Visualization of Nucleolar Genes

Abstract

The presence of extrachromosomal nucleoli in amphibian oocytes has permitted isolation and electron microscopic observation of the genes coding for ribosomal RNA precursor molecules. Visualization of these genes is possible because many precursor molecules are simultaneously synthesized on each gene. Individual genes are separated by stretches of DNA that apparently are not transcribed at the time of synthesis of precursor rRNA in the extrachromosomal nucleoli.

In embryonic chicken erythrocytes actively transcribed alpha globin genes are not associated with the nuclear matrix

“…Thus, in chicken and amphibian oocytes RNA polymerases transcribing lampbrush chromosomes are not associated with any supporting structure [Doyle et al, 2002;Morgan, 2002]. Similarly, in amphibian oocytes the ribosomal genes transcribed by RNA polymerase I do not seem to be associated with the nuclear matrix [Miller and Beatty, 1969]. A drastic change has been shown to occur in the specificity of association of ribosomal genes with the nuclear matrix during early development and in somatic cells in amphibians [Vassetzky et al, 2000;Lemaitre et al, 2005;Hair and Vassetzky, 2007].…”

Lampbrush-type chromosomes in the primary nucleus of the green alga Acetabularia mediterranea

“…b) Electron M icro8COPY' Fixation and embedding procedures for electron microscopy were as previously described (Franke et al, 1971. Spread and positively stained preparations of total nuclear contents and of nucleolus-free nuclear material (i.e., supern atants obtained after sedimentation at 200 g for 1 m inute or for 3-5 minutes at gravity) were performed according to our modification of the technique by Miller a nd his associates (Miller and Beatty, 1969;Miller andBakken, 1972, 1973; see a lso Scheer et al, 1973;Trendelenburg et al, 1974;Spring et al, 1974 ;Berger a nd Schweiger, 1975). Specimen grids were observed in electron microscopes Zeiss EM 10 or Siemens 101.…”

“…Miller and his assoc ia tes (Miller and Beatty, 1969;Miller and Hamkalo, 1972 ;H a mkalo and Miller, 1973 ;Miller and Bakken , 1973) have demonstrated , in amphibian oocyte nuclei, the electron microscopical appearan ce of spread and pos itively stained chromosomal structures whi ch th cy inte rpret ed as images of the transcriptional units contained in lamp brush chromosome loops. Th ese are characterized by very long ma trix units, correspondingly very long lateral fibrils in the t erminal intercepts of th ese matrix units, a hi gh package density of the points of a ttachment of the la teral fibril s t o the deoxyr ibonucleoprotein axis, i.e., of the puta tive RNA-polymerase B containing transcriptional compl exes.…”

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

“…In order to understand the functional organization of the nucleolus, it is of fundamental importance to know where exactly the active rRNA genes or transcription units are located. Although transcriptionally active rRNA genes can be visualized by the chromatin spreading technique introduced by O. Miller (Miller and Beatty, 1969;Fig. 2a), this approach does not provide an answer as to their intranucleolar localization.…”