Distribution of 5S ribosomal RNA genes in somatic and germ cells of the house cricket,Acheta domesticus

In the house cricket,Acheta domesticus, the 110 genes per haploid genome encoding 18S and 28S rRNA are contained within rDNA repeats which are amplified during oogenesis. The 5S rRNA coding sequences of this cricket are found in two sizes of 5S DNA repeating units (measuring 2.1 and 3.0 kb). The 3.0 kb repeats account for more than 90% of the totalAcheta 5S DNA. We have determined the number of cricket 5S rRNA genes by RNA-DNA hybridization analysis: 310 5S DNA repeats/haploid genome clearly approximates the number of 18S and 28S rRNA genes. Because of the relatively low copy number of 5S rRNA genes the possibility of 5S DNA amplification in oocytes ofA. domesticus was also examined. Although amplification of rDNA is readily detectable, amplification of 5S DNA is not observed in oocytes ofA. domesticus. Unlike the genes coding for 18S and 28S rRNA which are localized at a single chromosomal site in the genome ofA. domesticus, the 5S rRNA genes occupy numerous sites distributed along the length of most chromosomes.     

The 5S ribosomal RNA gene in Pythium species: two different genomic locations.

The 5S ribosomal RNA (rRNA) genes in eukaryotes may occur either interspersed with the other rRNA genes in the ribosomal DNA (rDNA) repeat, or in separate tandem arrays, or dispersed throughout the genome. In Pythium species and in several related Oomycetes, polymerase chain reaction (PCR) amplification of the nontranscribed spacer (NTS) region with one primer specific for the 5S gene revealed, with several exceptions, that the 5S rRNA gene was present in the rDNA repeat of those species with filamentous sporangia and was absent from the rDNA repeat of those with globose or unknown sporangia. When present, the gene was located approximately 1 kb downstream of the large-subunit rRNA gene and on the strand opposite that on which the other rRNA genes were located. This was confirmed in P. torulosum by sequencing of the gene and its flanking regions. The 5S rRNA genes of P. ultimum were found in tandem arrays outside the rDNA repeat. Evidence of dispersed 5S rRNA sequences was also observed. For many of the species of Pythium having the 5S gene in the NTS, the region between the large-subunit rRNA gene and the 5S gene was found to have length heterogeneity. Oomycetes related to Pythium were also found to have the 5S gene in the NTS, although sometimes in the opposite orientation. This may mean that the presence of the gene in the NTS is ancestral for the Oomycetes and that the absence of the gene in the NTS in those Pythiums with globose sporangia is due to loss of the gene from the rDNA repeat in an ancestor of this group of species. These results favor the view that 5S rRNA gene linkage to the rRNA cistron existed prior to the unlinked arrangement seen in most plants and animals.     

Euglena gracilis chloroplast ribosomal RNA transcription units. Nucleotide sequence polymorphism in 5 S rRNA genes and 5 S rRNAs

The three tandemly repeated ribosomal RNA operons from the chloroplast genome of Euglena gracilis Klebs, Pringsheim Strain Z each contain a 5 S rRNA gene distal to the 23 S rRNA gene (Gray, P.W., and Hallick, R.B. (1979) Biochemistry 18, 1820-1825). We have cloned two distinct 5 S rRNA genes, and determined the DNA sequence of the genes, their 5'- and 3'-flanking sequences, and the 3'-end of the adjacent 23 S rRNA genes. The two genes exhibit sequence polymorphism at five bases within the "procaryotic loop" coding region, as well as internal restriction endonuclease site heterogeneity. These restriction endonuclease site polymorphisms are evident in chloroplast DNA, and not just the cloned examples of 5 S genes. Chloroplast 5 S rRNA was isolated, end labeled, and sequenced by partial enzymatic degradation. The same polymorphisms found in 5 S rDNA are present in 5 S rRNA. Therefore, both types of 5 S rRNA genes are transcribed and are present in chloroplast ribosomes.     

5S ribosomal and U1 small nuclear RNA genes: a new linkage type in the genome of a crustacean that has three different tandemly repeated units containing 5S ribosomal DNA sequences.

