Length and Sequence Heterogeneity in the Mitochondrial Internal Transcribed Spacer of Acanthamoeba spp.

ABSTRACT. The internal transcribed spacer (ITS) between the mitochondrial large (23S rRNA; rnl ) and small (16S rRNA; rns ) subunit ribosomal RNA genes of Acanthamoeba castellanii strain Neff was sequenced previously and was uniquely interesting because it contained tRNA genes with acceptor stem mismatches that underwent RNA editing repair. Our interest in this ITS region was to determine its phylogenetic potential in differentiating between closely related isolates. We analyzed the mitochondrial ITS region for 17 Acanthamoeba isolates and observed extensive sequence and length variability, making this region difficult to align. Acanthamoeba griffini strain S-7 had the shortest ITS (i.e. 559 base pairs [bp]) compared with Acanthamoeba palestinensis strain Reich, which had the longest (i.e. 1,360 bp). The length disparity occurred predominantly between the spacer region of the aspartic acid ( trnD ) and methionine ( trnM ) tRNA genes. Unexpectedly, this region in A. palestinensis Reich was found to contain a duplication of the trnM gene. Additionally, like A. castellanii strain Neff, all isolates examined had tRNAs with mismatches in their acceptor stem. Also, the potential for an additional type of editing not described previously for Acanthamoeba , involving purine to pyrimidine transversions was observed.     

Intragenomic Variation and Evolution of the Internal Transcribed Spacer of the rRNA Operon in Bacteria

Variation in the internal transcribed spacer (ITS) of the rRNA (rrn) operon is increasingly used to infer population-level diversity in bacterial communities. However, intragenomic ITS variation may skew diversity estimates that do not correct for multiple rrn operons within a genome. This study characterizes variation in ITS length, tRNA composition, and intragenomic nucleotide divergence across 155 Bacteria genomes. On average, these genomes encode 4.8 rrn operons (range: 2–15) and contain 2.4 unique ITS length variants (range: 1–12) and 2.8 unique sequence variants (range: 1–12). ITS variation stems primarily from differences in tRNA gene composition, with ITS regions containing tRNA-Ala + tRNA-Ile (48% of sequences), tRNA-Ala or tRNA-Ile (10%), tRNA-Glu (11%), other tRNAs (3%), or no tRNA genes (27%). Intragenomic divergence among paralogous ITS sequences grouped by tRNA composition ranges from 0% to 12.11% (mean: 0.94%). Low divergence values indicate extensive homogenization among ITS copies. In 78% of alignments, divergence is <1%, with 54% showing zero variation and 81% containing at least two identical sequences. ITS homogenization occurs over relatively long sequence tracts, frequently spanning the entire ITS, and is largely independent of the distance (basepairs) between operons. This study underscores the potential contribution of interoperon ITS variation to bacterial microdiversity studies, as well as unequivocally demonstrates the pervasiveness of concerted evolution in the rrn gene family. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Margaret Riley     

Phylogenetic Relationships of Yessotoxin-Producing Dinoflagellates, Based on the Large Subunit and Internal Transcribed Spacer Ribosomal DNA Domains

Yessotoxin (YTX) is a globally distributed marine toxin produced by some isolates of the dinoflagellate species Protoceratium reticulatum, Lingulodinium polyedrum, and Gonyaulax spinifera within the order Gonyaulacales. The process of isolating cells and testing each isolate individually for YTX production during toxic blooms are labor intensive, and this impedes our ability to respond quickly to toxic blooms. In this study, we used molecular sequences from the large subunit and internal transcribed spacer genomic regions in the ribosomal operon of known YTX-producing dinoflagellates to determine if genetic differences exist among geographically distinct populations or between toxic and nontoxic isolates within species. In all analyses, all three YTX-producing species fell within the Gonyaulacales order in agreement with morphological taxonomy. Phylogenetic analyses of available rRNA gene sequences indicate that the capacity for YTX production appears to be confined to the order Gonyaulacales. These findings indicate that Gonyaulacoloid dinoflagellate species are the most likely to produce YTX and thus should be prioritized for YTX screening during events. Dinoflagellate species that fall outside of the Gonyaulacales order are unlikely to produce YTX. Although the rRNA operon offers multiple sequence domains to resolve species level diversification within this dinoflagellate order, these domains are not sufficiently variable to provide robust markers for YTX toxicity.     

