A 7.1 kb linear DNA molecule of Theileria parva has scrambled rDNA sequences and open reading frames for mitochondrially encoded proteins.

Theileria parva, an intralymphocytic protozoan parasite of cattle, contains a linear 7.1 kb DNA element with terminal inverted repeat sequences. The molecule is transcribed into low molecular weight RNA, and both DNA strands encode short stretches of unique sequences, usually < 100 nucleotides, which are similar to large (LSU) or small (SSU) ribosomal subunit RNA. Phylogenetically conserved conformational rRNA domains were assembled from the discontinuous rDNA sequences using comparative secondary structure modelling. For example, a minimum of four predicted sequences, two derived from each DNA strand, is required to assemble domain V of LSU rRNA which participates in peptidyl transferase activity. The discontinuities in the identified rRNA domains fall within regions of no known functional significance. Hence, it is likely that the element encodes fragmented rDNA genes and the mature rRNA is unconventional, consisting of several fragments of RNA, primarily held together by intermolecular and intramolecular base pairing. The element also has ORFs for components of the last two mitochondrial electron transport enzyme complexes. The structure of the parasite DNA element, its protein coding capacity and scrambled rDNA gene sequences, are reminiscent of the mitochondrial genome of Chlamydomonas reinhardtii. We propose that the 7.1 kb element is equivalent to the mitochondrial DNA of T. parva, although a number of its features are unusual for this family of extrachromosomal DNA molecules.     

Phylogenetic analysis of Glomeromycota by partial LSU rDNA sequences

We analyzed the large subunit ribosomal RNA (rRNA) gene [LSU ribosomal DNA (rDNA)] as a phylogenetic marker for arbuscular mycorrhizal (AM) fungal taxonomy. Partial LSU rDNA sequences were obtained from ten AM fungal isolates, comprising seven species, with two new primers designed for Glomeromycota LSU rDNA. The sequences, together with 58 sequences available from the databases, represented 31 AM fungal species. Neighbor joining and parsimony analyses were performed with the aim of evaluating the potential of the LSU rDNA for phylogenetic resolution. The resulting trees indicated that Archaeosporaceae are a basal group in Glomeromycota, Acaulosporaceae and Gigasporaceae belong to the same clade, while Glomeraceae are polyphyletic. The results support data obtained with the small subunit (SSU) rRNA gene, demonstrating that the LSU rRNA gene is a useful molecular marker for clarifying taxonomic and phylogenetic relationships in Glomeromycota.     

Genetic and chromosomal localization of the 5S rDNA locus in sugar beet (Beta vulgaris L.).

A digoxigenin-labelled 5S rDNA probe containing the 5S rRNA gene and the adjacent intergenic spacer was used for in situ hybridization to metaphase and interphase chromosomes of a trisomic stock from sugar beet (Beta vulgaris L.). Three chromosomes of primary trisomic line IV (T. Butterfass. Z. Bot. 52: 46-77. 1964) revealed signals close to the centromeres. Polymorphisms of 5S rDNA repeats in a segregating population were used to map genetically the 5S rRNA genes within a cluster of markers in linkage group II of sugar beet. The concentration of genetic markers around the centromere presumably reflects the suppressed recombination frequency in centromeric regions. The correlation of physical and genetic data allowed the assignment of a linkage group to sugar beet chromosome IV according to line IV of the primary trisomics.     

Precise characterization of rDNA genes by intraspecies and inter-loci comparison of rDNA sequences and biochemical analysis of ribosomal RNA molecules in Agrobacterium tumefaciens

