Structure and variation in ribosomal RNA genes of pea

Summary A complete ribosomal DNA (rDNA) repeat unit has been cloned from the genome of Pisum sativum (garden pea) and used to construct a map containing a total of 58 cleavage sites for 23 different restriction enzymes. Regions encoding 18s and 25s ribosomal RNA (rRNA) were identified by R-loop analysis. A 180 bp sequence element is repeated eight times in the intergenic nontranscribed spacer (NTS) region, as defined by eight evenly spaced RsaI cleavage sites. Sequence heterogeneity among these elements (subrepeats) is indicated by the presence of an NcoI site within the five RsaI subrepeats distal to the 25s rRNA gene but not in the three subrepeats proximal to this gene, and also by the presence of an additional RsaI cleavage site in one subrepeat.The approximately 4000 copies of the rDNA repeat in the pea nuclear genome show considerable heterogeneity with respect to the length of the NTS region, and differences are also frequently observed between different genotypes. In both cases the length variation appears to be due primarily to differences in the number of subrepeat elements.Comparison of rDNA restriction maps for two pea genotypes separated for hundreds or perhaps thousands of generations reveals that they contain many rDNA identical repeat units. This data is consistent with the view that new rDNA variants are fixed only infrequently in the evolution of a species.Differences also exist between the rDNA repeats of a single genotype with respect to the degree of base modification at certain restriction sites. A large number of sites known to exist in the pea rDNA clone are not cleaved at all in genomic rDNA, or are cleaved in only some copies of the rDNA repeat. We believe these examples of incomplete cleavage results mostly from methylation, although it is difficult to rule out the possibility of sequence variation in all cases. Most putative modifications are best interpreted in terms of cytosine methylation in CG and CXG sequences, but at least one example is more consistent with adenine methylation.We also have constructed a more detailed restriction map of the wheat rDNA clone pTA71 and present a comparison of this map to our map of pea, pumpkin, and wheat in order to assess the amount of useful evolutionary information that can be obtained by comparison of such maps.     

Intragenomic variation in ribosomal RNA gene of the sea urchin <Emphasis Type="Italic">Lytechinus variegatus</Emphasis>

The first series of studies on the rDNA satellite of the sea urchin, Lytechinus variegatus, based on saturation hybridization of rRNA-rDNA and renaturation kinetics, showed that repeat length of rRNA gene was of about 8 kb in which there was no provision for NTS. The EM denaturation mapping, however, revealed (1) that the gene was 75% larger (longer) than 8 kb, within which there was a NTS whose length varied in repeating units, (3) and there was a region of high GC almost in the middle of the transcribed part. The suggestion of length and sequential heterogeneity in the gene copies coming from the first denaturation mapping prompted further studies with techniques so that the conclusions of the previous results could be stated with finality. The results that emanated from further studies established that the rDNA repeat length of L. variegatuswas of about 12 kb and that the NTS ranged from 3.8 to 6.4 kb. Earlier demonstration of a moderately high-GC segment within the transcribed part was also confirmed by sequence analysis. However, the stipulations on the NTS regarding sequential and length heterogeneity, still awaits elucidation by sequence analysis.     

Bryophyte 5S rDNA was inserted into 45S rDNA repeat units after the divergence from higher land plants

The 5S ribosomal RNA genes (5S rDNA) are located independently from the 45S rDNA repeats containing 18S, 5.8S and 26S ribosomal RNA genes in higher eukaryotes. Southern blot and fluorescence in situ hybridization analyses demonstrated that the 5S rDNAs are encoded in the 45S rDNA repeat unit of a liverwort, Marchantia polymorpha, in contrast to higher plants. Sequencing analyses revealed that a single-repeat unit of the M. polymorpha nuclear rDNA, which is 16103 bp in length, contained a 5S rDNA downstream of 18S, 5.8S and 26S rDNA. To our knowledge, this is the first report on co-localization of the 5S and 45S rDNAs in the rDNA repeat of land plants. Furthermore, we detected a 5S rDNA in the rDNA repeat of a moss, Funaria hygrometrica, by a homology search in a database. These findings suggest that there has been structural re-organization of the rDNAs after divergence of the bryophytes from the other plant species in the course of evolution.     