Genetic Analyses of the Internal Transcribed Spacer Sequences Suggest Introgression and Duplication in the Medicinal Mushroom Agaricus subrufescens

The internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster is widely used in fungal taxonomy and phylogeographic studies. The medicinal and edible mushroom Agaricus subrufescens has a worldwide distribution with a high level of polymorphism in the ITS region. A previous analysis suggested notable ITS sequence heterogeneity within the wild French isolate CA487. The objective of this study was to investigate the pattern and potential mechanism of ITS sequence heterogeneity within this strain. Using PCR, cloning, and sequencing, we identified three types of ITS sequences, A, B, and C with a balanced distribution, which differed from each other at 13 polymorphic positions. The phylogenetic comparisons with samples from different continents revealed that the type C sequence was similar to those found in Oceanian and Asian specimens of A. subrufescens while types A and B sequences were close to those found in the Americas or in Europe. We further investigated the inheritance of these three ITS sequence types by analyzing their distribution among single-spore isolates from CA487. In this analysis, three co-dominant markers were used firstly to distinguish the homokaryotic offspring from the heterokaryotic offspring. The homokaryotic offspring were then analyzed for their ITS types. Our genetic analyses revealed that types A and B were two alleles segregating at one locus ITSI, while type C was not allelic with types A and B but was located at another unlinked locus ITSII. Furthermore, type C was present in only one of the two constitutive haploid nuclei (n) of the heterokaryotic (n+n) parent CA487. These data suggest that there was a relatively recent introduction of the type C sequence and a duplication of the ITS locus in this strain. Whether other genes were also transferred and duplicated and their impacts on genome structure and stability remain to be investigated.     

核糖体DNA的内转录间隔区序列标记在真菌分类鉴定中的应用

传统的真菌分类主要根据真菌菌株的形态特征、生长特性与生理生化指标进行,而分子生物学技术的发展提升了真菌分类鉴定研究的手段。真菌核糖体DNA内转录间隔区(ITS)在进化上比编码区快,种内的不同菌株之间高度保守,但在种间变化极大,故可为真菌学的研究提供丰富的遗传信息。简要综述了ITS序列分析技术在真菌分类鉴定中的应用现状、相关问题及前景。     

Diverse and Unique Picocyanobacteria in Chesapeake Bay, Revealed by 16S-23S rRNA Internal Transcribed Spacer Sequences

rRNA internal transcribed spacer phylogeny showed that Chesapeake Bay is populated with diverse Synechococcus strains, including members of the poorly studied marine cluster B. Marine cluster B prevailed in the upper bay, while marine cluster A was common in the lower bay. Interestingly, marine cluster B Synechococcus included phycocyanin- and phycoerythrin-rich strains.     

Sequence Variation in the Ribosomal DNA Internal Transcribed Spacer of Tridacna crocea

DNA-based genetic markers are needed to augment existing allozyme markers in the assessment of genetic diversity of wild giant clam populations. The dearth of polymorphic mitochondrial DNA regions amplified from known universal polymerase chain reaction (PCR) primers has led us to search other regions of the genome for viable sources of DNA polymorphism. We have designed tridacnid-specific PCR primers for the amplification of internal transcribed spacer regions. Sequences of the first internal transcribed spacer segment (ITS-1) revealed very high polymorphism, showing 29% variation arising from base substitutions alone. Preliminary restriction analysis of the ITS regions using 8 restriction enzymes revealed cryptic changes in the DNA sequence. These mutations are promising as marker tools for differentiating geographically separated populations. Such variation in the ITS region can possibly be used for population genetic analysis. Received February 1, 2000; accepted May 8, 2000.     

A Conserved Motif in the 5.8S Ribosomal RNA (rRNA) Gene is a Useful Diagnostic Marker for Plant Internal Transcribed Spacer (ITS) Sequences

The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has become an important nuclear locus for molecular systematic investigations of angiosperms at the intergenic and interspecific levels. Universal PCR primers are positioned on the conserved rRNA genes (18S, 5.8S, 26S) to amplify the entire ITS spacer region. Recent reports of fungal and algal contaminants, first described as plant ITS sequences, stress the need for diagnostic markers specific for the angiosperm ITS region. This report describes a conserved 14 base pair (bp) motif in the 5.8S rRNA gene that can be used to differentiate between flowering plants, bryophytes, and several orders of algae and fungi, including common plant pathogenic and non-pathogenic fungi. A variant of the motif (found in fungi and algae) contains a convenient EcoRI restriction site that has several applications for eliminating problematic contaminants from plant ITS preparations.     

