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.     

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.     

Sequence Analysis of the Internal Transcribed Spacer 2 (ITS2) from Yeast Species Within the Genus Candida

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) regions were determined for 13 species within the genus Candida, representing a collection of those species pathogenic for humans. No two species had identical sequences and the sizes of ITS2 varied fourfold, representing an apparent continuous gradient of nucleotides. When present, sequence homologies were observed in the 5′ end of ITS2, and many species exhibited more limited homologies within three known conserved domains found in other yeasts. Cluster analysis of primary sequence revealed a concordance with a known taxonomic subfamily and suggests that certain species within the genus form a similar grouping. A majority of species exhibited similar presumptive RNA secondary structures, consistent with the hypothesis that these spacer regions are essential for correct processing of the 5.8S and 28S subunits. Received: 14 April 1997 / Accepted: 22 July 1997     

Internal Transcribed Spacer Sequence Analysis of Puccinia helianthi Schw. and Its Application in Detection of Sunflower Rust

Two basidiomycete‐specific primers ITS1‐F and ITS4‐B were used in identification of the genus Puccinia. The primers showed good specificity for the genus with an 816‐bp product that was amplified exclusively. Twenty sequences of internal transcribed spacer (ITS) regions of Puccinia helianthi isolates from China remain unchanged. The whole ITS length (including ITS1 sequence 194 bp, 5.8S rRNA gene 156 bp, ITS2 sequence 206 bp) was 556 bp. By comparing the aligned ITS sequences of several Puccinia isolates from China, Spain and the United States, ITS homogeneity among these sunflower rust isolates was >99%. Genetic homology and phylogeny of P. helianthi with other Puccinia spp. was investigated. Nineteen sequences of rDNA ITS1 and ITS2 were determined and used as phylogenetic markers. Phylogenetic analysis of ITS regions showed that Puccinia spp. of sunflower was clustered in one clade with P. komarovii and P. violae, divergent from Puccinia spp. of Chrysanthemum, P. tenaceti of tansy (Tanacetum vulgare) and Puccina spp. of big sagebrush (Artemisia tridentate) indicating sunflower rust had distant phylogenetic relationships with other Compositae rusts. With the specified primers SR‐1 and SR‐2, either from purified urediniospores or symptomless (but infected) sunflower leaves could be examined specifically. Therefore, results of this study help in detection and polygenetic study of rust fungi occurring on sunflower.     

Genetic Variations in the Internal Transcribed Spacer and Mitochondrial Small Subunit rRNA Gene of Naegleria spp.

ABSTRACT: Naegleria spp. are widely distributed free-living amebas, but one species in the genus, N. fowleri , causes acute fulminant primary amebic meningoencephalitis in humans and other animals. Thus, it is important to differentiate N. fowleri from the rest in the genus of Naegleria , and to develop tools for the detection of intra-specific genetic variations. In this study, one isolate each of N. australiensis, N. gruberi, N. jadini , and N. lovaniensis and 22 isolates of N. fowleri were characterized at the internal transcribed spacers (ITS) and mitochondrial small subunit rRNA (mtSSU rRNA) gene. The mtSSU rRNA primers designed amplified DNA of all isolates, with distinct sequences obtained from all species examined. In contrast, the ITS primers only amplified DNA from N. lovaniensis and N. fowleri , with minor sequence differences between the two. Three genotypes of N. fowleri were found among the isolates analyzed in both the mtSSU rRNA gene and ITS. The extent of sequence variation was greater in the mtSSU rRNA gene, but the ITS had the advantage of length polymorphism. These data should be useful in the development of molecular tools for rapid species differentiation and genotyping of Naegleria spp.     

ERIC and REP-PCR Banding Patterns and Sequence Analysis of the Internal Transcribed Spacer of rDNA of Stemphylium solani Isolates from Cotton

The genetic diversity of the Stemphylium solani isolates from cotton was assessed by Enterobacterial Repetitive Intergenic Consensus (ERIC) and Repetitive Extragenic Palindromes (REP)-PCR fingerprinting. Twenty eight monosporic isolates of S. solani from cotton were used along with five isolates from tomato and one isolate of Alternaria macrospora from cotton for comparison. The dendrogram obtained revealed clear differences between the cotton and tomato isolates as well as between the tomato isolates from different geographic regions. The genetic relationships among S. solani isolates were also analyzed by sequencing the internal transcribed spacer (ITS) region of four isolates representing the three ERIC and REP groups. The tomato isolate from the State of S?o Paulo showed a distinct ITS sequence from that of the cotton isolates and tomato isolate from the State of Goiás, giving evidence that it belongs to a different genotype of S. solani. This is the first report of the entire sequence of the ITS1-5.8S-ITS2 regions of S. solani.     

