Metabolism of 5S RNA in the absence of ribosome production

The results presented in this report show that during early development of Xenopus laevis the synthesis of 5S RNA occurs in blastula embryos, whereas the synthesis of 18S and 28S RNA cannot be detected until gastrulation. Thus the initiation of synthesis of the three ribosomal RNAs is not coordinate during early development. Blastula embryos are similar to anucleolate mutants of Xenopus laevis, in that they both synthesize 5S RNA, but are unable to assemble new ribosomes because they do not synthesize 18S and 28S RNA or ribosomal proteins. The blastula and anucleolate embryos thus provide a unique opportunity to determine if newly synthesized soluble 5S RNA can exchange with the 5S RNA present in existing ribosomes. The results show that newly synthesized 5S RNA is not incorporated into the ribosomes of blastula or anucleolate embryos. Furthermore, the 5S RNA synthesized by anucleolate mutants has a shorter half-life than the 5S RNA made by normal embryos. The synthesis of excess 5S RNA and its subsequent degradation in the absence of ribosome production appears to be another example of the phenomenon of wastage of newly synthesized ribosomal RNA.     

The Synthesis of 5s RNA and Its Relationship to 18s and 28s Ribosomal RNA in the Bobbed Mutants of DROSOPHILA MELANOGASTER

Ribosomes contain one molecule each of 5S, 18S and 28S RNA. In Drosophila melanogaster although the genes for 18S+28S are physically separated from the 5S RNA genes, the multiplicity of various ribosomal RNA genes is roughly the same. Thus a coordinate synthesis of these three molecules might seem feasible. This problem has been approached by determining the molar ratios of various RNA's in ovaries and in adult flies. In ovaries there is a slight excess of 5S RNA molecules over other rRNA's, but in adult flies no such differences exist. Bobbed mutants also have the same molar ratios as wild-type flies. Results on 5S RNA synthesis in both in vitro and in vivo studies show that it is reduced in coordination with 18S+28S rRNA in the bobbed mutants of Drosophila melanogaster. Various possibilities are discussed in considering the implications of these results.     

Ribosomal RNA Turnover in Contact Inhibited Cells

CONTACT inhibition of animal cell growth is accompanied by a decreased rate of incorporation of nucleosides into RNA^1–3. Contact inhibited cells, however, transport exogenously-supplied nucleosides more slowly than do rapidly growing cells^4,5, suggesting that the rate of incorporation of isotopically labelled precursors into total cellular RNA may be a poor measure of the absolute rate of RNA synthesis by these cells. Recently, Emerson⁶ determined the actual rates of synthesis of ribosomal RNA (rRNA) and of the rapidly labelled heterogeneous species (HnRNA) by labelling with ³H-adenosine and measuring both the specific activity of the ATP pool and the rate of incorporation of isotope into the various RNA species. He concluded that contact inhibited cells synthesize ribosomal precursor RNA two to four times more slowly than do rapidly growing cells, but that there is little if any reduction in the instantaneous rate of synthesis of HnRNA by the non-growing cells. We have independently reached the same conclusion from simultaneous measurements on the specific radioactivity of the UTP pool and the rate of ³H-uridine incorporation into RNAs (unpublished work of Edlin and myself). However, although synthesis of the 45S precursor to ribosomal RNA is reduced two to four times in contact inhibited cells, the rate of cell multiplication and the rate of rRNA accumulation are reduced ten times. This suggests either “wastage”⁷ of newly synthesized 45S rRNA precursor, or turnover of ribosomes in contact inhibited cells Two lines of evidence suggest that “wastage” of 45S RNA does not play a significant role in this system. (1) The rate of synthesis of 45S RNA in both growing and contact inhibited cells agrees well with that expected from the observed rates of synthesis of 28S and 18S RNAs (unpublished work of Edlin and myself). Emerson has made similar calculations⁶. (2) 45S RNA labelled with a 20 min pulse of ³H-uridine is converted in the presence of actinomycin D to 28S and 18S RNAs with the same efficiency (approximately 50%) in both growing and contact inhibited cells. These results indicate that, in order to maintain a balanced complement of ribosomal RNAs, contact inhibited cells must turn over their ribosomes. We present evidence here that rRNA is stable in rapidly growing chick cells, but begins to turn over with a half-life of approximately 35–45 h as cells approach confluence and become contact inhibited.     

