Characterization of Two Perkinsus spp. from the Softshell Clam, Mya arenaria Using the Small Subunit Ribosomal RNA Gene

Sequence analysis and riboprinting of the small subunit ribosomal RNA genes were used to characterize two morphologically different Perkinsus species isolates from the gill (G117) and the hemolymph (H49) of the softshell clam, Mya arenaria. Sequence data of the polymerase chain reaction amplified ribosomal RNA loci of G117 and H49 indicated that these genes are 1803 and 1806 base-pair long, respectively. A sequence similarity of > 98.9% was calculated among ribosomal RNA sequences of the two isolates of this study and the published sequences of Perkinsus marinus from the American eastern oyster, Crassostrea virginica, and Perkinsus sp. from the blood cockle of the Australian mollusc, Anadara trapezia. From a phylogenetic tree obtained from Jukes-Cantor distances of the aligned ribosomal RNA gene sequences of 13 eukaryotic taxa using the Neighbor-Joining method, we showed that G117 and H49 clustered within the genus Perkinsus. Guided by the sequence data of Perkinsus marinus (accession # X75762) and Perkinsus sp. (accession # L07375), restriction endonucleases were selected for restriction fragment analysis of polymerase chain reaction products of the small subunit ribosomal RNA genes (riboprinting). Riboprinting was used to distinguish the four members of the genus Perkinsus from each other.     

Molecular Characterization of Human Rotavirus VP4 Genes by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Assay

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic variability and similarity of the VP4 genes of human rotaviruses. The VP4 genes of 14 human rotavirus strains, including VP4 serotype P1A strains (Wa, P, VA70), serotype P1B strain (DS-1), serotype P2 strains (M37, 1076, McN, ST3) and serotype P3 strains (AU-1, AU228, K8, PA151, PCP5, MZ58), and those of 2 feline strains (FRV-1 and Cat2) were reverse-transcribed and amplified by the polymerase chain reaction (PCR). The amplified VP4 cDNAs were then digested with a panel of restriction endonucleases (HindIII, NruI, HaeIII, and EcoRI), resulting in the identification of at least one enzyme with which digestion produced an RFLP profile specific for a particular P serotype. Of interest was the presence of two distinct RFLP patterns within the serotype P3 VP4 genes: one corresponding to the VP4 gene carried by the members of the AU-1 genogroup and the other corresponding to the VP4 genes carried by naturally-occurring reassortants between members of the AU-1 and other genogroups.     

Molecular Identification of Nanoplanktonic Protists Based on Small Subunit Ribosomal RNA Gene Sequences for Ecological Studies1

Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.     

A Three Nucleotide Signature Sequence in Small Subunit rRNA Divides Human Giardia in Two Different Genotypes

ABSTRACT. The nucleotide sequence of the 16S rRNA gene, part of the 23S rRNA gene and the spacer DNA region was determined for Giardia duodenalis , obtained from humans in The Netherlands (AMC-4) and Washington State (CM). These rDNA sequences differ from other G. duodenalis isolates (Portland-1 and BRIS/83/HEPU/106) both of which have virtually identical rDNA sequences. The most characteristic feature was found close to the 5'end of the 16S rRNA. The Portland-1 - Bris/83/HEPU/106 type has GCG in position 22–24, while AMC-4 and CM have AUC in this position. These two sequences, present in an otherwise conserved region of the 16S rRNA, are "signature" sequences, which divide Giardia isolates into two different groups.     

末端限制性片段长度多态性(T-RFLP)技术的系统误差分析

T-RFLP技术是一种新近发展起来的分析微生物多样性的分子生物学方法,与其它多样性分析技术相比,具有一些不可比拟的优势,但T-RFLP技术操作流程将对结果产生系统误差的程度鲜有报道。实验以紫茎泽兰入侵过程中4种土样中的nifH基因多样性分析为例,进行了只改变T-RFLP操作流程中一个步骤的3次重复分析,结果表明:限制性内切酶种类对T-RFLP分析结果的可重复性影响最大,PCR次之,而毛细管电泳对结果的可重复性几乎没有影响。     

Separation of Three Species of Ditylenchus and Some Host Races of D. dipsaci by Restriction Fragment Length Polymorphism

This study examined the ribosomal cistron of Ditylenchus destructor, D. myceliophagus and seven host races of D. dipsaci from different geographic locations. The three species showed restriction fragment length polymorphisms (RFLPs) in the ribosomal cistron, the 18S rDNA gene, and the ribosomal internal transcribed spacer (ITS). Southern blot analysis with a 7.5-kb ribosomal cistron probe differentiated the five host races of D. dipsaci examined. Polymerase chain reaction (PCR) amplification of the ITS, followed by digestion with some restriction endonucleases (but not others), produced restriction fragments diagnostic of the giant race. Because the PCR product from D. myceliophagus and the host races of D. dipsaci was about 900 base pairs and the ITS size in D. destructor populations was 1,200 base pairs, mixtures of populations could be detected by PCR amplification. ITS fragments differentiated between D. dipsaci and Aphelenchoides rhyntium in mixed populations. This study establishes the feasibility of differentiation of the host races of D. dipsaci by probing Southern blots with the whole ribosomal cistron.     

