srRNA evolution and phylogenetic relationships of the genus Naegleria (Protista: Rhizopoda)

A rapid RNA sequencing technique was used to partially sequence the small-subunit ribosomal RNA (srRNA) of four species of the amoeboid genus Naegleria. The extent of nucleotide sequence divergence between the two most divergent species was roughly similar to that found between mammals and frogs. However, the pattern of variation among the Naegleria species was quite different from that found for those species of tetrapods characterized to date. A phylogenetic analysis of the consensus Naegleria sequence showed that Naegleria was not monophyletic with either Acanthamoeba castellanii or Dictyostelium discoideum, two other amoebas for which sequences were available. It was shown that the semiconserved regions of the srRNA molecule evolve in a clocklike fashion and that the clock is time dependent rather than generation dependent.     

Amino acid sequence of Acanthamoeba actin

By amino acid sequence studies, only one form of cytoplasmic actin was detected in Acanthamoeba castellanii. Its amino acid sequence is very similar to the sequences of Dictyostelium and Physarum actins, from which Acanthamoeba actin differs in only nine and seven residues, respectively, including the deletion of the first residue. Acanthamoeba actin is unique in containing a blocked NH2-terminal neutral amino acid (glycine), while all other actins sequenced thus far have a blocked acidic amino acid (aspartic or glutamic) at the NH2 terminus. Acanthamoeba actin is also unique in that it contains an N epsilon-trimethyllysine residue at position 326. Like other actins, Acanthamoeba actin contains an NT-methylhistidine residue at position 73. The protein sequence is in complete agreement with the sequence derived from the nucleotide sequence of an expressed actin gene.     

Subgenus Systematics of Acanthamoeba: Four Nuclear 18S rDNA Sequence Types

ABSTRACT Classification of Acanthamoeba at the subgenus level has been problematic, but increasing reports of Acanthamoeba as an opportunistic human pathogen have generated an interest in finding a more consistent basis for classification. Thus, we are developing a classification scheme based on RNA gene sequences. This first report is based on analysis of complete sequences of nuclear small ribosomal subunit RNA genes ( Rns ) from 18 strains. Sequence variation was localized in 12 highly variable regions. Four distinct sequence types were identified based on parsimony and distance analyses. Three were obtained from single strains: Type T1 from Acanthamoeba castellanii V006, T2 from Acanthamoeba palestinensis Reich, and T3 from Acanthamoeba griffini S-7. T4, the fourth sequence type, included 15 isolates classified as A. castellanii, Acanthamoeba polyphaga, Acanthamoeba rhysodes , or Acanthamoeba sp., and included all 10 Acanthamoeba keratitis isolates. Interstrain sequence differences within T4 were 0%–4.3%, whereas differences among sequence types were 6%–12%. Branching orders obtained by parsimony and distance analyses were inconsistent with the current classification of T4 strains and provided further evidence of a need to reevaluate criteria for classification in this genus. Based on this report and others in preparation, we propose that Rns sequence types provide the consistent quantititive basis for classification that is needed.     

Discovery of group I introns in the nuclear small subunit ribosomal RNA genes of Acanthamoeba.

The discovery of group I introns in small subunit nuclear rDNA (nsrDNA) is becoming more common as the effort to generate phylogenies based upon nsrDNA sequences grows. In this paper we describe the discovery of the first two group I introns in the nsrDNA from the genus Acanthamoeba. The introns are in different locations in the genes, and have no significant primary sequence similarity to each other. They are identified as group I introns by the conserved P, Q, R and S sequences (1), and the ability to fit the sequences to a consensus secondary structure model for the group I introns (1, 2). Both introns are absent from the mature srRNA. A BLAST search (3) of nucleic acid sequences present in GenBank and EMBL revealed that the A. griffini intron was most similar to the nsrDNA group I intron of the green alga Dunaliella parva. A similar search found that the A. lenticulata intron was not similar to any of the other reported group I introns.     

Phylogenetic analysis of five medically important Candida species as deduced on the basis of small ribosomal subunit RNA sequences.

