Organization of the nuclear ribosomal DNA of Chlamydomonas reinhardii

Summary Hybridization of cytoplasmic ribosomal RNA (rRNA) to restriction endonuclease digests of nuclear DNA of Chlamydomonas reinhardii reveals two BamHI ribosomal fragments of 2.95 and 2.35×10⁶ d and two SalI ribosomal fragments of 3.8 and 1.5×10⁶ d. The ribosomal DNA (rDNA) units, 5.3×10⁶ d in size, appear to be homogeneous since no hybridization of rDNA to other nuclear DNA fragments can be detected. The two BamHI and SalI ribosomal fragments have been cloned and a restriction map of the ribosomal unit has been established. The location of the 25S, 18S and 5.8S rRNA genes has been determined by hibridizing the rRNAs to digests of the ribosomal fragments and by observing RNA/DNA duplexes in the electron microscope. The data also indicate that the rDNA units are arranged in tandem arrays. The 5S rRNA genes are not closely located to the 25S and 18S rRNA genes since they are not contained within the nuclear rDNA unit. In addition no sequence homology is detectable between the nuclear and chloroplast rDNA units of C. reinhardii.Abbreviations used rRNA ribosomal RNA - rDNA ribosomal DNA d, dalton     

Heterogeneity in Chinese hamster ribosomal DNA

A discrete heterogeneity has been detected in Chinese hamster ribosomal DNA after Eco R1 digestion of total DNA followed by a Southern transfer and hybridization with [¹²⁵I]18S or [¹²⁵I]28S ribosomal RNA. Digestion with Eco R1 produces three fragments, 4.3, 6.0 and 9.5×10⁶ daltons respectively, which hybridize with 18S RNA. The smallest fragment also hybridizes with 28S RNA. Either length heterogeneity or sequence heterogeneity (i.e. presence of an additional Eco R1 site in some of the rDNA molecules) must be invoked to account for the two larger Eco R1 fragments that contain 18S but not 28S sequences. Eco R1 and Hind III maps, consistent with either length or sequence heterogeneity, are presented. The data at this time, however, do not distinguish between the two alternatives.     

Free genes for rRNAs in the macronuclear genome of the ciliate Stylonychia mytilus

When separated on an agarose gel, macronuclear DNA of the hypotrichous ciliate Stylonychia mytilus gives rise to many well-defined bands ranging in molecular weight from 0.3×10⁶ to 14×10⁶ dalton. Hybridization of 25 S rRNA, 17 S rRNA or 5 S RNA to such a gel revealed sharp hybridization bands. This suggests that this banding pattern is not an artefact due to nonspecific degradation of macronuclear DNA but that the DNA in the macronucleus of Stylonychia occurs in discrete fragments, each coding for at least one gene. The size of the DNA fragment coding for rRNA was found to be 4.5×l0⁶ dalton, the fragment coding for 5 S RNA has a molecular weight of 150,000–250,000 dalton.     

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.     

Detection of specific sequences among DNA fragments separated by gel electrophoresis.

Ribosomal RNA and precursor ribosomal RNA from at least one representative of each vertebrate class have been analyzed by electron microscopic secondary structure mapping. Reproducible patterns of hairpin loops were found in both 28 S ribosomal and precursor ribosomal RNA, whereas almost all the 18 S ribosomal RNA molecules lack secondary structure under the spreading conditions used. The precursor ribosomal RNA of all species analyzed have a common design. The 28 S ribosomal RNA is located at or near the presumed 5′-end and is separated from the 18 S ribosomal RNA region by the internal spacer region. In addition there is an external spacer region at the 3′-end of all precursor ribosomal RNA molecules. Changes in the length of these spacer regions are mainly responsible for the increase in size of the precursor ribosomal RNA during vertebrate evolution. In cold blooded vertebrates the precursor contains two short spacer regions; in birds the precursor bears a long internal and a short external spacer region, and in mammals it has two long spacer regions. The molecular weights, as determined from the electron micrographs, are 2·6 to 2·8 × 10⁶ for the precursor ribosomal RNA of cold blooded vertebrates, 3·7 to 3·9 × 10⁶ for the precursor of birds, and 4·2 to 4·7 × 10⁶ for the mammalian precursor. Ribosomal RNA and precursor ribosomal RNA of mammals have a higher proportion of secondary structure loops when compared to lower vertebrates. This observation was confirmed by digesting ribosomal RNAs and precursor ribosomal RNAs with single-strandspecific S₁ nuclease in aqueous solution. Analysis of the double-stranded, S₁-resistant fragments indicates that there is a direct relationship between the hairpin loops seen in the electron microscope and secondary structure in aqueous solution.     