Genetic Variation of Nuclear Ribosomal DNA in Cockroaches (Order Blattaria): Phylogenetic Analysis of Restriction Fragment Length Polymorphism

Species-specific characteristics of nuclear ribosomal DNA (rDNA) have been determined for the first time for six insect species of order Blattaria (Insecta, Dictyoptera) with the use of restriction analysis and Southern blotting. Probes containing highly conserved fragments of 18S- and 28S-like genes of the ribosomal RNA (rRNA) of Tetrahymena pyriformis were tested in this study. The genetic similarity tree constructed for the species studied agrees with evidence from classical taxonomy based on morphological, ecological, anatomical, and physiological characters.     

Restriction Fragment Length Polymorphism and Random Amplified Polymorphic DNA Analysis of Chickpea Accessions

Genetic diversity analysis was carried out in chickpea accessions using restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) techniques. RFLP analysis using 26 Pst I sub-genomic clones on ten chickpea accessions in 130 probe-enzyme combinations detected polymorphism with only two clones. Pst I clones, CG 141 detected polymorphism in ICC 4918 and Pusa 209 while CG 500 detected polymorphism in Pusa 261, ILC 26 and in ILC 13326. These clones detected very few polymorphic markers. Analysis using 10 Eco RI clones on twelve chickpea accessions have shown better hybridisation signal and one clone detected polymorphism in Pusa 256. RFLP analysis of both cultivated and wild Cicer species using heterologous DNA probe Cab3C revealed polymorphism only in wild Cicer species (Cicer reticulatum L., JM 2100). RAPD analysis of 13 chickpea accessions which includes mutants of C 235 and E100Y showed greater degree of polymorphism with 1 - 5 unique DNA bands for all the accessions. Phylogenetic analysis of the RAPD data helped to group the accessions. C 235 and its mutants were found to be closely grouped while E100Y and its mutant E100Ym grouped apart. Desi and kabuli chickpea accessions however, could not be separately grouped. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rapid Identification of Rhizobia by Restriction Fragment Length Polymorphism Analysis of PCR-Amplified 16S rRNA Genes

Forty-eight strains representing the eight recognized Rhizobium species, two new Phaseolus bean Rhizobium genomic species, Bradyrhizobium spp., Agrobacterium spp., and unclassified rhizobia from various host plants were examined by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by polymerase chain reaction (PCR). Twenty-one composite genotypes were obtained from the combined data of the RFLP analysis with nine endonucleases. Species assignments were in full agreement with the established taxonomic classification. Estimation from these data of genetic relationships between and within genera and species correlated well with previously published data based on DNA-rRNA hybridizations and sequence analysis of 16S rRNA genes. This PCR-RFLP method provides a rapid tool for the identification of root nodule isolates and the detection of new taxa.     

Genetic Diversity in Ralstonia solanacearum Strains from Mauritius using Restriction Fragment Length Polymorphisms

Race 1, biovar III of Ralstonia (synonym Pseudomonas ) solanacearum , causal organism of bacterial wilt, has been reported in Mauritius on several crops and plant species. The genetic relationship among 38 strains isolated from potato, tomato, bean and anthurium was determined by restriction fragment length polymorphisms (RFLPs). After hybridization with probe 5a67, five RFLP patterns could be distinguished. Types V and I were most commonly encountered. A common band of approximately 6.5 kb was found in 35 strains. Type I pattern consisted of only this band and was observed in 12 out of 16 anthurium strains tested. Type V was associated with 12 out of 16 potato strains and consisted of a band of approximately 3.3 kb in addition to the one observed in type I. RFLP patterns II, III and IV were less frequently encountered. The RFLP analysis showed that genetic diversity was present in race 1, biovar III strains. The relationship between the host and RFLP pattern is discussed.     