We investigated the 5S ribosomal RNA (rRNA) genes of the isopod crustacean Asellus aquaticus. Using PCR amplification, three different tandemly repeated units containing 5S rDNA were identified. Two of the three sequences were cloned and sequenced. One of them was 1842 bp and presented a 5S rRNA gene and a U1 small nuclear RNA (snRNA) gene. This type of linkage had never been observed before. The other repeat consisted of 477 bp and contained only an incomplete 5S rRNA gene lacking the first eight nucleotides and a spacer sequence. The third sequence was 6553 bp long and contained a 5S rRNA gene and the four core histone genes. The PCR products were used as probes in fluorescent in situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus. The possible evolutionary origin of the three repeated units is discussed.     

Unique arrangement of coding sequences for 5 S, 5.8 S, 18 S and 25 S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis.

The arrangement of the coding sequences for the 5 S, 5.8 S, 18 S and 25 S ribosomal RNA from Saccharomyces cerevisiae was analyzed in λ-yeast hybrids containing repeating units of the ribosomal DNA. After mapping of restriction sites, the positions of the coding sequences were determined by hybridization of purified rRNAs to restriction fragments, by R-loop analysis in the electron microscope, and by electrophoresis of S₁ nuclease-treated rRNA/rDNA hybrids in alkaline agarose gels. The R-loop method was improved with respect to the length calibration of RNA/DNA duplexes and to the spreading conditions resulting in fully extended 18 S and 25 S rRNA R-loops. The qualitative results are: (1) the 5 S rRNA genes, unlike those in higher eukaryotes, alternate with the genes of the precursor for the 5.8 S, 18 S and 25 S rRNA; (2) the coding sequence for 5.8 S rRNA maps, as in higher eukaryotes, between the 18 S and 25 S rRNA coding sequences. The quantitative results are: (1) the tandemly repeating rDNA units have a constant length of 9060 ± 100 nucleotide pairs with one SstI, two HindIII and, dependent on the strain, six or seven EcoRI sites; (2) the 18 S and 25 S rRNA coding regions consist of 1710 ± 80 and 3360 ± 80 nucleotide pairs, respectively; (3) an 18 S rRNA coding region is separated by a 780 ± 70 nucleotide pairs transcribed spacer from a 25 S rRNA coding region. This is then followed by a 3210 ± 100 nucleotide pairs mainly non-transcribed spacer which contains a 5 S rRNA gene.     

Bombyx mori 28S ribosomal genes contain insertion elements similar to the Type I and II elements of Drosophila melanogaster.

We have examined the 28S ribosomal genes of the silkmoth, Bombyx mori, for the presence of insertion sequences. Two types of insertion sequences were found, each approximately 5 kb in length, which do not share sequence homology. Comparison of the nucleotide sequences of the junction regions with the uninserted gene reveals that one type of insertion has resulted in a 14 bp duplication of the 28S coding region at the insertion site. The location of this insertion and the 14 bp duplication are identical to that found in the Type I ribosomal insertion element of Drosophila melanogaster. The second type of insertion element is located at a site corresponding to approximately 75 bp upstream of the first type. The location of this insertion, the variability detected at its 5' junction, and a short region of sequence homology at its 3' junction suggest that it is related to the Type II element of D. melanogaster. This is the first example of a Type II-like rDNA insertion outside of sibling species of D. melanogaster, and the first example of a Type I-like rDNA insertion outside of the higher Diptera.     

5S Ribosomal RNA Database

Ribosomal 5S RNA (5S rRNA) is an integral component of the large ribosomal subunit in all known organisms with the exception only of mitochondrial ribosomes of fungi and animals. It is thought to enhance protein synthesis by stabilization of a ribosome structure. This paper presents the updated database of 5S rRNA and their genes (5S rDNA). Its short characteristics are presented in the Introduction. The database contains 2280 primary structures of 5S rRNA and 5S rRNA genes. These include 536 eubacterial, 61 archaebacterial, 1611 eukaryotic and 72 organelle sequences. The database is available on line through the World Wide Web at http://biobases.ibch.poznan.pl/5SData/.     

Primary structure of the 5.8S gene of rRNA and internal transcribed spacers of rDNA in diploid wheat Triticum urartu thum. ex. gandil

Nucleotide sequences of 5.8S rRNA gene and rDNA internal transcribed spacers ITS-1 and ITS-2 were determined in diploid wheat Triticum urartu. It was shown that 5.8S rRNA gene of this wheat species consists of 163 base pairs and GC-content is 59.5%. When comparing 5.8S rRNA sequences in diploid wheat, rice and lupine and also 5.8S rRNA in hexaploid wheat and horse beans a high evolutional conservatism of its structure was revealed. The size of ITS-1 and ITS-2 in Tr. urartu is 219 and 225 base pairs long correspondingly. While comparing structures of similar rDNA regions of Tr. urartu, rice and maize a high level of homology was found only between nucleotides adjoining genes of high molecular rRNAs. In ITS-1 of Tr. urartu an insertion of 5'-GACGACGACATTGTCCGTC-3' was found, which is absent in maize and rice.     

