Functional analysis of internal transcribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA.

Using the previously described "tagged ribosome" (pORCS) system for in vivo mutational analysis of yeast rDNA, we show that small deletions in the 5'-terminal portion of ITS2 completely block maturation of 26 S rRNA at the level of the 29 SB precursor (5.8 S rRNA-ITS2-26 S rRNA). Various deletions in the 3'-terminal part, although severely reducing the efficiency of processing, still allow some mature 26 S rRNA to be formed. On the other hand, none of the ITS2 deletions affect the production of mature 17 S rRNA. Since all of the deletions severely disturb the recently proposed secondary structure of ITS2, these findings suggest an important role for higher order structure of ITS2 in processing. Analysis of the effect of complete or partial replacement of S. cerevisiae ITS2 with its counterpart sequences from Saccharomyces rosei or Hansenula wingei, points to helix V of the secondary structure model as an important element for correct and efficient processing. Direct mutational analysis shows that disruption of base-pairing in the middle of helix V does not detectably affect 26 S rRNA formation. In contrast, introduction of clustered point mutations at the apical end of helix V that both disrupt base-pairing and change the sequence of the loop, severely reduces processing. Since a mutant containing only point mutations in the sequence of the loop produces normal amounts of mature 26 S rRNA, we conclude that the precise (secondary and/or primary) structure at the lower end of helix V, but excluding the loop, is of crucial importance for efficient removal of ITS2.     

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.     

Analysis of ribosomal DNA internal transcribed spacer sequences of the Leishmania donovani complex

To understand phylogenetic relationships of species and strains within the Leishmania donovani complex, we have analyzed the ribosomal DNA internal transcribed spacer (ITS) sequences of 27 Leishmania infantum, 2 Leishmania chagasi, 18 L. donovani and 5 Leishmania archibaldi strains of different zymodemes and geographical origin. Eight ITS sequence types were found. All detected sequence variation within ITS1 and ITS2 was based on 12 polymorphic microsatellites. The L. infantum strains from the Mediterranean region, China and L. chagasi from the New World formed a phylogenetic group well separated from the second main group including all strains from East Africa and India. Within the latter group three distinct phylogenetic subgroups could be differentiated: (1) L. donovani (Sudan/Ethiopia, China) + L. archibaldi (Sudan), (2) L. donovani (Sudan/Ethiopia) + L. infantum (Sudan) + L. archibaldi (Sudan/Ethiopia), and (3) L. donovani (Kenya, India). These groups are not consistent with previous species definitions based on isoenzyme analyses, e.g. L. infantum is polyphyletic and L. archibaldi is not supported as a distinct species. Two groups of Indian strains could be differentiated, one of which has an identical sequence type to the strains from Kenya. Three main lineages of strains can thus be differentiated in East Africa: two quite distantly related groups of strains from Sudan/Ethiopia, and a third group including all strains from Kenya, which is more closely related to part of the Indian strains than to any of the Sudanese/Ethiopian groups. The ITS sequence analysis presented here supports the need for revision of the taxonomy of the L. donovani complex.     

Evolutionary divergence of ribosomal internal transcribed spacer 2 in lizards

Internal transcribed spacers 1 and 2 (ITS1 and ITS2) are known to play an important role in rRNA maturation, yet the mechanism of their action is still not completely understood. Comparison of the ITS1 and ITS2 nucleotide sequences for various organisms reveals conserved regions, which are potentially involved in rRNA biogenesis, and yields new information about the evolutionary divergence of the corresponding region of the genome. The rDNA fragments containing ITS2 were amplified, cloned, and sequenced for three lizard species: Darevskia armeniaca, Lacerta strigata (Lacertidae), and Agama caucasia (Agamidae). The lizard ITS2 sequences were compared with their counterparts from other organisms and proved to contain not only universally conserved elements characteristic of the consensus secondary structure of vertebrate ITS2, but also lizard-specific regions. Comparison of the ITS2 size and the distribution of homologous regions for the two lizard families made it possible to assume that evolution of the modern species involved duplication of ITS2 in the genome of their common ancestor.     

