3-D organization of ribosomal transcription units after DRB inhibition of RNA polymerase II transcription.

In each bead of the nucleolar necklace, using adenosine analog DRB-treated PtK1 cells, we investigated the three components of rDNA transcription, i.e. the gene, transcription factor UBF and transcripts. In situ hybridization revealed the unraveling and 3-D dispersion of most of the rDNA coding sequences within the nucleus. The signals were small, of similar intensity and tandemly organized in the necklace. This observation is compatible with the fact that they might correspond to single gene units. Active transcription was visualized in these units, demonstrating that they were active functional units. Transcript labeling was not similar for each unit, contrary to UBF labeling. UBF and rRNA transcripts were only partially colocalized, as demonstrated by 3-D image analysis and quantification. As visualized by electron microscopy, the necklace was composed of a small fibrillar center partially surrounded by a dense fibrillar component. The 3-D arrangement of this individual unit in the necklace, investigated both by confocal and electron microscopy in the same cells, showed that the individual beads were linked by a dense fibrillar component. The reversibility of this organization after removal of DRB indicated that the beads in the necklace are certainly the elementary functional domain of the nucleolus. In addition, these results lead us to suggest that the organization of a functional domain, presumably corresponding to a single gene, can be studied by in situ approaches.

Involvement of in situ conformation of ribosomal genes and selective distribution of upstream binding factor in rRNA transcription.

The distribution of the ribosomal genes (rDNA) and the upstream binding factor (UBF), correlatively with their RNA transcripts, was investigated in G1, S-phase, and G2. rDNA was distributed in nucleoli, with alternate sites of clustered and dispersed genes. UBF was found associated with some but not all clustered genes and proportionally more with dispersed genes. It was distributed in several foci that were more numerous and heterogeneous in size during G2 than G1. We suggest that UBF associated with rDNA during S-phase because its nucleolar amount increased during that time and remained stable in G2. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole treatment indicated a similar amount of UBF per transcription unit, and consequently heterogeneous size of the UBF foci can represent a variable number of transcription units per foci. Direct visualization of the transcripts demonstrated that only part of UBF is associated with active transcription and that rDNA distribution varied with transcription. We propose that in the same rDNA locus three types of configuration coexist that are correlated with gene activity: 1) clustered genes without UBF; 2) clustered genes with UBF, of which some are associated with transcription; and 3) dispersed genes with UBF and transcription. These results support the hypothesis that rDNA transcription involved several steps of regulation acting successively and locally in the same locus to promote the repressed clustered genes to become actively transcribed dispersed genes.

The three-dimensional organization of ribosomal genes and the architecture of the nucleoli vary with G1, S and G2 phases.

The three-dimensional (3-D) organization of the nucleolus, a defined nuclear territory containing tandem repeats of the ribosomal genes (rDNA), was investigated in PtK1 cells. Identification of the interphase stages was performed in single cells using DNA quantification by cytometry for the G1 and G2 phases while the S phase was identified by immunolabelling of the proliferating cell nuclear antigen (PCNA). The 3-D organization of the rDNA in the nucleolus was analyzed by fluorescence in situ hybridization using confocal microscopy. All the rDNA was located inside the nucleolar structures during all stages and the two rDNA loci were orthogonal. The rDNA was heterogeneously distributed in each nucleolus during G1, S and G2, with alternate sites of clustered genes (spots) and of genes in more extended configurations. The number of spots (4 to 6 in G1) increased during S phase (7 to 12) and their 3-D organization was progressively relaxed from G1 to G2. Double spots in G2 could reflect a similar gene organization of two chromatids. During mid-S phase, PCNA co-localized with some clustered genes (spots), indicating that rDNA replication occurs inside nucleoli and at different sites of the same locus simultaneously. The evaluation of the rDNA transcription units in 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)-treated cells indicated a mean of 16 units per G1 nucleus and 25 units per G2 nucleus. For G1 and G2, the fine 3-D structure of nucleoli was reconstructed using ultrathin serial sections after specific contrast of DNA and RNA, digitization of the serial section images and computer-assisted 3-D architecture. Fibrillar centers (FCs) formed discrete structures (about 10 in G1 and 20 in G2) connected by a network of the dense fibrillar component. The 3-D arrangement of the FCs in G1 and G2 are similar to the rDNA spots. In conclusion, the architecture of the nucleoli during interphase reflects the distribution of the rDNA that is characterized by alternation of clustered and extended genes.

Three-dimensional organization of the ribosomal genes and Ag-NOR proteins during interphase and mitosis in PtK1 cells studied by confocal microscopy.