A comparison of the structural organization of amplified ribosomal DNA from Xenopus mulleri and Xenopus laevis.

Secondary structure maps of long single strands of amplified ribosomal DNA from two closely related species of frogs, Xenopus laevis and X. mulleri, have been compared. The secondary structure pattern of the gene region is identical in both ribosomal DNAs while the patterns in the non-transcribed spacers² differ. In X. mulleri, the spacer shows an extended region without any secondary structure adjacent to the 28 S ribosomal RNA sequence. In contrast, the same region in the X. laevis spacer has extensive secondary structure. A comparison of secondary structure maps and denaturation maps of these two ribosomal DNAs (Brown et al., 1972) reveals that the portion without secondary structure in the X. mulleri spacer corresponds to an early melting A + T-rich region. As in X. laevis ribosomal DNA, Escherichia coli restriction endonuclease (EcoRI) makes two cuts in each repeating unit of amplified ribosomal DNA from X. mulleri. The position of the cleavage sites is identical in the two species as judged from secondary structure mapping of the two classes of EcoRI fragments generated. The small fragments of X. mulleri ribosomal DNA are homogeneous in size with a duplex molecular weight of 3.0 × 10⁶, and contain about 85% of the 28 S ribosomal RNA gene and about 17% of the 18 S ribosomal RNA gene. The large fragments are heterogeneous in size with molecular weights ranging from 4.2 to 4.9 × 10⁶, and contain the remaining portions of the gene regions and the nontranscribed spacer. Heteroduplexes made between large fragments of different lengths show only deletion loops. The position of these loops indicates that the length heterogeneity resides in the non-transcribed spacer region. Electrophoretic analysis of EcoRI digests of chromosomal ribosomal DNA from X. mulleri demonstrates that this DNA is heterogeneous in length as well.     

Evidence that in higher plants the 25S and 18S rRNA genes are not interspersed with genes for 5S rRNA

DNA samples from various higher plants (Phaseolus aureus, Glycine max, Matthiola incana, Brassica pekinensis, Cucumis melo) were centrifuged in actinomycin-caesium chloride gradients and the genes coding for the ribosomal RNAs were detected by hybridisation with tritium labelled 5S and 25S+18S rRNA, respectively. With DNA of low molecular weight (< 5×10⁶ daltons) the 5S and 25S+18S rRNA genes are often fractionated together. A good separation of the genes for 25S+18S rRNA from the 5S rRNA genes occurred only with high molecular weight DNA (> 10×10⁶ daltons) indicating that at least most of the 5S rRNA genes are not linked to, or interspersed with, the genes coding for 25S and 18S rRNA. This result is in agreement with the situation in animal cells and in contrast to that reported for bacteria, lower eukaryotes and chloroplasts.     

Ribosomal RNA synthesis in mitochondria of Neurospora crassa

Ribosomal RNA synthesis in Neurospora crassa mitochondria has been investigated by continuous labeling with [5-³H]uracil and pulse-chase experiments. A short-lived 32 S mitochondrial RNA was detected, along with two other short-lived components; one slightly larger than large subunit ribosomal RNA, and the other slightly larger than small subunit ribosomal RNA. The experiments give support to the possibility that 32 S RNA is the precursor of large and small subunit ribosomal RNA's. Both mature ribosomal RNA's compete with 32 S RNA in hybridization to mitochondrial DNA. Quantitative results from such hybridization-competition experiments along with measurements of electrophoretic mobility have been used to construct a molecular size model for synthesis of mitochondrial ribosomal RNA's. The large molecular weight precursor (32 S) of both ribosomal RNA's appears to be 2.4 × 10⁶ daltons in size. Maturation to large subunit RNA (1.28 × 10⁶ daltons) is assumed to involve an intermediate ~1.6 × 10⁶ daltons in size, while cleavage to form small subunit RNA (0.72 × 10⁶ daltons) presumably involves a 0.9 × 10⁶ dalton intermediate. In the maturation process ~22% of the precursor molecule is lost. As is the case for ribosomal RNA's, the mitochondrial precursor RNA has a strikingly low G + C content.     

Physical mapping of Nicotiana tabacum chloroplast DNA

Summary Nicotiana tabacum chloroplast DNA was digested with several restriction endonucleases chosen for their potential usefulness in distinguishing between species of the genus, Nicotiana. The resulting fragments were ordered into a circular configuration of about 160 kilobase pairs, equalling about 100x10⁶ daltons. The physical map features an inverted, repeated unit of about 24 kilobase pairs separated by a unique sequence region with a mean size of 13 kilobase pairs on the short side. The cistrons coding for chloroplast ribosomal RNA are contained within the inverted repeat and have the arrangement: 16S, spacer, 23S, 4.5S/5S. Restriction endonuclease maps obtained with the enzymes PvuII, XhoI, and BglI are presented.     