Diversity In Symbiotic Dinoflagellates (Pyrrhophyta) from Seven Scleractinian Coral Species: Restriction Enzyme Analysis of Small Subunit Ribosomal RNA Genes

ABSTRACT The diversity of symbiotic dinoflagellates (SD) from seven coral species ( Fungia scutaria, Fungia paumotensis, Lep-tastrea transversa, Pavona cactus, Pocillopora verrucosa, Montastrea curia , and Acropora fonnosa ) was studied in a restricted geographical area, the Lagoon of Arue on the island of Tahiti. Their diversity was explored by small subunit ribosomal RNA gene (SSU rDNA) restriction fragment length polymorphism (RFLP). After a nested amplification with SD specific primers, RFLP analyses were performed directly and after a cloning step. The diversity of these different SSU rDNA was estimated in respect to possible technical artifacts. In an axenic culture of SD from the coral Galaxea fascicularis , both heterogeneous SSU rDNAs and artifact molecules were observed as in our SD samples. According to the number of patterns observed, corals Fungia paumotensis, Leptastrea transversa. Pavona cactus, Montastrea curia, and Acropora fonnosa contained one class of SD SSU rDNAs. whereas Fungia scutaria and Pocillopora verrucosa contained three and two classes of SD SSU rDNAs respectively. In the limited geographic area studied. SD from different coral species shared the same pattern, except SD from Montastrea curta , which showed a unique pattern. In addition to the possibility of SD flux among different coral species, specific mechanisms could also be involved in the establishment of a symbiosis.     

Differentiation of Rickettsia japonica by Restriction Endonuclease Fragment Length Polymorphism Using Products of Polymerase Chain Reaction Amplification with Rickettsia rickettsii 190-Kilodalton Surface Antigen Gene Primers

Restriction fragment length polymorphism of polymerase chain reaction (PCR) amplification products differentiated Rickettsia japonica, a causative agent of Oriental spotted fever, from other spotted fever group (SFG) rickettsiae. Primer pair Rr190. 70p and Rr190. 602n of R. rickettsii 190-kDa antigen gene sequence primed genomic DNAs obtained from R. japonica, type strain YH and strains NT, NK, YKI, and TKN. The products were cleaved by PstI but not by AfaI restriction endonuclease. The PstI digestion pattern of PCR-products amplified from all strains of R. japonica was identical and easily differentiated from that of other SFG rickettsiae. The present study demonstrated a genotypic difference between R. japonica and other pathogenic SFG rickettsiae.     

末端限制性片段长度多态性技术分析硝化细菌微生物多样性

利用T_RFLP(末端限制性片段长度多态性)技术,分析硝化细菌富集反应器中的微生物群落结构,并对硝化细菌的丰度进行半定量研究。结果表明,培养48h后,硝化细菌富集效果最佳,多样性指数与初始培养相比下降了62.80%,富集出的硝化细菌主要为亚硝酸盐氧化菌(Nitrobacter)。同时对投加该硝化细菌前后的对虾养殖水体进行微生物多样性的动态研究,并推测了虾塘水中可能稳定存在的几种主要细菌种类,其中投加富集硝化细菌前后均存在的细菌种类包括短芽孢杆菌Brevibacillus brevis、微杆菌Microbacterium lactium、固氮弧菌Azoarcus indigens或者霍氏鲍特菌Bordetella holmesii。     

Restriction Fragment Length Polymorphism Analysis of Epidemiologically Related Mycobacterium tuberculosis Isolates

Restriction fragment length polymorphism (RFLP) analysis of a large number of Japanese isolates of Mycobacterium tuberculosis, containing isolates from small outbreaks of M. tuberculosis infection, and clinical isolates of M. bovis BCG, was carried out using a DNA probe derived from the insertion sequence IS986. Clinical isolates of M. tuberculosis had a high degree of RFLP. The occurrences of the IS element varied from 1 to 19, the majority of isolates having 8 to 15 copies. Very similar fingerprints, however, were seen among strains isolated in the Kanto district. In particular, 3 strains were of the same pattern with or without an additional band. Similarity of the banding patterns of strains islated in the same district was observed in other areas. Six groups of strains, each group arising from a suspected common source of infection, were analyzed. Of these, 5 showed identical fingerprints within each group, but one showed different fingerprints. RFLP patterns of three strains isolated from individuals with lymphadenitis developed about two months after BCG vaccination, and one strain isolated from a bladder cancer patient with BCG instillation therapy were identical to those of BCG-Tokyo which had been used for the vaccination and therapy. These results confirm that RFLP analysis using IS986 is a suitable tool for epidemiology of tuberculosis.     

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.     