Distribution of 18+28S ribosomal genes in mammalian genomes

In situ hybridization with ³H 18S and 28S ribosomal RNA from Xenopus laevis has been used to study the distribution of DNA sequences coding for these RNAs (the nucleolus organizing regions) in the genomes of six mammals. Several patterns of distribution have been found: 1) A single major site (rat kangaroo, Seba's fruit bat), 2) Two major sites (Indian muntjac), 3) Multiple sites in centromeric heterochromatin (field vole), 4) Multiple sites in heterochromatic short arms (Peromyscus eremicus), 5) Multiple sites in telomeric regions (Chinese hamster). — The chromosomal sites which bind ³H 18S and 28S ribosomal RNA correspond closely to the sites of secondary constrictions where these are known. However, the correlation is not absolute. Some secondary constrictions do not appear to bind ³H ribosomal RNA. Some regions which bind ribosomal RNA do not appear as secondary constrictions in metaphase chromosomes. — Although the nucleolus organizing regions of most mammalian karyotypes are found on the autosomes, the X chromosomes in Carollia perspicillata and C. castanea carry large clusters of sequences complementary to ribosomal RNA. In situ hybridization shows that the Y chromosome in C. castanea also has a large nucleolus organizing region.     

Evidence for ribosomal RNA synthesis in pollen tubes in culture

The evidence is presented that pollen tubes ofNicotiana tabacum L. cultivated in shaken suspension do synthesize 5S, 18S and 28S RNA. Following incubation of pollen tubes in the presence of radioactive uracil or uridine, RNA was isolated from total pollen tube material after the removal of 4S RNA, from polysomes and from 80S ribosomal particles, and fractionated by density gradient centrifugation and MAK column chromatography. The results obtained further suggest a higher rate of 5S RNA synthesis with respect to 18S+28S RNA.     

An electron microscope heteroduplex study of the ribosomal DNAs of Xenopus laevis and Xenopus mulleri

The arrangement of the genes and spacers has been analyzed in ribosomal DNA of Xenopus laevis and Xenopus mulleri by heteroduplex mapping and visualization of ribosomal RNA-DNA hybrids. Heterologous reassoeiated molecules show a characteristic pattern in which two perfectly duplexed regions, whose lengths are those predicted by the known lengths of the 18 S and 28 S genes, are separated by a small substitution loop of about 0.23 × 10⁶ daltons and a large region of partial homology which averages 3.24 × 10⁶ daltons. These mismatched regions are entirely consistent with the known sequence divergence previously described (Brown et al., 1972) for the transcribed and non-transcribed spacer regions of the two rDNAs, respectively. Hybrids of X. laevis rDNA with 18 S and 28 S rRNA contain two duplex regions of the expected lengths for the 18 S and 28 S genes separated by 0.49 × 10⁶ daltons of single-strand DNA. This latter value is the length of the transcribed spacer region between the 18 S and 28 S RNAs that has been measured within the 40 S RNA precursor molecule by secondary structure mapping (Wellauer &; Dawid, personal communication). There is also a longer single-strand region separating one 18 S + 28 S gene set from the next; this is considered to be mainly non-transcribed spacer.We conclude that the 18 S and 28 S genes are separated by about 0.5 × 10⁶ daltons of DNA of which about half is homologous in the two Xenopus species. This region is part of the transcribed spacer. In addition, the longer non-transcribed spacer can be seen to have some homology between the two species; the location of this homology is fairly reproducible between molecules and has been carefully documented by contour length measurements.     

Genetic Analysis of the 5s RNA Genes in DROSOPHILA MELANOGASTER

The 5S RNA genes of Drosophila melanogaster in either an isogenic wild-type or a multiply inverted (SM1) chromosome 2 increase their multiplicity when opposite a deficiency for the 5S gene site. This is analogous to the compensation phenomenon previously described for the 18S and 28S ribosomal RNA genes of the X chromosome nucleolus organizer region. Molecular hybridization of 5S RNA to DNA containing various doses of the 56F1-9 region of chromosome 2 demonstrates that most, if not all, of the 5S genes reside in or near this region. Also, a deficiency missing approximately one-half of the wild-type number of 5S genes was isolated and genetically localized. This mutant has a phenotype like that of bobbed, a mutant known to be partially deficient in 18S and 28S ribosomal RNA genes. Finally, we report the existence of a chromosomal rearrangement which splits the second chromosome into two segments, each containing 5S DNA.     

