CONTROL OF 5S RNA SYNTHESIS DURING EARLY DEVELOPMENT OF ANUCLEOLATE AND PARTIAL NUCLEOLATE MUTANTS OF XENOPUS LAEVIS

Ribosomes of all eukaryotes contain a single molecule of 5S, 18S, and 28S RNA. In the frog Xenopus laevis the genes which code for 18S and 28S RNA are located in the nucleolar organizer, but these genes are not linked to the 5S RNA genes. Therefore the synthesis of the three ribosomal RNAs provides a model system for studying interchromosomal aspects of gene regulation. In order to determine if the synthesis of the three ribosomal RNAs are interdependent, the relative rate of 5S RNA synthesis was measured in anucleolate mutants (o/o), which do not synthesize any 18S or 28S RNA, and in partial nucleolate mutants (p^l-1/o), which synthesize 18S and 28S RNA at 25% of the normal rate. Since the o/o and p^l-1/o mutants have a complete and partial deletion of 18S and 28S RNA genes respectively, but the normal number of 5S RNA genes, they provide a unique system in which to study the dependence of 5S RNA synthesis on the synthesis of 18S and 28S RNA. Total RNA was extracted from embryos labeled during different stages of development and analyzed by polyacrylamide gel electrophoresis. Quite unexpectedly it was found that 5S RNA synthesis in o/o and p^l-1/o mutants proceeds at the same rate as it does in normal embryos. Furthermore, 5S RNA synthesis is initiated normally at gastrulation in o/o mutants in the complete absence of 18S and 28S RNA synthesis.     

Variation in the activity of nucleolar organizers and their ribosomal gene content

In metaphase preparations from leucocytes of the toad, Bufo marinus, conspicuous secondary constrictions are present in the number 7 pair of chromosomes. These constrictions were considered to be the nucleolar organizers since they were associated with nucleoli during prophase. In 35 out of 60 individuals taken from natural populations, the homologous nucleolar organizers produced two equal-sized nucleoli and secondary constrictions (Group I animals). Pour animals (Group II) had only one very large secondary constriction in the majority of their metaphase preparations and an abnormally high frequency of cells containing one nucleolus. The remaining 21 animals (Group III) had unequal-sized constrictions in most of their metaphases but were more variable than the individuals of Groups I or II since they also had metaphases with two equal constrictions or only one constriction.The DNA from individuals of each group was hybridized with radioactive ribosomal RNA in order to correlate the size of nucleoli and constrictions with the amount of DNA (rDNA) homologous to ribosomal RNA. The two animals of Group II which were studied contained 0.056% of their genome homologous to ribosomal RNA a value considerably higher than those found for any of the animals of Groups I or III. These high values for rDNA coupled with the morphological appearance of the nucleolar homologues suggested a duplication of the nucleolar organizer in the homologue with the long constriction. The amount of rDNA in animals of Group I and III varied between 0.025 and 0.048% of the genome. Although the animals with unequal-sized constrictions (Group III) had generally lower contents of rDNA than those with equal-sized constrictions (Group I), the values overlapped between the two groups. Further evidence which correlates the size of nucleoli with the number of ribosomal RNA genes comes from studies with a small nucleolar mutant of the Mexican axolotl (Ambystoma mexicanum). Animals homozygous for this deletion were found to contain only 55% of the complement of rDNA present in the wild type. It is concluded that partial deletions and duplications of the nucleolar organizer as well as highly variable contents of rDNA are common in the genome of these amphibians.Presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Biology Department, University of Rochester.     

Quantitative micro-assay for RNA/DNA hybrids in the study of nucleolar RNA from Chironomus tentans salivary gland cells

A microassay for RNA/DNA hybrids has been designed for the study of RNA from different nuclear components of Chironomus tentans salivary gland cells. The procedure comprises a scale reduction of the conventional filter method for hybridization, using ultraviolet microphotometry for quantitation of RNA and DNA. Hybridization is performed in 0.3 μl of 2 × SSC containing 1–2 × 10^-2 μg DNA, immobilized on a 0.2 mm² ‘micro-filter’, and 0.5–5 × 10⁻² μg RNA, with a specific activity of more than 10⁶ cpm/μg. Results obtained by the microtechnique are found to agree with results obtained by a large-scale, standard procedure. The applicability of the microtechnique is demonstrated in saturation and presaturation-competition experiments. RNA from micro-isolated nucleoli hybridizes a maximum of 0.22% of Chironomus tentans DNA, which corresponds to about 100 cistrons for the 38S ribosomal precursor in the haploid genome. The hybrids show a steep thermal dissociation profile with a T_m of 79 °C, close to the value expected for hybrids with a G + C content of 42%. Presaturation of filter-bound DNA by total unlabelled nucleolar RNA prevents 80% of the subsequent hybridization by labeled nucleolar Presaturation by RNA from one of the two nucleolar organizers prevents to a similar degree the subsequent hybridization by RNA from the other nucleolar organizer. This result indicates a sequence similarity of RNA transcribed in different nucleolar organizers. Further applications of the microtechnique are presented in the accompanying paper where the hybridization properties of chromosomal and nuclear sap RNA are investigated.     

