Nicole Angelier

Visualization of Ag-NOR proteins on nucleolar transcriptional units in molecular spreads

Application of NOR silver staining to Pleurodeles oocyte nuclei showed selective silver deposits on the fibrillar part of the nucleoli in situ. This staining was adapted to nucleoli spread on grids, by a procedure which allowed the spreading of transcription units from both nucleoli and lampbrush chromosomes on the same grids. This permitted localization of the predominant nucleolar Ag-stained proteins on the nucleolar transcriptional units and not on the lampbrush chromosomes. These proteins were found exclusively on the transcribed part of the nucleolar genes and were not seen in apparently untranscribed spacer regions. The proteins seemed preferentially located on the DNP axis rather than on the RNP fibrils.

DNA methylation and RNA transcriptional activity in amphibian lampbrush chromosomes

Lampbrush chromosomes simultaneously exhibit two types of chromatin: the actively transcribed chromatin of lateral loops and the transcriptionally inactive chromatin of the chromosome axis. In situ localization of 5-methylcytosine in lampbrush chromosomes of the amphibian Pleurodeles waltlii, using specific antibodies to 5-methylcytosine, allowed us to demonstrate an inverse relationship between RNA transcriptional activity and the level of DNA methylation by light and electron microscopy using immunofluorescence and immunogold staining. The 5-methylcytosine was exclusively located in transcriptionally inactive chromatin in the chromosome axis and in the untranscribed spacers of some loops when the deoxyribonucleoprotein (DNP) axis was not wholly transcribed. In the vast majority of loops where the DNP axis was wholly transcribed, no methylated cytosine residues were ever detected. These results indicate a close relationship between the functional state of chromatin and the level of DNA methylation and suggest that DNA methylation is closely linked to gene control mechanisms in lampbrush chromosomes, i.e. at a definite time during oogenesis.

Evidence for a particular mode of transcription in globular loops of lampbrush chromosomes of the newt Pleurodeles waltlii

In amphibian lampbrush chromosomes, many loops have a specific morphology; this is the case for globular loops in the newt Pleurodeles. We have previously shown that the specific morphology of these loops is linked to an extreme compactness of the transcription products which make up their matrix. — We investigated RNA synthesis in this type of loop by carrying out autoradiographic and transcription inhibition studies. We also analysed the organization of transcriptional complexes in these loops in the electron microscope using spread preparations. These studies revealed the presence of several transcription units in the same loop and asynchronous variations in RNA synthesis in these transcription units. We propose and discuss several hypotheses in order to explain this asynchronous RNA synthesis. We also discuss these results in the context of loop morphology and transcription mode.

Effects of cold shock treatment on lampbrush chromosomes of amphibian oocytes.

When females of the newt Pleurodeles waltl, normally raised in our laboratory at 20 degrees C, were placed in 8 degrees C water for several days, striking modifications occurred in the structure of oocyte lampbrush chromosomes: numerous normal-type lateral loops were partially reduced in size and number, while hyperdeveloped loops of a new morphological type occurred at constant, reproducible loci. However, induction of these cold loops apparently was not accompanied by preferential RNA synthesis at their levels. Cold loops resulted from the decompaction of specific landmarks, the granular loops. Autoradiography revealed a significant drop in lampbrush chromosome transcriptional activity at 8 degrees C. Both structural and transcriptional modifications were fully reversible when oocytes were returned to normal temperature. These modifications are discussed in relation to processes which could determine the morphological appearance of landmark loops.

Microdissection and cloning of DNA from landmark loops of amphibian lampbrush chromosomes

Microdissection of the “globular” and “granular” landmark loops of Pleurodeles lampbrush chromosomes and subsequent cloning of their DNA yielded several recombinant clones. The 6.6-kb insert of one of them was subcloned and the 600 bp of one subclone was characterized by Southern and slot hybridizations as well as by sequencing. This sequence, designated p130B, was shown to belong to a class of moderately repetitive DNA. RNA expression of this sequence was investigated by in situ hybridization of p130B to the nascent transcripts of lateral loops. Results showed that: (1) the same transcripts were not always found in matrices of landmarks exhibiting the same morphological features; (2) the same transcripts were expressed in loops of different morphological types. Based on these results we suggest that even if there is a morphological similarity of landmark loops, this does not reflect total similarity of their transcripts.