The classification of species belonging to the genus Candida Berkhout is problematic. Therefore, we have determined the small ribosomal subunit RNA (srRNA) sequences of the type strains of three human pathogenic Candida species; Candida krusei, C. lusitaniae and C. tropicalis. The srRNA sequences were aligned with published eukaryotic srRNA sequences and evolutionary trees were inferred using a matrix optimization method. An evolutionary tree comprising all available eukaryotic srRNA sequences, including two other pathogenic Candida species, C. albicans and C. glabrata, showed that the yeasts diverge rather late in the course of eukaryote evolution, namely at the same depth as green plants, ciliates and some smaller taxa. The cluster of the higher fungi consists of 10 ascomycetes and ascomycete-like species with the first branches leading to Neurospora crassa, Pneumocystis carinii, Candida lusitaniae and C. krusei, in that order. Next there is a dichotomous divergence leading to a group consisting of Torulaspora delbrueckii, Saccharomyces cerevisiae, C. glabrata and Kluyveromyces lactis and a smaller group comprising C. tropicalis and C. albicans. The divergence pattern obtained on the basis of srRNA sequence data is also compared to various other chemotaxonomic data.     

羊驼线粒体12srRNA/tRNA-Val/16rRNA 基因序列测定及分子进化研究

对GaneBank中登录的部分骆驼科动物线粒体12 srRNA/tRNA-Val/16 rRNA基因序列进行同源性比较,并借助DNAstar软件设计引物,扩增并进行序列分析,旨在揭示其遗传变异的规律。PCR条件优化后成功扩增出长度为1 014 bp的DNA片段,b lastn分析显示,该片断与骆驼科动物的同源性高于95%,所获得片断包括30bp的12 srRNA基因部分序列,71 bp的tRNA-Val基因全序列和913 bp的16 srRNA基因部分序列。利用RNA-structure 4.2 RNA分析软件绘制了部分骆驼科动物的线粒体tRNA-Val推导性二级结构图,比较结果显示,羊驼线粒体tRNA-Val氨基酸臂和反密码子环呈属间特异性遗传。     

Evolutionary relationships of eukaryotic kingdoms

The evolutionary relationships of four eukaryotic kingdoms—Animalia, Plantae, Fungi, and Protista—remain unclear. In particular, statistical support for the closeness of animals to fungi rather than to plants is lacking, and a preferred branching order of these and other eukaryotic lineages is still controversial even though molecular sequences from diverse eukaryotic taxa have been analyzed. We report a statistical analysis of 214 sequences of nuclear small-subunit ribosomal RNA (srRNA) gene undertaken to clarify these evolutionary relationships. We have considered the variability of substitution rates and the nonindependence of nucleotide substitution across sites in the srRNA gene in testing alternative hypotheses regarding the branching patterns of eukaryote phylogeny. We find that the rates of evolution among sites in the srRNA sequences vary substantially and are approximately gamma distributed with size and shape parameter equal to 0.76. Our results suggest that (1) the animals and true fungi are indeed closer to each other than to any other crown group in the eukaryote tree, (2) red algae are the closest relatives of animals, true fungi, and green plants, and (3) the heterokonts and alveolates probably evolved prior to the divergence of red algae and animal-fungus-green-plant lineages. Furthermore, our analyses indicate that the branching order of the eukaryotic lineages that diverged prior to the evolution of alveolates may be generally difficult to resolve with the srRNA sequence data.     

家蚕传染性软化病病毒（桐乡株）VP1基因片段的克隆及序列分析

以首株在中国分离到的家蚕传染性软化病病毒（Bombyx mori infectious flacherie virus,BmIFV）BmIFV-CHN001基因组为模板,扩增了编码主要结构蛋白的VP1基因。克隆测序后得到VP1基因片段906 bp。该序列与已发表的日本毒株相比,核苷酸序列的相似性为99.3%,编码氨基酸的相似性为100%,证明该毒株与家蚕传染性软化病病毒日本株的同源性较高。把BmIFV-CHN001的VP1序列与同属的另外6个昆虫小RNA病毒的结构蛋白进行序列比对,构建系统发育树,对其进化关系进行了初步分析,结果显示这7种病毒具有相近的亲缘关系,而BmIFV-CHN001与蜜蜂囊雏病毒的亲缘关系最近。     