珍珠菜rDNA ITS序列扩增及分析

以贵州境内珍珠菜属植物为材料,对其rDNA转录间隔区(ITS)序列进行PCR扩增和序列测定。实验共得到7个种的ITS序列,它们分别是:过路黄(Lysi machia christinae,GenBank登录号FJ362382),矮桃(L.cle-throides,GenBank登录号FJ362383),叶头过路黄(L.phyllocephala,GenBank登录号FJ362386),临时救(L.con-gestiflora,GenBank登录号FJ362387),显苞过路黄(L.rubiginosa,GenBank登录号FJ362388),茂汶过路黄(L.stellarioides,GenBank登录号FJ362384),腺药珍珠菜(L.stenosepala,GenBank登录号FJ362385),其中后2个种是国际上首次得到的。采用Blast方法将测序结果进行同源搜索,采用邻接法构建与其相关植物的ITS序列系统发育树。结果表明,珍珠菜属7种植物ITS序列总长度为613~620 bp;ITS1区序列长度为234~239 bp,5.8SrDNA区序列长度163 bp,ITS2区序列长度216~219 bp,7种植物的ITS序列差异主要集中在ITS1与ITS2区。聚类分析将茂汶过路黄聚为一支,其它6种植物聚为一支,表明茂汶过路黄与其它6种植物的碱基差异较大,从分子水平上支持据形态特征把花辐射生长的茂汶过路黄另立一类。     

蓼属头状蓼组rDNA-ITS的序列扩增及分析

以贵州境内蓼属头状蓼组6种(含1变种)植物为材料,对其rDNA的内转录间隔区(ITS)序列进行PCR扩增,得到6种植物的ITS序列,分别为:赤胫散2个居群(Polygonum runcinatum var.sinense,GenBank登录号FJ606887、FJ648802),平卧蓼(P.strindbergii,GenBank登录号FJ648803 ),尼泊尔蓼(P.nepalense,GenBank登录号FJ648804),羽叶蓼(P.runcinatum,GenBank登录号FJ648805),火炭母(P.chinense,GenBank登录号FJ648806)和头花蓼(P.capitatum,GenBank登录号FJ648807).其中赤胫散与平卧蓼的ITS序列为首次报道.序列分析结果表明,蓼属头状蓼组6种植物ITS序列总长度为661～666 bp,ITS1区序列长度为243～246 bp,5.8 S rDNA区序列长度165 bp,ITS2区序列长度253～258 bp,6种植物的差异主要集中在ITS1和ITS2区.聚类分析显示,6种头状蓼组植物具有共同起源,结果支持赤胫散从羽叶蓼变种上升为独立物种.     

樟属植物ITS序列多态性分析

对樟科樟属(Cinnamomum Schaeffer) 17个代表样本的核糖体DNA内转录间隔区(nrDNA ITS)进行克隆测序。对获得的87条不同ITS序列的长度变异、GC含量、5.8S区二级结构的稳定性、遗传距离、进化模式以及系统发育关系进行了相关分析。研究结果显示, ITS序列在樟属植物内存在明显的多态性, 87条序列中的22条序列被鉴定为假基因序列, 其余65条序列为功能基因序列; 假基因序列采用中性进化模式, 变异明显大于功能序列。ITS序列在樟属植物中出现一致性进化不完全和假基因现象也可能发生在樟科其它类群中, 这可能是导致樟科植物ITS序列直接测序方式成功率低的重要原因。     

From Genus to Phylum: Large-Subunit and Internal Transcribed Spacer rRNA Operon Regions Show Similar Classification Accuracies Influenced by Database Composition

We compared the classification accuracy of two sections of the fungal internal transcribed spacer (ITS) region, individually and combined, and the 5′ section (about 600 bp) of the large-subunit rRNA (LSU), using a naive Bayesian classifier and BLASTN. A hand-curated ITS-LSU training set of 1,091 sequences and a larger training set of 8,967 ITS region sequences were used. Of the factors evaluated, database composition and quality had the largest effect on classification accuracy, followed by fragment size and use of a bootstrap cutoff to improve classification confidence. The naive Bayesian classifier and BLASTN gave similar results at higher taxonomic levels, but the classifier was faster and more accurate at the genus level when a bootstrap cutoff was used. All of the ITS and LSU sections performed well (>97.7% accuracy) at higher taxonomic ranks from kingdom to family, and differences between them were small at the genus level (within 0.66 to 1.23%). When full-length sequence sections were used, the LSU outperformed the ITS1 and ITS2 fragments at the genus level, but the ITS1 and ITS2 showed higher accuracy when smaller fragment sizes of the same length and a 50% bootstrap cutoff were used. In a comparison using the larger ITS training set, ITS1 and ITS2 had very similar accuracy classification for fragments between 100 and 200 bp. Collectively, the results show that any of the ITS or LSU sections we tested provided comparable classification accuracy to the genus level and underscore the need for larger and more diverse classification training sets.     