Neurospora crassa ribosomal DNA: sequence of internal transcribed spacer and comparison with N. intermedia and N. sitophila

Using [32P]DNA probes from a clone containing 17S, 5.8S and 26S rRNA of Neurospora crassa, the remainder of the repeat unit (RU) for ribosomal DNA (rDNA) has been cloned. Combining restriction analysis of the cloned DNA and restriction digests of genomic DNA, the RU was found to be 8.7 kb. The nucleotide sequence was determined for the internal transcribed spacer (ITS) regions one and two, for 5.8S rRNA and for portions of 17S and 26S rRNAs immediately flanking the ITS regions, and compared to the corresponding region of Saccharomyces carlsbergensis. In addition, a comparative restriction analysis of two other Neurospora species was performed using twelve restriction endonucleases. Genomic DNA blots of rDNA from N. intermedia and N. sitophila revealed rDNA RUs of 8.4 kb. The majority of differences in restriction patterns were confined to sequences outside the mature rRNA regions. However, one SmaI recognition site was found in 26S rRNA of N. crassa and N. sitophila but not in N. intermedia.     

Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA

Background

Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns.

Methodology/Principal Findings

In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level.

Conclusions

Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.     

浙江4地桃花水母的rDNA-ITS序列分析

采用PCR和DNA测序技术,对浙江省4个地点桃花水母(Craspedacusta)的核糖体RNA基因内转录间隔区(rDNA-ITS)进行了扩增与测序,并与GenBank中已有的桃花水母ITS区基因序列进行比对分析,计算了它们的遗传距离,利用MEGA 4.1构建了系统发育树。结果显示:这4个地点的桃花水母与索氏桃花水母(Craspedacusta sowerbyi)的ITS区基因相似度极高,同源性都在97%以上,遗传距离保持在0～0.008之间,在进化树中与索氏桃花水母聚为同一支。研究结果表明,这4地的桃花水母都属于索氏桃花水母(C.sowerbyi)。     

Molecular systematics of dictyostelids: 5.8S ribosomal DNA and internal transcribed spacer region analyses

The variability and adaptability of the amoebae from the class Dictyosteliomycetes greatly complicate their systematics. The nucleotide sequences of the ribosomal internal transcribed spacers and the 5.8S ribosomal DNA gene have been determined for 28 isolates, and their utility to discriminate between different species and genera has been shown.     

a molecular phylogeny of apiaceae subfamily Apioideae: evidence from nuclear ribosomal DNA internal transcribed spacer sequences

Phylogenetic relationships among 40 New World and Old World members of Apiaceae subfamily Apioideae, representing seven of the eight tribes and eight of the ten subtribes commonly recognized in the subfamily, were inferred from nucleotide sequence variation in the internal transcribed spacer (ITS) regions of 18-26S nuclear ribosomal DNA. Although the sequences are alignable, with only 11% of sites excluded from the analyses because of alignment ambiguity, divergence values in pairwise comparisons of unambiguous positions among all taxa were high and ranged from 0.5 to 33.2% of nucleotides in ITS 1 and from 0 to 33.2% of nucleotides in ITS 2. Average sequence divergence across both spacer regions was 18.4% of nucleotides. Phylogenies derived from ITS sequences estimated using neighbor-joining analysis of substitution rates, and maximum likelihood and parsimony methods give trees of essentially similar topology and indicate that: (1) there is little support for any existing system of classification of the subfamily that is based largely on morphological and anatomical features of the mericarp; (2) there is a major phylogenetic division within the subfamily, with one clade comprising the genus Smyrnium and those taxa belonging to Drude's tribes Dauceae, Scandiceae, and Laserpitieae and the other clade comprising all other examined taxa; and (3) the genera Arracacia, Coaxana, Coulterophytum, Enantiophylla, Myrrhidendron, Prionosciadium, and Rhodosciadium, all endemic to Mexico and Central America, comprise a clade but their relationships to other New World taxa are equivocal. A phylogeny derived from parsimony analysis of chloroplast DNA rpoC1 intron sequences is consistent with, but considerably less resolved than, relationships derived from these ITS regions. This study affirms that ITS sequences are useful for phylogenetic inference among closely related members of Apioideae but, owing to high rates of nucleotide substitution, are less useful in resolving relationships among the more ancestral nodes of the phylogeny.     

