NUCLEOTIDE SEQUENCES OF SMALL-SUBUNIT AND INTERNAL TRANSCRIBED SPACER REGIONS OF NUCLEAR rRNA GENES SUPPORT THE AUTONOMY OF SOME GENERA OF THE GELIDIALES (RHODOPHYTA)

The lack of homogeneity in all previously proposed, distinguishing characteristics has left the relationships of taxa within the Gelidiales as one of the most enduring taxonomic uncertainties in the Rhodophyta. Although a precise knowledge of the taxonomy of commercially harvested members of the Gelidiales would assist resource management, agronomic practices, and marketing, even the distinction between two major groups, Gelidium and Pterocladia, has long remained controversial. In this study, the 18S ribosomal RNA (rRNA), internal transcribed spacer 1 (ITS1), 5.8S rRNA, and ITS2 regions of Gelidium latifolium (Greville) Bornet et Thuret, G. sesquipedale (Clemente) Thuret in Bornet et Thuret, G. vagum Okamura, Pterocladia lucida (Brown ex Turner) J. Agardh, and a recent segregate from Pterocladia, Pterocladiella capillacea (Gmelin) Santelices et Hommersand, were sequenced and analyzed. The ITS1, 5.8S rRNA, and ITS2 regions of G. arbuscula (Montagne) B=orgesen, G. canariensis (Grunow) Seoane-Camba, G. capense (Gmelin) Silva, and G. pristoides (Turner) Kützing were also sequenced. Phylogenetic analyses based on the 18S rRNA genes from four Gelidium species, Pterocladia lucida, and Pterocladiella capillacea , compared with 18S rRNA genes from several other red algae confirmed the division between Gelidium and the Pterocladia/Pterocladiella isolates and were consistent with the recently proposed segregation of Pterocladiella from Pterocladia. Analyses based on the ITS regions of seven Gelidium species, Pterocladia lucida, and Pterocladiella capillacea were completely consistent with the conclusions drawn from the 18S rRNA data. There were extensive length and sequence differences between Gelidium and Pterocladia/Pterocladiella . In addition, there were larger sequence differences between Pterocladiella and Pterocladia than exist among Gelidium isolates, in keeping with the recently proposed separation of the former two taxa.     

双壳纲动物核糖体RNA 18S-ITS1序列及其在分子系统发育研究中的应用

测定了双壳纲不同科、属、种及种内共11个个体的核糖体RNA 18S-ITS1序列。结果表明,该序列在种间存在很高的多态性,长度从558bp到784bp不等,碱基差异百分比在10．7％～61．7％之间,ITS1序列同源性很低,有片段的插入与缺失。种间18S部分序列碱基差异百分比在0．9％～23．7％之间,变化主要是碱基的转换。用邻接法(NJ)构建了8个种的18S部分序列(约240bp)的系统发育树,与传统形态学分类结果相符。马氏珠母贝(Pinctada martrtensi)4个不同地域个体间的序列差异百分比在0．6％～1．9％之间。分析指出：18S基因可以作为双壳纲动物高阶元系统发育的分子标记;ITS1序列种间变化很大,可以应用于该纲物种的分类及鉴别．在亲缘关系相近种及种内变异相对较小,但核苷酸变异位点信息量丰富,可用于属内种间、亚种和群体间的遗传多样性研究。     

鸡球虫18S rRNA基因序列的测定与分析

为了利用18S rRNA基因进行鸡球虫系统进化分析,对巨型艾美耳球虫(Eimeria maxima)、柔嫩艾美耳球虫(E.tenella)、堆形艾美耳球虫(E.acervulina)3种共8个不同来源的虫株,分别提取总DNA进行18S rRNA基因的扩增和测序;将得到的序列登录GenBank进行同源性和趋异性分析,并结合GenBank中其它原虫的18S rRNA基因序列构建进化树.结果显示扩增获得8株鸡球虫18S rRNA基因长度为1746～1756 bp,序列比对显示同种不同株间的同源性大于不同种间的同源性,其中3株E.maxima株间同源性在98.7%～99.3%之间,4株E.tenella株间同源性在99.7%～99.9%之间;不同种间同源性为96.5%～98.1%,其中E.maxima与E.tenclla的遗传距离最大,为0.038;E.maxima与E.acervulina的遗传距离最小,为0.021.顶复器门9个不同属所构建的进化树结果显示,E.imeria和等孢属(Isospora)聚为一支,说明亲缘关系比较近.与GertBank中其它5株不同鸡球虫的18S rRNA基因共同构建的进化树显示,3株E.maxima聚为一支,与E.brunetti、E.mitis、E.mivati、E.praecox和E.acervulina聚为一大分支;4株E.tenella与1株E.necatrix共同形成一个分支,说明E.tenella与E.necattix的亲缘关系最近.本研究证实了在鸡球虫系统进化研究中,18S rRNA基因不仅可以区分不同种,而且有可能成为区分同种不同株的理想靶基因.     

