Ribosomal DNA in spores of Physarum polycephalum

DNA was isolated from plasmodia, spores and newly hatched amoebae of the slime mould Physarum polycephalum. The DNA preparations were fractionated in CsCl gradients and each fraction hybridised to combined 19 S + 26 S rRNA. In all three DNA preparations hybridisation was found to be limited to satellite DNA (rho = 1.714 g/cm3) and at saturation was found to reach a level of 0.16--0.18 % of total DNA. The main band of nuclear DNA (rho = 1.702 g/cm3) did not hybridise appreciably. Further experiments using analytical CsCl gradients revealed that the ratio of satellite to main band DNA was similar in all three preparations. It is concluded that the genes for ribosomal RNA are equally reiterated in spores, hatching amoebae and in plasmodia. They appear to be similarly organised in all stages of the life cycle examined so far.     

Accessibility of the ribosomal genes to micrococcal nuclease in Physarum polycephalum.

In Physarum polycephalum most genes coding for ribosomal RNA are not integrated in chromosomes, but are located in many copies in the nucleolus as plasmid-like palindromic DNA molecules. To find out whether coding sequences of rDNA are organized in a chromatin-like structure similar to that of bulk chromatin, nuclei were treated with micrococcal nuclease and DNA fragments were isolated. From bulk chromatin multimers of a basic unit of 170-180 base pairs were obtained. Nuclease fragmented DNA hybridized with labelled 19-S + 26-S rRNA was found to give the same saturation value as did unfragmented control DNA. No preferential degradation of ribosomal genes to acid soluble products was observed. A more detailed analysis of the nuclease degradation products was carried out with fragments separated by preparative gel electrophoresis. DNA eluted from the gels was hybridized in solution with labelled 19-S + 26-S rRNA. The coding sequences of rRNA were found to be degraded to approximately nucleosome size slightly more quickly than was the DNA of bulk chromatin. However, the distribution of the rDNA fragments on the gels did not coincide with the distribution of the fragments derived from bulk chromatin nucleosomes and their oligomers. The amount of rDNA in the interband regions was about intermediate between that found in the two adjacent bands. These results lead to the conclusion that the ribosomal genes, most of which are presumably active during rapid growth, are protected by proteins, probably histones. However, the ribosomal genes are present in a structure differing in some way from that of bulk chromatin.     

Nucleotide sequences of wheat-embryo cytosol 5-S and 5.8-S ribosomal ribonucleic acids

The nucleotide sequences of wheat embryo 5.8-S and 5-S rRNAs have been determined with the use of several techniques, including classic analysis of oligonucleotides generated by ribonuclease T1 and resolution on gels of terminally labelled RNA partially degraded with ribonucleases or with chemical reagents. The sequence of wheat embryo 5.8-S rRNA was found to be (formula: see text). This sequence is compared to 5-S rRNA sequences previously published for wheat and several other angiosperms.     

Non-ribosomal nucleotide sequences in 7-S RNA, the immediate precursor of 5.8-S ribosomal RNA in yeast.

The topography and the length of the non-ribosomal sequences present in 7-S RNA, the immediate precursor of 5.8-S ribosomal RNA, from the yeast Saccharomyces carlsbergensis were determined by analyzing the nucleotide sequences of the products obtained after complete digestion of 7-S RNA with RNase T1. The results show that 7-S RNA contains approximately 150 non-ribosomal nucleotides. The majority (90%) of the 7-S RNA molecules was found to have the same 5'-terminal pentadecanucleotide sequence as mature 5.8-S rRNA. The remaining 10% exhibited 5'-terminal sequences identical to those of 5.9-S RNA, which has the same primary structure as 5.8-S rRNA except for a slight extension at the 5' end [Rubin, G.M. (1974) Eur. J. Biochem. 41, 197--202]. These data show that the non-ribosomal nucleotides present in 7-S RNA are all located 3'-distal to the mature 5.8-S rRNA sequence. Moreover, it can be concluded that 5.9-S RNA is a stable rRNA rather than a precursor of 5.8-S rRNA. The 3'-terminal sequence of 5.8-S rRNA (U-C-A-U-U-UOH) is recovered in a much longer oligonucleotide in the T1 RNase digest of 7-S RNA having the sequence U-C-A-U-U-U-(C-C-U-U-C-U-C)-A-A-A-C-A-(U-U-C-U)-Gp. The sequences enclosed in brackets are likely to be correct but could not be established with absolute certainty. The arrow indicates the bond cleaved during processing. The octanucleotide sequence -A-A-A-C-A-U-U-C- located near the cleavage site shows a remarkable similarity to the 5'-terminal octanucleotide sequence of 7-S RNA (-A-A-A-C-U-U-U-C-). We suggest that these sequences may be involved in determining the specificity of the cleavages resulting in the formation of the two termini of 5.8-S rRNA.     