Molecular phylogenetic analysis of ixodid ticks based on the ribosomal DNA spacer, internal transcribed spacer 2, sequences

An internal transcribed spacer (ITS2) sequence between the 5.8S and 28S rRNA genes was used to estimate the phyletic relationships among Ixodes spp. tick vectors of Lyme disease-causing Borrelia spirochetes. Analysis indicates that Borrelia burgdorferi sensu lato species associated with Lyme disease are found mainly in ticks of the Ixodes ricinus species complex. Other closely related tick species are not known to transmit the Borrelia-that cause Lyme disease in humans, but they appear to have a specific association with other closely related Borrelia species. There is a high degree of concordance in the phylogenetics of Borrelia taxa and the phylogenetic relationships among Ixodes ticks.     

Phylogenetics of Pinus (Pinaceae) based on nuclear ribosomal DNA internal transcribed spacer region sequences

A 650-bp portion of the nuclear ribosomal DNA internal transcribed spacer region was sequenced in 47 species of Pinus, representing all recognized subsections of the genus, and 2 species of Picea and Cathaya as outgroups. Parsimony analyses of these length variable sequences were conducted using a manual alignment, 13 different automated alignments, elision of the automated alignments, and exclusion of all alignment ambiguous sites. High and moderately supported clades were consistently resolved across the different analyses, while poorly supported clades were inconsistently recovered. Comparison of the topologies highlights taxa of particularly problematic placement including Pinus nelsonii and P. aristata. Within subgenus Pinus, there is moderate support for the monophyly of a narrowly circumscribed subsect. Pinus (=subsect. Sylvestres) and strong support for a clade of North and Central American hard pines. The Himalayan P. roxburghii may be sister species to these "New World hard pines," which have two well-supported subgroups, subsect. Ponderosae and a clade of the remaining five subsections. The position of subsect. Contortae conflicts with its placement in a chloroplast DNA restriction site study. Within subgenus Strobus there is consistent support for the monophyly of a broadly circumscribed subsect. Strobi (including P. krempfii and a polyphyletic subsect. Cembrae) derived from a paraphyletic grade of the remaining soft pines. Relationships among subsects. Gerardianae, Cembroides, and Balfourianae are poorly resolved. Support for the monophyly of subgenus Pinus and subgenus Strobus is not consistently obtained.     

Phylogeny of Rubus (rosaceae) based on nuclear ribosomal DNA internal transcribed spacer region sequences

We used nuclear ribosomal DNA internal transcribed spacer region (ITS 1 - 5.8S - ITS 2; ITS) sequences to generate the first phylogeny of Rubus based on a large, molecular data set. We sampled 57 taxa including 20 species of subgenus Rubus (blackberries), one to seven species from each of the remaining 11 subgenera, and the monotypic and closely related Dalibarda. In Rubus, ITS sequences are most informative among subgenera, and variability is low between closely related species. Parsimony analysis indicates that Rubus plus Dalibarda form a strongly supported clade, and D. repens may nest within Rubus. Of the subgenera with more than one species sampled, only subgenus Orobatus appears monophyletic. Three large clades are strongly supported: one contains all sampled species of nine of the 12 subgenera; another includes extreme Southern Hemisphere species of subgenera Comaropsis, Dalibarda, and Lampobatus; and a third clade consists of subgenus Rubus plus R. alpinus of subgenus Lampobatus. Rubus ursinus appears to be a hybrid between a close relative of R. macraei (subgenus Idaeobatus, raspberries) and an unidentified subgenus Rubus species. ITS sequences are generally consistent with biogeography and ploidy, but traditionally important morphological characters, such as stem armature and leaf type, appear to have limited phylogenetic value in Rubus.     

A phylogeny of Apiaceae tribe Scandiceae: evidence from nuclear ribosomal DNA internal transcribed spacer sequences

