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Notes: [Auszug] Fig. 1 a, Diagram of pXlr 101. This is a single rDNA repeat of Xenopus laevis inserted into the plasmid pMB9 at the HindIII site. The rDNA repeat (10,550 base pairs) is orientated with the non-transcribed spacer (NTrSp, 2,350 base pairs) next to the EcoRI and HindIII sites of the plasmid. The rDNA ...

Notes: Abstract  A survey of novel microscopic approaches for structural and functional analysis of subnucleolar compartments will be presented. Research on nucleolar structure and function concentrates predominantly on two distinct types of nucleoli: (1) nucleoli present during the interphase of the cell cycle in somatic tissue culture cells and (2) nucleoli present in meiotic cells, e.g. oocytes of amphibians. These nucleoli are found during meiotic prophase of oogenesis and are functional during several months of the diplotene stage of oogenesis. A further characteristic is the fact that these nucleoli are extra-chromosomal, since they originate by selective ribosomal DNA (rDNA) amplification during the early pachytene stage of oogenesis. Miller-type chromatin spread preparations using transcriptionally active nucleoli, to a major part, contributed to our understanding of the structural organization of polymerase I directed pre-rRNA transcription. Although the structural organization of the template-associated pre-rRNA transcript is known in some detail from chromatin spreads, relatively little is known about structural aspects of pre-rRNA processing. In order to investigate this intriguing question in more detail, we have developed a computer-based densitometry analysis of both template-associated and template-dissociated pre-rRNA transcripts in order to follow the structural modification of pre-rRNA transcripts during processing. Another line of experiments is devoted to the in situ structure of actively transcribing genes in the nucleolus. In order to bridge the gap between light microscopy and electron microscopy we started video-enhanced light microscopical analysis of actively transcribing genes. Although the dimensions of individual spread genes are critical for detection by optical microscopy, we succeeded in obtaining the first series of images of transcribing genes in their ’native’ hydrated state. An additional promising type of microscopy is transmission X-ray microscopy. Recent progress in instrumentation as well as in sample preparation has allowed us to obtain the first images of density distribution within intact, fully hydrated nucleoli using amplitude-contrast and/or phase-contrast X-ray microscopy of non-contrasted, fully hydrated nucleoli at different states of transcriptional activity. Whereas the above mentioned investigations using video microscopy and X-ray microscopy are predominantly applicable to the analysis of amplified nucleoli in amphibian oocytes, which are characterized by an extremely high transcription rate of 80–90% of rDNA genes per individual nucleolus, structural analysis of the in situ arrangement of actively transcribing genes in somatic nucleoli as present in the interphase nucleus is far more difficult to perform, mainly due to the much lower number of simultaneously transcribed active genes per individual nucleolus. Visualization of actively transcribed gene clusters is approached by an integrated experimental assay using video microscopy, confocal laser scan microscopy, and antibodies against specific nucleolar proteins.

Notes: Summary Methods for visualization of the ultrastructure of transcriptionally active eukaryotic genes have been developed using chromatin from giant nuclei of amphibian oocytes (Miller and Beatty 1969). Rapidly isolated chromatin is subjected to low salt treatment in order to dissociate most chromatin associated proteins. As a result, gene-chromatin with associated RNA polymerase particles and RNA transcripts can be directly analysed in electron microscope chromatin spread preparations. More recently, progress has been made in utilising living amphibian oocyte nuclei as a transcription system for cloned eukaryotic genes. In this article, an account of such experiments is given, with emphasis on results and problems of chromatin and transcription organization of microinjected cloned genes. The described transcription assay system possesses important potential for investigation of gene mutations and in particular for the elucidation of molecular aspects of experimental oncology and molecular human genetics.

Notes: Summary The ultrastructure of the growing and maturing primary nucleus ofAcetabularia mediterranea andAcetabularia major has been studied with the use of various fixation procedures. Particular interest has been focused on the details of the nuclear periphery and the perinuclear region. It is demonstrated that early in nuclear growth a characteristic perinuclear structural complex is formed which is, among the eukaryotic cells, unique toAcetabularia and related genera. This perinuclear system consists essentially of a) the nuclear envelope with a very high pore frequency and various pore complex associations with granular and/or threadlike structures some of which are continuous with the nucleolus; b) an approximately 100 nm thick intermediate zone densely filled with a filamentous material and occasional small membraneous structures from which the typical cytoplasmic and nuclear organelles and particles are excluded; c) an adjacent lacunar labyrinthum which is interrupted by many plasmatic junction channels between the intermediate zone and the free cytoplasm; d) numerous dense perinuclear bodies in the juxtanuclear cytoplasm which are especially frequent at the junction channels and reveal a composition of aggregated fibrillar and granular structures; e) very dense exclusively fibrillar aggregates which occur either in association with the perinuclear region of the lacunar labyrinthum or, somewhat further out, in the cytoplasmic strands between the branches of the lacunar labyrinthum in the form of slender, characteristic rods or “sausages”. A variety of other membraneous and non-membraneous structures characteristic of the juxtanuclear cytoplasm is described. The organization of the individual components in this special complex is summarized in a model drawing. The dynamic and transitory nature of this perinuclear complex apparatus is emphasized and its possible role in nuclear functions and in regulating nucleocytoplasmic interactions is discussed.