Structural features unique to the 5 S ribosomal RNAs of the thermophilic cyanobacterium Synechococcus lividus III and the green plant chloroplasts

The complete nucleotide sequence of the 5 S ribosomal RNA from the thermophilic cyanobacterium Synechococcus lividus III was determined. The sequence is: ^5′U-C- C-U-G-G-U-G-G-U-G-A-U-G-G-C-G-A-U-G-U-G-G-A-C-C-C-A-C-A-C-U-C-A-U-C- C-A-U-C-C-C-G-A-A-C-U-G-A-G-U-G-G-U-G-A-A-A-C-G-C-A-U-U-U-G-C-G-G-C- G-A-C-G-A-U-A-G-U-U-G-G-A-G-G-G-U-A-G-C-C-U-C-C-U-G-U-C-A-A-A-A-U-A- G-C-U-A-A-C-C-G-C-C-A-G-G-G-U_OH^3′This 5 S RNA has regional structural characteristics that are found in the green plant chloroplast 5 S RNAs and not in other known sequences of 5 S ribosomal RNAs. These homologies suggest a close phylogenetic relationship between S. lividus and the green plant chloroplasts.     

Synthesis and characterization of amplified DNA in oocytes of the house cricket,Acheta domesticus (Orthoptera: Gryllidae)

At a time in the life cycle when a large proportion of the oocytes of Acheta incorporate ³H-thymidine into an extrachromosomal DNA body, synthesis of a satellite or minor band DNA, the density of which is greater than main band DNA, is readily detected. Synthesis of the satellite DNA is not detectable in tissues, the cells of which do not have a DNA body, or in ovaries in which synthesis of extrachromosomal DNA by the oocytes is completed. The DNA body contains the amplified genes which code for ribosomal RNA. However, less than 1 percent of the satellite DNA, all of which appears to be amplified in the oocyte, is complementary to ribosomal 18S and 28S RNA. In situ hybridization demonstrates that non-ribosomal elements, like the ribosomal elements of the satellite DNA, are localized in the DNA body.Abbreviations used rRNA ribosomal RNA, includes 18S and 28S RNA - rDNA gene sequences complementary to rRNA - cRNA complementary RNA synthesized in vitro     

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos. V. Inability of inhibitor to repress 5S RNA synthesis.

A specific inhibitor of ribosomal RNA (rRNA) synthesis was partially purified from an acid-soluble fraction of Xenopus laevis blastulae. Effects of this inhibitor on 5S rRNA synthesis of isolated neurula cells of the same species were investigated. The results show that the synthesis of both 5S rRNA and 4S RNA proceeds normally when both 18 and 28S rRNA are almost completely inhibited. Failure of the inhibitor to suppress 5S rRNA synthesis suggests that it plays an important role in the regulation of 18 and 28S rRNA synthesis during development and that the synthesis of 5S rRNA is not coordinated to that of 18 and 28S rRNA.     

Biochemical studies of mammalian oogenesis: synthesis and stability of various classes of RNA during growth of the mouse oocyte in vitro

The synthesis of various classes of RNA in mouse oocytes at different stages of growth has been examined after incubating follicles in medium containing radiolabeled uridine. After fractionation on poly(U)-Sepharose of radiolabeled oocyte RNA, of which about 83% is associated with the nucleus after a 5-hr labeling period, revealed that about 40–50% of the radiolabeled RNA behaved as poly(A)-containing RNA. This value remained fairly constant during the period of oocyte growth in which oocyte diameter increased from about 35 to about 55 μm. After a 5-hr labeling, the percentage of radiolabeled poly(A)-containing RNA in either the fully grown dictyate oocyte, metaphase II oocyte, or one-cell embryo was about 20%. After a 5-hr labeling, agarose gel electrophoretic analysis of the radiolabeled species of oocyte RNA obtained after fractionation on poly(U)-Sepharose revealed the presence of a putative ribosomal RNA precursor, ribosomal (28 and 18 S) RNA, transfer plus 5 S RNA and heterodisperse poly(A)-containing RNA. A significant fraction of the radiolabeled RNA species was quite large (>40 S). The ratios of the relative proportions of the radiolabeled ribosomal RNAs and transfer plus 5 S RNA remained essentially constant during oocyte growth. The stability of various classes of RNA was examined by incubating follicles with radiolabeled uridine, washing the follicles free of radioactivity and culturing the follicles under conditions which support oocyte growth in vitro (Eppig, 1977). Under these conditions, total oocyte radiolabeled RNA was quite stable as determined by retention of acid-insoluble radioactive material ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 28}\text{days}$). However, under conditions in which oocytes are viable but do not grow, the half-life of total RNA was about 4.5 days. Poly(A)-containing RNA was also very stable; after 8 days in culture, about 50% of the radiolabeled poly(A)-containing RNA present after 5 hr of labeling was still present. Agarose gel electrophoretic analysis of radiolabeled RNA in oocytes after 4 days of culture and after fractionation on poly(U)-Sepharose revealed the presence of ribosomal (28 and 18 S) RNA, transfer plus 5 S RNA, and heterodisperse poly(A)-containing RNA. At this time, these RNAs are located in the oocyte cytoplasm. In addition, the molecular weight distribution of poly(A)-containing RNA was significantly lower than that after 5 hr of labeling. The ratios of the relative proportions of radiolabeled ribosomal RNAs and transfer plus 5 S RNA were quite similar to those after 5 hr of labeling.     

