Spectroscopic evidence for the uneven distribution of adenine and uracil residues in ribosomal ribonucleic acid of Drosophila melanogaster and of Plasmodium knowlesi and its possible evolutionary significance.

RNA was isolated from subribosomal particles of the malaria parasite Plasmodium knowlesi. The nucleotide composition (mole fraction) of the principal species was obtained (S-rRNA, 0.295A, 0.36U, 0.25G, 0.105C: L-rRNA, 0.326A, 0.31U, 0.228G, 0.144C). Ribosomal RNA was also isolated from Drosophila melanogaster. Optical properties of these A + U-rich species were measured. In all four cases analysis of the hypochromic effect revealed that adenine and uracil residues tended to form clusters along the polynucleotide chain. A substantial fraction of residues was located in bihelical regions of approx. 50% G-C base pairs or in regions of approx. 30-35% G-C base pairs. The possible evolutionary significance of these results was considered on the basis of comparison with properties of rRNA from bacteria (Escherichia coli) and a mammal (rabbit reticulocyte).     

A study of the influence of magnesium ions on the conformation of ribosomal ribonucleic acid and on the stability of the larger subribosomal particle of rabbit reticulocytes.

Mg2+ was shown to affect the conformation of rRNA over the range of 0.03-1.2M-KCl. The species studies were Escherichia coli S-rRNA and L-rRNA (the RNA moieties of the smaller and larger subribosomal particles respectively) and rabbits S-rRNA and L-rRNA. 2. The addition of Mg2+ to rRNA in reconstitution buffer (0.35M-KCl0.01M-Tris/HCl, pH7.2) at 20 degrees C let to an increase in bihelical secondary structure through the formation of additional (mainly A-U) base-pairs (e.g. an additional approx. 58 A-U base-pairs per molecule of E. coli S-rRNA as judged by u.v. difference spectrophotometry...     

The presence of a high-molecular-weight (guanine-plus-cytosine)-rich segment at the 3'' end of rabbit 28S ribosomal ribonucleic acid.

The 3' hydroxyl end of 28S L-rRNA (major RNA species of the larger subribosomal particle) was labelled by coupling its 2-hydroxy-3-naphthoic acid hydrazine with diazotized [3H]aniline. The RNA was hydrolysed partially with ribonuclease T1 and fractionated on Sephadex G-200. The results show that a highly structured segment with 78% G+C content and a number-average molecular weight of at least 1.0x10(5)-1.8x10(5) is located at the 3' hydroxyl end of the 28S rRNA molecule.     

Physicochemical characterization of the ribosomal RNA species of the Mollusca. Molecular weight, integrity and secondary-structure features of the RNA of the large and small ribosomal subunits.