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     

Inter- and intra-strain variation in the 5.8S ribosomal RNA and internal transcribed spacer sequences of Entamoeba histolytica and comparison with Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens

The ribosomal RNA genes in Entamoeba histolytica are located on circular DNA molecules in about 200 copies per genome equivalent. Nucleotide sequence analysis of the 5.8S rRNA gene and the flanking internal transcribed spacers was carried out to determine the degree of sequence divergence in the multiple rRNA gene copies of a given strain; amongst three different E. histolytica strains (HM-1:IMSS, Rahman and HK-9); and amongst four species of Entamoeba (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens). The results show that all rRNA gene copies of a given strain are identical. Few nucleotide positions varied between strains of a species but the differences were very pronounced amongst species. In general, the internal transcribed spacer 2 sequence was more variable and may be useful for strain- and species-identification. The 5.8S rRNA gene and the internal transcribed spacer 2 of E. invadens were unusually small in size.     

Some characteristics of processing sites in ribosomal precursor RNA of yeast.

The DNA sequences of the intergenic region between the 17S and 5.8S rRNA genes of the ribosomal RNA operon in yeast has been determined. In this region the 37S ribosomal precursor RNA is specifically cleaved at a number of sites in the course of the maturation process. The exact position of these processing sites has been established by sequence analysis of the terminal fragments of the respective RNA species. There appears to be no significant complementarity between the sequences surrounding the two termini of the 18S secondary precursor RNA nor between those surrounding the two termini of 17S mature rRNA. This finding implies that the processing of yeast 37S ribosomal precursor RNA is not directed by a double-strand specific ribonuclease previously shown to be involved in the processing of E. coli ribosomal precursor RNA [see Refs 1,2]. The processing sites of yeast ribosomal precursor RNA described in the present paper are all flanked at one side by a very [A+T]-rich sequence. In addition, sequence repeats are found around the processing sites in this precursor RNA. Finally, sequence homologies are present at the 3'-termini [6 nucleotides] and the 5'-termini [13 nucleotides] of a number of mature rRNA products and intermediate ribosomal RNA precursors. These structural features are discussed in terms of possible recognition sites for the processing enzymes.     

Metabolic stability of the extrachromosomal ribosomal RNA genes in the slime mould Physarum polycephalum

The rRNA genes of the slime mould Physarum polycephalum are located on free, linear DNA molecules of a discrete size, Mr=38X10(6). Using an isotope dilution technique we have examined the metabolic stability of these extrachromosomal genes during active, balanced growth. Microplasmodia, prelabelled with [3H]thymidine, were used to prepare synchronous surface plasmodial cultures which were subsequently grown on unlabelled medium. The gross synthesis of ribosomal DNA was then determined over three consecutive mitotic divisions from the ratio of 3H to 14C in a hybrid formed between the extracted ribosomal [3H]DNA and a [14C]rRNA probe. It was found that ribosomal DNA, like chromosomal DNA, is completely stable during active growth.     

Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.     

The organization of ribosomal RNA genes in the mitochondrial DNA of Tetrahymena pyriformis strain ST.