广东省水库3株水华微囊藻16s rRNA序列分析

微囊藻是有害淡水水华的主要藻种,但其形态分类困难,制约了相关研究的深入;16SrRNA序列分析广泛应用于微生物种类鉴定。测定广东3个水库的3株水华微囊藻16SrRNA基因部分序列,运用Mega3软件分析其遗传特征,结果表明,3株藻同源序列长度为1305bp,没有插入与缺失,序列中有3个变异位点,A T含量最大差异仅为0.2%,核苷酸相似性在99.85%以上,不能区分广东省水库同一藻种的不同藻株,表明水华微囊藻种内16SrRNA基因序列相当保守。要准确鉴定广东省水库微囊藻种类,丰富广东省水库微囊藻分子层面的分类资料,需要比较更多形态和地理来源的藻株,建立16SrRNA和其它基因序列核苷酸数据库,分析种间和种内差异,设计种专一性的分子探针。     

Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus

Acanthamoebae are increasingly being recognized as hosts for obligate bacterial endosymbionts, most of which are presently uncharacterized. In this study, the phylogeny of three Gram-negative, rod-shaped endosymbionts and their Acanthamoeba host cells was analysed by the rRNA approach. Comparative analyses of 16S rDNA sequences retrieved from amoebic cell lysates revealed that the endosymbionts of Acanthamoeba polyphaga HN-3, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39 are related to the Paramecium caudatum endosymbionts Caedibacter caryophilus, Holospora elegans a n d Holospora obtusa . With overall 16S rRNA sequence similarities to their closest relative, C. caryophilus , of between 87% and 93%, these endosymbionts represent three distinct new species. In situ hybridization with fluorescently labelled endosymbiont-specific 16S rRNA-targeted probes demonstrated that the retrieved 16S rDNA sequences originated from the endosymbionts and confirmed their intracellular localization. We propose to classify provisionally the endosymbiont of Acanthamoeba polyphaga HN-3 as ' Candidatus Caedibacter acanthamoebae', the endosymbiont of Acanthamoeba sp. strain UWC9 as ' Candidatus Paracaedibacter acanthamoebae' and the endosymbiont of Acanthamoeba sp. strain UWE39 as ' Candidatus Paracaedibacter symbiosus'. The phylogeny of the Acanthamoeba host cells was analysed by comparative sequence analyses of their 18S rRNA. Although Acanthamoeba polyphaga HN-3 clearly groups together with most of the known Acanthamoeba isolates (18S rRNA sequence type 4), Acanthamoeba sp. UWC9 and UWE39 exhibit < 92% 18S rRNA sequence similarity to each other and to other Acanthamoeba isolates. Therefore, we propose two new sequence types (T13 and T14) within the genus Acanthamoeba containing, respectively, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39.     

Detection of the antimicrobial peptide gene in different Amaranthus species

Using primers to amplify the gene AMP2 in Amaranthus caudatus, we found the gene to be present in seven other species of the Amaranthus genus (A. albus, A. cruentus, A. blitum, A. hybridus, A. hypochondriacus, A. retroflexus and A. tricolor), in which it had not been described previously. The PCR products were sequenced and it was established that all the sequences were identical, except for two polymorphisms. These single nucleotide polymorphisms occurred at nucleotide positions 45 and 246. This exchange of one nucleotide for another was manifested in an amino acid change in both cases. Due to the fact that both polymorphisms lay outside the region encoding the chitin-binding peptide domain, which is crucial for antimicrobial peptide function, they will not likely affect the proper functioning of the peptide. With the exception of the above-mentioned polymorphisms, all sequences were identical to the sequence of the AMP2 gene that codes for the A. caudatus Ac-AMP2 (antimicrobial peptide isolated from Amaranthus caudatus seeds). The detection of sequences with high degree of sequence similarity to A. caudatus AMP2 gene leads us to the assumption that an antimicrobial peptide could also be produced by other amaranth species.     

