I. Introduction.- II. Ribosomal Genes.- II. 1. Definitions.- II.2. Ribosomal RNA Genes.- II.2.1. Multiplicity.- II.2.2. Chromosomal Location.- II.2.3. Extrachromosomal rRNA Genes.- II.2.4. Organization and Structure.- II.2.4.1. Saccharomyces cerevisiae.- II.2.4.2. Tetrahymena.- II.2.4.3. Drosophila.- II.2.4.4. Xenopus laevis.- II.2.4.5. Higher Plants.- II.2.4.6. Mammalia.- II.2.5. General Features.- II.3. 5 S rRNA Genes.- II.3.1. Number and Chromosomal Location.- II.3.2. Organization and Structure.- II.4. Ribosomal Protein Genes.- II.5. Synopsis.- III. Transcription of Ribosomal Genes.- III. 1. Components of the Transcription Complex.- III. 1.1. RNA Polymerases.- III. 1.2. Nucleolar rDNA and r-Chromatin.- III.2. The Transcription Process >.- III.2.1. Topology of Primary Pre-rRNA.- III.2.2. Morphology of Transcribed rRNA Genes.- III.2.3. Transcribed and Non-Transcribed r-Chromatin.- III.2.4. Primary Transcripts and Primary Pre-rRNA.- III.2.5. Transcription Initiation and Termination.- III.2.5.1. Initiation.- III.2.5.2. Termination.- III.2.6. Transcription in vitro.- III.3. Transcription of 5 S rRNA Genes.- III.4. Transcription of r-Protein Genes.- III.5. Synopsis.- IV. Maturation of Preribosomes.- IV. 1. Structure of Primary Pre-rRNA.- IV. 1.1. Size and Primary Structure.- IV. 1.2. Modifications.- IV. 1.3. Conformation.- IV.2. Pre-rRNA Maturation Pathways.- IV.2.1. General Considerations.- IV.2.2. Common Pattern of Pre-rRNA Maturation.- IV.2.3. Multiplicity of Maturation Pathways.- IV. 2.4. Enzyme Mechanisms.- IV. 3. Preribosomes: Structure and Maturation.- IV. 4. Synopsis.- V. Molecular Architecture of the Nucleolus.- V. 1. Introduction.- V.2. Nucleolus Organizer.- V. 2.1. Chromosomes.- V.2.2. Interphase Nuclei.- V.3. Fibrillar and Granular Components.- V.3.1. The Fibrillar Component.- V.3.2. The Granular Component.- V.4. The Nucleolus and Other Nuclear Structures.- V.4.1. Nucleolus-Associated Chromatin.- V.4.2. The Junction with the Nuclear Envelope.- V.5. The Nucleolar Matrix.- V.6. Macromolecular Constituents.- V.6.1. DNA and RNA.- V. 6.2. Nucleolar Proteins.- V.6.2.1. General.- V.6.2.2. Ag-NOR Protein(s).- V. 6.2.3. Nucleolar Antigens.- V. 7. Outline.- VI. Regulation.- VI. l. General Considerations.- VI.2. Transscriptional Control.- VI. 2.1. Transitions in the State of Expression of rRNA Genes.- VI. 2.1.1. Inactive r-Chromatin.- VI.2.1.2. Potentialy Active and Transcribed rRNA Genes.- VI.2.2. Control of Transcription Rate.- VI.2.2.1. Role of RNA Polymerase I.- VI.2.2.2. Supply of Nucleoside-5'-Triphosphates...- VI.2.2.3. Role of Protein Synthesis.- VI.3. Posttranscriptional Control.- VI.3.1. Synthesis and Supply of r-Proteins.- VI.3.2. The Role of Pre-rRNA Structure.- VI.3.3. The Role of 5 S rRNA.- VI.3.4. Critical Control Sites.- VI.3.4.1. Alternative Processing Pathways and Intranuclear Degradation of Preribosomes and Ribosomes.- VI.3.4.2. Release From the Nucleolus and Nucleo- Cytoplasmic Transport of Ribosomes.- VI.3.4.3. Turnover of Ribosomes.- VI.4. Autogeneous Regulation of Ribosome Biogenesis in Eukaryotes: A Model.- VI.5. Synopsis.- VII. Ribosome Biogenesis in the Life Cycle of Normal and Cancer Cells.- VII. 1. Nucleologenesis and Nucleololysis.- VII. 1.1. Nucleoli and Ribosome Biogenesis During the Mitotic Cycle.- VII. 1.2. Nucleologenesis.- VII. 1.3. Nuclyeololysis.- VII.2. Inhibition of Ribosome Biogenesis.- VII.2.1. Inhibitors Interacting With DNA and Chromatin.- VII.2.2. Inhibitors That Act on RNA Polymerases.- VII.2.3. Inhibitors of Nucleoside-5'-Triphosphate Formation.- VII.2.4. The Effects of Analogues Incorporated into Polyribonu- cleotide Chains.- VII.2.5. Inhibitors of Protein Synthesis.- VII.2.6. Interpretation of Nucleolar Alterations.- VII.2.6.1. Nucleolar Segregation.- VII.2.6.2. Nucleolar Spherical Bodies and Perichromatin Granules.- VII.2.6.3. Microspherules.- VII.2.6.4. Nucleolar Fragmentation.- VII.3. Growth Transitions.- VII.3.1. Modulation of Growth Rates in Yeasts.- VII.3.2. Activation of Lymphocytes.- VII.3.3. Growth Stimulation of Cultured Cells.- VII.3.4. Differentiation of Myoblasts in Culture.- VII.3.5. Regeneration of Rat Liver.- VII.4. Senescent and Cancer Cells.- VII.4.1. Senscent Cells and Tissues.- VII.4.2. Cancer Cells.- VII.5. Synopsis.- References.