The evolutionary relationships among members of Apiaceae (Umbelliferae) tribe Scandiceae and representatives of all major lineages of Apioideae (including putatively allied Caucalideae) identified in earlier molecular studies were inferred from nucleotide sequence variation in the internal transcribed spacer regions (ITS1 and ITS2) of nuclear ribosomal DNA. In all, 134 accessions representing 18 genera commonly treated in Scandiceae were analyzed. Phylogenies estimated using maximum parsimony and distance methods were generally similar and suggest that: (1) Scandiceae form a well-supported clade, consisting of the genera Anthriscus, Athamanta (in part), Balansaea, Chaerophyllum, Conopodium, Geocaryum, Kozlovia, Krasnovia, Myrrhis, Myrrhoides, Neoconopodium, Osmorhiza, Scandix, Sphallerocarpus, and Tinguarra; (2) Athamanta is polyphyletic, with A. della-cellae allied with Daucus and A. macedonica placed close to Pimpinella; and (3) Rhabdosciadium and Grammosciadium find affinity with the Aegopodium group of umbellifers, whereas the placement of the monotypic Molopospermum cannot be inferred because of its high sequence divergence. The genus Bubon has been restored with two new combinations, B. macedonicum subsp. albanicum and B. macedonicum subsp. arachnoideum. Scandiceae arise within paraphyletic Caucalideae, the latter comprising two major lineages whose relationships to Scandiceae are not clear. Therefore, a broad treatment of Scandiceae is proposed, with subtribes Scandicinae, Daucinae, and Torilidinae (the latter two representing the Daucus and Torilis subgroups, respectively, of recent molecular systematic investigations).     

The second internal transcribed spacer of nuclear ribosomal DNA as a tool for Latin American anopheline taxonomy - a critical review

Among the molecular markers commonly used for mosquito taxonomy, the internal transcribed spacer 2 (ITS2) of the ribosomal DNA is useful for distinguishing among closely-related species. Here we review 178 GenBank accession numbers matching ITS2 sequences of Latin American anophelines. Among those, we found 105 unique sequences corresponding to 35 species. Overall the ITS2 sequences distinguish anopheline species, however, information on intraspecific and geographic variations is scarce. Intraspecific variations ranged from 0.2% to 19% and our analysis indicates that misidentification and/or sequencing errors could be responsible for some of the high values of divergence. Research in Latin American malaria vector taxonomy profited from molecular data provided by single or few field capture mosquitoes. However we propose that caution should be taken and minimum requirements considered in the design of additional studies. Future studies in this field should consider that: (1) voucher specimens, assigned to the DNA sequences, need to be deposited in collections, (2) intraspecific variations should be thoroughly evaluated, (3) ITS2 and other molecular markers, considered as a group, will provide more reliable information, (4) biological data about vector populations are missing and should be prioritized, (5) the molecular markers are most powerful when coupled with traditional taxonomic tools.     

Molecular evolution and phylogenetic implications of internal transcribed spacer sequences of nuclear ribosomal DNA in the Phaseolus-Vigna complex.

Molecular phylogeny based on internal transcribed spacer (ITS) sequences was studied to resolve the taxonomic contradiction in Vigna and its relation to Phaseolus. The ITS region of the 18S-26S nuclear ribosomal DNA repeat was sequenced for 29 Vigna species, selected from five of the nine subgenera, and 9 species of Phaseolus. The length of ITS-1 ranged from 187 to 243 bp and 217 to 290 bp, and that of ITS-2 from 187 to 219 bp and 225 to 243 bp, within Vigna and Phaseolus species, respectively. Phylogenies derived from ITS sequences based on maximum-parsimony and neighbor-joining methods gave trees essentially of similar topology. The ITS phylogeny was generally congruent with recent classifications based largely on morphological, biochemical, cytogenetical, and palynological features, except that subgenus Plectotropis of Neotropical origin was revealed to be closely related to subgenus Vigna instead of forming a link between African (subgenus Vigna) and Asiatic (subgenus Ceratotropis) vignas, and subgenus Sigmoidotropis, featuring morphological characters of both Vigna and Phaseolus, was placed as the sister group to the Phaseolus taxa. The ITS sequences were shown to be useful for identifying wild progenitors of V. mungo, V. radiata, V. umbellata, and V. unguiculata and for clarifying taxonomy-related problems in many previously controversial cases. This study also affirms that V. umbellata and V. angularis are the diploid progenitors of the only tetraploid species (V. glabrescens) known in the genus.     

Differentiation of two ovine Babesia based on the ribosomal DNA internal transcribed spacer (ITS) sequences

The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene for six Babesia spp. isolated from different geographic origins were characterized. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among these isolates. Phylogenetic analysis of the ITS1-5.8S gene-ITS2 region clearly separated the isolates into two clusters. One held an unidentified Babesia sp. transmitted by Hyalomma anatolicum anatolicum. The second held five other isolates, which were considered to be Babesia motasi. Each Babesia species cluster possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. The results showed that ITS1, ITS2 and the complete ITS1-5.8S-ITS2 region could be used to discriminate these ovine Babesia spp. effectively.     