Drosophila melanogaster has different ribosomal RNA sequences on S and Y chromosomes.

The primary structures of ribosomal RNAs transcribed from the nucleolus organizers on X and Y chromosomes of Drosophila melanogaster were compared by RNase T₁ fingerprints made with two different systems; i.e. homochromatography on DEAE-cellulose, and polyethyleneimine-cellulose thin-layer chromatography.Ribosomal RNA derived from the X-linked nucleolus organizer was obtained from a strain producing only female larvae and ribosomal RNA derived from the Y-linked nucleolus organizer was isolated from a mutant lacking the X-linked nucleolus organizer.No difference was detected between the fingerprints of 28 S RNA from these animals.In 18 S RNA, however, one oligonucleotide showed a remarkable difference in mobility. The structure of the X-linked organizer-specific oligonucleotide was 5′ U-C-U-U-U-U-U-U-C-C-U-A-U-G 3′, and that of the Y-linked organizer-specific oligonucleotide was 5′ U-C-U-C-U-U-U-U-C-C-U-A-U-G 3′, indicating one base substitution (U á3 C) between them.The absence of 5′-temninal phosphate in this oligonucleotide and available sequence data also suggest that these oligonucleotides did not come from either the 5′ or 3′ terminus of 18 S RNA.D. simulans, whose Y chromosome has no nucleolus organizer (Ritossa &; Atwood, 1966), showed an 18 S RNA fingerprint having only the X-linked organizer-specific oligonucleotide.We conclude from these results that in Drosophila the ribosomal RNA gene sequences are different for the two nucleolus organizers located on the X and Y chromosomes. The implications of those findings concerning the parallel evolution of these genes are discussed.     

The nucleolar organizer of Plethodon cinereus cinereus (Green)

The lampbrush nucleolar organizers in P. c. cinereus are on the shorter arms of the 7th longest bivalent, near to the centromeres. The organizer consists of a non-chromomeric, Feulgen-negative section of the half bivalent axis inserted between typically chromomeric regions with normal lampbrush loops. The main axis of the organizer may appear double or single. The distributions and lengths of the double regions are variable. The axial strand of the organizer can be broken with DNase. Nucleolus-like objects are attached at irregular intervals along the organizer axis. The attached nucleoli closely resemble the free nucleoli in the same nucleus. Where free and attached nucleoli appear as beaded rings, the general characteristics and range of lengths of the attached rings are similar to those of the free rings. The attached rings occur singly, in pairs, or in clusters. The point of attachment of a ring is sometimes marked by a granule on the organizer axis. Pairs of attached nucleolar rings sometimes form double bridges in which the nucleoli extend linearly across a gap in the organizer axis. The length of the organizer varies from 20 μm to 300 μm, depending, at least in part, on the stage of oocyte development. The difference is a function of the length of the axial strand. Nucleolar organizers similar to that described for P. c. cinereus have been seen on the lampbrush bivalents of 7 other species of Plethodon and 2 species of Eurycea. In all of these, the 2 organizers of the nucleolus bivalents differ in length.     

The relationship of ribosomal RNA synthesis to the formation of segregated nucleoli and nucleolus-like bodies

The relationship of ribosomal RNA (rRNA) synthesis to nucleolar ultrastructure was studied in partial nucleolar mutants of Xenopus laevis. These mutations are the result of a partial deletion of rRNA genes and therefore alow studies on nucleolar structure and function without using drugs that inhibit rRNA synthesis. Ultrastructural studies demonstrated that normal embryos have reticulated nucleoli that are composed of a loose meshwork of granules and fibrils and a typical nucleolonema. In contrast, partial nucleolar mutants in which rRNA synthesis is reduced to less than 50% of the normal rate have compact nucleoli and nucleolus-like bodies. The compace nucleoli contain granules and fibrils, but they are segregated into distinct regions, and a nucleolonema is never seen. Since other species of RNA are synthesized normally by partial nucleolar mutants, these results demonstrate that nucleolar segragation is related specifically to a reduction in rRNA synthesis. The nucleolus-like bodies are composed mainly of fibrils,and the number of such bodies are composed mainly of fibrils, and the number of such bodies present in the different nucleolar mutants is inversely related to the relative rate of rRNA synthesis. Although the partial nucleolar organizers produce segregated nucleoli in these mutants, they organize morphologically normal, but smaller, nucleoli in heterozygous embryos. Alternative explanations to account for these results are discussed.     