Annotation of rRNA genes has been incomplete in Agrobacterium species although a number of Agrobacterial rDNA fragments have been sequenced. In this study, precise characterization of rRNA operons (rrn) was carried out in two biovar 1 strains, C58 and MAFF301001. Complete DNA sequencing of four rrns in MAFF301001 indicated that each operon codes for 16S, 23S and 5S rRNA as well as three tRNAs, trn(Ile), trn(Ala) and trn(Met). The genes and 16S-23S ITS of a given locus were exactly identical with those in the other three loci, except for a T-base loss in the 23S rRNA gene of rrnA and in the 5S rRNA gene of rrnB. Comparison with the four C58 rDNAs available in the DNA database indicated extensive sequence and size variations in the 23S rRNA gene, suggesting the presence of an intervening sequence (IVS). Biochemical RNA analysis, including Northern hybridization and 5' end mapping, in MAFF301001 revealed 2886-base and 2571-base precursors, two 1.3-kb major fragments, a 150-base fragment and removal of an IVS for 23S rRNA. We confirmed similar biochemical characteristics in the C58 strain. The features of rDNA detected here enable correction of previously reported information about Agrobacterial rRNAs and rRNA genes and should be useful for phylogenetic considerations.     

Sequence analysis of the rDNA spacer ofParacentrotus lividus and observations about pre-rRNA processing

We have isolated and sequenced one intergenic region and a small part of the flanking regions (18S and 26S rRNA coding regions) of the rRNA-encoding genes (rDNA) from the sea urchinParacentrotus lividus. This region is about 3.8 Kb long. Northern blot hybridizations and S1 mapping experiments demonstrated the presence of a partially processed 21S rRNA precursor which has the same 5 terminus as the 32S primary precursor, also in developmental stages characterized by a low rate of rRNA synthesis.Abbreviations bp base pair(s) - Kb Kilobase(s) or 1000 bp - nt nucleotide(s) - rDNA DNA encoding rRNA - rRNA ribosomal RNA - S sedimentation constant     

Dynamics of 5S rDNA in the tilapia (Oreochromis niloticus) genome: repeat units,inverted sequences,pseudogenes and chromosome loci

In higher eukaryotes, the 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units composed of a coding region and a non-transcribed spacer sequence (NTS). These tandem arrays can be found on either one or more chromosome pairs. 5S rDNA copies from the tilapia fish, Oreochromis niloticus, were cloned and the nucleotide sequences of the coding region and of the non-transcribed spacer were determined. Moreover, the genomic organization of the 5S rDNA tandem repeats was investigated by fluorescence IN SITU hybridization (FISH) and Southern blot hybridization. Two 5S rDNA classes, one consisting of 1.4-kb repeats and another one with 0.5-kb repeats were identified and designated 5S rDNA type I and type II, respectively. An inverted 5S rRNA gene and a 5S rRNA putative pseudogene were also identified inside the tandem repeats of 5S rDNA type I. FISH permitted the visualization of the 5S rRNA genes at three chromosome loci, one of them consisting of arrays of the 5S rDNA type I, and the two others corresponding to arrays of the 5S rDNA type II. The two classes of the 5S rDNA, the presence of pseudogenes, and the inverted genes observed in the O. niloticus genome might be a consequence of the intense dynamics of the evolution of these tandem repeat elements.     

Phylogenetic analysis of the insect order Odonata using 28S and 16S rDNA sequences: a comparison between data sets with different evolutionary rates

Molecular phylogenetic analyses were conducted for the insect order Odonata with a focus on testing the effectiveness of a slowly evolving gene to resolve deep branching and also to examine: (i) the monophyly of damselflies (the suborder Zygoptera); and (ii) the phylogenetic position of the relict dragonfly Epiophlebia superstes. Two independent molecular sources were used to reconstruct phylogeny: the 16S rRNA gene on the mitochondrial genome and the 28S rRNA gene on the nuclear genome. A comparison of the sequences showed that the obtained 28S rDNA sequences have evolved at a much slower rate than the 16S rDNA, and that the former is better than the latter for resolving deep branching in the Odonata. Both molecular sources indicated that the Zygoptera are paraphyletic, and when a reasonable weighting for among‐site rate variation was enforced for the 16S rDNA data set, E. superstes was placed between the two remaining major suborders, namely, Zygoptera and Anisoptera (dragonflies). Character reconstruction analysis suggests that multiple hits at the rapidly evolving sites in the 16S rDNA degenerated the phylogenetic signals of the data set.     