Molecular characterization of the 5S ribosomal gene of the <Emphasis Type="Italic">Bradysia hygida</Emphasis>(Diptera:Sciaridae)

The rRNA genes are amongst the most extensively studied eukaryotic genes. They contain both highly conserved and rapidly evolving regions. The aim of this work was to clone and to sequence the Bradysia hygida 5S rDNA gene. A positive clone was sequenced and its 346 bp sequence was analyzed against the GenBank database. Sequence analysis revealed that the B. hygida 5S (Bh5S) rDNA gene is 120 bp long and is 87% identical to the aphid Acyrthosiphon magnoliae 5S rDNA gene. The Bh5S rDNA gene presents two unusual features: a GG pair at the 5' end of the gene sequence and the localization of the polyT signal immediately after the 3' end of the gene. In situ5S hybridization experiments revealed that the Bh5S rDNA gene is localized in the autosomal A chromosome     

Human ribosomal RNA gene cluster: identification of the proximal end containing a novel tandem repeat sequence

Human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters on the short arms of five pairs of acrocentric chromosomes. We have demonstrated that a majority of the rDNA clusters are detected as 3-Mb DNA fragments when released from human genomic DNA by EcoRV digestion. This indicated the absence of the EcoRV restriction site within the rDNA clusters. We then screened for rDNA-positive cosmid clones using a chromosome 22-specific cosmid library that was constructed from MboI partial digests of the flow-sorted chromosomes. Three hundred twenty rDNA-positive clones negative for the previously reported distal flanking sequence (pACR1) were chosen and subjected to EcoRV digestion. Seven clones susceptible to EcoRV were further characterized as candidate clones that might have been derived from the junctions of the 3-Mb rDNA cluster. We identified one clone containing part of the rDNA unit sequence and a novel flanking sequence. Detailed analysis of this unique clone revealed that the coding region of the last rRNA gene located at the proximal end of the cluster is interrupted with a novel sequence of 147 bp that is tandemly repeated and is connected with an intervening 68-bp unique sequence. This junction sequence was readily amplified from chromosomes 21 and 15 as well as 22 using the polymerase chain reaction. Fluorescence in situ hybridization further indicated that the 147-bp sequence repeat is commonly distributed among all the acrocentric short arms.     

The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris

Summary The total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined. The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp. The spacer contains long direct repeats and is heterogeneous in size. The LSU-rRNA of G. muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli. The LSU-rRNA has a size comparable to the 23S rRNA of E. coli but shows structural features typical for eukaryotes. Some variable regions are typically small and account for the overall smaller size of this rRNA. The structure of the G. muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.Offprint requests to: H. van Keulen     

Ribosomal DNA in Drosophila melanogaster. II. Heteroduplex mapping of cloned and uncloned rDNA.

Sequences in the cloned Drosophila melanogaster rDNA fragments described by Dawid et al. (1978) were compared by heteroduplex mapping. The nontranscribed spacer regions in all fragments are homologous but vary in length. Deletion loops were observed at variable positions in the spacer region suggesting that spacers are internally repetitious.Many rDNA repeats in D. melanogaster have a 28 S gene interrupted by a region named the ribosomal insertion. Insertions of 0.5, 1 and 5 kb were found in repeat-length EcoRI fragments. These DNA regions, named type 1 insertions, are homologous at their right ends. Although 1 kb insertions are quite precisely twice as large as 0.5 kb insertions they do not represent a duplication of the shorter sequence. Some insertions have at least one EcoRI site and therefore yield EcoRI fragments which are only part of a repeat. The sequences in two cloned right-hand partial insertion sequences are homologous, but the sequences in two lefthand partial insertions are not. None of the EcoRI-restrictable insertion sequences has any homology to any part of type 1 insertions; they are thus grouped together as type 2. Evidence for insertion sequences of at least two types in uncloned rDNA was obtained by annealing a cloned fragment with a 1 kb insertion to genomic rDNA. About 15% of the rDNA repeats show substitution type loops between the 1 kb type 1 insertion derived from the cloned fragment and type 2 insertions in the rDNA.     