Nature of the ribosomal RNA transcribed from the X and Y chromosomes of Drosophila melanogaster

In Drosophila melanogaster there is one nucleolar organizer (NO) on each X and Y chromosome. Experiments were carried out to compare the ribosomal RNAs derived from the two nucleolar organizers. ³²PO₄-labelled ribosomal RNA was isolated from two strains of D. melanogaster, one containing only the X chromosome NO, the other containing only the Y chromosome NO. 28 S and 18 S RNA from the two strains were subjected to a variety of “fingerprinting” and sequencing procedures. Fingerprints of 28 S RNA were very different from those of 18 S RNA. Fingerprints of “X” and “Y” 28 S RNA were indistinguishable from each other, as also were fingerprints of “X” and “Y” 18 S RNA. In combined “T₁ plus pancreatic” RNAase fingerprints several distinctive products were characterized and quantitated. Identical products were obtained from X and Y RNA, and the molar yields of the products were indistinguishable. Together these findings imply that the rRNA sequences encoded by the X and Y NOs are closely similar and probably identical to each other.Two further findings were of interest in “T₁ plus pancreatic” RNAase fingerprints: (1) in 28 S (as well as in 18 S) fingerprints several distinctive products were recovered in approximately unimolar yields. This indicates that 28 S RNA does not consist of two identical half molecules, though it does consist of two non-identical half molecules together with a “5.8 S” fragment. (2) Several methylated components in Drosophila rRNA also occur in rRNA from HeLa cells and yeast. This suggests that certain features of rRNA structure involving methylated nucleotides may be highly conserved in eukaryotic evolution.     

Nucleolus-like bodies in micronuclei of cultured Xenopus cells

Two of the 36 chromosomes in Xenopus laevis are known to carry nucleolar organizer loci. Partitioning of the chromosomes of cultured, early-passage Xenopus cells among variable numbers of micronuclei could be induced by extended colcemid treatment. A large, obvious nucleolus occurred in a maximum of 4 micronuclei per colcemid-induced tetraploid cell. The large, deeply-stained nucleoli incorporated [³H]uridine and appeared by electron microscopy to have typical nucleolar morphology with fibrillar and granular areas disposed in nucleolonema. In situ hybridization to radioactive ribosomal RNA (rRNA) resulted in heavy labelling of nucleoli in no more than 4 micronuclei per cell. The other micronuclei generally contained small bodies (blobs) which stained for RNA and protein as well as with ammoniacal silver. In the electron microscope, these appeared as round, dense bodies resembling nucleoli segregated by actinomycin D treatment. Nucleoplasmic RNA synthesis occurred in all micronuclei regardless of whether they contained definitive nucleoli. These observations suggest that micronuclei which formed large, typical, RNA-synthesizing nucleoli contained nucleolar organizer chromosomes, while the other micronuclei, which contained nucleolus-like “blobs” probably lacked nucleolar organizer loci. It is possible that the nucleolus-like bodies may have been aggregates of previously synthesized nucleolar RNA and protein trapped in micronuclei after mitosis.     

Ribosomal genes not integrated into chromosomal DNA in Drosophila melanogaster

DNA obtained by a gentle lysis procedure from adult Drosophila melanogaster was analyzed by sucrose gradient sedimentation. The major portion of the DNA has an estimated weight of at least 5–10×10⁹ daltons. All of the ribosomal genes are present in this high molecular weight DNA in adult males with one nucleolus organizer or in adult females with two nucleolus organizers as shown by hybridizing fractions of the gradient with ribosomal RNA. In female adults with one nucleolus organizer instead of the usual two, 68% of the ribosomal genes are found in high molecular weight DNA and 32% are found in DNA of smaller size (3×10⁸ daltons). We propose that these latter genes are not integrated into the DNA of the chromosome.     

