Structural components of the nuclear body in nuclei of Allium cepa cells.

Nuclear bodies have long been noted in interphase nuclei of plant cells, but their structural component, origin and function are still unclear by now. The present work showed in onion cells the nuclear bodies appeared as a spherical structure about 0.3 to 0.8 microm in diameter. They possibly were formed in nucleolus and subsequently released, and entered into nucleoplasm. Observation through cytochemical staining method at the ultrastructural level confirmed that nuclear bodies consisted of ribonucleoproteins (RNPs) and silver-stainable proteins. Immunocytochemical results revealed that nuclear bodies contained no DNA and ribosomal gene transcription factor (UBF). Based on these data, we suggested that nuclear bodies are not related to the ribosome or other gene transcription activities, instead they may act as subnuclear structures for RNPs transport from nucleolus to cytoplasm, and may also be involved in splicing of pre-mRNAs.

Ultrastructural rRNA localization in plant cell nucleoli. RNA/RNA in situ hybridization, autoradiography and cytochemistry.

The distribution of ribosomal transcripts in the plant nucleolus has been studied by non-isotopic in situ hybridization in ultrathin Lowicryl K4M sections and by high-resolution autoradiography after labelling with tritiated uridine. In parallel, cytochemical techniques were applied to localize RNA on different plant nucleolar components of Allium cepa L. root meristematic cells and Capsicum annuum L. pollen grains. For RNA/RNA in situ hybridization, several biotinylated single-stranded ribosomal RNA probes were used for mapping different fragments of the 18 S and the 25 S rRNA gene transcribed regions. Ribosomal RNAs (from pre-rRNAs to mature 18 and 25 S RNAs) were found in the nucleolus, in the dense fibrillar (DFC) and granular components (GC). Hybridization signal was found at the periphery of some fibrillar centres (FCs) with probes recognizing both 18 and 25 S rRNA sequences. A quantitative study was performed to analyze the significance of this labelling. Incorporation of tritiated uridine into roots was carried out and, later, after a long time-exposure, autoradiography revealed the presence of newly synthesized RNA mainly in the DFC and at the periphery of the FCs. The presence of RNA in these areas was also confirmed by the cytochemical techniques used in this study. Taken together, these data favour the hypothesis that transcription can begin at the periphery of the FCs, although we cannot exclude the possibility that the DFC plays a role in this process.

[Ultrastructural in situ hybridization: detection of mRNA on sections of tissue embedded in lowicryl resin]. / L'hybridation in situ ultrastructurale: détection d'ARNm sur coupes d'échantillons inclus en résine lowicryl.

In situ hybridization for the detection of RNAm in ultrathin tissue sections embedded in lowicryl resin is reported. This method proved suitable for both tissues and cell cultures, using either complementary DNA probes or synthetic oligonucleotides, labeled with a radioisotope or biotin. This method seems to strike a satisfactory balance between preservation of cell ultrastructure and sensitivity.

[Detection of mRNA in electronic microscopy: in situ hybridization of a biotin-labeled oligonucleotide prior to embedding]. / Détection d'ARNm en microscopie électronique: hybridation in situ d'un oligonucléotide biotinylé en pré-inclusion.

A technique for mRNA detection by ultrastructural in situ hybridization is reported. Vibratome sections are hybridized with a biotinylated oligonucleotide probe which is detected using an enhanced avidin-peroxidase technique. Sections are then osmicated and embedded in epoxy resin. This rapid, sensitive technique achieved ultrastructural detection of vasopressin mRNA in magnocellular neurons of rat hypothalamus.

[Ultrastructural in situ hybridization on ultrathin sections of frozen tissues]. / Hybridation in situ ultrastructurale sur couples ultrafines de tissus congelés.

In situ hybridization on ultrathin frozen sections can be performed with either complementary DNA or synthetic oligonucleotide probes, labeled with 35S or an antigen (biotin or digoxigenin) respectively revealed by ultrastructural autoradiographic and immunocytological techniques. Using this method, GH mRNA was found in somatotrophs, mainly in the cytoplasm (endoplasmic reticulum and cytoplasmic matrix). Ultrastructural deterioration is the main drawback of this method but is more than outweighed by high sensitivity.

Expression of the rat growth hormone-releasing hormone gene in placenta is directed by an alternative promoter.

Growth hormone-releasing hormone (GHRH) is a hypothalamic peptide that plays a critical role in controlling the synthesis and secretion of growth hormone by the anterior pituitary. GHRH has also been detected in other nonneural extrahypothalamic tissues, including rat placenta, although its role in the hormonal control of pregnancy and/or fetal development has not yet been defined. Here we present the isolation and characterization of cDNA clones corresponding to rat placental GHRH. The placental GHRH mRNA codes for a pre-pro-GHRH identical to that found in the hypothalamus, suggesting that the mature placental GHRH is identical to its hypothalamic counterpart. Nevertheless, the placental and the hypothalamic GHRH mRNAs differ in the region corresponding to the untranslated exon 1 because of the use of an alternative promoter in the placenta located 10 kilobases upstream from the hypothalamic promoter. A combined mechanism involving the use of tissue-specific alternative promoters and the differential splicing of exon 1 generates the mature GHRH transcript in placenta and hypothalamus. Multiple transcription initiation sites have been found in the placental GHRH mRNA, which correlates to the lack of a consensus TATA box in the promoter region.

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12.

Three different approaches were used to examine the regulatory effects of the amino acids specified by the peptide-coding region of the leader transcript of the ilvGMEDA operon of Escherichia coli K-12. Gene expression was examined in strains carrying an ilvGMED'-lac operon fusion. In one approach, auxotrophic derivatives were starved of single amino acids for brief periods, and the burst of beta-galactosidase synthesis upon adding the missing amino acid was determined. Auxotrophic derivatives were also grown for brief periods with a limited supply of one amino acid (derepression experiments). Finally, prototrophic strains were grown in minimal medium supplemented with single and multiple supplements of the chosen amino acids. Although codons for arginine, serine, and proline are interspersed among the codons for the three branched-chain (regulatory) amino acids, they appeared to have no effect when added in excess to prototrophs or when supplied in restricted amounts to auxotrophs. Deletions removing the terminator stem from the leader removed all ilv-specific control, indicating that the attenuation mechanism is the sole mechanism for ilv-specific control.

Selenoprotein A component of the glycine reductase complex from Clostridium purinolyticum: nucleotide sequence of the gene shows that selenocysteine is encoded by UGA.

The gene encoding the selenoprotein A component of glycine reductase was isolated from Clostridium purinolyticum. The nucleotide sequence of this gene (grdA) was determined. The opal termination codon (TGA) was found in-frame at the position corresponding to the location of the selenocysteine residue in the gene product. A comparison of the nucleotide sequences and secondary mRNA structures corresponding to the selenoprotein A gene and the fdhF gene of Escherichia coli formate dehydrogenase shows that there is a similar potential for regulation of the specific insertion of selenocysteine at the UGA codon.