The diversity of fungi in aerobic sewage granules assessed by 18S rRNA gene and ITS sequence analyses

Aerobic sewage granules are dense, spherical biofilms, regarded as a useful and promising tool in wastewater treatment processes. Recent studies revealed that fungi can be implemented in biofilm formation. This study attempts to uncover the fungal diversity in aerobic granules by sequence analysis of the 18S and 5.8S rRNA genes and the internal transcribed spacer regions. For this purpose, appropriate PCR and sequencing primer sets were selected and an improved DNA isolation protocol was used. The sequences of 41 isolates were assigned to the taxonomic groups Pleosporaceae, Xylariales, Theleobolaceae, Claviceps, Aureobasidium, Candida boleticola , and Tremellomycetes within the fungi. It turned out that the fungal community composition in granules depended on the wastewater type and the phase of granule development.     

Phylogeny of parasitiform mites (Acari) based on rRNA

Acari (mites and ticks) form one the most diverse lineages of arthropods, but basal relationships in the group are still poorly understood. The current study addresses this issue for one of its two main lineages, the order Parasitiformes. Relationships are examined at the subordinal and infraordinal level using complete 18S and partial 28S nuclear rRNA sequence data. Most currently recognized lineages are recovered with good support, suggesting that nuclear rRNA, and specifically 18S rRNA, is very well suited for analyzing relationships at this level in this lineage. These results were found despite quite variable rates of sequence evolution, with rates "ratcheting up" from relatively low in most non-mite arachnid lineages, to intermediate in Pseudoscorpiones, the mite order Acariformes, and the parasitiform suborders Opilioacarida, Holothyrida, and Ixodida, to high in the parasitiform suborder Mesostigmata. The most species rich mesostigmatid infraorder, Dermanyssina, shows huge distances to the outgroups, but remarkably low within-group divergence in nuclear rRNA. This suggests the possibility of a relatively recent origin of this lineage.     

Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.     

Changes in the rRNA levels of specific microbial groups in activated sludge during sample handling and storage

AIMS: To quantitatively analyse the changes in group-specific rRNA levels in activated sludge as a function of sample handling and storage procedure. METHODS AND RESULTS: Quantitative membrane hybridizations with (32)P-labelled oligonucleotide probes were used to analyse the effects of different sample handling and storage conditions on the relative rRNA levels of the alpha, beta, and gamma-Proteobacteria, the Cytophaga-Flavobacteria group, and the mycolic acid-containing actinomycetes in activated sludge. Group-specific rRNA levels, expressed as percentages of total 16S rRNA detected with a universal probe, in samples maintained at room temperature significantly changed after 48 h. Group-specific rRNA levels in samples treated with chloramphenicol showed significant change after 72 h. CONCLUSIONS: Sample storage at room temperature is a viable option if freezing or analysis can be performed within 24 h, while treatment with chlorampenicol can extend that time to at least 48 h. SIGNIFICANCE AND IMPACT OF THE STUDY: Handling, shipping, and storage of environmental samples under several conditions may result in inaccurate determination of the microbial populations in microbial ecology studies.     

Loss of rRNA modifications in the decoding center of the ribosome impairs translation and strongly delays pre-rRNA processing

The ribosome decoding center is rich in modified rRNA nucleotides and little is known about their effects. Here, we examine the consequences of systematically deleting eight pseudouridine and 2′-O-methylation modifications in the yeast decoding center. Loss of most modifications individually has no apparent effect on cell growth. However, deletions of 2–3 modifications in the A- and P-site regions can cause (1) reduced growth rates (∼15%–50% slower); (2) reduced amino acid incorporation rates (14%–24% slower); and (3) a significant deficiency in free small subunits. Negative and positive interference effects were observed, as well as strong positional influences. Notably, blocking formation of a hypermodified pseudouridine in the P region delays the onset of the final cleavage event in 18S rRNA formation (∼60% slower), suggesting that modification at this site could have an important role in modulating ribosome synthesis.     

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.     

用S1核酸酶作图法测定蓖麻蚕18S rRNA基因的5′端位置

测定基因5′端位置是研究基因转录调控的一个重要前提。本文将蓖麻蚕18S rRNA基因DNA的5′端用~(32)P标记,然后与18S rRNA杂交,再用S1核酸酶水解掉非杂交区的DNA和RNA。分析放射自显影的结果,测出18S rRNA基因5′端的位置。在18S rRNA基因的BglⅡ_2位点向EcoRⅠ,方向延伸约220bp处,从这一结果,可知道蓖麻蚕rRNA基因的转录方向是5′EcoRⅠ_2→BglⅡ_23′。     

Complete nucleotide sequence of mouse 18 S rRNA gene: comparison with other available homologs

We present the complete sequence of mouse 18 S rRNA. As indicated by comparison with yeast, Xenopus and rat, the conservation of eukaryotic 18 S rRNA sequences is extensive. However, this conservation is far from being uniform along the molecule: most of the base changes and the size differences between species are concentrated at specific locations. Two distinct classes of divergent traces can be detected which differ markedly in their rates of nucleotide substitution during evolution, and should prove valuable in additional comparative analyses, both for eukaryotic taxonomy and for rRNA higher order organization. Mouse and rat 18 S rRNA sequences differ by only 14 point changes over the 1869 nucleotides of the molecule.     