Genomic Cloning and Characterization of a Novel Lectin Gene from <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis>

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.     

Independent chromosomal localization of two different size 5S rDNA of Allium victorialis var. platyphyllum by sequential fluorescence in situ hybridization in accordance with sequence polymorphism

5S rRNA gene repeat units in a species are usually organized as either one relatively close size with numbers of intraspecific variations in NTS region or two different sizes with completely different sequence in NTS. Allium victorialis var. platyphyllum revealed two different size products of approximately 0.39 kb and 0.51 kb with highly conserved coding region of 120 bp. However, an extra sequences of approximately 120 bp between at 324 and 443 bp in long NTS region revealed, besides the remaining sequences of two NTS regions of short and long size were highly conserved giving the identity of 94.9%. To identify whether two different size 5S rDNA are occupied by a mixed state as random repeat or an independent group by each size in a particular locus, two rounds of FISH was sequentially performed using two probes of independent different size 5S rDNA and additional probe of only extra sequences of 120 bp in long NTS. Due to the highly conserved coding regions of both 5S rDNA, two different size 5S rDNA were detected in 3 loci in short arm of chromosome 6, however, extra sequences of long NTS was shown only in one locus within detected 5S rDNA from all examined chromosomes and interphase cells. This independent localization of two different size 5S rDNA suggests that 5S rDNA may be organized as a tandem repeat with random positions in a molecular level, but of cytogenetic view in chromosomes and interphase cells, they are organized as an independent group in a significant loci consisting of own size by the patterns of nucleotide variations.     

Direct repeats surrounding the ribosomal RNA genes of Physarum polycephalum

Sequence homology was found between the external transcribed spacer and the terminal non-transcribed spacer of Physarum polycephalum rDNA. The homologous sequences were located 2kb upstream from the 19s rRNA gene and 0.3kb downstream from 26S rRNA gene, respectively, and were arranged in a direct repeat manner. Sequence analyses showed that the direct repeats consisted of two parts: one was sequences of about 130bp which showed over 90% sequence homology with each other. The other consisted mainly of many tandem repeats of a 50 to 52bp unit. The direct repeat-rRNA genes-direct repeat unit was found to be flanked by short direct repetitious sequences. Based on these findings, the significance of the direct repeat is discussed in terms of evolution of rDNA.     

GENETIC VARIABILITY AND MOLECULAR PHYLOGENY OF DINOPHYSIS SPECIES (DINOPHYCEAE) FROM NORWEGIAN WATERS INFERRED FROM SINGLE CELL ANALYSES OF rDNA1

The objectives of this study were to determine rDNA sequences of the most common Dinophysis species in Scandinavian waters and to resolve their phylogenetic relationships within the genus and to other dinoflagellates. A third aim was to examine the intraspecific variation in D. acuminata and D. norvegica, because these two species are highly variable in both morphology and toxicity. We obtained nucleotide sequences of coding (small subunit [SSU], partial large subunit [LSU], 5.8S) and noncoding (internal transcribed spacer [ITS]1, ITS2) parts of the rRNA operon by PCR amplification of one or two Dinophysis cells isolated from natural water samples. The three photosynthetic species D. acuminata, D. acuta, and D. norvegica differed in only 5 to 8 of 1802 base pairs (bp) within the SSU rRNA gene. The nonphotosynthetic D. rotundata (synonym Phalacroma rotundatum[Claparède et Lachmann] Kofoid et Michener), however, differed in approximately 55 bp compared with the three photosynthetic species. In the D1 and D2 domains of LSU rDNA, the phototrophic species differed among themselves by 3 to 12 of 733 bp, whereas they differed from D. rotundata by more than 100 bp. This supports the distinction between Dinophysis and Phalacroma. In the phylogenetic analyses based on SSU rDNA, all Dinophysis species were grouped into a common clade in which D. rotundata diverged first. The results indicate an early divergence of Dinophysis within the Dinophyta. The LSU phylogenetic analyses, including 4 new and 11 Dinophysis sequences from EMBL, identified two major clades within the phototrophic species. Little or no intraspecific genetic variation was found in the ITS1–ITS2 region of single cells of D. norvegica and D. acuminata from Norway, but the delineation between these two species was not always clear.     