Sequence of the T4 recombination gene, uvsX, and its comparison with that of the recA gene of Escherichia coli.

We have determined the nucleotide sequence of the uvsX gene of bacteriophage T4 which is involved in DNA recombination and damage repair, and whose product catalyzes in vitro reactions related to recombination process in analogous manners to E. coli recA gene product. The coding region consisted of 1170 nucleotides directing the synthesis of a polypeptide of 390 amino acids in length with a calculated molecular weight of 43,760. Amino acid composition, the sequence of seven NH2-terminal amino acids and molecular weight of the protein deduced from the nucleotide sequence were consistent with the data from the analysis of the purified uvsX protein. The nucleotide sequence and the deduced amino acid sequence were compared with those of the recA gene. Although a significant homology was not found in the nucleotide sequences, the amino acid sequences included 23% of identical and 15% of conservatively substituted residues.     

The initiation of translation in E. coli: apparent base pairing between the 16srRNA and downstream sequences of the mRNA.

Bacteriophage T7's gene 0.3, coding for an antirestriction protein, possesses one of the strongest translation initiation regions (TIR) in E. coli. It was isolated on DNA fragments of differing length and cloned upstream of the mouse dihydrofolate reductase gene in an expression vector to control the translation of this gene's sequence. The TIR's efficiency was highly dependent on nucleotides +15 to +26 downstream of the gene's AUG. This sequence is complementary to nucleotides 1471-1482 of the 16srRNA. Similar sequences complementary to this rRNA region are present in other efficient TIRs of the E. coli genome and those of its bacteriophages. There seems to be a correlation between this sequence homology and the efficiency of the initiation signals. We propose that this region specifies a stimulatory interaction between the mRNA and 16srRNA besides the Shine-Dalgarno interaction during the translation initiation step.     

Immunological inter-strain crossreactivity correlated to 18S rDNA sequence types in Acanthamoeba spp

Various species of the genus Acanthamoeba have been described as potential pathogens; however, differentiation of acanthamoebae remains problematic. The genus has been divided into 12 18S rDNA sequence types, most keratitis causing strains exhibiting sequence type T4. We recently isolated a keratitis causing Acanthamoeba strain showing sequence type T6, but being morphologically identical to a T4 strain. The aim of our study was to find out, whether the 18S rDNA sequence based identification correlates to immunological differentiation. The protein and antigen profiles of the T6 isolate and three reference Acanthamoeba strains were investigated using two sera from Acanthamoeba keratitis patients and one serum from an asymptomatic individual. It was shown, that the T6 strain produces a distinctly different immunological pattern, while patterns within T4 were identical. Affinity purified antibodies were used to further explore immunological cross-reactivity between sequence types. Altogether, the results of our study support the Acanthamoeba 18S rDNA sequence type classification in the investigated strains.     

Intraspecific variation of Spirometra erinaceieuropaei and phylogenetic relationship between Spirometra and Diphyllobothrium inferred from mitochondrial CO1 gene sequences

Spirometra erinaceieuropaei is a diphyllobothriid cestode whose adult stage occurs mainly in cat-like carnivores, but occasionally in canids and humans. Although it is generally accepted that the distribution of S. erinaceieuropaei is cosmopolitan, it is controversial as to whether all of S. erinaceieuropaei reported are the same species. This study determined partial sequences of the CO1 gene from several isolates in Asian countries and compared them to sequence data from the GenBank/EMBL/DDBJ nucleotide sequence database. Then intraspecific variation of S. erinaceieuropaei and its phylogenetic relationship with Diphyllobothrium were evaluated. The level of nucleotide variation in the CO1 gene sequences within S. erinaceieuropaei was less than 2.6%. Although it was a little larger than that within each species of Diphyllobothrium (0.1-1.0%), it was much smaller than the interspecific variation within the genus Diphyllobothrium (6.2-14%). These facts indicate that all isolates of S. erinaceieuropaei used in this study, which were collected from Asia, Australia and New Zealand, belong to the same species. Based on CO1 gene sequences, genus Spirometra is clearly separate from the genus Diphyllobothrium. It seems that the genus Spirometra is not a synonym of the genus Diphyllobothrium. The phylogenetic relationship between S. erinaceieuropaei and Sparganum proliferum inferred from the CO1 gene clearly confirm the previous opinion that S. proliferum is a distinct species from S. erinaceieuropaei.     