Unintegrated ribosomal genes in diploid and polytene tissues of Drosophila melanogaster

We have examined DNA from polytene salivary glands and diploid brains and imaginal discs of male and female larvae having one or two nucleolus organizers. DNA having an estimated molecular weight of 5×10⁹ or greater was obtained by sucrose gradient sedimentation of gently prepared lysates. Hybridization of the gradient fractions with ³H-ribosomal RNA reveals that 42% of the ribosomal genes are found in DNA of lower molecular weight (approximately 3×10⁸ daltons) in the salivary glands of every genotype examined. In the brains and imaginai discs, by contrast, all of the ribosomal genes are found in the high molecular weight peak except in females with one nucleolus organizer where 42% are found in lower molecular weight DNA, as in the salivary gland. Thus unintegrated genes are not an exclusive feature of polytene tissue, but can occur in diploid tissue as well in at least one genotype.     

Variation in nucleolar organiser rRNA gene multiplicity in wheat and rye

In hexaploid wheat and diploid rye, different varieties have different numbers of ribosomal RNA genes as indicated by rRNA/DNA hybridisation. Wheat has four different chromosomes which carry nucleolar organisers. Analyses of DNA isolated from substitution lines in which each of these nucleolar organiser chromosomes of several varieties has been substituted one at a time into a common genetic background, have indicated that none of the four organiser chromosomes possess an invariant number of ribosomal RNA genes. The ribosomal RNA gene complement of the varieties investigated can be approximately accounted for by the sum of the ribosomal RNA genes on each of the four nucleolar organiser chromosomes.     

Amplification as a Rectification Mechanism for the Redundant rRNA Genes

IN eukaryotic cells multiple copies of ribosomal RNA (rRNA) genes are clustered in one or a few nucleolar organizers per haploid genome. These “redundant” or “reiterated” genes have all been shown to be, within the limits of available experimental techniques, identical within a given organism and species, producing homogeneous populations of rRNA molecules. On the other hand, rRNAs from organisms of different taxonomic position display evidence of considerable evolutionary change from the point of view of size, base sequence (revealed by RNA-DNA hybridization) and base composition^1,2.     

Cytosine methylation and nucleolar dominance in cereal hybrids

In wheat-rye hybrids the nucleolus organizer regions (NORs), the sites of ribosomal RNA genes, from rye are suppressed. Wheat and wheat-rye hybrid genetic stocks containing different numbers of wheat and rye nucleolus organizers, as well as addition lines and rye-barley hybrids, were used in Southern hybridization experiments to determine the cause of nucleolar dominance and suppression in cereal hybrids. Based on the use of restriction endonucleases that cleave near the ends of the spacer unit and an additional, methylation-sensitive enzyme, HpaII, which does not recognize the CCGG restriction site if the internal C is methylated, an indirect method of assaying NOR expression was established. The results indicated that cleavage by the HpaII enzyme of the rye NOR sequences, is reduced when major NORs from other cereals were present. The reduction in the number of rye rRNA genes containing an unmethylated CCGG site in the promoter was associated with the suppression of the rye nucleolus. These results are consistent with a model in which promoter and upstream regulatory repeats of ribosomal RNA genes compete for limited concentrations of regulatory proteins, and genes that are methylated at key binding sites fail to engage these regulatory proteins and thus remain inactive. Received: 15 November 1996 / Accepted: 19 March 1997     

Patterns of ribosomal DNA spacer lengths are inherited

The non-transcribed spacer regions in Xenopus laevis ribosomal DNA vary in length, even within a single nucleolar organizer. The pattern of spacer lengths is sufficiently different from one nucleolar organizer to another to allow the pattern to be used as a genetic marker. We have analyzed the spacer patterns of rDNA² from a total of 50 progeny from three separate matings. Spacer patterns were inherited with no detectable change in all but two cases. The reproducibility of the patterns among siblings and their stable inheritance between generations rule out sudden mechanisms for gene evolution, such as the master-slave model, and support more gradual mechanisms.Two animals out of 50 showed marked changes in their rDNA spacer patterns. It is not possible at present to decide which of several possible mechanisms were responsible for the observed changes.     

Localization of 5S RNA and rRNA genes in the Norway rat

The genes coding for the two classes of ribosomal RNA molecules, 5S RNA and 18+28S RNA, have been localized in the Norway rat (Rattus norvegicus). The 18+28S RNA cistrons are found on three chromosomes, at secondary constrictions on the short arms of chromosomes 3 and 12 and at the telomere of the short arm of chromosome 11. These sites were confirmed using the silver staining technique for nucleolar organizer regions. Two sites were found for the 5S RNA genes; one is closely linked to the 18+28S gene site on chromosome 12. The second site is at or near the telomere of the long arm of chromosome 19.