Reconsideration of systematic relationships within the order Euplotida (Protista, Ciliophora) using new sequences of the gene coding for small-subunit rRNA and testing the use of combined data sets to construct phylogenies of the Diophrys-complex

Comprehensive molecular analyses of phylogenetic relationships within euplotid ciliates are relatively rare, and the relationships among some families remain questionable. We performed phylogenetic analyses of the order Euplotida based on new sequences of the gene coding for small-subunit RNA (SSrRNA) from a variety of taxa across the entire order as well as sequences from some of these taxa of other genes (ITS1-5.8S-ITS2 region and histone H4) that have not been included in previous analyses. Phylogenetic trees based on SSrRNA gene sequences constructed with four different methods had a consistent branching pattern that included the following features: (1) the “typical” euplotids comprised a paraphyletic assemblage composed of two divergent clades (family Uronychiidae and families Euplotidae–Certesiidae–Aspidiscidae–Gastrocirrhidae), (2) in the family Uronychiidae, the genera Uronychia and Paradiophrys formed a clearly outlined, well-supported clade that seemed to be rather divergent from Diophrys and Diophryopsis, suggesting that the Diophrys-complex may have had a longer and more separate evolutionary history than previously supposed, (3) inclusion of 12 new SSrRNA sequences in analyses of Euplotidae revealed two new clades of species within the family and cast additional doubt on the present classification of genera within the family, and (4) the intraspecific divergence among five species of Aspidisca was far greater than those of closely related genera. The ITS1-5.8S-ITS2 coding regions and partial histone H4 genes of six morphospecies in the Diophrys-complex were sequenced along with their SSrRNA genes and used to compare phylogenies constructed from single data sets to those constructed from combined sets. Results indicated that combined analyses could be used to construct more reliable, less ambiguous phylogenies of complex groups like the order Euplotida, because they provide a greater amount and diversity of information.     

Cerastoderma glaucum 5S ribosomal DNA: characterization of the repeat unit, divergence with respect to Cerastoderma edule, and PCR-RFLPs for the identification of both cockles.

The 5S rDNA repeat unit of the cockle Cerastoderma glaucum from the Mediterranean and Baltic coasts was PCR amplified and sequenced. The length of the units was 539-568 bp, of which 120 bp were assigned to the 5S rRNA gene and 419-448 bp to the spacer region, and the G/C content was 46%-49%, 54%, and 44%-47%, respectively. Two types of units (A and B), differing in the spacer, were distinguished based on the percentage of differences and clustering in phylogenetic trees. A PCR assay with specific primers for each unit type indicated that the occurrence of both units is not restricted to the sequenced individuals. The 5S rDNA units of C. glaucum were compared with new and previously reported sequences of Cerastoderma edule. The degree of variation observed in C. edule was lower than that in C. glaucum and evidence for the existence of units A and B in C. edule was not found. The two cockles have the same coding region but displayed numerous fixed differences in the spacer region and group separately in the phylogenetic trees. Digestion of the 5S rDNA PCR product with the restriction enzymes HaeIII and EcoRV revealed two RFLPs useful for cockle identification.     