Organization of the 5S RNA genes in macro- and micronuclei of Tetrahymena pyriformis

The organization of the 5S genes in macro- and micronuclei of Tetrahymena pyriformis was studied using restriction endonucleases. After complete digestion of macronuclear DNA with BamH-I or Hpa I, 5S RNA hybridized to a DNA fragment of approximately 280 base pairs (bp). When macronuclear DNA was only partially digested with these enzymes, hybridization with ³²P-5S RNA demonstrated an oligomeric series with a spacing of 280 bp. These results indicate that the 5S genes are tandemly repeated in macronuclei and that the repeating unit is 280 bp (or 180,000 daltons). Since 5S RNA is 120 nucleotides, we conclude that the 5S repeat units contain a 120 bp transcribed region and a 160 bp spacer region. When macronuclear DNA was digested with Eco RI, Bgl I, or Eco RI + Bgl I, 5S RNA hybridized to DNA of molecular weight 3–4×10⁶, suggesting that these enzymes do not cleave within a 5S repeat. These 3–4×10⁶ dalton fragments define the maximum size of an average cluster of 5S repeated units. Assuming the size of the 5S repeat to be 0.18×10⁶ daltons, there are about 15–20 5S repeats per average tandem cluster, and since there are 350 5S-genes per haploid genome, there must be approximately 15–20 tandem arrays. Results obtained using micronuclear DNA suggest that organization of the 5S-genes is very similar in macro- and micronuclei. Macronuclear rRNA genes are extracnromosomal palindromic dimers. In contrast, 5S genes in Tetrahymena were found to be integrated within the genomes of both macro- and micronuclei and not linked to the rRNA genes. Moreover, it is unlikely that they are palindromes; rather they appear to be tandemly repeated in head-to-tail linkages. Thus, the organization of the 5S genes in Tetrahymena is similar to that of higher eukaryotes.     

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.     

Isolation and sequence organization of human ribosomal DNA.

The genes coding for 28 S and 18 S ribosomal RNA have been purified from leukemic leukocytes of one human individual by density gradient centrifugation. The purified ribosomal DNA was analyzed by restriction endonuclease digestion and electron microscopy. The location of cleavage sites for the restriction endonuclease EcoRI was established by R-loop mapping of restriction fragments by electron microscopy. The results are in agreement with gel analysis and gel transfer hybridization. One type of ribosomal DNA repeating unit contains four cleavage sites for EcoRI. Two of these cuts are located in the genes coding for 28 S and 18 S rRNA, while the other two are in the non-transcribed spacer. Thus, one of the restriction fragments generated contains non-transcribed spacer sequences only and is not detected by gel transfer hybridization if labeled rRNA is used as the hybridization probe. A second type of repeating unit lacks one of the EcoRI cleavage sites within the non-transcribed spacer. This indicates that sequence heterogeneity exists in human rDNA spacers. R-loop mapping of high molecular weight rDNA in the electron microscope reveals that the majority of repeats are rather uniform in length. The average size of 22 repeats was 43.65(±1.27) kb. Two repeats were found with lengths of 28.6 and 53.9 kb, respectively. This, and additional evidence from gels, indicates that some length heterogeneity does exist in the non-transcribed spacer. The structure of the human rDNA repeat is summarized in Figure 10.     

A denaturation map of sea urchin ribosomal DNA

The ribosomal RNA genes from the sea urchin Lytechinus variegatus have been studied with the electron microscope using the technique of denaturation mapping. A repeating pattern of denatured regions was found with an average repeat length of 3.87±0.24m. This corresponds to a DNA sequence of approximately 12,000 base pairs with a molecular weight of 8×10⁶ daltons.Abbreviations rRNA ribosomal RNA, including 26S and 18S RNA - Tris tris(hydroxymethyl)-aminomethane - EDTA ethylenediaminetetraacetate     

Characterization of cloned rat ribosomal DNA fragments

Summary Two Charon 4A lambda bacteriophage clones were characterized which contain all and part of the 18S ribosomal DNA of the rat. One clone contained two Eco RI fragments which include the whole 18S ribosomal RNA region and part of 28S ribosomal RNA region. The other clone contained an Eco RI fragment which covers part of 18S ribosomal RNA region. There were differences between the two clones in the non-transcribed spacer regions suggesting that there is heterogeneity in the non-transcribed spacer regions of rat ribosomal genes. The restriction map of the cloned mouse ribosomal DNA. Eco RI, Hind III, Pst I, and Bam HI sites in 18S ribosomal RNA region were in the same places in mouse and rat DNA but the restriction sites in the 5-spacer regions were different.     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

