MULTIPARAMETER MICROSCOPIC ANALYSIS OF NUCLEOLAR STRUCTURE AND RIBOSOMAL GENE-TRANSCRIPTION

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 dis tinct types of nucleoli: (1) nucleoli present during the interphase of the cell cycle in somatic tissue culture cells and (2) nucleoli prese nt in meiotic cells, e.g. oocytes of amphibians, These nucleoli are fo und during meiotic prophase of oogenesis and are functional during sev eral months of the diplotene stage of oogenesis, A further characteris tic is the fact that these nucleoli are extrachromosomal, since they o riginate by selective ribosomal DNA (rDNA) amplification during the ea rly pachytene stage of oogenesis. Miller-type chromatin spread prepara tions using transcriptionally active nucleoli, to a major part, contri buted to our understanding of the structural organization of polymeras e I directed pre-rRNA transcription, Although the structural organizat ion of the template-associated pre-rRNA transcript is known in some de tail from chromatin spreads, relatively little is known about structur al aspects of pre-rRNA processing, In order to investigate this intrig uing question in more detail, we have developed a computer-based densi tometry 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 i s devoted to the in situ structure of actively transcribing genes in t he nucleolus. In order to bridge the gap between light microscopy and electron microscopy we started video-enhanced light microscopical anal ysis of actively transcribing genes. Although the dimensions of indivi dual spread genes are critical for detection by optical microscopy, we succeeded in obtaining the first series of images of transcribing gen es in their 'native' hydrated state, An additional promising type of m icroscopy is transmission X-ray microscopy. Recent progress in instrum entation as well as in sample preparation has allowed us to obtain the first images of density distribution within intact, fully hydrated nu cleoli using amplitude-contrast and/or phase-contrast X-ray microscopy of non-contrasted, fully hydrated nucleoli at different states of tra nscriptional activity. Whereas the above mentioned investigations usin g video microscopy and X-ray microscopy are predominantly applicable t o the analysis of amplified nucleoli in amphibian oocytes, which are c haracterized by an extremely high transcription rate of 80-90% of rDNA genes per individual nucleolus, structural analysis of the in situ ar rangement of actively transcribing genes in somatic nucleoli as presen t in the interphase nucleus is far more difficult to perform, mainly d ue 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 sp ecific nucleolar proteins.