Cloning and structural analyses of partial nuclear rDNA fromBupleurum euphorbioides (Apiaceae)

For the cloning of nuclear ribosomal DNA (rDNA) fromBupleurum euphorbioides (Apiaceae), ten clones were screened by DNA-DNA hybridization method. Among them, two clones were strongly hybridized with a heterologous probe of rice rDNA and with an autologous probe of an internally-transcribed region ofB. euphorbioides amplified by PCR. We sequenced both ends of the two genomic clones aligned with a known sequence of rDNA. ITS2 sequences of the two clones showed 98% and 83% homology with the ITS2 sequence ofB. euphorbioides. Our clones showed 1 bp and 3 bp nucleotide substitutions in the 25S and intergenic spacer regions, respectively, and the ITS1 and 18S regions were both missing. Restriction enzyme sites and the orientation of both clones were analyzed for physical mapping purposes. Apart from the length difference between the two clones, we found restriction site variations in the 25S and intergenic spacer regions.     

Repeated DNA sequences found in the large spacer of Vicia faba rDNA

In the large spacer of the rDNA of Vicia faba, multiples of a 0.32 kilobasepair (kb) sequence reiterate to various degrees. We sequenced the repetitious region consisting of the repeating sequences and its flanking regions using two cloned plasmids, which contain V. faba rDNA segments encompassing the whole region of the large spacer. The repetitious region was found to consist of multiple complete copies and one truncated copy of a 325 bp repeat unit and to be flanked by direct repeat sequences of about 150 bp. The set of direct repeats located at either side of the repetitious region differed from each other with about 10% sequence heterogeneity. However, nucleotide sequences of the direct repeats were well conserved between the two clones examined. Southern blot hybridization indicated a widespread distribution within the whole V. faba genome of some related sequences with high homologies to the 325 bp repeat unit and to the direct repeats.     

Next generation sequencing from Hepatozoon canis (Apicomplexa: Coccidia: Adeleorina): Complete apicoplast genome and multiple mitochondrion-associated sequences

Extrachromosomal genomes of the adeleorinid parasite Hepatozoon canis infecting an Israeli dog were investigated using next-generation and standard sequencing technologies. A complete apicoplast genome and several mitochondrion-associated sequences were generated. The apicoplast genome (31,869?bp) possessed two copies of both large subunit (23S) and small subunit (16S) ribosomal RNA genes (rDNA) within an inverted repeat region, as well as 22 protein-coding sequences, 25 transfer RNA genes (tDNA) and seven open reading frames of unknown function. Although circular-mapping, the apicoplast genome was physically linear according to next-generation data. Unlike other apicoplast genomes, genes encoding ribosomal protein S19 and tDNAs for alanine, aspartic acid, histidine, threonine and valine were not identified. No complete mitochondrial genome was recovered using next-generation data or directed PCR amplifications. Eight mitochondrion-associated (215–3523?bp) contigs assembled from next-generation data encoded a complete cytochrome c oxidase subunit I coding sequence, a complete cytochrome c oxidase subunit III coding sequence, two complete cytochrome B coding sequences, a non-coding, pseudogene for cytochrome B and multiple fragmented mitochondrial rDNA genes (SSUA, SSUB, SSUD, LSUC, LSUG, RNA6, RNA10, RNA14, RNA18). The paucity of NGS reads generating each of the mitochondrion-like sequences suggested that a complete mitochondrial genome at typically high copy number was absent in H. canis. In contrast, the complete nuclear rDNA unit sequence of H. canis (18S rDNA to 28S rDNA, 6977?bp) had >1000-fold next-generation coverage. Multiple divergent (from 93.6% to 99.9% pairwise identities) nuclear 18S rDNA contigs were generated (three types with 10 subtypes total). To our knowledge this is the first apicoplast genome sequenced from any adeleorinid coccidium and the first mitochondrion-associated sequences from this serious pathogen of wild and domestic canids. These newly generated sequences may provide useful genetic loci for high-resolution species-level genotyping that is currently impossible using existing nuclear rDNA targets.     