Biochemical research on oogenesis. RNA accumulation during oogenesis of the dogfish Scyliorhinus caniculus

Four biochemical mechanisms have been shown to operate in the oocytes of amphibians and teleosts: (1) amplification of the 28 S and 18 S genes, (2) noncoordinate accumulation of 5 S RNA and 28 S + 18 S RNA, (3) storage of 5 S and transfer RNA made in excess by small oocytes within nucleoprotein particles, (4) expression of different 5 S genes in oocytes and somatic cells. We have tried to extend these observations to another group of vertebrates, i.e., selacians (Chondrichthya). Our data suggest that ribosomal gene amplification is low or absent in the oocytes of the dogfish Scyliorhinus caniculus. However, previtellogenic oocytes of this species accumulate more 5 S RNA than needed for ribosome assembly. Transfer and 5 S RNA present in small oocytes are probably not free in the cell sap. A substantial fraction of these RNAs sediments at 10 S when homogenates of immature ovaries are centrifuged in sucrose density gradients. In contrast to what we observed in amphibians and teleosts, 5 S RNA from ovaries of S. caniculus is identical in sequence to 5 S RNA from liver. Among the four mechanisms mentioned above, the second and probably the third one are used by the oocytes of S. caniculus. Mechanism (4) is absent in this species. No definitive conclusion can be drawn concerning mechanism (1), i.e., ribosomal gene amplification.     

Structural dynamics of bacterial ribosomes. III. Quantitative conversion of vacant ribosome couples into an initiation complex with R17 RNA as messenger

The arrangement of the DNA sequences coding for the ribosomal 5.8 S RNA in the genome of Xenopus laevis has been studied. In Xenopus the 5.8 S cistrons, like the ribosomal 28 S and 18 S cistrons, are reiterated some 600-fold (Clarkson et al., 1973a). When banded in caesium chloride, the 5.8 S cistrons separate from somatic DNA of high molecular weight and band as a distinct satellite, indicating a clustered arrangement in the genome. The buoyant density of this satellite (1.723 g cm^?3) corresponds to that of the ribosomal DNA satellite.It has previously been shown that the ribosomal DNA sequences have been deleted from the genome of the anucleotide Xenopus mutant. Our findings, first that the anucleolate mutant does not synthesize 5.8 S RNA and second that somatic DNA from this mutant does not detectably hybridize with 5.8 S RNA, demonstrate that the 5.8 S cistronic complement has been similarly deleted. This finding supports our contention that 5.8 S sequences are clustered on chromosomal DNA and further suggests that they are located close to or within the rDNA complements in the nucleolus organizer region.Pre-hybridization to saturation with unlabelled 5.8 S RNA results in only a slight increase in the buoyant density of denatured 5.8 S coding sequences from low molecular weight DNA. Since a contiguous arrangement of the 5.8 S sequences would give rise to a much larger increase in density, it follows that, although clustered, the sequences must be intercalated within stretches of other DNA. By contrast, pre-hybridization of the somatic DNA with unlabelled 28 S or 18 S ribosomal RNAs results in large shifts in the buoyant density of the 5.8 S sequences. These shifts indicate that the 5.8 S sequences are closely linked to both 28 S and 18 S coding sequences.It is concluded that the 5.8 S cistrons are interspersed along the ribosomal DNA sense strand and that each is located together with a 28 S and an 18 S cistron in a ribosomal repeat unit. Estimates, obtained from the pre-hybridization experiments, of the separations between the 5.8 S and the 28 S and 18 S sequences, are combined in a model of the ribosomal repeat unit. In this model the 5.8 S cistron is located within the transcribed spacer which links the 28 S and 18 S coding sequences.     

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.