The three-dimensional (3-D) organization of rDNA-containing chromatin and the set of protein markers of active ribosomal genes, the Ag-NOR proteins, were investigated by confocal laser scanning microscopy (CLSM). The rDNA genes of marsupial cells (PtK1) were mapped using biotinylated DNA probes for 45S rDNA sequences and the Ag-NOR protein distribution was revealed by specific Ag-NOR staining. We used PtK1 cells because each nucleolus possesses only one nucleolar organizer region (NOR). In metaphase chromosomes, nonisotopic in situ hybridization demonstrated the presence of rDNA in the secondary constriction of the X chromosomes with an axial distribution and also lateral expansions. 3-D reconstruction of the Ag-NOR protein signals revealed the presence of these proteins in the secondary constriction where they formed a crescent-shaped structure around the axial chromatin pedicule. The organization of the secondary constriction in PtK1 chromosomes is discussed. During interphase, nonisotopic in situ hybridization in intact cell monolayers and isolated nuclei showed the rDNA genes distributed as intense fluorescent spots linked by weak signals in the inner regions of the nucleoli. We conclude that the rDNA is not homogeneously distributed in the internal regions of the nucleoli. In the same nucleolar regions, the Ag-NOR proteins were revealed as granules linked by thin filaments. These images indicate similar 3-D distributions for rDNA probes and Ag-NOR proteins. The beaded organization of the transcriptional regions in the nucleoli is discussed.

Characterization and hormonal regulation of tissue and fluid proteins in the mouse epididymis.

The proteins of epididymal tissues and fluids recovered from different regions of the mouse epididymis from a natural population and an inbred line were examined by polyacrylamide gel electrophoresis under denaturing conditions. Two epididymal specific peptides on the order of 88 and 20 Kilodaltons (Kd), undetected in serum and testicular extracts, were identified in the initial segment, caput, corpus and cauda. Another specific 30 Kd peptide was localized in the cauda tissue and fluid. Castration caused the disappearance of the three specific epididymal bands and of a non-specific 34 Kd band. In contrast, a new band appeared at 14.5 Kd. Testosterone propionate administration only restored the three specific epididymal bands and had no effect on the 14.5 peptide. Variations in the staining intensity of the four bands, which were suppressed in castrated animals, were observed after ductuli efferentes ligation.

Characterization of regional proteins in tissues and fluids in the human epididymis.

The proteins of epididymal tissues and fluids recovered from five regions of the human epididymis were separated by polyacrylamide gel electrophoresis under denaturing conditions. Among the 60 peptides identified, eight appeared to be expressed solely in the epididymal duct when compared to serum and testis proteins. Three of these (92, 47 and 24 Kd) showed a degree of regional specificity in fluids. The 92 Kd peptide was found in the caput and proximal corpus, the 47 Kd in the distal corpus and cauda and the 24 Kd in the caput of the epididymis. Three of the specific epididymal proteins (39, 30, 26 Kd) displayed a remarkable analogy to those found in man and monkey in other conditions and which are present at the sperm surface in the epididymis cauda.

[Hormonal control of vitellogenin synthesis in the Amphipoda Crustacea Orchestia gammarella (Pallas)]. / Quelques aspects du contrôle hormonal de la synthèse de la vitellogénine chez le crustacé amphipode Orchestia gammarella (Pallas).

The presence of a vitellogenin stimulating ovarian hormone (VSOH) in Orchestia gammarella has been demonstrated by gonadectomy and ovary grafting. Vitellogenin synthesis is not affected by molting hormone injection; it decreases after Y-organ (molting gland) cauterization. Studies in progress seem to indicate that the destruction of the median area of the protocerebrum is followed by a decrease of this synthesis.

[Molecular weight of lipovitellins during egg development in the crustacean amphipod Orchestia gammarella (Pallas)]. / Le poids moléculaire des lipovitellines au cours du de'veloppement des oeufs chez le Crustacé Amphipode Orchestia gammarella (Pallas).

Egg lipovitellins of Orchestia gammarella tested by electrophoresis on gels of different acrylamide concentrations, following the procedure of Hedrick and Smith (1968), display a migration pattern identical to that of proteins with molecular weights of congruent to 3.2 x 10(5) (lipovitellin I) and congruent to 5.5 x 10(5) (lipovitellin II) respectively. Since their molecular weight remains constant during embryogenesis, the changes of their relative mobility in disc gels indicates alterations in their ionic charges.

[Determination of the molecular weight of vitellogenin and of lipovitellins of Orchestia gammarella, Crustacea, Amphipoda]. / Détermination du poids moléculaire de la vitellogénine et des lipovitellines d'Orchestia gammarella, Crustacé Amphipode.

Vitellogenin and lipovitellins of Orchestia gammarella, tested by electrophoresis on gels of different acrylamide concentrations, following the procedure of Hedrick and Smith (1968), displays a migration pattern identical to that of proteins of respectively congruent to 4 x 10(5) (vitellogenin), congruent to 3,5 x 10(5) (lipovitellins I and I') and congruent to 5 x 10(5) (lipovitellin II) molecular weights.

[Molting and vitellogenesis in Orchestia gammarella Pallas (crustacea amphipoda): study of the female-specific protein synthesis after ecdysterone administration (author's transl)]. / Mue et vitellogenèse chez Orchestia gammarella Pallas (crustacé amphipode); étude de la synthèse de la fraction protéique femelle après introduction d'ecdystérone

Ecdysterone, administered to Orchestia gammarella females (200 mug/g), has no positive effect on the female-specific protein synthesis and vitellogenesis.