1. The rRNA species of the Cephalopoda Octopus vulgaris and Loligo vulgaris were found to have unexpectedly high sedimentation coefficients and molecular weights. In 0.1 M-NaCl the L-rRNA (RNA from large ribosomal subunit) has the same s20 value as the L-rRNA of the mammals (30.7S), whereas the S-rRNA (RNA from small ribosomal subunit) sediments at a faster rate (20.1S) than the S-rRNA of both the mammals and the fungi (Neurospora crassa) (17.5S). The molecular weights of the L-rRNA were determined by gel electrophoresis in formamide and found to be 1.66 X 10(6) (Octupus) and 1.89 X 10(6) (Loligo); the mol.wt. of the S-rRNA of both species is 0.96 X 10(6), i.e. much larger than that of the mammals (0.65 X 10(6)) and almost coincident with that of the '23S' RNA of the prokaryotes. 2. By contrast, the less evolved Gastropoda and Lamellibranchiata (Murex trunculus and Macrocallista chione) have S-rRNA and L-rRNA species with mol.wts. of 0.65 X 10(6) and approx. 1.40 X 10(6).3. All the mature L-rRNA molecules of the cephalopoda are composed of two unequal fragments held together by regions of hydrogen-bonding having a similar, low, thermal stability in the two species; the molecular weights of the two fragments composing the L-rRNA are estimated to be 0.96 X 10(6) and 0.88 X 10(6) (Loligo) and 0.96 X 10(6) and 0.65 X 10(6) (Octupus). THe S-rRNA of both species is a continuous chain with exactly the same molecular weight (0.96 X 10(6)) as the heavier of the two fragments of the L-rRNA. 4. The secondary-structure features of the L-rRNA and S-rRNA species of the Caphalopoda were investigated by thermal 'melting' analysis in 4.0 M-guanidinium chloride; 60-70% of the residues are estimated to form short, independently 'melting' bihelical segments not more than 10 base-pairs in length. 5. Bases are unevenly distributed between non-helical and bihelical portions of the rRNA molecules, G and C residues being preferentially concentrated in bihelical comains. 6. The secondary-structure regions of the L-rRNA species of Octopus and Loligo are heterogenous, including two discrete fractions of independently 'melting' species that give rise to biphasic 'melting' profiles: a fraction consisting of shorter (G + C)-poorer segments (60-68% G + C, not more than 5 base-pairs in length) and a fraction consisting of longer (G + C)-richer segments (80-88% G + C, 5-10 base-pairs in length). No evidence for heterogeneity has been detected in the S-rRNa.     

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90 degrees C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70 degrees C) and Escherichia coli (max. growth temp., 47 degrees C) with respect to (a) bihelical content of rRNA; (b) G . C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. Subunits of C. acidophilia ribosomes (Tm = 90-93 degrees C) exhibit considerable thermal tolerance over their B. acidocaldarius (Tm = 77 degrees C) and E. coli counterparts (Tm = 72 degrees C). Based on the "melting' hyperchromicities of the intact ribosomal subunits a 51-55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67-70%, appears to be involved in hydrogen bonding in the naked rRNA species. The G . C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63-64% G . C, compared to 58.5% G . C for B. acidocaldarius and 55% G . C for E. coli. The increment of ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. Compared to E. coli the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

A study of the thermal stability of ribosomes and biologically active subribosomal particles

1. The ability of Escherichia coli ribosomes to function in poly(U)-directed protein synthesis was measured at elevated temperatures by using thermostable supernatant factors from Bacillus stearothermophilus. The amount of polyphenylalanine synthesized at 55 degrees C was about the same as at 37 degrees C, but the rate of synthesis was increased approximately fivefold. At 60 degrees C the activity of the ribosomes was halved. 2. E. coli ribosomes can sustain the loss of approx. 10% of the double-helical secondary structure of RNA without losing activity. 3. Within the active ribosome the double-helical secondary structure of the rRNA moiety is stabilized compared with isolated rRNA, as judged by enzymic hydrolysis and by measurements of E(260). 4. The main products, over the range 0-55 degrees C, of ribonuclease T(1) digestion of the smaller subribosomal particle of E. coli were two fragments (s(0) (20,w) 15S and 25.3S) of approximately one-quarter and three-quarters of the size of the intact molecule, revealing the presence of a ;weak spot' where intramolecular bonds appear insufficient to hold the fragments together. 5. Subribosomal particles of B. stearothermophilus were more stable to heating, by approx. 10 degrees C, than those of E. coli, and the stabilization of double-helical secondary structure of the RNA moiety was more striking. 6. Rabbit reticulocyte ribosomes were active in poly(U)-directed protein synthesis at 45 degrees C, and half the activity was lost after heating to 53 degrees C. Active subribosomal particles of rabbit reticulocytes and of oocytes of Xenopus laevis, like the bacterial subribosomal particles, underwent a conformational change to a slower-sedimenting form on heating. The temperature range of the transition depended on the species. 7. Slower-sedimenting particles, whether produced by EDTA treatment or by heating, had different ;melting' profiles compared with active subribosomal particles, providing another indication of conformational differences. 8. Comparison of the properties of the various subribosomal particles revealed greater variation in the secondary structure of the protein moieties (judged by measurement of circular dichroism) than in the secondary structure of the RNA moieties, which appeared to have features in common.     