5 S Rrna Is Involved in Fidelity of Translational Reading Frame

Chromosomal mutants (maintenance of frame = mof) in which the efficiency of -1 ribosomal frame-shifting is increased can be isolated using constructs in which lacZ expression is dependent upon a -1 shift of reading frame. We isolate a new mof mutation, mof9, in Saccharomyces cerevisiae and show that it is complemented by both single and multi-copy 5 S rDNA clones. Two independent insertion mutations in the rDNA locus (rDNA::LEU2 and rDNA::URA3) also display the Mof(-) phenotype and are also complemented by single and multi-copy 5 S rDNA clones. Mutant 5 S rRNAs expressed from a plasmid as 20-50% of total 5 S rRNA in a wild-type host also induced the Mof(-) phenotype. The increase in frameshifting is greatest when the lacZ reporter gene is expressed on a high copy, episomal vector. No differences were found in 5 S rRNA copy number or electrophoretic mobilities in mof9 strains. Both mof9 and rDNA::LEU2 increase the efficiency of +1 frameshifting as well but have no effect on readthrough of UAG or UAA termination codons, indicating that not all translational specificity is affected. These data suggest a role for 5 S rRNA in the maintenance of frame in translation.     

Review and re-analysis of domain-specific 16S primers

The Polymerase Chain Reaction (PCR) has facilitated the detection of unculturable microorganisms in virtually any environmental source and has thus been used extensively in the assessment of environmental microbial diversity. This technique relies on the assumption that the gene sequences present in the environment are complementary to the "universal" primers used in their amplification. The recent discovery of new taxa with 16S rDNA sequences not complementary to standard universal primers suggests that current 16S rDNA libraries are not representative of true prokaryotic biodiversity. Here we re-assess the specificity of commonly used 16S rRNA gene primers and present these data in tabular form designed as a tool to aid simple analysis, selection and implementation. In addition, we present two new primer pairs specifically designed for effective "universal" Archaeal 16S rDNA sequence amplification. These primers are found to amplify sequences from Crenarchaeote and Euryarchaeote type strains and environmental DNA.     

18S coding sequences in amplified ribosomal DNA from Xenopus laevis oocytes are highly homogeneous, unmethylated, and lack major open reading frames.

We have used two approaches to search for sequence variants in the 18S coding region of amplified ribosomal DNA (rDNA) from Xenopus laevis oocytes. First, using clones derived from amplified rDNA, we compared the equivalent of a complete 18S coding region from two clones and short regions from two other clones with the 18S sequence previously determined from a "reference" clone. The respective sequences in all the clones were identical. Secondly, we examined greater than 60% of the 18S sequence in "pooled 18S genes" in uncloned amplified rDNA. The predominant sequence corresponded to that in the reference clone and no heterogeneities were apparent. Since many chromosomal rDNA units contribute to rDNA amplification the findings indicate that 18S coding sequences in X. laevis are largely homogeneous. The previously established sequence is the predominant one, thus providing a reliable basis for studies on 18S rRNA. Sequencing gels on uncloned amplified rDNA confirmed the absence of methylated cytosine in this DNA. The 18S sequence lacks major open reading frames.     

Characterization of internal structure of the nucleolar organizing region in rice (Oryza sativa L.)

The genomic sequence of the nucleolar organizing region (NOR) in rice has not been characterized fully because of the difficulty in assembling repetitive sequences in silico. Here, we used a cytogenetic approach to elucidate the internal structure of the NOR. We detected one locus of the 18S rRNA genes on 'Nipponbare' chromosome. High-resolution fiber-fluorescence in situ hybridization made it possible to visualize each rRNA gene unit in the array of rRNA genes. Signals of pairs of alternating 18S and 25S rRNA genes were detected uniformly along the DNA fiber. Intergenic spacers were shorter than the transcribed region. The rRNA genes were infrequently interrupted. These and previous results based on the sequencing of genome fragments, PCR analysis and Southern blot hybridization suggest that the internal region of the NOR is filled with a uniform array of canonical rRNA genes separated by spacers carrying three 254-bp sub-repeats.     

