Library

Notes: Abstract Differential decondensation of isolated unfixed Chinese hamster metaphase chromosomes was obtained by decreasing the calcium ion concentration in the surrounding medium. A banded appearance of the swollen chromosomes could be observed either directly by phase contrast microscopy or after glutaraldehyde fixation and staining. There was a gradual transition from homogeneously dense to banded and finally to extensively decondensed chromosomes. The patterns induced at different stages were similar to those observed on fixed chromosomes after standard banding procedures (i.e., G-, C-, Cd−, Ag-NOR-staining). Chromosome decondensation could be reversed by the addition of calcium ions to the medium. Ca++-dependent reversible differential chromosome decondensation was not observed if the chromosomes were previously treated with 0.35 M NaCl. Chromosome regions which had incorporated BrdU into their DNA were more resistant to a decrease in calcium ion concentration than BrdU non-substituted regions.

Notes: Abstract A study of ultrathin sections of normal Chinese hamster cells and cells treated with decreasing concentrations of bivalent cations (Ca2+ and Mg2+) in situ revealed several discrete levels of compaction of DNA-nucleoprotein (DNP) fibrils in mitotic chromosomes and the chromatin of interphase nuclei. At concentrations ranging from 3 mM CaCl2 and 1 mM MgCl2 to ten times less, the chromosomes are found to contain fibrous elements (chromonemata) about 100 nm in diameter. As Ca2+ concentration is gradually decreased to 0.2–0.1 mM, the chromosomes decondense into a number of discrete chromatin structures, the chromomeres. As decondensation proceeds, these chromomeres acquire a rosettelike structure with DNP fibrils radiating from an electron-dense core. Upon complete decondensation of chromosomes, individual chromomeres persist only in the centromeric regions. The following levels of DNP compaction in mitotic chromosomes are suggested: a 10-nm nucleosomal fibril, a 25-nm nucleomeric fibril, and the chromonema, a fibrous structure, about 100 nm in diameter, composed of chromomeres. Interphase nuclei also contain structures which are morphologically similar to the chromomeres of mitotic chromosomes.

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.