Distribution of sequences common to the 25--28S-ribonucleic acid genes of Xenopus laevis and Neurospora crassa.

The extent of homology between the nucleotide sequence of L-rRNA (the major RNA component of the larger ribosomal subparticle) of a lower eukaryote (Neurospora crassa) and an amphibian (Xenopus laevis) was investigated by utilizing rDNA (DNA coding for rRNA) of X. laevis cloned in plasmids pMB9 and pML2, and rDNA of N. crassa cloned in bacteriophage lambda. Hybridization studies revealed that sequences common to both N. crassa and X. laevis L-rRNA comprise a total of approx. 1050 /+- 200 nucleotides. The thermal stability of the X. laevis rDNA.N. crassa L-rRNA hybrid was 5 degrees C lower than that of the X. laevis rDNA.X. laevis L-rRNA duplex, indicating the presence of fewer than 10% mismatches in homologous sequences. X. laevis rDNA was analysed by means of restriction endonucleases and hybridization with 125I-labelled N. crassa L-rRNA. Most (at least 95%) of the conserved sequences were present in a 3000-base-pair fragment produced by restriction with endonucleases HindIII and BamHI. This fragment, which includes the 3'-OH terminus of the L-rRNA-coding region, was used as an adaptor in the construction of a bacteriophage-lambda recombinant. One section of the recombinant phage terminating in a HindIII-specific site was obtained from bacteriophage lambda plac5 (after restriction with endonuclease HindIII). A second section terminating in a BamHi-specific site was obtained from bacteriophage lambda 540 (after restriction with endonuclease BamHI). These two parts were joined by means of the X. laevis rDNA fragment. Further analysis of cloned rDNA by means of restriction endonucleases confirmed that conserved sequences were widely distributed throughout the 3000-base-pair fragment produced by HindIII and BamHi endonucleases. A 3400-base-pair fragment of N. crassa rDNA cloned in a bacteriophage lambda [Cox & Peden (1979) Mol. Gen. Genet. 174, 17--24] was restricted with endonucleases. The products were hybridized with 125I-labelled X. laevis L-rRNA. Conserved sequences were shown to be distributed over a range of approx. 1600--2700 base-pairs. Hence, in neither X. laevis nor N. crassa L-rRNA can be conserved sequences from a single block; instead regions of high and low (or no) homology must be intermingled. Both N. crassa rDNA and X. laevis rDNA were found to hybridize with Drosophila melanogaster L-rDNA sequences. Those rDNA fragments with sequences common to X. laevis and N. crassa L-rRNA also hybridized with D. melanogaster L-rRNA probe. Thus the same set of conserved sequences may be present in all three species.     

Stability of Soybean Ribosomal RNA in Various Conditions

The stability of RNA preparations which were prepared from soybean cotyledons was examined by incubating the RNA solutions of high salt at 20°C in the presence or absence of PVS. Sedimentation profiles of incubated RNA were given by ultracenirifugal analysis and compared with that of original RNA. RNA retained its original size after incubating for 4 hr in the presence of PVS and 200 mM KCl, while RNA was completely degraded into small fragments in the absence of PVS after the same treatment.

The purified rRNA which was prepared from 3 day-old hypocotyls was treated with heating, EDTA or urea in the presence of PVS. L-rRNA component was obviously disappeared by heating at 50°C for 5 min. Partial disruption of L-rRNA component occurred by dialyzing against urea solution. L-rRNA separated by zonal ultracentrifugation was decomposed into components by heat treatment or leaving at 4°C for 20 hr in the buffer from which KC1 was omitted. No back-conversion of heated RNA to original L-rRNA occurred by gradual cooling at room temperature for 40 min. S-rRNA, however, seemed to be stable in these treatments compared with L-rRNA.     

Nucleotide sequence of an exceptionally long 5.8S ribosomal RNA from Crithidia fasciculata.