Phylogeny of the GenusTrichodermaBased on Sequence Analysis of the Internal Transcribed Spacer Region 1 of the rDNA Cluster

Sequences of the internal transcribed spacer region 1 (ITS1) of the ribosomal DNA were used to determine the phylogenetic relationships of species ofTrichodermasect.Pachybasium.To this end, 85 strains—including all the availableex-type strains—were analyzed. Parsimony analysis demonstrated that the section is nonmonophyletic, distributing the 85 strains among three main groups that were supported by bootstrap values. Group A comprises two clades (A1 and A2), with A1 includingT. polysporum, T. piluliferum,andT. minutisporum,while A2 includedT. hamatum, T. pubescens,andT. strigosumin addition to species previously included in sect.Trichoderma(i.e.,T. viride, T. atroviride,andT. koningii). Theex-type strain ofT. fasciculatumformed a separate branch basal to clade A. Clade B contained the sect.PachybasiummembersT. harzianum, T. fertile, T. croceum, T. longipile, T. strictipile, T. tomentosum, T. oblongisporum, T. flavofuscum, T. spirale,and the anamorphs ofHypocrea semiorbisandH. cf. gelatinosa.Sequence differences among clades A1, A2, and B were in the same order of magnitude as between each of them andT. longibrachiatum,which was used as an outgroup in these analyses. Sequence differences within clades A1, A2, and B were considerably smaller: in some cases (i.e.,T. virensandT. flavofuscum; T. strictipileandH. cf. gelatinosa), the ITS1-sequences were identical, suggesting conspecifity. In other cases (e.g.,T. crassumandT. longipile; T. harzianum, T. inhamatum, T. croceum, T. fertile,andH. semiorbis; T. hamatumandT. pubescens;andT. viride, T. atroviride,andT. koningii) differences were in the range of 1–3 nt only, suggesting a very close phylogenetic relationship. The sequence of a previously described aggressive mushroom competitor group ofT. harzianumstrains (Th2) was strikingly different from that of theex-type strain ofT. harzianumand closely related species and is likely to be a separate species.     

四种贺兰山紫蘑菇rDNA ITS序列分析

对4种紫蘑菇的rDNA ITS区段进行克隆测序和序列特征比较分析,并与GenBank检索获得的相似性最高菌株的ITS序列一起计算遗传距离并构建系统发育树,旨在探索紫蘑菇的分子鉴定方法及亲缘关系.结果显示,CM-1、CM-2、CM-3及CM-4四种紫蘑菇均属于丝膜菌(Cortinarius),分别与C.rufoolivaceus、C.coerulescens、C.humolens和C.calochrous序列进行比对后,均显示了较高的遗传相似性.贺兰山紫蘑菇隶属于丝膜菌属(Cortinarius).     

Evolution of Spore Release Mechanisms in the Saprolegniaceae (Oomycetes): Evidence from a Phylogenetic Analysis of Internal Transcribed Spacer Sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon.Saprolegniaemerges as the most basal genus, sister toAchlya, Thraustotheca,andDictyuchus. AchlyaandThraustothecaare most closely related, whileDictyuchusappears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited bySaprolegniarepresents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall.     

两个黄芪根瘤菌新类群代表菌株的16S rDNA序列分析

对黄芪根瘤菌两个新类群中心菌株CA8561和JL84进行了16S rDNA全序列测定,并进行了系统发育学分析。结果表明,CA8561位于Rhizobium分支中,JL84位于Sinorhizobium分支中。     

Phylogenetic Analysis and Polyphasic Characterization of Clavibacter michiganensis Strains Isolated from Tomato Seeds Reveal that Nonpathogenic Strains Are Distinct from C. michiganensis subsp. michiganensis

The genus Clavibacter comprises one species and five subspecies of plant-pathogenic bacteria, four of which are classified as quarantine organisms due to the high economic threat they pose. Clavibacter michiganensis subsp. michiganensis is one of the most important pathogens of tomato, but the recommended diagnostic tools are not satisfactory due to false-negative and/or -positive results. To provide a robust analysis of the genetic relatedness among a worldwide collection of C. michiganensis subsp. michiganensis strains, relatives (strains from the four other C. michiganensis subspecies), and nonpathogenic Clavibacter-like strains isolated from tomato, we performed multilocus sequence-based analysis and typing (MLSA and MLST) based on six housekeeping genes (atpD, dnaK, gyrB, ppK, recA, and rpoB). We compared this “framework” with phenotypic and genotypic characteristics such as pathogenicity on tomato, reaction to two antisera by immunofluorescence and to five PCR identification tests, and the presence of four genes encoding the main C. michiganensis subsp. michiganensis pathogenicity determinants. We showed that C. michiganensis subsp. michiganensis is monophyletic and is distinct from its closest taxonomic neighbors. The nonpathogenic Clavibacter-like strains were identified as C. michiganensis using 16S rRNA gene sequencing. These strains, while cross-reacting with C. michiganensis subsp. michiganensis identification tools, are phylogenetically distinct from the pathogenic strains but belong to the C. michiganensis clade. C. michiganensis subsp. michiganensis clonal complexes linked strains from highly diverse geographical origins and also strains isolated over long periods of time in the same location. This illustrates the importance of seed transmission in the worldwide dispersion of this pathogen and its survival and adaptation abilities in a new environment once introduced.     