Molecular systematics analysis of Lymantria dispar based on 18S rRNA and cox1 mtDNA sequence data

The complete sequence of the 18 S ribosomal RNA gene(18S rRNA) from Lymantria dispar was cloned and analysed here. 18 S rRNA and mitochondrial cytochrome c oxidasel(cox1) gene sequences of Lymantria dispar were compared with homologous sequences of other nine insects from different orders. Analytic results showed that 18 S rRNA of these insects had two conserved domains and the second domain was an even more conserved region. The phylogenetic trees based on the full-length sequence and the second domain fragment of 18 S rRNA as well as sequence of cox1 from different orders indicated that Lepidoptera and Trichoptera, which belongs to Amphiesmenoptera, had a closer phylogenetic relationship and fewer differences were observed comparing with traditional taxonomic results.     

Identification of waterborne bacteria by the analysis of 16S-23S rRNA intergenic spacer region

AIM: In this study, we evaluated, the use of universal primers, specific for the 16S-23S rRNA intergenic region, to detect and identify nine species that are of high interest for the microbiological control of water. METHODS AND RESULTS: The analysis of the fragments was carried out using a High Resolution acrylamide/bisacrylamide gels in a fluorescent automated DNA sequencer. The results showed specific profiles for each of the nine species but this technique failed to detect simultaneously micro-organisms in samples containing a mixed population. CONCLUSION: Nevertheless, the electrophoretic profiles obtained provided a very useful tool for the rapid and specific identification of water isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: A possible new methodology for a rapid identification of pathogens in water.     

Diplogonoporiasis in Japan: genetic analyses of five clinical isolates

Infection of the whale tapeworm Diplogonoporus balaenopterae (Diphyllobothriidae) is occasionally found in humans, especially among Japanese. In the present study, we analysed the nucleotide sequences of the 18S rDNA, ITS1 and cox1 genes of the immature and mature proglottids of Diplogonoporus species recovered from five Japanese patients. The nucleotide sequences of 18S rDNA, ITS1 and cox1 showed little, if any, intraspecific divergence. Phylogenetic analyses of several diphyllobothriid species revealed a close relationship of Diplogonoporus isolates with the cetacean tapeworm Diphyllobothrium stemmacephalum. The results suggest that the genus Diphyllobothrium is paraphyletic and raise a question regarding the validity of the genus Diplogonoporus.     

Crystal structure of the RluD pseudouridine synthase catalytic module, an enzyme that modifies 23S rRNA and is essential for normal cell growth of Escherichia coli

Pseudouridine (5-beta-D-ribofuranosyluracil, Psi) is the most commonly found modified base in RNA. Conversion of uridine to Psi is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Psi-synthase RluD modifies uridine to Psi at positions 1911, 1915 and 1917 within 23S rRNA. RluD also possesses a second function related to proper assembly of the 50S ribosomal subunit that is independent of Psi-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68-326; DeltaRluD) refined at 1.8A to a final R-factor of 21.8% (R(free)=24.3%). DeltaRluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DeltaRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of DeltaRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA-protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site.     

草鱼MYH mRNA表达量分析中采用的内参基因稳定性比较

本研究分别以β-actin、18S rRNA和GAPDH为内参基因,采用实时荧光定量PCR对草鱼早期发育时期肌球蛋白重链(myosin heavy light,MYH)基因的mRNA表达量进行分析,并比较不同内参基因对MYH基因mRNA表达水平检测结果的准确性.研究结果表明,以β-actin和GAPDH作为内参,MYH基因mRNA表达水平完全一致,其表达量从原肠到仔鱼阶段逐次递增,仔鱼与原肠期阶段相比表达量差异显著;当采用18S rRNA作为内参时,MYH基因mRNA在发育阶段的表达量呈不稳定状态.因此,β-actin和GAPDH均可作为内参基因,用于草鱼早期发育中MYH基因mRNA的相对定量研究:而18S rRNA作为内参时,可能会对检测结果造成偏差.本研究不仅准确的揭示了草鱼MYH基因mRNA的表达特征,并且为荧光定量PCR技术在鱼类基因表达研究方面提供了有价值的参考.