除虫菊和茼蒿核糖体ＤＮＡ ＩＴＳ区的序列

除虫菊(Pyrethrum cinerariifolium),又称白花除虫菊,是众所周知的著名杀虫植物,也是目前世界上唯一集约化栽培的天然杀虫剂原料,至今仍为肯尼亚、厄瓜多尔等一些国家的支柱产业。除虫菊的头状花序中含有6种0.4 %～2 %的杀虫成分,即除虫菊酯Ⅰ,Ⅱ(pyrethrin-I,II),瓜菊酯Ⅰ,Ⅱ(cinerin-I,II)和茉莉菊酯Ⅰ,Ⅱ(Jasmolin-I,II),6个成分组成的复合植物杀虫剂,对家蝇的杀虫活性LD50为15～20μg/g,对大白鼠的毒性LD50为2124～2416 mg/kg,对温血动物毒性更低。面对呼唤绿色产业的21世纪,除虫菊的产业开发又成为天然农药开发的一个热点。我们…     

Universal Primers for Amplification of Mitochondrial Small Subunit Ribosomal RNA-Encoding Gene in Scleractinian Corals

We describe the construction of polymerase chain reaction primers designed to amplify a portion of the mitochondrial (mt) small subunit ribosomal (SSU) RNA-encoding genes in scleractinian corals. Combinations of cloning and sequencing show that the amplified fragments are between 694 and 896 bp in length. Alignment of the amplified DNA sequences to the published mt SSU rRNA genes of Metridium senile and Sarcophyton glaucum indicates several conserved regions among actiniarian, corallimorpharian, octocorallian, and scleractinians, suggesting this primer set can successfully amplify over 80% of the mt SSU rDNA region of scleractinian corals. Surveys of sequence variation and estimation of the rate of evolution show an extremely slow divergence of the SSU rRNA gene in the family Acroporidae. Received June 11, 1999; accepted October 4, 1999.     

A Molecular Phylogeny of Lilium in the Internal Transcribed Spacer Region of Nuclear Ribosomal DNA

Phylogenetic relationships among 55 species of Lilium, Cardiocrinum giganteum, and Nomocharis saluenensis were inferred from nucleotide sequence variations in the internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA. The phylogeny derived from ITS sequences estimated using maximum-likelihood methods indicated that (1) most of the species construct their own clade according to the classification based on morphological features at the section level; (2) section Daurolirion is not independent of Sinomartagon, and it is appropriate to integrate two sections as Sinomartagon; (3) it is appropriate that L. henryi and L. bulbiferum are classified into subsection 6a and Sinomartagon–Daurolirion, respectively; (4) subsection 6b is much closer to Sinomartagon than subsection 6a and Archelirion, and it arose directly from Sinomartagon; and (5) Lilium is much closer to Nomocharis than Cardiocrinum. Phylogenetic estimation using sequences of the ITS region is suitable at the levels of genus, section, and most of subsection. Received: 18 December 1998 / Accepted: 14 March 1999     

Accession-Specific Haplotypes of the Internal Transcribed Spacer Region in Arabidopsis thaliana--A Means for Barcoding Populations

Eukaryote genomes contain multiple copies of nuclear ribosomal DNA (nrDNA) harboring both highly conserved and variable regions. This has made nrDNA the most popular genetic marker for phylogenetic studies and the region of choice for barcoding projects. Furthermore, many scientists believe that all copies of nrDNA within one nucleus are practically identical due to concerted evolution. Here, we investigate the model plant species Arabidopsis thaliana for intragenomic variation of the internal transcribed spacer (ITS) region of nrDNA. Based on a modified deep sequencing approach, we provide a comprehensive list of ITS polymorphisms present in the two most widely used accessions of A. thaliana-Col-0 and Ler. Interestingly, we found that some polymorphisms are shared between these genetically very distinct accessions. On the other hand, the high number of accession-specific polymorphisms shows that each accession can be clearly and easily characterized by its specific ITS polymorphism patterns and haplotypes. Network analysis based on the detected haplotypes demonstrates that the study of ITS polymorphism patterns and haplotypes is an extremely powerful tool for population genetics. Using the methods proposed here, it will now be possible to extend the traditionally species-bound barcoding concept to populations.