Paraphyly of Veronica (Veroniceae; Scrophulariaceae): Evidence from the Internal Transcribed Spacer (ITS) Sequences of Nuclear Ribosomal DNA

Veronica (Veroniceae; Scrophulariaceae) and segregated genera, such as Hebe from New Zealand has been debated intensively in the past. We conducted an analysis of sequence data from the internal transcribed spacer region of nuclear ribosomal DNA (ITS) to evaluate the validity of segregate genera and the monophyly of Veronica. According to the results presented here, Veronica is paraphyletic, with the Hebe complex, Synthyris, and Paederota nested within the larger Veronica clade. Pseudolysimachion is in a basal polytomy of the expanded Veronica clade in the strict consensus tree and might be nested within Veronica as well. Clades within Veronica do not correspond to sections traditionally recognized. This study provides a first estimation of the phylogeny of Veroniceae using molecular data and can serve as a starting point for future investigations of Veronica and relatives. Received 24 July 2000/ Accepted in revised form 19 October 2000     

Evidence suggests that modified setae of the crab spiders Stephanopis spp. fasten debris from the background

Crab spiders (Thomisidae) are known by their ability to change their body colouration via change in epithelial pigments. However, the crab spider genus Stephanopis appears to match the colouration of the bark they are sitting on by having debris attached to its dorsal cuticle. The functional morphology, colouration, and evolution of this phenomenon were investigated in Stephanopis cf. scabra and S. cambridgei. Analysis under the microscope revealed that debris originated from the bark they were sitting on. Using scanning electron microscopy, three different types of setae likely related in the retention of debris were found in S. cf. scabra and one in S. cambridgei. These setae are branched and possess barbs, unlike the more filiform setae found in other crab spider species. In addition, the presence of debris improved the brightness background matching of spiders against the bark, but not hue and chroma matching. Ancestral character state reconstruction suggested that presence of debris evolved two to three times within Thomisidae. The evolution of both masking and colour change among crab spiders indicates that they are under a strong selection to avoid detection.     

Comparative Sequence Analysis of the tuf and recA Genes and Restriction Fragment Length Polymorphism of the Internal Transcribed Spacer Region Sequences Supply Additional Tools for Discriminating Bifidobacterium lactis from Bifidobacterium animalis

The relationship between Bifidobacterium lactis and Bifidobacterium animalis was examined by comparative analysis of tuf and recA gene sequences and by restriction fragment length polymorphism analysis of their internal 16S-23S transcribed spacer region sequences. The bifidobacterial strains investigated could be divided into two distinct groups within a single species based on the tuf, recA, and 16S-23S spacer region sequence analysis. Therefore, all strains of B. lactis and B. animalis could be unified as the species B. animalis and divided into two subspecies, Bifidobacterium animalis subsp. lactis and Bifidobacterium animalis subsp. animalis.     

基于核糖体DNA内转录间隔区ITS序列的广义蓼属及近缘属分子系统学研究

通过对广义蓼属及近缘属共32个代表种内转录间隔区ITS序列的分子系统学分析,尝试研究备受争议的广义蓼属及近缘属的物种族、属、组级的划分问题,结果显示,广义蓼属在系统发育树上并不能形成一个单系类群,这些物种共聚为3大支,分别对应春蓼族、蓼族及荞麦族,其中荞麦属与翅果蓼属形成了一支独立于春蓼族及蓼族之外的类群。在春蓼族中,冰岛蓼属与分叉蓼组形成一个单系类群。     

Detection of the IAA Biosynthetic Pathway from Tryptophan via Indole-3-Acetamide in Bradyrhizobium spp.

The IAA biosynthetic pathway of tryptophan to IAA via IAM wasdetected in Bradyrhizobium spp. (slow-growing Rhizobium) butnot in Rhizobium spp. (fast-growing Rhizobium). A simple methodusing rapid HPLC analysis to measure the conversion from NAMto NAA was developed to detect indole-3-acetamide hydrolaseactivity in cultures of bacteria. Most of the Bradyrhizobiumstrains produce large amounts of NAA converted from NAM underour assay conditions. In addition, GC/MS analysis of purifiedextracts from cultures of B.japonicum wild-type strain J1063,grown in a tryptophan-supplemented liquid medium, demonstratedthe presence of IAM and IAA. The results strongly suggest thatbiosynthesis of IAA in Bradyrhizobium spp. involves the samepathway as that operating in Pseudomonas savastanoi and Agrobacteriumtumefaciens. (Received December 25, 1988; Accepted May 18, 1988)