Revisiting the Acanthamoeba species that form star-shaped cysts (genotypes T7, T8, T9, and T17): characterization of seven new Brazilian environmental isolates and phylogenetic inferences

Free-living amoebae of the genus Acanthamoeba are the agents of both opportunistic and non-opportunistic infections and are frequently isolated from the environment. Of the 17 genotypes (T1-T17) identified thus far, 4 (T7, T8, T9, and T17) accommodate the rarely investigated species of morphological group I, those that form large, star-shaped cysts. We report the isolation and characterization of 7 new Brazilian environmental Acanthamoeba isolates, all assigned to group I. Phylogenetic analyses based on partial (~1200 bp) SSU rRNA gene sequences placed the new isolates in the robustly supported clade composed of the species of morphological group I. One of the Brazilian isolates is closely related to A. comandoni (genotype T9), while the other 6, together with 2 isolates recently assigned to genotype T17, form a homogeneous, well-supported group (2·0% sequence divergence) that likely represents a new Acanthamoeba species. Thermotolerance, osmotolerance, and cytophatic effects, features often associated with pathogenic potential, were also examined. The results indicated that all 7 Brazilian isolates grow at temperatures up to 40°C, and resist under hyperosmotic conditions. Additionally, media conditioned by each of the new Acanthamoeba isolates induced the disruption of SIRC and HeLa cell monolayers.     

Analysis of Mitochondrial DNA Variation as an Approach to Systematic Relationships in the Genus Acanthamoeba1

Classification at the species level has been difficult in the genus Acanthamoeba. The taxonomic designations of a number of strains are in doubt and new approaches to classification are needed. We describe the use of electrophoretic patterns obtained with restriction enzyme digests of mitochondrial DNA as a basis for one new approach. Results from analysis of ten strains of A. castellanii, two of A. polyphaga and one of A. astronyxis are discussed. Examples both of nucleotide sequence diversity and of sequence conservation have been found among strains with the same species designation. Five strains from Europe, North America and New Zealand had identical digestion phenotypes with five enzymes; consequently, very similar nucleotide sequences are predicted. All are pathogenic to humans or mice. The mtDNA sequences of eight remaining strains are predicted to differ from this cluster and, in most cases, from each other at least as much as in sibling species of Paramecium aurelia.     

从12S rRNA和Cyt b基因部分序列研究13种猫科动物的分子系统关系

为探讨中国猫科动物(Felidae)的系统发生关系,本文对中国产13种猫科动物的12SrRNA基因(约371bp)和细胞色素b基因(Cytb)部分序列(约355bp)进行了分析,并采用“最大简约法”和“最大似然法”构建了分子系统树。结果表明:在Cytb基因序列中,有113个位点存在变异(约为总位点数的31.8%),高于12SrRNA基因序列的44个变异位点(约为总位点数的11.9%);构建的分子系统树显示,猞猁(Lynxlynx)可能是中国最早起源的猫科动物,与其它猫科动物之间的亲缘关系较远,支持将其立为猞猁属(Lynx)的观点;草原斑猫(Felislibyca)、丛林猫(Felischaus)、兔狲(Otocolobusmanul)和荒漠猫(Felisbieti)具有较近的亲缘关系,支持将兔狲划归于猫属(Felis)的观点;金猫(Caopumatemminckii)、云猫(Pardofelismarmorata)具有较近的亲缘关系,但它们与猫属物种之间的亲缘关系可能较远,不支持将它们划归于猫属;豹猫(Ponailurusribengalensis)、渔猫(Prionailurusviverrinus)具有较近的亲缘关系,支持将它们同归于豹猫属(Ponailurus);云豹(Neofelisnebulosa)、豹(Pantherapardus)、雪豹(Unciauncia)、虎(Pantheratigris)具有较近的亲缘关系,支持将它们同归于豹属(Panthera)的观点