The pattern of protein and nucleic acid synthesis in germinating spores of Phycomyces blakesleeanus

Summary Synthesis of proteins, RNA and DNA is measured by incorporation of labelled precursors at different times during germination of Phycomyces spores.RNA and protein synthesis increases immediately after activation. DNa synthesis begins at a later stage (± 8 h) of germination when germ tubes are already present. Nuclear division occurs earlier in germination (±4–5 h) and is accompanied by a decrease in RNA synthesis. It can be concluded that at least most of the dormant spores are in the G2 phase of the cell cycle.Analysis of ribosomal RNA after pulse-chase labelling shows only three labelled compounds: a precursor molecule (2.25×10⁶ daltons) and the two mature ribosomal RNA compounds (1.4×10⁶ and 0.7×10⁶ daltons). This suggests that the two rRNAs are formed directly from the precursor molecule. Cycloheximide totally blocks the transformation of the ribosomal precursor molecule into mature rRNA.     

Vorstufen der ribosomalen RNA in frei suspendierten Calluszellen der Petersilie (Petroselinum sativum)

Summary Six high molecular weight, rapidly labelled RNA species were detected in freely suspended callus cells of Petroselinum sativum by means of isotope labelling and electrophoretic separation in agarose-polyacrylamide gels. On the basis of their migration in the latter the RNA species were calculated to have the following molecular weights: 2.9×10⁶, 2,4×10⁶, 1.9×10⁶, 1.4×10⁶, 1.0×10⁶ and 0.75×10⁶ daltons. Thus they can clearly be distinguished from the two ribosomal RNA species (1.3×10⁶ and 0.7×10⁶ daltons). During incubation of the cells with [³H]methyl-methionine as a methyl donator all six components incorporated radioactivity rapidly. With [³H]nucleosides or [³H]orotic acid as precursors the 2.9×10⁶ and the 2.4×10⁶ daltons RNA were labelled within 10 min, while the other high molecular weight species appeared after about 20 min of labelling.Prolongation to 45–120 min resulted in accumulation of radioactivity preferentially in the 1.4×10⁶ and 0.75×10⁶ daltons RNA and in the ribosomal RNA species. The results of cell fractionation experiments provide evidence that these rapidly labelled high molecular weight RNA species are synthesized in the cell nucleus. The kinetics of their synthesis together with the other data obtained strongly support the suggestion that these RNA species function as precursors in the processing of ribosomal RNA. The possible mechanism of this process is discussed.

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Verwendete Abkürzungen EDTA Äthylendiamintetraessigsäure - DNase Desoxyribonuclease - Imp./min epm - MAK methyliertes Albumin an Kieselgur - POPOP 1,4- bis (4-Methyl-5-Phenyloxazol)-Benzol - PPO 2,5-Diphenyloxazol - RNase Ribonuclease - S Sedimentationskoeffizient in Svedberg-Einheiten - SDS Natriumdodecylsulfat - TPE Tris-Phosphat-EDTA-Puffer - Tris Tris-(hydroxymethyl)-aminomethan - Upm rpm     

Cloning and characterization of the rDNA repeat unit of Podospora anserina

Summary DNA coding for ribosomal RNA in Podospora anserina has been cloned and was found as a tandemly repeated 8.3 kb sequence. The cloned rDNA was characterized by restriction endonuclease mapping. The location of 5.8S, 18S and 28S rRNA coding regions was established by DNA-RNA hybridization and S1 nuclease mapping. The organization of P. anserina rRNA genes is similar to that of Neurospora crassa and Aspergillus nidulans. The rDNA unit does not contain the sequence coding for 5S RNA.     

Ribosomal DNA in a nuclear satellite of tomato

A satellite DNA of buoyant density 1.704 constitutes approximately 5%–6% of nuclear DNA isolated from cherry tomato leaves. Isolated satellite DNA exhibits a multi-component melting profile. Kinetic complexity measurements indicate that 37% of the satellite consists of repeating units of 10 ⁵ daltons, and 48% of it consists of repeating units of 5.5 x 10⁶ daltons. The latter component is identified as DNA coding for ribosomal RNA on the basis of its buoyant density, kinetic complexity, and abundance in nuclear DNA, 3.2% as determined by saturation hybridization measurements. Saturation studies show that the more rapidly reassociating component of the satellite does not code for 5S RNA. The question of genetic linkage between satellite components is not resolved by this study.