Preliminary note

Effects of α-amanitin on Triturus lampbrush chromosomes☆

Cajal bodies (coiled bodies) are nuclear organelles that contain a variety of components required for transcription and processing of RNA. Cajal bodies in amphibian oocytes are stained by mAb H14, which recognizes the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II when the heptapeptide repeat is phosphorylated on serine-5. Oocytes were treated with the transcription inhibitor 5,6-dichloro-1-β-$\text{d}$-ribofuranosylbenzimidazole (DRB), which prevents phosphorylation of the CTD. Cajal bodies from oocytes that had been treated for 2–3 h with DRB no longer stained with mAb H14, but staining reappeared when the inhibitor was washed out. Epitope-tagged transcripts of two small subunits of polymerase II, RPB6 and RPB9, were injected into the cytoplasm of Xenopus and Triturus oocytes. Newly translated RPB6 and RPB9 were specifically targeted to Cajal bodies within 4 h, and Cajal bodies remained the site of highest concentration of tagged protein during the next 2 days. These data suggest that polymerase subunits pass through the Cajal bodies with a transit time no greater than a few hours. We discuss the possibility that Cajal bodies are sites of assembly or modification of the transcription machinery of the nucleus.

The effects of pretreatment and simultaneous treatment with ethidium bromide (EB) on the clustering of chromatid translocations induced by hydroxyurea (HU) in barley nucleolus organizing regions (NORs) have been studied. EB significantly increased the involvement of barley NORs in chromatid translocations, while actinomycin D, α-amanitin and caffeine had been found to reduce it. Compaction (actinomycin D, α-amanitin, caffeine) and decondensation (EB) of rDNA, respectively, are assumed to be causally related to these differential effects.

Nuclei of amphibian oocytes contain large amounts of actin, mostly in unpolymerized or short-polymer form. When antibodies to actin or actin-binding proteins (fragmin and the actin modulator from mammalian smooth muscle) are injected into nuclei of living oocytes of Pleurodeles waltlii, transcription of the lampbrush chromosomes, but not of the rRNA genes, is inhibited. When transcription is repressed by drugs or RNA is digested by microinjection of RNAase into oocyte nuclei, an extensive meshwork of actin filament bundles is seen in association with the isolated lampbrush chromosomes. These observations indicate a close relationship between the state of nuclear actin and transcriptional activity and suggest that nuclear actin may be involved in transcriptional events concerning protein-coding genes.

Antibodies directed against RNA polymerase II (B) from Drosophila melanogaster were obtained from rabbit sera and, as monoclonal immunoglobulins, from mouse hybridomas and shown to cross-react with the amphibian enzyme protein. Localization by indirect immunofluorescence microscopy revealed the association of this enzyme with chromatin of interphase nuclei of amphibian cells and its absence in nucleoli. Purified immunoglobulins were microinjected into nuclei of living vitellogenic oocytes of Pleurodeles waltlii and Xenopus laevis and their effects on transcriptional processes were monitored by biochemical and light and electron microscopic studies. RNA polymerase II antibodies from rabbit sera caused a rapid and almost complete release of nascent transcripts from the chromatin axis of the loops of lampbrush chromosomes, followed by collapse of the loops and their retraction on the main chromosome axis. Monoclonal murine antibodies to the large RNA polymerase II subunits also inhibited transcription in chromosome loops but appeared to inhibit initiation rather than elongation events. Activities of class I and III RNA polymerases were not significantly affected by injection of antibodies to polymerase II, indicating immunological differences between the three RNA polymerases. The potential value of the in vitro test system described, as a very sensitive assay for detecting proteins involved in transcription in living cells, is discussed.

α-Amanitin interferes directly with transcription in eukaryotes by specific inhibition of RNA polymerase II. Administration of a sub-lethal dose of amanitin to adult chickens caused rapid but transient alterations in liver nuclei. Between 4.5 and 10 h after treatment, hepatocyte nuclei were swollen and exhibited extensive compaction of euchromatin, disaggregated nucleoli and segregated RNP components. By 48 h, however, the structural organization within nuclei was once more normal. The molecular composition of isolated nuclei was monitored in parallel with the structural analysis. Total RNA and residual NHP levels varied in concert with morphological changes in nuclei, decreasing sharply between 4.5 and 10 h after amanitin and returning to control levels by 48 h. DNA and histone quantities however, remained unchanged throughout. No large scale, qualitative changes in histone composition were detected by electrophoretic analysis, and only minor alterations in NHP patterns were evident. The results indicate that transcription, nucleolar activity and the structural state of chromatin are very closely integrated, and lend support to recent suggestions that the process of transcription may itself be causal in keeping chromatin physically dispersed or decondensed.

A hypothesis about lampbrush chromosome function is proposed which takes into account several aspects of their morphology and behavior. It is suggested that in lampbrush loops, alterations of the deoxyribonucleoprotein fiber take place which reprogram the chromosome for development. Such reprogramming could involve exchange or enzymatic modification of regulatory molecules (i.e., non-histone proteins). Since inhibition of RNA synthesis causes the loops to collapse, it is possible that transcription largely occurs to generate and maintain the loop, allowing the DNP fiber to “enter” the nucleoplasm. Lampbrush chromosomes are also involved in the production of “masked” mRNAs for early development. The giant granular loops could either be the sites where such mRNAs are packaged or they could be sites where specific alterations of the deoxyribonucleoprotein fiber take place.