Babesia Canis Canis, Babesia Canis Vogeli, Babesia Canis Rossi: Differentiation of the Three Subspecies By A Restriction Fragment Length Polymorphism Analysis On Amplified Small Subunit Ribosomal Rna Genes

The parasites Babesia canis and Babesia gibsoni (phylum Apicomplexa) are responsible for canine babesiosis throughout the world. Babesia canis was previously described as a group of three biologically different subspecies, namely B. canis canis, B. canis vogeli, and B. canis rossi. We report partial sequences of small subunit ribosomal RNA gene (ssu-rDNA) of each subspecies amplified in vitro with primers derived from a semi-conserved region of the ssu-rDNA genes in other Babesia species. The polymerase chain reaction combined with a restriction fragment length polymorphism analysis, using HinfI and TaqI restriction enzymes, confirmed the separation of B. canis into three subspecies. These sequences were compared with previously published sequences of other Babesia species. A phylogenetic approach showed that the three subspecies of B. canis belong to the clade of Babesia species sensu stricto where B. canis canis clusters with B. canis rossi whereas B. canis vogeli might form a monophyletic group with the cluster B. divergens and B. odocoilei. Our results show that the three subspecies of B. canis can readily be differentiated at the molecular level and suggest that they might be considered as true species.     

FMMU白化豚鼠线粒体DNA RFLP分析研究

目的研究FMMU白化豚鼠的mtDNA,并与花色豚鼠mtDNA进行多态性分析比较,以确定其独特的生物学特性是否与mtDNA相关。方法用碱变性法提取FMMU白化豚鼠以及花色豚鼠的mtDNA,并用AvaⅠ、BalⅠ等12种限制性内切酶进行酶切和限制性片段长度多态性分析。结果与结论FMMU白化豚鼠mtDNA和花色豚鼠mtDNA的相对分子质量相同,约为16.7×103;FMMU白化豚鼠与花色豚鼠两品系的mtDNA经AvaⅠ、BalⅠ等内切酶酶切后有3-8个酶切位点,酶切图谱完全相同,经RFLP分析FMMU白化豚鼠与花色豚鼠的mtDNA之间缺乏多态性。本实验没有发现FMMU白化豚鼠的独特的生物学特性与mtDNA相关。     

Thermal Adaptation of the Small Subunit Ribosomal RNA Gene: A Comparative Study

We carried out a comprehensive survey of small subunit ribosomal RNA sequences from archaeal, bacterial, and eukaryotic lineages in order to understand the general patterns of thermal adaptation in the rRNA genes. Within each lineage, we compared sequences from mesophilic, moderately thermophilic, and hyperthermophilic species. We carried out a more detailed study of the archaea, because of the wide range of growth temperatures within this group. Our results confirmed that there is a clear correlation between the GC content of the paired stem regions of the 16S rRNA genes and the optimal growth temperature, and we show that this correlation cannot be explained simply by phylogenetic relatedness among the thermophilic archaeal species. In addition, we found a significant, positive relationship between rRNA stem length and growth temperature. These correlations are found in both bacterial and archaeal rRNA genes. Finally, we compared rRNA sequences from warm-blooded and cold-blooded vertebrates. We found that, while rRNA sequences from the warm-blooded vertebrates have a higher overall GC content than those from the cold-blooded vertebrates, this difference is not concentrated in the paired regions of the molecule, suggesting that thermal adaptation is not the cause of the nucleotide differences between the vertebrate lineages. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Nicolas Galtier]     

Morel Phylogeny and Diagnostics Based on Restriction Fragment Length Polymorphism Analysis of ITS Region of 5.8S Ribosomal RNA Gene

Thirty-six cultures representing eight Morchella and related genera, namely, Morchella esculenta, M. crassipes, M. spongiola, M. vulgaris, M. angusticeps, M. conica, Mitrophora semilibera and Verpa conica were subjected to restriction analysis of ITS1-5.8SITS2 region of rDNA. Six restriction endonuclease enzymes viz TaqI, EcoRl, Mspl, Rsal, Hinfl and BsuRl were used to generate restriction fragments and analysis of phylogenetic relationships among morels. The Amplified Ribosomal DNA Restriction Analysis (ARDRA) not only distinguished yellow morels from black morels but also separated related genera Mitrophora semilibera and Verpa conica from true morels. Simultaneously, each morel species could be separated from each other exhibiting considerable phylogenetic distances. The unique restriction fragment profiles generated by the restriction endonucleases enabled us to identify marker fragments to distinguish each species within and amongst the morel group. Since no intra-specific variation in restriction profiles by the six restriction endonucleases could be visualized among monospores, the technique could be used for rapid identification of wild morel specimens as a cheap alternative to direct sequencing for germplasm cataloguing.     

The Sequence of the Hartmannella vermiformis Small Subunit rRNA Coding Region

ABSTRACT The Hartmannella vermiformis small-subunit rRNA coding region was amplified, and the amplified DNA was cloned and sequenced. The coding region is 1,840 nucleotides long, and is typical of eukaryotic rRNA genes in both size and composition. Different clones contained different nucleotides at three positions.