Time-lapse Microscopy and Fluorescence Resonance Energy Transfer to Analyze the Dynamics and Interactions of Nucleolar Proteins in Living Cells

The dynamics of proteins play a key role in the organization and control of nuclear functions. Techniques were developed recently to observe the movement and interactions of proteins in living cells; time-lapse microscopy using fluorescent-tagged proteins gives access to observations of nuclear protein trafficking over time, and fluorescence resonance energy transfer (FRET) is used to investigate protein interactions in the time-lapse mode. In this chapter, we describe the application of these two approaches to follow the recruitment of nucleolar processing proteins at the time of nucleolar assembly. We question the role of prenucleolar bodies (PNB) during migration of the processing proteins from the chromosome periphery to sites of ribosomal genes (rDNA) transcription. The order of recruitment of different processing proteins into nucleoli is the consequence of differential sorting from the same PNBs. The dynamics of the interactions between processing proteins in PNBs suggest that PNBs are preassembly platforms for ribosomal RNA (rRNA) processing complexes.

Effects of in vivo heat treatment on lampbrush chromosome structure in amphibian oocytes

When Pleurodeles (Amphibian, Urodele) females were subjected to high temperatures (32-35 degrees C) for varying periods of time (45 min to 7 days), lampbrush chromosome structure underwent striking modifications. These changes included a numerical reduction in normal loops and progressive disorganization of RNP matrices of various loops. The degree of such disorganization was a function of the intensity and duration of the stress. These modifications were completely reversible when females or oocytes were returned to a normal breeding temperature (20 degrees C). Results are discussed in comparison with previous studies on morphological changes induced by heat shock in lampbrush chromosomes carried out in vitro.

An analog of Xenopus N1N2 protein in Pleurodeles waltl

Summary— The oocyte nucleus of Pleurodeles waltl contains a major 185‐kDa protein analog of Xenopus N1N2. Rabbit antibodies were raised against the 185‐kDa protein. Affinity‐purified antibody directed against the acid part (pI 4.7) of the protein was prepared using antigens separated by two‐dimensional electrophoresis. Specificity of the antibody was controlled on two‐dimensional gels of nuclear proteins. It was shown that the 185‐kDa protein was separated into 2 forms: an acid form of pI 4.7–5.3, and a base form of pI 7. Peptide maps of the 2 spots revealed that they were closely related. The antibody was tested on: a) spread nuclear content, b) on sections of embryonic stages from stage 2 to stage 34, and c) the sections of adult tissies. The 185‐kDa protein appeared to be associated with the RNP matrix of a particular type of lampbrush chromosome loop, the granular loop. This protein was present in the nuclei of all embryonic cells. In adult tissues, it was present only in the nuclei of cells which presented high mitotic activity. These results confirm that, like N1N2, the 185‐kDa protein interacts with the constitution of chromatin; furthermore, they provide evidence for the role of this protein in transcriptional activity.

Comparison between in vivo and in vitro heat-induced changes in amphibian lampbrush chromosomes

At normal breeding temperature (20° C), amphibian lampbrush chromosomes are characterized by the presence of lateral loops which are related to the transcriptional process. Heat treatment induces changes in these loops, but the nature and timing of these modifications depend on hyperthermic stress conditions. Indeed, our data demonstrate that, at the same high temperature (34° C), lampbrush chromosome modifications induced by in vivo and in vitro gradual heat treatments are different from those induced by in vitro heat shock. In vivo and in vitro heat treatments lead to progressive disorganization of landmard loops, whereas in vitro heat shock results in chromosome condensation. The progressive adaptation of lampbrush chromosome structure in response to gradual heat stress is considered and discussed.

Ultrastructural similarity in landmark loops of amphibian lampbrush chromosomes

Summary— Simultaneous transmission and scanning electron microscopy studies were performed on lampbrush chromosomes of Notophthalmus viridescens and Xenopus laevis. The organization of their normal and landmark loop ribonucleoprotein (RNP) matrices was compared to that of Pleurodeles waltl lampbrush loops, previously described. Ultrastructural observations clearly showed that in the three species, the RNP matrix of normal and landmark loops displayed a common basic structure: an RNP fibril packed into tightly juxtaposed RNP particles of remarkably uniform size, ie 30 nm. Furthermore, analysis of the spatial arrangement of these constitutive RNP fibrils allowed us to establish ultrastructural similarities between the different types of loop matrices of the three species studied. Thus, granular loops with the same organization were found to be present in the three species, whereas Pleurodeles was the only one to exhibit, in its lampbrush chromosomes, the typical globular matrices previously described. “Sequential labelling loops” of Notophthelmus were shown to be similar of both “convoluted dense loops” of Xenopus and “dense loops” of Pleurodeles.