A molecular phylogeny of Arceuthobium (Viscaceae) based on nuclear ribosomal DNA internal transcribed spacer sequences

Ribosomal DNA (rDNA) internal transcribed spacer (ITS) and 5.8S rDNA sequences were obtained from 22 species of dwarf mistletoes (Arceuthobium — Viscaceae) to test phylogenetic relationships. Interspecific distances ranged from 0 to 21.4% between New World species, values two to five times higher than those measures for the ITS region in other plants. One Old World species (A. oxycedri) and one New World species (A. abietis-religiosae) were remarkably similar to each other but exhibited up to 41% sequence divergence from the remaining species. Minimum length trees support the concept of a verticillately branched subgenus Arceuthobium; however, interspecific distances indicate this group is extremely heterogeneous. Subgenus Vaginata, Section Vaginata, is centered in Mexico and encompasses all the taxa previously placed in this group but is expanded to include several species previously classified in Section Campylopoda (e.g., A. divaricatum, A. rubrum, and A. strictum). The sister group relationship between A. divaricatum and A. douglasii, first seen following isozyme analysis, is supported by ITS sequence data. Section Campylopoda s. s. is now composed of 13 mainly U.S. species that show a high degree of morphological and genetic similarity. The eastern dwarf mistletoe, A. pusillum, is not closely related to A. douglasii but rather with A. bicarinatum from Hispaniola, which suggests that these taxa represent highly modified relicts that shared an ancestor in the early Tertiary. Two endemic species from Mexico and Central America (A. guatemalense and A. pendens) formed a sister group and have been placed in a new Section (Penda). Rapid molecular evolution in Arceuthobium may be associated with the adaptive radiation of this genus on numerous conifer hosts.     

Vertebrate evolution reflected in the evolution of nuclear ribosomal internal transcribed spacer 2

Plastics are widely used in modern life, and their unbound chemicals bisphenol A and phthalates can leach out into the surrounding environment. BPA and PAEs have recently attracted the special attention of the scientific community, regulatory agencies and the general public because of their high production volume, widespread use of plastics, and endocrine-disrupting effects. In The Comparative Toxicogenomics Database, BPA and five most frequently curated PAEs (DEHP/MEHP and DBP/BBP/MBP) were found to have 1932 and 484 interactions with genes/proteins, respectively. Five of their top ten toxicity networks were found to be involved in inflammation, and their top ten diseases included genital, prostatic, endomentrial, ovarian and breast diseases. BPA and PAEs were found to exhibit similar toxicogenomics and adverse effects on human health owning to their 89 common interacting genes/proteins. These 89 genes/proteins may serve as biomarkers to assay the toxicities of different chemicals leached out from the widely used plastics.     

Vertebrate evolution reflected in the evolution of nuclear ribosomal internal transcribed spacer 2

In eukaryotes, mature rRNA sequences are produced from single large (45S) precursor (pre-rRNA) as the result of successive removal of spacers through a series of rapid and intricate actions of endo- and exonucleases. The excision of internal transcribed spacer (ITS2), a eukaryotic-specific insertion, remains the most elusive processing step. ITS2 is the element mandatory for all eukaryotic pre-rRNAs that contain at least three processing cleavage sites for precise 5.8S and 28S formation. Conserved core sequences (cis-elements) binding to trans-factors provide for precise rRNA processing, whereas rapidly diverging regions between the core sequences preserve internal complementarity, which guarantees the spatial integrity of ITS2. Characteristic differences in the formation of such insertions during evolution should reflect the relationships between taxa. The phylogeny of the reptiles and the relationships between taxa proposed by scientists are controversial. To delineate the structural and functional features preserved among reptilian ITS2s, we cloned and sequenced 58 ITS2s belonging to four reptile orders: Squamata, Crocodilians, Aves, and Testudines. We studied the subsequent alignment and folding of variable regions. The sizes and packing of the loop–stems between conserved consensus segments in reptiles vary considerably between taxa. Our phylogenetic trees constructed on the basis of the reptile ITS2s primary structural alignments revealed a split between Iguania clade and all other taxa. True lizards (suborder Scleroglossa) and snakes (suborder Serpentes) show sister relationships, as well as the two other reptilian orders, Crocodilia + Aves and Testudines. In summary, our phylogenetic trees exhibit a mix of specific features deduced or, to the contrary, rejected earlier by other authors.