In Crithidia fasciculata, a trypanosomatid protozoan, the large ribosomal subunit contains five small RNA species (e, f, g, i, j) in addition to 5S rRNA [Gray, M.W. (1981) Mol. Cell. Biol. 1, 347-357]. The complete primary sequence of species i is shown here to be pAACGUGUmCGCGAUGGAUGACUUGGCUUCCUAUCUCGUUGA ... AGAmACGCAGUAAAGUGCGAUAAGUGGUApsiCAAUUGmCAGAAUCAUUCAAUUACCGAAUCUUUGAACGAAACGG ... CGCAUGGGAGAAGCUCUUUUGAGUCAUCCCCGUGCAUGCCAUAUUCUCCAmGUGUCGAA(C)OH. This sequence establishes that species i is a 5.8S rRNA, despite its exceptional length (171-172 nucleotides). The extra nucleotides in C. fasciculata 5.8S rRNA are located in a region whose primary sequence and length are highly variable among 5.8S rRNAs, but which is capable of forming a stable hairpin loop structure (the "G+C-rich hairpin"). The sequence of C. fasciculata 5.8S rRNA is no more closely related to that of another protozoan, Acanthamoeba castellanii, than it is to representative 5.8S rRNA sequences from the other eukaryotic kingdoms, emphasizing the deep phylogenetic divisions that seem to exist within the Kingdom Protista.     

Primary and secondary structure of rat 28 S ribosomal RNA.

The primary structure of rat (Rattus norvegicus) 28 S rRNA is determined inferred from the sequence of cloned rDNA fragments. The rat 28 S rRNA contains 4802 nucleotides and has an estimated relative molecular mass (Mr, Na-salt) of 1.66 X 10(6). Several regions of high sequence homology with S. cerevisiae 25 S rRNA are present. These regions can be folded in characteristic base-paired structures homologous to those proposed for Saccharomyces and E. coli. The excess of about 1400 nucleotides in the rat 28 S rRNA (as compared to Saccharomyces 25 S rRNA) is accounted for mainly by the presence of eight distinct G+C-rich segments of different length inserted within the regions of high sequence homology. The G+C content of the four insertions, containing more than 200 nucleotides, is in the range of 78 to 85 percent. All G+C-rich segments appear to form strongly base-paired structures. The two largest G+C-rich segments (about 760 and 560 nucleotides, respectively) are located near the 5'-end and in the middle of the 28 S rRNA molecule. These two segments can be folded into long base-paired structures, corresponding to the ones observed previously by electron microscopy of partly denatured 28 S rRNA molecules.     

Mouse rDNA: sequences and evolutionary analysis of spacer and mature RNA regions.

Two regions of mouse rDNA were sequenced. One contained the last 323 nucleotides of the external transcribed spacer and the first 595 nucleotides of 18S rRNA; the other spanned the entire internal transcribed spacer and included the 3' end of 18S rRNA, 5.8S rRNA, and the 5' end of 28S rRNA. The mature rRNA sequences are very highly conserved from yeast to mouse (unit evolutionary period, the time required for a 1% divergence of sequence, was 30 X 10(6) to 100 X 10(6) years). In 18S rRNA, at least some of the evolutionary expansion and increase in G + C content is due to a progressive accretion of discrete G + C-rich insertions. Spacer sequence comparisons between mouse and rat rRNA reveal much more extensive and frequent insertions and substitutions of G + C-rich segments. As a result, spacers conserve overall G + C richness but not sequence (UEP, 0.3 X 10(6) years) or specific base-paired stems. Although no stems analogous to those bracketing 16S and 23S rRNA in Escherichia coli pre-rRNA are evident, certain features of the spacer regions flanking eucaryotic mature rRNAs are conserved and could be involved in rRNA processing or ribosome formation. These conserved regions include some short homologous sequence patterns and closely spaced direct repeats.     

Mitochondrial L-rRNA from Aspergillus nidulans: potential secondary structure and evolution.