1. We have constructed a physical map of the mtDNA of Tetrahymena pyriformis strain ST using the restriction endonucleases EcoRI, PstI, SacI, HindIII and HhaI. 2. Hybridization of mitochondrial 21 S and 14 S ribosomal RNA to restriction fragments of strain ST mtDNA shows that this DNA contains two 21-S and only one 14-S ribosomal RNA genes. By S1 nuclease treatment of briefly renatured single-stranded DNA the terminal duplication-inversion previously detected in this DNA (Arnberg et al. (1975) Biochim. Biophys. Acta 383, 359--369) has been isolated and shown to contain both 21-S ribosomal RNA genes. 14 S ribosomal RNA hybridizes to a region in the central part of the DNA, about 8000 nucleotides or 20% of the total DNA length apart from the nearest 21 S ribosomal RNA gene. 3. We have confirmed this position of the three ribosomal RNA genes by electron microscopical analysis of DNA . RNA hybrid molecules and R-loop molecules. 4. Hybridization of 21 S ribosomal RNA with duplex mtDNA digested either with phage lambda-induced exonuclease or exonuclease III of Escherichia coli, shows that the 21-S ribosomal RNA genes are located on the 5'-ends of each DNA strand. Electron microscopy of denaturated mtDNA hybridized with a mixture of 14-S and 21-S ribosomal RNAs show that the 14 S ribosomal RNA gene has the same polarity as the nearest 21 S ribosomal RNA gene. 5. Tetrahymena mtDNA is (after Saccharomyces mtDNA) the second mtDNA in which the two ribosomal RNA cistrons are far apart and the first mtDNA in which one of the ribosomal RNA cistrons is duplicated.     

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     

Direct amplification and sequencing of the 16S ribosomal DNA of an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia

DNA coding for the 16S rRNA of an intracellular bacterium was directly amplified from lysed cells of a host amoebae using the polymerase chain reaction and primers specific for eubacteria. The amoebae had been used to recover an uncultured bacterium observed in the sputum of a patient with pneumonia. The amplified DNA was sequenced directly and compared with published 16S rRNA sequences. The analysis revealed that the intracellular bacterium is a member of the genus Legionella and that it is different from species, including L. pneumophila, for which 16S ribosomal RNA sequence data are available.     

Location of the 5.8S rRNA gene of Saccharomyces cerevisiae.

Direct DNA sequence analysis of Saccharomyces cerevisiae ribosomal DNA cloned in an Escherichia coli plasmid revealed part of the structural gene for 5.8S rRNA at one end of a 700-base-pair EcoRI fragment. Taken with the previously established EcoRI restriction map of the ribosomal repeat unit, this sequence establishes that the yeast 5.8S RNA segment is located between the 18S and 28S segments in the 42S rRNA precursor and in the DNA which codes for it.     

DNA primase of human mitochondria is associated with structural RNA that is essential for enzymatic activity

DNA primase isolated from human mitochondria sediments in glycerol density gradients at 30S and 70S. These unusually high sedimentation coefficients are a result of association of the primase activity with RNA. Treatment of primase with nuclease not only affects its sedimentation behavior, but also inactivates the primase activity. The major RNA species that cofractionates with primase activity is shown by direct sequence analysis to be cytosolic 5.8S ribosomal RNA (rRNA). Specific degradation of endogenous 5.8S rRNA using ribonuclease H and oligonucleotides complementary to 5.8S rRNA results in reduction of primase activity. Other small RNAs may play a structural role in the formation of an active DNA primase complex.     

Tandemly arranged variant 5S ribosomal RNA genes in the yeast Saccharomyces cerevisiae.

Most of the ribosomal RNA genes of the yeast Saccharomyces cerevisiae are about 9 kilobases (kb) in size and encode both the 35S rRNA (processed to produce the 25S, 18S, and 5.8S species) and 5S rRNA. These genes are arranged in a single tandem array of 100 repeats. Below, we present evidence that at the centromere-distal end of this array is a tandem arrangement of a different type of rRNA gene. Each of these repeats is 3.6 kb in length and encodes a single 5S rRNA. The coding sequence of this gene is different from that of the "normal" 5S gene in three positions located at the 3' end of the gene.     

Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species

Genomes of 11 Quercus species were characterized using cytogenetic (Giemsa C-banding, fluorochrome banding), molecular-cytogenetic (fluorescence in situ hybridization, FISH, to ribosomal genes) and molecular (dot-blot for ribosomal gene-copy number assessment) techniques. Ribosomal genes are the first DNA sequences to be physically mapped in oaks, and the copy number of the 18S-5.8S-26 S rRNA genes is estimated for the first time. Oak karyotypes were analysed on the basis of DAPI banding and FISH patterns; five marker chromosomes were found. In addition, chromosomal organization of ribosomal genes with respect to AT- and GC-differentiated heterochromatin was studied. Fluorochrome staining produced very similar CMA/DAPI banding patterns, and the position and number of ribosomal loci were identical for all the species studied. The 18S-5.8S-26 S rRNA genes in oak complements were represented by a major locus at the subterminal secondary constriction (SC) of the only subtelocentric chromosome pair and a minor locus at paracentromeric SC of one metacentric pair. The only 5 S rDNA locus was revealed at the paracentromeric region of the second largest metacentric pair. A striking karyotypic similarity, shown by both fluorochrome banding and FISH patterns, implies close genome relationships among oak species no matter their geographic origin (European or American) or their ecophysiology (deciduous or evergreens). Dot-blot analysis gave preliminary evidence for different copy numbers of 18S-5.8S-26 S rRNA genes in diploid genomes of Q. cerris, Q. ilex, Q. petraea, Q. pubescens and Q. robur (2700, 1300, 2200, 4000 and 2200 copies, respectively) that was correlated with the size polymorphism of the major locus. Received: 26 February 1999 / Accepted: 16 March 1999     

Unusual ribosomal RNA of the intestinal parasite Giardia lamblia.

The anaerobic protozoan Giardia lamblia is a common intestinal parasite in humans, but is poorly defined at molecular and phylogenetic levels. We report here a structural characterization of the ribosomal RNA (rRNA) and rRNA genes of G. lamblia. Gel electrophoresis under native or non-denaturing conditions identified two high molecular weight rRNA species corresponding to the 16-18S and 23-28S rRNAs. Surprisingly, both species (1300 and 2300 nucleotides long, respectively) were considerably shorter than their counterparts from other protozoa (typically 1800 and 3400 nucleotides), and from bacteria as well (typically 1540 and 2900 nucleotides long). Denaturing polyacrylamide gel electrophoresis identified a major low molecular RNA of 127 nucleotides and several minor species, but no molecules with the typical lengths of 5.8S (160 nucleotides) and 5S (120 nucleotides) rRNA. The G. lamblia 1300, 2300, and 127 nucleotide RNAs are encoded within a 5.6 kilobase pair tandemly repeated DNA, as shown by Southern blot analysis and DNA cloning. Thus, the rRNA operon of this eukaryotic organism can be no longer than a typical bacterial operon. Sequence analysis identified the 127 nucleotide RNA as homologous to 5.8S RNA, but comparisons to archaebacterial rRNA suggest that Giardia derived from an early branch in eukaryotic evolution.     

Sequence arrangement of the rRNA genes of the dipteran Sarcophaga bullata

Velocity sedimentation studies of RNA of Sarcophaga bullata show that the major rRNA species have sedimentation values of 26S and 18S. Analysis of the rRNA under denaturing conditions indicates that there is a hidden break centrally located in the 26S rRNA species. Saturation hybridization studies using total genomic DNA and rRNA show that 0.08% of the nuclear DNA is occupied by rRNA coding sequences and that the average repetition frequency of these coding sequences is approximately 144. The arrangement of the rRNA genes and their spacer sequences on long strands of purified rDNA was determined by the examination of the structure of rRNa:DNA hybrids in the electron microscope. Long DNA strands contain several gene sets (18S + 26S) with one repeat unit containing the following sequences in order given: (a) An 18S gene of length 2.12 kb, (b) an internal transcribed spacer of length 2.01 kb, which contains a short sequence that may code for a 5.8S rRNA, (c) A 26S gene of length 4.06 kb which, in 20% of the cases, contains an intron with an average length of 5.62 kb, and (d) an external spacer of average length of 9.23 kb.