Strategies for Amplification by Polymerase Chain Reaction of the Complete Sequence of the Gene Encoding Nuclear Large Subunit Ribosomal RNA in Corals

The nearly complete nuclear large subunit ribosomal RNA (LSU rRNA) gene in corals was amplified by primers designed from polymerase chain reaction (PCR) strategies. The motif of the putative 3′-terminus of the LSU rRNA gene was sequenced and identified from intergenic spacer (IGS) clones obtained by PCR using universal primers designed for corals. The 3′-end primer was constructed in tandem with the universal 5′-end primer for the LSU rRNA gene. PCR fragments of 3500 bp were amplified for octocorals and non-Acropora scleractinian corals. More than 80% of the Acropora LSU rRNA gene (3000 bp) was successfully amplified by modification of the 5′-end of the IGS primer. Analysis of the 5′-end of LSU rDNA sequences, including the D1 and D2 divergent domains, indicates that the evolutionary rate of the LSU rDNA differs among these taxonomic groups of corals. The genus Acropora showed the highest divergence pattern in the LSU rRNA gene, and the presence of a long branch of the Acropora clade from the other scleractinian corals in the phylogenetic tree indicates that the evolutionary rate of Acropora LSU rDNA might have accelerated after divergence from the common ancestor of scleractinian corals. Received February 17, 2000; accepted June 12, 2000.     

Rice mitochondrial genome contains a rearranged chloroplast gene cluster

Summary We have previously reported the isolation and partial sequence analysis of a rice mitochondrial DNA fragment (6.9 kb) which contains a transferred copy of a chloroplast gene cluster coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL), and subunits of ATPase (atpB and atpE), methionine tRNA (trnM) and valine tRNA (trnV). We have now completely sequenced this 6.9 kb fragment and found it to also contain a sequence homologous to the chloroplast gene coding for the ribosomal protein L2 (rpl2), beginning at a site 430 bp downstream from the termination codon of rbcL. In the chloroplast genome, two copies of rpl2 are located at distances of 20 kb and 40 kb, respectively, from rbcL. We have sequenced these two copies of rice chloroplast rpl2 and found their sequences to be identical. In addition, a 151 bp sequence located upstream of the chloroplast rpl2 coding region is also found in the 3 noncoding region of chloroplast rbcL and other as yet undefined locations in the rice chloroplast genome. Hybridization analysis revealed that this 151 bp repeat sequence identified in rice is also present in several copies in 11 other plant species we have examined. Findings from these studies suggest that the translocation of rpl2 to the rbcL gene cluster found in the rice mitochondrial genome might have occurred through homologous recombination between the 151 bp repeat sequence present in both rpl2 and rbcL.     

Directed alterations of the X-chromosomal ribosomal gene repeats in mutant sc8 of Drosophila melanogaster

The patterns of the ribosomal DNA (rDNA) repeat units in seven Drosophila melanogaster inversional mutants have been studied. Among them, only the In(1)sc⁸ and its deletional derivative Df(1)mal¹² female rDNAs exibited significant reduction in the size of nearly all units, compared to the wild-type females (Canton S, Oregon R). Further investigation shows that each kind of repeat (insertion-free, insertion-containing) in the Xsc⁸ rDNA array is highly enriched with short (reduced to 4 kilobases) intergenic spacers (IGSs). We revealed two main types of rearrangements. Only part of the 4 kb IGSs display variable length deletions (0.2–0.6 kb) at the 5′ ends, within the so-called ‘1900’ base pair (bp) region, recognizable by restriction endonuclease AluI. The presence of additional 100–150 bp DNA in the start portion of this region has also been demonstrated. In contrast, the 3′ end spacer regions, corresponding to the external transcribed spacer, do not show any changes in size. These data indicate how reductions of approximately 1.1 kb DNAs in sc⁸ IGSs, carrying both the rearranged and non-rearranged ‘1900’ sequences, are achieved: the fixed decrease of a number of 240 bp AluI subrepeats, clustered in the central IGS portion, also contribute. None of the other similar inversional mutants examined has so many IGS variants. Therefore, alterations in the Xsc⁸ rRNA gene cluster seem not to be dependent on its inversional status. This revised version was published online in July 2006 with corrections to the Cover Date.