The alignment of gene sequences coding for A. nidulans mitochondrial L-rRNA and E. coli 23S rRNA indicates a strong conservation of primary and potential secondary structure of both rRNA molecules, except that homologies to the 5'-terminal 5.8S-like region and the 3'-terminal 4.5S-like region of bacterial rRNA are not detectable on mtDNA. The structural organization of the A. nidulans mt L-rRNA gene corresponds to that of yeast omega + strains: both genes are interrupted by a large intron sequence (1678 and 1143 bp, respectively) and by another smaller insert (91 and 66 bp) at homologous positions within domain V. An evolutionary tree derived from conserved L-rRNA gene sequences of yeast nuclei, E. coli, maize chloroplasts and six mitochondrial species exhibits a common root of organelle and bacterial sequences separating early from the nuclear branch.     

Evidence for the wide distribution of repetitive DNA sequences in the genusStreptomyces

Summary Repeated DNA sequences were detected as rapidly reannealing sequences in the chromosomal DNA of 13 out of 14Streptomyces species using either hypochromicity measurements or hydroxyapatite chromatography. These sequences made up between approximately 4% and 11% of the total DNA of these species; only inStreptomyces rimosus were repeated DNA sequences not detected. The repeated sequences fall into a number of distinct percentage G+C (%G+C) classes, many being of rather low %G+C. Analytical density ultracentrifugation of the DNA of these species indicated satellite bands of low %G+C, and high-resolution thermal denaturation profiles indicated the presence of blocks of DNA of low G+C content too. No such satellite band could be found inStreptomyces coelicolor and no low-%G+C DNA could be detected in its thermal denaturation profile. The possible relationship of this repeated DNA, an unusual occurrence in a procaryote, to genetic instability and genetic control mechanisms inStreptomyces is discussed.     

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila,Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90°C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70°C) and Escherichia coli (max. growth temp., 47°C) with respect to (a) bihelical content of rRNA; (b) G·C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. 1. Subunits of C. acidophila ribosomes (T_m = 90–93°C) exhibit considerable thermal tolerance over their B. acidocaldarius (T_m = 77°C) and E. coli counterparts (T_m = 72°C). 2. Based on the ‘melting’ hyperchromicities of the intact ribosomal subunits a 51–55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67–70%, appears to be involved in hydrogen bonding in the naked rRNA species. 3. The G·C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63–64% G·C, compared to 58.5% G·C for B. acidocaldarius and 55% G·C for E. coli. 4. The increment in ribosome T_m values with increasing thermophily is greater than the increase in T_m for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). 5. The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. 6. Compared to E. coli, the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

Reassembly of the peptidyltransferase centre of larger subparticles of rabbit reticulocyte ribosomes from a core-particle and split-protein fraction.

We report the reconstruction, from a core-particle and split-protein fraction, of the larger subribosomal particle of rabbit reticulocytes. The reassembled particle was active in polyphenylalanine synthesis and in the puromycin reaction. The core-particles and split-protein fractions were obtained by treatment of the larger subparticle with salt solutions containing NH4+ and Mg2+ in the molar ratio 40:1 over the range 2.25-2.75 M-NH4Cl/56-69mM-MgCl2 at 0 degrees C. This treatment led to the loss of about eight proteins (approx. 17% of the protein moiety), which were found wholly or largely in the split-protein fraction as shown by two-dimensional gel electrophoresis. The core particle retained 5S rRNA and had much decreased (no more than 10% of control) ability to function in the puromycin reaction or in poly (U)-directed polyphenylalanine synthesis. Activity was recovered when the recombined core-particle and split-protein fractions were dialysed overnight at 4 degrees C against 0.3M-NH4Cl/15mM-MgCl2/1mM-dithiothreitol/15% (v/v) glycerol/20mM-Tris/HCl, pH 7.6, and then heated for 1 h at 37 degreesEES C. The recovery was 40-80% of the original activity. Raising the concentration of MgCL2 to 300 mM in 2.5 M-NH4CL led to the removal of seven rather than eight proteins, and the core particle remained active in the puromycin reaction. We infer that the protein retained by raising the concentration of Mg2+ is an essential component of the peptidyltransferase centre of the ribosome.     