The 10 kb Drosophila virilis 28S rDNA intervening sequence is flanked by a direct repeat of 14 base pairs of coding sequence

Most repeat units of rDNA in Drosophila virilis are interrupted in the 28S rRNA coding region by an intervening sequence about 10 kb in length; uninterrupted repeats have a length of about 11 kb. We have sequenced the coding/intervening sequence junctions and flanking regions in two independent clones of interrupted rDNA, and the corresponding 28S rRNA coding region in a clone of uninterrupted rDNA. The intervening sequence is terminated at both ends by a direct repeat of a fourteen nucleotide sequence that is present once in the corresponding region of an intact gene. This is a phenomenon associated with transposable elements in other eukaryotes and in prokaryotes, and the Drosophila rDNA intervening sequence is discussed in this context. We have compared more than 200 nucleotides of the D. virilis 28S rRNA gene with sequences of homologous regions of rDNA in Tetrahymena pigmentosa (Wild and Sommer, 1980) and Xenopus laevis (Gourse and Gerbi, 1980): There is 93% sequence homology among the diverse species, so that the rDNA region in question (about two-thirds of the way into the 28S rRNA coding sequence) has been very highly conserved in eukaryote evolution. The intervening sequence in T. pigmentosa is at a site 79 nucleotides upstream from the insertion site of the Drosophila intervening sequence.     

Release of ribosome-bound 5S rRNA upon cleavage of the phosphodiester bond between nucleotides A54 and A55 in 5S rRNA

Reticulocyte lysates contain ribosome-bound and free populations of 5S RNA. The free population is sensitive to nuclease cleavage in the internal loop B, at the phosphodiester bond connecting nucleotides A54 and A55. Similar cleavage sites were detected in 5S rRNA in 60S subunits and 80S ribosomes. However, 5S rRNA in reticulocyte polysomes is insensitive to cleavage unless ribosomes are salt-washed. This suggests that a translational factor protects the backbone surrounding A54 from cleavage in polysomes. Upon nuclease treatment of mouse 60S subunits or reticulocyte lysates a small population of ribosomes released its 5S rRNA together with ribosomal protein L5. Furthermore, rRNA sequences from 5.8S, 28S and 18S rRNA were released. In 18S rRNA the sequences mainly originate from the 630 loop and stem (helix 18) in the 5' domain, whereas in 28S rRNA a majority of fragments is derived from helices 47 and 81 in domains III and V, respectively. We speculate that this type of rRNA-fragmentation may mimic a ribosome degradation pathway.     

The phylogenetic potential of entire 26S rDNA sequences in plants

18S ribosomal RNA genes are the most widely used nuclear sequences for phylogeny reconstruction at higher taxonomic levels in plants. However, due to a conservative rate of evolution, 18S rDNA alone sometimes provides too few phylogenetically informative characters to resolve relationships adequately. Previous studies using partial sequences have suggested the potential of 26S or large-subunit (LSU) rDNA for phylogeny retrieval at taxonomic levels comparable to those investigated with 18S rDNA. Here we explore the patterns of molecular evolution of entire 26S rDNA sequences and their impact on phylogeny retrieval. We present a protocol for PCR amplification and sequencing of entire (approximately 3.4 kb) 26S rDNA sequences as single amplicons, as well as primers that can be used for amplification and sequencing. These primers proved useful in angiosperms and Gnetales and likely have broader applicability. With these protocols and primers, entire 26S rDNA sequences were generated for a diverse array of 15 seed plants, including basal eudicots, monocots, and higher eudicots, plus two representatives of Gnetales. Comparisons of sequence dissimilarity indicate that expansion segments (or divergence domains) evolve 6.4 to 10.2 times as fast as conserved core regions of 26S rDNA sequences in plants. Additional comparisons indicate that 26S rDNA evolves 1.6 to 2.2 times as fast as and provides 3.3 times as many phylogenetically informative characters as 18S rDNA; compared to the chloroplast gene rbcL, 26S rDNA evolves at 0.44 to 1.0 times its rate and provides 2.0 times as many phylogenetically informative characters. Expansion segment sequences analyzed here evolve 1.2 to 3.0 times faster than rbcL, providing 1.5 times the number of informative characters. Plant expansion segments have a pattern of evolution distinct from that found in animals, exhibiting less cryptic sequence simplicity, a lower frequency of insertion and deletion, and greater phylogenetic potential.