anchoring the basis of chromatin loops to a chromosomal scafFold. Lampbrush chromosomes of am-phibian oocytes directly display this radial loop Organization in cytological preparations. In order to find out whether topo II may play a role in the Organization of these meiotic chromosomes, we performed immunofluorescence studies using antibodies against Xenopus topo II. Our results indicate that topo II is apparently absent from lampbrush chromosomes and is hence unlikely to act as a "fastener" of the numerous lateral chromosomal loops. Topo II was, however, enriched in the amplified nucleoli of Xenopus oocytes.
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.
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 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.
Using antibodies to various nucleolar and ribosomal proteins, we define, by immunolocalization in situ, the distribution of nucleolar proteins in the different morphological nucleolar subcompartments. In the present study we describe the nucleolar localization of a specific ribosomal protein (51) by immunofluorescence and immunoelectron microscopy using a monoclonal antibody (R5 1-105). In immunoblotting experiments, this antibody reacts specifically with the largest and most acidic protein of the small ribosomal subunit (51) and shows wide interspecies cross-reactivity from amphibia to man. Beside its localization in cytoplasmic ribosomes, this protein is found to be specifically localized in the granular component of the nucleolus and in distinct granular aggregates scattered over the nucleoplasm. This indicates that ribosomal protein 51, in contrast to reports on other ribosomal proteins, is not bound to nascent pre-rRNA transcripts but attaches to preribosomes at later stages of rRNA processing and maturation. This protein is not detected in the residual nucleolar structures of cells inactive in rRNA synthesis such as amphibian and avian erythrocytes. During mitosis, the nucleolar material containing ribosomal protein 51 undergoes a remarkable transition and shows a distribution distinct from that of several other nucleolar proteins. In prophase, the nucleolus disintegrates and protein 51 appears in numerous small granules scattered throughout the prophase nucleus. During metaphase and anaphase, a considerable amount of this protein is found in association with the surfaces of all chromosomes and finely dispersed in the cell plasm. In telophase, protein 51-containing material reaccumulates in granular particles in the nucleoplasm of the newly formed nuclei and, finally, in the re-forming nucleoli. These observations indicate that the nucleolus-derived particles containing ribosomal protein 51 are different from cytoplasmic ribosomes and, in the living cell, are selectively recollected after mitosis into the newly formed nuclei and translocated into a specific nucleolar subcompartment, i.e ., the granular component. The nucleolar location of ribosomal protein 51 and its rearrangement du'ring mitosis is discussed in relation to the distribution of other nucleolar proteins.