Environmental Shaping of Ribosomal RNA Nucleotide Composition

Ribosomal RNA (rRNA) is one of the most important macromolecules in the cell. It is well established that high-temperature environmental conditions destabilize rRNA, leading to a selection for G+C-rich stabilizing structures. Our knowledge about the nucleotide composition effect of other environmental conditions, however, is limited. In the present work, I addressed this by correlating the rRNA nucleotide composition to known environmental habitats for bacteria. The bacterial phyla Firmicutes, Actinobacteria, and Proteobacteria were chosen for in-depth analyses due to the abundance of information available in the databases. Major differences in nucleotide composition were identified between these phyla. In addition to the G+C-->A+T gradients, a main gradient of G+A-->C+T was identified for Firmicutes, while a G+T-->A+C gradient was identified for Actinobacteria. With respect to correlation to environmental conditions, the Firmicutes showed a main structure of high G+C being correlated to thermophilic conditions, high A+T to anaerobic conditions, and high C+T to halophilic conditions. The main patterns detected for Firmicutes can be explained by structural stability for high G+C, chemical instability of G under aerobic conditions, and structural stability by purine/pyrimidine skew for halophilic conditions. On the contrary, the correlations for Actinobacteria cannot easily be explained by chemical and/or structural stability. This may indicate interference with factors not included in my work. Finally, I found a main correlation between high A+T and endosymbiosis for Proteobacteria. High A+T probably reflects adaptation to cell internal growth. Further support for environmentally driven nucleotide composition shaping was found and that polyphyletic bacteria were associated with the same environment/nucleotide correlations. My conclusion is that environmental conditions and habitats have a major effect on rRNA nucleotide composition but that the effects may differ between the bacterial phyla.     

28 S ribosomal RNA in vertebrates. Locations of large-scale features revealed by electron microscopy in relation to other features of the sequences.

The 28 S rRNA from several vertebrate species, when examined by electron microscopy, is seen to contain regions of extensive secondary structure, as first reported for HeLa-cell 28 S rRNA by Wellauer & Dawid [(1973) Proc. Natl. Acad. Sci. U.S.A. 70, 2827-2831]. Here we correlate the locations of these regions, determined from the electron-microscopic data, with the primary structure of 28 S rRNA from human, mouse and Xenopus laevis determined by sequence analysis of rDNA. The secondary-structure features observed by electron microscopy correspond closely to phylogenetically variable G + C-rich regions that largely comprise the eukaryotic expansion segments in these three species. In most if not all cases the features can be identified with long G + C-rich helices deduced from sequence data. Correlations are given between the locations of the secondary-structure features and several 'landmark' restriction sites in 28 S rDNA. By correlating the locations of the rRNA methyl groups reported elsewhere [Maden (1988) J. Mol. Biol. 201, 289-314] with the present findings it is concluded that the rRNA secondary-structure features revealed by electron microscopy are largely or wholly unmethylated.     

Cloning and characterization of G+C-rich, highly repeated DNA sequences from Porphyra yezoensis (laver), Rhodophyta

G+C-rich sequences in the genomic DNA of Porphyrayezoensis (laver) were cloned and characterized. Sequence analyses of the genomic DNA inserted in fiveclones showed that the DNA contained long G+C-richstretches of more than 200 bp. These stretchesconsisted of more than 80% G+C residues. TheG+C-rich sequences were highly repeated andinterspersed throughout the genome of P.yezoensis and constituted about 6.0–6.6% of thegenome. Parts of these sequences were tandem repeatedin arrays. Hybridization experiments showed thatthese highly repeated, interspersed G+C-rich sequenceswere present in other species of Porphyra, butnot in species of the genera Grateloupia and Gelidium, suggesting that these sequences mightevolve rapidly among genomes, species and genera.