Molecular organization of 5S rDNA in bitterlings (Cyprinidae)

Molecular organization and nucleotide sequences of the 5S rRNA gene and NTS were investigated in freshwater fish, bitterlings (Acheilognathinae), including 10 species/subspecies of four genera, Acheilognathus, Pseudoperilampus, Rhodeus, and Tanakia, to understand the evolutionary trait of 5S rDNA arrays. Southern hybridization analysis revealed a general trend with tandem repeats of 5S rDNA in all the examined bitterlings. Sequence analysis demonstrated a conserved 120 bp sequence of the 5S rRNA gene and a short NTS of 56–67 bp with two distinct portions, a conserved (5′-flanking portion; at positions −1 to −38) and a variable part (3′-flanking portion), in 6 of 10 species/subspecies examined. The conserved NTS region was most likely an external promoter so far observed in various vertebrates, whereas the variable NTS region could be divided into two types due to its nucleotide polymorphisms. Molecular phylogeny using the 5S rRNA gene and NTS sequences suggested the occurrence of 5S rDNA duplication before speciation and a concerted evolution for the gene and conserved NTS regions, but a birth-and-death process to maintain the variable NTS region. Thus, the 5S rDNA in the examined bitterlings might have evolved under a mixed process of evolution.     

Tandem repeats of the 5' non-transcribed spacer of Tetrahymena rDNA function as high copy number autonomous replicons in the macronucleus but do not prevent rRNA gene dosage regulation.

The rRNA genes in the somatic macronucleus of Tetrahymena thermophila are normally on 21 kb linear palindromic molecules (rDNA). We examined the effect on rRNA gene dosage of transforming T.thermophila macronuclei with plasmid constructs containing a pair of tandemly repeated rDNA replication origin regions unlinked to the rRNA gene. A significant proportion of the plasmid sequences were maintained as high copy circular molecules, eventually consisting solely of tandem arrays of origin regions. As reported previously for cells transformed by a construct in which the same tandem rDNA origins were linked to the rRNA gene [Yu, G.-L. and Blackburn, E. H. (1990) Mol. Cell. Biol., 10, 2070-2080], origin sequences recombined to form linear molecules bearing several tandem repeats of the origin region, as well as rRNA genes. The total number of rDNA origin sequences eventually exceeded rRNA gene copies by approximately 20- to 40-fold and the number of circular replicons carrying only rDNA origin sequences exceeded rRNA gene copies by 2- to 3-fold. However, the rRNA gene dosage was unchanged. Hence, simply monitoring the total number of rDNA origin regions is not sufficient to regulate rRNA gene copy number.     

Copy Number of the Transposon,Pokey, in rDNA Is Positively Correlated with rDNA Copy Number in Daphnia obtusa

Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA) genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR) to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL) initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species.     

Informative characteristics of 12 divergent domains in complete large subunit rDNA sequences from the harmful dinoflagellate genus, Alexandrium (Dinophyceae)

The genus Alexandrium includes organisms of interest, both for the study of dinoflagellate evolution and for their impacts as toxic algae affecting human health and fisheries. Only partial large subunit (LSU) rDNA sequences of Alexandrium and other dinoflagellates are available, although they contain much genetic information. Here, we report complete LSU rDNA sequences from 11 strains of Alexandrium, including Alexandrium affine, Alexandrium catenella, Alexandrium fundyense, Alexandrium minutum, and Alexandrium tamarense, and discuss their segmented domains and structure. Putative LSU rRNA coding regions were recorded to be around 3,400 bp long. Their GC content (about 43.7%) is among the lowest when compared with other organisms. Furthermore, no AT-rich regions were found in Alexandrium LSU rDNA, although a low GC content was recorded within the LSU rDNA. No intron-like sequences were found. The secondary structure of the LSU rDNA and parsimony analyses showed that most variation in LSU rDNA is found in the divergent (D) domains with the D2 region being the most informative. This high D domain variability can allow members of the diverse Alexandrium genus to be categorized at the species level. In addition, phylogenetic analysis of the alveolate group using the complete LSU sequences strongly supported previous findings that the dinoflagellates and apicomplexans form a clade.     