The nucleolus and ribosome biogenesis

A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster.

DNA in prepared chromosomes from the larval salivary glands of Drosophila melanogaster was hybridized with [125I]-labeled 5S and tRNA from the same organism. Autoradiography revealed that radioactivity was frequently bound to all regions of the slides, masking labeling of the chromosomes. Acetylation of the preparations before hybridization prevented the formation of this background and revealed the specific chromosomal sites.

Acetylation of chromosome squashes of Drosophila melanogaster decreases the background in autoradiographs from hybridization with [125I]-labeled RNA.

The chromosomal locations of ribosomal DNA in wheat, rye and barley have been determined by in situ hybridization using high specific activity 125I-rRNA. The 18S-5.8S-26S rRNA gene repeat units in hexaploid wheat (cv. Chinese Spring) are on chromosomes 1B, 6B and 5D. In rye (cv. Imperial) the repeat units occur at a single site on chromosome 1R(E), while in barley (cv. Clipper) they are on both the chromosomes (6 and 7) which show secondary constrictions. In wheat and rye the major 5S RNA gene sites are close to the cytological secondary constrictions where the 18S-5.8S-26S repeating units are found, but in barley the site is on a chromosome not carrying the other rDNA sequences. — Restriction enzyme and R-loop analyses showed the 18S-5.8S-26S repeating units to be approximately 9.5 kb long in wheat, 9.0 kb in rye and barley to have two repeat lengths of 9.5 kb and 10 kb. Electron microscopic and restriction enzyme data suggest that the two barley forms may not be interpersed. Digestion with EcoR1 gave similar patterns in the three species, with a single site in the 26S gene. Bam H1 digestion detected heterogeneity in the spacer regions of the two different repeats in barley, while in rye and wheat heterogeneity was shown within the 26S coding sequence by an absence of an effective Bam H1 site in some repeat units. EcoR1 and Bam H1 restriction sites have been mapped in each species. — The repeat unit of the 5S RNA genes was approximately 0.5 kb in wheat and rye and heterogeneity was evident. The analysis of the 5S RNA genes emphasizes the homoeology between chromosomes 1B of wheat and 1R of rye since both have these genes in the same position relative to the secondary constriction. In barley we did not find a dominant monomer repeat unit for the 5S genes.

Molecular and chromosomal organization of DNA sequences coding for the ribosomal RNAs in cereals

3H-rRNA obtained from Xenopus laevis tissue cultured cells, or a 3H-cRNA made from Xenopus ribosomal DNA, was used for heterologous in situ hybridisation with human lymphocyte metaphase chromosomes. Prior to hybridisation, chromosome spreads were stained with Quinacrine and selected cells showing good Q-banding photographed; the same cells were then rephotographed after autoradiography and pairs of photographs for each cell were used to make dual karyotypes. The chromosomes within each karyotype were divided into equal sized segments (approx. 0.7 μ), with a fixed number of segments for each chromosome type. The distribution of silver grains between segments showed that the 3H-RNAs hybridised specifically to the nucleolar organising regions of the D and G group chromosomes with no other sites of localised labelling in the complement. Control experiments showed no localisation, with insignificant labelling, when metaphase spreads were incubated in a mixture containing Xenopus3H-rRNA and competing cold human (HeLa) rRNA. Filter hybridisation experiments on isolated human DNA showed that the Xenopus derived 3H-RNAs hybridised to a fraction of human DNA which was on the heavy side of the main DNA peak and that these RNAs were competed out in the presence of excess cold human rRNA, confirming the specificity of the heterologous hybridisation. In situ hybridisation experiments were also carried out on cells from individuals with one chromosome pair showing heteromorphism for either a very long stalk (nucleolar constriction) subtending a satellite, or a large satellite. It was shown that the chromosome with the large stalk hybridised four times as much 3H-rRNA as its homologue, whereas differences in the sizes of the subtended satellites did not materially affect hybridisation levels indicating that rDNA is located in the stalks and not the satellites. The amount of 3H-rRNA hybridised differs between chromosomes and individuals; these differences are heritable and rDNA can be detected by in situ hybridisation in all three chromosomes number 21 in cells from Down's patients and in translocated chromosomes conta.ining a nucleolar constriction. Different D and G group chromosomes which hybridised equal amounts of 3H-rRNA participated in rosette associations at metaphase in a random fashion in some individuals and in a non-random fashion in others. In all individuals studied chromosomes with large amounts of rDNA were not found to be preferentially involved in association. It was therefore concluded that the probability of a chromosome being involved in the formation of a common nucleolus is not a simple function of its rDNA content and other possible factors are considered.

Location of the genes coding for 18S and 28S ribosomal RNA in the human genome

[ 3H ] RNA with a high specific activity was prepared from larvae of Drosophila melanogaster grown 4 days in contact with [ 3H ] uridine. Purified tritiated 5 S RNA was annealed to the DNA of polytene chromosomes, which had been denatured in formamide. The 5 S RNA genes are placed within the region 56E-F of the right arm of chromosome 2. This localization was determined from autoradiographs, where the radioactivity from hybrids of [ 3H ] RNA and DNA was confined to the 56E-F segment.

https://science.sciencemag.org/content/sci/170/3958/639.full.pdf

D. M. Steffensen

Papers

Localization of 5S RNA Genes on Drosophila Chromosomes by RNA-DNA Hybridization