紫芝基因组rDNA序列特征及ITS2二级结构比较

紫芝栽培品种‘紫芝S2’(武芝2号)的ITS序列与NCBI数据库中5个紫芝菌株/分离株相似度高达99.79%-100%,在系统进化树上相聚成一类。本研究预测‘紫芝S2’基因组与参考基因组中的rRNA基因簇,分析rDNA结构及各构件序列间的多态性。从高质量‘紫芝S2’基因组中挖掘得到完整rDNA,序列全长40.377 kb,由4组串联重复的(18S、5.8S、28S、5S) rRNA基因簇组成,并含有完整的基因内间隔区(ITS1、ITS2)和基因间间隔区(IGS1、IGS2)。在紫芝S2的rDNA中,高度保守的28S rRNA基因间出现3个SNP和2个插入(1 bp,10 bp)位点;虽然第4条ITS2中有1个SNP位点,但紫芝S2的4条ITS2在二级结构上的分子形态高度一致,与ITS2数据库中其他紫芝菌株仅存在螺旋区间夹角的微小差异。由‘紫芝S2’基因组rDNA的ITS2生成的DNA条形码与二维码,可以作为该栽培品种鉴定与同源物种其他菌株鉴别的分子标记。     

Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis of specifically amplified 18s rDNA.

Marram grass (Ammophila arenaria L.), a sand-stabilizing plant species in coastal dune areas, is affected by a specific pathosystem thought to include both plant-pathogenic fungi and nematodes. To study the fungal component of this pathosystem, we developed a method for the cultivation-independent detection and characterization of fungi infecting plant roots based on denaturing gradient gel electrophoresis (DGGE) of specifically amplified DNA fragments coding for 18S rRNA (rDNA). A nested PCR strategy was employed to amplify a 569-bp region of the 18S rRNA gene, with the addition of a 36-bp GC clamp, from fungal isolates, from roots of test plants infected in the laboratory, and from field samples of marram grass roots from both healthy and degenerating stands from coastal dunes in The Netherlands. PCR products from fungal isolates were subjected to DGGE to examine the variation seen both between different fungal taxa and within a single species. DGGE of the 18S rDNA fragments could resolve species differences from fungi used in this study yet was unable to discriminate between strains of a single species. The 18S rRNA genes from 20 isolates of fungal species previously recovered from A. arenaria roots were cloned and partially sequenced to aid in the interpretation of DGGE data. DGGE patterns recovered from laboratory plants showed that this technique could reliably identify known plant-infecting fungi. Amplification products from field A. arenaria roots also were analyzed by DGGE, and the major bands were excised, reamplified, sequenced, and subjected to phylogenetic analysis. Some recovered 18S rDNA sequences allowed for phylogenetic placement to the genus level, whereas other sequences were not closely related to known fungal 18S rDNA sequences. The molecular data presented here reveal fungal diversity not detected in previous culture-based surveys.     

Molecular organization of 5S rDNA in fishes of the genus Brycon.

There are few reports on the genomic organization of 5S rDNA in fish species. To characterize the 5S rDNA nucleotide sequence and chromosomal localization in the Neotropical fishes of the genus Brycon, 5S rDNA copies from seven species were generated by PCR. The nucleotide sequences of the coding region (5S rRNA gene) and the nontranscribed spacer (NTS) were determined, revealing that the 5S rRNA genes were highly conserved, while the NTSs were widely variable among the species analyzed. Moreover, two classes of NTS were detected in each species, characterized by base substitutions and insertions-deletions. Using fluorescence in situ hybridization (FISH), two 5S rDNA chromosome loci that could be related to the two 5S rDNA NTS classes were observed in at least one of the species studied. 5S rDNA sequencing and chromosomal localization permitted the characterization of Brycon spp. and suggest a higher similarity among some of them. The data obtained indicate that the 5S rDNA can be an useful genetic marker for species identification and evolutionary studies.